Physicians Poster Sessions

EBMT 2009

Acute leukaemia P433 A survey on the outcomes following allogeneic haematopoietic stem cell transplantation for adult P432 patients with acute lymphoblastic leukaemia in fi rst Leukaemia-free survival of AML patients conditioned with relapse: a retrospective study on behalf of the ALWP FBM or other RIC protocols prior to allogeneic stem cell from the EBMT transplantation S. Santarone, M. Labopin, P. Di Bartolomeo, R. Arnold, R. Marks, M. Labopin, H. Bertz, T. de Witte, V. Rocha, J. Finke J. Finke, H.J. Kolb, C. Cordonnier, M. Baccarani, A. Fassas, on behalf of the Acute and Chronic Leukemia Working Parties M. Michallet, G. Ehninger, B. Samey, V. Rocha on behalf of the ALWP EBMT Conditioning with reduced intensity protocols (RIC) prior to allogeneic stem cell transplantation (SCT) has been proven The outcome after HSCT for adult patients with ALL is largely successful in the treatment of hematologic malignancies in dependent on disease status at the time of transplantation. patients with higher age or comorbidities, formerly regarded as Indication of allogeneic HSCT in patients with active disease at uneligible for treatment with SCT and standard conditioning. transplant is controversial. This retrospective study was aimed Nevertheless, control of advanced or active myeloid disease to analyze the results of HSCT in adult patients with ALL receiv- relies more on the dose intensity of the conditioning protocol ing HLA-identical or matched unrelated donor (MUD) myeloab- than on the graft versus leukemia effect and is hampered in lative HSCT while being in fi rst relapse of their disease between regimens with minimal intensity. Recently we described a 1990 and 2006. The primary and secondary endpoints of this conditioning protocol with reduced toxicity including fl udarab- study were the 3-year overall survival (OS) and the remission ine, melphalan and BCNU (FBM group), with particular activ- rate following HSCT. The EBMT registry received reports of 532 ity against advanced diseases with an acceptable rate of non patients (332 HLA-identical sibling and 200 MUD transplants). relapse mortality (Blood, 2008, 112: 415). Here we retrospec- Their median age was 29 years (16-65). There were 333 males tively compared FBM treated AML patients with patients treated and 199 females. The ALL immunological distribution at diag- with various TBI-, busulfan- or melphalan- containing RIC pro- nosis was: B-lineage 253, T-lineage 114, other 42, missing 123. tocols registered within the EBMT (RIC group). The two groups Sixty two patients were affected by Philadelphia chromosome differed signifi cantly not only in size (FBM group=130pt., RIC positive ALL. The median number of white blood cells at diagno- group=625pt.) but also in AML characteristics (sAML pt. in sis was 26x109/L (0.4-746). The median intervals from diagnosis FBM=47%, in RIC=88%), status at SCT (active disease/upfront to fi rst complete remission (CR1), from CR1 to relapse, and from SCT: 83% v. 17%), median patient age (63y v. 56y), and use CR1 to HSCT were 45, 140, 193 days respectively. The median of unrelated donors (67% v. 31%). Nevertheless, the 2-year blasts in the bone marrow (BM) at transplant was 8,5% (0-93). leukemia free survival (LFS) of AML patients in remission (CR1/ Total body irradiation (TBI) was used as conditioning regimen CR2) at SCT did not differ signifi cantly between the two groups in 400 out of 532 patients. Most patients (308, 58%) obtained a (FBM=32%(±10%) v. RIC=49%(±3%), p= 0,14). Patients with CR following HSCT. The 3-yr OS for all patients was 19+2%. In active disease at SCT (FBM=107pt., RIC=109pt.) differed in univariate analysis, 3 factors were signifi cantly associated with the use of unrelated donors (FBM=71% v. RIC= 36%). 2-year a better 3-yr OS: type of donor (HLA-identical sibling vs MUD, LFS in patients with active AML transplanted from a HLA iden- 21+3% vs 17+3%, p=0.03; no circulating blasts at transplant tical sibling was 30%(±5%) in the FBM group and 17%(±5%, (28+6% vs 14+5%, p=0,01) and lower number of BM blasts p=0.12) in the RIC group. Results for 2-year LFS with unrelated (<8% vs >8%, 33+6% vs 12+4%, p=0,004). There was no sig- donors were 38%(±6%) and 35%(±8%, p=0.41), respectively. nifi cant impact of other potential prognostic variables studied. Defi nitive conclusions between AML patients treated with FBM In multivariate analysis, performed for 102 patients with enough or other RIC protocols are diffi cult to make by this analysis information, there were 2 factors signifi cantly associated with due to the signifi cant heterogeneity of the two study groups. a decreased 3-yr OS: higher number of blasts in the BM >8% Anyway, despite the increased patient age in the FBM group (hazard ratio 0,49, 95% CI 0,31 to 0,76, p=0.002) and older treatment outcomes were comparable with a trend of better patients >29yr (HR 0,6, 95% CI 0,38 to 0,94, p=0.03). Our study LFS in AML patients with active disease and related donors. confi rms that HSCT is an effective salvage regimen for patients These data once more emphasize the fact that RIC protocols with ALL in fi rst relapse, mainly for young patients with lower show substantial differences in cytotoxicity and the choice of tumor burden. Half of patients achieve the CR after HSCT and the appropriate conditioning regimen should not only be guided a small but signifi cant proportion of them can benefi t in terms of by patient factors like age or comorbidities but also by charac- 3-yr overall survival either from related or unrelated donor. The teristics of the underlying malignant disease. number of blasts in BM has an important impact on OS.

S85 P434 analysis of 48 adults with high risk Ph negative ALL undergoing Fludarabine and PK-targeted intravenous busulfan before T- cell depleted (TCD) URD-HSCT in CR1 reported to the Brit- allografting for adult acute lymphoid leukaemia ish Society of Blood and Marrow Transplantation Registry from S. Santarone, M. Alsina, E. Ayala, T. Field, M. Kharfan-Dabaja, 1993 to 2005. Median follow-up of survivors was 56.3 months L. Ochoa, L. Perez, J. Perkins, J. Raychaudhuri, D. Sullivan, (range 18-160). Median age was 26.2 years (range 15.9-50). H. Fernandez, C. Anasetti Sixty seven percent of transplants were matched at 10 of 10 Department of Blood and Marrow Transplantation Moffi tt Cancer loci and 33% were mis-matched (27% at one allele only and 6% Center (Tampa, US) at two). TCD was carried out by in-vivo Campath administration in all recipients with additional ex-vivo TCD in 21% of patients. Remission consolidation with allogeneic blood or marrow The estimated actuarial overall survival (OS), disease free sur- transplantation improves survival of young patients with acute vival (DFS) and non relapse mortality (NRM) were 61%, 54% lymphoid leukemia (ALL), but the potential benefi t of transplan- and 13% at 5 years respectively. The overall incidence of Grade tation in older patients is offset by regimen toxicity and non- II-IV and III-IV acute GVHD was 26% and 8% respectively. The relapse mortality. Busulfan (BU) is not thought to be an effective actuarial estimate of extensive chronic GVHD was 25% at drug for ALL, presumably because of intrinsic resistance of ALL 5 years. Adverse cytogenetics at diagnosis was the only factor to alkylating agents, or perhaps because of the large variability associated with poorer 5 year DFS (41 vs 61%). T-cell depleted in BU pharmacokinetics and erratic drug exposure. Here we URD HSCT can result in good OS and low NRM for adults with report results of treatment with a PK-targeted intravenous BU high risk ALL in CR1 and merits prospective evaluation. regimen in 35 adults with ALL. Patient age was 19-55 (median 38) years, 21 were treated in fi rst complete remission (CR1), 4 in CR1 following primary induction failure (PIF), 9 in CR2, P436 and 1 in CR3. Treatment was with 4 consecutive daily doses High-resolution genome-wide analysis of copy number of fl udarabine (FLU) 40 mg/m2, followed by i.v. BU, given on abnormalities in adult acute myeloid leukaemia using days 1 and 2 at 130-145 mg/m2 daily over 4 hours with PK- single nucleotide polymorphism arrays identifi ed poor sampling and mass spectrometry assay. On days 3 and 4 risk pts candidate to allo-BMT BU dose was adjusted to target an average area under the I. Iacobucci (1), E. Ottaviani (1), F. Salmi (1), A. Astolfi (2), concentration curve of 5300+530 mMol*min/L for each of the N. Testoni (1), S. Luatti (1), C. Papayannidis (1), P. Giannoulia (1), four days. Donors were siblings (19), or unrelated (16). Grafts P. Paolini (1), PP. Piccaluga (1), D. Cilloni (3), F. Messa (3), were T-replete, fi lgrastim-mobilized hematopoietic blood cells. F. Arruga (3), C. Dell’Agnola (4), S. Cingarlini (4), D. Russo (5), Graft-versus-host disease (GVHD) prophylaxis was tacrolimus M. Baccarani (1), G. Martinelli (1) plus methotrexate or mycophenolate mofetil. Mortality from (1)Dpt. of Hematology/Oncology Seràgnoli (Bologna, IT); all non-relapse causes was 6% at 100 days, and 9% at one (2)Pediatric Oncology and Hematology “L. Seràgnoli” (Bologna, year. Causes of death were GvHD in 2 patients and infection IT); (3)Hematology (Orbassano, Turin, IT); (4)Policlinico GB in 1. Eight patients had leukemia relapse between day 20 and Rossi (Verona, IT); (5)Chair of Hematology and Unit of Blood 426 (median, day 82) post-transplant. Seven died of progres- Diseases and Cell Therapy (Brescia, IT) sive disease and 1 was reinduced back in to CR. The one-year relapse-free survival (RFS) for all patients is 68%, with a median Introduction: Acute myeloid leukemia (AML) is a heterogene- follow-up of 1.3 years for live patients. For the 21 patients trans- ous disease with various chromosomal aberrations. The karyo- planted in CR1, the one-year RFS is 86%, whereas it is 43% type at diagnosis provides important prognostic information that for the 14 patients transplanted in more advanced stage. The infl uences therapy and outcome of this disease. However, using one-year RFS is 72% in patients up to 40 years, and 64% in conventional chromosome banding techniques alone, karyo- patients 41-55 years old, with disease status and stage simi- type abnormalities are detected in only half of all AML cases. larly distributed in younger and older cohorts. With this treat- Aims: We sought to identify novel genomic regions of interest in ment protocol, the one-year non-relapse mortality is identical normal karyotype AML and to identify novel candidate regions to what is observed with non-transplant therapies. When com- and disease-related genes in patients with complex karyotypes pared to irradiation-containing regimens, FLU and PK-targeted using genome-wide high resolution single nucleotide polymor- BU appear much safer and similarly effective, thereby providing phism (SNP)-array. a treatment option for all adult patients with ALL. A multicenter Patients and Methods: 40 AML patients were analyzed until study comparing this transplant protocol against post-remission now. Cases included FAB-M0, M1, M2, M4, M5, miscellane- chemotherapy for adult ALL is warranted. ous cytogenetic abnormalities and normal karyotype. 250 ng of genomic DNA were processed on 500K SNP array according to protocols provided by the manufacturer. P435 Results: A wide spectrum of different genetic lesions (gains/ T-cell depleted unrelated donor stem cell transplants losses) involving complete chromosome arms (del 16q, appear to be of value for adult Philadelphia chromosome i(13q10), del 3p, del 7p, monosomy 9) or submicroscopic negative ALL patients and should be evaluated genomic intervals were identifi ed in a substantial proportion of prospectively in new large group studies cases without differences in the frequency of losses or gains. B. Patel, K. Kirkland, R. Pearce, R. Szydlo, R. Clark, Focal genetic alterations were detected at the breakpoints of C. Craddock, E. Liakopoulou, A. Fielding, S. Mackinnon, previously cytogenetically identifi ed chromosomal transloca- E. Olavarria, M. Potter, N. Russell, G. Cook, B. Shaw, tions, such as t(2;3)(p22-23)(q26-27) and t(1;11)(p32;q23). A. Goldstone, D. Marks on behalf of the British Society of Blood The most frequent genomic gains affected: 9p12 (ZNF658B, and Marrow Transplantation FOXD4L2), 22q11.1 (PPYR1), 5q31-q33 (CDX1) and 8p23.2 (CSMD1). The most frequent deletions were identifi ed in Approximately 40% of adults with Philadelphia chromosome ACTBL1 (22q11), NF1 (17q11.2) and often in regions lacking (Ph) negative acute lymphoblastic leukaemia (ALL) achieve annotated genes. Other recurring genetic lesions were uncom- long term survival following unrelated donor haematopoi- mon. Some lesions affected regions with a single gene, such etic stem cell transplantation (URD-HSCT) in fi rst complete as: ETAA1, FIGN, STK32B, PRAGMIN, PCM1, GLIS3, MRG- remission. Despite curative potential, the higher risk of severe PRX1, SESN3, BCL2L14. Marked differences in the combi- graft-versus-host-disease (GVHD) accompanying URD-HSCT nation of copy number anomalies were identifi ed across the remains a major barrier to achieving overall success. Although different genetic subtypes of AML. Patients with normal karyo- T-cell depletion of URD grafts decreases the incidence and type showed no relevant genetic alterations. severity of GVHD its impact on graft versus leukaemia effects Conclusion: These data demonstrated that, in contrast to adult in ALL is not known. We report a retrospective multicentre acute lymphoblastic leukaemia (ALL), AML is characterized by

S86 relatively few recurring copy number alterations, and that spec- We retrospectively evaluated 236 pat.with AML in two cohorts trum of genetic anomalies is signifi cantly associated with AML (108 pat. in 1st cohort transpl.from an HLA-ident.sibling donor, disease subtype. and 127 pat. transpl. from an HLA-ident.URD. Pat. were trans- Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, planted in Essen and Rostock. For the defi nition of a CMV-R Fondazione del Monte di Bologna e Ravenna, PIO project treatment with ganciclovir and the detection of pp65 pos.cells 2007, Strategico di Ateneo. in PMCs at minimum at two different occasions were required. For in vitro studies we used the cell lines Kasumi-1 (AML-M2), SD-1 (ALL) and K562 (CML in blast crises) and infected them P437 with the AD169 CMV strain. 14 days after infection we evalu- Sequential conditioning in high-risk leukaemia: ated the apoptosis rate by measuring annexin V by FACS, the a single-centre experience in 116 patients proliferation rate by BRDU assay, the expression of MRD and G. Kobbe, M. Kondakci, A. Czibere, I. Bruns, S. Balleisen, apoptosis marker p21, c-myc. R. Fenk, L. Distelmeier, T. Graef, U. Germing, R. Haas Infection of CMV in Kasumi-1 cells and SD-1 cells induced in Heinrich Heine University (Düsseldorf, DE) 99.8% and 31.3% of cells apoptosis, whereas no impact on the apoptosis rate was seen for K562 cells. HCMV-induced Allogeneic stem cell transplantation is a curative therapy for cell death in AML cells was mediated by a caspase-dependent many patients with leukemia. However, patients with high-risk mechanism and could be prevented by the specifi c caspase disease as defi ned by adverse genetic features, early relapse inhibitor zVAD.fmk. These results were in concordance with the or chemorefractory disease have a poor prognosis with conven- clinical observation of a CMV-R after transplant in AML, ALL, tional transplant protocols. Sequential conditioning regimens and CML. AML-Patients with a documented CMV-R had in both like the Munich FLAMSA approach have shown promising cohorts a markedly reduced risk for relapse (prob. at 5-year results in these patients. We here report a single center experi- 9.2% versus 52.6% in cohort 1 (p<0.0005) and 12.2% versus ence with a FLAMSA based conditioning regimen in myeloid 47.2% in cohort 2 (p<0.013), which resulted in a sign.improved leukemia and acute lymphoblastic leukemia. OS cohort 1 OS 73.6% versus 42.5% (p<0.04) and 48.0% ver- One hundret patients with high-risk AML, MDS or blast transfor- sus 33.5% in cohort 2 (p<0.04). There were no sign. differences mation of a myeloproliferative disorder and sixteen patients with in the characteristics of pat. with or without CMV-R. Patients high-risk ALL were treated within a sequential conditioning pro- with CMV-R had a higher incidence of acute GVHD grade 2 - 4 tocol at the Heinrich Heine University Duesseldorf. Patients fi rst (82% versus 38%, p <0.0001), but this was also seen in pat.with received a Fludarabine, HD-Ara C, Amsacrine (FLAMSA) based ALL and CML who did not have a reduced risk for relapse after induction therapy and directly proceeded to conditioning with a CMV-R. When stratifi ed according to the occurrence of acute Melphalan and Thiotepa (Mel/Thio, n=36), Melphalan (Mel/mono GVHD, the probability for risk of relapse remained sign.lower in n=64) or TBI (12Gy, n=16, all ALL) during FLAMSA induced pat.with a CMV-R compared to their counterparts (p<0.003). In cytopenia. Transplantation of PBPCs from matched related (n= multivariate analyses the sign.factors impacting the outcome 39) or unrelated (n= 77) donors was accompanied by a GvHD and relapse rate were disease stage, chronic GVHD, unfavora- prophylaxis with Tacrolimus and Mycophenolatemofetil. Patients ble cytogenetics and CMV-R. who had unrelated or mismatched donors received ATG. Our data indicate an unrecognized role of CMV reactivation As a consequence of the fi rst interim analysis after 50 patients after HSCT for AML, which is probably mediated by CMV Thiotepa was omitted from the conditioning protocol because of induced apoptosis. an unacceptable high late treatment related mortality in patients receiving Mel/Thio (p<0.05 vs Melphalan monotherapy). In a second analysis after a median follow up of 647 days (range P439 44-2159) the projected 2 year survival among 100 patients with Expression of different isoforms of the B-cell mutator myeloid disease (median age 51 years, range 17-67) was 41% activation-induced cytidine deaminase in BCR-ABL1- (47% with Mel/mono and 34% with Mel/Thio, p=ns). Relapse positive acute lymphoblastic leukaemia patients identifi ed rate was 25% with Mel/Thio and 42% with Mel/mono (p=ns). poor risk group pts candidate to an allo-BMT Among the 16 patients with high-risk ALL (median age 26 years, A. Lonetti (1), I. Iacobucci (1), A. Ferrari (1), M. Messina (2), range 18-54) 4 died due to relapse and 2 of treatment related D. Cilloni (3), S. Soverini (1), S. Chiaretti (2), E. Ottaviani (1), complications. After a median follow up of 1004 days (range N. Testoni (1), C. Papayannidis (1), F. Messa (3), F. Salmi (1), 194-1489) 10 patients are alive and free of disease including 3 A. Vitale (2), F. Arruga (3), F. Pane (4), PP. Piccaluga (1), patients who had refractory ALL before transplantation. C. Dell’Agnola (5), S. Cingarlini (5), A. Gnani (1), S. Paolini (1), We conclude that a sequential conditioning approach using G. Saglio (1), M. Baccarani (1), R. Foà (2), G. Martinelli (1) FLAMSA followed by either HD-Melphalan or full dose 12 Gy (1)Dpt. of Hematology/Oncology Seràgnoli (Bologna, IT); TBI is safe and effective in patients with poor risk leukemia. (2)University La Sapienza (Rome, IT); (3)Hematology Further studies will have to focus on early identifi cation of (Orbassano, IT); (4)CEINGE Biotecnologie Avanzate (Naples, patients who are at highest risk for relapse to initiate effective IT); (5)Policlinico GB Rossi (Verona, IT) post transplant therapies. Background and aim: Since at much lower frequency the acti- vation-induced cytidine deaminase (AID) enzyme can target P438 non-Ig genes and may even act as a genome-wide mutator, we Cytomegalovirus induces apoptosis in acute myeloid investigated AID expressed in BCR-ABL1-positive acute lym- leukaemia and thereby reduces markedly the risk for phoblastic leukaemia (ALL) and in chronic myeloid leukemia relapse after transplant (CML) at the time of progression to blast crisis. A.H. Elmaagacli (1), M. Koldehoff (1), T. Gromke (1), B. Opalka Patients and Methods: We analyzed 61 adult de novo BCR- (1), I. Hilgendorf (2), R.S Ross (1), R. Trenschel (1), S. Bauer ABL1-positive ALL patients (pts) and 60 CML (chronic phase (1), L. Kordelas (1), H. Ottinger (1), D. Beelen (1) and myeloid/lymphoid blast crisis) pts. AID cDNA was amplifi ed (1)University Hospital of Essen (Essen, DE); (2)University with two pairs of oligonucleotides, the forward primer of each Hospital of Rostock (Rostock, DE) couple conjugated with a fl uorescent dye (fl uorescein) at its 5’ end. PCR products were then loaded on the ABI Prism 3730 CMV-reactivation (CMV-R)after transplant occurs frequently DNA Analyzer for automated capillary gel electrophoresis and and is generally thought to be associated with an increased the results were plotted with the AbiPrism GeneMapper v3.5 TRM. We aimed to evaluate the cytotoxic effects of CMV on software (Applied Biosystems). AML in vitro, and further, the clinical outcome of patients with Results: On the 61 de novo adult BCR-ABL1-positive ALL AML with a documented CMV-R after transplant. pts, AID mRNA and protein were detected in 36 (59%); their

S87 expression correlated with BCR-ABL1 transcript levels and and 58% at 3 years. EFS at 3 years was signifi cantly better in disappeared after treatment with tyrosine kinase inhibitors at those with chronic GVHD (84% vs 49%; p=0.03) and in those the time of remission. AID expression was also found in lym- in complete remission at transplant (64% vs 36%; p=0.05). The phoid blast crisis CML (50%), but not in myeloid lineage or in use of alemtuzumab minimises toxicity from acute GVHD but chronic phase CML. Different isoforms of AID were identifi ed: permits a subsequent GVL effect in patients with high-risk AML, 13/61 (21%) pts expressed the full-length isoform; 19/61 (31%) resulting in successful transplantation using HLA matched and co-expressed the wild-type and different AID splice variants mismatched unrelated donors. with deaminase activity (AIDDeltaE4a, with a 30 bp deletion of exon 4; AIDDeltaE4, with the exon 4 deletion; AIDins3, with the retention of intron 3-4); 4/61 (7%) expressed the AIDDel- P441 taE3-E4 isoform without deaminase activity (deletion of exons Clofarabine ± fl udarabine with IV busulfan as 2 and 3). To investigate whether AID introduces DNA-single reduced-toxicity conditioning therapy for allogeneic strand breaks, we performed a genome wide analysis by 250K stem cell transplantation (Allo-SCT) in advanced myeloid NspI single nucleotide polymorphism (SNP) array (Affymetrix leukaemia/MDS Inc., USA). Patients who expressed wild-type AID had a higher B. Andersson, M. de Lima, L. Worth, P. Thall, U. Popat, number of alterations compared to AID-negative (median copy R. Jones, E. Shpall, C. Hosing, T. Madden, R. Kazerooni, number alteration of 14 versus 4. respectively, p<0.03). Recur- P. McAdams, A. Alousi, P. Kebriaei, R. Champlin ring copy number abnormalities were identifi ed in genes with an UT MD Anderson Cancer Center (Houston, US) established role in leukemogenesis, such as IKZF1, CDKN2A, CDKN2B, PAX5, MELK, BTG1 and MDS1. Introduction: To augment the antileukemic activity of the reduced Conclusions: Our fi ndings show that BCR-ABL1-positive ALL toxicity pretransplant regimen IV busulfan (Bu)-fl udarabine (Flu) cells aberrantly express different isoforms of AID that can act as we combined or replaced Flu with Clofarabine (Clo). Because mutator outside the Ig gene loci in promoting genetic instability the immunosuppressive/engraftment-promoting capacity of Clo in leukemia cells. is unknown, we used Bayesian adaptive randomization to pro- Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, tect patient safety while evaluating Clo±Flu with IV Bu. Fondazione del Monte di Bologna e Ravenna, Strategico di Patients and methods: Randomization: Arm I: Clo:Flu 10:30 Ateneo, GIMEMA Onlus. mg/m², Arm II 20:20 mg/m², Arm III 30:10 mg/m², Arm IV only Clo 40 mg/m². The Clo and Flu were infused over 1 hr each, Bu was given over 3 hr targeting a daily AUC of 6,000 mcMol-min P440 each given once daily for 4 days. The adaptive randomization Successful myeloablative transplantation in high-risk was based on a 2:1 weighted average of the posterior probabili- acute myeloid leukaemia using unrelated donors with ties of 30-day transplant-related mortality and response (alive at alemtuzumab for T-cell depletion day 30 w/o active disease, with >90% donor cells). 28 patients K. Thomson (1), P. Kottaridis (2), E. Morris (3), R. Chakraverty are enrolled, evaluable for engraftment and survival beyond (3), K. Peggs (3), S. Mackinnon (3) day +30. M/F: 17/11, med. age 45 yr (range: 6-59). 7 had CML (1)University College Hospital (London, UK); (2)Royal (BC: 1, AP2: 2, AP1: 3, late CP1: 1). 21 had AML (induct. failure: Free Hospital (London, UK); (3)University College London 7, refr. Relapse: 5, untx Rel-1: 1, untx Rel-2: 3, CR2: 2, high-risk (London, UK) CR1: 2, tx-refr MDS: 1). No AML/MDS patients had favorable cytogenetics (CG), 11 had intermediate, and 10 poor prognosis T cell replete transplantation from unrelated donors maximises CG. GVHD-prophylaxis was tacrolimus and mini-MTX, and with the graft-versus-leukemia (GVL) effect but at the cost of signifi - one Ag mism. or unrel. graft rabbit-ATG (Thymoglobulin™) (TD cant graft-versus-host disease (GVHD). We report 51 consecu- 4 mg/kg) was used. tive patients with acute myeloid leukemia (AML) at high risk of Results: All patients engrafted (3 progressed early). DNA chi- relapse, transplanted using unrelated donors with T cell deple- merism; Groups I+II (lower dose Clo, n=10) had a median of tion. Patients were considered high-risk on the basis of primary 96% (range 17-100) donor (T-cell)-derived DNA, groups III+IV induction failure (>5% blasts after at least 2 courses of inten- (higher dose Clo, n=17) a median of 100% (range 64-100) sive multi-agent chemotherapy; n=7); adverse cytogenetics (-5, donor-DNA by day +30. By day +100 both cohorts had a median -7, del(5q), abn 3q, >4 abnormalities; n=18); >CR1 (n=28), sec- of 100% donor-derived DNA, stable beyond 6 months in evalu- ondary AML (myelodysplastic syndrome, n=3; therapy-related, able patients. On December 15, 2008, 15 patients are alive in n=5). At transplant, 11 had refractory disease and 1 untreated CR (median F/u 11 mos, range 1-24). 11 pats died, none was relapse. Median follow-up was 30 months (3-84), donors were regimen-related; Progr Dx: 6, GVHD ± infection: 3 (2 before HLA-mismatched at up to 3 loci (HLA-A, B, C, DRB1 or DQB1) Day+100), infection alone: 1, CHF: 1, and 2 are alive with AML. in 20/51 (39%), 15 patients were female (29%), and median Main toxicity was mucositis grade 2-3 in half the patients. One age was 36 years (13-57). Conditioning was with fl udarabine patient had moderate liver toxicity, but there was no VOD or 30mg/sq.m days -9 to -7, cyclophosphamide 60mg/kg on day -6 signifi cant neurologic toxicity. and -5, 14.4 Gy TBI in 8 fractions over 4 days, day -3 to 0, and Conclusions: progenitor cells were mobilised from peripheral blood in 44/51 1) Clo- IV-Bu-based conditioning is safe in Chemo-refractory, (86%). Cyclosporin was given at 3mg/kg from day -1 and 20 mg high-risk patients, alemtuzumab was added to the progenitor cell bag 30 minutes 2) Clo is adequately immunosuppressive to support allo SCT, prior to infusion on day 0. All patients engrafted. Acute GVHD 3) additional studies are warranted to fully evaluate the anti- grade II-IV occurred in 7/51 (14%; grade II in 6, grade IV in 1), leukemic activity of Clo±Flu with - IV Bu ±- rabbit-ATG as pre- extensive chronic GVHD occurred in 10/44 assessable (23%; transplant conditioning therapy in myeloid leukemia / MDS. cutaneous only in 6) and cutaneous limited chronic GVHD in Supported by NIH grants CA55164 and CA49639. 10/44 (23%). CMV reactivation occurred in 21 of 27 patients at risk (78%). There were 10 non-relapse deaths, 9/10 within 10 months of transplant, giving 3 year NRM of 19%, with no sig- nifi cant impact of HLA-mismatching or the presence of GVHD. Eleven patients relapsed at a median of 6 months from trans- plant (2-15), giving a 3 year relapse risk of 24%. Relapse was signifi cantly reduced in those patients in complete remission at transplant (n=39; p=0.03) and in those with chronic GVHD (n=20; p=0.007). Overall survival was 65% at 1 year and 57% at 3 years, and event-free survival (EFS) was 63% at 1 year

S88 P442 At relapse, 15 patients receveid only palliative therapy; a Allogeneic transplantation in adult AML 1st CR: better reinduction therapy was offered to 38 patients, of whom 24 results with unrelated donors compared to HLA matched receveid fl udarabine and cytarabine-containing regimen (FLA) sibling transplants while 14 receveid FLA plus idarubicin (Ida-FLA). 5 (21%) of F. Bonifazi, G. Bandini, E. Colaci, M. Arpinati, M. Stanzani, 24 patients treated with FLA and 7 (50%) of 14 treated with S. Usai, A. Bontadini, A. Curti, S. Paolini, S. Rizzi, M.R. Motta, Ida-FLA achieved CR. 7 (29%) and 2 (14%) were early deaths V. Giudice, M. Baccarani in FLA and Ida-FLA groups respectively. Overall, 18 patients S. Orsola-Malpighi Hospital (Bologna, IT) performed a second HSCT of whom 9 (6 CR) after Ida-FLA and 9 (4 CR) after FLA. Comparing the two different reinduction The role of allogeneic transplantation in 1 st CR of adult AML, strategies, the Ida-FLA group showed longer interval between whether from HLA identical siblings or volounteer unrelated HSCT and relapse (11 versus 6 months, p=0.08) and larger pro- donors, is subject to debate. We present here ours results on portion of CR patients at fi rst HSCT (86% versus 56%, p=0.06) sixty one patients-37 HLA-identical siblings and 24 unrelated- in spite of a greater median age (41 versus 37, p=0.09). All transplanted between 1998 and 2008. Their main demographic 10 patients who were transplanted with advanced disease died chararcteristic were: median age 42 yrs (HLA identilcal sibling) with a median survival of 3 months. Of the 8 patients in CR at vs 39 yrs (VUD). 14 unrelated transplants were fully matched second HSCT, 4 are alive and disease-free with a median fol- (10/10 at high resolution), with the remaining patients being 9/10 low-up of 30 months. At any rate, all these 4 patients were in or 8/10. The source of stem cells was predominantly PBSC in CR1 at fi rst HSCT. the sibling group (83%) while it was bone marrow in the VUD Conclusions: Although retrospectively analyzed and not statisti- group (84%). The preparative regimens were myeloablative in cally signifi cant, the differences between the two reinduction nearly 90% of the cases: they were based on Busulfan and groups are due to the fact that a more aggressive therapy was Cyclophosphamide in 76% of the sibling group vs 50% in the adopted in patients that, whilst older, had greater probability to unrelated group, while TBI/ Cyclophosphamide was used only reach CR and to perform a second HSCT. Our results confi rm in 5% of the sibling and 35% of the VUD groups. Low dose ATG that a second HSCT, although the most effective treatment in (Fresenius, Bad Homburg) was used in all the VUD and 21% this prognostically dismal cathegory of patients, offers a real of the sibling transplants, from days -6 to day -2, at a total dose chance of cure only to patients who performed fi rst HSCT in of 15-30 mg/kg. Prophylaxis of GVHD consisted of CsA and CR1 and who achieved CR with reinduction therapy. short term, four doses MTX. Supportive measures consisted of protective isolation, HEPA air fi ltration, fl uconazole, acyclo- vir and cotrimoxazole/trimethoprim prophylaxis, weekly CMV P444 monitoring with preemptive intervention. All patients, except A single-centre experience with allogeneic one, engrafted. The incidence of aGVHD, grade III-IV was 8% haematopoietic stem cell transplantation for acute in both groups; the incidence of overall cGVHD was 43% and lymphoblastic leukaemia: analysis of factors leading to 29% in the sibling vs unrelated transplants, respectively and of improved outcome extensive cGVHD 27% vs 12.5%. The 5 year actuarial prob- P. Kaczmarek, M. Potter, M. Ethell, J. Treleaven, F. Davies, ability of TRM was 23% in the siblings and 17% in the unre- C. Dearden, G. Morgan, B. Shaw lated transplants. The 4 yr actuarial relapse risk was 25% in The Royal Marsden Hospital (Sutton, UK) the sibling group and 6% in the VUD group. With a median follow-up of 56 months, OS is 61% for the siblings vs 77% of Allogeneic haematopoietic stem cell transplantation (SCT) the VUDs. Within the VUD group, the patients matched at 10/10 remains the only curative option for many patients with acute fared better in term of TRM, relapse and OS compared to those lymphoblastic leukaemia (ALL). We reviewed the results of SCT who were matched at a lesser degree (8 or 9/ 10); the overall in a single institution treating both children and adults, over a survival of the 10/10 is 92% at 6 years. 16-year period (1991-2007). The study included 196 patients. These results show a better outcome of the VUD vs siblings, The median age was 16 years (range: 2-60). The donor was following a myeloablative regimen, because of slightly less a sibling in 99, unrelated (UD) in 93, mismatched relative in 4. TRM and much less relapse in the VUD population, despite SCT was performed in CR1 (71), CR2 (83), >CR2 (24) or with ATG-F pre-transplant; thus allogenic transplantation remains a persistent disease (16). 16% of patients were Philadelphia pos- strong indication as post remission therapy in adult AML. itive, 22% had T-cell ALL, 8% had a prior autograft. The major- ity of patients received TBI. 78% of the patients with an UD had in-vivo T-cell depletion. The overall survival in the whole group P443 was 48% and 42% at 5 and 10 years respectively. The relapse The outcome of adult patients affected by acute myeloid risk (RR) and non-relapse mortality (NRM) were 22% and 29% leukaemia relapsing after allogeneic transplant: at 3 years. Acute GvHD was seen in 64% of patients (grade a single-centre experience II-IV in 29%). There was an improvement in outcome depend- I. Cutini, S. Guidi, C. Nozzoli, B. Bartolozzi, F. Mannelli, ent on the time period over which the SCT was performed. The G. Gianfaldoni, S. Bencini, A. Bosi 3-year survival was 33%, 51% and 57% in those transplanted AOU Careggi (Florence, IT) from 1991-1995, 1996-2003 and 2004-2007 (p=0.03). This appeared to be due to a decrease in the NRM in more recent Objectives: The aim of the study was to evaluate the outcome years (40%, 32% and 18%, p=0.04). There was no signifi cant of patients affected by non-promyelocytic Acute Myeloid Leuke- difference in OS, NRM or RR based on the use of a sibling mia (AML) who relapsed after an allogeneic transplantation of or UD. SCT beyond CR2 was associated with a worse out- hematopoietic stem cells (HSCT). come in adults (p=0.01) but not in children. Survival was worse Methods: We retrospectively analyzed the characteristics of in patients who had previous autograft (p=0.0001). Although this group of patients and the therapeutic strategies offered at only a small number of patients (10) received reduced inten- relapse. sity conditioning SCT, the OS was not different to those receiv- Results: Since january 1989 to december 2006, 157 AML ing myeloablative conditioning (2 years: 45% vs 53%, p=0.4). patients receveid an HSCT of whom 53 (34%) relapsed. We did observe a signifi cant difference in OS in adult (>16) Median age of the relapsed patients was 38 (range 15-60). 30 compared to paediatric patients (16 and younger), (32% vs (60%) patients were in complete remission (CR) at fi rst HSCT 62% at 5 years, p=0.0004), but no difference in disease relapse (20 in CR1 and 10 in CR2) and 23 (40%) in advanced disease. (28% in adults vs 24% in children at 3 years). In adult patients Median time between HSCT and relapse was 6 months (1-55). only one factor impacted on OS in multivariate analysis: a SCT 45 (85%) and 8 (15%) patients received HSCT from sibling and undertaken in 2004-2007 compared to earlier years (p=0.009). matched unrelated donor (MUD), respectively. In paediatric patients two factors impacted on OS: SCT

S89 performed after 1995 resulted in a survival benefi t (p=0.024), donor was searched and a consolidating allogeneic transplant and T cell phenotype in worse outcome (p=0.031). In conclu- scheduled within 2 – 3 months. sion, we report outcomes in a large group of patients trans- Results: Twentytwo pts. with AML in fi rst relapse and a median planted for ALL. We show an improvement in OS and NRM age of 46 (range 29-64) years are evaluable. Eighteen of 22 over time despite the inclusion of older patients and RIC regi- pts. (82%) achieved a second CR and 20/22 pts. (91%) pro- mens. Unrelated and sibling donors can be used to achieve a ceeded to a consolidating allogeneic transplant in CR2 (n=14) similar long-term outcome. or PR2 (n=4) from a related (n=3) or unrelated (n=17) donor within a median of 2,4 (1,9 - 4,5) months after HD-Mel. Treat- ment-related mortality was 0/22 after HD-Mel and 5/20 (25%) P445 after subsequent allograft; 7 pts. died due to relapse and 10 GvHD prophylaxis using low-dose cyclosporine is safe patients are alive (8 in CR) with a median follow-up of 40 (range and reduces the risk of relapse and death in leukaemic 25 – 90) months. The projected survival at 2 and 5 years is 50% recipients of HLA-identical sibling transplants and 42%, respectively. R. Olsson, M. Remberger, J. Mattsson, Z. Hassan, O. Ringdén Relapse after allograft: Of 20 allografted patients, 8 relapsed Karolinska University Hospital (Sweden, SE) after a median of 11 (range 2 – 33) months in the marrow (n=4) after a median of 5,6 (2 -33) months or extramedullary (EM) Four doses of methotrexate (MTX) and low-dose cyclosporine (spine, heart, gut) after a median of 15,5 (6 – 20) months (n=4). (CsA), starting at 1 mg/kg/day i.v. with early discontinuation, Leukemia markers in patients with extramedullary relapse were were given to 171 consecutive HLA-identical sibling recipients inv16 (n=2), FLT3-ITD (n=1) and FLT3-TKD (n=1). with leukaemia. Apart from MTX, retrospective controls (n=40) Conclusions: received CsA starting at 5-7.5 mg/kg/day i.v., and discontinued 1. In patients with fi rst relapse of AML, HD-Mel is superior to 1 year post-transplant. The target CsA trough levels were 100 any other reported regimen in achieving a second CR and ng/ml and 200-300 ng/ml in the low-dose and the control group, allowing a sequential allograft. respectively. In the low-dose group, the risk of acute GVHD 2. Half of the relapses after sequential auto-allograft were grades I-II was augmented (66% vs. 49%, P<0.01), whereas extramedullary, refl ecting control of leukemia by GvL in the grades III-IV did not differ between the groups (9% vs. 8%). The marrow and escape at sanctuaries. risk of chronic GVHD was markedly increased in those receiving 3. The association of extramedullary relapse with markers low-dose CsA (50% vs. 29%, P<0.01), and the cumulative pro- as inv16 and FLT3-mutations needs to be studied in more portion of relapse at 5 years post-transplant was increased in patients. the control group (51% vs. 26%, P<0.01). Moreover, the 5-year survival rate was 63% in the low-dose group compared to 40% in the controls (P=0.04). In a multivariate analysis, low-dose P447 CsA was the only factor associated with acute GVHD grades Outcome of secondary AML following BCR-ABL negative I-IV (RH 2.40, P=0.02). Signifi cant predictors of chronic GVHD myeloproliferative disorders were low-dose CsA (RH 2.56, P<0.01), chronic myeloid leu- S. Ayari, P. Chevallier, T. Guillaume, R. Garant, V. Dubruille, kaemia (RH 1.92, P<0.01), and administration of donator lym- T. Gastinne, S. Le Gouill, N. Blin, B. Mahe, J.L. Harousseau, phocyte infusions (RH 1.66, P=0.03). The risk of relapse was M. Mohty, P. Moreau, J. Delaunay counteracted by chronic GVHD (RH 0.46, P<0.01), whereas Service d‘Hematologie Hôpital Hotel Dieu (Nantes, FR) chronic GVHD (RH 0.53, P<0.01) and acute GVHD grades III-IV (RH 4.35, P<0.01) were the strongest factors associated Introduction: Incidence of leukemic transformation in BCR-ABL with patient death. Importantly, the transplant-related mortality negative myeloproliferative disorders reaches 10 to 15% after at 5 years post-transplant was similar in the low-dose CsA and a median of 15 years of follow up. The prognosis remains very control groups (18% and 20%, respectively, P=0.58). poor and available therapeutic options depending on patient In conclusion, a low-dose CsA regimen in leukemic recipients of and disease characteristics are limited. Only few series have HLA-identical sibling transplants increases the rate of chronic investigated the outcome of this population. GVHD, which seems to attenuate the risk of relapse, thereby Patients and methods: From January 1992 to July 2008, 62 improving patient survival. patients (36M/26F) were diagnosed with blastic transformation secondary to a myeloproliferative disorder (MPD). Median age was 66 (range 44-85)years. Eleven (18%) patients had primary P446 myelofi brosis, 25 (40%) had PV, 18 (29%) had ET and 8 (13%) HD-Mel phase II study in fi rst relapse of AML: high rate had unclassifi able MPD. Patients had received a median of 2 of extramedullary manifestation in subsequent relapses (range 0-4) prior therapies. Prior exposure to alkylating agent after sequential auto-/allograft and radioactive phosphorus was present in 35 (56%) and 4 H. Martin (1), R. Schwerdtfeger (2), B. Wassmann (1), (6,4%) patients respectively. Cytogenetics analysis performed S. Mousset (1), H. Pfeifer (1), H. Serve (1), G. Bug (1) in 24 patients was as follow, unfavourable Karyotype:12 patients (1)Clinic J.W. Goethe-University (Frankfurt, DE); (2)DKD and intermediate: 12 patients. Seven patients out of 12 tested (Wiesbaden, DE) for JAK2V617K mutation were found positive. Results: Twelve (19,3%) patients received induction chemother- Background: Patients with AML in fi rst relapse were previously apy, 8 (13%) low intensity chemotherapy (low dose aracytine, n=3 reported to have a long-term survival ranging between 10-15%. and azacytidine, n=5) and 35 (56,4%) only supportive care. Ten To improve survival we introduced salvage therapy with high- (16%) patients received an allogeneic haematopoietic stem cell dose melphalan and autologous PBSC followed by a sequential transplantation (allo-SCT) either as fi rst line therapy (7 cases)or allotransplant (Bug et al. Ann Hematol 2005; 84:748-54.) Here as consolidation after induction chemotherapy (3 cases). we present a long-term follow-up of this patient cohort. Forty one percent (5/12)of patients who received induction Objectives: chemotherapy achieved complete remission, 3 of them were (1) To improve second CR rate in relapsed AML; transplanted and 2 relapsed at 2 and 3,5 months following CR. 2) to increase the proportion of AML fi rst relapse patients Median overall survival (OS) for the all cohort was 7,9 months. achieving an allogeneic transplant; Interestingly, for patients who received allo-SCT in the course (3) to improve long-term survival in pts. with relapsed AML. of the disease (n=10), median OS was 18 months compared to (4) to study the pattern of subsequent relapses. 4,7 months for patients who not received transplants (n=52), Methods: AML patients in fi rst relapse received salvage therapy p=0,03. with 200 mg/m² melphalan and autologous PBSC, which had One year OS was 64% in SCT group versus 26% only for the been cryopreserved in early CR1. Subsequently an allogeneic others.

S90 Conclusion: Overall, leukemic transformation of MPD is associ- P449 ated with very poor outcome. Allo SCT appeared to be the best Busulfan and melphalan as conditioning regimen for therapeutic option in this subgroup of patients and this strategy autologous stem cell transplantation in acute should be proposed as often as possible when patients are eli- myelogenous leukaemia patients in fi rst complete gible and have an HLA identical donor. remission: a Gruppo Italiano Trapianto di Midollo Osseo (GITMO) retrospective study R.M. Lemoli (1), A. D’Addio (1), G. Marotta (2), V. Mettivier P448 (3), E. Zuffa (4), M. Montanari (5), A. de Vivo (1), A. Bonini (6), Prognostic factors in relapsed acute myeloid leukaemia P. Galieni (7), A. M. Carella (8), S. Guidi (9), M. Michieli (10), patients receiving an allogeneic haematopoetic stem A. Olivieri (11), A. Bosi (9) cell transplantation as salvage therapy: a retrospective (1)University of Bologna (Bologna, IT); (2)Stem Cell analysis on behalf of the Northern Italy Leukemia Group Transplantation Unit (Siena, IT); (3)Ospedale Cardarelli (Naples, (NILG) experience IT); (4)S. Maria delle Croci Hospital (Ravenna, IT); (5)University I.M. Cavattoni (1), E. Morello (1), E. Oldani (2), T. Intermesoli of Ancona (Ancona, IT); (6)Santa Maria Nuova Hospital (2), E. Audisio (3), C. Minotto (4), E. Terruzzi (5), E. Borlenghi (Reggio Emilia, IT); (7)Ospedale Mazzoni (Ascoli Piceno, IT); (6), G. Rossi (6), E. Pogliani (5), F. Marmont (3), A. Rambaldi (8)IRCCS Ospedale “Casa Sollievo della Sofferenza” (San (2), R. Bassan (2), S. Cortelazzo (1) Giovanni Rotondo, IT); (9)University of Florence (Florence, IT); (1)Central Hospital (Bolzano, IT); (2)Ospedali Riuniti Bergamo (10)Cell Therapy and Stem Cell Transplantation Unit (Aviano, (Bergamo, IT); (3)AOU San Giovanni Battista (Turin, IT); IT); (11)Ospedaliera San Carlo (Potenza, IT) (4)Ospedale di Noale (Noale, IT); (5)New Hospital San Gerardo (Monza, IT); (6)Spedali Civili (Brescia, IT) Between 1997 and 2008, 129 acute myelogenous leuke- mia (AML) patients in fi rst complete remission (CR) received Only few acute myeloid leukaemia (AML) relapsed patients busulfan and melphalan (Bu/Mel) as conditioning regimen prior achieve a long term survival after allogeneic haematopoetic autologous stem cell transplantation (ASCT) in ten different stem cell transplantation (alloSCT). Adequate prognostic cri- Italian transplant centers. Melphalan was administered at 140 teria are required for selecting patients (pts) who can benefi t mg/m² and combined with busulfan at 16 mg/Kg orally or 12.8 from this procedure. Aim of this study was to assess prognostic mg/Kg intravenously for patients under 60 years (=97, 75.2%). factors affecting OS in a cohort of 50 AML pts who received Patients older than 60 years (=32, 24.8%) received melphalan alloHSCT as salvage treatment after a risk-oriented frontline at 120 mg/m² and busulfan at 12 mg/Kg orally or 9.6 mg/Kg chemotherapy (NILG AML01-00). Fifty out of 140 pts in fi rst intravenously. Eighty two patients (63.6%) received peripheral relapse received allograft as salvage therapy. Median age was blood stem cells (PBSC) and 47 patients (36.4%) received bone 50 yr (14 pts were >55). At diagnosis 31(62%) pts had high risk marrow (BM) cells. Median age of study patients was 50 years AML, defi ned by clinical and cytogenetic characteristics accord- (range 16-72). Cytogenetic categories distribution was con- ing to the NILG protocol. Median time from 1st CR to relapse ventionally defi ned as favorable (15.5%); intermediate (60.1%) (TTR) was 10 months (mo) and from relapse to allo was 3 mo. and unfavorable (24.3%). Patients were classifi ed at high risk At alloSCT 34 pts (85%) were in 2nd CR, and 16 were trans- (49% of evaluable patients) when the white blood cell count at planted with disease. The donor was matched related for 19 diagnosis was higher that 30×109 /L and/or cytogenetics was pts, matched unrelated for 22 and haploidentical for 9. Stem cell unfavorable and/or when AML was secondary to a myelodys- source was PB in 31, BM in 12 and CB in 7 pts. The conditioning plastic syndrome, otherwise were considered at standard risk regimen was myeloablative (MA) in 38 (74%) and reduced inten- (51%). Forty-four patients (36%) received only one post-remis- sity (RIC) in 12. The impact of risk factors on OS was analyzed sion chemotherapy course whereas 78 patients (64%) received by univariate and multivariate analyses. The clinical outcome two or more chemotherapy cycles after remission before trans- of the 50 transplanted pts was compared with 90 consecutive plantation. With a median follow-up of 31 months, the 8-years pts receiving other intensive salvage treatment. After alloSCT projected overall survival (OS) and disease-free survival (DFS) 44/50 (88%) achieved a 2nd CR. Relapse occurred in 22 pts at was 62% and 56%, respectively. The relapse rate was 45% a median time of 8 mo from alloSCT, and all died. Among the and the non-relapse mortality (TRM) was 4.65%. Patients who 16 pts transplanted with disease 3 are in CCR. Acute (grade>II) underwent two or more post-remission chemotherapy cycles or chronic GVHD occurred in 23 pts (46%). The 100dy- and showed a signifi cantly better OS and DFS than patients treated 1-yr TRM was 6% and 20%. Among transplant related deaths, with only 1 cycle (OS 74.3% vs 61.3% p=0.05; DFS 68% vs 75% were attributable to MA regimens. After a median follow up 54.5% p=0.05). As expected, there was a signifi cant better of 10 mo (range 3-68), median OS was 13 mo. The projected OS and DFS in younger patients (< 60 years) than in elderly 1 yr- and 3-ys OS was 60 and 33%. Thirteen (28%) were in CCR patients (OS 73.2% vs 59.3% p< 0.02; DFS 67.3% vs 51.6% at the end of follow-up. The 90 non transplanted pts showed a p<0.003). The stem cell source did not signifi cantly affect OS median OS of 3 mo, with a 1-yr OS of 16% (p=0.000). Univari- and DFS although PBSC transplantation led to a faster hemato- ate analysis performed on the 50 transplanted pts showed that logical recovery than BM transplantation. Of note, OS and DFS high risk AML, TTR<6mo, ECOG-PS>0 and disease status at were not different between high risk and standard risk patients transplant were predictive of a poor outcome (p<0.05). By Cox and this fi nding was also confi rmed when the data were ana- multivariate analysis TTR<6mo and PS>0 were independent lyzed according to cytogenetic categories alone. Our data sug- adverse prognostic factors for OS (p=0.04 and 0.01). Our data gest that Bu/Mel is an effective conditioning regimen for high confi rm that allograft can offer a better clinical outcome than risk AML patients in fi rst CR undergoing ASCT being associated other intensive regimen. Allograft should be proposed preferen- with a low toxicity profi le (mainly mucositis) and TRM. tially to pts with a TTR≥6 mo and a very good PS.RIC seems to be as effective and less toxic than MA conditioning regimen, but larger prospective studies are required for confi rming the role of RIC in AML salvage strategies.

S91 P450 AML-relapse (n=42). The projected pretransplant assessment Impact of molecular markers on long-term outcome after of mortality score (PAM-Score, Parimon T et al.) of this cohort allogeneic haematopoietic stem cell transplantation for revealed a median overall probability of death within 2 years acute myeloid leukaemia patients of 67% (range 49-85%). 40 pts received 140mg/m² and 10 pts M. Michallet (1), M. Sobh (1), X. Thomas (1), C. Charlot (2), 200mg/m² melphalan followed by a total body irradiation (TBI) F. Barraco (1), G. Cannas (1), F.E. Nicolini (1), based conditioning regimen in 20 pts (40%) or a chemotherapy- E. Nicolas-Virelizier (1), C. Plesa (1), Y. Chelghoum (1), based regimen in 30 pts (60%) within 10 days after melphalan J. Troncy (1), J.-P. Magaud (2), I. Tigaud (2), S. Hayette (2) application. 18 pts received grafts from sibling (ISD) (36%) and (1)Hôpital Edouard Herriot (Lyon, FR); (2)Hôpital Lyon-Sud 32 pts from matched unrelated donors (MUD) (64%). Complete (Lyon, FR) remission of AML was obtained in all but one patient (98%). The transplant-related mortality (TRM) estimate at 2 yrs after The principal objective of this study was to analyze the impact aSCT was 34%. The 2-year probability of death was 64% and of molecular markers on the long-term overall and disease-free the 2-year event-free survival estimate was 35% with the last survival (OS and DFS) after fi rst allogeneic haematopoietic event occurring at 1.5 yrs. Explorative data analysis identifi ed a stem cell transplantation (HSCT) for acute myeloid leukaemia MUD transplant, a melphalan-dose of 140mg/m², and chemo- (AML). From 1996 to 2007, only 63 out of 364 pts (27 M, 36 therapeutic conditioning using treosulfan 42g/m² combining F, median age of 41 years) had retrospectively available con- with fl udarabine 150mg/m² as favourable outcome predictors. served cells at diagnosis. The expression levels of WT1, Evi1, This resulted from a signifi cantly lower 2-year relapse estimate Flt3 and Hoxa9 were performed by quantitative RT-RQPCR. after MUD compared to ISD (21% vs. 81%, p<0.02) and a lower The mutational status of MLL duplication, FLT3 (internal tan- 2-year TRM after 140mg/m² melphalan combined with treosul- dem duplication or nucleotide substitutions) (ITD), NPM1 and fan + fl udarabine compared to other combinations (17% vs. CEBPá were determined by PCR, RFLP and/or sequenc- 54%, p<0.03). The 2-year event-free survival estimate for the ing analysis. Four (6%) pts had Flt3over-expressed (ov-ex), 18 pts with all favourable treatment was 68% opposed to 19% 19(30%) FLT3 ITD+, 3(5%) MLLdup, 10(16%) Hoxa9 ov-ex, in the 32 pts not fulfi lling all these characteristics (p<0.006). In 7(11%) Evi1ov-ex, 15(24%) NPM1mut+, 25 (40%) WT1 ov-ex conclusion, the concept of aplasiogenic high-dose melphalan and 1 CEBPAmut+ (this marker was evaluated only in12 pts). together with myeloablative conditioning can rescue more than Twenty three (36%) pts had no abnormal molecular markers 1/3 of otherwise refractory AML pts. The benefi cial infl uence and 10 (16%) 1 marker, 10(16%) 2 markers, 12(19%) 3 mark- of MUD in this study further supports a stronger antileukemic ers, 4(6%) 4 markers and 4(6%) 5 markers. Among the 23 potential compared to ISD in pts with refractory AML, which negative molecular pts, 9(41%) presented a normal, 12(50%) translates into a comparatively high event-free survival in com- a poor and 2(9%) a favourable karyotype,; and among the 40 bination with 140mg/m² melphalan followed by a conditioning positive pts, 16(40%) were normal, 8(20%) poor, 13(32.5%) regimen with treosulfan + fl udarabine. This sequential protocol intermediate and 3(7.5%) favourable. Concerning transplanta- deserves further evaluation against other sequential condition- tion, the median interval between diagnosis and transplantation ing approaches in refractory AML pts. was 6 months (2.6-68.5). Before conditioning, 41 pts were in CR, 8 in PR and 14 in relapse. Twenty fi ve (40%) pts received a non-myelo-ablative conditioning and 38(60%) a myelo-abla- P452 tive one. Twenty three (36.5%) pts received PBSC, 37 (59%) Allogeneic HSCT after prior treatment combining bone marrow and 4 (6.5%) cord blood cells from 47 (75%) HLA chemotherapy and gemtuzumab ozogamicin: a series of siblings and 16 (25%) unrelated donors. After transplantation, 44 cases 59 (94%) pts engrafted, 42 developed AGVHD and 13 devel- P. Chevallier (1), T. Prebet (2), C. Faucher (2), P. Turlure (3), oped cGVHD. At the last follow-up, 20 pts have relapsed, 29 M. Hunault-Berger (4), S. Vigouroux (5), T. Guillaume (1), pts are alive and 34 died. At the median follow-up of 48 months, F. Mechinaud (1), J. Delaunay (1), J.-L. Harousseau (1), the OS and DFS for the whole population were 40% (33-47) N. Ifrah (4), P. Moreau (1), D. Blaise (2), N. Vey (2), N. Milpied and 40% (34-46) respectively with a maximum follow-up of 130 (5), M. Mohty (1) months. The univariate analysis showed a signifi cant impact of (1)CHU Hotel-Dieu (Nantes, FR); (2)CHU (Marseille, FR); FLT3 ITD and over-expression of FLT3RQ on long-term DFS, (3)CHU (Limoges, FR); (4)CHU (Angers, FR); (5)CHU [25%vs47% p=0.03 and 50%vs34% p=0.02] and a trend on (Bordeaux, FR) long-term OS 27%vs46% p=0.08]. Regarding the karyotype (83% Fav, 50% Int and 24% Poor) and some other markers Purpose: No large series have comprehensively assessed the (Wt1:37%vs43%, EVI1:28%vs43%, NPM1:32%vs43% and outcomes and the risk of hepatotoxicity, especially Veno-Occlu- Hoxa9:50%vs38%), we did not observe signifi cant difference sive Disease (VOD), after allo-HSCT in patients who received because of small number of pts in subgroups. The known ben- prior treatment combining chemotherapy + Gemtuzumab Ozo- efi c impact of NPM1mut+, was erased because the majority of gamicin (GO) for CD33+ diseases. this group presented an associated FLT3 ITD+. Patients and Methods: This retrospective study included 44 patients who have been allo-transplanted after prior exposure to GO + chemotherapy. Median age was 50 years. There were P451 41 CD33+AML, 2 CD33+ ALL and 1 CD33+ myeloid sarcoma. Allogeneic stem cell transplantation after sequential Before transplant, most of the patients (n=36) had received the therapy using high-dose melphalan and myeloablative MIDAM regimen (GO 9 mg/m² day 4 + Ara-C 1g/m²/12 hours conditioning in patients with refractory acute myeloid days 1-5 + Mitoxantrone 12 mg/m²/day days 1-3). Median leukaemia interval between last administration of GO and the allograft N. Steckel, R. Trenschel, L. Kordelas, T. Gromke, H. Ottinger, was 4.2 months, including 19 patients allografted < 3.5 months M. Koldehoff, A.H. Elmaagacli, C. Schulte, D. Beelen from GO. At transplant, 33 patients were in CR. The majority Clinic of Bone Marrow Transplantation (Essen, DE) of patients (n=36) received a RIC conditioning regimen. All patients received low-dose heparin as VOD prophylaxis, except Patients (pts) with truly refractory acute myeloid leukaemia one. (AML) have a particular poor prognosis after allogeneic stem cell Results: Engraftment was observed in 34 patients (77%). With transplantation (aSCT). In this prospective analysis, high-dose a median follow-up of 15 (range, 1.1-63) months for surviving melphalan for induction of marrow aplasia followed by a myelo- patients, OS and DFS after allo-HSCT were 45% (95%CI, 30- ablative conditioning regimen before aSCT was evaluated in 50 61%) and 38% (95%CI, 24-54%) at 3 years respectively. The consecutive pts (median age 55, range 19-70 years) after failure incidence of Grade ¾ hyper-bilirubinemia was 13.5% (n=6), of primary double induction (n=8) or salvage chemotherapy for with this being 21% in patients with a short (<3.5 months)

S92 GO-allograft interval (n=4/19) vs. 8% in all others (P=NS). Overall, the incidence of VOD was 7% (n=3), with this being 10.5% (n=2/19) in patients with a short GO-allograft inter- val (<3.5 months) vs. 4% (n=1/25) for all others (P=NS) and 5.5% (n=2/36) in patients receiving a RIC regimen vs 12.5% for the others (n=1/8) (P=NS). In univariate analysis, no factors were signifi cantly associated with a higher risk of hepatotoxic- ity. Also, in this series, using a Cox multivariate analysis, high risk cytogenetics was the only signifi cant factor predictive of a worsened DFS after allo-SCT (P=0.0009; RR= 4.6; 95%CI: 1.9-11.2). Conclusion: These encouraging results suggest that GO-based therapy in combination with chemotherapy prior to allo-HSCT is feasible and does not result in an excessive rate of toxicity, especially VOD, after allo-HSCT. P454 Phase I-II induction intensifi ed regimens including P453 fl udarabine or mylotarg for acute myeloid leukaemia Comparison of busulphan (16 mg/kg) + fl uda versus patients: comparison by response and follow-up standard BU-CY in allogeneic transplantation of adult C. Papayannidis (1), A. Candoni (2), S. Paolini (1), patients affected with leukaemia: advantages of bus+fl uda E. Ottaviani (1), I. Iacobucci (1), M. Rondoni (3), M. Malagola (4), are evident also using oral route for busulphan PP. Piccaluga (1), D. Cilloni (5), R. Fanin (2), G. Visani (6), administration M. Baccarani (1), D. Russo (4), G. Martinelli (1) G. Milone, S. Leotta, E. Mauro, S. Coppoletta, M. Poidomani, (1)Oncology Seràgnoli (Bologna, IT); (2)Hematology (Udine, E. Marturano, A. Spadaro, S. Mercurio, A. Strano, IT); (3)Hematology Department (Siena, IT); (4)Hematology M.G. Camuglia, G. Avola, A. Di Marco and Unit of Blood Diseases and Cell Therapy (Brescia, Ospedale Ferrarotto (Catania, IT) IT); (5)Hematology (Orbassano, Turin, IT); (6)Hematology (Pesaro, IT) Background: The association of fl udarabine and full dose i.v. busulfan (BUS-FLUDA) in allogeneic transplantation deter- Background: Conventional induction treatment in young Acute mines, in comparison with BU-CY, a lessening of liver toxicity Myeloid Leukemia (AML) patients (≤ 60 years old) is still rep- and a lower TRM risk. However, it is not known to what extent resented by the association of antracycline and cytarabine, the clinical advantage of BUS-FLUDA is dependent by the i.v. namely the 3/7 schedule candidate to allo-BMT. route of BUS administration, that allows a more predictable Aim of the study. We evaluated the safety profi le and the plasma peak of the drug, or by the substitution of FLUDA for effi cacy of two four-drugs induction schedules, adding either CTX. We have conducted a retrospective analysis of patients fl udarabine (25 mg/sqm days 1-5) or mylotarg (3 mg/sqm day treated with oral BUS(16) + FLUDA or with oral BU(16)-CY. 6) to idarubicin (6 mg/sqm days 1, 3, 5), cytarabine (2 g/sqm Methods: Fifty-one patients (ALL, AML, CML) transplanted from days 1-5), etoposide (100 mg/sqm days 1-5) (FLAIE and matched sibling or UD donor were studied. Twenty-six patients MyAIE, respectively). received before allogeneic HSC transplantation BUSULPHAN Methods: Sixty-six consecutive AML patients were enrolled 16 mg/Kg p.o. and FLUDARABINE 150 mg/m² (BUS-FLUDA) either in the FLAIE (N=44, from 2002 to 2005) or in the MyAIE and results were compared to those obtained in a group of 25 (N=22, from 2005 to April 2007) schedule, with similar clinical patients that received BU-CY2 or BU-CY3. Majority of patients and biological characteristics. The median age was 45 and 48 in both groups were affected with AML. No differences between years, respectively. According to kariotype, WBC count and these two groups were found in percentage of UD donors FLT3 status, seventy and sixty-four percent of cases, respec- (p=0.6), nor in HSC source (BM versus PBSC, p=0.9), nor in age tively, were considered at high risk. Consolidation therapy con- (p=0.6). Pts were defi ned as “early disease” in 29% (B-F group) sisted of 2 cycles of HD-AraC and Ida. and 30% (BU-CY), p=0.9. Mean CD34+ cell dose infused was Results: The complete remission rate was 75% and 59% for comparable (p=0.29). GVHD prophylaxis was standard dose FLAIE and MyAIE (p=n.s.). Death during treatment rates were CSA + MTX short course. All patients had primary engraftment 5% and 0. After 1 consolidation course the overall CR rate was and mean time to reach N. count > 0.5 x 109/l was not different 80% and 73%. After a similar median follow up, 27 months (1- in the two groups. 62) and 21 months (5-42) respectively, 41% of patients are alive Results: In oral BUS-FLUDA group max score of oral mucosi- in CR in the FLAIE group (12 SCT and 3 ASCT) and 64% in the tis (DMS score) was lower during days 7-14 post transplan- MyAIE group (7 SCT and 4 ASCT) (p=n.s.; Chi-square, Fisher’s tation (p=0.008), BUS-FLUDA group had also a CHE serum exact test). Toxicity was comparable in the two regimens. The level higher than BU-CY group during week 1, 2 and 3 post- median time to ANC recovery (>1.0 x 109/L) was 31 and 23 transplant (p=0.002). Moreover pts of B-F group had a lower days for FLAIE and MyAIE; the median time to PLT recovery PLT transfusion requirement (p=0.004). Haemorrhagic cysti- (>100 x 109/L) was 28 and 24 days, respectively. The median tis was recorded in 20% of patients receiving BU-CY while in time of neutropenic fever episodes for patients was 1 and 1.4 in only 3.8% of pts treated with B-F (p=0.06), no cases of VOD the 2 groups, respectively. were registered in either groups. OS at 3 years was 80% in Conclusions: Four-drugs intensifi ed induction therapy including BUS+FLUDA versus 59% in BU-CY group (p=0.2) and DFS Mylotarg is a feasible approach in young AML patients candi- was 70% in BUS+FLUDA versus 51% in BU-CY group (p=0.3). date to allo-BMT. The limited number of patients involved in No difference in OS between BUS+FLUDA versus BUS+CTX this study, the low administration dosage of idarubicin, and the was observed also in the stratum of AML patients who had an relevant role of fl udarabine in induction therapy, can reason- OS of 70% versus 62% (p=0.9). ably justify the lower CR rate obtained in patients treated in the Conclusion: Oral BUS plus FLUDA showed in comparison to MyAIE schedule, if compared with FLAIE regimen and, above BUS plus CTX the same anti-neoplastic activity with a signifi - all, with standard chemotherapy. Fludarabine and Mylotarg, cantly lower toxicity in terms of mucositis, PLT requirement and in combination with conventional chemotherapy, represent a rate of hemorrhagic cystitis. promising induction regimen in young AML patients. Acknowledgements. Supported by: European Leukemia Net, COFIN 2005, AIRC, AIL, FIRB 2006, Fondazione del Monte di Bologna e Ravenna.

S93 P455 Myeloablative allogeneic stem cell transplantation in adults with acute lymphoblastic leukaemia 1985 – 2005. A single-centre study Y. Fløisand, L. Brinch, I. Dybedal, T. Gedde-Dahl, D. Heldal, P. Holme, T. Egeland, G. Tjønnfjord Rikshospitalet University Hospital (Oslo, NO)

Background/aims: Although the prognosis and long-term sur- vival in children suffering from acute lymphoblastic leukemia (ALL) has greatly improved in the last decades with long term disease free survival rates approaching 80%, treatment results in adults still need to be improved. We recently reported long term disease free survival of 50% in adults with ALL treated in our institution. Allogeneic stem cell transplantation (ASCT) following myeloab- lative conditioning is a treatment option available for patients under the age of 60-65 years, that may improve treatment results in adults. We present the results from the Norwegian stem cell transplant program 1985-2005, partly presented in Tidsskr Nor Legeforen. Patient material: In the 20-year period 1985–2005, 61 patients(m=38, f=23) underwent ASCT for ALL in our institu- tion.19 were transplanted in fi rst remission, the other 42 in later stages of disease. Myeloablative treatment consisted of busul- fan and cyclophosphamide(BuCy) in all but two patients, who received Cy and TBI 12Gy. Results: A matched family donor was used in 38 patients (62%). Ten of these patients had a haploidentical donor with 0-1 anti- gen mismatch. The remaining patients were transplanted with P456 cells from 10/10 or 9/10 MUD. 46 patients received bone mar- PET-CT scan for diagnosis of extramedullary relapse of row derived stem cells, whereas 15 were transplanted with AML after allogenic stem cell transplantation mobilized peripheral blood stem cells. T. Stuebig (1), D. Judith (1), B. Sauter (2), F. Gruenwald Acute graft vs host disease (GvHD) developed in 26 patient (2), R. Taube (1), M. Schleuning (1), H. Baurmann (1), (43%), grade II-IV in 22. Chronic GvHD developed in 21 patients R. Schwerdtfeger (1) (34%) with six patients experiencing extensive chronic GvHD. (1)Deutsche Klinik für Diagnostik (Wiesbaden, DE); (2)University In December 2006, 26 patients were alive, 21 without evidence Frankfurt (Frankfurt, DE) of disease. 10 patients, all transplanted with MUD, developed invasive fungal disease. Median survival was 1,5 years. Trans- Background: Allogeneic stem cell transplantation (alloSCT) is plant related mortality due to GvHD, infection, veno-occlusive the only curative treatment in patients with high risk leukaemia. disease or non-engraftment was 25%. The most important By using reduced intensity conditioning regimen (RIC) alloSCT cause of treatment failure was disease relapse in 23 patients can be offered to patients well beyond 50 years of age. The (38%).Relapse was the most common cause of death in lower rate of transplant related mortality (TRM) after RIC is off- patients transplanted with a family donor (67%), whereas death set in part by a higher rate of relapse. Since introduction of due to transplant related complication were more common in RIC in our center we have seen an increase in extramedullary patients who received grafts from MUD. 45% of patients with relapses (EMR). Treatment of EMR is diffi cult and requires a family donors relapsed with ALL, whereas relapse occurred in precise evaluation of extramedullary AML manifestations. Here 26% of patients with MUD transplants. we report about our experiences with [18F]Fluorodexyglucose Conclusion/discussion: ASCT is a potentially curative treat- (FDG) positron emission tomography (PET) and CT (PET–CT ment modality for adult patients with ALL where other treat- scan) for detecting sites of EMR. ment options have minor possibilities of success and should be Patients and Methods: In 2007 we observed 7 patients with offered to a greater proportion of patients in fi rst remission than histologically proven EMR after alloSCT in our center. PET-CT was common practice in the 20 year study period. scan was used in three for detection of solid extramedullary AML manifestations. PET-CT scans were performed using FDG and standard PET and CT scanner. Results: In all three patients the histology based EMR diagnosis was confi rmed by PET-CT scan. Furthermore in two Patients we were able to fi nd additional sites of EMR as compared to standard radiological methods as computed tomography (CT) and magnetic resonance imaging (MRT) only. Conclusion: PET-CT Scan seems to be of diagnostic value in AML patients with extramedullary relapse after allogeneic stem cell transplantation. In order to evaluate its sensitivity (and spe- cifi city) we are currently performing of PET-CT scan in these patients in a prospective, biopsy controlled manner.

S94 P457 Methods: since 2003 30 consecutive pts with median of age 52 Isolated extra-medullary relapse of acute leukaemia after years (range 28-59) with intermediate-risk AML in 1.CR under- allogeneic stem cell transplantation. Different kinetics and went alloSCT from related (43%) or unrelated (57%) donor better prognosis than systemic relapse after MAC (40%,BU/CY) or RIC (60%,FLU/MEL). The source A. Shimoni, A. Rand, I. Hardan, N. Shem-Tov, R. Yerushalmi, of stem cell was peripheral blood in 93%. GVHD prophylaxis A. Nagler were CSA/MTX. The control group consisted of 19 pts with Chaim Sheba Medical Center (Tel-Hashomer, IL) intermediate-risk AML in 1.CR indicated for alloSCT but treated by CHT due to lacking of donor or refusal of allo-SCT. The con- Allogeneic SCT is a potentially curative treatment for acute leuke- trol group except for the younger age did not differ in any prog- mia, however relapsing disease is the major cause of treatment nostic parameters. failure. Isolated extramedullary (IEM) relapse is considered a Results: all transplanted pts engrafted. 9 pts (30%) developed rare event and its characteristics and prognosis are less defi ned acute GvHD and among 25 evaluable pts 9 pts (36%) devel- than systemic relapse. We analyzed outcomes of 356 consecu- oped chronic GvHD. 3 pts (10%) relapsed and 5 pts (16%) died tive pts with AML/MDS (n=277) and ALL (n=79) given SCT in a due to TRM. The estimated probabilities of 3-years DFS and single institution. With a median follow-up of 30 months (range, OS were 84% and 60%. Type of donor or conditioning regi- 1-103), 149 pts relapsed (122 died). The overall survival rate was men did not have any statistical infl uence on transplant results. 34% (95CI, 27-41) and the cumulative incidence of relapse 47% Among CHT treated pts 2 (11%) of them died due to TRM and (95CI, 42-54). 17 pts had IEM relapse (11.4% of all fi rst relapses); 10 pts (53%) relapsed. The estimated probabilities of 3-years CNS (n=6), mediastinum (n=2), pleura (n=2), skin (n=2), breast DFS and OS were 31% and 40%. AlloSCT with comparable (n=2), bone (n=1), other soft tissue masses (n=2). IEM relapse TRM (p=0,69) signifi cantly improved DFS (p=0.001), cumula- occurred later after SCT than systemic relapse; median 14 (1- tive RI (p=0,002) and there was a trend for better OS (p=0,10) 38) and 3 (1-59) months, respectively (p=0.002). Pts with an IEM in comparison with CHT. relapse were younger, median age 38 and 46 years, respectively Conclusions:1. in comparison with CHT alloSCT with low TRM (p=0.02). IEM relapse occurred more commonly in ALL (23% in recent years and low RI improved DFS of pts with interme- of relapses) than MDS/AML (8%, P=0.02). There was a trend diate-risk AML in 1.CR. The smaller difference in OS can be for higher incidence in pts transplanted in remission (16% of explained by the effect of salvage treatment of relapsed pts in relapses) than in pts transplanted in active disease (8%, p=0.09). CHT group (80% salvage treatment, 50% alloSCT). 2. OS after There was no relation to donor or conditioning type. Interestingly, alloSCT was not infl uenced by type of donor or conditioning among 14 pts having IEM relapse more than 3 months after SCT, regimen. 11 had a history of chronic GVHD (79%), compared with 29 of 70 pts with systemic relapse (41%, p=0.01). Pts with IEM relapse were most often treated with systemic chemotherapy, followed P459 by radiation therapy and DLI when feasible. Pts with systemic Gemtuzumab ozogamicin, fl udarabine, ara-C and relapse were treated with chemotherapy and DLI or a second cyclosporineA (MFAC) regimen as induction therapy in SCT. Among the 17 pts with IEM relapse, 12 achieved CR (71%), relapsed or refractory CD33+ acute myeloid leukaemia 5 remained in continuous CR, 7 relapsed again, 2 systemic (both prior to allogeneic haematopoetic stem cell died), 5 EM (2 are in long-term remission). Among the 132 pts transplantation: a single-centre experience with systemic relapse, 35 achieved CR (27%), 14 died in remis- M. von Bonin, U. Oelschlägel, B. Mohr, U. Platzbecker, sion, 7 remained in continuous CR, 14 relapsed again, 9 sys- C. Thiede, M. Bornhäuser, G. Ehninger, T. Illmer temic (all died), 5 EM (1 is in long-term remission). The median TU Dresden (Dresden, DE) survival after relapse was 2.5 and 20 months, and the 2-year OS rates 7% and 38% after systemic and IEM relapse, respectively Gemtuzumab ozogamicin (GO) is an immunoconjugate con- (p=0.0002). In conclusion, IEM relapse of acute leukemia after sisting of a humanized CD33 monoclonal antibody linked to the SCT is relatively common. It occurs later than systemic relapse enediyene calicheamicin. GO is approved by the Food and Drug and more commonly in pts with chronic GVHD, suggestive that Administration (FDA) in the United States as single agent for GVL is able to prevent systemic recurrence but is less successful the treatment of CD33 positive AML in fi rst relapse in patients in EM sites. When treated aggressively prognosis is better with 60 years of age or older not eligible for other cytostatic therapy. IEM relapse and long-term survival is feasible. GO has been also used in combination with conventional chem- otherapy in CD33+ acute myeloid leukemia (AML). We retro- spectively analyzed 26 patients with CD33+ primary refractory P458 or relapsed AML receiving the MFAC regimen (GO, fl udarab- Favourable outcome of allogeneic stem cell ine, ara-C and ciclosporin CsA) directly followed in aplasia by transplantation for patients with intermediate-risk reduced intensity conditioning (RIC) for allogeneic hematopo- AML in 1.CR – A single-centre experience from 2003–2008 etic stem cell transplantation (HSCT). Early treatment assess- M. Karas, K. Steinerová, P. Jindra, D. Lysák, S. Vokurka, ment revealed an overall response (OR) of 65.4%. Grade III/IV T. Svoboda, L. Mohammad, V. Koza hepatic toxicity occured in 30.8% of the patients. The median Charles University Hospital Pilsen (Pilsen, CZ) time to allograft was 22.5 days. 77% of all patients proceeded to allogeneic HSCT. All patients except one obtained granulo- Objectives: allogeneic stem cell transplantation (alloSCT) cyte colony-stimulating factor-mobilized peripheral blood stem using myeloablative conditioning (MAC) and related donors cells. In 15 patients unmodifi ed blood stem cells were used, the for patients (pts) with intermediate-risk AML in 1.CR improves graft of 5 patients underwent ex-vivo CD3/CD19-depletion, four disease-free survival (DFS) and relapse incidence (RI) in com- out of fi ve in context of haploidentical transplantation. Unmodi- parison with pts treated by chemotherapy (CHT). The impact fi ed grafts (n=15) were derived from related and unrelated on overall survival (OS) was not so clearly demonstrated due donors in 5 and 10 cases, respectively. Donor and recipient to higher treatment-related mortality (TRM). Reduced-intensity were HLA-mismatched in 4 cases. Grade III/IV hepatic toxicity conditioning (RIC), the use of unrelated donor, new supportive after allografting was observed in 25%, including one patient therapy and diagnostic procedures introduced in recent years with veno-occlusive disease (VOD). The median neutrophil and can affect results of alloSCT. To evaluate the role of these platelet engraftement occured in patients receiving unmodifi ed factors we retrospectively analysed outcome of pts with inter- grafts on day 13 (range 8 – 27) and day 17 (range 9 – 103), mediate-risk AML in 1.CR transplanted in last 5 years at our respectively. Using ex-vivo CD3/CD19 depleted grafts, median transplant centre and we also compare their outcome with pts neutrophil and platelet engraftement was observed on day indicated for allo-SCT but treated by chemotherapy (CHT) due 17 (range 11 - 22) and day 32 (range 18 - 119), respectively. to lacking of donor or refusal of alloSCT. Median survival and 12-month overall survival (OS) was 8.1

S95 months and 53%, respectively. Actually, 10 patients (= 38.5%) P461 are still alive, 13 (= 50%) have died and 3 (= 11.5%) are lost Unrelated and related HSCT in elderly AML patients: is to follow up. The median follow up for surviving patients is 420 young matched unrelated donor comparable or superior days (range 274 - 943) and 396 days (range 257 - 922) after to older sibling donor? An analysis of 76 allografted MFAC and allogeneic HSCT, respectively. MFAC directly fol- patients aged above 50 years from the Czech Acute lowed by RIC for allogeneic HSCT seems to be active and fea- Leukaemia Clinical Register (ALERT) sible, but possibly with an increase in toxicity by adding CsA as P. Jindra (1), J. Muzik (2), K. Indrak (3), V. Koza (1), Pgp-Inhibitor with questionable effectivity. M. Karas (1), K. Chroust (4), F. Al Sabty (5), E. Demeckova (5), P. Cetkovsky (6), A. Jungova (1), P. Zak (7) (1)University Hospital (Pilsen, CZ); (2)Institut of Biostatistics P460 and Analyses (Brno, CZ); (3)University Hospital (Olomouc, Allogeneic stem cell transplantation for secondary acute CZ); (4)Institute of Biostatistics and Analyses (Brno, CZ); myeloid leukaemia: 7-year experience of a single centre (5)University Hospital an Health Centre (Bratislava, SK); Y. Koh (1), B.S. Kim (2), S.M. Bang (3), I. Kim (1), D.S. Lee (6)Institute of Hematology and Blood Transfusion (Prague, CZ); (1), S.S. Yoon (1), J.S. Lee (3), K.S. Han (1), S. Park (1), (7)University Hospital (Hradec Kralove, CZ) B.K. Kim (1) (1)Seoul National University Hospital (Seoul, KR); (2)Seoul AML affects mainly the elderly and the only curative option National University Boramae Municipal Hospital (Seoul, KR); is allo-SCT. However, due to the higher non-relapse mortal- (3)Seoul National University Bundang Hospital (Seongnam, ity (NRM), the unrelated SCT is offered reluctantly in the KR) absence of sibling donor. Moreover, the siblings of elderly pts are naturally older and often ineligible for stem cell collection. Background: Treatment most likely to cure secondary acute The question is whether younger age of unrelated donors myeloid leukemia (s-AML) is allogeneic stem cell transplanta- could compensate for the higher immune incompatibility. To tion (SCT). In this study, we report results of allogeneic SCT for answer this, we retrospectively analyzed allografted AML pts s-AML performed at single institution in recent 7 years. aged above 50 from the 5 Czech&Slovak centres and reported Methods: Retrospective medical record review between 2001 to ALERT. and 2008 was performed. Study population includes patients Patients: 76 consecutive patients aged ≥ 50 (median 56y, range treated with allogeneic SCT for s-AML. s-AML includes leuke- 50-68) allografted either with related (SIB group, n=47, median mia following aplastic anemia, myeloproliferative disease, age 56;50-68) or unrelated, 9-10/10 HLA allele-level matched myelodysplastic syndrome (MDS) and previous chemotherapy donors (MUD group, n=29, median age 57;50-63). Groups for other tumors (t-AML). We assessed survival after SCT (OS) were comparable in terms of age, remission status pre-SCT, and transplantation related mortality (TRM). cytogenetic-risk, follow-up, donor sex, CMV D-/R- status and Results: A total of 29 patients (Pts) (M:F=14:15, median age conditioning (RIC in 70% of SIB vs. 66% in MUD). As expected, 48.2 years) received allogeneic SCT in Seoul National Univer- the MUD have signifi cantly younger donors (median 30y, range sity Hospital for s-AML. 15 Pts developed s-AML from MDS, 20-46 vs. 55, range 39-68, p<0.001) and received more CD34+ while 11 developed s-AML following chemotherapy. 2 Pts (median 6.5 vs. 4.7x106/kg, p=0.006). Various conditionings developed s-AML from aplastic anemia, and 1 from idiopathic were used; ATG was administered in 48% of unrelated SCT. myelofi brosis. Median duration from diagnosis of original dis- Results: After median follow-up of 18 months (range 1-73), 39 ease (fi rst chemotherapy in t-AML) to the development of s- pts (51%) are alive. The 3-y Kap.-Meier OS and EFS probabili- AML was 12 months. Among the Pts in whom cytogenetic data ties were 42% and 40% (median survival 18 months), whereas were available, 6 belonged to poor, 19 to intermediate and 2 overall NRM and relapse rates were 25% (19/76) and 26% to good risk group. 18 Pts received SCT as consolidation in (20/76), respectively. After splitting the pts to SIB vs. MUD, the complete remission (CR), while 11 Pts received SCT not in CR. fi gures were as follows: number of surviving pts 24/47 vs. 15/29 19 Pts received SCT from sibling donors, while 10 Pts received resulting in median OS probabilities for SIB and MUD of 16 unrelated SCT. 19 Pts received NST and 10 Pts received mye- and 21 months (p=0,817). The relapse rates were nonsignifi - loablative SCT. 7 Pts (24.1%) experienced TRM during SCT cantly higher in the SIB (30% vs. 21%, p=0,433); whereas the and no single clinical factor was important for TRM. Median fol- NRM rate was similar (23% vs. 28%, p=0,77).The incidences of low up duration was 41.9 months. During follow up, 18 Pts died aGVHD were identical both for SIB and MUD (11% for aGVH- and median OS was 8.4 months. Three year OS rate was 30%. Dgr.III-IV). Comparable results were also reported for extensive Original disease causing s-AML was not related to prognosis cGVHD incidences (SIBxMUD: 17% vs. 21%, p=0,765). (p=0.460). However, cytogenetic risk grouping affected prog- Conclusions: Our results clearly indicate that unrelated donors nosis (p=0.042). Pts who were in CR survived longer than Pts did not adversely affect SCT outcome in the AML patients aged not in CR (median OS not reached vs. 3.7 months, p=0.027) at ≥ 50 and the younger allele-matched unrelated donor is at least the time of SCT. For Pts who received SCT in CR, survival was equivalent to an older HLA-identical sibling. We should not be the longer in Pts who received SCT after fewer cycle (≤ 1) of reluctant with MUD in older pts and the priority of sibling, yet consolidation chemotherapy (n=15) than Pts who received SCT older donor over readily available, molecularly matched young after more than 1 cycle of consolidation chemotherapy (n=3) unrelated donor should be tested in appropriately designed trial (median OS not reached vs. 3.7 months, p=0.002). with the larger patient’s cohort. Grant Support NR9481-3/2007. Conclusion: s-AML Pts receiving SCT shows poor prognosis with three year OS rate of 30%. Cytogenetic risk grouping and disease status at SCT has prognostic value, while original dis- P462 ease causing s-AML is not prognostic. For Pts in CR, upfront A novel resistant FIP1L1-PDGFRa mutation in acute SCT with fewer pretransplant consolidation chemotherapy may myeloid leukaemia, resistance to imatinib mesylate and be benefi cial. allogeneic haemopoietic stem cell transplantation Y. Sorour (1), C. Dalley (1), J.A. Snowden (1), N.C.P. Cross (2), J.T. Reilly (1) (1)Sheffi eld Teaching Hospitals NHS Foundation Trust (Sheffi eld, UK); (2)University of Southampton (Southampton, UK)

The FIP1L1-PDGFRa fusion gene has been described in eosi- nophilia-associated myeloproliferative neoplasias. Patients characterised by these disorders may benefi t from targeted therapy with tyrosine kinase inhibitors, and/or allogeneic

S96 haemopoietic stem cell transplantation (HSCT). We report the SCTC were not fi nally transplanted (50% vs 21%; p=0.03). At case of a FIP1L1-PDGFRa positive acute myeloid leukaemia last follow-up, the group of transplanted pts had superior OS (AML) who received both modalities of therapy. In December than those that fi nally do not undergo the procedure (33% vs 2006, a male age 25 years, presented to our institution with 0%; p=0.03) (Fig.1) and also better than the whole group of pts AML. Bone marrow (BM) examination demonstrated increased initially included in the program (33% vs 27%). eosinophil precursors, myeloblasts of 24%. BM karyotype was Conclusions: SCT was not performed in 25% of AL pts initially complex with structural aberrations of chromosomes 1,3,16 included in the SCT program mainly due to poor autologous and 18. Molecular genetics confi rmed FIP1L1-PDGFRa fusion stem cell mobilization, clinical complications and disease pro- product but no evidence BCR-ABL or JAK2 V617F mutations. gression. Our study identifi es the leukaemia status at the time The patient received two cycles of FLAG-Ida chemotherapy of inclusion in the SCT program as a crucial factor for the sched- on the UK MRC AML 15 trial. Although haematologic remis- uled transplants’ feasibility. The group of AL pts that fi nally do sion was achieved (BM myeloblasts <5%), the BM karyo- not undergo the SCT has very poor outcome and should be type demonstrated cytogenetic evolution, with persistence of considered when analysing AL therapeutic strategies. FIP1L1-PDGFRa. Imatinib mesylate (IM) (400 mg once daily) was commenced after the FLAG-Ida #2 until myeloablative matched unrelated allograft [Cyclophosphamide/TBI/Campth conditioning, GVHD prophylaxis methotrexate/ciclosporin] in May 2007. At the time of HSCT the patient was in haematologic and molecular remission and IM was discontinued. Progressive chronic GVHD (skin) grade III occurred at day +136 post HSCT and was managed with extracorporeal photopheresis and pred- nisolone. The patient remained in molecular and haematologic remission until 6 months post HSCT when FIP1L1-PDGFRa fusion product was detected; necessitating recommencement of IM at 400mg daily. Despite this intervention the FIP1L1-PDG- FRa fusion product remained detectable prompting mutation analysis at 12 months post HSCT. A D842V IM resistant muta- tion, not previously described in AML was detected. Although IM was discontinued the patient was unable to tolerate Dasatinib and died of AML 15 months post HSCT. Although FIP1L1-PDG- FRa AML is uncommon, patients in whom allograft is thought necessary should undergo mutation screening if molecular relapse is documented post HSCT.

P463 Reasons why acute leukaemia patients with established indication for stem cell transplantation do not fi nally undergo the procedure and its clinical consequences F.J. Márquez-Malaver, I. Espigado, J. Falantes, M. Carmona, P464 I. Montero, J. González, M.L. Martino, V. de la Osa, Continuous infusion idarubicin and intravenous Á. Urbano-Ispizua busulphan as conditioning regimen to autologous stem Hospital Universitario Virgen Del Rocío (Sevilla, ES) cell transplantation for patients with acute myeloid leukaemia Clinical results obtained with stem cell Transplantation (SCT) F. Ferrara (2), P. Musto (1), S. Palmieri (2), C. Copia (2), in acute leukaemia (AL) pts are compared with those obtained M. Pedata (2), T. Izzo (2), C. Criscuolo (2) with conventional chemotherapy. A potential bias for this com- (1)Division of Hematology and SC T (Rionero in Vulture, IT); parison is the possibility of a positive selection of pts in the SCT (2)Division of Hematology and SC T (Naples, IT) group since poorer performance individuals may not be fi nally transplanted. The current study aimed to evaluate the effi cacy and toxic- Aims: To prospectively analyze the pre-SCT program phase of ity of a combination of intravenous (iv) Busulphan (Bu) and a tertiary Institution, in order to identify: continuous infusion (c.i.) Idarubicin (IDA) as a conditioning to 1) the reasons why SCT in candidates with AL is not fi nally per- autologous stem cell transplantation (ASCT) in patients with formed, acute myeloid leukemia (AML). The protocol included IDA at 2) the clinical differences between AL transplanted pts and 20mg/sqm daily as 3 days continuous infusion (from day -13 those candidates that fi nally do not receive the procedure, and to -11) and intravenous BU at 3.2 mg/kg daily from day -5 to 3) to compare overall survival (OS) between AL transplanted -2. Patients aged over 60 years received a reduced schedule pts vs the whole group of pts initially accepted in the SCT pro- (two days IDA and 3 days BU at the same dose). Twenty-fi ve gram. patients with a median age of 51 years (28-72) were enrolled. Patients: Between 2005 and 2008 pre-transplant data on 341 All patients received peripheral blood stem cells. The median consecutive pts evaluated by the SCT Committee (SCTC) of interval between diagnosis and ASCT was 4 months. The our institution were prospectively collected and analyzed. median number of CD34+ cells infused was 5.9x106/kg. The Results: 101 AL pts were presented at the SCTC. SCT was median number of days to PMN >500/cmm and platelets considered not indicated in 13 out of the 101 candidates (13%). >20000/cmm was 10 and 13, respectively. In order to perform Diagnoses for the 88 pts accepted for SCT were ALL (34%) and a comparison in terms of hematological and non hematological AML (66%). Of those, 7 are awaiting SCT (8%), 59 pts have toxicity, a group of 30 patients, who were previously autografted been transplanted (67%) (16 auto and 43 allo), with a median after conditioning with IDA and oral Bu was considered. Selec- time from program inclusion to SCT of 4.1 m (1.07-14.2), and tion of factors for a matched pair analysis included median age, 22 pts (25%) were not fi nally transplanted due to clinical com- percentage of subjects aged over 60 years, median CD34+ plications and disease progression (10), poor autologous stem cell received, cytogenetic and molecular fi ndings and percent cell mobilization (5), patient’s refusal (2), absence of donor of secondary AML. In both subgroups no occurrence of trans- (1) and other reasons (4). A higher rate of pts with no recent plant related toxicity (TRM) was registered; in addition, results evaluation or active disease at the time of presentation in the in terms of neutrophils and platelet recovery, need of RBC and

S97 platelet transfusion were comparable. Finally, no difference was P466 found as far as occurrence of documented infections (either Allogeneic blood stem cell transplants for AML using the bacterial or fungal) as well as of FUO and liver, cardiac, renal Bu4Cy2 regimen in AML: long term experience or pulmonary toxic episodes. On the contrary, patients condi- L. Brinch, Y. Floisand, G. Tjonnfjord, T. Gedde-Dahl, D. Heldal, tioned with iv BU experienced much less frequently grade 3-4 P. Holme, T. Egeland oral mucositis as compared to the control group (12% vs 80%, Rikshospitalet National Hospital (Oslo, NO) p:<0.0001). This resulted in less frequent use and shorter dura- tion of TPN (20% vs. 83%, p: < 0.0001; 5 days vs. 11 days, Introduction: Allogeneic blood stem transplantation is a highly p: 0.02, respectively) and shorter duration of hospitalization effective and potentially curative treatment for acute myeloid (27 days vs. 31 days, p: 0.04). Finally, patients conditioned with leucaemia (AML). However, this is counterbalanced by a iv BU had signifi cantly lower need of narcotic analgesics (13% relatively high treatment related mortality (TRM). We started vs 80%, p:<0.0001). We conclude that replacement of oral with using the Busulfan 16 mg/kg, Cyclophosphamide 120 mg/kg intravenous BU results in a more favorable toxicity profi le. A (Bu4Cy2) regimen for this disease in 1990 and present our longer follow-up is required to assess a potential advantage in long-term experience from 1990-2008. terms of disease free survival. Family donors with up to one HLA- mismatch and unrelated donors with up to one of ten molecular mismatches were accepted. From 1999 the busulfan dosage was adjusted P465 according to farmaco-kinetic measurements. Effect of HLA disparity on the outcome of allogeneic No ATG was used for conditioning. GVHD prophylaxis was unrelated transplants in AML cyclosporin and metotraxate on day 1,3,6 and 11. The patients F. Bonifazi, G. Bandini, E. Colaci, M. Stanzani, M. Arpinati, transplanted in fi rst remission (CR1) had an intermediate or S. Usai, A. Bontadini, A. Curti, S. Paolini, S. Rizzi, M.R. Motta, high risk for relapse. M. Baccarani Results: 188 transplants were performed, 101 in CR1 and 87 S. Orsola-Malpighi Hospital (Bologna, IT) in relapse, later remissions or in patients with primary induc- tion failure. Median follow-up time is 8 years. The patients were The aim of the study was to evaluate the role of HLA match- 15-64 years of age, with a median of 41 years. Transplants in ing on the outcome of unrelated transplant for AML. 60 adult CR1 were performed in 83 with a family donor and in 18 with a pts where transplanted from 1998 to 2008. Median age was matched unrelated donor. 13 patients have so far relapsed and 39 yrs (range 16- 56). 24 were in 1st CR, 8 in 2nd CR and 28 have died, 18 (18%) of the patients died from TRM, 3 patients in more advanced phase. Patients and donors were typed for from other causes. 67 patients are alive in CR. In the group HLA-A, B, C, DR, DQ at the molecular level: 23 patients were beyond fi rst remission or with primary refractory disease, 41 fully matched, at 10/10 loci (group I); 10 had one or two allelic patients were transplanted with a family donor and 46 with a mismatches and 27 had antigenic mismatches and/or more matched unrelated donor. 49 patients are dead, 19 of these then 2 allelic mismatches (group II). Of the 23 fully matched pts from relapse. 28 patients died from treatment related complica- 14 were in 1st CR vs 10 of 37 of the mismatched transplants. tions, thus a TRM of 32%. 2 patients are alive in hematological The source of stem cells was predominantly bone marrow (50 remission after re-induction treatment, 36 alive in complete CR pts). The preparative regimen was myeloablative in 90% of the without relapse. cases, being TBI-based in 21 patients and Busulfan-based in Conclusions: We confi rm that Bu4Cy2 has considerable anti- 30. In all cases, ATG-Fresenius was added to the preparative leukemic activity in AML. Our long-term results concerning long- regimens, usually at a dose of 3-6 mg/kg /day for fi ve days term stable remissions and TRM confi rms the results reported (from days -6 to -2, total dose 15-30 mg/kg). The prophylaxis by other investigators. The regimen may be used safely in of GVHD was based on the association of CsA and MTX (four selected patients up to 65 years of age. doses). Engraftment: all patients but two engrafted aGVHD: overall incidence of grade II was 12%, grade III 6% P467 and grade IV 6%. Gemtuzumab ozogamicin prior to conditioning cGVHD: overall incidence of limited was 18%, extensive 10%. regimen before allogeneic haematopoietic stem cell The incidence of grade III-IV aGVHD and cGVHD was 4% and transplantation may lead to acceptable outcome in 22% in group I, and 19% and 32% in group II, respectively. high-risk acute myeloid leukaemia patients TRM and relapse were mainly related to the phase of the dis- M. Markova, V. Valkova, A. Vitek, H. Klamova, D. Pohlreich, ease, with the worst results observed in decreasing order from P. Cetkovsky the advanced phases to 2nd CR to 1st CR. However, even Institute of Haematology (Prague, CZ) within each category of disease phase, the degree of matching had an impact. Introduction: Although allogeneic haematopoietic stem cell 1st CR: 1yr TRM 7% in the fully matched group vs 30% in the transplantation leads to durable remission in many intermedi- remaining patients; 1yr relapse 0% in the 10/10 group vs 14% ate and high-risk acute leukemia patients, substantial number for the others. Overall Survival: 92% vs 60% at 4 years respec- of post transplant relapses especially in the high-risk group lead tively. to treatment failure. Advanced Disease: 4 yr TRM 45% for the fully matched trans- Potency of gemtuzumab ozogamicin (GO), to clear CD33+ blasts plants vs 46% for the mismatched; 4yr relapse 14% in the fully is well known. Usually this effect is only temporary in the majority matched group vs 77% in the others. Overall Survival at 4 years of non-transplanted patients. Concurrently the drug is known to was 52% for the 10/10 pairs vs 10% in the others. induce sinusoidal obstruction syndrome (SOS), which can be Results of myeloablative unrelated transplants in AML are very accented when connected with subsequent transplantation. successful when a 10/10 match is used, both in 1 st CR and Patients and methods: Between the years 2001-2008 GO was advanced disease. It is likely that a stronger GVL effect occurs administered prior to allogeneic transplant conditioning regi- with VUD transplants, not evidently related to an increase in men in 13 patients with high risk acute myeloid leukaemia. The GVHD. patients were either refractory or relapsing after conventional chemotherapy treatment. Following to GO administration 10 patients received full intensity conditioning regimen, 8 of them based on total body irradiation (12 Gy) and 2 on busulfan 8 mg/kg, 3 patients were transplanted in reduced intensity con- ditioning (RIC) regimen. Overall and disease free survival was evaluated by Cox regression analysis.

S98 Results: Estimated overall survival in 4 years was 42%. Non- P469 relapse mortality till day 100 after transplantation was 46.2% 17-year-old boy with secondary acute leukaemia presents (6 patients). Only one patient relapsed after transplantation. with Ewing’s sarcoma/PNET following allogenic-SCT Severe sinusoidal obstruction syndrome appeared in one L. Grkovic, P. Roncevic, R. Serventi-Seiwerth, M. Nola, patient only, surprisingly following RIC regimen, and was lead- D. Batinic, K. Gjadrov Kuvezdic, R. Lasan Trcic, D. Ozretic, ing to death. Other toxicity symptoms were comparable to simi- B. Labar lar conditioning regimens without GO. Infection was the major University Hospital Center Zagreb (Zagreb, HR) cause of death. Conclusion: These data suggest that GO administration prior Ewing Sarcoma is the second most common bone tumor in to allogeneic haematopoietic stem cell transplantation may be children and young adults. Primary vertebral Ewing sarcoma feasible when treating very high risk AML. (ES)/primary neuroectodermal tumor (PNET) is very rare, especially with intraspinal infi ltration. Most cases of ES con- tain somatic translocation t(11;22) (q24;q12). We present a P468 case of a boy who was diagnosed with Non Hodgkin lymphoma Sustained complete molecular response and low (B immunophenotype) of the stomach at the age of 12. He was treatment related mortality after allogeneic treated according to BFM 90 protocol. Complete remission haematopoietic stem cell transplantation for (CR) was achieved. At the age of 17 he was admitted to our Philadelphia-positive acute lymphoblastic leukaemia department because of the anemia, thrombocytopenia, fatigue patients previously treated with tyrosine kinase inhibitors and hypertrophy of the gums. The diagnosis of acute myeloid H. de Lavallade, T. Prebet, J. El Cheikh, C. Faucher, N. Vey, leukemia (AML-M4) was established cytomorfologically while A. Charbonnier, C. Chabannon, D. Blaise, S. Furst immunophenotypic analysis suggested pro T acute lymphob- Institut Paoli Calmettes (Marseille, FR) lastic leukemia (proT-ALL) with co-expression of myeloid mark- ers (due to presence CD2+,CD3-,citCD3+,CD7+,TdT+) with Although imatinib improves the CR rate in Ph-positive acute 60% of CD 117 marker. lymphoblastic leukemia (Ph+ALL), it remains possible that the Cytogenetic analysis revealed complex chromosome abnor- improved initial responses to imatinib will not translate into malities (46XY,t(q26;q27),del(7q22),t(8;19)(q23;p12),der(18q). improved survival. To date, myeloablative therapy (MAC) fol- Polymerase chain reaction (PCR) testing for the most common lowed by sibling or matched unrelated allogeneic hematopoi- mutations was negative. He received induction therapy with etic stem cell transplantation (AHSCT) represents the current Ara-C and idarubicine. CR was achieved and consolidation best available treatment option for appropriately aged patients therapy (NOVIA) followed. Allogenic bone marrow transplanta- (pt) with Ph+ALL in CR1. The use of reduced intensity regi- tion (allo-BMT) from the HLA identical family donor was per- mens (RIC) and alternative donors such as umbilical cord blood formed after standard conditioning regimen (Busulfan iv and (UCB) can offer an attractive treatment modality especially to Endoxan) in February 2007 with three lineage engraftment and older pt and those who lack a suitable matched donor. 100% chimera on the lymphocytes and granulocytes of the At our institution the policy was to offer an AHSCT to all CR1 peripheral blood. Eight months after allo-BMT he presented Ph+ALL pt from a matched sibling or unrelated donor or a UCB with paraparesis of the lower extremities. Magnetic resonance unit; a RIC regimen was used for elderly pt or pt receiving a imaging showed massive intraspinal infi ltration with tumorous UCB transplant. Between June 2003 and September 2008 16 tissue from L4 to S1. An operation proceeded and histologically consecutive Ph+ALL adult pt received an AHSCT at our insti- a small-blue-cell tumor (CD99 positive) was observed, indicat- tution, of whom 6 from a sibling donor, 4 from an unrelated ing ES/PNET. Immunophenotypic analysis of bone marrow 10/10 matched donor and 6 with one or two UCB units; 8 pt aspirate revealed CD45-CD90+CD117+ immunophenotype were transplanted after a MAC regimen, and 8 after a RIC regi- typical for tumors of neuroectodermal origin. No leukemia or men. Median age at transplant was 41 years (range, 19-59); lymphoma cells were found. Cytogenetic analysis showed all pt were in fi rst complete hematological remission at time of complex pseudodiploid clone with numeric and structural transplant [major cytogenetic response (MCyR, n=2), complete abnormalities (46-47,XY+del(1p22),+2del(6q),t(11;22)(q24;q13 cytogenetic response (CCyR, n=6), major molecular response .3),-16[cp10]/46XY) of which translocation t(11;22) is character- (MMR, n=4) and complete molecular response (CMR, n=4)]. istic for ES, although PCR analysis for (EWS/FLI1 type 1 and All pt received a tyrosine kinase inhibitors (TKI) before trans- 2) was negative. plant (imatinib=14 pt or dasatinib=2 pt)., and TKI were reintro- Conclusion: We presented a case of 17-year –old patient with duced after transplant in pt with satisfactory hematological three malignant diseases in a fi ve-year period, which is to our recovery (10 pt). knowledge not described in the literature. After a median follow-up of 790 (range, 98-1755) days after AHSCT, 14 pt are alive of whom 11 are in CMR. Objective responses following AHSCT without TKI were obtained in 5 pt. P470 Non-relapse mortality was 6% (one pt died of extensive chronic Allogeneic haematopoietic stem cell transplantation in GVHD in CMR) and 4 pt relapsed early after transplant at a AML t(8;21) in fi rst complete remission gives superior median time of 130 (range, 40-216) days, of whom 2 pt had relapse free survival over repetitive high-dose cytosine developed primary graft failure after UCBT. 8 pt developed arabinoside in certain populations acute GVHD (grade I=3, grade II=3, grade III=20), and 7 pt had S. Ahmed (1), F. Al-Qurashi (2), N. Chaudhri (1), M. Aljurf chronic GVHD (extensive, n= 4 and limited, n=3). (1), H. Al-Zahrani (1), F. Al-Sharif (1), S. Mohamed (1), Our results suggest that AHSCT after TKI therapy is feasible A.J.M. Saleh (1), M. Bakr (1), A. Nassar (1), S. Zaidi (3), without increased toxicity regardless of donor type and con- K. Enazi (1), M. Patel (1), W. Rasheed (1), K. Ibrahim (1), ditioning regimen. Postallogeneic TKI maintenance strategies R. Al-Nounou (1), E. Elghazaly (1), F. Al-Mohareb (1) may further improve results by eradicating minimal residual dis- (1)King Faisal Specialist Hospital (Riyadh, SA); (2)Sultan ease and needs to be investigated in clinical trials. Qaboos University (Muscat, OM); (3)King Fahad Medical City (Riyadh, SA)

Background: Acute Myeloid Leukemias with t(8;21) is a favoura- ble risk subtype, with higher rates of CR and superior responses to high dose Ara-C (HDAC). However, a subgroup of these patients do poorly and relapse, and re-induction failure rates are high. Racial differences in outcomes have been reported. We present a report on 29 racially homogenous patients with

S99 AML t(8;21) treated in our centre who received either induction takes about 3 hours. This method is suitably for the therapeutic chemotherapy followed by consolidation or allogeneic stem cell monitoring of busulfan in plasma following oral administration transplantation. of 1mg/kg b.w. of the drug. Patients And Methods: We retrospectively evaluated data for 29 consecutive patients who were treated in the adult hae- matology unit between 1990 and 2004 who were found to P472 have t(8;21) at diagnosis and were selected to receive either Paediatric high-risk AML: excellent early allogenic transplantation (ALLO, n=14) following achievement disease-free-survival using a fl udarabine-based of fi rst complete remission (CR1) or to continue with chemo- myeloablative preparative regimen therapy alone(CHEMO, n=15). Median age was 23.5 and 19 P.-L. Tan, A. Yeoh, T.-C. Quah respectively. All patients received standard induction chemo- University Children’s Medical Institute (Singapore, SG) therapy and 84% achieved remission with fi rst line therapy. In the ALLO group, 14 patients received a planned allogeneic Haematopoietic stem cell transplantation (HSCT) in paediatric transplantation following CR1; 4/14 (28%) proceeded straight patients with high risk AML such as non-Down de novo acute to allografting, while 10/14 (72%) received a further course of megakaryoblastic leukemia (AMKL), transformed myelodysplas- consolidation. In the CHEMO group, 46% received 3 or more tic syndrome (MDS), therapy-related AML (tAML) and refractory courses HDAC; 26% received 2 doses, 26% received 1 dose. AML (rAML) is limited by graft failure, regimen related toxicity Median dosage was 12g/m2 per cycle. and relapse. To minimize these risks and late effects related Results: The estimated survival for the ALLO group was 54% to total body irradiation (TBI), we treated 10 such consecutive at 7 years, and the mean event free survival was 60 months. patients (AMKL, n=2; MDS/ AML, n=4; tAML, n=1; rAML; n=3), In the CHEMO group, median survival was 28 moths and EFS median age 7.6 (range, 1.3 – 17.0) years with a non-TBI based was 12 months, and probability of survival was less than 30% myeloablative regimen that included 3 active agents against at 6 years. In the ALLO group 3 patients relapsed vs 13 in the AML with non-overlapping toxicities followed by bone marrow CHEMO group (21.4 vs 86.7% p=0.001). Number of patients (n=4), peripheral blood (n=5) or umbilical cord blood (n=1) surviving was 8 for ALLO vs 6 for CHEMO (57.1% vs 40% HSCT between September 05 and July 08. Three patients p=0.3). Of note, of 7 patients who received 3 or more cycles received stem cells (SC) from HLA 6/6 matched related donors of high dose Ara-C, 6 (85%) relapsed in less than one year of while 7 patients received SC from alternate donors (AD) (HLA completion of therapy. 5/6 matched related or HLA 6/6 matched unrelated donors). At Conclusion: We demonstrate signifi cantly lower rates of HSCT, 6 patients were in 1st complete remission (CR1), 2 in relapse and better disease free survival in a Middle Eastern CR2 and 2 in relapse. All received IV Fludarabine 125 mg/m2, cohort transplanted in CR1 compared to HDAC. The study IV Busulfan 0.8 – 1.2 mg/kg 6 hourly, IV Melphalan, 140mg/ is limited by the small numbers and the fi ndings would need m2. AD recipients also received ATG (Thymoglobuline) 5mg/ to be confi rmed by further studies in other non-white popu- kg. GVHD prophylaxis was with Cyclosporine/ Methotrexate lations. Allografting in CR1 should be considered in such (MTX) except for CBT where Mycophenolate Motfetil was used patients, especially in the presence of other high risk features. instead of MTX. Excluding the 2 patients who were in relapse, Autologous transplantation may be a less toxic alternative. all achieved myeloid engraftment at median of 14 (range, The biology of AML with t(8;21)in our population is under 9 – 16) days and sustained stable full donor chimera from 21 further study. days after HSCT. Signifi cant post-HSCT complications included engraftment syndrome (n=4) and reactivation of Herpes viruses (n=4). Incidence of grade I-III acute and chronic GVHD were P471 63% and 67%, respectively. All were alive at median follow-up High-performance liquid chromatographic assay of of 303 (range, 44 – 1037) days Karnosky/ Lansky scores 90 busulfan in plasma samples – Self-modifi cation – 100%. The 2 patients in relapse had secondary graft failures T. Burycz (1), L. Usnarska-Zubkiewicz (2), A. Wiela-Hojenska (2) and subsequently died of disease at a median of 138 (range, (1)Herbapol Inc., (Wroclaw, PL); (2)Wroclaw Medical University 77 – 198) days after HSCT. This FLU-based regimen was well (Wroclaw, PL) tolerated and provided prompt/ robust engraftment in children/ adolescent with high risk AML. In this small series, early dis- Busulfan, an alkylating drug is commonly used at high doses ease-free-survival of 80% appears to be superior compared to before bone marrow transplantation (BMT). Interpatient vari- historical experience with TBI-based or busulfan/ cyclophos- ability in pharmacokinetics and thus drug exposure are signifi - phamide regimens. The major drawback was the high rate of cant and does not always allow a safe and effi cient therapy. viral reactivation. Longer follow-up will defi ne the magnitude of High busulfan exposure is associated with toxicity, especially GVHD/ graft-versus-leukemia effects and if a high event-free- veno-occlusive disease (VOD), whereas low exposure results survival can be maintained with minimal late effects. in decreased effi cacy, which might result in lower engraftment rates. The purpose of our study was to implement analytical method based on liquid chromatography to routinely deter- P473 mination of busulfan concentrations in clinical practice. A Mobilisation of autologous PBSCT in patients with AML sensitive and specifi c assay, involving pre-column diethyldithi- in fi rst complete remission: choosing the optimal ocarbamate sodium (DDTC) derivatization was developed and mobilising regimen validated for the quantifi cation of busulfan in human plasma. A. Pivkova, S. Genadieva Stavrik, Z. Stojanoski, L. Cevreska, The reported assay requires only 600 µl of plasma for the B. Georgievski analysis, and its sensitivity is proper by monitoring samples University Hematology Hospital (Skopje, MK) at a wavelength of 280 nm. The internal standard was 1,6-bis- methansulfonyloxyhexane. The retention times of busulfan and A number of factors have been found to be associated with the internal standard derivatives were 12.5 min and 25.8 min. the likelihood of successful stem cell mobilization. Such prog- respectively. No interfeces were detected at the retention time nostic factors can assist clinicians in designing treatment regi- of this two compounds in plasma containing co-administred mens leading up to the transplant procedure and in identifying drugs, which may be present in clinical samples. The lower patients at high risk for failing the mobilization regimen. G-CSF limit of detection was 20 ng/ml with a limit of quantitation of 60 is the standard agent commonly administered to AML patients ng/ml. Calibration curves were linear from 60 ng/ml to 2500 undergoing PBPC mobilization and collection in a dose of ng/ml. Our modifi cation improves peak separation in chroma- 10 mcg/kg in a 4 days regimen. The additional Etoposide to tograms. The sample preparation was rapid, not complicated G-CSF proved effi cient primary mobilizing regimen for patients without special equipment. The preparation of 20 samples with lymphoproliferative malignancies, mainly NHL and HD.

S100 Since the kinetics of etoposide as mobilizing agent is sparsely P475 described, we evaluated its effectiveness in different dose Prognostic signifi cance of residual disease in bone schedule concerning the late effects of secondary malignan- marrow before allogenic transplantation in acute myeloid cies or myelodisplasia. leukaemia A total of 21 patients with acute myeloid leukemia (AML) in fi rst M.L. Amigo, A. Fernandez, E. López, M. Osma, E. Pérez, CR underwent chemotherapy+G-CSF mobilization for autolo- C. Castilla, I. Heras, J. Nieto, F. Ortuño, V. Vicente gous transplantation. 11 Pts received VP-16 (2gm/m²) followed Hospital Morales Meseguer (Murcia, ES) by daily G-CSF(10mcg/kg) and in 10 patient the dose of VP- 16 was lowered 1gr/m². All pts experienced a signifi cant WBC Objetive: To analize the impact on outcome of residual disease nadir on median day +6, and began aphaeresis in recovered (RD) determined by multiparametric fl ow cytometry (MFC) in WBC up to 5.0x109/L on day +12(median). All analyzed patients bone marrow (BM) before allogenic stem cell transplantation reached at least 2x106/kg CD34+ cells with median 3 (ranges (ASCT), in patients with acute myeloid leukemia (AML). 1-6) aphaeresis procedures. 16 (78%) of pts began collection Patients and methods: We performed a retrospective analysis on day +10, +11 or +12 and 4 (22%) pts did not begin aphaer- of 37 consecutive patients diagnosed with AML who underwent esis until day +17 or longer. One patient failed mobilization. In an ASCT in our Unit between June 1996 and April 2007. We statistical data we followed several variables concerning the collected data of RD on BM by MFC on the 30 days previous to platelet count on day 1 of aphaeresis which correlated with the the transplant procedure. The leukemia-associated phenotype ability to collect over 5x106CD34+cells/kg (p<0,001) and previ- was detected by MFC in all cases at diagnosis and/or in relapse ously received chemotherapy cycles of 6 pts(32%) who started and was used for monitoring RD. aphaeresis on median day +17 (p<0,001). We can conclude Results: Median age of patients in our cohort was 40 years that the VP-16+G-CSF mobilizing regimen for AML patients (range 10-66), 19 males and 18 females, 32 patients received showed effective with excellent yields results for median 3 day a transplant from an HLA-identical sibling donor and 5 from leukopheresis procedure. We didn’t observe statistical differ- an HLA-identical unrelated donor. Six patients received a ence between the dose schedule and the number of collected reduced intensity conditioning regimen. In 33 cases peripheral MNC in both groups. This confi rms the possibility for estab- blood was used as source of stem cells and bone marrow in lishing VP-16 1gr/m² + G-CSF 10mcg/kg regimen as primary the other 4. All the patients had received at least 2 cycles of mobilizing regimen for AML patients that undergo autologous chemotherapy before transplant. Twenty nine were in complete transplantation in fi rst complete remission. remission (CR) (26 in fi rst CR and 3 in second or >2ndCR) and 8 had active disease (2 in early relapse and 6 with refractory disease). Eleven patients had high-risk cytogenetics at diagno- P474 sis. With a median follow up after transplantation of 9 months Extramedullary relapse in cases of acute myeloid (range 5-161) 10 patients (27%) relapsed. At 4 years, disease leukaemia after allogeneic stem cell transplantation free survival (DFS) and overall survival (OS) were 59% and L. Perez-Bercoff (1), B. Gustafsson (2), O. Ringden (3), 75% respectively. On the group who underwent transplant with P. Ljungman (4) active disease (n=8). The median survival of this group was 24 (1)Karolinska Institutet (Stockholm, SE); (2)Barnens sjukhus months. Two of the 8 patients are alive and in CR. RD value (Stockholm, SE); (3)Karolinska Universitetssjukhuset were very varied, median 0,88% (0,2%-9%). Those patients Huddinge (Stockholm, SE); (4)Karolinska Institutet/Karolinska with a RD <0,4% presented a superior DFS than patients with Universitetssjukhuset Huddinge (Stockholm, SE) RD >0,4% (median not reached (MNR) versus 80 months; p=0,044). OS was higher in patients with RD <0,4%, although Relapse is the major cause of treatment failure following alloge- the survival advantage was not statistically signifi cant (MNR neic haematopoietic stem cell transplantation (HSCT) among versus 140 months; p>0,05). DFS and OS were better in the patients with acute myeloid leukaemia (AML). Most relapses group of intermediate risk cytogenetics than in the group of occur in the bone marrow. However, isolated extramedullary high-risk cytogenetics (MNR versus 30,5 months; p=0,04 and relapses (EMRs) occur. The aim of this retrospective study was MNR versus 106 months; p=0,015). to determine the incidence and outcome of EMRs in a con- Conclusions: Detection of high levels of RD before transplanta- secutive cohort (n=321) of AML patients who had undergone tion and high-risk cytogenetics at diagnosis are associated with allogeneic HSCT at Karolinska University Hospital – Huddinge high risk of relapse after ASCT. These factors should be help- from 1977–2008. Eighteen patients with EMR of AML (17) or ful to identify groups of patients who need new approaches to MDS-AML (1) were identifi ed. As the patient number is limited reduce postransplant relapses. and statistical power is diffi cult to obtain we used statistical analysis as a help to detect trends. The frequency of EMR was 5.6%, with a signifi cantly higher frequency among patients who P476 had undergone HSCT because of advanced disease (8.9% vs. Gemtuzumab-ozogamicin as maintenance therapy after 2.0%; p=0.01). Extramedullary relapse was more common in autologous stem cell transplantation in elderly patients children (12.8%) than in adults (3.3%; p=0.02), with an espe- with acute myeloid leukaemia cially high frequency in girls (14.3%). The most frequent loca- A.M. Carella, M. Greco, E. Merla, N. Cascavilla tions of EMR were in breast tissue (5) and skin (3). The median Casa Sollievo della Sofferenza (San Giovanni Rotondo, IT) time from HSCT to EMR was 17.8 months (range 4.3–127). Five of the eighteen patients subsequently developed bone marrow The role of ASCT in elderly patients (pts) with AML is under inves- relapses after a median time of 69 days (range 25–93). Six of tigation. We supposed that GO could have benefi cial effects as the 18 patients were alive at 2, 3, 4, 34, 42 and 177 months maintenance therapy after hematopoietic ASCT in high-risk pts. after EMR. However no signifi cant difference were seen in the We report on 9 (6 males and 3 females with a median age of overall mortality in females (46.6%), males (45.1%) or children 67) elderly AML pts in fi rst (8) or second (1) CR who received (43.6%) looking at all AML patients being transplanted at our 4 fractionated doses of GO as maintenance therapy following center. The mean survival time for all deceased AML patients ASCT. Patient 1 was in 2nd CR following an induction regimen post-HSCT was 24.4 months. Meanwhile the mean survival time of 2 cycles of fl udarabine (Flu) + cytarabine (Ara-C) + idarubicin for the deceased AML patients with EMR was 16.8 months. (Ida); he previously underwent ASCT, using a conditioning regi- Conclusions: Our study suggests that new larget multicenter men of Bu-Cy. One patient had an AML refractory to a standard studies are needed in the fi eld. induction chemotherapy (3 + 7 regimen) and rescued with 1 MEC regimen. The other pts were in 1st CR following an induc- tion regimen of 2 cycles of Flu + Ara-C. HLA-matched donors were not available for any pts. Three months after obtaining

S101 CR, all pts received a myeloablative conditioning regimen and Discussion: Allogeneic transplant is absolutely superior in treat- underwent ASCT. Two months after the complete engraftment, ment of AML pts over other two post remission options. To pts started treatment with GO. To date 7 of them are alive and in improve results of autologous SCT more attention should be CR at +35, +22, +16, +12, +8, +6 and +4 months respectively, given on the control of minimal residual disease and also prob- while the overall survival at the moment of reporting was +58, ably by using bone marrow as an origin of stem cells. +27, +28, +16, +11,+10 and +8 months. Two pts were relapsed at +12 e +14 respectively from the ASCT and one of them died due to disease progression at +18 months. One patient is alive P478 and in 2nd CR at +28 months from the diagnosis and to date First complete remission versus other than fi rst complete he is scheduled to undergo unrelated allogeneic transplant. Six remission in acute myelogenous leukaemia with patients completed all the scheduled therapeutic approach. allogeneic haematopoietic stem cell transplantation There were no cases of grade 3 or 4 liver toxicity and bleeding K. Alimoghaddam, F. Khatami, A. Jalali, M. Jahani, M. Iravani, was not observed in any cases. Thrombocytopenia (50,000– S. Mousavi, B. Bahar, A. Masnavi, E. Eini, A. Ghavamzadeh 100,000 cells/ìL) occurred in all pts and neutropenia (500–1000 Hematology-Oncology and Stem Cell Transplantation Research cells/ìL) in 2 patients. In conclusion, in our study, despite limited Center (Tehran, IR) in number, GO demonstrated a good effi cacy when adminis- tered in fractionated doses as maintenance therapy after ASCT Introduction: Allogeneic Hematopoietic Stem Cell Transplanta- in older patients with CD33+ AML in 1st or 2nd CR. GO showed tion (HSCT) for Acute Myelogenous Leukemia (AML) in fi rst an acceptable tolerability profi le, with no severe hepatotoxicity Complete Remission (CR1) is standard of care. In addition, the and no bleeding risk. Thrombocytopenia occurred in all patients studies show the cure when patients are transplanted in sec- and in all cases there was a rapid platelet recovery. A GO dose ond Complete Remission or in fi rst relapse. In this study, the of 3 mg/m2 appeared to be better tolerated than the higher outcome of AML patients with allogeneic HSCT in CR1 versus dose (6 mg/m2) and will be used in future studies. Our results other than CR1 was compared. could support a new therapeutic role for GO as a maintenance Patients and Methods: Since March 1991 until November 2008, therapy to better manage minimal residual disease for patients from 420 AML patients, 312 patients in CR1 with median age of in CR following hematopoietic ASCT. 27 years and 108 patients in other than CR1 with median age of 29 years have undergone allogeneic HSCT. Male/Female ratio was 168/144 in CR1 group and 55/53 in other than CR1. Totally P477 391 patients received Peripheral Blood, 27 patients Bone Mar- Transplant procedures in comparison to conventional row and 2 ones Cord Blood as sources of HSCT. therapy in the treatment of acute myeloid leukaemia: Result: At present 266 (85%) patients in CR1 and 73 (68%) long-term follow-up patients in other than CR1 are alive. The most common cause M. Elez, D. Stamatovic, L. Tukic, O. Tarabar, B. Balint, B. Todoric of death in CR1 group was Graft Versus Host Disease and in Zivanovic, O. Tasic, V. Skuletic, M. Malesevic, S. Marjanovic other than CR1 group was relapse. Median follow up time was Military Medical Academy (Belgrade, RS) 17 month (range: 1-158 month). Six month Disease Free Sur- vival (DFS) and Overall Survival (OS) in CR1 was 86% and Introduction: There are three approaches for consolidation of 91% (SE=2%). 2-years DFS and OS in CR1 was 78% (SE=3%) patients (pts) with acute myeloid leukemia (AML) (with excep- and 85% (SE=2%). Six month DFS and OS in other than CR1 tion of acute promyelocitic leukemia). Those approaches are was 64% (SE=5%) and 75% (SE=4%). In this group 2-years either allogeneic or autologous stem cell transplantation (SCT) DFS and OS was 50% and 64% (SE=5%). It is considerable or conventional therapy. that six month DFS and OS in transplanted patients with CR1 Aim: We have analyzed retrospectively 74 pts with AML divided was signifi cantly higher than in other than CR1 (p<0.001). Fur- into three groups- conventional, autologous stem cell trans- thermore, 2-years DFS and OS in patients with CR1 were bet- plantation (SCT) and allogeneic SCT group with respect to ter than second group (P< 0.001). relapses, time to progression (TTP) and overall survival (OS). Conclusion: Although results of HSCT in other than fi rst com- Material and Methods: We have treated 110 pts with AML from plete remission is not as good as in fi rst complete remission but 2001 till 2008. Nine of them had acute promyelocytic leukemia it seems that it is good enough to advice for doing allogeneic and 27 pts have showed primary resistance. We have enrolled HSCT for these patients. 74 pts into these analysis- 30 pts into “conventional” (M/F 23/7, median age 48,6 years), 30 pts in the “autologous transplant” (M/F 13/13, median age 33,5 years) and 18 pts in the “allo- P479 geneic transplant group”(M/F 13/5, median age 31,6 years). Dasatinib in the treatment of extramedullary relapse in After one or two courses of standard induction “7+3”), pts have BCR/ABL+ AML patients after allogeneic transplantation received three more cycles of consolidation (MAE, MidAC and R. Scimè, S. Tringali, A. Cavallaro, A. Indovina ICE) and last one was used for mobilization of autologous Ospedale V. Cervello (Palermo, IT) peripheral stem cells in approved complete remission. Both transplant groups immediately undergone conditioning up to We report two cases of extramedullary relapses after alloge- standard BuCy2±IDA regimen and “conventional group” have neic transplantation in BCR/ABL+ AML patients successfully received maintenance therapy with Thioguanin 160 mg daily treated with dasatinib. per os and Cyto ARA 100 mg subcutaneously daily for 5 days. Case 1: on July 06 a 56 yrs old lady was diagnosed with AML Prophylaxis of GvHD in allogeneic group is combination of BCR/ABL p210+. After induction she achieved CR; on Octo- Cyclosporine A and Metothrexate. ber 06 maintenance therapy with imatinib was started but she Results: On intention-to-treat analysis number of relapses was relapsed on December 06. 2° CR was obtained and on Febru- signifi cantly lower in allogeneic group (1/18- 5,55%, p<0,005) ary 07 she received an HLA related allogeneic transplant after while there were no difference between other two groups BUCY2 conditioning. She developed grade I aGVHD, respon- (autologous 12/26 – 46% vs 13/30 – 43,3%, ns). TTR was sive to steroid. She remained in molecular remission with longer in autologous group but not signifi cantly (9,4 range 2-42 a limited cGVHD until March 08, when she complained of a vs 8,7 range 1-48 months). There were signifi cantly better OS severe back pain: a CT scan showed a large abdominal mass in allogeneic group compared to others while there is no sig- and a thyroid enlargement; a diagnostic biopsy documented the nifi cance between autologous and conventional group (log rank presence of immature cells bcr/abl+ with a T cell phenotype test- au:al 2,88, p<0,01; con: al 2,69, p<0,01; au:con 0,57, ns). (TdT +, CD34+, CD79a+, CD10+, HLADR+, MPO-). Bone mar- Median survival in allogeneic group was 37,5 months (range row was negative for blasts but positive for bcr/abl and showed 7-86 months). a mixed chimerism. She received dasatinib, 70 mg/bid, with a

S102 dramatic improvement of the symptoms, a reduction of the thy- P481 roid enlargement and of the abdominal mass; after 1 month of Allogeneic transplantation from alternative donors is dasatinib the marrow molecular signal disappeared and donor feasible in elderly patients with poor prognosis acute chimerism was restored. After 8 months of treatment the patient myeloid leukaemia or myelodysplastic syndrome: is doing well with a signifi cative reduction of the mass and in a single-centre experience complete molecular remission. M. Tassara, M. Bernardi, J. Peccatori, A. Crotta, M.T. Lupo Case 2: on October 06 a 27 yrs old lady presented with AML Stanghellini, C. Messina, C. Corti, A. Assanelli, D. Clerici, BCR/ABL p210+; during induction she developed invasive S. Mastaglio, S. Malato, F. Ciceri fungal infection and no further chemotherapy was given but San Raffaele Scientifi c Institute (Milan, IT) imatinib 800 mg, without molecular response. She was then shifted to dasatinib 70mg/bid and transplanted from her HLA Background: allogeneic (allo) stem cell transplantation (SCT) identical sister on July 07, obtaining molecular remission. She is the only potentially curative strategy for poor prognosis developed grade I aGVHD responsive to steroid. On Novem- acute myeloid leukaemia (AML) and myelodysplastic syn- ber 07 molecular signal reappeared and she was given again drome (MDS). Patients (pts) older than 60 are rarely offered dasatinib 70 mg/bid and cyclosporine was stopped: a complete an alloSCT because of the procedure-related toxicities and the molecular response was obtained, but on February 08 dasat- diffi culty to fi nd a matched relative, fi t to donate; SCT from alter- inib was stopped due to toxicities and donor lymphocytes infu- native donors is usually controidicated in these pts because of sion was planned but she never received DLI because of an the high rate of transplant related mortality (TRM). Since 2002, extensive cGVHD. While on molecular remission with full donor we have transplanted 25 pts, age ≥ 60, with advanced or high chimerism, on August 08 an axillary mass (7x5cm) appeared risk AML/MDS, from a matched unrelated (URD) or haploideni- and consisted with a granuloblastic sarcoma (CD34+;CD117 cal related (HAPLO) donor. Data on feasibility and outcome are +;MPO+;Ki67+). Dasatinib was started again and the axyllary here reported. mass rapidly reduced. After 4 months of treatment the mass Aim: to retrospectively evaluate data on alloSCT from alterna- almost disappeared and she is in molecular remission. tive donors in elderly AML and MDS pts, at our Institute. In these 2 cases cGVHD was not effective in controlling Methods: period 10/2002 to 10/2008, 25 pts, median age extramedullary relapses, while dasatinib seemed to effectively 63.7 (60-72.1). Diagnosis (WHO): AML 6, AMLMD 7, RAEB- control the disease. 1 2, RAEB-2 4, MDS/MPD 2, bifenotipic AL 1, t-related AML/ MDS 3. Median number of chemo cycles before SCT: 2 (0-7). Donors:URD 5, HAPLO 20. Disease status at SCT: CR1 9, CR2 P480 2, upfront 2, refractory 6, relapsed 6. Conditioning regimens Two types of conditioning for HSCT in paediatric acute contained fl udarabine in 24 cases, treosulfan in 22, thiotepa lymphoblastic leukaemia in 2, cyclophosphamide, busulfan, melphalan in 1 case, ATG N. Minakovskaya, N. Kirsanova, O. Aleinikova in all cases. GvHD prophylaxis: T-cell depletion in 11 cases, Belarusian Center for Ped OncoHematology (Minsk, BY) CSA/MTX in 11, rapamycin/MMF in 3. All pts received SCT from peripheral blood. The aim of our study was the comparison of clinical outcome Results: TRM before day+30: 2 (8%); 23 pts (92%) engrafted after hematopoietic stem cells transplantation (HSCT) between and were in CR at day+30, included 13 (93%) with active dis- busulfan (BU)-contained and total body irradiation (TBI)-based ease before SCT. Relapses were 5 (21.7%). Overall TRM was regimens of conditioning in children with acute lymphoblastic 44% (11 pts), 9 for infections, 2 for other causes; 4 pts died for leukemia (ALL). disease progression, all deaths (15) occurring within the fi rst Patients and methods: 25 pts with ALL underwent the alloge- year after SCT. aGvHD grade>II: 4 cases; extensive cGvHD: 3 neic HSCT in one center from 2004 to 2008. The remaining cases. At last follow-up (FU) 10 pts (40%) are alive in CR with a 25 pts (median age, 11 (2,5-27) y, male/female – 12/13) were median FU of 770 (56-1375) days, median OS from SCT of all categorized according to protocol of conditioning. Regimen of 25 pts was 227 (3-1375) days. conditioning consists of BU for 13 pts (group I) and TBI for 12 Conclusions: alloSCT from alternative donors is feasible in eld- pts (group II). Twenty pts were transplanted in complete remis- erly pts with AML or MDS. Long term survival free from disease sion (CR) >I (10 pts included in group I and 10 pts – in group has been obtained in several pts; notably, about one third of II) and fi ve pts of high group risk with Ph-positive ALL (3 pts those transplanted with active disease are alive in CR at last from group I and 2 pts from group II). Graft versus host disease follow up. Reduced-toxicity conditioning regimens showed lim- GvHD prophilaxis included cyclosporine (CSA) alone 2 pts, ited early mortality; better prevention and management of infec- CSA + metothrexat (MTX)- 22 pts and 1 pts - tacrolimus. The tions could permit to reduce the overall TRM rate. In conclusion mean time of follow-up was 10 ± 2 months. We analyzed the alloSCT from an alternative donor is a therapeutic option for pts rate of GvHD (grade > II), transplant related mortality (TRM), with poor prognosis AML/MDS and advanced age. incidence of relapse (IR), overall survival (OS), event free sur- vival (EFS), relapse free survival (RFS) in both groups. Results: There were no differences in TRM and IR between two groups (p=0,3 and p=0,16; respectively). Evidence of GVHD was signifi cant higher for group II (8 out of 12pts, 12,6%) then for group I (2 out of 13pts, 15,3%) (p=0,009). Our data dem- onstrate that OS, EFS and RFS on TBI-based regimen (0,51 ± 0,19, 0,37 ± 0,19 and 0,80 ± 0,18 respectively) were better than for BU-contained (0,27 ± 0,15, 0,13±0,12 and 0,24 ± 0,21 respectively). We conclude that TBI-based regimen is better for OS and RFS than BU-contained (fi gure1); however, the rate of severe GVHD is signifi cantly higher in TBI-group. New modalities of chemo- based regimens in conditioning are needed.

S103 Stem cell source P483 Expansion potential of adipose tissue-derived mesenchymal stem cells: role of the addition of human platelet lysate P482 C. Almici (1), R. Verardi (1), A. Neva (1), G. Piovani (2), Results of two phase III, multicentre, randomised, A. Bianchetti (1), G. Santoro (1), D. Russo (2), S. Barlati (2), placebo-controlled trials of plerixafor + G-CSF versus M. Marini (1) G-CSF + placebo for mobilisation and engraftment of (1)Spedali Civili (Brescia, IT); (2)University of Brescia (Brescia, non-Hodgkin’s lymphoma and multiple myeloma patients IT) undergoing autologous transplantation J.F. DiPersio (1), I.N. Micallef (2), A.P. Nademanee (3), Mesenchymal stem cells (MSC) are multipotent cells that are P.J. Stiff (4), E.A. Stadtmauer (5), R.T. Maziarz (6), B.J. Bolwell (7), considered one of the most promising product for cellular ther- J. Angell (8), G. Bridger (8), G. Calandra (8) apy. MSC can be expanded from bone marrow and umbilical (1)Washington University (Saint Louis, US); (2)Mayo Clinic cord blood; recently, adipose tissue (AT) has been regarded as (Rochester, US); (3)City of Hope (Duarte, US); (4)Loyola a more suitable and easily accessible source of MSC. University Medical Center (Maywood, US); (5)University of We tested the effects of human platelet lysate (hPL) in com- Pennsylvania (Philadelphia, US); (6)Oregon Health & Science parison to fetal bovine serum (FBS) on expansion potential University (Portland, US); (7)Cleveland Clinic Foundation of AT-derived MSC. After informed consent and under local (Cleveland, US); (8)Genzyme Corporation (Cambridge, US) anesthesia, AT was obtained from patients undergoing liposuc- tion for plastic surgery pourposes (n=5, range 8-16 x 106 cells). We previously reported that plerixafor + G-CSF (G) was as safe After enzymatic digestion of the liposuction product, cells were as and more effective than placebo + G in mobilizing hemat- seeded (5 x 105/cm²) in fl asks in Iscove’s medium with the addi- opoietic stem cells for autologous transplant in non-Hodgkin’s tion of either 5% hPL or FBS for human MSC (StemCell Tech- lymphoma (NHL) and multiple myeloma (MM) patients. In nologies). Non-adherent cells were removed after 2-3 days the Phase III multicenter, placebo controlled randomized tri- and fresh medium changed twice weekly thereafter. On reach- als, 3101 and 3102, the European Medicines Agency (EMEA) ing confl uence, adherent cells were detached and replated at effi cacy objective was the ability to achieve a target number low density (2 x 103/cm²) in the same medium conditions. At of CD34+ cells/kg within a pre-specifi ed number of apheresis each passage, surface antigen expression was analyzed by days and having successful engraftment. fl owcytometry (CD45, CD90, CD105, CD73, CD166, CD144). Adult NHL and MM patients requiring an autologous transplant Expanded MSC were analyzed for differentiative capacities by were eligible. Patients received G (10mcg/kg/day) for 4 days; using specifi c differentiating medium for osteogenic (Mesen- on the evening of day 4 they received either plerixafor (240mcg/ Cult Human Osteogenic Supplement) and adipogenic (Mesen- kg) or placebo. Patients underwent apheresis on Day 5 after Cult Human Adipogenic Supplement) differentiation. Expanded a morning dose of G. Patients continued to receive evening MSC were cryopreserved and after thawing cultures restarted; study treatment followed by morning G and apheresis for up to moreover, expanded MSC were analysed for the presence of pre-specifi ed number of apheresis days or until a target number cytogenetic abnormalities. of CD34+ cells/kg were collected. In 3101, an EMEA-specifi c Confl uent growth was obtained at each passage after 7-10 composite endpoint was the proportion of NHL patients achiev- days, showing typical MSC phenotype (negativity for CD45 ing ≥5 x 106 CD34+ cells/kg in 4 or fewer days of apheresis and antigen, and positivity > 90% for CD90, CD73, CD105) and adi- engrafting successfully. In 3102, an EMEA-specifi c composite pogenic and osteogenic differentiative capabilities, irrespective endpoint was the proportion of MM patients achieving ≥ 6 x 106 of medium used. However, median fold expansion at fi fth pas- CD34+ cells/kg in 2 or fewer days of apheresis and engrafting sage was 15322 (range 1400-76199) for hPL cultures and 422 successfully. (range 16-827) for FBS cultures, recovering a median of 87 x A total of 298 NHL and 302 MM patients were enrolled and 109 MSC (range 9-206 x 109) and 1.3 x 109 MSC (range 0.1-2.5 randomized in 3101 and 3102, respectively. Baseline charac- x 109), respectively. teristics were similar between the active treatment and placebo Cryopreservation showed no detrimental effects on the expan- groups. In 3101, a signifi cantly greater number of NHL patients sion potential of AT-MSC. Karyotypic analysis did not disclose in the G + plerixafor group achieved ≥ 5 x 106 CD34+ cells/kg any cytogenetic abnormalities. in 4 or fewer days of apheresis and had successful engraft- In conclusion, AT represents a valid source of MSC that can ment compared with the G + placebo group (57.3% versus be easily expanded in high numbers for clinical applications. 18.9% of patients, respectively; p < 0.001). Similarly, in 3102, Moreover, hPL can be considered a valuable substitute of FBS a signifi cantly greater number of patients in the G + plerixafor for MSC expansion. The advantages are even superior consid- group achieved ≥ 6 x 106 CD34+ cells/kg in 2 or fewer days ering the compliance with the required criteria for somatic cell of apheresis and had successful engraftment compared with therapy applications. the G + placebo group (70.3% versus 34.4%, respectively, p < 0.001). Through 12 months follow-up, there were no differences in graft durability and hematology profi les between groups in P484 either study. Unrelated cord blood transplantation after G-CSF + plerixafor, compared with G + placebo, signifi cantly myeloablative conditioning for adult patients with increased the proportion of NHL and MM patients who were myelodysplastic syndrome able to mobilize the target number of CD34+ cells needed J. Ooi, S. Takahashi, N. Tsukada, S. Kato, A. Sato, F. Monma, for autologous transplant with equally prompt and durable A. Tojo, S. Asano engraftment. Institute of Medical Science (Tokyo, JP)

Recently, umbilical cord blood from unrelated donors have been used as an alternative stem cell source for adult patients with hematologic malignancies. We analyzed the disease specifi c outcomes of adult myelodysplastic syndrome (MDS) patients treated with unrelated cord blood transplantation (CBT) after myeloablative conditioning and identifi ed pre-transplant fac- tors related to the transplant outcomes. Between August 1998 and August 2008, 34 adult patients with MDS were treated with unrelated CBT in our institute. Diagnosis at transplantation

S104 included refractory anemia (RA) (n=4), refractory anemia with subgroup of patients was met in 29/57 (50.9%) and 17/67 excess blasts (RAEB) (n=7), RAEB-t (n=2), and MDS-related (25.4%) of the patients in the plerixafor plus G and G plus pla- secondary AML (n=21). All patients received four fractionated cebo groups, respectively (p<0.001). Median time to neutrophil 12 Gy total body irradiation and chemotherapy as myeloabla- engraftment in the plerixafor plus G group was 10 days and tive conditioning. All patients received standard cyclosporine 11 days in the G plus placebo group. Median time to platelet and methotrexate as a GVHD prophylaxis. The median age engraftment was 20 days and 21 days in the G plus plerixafor was 40 years (range, 19-52 years), the median weight was 56 and G plus placebo groups, respectively. kg (range, 43-75 kg), and the median number of cryopreserved Of the 302 patients enrolled in study 3102, 145 MM patients nucleated cells was 2.44 x 107/kg (range, 1.71-4.10 x 107/kg) were older than 60 years of age. The primary endpoint in this and the median number of cryopreserved CD34 positive cells subgroup of MM patients was met in 48/69 (69.6%) and 18/76 was 0.85 x 105/kg (range, 0.40-2.14 x 105/kg). 31 patients had (23.7%) of the patients in the plerixafor plus G and G plus pla- myeloid reconstitution and the median time to more than 0.5 x cebo groups, respectively (p<0.001). Median time to neutrophil 109/L absolute neutrophil count was 22 days. A self-sustained and platelet engraftment in both groups was 11 and 18 days, platelet count more than 50 x 109/L was achieved in 30 patients respectively. at a median time of 50 days. Grades III-IV acute GVHD occurred In these studies, the addition of plerixafor to G in NHL or MM in 5 of 31 evaluable patients and chronic GVHD in 26 of 29 eval- patients greater than 60 years old undergoing ASCT is superior uable patients. Among 26 chronic GVHD patients, in 13 patients to G alone. the disease was extensive. No factor was associated with hematopoietic recovery and the incidence of acute and chronic GVHD. The 5-year cumulative incidence of transplant related- P486 mortality (TRM) and relapse was 14.5%, 16.6%, respectively. Prospective analysis for antigen-specifi c cellular immune No factor was associated with TRM and relapse. 25 patients are reconstitution after cord blood transplantation: immune alive and free of disease at between 3.4 and 125 months after response to CMV is not affected by HLA disparity transplantation. Median follow-up was 69 months. The probabil- S. Takahashi, M. Ishige, N. Watanabe, T. Yamaguchi, J. Ooi, ity of event-free survival (EFS) at 5 years was 71.3%. Poor-risk N. Tsukada, S. Kato, A. Sato, F. Monma, R. Yamazaki, A. Fujita, karyotype at diagnosis was signifi cantly associated with worse N. Toki, H. Tanaka, T. Uehara, K. Fujimaki, K. Oshima, Y. Aisa, EFS (p=0.037). Cell dose and HLA matching had no impact K. Motohashi, R. Hyo, Y. Najima, H. Kanamori, Y. Nannya, on any transplant outcomes. These results suggest that adult R. Sakai, S. Takada, T. Kobayashi, T. Hoshino, K. Matsumoto, MDS patients without suitable related or unrelated bone marrow K. Hideyuki, J. Kato, A. Yokota, N. Nakamura, M. Tanaka, donors should be considered as candidates for CBT. T. Mori, H. Sakamaki, S. Okamoto on behalf of Kanto Study Group for Cell Therapy (KSGCT)

P485 Study purpose: Cytomegalovirus (CMV) infection is still serious Effi cacy of plerixafor plus G-CSF compared to G-CSF problem in cord blood transplant (CBT) and T cell immunity has plus placebo for mobilisation of CD34+ haematopoietic an important role in control of virus replication and prevention. progenitor cells in patients older than 60 years with The one of crucial questions in CBT is whether naivity of lym- non-Hodgkin’s lymphoma or multiple myeloma phocytes could gain antigen-specifi c cellular immunity during I.N. Micallef (1), J.F. DiPersio (2), A.P. Nademanee (3), early phase of human leukocyte antigen (HLA)-mismatched P.J. Stiff (4), E.A. Stadtmauer (5), R.T. Maziarz (6), B.J. Bolwell (7), transplant. To answer this, we have analyzed the CMV-specifi c J. Angell (8), G. Bridger (8), G. Calandra (8) immune reconstitution process for fi rst 6 months. (1)Mayo Clinic (Rochester, US); (2)Washington University (Saint Patients and Methods: During 2006 and 2008, 40 adults has Louis, US); (3)City of Hope (Duarte, US); (4)Loyola University received myeloablative regimens including 12 Gy of total body Medical Center (Maywood, US); (5)University of Pennsylvania irradiation followed by CBT and a standard cyclosporine and (Philadelphia, US); (6)Oregon Health & Science University methotrexate combination as GVHD prophylaxis in the Institute (Portland, US); (7)Cleveland Clinic Foundation (Cleveland, of Medical Science, University of Tokyo (IMSUT), for 19 patients US); (8)Genzyme Corporation (Cambridge, US) and in 9 different facilities which participated for the prospec- tive study using IMSUT regimen for 21 patients. CMV-specifi c Advanced age and intensive previous chemotherapy are two CD4+ and CD8+ T cell recoveries were assessed by detection independent risk factors that are associated with poor CD34+ of interferon-gamma (IFN-g) producing cells with CMV antigen hematopoietic stem cell (HSC) mobilization in patients under- stimulation using intracellular cytokine staining or tetramers for going autologous stem cell transplantation (ASCT). The pur- CMV pp65 in whom HLA-A0201, -A0206 or -A2402 positive pose of this report is to evalaute the effi cacy of plerixafor plus patients. The positive was defi ned as >0.03% IFN-g positive G-CSF (G) to G alone in non-Hodgkin’s lymphoma (NHL) and cells among CD4+ or CD8+ T cell population and >0.01% posi- multiple myeloma (MM) patients >60 years of age undergoing tive in tetramer assay. CD34+ HSC mobilization for ASCT. Results: CMV-reactive (IFN-g positive) CD4+ T cells were Adult NHL and MM patients requiring ASCT were eligible to par- detected in 65% at 1 month, 88% at 2 months, 92% at 3 ticipate in a phase III multi-center, randomized, placebo con- months, 92% at 4 months and 95% at 6 months after CBT trolled trial. Non-Hodgkin’s lymphoma patients participated in which were comparable to CMV-positive age-adjusted healthy study 3101 and MM patients in study 3102. In each of the trials, control (100%). CMV-reactive (IFN-g positive) and CMV-spe- patients were randomized to receive plerixafor plus G or G plus cifi c (tetramer-positive) CD8+ T cells were detected in 53/5% placebo. All patients received G (10mcg/kg/day) for 4 days; on at 1 month, 71/44% at 2 months, 68/36% at 3 months 75/50% the evening of day 4 they received either plerixafor (240mcg/ at 4 months, 65/50% at 6 months (39/67% in the control). Next, kg) or placebo. Patients underwent apheresis on day 5 after we looked the effect of HLA disparity (HLA-DR for CD4+ and a morning dose of G. Patients continued to receive evening HLA-A/ -B for CD8+ T cell) in graft-versus-host direction with study treatment followed by morning G and apheresis for up low resolution typing (LRT) and in high resolution typing (HRT). to a pre-specifi ed number of apheresis days or until a target CMV-reactive CD4+ T cells were detected in 94% with matched number of CD34+ cells/kg were collected. In 3101, the primary (0MM), 81% with one mismatched (1MM) in LRT and 100% endpoint was the percentage of patients who achieved 5x106 with 0MM, 89% with 1MM, 80% with 2MM in HRT at 2 months. CD34+ cells/kg in 4 or less apheresis days. In 3102, the pri- CMV-specifi c CD8+ T cells were detected in 33% with 0MM, mary endpoint was the percentage of patients who achieved 6x 38% with 1MM, 56% with 2MM in LRT and 38% with 1MM, 50% 106 CD34+ cells/kg in 2 or less apheresis days. with 2MM, 67% with 3MM in HRT at 2 months, respectively. Of the 298 patients enrolled in study 3101, 124 NHL patients Conclusion: Post-thymic naive T cells in cord blood might obtain were older than 60 years of age. The primary endpoint in this memory and effector function in vivo with antigen-specifi c

S105 manner during early phase of post-transplant without effect of the current expansion processes are laborious, expensive and HLA disparity. providing unwanted differentiated sub-populations. AIMES. As the ABC transporter Pgp (gene product of ABCB1) is over- expressed in various stem cells relatively to their differentiated P487 progeny, we reasoned that higher Pgp activity in CD133+ HSCs HHV6 infection is a hallmark of cord blood transplant would protect them from the anti-proliferative effect of the Pgp in adults and may participate to delayed engraftment: substrate colchicine (COL) and thus could be applicable to their a comparison with matched unrelated donors as stem selection and enrichment. cell source Methods: To this end, we isolated CD133+ HSCs from UCB by P. Chevallier, T. Guillaume, L. Planche, I. Hebia-Fellah, CD133-immunomagnetic separation (MACS). Pgp-expression C. Bressolette-Bodin, F. Rialland, M. Coste-Burel, S. Ayari, level was measured by fl ow cytometry using the Pgp-antibodies J. Delaunay, V. Horvais, M. Mohty, P. Moreau, J.-L. Harousseau, MRK-16, and its activity was measured by effl ux assay of the B. Imbert-Marcille Pgp-substrate Rh123. We further analyzed the relative distribu- CHU Hotel-Dieu (Nantes, FR) tion of various CD133+ subsets during 8 weeks of standard procedure of their expansion, in the absence or the presence Herpesvirus infections after cord blood transplant (CBT) in of COL. adults have been little studied thus far. To address this issue, Results: Analyses of freshly isolated CD133+ HSCs indicated we performed a comparison between 15 patients who received that 92% of CD133+ cells express functional Pgp on the cell a CBT (CBT group) with 40 patients who received an allogeneic surface. At 8 weeks of expansion, the CD133+ cell number transplant from a matched unrelated donor (MUD group) in our increased from 105 cells to 0.56±0.19 x109 and 1.60±0.41 x109 centre. in the absence and the presence of COL, respectively (2.9 ± The two groups were comparable except for the use of ATG and 0.5 fold increase, n=6). The long exposure of CD133+ HSCs to the median number of CD34+ cells infused and were stringently COL at the expansion process did not affect their ability to form monitored through CMV, EBV, and HHV6 DNA quantifi cations various hematopoietic colonies in semisolid culture after COL before and up to 9 months after transplant. An active infection removal (Table 1). Furthermore, FACS analyses indicated that was defi ned by a viral load >3 log/mL of blood or two consecu- ex-vivo expansion in the presence of COL preferentially enrich tive PCR between 2 and 3 log/mL. the CD133+/Pgp+ subset and specifi cally the CD34+/CD38- Considering the all cohort, 22, 29 and 29 patients experienced HSCs (Figure1). at least one positive PCR for CMV, EBV and HHV6. Incidence Conclusion: We established a novel expansion approach that of HHV6 infection was signifi cantly higher in the CBT group specifi cally enriches the CD133+/CD34+/CD38- fraction of (80% vs 42.5%, P<0.0001).Incidence of EBV infection was sig- UCB-derived HSCs. nifi cantly higher in the MUD group (62% vs 27%, P<0.0001). Incidences of CMV were similar between the 2 groups. In multivariate analysis, the use of a CBT and a myeloablative conditioning regimen were found to increase the risk of HHV6 infection (OR=5.4, 95% CI:1.2-23.0; P=0.02; OR=3.5, 95%CI: 1.03-12.05, P= 0.04) while the use of a MUD transplant was found to increase the risk of EBV infection (OR=0.31, 95%CI, 0.07-1.37; P=0.04). HHV6 infection started at a median of 36 days post-transplant in the CBT group vs 58 days in the MUD group (P=NS). In the CBT group, all patients with HHV6 infection (n=12/12, 100%) were still positive at the end of their follow-up (7 patients alive at 9 months post transplant and 5 patients dead before month +9) compared to only 7 out of 17 (41%) in the MUD group (P<0.009). Overall, four patients were still EBV positive at the end of the follow-up (1 patient in the CBT group, and 3 patients in the MUD group, P=NS). Interestingly, the occurrence of HHV6 reactivation translated towards delayed engraftment (median time to neutrophils and platelets recovery: 37 vs 16 days; P=0.03; 98 vs. 12 days; P=0.0001). Overall, these results show that after allogeneic transplant, the pattern of HHV6 and EBV infections is dependant on the source of stem cells. A specifi c relationship is suggested between HHV6 infection and the use of cord blood cells. In addition to a potential delayed engraftment, the clinical implications of the latter fi nding need to be refi ned and prospective screening and antiviral prophylaxis approaches are warranted in the context P489 of CBT. Clinical expansion of cord blood derived T-cells for use as donor lymphocyte infusion after cord blood transplantation P488 M. Okas, J. Gertow, O. Ringdén, J. Mattsson, M. Uhlin A novel P-glycoprotein (Pgp) -dependent ex-vivo Karolinska Institutet (Stockholm, SE) approach for expansion of human umbilical cord blood CD133+ cells signifi cantly enriches the Background: When no HLA-identical donor is available, cord CD34+/CD38- fraction blood transplantation (CBT) is an attractive option due to the H. Galski, I. Bar, A. Nagler rapid availability of the graft and its biological properties. One Chaim Sheba Medical Center (Tel Hashomer, IL) of the disadvantages associated with CBT is lack of possibility for donor lymphocyte infusion (DLI) after CBT. Here we report Introduction: While umbilical cord blood (UCB) is an attractive expansion and characterization of CD3 positive lymphocytes source for transplantation, major disadvantage is its relative low from CB grafts in connection to CBT. number of hematopoietic stem cells (HSCs). One approach thus Material and Methods: Lymphocytes from 13 CB grafts were involves ex-vivo expansion of UCB-derived HSCs. However, expanded with CD3/CD28 beads and 200 IU/ml rIL2. Expanded

S106 cells were cryopreserved in ready-to-use DLI doses. Immuno 7-AAD (up to 97% non-viable) in comparison to myeloma con- phenotyping and cytokine production assay were performed trols (0-32% non-viable). This problem appears to be correct- on expanded CB and ex vivo CB as well as peripheral blood able by immediate plasma depletion and resuspension. The mononuclear cells (PBMC) as controls. T cell receptor spectra- mechanism of reduced viability requires further investigation, typing was performed before and after expansion with aim to but unidentifi ed agents in SA plasma or increased fragility of determine usage of different TCR Vbeta. CD34+ cells in pts with SA may be contributory factors. Results: T cells have expanded with a median of 148-fold over a period of 8-11 days. Expanded preparations contained 99% of CD3+ cells without signs for signifi cant expansion of CD19+ P491 or CD3-/CD56+ cells. CD4/CD8 ratio of expanded T cells was Incidence of early infections after double cord blood not altered signifi cantly when compared to CB ex vivo (a mean transplantation preceded by a non-myeloablative of 1,7 to 2,02, respectively). Majority of T cells were positive conditioning regimen in adult patients for alpha-beta T cell receptor (93%). Up to 85% of expanded T J.A.E. Somers (1), E. Meijer (2), K. Sintnicolaas (1), cells were CD25+, 4% of CD3+ fraction were CD25+/FoxP3+. M. Oudshoorn (3), E. Braakman (2), A. Brand (1), Expanded T cells, when stimulated with CD3-beads, produced J.J. Cornelissen (2) IL-2, IFN-g, TNF-a in a cell to bead ratio in a dose-dependant (1)Sanquin Blood Bank (Leiden, NL); (2)Erasmus Medical Centre manner at higher levels, comparaed to peripheral T cells and (Rotterdam, NL); (3)Europdonor Foundation (Leiden, NL) CB ex vivo T cells. TCR spectratyping has shown a polyclonal pattern of TCR Vbeta-gene usage in the expanded T cell pool. Background: The rate of early (< day 100) infections after mye- Conclusions: We have successfully set up a clinically feasible loablative single umbilical cord blood transplantation (UCBT) is system of T cell expansion for use as DLI after CBT. Expan- high. It is caused by the limited number of hematopoietic stem sion procedure has not introduced major phenotypic changes cells infused, a protracted hematopoietic recovery, and by epi- to cells when compared to CB cells ex vivo. Expanded cells thelial barrier disruption caused by myeloablative conditioning. are functional in terms of cytokine production and display no Objective. To evaluate the incidence of early infections and oligoclonal pattern of TCR usage. Expanded CB T lymphocytes immune reconstitution in adult patients receiving a double may serve as a possibility for DLI after CBT. UCBT preceded by an non-myeloablative (NMA) conditioning regimen. Methods: Patients received a low-dose TBI-based NMA condi- P490 tioning regimen. GVHD prophylaxis consisted of cyclosporine Immediate plasma removal and cryopreservation in A and mycophenolate mofetil. All patients received antibacte- HAS/DMSO corrects reduced CD34+ cell viability in rial (ciprofl oxacin) and antiviral (valaciclovir) prophylaxis. After cryopreserved cell harvests from patients with systemic hematological recovery, prophylaxis for pneumocystis jiroveci amyloidosis and toxoplasmosis was added (cotrimoxazole). R. Krishna (1), V. Day (2), J.A. Snowden (1), D. Pawson (3), Results: 18 double UCBTs were performed in patients with C. Birchall (2), K. El-Ghariani (2), G. Cook (4), A. Lubenko (2) high-risk hematological diseases. Ten patients were CMV-IgG (1)Royal Hallamshire Hospital (Sheffi eld, UK); (2)NHSBT seropositive. The median number of total nucleated cells and (Sheffi eld, UK); (3)NHSBT (Leeds, UK); (4)St James institute total viable CD3+ cells infused was 5,4 x107/kg and 8,92 x105/ of Oncology (Leeds, UK) kg, respectively. To date, the median follow up of survivors is 17 months (4-29). The median time to neutrophil recovery was 28 Introduction: Since 2004, our stem cell processing laboratories days. None of the patients developed mucositis. Acute GVHD have routinely checked in vitro viability of CD34+ cells in stem developed in 6 (grade II: 5, grade III: 1) patients. 1). Febrile cell harvests, pre and post cryopreservation, using 7 Amino- neutropenia was observed in 4 patients. In 16 patients reach- Actinomycin D (7 AAD). Stem cell harvests from patients (pts) ing neutrophil recovery the following infections were observed with SA processed by routine methods were associated with during the fi rst 100 days post transplant: EBV reactivation poor CD34+ cell viability and recovery post cryo-preservation. (2), CMV-reactivation (3), central venous catheter associated This prompted us to investigate the processing method. bacteraemia (4). One patient, on treatment with steroids for Methods: Fourteen pts with SA (age 34-67) underwent stem acute GVHD, died 6 weeks post transplant due to a human cell harvesting with G-CSF alone or cyclophosphamide + G- herpes virus 6 infection. One patient died at day 82 due to a CSF. Viability testing was performed within 1 hour of collec- candida tropicalis candidemia. Total numbers of CD3+/CD19+ tion on fresh and thawed aliquots of the cryopreserved product and CD3-/CD16/56+ cells recovered to reference values within using standard ISHAGE gating protocol and single platform 2 months post transplant. To date, reference values of total methodology. Routine cryopreservation methods used standard CD3+ cells, CD3+/CD4+ and CD3+/CD8+ cells have not been procedures for processing with autologous plasma and Dimeth- reached. ylsulfoxide (DMSO) as cryoprotectant. A modifi ed processing Conclusion: These results suggest that the incidence of early method involved immediate centrifugation, plasma removal and severe bacterial infections in patients reaching neutrophil recov- resuspension of cells in 4.5% human albumin solution (HAS). ery is low after double UCBT preceded by a NMA conditioning Cells were then cryopreserved using standard procedures with regimen. While NK-cell and B-cell recovery was relatively rapid, 4.5% HAS and DMSO as cryoprotectant. CD3+ T-cell recovery appeared protracted. Results: With routine methods, mean pre-cryopreservation viability of harvests of SA pts (n=11) was 98.5% (range 92.9- 100%). However, the mean post-thaw viability was only 33% P492 (range 3.8%-100%). The mean percentage recovery of viable The use of HLA-specifi c monoclonal antibodies to CD34+ cells post cryopreservation (n= 12) was 25.8% (range determine graft kinetics in patients after double cord 3.8-58.5%). In comparison, a control group of myeloma pts blood transplantation (n=38), mean pre-cryopreservation viability was 99.1% (range J.A.E. Somers (1), Y. van Hensbergen (1), A. Mulder (2), 95.3% to 100%), and mean post- cryopreservation CD34+ J.J. Cornelissen (3), A. Brand (1) cell recovery was 88.1% (range 68%-100%). With the modi- (1)Sanquin Blood Bank (Leiden, NL); (2)Leiden University fi ed method used for harvests of SA pts, post-cryopreserva- Medical Centre (Leiden, NL); (3)Erasmus Medical Centre tion viability (n=4) was increased to a mean of 83.8% (range (Rotterdam, NL) 74-93%) and mean recovery of viable CD34+ cells (n=8) was 80.5% (range 54-100%). Background: Compared to single cord blood transplantation Discussion: In harvests from pts with SA use of viability testing (UCBT), double UCBT results in a higher proportion of engraft- post-cryopreservation demonstrated increased staining with ment in adult patients. Sustained hematopoiesis is usually

S107 derived from a single donor, however, the mechanism of pre- 2x106 CD34+ cells/kg or inability to achieve ≥ 10 CD34+ cells/ dominance of a particular unit has not been unravelled yet. ul without having undergone apheresis. The CUP mobilization Growth advantage of one of the units, immunological mecha- regimen consisted of G (10 mcg/kg) for 4 consecutive days. On nisms or both have been suggested to play a role. Early chimer- the evening of day 4, plerixafor (240 mcg/kg) was administered. ism studies in leukocyte subpopulations might contribute to a On day 5, G (10 mcg/kg) was administered followed by apher- better understanding of the mechanism of graft predominance. esis. This regimen of G, plerixafor and apheresis was continued Objective:To explore the feasibility of using discriminating HLA until the patient collected ≥ 2x106 CD34+ cells/kg. alloantigen-specifi c monoclonal antibodies (HLA-mAbs) to A total of 286 patients (164 NHL, 35 HD, 87 MM) were included study chimerism within leukocyte subpopulations in the early in this analysis. The mean age was 54.9 ± 13.4 years. Adminis- period after double UCBT. tration of G plus plerixafor resulted in a median CD34+ cells/kg Materials and methods: Fifteen patients received a double collection of 2.52x106, 2.31x106 and 3.52x106 in NHL, HD and UCBT preceded by a nonmyeloablative conditioning regimen. MM patients, respectively. The proportion of patients with NHL, Cord blood units (CBU’s) were typed and selected for HLA-A HD and MM that collected ≥ 2x106 CD34+ cells/kg were 67.1%, and B loci on serology split level and HLA-DRB1 on high reso- 65.7% and 81.0%, respectively. The proportion of patients lution level. The minimal required match grade was 4/6 both that proceeded to transplant were: 65.2%, 68.6% and 65.5% between recipient and CBU’s and between CBU’s. For each in NHL, HD and MM, respectively. A median of 3x106 CD34+ case we assessed whether the 3 different parties could theoreti- cells/kg were infused in all patients in this cohort. The median cally be discriminated with the available HLA-mAbs. In addition, time to neutrophil engraftment was 11 days, 12 days and 12 2 pilot experiments were performed in which chimerism was days in NHL, HD and MM, respectfully. Median time to platelet evaluated in peripheral blood by using fl uorochrome labelled engraftment was 21 days, 19 days and 21 days in NHL, HD and HLA-mAbs and lineage-specifi c mAbs. Analysis was performed MM, respectfully. at day 11,18,25 and 32 post transplant. These data demonstrate that G plus plerixafor mobilized Results: Based on the presence of HLA-mismatches between adequate CD34+ HSC in the majority of patients who failed recipient and CBU’s, we assessed that the discrimination of chemotherapy and growth factor mobilization. Plerixafor made the 3 different parties was feasible in 9 of 15 patient-donor it possible for these patients to receive potentially curative combinations (60%). In 6 patients (40%) it was not possible therapy. to distinguish all 3 parties due to a lack of discriminating HLA- mismatches. In both pilot experiments we were able to identify all 3 parties simultaneously in the total CD45+ fraction and in P494 the different sub-fractions (T-cells, myeloid cells and NK cells) Quantifi cation of mesenchymal stem cells in grafts adds at day 11 post transplant. As from day 18 post transplant a sin- to the benefi ts of haematopoietic stem cell and T-cell gle CBU was predominant in all subpopulations. Results at day analysis in prediction of the duration of aplasia but not 32 corresponded with data of chimerism analysis obtained by probability of acute graft-versus-host disease: preliminary polymerase chain reaction (PCR) in the total mononuclear cell results fraction. K. Dalva (1), T. Özçelik (2), M. Tol (1), E. Ayyildiz (1), H. Ersoy Conclusion: Our results suggest that the use of HLA-mAbs is (1), M. Ertem (1), A. Ikinciogullari (1), O. Ilhan (1), M. Beksac (1) a promising method to follow early engraftment kinetics in leu- (1)Ankara University (Ankara, TR); (2)Uludag University kocyte subpopulations in the majority of patients after double (Ankara, TR) UCBT. Its utility can be hampered by the lack of (serologically detectable) HLA mismatches in individual patients. Background and aim: For a successful haematopoietic stem cell(HSC) transplant. a minimum of CD34+ HSC is required. However there are other stem cells ie mesenchymal stem cells P493 (MSC) in the marrow (BM)and peripheral blood(PB) products. Effect of plerixafor plus G-CSF among patients who failed The MSCs are a source of HSC and known to be immunosup- to collect suffi cient haematopoietic stem cells after pressants . The aim of this prospective study is to quantitate the mobilisation attempt with chemotherapy plus cytokines MSC in the products used for SCT and to analyze their impact P.J. Shaughnessy (1), P.A. McSweeney (2), S. Solomon (3), on graft versus host disease(GVHD) and engraftment kinetics. J. McCarty (4), F. van Rhee (5), E.D. Jacobsen (6), G. Calandra Method: BM or PB samples collected for SCT were analysed (7), D. Huebner (7) for their CD34+ (ISHAGE protocol) and MSC quantity by fl ow (1)Texas Transplant Institute (San Antonio, US); (2)Rocky cytometry using the CD45, CD34,CD73, CD90, Stro-1, CD105, Mountain Cancer Centers (Denver, US); (3)Bone Marrow CD29,CD44 and HLA-DR antibodies. Transplant Group of Georgia (Atlanta, US); (4)Virginia Patient and Donors: 20 patients (aged 2 mo-50 y) diagnosed Commonwealth University (Richmond, US); (5)University of to have hematological malignacies or bone marrow failure Arkansas Medical Center (Little Rock, US); (6)Dana Farber syndromes received grafts (BM:13, PB:7) from healthy donors Cancer Institute (Boston, US); (7)Genzyme Corporation (gender missmatched 30% and aged 2-54 y). Two patients had (Cambridge, US) not received their grafts at the time of analysis. Engraftment (median)occurred on day 15(neutrophil) and 18 (platelet) in Autologous stem cell transplantation (ASCT) has been dem- PBSCTl patients 2 days shorter than following BMT. onstrated to be a potentially curative therapy for a variety of Results: BM MSC content (median: 0.15 (0.01-0.6 106/kg) was hematological malignancies. Successful ASCT is dependent on higher than PB (median: 0,057 106/kg) and 1 log less than BM harvesting suffi cient CD34+ hematopoietic stem cells (HSC) to HSC count . Pearson regression analysis showed a correla- ensure prompt and durable engraftment. The inability to mobi- tion between TNC and CD34 or MSC counts (r=0.71 or 0.51, lize adequate number of CD34+ HSC using chemotherapy in p=0.003 or 0.038, respectively). There was no correlation conjunction with cytokines ranges from 5.9%-18% in patients between MSC and CD34 counts. Table 1 summarizes the MSC with multiple myeloma (MM) and 4.2%-30% in patients with or TNC contents among early or late engraftmenters. Table 2 non-Hodgkin’s lymphoma (NHL) and Hodgkin’s disease (HD). compares the SC and CD3 cell counts in regard to aGVHD. The aim of this report is to evaluate the effect of plerixafor + Conclusions: This preliminary analysis is the fi rst report analys- G-CSF (G) among patients that previously failed to mobilize ing the role of graft MSC content in clinical transplantation . enough CD34+ HSC with chemotherapy and G. Although statistically not signifi cant, MSC counts were higher Patients enrolled in the plerixafor compassionate use program in BM vs PB grafts. BM MSC content is comparable to values (CUP) who had documented failure to harvest adequate CD34+ used in MSC therapy in human trials. TNC counts correlated HSC with prior chemomobilization were included in this analy- with MSC and HSC counts. HSC counts did not predict MSC sis. Mobilization failure was defi ned as the inability to collect content. Faster neutrophil recovery was associated with a

S108 tendency to result from grafts, regardless of stem cell source. Results Our analysis has shown a signifi cant relationship containing more MSCs. CD3 content was a stronger determi- between the QC performed on a thawed fragment and the val- nant of GVHD prediction compared to MSC or CD34. However ues both pre-freezing and at infusion of CBU, in terms of NC since PBSCT usually results with faster engraftment and more (p<0.01), CD34 (p<0.01) and CFU-GM (p<0.05), whereas no frequent GVHD compared to BMT, more data is required to be signifi cant correlation has been found for viability. The median able to analyze multiple factors simultaneously. We conclude time between CBU freezing and thawing was 5.2 years (0.26- that signifi cant amounts of MSCs are transfused with clinical 9.04) and did not affect the NC, CD34+ and CFU-GM recovery. BMT. MSC content analysis may be essential in future studies The viability of CBU at infusion signifi cantly correlates with the where engrafment kinetics and GVHD are addressed. speed of engraftment for PLTS (p=0.005). Conclusions: From this study we can draw the following prelimi- nary conclusions: 1) QC in terms of cell dose recovery and clonogenic assay are predictive for the values at infusion and refl ect the safety of cryopreservation procedure; 2) the viability of cells at infusion is related to the speed of PLTS engraftment; 3) CD 34+ and CFU-GM results of QC, performed on a thawed fragment, may replace pre-freezing missing data.

P496 Similar engraftment kinetics of cord blood transplantations of double units in adults, versus single units in children G. Goldstein (1), R. Elhasid (2), B. Bielorai (1), A. Toren (1), R. Yerushalmi (1), A. Shimoni (1), A. Nagler (1) (1)Sheba Medical Center (Ramat Gan, IL); (2)Rambam Health Care Campus (Haifa, IL)

Neutrophils engraftment kinetics is a key issue in Cord blood transplantations (CBT). By using double unit CB (DCB) suf- fi cient number of nucleated cells could be given for adults. We compared the engraftment kinetics in DCB versus sin- P495 gle CB transplantations. Thirty eight patients (pts)underwent Quality controls of cord blood units according to JACIE CBTs. Median age was 6 years (6 weeks -65 years). Thirty standards are highly predictive in unrelated cord blood patients were children and eight were adults. Twenty three transplant were males and 15 were females. Nineteen had hematologi- A. Picardi (1), A. Spagnoli (2), M. Caniglia (2), R. Pinto (2), cal malignancies –Acute Lymphoblastic Leukemia -7, Acute T. Dentamaro (2), I. Mangione (1), L. Cudillo (1), L. Cupelli (2), Meyeloid Leukemia - 6, Juvenile Meyelomonocytic Leukemia F. Zinno (2), A. Bruno (2), G. Del Proposto (1), G. Kloger (3), - 2, Meyelodysplastic syndromes - 2, Biphenotypic Leukemia - L. Lecchi (4), W. Arcese (1) 1, and Non-Hodgkins Lymphoma - 1. Nineteen had non malig- (1)Tor Vergata University and Rome Transplant Network (Rome, nant diseases –bone marrow failure syndromes - 5, immune IT); (2)Rome Transplant Network (Rome, IT); (3)Düsseldorf defi ciencies syndromes - 5, metabolic diseases - 4, hemo- Cord Blood Bank (Düsseldorf, DE); (4)Ospedale Maggiore globinopathies - 3, and histiocytic disorders - 2. Conditioning (Milan, IT) regimens were based on I.V. Busulfan (22), Fludarabine (9) and total body irradiation (6), or no conditioning (1). Nine pts Cord Blood Units (CBU) are increasingly used for allogeneic received DCB. CB units were from matched related donors in stem cell transplant and their selection is meanly done accord- 12, and unrelated donors in 26 pts. Of the unrelated single CB ing to value of pre-freezing cell dose and HLA compatibility. units - in 6, 9 and 2 pts there were 6/6, 5/6 and 4/6 antigens According to JACIE standard, the quality controls (QC) for matching, respectively. Of the nine DCB transplantations the evaluating the effi ciency of CBU before the recruitment include recipient-donor matching was- 6/6,5/6 - 1, 5/6,4/6 - 1, 4/6,4/6 - viability and CFU tests. 4, and 4/6,3/6 – 2, and 3/6,3/6 - 1. Median nucleated cells dose Objectives: Aim of our study was to evaluate the sensibility was 4.35X107 (0.65-50) cells/kg. RESULTS - Median follow up of QC, performed on pilot vial/fragment before CBU formal was 28 months (CI 95% 3-141). Cumulative incidence of neu- recruitment, as cell dose, clonogenic test and viability in order trophils engraftment was 75% (CI 95% 62-91). Ten pts did not to defi ne the predictive parameters for the graft. engraft (7 of them died). Median day of platelets engraftment Methods: We retrospectively analyzed 48 unrelated Cord Blood was 34 (14-126). Cumulative incidence of acute and chronic Transplant (CBT) performed with 52 CBU (4 double trans- graft versus host disease was 16% (CI 95% 8-34) and 19% (CI plants) at Rome Transplant Network (RTN) from September 95% 10-38) respectively. TRM cumulative incidence was 32% 2005 to September 2008. Patients underwent unrelated CBT (CI 95% 20-53). The probability of survival was 62% (CI 95% for malignant (n=45) or non malignant diseases (n=3) either in 46-79). Cumulative incidence of disease free survival was 49% 1st/2nd complete remission (n=18/11) or in advanced disease (CI 95% 32-66). Analysis of engraftment kinetics of the twenty (n=19). Median patient age and body weight were 20.4 years nine pediatric pts who received single CB units, versus the (0.6–60) and 50.5 kg (4 -96), respectively. The CBU were HLA 9 adults that underwent DCB transplantations revealed that matched in 4 cases and mismatched for 1 (n=12) or 2 HLA it was similar (fi gure). Median day of engraftment was 22 and loci (n=36) in 48 cases. According to RTN policy, CBU selec- 23.5 in the single CB and DCB, respectively. Similar engraft- tion was based on the following pre-freezing parameters: NC ment kinetics of DCB in adults and single CB transplantations dose > 2.5 x 107/kg, HLA compatibility >4/6 loci, CD34+cells in children should encourage clinicians to consider CB as >1x105/kg, CFU-GM >1x104/kg and AB0 matching. In order to an alternative source for a graft in adults with hematological confi rm the reproducibility of pre-freezing data, the cell recov- malignancies that are in need of transplantations and do not ery in terms of NC, CD34+, clonogenic assay and viability was have an HLA compatible donor. required after thawing of an aliquot before the CBU formal recruitment for transplant.

S109 P498 Evaluation of umbilical cord blood unit processing effi ciency in routine laboratory conditions V. Kindler (1), C. Troeger (2), G. Nicoloso de Faveri (3), A. Gratwohl (2), A. Tichelli (2), J. Passweg (1), S. Meyer-Monard (2) (1)Geneva University Hospital (Geneva, CH); (2)Basel University Hospital (Basel, CH); (3)Swiss Blood Stem Cell Foundation (Berne, CH)

Introduction: Umbilical cord blood (UCB) is an alternative stem cell source increasingly used for patients lacking an HLA matched sibling or unrelated donor. For UCB transplantation the total nucleated cells (TNC) and CD34+ cells greatly infl uence transplant outcome. UCB banks perform a white cell enrich- ment and red cell depletion prior to cryopreservation in order to decrease the stored product volumes. Various techniques have been evaluated in a restricted number of products; however the effi cacy of volume reduction under routine laboratory conditions is lacking. We report on the results of 1280 UCB processed in two banks using the same device and compare them with the P497 manufacturer’s qualifi cation reports. Cord blood stem cells enumerated using 7-AAD modifi ed Materials and methods: 1280 units were processed between ISHAGE guidelines may contain signifi cant numbers of 1994 and 2008, 456 in centre 1 and 856 in centre 2 using a non-viable (apoptotic) cells Sepax ® S-100 device (Biosafe, Eysins, Switzerland) piloted R.C Duggleby (1), S. Querol (1), R. Horton (2), S. Gomez (2), by proprietary software (version 1.17 to 264), according to the J.A Madrigal (1) manufacturer’s instructions. UCB were collected in 5 different (1)Anthony Nolan Research Institute (London, UK); (2)Anthony maternity wards and processed within 36 hours after collection. Nolan Cord Blood Bank (Nottingham, UK) TNC and CD34+ counts were performed before and after vol- ume reduction using standardized counting methods. One of the primary determinants of engraftment, independ- Results: Median volume obtained immediately after processing ent of genetic matching, has been demonstrated to be the was 21% of the initial volume (10th/90th percentile 15/29%). dose of viable stems cells that is transplanted. There is now Median TNC and CD34+ cell recoveries were 76% (10th/90th evidence that cellular viability predicts the speed of neutrophil percentile 63/86%) and 81% (10th/90th percentile 64/95%) and platelet engraftment and overall survival. Currently the respectively in both sites, whereas the device qualifi cation best measure of stem cell viability is the colony forming unit report proposes 88% and 96% for TNC and CD34+ cell recov- assay (CFU). Unfortunately CFU assays require 10-14 days ery respectively (p<0.0001 for each value according to one of incubation making them impractical as a releasing test for sample T-test). The recovery rates were stable over time in the assessment of cryopreserved cord units in a clinical set- both laboratories. Red cell depletion data were only available ting. In addition, there is only a weak association between CFU from centre 1 and corresponded to a median of 25% (10th/90th and CD34 numbers resulting in low clonogenic effi ciencies. percentile 10/44%) retained RBC. This suggests that signifi cant numbers of CD34+ cells are not Conclusions: Volume reduction of UCB with the Sepax® forming colonies. ISHAGE guidelines use CD45, CD34 and 7- device provides a satisfactory TNC and CD34+ cell recovery AAD to determine stem cell content. We hypothesize that there in a routine setting. The TNC and CD34+ cell counts after vol- are potentially large numbers of apoptotic cells present in the ume reduction are however inferior than these expected by the CD34+CD45low7-AAD- population; explaining the low clonal manufacturer. The stability of the cell recovery over time in both effi ciency in some samples. UBC banks excludes the contribution of a learning effect. Pre- Methods: Whole cord blood units and mononuclear cell isola- processing factors including the delay between collection and tions, (cryopreserved and stored at 4°C) were assessed for processing could be involved and have to be evaluated in the absolute CD34+ cell numbers using the ISAGE guidelines and future. These data demonstrate that stem cell processing within qualitatively with an additional annexin V labeling step; the via- a routine setting may provide relevant differences compared to ble stem cell number was assessed by CFU assay. data obtained within study conditions. Results: Many samples showed signifi cant evidence of apoptic cells present in the CD34+CD45low7AAD- gated population. Consequently whilst the CFU increased with increased CD34+ dose there is a good association with live (Annexin V-) CD34+ numbers (Pearson correlation R²=0.8). Conclusion: Live cell assessment by this method will enable the CFU to be predicted allowing a rapid enumeration, post-thaw, of the viable stem cells present in a cord unit. This would be an attractive releasing test for cord blood selection. We are cur- rently assessing the impact of time and temperature to optimize the storage and handling of cord blood units.

S110 P500 Microbial contamination: 10-year survey at a cord blood bank, Pavia P. Bergamaschi (1), R. Daturi (1), A. Marchesi (1), B. Romano (1), V. Genovese (1), T. Quaglini (2), M. Rossi (1), C. Del Fante (1), G. Viarengo (1), E. Giacobone (1), C. Perotti (1), L. Salvaneschi (1) (1)IRCCS Policlinico San Matteo (Pavia, IT); (2)Hospital of Voghera (Voghera, IT)

Cord blood (CB) use for unrelated stem cell transplantation is constantly encouraged by CB Banks spreading worldwide. Microbial contamination of the graft may cause severe clinical complications in immunosuppressed recipients; moreover cen- tral venous catheters may further enhance the risk of systemic infections. Despite accurate disinfection protocols, CB might be contaminated with bacteria at several steps from collection to cryopreservation. This is the reason why sterility still represents a major concern. All the more so because a rigorous policy of quality is essential for cell therapy protocols such as CB ex vivo expansion. Here we retrospectively reviewed the data of microbial contamination at our CB Bank in Pavia over 10 years activity. From 1997, 3025 units have been cryopreserved. Ste- rility testing was always performed at the end of manipulation before cryopreservation by inoculating a 2 ml sample into 30 ml media (BacT/ALERT FA/FN, Biomérieux Inc., Durham, North P499 Carolina, USA) to detect both aerobic and anaerobic bacteria. Reduced dose of lenograstim is as effi cacious as After 14 days incubation at 37ºC, positive cultures were subcul- standard dose for peripheral blood stem cell tured to identify the bacterial species. The corresponding anti- mobilisation and transplantation: a randomized study biotic sensitivity was also assessed. All contaminated CB units in patients undergoing autologous peripheral stem cell were removed from the inventory and discarded. 75/3025 units transplantation resulted positive, the overall contamination rate being 2.3%. M. Ozturk, F. Arpaci, S. Ataergin, A. Ozet, T. Cetin, O. Kuzhan, A single contaminant was demonstrated in 97.3% of positive B. Ozturk, S. Komurcu, S. Kilic, T. Guler, B. Budakoglu units (73/75). The list of pathogens species isolated by sub- Gulhane Medical Academy (Ankara, TR) cultures is provided in the table. Post-thawing sterility testing resulted positive for 13/21 units (62%), confi rming the same Background: 10 microg/kg/day of fi lgrastim and lenograstim species as previously detected on the fresh sample. The trend have been recommended for mobilization of CD34+ cells with- of bacterial contamination was analysed over the years (see out associated chemotherapy. However, in our previous rand- the graphic), showing a percentage of positive detections stably omized study we demonstrated that a 7.5 microg/kg/day dose under 3%, except of two spikes in 1997 and 2001. In conclu- of lenograstim has been as effi cacious as 10 microg/kg/day sion, our review over 10 years of banking activity demonstrated of fi lgrastim. In this study, we investigated whether a reduced the importance of constantly monitoring sterility as indicator dose of lenograstim is equavalent to standard dose for autolo- of CB quality and one personnel performance maintenance. gous peripheral blood stem cell (PBSC) mobilization and trans- Despite very low incidence of contamination on average, it plantation. appeared to be primarily due to skin fl ora and contaminants Material and method: A total of 49 consecutive patients were at site of withdrawal. Finally, multiple resistance to antibiotics randomized to either low dose (7.5 microg/kg/day, n = 24) or together with the ability to survive after cryopreservation and standard dose (10 microg/kg/day, n = 25) of lenograstim. These storage at -190°C, as shown for several pathogens, suggested two groups were similar in regard to disease, sex, body weight, the decision of promptly discarding any contaminated unit to be body surface area, conditioning regimens, previous chemo- still essential for quality assurance of the stem cell product. therapy cycles and radiotherapy. Each dose of lenograstim was administered for 4 consecutive days. The fi rst PBSC apheresis was done on the 5th day. In the posttransplant period, lenogras- tim was given at 5 microg/kg/day until leukocyte engraftment. Results: Successful mobilization with the fi rst apheresis, was achieved in 10/24 (42%) patients in low dose group versus 14/25 (56%) patients in standard dose group. No signifi cant difference was seen in the median number of CD34+cells mobilized, as well as the median number of apheresis, median volume of apheresis, percentage of CD34+ cells, and CD34+ cell number. Leukocyte and platelet engraftments, the number of days requiring G-CSF and parenteral antibiotics, the number of transfusions were similar in both groups in the posttrans- plant period. Lenograstim 7.5 microg/kg/day is as effi cious as Lenograstim 10 microg/kg/day for autologous PBSC mobiliza- tion and transplantation.

S111 P501 P502 Microbial contamination of peripheral blood stem cell Effi cacy, complication rates, and cost-effectiveness of products for autologous transplantation chemotherapy plus granulocyte-colony-stimulating factor P. Bergamaschi, C. Parisi, R. Daturi, E. Giacobone, A. Marchesi, conditioned mobilisation of peripheral blood B. Romano, V. Genovese, C. Del Fante, G. Viarengo, C. Perotti, haematopoietic stem cells in over 150 patients with L. Salvaneschi haematological malignancies IRCCS Policlinico San Matteo (Pavia, IT) I. Gabriel, J. Sharon-Richman, E. Olavarria, A. Rahemtulla, E. Kanfer, D. Marin, D. Milojkovic, K. Rezvani, D. MacDonald, Microbial contamination of peripheral blood stem cell (PBSC) R. Szydlo, J. Apperley products is a potential source of systemic infections following Imperial College NHS Trust (London, UK) transplantation causing severe clinical complications in immu- nosuppressed recipients. Several factors involving the patient, ASCT remains standard consolidation therapy for patients with the collection facility and the cryopreservation laboratory may multiple myeloma (MM) and chemosensitive relapsed lym- affect the risk of contaminating a PBSC product. Here we phoma (r-Ly). Peripheral blood as a source of stem cells (PBSC) report the data recorded at our Apheresis Unit in Pavia, Italy. has largely replaced marrow with the advantage of improved During 2001-2007 we have cryopreserved 375 autologous leu- engraftment rates. PBSC are routinely collected following kapheresis. All PBSC collections were obtained at our center, administration of chemotherapy in combination with GCSF. the patients coming from 5 distinct Transplant Centers. Sterility However, the resultant pancytopenia poses signifi cant risk to testing was performed at the end of manipulation before cryop- patients and additional chemotherapy prior to ASCT may lead reservation by inoculating 2 ml into 30 ml media (BacT/ALERT to increased end organ damage potentially precluding future FA/FN, Biomérieux Inc.) to detect both aerobic and anaerobic therapies. Novel agents can achieve PBSC mobilisation without bacteria. After 14 days incubation at 37°C, positive cultures were the use of cytotoxics. In the advent of such drugs, we reviewed subcultured to identify the pathogen specie. The correspond- the effi cacy of, and complications during PBSC mobilisation. ing antibiotic sensitivity was also assessed. For each contami- We also analysed the cost implications of adverse events. Of nated leukapheresis, one cryovial was thawed to repeat sterility 151 consecutive attempts, 13.2% failed to reach our criteria testing. 24/375 PBSC products resulted positive, the overall in order to attempt pheresis (1x104 CD34 cells/ml. Of those contamination rate being 6.4%. A single micro-organism was achieving target and undergoing pheresis, 6% did not achieve demonstrated in all cases, Staphylococcus epidermidis being an adequate cell dose for future ASCT (2x106CD34+cells/kg) isolated in 83.3% of positive units. In case of multiple PBSC col- an overall failure rate of 19.2%. Furthermore 17.9% failed to lections from the same patient, the same pathogen was found harvest our ideal of 4x106/kg (permitting >1 ASCT procedure). in all products. Post-thawing testing resulted positive for 13/24 Factors contributing to failure in achieving target CD34+ve units (54.2%), confi rming the same species as detected on the PB count on univariate analysis were; >2 lines of previous fresh sample in all cases except one. 12/24 positive leukapher- chemotherapy and occurrence of neutropenic sepsis(p=0.002, esis was infused to the patient with concomitant administration and p=0.005. These factors remained signifi cant on multi- of specifi c antibiotics as by the antibiotic sensitivity report. No variate analysis (RR:4.4 and 6.2). These factors also affected sepsis was documented. The trend of bacterial contamination CD34+ cell yield on both univariate and multivariate analysis over the years is depicted in the graphic. One of 5 Institutions (RR: 3.3 and 4.6). No differences were seen between MM and (TC no.1) accounted for about 63% of all positive results, as r-Ly. Overall, the complication rate was 34.4%,24.1% of patients detailed in the table. A catheter in the jugular vein was present suffering NS requiring admission. The mortality rate was 1.3% for all patients of TC no.1. Moreover 25% of collections for TC (NS and intra-cranial bleed). Of those developing NS, only 52% no.1 were found positive. In our experience, contamination harvested suffi cient cells, but with a median delay of 3 days. appeared to be primarily due to skin fl ora contaminants at site The median cost of PBSC collection was E12720.6 (E1448.30– of withdrawal, suggesting to implement disinfection protocols. E28802). NS signifi cantly increased the cost of mobilisation; In certain conditions, catheters enhance the risk of contami- median cost of E18686.2 vs E12069.7 (p≤0.0001). nation. Anyway, despite multi-drug resistance and the ability Conclusion: Our results suggest that potential candidates for to survive after cryopreservation and storage at -190°C, the ASCT should be harvested as near to achieving remission to administration of contaminated PBSC appeared feasible and prevent failure following additional therapy upon relapse. 1/5 uncomplicated if appropriate antibiotics are administered. patients will fail. The risks associated with current mobilisa- tion protocols are substantial, and also impact greatly on cost. Therefore these data suggest that transplant centres should consider the use of non-myelosuppressive agents either in place of, or as a dose reduction strategy for autologous stem cell procurement.

P503 Effi ciency of high-dose etoposide phosphate and G-CSF for primary and secondary stem cell mobilisation – A single-centre retrospective analysis of 66 patients K. Jost, M. Leithäuser, C. Grosse-Thie, I. Hilgendorf, C. Kahl, G. Kundt, M. Freund, C. Junghanss University of Rostock (Rostock, DE)

Introduction: In autologous hematopoetic stem cell transplanta- tion (AHSCT) a minimum stem cell dose of 2 x 106 CD34 + cells per kg body weight is needed to ensure engraftment. With com- monly used Cyclophosphamide-based mobilizing regimens a proportion of 10-20% of patients fail to mobilize suffi cient stem cell numbers. Here we report on effi ciency of Etoposide phos- phate/ G-CSF as a mobilization regimen with an acceptable toxicity profi le and HSC yields. Patients and methods: Data from 66 consecutive patients (mean age 51 years (18-73)) receiving high dose etoposide

S112 phosphate (2000 mg/m²; 22h c.i.; day 1) and G-CSF (10µg/kg in two procedures, but was unable to receive a third Plerixafor s.c.; starting day 3) for HSC mobilization between 1997 and dose due to diarrhoea (a recognised side-effect) – a second 2006 at our institution were analysed retrospectively. The study treatment cycle one month later with antidiarrhoeal cover was population consisted of patients who had already failed to mobi- successful. One patient was unable to undergo PBSC collec- lize suffi cient stem cell numbers on previous protocols or were tion because of intercurrent infection (not thought to be plerixa- not considered eligible for more intense mobilization protocols. for-related). No other immediate toxicities were seen. CD34 + cells in the peripheral blood were monitored daily after 7 patients have progressed to transplant. Median cell dose leucocyte recovery over 1 Gpt/l and stem cell harvest was transplanted was 3.39, range 2.68 – 4.72. All seven patients started as soon as the the CD34 cell count reached 10 /µl. A have achieved sustained neutrophil and platelet engraftment threshhold of 2 x 106 CD34 + cells/ kg body weight was defi ned (median 14 days to neutrophils >0.5; median 21 days to neu- as successful mobilization. trophils >1.0; median 24 days to platelets >50). Plerixafor Results: In total 47 patients (72.2%) mobilized suffi cient stem appears highly effective in the mobilisation of PBSC for trans- cell numbers with a mean number of 1.76 aphereses and a plant from patients failing to mobilise by conventional means, mean stem cell yield of 14.1 CD34+ cells/kg body weight. In with generally acceptable toxicity. patients with previous mobilization failures who constituted 47% of the cohort (31/66) a suffi cient stem cell yield was obtained in 52%. Previous mobilization failure was the strongest negative P505 predictive factor for insuffi cient stem cell harvest. Hematologic Immune reconstitution is signifi cantly impaired toxicity was moderate: mean time of leukopenia (defi ned as < following double umbilical cord blood transplantation in 1Gpt/l) was 3.9 days (0-22) and neutropenia (< 0.5 Gpt/l) of 4.8 adult patients days (0-16). The rate of neutropenic fever was as high as 47% A. Manaka, C. Balotis, M. Vagia, S. Vasiliou, Z. Poulopoulou, but clinically well manageable. M. Papageorgiou, E. Bika, M. Karatza, M. Garofalaki, I. Kakkas, Discussion: In this study group of mainly elderly, often comorbid C. Papasteriades, J. Apostolidis, N. Harhalakis, I. Baltadakis, patients with a signifi cant proportion of prior mobilization failures D. Karakasis, E. Nikiforakis Etoposide phosphate is a generally well tolerated mobilization Evangelismos Hospital (Athens, GR) agent with promising stem cell yields and should be considered as a salvage mobilization regimen after mobilization failure or Umbilical cord blood transplantation (UCBT) is associated with as a fi rst line strategy in elderly and comorbid patients. delayed immune reconstitution compared to bone marrow or peripheral blood transplantation, especially in adult recipients. UCBT with 2 partially matched units is increasingly utilized as P504 a means to increase probability of engraftment in this patient Plerixafor is highly effective in the mobilisation of PBSC population. Nevertheless, data on immune reconstitution with for autologous transplantation from patients failing to double UCBT are still scarce. We monitored reconstitution mobilise by conventional means: the initial Scottish of cellular and humoral immunity in 16 consecutive patients, experience in three transplant centres who underwent double UCBT at our center and achieved sus- W.C. Gordon (1), P.R.E. Johnson (2), P.H. Roddie (2), tained donor engraftment. Patients received UCBT for high-risk P.C.A. Shepherd (2), F.M. Scott (2), J.M. Davies (2), acute leukemia at a median age of 34 years (range, 16-60). L.M. Manson (3), D. Culligan (4), Y.-L. Chee (4), The majority of units (26/32) were 4/6 matched to recipient at A.N. Parker (1), I.G. McQuaker (1), A. Clark (1), R.L. Soutar (1), HLA-A, -B, and -DRB1. The conditioning regimen was abla- K.W. Douglas (1) tive in 11, and non-myeloablative in 5 cases. Only 1 patient (1)Beatson West of Scotland Cancer Centre (Glasgow, UK); received ATG. Cyclosporine plus mycophenolate was used as (2)Western General Hospital (Edinburgh, UK); (3)Royal acute graft-versus-host disease (GVHD) prophylaxis. Intrave- Infi rmary of Edinburgh (Edinburgh, UK); (4)Aberdeen Royal nous immunoglobulin was not routinely administered, unless Infi rmary (Aberdeen, UK) clinically required. Median nucleated and CD34+ cell doses at infusion were 4.2 x 107/kg (range, 2.6-5.4) and 1.35 x 105/kg The small molecule chemokine inhibitor Plerixafor has recently (range, 0.6-1.98). Neutrophil engraftment was achieved at a been used on a named-patient basis for PBSC mobilisation median of 19 days. Fifteen patients developed acute GVHD from patients failing to mobilise by conventional means. We (grade II: 12, grade III-IV: 3). Extensive chronic GVHD occurred present our initial experience with plerixafor in 18 mobilisation in 2 patients. Immunoglobulin concentrations and absolute attempts in 17 patients, all of whom had previously failed to counts of B, NK, and T lymphocytes were assessed at 1, 3, 6, mobilise a transplantable PBSC dose, at three Scottish trans- 9, 12 and 18 months post transplant. Median values of IgG at plant centres. the above time points were 961, 353, 382, 384, 973, and 1300 Patients had myeloma (n=10) or lymphoma (n=7). All had at mg/dl, respectively. The absolute counts of NK lymphocytes least one previous attempt at PBSC mobilisation which failed or reached normal values from the fi rst month (median count, resulted in an insuffi cient cell dose for transplant (2 attempts in 135/uL). By contrast, CD8+ and CD4+ T cells remained lower 2 patients). 5 of 17 patients had a pre-existing CD34+ dose from than normal for the entire follow-up period (median counts previous collections (range 0.98-2.32 x 106/kg; median 1.38); at 18 months, 233/uL and 488/uL, respectively). CD19+ cell 12 patients had no cells collected before receiving plerixafor. counts were also subnormal by 9 months, but recovered rap- As recommended by Genzyme, patients received four days idly thereafter (median counts at 9 and 12 months, 82/uL and of G-CSF 10 micrograms/kg followed by Plerixafor 240 micro- 784/uL, respectively). The relative percentage of naive helper grams/kg subcutaneously in the evening, 11 hours prior to fi rst T cells (CD4+/CD45RA+) increased marginally over 30% at 18 apheresis, except for one patient with dialysis-dependent renal months. CMV infection was detected in 7 of 11 seropositive failure who received 160 micrograms/kg. G-CSF and plerixa- patients. In addition, 4 patients developed HHV-6 reactivation, for were repeated daily until a transplantable CD34+ dose was and 2 EBV-related lymphoproliferation. In conclusion, immune obtained (if possible). Apheresis was started without waiting for reconstitution is considerably impaired after double UCBT in a day-of-collection peripheral CD34+ count. For patients with a adult patients. This could be attributed not only to graft source peripheral CD34+count of less than 20/microlitre, 3 (rather than but also to the increased incidence of acute GVHD and treat- 2) blood volumes were processed. ment with steroids. 16 of 17 patients achieved a transplantable PBSC dose (>2.5 x 106/kg CD34+ cells) after one (n=15) or two (n=1) courses of Plerixafor – a total success rate of 94%. The total number of apheresis procedures required was 1 (n=4), 2 (n=6), 3 (n=4) or 4 (n=2). One patient mobilised a cell dose of 1.89 x 106/kg

S113 P506 P507 Transplanted CD34+ cell dose is associated with Rescue from failed growth factor and/or chemotherapy long-term platelet count recovery following autologous haematopoietic stem cell mobilisation with G-CSF and haematopoietic stem cell transplant in patients with plerixafor: a single-centre experience non-Hodgkin’s lymphoma and multiple myeloma C.J. Fowler (1), A. Dunn (1), B. Hayes-Lattin (1), K. Hansen (1), P.J. Stiff (1), I.N. Micallef (2), A.P. Nademanee (3), L. Hansen (1), K. Lanier (1), V. Nelson (1), T. Kovacsovics (1), E.A. Stadtmauer (4), R.T. Maziarz (5), B.J. Bolwell (6), J. Leis (1), G. Calandra (2), R.T. Maziarz (1) G. Calandra (7), G. Bridger (7), J.F. DiPersio (8) (1)Oregon Health & Science University (Portland, US); (1)Loyola University Medical Center (Maywood, US); (2)Mayo (2)Genzyme Corporation (Cambridge, US) Clinic (Rochester, US); (3)City of Hope (Duarte, US); (4)Hospital of the University of Pennsylvania (Philadelphia, US); (5)Oregon Autologous hematopoietic stem cell transplantation (ASCT) Health & Science University (Portland, US); (6)Cleveland can be a curative procedure for a variety of hematological Clinic Foundation (Cleveland, US); (7)Genzyme Corporation malignancies. A number of patients fail to mobilize adequate (Cambridge, US); (8)Washington University (Saint Louis, US) number of peripheral blood stem cells (PBSC), thereby preclud- ing ASCT. The purpose of this report is to assess the effi cacy The impact of transplanted CD34+ cell dose on clinical out- of plerixafor plus G-CSF (G) in patients that previously failed to comes remains controversial. The purpose of this post-hoc mobilize suffi cient PBSC. analysis was to examine the relationship between CD34+ cell Patients meeting the criteria for the compassionate use proto- dose transplanted and platelet, neutrophil, and lymphocyte col (CUP) using plerixafor plus G were included in this analysis. counts at 100 days, 6, and 12 months following autologous Participants were eligible for the protocol if they had failed to col- transplantation in patients with non-Hodgkin’s lymphoma (NHL) lect enough CD34+ cells with a traditional mobilization regimen. and multiple myeloma (MM). Remobilization consisted of G at 10mcg/kg/day administered Data were obtained from two phase 3 clinical studies that com- on days 1-4 plus plerixafor 240 mcg/kg/day on the evening of pared the safety and effi cacy of plerixafor and G-CSF (G) to day 4. G-CSF 10mcg/kg was administered on day 5, one hour placebo and G in mobilization of hematopoietic blood stem cells before apheresis. If multiple days of collection were necessary, for autologous transplantation in NHL and MM patients. The the aforementioned combination regimen was repeated. details of the study designs and preliminary safety and effi cacy Twenty patients, 15 men and fi ve women, with a median age outcomes have been reported (DiPersio, ASH 2007). The fol- of 58 years were enrolled. Patients had the following malig- lowing data were obtained from the two clinical studies: number nancies: multiple myeloma (n=6), non-Hodgkin’s lymphoma of CD34+ cells transplanted (fi rst transplant only), absolute (n=10), Hodgkin’s disease (n=2), angioimmunoblastic lymphad- neutrophil, platelet, red blood cell, and lymphocyte counts at enopathy (n=1) and amyloidosis (n=1). Majority of the patients 100 days, 6, and 12 months post-transplant. The proportions of received cytokines only (n=15) as the initial mobilization regi- patients who had platelet ≥150 x 109/L or neutrophil ≥2.5 x 109/L men. Initial mobilization attempt in this cohort yielded a median or lymphocyte ≥0.5 x 109/L at 100 days, 6, and 12 months were of 0.21 x 106 CD34+ cells/kg (range 0-1.8 x 106 CD34+ cells/kg) compared among these transplanted CD34+ cell dose cat- in 2 (range 0-4) apheresis days. Eighteen of 20 patients who egories regardless of mobilization treatment: 2-4x106 cells/kg, were mobilized with plerixafor plus G were able to collect suf- >4-6x106 cells/kg, and >6x106 cells/kg. Two separate analyses fi cient PBSC to proceed to ASCT. Among patients who were were performed, one for NHL and one for MM patients. initially mobilized with cytokine only, plerixafor plus G resulted For the NHL study, 135/150 (90.0%) patients in the plerixa- in a median 3.7 x 106 CD34+ cells/kg (range 2-7.9 x 106 CD34+ for group underwent transplantation after initial mobilization cells/kg) in 2 apheresis days; among patients who were initially compared with 82/148 (55.4%) patients in the placebo group, chemomobilized, plerixafor plus G yielded 4 x 106 CD34+ cells/ p<0.001. For the MM study, 142/148 (95.9%) patients in the kg (range 2.5-6.2 x 106 CD34+ cells/kg) in 2 apheresis days. plerixafor group and 136/154 (88.3%) patients in the placebo Median time to neutrophil engraftment was day 14 (range 10-21 group underwent transplantation. A signifi cant linear trend with days) and platelet engraftment was day 25 (range 13-38 days). increasing proportion of patients with platelet count ≥150 x109/L These data demonstrate that plerixafor plus G is effective with increasing transplanted cell dose categories was observed among patients who previously failed traditional mobilization at 100 days, 6, and 12 months for NHL patients and at 100 days regimens. In those patients eligible for ASCT who have failed for MM patients (Table 1). There were no apparent signifi cant prior mobilization attempts, plerixafor can provide an opportu- relationships between transplanted cell dose categories and nity to still pursue a potentially curative procedure. other hematopoietic parameters. These ad hoc analyses confi rm previous reports that trans- planted cell dose is associated with better long-term platelet P508 recovery. Due to the small sample size in each transplanted cell High-dose G-CSF is safe and effective for mobilisation dose category and the nature of the analyses, further studies of haemopoietic cells for autologous stem cell are warranted to determine if cell dose has an effect on transfu- transplantation with clinical utility in the majority of sions, resource utilization and long-term survival. sub-optimal mobilisers A. Alfred (1), S. Mahmood (2), D.A. Jones (1), D. Hess (2), J. Wright (1), J.A. Snowden (1), A. Mijovic (2) (1)Sheffi eld Teaching Hospitals NHS Trust (Sheffi eld, UK); (2)King’s College NHS Foundation Trust (London, UK)

Aim: We report a retrospective analysis of our experience with the use of single agent High Dose (HD) G-CSF (≥16 mcg/kg/ day) for haemopoietic cell (HSC) mobilisation in patients (pts) for Autologous Stem Cell (ASCT). Methods: A total of 73 patients (36 MM, 27 NHL,HD 4,CML:2, Trophoblastic tumour:1,AML:2,ALL:1) received HD G-CSF between Sept 2001-Dec 2007 in two centres. G-CSF was administered as twice daily subcutaneous injections. 22 pts received 16mcg/kg/day, 50 pts 20mcg/kg/day and one received 40 mcg/kg/day. A decision was made to harvest when WCC ≥1.0x109/L and peripheral CD34+ ≥0.01x109/L. A successful target yield was defi ned as ≥2.5x106 /kg CD34+ cells.

S114 Patients: 12 pts had previously undergone ASCT and 30 pts P510 had received salvage chemotherapy at the median of 31 (range Mobilisation of peripheral blood stem cells in patients 16-369) days prior to mobilisation with HD G-CSF. 23/30 pts with malignant lymphomas with pegfi lgrastim following were in CR2 or beyond at the time of mobilisation. In 45/73 chemotherapy (62%), previous mobilisation attempts had been inadequate S. Alekseev, M. Estrina, E. Kochina, E. Babenko, M. Popova, for ASCT. Of these, 33/45 had received cyclophosphamide+G- N. Mikhaylova, L. Zubarovskaya, B. Afanasyev CSF and 11 had received single agent G-CSF at 10mcg/kg. Pavlov’s State Medical University (St. Petersburg, RU) Results: HD G-CSF was administered for 4 days in all pts. 31/73 (43%) pts achieved the target cumulative dose of 2.5 Background: Haemopoietic stem cell (HSC) transplantation x106 CD34+ cells/kg after HD G-CSF. Additional 17/73 (23%) has been widely performed to support high-dose chemother- pts achieved the cumulative target dose when combined with apy in haematological malignancies. In lymphoid malignancies, previous mobilisation episodes. 9 patients who did not reach autologous stem cell transplantation (ASCT) is the treatment the CD34+ threshold of ≥0.01x109/L were not harvested. 13/45 frequently employed in relapsed malignant lymphomas (ML) or pts (29%) had a successful yield having failed other forms of in very high-risk ML. A total of 10–12% of patients with ML do mobilisation. Ultimately, 31 patients proceeded to ASCT with not reach the minimum threshold of 2 x 106 CD34+ collected the median time to neutrophil engraftment (≥0.5 x109/L) of 14 cells/kg and are considered poor mobilizers. days (range 10-29) and the median time to platelet engraftment Patients and methods: We analysed data on 64 patients witn (≥20 x109/L) of 21 days (range 12-60). There were no discon- malignant lymphomas to whom mobilization and apheresis of tinuations or dose reductions with the higher doses of G-CSF HSC were performed in our clinic from January 2002 to Sep- and no unexpected toxicity was reported. tember 2006. 9 patients failed mobilization Hodgkin’s disease Conclusion: The use of HD G-CSF for HSC mobilisation is effec- (n=5), non-Hodgkin lymphomas (n=4)). All with advanced tive and well tolerated. HD G-CSF may be considered in heav- stage disease, previously received multiple courses of chemo- ily pre-treated patients and those who mobilised sub-optimally and radiotherapy and failed stem cell mobilization using G- with other regimens, enabling a majority (66% in this series) to CSF alone and were considered as poor mobilizers (average be considered for ASCT. Other advantages include avoidance number of CD34+ collected cells/kg 1.1 x 106). DHAP (cisplatin, of myelosuppressive chemotherapy, as well as outpatient self- Ara-C, dexamethazone) and pegfi lgrastim on D+1 after the end administration and fi xed-day apheresis scheduling. of chemotherarpy were used for remobilization. Mobilization was successful when there were more than 2.0 x 106 CD34+/kg in the fi nal product. PBPC were collected with a P509 continuous-fl ow blood cell separator (Fenwal CS3000 plus, Bax- Stable mixed donor-donor chimerism after double cord ter healthcare, Deerfi eld, IL, USA). Each apheresis procedure blood transplantation was performed for approximately 2 to 4 h, processing 10–14 l of J. Gertow, S. Berglund, M. Okas, O. Ringdén, M. Uhlin, whole blood volume. The total CD34+ count of the leukapher- J. Mattsson esis product were monitored daily following each collection. Karolinska Institutet (Stockholm, SE) Results: A total of 21 mobilization cycles were performed. The average number of CD34+/kg during one cycle was 1.1 x 106. 1 Umbilical cord blood is increasingly used as a source of stem patient (12%) was successful during the fi rst mobilization cycle. cells in allogeneic stem cell transplantation due to it’s naïve 3 patients (37%) were successful on a second cycle and 1 cell content and high permissiveness for HLA-mismatch. To patient (12%) on a third cycle. Four patients (40%) failed remo- overcome problems of limited cell numbers, double cord blood bilization and didn’t reach the fi nal target. During treatment we transplantation (DCBT) has proven both safe and effi cacious. observed mild hematological toxicity – thrombocytopenia grade Concerning chimerism analysis after DCBT, previous studies III (6 patients), anemia grade III (4 patients) (CIC AE). There have indicated single unit predominance early after DCBT. were no infectious complications and no signs of any other In the present study we evaluated the chimeric pattern in toxicity. The only grade 3 or higher non-hematologic toxicity T-, B- and myeloid cells using PCR based chimerism analysis directly related to pegfi lgrastim was bone pain. in patients after DCBT. Of the seven patients included in this Conclusions: Mobilization with DHAP and pegfi lgrastim seems study, fi ve had acute leukemia and two patients had lymphoma. to be effective, well tolerared and less toxic than using other Five patients received myeloablative conditioning and two disease specifi c chemotherapy regimens. It can be considered patients were given reduced intensity conditioning. the standard of care for the group of patients, who failed previ- Interestingly, three patients showed mixed donor chimerism ous attempts. in all cell lineages at 100 days post-transplantation, and two of them still at 25 and 29 months after DCBT, respectively. These two patients are doing clinically well, with no infectious P511 complications or signs of relapse, and neither of them devel- Monitoring of HPC-CB production process: oped acute GVHD after DCBT. All patients received high dose implementation of an internal quality control antithymocyte globulin (ATG) before DCBT, which could be an A. Pontari, D. Marcuccio, G. Pucci, I. Bova, A. Dattola, explanation for an increased tolerance between the cord blood C. Garreffa, E. Spiniello, D. Princi, R. Monteleone, G. Gallo, units. Immunological studies revealed phenotypic differences P. Scaramozzino, P. Iacopino between the two cord blood units. Among other things, antigen AO Bianchi Melacrino Morelli (Reggio Calabria, IT) presenting cells and T cells of memory phenotype predomi- nated in one cord blood unit, whereas natural killer cells were Calabria Cord Blood Bank (CCBB), at the Regional Bone Mar- found in higher frequencies in the other unit. row Transplant and Cell Therapy Centre “Alberto Neri”, Azienda In conclusion, in this study donor-donor mixed chimerism was Ospedaliera “Bianchi-Melacrino-Morelli” in Reggio Calabria, common after high dose ATG and DCBT, and in these cases obtained the certifi cate of quality ISO 9001:2000 related to phenotypical differences between the two cord blood units collection, manipulation and cryopreservation of cord blood regarding memory phenotype were found. units (CB) units in September 2007. The standardization of the process of the Haematopoietic Progenitor Cells-Cord Blood (HPC-CB) units is part of the Quality’s Policy, in order to assure effi cacy for the patients. Aim of our work is to defi ne an internal quality control (QC) of HPC-CB units as useful tool to monitor- ing the production process. From June 2008 to November 2008, n°68 cord blood units have been evaluated using samples cryopreserved with units

S115 and destined to internal QC. We implemented a QC protocol on or mice given MSC pre-transplant. This led us to hypothesise thawed out samples, including the following test: WBC count by that IFNgamma plays an important role in MSC-mediated cell count Dasit KX21; viability and recoveries of total nucleated immunosuppression. To investigate this, we removed T cells cells (TNC), mononuclear cell (MNC), CD34± cells and precur- as a major source of IFNgamma production in vivo by admin- sor cell cultures on Methylcellulose. In addition, sterility testing istering KT3 antibody to mice ± MSC, and found that although of the collected HPC-CB units before further processing was T cell depletion prolonged survival, MSC did not enhance this performed. survival further. We then showed that MSC from IFNgamma The viability is determinated using fl ow cytometric method receptor knockout mice or T cells from IFNgamma knockout (7AAD) and vital coloration by Trypan Blue. The determina- mice in T cell proliferation assays were unable to suppress T tion of CD34+ cells is by fl ow cytometric with BD FACS Calibur cell proliferation in vitro. Collectively, these results suggest that and monoclonal antibodies Becton Dickinson. Cell Cultures are IFNgamma is pivotal for MSC-mediated immunosuppression. incubated at 37° C in 5% CO2 and 95% humidity (Stem Cell We speculate that MSC remove IFNgamma in vivo preventing Technologies Methocult H4434 e H4534). The incubation time its downstream actions on GVHD effectors. These data also is 14 days. suggest that elevated systemic IFNgamma in HSCT recipients The above mentioned determinations are compared with the could be used as the trigger for MSC administration and there- same parameters performed on the sample before cryopreser- fore enhance their potency as cellular therapy for prophylaxis vation. and treatment of GVHD. To guarantee the reliability of the fl ow cytometric method, CTMO participates to VEQ programme, managed by UKNEQAS (National External Quality Assurance Scheme). P513 Our results in internal control of cryopreserved samples showed CD83 antibody prevents GvHD and does not impair GvL high recoveries for TNC (88%) and for viability CD34+ cells induction (82%). Recovery of total colony-forming units (CFU) was 76%. H. Cullup (1), J. Wilson (1), R. Lourie (2), Y. Shen (1), A. Palkova Microbiological contamination of the collected HPC-CB sam- (1), K. Radford (1), A.M. Dickinson (3), A. Rice (1), D. Hart (1), ples was negative. D. Munster (1) Quality and security of the production process of cord blood (1)Mater Medical Research Institute (South Brisbane, AU); units is an essential requisite of Quality System at CTMO- (2)Mater Health Services Pathology (South Brisbane, AU); CCBB. (3)Newcastle University (Newcastle-Upon-Tyne, UK) Quantifi cation of cell recovery post thawing out represents a basic side for our production process. Periodic implementation Introduction: The complication of acute graft versus host dis- of these simple tests is of fundamental importance for our inter- ease (GVHD) is a serious limitation to the wider application of nal QC. So we have always under control our production proc- allogeneic hematopoietic stem cell transplantation (alloHSCT) ess of HPC-CB units. for the treatment of leukemia and other hematological malig- nancies. GVHD is caused by dendritic cell (DC) stimulation of donor T cells and can be prevented by T cell depletion, but this impairs immunity to infections and the desired graft versus leukemia (GVL) effect. We have shown that activated human Graft-versus-host disease – preclinical DC depleting CD83 antibody inhibits alloproliferation and that and animal models treatment of human PBMC transplanted SCID (hu-SCID) mice with the antibody prevents GVHD. Objectives: To determine if CD83 antibody treatment of hu- P512 SCID mice, for the prevention of GVHD, impairs GVL precur- Interferon gamma is required for MSC-mediated sors or their induction to GVL effectors. immunosuppression in a murine model of GvHD Methods: (i) GVL effectors were induced in vitro by co-cul- M. Kambouris (1), B. Turner (2), L. Sinfi eld (1), H. Cullup (1), ture of irradiated leukemic cell lines with human cells recov- D. Hart (1), K. Atkinson (1), A. Rice (1) ered from CD83 antibody or negative control treated hu-SCID (1)Mater Medical Research Institute (South Brisbane, AU); mice. Responses were assayed in cytoxicity assays using 51- (2)University Medical School (Newcastle-Upon-Tyne, UK) Cr labelled leukemic cells as targets. (ii) GVL effectors were induced in vivo, by immunization of hu-SCID mice with irradi- Multipotent, mesenchymal stromal cells (MSC) are an emerg- ated human leukemic cells at the time of allogeneic human ing means of immunosuppression for patients with steroid PBMC transplant. At this time they were also treated with CD83 refractory graft-versus-host disease (GVHD). Despite clinical or negative control antibody. The mice were immunized again 7 use, pre-clinical data is lacking. In in vitro experiments, MSC days post-transplant. On days 9 or 10 post-transplant, when the suppress T cell proliferation, even at low ratios of 1 MSC per negative control antibody treated mice had developed symp- 100 T cells. We examined the effects of donor-derived, intra- toms of GVHD, all mice were sacrifi ced. Human cells recovered peritoneally injected MSC on GVHD severity and onset in a from bone marrow, spleen and peritoneal cavity were pooled myeloablative conditioned, MHC mismatched model of haemat- and used directly as effectors, without further stimulation, in 51- opoietic stem cell transplantation (HSCT) [UBI-GFP/BL6 (H-2b Cr release cytotoxicity assays with the leukemic cells used for to BALB/c (H-2d)]. Donor-derived MSC (4x10*5/mouse) were immunization as targets. injected 4hrs pre or 24hrs post HSCT then mice were moni- Results: (i) GVL effectors were successfully induced in vitro tored daily for GVHD. Only mice given MSC 24hrs post HSCT from human cells recovered from CD83 antibody treated hu- showed a signfi cant delay in death from GVHD, with median SCID mice for three out of the four leukemic cell lines tested. survival increased by 10 days (d) (d7 vs d17, p<0.001). To (ii) For two cell lines (U937, Raji), cytolytic effectors were suc- investigate the reason for this difference, we conducted timed cessfully induced in vivo in hu-SCID mice treated with CD83 sacrifi ce experiments to explore whether MSC altered cytokine antibody. secretion patterns and cellular effectors, such as dendritic cells Conclusion: CD83 antibody treatment is a potential new ther- (DC), known to play a role in GVHD. When we sacrifi ced trans- apy for the prevention of acute GVHD. Our in vivo preclinical planted mice on d+1 at the time they would normally receive data suggests that this treatment will not impair the desired an MSC infusion, we observed more activated splenic DC and GVL effect in alloHSCT. systemic IFNgamma compared to conditioned, untransplanted mice on d0. Despite this, MSC administered on d+1 post HSCT reduced the % and absolute numbers of activated DC and sys- temic IFNgamma (p<0.05) as compared to untreated controls

S116 P514 Our results show that NIK-defi cient T lymphocytes were unable Th17/Treg ratio in human gastro-intestinal of causing GVHD in the fully mismatched allogeneic setting. graft-versus-host disease The homing and proliferative capacities were not affected, G. Socié, P. Ratajczak, C. Leboeuf, A. Desveaux, R. Peffault de but at an increased apoptotic rate early after transplant was Latour, M. Robin, P. Bertheau, A. Janin detected among the NIK defi cient T lymphocytes compared to Hospital Saint Louis (Paris, FR) controls.

Experimental data suggest a reciprocal relationship between Th17-induced pathology, and Treg regulatory role. In murine P516 models, Th17 cells induce autoimmunity through tissue infl am- Donor HSP70-hom gene polymorphism as a risk factor of mation promotion, and innate immune system mobilization. chronic GvHD Murine models provide recent controversial results on the role A. Kuzyk (1), K. Bogunia-Kubik (2), G. Jackson (1), of Th17 in GvHD. The Th17 subset has never been explored in A.M. Dickinson (1) human GvHD. To investigate its potential implication in GvHD- (1)University of Newcastle Medical School (Newcastle upon induced infl ammation, we studied Th17 cells presence in the Tyne, UK); (2)Institute of Immunology and Experimental gastro-intestinal (GI) tract and their relationship with histologi- Therapy, PAS (Wroclaw, PL) cal and clinical parameters. In addition to characterization of the cellular infi ltrate, TNF, TNF receptors, Fas in situ expression, Objective: The involvement of heat shock proteins 70 (HSP70) and quantifi cation of apoptotic cell, CD4+/IL17+ cells (Th17) in peptide binding and presentation could suggest their poten- and CD4+/Foxp3+ cells (Treg) were quantifi ed in 40 patients tial role in the alloreactive process that leads to graft-versus- with suspected GvHD of the GI tract, who had biopsies before host disease (GvHD). HSP70 have been shown to be involved steroid treatment. A Th17/Treg ratio<1 correlated both with the in the pathology of GvHD in an animal model. More recent stud- clinical diagnosis (sensitivity 74%, specifi city 100%, p=0.001), ies in humans, have documented an association between the and 2 or more pathologic grade (sensitivity 64%, specifi city increased levels of antibodies to 70kDa and 90kDa heat shock 94%, p<0.001). A Th17/Treg ratio<1 also correlated with the proteins in serum and the incidence of acute GvHD. Further- intensity of apoptosis of epithelial cells (p=0.03), Fas expres- more a correlation between the expression of an inducible form sion in the cellular infi ltrate (p=0.003), TNF and TNF receptors of HSP70 and the degree of GvHR in an in vitro skin explant 1 and 2 expression (p<0.001 for all). Our data do not support model has been observed. The present study aimed to answer increased Th17 cells in human GvHD of the GI tract. the question whether two coding dimorphisms of the HSP70- hom gene identifi ed at positions +2437 and +2763, in addition to previously documented association with acute GvHD (Bogu- P515 nia-Kubik et al. Transplantation 2005, 2006) could affect the Donor T-lymphocytes defi cient for NF-KB inducing kinase risk of chronic GvHD in HSCT patients. do not cause graft-versus-host disease Methods: Polymorphisms within the HSP70-hom gene (+2763 M. Ramírez (1), L. Casanova (1), C. Sánchez-Valdepeñas G/A, +2437 C/T) were analysed in 232 HSCT patients and their (2), I. Colmenero (1), M. González-Vicent (1), M.A. Díaz (1), donors in relation occurrence and severity of graft-versus-host L. Madero (1), M. Fresno (2) disease (GvHD) and in 186 donor-recipient pairs for generation (1)Hospital Universitario Niño Jesús (Madrid, ES); (2)Centro de of graft-versus-host reactivity (GvHR) in an in vitro human skin Biología Molecular Severo Ochoa (Madrid, ES) explant model. Results: Acute GvHD was more frequently seen in patients with Graft versus host disease (GVHD) remains one of the major positive skin explant assay results (0.81 vs 0.67, p=0.046) and complications of allogeneic transplants. Donor T lymphocytes in those carrying the AA homozygous genotype (0.47 vs 0.22, are directly involved in GVHD. Molecules involved in T cell p=0.034). Moreover, the presence of the A allele was found to function may be targets for GVHD therapy and prophylaxis. be associated with higher GvHR in vitro (0.62 vs 0.45, p=0.045 We explored the effects of suppressing NF-kB Inducing Kinase for II-IV vs 0-I grade of GvHR, respectively). (NIK) in donor T lymphocytes in a murine model of GVHD: The incidence of chronic GvHD was higher in patients trans- H2Db T lymphocytes transplanted into fully mismatched H2Dd planted from donors carrying the A allele as compared to GG recipients. Donor mice were either aly/aly (NIK defi cient) or homozygotes (0.59 vs .039, p=0.041). After multivariate analy- C57BL/6 (control), and recipient mice were Balb/c. Mice recipi- sis donor A allele (OR=2.038, p=0.044), together with trans- ent of BL6 cells developed a severe form of GVHD and died plantation from female donor to male recipient (OR=3.561, in the third week after transplant. On the contrary, mice trans- p=0.006) and PBMC as a source of stem cells for transplanta- planted with aly T lymphocytes survived for 3 months and did tion (OR=2.092, p=0.049) were independent risk factors which not develop GVHD. Histopathological analysis of skin, gut and signifi cantly contributed to the development of chronic GvHD. liver of these surviving mice showed no sign of GVHD. We next Conclusion: HSP70-hom gene polymorphisms associate with studied the homing, proliferation and apoptosis of the infused T the development of post-transplant complications. Recipient lymphocytes in the early days postransplant, in order to ascer- and donor genotypes affect the risk of acute and chronic GvHD, tain the cause of the difference in GVHD survival. The same respectively. numbers of aly or BL6 donor T cells were recovered from the spleens of recipient mice 24-36 hours after transplant. How- ever, signifi cantly higher numbers of BL6 T cells were recov- P517 ered at day +5 compared to those of aly T cells. This difference Differential reactivity of rabbit anti T-lymphocyte globulin might be explained by impaired T cell proliferation, enhanced with peripheral blood mononuclear cell compartments T cell apoptosis, or a combination of both. Donor T lymphocyte in patients’ sera 21 days post allogeneic haematopoetic proliferation was studied by means of CFSE dilution. Analysis stem cell transplantation of in vivo T cell divisions showed the same kinetics for aly and C. Ziegler (1), J. Finke (1), C. Grüllich (2) BL6 T lymphocytes at 1, 3 and 5 days after transplant. There- (1)University Medical Center (Freiburg, DE); (2)National Center fore, aly T cells divided in vivo but the process was not produc- for Tumor Diseases (Heidelberg, DE) tive. We fi nally studied apoptosis among donor T lymphocytes at 1, 3 and 5 days after transplant. We found that the propor- Introduction: Polyclonal Anti T-Lymphocyte Globulins (ATG) tions of apoptotic aly T cells were higher than those of BL6, at are widely used for GvHD prophylaxis in allogeneic stem cell days 1 and 3, but not at day 5. The difference in total numbers transplantation (SCT). ATG have a wide epitope spectrum and of T cells at day 5 was associated to lower amounts of Th1 have been shown to react with all compartments of peripheral cytokines in the serum of mice receiving aly vs. BL6 T cells. blood monuclear cells (PBMNC). Furthermore, it has been

S117 demonstrated that ATG can induce apoptosis in PBMNC. Infused patients with a lower catalase increase (p=n.s.). The cumula- ATG can be detected in patients sera up to one year after trans- tive survival after 2.3 years was 56% (32-81%, 95% CI) for plantation. Here we investigated the reactivity of ATG in the sera patients without a catalase increase and 17% (0-46%, 95% CI) with PBMNC compartments from ten rabbit ATG treated patients for patients with an increase >1,5 (p=0,06). Interestingly, cata- after haematopoetic regeneration on day 21 post SCT. lase levels of six patients receiving dosis-reduced conditioning Patients and methods: Sera of ten patients who underwent unre- were, with two exceptions, at each time point higher than the lated allo SCT for haematologic malignancy and were treated catalase levels of the 16 patients treated with a myeloablative with anti-Jurkat rabbit ATG-Fresenius® were collected on day regimen suggesting that the intensity of the conditioning regi- 21 after SCT. All patients had achieved a full haematopoetic men is not infl uencing catalase levels. recovery. PBMNC of healthy volunteer donors were briefl y incu- In conclusion, this study revealed substantial catalase levels bated with heat inactivated patients sera followed by incubation in sera of patients undergoing allogeneic HSCT which were with FITC labelled anti-rabbit IgG. Then, subpopulations were enhanced during the occurrence of acute GvHD. It is clear labelled by lineage specifi c PE labelled antibodies (CD4, CD8, that larger patient cohorts and analysis of further antioxidative CD20, CD14 and CD56). ATG reactivity with cell compartments enzymes are needed in order to elucidate antioxidative proc- was detected by fl uorescence cytometry. esses and their relationship to GvHD during HSCT. Results: While the fresh ATG preparation virtually reacted with 100% of all PBMNC compartments, marked differences could be observed with day 21+ sera. The lymphoblastic leukemia P519 cell line Jurkat used for rabbit immunizing in ATG generation Human iNKT compartment homeostasis after still showed a 100% reactivity to ATG in sera. In PBMNC com- haematopoietic stem cell allograft partments only 44% of CD4+ T-lymphocytes, 58% of CD8+ T- A. Rossignol (1), A. Yip-Fa (1), C. Giraud (1), M. Charron (1), lymphocytes and 41% of CD56+ NK cells reacted with rabbit N. Maillard (1), A. Chauvineau (1), A. Barra (1), A. Herbelin (2), ATG still present in patients sera. In contrast, 82% of CD20+ J.M. Gombert (1), F. Guilhot (1) B-lymphocytes and 98% of CD14+ monocytes still reacted with (1)University of Poitiers (Poitiers, FR); (2)University Paris ATG in patients sera. Descartes (Paris, FR) Discussion: The change in reactivity with ATG in patients sera on day 21+ especially within the CD4+,CD8+ T-lymphocyte and Invariant Natural Killer T (iNKT) cells, expressing an invariant the CD56+ NK cell compartment compared to the fresh ATG TCR-a chain (Va24-Ja18) paired with a Vb11 TCR-b chain, preparation is most likely to be explained by specialized sub- recognize some glycosphingolipidic antigens presented by the groups within these compartments that have the highest cell MHC class I-like CD1d molecule. In murine models of HSC allo- turnover. Thus, antibody specifi cities directed against particular graft, they control both Graft Versus Host Disease (GVHD) and epitopes present only in those subgroups could be depleted Graft versus Leukaemia (GVL) phenomena. from the serum during haematopoetic regeneration. It can be In this study, we addressed the status of iNKT cells in patients speculated, wether these cells are represent activated cell sub- after HSC allograft with or without chronic GVHD treated by sets. To further characterize these subgroubs ATG reactivity immunosuppressive agents and corticoids or in patients resist- has to be evaluated against particular cell activation and dif- ant to this treatment treated by extra-corporal photo-chemo- ferentiation markers. therapy (ECP). Four groups of patients were designed: Group 1 corresponds to patients after HSC allograft without P518 cGVHD, n = 7 Serum catalase to predict graft-versus-host disease after Group 2 corresponds to patients after HSC allograft with a mild- allogeneic stem cell transplantation severe cGVHD controlled by conventional treatment, n=9 B. Kircher, P. Schumacher, J. Clausen, J. Auberger, Group 2 corresponds to patients after HSC allograft with severe D. Nachbaur cGVHD treated by ECP, n=5. Immunobiology and Stem Cell Laboratory (Innsbruck, AT) Group of healthy donors (HD), n=5. We fi rst compared the frequency, the phenotype and the pro- Oxidative imbalance may be involved in the pathogenesis of liferative potential of blood iNKT cells in patients and in HD, by complications after hematopoietic stem cell transplantation fl ow cytometry. (HSCT). The absolute blood iNKT cells number was signifi cantly In order to investigate possible repair mechanisms during com- decreased after HSC allograft in all groups of patients in com- plications we have analyzed the catalase levels of 22 patients parison with HD. The ex vivo phenotypic analysis showed with hematological malignancies undergoing allogeneic HSCT an enrichment in CCR7+CD161- iNKT cells in the group from HLA-matched donors. Catalase levels were measured in of allografted patients who did not develop a cGVHD. This serum at various time points before and after HSCT and were CCR7+CD161- phenotype had been described as a central expressed as median levels ± standard error. memory (CM)-like iNKT population. We further showed in the Serum catalase levels of 16 patients determined on days -4/-5 GVHD group 2 an inverse correlation between the expression before HSCT (146.9 ± 46.4 µM) were signifi cantly higher than of CCR7 and that of CD161, demonstrating the mutual exclu- those of eight healthy normal controls (46.7 ± 2.1 µM; p<0.005) sion of the CM phenotype (CCR7+) versus the effectors mem- indicating enhanced repair mechanisms present in leukemia ory (EM) phenotype (CCR7-CD161+). patients already during chemotherapy. After transplantation We showed that the proliferative response of iNKT cells to their catalase levels decrease and rise again when leukocyte counts cognate ligand was impaired in cGVHD patients (groups 2 and begin to increase. The catalase levels of patients developing 3) in comparison to iNKT cells from HD although there was no acute graft-versus-host disease (GvHD) are, with two excep- difference between group 1 and HD. This result was consist- tions, at each time point higher than the catalase levels of ent with the iNKT CM phenotype of group 1 patients versus a patients without this complication. The median increase of more EM iNKT phenotype in the patients from group 3, since catalase at onset of acute GvHD was 1.4 ± 0.3, whereas no EM cells have been characterized by decreased proliferation increase (1.0 ± 0.3) was observed for patients without GvHD capacities. on days +18 until +21 (represent the median days of GvHD Altogether our results demonstrate that the HSC allograft modify occurrence). Patients were further divided into a group with an the homeostasis of the iNKT compartment, generating a biased increase of catalase levels higher or lower than 1,5. The cumu- central memory iNKT compartment (CCR7+CD161-) which is lative incidence of developing acute GvHD was 83% (58-100%, lost in the patients with GVHD. From a therapeutic point of view, 95% confi dence interval (CI)) for patients with a 1,5 times higher it could be useful to preserve this EM iNKT compartment to catalase increase in comparison to 44% (25-76%, 95% CI) for maintain a GvL effect without developing a GVHD.

S118 P520 clinical practice). Thawing in 37°C water, then ECP on Vilber- Dysregulation of human plasmacytoid dendritic cells in Lourmat irradiator. Viability and apoptosis measured by trypan chronic graft-versus-host disease blue exclusion test and annexin V fi xation. Immunomodulatory A. Rossignol, A. Yip-Fa, C. Giraud, S. Noel, N. Maillard, effects assessed in haploidentic mixed lymphocyte reaction A. Chauvineau, A. Barra, F. Guilhot, J.M. Gombert (MLR) by measurement of non-treated cell proliferation (Car- University of Poitiers (Poitiers, FR) boxyfl uorescein succinimidyl ester = CFSE dye dilution). Results: cryopreservation induced a loss of about 10% of cells, The plasmacytoid dendritic cells (pDc) are antigen-presenting and triggered apoptosis. However, after ECP the rate of apop- cells and produce type-I IFN. They are defi ned by their co- totic cells was similar in fresh or frozen cells (one hour after expression of CD123, CD303 and CD4. PDc have a key role ECP: 24% of fresh cells were annexin V + vs 28% of cryop- in the control of viral infections, and could be implicated in the reserved cells, p=NS; after 48h culture: 61% vs 69%, respec- control of the immune tolerance. tively, p=NS). On the three fi rst experiments, the inhibition of Recent works showed a delay in the reconstitution of the pDc alloreactive cell proliferation in MLR induced by ECP-treated compartment after Hematopoietic Stem Cell (HSC) allograft cells was similar with fresh and frozen cells (– 21% of proliferat- associated with an increased susceptibility to herpes viruses ing cells vs – 17%, respectively). and apparition of acute Graft Versus Host Disease (aGVHD). Conclusion: these preliminary data suggest that frozen cells We analysed the blood pDc absolute numbers in fl ow cytometry retain their immunomodulatory effects after ECP. Other param- and the IFN-a production status of peripheral blood mononu- eters such as IL10 secretion and triggering of regulatory T cells clear cells (PBMC) in patients after HSC allograft with or without are currently investigated. These data will permit using cryop- chronic GVHD (cGVHD) and with severe cGVHD resistant to reserved cells to optimize and facilitate the treatment of GVHD conventional treatment (immuno-suppressors and corticoids), by ECP. treated by extra-corporal photo-chemotherapy (ECP). Four groups were designed: Group 1 corresponds to patients after HSC allograft without cGVHD, n = 7. Group 2 corresponds to patients after HSC allograft with a mild- severe cGVHD controlled by conventional treatment, n=6 Group 3 corresponds to patients after HSC allograft with severe cGVHD treated by ECP, n=6. Group of healthy donors (HD), n=9. In the three groups of patients we showed a decreased abso- lute blood pDc numbers, in comparison with HD (p<0.01 versus group 1 and p<0.001 versus groups 2 and 3 at T0 before initia- tion of ECP). We did not show any difference in the frequency and absolute numbers of myeloid dendritic cells (mDc) between the groups of patients and HD. Patients from groups 1 and 2 have a decreased in vitro IFN- a production by PBMC after stimulation by infl uenza virus in comparison with HD (p<0.05 for the comparison of group 1 and 2 with HD). We further analysed the pDc numbers along ECP and did not observed any modifi cation. At last, we show that, after 18 months post ECP initiation, the PBMC improved their IFN-a production in comparison with their production prior to initiation P522 of ECP (T0) or after 2 to 9 months (p<0.05 for the comparison of Genetic similarity of chromosome 6 between patients T0, 2 months and 9 months IFN-a production with the 18 month receiving haematopoietic stem cell transplantation and IFN-a production). This was correlated with a clinical improve- HLA-matched sibling donors ment of their cGVHD. H. Turpeinen (1), L. Volin (2), L. Nikkinen (1), P. Ojala (1), Altogether we showed a selective defi ciency of pDc compart- A. Palotie (3), J. Saarela (4), J. Partanen (1) ment in cGVHD patients, associated with a decreased produc- (1)Finnish Red Cross Blood Service (Helsinki, FI); (2)Helsinki tion of IFN-a. We showed an improvement of IFN-a production University Central Hospital (Helsinki, FI); (3)The Wellcome by PBMC after ECP without any modifi cation of the pDc com- Trust Sanger Institute (Cambridge, UK); (4)National Public partment. These results suggest that the pDc were a target of Health Institute (Helsinki, FI) the ECP therapy. Background: Matching for HLA genes located in chromosome 6 is required in hematopoietic stem cell transplantation (HSCT) to P521 reduce the incidence of graft-versus-host disease (GvHD). How- Immunomodulation induced by extracorporeal ever, a considerable proportion of patients still suffer from it, obvi- photochemotherapy: impact of cryoconservation ously due to genetic differences outside the HLA gene region. E. Merlin, R. Veyrat-Masson, P. Halle, M. Berger, F. Deméocq, Design and methods: We studied the similarity of almost 4000 J. Chassagne, J. Kanold single nucleotide polymorphisms (SNPs) on chromosome 6 CHU (Clermont-Ferrand, FR) between patients receiving HSCT and their HLA matched sib- ling donors. In the setting of Graft versus Host Disease (GVHD), the cryo- Results: We observed that as a result of routine HLA matching preservation of mononuclear cells could allow to maintain the the siblings in fact shared surprisingly long chromosomal frag- ECP schedule while reducing the number of apheresis. The aim ments with similar SNP genotypes – from 11.65 Mb to 134.66 of our work is so to assess whether cryopreservation impairs Mb. The number of genes mapped on these shared fragments the immunomodulatory function of Extracorporeal Photoche- varied from 402 to 1302. Considering the whole chromosome 6, motherapy (ECP) treated mononuclear cells. the HLA matched siblings were apparently identical for 65.2% Material and methods: Blood of haploidentic healthy donors. to 97.8% of the SNPs. Isolation of PBMC by centrifugation on Ficoll-Hypaque gradi- Conclusions: Potentially, genes similar in some transplantation ents. Cryopreservation in 3.5% diméthylsulfoxyde (DMSO) at pairs while different in others might have a signifi cant role in -80°C without controlled-rate freezing (same process we use in determining the outcome after HSCT.

S119 Graft-versus-host disease – clinical

P523 A comparison of 2 initiation timing of cyclosporine used as a single drug for GvHD prevention M.Y. Shapira, L. Dray, M. Aker, B. Gesundheit, S. Samuel, R. Or, I.B. Resnick Hadassah – Hebrew University Medical Cen (Jerusalem, IL)

Cyclosporine (CSA) is the backbone of GVHD prophylaxis in the last decades. It is established that CSA levels in the 1st weeks after transplant are critical for the rate and severity of GVHD. Initially, we gave CSA starting on day -1 in all our proto- cols. However, 7 years ago, we have changed CSA initiation in most of our protocols to day -4 in order to have stable, control- led therapeutic blood levels of CSA prior to transplant. Patients and methods: the records of 1716 patients that under- went allogeneic transplantation were analyzed. Out of them, we identifi ed 2 groups of patients that received T-cell repleted grafts in which CSA was used for GVHD prevention, starting on days -1 or -4 (n=219 and 261 respectively). The guidelines for CSA cessation and DLI were uniform in both groups. The groups were compared for engraftment, GVHD (both acute and chronic), GVHD associated death and overall survival. Results: The groups were equal for age, sex, donor type, matching, disease and disease status. The median time to ANC engraftment was 16 and 15 days in the CSA -1 and -4 groups respectively with a trend toward better engraftment with CSA -4 (fi gure 1A, P=0.07). However, platelet engraftment was signifi - cantly better with CSA -4, with a median of 14 and 12 days in the CSA -1 and -4 groups respectively (fi gure 1B, p=0.0005). One hundred and twelve and 138 patients developed aGVHD of any grade, respectively. Out of them 54% and 44% had severe (grade 3-4) aGVHD (p=0.45). The median time to aGVHD was similar, with a median of 29 and 28.5 days in the CSA -1 and -4 groups respectively (p=0.54). However, 64 patients devel- oped cGVHD in the CSA -1 group, while 102 did so in the CSA -4 group (fi gure 2A, p=0.0002. Hazard ratio 0.5893, 95% CI 0.3693 to 0.7328). Of these patients, 46.8% and 40.2% of the patients had extensive cGVHD (p=0.70), respectively. The mor- tality in the groups was higher in the CSA -1 group (67.6% and 50.5%, fi gure 2B, p=0.074. Hazard ratio 1.2370, 95% CI 0.9792 to 1.5828). Additionally, despite lower GVHD rate in the CSA -1 group, the proportion of GVHD associated death was more higher then in the CSA -4 group (41/148 and 17/132 patients, p=0.02). We conclude that initiating CSA on day -4 improves engraft- ment, conversely increases the risk for cGVHD of any grade (possibly through prevention of tolerance), but reduces the risk of GVHD associated death and improves overall survival.

S120 P524 P525 Comparison between an artifi cial neural network and Correlation of interleukin-23 receptor gene polymorphism logistic regression in predicting acute graft-versus-host with reduced incidence of acute GvHD disease following unrelated haematopoietic stem cell M. Wermke (1), S. Maiwald (2), R. Schmelz (1), C. Thiede (1), transplantation G. Ehninger (1), M. Bornhäuser (1), R. Wassmuth (2) G. Caocci (1), R. Baccoli (1), A. Vacca (1), E. Piras (1), (1)University Hospital (Dresden, DE); (2)DKMS Lifescience R. Littera (1), C. Giardini (2), F. Locatelli (3), C. Carcassi (1), Lab (Dresden, DE) G. La Nasa (1) (1)University of Cagliari (Cagliari, IT); (2)S. Salvatore Hospital Objectives: Occurrence and severity of infl ammation is infl u- (Pesaro, IT); (3)University of Pavia (Pavia, IT) enced by polymorphisms in genes coding for cytokines and their receptors. As an example a single nucleotide polymor- The prediction of acute graft versus host disease (aGVHD) is phism (SNP) at position 1142 of the Interleukin 23-receptor one of the crucial aspects of unrelated hematopoietic stem cell (IL23R) gene leading to an exchange of guanosin by adeno- transplantation (HSCT). A relatively high number of immuno- sine has been demonstrated to offer protection from Crohns biological variables have shown to play a role in aGVHD and disease. Recently, investigators suggested a protective effect research of a predictive algorithm is still ongoing. We investi- against acute Graft- versus-Host-Disease (aGvHD) after allo- gated the prognostic performance of an artifi cial neural network geneic transplantation. In this study we tried to further evaluate (ANN) and compared it with standard logistic regression (LR) IL23R-genotype for the prediction of aGvHD and respiratory in predicting aGVHD in a group of 71 beta-thalassemia major failure after allogeneic transplantation. patients (median age 11.4 years, range 1.5-29) transplanted Methods: Genomic DNA of 339 donor-recipient pairs was from an unrelated donor, having considered that these patients amplifi ed using whole-genome amplifi cation. Typing for have homogeneous immunobiological features. All patients IL23R(1142A>G)-SNP was done using a commercially avail- were administered a myeloablative conditioning regimen and able real-time PCR based genotyping assay. Suffi cient data GVHD prophylaxis with cyclosporine-A, short-term methotrex- regarding IL23R-mutational status and aGvHD was present ate and ATG. Thirty-one percent of the patients developed in 332 patients and their respective donors. The majority of grade II-IV aGVHD. Altogether, 30 different variables were patients (205 of 332 patients) were transplanted from matched evaluated: donor/recipient sex and age, Pesaro risk class, unrelated donors mainly using peripheral blood stem cells CMV serology, HCV-RNA positivity, conditioning regimen, (PBSC: n=314, bone marrow: n=17; cord blood: n=1). infused CD34 cell dose, HLA-DPB1 disparity, donor/recipient Results: At least one mutated IL23R-allele was present in 50 HLA-Cw ligand groups for killer immunoglobulin-like receptors of 332 (15.0%) recipients and 38 of 332 (11.4%) donors. Minor (KIR):C1/C2 heterozygosity or C2/C2-C1/C1 homozygosity, allele frequencies were in the range of published data (Donors: donor/recipient activatory or inhibitory KIRs, donor/recipient 0.059; Recipients: 0.078). The incidence of aGvHD °II-IV was HLA-G 14-basepair polymorphism, donor KIR AA haplotype. signifi cantly lower in patients with wild-type IL23R-gene trans- Patients were randomized into a learning data set (n=61) and planted from IL23R-mutated donors (15%) compared to cases a test data set (n=10). After training the neural network on the with wild-type status in donor and recipient (42%, p=0.044). learning data set, LR analysis and three-layer ANN were used Patients bearing the IL23R SNP receiving an IL23R wild-type to predict aGVHD. LR identifi ed three independent variables transplant as well as those with a mutation on donor- and with prognostic signifi cance for developing aGVHD: patients recipient-side, were demonstrated to have intermediate rates that were heterozygotes for HLA-Cw groups 1 and 2 (C1/C2); of aGvHD °II-IV(36% and 28% respectively). However, IL23- patients homozygous for the HLA-G 14-basepair deletion Receptor genotype did not correlate with overall-survival. Fur- (-/-14-bp); donors homozygous for KIR haplotype A (AA). The thermore, we found no association of IL23R-mutational status sensitivity of LR (performance in predicting aGVHD in patients and a deterioration of respiratory function as measured by oxy- who developed aGVHD) was 68.4% in the learning data set genation-index (paO2 / FiO2). and and 66.7% in the test data set; the specifi city (the ability to Conclusion: Transplanting IL23R-wildtype patients from donors predict the absence of aGVHD in patients who did not develop with the the IL23R(1142G>A)-SNP seems to be protective aGVHD) was 83.3% and 71.4% respectively. The sensitivity of against aGvHD. As this is the second study confi rming such ANN was 84.2% in the learning data set and 66.7% in the test benefi cial effects, a prospective evaluation of donor selection data set; specifi city was 100% and 71.4%, respectively (see according to IL23R-genotype is warranted. Table).The specifi city of ANN in predicting aGVHD following unrelated HSCT was signifi cantly higher than LR, but its sensi- tivity was lower. In this context, automated multivariate analysis P526 may prove to be a useful optimization tool. Patients with chronic graft-versus-host disease responding to extracorporeal photopheresis signifi cantly decrease immature/transitional B-cell numbers during therapy Z. Kuzmina, R Weigl, R. Knobler, N. Worel, H. Greinix, W. Pickl Medical University of Vienna (Vienna, AT)

Introduction: ECP is an effi cient and safe treatment for patients with cGVHD. Evaluation of response and distinction from irre- versible tissue damage can be diffi cult in patients with long- lasting cGVHD. We recently published that an elevation of immature/transitional CD19+/CD21- B cells is signifi cantly associated with active cGVHD (Greinix et al, BBMT 14:208, 2008). Aim of the study: We investigated the role of immature/transi- tional CD19+/CD21- B cells as potential cellular biomarkers for monitoring objectively response to ECP. Patients and methods: Twenty-eight patients (14 males and 14 females) with moderate (n=17) or severe (n=11) cGVHD and with 2 or less (n=14) or > 2 organs (n=14) involved were included. Seven had de novo onset, 17 quiescent and 4

S121 progressive onset of cGVHD. ECP was given as fi rst-line ther- alloreactive stimulation of the immune system, (3) Proportions apy in 12 patients and as salvage therapy in 16 ones. ECP of IL-17+ cells in CD4+ cells population decrease at the time of was performed initially on 2 consecutive days every two weeks full blown aGvHD, likely being marginalized in the infl amed tis- and monthly after improvement. CGVHD was assessed every sues, (4) IFNgamma producing cells among CD4+ cells popula- 3 months according to NIH Consensus Criteria. At the same tion do not increase at aGvHD manifestation (IFNgamma is not time points T cells, NK cells, dendritic cells and B cell subsets needed for aGvHD pathomechanism). (CD19+/21-, CD19+/IgD-/CD27+, CD19+/IgD+/CD27+) were Supported by the grant from the Polish Ministry of Science & analyzed from peripheral blood (PB) by fl ow cytometry. For Higher Education (2P05E 037 30). comparison of clinical response and PB cell numbers patients were divided into responders to ECP (n=20), and non-respond- ers (n=8). P528 Results: After 6 months of ECP 20/28 (71%) patients showed Once-weekly liposomal amphotericin B for prophylaxis of improvement of cGVHD including 14/17 with moderate and invasive fungal infection after graft-versus-host disease 6/11 with severe cGVHD. The number of immature/tran- in allogeneic haematopoietic stem cell transplantation: a sitional CD19+/CD21- B cells was signifi cantly (p <0.001) comparative retrospective monocentre study lower in patients with response to ECP with a mean of 6.8% J. El-Cheikh, L. Wang, B. Esterni, C. Faucher, S. Furst, (range 1-19) compared to ECP-non-responders with a mean P. Berger, D. Blaise of 25% (range 14-43). Whereas complete responders had a Institut Paoli Calmettes (Marseille, FR) mean of 5% (range 1-15), partial responders had 8% (range 1.7-19). In addition, the ratio between immature/transitional B Invasive pulmonary aspergillosis (IPA) is a signifi cant risk in cells and CD19+/CD27+ memory B cells was signifi cantly dif- patients with graft versus host disease (GvHD) after allo-trans- ferent (p<0.001) with a mean of 2.2% (range 0.1-6.5) in ECP- plantation. responders and 8.6% (range 1.2-20) in non-responders. One A comparative retrospective single centre study was conducted year after start of ECP immature/transitional CD19+/CD21- B to investigate the effi cacy and safety of liposomal amphotericin cells remained at a mean of 6% in CR patients compared to B (L-AmB, Ambisome®) prophylaxis of fungal infections in allo- 24% in ECP-non-responders (p<0.001). transplanted patients with GvHD. Discussion: We confi rm that ECP achieves high response rates A total of 125 patients receiving high-dose prednisone (2 mg/ in patients with cGVHD. In ECP-responders immature/transi- kg/day) therapy for acute and/or chronic GvHD after allo-SCT tional B cell numbers are signifi cantly lower suggesting that this were identifi ed. 118 pateints (94%) had a RIC regimen and 7 PB cell subset could serve as cellular biomarker for monitoring (6%) a myeloablative conditioning; 95 patients (76%) had a of cGVHD activity during ECP. family donor and 30 patients (24%) had an unrelated donor. Median age at transplant was 48 years (range 18-70). Forty-two patients received once-weekly high-dose (7.5 mg/kg/ P527 week) L-AmB as prophylactic treatment. Eighty-three patients Increased proportions of TH17+ cells in patients post of the control group received possibly other prophylactic anti- HSCT heralds overt aGvHD fungal drugs such as fl uconazole, voriconazole, itraconazole, D. Dlubek (1), E. Jaskula (1), M. Sedzimirska (2), J. Lange (2), posaconazole. A. Lange (1) The prophylactic median dose of L-AmB was 500 mg/week (1)L. Hirszfeld Institute of Immunology and Experimental (range 300-650) and the median duration of treatment was 7 Therapy, Polish Academy of Sciences (Wroclaw, PL); (2)Lower weeks (range 2-15). Silesian Center for Cellular Transplantation (Wroclaw, PL) The incidence of IPA disease was 8% in the prophylactic group at both one year and two years vs 36% and 44%, respectively, Alloreactivity with the presence of aGvHD in patients post in the control group (p=0.008). No fungal infection-related HSCT is a major factor affecting the outcome of haematopoi- deaths were observed post-transplantation in the prophylactic etic stem cells transplantation (HSCT). In this study we focused group vs 12 (14%) at one year, 14 (17%) at two years and 16 on the presence of IL-17 producing cells in patients post HSCT (19%) at three years post-transplantation in the control group in the context of cells with FoxP3 and IFNgamma producing (p=0.005). potential. The OS rate at one year was 69% for the prophylactic group We studied the cytoplasmic expression of IL-17, FoxP3 and vs 75% for the control group; at two years the OS was 55% in IFN-gamma in stimulated PBMC of alloHSCT pts (30 patients, the prophylactic group vs 64% in the control group (p = 0.60). median age: 45 years (from 1.0 to 59 years), 28 haematologi- There were no differences in the TRM rates between the two cal malignancies, 2 SCID). Fifteen of them developed aGvHD groups at two years (18% vs 19%) and at three years (18% vs including 9 pts with aGvHD seen at later stage post hemato- 21%) (p = 0.99). logical reconstitution. PBMC were stimulated, according to the Prophylactic treatment with L-AmB was well tolerated. Renal manufacturer procedure, with BD Leukocyte Activation Cocktail toxicity leading to treatment discontinuation was observed in (PMA, Ionomycin and BFA) in the presence of GolgiStop, and only fi ve patients (12%) who were concomitantly treated with then stained with CD4, IL-17A, IFN-gamma and FoxP3. Cells other nephrotoxic drugs; nephrotoxicity was reversible in all fi ve were analyzed in CD4+ and CD4- lymphocytes subpopula- patients. tions. These data suggest L-AmB prophylaxis is an effective and well We found: (1) Contribution of IL-17A+ producing cells to tolerated treatment for the prevention of invasive fungal infec- CD4+ lymphocytes increased from 1.16%+0.41 at the day of tion and can reduce the fungal infection related mortality in RIC hematological recovery to 5.16%+2.47 shortly before aGvHD allo-SCT patients presenting a severe GvHD; further prospec- manifestation (p=0.027, Wilcoxon Test) and then decreased tive clinical studies are required to confi rm these single center to 0.74%+0.27 (p=0.008, Wilcoxon Test) when patients had data. overt aGvHD, (2) aGvHD manifestation was associated with an increase of FoxP3+CD4+ cells (p=0.01, Wilcoxon Test), (3) IFN- gamma producing cells contribution to CD4+ cells populations is lower at aGvHD manifestation then at earlier time(p<0.04, Wilcoxon test). In conclusion: (1) Under PMA, Ionomycin and BFA stimula- tion CD4+ cells become in part IL-17, IFNgamma and FoxP3 positive in patients post HSCT, (2) FoxP3+CD4+ cells increase at the manifestation of aGvHD likely to counter-balance

S122 mTOR-I, 17 pts received a combination therapy with steroids and 6 pts received additional immunosuppressive drugs, no cal- cineurin inhibitors however. In 4 pts extracorporeal photopher- esis was performed as an additional therapy. To further reduce the risk of adverse events drug trough levels were monitored and the dosing was adjusted to low therapeutical levels (3-8 ng/ml). Five and 13 pts had a complete and a partial response, respectively, with an overall response rate of 67%. In 2 pts, sclerodermatous changes stayed without progression. Six pts displayed a progressive course of the disease despite mTOR-I therapy and therefore the treatment was discontinued. Steroids could be tapered and stopped in a signifi cant number of pts. No difference in response was observed in everolimus- and sirolimus-treated pts. Major adverse events possibly related to mTOR-I were hyperlipidaemia and impaired wound healing, especially in pts with ulcerative skin lesions. One patient devel- oped thrombotic microangiopathy and infectious complications were rare. Eight pts died, 5 of the non-responders either due to cGvHD (n=3) or infection (n=2). One of the responders died due to a relapse of the underlying malignancy, 1 died of secondary malignancy and 1 of unknown cause. The median follow-up for surviving pts is 19 months. Our retrospective analysis suggests that mTOR inhibitors are effective immunosuppressive agents for the control of sclero- dermatous cGvHD.

P530 Impairment of endothelial protein C pathway in tissues involved by acute and chronic graft-versus-host disease F. Toraldo, A. Bucalossi, M. Tozzi, C. Miracco, V. Mormouras, M.T. Pirrotta, E. Cosci, S. Machetti, F. Lauria, G. Marotta Azienda Ospedaliera Universitaria Senese (Siena, IT)

Graft-versus-host disease (GVHD) is the major cause of mor- bility and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Endothelial cells (EC) represent a barrier between recipient tissues and circulating T alloreactive lymphocytes after HSCT. Recent studies have demonstrated a role of Protein C (PC) pathway in the EC protection during infl ammatory diseases. In this study we evaluated the expression, by immunohis- tochemistry, of PC pathway’s components on EC in patients with acute and chronic GVHD. Particularly, we investigated the endothelial expression of thrombomodulin (TM), endothe- lial protein C receptor (EPCR) and protease-activated recep- tor-1 (PAR-1) in 18 acute and 5 chronic GVHD biopsies and P529 the results were compared with that observed in 7 normal skin mTOR inhibitors for treatment of sclerodermatous chronic biopsies, 5 normal intestinal biopsies, and 15 biopsies from graft-versus-host disease following allogeneic stem cell several infl ammatory lesions of skin and gut. transplantation The percentage of EPCR/TM/PAR1-positive EC was calcu- Z. Jedlickova, I. Burlakova, A. Cook, H. Baurmann, lated by evaluating 20 randomly-chosen fi elds in each biopsy. R. Schwerdtfeger, M. Schleuning Biopsies showing ≥75% of negatively-stained EC were scored Deutsche Klinik für Diagnostik (Wiesbaden, DE) 0; ≥50<75% scored 1; ≥25<50% scored 2; fi nally, score 3 was assigned to biopsies with <25% of negative EC. Scleroderma belongs to most severe manifestations of chronic In acute and chronic GVHD we observed a damage and loss of graft-versus-host disease (cGvHD). Sclerodermatous GvHD EC with a strong decrease of EPCR expression in EC compared strongly affects quality of life and survival of hematopoietic to normal controls. Furthermore, we observed a decreased transplant recipients. Since current therapeutic approaches positivity of TM in most EC and an increased expression of often fail, it is important to fi nd novel immunosuppressive strat- PAR-1. These fi ndings were mostly similar to those observed egies for refractory forms of sclerodermatous GvHD. Immu- in infl ammatory diseases. The Dunn test supplied statistically nosuppressants sirolimus (Rapamycin) and everolimus inhibit signifi cant differences (p<0.05) for all three scoring variables mTOR (the mammalian target of rapamycin). As mTOR con- between controls and all lesion groups. No differences were tributes to fi brotic overproduction of collagen, mTOR inhibitors found between lesion groups (table 1). (mTOR-I) are possible candidates for effective treatment of It has been proved that APC, through the involvement of cellular scleroderma. receptors EPCR and PAR-1, determines modifi cation of gene In this retrospective analysis we report 27 patients (pts) with expression, anti-apoptotic and anti-infl ammatory activities, as sclerodermatous cGVHD treated with mTOR-I (everolimus well as the improvement of endothelial barrier functions. n=16; sirolimus n=11). Nine pts had de novo cGVHD, 6 had The similar results observed in GVHD and infl ammatory dis- progressive cGvHD developing from acute GvHD and 12 had eases support the view that GVHD may be considered an cGvHD following donor lymphocyte transfusions. Whereas exaggerated manifestation of a normal infl ammatory mecha- 26 pts had cutaneous scleroderma, one patient developed nism. The cause of TM and EPCR reduction on EC may be oesophageal stenosis. Eight pts received a monotherapy of due to cytokines production. In fact, It has been demonstrated

S123 that TNF-alpha and IL-1 suppress both TM and EPCR on EC outcome of allogeneic stem cell transplantation in leukemia at transcriptional and post-transcriptional levels. Our data sug- patients. In an attempt to replicate this result in unrelated HSCT gest that in GVHD, both anti-coagulant and cytoprotective APC of thalassemia patients, we compared the genotypical distribu- pathway, might play a key role in the control of the process. tion of 3 polymorphisms of the CTLA-4 gene in 71 thalassemia patients and their unrelated donors with the clinical outcomes of the transplantation procedure. A signifi cant association was observed for the CTLA-4/CT60-AA genotype in recipients and the development of Grade II-IV acute graft versus host disease (aGVHD) (48% vs 15%; P=0.0058, OR=5.3) and Grade III-IV aGVHD (60% vs 20%; P=0.02; OR=6.1). In the donors, this analysis did not reach statistical signifi cance (36% vs 25%; P=0.3). Logistic regression demonstrated that the association observed in the patients did not depend upon other known or putative risk factors for Grade II-IV or Grade III-IV aGVHD. In our patient sampling, none of the 3 polymorphisms analyzed were associated with rejection. Overall, the data obtained in this study confi rm that the genetic variability of CTLA-4 is an important prognostic factor for the development of aGVHD after P531 HSCT. More specifi cally, our fi ndings suggest that the recipient Role of the HLA-G 14-base pair deletion-insertion and not the donor genotype is the main risk determinant. Other polymorphism in the development of severe acute studies on larger case reports will be necessary to confi rm this GvHD following unrelated haematopoietic stem cell association and to clarify its functional aspects. transplantation in beta-thalassaemia patients R. Littera (1), E. Piras (1), F. Alba (1), S. Lai (1), A. Vacca (1), L. Cappai (1), G. Caocci (1), C. Giardini (2), F. Locatelli (3), P533 C. Carcassi (1), G. La Nasa (1) The frequencies of T helper 1, T helper 2 in CD4+ T-cells (1)University of Cagliari (Cagliari, IT); (2)S. Salvatore Hospital and plasma interleukin-10 are good biomarkers of (Pesaro, IT); (3)University of Pavia (Pavia, IT) graft-versus-host disease S.-P. Yeh, Y.-M. Liao, C.-F. Chiu, W.-J. Lo, C.-L. Lin Non classical human leukocyte antigen (HLA)-G Class I mol- China Medical University Hospital (Taichung, TW) ecules are encoded by a gene mapped on the short arm of Chromosome 6, telomeric to the classical HLA-A, -B, and -C Objective: Graft-versus-host disease (GVHD) is the major genes. obstacle for successful allogeneic hematopoietic stem cell HLA-G allelic variants may be characterized by a 14-basepair transplantation and T helper1 (Th1), T helper 2 (Th2) and related (bp) deletion-insertion polymorphism located in the 3’-untrans- cytokines are key mediators. However, most of the Th1/Th2 lated (UT) region of the HLA-G gene. The presence of the 14- studies came from animal models and most of the cytokines bp insertion/deletion polymorphism seems to modify immune studies examined cytokine levels only at few time points after tolerance through enhanced synthesis of HLA-G molecules. transplant, which is diffi cult to refl ect the whole picture of real To investigate the impact of this polymorphism on allogeneic clinical situation. This study is designed to see the frequencies hematopoietic stem cell transplantation, we performed a retro- of Th1, Th2 and related cytokines in the whole post-transplant spective analysis of 77 thalassemia patients transplanted from course and to fi nd the correlation between changing levels of an unrelated donor. The donor/recipient pairs were selected for these immune parameters and clinical activity of GVHD. being completely identical for HLA class I and II and the HLA- Methods: Of consecutive 23 patients receiving allotransplant, G and HLA-G 14-bp genotypes. Twenty-fi ve patients devel- peripheral blood was collected weekly from Day 7 to Day oped grade II-IV acute graft versus host disease (aGvHD), 8 210 (patients without graft-versus-host disease) or Day 300 of whom had severe (grade III-IV) aGvHD. Patients that were (patients with chronic graft-versus-host disease). Plasma IL- homozygous for the 14-bp deletion had a higher risk of develop- 4, IL-10, IL-12, and IFN-gamma were determined by ELISA ing aGvHD compared to patients homozygous or heterozygous and the frequencies of Th1 and Th2 within CD4+ T cells were for the 14-bp insertion (13/25 – 52% were -14bp/–14bp). In determined by fl ow cytometry. Total 264 plasma specimens particular, 6 of the 8 severe cases of Grade III-IV acute GvHD and 44 buffy coats from 12 patients were thawed for examina- (75%) were -14bp/–14bp homozygotes (relative risk = 6.0; 95% tions. They were 2 patients of no GVHD, 1 patient who died of Confi dence Interval 1.12 – 32.10; p = 0.04). These data confi rm acute GVHD, 4 patients of both acute and chronic GVHD, and that the 14-bp polymorphism is one of genetic factors involved 5 patients of de novo chronic GVHD. in the development of aGVHD and may serve as a useful tool in Results: The plasma level of IL-10 and IFN-gamma (fi gure 1) predicting HSCT outcomes. and the frequency of Th1, Th2 (fi gure 2) were signifi cantly higher when acute and chronic GVHD developed. Interestingly, even without ex vivo phorbol 12-myristate 13-acetate [PMA] + iono- P532 mycin [I] stimulation, the Th1 and Th2 could be easily detected Recipient CTLA-4 genotype is a prognostic factor for when patients had GVHD and the Pearson correlation test acute GvHD in haematopoietic stem cell transplantation showed a perfect positive relationship between the frequency for thalassaemia of Th1 and Th2 (r=0.966, p<0.001). The dynamic changing S. Orrù (1), A. Vacca (1), N. Orrù (1), G. Caocci (1), L. Martorana pattern of plasma IL-10 was especially correlated well with (1), E. Piras (1), R. Littera (1), A. Ledda (1), C. Giardini (2), the clinical activity of GVHD. Besides, when patients¡¦ GVHD M. Bernardo (3), F. Locatelli (3), C. Carcassi (1), G. La Nasa (1) responded to immunosuppressive treatment, the decrease of (1)University of Cagliari (Cagliari, IT); (2)S. Salvatore Hospital plasma IL-10 level is prompt and is earlier than clinical improve- (Pesaro, IT); (3)University of Pavia (Pavia, IT) ment. The plasma level of IL-4, IL-12, and Th1/Th2 ratio did not signifi cantly correlated with GVHD. (Bars in Figure 1A and Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a member of Figure 2: 95% confi dence interval). the immunoglobulin superfamily and encodes a protein which Conclusion: The frequency of Th1, Th2 and plasma IL-10 level transmits an inhibitory signal to T cells that down-regulates closely related to the clinical course of GVHD. They are good T-cell activation. Polymorphisms of this gene have been asso- biomarkers of GVHD and can be used to monitor the therapeu- ciated with autoimmune diseases and it has recently been tic responsiveness. Furthermore, both Th1 and Th2 likely con- observed that donor genotypes correlate with the clinical tribute to the development of both acute and chronic GVHD.

S124 transplantations 20mg Alemtuzumab (10mg Alemtuzumab day-2 and -1) is suffi cient to achieve comparable results.

P535 Polymorphism in the fractalkine receptor CX3CR1 as a genetic risk factor for cutaneous acute graft-versus-host disease N. Dhédin, A. Ghadiri, M. Uzunov, S. Nguyen, J.P Vernant, P. Debré, C. Combadière Pitié-Salpêtrière (Paris, FR)

Background: Chemokines and chemokine receptors play criti- cal roles in migration of alloreactive donor T cells into graft- versus-host disease (GVHD) target organs. Recently, some published data have suggested a potential role of the chemok- ine fractalkine (CX3CL1) and its receptor CX3CR1, in a murine model of intestinal GVHD. (Ueha 2007). The aim of this study is to evaluate, in humans, the impact of the CX3CR1 genetic polymorphism in the occurence GVHD after allogeneic stem cell transplantation. Patients and methods: Single nucleotide polymorphisms of CX3CR1 were identifi ed by quantitative polymerase chain reac- tion (Taqman) in 99 patients transplanted from a HLA identical sibling donor. Twenty fi ve percent of the patients were hetero- zygous or homozygous for the I249 mutation and 14% for the M280 mutation. Median age at transplant was 50 years (range: 17-69). Forty percent of the patients had an advanced disease at the time of transplant. Fifty percent of the patients received a reduced conditioning regimen; the source of stem cell was peripheral blood in 56% of patients. Results: The incidence of grade II-IV and III-IV acute GVHD (aGVHD) were respectivelly of 45% et 16%; 41% of the patients P534 presented a cutaneous aGVHD and 17% an intestinal aGVHD. Low-dose alemtuzumab as GvHD-prophylaxis in matched In univariate analysis, patients with I249 or M280 mutations of and mismatched allogeneic sibling and unrelated donor CX3CR1 had a lower incidence of grade III-IV aGVHD (4% ver- transplantation sus 21% in the remaining patients p=0.04) and of cutaneous H. Bertz, A. Zerweck, D. Räpple, R. Waesch, R. Marks, aGVHD (23% versus 49% in the remaining patients p=0.02). In J. Finke univariate analysis, the only other factor signifi cantly associated Albert Ludwigs University Medical Center (Freiburg, DE) with the incidence of cutaneous aGVHD was the pre-transplant donor/recipient CMV serology. Impact on cutaneous aGVHD, Proposal: For GvHD prophylaxis we introduced low dose Ale- of CX3CR1 polymorphism remained signifi cant in multivariate mtuzumab (MabCampathTM) in combination with Cyclosporine analysis (p=0.03. HR= 0.39; SE: 0.44). A in matched (10mg Alemtuzumab) and HLA A, B and DRB1 Conclusion: These results suggest that fractalkine and its mismatched (20mg Alem- tuzumab) sibling or unrelated donor receptor CX3CR1 play a role in in the occurrence of aGVHD in alloHCT. To evaluate the effi ciency of these low doses of Ale- human and that CX3CR1 genetic polymorphism is associated mtuzumab in both groups, we compared the doses for the inci- with lower incidence of cutaneous aGVHD. dences of acute and chronic GvHD and reasons for death. Patients: Overall, 214 pts. (group 10mg n= 181; group 20mg n=33) received after a mainly fl udarabine-based reduced inten- P536 sity conditioning (group 10mg 87%; group 20mg 81%) a graft Assessment of liver damage with transient hepatic (PBSC in 99%) from a sibling (22%; 0%;) or UD (78%; 100%;). elastography (FibroScan) in patients with chronic Diagnosis were AML/MDS (72%; 67%;) or lymphoma/MM graft-versus-host disease (10%; 21%). In the mismatch-group n=25 had one and n=8 had C. Skert, A. Peli, K. Prestini, L. Biasi, C. Filì, M. Malagola, two major mismatches. Remission at TX was mainly advanced C. Bergonzi, M. Mendeni, A. Roccaro, M. Puoti, D. Russo disease stage/>CR1 (85%; 100%), also refl ected by the fact University of Brescia (Brescia, IT) that within both groups 59% and 66% had persistent induction failure or relapse. Median age was 59 (20-76) and 53 (23-74) Hepatic chronic GVHD (cGVHD) develops generally as an years, respectively. indolent cholestatic disease or as an autoimmune hepatitis, Results: In the day +30 diagnostic procedures 90% and 86% of being often refractory to immunosuppressive therapy. Liver the patients achieved CR; acute GvHD °II-°IV was seen in 27% biopsy is helpful to confi rm diagnosis, but it is not always feasi- and 33% and °III-°IV in 11% and 12%; limited (24%; 25%) and ble because of possible clinical complications. FibroScan is a extensive (16%; 21%) cGvHD show as well comparable results new, fast and non-invasive technique to measure liver stiffness. in both groups. Alive are 60% and 64% at a median of 375 days FibroScan has been validated to detect signifi cant positive rela- (8-893); main cause of death was relapse/progression (22%). tionship between liver stiffness values and histological fi brosis The non relapse mortality (NRM) was 18% in both groups; 16% in chronic liver diseases. Being fi brosis a hallmark of cGVHD, of all patients died due to infections/MOF and fi ve patients died we prospectively evaluated the usefulness of FibroScan as due to aGvHD (2%). None of the UD HCT recipients died from non-invasive tool to make diagnosis of liver cGVHD. acute GvHD. Liver stiffness measurements were performed on 10 healthy Conclusion: GvHD-prophylaxis with CsA and Alemtuzumab one subjects and 20 patients undergoing allogeneic SCT before single 10mg dose at day-1 absolutely is suffi cient and feasible and transplantation, then every 3 months and at the diagnosis of does show low incidences of °III-°IV aGvHD, extensive cGvHD cGVHD. One patient was HCV RNA-positive. No patients had and aGvHD-associated NRM. In HLA A,B or DRB1 mismatched any sign of liver disease at transplantation.

S125 In FibroScan, only procedures with at least ten successful Conclusion: Delayed engraftment and immune recovery were acquisitions and a success rate of at least 60% were consid- associated with the prolonged presence of C1H compared with ered reliable. The median value of successful measurements ATG. Low levels of ATG or C1H were associated with increased was considered representative of the liver stiffness in a given risk of aGvHD. Our results demonstrate the importance of mon- patient, only if the interquartile range (IQR) of all validated itoring levels of active ATG and C1H around SCT and during measurements was less than 30% of the median value. Wil- the fi rst two months thereafter. coxon test was used for statistical analysis. Nine patients developed cGVHD (67% extensive) at a median time of 5 months (range, 4-6). Liver was involved in 67% of P538 patients. Liver stiffness values did not differ signifi cantly in Kinetics of active and total thymoglobulin in paediatric healthy subjects, patients before allogeneic SCT and patients stem cell transplantation after allogeneic SCT without cGVHD. Patients with hepatic C.M. Jol-van der Zijde, A.M. Jansen-Hoogendijk, S. Raaijmakers, cGVHD had higher stiffness values than healthy subjects (7,7 ± R. Egeler, A.C. Lankester, R.G.M. Bredius, M.J.D. van Tol 4,6 vs 4,6 ± 1,5 kPa, p=0,03) and than patients without cGVHD Leiden University Medical Centre (Leiden, NL) (7,7 ± 4,6 vs 4,6 ± 1,9 kPa, p=0,03). Furthermore, liver stiffness values were higher in all patients with cGVHD than in healthy Introduction: Polyclonal anti-thymocyte globulin (ATG, Imtix) is subjects (7 ± 3,9 vs 4,6 ± 1,5 kPa, p=0,04) and patients without given prior to paediatric stem cell transplantation (SCT) to pre- cGVHD (7 ± 3,9 vs 4,6 ± 1,9 kPa, p=0,04). vent rejection and Graft-versus-Host disease (GvHD). Our prospective study suggests that FibroScan could be a Material and methods: In this study the course of the ATG con- reliable non-invasive technique for the early diagnosis of centration was measured in a cohort of 39 paediatric patients hepatic cGVHD. However, it was not possible to demonstrate who received their fi rst SCT at the department of Paediatrics of signifi cantly different stiffness values in the setting of cGVHD the Leiden University Medical Centre (LUMC) between August between patients with and without hepatic involvement. The ’06 and July ’08. All patients received 10 mg/kg divided over small number of patients included in the survey or a clinically 4 days, starting between 6 and 4 days prior to SCT. Unique silent involvement of liver in all cases of cGVHD may account in this study was that serum samples were taken frequently for this observation. before and after every ATG dose and continued until 3 months post SCT. The active ATG, capable of binding to T-cells, was measured by FACS, whereas the total amount of rabbit IgG P537 was measured by ELISA techniques. Serotherapy in paediatric allogeneic stem cell Results: It was found that the ATG concentration showed peak transplantation: comparison of ATG and Campath-1H as and off-peak values between every ATG dose. There was a part of conditioning huge variation between the individual patients, the median total C.M. Jol-van der Zijde, A.M. Jansen-Hoogendijk, R. Egeler, ATG concentration that was reached after the fi nal dose was A.C. Lankester, R.G.M. Bredius, M.J.D. van Tol 80 ug/ml with a range of 52 – 124 ug/ml, peak values after the Leiden University Medical Centre (Leiden, NL) subsequent doses were respectively 30-48-62 and 80 ug/ml. the active ATG concentration 12.0 U/ml with a range from 5.1 Polyclonal anti-thymocyte globulins (ATG) and monoclonal to 25.4 U/ml. humanized Campath-1H (C1H) are regularly administered It was found that there is no good correlation between the before allogeneic stem cell transplantation (SCT), to prevent amount of active ATG and total rabbit-IgG in a patient over the rejection and acute Graft versus Host Disease (aGvHD), different time points pre and post SCT, therefore FACS is the respectively. The dose of ATG and C1H is solely based on the most reliable method to measure the concentration of active total body weight (BW) of the patient. High concentrations of ATG. ATG or C1H post-transplant may delay immune reconstitution Although all patients received ATG in a dose of 10 mg/kg a sig- and may have an impact on cellular immunotherapy, scheduled nifi cant difference (P=0.002) was found between the patients early after SCT. In this single centre study serum levels of ATG with a bodyweight below or above 30 kg, therefore there could as well as C1H were related to immune recovery and aGvHD. be a risk of overdosing in this last group. Between April 2004 and October 2007, 104 children received The clearance of active ATG is much faster than the clearance their fi rst SCT. In 21 of them no serotherapy was used in of total rabbit-IgG with half-lifes of 2-7 days and 5-30 days the conditioning regimen, in 47 patients ATG (Imtix) and in respectively. In the fi rst days after ATG administration the half- 36 patients C1H (Alemtuzumab) was applied. ATG patients life of the active ATG is even faster. received a total dose of 10 mg/kg over 4 days, the total dose Patients with a low ATG concentration at the moment of trans- of C1H varied from 0.6 to 1 mg/kg over 3-5 days. Active ATG plantation were found to have a higher risk to develop GvHD, and C1H concentrations were measured in frequently collected some patients with high ATG concentration showed an impaired sera with a quantitative fl owcytometry assay (FACS), using the T-cell recovery. HUT78 T-cell line as target cells. Results were correlated to Discussion: It would be ideal to develop a pharmacodynamical pre- and post- transplant parameters. Immunophenotyping of model for the dosing of ATG in individual patients, but further lymphocytes (Ly) by FACS was done weekly. research with more patients is needed to make that possible. The clearance of ATG was much faster than of C1H. ATG treated patients reached the sub-therapeutic level of 1 U/ml at day +12 (range 2–41 days), whereas in C1H patients this level being 0.1 µg/ml was reached at day +37 (range 3-70 days). ATG treated patients engrafted faster and showed an earlier immune recovery of Ly and especially of T-cells than C1H treated patients. The serum ATG concentration at day of SCT did correlate with T cell recovery but not with engraftment. The serum ATG level after the fourth dose correlated with the total dose given, point- ing to the relative higher dosing in patients with a higher BW. The C1H dose given (total and per kg) correlated with the C1H concentration at day +20. A high C1H concentration at day +20 correlated with a delayed engraftment and recovery of Ly, T- and NK-cells. Patients with a C1H rest concentration below 1 µg/ml already at day +10 post SCT had a increased risk of aGvHD.

S126 accordance with NIH criteria (ref). Histopathological fi ndings included lichenoid infl ammation, epithelial apoptosis, ulcera- tion and fi brosis. The clinical diagnosis of probable cGvHD was confi rmed as probable or possible in biopsies of 15/17 cases, whereas in the group of pts clinically cathegorized as possible cGvHD 8/14 were graded as probable or possible. Depression as assessed by Beck Depression Inventory and sexual dysfunction as estimated by Female Sexual Distress Scale were established in 29% and 45% of pts, respectively. Conclusion: The prevalence of genital cGvHD after allo-grafting is unexpectedly high and appears to be associated with sexual dysfunction and mental depression. We hypothesize that early gynecological intervention may reduce the risk of severe symp- toms and sequelae, and propose that gynecological expertise should be part of the team around the allografted woman.

P540 Antithymocyte globulins as part of the myeloablative conditioning regimen can reduce the risk of severe graft-versus-host disease after allogeneic stem cell transplantation from matched-unrelated donors M. Mohty, M.L. Balère, G. Socié, N. Milpied, N. Ifrah, M. Kuentz, J.L. Harousseau, J.P. Vernant, M. Michallet, A. Buzyn, J.-Y. Cahn, J.H. Bourhis, D. Blaise, C. Raffoux, H. Esperou, I. Yakoub-Agha for the Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC)

Here, we report the results of a multicenter retrospective study analyzing the effect of ATG, incorporated within the MAC regimen for MUD-transplants in leukemic patients. The pur- pose of the study was to compare the incidence and severity of acute and chronic GVHD as well as overall outcome. 171 adult patients with acute leukemia and MDS, for whom detailed allelic HLA typing (4 digits) was available, were included. 81% of patients were transplanted from 10/10 allelic MUD, and 19% P539 from a MUD with at least one allelic difference. 120 patients Prevalence of genital graft-versus-host disease after (70%) did not receive ATG (no-ATG group), while 51 patients allogeneic stem cell transplantation received ATG (ATG group; Thymoglobuline* in all cases) as E. Smith Knutsson (1), A-K. Broman (1), Y. Björk (2), part of the MAC regimen. Except for a signifi cantly higher D. Hallberg (2), L. Helström (3), O. Nilsson (2), K. Sundfeldt (2), number of allelic differences between recipient and donor M. Styrenius (1), M. Brune (2) (33% vs. 13%; P=0.002), the no-ATG and ATG groups were (1)Northern Älvsborg Hospital (Trollhättan, SE); (2)Sahlgrenska strictly comparable. With a median follow-up of 30.3 (range, University Hospital (Göteborg, SE); (3)Södersjukhuset 2.6-68.1) months, grade 0-1 and 2-4 acute GVHD occurred in (Stockholm, SE) 74 (46%) and 88 patients (54%) respectively, with grade 3-4 acute GVHD being signifi cantly lower in the ATG group (18% Objectives: Chronic Graft-versus-Host Disease (cGvHD) is vs. 32%; P=0.04). Limited and extensive chronic GVHD were a recognized but unsatisfactorily described cause of geni- observed in 22 and 25% of assessable patients respectively, tal complications in female patients after allogeneic stem cell with extensive chronic GVHD being signifi cantly lower in the transplantation (SCT). In a population-based study, we have ATG group (5% vs. 33%; P=0.001). Interestingly, patients from assessed the prevalence, clinical features and histopathology the ATG group had a higher incidence of limited chronic GVHD of genital cGvHD. (33% vs. 18%; P=0.06). Moreover, infection-related mortality Methods: All women alive after allo-SCT 1996-2005 (n=53) was comparable between both groups (23% vs. 27%, P=NS). in the Western region of Sweden were invited to participate Also, NRM was comparable between both groups (30% vs. in a study comprising (i) gynaecological examination includ- 29%; P=NS). In multivariate analysis, an HLA allelic mismatch ing photo-documentation for the clinical diagnosis of genital and the non-use of ATG were associated with an increased risk cGvHD, (ii) biopsies for pathological examination and (iii) struc- of grade 3-4 acute GVHD (RR=2.80, 95%CI, 1.5-5.3, P=0.001; tured anamnesis and validated questionnaires for assessment and RR=2.4, 95%CI, 1.1-5.0, P=0.02 respectively). Similarly, of depression and sexual dysfunction. To exclude estrogen multivariate analysis showed that the absence of use of ATG defi ciency, all pts recevied local estrogen before cGvHD diag- was the unique parameter associated with an increased risk of nosis. extensive chronic GVHD (RR=6.9; 95%CI, 1.7-29.0, P=0.008). Results: Of 53 consecutive pts, 44 (83%) were examined with Finally, LFS and OS at 2 years were not signifi cantly different a minimal follow-up of 3 years post-transplant. Median age between the no-ATG and ATG group (48.8% vs. 41.3%, P=NS; was 47 (26-72) yrs and post-SCT 6 (3-12) yrs. Symptoms of and 53.6% vs. 54.3%, P=NS; respectively). genital cGvHD included dryness, smart, pain and dyspareunia; These results suggest a global long-term benefi cial effect of signs were dry, thin, sore mucosa, local white and red spots, ATG when used as part of the MAC regimen prior to allo-SCT lichenoid patterns, synechiae, stenosis and painful vaginal from MUD (especially in the HLA mismatch setting). Such strings. Clinical signs were categorized as probable, possible protective effect of ATG against severe GVHD can be likely or unlikely cGvHD. Probable genital cGvHD was diagnosed in achieved without an increased risk of infections or leukemia 17 pts (39%) of whom 13 had vaginal stenosis or adhesions recurrence. and 3 women had vulvar synechiae. Possible cGvHD was observed in another 14 pts. Biopsies were obtained from 34 pts and histopathological diagnosis of cGvHD was made in

S127 P541 alloHSCT are increasingly evaluated, little is known about suit- Chronic GvHD of the lung signifi cantly impairs quality able measurements for the physical performance and its impact of life and the activity profi le – Results of a prospective on the mental status. German multicentre validation trial Methods and Patients: 41 patients receiving an alloHSCT for D. Wolff (1), P. Herzberg (2), P. Heussner (3), F. Mumm (3), hematologic malignancies (n=40) or aplastic anaemia (n=1) I. Hilgendorf (4), S. von Harsdorf (5), A. Gerbitz (6), H. Greinix were evaluated for physical functioning before (t1), one month (7), G. Hildebrandt (1), C. Junghanss (4), E. Holler (1) (t2) and 3 months (t3) after alloHSCT. The median age was 45 (1)University of Regensburg (Regensburg, DE); (2)University years (range 16-69 yrs). The conditioning regime was standard of Leipzig (Leipzig, DE); (3)Ludwigs-Maximilians-University myeloablative (n=9), toxicity reduced myeloablative (n=30), or (Munich, DE); (4)University of Rostock (Rostock, DE); non-myeloablative (n=2). Twenty one patients developed no (5)University Hospital Ulm (Ulm, DE); (6)Charité – University acute GVHD (aGVHD) and ten patients developed aGVHD Hospital Berlin (Berlin, DE); (7)Medical University of Vienna grade 1-2 (n=4), grade 3-4 (n=6). Six patients died from GVHD (Vienna, AT) (n=2), relapse (n=2) or infection (n=2). The grip strength test was performed using a JAMAR dynamometer, 13 muscle The NIH staging and response criteria offer for the fi rst time groups were tested with the CITEC dynamometer, conditional the chance for uniform documentation of chronic graft-versus- status was tested with the two minute walk test. QOL was host disease (cGVHD) but require prospective evaluation with assessed using the HAP, the SF36, the HADS, the FACT-BMT, regard to clinical relevance and impact on QoL. We present the the BFI and the MFIS questionnaires. results of a German multicenter trial on the NIH staging criteria Results: A signifi cant loss of grip strength (t2) (p=0.005) and a in cGVHD. One-hundred-forty-seven patients (median age 44 signifi cant (p=0.04) lower grip strength in patients with GVHD years, range 18-64) after allogeneic hematopoietic stem cell (t3) was detected. The median muscle strength (t3) (in % of transplantation for hematologic malignancies were evaluated t1) of the upper limb was 106% (75-132%) and for the lower according to the NIH criteria based cGVHD activity assess- limb 103% (77-130%) for patients without GVHD and 72% ment, the Lee chronic GVHD Symptom-Scale (L-cGVHD-SC), (43-186%) of the upper and 88% (63-171%) of the lower limb FACT-BMT, HAP (human activity profi le), SF36, Berlin Social for patients with GVHD (no GVHD vs. GVHD for upper limb Support Scale (BSSS), 24 Item Adjective Measure (24–AM), p=0.04). The walking distance (t2) median 152 m (60-240) was HADS, and the NCCN-Distress-Thermometer. Enrolment signifi cant shorter compared to (t1) median 165 m (96-231) occurred between day 100 and 1 year after HSCT or in the (p=0.004). Patients with GVHD had signifi cant higher scores for presence of active cGVHD also at later time points. Follow-up anxiety (p=0.02), depression (p=0.004) and fatigue (p=0.009) surveys were conducted at 1, 2, 3, 5, 8, 12 and 18 months after (t3). The depression values correlated inversely with the HAP baseline survey. At all time points disease status, comorbidities adjusted activity score (AAS) (t1) (r=-0.6, p=0.0001), (t2) (r=- and medication were documented. Ninety-eight patients had 0.5, p=0.04), (t3) (r=-0.6, p=0.005). The fatique sum score (BFI) cGVHD (mild n=30, moderate n=43, severe n=25) including inversely correlated with the AAS (t1) (r= -0.4, p=0.01), (t2) (r=- patients with mild (n=18), moderate (n=11) and severe (n=1) 0.4, p=0.05), t3 (r=- 0.7, p=0.0001). bronchiolitis obliterans (BO). Forty nine patients had no cGVHD. Conclusion: AlloHSCT leads to a loss of physical function- Multiple regression analysis revealed that severity of cGVHD ing especially during time of hospitalisation. The presence of (ß=0.27, p=0.01), grade of distress measured by the distress GVHD leads to pronounced physical impairment and psycho- thermometer (ß=0.28, p<0.01) and extraverted personality (ß= logical distress like depression and fatigue. Strategies to care -0.27, p<0.01) signifi cantly correlated with QoL as measured for the physical impairment as well as the psychological status by the FACT-BMT. The HAP-maximum activity score correlated of the patients are essential. inversely with severity of cGVHD (ß= -0.25, p=0.015). Male sex (ß=0.24, p=0.02) as well as conscientious personality (ß=0.24, p=0.02) correlated positively with the HAP-maximum activity P543 score. Evaluation of the impact of specifi c organ manifestations Prophylaxis with mycophenolate mofetil and cyclosporine of cGVHD on QoL and the activity profi le detected a signifi cant can decrease the incidence of severe acute GvHD negative impact of BO on the activity profi le (p<0.01) as well as following reduced-intensity conditioning allogeneic stem QoL (p<0.001) already at an organ stage grade 2 which was cell transplantation from matched unrelated donors not detectable in specifi c other organ manifestations. BO cor- E. Brissot, P. Chevallier, T. Guillaume, J. Delaunay, S. Ayari, related signifi cantly (p<0.01) with impairment in the breath, eye, V. Dubruille, S. Le Gouill, B. Mahe, T. Gastinne, N. Blin, and energy subscale of the L-cGVHD-SC as well as with eye B. Saulquin, P. Moreau, J.L. Harousseau, M. Mohty involvement of cGVHD (p=0.02). The results demonstrate, that CHU Hôtel-Dieu (Nantes, FR) severity of cGVHD as assessed by the NIH consensus grad- ing correlates with impairment of physical functioning as well Acute GVHD remains a matter of concern after RIC allo-SCT, as QoL. The special weight of BO grade 2 in the overall grad- especially when using HLA-matched unrelated donors (MUD). ing of cGVHD is justifi ed by its impact on QOL and the activity Thus, the rapidly increasing use in elderly and high risk patients profi le. and evolving nature of RIC allo-SCT, emphasize the need for renewed clinical research of GVHD prophylaxis. This pilot report investigated Prophylaxis with mycophenolate mofetil P542 (MMF) and cyclosporine (CsA) combination in comparison to GvHD associated impairment of physical functioning, CsA alone for GVHD prophylaxis in 35 consecutive patients depression, and anxiety. Results from a prospective trial with hematological malignancies receiving RIC allo-SCT from a on physical and psychological functioning in patients MUD, and treated within the same period in a single institution. before and after allogeneic haematopoietic stem cell In this series, the median age was 52 years (range, 6-64; 20 transplantation males). The RIC regimen included fl udarabine, busulfan and M. Viehweg (1), P. Herzberg (2), H. Andree (1), I. Hilgendorf (1), ATG in all patients (with minor adjustments in 3 patients). The fi rst M. Leithäuser (1), C. Junghanss (1), J. Casper (1), M. Freund patients from this series (n=19; “CsA” group) received CsA alone (1), D. Wolff (3) for GVHD prophylaxis. The next patients (n=16; “CsA+MMF” (1)University Rostock (Rostock, DE); (2)University Leipzig group) received CsA and MMF. MMF was given at a fi xed oral (Leipzig, DE); (3)University Regensburg (Regensburg, DE) dose of 1 g x 2/day without any treatment adjustment to blood levels. The two groups showed no signifi cant differences as for Introduction: Allogenic hematopoetic stem cell transplantation demographic and disease features, GVHD risk factors, and allo- (alloHSCT) is associated with functional impairment and psy- SCT procedure characteristics (except for GVHD prophylaxis). chological stress. Although quality of live (QOL) issues after 31 patients (89%) received a 10/10 HLA matched graft at the

S128 allelic level. One allelic mismatch was observed in 4 cases, with Conclusions: Presence of the KIR2DS3 gene in the donor is this being comparable between the two groups. 3 patients from a potential risk factor for HSCT. Prospective KIR genotyping this series did not engraft (early disease recurrence) and were should be performed to exclude this gene in future HSCT donor not evaluable for acute GVHD. ANC >500/µL was achieved at selection. a median of 19 (range, 0-27) days. The cumulative incidence of grade 2-4 acute GVHD at day 100 was 67% (95%CI, 45-89%) in patients receiving CsA alone as compared to 53% (95%CI, 28- P545 78%) in those receiving the CsA and MMF combination (P=NS). Vascular endothelial growth factor gene polymorphisms However, the cumulative incidence of grade 3-4 acute GVHD may predict the risk of acute graft-versus-host disease was signifi cantly lower in the “CsA+MMF” group as compared to following allogeneic transplantation: preventive effect of the “CsA” group (13% vs. 56%; P=0.03). After a median follow- VEGF on acute GvHD up of 19.5 (range, 9-93) months for surviving patients, the one D.H. Kim (1), N.Y. Lee (2), M.H. Lee (2), S.K. Sohn (2), J.H. Jang year overall survival was signifi cantly higher in the “CsA+MMF” (1), K. Kim (1), S.J. Kim (1), W.S. Kim (1), C.W. Jung (1) group (81% vs. 47%; P=0.03). Overall, 4 deaths were attributed (1)Samsung Medical Center (Seoul, KR); (2)Kyungpook to refractory acute GVHD in the “CsA” group versus none in the National University Hospital (Daegu, KR) “CsA+MMF” group. Though a randomized trial is needed before drawing fi nal con- Background: Microvessel injury is associated with the develop- clusions, this pilot study suggests that adjunction of MMF to ment of graft-versus-host disease (GVHD), whereas high levels CsA can result in a signifi cant reduction of the incidence of of post-transplant vascular endothelial growth factor (VEGF) severe acute GVHD following ATG-based RIC allo-SCT using have a protective effect on severe acute GVHD (aGVHD) and MUD, and this may have a signifi cant impact on the probability transplant-related mortality. The current study aimed to deter- of a favorable outcome. mine the impact of Vascular endothelial growth factor (VEGFA) gene single nucleotide polymorphisms (SNPs) on the risk of acute GVHD following allogeneic stem cell transplantation P544 (SCT). Killer immunoglobulin-like receptor (KIR) 2DS3 gene and Methods: Four VEGF gene SNPs were analyzed in 98 survival outcome in identical HLA-matched sibling adult recipients using PCR/RFLP, i.e., -2578 C>A (rs699947), - haematopoietic stem cell transplantation 460 T>C (rs833061), +405 G>C (rs2010963) and +936 C>T J.A. Davidson (1), G. Lucas (1), J.A.L. Yin (1), E. Liakopoulou (rs3025039). (2), K.V. Poulton (1) Results: Strong linkage disequilibrium was noted between loci - (1)Manchester Royal Infi rmary (Manchester, UK); (2)Christie 2578, -460 and +405, but not between these and +936. Accord- Hospital (Manchester, UK) ingly, 4 haplotypes were generated based on the genotypes of -2578, -460, and +405 as follows: CTC (47.9%), CTG (26.7%), The objective of this study is to investigate the infl uence of the ACG (24.2%), and CCC (1.0%). The group with low produc- Killer Immunoglobulin-like Receptor (KIR) gene KIR2DS3 in ibility of VEGF (i.e., +936CT genotype and 2 copies of the ACG Haemopoietic Stem Cell Transplantation (HSCT). HLA and KIR haplotype) showed a higher incidence of acute GVHD: A sig- genes are located on different chromosomes so genetic identity nifi cant association was noted between the risk of grade 2-4 is not guaranteed. Thus the KIR gene and MHC-CI ligand may aGVHD and the + 936 CT (p=0.006), the -2578 AA (p=0.003), be mismatched even in HLA-identical related transplants. As and the -460 CC (p=0.002) genotypes, and the ACG haplotype yet the ligand for KIR2DS3 is unknown. (p=0.003). No association was observed between the VEGFA The study population included 177 consecutive HSC trans- gene SNPs and chronic GVHD. plants for adult HLA-identical recipient and sibling donors per- Conclusion: The VEGFA gene SNPs might predict a lower risk formed between January 1998 and July 2005. of acute GVHD. The current result suggests that VEGF might The method used to identify the KIR genes was molecular play a protective role in the pathogenesis of acute GVHD. genotyping by Luminex LABType® PCR-SSO system. Data were analysed by correlation to established clinical endpoints of HSCT outcome. Kaplan-Meier analysis was performed to P546 enable comparison of overall survival rates, Cox Regression The successful treatment of steroid-refractory liver analysis used to allow for covariates in our model, determining graft-versus-host disease with pulse cyclophosphamide: their signifi cance and relative risk. Cross tabulation tables were excellent long-term results used to analyse the relationship and signifi cance between vari- M. Krejci, J. Mayer, Y. Brychtova, M. Doubek, Z. Racil, ables by Chi-square or Fisher’s Exact Test. Z. Koristek, M. Navratil, M. Tomiska, J. Vorlicek Results: Presence of 2DS3 in the donor adversely infl uenced University Hospital Brno (Brno, CZ) overall survival (OS) from 65% if negative for the gene to 45% when positive for the gene (p = 0.02, RR 1.3). To identify a Background: Corticosteroid-resistant acute GVHD is diffi cult to potential ligand for KIR2DS3 investigations into the HLA type of manage, and is associated with high morbidity and mortality. No the recipients transplanted with 2DS3 positive donors were per- standard treatment exists. We had previously seen encourag- formed. Although no signifi cant alleles of interest were found, ing results with pulse cyclophosphamide (Cy) in the treatment poorer outcome was seen in C1 group positive individuals. A of liver GVHD in contrast to gastrointestinal GVHD, and we search was made of the common HLA-Cw allele sequences in report here the long-term results of pulse Cy for the treatment an attempt to fi nd shared epitopes in those recipients whose of steroid-refractory liver GVHD, with no association to the gut. survival outcome was reduced. Sites were identifi ed but none Methods: This is a retrospective study of 21 patients (pts) with were of signifi cance as possible ligands. Combined factors; hematological malignancies after allogeneic stem cell trans- presence of KIR2DS3 in the donor and recipient CMV serosta- plantation. Twelve pts had acute GVHD (2 pts grade I, 3 pts tus were analysed and showed signifi cant improved OS of 76% grade II, 7 pts grade III), 4 pts had chronic extensive GVHD when both parameters were negative compared to all other and 5 pts developed liver GVHD upon DLI. Two pts had only combinations when OS was reduced by up to 33% (p = 0.025). liver GvHD, 19 pts had GVHD with involvement of liver and/or Signifi cantly the presence of KIR2DS3 in the donor and sever- oral mucosa, and/or skin. All pts had hepatitic variant of liver ity of chronic GvHD showed that recipients experiencing mild GVHD (serum aminotransferase ALT or AST elevation > 10 chronic GvHD, regardless of donor 2DS3 status, have an OS of times the upper normal limit), classical GVHD with elevation 73%, but the presence of the 2DS3 gene in the donor reduces of bilirubine was observed at 9/21 pts. All pts were treated by OS from 57% to 20% in the presence of severe chronic GvHD cyclosporine A and steroids in dose 2 mg/kg before pulse Cy, (p = 0.024). fi ve pts had another previous therapy (mycophenolate mofetil,

S129 alemtuzumab). Steroid-refractory GVHD was defi ned as the P548 lack of response to steroids administered for at least 5 con- Extracorporeal photopheresis for the treatment of secutive days. steroid-resistant acute graft-versus-host disease Cy was infused at a dose of 1000 mg/m². Twenty-nine Cy S. Fritsch, J. Tischer, G. Ledderose, B. Maier, R. Reibke, administrations were given in 21 pts. The median time of GVHD A. Rank, H.J. Kolb onset and Cy administration after transplantation, or DLI, were Ludwig-Maximilians-University (Munich, DE) 58 and 69 days, respectively. Results: Eleven pts (52%) achieved CR and 6 pts (29%) Acute graft versus host disease (GvHD) still remains one major achieved PR. Four pts (19%) did not respond, however their cause of morbidity and mortality following hematopoietic stem condition stabilized and upon additional therapy, 3 achieved PR cell transplantation, especially in patients, who do not respond and one CR. Leukopenia and/or thrombocytopenia WHO grade to corticosteroid therapy. 4 developed after 5 Cy pulses, it was usually short-lived with a We report a retrospective analysis using extracorporeal pho- median of 7 days. Twelve infectious complications occurred in 8 topheresis (ECP) for second line treatment of steroid refrac- of 21 pts, all of them resolved after antimicrobial therapy. Neither tory acute GvHD in 30 patients. 8 patients (26,7%) received other signifi cant toxicity after Cy pulse nor infl uence of pulse Cy hematopoietic stem cells from a sibling HLA-identical donor, therapy to chimerism or disease status were observed. 10 patients (33,3%) from an unrelated HLA-identical donor, Three pts died, all deaths were without direct relationship to 2 patients (6,7%) from a sibling mismatched donor and 10 pulse Cy. Overall survival of all 21 pts is 86% with median and patients (33,3%) from a mismatched unrelated donor. Refrac- maximal follow-up of 33 and 81 months, respectively. toriness to corticosteroids was defi ned as progression or per- Conclusion: Pulse Cy is an effective treatment for steroid-refrac- sistence of GvHD after treatment with methylprednisolone 1-2 tory liver GVHD with overall response rate 81% and remarkably mg/kg every 8 hours over a period of 3 days. Median duration good toxicity profi le, which may favor its use instead of drugs of ECP was 54 days (range 8 – 227 days) with a frequency of with more pronounced immunosuppressive effects. two applications per week until clinical response and every sec- ond week thereafter. 9 out of 30 patients (30,0%) suffered from acute GvHD grade 2, 11 patients (36,7%) from grade 3 and 10 P547 patients (33,3%) from grade IV. Clinical signs of acute GvHD A phase II clinical trial using extracorporeal occurred after a median time of 20 days post transplantation photophoresis for chronic GvHD: evidence for (range 9 – 160 days). Median duration from onset of acute immune switching GvHD until start of ECP was 18 days (range 1 – 102 days). U. Holtick (1), X.N. Wang (2), S.R. Marshall (2), M. von Bergwelt- Complete response (CR) was defi ned as discontinuation of Baildon (1), C. Scheid (1), A. M. Dickinson (2) corticosteroid therapy following ECP, partial response (PR) as (1)University Hospital Cologne (Cologne, DE); (2)Newcastle reduction to a maximum of 10mg per day and minor response University (Newcastle upon Tyne, UK) (MR) was reduction by 50%. No possibility of steroid reduc- tion or a reduction less than 50% was named no response Graft versus Host disease (GvHD) is the major limitation to suc- (NR). Using these criteria 11 patients (36,7%) reached a cessful allogeneic haematopoietic stem cell transplantation and CR, 9 patients (30,0%) a PR and 4 patients (13,3%) a PR. contributes signifi cantly to transplant related mortality and mor- 6 patients (20,0%) did not respond (NR). Transplant related bidity. Steroid refractory or steroid dependent GvHD in particular mortality (TRM) on day 100 was 6,7% (2 out of 30 patients). is linked to poor survival and poor quality of life. Conventional One additional patient died because of relapse. One year after immunosuppression has limited success in these conditions transplantation 11 of 25 evaluable patients (44,0%) have died and increases susceptibility to infection and relapse. because of transplant related complications. Based on our four Extracorporeal Photopheresis (ECP) is a promising therapy for response groups the TRM after one year was 9,1% in the CR acute and chronic GvHD in patients not responding to conven- group, 22,2% in the PR group, 50,0% in the MR group and tional immunosuppression. ECP mechanisms are still poorly 100% in the NR group respectively. Overall survival (OS) after understood and although a number of studies using ECP treat- a follow up time between 45 and 994 days was 53,3% and ment for conditions including acute and chronic GvHD have median OS 304 days. been published, there is limited data on the variations in blood In conclusion ECP is a promising and valuable therapeutic cell subsets post ECP therapy. option for treatment of patients with steroid refractory GvHD. To In a phase II clinical trial, ten patients with steroid refractory assess the real benefi t a randomized prospective trial should chronic GvHD were treated with ECP. Monocyte, T cell, NK be performed. cell and dendritic cell subsets were analysed by fl ow cytometry prior to ECP and, at cycle two, six and three months after the end of ECP treatment. The overall response rate to ECP in this P549 study was 70% (3 Complete responders, 4 partial responders Extracorporeal photopheresis in the management of and 3 non-responders). steroid-refractory chronic graft-versus-host disease after A statistically signifi cant increase (p<0.05, Mann-Whitney test) allogeneic stem cell transplantation during ECP treatment and follow-up could be detected for total P. Kaloyannidis, A. Papalexandri, I. Sakellari, E. Yannaki, numbers of CD4+ T cells, regulatory T cells (CD4+ CD25bright I. Batsis, D. Mallouri, A. Barbouti, M. Ganidou, A. Fassas, foxP3+), CCR4+ CD4+ (Th2) cells and dendritic cells (lin- HLA- A. Anagnostopoulos DR+). Interestingly, the percentage of CD16+ CD14dim mono- G. Papanicolaou (Thessaloniki, GR) cytes was signifi cantly increased only in patients who responded to ECP treatment. There were no signifi cant changes in num- Conventional intensifi ed immunosuppressive therapy for refrac- bers or percentages of NK/NK-T cell and CD8+/CD4+ effec- tory chronic graft-versus-host-disease (cGvHD) increases tor/memory/naïve subsets. mortality and relapse rates after allogeneic hematopoietic cell In summary, the results suggest that ECP treatment alters transplantation. We retrospectively studied the effi cacy and the composition of immune cell subsets and switches the safety of extracorporeal photopheresis (ECP) in 44 patients blood cell compartment towards a regulatory immune reper- with corticoid-resistant cGvHD. Thirty-seven patients received toire. The role of CD16+ monocytes, which were only found grafts from siblings and 7 from matched unrelated donors. to be increased in patients responding to ECP, needs further Cyclosporine or tacrolimus and methotrexate±ATG were investigation. An immune monitoring platform has been estab- administered as GvHD prophylaxis. Patients had received 2(1- lished in Cologne to identify cellular subsets of importance for 4) lines of treatment for 13 months (median) before ECP. Ten immune responses after allogeneic haematopoietic stem cell patients had developed induced cGvHD post immunotherapy. transplantation. Fifteen patients had mucocutaneous disease only, 9 cutaneous

S130 sclerosis manifestations, 13 visceral involvement and 7 patients and 61.1% [37.4-84.8] in pts with alb < 35 gr/L, p=0.035). In skin sclerosis and liver. Patients completed a median number of multivariate analysis, initial liver involvement (HR: 2.45 (1.46 23 sessions of ECP/patient. The therapeutic schedule included to 4.12), p=0.0007) and a non-sibling donor (HR: 1.58 (1.02 to 2 sessions per week for 1 month followed according to the 2.43, p= 0.0039) were both associated with NRM. severity of cGvHD, by either a) one session per week for 2 Conclusion: Initial liver involvement was the most important months and 1 per 15 days for 2 months or b) 1 session per clinical predictor and may be considered in clinical manage- 15 days for 4 months, and then both cohorts received 1 ses- ment and need prospective validation. sion per month for 4-8 months. Response was evaluated at 3 months and at the end of the treatment. Five patients died due to early complications (<40 days): 2 early deaths occurred due P551 to GvHD or disease progression and 3 deaths due to existing Biomarkers associated with occurrence and severity of infections before ECP. Response at 3 months was 72% (28/39): graft-versus-host disease 90% in patients with mucocutaneous disease, 62% with skin S Ahmed (1), O Donze (2), E. Holler (3), X.N. Wang (1), sclerosis, 58% with visceral disease and 83% with cutane- A.M. Dickinson (1) ous sclerosis and liver manifestations. The cGvHD relapsed in (1)University of Newcastle upon Tyne (Newcastle, UK); 9/28(32%) in 2(1-15) months. Stable response was 48% (19/39) (2)Apotech Corporation (Epalinges, CH); (3)University of and 15 of 39 patients do not receive any immunosupression. Regensburg (Regensburg, DE) In multivariate analysis only visceral involvement affected the duration of response. Patients with de novo cGvHD seemed to The application of allogenic hematopoietic stem cell trans- respond better. In terms of immunological reconstitution, a 40% plantation (HSCT) is limited by life-threatening complications increase of CD4+ cells was observed after 12 months of treat- such as severe or acute graft-versus-host disease (GvHD). ment. Infections (gr? 3 WHO) were seen in 12 patients without Despite intensive prophylaxis with immunosuppressive agents, any mortality. No relapse of the underlying disease was docu- the incidence of GvHD occurs in 9-50% of patients undergo- mented. The 5-year disease free survival from the beginning ing transplant with an identical HLA sibling matched donor and of ECP was 80% with a median follow-up of 20 months. High 75% of patients undergoing unrelated HLA donors. Currently, response rates with acceptable toxicity and a low incidence of diagnosis of GvHD is based mainly on evaluation of clini- relapse of the underlying disease establishes the role of ECP cal symptoms including skin rash, diarrhoea and elevation of as a new, reliable therapeutic strategy in the treatment of resist- serum liver enzymes. To date, no validated biomarkers have ant cGvHD in earlier phase. been established for chronic GVHD, although several candi- date biomarkers have been identifi ed. The aim of this study was to characterize a number of candidate biomarkers in the P550 serum of patients pre and post HSCT with and without GvHD. Initial liver involvement in acute graft-versus-host Expression of the biomarkers were also analysed using the skin disease predicts severe acute GvHD and high non-relapse explant model. Blood samples were collected from patients mortality during pre-conditioning therapy (7 days prior to HSCT) and at M. Robin, R. Porcher, R. de Castro, G. Fisher, R. Peffault de 0, 14, 28 days 3,6,12 months post transplant. From the nine Latour, P. Ribaud, V. Rocha, A. Devergie, J.-Y. Mary, G. Socié candidate biomarkers (BAFF, IL-33, hTLA1, APRIL, Omentin, Hôpital Saint-Louis - APHP (Paris, FR) RANKL, OPG, NALP3, NALP1) tested, the results identifi ed two molecules, BAFF and IL-33 as potential GvHD markers Objectives: Current grading systems in acute GVHD can not which may be suitable for potential disease management. effectively identify patients with poor prognosis at time of GVHD BAFF (B cell activation factor) is a key regulator of B cell home- diagnosis. The aim of this study was to evaluate clinical or bio- ostasis and is also involved in regulation of T cell function. logical parameters at the onset of GVHD associated with poor BAFF has been shown to be elevated in autoimmune disorders prognosis of acute GVHD. and in cGvHD patients. Likewise, our results showed serum Methods: The study sample was composed of 146 patients (pts) BAFF levels were signifi cantly higher in patients with extensive who developed acute GVHD among the 257 who received an cGvHD (p<0.04). Furthermore, analysis using the skin explant allogeneic stem cell transplant (SCT) after a myeloablative con- model showed BAFF expression was signifi cantly increased in ditioning regimen between 1993 and 1999. Patients with acute patients with GvHR (p<0.04), confi rming the ELISA results. IL- GVHD were retrospectively analyzed for risk factors associated 33 is a member of the Interleukin 1 family, involved in infl am- with NRM using proportional cause-specifi c hazards models. mation, host defence and immune regulation. IL-33 is a ST2 Initial and maximal GVHD grade per organ (liver, skin, gut) were receptor ligand, upon binding it activates Th 2 cells, resulting recorded, as well as albuminemia. Characteristics of patients: in the release of Th 2 cytokines such as IL-5 and IL-13. Our Median age was 32 years (from 4 to 60 years). Source of stem results showed that patients who developed GvHD had higher cell was bone marrow in 106, peripheral blood stem cells in 23 IL-33 levels compared to control patients without GvHD. A sec- and cord blood in 13 pts. 87 patients received SCT from an ond independent cohort is currently being tested. The results HLA-identical sibling donor. suggest these two potential biomarkers may present a novel Results: At time of diagnosis, GVHD involved a single organ option of predicting the development of GvHD. in the majority of the patients (109 pts, 78%): skin for 63, gut for 35, liver for 11 pts. 37 patients had multiple organs affected by GVHD: 34 patients had 2 organs and 3 had 3 organs. Initial grade was I in 60 pts (23%), II in 83 pts (32%) and III in 3 pts (1%). Maximal grade was I in 31 pts, II in 72, III or IV in 43 pts. Overall 1-year NRM was 54.9% (95%CI 46.6-63.2). 57 patients experienced progression or stability 7 days after corticoster- oids initiation and were considered as “non-responders” (NR). Signifi cant (p < 0.05) risk factors for 5-y NRM were non sib- ling donor (71% vs. 54%), absence of methotrexate in GVHD prophylaxis (75% vs. 56%), initial liver involvement (80% vs. gut only: 54%, skin > grade I: 50%) and NR (85% vs. 44%). Patients who were responder to corticosteroids had same 5-y NRM than patients who did not develop any GVHD. Albumin level (alb) was associated with NRM in patients with initial gut involvement (NRM 21.4% [0.0-44.0] in pts with alb > or = 35

S131 6 patients. Nine patients died of aGVHD progression (n=7) and/ or severe infections (n=2) after a median of 40 days (range 4- 93); one patient in CR died due to relapse of acute myeloblastic leukaemia on day +188. According to these results, the study was prematurely closed. Overall, our results suggest that ster- oid-refractory severe aGVHD may be improved upon alemtu- zumab treatment. However, alemtuzumab does not overcome the dismal prognosis of patients with severe, steroid-refractory aGVHD, this pointing out to the necessity of alternative, more effective therapies for this clinical situation.

P553 Polymorphisms of MTHFR and clinical outcomes in allogeneic stem cell transplantation P. Chiusolo, S. Bellesi, S. Giammarco, S. Marietti, D. De Ritis, E Metafuni, S. DeMattteis, G. Leone, S. Sica Università Cattolica S. Cuore (Rome, IT)

Methotrexate (MTX) is an antifolate drug used to prevent graft versus host disease (GVHD) in allogeneic stem cell transplan- tation (alloSCT). Its function is due to inhibition of dihydro- folate reductase and limitation of function of other enzymes like MTHFR. It is well known that MTHFR 677 polymorphisms results in reduced activity of this enzyme and that the use of MTX in patients with 677 CT/TT variations may infl uence side effects like mucositis and bone marrow toxicity. Samples of 47 pts submitted to alloSCT from 2002 and respective donors were analyzed for 677CT polymorphism. Pts’characteristics were: 30M/17F, median age 37years (range10-58). Underlying disease were 23 AML, 17 ALL, 2 IMF, 2 SAA, 2 CML and 1 CLL. In 39 case pts were submitted to standard conditioning regimen and in 8 cases pts were submitted to reduced intensity condi- tioning regimen. All pts were submitted to GVHD prophylaxis with CSA and short-course MTX. Stem cell source was BM in 5pts, PBSC in 38pts and CBU in 4pts. The donor were HLA identical siblings or parents in 32 cases and MUD in 11 pts. All P552 CBU were unrelated. Seven pts died early after tx (14.8%) at a Alemtuzumab as treatment of steroid-refractory acute median time of 45d (range 30-90) for progression of disease. graft-versus-host disease: results of a phase II study Twenty-two out of 47 (46.8%) developed hepatic toxicity and C. Martínez, C. Solano, C. Ferrá, A. Sampol, D. Valcárcel, the median time for PMN (>0.5x109/L) and PLT (>20x109/L) J.A. Pérez-Simón on behalf of the Spanish Group for Stem Cell recovery was respectively 20 and 15 days (range 10-48 and Transplantation 11-55days). Twenty-two pts developed aGVHD grade II-IV at a median time of 26d after tx (range 10-70). Twenty-fi ve pts Acute graft-versus-host disease (aGVHD) is a signifi cant relapsed after tx at a median time of 4 months (range 1-34). At impediment to successful allogeneic stem cell transplantation this time 24pts are alive with a median follow-up of 31 months (SCT), particularly in patients not responding to primary therapy (range 1-51). OS, RFS, NRM and GVHD curves were obtained with steroids. There is no a good salvage therapy for steroid- by the Kaplan Meier method and statistically compared by refractory aGVHD. Based on prior reports, we conducted a log-rank test; while incidence of toxicities was evaluated with multicenter phase II study to investigate the safety and effi cacy chi2test. of the anti-CD52 antibody, alemtuzumab, in the treatment of Our data revealed that there was no correlation between steroid-refractory aGVHD > grade II. The treatment schedule 677CT polymorphisms and development of hepatic toxicity consisted of alemtuzumab 10 mg/d intravenously for 5 consec- and/or delayed engraftment (chi2test=ns); aGVHD was signifi - utive days followed by 10 mg/d on days 8, 15 and 22 if a com- cantly correlated with CC polymorphism in the donor (p=0.05) plete resolution of aGVHD manifestations was not achieved. and there was a trend toward a lower incidence of aGVHD in Response was assessed at days 7, 14, 21, 28 and at the last pts with TT polymorphism. Regarding NRM at 100days after follow-up after initiation of alemtuzumab. Ten patients (7 male / tx we found a strong correlation with CC polymorphism in pts 3 women; median age 57 years, range 19-65) were included in (p=0.006), whilst we found a trend toward a higher incidence of the study. Six patients had received an unrelated SCT and in relapse in pts with TT polymorphisms. 7 cases a reduced intensity conditioning regimen was used. At We conclude that greater immunosuppressive effect of MTX due study entry, 6 patients had grade III and 4 had grade IV aGVHD; to low MTHFR enzyme activity in mutated pts (TT) decreases the IBMTR severity index was B in 3 patients, C in 2, and D in the risk of GVHD but increases the risk of relapse. 5. Eight patients had 2 or 3 involved organs: gastrointestinal (GI) tract in 9, skin in 7, and liver in 5 patients. Median time from aGVHD diagnosis to alemtuzumab therapy was 8 days (range 4-22). All but one patient, who died on day 4 after treat- ment initiation, received 5 consecutive doses of alemtuzumab, 4 patients received the day 8 dose, and 2 patients completed the scheduled treatment. Main reasons to stop treatment were impairment of patient performance status or aGVHD progres- sion. Five (55%) patients responded to treatment (2 CR and 3 PR). Eight infectious events (4 of them grade 3-4) and 7 CMV reactivations were observed. Grade 3-4 cytopenias occurred in

S132 P554 density seeding of unmanipulated cells (100-200/cm2), obtained Kruppel-like factor 13 polymorphisms and clinical from 7 bone marrow harvests, allowed to prepare large quanti- outcomes after allogeneic haematopoietic stem cell ties of hMSCs, with only one in vitro passage. transplantation Results: 4 adults and 1 pediatric patients were treated for K.H. Lim (1), S.H. Park (1), E. Park (2), J.A. Song (3), I. Kim (1), aGVHD (grade III-IV) and 1 adult and 2 pediatric patients for S.S. Yoon (1), B.K. Kim (1), S. Park (1) extensive chronic GVHD (cGVHD), using 18 hMSCs certifi ed (1)Seoul National University Hospital (Seoul, KR); (2)Chung-Ang bags. Before hMSCs, second or third line treatments had been University Hospital (Seoul, KR); (3)DNA Link Inc. (Seoul, KR) given to patients with aGVHD, including Etanercept (n=4), Myc- ophenolate Mofetil (MMF, n=3) and Extracorporeal Photopher- Backgound: Graft-versus-host disease (GVHD) is a kind of esis (ECP, n=2). Patients with cGVHD were previously treated infl ammation, endothelialitis, in terms of target organ damage. with ECP (n=3), MMF (n=3), Imatinib (n=1), Rituximab (n=1) RANTES (regulated upon activation, normal T cell expressed and Etanercept (n=2). Each infusion contained a median dose and secreted) as chemokine plays an important role in the of 1x106/kg (range 0.7-1.2x106) hMSCs. As far as concerns pathogenesis of GVHD. Kruppel-like factor (KLF) 13 is one of patients with aGVHD, a single infusion was performed in the key regulators in RANTES expression. The aim of this study pediatric patient, while from 1 to 4 infusions were performed in was to analyze the association of KLF13 genetic polymor- 4 adult patients. The 3 patients with cGVHD received from 1 to 5 phisms and the clinical outcomes in patients treated with allo- infusions. All infusions were very well tolerated with no immedi- geneic hematopoietic stem cell transplantation (HSCT). ate or late adverse events according to WHO common criteria. Methods: Candidate single nucleotide polymorphisms (SNPs) Among pediatric patients, 3/3 complete responses were regis- associated with acute GVHD were selected using 100K DNA tered. A complete response was observed in 1 adult with grade chip analysis in 8 patients with acute GVHD and 7 patients III cutaneous aGVHD, although the patient rapidly relapsed and without acute GVHD, after allogeneic HSCT. Medical records died of leukemia progression. Three partial responses were of 251 patients with HSCT in Seoul National University Hospital observed in 3 adults, while only one adult showed no response between 1997 and 2006 were reviewed. and died of progressive grade IV gut and liver aGVHD. Results: The KLF13 polymorphisms, rs4779682, rs11070980, Conclusions: These data show that large numbers of third and rs4779520, were genotyped in 251 patients with allogeneic party hMSCs can be expanded in vitro with hPL-containing HSCT. Out of 3 SNPs, rs4779520 which has mutation within 3’ medium. Moreover, the clinical results and the toxicity profi le UTR was proven to be associated with acute GVHD incidence, confi rm those reported with hMSCs expanded in FBS contain- especially acute liver and gut GVHD. Acute GVHD incidence ing media. was signifi cant higher for the patients with CC genotype of rs4779520 than other genotypes (p=0.0086, HR=2.6584). TT genotype and co-dominant genotype of rs4779682 (p=0.0164, p=0.0293, respectively), which is an mutation within intron 1, had statistically signifi cant relevance to the increase of veno- occulsive disease, and CC genotype of rs4779682 showed higher treatment-related mortality compared with other geno- types (p=0.0306, HR=1.781). However, there was no signifi cant difference in relapse-free survival and overall survival accord- ing to KLF 13 polymorphisms. Conclusion: As the fi rst clinical study for the KLF13 genetic polymorphisms, this study suggested that KLF13 polymor- phisms would have the clinical value as a predictive parameter of acute GVHD, veno-occulsive disease, and treatment-related mortality after allogeneic HSCT, through the regulation of RANTES expression. P556 Chronic GvHD is associated with a defi ciency of P555 CD27+IgD+IgM+ B cells Human mesenchymal stromal cells expanded with human I. Hilgendorf (1), B. Mueller-Hilke (1), C. Junghanss (1), platelet lysate are safe and effective for the treatment of M. Leithäuser (1), M. Freund (1), D. Wolff (2) steroid-refractory graft-versus-host disease (1)University of Rostock (Rostock, DE); (2)University of M. Introna (1), C. Capelli (1), C. Micò (1), A. Algarotti (1), Regensburg (Regensburg, DE) A. Grassi (1), A. Salvadè (2), G. Gaipa (2), D. Belotti (2), P. Perseghin (2), G. D’Amico (2), A. Rovelli (2), A. Balduzzi Background: Chronic GVHD (cGVHD) is thought to be induced (2), J. Golay (1), J. Golay (1), A. Biondi (2), A. Rambaldi (1), by donor T-cells, but mechanisms of cGVHD and the critical E. Biagi (2) cellular subsets remain less well understood. In this study, we (1)Laboratory of Cellular Therapy G. Lanzani (Bergamo, IT); analysed T and B cell subsets in 44 patients (pts.) after alloge- (2)Ospedale San Gerardo (Monza, IT) neic haematopoietic stem cell transplantation (alloHSCT). Methods: Peripheral blood samples from 12 pts. who never Background: Very recently, encouraging results indicate that experienced cGVHD (group 1) and 12 pts. with resolved (group third party human mesenchymal stromal cells (hMSCs) are a 2) and 20 pts. with active cGVHD (group 3) were analysed for therapeutic tool for the treatment of severe steroid resistant lymphocyte subsets by FACS. Chronic GVHD was evaluated acute graft versus host disease (aGVHD). We have estab- using criteria and guidelines of the National Institute of Health. lished a highly effi cient protocol for in vitro expansion, under Results: The absolute CD19+ B cell count (in Gpt/l) in cGVHD strict GMP compliance, of bone marrow derived hMSCs using pts. was subnormal (group 2: median 0.14, range 0.008-0.69; human platelet lysate (hPL) in place of FBS. In this study, upon group 3: median 0.01, range 0.001-2.59); normal limits: 0.2- Ethical Committee approval and patient’s informed consent, 0.4 gpt/l) and less than that of group 1 (median 0.237, range hMSCs were administered on a compassionate basis for the 0.05-0.55). Furthermore relative and absolute numbers of the treatment of refractory GVHD. CD27- B cell compartment, which include immature and transi- Methods: hMSCs were prepared from washouts of bags and tional B cells, were lower in cGVHD pts. (group 2: 78%, median fi lters of bone marrow collection, from third party HLA-mis- 0.12Gpt/l, range 0-0.66Gpt/l; group 3: 68%, median 0.01Gpt/l, matched healthy donors. Cells were grown in the presence of range 0 -2.53Gpt/l) compared to pts. of group 1 (90%, median DMEM with 5% hPL. In a short period of time (10-33 days), low 0.20 Gpt/l, range 0.05-0.52Gpt/l). Although the relative number

S133 of cells of the CD 27+ memory B cell compartment was high- P558 est in the active cGVHD-group (22%) versus group 1 (10%) Bone marrow and intestinal but not peripheral blood and 2 (12%) due to the lower CD19+ B cell count (in Gpt/l), eosinophilia predicts graft-versus-host disease after the absolute number was lower in group 3 (median 0.02, haematopoetic cell transplantation with reduced-intensity range 0-0.96) compared to pts. of group 1 (median 0.17, range conditioning 0.04-0.65 gpt/l) and group 2 (median: 0.14, range 0-0.8). CD P. Roesch, G.-N. Franke, T. Lange, T. Aigner, D. Niederwieser, 27+IgD+IgM±cells (in 10E6/l) could not detected in pts. with N. Basara active cGVHD (except one patient with isolated cGVHD of the University of Leipzig (Leipzig, DE) oral mucosa and dry eyes) (5%, median 0, range 0-66.6) in contrast to pts. of group 1 (20%, median 1.69, range 0-6.26) Objectives: Acute GvHD remains often a severe complication and group 2 (18%, median 1.23, range 0-10.53). No differences after RIC-HCT. We report here the predictive value of bone in absolute CD4+ and CD8+ T cell numbers as well as regula- marrow and intestinal eosinophilia in patients transplanted from tory T cells were observed between the three groups. 1999 to 2005 by using RIC-HCT. Conclusion: This small series confi rms diminished B cell counts Patients and methods: One hundred and fi fteen patients (55 in pts. with cGVHD. Furthermore the loss of CD 27+IgD+IgM+ female) with haematological malignancies with the median age B cells in pts. with active cGVHD indicate functional asplenia in of 59 years (range 21 to 75) were treated with RIC-HCT for AML these pts. Analysis of B cell subsets can provide a diagnostic (n=76), ALL (n=7), CML (n=13) and MDS (n=19). Conditioning tool for monitoring cGVHD activity but has to be evaluated in a regimen consisted of 2 Gy total body irradiation at day 0 and prospective study. fl udarabine (30 mg/m2) at day -4 to -2 followed by treatment with MMF and CSA. Grafts were obtained from related (n=30), allele matched unrelated (n=76) or mismatched (n=9) donors. P557 Bone marrow and peripheral blood evaluation was performed A single nucleotide polymorphism in the intelectin before and on day +28 after RIC-SCT; Eosinophilia was gradu- 1 gene (ITLN1) is associated with acute intestinal ated from 0 to 4. Systematic colon and/or duodenal biopsies graft-versus-host disease have been performed immediately after fi rst symptoms of gut H. Hauser, O. Zach, O. Krieger, H. Kasparu, J. Koenig, GvHD. Analytic counts were performed for tissue eosinophils A. Weltermann, D. Lutz within the lamina propria and lamina submucosa. Elisabethinen Hospital (Linz, AT) Results: The incidence of acute GvHD was 51% (36% grade 1, 27% grade 2, 15% grade 3 and 22% grade 4 out of it). Most of Current concepts suggest a major role for innate immunity in the the patients developed one organ aGVHD (skin n=36, gut n=4, initiation of graft-vs.-host-disease (GvH-D) as well as in Crohn’s liver n=2). Two-organ aGvHD was diagnosed in 10 patients disease (CD). Identical single nucleotide polymorphisms (SNP) (skin and liver n=8, liver and gut n=2) and tree-organ aGvHD have been described as risk factors for both diseases. Recently, in 7 patients. The amount of bone marrow eosinophils was the noncoding C/T polymorphism rs2274910 in intron 3 of the increased after RIC-SCT in comparison to before HCT (6% vs intelectin 1 (ITLN1) gene (human lactoferrin receptor) has been 3,4%, p<0,001, respectively). Bone marrow eosinophilia after associated with CD. Thus, we tested whether this polymor- RIC-SCT was found in 66% of patients with aGvHD and was phism of the ITLN1 gene is also associated with gut GvH-D. a signifi cant predictive factor (p>0.03) for developing aGvHD. We retrospectively typed this polymorphism by TaqMan PCR However, eosinophil density did not correlated with aGvHD from peripheral blood from AML patients and their donors. severity. Peripheral blood eosinophilia was not predictive for A pilot study of 19 patients in 1st CR transplanted from HLA developing aGvHD. Intestinal eosinophils were found in 15 of identical sibling donors after myeloablative conditioning was 20 patients with clinical signs of gut GvHD which has been con- confi rmed in a second cohort including all other AML patients sequently histologically proven in 13 patients (87%, p<0.02). (n=40). A total of 59 consecutive patients (median age 43 yrs The degranulation of eosinophils, determined by using anti- (18-63); 31/28 female/male, 25/34 1st CR/advanced disease; MBP was present in all patients with gut aGvHD. 40 myeloablative and 19 reduced intensity conditioning; 54 Conclusions: Both gastrointestinal tract and bone marrow tis- peripheral stem cells) were tested. T-alleles were found at a sue eosinophilia after RIC-HCT predict aGvHD. In addition, gut frequency of 28.8% in recipients and 29.7% in donors. 50.8% eosinophil density and degranulation were increased in patients of patients had a CC genotype. with higher grades of aGvHD thus indicating their role as a bio- In the pilot study, 2 out of 12 patients with a CC genotype logical marker of GvHD. According to our knowledge this is the versus 5 out of 7 patients with a T allele had acute intestinal fi rst study showing that tissue eosinophils density might be a GvH-D (P=.045). These results were confi rmed in a second predictive marker for aGvHD after RIC-HCT. cohort. Acute intestinal GvH-D was found in 3/18 patients (16.7%) with a CC genotype and 12/22 patients (54.5%) with a T-allele (P = .014). In a combined analysis (n=59) we found P559 acute intestinal GvH-D in 58.6% of patients with a T-allele ver- The impact of cytokine genetic polymorphisms in acute sus 16.7% of patients with a CC genotype (P < .001). graft-versus-host disease in HLA-matched sibling bone The lower incidence of acute GvH-D grades II-IV in patients marrow transplants in the Sardinian population with a CC genotype (logrank P = .019) was only due to less M.G. Orofi no (1), M. Congia (2), M.A. Sanna (1), D. Macis intestinal GvH-D while no difference was seen with regard to (2), M. Badiali (1), M.C. Addari (1), A. Piroddi (1), F. Rizzo (1), skin or liver GvH-D. We did not see any association between F. Cossu (1), I. Ronch (1), F. Argiolu (2) donor genotypes and GvH-D. (1)Bone Marrow Transplant Unit (Cagliari, IT); (2)II Cl. Pediatrica The strong association (RR 3.52; 95% CI 1.5 – 8.2) between Università Studi (Cagliari, IT) this SNP and the incidence of acute intestinal GvH-D is in accordance with reports about SNPs in other genes associated The regulatory regions of cytokine polymorphisms (CPs) may with both acute GvH-D and CD. Surprisingly, we found a higher modulate the complexity of immunologic response, including incidence of GvH-D in patients with a T allele while the reported acute graft versus host disease (aGVHD) in allogenic haemat- risk allele for CD is the C allele. Our results further support the opoietic stem cell transplant setting. The imbalance between concept of an interaction between the gut associated innate pro-infl ammatory Th1 cytokine like IL-6, IL-1, IL-2, TNF-a, INF- immune system and GvH-D. g and Th2 protective IL-4, IL-10, TGF-beta1, promotes donor T cell activation and clonal expansion. The aim of this study was to determine if an association between single nucleotide polymorphisms SNPS of pro-infl ammatory or protective Th1/ Th2 cytokine and severe aGVHD existed in 41 donor and 41

S134 recipient pairs who received an HLA matched sibling bone (p=0.6). In the same period the probability of relapse was 27% marrow transplant (BMT). The correlation between IL-2, IL-4, in the ATG-G group vs 0% in the ATG-F group (p=0.08) (Figure IL-6, IL-10, TNF-a, and IFN-g cytokine levels in patient sera, 1). These data must be confi rmed in a randomised prospective and between cytokine genotype, both of which can result high, study and longer follow-up is necessary. intermediate or low producer, was also studied. Finally, the fre- quency of the cytokine haplotypes found in BMT patients was compared with that of 30 Sardinian healthy controls. Subjects and Methods: Forty-one patients and 41 donors were typed for IL-2, IL-1, IL-6, IL-10, IL-4, TGF-beta1, TNF-a and IFN-g polymorphisms. Thirty-seven patients underwent BMT for beta-thalassemia major, 1 for acute myelocytic leukemia, 1 for acute lymphocytic leukemia, 1 for congenital neutrope- nia, and 1 for histiocytosis. II-III° aGVHD was observed in 19 patients (46%), while 22 were aGVHD free. Twenty-six sera were collected from 13 patients with aGVHD and 13 without aGVHD, at various transplant times (pre BMT, 15, 30 and 60 days post BMT) and measured in an Elisa assay. Cytokine genotypes were performed using a commercially available kit (SSP Dynal Invitrogen). Results: Signifi cantly higher IL-6 levels were found in the group of patients with aGVHD compared to patients without aGVHD (p <0.03). At 30 and 60 days post BMT the high amount of IL-6 correlated with the IL-6 genotype –174 G/G. Thirteen out of 19 patients with aGVHD compared to 9 out of 22 without aGVHD showed -174G/G polymorphism (p<0.05) in the IL-6 promoter. Conclusions: The frequency of haplotype in the population of transplanted patients, not differed from healthy control. A cor- relation between the IL-6 -174G/G genotype, the IL-6 high pro- duction phenotype and a predisposition to severe aGVHD was observed in our study. These fi ndings could have clinical impli- cations including the use of Tocilizumab (a monoclonal antibody against IL-6) to prevent or cure aGVHD in a targeted manner.

P560 Retrospective evaluation between ATG-genzyme – ATG-fresenius in allogeneic HSCT setting; impact on GVHD, infections, relapse and survival D. Baronciani (1), C. Depau (1), M. Pettinau (1), F. Zaccheddu (1), F. Pilo (1), D. Derudas (1), A. Lai (2), E. Angelucci (1) (1)P.O. Businco (Cagliari, IT); (2)P.O. SS Trinita (Cagliari, IT)

Objective: This study aims to evaluate the effi cacy of 2 prepa- rations of ATG (ATG-Genzyme Thymoglobulin, ATG-G) and ATG-Fresenius (ATG-F) in two cohorts of transplanted patients in preventing GVHD and rejection, as well as their impact on infections, relapse, and overall survival. Patients and methods: Since July 2005 to November 2008, 50 consecutive patients undergoing myeloablative conditioning P561 from HLA identical or mismatch siblings (n=26) or unrelated Cytokine expression and genotype can affect outcomes donors (n=24), were retrospectively analysed. Underlying dis- after haematopoietic stem cell transplantation eases were advanced and/or high risk haematologic malignan- B. Turner, J.L. Harrold, J. Norden, E.A. Douglas, cies Thirty (30) patients received ATG-G at a total dose of 4,5 A.M. Dickinson mg/kg if HLA identical sibling transplants (n = 17) or 6 mg/kg Newcastle University (Newcastle upon Tyne, UK) if unrelated transplants (n=13). Twenty (20) patients received ATG-F at a total dose of 15 mg/kg if HLA identical sibling trans- Complications such as graft versus host disease (GVHD) plants (n=7), or 30 mg/kg if unrelated or mismatched trans- remain a major barrier to the successful application of haemat- plants (n=13). Both ATGs were administered on days -3,-2,-1. opoietic stem cell transplantation (HSCT). Cytokine expression GVHD prophylaxis was performed with Cyclosporin and sMTX. plays a major role in the pathogenesis of GVHD. Damage to Groups were comparable as regards age, underlying diseases mucosal tissues during the induction phase of acute GVHD is and conditioning. (Tab 1) accentuated by the release of proinfl ammatory cytokines, such Results: Infusion related side effects occurred more frequently as tumour necrosis factor-alpha (TNF-a). In contrast, the anti- in the ATG-G group (80% vs 65%). Time of engraftment was infl ammatory cytokine interleukin-10 (IL-10) is said to down similar. Acute GVHD II-IV occurred in 13% of ATG-G patients regulate the T-cell responses that are involved in intensifying versus 15% of ATG-F patients. Two patients rejected in the GVHD. In addition, polymorphisms in cytokine genes have ATG-G group, none in the ATG-F group. been associated with GVHD and patient outcome post-HSCT. Conclusions: In this retrospective analysis, a preliminary obser- We have looked at TNFd and IL-10 polymorphisms in a cohort of vation underlines no statistical difference in the incidence of allogeneic HSCT patient/donor pairs and compared these with Acute and Chronic GVHD among patients receiving the two patient cytokine levels and GVHD. Genotypes were determined types of ATG. Early post transplant bacterial infections and by polymerase chain reaction (PCR) and acrylamide electro- Cytomegalovirus infections were similar in the two groups. At 17 phoresis. Peripheral blood mononuclear cells from patients months post-transplant Overall Survival was 73% for patients pre-transplant were cultured in media alone, lypopolysaccaride in the ATG-G cohort vs 88% for patients in the ATG-F cohort (LPS) for 24 hours or phytohaemagglutenin (PHA) for 72 hours

S135 to facilitate activation. Levels of TNF and IL-10 were determined by enzyme linked immunosorbent assay (ELISA). LPS stimulated recipient TNF levels were signifi cantly increased in recipients with the TNF 3/3 genotype (p=0.043). However, in this cohort there was no statistical link between patient TNF 3/3 genotype and the development of GVHD (n=60). In addition, spontaneously released donor IL-10 levels were increased in donors with the IL-10 GCC haplotype (p=0.06). Furthermore, signifi cantly less, clinically signifi cant GVHD (p=0.047) was seen in patients with donors who had the IL-10 GCC/GCC P563 genotype (n=63). These results further substantiate the role of Ultrasonographic features of acute gastrointestinal cytokines in the development of acute GVHD and demonstrate graft-versus-host disease after allogeneic stem cell that functional studies can correlate with genotype and be of transplantation clinical relevance. G. Carnevale Maffè, R. Ciccocioppo, K. Markopoulos, S. Tirella, C. Alvisi, O. Luinetti, A. Colombo, F. Ripamonti, E.P. Alessandrino, G. Corazza P562 Foundation IRCCS Policlinico San Matteo (Pavia, IT) Methylation in the promoter region and expression of FOX-P3 correlate with GVHD, relapse and survival after Introduction: Gastrointestinal graft-versus-host-disease (GI- HLA-identical sibling allogeneic SCT GVHD) is one of the main causes of therapy-related morbility C. Manzano, P. Balsalobre, D. Serrano, G. Rodríguez-Macías, and mortality consequent to allogeneic haematopoietic stem J. Gayoso, A. Gómez-Pineda, J.L. Díez-Martín, I. Buño cell transplantation (HSCT), occurring in up to 80% of patients Hosp. G.U. Gregorio Marañon (Madrid, ES) and involving any part of the intestine from the oesophagus to the rectum. Early diagnosis is crucial for improving clinical Introduction: DNA Methylation controls the expression of cer- outcome. The clinical features of this pathological condition tain genes. FOX-P3 is a transcriptional regulator expressed are usually evident in an advanced phase and the histological constitutively in regulatory T lymphocytes CD4/25 (Treg) and examination of perendoscopic mucosal biopsies may be incon- transiently and with signifi cantly lower levels in effector T cells. clusive. Abdominal ultrasound with color Doppler (CD) allow the Objective: To analyze the association between FOX-P3 evaluation of single bowel wall layers with their vascularization mehtylation status and level of expression with the dynamics of throughout the small and large bowel. Up to now, only scanty chimerism and the development of complications after alloge- information are available about the role of ultrasound in the neic SCT (alloSCT). Patients and methods: FOX-P3 promoter diagnosis and staging of acute GI-GVHD. We aimed, therefore, methylation was assessed by quantitative methylation-specifi c to characterize the sonographic fi ndings of intestinal GVHD. real time-PCR from 32 peripheral blood (PB) samples obtained Patients and Methods: We retrospectively evaluated 11 patients within the fi rst month post-alloSCT. FOX-P3 mRNA expres- who developed acute GI-GVHD (M/F ratio 8/3, mean age 52.2 sion level was analyzed by real-time quantitative PCR in 18 years) after allogeneic HSCT for haematological malignan- patients. cies. Sonography was performed with both 5 MHz and 10 MHz Results: A higher degree of FOX-P3 methylation (“hi-meth”) was probes, and the evaluated parameters were: bowel loop trans- signifi cantly associated with a lower incidence of acute GVHD verse diameter (normal value <15 mm), wall thickness (normal (II-IV) and with a higher incidence of relapse. The observation value <3 mm), parietal stratifi cation and intraparietal blood fl ow of “hi-meth” would refl ect a lower amount of FOX-P3 express- by color Doppler, free abdominal fl uid, mesenteric lymph node ing cells, both Treg and effector cells. Since Treg comprises a size. In all patients stool samples resulted negative for bacte- minor population, such observation would result from relative rial, fungal and viral infections, whereas only in 5/11 patients low levels of effector T cells, which would explain the lower GVH the diagnosis of acute GVHD was confi rmed at histology. The and GVL effect observed in the “hi-meth” group. Surprisingly, remainder 6/11 patients resulted unable to undergo endoscopic when FOX-P3 mRNA levels were analyzed, an association with examination because of their critical condition. statistical signifi cance between “hi-meth” and a higher expres- Results: 54% patients showed an increased bowel wall thick- sion of FOX-P3 (“hi-exp”) was observed. It has been shown ness of both small bowel and colon (pan-intestinal disease), that FOX-P3 is mainly produced by Treg while effector T cells with the distal part of the ileum being more frequently involved produce very low amounts of FOX-P3 mRNA. In this sense, (73%); bowel wall thickening was in all patients the result of a higher amount of Treg cells produces high FOX-P3 mRNA increased thickness of the submucosa. In 73% patients an levels and reduces the number of effector T cells, resulting in increased intraparietal arterial blood fl ow was demonstrated. a “hi-meth” status in PB samples, in which most cells would The majority of patients presented hyperechoic mesenteric have methylated FOX-P3. FOX-P3 “low-exp” would, in turn, thickening (73%) and free abdominal fl uid (64%). indicate a lower amount of Treg to suppress the immune activa- Conclusions: ultrasonography with CD is a feasible and safe tion, resulting in higher amount of effector T cells and therefore method for the assessment of patients in whom intestinal would be associated with “low-meth”. Additionally, a signifi cant GVHD is suspected and it should be included in the diagnostic association between “hi-exp” and lower incidence of death was algorithm. Even if not specifi c, the fi ndings we have described observed. Moreover, acute GVHD was the main cause of death may help in both early detection and accurate staging of this (3/4) in the group with “low-exp”. The lower amount of effec- condition. tor T cells in PB samples with “hi-exp” would protect from the development of GVHD. Conclusions: Both FOX-P3 mRNA expression and promoter P564 methylation are of prognostic value for the development of Eye graft-versus-host disease in patients with allogeneic complications post-SCT, favoring an early establishment of the haematopoitic stem cell transplantation immunotherapeutic options for an improved management of A. Mikhaylova, Y. Astahov, B. Afanasyev transplanted patients. Pavlov State Medical University (St. Petersburg, RU)

The allogeneic hematopoietic stem cells transplantation (HSCT) can cause graft versus host disease (GvHD) with manifestation in different organs including eyes. Ophthalmologic GvHD does not infl uence life prognosis but can signifi cantly decrease the quality of life due to dry eye syndrome.

S136 The objectives: To evaluate the probability of eye acute and GvHD appearance to fi rst Rituximab administration was 24(4- chronic GvHD (aGvHD and chGvHD) occurrence and response 79) months. Twelve patients completed the fi rst phase, 8/14 the to local therapy. second phase and 6/14 are currently in the third phase. Partial Methods: 193 patients with different hematological malignan- or complete responses were observed in 10 patients (71%) at cies were treated with allogeneic HSCT and underwent oph- a median follow-up of 6 months. The time to response was 2 thalmologic examination in the period from 2006 till 2008. Three months in the majority of patients. Responses were mainly in stages of dry eye disease were diagnosed according to the fol- the skin (10/14) and mouth(7/14) and resolution of nephrotic lowing criteria: dry eye related clinical symptoms, positive stain- syndrome was observed in one patient. In responders (10/14), ing with fl uorescein, Schirmer’s I test results less than 10 mm steroids were tapered and GvHD reappeared in 2 of them. No or a tear fi lm break-up time value less than 15 seconds (Norn chimerism changes or hematologic toxicity were observed dur- test). The following treatment schedules of dry eye syndrome ing therapy. The main reason for treatment modifi cation (dose- associated with GvHD were used: interval) was infection (10/14, 71%). Eight patients developed Stage 1: Artifi cial tears (high viscosity) BID or TID; fungal pneumonia and one developed CNS toxoplasmosis, Stage 2: Artifi cial tears TID and Dexamethasone 0.1% BID or while one patient died from septic shock. The rituximab dosing TID topical; schedule was modifi ed or suspended until resolution of infec- Stage 3: Artifi cial tears 4-6 times a day, Dexamethasone 0.1% tion. Our data suggest that B-cell depletion by rituximab may TID topical and Cyclosporin A 0.05% 1 drop per eye BID. Wil- offer an effective alternative treatment for refractory cGvHD. coxon pair matched test, Spearman test and Pearson test were A major limitation seems to be the relatively high incidence used for statistical analysis. of infections. A possible earlier use of rituximab, when patient Results: Ophthalmologic complications occurred in 49,7% immune status is not severely immunocompromised or modi- (96/193) of HSCT patients. Eye GvHD was revealed in 24% of fi ed longer intervals between treatment courses may reduce all transplanted patients. 34 patients were eligible for assess- treatment-associated toxicity and improve outcome. ment of system chGvHD. Among them, 70% (24/34) of patients had signs of dry eye syndrome. All patients with aGvHD received systemic GvHD prophylaxis and eye component of P566 aGvHD responded quickly to local Dexametazone 0.1% treat- Impact of cyclosporine A concentration on the incidence ment. The median day of onset of eye chGvHD was D+145±29 of severe acute graft-versus-host disease after allogeneic (range 96-175). The eye chGvHD treatment results according stem cell transplantation to stages of dry eye syndrome are represented in Table 1. F. Mallard, E. Brissot, P. Chevallier, T. Guillaume, J. Delaunay, There was strong correlation between both tests used (p=0.004) S. Ayari, V. Dubruille, S. Le Gouill, B. Mahe, T. Gastinne, Two patients could not tolerate cyclosporine topical treatment N. Blin, B. Saulquin, P. Moreau, J.L. Harousseau, M. Mohty because of local irritation of conjunctive. CHU Hôtel-Dieu (Nantes, FR) Conclusion: Approximately half of patients treated with HSCT have signs of different eye complications. The frequency of Previous studies have demonstrated that the immunosuppres- eye chGvHD is 70% of patients with systemic chGvHD. The sive effects of cyclosporine A (CsA) may be correlated with CsA cyclosporine local therapy proved to be effective treatment of blood concentration, especially in the context of solid organ stage 3 dry eye syndrome caused by chGvHD. transplantation. This single centre report investigated the impact of CsA con- centrations in the early post allo-SCT period, on the incidence of severe acute GVHD, in 85 consecutive patients, for whom CsA concentrations were monitored weekly after the start of infusion. 85 patients received CsA (3 mg/Kg/d, 2-3 days prior to graft infusion) as a 24-h continuous infusion until hematopoietic recovery and switch to oral formulation. Dose modifi cations of CsA were performed to maintain adequate trough blood levels and to prevent nephrotoxicity. P565 Patients’ and donors characteristics were as follow: median Rituximab is an effective treatment for refractory chronic age: 51 (range, 18-67), 46 (54%) female donors, 33 (39%) graft-versus-host disease but is associated with high myeloid malignancies, 49 (58%) lymphoid malignancies, and incidence of infections 3 cases of SAA. The stem cell source was PBSC in 66 (78%) I. Batsis, A. Karpouza, I. Sakellari, E. Yannaki, P. Kaloyannidis, patients, while bone marrow was used in 19 (22%) patients. D. Mallouri, C. Smias, A. Fassas, A. Anagnostopoulos 37 (43.5%) were transplanted from a matched related donor, G. Papanicolaou (Thessaloniki, GR) and 48 (56.5%) from a matched unrelated donor. A myeloabla- tive conditioning regimen was used in 24 (28%) patients, and Chronic Graft versus Host Disease (cGvHD) is the main cause 61 (72%) received a reduced intensity regimen. The median of late morbidity and mortality following allotransplantation. concentrations of CsA in the blood at 1, 2, 3 and 4 weeks after Â-lymphocytes have been implicated in the pathogenesis as allo-SCT were 348 (range, 172-733), 284 (range, 137-535), antigen-presenting cells or by producing autoantibodies or 274 (range, 107-649), and 247 (37-695) ng/mL respectively. cytokines. We investigated the effect of rituximab in 14 patients All patients engrafted at a median of 17 (range, 0-42) days with refractory cGvHD to at least 2 therapy lines. Rituximab was after allo-SCT. With a median follow-up of 16 (range, 5-29) administered at 375 mg/m² per weekX4weeks (fi rst phase), 375 months, grade 2-4 acute GVHD occurred in 36 patients (42%) mg/m² every 2 weeksX4 times (second phase) and 375 mg/m² at a median of 29 (range, 6-100) days after allo-SCT. The inci- every 2 monthsX1 year (third phase). Nine male and 5 female dence of grade 3-4 acute GVHD was 23% (95%CI, 14-32%). patients, median aged 42 years, were included. Eleven had In multivariate analysis taking into account acute GVHD risk received grafts from siblings and three from unrelated donors. factors, we found that higher whole-blood CsA concentration The main graft source was mobilized peripheral blood(12/14) in the fi rst week following graft infusion, and before onset of and myeloablative conditioning regimens were used in 10 of 14 acute GVHD was the strongest parameter signifi cantly associ- patients. All patients had extensive cGvHD with organ involve- ated with a reduced the risk of severe grade 3-4 acute GVHD ment: skin (14 patients, 2 with cutaneous sclerosis), mouth(12), (P=0.01; RR=0.24; 95%CI, 0.08-0.73). eyes(9), liver(2), l ung(3), joints(1), gut(1), kidneys(1), bone Despite its retrospective nature, this data indicates a close marrow(1). The median therapy lines offered to patients before relationship between CsA trough blood concentration during rituximab were 3(calcineurin inhibitors plus steroids± mycophe- the early post allo-SCT period and the severity of acute GVHD. nolate mofetil, extracorporeal photopheresis). The time from Insuffi cient early exposure to CsA can be a serious risk for

S137 developing severe acute GVHD. Therefore, precise monitoring We enrolled 37 pts (23 allo-SCT, 14 auto-SCT), age 51 (21-73) of CsA concentrations and achievement of a high CsA target yrs, without relapse and with a median follow-up of 36 (7-70) concentration may be an effective tool to prevent the onset of months after last vaccination). Diagnosis were in the allo-SCT severe acute GVHD. group MDS and acute or chronic leukemias, and in the auto- SCT group myeloma and lymphomas. Sixteen allo-pts had GvHD and ongoing immunosuppresion (IS) at the time of the P567 fi rst two injections. Intracellular markers of eosinophils and mast cells in Results: In the auto-SCT group, 2 pts lacked immunity to diph- patients with chronic graft-versus-host disease theria/polio (n=1) and diphteria/tetanus (n=1). In the allo-SCT associated with high-grade eosinophilia group, three pts lacked immunity to diphteria (n=2) and diph- L. Komarova, N. Mikhaylova, B. Afanasyev theria/polio (n=1), all of whom had GvHD and IS at the point Pavlov State Medical University (St. Petersburg, RU) of the fi rst two immunizations. The seven allografted pts with- out GvHD and IS at immunization all had full immunity to polio, Chronic graft versus host disease (chGvHD) is the most com- diphtheria and polio. The difference observed with respect to mon late complication of allogeneic hematopoietic stem cell immunity between GvHD and non-GvHD pts was not statisti- transplantation. The peripheral blood eosinophilia was proven cally signifi cant (p=0.32, Fisher´s exact test). to be a prognostic factor of sclerodermatous form of chGvHD Conclusion: In spite of a high rate of GvHD and IS in the allo- (Skert C. et al, 2006) and occurred in 10-20% of patients. On SCT group, the results of reimmunization were good and simi- other hand the intracellular marker of eosinophils - eosinophil lar to what was found in the auto-SCT group, suggesting that cationic protein (ECP) and intracellular marker of mast cells ongoing GvHD does not abrogate a respons to vaccination. On – tryptase, are mediators of infl ammation and organ damage the other hand, it is noteworthy that all three allo-pts lacking full and could lead to development of sclerosis. immunity had ongoing GvHD at the time of vaccination. A larger Objectives: to determine the levels of ECP and tryptase in study is needed to clarify this issue. patients with chGvHD and compare these parameters in patients with other non-clonal hypereosinophilic conditions. Patients and Methods: The serum level of ECP and tryptase P569 were measured in three groups of patients with peripheral Limited effi cacy of low-dose imatinib in pulmonary blood eosinophils more than 0.4 x109/L: First group consisted chronic graft-versus-host disease of 6 patients with chGvHD, second group represented 18 M. Stadler, H. Diedrich, H. Kamal, R. Ahlborn, S. Buchholz, patients with bronchial asthma and in third group 20 patients A. Ganser, M. Eder with Hodgkin’s and nonHodgkin’s lymphoma were combined. Hannover Medical School (Hannover, DE) The second and third groups were used as controls. A com- mercial fl uoroimmunoenzyme assay was used (Pharmacia, Background: Chronic graft-versus-host disease (cGvHD) of the Uppsala, Sweden). lung, characterised by bronchiolar infl ammation and fi brosis, Results: The serum levels of total ECP were markedly increased is a devastating late-onset non-infectious pulmonary compli- in the patients with chGvHD (median: 39,8 ng/mL; range, 2-200 cation of allogeneic hematopoietic cell transplantation (HCT), ng/mL) to compare with second group (median: 18,8 ng/mL; with high morbidity and mortality. Treatment options are limited range, 2-197 ng/mL), p<0,03. The serum levels of tryptase were to immunosuppression, antiinfl ammatory and antiobstructive increased in group of patients with chronic GvHD (median: 12,3 drugs, or lung transplantation. Recently, platelet-derived growth ng/mL; range, 2,6-24,1 ng/mL), to compare with second group factor receptors (PDGFR) have been recognised as regulators (median: 6,8 ng/mL; range, 3,6-11,7 ng/mL), p=0,03.and third of proliferation and collagen deposition in fi brotic lung diseases. group (median: 5,95 ng/mL; range, 2,3-16,4 ng/mL), p<0,05. Imatinib, a PDGFR inhibitor, seems to exert antifi brotic actions Conclusion: The tryptase and ECP could take part in pathogen- at 200 mg daily, and has been reported to improve pulmo- esis of chGvHD with eosinophilia probably supporting infl am- nary arterial hypertension and bronchiolitis obliterans in single mation and secondary sclerosis. patients. Methods: Since November 2005, we employed Imatinib in eight patients with refractory pulmonary cGvHD. Median age was P568 45 years (range: 24 to 50); 3 patients were female and 5 male. Effects of graft-versus-host-disease and Peripheral blood HCT from related (n = 6) or unrelated (n = immunosuppressive treatment on immune response to 2) donors had been performed after myeloablative (n = 6) or vaccination after bone marrow transplantation reduced intensity (n = 2) conditioning, for AML, CML, or lym- S. Einarsdottir, P.O. Andersson, P. Horal, B. Kaijser, M. Brune phoma. All patients had already received combination therapy University of Göteborg (Göteborg, SE) with steroids, calcineurin inhibitors, mycophenolate, and/or extracorporal photopheresis. Additional Imatinib was started at Background and Objectives: Pretransplant immunity to bacte- 100 mg daily and increased in 100 mg steps, as tolerated. All rial agents (e g tetanus, dipheria) and viruses, eg polio, is fre- patients were evaluated monthly for toxicity and response (pul- quently lost after auto- and allo-SCT. Therefore, reimmunization monary function). of is routinely initiated after patients´ immune reconstitution, Results: Imatinib toxicity was generally acceptable (hemato- 6-12 months after transplant. Ongoing GvHD and its immuno- logic, nausea, or edemas), except in one patient who had to suppressive treatment may impair or delay antibody response discontinue the drug at 100 mg due to reversible dyspnea. to vaccination. Here, we account for the effect on antibody Dose increase was not possible in half of the patients; only one levels of our reimmunization practice in transplanted patients reached the 400 mg dosage. After a mean therapy duration of with and without GvHD. 6 months (range: 1 to 16), no signifi cant recovery of pulmonary Methods and Pazients: At our institution, pts are vaccinated function was noted. Best responses were minor improvements (regardless of ongoing GvHD) against diphteria, tetanus and facilitating steroid tapering in two patients treated for 16 and polio, using three injections given at +6, +8, +15 months after 10 months, four patients showed no change, and two patients auto-SCT, or at +8,+10, +18 months after allo-SCT. Titers of died due to progression of pulmonary cGvHD and/or leukemic neutralizing antibodies against poliovirus -1, -2 and -3 were relapse. assessed by the poliovirus-neutralizing antibody test. ELISA Conclusions: Imatinib at 100 to 400 mg daily had limited effi - technique was used to determine antibody levels against teta- cacy in this small cohort of patients with advanced refractory nus-toxoid. Titers of neutralizing antibodies against diphteria pulmonary cGvHD. We cannot exclude that patients might have were measured by diphteria-neutralizing test. Immune level for benefi ted from longer exposure to or higher doses of Imatinib, diphtheria and tetanus was determined to ≥0.01 IU/ml. but both were diffi cult to achieve. Conceivably, a stronger effect

S138 might also be seen if treatment was started before irreversible steroids. Patients had undergone stem cell transplantation from lung damage, or with more potent tyrosine kinase inhibitors. family donors (n=7) or matched unrelated donors (n=11). Five had 1 or 2 antigen mismatch. All patients had severe gut and/ or liver involvement. Initially, in three patients in rather critical P570 situations start doses of alemtuzumab in the range of 70mg to Effi cacy and tolerability of long-term inolimomab 80mg (total) were applied in fractions over several days and treatment for acute graft-versus-host disease repeated after 3 to 4 weeks. Impressive responses, such as L. Kammoun, M. Uzunov, S. Nguyen, N. Dhedin, L. Sutton, bilirubin levels of 48mg/dl returning to normal within several J.P. Vernant weeks, were encouraging, but virus reactivation and bacterial Hopital de la Pitié-Salpétriére (Paris, FR) infections were seen. This fi nding and considering the limited quantities of lymphoid tissue present early after transplantation Steroids are used for fi rst-line treatment of aGvHD grade >II. requiring less antibody, the starting dose was reduced to 20 to Steroid-resistant forms can be treated with monoclonal antibod- 33 mg in the second, and 5-10mg in the third cohort, repeated ies like inolimomab (anti-interleukin-2 receptor). Several studies every 2 to 3 weeks. Considerable improvement was obtained have shown the effi cacy of inolimomab, but the optimal regimen in most patients. Chronic GvHD was observed frequently. Pro- (dosing interval and duration) remains to be determined. We nounced lymphocyte depletion seems inevitable for effi cacy, reviewed the cases of 28 patients with aGvHD who received and CMV reactivation and other infectious complications were inolimomab for at least 2 months between 2000 and 2007. The seen. Thus, despite the need for close monitoring for signs of aim of this long-lasting treatment was to allow a quick decrease infections, alemtuzumab given in moderate doses has a sub- of steroids. Median age was 44 years (range 20-60 years). The stantial activity in severe steroid-refractory acute GvHD. conditioning was myeloablative in 22 cases. The source of pro- genitor cells was peripheral blood in 10 cases, bone marrow in 15 cases and cord blood in 3 cases. The donors were related P572 in 12 cases (1 mismatched) and unrelated in 16 cases (7 mis- Rituximab for chronic graft-versus-host disease matched). Acute GvHD was diagnosed a median of 20 days G. Nair, U. Schanz, G. Stüssi after AHSCT (range 7-39 days). University Hospital (Zurich, CH) AGvHD was grade II in 14 cases (all cutaneous), grade III in 11 cases (8 cutaneous, 1 digestive and 2 cutaneous+digestive) and Chronic Graft versus Host disease (cGvHD) is common after grade IV in 3 cases (2 digestive and one cutaneous+digestive). allogeneic stem cell transplantation (SCT) and is associated After a median of 16 days of steroid therapy, inolimomab was with a high morbidity and mortality. Traditionally, the therapy administered daily, for a median of 21 days, then two or three has been mainly directed against T-cells, but there is growing times a week. The median treatment period was 10 weeks (up evidence that B-cells are also important for cGvHD and that to 6 months). The following immunosuppressive treatments Rituximab may be benefi cial in such patients. were subsequently added in 16 cases (57%): mycophenolate We retrospectively evaluated the effect of rituximab in 13 mofetil (12 cases), infl iximab (7 cases), antithymocyte globulins patients with steroid-refractory cGvHD (375 mg/qm x 4 weeks). (2 cases) and phototherapy (1 case); 5 patients received two or Complete response (CR) was defi ned as resolution of all more of these treatments. GvHD-related symptoms; partial response (PR) as > 50% and Twenty complete responses (CR) and 5 partial responses (PR) no response (NR) < 50% reduction or exacerbation during or were obtained. Only cutaneous aGvHD responded completely. after therapy. During inolimomab administration, 11 of 21 CMV-seropositive All patients received HLA-identical SCT for AML (10) and CML patients had at least one episode of CMV reactivation. EBV (3). The stem cell source was peripheral blood (11) or bone mar- reactivation occurred in 3 patients. With a median follow-up of row (2) from related (8) or unrelated donors (5). Eleven patients 16 months after tranplantation (range 3-95 months), 57% of the received myeloablative and 2 patients non-myeloablative condi- patients (16/28) were alive. Six patients died of GvHD, 3 died tioning and GvHD prophylaxis consisted of cyclosporine A and of relapses and 3 died of bacterial infections (2 patients in CR methotrexate or mycophenolate mofetil. All patients received from aGvHD and one in PR). 1-2 cycles of rituximab for extensive cGvHD. The treatment In conclusion, long-term inolimomab therapy (≥ 2 months) is was well tolerated in all patients. One patient received less than effective on severe aGvHD, at least in its cutaneous forms. The 4 infusions because of early death from cGvHD. The median most signifi cant mid-term adverse effect of steroid-inolimomab duration of cGvHD prior to Rituximab was 12 (range: 1-36) combination therapy is viral infections, and especially CMV months with a median of 2.5 (0-4) previous immunosuppres- reactivation. The dosing schedule must be further optimized. sive treatments. One patient achieved a CR after Rituximab, 6 a PR and 6 showed no response (NR) to Rituximab. Prednison could be reduced in 5/12 patients (42%) with a mean reduction P571 of 91%. A total of 4/8 (50%) patients with skin cGvHD had a Effective therapy of steroid-refractory acute GvHD with benefi t from the treatment. The best responses were observed CD52 antibody alemtuzumab in patients with sclerodermiform (3/5, 60%), enoral (6/8, 75%), M. Gramatzki (1), N. Schub (1), A. Günther (1), C. Ehlert (1), and ocular cGvHD (4/7, 57%). Rituximab was administered in A. Schrauder (2), R. Repp (1) one patient without previous steroid therapy because of rap- (1)Div. of Stem Cell Transplantation and Immunotherapy (Kiel, idly progressive sclerodermiform cGvHD. No response was DE); (2)Dept. of Pediatrics (Kiel, DE) observed after one cycle of rituximab and the immunosuppres- sive therapy was completed with steroids and extracorporeal In recent years techniques of allogeneic hematopoietic stem photopheresis. One patient with obliterating bronchiolitis had a cell transplantation have improved considerably and allowed normalization of the FEV1 after 1 cycle of rituximab, whereas to use this procedure for older and less fi t patients. However, 2 patients died due to progressive obliterating bronchiolitis 1/3 severe graft-versus-host disease (GvHD) remains the main (33%). obstacle to apply this therapeutic modality. No standard ther- In conclusion, B-cell depletion with rituximab may be an alter- apy has been identifi ed once a patient has developed severe native treatment for steroid-refractory cGvHD. In particular, GvHD and is refractory to high-dose steroids. The pre-emptive patients with sclerodermiform cutaneous, ocular and enoral use of CD52 antibody alemtuzumab as part of the conditioning cGvHD seem to profi t from this treatment. regimen reduces the incidence of GvHD but leads to higher relapse and infection rates. CD52 antibody alemtuzumab was used in 18 patients now evaluable, age range 13-68 years, with GvHD grade III and IV refractory to at least 7 days of high-dose

S139 P573 Results: An 8-color panel was identifi ed for optimal resolution of Thymoglobulin prophylaxis of chronic GvHD after full T cell subsets by staining with monoclonal antibody conjugates matched peripheral blood SCT to CD3, CD4, CD8, CD25, CD127, CD45RA, CD62L and CCR7. Y. Zalyalov, B. Ganapiev, A. Zinchenko, L. Galenko, V. Kostorov, Sample sizes of CD4 T and CD8 T cells available for analysis B. Afanasyev of subpopulations were < 10000 on day 14 for most recipients, St. Petersburg State Medical University (St. Petersburg, RU) while on day 20, > 10000 events could be collected generally for both subpopulations. Regulatory CD4 T cells could be identifi ed Chronic GvHD (cGvHD) is the primary cause of late morbidity as a CD25high/CD127 low subpopulation in most patients start- and non-relapse mortality in transplant survivors. Probability of ing from day 20. Similar to CD4 and CD8 effector T lymphocytes, cGvHD is higher after peripheral blood SCT than after BM SCT. regulatory T cells with a memory phenotype were prevalent. CGvHD more frequently develops after MUD alloHSCT than Conclusions: We have found that 8-color fl ow cytometry allows SCT from matched related donors. Acute GVHD is also among to defi ne multiple T lymphocyte subsets starting from day 20 after the strong risk factors of this complication. CGVHD occurs even transplantation in most recipients despite low lymphocyte counts in SCT from a full matched donor, its severity and occurrence and the small size of clinical specimens available in paediatrics. doesn’t depend on conditioning intensity and it may deteriorate The estimation of T cell subsets including regulatory T cells early the outcome of alloHSCT. after HSCT may be useful to correlate phenotypic profi les with (1) The probability of advanced cGVHD after unrelated allograft the risk to develop severe GvHD or (2) with the responsiveness PBSCT from donors identical in HLA- A*, -B*, -C*, -DRB1* and - to therapeutic efforts undertaken to prevent or to cure GvHD. DQB1* loci were assessed. We analyzed data from 38 pts who underwent allo-PBSCT. For GVHD prophylaxis the patients received CsA with short courses Mtx or mofetil mycophenolate P575 with tacrolimus. All patients received ATG to fortify immunosu- Assessment of bioequivalence of a generic cyclosporine pression effect and decrease GVHD. Group 1 (11 pts) received (Equoral™) by a prospective randomised controlled trial Thymoglobulin with total dose of 7,5 mg/kg b.w.; Group 2 (27 in allogeneic stem cell transplant (ASCT) recipients pts) received ATGAM with total dose 60 mg/kg b.w. Age of T. Ben Othman (1), N. Ben Fredj (2), A. Klouz (2), A. Abdelkefi patients, intensity of conditioning regimens, number of CD34+ (1), S. Ladeb (1), L. Torjeman (1), A. Lakhal (2), M. Lakhal (2) cells in the graft and gender compatibility were comparable in (1)Centre National de Greffe de Moelle Osse (Tunis, TN); the both groups. (2)Centre National de Pharmacovigilance (Tunis, TN) In the follow-up period of 2 years OS was similar in the both groups (63% in Thymoglobulin group vs 55% in ATGAM group, Introduction: The bioequivalence of Equoral has been estab- p=0.8). There was no statistically signifi cant difference in fre- lished by measuring pharmacokinetic parameters in healthy quency of aGVHD II-IV in both groups: in Thymoglobulin group volunteers and in stable renal transplant recipients. To our it was 45% and in ATGAM group it was 63%, p=0.3. Neverthe- knowledge, no study was performed in ASCT recipients. The less, the rate of cGVHD was lower in patients, who received aim of our study is to compare the pharmacokinetics and safety Thymoglobulin (11%) compared to ATGAM group (52%) p= of Equoral solution (IVAX, USA) to Neoral solution (Novartis, 0.05; the most evident decrease was in the number of exten- USA) in ASCT recipients. sive forms (0 vs 43%), respectively (p= 0.02). Patients and methods: Our study was performed at the National Therefore, full HLA compatibility doesn’t eliminate the risk of Centre for Bone Marrow Transplantation (Tunis, Tunisia) cGVHD after allo MUD PBSCT. Thereby we conclude that the between June 2007 and November 2008. This Study consisted use of ATG leads to rate reduction of cGVHD, however Thymo- of an open label, two-way crossover, randomized controlled globulin shows reliably higher effectiveness than ATGAM. trial of Equoral versus Neoral in ASCT recipients. Dose conver- sion was 1:1. The duration of washout period was at least 7 to 14 days. A ten-point blood sampling from 0 to 12 hours was P574 done. The study measures included Cmax (extent of absorp- Eight-colour fl ow cytometry to establish the frequency tion), Tmax (rate of absorption) and AUC0-12h (Area under the of multiple T-lymphocyte subsets early after allogeneic concentration-time curve) calculated by linear trapezoid rule. haematopoietic stem cell transplantation in paediatrics The study protocol was approved by ethic committee. C. Winzler, E. Calore, M. Giordan, M. Gazzola, R. Destro, Results: Thirty patients enrolled the study. The median age was G. Tridello, M. Pillon, S. Cesaro, G. Basso, C. Messina 26 years (range: 6 to 47). The mean pharmacokinetics fea- Clinica Oncoematologia Pediatrica (Padua, IT) tures were: AUC0-12h: Equoral 4162±1231, Neoral 4370±1059 ng.ml/h (p=0.50); Cmax: Equoral 821±244, Neoral 834±298 ng/ Background: Graft-versus host disease (GvHD) is mediated ml (p=0.86); Tmax: 105 minutes for both formulations. Toxicity by donor effector T lymphocytes. Regulatory T cells mediate and acute GVHD were comparable in both groups. peripheral tolerance and protect from GvHD. Polychromatic fl ow Conclusion: This is the fi rst study demonstrating that Equoral cytometry allows to examine manifold antigens displayed by a solution and Neoral solution can be considered bioequivalent single cell and to identify fi ne lymphocyte subsets without the and interchangeable in ASCT recipients. need of multiple tubes and a huge number of cells. Unwanted spectral overlaps increase with the number of dyes employed and require to optimize instrument settings and to select stain- P576 ing combinations carefully. Addition of anti-thymocyte globulin could reduce the Objective: to develop an 8-color fl ow cytomety protocol to dis- GvHD and not increase mortality in unrelated stem cell tinguish T cell subsets including naïve, effector memory, central transplantation memory, effector memory RA+ and regulatory T cells. To evalu- T.-D. Tan ate this approach for clinical use in paediatrics early after allo- Koo Foundation Sun Yat-Sen Cancer Center (Taipei, TW) geneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Antigen groups were created according to the expres- Objective: Since the availability of matched sibling donors is sion modality. Selected dyes were assigned to each group and limited, the search of alternative, such as matched unrelated different combinations of conjugates were tested. Analysis donors is mandatory. Because of increased risk of graft rejec- were performed with a digital eight colour fl ow cytometer. The tion and graft-vs-host reaction, anti-thymocyte globulin (ATG) accuracy of the determination of individual subpopulations was is used to overcome the unrelated HLA barrier. Therefore, we estimated for different sample sizes with normal white blood analyzed how ATG prophylaxis affects the incidence and sever- cells. Clinical use was tested with ten specimens drawn for rou- ity of acute and chronic GVHD in our hematopoietic stem cell tine blood counts on day 14 and day 20 after allo-HSCT. transplantation recipients.

S140 Methods: 58 patients of various hematologic malignancies P577 underwent allotransplant (44 from related and 14 from unre- Alemtuzumab 20 mg only in vivo confers effi cient GvHD lated) between Mar 2001 and May 2008. Related myeloabla- prophylaxis in sibling and matched unrelated HCT tive preparative conditioning includes BUCY, TBI 12Gy+Cy, or A. Spyridonidis, M. Karakantza, A. Symeonidis, E. Triantafyllou, FluBu for AML, CML, and ALL; BEAM for lymphoma; Melpha- E. Tzouvara, A. Kourakli, M. Tiniakou, N. Zoumbos lan 200mg/m² for myeloma. Non-myeloablative conditioning Patras University (Patras, GR) includes BuFlu+ATG for CML; TBI 2Gy+ fl udarabine for lym- phoma. Among all transplant recipients, only patients received Despite standard CyA/MTX GvHD prophylaxis, severe acute myeloablative conditioning and hematopietic stem cells trans- GVHD occurs after ~35% of matched sibling HCT. MTX is plantation from matched unrelated or mismatched related associated with mucositis, delayed engraftment and pulmonary donors were treated with ATG before transplant for GVHD toxicity. Campath anti-CD52 antibody may reduce GvHD by tar- prophylaxis (n=11). The remaining patients received hemat- geting lymphocytes (less in CD56+cells) and dendritic cells (less opoieitc stem cells from matched related donors or non-myelo- in Langerhans cells). 100mg Campath1G (non humanized) in ablative conditioning regimen did not receive ATG for GVHD vivo has been shown to abrogate GvHD without increasing the prophylaxis (n=47). The incidence and the severity of these two risk of relapse (Blood 92:4581). Unfortunately, studies with sim- groups of transplant recipients were compared. ilar doses (100mg) of the humanized Campath1H (alemtuzu- Results: The risk stratifi catins in our related and unrelated mab) revealed increased incidence of relapse and severe viral hematooietic stem cell transplantaiton are cimparable. The infections which may be due to the prolonged in vivo half-life 3-year disease-free and overall survivals of related vs unrelated of Campath1H (21 days) vs Campath1G (13 hours). We report allotransplant are 77.1% and 62.6% versus 58.2% and 58.2%, our single centre experience of low dose (20mg) alemtuzumab respectively. The incidence of grade II~IV and III~IV acute as GvHD prophylaxis in 11 consecutive matched sibling (n=6), GVHD in patients without ATG prophylaxis treatment were related (n=1) or unrelated (n=4) transplantations, performed 48.8% and 26.8%, respectively. In contrast, the incidence of during 1/2007-1/2008. Median pt age was 39y (23-59). Diag- grade II~IV vs III~IV acute GVHD in patients with ATG prophy- noses were AML(n=8), ALL(n=2), MDS-RAEB(n=1). 7pts were laxis were 18.2% vs 0%. The incidence of chronic GVHD in transplanted in CR1 and 4 with advanced disease. Conditioning recipients without ATG prophylaxis was 42% and 10.0% for was myeloablative in 6 cases (BuCy=5, TBI/VP16/Cy=1) and unrelated patients with ATG prophylaxis. reduced toxicity in 5 (Flu/BCNU/Melphalan or Thiotepa). The Conclusions: The results of our study suggest that the use of stem cell source was peripheral blood in all cases. GvHD proph- ATG prophylaxis in recipients undergoing unrelated stem cell ylaxis included alemtuzumab 10mg iv absolute on d-2 and d-1, transplantation with ATG prophylaxis was quite effective to pre- posttransplant CyA with levels >200 until d+60 and thereafter vent the occurrence of acute and chronic GVHD and has com- tapering. All pts engrafted promptly (WBC>1000/µl on d+13(10- parable survival to related stem cell transplant. 19), PLT>50000/µl on d+11(10-14)) and revealed complete BM and PB chimerism on d+30. Stomatitis (Gr I-II) was observed in only 2pts. One patient, who discontinued Cya on d+25 experi- enced steroid/ATG resistant, fatal GvHD (d100 TRM 9%). From the remaining on protocol treated 10pts, none revealed gut or liver aGvHD whereas 5pts (50%) experienced steroid-sensible skin GvHD St I-III between d+16–d+36 (median d+18), result- ing in an overall incidence of aGvHD Gr I-II of 50% and Gr III-IV 0%. CMV reactivation was observed in 8 out of 10 pts at risk but no CMV disease occurred. Cya could be discontinued in all evaluable pts at median on d+135(72-210). 3pts revealed mild skin chronic GvHD and 2 moderate skin cGvHD which resolved with steroids. After a median follow up of 504 days (332-675), 7pts are alive and in CR and 4 died (relapse n=3, TRM n=1). Median Karnofsky is 100% and only 1 pt receives occasionally steroids for skin cGvHD. In conclusion, in vivo Campath 20 mg confers an effi cient schedule for GvHD prophylaxis in sibling and matched unrelated HCT.

P578 Prognostic signifi cance of elevated interleukin-13 levels in patients prior to haematopoietic stem cell transplantation J.L. Harrold, E.A. Leitch, G. Jackson, A.M. Dickinson Newcastle University (Newcastle upon Tyne, UK)

Interleukin-13 (IL-13) is an immunoregulatory cytokine secreted predominately by activated CD4+ T cells. It suppresses the cytotoxic action of macrophages, inhibits the production of pro- infl ammatory cytokines and induces major histocompatibility complex (MHC) class II expression on antigen presenting cells (APC). To examine the role of IL-13 in haematopoietic stem cell transplantation (HSCT) we measured serum IL-13 levels in 31 patients admitted for allogeneic HSCT using an enzyme linked immunosorbent assay (ELISA). All serum samples were obtained at the time of admission and prior to the administra- tion of any preparative treatment. IL-13 levels were correlated with HSCT outcome and in all statistical analyses P values <0.05 were regarded as being signifi cant. No associations were observed between IL-13 levels and the development of acute or chronic graft-versus-host disease (GVHD). Pre-transplant IL-13

S141 serum levels were, however, signifi cantly elevated in patients P580 who relapsed following HSCT (P < 0.001). Moreover, high IL- A pilot study of intra-arterial infusion of a relatively small 13 levels were observed mainly in patients with relapsed acute number of mesenchymal stem cells for treatment of myeloid leukaemia (AML); AML relapse occurred in 6 of 10 steroid-refractory acute graft-versus-host disease patients. Elevated IL-13 levels also correlated with decreased F. Nakamura (1), H. Ohgushi (2), H. Machida (2), A. Fukunaga survival (P = 0.0018), with 7 of 10 patients dying as a conse- (1), H. Hirata (1), N. Arima (1) quence of disease relapse. Due to the small nature of the study (1)The Tazuke Kofukai Medical Research Institute (Osaka, population, our results are currently being verifi ed in a larger JP); (2)National Institute of Advanced Industrial Science and cohort. Despite this, our data demonstrates the prognostic sig- Technology (Hyogo, JP) nifi cance of elevated IL-13 levels in patients prior to allogeneic HSCT and suggests a role for IL-13 in disease relapse. Backgrounds: Steroid-refractory acute graft-versus-host dis- ease (aGVHD) is associated with high mortality. Cell therapy with culture-expanded mesenchymal stem cells (MSCs) P579 infused intravenously was recently reported as a promising Allogeneic haematopoietic stem cell transplantation from treatment for severe aGVHD. Culture with donor’s serum offers the third party donor in a recipient of allogeneic kidney the advantage of safety, but results in the limited expansion graft of MSCs. We report the results of a-single-centre, pilot study E. Snarski, T. Torosian, M. Rokicka, M. Paluszewska, to treat aGVHD with intra-arterial regional administration of a A. Tomaszewska, A. Gronkowska, E. Urbanowska, M. Król, relatively small number of cultured MSCs. L. Koperski, W. Wiktor-Jedrzejczak Patients: Patients eligible for this pilot study were aged over 15 Medical University of Warsaw (Warsaw, PL) years and had grade 2-4 aGVHD that did not respond to steroid treatment. Patients with only skin GVHD or who were treated When allogeneic hematopoietic cells are transplanted to a solid with pulsed corticosteroid ≥1 mg per kg per day) or ATG within organ recipient there is a danger of organ graft rejection as 2 weeks were excluded. The clinical protocol and consent form these hematopoietic cells are HLA matched to organ recipient were approved by the Ethics Committees of the National Insti- and not to organ donor. We report a patient who after trans- tute of Advanced Industrial Science and Technology, and all plantation of the kidney from an unrelated donor, underwent participants in the study gave written informed consent. allotransplantation of peripheral blood stem cells from HLA- Treatment protocols: MSCs derived from each hematopoietic identical sister. stem cell donor’s bone marrow were expanded with the donor’s A 42 year old patient after renal transplantation performed one serum instead of bovine serum. MSCs at a dosage of 0.5 - 106 year earlier was diagnosed with acute myeloid leukemia with cells/kg were infused into the appropriate hepatic artery, and/or signs of myelodysplasia on April, the 8th.2004. Initial treat- gastroduodenal, superior mesenteric and inferior mesenteric ment consisted of induction chemotherapy and resulted in arteries. GVHD was evaluated using international criteria. complete remission. It was followed by consolidation chemo- Results: Three cases that received hematopoietic stem cell therapy (2 cycles) and maintenance chemotherapy (5 cycles). transplantation from their related donors (one was matched, the Leukemia relapse was diagnosed on October the 20th., 2005. others were haploidentical) and developed refractory aGVHD, Patient did not achieve remission after reinduction therapy. confi rmed by histopathology, were enrolled. All patients contin- In view of rapid progression of the disease, the availability of ued their treatment with steroids (n=3), cyclosporin A (n=1) or HLA-identical sister and poor general condition, the patient was mycophenolate mofetil (n=2) at the time of infusion. There were qualifi ed for salvage peripheral blood stem cell transplanta- no obvious side effects after arterial infusion. Two weeks later, tion with reduced intensity conditioning despite the fact that. two of the three patients achieved partial clinical remission from HLA antigens of both marrow donor and recipient were almost gastrointestinal symptoms, and maintained for two months. completely mismatched with the HLA antigens of renal graft. The other patient developed hepatic dysfunction and severe The conditioning regimen consisted of treosulfan and fl udara- cytomegalovirus infections. There were no obvious relapses of bine. There were no major complications observed in the early disease after infusions of MSCs, but adult respiratory distress posttransplant period and the engraftment was complete by the syndrome developed in the two cases. day +13. An acute graft versus host disease (aGvHD) (day +42) Conclusions: Local-arterial MSC infusion may be alternative and chronic graft versus host disease (cGvHD) (bronchiolitis treatment to systemic-intravenous MSC infusion. Further clini- obliterans, day + 97) were diagnosed during the posttransplant cal research is required to achieve satisfactory results with a treatment. On day +128 when outside hospital, patient has relatively small number of MSCs. developed severe pneumonia which lead to a septic shock and death on day + 138. Initially immunosupression consisted of tacrolimus and pred- P581 nisolone. Additionally from day –1 to day +23 posttransplanta- Survival benefi t and lower relapse rate in patients tion mycofenolate mofetil was administered. When symptoms experiencing graft-versus-host disease after allogeneic of aGvHD and cGvHD appeared they were treated with change retransplantation with dose-reduced conditioning from oral steroid to methylprednisolone at 2 mg/kg i.v. S. Eder, N. Worel, A. Schulenburg, W. Rabitsch, A. Rosenmayr, During the entire hematological treatment the transplanted G. Fischer, M. Mitterbauer, P. Kalhs, C. Zielinski, H. Greinix kidney was functioning properly. Creatinine values were at an Medical University Vienna (Vienna, AT) average of 1,6 mg/dL. Conclusion: We report a situation in which an almost complete Introduction: Relapse of malignant disease after high-dose therapy mismatch between the transplanted kidney and donor bone followed by hematopoietic cell transplantation (HCT) has a poor marrow did not result in allogeneic reaction directed against prognosis. Although allogeneic HCT with reduced-intensity con- transplanted organ, even in the presence of aGvHD and ditioning (RIC) is associated with lower transplant-related mortal- cGvHD. ity (TRM), it is controversial whether RIC improves survival after retransplantation due to high rates of recurrent malignant disease. Methods: We retrospectively compared the outcome of 56 patients (32 male and 24 female) who received either myelo- ablative (n=12) or reduced-intensity conditioning (RIC, n=44) for allogeneic retransplantation after fi rst (n=32) or second (n=3) autologous or fi rst allogeneic (n=21) HCT at our institu- tion. Twelve patients received myeloablative and 44 RIC for retransplantation, a median of 24 months after the fi rst one.

S142 Results: Eight patients (7 after MA and 1 after RIC) died within P583 35 days of stem cell infusion. Forty-three of 48 evaluable Analysis of incidence, risk factors, clinical presentation patients (90%, 38 after RIC, 5 after MA) achieved a complete and impact on outcome of chronic GvHD after remission after retransplantation. Eighteen patients experi- reduced-intensity conditioning. A single-centre enced relapse/progression after retransplantation (17 after RIC, experience one after MA). Kaplan-Meier probability of relapse at 5 years is G. Grillo, P. Marenco, E. Zucchetti, P. Brasca, V. Mancini, 50% after RIC and 25% after MA conditioning (p=0.32). Acute G. Bertani, M. Turrini, C. Gabutti, E. Morra and chronic graft-versus-host disease (GvHD) occurred in 24 Niguarda Cà granda Hospital (Milan, IT) patients. Incidence of relapse was 30% in patients experiencing GvHD compared to 62% in patients without GvHD (p= 0.01). Despite the wide use of allogeneic stem cell transplantation Kaplan-Meier probability of TRM is 67% after myeloablative (SCT) with reduced intensity conditioning (RIC), few papers and 21% after RIC HCT (p<0.001). Kaplan-Meier probability of describe chronic GVHD (cGVHD) risk factors, clinical features fi ve-year-survival is 43% after RIC and 25% after MA condition- and treatment responsiveness in this transplant setting. ing for retransplantation (p= 0.015) and 26% in patients without The aim of the study is to analyze risk factors and incidence GvHD compared to 55% in patients experiencing GvHD (p= of cGVHD as well as to investigate clinical characteristics of 0.017), respectively. this signifi cant complication, its response to treatment and its Conclusions: A signifi cantly lower probability of transplant-asso- impact on patients (pts) outcome in RIC transplant setting. ciated mortality was seen after reduced-intensity conditioning Between November 2003 and November 2007, 30 pts affected without a higher risk of relapse. An improved survival after RIC by myelodisplasia or acute leukemia (19), multiple mieloma (7), was observed. Occurence of graft-versus-host disease after lymphoma (4) underwent RIC SCT in our BMT Unit because of allogeneic retransplantation is associated with a signifi cantly age > 55 years or comorbidities. lower risk of relapse and signifi cantly improved overall survival. Conditioning regimen was: Thiotepa 5 mg/kg, Fludarabine 90 In summary, the importance of the graft-versus-leukemia-effect mg/m², Melphalan 100 mg/m². could be confi rmed. GVHD prophylaxis was Cyclosporine (CSA) and short term MTX (10 mg/m² on days +1, +3, +6). No ATG was given. CSA tapering was started on day +90 from transplant. P582 Stem cell source was PBSC in 29/30, from related donor (26) A new approach for the diagnosis of chronic or unrelated donor (4). graft-versus-host disease scleroderma-like: After a median time of 8 months (range: 6-9) 10/26 evaluable preliminary data and clinical implications pts developped cGVHD. E. Massara, M. Cuzzola, M. Martino, R. Monteleone, R. Fedele, Involved organs included skin and mucosae, lungs, muscles, C. Rigolino, E. Spiniello, F. Gatto, T. Moscato, P. Iacopino fascia and joints; cGVHD was categorized as extensive in U.O. Ematologia con Trapianto (Reggio Calabria, IT) 6 pts. Steroid treatment was given to 7 pts, for a median period of The chronic graft-versus-host disease (cGVHD) is the most 24 months, and lead to cGVHD resolution in 5 pts. important and disabling complication after allogenic haemopoi- Among all analyzed parameters, only donor gender (p= 0.009) etic transplantation; in recent years with the increased use of and the number of CD3-positive cells infused (p= 0.0269) were peripheral blood as a source of stem cells, there was a higher signifi cantly related to cGVHD, while no signifi cant association incidence of cGVHD, with major locations in the skin and lung was observed with recipient gender, donor or recipient age, (scleroderma-like). The pathogenesis of this disease is quite CD34-positive cells dose and previous acute GVHD. similar to autoimmune diseases, because of an immunological At a median follow up of 17 months (range 1-57) 18/30 pts were disorder that will result in irreversible damage to target organs alive. or tissues, often hesitating in a permanent disability. TRM was 10%. We observed 9 relapses, 8/9 occurring in pts The only investigation that can pose diagnosis of cGVHD is without clinical evidence of cGVHD. the biopsy of the organ or tissue affected, so that the immuno- Our conditioning regimen and GVHD prophylaxis allowed an suppressive therapy may be initiated only when the damage acceptable cGVHD rate (38%), even without ATG. is already well advanced. Our centre has undertaken a study Patients who developed cGVHD were successfully treated using the analysis of gene expression profi le (GEP) based on with steroid treatment, which, however, was prolonged in most macroarray method to identify and to diagnose early cGVHD cases. scleroderma-like. The purpose of this study is to recognize the Currently there is no consensus regarding the defi nition of risk early onset of cGVHD scleroderma-like and, then, apply the factors for cGVHD after RIC transplant, but they seem to be dif- necessary therapeutic measures, before an irreversible dam- ferent from the well-established ones for full dose setting. age. Starting from the evidence of similarities among patients cGVHD seems to be related to a better overall survival rate suffering from scleroderma and from cGVHD, we tested a panel (p=0.05). of transcriptome of genes associated to immune-infl ammatory/ These results may suggest that immuno-modulating rather than anti-infl ammatory activities, showing particular signal activity in immuno-suppressive agents may have a promising role in the both groups of patients. treatment of cGVHD after RIC transplant. The study selects patients undergoing to allogenic stem cell transplantation (allo-SCT) from sibling donor, HLA-identical, using HPC-A as a source of stem cells for haematological P584 malignancies; about 10 cc of peripheral blood in EDTA were Response of extensive sclerodermatous chronic taken and we performed the gene profi le and MAP-Kinase pro- graft-versus-host disease to an mTOR inhibitor-based teomic array. treatment So far, this method has been applied to six patients, four of them G. Bug, H. Pfeifer, S. Mousset, J. Schwaeble, H. Serve, subsequently developed a cGVHD scleroderma-like, confi rmed H. Martin by histological examination. During the congress will show the J.W. Goethe University Hospital (Frankfurt, DE) data of other patients included in the assessment. The new application of the GEP could allow detection of Sclerodermatous chronic graft-versus-host disease (ScGVHD) patients who are developing a sub-clinical cGVHD. Early diag- is a late complication of allogeneic PBSCT. Manifestations nosis and proper treatment approach may allow, in this light, include severe skin and subcutaneous fi brosis with contrac- starting soon more effective immunosuppressive therapy, so as tures, wasting and frequent infections, leading to substantial to limit the onset of incapacity and preserve the quality of life in morbidity. Besides high-dose corticosteroids, no effective ther- the patients long survivor after allo-SCT. apy has been established. The mTOR inhibitors sirolimus and

S143 everolimus combine immunosuppressive properties with anti- frequency of T (CD3+), CD3+CD4+, CD3+CD8+, activated T proliferative effects on fi brobroblasts and smooth muscle cells (CD3+HLA-DR+), T-regulatory (CD4+CD25+CD127lo) and and may benefi cially infl uence ScGVHD. We report a series NK (CD3-CD56+) cells in common lymphocyte population and of fi ve patients (pt) with extensive ScGVHD treated with an representation of different surface receptors (CD94, CD158a/h, mTOR-based therapy. CD158b, CD158e, CD226, NKp30, NKp44, NKp46) on the NK- Patient characteristics: All pt with a median age of 38 (range, cell membrane. 28-60) years received PBSCT for high-risk AML (n=3) or ALL There were no signifi cant changes of WBC numbers, frequency (n=2) using myeloablative (n=2) or reduced-intensity (n=3) con- of T-, CD3+CD4+, CD3+CD8+, or NK cells, and the expression ditioning. Acute GVHD of the skin (grade I-II; n=3) and/or gut of all investigated NK-cells surface markers (except NKp44) (grade I-III; n=4) was observed in all pt. Median time to onset of during lenalidomide therapy (Student’s t-test) (Table 1). In con- cGVHD was 435 (range, 128-806) days after PBSCT. Chronic trast, the number of Ly (p=0.012; Fig. 1, A), the frequency of GVHD occurred during tapering of immunosuppression in 3 pt activated (p=0.0048; Fig. 1, B), and regulatory (p=0.036; Fig. 1, and following DLI in 2 pt involving the skin (n=5) and mouth C) T-cells as well as of activated NK-cells (NKp44+) (p=0.0008; (n=5), but GI tract (n=2), eyes (n=4) and/or muscles/joints Fig. 1, D) were signifi cantly increased under lenalidomide. (n=3) were also affected. Early signs of cGVHD of the skin pro- These data suggest for the fi rst time that lenalidomide may gressed to ScGVHD in 4 pt within a median of 193 (range, 151- not only enhance the number of common lymphocyte popula- 261) days. Only one patient presented with ScGVHD. tion, but also signifi cantly increases the frequency of activated Everolimus (n=4) or sirolimus (n=1) was initiated at a median T- and NK-cells in peripheral blood of MM patients after allo- of 197 (range, 2-341) days after diagnosis of ScGVHD, aiming SCT. This immunostimulatory effect seems to be an important at trough plasma levels of 3-5ng/ml. Immunosuppressive treat- mechanism for antimyelomatous activity of lenalidomide. ment before or concomitantly with the mTOR inhibitor consisted of mycophenolate mofetil (n=4), prednisolone (n=2), cyclosporin A (n=1) and/or extracorporeal photophereses (n=1). One pt did not receive additional therapy. Results: All but one pt have continously been receiving an mTOR inhibitor for a median of 598 (range, 264-911) days. In one patient, everolimus was discontinued recently because of complete remission of ScGVHD. All other pt achieved signifi - cant clinical improvement and stopped additional immunosup- pressive therapy. No patient experienced progressive ScGVHD while on mTOR inhibitor treatment. Adverse events included infections (grade 3; n=3), hypertriglyceridemia (grade 2; n=2), deep vein thrombosis (grade 3; n=1) and hypothyreoidism (grade 2; n=1). None of the pt relapsed. Conclusion: For pt with extensive ScGVHD, mTOR inhibitor- based therapy appears to be safe and effective. Further studies including larger cohorts of patients are warranted.

Multiple myeloma

P585 Lenalidomide therapy increases the frequency of activated T- and NK-cells in patients with relapsed multiple myeloma following allogeneic stem cells transplantation M. Lioznov, P. Freiberger, Y. Hildebrandt, U. Bacher, A.R. Zander, N. Kröger University Medical Centre Eppendorf (Hamburg, DE)

Recent data suggest the signifi cant success of lenalidomide therapy in patients with multiple myeloma (MM) who show relapse after allogeneic stem cells transplantation (allo-SCT). This might be explained by its immunostimulatory properties. The ability of lenalidomide to enhance natural killer (NK) cells and to stimulate cytotoxic activity of T-lymphocytes was shown in vitro and in murine models but however has not been sys- tematically adressed in patients with MM. In this study we performed analysis of cellular immune response in eight MM patients (7 males and 1 females; median age: 54,3 years, range: 39-68) who relapsed ≥6 months (median: 19,6 month, range: 6-81) after allo-SCT and were treated with lenalidomide. Lenalidomide was administred for 21 days at 10-25 mg/day, followed by 1 week interruption. Peripheral blood samples were collected before and during lenalidomide therapy at two time-points: (i) between day 30 and 60 and (ii) between day 90 and 120 after start of treatment). Using cell counting and four-colour multiparameter fl ow cytometry (MFC) the following parameters were determined: the number of white blood cells (WBC) and lymphocyte (Ly) per µl whole blood;

S144 P586 We retrospectively studied a series of 23 patients with relaps- Thalidomide + dexamethasone as maintenance after ing MM who underwent allogeneic transplantation after NMA- single autologous stem cell transplantation improves C and compared their outcome with those of patients who progression-free survival in advanced multiple myeloma. relapsed but were not allografted. The propensity score (PS) A prospective Brazilian randomised trial methodology was used to correct for potential recruitment A. Maiolino (1), V. Hungria (2), G. Oliveira-Duarte (3), bias. The idea was to model, for each patient with MM alive D. Mercante (1), E.C.M. Miranda (3), A. Quero (2), A. Miguel at 6 months after relapse, the probability of receiving NMA-C Peres (2), P. Tanaka (2), L. Oliveira (4), R. Magalhães (1), transplantation, according to a set of baseline characteristics E. Rego (4), M Nucci (1), I Lorand-Metze (5), C. Lima (3), (age, serum beta2 microglobulin level (beta2M), and time to I. Zalcberg (5), E. Braggio (5), C. de Souza (3) progression (TTP) after the fi rst autologous SCT). This PS was (1)Universidade Federal do Rio de Janeiro (Rio de Janeiro, BR); estimated using logistic regression and was then used to match (2)Santa Casa da Misericordia (São Paulo, BR); (3)Unicamp 1:1 patients with similar propensity to receive allograft. Data of (Campinas, BR); (4)USP (Ribeirão Preto, BR); (5)Unicamp patients treated in the MAG-95 and -02 trials were used. (Campinas, BR); (5)INCA (Rio de Janeiro, BR) Twenty-three patients with MM in fi rst (n=21) or second (n=2) relapse were treated with high dose therapy followed by Autolo- Introduction: Autologous stem cell transplantation (ASCT) gous SCT preceding allogeneic SCT with a 2 Gy TBI NMA- remains the mainstay of treatment of multiple myeloma (MM) C. Donors were HLA-identical siblings in 13 (56%) patients. in patients <65 years old. However, most patients relapse after Median age at allograft was 50 years. Median follow-up after ASCT suggesting that additional treatment is needed. The Bra- allograft was 27.4 months. Post-allograft response was CR in zilian Multiple Myeloma Group designed a study to evaluate the 9 patients, VGPR in 8, PR in 4 and progressive disease (PD) impact of thalidomide maintenance after ASCT. in 2. TRM at one year was 13%. Two patients died from PD Methods: From October 2003 to July 2008, 212 untreated in the year after allograft. Acute GvHD occurred in 19 patients patients <70 years old were enrolled in a prospective rand- (15 grade I/II, 4 grade III/IV) and chronic GvHD in 11. Among omized multicenter study. All patients signed an informed con- the 10 patients (41.6%) who relapsed with a median of 10,7 sent and the protocol was approved by the Ethical Committees months, 6 are alive with a median survival of 38.3 months. So of each center. The treatment consisted of 3 phases: (1) induc- far, 8 patients (34,7%) including 5 in CR and 3 in VGPR are still tion with 3-5 cycles of VAD; (2) high-dose cyclophosphamide alive without relapse with a median follow-up of 36.8 months. (4g/m²) plus G-CSF for stem cell mobilization; (3) melphalan From the 23 allografted patients, 21 matched pairs were suc- 200 mg/m² and ASCT. On day +60 post ASCT patients were cessfully constituted. Based on these matched pairs, the esti- randomized to receive dexamethasone (40 mg/d x 4 days every mated hazard ratio of death was 0.35 (95% confi dence interval: 28 days) with (arm A) or without (arm B) thalidomide (200 mg 0.14-0.88, p=0.027) for allografted patients compared with non daily) for 12 months or until disease progression. allografted. In conclusion, PS matching method here suggests Results: The median age was 55 years (27-70), 52% were male, that NMA-C allograft in MM patients in fi rst relapse provides a the median serum beta-2 microglobulin was 3.66 mg/dl, 33% high and durable response rate with a low TRM. These promis- were ISS stage 3, 36% were ISS stage 2 and 24% had deletion ing results must be further evaluated in clinical trials. of chromosome 13. In July of 2008, 93 patients (44%) were randomized: 54 in arm A and 39 in arm B. Reasons for non- randomization were: treatment related deaths during phases 1-3 (n= 39), disease progression (n= 22), ineligible or refused ASCT (n= 7), SMD after ASCT (n= 1), protocol violation (n= 3), abandoned (n= 19), and still in phases 1-3 (n= 28). Clinical characteristics of each group were similar. The median follow- up from diagnosis was 15 months. Progression-free survival (PFS) in arms A and B were 42% (95% confi dence interval [CI] 22-62) and 25% (95% CI 5-45), p= 0.07. A multivariate analysis that included baseline serum beta-2-microglobulin and deletion of chromosome 13 showed that maintenance with thalidomide was signifi cantly associated with better PFS (hazard ratio 2.43, 95% CI 1.10–5.35, p=0.03). Overall survival was 65% in arm A (95% CI 35-95) and 74% in arm B (95% CI 44-100), p= NS. Conclusions: A high proportion of MM in Brazil has advanced disease at diagnosis, and this explains the high number of patients who did not reach the maintenance phase. This study shows that the addition of thalidomide to dexamethasone improves PFS after a single ASCT.

P587 Application of the propensity score matching method to the estimation of survival benefi t of non-myeloablative allogeneic transplantation in patients with multiple myeloma relapsing after a fi rst autologous transplantation L. Karlin, B. Arnulf, S. Chevret, M. Robin, R. Peffault de Latour, M. Malphettes, N. Kabbara, B. Asli, L. Ades, V. Rocha, J.-P. Fermand, G. Socié Saint-Louis Hospital (Paris, FR)

Despite recent advances, Multiple Myeloma (MM) is still an incurable disease with a poor prognosis at relapse. The use of conventional allogeneic hematopoietic stem cell transplan- tation (SCT) is limited by a high transplantation-related mor- tality (TRM). Allografting with nonmyeloablative conditioning (NMA-C) has therefore been considered to improve survival.

S145 P588 EU21 was a Phase II study in patients with MM and non-Hodg- Outcomes in patients with multiple myeloma following kin’s lymphoma (NHL) which examined the safety and prelimi- autologous stem cell transplantation using intravenous nary effi cacy of plerixafor when given in combination with G. busulfan and melphalan: a matched comparison to a Thirty-fi ve patients (31 MM and 4 NHL) received a mobilizing double autologous transplant strategy conditioned with regimen of G (10 mcg/kg) each morning for 4 days. Starting melphalan and CBV the evening of Day 4, patients received plerixafor 240 mcg/kg. M. Blanes (1), J. de la Rubia (1), J.J. Lahuerta (2), J.D. González Apheresis was initiated in the morning, 10 to 11 hours following (3), P. Ribas (4), C. Solano (5), A. Alegre (6), M.A. Sanz (1) the dose of plerixafor. Tumor cell mobilization was evaluated (1)Hospital La Fe (Valencia, ES); (2)Hospital 12 de Octubre using a very sensitive polymerase chain reaction (PCR)-based (Madrid, ES); (3)Hospital Insular (Las Palmas, ES); (4)Hospital technique in a sub-population of 7 patients with MM. Patient Doctor Peset (Valencia, ES); (5)Hospital Clínico (Valencia, ES); bone marrow (BM) samples were collected to establish the (6)Hospital La Princesa (Madrid, ES) primer for the PCR assay. Patient BM and peripheral blood (PB) samples were collected at three time points to assess Introduction: In multiple myeloma (MM) we have reported a high TCM: screening (pre-G), after G treatment but before plerixa- response rate with a novel conditioning regimen using intrave- for, and following G and plerixafor. Quantitative allele specifi c nous busulfan and melphalan (ivBuMel). We present results oligonucleotide PCR was performed to detect and assess the of a retrospective matched-pair analysis comparing outcomes mobilization of MM tumor cells for each patient at all three time of MM patients transplanted using the ivBu-Mel conditioning points. This result was then used to calculate the frequency regimen with matched controls receiving an autologous hemat- of tumor cells compared to total peripheral blood mononuclear opoietic stem cell transplant (ASCT) after standard melphalan cells (PBMCs). followed by a second ASCT for patients not achieving complete In the overall patient population, all patients collected ≥ 2×106 remission (CR) after the fi rst transplant (Control group). CD34+ cells/kg. Following treatment with G alone, just prior to Material and Methods: Forty three cases and 86 matched con- administering plerixafor, mobilization of tumor cells was detected trols were available for comparison. Patients in the Control with a minimum of 9.70×10-6 and a maximum of 1.38×10-3 tumor group were transplanted from 2002 to 2005, while patients in the cells per total PBMC. After plerixafor treatment, 3/7 patients ivBuMel group underwent transplant between 2005 and 2008. had a small increase and 4/7 patients a small decrease in PB Controls were selected to match on sex, age, Durie-Salmon tumor cells. The observed fold increase in tumor cells from and ISS stage at diagnosis, and disease status at transplant. before plerixafor administration to pre-apheresis ranged from ivBuMel conditioning regimen consisted of a single dose of 3.2 0.11 fold to 1.08-fold. mg/kg (days -5 to -3) followed by Mel at a dose of 140 mg/m² In a sub-group analysis, plerixafor did not appear to contrib- (day -2). Standard melphalan 200 mg/m² was the conditioning ute to TCM above that which occurs with G mobilization alone. regimen administered to the Control group in the fi rst ASCT. These fi ndings are consistent with other phase II clinical stud- The preparative regimen administered to those patients in the ies, which reported negligible TCM after plerixafor treatment. Control group undergoing a second transplant consisted of cyclophosphamide, BCNU and etoposide (CBV). Results: There were no differences in the hematopoietic recov- P590 ery and mild or moderate mucositis was the toxicity most fre- Outpatient stem cell transplantation for multiple myeloma quently observed in both groups. Fever was seen in 35 cases A. Ghavamzadeh, K. Alimoghaddam, A. Karimi, A. Manookian, in the ivBuMel group and in 66 cases in the Control group, M Asadi, R. Maheri, A.R. Shamshiri (p = NS). No patient developed sinusoidal occlusive syndrome. Hematology-Oncology and Stem Cell Transplantation Research Two patients died due to transplant-related complications in the Center (Tehran, IR) ivBuMel group and 1 in the Control group. Overall, 14 (16%) patients in the Control group underwent the planned second Introduction: The aim of this study was to explore the feasibility ASCT. After ASCT, 49% of patients were in complete remission and safety of performing aoutologous stem cell transplantation in the ivBUMel group vs. 51% in the controls. Thirty-months (ASCT) on an outpatient basis. overall and progression free survival was 79±8% and 47±1% Methods: Total of 134 patients affected by multiple myeloma in the ivBuMel and 74±5% and 46±1% in the Control group, (MM) in complete remission (CR) or partial remission (PR) respectively. were selected to receive ASCT on an outpatient or inpatient Conclusions: These results suggest that the use of ivBu-based basis. Our analysis consist of 100 patients. Median age was regimen for ASCT in patients with MM is a well tolerated con- 50.2 years (ranged 27-68) with 70% male. In the inpatient ditioning regimen associated with a low transplant-related mor- group 34 patients received 200 mg/m² and 21 patients received bidity and mortality that compares favourably with a double 140 mg/m² melphalan as conditioning regimen respectively. transplant strategy with melphalan and CBV. Further follow-up In outpatient group 13 patients received 140 mg/m² and 32 is necessary to ascertain whether ivBu will favourably infl uence patients received 200 mg/m² melphalan. In outpatient group all time to progression and overall survival. the patients were programmed to go home the day after ASCT and to be re-hospitalized in the case of febrile neutropenia or other sever toxicities. We used caregiver, general physician, P589 staff nurse as an outpatient and visit team and also unequipped Effect of plerixafor plus G-CSF on tumour cell routine house of the patients during neutropenia. mobilisation in patients with multiple myeloma Results: Median hospital stay were 25 days in inpatient and. S. Fruehauf (1), J. Topaly (1), S. Muller (1), M. Moos (1), 4.8 days in outpatient respectively (p<0.01). There were not H. Goldschmidt (1), A.D. Ho (1), G. Calandra (2) signifi cant differences between these groups in aphresis days, (1)University of Heidelberg (Heidelberg, DE); (2)Genzyme granulocyte colony stimulating factor requirement for mobiliza- Corporation (Cambridge, US) tion and mononuclear cell, There were also signifi cant reduction (p<0.001) in parenteral antibiotic, blood product requirement Tumor cell mobilization (TCM) can occur following G-CSF (G) and need for total parenteral nutrition. The most frequent mobilization of hematopoietic stem cells (HSC), as well as other causes of re-admission in 6 patients (outpatient group) were mobilization methods such as chemotherapy. Given that plerixafor febrile neutropenia and sever mucositis need TPN. 2 years is administered in combination with G to enhance HSC mobiliza- overall survival rate was 98.2% (SE=2%) in inpatient groups tion, it is important to understand whether or not plerixafor results and 86.6% (SE=5.7) in outpatient groups. in TCM at levels above that observed with G alone. The purpose Conclusion: The ease of administration of high dose melphalan of this report is to assess the effect of plerixafor plus G on TCM in as well as the lack of excessive extramedullary toxicity including patients with multiple myeloma (MM) enrolled in EU 21. nausea and vomiting renders patients with MM more suitable

S146 for outpatient management, in the present study, we describe Objective: To report the long-term outcome of patients who an outpatient program based on management of the patient in underwent a single ASCT at our institution. his/her house during aplastic phase. Our results clearly indicate Patients and Methods: From March 31st 1994 to December 31st that such a procedure is feasible and safe in a patient popula- 2003, 95 patients (median age: 54 yrs; M: 57, F: 38; IgG: 55, tion with an assessable caregiver. IgA: 21, light chain: 15, others: 4) underwent a single ASCT at our institution and had a minimum possible follow-up of 5 years. The initial chemotherapy treatment consisted of VBMCP/VBAD P591 (40 patients), VCMP/VBAP (15 patients), VAD (11 patients), Detrimental effect of platelet contamination on the quality others (27 patients). Sixty-seven patients received the ASCT of stem cells products collected to autograft patients with as intensifi cation of an initial response (upfront group) while in multiple myeloma 28 the ASCT was performed for relapsed or refractory disease A. Leon (1), L. Garderet (2), C. Soler (1), H. Rouard (1), (relapse/refractory group). The high-dose regimens consisted N.C. Gorin (2), P. Bierling (1) of: MEL-200 (49 patients), MEL140-TBI (31 patients) and (1)EFS Ile de France (Paris, FR); (2)Hopital Saint-Antoine Busulphan-12/MEL-140 (BU/MEL) (15 patients). Response, (Paris, FR) relapse and progression were evaluated according to the EBMT criteria. Patients with Multiple Myeloma (MM) in our institution receive Results: The response after ASCT was: CR (31%), PR (63%), Velcade and Dexamethasone (VD) for induction and are then MR (2%), SD or PD (3%). The median time to progression consolidated with high dose melphalan followed by one or two (TTP) from transplant was 30 months (40 vs. 17 months for autografts according to the IFM protocols. the upfront and the relapsed/refractory groups, respectively, We monitor the ratio CFUGM 104/ CD34 106/Kg (sup 8) in all p = 0.01) and the median overall survival (OS) 59 months apheresis products as an indicator of quality for the graft. In (78 vs. 30 months for the upfront and the relapsed/refractory recent months, we were alarmed by several unexpected low groups, respectively, p = 0.03). The median TTP for patients ratios (< 5) which led us to cancel the graft procedure and initi- who achieved CR and PR was 78 vs. 18 months, repectively, ate new aphereses. p < 0.001). After a median follow-up of 8.2 years, 11 of the 95 We retrospectively studied the quality of the apheresis products patients (11%) remain in continued CR from 8 to more than 14 in relation to the induction treatment VD or VAD (vincristine, years post-ASCT, this resulting in a PFS and OS plateau. Eight adryamycine and Dexamethasone) and the mobilisation proce- of these 11 patients received the ASCT as part of the upfront dure Cyclophosphamide (CY) (4g/m2) plus growth factor (GF). therapy (in continued CR from 8 to 14 years) while the remain- We focussed on the leukaphereses (LK) in the different groups ing three underwent the ASCT for relapsed/refractory disease with a ratio CFUGM 104/ CD34 106/kg under 5. (in continued CR from 10 to 14 years). Patients and methods: 114 MM treated between the years Conclusions: The long-term results of the present series with 2000-2006 at Hopital Saint-Antoine Paris were analyzed.. 11% of patients surviving in continued CR at 8 to 14 years from Three groups were identifi ed: group 1 (CY and GF) – Group 2 ASCT suggest that, a portion of patients with MM can be cured (VAD and 5 days GF) – Group 3 (VD and 5 days GF). During with a single ASCT. this period the procedure to harvest cells was not modifi ed and performed on a Spectra equipment (Gambro BCT). Results (median): Group 1/2/3. Number of patients 47/29/38. P593 Number of LK : 73/59/79. Granular cells % 44.5/21/29. Total Autologous peripheral blood stem cell transplantation in granular cells per LK : 149.5/91.13/143.63. CD34 106/Kg/LK: 5 patients affected by POEMS syndrome 2.81/3.37/2.70. Total CD34 106/mobilization: 6.76/8.21/7.69. L. Laurenti (1), S. De Matteis (1), P. Chiusolo (1), N. Piccirillo Ratio CFUGM/CD34: 9.53/9.55/8.24. Platelet 1011/LK: (1), F. Sorà (1), M. Tarnani (1), S. Bellesi (1), C. Giannotta (2), 2.28/5.71/5.56. E. Nobile Orazio (2), G. Leone (1), S. Sica (1) Patients presenting a Low ratio (<5) were identifi ed in each (1)Universita’ Cattolica del Sacro Cuore (Rome, IT); (2)Milan group: number of patients: 3/1/8. ratio: 1.72/3.85/3.27. platelets University (Rozzano Milan, IT) 1011/LK: 6.40/8.62/9.19. Stored overnight before freezing LK: 2/1/8. POEMS is a multisystemic paraneoplastic syndrome, the acro- Conclusions: Leukapheresis products with a very low ratio nym refers to Polyneuropathy, Organomegaly, Endocrinopathy, are highly contaminated with platelets. The overnight storage M protein, Skin changes. We treated 5 pts affected by POEMS at 4°C of such a quantity of platelets in a small volume may syndrome with high dose chemotherapy and autologous periph- induce various damages, such as modifi cation of the cell oxy- eral blood stem cell transplantation (aPBSCT). Pts were M/F gen metabolism and diminution of pH . We recommend har- 3/2, median age 54ys (range 44-62). All pts had a severe, rap- vesting and freezing the MM apheresis products the same day, idly progressive sensory-motor peripheral neuropathy, involv- especially for patients under VD who have a higher level of ing extremities, with inability to walk. All pts had M component platelets before harvesting. IgA-lambda and 1 had also M component IgG-lambda, all had plasmacytosis (7-10%) in bone marrow. Endocrinopathy was present in all pts as thyroid disease and in 1 patient as hypogo- P592 nadotropic Hypogonadism, in 1 pts as hypophysary adenoma, Is there a curative potential of autologous stem cell in 1 pts as glucose intolerance. All pts presented melanosis. 2 transplantation in multiple myeloma? Long-term results patient had splenomegaly, and 3 hepatomegaly. A patient had from a single-centre series sclerotic bone lesion. One patient had signifi cantly abnormal M. Rovira, L. Rosiñol, F. Fernández-Avilés, C. Martínez, pulmonary function before aPBSCT. All pts received Cyclo- E. Giné, J. Esteve, M.T. Cibeira, P. Marín, E. Carreras, phosphamide 1500 mg/m² on day 1,3 and Methylprednisolon J. Bladé 250 mg from day 1-4 for 2 cycles and G-CSF 5 mcg/kg/day Hospital Clínic (Barcelona, ES) was added after 2° cycle for mobilization. 3 pts were previously treated with high dose Ig i.v. and steroids in the neurologic unit. Background: Autologous stem cell transplant (ASCT) is the gold Time from diagnosis to aPBSCT was 4 months (range 3-7). standard in the upfront therapy of younger patients with multiple Conditioning regimen was HDMel (Melphalan 100 mg/m2 for 2 myeloma (MM). It is considered that ASCT is not curative. How- consecutive days). Engraftment was rapid and sustained. After ever, Barlogie et al (Br J Haematol 2006;135:158) reported that a median follow-up of 55 months (range 3-67), all pts are alive 16 out of the 231 (7%) patients enrolled in the Total Therapy I, with slow but progressive improvement in neurological disease, including tandem ASCT, were in continued CR after a median skin changes, organomegaly, performance status and without follow-up of 12 years, suggesting a possible cure. evidence of plasmacytosis. Negativization of M component was

S147 observed in 4 pt. Pt with sclerotic bone lesion also received with lenalidomide received a median number of 5 courses radiotherapy. VEGF level was only performed after aPBSCT, (range 2-14), in all cases with dexamethasone. The responses but in our results (see table) this parameter was not correlated to lenalidomide were as follows: 2 CR, 2 VGPR, 6 PR, one with clinical and laboratoristics improvement. Our experience SD, and 4 PD. The median DOR was 6 months (range 2-14). confi rms that HDMel and aPBSCT is feasible and effi cacious Toxicities were neuropathy for bortezomib, and neutropenia for and should be the treatment of choice for POEMS, arresting and lenalidomide. Only a reversible grade III lenalidomide-associ- even reversing the disease course. Early diagnosis is important ated hepatic toxicity was reported. No grade IV toxicities were to obtain best response and improve clinical outcome. aPBSCT observed. In one case GVHD worsened during treatment with might be safety be performed at experienced transplant centres bortezomib. In conclusion bortezomib and lenalidomide are combined to neurological expertise. We did not observe cor- promising treatments for MM patients in the allo-HSCT setting. relation between VEGF level and clinical improvement, but this It is reasonable to explore their use, also in combination with data should be confi rmed in the follow-up to clarify the role of immunological manoeuvres. bevacizumab, anti-VEGF antibody, as new therapeutic option for patients who can not perform transplant or relapse after aPBSCT. P595 Ethnicity and autologous stem cell transplantation in myeloma H. Auner, C. Giles, J. Pavlu, E. Olavarria, D. Macdonald, E. Kanfer, J. Apperley, A. Rahemtulla Hammersmith Hospital (London, UK)

The results of previous observations indicate that access to and outcome of autologous stem cell transplantation (ASCT) for mul- tiple myeloma may be different for African Americans compared to white Americans. The effect of Asian ethnicity is unknown, and the role of ethnic background on myeloma treatment has not been investigated outside the US. We have analysed the impact of ethnicity on ASCT in all myeloma patients referred to a large UK transplantation centre over a 15-year period. Hammersmith Hospital is a tertiary open-access referral centre P594 for SCT with a catchment area that primarily covers Northwest Bortezomib and lenalidomide for multiple myeloma London. A total of 311 patients with a diagnosis of myeloma were patients relapsed after allogeneic stem cell referred to our institution between 1994 and 2008 for ASCT. 235 transplantation were Caucasians, 47 were of African, and 29 of Asian back- P. Corradini (1), V. Montefusco (1), B. Bruno (2), E. Merla (3), ground. All patients received single Melphalan-conditioned R. Sorasio (2), A. Olivieri (4), A. Falcone (3), L. Giaccone (2), ASCTs. There were no differences in baseline patient demo- L. Farina (1), F. Spina (1), A. Dodero (1), R. Milani (1), graphics and disease status at the time of ASCT between the eth- A. M. Carella (3), N. Cascavilla (3) nic groups. The proportion of ethnic minorities undergoing ASCT (1)Istituto Nazionale Tumori (Milan, IT); (2)Ospedale San for myeloma more than doubled from 14% in the fi rst to 31% in Giovanni Battista (Turin, IT); (3)”Casa Sollievo della Sofferenza” the last 5-year observation period, while the absolute number of Hospital (San Giovanni Rotondo, IT); (4)Ospedale San Carlo Caucasians receiving ASCT for myeloma remained stable. For (Potenza, IT) the entire study group, PFS after ACST was 19.7 months, and OS from diagnosis was 71.3mo. Progression-free survival (PFS) Allogeneic stem cell transplantation (allo-HSCT) represents an after ASCT was signifi cantly shorter in Africans (14.9m) com- effective treatment for multiple myeloma (MM). However, clini- pared to Caucasians (21.7m, p=0.02). In Asians, there was a cal relapse or progression after allo-HSCT are common and trend towards shorter PFS after ASCT (15.9m), but this was not new therapeutic strategies are needed. New drugs, in par- signifi cant. Overall survival (OS) from diagnosis (Africans, 65m; ticular bortezomib and lenalidomide, are appealing for several Asians, 76m; Caucasians, 73.8m) and from the time of ASCT reasons: 1) their toxicity is not overlapping with chemotherapy; (Africans, 46.1m; Asians, 36.4m; Caucasians, 56.9m) were not 2) the mechanism of antitumor activity is different from that of signifi cantly different between the ethnic groups. chemotherapy; 3) their activity encompass also modulation of These observations suggest that access to ASCT for myeloma immune cells, with potential effects on the graft versus-mye- has improved for ethnic minorities in the UK, probably because loma effect. In order to evaluate the clinical role of bortezomib of improved referral rates rather than changes in population and lenalidomide after allo-HSCT, we reviewed data from 23 demographics. Although patients of African ethnicity have a patients treated at 4 Italian institutions. Median age at trans- signifi cantly shorter PFS after ASCT, OS after transplantation plantation was 53 years (range 35-64). Twenty-one patients and from diagnosis is not different. Further studies are required underwent a reduced-intensity, and 2 a myeloablative con- to investigate if myeloma patients of African background benefi t ditioning regimen. Donors were HLA-identical siblings in 17 from ASCT to the same extent as other ethnic groups. patients, and matched unrelated donor in 6 patients. Eleven (48%) patients developed acute GVHD, and 15 (65%) chronic GVHD (9 limited, 6 extensive). All patients relapsed or pro- P596 gressed after allo-HSCT. Eight patients were treated with bort- Vinorelbine and pegfi lgrastim for mobilization of ezomib, 7 with lenalidomide, and 8 received bortezomib and, peripheral blood progenitor cells in patients with multiple subsequently, lenalidomide. The median interval between allo- myeloma: a predictive and cost-effective procedure HSCT and bortezomib was 31 months (range 7-144), and 27 M.J. Bargetzi (1), T. Pabst (2), A. Schoenenberger (1), months (range 7-71) for lenalidomide. The 16 patients treated J. Burger (1), P. Fernandez (1), P. Moosmann (1), A.R. Huber with bortezomib received a median number of 3 courses (range (1), M. Wernli (1), M. Heizmann (1) 1-7), in all cases with dexamethasone, in 8 cases also with tha- (1)Kantonsspital (Aarau, CH); (2)University Hospital (Berne, lidomide, and, in one case, in association with doxorubicin. The CH) responses to bortezomib were as follows: 5 CR, 4 VGPR, 4 PR, 2 SD, and 1 PD. The median duration of response (DOR) Several mobilization regimens are used to mobilize stem cells was 10 months (range 1-43). Longer DOR were observed in for transplantation in multiple myeloma, but timing of collection patients also receiving thalidomide. The 15 patients treated is often diffi cult and the rate of side effects is quite high. To

S148 optimize timing of collection of peripheral blood stem cells, to 55.5% of the cases no partner gene has been identifi ed. Three reduce side effects and costs of the procedure, we evaluated prognostic categories were distinguished: good prognosis vinorelbine, a drug shown to have activity in multiple myeloma, (t(11;14)), intermedier prognosis (normal FISH results, IgH gene with a single dose of pegfi lgrastim as mobilizing regimen. abnormalities without identifi cation of a partner gene, t(11;14) + Thirty consecutive patients with newly diagnosed stage I to III the presence of a poor prognostic marker) and poor prognosis multiple myeloma received 2 - 4 cycles of VAD (vincristine, adri- (the presence of any unfavorable genetic prognostic marker). amycin, dexamethasone) induction therapy at two transplan- 22% of the patients belonged to the good, 33% to the poor and tation centers. This was followed by one dose of vinorelbine 45% to the intermedier prognostic category. There were signifi - 35mg/m² iv in an outpatient setting on day one and a single cant survival differences (p= 0.01) between the different genetic dose of pegfi lgrastim 6mg s.c. on day 4. prognostic groups of patients. On day eight a median count of 52.5 CD34+ stem cells/micro- Autologous stem cell transplantation defi nitely improves survival liter (range 2 - 320) was measured in the blood of 28 patients. in patients with multiple myeloma. Cytogenetical examinations Two patients already showed CD34+ cells of 51 and 60 at day play an important role in the defi nition of different prognostic 7. One stem cell apheresis was suffi cient to collect a median of categories that represent signifi cant survival differences of 8.9 x106 CD34+ cells/kg body weight (range 1.2 –36.6). Four these patients. patients (13%) needed two stem cell aphereses to achieve a suffi cient number of stem cells for transplantation. Twenty-one (70%) of the 30 patients mobilized had their fi rst apheresis on P598 day 8, 2 (7%) on day 7, and 7 patients (23%) on day 9. No CD66A expression on plasma cells from multiple episodes of fever in neutropenia or other side effects were myeloma patients observed during mobilization. After high dose chemotherapy C.H. Lee, B. Guinn, S. Brooks, D. Richardson, K. Orchard with melpahalan and stem cell transplantation, recovery of neu- Southampton General Hospital (Southampton, UK) trophils was within the expected range, indicating no harmful effect of the mobilization scheme to the stem cells collected. Background: Expression of CD66, a member of the carcinoem- The combination of vinorelbine and pegfi lgrastim is an effec- bryonic antigen (CEA) and immunoglobulin superfamilies, has tive alternative to cyclophosphamide in the mobilization of been reported on granulocytes. Phase I and II clinical trials at stem cells for autologous transplantation in multiple myeloma. our centre utilise this property for the delivery of targeted radio- Results are reliable and day of apheresis is highly predictable. therapy as part of the conditioning regimen for transplantation As all the mobilizing and collection procedures were done in in acute leukaemias and multiple myeloma. In these studies an out patient setting, this mobilization scheme is cost saving the vector for the application is conjugated to one of two radio- compared to the mobilization with cyclophosphamide, that usu- isotopes: Indium-111, a gamma radiation-emitting isotope for ally requires hospitalization. In addition, a single dose of pegfi l- imaging to demonstrate bone marrow targeting and Yttrium- grastim averts the problem of non-compliance of daily fi lgrastim 90, a beta-emitter which delivers therapy. Previous stud- injections and improves acceptance of the treatment by the ies revealed abnormal expression of CD66 and its isoforms patient. (CD66a to CD66e) in tumour cells. Preliminary studies at our centre suggested the presence of CD66 antigen on normal and malignant plasma cells. However, little is known about the dif- P597 ferential expression pattern of its variants on plasma cells from Genetic prognostic factors and the outcome of patients with multiple myeloma. autologous stem cell transplantation in plasma cell Objective: We performed fl ow cytometry on myeloma cell lines disorders and plasma cells from patients with multiple myeloma to evalu- M. Dávid, S. Kosztolányi, Á. Szomor, D. Alpár, B. Kajtár, Á. Nagy, ate the expression of the CD66 variants. G. Kovács, R. Csalódi, J. Hermesz, J. Tábori, C. Szalontay, Methods: Fresh bone marrow samples were obtained from H. Losonczy, L. Pajor patients at Southampton General Hospital between 2007 and University of Pécs (Pécs, HU) 2008. Mononuclear cells expressing CD138, a specifi c plasma cell marker, were either positively selected (Easysep magnetic Between December 1999 and May 2008 104 autologous stem separation kit) or incubated with saturating amounts of CD138 cell transplantations were performed in 103 patients with plasma and a single CD66 monoclonal antibody (CD66a and d: R&D, cell disorders in our transplant center in Hungary. The patients Genovac; CD66b and e: AbD Serotec; CD66c- Santa Cruz). mean age was: 54.48 years. Sex distribution: 56 males, 47 Similar analysis was performed on two human myeloma cell females. The diagnosis was multiple myeloma in 98, multiple lines, U266 and ARH77. plasmocytomas in 4, acute plasma cell leukemia in 1 patient. Results: Data acquisition and analysis were performed using The conditioning regimen was 200 mg/m² melphalan in the FACScalibur, CellQuest Software (Becton Dickinson). 100 cases, 120-160 mg/m² melphalan in 3 patients with kid- Plasma cells were gated according to forward and side scatter ney failure and in 1 case with a second transplantation. We characteristics and CD138 expression. administered the median of 4.9x106/kg CD34 positive cells on In all clinical bone marrow specimens, positive expression for transplantation. Neutrophil engraftment (ANC>1.0 G/l) occurred CD66a was identifi ed (range 69% to 100%) with a median fl uo- on day +10, platelet engraftment (PLT > 20.0 G/l) on day +13. rescence ranging from 54 to 673. Expression of CD66a was According to bone marrow morphology on day 100 and immun- also identifi ed in cell lines, with no detectable expression of ofi xation CR rate was 76%, VGPR occurred in 21%, 2 patients the other isoforms of CD66. Positive but weaker expression were considered as non-responders. 80% of the 103 patients was seen with utilisation of the panCD66 monoclonal antibody are still alive. The median observation time from diagnosis was (CD66a/b/c/e) in primary samples and the cell lines. 38.4 months and from transplantation 28.9 months. The day Conclusion: CD66 expression, in particular CD66a expression, 100 non-relapse mortality was 0%. The progression free sur- was detectable on cell lines and CD138+ plasma cells. These vival according to Kaplan-Meier analysis was 63.17 ± 6.1 (95% fi ndings may help in optimisation of future radio-immunothera- CI: 51.0 – 75.3) months, the overall survival was 83.2 ± 5.7 peutic strategies in patients with multiple myeloma. (95% CI: 71.9- 94.5) months. The results of interphase cytogenetical examinations (FISH) were available in 45 patients. In 74% of them genetical abnor- malities have been identifi ed. Del(13q) was proved in 41.9%, del(17p) in 9.7%, abnormalities in the IgH gene in 58% of the cases (overlaps). Among IgH gene abnormalities t(11;14) occurred in 16.7%, t(4;14) in 22.2% and t(14;16) in 5.6%, in

S149 P599 Methods: Prospective audit. The regime used initially consisted Regional differences in haematopoietic stem cell of Plerixafor 160 micrograms/kg/day following 4 days of G-CSF activity in South East England: a multi-centre 10 micrograms/kg/day, with apheresis initiated at 11-12 hours investigation of inequalities in utilisation and inequity of after each plerixafor dose, and plerixafor and G-CSF repeated access to treatment for multiple myeloma daily until a transplantable PBSC dose was achieved (where H. Owiti (1), J. Apperley (2) possible). Plerixafor was administered post-dialysis, as it is pre- (1)Imperial College London (London, UK); (2)Hammersmith dicted to be removed by dialysis. Hospital (London, UK) Results: All 3 patients were female, aged 58-61, & had mye- loma presenting in dialysis-dependent renal failure. All had International variations in haematopoietic stem cell transplant failed to mobilise PBSC by conventional means (cyclophospha- (HSCT) rates are widely documented but few comparative mide plus G-CSF in 1 patient; G-CSF only in 2 patients). Fol- studies exist at national levels. Studies from the USA have lowing plerixafor plus G-CSF on the regime described above, examined differences in HSCT rates between States, often in 2 patients mobilised a transplantable PBSC dose of >2x106/kg the context of socioeconomic and ethnic differences between CD34+ cells after 1 and 2 apheresis procedures respectively. populations. Little is known about the differences in transplant The third patient mobilised a suboptimal CD34+ dose of 0.36 activity within a universal healthcare system such as the UK. x 106/kg after the fi rst apheresis procedure, and the plerixa- This study investigated inequalities in HSCT activity between for dose was therefore increased to 240 micrograms/kg/day. 9 strategic health authorities in the Pan Thames region of the After 3 apheresis procedures, the total dose collected remained UK, and the extent to which this is suggestive of an inequity of inadequate for transplant at 0.86 x 106/kg. In view of this, a access. Data was obtained from the British Society for Blood second treatment cycle of G-CSF and plerixafor was initiated and Marrow Transplant (BSBMT) for transplants carried out approximately 4 weeks later, with the plerixafor dose escalated from 2004 to 2007 inclusive, in 4 South East England and 5 to 240 micrograms/kg/day. On this occasion, a total CD34+ London health authority regions. Each region is served by a dose of 2.12 x 106/kg was collected in 3 apheresis procedures, single transplant centre. Collectively, these centres perform giving a total cumulative CD34+ dose of 2.98 x 106/kg which is 20% of the annual volume of HSCT in the United Kingdom. adequate for transplant. No immediate plerixafor toxicities were Transplant rates per million population were calculated for 1900 experienced by any of the three patients. All 3 patients are now patients aged 18-72, receiving fi rst transplants for haematologi- scheduled for transplant in early 2009. cal malignancies. Rates were directly age and sex standardised Conclusions: In our inital experience, plerixafor is effective to the 2001 UK census population. Utilisation was examined in PBSC mobilisation from myeloma patients with dialysis- in relation to regional deprivation scores, ethnicity and level of dependent renal failure, without undue toxicity. educational attainment of the patient. Transplant rates in gen- eral, increased during this period but differences were noted between regions seemingly matched for size and economic P601 status. The transplant rate in one particular region increased in Severe renal impairment is not a major limit for association with the expansion of the local transplant unit. There autologous stem cell transplantation in patients with was also an overall increase in transplantation for ethnic minori- multiple myeloma or amyloidosis ties. Following standardisation, we found inequalities in stem V. Fiaccadori, F. Ripamonti, A. Colombo, P. Bernasconi, cell transplantation rates for different indications between age D. Caldera, M. Merli, C. Elena, G. Pica, E.P. Alessandrino and ethnicity matched regions. Low educational attainment was Foundation IRCCS Policlinico San Matteo (Pavia, IT) associated with low transplant rates in both deprived and affl u- ent regions and in regions with different ethnic compositions. High-dose therapy using Melphalan (200mg/mq) followed by Our results suggest that factors related to ethnic background, autologous stem cell transplantation (auto-HSCT) is the most education level and the referral process may be associated common treatment for multiple myeloma (MM) and Amyloido- with underuse of HSCT, particularly by populations resident in sis. Reducing Melphalan dose allows to offer this procedure Essex and North East London. Further research with emphasis to patients with severe renal impairment. From 2003 to 2008, placed on quantifying need, is required in order to assess the nine patients (6 MM, 2 Amyloidosis, 1 Crystalcryoglobulinemia) extent to which these factors can be interpreted as evidence of with renal impairment underwent auto-HSCT: six were males, inequity of access to HSCT in the Pan Thames region. three females, median age was 55 years (range 30-65), median creatinine value was 5.4 mg/dl (range 1.5-8.65), median cre- atinine clearance was 19 ml/min (range 10-48). Status of dis- P600 ease at transplant was near complete remission (nCR) in two Plerixafor plus G-CSF is effective without signifi cant patients, very good partial remission (VGPR) in one patient, toxicity in PBSC mobilisation from myeloma patients partial remission (PR) in three patients, while three patients had with dialysis-dependent renal failure who have failed to active disease. Median Melphalan dose was 140 mg/mq (range mobilise by conventional means: an initial series of three 100-160), administered at day -2 and followed by autologous patients peripheral stem cells infusion at day 0 after DMSO removal K.W. Douglas (1), P.J. Hayden (2), M.E. O’Dwyer (2), (median number of CD34+ : 4.61 x 106 cells/Kg, range 3.74- A. Rahemtulla (3) 7.87). All patients engrafted, with a median time to engraftment (1)Beatson West of Scotland Cancer Centre (Glasgow, UK); of 12 days (range 10-15); they all developed fever with only two (2)University College Hospital (Galway, IE); (3)Hammersmith positive blood coltures (Staphylococcus aureus and Staphy- Hospital (London, UK) lococcus warnerii). Median duration of hospitalization was 22 days (range 18-30). We observed grade II oral mucositis in four Objectives: Myeloma patients with dialysis-dependent renal failure patients, ARDS-like pulmonary toxicity in one patient, severe may benefi t from autologous transplant, but PBSC mobilisation gut mucositis in one patient. At day +90 (data available for 7 may prove challenging. The unlicensed small molecule chemo- patients) six patients achieved a favourable response (2 CR, kine inhibitor plerixafor is available on a named-patient basis for 3 VGPR, 1 PR). All patients are alive with a median follow up PBSC mobilisation from patients failing conventional mobilisa- of 12 months (range 1-60 months); three out of six patients on tion. Plerixafor is renally excreted & has not initially been used in haemodyalisis at transplant became haemodyalisis independ- patients with renal failure. However, on the basis of encouraging ent (50%). Median duration of response (available for 7 patients) toxicity data in patients without renal failure, we have recently is 14 months (range 4-27). This study confi rms that Melphalan used plerixafor at reduced dosage for PBSC mobilisation from followed by auto-HSCT is feasible in patients with severe renal three myeloma patients with dialysis-dependent renal failure who impairment; a dose of 140 mg/mq is well tolerated and this had failed to mobilise PBSC by conventional means. reduction doesn’t jeopardize the effi cacy of the procedure.

S150 P602 Bu-Mel, 1x twin sibling). Median age of patients was 56 years Short-course pretransplant velcade, thalidomide and (27-74). We analyzed overall survival (OS) and progression- dexamethasone regimen induces high complete response free survival (PFS) regarding conditioning, stage, complete or rate without compromising CD34+ cell collection in very good partial remission (CR, VGPR) achievement, renal untreated multiple myeloma patients: preliminary results impairment, single vs. double transplant and use of new drugs of a single-centre experience (thalidomide and bortezomib). E. Pennese, M. Dargenio, M.R. De Paolis, P. Forese, Results: Estimated 10-years survival of the whole set of patients R. Matera, G. Pugliese, G. Reddiconto, A. Valacca, C. Vergine, is 38% (median survival 55 months). Patients who underwent N. Di Renzo allogenic transplantation upfront had lower PFS (p= 0.0001) Vito Fazzi Hospital (Lecce, IT) and OS (p=0.018) compared to those who had only ASCT. Patients with renal impairment had signifi cantly shorter OS Background: High-dose chemotherapy with autologous stem- than those without (p=0.03). Patients with Mel100 conditioning cell support has signifi cantly improved progression-free and had trend towards worse OS than those with Mel200, however overall survival of patients with untreated multiple myeloma statistical signifi cance was not reached (p=0.08). We observed (MM). Pre-transplant complete response (CR) and tumor reduc- trend towards better outcome in patients treated with new drugs tion have been identifi ed as most relevant prognostic factors for but signifi cance was not reached (p=0.052). Reaching CR or long-term survival in untreated MM. Recently, thalidomide and VGPR was surprisingly not translated into better OS (p=0.29) bortezomib both given as single agent shown to be effective in and PFS (p=0.27). Also stage of the disease and whether sin- previously treated MM. Since they have target different molecu- gle or double trasnplant was used did not make any signifi cant lar pathways their combination is waited to be highly effective difference in the outcome. to induce high CR rate and tumor reduction before HDT with Conclusion: Stem cell transplantation greatly improves out- stem-cell support. We present early data of this regimen in come of patients with MM. Poor outcome of allogenic trans- untreated symptomatic MM patients. plantation in our group of patients is related to high transplant Patients and Methods: From March 2007 to October 2008 16 related mortality (20% vs. 0.4%) and unexpected high relapse pts (M/F: 12/4) were enrolled in the study; median age was 57 rate. However in this group are two long term survivors who are years (range: 42-71), 47% were older 60 years; Most of pts had disease free for more than 10 years. There is a trend towards stage III according to Durie & Salmon (76%) and ISS score better survival, when new drugs are incorporated at any time in 2-3(76%). M-component was IgG 69% (11/16), IgA 19%, and the course of the disease as an addition to ASCT. This fact sup- non secretory 12%. ports hypothesis that use of new drugs with ASCT should trans- Regimen: Bortezomib was given at 1.3 mg/m² on days 1,4, 8, late into better long-term outcome. Our results also suggest 11, thalidomide at dose of 100 mg PO daily, and dexametha- that Mel100 regimen is insuffi cient and other treatment options sone (40 mg/die PO) was given the day of bortezomib and the should be offered to older patients. This is in good agreement day after (320 mg td for each cycle). All patients received 3 with results of recent clinical trials. cycles of WTD every 4 weeks before CD34+ cell collection and Supported by project MZO00179906. high-dose melphalan (200 mg/m²). Deep-venous thrombosis prophylaxis was established in all pts consisting of aspirin 100 mg daily in 44% of pts, low-molecular weight heparin (28%) or P604 low dose warfarin (28%). Autologous haematopoietic progenitor cell Results: all pts are evaluable for response to Velcade, Thalido- transplantation followed by a reduced-intensity mide and Dexamethasone (VTD) regimen and PBSC collec- conditioning and allotransplantation in de novo multiple tion; 13 pts have been transplanted. After three VTD 88% of myeloma patients: an update pts achieved PR including 47% of CR. VTD regimen was well M. Martino, G. Console, E. Spiniello, D. Marcuccio, tolerated with main toxicity consisting of WHO grade III periph- G. Messina, G. Irrera, R. Fedele, E. Massara, I. Bova, T. Moscato, eral neuropathy in 12% of pts. There was no chemotherapy M. Cuzzola, P. Iacopino reduction or delay due to toxicity. A median of 6.5 x 106/kg of U.O. Ematologia con Trapianto (Reggio Calabria, IT) CD34+ cells (range: 2.7-11.6) was collected in 15 of 16 pts. The median CD 34+ cells infused was 3.2 x106/kg (range: 2.0 - 4.3). We evaluated an up-date of the combination of high-dose ther- Times to platelet (20x109/L) and granulocyte (500x109/L) recov- apy and autologous hematopoietic progenitor cells transplanta- ery were 13 and 10 days respectively. After VTD and transplant tion (AHPCT) followed by a reduced intensity conditioning and 10 of 13 patients achieved CR (75%) and 2 (15%) a VGPR. allotransplantation (RICT) in de novo multiple myeloma (MM) Conclusion: These very preliminary data suggest that VTD is patients (Martino et al. AJH 81:973–978, 2006). 20 subjects effective and well tolerated regimen; high response rate without with stage III MM (median age 52 years, range 40–64) received compromising PBSC collection makes it a good option for initial high dose melphalan (200 mg/m²) followed by AHPCT previ- treatment of MM although more pts and longer follow-up are ously collected after cyclophosphamide (4 g/m²) and granulo- required. cyte colony-stimulating factor (G-CSF). After 3–4 months from APBSCT, the patients underwent RICT, consisting of fl udarab- ine 90 mg/m² + cyclophosphamide 900 mg/m² on days. Graft- P603 versus-host disease (GVHD) occurred in 12 patients (8 patients 11 years of single-centre experience with stem cell developed chronic GVHD); 5 patients developed CMV anti- transplantation for multiple myeloma genemia and were treated pre-emptively with ganciclovir. No J. Radocha, V. Maisnar, P. Zak, A. Zavrelova, M. Cermanova, transplant related mortality was shown. Response was simul- M. Kmonicek, L. Jebavy, J. Maly taneously measured by both electrophoresis (EP) and immun- Fakultni nemocnice Hradec Kralove (Hradec Kralove, CZ) ofi xation (IF); when IF was negative, patients were classifi ed in complete remission (CR) and when it remained positive, Introduction: Autologous stem cell transplantation (ASCT) near CR (nCR). After a median follow up of 62,2 months post became standard of care for patients with multiple myeloma AHPCT, the OS and EFS are 62.2 and 36.2 months, respec- (MM) under the age of 65 years. We routinely perform ASCT for tively. Overall, the CR + nCR rate after dose-reduced allograft newly diagnosed MM since 1996 in our department. was enhanced from 30 to 70%. A correlation not statistically Patients and methods: We retrospectively analyzed all 225 signifi cant between GVHD and EFS was found. In conclusion, transplants in 136 patients done for MM from 1996 till 2007. this up-date confi rm that an up-front tandem strategy with RICT 210 transplants were autologous (195x Mel200, 15x Mel100 for following autografting is feasible and induces high CR/nCR rate elderly patients), 15 transplants were allogenic (13x reduced- in MM. It is open the debates around the results gotten in terms intensity conditioning Bu-Flu-ATG or Flu-Cy, 1x myeloablative of OS and EFS.

S151 P605 phosphamide alone in 53 cases or associated with other drugs Impact of low-dose thalidomide as maintenance therapy in 9 cases. The number of apheresis were 1 (n=65), 2 (n=31), 3 in advanced multiple myeloma patients following (n=16), 4 (n=18), 6 (n=1) and 8 (n=1). During mobilization, we high-dose therapy used GCSF in 179 cases, GM-CSF in 5cases, SCF in 4 cases A. Andreini, F. Randon, C. Tecchio, G. Quaresmini, F. Frattini, and associations of GM-CSF or SCF with GCSF in 9 (4.5%) R. Di Bella, M. Sorio, S. Ledro, C. Perbellini, D. de Sabata, cases. The median number of infused cells were: TNC 5x107/kg F. Benedetti (1-59), CFU-GM 70.5x104/kg (0-2616) and CD34+cells 3x106/ Bone Marrow Transplant Unit (Verona, IT) kg (0-27); 115 (58%) received a number of CD34+cells<4x106/ kg and 82 (42%) ≥ 4x106/kg. After transplantation, 156(79%) Introduction: Multiple Myeloma (MM) is a fatal disease with a received growth factors [1 GM-CSF, 148 G-CSF and 7 SCF]. median survival of 3-4 years. The standard therapy in pts aged The median number of RBC and Pt transfusions were 0 [0-23] <65 years was, in the last years, VAD or VAD-like regimens and 1 [0-20] respectively. The median number of days with neu- followed by high dose Cyclophosphamide (Cy) and Alkeran trophils <0.5G/L was 6 (0-33) and with Pt<50G/L 17 (2-104) and (L-PAM) with support of peripheral stem cells (PBSC). Tha- the median length of hospitalization was 18 days (14-54). The lidomide (Thali) alone or associated to chemotherapy (CT) probability of 5-year overall and event-free survival were 64.3% has showed considerable activity. Its effi cacy as maintenance (56.3-73.4%) and 32.4% (24.9-42.2%). The variables studied therapy is still under investigation. were age, disease status at transplant, infused TNC, CD34+cell Patients and methods: We considered a conventional CT and CFU-GM, growth factor use during mobilization and after (3 cycles of VAD or VAD-like regimen), high dose Cy (7 g/m² transplantation, mobilization chemotherapy and interval Diag- i.v.) and PBSC harvest, followed by single or tandem (when T. Using a conditional logistic-regression model, we observed disease still present) high dose L-PAM (200 mg/m² i.v.) with a signifi cant negative impact of interval diagnosis-T (p=0.05) rescue of PBSC (CD34+>4x106/Kg). All pts were in stage II/III, on length of hospitalization and RBC transfusions. A multivari- according to Durie and Salmon. Thali (100 mg/die) was given to ate analysis showed a signifi cant positive impact of CFU-GM all pts regardless the response and discontinued at the time of number [HR=1 (1000-1.002) (p=0.03)] and growth factor use relapse or progression or because of toxicities. No anti-throm- after transplantation [HR=0.55 (0.36-0.85) (p=0.005)] on the botic prophylaxis was administered. Between January 1999 number of days with less than 0.5 G/L neutrophils and a signifi - and October 2007, 95 consecutive pts were treated, 90 valu- cant negative impact of CD34+cell<4 x106/kg on the number of able, median age 55 (33-66), male/female 50/40, M component days with less than 50G/L Pt ([HR=1.65 (1.09-2.50) (p=0.01)]. IgG/IgA/IgD/monoclonal light chain and non-secretory MM were In conclusion, we did not show any apparent impact of the pre- 51/22/3/12 and 2, respectively. All pts completed induction and transplant, mobilization and graft variables but a signifi cant high dose CT. One toxic death occurred. Thali (100 mg/day) infl uence of the diagnosis-T interval on platelet transfusion and was started within 3 months from transplant in 68 pts; 22 could of the use of GCSF post-transplant, the CD34+ and CFU-GM not be treated because of refusal (4), progression disease (9), numbers on the length of aplasia. allergic reactions (3) or neurological toxicities (6). Results: At transplant, 13/90 (14%) were in very good partial remission or complete remission (VGPR+CR according to P607 EBMT criteria), further 33 pts (30%) reached the CR after trans- Lenalidomide as salvage therapy after allogeneic plant, for a total of 46/90 CR (51%) at the end of the program. transplant for primary plasma cell leukaemia: a case We compared the number of responses (VGPR+CR), the treat- report ment-free interval (TFI) and overall survival (OS) in the 2 groups I. Attolico (1), M. Cimminiello (1), G. Discepoli (2), R.A. Cifarelli of pts. The median follow up was 42 months. The VGPR+CR (3), M. Pizzuti (1), N. Filardi (1), M. Poggiaspalla (1), D. Vertone rate was 64% (44/68) in the group treated with Thali versus (1), A. Olivieri (1) 50% (11/22) in the remaining pts. With Thali we obtained fur- (1)U.O.di Ematologia Ospedale San Carlo (Potenza, IT); ther 9 CR+VGPR/33 pts not in remission after transplant (27%). (2)Laboratorio di Citogenetica e Genetica Molecolare, Ospedale Comparing the 2 groups, the TFI was 37 and 22 months, the “G.Salesi” (Ancona, IT); (3)Unità di Genomica, Metapontum OS was 47 and 37 months, respectively (p<0.08). Agrobios (Metaponto (MT), IT) Conclusions: Low dose Thali following single or tandem autotransplant appears to be a safe and feasible treatment Primary Plasma Cell Leukemia (PPCL) is an aggressive and not only as maintenance, but also as consolidation therapy, rare variant of Multiple Myeloma (MM). Allogeneic haematopoi- improving the rate of response (VGPR+CR), delaying the need etic stem-cell transplantation (HSCT) results in sustained long- of subsequent therapy and improving the OS, with an accept- term OS, suggesting a possible graft-versus-leukemia (GVL) able toxicity. effect. Relapse or progression after HSCT are common and outcome is poor. No data are available about Lenalidomide as salvage therapy for PPCL relapsed after HSCT. We report a P606 41 year-old woman with PPCL (IgA kappa monoclonal compo- Impact of pretransplantation, mobilisation and graft nent (MC), 32% immature plasmacells in peripheral blood, 50% variables on transplant outcome after autologous immature plasmacells CD38/CD138/sIgkappa+, CD19/CD56/ haematopoietic stem cell transplantation for patients with CD45- in the bone marrow). She received six Hyper-CVAD multiple myeloma courses achieving Complete Remission (CR) and, after blood M. Michallet, M. Sobh, Q.-H. Le, F. Barraco, C. Dumontet, stem cells collection, underwent ASCT (Melphalan 200mg/m² Y. Chelghoum, E. Nicolas-Virelizier, X. Thomas, A. Praire, followed by reduced intensity conditioning HSCT from HLA C. Plesa, G. Cannas, F.E. Nicolini, J. Troncy identical brother (thiotepa 5mg/Kg, fl udarabine 30mg/m², mel- Hôpital Edouard Herriot (Lyon, FR) phalan 140mg/m². Full donor chimerism was documented at day +90. At day +138 she relapsed with cytogenetics showing We studied 197 autologous HSCT performed in 132 patients a complex hyperdiploid karyotype. Bortezomib (1,3mg/m²) and (53 F, 79 M, median age of 57 years) treated for multiple mye- Dexamethasone (40mg) (days 1,4,8,11) were started without loma in our center. At diagnosis there were 71 IgG (49 kappa, response; as a second-line, cyclophosphamide (300 mg/m² on 22 lambda), 26 IgA (15 kappa,11 lambda), 2 IgD (1 kappa, 1 days 1,8,15) was added to Bortezomib and Dexamethasone. lambda, 27 light chain (18 kappa, 9 lambda), 2 plasma cell Nevertheless disease progressed and full donor chimerism was leukemia, 3 non secretory, 1 non secretory with 11 stage I, 12 lost; therefore Lenalidomide (25mg/day, 1-21) and Dexametha- II, 96 III (13 were not classifi ed). At diagnosis, 24/98pts had sone (40 mg/day 1,8,15,22) were administered. At the end of a del(13), and 65/179 had high levels of beta2microglobulin. the fi rst course the patient developed a skin rash, hyperbiliru- PBSC were mobilized in steady state in 135 cases, after cyclo- binemia and increased liver enzymes, compatible with acute

S152 GVHD, treated with methilprednisolone (2 mg/Kg/day). The ment. Testing for infectious agents was uneventful. Findings in patient showed a quick improvement with reduction of MC and the latter examinations were consistent with unspecifi c infl am- abnormal plasmacells in the bone marrow. After six courses mation of the orbital apex. The patient received local irradiation of Lenalidomide the patient is in CCR, with complete donor that allowed cessation of corticosteroid treatment. We conclude chimerism and normal cytogenetics; a mild cGVHD persists, Tolosa Hunt-like syndrome in this case to be secondary due requiring low dose steroids. We describe here the fi rst case of to a specifi c pharmacologic adverse effect of palifermin rather a patient with relapsed and chemorefractory PPCL after alloge- than an immune-mediated infl ammation since myeloablative neic HSCT, who achieved a CCR with Lenalidomide; moreover, treatment is considered to be one of the most effective means this is the fi rst case of response to Lenalidomide associated of immunosuppression. with development of GVHD. The mechanism of the GVL/GVM is largely unknown. T and NK cells might exert an alloimmune reaction. In this case we hypothesize that the strong antiprolif- P609 erative effect of Lenalidomide could have reduced the leukemia Autologous peripheral blood stem cell transplantation in burden, making the GVL effect stronger. Furthermore Lenalido- patient in advanced stage of POEMS syndrome mide could have stimulated donor T/NK cells, as suggested by M. Cioch, J. Manko, A. Dmoszynska experimental data. Medical University of Lublin (Lublin, PL)

Introduction: POEMS syndrome is a rare plasmatic cell dyscra- sia, which is associated with overproduction of VEGF and char- acterized by polyneuropathy, organomegaly, endocrinopathy, monoclonal gammapathy, and skin changes. A specifi c treat- ment for this disease is not established. In recent reports, high dose chemotherapy followed by autologous peripheral blood stem cell transplantation (APBSCT), performed in early stage can improve clinical symptoms, especially for polyneuropathy. We describe the clinical course of a patient with POEMS syn- drome in advanced stage and unresponsive to standard chem- otherapy, treated successfully with APBSCT. Case report: A 47-year-old man was admitted to Department of Haematooncology and Bone Marrow Transplantation of Medical University in Lublin with quadriparesis, hepatosplenomegaly, sclerotic bone lesion, acrocyanosis, IgG lambda gammapathy and thrombocytosis. There was no increased number of plas- matic cells in bone marrow smear. The fi rst symptoms of dis- P608 ease were appeared three years ago and diagnosis of POEMS Tolosa-Hunt-like syndrome in a myeloma patient receiving was established 18 months ago. The patient was unresponsive palifermin shortly after high-dose melphalan therapy to chemotherapy consisted with cyclophosphamide and pred- S. Mueller, H. Steffen, F. Wendel, M. Kreissl, R. Seggewiss, nisone, so we decided to perform APBSCT. Peripheral blood H. Einsele, S. Knop stem cells were collected after mobilization by cyclophospha- University Hospital (Würzburg, DE) mide (4g/m²) and G-CSF. The number of collected CD34+ cells was 12x106/kg. The conditioning regimen was high dose Oral mucositis is a common adverse effect in subjects with melphalan (140 mg/m²). The number of transplanted CD34+ haematological cancers receiving high-dose (HD) therapy and cells was 4x106/kg. The post-transplantation course was com- autologous peripheral blood progenitor cell (APBC) transplan- plicated by fever with no identifi able infectious etiology, diar- tation. Palifermin, a recombinant human keratinocyte growth rhoea, cutaneous rash and swelling without hipoalbuminaemia factor, has been approved to decrease incidence and severity (engraftment syndrome?). Neutrophil (0.5 G/l) and platelet (20 of oral mucositis and is associated with rash, edema/erythema G/l) engraftment occurred on days 11 and 13, respectively. The and sensations of increased tongue thickness. We report on improvement was manifested by disappearance of gammapa- a 65 year-old female patient with IgG kappa myeloma in ISS I thy and organomegaly. The correction of neurological symp- stage who underwent chemomobilization of APBCs followed by toms was unsatisfactory. Therefore thalidomide in a dose of 50 HD melphalan 140 mg/m². She was included in an open-label mg/day was applied. The second autologous transplantation is study to receive palifermin from day -6 to +2 with APBC retrans- planned in January 2009. fusion on day 0. On day +10 the patient complained of severe Conclusion: Our experience suggests that APBSCT may be val- unilateral headache and diplopia. On clinical examination she uable method of treatment even in advanced stage of POEMS had ophthalmoplegia, partial visual loss down to 0.1 and exoph- syndrome. The effect of thalidomide and second transplanta- thalmos of the left eye. Initial cranial MRI revealed infl amma- tion will be presented during EBMT meeting. tion in the ethmoid and sphenoid sinuses while the subsequent one showed diffuse enhancement of superior orbital fi ssure and orbital apex consistent with Tolosa Hunt syndrome. Painful oph- thalmoplegia as well as exophthalmos responded to high-dose dexamethasone with a quick recovery of visual acuity to 0.8. Extensive radiographic and laboratory work-up including CSF did reveal neither signs of specifi c infection nor of recurrence of myeloma. Following haematological recovery both typical signs of palifermin-induced adverse effects as well as elevated CRP levels normalized while several attempts in tapering cor- ticosteroid doses were followed by prompt worsening of visual loss. Eight weeks from APBC transplantation the patient was in excellent clinical shape except persistent Tolosa Hunt syn- drome. We repeated bone marrow and serological assess- ments of myeloma that was confi rmed to be in VGPR, cranial MRI and, eventually, performed an 18F FDG positron emission tomography (PET) to exclude possible extramedullary involve-

S153 Solid tumours 10/10 (1 pt). RIC allo-HSCT conditioning was : Fludarabine 180 mg/m², Busulfan IV 6.4 to 9.6 mg/kg, and rabbit anti-thymocyte globulin 2.5 mg/kg for related and 5 mg/kg for unrelated recipi- ents. One pt received exclusively TBI 2 grays as conditioning P610 regimen. GVHD prophylaxis was cyclosporin (CSA) alone 3 High-dose chemotherapy with autologous haematopoietic mg/kg IV starting on day –1. In the absence of grade > II acute stem cell transplantation for metastatic breast cancer GvHD, CSA was tapered 25% weekly starting on day+30. After in Italy: evaluation of data from the GITMO registry RIC allo-HSCT four pts responded, and 2 pts progressed. Maxi- 1990–2005 mal peripheral chimerism was 100% at day+60. Four patients M. Martino, S. Secondino, A. Zambelli, R. Schiavo, M. Aieta, had evidence of GVHD and 4 had “evidence” of graft-versus A. Ballestrero, C. Zamagni, M. Musso, A. Capaldi, G. Azzarello, neuroblastoma effect (disappearance or decrease of blood/ B. Bruno, P. Pedrazzoli on behalf of Gruppo Italiano per il marrow tyrosine hydroxylase transcript and /or MIBG spots Trapianto di Midollo, di Cellule Staminali Emopoietiche e di after the induction of aGvHD by withdrawal of immunosuppres- Terapia Cellulare (GITMO) – Sezione Tumori Solidi sive drugs or by DLI and reappearance of neuroblastoma after the control of GvHD by immunosuppressive drugs). Maximal Aim of this study is to assess survival in a large cohort of grade for aGVHD was III skin and liver (1pt). One patient had metastatic breast cancer (MBC) patients receiving high-dose chronic skin GVHD. chemotherapy (HDC) with autologous hematopoietic stem cell Four patients are alive, with no evidence of disease, with transplantation (HSCT) in Italy between January 1, 1990 and median time after RIC allo-HSCT of 21.8 months [3.2 – 52.7] December 31, 2005. Over this period 563 patients were given [Table 1]. HDC and HSCT, in 36 Italian Centers. General information Conclusion: RIC allo-HSCT appears feasible and safe in regarding these patients was obtained from the GITMO Reg- patients with advanced NB. Additional studies of therapeutic istry and all Centers were then contacted for additional data. strategies used donor-derived effector cells (alloreactive NK During the nineties the number of HSCT, along with the number cells or T cells) and possible graft versus-tumor effects are war- of reporting centers for BC, increased progressively until 1999. ranted in this group of patients. In the following years a progressive decline in the number of HSCT for BC has been observed. In the nineties a dramatic shift towards a widespread use of blood progenitors occurred. Transplant related mortality, i.e. any death not related to the disease occurring within the fi rst 100 days after transplant, has been 2%. Complete data set has been obtained so far from 17 centers for 330 patients (1 male). Median age at transplant was 43 years; premenopausal patients were 68%, HR+ 66%. Fifty % of patients had visceral metastases at time of transplant. Analysis of clinical parameters including progression-free sur- vival (PFS) and overall survival (OS) in the whole population and in subsets of patients based on prognostic factors (ie sta- tus at transplant, menopausal status, hormonal status) dem- onstrated: i) PFS and OS at 5 years in the whole population were 18% and 36%, respectively. ii) HDC provided signifi cantly more benefi t in younger patients, in patients harbouring hormone receptor positive tumors and P612 in women with chemosensitive disease (48% OS at 5 years in Increased frequency and responsiveness of patients transplanted in complete remission). PSA-specifi c T cells after allogeneic stem cell Full data will be presented at the meeting. transplantation for prostate cancer J. Mattsson (1), M. Okas (1), J. Gertow (2), M. Uhlin (2) (1)Centre for allogeneic stem cell transplantation (Stockholm, P611 SE); (2)Clinical Immunology (Stockholm, SE) Reduced-intensity conditioning allogeneic stem cell transplantation for high-risk neuroblastoma: French Therapies for localized prostate cancer include curative surgery experience and radiotherapy while treatment of metastatic disease is often C. Paillard (1), P. Lutz (2), A. Tchirkov (3), N. Cojean (2), insuffi cient. Therefore, we started to investigate the potential A. David (1), E. Dore (1), F. Isfan (1), E. Merlin (1), A. Marabelle of allogeneic SCT as a treatment for non-curable prostate (4), R. Rousseau (4), F. Deméocq (1), J. Kanold (1) cancer. (1)CRCTCP CHU Hôtel Dieu (Clermont Ferrand, FR); (2)CHU A patient underwent allogeneic SCT from his HLA-identical Strasbourg (Strasbourg, FR); (3)CHU Clermont Ferrand sister after a non-myeloablative conditioning regimen as treat- (Clermont Ferrand, FR); (4)IHOP (Lyon, FR) ment for his metastatic prostate adenocarcinoma. The patient was treated with donor lymphocyte infusions after SCT due to Autologous haematopoietic stem cell transplant has improved threatening rejection. Frequencies of prostate-specifi c T cells the outcome for children with high risk neuroblastoma (NB) but in the peripheral blood of the patient, sibling donor and a group long term event free survival remains lower then 50%. Alloge- of control individuals were determined by fl ow cytometry using neic haematopoietic stem cell transplantation (allo-HSCT) has tetrameric and pentameric HLA A2 complexes containing pep- the potential to confer a durable graft versus tumor immunity, tides derived from the prostate specifi c antigen (PSA). Cytotoxic but may be limited by signifi cant toxicity in heavily pre-treated activity of PSA-peptide-specifi c T-cells against peptide-pulsed children. Therefore we have investigated the feasibility of target cells was analyzed ex vivo by 51Cr-release assays. reduced intensity conditioning (RIC) allo-HSCT in 6 patients Stable clinical and laboratory remission lasting for more than 5 with refractory or relapsed NB. years was observed after SCT. Using HLA containing pentam- All patients received at least 4 lines of chemotherapy including ers with PSA-derived peptides we could detect prostate-spe- auto-HSCT with melphalan 140mg/m² + busulfan 600 mg/m² cifi c CD8+ T cells in this patient in high frequencies over several regimen. Patients were in PR or VGPR at the time of RIC allo- months. Furthermore, higher frequencies of PSA-specifi c T- HSCT. Donor cell sources were: related CB 6/6 (1pt), related cells were revealed in the peripheral blood of the patient and BM 10/10 (3 pts), unrelated BM 10/10 (1 pt) unrelated PBSC female controls as compared with male healthy controls.

S154 Recently the PSA levels in the patient started to increase and P614 prostate biopsies showed prostate carcinoma in 3/8 biopsies, Treosulfan plus melphalan as megachemotherapy for Gleason 4+4, totally 3.5 mm of cancer (of 100 mm totally). Due autologous haematopoietic stem cell transplantation in to this progression we have initiated a protocol for adoptive children and young adults with high-risk or relapsed transfer of allogeneic PSA-specifi c T cells from the patient. We neuroblastoma and Ewing/PNET will, by using pentamers and fl ourocrome specifi c beads sepa- M. Choma (1), K. Drabko (1), A. Zaucha- Prazmo (1), B. Wojcik rate out PSA specifi c T cells from the patient. In order to break (1), M. Leda (2), J. Wachowiak (2), J. Kowalczyk (1) a possible tolerance we will keep them in high concentration (1)Medical University of Lublin (Lublin, PL); (2)Poznan of IL-2 and the respective peptides in vitro culture over night University of Medical Sciences (Poznan, PL) before injection back into the patient. Clinical data of this novel therapy will be presented at the meeting. Treosulfan (TREO) reveals cytotoxic activity not only against Our data suggest that allogeneic SCT led to the generation of hemopoietic stem cell (Pleomacher 2000), but also against a T cell mediated prostate-specifi c immunity in the reported important childhood solid tumors (Munkelt 2004; Lanvers- patient. The in vitro and ex vivo immunological monitoring per- Kaminsky 2006). Its low organ toxicity was demonstrated in chil- formed indicate an adjuvant anti-tumor effect of PSA-specifi c T dren with non-malignant disorders and in those with advanced cells. This report presents a novel treatment approach involving hematological malignancies (Wachowiak et al. 2005, 2008). allogeneic SCT in prostate cancer patients who do not respond Objectives: To evaluate safety and anti-tumor effi cacy of meg- to chemotherapy and/or cannot undergo prostectomy. achemotherapy (M-CHT) containing TREO and melphalan (MEL) for autologous hematopoietic stem cell transplantation (auto-HSCT) in children with high-risk and relapsed neuroblast- P613 oma (NBL) and Ewing/PNET (ES/PNET), and contraindications Ewing’s sarcoma at relapse: sequential high-dose for busulfan (BU) administrations. cyclophosphamide, etoposide, mitoxantrone+melphalan Patients and methods: Between 2001-2007 TREO was admin- followed by autologous peripheral blood stem cell rescue istered instead of oral BU in M CHT before auto-HSCT in 10 in a cohort of paediatric patients patients aged from 8 months to 21 year (median age: 4.5 year). R. Luksch (1), M Podda (1), L. Gandola (1), S. Mapelli (2), There were 7 girls and 3 boys. Diagnosis were as follows: NBL P. Daolio (2), F. Ravagnani (1), M. Casanova (1), G. Cefalo (1), in 7 children (5 in CR1, 1 in CR2, 1 in PR) and ES/PNET in M. Massimino (1), D. Polastri (1), A. Ferrari (1), F. Spreafi co (1), 3 children (2 in CR1,1 in PR). All patients demonstrated prior M. Terenziani (1), C Meazza (1), P. Coluccia (1), V. Bedini (1), auto-HSCT either contraindication for use of BU (4/10) or were L. Piva (1), F. Fossati-Bellani (1) too young for its oral administration (6/10), while BU i.v. was (1)Fondazione IRCCS Istituto Nazionale dei (Milan, IT); unavailable at that time. In all patients M-CHT consisted of (2)Istituto Ortopedico G. Pini (Milan, IT) TREO in total dose of 36 g/m² (12 g/m²/day i.v. for 3 consecu- tive days) and melphalan 140 mg/m² i.v. in one dose. Support- A treatment program was opened in the period 1988-1999 ive care was carried out according to HR NBL-1/SIOPEN and for children with previously untreated nonmetastatic Ewing’s Euro-Ewing protocols. sarcoma, consisting of 8-monthly cycle vincristine+etoposid Results: All children demonstrated engraftment and were dis- e+cisplatinum and ifosfamide alternated with epirubicin, and charged from hospital after procedure. None patient experienced local treatment with surgery and/or radiotherapy. Eighty-four organ toxicities greater then II WHO or live-threatening infection. consecutive children entered this program. All patients at fi rst 5/10 children relapsed (3 with NBL-2 in CR 1 and 1 in CR 2, 2 with relapse (with the exception of late relapse and isolated single ES/PNET - 1 in CR1, 1 in PR) at median time of 13 months from pulmorary metastasis) were enrolled in the period 1994-2004 in auto-HSCT (range 6-24 months) and 4 of them subsequently a protocol including sequential high-dose chemotherapy: cyclo- died of disease progression. 6/10 patients are alive, including phosphamide 7g/sqm+G-CSF and PBSC apheresis, etoposide 5 in CCR (4 with NBL and 1 ES/PNET) since auto-HSCT with 2g/sqm + G-CSF ± further apheresis, and mitoxantrone 60mg/ median observation time of 49 months (range 14-76 months). sqm+melphalan 180mg/sqm followed by peripheral blood stem Conclusions: cell rescue (PBSC) rescue. Radiotherapy was given on indi- 1. M-CHT with TREO and MEL for auto-HSCT in patients with vidual basis on the site(s) of relapse. Twenty-one consecutive NBL and ES/PNET and high risk of BU-related complications patients were treated according to this program. The median demonstrates low organ toxicity. age at relapse was 16 years, and the median time to relapse 2. Results of auto-HSCT after M-CHT consisted of TREO and was 28 months (range 12-126). The site of relapse was: skel- MEL in children with NBL and ES/PNET seems to be compara- eton only in 7 patients, lungs only in 6, skeleton+lungs in 7, ble with those achieved with BU, but this observation need to be local in 1. Neither disease progression, nor treatment discon- confi rmed within prospective trial in larger group of patients. tinuation due to toxicity occurred during the treatment. An ade- quate PBSC harvesting (>3x106 CD34+/kg) was obtained in 20 patients (95%) who were included in the present analysis. P615 Mean time for take of PMN and platelets after PBSC was 11 and Salvage high-dose chemotherapy and autologous 19 days, respectively. No toxic death was recorded after the peripheral blood stem cell transplantation in patients with stem cell rescue. As to September 2008, median follow-up from relapsed or refractory germ cell tumours: a Turkish relapse was 88 months. The actuarial 3-year overall survival oncology group study probability after PBSC rescue is 0.30, 0.45 for the subgroup F. Arpaci (1), M. Ozturk (1), E. Ozdemir (2), A. Ozet (1), with a „late“ relapse (>24mos) and 0.10 for the subgroup with S. Ataergin (1), S. Dagdas (3), O. Kuzhan (1), S. Kilic (1), an early relapse ≤24 mos from diagnosis). None of the other B. Ozturk (1), S. Komurcu (1), G. Ozet (3) variables analyzed (sex, age, site of disease at diagnosis, site (1)Gulhane Medical Academy (Ankara, TR); (2)Hacettepe of relapse) infl uenced the outcome in this patient cohort. The Medical Academy (Ankara, TR); (3)Numune Medical Hospital results suggest that patients with a „late“ relapse could benefi t (Ankara, TR) from this intensive sequential high-dose treatment. Objectives: Germ cell tumors are potentially curable by means of high-dose chemotherapy (HDC) and autologous peripheral blood stem cell transplantation (APBSCT), even when this treatment is used as third-line or later therapy or in patients with platinum-refractory disease. Methods: We conducted a retrospective review of fourtynine patients with metastatic germ cell tumor that had progressed

S155 after they received at least two lines therapy in which cisplatin- Finally, these studies should help to elucidate the impact of containing combination chemotherapy between 1991 and 2008 xenoreactivity on the induction of GVH immunity by human T at three centers in Turkey. Fourtysix patients were evaluable: cells in humanized mice. 36 (78.30%) testicular, and 10 (21.70%) extragonadal prima- ries. HDC regimens were ifosfamide + carboplatin + etoposide in 29 patients, carboplatin + melphelan in 11 patients and car- P617 boplatin + melphelan + thiotepa in 6 patients. Pharmacokinetics of ATG-fresenius S in patients Results: The median age was 25.29 years (range:12 to 57). undergoing allogeneic stem cell transplantation: Transplant related mortality was 4.3% (2/46). During follow-up implications for adoptive immunotherapy period (range: 23 days to 123 months), 17 of 46 patients (37%) S. Mastaglio, J. Peccatori, M. Bernardi, D. Clerici, A. Crotta, were continuously disease-free. The median disease-free and C. Messina, A. Assanelli, C. Corti, M.T. Lupo Stanghellini, overall survivals were 34.30 and 52.80 months, respectively. M. Marcatti, C. Bonini, F. Ciceri The 10-year projected disease-free and overall survivals rates San Raffaele Scientifi c Institute (Milan, IT) were 41% and 48%. Conclusion: Salvage HDC and APBSCT can induce long-term ATG-Fresenius S (ATG-F) is a product of purifi ed, concen- remissions or cure in patients with relapsed or refractory germ trated polyclonal immunoglobulin G from hyperimmune serum cell tumors. of rabbits immunized with the human lymphoblast T-cell line. It induces immunosuppression; very recently, the addition of ATG-F to CyA/Mtx prophylaxis resulted in lower incidence of acute and chronic Graft-versus-Host Disease (GvHD) without increase of relapse or mortality rates in a phase III trial of HLA- Experimental stem cell transplantation matched unrelated stem cell transplantation (SCT). The poly- clonality of ATG-F is responsible for its effects on the immune system through different mechanisms: T-cell depletion through P616 complement-dependent lysis and T-cell activation and apopto- Evaluation of alloimmune responses in humanised NOD/ sis; modulation of molecules involved in leukocyte/endothelium SCID/IL2Rgnull mice following human CD34+ stem cell interactions; induction of apoptosis in B-cells; interference with transplantation dendritic cells and induction of regulatory immune cells. How- U.F. Hartwig (1), M. Nonn (1), S.A. Khan (1), A. Brunk ever, the slow clearance of ATG-F administered before alloge- (1), J. Hemmerling (1), R.G. Meyer (1), M.C. Andre (2), neic SCT may delay immune reconstitution, increase the risk of R. Handgretinger (2), C. Huber (1), W. Herr (1) disease relapse and impair the activity of a donor lymphocyte (1)Johannes Gutenberg-University (Mainz, DE); (2)Eberhard- infusion (DLI). In vivo pharmacokinetics of ATG-F specifi c anti- Karls University (Tübingen, DE lymphocyte fraction is warranted for the planning of appropriate timing of post-SCT adoptive immunotherapy with donor T-cells. Immunodefi cient NOD/SCID/IL2Rgnull (NSG) mice can be We studied 15 patients with hematologic malignancies, who effi ciently used to establish human lymphohematopoiesis and underwent allo-SCT from mismatched family or unrelated lymphohematological neoplasias in a murine environment. donors, after a conditioning regimen based on treosulfan and Thus, upon engraftment of human CD34+ hematopoietic stem fl udarabine. 13 patients received ATG-F (10 mg/kg/d on days cells (HSC) or acute leukemia blasts and allogeneic donor lym- -4,-3,-2 before SCT), 2 patients did not receive in vivo T-cell phocyte infusions these mice might become a powerful tool to depletion. We collected serum samples from the fi rst ATG-F provide an allogeneic HSC transplantation model for investigat- dose to 54 days after SCT. We used a fl ow cytometry-based ing human graft-vs-host (GVH)- and graft-vs-leukemia (GVL)- assay to detect the concentration of free T-cell specifi c rabbit immunity in vivo. IgG (SRIgG), corresponding to the serum biological activity In the current study, we engrafted NSG mice with human CD34+ against human T-cells and compared it to the levels of unspe- HSC to provide a model for analyzing the role of patient-derived cifi c rabbit IgG (RIgG), that lack anti-T-cell function. antigen presenting cells for the induction of GVH- and GVL-reac- Preliminary data show that both SRIgG and RIgG reached their tivity and to evaluate reactivity to human alloantigens of ex vivo concentration peak 64 hours after the fi rst ATG administration. engineered T cell allografts for improved immunotherapy. Fol- However, the terminal elimination half-life of SRIgG is signifi - lowing reconstitution of busulfan conditioned neonatal or irradi- cantly shorter than the half-life of RIgG: 15 vs 69 days, indi- ated (300 cGy) adult NSG recipients with 1x105 to 1x106 purifi ed cating that 10 days after SCT SRIgG titers may have reached CD34+ adult HSC and weekly i.v. injections of human IL-7 linked sub-therapeutic levels. to the Fc-domain (Fc-IL7), human CD45+ peripheral blood These results suggest that in vivo specifi c activity of rabbit ATG mononuclear cells were detected as early as 30 and 60 days (d) Fresenius against T-cells disappears from circulation between post reconstitution of newborn and adult mice, respectively. Fol- days +7 to +10 after the last administration. According to these lowing monocyte recruitment signifi cant numbers of T-, B-, NK- results current policy of delaying DLI after day +21 post-SCT and dendritic cells (DCs) were found in spleen (SPL) and bone should be revisited. marrow of both newborn and adult recipients by fl ow cytometry and immunhistology 30d to 60d after initial screening. However, as reconstitution of newborns resulted in thymopoiesis of human CD3+ cells and better lympho- and myelopoiesis as compared to adult mice, further studies were performed in HSC engrafted newborn recipients. Functional analyses of lymphocytes grown in humanized NSG mice revealed that human CD3+ T cells isolated from SPL and challenged ex vivo with i) murine NSG derived DCs, ii) human HLA-mismatched DCs or iii) HSC donor derived (autologous) DCs elicit immune reactivity to human but not murine alloantigens. These results and further studies on ex vivo analyzed allo- and xenoimmunity of naive HLA-matched and HLA-mismatched CD8+ T cells transferred into HSC engrafted NSG recipients will be presented to demonstrate T cell alloim- mune responses in humanized mice and to show that our model can be used to evaluate residual GVH reactivity of CD8+ donor T cells in HLA-matched or haploidentical settings.

S156 P618 and within the nucleus of the HaCaT cells as evaluated by Bimodal immunomodulatory effect of multipotent adult confocal microscopy. Metaphases from the BrdU+ HaCaT progenitor cells on alloreactive T-cell responses cells confi rmed that the BrdU signal was located within the A. Luyckx, L. De Somer, O. Rutgeerts, F. Ulloa-Montoya, isolated chromosomes suggesting the creation of hybrid chro- M. Geeraerts, C. Lenaerts, M. Waer, C. Verfaillie, A.D. Billiau mosomes. Incorporation of the transferred genomic material in University of Leuven (Leuven, BE) the HaCaT host genome was inhibited by cytochalasin (inhibi- tor of phagocytosis), increased 3-fold by bafylomicin (inhibition Multipotent Adult Progenitor Cells, a novel type of bone mar- of lysosomal degradation) and 2-fold when HaCat cells were row stem cells with extensive proliferation potential and a broad repeatedly exposed to Jurkat apoptotic bodies. In order to eval- differentiation capacity (Mesoderm including a.o. endothelium, uate expression of the transferred genomic material, we co- hepatocyte like cells), are very promising as a source for regen- cultivated HacaT cells with intact or apoptotic GFP-transfected erative cell therapy. In analogy with classical lineage-restricted JvM cells. Cocultivation with non-apoptotic GFP+ cells resulted mesenchymal stem cells (MSC), of which the immunosup- in no GFP expression in Hacat cells. In contrast, cocultivation pressive behavior is well documented and is being clinically of Hacat cells with GFP+ apoptotic bodies resulted in a 6-fold explored, we investigated the immunomodulatory properties increase in the mean GFP fl uorescence in HaCaT cells. Taken of mouse MAPC (mMAPC) in in vitro/in vivo assays of T cell together, our in vitro model suggests horizontal gene transfer alloreactivity. through apoptotic bodies as a possible mechanism explaining We used mMAPC (mMAPC-2) and mClone-1 (cell population epithelial chimerism after allogeneic HCT. isolated under MAPC conditions with phenotypic and functional characteristics of MSC) (C57BL/6 background), described in Ulloa et al, Genome Biol 2007. Mit-C-treated mMAPC-2/ P620 mClone-1 were added to MLRs (C57BL/6 CD4+ T-cells stimu- Radiation dose, recipient gender and cell dose as risk lated with Mit-C-treated CD11c+ BALB/c DCs) at increasing factors for graft survival in a canine non-myeloablative suppressor/effector (S:E) ratios. mClone-1 mediated a dose- HSCT model dependent profound suppression of T-cell proliferation. By S. Lange, A. Knueppel, D. Killian, G. Knuebel, A. Sekora, contrast, mMAPC-2 exhibited a bimodal immunomodulatory G. Kundt, M. Freund, C. Junghanss effect, with immunostimulation at low and immunosuppression University of Rostock (Rostock, DE) at high S:E ratios. Transwell assays showed that the mMAPC- 2 immunostimulatory effect is cell-contact dependent, whereas The canine non-myeloablative stem cell transplantation (NM- the immunesuppressive effects (of mMAPC-2 ánd mClone-1) SCT) model has become accepted during the last decades as a are contact-independent. Inhibition assays demonstrated that good preclinical model for the development of new transplanta- NO and PGE2 - in part - are involved in mClone-1-mediated, tion strategies. Information on factors associated with outcome but not mMAPC-2-mediated suppression. Other mediators are after allogeneic SCT are a prerequisite for designing new risk being tested. Neither mMAPC-2 nor mClone-1 suppressed the adapted transplantation protocols. Here we report on a retro- development of graft-versus-host disease induced by C57BL/6 spective analysis aimed at identifi cation of risk factors of graft T cells in NK-cell-depleted CB17SCID mice. In Popliteal Lymph survival in the canine SCT model. Node Assays, mClone-1 consistently suppressed the expansion Data from 39 consecutive MHC-identical allogeneic NM-SCT, of alloreactive C57BL/6 T-cells in NK-cell-depleted CB17SCID performed between 2003-2008 on different protocols, have mice, whereas mMAPC-2 in this assay mediated either stimula- been evaluated in regards to allograft survival. 23 recipients tory or suppressive effects. were male (59%). The median age was 14 months (range 7-29) In conclusion, mouse MAPC exhibit complex immunomodula- and the median weight was 12.4 kg (9.8-19.0). Conditioning tory activities, in part comparable to those of classical MSC. consisted of 1 Gy (n=20) or 2 Gy (n=19) total body irradiation Further study is focused on the environmental factors infl uenc- (TBI) based regimen. CSA (days -1 to +35) in combination with ing the bimodal mMAPC immune behavior. MMF (days 0 to +27) or everolimus (days 0 to +27) were used for immunosuppression. A median cell dose of 3.6 total nucleated cells (TNC)/kg (1.6-11.4) was infused. Follow-up was restricted P619 to 26 weeks after SCT. Uni- and multivariate Cox analyses were Horizontal gene transfer through apoptotic bodies used to calculate the infl uence of age, weight, TBI dose, gender confers a novel mechanism of epithelial chimerism after of donor/recipient, type of immunosuppression and cell dose of allogeneic HCT TNC and CD34+ cells on the outcome of SCT. M. Waterhouse (1), M. Themeli (2), A. Spyridonidis (2), Initial engraftment occurred in all dogs. Animals that received a J. Finke (1) 1Gy conditioning eventually rejected their graft after median 10 (1)Freiburg University (Freiburg, DE); (2)Patras University weeks (7-16). After 2Gy TBI 9/17 dogs (53%) achieved stable (Patras, GR) long-term engraftment (>26 weeks). 2 dogs died in week 10. Univariate analysis revealed TBI dose, gender, type of immuno- Animal and human studies have shown that after allogeneic suppression and TNC dose as factors infl uencing graft survival hematopoietic cell transplantation (HCT) low percentage of (p<0.2 for each). In multivariate analysis 3 factors were iden- epithelial cells containing donor-derived genome emerge. The tifi ed as independent risk factors for graft rejection: TBI dose mechanisms underlying this phenomenon are unknown. We (1Gy vs. 2 Gy; HR 12.5, CI 2.6-59.8, p=0.002), gender (female investigated using an in vitro model if horizontal gene trans- vs. male recipients; HR 3.2, CI 1.2-8.1, p=0.016), TNC cell fer could account as a possible mechanism for epithelial chi- count (<3x108 TNC/kg vs. >7x108 TNC/kg; HR 7.4, CI 0.9-64.4, merism after allogeneic HCT. Briefl y, keratinocyte HaCaT cells p=0.069). PBMC chimerism >30% and granulocyte chimerism (Y-chromosome neg) were cocultivated with non-apoptotic Jur- >70% 4 weeks after SCT were identifi ed as independent predic- kat cells (Y chromosome pos) or Jurkat cells in which apoptosis tors for stable engraftment (p=0.005 and 0.010, respectively). was induced with Camptothecin. Jurkat cells were labeled with In summary, these data support that even in low dose TBI CMFDA or BrdU in order to track the fate of apoptotic bodies based regimen irradiation dose is important. In addition female and genomic material respectively. HaCaT cells co-cultivated sex as recipient and TNC dose were identifi ed as risk factors of with non-apoptotic Jurkat cells for 24-72h did not show any graft survival. Chimerism at 4 weeks post SCT was predictive CMFDA, BrdU or FISH-Y chromosome signal. In contrast, co- for long-term engraftment in the canine SCT model. cultivation of HaCaT cells with Jurkat apoptotic bodies resulted in CMFDA signal in 28% of the HaCaT cells, BrdU signal in 18% and Y-chromosome signal in 3% of the cells. Importantly, BrdU and Y-chromosome signals were observed in the cytoplasm

S157 P621 melphalan (140mg/m²) and anti-thymocyte immunoglobulin. CD34-selected stem cell boost for poor graft function after GvHD-prophylaxis consisted of cyclosporinA and mycophe- allogeneic stem cell transplantation for myelofi brosis nolate. Both patients had matched unrelated donors (10/10). E. Klyuchnikov, M. Lioznov, U. Bacher, A.R. Zander, N. Kröger Patient 1 received blood stem cells with a reduced T-cell University Cancer Center (Hamburg, DE) number, patient 2 stem cells from red blood cell depleted bone marrow. The low bone marrow milieu by myelofi brosis (MF) may Results: In both patients hematological reconstitution was predispose to poor graft function (PGF) after allogeneic stem uneventful, toxicity was mild, one patient showed GvHD °I of cell transplantations (allo-SCT). We investigated the effi cacy of the skin. Infectious complications included EBV-reactivation in CD34+ stem cell boost (SCB) in MF patients with PGF. The PGF patient 1 and brain abscesses in patient 2, which resolved with- was diagnosed in patient with 2 or 3 cytopenic counts (Hb<10g/ out clinical residuum. dL; neutrophil count <1.0x109/L; platelet count <30x109/L) for Follow-up period after SCT is 3 years in patient 1 and 7 months at least 2 consecutive weeks beyond day +14 post-transplant, in patient 2. They are both in good clinical condition, showing with transfusion requirement and in the absence of severe graft full donor chimerism. Patient 1 is leading a normal life, free of versus host disease (GvHD) and relapse. A total of 8 patients severe infections. Infection by mollusca contagiosa resolved, (5 males and 3 females) with median age 61 year (range, eosinophilia disappeared, IgE levels, T-cell-number and -func- 53 – 66 y) underwent allo-SCT for MF (primary n=6, secondary tion normalized. Patient 2, now 7 months after SCT, shows a n=2). Three patients were JAK2V617F mutated. All patients had markable reduction of the mollusca contagiosa. She is still on received HLA-matched grafts from unrelated donors (periph- antibiotics as treatment for the brain abscesses. Immunological eral blood stem cells, n=8) after reduced intensity conditioning reconstitution is progressing but not complete yet, eosinophilia regimen (RIC) based on busulfan (10mg/kg) combined with disappeared, IgE levels decreased signifi cantly. fl udarabine (180mg/kg) and antithymocytic globuline (ATG) Conclusion: The clinical post transplant course suggests SCT to (60mg/kg). All patients succefully engrafted with leukocytes be a curative therapeutic approach in patients with AR-HIES. (median day 18), while platelet engraftment was observed in one patient at day 16 from allo-SCT. SCB was performed at median time point of 150 days after allo-SCT, when P623 the median leukocyte and platelet counts were 2.3x109/L Toll-like receptor 2 is responsible for enhanced and 38x109/L, respectively and mean chimerism level was engraftment of G-CSF-mobilised peripheral blood 95% (range, 80 – 100%). stem cells Positive selection of CD34+ stem cells was performed on a Y.D. Joo (1), H.J. Won (2), S.M. Lee (1), J.H. Park (3), S.K. Park CliniMACS device, achieving a median of 98.5% purity. The (3), I. Choi (2), S.K. Seo (2), H. M. Ryoo (4) patients received a median of 3.8 x 106/kg (range, 2.2-7.0x106/ (1)Busan Paik Hospital (Busan, KR); (2)Center for Viral Disease kg) CD34+ cells and 0.16x105/kg CD3+ T-lymphocytes (range, Research (Busan, KR); (3)Ulsan University Hospital (Ulsan, 0.01-0.5x105/kg). Hemograms at days 30, 60, and 90 from SCB KR); (4)Daegu Catholic University Medical Center (Daegu, KR) showed steadily increasing median leukocyte (0.6, 1.2, and 1.9 x109/L) and platelet (58, 81, and 103 x109/L) counts. Five of Peripheral blood stem cells (PBSCs) obtained from granulocyte- eight patients showed an increase in leukocyte count at day colony stimulating factor (G-CSF)-mobilized donors have been 30 from SCB, three of them showed also an increase in throm- used more frequently than bone marrow stem cells (BMSCs) bocyte count. Of those, two patients showed the increase in as the source of cells in allogeneic hematopoietic stem cell leukocyte and thrombocyte counts further up to day 90 from transplantation (allo-HSCT) because of more rapid engraftment SCB. Moreover, with those fi ve patients, the average time lag without increasing the incidence of acute graft-versus-host dis- between transfusions increased from a 8 day to a 35 day time ease (GVHD). However, it remains unclear whether mecha- interval before and after SCB, respectively. Two patients devel- nisms are responsible for enhanced engraftment. Recently, oped acute GvHD: hepatic GvHD grade II and gut GvHD grade it was reported that innate immune sensor toll-like receptor 2 II 30 and 90 days from the SCB, respectively. Three patients did (TLR2) and TLR4 were expressed on hematopoietic progeni- not show any signs of response. tor cells (HPCs). These receptors greatly drove of HPCs to These preliminary results point to the possibility of safely mature innate immune cells. Here, we investigated that TLR2 is improving graft function using CD34+ SCB in patients with PGF responsible for rapid engraftment of PBSCs in allo-HSCT using post allo-SCT for MF. murine model of PBSC transplantation (PBSCT). We found that TLR2 expression was dramatically up-regulated on G- CSF-mobilized PBSCs (gmPBSCs, lineage-c-kit+) compared P622 with BM stem cells (BMSCs). However, TLR4 was very weakly Stem cell transplantation in autosomal recessive expressed and there was no change. It was confi rmed that hyper-IgE-syndrome TLR2 was directly up-regulated by G-CSF on BMSCs. Treat- U. Benninghoff, S. Gatz, C. Schütz, M. Hönig, F. Speth, ment of TLR2 ligand Pam3CSK4 directly augmented differen- K. Schwarz, A. Schulz, W. Friedrich tiation of gmPBSC to mature monocytes/macrophages. This is Children`s Hospital, University of Ulm (Ulm, DE) much higher than BMSCs. These responses did not occur with both stem cells from TLR2-defi cient mice. Our results demon- Objectives: We present 2 patients, a 10-years old boy and a 17- strate that TLR2 signaling in gmPBSCs correlates with their years old girl with autosomal recessive Hyper-IgE-Syndrome differentiation resulting in improvement of engraftment, sug- (AR-HIES), who were treated by allogeneic SCT. So far there gesting TLR2 is novel target for increasing allo-HSCT effi ciency are no reports on SCT in AR-HIES. to overcome engraft failure. In both patients the main clinical features were recurrent bacte- rial infections (pneumonia, bronchitis, sinusitis) and uncontrol- lable infections by mollusca contagiosa, particular in the face P624 and genital region, leading to considerable morbidity and psy- Phenotypic and functional characterisation of canine chological problems. Pathological laboratory values included regulatory T-cells in the allogeneic setting eosinophilia, elevation of serum IgE (9.000-21.000 IU/ml), A. Knueppel, S. Lange, A. Sekora, D. Killian, M. Freund, T-cell lymphopenia and impaired T-cell function. Indication for C. Junghanss SCT was based on the risk of potentially lethal infections in this University of Rostock (Rostock, DE) primary immunodefi ciency. Methods: A reduced-toxicity conditioning regimen was applied, Background: CD4+CD25high regulatory T-cells (Tregs) can including radioimmunotherapy, fl udarabine (160mg/m²), modulate immune responses following allogeneic hematopoietic

S158 stem cell transplantation (HSCT). Canine Tregs have not been when compared to hES and EB cells. Importantly, these cells well characterized despite the fact that the canine allogeneic could be differentiated to hematopoietic cells forming CFU-M HSCT model has served as important preclinical model for dec- colonies containing macrophages, BFU-E containing nucle- ades. Here, we report on phenotypic and functional properties ated red blood cells, and CFU-EM colonies composed of mac- of canine Tregs. rophages and nucleated erythrocytes. Cells of CFU-EM and Methods: Canine peripheral blood mononuclear cells (PBMCs) BFU-E expressed both epsilon- and gamma-globin genes, but were labeled with CD4, CD25 and Foxp3 for phenotypic not adult-type gamma-globin. The BLCs could differentiate to analysis. CD4+CD25high cells were enriched from PBMCs endothelial cells which had the ability to take up Dil-Ac-LDL and for functional assays by using magnetic cell sorting (Miltenyi, formed vascular networks in Matrigel. enrichment for CD25high). Expansion of Tregs was done by Conclusions: culturing cells in IMDM supplemented with IL-2 (100 I.U./ml) for 1) Our EB system recapitulates early yolk sac hematopoiesis. one week. For allogeneic mixed lymphocyte cultures (MLCs) 2) It enables investigation of human hemangioblast and its irradiated stimulator cells were cultured with allogeneic T-cells progeny. ± freshly isolated or expanded Tregs. 3) Differentiation of hemangioblasts can be tracked using cho- Results: Canine Tregs can be characterized by their immune sen molecular markers. phenotype as CD4+, CD25high and Foxp3+. The median per- 4) BLCs are functional and can be potentially used in regenera- centage of CD4+CD25highFoxp3+ Tregs within the CD4+ com- tive medicine. partment of PB was 0.8% (range: 0.3 – 4.6%, n=11). Expansion of canine Treg could be achieved up to median 12.4% (range: 2.2 – 34.2%, n=6) after one-week-cultivation. In allogeneic MLCs P626 freshly isolated Tregs as well as expanded Tregs suppressed Novel system for development of ectopic bone marrow: signifi cantly allogeneic T-cell proliferation (48% and 88%, prolif- identifi cation and isolation of the haematopoietic stem eration reduction compared to controls, respectively). cell niche initiating cell population Conclusion: Canine regulatory T-cells display similar character- C. Chan, D. Kraft, C. Chen, C. Luppen, J. Kim, I. Weissman istics as Tregs of other species such as a CD4+CD25highFoxp3+ Stanford University (Stanford, US) phenotype and their ability to suppress allogeneic reactions. These data may pave the way to invesigate Tregs in the pre- Introduction: Little is known about the formation of niches, local clinical canine outbred allogeneic HSCT model. micro-environments required for stem-cell maintenance. Here we develop a novel in-vivo assay for adult haematopoietic stem-cell (HSC) niche formation in which isolated cells from fetal bone can form a functional ectopic bone marrow environ- ment following transplantation under the kidney capsule. Stem cell research Results: With this assay, we identifi ed a population of progenitor cells with surface markers CD45-Tie2-alphaV+CD105+Thy1.1- (CD105+Thy1-) that, when sorted from 15.5 days post-coitum P625 fetal bones and transplanted under the adult mouse kidney Human embryonic stem cell ex vivo differentiation capsule, could recruit host-derived blood vessels, produce system is a new tool for investigation of early yolk sac donor-derived ectopic bones through a cartilage intermediate haematopoiesis and generate a marrow cavity populated by host-derived long- G.W. Basak (1), S. Yasukawa (2), B. Minev (2), E. Carrier (2) term reconstituting HSC (LT-HSC). In contrast, CD45-Tie2- (1)Medical University of Warsaw (Warsaw, PL); (2)University of alphaV+CD105+Thy1+ (CD105+Thy1+) fetal bone progenitors California, San Diego (La Jolla, US) form bone that does not contain a marrow cavity. Suppress- ing expression of factors involved in endochondral ossifi cation, Rationale: It was hypothesized that yolk sac hematopoiesis such as osterix and vascular endothelial growth factor (VEGF), derives from hematoendothelial precursor called hemangiob- inhibited niche generation. CD105+Thy1- progenitor popula- last. While investigation of early steps of hematopoiesis in tions derived from regions of the fetal mandible or calvaria that human embryos meets technical and ethical limitations, we do not undergo endochondral ossifi cation formed only bone propose the embryoid body (EB)/blast cell differentiation sys- without marrow in our assay. tem that provides an outstanding alternative to early embryo for Conclusions: Collectively, our data implicate endochondral identifi cation of hemangioblast and provides an easy access ossifi cation, bone formation that proceeds through a cartilage to a large number of cells representing earliest stages of intermediate, as a requirement for adult HSC niche formation. hematopoiesis. In addition to identifying the limb-derived skeletal progenitor Methods: We used two-step EB differentiation system that capable of endochondral ossifi cation and the basic mecha- involved induction of mesoderm followed by stimulation of nisms of HSC niche initiation, our study provides a functional hemato/vasculopoiesis. The pattern of gene expression was framework by which future studies on HSC-niche interactions monitored by semi-quantitative RT-PCR. Cell suspensions from at the cellular level can be carried out. EB culture (day 0-6) were seeded in semisolid medium sup- plemented with cytokines. Obtained colonies have been char- acterized by Wright-Giemsa staining, immunofl uorescence, P627 FACS and RT-PCR. Their hematopoietic potential was further Expression, regulation and potential function of NFAT assessed in CFU assay and in endothelial cell differentiation transcription factors in human mesenchymal stem cells culture. S. Quick, F. Hauck, K. Mueller, I. Habermann, M. Bornhäuser, Results: Cells obtained from EBs formed homogenous grape- M. Suttorp, G. Ehninger, A. Kiani like colonies composed of 30-50 cells (blast-like cells, BLCs) Dresden University of Technology (Dresden, DE) on day 6 of culture. The occurrence of colony precursor cells was transient and peaked on day 3 of EB culture in associa- Introduction: Human mesenchymal stem cells (hMSCs) display tion with expression of T (early mesoderm gene) and dramatic immunomodulatory effects partially mediated by soluble factors increase in hematopoietic/endothelial genes: CD34, CD31 and like prostaglandin E2 (PGE2), and have been successfully used KDR. BLCs had homogenous morphology in Wright-Giemsa in the treatment of graft-versus-host-disease. Information about stain, but they differed in expression of markers of both imma- transcriptional mechanisms regulating hMSC function is scarce. ture hematopoietic and endothelial cells (CD31, CD34, VE- Nuclear Factor of Activated T cells (NFAT) is a transcription cadherin, Flt-1) and mature differentiated cells (CD45, CD33, factor known to control T-cell-dependent immune responses. CD146). They expressed higher level of HLA class I molecules In resting cells, NFAT is phosphorylated, cytoplasmic and

S159 inactive, while upon stimulation NFAT is dephosphorylated by lymphocyte proliferation; ii) decrease alloantigen-induced cyto- the phosphatase calcineurin, leading to nuclear translocation toxic activity (CA); iii) increase secretion of IL-6 and IL-10 in and activation of the protein. Inactivation of NFAT is mediated MLC supernatant. While the addition of BM-MSCs to MLC by several kinases, e.g. glycogen synthase kinase 3b (GSK3b). increased the percentage of CD4+CD25+FoxP3+T cells, the Little is known about expression, regulation or function of NFAT addition of UCB-MSCs did not result in any increase of this cell in hMSCs. subset. With the aim of understanding the biological mecha- Methods: Bone marrow-derived primary hMSCs were used in nisms responsible for the immunosuppressive effect exerted all experiments. NFAT and cyclooxygenase 2 (COX2) expres- by UCB-MSCs, in a second set of experiments, MLCs were sion was determined by real time PCR and western blotting. carried out in the presence of IDO- or PGE2-specifi c inhibitors Subcellular localization of NFAT was analyzed by confocal (INs). Results obtained in terms of alloantigen-induced lym- microscopy. NFAT and COX2 promotor activity was measured phocyte proliferation documented that neither IDO-specifi c IN by luciferase assays. PGE2 concentration was analyzed by nor PGE2-specifi c IN were able to reverse the MSC-induced ELISA. suppressive effect. At difference, a clear-cut effect of PGE2- Results: hMSCs express considerable levels of all NFAT fam- specifi c IN was observed when alloantigen-induced CA was ily members (predominantly NFATc3 and NFATc4), with the evaluated, as addition of PGE2-specifi c IN to MLC was able to exception of NFATc2. In untreated hMSCs, NFAT proteins are reverse the suppressive effect exerted by both UCB-MSCs and partially constitutively active, as judged by their phosphoryla- BM-MSCs; on the contrary, IDO-specifi c IN was not effective tion and subcellular localization status. Dephosphorylation, for both cell source. nuclear translocation and transcriptional activity of NFAT are The differences between UCB- and BM-MSCs in terms of clo- further induced by treatment with ionomycin and reversed by nogenic effi ciency, proliferative capacity, mechanisms of action cyclosporin A, indicating that the regulation of NFAT in hMSCs and immunomodulatory properties may be relevant for their occurs in a calcineurin-dependent manner. Inhibition of GSK3b clinical application. leads to cyclosporin A-dependent nuclear accumulation of NFAT, emphasizing the presence of a constitutive active cal- cineurin pathway in hMSCs. Treatment of hMSCs with iono- P629 mycin and phorbol ester induces, while CsA attenuates, the No evidence of the JAK2V617F mutation in mesenchymal expression of COX2 and PGE2, suggesting that these genes stromal cells in mutated patients with primary are transcriptional targets of NFAT in hMSCs. myelofi brosis Conclusion: hMSCs show an NFAT expression profi le which is S. Asenova, F. Ayuk, A. Badbaran, A.R. Zander, U. Larsen, clearly distinct from that found in Jurkat T cells or CD34+ cells. S. Hannemann, N. Kroeger, C. Lange, U. Bacher Constitutive activation of NFAT in hMSCs suggests it may be University of Hamburg (Hamburg, DE) involved in mediating biological properties of these cells, e.g. the modulation of lymphocyte function through the expression Introduction: The JAK2V617F mutation (JAK2V617Fmut) is of PGE2. Dissection of NFAT-regulatory pathways and target frequent in the chronic myeloproliferative neoplasms (MPN). genes in hMSCs will allow to identify and specifi cally manipu- An open question is the level of cell differentiation, where the late hMSC functions. JAK2V617Fmut occurs. The existence of a common meso- derm cell as origin of both hematopoietic and mesenchymal progenitor cells was postulated e.g. by experiments in the P628 mouse model as mesenchymal stromal cells (MSC) were dem- Immunomodulatory properties of umbilical cord onstrated to be able to initiate hematopoietic reconstitution and blood-derived mesenchymal stromal cells expanded stroma cell formation in syngeneic mice (Lange et al., 1999). in the presence of platelet lysate: potential for clinical Screening for the JAK2V617Fmut in MSC derived from MPN application patients carrying the mutation might contribute to insights in the M. Bernardo (1), M. Avanzini (1), C. Perotti (1), A. Cometa (1), differentiation level where the mutation occurs. In a series of 5 N. Zaffaroni (2), F. Novara (3), A. Moretta (1), R. Maccario (1), JAK2V617F mutated myelofi brosis patients of Pieri et al. (Leuk W. Fibbe (4), F. Locatelli (3) Res, 2007), MSC were tested negative for the mutation. (1)Fondazione IRCCS Policlinico San Matteo (Pavia, IT); Methods: We performed screening for the JAK2V617Fmut by (2)Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, IT); quantitative real-time polymerase chain reaction (qPCR) in (3)Università di Pavia (Pavia, IT); (4)Leiden University Medical unseparated bone marrow (BM)/ peripheral blood (pB) (sensi- Center (Leiden, NL) tivity: 0.01%) of MPN patients. From these, we selected 7 aspir- able patients with myelofi brosis (primary: n=3; secondary: n=4; We isolated and expanded ex vivo MSCs from full-term UCB 5 males, 2 females) before/after allogeneic stem cell transplan- (UCB-MSCs) in the presence of a platelet lysate- (PL, 5%) tation (SCT) (who all showed evidence of the JAK2V617Fmut additioned medium. UCB-MSCs were phenotypically and func- at the time of analysis in BM/pB) for parallel screening of in tionally characterized, in comparison to bone marrow-derived vitro expanded MSC for the mutation. MSC were expanded (BM) MSCs. until reaching confl uence of 90-95% (Lange et al., Journal of Ten UCB units (median volume 45 ml, range 40-60), from Cell Physiol, 2007). Quantifi cation of the JAK2V617Fmut was full-term deliveries, were processed. Two of the 10 UCB units performed by correlation with the HCK gene for normalization (UCB3 and UCB6) gave rise to MSC-like clones, which were (Kröger et al., Blood, 2007). expanded ex vivo and characterized. UCB-MSCs displayed Results: MSC cultivation was performed in one patient before the typical morphology, immunephenotype and differentiation and in 6 patients after SCT with dose reduced conditioning capacity. Although displaying a rather low clonogenic effi ciency, and peripheral blood stem cells from related/unrelated donors. UCB-MSCs showed a higher proliferative potential as compared Six cases showed negative results after the fi rst passage of to BM-MSCs. UCB3 and UCB6-MSCs entered senescence MSC, although all patients showed systemic evidence of the at passage (P) 10 and P11, respectively. Telomerase activity JAK2V617Fmut in BM/pB (median 3.85%; range: 0.005%- and hTERT transcripts analysis, as well as molecular karyo- 32.7%). One case showed a high JAK2V617F/HCK ratio of typing through array-CGH demonstrated lack of spontaneous 32.7% in bone marrow, while MSC revealed a mutation ratio of transformation into tumor cells. The immunoregulatory effect of 0.004%, which was probably due to contamination of MSC with UCB-MSCs on alloantigen-specifi c immune response in mixed remaining hematopoietic cells. lymphocyte culture (MLC) was investigated, together with the Discussion: We here found no evidence of involvement of the mechanisms potentially responsible for this effect (PGE2 pro- MSC in the JAK2V617F mutation using a highly sensitive real- duction, IDO activity). We found that, similarly to BM-MSCs, time based PCR assay. This study gives further confi rmation to UCB-MSCs are able to: i) strongly inhibit alloantigen-induced the hypothesis that MSC are not affected by this mutation and

S160 do not serve as common progenitor cell for stromal and hemat- opoietic cells in MSC.

P630 Global comparative transcriptome analysis of human cord blood stem cells and adult stem cells: differential stemness state M. Cuzzola, G. Pucci, A. Pontari, D. Marcuccio, I. Bova, C. Rigolino, M.C. Cannatà, A. Meliadò, I. Callea, T. Del Vecchio, O. Iacopino, P. Scaramozzino, R. Surace, E. Spiniello, C. Garreffa, A. Dattola, P. Iacopino Bone Marrow Unit (Reggio Calabria, IT)

Background: The genome-wide analysis provides a modern approach to reveal intrinsic properties of stem cells such as the pluripotency, self-renewal and commitment. To elucidate simi- larities and differences about transcriptome we conducted a direct stringent comparison between stem cells of different cell source: human cord blood (HUCB) and classical adult periph- eral blood stem cells (PBSCs). Materials and Methods: PBSCs were obtained from 10 healthy donors treated with human (rh) G-CSF, HUCBs were col- lected from 20 umbilical cord. RNA extraction and reverse transcriptase was performed in all samples obtaining identical cDNA concentration (1000ng/ml). The pool of PBSCs-cDNA was used to normalize HUCB and the pool of HUCB was used to normalize PBSCs in different analysis. TaqMan® Low Den- sity Array based on comparative ddCT method was employed to estimate relative quantifi cation of 48 genes that are known to be associated with stemness functions. The list included key human Stem Cells (SCs) pluripotency genes such as Lefty 2, FG2, TDGF1, POU5F1(OCT4), Nanog and Sox2, thus provid- ing a positive control for the quality of the cells and the data obtained. Results: We were able to identify at least three gene blocks with different expression among the two SCs of different origins (table 1). The fi rst and second blocks included genes with higher and lower expression of mRNA-copies in HUCB than PBSCs, respectively. In the third block the genes were expressed in a similar manner. As expected, the most genes with self- renewal property were all up-expressed in HUCB (CD34, GATA1, GATA2, HOXA5, HOXA9, TGB1, PBX1, M1ES1) together with the pluripotency pattern genes. In addition, as it’s note- worthy, the genes involved in homing functions were highly over-expressed in HUCB (CXCR4, IL-8, ICAM-1). Of course, P631 the gene pattern associated with hematopoietic erythroid and Complement compatibility of human mesenchymal stem myeloid lineage commitment showed interesting increases in cells during clinical infusion into whole blood for adult SCs (LMO2, HOXA10, CUX1). Finally, MAP4K1 LEFTY2 treatment of graft-versus-host disease genes involved in TGFB signalling pathway were analogously G. Moll (1), I. Rasmusson (2), G. Elgue (2), B. Sundberg (1), regulated in either SCs. K. Nilsson Ekdahl (2), K. Le Blanc (1), B. Nilsson (2) Conclusion: Comparative analysis of gene expression among (1)Karolinska Institutet (Stockholm, SE); (2)Uppsala University various SCs sources showed differential profi le. We believe (Uppsala, SE) that stemness transcriptome may be the base for the activation of a molecular program to characterize the effi ciency state of Infusion of third-party mesenchymal stem cells (MSC) appears different cell products employed in stem cell based-therapy. to be a promising therapy for graft-versus-host disease (GvHD). To date, little is known about the interaction of MSC with the innate immune defense mechanisms after infusion and in par- ticular the effect of complement (C) on cell fate. In this study we characterized the C-compatibility of MSCs as compared to endothelial cells (EC). Studies of C-component C3 cell surface deposition, as well as C3a generation in vitro and in a whole blood system showed increased C-activation for MSCs. In presence of whole blood, FCS cultured MSCs induced elevated activation of Lymphocytes, Monocytes and upregulation of CD11b on PMNs which could be abrogated in the presence of compstatin or C5aR-antagonist. Differential analysis of C-regulatory proteins MCP (CD46), DAF (CD55) and protectin (CD59) as well as antibody binding to cells iden- tifi ed the lack of MCP and DAF in MSCs, whereas IgG- and IgM-binding was comparable to ECs. MSCs showed classical and alternative pathway activation, but were protected from C- lysis via CD59. C-activation could be reduced to the level of

S161 ECs when cells where cultured in human AB-serum or early channels came up as an alternative method to count haemat- passage clinical MSCs were tested and completely abrogated opoietic progenitor cells (HPC), with several studies showing a by C-antagonists. correlation between CD34+ cells and HPC. These observations provide a fi rst characterization of C-com- The aim of this ongoing study is to compare which of the two patibility of culture expanded MSC for clinical infusion, which parameters (CD34+ cells or HPC) better predicts the success shows the importance of serum choice on cell fate in vivo. of the collection. We retrospectively analysed PBPC grafts from 81 healthy donors and 103 patients with haematological malignancies P632 (MM n=49, NHL n=37 and HD n=17), who underwent G-CSF Comparison of bone marrow buffy coat-, bone marrow mobilization between January 2007 and October 2008. On fat- and adipose tissue-derived mesenchymal stromal the 5th day of mobilization, PB samples were collected from cells all individuals for WBC and HPC counts in the Sysmex XE- B. Mazzanti (1), S. Dal Pozzo (1), S. Benvenuti (2), I. Cellai (2), 2100 analyzer and CD34+ cell enumeration by fl ow cytometry. G. Gerlini (3), S. Urbani (1), C. Ballerini (2), M. Santosuosso All individuals started apheresis and PBPC counts were also (1), A. Bosi (1), R. Saccardi (1) performed using the same parameters as for PB. (1)Careggi Hospital (Florence, IT); (2)University of Florence Results are shown as median and quartile (Q) ranges. Median (Florence, IT); (3)SS. Annunziata Hospital (Florence, IT) number of CD34+ cells and HPC obtained/µL in the PB of patients were 16 and 71 respectively and for donors values Objective: Mesenchymal stem cells (MSC) are a heterogene- were 65 and 130 cells/µL. Most donors were good mobilizers, ous population of stromal stem cells that can be isolated from collecting >4x106 CD34+ cells/kg of recipient body weight (bw). different adult tissues. We compared the characteristics of For patients a successful collection was defi ned as >2.5x106 freshly isolated and of in vitro expanded cells derived from bone CD34+ cells/kg bw collected in up to 4 apheresis performed in marrow (BM) buffy coat (BC), BM fat and adipose tissue (AT). consecutive days. Methods: BM aspirates were obtained from 7 BM donors and Patients’ PB results and the predictive value for the total of 5 patients who underwent stem cell infusion for the treatment CD34+ cells collected are summarised in Table 1. None of the of osteopeneic disorders. AT was isolated from 3 patients patients with CD34+ cells below 8 (n=23) (1st Q) had an effi - undergoing reconstructive plastic surgery. All procedures were cient mobilization, whereas 37% of patients with CD34+ cells approved by the local Ethics Committee. After the centrifuga- within the 2nd Q had a successful collection. For patients with tion of BM samples, a layer of nucleated cells (BC) at inter- PB CD34+ cells values above median, collection was success- phase between the plasma and red cells pellet was collected. ful in 78% of cases for the 3rd Q and 100% for the 4th Q. For Cells from BM fat or AT were isolated by collagenase digestion. HPC counts there was no defi ned pattern of success, as we CFU-F determination, MSC in vitro expansion, fl ow cytometric observed in all Q collections above the defi ned PBPC target. characterization, differentiation potential and immunomodula- Even though we found a correlation between PB CD34+ cells tory properties were evaluated as previously described (Urbani and HPC and the total of collected cells/kg patient bw, PB et al., Cytotherapy 2006; Mazzanti et al., J. Neuroimmunol CD34+ cells are a better predictor of PBPC harvests’ success 2008). than HPC. Results: Highest frequency of CFU-F/106 TNC was observed for AT-, followed by BM fat-derived SC (803.3 ±753.0 and 652.4 ±516.1 respectively). In addition, BM fat had a 17.1 fold increase in the mean number of CFU-F/106 TNC in comparison with BM BC of the same donor. Flow cytometric analysis of freshly isolated cells from AT showed a high percentage of CD34 (61.5%), CD90 (61.3%) and CD271 (19.4%) positive cells. CD45lowLNGFR+ stromal progenitor cells frequency in AT (20.3%) and BM fat (2.9%) is higher than BM BC (0.04%). BM BC and fat derived CD45low- LNGFR+ cells showed a high expression of the MSC markers CD90, CD73 and CD105; however only AT-derived popula- tion expressed the CD34+ haemopoietic marker. Proliferative potential of AT derived MSC was lower in comparison to BM BC and BM Fat derived MSC. MLR experiments indicated a higher suppression of lymphocyte proliferation by AT- derived MSC. P634 Conclusions: Results from our study demonstrated that stem Immunophenotype of human bone marrow MSCs on early cells isolated from AT and BM showed different ex vivo and in and late passages during ex vivo expansion vitro properties in terms of phenotype, proliferation and immu- E. Osipova (1), T. Astrelina (1), B. Purbueva (1), A. Ystiugov nomodulation properties. These fi ndings are relevant for the (1), E. Skorobogatova (1), Z. Dishleva (1), S. Roumiantsev (1), selection of stem cell source and are a critical issue for clinical M. Yakovleva (2), O. Mayorova (1) applications. (1)Research Centre of Pediatric Hematology, Oncology and Immunology (Moscow, RU); (2)Stem Cell Bank (Moscow, RU)

P633 Objectives: MSCs are multipotent cells with many potential clin- PB CD34+ cells are better predictors than HPC of the ical applications. However, the lack of a precise defi nition of the success of PBPC harvest cells preparation and the heterogeneity of the obtained product I.L. Barbosa, C. Carvalho, F. Campilho, S.M. Lopes, S. Roncon, render validation criteria of the fi nal product of the expansion. A.P. Gonçalo, T. Sousa, C. Mendes, R.B. Ferreira, C.P. Vaz, During the culturing procedures immunophenotype changes A. Campos, A. Carvalhais can arise which can lead to long term consequences of compli- Instituto Português de Oncologia (Porto, PT) cations of MSC transplantation. Aims: to estimate immunophenotype of MSC from healthy For both healthy donors and patients, peripheral blood (PB) donors on early (2-4) and late (10-12) passages. CD34+ cell count is the parameter currently used to monitor Methods: MSC (n=25) were prepared by seeding bone marrow G-CSF mobilization and to predict the yield of harvested periph- MNC in 75 cm² culture fl asks in low glucose DMEM with 20% eral blood progenitor cells (PBPC). Recently, last generation FCS. After 72 hours the non-adherent cells were discarded and haematology analyzers with «immature information» (IMI) cells were passed every 7 days by trypsinization and replanting

S162 at the density of 0.5x106 cells/75 cm² fl ask until the end of the was signifi cantly reduced (p<0.01) and resembled that seen culture. The immunophenotype of MSC (CD3, CD13, CD14, in patients treated with in vivo chemotherapy. It is concluded CD19, CD25, CD29, CD31, CD34, CD38, CD44, CD45, CD73, that co-culture with liver spheroids can enhance the therapeutic CD90, CD105, CD106, CD166 è HLA-DR) was analyzed by effectiveness of CY in vitro and provide a physiologically rel- fl ow cytometry. evant model to study chemotherapeutic damage. Results: We found that in vitro expanded bone marrow MSC demonstrate higher proliferative activity on 2-4 in comparison with 10-12 passages (5,9-fold increase and 2,0-fold accord- P636 ingly, p=0.049). Viability of CD34+ cells in cryopreserved cord blood On the 4-th passage the cells were high positive (median Y.W. Eom (1), S.H. Jeong (2), J.H. Choi (2), S.Y. Kang (2), >90%) for CD90, CD105, CD73, CD166, CD44, median posi- H.W. Lee (2), H.C. Kim (2), J.S. Park (2) tive (median 30-90%) for CD13 è CD29 and negative (median (1)Biomedical Research Institute, LifeCord Inc. (Suwon, KR); < 5%) for CD45, CD34, CD133, CD3, CD19, CD25, CD38, (2)Ajou University Hospital (Suwon, KR) CD45, CD106, CD31. We found that after 10-12-th passages the expression of CD90 and CD166 on MSC became lower in Background: On performing umbilical cord blood (UCB) trans- comparison with MSC of 2-4 passages (p=0.049 and p=0.05 plantation, faster engraftment may lead better clinical outcome. accordingly). We have not seen the increasing of CD133+ cells Because transplanted viable cell count in UCB is related to the after 10-12-th passages of MSC. engraftment, we examined cryopreserved UCB cells with sev- After 10-12-th passages in despite of the decreasing of the num- eral methods after thawing. bers of CD90+ and ÑD31+ cells, the intensity of the expression Methods: Viability of cryopreserved cells were examined with of these antigens increases. trypan blue, DNA contents analysis, caspase-3 activation test, Conclusions: Our results showed, that linear homogeneity of intracellular esterase activity and Annexin-V/PI staining. MSCs on 3-4 passages is stabile then cells are cultured up to Results: A total of 60 samples were used in this study. After 10-12 passages. For cell therapy with MSC is necessary to use thawing, 89% of the total MNCs and 84% of CD34+ cells were well tested, homogeneity cells population with known immu- viable as identifi ed by trypan blue exclusion assay. In the nophenotype. Further research is required concerning different CD34+ cell population, the cell death rate was found to be 47% passaging techniques and culture conditions to better under- by Annexin-V/PI staining and less than 5% by DNA contents stand their effects on MSC’s immunology characteristics. analysis. Caspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10 - 20% at 2, 4, and 6 hours after thawing. P635 Conclusion: Viable cells in UCB should be measured by several Mesenchymal stem cell response to chemotherapeutic compensatory techniques rather than a single method. Discord- damage ance among Annexin-V/PI staining versus trypan blue exclu- J. May (1), J. Xu (1), R. Morse (1), N. Avent (1), C. Cox (2), sion, DNA contents analysis, and the caspase-3 activation test S. Wexler (2), C. Donaldson (1) or intracellular esterase activity should be clarifi ed in order to (1)Centre for Research in Biomedicine, University of the West apply these techniques for actual cord blood transplantation. of England (Bristol, UK); (2)Royal United Hospital (Bath, UK)

Mesenchymal Stem Cells/Stromal Cells (MSC) form the bone marrow microenvironment and are essential in supporting hae- mopoiesis. Following stem cell transplantation (SCT), whilst Early side effects/Late effects and haemopoietic cells are replaced, MSC remain of recipient ori- quality of life gin. However, MSC have previously been shown to be dam- aged by chemotherapeutic treatment, which is administered prior to SCT. Therefore, if damage is severe it may contribute P637 to lack of engraftment in some transplants, one of the leading Genetic polymorphism of cytochrome P450 1B1 (C432G) causes of mortality in this setting. To elucidate this damage, a is associated with an increased treatment-related physiologically relevant model is needed, particularly to allow mortality and lower overall survival in patients undergoing study of prodrugs where cytochrome P450 enzymes are neces- allogeneic haematopoietic stem cell transplantation sary for metabolism. M. Koldehoff (1), L. Kordelas (1), U. Graeven (2), D. Beelen (1), A co-culture model utilising HepG2 liver spheroids and MSC A.H. Elmaagacli (1) has been developed to enable study of chemotherapeutic dam- (1)University of Duisburg-Essen (Essen, DE); (2)Clinics Maria age, currently focussing on cyclophosphamide (CY) which Hilf (Mönchengladbach, DE) requires metabolism to the main cytotoxic agent phospho- ramide mustard. This co-culture model has been evaluated Introduction: The human cytochrome P450 1B1 (CYP 1B1) is a by comparing results observed in haematological malignancy key enzyme involved in the production of reactive metabolites patients who have received prior chemotherapeutic treatment and in the activation of environmental carcinogens. Several in vivo. Following chemotherapeutic treatment MSC morphol- polymorphisms were identifi ed in CYP 1B1 gene. The CYP 1B1 ogy is less uniform, proliferative capacity reduced (p<0.01) and codon 432 polymorphism leads to a three-fold higher 4-hydrox- an altered adhesion molecule expression (e.g. CD44) can be ylase activity for the variant CYP 1B1 isozymes than the wild demonstrated. types. Never before the infl uence of genetic polymorphisms of In preliminary experiments CY treatment was modelled in vitro CYP 1B1 on patients who underwent allogeneic hematopoietic using an S9 liver extract as a source of P450 enzymes. The initial stem cell transplantation (HSCT), was evaluated. results raised concerns over the toxicity of S9 itself, particularly Methods: Here we genotyped in a retrospective study 384 over extended periods in culture (e.g. 48 hours), demonstrating recipients (R) (and their donors (D)) for CYP 1B1 expression the need for a more physiologically relevant model. MSC were that underwent allogeneic HSCT for various diseases and ana- co-cultured with either S9 or HepG2 spheroids and exposed to lyzed their outcome. Genotyping of CYP 1B1 (C432G) was per- 0.5mM CY for 3hrs or 48hrs. Following 48hr exposure, expan- formed by real-time PCR. sion of MSC was greatly reduced in the presence of both S9 Results: 170 R (44.3%) were genotyped as homozygous wild- (p<0.01) and HepG2 spheroids (p=0.051). However, following type gene C/C, 157 R (40.9%) was genotyped as heterozygous 3hr treatment, subsequent expansion of MSC exposed to CY in genotype C/G and 57 R (14.8%) were genotype as homozygous the presence of S9 was no different to untreated MSC, whereas gene mutation G/G. From the 167 D (43.5%) were C/C, 164 D expansion of those exposed in the presence of liver spheroids (42.7%) were C/G, and 53 D (13.8%) had a homozygous gene

S163 mutation G/G. A homozygous CYP 1B1 gene mutation G/G was We conclude that the uptake of gamete and embryo storage found on 18 R/D side (4.7%). Five-year estimate for treatment- was low in this group of patients. Nonetheless when stored related mortality (TRM) and overall survival (OS) were different gametes or embryos were used to attempt pregnancy, the suc- in genotype C/G- and G/G- R with 33 + 4%, and 49 + 4% com- cess rate was high. pared to homozygous wild-type gene C/C- R (23 + 4%, and 59 + 4%, respectively, [p<0.03]), whereas the fi ve-year estimate for relapse rate (RR) was not different between the groups. No P639 differences for fi ve-year estimates for TRM, RR, or OS were Hepatic veno-occlusive disease in children after seen in R with either genotype C/C-, C/G- or G/G- D. No sta- haematopoietic stem cell transplantation: incidence, risk tistic differences were found in the incidence of acute GVHD factors, and outcome grade ≥ 2-4 or chronic GVHD on R- or on D- side with variant S.H. Lee (1), Y.J. Kwon (1), H.J. Park (1), B.K. Park (1), CYP 1B1 polymorphisms. Surprisingly, the fi ve-year estimate J.A. Park (2), H.J. Im (2), J.J. Seo (2), K.H. Yoo (3), K.W. Sung for TRM, RR, and OS were different in homozygous gene muta- (3), H.H. Koo (3), H.J. Kang (4), H.Y. Shin (4), H.S. Ahn (4), tion G/G on R/D site with 57 + 16%, 78 + 17%, and 16 + 10% K.S. Lee (5), T.J. Hwang (6) compared to all other CYP 1B1 genotypes with 28 + 3%, 32 + (1)National Cancer Center (Goyang, KR); (2)University of 3%, and 55 + 3%, respectively [TRM, p<0.01; RR, p<0.003 and Ulsan (Seoul, KR); (3)Sungkyunkwan University (Seoul, OS, p<0.001]). Multivariate analysis confi rmed that CYP 1B1 KR); (4)Seoul National University (Seoul, KR); (5)Kyungpook homozygous gene mutation G/G on R/D site had an increased National University (Daegu, KR); (6)Chonnam National risk for TRM and RR (p<0.02), whereas the mutation G/G on University (Hwasun, KR) R/D site revealed a worse OS (p<0.01). Conclusions: These results suggest that recipients with genetic Objectives: We conducted this study to describe recent char- polymorphism of CYP 1B1 do have an increased TRM, RR acteristics of incidence, risk factors, and outcome of veno- and lower OS after transplantation. Genotyping for CYP 1B1 occlusive disease (VOD) in children undergoing autologous or (C432G) might help to identify patients with higher risk for allogeneic hematopoietic stem cell transplantation (HSCT). HSCT. Methods: Four hundred sixty-seven HSCTs (217 autologous and 250 allogeneic) performed in 374 patients from January 2005 to December 2007 were evaluated. VOD was defi ned P638 according to McDonald criteria and classifi ed as severe on the Uptake and outcome of artifi cial reproductive techniques basis of persistent symptoms after day 100 or death before day following SCT for leukaemia: a single-centre experience 100 with ongoing VOD. N. Farah, H. Auner, E. Kanfer, A. Rahemtulla, D. Marin, Results: Among 467 transplants, VOD developed in 72 trans- J. Apperley, N. Salooja plants (15.4%) at median 10 days (1-64) after HSCT. VOD Hammersmith Hospital (London, UK) was graded as mild or moderate in 62 and severe in 10 trans- plants. For VOD prophylaxis, heparin was used in 116 trans- Infertility is common following SCT for leukaemia. Very little data plants, heparin + prostaglandin E1 (PGE1) in 230 transplants, is available on the use and success of artifi cial reproductive PGE1 ± ursodeoxycholic acid in 86 transplants, and defi brotide techniques in patients who have received chemoradiotherapy + heparin or PGE1 in 35 transplants. The median duration of as treatment for cancer. We have performed a retrospective VOD was 12 days (3-80). Hepatomegaly was the most com- survey to assess the uptake of gamete/embryo storage prior mon sign of VOD (87.5%). Ascites, respiratory dysfunction, to SCT, their use following SCT and the success rate of these and renal dysfunction were more common in severe VOD com- techniques. pared to mild or moderate VOD. Multivariate analysis showed 63 patients (28 female) responded to a questionnaire a median that total body irradiation (TBI) or busulfan containing regimen of 16 years (range 2-26) post transplant. All had received total (P=0.002), VOD prophylaxis without PGE1 (P=0.010), number body irradiation at a median dose of 13.2 Gy (range 10-14.4) of previous HSCT (P=0.012), and pre-transplant serum fer- together with cyclophosphamide 120mg/kg . The median age at ritin (P=0.014) were independent risk factors for developing transplant was 34 years (range 19-58). VOD. Mean serum ferritin level was higher in HSCT with VOD The possibility of infertility was discussed with 44/63 patients (2109.6 ± 2842.5) than in HSCT without VOD (1315.9 ± 1094.4) (28M,16 F) prior to SCT. Of 28 male patients with whom infertil- (P<0.001). Deaths within 100 days after HSCT occurred in 13 ity was discussed, all were offered sperm storage. 15 declined of 72 VOD-positive transplants, the cause of death being VOD- because they had completed their family (n=12) or because related multi-organ failure in 5 cases. The risk of death within there was insuffi cient time to organise sperm storage prior to 100 days after HSCT was 2.8 times higher (95% CI: 1.718, transplant (n=3). Of 13 patients who attempted to store sperm, 4.563) for VOD-positive transplants (P<0.001). 5 provided samples of insuffi cient quality for storage. Of 8 Conclusion: TBI or busulfan-based conditioning regimen, VOD- patients with stored sperm, one decided not to attempt parent- prophylaxis without PGE1, repeated HSCT, and pre-transplant hood after he relapsed from his original disease. The remaining ferritin increased signifi cantly the incidence of VOD in children 7 patients used their stored sperm to attempt pregnancy. Six out after HSCT. The encouraging result of our study is to justify the of seven patients were successful in fathering children; in three role of PGE1 in the prophylaxis of VOD, however prospective cases intracytoplasmic sperm injections were used. The median randomized trials are needed to confi rm the superior effi cacy number of attempts was 2 (range 1- 10) and the median duration of PGE1 in preventing VOD. Despite the combination of sup- of storage prior to successful use was 9 years (range 2-11y). portive cares and VOD therapy, signifi cant numbers of patients Of 16 women with whom infertility was discussed, embryo stor- are still suffering from VOD. Continued research for prevention age was arranged for one; this patient had received hydroxy- and effective treatment of VOD will be necessary to improve the carbamide prior to egg collection. A fi rst pregnancy using frozen outcome of HSCT. embryos miscarried at 9 weeks but a second pregnancy was succesful. A second patient had embryos stored prior to her diagnosis of CML and had already had a successful pregnancy using IVF. Following transplant, however, a single attempt at IVF failed. Reasons for failure to discuss infertility with these women or to offer embryo storage were not always clear but in the majority of patients were likely to have been related to an age greater than 40 at the time of transplant or else lack of availability of embryo storage in patients transplanted prior to 1990.

S164 P640 by non-compartmental and compartmental analysis using Win- Clinical signifi cance of autoantibody expression and Nonlin 5.0.1 software (Pharsight Corporation, Mountain View, autoimmune disease-like manifestations in allogeneic CA, USA). Bu PK are dependant on the rate of infusion, which stem cell transplantation recipients should remain constant during and between doses. We chose J.H. Moon, J.S. Gyung, S.J. Lee, J.G. Kim, Y.S. Chae, an infusion rate of 80mg/hr (160cc/hr) and rely on the fact that S.N. Kim, J.S. Suh, K.S. Lee, S.K. Sohn clearance of the test and treatment dose should be equal in Kyungpook National University Hospital (Daegu, KR) order to predict a therapeutic dose. Eleven patients required a dose reduction (the test dose predicted >6000uM*min) where We analyzed the association of the occurrence of autoantibod- an AUC of 5000uM*min was targeted to allow a safe margin of ies or autoimmune diseases with chronic graft-versus-host dis- error. Within those patients 82% of the adjustments were within ease (cGVHD) and survival in allogeneic stem cell recipients. 15% of the error margin for the targeted AUC. A mean reduc- From Nov. 2001 to Mar. 2008, 121 patients with hematological tion of 22% of the originally prescribed dose was observed. We diseases who survived at least 3 months after allogeneic stem found 91% of the 166 patients to be within 15% of the predicted cell transplantation (SCT) were enrolled in the current study. therapeutic dose as determined from the test dose analysis. Forty-seven patients (38.8%) expressed at least one of various Furthermore, 83% of the 166 patients fell within a safe AUC autoantibodies after transplantation. ANA was positive in 22 window of 3000 to 5000uM*min. Since our move to therapeutic patients (18.2%), anti-dsDNA in 7 patients (5.8%), anti-smooth drug monitoring by test-dose PK analysis, none of our patients muscle (Sm) antibody in 6 patients (5%), rheumatoid factor (RF) have been exposed to AUC > 6000uM*min. These data confi rm in 17 patients (14.0%), and a positive Coombs test recorded for the value of an individual dose monitoring regimen by test-dose 12 patients (9.9%). Autoimmune disease-like manifestations PK analysis. were observed as follows; 2 membranous glomerulonephritis, 8 AIHAs and 1 ITP, 2 sclerodermas including 1 with esophageal involvement, 2 lichen planus-like skin features, and 2 vitiligos. P642 The positive rate of autoantibodies was higher in the patients The incidence and morbi-mortality of veno-occlusive with cGVHD (47.8% vs. 27.8%; p=0.025). The expression of syndrome after allogeneic HSCT have decreased in the autoantibodies had a statistical correlation to the specifi c organ last decade. A single-centre experience involvement of cGVHD; ANA to sicca syndrome (p=0.010), M. Diaz-Beya, L. Rosiñol, M. Rovira, C. Martínez, anti-Sm antibody to hepatic GVHD (p=0.003), and RF to sicca F. Fernández-Avilés, E. Carreras syndrome (p=0.039) and musculoskeletal GVHD (p=0.026). Hospital Clínic (Barcelona, ES) The patients expressing autoantibody had a better survival than those without autoantibody expression. The 5-year overall Introduction: Since the classical series of Seattle, Baltimore, survival (OS) was 70.2% and 47.9% in autoantibody positive Huddinge, IBMTR and EBMT establishing the frequency and and negative patients, respectively (p=0.002). Especially, the risk factors for VOD in allogeneic HSCT many changes have patients expressing autoantibody without cGVHD had a bet- been introduced in this therapeutic procedure. At that time, ter survival (OS = 100%) than other groups. The 5-year OS peripheral blood stem cells, reduced-intensity conditioning rate were 63.1% in both autoantibody and cGVHD positive (RIC), unrelated donors, cord blood, mycophenolate mofetil group, 59.6% in autoantibody negative and cGVHD positive and defi brotide were not routinely used. For that reason we group, and 36.6% in both autoantibody and cGVHD negative analyzed the evolution of VOD in the last 24 years in a single group. Limited cGVHD (p=0.025, HR=0.353) and autoantibody centre. expression (p=0.007, HR=0.371) were identifi ed as a good Patients and Methods: Between 01/1987 and 07/2008, 845 prognostic factors affecting survival. In conclusion, the occur- allo-HSCT were performed. Primary diseases were AML or rence of autoantibodies after allogeneic SCT was found to be ALL (n=363), CML (n=238), MDS (n=55), or NHL/HL/MM (n= related to cGVHD, and patients expressing autoantibodies had 149). Their median age was 36 years (range, 4-67). Alterna- a better survival. tive donors were used in 197 cases (23%). 121 patients had received a previous auto- (n= 69) or allo-HSCT (n=52). RIC was used in 135 cases (16%). The Seattle and Baltimore clini- P641 cal criteria were used for VOD diagnosis. Severity of VOD was Safe dosing of once-daily intravenous busulphan by classifi ed according with the retrospective Seattle criteria and pharmacokinetic test-dose analysis in adult allogeneic by the presence of multi-organ failure (MOF). Since 2000 all haematopoietic stem cell transplant patients VOD cases were treated with defi brotide. S.B. Kangarloo (1), F. Naveed (1), N.J. Bahlis (2), C. Brown (2), Results: Applying the Seattle and Baltimore criteria 117 (14%) M.A. Chaudhry (2), A. Daly (2), P. Duggan (2), M. Geddes (2), and 73 (9%) patients developed VOD, respectively. VOD was M.L. Savoie (2), D. Stewart (2), J. Storek (2), D. Quinlan (1), mild, moderate or severe in 37 (32%), 53 (45%) and 25 (21%) M. Yang (1), N. Zacarias (2), J. Russell (2) cases. Twenty-six (22%) patients developed MOF and 20 died (1)Alberta Blood Cell & BMT Program (Calgary, CA); (2)Alberta of VOD (67% of severe VOD, 17% of VOD, and 2% of the whole Blood Cell & BMT Program, and Division of Hematology and series). Twenty-seven additional patients with VOD (23%) died Hematologic Malignancy (Calgary, CA) before day +100 due to other causes. To analyze the incidence of VOD along the years we only considered cases fulfi lling the Busulphan (Bu) is a bifunctional alkylating agent used in con- more strict Baltimore criteria. Considering the whole series the ditioning regimens for hematopoietic stem cell transplants. As incidence of VOD in last decade seemed to be lower. Of note, previously seen from oral Bu data early dose adjustments are when excluding RIC HSCT it was very similar in both periods, key in preventing toxicity due to overdosing. We report on 166 except for HSCT from alternative donors that showed a nota- patients with hematological malignancies who were given a ble reduction in VOD incidence. However, multivariate analysis once-daily intravenous (i.v.) Bu (3.2mg/kg days -5 to -2) and disclosed that in addition to well known risk factors (previous fl udarabine (Flu, 50mg/m2 days -6 to -2) regimen. Using this liver disease, low Karnofsky index, myeloablative conditioning, regimen we have shown that non-relapse mortality is increased alternative donors) time of HSCT had a clear impact on VOD, by exposure to an area under the concentration-time curve with a signifi cant lower risk in the last decade. (AUC) for Bu of >6000uM*min. By giving a small test dose Conclusions: The incidence of VOD has decreased along the (0.8mg/kg day -7) during the myeloablative prophylaxis, we years probably as a consequence of a better management of were able to adjust patient doses by day -5 (fi rst Bu dose). Bu patients. However, the most remarkable fi nding in our historical levels were measured in plasma using high performance liquid analysis has been the notable reduction in the cases of VOD chromatography (HPLC) and quantifi ed by the AUC in uM*min. that evolve to MOF or die, probably due to the better manage- The pharmacokinetic (PK) data were modeled and analyzed ment of VOD cases and the use of defi brotide.

S165 ity in international banks. Long-term survival is important in this group of pts because they could have a long life expectancy if they were not affected by the disease. Objectives: Analyze the long-term survival and causes of late mortality in children and adolescents submitted to an allogeneic HSCT in a single institution. Materials and Methods: The information about survival and causes of death of 507 pts aged below 18 years was retrieved P643 from our database. 201 (39%) pts survived more than 5 years Cognitive development, self physical function and and were included in this analysis. Gender: M/F: 126/75. satisfaction profi le in long-term survivors of paediatric Median age at HSCT: 9ys (range: 1-17ys). Median follow-up HSCT after HSCT: 9ys (range: 5- 22ys). Most pts received bone mar- M. Faraci, M. Miano, M.G. Calevo, G. Hanau, F. Fioredda, row (93%) from their related donors (90%). 54 pts had malig- G. Dini, E. Lanino, M. Canepa nant disease while 147 had non-malignant diseases (51 genetic G. Gaslini Institute (Genoa, IT) diseases). The 5 year-mortality in this cohort of pts was com- pared to the mortality observed in normal population (based on Introduction: the evaluation of health-related quality of life rep- data published by IBGE in 1991). We used the Kaplan-Meyer resents an important instrument of outcome in transplanted estimator and standardized mortality ratio (SMR) for statistical patients especially in those who received HSCT during pae- analysis. diatric age. The aim of this study is to evaluate the subjective Results: Survival of children undergoing HSCT was worse cognitive development, self physical function and satisfaction than the general population and remained in that way even for profi le in a group of adolescents (age >18 yrs) transplanted dur- children surviving 5ys or more after HSCT. The SMR (95% CI) ing childhood. of the children who survived at least 5 years post-HSCT was Material and Methods: 32 survivors (18 males, median age 5.437 (1.977-8.896). 25 yrs, range 18-33) at a median of 15 yrs (range 10-20) from The death of rejection or relapse (table) occurred in 3pts (all HSCT performed at a median age of 9 yrs (range 0-16 yrs) before the 10 years post-HSCT). All deaths after 10 years of responded to questionnaires on Cognitive Behaviour Assess- HSCT occurred in children with genetic diseases (Cirrhosis ment (CBA, evaluating global personality), Self Physical Func- secondary to hepatitis C in one pt with Fanconi´s Anemia and tion (SF-36), and Satisfaction Profi le(SAT-P). The source of progressive pulmonary fi brosis in one pt with Dyskeratosis HSCT was autologous in 19 pts. The conditioning regimen Congenita). Two pts with Fanconi’s Anemia developed second- included TBI or TAI in 21 pts. Late effects included sub-capsular ary tumors and died of progressive disease. opacity (n=13), hypogonadism (n=23), hypothyroidism (n=7), Conclusion: Compared to the normal population, children thyroidal nodules (n=13), secondary solid tumours (n=10), undergoing an allogeneic HSCT had an increased chance of skeletal problems (n=13), restrictive respiratory insuffi ciency dying from any cause even after 5ys of transplantation. In our (mild and severe in 16 and 1, respectively), C hepatitis (n=5), country, these pts go back to their home town and may not have cavernomatous angiodysplasia (n=7), and metabolic syndrome adequate access to health care. Brazil is a huge country and (n=6). Karnosky scores ranged from 70 to 100 (median 90). despite our limited resources we are developing a national pro- Results: All subjects resulted within the normal range accord- gram to educate our pts and primary care physicians about the ing to the questionnaires control values. Females resulted more importance of maintaining a lifelong surveillance after HSCT. sensible to pain (P=0.004), and more impaired in their social activities (P=0.04), emotional status (P=0.04) and instability (P=0.007) compared with males. Patients transplanted at age > 5 yrs resulted more compro- mised in their work activity (P=0.02). As for conditioning regi- men, patients irradiated with TBI o TAI had a major impairment of physical activity (P=0.02). The relationship between subjective perception of quality of life and late effects demonstrated a correlation between hypogo- P645 nadism and social activity (P=0.03), emotional status(P=0.01) Late-onset neutropenia in 53 patients treated with and mental health (P=0.01). Patients with skeletal problems radioimmunotherapy and autologous stem cell seem to have a higher perception of physical pain (P=0.007) transplantation for non-Hodgkin’s lymphoma and vitality (P=0.01). V. Pavone (1), A. Rana (1), A. Mele (1), C. Del Casale (1), Conclusion: in our experience the subjective perception of sat- A. Messa (1), G. Greco (1), S. Sibilla (1), R. De Francesco isfaction profi le and quality of life of a restrict group of adults (1), V. Frusciante (2), A. Varraso (2), B. Botto (3), U. Vitolo (3), receiving HSCT in childhood is within the normal range. Female G. Milone (4), S. Leotta (4), P. Iacopino (5), G. Console (5), gender, and hypogonadism represent the variables that affected A. Olivieri (6), M. Cimminiello (6), V. Mettivier (7), L. Pezzullo more on subjective perception of quality of life. (7), D. Baronciani (8), E. Angelucci (8), M. Musso (9), N. Cascavilla (2), A. Guarini (10), S. Capalbo (11), G. Loseto (12), G. Quarta (12), F. Gaudio (13), G. Specchia (13), V. Liso P644 (13), A. Ostuni (1) Late mortality in children and adolescents undergoing (1)Hospital Cardinale G. Panico (Tricase, IT); (2)Hospital haematopoietic stem cell transplantation: impact on life Casa Sollievo della Sofferenza (San Giovanni Rotondo, IT); expectancy of 507 patients (3)Hospital San Giovanni Battista (Turin, IT); (4)Hospital S. Fortier, J. Morando, F. Piazera, J. Ruiz, L. Medeiros, Ferrarotto (Catania, IT); (5)Department of Haematology (Reggio M. Oliveira, E. Nunes, D. Setúbal, V. Funke, M. Bittencourt, Calabria, IT); (6)Hospital San Carlo (Potenza, IT); (7)Hospital R. Pasquini, C. Medeiros, J. Zanis Neto, C. Bonfi m on behalf Cardarelli (Naples, IT); (8)Hospital A Businco (Cagliari, IT); of the Bone Marrow Transplantation Service of the Clinical (9)La Maddalena (Palermo, IT); (10)Istituto Oncologico (Bari, Hospital - Federal University of Paraná IT); (11)Hospital Riuniti (Foggia, IT); (12)Hospital Perrino (Brindisi, IT); (13)Policlinico (Bari, IT) Introduction: Over the past decade, the number of transplants in children increased considerably. In our BMT unit this occurred Background: Late onset neutropenia (LON) following chemo- mainly because of the inclusion of pts with genetic diseases immunotherapy have been reported in 10-50% of patients (pts). and the possibility of fi nding donors with appropriate compatibil- It appears from 1 to 6 months after the end of chemotherapy

S166 (CHT) and it is severe (<0.5-1 x109/L) but spontaneously P646 reversible. Recent studies suggests that LON is due to pertur- EBV-driven post-transplant lymphoproliferative disorder bations of Stromal-derived Factor 1 (SDF-1) and coincides with following allogeneic stem cell transplant: A review of one the time of maximum B-cell depletion in periferal blood (PB) centre’s experience according with the hypothesis that LON is due to a promotion J. Laird (1), A.N. Parker (1), G. MacQuaker (1), A. Clark (1), of B-cell lymphopoiesis over granulopoiesis in the bone marrow C. Aitken (2) (1-2). (1)Beatson West of Scotland Cancer Centre (Glasgow, UK); Aims: Neutropenia and thrombocytopenia are the most com- (2)Depy. Virology (Glasgow, UK) mon hematological toxicities in NHL pts treated with RIT (Zevalin®). The median days to nadir of the neutropenia and EBV-driven post-transplant lymphoproliferative disorder (PTLD) thrombocytopenia is around day +60 after Zevalin® treatment is a well recognised complication of allogeneic stem cell trans- without stem cell rescue, but poor information exists about late plantation. Risk of developing early PTLD is associated with hematological toxicity in pts treated with RIT plus high dose reactivation of EBV which can be detected by rising EBV copy CHT and ASCT (Z-BEAM). METHODS: We evaluated LON in numbers. Patients in our unit are screened weekly using plasma 53 high risk NHL pts, all pretreated with Z-BEAM and ASCT in TaqMan realtime PCR, increasing to twice weekly if a positive 14 italian centers from February 06 to June 2008. In 13 pts the result is found. Those with suggestive clinical features, rising immunological recovery was evaluated monthly. copy number by >1 log in a week or an absolute value >5 log Results: All pts engrafted. Median CD 34+ cells infused was 5.5 have a CT scan and biopsy if possible. Patients are given pre- x106/Kg (range 2.55-34). Median time to ANC &≥ 0,500 x109/L emptive rituximab 375mg/m² if EBV copy number reaches >log was 10 days (range 8-20), median time to platelets (plt) ≥ 20 6 or clinical features with rising copy number are found. Patients x109/L was 14 days (range 9-60). Median follow up was 247 with new lymphadenopathy and rising EBV copy number are days (range 125-818). 39/53 pts with > 6 months follow up were treated with rituximab 375mg/m². evaluated for LON (fi g.1). 6/39 (15,3%) pts experienced grade A retrospective case note review of all allografts in our unit was 3-4 neutropenia. Median time of onset was day + 87 (range carried out between 1/6/04 and 31/7/08 to identify patients who 30-147). Median duration of neutropenia was 29 days (range had been treated with rituximab pre-emptively or for suspected 3-87). 3 CMV reactivation, 3 urine infection, 2 blood colture + PTLD. There were 165 fi rst allografts during this period; 64 for bacteria and 1 pneumonia was demonstrated during neu- sibling, 100 VUD, 1 Cord. A total of 11 patients (6%) received tropenia (tab.1). In 6 pts with LON, a median CD 19 counts of rituximab, 5 for biopsy-proven PTLD, 6 were treated pre-emp- 15 cells/mmc (range 0-40) was documented. In the control his- tively for rising EBV titres and clinical features only. Treatment tory group treated with BEAM +ASCT in the same period a CD was initiated at a median EBV copy number of log 5.2 copies 19 count of 100 cells/mmc (range 10-300) was documented, in (range 3.2 to 6.91). The number of treatments ranged from 1 to agreement whit the hypothesis that LON coincides with the time 5, with a median of 2. Four of the eleven patients (36%) treated of maximum B-cell depletion in PB. for PTLD have died, two (18%) from PTLD and two from under- Conclusions: In our Institutions 53 pts were treated with Z- lying disease relapse. The remaining 7 patients (64%) remain BEAM + ASCT for NHL from February 06 to June 08. After alive and well. engraftment 6/39 (15,3%) pts experienced grade 3-4 neutrope- On analysing possible risk factors we have identifi ed that VUD nia. The time of emergence of neutropenia seems to be longer recipients 9/100 (9%) had a higher incidence than sibling trans- (g+87) than in pts treated with Zevalin alone and coincides with plants 2/64 (3%). In our unit in vivo T depletion with Alemtuzu- the time of maximum B-cell depletion in PB as demonstrated by mab 100mg is used in all reduced intensity conditioning (RIC) PB immunophenotipe. regimens, and in myeloablative (MA) regimens for VUD recipi- ents and sibling HLA mismatches. No sibling conditioned with a MA regimen (29/64) developed PTLD. Recipients of a RIC fl udarabine based regimen had an incidence of 11% (10/89) compared to 2% (1/44) for an Alemtuzumab MA regimen for VUD recipients. The dose of alemtuzumab was identical in both RIC and MA regimens suggesting that the addition of fl udarabine increases the chance of EBV reactivation and development of PTLD. However, the use of PCR monitoring and preemptive therapy with rituximab has been a successful strategy in our patients, even when biopsy positive.

P647 Major ABO-incompatibility, reduced-intensity conditioning regimens, and patient age are associated with prolonged red cell transfusion dependency after allogeneic haematopoietic stem cell transplantation D. Dahl, C. Koenecke, H. Heuft, E. Dammann, M. Stadler, S. Buchholz, J. Krauter, M. Eder, K. Sykora, C. Klein, A. Hahn, A. Ganser, M. Sauer Mediacl School Hannover (Hannover, DE)

Background: This study aimed to identify factors infl uencing the duration of isolated red cell transfusion dependency after allo- geneic stem cell transplantation (SCT). Methods: We retrospectively analyzed a cohort of 487 patients transplanted at Hannover Medical University between 2000 and 2006. Patients were identifi ed by their requirements of red cell transfusions beyond day 60 after SCT. Median follow-up was 51 months (range 18 -96 months). Median patient age was 37 years (range 0 – 70 years). Stem cell source were peripheral blood stem cells (n = 344, 71%), bone marrow (n = 138, 28%),

S167 and cord blood (n = 5, 1%). 57% (n = 278) had an unrelated ited from returning to society because of neuropsychological donor including 59 human leukocyte antigen (HLA)-mismatched disorders including anterograde amnesia and seizure. transplantations. 43% (n = 209) had family donors out of which Conclusions: Although HHV-6 encephalitis after SCT is becom- 17 were mismatched. Factors analyzed were age, underlying ing a curable complication, the aftereffect of encephalitis such disease (malignant versus non malignant), transfusions pre as neuropsychological disorders has infl uence on quality-of-life SCT, time between diagnosis and SCT, conditioning regimen in long-term survivors. (full versus reduced intensity), donor type, graft source, T cell depletion, mixed chimerism, and graft versus host disease. Results: 47 patients (9.6%) showed a prolonged isolated red P649 cell transfusion dependency. Multivariate analysis identifi ed Donor-derived alveolar macrophages are the effecter cells ABO-incompatibility between donor and recipient as major risk in cryptogenic organizing pneumonia after SCT factor (incidence of 16.4% for major mismatch, 2.9% for minor M. Ito, M. Fujino, D. Kajiura, A. Kominami, T. Yokoyama, mismatch, and 9.1% for AB0-compatible transplantations (p = F. Nomura, K. Miyamura 0.002)). Independently of ABO-incompatibility reduced intensity Japanese Red Cross Nagoya 1st. Hospital (Nagoya, JP) conditioning regimens (RIC) were associated with prolonged red cell transfusion dependency (15.2% versus 7%; p = 0.005). Aim: Non-infectious pulmonary complication after SCT is a Although inseparably linked to RIC, patient age was associated signifi cant morbidity and mortality, and its diagnosis is some- with prolonged red cell transfusion dependency in univariate times diffi cult. We investigated cryptogenic organizing pneumo- analysis (18.3% > 55 years, 10.2% from 13 to 54 years, and nia (COP, synonym of bronchiolitis obliterans with organizing 2.0% < 12 years (p = 0.001)). The remaining parameters were pneumonia (BOOP)) which is one of the subacute typical SCT not associated with prolonged red cell transfusion dependency pulmonary complications, clinicopathologically by using biopsy in our series. Interestingly, overall survival was not affected. material. The characteristic histopathologic lesions of COP are Conclusions: Major AB0-incompatibility is a major factor for iso- excessive proliferations of granulation tissue within small air- lated prolonged red cell dependency after SCT. The increasing ways and alveolar ducts associated with chronic infl ammation use of RIC in younger patients will show whether RIC and older by large amount of macrophages infi ltration. The aim of this age can be identifi ed as independent risk factors as patient study was to clarity the origin of macrophages which are the numbers grow. key infl ammatory cells in COP lesions. Cases and methods: We have selected the pulmonary biopsy cases from our SCT data fi les of Department of Pathology, from P648 1995 to 2008. Fifteen cases of COP by histologically confi rmed Long-term outcome of patients with human herpesvirus-6 from among 41 biopsy (transbronchial biopsy (TBB) 27 cases encephalitis after allogeneic stem cell transplantation and surgical lung biopsy 13 cases) were used in this study. Par- R. Sakai (1), H. Kanamori (2), K. Motohashi (2), W. Yamamoto affi n sections were examined histological, immunohistological (1), S. Matsuura (1), A. Fujita (1), R. Oshima (1), H. Kuwabara and tissue FISH examination using X, Y gene probes. (1), A. Maruta (2), Y. Ishigatsubo (3), S. Fujisawa (1) Results: COP cases were 4-62 y.o (median 33.0), 11 cases of (1)Yokohama City University Medical Center (Yokohama, JP); male and 4 of female. Primary diseases were 14 of hematologi- (2)Kanagawa Cancer Center (Yokohama, JP); (3)Yokohama cal malignancy and one of aplastic anemia. Stem cells sources City University Graduate School of Medicine (Yokohama, JP) were 5 of cord blood and 10 of bone marrow (2 of related and 8 of unrelated donor). Onset of these cases was 35-450 days Background: Human herpesvirus-6 (HHV-6) encephalitis has (median 192.0 days) after SCT. All of them were cured by ster- been recognized as a relatively rare but sometimes lethal com- oid therapy after histological diagnosis. Histopathologically, plication following allogeneic stem cell transplantation (SCT). COP with some different disease phases were all characterized Although the development of diagnostics and antiviral therapies by CD 163+ alveolar macrophages and small number of CD3+, improved prognosis, the consequence of encephalitis is still 8+ T lymphocytes infi ltrations. Seven cases of COP were sex- unclear. The purpose of this study is to clarify neuropsychologi- mismatched SCT and 4 of them could work tissue FISH exami- cal disorders in survivors with HHV-6 encephalitis. nation. All of 4 cases could be demonstrated donor type sex Patients and Methods: Patients receiving allogeneic SCT gene signals in infi ltrated macrophages. between January 2004 and March 2008 were retrospectively Conclusions: We could demonstrate that the donor derived analyzed. The patients who developed unexplained neuropsy- macrophages were dominant infl ammatory cells in COP after chological disorders with a positive reaction of quantitative SCT cases. COP after SCT is the distinctive disorder in early polymerase chain reaction (PCR) for HHV-6 in the cerebrospi- to late onset complication with favorable prognosis. Increased nal fl uid (CSF) were diagnosed with HHV-6 encephalitis. number of alveolar macrophages donor origin was the clue of Results: Among165 recipients, eight (4.8%) were diagnosed as pathological diagnosis of COP after SCT. HHV-6 encephalitis. Five were male and three were female with a median age of 40.5 years (range, 22-56). Underlying diseases included 3 AML and 5 ALL, and 6 were in a state of remission P650 at SCT. All of them received stem cells from unrelated donors The signifi cance of the specifi c comorbitity index (HCT-CI) (4 cord blood and 4 bone marrow). Myeloablative conditioning in predicting non-relapse mortality following allogeneic regime was carried out on all but one patient. Graft-versus-host stem cell transplantation with myeloablative regimens disease (GVHD) prophylaxis was attempted with tacrolimus in P. Kaloyannidis, D. Mallouri, C. Apostolou, I. Sakellari, all patients. The median onset of HHV-6 encephalitis was day E. Yannaki, C. Smias, I. Batsis, S. Tsimperis, R. Saloum, +18 (range, day +14 to +27) after SCT. The median number A. Anagnostopoulos, A. Fassas of HHV-6 DNA copies in CSF at the onset of encephalitis was G. Papanicolaou (Thessaloniki, GR) 84,400 copies/ml (range, 590 to 930,000). Four patients were treated with ganciclovir and the others were treated with foscar- Non-relapse mortality (NRM) following allogeneic stem cell net. The median period of the antiviral therapy was 41days transplantation (alloTx) remains a signifi cant factor of treatment (range, 16 to 77). The examination of PCR for HHV-6 in the failure. Recently, the HCT-specifi c comorbidity index (HCT-CI) CSF was negative in all patients by the end of the antiviral ther- has been reported to be important predictive marker for NMR. apy. The median survival from the onset of encephalitis was To evaluate the impact of HCT-CI in terms of NRM and overall 360 days (range, 82 to 1636), and three patients died of irrel- survival (OS), we retrospectively analyzed 242 patients (pts) evant causes of encephalitis (sepsis on day 89, pneumonia on aged 4-59 years (median 30), allografted for haematological day 139 and gastro-intestinal tract bleeding with chronic GVHD disease with myeloablative regimens during a 10-year period on day 327 after SCT). Four patients in survivors were prohib- (1998-2007). One hundred sixty seven pts were transplanted

S168 for acute leukaemia (AML: 92, ALL: 75), 26 for myeloprolifera- Three/6 pts received treatment for chronic GVHD, unmodifi ed tive disease, 21 for aggressive NHL, 12 for aplastic anemia, 8 for LONIPC. for MDS and 7 for multiple myeloma. At the time of transplant, Conclusions: pulmonary non-infectious complications appear 132 pts were in complete remission (CR) (70 in CR1) and 110 early after transplant, even if with mild clinical or spirometric had relapsed or primary refractory disease. Thirty-six received signs; the cooperation with pneumologists and breathing physi- marrow while 206 peripheral blood grafts harvested from 179 otherapists allows the early recognition of pulmonary complica- siblings, 52 matched unrelated and 11 relative (2-3 antigen-mis- tions and specifi c treatments with a good response, avoiding matched) donors. Calcineurin inhibitors plus methotrexate were clinical evolution with a better quality of life and reducing the administered as GvHD prophylaxis. With regard to HCT-CI, 147 employment of immunosuppressive anti-GVHD therapy. pts had score 0, 66 had score 1-2 and 29 had score ? 3. HCT-CI scores were not correlated with patients’ age (÷2 test: ns). The NRM and the long-term OS were 19% and 60%, respectively, P652 for pts transplanted in CR, compared to 33% and 25% for pts Platelet transfusion refractoriness in paediatric patients transplanted in advanced disease (p< 0.02). Patients aged <30 following allogeneic haematopoietic stem cell and 30-45 years had a similar NRM of 20%, while pts >45 years transplantation had a NRM probability of 53% (p=0,002). The NRM rates were T. Ileri, M. Ertem, E. Unal Ince, A. Sayili, Z. Uysal, N. Solaz 20%, 30% and 35% (p=0.1) while long term OS rates were Ankara University Pediatric BMT Unit (Ankara, TR) 45%, 37%, and 36% (p=0.09) for pts with HCT-CI score 0, 1- 2 and ? 3 respectively. To evaluate the possible factors which Prophylactic platelet transfusions reduce the thrombocytopenia independently affected the NRM and OS, we incorporated in a related complications in children following allogeneic hematopoi- Cox-regression model patient’s age, sex, body mass index and etic stem cell transplantation (allo-HSCT). Insuffi cient response HCT-CI score, disease status and donor’s histocompatibility. In to platelet transfusion is common in this period and diagnosis our study age <45 years, CR at the time of transplant, and allo- and management of this complication is very challenging. A graft from a sibling donor were identifi ed as signifi cant factors prospective study was planned to evaluate the frequency and for lower NRM and higher OS rates while a HCT-CI score ?3 the causes of platelet refractoriness following allo-HSCT since exhibited a trend to be signifi cant (p=0.06) indicating that HCT- very limited reports have specifi cally addressed this issue in CI score should possibly be taken into consideration before the children. The impact of the platelet refractoriness to the bleed- decision of a treatment approach. ing complications, diagnosis and practical options for manage- ment were evaluated. Eighty-six pediatric patients who received allo-HSCT for hematological disorders were evaluated. Platelet P651 refractoriness was defi ned as 20-hr CCI<2500 and developed Late-onset non-infectious pulmonary complications after in 44 patients (50%). In case of platelet refractoriness, immune allogeneic stem cell transplantation: results of a treatment and nonimmune factors were evaluated. Alloimmunization was strategy with multi-disciplinary cooperation defi ned as 1-hr CCI<5000 and developed in 30 of 44 children M. Brunori (1), G. Biscione (2), I. Mininni (1), F. Oriolo (1), (68%) with platelet refractoriness. The possible nonimmun G.F. Torelli (1), W. Barberi (1), B. Lucarelli (1), V. Valle (1), causes were sinusoidal obstruction syndrome in 3, infection in E. Iannella (1), F. Natalino (1), F. Pasqua (2), A. Perrone (1), 4, splenomegaly in 2, drugs in 1, graft vs host disease in 1 and R. Foà (1), A.P. Iori (1) HSCT in 3 patients with platelet refractoriness. Hemoglobinop- (1)Sapienza University (Rome, IT); (2)S.Raffaele Institute athy was the diagnosis in 77% of the cases with platelet refrac- (Rome, IT) toriness and 23% of patients had other diagnosis requiring transfusions prior to HSCT (P<0.01).Transfusion of whole blood Despite the risk of mortality after allogeneic stem cell trans- components without leukoreduction before HSCT was found as plantation (HSCT), nowadays a large number of patients(pts) the most important factor for the development of platelet alloim- are long-term survivors; Late-Onset Non-Infectious Pulmonary munization. Platelets from HLA-matched HSCT donors (n:11) Complications (LONIPC) arise, therefore, increasing interest. and haploidentical parents (n:9) were transfused to alloimmu- With the aim of a better management of LONIPC, from Septem- nized patients and 100% and 55% of them improved platelet ber 2006 we started a cooperation with the Respiratory-Patho- responses, respectively. Transplant related mortality rate was physiology Department of our University and with the Respiratory 9% and none of the patients died because of bleeding. There Rehabilitation Division of S. Raffaele Institute. We analyzed 25 was no signifi cant difference in the frequency of hemorrhage consecutive adult outpatients in follow-up visit at a median time between the patients with and without platelet refractoriness of 35 months post-transplant with a normal lung function before (26% vs 23%). Patients with platelet refractoriness required HSCT; 14/25 pts had a ventilatory defect that was obstructive more frequent platelet and red blood cell support (P<0.01). In (OD) in 8, restrictive (RD) in 5 and mixed (MD) in 1. They were conclusion, platelet refractoriness associated with alloimmuni- treated by inhaled formoterol, budesonide and tiotropium bro- zation is quite high in children undergoing allo-HSCT. Platelet mide for mild/moderate OD (4 pts); as above + oral prednisone alloimmunization is more frequent among the multitransfused (PDN) for severe OD (4 pts); oral PDN + acetylcystein for RD hemoglobinopathy patients. Transfusion from the HSCT donor (5pts); oral PDN + inhaled bronchodilators for MD. All patients and/or other relatives could be an option for these patients were trained to the use of the inhalers, 8 received respiratory especially in developing countries where the selection of HLA- devices for home pulmonary rehabilitation care. Seven were matched platelet donors may not be possible. in treatment for chronic GVHD that remained unmodifi ed. The 2-year cumulative incidence of response to therapy was 76%; 3/14 pts died of LONIPC, with a 15 year overall survival of 35%. P653 The only variable associated with a worse response to therapy High burden of late effects after haematopoietic stem cell was the use of TBI pre-transplant. With the aim of early recogni- transplantation in childhood. A single-centre study tion and treatment of respiratory complications, from Jan. 2007 D. Bresters, I.C.M. van Gils, W.J.W. Kollen, L.M. Ball, to Aug. 2008 we analyzed prospectively 11 consecutive pts. W. Oostdijk, J.G. van der Bom, R. Egeler Lung function tests were performed pre-transplant and after 3, LUMC (Leiden, NL) 6 and 12 months. At a median of 5 months, 6 pts (54%) showed LONIPC. All patients received early therapy according to the Survival after hematopoietic stem cell transplantation (HSCT) protocols reported above and pulmonary physical treatments; in childhood has improved, but many survivors develop late 4/6 showed a clinical and spirometric response, one of these effects after treatment. The burden of late effects has been underwent a pulmonary rehabilitation program with legs and described in childhood cancer survivors, but only few studies arms exercises, chest physiotherapy and mucous evacuation. focused on HSCT survivors.

S169 Objectives of our study are to establish the cumulative inci- > grade 2 and chronic GVHD extensive disease appeared in dence and severity of late effects in childhood HSCT survivors 48% and 67% respectively. Thus, these pts must be treated in a single centre as well as risk factors for severe late effects. with prolonged immunosupressive therapy. 2/21 (9,5%) died Patients and methods: Survivors of HSCT in childhood who of secondary malignancies (carcinoma of small intestine and were alive at least 2 years after HSCT and were seen by a colon) and one pts of accidential infection. Pediatric hemato-oncologist in our Pediatric Late Effects out- Conclusion: Long term surviver of allogeneic stem cell trans- patient clinic, according to national follow-up guidelines for late plantation are at increased risk of a secondary malignancies. effects, were eligible for this study. Cumulative incidence and Most of the pts received TBI-based conditioning and suffered severity of late effects was assessed according to the Com- from chronic GvHD extensive disease with consecutive immu- mon Terminology Criteria for Adverse Events (CTCAE) v3.0 nosupressive therapy. Life-long follow-up will be needed to and graded from 0 to 5. Burden of late effects (LE) was defi ned detect secondary malignancies in early stage of disease which as: mild: LE grade 1 only and < 3 LE, moderate: LE grade 2 might offer curative therapeutical options. Therefore, in our and/or 4-6 LE, severe: LE grade 3 and/or > 7 LE and disabling/ institution all pts undergo annual skin screening program and life-threatening: any LE grade 4. Risk factors for severe or disa- are sensitized for possibility of secondary malignancies. bling burden of LE were assessed by univariate and multivari- ate logistic regression analysis. Results: 162 survivors of HSCT were included in this study and P655 had a median age at HSCT of 6.3 years, 63% were male, and Prognostic factors and outcomes of patients admitted to the majority underwent HSCT for a hematological malignancy the ICU after haematopoietic stem cell transplant (55%). Half of the patients were treated with a conditioning regi- C. Martínez, A. Carrillo, E. López, E. Pérez, J. Sánchez-Blanco, men including either total body or thoraco-abdominal irradiation J. Nieto, C. Castilla, I. Heras, V. Vicente (TBI/TAI). Fifteen children (9.3%) had chronic graft versus host Hospital Morales Meseguer (Murcia, ES) disease (cGVHD). Cumulative incidence of late effects was 93.2% (151/162) with Hematopoietic stem cell transplant (HSCT) is the only curative a median follow-up time of 7.2 years after HSCT. The burden approach for many patients with haematological malignancies of late effects was mild, moderate, severe and disabling in 45 (HM). Despite this, successful treatment of malignant disease by (28%), 66 (41%), 38 (24%) and 2 (1%), respectively. HSCT frequently depends upon the ability to control transplant Risk factors for a severe or disabling burden of late effects related toxicities (TRT). These TRT can be severe and require were older age at HSCT (p for trend <0.001) and a conditioning the admission of patients to the Intensive Care Unit (ICU). We regimen including TBI/TAI (OR 2.2 CI: 1.1-4.7, p=0.03). Sex, analyzed outcomes among HSCT patients admitted to the ICU diagnosis, donor and stem cell source and cGVHD were not in our center to identify the prognostic factors that could be used signifi cant risk factors, but for the latter analysis was hampered to inform the design of clinical trials for these patients. A ret- by low patient numbers. rospective analysis was performed of 34 consecutive patients Conclusion: A high burden of late effects is found in childhood who were admitted to the ICU after HSCT between 2000 and HSCT survivors after a median follow-up of only 7 years. Older 2007. Data were collected for disease, cause of admission, pre- age at SCT and TBI/TAI are independent risk factors for more vious hospitalization, biologic data on admission, during ICU severe late effects. stay and at the time of discharge, SOFA and SAPSII scales scores and critical support care. Survival data during hospitali- zation, 30 days and 1 year after discharge were also collected. P654 Median age of patients was 38 years. 21 (63.6%) were patients High incidence of secondary malignancies after who underwent allogeneic HSCT, 16 of whom had GVHD on allogeneic haematopoietic stem cell transplantation admission to the ICU. 12 patients were in relapse after trans- S. Neuburger (1), P. Hemmati (1), G. Massenkeil (2), T. Terwey plant and before admission to the ICU. Median time from HSCT (1), T. Kim (1), P. le Coutre (1), B. Dörken (1), R. Arnold (1) to admission on the ICU was 16.5 months (range 1-73). Median (1)Charité Virchow Klinikum (Berlin, DE); (2)Städtisches stay at the ICU was 6 days (range 1-28). The most frequent Klinikum (Gütersloh, DE) cause of admission to the ICU was acute respiratory failure (ARF) (53%): 14 patients (41%) were intubated, 16 patients Introduction: Allogeneic stem cell transplantation is a curative (47%) needed non invasive mechanical ventilation (MV) and 4 therapeutical option for patients (pts) with hematological dis- patients (11.8%) voluntary denied intubation. The mean SAPSII eases. As result of long term survival, late complications such score on admission was 49 ±19 and the maximum SOFA score as secondary malignancies are emerging. was 11 ±6. Overall survival (OS) during hospitalization, 30 days Methods: Here we present a retrospective analysis of 589 pts and 1 year after discharge was 50%, 50% and 37% respec- (median age at transplantation 41 years, range 16 - 75, male tively. No statistically signifi cant differences were observed in 339, female 250) who underwent allogeneic stem cell trans- OS between allogeneic and autologous transplant patients, and plantation in our institution between 1995 and 2007 (siblings neither was observed when comparing patients with or without n= 285, unrelated donors n= 304). Pts suffered from acute GVHD after HSCT. Non relapse mortality was higher in those leukemia (n=257), chronic myeloproliferative disorders (n=120), patients requiring MV and intubation versus those not requir- myelodysplastic syndrome (n=39), non-hodgkin-lymphomas ing these measures (85.7% versus 25%). The need of vasoac- (n=41) and others (n=132). Pts received conditioning with tive drugs was also an adverse prognostic factor for OS. ARF (n= 436) or without (n=152) 12 Gy total body irradiation (TBI). was the main cause of admission to the ICU after HSCT in our Results: Up to 2007, 283 of 589 pts (48%) died of relapse or cohort and the need of intubation an independent prognostic transplant related mortality (n=135 relapse, n=84 infection, factor. Larger sample studies are needed to identify prognostic n= 36 graft-vs-host-disease (GVHD), n=8 organ toxicity, n=20 factors for patients admitted to the ICU after HSCT and design others or unknown). 305 out of 589 pts (52%) had survived scores that could predict outcomes for this group. with a median follow-up of 40 months (range 1 – 144 months). 21 out of 305 pts (6,9%) developed secondary malignancies at a mean of 5,1 years (range 1-10 years) after allogeneic stem cell transplantation. Localizations of secondary malignancies were skin (n=11 basalioma, n=2 melanoma), gut (n=2 aden- ocarcinoma of the small intestine, n=1 coloncarcinoma), oral cavity (n=1 squamous cell cancer) and 3 lymphomas. 17/21 pts (81%) received a myeloablative conditioning with 12 Gy TBI, 4/21 pts (19%) a reduced intensity conditioning. Acute GVHD

S170 P656 Results: Since 1996 till 2007, 103 patients underwent HSCT Long-term follow-up of nutritional status after with conditioning regimen of busulfan (n=32 for poBuCy = 32; haematopoietic stem cell transplantation in infants n=71 for ivBuCy) at the Samsung Medical Center, Seoul, Korea. D.J. Campos, A. Koerich, R. Jung, A.M. Ordoñez, S. Fortier, The median age of recipients was 35 (range, 16 ?57) years. J. Zanis Neto, C. Bonfi m The diagnoses for HSCT were acute myeloloid leukemia (n=39; Universidade Federal do Parana (Curitiba, BR) 38%), chronic myeloid leukemia (n=36; 35%), myelodysplatic syndrome (n=13; 13%) and others (n=15; 14%). With median Introduction: The hematopoietic stem cell transplantation 18.8 months of follow up, ivBuCy group showed statistically (HSCT) may lead to an increase in energy expenditure, reduc- lower non-relapse mortality rate (19.8±0.3% vs 42.9±0.1% at ing food intake and absorption of nutrients. The protein-caloric 3 years, p=0.018; 4.2±0.4% vs 18.9±0.9% at 1 year, p=0.008). malnutrition in infants can cause irreversible impacts on growth The liver toxicity within day 30 was signifi cantly lower in ivBuCy and development. group compared to poBuCy group; peak level bilirubin and alka- Objective: Evaluate the long-term impact of HSCT in the nutri- line phosphatase was 2.49±0.52 vs 8.00±0.99 mg/dl (p=0.01), tional status of infants. and 138±3 vs 187±2 IU/L (p=0.06), respectively. However, the Methods: Data was collected retrospectively, considering the difference with respect to overall survival or VOD incidence was anthropometry at the time of hospitalization and last outpatient not observed in the current study. visit. Conclusion: The ivBuCy for allogeneic HSCT decreases liver Period: December 1990 to November 2007. All pts who had toxicity within day 30 and non-relapse mortality, but the benefi - less than 24 months-old at time of transplantation and had sur- cial effect of ivBuCy on improving overall survival or reducing vived more than one year after this procedure were included VOD was not demonstrated. Further study is warranted to dem- in this analysis. We used the World Health Organization clas- onstrate the benefi cial effect of intravenous busulfan together sifi cation (WHO, 2006) to assess the nutritional status. Survival with other intensive immunosuppressive conditioning such as was analyzed using the Kaplan-Meyer method. fl udarabine on overall survival following allogeneic HSCT. Results: 53 infants were transplanted in this period and 33pts survived at last one year, with a median follow up of 2540±1448 days after HSCT. The median age at admission was 13.8±5.9 months and 6.4±3.9 years at the latest evaluation, 73% (22pts) were male, 79% (26pts) had non malignant diseases and 88% (29pts) received a chemotherapy based conditioning. Sixty one percent (20pts) received HSCT from unrelated donors and 33% (11pts) developed graft-versus-host-disease (GVHD) after transplant. At the time of admission, 76% (25pts) had short stat- ure (based on the indicator height/age), 27% (9pts) had mild to moderate malnutrition and 18% (6pts) severe malnutrition (based on the indicator weight/age). Regarding the current nutritional status, 58% of patients (19pts) had short stature, 39% (13pts) showed mild to moderate malnutrition and 12% (4pts) had severe malnutrition. Analyzing the height/age curve, 45% (15pts) did not reach an ascendant growth curve after HSCT and 42% (14pts) did not reach an ascendant weight/ age curve. In this small cohort, infants who had GVHD did not develop signifi cant long-term malnutrition. Conclusion: We observed a high prevalence of malnutrition prior to HSCT and many pts persisted with defi cits in height and weight after transplant. We should intervene as early as possible in order to prevent or diminish nutritional long-term complications. In developing countries we urgently need more investments to ensure nutritional support to infants after hos- pitalization.

P657 The use of intravenous busulfan combined with cyclophosphamide reduces early hepatic toxicity and non-relapse mortality following allogeneic haematopoietic stem cell transplantation compared to oral busulfan- containing regimen H.J. Jun, K.H. Kim, M.H. Chang, S.Y. Yi, S.J. Kim, J.H. Jang, K. Kim, W.S. Kim, D.H. Kim, S.J. Kim, W.S. Kim, C.W. Jung Samsung Medical Center (Seoul, KR)

Background: Busulfan has been used as a key conditioning agent for successful hematopoietic stem cell transplantation (HSCT). Before the recent introduction of intravenous formula of busulfan, oral busulfan was only available. Previous reports suggested that the use of intravenous formula of busulfan permits stable concentration of busulfan in the plasma, thus reducing regimen-related toxicities, especially veno-occlusive disease (VOD). Methods and patients: The current study aimed to compare the transplant outcomes of allogeneic HSCT following intravenous busulfan with cyclophosphamide (ivBuCy) versus oral with cyclophosphamide (poBuCy), retrospectively.

S171 P658 P659 Usefulness of a simplifi ed scoring system to predict early Incidence and clinical presentation of disreactive immune non-relapse mortality after stem cell transplantation reconstitution after stem cell transplantation in PID: the P. Balsalobre, C. Muñoz, D. Serrano, J. Gayoso, G. Rodriguez, experience of a children’s hospital, Brescia I. Buño, Y. Martinez, A. Gomez-Pineda, J.L. Diez-Martin F. Porta, R. Schumacher, L. Notarangelo, C. Forino, Hosp. Gen. Univ. Gregorio Marañón (Madrid, ES) D. Demartiis, V. Bennato, V. Grassi, L. Lorenzi, F. Bolda, A. Bosi, S. Berta, R. Baffelli, E. Mazzolari, A. Lanfranchi Several scoring systems have been developed to assess the Oncohaematology and BMT Unit (Brescia, IT) risk of toxic mortality after stem cell transplantation (SCT). Basu et al (BMT Tandem Meetings, San Diego CA, 13-17 Feb 2008) Since 1990 more than 200 stem cell transplants in PID both preliminary reported the usefulness of a simplifi ed scoring sys- from MUD/HLA identical or haploidentical donors have been tem to predict the non-relapse mortality (NRM) at day 100 after realized. The clinical results have improved thanks to the new SCT. We have checked the usefulness of the Basu scoring sys- stem cell manipulation techniques and to the improved HLA typ- tem in our patients. ing techniques. At present the overall survival exceeds 75% in Methods: All consecutive transplants performed in our institu- HLA ID/MUD group and 70% in the haplo group. Since haploi- tion between Jun-04 and Jun-08 were selected. Patients with a dentical are realized previous positive selection of CD34 cells, previous SCT were excluded from the analysis as those alive depleting the graft from accessory cells such as stromal or den- with a follow-up lower than 100 days. Follow-up was censored dritic cells, the assumption was that clinically the patients could at time of last visit or subsequent SCT. Variables included in have behaved differently as concerns immunological reconsti- the scoring system are included in table 1. For the purpose of tution. Therefore we analyzed 114 SCID children who received this analysis the use of CB cells, not included in the original in 70 cases an haploidentical BMT and in 44 a MUD BMT, and scoring system, was scored as 2. Following the sum of the val- 94 children affected by Inborn Errors who received in 79 cases ues given to the scoring variables, each patient was classifi ed an haploidentical BMT and in 15 a MUD BMT. The result of the within one of the tree risk groups: Low (total score 0-1), Moder- analysis pointed out that autoimmunity was present in aggre- ate (total score 2) y High (total score >2). NRM was defi ned as gate only in 17 cases evenly distributed by underlying disease. death from any cause without a previous disease relapse or No major differences were evidenced as concerns incidence on progression. the type of transplant (6 MUD versus 10 haplos). At the same Results: 141 patients were selected, of which 112 (52% female, time, if donor-recipient chimerism is analyzed, it is not T-B split age 44.7 (19-68) years old) were valuables. chimerism responsible for the disregulated immune reconstitu- Diagnosis: Lymphoma (48), AL/MDS (38), MM (14), Other (12). tion. Infact in the haplo group only 2 out of 10 children had a SCT type: Auto (73)/Allo (39), including 6 with non-myeloab- mixed T-B chimera, while in MUD the mixed chimera were 2 out lative conditioning regimen, 22 from an unrelated donor (59% of 7. As concerns cell transplanted doses in all cases, CD34 with cord blood progenitors). Pre-SCT risk: Low (79), Moderate positive cell numbers, T cell infused in MUD were statistically (10), High (23). The global cumulative incidence (CI) of NRM at not different in the group of children who reconstitute a normal day +100 was 10% (IC 95%:4.5-15.5). When analyzing accord- immune function and in children who present autoimmunity. ing to the pre-SCT risk (Moderate/High vs Low) the relative risk Out of the 17 cases, 10 presented autoimmune haematolytic (RR) of NRM at day +100 was 1.4 (IC 95%:1.13-1.77) p<0.001; anaemia, 4 hypothyroidism and 3 vasculitis. CI of early NRM was 37% (20-53.5) vs 4% (IC95%: 0.1-8) p<0.001, respectively. Differences in RR and CI of NRM at day +100 between patients with Moderate and High risk were found P660 not statistically signifi cant. Impact on engraftment, transfusional requirements and Conclusions: In our experience, the simplifi ed scoring system outcomes of AB0 incompatibility in allogeneic stem cell proposed by Basu et al seems to be useful to identify patients transplant with Low vs Moderate-High risk of NRM. This tool could be E. López, J. Nieto, S. Palacios, A. Vaccaro, M.L. Lozano, of help in the decision-making process regarding candidates V. Roldán, N. Navarro, C. Castilla-Llorente, I. Heras, to SCT. V. Vicente Hospital Morales Meseguer (Murcia, ES)

ABO donor-receptor incompatibility is not considered an obsta- cle for allogeneic stem cell transplant (ASCT) but it can have an impact on the success of this procedure. We analyzed outcomes among ASCT patients in our center to identify the infl uence of ABO incompatibility on engraftment, transfusional requirements (TR) and survival. A retrospective analysis was performed of 151 consecutive patients that underwent ASCT from 1995 to 2007. Data were collected for TR and other clinical and biological values from time of admission for ASCT to last follow up. Hematopoietic stem cell source was peripheral blood in 138 cases (91.4%), bone marrow in 12 (7.9%) and umbilical cord blood in 1 (0.7%). The majority of patients received pro- genitors from a related donor (89.4% related versus 10.6% non related donors), and in 138 cases the HLA status was identical. 36 patients received a reduced intensity conditioning (RIC). 97 were isogroup (64.4%), 29 had a mayor ABO incompatibility (19.2%), 33 (21.9%) a minor and 8 patients (5.3%) bidirectional ABO incompatibility. 8 cases with mayor ABO incompatibility needed pretransplant plasmapheresis due to high antibody titles. Postransplant hemolysis was documented in 10 patients and 1 developed a selective eritroblastic aplasia. Median RBC TR for the group with ABO incompatibility was 7.74 units and for the ABO identical 6.44 units, median platelets TR was 9.11 and 7.45 units for the ABO incompatible and identical group respectively. Median time to neutrophile, platelets and

S172 RBC engraftment was 17.3, 15.2 and 34.4 days in the ABO P662 incompatibility group and 15.7, 13,1 and 27.2 days for the ABO Autologous stem cell transplantation in >70-year-old indentical group (p=0.016; p=0.029 and p=0.083). The overall patients: one hospital experience survival (OS) of our cohort is 62.9%. Median survival time is J. Nuñez, C. Montes, A. Bermudez, L. Yañez, A. Iriondo, 44.3 months in the whole group (range 0.3 to 155.5 months), A. Insunza 39.5 and 47.3 months for the ABO incompatible and ABO iden- HUMV (Santander, ES) tical groups respectively. In the RIC group median time of sur- vival was 20.2 (range 1.3 to 82.5), 15.4 and 24.5 months for Background: Although Autologous stem cell transplantation those with ABO incomptatibility and ABO identical graft respec- (ASCT) is an effective therapeutic option for several hemato- tively. There is a tendency for higher TR among the group of logical disorders, there is limited experience on the feasibility patients receiving an ABO incompatible graft versus those ABO and effi cacy of ASCT on elderly patients. identical. Neutrophile, platelets and RBC engraftment are faster Objectives: to analyze toxicity and effi cacy of ASCT in patients in the ABO identical group. There are no statistically signifi cant >70 years old differences in OS regarding the ABO status although there is a Design and methods: We analyzed the outcome of 24 patients favorable tendency for those patients ABO identical, this should >70 years old (median, 70 years) treated with ASCT between be considered when an AHSCT is programmed. 1997 and 2007. The baseline pathologies were: 7 AML, 11 lym- phomas, 5 myelomas and 1 amyloidosis. 12 patients received at least two treatment lines before ASCT. 12 patients were at P661 fi rst or second complete remission (CR)at the time of transplan- Prevention of treatment-related ovarian failure by tation, 2 at >3rd CR, 8 at partial remission and 2 with activated gonadotropin-releasing hormone analogue is ineffective disease. 17 patients had some co-morbidity (cardiac, hepatic, in young women with relapsed/refractory Hodgkin’s renal, CNS or pulmonary); 9 patients with >1 afected organs. lymphoma treated with peripheral blood stem cells Stem cell precursors were obtained from peripheral blood in autografting 22 patients and from peripheral blood plus bone marrow in S. Falorio, S. Santarone, F. Fioritoni, F. Angrilli 2 patients. The conditioning regimens were BUCY in 7 Civic Hospital (Pescara, IT) patients, BEAC/BEAM in 11, and Melphalan ± Busulphan in 6. 19 patients received Granulocyte Colony-Stimulating Factor Introduction: Treatment related (TR) ovarian failure causes after transplantation. amenorrhea and infertility in young women affected by Hodgkin Results: Early toxicity: all of patients suffered mucositys (III/IV Lymphoma (HL), depending on the type and intensity of treat- grade in 10 patients), 10 patients developed cardiac complica- ment. Alkylating drugs (particularly cyclophosphamide, iphos- tions, and 5 patients mild encephalopathy. A median of 9 and phamide, nitrosureas, procarbazine, busulfan, melphalan), 11 days were necessary to recovery 100 and 500 granulocytes, especially when used at higher doses as conditioning for stem and 12 and 15 days to recovery 20000 and 50000 platelets. cell transplantation, are most frequently associated with irre- The median of bed-days were 16 . 23 patients suffered neu- versible gonadic toxicity. Oral contraceptives or gonadotropin- tropenic fever, and 15 patients suffered later non neutropenic releasing hormone analogue (GnRHa) are generally used with fever. Only 3 patients died 100 days before due to sepsis, and the aim of preventing ovarian damage. 8 patients died due to relapse of the disease. There was one Patients and Methods: From 1994 to august 2008, we treated death in CR at 79th month due to an unknown cause. With a 91 consecutive women with a median age of 26 years (14- median follow up of 29 months the overall survival was 50%, 40). Patients received fi rst-line polychemotherapy both with- and the progression free survival was 37%. out alkylating drugs (ABVD or VBM) and with alkylating drugs Conclusions: Autologous Stem Cell Transplantation is feasi- (BEACOPP or COPPEBVCAD), followed or not by radiother- ble in elderly patients, and may be curative in selected elderly apy. Refractory/relapsed patients received peripheral blood patients with hematological diseases. The cardiac complica- stem cell autografting (PBSCA) after salvage polychemother- tions are the most notable but manageable toxicity. apy. To avoid TR ovarian damage, all patients received monthly Triptorelin 3.75 mg until treatment conclusion. The aim of this study is to evaluate the effi cacy of GnRHa in preventing TR P663 ovarian damage in young women receiving high dose therapy Adverse impact of iron overload on transplant outcome and PBSCA. G. Sucak, Z.A. Yegin, Z. Özkurt, Z. Aki, M. Yagci Results: Today 88 patients are alive and 3 died of HL. Before Gazi University Faculty of Medicine (Ankara, TR) the treatment, all patients had normal menses. After a minimum of 6 months from the end of the therapy, 79 (90%) patients Introduction: Iron overload(IO) is a well-recognized prob- reported regular menses and 9 (10%) patients became meno- lem in patients undergoing hematopoietic stem cell pausal. The last group consisted of 9 patients with refractory/ transplantation(HSCT) for underlying hematologic malignan- relapsed HL who received PBSCA with BEAM conditioning cies. Pretransplant iron burden is a signifi cant contributor to regimen, after salvage treatment with IGEV (iphosphamide, transplant-related morbidity and mortality. This retrospec- gemcytabine, vinorelbine) polychemotherapy. The cumulative tive review is planned to assess the impact of pretransplant doses of iphosphamide, carmustine and melphalan adminis- iron status on overall survival(OS) and transplant-related tered in these patients were 32-48 g/m2, 0,3 g/m², 0,14 g/m², complications(TRC). respectively. Methods: The charts of 250 patients [M/F:162/88; median age Conclusions: In our experience, GnRHa is able to prevent TR 34(16-71)] who underwent HSCT[148 allogeneic (allo), 102 ovarian failure in patients receiving a single line of polychemo- autologous (auto)] at Gazi University SCT Unit were retrospec- therapy with or without alkylating drugs. tively reviewed. Median follow-up was 315(1-1809) days. We On the contrary, it is ineffective during salvage polychemother- evaluated the effect of IO, as measured by pretransplant serum apy followed by PBSCA, because of the administration of high iron, iron binding capacity, ferritin and transferrin saturation(TS) cumulative doses of alkylating drugs. Consequently, patients on survival, mortality and toxic-infectious complications. with refractory/relapsed HL need alternative approaches to Results: Median pretransplant ferritin level was 576,2(2,1- prevent TR ovarian failure. In this regard, cryopreservation of 26971)ng/ml [231,36(2,1-9066);1001,8(11,4-26971) for auto ovarian tissue before the treatment followed by its reimplan- and allo, respectively].Patients with grade 3 and 4 mucositis tation after treatment should be one of the most promising had higher pretransplant ferritin in auto and allogroups(p=,009; modalities. p=,009).Patients who developed fungal infections had elevated pretransplant ferritin (p=,003;p=,028). Acute graft versus host disease(aGVHD) was diagnosed in 30(20,3%) of allosubjects

S173 median 43,5(12-94) days after HSCT. Pretransplant TS was P665 higher in hepatic GVHD group(p=,04). Pretransplant serum fer- Activation of haemostasis after transfusion of ritin was signifi cantly higher in SOS group(p=,01). A positive cryopreserved haematopoietic stem cells containing correlation was found between pretransplant ferritin, TS and dimethyl-sulfoxide Sorror’s comorbidity index(CI) in allogroup (p=,0001,r=,314;p= J. Studt, S. Meyer-Monard, C. Arber, M. Stern, D. Heim, ,01,r=,209). The impact of ferritin on survival was independent G. Marbet, A. Gratwohl, D. Tsakiris from CI. A strong relationship was found between pretransplant University Hospital Basel (Basel, CH) ferritin and day30, day100 and OS(p=,001;p<,0001;p<,0001). F erritin had a signifi cant impact on OS in auto(p=,002),and Objectives: Investigation of the toxicity of Dimethylsulfoxide on day30,day100 and OS in alloHSCT (p=,004;p=,001;p=,001). (DMSO)-containing cryopreserved hematopoietic stem cells Survival probability was found signifi cantly lower when ferri- (HSC) on hemostasis by determination of platelet function and tin>1000 ng/ml(p=,0001). of coagulation and endothelial cell activation in patients receiv- Conclusion: IO is a strong prognostic marker for OS and mor- ing autologous HSC transplants (HSCT). tality in allo and autoHSCT survivors. Elevated pretransplant Patients and methods: 40 patients (16 female, 24 male, age ferritin may contribute to development of toxic-infectious com- 54±13 years) received autologous HSCT containing 7,5% plications like SOS, aGVHD, mucositis and fungal infections DMSO. Underlying diseases were multiple myeloma (n=21), through the pathway of oxidative stress and tissue damage lymphoid neoplasia (n=10), acute leukemia (n=5), others caused by free oxygen radicals. Current study addresses the (n=4). Blood samples were taken immediately before, 15 min adverse impact of IO on survival and TRC. Our results should and 16-18 hours after HSCT. We tested whole-blood platelet be validated with further prospective studies and methods to aggregation (adenosine-diphosphate [ADP 6.4mcM], collagen modify IO in patients who are candidates for HSCT should be [COL 3.2mcg/ml], thrombin receptor activating peptide [TRAP developed. 32mcM], and arachidonate [AA 0.5mM]), fi brinogen (FBG), d-dimers (DD), thrombin-antithrombin complex (TAT), von Willebrand factor antigen (VWF:Ag), and cell-membrane micro- P664 particle activity (MPA) measured as thrombin-generation. Prognostic impact of serum ferritin concentration on Results: Mean MPA (19±9nM, 32±19nM, 16±8nM, p<0.001) survival following reduced-intensity conditioned and TAT (4±2, 15±11, 5±5 mcg/L, p<0.001) increased signifi - allogeneic haemopoietic stem cell transplantation cantly immediately after HSCT, returning to baseline the day R. Oakes (1), N. Sood (1), R. Pearce (2), G. Cook (1), after. No signifi cant changes were seen in FBG, DD, VWF: M. Gilleece (1) Ag. Only a trend for reduction of platelet aggregation with ADP (1)Leeds Teaching Hospitals (Leeds, UK); (2)British Society of (449±263, 380±277 AU, p=0.06) was found immediately after Blood and Marrow Transplantation (London, UK) HSCT. Eight of 36 (22%) patients presented fever and/or urti- caria immediately after transfusion of HSC, accompanied by Recent reports have identifi ed high serum ferritin levels pre- higher DD (p<0.05) and TAT (only trend) changes. transplant as an adverse prognostic factor in Full Intensity Discussion: We observed an activation of hemostasis, meas- (FI) allogeneic stem cell transplantation (AlloSCT). We report ured as increase of MPA and TAT immediately after HSCT. This a retrospective review of the effect of ferritin level (performed effect is short-lasting and might be attributed to components during assessment for AlloSCT) on the post-transplant survival of the HSC preparation including DMSO. Additional experi- of patients selected for AlloSCT at our centre. Of 106 patients ments addressing the infl uence of DMSO on platelet function consecutively transplanted since 2005, 64 had pre-AlloSCT fer- in vitro and on direct endothelial toxicity are needed. Known ritin levels available. Of these, 47 underwent Reduced Intensity adverse reactions to transfusion of cryopreserved HSC, such Conditioned (RIC) AlloSCT (median age 53; range 20-64). The as fever and/or urticaria, were associated with higher DD and range of diagnoses included 13 patients with acute myeloid leu- TAT changes. kaemia, 2 with acute lymphoblastic leukaemia, 8 with myelod- ysplasia, 3 with chronic myeloid leukaemia, 4 with Hodgkin’s lymphoma, 4 with myeloma, 10 with other chronic lymphopro- P666 liferative disorders, 1 with idiopathic myelofi brosis and 2 with Retrospective comparison of oral and intravenous severe aplastic anaemia. Three groups were arbitrarily defi ned busulphan based myeloablative conditioning regimens in by serum ferritin: Group 1 (ferritin <1000 micrograms/litre allogeneic stem cell transplantation (mcg/L), Group 2 (ferritin 1000-2500 mcg/L) and Group 3 (fer- K. Liapis, F. Panitsas, F. Michelis, M. Vagia, K. Manaka, ritin >2500 mcg/L). Two-year survival for patients in Group 1 V. Delistrati, I. Baltadakis, D. Karakasis, J. Apostolidis, was 77% (3 deaths among 18 patients); Group 2, 43% (9/21), N. Harhalakis, E. Nikiforakis and Group 3, 0% (8/8); p=0.00035 (by Cox proportional haz- Evangelismos Hospital (Athens, GR) ard regression). The distribution of ages was similar between the three ferritin groups (median age 52, 53 and 49 respec- Background: High-dose busulphan (Bu) is widely used in pre- tively). The types of stem cell donor were also similar (pro- parative regimens for AlloSCT. Intravenous Bu (ivBu) offers portions of matched related donors were 39%, 33% and 38% more predictable bioavailability than oral (poBu), avoiding respectively). Transplant (non-relapse) Related Mortality (at 1 erratic fl uctuations of serum concentrations. It has been pro- year) of the three groups was 6%, 27% and 63% respectively posed that this results in less toxicity without compromising (p=0.0128), whereas disease-related mortality was 18%, 30% effi cacy. and 25% respectively (p=0.69). A multivariate analysis incor- Aim: The purpose of this study was to retrospectively compare porating ferritin group, age, diagnosis and donor type showed poBu with ivBu in patients undergoing AlloSCT for AML or MDS only ferritin to be signifi cant (p=0.02) for overall survival in RIC in terms of regimen related toxicity (RRT), treatment related AlloSCT recipients. No signifi cant effect of ferritin on progno- mortality (TRM), rate of relapse (RR), GVHD, disease-free sur- sis was seen in our 17 FI conditioned AlloSCT recipients. We vival (DFS) and overall survival (OS). conclude that elevated ferritin pre-AlloSCT is an adverse prog- Methods: We retrospectively studied 172 adult patients allo- nostic indicator for RIC HSCT recipients, particularly in terms of grafted with myeloablative regimens based on poBu (n=135) or transplant related mortality. ivBu (n=37) during the period 1992-2008. Median follow up was 51 months for the poBu group and 13 months for the ivBu group. In the ivBu group median age was higher, high risk karyotype was more frequent, more donors were unrelated, and all grafts were peripheral blood stem cells (PBSC). In the poBu group HLA typing was low resolution in 2/3 of patients receiving unrelated

S174 grafts, while marrow and PBSC grafts were equally used. Dis- P668 tribution of diagnostic categories and stage of disease prior to Melphalan dose per kilogram of body weight has transplantation were similar between both groups. Use of ivBu signifi cant infl uence on oral mucositis after allogeneic was initiated in 2005. We adjusted for these potential confound- stem cell transplantation with FLU/MEL conditioning ing factors by employing multivariate statistical analysis. regimen Results: Regarding RRT, poBu was associated with greater S. Vokurka, K. Steinerova, M. Karas, V. Koza liver, gastrointestinal and renal toxicity, but this was of grade University Hospital (Pilsen, CZ) ≤ 2. The incidence of venoocclusive liver disease (VOD) did not differ between the two groups, although severe VOD devel- Introduction: Conditioning regimens with reduced toxicity are oped in 2 patients in the poBu group vs 0 in the ivBu group. No suitable for older and pretreated patients. The FLU/MEL regi- differences in engraftment were observed. ivBu predicted for men comprising of fl udarabine total dose 125-150 mg/m2 and longer OS (HR 0.33, 95% CI 0.14-0.78), however no signifi cant melphalan total dose 140-180 mg/m2 appears well-effective difference was detected in the RR (cumulative incidence 30%), and accompanied by minimum of fatal toxic complications. To acute GvHD (54% overall), TRM (overall 7% at 100 days) and explore characteristics of oral mucositis (OM) and individual DFS (overall 64%) between the two groups. risk factors in widely used conditioning regimens is important Discussion: Given the limitations of our study, the two routes issue that can help fi nd toxicity reducing approaches, high risk of Bu administration did not differ in antileukemic activity and patients and candidates for novel therapeutical agents. TRM. The OS benefi t probably refl ects improved results in the Goal: To explore characteristics and individual factors of OM in more recently transplanted patients and will have to be ascer- FLU/MEL patients. tained with longer follow-up. In pursuing a cost-effective trans- Methods: Prospective observation. FLU/MEL conditioning regi- plant policy, the higher cost of ivBu should be accounted for men (fl udarabine 30 mg/m² i.v. once daily for 4 days in total when choosing formulation. ivBu may be more appropriate for dose 120 mg/m², melphalan 140 mg/m² i.v. once daily on one patients with an increased risk of liver toxicity and VOD. day prior to transplantation). Amiphostine, palifermin and cryo- therapy were not used for OM prevention. OM assessed by WHO grading 0-4. P667 Results: 71 patients after allogeneic stem cells transplantation Differences in endocrine sequelae after non-myeloablative with FLU/MEL conditioning in 1/2005-12/2007, median age 56 and full-intensity conditioning in patients who underwent (23-68) years, gender: males 51%, graft: peripheral stem cells haematopoietic stem cell transplantation in childhood or 100%, number of CD34+ cells in graft: median 4,95 (1,6-15,2) adolescence x106/kg, creatinine clearance: median 1,5 (0,4-3,0) ml/s, gran- L. Papusha, D. Balashov, A. Tulpakov, Y. Skvortsova, ulocytes ≥1x109/l on day: median 13 (0-26) post-transplant. E. Skorobogatova, A. Maschan Melphalan dose per kilogram of actual body weight ranged Research Center for Pediatric Hematology (Moscow, RU) between 2,5–5,2 mg/kg (median 3,5 mg/kg) due to body sur- face area based dosing. OM incidence was 78%, grade 3-4 Background: Conditioning regimens and complications of WHO 38%, OM onset on median day 6 (2-10) post-transplant, HSCT such as “graft-versus-host” disease (GVHD) may cause duration 12 (1-34) days, OM resolution on day 18 (9-37) post- multiple endocrine dysfunctions in children. We analyzed risk transplant. OM in males vs. females: 67% vs. 89% (p=0,04), factors for endocrine complications after nonmyeloablative- OM in melphalan dose per kg <3,5 vs. ≥3,5mg/kg: 65% vs. 88% cyclophosphamide (Cy), fl udarabine (Flu) and ATG or myelo- (p=0,03). No correlations were observed between OM and age, ablative Busulfan (Bu) or Melphalan (Mel)-based conditioning patient-donor HLA identity and creatinine clearance (range regimens in patients after HSCT. 0,4–3,0 ml/s, median 1,5). In logistic regression analysis higher Patients and Methods: We studied 50 pts (27M/23 F) with acute melphalan dose per kilogram of body weight remained signifi - myeloid leukemia (AML) and severe acquired aplastic anemia cant predictor of OM (p=0,008). (SAA), 1-12 y after HSCT. All pts were divided according to Conclusion: conditioning regimen FLU/MEL can be considered receiving myeloablative (group I) or nonmyeloablative (group II) as high risky in respect to OM. The major factor infl uencing conditioning. Group I included 23 pts (12M,11F) with SAA, who OM in FLU/MEL patients is melphalan dose administered received Cy, Flu and ATG. Group II included 27 pts (15M,12 per kilogram. F) with AML, who received conditioning with Bu 16 mg/kg or Mel 140 mg/kg with Cy or Fludarabine. Endocrine function tests included TSH and free thyroxin, insulin-like growth factor I (IGF- P669 I) LH, FSH, estradiol and testosterone. Acute and chronic renal insuffi ciency after allogeneic Results: All children in group I had normal pubertal development, haematopoietic stem cell transplantation in children whereas in those from group II high incidence of gonadal dys- T. Ileri, M. Ertem, Z.B. Ozcakar, E. Unal Ince, Z. Uysal, M. Ekim, function was revealed: ovarian failure was found in all but two F. Yalcinkaya patients in group II. Mean levels of FSH, LH and estradiol were 70 Ankara University Pediatric BMT Unit (Ankara, TR) (45,6-89,2) UI; 35 (20-89,3) UI and 50 (10,9-80,2) pmol/l respec- tively. Only one girl of 10 recovered menstrual cycles after 1 year Acute and chronic renal impairments are important complica- of hormone replacement therapy; in 80% of boys in group II ger- tions after hematopoietic stem cell transplantation (HSCT). A minal epithelial damage was observed (FSH-20 (15-29,5)UI/l), prospective study was conducted to investigate the glomerular but Leydig cell function was normal in all pts (LH-6,7 (3,4-8) UI/l, renal function in children who received allogeneic HSCT from testosteron 20,5 (9-30) nmol/l). No patients developed primary matched related donors. Non-TBI conditioning regimens were hypothyroidism. Subclinical hypothyroidism (TSH-5,5 (4,2-6,7) used in all but one patient. Glomerular fi ltration rate (GFR) and mU/ml, free thyroxin- 12 (10-13,8) pmol/l) was found in 3 pts with serial measurements of serum creatinine were evaluated prior AA and 2 pts with AML. Growth retardation (mean height SDS=- to HSCT, within the fi rst 100 days and one year after. Acute 2,5) was evident only in 4 patients with extensive chronic GVHD kidney injury (AKI) was defi ned as at least 1.5 fold rise in pre- who received corticosteroids for > 1 year, independently of the HSCT serum creatinine within the fi rst 100 days and classifi ed type of conditioning. IGF- levels were normal in all pts. as grade 1 to 3 according to the new defi nition criteria proposed Conclusions: Myeloablative conditioning regimens produce by “Acute Kidney Injury Network”. Fiftyseven patients (hemo- gonadal damage in vast majority of children recipients of HSCT. globinopathy,29; aplastic anemia,11; malignancy,17) were cGVHD and prolonged steroid therapy predispose to growth enrolled in the study and 24 patients (42%) had AKI. Grade 1, retardation. 2 and 3 AKI was detected in 12 (21%), 9 (16%) and 3 patients Key words: stem cell transplantation, endocrine complications, (5%) respectively. Cyclosporine, amphotericin B and sinusoi- conditioning regimens, chronic GVHD, children. dal obstruction syndrome were found as risk factors for AKI.

S175 One year after HSCT only three patients (6%) had chronic P671 kidney disease (CKD) and none of the parameters, including Transplant conditioning toxicity: a comparison of 3 acute kidney injury, were found as a predictor for CKD in our myeloablative protocols series. All children with CKD had normal serum creatinine and M.Y. Shapira, R. Or, M. Aker, L. Dray, B. Gesundheid, none required dialysis. None of the patients had hypertension S. Samuel, I.B. Resnick or anemia. We conclude that renal impairment is an important Hadassah – Hebrew University Medical Centre (Jerusalem, IL) complication of HSCT and frequently occurs during the early phase of transplantation. Careful monitoring of renal function, Transplant toxicity, side effects etc., are known to vary in rela- minimizing the use of nephrotoxic medication, prophylaxis and tion to the conditioning protocols. In order to assess the toxicity effective treatment of SOS might be effective preventive meas- of 3 myeloablative conditioning protocols (CY-TBI (cyclophos- ures to decrease the incidence of AKI. phamide 60mg/kg/day x2 and fractionated total body irradia- tion (TBI) 2 Gy x6); Flu-Bu4 (fl udarabine 30mg/m²/day x6, oral busulfan 4 mg/kg/day or i.v. 3.2 mg/kg/day busulfex x4 ± i.v. P670 ATG) or Flu-Bu2-TT2 (fl udarabine 30mg/m²/day x6, oral busul- Translation and validation of the Functional Assessment fan 4mg/kg/day or i.v. 3.2 mg/kg/day busulfex x2 and i.v. thi- of Cancer Therapy-Bone Marrow Transplant (FACT-BMT) otepa 5 mg/kg/d x2 ± i.v. ATG), all patients in our registry that Version 4 quality of life instrument into Arabic among underwent bone marrow transplantation from a fully matched lymphoma patients who underwent high-dose chemo- siblings or fully matched unrelated donor using one of these therapy and autologous stem cell transplant protocols were reviewed. Patients with non-malignant indica- A. Al-Zahrani, H. Soudy, E. Colcol, E. Abdalla, I. Maghfoor, tions for transplant were excluded from this analysis as were S. Akhtar patients that had a 2nd or more allogeneic transplant. King Faisal Specialist Hosp. & Res. Ctr (Riyadh, SA) Results: 55, 47 and 36 patients were transplanted using the CY- TBI, Flu-Bu4 and Flu-Bu2-TT2, respectively. Non-relapse mor- Objective: To translate into Arabic and validate the “Functional tality (NRM) occurred in 15/55 (27.2%, p=0.59), 9/47 (19.1%) Assessment of Cancer Therapy –Bone Marrow Transplant” and 16/36 (44.4%, p=0.11) patients in the CY-TBI, Flu-Bu4 (FACT-BMT) quality of life questionnaire, among patients who and Flu-Bu2-TT2 conditioning protocols respectively (signifi - underwent high dose chemotherapy and autologous stem cell cance compared to Flu-Bu4 that the lowest NRM). The patients transplant (HDC-ASCT) for non Hodgkin’s lymphoma and treated with Flu-Bu2-TT2 had signifi cantly more total parenteral Hodgkin’s lymphoma. nutrition (TPN) days then the patients in the Flu-Bu4 group Methods: The Arabic translation followed the standard Func- (p=0.03) which may refl ect the general toxicity of the condition- tional Assessment of Chronic Illness Therapy (FACIT) transla- ing as they express the patients’ inability to either eat or tolerate tion methodology. Provisional Arabic FACT-BMT was pre-tested food. There was no signifi cant difference in the frequency or by personal interview of 20 native Arabic speaking patients. grade of acute GVHD, although patients conditioned with Flu- This version was then applied to the next 40 patients through Bu2-TT2 had slightly higher rate (p=0.1) and grade (p=0.08) of telephonic interview for validation. One lymphoma and autolo- acute GVHD. There was no difference in the myelosuppres- gous stem cell transplant expert (S. Akhtar) also developed sion, blood loss, cGVHD, vital organs toxicity or infections. 17 additional culturally adapted questions (Cultural Adapted Conclusion: With minor variations, there is no signifi cant differ- FACT-BMT) for relevant cultural issues. ence in the frequency and level of side effects and complica- Preliminary result: Sixty patients, males 39 (65%), females 21 tions between CY-TBI, Flu-Bu4 and Flu-Bu2-TT2 myeloablative (35%) participated in the study. At the time of study, median age transplant conditioning protocols although days in need for TPN was 32 years (15 -62 years) and ECOG performance status were signifi cantly higher in patients conditioned with Flu-Bu2- was 0 in 73% and 1 in 22%. First 20 patients interviewed in TT2 and a trend toward higher NRM, and the higher rate and pre-testing phase. All patients understood the questions well grade of acute GVHD was seen in this group. and no modifi cation has to be undertaken. The pre-test results indicated good content coverage and overall comprehensibil- ity. All patients easily understood all items of Arabic version of P672 FACT-BMT and high internal consistency in pre-testing phase Nitrogen balance in patients undergoing autologous is expected. Patients have high scores in all domains of quality haematopoietic stem cell transplantation of life including physical, social, emotional and functional well- A.A. Novik (1), V.N. Matyshevskaya (2), V.Y. Melnichenko (2), being, indicating that most patients were leading a normal life. D.A. Fedorenko (2), T.I. Ionova (1), S.I. Trifonov (2), K.F. Kim Patients scored > 70% for not at all (0) or a little bit (1) for lack (2), T.S. Sysoeva (2), G. Gorodokin (1) of energy, nausea, trouble meeting needs of the family, having (1)International Clinical Nutrition Study Group (New pain or feeling ill. Support from family and friends were > 80%. Jersey, US); (2)National Pirogov Medical Surgical Center Quite a bit and very much satisfaction with sex life was 60%. (Moscow, RU) Feeling sad was not at all (0) or a little bit (1) in 83%. Twenty percent patients were concerned about their ability to have chil- We aimed to study the impact of high dose chemotherapy dren; this is a refl ection of younger patients being transplanted (HDCT) with autologous hematopoietic stem cell transplanta- and also the social set up where the average family size usually tion (ASCT) on nitrogen balance in patients with autoimmune exceeds 5 children. Statistical analysis is underway. diseases and hematological cancer. Conclusions: These data validates the fi rst Arabic version Patients and methods: 41 patients (male/female – 22/19) FACT-BMT. This is likely to help large number of Arabic speak- undergoing HDCT+ASCT were enrolled in the study. The sam- ing patients, not only in the Middle East and Arab countries, but ple consisted of 26 patients with autoimmune diseases (male/ also in Europe and North America. Most of our patients enjoyed female – 11/15) and 15 patients with hematological cancer a good quality of life post HDC-ASCT. (male/female – 11/4). Mean patient age was 34 (range 17-63). Three conditioning regimens were used: BEAM (BCNU, etopo- side, cytarabine and melphalan), mini BEAM (30% reduced BEAM), and BM (BCNU+melphalan). The following parameters were studied before transplantation and after stem cell rein- fusion (D0, D+7, D+14): body mass, protein intake with food, daily urea excretion, and nitrogen balance. Results: After HDCT+ASCT dietary caloric intake decreased in 93% of patients, which is related to the loss of appetite, taste abnormalities, and nausea in this patient population. In 61% of

S176 cases the caloric intake provided less than 70% of the total daily This case indicates a possible immunomodulatory effect of caloric need. The decrease in body mass was observed in 78% of Lenalidomide in the immunomediated PRCA. patients. Insuffi cient protein intake (less than 1.2 g/kg body mass) was registered in 63% of patients. After HDCT+ASCT the increase in daily urea excretion as compared to baseline was observed P674 in 73% of cases with the value more than doubling in 34% of Comparison of acute renal failure in unmanipulated and patients. By D+7 the increase of daily urea excretion was noticed CD34+ cell-selected myeloablative allogeneic stem cell in 81% of patients (more than doubling in 27% of patients). By transplantation: a single-centre experience D+14 daily urea excretion decreased but in 59% of cases it was F. Fernández-Avilés, L. Quintana, P. Abrisqueta, M. Rovira, higher than before HDCT+ASCT. Negative nitrogen balance was C. Martínez, L. Rosiñol, E. Carreras, J. Campistol, observed in HDCT+ASCT patients: in 81% of patients at D0, in E. Montserrat 93% of patients at D+7, and in 59% of patients at D+14. Hospital Clinic (Barcelona, ES) To ensure adequate and balanced diet, enteral and parenteral nutritional support was provided for patients undergoing Objective: We compared the incidence, kinetic and clinical HDCT+ASCT. impact of postransplant acute renal failure (ARF) in patients Conclusion: HDCT+ASCT leads to abnormalities in nitrogen undergoing unmanipulated (allo-SCT) and CD34+ cell-selected balance in the majority of patients. The question of optimization (allo-SCT/CD34+) myeloablative allogeneic stem cell trans- of nutritional support for this patient population needs further plantation. investigation. Methods: 120 patients were included in this study. Sixty received an allo-SCT (group A: 41 sibling and 19 unrelated donors) and sixty a sibling allo-SCT/CD34+ (group B). Conditioning regimen P673 consisted of cyclophosphamide (120 mg/kg of body weight) Resolution of pure red cell aplasia after reduced-intensity and total body irradiation (12 Gy in 4 fractions in group A and allogenic stem cell transplantation with major ABO 13 Gy in 4 fractions in group B), and graft-versus-host disease mismatch during therapy with lenalidomide prophylaxis of cyclosporine A (CsA) plus methotrexate (group F. Pilo, D. Derudas, D. Baronciani, C. Depau, R. Murru, A) and CsA with or without methylprednisolone (group B). Prior E. Angelucci chronic renal dysfunction (CRD) was defi ned as follows: esti- Ospedale Oncologico “A. Businco” (Cagliari, IT) mated glomerular fi ltration rate (GFR) by Modifi cation of Diet in Renal Disease (MDRD) Study equation < 90 ml/min/1.73 We report a case of pure red cell aplasia (PRCA) following a m². ARF was divided into four categories: Grade 0: ARF <25% reduced intensity allogeneic stem cell transplantation with major decrease in GFR from baseline, Grade 1: less than twofold rise ABO mismatch, resistant to standard treatment and resolved in baseline serum creatinine and decrease in GFR between during lenalidomide therapy. 25-50% from baseline, Grade 2: greater than twofold rise in A 57 years old man was diagnosed of Multiple Myeloma (IgD / baseline serum creatinine without the need for haemodialysis, lamda, stage IIIA) in August 2003. After treatment with 4 cycles Grade 3: greater than twofold rise in baseline serum creatinine of VAD regimen and autologous stem cell transplantation, he and the need for haemodialysis. received a reduced intensity allogeneic transplant from his Results. Patient’s median age was 34 years (18-54) in group HLA identical sister with major ABO incompatibility (donor B+ A and 42 (18-64) in group B (p<0.001). Advanced disease was / recipient 0+). more frequent in group A (87% vs. 0%, p<0.00001). In our study, Post transplant period was uneventful but 40 days after trans- prior CRD did not infl uence the development of ARF (group A plantation patient progressively developed a pure red cell vs. B: 50.9% vs. 43.3%, p=NS). The incidence of ARF grades aplasia with high immune anti-donor isohemagglutinin titers 3 and 2 (A vs. B) were 11.6% vs. 0% (p=0.01) and 56.6% vs. (immune anti B, 1:512). Erithropoietin level was 203 mU/ml. 73.3% (p=0.05), with a median (range) day of presentation of Analysis of chimerism revealed allogeneic engrafment. In vitro 16 (1-210) vs. 70.5 (6-390), respectively (p=0.00003). Patients bone marrow cultures showed CFU-E absence. Since trans- of group A that developed ARF grades 3 and 2 had an increased plant patient remain transfusion dependent receiving 4 units/ risk of non-relapse mortality (NRM) at 6 months, as compared months of 0 + RBCU and chelation. with those that developed ARF grade 0-1 (85.7% vs. 10.5%, Subsequently he was treated with several immunosopres- p=0.0008; 38.2% vs. 10.5%, p=0.05, respectively). In group sive treatment: cyclosporine and steroids, plasma exchange B, patients with ARF grade 2 do not show an increased NRM (4 courses), 4 cycles of Rytuximab plus Cyclophosphamide, (18.2% vs. 12.5%, p=0.7). and fi nally with escalation doses of donor lymphocyte infusions Conclusion. In this series, ARF grade 3 after allogeneic trans- (DLI) in association with 150 ìg /weekly of erythropoietin (EPO). plant was related with a very poor prognosis. ARF grade 2 was No clinical or laboratoristic improvement was observed. signifi cantly associated with higher NRM only in patients under- Extramedullary Myeloma relapse occurred in February 07’; he going unmanipulated allo-SCT, corresponding to those who was treated with Bortezomib, Liposomial Doxorubicin and Dex- developed ARF in an early and critical period after transplant, amethasone for 8 cycles. Complete remission was obtained. in contrast to those that received an allo-SCT/CD34+. PRCA was unchanged. A second extramedullary relapse was occurred in December 07’. He was submitted to a local radiotherapy and Lenalidomide P675 (25 mg/day for 3 weeks) associated with low dose of Desam- Safety and pharmacokinetics of intravenous versus oral ethasone (Rd). busulfan used in pretransplantation myeloablative During the forth Rd cycle (in September 08’), together with conditioning therapy Myeloma response, a stable increase of the hemoglobin value M.L. Battista, F. Patriarca, M. Medeot, A. Sperotto, M. Cerno, was observed (about 5 grams in two month). A. Geromin, F. Florio, R. Fanin The anti-donor titers isohemagglutinin became undetectable, Division of Haematology (Udine, IT) the direct ABO blood group converted to B+. Analysis of chi- merism revealed a permanent allogeneic engrafment. Erithro- Objectives: We conducted a retrospective analysis of intrave- poietin level was 35.8 mU/ml. nous (i.v.) versus oral (p.o.) busulfan in patients undergoing This patient has been remaining transfusion–dependent 47 allogeneic hematopoietic stem cell transplantation (HSCT) with months after transplant, (total units of 0+ RBCU 184 since the aim of evaluating toxicity, clinical outcome and busulfan transplant), without conversion to the donor ABO blood group; pharmacokinetics. only after the Lenalidomide treatment he was able to recover a Patients and methods: Twenty-three consecutive patients with normal hemoglobin value. haematological malignancies (mainly acute myeloid leukaemia)

S177 underwent HSCT in our Centre. All patients received busulfan pts with NHL (14), HD (11), MM (11) and AML (4) were treated over 4 days followed by cyclophosphamide 60 mg/kg i.v. for 2 with autologous PBSCT (36) or BMT (4). Elevated ferritin level days as standard myeloablative conditioning regimen. Busulfan >300 ug/dL was found in 54 (88,5%) pts: 300-1000 in 25 (41%); was administered in the 4-times-daily dosing schedule: group 1000-2000 in 15 (24,5%); 2000-3000 in 7 (11,5%) and >3000 1 (14 patients, median age 45 years, range 16-66) received i.v. in 7 (11,5%) pts. busulfan 0.8 mg/kg every 6 hours, group 2 (9 patients, median Results: neutrophil recovery occurred in 57 pts at 16 (12-54) age 37 years, range 25-50) received p.o. busulfan 1 mg/kg days; 4 pts died before hematologic reconstitution due to every 6 hours. In both groups the majority of the donors were septic shock (1) or multiorgan failure (3). Infection complica- unrelated (57% group 1; 67% group 2). The major source of tions up to 100 days occurred in 45 (74%) pts including: FUO stem cells was peripheral blood (78% group 1; 67% group 2). At (27), bacterial (17), fungal (5); CMV (10), and VZV (2) infec- the time of transplantation 64% of the patients in group1 were tions. Organ dysfunction (renal, hepatic, lung) was diagnosed in complete remission, 55% in group 2. Blood samples were in 7 patients. GVHD>2-4 was diagnosed in 4 (19%) pts from collected for pharmacokinetic analysis on the fi rst and last day allogeneic group. Eight (13%) pts died up to 100 days due to of busulfan administration. bacterial infection (1), invasive aspergillosis (2), GVHD (1) or Results: Table 1 provides a summary of the time of engraftment, multiorgan failure (4). Ferritin level >1000 ug/dL was associ- regimen-related toxicity, incidence of acute GvHD, relapse, 100- ated with signifi cant higher rate of organ toxicity and TRM day transplant-related mortality (TRM) and pharmacokinetic (p=0.046 and p=0.083 respectively). Factors signifi cant for data. All patients in group 1 achieved engraftment, while 1 patient unfavourable outcome at day +100 by Kaplan-Meier analysis in group 2 experienced engraftment failure and another patient were: ferritin level >1000 ug/dL (0.724±0.083 vs 0.938±0.043, died before engraftment could be evaluated. Median time to neu- p=0.025), advanced disease (0.730±0.073 vs 1.000, p=0.006), trophil and platelet engraftment was similar. Group 2 patients and alloHSCT (0.571±0.108 vs 0.975±0.025, p<0.001). Factor experienced a higher incidence of 100-day TRM (44% group 2; predicting overall survival by multivariate analysis in Cox model 0% group 1; p 0.03) and grade III-IV mucositis (78% group 2; was alloHSCT (p=0.029, HR=6.5, 95%CI=1.3-166). 64% group 1; p 0.02). Median busulfan AUC on the fi rst and last Conclusions: Elevated level of pretransplant ferritin >1000 ug/ day of oral administration in group 2 was higher than the tar- dL is associated with increased risk for multiorgan failure and geted therapeutic window (AUCs between 900-1500 microMol/ toxic deaths following HSCT. min). Differences in relapse rate and incidence of hepatic veno- occlusive disease (VOD) and grade II-IV acute GvHD were not signifi cant. No seizures were recorded in any of the patients. P677 Conclusions: Our experience demonstrates that the condition- Immune reconstitution more than one year after ing regimen based on i.v. busulfan can be safer than the oral allogeneic haematopoietic stem cell transplantation route considering the reduced incidence of severe mucositis S. Narayan, J. Adams, G. Lucas, J. Burthem, E. Tholouli, and 100-day TRM, most likely because of dose assurance H. Lenehan, J.A.L. Yin with predictable pharmacokinetics. No signifi cant difference in Manchester Royal Infi rmary (Manchester, UK) relapse rate and incidence of VOD was observed. Duration of immune defi ciency post haemopoietic stem cell transplantation (HSCT) is highly variable. We enumerated cir- culating natural killer cells & lymphocyte subsets post trans- plant in association with stem cell source, chronic graft versus host disease (cGVHD), conditioning and recipient age. We studied 63 patients more than one year post transplant. Peripheral blood samples were analysed using fl ow cytometry. The absolute counts of T & B lymphocytes, NK cells, CD4+ & CD8+ T cell subsets were determined. Results were correlated with graft, host & transplant related factors. Of the 63 patients studied, 43(68%) received sibling allografts and 20(32%) matched unrelated transplants for predominantly malignant haematological conditions. The median age at trans- plant was 43 years (range 19-66 years). The median time of sampling post HSCT was 42 months (12-262 months). Twenty three (37%) had a reduced intensity transplant while 37 of the remaining 40 received TBI based myeloablative conditioning. Twenty fi ve (40%) received Alemtuzumab (Campath) pretrans- plant. 38(60%) received haemopoietic progenitor cells collected by apheresis (HPC-A). Of the 63 patients, 8(13%) were lym- phopenic; 5 of whom had cGVHD. Ten (16%) had low NK cells. 29 (46%) patients had an inverted CD4/8 (<1) ratio (even when CD4 numbers were within our reference range), 11 of these patients were 3-5 years & 6 patients >5years post transplant. P676 Persistent CD4 lymphopenia was seen in 12 patients, 3 of Increased risk for transplant-related toxicity and mortality whom were >5 years post transplant (although more common in patients with iron overload in the group with cGVHD, the difference was not statistically L. Gil, A. Lojko-Dankowska, A. Nowicki, A. Czyz, D. Dytfeld, signifi cant). Fewer patients in the group receiving HPC-A had M. Komarnicki low NK and T lymphocyte numbers compared with recipients of University oF Medical Sciences (Poznan, PL) marrow stem cells (p<0.05 and 0.001 respectively). Recipients of matched unrelated transplants had lower NK cells (p<0.05), Background: Iron overload is associated with increased mor- lymphocytes (p<0.01), and particularly CD4+ counts (p<0.05) bidity and mortality among HSCT recipients. We evaluated the compared with sibling donors (the proportion of HPC-A to HPC- risk of infections, organ toxicity and TRM within 100 days after M was similar in both groups). Age at transplant, intensity of HSCT with respect to pretransplant serum ferritin levels. conditioning including TBI & Campath did not demonstrably Methods: A total of 61 patients (median age 42; range 19-68) affect reconstitution in our limited cohort of patients. were analyzed. Twenty one pts with AML (10), ALL (3), AA (3) Immune reconstitution post HSCT is variable, with low CD4 and HD (5) underwent allogeneic PBSCT (14) or BMT (7). Forty counts and an inverted CD4/8 ratio commonly seen; this may

S178 result from ongoing thymic insuffi ciency post transplant. In our P679 study the source of stem cells and chronic GVHD were the main Motor disability as early complication of haematopoietic contributory factors, however the signifi cance of an inverted stem cells transplantation CD4/8 ratio remains to be fully evaluated. A. Tendas, P. Niscola, S. Delogu, T. Dentamaro, L. Cupelli, A. Siniscalchi, L. Scaramucci, M. Giovannini, M. Ales, G. Natale, R. Cinque, A.P. Perrotti, P. De Fabritiis P678 S. Eugenio Hospital (Rome, IT) Busulfex (i.v. BU) and CY regimen before SCT: a phase II pharmacokinetics combined study Background. Motor disability (MD) is a reduction of motor func- S.-W. Kim (1), S.I. Mori (1), R. Tanosaki (1), T. Fukuda (1), tion when compared with normal condition. Althougth frequently M. Kami (2), H. Sakamaki (3), T. Yamashita (3), Y. Kodera (4), observed in hematological malignancies, MD was rarely S. Terakura (5), S. Taniguchi (6), S. Miyakoshi (7), N. Usui (8), explored and reported in patients (pts) undergoing stem cells S. Yano (8), Y. Kawano (9), Y. Nagatoshi (10), M. Harada (11), transplantation (BMT). Y. Morishima (12), S. Okamoto (13), AM. Saito (14), Y. Ohashi Patients and methods. We defi ned MD incidence, etiology and (14), R. Ueda (15), Y. Takaue (1) pathophysiology in 24 pts, undergoing 25 BMT (20 autologous (1)National Cancer Center Hospital (Tokyo, JP); (2)Institute and 5 allogeneic, standard conditioning regimen in all cases). of Medical Science, University of Tokyo (Tokyo, JP); Median age was 42 years (18-70). Diagnosis at BMT was: acute (3)Komagome Hospital (Tokyo, JP); (4)Aichi Medical University leukemia in 5, multiple mieloma in 11, lymphoma in 8, myelo- (Aichi, JP); (5)Nagoya University (Nagoya, JP); (6)Toranomon displasia (MDS) in 1. Disease phase was: complete remission Hospital (Tokyo, JP); (7)Tokyo Metropolitan Geriatric Hospital in 14, partial remission in 10, at diagnosis in 1 (MDS). Pts dis- (Tokyo, JP); (8)Jikei University School of Medicine (Tokyo, JP); ability was assessed weekly by Barthel index (BI), till discharge. (9)Kagoshima University (Kagoshima, JP); (10)National Kyushu Mechanism of MD was defi ned with a scale derived by NCI 3.0 Cancer Center (Fukuoka, JP); (11)National Omuta Hospital toxicity criteria, adjusted for motor functioning (Table 1). (Omuta, JP); (12)Aichi Cancer Center Hospital (Nagoya, JP); Results. At entry MD (grade 2 or more) was present in 3/25 (13)Keio University (Tokyo, JP); (14)Nagoya Medical Center pts. During BMT disability (grade 2 or more) was increased to (Nagoya, JP); (14)University of Tokyo (Tokyo, JP); (15)Nagoya 10/25 transplants; among 10 disable pts, grade 2 MD (without City University (Nagoya, JP) ADL impairment) and grade 3-4 MD (ADL impairment) were observed in 4/10 and 6/10, respectively. At discharge, MD was Background: Although i.v. BU has been recently introduced into still present in 6 pts (Figure 2). Single or multiple mechanisms clinical use worldwide, drug profi les of i.v. BU preparation have of MD were: muscular defi cit in 8 pts (fatigue/muscle wasting in not been fully evaluated in different races, who may have dif- 6, CNS damage related hypotrophia in 2); bone/joint damage ferent PK. As part of our pivotal study in Japan, we conducted with movement-related pain in 3 pts; respiratory tract infection a phase II study with PK analysis of a combined i.v. BU and CY in 2 pts; others central or peripheral nervous system alterations regimen administered before allo- or auto-SCT. in 3 pts; movement-related pain alone in 1; visual defi cit in 1. Patients and Methods: Patients with hematological malignan- Among 18 MD episodes in the 10 MD pts, 14 were due to treat- cies were eligible for this study (age, 5 to 55 years). The effi - ment and 4 were due to cause not related with disease. cacy variables were engraftment, relapse, OS and DFS. The Conclusion. In our experience motor ability impairment is a safety variables were NRM and adverse events including sei- frequent feature in the setting of hemopoietic stem cells trans- zure, VOD, GVHD. The conditioning regimen consisted of i.v. plantation, with 40% of pts experiencing a worsening on motor BU (at 0.8 mg/kg for 2 h every 6 h at a total of 16 doses, days functioning during procedure, mainly due to treatment-related -7 to -4) and CY (at 60 mg/kg for 3 h at a total of 2 doses, complications. Larger studies are needed to understand dis- days -3 and -2). Seizure prophylaxis was phenytoin from day ability risk factors and to plan rehabilitative programs to prevent -9. For allo-SCT patients, GVHD prophylaxis consisted of CYA and treat disability. Effects on both pts quality of life and car- and short-term MTX. Multiple PK samples were taken on 1st egiver work-load are strongly expected. and 9th doses. BU levels were determined by a gas chroma- tographic-mass spectrometric detection method. PK modeling was performed using WinNonlin. Results: Thirty patients (28 HLA-matched allo-SCT, 2 auto- SCT; median age, 30 years) were enrolled between 7/2002 and 10/2003. The diseases were 13 AML (11CR), 5 ALL (4CR), 5 CML-CP, 4 NHL (3CR) and 3 MDS. There were no signifi cant toxicities, and all but one patient showed evidence of neutrophil engraftment at a median of 14 days for allo-SCT and 11 days for auto-SCT. Grade II-IV acute and chronic GVHD occurred in 9 (33%) and 16 patients (59%), respectively. Four patients (15%) died of non-relapse causes: MOF, chronic GVHD, hepatic fail- ure and viral pneumonia. The relapse rate at 1-year was 26%, and 4 patients (15%) died of relapse. OS and DFS at 1-year in allo-SCT was 78% and 63%, respectively (median follow-up: 413 days). The 2 auto-SCT patients were alive disease-free at 1-year. The PK of i.v. BU showed close inter- and intrapatient consistency. The mean AUC for dose 1 and dose 9 was 1171 micromol min/L and 1242 micromol min/L, and the mean Cmax was 994 ng/mL and 1311 ng/mL, respectively. The AUC of the 1st dose was below 1500 micromol min/L in 27 patients (90%), and this was within the range of 900 to 1350 micromol min/L in 21 patients (72%). Conclusions: Since all of the profi les overlap with non-Japa- nese data, we conclude that racial factors may not seriously infl uence the bioactivity of i.v. BU and a combined i.v. BU and CY regimen may be practically useful.

S179 P680 years after transplantation. Prophylactic use of zoledronic acid Quality of life in adult survivors of blood and marrow signifi cantly prevented bone loss in the femoral neck, as well as transplantation correlates strongly with physical and the spine (OR=0.18, 95% CI=0.05-0.69, p=0.012). Therefore, psychological late effects of treatment: a mixed methods BMD measurements and the use of zoledronic acid are recom- approach mended in the case of GVHD or long-term steroid use after L. Fredrick (1), R. Beeken (2), N. Taylor (2), C. Eiser (2), Allo-SCT to improve the QoL. J. Wright (1), J. Snowden (1), C. Dalley (1) (1)The Royal Hallamshire Hospital (Sheffi eld, UK); (2)The University of Sheffi eld (Sheffi eld, UK) P682 The impact of bone marrow fi brosis in the outcome of Objectives: Quality of life (QOL) may be compromised follow- haematopoietic stem cell transplantation ing bone marrow transplantation (BMT). We report a mixed- G. Sucak, E. Suyani, Z. Aki, N. Akyürek, Z. Özkurt, S. Altindal, methods approach to explore 1) QOL in adult survivors and 2) A. Yegin psychosocial variables associated with QOL and coping. Gazi University Faculty of Medicine (Ankara, TR) Methods: Study 1: Qualitative interviews were conducted with 19 patients (18-65years), post-BMT to explore QOL after trans- Objectives: To investigate the effects of bone marrow fi brosis plant and the role of psychosocial variables. (BMF) on hematopoietic stem cell transpalantation (HSCT). Study 2: 15 patients (18-45 years), at least 2 years post-BMT, Methods: In this study, we retrospectively analysed the data completed standardised measures of QOL (physical (PCS) and of 175 patients with a diagnosis of hematological malignancy, mental (MCS)), posttraumatic stress (PTS), optimism, self effi - aplastic anemia, myelodysplastic syndrome and primitive neu- cacy and late-effects. Information was also taken from medical roectodermal tumor, who underwent autologous or allogeneic records. HSCT (69 autologous HSCT, 106 allogeneic HSCT) at the Results: Study 1: Interviews were analysed using template Stem Cell Transplantation Unit of Gazi University. At the time analysis. Patients describe their QOL as good; but more com- of HSCT, the median age of patients was 33 years (range promised where patients experience late effects and transplant- 16-68) and, 56 were female and 119 male. The median time related complications. Patients experience worsened physical from diagnosis to HSCT was 332 days (range 38-3107 days). QOL, due to decreased mobility and increased fatigue. Psy- Post transplantation follow-up was 318 days (range 10-1433) chosocial variables including optimism and social support were in autologous HSCT patients, and 265 days (range 7-1307) in important for managing compromised QOL. allogeneic HSCT patients. Bone marrow fi brosis was evaluated Study 2: Quantitative data were analysed using t-tests and immediateley before the HSCT. The effects of BMF on engraft- correlations. Patients’ PCS and MCS scores did not differ sig- ment, graft versus host disease (GVHD), early post-transplan- nifi cantly from population norms. Patients with more late effects tation complications and survival were evaluated. recorded in medical notes had lower PCS scores (p<0.005). Results: Nineteen (27.5%) of the autologous HSCT patients, High self-reported late effects and PTS symptoms were associ- and 38 (35.8%) of the allogeneic HSCT patients had BMF before ated with lower MCS scores (p<0.005; p<0.01). Higher MCS HSCT. Pre-transplantation BMF did not cause delayed engraft- scores were associated with higher optimism (p<0.005) and ment, and had no impact on early post-transplantation compli- self effi cacy (p<0.05). There was no correlation between PCS cations. Development of acute and/or chronic GVHD were also scores and psychosocial variables. not affected by the presence of fi brosis in the bone marrow. Conclusion: These fi ndings suggest that following BMT, patients Overall survival (OS) was 76.7% in patients with and 88.6% in experience good QOL. However late-effects and PTS may patients without fi brosis (p>0.005) while progression free sur- compromise this. Psychosocial variables help patients to cope vival (PFS) was 26.33% in patients with and 16.5% in patients and may act as a buffer, enabling patients to experience good without fi brosis (p>0.05) in autologous HSCT recipients with a QOL despite the negative effects of BMT. follow up period of median 318 days (range 10-1433). Although The advantages of the mixed methods approach include greater the presence of fi brosis seems to decrease OS an PFS, this insight into QOL effects and patients adaptation to limitations, decrease did not reach signifi cant levels in autologous HSCT coupled with the ability to compare against population norms. patients. OS (35.2% vs. 48.9%) (p=0.004) and PFS (27.8% We conclude there is a need for a BMT specifi c measure of vs 51.2%) (p=0.0008) decreased signifi cantly with BMF with a QOL sensitive to the issues identifi ed in the interviews, includ- follow up period of 265 days (range 7-1307) in patients who ing late effects of treatment. received allogeneic HSCT. Cox regression analysis showed that the effect of BMF on survival in allogeneic HSCT reciepi- ents was independent of age, Sorror comorbidity index, primary P681 disease and disease status during HSCT (p=0.05). Pilot study on prophylactic use of zoledronic acid to Conclusion: Pretransplant BMF does not affect engraftment, prevent bone loss in allogeneic recipients early post transplantation complications and mortality, in allo- S.K. Sohn, Y.S. Chae, J.G. Kim, J.H. Moon, S.N. Kim, S.J. Lee, geneic HSCT patients, However it seems to cause a relatively J.S. Suh, K.S. Lee poor prognosis in terms of overall survival. Kyungpook National University Hospital (Daegu, KR)

Bone loss is recognized as worsening the quality of life (QoL) in long-term survivors of allogeneic SCT (Allo-SCT). There- fore, this study evaluated the risk factors associated with bone loss and the role of zoledronic acid in preventing bone loss in allogeneic recipients. Fifty-three patients who underwent HLA-matched Allo-SCT were evaluated for their bone mineral density (BMD) in the lumbar spine and femoral neck at regular intervals. Zoledronic acid (4 mg) was given intravenously to 18 patients (ZA patients) at 2 months after SCT and then every 3 months until 2 years. Grade 2-4 acute GVHD was associ- ated with bone loss (OR=4.90, 95% CI=1.41-16.99; p=0.012) at 1 year after SCT, while the existence of extensive chronic GVHD and steroid use were both unfavorable prognostic fac- tors (OR=9.00 and 7.22, 95% CI=1.52-53.40 and 1.44-36.22; p=0.016, respectively) in terms of osteopenia/osteoporosis at 2

S180 Inborn errors dominant Hyper IgE syndrome (HIES). We have previously described successful HSCT with no amelioration of disease in a patient with HIES1. We re-evaluate our experience. Patient 1 suffered severe infected eczemoid dermatitis, recurrent staph & P683 pseudomonas infection, staphylococcal pneumatoceoles, and Assessment of reduced-intensity conditioning regimens ankle + wrist fractures. She had novel heterozygous STAT3 for haematopoietic stem cell transplantation in primary mutation (g.1771A>G, p.K591E), predicted to disrupt the SH2 immunodefi ciency domain. She underwent 10/10 HLA-matched URD HSCT after H. Robinson (1), M. Slatter (2), D. Barge (2), N. Bown (2), conditioning with Bu16/Cy200, aged 7 years. After 12 yrs follow A. Jackson (2), H. Harvey (2), T. Flood (2), A. Cant (2), up, she is 100% donor, has had infected eczema (St. aureus, M. Abinun (2), A. Gennery (2) pseudomonas) controlled and resolving over 7-8 years, soften- (1)Newcastle University (Newcastle upon Tyne, UK); ing of facial features, normalization of lumbar spinal bone den- (2)Newcastle upon Tyne Hospitals NHS Foundation Trust sity over 8 years (previously –2SD below normal), no further (Newcastle upon Tyne, UK) signifi cant lung infections and normal lung function. She has ankle and knee instability, but no new fractures, and developed Optimal conditioning needed for stable engraftment and full thoracolumbar scoliosis 10 yrs post HSCT. Her IgE remains immune reconstitution (IR) after hematopoietic stem cell elevated. Patient 2 experienced Severe bilateral lower, middle, transplantation (HSCT) in primary immunodefi ciency (PID) is and lingular lobe bronchiectasis, recurrent pseudomonal chest undetermined. Bu/Cy conditioning is associated with short and infections despite antibiotic and immunoglobulin trearment, long-term treatment-related toxicity, particularly in children with recurrent severe impetigo & chronic dermatitis and osteopenia pre-existing end-organ damage. Reduced intensity (RIC) regi- with vertabral crush fractures T10-L2. He had a heterozygous mens have been proposed, with good results in some centres. STAT3 mutation (g.1144C>T, p.R382W) predicted to disrupt We present our results. 33/135 patients transplanted 1999- the DNA-binding domain. He underwent a 10/10 HLA identical 2007 received Flu/Melph based RIC. 5 had SCID (mean age URD HSCT after conditioning with C-1H, Flu/Melph, aged 13 at HSCT 0.7 months, range 6-12), 28 other PID (mean age yrs. After 3 yrs follow up, he has had 3 episodes of bronchop- 8.5 yrs, range 0.16-17 yrs). All received Flu 150mg/m², Melph neumonia, reduced but stable lung function, his bone density 140mg/m²; 6/33 also received ATG and 27/33 Campath 1-H. has normalized and he remains 100% donor. Functional stud- All received CSA as graft-vs host disease (GvHD) prophylaxis. ies of fi broblasts, lymphocytes and osteoclasts are in progress. 18 had pre-existing chest-, 4 hepatic- and 10 severe gastro- HSCT does appear to modify disease process in STAT3- intestinal- (GI) disease. HSC source was matched sibling or defi cient HIES. The effect may be more like that of HSCT for family donor bone marrow (BM) in 10, 10/10 HLA-matched vol- metabolic disease – with slow onset of effect and resolution unteer unrelated donor (VUD) BM in 13, VUD peripheral blood of pathology. The effect on development of lymphoma is yet (PBSCT) in 4, VUD cord blood (2 9/10 HLA-matched) in 5 and to be determined, and long-term follow up is required to deter- T cell depleted haplo-identical parental BM in 1. mine benefi t. Further studies are required to determine which There was an average 18 days until neutrophil and platelet patients would benefi t from HSCT, but it may have a role in engraftment. 1 yr survival was 28/33 (85%), with survival at last treatment for selected patients. follow up (FU) of 25/33 (78%) (median FU 28 months, range 1. Gennery AR et al. Bone Marrow Transplant 2000;25:1303-5 0.5 – 6 yrs) - SCID 80%, non-SCID 75%. 5 deaths occurred within 1 yr of HSCT from infection (3) and GI complications (2). 3 late deaths were due to GvHD. 7/8 deaths were amongst the P685 fi rst RIC transplants performed in our unit. 23/33 (70%) had Stem cell transplantation for Nijmegen breakage GvHD, only 5 > grade II. 13/33 (40%) reactivated viruses (CMV syndrome – Experience in 6 patients 4, HHV6 4, Adenovirus 2, EBV 3). 11/25 survivors (44%) had M.H. Albert (1), A. Gennery (2), J. Greil (3), C.M. Cale (4), 100% donor chimerism, 14 (56%) had stable mixed chimerism, K. Kalwak (5), I. Kondratenko (6), G. Notheis (1), M. Führer (1), 7 (28%) required boost infusions within 12 months of HSCT for B.H. Belohradsky (1) falling chimerism. 16/25 are >1yr post HSCT; all have normal T (1)Dr. von Haunersches Kinderspital der LMU (Munich, DE); lymphocyte function, 12/16 are IVIG independent, with normal (2)Newcastle General Hospital (Newcastle, UK); (3)Zentrum vaccine antigen responses. fuer Kinder und Jugendmedizin (Heidelberg, DE); (4)Great These results are excellent with overall survival compara- Ormond Street Hospital (London, UK); (5)Pediatric Hematology/ ble with current European data. IR was good, with surviving Oncology (Wroclaw, PL); (6)Russia Clinical Children›s Hospital patients cured of underlying immunodefi ciency. Viral re-activa- (Moscow, RU) tion is a recognised complication of RIC HSCT: PCR surveil- lance and prompt pre-emptive treatment with anti-viral agents Nijmegen breakage syndrome (NBS) is a autosomal recessive should reduce mortality. Late aGvHD remains a problem, disorder characterized by immunodefi ciency, characteristic although in most patients it was ≤ grade II. RIC has a role in facial appearance, chromosomal instability, X-ray hypersen- successful HSCT of the sickest PID patients; viral reactivation sitivity, and predisposition to malignancy. Patients harbour and GvHD remain risks. FU studies are required to determine mutations in NBS1 which encodes for nibrin, a protein critically long-term outcome. important in DNA double strand repair. About 40% of patients develop malignancy mostly of lymphoid origin before age 21. Traditionally NBS patients have not undergone HSCT owing to P684 concerns about chromosomal instability and increased toxicity. Haematopoietic stem cell transplantation for STAT3 Only 2 cases have been reported, both included in this series. defi ciency We therefore attempted to summarize the limited transplant A. Gennery (1), M. Slatter (1), D. Barge (1), C. Woellner (2), experience in NBS patients in Europe. B. Grimbacher (2), S. Jolles (3), H. Harvey (1), T. Flood (1), A total of six patients could be included. All carried homozygous A. Cant (1), M. Abinun (1) NBS1 mutations. Median age at diagnosis was 7.6 years (1)Newcastle upon Tyne Hospitals NHS Foundation Trust (range 1-18) and 14.5 years (2.3-20.3) at transplant. The rea- (Newcastle upon Tyne, UK); (2)University College London son to attempt SCT was resistant or secondary malignancy in (London, UK); (3)University Hospital of Wales (Cardiff, UK) four cases. The other two were transplanted because of severe immunodefi ciency or suspected Fanconi anaemia (FA) with Mutations in the signal transducer and activator of transcription immunodefi ciency. All had a T-cell defect and 4/6 had defi cient 3 (STAT3), a key protein in the signal transduction pathway of a humoral immunity before transplant. As expected the con- broad range of cytokines, are found in patients with autosomal ditioning regimes varied widely, however 5 patients received

S181 reduced intensity regimens with in vivo T-cell depleting agents. P687 The donors were matched family members in 3 patients, one Neonatal pancytopenia and severe hepatic failure haploidentical parent and two matched unrelated donors. revealing an intrauterine twin-to-twin haematopoetic stem At a median follow up of 2.2 years (1.6-8.1) fi ve of six patients cell transfer are alive and well. Three patients had no relevant transplant D. Moshous (1), I. Radford Weiss (1), S. Caillat-Zucman (1), related complications. Three had severe mucositis and infec- N. Brousse (1), M.-D. Dumont (1), C. Picard (1), O. Ackermann tious complications, one of whom died on day +5 from sepsis. (2), M. Fabre (2), F. Méchinaud (3), A. Fischer (1) The latter one was the patient who had received BMT from a (1)Hôpital Necker-Enfants Malades (Paris, FR); (2)CHU de matched sibling after a myeloablative, busulfan containing Bicêtre, Le Kremlin Bicêtre (Paris, FR); (3)CHU Nantes (Paris, conditioning regimen. Acute GVHD grade I-II occurred in 3/5 FR) patients, mild chronic GVHD in one. Four patients are either complete or nearly complete donor chimeras, one has mixed Patient: We report on a male twin born at a gestational age of chimerism, all fi ve exhibit restored T-cell immunity. Four patients 38+3. At day 6 of life, the patient presented fever, abdominal have normal humoral immunity and one remains on IVIG at 1.6 symptoms and pancytopenia requiring repeated platelet and years post transplant. red blood cell transfusions. Neutropenia persisted despite G- Although very limited, the experience in these six patients sug- CSF. Several myelograms showed empty bone marrow with gests that SCT in NBS is feasible, can correct the immunode- exception of lymphoid cells, also found in the peripheral blood fi ciency and effectively treat malignancy. Acute toxicity seems being exclusively CD4+ without signs of activation. The patient to be reasonable with reduced intensity conditioning regimens. showed hepatomegaly, severe hyperbilirubinaemia, cutaneous However, valid concerns remain about increasing the risk for eruption and repeated gastrointestinal bleedings. Digestive subsequent malignancy through cytotoxic agents in these endoscopy at 2,5 months of age evidenced gastritis and subto- patients. tal villous atrophy of the duodenum. Liver biopsy could not be performed initially because of profound thrombocytopenia. Evolution: At about two months of age, neutrophils appeared, P686 as did eosinophils and monocytes. HLA typing on peripheral Haematopoietic stem cell transplantation in Griscelli blood revealed the presence of three alleles. Fluorescence in syndrome type 2: a single-centre report of 10 patients situ hybridisation found 100% XY cells in the peripheral blood, J. Pachlopnik Schmid (1), D. Moshous (2), M. Cavazzana-Calvo thus ruling out materno-fetal GVHD. The origin of the third allele (3), S. Blanche (2), G. de Saint Basile (1), A. Fischer (1) was compatible with the presence of cells of his twin brother. (1)INSERM U768 (Paris, FR); (2)Unite d’Immunologie et HLA typing on sorted cells revealed that the CD3 positive cells Hematologie Pediatrique (Paris, FR); (3)Departement de belonged to both twin brothers, whereas the CD3 negative cells Biotherapie (Paris, FR) were predominantly of “donor”-origin. At about three months of age, reticulocytes were present and the patient’s blood group Griscelli syndrome type 2 is a rare immune disorder causing changed into the group of his twin brother. At the age of 10 potentially fatal hemophagocytic lymphohistiocytosis (HLH). months platelet counts were > 100.000/µl. Allogeneic hematopoietic stem cell transplantation (HSCT) is Severe liver damage led to hepatocellular failure requiring an the only curative treatment. Only few cases of successful HSCT urgent liver transplantation at 7 months of age. Histological have been described and optimal modalities are not yet well fi ndings of the explanted liver are compatible with GVHD driven known. We retrospectively analyzed the outcome of HSCT liver destruction, HLA typing on liver tissue confi rmed the pres- that was performed in 10 consecutive patients with Griscelli ence of alleles of the twin brother. syndrome type 2 who were treated in a single center between Discussion: Maternal microchimerism can be detected quite 1996 and 2008. Seven patients (70%) survived and were cured frequently in healthy individuals, but it is usually not associated from the primary immune defect with a median follow-up of with disease. Materno-fetal GVHD is observed in immunodefi - 3.4 years (147 days – 11 years), death occurred in 3 patients cient children. Chimerism involving all peripheral blood cell sub- within 110 days after HSCT. One patient received 2 transplants sets has been frequently described in healthy chimeric twins, because of graft failure that had led to HLH relapse. Grade II- who usually present immunological tolerance as evidenced by III acute and chronic graft-versus-host disease occurred after mutual absence of alloreactivity in mixed lymphocyte culture. 7 and 1 transplants, respectively. Veno-occlusive disease was To our knowledge, we report the fi rst patient with feto-fetal cell observed after 5 transplants. Two patients experienced EBV- transfer presenting deleterious consequences due to intrauter- induced lymphoproliferative disease after HSCT. Out of seven ine GVHD. The question whether the cell-transfer occurred on patients with neurological involvement prior to HSCT, four sur- the basis of a primary immunodefi ciency in the recipient is cur- vived and two presented neurological long-term sequelae. Fur- rently under investigation. thermore, one patient without neurological involvement prior to HSCT developed long-term neurological sequelae. Taken together, this survey demonstrates effi cacy of HSCT to cure P688 the immune disorder of Griscelli syndrome type 2. It shows that Bone marrow transplantation in adult cerebral X-linked neurological involvement, which is common in these patients adrenoleukodystrophy: an update prior to HSCT, is, however, a major concern, also after suc- J.-S. Kühl (1), W. Köhler (2), O. Bandmann (3), S. Buchholz (4), cessful HSCT. Therapy-associated immunosuppression might P. Hemmati (1), J. Snowden (3), P. Sokolowski (2), M. Stadler contribute to the emergence of EBV-induced lymphoprolifera- (4), T. Terwey (1), R. Arnold (1) tive disease. Additional studies are required to improve the (1)Charite Campus Virchow Klinikum (Berlin, DE); transplantation procedure and reduce its toxic effects. It is likely (2)Fachkrankenhaus Hubertusburg (Wermsdorf, DE); that preemptive HSCT, i.e. before the occurrence of HLH is to (3)Royal Hallamshire Hospital (Sheffi eld, UK); (4)Medizinische be favorized. Hochschule Hannover (Hannover, DE)

Background: The acute infl ammatory cerebral form of X-linked adrenoleukodystrophy in adult men (ACALD) is as devastating as in childhood. While HSCT is an accepted long-term treat- ment method in boys with cerebral disease (C. Peters, Blood, 2004), there are only anecdotal reports in men. Based on neu- rologic criteria, we defi ned a group of ACALD patients who were eligible for HSCT on a compassionate use basis in order to prevent complete dementia.

S182 Patients: Since 01/2006, 4 men with ACALD (age: 33-48 years) delta T-cells due to the same hypomorphic del368/369AA muta- secondary to adrenomyeloneuropathy (AMN) were trans- tion. There were 3 girls and 7 boys aged from 1 to 48 months planted from a 10/10-HLA-identical sibling (n=2) or unrelated (median 9 months), who underwent primarily haploidentical donor (n=2). Pts. were able to walk (Expanded Disability Status CD34±enriched T-depleted PBSCT (n=6), (m)MUD-PBSCT Score (EDSS) <7.0), had no major bladder dysfunction and no (n=3) or MSD-BMT (n=1). Conditioning regimens were myelo- severe impairment of cortical functions incompatible with nor- ablative, incl. Bu or Treo ± Cy ±Flu ±Thiotepa except for one mal daily life. Infl ammatory CNS disease was demonstrated by pt. given RIC. Two patients with hypomorphic mutations result- contrast enhancement in MRI. The preparative regimen con- ing in two different phenotypes (Omenn and atypical SCID with sisted of oral busulfan (4 x 4 mg/kg), cyclophosphamide (2 x 60 gamma delta T-cells) rejected haploidentical T-cell depleted mg/kg), and ATG. PBSCT and were successfully retrasplanted from 7/10 HLA- Results: At a minimum follow-up of 9 months all 4 pts. were allele-mismatched unrelated donor (UD) in one case and from alive after BMT. All patients engrafted well demonstrating a fully matched UD in another case. The pt with atypical SCID complete or predominant donor chimerism. 3 pts. had no unex- was conditioned with RIC, incl. Flu, Mel, Campath-1H, the pt pected toxicities, only bacterial infections in neutropenia (2x II°, with Omenn S. was given Bu, Flu, TT, OKT-3 and ATG. Another 1x III°), and no GvHD. Pt. #2, however, developed high fever, pt with asymptomatic Omenn S. given only Bu, Cy developed hypotension and an epileptic seizure under ATG. Furthermore, Omenn-like symptoms post haplotransplant incl. commencing he had an acute renal failure and was on long-term artifi cial rejection with autologous T cells, but was successfully recondi- ventilation. Only this pt. experienced limited acute and chronic tioned with OKT-3, steroids. GvHD. The neurologic outcome of all pts. is summarized in Three pts died early posttransplant due to sepsis (n=2) or CMV the table below: In 3 of 4 pts., CNS infl ammation was stopped infection (n=1). Remaining 7 children, incl. both with atypical within 6 months post BMT. In these pts. a complete loss of intel- SCID are alive and well from 3 to 111 months posttransplant lectual functions could be prevented after an initial phase of (median 38 months). mild to moderate deterioration. Their loss of motor function post In conclusion: Haploidentical T-depleted PBSCT in patients BMT correlated with their initial impairment. In contrast, the pt. with hypomorphic RAG mutations remains associated with #2 with GvHD had evidence of persistent cerebral infl amma- increased risk of graft rejection due to partial autologous T-cell tion. Despite a favourable baseline status, the pt. showed an function. Pretransplant immunosuppressive therapy should unexpected degree of deterioration both in motor as well as in be performed in all symptomatic pts. We strongly recommend cerebral function. myeloablative conditioning regimens for this specifi c cohort of Conclusion: These preliminary results indicate: 1) The above pts, incl. Bu, Flu, TT, OKT-3/ATG in haploidentical setting and approach seems to be safe and feasible for ACALD pts. 2) Pre- TREO, CY/FLU ±ATG in MUD/MSD-setting. vention of GvHD and severe tx complications is crucial for halt- ing CNS infl ammation as early as possible. 3) Progression of advanced neuropathy appears to be poorly infl uenced by BMT. P690 Until more data are available HSCT should only be offered in Targeting to an optimal AUC of intravenous busulfan experienced centers to carefully chosen pts. prevents graft failure in transplantation in children with non-malignant diseases I.H. Bartelink (1), R.G.M. Bredius (2), S.V. Belitser (3), M. Bierings (1), C.A.J. Knibbe (4), M. Egeler (2), A.C. Lankester (2), A.C.G. Egberts (1), J. Zwaveling (2), J.J. Boelens (1) (1)UMCU (Utrecht, NL); (2)LUMC (Leiden, NL); (3)UU (Utrecht, NL); (4)LACDR (Leiden, NL)

Background: Busulfan combined with therapeutic drug moni- toring guided dosing is associated with higher event free sur- vival (EFS) rates due to less graft-failure/relapses and lower toxicity in haematological stem cell transplantation (HSCT). In an earlier study, our group showed an optimal AUC between P689 74-82 mg*h/L in a group of children with malignant and nonma- How to condition patients with Omenn syndrome or lignant indications. Nonmalignant diseases, especially inborn atypical SCID with gamma delta T-cell predominance? error of metabolism (IEM) are known to be associated with high Single-centre experience on 10 patients graft-failure rates. It is not known whether there is an optimal K. Kalwak (1), E. Gorczynska (1), M. Ussowicz (1), target AUC in nonmalignant diseases. Historically intravenous J. Owoc-Lempach (1), A. Dyla (1), D. Pazdzior (1), J. Musial busulfan has been used in the two Dutch pediatric stem cell (1), M. Skarznska (1), D. Wójcik (2), J. Porwolik (1), M. Van der transplantation centers in various myelo-ablative regimens with Burg (3), K. Schwarz (4), A. Chybicka (1) different targets for the area under the curve (AUC) and differ- (1)Wroclaw Medical University (Wroclaw, PL); (2)Haukeland ent dosing regimens of busulfan. We retrospectively analyzed Medical University (Bergen, NO); (3)Erasmus Medical Center the association between busulfan AUC and clinical outcome. (Rotterdam, NL); (4)University of Ulm (Ulm, DE) Methods: All children, transplanted between 2001-2008, receiv- ing intravenous busulfan as part of a myeloablative regimen Hypomorphic mutations of the RAG1/2 genes may generate for non-malignant indications, were included. The association different phenotypes, characterized by residual T±B-cell differ- between an AUC below or above the optimum of 74mg*h/L and entiation. They impair but do not abolish the V(D)J recombi- the endpoints overall survival (OS), treatment related mortal- nation process and are responsible for the Omenn phenotype ity (TRM), event free survival (EFS) and toxicity (acute-Graft- or atypical SCID with gamma delta T-cell predominance. It versus-Host Disease grade 2-4 (aGvHD) and Veno-occlusive remains controversial, which conditioning regimen is optimal Disease (VOD), were tested using uni- and multivariable Cox for this specifi c cohort of immunodefi cient children with residual regression analysis. T cell function. Another question is, whether these pts have to Results: 28 patients with IEM, 36 with immune defi encies and 5 be given conditioning in matched sibling donor (MSD) setting, patients with bone marrow failures were included, with a median or not. follow up time of 2.5 years. EFS was negatively infl uenced by Here we report on ten patients: eight pts with either symptomatic Graft-failure below the lower limit of the AUC (18% in patients or asymptomatic Omenn Syndrome due to del368/369AA hypo- receiving <74mg*h/L, vs 0% in patients receiving > 74mg*h/L, morphic mutation (n=3), Artemis mutation (n=1) or unknown HR=0.2, P=0.016). OS was similar in both groups (OS 20% mutation (n=4) and two pts with atypical SCID with gamma vs 19%). All but one patients died due to TRM. EFS was 72%

S183 vs 82% (not signifi cantly different). Acute graft-versus-host dis- with bone marrow failure syndrome (BMF). An alternative donor ease (aGvHD) grade 2-4 did occur more frequently in patients is required in patients who do not have a matched related fam- receiving a high exposure of busulfan (5% vs 20%), but this ily donor. was not signifi cantly different. VOD was seen in 13% vs 17% of In this study we report our experience over ten years. the two AUC-groups. Patients and Method: Between August 1998 and October 2008, Conclusion: Dose targeting of busulfan to a high therapeutic 31 patients with BMF underwent transplantation from 22 related range decreases the amount of graft failures in patients with and 9 unrelated donor. Patient group consists of 17 Fanconi non-malignant diseases, without compromising the treatment aplastic anemia (FAA), 8 aplastic anemia (AA), 2 amegakaryo- due to treatment related mortality. cytic thrombocytopenia, 2 Kostman syndrome, 1 Diamond Blackfan anemia, 1 Diskeratosis conjenita. The median age was 10 year (range 1-17) and 21 were male. All patients with P691 anemia were transfusion dependant. Bone marrow, periph- Allogeneic stem cell transplantation in Farber’s disease eral blood and cord blood were used as stem-cells source in with bone involvement: long-lasting and substantial patients, 4, 24, 3 respectively. improvement We have used four different conditioning regimen. Fludarab- A. Jarisch, J. Soerensen, L. Porto, M. Kieslich, T. Klingebiel, ine based regimen or CY-ATG with thoraco-abdominal irradia- P. Bader tion (TAI) regimen for FAA patients, CY+ATG regimen for AA Goethe University Frankfurt (Frankfurt, DE) patients and Busulfan+CY for DBA and Kostman syndrome were performed. Graft versus host disease (GVHD) prophy- Introduction: Farber disease (FD) is a rare, lysosomal storage laxis was cyclosporine alone for FAA patients (MMF for two disorder caused by a defi ciency of acid ceramidase and sub- FAA patients) and methotrexate plus cyclosporine A for other sequent accumulation of ceramide in various organs and tis- patients. Neutrophil and platelet engraftment occurred in all sues. Clinical features are characterized by subcutaneous skin patients on day 12 (8-66) and day 17 (15-49), respectively. nodules mostly near the joints leading to stiffness, contractures Post-transplant graft failure was occurred in two patients. Only and deformation and progressive hoarseness. 7 different phe- one patient succumbed because of transplantation related notypes are reported due to additional organ involvement, like problem in fi rst 100 days. Acute GVHD and chronic GVHD were the lungs, nervous system, heart and lymph nodes. Patients found in 7 patients (6 patients grade II-III, one patient grade IV) with neurological involvement mostly die in early childhood and one patient, respectively. Overall and disease free survival whereas in other phenotypes mostly respiratory insuffi ciency were found 81%. leads to death in the second decades of live. Allogeneic hemat- Conclusion: We conclude that SCT the only curative treatment opoietic stem cell transplantation (HSCT) may offer the only modality for BMF patients. Fludarabine based regimen was curative treatment for FD patients and only few patients are found more tolerable by patients and successful than regimen transplanted so far. with TAI in FAA patients. Transfusion related complications and Case report: We present a patient with FD type 3. She showed severe infections that worsens post-transplant outcome should typical features like hoarseness, failure to thrive and multi- be considered for each patient. The planning of expedited SCT ple subcutaneous skin nodules. HSCT was discussed but no might be more cost-effective and feasible before occurrence of matching donor was found. At the age of 9 years she presented complications for BMF patients. with contractures and joint deformations restricting her to wheelchair. In cranial MRI no neurological manifestation was seen but a P693 nearly complete destruction of dens axis with spinal compres- Successful allogeneic peripheral blood stem cell sion due to bone involvement of the underlying disease proven transplantation from an HLA-identical sibling in a by biopsy. The patient was in danger to develop a tetraplegia patient with mitochondrial neurogastrointestinal and at this time a matched unrelated donor could be identifi ed. encephalomyelopathy She underwent a myeloablative matched unrelated SCT. Subcu- K.S. Hill, D. Richardson, S. Hammans, M. Stroud, D. Fine, taneous skin nodules resolved completely after 3 months. The V. Walker, J. Newman, N. McKeag, C. Hurlock, K. Orchard acid ceramidase activity in white cells was 0.28 (range 0.33- Southampton University Hospital (Southampton, UK) 2.42) and b-galactosidase activity was normal. The MRI showed an exceptionally improvement of bone structure and density of Background: Mitochondrial neurogastrointestinal encephalo- dens axis without further stenosis and spinal compression. myelopathy (MNGIE) is an autosomal recessive condition char- Although treated with a myeloablative conditioning regimen the acterised by gastrointestinal (GI) dysmotility, cachexia, ptosis, patient remained mixed chimeria with increasing autologous ophthalmoplegia, demyelinating peripheral neuropathy and cells. Several donor lymphocyte infusions (n=5) were given, leucoencephalopathy. The genetic defect resides in the TYMP the patient complete rejected the graft 2 year post transplant gene (previously ECGF1) encoding thymidine phosphorylase and became aplastic. A second SCT from her haploidentical (TP) resulting in marked reduction of TP activity in all tissues. father was performed. 21 month post 2nd transplant she is well, This results in marked increases in plasma thymidine (Thd) and complete donor chimerism, is able to walk and is showing a deoxyuridine (Urd). The altered mitochondrial (Mt) nucleotide continuously improvement of all physical abilities. pool leads to mutations, depletion and deletions of MtDNA. Conclusion: We report the fi rst patient with proven bone It has been postulated that reductions in Thd and Urd levels involvement of Farber disease. In our patient HSCT improves could be therapeutic in MNGIE. It has been reported that allo- the manifestation of FD even though SCT was done at the age geneic stem cell transplant (SCT) can correct the biochemical of 9 years. derangements in MNGIE. Patient: A 41-year-old woman presented at age 18 with anorexia and vomiting and developed ptosis and ophthalmoplegia 10 years P692 later. Cerebral MRI revealed leucoencephalopathy and electro- Haematopoietic stem cell transplantation in children with myography (EMG) was suggestive of a demyelinating neuropa- bone marrow failure: a single-centre experience thy. MNGIE was confi rmed genetically by age 33. By age 40 she A. Kupesiz, G. Tezcan Karasu, V. Uygun, P. Kurt, Z. Ozturk, was wheelchair bound and despite long-term percutaneous endo- M. Kazik, V. Hazar, A. Yesilipek scopic gastrostomy (PEG) and total parenteral nutrition (TPN) Akdeniz University Faculty of Medicine (Antalya, TR) feeding was signifi cantly underweight at 28.5kg (height 163cm; body mass index 10.7kg/m²) and a Karnofsky score of 40%. Background: Stem cell transplantation (SCT) is the only treat- Methods: A peripheral blood SCT from an HLA-identical sis- ment that defi nitively restores normal hematopoiesis in patients ter (with normal TP activity) was performed in September

S184 2007. Conditioning consisted of 6.4mg/kg Busilvex, 150mg/m² lymphoma. He received 10/10 HLA MUD bone marrow infusion. Fludarabine and 30mg Alemtuzumab with cyclosporin and 3- On D+14 he had 422 T cells/ul and developed pneumonitis. He day methotrexate as GVHD prophylaxis. The CD34+ cell dose received one dose of vinblastine, methylprednisolone and anti- infused was 12.1x106/kg. TNFa. He improved and is now well ≥ aged 10 years. Results: Neutrophil and platelet engraftment was achieved by A 5 month child with PCP had a large soft tissue lesion on the day +15. By day 25 her TP activity in white cells and plate- left forehead, enlarging over 2 months. He was lymphopenic lets were within normal ranges and full donor chimerism was with hypogammaglobulinaemia. He had a c.676C>T mutation achieved by day 28. These levels have been sustained at 14 in the CgC gene. Initially neurologically normal, he became less months post SCT. Complications included idiopathic throm- alert, with abnormal eye movements. CSF protein was raised bocytopenic purpura successfully treated with Rituximab, GI at 4.52g. The lesion showed bone erosion, thickened, infi ltrated haemorrhage managed conservatively and adenovirus infec- dura, but no cerebral infi ltration, with a small extra- and larger tion. She has had no evidence of GHVD. Recent abdominal intra-cranial component. Histology revealed pleomorphic spin- MRI shows persistent GI pseudo-obstruction but her oral intake dle shaped cells surrounding a central area of necrosis with has improved and her weight has increased to 32.3kg. Perform- peripheral reactive lymphoid cells and foamy histiocytes with ance status, repeat EMG and cerebral MRI are unchanged. no osteoid production, diagnosed as malignant fi brous histiocy- Conclusion: Allogeneic SCT can be delivered successfully to toma, storiform-pleomorphic variant. Chest CT showed bilateral patients with MNGIE with sustained full donor chimerism and infi ltrates. Despite 10/10 HLA matched maternal bone marrow normal TP activity. Despite symptomatic improvement in the infusion, he died on D+ 8 from pulmonary haemorrhage. Pul- patient signifi cant physical improvement may take more time. monary and hepatic malignant fi brous histiocytoma were identi- fi ed at necroscopy. Immunodefi ciency-associated malignancy is usually secondary to EBV-associated B lymphocyte proliferation: these tumours were unusual and not previously reported in CgC SCID.

P695 Haematopoietic stem cell transplantation for DNA-DSB repair defects using a modifi ed Fanconi anaemia conditioning regimen M. Slatter (1), D. Barge (1), P. Jeggo (2), H. Harvey (1), T. Flood (1), A. Cant (1), M. Abinun (1), A. Gennery (1) (1)Newcastle upon Tyne Hospitals NHS Foundation Trust (Newcastle upon Tyne, UK); (2)University of Sussex (Brighton, UK)

Normal lymphocyte receptor generation requires functional pro- teins in the non-homologous end-joining pathway. DNA ligase IV (LIG4) and Cernunnos-XRCC4-like factor (C-XLF) are key components required for the ligation reaction that forms lym- phocyte receptors, but are more widely required for DNA-dou- ble strand break repair. Defi ciency in these enzymes confers radiosensitivity and immunodefi ciency. Patients identifi ed with SCID or combined immunodefi ciencies due to LIG4 or C-XLF defi ciency are increasingly being identifi ed and offered HSCT. P694 Many of the conditioning regimens damage DNA and patients Malignancy as a presentation of common gamma chain with these disorders die from conditioning toxicity; less toxic defi cient severe combined immunodefi ciency regimens are required to successfully treat these patients. We M. Slatter (1), K. Windebank (1), B. Angus (1), A. Barborie report 2 patients successfully treated using a modifi ed Fan- (1), A. Perry (1), T. Lester (2), G. Norbury (2), M. Abinun (1), coni anaemia protocol. Patient 1 presented with microcephaly, T. Flood (1), A. Cant (1), A. Gennery (1) developmental delay, severe lymphopenia and hypogamma- (1)Newcastle upon Tyne Hospitals NHS Foundation Trust globulinaemia, recurrent respiratory and CMV vireamia. He (Newcastle upon Tyne, UK); (2)Great Ormond Street Hospital had a homozygous C-XLF mutation (c.169C>T, p.R57X). She (London, UK) underwent 9/10 (Amm) HLA-mis-matched URD HSCT after conditioning with Flu 150mgm2/Cy20mg/kg and C-1H 1mg/kg, A 3 mth boy with diarrhoea and Parainfl uenza 3 respiratory aged 3.5 years. CSA and MMF were used as GvHD prophy- infection was hypogammaglobulinaemic with CgC SCID due to laxis. Initial donor chimerism was 25%, but by 2 months post IVS3-11bp C>G eleven bases into the CgC gene intron 3 splice HSCT was 100%. The transplant course was complicated by acceptor site, in both patient and mother, who has non-random grade 2 lichenoid skin GvHD and CMV reactivation. There was T lymphocyte X-inactivation. He developed hepatomegaly, no conditioning-related toxicity and immunoreconstitution at 4 pyrexia and erythematous axillary induration. Hepatosplenic months is good although follow up is short. Patient 2 presented lesions were present and a circumscribed lytic rib lesion on CT with failure to thrive, persistent diarrhoea, recurrent resoira- scan. Bone marrow examination revealed no lymphocytes or tory infection and severe lymphopenia with hypogammaglob- malignancy. Axillary nodule histology demonstrated granuloma- ulinaemia. She had 2 heomozygous LIG4 mutations (p.A3V, tous infi ltrate of large uni or binucleated cells containing promi- p.R278H). She underwent a 9/10 (Amm) HLA mis-matched nent nucleoli and abundant eosinophilic cytoplasm confi rmed to parental HSCT after conditioning with Flu 150mgm2/Cy20mg/ be of T cell origin with some B cell infi ltrate, resembling Reed- kg and C-1H 1mg/kg, aged 4.5 years. CSA and MMF were Sternberg cells. Ki67 staining showed marked proliferative activ- used as GvHD prophylaxis. Initial donor chimerism was 12%, ity. There was no evidence of EBV. EMA and p80 markers were but by 6 weeks post HSCT was 100% T cell and 85% myeloid. negative. Rib biopsy demonstrated extensive reactive small The transplant course was complicated by CMV reactivation. lymphocyte infi ltrate with pleomorphic blast-type large CD30+, There was no conditioning-related toxicity. Longer follow up leucocyte common antigen negative cells with large vesicular is required to demonstrate long term immunoreconstitution. nuclei, suggestive of a Ki1+ lymphoma, described as a large These cases, in addition to our previously patient transplanted cell anaplastic lymphoma or poorly differentiated Hodgkins with Nijmegen Breakage Syndrome1, demonstrate safety and

S185 effi cacy of this conditioning protocol. Long term studies will be treated according HLH-2004 protocol and at 2 years of age he required to assess the longer term outcome. had transplantation from UCB after receiving conditioning con- 1. Gennery et al. J Ped Haem Oncol 2005;27:239 sisted of Bu,Eto and CYM. GVHD prophylaxis was consisted of cyclosporine. The infused NC was 12,6X107/kg. Neutrophil engraftment occured at 33th day after transplantation. He P696 developed intestinal GVHD treated with steroid, tacrolimus and Haematopoietic stem cell transplantation for cerebral MMF.16 months after transplant autoimmune hemolytic anemia X-linked adrenoleucodystrophy in adult (AHA) developed that was treated with steroid and MMF.19 E. Paubelle (1), F. Suarez (1), D. Ghez (1), N. Maillard (1), monthts after HSCT, the patient is well, is not receiving any M. Rubio (1), R. Delarue (1), I. Hirsch (1), B. Varet (1), immunsupression and AHA recovered. Case 2:18 months old C. Bellesmes (2), P. Aubourg (2), A. Buzyn (1) with GS and HLH had transplantation from UCB. The condi- (1)Hopital Necker-Enfants Malades (Paris, FR); (2)Hopital Saint tioning and GVHD prophylaxis was same as other patients’. Vincent de Paul (Paris, FR) NC count was 10,5X107/kg. Neutrophil engraftment occured at posttransplant day 22.At 25th day, AHA developed. Improve- X-linked adrenoleukodystrophy (ALD) is a rapidly progressive ment could not be achieved by steroid and plasmaphresis. neurodegenerative disorder that affects the central nervous Finally rituximab was used and AHA recovered. 2 years after system (CNS) and the adrenal cortex. It is caused by mutations transplant, the child is well. Discussion: Allo-HSCT is the only of the ABCD1 gene, that result in an accumulation of saturated curative treatment for GS. If family donor is not avaliable, unre- very long chain fatty acids in all tissues. lated donor should be searched. Although papers indicating ALD presents with two main phenotypes: the childhood cerebral successfull treatment of GS with unrelated HSCT is available form with onset between 5-7 years that leads rapidly to demise in the literature, the experience is very limited and for all we of the CNS and ends in dementia then death; the adult form, know transplantation with UCB has not been published yet. The called adrenomyeloneuropathy (AMN), with onset between cases described here are the fi rst published GS cases treated 20-40 years, characterized by progressive spastic paraparesis successfully with UCB transplantation. Besides this, developing with chronic course. However 35% of patients with AMN may of AHA at postransplant period in both cases are remarkable. develop a cerebral demyelinating disease whose evolution is It has been suggested in the literature that immunsupressive similar to the childhood form. Paediatric series show that hae- therapy given in posttransplant period can create immun dys- matopoietic stem cell transplantation (HSCT) is effective in regulation and this may lead to autoimmune cytopenias. In early symptomatic boys with cerebral ALD, by restoring partial patients with immune dysregulation like Griselli syndrome or enzymatic activity. HLH, the risk may be higher and it may be benefi cial to follow We report for the fi rst time 3 cases of HSCT performed in adult up these patients during posttransplantation period in respect patients (15 to 28 years). All three had infl ammatory cerebral to AHA. The role of CB transplantation in treatment and whether demyelination that developed after AMN. One had only electro- having CB transplantation increase the risk for AHAin this group physiological signs of AMN without clinical symptoms. of patients should be evaluated in larger series. Two patients received myeloablative conditioning regimen including busulfan, cyclophosphamide and antithymocyte glob- ulin and one a reduced intensity conditioning regiment (RIC) P698 aimed at decreasing neurological toxicity (clofarabine 40 mg/ Graft failure in major histocompatibility complex class II m2, alemtuzumab, busulfan 4 mg/kg, and melphalan 140 mg/ defi ciency kg). The source of the graft was bone marrow (one from an C. Forino (1), D. De Martiis (1), R. Marzollo (1), S. Cavagnini match unrelated donor and one from a sibling donor) and a (1), A. Lanfranchi (1), E. Mazzolari (1), R.M. Lemoli (2), cord blood for the patient receiving a RIC. The latter patient L.D. Notarangelo (3), F. Porta (1) died because of infectious complications after engraftment fail- (1)Spedali Civili (Brescia, IT); (2)Policlinico S.Orsola (Bologna, ure. The 2 remaining patients developed a grade 1 and grade 2 IT); (3)Children Hospital (Boston, US) acute graft versus host disease (GVHD) and no chronic GVHD. As seen in transplanted ALD boys, cerebral demyelination pro- We describe the case of Sabrine, affected by MHC Class II gressed during the fi rst 18 months after HCT then stabilized Defi ciency. during the following 5 years after HCT in strictly normal cogni- Parents were related and came from North Africa; both their tive function in one patient and moderate visuo-spatial defi cit previous children died in the fi rst two years of a severe respi- in the second. Spastic paraparesis deteriorated however sig- ratory distress. The latter performed immunological evaluation nifi cantly during the conditioning regimen in the second patient before dying, showing a low lymphocytes DR+ count. and then stabilized. Cordocentesis performed during Sabrine pregnancy showed In ALD adult patients, HSCT seems as effective as in children lymphocyte DR+ absence, while molecular studies demon- to stop demyelination but with a risk of aggravation of the mye- strated homozygotic mutation of CIITA gene (IVS13+1g>a). lopathy. Progresses have to be made to determine a less neu- The deliver was uneventful and at birth Sabrine went in isola- rotoxical conditioning regimen. tion under Laminar Air Flow. At fi rst clinical examination the child was eupnoic with normal cardio-respiratory activity. WBC were 12.080/mm3 (L 9340/ P697 mm3), Hb 10.6 gr/dl, PLT 375.000/mm3. Lymphocyte pheno- Successful treatment of Griscelli syndrome with unrelated type showed normal CD19 and CD3 level (low TCD4 and high cord blood transplantation TCD8) and absent DR+ cells. Genetic diagnosis was confi rmed M. Kazik, G. Karasu, V. Uygun, A. Kupesiz, V. Hazar, and the research for a Matched Unrelated Donor (MUD) was A. Yesilipek started, not being available a HLA-identical sibling. Owing the Akdeniz University (Antalya, TR) absence of a MUD, the option of a haploidentical Bone Marrow Transplantation (BMT) was therefore considered. Griscelli syndrome (GS) is rare genetic disorder characterized Experiences in other centers showed an elevated risk of Graft by partial albinism, neurological manifestations and immuno- versus Host Disease (GvHD) following this procedure, prob- defi ciency. In most patients with immunodefi ciency accelerated ably due to an incorrect negative/positive intrathymic selec- phase known as hemophagocytic lymophohistiocytosis (HLH) tion of TCD4. To avoid this, the infusion of maternal T depleted develop. Allogenic transplantation (HSCT) in early period is the CD34 infusion (19.1 x 106/kg), was followed, after 2 wks, by only curative treatment modality. We describe here two cases three infusion of immature donor dendritic cells (10 x 106/kg), with GS cured with unrelated cord blood (UCB) transplantation. who could eventually migrate in the thymus to perform T cell Case 1:3,5 months old boy diagnosed as GS and HLH was selection.

S186 Immune reconstitution was slow with weak DR+ expression; P700 engraftment on D+30 was 52% donor on lymphocytes and 73% Evidence for genomic stability of bone marrow-derived on granulocytes. No laboratory or clinical signs of GvHD were MSCS after large-scale expansion in a humanised system ever seen. Sabrine was dismissed on D+49, in good clinical K. Schallmoser, E. Rohde, A. Reinisch, A. Obenauf, conditions with prophylactic therapy. Unfortunately, her DR+ C. Bartmann, G. Lanzer, W. Linkesch, D. Strunk cells count progressively decreases to become nearly undetec- Medical University of Graz (Graz, AT) table 4 months after BMT, while donor chimerism became 0% (CD14, CD19, CD3), showing total graft rejection. Human multipotent mesenchymal stromal cells (MSCs) are cur- Considering the excellent clinical condition and the high risk for rently tested in a growing number of clinical trials to determine severe GvHD after a second haplo-BMT, we decided to wait their safety and effi ciency for immune modulation and regen- and postpone other therapeutic options. Even with virtually erative therapy. Repeated observations of genomic instability absent immunity, Sabrine during last year of follow-up showed in commonly used fetal bovine serum (FBS)-driven MSC cul- no major infectious morbidity and satisfactory growth. tures and consecutive tumor formation by transformed MSCs in This case confi rms that MHC-II Defi ciency is a subtype of SCID experimental animals have raised serious safety concerns. We still very diffi cult to transplant, probably owing to congenital lack and others have recently established animal serum-free clini- in thymic selection. cal scale MSC expansion protocols with pooled human platelet lysate (pHPL) as a substitute for FBS. This study was per- formed to determine the genomic stability of MSCs expanded under humanized conditions ex vivo. Small volume (14-17mL) bone marrow aspirates of 4 donors Cellular and gene therapies (3 male; age: 30, 36, and 47 years; 1 female; age 13 years) were seeded without manipulation directly in pHPL and L- glutamine supplemented minimum essential medium. Serial P699 limited dilutions were performed to analyze the frequency of JAK2-V617F-triggered pre-emptive and salvage colony-forming MSCs within marrow samples. Colonies con- adoptive immunotherapy with donor-lymphocyte infusion sisting of more than 50 cells were defi ned as colony forming in patients with myelofi brosis following allogeneic stem unit-fi broblasts (CFU-F) and were counted after staining. Clini- cell transplantation cal-scale expanded MSCs were harvested directly after primary H. Alchalby (1), E. Klyuchnikov (1), A. Badbaran (1), culture. Population doublings were calculated based on primary Y. Hildebrandt (1), F. Ayuk (1), U. Bacher (1), O. Bock (2), CFU-F frequency. Array-CGH was carried out using a whole M. Kvasnicka (3), B. Fehse (1), A. Zander (1), N. Kröger (1) genome oligonucleotide microarray platform with female refer- (1)Univerity Hospital Hamburg (Hamburg, DE); (2)Medical ence DNA. Samples were labeled and scanned images were School Hannover (Hannover, DE); (3)Universtiy Hospital analyzed using CGH Analytics software. Cologne (Cologne, DE) We found a strong inverse correlation between the MNC seed- ing density and the recovery of CFU-F indicating a profoundly Allogeneic stem cell transplantation after dose-reduced condi- higher frequency of colony-forming MSCs in BM aspirates than tioning has become a reasonable, curative treatment option for previously described (app. 1/1,000 BM-MNCs). Genomic anal- myelofi brosis during the last few years. However, relapse after ysis after large scale expansion revealed balanced profi les for allogeneic stem cell transplantation remains a major concern. all propagated MSC products despite 5 copy number variations Single cases reported about successful donor-lymphocyte (CNVs; >60kb) that were not documented in the database of infusion (DLI) for relapsed patients after allogeneic stem cell genomic variants. Several small (7kb–1.8Mb; n=33) autosomal transplantation and provided evidence of a graft-versus-mye- CNVs were observed in normal individuals and were not asso- lofi brosis effect. We investigated the effect of escalating donor- ciated with phenotype changes. lymphocyte infusion (DLI) in 17 patients with myelofi brosis who Our data support the notion that despite high proliferation rate had clinical relapse (salvage-DLI, n=9) or molecular relapse MSCs propagated without FBS are genomically stable in array- or molecular residual disease (pre-emptive DLI, n=8) after CGH. This extends earlier results showing that MSCs expanded dose-reduced allogeneic stem cell transplantation. Molecular under humanized conditions did not form tumors in experimen- disease was monitored by highly sensitive JAK2-V617F-PCR. tal animals in vivo and indicates superior safety of the rapidly Thirty-fi ve DLIs from related (n=5) or unrelated donors (n=12) available humanized MSC transplants compared to currently were given with a median dose of 1x106 for the fi rst and 5 x utilized FBS-expanded MSCs. 106 CD3+cells/kg for the second DLI. The complete molecular response rate was 68%, and signifi cantly higher for patients who received DLI for molecular than for clinical relapse (100% P701 vs. 44%) (p=0.04). In comparison to molecular relapse, clini- KIR/KIR-L mismatched NK cells reconstitution after cal relapse required more donor-lymphocyte infusions (two allogeneic SCT needs T-cell depletion and contributes to vs. one) and was associated with a higher incidence of acute GvL effect GvHD (22% vs. 0%). From the Patients in the salvage group J. Hasenkamp (1), A. Borgerding (1), G. Wulf (1), W. Jung (1), achieved 4 a complete molecular remission and 2 of them had L. Trümper (1), B. Glass (2) developed an acute GvHD (grade III). In non-responders of (1)Georg-August-University (Göttingen, DE); (2)AK St. Georg the salvage group was no GvHD documented. This provid evi- (Hamburg, DE) dence for a strong donor-T-cell-mediated graft-versus-myelofi - brosis effect. Molecular monitoring with highly sensitive PCR for Allo-reactive Natural killer (NK) cells contribute to graft-versus- JAK2-V617F-mutation to detect residual disease and to guide leukemia (GvL) effect. Target cell recognition is assumed to rely adoptive immunotherapy seems to be more effi cient and less on Killer cell immunoglobuline-like receptor (KIR)/KIR-ligand (L) toxic than using donor-lymphocyte infusion for clinical relapse mismatch of donor NK cells and human leukocyte antigen class and should be implemented in further clinical trials. I (HLA-I) host tissue. Reconstitution of NK cells repertoire and conditions that contribute or disturb GvL effect of NK cells after allogeneic stem cell transplantation (SCT) are incompletely understood and studied. We investigated clinical course and NK cells repertoires of inhib- itory receptors in 49 patients at 1, 2, 3, 6 and 12 months after all- ogeneic SCT by multi-color fl ow cytometry. Patients suffer from NHL (18), AML (17), CLL (5), MPS (5), MDS (3) and ALL (1).

S187 In donor-recipient situations with KIR/KIR-L mismatch in GvH of the functional scales or maintained basal FVC values. Serial direction NK cells with allo-reactive phenotype – missing an MRI studies did not show any spinal cord damage. There were inhibitory receptor for patients HLA-I – were detectable with two severe adverse reactions (AE): 1 syncope secondary to mean frequencies from 3 to 7%. Peak frequencies are two constipation, and 1 high tract respiratory infection. AEs of WHO months after SCT slightly decreasing in further time course. grade 1 or 2 and less than 2 months of duration were: constipa- In KIR/KIR-L match situations mean frequencies of these NK tion, intercostal pain, CSF hypotension and lower limbs par- cells were always <3%. In vivo T cell depletion with ATG, as esthesias (1 of them persistent). After 9 months of follow-up, part of the conditioning regimen, leads to mean allo-reactive all patients had asymptomatic abolition of the somato-sensorial NK cell frequencies from 2 to 6%. Without ATG allo-reactive potentials of the posterior tract. NK cells could hardly be detected. 12 patients (25%) devel- Conclusions: The procedure was safe and feasible. No major oped progress or relapse of their disease within one year post complications or signifi cant morbility were observed. Stabili- SCT. Allo-reactive NK cell frequencies in these patients were zation of the disease was achieved in 6 of the fi rst 7 patients in mean maximal 1.6% at 2 months post SCT, dropping over included in the protocol. time below 1%. Cases without reoccurrence of their disease had constantly mean allo-reactive NK cell frequencies of ~2%. In cases with relapse more NKG2A+(KIR±) NK cells are found. P703 Cases with long-term remissions show more KIR+NKG2A- NK SCT recipients have specifi c tolerance to MSC but not to cells. The underlying disease – of lymphoid or myeloid origin the MSC donor – had no infl uence as well as the presence or absence of acute M. Sundin (1), J. Barrett (2), O. Ringdén (1), M. Uzunel (1), GvHD on the frequencies of NK cells with allo-reactive pheno- H. Lönnies (1), Å. Dackland (1), B. Christensson (1), type. K. Le Blanc (1) KIR/KIR-L mismatch favors the occurrence of allo-reactive NK (1)Karolinska Institutet (Stockholm, SE); (2)National Institutes cells in peripheral blood after allogeneic SCT. This contributes of Health (Bethesda, US) to control of malignancies. But, reconstitution of the NK cell rep- ertoire is crucially infl uenced by T cells. T cell depletion e.g. with Multipotent mesenchymal stromal cells (MSC) are increasingly ATG seems to augment frequencies of allo-reactive NK cells. used to treat refractory graft-versus-host-disease and other complications following hematopoietic stem cell transplanta- tion (SCT). We evaluated immunogenicity of HLA-mismatched P702 MSC infused post-transplant to SCT recipients. Recipient lym- Phase I-II clinical trial on the use of bone marrow phocyte response to MSC and peripheral blood lymphocytes mononuclear cells in the treatment of amyotrophic lateral (PBL) from the MSC or third party donors was measured before sclerosis. Nine-month follow-up and after infusion. In vitro primary and rechallenge lymphocyte M. Blanquer (1), F. Iniesta (1), F.J. Ruiz (1), J. Meca (1), responses of healthy individuals to MSC and to PBL from the J. Gómez Espuch (2), R. Villaverde (2), J. M. García Santos MSC donor were similarly studied. SCT recipients given MSC (2), S. Torres del Rio (2), C. Funes (1), V. Izura (1), P. de Mingo responded to third party allostimuli, but showed no response Casado (1), A. Sánchez Salinas (1), V. Sáez (1), R. Carles (1), to infused MSC before and up to 6 months after infusion, while C. Antúnez (1), L. Vivancos Moreau (1), J. Hernández Palazón maintaining an alloresponse to the MSC donor. This indicates (1), P. Bleda (1), M. J. Majado (1), M. A. Pérez Espejo (1), immune unresponsiveness was restricted to MSC, since the S. Martínez (3), J. M. Moraleda (1) SCT recipient was not tolerized to the MSC donor. In vitro we (1)Hospital Virgen de la Arrixaca (Murcia, ES); (2)Hospital confi rmed that MSC failed to prime responder lymphocytes to Morales Meseguer (Murcia, ES); (3)Universidad Miguel rechallenge with PBL from the MSC donor, and lymphocytes Hernández (Murcia, ES) primed with MSC donor PBL and rechallenged with MSC only showed weak responses at high stimulator-responder ratios. Introduction: Patients with Amyothrophyc Lateral Sclerosis Although MSC upregulated lymphocyte gene expression of (ALS) endure a progressive paralysis due to the continued loss CD25, IFN-g, FoxP3, CTLA-4 and IL-10, they failed in both of motoneurons that leads them to death in < 5 years. No treat- unprimed and primed responders to induce CD25+ (activated) ment has changed the natural history of the disease. Intraspinal or CD57+ (effector) CD4+ or CD8+ subsets and only inconsist- injections of bone marrow mononuclear cells (MNC) have been ently induced FoxP3+ regulatory T lymphocytes. These results able to ameliorate the course of ALS in murine models. We show for the fi rst time in vivo that infused MSC are only weakly have designed a phase I/II clinical trial to check the feasibility of immunogenic and validate the clinical use of MSC from HLA- this approach in humans. mismatched donors. Methods: The intended recruitment was 11 patients. Inclusion criteria required a medullar onset of the disease, a forced vital capacity (FVC) >50% and T90 < 2%. Sixty mL of bone mar- P704 row were harvested under sedation. A fi coll procedure was Contribution of endothelial and mesenchymal niche cells performed. MNC were resuspended in 2 mL of saline. After to haematopoietic progenitor expansion in a humanised laminectomy, the MNC were infused through a spinal needle system 10 and 6 mm deep in the posterior tract of T3-T4 under elec- M. Fruehwirth, D. Thaler, A. Reinisch, K. Schallmoser, trophysiological surveillance. This level was chosen aiming the E. Rohde, N. A. Hofmann, W. Linkesch, D. Strunk preservation of the lower intercostals function as a mean to Medical University of Graz (Graz, AT) stop the deterioration of the FVC. Patients were followed for 6 months before the infusion, to establish the individual evolu- Distinct stromal and vascular niches within bone marrow are tion of the disease, and every three months for 1 year after the thought to regulate SC (stem cell) proliferation, differentiation procedure. and self renewal. This study compares mesenchymal stromal Results: Seven patients have been infused so far. Median time cell (MSC) and vascular endothelial colony-forming cell (ECFC) from diagnosis to cellular infusion was 20 months. We infused contribution to niche functions in a novel humanized co-culture a total of 402 x106 (240-602.8) MNC, including 3.16 x106 (0.96- system. 10.25) CD34+ cells. After 9 months of follow-up, assessment Autologous MSC-ECFC pairs were established as stromal of the FVC’s evolution and the score points of the international stimulators under animal protein-free conditions. Purifi ed ALS-FRS, Norris and MRC scales, revealed that of two rapidly CD34+ hematopoietic progenitor cells (HPCs) were used as evolving patients, one achieved stabilization of the progres- responders in a cytokine-driven (IL-3/Flt-3-L/SCF) compared sion and one was unaffected by the intervention. Five patients to external cytokine-free niche cell-regulated HPC co-culture. whose disease evolved more slowly also achieved stabilization Serum-free conditions were compared with humanized cultures

S188 supplemented with pooled human platelet lysate (pHPL) as a source of human growth factors present within the niche. Pri- mary expansion culture with and w/o niche cell support was followed by colony-forming cell (CFC) assays to determine maintenance of clonogenicity. Humanized liquid cultures supplemented with 10% pHPL were more effi cient than parallel serum-free cultures under three of four medium conditions resulting in total nucleated cell and CFC increase. Both MSCs and ECFCs further amplifi ed HPC prolif- eration. In the absence of external cytokines the MSC/ECFC combination was the most effi cient in amplifying HPC (mean 180-fold). MSCs were more effi cient than ECFCs in expanding HPCs and CFCs under all other conditions tested. The addition of external cytokines amplifi ed the HPC proliferation (500-fold) and CFC expansion (30-fold) but abrogated the additive effect of MSCs plus ECFCs. The stromal effect was more pronounced in pHPL-supple- mented compared to serum-free cultures, indicating a peculiar role of natural platelet-derived growth factors in this process. We demonstrate for the fi rst time that HPC expansion can be P706 modulated in a fully humanized co-culture system based on Outcomes of the patients with myelodysplastic syndrome, the use of pHPL as a natural source of human platelet-derived acute myeloid leukaemia with multilineage dysplasia and growth factors. Our data indicates that the combination of MSCs treatment-related acute myeloid leukaemia after + ECFCs is at least as effi cient in supporting HPC proliferation allogeneic stem cell transplantation. A single-centre and CFC amplifi cation as either niche cell compartment alone. experience We speculate that human platelet-derived factors may avoid K. Steinerova, V. Koza, P. Jindra, D. Lysak, M. Karas, premature HSC exhaustion in this system. The humanized T. Svoboda co-culture thus provides a novel model system for subtractive Charles University Hospital (Pilsen, CZ) analyses of HPC and SC-niche cell interactions. It also builds the basis for further developments towards more effective ani- The myelodysplastic syndrome (MDS) and its related condi- mal serum-free HSC expansion strategies. tions such as acute myeloid leukemia with trineage dysplasia (TLD-AML) and treatment-related myelodysplasia/acute leu- kaemia (t-MDS/t-AML) usually carry a poor prognosis after P705 conventional chemotherapy. Allogeneic stem cell transplanta- The incidence of fi rst mobilisation failure with tion (SCT) may offer the chance of cure for adult patients (pts) chemotherapy and G-CSF in 594 patients with with this diagnoses (dg.). We retrospective analyzed a cohort of lymphoproliferative disease: potential for intervention pts transplanted at our department for high risk AML/MDS. with Plerixafor™ Materials and methods: 50 pts with dg. TLD-AML (32 pts, 65%; M.J. Watts, D. Linch 9 pts with prior MDS and 23 pts with de novo AML), t-AML Wolfson Cellular Therapy Unit (London, UK) (7 pts, 14%), MDS (7 pts, 14%) and t-MDS (4 pts, 8%) under- went 51 allogeneic SCT at our institution between the years Failure to harvest an apheresis CD34+ cell target dose of 2x106/ 2003 – 2008. The median age in whole group was 53 years kg for autologous transplant procedures remains a signifi cant (range 24-68 years). 2 pts (4%) had favorable cytogenetics, 33 problem for many transplant centres. The CXCR4 antagonist pts (66%) intermediate and 16 pts (32%) poor cytogenetic prog- Plerixafor™ has been used on a compassionate basis with G- nosis. 20 pts (40%) were transplanted with related donors, 30 CSF alone to augment progenitor yields where mobilization has pts (60%) with unrelated donors. 17 pts (34%) have undergone previously failed. However, the aim of this study is to identify a myeloablative conditioning (Bu/Cy/±ATG) and 33 pts (66%) target group of patients in which we can utilize the short-term reduced intensity conditioning (FAMP/Mel). Posttransplant action of this drug to evaluate intervention during standard graft-versus-host disease prophylaxis included cyclosporine chemotherapy/G-CSF mobilization procedures for those who and “short” methotrexate in all pts. fail or are at high risk of apheresis harvest failure. This could Results: 26 pts (52%) alive in complete remission and com- reduce the need for remobilization and avoid its associated plete chimerism with the median of follow-up 15 months (1-63 treatment delays and expense. Between January 2003 and 1st months) and the probability of 2-years overal survival (OS) 46% September 2008, 594 patients with lymphoproliferative disease (42% related and 49% unrelated, p=0,6552). 24 pts (48%) died received mobilizing chemotherapy regimens and G-CSF for the with the median of 6 months (1-50 months). The nonrelapse fi rst time. Of these patients, 26 were not harvested due to sepsis mortality (NRM) at day 100 was 14% (6% related and 8% unre- and disease complications and were excluded from analysis. A lated), NRM at 1 year was 24% (6% related and 18% unrelated). further 27 patients were not apheresed as the peripheral blood 10 pts (20%) -7 (14%) related and 3 (6%) unrelated, p=0,0673 CD34+ cell count did not rise above 10/ul and these were clas- - relapsed after SCT with the median of relapse 5 months (3-48 sifi ed as mobilization failure. The overall failure rate to achieve months) and with 2-years disease free survival (DFS) probabil- a total apheresis CD34+ cell dose of 2x106/kg by diagnostic ity of 47% (43% related and 50% unrelated, p=0,5116). group at the fi rst attempt was as follows; Hodgkin Disease Discussion: our data indicate, that allogeneic SCT is an effec- 11/85(13%), NHL(high grade) 34/178(19%), NHL(low grade) tive treatment option for pts with high risk AML and MDS. Com- 21/91(23%) and Myeloma 32/214(15%). The harvest outcome paring the group of pts transplanted with related and unrelated fl owchart and failure to achieve the target CD34+ cell dose for donor no statistical signifi cant difference in OS and DFS were the 568 evaluable patients is summarised in fi gure 1. Notably, found. However, our results suggest better overall survival, of the 88 patients who had a poor fi rst apheresis CD34 yield of disease free survival and lower relapse rate for patients after <1x106/kg, 72/88(82%) failed to collect a total CD34 cell dose of unrelated SCT due to supposed GVL effect. 2x106/kg in two days of apheresis and 55 of the 88(63%) failed in up to three harvest days. The ideal starting point to evaluate the impact of Plerixafor™ therefore is in those patients who fail to achieve a blood CD34+ cell count of 10/ul and where the fi rst apheresis harvest CD34+ cell dose is <1x106/kg.

S189 P707 surface markers and have different differentiation capacities. Imatinib mesylate in the treatment of sclerodermatous Cell surface markers analysis showed that adherent cells pos- chronic graft-versus-host disease sess high percentage of CD14 (28.1±6.13), CD90 (4.24±0.94), I. Yakoub-agha (1), L. Magro (1), B. Catteau (2), V. Coiteux (1), CD105 (42.19±8.42), CD117 (9.89±5.99) expression and L. Terriou (1), J.P. Jouet (1) reduction of CD45 (46.3±8.14), CD31 (5.9±1.9) and CD34 (1)UAM Allo-CST (Lille, FR); (2)Dermatologie (Lille, FR) (0.18±0.03) compare to non-manipulated PBMNCs. On the other hand, the non-adherent sub-population expresses more Outcomes in the treatment of sclerodermatous chronic graft- CD34 (1.2±0.02), KDR (3.62±0.82), and CD45 (88.7±0.51) versus-host disease (cGVHD) are generally disappointing. compared to non-manipulated PBMNCs. The adherent cells Imatinib mesylate enables selective, dual inhibition of the subpopulation showed higher differentiation potential into transforming growth factor beta (TGFbeta) and platelet-derived endothelial (116±23.1 EC colonies/106 cells) cells compare to growth factor (PDGF) pathways. Recently, the drug’s effects non-manipulated PBMNCs (11.4±1.4 EC colonies/106 cells). on fi broblasts have been reported. Inhibiting fi broblast growth The non-adherent subpopulations showed higher differen- (and thus decreasing collagen production in dermal fi broblasts) tiation potential into hematopoietic colonies (445±75 CFU/106 is thus a logical therapeutic approach. Here, we report on our cells) and mesenchymal (18.65±4.1 CFU-F/106 cells) line- experience with 12 patients who received imatinib mesylate for ages compared to the non-manipulated PBMNCs (373±39.7 refractory sclerodermatous cGVHD following allogeneic stem CFU/106 cells, 6.98±2.7 CFU-F/106 cells) of total colony num- cell transplantation (allo-SCT). The patients’ characteristics bers. Recently, we performed similar studies with PBMNCs were as follows: median age, 35 years (range: 15-59); 7 male from consenting ischemic heart disease patients (n=9). En- recipients, 6 female donors; 4 cases of CML, 4 MDS, 2 ALL dothelial, mesenchymal and hematopoietic progenitors were and 2 Hodgkin’s lymphoma. The patients had received either similar although in sub-normal frequencies. myelo-ablative conditioning with standard GVHD prophylaxis In summary, our ex-vivo enrichment methodology yields two based on cyclosporine and short-course MTX (n=9) or nonmy- different subpopulations with different differentiation capacities. elo-ablative conditioning with cyclosporine and MMF. Seven The ability to enriched populations of endothelial, mesenchy- patients received a marrow graft and 5 received a peripheral mal and hematopoietic progenitors from non mobilized periph- blood graft. All displayed refractory, chronic, sclerodermatous eral blood cells obtained from healthy individuals and cardiac GHVD with at least 3 lines (range 3-6) of prior immunosup- patients may have an important clinical application. pressive therapy. The modifi ed Rodnan skin score was used to assess the extent of skin damage. Glivec was initiated at a dose of 400 mg/day between 16 and 119 months post-transplanta- P709 tion (median: 44). Despite an imatinib dose reduction and the Expansion of human mesenchymal stem cells seeded in administration of various symptomatic treatments, 4 patients biological scaffods (33%) had to discontinue their treatment soon after its initiation E. Michalopoulos, C. Saliagopoulou, T. Chatzistamatiou, (range: 16-64 days) because of intolerance (especially muscle A. Thiakos, A.C. Papassavas, C. Stavropoulos-Giokas cramps) and were not evaluable in terms of the effi cacy crite- Biomedical Research Foundation (Athens, GR) rion. Other side effects reported were parenthesis, diarrhea and edema. In the remaining patients, the scleroderma symptoms There is considerable evidence that mechanical stimuli affect improved within three months of treatment initiation. At the time gene expression in a range of differentiated cell types and sim- of this report, all patients were alive and those who tolerated ulation systems to re-cellularise tissues in vitro in a functional imatinib mesylate have experienced a complete or near-com- manner. plete response (n=4) or partial response (n=4). All responders Based on this principle, the approach taken on this study was (except for one who discontinued the drug 157 days after initia- to construct a 3D scaffold from Collagen/Alginate Gel and tion, due to cramps) were still on the treatment, after a median reseed it with autologous human mesenchymal stem cells in time period of 216 days (range: 80-2053). This retrospective vitro. The main aim was to determine whether the cells would report shows that when imatinib mesylate is well tolerated, it proliferate in the scaffolds and determine if they remain viable is effective in patients with refractory sclerodermatous cGVHD over prolonged culture periods and retain their undifferentiated and is thus a promising candidate for the treatment of this phenotype. complication. Collagen type I (2.8% w/v) was extracted from rat tails and puri- fi ed, then cast into gels of 1.5 mm thickness in 24-well-plates. The gels were then freeze-dried and D7-FIB+ MSCs were P708 delivered onto the sponges by mixing. Optimisation of the 3D Two distinct populations of enriched non-mobilised cell culture techniques in collagen sponges/alginate gels was peripheral blood mononuclear cells with different achieved and 3D collagen/ alginate composites were seeded functional capacities with hMSCs. Cell proliferation was assessed using the ATPlite N. Bloom, T. Kniazhansky, A. Treves, A. Nagler assay and the cell viability was assessed using the live/dead Sheba Medical Center (Ramat-Gan, IL) cell assay upon serial sections of seeded scaffolds at different depths. Results demonstrated that the scaffolds supported the Pluripotent stem cells are valuable sources for transplanta- growth of MSCs over the extensive period (60 days) of culture. tion and tissue repair. Non-mobilized peripheral blood contains Histological evaluation demonstrated that cells attached on the mostly committed cells but recent studies suggest the pres- collagen fi bers and spread within the pores. Finally, potential ence of early progenitor and stem cells as well. Here we aim changes in the phenotype of hMSCs within the 2.8% (w/v) col- to develop a method to enrich and recover functional progeni- lagen/alginate scaffolds were determined by immunostaining tor cell populations from non-mobilized peripheral blood. The of HLA-DR, CD-117, CD-45, CD-34, alkaline phosphatase, ex-vivo enriched non-mobilized PBMNCs were tested by FACS osteocalcin, D7-FIB, CD-90, CD-13 and CD-105 by the immu- analysis of specifi c cell surface markers and by functional anal- noperoxidase method. Results demonstrated that hMSCs were ysis of differentiation. positively stained only for CD-90, D7-FIB, CD-13 and CD-105 Non mobilized PBMNCs obtained from consenting healthy indicating that the cells retained their undifferentiated state dur- donors (n=18) cultured for 7 days in a defi ned cytokine cocktail ing the prolonged period of 3D culture. media were analyzed by FACS and tested for their differentia- In conclusion, MSCs are an attractive source for tissue engi- tion capacity into hematopoietic, endothelial and mesenchymal neering applications due to their ease of isolation proliferation cells lineages compared to non-manipulated PBMNCs. We and multilineage differentiation ability. A promising approach in found that enriched PBMNCs resulted in two distinct subpopu- the tissue engineering fi eld might be the cellularisation of tissue lations, adherent and non adherent, that present different cell scaffolds using in vitro repopulation with autologous hMSCs

S190 coupled with physically appropriate conditioning prior to implan- Median age was 51 years (range, 19-70), with 44 donors aged tation. Therefore, in future the basic principle will be seeding of ≥55 years. All donors received 4-5 days of G-CSF given once cells onto a matrix scaffold and use of physically interactive bio- daily in the evening, with the fi rst leukapheresis initiated on the reactors for the functional differentiation of mesenchymal stem morning of day 5. Although the recommended dose for rhG- cells within 3-dimensional scaffolds in vitro. CSF is 10 µg/kg/day for stem cell mobilization, most donors received a total dose of rhG-CSF that was rounded to the low- est multiple of 300 µg, thus allowing the daily use of a whole P710 number of vials: as a consequence, the median received dose/ Three-year clinical follow-up of 5 patients treated with kg of rhG-CSF was 8.9 µg, which may contribute to the lower suicide gene modifi ed donor lymphocyte infusion therapy number of circulating CD34+ cells observed in our cohort of in Japan patients (59.5/µL). S. Kaneko, Y. Okoshi, M. Otsu, N. Nemoto, K. Suzukawa, In order to identify factors affecting circulating CD34+ cell Y. Hasegawa, H. Kojima, T. Fukushima, R. Sumazaki, counts as a surrogate maker for stem cell mobilization, we fi rst M. Onodera, Y. Harada, H. Sakamaki, M. Tsuchida, S. Kato, performed a univariate analysis, including age, gender, weight, C. Bonini, C. Bordignon, T. Nagasawa, S. Chiba, H. Nakauchi height, total G-CSF dose and G-CSF dose per kg. Ethnicity was on behalf of the Tsukuba TK-DLI Gene Tharapy Group not tested as an explanatory factors for stem cell mobilization, because our cohort of donors was smaller and more homoge- Suicide gene, herpes virus thymidine kinase gene (hsv-tk), neous, mostly with individuals of Caucasian ascent Variables modifi ed donor T lymphocyte infusion (TK-DLI) is one of the associated with higher post-G-CSF CD34+ cell counts were promising approach to high risk leukemia patients who are donor weight (p=0.0026) and the total dose of G-CSF received going to have T cell depleted haploidentical hematopoietic (p<0.001), thus reproducing recently published results. By stem cell transplantation (1). Despite that success of the T lym- multivariate analysis, only the total dose of G-CSF received phocyte based gene therapy in haplo SCT setting to reduce remained signifi cant (p=5.49*105). relapse unrelated mortality with control of graft-versus-host dis- Interestingly, our group of recent donors was heterogeneous ease is becoming clear, there remains some diffi culties to be in terms of age, and therefore might accurately refl ects current solved for inducing remission to patients with relapsed leuke- practices in allogeneic transplantation from related donors. It mia after SCT. The solutions would be 1) very early detection is important to stress that age did not signifi cantly infl uence of leukemia relapse and enough reduction of leukemia burden CD34+ cell counts (p=0.3441). When looking at subgroups before TK-DLI, 2) making a “niche”, space for lymphocytes, for according to age, no signifi cant difference was found between TK-T lymphocytes, and 3) maintaining T lymphocytes to have the mobilization procedure and endpoints for “elderly donors” potentials of proliferation and survival such as naive and central (age ≥ 55, n=44, of which 10 were older than 70 years) as com- memory T cells. pared to “younger donors” (age <55, n=85). A phase I/II clinical trial of TK-DLI have been performed in Our results suggest that the absence of deleterious effect of the Tsukuba University Hospital to 5 patients (2 AML, 2 ALL, older age on progenitor cell mobilization is important to con- and 1 MDS) since 2002. Actually our fi ve patients for relapsed sider in view of the increasing use of older donors, in the con- leukemia had one or more diffi cult points as listed above, and text of RIC allo-SCT. it results only temporal and insuffi cient clinical response in 4 of 5 patients. Different from the other patients in treatment failure, only one MDS patient survives more than 3 years after TK-DLI without any sign of relapse. He was in hematological remis- sion at the time of TK-DLI and had been in immunosuppressive status of 6 month after SCT, suggests that he had been in ideal clinical conditions for TK-DLI. But TK-T cells disappeared within 4 month after TK-DLI same as the other patient, suggests TK-T cells lacked potentials of proliferation and survival in a patient. According to the result, we optimized clinical and manipulation protocols. They include 1) detection of molecular relapse and perform a TK-DLI at the level, 2) addition of lympho-depletion conditioning with Fludarabine and Cyclophosphamide before TK-DLI, and 3) usage of CD3/CD28 stimulation + low dose IL- 2, instead of OKT3 + high concentrated IL-2, to obtain suicide gene transduced central memory TK-T cells(2,3). Clinical outcome of fi ve TK-DLI patients and result of protocol modifi cation will be discussed in the meeting. P712 1. Bonini C et al, Mol. Ther. 15:1248-52. 2007 Use of amniotic membrane as a reparative wound 2. Bondanza A et al, Blood 107(5):1828-36 dressing in patients with post-traumatic wounds. 3. Kaneko S et al, Blood Oct 31, 2008 [Epub ahead of print] Preliminary results C. García de Insausti, G. Castellanos, M. Blanquer, M. J. Majado, P. Bleda, F. Iniesta, F. J. Rodríguez Lozano, P711 F. J. Nicolás, J. M. Moraleda Older age does not infl uence allogeneic peripheral blood H. U. Virgen de la Arrixaca (Murcia, ES) stem cell mobilisation in a population of mainly Caucasian donors Objectives: To evaluate effects on re-epithelialization of amni- H. de Lavallade, P. Ladaique, C. Lemarie, S. Furst, C. Faucher, otic membrane (AM) as a wound dressing in patients with D. Blaise, C. Chabannon, B. Calmels chronic traumatic ulcers. Institut Paoli Calmettes (Marseille, FR) Material and methods: Amniotic membrane processing Term placentas from healthy donor mothers were obtained from So far the question whether age has a negative impact on uncomplicated cesarean section after informed consent. Under peripheral blood stem cell (PBSC) mobilization in healthy sterile conditions, AM was mechanically peeled from chorion, donors remains controversial. We retrospectively evaluated washed with PBS and stored at -196 ºC. After thawing, seg- 129 consecutive related adult donors who underwent PBSC ments were washed with PBS and used as dressing in post- mobilization and collection at the Institut Paoli-Calmettes (Mar- traumatic wounds. Bacteriology tests and quality controls for seille, France) between January 2005 and December 2007. maternal infectious markers (syphilis, HBV, HCV, HIV, and

S191 HTLV on cesarean day and after a quarantine period of 90 days in absence of risk factors as unfavourable cytogenetic, initial were performed. hyperleukocytosis, need of ≥3courses of chemotherapy or M5 Case report 1: A 49 year old woman presented with general- subtype. ized polytrauma and a wound of 17x15 cm in the right tight. Three weeks later the wound had failed to heal and an infec- tion with Acinetobacter baumanii was documented. Wound was debridated, treated with antibiotics and vacuum therapy. No epithelialization was observed sixty days later. Wound bed was dressed with AM fragments. Ulcer evaluation was performed every 3 - 4 days until complete healing. Case report 2: A 76 old year lady suffered a crush trauma in the gemelar area of the right leg. She developed local infl amma- tory signs and the evolution was not satisfactory. One month later, an ulcer of 17 x 10 cm affecting soft tissues and muscles appeared. The wound was debrided and subjected to vacuum therapy. Forty fi ve days later there was no epithelialization. As previously described, wound was completely dressed with AM fragments and covered with the vacuum device. Results: Effect of AM on reepithelialization in massive wounds. Case report 1: After eight days AM was adhered to ulcer bed and re-epithelialization from the ulcer margins had started. Wound size gradually reduced and wound re-epithelialization was successfully established by the day +193. Case report 2: Eight days after AM application some re-epithe- lialization from the edges was observed. Even though epitheli- alization developed appropriately for the following 6 months, a second application of AM was decided and 2 months later the wound completely healed. Histopatological evaluation of induced newly formed skin, showed a normal histological skin structure without glands and P714 hair. A new scaffold for bone tissue engineering based in Conclusions: These preliminary results suggest that healing of mesenchynal stem cells and glass-ceramics extensive and chronic wounds may improve remarkably after L. Meseguer Olmo, C. García de Insausti, A. Bernabeu treatment with amnion, resulting in total re-epithelialization of Esclapez, F. J. Rodríguez Lozano, M. Vallet Regi, M. Blanquer, the ulcers. J. M. Moraleda, M. Clavel Hospital Virgen de la Arrixaca (Murcia, ES)

P713 Introduction: Treatment of bone defects of any etiology remains Autologous stem cell transplantation for acute myeloid a challenge in the bone reconstructive surgery. Nowadays the leukaemia: a 12-year, single-centre follow-up study standard technique is the use of bone grafts. However this M.J. Garcia-Rodriguez, R. Arrieta, M.A. Canales, F. Hernandez- approach has well documented drawbacks. An alternative Navarro worth to be explored are the hybrid materials. Hospital La Paz (Madrid, ES) Objectives: we intend to demonstrate the effi cacy of a 55S bio- active glass as a substrate for the induction of the differentiation ASCT has been a reasonable therapeutic option in patients of bone marrow mesenchymal stromal cells (bmMSC) with the with AML non-candidates to allogeneic transplantation. The requirements to be considered osteoinductive and osteogenic. aim of our study has been to analyse the infl uence of different Materials and Methods: characteristics in long-term outcome of patients diagnosed with • Material preparation: The glass ceramic was fabricated by AML who undergone an ASCT. heat treatment of a bioactive glass (55-SiO2; 41-CaO; 4- We have analysed 36 patients (18 male) following ASCT between P2O5; mol %), obtained by the sol-gel method. September 1996 and January 2008. Patient characteristics are • Isolation and culture of bmMSCs: The adult bmMSCs were described in table 1. Median follow-up was 11 months (range 0-140 isolated from adult human bone marrow obtained by direct mo). All patients received G-CSF for mobilization and peripheral aspirations of ileac crest. The cells were plated out in culture blood stem cells were infused with a median of 2.77±106/kg CD34 and they were incubated at standard conditions. cells (range 1.02-13.42). Conditioning regimen was Busulphan- • Isolation and culture of osteoblasts (OB): The OBs used as Cyclophosphamide. ASCT was performed in CR in 88.9% patients control were obtained by the method of enzymatic digestion. with median of 3 months (range 1-6 mo) from last chemotherapy • Behavior of bmMSCs on 55S glass ceramic: The behavior of and 6 months (range 3-16 mo) from diagnosis. The actual 12-year isolated bmMSCs on the glass ceramic, two series of 96-well relapse probability was 27.8%, event-free survival 41.7% and over- plates were seeded, with GM and OM. Then 10 x 103 cells all survival 44.4%. Relapse occurred with median of 6.5 months were seeded. At 24 hours, 7, 15, 21 and 27 days cell adher- from transplantation (range 2-19 mo) and 90% of them died with ence and growth were analyzed. median of three months (range 0-11 mo). A total of twenty patients • Statistical analysis: A variance analysis (ANOVA) was carried have died (55.6%) with median of 7 months (range 0-21 mo) after out with a minimal signifi cance of p<0.05. ASCT. We have analysed the infl uence of sex, age, AML sub- Results: The 55S glass ceramic was an excellent substrate for type, cytogenetic abnormalities, WBC at diagnosis, chemotherapy the osteoblastic differentiation of the bmMSC as assessed by courses before ASCT and time between diagnosis/last chemother- the increment in the production of osteocalcin and phosphatase apy and transplantation on relapse risk and survival. Unfavourable alkaline and the reduction in the CD90 expression. Adhesion cytogenetic, hyperleukocytosis and M5 subtype were associated and proliferation of the cells on the substrate was observed by with poor outcome and early mortality. A signifi cant correlation scanning electron microscopy as well as their ability to produce between mortality and number of chemotherapy courses was calcifi ed bone extracellular matrix. evidenced (p<0.02). Conclusions: The results obtained allow us to assert that the Our results suggest that ASCT could be an effective treatment 55S glass ceramic provides the adequate microenviroment to in AML patients non-candidates to allotransplant, especially support the adhesion, proliferation and differentiation of the

S192 bmMSC to osetoblasts. Thus, it can be considered as an thera- peutic option for tissue engineering techniques in the fi eld of bone tissue reconstruction.

P715 Impact of mesenchymal stem cell transfusion on thrombotic microangiopathy post haematopoietic stem cell transplantation M. Ansari (1), V. Kindler (1), C. Delcò (1), V. Salah (1), Y. Chalandon (1), F. Gumy-Pause (1), A. Rougemont (1), S. Moll (1), P. Parvex (1), H. Chehade (1), D. Strunk (2), J. Passweg (1), H. Ozsahin (1) (1)University Hospital Geneva (Geneva, CH); (2)Medical University of Graz (Graz, AT)

Background: Thrombotic microangiopathy (TAM) post hemat- opoietic stem cell transplantation (HSCT) has an incidence of 4–13%, and is a life-threatening complication with a mortal- ity rate of approximately 60%. Clinical manifestations include severe thrombocytopenia, microangiopathic hemolysis, and frequent renal dysfunction with neurological complications. The reported risk factors of TAM include: female gender, total body irradiation, lymphoid malignancy, unrelated or mismatched donor, calcineurin inhibitors, presence of infection and graft- versus-host disease (GvHD). There is no consensus for treat- ment of post-transplantation TAM. Description: A 9-year-old girl with severe refractory aplas- tic anemia received an unrelated HSCT (9/10) with HLA-Cw mismatch. The conditioning regimen included fl udarabine 150 mg/m2, cyclophosphamide 200 mg/kg and ATG. Cyclosporine (CSA) was administered to prevent GvHD. The graft was T-cell depleted with Campath-1H in vitro and followed by an addback of CD3+ cells of 5x106/kg on day+1. On day+2 she developed a Coomb’s negative anemia, with schistocytes, elevated LDH, thrombocytopenia together with an acute renal failure. ADAMTS 13 antibody was negative. The renal biopsy confi rmed TAM. P716 She then developed a grade III intestinal GvHD. The TAM was Cyclosporin monitoring at 2 hours from oral intake leads treated with plasma exchange, basiliximab, anti-TNF, heparin, to less toxicity and better compliance in patients and defi brotide. CSA was replaced with methylprednisolone/ undergoing haematopoietic stem cell transplantation tacrolimus, then with methylprednisolone/sirolimus and ster- and immunosuppressive treatment for severe aplastic oids only. None of these drugs decreased TAM intensity. The anaemia patient clinical status further worsened with CMV, HHV-6 reac- B. Montante, M.B. Pinazzi, A. Severino, A. Proia, I. Majolino, tivation, cardiac and pleural effusions. Considering TAM as a A. Locasciulli form of GvHD, allogenic fully mismatched third pary mesenchy- San Camillo (Rome, IT) mal stem cell (MSC) were infused (2.22x106/kg). Two weeks later, the biological parameters went back to normal without Background: Nephrotoxicity derived from Cyclosporin treat- any need of thrombocyte transfusion (fi gure 1, 2). Renal biopsy ment is a well known complication and for this reason drug still showed TAM with global interstitial fi brosis. The biological level baseline is usually monitored (before morning oral intake). signs of TAM reappeared 10 weeks after MSC infusion, associ- Recently, data in the literature demonstrated a higher reliability ated with viral reactivation, pleural and cardiac effusions. The of drug serum level after 2 hours from oral intake as regards pulmonary biopsy showed medium-sized artery thrombosis, but to absorption and needed doses in the specifi c patient. These no signs of pulmonary TAM. The issue ultimately proved to be reports are obtained among Solid Organ Transplant settings. fatal. Aim: To assess if cyclosporin blood level at 2 h from oral intake Conclusion: This case illustrates a possible, benefi t of allogenic is a more reliable tool than the pre-intake level to prevent or MSC infusion for the treatment of TAM. The timing of admin- reduce nephrotoxicity in Hematopoietic Stem Cells Transplan- istration, the quantity and the number of doses is not defi ned tation (HSCT)and Severe Aplastic Anemia (SAA) patients, and yet. Further studies need to be done in order to confi rm this to tailor the required dose in each patient. fi nding. Materials and methods: Patients. From February to November 2008 we prospectively enrolled 11 patients post HSCT and 4 under Immunosuppressive treatment (IS) for SAA. We monitored serum level of cyclosporin at time 0 and after 2 hours from oral intake, creatinine and urea serum levels, drug toxicity, and Graft versus host disease (GvHD) in HSCT group. Oral dose was then modifi ed according to the 2 hours post cyclosporin results. Results: The incidence of hypercreatininemia, renal failure, hypomagnesiemia, headache and tremors was trivial and drug withdrawal was very seldom required. Creatinine serum level was ≥ 1.5 in 3 patients (20%). Only 2 out of 15 patients (13%) needed cyclosporine withdrawal: one for renal toxicity, the other one for trombotic trombocitopenic purpura; both of them were in HSCT group. Other side effects (headache and tremors) were evident only in 2 (13%) patients, one with SAA, the other one in HSCT

S193 group. Cyclosporin level after 2 hours seemed to be independent spectrum of cellular therapies. They can be isolated from differ- from the dosage/Kg of body weight, especially in children. ent biological sources and expanded in colture but a reproduc- In 7 out of 11 HSCT patients (63%) acute GvHD (grade I-II) was ible and effi cent method to obtain hMSCs feasible for clinical evident whereas limited chronic GvHD in 4 out of 11 patients use is diffi cult to perform. Aim of this study was to validate a (36%) was found. FBS-free two-steps protocol expansion starting from both bone Both SAA and HSCT patients tolerated cyclosporin with 2 hour level marrow aspirates and washouts of bone marrow collection bags monitoring very well, with apparent less toxicity. The two patients and fi lters in compliance to Good Manufacturing Practice. requiring cyclosporin withdrawal were both in the HSCT group. Methods: Total nucleated cells were collected and seeded Conclusions: Our data suggest that dose adjustment of without any manipulation in 5% human platelet lysate (hPL) cyclosporin according to 2-hour level is more accurate than the supplemented alpha-MEM. After 48 hours nonadherent cells basal one and more effective to prevent toxicity and side effects. were removed and the adherent cells were expanded for 7- Furthermore, it also provides a better compliance. The scarce 14 days with periodic feeding. The cells were then harvested number of patients doesn’t allow to draw defi nite and fi rm con- and seeded at low density (100-200 cells/cm²) and allowed to clusions, prompting the need to further prospective studies. expand for additional 10-20 days. Finally the cells were har- vested and frozen. Results: In a median of 21 days (range 17 to 33), 19 “adults”(mean P717 69x106 hMSCs/bag) clinical bags could be prepared from the Rapid immunological reconstitution after infusion of expansion of a portion of the cells recovered from 4 independ- allogeneic, cytokine induced killer cells: a case report in a ent washouts and from 4 bone marrow harvests. 35 and 8 vials patient with Sezary syndrome could be frozen from the remaining cells recovered from the C. Micò, A. Grassi, G. Borleri, A. Algarotti, C. Capelli, M. Introna, washouts and bone marrow harvests, respectively. The theo- A. Rambaldi retical full expansion of the frozen vials (validated by the expan- Ospedali Riuniti di Bergamo (Bergamo, IT) sion of 2 independent vials) could have allowed the production of 173 bags from 4 independent washouts and 18 bags from 4 Background: CIK cells are naturally occurring cytotoxic cells active bone marrow harvests. Clinical scale expanded hMSCs identity against a variety of leukemia and lymphoma targets and with low and purity were assessed by testing viability and expression or absent activity against normal BM stem cells. These cells can of CD14, CD34, CD45, CD73, CD90 and CD105. Cytogenetic be easily expanded in vitro under adherence to GMP conditions analysis was performed and no chromosomal alteration has and safely used after allogeneic stem cell transplantation. been revealed. Bacterial, fungal, mycoplasma and endotoxin Case report: A female patient with a diagnosis of cutaneous T-cell lym- contamination were tested by validated tests according to Euro- phoma (2000) was sequentially treated with steroids, Cyclosporine pean Pharmacopea guidelines and always found negative. and PUVA therapy for 2 years. In 2002 she received chemotherapy Discussion: Starting from both bone marrow aspirates and bone program VAPEC-B and in 2003, she underwent a Bone Marrow marrow collection bags and fi lter, a minimally manipulated cul- stem cell allogeneic transplantation (TNC 2.8x108/kg,CD34+ cells ture procedure allows to produce suffi cient numbers of hMSCs 6x106 cells/kg and CD3+ cells 34x106/kg).The conditioning regi- for therapeutic purpose using only clinical grade reagent and men was based on Melphalan,Alemtuzumab,Fludarabine and TBI. avoiding animal serum and to establish a frozen bank of early From June 2003, for progression to large T cell lymphoma of the passages cells which can be subsequently recovered and skin, she was treated with local radiotherapy, CHOP chemotherapy further expanded. Furthermore it has been demostrated the and Alemtuzumab,Fludarabine and Cyclophosfamide and fi nally normally discarded collection devices such as BM washouts Gemcitabine. On day +1016 from transplant (2005), she devel- represent a convenient source of large amount of hMSCs. oped pancytopenia with severe neutropenia, fever and pneumonia. Hospitalization was required. Peripheral blood immunophenotype showed a marked lymphocyte depletion(total lymphocyte count P719 112/µl,CD3+ 64/µl,CD4+ 24/µl,CD8+ 39/µl,CD16/56+10/µl,). For Comparison between BEAM and lomustine, Ara-C, this reason, donor CIK cells (1490x106 CD3+ cells;782x106 CD3+/ etoposide and melphalan as conditioning regimen before CD56+)were infused. The clinical, hematological and immune stem cell transplantation. A single-centre experience situation of the patient rapidly recovered allowing the hospital dis- G. Grillo, P. Marenco, G. Bertani, E. Zucchetti, M. Turrini, charged after 2 weeks. One month after the infusion of CIK cells, P. Brasca, C. Gabutti, E. Morra the absolute number of peripheral blood lymphocytes was 2083/ Niguarda Cà granda Hospital (Milan, IT) µl,(with CD3+1595/µl,CD4+226/µl,CD8+1422/µl,CD16+/56+ 440/ µl).No evidence of aGVHD was registered. In our Centre the standard conditioning regimen for autologous Conclusion: In a clinical setting characterized by severe deple- SCT in the setting of lymphoma is the combination of Carmus- tion of peripheral blood lymphocytes, the infusion of CIK cells tine, Etoposide, Ara-c and Melphalan (BEAM). In the last few confi rmed to be well tolerated and allowed a rapid recovery of years an impairment in carmustine availability, at least in Italian the whole immune system including not only cytotoxic CD8+ hospitals, has occurred. cells but also all the other main lymphocyte subpopulations. Thus, we modifi ed the standard BEAM regimen by replacing the It is tempting to speculate that CIK cells and possibly some iv administered Carmustine with its oral analogous Lomustine. cytokines massively released in the setting of severe immune The aim of the study is to evaluate feasibility and toxicity of the depletion may play such a major role to stimulate the immune conditioning regimen combining CCNU 200 mg/m², Ara C 1600 recovery. The possible direct role of CIK cells in controlling viral mg/m² Etoposide 800 mg/m², and Mel 140 mg/m² (CEAM), and fungal infections in vivo is currently under investigation. and to compare these data with those of our historical group of patients conditioned with BEAM. We analized 94 consecutive patients who underwent autolo- P718 gous SCT in our BMT Unit between Jan 2005 and May 2008; Clinical scale serum-free expansion of human 47 patients (pts) were conditioned with CEAM. Among these, mesenchymal stromal cells: validation of a “two-steps” 16 were affected by HD, 24 were diagnosed as high grade NHL protocol under GMP conditions and 7 as FCL. Disease status at time of SCT was 1st CR in 15 C. Capelli, V. Barbui, O. Pedrini, E. Gotti, G. Borleri, A. Rambaldi, pts and 2nd or subsequent CR in 10; 21 pts had PR or active J. Golay, M. Introna disease. Median age was 45 y (range 22-61). Median level of Laboratory of Cellular Therapy “G.Lanzani” (Bergamo, IT) CD34-positive cells infused was 10.83 x 106/kg (range 3.44- 41.6). Median time to neutrophil engrafment was 9 days (range Objectives: Human Multipotent Mesenchymal Stromal Cells 7-11). 27 pts had at least one episode of febrile neutropenia (hMSCs) are considered good candidates for a growing (57.4%) and in 12 cases an infection was microbiologically doc-

S194 umented. Gastrointestinal mucositis requiring major analgesia disease. This profi le can be matched against existing gene pro- occurred in 2 pts. No other unexpected toxicities were recorded. fi les. Based on similarity, it can be predicted with certain prob- Median hospitalization period was 18 days (range 15-30). ability if the patient is in an early phase of a disease or he/she In the other group of 47 pts treated with BEAM, 14 were affected is at risk of developing the disease in the future with certain by HD, 30 by HG NHL and 11 by FCL. Disease status at time of probability. We apply a Computational Neurogenetic Modeling transplant was 1st CR in 14 pts; 2nd or subsequent CR in 14; (CNGM) technique to confi rm clinical diagnosis of acute Graft PR or active disease in 28. Median age was 46 y (range 17- vs Host Disease (aGVHD) and to predict the occurrence of this 62). Median level of CD34-positive cells infused was 14 x 106/kg complication using a specifi c profi le of a immune gene panel. (range 7.48-60). Median time to neutrophil engrafment was 9 days Experimental data: Expression changes of 48 genes were eval- (range 7-11). 29 pts had at least one episode of febrile neutrope- uated in 21 patients affected by haematological malignancies nia (61.7%) and in 12 cases had a microbiologically documented submitted to allo-SCT from HLA-related (n=19) and unrelated infection. Gastrointestinal mucositis requiring major analgesia donors (n=2) for a total of 82 single observations. We have occurred in 4 pts. No other unexpected toxicities were recorded. used a TaqMan® Low Density Array based on comparative Median hospitalization period was 19 days (range 15-42). ddCT method to perform a relative quantifi cation of mRNA. These data seem to demonstrate a good safety and feasibility pro- In all patients serial samples of peripheral blood mononuclear fi le for CEAM schedule as conditioning regimen. Regimen-related cells were collected between 10-90 days after transplant and in toxicities are similar to those associated with BEAM. Therefore each time when a clinical feature compatible with aGVHD was these conditioning schemes seems to be exchangeable. present. Ten patients did not experience aGVHD, 11 patients had II-III grade of aGVHD. Result: In this study we have applied Principal Component P720 Analysis (PCA) method to reduce numbers of variables, but Clinical impact of bronchoalveolar lavage in stem cell also to select variables with major weight for each principal transplanted patients with pneumonia component (PC). We found: a) for diagnosis GVHD (Yes) 2 PC, U. Forslöw with cumulative variance of 64% and a third PC lower, b) for Laboratory Medicine (Stockholm, SE) diagnosis GVHD (No) 2 PC, with cumulative variance of 57% and a third PC very lower. The variables compose a subset of Background: Patients receiving allogeneic hematopoietic stem 23 genes present in the fi rst two principal components of GVHD cell transplantation (HSCT) are prone to pulmonary infections Yes/ No. Variables of the fi rst PC of GVHD (No) is included in caused by a wide spectrum of micro-organisms. the subset of the fi rst two principal components GVHD (YES). Methods: Between 1998 and 2004, 450 patients received Therefore we believe that this presence strengthens the impor- HSCT at Karolinska University Hospital, Huddinge. Pneumonia tance of sub-set genes for training an intelligent system. was diagnosed in 167 (37%) patients including 42 children. All Conclusion: Using the PCA a sub-set of genes with largest patients were investigated by chest X-ray and/or computer tom- information was extrapolated from immune panel. Starting from ography. Bronchoalveolar lavage (BAL) was performed on 68 this analysis it’s natural to develop an Artifi cial Neural Network occasions in 57 patients (6 children). In 110 patients including (ANN) where genes are inputs and output is the evaluation of 36 children with pneumonia, BAL was not performed. syndrome. We have developed different types of ANN, and Results: BAL contributed to the diagnosis in 43 (63%) cases have compared them to improve results. The fi nal obtained with 53 considered relevant fi ndings; bacteria 13 (24%), viruses results are good, and tell us that it’s possible predict the aGVHD 28 (53%), and fungi 12 (23%). In 25 cases BAL was negative. using a restrict number of variables with a sensitivity of 92,8%, In 15 of these, BAL was performed ≥ 4 days after chest X-ray, fi g.1,2. in 4 not in the same segment as the infi ltrations. Median time between radiographic fi ndings and a positive BAL was 2 (0-15) days and a negative BAL 6 (1-30) days, p<0.001. Antimicro- bial treatment was administered to 84% patients with positive and 92% with negative fi ndings at the time of BAL. No serious complication due to the procedure was reported. BAL resulted in a changed treatment in 31 (72%) episodes of pneumonia. In the BAL-group 63% and in the non-BAL group 71% recovered, respectively. However, the latter group included 23% more chil- dren, more likely to recover. Conclusions: We conclude that BAL is a safe and useful diag- nostic procedure for the evaluation of pulmonary complications and should be performed early at the onset of pneumonia fol- lowing allogeneic HSCT.

Graft engineering

P721 A computational intelligent method for diagnosis of acute graft-versus-host disease based on gene expression data M. Cuzzola (1), C. Rigolino (1), R. Fedele (1), E. Massara (1), M. Martino (1), G. Irrera (1), G. Messina (1), G. Console (1), D. Princi (1), M. Fiaschè (2), A. Meliadò (1), F.C. Morabito (2), P. Iacopino (1) (1)A.O. Bianchi-Melacrino-Morelli (Reggio Calabria, IT); (2)University Mediterrarea (Reggio Calabria, IT)

Rationale: A gene profi le is a pattern of expression genes that is typical for all, or for some of the known samples of a particular

S195 P722 able WT1 expression levels in peripheral blood remained in com- Evaluation of a tubing system with an incorporated plete remission at a median of 1925 days after HSCT. In contrast, DMSO-resistant sterile fi lter for cryopreservation of all 31 patients who suffered from hematological relapse after a cellular products outside of cleanroom facilities median of 151 days presented with high levels of WT1 expres- A. Humpe (1), S. Schubert (2), A. Ketels (1), M. Gramatzki (1) sion (p<0.001). In 18 patients, we observed an increase of WT1 (1)Section of Stem cell and Immunotherapy (Kiel, DE); expression levels at a median of 34 days before hematological (2)Institute of Infection Medicine (Kiel, DE) relapse. In 14 of these 18 patients, DNA was available at the time of increase of WT1 expression level to perform analysis of hemat- Objectives: Processing of cellular therapeutics in an open sys- opoietic chimerism using a semi-quantitative short-tandem-repeat tem according to GMP guidelines requires a cleanroom grade PCR. Interestingly, 10 of the 14 patients (71%) revealed a complete A with surrounding grade B conditions. In a validation project a donor chimerism, whereas only 4 of the 14 patients (29%) showed newly developed tubing system with an incorporated dimethyl a mixed chimerism. In the remaining 13 patients with relapse, no sulfoxide (DMSO)-resistant sterile fi lter (Closed Cryo Prep Set, increase of WT1 expression levels could be detected because Cell•Max, Germany) for cryopreservation of cellular products the time interval between the last sample harvest and hemato- was evaluated by challenge experiments and media fi ll runs. logical relapse was to long (median, 66 days). In fi ve of these 13 Methods: In a fi rst series, fi lter of 3 systems were challenged patients, DNA was available at the time of the last sample harvest with spiked DMSO. 20 ml of DMSO were spiked with a defi ned before relapse and showed a complete donor chimerism. In two solution of Bacillus subtilis (strain ATCC 6051) leading to a con- patients, we diagnosed a molecular relapse using WT1 expres- centration of 1×10E+06 colony forming units/ml. Prior fi ltration sion. At the time of molecular relapse, both children revealed a 100 µl were used to titrate bacteria counts of the inoculum in complete donor chimerism. In both patients, molecular remission log 10 steps using BHI agar plates. 20 ml of the bacterial sus- was achieved by immunotherapy. Both children are alive and well pension were applied to the system. The fi ltrate was collected without relapse at 10 and 9 years after transplantation. and fi nally titrated in log 10 steps on BHI agar plates. The infl u- In conclusion, quantitative analysis of WT1 expression is a ence of DMSO on the bacterial growth was examined by spik- valuable tool for monitoring of MRD after HSCT. Increasing lev- ing DMSO and analysis of bacterial growth after different times. els of WT1 expression strongly predict hematological relapse. In a second series, fi lter of 3 systems were challenged in the Therefore, this approach is very useful for early diagnosis and same way but NaCl 0.9% was used as medium. Additionally, 3 treatment of molecular relapse after HSCT. MRD measurement media fi ll runs with CSL medium were performed to detect even using WT1 expression is more sensitive for the detection of smallest bacterial contaminations during processing. relapse than the analysis of hematopoietic chimerism. Results: In the DMSO series, after spiking but before fi ltration the number of detectable bacteria was already diminished by 4 to 5 log leading to detectable concentrations of 1×10E+01-10E+02/ml. P724 After fi ltration, bacterial growth was not detectable. Bacteria spiked Kinetics of CD3+ T-cell engraftment correlates with in DMSO exhibited a time dependent decline of growth with a com- development of acute GvHD and relapse in patients plete growth inhibition after 5 minutes of incubation. In the NaCl treated with reduced-intensity conditioning allogeneic series, 35 to 50% of the spiked bacteria could be detected after stem cell transplantation spiking and before fi ltration. After fi ltration, bacterial growth was no E. Toffoletti, F. Patriarca, A. Chiarvesio, A. Michelutti, longer detectable. All media fi ll runs led to sterile products. A. Sperotto, M. Cavallin, M.L. Battista, F. Zaja, R. Fanin Conclusions: Although certainly most of the potential bacterial Hematological Division of Udine (Udine, IT) contaminants of hematopoetic progenitor cell (HPC) grafts are not viable after treatment in 99% DMSO (similar results were obtained Objectives: This study investigated the kinetics of whole periph- for S. epidermides as published recently) the sterile fi lter offers an eral blood (PB) and CD3+ T-cells chimerism in patients receiving additional security measure to eliminate potential contaminants. reduced-intensity conditioning (RIC) allogeneic stem cell transplan- Final validation runs processing leukaphereses from unmobilized tation (SCT). The T-cell chimerism has been correlated with risk of healthy donors and evaluating cellular integrity, proliferative capac- grade II-IV acute graft-versus host disease (GvHD) and relapse. ity and sterility of the product are the next task before implementa- Materials and methods: Twenty-eight patients with a median age tion of the system in routine cryopreservation of HPC grafts. of 56 years (range 17-65) affected by lymphoma (19), multiple myeloma (5), acute myeloid leukaemia (3) or idiopathic myelofi - brosis (1) received RIC allogeneic SCT at Hematology Division of Udine between January 2007 and August 2008. Source of Minimal residual disease stem cell was PB for all patients out one, that received one cord blood unit, and donors were matched unrelated for 21/28 patients. Conditioning regimens were: thiotepa plus cyclophos- P723 phamide (13), 2 Gy total body irradiation (TBI) plus fl udarabine Relevance of minimal residual disease and chimerism for (3), melphalan plus fl udarabine (3), total lymphoid irradiation the detection of relapse after haematopoietic stem cell (1). In 18 cases anti-thymocyte globulin was used as part of transplantation GVHD prophylaxis. Hematopoietic chimerism has been serially B. Gruhn (1), J. Sanft (2), N. Pfaffendorf (1), I. Wolff (1), F. Zintl assessed at 30, 60, 90 and 180 days after SCT in whole PB (1), J. Beck (1) and sorted CD3+ T-cells. The analysis have been performed by (1)Department of Pediatrics (Jena, DE); (2)Institute for Forensic polymerase chain reaction (PCR) based amplifi cation of short Medicine (Jena, DE) tandem repeats (STR) sequences using the Ampfl STR identi- fl er kit (Applied Biosystems). Full donor chimerism (FDC) was The relevance of both minimal residual disease (MRD) and chi- defi ned as the presence of at least 95% donor cells. merism after hematopoietic stem cell transplantation (HSCT) for Results: The percentage of patients achieving FDC was lower the detection of relapse has not yet been extensively studied. We in CD3+ T-cells in comparison with whole PB at day 60 (74% vs investigated MRD in 121 consecutive children with ALL (n=56), 79%) onward. Patients with grade II-IV acute GVHD had more AML (n=36), MDS (n=22), or CML (n=7) who underwent HSCT. frequently FDC in CD3+ at day 30 in respect to patients without The Wilms’ tumor gene (WT1) expression was used for the detec- acute GvHD or with grade I (91%vs 61%, p=0.07), while there tion of MRD because WT1 is overexpressed in the vast majority were no signifi cant differences at 60 and 90 days. Patients of patients with leukemia. We performed a quantitative reverse who subsequently relapsed had a signifi cant lower incidence transcriptase polymerase chain reaction (PCR) to examine the of CD3+ FDC at day 30 (55% vs 87%, p= 0.07), at day 60 (50% level of WT1 expression. All 88 patients with continuous normal vs 88%, p= 0.03) and at day 90 (66% vs 100%, p= 0.01) in WT1 expression levels in bone marrow and continuous undetect- comparison with patients with sustained remission.

S196 Conclusions: We conclude that mixed chimerism at days 30, 60 and 90 was associated with an increased risk of relapse and the development of acute GvHD was correlated with FD T-cell chi- merism at 30 day. These fi ndings can help the management of immunotherapy after RIC allogeneic stem cell transplantation.

P725 The predictive value of [18f]fdg -pet/ct after autologous stem cell transplantation F. Sorà, N. Piccirillo, P. Chiusolo, L. Laurenti, M. Lavalle, I. Innocenti, G. Ausoni, S. De Matteis, V. Rufi ni, G. Leone, S. Sica Catholic University of Rome (Rome, IT)

Autologous stem cell transplantation (ASCT) is a standard treat- ment for refractory or relapsed lymphoma. Defi ning response cri- teria based on conventional radiographic characteristics remains diffi cult because anatomical technique, as computed tomography (CT), cannot differentiate between active tumor and fi brosis. 18- fl uorodeoxyglucose positron emission tomography ([18F]FDG- PET/CT), has been clinically accepted and widely use to optimize lymphoma restaging during and after chemotherapy. The aim of this study is to evaluate the role of [18F]FDG-PET/CT to detect residual disease or early relapse after ASCT in order to design treatment intensifi cation or complementary radiotherapy. From December 2004 to June 2008 we enrolled 49 patients (31 males and 18 females, median age: 50 years, range 15-66) affected by lymphoma (16 large B cell, 10 follicular, 8 mantle cell, 2 anaplas- tic CD30, 5 peripheral T cell, 2 other subtypes and 6 Hodgkin’s lymphoma). Patients were homogeneously treated with salvage P726 chemotherapy and received ASCT, respectively 27 patients in In multiple myeloma, stringent CR utilising in situ nucleic CR, 17 patients in PR, and 5 in SD/PD. [18F]FDG-PET/CT was acid hybridisation provides a cheap, robust alternative for performed prospectively on day +100 after ASCT. FDG-PET/CT patient stratifi cation post autograft was negative in 37 patients; 34 are alive without disease progres- K. Lund (1), G. Marron (1), A.N. Parker (1), I.G. McQuaker (1), sion (median follow-up 23,5 months, range 5-38), 2 died in CR R. Jackson (2), D. Moffat (3), A. Clark (1) 5 and 8 months after ASCT respectively from sudden death with (1)West of Scotland Cancer Centre (Glasgow, UK); (2)Glasgow no evidence of lymphoma and from CMV/Pneumocystis Carinii Royal Infi rmary (Glasgow, UK); (3)The Institute of Medical and pneumonia. One patient, affected by T lymphoma, relapse at Veterinary Science (Adelaide, AU) 16 months requiring treatment and is alive at 18 months after ASCT. Twelve patients were FDG-PET/CT positive, respectively The most useful means of assessing response to therapy in 7 in NHL group and 5 in HD group. In this group 5 patients died myeloma is controversial. Multiparameter fl ow cytometry (MFC) from disease progression at a median of 13 months (range 10- and PCR based techniques can accurately predict survival post 39) after ASCT, 5 patient received radiotherapy on involved fi elds autograft. However, the additional impact of such sensitive tests and are alive in CR after a median of 21 months (range 6-37). of MRD on practical decision making in the clinical setting is One patient affected by T lymphoma went on to have relapse/ still unproven. In addition, these techniques are expensive, progression at 8 months after ASCT and required further treat- time-consuming and not accessible in many centres. We have ment. One patient was lost to follow up. found that in situ hybridization (ISH) defi ned “stringent Com- One and 2 years after ASCT OS rates were 94% in PET-nega- plete Remission” (sCR) is simple to calculate and provides tive and 80% and 70% respectively in PET-positive patients excellent prognostic information, comparable to more complex (p=0.011) (Figure 1); PFS rates were 100% and 96% at 1 and tests. Requiring only two informative probes, ISH clearly identi- 2 years respectively in PET/CT-negative patients, meanwhile fi es plasma cell populations and allows accurate assessment of PFS rates were 39.7% at 1 and 2years in PET/CT -positive light chain restriction, in contrast to immunocytochemistry where patients (p<0.0001) (Figure 2). background staining hampers interpretation. We validated this [18F]FDG -PET/CT is able to predict PFS and OS in LNH and technique in 110 patients who underwent autologous stem cell HD after ASCT. Interestingly radiotherapy was able to induce transplant between 1998 and 2004. Informative biopsies were durable CR in some of them. available in 105 patients (>95%). ISH was used to measure light chain restriction of residual plasma cells on trephine biopsy (> 4: 1 or 1: 2 Kappa: Lambda ratio was defi ned as abnormal). CR by standard EBMT criteria was associated with improved pro- gression-free survival (p=0.011). This was more marked for ISH negative patients compared to ISH positive patients (p=0.001). Those in CR with negative ISH results (sCR) had the best out- come with median event-free survival of 81 months compared to 30 months in those that did not attain sCR (p=0.008). Of interest MRD negative/paraprotein positive patients show a trend towards a longer progression free interval than their MRD positive/paraprotein negative counterparts. A similar result is seen in relation to overall survival (sCR vs others = median sur- vival not yet reached vs 86 months (p=0.026)). We do develop informative MFC panels at diagnosis but, since there is no data supporting alteration of therapy post transplant at present and pending further clarifi cation of the clinical utility of these more

S197 expensive tests, we currently use ISH based sCR as a prag- value of fl ow cytometric immunophenotyping (FCM) applica- matic way to stratify patients to enable therapeutic decisions to tion of detecting MRD in stem-cell products compared to that be made and inform patients of their likely prognosis. of seminested polymerase chain reaction (PCR) analyses in patients with indolent lymphoma receiving ASCT. Subjects were 35 patients with bone marrow/peripheral blood involvement of P727 indolent lymphoma before induction chemotherapy who eventu- Early intervention at the stage of molecular relapse after ally underwent ASCT (20 relapsed follicular lymphoma(FL), one allogeneic haematopoietic stem cell transplantation newly diagnosed FL, three relapsed or refractory mantle cell M. Krejci, J. Mayer, M. Doubek, D. Dvorakova, O. Horky lymphoma(MCL) and eleven newly diagnosed MCL). Twenty- University Hospital Brno (Brno, CZ) three patients were male. Median-age was 49 years old. Ten patients received ex-vivo purged ASCT, and 25 received in- Background: The relapse of myeloid hematological malig- vivo purged ASCT. Twenty-nine patients archived complete nancies after allogeneic stem cell transplantation (SCT) is a remission and six archived partial remission. The maximum major problem. The early detection of minimal residual dis- sensitivity of FCM usage of light chain restriction in CD10 and ease (MRD) may provide the opportunity to implement some CD19 positive for FL and in CD5 and CD19 positive for MCL preemptive therapies prior to overt hematological relapse. Here was 1.5 - 10-4, and that of PCR was 1.0 - 10-6. The fi rst fi ve FL we present our experience with treatment of molecular relapses patients receiving in-vivo purging was evaluated according to (MR) after SCT. morphologic, FCM and PCR examinations, which all confi rmed Methods: We analyzed 15 patients (pts) with MR of myeloid tumor-free graft. In the group of in-vivo purging, lymphoma cells hematological malignancies (AML, n=6; CML, n=9) after in marrow and/or peripheral blood samples of all patients were SCT. Molecular relapse in AML was defi ned as the at least not detected before harvest by each examination except one once confi rmed reappearance of fusion transcript detection patient who showed MRD in marrow detected by FCM exami- (CBFB/MYH11, n=1; MLL/AF6, n=2; MLL/ENL, n=2; RUNX1/ nation. With a median follow-up of 41 months, eight patients RUNX1T1, n=1;) or its 10-fold increase, when detected were relapsed (two FL and six MCL). Estimated 3-year overall repeatedly using RQ PCR, and while the bone marrow mor- and relapse-free survival was 87.3% (95%CI: 69.5 to 95.0) and phology, immunophenotype, and cytogenetic examinations 77.1(95%CI: 57.5 to 88.5), respectively. In the patients with FL, remained normal. Molecular relapse in CML was diagnosed 3-year estimated overall and relapse-free survival was 85.0% when a patient had, over a period of at least 4 weeks, (1) (95%CI: 60.4 to 94.9) and 80.0% (95%CI: 55.1 to 92.0), respec- three consecutive samples with BCR-ABL/ABL ratio greater tively. In the patients with MCL, estimated 3-year overall and than 0.02%; or (2) three samples with rising ratio, the last two relapse-free survival was 90.9% (95%CI: 50.8 to 98.7) and greater than 0.02%; or (3) two samples greater than 0.05% 71.6% (95%CI: 33.7 to 90.3), respectively. In conclusion, three- without cytogenetic relapse. The following procedures were color FCM analyses were reliable for detecting MRD. Consid- used to treat the AML MR: chemotherapy (2/6 pts), inter- ering the less expensiveness than PCR method, FCM is the leukin 2 (4/6 pts), donor leukocyte infusions (DLI) (6/6 pts). valuable techniques for detecting MRD in stem cell products. The CML MR were treated by imatinib (4/9 pts) and/or DLI (3/9 Long-term follow up are needed to confi rm the clinical value. pts) or discontinuation of immunosuppression only (3/9 pts). Median age of pts was 46 years (range 21-62). The median fol- low up from start of MR treatment was 28 months (range 8-61). P729 Results: Molecular remission was achieved in 89% of CML pts The time to complete donor haematopoietic chimerism (8/9) and 83% of AML pts (5/6). Next MR were observed in 27% may predict the outcome of allogeneic stem cell of pts (CML, n=2, and AML, n=2) after 2, 10, 11, and 20 months. transplantation With median follow-up of 28 months, 80% of pts (12/15) were C. Vadikolia, I. Sakellari, D. Mallouri, I. Batsis, A. Bitzioni, alive and disease free, 20% of pts died (2 deaths from relapse B. Tahinopoulou, E. Stalika, I. Zorbas, A. Anagnostopoulos, of AML, one death from GVHD without link with treatment of A. Fassas MR). Toxicity of treatment was evaluated according to CTCAE, G. Papanicolaou (Thessaloniki, GR) version 3.0. Hematologic toxicity grade 3 or 4 and febrile neu- tropenia grade 3 were observed in 2 pts treated by chemother- The role of hemopoietic chimerism analysis as a prognostic apy. Other toxicities were moderate to grade 1-2. GVHD grade indicator of relapse after conventional haemopoietic stem cell 3 developed in 3 pts after DLI. transplantation (HCT) is under investigation. We monitored Conclusion: Early intervention at the stage of MR after SCT the chimerism status by serial examination of short tandem seems to be effective treatment in both CML and AML pts. repeat-based polymerase chain reaction (STR) and evaluated Response rate is high (87%) and the toxicity moderate. This the clinical outcome in 93 consecutive patients who underwent strategy is worth to be pursuit in more pts and therefore, new unmanipulated HCT with myeloablative conditioning .Thirty fi ve reliable markers for MRD in AML pts are urgently needed. patients had ALL, 53 AML,2 blast crisis of CML, 2 non- Hodgkin Lymphoma, and 1 had NK leukemia. Thirty six patients were in fi rst CR, 27 in second or third CR and 28 had relapsed or primary P728 refractory disease. The number of infused CD34 + cells were 5,3 Clinical value of fl ow cytometric immunophenotyping (0,99- 12,9) x106/kgr, while CD3+ cells were 3,77 (0,25-12,3) analyses for minimal residual disease detection in x108/kgr. All patients had rapid and successful engraftment. stem-cell products in patients with follicular and mantle Complete donor chimerism (CC) was achieved in 91/93 patients cell lymphoma: comparison to IgH-polymerase chain (98%) at day +18(14-100). Early CC (≤ months) was observed in reaction 75(80%) while late (≥2 months in 18 patients(19,5%). Post-rans- H. Kato (1), K. Yamamoto (1), Y. Oki (1), D. Chihara (1), S. Ine plant fl uctuated values of mixed chimerism(MC) were observed (1), H. Taji (1), M. Seto (2), Y. Kagami (1), Y. Morishima (1) at least once in 39/ 92 patients (42%) at a median time of 10 (1)Aichi Cancer Center Hospital (Nagoya, JP); (2)Aichi Cancer months. Twenty three of them were in hematological relapse. Center Research Institute (Nagoya, JP) Donor lymphocyte infusions or immunotherapy with IFN-g or IL- 2 was given in 28/39 patients. Estimates of disease free survival Lymphoma cell contamination in stem-cell source is consid- (DFS) and overall survival (OS) were 50% and 52% respectively, ered to have a role in eventual relapse in patients receiving at 67 months. In terms of immune reconstitution after HCT, the autotransplant (ASCT). However, most appropriate methods time to complete immunological recovery was 7 months (6- for detecting minimal residual disease (MRD) in autograft have 60). In multivariate analysis we evaluated the time of obtaining not been well described in clinical outcome or cost-effective- CC with the following parameters :DFS, OS, relapse rate(RR), ness. We retrospectively evaluated usefulness and clinical treatment related mortality(TRM), CD34+and CD3+cell dose of

S198 the graft, status of disease pre-HCT, acute and chronic GVHD, and 2008 were retrospectively studied. Fifteen pts were and TBI, or BUCY as conditioning regimen. Early occurrence excluded from the analysis (early death, lack of chimerism of CC was associated with better DFS and OS (51% and 55%) data or prior allogeneic SCT). Pts characteristics are shown in (p=0,03). DFS and OS in patients with late CC was 43% and Table 1. In our standard GVHD prophylaxis, CsA was aimed 33% respectively(p=0,03).The RR was signifi cantly higher in to be tapered from day +50 if no signs of GVHD were present. patients with late attainment of CC(p:0,02). In conclusion, fre- Chimerism analysis was performed by FISH or STR-PCR. quent monitoring of chimerism status by highly sensitive meth- Sequential studies were attempted to be performed in BM and ods might identify impending relapse and allow early therapeutic PB on day +15, +30, +100, +180 and +365, as well as every intervention which could be benefi cial in low disease load. other week in PB in cases with mixed chimerism (MC). Results: Out of the 34 evaluable pts, 26 showed stable complete chimerism (CC) or MC with decreasing recipient cells, therefore P730 allowing standard management with progressive reduction of Quantitative analysis of chimerism by real-time CsA. In this group, 20 patients showed CC in all samples since polymerase chain reaction of insertion/deletion day +30 while MC after day +30 was seen in 6 patients (Table polymorphism is a useful predictor of relapse after 2). Acute GVHD (aGVHD) rate was 46% (15% grades III-IV). allogeneic stem cell transplantation for acute leukaemia Relapse rate was 26% in a median time of 9 months after SCT, N. Jacque (1), N. Dhedin (1), M. Uzunov (1), S. Nguyen Quoc (1), with a median follow-up of 56 months (4-132). Interestingly, the J. Lazarovici (1), L. Sutton (1), J.P. Vernant (1), D. Bories (2) subgroup of pts with MC after day +30 showed a higher relapse (1)Pitie Salpetriere (Paris, FR); (2)Henri Mondor (Paris, FR) rate in spite of similar GVHD rate compared to pts with CC. On the other hand, from the 34 pts, 4 (11%) showed MC with increas- Background and objectives: Quantitative analysis of chimerism ing recipient cells which caused a change in the immunossupre- after allogeneic stem cell transplantation (allo-SCT) for acute sion management (withdrawal of CsA followed eventually by DLI leukemia is helpful to monitor the kinetic of engrafment. Usu- or 2nd SCT). A response, return to CC in PB and/or BM was seen ally, chimerism is assesed by variable number tandem repeat in 2. Finally, chimerism analysis did not predict clinical outcome in (VNTR) or short tandem repeat (STR) amplifi cation by polymer- additional 4 pts: 3 pts showed relapse after day +80 in whom MC ase chain reaction (PCR), which have a sensitivity of 1 to 5%. was detected after relapse showing CC in the immediate prior Insertion/Deletion (InDel) polymorphism analysis by real-time sample, and 1 pt presented graft failure showing CC (day +20). quantitative PCR (InDel-QPCR) is a more sensitive method, so Conclusions: The observation of MC with increasing recipient it should be able to predict relapse earlier. cells was useful for decision making in a small subgroup of pts. Design and methods: We conducted a unicentric retrospective Most pts showed stable CC or MC with decreasing recipient cells study and we evaluated the fi les of all consecutive patients trans- allowing reduction of immunossupresion. However, high relapse planted from May 2004 to April 2008 which relapsed of an acute rate is observed in spite of stable CC, especially in the subgroup leukemia after allo-SCT. Sixteen patients (12 myeloblastic and 4 who show MC after +30. This pt population could benefi t from lymphoblastic acute leukemia) were included in the study. Median earlier withdrawal of immunossupresion or preemptive DLI. age was 45 years (22-60). Conditionning regimen was myeloab- lative in 12 patients, the source of stem cell was bone marrow in 8 patients. Quantitative analysis of chimerism by InDel-QPCR method was usually performed every month post transplant. Results: The median delay between transplant and cytological relapse was 8.5 months (2-32). In three patients the quantifi ca- tion of recipient DNA rate was always superior to 1% at each point post transplant and they presented an early relapse (at 2, 3 and 4 months after transplant). The 13 remaining patients achieved a recipient DNA rate inferior to 1% at a median time of 35 days, in 11 patients recipient DNA rate was less than 0.1% at 56 days and in 7 patients recipient DNA rate was less than 0.01% at a median time of 189 days. The fi rst 13 patients presented an increased chimerism preceding cytological relapse, at a median interval of 52 days (5-154). At the time when an increased chimerism was detected, the peripheral blood count was normal in 12 patients, one had persistent thrombocytopenia since the allo-SCT. Conclusion: The 0.01% to 1% range was found to be valuable for an early relapse prediction. Thus, chimerism evaluation by InDel-QPCR is a necessary method for detection of post trans- plant relapse before cytological abnormalities in acute leuke- mia, allowing early therapeutic measures.

P731 Clinical management of patients in mixed chimerism after myeloablative allogeneic stem cell transplantation from an HLA-identical sibling donor M. Kwon, I. Buño, P. Balsalobre, D. Serrano, G. Rodriguez- Macias, J. Gayoso, A. Pineda, J.L. Diez Martín Gregorio Marañon G. U. Hospital (Madrid, ES)

Monitoring of chimerism kinetics after allogeneic SCT may facil- itate clinical decision making regarding immunomodulation. The objective of this study was to analyze how chimerism dynamics drives clinical decisions in HLA-identical sibling myeloablative allogeneic SCT patients. Methods: 49 consecutive patients (pts) who underwent HLA- identical sibling myeloablative allogeneic SCT between 1996

S199 Graft versus malignancy this study were acute myelogenous leukemia (AML, 188 pts), non-Hodgkin´s lymphoma (NHL, 80 pts), myeloproliferative disease (MPS, 54 pts), acute lymphoblastic leukemia (ALL, 47 pts). The overall survival (OS) and disease free survival P732 (DFS) were estimated according to Kaplan-Meier method. NK-cell education after allogeneic haematopoietic stem Differences between curves were assessed by Mantels’s cell transplantation in humans log-rank test. The Cox proportional hazards model was used P. Haas (1), P. Loiseau (1), R. Tamouza (1), J.-M. Cayuela to assess the independence of OS and DFS from prognostic (2), G. Henry (1), C. Falk (3), D. Charron (1), G. Socié (2), factors in a multivariate model. A. Toubert (1), N. Dulphy (1) The 2 year OS and 5 year OS in this group were 36.6% and (1)INSERM (Paris, FR); (2)Hôpital Saint-Louis (Paris, FR); 26.9% respectively. The DFS at 2 years and 5 years were (3)National Centre for Tumor Diseases (Heidelberg, DE) 50.0% and 42.4%. Diagnosis was associated with a signifi - cant impact on both, OS and DFS (p<0.001 and 0.006), with Natural killer (NK) inhibitory receptors for self-human leuko- NHL and MPS having the best outcome. In pts who survived cyte antigen (HLA) play a central role in the acquisition or more than 100 days (60.8%), limited chronic graft-versus-host maintenance of NK-cell functional competence. This NK-cell disease (GvHD) improved the OS signifi cantly compared to education process relies on interactions with a partner cell extensive chronic GvHD or no chronic GvHD (p=0.002). Inten- whose identity remains undefi ned at present. As NK cells sity of conditioning regimen had no impact on DFS, there was have been reported to exert a potent graft-versus-leukemia a trend for longer OS in pts who received RIC compared to (GvL) effect after allogeneic hematopoietic stem cell trans- those with myeloablative conditioning (p=0.08). In multivariate plantation (HSCT), it is important to determine how NK-cell analysis underlying malignancy, intensity of conditioning regi- education takes place after HSCT. Therefore, we studied the men, acute and chronic GvHD and performance status were process of NK-cell education in a cohort of 28 recipients of associated with OS. unrelated T-cell-replete bone marrow or peripheral stem cell A proportion of pts with advanced hematological diseases graft. Fifteen transplants were fully matched (10/10 allelic achieve long term remission after AHSCT. CGVHD but not the match on the HLA-A, -B, -C, -DQ, and -DR loci), whereas 13 intensity of conditioning has impact on outcome in this setting. showed a partial HLA mismatch (8/10 or 9/10), resulting in Development of transplant protocols with focus on immunologi- a killer immunoglobulin-like receptor (KIR)-ligand mismatch. cal rather than cytotoxic effects is necessary. Using the CD107a mobilization assay, we measured the responsiveness of different NK-cell subsets towards the K562 target cell line, as a function of their expression of inhibitory receptors for self. We show that allogeneic HSCT is followed P734 by a fully effi cient NK-cell education process that is achieved Improved overall survival in patients recovering with high by 3 months after transplant, and lasts for at least 2 years gamma/delta T-cells after allogeneic haematopoietic stem after transplant. In addition, HLA-mismatched HSCT leads to cell transplantation a stable education pattern that is determined by donor- and L. Kordelas, M. Junge, T. Gromke, N. Steckel, R. Trenschel, not by recipient- HLA ligands. This result suggests that the H. Ottinger, A.H. Elmaagacli, D. Beelen NK cell’s partner in education is from hematopoietic rather Universität Essen (Essen, DE) than extra-hematopoietic origin. From a clinical point of view, this donor-ligand-driven NK-cell education model provides the Introduction: T-cells play a crucial role in the Graft-versus- fundamental basis for a sustained and long-lived GvL effect Leukaemia (GvL)-effect. Unfortunately, the GvL-effect is often after HLA-mismatched HSCT. associated with Graft-versus-Host-Disease (GvHD). T-cells can be distinguished into two phenotypic sub-groups by the expression of specifi c alpha/beta- and gamma/delta (GD) T- cell receptors. P733 Methods: Only few studies have investigated the impact Limited chronic GvHD signifi cantly improves outcome of recovery with increased GD-T-cells following allogeneic after allogeneic haematopoietic stem cell transplantation hematopoietic stem cell transplantation (HSCT). A recent study in advanced haematological diseases (Godder et al., BMT 2007) indicated a survival advantage for C. Kahl (1), D. Wolff (2), A. Nogai (3), G. Kundt (4), J. Hahn (2), patients (pts) recovering with high GD-T-cells following HSCT. E. Holler (2), L. Uharek (3), C. Junghanss (1), M. Leithäuser (1), The data presented here emerge from a single-centre analy- E. Thiel (3), M. Freund (1) sis evaluating the role of high GD-T-cells following HSCT in (1)Division of Hematology and Oncology (Rostock, DE); pts with hematologic malignancies. We included all patients (2)Division of Hematology and Oncology (Regensburg, with at least three consecutive analyses of alpha/beta- and DE); (3)Charite CBF (Berlin, DE); (4)Department of Medical GD-T-cells within the fi rst year after HSCT. This cohort of 107 Informatics and Biometry (Rostock, DE) pts. includes the following entities: AML (n=40), CML (n=19), ALL (n=13), MDS (n=11), OMF (n=9), NHL (n=7), and other Despite the fact that the outcome after allogeneic stem cell diseases (n=8). transplantation (AHSCT) for advanced or relapsed hematologic Results: The threshold for the GD-high-group was defi ned as malignancies is poor, it is often performed since it remains the follows: 3 x above the median of all GD-T-cells of the study only curative therapeutic option. We conducted a multicentre, cohort within the fi rst 12 months after HSCT. According to this retrospective analysis in order to analyse variables associated threshold 29 pts (27%) recovered with increased GD-T-cells with outcome in this setting. at 3 or more time points after HSCT. The pts recovering with The median age of the 406 patients (pts) included in this study increased GD-T-cells achieved a signifi cantly higher overall was 46 years (range, 13-70 years), 174 pts (42.5%) received survival (88% ± 12%) compared to pts with lower GD-T-cells a graft from a HLA-identical sibling, whereas 229 (56.4%) (69% ± 6%, p 0.029). There was a trend (p=0.180) to less received an unrelated graft. The median follow up was 282 relapse in the GD-high-group, but also a trend (p=0.083) to days (range 3-3607 days). A variety of conditioning protocols more acute GvHD in the GD-high-group. The multivariate were used. In 53 pts (13.1%) a myeloablative approach was Cox-analysis confi rmed the overall survival advantage for the used and 338 pts (83.3%) received reduced intensity condi- GD-high-group, however, without statistic relevance (p=0.105). tioning (RIC). Advanced stage of a disease was defi ned as Similar sub-group analyses for the recovery with increased refractory disease, partial remission or more than 2nd com- alpha/beta T cells or with increased T helper cells did not indi- plete remission at time of transplant. Diseases included in cate any survival advantage. In addition, elevated alpha/beta

S200 T cells or elevated T helper cells were not directly correlated unrelated donors, 7 from mismatched donors and 2 from with elevated gamma/delta T cells. These analyses support that unrelated cord blood units. Pretreatment acute GVHD grade the benefi cial effect on overall survival is due to the recovery ranged 2-4 (median 3) and involved the skin (25), gut (16) with high GD-T cells. and liver (5). All the patients with chronic GVHD had exten- Conclusions: This study confi rms the hypothesis of a ben- sive involvement prior to therapy. The median time from trans- efi cial effect of high GD-T-cells recovery following HSCT plantation to alefacept was 42.5 days and 13 months in acute regarding overall survival. Further research is warranted to and chronic GVHD respectively (range 18-110d and 3-47.5m) determine more accurately the importance of GD-T-cells to and a median of 9 (range 1-25) injections that were given per possibly develop new therapeutic options in HSCT as e.g. graft patient. engineering. Results: Twenty-eight out of the 37 patients (75.6%) responded to the treatment (19 and 9 in the acute and chronic group respectively). Despite this high response rate, demonstrating the deep immunosuppressive effect of ale- P735 facept, only 3/35 evaluable patients (fi gure 1) have relapsed Reconstitution of KIR-expressing natural killer cells and (with a median follow-up of 32.5 months on the 13 survivors). survival after haplo-identical stem cell transplantation for Other than the 3 patients that relapsed, full-donor chimerism acute myeloblastic leukaemia (100% donor cells and no residual host-type DNA) was sta- M. Stern, L. Ruggeri, C. de Angelis, A. Mancusi, A. Velardi ble throughout the treatment period and later in all but one University Hospital (Perugia, IT) patient, that developed mixed chimerism under alefacept treatment. His chimerism returned to full-donor chimerism Natural killer (NK) cell alloreactivity has been demonstrated with taper-down of immune suppression. Currently, 13/37 to exert powerful antileukemia effects after T-cell depleted patients are alive (fi gure 2), most with improved or stable haploidentical stem cell transplantation. Functional analy- GVHD. Twenty-four patients died due to GVHD progression sis of clonally expanded NK cells is the gold standard to (13), progression of the basic disease (4), infections (4) or assess alloreactivity, but is labor intensive and time con- other causes (3). suming. We investigated the reconstitution of inhibitory NK Conclusion: alefacept is effective and safe for the treatment of cell receptor repertoire by flowcytometrical study of Killer- acute or chronic steroid resistant/dependent GVHD and may cell immunoglobulin-like receptor (KIR)/natural killer-cell carry some discrimination between GVHD and GVL. group-2-antigen (NKG2A) expression on donor derived natural killer cells in 39 patients after haploidentical stem cell transplantation (SCT). The KIR/NKG2A repertoire was skewed in the fi rst 6 months after SCT with reduced numbers of NK cells expressing a single KIR without co-expression of other KIR/NKG2A. Generation of such potentially alloreactive single KIR expressing subpopula- tions was faster for KIR binding HLA-C1, than for slower for KIR binding HLA-C2 or HLA-Bw4. In agreement with the repertoire studies, a retrospective analysis of transplant outcome of 115 patients transplanted for acute myelod leukemia showed bet- ter survival in patients transplanted from HLA-C1 mismatched donors than in patients grafted from donors mismatched for HLA-C2 or HLA-Bw4. In conclusion, these results appear to indicate that the temporal dynamics of reconstitution of NK cells of different allospecifi ci- ties may impact upon survival after haploidentical SCT. As fl owcytometrical studies are limited by several factors, e.g. the fact that antibodies discriminating between activat- ing and inhibitory KIR are not universally available, these studies need to be corroborated by post-transplant functional evaluations of the reconstituting alloreactive NK repertoires by NK cell cloning and cytotoxicity assays against recipient targets.

P736 The effect of alefacept given for steroid refractory/ dependent graft-versus-host disease on relapse rate M.Y. Shapira, L. Dray, B. Resnick, M. Aker, P. Stepansky, B. Gesundheid, R. Or Hadassah – Hebrew University Medical Center (Jerusalem, IL)

Introduction: In general it is expected that strong immune sup- pression that alleviates GVHD, will increase the risk of relapse. Alefacept (Amevive®) is an immunosuppressive dimeric fusion protein that is used for psoriasis control. We showed its remark- able effect in acute steroid resistant/dependent and chronic extensive GVHD. Patients and methods: To date, 37 patients with a median age of 30 years (range 3-66) were treated by us with alefacept due to acute (n=25) or chronic (n=12) steroid resistant/dependent GVHD (23 males, 14 females). Eighteen were transplanted from HLA matched family members, 10 from HLA matched

S201 P737 Results: 132 PBSCT patients received a median of 4 DLIs Escalated lymphodepletion followed by donor lymphocyte (range 1 - 14) starting day 203 (range 25 - 2202) after PBSCT. infusion can induce a potent graft-versus-tumour Average follow up was 816 days post DLI. 27% of the patients response had objective response (CR, CC, PR), 25% had stable disease, T. Guillaume, P. Chevallier, S. Ayari, J. Delaunay, F. Mechinaud, and 38% progressed. 10% of the patients were not assessible P. Moreau, J.L. Harousseau, M. Mohty for response. Objective response was observed independent CHU Nantes (Nantes, FR) of the underlying disease but was more obvious for molecular relapse or MC compared to haematological relapse. Median Donor lymphocyte infusion (DLI) to treat relapse of non-CML overall survival was 1.1 years after fi rst DLI. Patients respond- hematologic malignancies following alloSCT has relatively low ing to DLI had a markedly better prognosis (median survival effi cacy. We recently reviewed our experience of DLI for relapse not reached, p<0.0001) than patients who had stable disease in non-CML diseases: 64 pts received DLI±chemotherapy for (median survival 409 days) or disease progression (median relapse (25 MAC, 39 RIC; 56 Sib., 8 MUD). A response could survival 83 days). Likewise, survival depended on the indica- be achieved in 19 (26%): 7/21 AML, 5/18 MM, 4/10 NHL, 1/5 tion for DLI. Patients who had received DLI for treatment of a HD, 1/5 ALL, 1/3 other. Although these results indicated a GVT full-blown hematological relapse had the worst prognosis with effect, they remain relatively disappointing. Several factors may a median survival of 120 days post fi rst DLI. Acute GvHD was account for this lower effi cacy namely inhibition of effector cells generally mild, with a dose dependence for aGvHD grade I+II. by host-derived regulatory sub-populations. DLI results might be Only one patient died due to DLI-induced aGvHD in CR. improved through manipulation of the recipient immune environ- Conclusion: DLI of stimulated lymphocytes derived from ment to allow for in vivo expansion of donor T cells from the DLI. frozen allogenic grafts after PBSCT is safe and effi cient to For this purpose, we designed a dose-escalation protocol of DLI induce long-term remissions. Timely DLI in beginning relapse preceded by lymphodepletion (LD) using fl udarabine (Flu) and (MC, molecular/cytogenetic) is generally more effective than cyclophosphamide (Cy). Thus far, we treated 12 pts with LD-DLI in full hematological relapse. for post-transplant relapse (3 ALL, 5 AML/MDS, 2 HD, 2 T-NHL; 2 MAC, 10 RIC; 6 Sib., 6 MUD). To avoid overwhelming toxicity due to severe GVHD, we proceeded by sequentially escalating P739 both LD and T cell doses infused. LD consisted in Flu 25mg/m²/d Killer-cell-immunoglobulin-like receptor/HLA mismatches for 3 d followed by infusion of 1x107 CD3+cells/Kg. If no GVHD improve survival after stem cell transplantation of developed and disease persisted or progressed, the same LD patients with acute myeloid leukaemia was followed by a second DLI at 5x107 CD3+cells/Kg. At the next R. Richter, G. Bug, S. Mousset, E. Seifried, H. Serve, C. Seidl, step, LD combined both Cy 600mg/m² for 1 d and Flu 25mg/m²/d H. Martin for 3 d followed by DLI at 5x107 CD3+cells/Kg. Eight, three and Clinic of J.W. Goethe-University (Frankfurt/Main, DE) one pt(s) received respectively one, two or three LD-DLIs. The fi rst LD-DLI was given at a median of 204 d (62-1917) follow- Background: Human class I leukocyte antigens are ligands for ing alloSCT. Of note, chemotherapy to treat relapse prior LD-DLI killer cell immunoglobulin receptors (KIRs). These receptors was administered in 7 pts with only one pt responding success- are expressed by NK and T-cells and thus modulate innate and fully. Gr II-IV acute GVHD following LD-DLI was observed in 5/12 adaptive immunity. KIR mismatches have been suggested to pts (45%) while in our control group of 64 pts without LD, the modify the outcome after stem cell transplantation. The clinical incidence was 36.8% (P=ns). The median onset of acute gr II-IV impact of killer cell receptors may vary between disease entities GVHD following the last LD-DLI was 31 d (range 15-59) not sig- and pre- and post transplant therapeutic regiments. We have nifi cantly different from the control group (34 d; 12-120). At last therefore performed a retrospective study with patients trans- follow-up, 6 pts died: 4 relapses, 1 sepsis, 1 lung carcinoma. No planted in our centre between 1996 and 2007. pt died due to GVHD. Tumor response to LD-DLI was observed Patients and Methods: Samples from 54 donor/patients pairs in 3 pts, all 3 achieved CR. Study of the immunomodulatory out of a consecutive cohort of 147 allografted AML-patients, effects of LD (pre- and post-LD-DLI T cells including Treg and pairs were evaluable for retrospective KIR-ligand matching. cytokine secretion) are currently being further characterized and The median patient age was 47 years (25-66). Patients were will be presented. We conclude that LD-DLI has an acceptable transplanted with G-CSF-stimulated peripheral blood stem cells toxicity if used in a stepwise manner or adapted to the severity (n=51) or bone marrow (n=3) from HLA matched unrelated of the disease. (n=31) or related donors (n=23). KIR typing was performed as previously described. Patients were categorized according to their HLA inhibitory KIR ligand group C1, C2, Bw4, A3/A11 and P738 presence or absence of KIR. Effective induction of graft versus malignancy effect after Results: The overall survival (OAS) of all 54 evaluable patients unrelated allogeneic HCT using donor lymphocyte at 5 years is 53% with a median follow up period of 2,6 years infusions derived from frozen aliquots of the original graft (range 1 – 6.2 years). Twentythree patients died due to relapse J. Hasskarl, A. Zerweck, J. Isernhagen, H. Bertz, J. Finke (n=14) or nonrelapse mortality (NRM, n=9). Patients with a KIR- University Clinic Freiburg (Freiburg, DE) ligand mismatch (KIR-mm) had a trend towards lower relapse and nonrelapse mortality (3/31 vs. 5/23, p=0.12 and 6/31 vs. Purpose: To evaluate the safety and effi cacy of infusions of 8/23, p=0.15, respectively). Overall survival was superior in donor lymphocytes (DLI) derived from frozen aliquots of the pri- patients with KIR-ligand mismatches (KIR-mm) compared to mary graft after PBSCT. the group of patients without mismatches (70% versus 28%, Patients and methods: We retrospectively analyzed the data of log rank p= 0.005). Patients with 2 KIR mismatches (C1 and/or 121 patients with hematological malignancies after allogeneic C2) had even a better survival compared to patients with single PBSCT who were treated with donor lymphocyte infusions. 11 KIR mismatches. of these 121 pts received second PBSCT, each from a different Conclusions: Our results demonstrate that allografted AML- unrelated donor, resulting in 132 transplantations with consecu- patients with KIR mismatch have a signifi cantly superior sur- tive indication for DLI. Lymphocytes were derived from frozen vival. The mortality due to both relapse and as well as NRM is samples of the original (rh-G-CSF stimulated) graft from unre- reduced. Patients with AML benefi t from KIR-ligand mismatched lated donors. Indication for PBSCT was predominantly AML/ allografts. KIR typing could be a useful tool to be included to MDS, followed by ALL, lymphoma and MM, and MPS. Reasons defi ne optimal histocompatibility of donors and patients. for DLI were hematological relapse (70%), isolated cytogenetic/ molecular relapse (4%) or mixed chimerism (13%), pre-emptive DLI (10%), and other reasons (4%).

S202 existence of effective GVL activity. Therefore NST seems to be a promising therapeutic option in this otherwise incurable disease.

P741 The impact of NOD2/CARD15 SNP 5 on the outcome of unrelated donor haematopoietic stem cell transplantation for acute lymphoblastic leukaemia N. Mayor, B.E. Shaw, L. Cooke, S. Marsh, J.A. Madrigal Anthony Nolan Research Institute (London, UK)

The NOD2/CARD15 gene encodes the NOD2 protein which is critically involved in innate immunity. We have recently shown that NOD2/CARD15 Single Nucleotide Polymorphisms (SNPs) 8, 12 and 13 within the genotype of an Unrelated Donor (UD) Haematopoietic Stem Cell Transplant (HSCT) pair result in sig- nifi cant increases in mortality and relapse for acute leukaemia patients. There are over 100 additional NOD2/CARD15 SNPs but knowledge of their functional impact is limited and their affects on HSCT outcome have yet to be established. Here we P740 report the impact of the frequently seen NOD2/CARD15 SNP Molecular evidence for graft-versus-leukaemia effects 5 (P268S) on transplant outcome in addition to SNPs 8, 12 in T-prolymphocytic leukaemia after non-myeloablative and 13. allogeneic stem cell transplantation A sequencing protocol for the entire NOD2/CARD15 exonic M. Rieger (1), M. Brüggemann (2), S. Dietrich (1), U. Hegenbart region was established and used to genotype 87 Acute Lym- (1), M. Hensel (3), M. Moos (1), A.D. Ho (1), M. Kneba (2), phoblastic Leukaemia (ALL) recipients and their UDs trans- P. Dreger (1) planted between 1996 and 2003 in the UK. 66% of pairs were a (1)University of Heidelberg (Heidelberg, DE); (2)University 10/10 HLA allele match (8% 12/12 matched). 81% used myelo- of Kiel (Kiel, DE); (3)Onkologische Schwerpunktpraxis ablative conditioning regimens, 72% included T-cell depletion (Mannheim, DE) with in-vivo alemtuzumab being the usual method. Grafts were bone marrow (82%) and peripheral blood stem cells (18%). T-prolymphocytic leukaemia (T-PLL) is a T-cell malignancy Immunosuppression was Cyclosporine A alone (30%) or with with an aggressive clinical course. Only a small proportion of Methotrexate (46%). patients respond to conventional chemotherapy. Although anec- The observed frequency of SNPs 5, 8, 12 and 13 in this cohort dotal reports suggest that allogeneic stem cell transplantation were 27%, 7%, 1% and 1% respectively. The presence of SNP can provide prolonged disease control in patients with T-PLL, 5 within the pair genotype resulted in signifi cantly higher mor- evidence that this disease is sensitive to graft-versus-leukae- tality (6 years: WT 60%, SNP 24%; P=0.017) due to signifi - mia (GVL) activity is still lacking. Here we describe for the fi rst cantly increased disease relapse (6 years: WT 8%, SNP 47%; time the observation of molecular clearance of residual disease P=0.002). These effects persisted in multivariate analysis (OS: upon onset of chronic graft-versus-host disease (cGVHD) in a P=0.05, RR 2.232; Relapse: P=0.032, RR 5.007). In contrast patient who had undergone non-myeloablative allogeneic stem to previous data, there were no signifi cant effects of SNPs 8, cell transplantation (NST) for T-PLL. 12 or 13 individually or in combination in any of the models Case report: A 58-year old man presented with rapidly progres- analysed. Of the individuals genotyped as having SNP 8, 12, sive monoclonal (V beta 14) CD3+ CD8+ T lymphocytosis, and/or 13, 88% were also positive for SNP 5 indicating that thrombocytopenia and splenomegaly consistent with T-PLL. He there is likely Linkage Disequilibrium (LD) between the NOD2/ achieved complete remission after 1st-line treatment with alem- CARD15 SNPs. tuzumab by clinical criteria, fl ow cytometry, and TCD beta multi- We conclude that the presence of NOD2/CARD15 SNP 5 plex PCR (BIOMED-2 primers, genescan, sensitivity 1-5%). Six within an UD-HSCT pair genotype results in increased disease weeks after the end of alemtuzumab therapy the patient under- relapse and mortality. There were no effects of any of the three went NST from an HLA-identical sibling donor after condition- previously studied NOD2/CARD15 SNPs (8, 12 or 13) on trans- ing with fl udarabine (30mg/m² day 1-5) and cyclophosphamide plant outcome suggesting that they may be markers for a nega- (500mg/m² day1-5). He experienced successful engraftment tive outcome post-transplant and may be in LD with other SNPs and there was no relevant immediate transplant-related toxicity. which are causative of these complications. These data dem- However, full donor chimerism was not achieved unless com- onstrate the need for identifying NOD2/CARD15 haplotypes plete withdrawal of systemic immunosuppression at day +134 and establishing their functional consequences and impact on post transplant. From day +181 onwards, the patient developed transplant outcome. symptoms of limited cGVHD which did not require resumption of immunosuppressive therapy. Post transplant molecular mini- mal residual disease (MRD) monitoring of blood and bone mar- row samples showed reappearance of clonal TCD beta PCR signals starting at day +46. Taqman-based clone-specifi c quan- Non-haematopoietic tissue repair titative TCR beta PCR using 2 markers (J beta 1.3, sensitivity 0.001%; J beta 2.2, sensitivity 0.05%) confi rmed identity with the original T-PLL clone. MRD level was 0.4% at day +134 but P742 became undetectable by J beta 2.2. and decreased to ongo- Pro-atherogenic risk of cellular therapy for vascular ing low-level positivity by J beta 1.3. (<0.01%) from day +209 regeneration? onwards subsequent to onset of cGVHD. 14 months after NST, E. Rohde, T. Pfeifer, K. Schallmoser, A. Reinisch, N. A. Hofmann, the patient is in ongoing clinical remission and shows sustained E. Froehlich, G. Rechberger, G. Lanzer, D. Kratky, D. Strunk molecular response with decreasing signs of mild cGVHD. Medical University of Graz (Graz, AT) Conclusions: This is the fi rst report showing disease response at the molecular level subsequent to immunosuppression with- Cellular therapy for vascular regeneration is the subject of drawal and onset of cGVHD in T-PLL, strongly suggesting the intense clinical research. Ongoing clinical trials mainly use

S203 blood- or marrow-derived cell fractions for transplantation to treated group in the fi rst six treatment months. Shown by lower treat cardiovascular disease. Endothelial colony-forming cells slopes of ALSFRS-R (monthly decrease of 0.77 vs 1.08 points (ECFCs), monocytes and mesenchymal stromal cells (MSCs) in the G-CSF vs controls, respectively, which means about within these transplants are thought to contribute to vessel 30% reduction) and the McGill QoL [decline by only 0.5 point /6 regeneration. Safety concerns regarding atherosclerosis aggra- months vs a decline of 3 fold higher (1.4 points) in the controls], vation have emerged because all three cell types can also con- respectively. The treatment had no major side effects. tribute to atheroma formation. We therefore examined the foam Conclusion: G-CSF administration in ALS patients caused suc- cell capacity of clinically applied vascular regenerative cells as cessful mobilization of autologous stem progenitor cells, within a surrogate marker for potential pro-atherogenic side effects. the normal range. A trend of slowing down disease deterioration Foam cell formation was tested with human ECFCs, CD14+ at six months was observed, although it did not reach statistical monocytes and MSCs which were either immediately exposed signifi cance due to the small number of patients enrolled and for 12 to 84 hours to acetylated low-density lipoprotein (acLDL) the high drop-out rate. No major side effects were observed. or subjected to a 3-day pro-angiogenic induction prior to the A further study with a higher G-CSF dose, possibly a differ- acLDL exposure according to clinical trial protocols. Intracel- ent dose schedule, a combination with an intervention with lular lipid accumulation was detected by Nile Red staining with changes permeability of the blood-brain-barrier, or with one of fl uorescence laser-scanning microscopy. After 3D mapping the novel CXCR4 based emerging cell mobilizing drugs, in a 0.5µm sections along the z-axis, the number of lipid droplets larger number of patients is warranted. (LDs) per cell was determined using ImageJ software. Morpho- metric observations were confi rmed by fl ow cytometry. We found an accumulation of bright fl uorescent LDs in cul- P744 tured monocytes typical for foam cells. Twelve hours of acLDL EPO and MIP-1 alpha are associated with increased levels exposure after a 3-day pro-angiogenic induction resulted of vascular progenitors in autologous haematopoietic in increased numbers of LDs/cell, compared to 12h acLDL- stem cell grafts exposed fresh monocytes (mean±SD: 42±14; range: 28–58 L. Labonte (1), C. Li (1), L. Yang (2), A. Gillingham (2), versus 5±7; 0-18 LDs/cell). A prolonged acLDL exposure for M. Halpenny (2), A. Giulivi (2), D. Allan (1) 84h after pro-angiogenic induction even more clearly enhanced (1)University of Ottawa (Ottawa, CA); (2)Canadian Blood lipid accumulation in monocytes as compared to 84h of acLDL Services (Ottawa, CA) exposure without pro-angiogenic conditioning (100±34; 49-138 LDs/cell; versus 20±7; 13-32 LDs/cell). Despite acLDL uptake, Background: Increased levels of endothelial-like vascular pro- MSCs and ECFCs displayed no obvious LD accumulation as genitor cells (VPCs) in PBSCs used in HSCT have been asso- confi rmed by fl ow cytometry within a seven day observation ciated with reduced transplant-related toxicity. In this study, period. angiogenic and infl ammatory plasma proteins were quantita- Monocytes clearly displayed foam cell formation in our test tively analyzed in patients undergoing autologous HSCT and system that was increased after pro-angiogenic culture. This correlated with graft VPC levels, graft CD34+ levels and trans- data raises serious concerns that cellular therapy with foam plant-related toxicity to identify informative biomarkers for VPC cell-skewed monocytes or monocyte-containing hematopoietic mobilization. cell preparations may be counterproductive and even aggra- Methods: Patients undergoing autologous HSCT were enrolled vate atheroma formation in patients with cardiovascular dis- following informed consent. Using plasma samples from base- ease if underlying pathologic conditions are not appropriately line and on day of PBSC collection, 19 factors were quantifi ed reverted. using multianalyte fl uorescence and/or ELISA. VPC clusters were enumerated using a standard cell culture assay, and trans- plant-related organ toxicity was determined using the Seattle P743 criteria. Variables were dichotomized based on mean values Recombinant human granulocyte colony-stimulating and outcomes were dichotomized using established thresholds factor administration for treatment of amyotrophic lateral (VPCs > or < 2.0x103/kg, CD34 > or < 5.0x106/kg). sclerosis: a double blind randomised study Results: 36 patients were enrolled (mean age 51, 42% female) B. Nefussy (1), I. Artamonov (1), V. Deutsch (1), E. Naparstek and had myeloma (10), non-Hodgkin‘s lymphoma (14 pts), (3), V. Drory (1), A. Nagler (3) Hodgkin‘s Disease (7 pts) and other diagnoses (5 pts). 30 pts (1)Tel Aviv Sourasky Medical Center (Tel Aviv, IL); (3)Tel Aviv underwent autologous HSCT with a median of 7.0 ± 3.9x106 Sourasky Medical Center (Tel Hashomer, IL); (3)Chaim Sheba CD34+ cells/kg and 6.9 ± 10.3x103 VPCs/kg in the PBSC graft. Medical Center (Tel Hashomer, IL) Only plasma EPO levels increased signifi cantly on the day of PBSC collection in comparison with baseline plasma levels Background: Granulocyte Colony-Stimulating Factor (G-CSF) (p<0.05). IL-2, -10, epidermal growth factor (EGF), IFN-alpha has been previously shown to be a neuroprotective factor in and angiopoietin-1 all decreased signifi cantly between base- models of cerebral ischemia and Parkinson’s disease. In addi- line and day of PBSC collection (p<0.05). At baseline, elevated tion it has been demonstrated to have a direct anti apoptotic macrophage infl ammatory protein-1 alpha (MIP-1alpha) levels neuronal effects and also to stimulate neurogenesis in ani- correlated with high graft VPCs (p=0.05) and elevated IL-6 con- mal stroke models. We proposed to use cell population sub- centrations correlated with high graft CD34+ levels (p=0.04). At sets induced by G-CSF to slow down disease progression in date of PBSC collection, elevated erythropoietin (EPO) concen- patients with amyotrophic lateral sclerosis (ALS). trations correlated with high graft VPCs (0.02) while elevated Methods: Patients with defi nite or probable ALS were rand- EGF concentrations correlated with low CD34+ levels (p=0.03). omized in a double-blind fashion to receive G-CSF 20µg/kg High tumour necrosis factor-alpha concentrations on day of (5µg/kg x 4d) or placebo every three months for a year. The pri- PBSC collection correlated with high transplant-related toxicity mary outcome measure was the functional decline as defi ned (p=0.03). by the revised ALS Functional Rating Scale score (ALSFRS-R). Conclusions: The association of different plasma proteins with Secondary effi cacy measures included changes in vital capac- graft VPC and CD34+ cell mobilization suggests VPC and ity, manual muscle strength, compound muscle action potential HSCs mobilize through independent mechanisms. Patients amplitudes, neurophysiological index, McGi11 single item qual- with low MIP-1alpha at baseline may benefi t from interventions ity of life score (QoL). that increase graft VPC levels. Strategies that increase plasma Results: Thirty-nine patients were enrolled (study arm – 19, con- EPO to augment the regenerative properties of PBSC products trol arm – 20). G-CSF was effective in mobilizing CD34+ cells should be considered. to blood. The results showed a trend of slowing disease pro- gression following two cycles of G-CSF treatment in the G-CSF

S204 P745 Bio-Sciences AB, Sweden) according to their different densi- Adipocyte tissue-derived stem cells to treat severe ties: 1.077 versus 1.073 g/ml. cutaneous radiation syndrome: preliminary study in a rat Methods: Samples (n=13) were obtained by BM aspiration pro- model cedures. BM suspensions were scored, mixed with PBS (1:1) D. Agay (1), P. Garrigou (1), F. Forcheron (1), N. Grenier (1), and gently layered on both gradients. Centrifugation was per- L. Touvard (1), Y. Chancerelle (1), V. Meineke (2), F. Hérodin formed at 623g for 20 minutes at RT. The BMMNC were then (1), M. Drouet (1) exposed to the following assays: viability (by 7AAD), FACS (1)CRSSA (La Tronche, FR); (2)Bundeswehr Institute of analyses (CD45, CD14, HLA-DR, CD105, CD90, CD73, GD2, Radiobiology (Munich, DE) CD140, CD146, CD200), clonogenic MSC assay (CFU-F), MSC ex-vivo expansion and differentiations (4). The cutaneous radiation syndrome (CRS) is the well described Results: After separation, there was a similar cell recovery in delayed consequence of localized skin exposure to high doses the 1.077 (25,8±14,5%) and 1.073 (26,5±17,6%) groups. No of ionizing radiations. Based on experimental mouse studies, difference in cell viability was noticed between groups (7AAD+: the benefi t of local injection of bone marrow multipotent mesen- 4,4±5,3% in 1.077 and 4,8±4,5% in 1.073). The FACS analy- chymal stem cells to favour wound healing has been suggested ses on freshly isolated BMMNC indicate that 1.073 signifi cantly after grafting 3 burned patients. However, further studies are reduces the CD45+ cell fraction and enriches CD45-/CD105+/ required to validate these promising results and to elucidate the CD90+/GD2+ cell fractions by approximately 1.4 fold. Moreo- mechanisms involved. The aim of this study was to set up a rat ver, the average number of CFU-F was 1.5 fold higher in 1.073 model of severe CRS in order to clarify some of these points. versus 1.077 samples; similarly all parameters related to cell We especially focused on Adipocyte Derived Stem Cells (ASC) expansion were signifi cantly higher in the 1.073 group. In which represent an alternative source of multipotent cells in particular, after 4 passages, the average MSC number in the regenerative medicine. Following injection, they may favour 1.073 group was 1.8 fold higher than in the 1.077 group. Both re-vascularization of damaged tissues via local trophic factor reagents could isolate MSC showing an expected phenotype secretion. Moreover, their capacity to transdifferentiate remains (CD45-/CD14-/HLA-DR-/CD200low/CD140low/CD73+/CD90+/ debated. CD105+/GD2+/CD146+) and capable of differentiating into Syngeneic Fisher rats were locally irradiated at the back (50 Gy bone (Alizarin-red stain), adipose cells (Oil-red-O stain) and gamma) using a 60Co source (n=4). Pinching the skin above a cartilage (Alcian-blue stain). lead screen during exposure prevents global irradiation. After Conclusions: Our data confi rm that BMMNC fractions contain a 10 days long phase of latency, the infl ammatory processes functional MSC precursors and show that Ficoll-Paque PRE- were clinically revealed by acute pain and erythema. Then MIUM 1.073 provides signifi cantly higher yield of MSCs than hair loss and dry then moist desquamation were successively the standard Ficoll-Paque PREMIUM. observed (weeks 1 to 3). At week 3-4, all the irradiated area (i.e. 1 Castro-Malaspina H Blood 1980; 2 Dominici M Int. J. Cancer about 25 cm²) exhibited a radiation ulcer damage aspect. This 1999; 3 Horn P Cytotherapy 2008; 4 Dominici M Cytotherapy indicates a second-degree burn. During the next three months, 2006. wound healing remained incomplete with iterative infl ammatory waves and moist desquamations which led to partial wound re-opening. General state of the irradiated rats was impaired (prolonged body weight loss). 4 supplementary irradiated Fischer rats were locally injected Lymphoma with Syngeneic ASC (previously cultured in presence of Fibrob- last Growth Factor 2 at 2 ng/ml) at day 7 and day 13 (10x106 twice). Pain was reduced and wound healing improved 2 P747 months after irradiation in all treated rats when compared with Analysis of peripheral blood stem cells mobilisation in controls. In one of them, only hair loss and a localized (3 cm²) patients with non-Hodgkin’s lymphoma treated with the painless fi rst-degree burn at day 30 was observed followed by DHAP regimen plus fi lgrastim or pegfi lgrastim ultimate healing. F. Gaudio, A.M. Giordano, T. Perrone, D. Pastore, M. Delia, Our study suggests the benefi t of the early localized injection C. De Risi, A. Spina, V. Liso, G. Specchia of ASC to favour healing in case of CRS. Work is in progress to Hematology (Bari, IT) manipulate ASC ex vivo to improve their graft effi cacy. High-dose chemotherapy and autologous stem cell transplan- tation play an important role in achieving long-term remission in P746 certain groups of patients with non-Hodgkin’s lymphoma. Refi ning GMP-manufactured density gradient media for A potential advantage of using mobilized peripheral blood stem optimised mesenchymal stromal/stem cell isolation and cells (PBSC) is the possibility of harvesting a great number of expansion hematopoietic stem cells capable of more rapid engraftment. G. Grisendi (1), C. Annerén (2), L. Cafarelli (1), E. Veronesi (1), The DHAP regimen was thus integrated into various treatment R. Sternieri (1), GL Cervo (1), S. Luminari (1), A. Frassoldati plans tailored for NHL patients as a salvage chemotherapy and (1), M. Maur (1), G. Palazzi (1), P. Paolucci (1), P. Conte (1), mobilizing regimen. A a single injection of pegfi lgrastim has M. Dominici (1) been shown to be equivalent to daily fi lgrastim in enhancing (1)University-Hospital of Modena and Reggio Emilia (Modena, neutrophil recovery after chemotherapy, whereas the experi- IT); (2)GE Healthcare Bio-Sciences AB (Uppsala, SE) ences with pegfi lgrastim in mobilization of PBSC are limited. The study included 72 patients with NHL (42 follicular and 30 Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are large cells). The mean age was 37 years (range 17-60). Sixty- valuable therapeutic tools in regenerative medicine and mar- four patients (88.9%) had stage III-IV disease. Forty-eight row transplantation. For decades MSC have been enriched by patients (66.7%) had bone marrow involvement. Mobiliza- density gradient media (DGM) centrifugation using, for exam- tion chemotherapy regimens were DHAP plus fi lgrastim in 38 ple, Ficoll-Paque™ type products with a density of 1.077 g/ml, patients (52.7%) or pegfi lgrastim in 34 (47.2%). Pegfi lgrastim 6 which isolates the BM mononucleated cells (BMMNC)(1-3). mg was given subcutaneously on days +6; fi lgrastim 5 microg/ Objectives: We hypothesized that the use of a lower density kg/days from day +6. may be associated with a further enrichment of MSC progeni- Sixty-fi ve out of 72 patients harvested a median of 12.3 x tors having distinct physical and biological properties. Thus, we 106/CD34+ cells (range 2.5-28.9) after a median of 13 days compared two novel GMP-manufactured DGM (Ficoll-Paque (range 8-16 days), with a single apheresis procedure in 58 PREMIUM and Ficoll-Paque PREMIUM 1.073 GE Healthcare (80.6%) cases. Failure to mobilize, defi ned as failure to reach a

S205 circulating CD34+ cell count of 10/mcl, occurred in 5 patients P749 (13.2%) in the Filgrastim group and 8 (23.5%) in the pegfi lgras- Long-term outcome of high-dose chemotherapy with tim group (p=ns). The mean number of CD34+ cells collected autologous haemopoietic progenitor cells transplant in was 13.2x10e6/kg in the pegfi lgrastim group and 12.3x106/kg high-risk lymphoma in the fi lgrastim group (p=ns). A median of CD34+ >10 cells/ T. Moscato (1), M. Martino (1), G. Console (1), R. Fedele (1), microl in peripheral blood was reached in 12 days (range 8-14) G. Irrera (1), E. Massara (1), G. Messina (1), C. Stelitano (2), in fi lgrastim group and in 15 days (range 12-18) in pegfi lgrastim S. Molica (3), M. Brugiatelli (4), A. Peta (5), C. Musolino (6), group (p=n.s.). A. Abbadessa (7), P. Iacopino (1) The median duration of CD34+ >10 cells/microl in peripheral blood (1)U.O. Ematologia con Trapianto (Reggio Calabria, IT); was 4 days (1-5) in fi lgrastim group and 7 days (3-12) in pegfi lgras- (2)U.O. Ematologia (Reggio Calabria, IT); (3)U.O. Oncologia tim group (p<0,05). To date, 65 patients have been autografted (Catanzaro, IT); (4)U.O. Ematologia (Messina, IT); (5)U.O. with a median of 5.4 x106 CD34+ cells/kg (range 2.5-12.9) with no Ematologia (Catanzaro, IT); (6)U.O. Ematologia Universitaria difference in engrafment speed between two groups. (Messina, IT); (7)U.O. Ematologia (Caserta, IT) The results confi rm the effi cacy and feasibility of PBSC mobi- lization with chemotherapy and single-dose pegfi lgrastim in The purpose was to assess long-term outcome of patients with patients with non Hodgkin lymphomas. Six mg of pegfi lgrastim high risk or relapsed/refractory Lymphoma who have received after chemotherapy induced an adequate mobilization, whereas high-dose chemotherapy with autologous hemopoietic pro- dose and schedule in heavily pretreated patients need further genitor cells transplant (AHPCT). So far, few large studies with investigation. extensive follow-up are available for analysis. Primary end-point was overall (OS) and progression free survival (PFS). OS was defi ned as death from any cause measured from date of trans- P748 plant and PFS defi ned as disease progression or death from The absolute lymphocyte count on day 15 after any cause. Data on 138 pts undergoing AHPCT at our institute, autologous peripheral blood haematopoietic stem from September 1992 through July 2007. The median age of cell transplantation is associated with the clinical the pts was 48 y (range 14-73). Median time from 1st treatment outcomes in non-Hodgkin’s lymphoma patients to AHPCT was 19 mo. (range 4-276). Forty-four pts (31.9%) T. Moscato, M. Martino, G. Messina, G. Irrera, G. Console, had Hodgkin Disease (HD), 94 (68.1%) had Non Hodgkin Lym- D. Marcuccio, A. Dattola, R. Fedele, E. Massara, M. Cuzzola, phoma (28 indolent and 64 aggressive). The majority of the pts, I. Bova, E. Spiniello, P. Iacopino 123 (89.2%) were undergoing transplant as part of savage treat- U.O. Ematologia con Trapianto (Reggio Calabria, IT) ment for their refractory/relapsed disease. One hundred-twenty- four pts (82%) were chemosensitive to their last chemotherapy The absolute lymphocyte count (ALC) recovery of 500 cells/ regimen. At time of transplant, 123 pts had responding disease ml or more at day 15 (ALC-15) after autologous peripheral (54 CR, 69 PR), 15 pts had stable/untested relapse/refrac- blood hematopoietic stem cell transplantation (ASCT) has tory disease. Seventy-seven percent of pts received BEAM as been reported as a powerful, independent prognostic indica- conditioning treatment and the peripheral blood was source of tor of clinical outcomes in multiple hematologic malignancies. stem cells in 86.2%. Post-AHPCT, 67% (93/138) pts attained However, few large studies with extensive follow-up are so far CR with an overall response (CR+PR) of 92.8%. Four pts had available for the analysis of the impact of ALC-15 on the out- non-relapse mortality. Of 93 pts attaining CR, 27 relapsed; 5 come of pts with high risk or relapsed/refractory Non Hodgkin’s after attaining CR for >2 y and 1 after attaining CR for> 5 y. The Lymphoma (NHL). One hundred four patients (61 males and 43 median follow-up of surviving pts was 48 mo. (range 8.0-175.0). females) with median age of 49 years (16-73) were include in Median OS/PFS from ASCT was 10 y/10 y. Five-year OS/PFS the study. The median number of previous cycles of chemother- was 62.9/58.7% and 10-y OS/PFS was 49.1/42.9% for whole apy was 2. Stem cell mobilization consisted of various types group. Five-years PFS for pts with HD, indolent and aggressive of chemotherapy associated with G-CSF 5 µg/kg/day. Forty- NHL was 61.6%, 48.7% and 60.1% (P=NS), respectively. Cox- nine out 104 pts (47.1%) were in CR at the transplant time; regression analysis identifi ed only the disease status at trans- the others showed an advanced status of disease. The main plant having independent impact on OS (P=0.003). Regarding conditioning regimens were BEAM (78 pts) and Melphalan plus PFS in multivariate analysis, responding disease at transplant Mithoxantrone (16 pts). Variables analyzed with regard to PFS and female sex were associated with a better prognosis. These and OS were age, sex, LNH histotypes, previous number of data provide a long follow-up for pts receiving AHPCT for high chemotherapy lines, previous treatment with MabThera, dis- risk and relapsed/refractory lymphoma. In particular, long-term ease status at transplant, number of infusedCD34+ and mono- OS/PFS is possible following AHPCT for indolent lymphoma. nuclear cells, ALC pre-apheresis, ALC-15. Median number of Overall, the type of lymphoma does not affect the results of infused CD34+cells was 4.8 x 106/kg (2.0-16.0). The median the transplant. In addition, the disease status at the time of the time to reach PMN >500/ml and PLT >30.000 was 11 days (8- transplant and sex affect outcome with the best results seen in 21) and 13 days (7-35), respectively. After a median follow-up responding and in female pts. of 28.1 mo. (1-181), 75 (72.1%) and 69 pts (66.3%) were alive and without progression or relapse CR, respectively. Univariate analysis demonstrated a remarkable impact of ALC-15 on OS P750 (0.0051) and PFS (0.0026). Cox regression analysis showed Modifi ed BEAM schedule (lomustine instead carmustine) that ALC-15 (p=0.017) and disease status at the time of trans- in the conditioning of ABMT for lymphoid malignancies plant (p=0.036) were the predictive factors infl uenced OS. P. Perfetti, C. Scollo, E. Terruzzi, M. Parma, E. Pogliani, In addition, ALC more than 500/mL and a high number of infused P. Pioltelli CD34+ cell were predictive for longer PFS. Our study confi rms AO San Gerardo (Monza, IT) that the number of ALC on day 15 was related with PFS and OS in NHL patients. A threshold number of CD34+ cells should not In the autologous setting BEAM is the most widely used condi- be the only parameter considered for an adequate PBSC col- tioning regimen to treat lymphoid prolypherative disorders, but in lection, probably the number of lymphocytes should be aimed recent years the availability of BCNU (carmustine), which needs for as well. an alcoholic solvent to be infused intravenously, seems ham- Finally, this study may contribute to support the hypothesis pered by technical problems in its production. CCNU (lomus- that the immune system may be important in the disease con- tine) another nitrosurea, available for oral administration, is the trol also in the ASCT setting, but to better understand these most logical drug to overcome these diffi culties and we test results, an immunological study is needed to evaluate the vari- the feasibility of such a variance at the BEAM schedule. From ous subset of lymphocytes. 1995 to 2008 we uniformly treated 127 patients (74 females

S206 and 53 males) with poor prognosis lymphoma, both non-Hodg- (38%) had died, 21 due to disease and one CR patient died of kin’s (NHL) and Hodgkin’s (HD), with a modifi ed BEAM using drowning. 5 (9%) are alive with disease. OS is 62%. Median OS lomustine instead of carmustine (lomustine 200mg/m² on day has not reached yet. Thirty-one (53%) patients had an event 1, etoposide 200mg/m² + cytarabine 200mg/m² b.d. on days 2- and EFS is only 47%. Probability of EFS and OS is 45% and 5 and melphalan 140mg/m² on day 6) before autologous stem 55% at 11 years. Negative prognostic factor for OS was pres- cell transplant (ASCT). In our series the overall median age was ence of B symptom at relapse / progression, P = 0.032 (prob- 45.42 years (range 16.6 – 68.52), 45.79 (range 16.6 – 68.52) ability of survival 27% vs. 60%, P = 0.0025). Despite high CR, in the males group and 45.17 (16.8 – 68.04) in the females EFS is only 47%. one. Fifty-seven patients were in complete remission (RC) at Conclusion: We have reported high response rate with 76% transplantation, 36 patients had a partial response to previ- CR. Almost quarter of these patients with CR has relapsed. ous therapies (RP) and 34 a progressive disease. The median EFS is only 47%. B symptom at relapse was a signifi cant nega- number of CD34+ cells infused was 5,04x106/Kg (range 1,5- tive prognostic factor for OS. Better treatment strategies are 21,2). The median time to neutrophil (>500x106/L) and platelet needed for this group of patients. (>20x109/L) engraftment was 11 days (range 8-25) and 14 days (range 6-35) respectively. Granulocyte-colony stimulating fac- tor (G-CSF) was given to 105 patients (83%). Oral mucositis was observed in 75 patients (59%), 32 had grade II (25%), 22 grade III (17%) and 16 patients grade IV (12%). Forty-seven (37%) patients were supported with parenteral nutrition. Fifty- six (44%) patients had grade I-II diarrhea, 5 (4%) grade III. No grade IV diarrhea was observed. Fever of unknown origin (FUO) was observed in 56 (44%), in 32 patients (25%) the infectious agent was identifi ed. Three patients had a bacterial pulmonitis. At a median follow-up of 3.66 years (range 0.1- 9.4 ) the overall survival (OS) is 72% and disease free survival (DFS) is 66%. The most frequent cause eof death was disease progression, two patients (1.5%) developed an acute leukemia and four patients (3%) a fatal infection. Our data seem demonstrate that lomustine can be safely used as part of BEAM with outcomes comparable with those reported for the original schedule.

P751 High-dose chemotherapy and autologous stem cell transplant for relapsed or refractory Hodgkin’s lymphoma in 58 adolescent patients. Evaluation of prognostic P752 factors and their impact on survival Tandem auto-SCT and reduced-intensity conditioning S. Akhtar (1), A. El Weshi (2), M. Rahal (3), M. Abdelsalam (1), allo-SCT for relapsed or transformed aggressive B cell H. Al Husseini (1), I. Maghfoor (1) non-Hodgkin’s lymphoma (1)King Faisal Specialist Hosp. & Res. Ctr (Riyadh, SA); A. Clavert, E. Brissot, V. Dubruille, B. Mahe, T. Gastinne, (2)Centre Hospitalier Public du Cotentin (Octeville., FR); N. Blin, P. Chevallier, T. Guillaume, J. Delaunay, S. Ayari, (3)King Fahad Specialist Hospital (Dammam, SA) B. Saulquin, A. Moreau, J.L. Harousseau, P. Moreau, S. Le Gouill, M. Mohty Objectives: To review clinical outcome and prognostic factors CHU Hôtel-Dieu (Nantes, FR) for over all survival (OS) and event free survival (EFS) in ado- lescent patients with recurrent and/or primary refractory Hodg- Allo-SCT can provide a potentially curative graft-ver- kin’s lymphoma (HL) after high dose chemotherapy (HDC) and sus-lymphoma effect. However, such benefi t is usu- autologous stem cell transplant (ASCT). ally offset by a signifi cant incidence of non-relapse Methods: From 1996 to May 2007, 135 consecutive patients mortality (NRM), especially in advanced and/or elderly with HL had HDC ASCT, 58 of these were between the ages patients. With this background, RIC regimens for allo-SCT of 14-21 years. Impact of various prognostic factors (table) have arisen as an attractive modality in patients with NHL, but prior to the initiation of salvage chemotherapy on EFS and OS the role of RIC allo-SCT in aggressive B cell NHL remains a was evaluated using multivariate regression analysis. Primary matter of debate because of a higher incidence of disease refractory HL (PR-HL) is defi ned as patients who failed induc- relapse/progression. tion chemotherapy i.e. only partial response (PR), no response This single centre study aimed to assess the potential benefi t (NR) / stable disease (SD), progressive disease (PD) or relaps- of a tandem auto-allo-SCT approach as salvage therapy in 23 ing within 3 months. consecutive patients with relapsed or transformed aggressive Results: 29 male (50%), 29 female (50%), Median age at diag- B cell NHL. Median age at time of allo-SCT was 55 (range, nosis 15 years (6 to 20) and at ASCT 17 years (14 to 21). Prior 44-65) y. NHL histology at diagnosis included 5 cases (22%) of to salvage chemotherapy, stages I:II:III:IV were 4:20:10:24, DLBCL, 12 (52%) follicular NLH, and 6 (26%) other subtypes. bulky disease (≥8 cm) in 17 (29%), involvement of mediastinum Aggressive transformation was documented in 18 patients in 39 (67%), spleen in 12 (21%) and extranodal involvement in (78%; 26% of primary and 52% of secondary transformation). 25 (43%) patients. Relapsed disease in 24 (41%) and PR-HL Patients received a median of 2 (range, 1-4) lines of previ- in 34 (59%) patient. 39 patients (67%) had tissue confi rmation ous chemotherapy regimens prior to allo-SCT, and all patients at relapse /progression. ESHAP was used as salvage; median (n=21; 91%) but 2, could actually proceed to auto-SCT prior of 3 cycles. Post ASCT, overall response in 48 patients (83%); to allo-SCT. At time of allo-SCT, 9 patients (39%) were in fi rst complete response (CR) / CR-u in 41 (71%), PR in 7 (12%), CR, 6 patients (26%) were beyond fi rst CR and 8 (35%) in PR. NR / SD in1 (2%) and PD in 9 (15%) patients. Three patients Median times between diagnosis and allo-SCT and auto-SCT achieved CR after XRT post HDC. No treatment related death. and allo-SCT were 25 (range, 7-131) and 4 (range, 2.4-71) m. Combining all the CRs, total of 44 (76%) patients achieved CR. respectively. The RIC regimen consisted of Fludarabine, Busul- Median follow up from diagnosis is 63 months and from ASCT fan and ATG in 20 patients (87%) and Fludarabine and low dose 43 months. Currently 14 patients (24% of 58 patients and 32% TBI (2 Gy.) in 3 patients (13%). After RIC allo-SCT, engraftment of 44) who achieved CR had relapsed and 31 (53%) in CR. 22 was achieved in all patients. With a median follow-up of 38

S207 (range, 3-86) m., 13 patients had grade 2-4 acute GVHD (56%) and 10 patients had extensive chronic GVHD (48%). Overall, 9 patients died (3 from progression, 2 from acute GVHD, 2 multi- organ failures, and 2 other causes). The incidence of NRM was 26%, (95%CI, 8-44%). At last follow-up, 13 patients (57%) were in sustained CR. The Kaplan-Meier estimate of progression- free and overall survival rates were 51% and 64% respectively at 4 years. We conclude that a tandem auto-RIC allo-SCT approach is a potentially effi cient salvage therapy for relapsed or transformed aggressive B cell NHL, with a relatively low toxicity, likely over- coming the poor prognosis usually associated with this pheno- type. These results can also likely be improved with strategies aiming to enhance the GVL effect (e.g. inclusion of Rituximab as part of the RIC regimen, and/or as maintenance therapy).

P753 Long-term results of fl udarabine/melphalan as reduced-intensity conditioning regimen in mantle cell lymphoma: age matters J. Gayoso (1), R. Martino (2), P. Balsalobre (1), I. Heras (3), J.L. Piñana (2), D. Serrano (1), J. De la Serna (4), J.F. Tomas (5), A. Fernandez de Sevilla (6), A. Sureda (2), J.L. Diez-Martin (1), D. Caballero (7) P754 (1)Hospital Gregorio Marañon (Madrid, ES); (2)Hospital Sant High-dose sequential chemotherapy with autologous Pau (Barcelona, ES); (3)Hospital Morales-Meseguer (Murcia, stem cell transplant in relapsed or refractory Hodgkin’s ES); (4)Hospital Doce de Octubre (Madrid, ES); (5)MD lymphoma: long-term results and late toxicity. A single Anderson (Madrid, ES); (6)ICO (Barcelona, ES); (7)Hospital center experience Clinico (Salamanca, ES) C. Tecchio, F. Randon, G. Quaresmini, F. Frattini, A. Andreini, R. Di Bella, M. Sorio, F. Benedetti Myeloablative conditioning for allogeneic transplantation has Policlinico G.B. Rossi (Verona, IT) been reserved to young patients(pts) without co-morbidities due to its high toxicity. Reduced intensity conditioning (RIC) Background: A minor part of patients affected by Hodgkin’s regimens have been used trying to reduce this, while preserv- lymphoma (HL) fail to respond or relapse after primary con- ing the establishment of a graft-vs-tumor effect (GVT). Few ventional treatments. Although high-dose chemotherapy with homogeneus series have been reported focused on MCL. We autologous peripheral blood stem cell transplant (PBSCT) is report the outcome of consecutive MCL pts who received a the standard of care, long-term results and late toxicity still peripheral blood allo-RIC from an HLA-identical sibling donor. remain unsatisfactory. The RIC consisted in fl udarabine 125-150 mg/m² from day -8 to Objective: To retrospectively analyze the response rate, free- -4 and melphalan 80-140 mg/m² on days -3 and -2. Rituximab dom from treatment failure (FFTF), long-term outcome and late 375 mg/m² was added to RIC in 8 pts on days -9, +1, +8 and toxicity in a series of 71 consecutive HL patients treated with +15. GVHD prophylaxis consisted in cyclosporine+methotrexat high-dose sequential chemotherapy (HDSCT) and autologous he. Cyclosporine tappering was anticipated at day +50 if mixed PBSCT following relapse or failure to conventional therapies. chimerism or persistent disease were present. Patients and Methods: From march 1986 to october 2008, 71 Results: Between 2000 and 2008, 21 MCL pts were included: patients affected by relapsed or refractory HL were recruited to Median age 56 years (40-68), 16 males, median pre-alloRIC received HDSCT (cyclophosphamide 7g/m², day 0, followed by treatment lines 2 (1-4), 3 previous autoHSCT. Before allo-RIC, PBSC harvest; methotrexate 8 g/m² and vincristine 1.4 g/m², 15 pts (71%) were in 1st or later CR, 6 had chemo-sensitive PR +20; vepesid 2 g/m², +28; melphalan 140 mg/m² and mitox- (29%). Median CD34+/kg infused cells were 5,8x106(2,5-10,8) antrone 60 mg/m² +48-50) and autologous PBSCT following and 2,0x108(0,7-4,1) CD3+lymphocytes/kg. All pts engrafted second-line therapy. Eligibility criteria were age between 14 and reaching ANC>500 on day +16(13-20) and platelets >20.000 65 years, adequate organ function, biopsy-proven relapsed or on day +12(8-32). Early toxicity(

S208 treatment presents a signifi cant late toxicity. Further efforts are patients had HD (BEAM-21, LACE-10) while 13 had NHL awaited to improve this treatment approach in order to avoid (BEAM-8, LACE-5). Six patients in BEAM had chemorefrac- late adverse effects. tory relapse compared to 2 in LACE. The body mass index and serum albumin level at the time of transplant were com- parable. The mononuclear count (MNC) of the graft was com- parable (BEAM- 5 x 108 /kg, LACE-6.1 x 108/kg), though all patients in the LACE group received PBSC grafts (100% Vs 70%; P=0.018). Grade 3 and 4 OM was more commonly seen in BEAM (60% Vs 6%; P=0.04) and more patients in BEAM received parenteral nutrition (69% Vs 13%; P=0.0005). Seven patients in LACE did not develop any OM (P=0.001). The maximum grade and duration of diarrhea were comparable. No patient in LACE developed VOD compared to 4 in BEAM. Sep- sis leading to death occurred more frequently in BEAM (17% Vs 6%; P=0.462). The median days to NE (10 Vs 12; P=0.001) and PE (12.5 vs 19; P=0.002) were shorter in LACE. This dif- ference remained statistically signifi cant even after excluding patients who did not receive PBSC grafts in BEAM. No patient in either group developed pulmonary toxicity attributable to chemotherapy. The complete response rates at day 100 were comparable (BEAM-58%, LACE-60%). Conclusion: LACE is better tolerated than BEAM in lymphomas. The follow up in the LACE group is short to compare survival rates.

P756 Clinical outcomes of high-dose chemotherapy using mitoxantrone, etoposide, cytarabine, and melphalan (NEAM) followed by autologous stem cell transplantation in chemosensitive relapsed or primary refractory aggressive non-Hodgkin’s lymphoma patients J.W. Kim (1), B.S. Kim (1), S.M. Bang (1), J.S. Kim (2), S.T. Lee (2), I. Kim (1), S.S. Yoon (1), J.S. Lee (1), K.S. Han (1), S. Park (1), B.K. Kim (1) (1)Seoul National University College of Medicine (Seoul, KR); (2)Yonsei University College of Medicine (Seoul, KR)

Chemosensitive relapsed or primary refractory aggressive non-Hodgkin’s lymphoma (NHL) patients (pts) could benefi t from high-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT). We report clinical outcomes of HDC using novel regimen consisting of mitoxantrone, etoposide, cytarabine, and melphalan (NEAM) with ASCT in these pts. Between December 2005 and November 2008, 45 pts (male, 60.0%) from 2 hospitals were included. Median P755 age was 43 years (range, 20-71). Median follow-up duration Comparison of toxicity of BEAM vs. LACE regimen in was 11.4 months (range, 0.6-35.5). Treatment response was patients of lymphoma evaluable in 39 pts. Overall response rate (RR) was 82.1%: N. Kumar, A. Joshi, M. Shah, S. Kannan, S. Gupta, R. Nair, 56.4% attained complete remission and 25.6% attained par- N. Khattry tial response. Median event-free survival (EFS) was 6.9 Tata Memorial Center (Mumbai, IN) months (95% CI, 4.2-9.6). Median overall survival (OS) has not been reached and the estimated 1-year survival rate was Background: BEAM (carmustine, etoposide, ara-c and melpha- 60.5%. There was no signifi cant difference in RR, EFS, and lan) is commonly used high dose regimen for Hodgkin’s disease OS between chemosensitive relapsed pts (n=14) and pri- (HD) and non-Hodgkin’s (NHL) lymphomas. LACE (lomustine, mary refractory pts (n=31). Median duration of hospitalization ara-c, cyclophosphamide and etoposide) is a well tolerated and was 31 days (range, 19-70). Median number of CD34+ cells effective regimen. We retrospectively compared the two regi- infused was 3.92x106/kg. Median time to recovery of neutrophil mens for early toxicity. (>500/microliter) and platelet (>20,000/microliter) was 13 days Methods: Patients of HD and NHL with primary refractory or (range, 9-50) and 21 days (range, 0-235), respectively. Forty relapsed disease from August 1994- August 2008 were included. pts (88.9%) had febrile neutropenia and 16 pts (35.6%) had BEAM (carmustine-300 mg/m² x 1 day(d), etoposide 200mg/m² microbiologically documented sepsis. Grade 3-4 hepatic toxicity x 4 d, ara-c- 200 mg/m² 12 hourly x 4 d and melphalan-140 developed in 11 pts (24.4%) and grade 3-4 renal toxicity in 2 pts mg/m² x 1d) was used between August 1994-March 2007. April (4.4%). Grade 3-4 cardiac toxicity developed in 3 pts (6.7%): 2 2007 onwards, all patients received LACE (lomustine-200 mg/ pts had new symptomatic dilated cardiomyopathy and 1 pt had m² x 1d, etoposide 1000mg/m² x1d, ara-c 2000 mg/m² x 2d aggravation of preexisting dilated cardiomyopathy. Transplant- and cyclophosphamide 1800mg/m² for 3d).Oral mucositis (OM) related mortality (TRM) occurred in 4 pts (8.9%): all resulted and diarrhea were graded according to WHO scale. Neutrophil from uncontrolled infection. Pts with low baseline serum albu- engraftment (NE) and platelet engraftment (PE) were defi ned min level (<4.0g/dL) or low baseline hemoglobin (Hb) level according to standard defi nition. Veno-occlusive disease (VOD) (<9.0g/dL) had signifi cantly shorter EFS (P=0.029, P=0.001, of the liver was recorded according to Seattle criteria. respectively) or OS (P=0.004, P<0.001, respectively). In the Results: Twenty- nine patients received BEAM and 15 received multivariate analysis, these variables maintained independent LACE with comparable median age (26y Vs 30y). Thirty-one prognostic value. Low baseline Hb level (<9.0g/dL) was also

S209 associated with signifi cantly longer duration of hospitalization who did not respond to MINE-ESHAP had signifi cantly lower (P=0.034) and higher incidence of TRM (P=0.012). In conclu- OS and EFS that those in CR or PR (46% vs 74%; and 35% sion, HDC using NEAM with ASCT demonstrated considerable vs 69%, respectively). In conclusion, MINE-ESHAP results in effi cacy with relatively modest toxicity as a salvage treatment of a high response rate with acceptable toxicity in patients with chemosensitive relapsed or primary refractory aggressive NHL HL having failed ABVD. Intensifi cation with HDT and ASCT pts. Baseline serum albumin and Hb levels were independent results in a high remission rate and long-term EFS, particularly prognostic factors in this group. in patients who respond to MINE-ESHAP.

P758 Outcome of 132 autologous stem cell transplantations in relapsed or refractory Hodgkin’s lymphoma Z. Csukly, A. Barta, A. Batai, V. Goda, L. Gopcsa, L. Lengyel, P. Remenyi, M. Reti, A. Sipos, E. Torbagyi, A. Tremmel, T. Masszi St. Istvan and St. Laszlo Hospital of Budapest (Budapest, HU)

The purpose of this study was to assess prognostic factors of overall survival (OS) and disease free survival (DFS) of patients with relapsed/refractory Hodgkin’s lymphoma after autologous stem-cell transplantation (ASCT). Data were reviewed of 132 consecutive patients 1994 and 2008. Patients caracteristics was 81 men and 51 women, median age at ASCT 29 years (18- 60), stage (Ann-Arbor) at diagnosis: stage I n=5 (4%), stage II n=60 (60%), stage III n=28 (25%), stage IV n=21 (18%), bulky disease at diagnosis n=26 (20%), median time to ASCT 30 month ( 9-130 ), primer therapy refractory n=41 (36%), relapsed < 1 year n=34 (30%), relapsed > 1 year n=38 (34%), tumor status at ASCT: complete remission (CR) n=27 (37%), residual tumor n=46 (63%), type of stem cell graft: periferial blood stem P757 cell n=108 (82%), bone marrow n=18 (14%), mixed n=6 (4%), Salvage chemotherapy with alternating MINE-ESHAP conditioning: BEAM n=111 (84%), Bu/Cy n=21 (16%), median regimen for relapsed or refractory Hodgkin’s lymphoma follow up from ASCT 501 day (6-4526). Before the year 2006 previous to autologous stem cell trasplant: tumor stage at ASCT was defi ned with computer tomography, a single-centre experience after 2006 with positron emission tomography (PET-CT). C. Fernández de Larrea, C. Martínez, A. López-Guillermo, Results: Three-year OS/DFS was 82/76%. Three-year OS/DFS M. Rovira, F. Fernández-Avilés, E. Montserrat, E. Carreras was in the primer therapy refractory group (PT) 74/62%, in the Hospital Clinic (Barcelona, ES) group relapsed < 1 year (ER) 79/74% and in the group relapsed > 1 year (LR) 100/94%. Three-year OS/DFS was in the non- High-dose chemotherapy (HDT) and autologous stem-cell bulky group 82/73% and in the bulky group 81/46%. Accord- transplantation (ASCT) are considered the standard of treat- ing to CR and to residual tumor group at ASCT the three-year ment for most patients with relapsed or refractory Hodgkin lym- OS/DFS was 100/80% and 73/65% respectively. 96% of the phoma (HL). Response to pre-transplant salvage chemotherapy PT, 56% of the ER and 24% of the LR group had residual tumor remains the most important prognostic factor in these patients. at ASCT. Median time to progression after ASCT was 305day However, the optimal salvage regimen has not yet been estab- (22-745). Allo-PBSCT (sibling) after ASCT were in 7 and MUD lished. We retrospectively analysed the effi cacy and toxicity PBSCT in 5 cases because of disease progression. of MINE (ifosfamide, vinorelbine, etoposide) alternated with Conclusion: Post-ASCT, there was no signifi cant difference nei- ESHAP (etoposide, high dose cytarabine, cisplatin, metylpred- ther in OS nor DFS between the early ( I, II ) and late ( III-IV ) nisolone) in the treatment of relapsed or refractory HL after fi rst- stage groups. One of the reasons can be the incorrect stage at line chemotherapy. Sixty-one patients (median age 36 years, diagnosis not defi ned by PET-CT. There is signifi cantly better range 18-63; 31F/30M) who either did not achieved a complete OS and DFS in LR than ER and PT groups. Patients without response (CR) (partial response, PR n=13, or refractory dis- bulky disease showed similar OS and not signifi cantly better ease n=17) or relapsed (n=31) after ABVD based chemother- DFS ( p=0,091 ) than patients with bulky. There was signifi - apy were included in this study. After MINE-ESHAP (median cant difference both in OS and DFS between the patients with number of cycles 4, range 1-6), 25 patients (41%) achieved a residual disease at ASCT and patients in CR. Early PET-CT CR, 23 (38%) a PR, four (7%) a stable disease (SD) and eight should be done to defi ne PT group of patients. Theese patients (13%) progressed for an overall response rate (ORR) of 79%. need early intesifi cation of chemotherapy followed by ASCT In the univariate analysis, response to fi rst-line chemotherapy to improve OS and DFS. In case of relapse/progression after was the most important prognostic factor for response to MINE- ASCT allo-PBSCT could be curative. ESHAP (ORR 70% for patients with PR, SD or refractory HL vs 87% for patients relapsing after fi rst CR, p>0.0001). Grade 3-4 neutropenia, thrombocytopenia, and anemia occurred in P759 26 (46%), 23 (40%), and 22 (38%) patients. Eleven episodes Long-term outcome of autologous haematopoietic stem of grade 3 non-hematologic toxicities (infectious n=8, renal cell transplantation following a uniform conditioning n=2, etoposide allergic reaction, n=1) were observed. No toxic regimen in patients with Hodgkin’s lymphoma – A deaths were seen. Adequate peripheral blood stem cell collec- single-centre experience tion was achieved in 56 of 58 (96.6%) mobilized patients. After A. Czyz, A. Lojko, L. Gil, D. Drozdowska, D. Dytfeld, A. Nowicki, MINE/ESHAP, two patients with progressive disease received M. Kozlowska, K. Sawinski, M. Komarnicki only palliative care and an additional patient died of a HL-unre- University of Medical Sciences (Poznan, PL) lated disease. As a result, 58 patients eventually received HDT and ASCT. After ASCT, 48 patients achieved CR and three PR. Autologous hematopoietic stem cell transplantation (autoHCT) Overall survival (OS) and event free survival (EFS) after all is considered the treatment of choice for patients with relapsed treatments were 68% and 61% at 5 years, respectively. Patients Hodgkin lymphoma (HL). However, the optimal therapy for

S210 primary refractory disease and chemoresistant relapse has Results: After (R-)DHAP the ORR was 42% in 43 evaluable pat. not been established. We retrospectively evaluated the long- (CR: 7%, PR 35%). The ORR was 60% in 55 evaluable pat. term outcome of 156 pts, median age 28 (16-59) years with treated with (R-)IEV (CR: 25.5%, PR 34.5%). advanced HL (stage IIB-IVB) treated with autoHCT between Stem cells were mobilized in 94.4% after (R-)DHAP com- 1992 and 2007 in our department. The identifi ed indications pared to 94.5% after (R-)IEV. Transfusions were needed after for autoHCT included: partial response to 1st line therapy (75 29% of (R-)DHAP cycles. Transfusion dependency was 0,75 pts), relapse (53 pts), primary refractoriness to chemotherapy erythrocyte and 0,30 platelet units/cycle. Fever in neutrope- (8 pts) and high risk disease at diagnosis (17 pts). The disease nia occurred in 26% of patients with no septic death. 3/61 pat. state at transplant was: CR (64 pts), PR (74 pts) and less then developed acute renal failure. PR (9 pts). All patients were conditioned with modifi ed BEAM (R-)IEV: Transfusions were needed after 33% of cycles. Overall regimen (BCNU 300 mg/m², etoposid 800 mg/m², cytarab- transfusion dependency was 0,74 erythrocyte and 0,26 plate- ine 6000 mg/m², melphalan 140 mg/m² and dexamethasone let units/cycle. Fever in neutropenia occurred in 44% of the 168 mg/m²). Eleven pts were treated with tandem autoHCT patients, causing death of 3 patients. and received different preparative regimen before second Conclusion: In both groups the addition of R neither infl uenced transplant. The source of progenitor cells was PB (79 pts), stem cell mobilisation nor the frequency of neutropenic fever. BM (65 pts) and BM plus PB (12 pts). The median follow-up The main toxicity in treatment with (R-)IEV was neutropenic of the living patients is 65 (12-196) months. There was 6% fever and sepsis. In the (R-)DHAP group the main toxicity was (9/156) early deaths due to infections (8 pts) and primary graft acute renal failure. The higher ORR achieved with (R-)IEV failure (1 patient). OS was 82% (95% CI 62-100), 70% (95% compared to (R-)DHAP should be interpretated with care in this CI 57-84) and 44% (95% CI 7-80) at 7 years for patients trans- retrospective analysis. planted due to partial response to 1st line therapy, relapse and primary refractory disease respectively estimated with the Kaplan-Meier method. The 7-yrs DFS was 68% (95% CI 51- P761 86), 38% (95% CI 18-58) and 43% (95% CI 0-88) for these Single or double autologous stem cell transplantation for three groups respectively. The 7-yrs DFS for a subgroup of 6 transformed aggressive B-cell non-Hodgkin’s lymphoma pts with chemorefractory relapse was 22% (95% CI 0-59). The A. Clavert, V. Roland, S. Le Gouill, V. Dubruille, B. Mahe, pts with partial response to 1st line therapy as the indication T. Gastinne, N. Blin, P. Chevallier, T. Guillaume, J. Delaunay, for autoHCT had a superior OS (p=0.017) and DFS (p=0.014) S. Ayari, A. Devys, G. Flandrois, B. Saulquin, A. Moreau, to the other group. The 16/17 pts with high risk disease trans- P. Moreau, J.L. Harousseau, M. Mohty planted in CR1 live in remission, one died due to relapse. The CHU Hôtel-Dieu (Nantes, FR) secondary malignancies developed in 5/156 (3%) pts, includ- ing 3 MDS/AML, 1 NHL and 1 solid tumor. We conclude that Transformed aggressive non Hodgkin’s lymphoma (TANHL) high proportion of patients with advanced HL who achieved has a poor outcome with conventional therapies. Patients with only partial response to standard treatment can be cured with adverse prognostic factors or who didn’t achieve complete autoHCT following modifi ed BEAM regimen. The outcome remission after initial treatment may benefi t from intensive of pts with relapse and primary refractory disease is inferior, chemotherapy approaches. However, the role of autologous but approximately 40% of patients can achieve long lasting stem cell transplantation (auto-SCT) in TANHL is not yet well response. The survival of pts with chemorefractory relapse is defi ned. not satisfactory. This single centre report investigated the potential benefi t of a single or double auto-SCT in 25 patients with TANHL. Median age at time of fi rst auto-SCT was 55 (range, 32-65) years. P760 TANHL histology was confi rmed in all cases (22 cases of trans- (R-)DHAP vs. (R-)IEV as salvage treatment for relapsed formed follicular NHL and 3 cases of Richter following chronic or refractory B-cell NHL: comparison of response rates, lymphocytic leukemia). Patients received a median of 1 (range, stem cell mobilisation and toxicity 1-3) line of previous chemotherapy regimen prior to the fi rst B. Dengler (1), A. Müller (2), U. Keller (1), C. Peschel (1), auto-SCT. Among the 25 patients, 14 patients (56%) received a J. Duyster (1), H. Menzel (1) single auto-SCT, while 11 (44%) received 2 auto-SCT. Median (1)Klinikum rechts der Isar (Munich, DE); (2)Med. Klinik time between diagnosis of transformation and fi rst auto-SCT V (Erlangen, DE) was 4.6 (range, 2.6-20.1) months. At time of fi rst auto-SCT, 10 patients (40%) were in fi rst complete remission (CR) and 15 High dose Chemotherapy and auto-SCT is used for consoli- patients (60%) in partial remission (PR). The high dose chemo- dation of relapsed/refractory B-NHL after response to salvage therapy administered prior to auto-SCT consisted of Carmus- treatment. The ideal salvage protocol for stem cell mobilization tine, Etoposide, Aracytine and Melphalan (BEAM) for the 14 and cytoreduction remains controversial. Here we report data patients undergoing a single auto-SCT. In the double auto-SCT of a retrospective analysis from a single center regarding the group, patients received Novantrone-Aracytine followed by Overall Response Rate (ORR), the stem cell mobilisation, and BEAM (n=7; 28%), Novantrone-Aracytine followed by TBI/Cy the toxicity with regard to the salvage chemotherapy used. (n=3; 12%) and BEAM followed by TBI/Cy in one case (4%). Methods: The following protocols were used: Ifosfamide, Epi- In this series, the median follow-up is 23 (range, 3-63) months. rubicin and VP16 (IEV), q21; R-IEV: addition of Rituximab (R) Overall, 3 patients died, all from disease progression. All of 375 mg/m². Cisplatin, Cytarabin, and Dexamethason (D-HAP) these 3 patients could only achieve PR after the fi rst and sec- q21; R-DHAP: addition of R. 375 mg/m². G-CSF was given at a ond auto-SCT. At last follow-up, the remaining alive 21 patients dose of 5-10micg/kg. (R-)DHAP: 61 patients (pat.) were treated (84%) were in CR and 1 (4%) in PR. The Kaplan-Meier esti- between 9/98 and 4/08. 32/61 pat. received R.. Pat. suffered mate of overall survival was 88% at 4 years after auto-SCT, with from B-cell NHL (25 DLBCL, 12 FL, 12 MCL, 12 other NHL). this being comparable between patients who received a single 21 pat. had refractory disease, and 17 early relapse. (R-)DHAP auto-SCT versus those who received a double auto-SCT. was administered as 2nd line therapy in 34, and ≥ 3rd line ther- These results suggest that high-dose chemotherapy and auto- apy in 27 pat. 52 DHAP cycles were given in 29 pat. and 68 R- SCT is an effi cient therapy for TANHL. Moreover, double auto- DHAP cycles in 32 pat.. (R-)IEV: 82 pat. were treated between SCT is feasible in this group of patients without any added 12/97 and 11/03. 14/82 received R.. Pat. suffered from B-cell toxicity. However, it remains unclear whether the double auto- NHL (26 DLBCL, 44 FL, 12 pat. other NHL). 17 had refractory SCT approach may result in any long term increased effi cacy. disease, and 35 relapsed early. (R-)IEV was given as 2nd line therapy in 59, and ≥ 3 rd line therapy in 23 pat.. 142 IEV cycles were given in 68 pat. and 30 R-IEV cycles in 13 pat.

S211 P762 response in 32 pts (74%) and a stable or progressive disease in Intermediate VP-16 (1gr/m²) plus G-CSF (10mcg/kg) or 11 pts (26%), while 6 pts did not perform pre-ASCT CT. Overall cyclophosphamide (3gr/m²) plus G-CSF (10mcg/kg) for survival (OS) and progression-free survival (PFS) at 4 years progenitor cell mobilisation in lymphoma patients were 69% and 66%, respectively. Pre-ASCT PET and CT were A. Pivkova, S. Genadieva Stavrik, Z. Stojanoski, L. Cevreska, statistically signifi cant for both OS (p.004 and p.006, respec- O. Karanfi lski, V. Milenkov, B. Georgievski tively) and PFS (p.000 and p.000, respectively). Considering University Hematology Hospital (Skopje, MK) pts by histology, pre-ASCT PET showed a defi nite prognostic role in NHL (OS p.0004; PFS p.0001) but not in HD (OS p.6409; Cyclophosphamide (CTX) 3gr/m² plus G-CSF 10mcg/m² is PFS p.067), where pre-ASCT CT was superior (OS p.036, PFS commonly used regimen for mobilization of peripheral blood p.0000)). Considering PFS in all pts, pre-ASCT PET positive progenitor cells (PBPC) to support high-dose chemotherapy and negative predictive values (PPV; NPV) were 63% and 90%, (HDT) followed by autologous stem cell transplantation (ASCT) respectively, pre-ASCT TC PPV and NPV were 91% and 87%. in patients with lymphoproliferative malignancies, although this Multivariate analysis showed pre-ASCT PET as the only prog- schedule is associated with higher rate of hemorrhagic cystitis, nostic factor for OS, but not for PFS. prolonged neutropenia, infective complications and other tox- Our data showed a prognostic role of pre-ASCT PET in pts with icities. Therefore we evaluated the effi ciency of VP-16 (1 gr/ NHL, while its role is less defi nite in HD pts, probably due to a m²) +G-CSF 10mcg/kg for PBPC mobilization, as possibility to small cohort. The use of PET before ASCT in HD still needs lower the underlying toxicities, since the optimal dose for CTX further investigation. and VP-16 as mobilizers of PBPC is still unknown. We analyzed 55 patients with lymphoproliferative malignancies treated with HDT/ASCT. The patients were mobilized either with P764 cyclophosphamide 3gr/m² + G-CSF 10mcg/kg starting from day Autologous stem cell transplantation improve outcome in 5 of chemotherapy regimen or VP-16 (1 gr/m²)+G-CSF 10mcg/ non-Hodgkin’s lymphoma: results of a computer science kg. Both regimens were effective in the progenitor cell mobili- analysis zation and almost 84% of analyzed patients reached at least M. Pelosini, F. Baronti, S. Galimberti, F. Caracciolo, E. Benedetti, 2x106/kg CD34+ cells with median 3 (ranges 1-6) apheresis F. Papineschi, A. Starita, M. Petrini procedures. Only two patients with HD failed mobilization in University of Pisa (Pisa, IT) the CTX+G-CSF group and none in the group mobilized with VP-16+G-CSF. Patients mobilized with VP-16+G-CSF revealed Objective: Detection of prognostic factors in Non Hodgkin lym- less toxicity; lower duration in hospital days (6 vs 10, p<0.001), phoma (NHL) patients trough an application of a partitioning lower infective complications (30% vs 69%, p<0.001), less recursive algorithm. supportive treatment including Plt transfusions (2% vs 17%, Methods: Dataset comprised 651 patients affected by NHL p<0.004), days on parenteral antibiotics after mobilization (0 treated from 1990 to 2005, divided in High grade (HG NHL, vs 5 days, p<0.001). We can conclude that these two regimens n=343; 52,7%) and Low grade Lymphoma (LG NHL, n= 308; are equally effective in terms of PBPC mobilization in patients 47,3%). Only responsive patients were considered. with lymphoproliferative diseases, but intermediate VP-16+G- We evaluated as variables for algorithm analysis: age, sex, CSF schedule revealed less toxicity, lower hospital days, less histological subtype, IPI status and bone marrow involvement, supportive care. These results encourage the study of such treatment approaches (poli-chemotherapy, mono-chemoterapy, schedule in a randomized trial, as well as evaluating its role in radiotherapy, surgery, purine based chemotherapy, monoclonal the onset of the posttransplant secondary malignancies. antibodies, transplant approach, oral chemotherapy), response to therapy, previous successful treatments, previous relapses, previous failed therapies. P763 Data were analyzed with the Hypothesis testing Classifi er Pretransplantation positron emission tomography as System algorithm (HCS), which belongs to the family of recur- predictive factor in malignant lymphomas sive partitioning algorithms. Such an algorithm is thought to L. Nassi, S. Bassi, M. Negri, L. Travaini, E. Cocorocchio, splits data in different subgroups that behave in a different F. Gigli, A. Vanazzi, L. Preda, P.R. Rafaniello, L. Orlando, way. It works starting from data and utilized them to create D. Laszlo, G. Martinelli all the possible combinations of splits available. Among them European Institute of Oncology (Milan, IT) it chooses the best one by statistics and fi nally applies it to patients’ datasets. For these reason it is defi ned as “partition- Autologous stem-cell transplantation (ASCT) is a well established ing”. Afterwards the algorithm starts again the analysis on treatment in both Hodgkin’s (HD) and non-Hodgkin’s lympho- the subset previously detected and that’s because it is called mas (NHL). Chemosensitivity, usually assessed with computed “recursive”. tomography (CT), is a major prognostic factor in ASCT; Positron Results: The most important split was found between patients emission tomography (PET) has been proposed for evaluation in partial remission (PR) and complete remission (CR). of chemosensitivity before ASCT. We present our fi ndings with Among PR patients, two subsets with worse prognosis were pre-ASCT PET and CT in HD and NHL patients (pts). found: both comprised patients with HG NHL not treated with Forty-nine pts (22 NHL (11 diffuse large B-cell, 8 mantle cell, 2 monoclonal antibodies and/or transplant. Therefore the remain- follicular, 1 Burkitt) and 27 HD) were evaluated in this retrospec- ing PRs patients, treated by transplant approach and immuno- tive study. Median age at diagnosis was 36 years (range 18- therapy, had a better outcome. 67); 22 pts were stage I-II, 27 pts stage III-IV; B-symptoms were In CR group two subset (different only for age) with HD NHL present in 53% of pts, and 23 pts had bulky disease. 41 pts were diagnosis not treated by oral chemo alone were detected. Those considered for ASCT because of relapse (18 pts; median time patients, who had a better outcome, had been treated aggres- to relapse 14 months (range 2-64)) or refractory disease after sively with polichemotherapy and/or autologous transplant. Dif- induction therapy (23 pts); eight pts with NHL (6 mantle cell and ferences between detected groups are statistically signifi cant 2 diffuse large B-cell) received ASCT in fi rst complete remission with p.value 0,03 maximum. according to internal guidelines. Conditioning consisted of BEAM Conclusion: Application of computer science analysis to NHL in 37 pts, Mitoxanthrone-Melphalan in 9 pts, high-dose Zevalin in patients has been successfull. Quality of response emerged as 2 pts, standard-dose Zevalin-Busulphan-Melphalan in 1 patient; the most important prognostic factor but among both PRs and Rituximab was added to conditioning in 20 NHL pts. Median time CRs patients those with better outcome had HG NHL diagnosis between PET and ASCT was 15 days (range 2-36). and were treated by autologous transplantation or/and immuno- Pre-ASCT PET was negative in 30 pts (61%) and positive therapy. This analysis confi rms data available about transplant in 19 pts (39%); pre-ASCT CT showed a partial or complete as a good approach for NHL patients.

S212 patients has shown disease free status of 72.1 months and 2 of 3 patients had shown relapse or progression within 2.0 months. When response was re-assessed with CT images, post-ASCT response did not correlated with patients’ survivals in 7 patients (33.3%) but, with PET study, post-ASCT response did not corre- lated with patients’ survivals in 4 patients (19.0%). According to PET results, the 2-year EFS rate was 60.0 ± 18.2% in patients with negative post-PET result and 15.4 ± 10.0% in patients with positive post-PET result (P=0.026). The 2-year OS rate was 70.0 ± 18.2% in patients with negative post-PET result and 23.1 ± 11.7% in patients with positive post-PET result (P=0.062). Conclusion: post-ASCT PET results after ASCT had substan- tial improvement in response estimation and patient’s out- comes in T- cell NHL. Although routine PET study in T-NHLs is currently not recommended, it seems to have signifi cant benefi t in prediction of patients’ prognosis after ASCT as it does in B-cell NHL.

P766 Rituximab plus donor lymphocyte infusion to prevent or treat relapse for B-cell malignancies after allogeneic haematopoietic stem cell transplantation I.M. Cavattoni, H. Schieder, T. Zabelina, U. Bacher, F. Ayuk, C. Wolschke, A. Zander, N. Kröger University Medical Center Hamburg Eppendorf (Hamburg, DE)

Dose-reduced conditioning followed by alloHSCT is a poten- tially curative therapy in B non Hodgkin lymphomas (NHL). However, the number(no) of patients(pts) who experienced an early relapse is signifi cant, and the achievement of a pro- longed second remission is usually a rare event. Post trans- plant immunotherapy with DLI to induce graft versus lymphoma (GVL) effect is a reasonable option to prevent or treat relapse, however the risk of graft versus host disease (GvHD) is high. Since B-cells might serve as antigen presenting cells to induce GvHD, additional B-cell depleting therapy with monoclonal anti- body (antiCD20) might reduce the risk of GvHD while inducing additional cytotoxicity to B-cells originating from B-cell lym- P765 phoma. In the current pilot study we investigated the feasibility The role of post-transplantation 18F-fl uoro-deoxyglucose of a combined Rtx plus DLI therapy after alloHSCT to prevent positron emission tomography in T-cell non-Hodgkin’s (n=10) or treat relapse (n=2). Twelve consecutive pts, affected lymphoma patients by B cell malignancies and transplanted between July 2002 B.S. Sohn, S. Kim, D.H. Lee, S.W. Kim, I.K. Hong, J.S. Ryu, and February 2007, were included in this study. The median J. Huh, J.S. Lee, C. Suh age at alloHSCT was 58 ys (range 27-64). The conditioning Asan Medical Center (Seoul, KR) regimen consisted of melphalan, fl udarabine and ATG (n=9) or busulfan, fl udarabine (n=3). Donors were HLA identical sibling Introduction: Although there had been numerous studies about in the half of the group, with one HLA mismatched-unrelated the usefulness of 18F-fl uoro-deoxyglucose positron emission donor. Seven pts were not in disease remission at the time of tomography (FDG-PET) for predicting patients’ outcomes fol- HSCT. The indication of Rtx-DLI was prophylaxis of relapse in lowing autologous stem cell transplantation (ASCT), there all but two pts. The schedule of this approach was: 4 consecu- had been little reports about T-cell Non-Hodgkin’s Lymphoma tive weekly Rtx(375mg/mq), started at a median time of 194 dy (NHL). from HSCT(range 77-895).DLI in single dose was administered Patients and Methods: After searching registry, we identifi ed after Rtx (median dose 1x106/Kg), without ongoing immunosup- 21 patients with T-cell NHL, in whom FDG-PET was performed pression and signs of GVHD. Three pts developed acuteGVHD after ASCT. The usefulness of FDG-PET study for predicting after DLI, 2 gastro-intestinal and 1 cutaneous, whereas 6/12 pts patients’ outcomes, including event-free survival (EFS) and experienced chronic GVHD, limited in all but two. Two cases overall survival (OS), were re-assessed and compared with of bacterial and one case of Pneumocystis jirovecij pneumonia conventional procedures including computed tomography (PJP) were diagnosed, in 3 different pts, and 2 died in complete (CT). remission due to infection. The median no of B lymphocytes Results: In 21 patients, there were 14 patients who had con- before and after Rtx was 42/µL and 4 respectively, and the dif- cordant results from both CT and PET result; 5 patients had ference is not statistically signifi cant in terms of incidence of complete response (CR) at CT and negative PET result, 9 infection (µ2 test). After a median follow up of 21 mo (range patients had Not-CR at CT and positive PET result. Seven of 4-37) from the 1st Rtx infusion, 8/12 pts are alive at the last twenty-one patients (33.3%) showed discordant response esti- follow-up, six of them without disease, whereas 2 pts died due mation between CT image and PET study. In these patients, to progressive disease. Rtx and DLI post alloHSCT seems to post-ASCT PET detected additional abnormal uptake in 4 of be a safe and effective approach to prevent early relapse in B 7 patients, which suggesting new pathologic lesions and not malignancies in complete remission after alloHSCT. reported by CT images; two cases of new extranodal involve- ments and two cases of nodal involvements. All of 4 patients showed relapse or progression within 4.2 months. Remaining 3 patients showed negative PET result, although their lesions were evaluated as Not-CR according to CT images, 1 of 3

S213 P768 Vinorelbine and gemcitabine as a salvage regimen prior to stem cell transplantation in refractory or relapse Hodgkin’s disease Z. Aki, G. Sucak, A. Yegin, Z. Özkurt, M. Yagci Gazi University Faculty of Medicine (Ankara, TR)

Background: The current practice for the management of relapsed or refractory Hodgkin’s disease (HD) patients is high dose chemotherapy followed by autologous stem cell trans- plantation (ASCT). However, there is no standart salvage chemotherapy prior to transplantation. In the present study we evaluated the effi cacy and toxicity of the combination chemo- therapy vinorelbine and gemcitabine as salvage therapy prior to SCT in relapsed or refractory HD patients. Patients and Methods: A total of 30 HD patients [7 women and 23 men; median age 28 years (range 17- 61)] with relapsed (n= 13) or refractory (n=17) disease were treated with vinorelbine 30 mg/m² and gemcitabine 1000 mg/m² on days 1 and 8 and 15 of each 28 day cycle with granulocyte colony stimulating factor (G- CSF) support. One patient (3,3%) died during chemother- P767 apy due to septic shock. Second cycle of chemotherapy was TEAM (thiotepa, etoposide, cytarabine, melphalan) as used as stem cell mobilization regimen in 16 patients (53,3%). conditioning regimen for lymphoma treatment with Response was assessed after 2 cycles of therapy and then 22 autologous haematopoietic stem cell transplantation patient underwent ASCT, fi ve patients (16,6%) with prior ASCT A.M. Carella (1), G. Palumbo (2), M. Greco (1), E. Merla (1), and 1 patient (3,3%) with bone marrow involvement underwent M. Dell’Olio (1), P. Scalzulli (1), S Capalbo (2), G Pisapia (3), allogeneic SCT. P. Mazza (3), F. Ferrara (4), A. Guarini (5), A. Iacobazzi (5), Results: Twenty nine of the 30 patients, 29 were eligible for P. Musto (6), N. Cascavilla (1) assessment of response prior to SCT. Eleven patients (36,7%) (1)IRCCS “Casa Sollievo Della Sofferenza” (San Giovanni achieved complete remission (CR) and 8 patients (26,7%) had Rotondo, IT); (2)Ospedali Riuniti (Foggia, IT); (3)Ospedale partial remission (PR), with an overall response rate of 63,4%. G. Moscati (Taranto, IT); (4)Ospedale Cardarelli (Naples, IT); All the patients (100%) were succesfully collected during recov- (5)IRCCS Istituto Tumori (Bari, IT); (6)IRCCS CROB (Rionero ery from second cycle of chemotherapy [median of 5,97 x 106 in Vulture, IT) CD34 cells/ kg (range 1,78- 27]. After a median follow up of 16 months (range 1- 48) progression free survival (PFS) was Background: Standard conditioning regimen before an autolo- 48,5% and overall survival (OS) was 73,7%. Hospital admis- gous haematopoietic stem cell transplantation (AHSCT) for sions were required in 4 patients (13,3%) due to lymphoma chemosensitive relapsed Hodgkin Lymphoma (HL) and Non related poor performance status. Among non hematological Hodgkin Limphoma (NHL) patients (pts) is still represented by toxicities grade III- IV hepatotoxicity was observed in 7 patients a scheduled combination of carmustine, etoposide, cytarabine (23,3%) and infections in 3 patients (10%). Grade III- IV hema- and Melphalan (BEAM ). Despite the effi cacy of this regimen, it is tological toxicity was observed as anemia in 12 patients (40%), disadvantaged by the presence of carmustine, which is respon- neutropenia in 22 patients (73,3%) and thrombocytopenia in 10 sible of a high pulmonar toxicity with fi brosis and progressive patients (33,3%). reduced diffusion capacity. In order to prevent pulmonar toxic- Conclusion: Stem cell collection is sucessfull in all patients ity, we replaced carmustine with Thiotepa in 26 chemosensitive attempted with Vinorelbine and Gemcitabine which can be relapsed HL and NHL pts. We have also evaluated the effi cacy administered in an outpatient setting and shows signifi cant effi - and tolerability of this new regimen. cacy against refractory or relapse lymphoma. Patients: At day -7 26 pts received 10 mg/kg thiotepa (5 mg twice every 12 hours) followed by cytarabine 200 mg/m2 die (-5 to -3) etoposide 200 mg/m2 die (-5 to -3) and melphalan P769 140 mg/m2 (day -2). Out of these pts, 16 were male and 11 GEMOX is an effective and low-toxicity salvage protocol female. Fifteen pts were affected by da NHL and 11 by HL. for refractory and relapsed advanced Hodgkin’s At the moment of transplant only 8 pts were in CR, 14 were lymphoma in PR and 4 were in progression. All patients were in second L. Facchini (1), B. Sanchez-Gonzalez (2), D. Valcarcel (1), or sebsequent line of therapy. The median time of neutrophil A. Salar (2), A. Ferrari (1), J. Delgado (1), J. Briones (1), >500 and platelets >20000 recovery was of 10 (range 8-12) J. Sierra (1), A. Sureda (1) and 12 (range 10-20) days respectively. No severe infectious (1)Hospital de la santa Creu y sant Pau (Barcelona, ES); event has been observed. Eight pts maintain the CR, 12 have (2)Hospital del Mar (Barcelona, ES) reached CR, 3 have a stable disease and 3 pts did not respond to the treatment. Three pts that were in CR have subsequently Introduction and objectives: The treatment of refractory and relapsed. relapsed advanced Hodgkin’s lymphoma (HL) is challenging. Conclusions: Our experience with TEAM protocol has shown Gemcitabine-containing regimens have been used as salvage a signifi cant effi cacy not lower to our historical experience with treatment although little is known about their effi cacy, toxicity BEAM protocol. The tollerability was signifi cantly accetable and profi le and ability to mobilize progenitor cells into the peripheral no serious adverse event has been documented during the blood (PBPCs) for autologous stem cell transplantation (ASCT) treatment or during aplastic phase, therefore the TRM at 100 procedure. Herein, we report our experience with the combina- days has been equal to zero. In no case, oral mucositis was tion of gemcitabine-oxaliplatine (GEMOX) as salvage protocol higher than grade 2, even if most of the pts underwent total for relapsed or refractory HL patients in terms of effi cacy, toxic- parenteral nutrition. Finally no cases of serious organ toxicity ity and stem cell mobilization ability. was observed (pulmonar, hepatic, cardiac and renal). Hemat- Matreial and methods: From 1/2007 to 6/2008, 19 consecutive opoietic recovery, stimulated with G-CSF, has been reached patients with advanced refractory or relapsed HL have been as scheduled and no cases of prolonged neutropenia were included in two different institutions. The GEMOX protocol con- observed. sisted on the combination of gemcitabine 1000 mg/m² iv (day

S214 1) and oxaliplatin 130 mg/m² iv (day 2) every 28 days. G-CSF living in CCR 2 after alternative therapy, 2 patients relapsed was administered from day +7 until granulocytes > 0.5 x 109/l after 1,4 and 2,1 years and died 4,8 and 2,8 years after trans- x 3 consecutive days in the peripheral blood. Response rates plantation. were evaluated as complete remission (CR), partial Remission Conclusion: A conditioning high dose protocol containing Rituxi- (PR) and progressive or refractory disease (PD) following Che- mab is a safe and effective option for elderly patients with man- son criteria (JCO, 1999). WHO criteria were used to evaluate tle cell lymphoma. toxicity. Results: Nineteen patients (11 males, 9 females), aged 17 - 65 years (median 30 years) were evaluated. At diagnosis, two P771 patients presented in early stages and 17, with advanced dis- Haematopoietic stem cell transplantation in mantle cell ease. Median number of chemotherapy lines before receiving lymphoma patients between 2001 and 2008 – A single- the GEMOX protocol were 3 (range, 2 – 5) and time interval centre experience between diagnosis and the salvage protocol, 24 (range, 8 – 36) A. Barta, Á. Bátai, Z. Csukly, V. Goda, L. Gopcsa, months. Eleven patients had previously failed an ASCT. At the B. Kapas, L. Lengyel, N. Lovas, S. Lueff, Z. Matrai, G. MIkala, time of GEMOX all the patients were in progressive disease S. Nahajevszky, M. Peto, A. Sipos, E. Torbagyi, R. Rasonyi, or in untreated relapse. Median number of administered cycles M. Reti, A. Tremmel, P. Remenyi, T. Masszi was 2 (2 – 6). Grade 3-4 haematological toxicity was present in St. Istvan and St. Laszlo Hospital of Budapest (Budapest, HU) 10 patients (neutropenia – 6 patients and thrombocytopenia – 4 patients). There was no grade 3-4 extra-haematological toxic- Background: In this study we present our results of haemopoi- ity. Treatment related mortality was 5%. In 7 patients, PBPCs etic stem cell transplantation (HSCT) in mantle cell NHL. [median 4.3 x 106/kg (range, 2.5 – 14.3)] were collected after Methods: Between 2001. and 2008. we performed 42 HSCT in the second cycle of GEMOX. Median number of apheresis pro- MCL: 35 autologous, and 7 allogeneic. Gender ratio: 29 male cedure was one. Response rate was 53% (4 CR; 6 PR). Seven and 13 female. At the time of transplant the median age was patients did not respond to the salvage protocol. 48,6 years old. The median follow-up was 33 months (3-84). In Comments: The GEMOX protocol shows promising response 29 cases 12 Gy Totalbody irradiation + 120 mg/bw Cyclophos- rate and an adequate toxicity profi le in heavily pretreated phamide (TBI/CY), in 13 cases other chemotherapy (BEAM, relapsed or refractory HL patients. In addition, it shows a BU/CY) were applied for conditioning regimen. In 39 cases good capacity to mobilize PBPCs for an ulterior autologous mobilized peripheral blood, in 3 bone marrow were used for procedure. stem cell source. Results: 31 out of 35 autologous transplanted patients alive (88,6%), 4 patients died (11,4%). 25 patients are in complett remis- P770 sion (80,6%), 3 patients live in relapse, and further 3 relapsed Low toxicity after chemoimmuno-conditioning in mantle patients underwent a second allogeneic transplantation. cell lymphoma The conditioning regimen before allogeneic SCT was TBI/ M. Binder, R. Ziebermayr, M. Girschikofsky, H. Kasparu, CY in 2 cases, and the reduced intensity conditioning regi- J. Koenig, O. Krieger, A. Weltermann, D. Lutz men included Fludarabine/Melphalane/Rituximab in 5 cases. Elisabethinen Hospital (Linz, AT) The graft versus host disease (GVHD) prevention was Cyclosporine-A + Methotrexate in 2, and Mycofenolate mofetil Background: Intensive therapeutic programs improve the clini- + Cyclosporine-A in 5 cases. Among 7 allotransplanted patients cal outcome of mantle cell lymphoma, but toxicity could be a in 3 the allogeneic HSCT were performed following relapse problem especially in elderly patients because of comorbid- after autologous HSCT, in further 4 cases the allogeneic HSCT ity. We evaluated the toxicities and side effects after immuno- was the fi rst transplant. The median follow-up was 24 months therapy combined with high dose chemotherapy followed by (4-80). Acute GVHD (skin, gut, liver) gr.IV. developed in 1 case. autologous stem cell transplantation in patients with MCL. Chronic extensive GVHD occured in 1 case. 5 out of 7 patients Methods: Since 2000 21 consecutive patients with newly diag- alive in complett remission after allogeneic HSCT. 1 patient nosed or pre-treated MCL of stage III- IV were eligible for high died of bacterial sepsis before engraftment, further 1 patient dose chemotherapy followed by autologous stem cell transplan- died 5 months following allogeneic HSCT in steroid-resistant, tation. After an induction with CHOP like regimen or intensive gr.IV. GVHD. chemoimmunotherapy (Nordic Lymphoma Study Group) autol- Conclusion: Autologous HSCT following high dose radio-chem- ogous stem cell transplantation was performed at a median of otherapy in MCL can provide favorable DFS. Allogeneic stem 9 (4-70) months from diagnosis. In addition to high dose condi- cell transplant might play a role in special cases, and can be tioning chemotherapy (BEAM) 4 doses of Rituximab (d -9, -1, recommended even in older age. +48 and +55) should be administered. Results: 21 patients (10 female, 11 male) at a median age of 59 (49-67) years were treated. 12 reached CR, 3 VGPR and 5 P772 PR (+1 responding relapse) before receiving ASCT. Rituximab Successful outcome of patients with relapsed/refractory could be performed in 17 of 21 patients. Due to infectious com- Hodgkin’s lymphoma treated with high dose therapy at a plications third and fourth dose of Rituximab was not adminis- national adult bone marrow transplant unit, Dublin tered in 4 patients. A median number of 6,88 x 106/kg CD 34 C. Bacon, P. Daly, D.O. Mahony, P. Browne, E. Conneally, positive cells (0,82- 22,42) were reinfused. The engraftment C. Flynn, N. Chadwick, N. Gardiner, E. Higgins, S. O Briain, was generally prompt and durable (granulocytes > 0,5 G/l day R. Mc Dermott, S. McCann, E. Vandenberghe +10 (9- 11), platelets > 50 G/l day +13 (10- 17)). Only in one St James Hospital (Dublin, IE) case (male patient, age at TX: 55 years) a delayed regenera- tion (d +140; granulocytes > 0,5 G/l +11, platelets day +21) was Most patients (80-90%) with Hodgkin Lymphoma (HL) attain a observed. Side effects were mild in 20 of 21 patients. In 12 durable complete remission (CR) with fi rst line therapy. Patients patients mucositis grade I- II was seen, 10 suffered from eme- with primary refractory or relapsed disease have various treat- sis grade I- III and 10 from enteritis grade I- II. Grade 4 toxici- ment options including salvage chemotherapy ± radiotherapy ties and treatment related deaths were not observed. Infectious or high dose therapy with autologous stem cell transplant (HDT- complications such as fever of unknown origin occurred in 5, ASCT). septicaemia in 3 and pneumonia in 3 patients. Following trans- We reviewed the outcome of 41 patients with relapsed/refrac- plantation all patients reached clinical and molecular CR. 18 of tory HL referred for HDT-ASCT to St James Hospital from 9 21 patients are still in CCR with a median observation period referring hospitals between 2000 and 2007. Patients were of 33 months (1- 83). 1 patient relapsed after 3,7 years and is included in the programme if they had HL refractory to primary

S215 chemotherapy (n=13), relapsed HL following chemotherapy developed acute GVHD. Following allo-HSCT, 3 patients alive (n=13) and patients who had > 2 lines of treatment prior to and 2 patients are in CR. Both patients with CR received RIC referral to the transplant program (n=15). The M:F ratio was conditioning regimen. 22:19 with a median age at transplant of 33yr (range 15 - 46yr). Conclusion: Patients with T cell neoplasm who respond com- Radiological assessment of disease was determined at relapse, pletely following autologous-HSCT have an excellent prognosis. post- salvage chemotherapy and at 100 days post HDT-ASCT Those who only achieved pre-transplant PR but then obtained by Computerised Tomography (CT) or CT- positron emission a CR following autologous-HSCT have similar outcome. More tomography (PET) from 2004. than half of patients (61%) with ALCL and approximately one All patients received 3 courses of salvage chemotherapy includ- third of the patients (30%) with PTCL-U may enjoy prolonged ing one peripheral blood stem cell (PBSC) mobilising course DFS following autologous-HSCT. In refractory or relapsing T cell prior to HDT-SCT. The median time from initial diagnosis to lymphoma, the allogeneic-HSCT might improve the outcome HDT-ASCT was 20.1 months (range 6 – 145mo). Post-salvage for eligible patients, especially when using RIC conditioning. chemotherapy 16 patients were in complete remission (CR) and the remaining 25 patients had a partial response (PR). The median CD34 cell dose administered was 3.65 x 106/Kg, (range P774 0.6-10.45 x 106/Kg). The median time for neutrophil and platelet Correlation of lymphocyte recovery and progression-free engraftment was 10 days with a range of 8-25 and 6-35 days survival after autologous stem cell transplantation in respectively. At day 100 post-HDT, 36 patients were in CR, 4 patients with Hodgkin’s lymphoma were in PR, 1 died of progressive disease and there were no H. Mocikova (1), B. Vackova (1), R. Pytlik (1), P. Obrtlikova (1), toxic deaths. 27 of 29 patients were FDG-PET negative at day P. Kobylka (2), M. Trneny (1) 100 post-HDT of whom 26 remain in CR. All patients who were (1)Charles University General Hospital (Prague, CZ); (2)Institute PET +ve pre-HDT subsequently relapsed. The calculated OS of Hematology and Blood Tranfusion (Prague, CZ) at 5 year was 84%. The clinical outcome of the patients treated in the National Adult Objectives: The aim of this study was to correlate lymfocyte Transplantation unit compares favourably with international fi g- recovery and progression-free survival (PFS) after autologous ures. Further improvement will depend on better patient selec- stem cell transplantation (ASCT) in patients (pts) with primary tion as relapsed disease remains the only cause of death. The progressive or recurrent Hodgkin lymphoma (HL) and to ana- routine use of FDG-PET scanning may permit the selection lyze factors associated with outcome. of patients who will not benefi t from HDT and need an alter- Patients and methods: We retrospectively analyzed 33 pts native management pathway which may include intensifi ed with HL who underwent ASCT between 2000 and 2008. Fac- salvage therapy, different conditioning regimens or allogeneic tors analyzed: clinical stage at relapse, relapse <12months and transplantation. >12months, chemosensitivity of relapse, infused lymphocytes and CD34 cells, pre-apheresis absolute lymfocyte count (ALC), day 15 and day 90 ALC following ASCT, engraftment character- P773 istics and infections within fi rst 6 months post-transplant. Haematopoietic stem cell transplantation in T-cell Results: At the time of ASCT 14/33 patients (pts) were in CR, neoplasms: the Budapest experience 7/33 in PR, 8/33 in SD and 4/33 pts were primary progressive L. Gopcsa, A. Barta, A. Batai, Z. Csukly, V. Goda, B. Kapas, HL. Median age:34 years (range 19-57). Median CD34 cell L. Lengyel, N. Lovas, S. Lueff, Z. Matrai, G. Mikala, infused: 6.8x106/kg (range 4.1-34.7). Median pre-apheresis S. Nahajevszky, M. Peto, R. Rasonyi, M. Reti, A. Sipos, ALC: 1.2x109/L, day 15 ALC: 0.7x109/L, day 90 ALC: 1.35x109/ A. Tremmel, E. Torbagyi, P. Remenyi, T. Masszi L. Median follow-up: 3.6years. 3 year median overall survival St Istvan and St Laszlo Hospital (Budapest, HU) (OS) and PFS are 82% and 71%. There was no association between PFS and remission status at the time of ASCT, infused Objectives: The impact of hematopoietic stem cell transplanta- lymphocytes and CD34 cell number, pre-apheresis ALC, day 15 tion (HSCT) in T cell neoplasms is poorly defi ned. ALC, or engrafment times. Day 90 ALC (HR=0.205, p= 0.017) Methods: Medical records of HSCT patients at Budapest from was signifi cantly associated with PFS in univariate analysis and 1995 to 2008 were reviewed. We retrospectively analyzed the multivariate analysis. results of all 33 T cell lymphoma patients treated with HSCT. Conclusions: In our study only ALC day 90 was signifi cantly Results: Among the 33 patients, we performed 29 autologous associated with improved PFS suggesting long-term lymphoid and 6 allogeneic HSCT. Patients had a median age of 28 (range, reconstitution may be an important factor for tumor control. 15 to 60 years). Eighteen patients (54,5%) had anaplastic large cell lymphoma (ALCL) including 10 anaplastic lymphoma kinase (ALK) positive and 8 ALK negatív cases, 10 (33,3%) P775 peripheral T cell lymphoma unspecifi ed (PTCL-U), 2 T cell lym- Reduced-intensity allogeneic haematopoietic stem cell phoblastic lymphoma, 1 angioimmunoblastic T cell lymphoma transplantation for lymphomas and 1 hepatosplenic T-cell lymphoma. Disease status at autolo- F. Bonifazi, E. Colaci, G. Bandini, M. Arpinati, M. Stanzani, gous HSCT was complete response (CR) in 12 patients, partial S. Usai, P. Tazzari, A. Bontadini, F. Ricci, E. Dan, S. Taioli, remission (PR) in 12, and refractory in 5. Preparative regimens M.P. Fina, C. Pellegrini, P. Zinzani, M. Baccarani were BEAM (89%) and TBI-CY (11%). Following autologous S. Orsola-Malpighi Hospital (Bologna, IT) HSCT, 21 (72%) achieved CR, 2 PR and 6 unresponsive. Of the 12 patients who were in pre-transplant PR, 8 achieved CR fol- We analyzed data on 46 patients with lymphomas, treated with lowing HSCT. The median follow-up of the 20 surviving patients reduced intensity conditioning and allograft between 2002 and is 27 months (1 to 124) 15 of whom are free of disease. Accord- 2007 at our institution. Twenty-three patients had HL, seven ing to disease entities the overall survival (OS) and disease high-grade lymphomas, twelve low-grade and 4 T-cell lympho- free survival (DFS) were higher in ALK+ ALCL group and lower mas. Median age was 37.5. in ALK- ALCL and PTCL-U groups. Among the 6 allo-grafted 29% patients had a chemosensitive disease and 87% had been patients, 4 received myeloablative (TBI-CY 3, BU-CY 1) and 2 treated before with autologous transplantation. underwent HSCT after reduced intensity (Fludarabine-Melpha- 59% received stem cell from peripheral blood and 41% from lan-Alemtuzumab) conditioning (RIC). The source of stem cells bone marrow. The donor was a related sibling for 27 patients was mobilized PB in 5 patients and BM in 1 case. Graft-versus- and unrelated for 19. Conditioning regimen was the asso- host disease (GVHD) prophylaxis consisted of cyclosporine A ciation Tiothepa (10mg/kg), Fludarabine (60 Mg/kg), Endoxan and methotrexate in 3 patients, tacrolimus+mycofenolate mofetil Cyclophosphamide (60 mg/kg) ± ATG-F (total dose 15 mg/kg) in 2 cases and tacrolimus+rapamycine in 1 case. Two patients for patients who received a related allograft, and Tiothepa

S216 (10 mg/kg), Melphalan (30 Mg/mq), Endoxan (100 mg/mq) + was older and had less patients than rituximab negative group. ATG-F Fresenius for the unrelated transplant. All patients, Further randomized, prospective studies with larger patient except one who died at day 7 after transplantation, engrafted. groups are needed. Overall survival was 51% at 3 yrs and it was signifi cantly asso- ciated with chemoresistant vs chemosensitive disease ( 31% vs 93%). Transplant related mortality was 23% at 3 yrs, and we observed an association with chemoresistance (33% vs 8% in chemo- Paediatric issues sensitive group). Relapse was 66% at 3 yrs, and signifi cantly higher for patients with chemoresistant disease (74% vs 34%). We also tried to analyze the Graft-versus-Lymphoma effect, P777 looking for association between complete remission (CR) and NOD2/CARD15 mutations have no impact on the chronic GVHD: of 24 patients who obtained CR, 17 had experi- clinical outcome of paediatric patients after mented cGVHD, but of 13 who did not obtain response only 4 allo-SCT – A retrospective multicentre study had cGVHD (chi squared=6.072, p=0.048). However, different H. Kreyenberg (1), A. Jarisch (1), C. Niemeyer (2), B. Strahm diseases had different response to the immunological effect: HL (2), B. Kremens (3), J. Beck (4), A. Schrauder (5), S. Burdach and high grade NHL appeared to have a very limited response (6), M. Führer (7), C. Roessig (8), H. Kabisch (9), E. Holler (10), to GVL effect, but in low grade NHL and peripheral T-cell Lym- T. Klingebiel (1), P. Bader (1) phomas there was an higher rate of response with a minimal (1)Goethe University (Frankfurt, DE); (2)University Children’s relapse risk at 3 yrs. Hospital (Freiburg, DE); (3)University Children’s Hospital In conclusion, we can say that allogeneic stem cell transplanta- (Essen, DE); (4)University Children’s Hospital (Jena, DE); tion represents an effective therapeutic option for Lymphomas, (5)UK-SH Campus Kiel (Kiel, DE); (6)Univesity Children’s even if best results are obtained in low grade and peripheral Hospital (Munich, DE); (7)Von Hauner Kinderspital (Munich, T-cell Lymphomas. More studies will be required to better DE); (8)University Children’s Hospital (Münster, DE); understand the role of allograft in the treatment of HL and high (9)University Children’s Hospital (Hamburg, DE); (10)University grade NHL. Medical Center (Regensburg, DE) In addition, the importance of chemosensitivity/ chemoresist- ance suggests questions about the best timing to subject Purpose: In recent years, several studies have pointed towards patients to allo-transplantation. an association between certain single nucleotide polymor- phisms (SNP8, SNP12, and SNP13) in the NOD2/CARD15 gene on incidence of severe acute GvHD and TRM after alloge- P776 neic stem cell transplantation (SCT) in adult patients. No studies The effect of rituximab in fi rst line therapy to results of were published so far, evaluating the impact of these mutations autologus haematopoietic stem cell transplantation in in pediatric recipients of allogeneic SCT. Here we report the relapsed non-Hodgkin’s lymphoma patients results of a retrospective multicenter trial in 421 children and S. Civriz Bozdag, P. Topcuoglu, S. Bakanay, M. Arat, adolescents who received allogeneic stem cell transplantation O. Ilhan, M. Beksac, A. Uysal, O. Arslan, T. Demirer, G. Gurman, for leukemia or myelodysplastic syndromes. N. Konuk, M. Ozcan Patients: Between February 1997 and March 2008, 421 chil- Ankara University School of Medicine (Ankara, TR) dren (median age=10.7) with ALL (n=212), AML (n=106), and MDS (n=103) underwent an allogeneic SCT and were included Introduction: The response rates to salvage therapy and high into the analysis. The study was approved by the Ethical Com- dose chemotherapy with autologus hematopoietic transplanta- mittee of the Goethe University Frankfurt. Data were obtained tion of relapsed non Hodgkin lymphoma(NHL) patients treated for analysis until November 2008. with rituximab in fi rst line therapy, is still debate. Patients received their grafts either from a matched family donor Patients and methods: We retrospectively analyzed 76 patients (MFD, n=108), from a mismatched family donor (MMFD; n=46), (median age 42(17-63), Male/Female:48/28) who relapsed or a matched unrelated donor (MUD; n=223) or a mismatched progressed after fi rst line therapy and had high dose chemo- unrelated donor (MMUD; n=44). NOD2/CARD15 mutations therapy, autologus transplantation. We grouped them as were assessed using RT-PCR as described previously. Diagno- patients treated with rituximab (R(+) n:21) and without rituximab sis of aGVHD was based on clinical signs or histopathological (R(-) n:54) in fi rst line therapy. The major group of the patients fi ndings. Grading was performed based on the Seattle criteria. were diffuse large B cell lymphoma. Median time from diagno- Results: In the 421 transplant pairs 122 mutations (21%) could sis to transplantation was12 months (5-151) in R (-) group and be identifi ed in either the recipient (n=52) or in the donor (n=52), 20 months (5-54) in R (+) group. in 18 pairs both, recipient and donor revealed mutations. Two Results: The median age of R (+) patients was slightly older recipients were positive for two mutations and one donor was than R(-) patients. homozygous for SNP 12. Overall response (CR+PR) to salvage therapy before trans- The cumulative incidence (CI) of severe aGVHD III-IV was plantation was 62% in R (+) group and 44% in R (-) group, higher in patient donor pairs (0.17) with wildtype genes com- p=0.448. Overall response after transplantation was 76% in pared to 0.08 in patient/donor pairs who showed mutated genes both groups. Posttransplantation 2 years survival of all patients (p=0.026). TRM in transplantations without mutations was 0.24 was 50%±7% with median 21 months of follow up. Posttrans- compared to 0.25 with mutations (P=0.9). plantation 2 years survival according to groups was statisti- In matched donor transplants CI of TRM was 0.20 in wildtype cally insignifi cant between two groups R (+); 35%±26%, R(-) pairs compared to 0.22 in transplant pairs showing a mutanted 51%+8%, p=0.72. Diffuse large B cell lymphoma patients were gene in either the recipient or the donor (p=0.72). In these analyzed spesifi cally; rituximab was found with no effect to patients the CI of severe aGVHD III-IV was also higher in the the posttransplantation responses and survival rates; 1 year wildtyp pairs (0.16) compared to 0.08 in transplant pairs show- survival was 71% ±14% in R (+) group and 61%± 8% in R (-) ing a mutation in the NOD2CARD15 gene (p=0.044). group, P=0.838. Twenty nine patients (RR+) treated with rituxi- Conclusion: On the basis of these fi ndings we conclude that mab both in fi rst and second line therapy was compared with consideration of NOD2/CARD15 mutations are not pertinent 46 patients(RR-) who did not; 2 years survival was 40%±15% in when selecting a donor for pediatric patients. RR(+) group and %55±8% in RR (-) group, p=0,72. The Study was supported by the German José Carreras Leuke- Conclusion: In conclusion, we did not fi nd a positive effect of mia Foundation. rituximab in fi rst line treatment to responses of salvage therapy and posttransplantation period. Our rituximab positive group

S217 P778 p<0.043). Independent risk factors in multivariate binary logistic Reconstitution of NK receptors after haploidentical analyses were age > 12 years and the combination of busulfan transplantation with CD3/CD19 depleted grafts in children: and cyclophosphamide. Furthermore, we analyzed the BK virus fast recovery of CD16 positive NK cells enables early load in urine by real-time polymerase chain reaction. We found ADCC activity in patients without HC a signifi cantly increased number of BK M. Pfeiffer, T. Feuchtinger, H. Teltschik, I. Müller, virus copies in urine in children older than 12 years (p<0.009) R. Handgretinger, P. Lang and in children who received antithymocyte globulin (p<0.001). University Tübingen (Tübingen, DE) In addition, BK virus load in urine was signifi cantly increased in children who suffered from HC. Thirteen of 14 children with NK activity is regulated by signaling through activating and HC had a BK virus load in urine > 107 copies/ml (p<0.001). inhibitory receptors. Acquisition of both inhibiting and activating We observed in individual BK virus profi les an increase of BK receptors on developing NK cells is an important step in their virus copies in urine before the onset of HC. 13 of 19 children functional maturation after transplantation. (68.4%) with HC and 43 of 146 children (29.4%) without HC Here we report for the fi rst time on the reconstitution of NK- died after HSCT (p<0.003). All 6 children (100%) who suffered receptors after haploidentical transplantation of CD3/CD19 from grade IV HC died after transplantation. We conclude that depleted grafts in children. KIR reconstitution followed an HC in children is a disease of multiple etiologies and leads to already known pattern with higher percentage of KIR nega- a high mortality. Allogeneic HSCT, the combination of busul- tive NK cells early after transplantation and a higher and faster fan and cyclophosphamide, age > 12 years, and an unrelated expression of CD158b compared to CD158a and CD158e inde- donor are risk factors for the development of HC in childhood. pendent of presence or absence of the corresponding KIR lig- Increased BK virus load in urine > 107 copies/ml may lead to ands in donors or recipients. This pattern was also seen for the HC. Therefore, it is useful to quantify BK virus in urine in those subpopulation of KIR single positive NK cells, which express children with above mentioned risk factors to initiate early only one of the above mentioned inhibitory KIRs and which are treatment or to prevent the development of HC and potentially thought to be involved in NK alloreactivity. decrease the HC-related mortality. Whereas some patients showed reduced NKG2D expression in the fi rst hundred days after transplantation almost all patients soon showed nearly normal levels of NKp30, NKp46, CD226, P780 CD85j and CD244 and elevated levels of NKp44 and NKG2A. Differences in immune reconstitution in children In contrast to published results in adult patients after haploi- receiving either bone marrow or cord blood stem cell dentical transplantation of positive selected grafts we found a transplantation fast recovery of CD56+/CD16+ NK cells with cytolytic activity A.P.J. de Pagter (1), L. Keukens (1), S. Belitzer (2), E. Baeten against K562. Cytotoxic activity against K562 of both resting (1), A.P. de Gouw (1), A. Bloem (1), J.J. Boelens (1) and IL-2 activated NK cells reached already the same level (1)University Medical Center (Utrecht, NL); (2)Pharmaceutical in the fi rst 30 days after transplantation as compared to later Science Department (Utrecht, NL) time points. Furthermore these NK cells showed strong ADCC activity against neuroblastoma cell lines and leukemic blasts Objective: Early immunereconstititution after haematopoietic as early as day fourteen after transplantation using antibodies stem cell transplantation (HSCT) is important for controlling against disialoganglioside 2 (for neuroblastoma) or CD19 (for infectious complications. We have prospectively analyzed dif- pediatric leukemic blasts). It is not clear from published results ferences in immunereconstitution after matched sibling trans- if the fast recovery of CD56+/CD16+ NK is an effect of CD3/ plantation (id-SIB) and unrelated bonemarrow transplantation CD19 depletion or a general difference between children and (MUD) or cordblood (u-CB) HSCT. adults. Our results indicate a fast recovery of functional CD56+/ Methods: In a cohort study among allogeneic pediatric HSCT CD16+ NK cells after haploidentical transplantation with CD3/ patients (May 2004-October 2008), immunophenotyping was CD19 depleted grafts which can mediate ADCC and therefore performed on whole blood samples every other week if leu- enable an early use of antibodies to target residual malignant cocyte > 0.4E9/L. Numbers of T- (CD3+), B-(CD19+) and NK- cells. (CD3-CD16+CD56+) cells and subsets were determined by fl ow cytometry. Differences in maximum and time to maximum of T- and B-cell numbers during the fi rst 100 days after HSCT P779 between recipients of the various grafts (id-SIB, MUD and u- Risk factors, BK virus association, and outcome of CB) were analyzed using Wilcoxon tests. haemorrhagic cystitis in children after haematopoietic Results: 107 patients were included, with a median age of 4.7 stem cell transplantation (0.1-21.2) years. 23 recipients received id-SIB, 47 MUD and 37 B. Gruhn (1), L. Maier (1), R. Egerer (2), F. Zintl (1), J. Beck (1) u-CB graft. The median follow up after HSCT was 57,8 (2,6– (1)Department of Pediatrics (Jena, DE); (2)Institute for Virology 220,4) weeks and 29 (27%) patients deceased. Maximum and Antiviral Therapy (Jena, DE) CD8+T-cell numbers were higher after id-SIB- and MUD compared to u-CB-HSCT (151 and 373 versus 47 CD8+T- Little is known about the development and outcome of hem- cells/uL, p=0.000). The proportion of naïve CD8+T-cells orrhagic cystitis (HC) in children after hematopoietic stem cell (CD45RA+CD27+) was signifi cantly higher in id-SIB com- transplantation (HSCT), especially about the association with pared to MUD and u-CB-recipients (22% versus 10% and BK virus infection. Therefore, we analyzed the incidence, risk 4%, p=0,001). CD4+T-cell numbers did not signifi cantly dif- factors, BK virus association, and mortality of HC in 165 con- fer between id-SIB, MUD and u-CB recipients (138, 159 and secutive children who underwent HSCT between 1/2000 and 68 CD4+T-cells/uL, p=0.160), but the proportion of naïve 12/2006 in a single center. Fifty nine patients received autolo- CD4+Tcells (CD45RA+CD27+) was higher in id-SIB com- gous HSCT and 106 patients underwent allogeneic HSCT. pared to MUD- and u-CB-recipients (17% versus 2% and 1%, Nineteen of the 165 patients (11.5%) developed HC after a p=0,000). NK-cell and B-cell numbers were higher in u-CB com- median of 33 days. All 19 patients with HC underwent allo- pared to id-SIB and MUD recipients (577 versus 253 and 385 geneic HSCT and showed BK viruria after transplantation. An NK-cells/uL, p=0,001 and 538 versus 92 and 176 B-cells/uL, acute graft-versus-host disease was signifi cantly more frequent p=0,001). These B-cells mainly showed immature B-cells phe- in children with HC (p<0.001). Signifi cant risk factors for HC notype (SIgD+CD10+CD38++). No difference in median time to development in univariate binary logistic analyses were age > maximum CD3+, CD4+, NK- and B-cell numbers was observed, 12 years (OR, 3.275; p<0.031), use of busulfan (OR, 3.514; except for maximum CD8+T-cell numbers, which was earlier in p<0.013), use of busulfan and cyclophosphamide in combina- id-SIB compared to MUD and u-CB recipients (30 (4-90) versus tion (OR, 4.935; p<0.002), and an unrelated donor (OR, 3.309; 77 (3-99) and 66 (6-100) days after HSCT, p=0.003).

S218 Conclusion: Id-SIB and MUD recipients show higher CD8+T- plication with high morbidity and mortality rate. By intensive cell numbers after HSCT compared to u-CB HSCT recipients. surveillance patients at high risk for developing HAdV disease U-CB recipients showed higher B-cell and NK-cell numbers can early be identifi ed in order to start preemptive therapy with compared to BM recipients. The latter may indicate a faster in antiviral medication in an early stage. vivo proliferative capacity of CB stem cells compared to BM Methods: In a prospective study, we determined the predictive derived stem cells. value of HAdV DNA positivity in nasopharyngeal fl uid preceding HSCT to identify patients at risk for a HAdV reactivation after HSCT. We weekly monitored Adenovirus DNA loads in plasma P781 after HSCT. Secondly, the association between HAdV reactiva- High percentage of perforin-expressing T-cells is tion and alloreactive disease (Graft-versus-Host disease and/or associated with herpesvirus reactivations clearance early idiopathic pulmonary syndrome) was analyzed. after haematopoietic stem cell transplantation in children Results: A total of 62 patients after allogeneic HSCT were A.P.J. de Pagter, J.J. Boelens, R. Jacobi, E.A.M. Sanders, included: 37 (60%) recipients received bone marrow (17/37, R. Schuurman, D. van Baarle 46%, matched family donor) or unrelated peripheral blood stem University Medical Center (Utrecht, NL) cells while 25 (40%) recipients received unrelated cord blood stem cells. 42/62 (68%) recipients received HLA-matched stem Background: Haematopoietic stem cell transplantation (HSCT) cells (for BM/PBSC high resolution typing, for CB intermedi- is frequently complicated by human herpesvirus type 6 (HHV6), ate: HLA-A and B on low and HLA-DR on high resolution). All cytomegalovirus (CMV) or Epstein Barr virus (EBV) reactiva- patients received myeloablative conditioning and standardized tions. Herpesvirus reactivations are associated with severe Graft-versus-host disease prophylaxis. The median follow-up morbidity and poor survival after HSCT. T-lymphocytes play an time was 47 (5-150) weeks. important role in the clearance of viral reactivations after HSCT, Prior to HSCT, HAdV DNA could be detected in nasopharyngeal however an easy measure of potent T cell responses for pre- fl uid of 8/62 patients (13%). In all these patients (100%), plasma dicting outcome of viral reactivations is lacking. In this study, we HAdV reactivation occurred at a median time of 12.5 days analyzed expression of perforin as a marker for cytotoxic poten- (range 5-72 days) after HSCT. Additionally, 11 (18%) patients tial in CD8+T-cells in children and related this to the clearance developed HAdV reactivation at a median time of 40 (14-160) of viral reactivations early after allogeneic HSCT. days after HSCT. In multivariate analysis HAdV DNA positivity in Methods: Prospectively, we weekly analyzed HHV6, CMV and nasopharyngeal fl uid was the only signifi cant predictor for HAdV EBV DNA loads in plasma by quantitative realtime PCR. Her- reactivation after HSCT (HR 9,7; 95%CI 3,4-27,4; p=0.000). In pesvirus reactivation was defi ned as DNA load >1000 cp/mL. patients with HAdV reactivation, HadV was a predictor for allo- CMV- and EBV reactivation were pre-emptive treated accord- reactive disease (HR 2.6; 95% CI 1,2-5,4; p=0.013). ing to local guidelines. HHV6reactivation was treated when Conclusions: HAdV positivity in nasopharyngeal fl uid pre-HSCT there was clinical suspicion of disease. is a very strong predictor for the development of HAdV reactiva- Additionally, we have weekly analyzed Perforin expression in tion after HSCT. Prevention or early pre-emptive treatment with CD8+T-cells by whole blood FACS analysis, until 4 months antiviral therapy might contribute to prevent HAdV disease and after HSCT. / or SCT associated problems after HSCT. Results: A total of 27 patients were included; median age of 4,3(0.3-20,1) years. 15/27 (56%) recipients received bonemar- row (7/15 from matched family donor) or unrelated peripheral P783 blood stemcells while 12/27 (44%) recipients received unre- A prognostic score at diagnosis for Ewing tumour lated cordblood stemcells. All patients received myeloabla- patients with metastatic disease at extra-pulmonary sites tive conditioning and standardized Graft-versus-host disease R. Ladenstein (1), U. Pötschger (2), J. Whelan (3), O. Oberlin prophylaxis. (4), M. Paulussen (5), M.C. Delay (4), I. Lewis (6), H. van den During the fi rst 4 months after HSCT, 16 patients developed Berg (7), H. Jürgens (8) HHV6reactivation, 2 patients developed CMVreactivation and (1)St. Anna Children’s Hospital (Vienna, AT); (2)Children’s 1 patient developed EBV reactivation. The median time of her- Cancer (Vienna, AT); (3)University College Hospital (London, pesvirus reactivation peak was 2,4 weeks (range 1,3-15,6) after UK); (4)Institut Gustave Roussy (Villejuif, FR); (5)University HSCT. Viral clearance was marked by a perforin peak in CD8+ Children’s Hospital Basel (Basel, CH); (6)CCLG Data Centre T-cells (median time of maximum perforin expression in CD8+T- (Leicester, UK); (7)University of Amsterdam (Amsterdam, NL); cells was 4 weeks (range 1,4-15,9). Patients with herpesvirus (8)University Children’s Hospital (Münster, DE) reactivation (HHV6, CMV or EBV) showed signifi cantly higher perforin-expression in CD8+T-cells during viral load clearance Purpose: To assess prognostic factors at diagnosis in prospec- than patients without herpesvirus reactivation (17,2% (range tively treated patients with primary extra-pulmonary metastatic 0-63%) versus 6,8% (range 0-16%) respectively; p=0.003). Ewing tumors (EPM-ET) of the EURO-E.W.I.N.G. 99 Study. Conclusion: Herpesvirus reactivation clearance was signifi - Patients and Methods: From 1999 to 2005, 281 patients were cantly associated with a peak in perforin-expressing CD8+T- enrolled. Median age was 16.2 years (0.4-49). Primary site was cells. Although the early virus specifi c immune responses after extremity in 84 patients and axial in 197 (115 pelvic sites), with HSCT have to be elucidated, perforin expression in T-cells is a a tumor volume >200ml in 171 patients. Treatment consisted useful marker for antiviral T-cell responses and viral load clear- of 6 VIDE cycles, one VAI/VAC cycle, local treatment (surgery ance after HSCT. and/or radiotherapy), and high-dose busulfan-melphalan fol- lowed by peripheral stem cell transplantation (HDT/SCT). Results: After a median follow up of 3.8 years, event-free sur- P782 vival (EFS) and overall survival (OS) at 3 years for all 281 Adenovirus DNA positivity in nasopharyngeal fl uid patients were 27%±3% and 34%±4%. Six VIDE cycles were preceding haematopoietic stem cell transplantation: completed by 250 patients (89%); 169 (60%) received HDT/ a very strong predictor for adenovirus reactivation in SCT. Cox regression analyses demonstrated increased risk for paediatric patients patients with more than two bone metastatic sites (hazard ratio: A.P.J. de Pagter, L. Haveman, R. Schuurman, M. Bierings, HR 2.0), a primary tumor volume >200ml (HR 1.8), bone mar- J.J. Boelens row metastases (HR 1.6), age >14 years (HR 1.6), and addi- University Medical Center (Utrecht, NL) tional lung metastases (HR 1.5). A risk score based on these HR identifi ed three risk groups with EFS rates of 50% for scores Objective: After paediatric haematopoietic stem cell transplan- ≤3 (82 patients), 25% for scores >3 to <5 (102 patients), and tation (HSCT) adenovirus (HAdV) infection is a severe com- 10% for scores ≥5 (70 patients), p< 0.0001.

S219 Conclusions: A proportion of EPM-ET patients may survive with identical donor (MUD) in neuroblastoma (NB) poor responder intensive multimodal therapy. Age, tumor volume, and extent of to front line therapy or relapsed after a previous autologous metastatic spread are relevant risk factors. A score based on stem cell transplantation. these factors identifi es EPMD-ET patients with a more favora- Methods: 19 patients (pts), aged 3-17 years, affected by resist- ble outlook at diagnosis and may facilitate risk adapted treat- ant (5) or relapsed (14) NB were enrolled and submitted to an ment approaches. SCT after a RIC consisting of Thiotepa 15 mg/kg and Melpha- lan 140 mg/sqm. The donor was an identical sibling in 11 cases or a MUD in 8. At time of transplant 14 pts were in any kind of P784 remission of disease and 5 in progressive disease. Graft versus Rituximab is effective in refractory autoimmune hemolytic host disease (GVHD) prophylaxis consisting of Cyclosporin A + anaemia following allogeneic haematopoietic stem cell Anti-lymphocytic serum and short term methotrexate in MUD transplantation setting. SC sources were bone marrow in 15 cases and periph- R. Formankova (1), P. Sedlacek (1), P. Keslova (1), eral blood in 4. E. Mejstrikova (1), M. Pisacka (2), E. Linhartova (1), H. Zizkova Results: The reconstitution of bone marrow function was (2), J. Stary (1) obtained in all the 19 pts after a median time of 12 and 17 days (1)University Hospital Motol (Prague, CZ); (2)Institute of for PMN and PLT respectively in sibling setting, and 14 and 17 Hematology and Blood Transfusion (Prague, CZ) days in MUD setting. Acute GVHD of grade II-III occurred in 7 pts and a complete marrow donor chimerism was observed Autoimmune hemolytic anemia (AIHA) is a recognized compli- after 40 and 60 days in sibling and MUD setting respectively. cation of allogeneic stem cell transplantation (SCT). After a median follow-up of 15 (1-35) months, 9 pts relapsed, Between I/2000 and VI/2008 214 children underwent allogeneic 6 dead for progressive disease and 10 are alive and well. The SCT at our center for malignant (146; 68%) or non-malignant (68; median time of relapse from SCT was 9 (2-17) months. No pts 32%) disease. Donors were HLA-identical siblings (60; 28%), dead for treatment related causes (TRM). The 2 years prob- unrelated donors (144; 67%) or mismatched family donors (10; ability of OS and EFS of the entire cohort of pts were respec- 5%). Eight grafts were T-cell depleted. In 10 patients (4.6%) tively 0.59 (0.13) and 0.38 (0.13), with a better EFS for pts who AIHA with signifi cant hemolysis and a positive direct antiglobu- developed grade II-III acute GVHD (0.44 vs 0.25), were in any lin test (DAT) was diagnosed at a median time of 201 days (76 kind of remission of disease (0.46 vs 0), received a sibling graft - 407) after SCT. Warm-reacting antibodies were detected in all (0.46 vs 0.20). 10 patients, in 6 in combination with cold antibodies. AIHA was Conclusions: Our experience show the feasibility and effi cacy diagnosed exclusively in patients following SCT from unrelated of a RIC with SCT from HLA identical sibling donor or MUD in donor (9 unmanipulated) and predominantly (9/10) in patients the treatment of relapsed or refractory NB. In fact no patient transplanted for non-malignant disease (metabolic disorder suffered TRM. Moreover in a setting of pts who the 2 years 4, primary immunodefi ciency 4, severe aplastic anemia 1). 8 probability of survival is nearly to zero, in our experience more children were transplanted after fully myeloablative condition- than 50% are alive and well. The observation that the develop ing regimen (busulfan 16/20 mg/kg), one after reduced inten- of acute GVHD is related to a better outcome may offer the sity conditioning and one received antithymocytic globulin only. evidence of GVT in NB. AIHA was associated with mixed chimerism (10-90% of autolo- gous hematopoiesis) in 7 patients. Therapy of AIHA consisted of corticosteroids (10/10), intrave- P786 nous immunoglobulin (8/10) and rituximab (8/10). Rituximab Feasibility of intensive care for paediatric patients in was administered at the dose of 375mg/m2 (1 - 13 doses; HSCT setting median 5), mostly weekly x 3, starting at AIHA fi rst manifesta- P. Stepensky, I. Yatsiv, J. Baron, M. Aker, M. Weintraub, tion (7) or reactivation (1). 4/10 patients suffered from recur- S. Revel-Vilk, M. Shapira, R. Or, I. Resnick rent AIHA (1-4 reactivations), therefore 3 of them were treated Hadassah Hebrew University (Jerusalem, IL) with maintenance courses of rituximab (every 2 months; 2 - 8 doses). Two of them achieved DAT negativity and are well 23 Introduction: In some publications the feasibility of intensive and 7.5 months after the treatment discontinuation, in 1 child care in the HSCT setting is in doubt because of an extremely the treatment is ongoing. high mortality rate which approaches 100%. These poor results All 10 patients are alive without clinical signs of hemolysis. have improved in recent years, particularly in the pediatric age 6 children achieved negativity of DAT 7.5 - 75 (median 43) group [1,2]. months after discontinuation of therapy for AIHA, in 4 children Aim: Assessment of risk factors for mortality and outcome in treatment is still ongoing. a cohort of children undergoing hematopoietic stem cell trans- In our series SCT from unrelated donor, non-malignant disease plantation (HSCT) who were admitted to a pediatric intensive and mixed chimerism were considered as risk factors for devel- care unit (PICU) from 2002 till September 2008. opment of post-transplant AIHA. Therapy including repeated Methods: Retrospective case study. infusions of rituximab was well tolerated without any signifi cant Results: 95 HSCT were performed in 91 children. Twenty one infections. We conclude that rituximab is an effective modal- (23%) experienced 25 admissions to PICU (table); 12 patients ity in patients suffering from post-transplant AIHA and mainte- were diagnosed with non-malignant and 9 patients with malig- nance courses could prevent recurrence. nant diseases, all but one child underwent allogeneic HSCT. Supported by VZ FNM MZ 64203. Eleven (52%) admissions resulted in death in PICU and overall 1-year survival was 38%. Of the 11 deceased patients 4 suf- fered from acute GVHD (grade 3-4) and 8 required endotra- P785 cheal mechanical ventilation. All 10 children who developed Reduced-intensity conditioning regimen and allogeneic multiple organ failure died. Of the 10 surviving patients, 3 were stem cell transplantation from related or unrelated ventilated; all suffered from SCID and were transplanted in HLA-identical donor in high-risk neuroblastoma PICU. One year survival in children discharged from PICU was A. Prete, R. Rondelli, E. Lanino, C. Favre, M. Rabusin, 80% (8/10). F. Locatelli, A. Pession, F. Fagioli on behalf of the AIEOP-BMT Conclusions: Group 1. Effectiveness of treatment in PICU was similar in children suffering from malignant and non-malignant diseases. Objectives: To evaluate the feasibility and effi cacy of a reduced 1. The most prominent risk factor associated with immediate intensity conditioning regimen (RIC) followed by allogeneic mortality was development of multiple organ failure (p=0.00003). stem cell transplantation (SCT) from related or unrelated HLA For 1-year survival it was aGVHD (p=0.039). The need for

S220 mechanical ventilation (p=0.063) as well as HSCT from mis- had an increase in CD20 lymphocytes were treated with rituxi- matched donors (p=0.094) were additional parameters that can mab. After rituximab, only one patient developed PTLD requir- predict a bad outcome for children admitted to PICU. ing chemotherapy and infusion of donor specifi c EBV cytotoxic 2. One year survival of all patients admitted to PICU and those T-lymphocytes. The remaining 3 had EBV load ≥ 20,000 cop- discharged make this treatment feasible for the pediatric group ies/105 PBL, but with no increase in CD20/CD19. They did not in the HSCT setting. receive rituximab and they did not develop PTLD. The patients References: treated with rituximab needed immunoglobulin replacement 1. van Gestel JP, Bollen CW, van der Tweel I, Boelens JJ, van therapy for a mean of 566 days (range 41-1356 days). None Vught AJ. Intensive care unit mortality trends in children after of the remaining 20 HSCTs with EBV load between 1000 and hematopoietic stem cell transplantation: a meta-regression 20,000 copies/105 PBL developed PTLD. analysis. Crit Care Med. 2008 Oct;36(10):2898-904. Review. Conclusions: our data suggest that in allogeneic paediatric 2. Kache S, Weiss IK, Moore TB. Changing outcomes for chil- recipients, an EBV viral load ≥20,000/105 PBL associated with dren requiring intensive care following hematopoietic stem cell CD20/CD19 lymphocyte count monitoring may represent a reli- transplantation. Pediatr Transplant. 2006 May;10(3):299-303. able cut off for the administration of rituximab to prevent the EBV-PTLD.

P788 Targeting to optimal busulfan exposure is associated with a low relapse rate and high acute-GvHD 2-4 rates in MDS and JMML patients I.H. Bartelink (1), R.G.M. Bredius (2), M. Bierings (1), C.A.J. Knibbe (3), M. Egeler (2), A.C. Lankester (2), A.C.G Egberts (1), J. Zwaveling (2), J.J. Boelens (1) (1)UMCU (Utrecht, NL); (2)LUMC (Leiden, NL); (3)LACDR (Leiden, NL)

Background: Busulfan combined with therapeutic drug moni- toring and guided dosing is associated with higher event free survival (EFS) rates due to less graft-failure/relapses and lower toxicity in haematological stem cell transplantation. We previ- ously demonstrated an optimal AUC between 74-82 mg*h/L in a group of children with (non)-malignant indications. Therapeutic drug monitoring of busulfan is still controversial. The optimal P787 target AUC in these children is not known. We retrospectively Management of Epstein-Barr virus infection after analyzed the association between busulfan exposure (AUC) haematopoietic stem cell transplantation in 80 paediatric and clinical outcomes in MDS and JMML patients. allogeneic recipients Methods: All children, transplanted for MDS and JMML between M. Faraci, I. Caviglia, G. Morreale, E. Lanino, D. Cuzzubbo, 2001-2008, receiving intravenous busulfan as part of a myelo- S. Giardino, E. Di Marco, C. Cirillo, F. Scuderi, G. Dini, ablative regimen, were included. 34 patients received busulfan, P. Terranova, E. Castagnola cyclophosphamide 120mg/kg, and melphalan 140mg/m². Based G. Gaslini Children’s Research Institute (Genoa, IT) on the fi ndings of these patients the most recent transplanta- tions (n=3) were performed using busulfan and cyclophos- Introduction: Epstein Barr virus (EBV) related lymphoprolifera- phamide 120mg/kg only (AUC of >74mg*h/L).The association tive disease (PTLD) is a serious complication of haemopoietic between an AUC below or above the previously found lower stem cell transplantation (HSCT). Measuring EBV viral load limit of the optimum of 74mg*h/L and the main endpoints; by semiquantitative polymerase chain reaction (PCR) is use- relapse rate, overall survival (OS) and event free survival (EFS) ful for detecting EBV-PTLD in patients at risk before the onset and the toxicity endpoints: acute-Graft-versus-Host Disease of clinical symptoms. Rituximab given at 375mg/m² when the (aGvHD) grade 2-4; and Veno-occlusive Disease (VOD), were EBV viral load is >1000 copies/105 PBL is reported to prevent tested using Cox regression analysis and log-rank statistics. EBV-PTLD. Results: 38 patients (27 MDS, 11 JMML) were transplanted Materials and methods: between June 2004 and June 2008 (1 patient was excluded because of unpredictable pharma- at the HSCT Unit of the Gaslini Children’s Hospital, patients cokinetics). Median follow up time was 2.2 (range 1-18) years. undergoing allogeneic HSCT were monitored for the risk of EFS in the <74mg*h/L and >74mg*h/L was 58% and 82% PTLD by weekly EBV load assessment and evaluation of CD20 (p=0.13), resp. OS rates were respectively 51% and 82% for and CD19 absolute lymphocyte counts. EBV-DNA extracted these AUCs. EFS and OS were negatively infl uenced by higher from peripheral blood leukocyte (PBL) load was evaluated by rates of graft-failures/relapses in the <74mg*h/L group (38% vs. semi quantitative PCR assay. Only patients with EBV copies ≥ 0%, P=0.047). Regarding the toxicity endpoints, high busulfan 20,000/105PBL or between 10,000 and 20,000 copies/105PBL exposure was associated with more aGvHD grade 2-4: 20% vs but with an increase in CD20/CD19 cells in the peripheral blood 58% (p=0.037), and showed a trend to a higher VOD-rate: 12% were eligible for rituximab therapy. vs 34%, (p=0.105). Results: 80 children undergoing allogeneic HSCT (62 from Conclusion: Higher busulfan exposure (>74mg*h/L) was asso- unrelated donors,17 from HLA family matched donors and ciated with higher EFS / OS rates due to lower rates of graft- 8 from HLA family mismatched donors)were evaluated. Six failure/relapses. A concern however is the incidence of toxicity patients received more than one HSCT, for a total of 87 trans- (aGvHD grade 2-4 and VOD) in the higher exposure group in plants. No EBV reactivation was observed in 34 HSCTs. EBV patients receiving a combination of busulfan, cyclophospha- reactivation was documented in 53 other transplants, but EBV mide and melphalan. More precise targeting (defi ning the upper copies remained <1000/105PBL in 21 cases, while EBV viral limit for AUC) might further optimize the outcomes in SCT for load was ≥ 1000 copies/105 PBL in 32 (60%).These patients MDS/JMML. were more strictly monitored (twice a week). We observed an increase in EBV load ≥ 20,000 copies/105 PBL after a mean of 54 days from HSCT in 12 transplants(SD 25.9). Nine of them (8 unrelated HSCTs and 1 HLA familiar mismatched donor) who

S221 P789 kin disease =1. Nine patients were in CR2 (4/9 after clofarabine Results of a phase II study on the use of peg-fi lgrastim for containing rescue therapy), one in CR >2 and three patients the collection of autologous peripheral CD34+ stem cells were transplanted in relapse. The fi rst SCT was autologous in all in paediatric patients cases and the median gap between transplants was 17 months S. Cesaro (1), G. A. Zanazzo (2), G. Tridello (1), S. Frenos (3), (9-54). The source and HLA identity of second SCT were: Umbil- A. Bellan (1), S. Dallorso (4) ical cord blood =6 (1 related), peripheral blood =6 (MRD =1, (1)University of Padua (Padua, IT); (2)Istituto Burlo Garofalo MMRD =2, MUD =3), match unrelated bone marrow =1. (Trieste, IT); (3)Ospedale Meyer (Florence, IT); (4)Istituto G. Conditioning regimen were chemotherapy based in 9 cases Gaslini (Genoa, IT) (RIC =1); and TBI-based in 4. ATG was administered to 11 patients. From May 2007 to July 2008, Peg-f was administered to 34 All patients engrafted at a median time of 16 days (9-36). Acute consecutive patients from 4 Italian pediatric centres at a dos- GVHD = 2 and Chronic limited GVHD =1. age of 100 ug/kg (max 6 mg) for PBSC purposes. They were Infectious complications were the main morbidity cause: viral 21 male and 13 female, median age at diagnosis of 10 years =8, fungal =1 (Candida spp), bacterial sepsis =5. (range 3-18), affected by solid tumour, 30, acute lymphoblastic One patient presented grade II veno-occlusive disease. We leukaemia or non-Hodgkin lymphoma, 4. The remission status report lung infi ltrations in 4 cases and Post transplant lympho- at PBSC was: CR 8 (24%), VGPR 5 (15%), PR 19 (56%), SD or proliferative disorder (PLPD) in two patients. not known 2 (6%). The median weight was 35.5 kg (range 13.5- Five patients died (disease progression =2, LPD =2, toxicity 86) and the median number of planned infusion was 1, range =1), 3 before day +100. 1-3. Different regimens of mobilizing chemotherapy were used, Eight patients remain alive and in CR at a median follow-up of etoposide, cyclophosphamide and ifosphamide being the most 47 months post SCT (3 - 104). frequent drugs administered. The median time to fi rst PBSC Conclusions: In our experience second transplant can be cura- was 10 days, range 6-15. The least threshold for CD34+ col- tive for a selected group of patients. Morbidity and mortality are lection (20 cell/ul) was obtained in 28 of 34 patients (82%), the acceptable. A close surveillance and preemptive treatment in median value of CD34+ peak being 140 (range 20-1988). Suc- viral infection might explain our good results. cessful PBSC was obtained in 27 patients (79%) because one very low weight child failed it for suboptimal vascular access. Sixteen of 27 patients (59%) achieved the target PBSC col- P791 lection with 1 leukapheresis whilst 10 patients required a sec- Comparing effi cacy and toxicity of conditioning ond leukapheresis. The median collection yield was 8 (range regimen of treosulfan + cyclophosphomide versus 1.9-116) and 2.45 (range 1-6) CD34+ x 106/kg) for the fi rst and busulfan + cyclophosphomide in children with second leukapheresis, respectively. No Peg-f related adverse haematological malignancies after allogeneic effects were reported. haematopoietic stem cell transplantation At July 31st 2008, 15 of 27 patients (56%) underwent a fi rst N. Stancheva, E. Semenova, Y. Vasilieva, N. Zubarovskaya, autologous transplant whilst 4 and 1 underwent a second and V. Vavilov, O. Goloschapov, I. Mushchitskaya, L. Zubarovskaya, third transplant, respectively. The median time from PBSC to fi rst B. Afanasyev transplant was 64 days (range 10-154) and the median value SPb State I. Pavlov Medical University (St. Petersburg, RU) of CD34+ x 106/kg infused was 7 (range 3-299). After a median f-up of 29 days from fi rst transplant (range 16-176), all patients Background: Allogeneic hematopoietic stem cell transplant achieved a PMN count > 0.5 x 109/l in a median time of 13 days (allo HSCT) is a curative approach for children with refractory (range 5-23) whilst 14 and 12 achieved a transfusion-unsup- or recurrent hematological malignancies but is associated with ported PLT count > 20 and > 50 x 109/l in a median time of 13 high treatment related morbidity and mortality. Conditioning (range 5-23) and 15 days (range 12-36), respectively. Prophylac- regimen with reduced toxicity is a potential option used in these tic post-transplant G-CSF was used in 7 of 15 patients (47%) for a cases. Treosulfan is an alkylating agent with high antileukemic, median time of 7 days (range 1-11). All 5 patients who performed myeloablative activity and low toxicity. We compared effi cacy a second or third transplant successfully engrafted for PMN and and toxicity of two conditioning regimens in allo HSCT: treosul- PLT. All 15 transplanted patients were alive at latest f-up. fan (TREO) +cyclophosphamide (CY) (1-st group) and busulfan We conclude that Peg-f single-shot CD34+ mobilisation is safe (BU)+CY (2-nd group). and effi cacy also in pediatric patients. Further prospective stud- Patients and methods: First group -11 patients (pts), follow-up ies are needed to investigate the non-inferiority or superiority of is from Feb, 2004 till Nov, 2007: 6 boys, 5 girls, with mediana Peg-f vs. fi lgrastim in PBSC. of age–9 y.o., ALL-10 pts (relapse- 3 pts, 1 complete remission (CR) –1 pts, 2 CR –6 pts), AML in 2 CR -1 pt. Conditioning regimen: TREO 30-42 g/m² and CY 120mg/kg. HSC donors: P790 2 matched related donors (MRD), 8 matched unrelated donor Outcome of second transplant for relapsed (MUD), 1 mismatch (8/10) unrelated donor (MMUD). Follow –up haematological paediatric malignancies 2-nd group was from Nov 2000 till Nov 2006 and consisted of 31 M. Andres, J. Fernández-Navarro, F. Bautista, L. Moreno, pts (19 boys, 12 girls), mediana of age –12 y.o. AML –7 pts (1CR A. Verdeguer –4 pts, 2 CR-2 pts, and relapse -1 pts), ALL –24 pts (1 CR-1 pt, Hospital La FE (Valencia, ES) 2 CR-11 pts, 3 CR -8 pts, relapse- 4 pts). HSC donors: 11 MRD, 20 MUD. Conditioning regimen: BUCY (BU 16 mg/kg days and Purpose: Second transplant has been considered the only CY 120mg/kg). In both groups prophylaxis of acute graft-ver- rescue option for patients relapsing after stem cell transplant sus-host-disease (aGvHD) included CsA + short course of MTX. (SCT). High morbidity and mortality rates are the major limiting Unrelated allo-HSCT pts received ATG (“Pfi zer”) 60 mg/kg. factors when confronting this treatment. Results: 1-st group vs 2-st group: engraftment at day +17 vs +21 Patients and methods: Since 1989 a total of 293 SCT have been (p>0,05), primary non engraftment 0% vs 6% (p<0,05), hemor- carried out in our Unit. During this period 21 patients received rhagic complication 18 vs 68% (p<0,05), administration G-CSF a second graft. In order to have a more homogeneous sample 36% vs 52% (p>0,05), median quantity of platelet transfusion- regarding surveillance of both viral and fungal infection and their 4,3 vs 9,3 (p>0,05); erythrocyte transfusion-5 vs 7,8 (p>0,05). treatment, conditioning and graft versus host disease (GVHD) Common toxicity criteria (CTC) II/IV: VOD 0% vs 3% (p<0,05), management, we retrospectively review our experience in sec- mucositis 18% vs 68% (p<0,05), neurology symptoms 9% vs ond transplant from January 2000 to November 2008. 23% (p<0,05), hepatic toxicity 18% vs 26% (p>0,05). aGvHD Results: Thirteen patients underwent second SCT at a median III/IV 36% vs 20% (p>0,05), relapse 27% vs 29% (p>0,05). 3- age of 9 years (2.1-19). Diagnoses were: AML =7, ALL =5, Hodg- year overall survival (OS) 37% vs 40%, respectively.

S222 Conclusion: Treosulfan-based conditioning regimen is well tol- Material and Methods: 46 children received SCT after BU-based erable, safe, and effi cient, has lower toxicity and can be used conditionig regimens at a median age of 7,2 years (yrs) (range in heavy-pretreated children with severe complications after 0,5-16,4) for leukaemia (29), solid tumours (15) and non onco- previous chemotherapy, but in comparing of effi cacy both regi- logic hematological diseases (2). 28/46 pts underwent ALLO ments have no differences. SCT, 18/46 AUTO SCT. During follow-up (median 6,3 yrs, range 1,4-15,3 yrs) TSH, FT3, FT4, t-peroxydase (TPO)/ thyroglobu- lin (TG) autoantibodies (ab) were regularly evaluated in all pts; P792 thyroid ultrasonography (US) was obtained in 30/46 pts. Osteochondroma after HSCT in childhood. An Italian Results: 37/46 pts, (21 pts after ALLO SCT) showed at least study on behalf of the AIEOP-HSCT Group one thyroid status abnormality. In particular 20/46 pts (12 pts M. Faraci, F. Bagnasco, P. Corti, C. Messina, F. Fagioli, after ALLO SCT) developed subclinical hypothyroidism (SH) at M. Podda, A. Prete, D. Caselli, L. Edoardo, G. Dini, R. Rondelli, a median age of 8 yrs (range 2-21), with a median time of onset R. Haupt on behalf of the AIEOP-HSCT group after SCT of 2 yrs (0,2-10,9). 2/46 patients developed primary hypothyroidism, after ALLO SCT for hemophagocytic lympho- Introduction: osteochondromas (OS) are benign bone masses histiocytosis. documented after Total Body Irradiation (TBI). The aim of this Anti TPO/TG ab positivity was found in 28 pts (17 pts after study was to establish the implications of any other risk factors ALLO SCT) and in 16 pts (10 after ALLO SCT) of 22 who devel- in the development of OS after HSCT in childhood. oped hypothyroidism. Materials and methods: a retrospective study was organised in US thyroid abnormalities were found in 15/30 examined pts: the framework of the Italian paediatric HSCT group (AIEOP) to reduction in size (7 pts), structural dyshomogeneity (8 pts) and evaluate the incidence and risk factors of OS in children after nodes (3 pts). allogeneic and autologous HSCT. Nine AIEOP-HSCT centres Nine out 22 patients started l-thyroxine therapy after a variable participated in the study and 1632 children were eligible for this time after a median time from SCT of 2,2 yrs (range 1-8), while study. There was a slight prevalence of males (59%) and of 14/22 pts showed a transient SH and did not receive any sub- autologous HSCT(52%). The median age at HSCT was 7.6 yrs stitutive therapy. (range 0.1-18.9). 49% had haematological malignancies, 38% Conclusions: In our experience 80% of pts receiving SCT after had solid tumours and 13% showed non malignancies. preparative regimen with BU showed an altered thyroid status. Results: 27 children who developed OS after the fi rst HSCT were SH was the most frequent thyroid abnormality occurred in our identifi ed (1.6%). There was a prevalence of males (81%) and children (45%), who in some cases needed l-thyroxine treat- of autologous HSCT (70%). Median age at HSCT was 4.3 yrs ment. The presence of anti TPO/TG ab seems to indicate auto/ (range 0.7-10.6), while median interval between HSCT and OS allo immune pathogenesis of thyroid hypofunction at least in was 8.9 yrs (range 1.9-13.8). None had a family history of OS. 70% of our pts. The underlying disease was haematological malignancy in 17 These data suggest that thyroid function should be assessed cases (63%). TBI was included in the conditioning regimen in 26 regularly even in pts who received Bu-based conditioning cases (96%). Length of follow-up ranged between 1.1 and 29.1 regimens. yrs (median, 4.8 yrs) and the estimated 5,10, and 20 year cumu- lative risk of developing OS for the entire cohort was 0.5%, 3.2%, and 6.1%, respectively. An analysis of cumulative risk stratifi ed by P794 the various risk factors, i.e., male gender (P=0.026), autologous Infl ammatory BCG adenitis associated with immune HSCT (P=0.0007), age at HSCT (≤ 3 yrs) (P<0.0001) and TBI reconstitution following allogeneic haematopoietic stem (P<0.0001)showed that they signifi cantly affect the risk of OS. In cell transplant in infancy the Cox proportional hazard model the factors that were found to E. Searle (1), H. Patel (1), F.J. Vilar (2), G. Batra (1), R.F. Wynn be signifi cantly associated with increased risk of OS were: hav- (1) ing received HSCT at an earlier age (≤ 3 yrs) (P<0.0001); hav- (1)Manchester Children’s Hospital (Manchester, UK); (2)North ing undergone autologous HSCT (P=0.0191); or having been Manchester General Hospital (Manchester, UK) treated with TBI (P<0.0001). Among the 19 autologous HSCT patients with OS, 18 received TBI as part of their conditioning Introduction: Since 2006 we have observed a new phenom- regimen and 10 of them were ≤ 3 yrs old at transplant. enon in a small minority of patients undergoing allogeneic Conclusions: this is the largest analysis and description of OS HSCT (Haematopoietic Stem Cell Transplant); BCG (Bacillus in paediatric recipients, and it demonstrates that besides radio- Calmette-Guérin) adenitis occurring with immune reconstitu- therapy, other independent risk factors for OS are early age at tion. Review of the published literature has not revealed any HSCT and autologous transplant. Careful clinical and anam- previously described cases of BCG-adenitis in the post trans- nestic examination of these patients at risk could help us to plant setting. perform radiological evaluation in suspected cases. Localised infl ammatory reactions at the site of BCG vaccina- tion with ipsilateral axillary lymphadenopathy are well described following the initiation of highly active anti-retroviral therapy P793 (HAART) in children with HIV (Human Immunodefi ciency Incidence of thyroid dysfunction after busulphan-based Virus). This represents a manifestation of Immune Reconsti- conditioning regimens for haematopoietic stem cell tution Infl ammatory Syndrome (IRIS) and occurs as the viral transplantation load declines and the immune system recovers with rising CD4 R. Masetti, F. Baronio, K. Kleinschmidt, S. Bonetti, A. Pasini, counts. R. Rondelli, A. Prete, A. Cicognani, A. Pession Cases: 4 cases of infl ammation at the site of previous (at birth) Università degli Studi di Bologna (Bologna, IT) BCG vaccination associated with localised axillary lymphad- enopathy in children post- HSCT performed in infancy have Objectives: It is generally assumed that busulphan (BU)-based occurred in our unit (See Table 1 for case details). In 2 of the conditioning regimens for hematopoietic stem cell transplan- cases pathological samples have demonstrated an infl am- tation (SCT) affects thyroid function in children less than total matory response and in all cases symptoms were associated body irradiation-based regimen. The incidence reported by lit- with immune reconstitution as measured by increasing T cell erature of thyroid dysfunction in children receiving BU-based numbers in the peripheral blood. As such it is the transplant regimen for allogeneic (ALLO) and autologous (AUTO) SCT is equivalent of IRIS. between 10 and 25%. We retrospectively evaluated the inci- All the 4 cases suffered associated ipsilateral, painful lym- dence of thyroid abnormalities in a series of 46 patients (pts) phadenopathy which in cases 1 and 2 required surgical man- treated with SCT with BU-based preparative regimens. agement. Case 3 resolved spontaneously but cases 1, 2 and

S223 4 required prednisolone to control the local infl ammatory died, p=0.14). No toxicity grade 4 according to NCI-CTC grad- response and lymphadenopathy. 2 of the children were treated ing system occurred in both groups apart from mucositis. with anti-mycobacterial cover during steroid treatment. Conclusions: clofarabine was well tolerated as part of a con- Conclusion: Paediatric transplant and their local infectious dis- ditioning regimen. Compared to our standard regimen with eases teams need to be aware of the previously un-described fl udarabine, thiotepa, melphalan, the introduction of clofarabine phenomenon of BCG-adenitis occurring with immune reconsti- did not result in any unexpected toxicity. Immunosuppressive tution following HSCT. There are distinct parallels between this effects appeared to be similar to those of fl udarabine, since pri- pathological process and that seen in children with HIV follow- mary engraftment was observed in 92%. A tendency for a lower ing the initiation of HAART and the subsequent immunological TRM and a reduced risk of relapse in patients with refractory improvement. leukemias was observed. Our treatment of this complication has been described above and all of our patients have either fully recovered or are making a good recovery. However further research is required to deter- P796 mine the frequency of this complication and the best methods Effective immunotherapy of donor-derived AML after of management, in particular the place of anti-TB therapy and unrelated stem cell transplantation surgical intervention. I. von Luettichau (1), C.V. Denne (1), P. Bader (2), S. Schnittger (3), I. Furlan (4), B. Strahm (4), C. Peters (5), A. Wawer (1), H.-J. Kolb (6), S. Burdach (1) (1)Technical University (Munich, DE); (2)Johann Wolfgang Goethe University (Frankfurt, DE); (3)Munich Leukemia Laboratory GmbH (Munich, DE); (4)Albert Ludwigs University (Freiburg, DE); (5)St. Anna Children›s Hospital (Vienna, AT); (6)University of Munich & Helmholtz Zentrum (Munich, DE)

Donor derived acute myeloid leukaemia (AML) was diag- nosed in an 8 year old boy 4 years after matched unrelated stem cell transplantation (SCT) of a female donor for high risk acute lymphoblastic leukemia (ALL). The patient suffered from extensive chronic graft versus host disease (GvHD) demand- ing high doses of steroids and immunosuppressive therapy. Good response was achieved with Psoralen plus UV-A (PUVA) and extracorporal photophoresis. In the fourth year after SCT the boy presented with progressive pancytopenia. Bone mar- row analysis revealed myelodysplasia with excess of blasts (20%) and Auer rods consistent with AML M2 (WHO criteria). Donor origin of the leukemic cells was confi rmed by Y-FISH P795 (fl uorescent in situ hybridization) revealing female caryotype in Clofarabine versus fl udarabine for haploidentical stem all analyzed metaphasic nuclei. Short-Tandem-Repeat analysis cell transplantation in paediatric patients with refractory (STR) confi rmed the 100% donor chimerism excluding second- leukaemias ary AML of recipient cells. The adult donor type of leukemia H. Teltschik, I. Müller, M. Pfeiffer, T. Feuchtinger, M. Schumm, was further supported by the detection of the NPM1B muta- R. Handgretinger, P. Lang tion typical for a subtype of adult AML as well as a deletion University Children`s Hospital (Tübingen, DE) of chromosome 9q. Donor derived leukemia may be due to a leukemogenic host microenvironment. There is no standard Studies have shown the effi cacy of clofarabine in resistant therapy regimen. Considering the low blast count in the bone leukemias. We compared clofarabine with fl udarabine in a marrow and the benign clinical course of the disease in our melphalan based conditioning regimen for haploidentical stem patient we started a combination of low dose chemotherapy fol- cell transplantation in pediatric patients with refractory leuke- lowed by adoptive immunotherapy. The regimen comprises the mias. Patients after standard treatment (n=5) or after previous transfusion of increasing numbers of donor lymphocytes (DLI) allogeneic transplantation (n=8), 9 with ALL (NR1=3, NR2=2, and the simultaneous application of low dose Interferon-alpha CR3=4), 3 with AML (NR 1=3) and 1 with MDS (NR2=1) and GM-CSF following a 14 day course of low-dose cytosine received clofarabine 4x50mg/m² (day -8 to -5), thiotepa 10mg/ arabinosid (10mg/m2 sc BID). 4 weeks after the fi rst DLI, < 5% kg (day -4), melphalan 2x70mg/m² (day -3 to -2) and OKT3 blasts were found in the bone marrow. Dysplasia, however, was (0,1mg/kg day -8 to +14; n=13), followed by infusion of haploi- persistent. The peripheral blood count improved, resulting in dentical stem cells. We compared them with a historical group transfusion independency. We plan to continue the treatment of patients (n=9) with refractory leukemias after standard treat- until NPM1B mutation carrying cells are no longer detectable. ment (n=6) or after previous transplantation (n=3), 1 with ALL So far the treatment is well tolerated and no GvHD is apparent. (CR2=1), 7 with AML (NR1=2, NR2=4, CR3=1) and 1 with MDS Taken together, adoptive immunotherapy in combination with a (NR3=1). Depletion of T and B cells was carried out with CD3/ mild chemotherapy regimen might be feasible for this rare con- CD19 coated magnetic microbeads. Primary engraftment in the dition considering that the leukemic clone developed in a highly clofarabine group (12/13 patients) was comparable with the immunosuppressed setting resulting in a potentially higher sen- fl udarabine group (9/9 patients). One patient of the clofarabine sitivity for induced immunological control of donor leukemia. group was successfully retransplanted from a second parental donor. Sustained engraftment could be obtained in all patients. Median time to ANC >500/µl with G-CSF stimulation was 10 days (9-11) in the clofarabine group vs. 11 days (9-12) in the Fludarabine group. Independence from platelet transfusion was reached after 9 days in both groups. Relapse rate was lower in the clofarabine group (9/13 patients vs. 8/9 patients, p=0.21) than in the fl udarabine group (median time to relapse: 120 days vs. 114 days). One year EFS was 23% in the clofarabine group (3/13 patients are disease free with a median follow up of 1.2 years) compared with 0% in the fl udarabine group (9/9 patients

S224 P797 Patients: In four pediatric BMT centers in Poland, between Allogeneic haematopoietic stem cell transplantation for 2000 and 2008, twenty three children with NHL received allo secondary haematological malignancy in children and SCT. There were 17 males and 6 girls, aged from 6,1 to-17,2 adolescents – Results of the Polish Paediatric Group for years (median 13,5). Among those children 13 suffered from HSCT B-NHL, 7 -from T-NHL and 3 children had Large Cell Anaplastic J. Wachowiak (1), G. Grund (1), W. Gabor (1), A. Chybicka (2), Lymphoma (LCAL). E. Gorczynska (2), J. Owoc-Lempach (2), J. Kowalczyk (3), At the moment of SCT 10 patients were in second complete K. Drabko (3), M. Choma (3) remision (2CR), 4- in 1 CR, 3 children –in ≥ 2 CR, 5 patients (1)University of Medical Sciences (Poznan, PL); (2)Medical had partial remision (PR) and one boy had progressive disease University (Wroclaw, PL); (3)Medical University (Lublin, PL) resistant to therapy. Conditioning before SCT was myeloablative in all and consisted Secondary hematological malignancy (sHM) is usually con- of total body irradiation (TBI) with chemotherapy in 16 children sidered as high risk one, however there are no standardized and only chemotherapy –in 7 procedures. Thirteen children recommendations concerning indications and preparative were transplanted from matched sibling donors (MSD) and ten- regimens for allogeneic stem cell transplantation (allo-HSCT) from matched unrelated donors (MUD). for them. Apart from that there are no available data in the lit- Results: 22/23 patients engrafted, and in 1 child autologous erature concerning specifi cally results of allo-HSCT for sHM in recovery was observed. children and adolescents. 11/23 children are alive (48%) and they are still in CR. Among Objectives: To evaluate engraftment rate, non-relapse mortality those patients: 8 were in 2CR, 2-in 1CR and 1-in 1PR befor SCT. and anti-leukemic effect of allo-HSCT in children with second- 12/23 patients died: 4 children (17%) died due to relapse, 3- ary hematological malignancies. due to progression and 5 children (22%)- due to transplantation Patients and methods: Retrospective analysis was performed in -related complications (TRM). Overall survival (OS) was 48%, 12 children and adolescents (7 female, 5 male), who underwent with the observation time from 14 to 104 months (median 40). allo-HSCT for sHM since year 2000 within transplant centers Conclusions: Our study suggests, that allo SCT may improve of the Polish Pediatric Group for HSCT. Median age was 11.5 prognosis in children with recurrent NHL, when transplantation years (range 6-17 years). Diagnosis consisted of secondary is being performed in status CR of disease. However, allo SCT AML (n = 8; 4 in CR1, 4 in CR2), secondary MDS (n = 3), and doesn’t improve results in patients with partial remision. The secondary NHL (n = 1). Nine patients were transplanted from conclusion should be evalueted in a larger series of patients. matched unrelated donor (MUD) and only 3 from matched sib- Allo SCT in children with resistant or recurrent NHL is burdened ling donor (MSD). In 6 patients preparative regimens for HSCT with high risk of complications in those heavily pre-treated were based on busulfan (total dose 16 mg/kg), and in remaining patients. 6 on treosulfan (total dose 30-36 g/m2). In addition, prior MUD- HSCT, antithymoglobulin was given for T depletion in vivo. As GvHD prevention CsA+MTX was given after MUD-HSCT, and P799 CsA alone after MSD-HSCT. Loss of chimerism following allogenic haematopoietic Results: Non-relapse death occurred in one out of evaluated progenitor cell transplantation in children: a silent alarm 12 patients (day +6, ARDS in course of sepsis and pneumonia F.J. Bautista Sirvent, L. Moreno Martin Retortillo, A. Verdeguer, in child transplanted from MUD for sAML in CR1 after PNET). M. Andres Moreno, J.M. Fernandez Navarro, V. Castel Except the last one patient, who died too early, engraftment Hospital Infantil La Fe (Valencia, ES) was achieved between day (+12) and (+19) (median: day +14) in all others recipients. Relapse occured in 5 patients from 3 to Introduction: Allogenic haematopietic progenitor cell transplan- 14 months (median: 9 months) after HSCT, including 3 out of tation (Allo-HPCT) is the cornerstone in the treatment of a wide 8 patients with sAML (one in CR1, 2 in CR2), and in 2 out of 3 spectrum of hematological malignancies in children. Its suc- with sMDS. Six recipients are alive in CCR for 25-75 months cess is highly affected by recurrence of the underlying disease. (median: 40 months) after transplantation. Chimerism follow up is essential to fi nd out patients at higher Conclusions: In children with sHM the allo-HSCT after condi- risk of recurrence. tioning with busulfan- or treosulfan-based regimens is feasible. Objective: To analyze the impact of chimerism status at days However, post-transplant relapse occurs in around half of them +30, +60 and +90 post Allo HPCT regarding clinical outcome and represents the most frequent cause of treatment failure. Material and Methods: We retrospectively reviewed 91 patients Therefore, there is a need for further studies concerning allo- (male=51 and female=40) with underlying malignancies who HSCT in children suffering from sHM to defi ne in the future indi- underwent Allo-HPCT between November 1987 and September cations for HSCT and optimum transplant procedure in this very 2008. Patient age ranged from 0.5 to 19 years (median=7.58). special subgroup of pediatric patients. Diagnosis included Acute Lymphoblastic Leukemia (ALL=53), Acute Myeloid Leukemia (AML=30) and others (O=6). Chimer- ism status was determined by quantitative polymerase chain P798 reaction (PCR) of variable number of tandem repeats (VNTR) Allogeneic stem cell transplantation in children with at days +30, +60 and +90 after Allo HPCT. Chimerism was non-Hodgkin’s lymphoma – Polish experience defi ned as Complete Chimerism (CC) when there was no evi- B. Wojcik (1), M. Choma (1), K. Drabko (1), A. Zaucha-Prazmo dence of autologous cell at any time post transplant, as Mixed (1), J. Kowalczyk (1), E. Gorczynska (2), J. Owoc (2), K. Kalwak Chimerism (MC) when both recipient and donor cells where (2), M. Leda (3), J. Wachowiak (3), J. Gozdzik (4) found and Null Chimerism (NC) when complete autologous (1)Children’s University Hospital (Lublin, PL); (2)Children’s reconstitution was seen. University Hospital (Wroclaw, PL); (3)Children’s University Results: 31 out of 91 patients (ALL=18, AML=12, O=1) relapsed Hospital (Poznan, PL); (4)Children’s University Hospital after Allo-HPCT. Median time to relapse was 114 days (25-844). (Cracow, PL) All relapsed patients eventually died (Disease progression (DP) =27, Sepsis (S) =2, Graft Versus Host Disease (GVHD) =2). Conventional chemioterapy for non-Hodgkin’s lymphoma (NHL) 28 other children died in remission (S=4, GVHD=2, Viral Infec- are very successful-up to 70-90% children make a full recovery. tions=3, Other=19). Mean follow up was 833.5 days ± 1238.1. However, prognosis for patients with refractory and recurrent At day +30 median relapse-free-survival was 14,53 months disease are not so much promising. in patients with CC, 12.3 months with MC and 2.07 months in Objective: we retrospectively analyzed the therapeutic effi - children with NC (p<0.05). Median Overall-survival was 15.02 cancy and safety of allo SCT in children with recurrent or resist- months in patients with CC at day +30, 12.37 months with MC ant NHL. and 2.07 months in children with NC (p<0,05).

S225 Conclusions: Conclusions: • Our series confi rm how patients with NC have a very poor • HPCT is a useful therapeutic strategy, but special consid- outcome. In our cohort patients with MC did as well as erations should be taken into account when transplanting patients with CC at day +30, but at day +60 and +90, only infants. patients with CC had a favorable outcome. • ALL behaves very aggressive in this age group. • VNTR-PCR is a useful technique to detect chimerism in Allo • The role of auto HPCT for infant leukaemia has yet to be HPCT and is able to predict relapse. established. • Serial characterization of post transplantation chimerism • Infants with NB have a favourable biological feature and thus, might identify those patients at higher risk to develop relapse prognosis is better compared to older children. that could benefi t from other therapies to be rescued. • A group of patients might be rescued with second transplant. • Allogenic HCPT is a risky procedure in infants, with raised MRT, related to GVHD and viral infections.

P801 High-dose chemotherapy in patients with high-risk P800 Ewing’s sarcoma of pelvis. A single-centre experience Haematopoietic progenitor cell transplantation for infants: I. Dolgopolov (1), V. Boyarshinov (1), R. Pimenov (1), our institutional experience N. Subbotina (1), I. Visotchin (1), S. Siegel (2), G. Mentkevich (1) F.J. Bautista Sirvent, L. Moreno Martin Retortillo, A. Verdeguer, (1)Institute of Ped. Oncology/Hematology (Moscow, RU); M. Andres Moreno, J.M. Fernandez Navarro, V. Castel (2)Children’s Hospital (Los Angeles, US) Hospital Infantil La Fe (Valencia, ES) This study was initiated in an attempt to improve EFS in pts Introduction: Haematopoietic progenitor cell transplantation with high-risk pelvic ES using an intensive treatment program (HPCT) is a diffi cult procedure in children, and might be even of short duration and HDCT followed by autoPBSCT. From harder in infants. We analyze our experience in a specialised January 1996 to November 2008 27 pts (M/F-11/16) with high- children’s hospital performing HPCT in infants. risk pelvic ES (localized 21 pts, metastatic 9 pts (only lungs-2, Objectives: combined-7) received HDCT with autologous transplantation • To describe the outcome after HPCT in infants at our institu- as a consolidation. The median primary tumor volume (PTV) tion was 739 cm³. CT consisted of 5 courses: 1, 3, and 5 included • To identify major complications in this subset of patients. cyclophosphamide 2100 mg/m²/day on days 1,2, doxorubicin • Material and Methods: We retrospectively reviewed 30 37.5 mg/m² as a 24-hour infusion on days 1,2, and vincristine patients (20 males and 10 females) treated for malignan- 1.5 mg/m² on days 1, 8, and 15. Cycles 2 and 4 consisted of cies who underwent HPCT from 1992 to 2008. Age ranged ifosfamide 2400 mg/m²/day on days 1-5 and VP-16 100 mg/ from 6 to 22 months (median=15). Diagnosis included Acute m²/day on days 1-5. No G-CSF was given routinely between Lymphoblastic Leukemia (ALL=8), Acute Myeloid Leuke- cycles. Radiation therapy was given after the fi fth cycle of CT at mia (AML=12), Neuroblastoma (NB=7), Non-Hodgkin Lym- athe median dose of 52 Gy (range, 50-56). Patients with lung phoma (NHL=1), Myelodisplastic Syndrome (MDS=1) and involvement uniformly received lung irradiation after the sec- Meduloblastoma (MB=1). 21 patients underwent Autologous ond cycle of CT at a dose of 10.8-12 Gy. As a consolidation all HPCT (ALL=4, AML=9, NB=7, MB=1) and 9 Allogenic HPCT pts received busulfan 16 mg/kg, melphalan 140 mg/m²-based (ALL=4, AML=3, NHL=1, MDS=1). high-dose chemotherapy (HDCT) (n=8); with the addition Results: of thiotepa (TT) 600-900 mg/m² (n=10) or etoposide (VP16) • 9 patients undergoing Auto HPCT recurred (3 ALL, 4 AML, 1 1400 mg/m² (n=9) followed by autologous stem cell rescue NB, 1 MB) and 3 patients recurred after Allo HCPT (2 ALL, (6.4 (1.9-25.3)x106 CD34+ cells/kg). Median number of days 1 AML). Median time to recurrence was 197 days for Auto to WBC>1.0x109/l and Plt>20 and 50x109/l was 10 (8-14), 16 HPCT and 178 days for Allo HPCT. 5 recurred patients after (0-52) and 28 (11-66) days, respectively. TRM was 11% (3 pts Auto HPCT underwent subsequent Allo HPCT. 1 recurred out of 27). Eight pts relapsed (5 of them with primary metastatic patient after Allo HPCT underwent second Allo HPCT but disease). EFS and DFS were 58% and 66% with a median fol- eventually he died. low-up of 86 and 96 mo, respectively. EFS and DFS in TT, VP16 • 9 patients undergoing Auto HPCT died (3 ALL, 3 AML, 2 NB, and BuMel groups were 56%, 50% and 73% and 63%63% and 1 MB) and 6 patients died after Allo HPCT (4 ALL, 1 AML, 73%, respectively. One pt had ileus 3 mo after BMT and had 1NHL). Most frequent cause of death in both groups was dis- successful surgery. ease progression. 2 out of 5 relapses after Auto HPCT where Our results support the hypothesis that pts with high-risk pelvic rescued with Allo HPCT. ES may benefi t from BuMel-based regimens and that an addi- • 40% of patients undergoing Allo HPCT developed Acute tion of any drug to BuMel regimen increases a toxicity with no Graft Versus Host Disease (GVHD) and 53% viral infections. infl uence to DFS. • Median follow up was 26.1 months. 1 out of 8 (12.5%). ALL Supported by Ronald McDonald House Charities. patients remains alive, 8 out of 12 (66%) AML patients do and 5 out 7 (71%) NB patients do. • Mortality related transplant was 4,1% for Auto HPCT and 20% for Allo. • 57% of NB patients were in complete remission at the time of transplant. Only one out of 7 relapsed.

S226 P802 based on defi cient arylsulfatase A (ARSA) activity in fi broblasts, Allogeneic transplantation using adjusted blood busulfan elevated urinary sulfatides, and the presence of the genetic levels results in a very low incidence of veno-occlusive ARSA defect responsible for MLD in her sister. disease, irrespective of the presence of the glutathione The decision to perform an hematopoietic stem cell transplan- S-transferase M1 null genotype tation (HSCT) in both patients was made to stop the progres- R. Elhasid (1), E. Efrati (2), A. Khalil (1), J. Stein (3), I. Zaidman sion of MLD. Both patients received an equal number of cells (1), M. Ben Arush (1), J.M. Rowe (1), N. Krivoy (1) from one single bone marrow harvest (MUD, 3,46 million CD (1)Rambam Medical Center and Technion (Haifa, IL); 34 + cells per kg body weight each). Conditioning consisted of (2)Bruce Rappaport Faculty of Medicine, Technion (Haifa, IL); Flud/Bu/Mel. ATG, MTX and cyclosporine were given as GVHD (1)Rambam Medical Center (Haifa, IL); (3)Shneider Children’s prophylaxis. Medical Center of Israel (Petach-Tikva, IL) Both patients had a concurrent take at day 15 after HSCT and an uneventful post transplantational course. But from day 30 Optimal busulfan (BU) pharmacokinetic data have been estab- after HSCT one twin developed GVHD Grade II-III involving lished for stem cell transplantation (SCT) setting, and dose skin, gut and conjunctiva, requiring corticosteroids up to 2mg/ adjustment is made based on “therapeutic” BU area under kg/d. The gut GVHD was proven by biopsy and revealed a concentration-time curve (AUC) calculation for individual grade 2 GVHD according to Lerner. In the following weeks this blood level determination. Srivastava (Blood 2004) assessed patient had intermittend episodes of fever, rash and diarrhea BU pharmacokinetic analysis and impact of glutathione S- due to his GVHD showing more features of acute than chronic transferase (GST) genes polymorphism on the risk of hepatic GVHD. veno-occlusive disease (HVOD). In that study, pharmacokinetic GVHD resolved and GVHD treatment was stopped on day 270 analysis showed higher clearance of BU among patients with after HSCT. The clinical course of the other transplanted twin VOD and higher frequency of GSTM1-null genotype, suggest- was uneventful. The GVHD prophylaxis was stopped on day ing that GSTM1-null predisposes to HVOD. In a pilot study of 198 after HSCT. the Rambam Medical Center, BU pharmacokinetics were meas- This is the fi rst report on identical twins transplanted from a ured in 25 children with congenital hemoglobinopathies under- single BM harvest. This was a save procedure in respect to the going SCT; 5 of them with biopsy-proven liver cirrhosis. Dose cell dosage since the recipients weighed 22 kg. The difference adjustments were performed according to the BU AUC. Only in GVHD in both patients was unexspected, since cells of a sin- 1/25 patients had HVOD, a young child with no cirrhosis and gle donor came in contact with genetically identical hosts and low BU level. This fi nding was in discrepancy with the higher induced a very different immunological reaction (GVHD). incidence of VOD (29%) reported by Srivastava. The purpose Our HSCT might point to host factors (virus infections, cytokines of the present study was to examine the effect of BU disposition etc.) leading to different immune reactions in otherwise identi- and GST-M1 polymorphism on occurrence of VOD in children cal donor recipient pairs. receiving BU as conditioning regimen prior to SCT. Patients: 25 children (13 females and 12 males) received BU (12-16 mg/kg) before SCT; individual BU levels were analyzed P804 after a second dose, and dose adjustments were performed to Prospective, multicentre, open trial in high-risk reach AUC between 900-1100 µM* min. Sixteen children had paediatrics using a reduced intensity with intravenous congenital hemoglobinopathy, 3 immunodefi ciency, 3 neuro- busulfan and fl udarabine blastoma, 2 Hurler syndrome, and 1 AML. Median age at trans- C. Paillard (1), P. Lutz (2), G. Leverger (3), G. Michel (4), plantation – 4 years (range 3 m.-16 y). Genetic analysis of GST P. Bordigoni (5), M. Poirée (6), D. Plantaz (7), J.P. Vannier (8), M1 polymorphism was determined by PCR of DNA extracted P. Halle (1), F. Deméocq (1), J. Kanold (1) from peripheral blood taken prior to SCT. (1)CRCTCP CHU Hôtel Dieu (Clermont Ferrand, FR); (2)CHU Results: GSTM1-null genotype was found in 65.4% of the chil- Strasbourg (Strasbourg, FR); (3)Hopital Trousseau (Paris, FR); dren. Only 2 patients developed VOD, one with thalassemia (4)Hopital la Timone (Marseille, FR); (5)CHU Nancy (Nancy, major and wild type/heterozygote GSTM1, and the other with FR); (6)Hopital Archet (Nice, FR); (7)CHU Grenoble (Grenoble, Hurler syndrome and GSTM1-null genotype. Both had low BU FR); (8)Hopital Charles Nicolle (Rouen, FR) AUC and dose adjustments were done. Conclusions: The incidence of GSTM1-null genotype (65.4%) Objectives: The purpose was to evaluate engraftment, chimer- was signifi cantly higher than previously reported (table). ism of a reduced intensity conditioning in high-risk children Despite this, the incidence of VOD was very low, suggesting undergoing allogeneic stem cell transplantation but who can’t that adjusting BU to well defi ned therapeutic levels may amelio- benefi t of a myeloablative therapy or who failed previous stand- rate the effect of GSTM1-null genotype on BU pharmacokinet- art treatment. ics, allow greater exposure to the drug, prevent its toxicity and Methods: This phase II trial started in april 2007 and 30 children occurrence of VOD. are planned to be enrolled. 20 patients were enrolled between april 2007 and november 2008. The conditioning regimen con- sisted of intravenous busulfan 3.2 mg/kg in once daily infusion for 2 days, and fl udarabine 30mg/m² 6 days, antithymoglob- uline 2.5 mg/kg, 1 d for related and 2 d for unrelated donor. GVH prophylaxis consists in cyclosporine alone. Results: This is an intermediate analysis in 20 heavily pre- treated children with a median age of 13.8 years at transplant (4.8-20.2) with 14 males and 6 females. Six patients had malig- nant solid tumors (neuroblastoma, rhabdomyosarcoma), 5 had P803 acute myeloblastic leukemia, 2 acute lymphoblastic leukemia, Divergent GvHD in identical twins receiving bone 7 Hodgkin’s lymphoma. marrow from a single donor for halting metachromatic 16 patients had more than 3 lines of chemotherapy; 8 patients leukodystrophy had previous autologous stem cell transplantation. 12 children J. Schrum, A. Bley, H. Kabisch, A. Zander were allografted in active disease (partial remission, relapse, University Hospital Hamburg-Eppendorf (Hamburg, DE) progression or refractory disease) and 8 were in complete remission. 13 patients had an HLA-matched sibling donor,7 A pair of healthy 4-year old identical twins, brothers of a girl had an unrelated donor. Graft source was PBSC in 8 cases and suffering from metachromatic leukodystrophy (MLD), were bone marrow in 12 cases. The median time from diagnosis to diagnosed with the preclinical stage of MLD. The diagnosis was allograft was 22 months. Concerning the hematologic recovery,

S227 the median time to reach absolute neutrophil count (ANC) ≥ P806 500 cells/mm3 is 16 days. The median days under 25 000/mm3 CB transplant in a haemophilic child for platelet is 5 days. Evaluation of chimerism is available for D. Caselli (1), V. Tintori (1), F. Bambi (1), S. Frenos (1), 18 children. At day 30 post-transplant, chimerism is mainly M. Veltroni (1), M. Morfi ni (2), S. Guidi (2), M. Aricò (1) donor for sixteen children (14 with complete chimerism ≥ 95% (1)AUO Meyer (Florence, IT); (2)AUO Careggi (Florence, IT) of donor’s cells; 2 children with mixed chimerism and ≥ 80% of donor’s cells). At day 60 post-transplant, the 2 previous children Introduction: Haemophilia A (OMIM 306700) is a X-linked, had a complete chimerism. Only one patient had primary graft recessive, bleeding disorder caused by defi ciency of coagula- failure at day 30. 8 children experienced acute GVHD grade tion factor VIII (FVIII); mutations in FVIII gene (F8, F8C, HEMA) I-II and 2 experienced grade IV. At the date of the cut-off, 15 mapping to Xq28 are heterogeneous and phenotype is variable children are alive. with hemorrhage into joints and muscles, bruising, prolonged Conclusion: Even if further follow-up is needed the reduced bleeding from wounds. Liver is the main source of FVIII, and lit- intensity conditioning combining i.v. busulfan and fl udarab- tle is know about the potential for synthesis by other tissues and ine is feasible, safe and effective. This regimen enables to organs. Studies of organ transplant recipients raise the pos- perform allo HSCT in children not eligible for a myeloablative sibility that lymphatic tissue, spleen, lung may compensate for conditioning. defi cient hepatic FVIII production. We report a boy with severe hemophilia A (mutation of exon 14) and relapsed anaplastic large cell lymphoma, successfully treated by transplantation of P805 unrelated cord blood. A.K. hemophilia A diagnosed at 18 months Immune reconstitution and immunisation in paediatric in his country, treated with blood transfusion and cryoprecipitate allogeneic bone marrow transplant recipients and at 6 years, upon immigration in Italy, with FVIII. At 12 years H. Olkinuora (1), S. Siitonen (2), E. von Willebrand (2), he developed a localized scapular mass, excised and treated U. Pihkala-Saarinen (1), J. Kantele (3), O. Vainio (4), with chemotherapy followed by consolidation with autologous H. Käyhty (5), R.-M. Ölander (5), M. Roivainen (5), I. Davidkin (5), HSCT. At months +18 shows systemic lymphadenomegaly; his- K. Vettenranta (1) tological diagnosis was ALCL. Treated according to the EICNHL (1)Hospital for Children and Adolescents (Helsinki, FI); protocol, for Relapsed ALCL. After 3 chemotherapy blocks, he (2)Helsinki University Central Hospital (Helsinki, FI); (3)Turku underwent HSCT from an unrelated cord blood unit; HLA match University Central Hospital (Turku, FI); (4)University of Oulu was 4/6, CD34 count 2x105/kg. Conditioning regimen included (Oulu, FI); (5)National Public Health Institute (Helsinki, FI) Thiotepa 10 mg/kg, Etoposide 40 mg/kg and TBI 12 Gy. During the entire transplant phase, platelets were infused every other Background: Lymphocyte recovery constitutes an essential day, aiming at count >30,000/µl, RhFVIII 1,000 IU every 12 part of immunoreconstitution post hematopoietic stem cell hours. Neutropenia was complicated by Pseudomonas a. and transplantation (HSCT). Delayed and/or insuffi cient recovery Klebsiella septicemia. Stable engraftment was documented for leads to an increased risk of post-SCT complications. The aim neutrophils since day +30 and for platelets (>50 000/ml) since of this study was to evaluate immunological reconstitution and day +42. On day +44 he was readmitted for hemorrhagic cys- antibody responses to vaccines post HSCT and prospectively titis. At present, on day +65, he is doing well. VNTR analysis evaluate the two processes among recipients of pediatric allo- showed 100% donor engraftment. Clotting test results and the genic stem cell grafts. FVIII level are similar to pre-BMT values. No inhibitors were Material and methods: Twenty-three long-term (> 18 mos) sur- present before or after transplantation. vivors of pediatric HSCT with a mean age of 8,5 yrs with either a Conclusion:To our knowledge this is the third case of HSCT in malignant (18) or non-malignant (5) disease were transplanted hemophiliac patients, after one child with severe aplastic ane- using a matched unrelated (11) or sibling donor (12). T-cell mia transplanted from his HLA-identical unaffected 5-year-old recovery was evaluated by analyzing lymphocyte subpopula- brother, and a 26-year-old HIV-infected man with Burkitt-type tions by fl ow cytometry, determining T-lymphocyte responses ALL transplanted from his homozygous twin brother. In both to key mitogens and measuring antibody responses to estab- the FVIII level did not change after transplant, suggesting that lished antigens. B-cell recovery was studied by fl ow cytometry bone marrow cells does not contribute signifi cantly to FVIII and functionally by ELISPOT. The effects of scheduled passive production. immunization following HSCT were also analysed. Results: The absence of chronic GVHD (cGVHD) associated with a brisk recovery of both cellular and humoral immunity by P807 18 months post HSCT among the pediatric recipients of alloge- Cord blood transplantation at King Hussein Cancer neic grafts. However, the presence of cGHVD associated with Center: an evolving experience in a newly developed a slowing down in the process and only 40% of these recipients programme reached the values of the immunosuppressed, non-transplant Ayad A Hussein, A. Al-Zabin, S. Musleh, R. Rihani, controls during the follow-up in the multitude of parameters F. Abdul-Rahman, H. Al-Ta’ani, M. Sarhan analysed. King Hussein Cancer Center (Amman, JO) Prior to revaccination protective antibody levels were found for tetanus in 66% of recipients, for Hib in 44%, for measles in Purpose: To describe the outcome of patients who received 36%, for all 3 poliovirus serotypes in 75% and for all 7 pneu- related and unrelated umbilical cord blood transplantation mococcal serotypes in 0%. Post revaccination protective anti- (UCBT) at King Hussein Cancer Center (KHCC). body levels were found for tetanus in 96% of recipients, for Patients and Methods: Between October, 2006 and September, Hib in 100%, for measles in 88%, for all 3 poliovirus serotypes 2008; ten pediatric patients less than 18 year of age received in 83% and for all 7 pneumococcus serotypes in 48%. None UCBT at KHCC in Jordan. Charts review was performed to of the parameters analysed to illustrate the immunological analyze the outcome of those patients. recovery process was predictive of immunization responses Results: The median age of the patients was 7 years (1-16), post HSCT. 7 were males & 3 were females. Primary diagnosis was Acute Conclusions: Among pediatric recipients of allogeneic stem Lymphoblastic Leukemia (ALL) (n=5), Chediak Higashi Syn- cell grafts cGVHD, particularly in its extensive form, and its drome (n=3), Fanconi Anemia (FA) (n=1) and chronic mye- therapy appear to have a profound tampering effect on a multi- loid leukemia (CML) (n=1). Eight patients received unrelated tude of aspects in the immunological recovery post HSCT. Yet, UCBT while two patients received related UCBT. The median as a response to passive immunization most of these children Total nucleated cell dose was 6.2 X107/kg(3-16). The median appeared to attain protective antibody levels without complica- CD34 count was 1.75 X105/kg(0.89-8). All patients received full tions attributable to the revaccination program. myeloablative conditioning according to primary disease. Five

S228 patients received Cyclosporine A (CSA) and Mycophenolate as P809 Graft versus Host Disease (GvHD) prophylaxis, while 4 patients Outcome of high-dose chemotherapy and autologous received CSA, methotrexate (MTX) and steroid, and one patient haematopoietic stem cell transplantation in children with received CSA and MTX. After a median follow up of 9 months high-risk primary central nervous system tumour (3-26); 7 patients survived. Two patients died before 100 days S.Y. Kwon, S.C. Won, H.S. Kim, C.J. Lyu post unrelated UCBT due to sever sepsis while one patient died Yonsei University College of Medicine (Seoul, KR) 6 months post related UCBT due to relapse of Philadelphia chromosome ALL. The overall survival was 70%.The median Background: Primary central nervous system (CNS) tumors stay in the hospital was 53 days (26-120). Five patients (50%) are known to be the most common solid tumor in children. were admitted to the Intensive Care Unit, 2 of them died with Even though the long term survival rates are increasing, still sever sepsis, while 3 patients survived. Three patients (30%) considerable portion of patients experience relapse and dis- had primary graft failure, one with FA and he is still alive after ease progression. High dose chemotherapy (HDCT) followed haploidentical allogenic hematopoietic stem cell transplanta- by autologous hematopoietic stem cell transplantation (HSCT) tion at day 75 post unrelated UCBT, the second patient with has been applied to high risk solid tumor patients including high CML had auto recovery and still alive, while the third patient risk CNS tumors with the background of its chemosensitivity had ALL and died with sever sepsis. Seven patients (70%) had and dose dependent relationship. neutrophil and platelets engraftment. The median time for neu- Purpose: To evaluate the response and survival effi cacy of trophil engraftment was 20 days (15-30), and 40 days (21-84) HDCT followed by autologous HSCT for children with high risk for platelets engraftment. Five patients (50%) had acute GVHD primary CNS tumor or young patients with limited use of radio- of skin that necessitate systemic steroid treatment, 2 of them therapy. had additional liver GvHD, and third patient had gut and liver Methods: Twelve patients with newly diagnosed high risk or GVHD. No patient had chronic GVHD. recurred CNS tumors underwent total 22 HSCTs. Six patients Conclusions: UCBT can be performed in developing countries received single HDCT and another six patients received twice with considerable success. The evolving experience will lead or more cycles of HDCTs each followed by autologous HSCTs. to better outcome in the coming years. There is an increas- Results: Patients were consisted of nine medulloblastoma ing need for establishing public cord blood banks in our area; patients, two pineoblastoma and one germ cell tumor patients. to save more lives through having more readily available cord Six patients were under age of four years. Ten patients were blood units for transplantation. in complete response (CR) status and two patients in partial response at fi rst HDCT. During the 17 months of median follow up period (range 2~47 months), six patients are alive in CR, six P808 patients failed to survive owing to relapse and disease progres- Improved outcomes of paediatric allogeneic sion after HDCTs. There were two treatment-related mortalities haemopoietic stem cell transplantation following (TRMs) during 22 HDCTs. Myelosupression, stomatitis and implementation of standardised transplant procedures infections were most frequent major acute toxicities, and most in preparation for JACIE accreditation common long term complications of the survivors were growth Y. Sorour, J. Williams, S. Rush, S. Barrott, A. Vora hormone defi ciency (4/6), thyroid hormone defi ciency (3/6) and Sheffi eld Children’s Hospital (Sheffi eld, UK) alopecia (3/6). Both two patients with TRM received more than seven cycles of chemotherapy before HDCT since they were Introduction: The Joint Accreditation Committee-ISCT & EBMT relapsed cases. Three survivors who were under age of four at (JACIE) requires establishment of standardised policies and diagnosis received radiotherapy after completion of HDCTs. procedures within a quality management system. We are a Conclusion: HDCT followed by autologous HSCT was tolerable small paediatric centre performing, on average, 12 allografts/ in childhood high risk and relapsed CNS tumor patients. It was annum and were JACIE accredited in 2006. In preparation for also tolerable and effective in young patients who had limita- the inspection in November 2005, we reviewed and revised tions of receiving radiation therapy. Double tandem or more many of our existing policies in regard to donor selection, con- HDCTs seems also tolerable, however, further study and long ditioning, graft-versus-host disease (GVHD) prophylaxis and term follow up should be performed to establish the effi cacy. therapy, as well as, monitoring and treatment of post-trans- plant viral infections. We retrospectively compared outcomes of allogeneic HSCTs undertaken at our centre between Janu- P810 ary 2000-August 2006 and following JACIE accreditation in Severe haemorrhagic cystitis in children undergoing November 2006. haematopoietic stem cell transplantation Methods: In the period leading up to JACIE accreditation A.A. Hamidieh, M. Jalili, A. Ghavamzadeh, M. Jahani, we introduced standardised donor selection based on high K. Alimoghaddam, A. Khodabandeh, B. Bahar, A. Mousavi, resolution 10 antigen DNA tissue typing, changes to indica- M. Iravani tion specifi c conditioning and GVHD prophylaxis protocols Hematology-Oncology and SCT Research Center (Tehran, IR) incorporating the use of Treosulphan and restricting pre-trans- plant serotherapy to mismatched unrelated donor transplants, Introduction: Hemorrhagic cystitis (HC) is a major complica- GVHD treatment involving early use of anti-cytokine antibod- tion in Pediatric patients undergoing hematopoietic stem cell ies for steroid refractory disease and routine PCR surveillance transplantation (HSCT). HC has a spectrum of manifestations with pre-emptive treatment of cytomegalovirus, Epstein-Barr that range from microscopic hematuria to severe hemorrhage. virus, herpes simplex virus, varicella zoster virus and adeno- When severe, HC causes signifi cant morbidity, leads to renal virus infections. complications, prolongs hospitalization, and occasionally con- Results: Between January 2000-August 2006 (period A), 70 tributes to death. allogeneic HSCTs were performed (22 match sibling, 48 unre- Materials and Methods: We retrospectively studied the medi- lated) while between November 2006-October 2008 (period B) cal records of 598 children (<15 years), undergoing HSCT at there have been 33 (8 match sibling, 25 unrelated). Event free Hematology-Oncology and SCT Research Center (Tehran, survival and non-relapse mortality were 49% and 24% in period Iran) between 1991 and 2008 to describe the incidence, clinical A compared with 83% and 3% in period B, respectively. course, and outcome of HC in all patients and to identify the Conclusion: Introduction of standardised protocols incorporat- risk factors for HC. Conditioning regimens varied according to ing a state of the art with regard to donor selection, condition- disease and clinical status. ing, GVHD prophylaxis and management, and PCR based Results: 26 recipients (4.3%), 21 boys and 5 girls devel- pre-emptive therapy of viral infections has led to an improve- oped severe HC. The indications for HSCT (24 allogeneic, ment in transplant outcomes. 2 autologous) were Thalassemia major in 12 patients, Acute

S229 Myelogenous Leukemia in 5, Fanconi anemia in 3, Non-Hodg- P812 kin’s Lymphoma in 3, Acute Lymphoblastic Leukemia in 2, and Malnutrition and use of nutritional support in infants Chronic Myelogenous Leukemia in 1. 24 recipients received submitted to haematopoietic stem cell transplantation Cyclophosphamide for conditioning regimen. Busulfan was part R. Jung, A.M. Ordoñez, D.J. Campos, A. Koerich, S. Fortier, of the pretransplant conditioning regimen in 9 patients. Alloge- J. Zanis Neto, C. Bonfi m neic recipients received cyclosporin for prophylaxis of graft-ver- Universidade Federal do Parana (Curitiba, BR) sus-host disease (GVHD). 21 recipients developed acute GVHD and 4 chronic GVHD. Introduction: The hematopoietic stem cell transplantation Although 15 patients were suffering from serious micturition (HSCT) promotes deleterious consequences on nutritional pain symptoms were well controlled by hydration, three patients status of young patients (pts). In the fi rst two years of life the required bladder irrigation. One went on to develop acute renal maintenance of adequate nutritional status is crucial to ensure failure due to urinary obstruction and required cystotomy. From full growth and development. these three patients, two deaths were directly linked to the HC. Objectives: 1-Evaluate changes in nutritional status of infants Discussion: In our series, severe HC occurred in 26 of 598 undergoing HSCT in Curitiba-Brazil. 2- Analyze the nutritional patients (4.3%), an incidence lower than that of adult reports. therapy used during HSCT and the implications of this interven- This study shows that young age correlated with a lower inci- tion in the outcome of pts. dence of severe HC. A prospective study is necessary to clarify Methods: Data was collected retrospectively from medical the association between clinical factors in the development of records. All pts less than 2 years-old, transplanted between severe HC following HSCT in children. December 1990 and November 2007 were included in this analysis. We used the World Health Organization classifi ca- tion (WHO 2006) to evaluate the nutritional status of this group P811 of pts. Survival was analyzed using the Kaplan-Meyer method. Estimation of glomerular fi ltration rates in paediatric Results: 53pts were included in this study, 72% (38 pts) were patients undergoing stem cell transplantation: the role of male. The mean age was 13.5±6.2 months, 19% (10pts) had cistatine plasma levels malignant diseases and 81% (43pts) had non malignant dis- R. Mar, J. Fernández-Navarro, M. Sanahuja, J. Lucas, eases. At hospitalization 32% (17pts) showed mild to moder- M. Matoses, M. Andres, A. Verdeguer ate malnutrition and 15% (8pts) severe malnutrition based on Hospital La FE (Valencia, ES) z-score weight/age. There was no relationship between the baseline nutritional status and survival. At hospital discharge, Background: Creatinine clearance can overestimate renal func- 40% (21pts) showed mild to moderate malnutrition and 17% tion in malnourished patients. Cistatine C is a protein produced (9pts) severe malnutrition. During hospitalization 6 (11%) in all nucleated cells. Its elimination depends exclusively upon patients lost 5-10% of body weight and 3 (6%) lost more than glomerular fi ltration, which makes it a good target for glomeru- 10%. There was no relationship between pathology, length lar function evaluation. of hospital stay and weight loss. Fourteen (26%) patients Patients and methods: Thirteen consecutive patients undergo- received enteral nutritional (EN) via tube for an average of ing SCT were included in this prospective observational study 12.7±11.5 days, to supply an average of 30.4 kcal/kg/day aimed to compare the glomerular fi ltration rates (GFR) obtained and 0.9g protein/kg/day. Twenty-two (42%) patients received through both, creatinine clearance and cistatine plasma levels. parenteral nutrition (PN) for an average 15.7±6.1 days to sup- Main diagnosis, characteristics of patients and type of trans- ply 49.8 kcal/kg/day and 1.5g protein/kg/day. There was no plant are reported in table I. Blood samples were obtained in statistical difference in the incidence of vomiting, diarrhea and the same day for creatinine and cistatine, 24h after urine col- hyperglycemia among patients who received EN, PN or exclu- lection, in three separate time-points, so a total of 39 samples sively oral feeding. were recruited on each arm. GFR was calculated in the regu- Conclusion: We observed a high prevalence of malnutrition lar way for creatinine clearance and then corrected for body prior to HSCT. In this cohort of pts the use of enteral nutrition surface. The following formula (74,835/CistCexp 1,333) was did not lead to increased gastrointestinal complications and applied to extrapolate GFR from cistatine plasma levels, and could be used earlier in the transplant course in order to pre- no further corrections were applied. GFR results obtained with vent weight loss during HSCT. both methods were compared with T student and Pearson cor- relation tests (SPSS). Results: We have not found a signifi cant difference between P813 GFR obtained through both techniques (T student, table II) and Early multiple viral infections may predict chronic GvHD moreover, there is a positive correlation between them (Pear- among paediatric stem son correlation 0.003, table III). H. Olkinuora, M. Taskinen, U. Pihkala-Saarinen, Comment: In our transplanted patients, cistatina plasmatic K. Vettenranta levels are a good alternative to evaluate GFR although no Hospital for Children and Adolescents (Helsinki, FI) signifi cant differences were found with creatinine clearance. Background: Delayed immune reconstitution and opportunistic infections following pediatric hematopoietic stem cell transplan- tation (HSCT) associate with an increased treatment-related morbidity and mortality. We retrospectively evaluated in a single institution setting the impact of viral infections on the post-trans- plant recovery of pediatric recipients of stem cell grafts as an indicator of their post-transplant immunoreconstitution. Material and methods: Between 1/1999 and 9/2006 a total of 180 (56 autologous and 124 allogeneic) patients with a mean age of 7.9 yrs with either a malignant (160) or non-malignant (20) disease were transplanted using a matched unrelated (79) or sibling donor (45) or given autologous stem cell rescue (56). TBI was included in the conditioning in 12/56 autologous and 116/124 allogeneic cases. No method of T cell depletion was employed. Cyclosporine and MTX were given as GVHD proph- ylaxis. No viral prophylaxis was employed. The medical histo- ries were retrospectively reviewed for single or multiple viral

S230 infectious episodes during the fi rst two years post-transplant dren who received tacrolimus and minidose MTX as prophy- and correlated with the respective clinical parameters. laxis were reviewed, with median age of 6.8 years (range 1.4 Results: 42% of the allogeneic stem cell recipients developed – 17 years). Nine patients underwent related donar transplants chronic GVHD: 21/52 limited and the rest extensive. 33/56 and 8 underwent unrelated donar transplants. All patients (59%) of the autologous and 109/124 (76%) of the allogeneic received tacrolimus beginning the day prior to transplant at a patients had at least one viral reactivation/clinical infection dose of 0.03 mg/kg/day by continuous iv infusion. Tacrolimus post-transplant. The viruses were categorized into two groups: levels were monitored every other day and dosages adjusted to latent (117/180, 65%) and nosocomial viruses (62/180, 35%). maintain serum levels 5-15 ng/ml. MTX 5 mg/m² i.v. was given In a multivariate analysis of the allogeneic group chronic GVHD on days 1, 3, 6 and 11. The dose was reduced or omitted for (P=0,001; Risk Rate =4,7) and secondary graft failure (P=0,02; severe mucositis. The median follow-up for patients was 22 Risk Rate=4,9) were signifi cantly associated with early multiple months. All patients except one who had a unrelated cord blood viral infections. transplant were successfully engrafted. The median time to Conclusions: Our data indicate that viruses are common clinical ANC recovery ( ANC ≥ 500 x 106/l) was 15 days. The incidence pathogens among pediatric recipients of stem cell grafts. How- of acute GVHD was 22.2% with related donar group and 42.8% ever, multiple viral infections early on seem to refl ect a more with unrelated group. The incidence of grade III-IV GVHD was severe degree of immunological derangement in the recipient 0% with related donar group and 14.2% with unrelated group. and appear to identify a group of recipients with an increased There was no case with chronic GVHD on both groups. Two risk of chronic GVHD. patients had documented relapse post-transplant. The actual relapse rate at 1 year was 14%. Day-100 survival was 82%, and the survival at 1 year was 70%. The administered dose P814 of tacrolimus to achieve therapeutic level (5-15 ng/ml) was Allogeneic HSCT in children and adolescents with 0.042 mg/kg/d for younger age group (< 8 yrs) compare to relapsed or refractory Hodgkin’s lymphoma 0.028 mg/kg/d for older age group ( ≥ 8 yrs). Common adverse I. Badell, M. Torrent, M. Gonzalez-Vicent, C. Diaz de Heredia, effects of tacrolimus included hypomagnesemia (88%), neph- A. Martinez, J.M. Perez-Hurtado on behalf of GETMON rotoxicity (23%), hyperglycemia (23%), hypertension (5%). Overall, tacrolimus and minidose MTX was well tolerated and We evaluated the results of allogeneic hematopoietic stem effective as prophylaxis for acute GVHD in pediatric patients cell transplantation (HSCT) in eleven patients ≤ 21 years with undergoing allogeneic transplantation. And children < 8 years relapsed or refractory Hodgkin disease. All of these 11 patients old undergoing HSCT should receive a higher dose of tac- had been treated with ≤ 3 lines of chemotherapy including autol- rolimus in order to avoid the subtherapeutic level. ogous HSCT in 8 of them. Allogeneic HSCT were performed between 2000 and 2008, in six GETMON centers. The median age at transplant was 16.9 years (13-21). They were 6 males and 5 females. Ten patients received reduced intensity condi- tioning and only one myeloablative. Five patients were trans- Haemoglobinopathy planted from HLA-identical sibling, 3 from mismatched family donor and 3 from unrelated donor, who were one matched and 2 mismatched. Graft was demonstrated in 10 patients. Six P816 patients died after allogeneic HSCT without relapse or progres- Optimal thalassaemia-free survival and minimal sion, and one after disease progression. The causes of death regimen-related toxicity in 50 consecutive high-risk beta were acute and chronic graft-versus-host disease associated thalassaemia paediatric patients using myeloablative with infection and multiorgan failure in 3 patients, veno-occlu- therapy with intravenous busulphan sive disease in 2 patients and interstitial pneumonia in one. The R. Chiesa, B. Cappelli, R. Crocchiolo, E. Biral, A. Noè, I. Frugnoli, cumulative incidence of transplant-related mortality (TRM) at T. Roccia, C. Evangelio, M. Fossati, F. Ciceri, M.G. Roncarolo, one year was 0.43 ± 0.15 at 12 months. The actuarial over- S. Marktel all survival and event-free-survival was 0.29 ± 0.15 and 0.22 ± Scientifi c Institute Osp. San Raffaele (Milan, IT) 0.13 at 57 months respectively. These patients with recurrent or refractory disease have a fair chance of cure. We observe very Busulfan(Bu), associated with Cyclophosphamide(Cy), is high TRM in this study, instead of reduced intensive condition- part of standard conditioning regimen prior to stem cell ing. Three patients are long survivors without disease at 8, 13 transplantation(SCT) in beta thalassemia(bthal). Intravenous and 57 months. Two are survivors after related and one after (iv) formulation of Bu should reduce intra-inter individual vari- unrelated transplant. ability in drug systemic exposure, limiting the risk of regimen We need prospective trials to evaluate the role of allogeneic HSCT related toxicity and graft failure. Between June 2005 and May in children and adolescents with recurrent or refractory Hodgkin 2008, 50 consecutive Bthal patients from the Middle East under- disease. went SCT from an HLA identical sibling in our center with an iv Bu/Cy based conditioning regimen. iv Bu dosage was given according to weight and adjusted from the fi th dose to a tar- P815 get AUC 1250 umol/min. Median age was 8 years (2-15), one The effectiveness of tacrolimus and minidose patient classifi ed as Pesaro class I, 24 class II and 25 class III. methotrexate for prevention of acute graft-versus-host Most patients had severe iron overload evidenced by irregular disease after an allogeneic stem cell transplantation in iron chelation (42/50) and high median ferritin 2973 ug/L(956- children: a single-centre experience in Korea 14280). Median ALT and AST level was 71 U/L(12-545) and 59 S.S. Park, S.E. Jun, Y.T. Lim U/L(18-371), liver fi brosis median Ishak stage 3 (0-5) and 16/50 Pusan Natational University Hospital (Pusan, KR) patients were HCV pos (32%). Class I-II patients were condi- tioned with iv Bu and Cy200mg/kg(n19) with thiotepa(TT 10mg/ Currently, limited data exist on the role of tacrolimus and mini- kg) if <4 years(n6). Class III patients received fl udarabine(Flu) dose methotrexate (MTX) in pediatric allogeneic hematopoi- 100(n6) or 150mg/m2(n16), iv Bu and Cy 160mg/kg(n22) and etic stem cell transplantation (HSCT). We report the outcomes ATG(n16); patients receiving a second SCT following graft for allogeneic hematopoietic stem cell recipients, evaluating rejection (n3) received Flu 150mg/m2, iv Bu, Cy 200 mg/kg, engraftment status, incidence of acute and chronic graft ver- TT 10 mg/kg and ATG. All patients received anti veno-occlu- sus host disease (GVHD) and toxicities after use of tacrolimus sive disease (VOD) prophylaxis with defi brotide. GVHD proph- and minidose MTX for GVHD prophylaxis in a single pediatric ylaxis consisted of cyclosporine A(CyA) and methotrexate, center of Korea. From March 2003 to February 2008, 17 chil- with methylprednisolone given to patients not receiving ATG.

S231 33 patients(66%) achieved Bu AUC in the expected range of patients in class I, 132 patients in class 2 and 120 patients in 900-1500 umol/min following the fi rst dose. Seventeen patients class 3) and 23 patients were thalassemia intermediate. 366 (34%) required dose adjustment because either above (n9) or patients received stem cell from healthy full human leukocyte below (n8) the expected range. antigen (HLA) matched siblings, 5 patients from other related Post transplant toxicity was mild and characterized by: hemor- (HLA-matched confi rms with high resolution method) and 10 rhagic cystitis(n4), CyA neurotoxicity(n3), VOD(n1), grade II-IV patients from other related with one antigen mismatch. The graft stomatitis(n7), reversible lung infi ltrates of unknown origin(n4). source was bone marrow in 165 patients, peripheral blood stem The incidence of acute GvHD II-IV was 5/47 (4 grade II, 1 grade cells (PBSC) in 207 patients and cord blood in 9 patients. For III). Limited chronic GvHD was observed in 3/47 patients. At a 10 patients second transplantation was performed. Median age median follow-up of 528 days (112-936), 98% pts are alive and at HSCT was 6 years (range, 2-28 years). 272 patients were current thalassemia free survival is 94% of which 100% in class I- conditioned with Busulfan and Cyclophosphamide, 59 patients II pts and 88% in class III. In conclusion a myeloablative condition- with antithymocyte globulin (ATG), cyclophosphamide and ing based on iv Bu resulted in limited regimen related toxicity and Busulfan and 50 patients with ATG, Busulfan and Fludarabine. excellent bthal free survival in patients at high risk; we recommend No radiation therapy was given. For graft-versus-host disease to perform Bu pharmacokinetic studies and target Bu dose. (GVHD) prophylaxis, we used cyclosporine and Methotrexate. Results: Median follow-up was 2.5 years. 319 (83.7%) patients are alive and 62 (16.3%) patients died. Most common causes of P817 death include GVHD in 25 patients, infection in 11 patients and Purifi ed T-depleted peripheral blood and bone marrow graft failure in 7 patients. 5-year overall survival and disease CD34 transplantation from haplo identical mother to child free survival was 80% (se=2.4%) and 70% (se=2.8%) respec- with thalassaemia tively. Median time to ANC engraftment (ANC>0.5 x 109/I) was P. Sodani 15 days (range 6-73 days) and for platelets engraftment (PLT Mediterranean Institute of Hematology (Rome, IT) >20 x 109/l) was 22 days (range 6-92 days). Acute GvHD was seen in 67% of patients. Purpose: Feto-matenal microchimerism suggests that immuno- Conclusions: Allogeneic HSCT remains the only cure for severe logical tolerance exists between mother and foetus. Based on beta thalassaemia. Now we are the second after Pesaro team, this hypothesis we analyzed the outcome of primary hematopoi- regarding large number of transplanted thalassemia patients. etic stem-cell (HSC) transplantation from mismatched mother In order to make this treatment available to more patients, we to thalassemic patients without an HLA identical donor. should enlarge the donor pool. Patients and Methods: A total of 20 patients with Thalassemia As there are marriages among relatives in some countries, we Major (14 in risk class 3; 5 in risk class 2 ; 2 in risk class 1) were may fi nd HLA-identical relatives. It should consider that patients conditioned with hydroxyurea (OHU) 60 mg/kg and azathioprine with thalassemia who lack sibling donor but have HLA-identical 3 mg/kg from from day -59 to day-11, fl udarabine (FLU) 30 mg/ relatives should be transplanted before they are in class 3. m² from day -17 to day -11, busulfan (BU) 14 mg/kg starting on day -10, and cyclophosphamide (CY) 200 mg/kg, Thiotepa 10 mg/kg and ATG 12,5 mg/kg daily from days -5 to -2 (Fre- P819 senius S). Twelfe patients received positively-selected CD34+ Donor’s NK cells may infl uence the engraftment in mobilized peripheral and bone marrow progenitor cells using paediatrics patients after T-cell depleted haploidentical the CliniMacs procedure. In a second court of eight patients in stem cell transplant for thalassaemia order to retain NK cells in the graft, negative selection of B and A. Isgro (1), M. Marziali (1), P. Sodani (1), J. Gaziev (1), T-lymphocytes (CD3/CD19 depletion) of haploidentical marrrow P. Polchi (1), G. De Angelis (1), K. Paciaroni (1), C. Alfi eri (1), was employed rather than CD34+ selection. Total T-cell dose M.D. Simone (1), D. Fraboni (2), G. Lucarelli (1) was adjusted to 2 x 105/kg by fresh bone marrow donor cell add (1)Mediterranean Institute of Hematology (Rome, IT); back at the time of transplant. Both groups received cyclosporin (2)Policlinic “Tor Vergata” (Rome, IT) after transplant for graft versus host disease (GVHD) prophy- laxis during the fi rst two months after transplantation. In this study we analysed the potential role of NK cells in hap- Results: Two patient died during post-transplant day, one on loidentical transplant for beta-thalassemia patients. A total of 11 day +114 of cerebral EBV lymphoma, the second on day +92 of pediatric patients with beta-thalassemia received T and B cell CMV pneumonia. Six patients reject their grafts, and 12 showed depleted transplants from their haploidentical mothers. T and B full chimerism with functioning grafts at a median follow-up of 38 cell depletion was carried out with CD34+ CliniMACS selection months. None of the ten patients showed AGVHD or CGVHD. (Miltenyi Biotec©) from peripheral blood and bone marrow of Conclusion: These preliminary report suggest that maternal donors (the mothers) and resulted in grafts consisting of stem haploidentical HSC is feasible therapeutic option for thalassemic cells and effector cells (NK cells, monocytes) with the addition patients lacking a matched related donor. With the increasing of bone marrow mononuclear cells (BMMNCs 3 x 105/kg of the availability combined cell therapies with the potential to improve recipient). To analyse the mechanisms involved in immunologi- engraftment and immune recovery such as NK cells, regulatory cal reconstitution post transplant, we analysed T cell subsets by T cells, mesenchymal stem cells, haploidentical transplantation fl ow cytometry, particularly NK cells at day + 20 and + 60 post will play an increasing role in allogenic transplantation. transplant. Day + 20 post transplant, the patients had signifi - cantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), whereas CD8+ T cells num- P818 bers did not statistically differ between patients and controls Haematopoietic stem cell transplantation from related (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the fi rst lym- donors in severe beta thalassaemia phocytes to repopulate the peripheral blood, and up to 70% of A. Ghavamzadeh, A.A. Hamidieh, M. Jahani, K. Alimoghaddam, these cells were CD3-CD56+bright cells. Interestingly, a direct A. Mousavi, M. Iravani, B. Bahar, A. Khodabandeh, M. Jalili correlation has been observed between the percentages of Hematology-Oncology and SCT Research Center (Tehran, IR) CD56+CD16+ NK subset and the BM engraftment (in mean 71 ± 21% CD56+CD16+ in the four patients with full engraftment, Objective: Follow-up of patients with Severe Beta Thalassemia 27 ± 28% in the three patients with a stable mixed chimerism transplanted in Hematology-Oncology and Stem Cell Trans- after BM transplant (70-80% of donor cells) and 1.4 ± 1% in the plantation Research Center, Tehran, Iran. four patients with rejection). In all the patients the origin of the Methods: 381 patients (165 female, 216 male) received alloge- NK subsets was from the mothers. Day + 60 post transplant neic Hematopoietic Stem Cell Transplantation (HSCT) between an increase in the percentages of CD4+ T cells, naïve CD4+ 1991 and 2008. 358 patients were thalassemia major (106 cells and in thymic naïve Th cells were observed (3 ± 1.2%,

S232 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also not essential for sustained engraftment and the simultaneous increased (in mean 35 ± 27.5%), in parallel with the increase presence of haematopoietic cells of both donor and recipient of the CD3-CD16+ NK cells (potent cytotoxic effector cells) origin is not a rare event after a transplant. However there is especially in the patients with full engraftment (in mean 47 ± evidence that MC is associated with an increased risk of graft 20% vs. 28 ± 31% in mixed chimerism).We observed higher failure and/or disease recurrence and the presence of more percentages of NK subsets just twenty days post transplant than 25% residual host cells within the fi rst 2 months is highly in the patients with full engraftment respect the mixed chimer- indicative for graft rejection. Patients with MC may benefi t from ism and the rejection, suggesting a role of donor NK cells on donor lymphocyte infusion (DLI) by eradication of residual host engraftment and on prevention of the rejection with the attack of cells. In this study, we retrospectively review transplanted tha- the host lympho-hematopoietic cells especially in haploidentical lassemia patients with MC in whom DLI was used to reverse recipients. relapse. In our center, DLI was used in 17 transplanted tha- lassemia patients. Totally 52 DLI was used and the median infusion number per patient was 3 range 1-8). Median dura- P820 tion between the transplantation and fi rst DLI was 5 months Co-transplantation of HLA-identical related donors (1.5-13.5 m). DLI was done in fi ve patients due to presence of mesenchymal stromal cells and haematopoietic stem more than 25% of residual host cells within 2 months after BMT cells in class III thalassaemia major patients and in rest of the patients due to gradual increase in residual A. Ghavamzadeh, K. Alimoghaddam, A.A. Hamidieh, A. Karimi, host cells and/or decrease in hemoglobine levels. Eleven out N. Valizadeh, M. Bashtar, A.R. Shamshiri of 17 patients have been followed as transfusion independ- Hematology-Oncology and Stem Cell Transplantation Research ent. The median hemoglobine levels in transfusion independ- Center (Tehran, IR) ent patients before and after DLI were 7.25 gr/dL(6.3-9.9) and 9.55 gr/dL(7.3-12.9), respectively. In this group of transfusion Objective: Following a positive phase II study in all classes of independent patients the median percentage of donor-derived thalassemia major, in this study we aimed to show that co-trans- cells before and after DLI were 41% (23-64) and 49% (30- plantation of mesenchymal stem cells (MSC) and hematopoi- 100) respectively. Two out of fi ve patients who had DLI within etic stem cells (HSC) from HLA-identical related donors after two months after BMT due to presence of more than 25% conditioning regimen is safe and could facilitate engraftment of residual host cells became transfusion independent. The and decrease graft versus host disease (GVHD). median CD3 cell count per DLI was 1.5x107/kg (range 1.5x107/ Methods: Between November 2006 and December 2008, 28 kg-1x108/kg). All fi ve patients in whom cell counts higher than class III thalassemia major patients were enrolled. In this double- 1.5x107/kg were used became transfusion independent. In blind randomized clinical trial, HLA-identical related donors HSC three patients two of which were receiving higher cell counts, were transplanted (non-MSCs group, n=16 with median age grade II-IV GVHD developed at 2.5, 3, 4 months after DLI. of 14.5yrs and F/M=5/7) or co-transplanted with MSCs (MSCs Our results showed that patients with MC characterized by the group, n=12 with median age of 14.0yrs and F/M=3/13) in tha- presence of high numbers of residual host cells even within the lassemia major patients. Patients received Cyclophosphamide- fi rst two months after transplantation benefi t from DLI. Although based or Fludarabine-based conditioning regimens and short the proposed cell count for DLI is 1.5x107 CD3 cells/kg, the course methotrexate and cyclosporine as GVHD prophylaxis. On higher doses may add benefi t in changing the balance between day 0, MSCs group patients were given MSCs intravenously 4 thalassemic and donor cells but should be evaluated in larger hours before infusion of either bone marrow or peripheral blood group of patients. stem cells. The number of MSCs infused was 1.45-1.75x106/ kg. Results: MSCs infusions were well tolerated. The median time to neutrophil engraftment (absolute neutrophil count >0.5x109/ P822 L) was 18 days for MSCs group and 20 days for non-MSCs Cyclosporine neurotoxicity in beta-thalassaemic children group (p-value=0.31). The median time to platelet engraft- undergoing myeloablative BMT ment (platelet count >20x109/ L) was 23 and 25 days, respec- A. Noè, S. Marktel, R. Chiesa, C. Evangelio, I. Frugnoli, tively (p-value=0.57). Acute GVHD was observed respectively, M. Fossati, E. Biral, S. Napolitano, F. Ferrua, A. Biffi , T. Roccia, in 10(62.5%) and 5(41.7%). Chronic GVHD was found in M.G. Roncarolo, F. Ciceri, B. Cappelli 3/7(42.9%) and 5/11(45.5%) evaluable patients (p-value=1.0). Scientifi c Institute Osp. San Raffaele (Milan, IT) Median follow-up duration for alive patients was 5 months (ranged 1-19). The number of patients who not engrafted were Severe neurotoxicity (tremors, insomnia, agitation, headache, 1(6.3%) and 2(16.7%) (p-value=0.56), and mortality rates were hallucinations, seizures, cortical blindness, aphasia and ataxia) 3(18.8%) and 4(33.3%), respectively (p-value=0.42). is a recognized complication of Cyclosporine A (CSA). We Conclusion: In this study we demonstrated that co-transplan- describe 6 cases of CSA related neurotoxicity out of 67 HLA tation of HLA-identical related donors MSCs with HSCs is identical sibling BMT performed between June 2005 and Nov seems to be safe. Although the above mentioned differences 2008, in middle east patients (pt) affected by haemoglobinopa- are weighing a partial superiority of MSC group, but we didn‘t ties (pts characteristics in tables). fi nd statistical signifi cant difference in acute GVHD incidence, There was no correlation between serum CSA levels and sei- rate of non-engraftment, mortality rate and median time to neu- zures occurrence. The clinical and radiological fi ndings dis- trophil and platelet recovery between two groups. Most prob- appeared discontinuing CSA and switching to Tacrolimus. All ably explanations are small number of patients in study groups patients are symptoms-free at a median of 460 days of follow and short follow-up period. up (range 72-866). Conclusions: Our experience suggests that pts with thalassemia have a high incidence of CSA related neurological events but P821 that there is no correlation between serum CSA levels and sei- Donor lymphocyte infusions after allogeneic bone marrow zure occurrence. Prognosis is good following CSA removal. transplantation for thalassaemia: a single-centre Specifi c prodromes such as headache, hypertension or visual experience disturbances occurring in the early posttransplant period should G. Karasu, V. Uygun, M. Kazik, A. Kupesiz, P. Kurt, Z. Öztürk, be carefully evaluated with electrophysiological and imaging V. Hazar, A. Yesilipek strategies in order to start promptly effective treatment and Akdeniz University School of Medicine (Antalya, TR) reduce possible irreversible sequaelae.

Allogeneic transplantation (BMT) is the only curative option for thalassemia major (TM). Complete donor haematopoiesis is

S233 Conclusions: these data confi rm that SCT from HLA-matched related donors offers high probability of cure with acceptable transplant-related complications. In our center a program for SCT allogenic gene therapy in SCA has been initiated in coop- eration with the National Hospital Abuija, Nigeria.

P824 Immunological evaluation after haematopoietic stem cell transplantation for beta-thalassaemia M. Marziali (1), A. Isgro (1), J. Gaziev (1), D. Fraboni (2), P. Sodani (1), K. Paciaroni (1), C. Gallucci (1), A. Montuoro (1), P. Polchi (1), A. Roveda (1), G. Lucarelli (1) (1)Mediterranean Institute of Hematology (Rome, IT); (2)Policlinico Tor Vergata (Rome, IT)

Background: Haematopoietic stem cell transplantation (HSCT) remains the only curative option for patients with thalassemia. After HSCT is particularly important to recover a complete immune reconstitution for prevention of opportunistic infec- tions. Methods: The immunological phenotype of peripheral blood mononuclear cells in 110 consecutively subjects after HLA identical HSCT performed for beta- thalassemia were studied. Legend: SC: sickle cell anemia; BT: beta thalassemia major; CD3+CD4+, CD3+CD8+, CD3-CD19+, CD3-CD16+CD56+ During Bu therapy, Clonazepam was administered 0.3 mg/kg/d; were evaluated by Flow Cytometry at 6 and 12 months after MTX: Methotrexate; #MPD: metilprednisolone 0.5 mg/kg/die; HSCT in 70 patients followed. In a part of patients CD4 naïve T GenSeiz: generalized seizures; ° CSA levels were evaluated cells was also evaluated at 12 months after HSCT. routinely twice weekly, target 150-250 ng/ml; MRI: Magnetic Results: at 6 months after HSCT we observed reduction of resonance imaging; EEG: electroencephalography; PRES: the mean of CD4 T cell percentage in patients respect normal posterior reversible encephalopathy syndrome; Occip epilep: value: in mean 17, 5% ± 9 vs 45% ± 10 respectively. After 1 occipital epileptiform abnormalities; L: Lorazepam; Ph: Pheni- year the rate of CD4+ T-cell population progressively increase: toine; PB: Phenobarbital; CL: Clonazepam 24 ± 9 at 12 months vs 17, 5% ± 9 at 6 months, but the mean of CD4+ T-cell count was persistently lower than in controls. The mean of CD8 T cell is stable during the follow up and the CD4/C8 ratio decreased. The mean of the CD19 increase dur- P823 ing the follow up ( 4,5% at 6 months vs 17% at 12 months). At Stem cell transplantation in sickle cell anaemia 1 year after HSCT the percentage of NK (CD3-CD16+CD56+) G. De Angelis (1), C. Gallucci (1), K. Paciaroni (1), C. Alfi eri was normalized. At 1 year after HSCT reduction of naïve CD4 (1), A. Isgro (1), M. Marziali (1), A. Roveda (1), M. Simone (1), T cell we observed. A. Montuoro (1), P. Sodani (1), P. Polchi (1), T. Wakama (2), Discussion: At 1 year after HSCT the immunological reconstitu- J. Gaziev (1), G. Lucarelli (1) tion is not still complete. The time and the extent of immune (1)MIH Foundation (Rome, IT); (2)National Hospital Abuja reconstitution depend on several factors. It is very important (Abuja, NG) to continue the immune surveillance after HSTC and to evalu- ate other immunological parameters (i.e. thymic output) for to Objectives: Stem Cell Transplantation (SCT) is the only defi ni- prevent infectious complications and elaborate an appropriate tive cure for Sickle Cell Anemia (SCA). We report the outcomes therapeutic strategy. of SCT from HLA-matched related donors in 7 SCA patients (pts), performed in our Institute (Mediterranean Institute of Hematology) in Rome. Methods: between October 2004 and December 2006, 7 pts affected by SCA (median age of 13 years; range 2-16 years) Infectious complications underwent SCT. The indications for SCT were: recurrent severe pain crisis and/or chronic blood transfusions (4 pts), acute chest syndrome and pain crisis (2 pts), avascular necrosis of P825 multiple joints and pain crisis (1 pt). Two patients were HCV Impairment of anti-leukaemia and anti-tumour CD8+ positive. Patients on chronic blood transfusions have received cytotoxic T-lymphocyte function by antifungal agents in cytoreductive/immunosuppressive treatment with hydroxyurea, vitro azathioprine and fl udarabine before conditioning regimen. The E. Distler (1), H.H. Chapin (1), E. Koch (1), C. Lass-Flörl (2), conditioning regimen consisted of Bu 14, Cy 200 and ATG for A. Ullmann (1), C. Huber (1), W. Herr (1) 6 pts and Bu14 and Cy 160 for 1 pt. As GVHD prophylaxis, all (1)Johannes Gutenberg-University (Mainz, DE); (2)University patients received “short MTX”, cyclosporine (CsA) and methyl- of Innsbruck (Innsbruck, AT) prednisolone (MP). Results: all patients obtained full engraftment. Acute grade Antimycotic agents are increasingly used to prevent and treat 2-3 GVHD was observed in 4 pts. Three pts had moderate fungal diseases in immunocompromised patients, especially chronic GVHD. CMV reactivation was observed in 5 pts. One during chemotherapy-induced neutropenia or after stem cell pt died 1 year after SCT in her home country, likely due to transplantation. Little is known about possible infl uences of septic shock, while she still was on immunosuppressive treat- antifungal drugs on human T cell function. We investigated ment for chronic GVHD. The other pts are alive, 4 pts in good the effects of the antimycotics fl uconazole, amphotericin B, clinical conditions and 2 still on tapering immunosuppressive caspofungin, voriconazole, and posaconazole on the prolif- treatment for chronic GVHD. None of the patients have expe- eration and functional activity of anti-leukemia and anti-tumor rienced painful events or other clinical complications related to CD8+ cytotoxic T lymphocyte (CTL) clones in vitro. Five dif- SCA after SCT. ferent CTL clones (anti-acute myeloid leukemia CTL 1C6;

S234 antimelanoma CTL IVSB and 14/35, anti-renal cell carcinoma outcome in allogeic SCT. Further studies are needed to confi rm CTL BC-I and BC-V) were weekly stimulated with leukemia these fi ndings. blasts or tumor cells in the presence of drugs titrated between 0.1-100µg/mL. We found that the in vitro proliferation of CTL clones was inhibited in a dose-dependent manner at amphoter- P827 icin B and posaconazole concentrations of 0.25µg/mL and 3µg/ Prospective evaluation of Candida mannan and mannan mL, respectively, or higher. Doses of 10-25µg/mL amphotericin antibody to diagnose candidiasis in febrile neutropenic B and 10-30µg/mL posaconazole were even able to completely patients prevent CTL growth. Both drugs also changed the cell mor- M. Ellis, B. al-Ramadi, R. Bernsen, J. Kristensen, H. Alizadeh, phology into a less blast shape type. In contrast, caspofungin, U. Hedstrom fl uconazole, and voriconazole did not induce growth inhibition Faculty of Medicine and Health Sciences (Al Ain, AE) in the analyzed dose range. To determine the effect of antifun- gal drugs on CTL function, 51Cr release and IFNg ELISPOT Objective: Antifungal (AF) therapy given to all pts with febrile assays were performed in the presence of drugs following pre- neutropenia (FN) is costly and toxic. Beta-D-glucan assay is fun- cultivation of CTL in drug-containing medium for 11 days. We gal-non-specifi c; galactomannan is useful only for aspergillosis. observed that high concentrations of amphotericin B (≥25µg/ This study focused on serial assay of mannan (M) and mannan- mL) and posaconazole (≥10µg/mL) strongly impaired target cell antibodies (MA) to diagnose invasive candidiasis (IC) in pts with lysis as well as IFNg spot production by CTL. In assays using FN, hence to aid selection of pts for AF. Previous experience previously untreated CTL clones as effectors, only posacona- with this assay has been limited to non-neutropenic pts, based zole at concentrations >10µg/mL reduced CTL function. This on retrospective infrequent sampling. effect was most pronounced in the ELISPOT compared to the Methods: 100 patients with acute leukemia undergoing chem- 51Cr release assay, presumably due to the longer incubation otherapy complicated by FN, given liposomal amphotericin period (20h vs. 4-5h). Again, impairment of functionality was not B, were studied prospectively with clinical, microbiological seen if CTL clones were cultured in medium containing caspo- (blood culture), and radiological (CT scans chest, liver, spleen, fungin, fl uconazole, or voriconazole, respectively. We conclude sinuses) evaluations for the development of IC, based on from these data that posaconazole and amphotericin B at high revised EORTC/MSG diagnostic criteria. M + MA were meas- concentrations impair proliferation and functional activity of ured daily using Platelia Candida-specifi c antigen/antibody anti-leukemia and anti-tumor CTL clones in vitro. Our results ELISA kits (Bio-Rad). Diagnostic cut-offs were determined may have important implications for T-cell mediated immuno- using ROC curves. surveillance of tumors and leukemias in vivo, particularly if peak Results: 12 of 86 (14%) eligible pts had IC [C. albicans candi- levels of antifungal agents exceed the concentrations defi ned demia (1), C. tropicalis candidemia (4), hepatosplenic candidia- in this study. sis (7)], 24 had invasive mold, 50 persistent FN. These last 2 groups served as the comparison group. Cut-offs were 0.25ng/ml and 2.5AU/ml for M and MA, lower than P826 manufacturer’s recommendations. All pts with IC developed Genetic polymorphisms in the genes encoding human ≥ 1 +ve diagnostic M or MA during persistent FN (FIG1).Optimal Toll-like receptor 1-10 overall performance occurred when 2 consecutive positive tests C. Enevold, I. Christensen, B. Kornblit, K. Bendtzen, C. for both M and MA were used. Sensitivity, specifi city, PPV and Heilmann, N. Jacobsen, K. Müller NPV [95% CI] were 0.73[0.39-0.94], 0.80 [0.69-0.89], 0.36[0.17- Rigshospitalet (Copenhagen, DK) 0.59], 0.95[0.86-0.99] respectively. A positive correlation (r=0.28, p=0.01) was seen with previously Objective: In humans ten different Toll like receptors (TLR1- determined beta-D-glucan (BDG) concentrations in these pts. 10) mediate the activation of innate effector cells by molecules The fi rst +ve M test occurred at a mean, bs.d. of 8.8, b8.5 (2 to of microbial origin. A number of polymorphisms have been 23) days prior to clinical/mycological diagnosis of IC. described in the genes encoding these receptors, and previous High MA concentrations were delayed until leucopenia studies indicate that SNPs within TLR coding genes may be resolved. associated with the course of infl ammatory disorders. The aim The candidal colonisation index (CCI) was ≥0.5 in 60% of the of the present investigation was to evaluate the signifi cance of comparison group. SNPs within TLR1-10 for adverse outcomes in allogeneic SCT. Conclusions: Using institution-derived cut off values, early Patients and methods: Thirty-four SNPs within the genes serial determination of combined M+MA is useful to diagnose encoding TLR 1-10 were determined by a SSP-PCR system IC in FN pts. Low PPV may refl ect low prevalence of IC, whilst in genomic DNA from donors and recipient pairs of transplants the high NPV confi dently excludes IC. Negative M+MA tests in with either matched sibling donors or matched unrelated donors the presence of +ve BDG test indicates IC rather than mold. (MUD). The indication for SCT included ALL (n=67), CML Specifi city may be low as a result of subclinical candida infec- (n=55), AML (n=75), MDS (n=19) and other leukaemias (n=10). tion in the comparator group (high CCI). This assay could be Outcome in terms of grade 2-4 acute graft versus host disease used as part of a broad fungal diagnostic strategy aimed at tai- (aGvHD) as well time to relapse, overall survival, disease free loring AF (FIG2). survival and treatment related mortality were recorded. Signifi - cance was tested by chi-square test for aGvHD; survival data were analysed using a Cox model. Results: In transplants with sibling donors recipient genotype of TLR2 (rs1898830) was associated with overall survival (p=0.0100; HR=0.34, 95% CI: 0.15-0.79 for A/A vs. G/G; HR=0.25, 95% CI: 0.10-0.66, A/G vs. G/G) and treatment related mortality (p=0.0096; HR=0.22, 95% CI: 0.07-0.68, A/A vs. A/G; HR=0.18, 95% CI: 0.05- 0.66, A/G vs. G/G). Recipient type of TLR5(rs5744168) was asso- ciated with aGvHD (p=0.015; OR=2.63, 95% CI: 1.21-5.73, C/C vs. C/T). In MUD transplants donor type of TRL2 (rs1898830) was related to aGvHD (p=0.0279; OR=1.81, 95% CI:0.96-3.43, A/A vs A/G; OR=0.98, 95% CI:0.40-2.38, G/G vs A/G). Conclusion: The presented data suggest that SNPs within genes of several TLR receptors may be associated with adverse

S235 one patients had a second transplant. The following NTD were identifi ed in R and/or D: 2 cases of tungiasis (R), 1 visceral leishmaniasis (R), 3 pre-transplant leprosy (2 R and 1 D), 6 pre-transplant positive serology for Chagas disease (4 R, 1 D and 1 D+R), 7 tuberculosis (3 R before HSCT, 3 R after and 1 D before and respective R after HSCT), 6 dengue fever (4 R after, 1 R before HSCT, 1 D), 6 pre-transplant malaria (4 D, 2 R). Donor infection and subsequent recipient infection was seen in one case of leprosy and 1 case of tuberculosis. One patient died as a consequence of Chagas myocarditis 8 months after HSCT. Yellow fever (YF) serology was performed in 27 HSCT recipients living in endemic areas. Seven patients were suscep- tible to YF despite of pre-transplant vaccination in two of them. Four HSCT recipients were vaccinated against YF in 2008. Conclusions: Pre-transplant investigation of NTD is neces- sary to better estimate the risk of post transplant reactivation. Pre-emptive strategies should be prospectively investigated in transplant candidates at risk.

P829 Each herpesvirus elicits a unique T-cell response in chronically infected healthy individuals F. Khan, S. Sy, S. Liu, F. Zhou, Y. Liu, A. Ugarte-Torres, J. Storek University of Calgary (Calgary, CA)

Background: Herpes viruses cause morbidity and mortality of hematotoeitic cell transplant (HCT) recipients. Three com- mon herpesviruses: cytomegalovirus (CMV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV) belong to three subfamilies - alpha, beta and gamma respectively. Since the three subfamilies differ in their biological properties, we hypoth- esized that anti-viral T cell response (leading to the generation of degranulating, cytokine-producing or proliferating T cells) will also differ for each of the 3 viruses. Aim: To determine in healthy, chronically infected individuals whether different T cell subsets dominate the immune response to the 3 viruses. P828 Methodology: Blood mononuclear cells from 15 healthy individ- Neglected tropical diseases in haematopoietic stem cell uals (all seropositive for EBV and VZV, 9 seropositive for CMV) transplantation: a single-centre experience in Brazil were stimulated with EBV, VZV or CMV lysate or with over- T.C. Martins (1), I. Colturato (2), M. S. Leite (1), A.J. Simione lapping peptides (CMV-pp65 or EBV-LMP1+2). Cytokine (IFNg (1), V. Colturato (1), M.P. de Souza (1), M.A. Mauad (1), and IL-2) production and degranulation (CD107a expression) W. Azevedo (1), C.M. Machado (2) were assessed after 6-18 h using multicolor fl ow cytometry. The (1)Fundação Amaral Carvalho (Jaú, BR); (2)University of São subsets shown in the Table have enumerated. Paulo (São Paulo, BR) Results: As shown in the Figure, CMV-specifi c CD4 T cells were mainly IFNg producers, both alone and in combination with IL2. Introduction: Infections are major obstacles to successful hemat- EBV-specifi c CD4 T cells were mainly either IFNg or IL2 sin- opoietic stem cell transplantation (HSCT) worldwide. Transplant gle cytokine producers. VZV-specifi c CD4 T cells were almost programs in developing countries have the additional challenge entirely double cytokine producers (IFNg and IL2). CD8 T cells of management of neglected tropical diseases (NTD). About stimulated with pp65 peptides or EBV lysate showed similar one billion people are affected by one or more NTD globally, cytokine production as CD4 T cells (very few CD8 T cells were which cause approximately 534,000 deaths annually. The most detected using stimulation with VZV lysate or CMV lysate). prevalent NTDs in Brazil are: tuberculosis, leprosy, dengue Degranulating pp65-specifi c CD8 T cells frequently produced fever, leishmaniasis, malaria, Chagas disease, schistosomia- IFNg whereas degranulating LMP1/2-specifi c CD8 T cells did sis, yellow fever (YF) and soil-transmitted helminth infections. not. Absolute count of IFNg or IL2-producing (single and double We retrospectively reviewed the experience concerning to the cytokine producers combined) EBV-specifi c CD4 T cells was management of NTDs at the HSCT program from Fundação six fold higher than that of VZV-specifi c CD4 T cells and two fold Amaral Carvalho, the greatest program for allogeneic trans- higher than that of CMV-specifi c CD4 T cells. Absolute count plants in Brazil. of IFNg or IL2-producing or degranulating pp65-specifi c CD8 Methods: Information was obtained from both recipients (R) T cells was ~2 fold higher than that of LMP1/2-specifi c CD8 T and donors (D). Search strategy included chart revision, pre- cells. transplant visit forms and serologic data review. Phone calls Conclusions: Different T cell subsets are dominant for different were used to get information on YF vaccination. Soil-transmit- herpes viruses. The relevance for pathogenesis of herpesviral ted helminth infections were not included as empiric treatment diseases needs to be elucidated. with albendazole and mebendazole is performed in all trans- plant candidates. Results: NTDs were not spontaneously reported in pre-trans- plant visit forms. Thus, only serologic information could be obtained pre-HSCT. Donor information was only obtained if a NTD was suspected after HSCT. 950 charts were reviewed from 296 autologous, 543 allogeneic and 70 URD HSCT. Forty-

S236 P830 The impact of Dectin-1 polymorphism Tyr238Stop on the occurrence of Candida colonisation and disease of haematopoietic stem cell transplantation recipients P831 W.J.F.M. van der Velden, T.S. Plantinga, B. Ferwerda, The natural history and control of cytomegalovirus P. Donnelly, M.G. Netea, N.M.A. Blijlevens infection following allogeneic stem cell transplantation Radboud University Nijmegen Medical Center (Nijmegen, NL) G. Garnett (1), M. Boeckh (2), H. Einsele (3), V. Emery (4) (1)Imperial College (London, UK); (2)Fred Hutchinson Cancer Background: Haematopoietic stem cell transplantation (HSCT) Research Center (Seattle, US); (3)University of Würzburg is accompanied by a high incidence of invasive fungal dis- (Würzburg, DE); (4)Royal Free and University College Medical ease (IFD) including those due to Candida species. There is School (London, UK) increased interest in the molecular determinants of suscepti- bility to such infections. A new polymorphism, Tyr238Stop has Background: Despite current treatments, cytomegalovirus been characterised in Dectin-1, a C-type lectin that recognises (CMV) remains an important pathogen in allogeneic stem cell the 1,3-beta-glucan of fungi. transplant (SCT) recipients. Both the direct and indirect effects Aim: Study the association of the Dectin-1 Tyr238Stop polymor- of CMV can lead to poor patient outcomes. Given the lack of phism with the occurrence of Candida colonization and disease robust epidemiological data on the natural history of CMV in in HSCT recipients. SCT, the objective of this work was to develop a model which Methods: We performed a retrospective analysis in a 142 depicts this natural history. Dutch patients admitted for a sibling donor allogeneic T cell- Methods: An ordinary differential equation model describing the depleted HSCT following myeloablative conditioning. Patients fl ows between risk, infection and disease categories in an adult only received fl uconazole (200 mg daily) if they were colonised population undergoing allogeneic SCT was developed. Model with Candida spp. (except C. glabrata and C. krusei) defi ned parameters were derived from the literature as well as cohort as the same yeast being present in both mouthwash and fae- data from two transplant centres. The model can be solved cal samples obtained on the same day, or from the same site numerically to predict the expected rates of disease and death on two consecutive occasions. Only those with data on colo- in a cohort with specifi c characteristics. nisation and those not receiving prophylaxis with itra-, vori- or Results: The model divides the population of SCT recipients into posaconazole before day 0 were included in the analysis of three categories according to serotype: recipient positive(R+), Candida colonization. In the analysis of candidaemia patients recipient negative but donor positive(R-/D+) and recipient neg- not receiving anti-mould prophylaxis were included in the analy- ative and donor negative(R-/D-) with their associated risks of sis. Genotyping for the presence of the Tyr238Stop mutation in CMV viremia. In addition, the population is stratifi ed by risk: the Dectin-1 gene was performed by employing a TaqMan RT- – very high risk (haplotypic donors or with cord blood source PCR SNP assay. In addition functional studies were performed cells); high risk (unrelated donors) and moderate risk (related with monocytes homozygous for the polymorphism, obtained donors). The nine resulting categories determine the param- from three individuals outside this cohort. eters of the model. For all of the categories, the fl ows through Results: The allele frequency the Tyr238Stop polymorphism in infection and disease are illustrated. A fraction of CMV disease the Dectin-1 gene was 6.6% in our cohort and these patients is not preceded by viremia, but in a majority of cases, viremia were more commonly colonized with Candida spp. on admission provides an opportunity for screening and pre-emptive treat- and on the day of HSCT, and therefore more often received fl u- ment. Three signifi cant CMV diseases are included, pneumo- conazole (Table 1). In contrast, the incidence of candidaemia up nia, gastrointestinal disease and retinitis, along with secondary to day 21 was not signifi cantly infl uenced by this Dectin-1 poly- infections, which can result from other causes associated with morphism (Table 1), but the use of fl uconazole was most likely neutropenia or be can result directly from CMV. The model a confounding factor. In vitro functional studies with monocytes allows calculation of the number of days over which treatment homozygous for the polymorphism demonstrated ‘’loss of func- is received, the duration spent with different diseases and tion’’ occurring with this mutation resulting in impaired cytokine the pattern of mortality associated with different causes since responses and Candida binding by innate immune cells. transplantation. Conclusion: These data suggest Dectin-1 plays a role in the Conclusions: This model represents the pathways by which host defences against Candida infection and screening for the CMV causes disease in allogeneic SCT recipients. The model Tyr238Stop polymorphism might help select those high-risk can also be used to look at future trends, such as the increas- patients who most likely to profi t from antifungal prophylaxis. ingly older transplant patient population and the projected increases in the use of cord blood cells in SCT. Finally, when available, estimates of the effi cacy of new and existing treat- ments can be incorporated into the model, allowing alternative management strategies to be compared.

S237 of the episodes were caused by an epidemic clonal strain. 7 patients had strong evidence for bloodstream infections (BSIs) with P.aeruginosa, 2 of them died. The epidemic strain was identifi ed from the nanofi ltered water and later on, in multiple samples from toilet drains. MRPA was not recovered from HCWs, surfaces or food items. Contami- nated drinking water was considered as the probable source. However, molecular typing with pulsed-fi eld electrophoresis demonstrated a closer match with strains isolated from toilet drains than from freshwater. Spillage of Pseudomonas contam- inated toilet drain water was the most likely route of transmis- sion, possibly during fl using the toilet. Continuous high-dose chlorination resulted in decontamination of the toilets, but was poorly tolerated. After introduction of con- tinuous bromide disinfection of the toiled drains, water cultures were continuously negative and the rate of MRPA infections P832 signifi cantly declined to < 0.1 episodes/1000 patient days. Rou- Lower respiratory tract respiratory virus infections tine surveillance cultures failed to isolate the epidemic strain increase the risk of invasive aspergillosis following a with a follow up of 4 years. reduced-intensity allogeneic haematopoietic stem cell Conclusions: MRPA is capable of persistently contaminating transplantation toilet drains, thus colonizing and infecting immunocompromised R. Martino, R. Parody, J.L. Piñana, D. Valcarcel, A. Sureda, patients. Nanofi ltration of drinking water and continuous disin- S. Brunet, J. Briones, J. Delgado, F. Sanchez, N. Rabella, fection of toilet drains in addition to active surveillance, contact J. Sierra isolation of infected patients contained the epidemic. Untreated Hospital de Sant Pau (Barcelona, ES) drinking water is an important source for P. aeruginosa infec- tions in the severely immunocompromised patient. We have analyzed the incidence and risk factors for the occur- rence of invasive aspergillosis (IA) among 219 consecutive recipients of an allogeneic hematopoietic stem cell trans- P834 plantation following a reduced-intensity conditioning regimen Children with acute leukaemia have an increased risk for (AlloRIC). Twenty-seven patients developed an IA a median Varicella zoster reactivation after allogeneic stem cell of 218 days (range 24-2051) post-AlloRIC, for a 4-year inci- transplantation: a possible role for fl udarabine dence of 13% (95% confi dence interval 4-24%). In multivari- A.C. Lankester, C.M. Jol-van der Zijde, P. Hissinkmuller, ate analysis risk factors for developing IA were steroid therapy A.C.T.M. Vossen, L.M. Ball for moderate-to-severe GVHD (Hazard Ratio [HR] 2.9, P= Leiden Univ Medical Center (Leiden, NL) 0.03), occurrence of a lower respiratory tract infection (LRTI) by a respiratory virus [RV] (HR 4.3, P<0.01) and CMV disease Background/introduction: Varicella Zoster virus (VZV) belongs (HR 2.8, P = 0.03). Variables which decreased survival after to the herpes family and reactivation is seen in immune com- AlloRIC were advanced disease phase (HR 1.9, P = 0.02), ster- promised hosts. VZV reactivation, especially when presenting oid therapy for moderate-to-severe GVHD (HR 2.2, P<0.01), early after hematopoietic stem cell transplantation (HSCT), not developing chronic GVHD (HR 4.3, P<0.01), occurrence of may cause signifi cant morbidity. In recent years, (re-) induction LRTI by a RV (HR 3.4, P<0.01) and CMV disease (HR 2, P = treatment of acute leukemia (AL) has been intensifi ed including 0.01), while occurrence of IA had no impact on survival (P=0.5). immunosuppressive agents eg. fl udarabine. Based on our initial Our results show that IA is a common infectious complication clinical observations of early VZV reactivation in children under- after an AlloRIC, which occurs late post-transplant and may not going HSCT in our unit, we undertook a retrospective study to have a strong impact on survival. An important observation is determine the frequency and the possible factors associated the possible role of LRTI by conventional RVs as risk factors with the frequency and timing of VZV reactivation. for IA. Patients and Methods: Between 2002 and 2008, all children undergoing a fi rst HSCT (n=166) in our unit were included. HSCT indications included malignant (63%) and non- P833 malignant diseases (37%). Antiviral prophylaxis (incl. Acyclovir) Outbreak of waterborne Pseudomonas aeruginosa was not routinely administered to our patients. VZV diagnosis infections in a reverse isolation haematology unit was based on clinical features supported by Q-PCR and viral A.F. Widmer, R. Frei, M. Stern, D. Heim, C. Arber, M. Battegay, culture. U. Flückiger, A. Gratwohl Results: Within the cohort of 166 patients, 40 (24%) patients University Hospital Basel (Basel, CH) developed VZV reactivation during the fi rst year after trans- plantation. VZV reactivation occurred more often within the Background: Pseudomonas aeruginosa infections in immuno- subgroup of AL patients compared to the remainder of patients compromised patients are associated with high morbidity and (n=26/67, i.e. 39% vs. n=14/99, i.e. 15%, p<0.01 respectively). mortality. We report an outbreak with an epidemic multi-resist- In children greater then 4 yrs VZV occurred more often in the ant Pseudomonas aeruginosa (MRPA) clone in a 12-bed hae- AL group compared to all others (40% vs 19%, respectively). matology unit after a new centralized water treatment system Donor type, use of serotherapy or conditioning regimen did not with nanofi ltration and continuous fl ow was installed. infl uence VZV occurence. Methods: An epidemiological investigation was begun after VZV reactivation was signifi cantly increased in AL patients who identifi cation of a cluster of P. aeruginosa including extensive had previously received fl udarabine compared to the remain- environmental cultures and a case control study. Samples of der of the SCT patients (n=18/41, i.e. 44% vs. n=22/125, i.e. water from all faucets, showers and fi lters were taken at regu- 18%, p< 0.01). In addition, VZV reactivation presented earlier lar intervals. Screening of patients included rectal and perineal after transplantation in patients that had received fl udarabine swabs, supplemented with environmental cultures from water (median day + 36 vs day + 72). Notably, children with myelo- and surfaces and cultures from hands of health care workers dysplastic syndrome, not previously treated with fl udarabine, (HCWs). and although having the same median age as the AL patients Results: A total of 74 episodes of P. aeruginosa colonization (11.2 and 11.5 yrs, respectively) VZV reactivation was found in or infection occurred between Jan 1, 1999 and Dec, 2004. 24 only 21% of the patients.

S238 Conclusion: VZV reactivation is non-randomly distributed re-evaluate routine bacterial prophylaxis in cytotoxic chemo- among pediatric alloSCT recipients and predominates within the therapy or hematopoietic stem cell transplantation (SCT). Here, subgroup of AL patients. Patients that have received fl udarab- we retrospectively analyzed the impact of restricting the use ine during the (re-)induction therapy prior to alloSCT seem of bacterial prophylaxis. We evaluated 126 consecutive adult particularly at risk. These observations may be instrumental to patients (pts) with hematologic malignancies who had under- improve pre-and posttransplant VZV prophylactic strategies. gone myeloablative allogeneic marrow or peripheral blood SCTs between January 2000 and September 2008 at our institution. Until August 2004, patients received oral fl uoroquinolones (FQs) P835 as bacterial prophylaxis (prophylaxis group). In pts who could Different CMV and EBV reactivation among alternative not ingest oral drugs, oral FQs were temporarily substituted with donor transplants depending on the type of stem cell intravenous administration of antipseudomonal beta-lactams. source: a single-centre analysis Since September 2004, any bacterial prophylaxis including oral M. Mikulska, A. Ibatici, A. Dominietto, A.M. Raiola, V. Pinto, FQs was discontinued (non-prophylaxis group). In both groups, M. Soracco, F. Gualandi, V. Del Bono, T. Lamparelli, M.T. Van intravenous antibiotics were promptly administered to treat epi- Lint, C. Viscoli, A. Bacigalupo, F. Frassoni sodes of febrile neutropenia or suspected bacterial infections. Ospedale San Martino (Genoa, IT) We estimated the cumulative incidence of microbiologically doc- umented bacterial infection (DBI) within 30 days after SCT and Background: Cytomegalovirus (CMV) and Epstein-Barr virus early mortality within 100 days in each group, and compared (EBV) reactivation have an higher incidence after allogeneic the results of non-prophylaxis group with those of prophylaxis stem cell transplant (ASCT) using alternative sources such as group using a competing risk regression analysis. Of the126 pts cord blood (CB) or unrelated (URD)/related mismatched (rel- included in the analysis (median age, 42 years; range, 17-61 mm) donors. This is due to several factors including the use years), 61 received stem cell grafts from related donors, while of anti-thymocyte globulin (ATG) and the donor/recipient HLA 65 from unrelated donors. Background patient characteristics mismatch. Routine monitoring and pre-emptive therapy have did not signifi cantly differ between the prophylaxis arm (87 pts) limited the clinical impact of CMV and EBV on outcomes. How- and non-prophylaxis arm (39 pts). The median day of onset of ever, duration of monitoring and the need of pre-emptive ther- DBI after SCT was 5.5 (range, 2-11) in the prophylaxis group apy might differ depending on the type of donor source. and 7 (range, 1-30) in the non-prophylaxis group (P = 0.14). Aims: To evaluate retrospectively the frequency and duration of The cumulative incidence of DBI was much higher in the non- CMV and EBV reactivation and its relevance on outcomes after prophylaxis group than in the prophylaxis group (0.18 [95% ASCT from 1) CB and 2) URD/rel-mm donors. Post-ASCT vari- confi dence interval, 0.08-0.31] vs. 0.07 [0.03-0.12]), with mar- ables were compared by time-dependent analyses and cumu- ginal signifi cance in univariate analysis (P = 0.06). However, lative incidences (CI) were calculated. early mortality did not signifi cantly differ. Further, organ failure Methods: Since Jan 06 through Sept 08, 119 patients were ana- and septic shock due to bacterial infection were not observed, lysed (CB=58; URD/rel-mm=61), the majority having received except in two pts in the prophylaxis group who died because of a myeloablative conditioning (72%). GVHD prophylaxis con- infection with multi-drug resistant organisms. Bacterial prophy- sisted of CSA-based regimens and ATG (Thymoglobulin) for 2 laxis can be restricted without signifi cant adverse infl uence on days (3.0 to 3.75mg/kg dd-3, -2). All pts received CMV prophy- the early mortality after allo-SCT, if appropriate antibiotic treat- laxis with Foscarnet, as previously reported and EBV prophyl- ment is immediately initiated in the case of febrile neutropenia. axis with Rituximab 200mg total dose on day +5. EBV viremia was defi ned as more than 1000 copies of EBV-DNA/ml of whole blood. P837 Results: The median FU was 165 (3-732) and 245 (1-942) days Functional immune reconstitution after high-dose for the CB and URD/rel-mm group, respectively (p=NS). Gen- chemotherapy parallels control of endogenous viral der, median age, disease phase, type of conditioning were not reactivation: the torquetenovirus model different between the 2 groups, whereas GVHD prophylaxis D. Focosi, F. Maggi, M. Albani, J. Rocchi, V. Ricci, S. Gragnani, was (p=0.001). Acute GVHD I-IV was less frequent in the CB M. Ghimenti, F. Papineschi, M. Bendinelli, M. Pistello, group (54 vs 82% p=0.001), but no difference in chronic GVHD L. Ceccherini-Nelli, M. Petrini (p=0.87). TRM and OS did not differ between the 2 groups. University of Pisa (Pisa, IT) The CI of CMV reactivation was higher in the CB group (82 vs 57% p=0.025) and there was a trend for a longer duration (99 Background: It is common experience that retreating patients vs 35 days p=0.09). EBV reactivation (CI at 1 year) was more too early after a course of intensive chemotherapy predispose frequent in the URD/rel-mm group (69 vs 38 p<0.001) and the to opportunistic infections despite apparently normal lym- number of pts with EBV viremia was higher (23 vs 10% p=0.09 phocyte levels. The kinetics of reactivations of endogenous trend) as well. viruses that cause no obvious disease (and hence need no Conclusions: In our series, CMV and EBV reactivation are infl u- treatment) can serve as a surrogate marker to better under- enced by the type of source. CMV reactivation is higher after CB stand when a patient has recovered from this functional transplantation and seems to last longer, requiring extra-treat- immune defi ciency. ment for control. Conversely, EBV reactivation has a greater Methods: We used real-time polymerase chain reaction to impact on URD/rel-mm despite both groups had day+5 Rituxi- monitor the kinetics of serum Torquetenovirus (TTV) viremia in mab prophylaxis. These observation may lead to a better defi ni- 44 hematological patients undergoing high-dose chemotherapy tion of recommendations for CMV and EBV management. supported by autologous hematopoietic stem cell transplanta- tion, followed for up to 3 years. Results: Independently from underlying haematological disease P836 or chemotherapy regimen, TTV viremia fl uctuations parallel Reappraisal of bacterial prophylaxis in allogeneic marrow those of other known immunophenotypic markers of functional or peripheral blood stem cell transplantations after immune defi ciency. Furthermore, we suggest here that the myeloablative conditionings maximal increase in plasma TTV level reached after a chemo- J. Kanda, T. Ichinohe, T. Saito, K. Yamashita, T. Kondo, therapy course determines the time required before returning T. Ishikawa, S. Ichiyama, T. Uchiyama to basal TTV levels, allowing prediction of the appropriate time Kyoto University (Kyoto, JP) gap before delivery of the next chemotherapy course. Conclusions: We suggest here that monitoring of TTV viremia Widespread emergence of multidrug-resistant microorgan- represent a unique opportunity to follow functional immune isms in hematology-oncology units has raised the necessity to reconstitution.

S239 after 8 months due to intolerance. Hepatitis developed in 3 untreated patients and in none of the patients on prophylaxis. All patients with hepatitis received a RIC allo-SCT. In conclusions, we observed: 1) a null risk of hepatitis B in patients serologically negative for HBV, transplanted with HBsAg-negative donors; 2) the effi cacy of LAM in controlling HBV replication and hepatitis B reactivation in both HBsAg- positive recipients from negative donors and in HBsAg-positive donors used in negative recipients; 3) the effi cacy of prophy- lactic LAM in HBsAg-negative and antiHBc-positive recipients in preventing HBV related hepatitis; 4) the lack of a protective effect of RIC for patients at risk of HBV reactivation.

P839 Posaconazole and cyclosporine drug interaction in allogeneic HSCT: dose adjustment, interaction kinetics and safety profi le I. Sánchez-Ortega (1), T. Caballero (2), C. Montes (3), B. Patiño (1), J. Villa (1), M. Arnan (1), L. Yáñez (3), A. Iriondo (3), L. Vázquez (2), R.F. Duarte (1) (1)Institut Catalá d’Oncologia (Barcelona, ES); (2)Hospital Universitario (Salamanca, ES); (3)Hospital Marqués de Valdecilla (Santander, ES)

Posaconazole (POSA) is a potent inhibitor of CYP3A4 and increases exposure to its substrates, such as Cyclosporine A (CSA). Prescribing information for POSA recommends a reduction of CSA dose at initiation of POSA therapy (www. noxafi l.com). CSA & POSA drug interaction has not been for- mally studied in allogeneic HSCT (alloHSCT) yet. Here, we present a pharmacokinetic analysis in 41 alloHSCT recipients on CSA for GVHD prophylaxis who initiated POSA (200mg/8h PO) for primary antifungal prophylaxis: 25 men; median age 51, 18-67; 21 AML-MDS, 11 lymphoma, 3 ALL, 3 Myeloma, 3 others; 25 related, 16 unrelated donors; 12 myeloablative, 29 P838 reduced-intensity conditioning. For this study, CSA dose was Risk of hepatitis B development and effi cacy of not reduced at initiation of POSA. Rather, CSA levels were prophylaxes with lamivudine after allogeneic stem cell monitored (x2-3/week) and the dose adjusted as required to transplantation keep levels within range or if CSA-toxicity occurred. Kinetics of L. Giaccone, I. Resta, A. Marengo, R. Sorasio, F. Fiore, CSA plasma levels, dose-adjustment and ratio of levels to dose M. Rizzetto, M. Boccadoro, A. Marzano, B. Bruno were studied. Results are summarised in the table for base- University of Turin (Turin, IT) line (pre-POSA) and days 7, 14 and 30 of combined treatment. They show that POSA increases CSA levels in alloHSCT recipi- Hepatitis B virus (HBV) positive patients undergoing allogeneic ents (p=.011), with a signifi cant effect already present within the stem cell transplantation (allo-SCT) and recipients of allo-SCT fi rst week of treatment (225.8 pre-POSA vs 293.1 day 7 POSA; from HBV positive donors are at risk of hepatitis reactivation p=.028). Hence, POSA induces a 50% overall reduction in CSA and fatal liver failure. In this setting, the impact of reduced dose (p<.001). This dose reduction however is not required intensity conditioning (RIC) and the role of prophylaxis with during the fi rst week of combined treatment (p=.857), and nor- lamivudine (LAM) are still unclear. Ninety-nine patients under- mally occurs after day 7 (p=.082), and most frequently after going allo-SCT between 1999 and 2007 with at least 3 months day 14 (p<.001). Ratio of CSA levels to dose show a steady of follow-up (median 32 months, range 4-97) entered the study. increase with time (p<.001). Combined treatment with CSA & Patients, median age of 51 years (20-67), were transplanted for POSA was safe. Only 7 patients had CSA-attributed toxicities: multiple myeloma (54), acute myeloid leukemia (16), chronic 4 mild increases in creatinine, 1 hypertension, 1 microangio- lymphatic leukemia (11), lymphoma (10), chronic myeloprolif- pathy, 1 tremor. Two patients stopped POSA for abnormal liver erative disease (7) and aplastic anemia (1). The conditioning function tests that resolved upon discontinuation. Our results regimen was myeloablative in 12 patients and reduced intensity confi rm that POSA treatment increases CSA therapeutic levels in 87; 2 patients received 2 allo-SCT from the same donor for in alloHSCT recipients. Interestingly, this effect is evident from disease progression. Stem cell source was peripheral blood in the fi rst few days of POSA treatment, but does not require CSA 96 patients and bone marrow in 3. Seventy-six patients had a dose-reduction for 1 to 2 weeks. The initial recommendation to HLA-matched sibling donor and 23 an unrelated donor. Recipi- reduce the dose of CSA upfront should be reviewed, as it may ents negative for hepatitis B surface antigen (HBsAg) and cause subtherapeutic CSA levels in some patients that would anti-hepatitis B core antigen antibodies (antiHBc), with HBsAg- be posed at a higher risk of GVHD. Our recommendation would negative donors, were not considered at risk of HBV reacti- be for close monitoring of CSA plasma levels and CSA dose- vation (72). None of them experienced HBV related hepatitis reduction not upfront but according to plasma levels or toxicity. during follow-up. The other 27 patients were considered at risk. In this study, such a strategy has shown to be safe to manage All HBsAg-negative recipients from HBsAg-positive donors (3) these patients. and HBsAg-positive recipients from negative donors (2) were treated with LAM. None of them developed hepatitis B after a median follow-up of 21 months (13-30).Twenty-two patients were antiHBc-positive. Twelve/22 patients were not treated with LAM and 10 received prophylactic LAM for a median time of 19 months (range 4-36). Of these, one discontinued LAM

S240 P841 Respiratory syncytial virus infection in recipients of allogeneic stem cell transplantation: retrospective study of the incidence, clinical features and outcome G. Avetisyan (1), J. Mattsson (2), P. Ljungman (3) (1)Karolinska Institutet (Stockholm, SE); (2)Karolinska University Hospital (Stockholm, SE); (3)Karolinska Institutet, Karolinska University Hospital (Stockholm, SE)

Objectives: Respiratory syncytial virus (RSV) is a common cause of serious respiratory infections (mostly bronchiolitis and pneumonia) in pediatric and adult stem cell transplant recipi- ents. We aimed to determine the frequency, risk factors, and outcome of RSV infection in the group of allogeneic stem cell transplant (allo-HSCT) recipients. Methods and Results: Data were collected retrospectively from a total of 275 allo-HSCT recipients and identifi ed 33 patients (12%, 27 adults and 6 children) with laboratory confi rmed RSV infection; 18 had upper tract and 15 lower respiratory tract infection (LRTI). The duration of viral shedding was long P840 (20 days; 7-83). 28 patients received ribavirin (6 iv, 11 oral, Tetramer-screening to predict the risk of recurrent 9 both, 1 oral + aerosolized, and 1 received all three forms). The CMV-reactivation after HSCT: a 2-year screening and median duration of therapy was 22.5 days (7-54). Four (1.5% follow-up phase of the entire cohort, 12% of infected patients; 27% of patients S. Borchers, S. Luther, N. Hahn, J. Kontsendorn, M. Stadler, with LRTI) patients died from RSV (attributable mortality) while H. Diedrich, A. Ganser, E.M. Weissinger 4 patients died from other infections 38 days – 5 months after Hannover Medical School (Hannover, DE) diagnosis of RSV. Long-term respiratory function was assessed in surviving patients. No patient developed severe respiratory One of the major problems after hematopoietic stem cell trans- dysfunction after RSV. Mild respiratory dysfunction was com- plantation (HSCT) are infectious complications in aplasia or mon after RSV infection but lack of pre-infection information during immunosuppression post HSCT. Reactivation of latent makes interpretation diffi cult. viruses, like cytomegalovirus (CMV), are major causes of mor- Conclusions: RSV infection results in a low overall attributable bidity and mortality. mortality after allo-HSCT but LRTI infection is associated with Virus-specifi c T-cells control the infection/reactivation of CMV, a signifi cant risk for death. Future studies are required to iden- but during the recovery of the immune system of the patients tify prognostic factors for developing LRTI, and to optimize the these may be scarce. Recently, the screening for virus-epitope- therapy of RSV. binding CD8+ T-cells using MHC-I-peptide-complexes (tetram- ers) was established. We used this technique to gain insight in the immune-reconstitution of CMV-reactive T-cells. We P842 monitored all patients undergoing HSCT in our department, CMV infection and disease after allogeneic stem cell matching at least one of 6 commercially available tetramers transplantation with reduced-intensity conditioning for common HLA-types. Samples were taken prior to HSCT, G. Avetisyan (1), H. Hägglund (2), Z. Hassan (2), K. LeBlanc (2), on days +50, +80, +100, +180 and +365 or weekly in case of J. Mattsson (2), O. Ringdén (2), J. Winiarski (2), P. Ljungman (3) CMV reactivation and/or increased immunosuppressive ther- (1)Karolinska Institutet (Stockholm, SE); (2)Karolinska apy. Of 134 patients 114 could be included in the screening. University Hospital (Stockholm, SE); (3)Karolinska Institutet, The median age was 54.5 years (range: 18 to 70). Sixty-seven Karolinska University Hospital (Stockholm, SE) patients had AML (de novo: n=42; secondary AML n=25). Fifty- six were transplanted from matched unrelated (MUD; 49.1%), Objectives: Cytomegalovirus (CMV) is one of the most frequent 35 from matched related donors (MRD; 30.7%), and 23 patients pathogens after allogeneic hematopoietic stem cell transplanta- received HSCT from mismatched donors (MMUD: n=19; tion (allo-HSCT). Recipients of reduced intensity conditioning MMRD: n=4). The CMV-serostatus for recipients (R) and donors (RIC) experience less transplantation-related toxicity and ear- (D) was: R+/D+ in 55, R+/D- in 14, R-/D+ in 10 and R-/D- in 35 lier recovery of CMV-specifi c immunity. However, it is unknown recipient/donor pairs. Thirty-seven patients (32.5%) reactivated if these transplants are associated with reduced risk of post- CMV at least once within a median of +38 days (range: day +11 transplantation infections. The aim of this retrospective study to +93), 16 patients reactivated CMV more than once. In the was to analyze incidence, timing, and risk factors for CMV after R+/D+ group tetramer binding cells were detected in all patients allo-HSCT with RIC. yielding to protection against CMV-reactivation in the majority Patients and methods: 168 consecutive patients receiv- of recipients. In the R+/D- group recurrent reactivation occurred ing RIC allo-HSCT between June 2000 and May 2008 were more frequently, 5 of 7 patients transplanted from MUD and included. Five patients received two transplants due to rejec- 4 of 6 transplanted with MMUD reactivated CMV at least once, tion or relapse. The median age was 53.0 ys (2.7-69.0). 78 4 and 3, respectively, reactivated CMV more than once. In the transplants were from HLA-identical siblings and 95 from R-/D+ group no reactivation occurred. unrelated or mismatched family donors. 140 grafts were In order to identify risk factors besides the number of CMV- PBSC, 24 bone marrow, and 9 were cord blood. CMV-sero- binding T-cells, we analysed all patients receiving HSCT in logic status in donor/recipient pairs was as follows: 15 D-/ our centre in the last 10 years (n=603) retrospectively. Next to R-, 15 D+/R-, 52 D-/R+, and 91 were D+/R+. Patients were CMV-serostatus, HLA-matching infl uenced the risk of recurrent monitored weekly by a quantitative real-time PCR for CMV CMV-reactivation signifi cantly. Therefore chimerism analyses DNA and preemptive therapy was given at pre-specifi ed viral of CMV-binding T-cells are performed. Our results indicate that loads. Risk factors for CMV DNA positivity, repeated CMV tetramer binding T-cells can be used as an additional param- replication episodes, and CMV disease were analysed by eter to identify patients at risk for recurrent CMV-reactivations. multivariate logistic regression. Results: CMV DNA was detected after 113/173 (65.3%) HSCT. The only signifi cant risk factor for CMV DNA detection was the recipient serological status. With the D-/R- cases excluded, the

S241 rate of CMV DNA detection was 70.8%. More than one CMV P844 episode was observed after 40 (23%) HSCT. With the D-/R- Voriconazole plasma levels monitoring in allogeneic cases excluded, a CMV seropositive donor reduced the risk haematopoietic stem cell transplant recipients for repeated CMV episodes (OR 0.45; CI 0.22-0.95; p=.02). J. Winterová (1), Z. Rácil (1), L. Malášková (2), I. Kocmanová Ten cases of CMV disease were documented of which 2 were (3), B. Weinbergerová (1), J. Mayer (1) fatal. No case occurred in D-/R- patients. The CMV disease (1)Dept. of Internal medicine Hemato-oncology (Brno, CZ); rate was 6.3%. Risk factors for CMV disease were either 1st (2)Dept. of Biochemistry and Hematology (Brno, CZ); (3)Dept. episode log10 initial viral load (OR 2.5; CI 1.3-5.0; p=.006) or of Microbioogy (Brno, CZ) peak viral load (OR 2.3; CI 1.03-5.1; p=.04). Non-signifi cant factors were acute GVHD grades II-IV, CMV serological status Objectives: Voriconazole plasma levels measurement of donor or recipient, repeated CMV episodes, donor type, stem enables to optimize dosing and improves the effi cacy of antifungal cell source. treatment. Conclusions: In this cohort of RIC allo-HSCT patients, CMV Methods: Retrospective analysis of documentation and labora- infections were observed in 65% of all cases. The risk for CMV tory results of patients after allogeneic transplantation treated disease was similar to what was previously reported in myelo- with voriconazole from August 2005 to November 2008 was ablative HSCT (6.3 vs. 5.2%). Higher initial and peak viral loads performed. Steady-state plasma Voriconazole levels were during the fi rst episode of CMV replication are risk factors for obtained using a high-performance liquid chromatography development of CMV disease. assay. Results: 392 plasma samples from 51 patients(pts.) were ana- lyzed; 1-29 samples per patient. Voriconazole was adminis- P843 tered in 59% as prophylaxis, in 25% as an empirical antifungal RSV infection after allogeneic haematopoietic stem cell treatment and in 16% as a preemptive treatment of invasive transplantation from a single centre fungal infection. In 48 pts. (94%) voriconazole was adminis- F. Piazera, S. Fortier, J. Morando, C. Bonfi m, M. Bitencourt, trated orally, only in 3 patients intravenously and the total daily V. Funke, D. Setubal, J. Ruiz, C. Arns, R. Silva, R. Pasquini, dose varied from 200 to 800 mg. The mean trough voricona- C. Medeiros, J. Zanis-Neto zole plasma concentration after the standard daily dose 400 HC-UFPR (Curitiba, BR) mg orally was 1,44 µ/ml (0,2-8,89 µg/ml). In 8% (23) of plasma samples from 20 (40%) patients the voriconazole plasma con- Background: Respiratory syncytial virus (RSV) causes signifi cant centration was undetectable(<0,2 µg/ml). In 42,3% (267) of mortality in pts submitted to SCT. Previous data reported a fre- samples from 31 (63%) patients after 400 mg/day the plasma quency of 3,5% of RSV infection in allogenic SCT. Despite the concentration was under 1 ug/ml = below the level associated use of ribavirin aerosol (RA), mortality rates is still between 30 and with a better response to therapy and better prognosis of inva- 40% in many centers. sive aspergillosis. The increase in daily voriconazole dose from Objectives: Analyse the clinical course and outcome of 59 pts 400 to 600 mg in 11 patients led to elevation of voriconazole who developed RSV infections after SCT in a single institution trough plasma level above 1,0 µg/ml in 4 cases and above (Federal University of Parana – Curitiba-Brazil). 0,5 µg/ml in 3 cases. On 4 cases the concentration didi not Methods: In retrospective analysis, diagnosis of a RSV infection changed or decreased. was confi rmed in 59 pts submitted to hsct. All pts had symptoms Conclusion: Our results demonstrate that high percentage of upper or lower tract respiratory infection. From 1991-2004: of patients after allogeneic stem cell transplantation did not 51 pts indirect immunofl uorescence method (IFI). rom 2004 - achieved suffi cient voriconazole plasma concentration. Moni- 2008 : 8 pts qualitative PCR. RSV infection was defi ned as a toring of plasma levels enables an individual dosage adjust- laboratory identifi cation of the virus in the nasopharyngeal aspi- ment and lead to improving response to antifungal treatment in ration, bronchoalveolar lavage or coryza. RA was administrated this group of very high risk patients. during 12 hours, in the dose of 5g diluted in 200ml of distillated water, for 5 days. Male: 37, Female: 22. Median age of patients was 19,9 years (2,8 – 54,02). Donor: 48 related, not related 11. Detection method: IFI – 51, PCR- 8. 15 patients presented upper respiratory tract (URT) infection, 44 patients presented lower respiratory tract (LRT). Diagnoses: SAA- 17, CML- 14, AML- 5, ALL- 3, MDS- 5, others- 5. SPSS program was used for statistical analysis. OS was esti- mated with the use of Kaplan-Meier method. Multivariate analy- P845 sis was performed by Cox method. Prevalence of multiresistant pathogens in surveillance Results: Median time after transplantation for the diagnosis of and clinical isolates from patients undergoing RSV infection was 17 days (1 - 435). In the group of patients haematopoietic stem cell transplantation in Hungary: who received RA (n=50), 40 had URT infection and 10 had LRT. a single-centre experience In patients who did not receive RA, 3 had URT infection and 6 C. Kassa, J. Sinkó, K. Kállay, A. Kertész, M. Konkoly-Thege, had LRT. 20 patients died (33,8%), and the main cause of death G. Kriván in 13 pts was RSV infection (all this pts have need mecanic St. István and St. László Hospital (Budapest, HU) ventilation). 9 patients died before the use of RA as standard treatment for RSV (before 1992). The overall survival (OS) of Introduction: Growing prevalence of multiresistant bacte- pts treated with RA was 66%. In the univariate analysis: type of ria (MRB) in stem cell transplant patients has gained special engraftment, method of identifi cation of RSV, time for diagno- importance due to the subsequent increase in mortality rate sis, type of donor, stem cell source or baseline disease did not and limited options to treat infections caused by these strains. interfere in the OS. However, OS was inferior in patients who Patients and methods: A two years’ retrospective epidemiologi- had LRT infection (37,5%) compared with those who had URT cal survey was performed among patients undergoing stem cell infection (67,5%), p=0,007. In the multivariate analysis, only the transplantation at our center between March 1, 2005 and Feb- use of RA affect OS (p=0,001). ruary 28, 2007. Aim of our study was to determine the rate of Conclusions: In this group of pts RSV infection resulted in an colonization and infections, caused by MRB. At the same time overall mortality of 34%. All pts who were not treated with RA to investigate outcomes and the potential role of surveillance died. cultures in predicting serious infections. During the observation period 144 hematopoetic stem cell transplantations (autologous: 83, allogeneic: 61) were performed in 140 patients (males: 79,

S242 females: 61; children: 56, adults: 84). The age of patients aver- metronidazole. Although the data are limited in number, C. diff aged 26 years (from 2 months to 68 years). Respiratory tract, infection may be associated with a higher incidence and sever- fecal and urine surveillance cultures were taken once weekly. ity of gut aGvHD. Prospective studies are required to defi ne In case of fever and/or symptoms of an infection appropriate optimal prophylaxis of C. diff. infection and its impact on gut clinical sampling was performed. aGvHD. Results: From 80 pts. with positive surveillance cultures (231 isolates) 37 pts were colonized by MRB (115 isolates). Most frequently multiresistant Pseudomonas aeruginosa (57,4%), P847 extended spectrum beta lactamase (ESBL) producing Gram- Multiple co-pathogens are associated with increased negative bacteria (22,6%) and multiresistant Stenotrophomonas morbidity and mortality in parainfl uenza virus 3 infection maltophilia (13,9%) were isolated. In 31 pts. we have found 78 A. Hodson, M. Kasliwal, A Retter, K. Cuthill, M. Streetly, positive clinical cultures. These samples were taken from blood M. Kazmi, E. MacMahon, K. Raj (78,6%), wound (9,5%), bronchoalveolar lavage (7,2%) as well Guy’s Hospital (London, UK) as other sites (4,7%). MRB grew from 42 samples, dominated by P. aeruginosa (83,3%). MRB caused infection in 12 patients. The estimated frequency of parainfl uenza virus 3 (PIV-3) infec- From the 9 individuals with bloodstream infections 7 died. High tions following haemopoietic stem cell transplant (HSCT) is 2- mortality was primarily attributable to P. aeruginosa. Primary 5% with mortality discrepantly reported as between 17-75%. immunodefi ciency, allogeneic stem cell transplantation and It is the policy of our unit to test HCST recipients with upper CMV reactivation were the main risk factors for MRB coloni- or lower respiratory tract symptoms for respiratory viruses by zations and infections. While positive predictive value of sur- immunofl uorescence of nasopharyngeal aspirate (NPA) or veillance cultures was poor (21,6%), negative predictive value broncho-alveolar lavage (BAL) samples. We report a retrospec- seemed to be fair (96,1%). tive analysis of the outcomes of PIV-3 infection in our unit over Conclusions: The majority of patients with negative surveillance an 8 month period. cultures for MRB do not develop multiresistant infections. In Sixteen HSCT patients developed a PIV-3 infection (allogeneic MRB-positive pts., however, the choice of empirical antibiotics =15, autologous =1). Nosocomial transmission occurred in 14 should be guided by the result of surveillance cultures. cases. Infection between 3-568 days from stem cell return, in 3 cases pre-engraftment. All patients had upper respiratory tract infection (URI) while lower respiratory tract infection (LRI) with P846 virus detected on BAL occurred in 6. Thirteen patients were Incidence of Clostridium diffi cile infection in patients treated with aerosolised ribavirin (2g tds for 5 days) and IV undergoing allogeneic stem cell transplantation – immunoglobulin (0.5g/kg) as per standard protocol. One patient A single-centre analysis refused treatment while two with full immune reconstitution G. Beutel, S. Buchholz, J. Krauter, E. Dammann, H. Diedrich, were not treated. M. Stadler, P. Kirschner, A. Ganser, M. Eder Overall mortality was 50%. Seven of the 13 patients (54%) Hannover Medical School (Hannover, DE) treated with ribavirin/IVIG died. Risk factors - respiratory fail- ure (n=9, mortality 89%), multiple co-pathogens (n=8, mortality Background: Clostridium diffi cile (C. diff.) causes antibiotic- 75%), septic shock/multi-organ failure (n=7, mortality 86%), LRI associated colitis upon alterations in colonic microfl ora. Bacterial (n=6, mortality 66%). acute GVHD on prednisolone ≥ 1mg/kg exotoxins bind to receptors on intestinal epithelial cells leading (n=5, mortality 80%), infection pre-engraftment (n=3, mortality to infl ammation and diarrhea. New C. diff strains (ribotype 27) 100%). The pulmonary co-pathogens isolated were predomi- and fl uorquinolones often used prophylactically in allogeneic nantly Gm–ve bacteria (n=8) but also included Gm+ve bacteria SCT have been linked to increasing C. diff. infections. (n=2) and fungi (n=2). Aims: The aim of our study was to evaluate the incidence and Our series confi rms the high mortality associated with PIV- clinical manifestation of C. diff. infections in patients undergoing 3 infection in HSCT recipients particularly in those with LRI, allogeneic SCT. secondary bacterial/fungal infections and those receiving ster- Methods: From 01/07 to 10/08, 126 adult patients with haema- oids for GVHD. A similar case series reported a lower mortal- tological malignancies were transplanted at Hannover Medical ity although over half the patients receiving broad spectrum School. For 18 patients (AML n=8, ALL n=5, MDS n= 2, CML antibiotics/antifungals at diagnosis (Dignan et al, J Hosp Inf, n=1, MPS n=1, NHL n=1), C. diff. toxin was detected in stool 2006 (63), 452-458). PIV-3 predisposes to secondary bacte- specimen at any time upon SCT (14.3%, median day + 52, rial infection by increasing expression of bacterial receptors on range 0 – 461 days). So far, no ribotype 27 C. diff. strain has viral infected respiratory cells. Strategies to disrupt the bind- been isolated. At time of C. diff. toxin detection, fi ve patients ing of PIV-3 Haemagglutin-Neuraminidase to respiratory cells were under prophylactic metronidazole treatment, and two are in development. Meanwhile in patients who develop PIV-3 additional patients were diagnosed within a week after end of infection despite strict enforcement of infection control policies, treatment. All patients were subsequently treated either with the best strategy may be careful risk assessment, with effec- metronidazole or oral vancomycin, and C. diff. infection and tive broad spectrum anti-microbials in those at risk of secondary toxin detection fi nally resolved in all cases. Interestingly, 12 out infection. of 18 patients developed aGvHD including 4 patients suffering from III° - IV° aGvHD of the gut (22.2%). Two of these patients died due to refractory GvHD. C. diff infection was diagnosed P848 before or at onset of GvHD in 7 patients, and after GvHD diag- Refractory late onset haemorrhagic cystitis following nosis in two patients, respectively. Three patients developed fl udarabine and alemtuzumab conditioned allogeneic cGvHD (2 extensive, 1 limited). In 108 C. diff. negative patients haematopoietic stem cell transplant (AML n=63, ALL n=9, MDS n=13, CML n=3, MPS n=13, NHL A. Hodson, M. Streetly, P. Fields, K. Raj, E. MacMahon, including multiple myeloma n=7) 52 developed aGvHD. Among M. Kazmi those fi ve suffered from gut aGvHD III-IV° (4.6%) which was Guy’s Hospital (London, UK) signifi cantly less than in the C. diff. positive group (p=0.038). 25 evaluable patients developed cGvHD (9 extensive, 16 limited). The incidence of Haemorrhagic Cystitis (HC) post haemat- Both groups were comparable in terms of conditioning regimen opoietic stem cell transplant (HSCT) varies from 5-40%. Most and CSA-based GvHD prophylaxis, respectively. early HC (day 0-20) can be attributed to chemotherapy while Conclusions: Clostridium diffi cile infection can be detected viral infections (polyomaviruses (BK/JC), adenovirus) are impli- in a reasonable proportion of patients undergoing allogeneic cated in late HC (>day 20). BK viruria is detected in 50-100% of SCT even in the presence of prophylactic treatment with patients following HSCT with or without HC. Graft versus host

S243 disease (GVHD), fl udarabine and T-cell depletion are risk fac- Furthermore, after 6h co-cultivation of PMNs and A. fumi- tors for late onset HC. gatus germ tubes, 195 (1.273) genes showed an at least Our aim was to estimate the frequency of HC associated with 4fold (2fold) altered gene expression. We observed upregu- BK virus reactivation following myelo-ablative (MA) and non- lation of genes (hemoxygenase, heat shock 70kDa protein, myeloablative (NMA) transplantation and to review the effi cacy HSPA8, HSPA1B, HSP90AB1, Ferritin) involved in self- of Cidofovir in these patients. protection against radicals. Additionally, genes involved in Over 24 months 106 patients underwent 108 transplant pro- inflammatory responses (IL-8, CCL3, CXCL2, IL1RN) were cedures (60 autologous (55%) and 48 allogeneic (45%)). HC significantly upregulated. Luminex analysis was performed occurred in 9 of 108 (8%) transplant recipients all allogeneic for TNF-alpha, IL-12, GM-CSF, IFN-gamma, IL-6, IL-8, IL-10 transplants. The frequency following allogeneic transplant was and IL-1beta to identify secreted cytokines, thereby confirm- 19% (9/48). Conditioning was myelo-ablative (MA) in 5 and ing array data. non-myeloablative (NMA) in 4 cases. Early HC occurred in Conclusions: In conclusion, A. fumigatus had substantial effects 4/48 (8%) all following MA conditioning and without associated on the activation of human PMNs. Various defence strategies BK viraemia. were initiated, including phagocytosis, ROS release and mobi- Late HC associated with BK viraemia and viruria occurred in lization of other immune effector cells by secretion of chemoat- 4/48 patients (8%). Three patients received NMA condition- tractants. ing with Alemtuzumab in all and Fludarabine in 2 cases. One patient received MA conditioning with Fludarabine and Cyclo- phosphamide. Initially Cidofovir 5mg/kg was used in 2 patients and BK virus P850 DNA became undetectable in the blood with resolution of hae- The rate of cytomegalovirus infection and disease in maturia. However in view of the apparent effi cacy of a low dose correlation with the type of haematopoietic stem cell approach, 1mg/kg without probenecid (Savona et al, Bone Mar- transplantation in the era of pre-emptive antiviral therapy row Transplant 2007; 39:787-787), two patients received Cido- B. Gesundheit, R. Or, E. Budowski, M. Shapira, I. Resnick, fovir 1mg/kg. With this approach treatment was ineffective and S. Samuel, L. Dray, O. Kaplan, D. Wolf HC persisted, although both patients were on immunosuppres- Hadassah Hebrew University Hospital (Jerusalem, IL) sive therapy for acute GVHD. In this small series BK virus associated HC was more frequent Background: Despite diagnostic and therapeutic advances, following NMA transplantation, in contrast to a recent report cytomegalovirus (CMV) has remained a signifi cant complica- (Giraud et al, Bone Marrow Transplant (2008) 41, 737-742). tion after hematopoietic stem cell transplantation (HSCT). The This may relate to the frequent use of Fludarabine and Alemtu- widespread use of preemptive antiviral therapy has reduced the zumab as T cell depletion in our centre. occurrence of early CMV disease and changed its epidemiol- Cidofovir (5mg/kg) appeared more effective at clearing BK ogy. In order to defi ne the current trends and to establish future virus DNA from the blood and urine leading to resolution of guidelines, we retrospectively analyzed the rate of CMV infec- HC. In cases with risk factors such as GVHD or high dose tion and disease in the following types of HSCT: (A.) autologous, immunosuppression with rising BK DNA in the blood or urine (B.) fully matched-, (C.) mismatched- and (D.) haploidentical- low dose Cidofovir could be considered as pre-emptive allogeneic HSCT. treatment. Patients & Methods: Patients receiving HSCT in the Hadassah Hebrew University Medical Center over a 5-year period (April 2003-March 2008) were retrospectively reviewed for the occur- rence of CMV infection and disease in relation to the 4 types P849 of HSCT. Infection was defi ned by the presence of positive Immune response of human polymorphonuclear CMV antigenemia and/or real time PCR assays, and patients neutrophils to Aspergillus fumigatus received preemptive antiviral treatment upon detection of CMV J. Loeffl er, M. Mezger, I. Wozniok, H. Einsele infection. Labor Professor Einsele (Würzburg, DE) Results: Of 634 patients who received HSCT during the study period, 276 (43.5%) developed CMV infection and 16 (2.5%) Background: Invasive fungal infections with Aspergillus fumiga- had evidence for CMV disease, with the vast majority of dis- tus show an increasing incidence due to the growing number ease occurring among haploidentical HSCT recipients. The of severely immunocompromised patients. Polymorphonuclear occurrence of CMV infection and disease by transplant type is neutrophils (PMNs), as part of the innate immune system, are presented in the table. key players in antifungal immune responses, recognizing fun- No statistically signifi cant differences were found between gal pathogens at an early step of infection. Besides phagocytic the CMV-donor and recipient serostatus in the 3 categories of mechanisms, PMNs kill pathogens by the release of reactive allogeneic HSCT. The rate of CMV infection was signifi cantly oxygen species (ROS). higher in allogeneic compared to autologous HSCT (p < 0.001), Methods: Human PMNs were isolated from blood of healthy with no signifi cant differences between the 3 types of allogeneic donors using Biocoll separation. Cells were co-cultivated with HSCT. However, the rate of disease was signifi cantly higher conidia, germlings and hyphae of the clinical strain ATCC 9197. in haploidentical HSCT (p < 0.001). 15 of the 16 patients with The oxidative burst was determined in a kinetic measurement CMV disease had a fatal outcome. We are presently analyzing quantifying dichlorfl uorescein production. Chemotaxis was the viral load kinetics and the associated risk factors in the dif- analyzed by transwell assays. Furthermore, total RNA was ferent HSCT categories. extracted and gene expression profi ling was performed using Conclusions: Preemptive antiviral approach proved to be Affymetrix U133Plus2.0 arrays. effective in the prevention of CMV disease in autologous, fully Results: We could demonstrate that A. fumigatus represents a matched and mismatched allogeneic HSCT recipients. strong stimulus releasing ROS, depending on the morphotype; The increased rate of CMV disease and associated mortality in germlings revealed high ROS release, whereas resting conidia recipients of haploidentical HSCT despite preemptive antiviral showed low stimulation capacity. PMNs actively tracked germ- therapy refl ects their delayed immune reconstitution and raises lings and directly attached to fungi as demonstrated by real- concern regarding the effectiveness of the current preemptive time microscopy. antiviral approach. Based on our fi ndings, close monitoring Transwell assays revealed that chemotaxis of PMN is strongly and prophylactic antiviral treatment should be employed in this dependent on the fungal morphotype; PMN showed only weak high-risk subgroup of HSCT recipients. chemotaxis in the presence of conidia, whereas in the presence of germlings, high chemotactic activity was achieved.

S244 are frequently observed following intensive chemotherapies (ctx). Mortality is high and previous IFI enhance the risk of consecutive fungal infections if an allogeneic hematopoietic stem cell transplantation (SCT) is performed. Here, we report on patients with proven, probable or possible IFI prior to SCT, thus receiving antifungal secondary-prophylaxis (SP) during their SCT. Methods: Data of 202 consecutive patients (pts) who received SCT during 1/99 to 12/06 were retrospectively analysed with regards to outcome of pts with prior proven, probable or pos- sible IFI before SCT. Other factors included were viral infec- tions, gender, age, underlying disease, disease status, donor P851 type, gender-matching between donor and recipient, stem cell Neutropenic enterocolitis: usefulness of ultrasound source, conditioning-regimen and acute and chronic GvHD. sonography. Single-centre experience Results: Median follow-up was 9.2mths (8 days to 95.3mths). E. Benedetti (1), F. Simonetti (1), F. Caracciolo (1), F. Papineschi Median pt age was 45yrs (15 to 69 yrs). Underlying diseases (1), E. Orsitto (2), M. Tonerini (2), B. Bruno (3), M. Pelosini (1), were Leukaemias (n=122), Lymphomas (n=45), MDS (n=21), D. Focosi (1), M. Petrini (1) OMF (n=5), others (n=9). 137 pts received reduced intensity (1)Hematology Unit (Pisa, IT); (2)UO Emergency Radiology conditioning most commonly based on treosulfane/fl udara- (Pisa, IT); (3)Hematology Unit (Turin, IT) bine (n=112). 65 pts received myeloablative condition- ing mainly based on 12Gy TBI containing regimen (n=48). Neutropenic enterocolitis (NEC) is a life threatening complication of GvHD-prophylaxis consisted of CsA/MTX (n=106) or other patients treated with chemotherapy. The cecum is almost always CsA based regimen (n=83). 22 pts received SP consisiting affected, but the terminal ileum, other parts of the small bowel and of Vori- (n=12), Posaconazole (n=2), Caspofungin (n=3) or right and left colon can also be involved by the disease. Perfora- Amphotericine B (n=5), respectively, due to possible/prob- tion occurs in 5%-10% of cases. Early diagnosis is crucial to start able (n=21) or proven (Aspergillus n=1) IFI before SCT. 178 conservative medical management which appears the optimal pts received PP mainly consisting of Flu- (n=90) or Itraco- strategy for most cases. Despite aggressive management mortal- nazole (n=87). Median survival of pts receiving SP was 4 ity rate are high up to 21-48%. NEC should be always suspected in mths (15 days to 54 mths) compared to a median survival of Neutropenic patients with abdominal pain, fever and diarrhoea. 24 mths (8 days to 95 mths) in pts receiving PP (p=0.003). Ultrasound (US) was used to evaluate bowel-wall thickening 15/22 (68%) pts receiving SP and 86/180 (48%) of pts receiv- (BWT). The degree of BWT correlated with the outcome in one ing PP died during the study period. In the SP group causes study and 60% of patients with BWT > 10 mm died from this com- of death were: 9 infection, 2 relapse, 3 relapse/infection, 1 plication compared with 4.2% of those with BWT < 10 mm. We GvHD. In multivariate analyses SP remained to be associ- evaluated retrospectively NEC cases occurred in the last two years ated with inferior survival (p=0.004, HR=2.28, CI 1.29–4.02), and usefulness of US and 24 fi t patients were identifi ed. Disease besides pts undergoing unrelated donor SCT (p=0.015, diagnosis were: MH 8, ALL 3, AML5 MM 1 and NHL 7. Treatment HR=1.71, CI 1.11–2.65), high-risk pts (p=0.,044, HR 1.57 received was: intensive chemotherapy(10), allogenic transplant CI 1.01-2.43) and pts developing aGvHD III-IV (p=0.011, with bu/cy (1) and cy/tbi (1), autologous transplant with bu/cy (1), HR=1.73, CI 1.13–2.65). BEAM (10) and, MEL 200 (1). All 24 patients were neutropenic at Conclusion: Despite highly effective antifungal secondary time of diagnosis. Abdominal pain was present in 23/24 patients. prophylaxis, proven or suspected IFI prior to SCT showed sig- Diarrhoea was present in all patients. Positive culture were found nifi cant infl uence on overall survival after SCT. in 12.5% (stool) and 25% (blood) of patients. Our internal proto- col changed from 2007 to 2008 were neutropenic patients were US-scanned immediately as one symptom presented. US signs of P853 NEC were considered thickening or dilation of small and/or large Cytomegalovirus pre-emptive strategy guided by Ag intestine. US allowed to detect NEC in one patient with abdominal pp65 or quantitative PCR after reduced-intensity pain and diarrhoea without fever and furthermore allowed diag- conditioning allogeneic stem cell transplantation: nosis in 6 out of 9 patients within 12 to 24 hours from the start of a single-centre experience symptoms in respect to 2007 were US was applied later in the J.L. Piñana, R. Martino, N. Rabella, P. Barba, N. Margall, course. Two patients within 12 hours from diagnosis underwent M. Roig, D. Valcarcel, J. Sierra succesful colon surgery guided by US features of potential wall Hospital de la Santa Creu i Sant Pau (Barcelona, ES) rupture. Two patients with previous diagnosis of NEC relapsed during subsequent chemotherapy induced neutropenia and one With the purpose of reducing cytomegalovirus (CMV) morbidity died of sepsis. Overall 3/24 patients died, and 19/24 had complete and mortality pre-emptive therapy guided by sensitive methods remission with conservative medical management. In conclusion have been developed. The aim of this study was to analyse our early intestinal US in neutropenic patients performed in suspicion 8 years single-centre experience, with 2 different pre-emptive of NEC helped to detect early signs consistent with NEC and to therapy protocols in patients who underwent reduced intensity start immediate treatment of this life-threatening complication. conditioning allogeneic HSCT (AlloHSCT-RIC). We have retrospectively included 186 consecutive AlloHSCT-RIC adult patients at risk for CMV reactivation. Conditioning regimen was P852 mainly based on Fludarabine plus an alkylating agent. CMV was Outcome of allogeneic haematopoietic stem cell monitored by pp65 Antigenemiag (pp65 Ag) in 116 patients while 70 transplantations in patients with secondary antifungal patients were monitored by quantitative PCR (quantPCR). prophylaxis due to proven or suspected prior fungal Alternative donors (AD), micophenolate mophetil as a part of infection GVHD prophylaxis and T-cell depleting agents were more fre- S. Rohde, M. Leithäuser, G. Kundt, M. Haversath, D. Wolff, quent in quantPCR group (p< 0.05). The incidence at 2-year of I. Hilgendorf, H. Andree, J. Casper, C. Kahl, M. Freund, CMV infection (CMV-I) and disease (CMV-D) was 15% in the C. Junghanss pp65 Ag and 39% in the PCR period (P<0.01). This difference University of Rostock (Rostock, DE) was due to a higher asymptomatic CMV-I in the quantPCR group (5% vs 27%, (P<0.01). However a 2-year CMV-D inci- Introduction: Despite highly effective primary-prophylactic dence of 10% and 12%, are still similar between Antigenemia (PP) antifungal strategies, invasive fungal infections (IFI) and quantPCR respectively, (P= 0.5). Patients monitored by

S245 pp65 Ag had more often isolate CMV pneumonia (64%) than P855 patients followed by quantPCR (20%) (p=0.01). The proportion NOD2/CARD15 polymorphism evaluation in autologous of patients with isolated CMV pneumonia with negative screen- stem cell transplantation ing test was unusually high (66%), especially for patients moni- S. Giammarco, S. Bellesi, P. Chiusolo, S. Marietti, D. De Ritis, tored with pp65 Ag (77%). Multivariate analysis showed that L. De Padua, E. Metafuni, G. Leone, S. Sica steroids and alternative donors were independent risk factors Università Cattolica S. Cuore (Rome, IT) for CMV-D (HR 4.7, 95%C.I 1.7-13, p=0.02 and HR 2.7, 95%C. I. 1.2-6.5, p= 0.002, respectively). NOD2/CARD15, member of the NOD family, encodes a pro- Our fi ndings suggest that quantPCR allow us to treat pre- tein which is involved in the interaction with infecting bacterial emptively a larger proportion of patients at higher risk for lipopolysaccharides (LPS) and so it favour apoptosis and the CMV-D, achieving similar incidence of CMV-D compared to NFkB signaling pathway inducing the transcription of several those monitored by pp65 Ag. genes encoding proinfl ammatory cytokines. R702W, G908R, The author is supported by grants from the Instituto de Salud L1007f insC polymorphisms are supposed to alter the recogni- Carlos III (expedient CM06/00139, Ministerio de Sanidad). tion of the bacterial LPS. Supposing an abnormal infl ammatory response to bacterial infections due to these polymorphisms we performed a retrospective analysis of infl uence of them on P854 outcome of pts submitted to autologous HSCT, and we corre- Adoptive transfer of cytomegalovirus-specifi c CD8+ lated them to transplant complications, as sepsis, organ toxic- T-cells powered by streptamer technology leads to ity, non relapse mortality (NRM) and overall survival (OS). We enduring virus clearance in patients after allogeneic stem studied 111 patients submitted to autologous SCT between cell transplantation 1994 and 2008. Allelic variants at SNP12 (G908R) and SNP13 A. Schmitt (1), T. Tonn (2), D. Busch (3), G. Grigoleit (4), (L1007fi nsC) of the NOD2/CARD15 gene were genotyped by H. Einsele (4), M. Odenthal (3), L. Germeroth (5), M. Ringhoffer PCR. Incidence of sepsis and organ toxicities were analyzed by (6), S. Ringhoffer (6), M. Wiesneth (7), D. Michel (7), T. Mertens beta-2 test while OS and RFS curves were obtained by the Kap- (7), M. Rojewski (7), M. Marx (7), S. von Harsdorf (6), H. Döhner lan Meier method and statistically compared by log-rank test. (6), E. Seifried (2), D. Bunjes (6), M. Schmitt (1) Pts’characteristic were: M66/F45, median age 46 years (range (1)University Clinic (Rostock, DE); (2)German Red Cross 15-66).Underlying disease were 16 AML, 55 lymphoproliferative (Frankfurt, DE); (3)Technical University (Munich, DE); disease, 32 MM, 4 POEMS, 4 solid tumor. The median follow-up (4)University Clinic (Würzburg, DE); (5)IBA Ltd. (Göttingen, was 33 months (range 1-129).The most relevant complications DE); (6)University Clinic (Ulm, DE); (7)University of Ulm in the post-transplant phase were: FUO (55%), documented (Ulm, DE) sepsis (28%) and hepato-renal toxicity (NCI grade II-IV) (48%). Ninety patients relapsed after a median time of 6 months (range Background: After allogeneic peripheral blood stem cell trans- 4-85); 97 (87%) out 111 are alive while 14 (13%) died. NOD2/ plantation (allo-PBSCT), cytomegalovirus (CMV) disease con- CARD15 mutations occurred with a frequency of 13%. For stitutes a serious complication. CD8+ T cells are pivotal for the SNP12 108 out 111 (97%) patients were unmutated and 4 (3%) clearance of CMV. Adoptive transfer of CMV specifi c T cells patients were heterozygotes, no one was found homozygous; might constitute the key to overcome this problem. The novel for SNP13 102 (91%) patients were normal, 9 (8%) had het- technology of “streptamers” allows the selection of CMVpp65 erozygotes genotype and 1 (1%) patient was homozygous. In specifi c CD8+ T cell up to 98% purity without altering the func- our casistic we found no correlation between NOD2/CARD15 tional properties of the selected T cells and without requiring polymorphisms and incidence of sepsis or organ toxicities labor and time consuming T cell cultures. after tx; moreover we demonstrated that the presence of NOD- Materials and Methods: Here, the novel streptamer technology 2CARD15 polymorphisms did not infl uence OS and NRM after was used for adoptive transfer of CMV specifi c T cells into three tx. There are no data in literature regarding NOD2/CARD15 acute leukemia patients with recurrent high CMV antigenemia in autologous transplant setting, even if these polymorphisms after allo-PBSCT, in the case of the third patient in a haploidenti- could infl uence the toxicity grading and the incidence of com- cal setting. Standard peripheral blood mononuclear cell apheresis plications after high dose chemotherapy, altering the interaction was performed on the former stem cell donors of three patients between bacterial toxins and monocytes-macrophages system with acute leukemia. Isolation of CMV specifi c donor lymphocytes in mucosaes. Probably our results are infl uenced by the low was performed using a Good Manufacturing Product (GMP)- prevalence of these polymorphisms in the Italian gender. grade Streptamer selection kit on a CliniMacs™ device. Results: A single specifi c donor lymphocyte infusion (sDLI) of 2.2, 0.4 or 0.4 x105 CMVpp65 specifi c T cells per kg body weight was P856 performed in an AML or ALL patient respectively, after allogeneic Prospective study of oral valganciclovir for pre-emptive PBSCT developing a CMVpp65 antigenemia with a maximum therapy of cytomegalovirus after allogeneic stem cell of 959 or 716 or 190 CMVpp65 positive/500,000 cells and treat- transplantation ment with foscarnet, ganciclovir and valganciclovir. After sDLI, R. de la Camara (1), L. Vazquez (2), J. Lopez (3), C. Solano (4), the CMV antigenemia was cleared and remained persistently D. Serrano (5), A. Garcia Noblejas (1), J.M. Ribera Santasusana controlled even after discontinuation of valganciclovir therapy in (6), C. Ferra (6) both patients. No acute or chronic toxic side effect, particularly no (1)Hospital Universitario de la Princesa (Madrid, ES); (2)Hospital aggravation of graft-versus-host disease (GvHD) was observed. Universitario (Salamanca, ES); (3)Hospital Universitario A strong and sustained increase of the absolute count of CMV- Ramon y Cajal (Madrid, ES); (4)Hospital Clinico Universitario specifi c CD8+ T cells was detected. CMV-specifi c CD8+ T cells (Valencia, ES); (5)Hospital Universitario Gregorio Marañon were CCR7-/CD45RA+, indicating a effector T cell phenotype. (Madrid, ES); (6)Hospital Universitario Germans Trias i Pujol The chimerism analysis of the in vivo expanded CMV-specifi c (Barcelona, ES) CD8+ T cells demonstrated a 100% donor chimerism. T cell receptor excision circle (sjTRECs) analysis revealed a fre- Intravenous ganciclovir (iv-Gcv) is the standard drug for pre- quency of sjTRECs two logs lower than expected, indicating emptive therapy of cytomegalovirus (CMV) infection in alloge- peripheral expansion rather than thymic proliferation of CMV neic stem cell (allo-SCT) patients. Valganciclovir (Vgcv), an oral specifi c CD8+ T cells. Clonality of transferred cells was proven prodrug of ganciclovir, is used very often in practice although by PCR of the respective CDR3 region. there is no approved indication for this specifi c use. Conclusion: Streptamer technology constitutes an advanta- We performed a prospective phase II multicentric study with geous technology for selecting CMV specifi c CD8+ T cells at Vgcv in adult patients to evaluate its effi cacy and safety in the GMP level for adoptive T cell transfer. pre-emptive treatment of CMV-Ag or DNAemia within the fi rst

S246 180 days after SCT. The results will be compared with those the correlation coeffi cient r being 0.998 (p<0.0001). But also obtained with iv-GCV in a retrospective control group. The pri- the EBV DNA levels in WB correlated with those in PBMCw/o or mary study endpoint was the achievement of negative CMV-AG/ in PBMCw (r=0.81 [p<0.0001] or 0.81 [p<0.0001], respectively). PCR on day 14 of treatment. Secondary end-points were nega- EBV detection in plasma was less sensitive than in WB or PBMC. tive CMV-AG/PCR, rate of neutropenia (<500 neutrophils/mm3) Thus, correlations between plasma and WB or between plasma and nephrotoxicity, until 35 days after the beginning of treat- and PBMC were lower (plasma versus WB: r=0.57 [p<0.0001]; ment. A minimum patient weight of 50 kg was required. Here plasma versus PBMCw/o: r=0.51 [p<0.0001]; plasma versus we presented the results on the fi rst 53 prospective patients PBMCw: r=0.50 [p<0.0001]). treated with Vgcv. Conclusions: The three blood compartments (whole blood, Vgcv was given at 900 mg/12h for 14 days (induction therapy) PBMC and plasma) show different kinetics of their EBV DNA followed by 900 mg/day for another 14 days (maintenance levels after allogeneic HSCT. Monitoring of EBV DNA levels treatment), in both cases adjusted according to renal function. in whole blood appears to be a valuable alternative to PBMC. If CMV was positive at day +21, the treatment was considered Normalization of EBV DNA in PBMC towards an endogenous a failure and Vgcv was stopped. standard does not seem to provide additional information. The median age and weight of the patients was 48.2 years and 72 kg. Type of donor: 31 HLA identical siblings, 21 unrelated and 1 related mismatch. Mieloablative conditioning regimen P858 was used in 20 cases and reduced intensity in 33 cases. Twenty Use of saliva in the detection of HCMV DNA in allogeneic cases (38%) had active GVHD and 51 (96%) where receiving stem cell transplantation and its comparison with viral immunosuppression at CMV viremia presentation. Median time load in blood by real-time PCR for CMV-Ag/PCR positivity was 46,5 days after SCT. J. Correa-Silva (1), O. Bruna-Romero (2), R. Resende (1), At day +14, 33 patients remained on Vgcv and had a negative L. Miranda (1), F. Oliveira (1), F. Costa (1), S. Xavier (3), CMV-Ag/PCR. Twenty patients failed: 9 remained CMV positive; S. Figueiredo-Neves (3), R. Gomez (1), H. Bittencourt (4) 5 had changed to another antiviral due to an increase in viral (1)School of Dentistry (Belo Horizonte, BR); (2)Institute of load (between 6-13 days of treatment); 5 had Vgcv stopped due Biological Sciences (Belo Horizonte, BR); (3)School of Medicine to adverse events (4 neutropenia and 1 digestive intolerance); (Belo Horizonte, BR); (4)Hospital das Clinicas / School of 1 patient developed a proved gastric CMV disease on day +9. Medicine UFMG (Belo Horizonte, BR) At day +35, 32 patients (60%) remained CMV-Ag/PCR nega- tive, 7 patients (13%) developed neutropenia, but none devel- Human cytomegalovirus (HCMV) infection still remains the oped nephrotoxicity. At 2 months of follow-up no more cases of most frequent viral complication of the post transplant period CMV disease were diagnosed. in allogeneic hematopoietic stem cell transplant (allo-HSCT) Oral valganciclovir therapy given at a fi xed dose in patients recipients. The aim of this study was to investigate the ability over 50 kg for the pre-emptive therapy of CMV in allogeneic of real time PCR methods to detect HCMV in saliva samples patients, was effective and with similar toxicity compared with of allo-HSCT recipients and to compare it with blood HCMV the previous published experience with iv-gcv. detection by PCR. Thirty consecutive allo-HSCT recipients, admitted between October 2006 and November 2007, were included in this study. Patients were sampled for PCR analysis P857 from seven days before transplantation (day -7) to 100 days Towards standardisation of Epstein-Barr viral load after HSCT (day +100) or until death of recipient. One saliva quantifi cation: comparison of four different ways of and one blood samples were attempted to be simultaneously measurement in peripheral blood of paediatric patients obtained from each recipient once a week during the evalua- after allogeneic haematopoietic stem cell transplantation tion course. HCMV DNA load in saliva samples showed a high S. Volke (1), B. Gruhn (2), W. Woesmann (1), E. Landmann (1), correlation with blood viral levels, as detected by real time PCR J. Beck (2), A. Reiter (1), H. Wagner (1) (correlation coeffi cient of 0.858; p<0.0001). Virus DNA levels (1)Pediatric Hematolgy and Oncology (Giessen, DE); in blood also showed a positive correlation with antigenemia (2)Pediatric Hematolgy and Oncology (Jena, DE) assays (correlation coeffi cient of 0.773; p<0.0001). One HCMV DNA copy / 200ng DNA in saliva samples corresponded to 41 Epstein-Barr-Virus (EBV) reactivations are serious complica- HCMV DNA copies / 200ng DNA in blood samples. The best tions affecting the recipients after allogeneic hematopoietic possible prediction point set by a receiver-operating character- stem cell transplantation (HSCT) as they may lead to post istic (ROC) curve analysis, determining positive results in saliva, transplant lymoproliferative disorders (PTLD). EBV load meas- was the cut-off point of 2 HCMV DNA copies /200ng DNA, with urement in peripheral blood has been shown to be essential sensitivity of 70% and specifi city of 75%. Real time PCR assays for the monitoring of patients; however, the optimal sample were able to clearly distinguish levels of HCMV replication in type and way of PCR quantifi cation remains uncertain. Aim of saliva (p = 0.015) and blood (p = 0.008) before, at the beggining the study was to compare the EBV DNA levels in whole blood and after anti-viral therapy. It was worth noting that HCMV DNA (WB), in plasma and in peripheral mononuclear cells (PBMC) levels in survival patients were lower than in individuals who without (PBMCw/o) or with (PBMCw) normalization towards an did not survive. Despite the preliminary nature of our data, this endogenous control. Therefore, we examined the blood of 213 study shows that the highly sensitivity real time PCR test could paired samples from 37 children and adolescents (median age be useful to identify HCMV DNA in saliva samples and to moni- 10 years; age range 1 to 21 years) who underwent allogeneic tor patients at risk of HCMV disease after allo-SCT. HSCT by using real time duplex PCR (ABI Prism 7500). 21/37 (56.8%) patients became EBV positive (EBV+) in periph- eral blood following HSCT. The median day on which patients P859 were EBV+ after HSCT for the fi rst time was day +49 (range, EBV and HHV-6 reactivation associate with mental day +11 to day +180). In 11/37 (29.7%) patients we observed a disturbances in alloHSCT patients total of 14 signifi cant EBV-reactivations, defi ned as >100 cop- A. Burniak (1), E. Jaskula (2), S. Madej (1), A. Lange (2) ies/ml plasma or >500 copies/µg PBMC DNA. All reactivations (1)Lower Silesian Center for Cellular Transplantation with were found in WB, but only in 11/14 (78.6%) patients in their National Polish Bone Marrow Donor Registry (Wroclaw, PL); plasma or in 10/14 (71.4%) patients in PBMCw/o. EBV reacti- (2)L. Hirszfeld Inst. Immun & Exp. Ther. (Wroclaw, PL) vations were frequently earlier detectable in WB than in PBMC or plasma. In other our study encephalitis was found to be more frequent Comparing the four ways of EBV quantifi cation, we found in patients positive for EBV and to lower extent for HHV-6 strongest correlations in PBMC with and without normalization, DNA copies in blood. Therefore, we reanalyzed our standard

S247 psychological observation charts to verify the hypothesis on bodies prior to HSCT (34/84 vs 1/10 p=0.08) and (iii) in those association between Herpes viruses reactivation and psycho- receiving HLA mismatched graft (9/16 vs 26/86 p=0.08). logical status of alloHSCT patients. The present group included EBV reactivation was associated with encephalitis (7/12 vs 89 adults and 11 children receiving allograft from sib (45 pts) 21/80 p=0.01) and relapse (6/12 vs 22/90 p=0.08), the pres- and alternative donors (55 pts). ence of EBV IgG antibodies in donors (28/84 vs 0/10 p=0.03), Routine psychological observation included personal conversa- female donor (18/47 vs 10/55 p=0.03), and female recipient tion and psychological tests enabling assessment of emotional, (16/42 vs 12/60 p=0.07). cognitive and behavioral status of the patients. HHV6 reactivation was associated with encephalitis (6/12 vs We found that patients: 21/90 p=0.08), but in contrast to EBV and CMV, was rather rare (i) with EBV reactivation (number of DNA copies > 100 / 105 in patients with extensive cGvHD (22/61 vs 1/23 p<0.01) and in blood cells) showed more frequently cognitive abnormalities those receiving HLA matched graft (26/86 vs 1/16 p=0.06). (patients were rather diffi cult in contact and produced illusions Both CMV and EBV reactivation were associated with a low and hallucinations) than others (0.54 vs 0.29, p=0.028); proportion of CD8high+ CMV pp65 reactive (0.38%±0.07 vs (ii) with HHV-6 reactivation (> 10 DNA copies/105 blood cells) 0.88%±0.12, p=0.03) and CD8high+ EBV BLMF-1 reactive had behavioral disturbances as compared to other patients cells (0.284%±0.047 vs 0.48%±0.06, p<0.01). having or lacking other than HHV-6 Herpes viruses in blood Multivariate analysis proved that the factors listed above play an (0.55 vs 0.3, p=0.012); independent role in reactivation of investigated herpes viruses. (iii) the presence of CMV reactivation did not signifi cantly asso- Supported by the MNiSW grant N401 169 32/3358. ciate with mental disturbances. In multivariate analyses we analyzed the presence of emo- tional, behavioral and cognitive abnormalities against EBV P861 (>100 DNA copies/105 blood cells) and HHV-6 (>100 DNA cop- Levofl oxacin prophylaxis after high-dose chemotherapy ies/105 blood cells) reactivations and the presence of aGvHD, and autologous peripheral stem cell support: does it encephalitis, hepatitis, cystitis, pneumonia, relapse, sex and work? A single-centre experience age of patients and also donor-recipient gender relation. EBV A. Anastasia, L. Castagna, S. Bramanti, B. Sarina, R. Mazza, reactivation together with age and the presence of aGvHD were E. Todisco, L. Giordano, A. Santoro independently associated with the presence of any of psycho- Humanitas (Rozzano, IT) logical abnormalities analyzed. When HHV-6 was considered it became apparent that this virus reactivation contributes to all Introduction: Antibacterial prophylaxis after high-dose chemo- those abnormalities individually, similarly to the situation seen therapy (HDC) and autologous peripheral stem cell (PBSC) in EBV study, with age and the presence of aGvHD. support is still controversial. However, a recent randomized, From this study it is apparent that psychological status of HSCT double blind, placebo-controlled trial showed that prophylactic patients is affected by reactivation of EBV and HHV-6 but not levoxacin reduced the incidence of fever, probable infections CMV. with shorter hospitalization in patients treated with HDC. The aim of this work is to evaluate the role of levoxacin analyz- ing two consecutive cohorts of patients, receiving HDC, with or P860 without prophylaxis. Reactivations of CMV, HHV6 and EBV differ with respect Patients ad methods: From October 2004 to November 2006, to the risk factors and the clinical outcome in patients 98 HDC was performed in 74 patients. Patient characteristics post haematopoietic stem cell transplantation are showed in Table 1. Only patients reinfused with <5x106 E. Jaskula (1), D. Dlubek (1), A. Tarnowska (2), D. Duda (3), CD34+/Kg received G-CSF from day +5. The fi rst cohort (A) of A. Lange (1) patients without prophylaxis was treated until 2005. The second (1)L. Hirszfeld Institute of Immunology and Experimental cohort (B) with prophylaxis was treated from 2005 and there- Therapy, Polish Academy of Sciences (Wroclaw, PL); (2)Lower after. Prophylaxis with levofl oxacin was started during con- Silesian Center for Cellular Transplantation (Wroclaw, PL); ditioning regimens and stopped in case of fever. At this time, (3)Lower Silesian Center for Cellular Transplantation & National an empiric antibiotic treatment was administered, preceded Polish Bone Marrow Donor Registry (Wroclaw, PL) by blood cultures, chest X-ray and all others diagnostic tools. Microbiological sample analysis were performed weekly for Herpes viruses reactivation constitutes severe complication each patients during aplasia period. with a high rate of mortality especially in patients on immuno- Primary end points were incidence and duration of fever, sec- suppression post haematopoietic stems cell transplantation. ondary end points were documented infections, time to dis- One hundred and two (45 SIB, 57 MUD) HSCT recipients were charge, prevalence of quinolone-resistance strains in stools, followed for CMV, HHV6 and EBV reactivation (qPCR) in the mortality due to infection. context of immunological reconstitution and post transplant Results: In cohort A incidence of fever was 77,8%, with median complications. All tests were performed beginning from hae- duration of 2,8 days. Incidence of documented infections was matological reconstitution and then in one week intervals while 47,2%. Incidence of fever in severe mucositis 72,7%. Preva- patients in the hospital, at later time each time while seen in our lence of quinolone-resistance strains 80%. In cohort B inci- outpatient department and when clinical symptoms were sug- dence of fever was 62,9%, median duration 4,1. Incidence of gestive of viral reactivation (usually 8 observations during fi rst documented infections 48,4%. Incidence of fever in severe year post HSCT). More than 100 viral DNA copies/105 blood mucositis was 80,6%. Prevalence of quinolone-resistance was cells seen at least on one occasion during one year post trans- 94%. No statistical differences were seen between the two plant period was signifi cantly associated with clinical complica- cohorts. tions (29/66 vs 6/36, p<0.01 and 24/66 vs 4/36, p=0.01 for EBV Discharging day after reinfusion was 16 in both groups. Overall and CMV, respectively). mortality due to infections was null in the fi rst 100 days after CMV, EBV and HHV6 DNA copies (above 100 copies/100 000 HDC. cells) were detected in 34%, 27% and 26% of patients, respec- Microbiological data suggest that cohort B has a higher inci- tively. dence of resitant pathogens. Acute GvHD (grade ≥I) constituted the main risk of CMV (17/35 Conclusion: Furthermore, our results suggest that antibacte- vs 18/67, p<0.05) but not of EBV and HHV6 reactivation. CMV rial prophylaxis will not be a must for neutropenia after HDC. reactivation was associated with higher rate of bacterial pneu- monia (7/11 vs 28/91 p=0.04) and was more often in (i) adults (2/16 vs 33/86, p<0.05), (ii) patients positive for IgG CMV anti-

S248 2 patients Posaconazole. After RIC transplantation only 4 patients (14%) had reactivation of their IPA. The latter 4 patients were experiencing severe acute GVHD treated with high dose corticosteroids. None of these patients died of IPA. Actually 18 patients (64%) are still alive with a median follow up of 23.5 months (12.6–48.5). Overall survival at 2 years was 59% [95%CI, 43–83]. In all, 3 patients died from other causes not directly related to IPA, 2 patients because of relapse or disease progression, and one due extensive chronic GVHD. In comparison to literature in standard myéloablative allo-SCT setting, these data suggest that adequately treated and con- trolled IPA prior to RIC Allo-SCT do not worsen outcome. The latter is likely due to multiple changes in transplantation prac- tice, including the RIC nature of the conditioning regimens, the infusion of peripheral blood stem cells, rapid diagnosis of IPA, and use of modern effi cient antifungal drugs.

P863 Use of “high fl ow” totally implantable central venous access ports for myeloablative conditioning followed by allogeneic stem cell transplantation: a prospective study A. Abdelkefi (1), O. Ben Gaied (1), S. Laabidi (2), H. Allouche (1), S. Ladeb (1), N. Ajmi (3), M. Maamar (3), L. Torjeman (1), A. Lakhal (1), A. Mezlini (2), M. Elloumi (3), N. Ben Romdhane (5), T. Ben Othman (1) (1)Centre National de Greffe de Moelle Osse (Tunis, TN); (2)Instiut Salah Azaiz (Tunis, TN); (3)Hôpital Hédi Chaker (Sfax, TN); (3)Centre National de Transfusion Sanguine (Tunis, TN); (5)Hôpital La Rabta (Tunis, TN)

Objectives: A central venous access is always necessary for the management of patients receiving myeloablative condi- tioning followed by allogeneic stem cell transplantation (SCT). While the wide use of quinolones will increase prevalence of Tunnelled, cuffed silastic catheters are the device of choice quinolone-resistance germs. for these patients. To our knowledge, there are no prospec- tive studies investigating the use of totally implantable central venous access ports (TIAP) in patients receiving myeloablative conditioning followed by allogeneic SCT. The aim of this pro- P862 spective study was to investigate the usefulness of a new “high Impact of prior invasive pulmonary aspergillosis on fl ow” single lumen port device in these patients. outcome in patients receiving reduced-intensity Methods: Between December 2007 and November 2008 at the conditioning allogeneic haematopoietic stem cell National Centre for Bone Marrow Transplantation (Tunisia), transplantation patients with haematological malignancies received a TIAP J. El-cheikh, L. Wang, C. Faucher, S. Furst, B. Esterni, prior to allogeneic SCT. All patients received the same type P. Berger, D. Blaise of port [silicone, 10 French, high fl ow rate (3100ml/h), Refer- Institut Paoli Calmettes (Marseille, FR) ence 40010, Laboratoires Perouse, Ivry le Temple, France]. All devices were inserted under local anesthesia through direct Invasive Pulmonary Aspergillosis (IPA) is a major cause of puncture of the right subclavian vein using the Seldinger tech- morbidity in patients with hematological malignancies. The nique. A chest X-ray was always obtained to document correct aim of this single centre retrospective study was to deter- positioning. All infusions, including the graft itself and all blood mine the impact of prior IPA on outcome after RIC Allo-SCT. drawings, were performed via the port. Port-related blood- All cases of proven or probable IPA diagnosed prior to per- stream infection was defi ned according to Infectious Disease formance of RIC Allo-SCT at our Cancer Centre. 28 patients Society of America guidelines. All patients were examined by were identifi ed among 434 patients undergoing Allo-SCT. ultrasonography in case of clinical signs of thrombosis and sys- Gender: M/F (16/12); median age at diagnosis was 53 years tematically after discharge. (18–65). 23 patients (82%) had acute myeloid leukemia. IPA Results: Thirty fi ve TIAP were placed in 35 patients [median was diagnosed according to standard procedures. IPA therapy age: 26 years (20-40 years); 14 female and 21 male], and included: 20 patients (71%) were receiving Voriconazole; 4 remained in place for a cumulative duration of 7302 days in situ patients Itraconazole, and 4 patients with an association of (range 30-350 days). No pneumothorax occurred. Port-related different antifungal drugs including Caspofungine, Liposomal bloodstream infection (candida parapsilosis) occurred in only Amphoteracine-B, or Posaconazole. The median duration of 1 case (1/35, 2.8% ; 0.1 event per 1000 days). This port was treatment prior to RIC was 8 months range (1–16). The median removed. No port-related thrombosis occurred. No port-related time between the diagnosis of IPA and transplantation was deaths were observed. 6 months (1–27); 20 patients (71%) received a graft from a In conclusion, the use of “high fl ow” totally implantable ports family donor, 4 patients had a MUD, and 4 patients received has resulted in a good option for long-term access to central cord blood cells donors. 21 patients (75%) received a condi- veins and delivery of myeloablative conditioning followed by all- tioning regimen with Fludarabine Busulfan and ATG, and 4 ogeneic SCT, in spite of severe neutropenia and increased risk patient (14%) received a conditioning with Fludarabine Cyclo- of sepsis in this category of patients. Although multicentre rand- phosphamide and TBI, 3 patients (11%) Fludarabine and TBI, omized clinical trials are needed to defi ne the optimal device in Most patients (n=25; 89%) received or continued a secondary this clinical setting, the results of this prospective study support prophylaxis against aspergillosis at time of Allo-SCT: 17 (68%) the wider use of TIAP in patients receiving myeloablative condi- had a prophylaxis with Voriconazole; 3 patients Itraconazole, tioning followed by allogeneic SCT.

S249 P864 From Jan. 2001 to Mar. 2008, the data of 124 consecutive Graft-versus-host disease and fungal infections are the patients with hematological diseases who received allogeneic main outcome predictors after allogeneic stem cell PBSCT were retrospectively reviewed. Prophylactic immu- transplantation using reduced-intensity conditioning noglobulin-G was given biweekly for 3 months, then monthly up regimens to 6 months after allogeneic PBSCT and serum Immunoglobu- F. Patriarca (1), M.l. Battista (1), A. Sperotto (1), M. Medeot (1), lin-G (IgG) and immunoglobulin-M (IgM) levels were checked A. Geromin (1), M. Cerno (1), E. Toffoletti (1), S. Buttignol (1), at 3 monthly for 1 year then yearly. Prophylactic use of immu- C. Savignano (2), C. Rinaldi (2), V. Miotti (2), R. Fanin (1) noglobulin and maintaining high levels of IgG prevented infec- (1)Division of Haematology (Udine, IT); (2)Dpt Transfusion tious complications (p=0.026), also early recovery of humoral Medicine (Udine, IT) immunity represented by high IgM levels reduced infectious complications (p=0.001). The patients with high IgM levels at Objectives: In order to identify prognostic factors, we analyzed 3 months showed lower viral or fungal infection rate. Acute the outcome of 116 patients with haematological malignancies GVHD (grade II-IV) and steroid use affected negatively on IgM who underwent allogeneic SCT with reduced-intensity condi- recovery (OR=0.369, p=0.027). In addition, overall survival was tioninig (RIC) in our Centre between January 2002 and June better in patients with high IgM levels at 3 months compared to 2008. low IgM levels (5-year OS=72% vs. 48%; HR=0.425, p=0.034). Patients and methods: Median age was 56 years (range However, prophylactic IgG administration had no benefi t on sur- 19-69). Haematological diseases were: acute leukemia 28, vival. In conclusion, early humoral immune recovery is related aggressive non-Hodgkin lymphoma 22, indolent lymphoma with favorable transplantation outcomes but prophylactic use of 18, Hodgkin lymphomas 19, multiple myeloma 21, idiopathic immunoglobulin had no benefi t in terms of survival. myelofi brosis 8. Pretreatment included autologous SCT (50) or more than 3 previous chemotherapy regimens (49). Dis- ease status before SCT was: complete remission (41), partial P866 response (26), refractory disease (49). Comorbidity index (CI) Infectious complications after cord blood transplant: was higher than 1 in 57 patients (49%). Conditioning regimens impact on mortality and analysis of risk factors for were mainly based on thiotepa plus cyclophosphamide plus bacterial, fungal and viral infections fl udarabine. Stem cells came from sibling donors (53) or from C. Girmenia, L. Malandruccolo, A.P. Iori, W. Barberi, matched unrelated donors (63). Ninety-seven patients (84%) G.F. Torelli, V. Valle, E. Iannella, B. Lucarelli, M. Screnci, received peripheral blood stem cells (PBSC). GVHD prophy- G. Gentile, A. Micozzi, R. Foà laxis was based on cyclosporine, short-term methotrexate and Azienda Policlinico Umberto I (Rome, IT) ATG in unrelated transplants. Fluconazole was administered as antifungal prophylaxis. We analyzed the infl uence of pre-trans- Objectives: Infections represent a major cause of morbidity and plant and post-transplant variables on TRM and OS by means mortality in cord blood transplant (CBT) recipients. We ana- of the univariate and multivariate analysis. lyzed the impact of infections on mortality and the factors asso- Results: The cumulative incidence of engraftment was 93%. ciated with an increased infectious risk in patients who received The estimated 1- and 3-year TRM were 23% and 26% respec- a CBT at our Center. tively. The estimated 3 year- OS and disease-free-survival Methods: Between 1996 and 2007, 69 consecutive patients with (DFS) were 53% and 48%. Grade II-IV acute GVHD and chronic a high risk hematologic malignancy who underwent CBT were GVHD developed in 35% and 45% of the patients. Twenty-one evaluated. Patients received two types of conditioning regimen patients (18%) presented a proven or probable mycotic infec- (with or without TBI) and the same supportive care. The median tion after SCT. Among pretransplant factors, only CI higher than age of the patients was12 years (range, 1-42 years); 46 were 1 signifi cantly increased TRM and shortened OS, while patient children (age <18 years) and 23 adults. age, disease type and status, donor and stem cell features did Results: Overall, 88.4%, 18.4%, 58% and 11.6% of patients not affect outcome. Among post-transplant factors, develop- developed at least one bacterial, fungal, CMV or EBV infec- ment of grade II-IV acute GVHD or extensive chronic GVHD or tion, respectively. Most infections occurred in the fi rst 100 mycotic infections signifi cantly increased TRM rate and short- days from transplant, in particular 88.8% of bacteremis, 69.2% ened OS. of fungal infections, 95% of the fi rst CMV infections and 37.5% Conclusions: Our experience demonstrated that post-transplant of the fi rst EBV infections were documented during this period. factors such as GVHD and fungal infections were more likely to 77% of H. zoster infections occurred after 1 year post-trans- affect outcome after RIC transplant than pre-transplant features plant at discontinuation of antiviral prophylaxis. No difference of patients, donors and stem cell source. Only pre-transplant in the incidence of all types of infection was observed between patient CI higher 1 was a signifi cant unfavourable outcome children and adults. The 10 year actuarial overall survival is predictor. Therefore, every effort should be made to potentiate 40% (50% in children and 18% in adults). Infections were con- GVHD and infection prevention after RIC transplants. sidered the primary or secondary cause of death in 28.6% of recipients who died. At multivariate analysis, adult age repre- sented the only factor signifi cantly associated with mortality. P865 The occurrence of any infection had no effect on the overall Early recovery of immunoglobulin levels at 3 months after survival. Univariate analysis failed to identify any signifi cant allogeneic peripheral blood stem cell transplantation can risk factor for bacterial infections. At multivariate analysis, the predict favourable transplantation outcomes only signifi cant risk factor for fungal infections was the period J.H. Moon, S.J. Lee, J.G. Kim, Y.S. Chae, S.N. Kim, J.S. Suh, of transplant (CBT performed in the years 2002-2007 were K.S. Lee, S.K. Sohn more frequently complicated by fungal infections compared to Kyungpook National University Hospital (Daegu, KR) CBT performed in the years 1996-2001). Factors signifi cantly associated with an increased risk of CMV and EBV infections Patients undergoing allogeneic peripheral blood stem cell at multivariate analysis were a positive CMV serology of the transplantation (PBSCT) have an increased risk of infectious recipient at transplant and the development of acute GVHD, complications due to secondary hypogammaglobulinemia. Pro- respectively. phylactic use of immunoglobulin has been used for the purpose Conclusions: Our study confi rms the relevance of infections as of decreasing the infection-related morbidity or mortality in allo- a source of severe morbidity in CBT, particularly in the fi rst 100 geneic PBSCT recipients. We studied to determine whether days post-transplant; however, unlike previous experiences, age prophylactic use of immunoglobulin or early immunoglobulin was not associated with an increased risk of any type of infec- recovery could affect transplantation outcomes. tion and infections did not represent the major cause of death.

S250 P867 staphylococcus heamolyticus (%14.3) and all of the gr(-) bacilli Serum vascular endothelial growth factor in adult isolated were pseud. aeroginosa. The stages of bacterial haematological patients with neutropenic fever: growth are depicted in table 1. The major stage for contamina- a prospective comparison with C-reactive protein tion was leukopheresis procedure, followed by preparation of E. Jantunen (1), S. Hämäläinen (2), I. Matinlauri (3), T. Kuittinen cryosolution and thawing. The majority of the MO grown at the (4), I. Koivula (1), A. Juutilainen (1) primary stages were not identifi ed after thawing. Only in 4/30 (1)Kuopio University Hospital (Kuopio, FI); (2)Department of procedures we identifi ed MO in infused PBSC. They did not Medicine (Kuopio, FI); (3)Turku University Central Hospital develop any relevant infectious complications during peritrans- (Turku, FI); (4)Helsinki University Central Hospital (Helsinki, FI) plant period. Conclusion: Collection of autoPBSC and transplantation are Elevated vascular endothelial growth factor (VEGF) concentra- multistage consecutive procedures, which are prone to bacte- tions have been observed in patients with severe sepsis. No rial contamination. The contamination risk during leukopheresis data are available on VEGF kinetics in haematological patients was higher than expected and gr(+) MO in the majority. After with neutropenic fever. We evaluated prospectively serum cryopreservation only in a minority of infusions we detected VEGF and CRP kinetics in 42 adult haematological patients (26 bacterial contamination which did not develop signifi cant clini- males, 16 females, median age 57 years) with fever. Twenty- cal infectious episodes of the same MO. seven patients had received an autologous stem cell transplant (non-Hodgkin lymphoma 13, multiple myeloma 7, Hodgkin lym- phoma 6, chronic lymphocytic leukaemia 1) and 15 patients had received induction chemotherapy for acute myeloid leukaemia. The blood samples were taken at the onset of fever (d 0) and then daily for three days. Time from d0 to peak serum VEGF concentration (mean 1.02 with SE 0.18 d) was shorter that to the peak of CRP concentration (mean 1.93 with SE 0.18 d) (p=0.002). In general VEGF values were low in comparison to P869 previous studies. Patients with severe sepsis (n=5) had higher Monitoring strategy of adenovirus infections in allogeneic VEGF values on d0 (77 pg/ml vs 52 pg/ml, p=0.06) and on d1 stem cell transplant patients (82 pg/ml vs. 56 pg/ml, p=0.05) than patients without severe H. Omar, G. Avetisyan, P. Ljungman sepsis (n=37). No signifi cant differences were observed in Karolinska Institute (Huddinge, SE) median CRP values between patients with and without severe sepsis at any time points analysed. This study shows that also Background: Adenoviruses have emerged as important patho- neutropenic and thrombocytopenic haematological patients gens after allogeneic stem cell transplantation (SCT) with high with fever are capable of producing VEGF during sepsis. High mortality in disseminated disease especially in children. The levels were associated with development of severe sepsis in fi rst 3-6 months after SCT is considered as the most critical patients with neutropenic fever. VEGF might be more rapid indi- period due to severe immuosuppression and poor T cell immune cator for severe sepsis than CRP in haematological patients reconstitution. Monitoring of patients against adenovirus infec- with neutropenic fever. Further studies on VEGF in febrile hae- tions might be a useful strategy to decrease the morbidity and matological patients are warranted. possibly mortality. Aim: To study adenovirus specifi c T-cell responses in adult SCT recipients P868 Patients and methods: 24 allogeneic SCT patients were Risk of bacterial contamination at autologous peripheral included. All patients were adults; age ranged 19-66 years. 23 blood stem cell collection and infusion patients had hematological malignancies. The patients were A. Yilmaz, E. Ayyildiz, M. Bay, A. Azap, G. Gürman, Ö. Arslan, monitored with weekly blood samples by a quantitative adeno- O. Ilhan, M. Arat virus PCR on plasma during the fi rst three months. Adenovi- Ankara Un. Fac. of Med. (Ankara, TR) rus specifi c T cell responses were assessed by ELISPOT after stimulation with adenovirus lysate at 4, 8 and 12 weeks after Background: Peripheral blood stem cells (PBSC) are the major SCT. 20 spots/1 x 106 peripheral blood mononuclear cells were stem cell source for autologous stem cell transplantation. Bac- regarded as a positive result. terial contamination (BC) of the collected PBSC is a major prob- Results: 215 samples were analysed by adenovirus PCR lem, which is seldomly reported. The multi-stage processing of (median 8 samples/patient). All were negative. 59 ELISPOT autoPBSC till infusion incapacitates hazards for BC. There are assays were performed. 20/24 (83%) patients were positive; several published reports about BC but its impact on mortality 53% at 4 weeks, 55% at 8 weeks and 43% at 12 weeks after and morbidity is seldomly shared. In our single center prospec- SCT. There was a tendency for the response to become weaker tive non-controlled study we aimed to determine the source of over time (8 w vs. to 4 w; p=ns; 12 w. vs 8 w; p = .058). Patients contamination at all possible stages and its clinical relevance. who received PBSC grafts were more likely than other patients Patients and Methods: Our center is performing more than 100 to have an adeno T-cell response at 4 weeks (6/10 vs 1/4; p = leukopheresis and SCT per year. Candidates for autoPBSC ns) and 8 weeks after SCT (9/13 vs. 1/5; p=.058). There were transplantation are consecutively entered into this study. We ana- no effects by conditioning intensity, donor type or severity of lyzed 30 completed leukopheresis procedures on 18 patients, acute GVHD. whose median age was 52 y (range, 25-68) with diagnoses of Conclusion: A majority of allo SCT patients had detectable ade- lymphoma and myeloma. One ml. samples were collected into novirus specifi c T cell immune response during the fi rst three the BD Bactec Ped/Plus Aerobic&Anaerobic blood culture bot- months after SCT. tles at the following stages in aspetic conditions: before and after leukopheresis from peripheral vein or from both catheter arms, in collected PBSC, just before cryopreservation from the cryobags, just after thawing at the time of infusion. Bacteriologi- cal examination was performed by BACTEC 9050 blood culture system. Growing microorganisms (MO) were identifi ed using conventional methods and gr(+) crystal ID Kit. Results: We identifi ed MO in 12 PBSC procedures of 10 patients. The majority was gr(+) cocci (%77.7) as expected and gr(-) bacilli (%22.3). Gr(+) MO were staph. epidermidis (%85.7),

S251 P870 4, ALL 3, MDS 2, MM 1; lymphoma 1; median age was 43 yrs No difference in the risk of invasive fungal infections after (range 19-63); 14 were male/9 female; 16 pts conventional con- HLA identical sibling or unrelated donor transplantation ditioning and 7 reduced intensity. 6 pts received a TBI based receiving low-dose ATG-F in the preparative regimen conditioning; 11 pts received ATG and 1 Campath-1H as part of before transplant. Results from a single centre conditioning regimen. Stem cell source was peripheral blood in F. Bonifazi, G. Bandini, S. Usai, E. Colaci, M. Stanzani, 14 pts, bone marrow in 4 and cord blood in 5 pts (at least 4/6 M. Arpinati, N. Vianelli, M.R. Motta, S. Rizzi, M. Baccarani mismatched). Recipients CMV serostatus was positive in all. S. Orsola-Malpighi Hospital (Bologna, IT) CMV Q-PCR was weekly performed from the engraftment and ELISPOT assay was done at day +30,+60,+90 and at the time Polyclonal ATG antibodies incorporated into the preparative of CMV reactivation. Valganciclovir was employed in preemp- regimens have resulted in a reduced incidence of GHVD, but tive treatment. the risk of an increased incidence of IFI could theoretically be Results: 12/23 (52%) patients experienced CMV reactivation expected. We report our results in sibling HLA identical and in within a median time from HSCT of 43 days (range 22-60). The VUD transplants, all receiving low dose ATG Fresenius (Bad median CMV DNA copies was 19249/ml (range 4030-565.000 Homburg, D); a substantial reduction of GVHD, especially the viral copies/ml). CMV reactivation developed among allo IS chronic form, has been observed without an increase in infec- HSCT in 5/11 (45%) vs 7/12 (58%) of alloUD HSCT recipi- tions. From 2001 to 2005, 179 transplants have been performed ents, with no differences in interval and median CMV DNA viral here in patients with hematological malignancies. 77 patients load (42 vs 43 days; 18480/ml vs 20000/ml). ELISPOT assay received VUD transplants and 102 HLA identical sibling trans- performed at +30 days revealed a median value of 52 CMV T plants. The BM was the source of HSC in 50% of the cases, lymphocytes/200.000 cells (range 0-50.000) in all allo HSCT PBSC in 48% and cord blood in 2%. Median age was 40 yrs recipients, 26 in pts reactivating CMV (range 0-338) and 96 (range, 18-61); one third had AML/MDS. One third had early (range 1-50000) in those who did not reactivate. Particularly disease. The preparative regimen was myeloablative in 73% at the time of CMV reactivation, ELISPOT showed 90 CMV T of the cases. GVHD prophylaxis was attempetd with CsA and lympocytes/200.000 cells (range 0-419) in all group, with differ- short term MTX. The VUD patients received, in all cases, ATG- ences between allo IS and allo UD (96 vs 27) and CMV viral Fresenius, usually at a dose of 3-6 mg/kg /day for fi ve days clearance of 21 vs 41 days respectively. (from days -6 to -2, total dose from 15 to 30 mg/kg). All patients Conclusion: CMV-specifi c T-cells response in allo HSCT at high received Fluconazole as prophylxis. There were 11 possible risk of reactivation for CMV recipient serostatus, seems to be cases, 1 probable and 7 proved, of which 5 were autoptic. Can- related to type of donor and T-depletion and appear to predict dida ans Pneumocystic occurred in one case, each, Aspergillus the CMV reactivation rate and the response to therapy. fumigatus in 7 and A. Terreus in 2, one patient having Aspergil- lus + Candida, Main site of involvement was the lung (19), the rest being CNS and para-nasal sinuses(1 each), kidney (2) P872 and myocardium (1). The mean day of onset was + 25; in 11 Treatment of respiratory syncytial virus infection with patients infection occurred during cytopenia, in 8 after cytope- nebulised ribavirin and the humanised monoclonal nia, always associated with high dose steroids (5 cGHVD, 3 antibody palivizumab in eight haemopoietic stem cell no GVHD). 8% of the infections occurred in VUD transplants, transplant recipients 12% in sibling transplants; 67% occurred in advanced disease D.A. Tsitsikas, H. Oakervee, J. Cavenagh, S. Agrawal, and there was no difference between myeloablative and RIC J. Gribben, F. Mattes regimens, 28% and 27%, respectively. Therapy had a mean Barts and the London NHS Trust (London, UK) duration of 30 days (3-120), was with a single agent in 13 pts and multiple drugs in 6(sequential 5, combined 1). Response Objectives: To assess the safety and effi cacy of Palivizumab in to therapy- lyposomal Amphotericin, caspofungin, voricona- haemopoietic stem cell transplant (HSCT) recipients with respi- zole was: failure in 9 cases who died, 4 no responses and 6 ratory syncytial virus (RSV) who received Palivizumab between improvement/resolution. No difference was observed between 2005 and 2007 in our centre. VUD and sibling transplants. Background: RSV represents a common infectious complication Our results underline the prognostic importance of advanced of HSCT, with signifi cant short and long term morbidity. If left disease and steroid use in the risk of developing IFI, and no untreated, the mortality from RSV lower respiratory tract infec- difference between family and VUD transplants. Thus, the use tion (LRTI) approaches 100%. Aerosolized Ribavirin has been of low dose ATG-F doesn’t increase the risk of IFI after trans- reported to improve survival from 0% to 60-70% and increased plantation. further to 80-90% with the addition of high titre RSV IVIg or Pal- ivizumab. Palivizumab is a humanized monoclonal antibody against RSV that has been shown to be safe and well tolerated P871 when used as prophylaxis in high risk infants (prematurity or Cytomegalovirus-specifi c immune reconstitution bronchopumonary dysplasia), leading to a 55% reduction in hos- monitored by ELISPOT assay in allogeneic pitalisation. Data on its use in the setting of HSCT are limited. haematopoietic stem cell transplantation Methods: 8 HSCT recipients (table 1) received aerosolised Rib- C. Nozzoli (1), L. De Risi (2), S. Guidi (1), A. Orsi (2), avirin and a single intravenous dose of 15mg/kg Palivizumab B. Bartolozzi (1), A. Gozzini (1), L. Lombardini (1), within three days of diagnosis. The choice of dose and route of R. Saccardi (1), A. Bosi (1) administration were based on a previous phase I study in HSCT (1)BMT UNIT (Florence, IT); (2)Microbiology (Florence, IT) recipients. The use of Palivizumab was an individual decision of the treating physician in each case. Background: Cytomegalovirus (CMV) infection represents the Results: No side effects, haematological or other organ toxici- most frequent viral complication in patients undergoing alloge- ties were documented for any of the patients. neic hematopoietic stem cell transplantation (allo HSCT). We By polymerase chain reaction (PCR), 6 of the 8 patients investigated the development of CMV–specifi c T-cells reconsti- became RSV negative after treatment. One tested positive 3 tution in adult recipients of allo HSCT. days after treatment and was not repeated subsequently, and Patiens and methods: Since January 2008 23 consecutive the other, showed reduction of the number of RSV infected cells recipients of allo HSCT were monitored for CMV reactivation by by immunofl uorescence but again, was not repeated subse- Q-PCR and for CMV-specifi c T-cell recovery using interferon- quently. Both patients improved clinically and were discharged gamma-enzyme-linked immunospot (ELISPOT) assay. 12 pts from hospital. received an allo HSCT from an unrelated donor (UD) and 11 Two patients died; the fi rst of neutropenic sepsis and progres- from an identical sibling (IS) one. Diagnosis were: AML 12, CML sion of his primary haematological disease. At the time of death

S252 he was negative for RSV by PCR. The other, who had devel- who received anti-thymocyte globulin or T-cell depletion were oped severe RSV LRTI requiring intubation, was negative for excluded. Patients received oral acyclolovir 1,000 mg/day until RSV by PCR after treatment, improved clinically and was even- day 35 after HSCT. Oral valacyclovir 500 mg/day three times tually extubated, but subsequently developed Pseudomonas a week was started on day 36 and continued until 1 year after pneumonia to which he fi nally succumbed. There were no HSCT. Development of VZV reactivation was monitored until 2 deaths attributed to RSV. (table 2) years after HSCT. Conclusion: Our experience supports the existing evidence Results: Forty patients with a median age of 43 years (18 - that Palivizumab is safe in HSCT recipients, and for the limited 61) were enrolled. Three patients had aplastic anemia and number of patients that were treated, there was evidence of the remaining 37 patients had hematological malignancies. viral clearance while no RSV related deaths occurred, suggest- Valacyclovir was well torelated in all but one patient who dis- ing that further studies are warranted in this setting. continued it on day 224 due to thrombocytopenia of unknown cause. Seven patients developed VZV reactivation at a median of 479 days (range 145 - 651 days) after HSCT, with a cumu- lative incidence of 18.5%. With this prophylaxis, visceral dis- semination and serious complications other than post-herpetic neuralgia was completely eliminated. All patients who experi- enced VZV reactivation responded well to the therapeutic dose of valacyclovir or intravenous acyclovir. Two patients who were receiving steroids for acute GVHD developed breakthrough reactivation during the prophylaxis. The other 5 patients devel- oped VZV reactivation after the discontinuation of valacyclovir, four of whom had developed extensive chronic GVHD. As for the evaluation of risk factors, VZV reactivation was more fre- quently observed in patients who developed grade II-IV acute GVHD, but it was not statistically signifi cant (13.1% vs 26.7%, P=0.24). Discussion: Long-term low-dose valacyclovir can be safely and effectively administered for the prevention of VZV-related com- plications after allogeneic HSCT. However, VZV reactivation cannot be completely eliminated and thus close monitoring is still required, especially for patients with GVHD.

P874 Low-dose amphotericin B lipid complex or liposomal amphotericin B prophylaxis of invasive fungal infections in children following stem cell transplantation for haemoglobinopathies J. Gaziev, C. Alfi eri, M. Marziali, A. Isgro, P. Sodani, P. Polchi, M.D. Simone, C. Gallucci, K. Paciaroni, A. Roveda, G. De Angelis, A. Montuoro, G. Lucarelli Mediterranean Institute of Hematology (Rome, IT)

Invasive fungal infection (IFI) is a leading cause of non relapse mortality in allogeneic SCT recipients. This is the fi rst study evaluating low-dose amphotericin B lipid complex (Abelcet) as antifungal prophylaxis in patients undergoing SCT. The pur- pose of this study was to evaluate the safety, tolerability and effi cacy of low-dose Abelcet or Ambisome antifungal prophy- P873 laxis in 87 patients undergoing SCT for thalassemia (n=81) or Long-term low-dose valacyclovir against Varicella-zoster sickle cell disease (n=6): 38 patients in the Ambisome arm and virus reactivation after allogeneic haematopoietic stem 49 patients in the Abelcet arm. The two study arms were bal- cell transplantation anced for baseline demographic characteristics. Culture tests K. Oshima (1), T. Takahashi (2), T. Mori (3), T. Matsuyama (4), for fungal organism were performed before the start of prophy- K. Usuki (5), Y. Asano-Mori (2), F. Nakahara (2), S. Okamoto laxis and, thereafter once weekly and were taken from blood, (3), M. Kurokawa (2), Y. Kanda (1) urine, throat and stool. Blood samples for Aspergillus galacto- (1)Saitama Medical Center, Jichi University (Saitama, JP); mannan and PCR for Aspergillus DNA were taken once a week. (2)University of Tokyo (Tokyo, JP); (3)Keio University School of Patients in both arm received the study drug after SCT from Medicine (Tokyo, JP); (4)Jichi Medical University (Tochigi, JP); day of profound neutropenia (ANC <200) until discharge. In the (5)NTT Kanto Medical Center (Tokyo, JP) Ambisome arm patients started on study drug 1 mg/kg every other day until the fi rst fungal colonization and thereafter they Background: Varicella-zoster virus (VZV) reactivation remains received the drug 1 mg/kg per day. In the Abelcet arm patients a common complication after allogeneic hematopoietic stem received the drug at a dose of 1 mg/kg per day. The median cell transplantation (HSCT). Long-term prophylaxis with low- total dose of the study drugs and their median consumption dose acyclovir against VZV reactivation has been reported to by patients, and median duration of prophylaxis were similar be effective. We carried out a trial of prophylaxis with long- in both treatment arms. Patients in the Ambisome arm had sig- term low-dose valacyclovir, which is a prodrug of acyclovir with nifi cantly more post transplant fungal colonization compared higher bioavailability, against VZV reactivation after allogeneic to the Abelcet arm [15 (44%) vs 10 (21%); p=0.030]. Candida HSCT to evaluate its effi cacy and safety. species comprised the vast majority of isolates. Median time to Methods: Adult patients seropositive for VZV-IgG antibody development of colonization was signifi cantly short in the Ambi- before HSCT were enrolled in this prospective study. Under- some than in the Abelcet arm (8 days vs 20 days; p=0.033). lying diseases, preparative regimen and graft-versus-host dis- Duration of neutropenia was similar in both arms and there was ease (GVHD) prophylaxis were not concerned, but patients not signifi cant difference between colonization and duration

S253 of neutropenia. There was no correlation between disease P876 related covariates such as serum ferritin, liver iron concentra- The role of prophylactic antimicrobials during autologous tion, number of red blood cell transfusion units, risk classes, stem cell transplantation: a single-centre experience and post transplant fungal colonization or IFI in both arms. In B.S. Sohn, D.H. Yoon, S. Kim, D.H. Lee, S.W. Kim, J. Huh, each arm 2 patients had probable or proven IFI. One patient J.S. Lee, C. Suh in the Ambisome arm and 2 patients in the Abelcet arm had Asan Medical Center (Seoul, KR) moderate adverse events as fever and chills. There was not signifi cant difference between the Ambisome and the Abelcet Introduction: The aim of this retrospective study was to inves- arm in any laboratory values measured, including nephrotoxic- tigate the effi cacy of antibiotic prophylaxis during autologous ity and hypokaliemia. In conclusion, low-dose Abelcet could be peripheral stem cell transplantation (ASCT) in patients with successfully used for antifungal prophylaxis in post transplant multiple myeloma (MM) and non-Hodgkin’s lymphoma (NHL). patients. Patients and Methods: We searched Asan Medical Center Reg- istry for NHL and MM. Total 114 cases received antimicrobial prophylaxis consisted of ciprofl oxacin (500mg twice daily p.o.), P875 fl uconazole (100mg twice daily p.o.) and acyclovir (400mg Secondary prophylaxis and pre-emptive antifungal every 8hr p.o.), starting 1 day before HDT (D -1), and contin- therapy are effective in allogeneic transplanted patients ued until neutrophil engraftment: when the absolute neutrophil nursed in highly protective environment count (ANC) reached ≥ 500/mm³ after nadir, or switching to P. Marenco, G. Grillo, G. Bertani, C. Gabutti, V. Mancini, intravenous (IV) broad spectrum antibiotics at the occurrence of M. Turrini, A. Brizio, E. Morra signs of infection; while 118 cases did not receive antimicrobial Ospedale Niguarda Ca’ Granda Milano (Milan, IT) prophylaxis during ASCT. Results: In prophylaxis group, 80 of 114 (70.2%) patients had Introduction: Antifungal treatment is a main issue in haema- experienced febrile episodes at median day +6 after transplan- tological patients (pts) due to costs in terms of toxicity and tation with a statistically signifi cant difference (P<0.001). In economical resources. The reported incidence of invasive no-prophylaxis group, 111 of 118 (94.1%) patients had expe- fungal infections (IFI) varies widely between BMT Units, rienced at median day +5. Documented infection occurred in due to the variability in isolation and other preventive pro- 14 of 114 (12.3%) patients in prophylaxis group, and 16 of 118 cedures. These range from use of conventional rooms to (13.6%) patients in no-prophylaxis group (P=0.846). In these highly protective environment as Laminar Air Flow (LAF) patients, the positive blood culture was seen in 12 (10.5%) rooms which could be considered a real primary antifungal of 114 patients in prophylaxis group, and 12 (10.7%) of 118 prophylaxis. patients in no-prophylaxis group (P=1.000). Documented viral Materials: In our BMT Unit all allogeneic transplant patients are infection or reactivation was not observed in prophylaxis group, managed in highly protective environment, usually LAF, with but observed in 4 patients of no-prophylaxis group. Both groups highly sterile nursing procedures and receive fl uconazole iv showed no invasive fungal infection or serious adverse event after transplant. Patients with previous possible, probable, or during ASCT. The day of infection resolved was a median day proven fungal infection receive appropriate secondary proph- +15 (range, 3-29) in prophylaxis group and day +14 (range, ylaxis starting from aplasia. Empirical antifungal treatment is 2-70) in no-prophylaxis group (P=0.945). The duration of anti- not routinely used. Galactomannan Antigenemia is periodically microbial treatment was median 10 days both in prophylaxis checked as chest X-ray. CT scan is evaluated only for selected group and in no-prophylaxis group (P=0.565). cases to minimise patient contamination. Conclusions: In our experience, the antimicrobial prophylaxis We reviewed the 178 allogeneic transplants performed from seems to decrease the incidence of febrile episodes during January 1997 to December 2007 and evaluated the propor- ASCT, but seems to have no benefi cial effect on reducing infec- tion of patients treated with prophylactic, empirical, or targeted tious complications. The antimicrobial prophylaxis of our study therapy. did not show the difference in the detection of causative organ- Median age was 42 y (range: 17-65); stem cell source was ism as an infective agent, duration of antimicrobial therapy and unrelated donor in 58 pts; reduced intensity conditioning (RIC) hospitalization between two groups. was used in 34 cases. GVHD prophylaxis: CSPA and short term MTX. No T-depletion or ATG have been used. P877 Results: 34/178 patients (19%) received systemic anti-mould Rota- and calicivirus infections following allogeneic treatment. Of whom: haematopoetic stem cell transplantation • 19 for secondary prophylaxis for previous proven (7), prob- K. Kállay, J. Sinkó, G. Benyó, Á. Tóth, V. Goda, G. Kriván able (5) or possible (7) infection (only 1/19 subsequently St. István and St. László Hospital (Budapest, HU) developed a possible infection). • 1 for primary prophylaxis (due to fl uconazole intolerance). Objectives: Rota- and calicivirus infections represent a chal- • 9 for empirical antifungal therapy, started on the basis of a lenge in differential diagnosis. The authors retrospectively FUO lasting >5 days and additional clinical factors (reported analyzed the incidence and outcome of enteric viral infections in table 1). No subsequent documentation of IFI in this in allogeneic transplant patients during a period of one year cohort. (between 01/11/2007 and 31/10/2008) at their centre. • 5 for preemptive (3 cases) or targeted (2 cases of non Results: During the observation period 23 allogeneic hemato- Albicans Candidemia) therapy. poetic stem cell transplantations were performed in 19 patients. One of these died for Candida septic shock. In case of enteral symptoms bacterial cultures, Clostridium dif- Conclusions: fi cile -toxin determinations and viral tests were performed to 1) Secondary prophylaxis makes allogeneic transplantation detect infectious origin. From the 8 symptomatic patients an possible in pts with previous IFI. underlying viral infection could only be identifi ed in 4 cases (2 2) in a highly protective environment a very selective risk- rota- and 2 calicivirus). In the remaining 4 patients symptoms adjusted empirical antifungal treatment seems safe and were related to graft versus host disease (GvHD), mucositis, reduces toxicity and economic burden without increasing IFI CMV-colitis and malabsorption. The median observation time related TRM (0,6%). These results, to be confi rmed by larger was 92 days (53-265). We experienced two major complica- prospective studies, suggest a signifi cant role of air treatment tions: one sepsis caused by an ESBL producing E. coli strain as primary antifungal prophylaxis in HSCT wards. and one enteropathic acrodermatitis due to the excessive loss of zink. In one of the rota-positive patients with progressive symp- toms a CMV-colitis has also been confi rmed by a gut biopsy.

S254 Two patients have died but no fatal outcome was related to the P879 enteral infection (relapse: 1, pulmonary infection: 1). Level of Catheter-related infections in tunnelled central immunosuppression could be decreased in two cases resulting venous devices in haematologic malignancies and in the cessation of enteral symptoms. haematopoietic stem cell transplantation patients. Discussion: Rota- and calicivirus infections are a maior threat to A single-centre experience this patient population, however, not much has been published E. Carrillo Cruz, J. Falantes González, J. González Campos, on this issue in the transplant literature so far. The long-lasting M. Ruiz Seixas, A. Urbano Ispizua, I. Espigado Tocino damage of the intestinal barrier can facilitate bacterial invasion Hospital Virgen del Rocio (Sevilla, ES) and can lead to sepsis. Patients may require very long hospital care for enteritis thus becoming candidates for further nosoco- Background: Tunnelled central venous catheters (CVC) are mial infections. There is no antiinfective treatment for rota- and routinely used in haematological patients. CVC include total calicivirus but decreasing or, - if possible -, stopping immuno- implantable devices (ports) and Hickman catheter. Catheter- suppression can help. related bloodstreams infection (CRBI) is reported approximately Conclusions: When evaluating patients presenting with enteri- as 1.6 episodes per 1000 catheter days in tunneled CVC. tis after HSCT a rota- or calicivirus infection should always be Objectives: Prospectively analyse incidence, morbidity and considered. Transplant centers should be able to provide long mortality related to tunnelled CVC in hematologic malignancies hospital care for affected individuals. Ceasing immunosuppres- and hematopoietic stem cell transplant (HSCT) patients. sion can be benefi cial. Patients and methods: From january 2008, 62 CVC were placed in 57 pts (ports devices, n=19 and Hickman, n=43). 35 pts planned for HSCT and 27 pts for intensive chemotherapy. P878 Observation period included chemotherapy treatment, hospital Preliminary monocentric survey on the prevalence of HBV admissions and as outpatien follow-up. seropositivity in HSCT recipients Results: Infection episodes occured in 26 CVC (41,9%): 17 P. Usardi (1), C. Annaloro (1), M. Viganò (2), P. Lampertico (2), were local (16 in Hickman and 1 in ports) and 10 episodes of F. Onida (2), E. Tagliaferri (1), A. Della Volpe (1), G. Lambertenghi CRBI (3 in ports and 7 in Hickman) for a global systemic cath- Deliliers (2) eter-related infection of 16,1% (1,5 episodes of CRBI per 1000 (1)Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli catheter days: 1,15 per 1000 catheter days in ports and 1,7 in e Regina Elena (Milan, IT); (2)Fondazione IRCCS Ospedale Hickman). 17/27 episodes of CVC related infection occured in Maggiore Policlinico, Mangiagalli e Regina Elena - University of recipients of HSCT (10 autoSCT and 7 alloSCT) and 10 occured Milan (Milan, IT) in patients after intensive treatment (acute leukemia=4, lym- phoma=5 and multiple myeloma=1). 60% of CRBI were due to Hepatitis B (HBV) infection prevalence is frequently reported gram-positive and 40% to gram-negative bacteria. In patients as increased among hematological patients. This enabled us with clinical suspected CVC related infection, microorganisms to start reviewing HBV serology in our HSCT recipients. Mov- isolated in blood cultures obtained from CVC were: bacillus ing back over the last four years we collected a series of 238 cereus (n=3), enterococcus faecalis (n=2), pseudomona aeru- consecutive HSCT recipients (male 129, female 109, median ginosa (n=2), staphilococcus aureus (n=2), staphilococcus epi- age 50 years, range 18-70, autologous 151, allogeneic 87). The dermidis (n=1), serratia marcenses (n=1), stenotrophomona baseline diagnoses were lymphoma in 96, acute leukemia in maltophilia (n=1), candida kruzei (n=1). All patients recieved 59, CLL and myeloproliferative disorder in six each, multiple empirical and specifi c antibiotic directed by the in vitro suscep- myeloma in 47, PNH in 5 and autoimmune disease (AD) in 19 tibility of the isolated pathogen. Infectious events were solved cases. Five patients were HBSAg positive, 54 had isolated anti- in 9/10 patients. 16 catheters were removed. One patient with HBS antibodies (14 vaccinated), 38 were simultaneously anti- infection by candida kruzei died with systemic dissemination in HBS and anti-HBC positive, and eight had anti-HBC without spite of treating with properly antifungical drugs after 23 days anti-HBS antibodies. Anti-hepatitis C (HCV) antibodies were from the infectious diagnosis. observed in fi ve patients. As a whole, 91/224 not vaccinated Conclusion: In this cohort, incidence of CRBI was 1,5 per 1000 patients (40.62%) showed some kind of HBV positivity. Of 19 catheter days in tunnelled CVC with a signifi cant predomi- patients with AD, three were vaccinated, three had isolated nance of gram-positives microorganisms. It was observed a anti-HBS and one isolated anti anti-HBC positivity. Therefore, higher incidence of systemic infection in Hickman CVC than in 87/208 not vaccinated neoplastic patients (41.82%) showed port devices. Global mortality was 1.7%, and 10% of CRBI. some kind of anti-HBV positivity, with 46/208 having anti-HBC There is a need for further prospective studies to determinate antibodies without HBSAg positivity (22.11%). Even dividing the more exactly the impact of the CVC infections in this kind of series by ten-year age cohorts, the prevalence of anti-HBC pos- population. itivity was higher in comparison with the reference populations in European prevalence studies, showing median values below 10% and 16-17% in the worst age cohorts; no reliable compari- P880 sons are available for isolated anti-HBS positivity. Hematologic High effi cacy and low toxicity of short-course oral patients have an up to 40% rate of HBV positivity, including valganciclovir given as one-week BID treatment and both HBSAg and anti-HBS/anti-HBC. Nevertheless, a deeper one-week QD maintenance as pre-emptive therapy for understanding is hindered by the quality of data in the refer- post-HSCT CMV infection ence populations, since prevalence study either report blood A.J.M. Saleh, F. Al-Mohareb, F. Al-Rabiah, N. Chaudhri, donors series, a somewhat selected population, or encompass F. Al-Sharif, H. Al-Zahrani, S. Mohamed, K. Al-Anazi, M. Patel, relatively small series. The imbalance between general popula- W. Rasheed, A. Elghazaly, M. Bakr, S. Ahmed, A. Nassar, tion and hematologic patients is a persistent fi nding over time, F. Hussain, Z. Nurgat, H. Al-Abdely, M. Aljurf despite the evolving pattern of HBV diffusion and the increas- King Faisal Specialist Hospital & R C (Riyadh, SA) ing proportion of European population accounted for by people coming from countries at high HBV endemics. The contrast is Introduction: Cytomegalovirus(CMV) infection is one of the lead- somewhat surprising between the interest in the relationships ing infectious complications post-allogeneic hemopoietic stem between HCV and lymphomas, and the matter of HBV infec- cell transplantation (HSCT) observed in 60%-70% of cases in tion in hematology, mainly focusing on the prevention of HBV the fi rst 3 months. CMV seropositivity in East Mediterranean complications. The question may be raised as to whether HBV and certain Asian countries reported to be close to a 100%, infection should also be considered as a risk factor in the devel- hence, the need for a successful pre-emptive treatment strat- opment of hematologic neoplasms. egy that has low toxicity. Extended treatment with Ganciclovir has been shown to be associated with increase toxicity of late

S255 CMV infection and also may interfere with the anti-CMV T-cell removal of the catheter occurred only in one pt. Infections were specifi c immunity. Oral Valganciclovir(VGC) is monovalyl ester the most relevant late complication. Two out of 24 pts presented of Ganciclovir with highly improved bioavailability. a catheter related bloodstream infection (CRBSI). In both cases Materials and Methods: We have treated 47 patients as out- cultures of blood and catheter samples documented a concord- patient follow-up after discharge with VGC 900mg BID for 1 ant growth of the same micro-organism, Staphylococcus Epi- week. Those who were antigenemia negative after one week, dermidis. For this reason PICC was removed in one pts. The received 900mg daily (QD) for another week then treatment infection rate of 8% equates to 4.0 infections per 1000 PICC- was discontinued. Those who were positive after one week, days. Twenty of PICCs (83%) were removed due to comple- continued on BID schedule for another week and changed to tion of therapy, 1 PICC (4%) was removed for CRBSI, 1(4%) QD schedule once they converted to antigenemia negativity. for fever of unknow origin (FUO) and 2 out 24 (8%) for other Those remained positive after 2 weeks of treatment, consid- causes (accidental removal and malfunctioning). In our expe- ered refractory and offered alternative treatment. rience PICCs are associated with a rate of CRBSI similar to Results: From 1/2004 to 12/2007 a total of 47 patients under- conventional central venous catheter but with a good pts’ com- went HSCT received preemptive treatment with VGC. All pliance and reduced risk for early complications. donors and 44 of the recipients were seropositive for CMV. Donor source was: bone marrow in 30, peripheral blood in 13 and cord blood in 4 patients. Preparative regimens - Myeloabla- P882 tive in 33 and reduced intensity in 14 patients. Disease pattern - Frequency and mains causes of the febrile episodes neoplastic in 34 and non-neoplastic in 13 patients. Twenty nine during myeloablative chemotherapy neutropenia induced (65%) patients had graft-versus-host disease (GVHD) requir- after bone marrow transplant ing steroid therapy; additional GVHD treatments were added in A. Talbi, R. Ahmed Nacer, M. Benakli, R. Belhadj, F. Mehdid, steroid refractory cases. M. Baazizi, N. Rahmoune, K. Saidani, D. Ait Ouali, S. Madene, Sixty one episodes of CMV infection (defi ned by positive anti- S. Abderrahmani, R.M. Hamladji genemia) were observed in the 47 patients. Antigenemia var- Pierre and Marie Curie Center (Algiers, DZ) ied from 1 to 700 infected cells/slide. After fi rst week, complete response(CR) was observed in 92% episodes, 5% of the epi- Introduction: The fever is a call symptom of making an infection sodes required 2 weeks treatment to achieve CR and 3% epi- during the chemotherapy (CT) induced neutropenia. The infec- sodes were refractory required alternative therapy. WBC count tions are mostly bacterial and fungal or due to Herpes virus. We prior to therapy was 1.75 to 11.0X109/L. G-CSF was required in estimate, in this study, the frequency and the mains causes of 8 episodes ( range 1 to 5 doses). No patients had interruption the febril episodes (FE) during myeloablative CT neutropenia of therapy. induced in 762 patients (pts). Conclusions: Treatment of CMV infection with “short course Material and methods: from April 1998 to December 2006, 777 oral Valganciclovir” is highly effective with minimal toxicity, very myeloablative stem cell tranplantation for hematologic disease convenient to the patient and has the potential to make a sig- were realized (allografts: 541, autografts: 236). Condition- nifi cant saving in administrative and labor cost. ing regimen or intensifi cation were based on CT only. In our study, 762 aplastic episodes after CT are observed (allograft: 533, autografts: 229) for 590 pts with hematologic malignant P881 diseases and 172 pts with no malignant diseases; median age Peripherally inserted central venous catethers in the 31 years (3-65); sex ratio M/F 1,3. Infectious prophylaxis con- management of autologous haematopoietic stem cell sisted of isolation of the pt (Flow laminar for allograft), a gut transplantation decontamination with sterile food, strict aseptia;Aciclovir and S. Bellesi, G. De Pascale, P. Chiusolo, G. Scoppettuolo, Fluconazole when mucositis mucosae. All FE are investigated M. Pittiruti, F. Sorà, L. Laurenti, G. Leone, S. Sica clinically and by lung radiology, blood cultures (on peripheral Università Cattolica S. Cuore (Rome, IT) blood and on central venous catheter), bacteriological study of the urines, stool parasitology, sampling according to the obvi- Patients (pts) undergoing haematopoietic stem cell transplan- ous home. The empirical anti-infectious treatment contained 4 tation (HSCT) require a reliable venous access. Peripherally lines which follow each other: Piperacilline-Netromycin; Fortum- inserted central venous catheters (PICC) offer certain advan- Amikacin-Vancomycin; Tienam-Quinolon-Vancomycin; addition tages over other forms of long-term venous central access. of an antifungal after 7 days of resistant fever to anti bacterial They are inserted via peripheral veins such as cephalic, basilic treatment. or brachial vein without general anaesthetic or sedation and Results: all pts presented neutropenia grade 4 (OMS) with a surgical procedure is not required. The risk of pneumotho- median duration 11 days (1-64). One thousand four hundred rax and haemothorax from PICC insertion is minimal. They are thirty one FE were observed in 759 pts (99,6%) with median also easy to remove and less expensive than central venous episode/pt 1,9. Infectious clinically documented in 391 FE catheters. (27,3%) with several localizations (central venous catheter: A total of 24 PICC lines (single-lumen silicone catheter with Gro- 119, gut : 137, lung: 55, cutaneous 58, others: 32). Infectious shong valve, 4 Frx60cm, 18Ga.lumen) were inserted between microbiologically documented (which 131 with clinical localiza- May and November 2008 by expert surgeon under ultrasound tion) observed in 263 FE (18, 4%): blood cultures: 248, ECBU: guidance bedside in 24 pts, undergoing high dose chemo- 11, stool: 3, vaginal: 1; dominated by cocci gram + (n=185) and therapy followed by autologous SCT for onco-haematological bacillus gram-(n=76), Candidose (n:2). The FE of unknown ori- malignancies. Pts’ characteristics were: M/F 15/8, median age gin (FUO) represent around 63.5%. 237 pts (31%) required an 48 years (range 19-62). Underlying disease were: AML 2, MM anti infectious treatment beyond the second line. The mortality 7, NHL 9, HD 3, POEMS 1 and germ cell tumours 2. Chest bound to an infectious cause is 4.7% (36 pts). radiograph was routinely performed in all pts. At insertion pts’ Conclusion: in this study FE of unknown origin (FUO) represent disconfort was minimal. No early complication as haematoma around 63.5% accordingly to the data of the literature (60-70%) and pneumothorax were observed. PICCs were in place for and also the predominance of bacterial cocci gram positive. an inclusive period of 468 days (median time 19 days, range 6-32). Three out 24 of PICCs (12.5%) were removed due to bad positioning and for this reason inserted again. Six out of 24 pts (25%) presented a transient malfunction during stem cell infusion because PICCs resulted not adequate despite the low hematocrit of stem cell product infused. No pts developed clinically evident catheter-relater thrombosis (CRT). Accidental

S256 P883 1– MDS. 5 were with relapsed or refractory disease and 1 in Cytomegalovirus DNAemia and treatment following 3rd complete remission (CR). Median age was 29.5 years. allogeneic stem cell transplantation with focus on 5 patients underwent previous SCT (2 allo, 1 haplo and 2 auto) long-term outcome and 3 patients had a 2nd transplant (2 haplo, 1 allo) following/ J. Lindahl during diagnosis of zygomycosis. The infection involved lung Infectious Diseases (Göteborg, SE) -1, maxillary bone – 2, mandibular bone –1, oral mucosa – 1 and esophagus – 1. Diagnosis was established by histology Background: The aims were to describe the incidence of CMV (5/6) and microbiology (6/6). Treatment was combined surgi- infection, CMV disease, CMV related death and the prognostic cal and antifungal in 3/6. Posaconazole was given to 4 and factors associated with long-term outcome after allogeneic hae- isavuconazole to 2 patients. Three patients recovered from matopoetic stem cell transplantation (SCT). the infection and 3 died from progression of underlying dis- Methods: Cases of proven and probable CMV infection and eases with evidence of controlled fungal infection. None of the disease diagnosed among 97 allogeneic SCT recipients at the patients died from progressive fungal infection. Retrospective Sahlgrenska University Hospital, Gothenburg, Sweden from analysis revealed antecedent neutropenia of > 2-week dura- January 1997 through December 2001 were included. Patient tion in 5 of 6, prolonged steroid use in 3 of 6 and prior use of data were collected by retrospective clinical chart review. voriconazole in 5 of 6 patients. Infection rate was 0.17% of Results: CMV DNAemia was detected among 60 patients at a 2904 treatment days versus 0.03% of 3319 treatment days on mean of 45 days after SCT. Three patients had proven CMV fl uconazole. disease. Three additional patients had probable disease, but In conclusion: Previous exposure to voriconazole may increase adequate laboratory samples were not taken. Two out of the the incidence of the infection and should raise suspicion in the 6 patients (one proven, one probable) died in severe CMV dis- proper context. Use of new antifungal treatment with good anti- ease even though CMV was treated promptly. In one additional zygomycosis activity enables managing this serious infection patient, CMV was considered to contribute to the patient’s death. even in the transplantation setting. Direct CMV related mortality was 2%. Fifty patients (51%) were treated in a total of 93 treatment episodes, for proven or suspected CMV infection. Treatment was started at a mean of 104 days P885 after transplantation and many patients developed repeated epi- Invasive fungal infection in patients after allogeneic sodes of CMV reactivation. Our regimen of therapy was short (21 haematopoietic stem cell transplantation. A single-centre days). The overall one-year survival was 76% and the fi ve-year experience survival 56%. In our study, the CMV serology of donor and recipi- N. Zubarovskaya, Y. Vasilieva, E. Semenova, N. Stancheva, ent and the conditioning regimens are related to CMV infection, E. Morozova, N. Klimko, A. Chukhlovin, B. Afanasyev CMV treatment, CMV disease and mortality. Risk factors inde- SPb State I. Pavlov Medical University (St. Petersburg, RU) pendently associated with CMV morbidity and mortality includes positive serostatus for CMV of the recipients before SCT, acute Invasive fungal infection (IFI) is a common cause of mortality graft-versus-host disease (GVHD), cortisone treatment and older in patients undergoing allogenic hematopoietic stem cell trans- age. Receiving ganciclovir/foscarnet was independently associ- plantation (allo-HSCT). ated with protection from CMV-related death. Aim: to evaluate the incidence of IFI, to determine risk factors Conclusions: There has been a signifi cant decrease in morbid- of IFI and to study their infl uence to overall survival (OS) in ity and mortality in patients related to CMV disease following patients (pts) undergoing allo-HSCT. allogeneic SCT in the past ten years due to CMV surveillance Patients and methods: 221 pts. after allo-HSCT were included and early antiviral treatment. Thus, only few patients develop from 2000 till July 2008. Age:1-66 (median 21 y.o.). ALL, AML- CMV disease and a decreased number of cases with CMV 150 pts, CML, CMF, CLL - 27 pts, AA- 11 pts, congenital dis- viremia are observed. orders-8pts, NHL/HD-17pts, MDS-6pts, solid tumors-3pts underwent myeloablative conditioning – 86pts and non-myelo- ablative conditioning (RIC) -135pts followed by alloHSCT. At P884 the moment of alloHSCT 134 pts were in CR, 87 pts -in relapse. Emergence of uncommon infection in bone marrow Donor: MRD- 77pts, MUD- 135pts, haploidentical–9pts. transplantation: the modern Trojan horse? Sources of HSC:PBSC-142pts, BM-67pts, BM+PBSC- 12pts. T. Zuckerman (1), N. Benyamini (2), N. Haddad (2), R. Fineman Median CD34± 8 x 106/kg. Acute GVHD prophylaxis: CsA or (2), I. Avivi (1), E. Sabo (2), J.M. Rowe (1), I. Oren (1) tacrolimus and short course of MTX or MMF. (1)Rambam Medical Center and Technion (Haifa, IL); Results: The incidence of IFI before D+100 was 27%, after (2)Rambam Medical Center (Haifa, IL) D+100- 36%. Possible invasive aspergillosis (IA) was detected in 27 pts, probable IA- 34 pts, probable IA and Cryptococco- Background: Invasive fungal infections are a signifi cant cause sis-1pts, proven wasn’t diagnosed, disseminated candidosis- 2 of morbidity and mortality in recipients of hematopoietic stem pts(c. crusei- 1 pts), candidemia with c. parapsilosis- 2 pts The cell transplant (HSCT). Predisposing factors include: prolonged onset of IFI was on D+1-940 (median D+86). The incidence neutropenia, mucositis, graft-versus-host disease (GVHD), pro- of IFI under prophylaxis by intraconasole was 21%(4/19), fl u- longed immune suppression and steroid use. Zygomycosis is a conasole 31%(48/155), voriconasole 43%(16/37). In pts who rare infection with increasing frequency and a high fatality rate, received caspofungin(0/4), and caspofungin+posaconasole( affecting both immunocompetent and immunocompromised 0/1) IFI wasn’t detected. In multivariable analyses the follow- patients. An increase in zygomycosis infection among HSCT ing risk factors were revealed: age greater 10 years(RR=1.2; patients has been recently noted. Presented herein are our 95%CI, 1.01-1.6), usage of ATG in conditioning in pts up 21 yo experience and possible explanations for the increased inci- (RR=0.7; 95% CI, 0,59-0,88, Ð<0,05), mucositis I-IV(RR=2.1; dence of the infection. 95% CI, 1.2-3.8), chGVHD in patients under 21yo(RR= 2.1; Patients and methods: Analysis of the prevalence of zygomyco- 95% CI, 1.3 -3.4, Ð<0,05). Conditioning, time of engraftment, sis infection was performed between 09/2007 and 09/2008 at stage of the diseases and sources of HSC weren’t independent our department. During this period 50 new patients were treated risk factors for IFI. In pts under 21 years synergistic effect of for AML and 10 for ALL. 101 patients underwent stem cell trans- following factors was detected: relapse, myeloablative condi- plantation (56 – auto and 45 allo). Antifungal prophylaxis con- tioning, BM as source of HSC (RR=0.4; 95% CI, 0,21-0, 76, sisted of fl uconazole. Voriconazole was given to patients with Ð<0,05). In pts up 21 years with MRD after RIC and source suspected or proven aspergillus infection. of HCS PBSC the incidence of IFI was lower (RR=1.59; 95% Results: During this period 6 (4%) patients with zygomycosis CI, 1.01 - 2.51, Ð<0,05). OS in pts undergoing HSCT with IFI infection were identifi ed; 4 were diagnosed with AML, 1– ALL, versus without IFI is 25%vs53% (p<0,005).

S257 Conclusion: The rates of IFI after HSCT is high, it impairs OS. In defi ned antifungal prophylaxis period. Unit costs were obtained spite of usage different anti-fungal drugs there are no adequate from the Hospital Pharmacy and the Soikos database, and are regimens of IFI prophylaxis in MRD or MUD allo-HSCT. expressed in Euros 2008. More patients in the POSA group were T-cell depleted (10 vs 0; p<.01), had a reduced intensity conditioning (19 vs 6; p=.03), and an unrelated donor graft (10 P886 vs 2; p=.08). Otherwise, age (52, 20-68), sex (58% men), dis- The utility of screening swabs in allogeneic ease (30 MDS-AML, 6 lymphoid, 4 CML, 3 ALL), days to neu- haematopoeitic stem cell transplant recipients: trophil engraftment (17, 13-33), and incidence of acute GVHD a retrospective evaluation (30.2%) were similar in both groups. ITRA patients received oral S. Ager, N. Young, H. Inns, G. Cook, M. Gilleece, S. Liebersbach, suspension or IV drug based on their tolerance. POSA patients G. Shenton, T. Collyns had no problems with oral compliance or toxicity. Patients in LTH NHS Trust (Leeds, UK) the POSA group had a lower incidence of proven or probable IFI (0 vs 13%; p=.06), a higher probability of IFI-free survival Patients undergoing allogeneic haematopoeitic stem cell (85% vs 56%; p=.01) and a trend towards an improved over- transplantation at Leeds Teaching Hospitals (LTH) are regu- all survival (85% vs 63%; p=.09) compared with ITRA patients. larly screened during their in-patient stay with weekly swabs Total cost per patient up to day +100 were €46,562 in the POSA of nose, mouth and perineum for the presence of certain anti- group (3% conditioning costs, 67% hospitalization and labora- biotic-resistant pathogens. Urine and faeces are also cultured tory tests costs, 20% antifungal drug costs and 10% other drug and Hickman line exit sites are swabbed if clinically indicated. costs) and €45,079 in the ITRA group (6% conditioning costs, We analysed the results of these screens to assess their rele- 73% hospitalization and laboratory tests costs, 11% antifungal vance in predicting any subsequent infections for each patient. drug costs and 10% other drug costs). Incremental cost-effec- We also looked for any organisms that represented a noso- tiveness of POSA vs ITRA per IFI avoided was €11.858. In con- comial infection transmission risk or required a change in the clusion, our experience shows that in addition to neutropenia standard empirical treatment regime for febrile neutropenia at and GVHD, as established by the registration trials, POSA may LTH (piperacillin-Tazobactam + an aminoglycoside). be superior to ITRA as primary antifungal prophylaxis during Between January 2007 and December 2007, 26 adult patients the early high-risk neutropenic period after alloSCT in terms of (age 18 – 57 years) and 11 paediatric patients (age 6 months clinical effi cacy and cost-effectiveness. In the absence of ran- – 16 years) underwent allogeneic haematopoeitic stem cell domized trials with POSA in this indication, our single-centre transplantation (19 peripheral blood stem cell, 14 bone mar- study provides supporting evidence for the use of POSA for row, 4 cord blood). Four adults and 2 paediatric patients had antifungal prophylaxis in this important clinical setting. organisms identifi ed in screening samples that represented an infection control hazard: 2 patients (1 adult and 1 paediatric) had a Vancomycin-Resistant Enterococcus (VRE) isolated in P888 pre-transplant perineal swabs, 1 paediatric patient had a VRE in Is serological or PCR positivity of Toxoplasma gondii in a perineal swab taken 24 days post-transplant, 2 patients had donor or recipient the reason for haematopoietic stem cell a ciprofl oxacin-resistant, aminoglycoside-resistant Escherichia transplantation delay? coli (E Coli) in pre-transplant perineal swabs and 1 patient had L. Krol, M. Matulova, P. Hubacek, P. Keslova, R. Formankova, a gentamicin-resistant, ciprofl oxacin-sensitive E coli present in P. Sedlacek a urine sample. None of these patients developed subsequent Charles University 2nd Medical School (Prague, CZ) bacteraemias with the organism. Two patients developed bacter- aemias with ‘infection control hazard’ organisms in the 4- month T. gondii is obligate intracellular parasite causing infection period post-transplantation, one with a gentamicin-resistant known as toxoplasmosis. In immunocompetent host it causes Enterobacter cloacae and one with an Extended-spectrum beta- an asymptomatic infection or fever with lymphadenopathy. lactamase-positive, gentamicin-resistant Klebsiella oxytoca, Primary infection leads to latency and development of tissue neither of which had been identifi ed on screening samples. cysts in muscles and other organs. Most frequently, humans These results suggest that screening samples have little pre- become infected by ingesting meal contaminated with oocysts dictive usefulness for the development of subsequent infections from infected cat faeces. The other ways are transplacentar in this patient group. transmission or via infected blood products or hematopoietic stem cells from donor with primary infection (seroprevalence of IgM antibodies). T. gondii causes an infection with fulminant P887 course or end organ disease (central nervous system, lungs, Posaconazole has superior clinical effi cacy and liver or myocard) in immunocompromised host. Encephalitis is cost-effectiveness to itraconazole for antifungal the most common manifestation of toxoplasmosis in immuno- prophylaxis in allogeneic stem cell transplant recipients: compromised patient. Typical symptoms are fever, coma and a single-centre experience general seizures based on necroses fi lled with tissue cysts R.F. Duarte (1), B. Patiño (1), C. Muñoz (1), M. Arnan (1), in central nervous system. Mortality in immunocompromised T. Peralta (1), C. Gudiol (1), I. Sánchez-Ortega (1), patients is 60 – 90%. High mortality is caused by non-specifi c R. Parody (1), A. Clopés (1), J.L. López-Belmonte (2), clinical signs leading to late diagnosis assessment. Although F.J. Sabater (2), A. Fernández de Sevilla (1) toxoplasmosis is common in patients with advanced AIDS, in (1)Institut Catalá d’Oncologia (Barcelona, ES); (2)Health hematopoietic stem cell transplant (HSCT) patients it is rare but Economics Unit, Schering-Plough España (Madrid, ES) often fatal complication. Serological positivity of T. gondii in allogeneic HSCT is routinely Posaconazole (POSA) has shown to be superior to fl ucona- tested in donor only. In our department we have observed IgM zole and/or itraconazole (ITRA) as antifungal prophylaxis seropositivity in 3 patients and 1 donor pre-HSCT. RT-PCR was in patients with prolonged neutropenia or GVHD. Here, we performed in all cases. RT-PCR provides specifi c evidence of present our experience with POSA prophylaxis during the neu- tachyzoites in examined material such as peripheral blood or tropenic phase post-allogeneic stem cell transplant (alloSCT). stem cell graft. Detection limit of RT-PCR is 30 tachyzoites/ Forty three consecutive alloSCT recipients with no prior history 1mL, sensitivity 30% and specifi city over 90%. of invasive fungal infection (IFI) received either POSA (n=27; Preventive administration of clindamycine was provided in all 200mg/8h oral; from May 2007) or ITRA (ITRA; n=16; oral patients during neutropenia and after engraftment patients and/or iv; up to May 2007) for primary antifungal prophylaxis. received TMP/SMZ three days a week in a long term. We have Clinical effi cacy and total costs (including medical resource not seen the reactivation of T. gondii in our cohort yet. Median use and drugs) were assessed for 100 days post-alloSCT, our follow up is 34.5 months (3-67 months). All patients are alive.

S258 The most common mechanism of toxoplasmosis development P890 in HSCT patient is latent infection reactivation. Transmission Use of ciprofl oxacin to prevent bacterial infection in of T. gondii via infected blood or stem cells is less frequent. patients receiving high-dose chemotherapy and autolo- Preemptive administration of antibiotics reduces risk of reacti- gous stem cell transplantation for multiple myeloma, vation or transmission. RT-PCR monitoring in peripheral blood AML, and lymphoma/solid tumours is warranted in high risk patients. The potential risks of infection E. Bauernschmitt, T. Szislo, C. Peschel, H. Menzel development in patient screened and prophylactically treated Klinikum rechts der Isar (Munich, DE) highly positively outweigh the negative impact of HSCT delay. Supported by VZ MZ CR 64203. Background: Antimicrobial prophylaxis with ciprofl oxacin (cipro) is used for patients (pat.) at high risk for neutropenic fever after chemotherapy. 2005 Bucaneve et al. published data about the P889 use of levofl oxacin (levo) vs. placebo to prevent bacterial infec- Ciprofl oxacine as an antibacterial prophylaxis for patients tion in pat. with cancer and neutropenia, concluding that levo undergoing high-dose chemotherapy and autologous is effective in preventing febrile episodes, lowers the rate of stem cell transplantation – A single-centre experience microbiologically documented infections (MDI) and bacterae- A. Wolska, A. Wierzbowska, A. Szmigielska-Kaplon, A. Pluta, mia. To compare these data with the standard use of cipro we T. Robak have already re-evaluated the data of pat. with AML and with Medical University of Lodz (Lodz, PL) lymphoma/solid tumours receiving HD-CTx and auto-SCT. Now we add the data of pat. with Multiple Myeloma (MM). Objective: The objective of the study was to evaluate the effi - Methods: We analyzed 100 cycles of HD-Melphalan (HD-Mel) cacy of fl uoroquinolone prophylaxis for patients undergoing and auto-SCT in 69 pat. with MM who were at risk for prolonged high dose chemotherapy and autologous stem cell transplanta- neutropenia. In 38 pat. one cycle and in 31 pat. 2 cycles of HD- tion (autoSCT). Mel. were analyzed. Pat. after allogeneic SCT, fever before start Methods: We analysed the data of 104 patients transplanted of conditioning, antimicrobial therapy or documented infections at the Department of Hematology, Medical University of Lodz were excluded. Prophylaxis consisted of 500 mg cipro orally TID between January 2002 and October 2008. The cohort was until the development of fever or until neutropenia resolved. Fol- divided into two subgroups, depending on the administered cip- lowing the IDSA – guidelines we analyzed the reason of fever rofl oxacine prophylaxis (50 patients in the prophylaxis group and classifi ed pat. with fever of unknown origin (FUO), and clini- and 54 in the no-prophylaxis group). The groups did not differ cally (CDI) or microbiologically documented infections (MDI). signifi cantly in terms of age, gender and conditioning regimens. Results: The mean duration of cipro application was 13d. The Multiple myeloma prevailed as an indication for autoSCT in the mean age of the MM pat. was 58y. All pat. had a CVL. The ciprofl oxacine treated (30 cases), as well as in the no-prophy- mean duration of leukopenia <1G/l was 7d (AML: 16d, Lym- laxis group (28 cases), and other diagnoses included Hodgkin phoma/solid tumours: 8d). The incidence of fever in neutro- lymphoma (13 and 16 cases respectively), diffuse large B-cell penia was 62% after the 1st cycle. After the 2nd cycle febrile lymphoma (2 and 6 cases respectively), acute myeloid leuke- neutropenia occurred in 77% (all cycles: 67%). In AML-pat. the mia (4 and 3 cases respectively) and multiple sclerosis (2 cases incidence of fever was 93%, and in lymphoma/solid tumours in total). 75%. Of the 67 febrile episodes after 100 cycles of HD-Mel 58% Results: Ciprofl oxacine prophylaxis resulted in a statistically were FUO, 4% CDI and 39% MDI. 25% of the MDI were cen- signifi cant reduction in the number of days intravenous antibiot- tral venous line (CVL) infections and 38% were bacteraemia. In ics were administered to patients in the group with prophylaxis only 2/100 cycles HD-Mel. we documented gram- bacteraemia. (p<0,01). A trend was observed towards a lower prevalence of The TRM at d+30 was 0% in MM and 1% in AML/Lymphoma/ days with fever related to infection in the prophylaxis group. solid tumours. Other correlations, e.g. the number of days with fever in total, Conclusions: The lower incidence of neutropenic fever in MM- the number of days with neutropenic fever, the percentage of pat. refl ects the duration of neutropenia in the different groups. patients receiving granulocyte-colony stimulating factor (G- In MM pat. the incidence of fever rised with the 2nd cycle. CSF), and the number of G-SCF doses were insignifi cant, prob- Compared with the levo-data (neutropenic fever: 67% in AML, ably due to the low number of cases in the study groups. 62% in lymphoma/solid tumours) results with cipro seem to be Conclusion: These results demonstrate that ciprofl oxacin unfavourable – but the low incidence of gram- bacteraemia with prophylaxis is benefi cial for patients treated with autoSCT fol- cipro (3/200 cycles) and the low mortality (1/200 cycles) is not lowing a high dose chemotherapy regimen, in terms of mini- prompting us to change to levo. mising both the duration of the hospital stay, and intravenous antibiotics usage. Moreover, this clearly translates into an eco- nomic advantage. Chronic leukaemias

P891 In vivo T-cell depletion in allogeneic haematopoietic cell transplantation for chronic lymphocytic leukaemia: a retrospective EBMT analysis J. Schetelig, D. Milligan, D. Niederwieser, L. Volin, J. Maertens, N. Russell, A. Gratwohl, M. Bornhäuser, M. van Gelder, A. van Biezen, R. Brand, T. de Witte, P. Dreger on behalf of the Chronic Leukemia Working Party

Objectives: In vivo T-cell depletion (TCD) is discussed contro- versially for patients with chronic lymphocytic leukemia (CLL). While it is a powerful tool to prevent graft-versus-host disease (GVHD) there is concern about increased incidences of relapse and infections. The aim of this analysis was to study the impact of in vivo TCD in a large cohort of patients with CLL.

S259 Patients and Methods: Patients with B-CLL who received allo- genes is ABCC4, an ATP-binding cassette (ABC) transporter geneic hematopoietic cell transplantation (HCT) from a matched and among the most signifi cantly down-regulated genes is sibling or unrelated donor between 2001 and 2008 were eli- PLCB1, which we recently described as being deleted in myelo- gible. Only patients with cyclosporine-based GVHD prophy- dysplastic syndromes and in myeloid acute leukemia. laxis were selected, whereas patients with ex vivo TCD were In the second part of the study, the 89 probes set differentially excluded. The outcome of three major groups of patients was expressed in the training set were tested on an independent test compared: those who were transplanted without TCD, those set of 13 CML pts, including 4 high, 4 intermediate and 5 low who received anti-thymocyte globulin (ATG) or alemtuzumab Sokal risk pts. The test set GEP clustering displayed the same (CAM). Baseline and follow-up data were downloaded from the trend observed in the training set and, while low and intermediate EBMT database. risk pts resulted quite scattered, high risk pts clustered together. Results: 413 patients were eligible. At HCT 64% of the patients Overall, our data suggests that the expression at diagnosis were in complete or partial remission. Donors were HLA-identi- of a particular array of genes might drive the evolutive risk of cal siblings in 74% and matched unrelated donors in 26% of CML pts. the patients. 82% of patients had been treated with a reduced Supported by: CML Correlative Studies Network (Novartis intensity conditioning regimen. No TCD was applied in 57% Oncology), European Leukemia Net, PRIN, AIRC, AIL, FIRB. of the patients while 19% received ATG and 24% CAM. The median follow-up of living patients was 22 months (1 to 92). For the whole cohort of patients 4-year overall survival (OS) P893 and progression-free survival (PFS) were 57% (95% CI, 50% Apoptosis induction by treosulfan is superior to busulfan to 57%) and 44% (95% CI, 37% to 51%). The cumulative inci- in risk-defi ned human CLL cells dence of acute GVHD II-IV was 36% without TCD, 28% with V. Ristovska (1), N. Wagner (1), M. Herling (1), T. Herold (2), ATG and 24% with CAM (gray test, p=0.13). Patients without M. Hallek (1), G. Fingerle-Rowson (1) TCD received less frequently DLI with a 4-year cumulative (1)University Hospital Cologne (Cologne, DE); (2)University chance of 13% compared to 24% with ATG and 32% with CAM Hospital Grosshadern (Munich, DE) (p=0.03). At four years the incidence of extensive chronic GVHD was reported to be 44%, 34% and 31% (p=0.09), respectively. Introduction: The conditioning regimen used prior to alloSCT The relapse incidences at 4 years were 28%, 26% and 31% infl uences the clinical outcome since it adds to toxicity/mortal- (p=0.44), respectively. Acute GVHD II-IV or chronic GVHD had ity and affects relapse rates and incidence of GvHD. Alkylating a protective effect against relapse (RR 0.6, p=0.01). 4-year OS agents are used as part of the conditioning regimen in pts with and PFS was 59% and 46% without TCD, 51% and 42% with CLL. While cyclophosphamide possesses a well defi ned activ- ATG and 58% and 42% with CAM (p= 0.28 and p=0.21). In ity profi le, the benefi t of administering busulfan to CLL patients multivariate analysis TCD did not impact on OS or PFS, while is unclear since its cytostatic potential has not been evaluated important predictors were remission status at HCT and age. in comparison to other alkylating agents. Treosulfan is a water- Conclusion: In patients with CLL the combination of in vivo TCD soluble alkylating agent which offers suffi cient stem cell toxicity, and DLI appears to result in comparable rates of extensive a favourable safety profi le and is increasingly used prior to allo- chronic GVHD, OS and PFS compared to T-cell replete HCT. SCT. This analysis was performed in order to compare the cyto- toxic potential of busulfan (B) and treosulfan (T) in the induction of apoptosis in cytogenetically-defi ned human CLL cells. P892 Methods: PBMCs from 25 pts with the diagnosis of CLL, a WBC CD34+ from high Sokal risk chronic myeloid leukaemia > 30 G/l and defi ned cytogenetic risk (5 x del 17p-, 5 x del 11q-, patients expresses gene profi le signifi cantly different 5 x trisomy +12, 5 x del 13q-, 5 x no aberration) were cultured from CD34+ obtained from low and intermediate Sokal in the presence of IL-4/CD40L. Apoptosis was induced by the risk pts: a subgroup of CML pts candidate to upfront addition of busulfan (B, 3-100µM), treosulfan (T, 3-100µM), search for BMT donor? 4-hydroperoxycyclophosphamide (4-hpC, 5-80µM), fl udarabine C. Terragna (1), S. Durante (1), A. Astolfi (2), F. Palandri (1), (F, 5-50µM) or cytarabine (Ara-C, 5-10µM). Rates of apoptosis F. Castagnetti (1), G. Rosti (1), N. Testoni (1), S. Luatti (1), were determined using annexinV/PI staining and caspase-3 A. Poerio (1), I. Iacobucci (1), R.M. Lemoli (1), S. Soverini (1), production after 24 and 48 hs. D. Russo (4), M. Malagola (4), M. Baccarani (1), G. Martinelli (1) Results: In the entire cohort as well as in cytogenetic sub- (1)Dpt. of Hematology/Oncology Seràgnoli (Bologna, IT); groups, B induced apoptosis of CLL cells only to a minimal (2)Pediatric Oncology and Hematology “L. Seràgnoli” (Bologna, extent (< 5% at 24h, < 10% at 48 h). In contrast, T, 4-hpC, F and IT); (4)Hematology and Unit of Blood Diseases and Cell Therapy AraC were potent inducers of apoptosis and induced cell death (Brescia, IT) via caspase-activation. The effi cacy of T was comparable to that of F. The relevance of cytogenetic aberrations was clearly Even after the introduction of imatinib, the calculation of the evident since apoptosis induction was greatest in subgroups Sokal and the Euro prognostic scores has remained essential with favorable prognosis (no aberration, 13q-) and reduced in in clinical practice, since allow to stratify CML pts at different subgroups with unfavorable prognosis with 17p- being the most evolutive risk at diagnosis, guiding therapeutic decisions and drug-resistant aberration (rate of apoptosis +12 > 11q- > 17p-). upfront search for an allo-BMT donor. The combination of T with F or 4-hpC resulted only in limited Here we reported GEP experiments aimed at the identifi cation additive effects. Results of additional in vivo-experiments in of genes and able to predict the disease course of CP-CML pts mouse xenograft models will be presented. at the onset of the disease were performed on highly enriched Conclusions: Our work identifi es treosulfan as an alkylating CD34+ cells from 27 pts with untreated CML in CP by Affymetrix agent with effi cient cytotoxic potential in human CLL. Since HG-U133 Plus 2.0 platform. induction of cell death by busulfan appears to be minimal and In the initial part of the study, the fi rst 14 CML pts (the “training given the suitability of treosulfan in alloSCT, the use of treo- set”) were successfully assayed for global GEP and microarray sulfan instead of busulfan may be considered to improve the data were used to defi ne genes differentially expressed in high effi cacy of conditioning therapy prior to alloSCT in CLL pts. (6 pts) vs. low (8 pts) Sokal risk pts, thus identifying 89 probes set; clustering of their GEP showed an homogeneous pattern in high Sokal risk pts, where up-regulated genes are mainly related to the positive regulation of immune response and to the induction of apoptosis, whereas down-regulated genes are mainly involved in the negative regulation of metabolic proc- esses. Of note, among the most signifi cantly up-regulated

S260 P894 Methods: A retrospective analysis of the outcome of 12 imat- Allogeneic haematopoietic stem cell transplantation as a inib-resistant CML patients treated with dasatinib (5) or nilotinib curative therapy in primary and secondary myelofi brosis (6) or both (1) before allo-HSCT, with the aim to investigate the S. Lissandre (1), J.-O. Bay (2), J.-Y. Cahn (3), V. Cacheux (2), infl uence of second-generation TKIs on transplant engraftment, A. Cabrespine (2), J. Cornillon (4), B. Cassinat (1), R. Peffault toxicity and long-term effi cacy. de Latour (1), G. Socié (1), M. Robin (1) Results: Patients were treated with second generation TKIs (1)Hôpital Saint-Louis - APHP (Paris, FR); (2)Hôpital Hôtel-Dieu for 1 to 17 months (median, 8): all patients but one achieved (Clermont-Ferrand, FR); (3)Hôpital Michallon (Grenoble, FR); a complete hematologic response (CHR); 6 patients obtained (4)Institut de Cancérologie de la Loire (Saint-Etienne, FR) also a Major Cytogenetic Response, which was complete (CCgR) in 3 patients. Aim of the study: To describe the outcome of pts who underwent Donors were HLA-matched related in 4 cases and unrelated in HSCT from 1994 to 2008 in 4 hematological French centers. 8 cases. Stem cell source was peripheral blood, bone marrow Method: 32 pts with MF were identifi ed. Overall survival (OS) or cord blood in 6, 5 and 1 cases, respectively. Conditioning and relapse free survival (RFS) were estimated by Kaplan- regimen was full for all patients. All patients engrafted success- Meier method and cumulative incidence of non-relapse mortal- fully: median time to neutrophil and platelet recovery was 21 ity (NRM) with relapse as a competing event. days (range, 11-41) and 22 days (range, 14-86), respectively. Patient and disease characteristics: Median age of pts at time of All patients but one achieved a full donor chimerism. Best HSCT was 49 years (15-65). Twenty-seven pts had primary MF response to allo-HSCT was Complete Molecular Response whereas 12 patients had MF secondary to polycythemia vera (CMR) in 9 patients, Major Molecular Response (MMR) in 1 (PV) (n=7) or to essential thrombocythemia (ET) (n= 5). Among patient and CCgR in 2 patients. Four patients relapsed: one 25 pts with a cytogenetic analysis, 12 had clonal abnormalities. patient at a molecular level, one patient at a cytogenetic level A JAK2 mutation was detected in 6 out of 22 tested patients. and two patients had a hematological relapse (sudden blast Treatment before transplantation mainly consisted of hydroxyu- crisis). The patient who presented a molecular relapse recov- rea or busulfan (26 pts). Before HSCT, 12 pts required platelet ered a molecular negativity after discontinuation of the immu- transfusions and 19 required blood transfusions. Twenty-three nosuppressive therapy, whereas the patient who experienced pts experienced a splenectomy. Ten patients were transformed a cytogenetic relapse became RT-PCR negative after Donor in AML before HSCT. Dupriez score was low in 9, intermediate Lymphocytes infusions. At last contact, 9 patients were in con- in 16 and high in 14 pts at time of transplantation. tinuous CMR and 1 patient in MMR; 2 patients died of disease Results: 25 pts received a HSCT from an HLA-identical sibling progression. Median overall survival was 16.5 months. and all pts were transplanted with an HLA matched donor. Fif- Conclusions: Our report confi rms that second-generation teen pts received a myeloablative conditioning regimen (MAC) TKIs before allo-HSCT does not negatively affect transplant and 24 received a fl udarabine-based reduced intensity con- engraftment and response rate, nor increases transplant- ditioning regimen (RIC). Graft-versus-Host disease (GVHD) related toxicity. prophylaxis consisted of cyclosporine plus mycophenolate in Supported by: AIL, AIRC, FIRB 2006, European LeukemiaNet. 21, cyclosporine plus methotrexate in 13 and cyclosporine alone in 5 pts. All but one pts engrafted in median 15 days (range:0- 129) after transplantation. Thirty-one pts developed grade I-IV P896 acute GVHD. Among 35 evaluable pts, 18 developed a chronic Cancer-testis antigen expression and immunogenicity in GVHD. Median time to discharge was shorter with RIC regi- chronic myeloid leukaemia men (23 days) than with MAC regimen (46 days). Three-year T. Luetkens, F. Uhlich, T. Stasche, R. Akblukak, Y. Hildebrandt, OS was 61% (95%Confi dence Interval (CI): 47-80). 3-year RFS S. Kobold, K. Bartels, T. Brümmendorf, P. Schafhausen, was 55% (41-75). 3-year NRM was similar with RIC or MAC N. Kröger, C. Bokemeyer, D. Atanackovic (32% vs. 20%). Splenectomy, age, sex mismatch, conditioning University Medical Center Hamburg (Hamburg, DE) regimen (RIC vs MAC), Dupriez score and transformation into AML had no impact on outcome. Patients who received platelet Background: Expression of cancer-testis (CT) antigens is char- transfusions before HSCT or who was CMV+ had signifi cant acteristically restricted to cancer and the human germline and poorer OS. their distinctive immunogenicity renders them potential targets Conclusion: In these high risk MF pts, median OS after HSCT in immunotherapeutic settings. In order to identify such tar- is much better than reported with alternative treatment. The gets in CML, we performed a comprehensive analysis of the main risk factor for poor outcome is thrombocytopenia requiring expression and immunogenicity of CT antigens in cell lines and platelet transfusion before transplantation whereas other usual patients with CML. risk factors have no more impact after HSCT. Methods: 10 CML cell lines and bone marrow (BM) samples from 10 healthy donors were screened by RT-PCR for the expression of 30 CT antigens. RNA levels of PRAME were also P895 evaluated in 20 healthy tissues by quantitative RT-PCR. Cell Outcome of allogeneic stem cell transplantation in lines were analyzed before and after stimulation with 5’-Aza-2’- patients with chronic myeloid leukaemia treated with Deoxcytidine and Trichostatine. Expression of 15 selected CT second generation tyrosine kinases inhibitors after antigens was then analyzed in BM and peripheral blood (PBL) imatinib failure samples from 84 patients with CML by RT-PCR. Protein expres- F. Palandri (1), M. Breccia (2), A.P. Iori (2), F. Bonifazi (1), sion was confi rmed by Western Blot for 10 CT antigens. Sera of R. Latagliata (2), E. Colaci (1), GF. Torelli (2), F. Castagnetti 45 patients with CML were screened for antibodies against 15 (1), V. Valle (2), S. Usai (2), G. Martinelli (1), C. Dell’Agnola (3), CT antigens by ELISA. S. Cingarlini (3), G. Rosti (1), M. Baccarani (1), G. Alimena (2) Results: RT-PCR showed expression of CABYR, CT45, DKK1, (1)Oncology Seràgnoli (Bologna, IT); (2)University La Sapienza HAGE, KM-HN-1, MPHOSPH1, PASD1, PBK and SP17 in BM (Rome, IT); (3)Policlinico GB Rossi (Verona, IT) samples from healthy donors. 16 of the remaining 21 antigens were expressed in at least one untreated cell line and treat- Objectives: In Philadelphia-positive chronic myeloid leukemia ment with 5’-Aza-2’-Deoxycytidine led to a two-fold increase (Ph+ CML) resistance to imatinib is a well-recognized problem. in the average number of antigens expressed per cell line. Second generation tyrosine kinase inhibitors (TKIs), such as Treatment with Trichostatine had a less pronounced effect. dasatinib and nilotinib, have been designed to overcome this Analyzing patient samples, PRAME was found in 32.1% and phenomenon. Limited data exist on the possible effects of expression correlated with stage of disease (p=0.02), blast cell these drugs on subsequent allogeneic hematopoietic stem cell count (r=0.38; p=0.01), and reduced overall survival (p=0.021). transplantation (allo-HSCT). Sporadic expression was only detected for BAGE2 (N=2) and

S261 SLLP1 (N=1). Importantly, among 20 healthy tissues PRAME the fi rst allo-SCT and who failed to DLI. There were three males was indeed characteristically restricted to germ-line tissues. and two females (median age 58 years; range, 49 – 64). First While none of the patients showed PRAME-specifi c serologi- transplantation had been performed in all cases by a reduced cal immune responses, one patient who had not expressed conditioning regimen based on a combination by busulfan NY-ESO-1 showed a signifi cant antibody response against this (cumulative dose 10mg/kg) and fl udarabine (cumulative dose antigen. 180 mg/kg). The maximal response after the fi rst allo-SCT was Conclusions: CT antigens are frequently expressed in CML complete remission (CR) in two patients and partial remission cell lines and expression can be increased by application (PR) in three patients. All patients received DLI at 1-4 time of demethylating agents and histone deacetylase inhibitors. points in median dose 2.2x107/kg (range, 1x106 - 1x108/kg) due Most frequently detected in patient samples was PRAME, to clinical relapse after the fi rst allo-SCT. The best response the expression of which correlated signifi cantly with multi- after the DLI according to IWG-MRT criteria was stable disease ple clinicopathological parameters. One patient who had not (SD) in three patients, clinical improvement (CI) in one patient, expressed NY-ESO-1 showed an antibody response against while the fi fth patient showed progressive disease (PD). The this antigen, possibly indicating spontaneous immunity lead- median interval between the fi rst and second allo-SCT was ing to the eradication of CML clones expressing this antigen 17 months (range, 11 – 25). Second allo-SCT was prepared in vivo. in all patients with a reduced-intensity conditioning (RIC) regimen consisting of a combination of fl udarabine (median cumulative dose 5x30mg/kg), and treosulfan (3x12mg/kg). All patients received peripheral blood stem cells (median cell dose 8.3 x 106 CD34+ cells/kg bw, range, 5.4 – 11.1) from HLA- matched unrelated donors. All patients successfully achieved engraftment; median leukocyte engraftment was observed on day +16 (range, 10 – 20) after second allo-SCT. Acute graft- versus-host disease (aGvHD) grade II-IV was observed in two patients. Three patients (60%) are alive, 4, 7, and 25 months from second allo-SCT in complete remission (CR) according to IWG-MRT criteria, including molecular remission. Two patients (40%) achieved PR and SD but relapsed and died 11 and 15 months from the second allo-SCT. In conclusion, the use of a second allo-SCT, based on a combina- tion of fl udarabine and treosulfan is a effective and well tolerated approach for the patients, who failed to DLI after fi rst allo-SCT.

P898 Innovative phase I-II study of concomitant and consecutive treatment with dasatinib and MK-0457 in refractory Ph+ CML and ALL patients candidates for allogeneic bone marrow transplantation of savage C. Papayannidis (1), I. Iacobucci (1), S. Soverini (1), S. Paolini (1), S. Santucci (1), F. De Rosa (1), D. Cilloni (2), F. Messa (2), F. Pane (3), V. Meneghini (4), P. Giannoulia (1), E. Ottaviani (1), N. Testoni (1), B. Lama (1), M. Pantaleo (1), M. Baccarani (1), G. Martinelli (1) (1)Oncology Seràgnoli (Bologna, IT); (2)Hematology (Orbassano, Turin, IT); (3)CEINGE Biotecnologie Avanzate (Naples, IT); (4)University of Verona (Verona, IT)

Background: MK-0457 is a pan-aurora kinase inhibitor with demonstrated activity against wild-type and mutated BCR- ABL, including the T315I form, as well as FLT3 and JAK-2. It is a promising molecule for the management of Ph+ leuke- mias, in which the emergence of mutations in the ABL kinase domain still represents the main mechanism of resistance to TK inhibitors. Aim: We conducted an innovative and proof of concept Phase I clinical study of sequential and concomitant treatment with Dasatinib, previously administered for three months, and MK- 0457. This combined activity suggests that MK-0457, in asso- P897 ciation with Dasatinib, would suppress the emergence of T315I Second allogeneic stem cell transplantation for and other resistant clone, improving upon the response rate for osteomyelofi brosis relapsed after initial allogeneic stem Dasatinib and the durability of response. The trial investigated cell transplantation two schedules of therapy: patients who achieved and main- E. Klyuchnikov, F. Ayuk, U. Bacher, A. Zander, N. Kröger tained a major hematologic response (MHR) after three months University Cancer Center, Hamburg (Hamburg, DE) of therapy with Dasatinib (70 mg twice daily) received a 6-hour biweekly infusion of MK-0457 at 64 mg/m²/hr, whereas patients Therapeutic options for patients with osteomyelofi brosis whose who failed to achieve a MHR received a 5-days continuous infu- disease has relapsed after a prior allogeneic stem cell trans- sion of MK-0457 at 10 mg/m²/hr, every 4 weeks. plantation (allo-SCT) include donor lymphocyte infusion (DLI) or Results: Two patients with Ph+ ALL and one patient with CML a second transplant. There are no large studies addressing the in myeloid blast crisis, previously unsuccessfully treated with safety and effi cacy of a second allo-SCT for relapsed disease. imatinib, were enrolled in the protocol. The fi rst two patients, We report our experience with a second allo-SCT being per- both in hematologic response after three months of treatment formed in fi ve patients with relapse of osteomyelofi brosis after with Dasatinib, subsequently received the 6-hour biweekly

S262 schedule, maintaining the haematological response. No hae- These fi ndings indicate that WT1 peptide-specifi c T cells might matological toxicity was described. The third patient, enrolled represent ideal candidates for post transplant immunotherapy in progression disease, received the 5 days MK-0457 schedule in patients with WT1-expressing malignancies. of treatment. His peripheral blood count was consistent with a Supported by “Patenschaftsmodell” of Goethe University Frank- severe pancytopenia, which required frequent platelets and red furt (GW). blood cells transfusions. His bad clinical performance status was compromised by a severe hemorrhagic pleural effusion, responsible for moderate dyspnoea and severe asthenia. After P900 one cycle of MK-0457, a complete recovery of the pulmonary Human alloreactive CD4+ T-cells as potent effector cells disease and a complete hematologic response were obtained. and sole mediators of anti-tumour responses in a NOD/ In all three patients an allogeneic bone marrow transplantation Scid mouse model for human acute leukaemia of savage from HLA identical donor could be performed. S. Stevanovic, M. Griffi oen, B.A. Nijmeijer, M.L.J. Van Schie, Conclusions: The sequential and concomitant administration R. Willemze, J.H.F Falkenburg of Dasatinib and MK-0457 represents a promising therapeutic Leiden University Medical Centre (Leiden, NL) strategy for refractory Ph+ CML and ALL candidate to an BMT of savage. Donor T cells administered by donor lymphocyte infusion (DLI) Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, for the treatment of relapsed leukemia after allogeneic stem Fondazione del Monte di Bologna e Ravenna, Merck Sharp & cell transplantation (alloSCT) mediate graft-versus-leukemia Dohme. (GvL) reactivity as well as graft-versus-host disease (GvHD). Since HLA-class II molecules are predominantly expressed on hematopoietic cells, adoptive transfer of donor CD4+ T cells may selectively mediate GvL reactivity without GvHD. This is Immunotherapy supported by clinical studies with CD8+ T cell-depleted DLI, but it remains unknown whether GvL reactivity and remain- ing GvHD observed in these patients is mediated by CD4+ P899 T cells or by contaminating or residual CD8+ T cells. We Generation of WT1 peptide-specifi c T-cells out of healthy assessed the capacity of highly purifi ed CD4+ T cells to exert volunteer donors for adoptive immunotherapy after GvL reactivity in a NOD/Scid mouse model for human acute allogeneic stem cell transplantation leukemia. Mice inoculated with primary human acute lym- G. Weber (1), J. Karbach (2), S. Kuci (1), H. Kreyenberg (1), phoblastic leukemic cells from different patients were treated A. Willasch (1), T. Klingebiel (1), W. Wels (3), E. Jäger (2), with purifi ed human CD4+ DLI (consisting of CD4+ T cells) P. Bader (1) or CD3+ DLI (consisting of CD4+ and CD8+ T cells) from dif- (1)Goethe University (Frankfurt, DE); (2)Krankenhaus Nordwest ferent donors. In mice treated with CD4+ DLI, the expansion (Frankfurt, DE); (3)Georg-Speyer-Haus (Frankfurt, DE) of activated CD4+ T cells in peripheral blood coincided with disappearance of leukemic cells, showing similar kinetics as Introduction: Allogeneic SCT has become an important treat- for CD3+ DLI. In the presence of these activated T cells after ment modality for patients with high risk leukemia and is also treatment with CD4+ DLI, the leukemic cells acquired in vivo increasingly used in the treatment of children and adolescents an antigen presenting cell (APC) phenotype, characterized by with solid malignancies. Nevertheless, relapse remains the upregulation of HLA-class II, adhesion and costimulatory mol- major cause for treatment failure. Immunotherapeutic treat- ecules, whereas leukemic cells from untreated mice remained ment approaches targeting tumor antigens might help to pre- unchanged. The specifi city of the GvL effect of CD4+ DLI was vent relapse. A possible tumor antigen is the Wilms tumor investigated by clonal isolation of CD4+ T cells, showing that antigen WT1 which is over expressed in various leukemias and anti-tumor immunity was mediated by a polyclonal response solid tumors. Cytotoxic T-cell responses have been detected in comprising T-helper and T-effector cells directed against the leukemia patients. Therefore, we investigated the feasibility to mismatched HLA class II molecules of the patient. The data generate WT1-specifi c T cells from healthy donors which is a showed that 84% of isolated CD4+ T cell clones were reactive pre-requisite for an application as adoptive immunotherapy in against leukemic cells that were in vitro modifi ed into leukemic the transplantation setting. APC, whereas only 28% of the clones also directly recognized Methods: Peripheral CD8+ cells were stimulated by autologous unmodifi ed leukemic cells. Therefore, we hypothesize that WT1 peptide-pulsed antigen presenting cells. Peptide-specifi c crosstalk between leukemic cells and specifi c CD4+ T cells is T-cell clones were generated by limiting dilution assay. Peptide the fundamental basis for induction of leukemic cells with APC recognition and cytotoxicity were tested in Interferon-g Elispot phenotype and effi cient anti-tumor immunity. In conclusion, and Europium-release assays. our data illustrate that CD4+ T cells can be potent effector cells Results: WT1 peptide-specifi c T cells could be found in 9 out of and sole mediators of anti-tumor immunity, thereby emphasiz- 10 healthy HLA-A2+ volunteers. T-cell clones specifi c for two ing the clinical relevance of CD4+ T cell based immunothera- WT1-derived peptide epitopes (WT1 p37-45: VLDFAPPGA, pies after alloSCT. WT1 p126-134: RMFPNAPYL) could be generated. Effi cacy of in vitro generated T-cell clones to recognize and lyse peptide-pulsed tumor cell lines as well as endogenously WT1- P901 expressing tumor cell lines Rh1 and MDA-MB-231 was demon- The composition and quality of leukaemia-derived strated. Thereby, blocking with HLA class I and CD8 antibodies dendritic cells, T-cells and the cellular microenvironment inhibited lytic activity and peptide recognition. Peptide dose titra- is predictive for the antileukaemic T-cell cytotoxic tion demonstrated the ability of T-cell clones to recognize even reactions of DC-primed T-cells and the response to low nM peptide concentrations. No allo-reactive effects against therapy HLA-different PBMCs or HLA-A2+ CD34+ peripheral stem cells H. Schmetzer (1), A. Liepert (1), C. Grabrucker (1), could be observed. In vitro generated WT1-specifi c T-cell clones D. Fischbacher (1), M. Merle (1), M. Freudenreich (1), were CD8+/CD45R0+/CD45RA-/CCR7- effector-memory T cells F. Schuster (1), S. Reuther (1), A. Kremser (1), J. Loibl (1), with a purity of over 95% WT1-tetramer positive cells. R. Reibke (1), C. Schmid (2), T. Kroell (1), H.-J. Kolb (1) Conclusion: We could demonstrate that the generation and in (1)University Hospital of Munich (Munich, DE); (2)Municipal vitro expansion of functionally active WT1-specifi c T cells from Hospital of Augsburg (Augsburg, DE) peripheral blood of healthy HLA-A2+ volunteer donors is feasible. These in vitro expanded effector cells displayed a peptide- specifi c Conversion of leukemic cells to leukemia-derived DC (DCleu) cytotoxicity against WT1-expressing solid tumor cell lines. improves antigenpresentation and generation of leukemia-specifi c

S263 ACTR. We studied the role of the composition/quality of DC and Results: The median frequencies of KIR2DL1+ NK cells on days (DC-primed) Tcells, chemokines/cytokines in the mediation of leuke- +28,+56,+100,+180,+365 equaled 4.7%,5.6%,5.9%,9.1%, and mia-cytotoxic reactions ex vivo or to predict the response to DC/DLI- 12.6%, respectively, and were signifi cantly lower compared to based immunotherapy in vivo. 20.1% in donors. Expression of KIR2DL2-3 in subsequent time- 1) We generated DC/mature DC/DCleu from 30 AML/MDS pts points was 14.4%,20%,19.6%,20.9%, and 25.4%, compared (on average 23/43%/66%) and primed Tcells (in 17 cases) with to 24.5% in donors (p<0,05 up to day +100). The frequen- DC or blasts in MLC. cies of KIR3DL1 were 16.8%,13.8%,10.9%,10.1%, and 9.2% 2) After DCpriming on average 47% of Tcell-cases gained an vs. 18.1% in donors. The expression of NKG2A on day +28 ACTR, but only 23% after blast-priming. equaled 89.9% and decreased to 79%, 68.9%,64% and 51.9% 3) The ACTR effi ciency of DCpriming was superior to blast- (up to day +180, p<0.05 compared to 46.8% in donors). priming (on average 44%vs34% lysed blasts). At all time-points DI was signifi cantly lower compared to the 4) About 70% of cases gained an ACTR after DCpriming initial donor-recipient phenotypic difference. Median DI continu- with >45% proliferating/>65% CD4+/>42% memory Tcells ously decreased from 5.9 on day +28 to 4.1 on day +365. The or >40% mature DC/>65% DCleu and if >95g IFN gamma or use of steroids was associated with higher DI and the effect >15g IL6/106cells were in the DC-MLC. 80% of cases gained was signifi cant on days +56 (p=0.01), and +100 (p=0.04). Up ACTR after priming with DC that produced >2000pgCXCL8 or to day +100, DI correlated adversely with the absolute number >100pgCCL2/106 cells. of circulating T, Th, and Tc cells. Other factors including T-cells Differences were most distinct in the group with DC primed in the graft, source of stem cells, donor type, GVHD had no Tcells prepared at relapse after SCT. 5) Cases with a response impact on DI. to therapy showed higher proportions of DCleu, proliferating, We conclude that: memory or CD4+ Tcells. Pts presenting with a relapse after SCT 1) acquisition of KIRs by NK cells after alloHSCT is sequen- showed better ex vivo convertibility of blasts to DCleu if they tial and starts with KIR3DL1, followed by KIR2DL2-3 and had responded to a GMCSF/DLI based therapy of their relapse KIR2DL1; after SCT compared to pts with no response (72 vs 36% blasts 2) NKG2A is overexpressed within 6 months after transplanta- convertible to DCleu; 44vs 29% generable DC). Moreover we tion; found, that cases with more vs less than 60% blasts convertible 3) NK cell phenotypic pattern tends to recapitulate the donor to DCleu the overall survival from SCT to endpoint was 612 vs type; 168 days. 7) Spectratyping of the VbetaTCR region in an AML pt 4) Phenotypic maturation is disturbed by the use of steroids; reveiled a more extended clonal restriction of donor Tcells after 5) T-cell recovery occurs in parallel to NK cell maturation. Our DCpriming of Tcells compared to blast-primed Tcells and the observations should be taken into account when trying to pre- restricted pattern was also found in Tcells from the pt after SCT. dict potential benefi t from NK cell alloreactivity. Sum:DC/DCleu can be generated in any given pt independent from karyotype. DCpriming of Tcells improves the antileukaemic CTL, but can also mediate Tcell anergy. The composition of DC/ P903 Tcells/culture supernatants is predictive for the lytic effi ciency of Chemotherapy before donor lymphocyte infusion primed Tcells and the patients’ response to therapy. Tcell clones enhances lymphocyte activation and induces clinical are enriched after DC- compared to blast priming and are found responses in vivo selected Tcells. We contribute to understand cytotoxic/ G.F. Torelli, R. Maggio, W. Barberi, N. Peragine, escape mechanisms and detect pts qualifying for DCbased M.S. De Propris, A.P. Iori, V. Valle, E. Iannella, F. Natalino, immunotherapies. Possibly DCprimed, specifi c Tcells could be E. Arleo, A. Guarini, R. Foà generated in vivo or selected from ex vivo cultures and trans- Sapienza University (Rome, IT) ferred to pts in therapeutic or prophylactic settings. Animal models have demonstrated that the serum peak concen- tration of activating cytokines and hematopoietic growth factors P902 is reached 48 hours after a chemotherapeutic treatment. We The use of steroids but not T-cell recovery disturbs hypothesized that patients affected by resistant diseases who reconstitution of NK cell receptor repertoire after have already undergone an allogeneic SCT may benefi t from alloHSCT adoptive cell transfer performed after chemotherapy. S. Giebel (1), J. Dziaczkowska (2), T. Czerw (2), J. Wojnar Four high risk patients affected by AML evolved from a MDS, (2), M. Krawczyk-Kulis (2), I. Nowak (3), A. Holowiecka-Goral who already presented a disease recurrence after an allo- (1), A. Segatti (4), S. Kyrcz-Krzemien (2), J. Holowiecki (1), geneic SCT (1 from haplo, 1 from MUD and 2 from siblings), P. Kusnierczyk (3) completed 2 cycles of DLI (CD3 = 1x107/Kg of recipient BW for (1)Institute of Oncology, Branch Gliwice (Gliwice, PL); haplo and MUD, and 1x108/Kg for sibling) performed 2 days (2)Silesian Medical University (Katowice, PL); (3)L. Hirszfeld after a lymphodepleting chemotherapeutic treatment. These Institute of Immunology and Experimental Therapy (Wroclaw, patients underwent cytofl uorimetric analysis of the PB for CD3, PL); (4)University of Pavia (Pavia, IT) CD4, CD8, CD25, CD16, CD56, CD20, CD7, CD38, HLA-DR and intracytoplasmic staining for IFNg, TNFa, IL-2, IL-10 at Although incompatibility of KIR ligands was shown to result in days 0, 2, 5, 10, 20, 30 from DLI; two patients who underwent graft-vs.-leukemia reaction, clinical data are inconsistent sug- 2 cycles of standard DLI served as controls. gesting the impact of various procedure-related factors on NK The results demonstrated that from day 2 throughout the entire cell maturation and their possibility to display alloreactivity. Our study period, patients who received DLI after chemotherapy goal was to prospectively evaluate reconstitution of NK cell showed an increased presence of reactive elements, as docu- receptor repertoire after alloHSCT and to identify factors affect- mented by lymphocytes expressing the CD7/CD38/HLA-DR ing this process. profi le when compared to controls (p 0.0001). The difference 83 adults treated with myeloablative T-replete alloHSCT reached its maximum at day 5 (mean %±S.D. 40±21 vs 8±3; from sibling (n=32) or unrelated donor (n=51), were exam- p 0.02). The production of IFNg was also superior in patients ined NK cell phenotype before transplantation and on days from the study group (p 0.01), achieving the peak at day 10 +28,+56,+100,+180,+365. In addition, for each patient and (mean %±S.D. 36±11 vs 16±-4; p 0.01). donor, a phenotypic pattern was created based on receptor fre- Three of the 4 patients from the study group presented for quencies and median fl uorescence intensity (MFI). Discrepancy the fi rst time a clinical picture of GVHD in association with the index (DI) calculated as a sum of differences between patient chemo-immunotherapeutic treatment (despite having previously and donor in frequencies and MFIs for each receptor was used undergone many cycles of standard DLI); in these 3 patients, as a measure of NK cell maturation. the onset of GVHD was associated with an hematologic CR.

S264 The fi rst patient is now in CR 19 months later (which represents P905 the longest CR from the transplant performed 5 years ago); the CXCL8 and CCL2 secretion by dendritic cells generated second patient remains in CR after more than 5 months; the from AML-blasts (DCleu) as well as IFN-g or IL-6 release in third patient obtained a CR, but died of pneumonia 2 months mixed lymphocyte cultures from T-cells after DC-priming after the treatment; the fourth patient, who did not show GVHD, are predictive for antileukaemic T-cell reactions has relapsed. D. Fischbacher (1), M. Merle (1), A. Liepert (1), C. Grabrucker These observations suggest that donor lymphocytes may (1), A. Kremser (1), J. Loibl (1), C. Schmid (2), T. Kroell (1), undergo a process of activation when infused into patients who H.J. Kolb (1), H. Schmetzer (1) have received a chemotherapy in the last 2 days and advocate (1)University Hospital of Munich (Munich, DE); (2)Municipal a possible new chemo-immonotherapeutic schedule that can Hospital (Augsburg, DE) be taken into consideration in the case of resistant/residual dis- eases for patients who have undergone an allogeneic SCT. DC as well as specifi c T-cells are pivotal mediators of CTL responses. We could show that autologous T-cells obtained from AML-patients or allogeneic Donor-T-cells can be primed P904 by DCleu resulting in a very effi cient lysis of naive blasts, how- Clinical grade generation of human anti-adenovirus- ever not in every case - resulting in anergic T-cells or even in cytotoxic T-cells for adoptive immunotherapy blast stimulation. To enlight the interactions between DC/MNC L. Aissi-Rothe, V. Decot, C. Mathieu, A. Kennel, V. Venard, and T-cells and to predict antileukemic or non-antileukemic T- H. Jeulin, A. Salmon, L. Clement, P. Bordigoni, J.F. Stoltz, cell reactions we investigated chemokine and cytokine release D. Bensoussan profi les in supernatants collected during DC-generation (n=91) CHU Nancy (Vandoeuvre Les Nancy, FR) and from MLC-cultures of T-cells with MNC (n=55) or DC (n=56) from 39 AML/MDS-cases. Cytokines and chemokines Adenovirus (ADV) infections represent one of the major cause of were quantifi ed by cytometric bead array. The lytic activity of morbidity and mortality following Hematopoietic stem cell trans- DC/MNC-trained T-cells against naive blasts was quantifi ed plantation. The incidence of ADV infections ranges between after 3/24 hours. 5 to 30%, with paediatric recipients showing the highest rate 1) Lytically active T-cells after DC/MNC-priming secernated of infection. The mortality rate is very high in those patients: higher levels of IL-6 (median release 27/44pg per 106cells) 60 to 73%, despite new antiviral treatment strategies. Actually, than lytically inactive T-cells (median release 14.5/15pg per it has been demonstrated that a suffi cient host T-cell response 106cells). We showed that more han 75% of all cases gained is essential to clear the virus. lytic T-cell activity if >15/20pg IL-6 per 106cells were released We describe here a complete clinical grade generation of after DC/MNC-priming. Lytically active T-cells secernated Human anti-Adenovirus cytotoxic T cells in order to propose an higher amounts of IFN-g than lytically inactive T-cells after DC- adoptive immunotherapy to the recipient. priming (95/<5pg per 106cells). After MNC-priming however the Healthy donor mononuclear cells (PBMC), known for their good median release of IFN-g of lytically inactive T-cells was higher cellular immunity against ADV, are stimulated during 6 hours than that of lytically active T-cells (110/56pg per 106cells). We with the Peptivator-ADV5 (Miltenyi Biotec) which is a synthetic found no differences in secretion profi les of IL-2, IL-4, IL-10 and peptide pool covering the ADV5 Hexon protein. Gamma Inter- TNF-a between the lytically active or inactive T-cells after DC/ feron (IFNƒ×) secreting cells are isolated on the CliniMACS MNC-priming. device using the Cytokine Capture System (Miltenyi Biotec) 2) DC able to prime lytically active T-cells expressed higher (Feuchtinger et al, 2007). The results of 4 immunomagnetic levels of CXCL8, CCL2, CXCL10, CCL5 and CXCL9 (median selections are presented in the table below: release 864/30/5/3/2pg per 106cells) than DC unable (median Isolated T lymphocytes (CTL) are cultured with IL2 and autolo- release all 0pg/106cells). We observed that 75% of all cases gous feeder cells (irradiated cells from the negative fraction) in gained lytic T-cell activity after priming with DC that produced order to perform the functional quality controls. >800pg/106cells CXCL8 or >200pg/106cells CCL2. We fi rst controlled the ability of the amplifi ed CTL to secrete That means that the secretion-profi le of CXCL8 and CCL2, both IFNƒ× when restimulated with the Peptivator ADV. A cytotocity T-cell chemoattractants, of DC as well as the release of IL-6 of 40% against autologous dendritic cells (DC) as target cells and IFN-g (mediators of CTL) in MLC of DC-primed T-cells is (10/1) loaded with ADV5 or ADV 2 lysates could be observed predictive for the antileukemic capacity of T-cells. when a cytotoxicity of 6% was reported with non loaded DC. However, after MNC-priming of T-cells the IFN-g release was Finally, we could observe a low allogeneic reaction with CTL not associated with a lytic activity thereby confi rming the central against non HLA identical healthy donor PBMC, decreased of role of DC in antileukemic T-cell response. more than one log compared to the autologous PBMC. Good Manufacturing Practice-grade generation of ADV-specifi c T cells for adoptive immunotherapy could be achieved with a P906 synthetic antigen. This technology presents the advantages to Immune response of human propagated gd-T-cells to be fast, without any in vitro amplifi cation before infusion, and neuroblastoma recommend the Vd1 subset for gd-T-cell to allow a good reactivity to propose immunotherapy in case of based immunotherapy anti-viral treatment failure. K. Schilbach, K. Frommer, S. Meier, R. Handgretinger, M. Eyrich University of Tübingen (Tübingen, DE)

Objective: Human peripheral gd-T-cells effi ciently lyse neurob- lastoma tumor cells. Besides innate effector functions against pathogens and tumors, gd-T-cells are involved in T-helper Th1/ Th2 differentiation of ab-T-cells. However, since different gd-T- cell subsets vary considerably in their functional properties, the aim of the present study was to defi ne repertoires of cytokines, chemokines and angiogenic factors of in vitro expanded Vd1- and Vd2-T-cells in response to neuroblastoma. Methods: gd-T-cell culture: Freshly isolated gd-T-cells from PBMCs of healthy individuals were resuspended (1.5 x 106 cells/ml) in IMDM (10% FCS, 2 mM Glutamine, 200 IU/ml IL-2, 0.5µg/ml PHA) and plated in 96-well plates. 100 µM

S265 pamidronate was added to 50% of the cell-pools to facilitate Our data also suggested that the phenotype of CTL varies preferential outgrowth of Vd1+ T-cells. Since no feeder or upon different triggering of co-stimulatory pathways via aAPC. accessory cells were added, the percentage of Vd1+ T-cells We could detect a higher amount of central memory T-cells increased due to decay of Vd2+T-cells in these cultures. gd-T- (CCR7+/CD45RA-) in samples co-stimulated with the 4-IBB cell neuroblastoma challenge. Irradiated Nb-cells (50Gy) were (AB) as compared to those treated with other co-stimulations seeded in 24-well plates. 24 hours later, when they were adher- [e.g. MHC/CD28: 6.2% of CD8+/Tet+ vs. MHC/CD28/4-IBB ent, culture medium was displaced and cells washed twice with (AB): 23.5% of CD8+/Tet+]. PBS. RPMI 1640defi cient medium (no additives but serum and Likewise, samples co-stimulated with OX40 had a higher con- L-glu) was added with gd-T-cells in a 1:10 ratio to Nb-cells. After tent of non-activated (CD25-) T-cells with less replicate capa- 2 hours supernatants were harvested for protein array and gdT- bility (CD57-) than those co-stimulated with CD28(AB) [e.g. cells for mRNA preparation. MHC/CD28: 23% of CD8+/Tet+ vs. MHC/CD28/OX40 (L): Results: After short term culture, both gd-subsets released 45.2% of CD8+/Tet+]. Th1 (IL-2,IFN-g,IL-12,TNF-a,TNF-b) and Th2-cytokines (IL- In conclusion, our preliminary fi ndings suggest that the CD28 4,-5,-6,-10,-13, Vd1 also TGF-b), chemokines (I-309,MCP1- signal on aAPC is indispensable for antigen-specifi c CTL 3,RANTES), interleukins (IL-1,-8,-15), chemokines expansion. However, the phenotype of CTL which determines (leptin) as well as angiogenic growth factors (ANG,VEGF,EGF, the function and survival in vivo may be controlled by different IGF-1). These molecules were expressed at higher levels defi ned co-stimulatory signals. in Vd2- than Vd1-T-cells. Neuroblastoma-challenge blocked Th2-cytokine and IFN-g release in both gd-T-cell subsets. In Vd2-T-cells, Th1-cytokines were down- and tumor growth- P908 promoting factors (ANG,VEGF, EGF, and IGF-I) were strongly Assessment of signalling events in antigen-specifi c upregulated. In contrast, Vd1-T-cells stopped the release of T-cells by combining phosphoepitope specifi c fl ow tumorsupportive factors and tolerogenic TGF-b, and strongly cytometry and MHC-multimer staining upregulated TNF-a and -b, MCP-1 and -2 and maintained R. Thiele, M. Kapp, E. Baumeister, K. Fick, G. Stuhler, A. Opitz, their IL-2 production. H. Einsele, G. Grigoleit Conclusions: Vd1- rather than Vd2-T-cells support antitumor Julius-Maximilians-University (Würzburg, DE) responses against Neuroblastoma by secreting proinfl am- matory cytokines. Furthermore Vd1-T-cells do not sustain a Flow cytometry has become a routine method in both clinical growth-promoting or tolerogenic microenvironment. These and basic immunological research. A main advantage is its abil- results make Vd1-T-cells an ideal candidate for upcoming ity to differentiate between distinct populations of cells by sur- immunotherapy trials in neuroblastoma. face staining of various parameters. For example, we have the possibility to identify antigen-specifi c T-cells by fl ow cytometry through the development of soluble multimeric peptide–MHC P907 complexes. Nevertheless, surface staining does not provide Priming and expansion of HLA-B*0702/CMV_pp65 information about the functionality of the respective cell popula- restricted CTL for adoptive immunotherapy by use of tions towards stimuli that are immediately refl ective of intracel- artifi cal antigen presenting complexes is essentially lular events. Hence, further methods have been described to infl uenced by differential triggering of co-stimulatory defi ne cells by detection of intracellular epitopes. These assays pathways include the intracellular staining of distinct cytokines or phospo- M. Kapp (1), K. Fick (1), S. Jackob (1), M. Oelke (2), R. Thiele rylated signaling molecules (Phosfl ow). (1), E. Baumeister (1), S.M. Tan (1), H. Wajant (1), J.P. Schneck MHC-multimer approaches combined with intracellular stain- (2), A. Opitz (1), H. Einsele (1), G. Grigoleit (1) ing are routinely used, whereas the detection of intranuclear (1)Julius-Maximilians-University (Würzburg, DE); (2)Johns p-kinases under MHC-multimer staining applying the Phos- Hopkins University (Baltimore, US) fl ow-protocols has not been realized so far. The use of phos- phoepitope analysis in antigen-specifi c T-cells is of high interest The ex-vivo priming and expansion of antigen-specifi c cytotoxic in infections or especially during immunosuppressive drug T cells (CTL) for transfer is fundamental in adoptive immuno- treatment. Therefore, we aimed to establish a dual multimer- therapy. However, the obstacles in generation of CTL using phospho-staining protocol to provide a method to get insight autologous APC namely dendritic cells are labor intensive and into the biochemical signaling processes in antigen-specifi c expensive besides it’s highly variable in quality and quantity. T-cells. We chose CTL responses against CMV as model sys- This has triggered the interest in the development of artifi cial tem due to well established epitopes and high frequency in antigen presenting complexes (aAPC) for reliable antigen-spe- healthy donors. The original Phosfl ow-protocols (Chow et al, cifi c CTL expansion. 2001; Nolan & Krutzik, 2003) did not turn out to be suitable for a In our study, we explored various co-stimulatory signals for the combination with the MHC-multimer approach. The very harsh possibility to induce higher stimulatory capability in aAPC. fi xation and permeabilization procedures largely or completely We loaded epoxy surface activated magnetic beads with HLA- abrogated the antigen-specifi c staining. We have been able to B*0702/CMV_pp65 (TPRVTGGGAM) monomer and activat- stain both the CMV-specifi c T-cell-receptor and the phosphor- ing antibodies (AB) against CD28 co-stimulatory molecules ylated kinase following polyclonal stimuli (e.g. PMA, IL-2 etc.) and/or ligands (L) of the TNF-receptor (TNFR) family namely using different protocols for some p-kinases. As far as we could CD28 (AB), ICOS (AB), 4-IBB (AB+L), CD27 (L), OX40 (L). Cell determine, these protocols for the fi rst time facilitate specifi c T- cultures were analyzed weekly by staining with HLA-B*0702/ cell-receptor staining with that of intranuclear phosphoepitopes CMV_pp65 (TPRVTGGGAM) tetramers and surface antibodies after polyclonal stimulation. We currently evaluate these proto- against CD3, CD8, CD45RA, CCR7, CD57 and CD25. cols under ex vivo conditions with CMV-specifi c artifi cial anti- Our results demonstrated that triggering of CD28 co-stimula- gen presenting cells and antibody-coated plates. As mentioned tory molecules is pivotal for aAPC besides the antigen-specifi c above, the use of phosphoepitope analysis in antigen-specifi c signal to ensure an optimal expansion of CMV-specifi c CTL. T-cells may offer the possibility to get insight into complex sig- This signal could not be replaced by neither ICOS nor 4-IBB naling processes in defi ned clinical situations, e.g. in immuno- alone or in combination. The addition of ICOS and/or 4-IBB to suppressed patients post Allo-SCT. CD28 co-stimulatory molecules did not signifi cantly improve the percentage of CTL after 3 weeks of culture. However, a trend towards higher cell numbers and specifi city was observed when combining all three CD28 (AB), ICOS (AB) and 4-IBB (AB).

S266 P909 MSCs often involve patients with malignant diseases, to obtain Escalating doses of donor lymphocytes to rescue from further insights on this issue is of crucial importance. We evalu- rejection following bone marrow transplantation for ated the effect of both autologous and third-party bone-marrow thalassaemia derived MSC on in vitro induction of leukemia-reactive cytotoxic I. Frugnoli, B. Cappelli, R. Chiesa, A. Noè, E. Biral, S. Gattillo, T-lymphocytes (CTL). The effect of MSCs was evaluated on T. Roccia, C. Evangelio, M. Fossati, S. Napolitano, A. Giardelli, fi ve different donor-derived anti-leukemia CTL lines generated C. Soliman, F. Ciceri, M.G. Roncarolo, S. Marktel through an experimental approach that we previously set-up. Scientifi c Institute H S Raffaele (Milan, IT) Anti-leukemia CTL lines were generated both in the presence and in the absence of MSCs (MSC/PBMC ratio 1:20). We found Mixed chimerism (MC) and secondary graft failure are frequent heterogeneity in terms of MSC capacity to affect the emergence events following BMT for thalassemia. There is limited informa- of anti-leukemia CTL response in vitro, irrespectively of the fact tion regarding the outcome of donor lymphocytes infusion (DLI) that employed MSCs were either autologous or third-party. to rescue from rejection, mainly from case reports describing The addition of autologous MSCs determined an enhance- only successful cases. ment of cytotoxic activity (mean increase: 45%) in three anti- We report our series of 6 middle east thalassemia patients leukemia CTL lines and an inhibition of cytotoxic activity in two (median age 7 years, range 4-13); Pesaro class II (N=2) or III lines (mean inhibition: 33%). Third-party MSCs determined an (N=4) treated with DLI for grade 2-3 (<90% donor) MC following enhancement of cytotoxic activity in three CTL lines (mean myeloablative Bu-Cy based BMT. In accordance to the sched- increase: 26%) and an inhibition in two lines (mean inhibition: ule used in chronic myeloid leukemia, patients transplanted 22%). These results were observed at an effector/target ratio from a sibling donor (N=5) received a fi rst dose of 107 CD3/kg of 10:1. When present, the reactivity of anti-leukemia CTL lines when MC was documented and, in the absence of side effects, against patient non-malignant cells was always reduced in the full response or rejection, a second dose of 5*107 CD3/kg presence of MSCs (mean inhibition: 77%). In four anti-leuke- one month later; 1 patient transplanted from a phenotipically mia CTL lines the addition of both autologous and third-party matched marrow was treated with 106 CD3/kg. Cyclosporin- MSCs inhibited the expansion rate of CTLs (mean inhibition: A was gradually tapered off and discontinued 1 year following 35%), while in one case a great expansion of CTLs was docu- BMT according to the transplant protocol with no dose modifi - mented (increase= 120%). The regulatory effect of MSCs was cation in DLI treated patients. Patients were evaluated monthly not related to an expansion of either CD4+ or CD8+ Treg. The on peripheral blood, CD3, CD19, CD56, CD15 subpopulations evaluation of cytokines production and of the level of differ- and bone marrow for donor chimerism. ent soluble mediators is in progress with the aim of clarifying In terms of safety, no graft versus host disease, aplasia or the biological mechanisms underlying the discordant results other severe adverse event were documented. Only one obtained on the immune modulatory effects of MSCs on anti- patient, infused when donor chimerism was 65%, experienced leukemia CTLs. transient grade 3 thrombocytopenia followed by autologous reconstitution. In terms of effi cacy, we observed a different pattern of response P911 in class II and class III patients. Among the 2 patients in class II, Pre-treating human NK cells with dasatinib increases the fi rst patient, transfusion independent post BMT, was infused cytotoxicity against resistant Daudi but not sensitive on day 90 (MC 65%, CD3+ 40%) and day 176 (MC 50%, CD3+ K562 or Jurkat cells – An example for differential 60%), and converted to full donor following the second DLI. immunomodulation The second patient received DLI on day 174 (MC 82%, CD3+ N. Hassold, K. Seystahl, C. Dix, J. Wischhusen, H. Einsele, 90%) when transfusion dependent and converted to full donor R. Seggewiss 1 month later becoming transfusion independent. These results University of Würzburg (Würzburg, DE) were not confi rmed in class III patients (N=4)where DLI were never successful in preventing secondary graft failure. Objectives: Immunotherapies have not been as successful as In conclusion, escalating doses of DLI was safe in a cohort of 6 expected. Thus, pharmacologic approaches to enhance NK cell patients with MC following myeloablative BMT for thalassemia cytotoxicity and/or increasing tumor ligand expression are war- but effi cacy was achieved only in class II patients. We specu- ranted. Tyrosine kinase inhibitors such as dasatinib may offer late that class II patients are less alloimmunized and therefore a such an approach. While Blake et al. (2008) have reported that longer coexistance of host and donor marrow allows time to DLI dasatinib inhibits NK cell cytotoxicity, we have observed differ- to eliminate residual host hemopoiesis; class III patients prob- ential effects of clinically relevant doses (10-50nM) on NK cell ably have stronger alloimmunity hesitating in immune mediated functions against different tumor cell targets. rejection of donor cells despite DLI. Methods: NK cells from healthy human donors were expanded in coculture with RPMI 8866 feeders yielding a median NK cell purity of 77% (range 65-91%). Functional outcomes assessed P910 included cytotoxicity, CD107a/b expression, TNFa/INFg Infl uence of bone marrow-derived human mesenchymal production and apoptosis/necrosis induction. stem cells on the in vitro induction of anti-tumour T-cell Results: Expansion of NK cells was completely inhibited at responses 50nM (n=4, p<0.001) as well as dose dependently NK cyto- D. Montagna (1), F. Locatelli (1), E. Montini (2), M. Avanzini (2), toxicity against Daudi, Jurkat and K562 cells (n=3, p<0.001 for L. Caliogna (2), I. Turin (2), A. Moretta (2), A. Mastronuzzi (2), all cell lines) when dasatinib was added in the 4h cytotoxicity V. Spartà (2), I. Possenti (2), M. Bernardo (2), R. Maccario (2) assays. 24h pre-treatment of K562 cells with 50nM dasatinib led (1)University of Pavia (Pavia, IT); (2)Fondazione IRCCS Pol. to signifi cantly reduced cytotoxicity (n=3, p<0.001) in opposite San Matteo (Pavia, IT) to pre-treating Daudi and Jurkat cells (n=3). Instead, 24h pre- treatment of NK cells led to an increased cytotoxicity against Human mesenchymal stem cells (MSCs) are endowed with Daudi (n=3, p<0.001) but not against K562 and Jurkat cells. immune modulatory properties. Because of these proper- TNFa/INFg production and CD107a/b expression was inhibited ties, MSC have been tested in the fi eld of hematopoietic stem by dasatinib when NK cells were co-cultured with K562 cells cell transplantation where they proved to be able to improve (n=3) but unaffected after PMA/calcium-ionophore induced engraftment of donor haematopoietic stem cells and to res- stimulation (n=3). Interestingly, the K562 induced TNFa/INFg cue patients with steroid-resistant graft-versus-host disease production was increased in dasatinib pre-treated NK cells (GVHD). Despite these encouraging clinical data, limited and (n=3). The observed effects were not due to a decreased via- confl icting results are available on the effect of MSCs on anti- bility of NK cells as apoptosis/necrosis rates were unaffected tumor immune surveillance. As therapeutic applications of (n=4).

S267 Conclusion: SRC kinase-dependent cytokine production and P913 degranulation was reduced raising concerns regarding NK Developing a murine model to study B cells as APC in cell function impairment. Hampered NK cell cytotoxicity when vivo dasatinib was present in the cytotoxicity assay may be due to T. Liebig (1), A. Shimabukuro-Vornhagen (1), N. Klein-Gonzalez an inhibition of SRC kinase-dependent RAS/RAF/MEK/ERK- (1), E. Kondo (1), C. Wickenhauser (2), M. von Bergwelt- pathway. Interestingly, dasatinib pre-treated NK cells showed Baildon (1) increased cytotoxicity against Daudi but not against Jurkat (1)Lab for Tumor /Transplantation Immunol (Cologne, DE); and K562 cells, which suggests that dasatinib may block sig- (2)Pathology (Cologne, DE) nals from certain activating and inhibitory receptors, making the overall effect dependent on cell line specifi c receptor/lig- Introduction: As Dendritic cells (DC) are effective conductors of and expression. Our fi ndings argue for a careful evaluation of adaptive and innate immune responses, they are used as cellu- the timing of dasatinib application during tumor treatment to lar adjuvant for active immunotherapy. Nevertheless, challenges enhance immunotherapeutic efforts. to this approach have been identifi ed. Recently CD40-activated B cells (CD40-B) have been studied as complementary antigen presenting cells. They can be expanded from small amounts P912 of PB at >95% purity under GMP-like conditions and effi ciently Treatment of threatening rejection after umbilical cord prime and expand naïve T cells in vitro. Importantly these cells blood transplantation with ex vivo expanded CB derived are available at virtually unlimited amounts for high-dose, high- T-cells frequency vaccination considered crucial for the control of can- M. Uhlin, M. Okas, J. Gertow, J. Mattsson cer in vivo. Karolinska University Hospital (Huddinge, SE) Methods and results: For preclinical in vivo testing using high- dose, repetitive vaccinations with CD40-B we developed a For patients lacking a human leukocyte antigen (HLA)-matched murine system to study their ability to induce immune responses donor, umbilical cord blood (UCB) is a promising source of in vivo. mCD40B are generated from murine splenocytes by co- hematopoetic stem cells. Greater HLA disparity can be toler- culture with murine CD40-Ligand and addition of IL-4. mCD40B ated between the recipient and donor UCB compared with bone cells could be expanded more than 6-fold within 14 days and marrow or peripheral blood stem cells because of the naive and >90% purity. mCD40B were used to establish a vaccination immature phenotype of UCB derived T cells. The risk of rejec- model to study induction of tumor- and auto-antigen specifi c tion is increased after UCB transplantation. After HLA-identical immune responses as well as toxicity of very-high-dose injec- sibling or matched unrelated SCT the graft-versus-leukemia tions (VHD) in B6 mice. To determine long-term toxicity mice (GVL) effect may be increased by donor lymphocyte infusion were vaccinated twice weekly for 5 weeks using high-doses (DLI) after SCT. However, after UCB transplantation DLI is not (3x106 ~ 1x108/kg) of mCD40B. This is about 2-3 log higher possible. than currently used in DC vaccinations in humans. Acute toxic- This raised the question of whether ex vivo expanded CB lym- ity was assessed by vaccinating mice once with VHD (1x107 phocytes (CBL) also can be used as a tool for adoptive immu- ~ 3.3x108/kg). There was no difference in survival, weight and notherapy after CB SCT. clinical appearance compared to control mice. Histo-pathologi- We have managed to establish a protocol for massive expan- cal assessment did not reveal any pathologic changes. Murine sion of CB derived T cells making them usable in the clinic Mixed Lymphocyte Reaction was established to investigate the suitable for DLI after CB transplantation. We have further been immune-stimulatory capacity of mCD40B in comparison to mDC able to show that the expansion protocol doesnt skew the T through detection of T cell proliferation in vitro. Immune monitor- cell population regarding the phenotype, CD4:CD8 ratio as well ing by intracellular cytokine staining for IFN-g and assessment as TCR usage profi le measured by spectratyping. By activat- of in vivo cytotoxicity revealed that mCD40B vaccination leads ing the cells we have further investigated and confi rmed the to increased IFN-g production by activated T cells and specifi c expanded T cells capacity to effi ciently produce pro-infl amma- lysis of CFSE-labelled peptide-pulsed target cells. tory cytokines and respond in an allogeneic setting. Conclusion: Based on these fi ndings that mCD40B induce We have now tried expanded CBLs in an adult patient with AML T cell responses in vitro and in vivo we are now developing with threatening rejection after double UCB transplantation. the optimal mCD40B vaccination algorithm for preventive and The patient received Bu/Cy and ATG as conditioning therapy. therapeutic treatment of B16-Melanoma in B6 mice as a next He was transplanted with double UCB with a total nucleated step towards future clinical application in post transplant donor- cell dose of 5 x 107/kg. Both UCB units were 5/6 matched for lymphocyte-infusion or vaccination. HLA-A,-B and -DRB1 with low resolution HLA-typing. Chimer- ism analysis one month after transplant showed mixed chimer- ism in T-, B- and myeloid cells of both donor units and recipient. P914 At four months after transplant the patient had 85% recipient Cytokine-induced killer cells are terminally differentiated cells in both T cells and myeloid cells indicating a threatening activated CD8 cytotoxic T EMRA lymphocytes rejection. However, in bone marrow CD34+ cells were 90% of A. Pievani (1), M. Franceschetti (1), G. Borleri (1), L. Vago (2), donor origin. Due to anticipated rejection the patient was treated K. Fleischhauer (2), J. Golay (1), M. Introna (1) with expanded CBLs, 5 x 103/kg at 4 months, 1 x 104/kg at 5 (1)Laboratory of Cellular Therapy G. Lanzani (Bergamo, IT); months and 1 x 105/kg at six months. Immunusuppression was (2)San Raffaele Hospital (Milan, IT) tapered at six months. The immunemodulatory treatment was well tolerated with no development of GVHD. So far no change Objectives: Cytokine induced killer (CIK) cells are CD3+CD56+ in chimeric pattern has been shown. The immunemodulatory cells obtained by in vitro stimulation of peripheral blood mono- treatment will be continued with increasing doses of CBLs. nuclear cells (PCMCs) with INF-gamma followed by anti-CD3 antibody and expanded for 21-28 days in the presence of IL-2. CIK cells show considerable NK-like cytotoxic activity against tumor cells of several lineages, in particular hematologic neo- plasms, and potent anti-tumoral activity in vivo when injected in mice carrying either murine or human tumors with little GVHD. We aimed to obtain a full ontogenic and functional characteri- zation of CIK cells that represent a valid tool for the immuno- therapy of tumors. Methods: CIK cells were generated in vitro by stimulation of PCMCs or sorted T cell subsets with IFN-gamma, anti-CD3 and

S268 IL-2. They were fully characterized in terms of phenotype, cyto- apoptosis are underway. In contrast detection of alloreactivity toxic activity and gene expression in comparison with CD56- T was negligible. The reason for that will also be part of further cells and NK cells also present at the end of CIK cultures, and investigations. with circulating CD3+CD56+ cells. Conclusion: Based on their anticancer activity and non-allore- Results: We demonstrate that CIK derive from proliferating activity, CIK cells might represent an effective tool for cellular CD3+CD56-CD8+ T cells and not from the few CD3+CD56+ therapy in patients with acute leukemia, rhabdomyosarcoma cells present in the starting culture. They express polyclonal T and ewing sarcoma undergoing an allogeneic transplantation. cell receptor Vbeta chains and have acquired CD56, NKG2D Supported by the Kind-Philipp Stiftung (BV) and Else Kröner- and large granular lymphocyte morphology, but lack expres- Fresenius-Stiftung (PB). sion of most NK specifi c activating (NKp30, NKp44, NKp46) and inhibitory (KIR2DL1, KIR2DL2, KIR3DL1, NKG2A, CD94) receptors, and can kill K562 targets. Circulating CD3+CD56+ cells are also CD8+CD16- but are oligoclonal, poorly cytotoxic for K562 and express lower levels of CD56 and NKG2D. Gene Aplastic anaemia profi ling of CIK, CD56- T and NK cells present at the end of culture shows that differences are much more limited between CIK and CD56- T compared to CIK and NK cells. CIK cells P916 express many genes shared by the “infl ammatory” memory T Bone marrow transplantation for children and cells. Interestlingly, most of the genes up-regulated in CIK cells adolescents with severe aplastic anaemia: analysis of compared to CD56- T cells are part of the TNF gene network. 75 pts heavily transfused patients treated with Conclusion: CIK cells are terminally differentiated CD8 T cells. busulfan + cyclophosphamide They differ from CD56- T cells present at the end of culture in C. Bonfi m, S. Fortier, M. Bitencourt, L. Medeiros, J. Ruiz, terms of expression of CD56, NK cytotoxicity and proliferation. M. Oliveira, A. Koliski, D. Setubal, V. Funke, E. Nunes, CIK phenotype, that is CD45RA+, CCR7-, CD62L weakly posi- J. Morando, C. Medeiros, R. Pasquini, J. Zanis-Neto tive, CD11a+, CD27+, CD28-, MIP1alpha+, Perforin+, FASL+ Federal University of Parana (Curitiba, BR) coincides almost exactly with that described for the T effector memory RA+ (EMRA) CD27 single positive subset of terminally Introduction: Bone Marrow Transplantation (BMT) is the treat- differentiated human memory T cells. Therefore, CIK cells may ment of choice for pts with Severe Aplastic Anemia(SAA). Unfor- represent an even more advanced stage of differentiation of tunately rejection is still very high in many countries and this is CD56- T cells. probably related to the quality and the number of previous blood transfusions. Since 1993 we added Busulfan to the prepara- tory regimen of our pts in order to decrease the incidence of P915 rejection. In this study we will analyze the overall survival(OS) Generation of cytokine-induced killer cells for adoptive and risk of rejection in 75 children and adolescents with SAA immunotherapy submitted to a BMT using HLA matched siblings. E. Rettinger, S. Kuci, G. Weber, B. Voss, S. Kloess, A. Willasch, Material and Methods: Period: 01/1993- 01/2007. Age: H. Kreyenberg, U. Koehl, P. Bader, T. Klingebiel 2-18y(M:13y). Gender: 26F/49M. Neutrophils/µl before BMT: Goethe University Frankfurt (Frankfurt, DE) 7-1705/µL (M:322). Previous blood transfusions: 16-103U(M: 22). Disease duration: 1-114m(M:4m). Total nucleated cels:4-7,0x Introduction: Allogeneic SCT has become an important treat- 10*8/kg(M:3,4x10*8/kg). All patients received bone marrow from ment modality for patients with high risk leukemia and is also their HLA identical siblings. Preparatory regimen: Bussulfan12mg/ increasingly used as a therapeutic tool for children and ado- kg+ Cyclophosphamide120mg/kg and GVHD prophylaxis with lescents with solid malignancies like rhabdomyosarcoma cyclosporine and methotrexate. Survival was analyzed by Kap- and ewing sarcoma. Nevertheless, relapse still remains the lan-Meyer, the log-rank test and Cox regression, and categorical major cause for treatment failure. Adoptive immunotherapy variables where tested by X2 and Multinomial logistic regression. approaches using cytokine induced killer (CIK) cells might help Results: 52/75pts are alive between 1,9-15,9years (M:9,9ys) to prevent relapse in these cases. after BMT with an overall survival of 70%(5ys). 71pts sur- CIK cells are ex vivo activated and expanded non-MHC vived more than 28 days and were evaluable for engraftment. restricted lymphocytes including basically “NK-like” T cells, T Primary graft failure(PGF) occurred in 4% while late graft helper cells, cytotoxic T cells and NK cells. The entire popula- failure(LGF) occurred in 22% between 218-3665 days after tion is known for a notable anticancer toxicity compared to a BMT (M:540d). Children with age ≤ 10ys had more LGF than moderate alloreactivity. older pts(42%x22% p<0,009). Pts admitted to the BMT unit Methods: For CIK cell generation peripheral blood mononu- with ANC>320/µ/l had more LGF than those with ANC <320/µl clear cells were expanded over 2 to 3 weeks by addition of (p<0.001). All pts with PGF died while 60% of pts with LGF IFN-g (1000U/ml) on day one and OKT-3 (50ng/ml) as well as were rescued with a 2nd BMT or immunosupression and are IL-2 (500U/ml) within the following 24 hours of culture. Culture alive and well. Other complications: Mucositis grade III-IV:38%; medium and IL-2 were changed every 3-4 days. By the time the hemorrhagic cystitis:9%; Acute GVHD grade III-IV:20%; percentage of CD3+ CD56+ cells increased the whole popula- Extensive chronic-GVHD:17%. Severe bleeding:20%.Twenty- tion and all subgroups were analyzed for their phenotype and three pts died between 15-1455days after BMT(M:154d). cytotoxic mechanism against rhabdomyosarcoma and leuke- Causes of death: Rejection:8pts; fungal or bacterial infec- mia cell lines. In addition alloreactivity was tested by europium tions: 8pts; GVHD+infections: 4pts; severe bleeding:2pts and release assay. bronchiolitis:1pt.TRM at 100 days:12%. Results: By using this protocol easily expanded CIK cells Conclusions: 1) Overall survival was adequate for this group of showed an excellent cytotoxicity against alveolar rhabdomy- heavily transfused pts especially for pts above the age of 10. 2) osarcoma (RMS13), T-ALL (H9, Molt4/8), B-ALL (Raji) and Children with age ≤ 10 had a higher incidence of LGF as well AML (THP-1) cell lines even within 7 to 10 days of culture. as all pts admitted with ANC> 320/µl. For this group of pts, this Using the europium release assay up to 60% of RMS13 cells, regimen was not suffi cient to reduce the incidence of LGF and 75% of Molt4/8 cells, 85% of H9 cells, 60% of THP-1 cells and other approaches should be used. 35% of Raji cells were killed. Particularly the NK cell subgroup appeared to have a main part in antitumor and antileukemia activity. In this regard blocking MICA/B and ULBP2 resulted in a more than 50%-reduction of cell lysis. Further experiments concerning the killing mechanism of CIK cells, e.g. the role of

S269 P917 These results suggest that alternative donor transplants can be Results of haematopoietic stem cell transplantation safely performed in patients with acquired aplastic anemia, also in children with severe aplastic anaemia in Hungary in adults. The persisting risk of rejection may be due to the low (1992–2008) dose of cyclophosphamide (CY) which could be signifi cantly G. Krivan (1), K. Kállay (1), G. Vértesi (2), G. Benyó (1), increased. G. Márton (2), R. Simon (2), Á. Tóth (1), K. Nagy (2) (1)St. László Hospital (Budapest, HU); (2)Postgraduate Medical School (Miskolc, HU) P919 Allogeneic haematopoietic stem cell transplantation with Objectives: The authors have summarized retrospectively cyclophosphamide and alemtuzumab for severe aplastic the results of allogeneic stem cell transplantation in pediat- anaemia ric patients (<18y) with severe aplastic anaemia performed F. Lodi (1), M. Laercio (2), G. Fischer (3), C. Rodrigues (4), between 01.01.1992. and 30.09.2008. in Hungary. A. Macedo (1), S. Magalhães (1), R. Lamego (1), L. Fogliatto Results: During this period 43 transplants were performed in (3), J. Oliveira (2), H. Bittencourt (1) 38 pts (5 re-transplantation: 3 graft rejection, 1 graft failure, (1)Hospital das Clinicas (Belo Horizonte, BR); (2)HSP/UNIFESP 1 late clonal disease /paroxysmal nocturnal haemoglobinuria/). and Hospital Santa Marcelina (Sao Paulo, BR); (3)Complexo Median age of pts. was 10 years (1,8-17,8). 27 sibling and 16 Santa Casa (Porto Alegre, BR); (4)HSP-UNIFESP (Sao Paulo, alternative (9 matched unrelated, 5 mismatched unrelated, 2 BR) haplo) donors were used. Median elapsed time from diagnosis to transplant was 85 days (17-1826) (matched related donor/ Background: Allogeneic Hematopoietic Stem Cell transplanta- MRD/: 42 days, alternative donor 294 days). Source of graft tion (AlloHSCT) with cyclophosphamide (Cy) and antithymo- was bone marrow in 25 transplants, peripheral blood in 17 cyte globulin (ATG) as conditioning regimen is the treatment of cases and umbilical cord blood in one patient. Conditioning choice for young patients with severe aplastic anemia (SAA). regimen consisted of mainly anti-thymocyte globulin (ATG)- In developing countries, and particularly in Brazil, ATG costs Cytoxan (19), Fludara-Cytoxan-ATG (7) or Cytoxan alone (5). limit its use in AlloHSCT for SAA patients. Alternative low-cost Short course of cyclosporin A-methotrexate combination was regiments, like low dose busulfan with Cy as conditioning regi- used as graft versus host (GvH) profi laxis in 34 cases. 39/43 men, has been used but is still associated with a higher rate transplants have engrafted (absolute neutrophil counts > 0,5 G/ of rejection, especially in heavily transfused patients. Recently, l: 19 days; platelet > 20 G/l: 20 days), rejections were observed alemtuzumab (Cam) was reported as an alternative to ATG for in 4 cases. Acute (II-IV) and chronic GvHD were developed in SAA patients with similar activity and a lower cost. 16/38 and 5/31 cases, respectively. 8/38 pts. have died (bacte- Aims and Methods: In order to study the effect of the combination rial septicaemia 2, fungal infections 2, tuberculosis 1, posttrans- Cam+Cy, we reviewed all AlloHSCT performed for SAA using plant lymphoproliferative disease 1). After a median of 1553 this conditioning regimen. Between April 2007 and November days 30/38 pts (79%) are alive; overall survival of patients with 2008, thirteen patients with SAA (defi ned by Camitta criteria) MRD and alternative donor was 21/24 (88%) and 9/14 (64%), underwent an AlloHSCT in four different institutions in Brazil. respectively. Median age at transplantation was 30 (range 11-42) years. All Conclusions: While the results of MRD transplants are com- patients had a positive CMV serology. Median number of trans- parable with other national registries, the unacceptably long fusions was 16 (range 0-172). One patient received Cam+Cy elapsed time from diagnosis to transplant has infl uenced unfa- as a second allo-HSCT. Patients received an unmanipulated vourably the outcome of transplants with alternative donors. bone marrow (n=11) or peripheral blood (n=2) graft as stem cell source and all but one patient were transplanted with an HLA-identical sibling. Median number of nucleated cell infused P918 was 3.5 (range 1.65-4.80)x108/kg. Cyclosporin alone (n=8) or Fludarabine, cyclophosphamide with or without low dose in combination with methotrexate (n=5) was used as GVHD TBI for alternative donor transplants in acquired aplastic prophylaxis. anaemia: a report from the EBMT-SAA Working Party Results: twelve out of 13 patients presented neutrophil recovery A. Bacigalupo (1), F. Locatelli (2), E. Lanino (3), J. Marsh (4), with a median time to > 0.5x109 neutrophil/L of 23 (range 13- G. Socié (5), S. Maury (6), A. Ibatici (1), A. Prete (7), 28) days. Platelet recovery (> 20x109 platelets/L) occurred in all A. Locasciulli (8), S. Cesaro (9), J. Passweg (10) patients with a median time of 15 (range 9-45) days. Acute graft (1)Ospedale San Martino (Genoa, IT); (2)Ospedale San versus host disease (GVHD) was observed in just one patient Matteo (Pavia, IT); (3)Ospedale Gaslini (Genoa, IT); (4)St (grade II). One of 11 patients alive 100-days after AlloHSCT George Hospital (London, UK); (5)Hopital St Louis (Paris, presented a limited chronic GVHD. Seven patients presented FR); (6)Hopital Henry Mondor (Créteil, FR); (7)Ospedale Sant’ a CMV reactivation and none presented CMV disease. With a Orsola (Bologna, IT); (8)Ospedale San Camillo (Rome, IT); median follow-up of 293(range 32-544) days, only one patient (9)Clinica Pediatrica (Padua, IT); (10)Hopitaux Universitaires died of AlloHSCT complications. (Geneva, CH) Summary: In conclusion, the combination of cyclophospha- mide and alemtuzumab is well-tolerated and effective in SAA We are reporting the outcome of 80 patients with acquired patients undertaking AlloHSCT, with lower rates of acute and severe aplastica anemia (SAA) allografted from an alternative chronic GVHD. A longer follow-up is required, however, to prop- donor, after conditioning with fl udarabine, cyclophosphamide erly evaluate late rejection incidence. (FLU-CY), with or without the addition of low dose total body irradiation (2 Gy TBI). All patients also received rabbit ATG and cyclosporine- methtrextae for GvHD prophylaxis. The FLU-CY regimen was given to 40 patients (median age 13) and the FLU- CY-TBI to 35 patients, with a median age of 30 GvHD grade III- IV was seen in 1 patient in each group. Rejection was the cause of death in 3 patients in the FLU-CY group and in 2 patients in the FLU-CY-TBI group. The overall crude survival (80%) was excellent in both groups: the actuarial 5 year survival is 79% and 77%. The median interval from diagnosis to transplant was 582 days for all patients: survival was 87% vs 70% for patients grafted before or beyond that cut off. Causes of death were rejection (5), PTLD (2), infections (5), hemorrhage (2).

S270 P920 myelodisplastic syndrome, at median interval of 181 months A pilot study of anti-interleukin-2 receptor antibody (limits 5 years 10 months and 24 years 5 months). Overall sur- daclizumab combined with cyclosporine as salvage vival was 166 months (limits 5 years and 27 years 7 months). immunosuppressive therapy for refractory or relapsed In conclusion, a long survival, in selected patients with aplastic aplastic anaemia anemia could be obtained also with nontransplant regimens of A. Kulagin (1), I. Lisukov (1), I. Kruchkova (2), V. Sergeevicheva therapy. (2), A. Gilevich (2), S. Sizikova (2), O. Korolkova (2), V. Kozhevnikov (2), V. Kozlov (2) (1)Novosibirsk State Medical University (Novosibirsk, RU); (2)Institute of Clinical Immunology SB RAMS (Novosibirsk, RU) Autoimmune diseases

Objective: To evaluate effi cacy of daclizumab combined with CsA in adult patients with refractory or relapsed AA. P922 Patients and Methods: We treated 6 AA patients (VSAA - 1, Autologous stem cell transplantation for refractory SAA - 4, NSAA - 1) with daclizumab (1 mg/kg every 2 weeks systemic lupus erythematosus: clinical effects and for a total 6 doses) and CsA (5 mg/kg/d) who relapsed (n=1) or immune reconstitution did not achieve PR (n=5) after one (n=2) or two (n=4) courses I. Lisukov (1), V. Sergeevicheva (2), S. Sizikova (2), A. Kulagin of ATG and long-term therapy with CsA ± MMF. Median age (1), I. Kruchkova (2), A. Gilevich (2), A. Sizikov (2), L. Konenkova was 20 years (range 19-32); median disease duration was 19 (2), E. Chernykh (2), E. Kurganova (2), V. Kozhevnikov (2), months (7-21) in non-responders and 36 months in relapsed N. Pronkina (2), E. Blinova (2), V. Borisov (2), T. Sentyakova patient; median period from fi rst and second ATG course were (1), A. Demin (1), V. Kozlov (2) 12 (6-18) and 6 months (2-9) respectively. Minor PNH clone (1)Novosibirsk State Medical University (Novosibirsk, RU); was detected in 4 of 6 patients. (2)Institute of Clinical Immunology SB RAMS (Novosibirsk, Results: Daclizumab was well tolerated in all cases. At median RU) follow-up of 36 months all 6 patients are alive. Three patients (50%) achieved a good PR and the last treated patient cur- High-dose immunosuppression and autologous hemopoietic rently does not meet criteria for a PR but continues to improve. stem cell transplantation (ASCT) has been proposed as an Two patients (including relapsed) did not respond to daclizu- investigational therapy for patients with refractory autoimmune mab. The patient with relapse received second course of ATG diseases including SLE. We report the results of a single-center and PR was achieved. Another non-responder was success- study of immune reconstitution following ASCT in 15 female’s fully transplanted from MRD. Median time to response was 4 patients in our Institution from 1998 to 2008. months (range 3-6). One patient relapsed in the 21 months Methods: Autologous hematopoietic stem cells were collected after the course of daclizumab and responded again to rabbit from bone marrow (n=4) or mobilized from peripheral blood ATG. Some minor laboratory improvements which not fulfi lling with either granulocyte colony-stimulating factor (G-CSF) (n=2) PR to previous intensive IST and subclinical PNH were present or Cy and G-CSF (n=9). Pretransplant conditioning regimens in all three responders before start of daclizumab. included BEAM (n=1), BEAM + ATG (n=1) Melphalan 140 mg/ Conclusion: Moderate and prolonged immunosupression with m² + Etoposid 1600 mg/m² (n=2), Melphalan 120 mg/m² (n=1), daclizumab and CsA might become a new salvage protocol Cy 200 mg/kg (n=2), Cy 200 mg/kg + ATG (n=1), Cy 120 mg/kg for AA, especially for slowly responding to standard IST PNH+ (n=7). In one patient we used co-transplantation of PBSC and patients. expanded mesenhymal stromal cells. Results: Three patients died due to transplant-related com- plications (sepsis, CMV infection). These patients had long P921 history of the corticosteroid treatment, multiple and severe More than fi ve years survival of aplastic anaemia patients episodes of infections pre-ASCT. All of the alive transplanted receiving non-transplant treatment patients showed improvement in disease activity: CR (n=11), A. Moicean, A.M. Popp, M. Brinza, C. Calugaroiu, T. Puscariu PR (n=1). One patient died after 8 years due to relapse and on behalf of the Romanian Working Group for Aplastic Anemia one patient in PR developed a relapse 3 years after ASCT. Long-term clinical remissions observed in our SLE patients According to World Bank data, released in the 2008 report, are accompanied by the disappearance of anti-ds DNA and Romania has an upper-middle-income economy. The health ANA antibodies and increasing of number of CD4+CD45RA+ budget is low and most of expansive treatment for hematologi- T cells, CD4+CD25+bright T cells and CD4+Foxp3+ cells. We cal diseases are not large available. also demonstrated the signifi cant increase of CD4+ and CD8+ Nine three patients with aplastic anemia (AA) was taken in T cells in S/G2M phase of the cell cycle until 1 year after ASCT. charge by our institution between 1986 and 2002. Eighteen of This data may demonstrate that ASCT can induce the homeo- 93 patients survived more than 5 years. We have studied the static proliferation of T cells and may form the basis for immune clinical characteristics and treatment of them. reconstitution. There were 8 female and 10 male, aged between 15 and 54 Conclusion: Our data demonstrate that ASCT in refractory years, median 25 years. Eleven patients had severe AA and SLE can induce stable long-term remission. The assessment 7 patients had moderate AA. Fifteen out of 18 patients were of immune reconstitution can be important to understand the transfused erythrocytes dependent and 8 out of 18 patients mechanisms of self tolerance re-establishing. International were transfused thrombocytes dependent. First line treatment multicenter clinical trial would be required to clarify these was in 9 patients with Cyclosporine A (CsA), in 5 patients with questions. danazol and in 3 patients with methyl-testosterone. Eight out of 18 patients had complete remission (3 with CsA, 3 with dana- zole and 2 with methyl-testosterone). Ten patients had partial response (6 with CsA, 2 with danazole and 2 with methyltes- tosterone). Average time until response was 231 days, limits 9 to 950 days (~32 months). Two patients relapsed, both after a fi rst response obtained with CsA, at 3 years and at 5 years and 9 months respectively. The second response was obtained in both cases with CsA too. Five out of 18 patients had a sec- ond disease: 1 paroxysmal nocturne hemoglobinuria and 4

S271 P923 matory response. Here we report two cases of a 6-year-old Development of antinuclear antibodies with new boy and a 24-year old women with coeliac disease (CD) and specifi cities in systemic lupus erythematosus after thalassaemia major who received allogeneic HSCT. Patients autologous haematopoietic stem cell transplantation were assigned to risk class 1 and 3 of the Pesaro classifi cation, suggests de novo development of disease rather than respectively. The donor was an HLA-identical sibling and an lupus reactivation unrelated donor (UD), respectively; the latter was typed using T. Alexander, A. Thiel, O. Rosen, G. Massenkeil, G. Burmester, high-resolution techniques for both class I and II HLA loci. Con- H. Radtke, R. Arnold, F. Hiepe ditioning regimen included busulfan (16 mg/Kg), thiotepa (8 mg/ Charite University Medicine (Berlin, DE) Kg) and fl udarabine (40 mg/m2). GvHD prophylaxis was based on the use of Cyclosporine (CsA) and short term methotrexate. Introduction: In recent years, clinical trials have indicated that Pre-transplantation antithymocyte globulin was administered immunoablation followed by autologous hematopoietic stem in the patient transplanted from a UD. CsA was given for 12 cell transplantation (ASCT) has the potential to induce long- months after HSCT. DC and lymphocyte populations, includ- term clinical remission in patients with severe autoimmune ing FoxP3+ Treg, as well as the proliferative T cell response to diseases, including refractory systemic lupus erythematosus chymotrypsin-digested gliadin (1-10-20 ug/ml) were studied at (SLE). However, relapse of disease may occur in a fraction of time of CD diagnosis and after HSCT, during both gluten-free these patients. and gluten-containing diet. Methods: As part of a monocentric phase I/II clinical trial, seven The 2 patients successfully engrafted, showing 100% donor patients have received immunoablation with cyclophosphamide chimerism. After discontinuation of immunosuppressive therapy, and antithymocyte-globulin and CD34-ASCT for refractory the establishment of a gluten-containing diet did not reinduce SLE. Peripheral blood lymphocytes were analyzed using mul- positivity of both serological and histological markers of CD tiparameter fl ow cytometry. In addition, autoantibody titers and after a 2 year follow-up. In both patients, the cytometric char- specifi cities were monitored during follow-up. Disease activity acterization of peripheral blood cells showed a depletion either was measured by SLEDAI. in the % or in the absolute value of both DC and Treg at time Results: With a median follow-up of 80 months, clinical (SLEDAI of diagnosis (22-24 cells/ul, 0.2-0.2% and 96-83 cells/ul, 8-7%, < 3) and serologic remission (disappearance of anti-dsDNA respectively) in comparison to values found after HSCT dur- antibodies) could be achieved in all patients. Three patients ing both diet regimens (41/54-39/48 cells/ul, 0.6/0.7%-0.7/0.7% suffered relapse of SLE after being free of clinical symptoms for and 196/222-177/191 cells/ul, 18/20%-17/18%, respectively). 18, 30 and 80 months, respectively. Immunophenotypic analy- A critical reduction of proliferative T cell response to gliadin at ses revealed signifi cant expansion of circulating B cells with any doses tested was observed after HSCT. Our data suggest memory (IgD-CD27+) and plasmablast phenotype (CD27high that substitution of patients’ immune system with the donor CD20-), and increase in CD45RO+ memory T cells six months one, possibly together with phenomenon of recapitulation of prior to onset of relapse. Recurrence of antinuclear antibod- immune system ontogeny, may result into induction of immune ies (ANA) preceded clinical symptoms in two patients by 2 and tolerance towards gluten. The recovery of a normal value of 36 months, respectively, while in one patient ANA persisted circulating DC and Treg may play a major role in silencing glia- after ASCT. Serologic analysis revealed changes in the ENA din-reactive T cells. pattern in all three patients. One patient developed antinuclear antibodies specifi c for Smith-Antigen (Sm) while anti-Ro/SSA and anti-La/SSB antibodies disappeared. In contrast, the other P925 two patients newly developed anti-Ro/SSA and anti-La/SSB Reversal of neurologic disability after non-myeloablative antibodies while anti-Sm and anti-Histone antibodies disap- autologous haematopoietic stem cell transplantation for peared after the fl are. relapsing-remitting multiple sclerosis Conclusion: The disappearance of pathogenic serum antibod- R.K. Burt, J. Bucha, F. Milanetti, A. Testori ies points to the effi cient depletion of immunological memory Northwestern University (Chicago, US) while recurrence of naïve B- and T cell subsets suggests a complete reconfi guration of the adaptive immune system We evaluated the safety and clinical outcome of autologous after ASCT. The recurrence of lupus activity observed in three non-myeloablative HSCT in relapsing-remitting multiple sclero- patients after ASCT was accompanied by the development sis (RRMS) failing interferon therapy. of antinuclear autoantibody with new specifi cities suggesting Eligible patients had RRMS and despite interferon beta had 2 the de novo development of SLE rather than fl are of disease. corticosteroid treated relapses within the prior 12 months, or We propose that ASCT has the potential to reinduce self- 1 such relapse and gadolinium enhancing lesion(s) on mag- tolerance in SLE, which is apparently not maintained in all netic resonance imaging (MRI) demonstrated on an occasion patients maybe by the presentation of autoantigens in a toler- distinct from the relapse. Peripheral blood hematopoietic stem ance-breaking form in genetically susceptible individuals. cells (HSC) were mobilized with cyclophosphamide (2.0 g/m²) and granulocyte colony stimulating factor (10 ug/kg/day). HSCT conditioning regimen was cyclophosphamide (200 mg /kg) and P924 either alemtuzumab (20 mg) or rabbit anti-thymocyte globulin (6 Induction of immune tolerance to gluten in coeliac mg/kg). Twenty-one patients were treated. Engraftment of white disease after allogeneic haematopoietic stem cell blood cells and platelets was on mean day 9 and hospital dis- transplantation for thalassaemia major charge on mean day 11. The only infections were one episode of M. Bernardo (1), R. Ciccocioppo (1), G. Giorgiani (1), A. Cometa Clostridium diffi cile diarrhea and 2 cases of dermatomal zoster. (1), M. Valli (1), A. Mastronuzzi (1), R. Maccario (1), G. Corazza Two patients receiving alemtuzumab developed late immune (1), F. Locatelli (2) thrombocytopenic purpura (ITP) that remitted with standard (1)Fondazione IRCCS Policlinico San Matteo (Pavia, IT); therapy and all patients are beyond 2 years (mean 3 years). All (2)Fondazione IRCCS Policlinico San Matteo, Università di patients are surviving, 17 patients (81%) improved by at least Pavia (Pavia, IT) 1.0 or more EDSS points, and fi ve patients (24%) relapsed but achieved another remission after further immunosuppression. Several case reports have already shown that patients under- After a mean of 3 years, progression-free survival is 100%, going allogeneic HSCT for haematological disorders resolved and relapse-free survival is 76%. Signifi cant improvement concomitant autoimmune disorders. FoxP3+ regulatory T cells occurred in neurologic disability determined by extended dis- are responsible for immunological tolerance to both self and ability status scale (EDSS) (P < 0.0001), neurologic rating score foreign antigens; dendritic cells (DC) play a pivotal role in (NRS) (P=0.0001), paced auditory serial addition test (PASAT) addressing immune system towards a tolerogenic or infl am- (P=0.009), and 25 foot walk (P < 0.0001), as well as quality of

S272 life as measured by the short form-36 (SF-36) (P < .0005). Non- showing good effi cacy and acceptable safety. We updated our myeloablative autologous HSCT performed in RRMS appears fi rst experience with unselected stem cell autotransplantation capable of reversing neurologic defi cits but results need to be in refractory CD. confi rmed in a randomized trial. The Multiple Sclerosis Inter- Seven patients (3 male, 4 female; age 26-45 years) with active national Stem Cell Transplant (MIST, www.clinicaltrials.gov, moderate-severe CD (median CDAI 336, range 272-395; four NCT00273364) trial that randomizes patients with relapsing- with perianal disease), refractory or intolerant to several lines of remitting MS failing interferon between standard therapy versus treatment (2-6, median 4) including corticosteroids, infl iximab autologous non-myeloablative HSC transplant. and immunosuppressant agents, were enrolled. Unselected PBSCs were collected after mobilisation with CTX 1.5 g/m² and G-CSF 10 micrograms/kg. The conditioning regimen included P926 CTX 50 mg/kg on days -5 to -2 and rabbit ATG 2.5 mg/kg on Incidence of cytomegalus virus infection in autoimmune days -4 to -2. Toxicity, clinical response (clinical remission = diseases after autologous stem cell transplantation CDAI < 150), endoscopic response (SES-CD) and extramu- F. Pieroni, A.B.P.L. Stracieri, M.C. Rodrigues, D.A. Moraes, cosal response (ultrasound sonography [US]) were assessed G.M.N. Barros, B.P. Simões, J.C. Voltarelli after mobilisation, at 3, 6, 9, 12 months and then every 6 months Clinical Hospital of Ribeirão Preto (Ribeirão Preto, BR) after stem cells reinfusion. No improvement was observed after mobilisation (median There is no report in the literature regarding the incidence of CDAI 363, range 258-404). Six patients were in clinical remis- CMV infection in autoimmune diseases patients treated with sion at the fi rst month (median CDAI 141, range 90-198). At high-dose immunosuppression followed by ASCT. We report the third month, all patients were in clinical remission (median here the incidence of CMV infection in 88 consecutive ASCT CDAI 98, range 56-132) despite discontinuation of all medica- for autoimmune diseases transplanted between January 2003 tions. After a median follow-up of 28 months (range 13-37), two to May 2008 (Multiple Sclerosis (MS)=45; Type 1 Diabetes relapses occurred after 4 and 12 months, respectively, while Mellitus=22; Systemic Sclerosis=9; Neuromyelitis Optica=4; all other patients maintained clinical remission. At the last fol- Systemic Lupus Erithematous=4; Amiothrophic Lateral Sclero- low-up visit, complete mucosal healing was achieved in 5 out sis=2; Vulgar Pemphigus=1; and Takayasu Arteritis=1). Before of 6 evaluable patients, with complete endoscopic remission ASCT, all patients were seropositive for CMV. The used con- in 3/3 patients evaluable at 24 months. Bowel thickening as ditioning regimens were BEAM + hATG 30mg/Kg for 11 MS assessed by US was slower to decrease, but improved in all patients, and Cy 200mg/Kg + rATG 4,5mg/Kg for the other patients and disappeared in 4/6 patients. Complete perianal patients. All patients received peripheral blood as source of fi stulas closure was observed in 3/4 patients. No deaths or life- stem cells. Blood red cells and platelets infused were all irra- threatening infections occurred. Major adverse events included diated and fi ltrated. Blood CMV pp65 antigen test was based a perianal abscess after mobilisation in one patient, pleural and on monoclonal antibody against the virus and immunofl uores- pericardial effusions in another, BK virus-related hemorrhagic cence. CMV infection was defi ned as a positivity of at least cystitis in one case, and acute pyelonephritis in another patient, one pp65 antigenemia assay at any level. In all patients, a all rapidly resolved with conservative treatment. CMVpp65 antigenemia assay was determined weekly, starting Unselected CD34+ cells transplantation has the potential of from the day when the absolute neutrophil count went above inducing and maintaining both clinical and endoscopic remis- 500 x 10³/ul, and until day 60 after ASCT. Patients with fi ve sion in refractory CD patients. Our data encourage the enroll- or more positive CMV cells, patients in use of corticosteroids ment of patients with refractory CD into the currently ongoing with any number of positive CMV cells, and patients with any EBMT multicenter trial (ASTIC). clinical signs of CMV disease with any number of positive CMV cells were treated with Gancyclovir. Among the 88 transplanted patients, 18 (20,5%) presented a positive antigenemia. The P928 fi rst positive antigenemia presented a median of 19 days after Haematopoietic stem cell transplantation for severe stem cell reinfusion (range 05 to 52). The median antigenemia autoimmune diseases. A single-centre 12-year experience level at the fi rst appearance was 2 infected cells (range 1-40). F. Gualandi, B. Bruno, S. Bregante, A. Dominietto, M.T. van Lint, Overall, 07 patients were treated with Gancyclovir. Five were L. Grassia, A. Uccelli, E. Capello, G.L. Mancardi, A. Bacigalupo, treated preemptively. Two patients had CMV pneumonits, and A.M. Marmont one died from it at D+17 after transplantation. In 61% of the Azienda Ospedaliera-Universitaria S. Martino (Genoa, IT) cases (11 patients), CMV reactivation was asymptomatic with less them 5 cells positive and antigenemia has cleared spon- Starting from 1996, 45 patients with SADs were treated with taneously. Our study shows that CMV infection can reactivate high-dose immunosoppression (HDIS) in Genoa. Of these, in a signifi cant proportion of seropositive patients after ASCT. 2 with systemic lupus erythematosus (SLE) achieved complete However, despite the use of Antimocyte Globulin in the condi- remission already after mobilization (Cy 4g/sm) and received tioning regimen, the rate of CMV reactivation in this subset of no further treatment. 37 pts received autologous stem cell patients seens not be different from the 29% rate of reactivation transplantation (ASCT), 4 received allogeneic SCT (2 after recently encountered after ASCT for hematologic malignancies ASCT) and 1 syngeneic. One patient with severe pulmonary (Rossini, F., et al, Transpl Infect Dis 2005: 7: 122-125). scleroderma (SSc) died after mobilization and 1 with multiple sclerosis (MS) died 70 days after ASCT of thromboembolic complications. P927 Twenty-three patients with MS were treated with ASCT, and Autologous haematopoietic stem cell transplantation for achieved total regression of Gd-enhancing CNS lesions, and refractory Crohn’s disease: updating the Milan experience 1-2 degrees of improvement on the EDSS scale. Both only without CD34+ cell selection mobilized and autotransplanted SLE patients achieved com- F. Onida (1), A. Cassinotti (2), C. Annaloro (3), A. Della Volpe plete clinical remission (SLEDAI) and signifi cant reduction of (3), P. Usardi (3), B. Motta (3), S. Ardizzone (2), E. Tagliaferri biomarkers but all of them had to receive maintenance treat- (3), G. Bianchi Porro (1), G. Lambertenghi Deliliers (1) ment. Allogeneic transplants were performed for 4 patients (Beh- (1)University of Milan (Milan, IT); (2)Ospedale Luigi Sacco cet’s, Evans, Pure White Cell Aplasia-PWCA and Sjogren-SLE (Milan, IT); (3)Ospedale Maggiore Policlinico, Mangiagalli e overlap with chronic infl ammatory demyelinating polyradicu- Regina Elena (Milan, IT) loneuropathy (CIPD). Two of them died 5 years (haemolytic- uremic syndrome-HUS) and 6 years (hepatitis-polyneuritis) Autologous haematopoietic stem cell transplantation has after transplant. One patient with severe rheumatoid arthritis recently been used to treat refractory Crohn’s disease (CD), (RA) received a syngeneic transplant, had a marked decrease

S273 of anti-cyclic citrullinated peptide (CCP), but relapsed clinically 3 months later. Our experience indicates that ASCT, potentiated by prior stem cell mobilization with 4g/sm of cyclophosphamide, has a power- ful remitting effect in SADs. More intense conditioning regimens have resulted in longer and better remissions. Immunological cure was not achieved, and MRD could not be evaluated. After allo-SCT, in 2 cases DLI were necessary to achieve full donor chimerism coincident with CR, in 1 case relapse occurred not- withstanding full donor chimerism.

P929 Thiotepa-cyclophosphamide high-dose immunosuppressive therapy with autologous haematopoietic stem cell transplantation in aggressive forms of multiple sclerosis: results in 9 patients R. Greco, L. Moiola, T. Roccia, M. Radaelli, J. Peccatori, C. Bonini, C. Corti, M. Bernardi, M.T. Lupo Stanghellini, G. Comi, F. Ciceri San Raffaele Scientifi c Institute (Milan, IT)

Background: Autologous stem cell transplantation (ASCT) has been performed for aggressive Multiple Sclerosis (MS) with rais- ing frequency; standard myeloablative chemotherapy (BEAM) ± in vivo T-cell depletion produces long-term remission of dis- ease, with signifi cant morbidity and mortality. We assessed the feasibility of high-dose immunosuppression with Thiotepa and cyclophosphamide (Cy) with ASCT in patients with aggressive forms of MS. P930 Methods: From December 2005 to October 2007, 9 patients Factor VIII haemophilia acquired as secondary affected by MS, unresponsive to conventional therapies, with autoimmune disease after haematopoietic stem cell elevated infl ammatory disease activity, underwent ASCT in transplantation (ASCT) for refractory systemic lupus our institution. Five patients had a relapsing-remitting, 3 a sec- erythematosus (SLE) dissolves with relapse of SLE – ondary progressive and 1 an hyper-acute MS. Patients had a A case report median of 26 years, 6 (range 2.5-8) points in EDSS disability T. Alexander, S. Schneider, S. Ziemer, U. Schneider, H. Radtke, score and 15 enhancing lesions (range 1-67) on MRI before G. Burmester, A. Thiel, R. Arnold, F. Hiepe ASCT, with a median of 2 relapses in the 2 years before ASCT. Charite University Medicine (Berlin, DE) Autologous hematopoietic stem cells were mobilized with Cy 4 gr/mq at day 0 followed by granulocyte colony stimulating Introduction: Stem cell therapy for autoimmune diseases is factor 5mcg/Kg/day from day +2 to stem cell harvest. Harvest rapidly developing with promising clinical results. However, it was not manipulated before cryopreservation. The conditioning is increasingly recognized these patients have an added pro- regimen before ASCT consisted of Thiotepa 5mg/Kg bid on day pensity to develop secondary autoimmune disorders, distinct -5 and Cy 50 mg/Kg on days -3 and -2. The median follow-up from the underlying autoimmune disease, given their genetic was 29 months. predisposition to autoimmunity. Results: Mobilization was successfull in all cases, with a median Methods: As part of a monocentric phase I/II clinical trial at of 12.91x106 collected and 5.7x106 infused CD34+cells/Kg. the Charité University Medicine, seven patients have received Hematopoietic recovery was documented safely in 9/9 patients, immunoablation with cyclophosphamide and ATG and CD34- with a median of 10 days for neutrophil and platelet engraft- ASCT for SLE. We now report the case of acquired factor VIII ment. The median time of hospitalization was 24 days. Six hemophilia in a patient developing nine months after receiving patients developed febrile neutropenia, lasting a median of immunoablation and CD34-ASCT for life-threatening SLE. 4.5 days. There were no major adverse events. TRM was 0%. Results: The 21 year old male patient received immunoab- Seven patients were stable or improved at the last follow-up: 5 lation and CD34-ASCT in 2005 for persistent active SLE improved EDSS by at least 0.5 points, 2 were stable. Relapses despite the usage of various immunosuppressants including were observed in 5 patients (4 at 1 year, 1 at 2 years after ASCT) cyclophosphamide and mycophenolate. Clinical and sero- with complete recovery after steroids. The confi rmed progres- logic remission was achieved within 1 month from ASCT sion free survival is 77.8%, the disease activity free survival and immunophenotypic analyses revealed the recurrence of is 33%. A signifi cative reduction by the 79% of the annualized thymic-naïve CD31+ T cells, FoxP3 regulatory T cells, naïve relapse rate and 99,6% of the enhancing lesions occurred. B cells with similar kinetics compared to other patients under- Conclusions: High-dose immunosuppressive therapy with Thi- going ASCT for SLE. While in persistent clinical and sero- otepa and Cy followed by ASCT reduced disease activity and logic remission he presented with signs of hemorrhage nine disability progression of aggressive MS with safe profi le and months after ASCT and factor VIII (fVIII) hemophilia was diag- low toxicities. Larger number of patients and longer follow-up nosed with high titers of fVIII-inhibitor and fVIII-activity of 2%. will be necessary to establish the value of this regimen in pro- Treatment regimens with cyclophosphamide, glucocorticoid ducing long-term disease remissions. pulse therapy combined with IVIG as well as B cell deple- tion and a combination of immunoadsorption and immuno- suppressive therapy according to the modifi ed Bonn-Malmö protocol failed to eradicate the fVIII-inhibitor. At 30 months after ASCT the patient suffered a complete clinical and sero- logic relapse of SLE. To our surprise, titers of fVIII-inhibitor completely vanished during the recurrence of ANA and anti- dsDNA antibodies and were not detectable since then with persistent normalization of fVIII activity.

S274 Conclusion: The acquired factor VIII hemophilia after ASCT P932 is regarded as a secondary autoimmune disorder rather than Autologous stem cell transplant for severe refractory lupus-associated given the clinical and serologic remission at the Crohn’s disease: The UK experience time-point of development. We propose that the disappearance M. Kazmi (1), A. Ansari (2), J. Sanderson (1), G. Jackson (3), of fVIII-inhibitor during the lupus-fl are illustrates the competition A. Lobo (4), N. Thompson (5), J. Snowden (4) of two antibody mediated autoimmune disorders suggesting a (1)Guy’s & St Thomas’ Hospital (London, UK); (2)Chelsea & direct competition for survival niches of plasma cells secreting Westminster NHS Trust (London, UK); (3)Newcastle University these antibodies with polyclonal antibody responses in SLE Hospitals Trust (Newcastle, UK); (4)Sheffi eld Teaching Hospitals overcoming the single fVIII inhibitor. NHS Trust (Sheffi eld, UK); (5)Newcastle upon Tyne Hospitals NHS Trust (Newcastle, UK)

P931 Background: The use of autologous stem cell transplantation A two step approach to autologous peripheral blood stem (ASCT) as an option for severe, treatment refractory Crohn’s cell transplantation for refractory chronic infl ammatory disease has been shown in 2 studies to confer clinical benefi t. bowel diseases – The Freiburg Pilot Study We report the summary of the UK experience to date: 5 trans- W. Kreisel, K. Potthoff, H. Bertz, P. Hasselblatt, T. Baumert, plants in 3 centres over the last 5 years. C. Arnold, J. Finke Methods: Between 2003 and 2007, 5 autologous stem cell University Hospital Freiburg (Freiburg, DE) transplants were performed on patients with a median age of 36 years (25-45 years), with severe Crohn’s disease refractory Introduction: In about 5% chronic infl ammatory bowel diseases to all available standard therapy. Stem cell mobilisation was (IBD) are refractory to current medical or surgical treatment. achieved by a combination of cyclophosphamide and G-CSF in Ubiquitous bacterial antigens are supposed to trigger an uncon- 4 patients and G-CSF alone in 1 patient. Pre transplant condi- trolled immune reaction in IBD based on a genetic predisposi- tioning was achieved with a combination of ATG (total dose 7.5 tion (e.g. NOD2-gen mutations). Therefore it is intriguing to try mg/kg) and cyclophosphamide (200 mg/kg). to eliminate the disturbed immune reactions and to redevelop a Response was measured endoscopically, and by calculation of better immunological tolerance. the Crohn’s Disease Activity Index (CDAI). Methods: We used a two step approach towards autologous Results: The median number of prior lines of medical treatment peripheral blood stem cell transplantation (aPBSCT) in patients was 6 (range 5-8) and median number of prior surgical proce- with IBD refractory to corticosteroids, aminosalicylates, immu- dures was 5 (range 3-9). 3/5 patients were on long term PN pre nosuppressors, antibiotics and anti-TNF-antibodies. Remission ASCT. With a median FU of 27 months; 5/5 patients had clini- is induced with cyclophosphamide (2.0 g/m²/d on two succes- cal improvement in their disease activity. The median duration sive days), and peripheral blood stem cells are harvested. APB- of response however was only 8 months (range 3-27 months) SCT is performed after high-dose cyclophosphamide (50 mg/kg as defi ned by time from ASCT to next therapeutic intervention. bw/d on four successive days). In 4/5 patients CDAI was assessable and showed a reduction Results: 8 patients were included in this pilot study. In the two from a median score of 403 (220-550) pre transplant to 134 patients with severe ulcerative colitis stem cell mobilisation (120-183) at 6 months post transplant. therapy induced a transient clinical remission. In one patient In terms of transplant related complications 4/5 patients devel- colectomy was performed for safety reasons 4 months later. In oped a febrile neutropenic event which was prolonged in 3/5 the other one urgent colectomy was necessary after 8 weeks patients. No patient required Intensive care support. There due to severe relapse. In all 6 patients with Crohn’s disease were no procedure related deaths. (CD) mobilisation chemotherapy induced an endoscopical and Conclusion: Our experience suggests that ASCT may have clinical remission, which lasted between 2 and 18 months Two a transient benefi cial role in treatment of severe, refractory have a heterozygous mutation of the NOD-2-gene (3020insC- Crohn’s disease. Treatment-associated morbidity in this series mutation, C2104T mutation). In one of them, there was an was low with no procedural mortality. No patients attained a early relapse in the terminal ileum. Resection was performed sustained remission and the duration of response varied con- followed by maintenance therapy with budesonide and adali- siderably between patients involved in the study. The reasons mumab. In the other patient with involvement of the total colon, behind this require further evaluation. It is possible that this rep- after complete remission the sigmoid colon was resected due resented a particularly resistant cohort of patients with Crohn’s to a local relapse. After development of perianal abscesses and disease as evidenced by the need for prolonged PN and multi- fi stulae surgical interventions must be performed. After healing ple previous surgical procedures. of the fi stulae (prednisolone, antibiotics, adalimumab) aPBSCT Interestingly all patients displayed an ameliorated form of will be performed. In two of the 6 patients with CD aPBSCT is relapse often localised with sensitivity to previously unsuc- scheduled for 02/2009.In two CD patients aPBSCT was per- cessful therapy suggesting some change in the biology of the formed 9 and 33 months after stem cell mobilization, respec- disease. tively. This therapy induced a complete clinical, endoscopical and histological remission, which lasts 78 and 10 months, respectively, until now. Conclusions: Stem cell mobilisation with cyclophosphamide induces a transient remission in refractory IBD. High-dose cyclophosphamide followed by aPBSCT may lead to a long- lasting steroid-free complete remission in patients with refrac- tory Crohn’s disease.

S275 Myelodysplasia This may have consequences for decision making regarding transplant indications or procedures. We evaluated the results of 172 consecutive pts with primary MDS (N=131) or t-MDS arising from malignant lymphoma (N=24) or solid tumor (N=17) P933 treatment, who underwent alloSCT during a 10-year period, for Prognostic impact of pretransplantation whom complete information of all disease features was avail- transfusion-dependency and serum ferritin level in able. Median age was 48 (16 – 69) years (yrs) and was not patients with myelodysplastic syndrome undergoing different between primary and t-MDS. Eighty-two pts (48%) allogeneic stem cell transplantation: a study from the received transplants from HLA-identical sibling donors and 90 Gruppo Italiano Trapianto di Midollo Osseo (GITMO) pts (52%) from matched unrelated donors after myeloablative E. P. Alessandrino, M. G. Della Porta, A. Bacigalupo, M. T. Van conditioning regimens. Of the 131 pts with primary MDS, 96 Lint, M. Falda, E. Angelucci, F. Onida, M. Bernardi, A.P. Iori, pts had an HCT-CI of ≤2 (73%). In order to test, whether the A. Rambaldi, R. Cerretti, P. Marenco, P. Pioltelli, L. Malcovati, HCT-CI adequately separates “low risk” primary MDS pts from C. Pascutto, R. Oneto, R. Fanin, A. Bosi on behalf of Gruppo t-MDS pts with ≤ 2 index points resulting from other comorbidi- Italiano Trapianto di Midollo Osseo (GITMO) ties than the pre-existing malignant disease (n=30 [73%]), we compared these two cohorts in terms of major transplant out- We evaluated the impact of pretransplantation transfusion- come endpoints. This analysis revealed no differences between dependency and serum ferrtin level on the outcome of patients “low risk” primary and t-MDS pts with an overall 5- year OS with myelodysplastic syndrome (MDS) receiving allogeneic estimate of 45% ± 5% for primary and 40% ± 9% for t-MDS pts stem-cell transplantation (allo-SCT). We studied 360 patients (ns). In contrast, when “high-risk” pts of each cohort were com- reported to the GITMO between 1997 and 2007. According to pared, a signifi cantly inferior 5-year survival estimate for t-MDS the WHO criteria, 23 patients were diagnosed as refractory ane- could be demonstrated (34% ± 9% vs. 0%, p < 0.006). This mia (RA) with or without ringed sideroblasts (6%), 61 patients difference resulted from a signifi cantly higher 5-year estimate as refractory cytopenia with multilineage dysplasia (RCMD; of TRM, which was 43% ± 11% for “high-risk” primary MDS pts 17%), 53 patients as RA with excess blasts, type 1 (RAEB-1; and 100% for “high-risk” t-MDS pts. Further, no differences in 15%), 107 patients as RAEB-2 (30%) and 116 patients as AML the outcome of pts with t-MDS arising from either malignant from MDS (31%). There were 229 HLA-matched sibling and lymphoma or solid tumors was detectable after adjustment for 131 unrelated donor SCT. The source of hematopoietic stem the stratifi ed HCT-CI. cells was peripheral blood in 224 patients and bone marrow In multivariate analysis the outstanding prognostic factor for in 136. Two hundred nineteen patients received a myeloabla- OS and TRM was the pretransplant IPSS, whereas the HCT- tive conditioning regimen, whereas a reduced-intensity regimen CI reached no statistical signifi cance after adjustment for the (RIC) was performed in 141 patients. Regular transfusion need IPSS. In summary, the HCT-CI may overestimate TRM of t- was reported in 223 of 328 evaluable subjects (68%). Transfu- MDS pts with an otherwise “low risk” comorbidity profi le. This sion-dependency was independently associated with a reduced analysis suggests a prognostic heterogeneity of t-MDS pts overall survival (OS, HR=1.48, P=.017) and increased probabil- which is best refl ected by the pretransplant IPSS. Therefore it ity of transplant-related mortality (TRM, HR=1.68, P=.024). The appears questionable whether the HCT-CI alone allows valid negative impact of transfusion-dependency on survival was prognostic stratifi cation in pts with t-MDS. noticed only in patients receiving myeloablative conditioning (OS HR 1.76, P=.003; TRM HR 1.70 P=.02) with an inverse relationship between severity of transfusion requirement and P935 both OS (P=.022) and TRM (P=.021). In transfusion-dependent Chimerism analysis as a predictor of relapse of patients receiving myeloablative allo-SCT, pretransplantation myelodysplastic syndrome after allogeneic stem cell serum ferritin level showed a signifi cant effect on OS (P=.02) transplantation and TRM (P=.04). This effect was maintained adjusting for both M. Tobiasson (1), R. Olsson (2), M. Remberger (2), E. Hellström- transfusion burden and duration (P=.038 and P=.04 for OS Lindberg (2), J. Mattsson (2) and TRM respectively), suggesting that the negative impact of (1)Karolinska Institutet (Eskilstuna, SE); (2)Karolinska transfusion-dependency might be determined at least in part by University Hospital-Huddinge (Stockholm, SE) iron overload. Finally, transfusion-dependency was found to be an independent risk factor for acute graft versus host disease in Introduction: SCT is a potentially curative treatment for myelo- a multivariate regression analysis (P=.04). dysplastic syndrome (MDS), yet associated with a high risk of In conclusion, pretransplantation transfusion-dependency and transplantation related mortality (TRM) and relapse. Chimerism serum ferritin have prognostic value in MDS patients undergoing analysis (CA) of peripheral blood (PB) or bone marrow (BM) myeloablative allo-SCT, through a signifi cant increase in TRM. may predict relapse after SCT, but has not been evaluated spe- These results suggest that transfusion dependency should be cifi cally for MDS patients. taken into account in transplant decision-making: patients with Materials and methods: The cohort consisted of 75 consecutive a long history of blood transfusion and evidence of iron over- patients (40y, 0-67) with MDS who underwent SCT between load might benefi t from a reduced-intensity conditioning. 1998 and 2008. WHO subclasses were RARS (2), RCMD (21), RAEB-I+II (36), MDS/AML (6), CMML (3), JMML (6), and MDS 5q- (1). Fourteen patients had IPSS Low/Int-1 and 48 INT-2/ P934 High. Myeloablative (MAC) conditioning regimens were used in Allo SCT in therapy-related MDS arising from solid tumour 59% of patients (23y, 0-57), and reduced intensity (RIC) in 41% or malignant lymphoma treatment without additional (55y, 27-67). Median follow up time was 36 months (4-110). adverse prognostic factors of the HCT-CI Index leads to Linear-specifi c CA was used analyzing CD33 in PB (n=53) and comparable results as in primary MDS CD34 in BM (n=37). Sensitivity and specifi city were calculated R. Trenschel, N. Steckel, L. Kordelas, T. Gromke, for samples analyzed within 3 months prior to morphological A.H. Elmaagacli, C. Schulte, D. Beelen relapse (MR). University Hospital of Essen (Essen, DE) Results: Twenty-fi ve% of patients relapsed after a median of 5 months (1-31); 53% after RIC and 18% after MAC (p=0.02.) The HCT-CI has gained interest as a predictor of transplant out- TRM was 34%; 40% for MAC and 27% for RIC (p=0.39). Over- come especially in pts with MDS and AML. The HCT-CI classi- all survival at 5 y was 45%; 55% for MAC and 34% for RIC fi es MDS pts surviving a pre-existing solid tumor with 3 index (p=0.67) Acute GVHD grades I-II and III-IV occurred in 27% points which leads to categorization to an adverse prognostic and 16% of patients, respectively. Complete donor chimerism group according to the index stratifi cation (0 - 2 versus >2). (CC) was observed in 27 patients whereas MC, defi ned as

S276 ≥ 5% recipient cells in either CD33 PB, CD34 BM at any point P937 was observed in 30 patients. We used a cut-off of 5% recipi- Allogeneic haematopoietic cell transplantation for ent cells to discriminate CC from MC, and analyzed MC as a myelodysplastic syndrome: prognostic signifi cance of predictor of MR. The sensitivity of CD33 PB and CD34 BM was pretransplant IPSS score and co-morbidity 61% and 92%, respectively. The specifi city was 88% (CD33 PB) J.H. Lee, J.H. Lee, S. Lim, D.Y. Kim, S.H. Kim, Y.S. Lee, and 68% (CD 34 BM). CD34 BM was analyzed prior to MR in 6 Y.A. Kang, S.I. Kang, S.G. Ryu, M.J. Jeon, M. Seol, E.J. Seo, relapsed patients. Five of these showed MC at a median time H.S. Chi, C.J. Park, S. Jang, S.C. Yun, K.H. Lee of 2,5 months (0,5-7) before MR. Eight of 18 patients had PB Asan Medical Center, Ulsan University (Seoul, KR) CD33 MC prior to MR with median of one month (0,5-2) before MR. Eight patients were treated with DLI due to MR. None of International Prognostic Scoring System (IPSS) score at diag- these resulted in CR. Another fi ve patients were treated with nosis has been shown to be effective in predicting the out- DLI due to MC of whom three are still in CR. comes of hematopoietic cell transplantation (HCT) in patients Conclusion: CA is a useful method to predict relapse of MDS with myelodysplastic syndrome (MDS), but the signifi cance of after SCT. CD34 MC over 5% is a highly sensitive method to IPSS score at the time of HCT has not been evaluated. Pre- predict relapse and may be combined with CA on PB CD33 transplant comorbidity was recently shown to be an independent to enhance specifi city. CA on PB alone is not suffi cient to pre- predictor of HCT outcomes in patients with a variety of hemato- dict relapse, probably due to the disease biology where apop- logic diseases. We therefore analyzed the clinical signifi cance tosis of marrow progenitors prevent these from entering the of IPSS score at the time of HCT and pre-transplant comor- circulation. bidity in 68 patients who underwent allogeneic HCT for MDS (n=48) or acute myeloid leukemia evolved from MDS (n=20) between December 1995 and January 2008 at a single insti- P936 tute. Prior to HCT, 23 patients were given chemotherapy and 12 Update on immunosuppressive therapy for children with were in complete remission (CR) at the time of HCT. Condition- refractory cytopenia ing regimen was busulfan-cyclophosphamide in 38, busulfan- A.M.J. Peters (1), A. Yoshimi (1), I. Baumann (2), B. Strahm (1), fl udarabine in 2, busulfan-fl udarabine-ATG in 24, and others in I. Furlan (1), E. Bergsträßer (3), A. Fischer (1), F. Locatelli (4), 4. Bone marrow was used for hematopoietic cell grafts in 48 M. van den Heuvel-Eibrink (5), M. Führer (6), C. Niemeyer (1) and G-CSF mobilized peripheral blood mononuclear cells in (1)University Hospital Freiburg (Freiburg, DE); (2)Medical 20. The cumulative incidence of engraftment failure was 12.0% Center Bayreuth (Bayreuth, DE); (3)University Children´s with primary failure in 2 and secondary failure in 6. During a Hospital (Zurich, CH); (4)University of Pavia (Pavia, IT); median follow-up period of 41.0 months (range, 3.2 to 132.0 (5)Sophia Children´s Hospital (Rotterdam, NL); (6)Dr. von months), 27 patients died and 7 relapsed. Twenty-one deaths Hauner´sches Kinderspital (Munich, DE) were not related to relapse of MDS. The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 60.0% Refractory cytopenia (RC) of childhood is characterized by per- and 57.4%, respectively, and the 5-year cumulative incidences sistent cytopenia with less than 5% blasts in the bone marrow of non-relapse mortality (NRM) and relapse were 32.7% and and less than 2% blasts in the blood. It is the most common 9.9%, respectively. Multivariate analyses demonstrated that subtype of MDS in childhood accounting for about half of the IPSS score at the time of HCT and pre-transplant comorbidity cases. Because early bone marrow failure can at least in part were signifi cant independent prognostic factors for OS, EFS, be mediated by T-cell immunosuppression of haematopoiesis, and NRM. Post-transplant outcomes were signifi cantly affected immunosuppressive therapy (IST) can be a successful therapy by the presence and severity of acute GVHD, but not by chronic strategy for some children with RC. We have previously pub- GVHD. Patients who developed grade I or II acute graft- lished the results of IST with antithymocyte globuline (ATG) and versus-host disease (GVHD) showed superior outcomes, as cyclosporine for childhood RC based on the recommendations assessed by OS, EFS, and NRM, compared with patients with- for severe aplastic anemia (SAA) (Yoshimi A, et al. Haemato- out acute GVHD or those with grade III or IV acute GVHD. No logica 2007, 92:397). Here, we present a longer-term follow-up relapse occurred in patients who developed acute GVHD. In (2.7 – 7.8 years, mean 4.4 years) of the described cohort of 31 conclusion, IPSS score at the time of HCT and pre-transplant patients. Response to IST was evaluated according to a con- comorbidity may stratify the risk of post-transplant outcomes in sensus between the Japanese and European Working Groups MDS. Our results also indicate that graft-versus-MDS effects on SAA. In brief, response to treatment was defi ned as com- occur after allogeneic HCT. plete response (CR) if the following criteria were fulfi lled: ANC >1.5 x 109/l, Hb > age-adjusted cut-off value, platelet count > 150 x 109/l. In the absence of CR, PR was defi ned when all P938 following criteria are fulfi lled: ANC> 0.5 x 109/l, no platelet or Allogeneic haematopoietic stem cell transplantation for red cell transfusion, platelet count >20 x 109/l. At 12 months acute myeloblastic leukaemia or myelodysplastic following IST, 5 patients (16%) were in CR and 16 (52%) in PR. syndrome in patients older than 50 years This response rate is inferior to what had been described for M. Robin (1), R. Porcher (1), L. Adès (2), E. Raffoux (1), SAA in the German study SAA 94 with 40% CR and 41% PR. At R. Peffault de Latour (1), A. Petropoulou (1), N. Boissel (1), 60 months, data of 15 of the 31 RC patients were available: 7 P. Ribaud (1), A. Devergie (1), V. Rocha (1), P. Fenaux (2), patients were in CR, 3 in PR and 8 had relapsed or developed H. Dombret (1), G. Socié (1) chromosomal abnormalities/increased blast count (clonal evo- (1)Hôpital Saint-Louis - APHP (Paris, FR); (2)Hôpital Avicenne lution). Defi ning toxic death, non-response, late response (later - APHP (Bobigny, FR) than 6 month after initiation of ATG), secondary non-response, and clonal evolution as event, 15/31 patients remained event- Aim: Analyse the outcome of patients older than 50 years who free. Early or secondary non-response were the most frequent received an allogeneic hematopoietic stem cell transplantation events (n = 11). Eight of the 22 patients defi ned as responders (HSCT) for MDS or AML, in Saint-Louis Hospital from January at 6 months experienced disease recurrence or clonal evolu- 1997 to December 2007. tion. In total, 5 patients suffered clonal evolution. The estimated Method: 48 patients (pts) aged from 50 to 68 years (median: 56) event-free survival at 60 months is 0.44% (0.33-0.54). Since the received an HSCT for MDS (N=28) or AML (N=20) during this results of hematopoietic stem cell transplantation (HSCT) from period. AML pts had intermediate (n=15) or high risk cytogenet- matched unrelated donors in RC indicate a cure rate of approxi- ics (n=3). 8 pts had failure to initial induction chemotherapy. FAB mately 70% to 80%, it is reasonable to recommend that, in the classifi cation for MDS was: RAEB (n=8), RAEBt (n=6), second- presence of a suitable alternative donor, IST non-responders ary AML (n=2) or refractory anemia (n=2). Maximal IPSS score should proceed to HSCT early in the course of their disease. was high, intermediate-2 and intermediate-1 in 11, 14 and 4 pts.

S277 Ten pts with MDS received an “AML-like” chemotherapy and 5 44%, respectively. Univariate analyses showed that RR, RFS received demethylating agents before transplant. 18 of the AML and OS were signifi cantly infl uenced by disease classifi cation pts were in complete remission (CR) at time of transplant (CR1, (p=0.02, p=0.004 and p=0.02). NRM was not infl uenced by dis- n=15; CR2, n=3) and 2 were in relapse at time of HSCT. 10, 9 ease stage (p=0.18). A low number of transfusions (<20) led to and 9 patients with MDS had < 5%, 5-10% and > 10% blasts in a decrease of the relapse risk (p=0.05). The other assessed bone marrow at time of HSCT. variables, including age, donor type and the interval between Results: 19 patients with AML (95%) and 16 patients with MDS diagnosis and SCT, had no signifi cant impact on all disease (57%) received a HSCT from an HLA-identical sibling donor. outcomes. The multivariate Cox-regression analyses showed Other pts received a matched unrelated donor. Thirty-two pts, again a signifi cant impact of disease classifi cation at SCT, but including 9/20 and 23/28 had at least one co-morbidity accord- now for all endpoints, including NRM (p=0.04). An early SCT ing to Sorror score. The conditioning regimen was myeloab- (interval between diagnose and disease < 6 months) correlated lative (MAC) in 14 pts (29%). Reduced intensity (RIC) was with an increased survival rate (p=0.04). fl udarabine based in 34 pts, associated with either 2 Gy TBI Conclusion: These preliminary data show that disease stage (n=25) or chemotherapy (n=9). All pts engrafted. 35 pts had has a signifi cant impact on outcome. More data are collected acute graft-versus-host disease (GVHD): grade I in 11, grade II to quantify the impact of the number of transfusions and of iron in 18 and grade III in 6. GVHD incidence did not differ between toxicity. pts who received a MAC or a RIC regimen. Two-year overall survival (OS) was 41% (95% CI: 26-57). OS and relapse-free- survival (RFS) were similar after MAC or RIC regimen (OS: P940 29% vs 35% and RFS: 29% vs 30%). Short-term non-relapse Allogeneic haematopoietic stem cell transplantation as mortality (NRM) was lower in patients who received a RIC as front line therapy for patients with “de novo” or compared to patients who received a MAC regimen but was secondary myelodysplastic syndrome: a single-centre similar at long-term (6-month NRM = 21% vs 9% and 12-month experience NRM = 21 vs 19%). Patients with AML or MDS had similar OS M. Sorio, R. Di Bella, A. Andreini, C. Tecchio, F. Frattini, and RFS (OS: 42 vs 41%; RFS: 37 vs 26% at 2 years for AML G. Quaresmini, F. Randon, G. Ruggeri, A. Al-Kafaff, S. Ledro, and MDS, respectively). NRM was not signifi cantly higher in pts D. de Sabata, F. Benedetti with MDS (26 vs 10% at one year) whereas relapse rate was Bone Marrow Transplant Unit (Verona, IT) not signifi cantly higher in pts with AML (13 vs 6%). Conclusion: HSCT for AML or MDS after 50 years is a cura- Introduction Myelodisplastic syndromes (MDS) are a heteroge- tive option for pts with related or unrelated HLA-identical donor neous group of hematologic malignancies with an elevated risk regardless kind of conditioning regimen and co-morbidity at of developing acute myeloid leukemia. Conventional chemo- time of transplantation. therapy is unable to cure the disease. Allogeneic transplant is the only curative treatment, but very often it’s not suitable because of age and comorbidities. P939 Patients characteristics 47 consecutive pts with “de novo” and The effect of transfusions and iron toxicity on non-relapse secondary MDS, including transformed acute myeloid leukemia mortality in patients with untreated adult MDS treated with (t-AML) were treated between 1993 and 2007. myeloablative alloSCT: a retrospective study of the MDS At the diagnosis, according to the FAB, the pts were classifi ed subcommittee of the Chronic Leukaemia Working Party of as: 14 RA, 12 RAEB, 18 RAEB-T, CMML 1, SAA 1. According to the EBMT WHO the same patients resulted classifi ed as: 1 RA, 12 RCMD, L. Spinnewijn, A. van Biezen, R. Brand, J. Finke, 6 RAEB-1, 11 RAEB-2, 3 MDS-U and 1 CMML. U. Platzbecker, T. Ruutu, D. Beelen, R. Schwerdtfeger, Y. Floisand, Median age was 51 years (range 11-66), M/F 26/21. When R. Martino, D. Niederwieser, A. Atienza, K. Steinerova, N. Kröger, enrolled in the transplant program, 23/47 pts had already devel- T. de Witte on behalf of the MDS subcommittee of the Chronic oped an overt leukemia. The median time of transformation to Leukaemia Working Party of the EBMT AML was 4.6 months Seventeen/23 of these pts (74%) were treated with at least one course of conventional chemotherapy. Background: Standard treatment for MDS includes blood prod- Nevertheless a complete remission was reached in a minority uct transfusions, in some cases followed by allogeneic stem of pts. cell transplantation (alloSCT) after a standard myeloablative The conditioning regimen consisted of FTBI (200 cGy x 6) + regimen. This treatment approach leads to considerable treat- CTX or BUCy for 35 pts. Reduced Intensity Conditioning (RIT), ment-related morbidity and non-relapse mortality (NRM) during including Thiotepa, Fludara, Cyclophosphamide, was chosen the fi rst 6 months after treatment with alloSCT. This increased for 12 pts over 60 years or suffering comorbidities. The source mortality of transfused patients can partly be attributed to of stem cells was identical sibling for 28 pts, unrelated donor for increased iron toxicity, caused by frequent erythrocyte transfu- the remaining cases. sions, release of toxic iron radicals by the intensive treatment Outcomes We observed 11/47 (23%) cases of transplant related itself and its associated ineffective hematopoiesis and some mortality within 100 days (3 infections, 2 CNS bleeding, 2 Multi other less well defi ned processes. Most information about Organ Failure, 2 VOD, 2 acute GVHD), 8/11 cases in the group iron overload and chelation originates from non-MDS patients. of pts treated with myeloablative regimens, and 3/11 in the RIT Therefore this study aims to quantify the effects of iron overload group. After day +100, relapse was the most important cause of in MDS patients. death: 9 pts, 3 pts in RIC group and 6 in the group transplanted Methods: A retrospective analysis of data from the EBMT regis- with conventional regimens, in most cases within 6 months. try was carried out with an additional survey within EBMT cent- The overall incidence of acute GVHD of clinical grade >II, and ers, covering in total 206 adult MDS patients who were treated the chronic GVHD, was 55% (26/47) and 26% (12/47), respec- with alloSCT between 2000 and 2005. Only patients were tively. included with alloSCT as their primary treatment, and who were The overall survival (OS) was 47% (median follow-up 14 classifi ed as MDS patients or as patients with MDS transformed months). The sibling donor (28 cases) still seems the best to acute myeloid leukemia (sAML) at time of SCT. source of stem cells: in fact, as compared with unrelated donor Results: 63 patients were categorized as refractory anaemia (18 cases), we obtained an OS of 53% and 33%, respectively. with or without ring sideroblasts (RA/RARS), 100 patients as Conclusions Allogeneic HCT from a suitable HLA-matched RA with excess of blasts (RAEB), and 38 patients as RAEB donor is o good curative option for patients with MDS, includ- in transformation (RAEB-t) or sAML. The estimated 3-years ing those with t-AML (about 50% of cured cases). Despite the NRM, relapse rate (RR), relapse free survival (RFS) and over- elevated median age of these pts, an acceptable related mor- all survival (OS) for the whole group were 44%, 17%, 39% and tality was observed.

S278 Reduced-intensity transplants There were 287 males and 216 females with a median age of 36 (1-69) years. Most patients (n=312) received PBSC while 155 were given BM and 36 CB. Results: Median level of CRP before conditioning among all P941 patients were <10 mg/L (range <10-142). Increased CRP levels Role of co-morbidity score, age and disease status at (>10 mg/L) were detected in 129 (26%) patients. Overall sur- transplant in predicting survival and non-relapse mortality vival in the whole population was signifi cantly inferior in patients in patients with MDS and leukaemia undergoing with increased CRP (63% vs. 53%, p<0.05). However, if ana- reduced-intensity conditioning transplantation lysing RIC and MAC separately, this difference only persisted S.W. Bokhari, L. Watson, E. Das-Gupta, J. Byrne, N. Russell among RIC patients (67% vs. 42%, p=0.004) while no differ- Nottingham City Hospital (Nottingham, UK) ence was seen among MAC patients (61% vs. 63%, ns). Fur- thermore, in the RIC cohort, transplant-related-mortality (16% Allogeneic stem cell transplantation is the favoured treatment vs. 30%, p=0.036) and relapse-free survival (53% vs 27%, for high risk leukemias and MDS, but its utlility is limited to p=0.015) were worse in patients with increased CRP, while no younger fi tter patients. Reduced intensity conditioning (RIC) difference in acute GVHD was seen. In multivariate analysis regimens have allowed older patients, often with co-morbidities, CRP still remained signifi cantly associated to OS (HR 1.62, CI to undergo HSCT. The HCT-comorbidity index has been vali- 1.01-2.59, p<0.05), and RFS (HR 1.62, CI 1.04-2.51, p=0.03). dated in predicting non-relapse mortality(NRM) in some stud- No correlation between any outcome variable and CRP was ies comparing myeloablative versus RIC conditioning. Patients seen among the MAC treated patients. receiving RIC allografts are however very heterogenous as far Conclusion: CRP may be a good prognostic factor for outcome as their co-morbidities are concerned and it is uncertain whether in allogeneic stem-cell transplantation after reduced intensity one can further delineate groups of RIC allograft recipients with conditioning. signifi cantly different mortality and outcomes. This retrospec- tive single centre study was done to assess the outcome of RIC transplants in patients with MDS and leukemia based on their co-morbidity scores in addition to other prognostic factors. 90 RIC allografts were performed between 2002 and 2008. Fac- tors analysed in addition to HCT-comorbidity index were age of >60 years, disease status at transplant, CMV status, stem cell source and donor type(related/unrelated). The median age at transplant was 58.5 years(29-71.1) with a median follow-up of 23 months(2-96). The number of patients with scores 0-2 and ≥3 was 53 and 33 respectively(scores not available in 4). The commonest co-morbidity was moderate pulmonary function impairment. The overall survival(O.S) and NRM of the whole cohort was 51% and 28% at 2 years respectively. NRM of patients with HCT-CI of ≥3 was higher as compared to those with score 0-2 reaching statistical signifi cance both on uni- and multivariate analyses(p 0.02). Age >60 years was also a signifi cant factor on multivariate analysis affecting non-relapse mortality(p 0.01) as well as overall survival(p 0.004). Disease status at trans- plantation of CR2 or PR had a lower overall survival (p 0.001) but no signifi cant impact on NRM. None of the other factors were signifi cant. Hence, age of < or >60 years and HCT-co-morbidity index of < or ≥3 can be combined to further delineate RIC allograft P943 recipients into 3 groups; group 1(age<60years and HCT-CI 2 or A comparison of campath and thymoglobulin as part of less), group 2(age>60 years and HCT-CI 2 or less) and group conditioning before allogeneic HSCT 3 (age >60 years and HCT-CI ≥3) with 2 year NRM of 8.4%, M. Remberger, A.-C. Norlin 40% and 55% respectively (p 0.001) and O.S of 76%, 40% and Clinical Immunology (Stockholm, SE) 30% respectively (p 0.01).This data confi rms the effi cacy of RIC allografts in group 1 patients but shows high NRM in other Background: Anti-thymocyte globuline (ATG) are frequently two groups in whom different treatment/conditioning strategies used as prophylaxis against rejection and GVHD after allo- should be considered. geneic hematopoietic stem-cell transplantation (HSCT) using unrelated donors. Patients: We compared 30 patients given Campath (alemtuzu- P942 mab) as part of the conditioning with a matched cohort of 30 C-reactive protein predicts outcome after reduced patients receiving Thymoglobulin. Most patients had a hemato- conditioning allogeneic stem cell transplantation logical malignancy beyond fi rst remission. Median age was 56 M. Remberger, J. Mattsson (17-67). A majority of the patients had an unrelated donor (60%) CAST (Stockholm, SE) and were given PBSC (87%). Most patients received reduced intensity conditioning (RIC). Purpose: The prognostic value of CRP taken before condition- Results: Graft failure occurred in three patients in each group. ing for allogeneic stem-cell transplantation was evaluated in Engraftment of neutrophils and platelets occurred at a median 503 patients transplanted at Karolinska University Hospital, of 19 days (range 11-50) and 14 days (0-106) after HSCT Huddinge. with no difference between the two groups. The cumula- Patients: Reduced intensity conditioning (RIC) was given to 204 tive incidence of GVHD any grade was 66% and 31% in the patients and conventional myeloablative conditioning (MAC) Thymoglobulin and Campath cohort, respectively (p=0.017). to 299 patients. Most patients (n=425) had a haematological The incidence of grade II-IV GVHD was 31% and 24% (ns) in malignancy while 78 patients had a non-malignant disease. An the two group, respectively. No differences in TRM (21% vs. HLA-compatible related/unrelated donor was used in 196/229 34%), OS (45% vs. 49%) and RFS (42% vs. 42%) were found cases and an HLA-A, -B or –DR mismatched donor in 78 cases. between the Thymoglobulin and Campath groups. Furthermore,

S279 there were no difference in bacterial and viral infections while P945 there was a weak trend for more fungal infections (6% vs. 20%, Radioimmunotherapy with yttrium-90-ibritumomab p=0.2) among the Campath patients. Thymoglobulin treatment tiuxetan as part of a reduced-intensity conditioning was associated with a higher proportion of T-lymphocytes at regimen for allogeneic haematopoietic cell 6 months and a lower proportion of B-lymphocytes at 3, 6 and transplantation in patients with advanced indolent 12 months. No difference in IgG levels were seen between the non-Hodgkin’s lymphoma two groups during the fi rst year after HSCT. W.A. Bethge (1), T. Lange (2), S. von Harsdorf (3), Conclusion: Thymoglobulin and Campath as part of the con- M. Bornhäuser (4), B. Federmann (1), M. Stadler (5), L. Uharek ditioning produce similar results. Campath was associated (6), S. Knop (7), G. Wulf (8), H. Dittmann (9), C. Faul (1), with less overall acute GVHD without a higher relapse rate. W. Vogel (1), L. Kanz (1), D. Bunjes (3) Thymoglobulin lead to a higher proportion of T-lymphocytes (1)Medical Center University of Tübingen (Tübingen, DE); and a lower proportion of B-lymphocytes. (2)Medical Center University of Leipzig (Leipzig, DE); (3)Medical Center University of Ulm (Ulm, DE); (4)Medical Center University of Dresden (Dresden, DE); (5)Medical Center P944 University of Hannover (Hannover, DE); (6)Charite-Campus Factors affecting thymopoiesis after non-myeloablative Benjamin Franklin (Berlin, DE); (7)Medical Center University conditioning of Würzburg (Würzburg, DE); (8)Medical Center University E. Castermans (1), M. Hannon (1), R. Cheynier (2), E. Willems of Göttingen (Göttingen, DE); (9)University of Tübingen (1), A. Gothot (1), V. Geenen (1), Y. Beguin (1), F. Baron (1) (Tübingen, DE) (1)University of Liège (Liege, BE); (2)Pasteur Institute (Paris, FR) Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a poten- Background: Nonmyeloablative conditioning followed by alloge- tial curative therapy to patients with advanced indolent NHL. neic hematopoietic cell transplantation (HCT) is used in elderly RIC HCT induces potent graft-versus-lymphoma effects, with patients with hematologic malignancies. It has been suspected best results in patients with low tumor burden at time of HCT. that reconstitution of T-cell numbers would be impaired in eld- Combined use of radioimmunotherapy (RIT) with RIC may erly patients given nonmyeloablative conditioning because of increase anti-lymphoma activity of RIC. Forty patients have age-related thymic atrophy. Here, we investigated long term been enrolled in a multicenter phase II study of allogeneic HCT lymphocyte reconstitution and thymic function in 64 patients combining RIT using yttrium-90-ibritumomab tiuxetan (Y90- given allogeneic peripheral blood stem cells (PBSC) after non- CD20, Zevalin) with 0.4 mCi (15 MBq)/kg on day -14 combined myeloablative conditioning. with RIC using fl udarabine (30 mg/m² day -4 to -2) and 2 Gy Patients and Methods: Median age at transplant was 56 years TBI (day 0) followed by allogeneic HCT from matched related (range 10-69). Conditioning regimen consisted of 2 Gy total or unrelated donors. Postgrafting immunosuppression con- body irradiation with or without added fl udarabine (n=57), or sisted of cyclosporine and mycophenolate mofetil. Diagnoses cyclophosphamide plus fl udarabine (n=7). Thirty-nine patients were follicular lymphoma (n=17), chronic lymphocytic leuke- received grafts from related and 25 from unrelated donors. mia (n=13), mantle cell lymphoma (n=8), marginal zone lym- GVHD prophylaxis consisted of mycophenolate mofetil and phoma (n=1) and immunocytoma (n=1). Median age was 55 cyclosporine or tacrolimus. Immune recovery was assessed (range, 34-68) years. PBSC grafts were either from matched between 1 and 6.5 years after HCT by signal-joint T-cell recep- related (n=13) or matched unrelated donors (n=27). All patients tor excision circle (sjTREC) quantifi cation (211 samples), and were high risk with refractory disease or relapse after preced- fl ow cytometry. Further, in order to demonstrate a potential ing autologous HCT. Disease stage at time of HCT was CR=7, thymic recovery, sjTREC level changes from day 100 to day PR=27, SD=6. Engraftment was rapid and sustained with no 365 were also assessed. graft rejections. Median time to >500 granulocytes/µL was 15 Results: There was a close correlation between sjTREC levels (range, 0-69) days, and to >20000 platelets/µL 9 (range, 0-69) and naive CD4+ T-cells (defi ned as CD4+CD45RA+) counts days. In 14 patients platelets never dropped below 20000/µL (P<0.0001). An inverse correlation was observed between and in 8 patients granulocytes never below 500/µL, illustrating the levels of sjTREC/ml and the recipient’s age (R=-0.37, the non-myeloablative intensity of the conditioning regimen. p<0.0001). Further, sjTREC levels increased signifi cantly from TRM in the fi rst 100 days was 10% (n=4) and overall 35% day 100 to 1 year after transplantation, this was more promi- (n=14). No additional toxicity due to RIT in comparison to our nent in younger recipients (p<0.01 for patients <50; p=0.02 previous experience with the same RIC alone was observed. for patients 50-60) and absent in patients >60. Naïve T-cell Incidence of grade II-IV GVHD was 40% (II=2, III=11, IV=3). To counts also increased signifi cantly over time. SjTREC levels date, extensive chronic GVHD occurred in 9 patients. Deaths still increased from 1 year to 2 years after HCT in patients < 50 occurred due to infections=8, GVHD=6 and relapse=2. 24/40 years old (p=0.02). In multivariate analyses, younger patient (60%) of all patients are alive with a median follow up of 454 age (P<0.001 and P=0.01), and absence of extensive chronic (range, 77-847) days, resulting in a Kaplan-Meier estimate 1 GVHD (P<0.001 and P=0.001) were the main factors associ- year survival of 62%. Disease status of patients alive is CR=16, ated with high sjTREC levels and high number of naive CD4+ PR=6 and Relapse=2. T-cells after nonmyeloablative conditioning. In conclusion, a combination of RIT with RIC is feasible with Conclusions: Our data suggest that thymic neo-generation of no additional toxicity due to RIT and with stable engraftment T-cells occurred from day 100 in patients under 60. However, in all patients. Disease response and treatment related mortal- the levels of sjTREC remained low for patients above 60. Fur- ity seems promising even in this elderly and heavily pretreated ther, chronic GVHD has a dramatic impact on thymic function, cohort but requires further follow-up. as observed after myeloablative conditioning. The impact on sjTREC levels on infectious complications will be presented.

S280 P946 with MDS/AML (n=277) and ALL (n=79) given SCT over an Dose-escalated radioimmunotherapy with 8-year period in a single institution. The median age was 51 yttrium-90-ibritumomab tiuxetan as part of a (range, 17-75). The donors were HLA-matched siblings (n=191), reduced-intensity conditioning regimen for allogeneic matched unrelated (n=139) and alternative ( n=26). Pts meeting haematopoietic cell transplantation in patients with standard eligibility criteria were routinely given myeloablative advanced aggressive non-Hodgkin’s lymphoma conditioning (MAC, Cy/TBI or BuCy, n=141). Pts non-eligible W.A. Bethge (1), S. von Harsdorf (2), M. Bornhäuser (3), for MAC were given either RIC (fl udarabine and reduced doses D. Beelen (4), M. Stelljes (5), R. Schwerdtfeger (6), L. Uharek of busulfan or melphalan, n=116) or modifi ed myeloablative (7), B. Federmann (1), C. Faul (1), W. Vogel (1), H. Dittmann conditioning (modMAC, fl udarabine with myeloablative doses (8), L. Kanz (1), D. Bunjes (2) of busulfan or treosulfan, n=99). Disease status at SCT was (1)Medical Center University of Tübingen (Tübingen, DE); CR1/ CR2 (n=176), previously untreated or untreated relapse (2)Medical Center University of Ulm (Ulm, DE); (3)Medical (n=63) and chemo-refractory (n=117). With a median follow-up Center University of Dresden (Dresden, DE); (4)Medical Center of 30 months (range 1-103), 159 pts are alive and 197 died; 75 University of Essen (Essen, DE); (5)Medical Center University of treatment-related causes and 122 of relapse. The estimated of Münster (Münster, DE); (6)Deutsche Klinik für Diagnostik 5-yr overall survival (OS) in this relatively high-risk pt group was (Wiesbaden, DE); (7)Charite-Campus Benjamin Franklin 34% (95%CI, 27-41). The status of disease at SCT was the (Berlin, DE); (8)University of Tübingen (Tübingen, DE) most important factor predicting OS; 48, 31 and 16% for pts in CR, untreated or chemo-refractory leukemia, respectively Allogeneic hematopoietic cell transplantation (HCT) using (p<0.001). Multivariable analysis (MVA) identifi ed SCT not in reduced intensity conditioning (RIC) regimens offers a potential CR, SCT from unrelated and alternative donors as adverse curative therapy to patients with advanced aggressive NHL. RIC prognostic signs with hazard ratios (HR) of 2.7, 1.5, and 2.2, HCT induces potent graft-versus-lymphoma effects with best respectively. The conditioning regimen used was not a signifi - results in patients with low tumor burden at time of HCT. Com- cant factor in the whole pt group with 5-yr OS of 38, 35 and 31% bined use of radioimmunotherapy (RIT) with RIC may increase after MAC, modMAC and RIC, respectively (p=NS). However, anti-lymphoma activity of RIC. In addition to the graft versus RIC was associated with reduced OS in 2 subgroups. Among lymphoma effect, HCT provides rescue from hematologic toxic- pts with MDS/AML given SCT not in CR, 5-yr OS was 34, 24 ity of RIT which may allow dose escalation of RIT. 20 patients and 13% after MAC, modMAC and RIC, respectively (p=0.03) have been enrolled in a multicenter phase I/II dose escalation with HR of 1.6 for RIC in MVA. In the group of ALL pts, MVA study of RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20, identifi ed RIC and SCT not in CR1 as adverse prognostic signs Zevalin) at two dose levels (0,6 (22 MBq) and 0,8 (30 MBq) with HR of 2.0 (p=0.04) and 2.0 (p=0.03), respectively. mCi/kg) in 10 patients each, combined with RIC for treatment of In conclusion, MAC should still be considered the standard of patients with aggressive NHL. Patients received dosimetry with care for SCT in acute leukemia in eligible pts. RIC is associated In111-CD20 on day -21 and RIT with an escalated dose of Y90- with inferior outcome in pts with ALL and pts not in CR at SCT. CD20 (0,6-0,8 mCi (22-30 MBq)/kg) on day -14 followed by RIC Randomized studies are needed to determine the role of RIC in using fl udarabine (30 mg/m² day -8 to -4), melphalan (140 mg/ MDS/AML pts in CR at SCT. The novel modMAC regimens are m² day -3) and alemtuzumab (20-30 mg day -3 to -2). For post- relatively well tolerated even in pts not eligible for MAC and may grafting immunosuppression cyclosporine was administered. be more effective than standard RIC in refractory disease. Diagnoses were diffuse large B-cell lymphoma (n=13), trans- formed chronic lymphocytic leukaemia (n=4), blastoid mantle cell lymphoma (n=2) and follicular lymphoma grade 3 (n=1). P948 Median age was 51 (range, 30-69) years. PBSC grafts were Cytoreduction with clofarabine/Ara-C combined with either from matched related (n=5) or matched unrelated donors reduced-intensity conditioning and allogeneic stem cell (n=15). All patients were high risk with refractory disease or transplantation in patients with high-risk, relapsed, or relapse after preceding autologous HCT. Disease status at time refractory acute leukaemia of HCT was CR=4, PR=15 and SD=1. No additional toxicity due S. Buchholz, J. Krauter, E. Dammann, G. Beutel, M. Stadler, to RIT was observed in comparison to our previous experience M. Eder, A. Ganser with the same RIC alone. Engraftment was rapid and sustained Hannover Medical School (Hannover, DE) with no graft rejections. Median time to >500 granulocytes/ µL was 15 (range, 9-32) days and to >20000 platelets/µL 11 Background: The combination of cytoreductive chemo- (range, 9-56) days. TRM at day +100 was 0% and overall 20%. therapy with reduced intensity conditioning (RIC) is a highly- Incidence of grade II-IV GVHD was 50% (II=8, III=1, IV=1). To effective anti-leukemic therapy. Since leukemic relapse after date, chronic extensive GVHD occurred in 6 patients. Causes SCT still remains a major problem we are using Clofarabine/ of death were relapse=6, suspected suicide=1, infection=3 and Ara-C (ClaraC) treatment followed by RIC and allogeneic SCT GVHD=1. 9/20 patients (45%) are alive with a median follow-up to treat high risk leukemia. of 258 (range, 81-497) days. Disease status of patients alive is Aims: To evaluate the anti-leukemic effi cacy of ClaraC followed CR=7, PR=2 and Relapse=1. In conclusion, dose escalation of by RIC allogeneic SCT. RIT and combined use with RIC is feasible with no additional Methods: Up to 11/08, 25 patients (median age: 59 years, range toxicity due to RIT and with stable engraftment in all patients. 28 – 69) with high-risk, relapsed or refractory acute leukemia or Long-term disease-free survival remains to be determined. MDS (AML n=15, ALL n=3, MDS n=5; atypical CML n=1, NK- cell NHL n=1; high-risk cytogenetics n=8) were in CR (n=6), in relapse (n=9), refractory (n=6), and untreated (n=4) at time P947 of SCT. Therapy consisted of clofarabine 30 mg/m² and ara-C Conditioning regimens prior to allogeneic stem cell 1000 mg/m² for 5 days and, after 4-5 days of rest, RIC with 4 transplantation in acute leukaemia: the role of dose Gy of TBI, 80 and 120 mg cyclophosphamide/kg for related and intensity unrelated donors, and ATG. Grafts were unmanipulated G-CSF A. Shimoni, A. Rand, I. Hardan, N. Shem-Tov, R. Yerushalmi, mobilised PBSC from matched (n=15) and mismatched (n=4) A. Nagler unrelated or matched related (n=6) donors. GvHD prophylaxis Chaim Sheba Medical Center (Tel-Hashomer, IL) consisted of CSA and MMF. Results: ClaraC cytoreduction followed by RIC allogeneic SCT Allogeneic SCT with both myeloablative and reduced-intensity is well tolerated overall with good anti-leukemic effi cacy in high- conditioning (RIC) is effective therapy in acute leukemia. To bet- risk patients: 15/25 patients are in ongoing CR, 2/25 relapsed ter defi ne the role of dose intensity in SCT, we retrospectively (day + 87 and +382; one patient in 2. CR upon DLI treatment), analyzed SCT outcomes in 356 consecutive adult patients (pts) and 8/25 died (median follow-up 7 months, range 0.1 – 19).

S281 All but four (early death) patients engrafted (median day + 15 P950 for ANC > 0.5 G/l, range: +10 to +31). So far, no acute GvHD Long-term clinical and molecular outcomes following > II° was observed with follow-up > 100 days for 18 patients. RIC-allogeneic HSCT from HLA-identical sibling in Limited and extensive cGvHD occurred in 2 and 3 out of 17 patients with advanced mycosis fungoides and Sézary evaluable patients. Non-hematological toxicity included revers- syndrome ible hepatotoxicity (increase in ALT and AST CTC II - IV°) in 20 F. Onida (1), E. Berti (2), G. Saporiti (2), L. Corti (3), C. Annaloro patients, hyperbilirubinemia CTC III°-IV° in 7 patients, revers- (2), E. Tagliaferri (2), A. Della Volpe (2), P. Vezzoli (2), P. Usardi ible skin toxicity (CTC II° - III°) including mild to severe hand- (2), G. Lambertenghi Deliliers (1) foot-syndrome in 16 cases, hemorrhagic cystitis in one patient, (1)University of Milan (Milan, IT); (2)Ospedale Maggiore and seizures after SCT in one patient. Causes of death include Policlinico (Milan, IT); (3)Fondazione Matarelli (Milan, IT) septicaemia (n=4), pneumonia (n=1), EBV-PTLD (n=2), and cerebral hemorrhage (n=1), respectively. Therapeutic options for pts with advanced tumor-stage mycosis Conclusions: Cytoreduction with clofarabine/Ara-C followed fungoides (MF) and Sézary syndrome (SS) are very limited and by RIC allogeneic HSCT has good anti-leukemic effi cacy even mainly palliative. Allogeneic hematopoietic stem cell transplanta- in elderly patients with high-risk acute leukemia or MDS with tion (allo-HSCT) represents the only curative strategy in selected engraftment and GvHD-incidence comparable to other RIC- pts. However, because pts with MF/SS are often elderly and in regimen. Non-hematological toxicities of this regimen mainly poor conditions, high morbidity and mortality counterbalance affect liver and skin and are reversible. survival benefi t. Reduced-intensity conditioning (RIC) regimens signifi cantly decrease TRM, allowing gradual establishment of full donor chimerism and possible GVL effect. Evaluation of P949 MRD is particularly useful to guide post-transplant strategies, Reduced-intensity conditioning umbilical cord blood such as DLI. Due to its extensive combinatorial repertoire and transplantation in adults: age, disease status and large hypervariable regions, rearrangements of TCRb represent infectious complications are important risk factors for the best target for MRD monitoring in T-cell malignancies. outcome In our Institution, between 09/2000 and 12/2007, 13 pts under- S. Fürst, C. Faucher, J. El Cheikh, J. Boher, P. Ladaique, went allo-HSCT from a HLA-identical sibling following a RIC C. Lemarié, N. Vey, A.M. Stoppa, R. Bouabdallah, regimen including fl udarabine/cyclophosphamide/TBI200 (up to H. de Lavallade, M. Mohty, C. Chabannon, D. Blaise 2001, 3 pts) or pentostatin/TBI200 (from 2002 to present, 10 Institut Paoli Calmettes (Marseille, FR) pts). GVHD prophylaxis included Cy-A and MMF. At the time of transplant all pts (10 males and 3 females; median age 48 years, We report the outcome of 35 patients (pt) who received a range 37-66) had stage III/IV refractory MF (n=9) or refractory Reduced intensity conditioning umbilical cord blood trans- SS (n=4). Median time from diagnosis to HSCT was 36 months plantation (RIC-UCBT) in a single centre between 2005 and (range 13-252). Source of stem cells was PB in all pts. 2008. All pt had high risk hematological malignancies (AML=19; Full donor chimerism was achieved in 38% of pts at day +30 ALL=9; NHL=5; CML=1; and Hodgkin disease=1). 27 pt (77%) and in 87% at 6 months. Clinical CR was obtained in 11 pts. were in CR (CR1=18; CR2=8; CR3=1), whereas 8 had a more Acute GvHD occurred in 7 pts (6 grade I-II and 1 grade IV); advanced disease (PR=2; refractory=6) at time of UCBT. The chronic GvHD (extensive in 2 cases) occurred in 6 pts. 4 pts median age was 44 (range, 17-62) years. For age we distin- have died, 2 in CR (1 from sepsis and 1 from aGVHD), and 2 guished 2 groups: group 1 (n=13, age > 50 years) and group 2 with progressive disease. (n=22, age ≤ 50 years). 13 pt (37%) received a single CB unit, After a median follow-up of 43 months (range 2-99), 9 pts are whereas 22 (63%) received 2 CB units in order to achieve a alive and CR is maintained in 8 (Fig.1). minimum required cryopreserved cell dose of 3.0x107 TNC/kg. Skin biopsy and PB samples at diagnosis and at different time For the entire group the median infused cell dose was 3.7x107/ points after HSCT were obtained in 7/9 evaluable pts and mon- Kg (range, 1.9-5.5). Neutrophil engraftment occurred in 33 pt oclonal TCRb rearrangement has been sequenced obtaining (94%) at a median of 20 (range, 6-45) days and a sustained clone-specifi c primers for PCR assays. Molecular remission platelet recovery was observed in 25 pt (71%) at a median of was documented in all pts. 1 pt experienced clinical relapse 36 (range, 23-136) days. The overall incidence of grade II-IV 52 months after HSCT. Retrospective clone-specifi c analysis acute GVHD was 54% (95%CI=[37-71]; 7 grade II, 10 grade III of TCRb rearrangements unveiled the presence of molecular and 2 grade IV) and 37% for chronic GVHD (95%CI=[21-55]; relapse already 24 months before. 8 limited and 5 extensive cases) with no difference between Our results suggest that RIC-HSCT represents an effec- the 2 groups. 21 pt (60%; 95%CI=[37-71] experienced at least tive strategy of cure in advanced stages of refractory MF/SS. one episode of a severe infectious complication (SINC) (virus, Detection of MRD by TCRb analysis is feasible, allowing earlier n=11; bacteria, n=10; fungal, n=4), requiring long-term hospi- identifi cation of relapses and possible adoption of pre-emptive talization. With a median follow-up of 468 (95%CI=[201-681]; treatment such as DLI. range, 50-1170) days, 10 pt (28%) had relapsed or progressed with this being signifi cantly lower in younger pt (P=0.045) and in those transplanted in CR (P=0.007). Regarding the 2 groups, 7 pt (54%) in group 1 died (infection=3; GVHD=2; dis- ease=2; TRM=38%) whereas 7 (32%) in group 2 (infection=2; GVHD=1; disease=4; TRM=14%).The KM estimate of OS and DFS was 61% (95%CI=[42-75]) and 52% (95%CI=34-67) at 2 years respectively for the entire population with signifi cant better outcome in pt in CR (OS, P=0.004; DFS, P=0.007) and pt in group1 (OS, P=0.052; DFS, P=0.041). The multivariate analysis showed a signifi cant impact of 2 factors on DFS : age (group 1 vs group 2: HR 3.05, 95%CI=[1.07-8.73], P=0.037) and disease status at transplant (No CR vs CR: HR 6.33, 95%CI=[1.77-22.6], P=0.005). Age and disease status at trans- plant are crucial for patients outcome and further efforts are needed to defi ne risk factor specifi c transplant procedures. Infectious complications and GVHD are still a matter of concern warranting better strategies to provide optimal prophylactic and therapeutic approaches.

S282 P951 45RA/R0), TCR-Va repertoire and NK-cell receptors (NKP30, Double reduced-intensity allogeneic haematopoietic stem NKP44, NKP46, NKG2D, CD158a/b/e, CD85j, NKG2A, CD161) cell transplantation were analyzed by FACS. J.-O. Bay, A. Cabrespine-Faugeras, C. Faucher, R. Tabrizi, Grafts contained 8.6x106 CD34+ (range 4.3-18.0x106), 2.9x104 P. Bordigoni, K. Bilger, J.P. Jouet, M. Renaud, V. Mialou, CD3+ (range 1.9-9.2x104) and 3.3x107 CD56+ cells/kg (range M. Robin, M. Kuentz, P. Chevallier, N. Dhédin, A. Huynh, 0.02-23.0 x107). Engraftment was rapid with a median of 11 F. Garban, F. Witz, A. Buzyn, T. De Revel, C. Galambrun, days (range, 9-15) to >500 granulocytes/µl and 11 days (range, E. Deconnick, N. Contentin, S. Francois, D. Blaise, M. Michallet 8-23) to >20 000 platelets/µl. Full chimerism was reached on on behalf of the Société Française de Greffe de Moelle et de day 14 (range 9-26). NK-cell engraftment was rapid reach- Thérapie Cellulaire, SFGMTC ing normal values on day 20 (median 248 CD16+56+3- cells/ µl, range 1-886) with NK cells comprising up to 70% of lym- Aim of the study: Describe the outcome of patients who phocytes. T-cell reconstitution was impaired with 50 (range 0- underwent double reduced-intensity allogeneic hematopoietic 586) and 200 (range 35-536) CD3+ cells/µl on day 60 and day stem cell transplantation (AHSCT) Methods: Fifty four double 150, respectively. We observed an increase of CD3+8+ cells in reduced-intensity AHSCT performed between 1997 and 2006 contrast to CD3+4+ cells with a median of 41 (range 0-1277) vs were retrospectively studied. Results: Diagnosis was 21 acute 33 cells/µl (range 0-301) on day 80 and 247 (range 0-558) vs myeloid leukemia (39%), 7 aplasic anemia (13%), 5 myelod- 175 (0-284) cells/µl on day 250, respectively. CD4+45RA+ T- ysplasic disorder and/or myeloproliferative syndrome (9.3%), 5 cells increased slowly while CD4+45RO+ T-cells reconstituted acute lymphoblastic leukemia (9.3%), 5 chronic myeloid leuke- faster with a median of 68 CD4+45RA+ (range 0-310) vs 28 mia, 2 myeloma (3.70%), 1 ovarian cancer (1.8%), 5 lymphoma CD4+45R0+ cells/µl (range 0 to 152) on day 100. B-cell recon- (9.3%), 2 chronic lymphoblastic leukemia (3.70%) and 1 acute stitution was delayed with 120 (range 0-489) and 181 (range leukemia (1.8%). Major graft source was PBSC (n=39, 72%). 11-452) CD19+20+ cells/µl on days 150 and 400, respectively. Donor histocompatibility was identical sibling (n=37), match TCR-Va-repertoire was analyzed in 15 and NK-cell receptor unrelated (n=10), mismatch unrelated (n=6) or syngeneic reconstitution in 8 patients. We found a skewed T-cell repertoire (n=1). Second AHSCT indication was relapse (n=28), lost of with oligoclonal T-cell expansions to day 100 and normalization graft (n=11), no engraftment (n=9), residual disease (n=2), after day 200. An increased NCR and NKG2A, but decreased refract disease (n=1) or scheduled therapeutic sequence (n=1). NKG2D and KIR-expression was observed on NK-cells until Median time between both AHSCT was 7.8 months [1.1-61.0]. day 100. For AHSCT 2, conditioning regimen, source of stem cells or In summary, after HHCT using CD3/CD19 depleted grafts, donor were changed for 79% (n=43), 32% (n=17), and 39% NK-cell reconstitution occurs early and fast while T- and B-cell (n=21) of patients respectively. The median delay to reach a reconstitution is delayed but seems faster compared to data neutrophil count of 1x109/l was signifi cantly smaller after sec- published with CD34 selected grafts. A combination of reduced ond AHSCT (21 days [range 6.1-250] versus 16 days [range intensity conditioning with CD3/CD19 depleted grafts appears 6.1-104], p= 0.026). Median time to reach a platelet count of to accelerate the immune recovery after HHCT. 50x109/l was 18.5 days [range 6.1-250] versus 14 days [range 3-85.4] but difference failed to reach statistical signifi cance (p=0.084). Overall incidence of acute GvH was similar after P953 both AHSCT (33% versus 37%). However, acute GvH grade Acute graft-versus-host disease prophylaxis after III-IV proportion seems to be higher after second AHSCT (11% reduced-intensity conditioning in allogeneic peripheral versus 37%, p=0.12). Complications rate was signifi cantly blood stem cell transplantation from HLA-identical sibling higher after AHSCT 2 (24% versus 45%, p = 0.027). Response donors. Comparison of CsA and MMFvs CsA and MTX was improved by second AHSCT for 28% of patients who had J.L. Piñana (1), D. Valcarcel (1), F. Fernandez-Avilés (2), no response after AHSCT 1 (8/28) and lead to engraftment for R. Martino (1), M. Rovira (2), P. Barba (1), A. Sureda (1), 40% of patients who had no engraftment or lost of graft after E. Carreras (2), J. Sierra (1) AHSCT 1 (8/20). (1)Hospital de la Santa Creu i Sant Pau (Barcelona, ES); Fifteen patients are still alive with 67% with complete response (2)Hospital Clínic de Barcelona (Barcelona, ES) status (n=10). Twenty one patients died from progression and 16 of TRM (43%). With a median follow-up of 61.6 months [27- Background: The data comparing cyclosporine A/Methotrexate 133], median overall survival was 25 months [3-105]. and Cyclosporine/MMF in reduced intensity conditioning Allo- Conclusions: Double reduced-intensity AHSCT is a feasible HSCT (Allo-RIC)as GVHD prophylaxis is still limited. In this procedure with satisfactory graft performance which allows to case-control study we retrospectively analyzed the introduc- response or engraftment not observed after AHSCT 1. Main tion of MMF as GVHD prophylaxis in comparison with a short indication is relapse. However, TRM remains high. course of MTX in a large series of patients who underwent HLA identical sibling donor Allo-RIC. Patients and methods: We included 145 consecutive Allo-RIC P952 patients who received peripheral blood (PB) as source of stem Immune reconstitution after haplo-identical haematopoi- cells, between April 2000 and August 2007. The median fol- etic cell transplantation: impact of reduced-intensity low-up for the whole group was 514 days (8 to 3170 days). The conditioning and CD3/CD19 depleted grafts study group included 91 males and 54 females. Median age was B. Federmann, M. Haegele, C. Faul, W. Vogel, L. Kanz, 55 years (range 18 to 71 years). Diagnoses were acute leuke- W.A. Bethge mia/ myelodisplastic syndrome/ myeoloproliferative disorders Hematology & Oncology (Tübingen, DE) (n=37/15/13), multiple myeloma (n= 32), Hodgkin lymphoma (n= 6), chronic lymphocytic leukemia (n=15) and non-Hodgkin Haploidentical hematopoietic cell transplantation (HHCT) using lymphoma (n= 27). GVHD prophylaxis consisted of CsA/MMF in CD3/CD19 depleted grafts and reduced intensity condition- 52 and CsA/MTX in 93 patients. The conditioning regimen was ing (RIC) may lead to fast engraftment and immune reconsti- based on fl udarabine in combination with busulfan (n= 59) or tution since grafts contain in addition to stem/progenitor cells melphalan (n=86).Results: The occurrence of mucositis grade also NK-cells, graft-facilitating-cells, dendritic-cells and CD34- 2-4 was higher in the CsA/MTX group than in the CsA/MMF progenitors. group (57% vs 23% p= 0.001). Cumulate incidence of acute 27 adults received HHCT with CD3/CD19 depleted grafts after and chronic GVHD were similar 50% vs 48% and 68% vs 71% RIC(fl udarabine, thiothepa, melphalan and OKT-3)and were for CsA/MMF and CsA/MTX, respectively (p> 0.7). Multivariate prospectively evaluated for engraftment and immune reconsti- analysis showed that risk factors for early (at day +100) non- tution. NK-, B-, T- cells and T-cell-subsets (CD3/8, CD4/8, CD4/ relapse mortality (NRM) were CsA/MTX combination (HR 3.3,

S283 95% C.I. 1-11.3, p= 0.05), advanced disease (HR 5.2, 95% C.I. P955 1.2-22, p= 0.03), ECOG >1 (HR 2.9, 95% C.I. 1.2-7, p= 0.02) Pretransplant characteristics could predict full donor and grade II-IV aGVHD (HR 2.8 95% C.I. 1.2-8, p= 0.02), while chimerism on day +90 and day +180 and outcomes in for 2-years NRM they were grade II-IV aGVHD, ECOG >1 and reduced-intensity alemtuzumab conditioning allogeneic lymphoid malignancy (HR 3, 95% C.I. 1.5-6, p= 0.002), (HR 2.1, transplantation can be infl uenced by chimeric status 95% C.I. 1.1-4, p= 0.02) and (HR 2, 95% C.I. 1-4.1, p= 0.05), E. Nikolousis (1), S. Tauro (2), C. Brookes (3), S. Paneesha (1), respectively. Relapse and overall survival at median follow-up I. Novitzky Basso (1), P. Mahendra (3), M. Cook (3), F. Clark (3), were similar between CsA/MTX and CsA/MMF groups (25% vs D. Macmullan (4), M. Griffi ths (4), R. Lovell (1), S. Chaganti (1), 18%, p= 0.4 and 52% vs 51%, p= 0.7, respectively). S. Chaganti (1), D. Milligan (1), S. Robinson (5), C. Craddock Conclusion: Our results support the use of the CsA/MMF com- (3) bination as GVHD prophylaxis in HLA- identical sibling donor (1)Birmingham Heartlands Hospital (Birmingham, UK); Allo-RIC. It provides a better toxicity profi le, refl ected by a sig- (2)University Hospital Dundee (Dundee, UK); (3)University nifi cantly lower incidence of moderate-severe mucositis. Hospital Birmingham (Birmingham, UK); (4)Women’s Hospital The author is supported by grants from the Instituto de Salud Birmingham (Birmingham, UK); (5)Bristol Royal Infi rmary Carlos III (expedient CM06/00139, Ministerio de Sanidad). (Bristol, UK)

The use of reduced intensity conditioning transplants gained P954 momentum over the last 10 years enabling older patients with Regulatory T-cells and chronic GvHD after haematological malignancies to go through a potentially cura- non-myeloablative conditioning tive treatment option. S. Humblet-Baron, E. Castermans, J-F. Vanbellinghen, We have analysed whether pretransplant characteristics could M. Hannon, N. Jacobs, E. Willems, S. Ormenese, Y. Beguin, predict full donor (FD)chimerism on whole blood(WB) and T-cells F. Baron on day +90 and +180 and also the effect of the chimeric status University of Liège (Liege, BE) on the transplant outcomes. This study included 70 patients with lymphoid (n=43) and myeloid (n=27) malignancies with Purpose: We investigated the association between regula- a median age of 47 years (9-63). Median follow up was 22.3 tory T-cell (Treg) levels and chronic graft-versus-host disease months (range 2.2–49.6 months). Pretransplant characteristics (GVHD) after allogeneic hematopoietic stem cell transplanta- included age, sex, donor type, previous stem cell transplant, tion (HCT) following nonmyeloablative conditioning. CD34 dose, CD3 dose, disease status at transplant, alemtuzu- Methods: Data from 74 patients given nonmyeloablative con- mab dose (50 vs. 100mg) and conditioning regimen. ditioning as treatment for hematological malignancies or renal Multivariate analysis showed CD34 count was signifi cant in pre- cell carcinomas were analyzed. Conditioning regimens con- dicting FD T cell chimerism at 90 days (p=0.05). High CD3 cell sisted of low-dose TBI with (n=45) or without (n=17) fl udara- dose (p=0.01) predicted FD whole blood chimerism at 90 days. bine, or cyclophosphamide plus fl udarabine (n=12). T-reg High CD34 cell count (median 6.0 vs. 5.0, p=0.02) and type of (CD4+FoxP3+) levels on days 40, 100, 180 and 365 were donor (Unrelated vs. sibling: p=0.003) were predictors for FD determined by fl ow cytometry. Chimerism levels among total chimerism at 180 days. FD T cell chimerism at 180 days was white blood cells, CD3+ T-cells and CD4+CD25+CD127dim/ predicted by high CD34 count (p=0.02) and type of donor (Unre- neg regulatory T-cells were determined by multiplex STR PCR lated vs. sibling: p=0.006). Donor type (unrelated vs. sibling; or X-Y FISH. Thymic function was determined by assessing 68% vs. 33%: p=0.01), high CD3 cell dose (median 130 vs. 2, sjTREC levels. p=0.0001) and dose of alemtuzumab (100mg vs 50mg p=0.006) Results: Mean Treg ( SD) levels in controls were 31 18 cells/ L. were predictors for FD whole blood chimerism at 180 days. Mean (SD) Treg levels on day 100 were 20 24 cells / L (P<0.01 FD and mixed T cell chimerism at 90 days did not impact on in comparison to controls) in patients without grade II-IV acute overall survival (OS), disease free survival (DFS), relapse free GVHD before day 100, and 27 21 cells/ L (NS) in patients with survival (RFS) and chronic graft versus host disease (GvHD). an antecedent of grade II-IV acute GVHD. Mean donor CD3+ FD chimerism at 90 days (p=0.001) was associated with acute T-cells and Treg chimerism levels on day 100 were 75 29% GvHD. T cell mixed chimerism at 180 days was a predictor for and 81 18%, respectively (NS). Day 100 chimerism levels relapse (HR=7.6, p=0.02). Full donor whole blood chimerism among CD3+ T-cells and Tregs were highly correlated (r=0.78, at 180 days was a predictor for both acute (OR=3.3, p=0.04) P<0.01). The 1-year probability of grade II-III NIH chronic GVHD and chronic GvHD (OR=4.6, p=0.02). However whole blood in patients with day 100 Treg levels below or above median was chimerism at 180 days did not impact OS, DFS, RFS and acute 53% and 36%, respectively (P=0.13). SjTREC levels signifi - or chronic GvHD, but it showed a trend for increased relapse cantly increased from day 100 to day 365 after HCT (P<0.01), (HR=2.52, p=0.05). demonstrating thymic recovery. Finally, Treg and sjTREC lev- Our study revealed CD3 & CD34 cell dose along with unrelated els correlated on days 100 (r=0.48, P<0.01) and 365 (r=0.47, donor type were predictors for whole blood & T cell chimerism at P<0.01) after HCT. days +90 and +180 in reduced intensity alemtuzumab transplan- Conclusions: Our data did not show thus far a signifi cant cor- tats. It also showed donor chimerism may be a predictor for GvHD. relation between Treg levels and occurrence of chronic GVHD. More studies are warranted to understand the predictors for donor The association between Treg and TREC levels on days 100 chimerism and to establish optimal transplant strategies. and 365 might suggest a role for the thymus in regulating Tregs levels after HCT, or that similar factors affect thymic function and Treg levels after nonmyeloablative conditioning. Data P956 including higher number of patients will be presented. Effect of donor and recipient ABO group on overall survival post allogeneic HSC transplant: is minor-side ABO incompatibility still associated with adverse outcome in reduced-intensity conditioning allograft despite the use of alemtuzumab in conditioning? E.K. Nicholson, A. Clark, I.G. McQuaker, A.N. Parker, K.W. Douglas Beatson West of Scotland Cancer Centre (Glasgow, UK)

In reduced-intensity conditioning (RIC) allogeneic hemopoi- etic stem cell transplant (HSCT), minor-side ABO blood group incompatibility (mns-ABOi) can lead to delayed haemolysis of

S284 recipient red cells following antibody production by passenger allo-HSCT was 16% (n=15), at the same time OS after RIC allo- donor-derived lymphocytes. Some studies have suggested that HSCT was 0% (n=11). All relapsed or primary resistant patients mns-ABOi RIC transplants are consequently associated with that undergone RIC allo-HSCT died within 8 months. adverse outcome, and that this may be prevented by prophylac- Conclusion: Therefore, our data showed that RIC may improve tic apheresis red cell exchange on the recipient. Because alem- OS after allo-HSCT performed in remission of pediatric ALL. tuzumab is part of RIC regimens in our unit, depleting both B and The feasibility of RIC HSCT in relapsed or primary resistance T lymphocytes unlike anti-thymocyte globulin, and because of a cases of pediatric ALL requires further investigations. It is nec- low reported incidence of post-transplant haemolysis, this has essary to pay special attention to posttransplantation therapy not been thought necessary when using alemtuzumab-contain- (chemotherapy, immunotherapy and target therapy). ing conditioning regimens. However, this has never been sub- ject to formal audit. We have audited overall survival (OS) for all patients with mns-ABOi versus no mns-ABOi undergoing HSCT P958 at our centre from January 2000 to January 2008. Outcome of sequential regimen of chemotherapy and Methods: Retrospective audit of local transplant database. reduced-intensity conditioning in patients with advanced Results: There were 277 patients in total; median age 42 years; haematological malignancies male: female ratio 162//115; 133 sibling allografts and 144 VUD M. Krejci, J. Mayer, Y. Brychtova, M. Doubek, Z. Racil, transplants. 160 transplants used myeloablative conditioning Z. Koristek, M. Navratil, M. Tomiska, J. Vorlicek (MAC; median age 36.92 years) and 117 were RIC transplants University Hospital Brno (Brno, CZ) (median age 49.51 years). In total 72/277 (26%) patients had mns-ABOi and 205/277 (74%) did not. Of the RIC transplants, Background: Sequential use of intensive chemotherapy and 26/117 (22%) had mns-ABOi and 91/117 (78%) did not. The reduced-intensity conditioning (RIC) for allogeneic stem cell mns-ABOi RIC transplants had a trend towards reduced OS transplantation (SCT) in high-risk patients (pts) represents a of 297 days compared to 856 days for RIC without mns-ABOi promising approach (Schmid et al., JCO 23, 2005: 5675-87). (p=0.261). In contrast, among the MAC transplants (n=160), Here, we wanted to explore if these results are reproducible at there was no difference in OS between mns-ABOi transplants another group of pts. (median OS 683 days; n=46) and non-mns-ABOi transplants Methods: We retrospectively analyzed 36 pts with advanced (median OS 467 days; n=114; p=0.473).There was a higher hematological malignancies (AML, n=22; ALL, n=4; MDS, n=3; proportion of VUD, HLA mismatch and older patients within the others, n=7) undergoing chemotherapy and RIC SCT in our mns-ABOi RIC transplants as compared to the non-mns-ABOi centre from March 2006 to May 2008. Fludarabine (30 mg/m²), RIC transplants which may contribute to the inferior OS seen. cytarabine (2 g/m²), and amsacrine (100 mg/m²) for 4 days However, on considering only fully-HLA-matched sibling RIC (FLAMSA) were used for cytoreduction. Amsacrine was not allografts, there was still at trend towards inferior OS for mns- administered to pts with lymphoid malignancies (8/36). After 3 ABOi transplants (median OS 433d, n=7) compared with non- days of rest, RIC consisting of 4 Gy TBI, ATG (Fresenius) 10-20 mns-ABOi transplants (median OS 1056d, n=47, p=0.38). mg/kg/day for 3 days, and cyclophosphamide 40-60 mg/kg/day Conclusions: Although patient numbers are low, there is a trend for 2 days followed. Disease status before SCT was: CR1, n=5; towards poorer outcome with minor-side ABO incompatible RIC CR2, n=9; CR3, n=1; refractory disease or relapse, n=21, type allografts despite the use of alemtuzumab with conditioning of donors: HLA identical sibling, n=12; unrelated donor, n=24, therapy. This may merit audit at national level. and grafts used were: PBSCs, n=35; BM, n=1. Median age of pts was 49 years (range 19-61). Results: The neutrophil engraftment (above 0.5x10E9/L) was P957 achieved at a median time of 18.0 days. No graft rejection Allogeneic HSCT with reduced intensity versus was observed. Transplant-related mortality (TRM) to the day myeloablative conditioning regimens in paediatric +100 was 25% (9/36). Causes of death were GVHD (n=2), acute lymphoblastic leukaemia septic shock (n=3), multiorgan failure (n=2), and brain hemor- E. Semenova, N. Stancheva, I. Kazantsev, Y. Vasilieva, rhage (n=2). All early deaths were seen in very high risk pts V. Kostorov, S. Shiryaev, B. Afanasyev, L. Zubarovskaya only: relapse or refractory disease, n=8; CR3, n=1. Incidence of SPb State I. Pavlov Medical University (St. Petersburg, RU) acute GVHD was evaluated in 29 pts: 55% (16/29) of pts had GVHD (grade I+II in 14 pts, grade III in 2 cases). Incidence of Background: Reduced-intensity conditioning (RIC) regimens chronic GVHD was evaluated in 24 pts, 58% (14/24) of pts had prior to allogeneic hematopoietic stem cell transplantation (allo- GVHD (limited in 11 cases, extensive in 3 cases). Treatment HSCT) are used in patients (pts) not eligible for conventional response was evaluated in 29 pts: remission was achieved conditioning therapy due to advanced disease, toxic complica- in 27 pts (93%), only 2 pts had progression of leukemia. With tions after previous chemotherapy or signifi cant comorbidity. median follow-up 225 days (range 1-900), 56% of all pts (20/36) Outcome of alloHSCT in pediatric patients with acute lymphob- were alive (19 pts in remission, 1 pt with relapse), 16 pts died lastic leukemia (ALL) retrospectively was analyzed. (9 deaths from TRM, 7 deaths from relapse or progression of Patients and methods: 73 children and adolescences (median leukemia), nine relapses (31%; 9/29) occurred. age, 8.8, range 1–20 years) suffering from ALL were underwent Conclusion: FLAMSA-RIC protocol seems to be effective sal- alloHSCT. Myeloablative conditioning was used in 51 pts: 2 CR vage treatment in pts with advanced both myeloid and lymphoid -36 pts, relapse -15 pts, RIC in 22 pts: 2 CR-11 pts, relapse malignancies. Response rate is high (93%) and the toxicity -11 pts. moderate with low frequence of serious GVHD. Early deaths Results: 7-year overall survival (OS) in patients at all disease in very high risk pts call for careful indication of this treatment. stages including relapsed and primary resistance cases was However, longer follow-up is needed. 32%. Comparative analyzes showed a signifi cant difference (p<0.05) in OS depending on disease status (41% 7-years OS after allo-HSCT in remission versus 11% in relapsed or primary P959 resistance cases). Also, it was revealed that use of RIC may Improved outcome following reduced- improve OS after allo-HSCT performed in remission of disease. intensity conditioning for second allogeneic OS after RIC regimens (n=11) was 64% versus 37% after mye- stem cell transplantation loablative conditioning (n=36). Survival improvement could be D. Nachbaur, J. Auberger, J. Clausen, B. Lindner explained by lower transplant related mortality (TRM) in RIC Hematology and Oncology (Innsbruck, AT) group. Nevertheless, according to our data, RIC regimens had poorer prognosis than myeloablative allo-HSCT in the case of Objectives: Second conventional myeloablative allogeneic relapsed or primary resistant ALL. 3 - year OS after myeloablative transplants for patients relapsing after a fi rst allogeneic or

S285 autologous transplantation are associated with a dismal out- (1.1-9.0) x 107/kg. Except one patient dying to early all patients come due to a high non-relapse mortality and a high relapse engrafted after a median of 18 (13-50) days. All but one patient rate. were >85% donor chimeras by d+30. Clinically signifi cant acute Patients: Forty nine patients receiving a second allogeneic graft-versus-host disease occurred in 7/13 (cumulative inci- SCT for relapse/progression after a fi rst autologous or alloge- dence 54%) and grade III/IV in 4/13 patients. CMV infection neic transplantation were studied. Underlying diagnoses were occurred in 7/12 patients at risk (cumulative incidence 58%). acute leukemia (n=21), lymphoma (n=11), myeloma (n=11), The relapse incidence was 38% (19%-77%, 95% confi dence and other diseases (n=6). Median patient age at the time of interval) and the non-relapse mortality was 61% (40%-94%, second transplant was 42 (range, 18-62) years. Ten patients 95% CI). The median overall survival for the entire cohort was had a prior allogeneic transplantation, 33 had an autologous 73 (9-482) days. Causes of death were relapse/progression in transplant, and 6 patients had a relapse/progression after a fi ve (38%), toxic multiorgan failure in one (8%), and infection in double autotransplant. In patients receiving a second alloge- six patients (46%). neic transplantation eight patients had an alternative stem cell Conclusion: Reduced intensity haploidentical SCT with post donor. Thirty patients were grafted from a sibling donor and 19 transplantation cyclophosphamide results in a high rate of patients received grafts from volunteer unrelated donors. Con- engraftment and donor chimerism. However, long-term survival ditioning was myeloablative in 21 patients and of reduced inten- in this high-risk cohort was not observed due to a high relapse sity in the remaining 28 patients. rate and an unacceptable high non-relapse mortality. Better Results: Nine (18%) patients died before hematopoietic patient selection would help to identify the value of this haploi- engraftment either due to toxicity or infection. All other patients dentical transplantation protocol. engrafted after a median of 11 (range, 8-25) days. Overall sur- vival for the entire cohort was 13% (2%–24%, 95% confi dence interval, CI). Survival was signifi cantly better in patients receiv- P961 ing a second transplant after reduced intensity conditioning Clinical course and outcome of 20 patients with two or (18% vs. 0%, p=0.0276 log rank test) due to a signifi cantly lower more allogeneic haematopoetic stem cell transplantations non-relapse mortality (80% vs. 62%, p=0.04 log rank). The C. Grosse-Thie, K. Borchert, K. Jost, I. Hilgendorf, H. Andree, relapse incidence following a second allogeneic SCT was 28% M. Leithauser, C. Kahl, M. Freund, C. Junghanss (17%–47%, 95% CI), with a trend for a high relapse incidence University of Rostock (Rostock, DE) following reduced intensity transplants (33% vs. 22%, p=ns.). Even by multivariate analysis including time interval between Introduction: Allogeneic transplantations of hematopoietic stem fi rst and second transplant as numerical variable and donor cells (alloHSCT) are common procedures to cure haemato- type, conditioning, prior allogeneic SCT, HLA mismatch, male logical malignancies. Data on second alloHSCTs in the same recipient/female donor, and the diagnosis of acute leukemia as patient (pt) are limited and little is known about outcome and categorical variables, receiving a second transplant following complications. Here we report on a series of patients (pts) that reduced intensity conditioning was associated with improved received more than one alloHSCT. outcome (RR for death 0. 45, p=0.03, Cox regression). Method: Data from alloHSCT recipients that were transplanted Conclusion: Second allogeneic stem cell transplantation follow- between 7/1997 and 10/2008 at our institution were retrospec- ing reduced intensity conditioning results in a better outcome tively analysed. All pts receiving another alloHSCT following than following conventional myeloablative conditioning. How- fi rst alloHSCT were analysed in regards to disease characteris- ever, non-relapse mortality and relapse still remain challenging tics, age, donor types and outcome. problems. Results: A total of 20 pts with a median age at second HSCT of 44 yrs (16-64 yrs) received 2 or more alloHSCTs. Five pts got a total of 3 alloHSCTs. Underlying diseases were: acute myeloic P960 leukemia (14 pts), chronic lymphocytic leukemia (2 pts), T-cell- Reduced-intensity HLA-haplo-identical stem cell lymphoma (1 pt), osteomyelofi brosis (1 pt), myelodysplasia (1 transplantation with post transplantation pt) and acute lymphoblastic leukemia (1 pt). Second and third cyclophosphamide in patients with advanced HSCT were performed with a mean of 310 days (69- 1191) and haematological malignancies 827 days (145-1991) after fi rst HSCT, respectively. Indications D. Nachbaur, J. Auberger, R. Muehlmann, B. Kircher, for second HSCT were relapse of disease (15 pts, 75%) or sec- J. Clausen ondary graft failure (5 pts, 25%), all pts with three HSCTs had Hematology and Oncology (Innsbruck, AT) relapse of disease. Graft source (1st, 2nd, 3rd HSCTs, n=x) were PBSC (14, 18, 5) and BM (6, 2, 0). Most donors were Objectives: Potentially curative allogeneic stem cell transplan- full matched related (VRD, 9 pts), unrelated (VUD, 9 pts), mis- tation is often limited by identifying a suitable, ideally HLA- match VUD (2pts). Donor of fi rst and second HSCT were identi- identical, donor in time. In patients who lack an HLA-identical cal in 17 cases, 3 pts had a donor change. In addition, donors sibling or volunteer unrelated donor and who are at high risk for were changed in 3/5 pts for the third HSCT. Conditioning regi- disease progression the use of mismatched related family (i.e. men (1st, 2nd, 3rd HSCT, n=x) were none (0, 4, 0), reduced haploidentical) donors is a valuable alternative approach. intensity conditioning (RIC, most often treosulfan based) (11, Patients: Between Oct 2002 and Apr 2008, thirteen patients 7, 3), myoablative (9, 2, 0) or conventional chemotherapy (0, underwent haploidentical SCT at our institution (acute leukemia, 7, 2). Median overall survival was 9 mths (7d-120 mths). 8/20 n=9; myeloma n=2; lymphoma n=2). Median patient age was pts (40%) are still alive and in complete remission. 2/5 pts 51 (20-66) years. All patients had advanced or chemorefractory (40%) with secondary graft failure and 10/15 pts (67%) with disease. Seven patients had failed prior allogeneic (n=6) or relapsed disease as reason for HSCT died. Causes of death autologous transplant (n=1). Conditioning consisted of fl udara- were: relapse/refractory disease (7 pts, 35%), graft versus host bine 30 mg/m²/d i.v. on days -6 to -2 followed by 200 cGy TBI disease (GvHD)/infection (4 pts, 20%), other (1 pt, 5%). The on day -1. On day +3, 50 mg/kg cyclophosphamide was admin- occurence of a GVHD was more often during second transplan- istered followed by tacrolimus and mycophenolate mofetil from tation compared to fi rst (45% vs 25%). day 4 (P. O Donnell, BBMT 2002;8:377). Donors for all patients Conclusions: Second allogeneic HSCT is a feasible procedure were HLA-mismatched at least in 3/12 loci. that rescues pts with secondary graft failure and – to a lesser Results: Ten patients received unmodifi ed G-CSF-mobilized extent – pts with relapse after 1st alloHSCT. peripheral blood stem cells and three patients received unma- nipulated bone marrow stem cells with a median CD34 number of 5.63 (0.78-24.91) x 108/kg, a median CD3 number of 5.02 (2.71-21.65) x 108/kg, and a median NK cell number of 3.7

S286 P962 after the transplant. With a mfu of 43 mo (29-53), 9 patients Reduced-intensity conditioning with fl udarabine/ (50%) are alive, all in CR, one after 3 DLI and the development cyclophosphamide has remarkably high activity in of cGVH; 9(50%) have died, 6 (26%) due to progression and 3 relapsed or refractory lymphoid malignancies (16%) due to NRM.with a 3 y projected of 18%. Median OS and F.S. Dijkstra, O. Visser, S. Zweegman, P.C. Huijgens, DFS have not been reached ; 4 years OS and DFS are 50% J.J.W.M. Janssen and 68% with a plateau phase from 12 months; None of the Vrije Universiteit (Amsterdam, NL) variables analyzed infl uenced on OS or DFS; NRM in patients <45 years was 0vs 54% in ≥45(S). Several studies suggest that non-myeloablative allogeneic stem Our results in one of the largest reported series confi rm the effi cacy cell transplantation (alloSCT) with reduced intensity condition- of allogeneic transplant in these poor prognosis patients; early pro- ing (RIC) has encouraging effi cacy in the treatment of lymphoid gression observed in our patients needs different strategies. malignancies. However, large series are lacking. Therefore we retrospectively analyzed data of 43 patients with indolent lym- phoma (n=13), aggressive lymphoma (n=18: DLBCL n=3, MCL P964 n=3, transformed follicular lymphoma n=10, ATLL n=1, Richters’ Long-term follow-up after non-myeloablative allogeneic transformation n=1) and B-CLL (n=12) receiving alloSCT after related transplant with melfalan and fl udarabine in a fl udarabine/cyclophosphamide (Flu/Cy)-based conditioning patients with aggressive B-cell lymphoma: results of three regimen. Previous autologous stem cell transplants (autoSCT) consecutive multicentre trials from the GEL/TAMO Group had been performed in fi ve patients with indolent lymphoma M. Caballero, R. Martino, M.L. Vasquez, J.L. Piñana, I. Heras, and in six with aggressive lymphoma. Disease status at allo- C. Ferra, J.F. Tomas, R. Arranz, J. Perez-Oteiza, J. Briones, transplant was CR in 9, PR in 26, SD in 6 and chemoresistant J. San Miguel on behalf of the GEL/TAMO group disease in 2 patients. At a median follow-up of 653 days (range 98-2282 days) after transplantation, OS was 51.7% (95%-CI: Although autologous transplant(ASCT) is the election treatment 16.5%-86.9%), 2-yr PFS was 62% (95%-CI: 45.3%-78.8%) in sensitive relapsed DLBCL other strategies are needed for and 2-yr DFS was 55% (95%-CI: 36.3%-73.7%) No signifi cant those relapsing after ASCT or transplanted with active disease. differences in OS were found between the different patient From 1999 24 patients were included in these trials.18 (75%) groups. Overall survival according to having had a previous had a DBLCL,3 (13%). Follicular grade 3 lymphoma, 2 (8%) a autologous SCT or not showed no signifi cant difference (P = transformed MZL and 4% a BL Patients received fl udarabine 0.814). Six patients (14%) relapsed. Frequencies of grade III-IV 30 mg/m2 IV ( 8 to 4) plus melfalan 70 –140 mg/m2 (3 and 2); acute and extensive chronic graft-vs-host disease were 16.3% 12 patients (50%) enrolled in the latest trial received rituximab and 51.1%, respectively. 375mg/m2; on days –8,-1,+8 and +15 and CsA plus MTX 10mg/ RIC with Flu/Cy therefore seems a feasible strategy in patients m2(+1, +3, +6). Median age was 51 y (26-62); 4 patients (16%) with lymphoid malignancies, with remarkably high activity in were in >CR1 and 84% had active disease -10 (41%) sensi- relapsed/refractory indolent as well as aggressive lymphoid tive and 10 (41%) refractory; median number of previous lines neoplasms. was 3(2-7) and 43% had received a previous ASCT. Acute GVH was 37%(8% grade III-IV). 65% of patients at risk developed cGVH, (ext 59%). At day +100 21 patients were evaluated for P963 response: 67% responded (13 CR and 1 PR) and 7(33% pro- Long-term follow-up after non-myeloablative allogeneic gressed); in the 20 patients transplanted with active disease 11 related transplant with melfalan and fl udarabine in responded (55%) (10 CR and one PR, later converted into CR), patients with aggressive T-cell lymphoma. Results from 7 didn’t respond (35%) and three (12%) were not evaluable due three prospective trials. The GELTAMO experience to early death. With a mfu of 35 months (range: 4-96 months), D. Caballero, J. Gayoso, R. Arranz, A. Sampol, 8 patients (33%) are alive, 7 (29%), disease free and 16 (66%) J. Perez-Oteiza, R. Martino, J.F. Tomas, I. Heras, C. Solano, died, 8(33%) due to progression and 7 (29%) due to NRM, 2 J.L. Diez, L. Vazquez, J. San Miguel on behalf of the GEL/ of them in progression. Median OS is 22 mo(1-58) with a pro- TAMO group jected OS at 5 years of 30% and a plateau from 44 mo; regard- ing DFS, median has not been reached and estimated DFS at T Cell lymphomas have a worse prognosis as compare with 5 y is 56% with a plateau phase from 10 mo. NRM at 1 year DLBCL; Standard therapy is not well defi ned with less than is 20%. No variables infl uenced OS or DFS although patients 40% of long term survival. Recently a potential graft versus receiving <4 or <4 lines has an OS of 32 months and 4 months. lymphoma effect(GVL) has been suggested, although still (NS). This is one of the largest reported series of Non Myeloab- published series include a reduce number of patients. From lative allogeneic transplant in Aggressive B Cell lymphoma; our 1999 18 patients with poor prognosis T Cell Lymphomas were results with this intermediate conditioning including melfalan included in three consecutive multicenter trials. From them 6 and fl udarabine ± Rituximab indicates a good response early (33%) had an Anaplastic T Cell lymphoma(ATCL), 2 (11%) a after transplant, however most of the patients transplanted with cutaneous T cell Lymhoma (1 M F and 1 S S), 1(5%) a nasal progressive disease do not benefi ciate from this strategy; other Natural Killer type,1 (5%) a gamma/delta and 7 (39%) a Periph- approaches improving conditioning as well as perhaps some eral T Cell lymphoma non specifi ed (PTCLNOS). Conditioning kind of maintenance will be tested in the future. Also a better regimen consisted of fl udarabine 30 mg /m2 IV 8 to 4 + melpha- control of acute and chonic GVH is needed. In summary, results lan 70 –140 mg/m2 3 and 2 and CsA +MTX) (10mg/m2, days are encouraging with a third of the patiens showing a long term +1, +3, +6). Median age was 47 years (range 26-58) and at disease control suggesting a clear graft versus Lymphoma transplant 4 patients (22%) were in CR1, 6 (33% ) in a later CR effect (GVL) in patients with B aggressive histologies. and 8 (44%) had active disease( 5 sensitive (28%) and 3 (17% ) refractory disease). Median number of previous lines was 2(1-5) with ASCT in 50% of the patients. Median number of days to reach >500 x109 granulocytes and > 20000 and >50 x109 platelets were 17(range 10-27),12 (8-23) and 13(10-30). At a median time of 23 days (range 10-74), 8 patients (44%) devel- oped acute GVHD (4 patients (22%) grade II-IV).At a median time of 219 days(156-372), 6 out of 14 (43%) patients at risk developed cGVH being extensive in 5 (36%). At day + 100 10 / 16 evaluable patients( 62%) were in CR (5 reached CR after the transplant) and 6 (37%) progressed (3p) or relapsed (3 p)

S287 P965 count >0.5x109/L was 11-22d (median 16d, fi gure 1A). The Reduced-intensity transplant for MDS/AML – A single- time interval to platelet recovery >20x109/L was 8-35d (median centre experience and retrospective analysis 11d, fi gure 1A). Twenty-one/33 patients (63.6%) developed S. McLean, M. Ni Chonghaile, E. Lawlor, C. Flynn, acute GVHD, out of them 6 (18%) had grade 3-4 GVHD. The E. Vandenberghe, P. Browne, E. Conneally time to the development of acute GVHD was 28 days (fi gure St. James Hospital (Dublin, IE) 1B). Eighteen/25 patients developed chronic GVHD (extensive in half of them). With a median follow-up of 27.7 months, 16 Optimal therapy for many cases of acute myeloid leukaemia patients (44.4%) are alive (fi gure 2A)and 15 are disease free. (AML) and myelodysplastic syndrome (MDS) is allogeneic Transplantation in remission was a predictor of survival (fi gure bone marrow transplantation, however many patients are 2B, p<0.0001). not candidates for myeloablative transplants due to their age Conclusion: We conclude that NST without ATG seems safe or performance status. Reduced intensity conditioning (RIC) and effective. transplantation is an option for these patients but may be asso- ciated with higher relapse rates. To evaluate this, we studied 48 patients who underwent RIC allogeneic transplantation for MDS/AML in our institution between 1999 and October 2008. The cohort of 20 males and 28 females had a median age of 54 years (26-66). Diagnoses at transplant were: AML in 1st complete remission (CR) (n=15), AML in 2nd CR (n=19), MDS (n=7), MDS with secondary AML (n=5), and therapy related AML (tAML) (n=2). Cytogenetic data was available in 45/48. Of the 32 AML patients 3 had good risk (transplanted in 2nd CR), 23 had intermediate risk, and 6 had poor risk karyotypes. In the 11 MDS or MDS/AML patients 9 had a normal karyotype and 2 had poor risk cytogenetics. The 2 patients with tAML both car- ried an 11q23 rearrangement. 18 patients (38%) had comorbidities, notably cardiac disease, diabetes, and chronic obstructive pulmonary disease. One patient had a prior RIC allograft, and 3 had autografts. Median time from diagnosis to transplant was 16 months. Peripheral blood stem cells were used in all but one patient (pt) with an engraftment rate of 98%. The one pt that recieved bone marrow derived stem cells failed to engraft. A sibling donor was used in 79%, and 21% recieved a graft from an unrelated donor. All pts received fl udarabine / busulfan and ATG based conditioning. GVHD prophylaxis was cyclosporin in 69% and tacrolimus in 31%. Median time to neutrophil engraft- ment was 15 days (8-28). Acute GVHD occurred in 12 pts (25%): skin (n=8), gut (n=1), skin and gut (n=1), and liver (n=2). Four pts developed chronic GVHD. Seven pts had CMV reactivation. The treatment related mortality was 17%. Late complications included chronic GVHD, iron overload, and osteoporosis. Fourteen pts (29%) relapsed and 11 of these have died. Overall 26 pts (54%) remained dis- ease free at a median follow up of 29 months (1-100 months). This data supports the use of reduced intensity transplantation for myeloid malignancies with an outcome comparable to mye- loablative transplantation and warrants further study.

P966 Non-myeloablative stem cell transplantation (NST) conditioning protocol without anti-thymocytic globulin M. Shapira, L. Drey, I. Resnick, P. Tsirigotis, B. Gesundheit, R. Or Hadassah – Hebrew University Medical Center (Jerusalem, IL)

Introduction: classically, the non-myeloablative stem cell trans- plantation (NST) protocol was consistent of fl udarabine, busul- fan and ATG. Here we describe the outcome of the use of NST conditioning without ATG for allogeneic transplantation. Patients and methods: Thirty-six patients (median age 46 years, 19 males) that underwent transplant from 27 matched sibling donors, 5 matched unrelated and 4 other family members, were included. They were conditioned with i.v. fl udarabine 30mg/m²/ day x6 days, oral busulfan 4mg/kg/day (n=13 patients), or i.v. 3.2 mg/kg/day busulfex (n=25 patients) x 2 days. GVHD proph- ylaxis was consisted of short-term single-agent i.v. cyclosporine 3 mg/kg daily in two divided doses, starting on day -1 or -4. Cyclosporine dosage was tapered during the second-third month post transplant, according to chimeric status and GVHD evidence. T-cell depletion was not done. Results: 33/35 evaluable patients (94.3%) displayed three- lineage engraftment. Time to recovery of absolute neutrophil

S288 cyclosporine (CsA)/methotrexate in 18 pts, CsA/mycophenolate mofetil in 1 pt and CsA alone in 2 pts Results: Eleven pts remain alive (47.5%) with a median survivor follow-up of 28.5 months. At last follow-up, OS, EFS and PFS are 42%, 31% and 60%. Day 100 and 2-year TRM was 4.7% and 28%. Patients with day-100 TRM underwent SCT in 2001-2003 period. Day 100 TRM was 0% from 2004-2008. Cumulative incidence of acute and chronic GVHD was 38% each. There was a statistically signifi cant impact of acute GVHD (grade III-IV vs 0-I; p=0.012) on OS and a trend of chronic GVHD favouring OS, PFS (p=ns) and EFS (44% vs 23%; p=0.044). Response status before transplant also had a favourable impact on OS (OS for refractory relapse was 14 vs 51 months for sensitive relapse group, p=0.002). Conclusion: RIC alloSCT with intensifi ed fl udarabine regimen is associated with reduced early TRM and favourable OS and PFS in pts with advanced lymphoid malignancies non eligible for myeloablative regimens. Disease status and chronic GVHD seems to have a positive impact on OS. Efforts may be directed to identify patients who achieved response to previous regi- mens prior to move to RIC alloSCT.

P967 Results of reduced-intensity conditioning stem cell transplantation with intensifi ed fl udarabine-based regimen for advanced lymphoid malignancies J. Falantes, M. Rodríguez, F.S. Márquez, M. Carmona, E. Ríos, A. Marín, M. Martino, I. Montero, J. González, R. Parody, A. Urbano-Ispizua, I. Espigado Hospital Virgen del Rocío (Seville, ES)

Background: Reduced-intensity conditioning (RIC) seems to offer a reduction in transplant related mortality (TRM) in patients (pts) not eligible for myeloablative regimens. We report an update from a single-center experience with intensifi ed fl udara- bine (150 mg/m²) regimen in pts with lymphoid malignancies Objectives: Primary end-point: Overall, event and progression free survival (OS, EFS, PFS). Additional end-points: Variables associated to OS and EFS. Design and Methods: Twenty-one pts with lymphoid malignancies (HL=8, NHL=13) underwent RIC allogeneic stem cell transplanta- tion (AlloSCT) from a matched related donor from 2001-08. Median age at transplantation: 41y (range 23-52). Median number of pre- vious chemotherapy regimens: 2.6 (range 1-5). Four pts (19%) had undergone prior autologous SCT. Disease status before transplant was sensitive relapse (complete or partial response to previous chemotherapy, n=17) or refractory relapse (progressive disease, n=4). Stem cell source was peripheral blood in all cases. Conditioning regimen consisted of fl udarabine (30 mg/m² daily for 5 days) and TBI (200 cGy) in 19 pts. One pt received fl udarab- ine and melphalan (70 mg/m² daily for 2 days) and 1 pt received reduced-BEAM (carmustine, etoposide, cytarabine and melpha- lan). Graft-versus host disease prophylaxis (GVHD) included

S289 P968 Reduced-toxicity conditioning with busulfan and fl udarabine and allogeneic stem cell transplant: chimer- ism evaluation and global outcome of 26 consecutive patients A. Algarotti, C. Micò, A. Salvi, C. Zanotti, A. Grassi, A. Rambaldi Ospedali Riuniti di Bergamo (Bergamo, IT)

Background: Reduced intensity conditioning (RIC) regimens have been widely used to offer an allogeneic transplant to otherwise ineligible patients. Although well tolerated, RIC regimens are often associated with an increased proportion of disease relapse. We investigated a reduced toxicity condi- tioning regimen consisting of a myeloablative dose of Busul- fan (3.2 mg/kg/day for 4 days) and Fludarabine (30mg/m² for 4 days). Patients and Methods: From January 2006 to June 2008, 26 patients with a median age of 53 years and a diagnosis of Acute Myelogenous Leukemia (AML,n=12), Acute Lymphoblastic Leukemia (ALL,n=1), Myelodysplastic Syndrome (MDS,n=1), P969 Multiple Myeloma (MM,n=7), Hodgkin’s disease (HD, n=3) and Long-term results of reduced intensity compared to non-Hodgkin’s Lymphoma (NHL,n=2) entered this program. At myeloablative conditioning before stem cell the time of ASCT, 9 patients (5AML, 1ALL, 2NHL and 1HD) transplantation were in complete remission (CR), 7MM patients were all in 2nd Y. Brychtova, M. Doubek, M. Krejcí, M. Navrátil, O. Horký, good partial remission, 1HD patient was in partial remission T. Jurcek, J. Mayer while the remaining 9 patients (7AML, 1MDS and 1HD) had University Hospital Brno (Brno, CZ) active disease. Results: The median dose of CD34+ and CD3+ cells infused Background: In this retrospective analysis, we summarized our was 4.4 x 106/kg and 202 x 106/kg, respectively. The median experience with RIC and MC after the fi rst HCT in patients (pts) time to leukocyte and platelet engraftment was 16 days and with AML and CML in order to compare the longer-term out- 14 days respectively. At day +30, + 60 and +100 all the evalu- come of these treatment procedures. able patients showed a full donor chimerism when BM mono- Methods: 70 pts were transplanted during the 1998-2007 using nuclear cells were tested. Peripheral blood CD4+ cells were PBSC from matched sibling donors. The RIC (37 pts) and MC of donor origin in 16/19 and 17/17 patients tested at day + 60 (33 pts) groups were balanced regarding the diagnosis and and +100 respectively. With a median follow-up of 370 days, disease stage. The RIC patients, however, signifi cantly differed 18 patients are alive (15 patients in CR, 3 with active disease) from MC patients in median age (51 y vs. 35 y). The RIC con- and 8 patients are dead. Fourteen patients experienced acute sisted of fl udarabine (30 mg/m2/d; 5 days), busulfan (total dose GVHD (grade I, n=4; grade II-IV, n=10) after a median of 8-12 mg/kg), and ATG Fresenius (10 mg/kg/d; 4 days), post- 47 days from ASCT. Disease relapse or progression occurred transplant immunosuppression was mostly cyclosporine alone. in 10 patients and was the primary cause of treatment failure. The conventional regimen was busulfan (total dose 14-16 mg/ Only one patient died for a transplant-related reason (grade kg) + cyclophosphamide (120 mg/kg) followed by post-trans- 4 acute GVHD). Among the 23 evaluable patients, a mild plant cyclosporine and methotrexate. chronic GVHD was diagnosed in 7 while an extensive presen- Results: The RIC vs. MC group had a median follow up 1561 tation was evident in 5 patients. (4,3 y) vs. 1898 days (5,2y), respectively. None or mild RRT Conclusion: The association of a myeloablative dose of Busul- was more frequent and severe RRT less frequent in RIC group fan and Fludarabine confi rm its strong anti neoplastic activity in comparison with MC group (Tab 1). Eleven percent and 22% and its remarkable tolerability in most patients who are inel- of patients in the RIC and MC group did not achieve a complete igible to a full dose conditioning regimen because of age or chimerism, usually for early disease progression. Median time comorbidities. to achievement a complete chimerism was 166 vs.108 days for RIC and MC, respectively. Concerning GvHD, the incidence and severity was less in the RIC group (Tab. 1). Nine/15 AML pts (60%) in RIC group relapsed and 6 of them died (40%), whereas 8/16 AML pts (50%) relapsed and 7 of them died (46%) in MC group. Twenty/22 (91%) RIC CML pts were alive, all in molecular remission (MR), versus 14/17 (82%) CML living pts from MC group. Whereas only 1/17 CML pts (6%) relapsed in MC group, 10/22 CML pts (45%) in RIC group relapsed (4 pts once) and 10/10 achieved new MR after treatments (DLI, imatinib, dasatinib, retransplantation) and 2/10 of them died (1 for new progression and 1 for GVHD after DLI). Mortality from any cause was 24% vs. 36%, NRM was 5% vs. 15%, early TRM was 5% (for disease progression) vs. 15%, late TRM was 5% vs. 3% for RIC and MC patients, respectively. Conclusion: Allogeneic SCT after RIC conditioning using Flu- Bu-ATG ( Fresenius) is well tolerated, after this procedure, all CML pts achieved CMR, however, quite frequent interventions for molecular relapses are required than conventional myeloab- lative regimen. This regimen can completely replace standard conditioning in AML pts. This work was supported by grant IGA MZ CR NS9683- 4/2008.

S290 occurred in 5 cases and 2 cases in each group. Three patients in CD56+ supplement group relapsed but soon achieved CR after salvage treatment. Two patients relapsed in control group and died of disease progression. DFS showed no difference but OS was longer in CD56+ supplemented group. Out data had limits to show benefi ts of supplementary infusion of CD56+ lym- phocyte in non-myeloablative transplantation. Optimal doses of NK cells and T cell subsets needs to be determined by larger scale data.

Stem cell donor

P971 Military induction centres as stem cell donor registration sites: effects on donor recruitment J. Stein (1), A. Peles-Bortz (1), N. Shriki (2), A. Sinai (2), B. Zisser (2), I. Yaniv (1) (1)Schneider Children’s Medical Center (Petach Tikva, IL); (2)Ezer Mzion National Registry (Petach Tikva, IL)

The ideal stem cell donor registry should contain a young, healthy, and motivated donor pool with ethnic diversity refl ect- ing that of potential transplant recipients. Many ethnicities remain under-represented in major registries, and marked genetic diversity within some ethnic groups demands a higher ratio of registrants to recipients to insure successful donor searches. The Israeli population is ethnically diverse, with large numbers of second-generation emigres from Middle-Eastern countries, Ethiopia, Baltic States, etc., prompting us to diversify the ethnic representation within our national registry. To achieve P970 this goal, we opened a single stem cell donor recruitment site Effect of supplementary CD56+ lymphocyte infusion in at the Israeli National Armed Forces Induction Center (Israeli HLA-matched non-myeloablative stem cell transplantation law mandates on-site registration of all potential military con- S.J. Kim, H.W. Lee, J.S. Kim, J.W. Cheong, Y.H. Min scripts regardless of their eventual draft status). Information Yonsei University (Seoul, KR) about the registry and stem cell donation was provided to all conscripts, but coercion to register was not sanctioned. 70% of Transplanted doses of T cell subsets are known to correlate eligible individuals agreed to participate, with higher registra- with transplantation outcome. Among them, natural killer (NK) tion rates among potential combat conscripts vs. non-combat cells express anti-leukemic effect via alloreactivity and mitigate inductees. Since May 2005, 80392 military conscripts have graft-versus-host disease (GVHD). However optimal dose of entered our registry vs. 121000 individuals recruited through NK cells are not yet derived in non-myeloablative stem cell costly and cumbersome volunteer donor drives organized in transplantation (NST). Therefore we tried to evaluate the effect the civilian population during the same period (current registry of the CD56+ cell dose on NST by giving additional CD56+ cells size 388888 donors). The median age of donors in our registry using immunomagnetic process. Nine patients were enrolled in dropped from 37 years in 2005 to 32 years (as of November the protocol from January 2005 to December 2006. Transplan- 2008) since we have started conscript registration. We com- tation outcome was compared to 7 patients who did not consent pared requests for donor confi rmatory typing (CT) between the protocol during same period. All patients had AML or ALL or military and civilian donors as a surrogate for the “matchability” high degree MDS. HLA-identical siblings or unrelated donors of these donors. During this period, CT requests were received were mobilized using G-CSF. All patients received whole leu- for 953 out of 308496 civilian donors and for 302 out of 80392 kapheresis product on Day 1, and purifi ed CD56+ cells from military conscript donors, ratios of 0.31% and 0.38% (p<0.01 second day leukapheresis were given to Day 2. Selection of by chi squared). Specifi c data on ethnic representation is diffi - CD56+ cells were performed with CliniMACS system by manu- cult to obtain as many conscripts listed “Israeli” or “mixed” (par- facturer’s protocol. Conditioning regimens were fl udarabine ents of different ethnicities) as their ethnicity. Nonetheless, we plus busulphan or fl udarabine plus idarubicin plus melphalan. increased the number of Ethiopian donors from nearly none to All patients received cyclosporine A or tacrolimus for GVHD nearly 2000. The decrease in median age of our donors contrib- prophylaxis. The numbers of transplanted total nuclear cells utes to a longer donor dwell time within the registry. Conscripts showed no signifi cant difference (supplement group; 10.95x108/ are a highly motivated population and are generally of robust kg, range 9.32-18.27, non-supplement group; 10.28x108/kg, health, increasing the likelihood that they will be willing and eli- range 8.25–12.45). Also transplanted doses of CD3+, 4+, 8+ gible stem cells donors. The higher frequency of matching in cells showed no statistical difference except for CD56+ doses this population refl ects its ethnic diversity. (106.24x106/kg, range 73.59-240.95 vs. 73.02x106/kg, range 46.06–112.09; P=0.04). Ratio of CD56+ cells to CD34+ cells and CD3+ cell were higher in CD56+ supplemented group. Purity of CD56+ selection was mean 96.6% (range 93.1–98.9) and yield was mean 48.3% (range 28.8-82.3). Mean period to engraftment was earlier in CD56+ group but without statisti- cal signifi cance (mean 12.5 to 14.7 days, P=0.2). Two cases of acute GVHD(>-grade 2) occurred in CD56+ group and one case in non-supplement group. Chronic GVHD (extensive)

S291 P972 unrelated donor (UD) selection in cases with many HLA Natalizumab treatment modulates phenotypic profi le and matched UD available; typically in patients with common HLA expression of adhesion molecules in bone marrow and haplotypes, e.g. in the most frequent haplotype A1B8DR3 (also peripheral blood CD34+ cells of patients with multiple termed AH8.1). sclerosis Methods: 41 unrelated individuals from the Czech National U. Oelschlägel, T. Ziemsen, C. Garten, M. Markgraf, Marrow Donors Registry (CNMDR) with confi rmed HLA geno- G. Ehninger, M. Bornhäuser type A*0101-Cw*0701-B*0801-DRB1*0301-DQB1*0201 (PCR- University Hospital Dresden (Dresden, DE) SSP), i.e. unequivocally composed of the 2 AH 8.1 haplotypes, were genotyped for the complete set of 14 KIR genes and 2 Objective: The alpha 4 integrin inhibitor Natalizumab is used in pseudogenes. the treatment of highly active multiple sclerosis (MS). Elevated Results: 14 different KIR genotypes were found, with 9 (22%) CD34+ counts in peripheral blood have been described in MS occurred only once. The most frequent genotype found in 39% patients during this treatment. In the present study CD34+ individuals corresponds to A haplotype homozygosity (AA). The counts and expression of adhesion molecules on hematopoi- remaining possessed more than 1 activating KIR and were etic progenitor cells in bone marrow and peripheral blood of either AB heterozygous or BB homozygous. All individuals pos- MS patients under Natalizumab (MS-BM, MS-PB) should be sess inhibitory KIRs for self class I ligand (C1group) and only compared with those in healthy BM and mobilized blood after 4% lack either the 2DL1 or 3DL1, i.e. KIRs for not possessed G-CSF stimulation. inhibitory epitopes (i.e. C2 group and Bw4). Median number Methods: BM (n=11) and leucapheresis samples of (n=11) of of all possessed KIRs was 9 (range 7-14), fi gures for S and L healthy donors and BM (n=4) and PB (n=6) of MS patients were genes were 2 (1-6) and 7 (5-8). While AA genotypes individuals investigated fl ow cytometrically combining CD45 PerCP5.5/ possess solely uniform 7 KIRs, AB/BB haplotypes were more CD34 APC with further antibodies in FITC and PE (CD11a, heterogeneous – median of 11 KIRs (range 7-14), 13 different CD62-L, CD44, CD31, VLA4, CXCR4, or CD133). Measure- genotype observed. The L genes frequencies: 2DL4, 3DL1, ments have been performed on a FACSCanto II. If possible, 3DL2 and 3DL3 -100%, 2DL3-98%, 2DL1-95%, 2DL2-51%, 2000 CD34+CD45dim cells with overall ca. 500,000 cells were 2DL5-37%. S gene frequencies: 2DS4-100%, 2DS2-51%, measured. 2DS1-29%, 3DS1-27%, 2DS3-24% and 2DS5-17%. Results: Of note, the absolute CD34+ cell count was signifi - Conclusion: We confi rm the variability in terms of KIR geno- cantly higher in MS-BM compared to control samples (1334/ types and gene diversity even in individuals with most con- µl vs. 109/µl; p=0.0050). An elevated CD34+ count was also served HLA haplotype (A1B8DR8 homozygous). The selection detectable in MS-PB vs. healthy steady-state PB (7/µl vs. <1/ of HLA matched UD according the KIRs contents could thus be µl). The expression of most adhesion molecules was lower in applied in patients with common HLA phenotypes. The benefi ts MS patients compared to healthy donors, BM: CD62-L 6% vs. of NK alloreactivity could be accomplished in such a group of 74% (p<0.05); CD11a 31% vs. 48% (p<0.05); PB: CD62-L 43% patients where many perfectly HLA matched donors are usually vs. 85% (p<0.01) and MFI of CD11a (p<0.05), CD31 (p<0.05), available. and CD44 (p<0.01). Remarkably, the CD133 expression on CD34+ cells was also clearly lower in MS-BM and PB (26% vs. 82%, p<0.01; 26% vs. 48%, p<0.01) compared to healthy P974 donors. Repetitive measurements, done before and during 5 Results of a prospective, monocentric study on the days after the 1st administration of Natalizumab in two patients, search of an unrelated donor for patients with high-risk revealed the maximum CD34+ count between day 3 and 4. The acute lymphoblastic leukaemia blocking of VLA4 via Natalizumab was refl ected as a decrease A.P. Iori (1), V. Valle (1), A. Piciocchi (1), G.F. Torelli (1), in mean VLA4 fl uorescence intensity from 8157 before to 3526 G. Meloni (1), A.M. Testi (1), W. Barberi (1), A. Vitale (1), at day 4. Furthermore, the expression of CD44 was also down L. Malandruccolo (1), E. Iannella (1), B. Lucarelli (1), E. Arleo regulated during the same time period. (1), M.P. Perrone (1), L. Laurenti (1), M. Screnci (1), R. Ricci Conclusion: The mobilization of CD34+ cells in MS patients after (1), W. Arcese (2), R. Foà (1) Natalizumab administration in PB and even more in BM was (1)University La Sapienza (Rome, IT); (2)University Tor Vergata shown to be accompanied by a down regulation of adhesion (Rome, IT) molecules. The lower CD133 expression suggests that a more mature progenitor subset is mobilized by this treatment. Further Allogeneic stem cell transplantation (HSCT) is potentially cura- investigations should evaluate whether these differences cor- tive for patients (pts) with high risk acute lymphoblastic leukemia relate with distinct biological features and whether Natalizumab (HRALL), and the search of an unrelated donor is a common could be clinically useful as an adjuvant mobilization agent in strategy for pts lacking a family donor. To assess the effect of heavily pretreated patients with hematologic diseases. the search on the outcome of pts with HRALL, we analyzed prospectively 136 pts, median age 11 yrs (1-59), on search of an unrelated donor through the BMDWW Registry and Cord P973 Blood (CB) Banks, between 1995 and 2008. At the start of the Could donor selection according the KIR repertoire be search, 37 pts were in ICR, 70 in IICR, 3 in >IICR, 26 in relapse. feasible? Analysis of KIR genes diversity of the HLA The cumulative incidence (CI) of fi nding a donor (CB or volun- haplotype A1B8DR3 (AH 8.1): Study on Czech National teer donor, VD) was 62% and 72% at 3 and 6 months, respec- Marrow Donors Registry donors tively; after this time no increase in the CI of fi nding a donor P. Jindra (1), P. Venigova (1), V. Koza (2), J. Navratilova (1), was observed. The median time from the start of the search K. Steinerova (2), H. Pittrova (1) to fi nd a donor was 1.5 months for CB and 3.5 months for VD. (1)Czech National Marrow Donors Registry (Pilsen, CZ); Fifty-one % of the 110 patients who entered into the study in (2)University Hospital (Pilsen, CZ) CR showed a relapse at a median of 4 and 2 months from the start of the search for pts in I-II CR and >II CR, respectively. Of The KIR genes (KIRs) and the HLA genes, located on differ- the 101 pts with a donor, 61% underwent a transplant: 25% in ent chromosomes, segregate independently of each other. a more advanced phase compared to the start of the search; Thus perfectly HLA-matched pairs are usually and typically 35% of pts who started the search in relapse obtained a CR and mismatched for KIRs. Both activating (S-genes) and inhibitory underwent an HSCT. Therefore, 44% of pts failed to undergo a (L-genes) KIRs probably infl uence the HSCT outcome and transplant because of lost eligibility due to disease progression. various studies suggest that number and/or composition of The 10-year probability of OS was 22.5%. Disease progression donor KIR genes repertoire may impact on transplant outcome. was the major cause of death. In univariate analysis, relapse vs This suggests that number of KIRs might be the criterion of no relapse during the search (HR:4.1; CI%95:2.75-6.12) and

S292 transplant vs no transplant (HR:0.62;CI%95:0.4-0.98) signifi - P976 cantly affected OS. For pts who underwent an HSCT, disease Distribution of killer cell immunoglobulin-like receptor phase at transplant was the most important factor affecting OS. genes in East China and Taiwanese populations In conclusion, by decreasing the length of the search (wait- H. Huang (1), G. Wu (1), X. Lai (1), Y. Tan (1), Y. Luo (1), ing for more than 3 months increases the probability of fi nding F. Zhu (2) a donor of just 10%, while half of the pts relapse) the risk of (1)Zhejiang Uninversity School of Medicine (Hangzhou, CN); relapse can be reduced, increasing the possibility of performing (2)Blood Center of Zhejiang Province (Hangzhou, CN) a transplant. To address the search at the same time for both VD and CB allows to increase the availability of stem cells for a Natural killer (NK) cells are a subset of lymphocytes involved in transplant to be carried out prior to the median time of relapse, primary immune response to recognize and mediate cytolysis of if the monitoring of the disease, during the search, shows an virus-infected, neoplastic and allogenic transplanted cells, with- increased risk of disease recurrence. During the search of a out prior sensitization. Killer cell immunoglobulin-like receptors donor for pts with HRALL, the major strategic factor must be (KIRs) are a family of inhibitory and activatory receptors and considering “the time” of the transplant, more than the type of expressed by most NK cells. The interaction between KIR and the transplant;according to this strategy, haploidentical trans- HLA molecules expressed on target cells is known to modulate plant need to be considered. the cytolytic activity of NK cells. At present, 17 KIR genes have been identifi ed. The KIR gene family is polymorphic including genomic diversity and allelic polymorphism. The number of P975 KIR gene loci has been reported to vary among individuals, Retrospective study of safety of peripheral blood stem resulting in a heterogeneous array of KIR genes in different cell donation by family members populations. Here, we compare the KIR gene content and the J. Wiersum-Osselton (1), A.M. van Walraven (2), W. Fibbe (2), genotypic structure of KIRs between two Chinese communities: A. Brand (3) East China (n=106) and Taiwanese (n=97). (1)Sanquin Bloodbank (The Hague, NL); (2)Leiden University Methods: 203 DNA samples were studied by a low resolution Medical Centre (Leiden, NL); (3)Sanquin Blookbank, Leiden PCR-SSP kit to identify all KIR genes. University Medical Centre (Leiden, NL) Results: All 17 KIR genes were observed in our two popula- tions. Framework genes KIR2DL4, KIR3DL2, KIR3DL3 and Background: Donation of peripheral blood stem cells (PBSC) KIR3DP1 were present in all individuals. The two populations by relatives is widely practised. Family donors are sometimes had very similar frequencies for most loci, however, signifi cant accepted outside the age range and strict medical criteria differences were noted in the frequencies of KIR3DS1 and which apply for unrelated donors and long-term follow-up (FU) KIR2DS4*003. A total of 35 genotypes were identifi ed in the is not routinely performed. This report describes a retrospective East China population and 29 genotypes in the Taiwanese pop- review of PBSC donations at a university hospital. ulation. The genotypes comprised of two homozygous groups. Method: Case notes and laboratory results were examined of Group A haplotype, showed high frequencies in individuals from all 268 (115 female, F; average age 42.7) family PBSC donors East China (45.3%) and Taiwanese (52.6%). Group A haplotype in the period May 1996 to May 2006. Medical screening results outnumbered group B haplotype in frequency by approximately were checked against the criteria for unrelated PBSC donation 2.2:1 in the East China population and 3.0:1 in the Taiwanese in The Netherlands and internationally (criteria of National Mar- population, respectively Some pairs of KIR show signifi cant row Donor Program, NMDP). The dose of granulocyte colony positive and negative linkage disequilibrium. stimulating factor (G-CSF) for mobilisation was 10 ug/kg body Conclusion: Our data demonstrated that there are some dis- weight subcutaneously for 5 days. In 2008 a FU questionnaire tinct in KIR gene frequencies, genotypes and LD between the on health as well as of psychological aspects was sent to the two populations or communities, which shed light on a possible donors. geographic genetic demarcation among different communities Results: 85 donors (32%; average age 50.8) would have been in China. The present study also provides a model to approach deferred (deferrable donors) under the NMDP criteria (n=49) or the effect of population migration on KIR genetic drift. It is very Dutch regulations (n= 36 of whom 12 only for age >55 years). A useful for knowing the KIR gene distribution in different commu- central venous catheter (CVC) was required in 20 donors (7%, nities or populations to select adaptable donors for unrelated 15 F, 10 deferrable). In 166 (62%, 57 F) collections one session hematopoietic stem cell transplantation. was suffi cient. One deferrable donor mobilised insuffi ciently and donated bone marrow instead. Five serious adverse reactions occurred (2%; 3 in deferrable donors). These were symptoms P977 of chest pain, severe pain requiring opiate treatment, extreme Clinical outcome of haematopoietic stem cell transplant fatigue, inguinal vein thrombosis after CVC and severe pain at from AB0-blood group incompatible donors the injection sites. C. Elena, F. Ripamonti, P. Bernasconi, D. Caldera, A. Colombo, FU contacts were recorded for 216 donors (81%; 71 deferrable) P. Isernia, M. Merli, V. Fiaccadori, G. Pica, C. Pascutto, 1-3 months after donation and for 160 (60%, 57 deferrable) also M. Lazzarino, E.P. Alessandrino roughly 1 year (y) after donation. Foundation IRCCS Policlinico San Matteo (Pavia, IT) 5 donors are known to have died (3 deferrable). Mail was unde- liverable to 6 abroad and 17 unknown at last recorded address. To evaluate the effect of AB0 incompatibility on overall survival Ninety-nine responses were returned (41%; 32 from deferrable (OS), transplant-related mortality (TRM), relapse rate (RR) and donors) on average 6.5 y after donation. Deferrable donors with post-transplant transfusion requirement (PTR) we analysed late health data were on average 15 y older at donation and 14 data of 421 consecutive adult patients with haematological y older at the time of late FU information. 6 malignancies (4 in malignancies (HM) who underwent allogeneic haematopoietic deferrable group) and 9 cardiovascular diagnoses (7 in defer- stem cell transplantation in a single centre from 1990 to 2007. rable group) have occurred. Of 421 transplants, 285 (68%) were from a related donor, and Conclusion: PBSC donation after stimulation with G-CSF has 136 (32%) from a matched unrelated donor. Patients were clas- an acceptable safety profi le but all donors should be carefully sifi ed as high risk (n=229, with active disease), or as standard screened. The conditions seen in former donors seem chiefl y risk (n=192, with low risk MDS or HM in complete remission). age-related. Prospective FU of donors and appropriate controls As to AB0 compatibility, 255 donors (61%) were compatible, is needed to further document absence of long-term hazards. 68 (16%) major incompatible, 72 (17%) minor incompatible, and 26 (6%) bi-directional incompatible. In patients with major AB0 incompatibility, bone marrow haemopoietic stem cells (HSC) were prepared by red blood cells (RBC) removal by

S293 separation on a Ficoll gradient; two patients with high isohae- These results indicate that a quick assessment of risk for poor magglutinins (IHA) titre had plasmaexchange at day -1 and day mobilization response in healthy donors can be achieved through 0 before transplant. Conditioning regimen was myeloablative simple demographic and routine parameters. Knowledge of (MA) in 348 patients (83%) and reduced intensity in 73 (17%). such predictive factors may be of high interest, with the develop- In univariate analysis, patients who had host-derived antibod- ment of newer mobilizing agents like CXCR4 antagonists, and ies against donor RBC antigens (major and bi-directional AB0 may result in overall benefi t both to donors and patients. incompatibility) showed higher PTR (p<0.001), delayed neu- trophil engraftment (p=0.01), more frequent occurrence of fever (p=0.01), higher TRM (p=0.009) and worse OS (p= 0.03). Plate- P979 lets engraftment, days of hospitalization, acute Graft versus Allele frequencies at HLA class I and class II loci in Host Disease and RR did not differ signifi cantly. We did not fi nd Iranian placental blood samples any association between IHA titre and PTR, OS, TRM and RR. A. Ghashghaie, K. Alimoghaddam, M. Ostadali, H. Ghaffari, As to conditioning regimens, our data showed a signifi cantly L. Khansari, M. Sadraee, E. Mir Rasekhian, I. Mohyedin, worse OS (p=0.03) and TRM (p=0.01) in patients with major F. Raoofi , Z. Noori, H. Eshaghi, H. Yaghmaian, or bi-directional incompatibility treated by MA conditioning. In A. Ghavamzadeh multivariate analysis, the effect of AB0 incompatibility was not HORCSCT (Tehran, IR) confi rmed as an independent risk factor; type of donor, status at transplant, and conditioning regimen were the only risk fac- Objective: Placental Blood, as an alternative source of hemat- tors for OS, while TRM was affected by type of donor, status opoietic stem cells for bone marrow reconstitution, has recently at transplant and HSC source. These data show that AB0 been shown to yield successful grafts in patients, especially incompatibility does not infl uence OS, TRM and RR. However, children. In comparison to adult bone marrow transplantation, patients with AB0 major or bi-directional mismatch have higher cord blood has the weak ability to induce severe graft-versus transfusion requirement, delayed neutrophil recovery and more host disease (GvHD) and HLA-matched grafts engraft faster frequent occurrence of fever. and reduce post-transplant morbidity and mortality. HLA Class I and Class II for 534 cord blood samples from Iran were types and the frequency of each allele were evaluated. Also we could P978 fi nd the most frequent haplotypes in this population. Factors predicting allogeneic peripheral blood stem cell Methods: In this study, 534 cord blood samples were collected mobilisation after G-CSF treatment in healthy donors from mothers with informed consent at shariati hospital. DNA of E. Brissot (1), P. Chevallier (1), T. Guillaume (1), J. Delaunay each sample was extracted using salting out method. HLA typ- (1), S. Ayari (1), V. Dubruille (1), S. Le Gouill (1), B. Mahe (1), ing was performed using sequence specifi c primer (PCR/SSP) T. Gastinne (1), N. Blin (1), B. Saulquin (1), G. Flandrois (2), method. The frequency of alleles was evaluated. A. Devys (2), A. Cesbron (2), V. Stocco (2), P. Moreau (1), Results: Allele frequencies at six HLA Class I and Class II loci J.L. Harousseau (1), M. Mohty (1) were evaluated. The most frequent alleles were A*02(36.51%), (1)CHU Hôtel-Dieu (Nantes, FR); (2)Etablissement Francais du A*24(31.08%), B*35(41.94%), B*51(24.71%), DRB1*11(45.5%) Sang (Nantes, FR) and DRB1*04(24.71%) respectively. Common three-locus hap- lotypes in this population were A*24-B*35-DRB1*11, A*30-B*13, The aim of this single centre report was to analyze factors DRB1*07, A*33-B*14-DRB1*11 and A*11-B*35-DRB1*11. associated with allogeneic peripheral blood stem cell (PBSC) Conclusion: The HLA data of our study was similar with the mobilization and yield in a homogeneous caucasian popula- other investigations on Iranian alleles which showed DRB1*07 tion (n=95; 53% males) of healthy allogeneic adult donors. All and -*11 in Kurds and Azeris and DRB1*03 and -*11 in random donors received G-CSF dosed at 10 µg/kg/d for 5 days fol- Iranian population. In other races, the frequent alleles are dif- lowed by large volume leukapheresis. Complete blood counts ferent. For example in Japan there are A*02,-*30, B*07,-*35, (WBC, Hb, Platelets) were measured for all donors at baseline DRB1*04,-*09 and in Jordan A*02,-*30, B*07,-*35, DRB1*03,- before G-CSF administration, before and after PBSC collection. *07 and most frequent haplotype is A*30, B*07, DRB1*03. In Peripheral blood CD34+ cell counts were also measured prior China, A*24,-*02, B*46,-*13, DRB1*09,-*12 is common. In to apheresis. 69 donors (73%) were healthy sibling donors, some previous report for USA, there are A*33-B*58, A*01, while 26 (27%) were healthy volunteer donors for HLA-identi- B*08, A*02, B*46 and A*11-B*40 haplotypes in different states. cal unrelated transplants. All donors were undergoing their fi rst Neighbor-joining Tree in some articles shows Iran in vicinity to PBSC mobilization. In this cohort, donors’ demographic charac- Italy and Greece which is concordant to HLA data. In Italy, A*30, teristics were as follow (median; range): age, 47 (18-81) years, B*42, DRB1*03,-*16 and in eastern Cicili DRB1*04,-*11 are the weight, 69 (43-106) kg, height, 170(150-187) cm, body-mass most frequent alleles. Comparison between this data of differ- index (BMI), 23.9 (15.2-35.7) kg/m². As per institutional policy, ent countries helps to fi nd HLA-Match donor for patients with the targeted total number of CD34+ stem cells was between 4 rare HLA alleles. and 8 x106/kg recipient body weight to be collected in a maxi- mum of 3 apheresis sessions. Overall, the median number of collected CD34+ cells was 6.25 x106/kg (range, 1.7-16.6), with P980 16 donors (17%) yielding less than 4 x106/kg CD34+ cells. In Mobilisation and harvest of peripheral blood stem cells univariate analysis, female gender, lower weight, height, pre- for allogeneic stem cell transplantation from 69 healthy G-CSF and post G-CSF Hb levels, and low CD34+ cell counts donors less than 18 years old prior to fi rst apheresis, were associated with signifi cantly lower M. Benakli, R. Ahmed Nacer, R. Belhadj, F. Mehdid, total CD34+ stem cells yields (<6.25x106/kg). In multivariate N. Rahmoune, F. Harieche, R.M. Hamladji analysis, male donor gender and higher post-G-CSF CD34+ Pierre and Marie Curie Center (Algiers, DZ) cell counts prior to the fi rst apheresis were most strongly asso- ciated with a higher total number of collected CD34+ stem Introduction: The PBSC are the source of haematopoietic stem cells (OR=6.17, 95%CI (2.39-15.93), P=0.0001; and OR=3.95, cells (HSC) currently most used in the allograft of malignant 95%CI (1.53-10.19), P=0.004 respectively). Also, when consid- and non malignant haematological diseases in our practice. ering the group of 16 “poor” mobilising donors (CD34+ stem If the procedure of mobilization and collection is similar for cells yield <4 x106/kg), in multivariate analysis, we found that adult and children, of many technical sides must be taken into a higher post-G-CSF CD34+ cell count prior to the fi rst apher- account in the paediatric apheresis because of extracorporal esis was the strongest parameter signifi cantly associated with volume related to the weak weight of the donors. We report our a higher total number of collected CD34+ stem cells (OR=6.36, experiment of collection of PBSC in minor sibling donors over 95%CI (1.68-24.15), P=0.006). one 3 years period for allogeneic stem cell transplantation.

S294 Material and methods: From January 2005 to December 2007, 69 minor donors (<18 years), including 37 of male sex, had a collection of PBSC by technique of apheresis after paren- tal assent. Their median age was 12 years (3–17) of whom 20 (29%) have less than 10 years. Their median body weight was 42 kg (16 - 79) of whom 8 (12%) were lower than 20 kg. The mobilization consists of a subcutaneous injection of grano- cyte colony-stimulating factor (G-CSF: 10 µg/kg/day) for fi ve consecutive days. The collection is carried out as from the 5th day on separator COBE Spectra according to a semi-automatic procedure. No donor profi ted from red blood cells priming the extracorporeal line before blood processing. Results: All of the donors tolerated the whole procedures. Only 8 donors (11,5%) required installation of a femoral catheter. Obtaining an adequate CD 34+ rate required 2 apheresis in 49 donors (71%), 3 apheresis in 9 donors (13%) and only one in 11 donors (16%). The intermediate duration of the procedure was 280 minutes (150 - 450) and the median volume of col- lection was 254 ml (128 - 414). The rates of CD34+ and MNC P982 obtained are respectively 4,9.106/kg (0,34 - 17,7) and 10.108/kg The importance of graft cell composition to outcome after (3,8 - 30,6). Symptoms of hypocalcaemia observed in 51 donors allogeneic stem cell transplantation (73,9%). After apheresis, 12 donors (17,3%) had haemoglobin P. Svenberg, O. Ringdén, M. Uzunel lower of 10 g/dl and only one had thrombopenia < 70000/mm3 Karolinska University Hospital (Stockholm, SE) without hemorrhagic signs. Conclusion: Our study shows that the PBSC obtained in the Background: Stem cell dose has been shown to be important sibling donors, less than 18 years, are an effective procedure for outcome in patients who undergo stem cell transplanta- of collection and without risks on the condition of being real- tion (SCT). Grafts may contain varying amounts of different ized thoroughly by a team specialized with a perfect control of cell populations like CD3+, CD19+, CD56+, CD4+ and CD8+ apheresis technics. immunocompetent cells, whose role for clinical outcome is less clear. Patients and methods: We analyzed 591 patients who under- P981 went SCT at Karolinska University Hospital, Huddinge between KIR gene and genotype frequencies in Russian stem cell 1998 to 2007 and correlated FACS results of the above men- donors tioned cell populations to clinical outcome. Most patients (76%) E. Khamaganova, Y. Lednev, N. Ovchinina, L. Golovkina had hematological malignancies. 58% of the patients received Hematological Research Center (Moscow, RU) a myeloablative conditioning regimen, 62% were given periph- eral blood stem cells (PBSC) and in 58% of the transplants an NK (natural killers) are the important effector lymphocytes unrelated donor was used. which participate in early immune response to pathogens. NK Results: As expected, PBSC contained much higher levels of are implicated in the suppression of graft vs. host disease, pro- all different cell populations as compared to BM. We found that motion of bone marrow engraftment, mediation of a graft vs. patients reciving high numbers of CD4+, and CD8+ cells had leukemia effect. These effects are mediated in a large part by signifi cantly more rapid engraftment. Also, a CD4+ level above donor-recipient KIR (killer Ig-like receptors) / HLA interrelations. 200 x 10(6)/kg was correlated to higher risk of developing acute The KIR gene family exhibits extensive diversity. The objective GVHD grades II-IV (41%) as compared to patients receiving of the present study was to estimate KIR gene and genotype below this level (24%) (p<0,037). While the incidence of bacte- frequencies in the Russian unrelated donors of hematopoietic ria sepsis was lower in patients receiving a high number of cell stem cells. dose (CD3, CD19, CD56), no correlation between cell dose and Materials and methods: KIR genotyping was performed by CMV reactivation was found. Relapse free- and overall surviv- PCR-SSP in 64 donors using the PEL-FREEZ KIR genotyping als were not signifi cantly affected by the different cell popula- SSP kit (distributor “Biochemmack”, Moscow). The kit identifi ed tions. 14 KIR genes and 2 pseudogenes. The KIR genotyping was Conclusion: Grafts containing high numbers of CD4+ cells is a performed as recommended by the manufacturer. Direct count- risk factor for developing severe acute GVHD but survival rates ing was used for the estimation of the observed frequencies of were not signifi cantly correlated to graft composition. each particular KIR gene. The determination of genotypes was based on the offi cial KIR nomenclature. Results: All donors carry 2DL4, 3DL2, 3DL3 genes and pseu- P983 dogene 3DP1. The frequencies of other inhibitory genes are Donor search for patients at a St.Petersburg from 51.6% (2DL2) to 98.4% (2DL1 and 3DL1). Activating gene medical university bone marrow transplantation frequencies are from 28.1% (2DS3 is the most rare activating clinic inassociation with HLA polymorphism gene in our donors) to 96.8% (2DS4). 2DS4*003-007 is the A. Alyanskiy, O. Paina, N. Ivanova, A. Golovacheva, most frequent variant of the 2DS4 activating gene, it has been L. Zubarovskaya, B. Afanasyev revealed in 81.2% of the donors. SPSMU named after I. Pavlov (St.Petersburg, RU) The A haplotypes (2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2, 3DL3, 2DP1, 3DP1) are the most frequent.31 different geno- Background: Number of patients, who has matched sibling isn’t types have been identifi ed. The AA genotypes present in 22 higher 15% and there’re not well functioning donors registries donors (34.4%). Among AB and BB genotypes 17 genotypes in Russian Federation. In Saint Petersburg State I. Pavlov have been found only once, 9 genotypes – twice, one genotype Medical University (SPSMU) BMT Clinic more then 650 pre- has been revealed tree times, and another one – four times. liminary unrelated donor searches in BMDW were performed Conclusion: In general the KIR gene and genotype distribution since 2000 till 2008. International Registers network allowed in our donors is similar to other Caucasoid populations. fi nding the unrelated donors for 50% of Russian patients. Nev- ertheless, the part of Russian’s recipients has not potentially matched unrelated donor in BMDW database.

S295 Materials and methods: Analyzed distribution of HLA -À,-B and the goal of increasing the number and genetic diversity of CB -DRB1 genes and haplotypes frequency for SPSMU BMT Clinic units available for matches. Initiatives included brochures and patients compare to the donors listed in BMDW and donors of promotional materials that were distributed to donation centres SPMU bone marrow donors registry. The HLA-typing was per- at free costs. Education materials and informed consent were formed in SPSMU BMT clinic HLA-typing laboratory using PCR translated into fi ve languages (English, French, Spanish, Arab, SSP “Protrans” technology. Albanian) and carry complete information about the donation Results: The Russian donors and recipients coincide on fre- procedure and purpose. Here we evaluate the impact of such quency of occurrence of defi ned HLA-A and -B haplotypes, a strategy on implementing the ethnic diversity among our CB thus we do not observe the same tendency at comparison of Bank. As a result of this programme (see the graphic), between haplotypes of our patients and BMDW donors (A26-B44;A03- 2006 and 2008, the number of non-Italians donors increased as B41;A30-B51;A02-B49;A33-B18;A11-B18;A26-B27;A24- referring to those coming from Europe but remained stable for B22;A23-B15;A03-B55;A03-B37;A31-B27;A26-B07) (Graf.1) those coming from extra-European countries. The proportional This data should also prove useful in the search for unrelated distribution of racial and ethnic groups among the CB collec- bone marrow donors in Russian population because the varia- tions is far away from approaching their proportional distribution bility of HLA-polymorphism in Russian population give a higher in population. Further efforts are required to increase not only chance of fi nding a suitable donor for Russian patients. As a understanding of the need of CB donations for minority donors result of the conducted research the difference in occurrence but also their motivation and opportunity to donate by establish- of certain genes and hyplotypes at the Russian patients and ing relationships with community centres in collaboration with donors in comparison with donors listed in BMDW is authenti- bilingual translators and cultural mediators. cally proved. Conclusion: There are certain differences in occurrence of sepa- rate genes and HLA-haplotypes in group of the Russian donors and recipients in comparison with donors listed in BMDW.

P985 Infant or young donors for bone marrow transplant in thalassaemic sibling recipients: a comparison of graft products P984 K. Paciaroni, C. Gallucci, C. Alfi eri, G. De angelis, A. Isgro’, Minority report. Representation of ethnic minority groups M. Marziali, A. Roveda, P. Polchi, P. Sodani, J. Gaziev, at a cord blood bank, Pavia G. Lucarelli P. Bergamaschi (1), C. Parisi (1), V. Genovese (1), E. Beolchini International Mediterranean Institute of (Rome, IT) (2), T. Quaglini (3), L. Gilardi (4), A. Campagnoli (5), G. Marchese (3), G. Viarengo (1), C. Del Fante (1), A. Marchesi Background: Data regarding the characteristics of graft cells (1), B. Romano (1), C. Perotti (1), L. Salvaneschi (1) products obtained by healthy infants are lacking. (1)IRCCS Policlinico San Matteo (Pavia, IT); (2)Hospital of Broni Objective: To compare the graft products obtained by infants (Broni, IT); (3)Hospital of Voghera (Voghera, IT); (4)Hospital of with that obtained by young matched HLA-identical sibling Codogno (Codogno, IT); (5)Hospital of Lodi (Lodi, IT) donors for thalassemic recipients. Methods: Consecutive healthy infant donors(<24 months of Concerns exist about underutilization and underrepresentation age) and 99 young donors (3-53 years, median 15,1 years) of ethnic minorities on donors Registries. Some ethnic groups undergoing bone marrow stem cells collection for their matched are poorly represented within the total number of donors lead- recipients entered the study. Main variables of interests ana- ing to unequal access to stem cell transplantation for all patients lyzed were donor weight, patient weight and the ratio donor in need. In fact some minority groups have rare and varied HLA weight/patient weight in order to identifi ed great unfavourable combinations, therefore fi nding a match is more diffi cult than differences between donors and recipients weights (ratio <1). among Caucasian donors. As HLA-matching requirements are Levels of CD34 cells and CD3 cells collected in the bone mar- less stringent, cord blood (CB) may offer to minorities with rarer row donations were assessed and compared between the two and more varied HLA types an increased chance of fi nding study groups. a compatible donor. Several factors contribute to discourage Results: The weight of infant donors was signifi cantly less minorities from participating to donors program (lack of informa- then the young donors (median 10.9 vs 43.5 Kg, p<0.001) and tion about: benefi ts of transplantation, existence of registries, consequently the bone marrow volumes harvest were signifi - need for donors, risk of donation and the lack of opportunity cantly smaller (median 258 ml vs 630 ml (p<0.001). Indeed we to donate). Common barriers to donation can be addressed observed that the median weight recipient is rather two-fold by educational strategies aimed at increasing knowledge and greater then the respective infant donors (18,8 kg patients vs educating minorities about the importance to people of their 10,9 kg donors) on the contrary in the young donor group the ethnic background of becoming a donor, as a match is more patient weight exceeds not much the half of the donor weights likely within the same ethnic group. For this reason, a project of (26,8 kg patient vs 43,6 kg donors). In order to quantifi ed minority recruitment was initiated by our CB Bank in 2006 with the unfavourable discrepancy between donor and recipients

S296 weights we calculated the ratio donor weight/patient weight. In P987 the infant group the median ratio was signifi cantly lower then Infl uencing factors on stem cell mobilisation and the yield the value observed in the young donor group (0,6 vs 1,8, p≤ of collection in paediatric malignancies 0.001). Despite of low ratio, the median counts per kg recipient C. Landefeld, K. Kentouche, A. Müller, F. Zintl, J. Beck weight of CD34 cells, CD3 cells were not different in the infant University of Jena (Jena, DE) bone marrow product compared to that of young donors (CD 34 cells:10.6 x 106/kg vs 7.7 x 106/kg, (p=0,08), CD3: 45.6x106/kg High-dose chemotherapy with stem cell transplantation is an vs 65.6 x 106/ kg (p=0,11)). No major adverse events occurred established procedure in the treatment of oncological diseases in the infant donors. Nevertheless red cells blood transfusions in childhood. We investigated retrospectively the apheresis in were more commonly used in the infants donors (50% vs 10%, our transplant center for infl uences on the stem cell yield. p<0.001) in the presence of similar post donation haemoglobin We analyzed 174 stem cell mobilizations and apheresis results levels(Hb 9,8 gr/dl vs 9,7 gr/dl, p=0,92). of 130 patients. Indications were Neuroblastoma (n = 28), Conclusion: The present study supports the feasibility to col- Ewing tumor (23), Hodgkin´s disease (14), Leukemia (34), lect an adequate number of progenitors cell from infant donors Autoimmune disorders (14) and others disease. The average despite their low weights. Higher rate of red blood transfusion age was 11.1 years, with a gender distribution of 55% female has been observed in the absence of more severe anemization and 45% male. suggesting that a more conservative approach may be imple- The middle CD 34 yield was 6.1x106/kg. In 69% the minimum mented. required amount of 2x106/kg and in 43% in the optimum amount of 5x106/kg were collected. The collection began on average 12.5 days after the end of the chemotherapy and following P986 7.5 days of G-CSF stimulation. In 25 cases we were unable A reassessment of the value of peripheral blood CD34 to collect after mobilization, because of insuffi cient blood CD enumeration prior to autologous peripheral blood 34. Mobilization at an early stage of the disease give better collection results for blood CD 34 on the beginning of the collection (68 vs S. Wadiwalla, S. Couban 30/µl*, p<0,05) and for the collected CD 34 (12.9 vs 5.5 x106*). QE II Health Science Centre (Halifax, CA) Relapse-malignancies showed a lower yield of blood CD 34 (19 vs. 39/µl*) and collected stem cells (4.3 vs.7.5 x106*). A pre- APB transplantation is an established therapy several hemato- vious radiation therapy also has a negative infl uence on the logical malignancies. Collection of an adequate graft is a pre- mobilization (19 vs. 37/µl) and collection (3.8 vs.7.4x106*) of requisite for successful transplantation and higher CD34 cell CD 34. There are also differences in collection and mobiliza- counts are associated with improved outcomes. Many centres tion, depending on the malignancies and used cytostatics. The measure the concentration of CD34 cells in the patient’s periph- stimulation of G-CSF in a dose of 5-10 µg/kg showed higher eral blood and proceed with collection only if the concentration blood CD 34 than stimulation with 10-15 µg/kg (38 vs. 22/µl*). is above a threshold level of 5 or 10 CD34 cells/ul. Experience Beginning the apheresis when blood CD 34 rise more than 20/ at our centre indicated that several patients had excellent col- µl increases the success rate. In 95% we collected so a suf- lections despite low CD34 concentrations in the peripheral fi cient number of CD 34. blood. Between 2006-07, consecutive patients undergoing We fi nd overall good results for the collection of stem cells. But APB collection proceeded to standard volume autologous there is still plenty of potential to improve this expensive and collection without consideration of the peripheral blood CD34 complex procedure. Great infl uence has the time of the col- concentration. Daily apheresis continued until at least 2.0 mil- lection. As early as possible in the course of therapy without lion CD34 cells/kg were collected. Patients with myeloma were prior irradiation showed signifi cantly better results. Improving apheresed until 5 million CD34 cells/kg were collected if this potential exists also in the process of mobilization. The effec- were possible. There were 64 patients who underwent APB col- tive use of G-CSF (5-10 µg/kg), and start the collection when lection during this period, 34 with myeloma, 27 with lymphoma enough blood CD 34 stimulated (> 20/µl), can increase stem and 3 with other malignancies. There were 54 patients who cell yield and reduce costs. Taking into account these factors underwent one set of collections, 10 patients who underwent and in combination with individual approach stem cell mobiliza- 2 collections. The median number of apheresis procedures per tion can be improved and more effective collection results can patient was 2 (range: 1-7 procedures). Using a threshold of 2.0 be achieved. million CD34 cells/kg, there were 53 successful collections of which 46 patients were completed with one attempt and 7 with 2 attempts. Among the patients who underwent successful col- lections, 15 of 53(28.3%) and 11 of 53(20.8%) would not have been collected if we had used a peripheral blood threshold of Regulatory issues 10 CD34/ul or 5 CD34/ul, respectively. Of the 11 patients with collections < 2.0 million CD34/kg, none had a peripheral blood CD34 count > 10 and 1 had a peripheral blood CD34 count > P988 5. Ten patients with peripheral CD34 count < 10 cells/ul, under- First European haematopoietic progenitor cell transplant went APB transplant. The median day to neutrophil recovery unit obtaining re-accreditation of a JACIE quality system: (ANC> 500 for 2 consecutive days) was 14 (range 9-19 days) experience and diffi culties for implantation and for platelets (platelet count >20,000 for 2 consecutive days) F. de Arriba (1), M.L. Lozano (2), J. Nieto (1), C. Castilla (1), was 12 (range 11-21 days, 1 patient did not engraft platelets). 9 J. Márquez (1), J. Tomás Martínez (1), J. Rivera (2), I. Heras patients achieved 100days survival and one patient died on day (1), V. Vicente (3) 25. 38 patients with peripheral CD34 counts > 10 cells/ul under- (1)Hospital Morales Meseguer (Murcia, ES); (2)Centro Regional went APB transplant. The median day to neutrophil recovery de Hemodonación (Murcia, ES); (3)Universidad de Murcia was 13 (range 7-21 days) and for platelets was 11 (0-16 days). (Murcia, ES) All patients achieved 100days of survival. In conclusion, almost 30% of successfully collected patients Directive 2004/23/EC of the European Parliament and of the had peripheral blood CD34 count < 10/ul. Council sets rules and standards of quality and safety for the donation, procurement, testing, processing, preservation, stor- age and distribution of human tissues and cells. On the fi eld of HPC transplantation, JACIE has established standards of quality regarding medical and laboratory practice. However, compliance is diffi cult and few units in Europe have achieved

S297 accreditation. Our transplant unit performs about 50 transplants accounted for the majority of these (€21,727, 47% per patient), a year, and it was accredited by JACIE in January 2004. Early followed by drug costs (€14,210, 31% per patient) and diag- this year, we became the fi rst European unit achieving JACIE nostic and laboratory tests costs (€10,072, 22% per patient). re-accreditation. We believe that it is worth to show here our Mean conditioning treatment costs per patient were €1,882 experience in the implementation and stabilization of a JACIE (4%, €4,464 myeloablative conditioning costs –MAC- and €521 program in our Transplant Unit. Our quality management sys- reduce intensity conditioning –RIC-), mean baseline disease tem (QMS) was designed according to a circular structure (plan drug costs per patient were €4,602 (10%) and mean fungal - do - review - plan) which pursues continuous improvement in prophylaxis or treatment drug costs per patient were €7,727 the HPC transplantation program; this design has been crucial (17%). Up to a 60% of all costs were incurred in the fi rst 30 days for establishing and maintaining the JACIE standards. Note- from hospitalization for alloSCT. Non pharmacological treat- worthy, our collecting and processing HPC Unit already had ment costs accounted for 69% of all costs, mainly due to hos- a QMS certifi cated by ISO 9000 and this represented a great pitalization costs (47%). Allogenic stem cell transplant poses a advantage while setting the QMS in the whole HPC transplant high burden on the patient and society, with total costs around program. €46,000 per patient for the fi rst 100 days. The key elements of our QMS are: 1) unequivocal leadership of the Director in the scheduled review and planning of quality objectives, as well as on dia- logue with administrative personnel of hospitals to ensure the additional requirements of human and material resources; Cytokines 2) designation of a Quality Coordinator, with reduced dues of care as to deal with QMS issues, 3) appropriate defi nition of the responsibilities of each position; P990 4) setting up a standardised documentation system, suffi ciently Polymorphism in the genes encoding human interleukin-7 controlled, and accessible to staff; receptor-alpha and outcome after allogeneic 5) initial and continuous training of personnel in technical and haematopoietic cell transplantation with matched quality management, unrelated donor 6) close communication between clinical, collection, and Z. Shamim (1), L. Ryder (1), M. Haagenson (2), S. Spellman processing areas. (3), T. Wang (4), S. Lee (5), K. Müller (1) Among these issues, both the release of the Coordinator of (1)National University Hospital (Copenhagen, DK); (2)Center Quality of care duties, and continuous updating of the docu- for International Blood and Marrow Transplant Research mentation system, have far been the most diffi cult tasks. The (Minneapolis, US); (3)National Marrow Donor Program greatest challenges achieved meanwhile have been systema- (Minneapolis, US); (4)Medical College of Wisconsin (Milwaukee, tisation of all operational and management activities, minimiz- US); (5)Fred Hutchinson Cancer Research Center (Seattle, ing the impact of changes in staff, and widespread commitment US) of staff with compliance with the standards and continuous improvement. Objective: Interleukin-7 (IL-7) is essential for T cell develop- The re-accreditation of our program demonstrates the ability of ment in the thymus and maintenance of peripheral T cells. The committed units to implement and sustain JACIE standards. alpha-chain of the IL-7R is polymorphic with the existence of 4 SNPs (+510C/T (rs1494558), +1237 A/G (rs1494555), 2087T/ C (rs6897932) and +3110A/G (rs3194051), that all give rise to P989 amino acid substitutions. We previously found an association Cost of allogeneic haematopoietic stem cell between donor +1237G and increased treatment related mor- transplantation tality (TRM) and acute graft versus host disease (aGvHD) after R.F. Duarte (1), B. Patiño (1), C. Muñoz (1), M. Arnan (1), matched unrelated donor (MUD) hematopoietic cell transplan- T. Peralta (1), A. Clopés (1), J.L. López-Belmonte (2), tation (HCT) as well as allergy. The C allele of +2087 has been A. Fernández de Sevilla (1), F.J. Sabater (2) associated with allergy and multiple sclerosis. The aim of the (1)Institut Catalá d’Oncologia (Barcelona, ES); (2)Health present study was to evaluate the prognostic signifi cance of IL- Economics Unit, Schering-Plough España (Barcelona, ES) 7Ra SNP genotypes in a large cohort of adults receiving MUD HCT for haematological malignancies. Allogenic haematopoietic stem cell transplantation (alloHSCT) Patients and methods: 590 Caucasian adult (>18 years old) is a well-established treatment for several haematological patients with AML (n=111), ALL (n=76), CML (n=373) or MDS malignancies. However, little is known regarding the real costs (n=30) in early stage disease receiving a myeloablative con- of this procedure in our setting. The aim of this study is to deter- ditioning regimen and an allele-level matched (HLA-A, B, mine the total costs of alloHSCT in patients with haematological C, DRB1 and DQB1) T cell replete HCT from an unrelated malignancies. Forty-three peripheral blood alloHSCT recipients Caucasian donor from the National Marrow Donor Program. were included in this analysis. We reviewed all clinical activ- IL-7Ra polymorphisms were determined by an SSP-PCR sys- ity and categorized it by in-patient admission, out-patient visits, tem in genomic DNA extracted from banked pre-transplant diagnostic procedures, laboratory tests, and medication use blood samples from the NMDP Research Repository. Univari- (divided by conditioning regimen drugs, antifungal prophylaxis ate and multivariate analyses were performed for overall (OS), and treatment drugs, and all other pharmacy expenditure). All transplant-related mortality (TRM), relapse, and acute grades costs were analysed for an initial period up to day +100 after II-IV and III-IV and chronic GvHD, adjusting for clinical variables alloHSCT. Unit costs for drugs were obtained from the hospi- in the multivariate analysis. tal pharmacy and for all other resources from the Soikos data- Results: Neither donor nor recipient SNP genotypes were sig- base. All costs are expressed in Euros 2008. Transplant donor nifi cantly associated with overall survival or TRM. Associations costs were not taken into account at this point. Patients in the between donor SNP genotypes, relapse and GvHD are sum- study had a median age 52 (20-68), 58% were male, 30 had an marized in the table. MDS-AML, 6 a lymphoid malignancy, 4 CML, 3 ALL. Eighteen Conclusion: Although the univariate analysis indicated an received a myeloablative conditioning and 25 a reduced inten- impact of the previously identifi ed risk alleles of SNP +2087 sity conditioning, 31 from a related and 12 from an unrelated (C) and +1237 (G) of the donor on GvHD or relapse, this was donor. Median time to neutrophil engraftment was 17 days (13- not confi rmed in the multivariate analysis. Our study does not 33), and incidence of acute graft versus host disease grades fi nd suffi cient evidence to support an association between II-IV was 30.2%. Patient overall survival at day +100 was 79%. the three examined IL-7Ra SNPs and adverse outcomes in Mean total cost per patient was €46,010. Hospitalization costs MUD HCTs.

S298 P992 TGFB1 Codon 10 polymorphisms are associated with worst outcome in allogeneic unrelated donor haematopoietic stem cell transplantation M. Berro (1), N. Mayor (1), L. Cooke (1), G. Kusminsky (2), S. Marsh (1), J.A. Madrigal (1), B. Shaw (1) (1)Anthony Nolan Research Institute (London, UK); (2)Hospital Universitario Autral (Pilar, AR)

Haematopoietic stem cell transplantation (HSCT) using unre- lated donors (UD) is a life-saving intervention for patients with haematological malignancies. TGF beta 1 has numer- ous immunomodulatory properties. Several functional poly- morphisms have been identifi ed, such as a single nucleotide polymorphism (SNP) at codon 10 (c.29T>C, p.L10P) of exon 1. TGFB1 genotyping was performed on 427 UD-HSCT pairs pro- vided by the Anthony Nolan Trust. Transplants were between 1997 and 2006 with a median follow up time of 4.8 years (0.23- 11 years). Diagnoses were chronic myeloid leukaemia, 26.9%, acute lymphoid leukaemia, 29.3% and acute myeloid leukae- P991 mia, 28.8%. All transplants received myeloablative condition- A prospective randomised multicentre trial of ing regimens and 86.4% included T-cell depletion. 65.6% were darbepoetin-alfa and I.V. iron administration after 10/10 HLA matched and 17.1% were 12/12 matched. Patients autologous haematopoietic stem cell transplantation and donors TGFB1 genotypes frequencies were similar to pre- Y. Beguin (1), J. Maertens (2), B. De Prijck (3), R. Schots (4), viously described. Patients homozygous for the SNP (CC) had P. Frère (1), C. Bonnet (1), K. Hafraoui (1), E. Willems (1), signifi cantly reduced overall survival (OS) compared to other G. Vanstraelen (1), M. Lejeune (1), K. Theunissen (2), G. Fillet genotypes (5 years 29.3% vs. 42.2, p=0.01) and increased non- (1), F. Baron (1) relapse mortality (NRM) (3 years 46.8% vs. 29.3%, p=0.01). (1)University of Liège (Liege, BE); (2)KUL (Leuven, BE); There was no impact of donor genotype alone although an (3)CHR Citadelle (Liege, BE); (4)VUB (Brussels, BE) additive effect was seen when analysed with patient genotype. Multiple SNPs within the pair (3-4 SNPs) were associated with Background: We report the results of a multicenter prospective signifi cantly decreased OS (5 years: 33.8% vs. 41.9%, p=0.03) randomized study analyzing the impact of darbepoetin alfa with and higher NRM (3 years 45% vs. 30%, p=0.02). In multivari- or without i.v. iron on erythroid recovery after autologous HCT. ate analysis there was a trend to decreased OS in patients Patients and Methods: 127 autologous HCT recipients with homozygous for the SNP (HR: 1.44; p=0.083). In the 10/10 HLA lymphoid malignancies were randomized 1:2:2 between no matched transplants (n= 280), non-wt donors (TC/CC) signifi - treatment (group 1, n=25), darbepoetin alfa (Aranesp®) 300 µg cantly increased the incidence of aGVHD II-IV (29.9% vs. 20%, QOW starting on day 28 after HCT for a total of 7 doses (group p=0.05). The same additive effect was seen when analysed 2, n=52), or the same regimen of darbepoetin alfa plus i.v. iron with patient genotype. Non-wt pairs signifi cantly increased the sucrose (Venofer®) 200 mg on days 28, 42 and 56 after HCT incidence of overall aGVHD (51% vs. 32%, p=0.018), aGVHD (group 3, n=50). Primary endpoints included proportion of com- II-IV (30% vs. 2.5%, p<0.0001) and a trend to increase the plete correctors (i.e. patients reaching Hb ≥ 13 g/dL) before day incidence of aGVHD III-IV (6.7% vs. 0% p=0.09). In the multi- 126 post-transplant and median time to achieve Hb correction variate analysis, the non-wt pairs signifi cantly increased overall in each arm. aGVHD (HR: 2.1; p=0.03) and aGVHD II-IV (HR: 15.5; p<0.01). Results: In intent to treat analyses, the proportion of complete We conclude that in UD-HSCT increasing numbers of SNPs in correctors was 24% in group 1, 81% in group 2 (P<0.001 com- the TGFB1 gene at codon 10 in patients and donors is associ- pared with group 1), and 92% in group 3 (P<0.001 compared ated with a worse outcome following UD-HSCT. Identifi cation of to group 1, and P=0.099 compared to group 2). Median time to these SNPs pre-transplant will allow for transplant conditioning achieve Hb ≥ 13 g/dL was not reached in group 1, 42 days in and immunosuppression regimens to be tailored to the individ- group 2 (P<0.001 compared to group 1), and 32 days in group ual patient, as well as assisting in the most appropriate choice 3 (P<0.001 compared to group 1 and P=0.127 compared to of donor. group 2). Mean ± standard deviation total doses of darbepo- etin-alfa administered were 1,445 ± 489 µg in group 2 vs 1,272 ± 443 µg in group 3 (P=0.06). Eight patients (2 in group 1, 4 in group 2, and 2 in group 3) required red blood cell transfusions on study, including 4 patients following early disease progres- sion. In per protocol analyses, the proportion of complete correctors was 21% in group 1, 80% in group 2 (P<0.001 com- pared with group 1), and 96% in group 3 (P<0.001 compared to group 1, and P=0.029 compared to group 2). Median time to achieve Hb ≥ 13 g/dL was 190 days in group 1, 44 days in group 2 (P<0.001 compared to group 1), and 31 days in group 3 (P<0.001 compared to group 1 and P=0.025 compared to group 2). There was no difference in ferritin levels, nor in rates of thrombo-embolic events, or other complications among the groups. Conclusions: This is the fi rst prospective randomized trial dem- onstrating that darbepoetin alfa is safe and highly effective to ensure full erythroid reconstitution after autologous HCT when started on day 28 posttransplant. I.v. iron sucrose tended (not statistically signifi cant in intent to treat analyses) to further fas- ten erythroid recovery with a lower dose of darbepoetin alfa required.

S299 P993 P994 A phase II randomised study comparing pegfi lgrastim vs. The G-CSF effect in the setting of splenomegaly and fi lgrastim after high-dose chemotherapy and autologous extramedullary haemopoiesis: a mobilisation study in a peripheral blood stem cell support thalassaemia mouse model as compared to normal strain L. Castagna (1), S. Bramanti (1), A. Levis (2), M. Michieli (3), E. Yannaki, N. Psatha, E. Athanasiou, G. Karponi, A. Anastasia (1), B. Sarina (1), E. Todisco (1), A. Nozza (1), D. Bougiouklis, V. Constantinou, M. Demertzi, P. Kaloyannidis, L. Giordano (1), A. Santoro (1) I. Batsis, A. Anagnostopoulos, A. Fassas (1)Istituto Clinico Humanitas (Rozzano, IT); (2)Ospedale SS George Papanicolaou Hospital (Thessaloniki, GR) Antonio e Biagio (Alessandria, IT); (3)Centro do Riferimento Oncologico (Aviano, IT) A G-CSF-associated risk of splenic rupture has been recog- nized in normal donors of hematopoietic stem cells (HSCs). The aim of this phase II randomized trial, was to demonstrate the Limited information is available on the G-CSF effect in the non-inferiority of one fi xed dose pegfi lgrastim (PEG) compared presence of splenomegaly and extramedullary hemopoie- to daily fi lgrastim (FIL), in patients receiving high-dose chemo- sis as in thalassemia. Gene therapy (GT) has recently been therapy (HDC) and peripheral stem cell support (PBSC). postulated as a therapeutic potential for thalassemia and the Patients and methods. From September 2006 to March 2008, mobilized autologous CD34+ cells may represent the prefer- 80 patients with hematological malignancies and solid tumors able source of stem cells for genetic modifi cation due to the were enrolled in this phase II open-label randomized study. higher yield of HSCs compared to bone marrow harvest. An Conditioning regimens were different based on disease and enlarged spleen may potentially pool HSCs from the periph- preference of each center. Autologous PBSC were reinfused ery and negatively affect mobilization. We explored the G-CSF at day 0. PEG was administered subcutaneously at fi xed dose effect in a thalassemic mouse model (HBBth-3) as compared (6 mg) 24h after autologous stem cell infusion (day +1). FIL to a normal strain (C57Bl6), in terms of safety, mobilization was administered subcutaneously at weight-based daily dose effi cacy and distribution of HSCs among hematopoietic com- (5 mcg/kg/d) from day +1. The primary end points were dura- partments. There was no death or clinical sequelae of splenic tion of severe neutropenia (absolute neutrophil count, ANC < rupture in G-CSF-treated animals of either strain, however, at 0.5 x 109/L) and the number of days to achieve an ANC > 0.5 x the time of sacrifi ce, hemorrhagic infarcts were detected only in 109/L starting from the days +1. Secondary end points were the the spleens of G-CSF-treated HBBth-3 mice (5/40). In contrast, number of days to achieve an ANC > 1.0 x 109/L starting from the G-CSF effect on spleen size was stronger in the normal the days +1, number of days with fever > 38 °C, duration of than in the thalassemic (thal) strain (133% vs 65% increase of antibiotic therapy, and number of documented infections. the spleen volume over baseline). HBBth-3mice mobilized less Results. Patient and disease characteristics were reported in Table effectively than C57Bl6 mice (Lin-sca-1+ckit+ cells/microl PBM- 1. No differences were observed between the two groups (FIL vs NCs:90±55 vs 255±174, p=0.01/CFU-GM/ml PBMNCs:390±262 PEG). The duration of ANC less than 0.5 x 109/L and the time to vs 1131±875, p=0.01) due to increased trapping of HSC and reach an ANC more than 0.5 x 109/L were not signifi cantly differ- progenitor cells into the spleen (Lin-sca-1+ckit+cells/spleen: ent (mean days 6 vs 6, and 11 vs 11, p value: 0.5, respectively). 487±35 vs 109±19.6, p=0.01/CFU-GM/spleen/102:1470±347 No statistically signifi cant differences were observed in terms of vs 530±425, p=0.0006). Splenectomy restored the mobilization mean time to reach an ANC more than 1.0 x 109/L (12 vs 12 days), profi ciency of thal mice at comparable levels to normal mice number of patients with fever (24 vs 22), number of documented (Lin-sca-1+ckit+ cells/microl PBMNCs:164±111 vs 223±99, infections (12 vs 10), mean number of days with fever (1.7 vs p=ns) and resulted in the development of a hematopoietic 0.97 days), duration of antibiotic therapy (5.7 vs 3.5 days). Finally, compensatory mechanism in the steady-state liver of splenect- no differences were documented regarding extra-hematological omized thal mice which was greatly increased after G-CSF. G- toxicities and time to discharge between the two groups (16 vs CSF didn’t signifi cantly affect Hb or reticulocytes in either strain 14). Time to reach a platelets count more than 20 x 109/L was sig- when spleens were intact. However, G-CSF caused anemia nifi cantly shorter in PEG group compared to FIL group (15 days after splenectomy in the C57 but not in the thal mice due to vs 11 days, p value 0.054). Considering the average wholesale intense compensatory liver hemopoiesis. Our data imply that, price, and the mean number of vials (20) used in FIL group, the in view of human GT for thalassemia, either multiple cycles of use of PEG reduced the per patient cost of 562 euros. mobilization or alternative ways (ie AMD3100+G-CSF), may Conclusions. This phase II randomized study shows that PEG be required for a suffi cient yield of transplantable gene-modi- was not inferior to FIL in terms of hematological reconstitution fi ed HSCs in non-splenectomized patients. Strategies also to (primary end point). Consequently, PEG could be safely used reduce the risk of G-CSF-induced splenic infarcts should be after PBSC infusion. explored in a clinical setting.

P995 The stromal cell-derived factor -1 (SDF-1/CXCL12) gene polymorphism as a marker of recovery of granulocytes and platelets after transplantation of haematopoietic stem cells A. Gieryng (1), K. Bogunia-Kubik (1), S. Madej (2), M. Mordak-Domagala (2), A. Lange (3) (1)Institute of Immunology and Experimental Therapy, PAS (Wroclaw, PL); (2)Lower Silesian Center for Cellular Transplantation & National Polish Bone Marrow Donor Registry (Wroclaw, PL); (3)Institute of Immunology and Experimental Therapy & Lower Silesian Center for Cellular Transplantatio (Wroclaw, PL)

We have previously reported that the presence of A at position 801 in the 3’-untranslated region (3’-UTR) within the stromal cell- derived factor -1 (SDF-1/CXCL12) gene was associated with a higher yield of CD34+ mobilized for haematopoietic stem cell transplantation (HSCT) in autologous patients and allogeneic donors. Initial work also showed the strong correlation between

S300 the A+ allel and HSC accommodation in marrow, refl ected by time of harvest the median peak of absolute CD34+ cells in the granulocytes recovery (expressed as the day post trans- the PBL was 68 microL (range 7-252) and the median CD34+ plant when the number of granulocytes exceeded 500/ul) in the cells collected for single patient were 9,4 x106/Kg (range 2,4- group patients after autologous HSCT. These studies prompted 47,6). No failure at collection was recorded and 28 pts obtained us to analyse the infl uence of the SDF-1 gene polymorphism on an optimal harvest (>5,0x106/Kg). Up to now 23 pts evaluable the granulocytopoiesis (>500/ul) and megakaryopoiesis (plate- for engrafment after BEAM regimen engrafted. The median let count > 20x103/ul) in patients undergoing autologous and time of grade 4 neutropenia was 8 days (range 6-10 days) and allogeneic HSCT. the recovery of platelet ≥20 x109/L was recorded in median 12 In total 162 recipients (40 auto and 122 alloHSCT) and 122 allo- days after the reinfusion (range 8-21 days). Our experience geneic donors were genotyped for SDF-1 alleles using a PCR- showed that a single injection of pegfi lgrastim in combination RFLP technique and digestion with MspI restriction enzyme. with CHT is a valid procedure for mobilization of CD34+ cells Number of transplanted CD34+ cells was associated with the into the PBL in pts with malignant lymphoma. Besides, a high pace of granulocytes and platelets recovery in autologous (R percentage of pts (82%) obtained an optimal collection just Spearman: –0.380; p<0.016 and –0.468, p<0.003 for granu- with a single leukapheresis. Finally, the pegfi lgrastim-mobilized locytes and platelets, respectively) and allogeneic (–0.369; CD34+ cells induced rapid and sustained engrafment after p<0.0001 and -0.0357, p<0.0001) transplant recipients. myeloablative CHT. Autologous transplant recipients carrying the SDF-1A allele char- acterised with faster recovery of granulocytes (median: 15 vs 17 day after HSCT, p<0.032) and platelets (median: 17 vs 21 day after HSCT, p=0.033). The multiple regression analyses (con- sidering: patients‘ age and sex, number of transplanted CD34+ cells and SDF-1 polymorphism) confi rmed the independent association of the SDF-1-A allele and number of CD34+ cells in the graft (p=0.032) with granulocyte (p=0.049 and p=0.032) and platelet (p=0.027 and p=0.019) recovery after autoHSCT. In allogeneic transplant recipients, all AA+ carriers and only 72% of those having G allele (p=0.037) were among patients that recovered in granulocytes prior to 19 day post alloHSCT independently of number of transplanted CD34+ cells. In conclusion, the presence of the SDF-1-A allele facilitates the mobilization of CD34+ cells from bone marrow to peripheral cir- culation and (in addition to the number of CD34+ transplanted cells) also signifi cantly infl uences the hematological recovery of granulocytes and platelets in patients after HSCT. Supported the MNiSW grants No. N-N401-008035 and N-N402-193335.

P996 Successfull autologous peripheral blood stem cells collection by a single dose of pegfi lgrastim in combination with chemotherapy in patients with malignant lymphomas S. Bassi, S. Steffanoni, P. Antoniotti, A. Babic, A. Alietti, L. Nassi, B. Lucchetti, S. Sammassimo, MT Lionetti, L. Roveda, P997 M. Negri, C. Rabascio, G. Martinelli Timing of G-CSF injection for effective autologous stem European Institute of Oncology (Milan, IT) cell collection J.E. Kim, C.-W. Suh, E.K. Kim, B.S. Sohn, I. Park, D.H. Yoon, Granulocyte colony-stimulating factors (G-CSF) alone or in C. Yoo, G. Jang, D.H. Lee, S.-W. Kim, J.S. Lee combination with CHT are standard procedures for mobilization Asan Medical Center (Seoul, KR) of peripheral blood stem cells (PBSC). Recently the pegylated form of G-CSF has been introduced, that leads to a larger mole- Objective: Granulocyte colony-stimulating factor (G-CSF) has cule with an increased half-life. While pegfi lgrastim is equivalent been given approximately 12 hours before leukapheresis in the to daily fi lgrastim in enhancing neutrophil recovery, its effi cacy patients who are planned to do autologous stem cell collec- in PBSC collection is still under evaluation. From January 2006 tion. The objective of this study was to identify better timing of to October 2008 37 patients (pts) with malignant lymphoma G-CSF administration which improves the effi cacy of autolo- were eligible for stem cell mobilization and ASCT. They were gous stem cell collection. relapsed or progressive disease or mantle-cell, T-cell lymphoma Methods: A total of 262 patients (157 male and 105 female at diagnosis. Pts received DHAP-modifi ed schedule (ESHAP) patients, ages from 15 to 66 years with median of 49 years) who as cytoreductive and mobilizing regimen. Twenty-four hours underwent autologous stem cell collection from January 2000 to after the chemotherapy they received a dose of 6 mg of peg- March 2008 were included. The patients were diagnosed with fi lgrastim. For the characteristics of pts see Table 1. Peripheral Hodgkin’s lymphoma (N=19), non-Hodgkin’s lymphoma (N=131) CD34+ cell evaluation started from the increase of WBC above and multiple myeloma (N=112). Peripheral blood stem cells 1x109/L. PBSC were collected using a Cobe Spectra separator. were mobilized with lenograstim (Choongwae Pharma Corp., Leukapheresis started when the number of circulating CD34+ Seoul, Korea) following chemotherapy, such as cyclophospha- cells was >7microL and performed daily until target number of mide (N=141), ESHAP (Etoposide, methylprednisolone, Ara-C PBSC ≥2x106 was reached. All pts were considered for time to and cisplatin, N=73) with (N=14) or without rituximab (N=59) neutrophil and CD34+ cells recovery, as for the engrafment after and other chemotherapy regimens (N=48). Before November high dose CHT. Thirty-fi ve pts (94%) performed successfully 2004, patients received lenograstim injection at a dose of 10 ug/ PBSC collection, that was completed after a single apheresis in kg subcutaneously at 8:00 PM and underwent peripheral stem 33 pts. The median time to a suffi cient number of CD34+ was cell collection on next day (N=129). After then patients received 10 days (8-12 days). Two pts required an additional procedure, lenograstim injection at 6:00 AM and underwent peripheral stem despite the satisfactory number of CD34+ circulating. At the cell collection at the same day (N=133).

S301 Results: Injection of lenograstim at 6 AM was superior to injec- NK attack on AML blasts currently observed after haplo-SCT. tion of lenograstim at 8 PM with greater numbers of total col- By contrast, at 2 years post-SCT and in donors, mature NK lected CD34+ cells/kg in shorter duration of leukapheresis cells were functionally, which is encouraging to develop clini- procedures (13.29 x 106 versus 8.51 x 106 CD34+ cells/kg; cal trials using adoptive transfer of healthy donor alloreactive p=0.001, 2 versus 3 leukapheresis procedures; p=0.035). The NK cells. median number of CD34+ cells/kg collected at fi rst leukapher- esis was also greater in 6 AM group (3.94 x 106 versus 2.47 x 106 CD34+ cells/kg; p=0.001). Stem cell collection effi cacy P999 defi ned as ratio of total collected CD34+ cells per days of leuk- Targeting the programmed death (PD)-1 / PD ligand paheresis were 5.34 and 2.96, respectively. It was signifi cantly 1 pathway to induce peripheral tolerance in allograft better in the group of patients received lenograstim injection at recipients 6 AM (p=0.001). The median number of patients who achieved P. Müller, E. Distler, R.G. Meyer, U.F. Hartwig, C. Huber, a total collected CD34+ cells > 5 x 106/kg was also greater in 6 W. Herr AM group (113 versus 96; p=0.002). Johannes Gutenberg-University (Mainz, DE) Conclusions: The present study shows that injection of lenogras- tim at 6 AM improves the effi cacy of stem cell collection with Several transplantation studies in animals show that ligation of greater number of collected CD34+ cells/kg in shorter duration the programmed death (PD)-1 receptor can decrease allograft of leukapheresis procedures compared to that of 8 PM group. rejection and graft-versus-host disease (GVHD) lethality, and that expression of its ligand PD-L1 contributes critically to the protection of tissues from GVHD. We have recently observed by functional studies on ex vivo isolated lymphocytes from patients with liver transplant-associated GVHD that alloreactive CD8+ Tolerance and alloreactivity T cells are negatively regulated by the PD-1/PD-L1 pathway (Schuchmann et al., Am. J. Transplant. 2008;8:2434-2444). We have now extended this work and found by fl ow cytometry P998 analysis that human alloreactive cytotoxic T lymphocyte (CTL) HLA-E up-regulation on IFN-g-activated AML blasts clones (n=9) express PD-1 after in vitro stimulation with target impaired CD94/NKG2A-dependent NK cytolysis following cells carrying mismatch HLA-A, -B or -C alleles. Maximum PD- haplo-identical haematopoietic stem-cell transplantation 1 expression level was observed 24-48 hours after alloantigen S. Nguyen (1), V. Beziat (1), N. Dhedin (1), M. Kuentz (2), contact. Interestingly, PD-1 expressing CTLs demonstrated M. Uzunov (1), J.P. Vernant (1), P. Debre (1), V. Vieillard (1) strong interferon-gamma production in cytokine secretion and (1)Hopital Pitie Salpetriere (Paris, FR); (2)Hopital Henri Mondor ELISPOT assays, indicating that recently activated cells are the (Creteil, FR) main PD-1 expressors. Preliminary experiments also showed that blocking the interaction between PD-1 and PD-L1 by a We previously reported that NK-cell generated after haplo- PD-L1 antibody results in increased IFN-gamma production, SCT in high risk AML patients are characterized, during the if T cells are stimulated with PD-L1 expressing allogeneic fi rst 6 months post-SCT, by specifi c phenotypic features and dendritic cells (DC). By using fl ow cytometry we analyzed the impaired functioning having potential impact for transplantation expression of the PD-1 ligands PD-L1 and PD-L2 on primary outcome (Nguyen et al, Blood 2005). In the present study, we cells isolated from various human tissues. We detected strong examined the impact of INF-g on the impaired recognition of PD-L1 expression on monocyte-derived DC, whereas staining AML blasts by NK cells after haplo-SCT. We studied NK cells was negative on B cells, hepatocytes and fi broblasts. One-third at 1 to 6 months (M1-M6; n=21patients) or at 24 months (M24; of acute myeloid leukemia blast samples expressed PD-L1, n=3 patients) post -SCT. Patients received CD34+ haplo- albeit with low staining intensity. We also found weak PD-L1 transplant after a myeloablative conditionning regimen for high expression on renal proximal tubular epithelial cells (PTEC). risk hematological diseases. The PD-L2 ligand was expressed only by DC and weakly by As previously observed, the subset of CD56brightCD94/ fi broblasts and PTEC. In conclusion, we demonstrate activa- NKG2A+ NK cells was increased and NK lysis of AML blasts tion-induced PD-1 expression on human alloreactive CTL was poor, suggesting an immature status of NK cells post clones and analyzed the expression of its ligands PD-L1 transplant. However, at M24, CD56brightCD94/NKG2A+ NK and PD-L2 on primary cells of various human tissues. Early subset and lytic functions restored and returned to donors lev- functional studies suggest that the PD-1/PD-L1 interaction els. Blocking, ex vivo, the inhibitory CD94/NKG2A receptor on infl uences the function of alloreactive CD8+ CTL in vitro. We NK cells restored the lysis at M3 but not at M24. Higher level of believe that interfering with this negative regulatory pathway intra-cellular IFN-g production was detected by FACS in CD94/ might be a novel therapeutic strategy to control GVH-reactive NKG2A+NK at M1-M6 as compared to the donors and at M24, CD8 T cells in allograft recipients. The PD-1/PD-L1 pathway after incubation with IL-12+IL-18 (70% vs 14%). IFN-g treat- might be also of relevance for graft rejection, where the induc- ment led to higher surface expression of HLA-E on AML blasts, tion of peripheral tolerance to the allotransplant is of paramount resulting in decreased killing by NK cells from healthy donors. importance. Actually, adjonction of IFN-g on AML blasts inhibited the lysis by healthy donor NK cells (12% specifi c lysis with INF-g, vs 37% without INF-g). Blocking CD94/NKG2A restored the lysis previ- P1000 ously inhibited by INF-g. Increasing probabilities of kir expression characterize the Neutralization of INF-g during the interaction of NK cells post- formation of inhibitory kir repertoires: evidence against SCT with mismatched AML blasts restored the lysis (16% lysis selection of human NK cells without INF-g neutralization versus 44% after IFN-g neutraliza- S. Andersson, C. Fauriat, J.A. Malmberg, H.G. Ljunggren, tion at M1). This effect was no more observed at M24 or in K.J. Malmberg healthy donors. Karolinska Institutet (Stockholm, SE) To conclude, increased secretion of INF-g by immature NK cells generated after haplo-SCT upregulates the cell surface Objectives and Methods: Inhibitory killer cell immunoglobulin- expression of HLA-E on AML blasts. Increased HLA-E expres- like receptors (KIRs) preserve tolerance to self and shape the sion protects AML targets from NK-mediated lysis through the functional response of human NK cells. Here, we used multi- inhibitory CD94/NKG2A receptor which is overexpressed on NK parameter fl ow cytometry and mathematical modeling to evalu- cells during the early period post-SCT. This suggests that HLA- ate the infl uence of selection processes in the formation of the E could be a component of an immune escape strategy against human inhibitory KIR repertoire.

S302 Results: Co-expression of multiple KIRs on NK cells was more transplant option when HLA-matched related donors are not frequent than expected from random association of independ- available, the disadvantages being essentially the increased ent events. Individuals having three or four of the major KIR risk for graft-versus-host disease (GvHD) and graft rejection. In ligands had similar frequencies of NK cells expressing multiple particular, ASCT from an inherited paternal HLA antigen (IPA)/ KIRs as those with only one KIR ligand. The distribution of self non inherited maternal antigen (NIMA)-mismatched donor has and nonself-KIRs at the cell surface refl ected a stochastic com- been previously demonstrated to be feasible decreasing GvHD bination of receptors rather than a selection process conferred and preserving graft-versus-leukaemia (GvL) effect. In fact, by cognate HLA class I molecules. Co-expression of KIR and maternal cells passing the placenta during pregnancy express NKG2A differed for individual KIRs but was independent on HLA molecules which are acknowledged by the foetus, who the presence of cognate HLA class I ligands. Together, these develops a tolerance to NIMAs maintained in adulthood. In the results indicate that KIR expression is independent of expres- setting of unrelated ASCT, the maternal haplotypes are easily sion of cognate HLA class I ligands in the host and regulated available for cord blood (CB) units while this information is not by a stochastic process characterized by increasing expression provided for adult donors. As NIMAs should be tolerated by the probabilities during KIR acquisition. CB immune system, one can argue that the knowledge of the Conclusion: The results provide new insights into the formation of donor’s NIMA haplotype (a third ghost haplotype) could further the KIR repertoire and argue against selection of human NK cells augment the chance for fi nding a compatible CB donor and conferred by HLA class I molecules. Better knowledge on how reduce immune complications of ASCT. For this purpose we inhibitory KIR repertoires are formed in new hematopoietic envi- fi rst identifi ed the 9 most frequent NIMAs and inherited mater- ronments may have implications for how to interpret and exploit NK nal alleles (IMAs) in the Cord Blood Bank of Pavia, aiming to cell mediated alloreactivity in allogeneic stem cell transplantation. establish if the NIMA haplotypes could be a common source of genotypic compatibility in our population. In our policy, low and high resolution DNA analysis was used to defi ne HLA-A, HLA-B P1001 and HLA-DRB1 alleles for each CB-mother couple participating Maintained hyporesponsiveness of NK cells expressing to the CB banking program. Typing was performed by PCR-SSP inhibitory KIR for non-self ligands in HLA-matched sibling and PCR-SSO. 6324 haplotypes for the HLA-A, HLA-B and stem cell transplantation HLA-DRB1 loci were considered retrospectively, corresponding A.T. Björklund (1), M. Schaffer (1), C. Fauriat (1), O. Ringdén to 1581 CB units and their mothers. The frequencies of HLA-A; (1), M. Remberger (1), P. Ljungman (1), H.G. Ljunggren (1), -B;-DRB1 haplotypes were obtained by direct counting. 7 hap- K.J. Malmberg (1) lotypes out of the 9 most frequent were present both as NIMAs (1)Karolinska Institute, Karolinska University Hospital and IMAs, as shown in the graphic. In our population NIMAs (Stockholm, SE); (1)Karolinska University Hospital appear to be neither rare genotypic combinations nor preferen- (Stockholm, SE) tially non-inherited haplotypes. Thus, the knowledge of the real tolerogenic potential of CB units, namely the third haplotype Objectives: Transplantation over HLA barriers involving killer that is the NIMA one, may further increment the immunologi- cell Ig-like receptor (KIR)-HLA mismatch may trigger donor cal privileges of this stem cell source, in particular for patients NK cell alloreactivity that contributes to graft-versus-leukemia affected with non-malignant diseases for whom GvL effect is effects and improved survival of patients with myeloid malig- not relevant, while reduced GvHD is highly desirable. nancies. Such effects have also been reported in HLA-matched stem cell transplantation (SCT) when donor NK cells express KIR for non-self HLA ligands. This is intriguing and indicates that mechanisms of NK cell tolerance and hyporesponsiveness may be set-aside during the post-transplantation period. Methods and results: To explore this possibility, we have per- formed a retrospective analysis of the outcome in a cohort of 110 patients with acute myeloid leukemia (n=82) and myelod- ysplastic syndrome (n=28), undergoing T cell-replete SCT with an HLA-matched sibling donor. Lack of ligands for donor inhibi- tory KIR had no effect on overall survival, incidence of relapse or graft-versus-host disease. Presence or absence of activating KIR genes in donors with KIR haplotype group B and A respec- tively, had no effect on outcome, nor did any specifi c activating KIR-HLA combination. In line with these clinical observations, functional analysis of the NK cell repertoire post SCT using mul- ticolour fl owcytometry revealed maintained hyporesponsiveness of NK cells expressing inhibitory KIRs for non-self HLA ligands. Conclusions: Our results provide evidence against the emer- gence of an alloreactive NK cell repertoire in HLA-identical allogeneic SCT. P1003 The effect of donor KIR and patient HLA genotypes on P1002 outcome following allogeneic haematopoietic stem cell Cord blood banking: the role of NIMA haplotypes on transplantation in a Chinese population searching for stem cell compatible donors H. Huang (1), G. Wu (1), X. Lai (1), Y. Tan (1), Y. Luo (1), C. Capittini (1), P. Bergamaschi (2), A.M. Pasi (2), C. Badulli (2), F. Zhu (2) I. Sbarsi (2), F. Garlaschelli (2), M. Guarene (2), A. Marchesi (1)Zhejiang Uninversity School of Medicine (Hangzhou, CN); (2), B. Romano (2), V. Genovese (2), M. Martinetti (2), (2)Blood Center of Zhejiang Province (Hangzhou, CN) L. Salvaneschi (2), M. Cuccia (1) (1)University of Pavia (Pavia, IT); (2)IRCCS Policlinico San Killer cell immunoglobulin-like receptors (KIRs) are a family Matteo (Pavia, IT) of inhibitory and activatory receptors and expressed by most NK cells. It is reported that KIR-ligand mismatch can alleviate The search of compatible donors still represents the major aGVHD, reduce relapse and improve disease-free survival in limit to widespread use of allogeneic stem cell transplantation mismatched allo-HSCT. However, the role of KIRs on outcomes (ASCT). Haploidentical siblings may provide an alternative of allo-HSCT remains controversial. The aim of this study is

S303 to investigate the infl uence of KIR-ligand mismatch and some to stimulus with PBMC, UCMC only produced cytokines when activatory receptors of donor KIR on the outcome in Chinese rechallenged with CD3+ antigen presenting cells (with high population. costimulatory molecule expression) but not the original PBMC. Methods: KIR genes were typed by using PCR-SSP, and HLA- These data confi rm that not only naïve T cells from neonates A, B, and C loci genes were used by PCR-SSP or PCR-SSO. and adults, but also adult memory T cells respond to alloPBMC. These patients received standard myeloablative (n=180) or To directly assess if T cells specifi c for DNA viruses such as nonmyeloablative (n=23) conditioning followed by stem cell EBV cross-react with alloantigens, we stimulated PBMC from transplantation from HLA matched (n=164) or mismatched three adults with EBV lysates and immunomagnetically sorted (n=39) donors. CD137+ cells. Cells expanded for three weeks were rechal- Results: 154 out of 203 (75.9%) donor-recipient KIR-ligand lenged with EBV-negative alloPBMC. We found a small but mismatch could be characterized by lack of recipient HLA-Cw signifi cant population of bi- reactive T cells producing IFNg or ligand for donor KIR2DL1, KIR2DL2/2DL3. KIR-ligand mis- TNFa. These fi ndings indicate that in man alloresponses are match result in a decreased incidence of grade II-IV aGVHD not confi ned to a particular subsets of post-thymic T cells, and (RR, 0.405; 95%CI, 0.233-0.704; P=0.001). But it did not affect that strategies to prevent GVHD by naïve T cell depletion may relapse (RR, 1.170; 95%CI, 0.538-2.545; P=0.693) and OS be ineffective. (RR, 0.697; 95%CI, 0.372-1.306; P=0.260). In the patients with myeloid malignancy, KIR ligand mismatch was associ- ated with a decreased incidence of grade II-IV aGVHD (RR, 0.419; 95%CI, 0.208-0.843; P=0.015), a lower rate of relapse (RR, 0.197; 95%CI, 0.089-0.434; P=0.000) and a better OS (RR, 0.533; 95%CI, 0.289-0.982; P=0.044). Transplants with KIR2DS2-positive donors (n=53) had a lower grade II-IV aGVHD (RR, 0.387; 95%CI, 0.175-0.857; P=0.019), and a bet- ter of OS (RR, 0.470; 95%CI, 0.239-0.923; P=0.028). But it did not affect relapse (RR, 0.839; 95%CI, 0.389-1.767; P=0.644). Transplants with KIR2DS5-positive donors (n=47) had a benefi t impact on the outcome of aGVHD (RR, 0.389; 95%CI, 0.166- 0.911; P=0.03), but did not affect relapse (RR, 1.180; 95%CI, 0.575-2.421; P=0.653), and had no signifi cant effect on OS P1005 (RR, 0.954; 95%CI, 0.535-1.702; P=0.873). Management of platelets transfusions in highly Conclusion: KIR-HLA mismatch can contribute to improved immunised beta thalassaemia children undergoing outcomes of patients with myeloid malignancy. Donor having bone marrow transplantation activatory gene whether KIR2DS2 or KIR2DS5 was associ- S. Napolitano, E. Zino, R. Chiesa, B. Cappelli, R. Crocchiolo, ated with lower aGVHD. The presence of KIR2DS2 gene in the E. Biral, A. Noè, I. Frugnoli, T. Roccia, C. Evangelio, M. Fossati, donor was also associated with a lower risk of mortality follow- A. Polselli, L. Barzizza, S. Rossini, F. Ciceri, P. Ronchi, ing allo-HSCT, resulting mainly from a decreased incidence of M.G. Roncarolo, K. Fleischhauer, S. Marktel grade II-IV aGVHD. Scientifi c Institute H S Raffaele (Milan, IT)

Between 2005 and 2008, 47 thalassemia patients from mid- P1004 dle east countries received a Bu-Cy based myeloablative Both naïve and memory lymphocyte subsets participate BMT in our unit. During the aplasia post BMT, we experi- in alloresponses to HLA mismatched stimulators – enced a high incidence of refractoriness to platelets (plt) implications for selective depletion of GVHD reactivity transfusions (trx). This is explained by the presence of anti J. Barrett, M. Smith, Z. McIver, N. Hensel, J. Melenhorst HLA antibodies (abs) in patients transfused in countries National Institutes of Health (Bethesda, US) using non leukodepleted blood units. Based on this, we set up a system (HLA specifi c trx) to identify the best plt donor by Since lymphocytes do not normally encounter cells from unre- avoiding HLA antigens against which the patient had specifi c lated individuals, it is presumed that alloresponses causing anti-HLA-abs. GVHD are mainly derived from naïve T cells. However, memory Patient characteristics: median age 8 years (range 2-17), 22 in cells may also participate in the alloresponse, following sensi- Pesaro transplant risk class II, 25 in class III. Median number tization through pregnancy, or through cross-reactivity, against (MN) of blood trx pre BMT for class II: 42 (18-225), class III: viral antigens. We used fl ow cytometry to track allorespond- 101 (6-230). Plt refractoriness to trx defi ned as corrected count ing T cell activation using CD38, and proliferation by CFSE increment < 4,5 at 24 h post trx calculated as body surface*(post dilution. Umbilical cord mononuclear cells (UCMC) from fi ve plt trx– pre plt trx)/number plt in transfused unit. donors were stimulated with an irradiated pool of HLA-dispa- All patients in class III were positive for anti HLA antibodies (MN rate donor peripheral blood mononuclear cells (alloPBMC). By 26, range: 9-54), whereas only 16/22 in class II (MN 18, range: day 8, over 80% of the cells were CD38+CFSE dim, indicat- 8-40). Trx of random plt to immunized patients resulted in 74% ing naïve T cells were alloresponsive. Next, PBMC from adult refractoriness compared to 25% in non immunized (p< 0.0001). donors were negatively immunomagnetically sorted into sub- Among immunized patients, splenectomized patients had a sets, using CD57+ and CD45RO+ for naïve (N) cells, CD45RA+ better plt trx outcome to random units (p < 0.01). In addition, and CD57+ for central memory (CM) cells, CD62L and CD57+ patients with more then 26 anti-HLA-abs had a higher number for effector-memory (EM) cells, and CD27+ and CD45RO+ for of failed plt trx (82%) compared to patients with < 26 abs (64%), effector (E) cells. Adult N, CM and EM cells – and to a lesser p< 0.01. When HLA specifi c trx were employed (in immunized extent, E cells – proliferated and acquired CD38 expression patients only), 68% trx were successful, compared to 26% with comparably to naïve T cells, indicating that all subsets can be random trx (p < 0.0001). When a plt donor negative for anti- alloresponsive. Electronically sorted T cell subsets, stimulated gens against which the patient had specifi c abs was not identi- with single donor alloPBMC confi rmed that CM + EM CD4+ and fi ed, 1 antigen mismatch was tolerated. Analysing trx outcome CD8+ T cells make proliferative alloresponses. PBMC subsets depending on the degree of HLA match and AB0 compatibility, from 3 adults were then primed for 8 days with alloPBMC and we documented that full HLA match is probably better then 1 tested for intracellular cytokine (IFNg; TNFa; IL-2) production antigen mismatch (p=0.08) and that AB0 compatible units are after rechallenge with the pool. CD4+ and CD8+ N, CM + EM probably better then incompatible (p < 0.4) and the best result cells (but predominantly CM cells) generated cytokines after is obtained when HLA matched and AB0 compatible plt were exposure to alloPBMC. While showing a proliferative response used (p< 0.07).

S304 In conclusion, we documented a high incidence of anti-HLA- Abs in middle east thalassemia patients. In this setting, the use of HLA specifi c trx following BMT resulted in improved transfu- sion outcome, limitation of costs, fl uid overload and exposure to multiple donors. We suggest that this method is more effective and worth cost wise.

P1006 HLA-DR15 and outcome of unrelated donor haematopoietic stem cell transplantation– An IHWG analysis M. Stern, A. Gratwohl, M. Malkki, Y. Morishima, S. Spellman, T. Gooley, E. Petersdorf on behalf of the International Histocompatibility Working Group in Hematopoietic Cell Transplantation

HLA-DR15 is associated with autoimmune bone marrow failure syndromes such as aplastic anemia, and in this setting predicts response to immunosuppressive treatment. A particular sus- ceptibility of DR15 expressing hematopoietic cells to autoim- mune attack has therefore been suggested. We hypothesized that DR15 may also play a role in allo-immune effects after allogeneic hematopoietic stem cell transplantation (HSCT). In a datafi le of 7521 10/10 matched unrelated donor transplants contributed to the International Histocompatibility Working P1007 Group (IHWG) in Hematopoietic Cell Transplantation (HCT) Soluble CD30 following allogeneic haematopoietic stem we investigated rates of survival, disease relapse and acute cell transplantation: an immunologic marker for an GvHD in patients with or without DR15. Transplants facilitated immunologic reaction? by the Japan Marrow Donor Programme (JMDP, n=2569) were K. Hübel, B. Cremer, E. Heuser, M. Hallek, H.P. Hansen analyzed separately from non-JMDP (n=4952) transplants due University of Cologne (Cologne, DE) to differences in the frequencies of the DR*1501 and 1502 alle- les. DR15 was present in 46% of JMDP and 34% of non-JMDP CD30, a member of the TNF receptor family, is preferentially transplants. Effects were analyzed for DR15, as well as for its expressed by activated or memory Th2 cells, regulatory T cells most frequent alleles, DR*1501 (more prevalent in non-JMDP and NK cells but not by resting B cells or T cells. In the trans- patients) and DR*1502 (more prevalent in JMDP patients). plant setting, CD30 plays an important role for the balance DR15+ and DR15- patients were well matched for disease between Th1/Th2 immune responses, modulating immune stage, age at transplant, rates of T-cell depletion, matching of responses and tolerance induction. CD30 is cleaved by met- HLA-DP, and donor/recipient gender. No statistically signifi cant alloproteinases and the ectodomain, soluble CD30 (sCD30), effects of the presence of DR15 were observed for survival, is released. In organ transplantation (heart, liver, kidney) sev- rates of relapse, incidence and severity of acute GvHD, with eral investigators documented the relevance of sCD30 in the the exception of a small reduction in rates of grade II-IV aGvHD peripheral blood as a predictive marker for allograft rejection. in DR15+ patients of borderline statistical signifi cance (hazard In fact, patients with high levels of sCD30 had a reduced graft ratio 0.89, 95% confi dence interval 0.79-0.99, p=0.03, see Fig- survival. ure 1 with univariate survival and cumulative incidence curves Considering these results, a signifi cant role of sCD30 also in and Table 1 with adjusted hazard ratios). hematopoietic stem cell transplantation could be assumed. In In conclusion, in a large population of well-matched unrelated our project we investigate sCD30 levels at different time points donor transplants, the presence of HLA-DR15 did not impact (day -1, day of engraftment, day 30, day 100, and in case of survival, risk of disease relapse, or acute GvHD incidence and graft-versus-host-disease (GvHD) development) in patients severity. These data suggest that DR15 does not appear to undergoing allogeneic stem cell transplantation. The primary confer a special susceptibility to allo-immune effects in the set- goal of this trial is the evaluation of possible correlations ting of matched unrelated donor allogeneic HSCT. between sCD30 levels and immunologic reactions defi ned as GvHD or graft-versus-leukaemia effect. At this time, a total of 10 patients have been observed until day 100 (AML: 4 pts; ALL: 2 pts; CML: 2 pts; CLL: 1 pt; T-NHL: 1 pt). Several fi ndings emerge from this investigation: 1.) In all patients, levels of sCD30 at baseline (day -1) were low (mean 24,9 U ± 9,44 U (SEM). 2.) There were 4 patients with no signs of GvHD until day 100. In these patients, no signifi cant changes in sCD30 levels were found. 3.) Mild to moderate GvHD which responded well to therapy did not show increased sCD30 levels. 4.) Two patients experienced high sCD30 levels. One patient developed severe lethal GvHD (CD30, min: 10 U; max: 134 U); the other patient had continuous moderate GvHD (CD30, min: 60 U; max: 145 U). 5.) One patient with mild GvHD showed elevated sCD30 levels in the relapse situation (CD30, min 10 U; max 70 U). In conclusion, these results suggest a critical role of sCD30 as predictive marker for immunologic reactions after allogeneic stem cell transplantation.

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