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Physicians Poster Abstracts

EBMT 2007

Acute leukaemia P411 The development of chronic GvHD is the most important factor in the outcome of reduced-intensity conditioning HLA identical sibling allogeneic transplantation P410 in patients with acute myeloid leukaemia or Persistent centre effect but improvement of leukaemia- myelodysplastic syndrome free survival of HLA identical haematopoietic stem cell D. Valcarcel (1), R. Martino (1), J. Martin (2), C. Ferrara (3), A. transplantation for adults with AML in first CR in Europe Sampol (4), J.B. Nieto (5), D. Carrera (6), J.L. Piñana (1), from 1987-2005 an analysis on behalf of the ALWP of M.D. Caballero (2), J.M. Ribera (3), J. Besalduch (4), J.M. EBMT Moraleda (5), M.T. Bernal (6), J.F. San Miguel (2), J. Sierra S. Katsahian (1), M. Labopin (1), F. Frassoni (2), V. Rocha (1) (1), S. Chevret (1) (1)H. Santa Creu i Sant Pau (Barcelona, E); (2)H. (1)AP-HP (Paris, F); (2)Ospedale San Martino (Genoa, I) Universitario (Salamanca, E); (3)H. Trias i Pujol (Badalona, E); (4)H. Son Dureta (Palma de Mallorca, E); (5)H. Morales We have shown that centre effect was an important factor Meseguer (Murcia, E); (6)H. Universitario Central de Asturias associated with outcomes for HLA identical sibling (Oviedo, E) hematopoietic stem cell marrow transplantation (HSCT) for adults with AML in first CR during the period 1987 to 1995 in Background: The use of reduced intensity conditioning European transplant centres (F Frassoni et al, Lancet 2000). regimen (RIC) reduces non-relapse mortality (NRM) allowing In order to determine the persistence or not of this centre long term disease free survival (DFS) and overall survival effect after 1995, we have analysed the outcomes of HSCT in (OS) through versus (GVL) effect in patients the same population (adults with AML in CR1 given an HLA considered not eligible for convenitonal Allogeneic HSCT identical HSCT), performed from 1996 to 2005 in the same (Allo-SCT). In this multicenter, prospective study we report our transplant centres. Thus, two cohort of patients transplanted in experience with RIC regimen based on fludarabine and the same 13 EBMT centres were analyzed: 1) 515 patients busulfan prior to allo-SCT as treatment of AML/MDS. (aged 16–54 years), transplanted between 1987 and 1995 Patients and methods: We included all 93 consecutive and 2) 455 patients transplanted (aged 16–55 years) between patients with AML and MDS who underwent RIC-Allo-SCT 1996 and 2005. Transplant related mortality (TRM), relapse from an HLA identical sibling, in 6 Spanish centers since 1998 incidence(RI) and leukaemia free survival (LFS) were to 2005. There were 59 AML and 34 MDS. Median age was analysed in regression models taking into account centre 58 (range 21-70)years. Follow up for survivors was 43 (3-89) effect adjusted for relevant covariates, such as age, WBC, months. Conditioning regimen consisted of fludarabine 150 FAB classification, time from CR1 to transplant, source of mg/m² and busulfan 8-10 mg/kg. GVHD prophylaxis consisted stem cells, T-cell depletion, female donor to male recipient. of cyclosporin A and a short course of methotrexate or Results: outcomes estimated at 5 year are listed in the table mycofenolate mofetil. below in cohort one and two. Range [minimum and maximum] Results: The patients received a RIC regimen for advanced of each outcome are reported according to the transplant age ± comorbidities (83%) previous SCT ± advanced age centres. (9%) or active infection, poor PS, or other comorbidities (8%). In conclusion, these findings show that LFS of adults given an 30% of patients were in advanced phase at SCT. Cumulative HLA identical sibling HSCT with AML in CR1 have improved Incicidence (Cu In) of acute and chronic GVHD was 33% over time in Europe. In addition significant centre-specific (95% CI 25-44) and 54% (95% CI 44-65%) respectively. The variation in the success of bone transplantation in Europe is 100-day and 1-year Cu In NRM was 8% and 17% observed. Further studies are needed to elucidate the causes respectively; grade II-IV acute GVHD was de most important of this variation, with the goal of developing strategies to variable associated with NRM HR (3.7 (95% CI 1.4-13) minimize the centre effect and ensure the best possible P=0.005) in multivariate analyses (MA). Relapse Cu In was outcomes for all transplant recipients. 37% (95% CI 28-50%); the absence of chronic GVHD (HR:6.5 (95%CI:3.1-13.8) P<0.001) and an advanced disease status (HR 2.2 (1.1-4.4) P=0.03) were related with a higher risk of relapse in MA. The 4-years DFS was 43% (33-53%); the absence of chronic GVHD (HR: 5.3 (2.4- 13.8) P<0.001) and an advanced disease status (HR 2.6 (1.2-5.4) P=0.01) showed worse DFS in MA. The 4-year probability of OS was 45% (95% CI 33-57%) for the whole group. OS for AML and MDS was 42% (95% CI 28-56%) and 49% (95% CI 31-76%) (P=0.4). The absence of chronic GVHD was the only factor associated with worse OS HR 6.1 (95% CI: 3.3-11.3) P<0.001 in MA. Conclusion:Our results supports the use of our RIC as a curative option for those patients with high risk NRM for conventional allo-SCT but who can benefit from GVL effect. The development of chronic GVHD was associated with better outcome (lower relapse rate and better DFS and OS)

S56 P412 by real-time quantitative polymerase chain reaction Use of high-dose melphalan for induction of aplasia (sensitivity, 10-5). before allogeneic stem cell transplantation in patients Results: At the time of enrollment (after induction with refractory acute myeloid leukaemia ), 41 patients (80.4%) achieved complete N.K. Steckel, R. Trenschel, M. Ditschkowski, H. Ottinger, M. remission (CR). After the first imatinib cycle, 39 of the 41 Koldehoff, A.H. Elmaagacli, C. Schulte, D. Beelen patients in CR remained in sustained first CR, while the other Universityhospital Essen (Essen, D) 2 patients relapsed. In the remaining 10 refractory patients, 7 achieved a new CR after the first imatinib cycle. The kinetics Patients (pts.) with refractory acute myeloid leukaemia (AML) of MRD correlated well with the patients' clinical course. after multiple chemotherapy cycles have a particular poor Overall, the BCR-ABL/ABL ratios were decreased in 45 prognosis. In this prospective study, 20 pts. (median age 55 patients (88.2%), which included 18 (35.3%) molecular CR years, range 19-67 years) with detection of blasts > 20 % in after the imatinib interim therapy. A total of 50 patients bone marrow aspirates before scheduled transplantation have underwent allogeneic SCT from matched sibling (n=33) or been enrolled for a sequential high-dose Melphalan therapy unrelated (n=17) donors, and among them, 46 (86.2%) followed by allogeneic stem cell transplantation after received transplants in first CR. With a median follow-up myeloablative conditioning. So far, 17 pts. have undergone duration of 40 months (range, 6+ to 67+ months) after SCT, the sequential therapy, while 3 pts. are still under treatment. the actuarial 3-year relapse, nonrelapse mortality, disease- Fourteen pts. received 140 mg/m² melphalan and 6 pts. 200 free survival, and overall survival rates were 17.1%±6.1%, mg/m². The median interval between melphalan and start of 15.9%±6.2%, 69.1%±7.4%, and 72.5%±7.4%, respectively. the conditioning regimen was 11 days (range 6-24 days). Multivariate analysis showed that an MRD level of greater Blast clearance was checked by bone marrow aspiration prior than 10³ after the first imatinib cycle was the most powerful to start of conditioning regimen. Eleven patients underwent a predictor of relapse (39.9%±14.8% versus 4.8%±4.6%, conditioning regimen with (TBI) and p=0.024, RR=12.0) and disease-free survival (39.1%±13.1% fludarabin and 6 pts. with treosulfan and fludarabin. Six pts. versus 86.7%±7.3%, p=0.007, RR=8.9). were transplanted with an identical sibling donor, 2 with a Conclusion: In the setting of allogeneic SCT after front-line haploidentical familiar donor, and 9 pts. with matched imatinib interim therapy, prospective MRD monitoring may unrelated donors. The acute toxicity was as follows: allow us to identify subgroups of Ph+ ALL patients at high risk nephrotoxicity grade 0 n=8, grade I-II n=8, grade III-IV n=1, of relapse at an earlier stage of treatment. hepatotoxicity grade 0 n=5, grade I-II n= 10, grade III-IV n= 2, mucocitis grade 0 n=6, grade I-II n=4, grade III-IV n=7 After a median follow up of 130 days for surviving pts. after P414 transplant, the overall survival estimate at 2 years after Allogeneic stem cell transplantation in adults with transplant was 65 % (11/17 patients). The treatment related Philadelphia chromosome-negative acute lymphoblastic mortality was 23 %, four pts. died due to infectious leukaemia: risk factors determining outcome and complications (n=3) and acute graft versus host disease grade evidence of antileukaemic activity of chronic IV (n=1). Three pts. (18 %) developed a relapse up to 150 graft-versus-host disease days after transplant. Two of these three pts. (66 %) died due S. Lee (1), J.Y. Kwak (2), J.-A. Kim (1), K.-S. Eom (1), Y.-J. to the relapse, while 1 patient survives in remission. Kim (1), C.-K. Min (1), H.-J. Kim (1), S.-G. Cho (1), D.-W. Kim Our data indicate that high-dose melphalan can be used for (1), J.-W. Lee (1), W.-S. Min (1), C.-C. Kim (1) pts. with refractory acute myeloid leukaemia to induce marrow (1)The Catholic University of Korea (Seoul, KOR); (2)Chonbuk aplasia without detectable blasts. The acute toxicity of this National University Medical School (Chonju, KOR) sequential protocol is acceptable and it leads to a complete remission rate of 100 %. The relatively low relapse rate in Purpose: Data from recent studies using risk-based otherwise refractory pts is promising and deserves further approaches suggest that allogeneic stem cell transplantation study in a larger population of pts with refractoty leukaemia. (SCT) clearly benefits certain patients with high-risk acute Therefore, we are currently launching a multicenter study lymphoblastic leukemia (ALL), such as those with Philadelphia protocol using this sequential approach. chromosome (Ph)-positive ALL. In contrast, the role of allogeneic SCT for Ph-negative ALL remains unclear because interpretation of transplantation outcome is complicated by the P413 criteria used to select patients and by the relatively small Minimal residual disease level after first imatinib cycle number of patients studied. The aim of the present study was may help to identify subgroups of patients at high-risk of to evaluate risk factors affecting clinical outcome and the relapse: updated results of allogeneic SCT following graft-versus-leukemia (GVL) effect for 149 adults with Ph- front-line imatinib interim therapy in adults with negative ALL who were treated with allogeneic SCT during the Ph-positive ALL last 10 years (1995 to 2004). S. Lee (1), J.-Y. Kwak (2), J.-A. Kim (1), K.-S. Eom (1), Y.-J. Patients and methods: The study population was 149 Kim (1), C.-K. Min (1), H.-J. Kim (1), S.-G. Cho (1), D.-W. Kim consecutive adults receiving an allogeneic SCT from matched (1), J.-W. Lee (1), W.-S. Min (1), C.-C. Kim (1) sibling (n=109) or unrelated (n=40; 27 matched, 13 allele- (1)The Catholic University of Korea (Seoul, KOR); (2)Chonbuk mismatched) donors. Their median age was 27 years (range, National University Medical School (Chonju, KOR) 15-61 years). One hundred fourteen (76.5%) patients had high-risk criteria. One hundred seventeen (78.5%) were Purpose: Recently, we demonstrated the positive impact of transplanted in first complete remission (CR1); 12 (8.1%) in imatinib interim therapy on the curative potential of allogeneic CR2; and 20 (13.4%) were resistant to chemotherapy before stem cell transplantation (SCT) in adults with Philadelphia transplantation. Most patients (n=140, 94.0%) received a chromosome-positive acute lymphoblastic leukemia (Ph+ preparative treatment of total body irradiation (TBI)-containing ALL) (Blood 2003;102:3068 & Blood 2005;105:3449). regimen (TBI/cyclophosphamide for CR1, However, a proportion of patients continue to eventually die as TBI/cytarabine/melphalan for >CR1). Graft-versus-host a result of disease recurrence. The aim of this study was to disease (GVHD) prophylaxis was attempted by administering evaluate risk factors affecting transplantation outcome, mainly calcineurin inhibitor plus methotrexate. focusing on the prognostic relevance of minimal residual Results: With a median follow-up of 53 months (range, 18+ to disease (MRD) level. 130+ months), the 5-year probability of disease-free survival Patients and Methods: Between 2000 and 2005, 51 adults (DFS) was 51.3%±4.2% for all patients; 62.9%±4.6% for with Ph+ ALL who completed induction chemotherapy and patients in CR1; and 9.4%±5.2% for patients in >CR1 at intended to undergo imatinib interim therapy followed by transplantation. There was no difference in DFS for sibling allogeneic SCT were enrolled in this study. For MRD and unrelated transplant patients in CR1 (64.4%±5.3% versus assessment, patients' bone marrow samples were analyzed 58.8%±9.3%). Multivariate analysis showed that the most powerful predictive factor affecting relapse and DFS was

S57 disease status at the time of transplantation (CR1 versus conducted by the German Cooperative Transplant Study >CR1, p<0.001). The presence of chronic GVHD was also Group (GCTSG) to investigate the outcome after such an found to be significantly associated with favorable outcome approach. (p=0.001). Data on 44 pts. (24m, 20f, median age 49y, range 19-66) with Conclusion: Our data suggest that allogeneic SCT should be high-risk MDS (9), relapsed (16) or primary refractory AML performed in CR1 in adults with Ph-negative ALL. The (19) were reported by 3 centers. Pts. received aplasia- presence of chronic GVHD results in better DFS, indicating a inducing chemotherapy with the following regimens: clinically important GVL effect. Further studies to develop idarubicine / fludarabine / ara-C (16), daunorubicine / ara-C treatment strategies to reduce leukemic cell burden and to (15), fludarabine / ara-C / amsacrine (5), or other ara-C-based enhance GVL effect are needed. schedules (8). Subsequent transplant conditioning therapy consisted of melphalan (150mg/m²) plus fludarabine (150mg/m²) in n=28, TBI (8Gy) plus fludarabine (150mg/m²) in P415 n=11, and TBI (4Gy) plus cyclophosphamide (80–120mg/kg) Outcome of patients with acute lymphoblastic leukaemia in n=5 and was commenced after a median of 15 days (8-40) in first complete remission treated with allogeneic from the start of chemotherapy. Allogeneic peripheral blood transplantation: an analysis of intention-to-treat based (41) or bone marrow (3) stem cells from sibling (16), matched clinical trials unrelated donors (18) or mismatched donors (10) were B. Bartolozzi, C. Orsi, A. Messori, E. Tendi, S. Guidi, A. Bosi infused thereafter. Graft versus host disease (GVHD) Careggi Hospital (Florence, I) prophylaxis was performed with cyclosporin A monotherapy (33) or by combination of a calcineurine inhibitor and Allogeneic transplantation in patients with acute lymphoblastic methotrexate (11), n=31 received additional anti-thymocyte leukaemia (ALL) in first complete remission (CR1) has been globuline. studied in a limited number of clinical trials. However, no 43 pts. achieved stable engraftment and 41 showed >90% systematic analysis has yet been done to assess whether this donor chimerism after 28-90 days. Grade 2-3 acute GVHD treatment option improves overall or event-free survival and occurred in 34%, whereas limited or extensive chronic GVHD to determine the magnitude of the survival benefit if any. was seen in 26% of evaluable pts. 100-day mortality rate was We conducted a survival meta-analysis to compare allogeneic 24%: 5 pts. succumbed to treatment related complications, 4 transplantation vs. standard chemotherapy (or autologous to disease relapse. The median follow up is 200 days (19- transplantation) in patients with ALL in CR1 on the basis of an 1455). Actuarial survival rates (Kaplan-Meier analysis) are intention to treat approach. 56.6% and 29.5% at 6 and 12 months, whilst actuarial Our study included the controlled clinical trials published in progression free survival rates are 42% and 30.8% at 6 and English from 2000 to 2005. The literature search was mainly 12 months, respectively. based on MEDLINE. The event-free individual survival data This multi-center retrospective analysis suggests feasibility of were reconstructed on the basis of published information and the approach combining leukemia burden reduction by Kaplan-Meier graphs. standard AML induction chemotherapy followed by RIC-SCT We generated the meta-analytic event-free survival curves for performed in aplasia. Treatment-related complications were patients given allogeneic transplant and for controls (standard comparable to those of a conventional transplant setting. chemotherapy or autologous transplantation) and determined Prospective studies are needed to assess whether durable statistical significance using log-rank test and Cox analysis. remissions are achievable in patients with these otherwise After including 4 studies in our meta-analysis, total of 293 fatal conditions. patients for the allogeneic transplantation group and 479 for the control group were examined. Our results showed that the event-free survival difference between the 2 treatments was P417 statistically significant (chi-square by log-rank test = 6.54, p = Haematopoietic chimerism and minimal residual disease 0.011). The Cox analysis calculated a relative risk for event in patients with AML after stem cell transplantation with occurrence of 0.79 for the experimental group vs. the controls conventional versus treosulfan-based conditioning (95%CI: 0.66 to 0.96; p=0.017). Secondary analyses gave O. Blau, I.W. Blau, K. Rieger, C. Gentilini, K.A. Nogai, A. essentially the same result even in the case where we Müssig, I.S.N. Köhler, K. Freiberg, E. Thiel, L. Uharek included two more small-scale negative studies in which the Charité (Berlin, D) experimental arm included allogeneic transplants from related or unrelated donors. The mean survival gain was 1 year per In this study we analyzed 43 patients (24 male, 19 female) patient; the cost per life-year gained was less than the with AML who underwent related (17) and unrelated (26) conventional threshold of 50,000 Euros. allogeneic stem cell transplantation (SCT). Fifteen patients On the basis of our meta-analysis, allogeneic transplantation received standard myeloablative and 28 Treosulfan-based in patients with ALL in CR1 confers a statistically significant reduced intensity conditioning (RIC). The mean age was 53 event-free survival benefit as compared with standard (33-66) in RIC group and 38 (17-56) in patients with standard chemotherapy or autologous transplantation. Its cost- conditioning. Genetic analysis was performed in all patients at effectiveness profile is acceptable. diagnosis and all through different stages of the disease. To detect genetic aberrations we used G-banding, FISH, and PCR. For quantitative chimerism investigation we used P416 AmpFISTR® Identifier® PCR Amplification KIT (Applied Treatment of high-risk AML and MDS with ara-C- Biosystems) contains fluorescent-labeled primer pairs for containing standard chemotherapy followed by reduced- simultaneous amplification of 16 different loci each. We intensity conditioning allogeneic stem cell transplantation evaluated chimerism in bone marrow aspirate (BM) and in during aplasia: a survey of the German Cooperative CD34+ cells on days +28, +56, +100, +180, +360, and at end Transplant Study Group (GCTSG) of investigation. FISH with specific probes for sex J.B. Dengler, M. Stelljes, C. Theuser, C. Schmid, U. chromosomes were exercised after sex-mismatch SCT. The Hegenbart, J. Kienast, A.D. Ho, G. Ehninger, P. Dreger, M. data was compared with minimal residual disease (MRD). Bornhäuser on behalf of the German Cooperative Transplant Both standard and RIC regimens were well tolerated. All Study Group (GCTSG) patients engrafted. Early chimerism analysis (day +28) showed that all patients with standard conditioning and 69% of Prognosis of patients (pts.) with refractory or relapsed acute patients with RIC had complete chimerism (CC) in BM. myeloid leukaemia (AML) or advanced myelodysplasia (MDS) Interestingly, all patients after RIC SCT with CC at day +56 in is poor. Preliminary reports suggest that induction of aplasia BM did not develop relapse. By contrast, 70% of patients by a standard AML regimen followed by reduced intensity which had a relapse showed mixed chimerism (MC) on day conditioned stem cell transplantation (RIC-SCT) in aplasia +56 after RIC SCT. We suggest that the chimerism data at may be an effective strategy in this situation. A survey was day +56 is important factor for relapse in patients after RIC

S58 SCT. We observed a trend to a higher level of chimerism on P419 days +28; +56, and +100 in patients after standard Successful interim therapy with imatinib prior to conditioning versus RIC. It is necessary to note that the allogeneic stem cell transplantation in Philadelphia relapse rate was significant above in RIC patients (39%) than chromosome-positive acute myeloid leukaemia in patients with standard conditioning (19%) (log rank, W.S. Min, B.S. Cho, H.J. Kim, S.Y. Kim, K.S. Eom, J.W. Lee, p<0,05). Quantitative chimerism analysis in CD34+ cells was C.C. Kim more sensitive to detect relapse in the early terms. MRD Catholic University of Korea (Seoul, KOR) assessment from days +28 till day +100 after SCT allowed identification of patients at risk of relapse. After a median Objectives: t(9;22)(q34;q11) in de novo AML is very rarely follow-up of 556 days, 28 (65%) out of the 43 patients remain found in only 1-2% of newly diagnosed patients. Ph+ AML alive, 26 (60%) patients keep complete remission and CC. In represents a clinically distinct entity with poor outcome and conclusion, simultaneous studies of both chimerism and MRD has been considered at high risk of treatment failure or early are a useful tool in order to predict risk of relapse in patients relapse with standard chemotherapy. undergoing SCT and so can be helpful for individualizing Methods: Imatinib (Glivec, STI571) has been successfully treatment strategies after transplant. used in CML and Ph+ acute lymphoblastic leukemia (ALL). We applied the concept of the imatinib interim therapy for newly diagnosed Ph+ AML and monitored the status of P418 minimal residual disease (MRD) by real-time quantitative Sustained effect of first-line sequential therapy with polymerase chain reaction (RQ-PCR). We performed 8 cases intensive consolidation chemotherapy and allogeneic of hematopoietic stem cell transplantations (HSCT) so far. In haematopoietic stem cell transplantation after all cases, the disease status before a first imatinib cycle was reduced-intensity conditioning for patients with complete remission (CR). Subsequently, all cases received AML in first complete remission 1~2 cycles of consolidation chemotherapy followed by a D. Blaise (1), R. Tabrizi (2), C. Faucher (1), M. Mohty (1), J.O. second imatinib cycle bridging the time to HSCT. The Bay (3), J.M. Boiron (2), G. Marit (2), S. Furst (1), A.M. Stoppa preparative regimen consisted of total body irradiation (1320 (1), A. Charbonnier (1), T. Prebet (1), C. Chabannon (1), N. cGy) and cyclophosphamide (120 mg/kg). Cyclosporin or Milpied (2), N. Vey (1) tacrolimus plus methotrexate were used to prevent graft- (1)Institut Paoli-Calmettes (Marseille, F); (2)CHU de Bordeaux versus host disease. For MRD monitoring, all cases were (Bordeaux, F); (3)Centre Jean Perrin (Clermont-Ferrand, F) analyzed by RQ-PCR and the samples were collected at diagnosis, before and after imatinib therapy, and then at 21- We have previously shown that RIC-based ASCT can be days and every 3-month after HSCT. safely used in AML pts after intensive consolidation Results: After the first imatinib cycle, all patients remained at chemotherapy. In the current update report, we asked whether least in sustained CR and the BCR-ABL transcript decreased leukemic control was maintained after a longer follow-up. compared to the level of post-induction period with a variable 37 pts (age:51 (range,26-60)) with high risk clinical degree. All patients received the second imatinib cycle characteristics (n=26;70%) (Age>50 (N=22,59%); associated following consolidation chemotherapy or during pre-transplant severe comorbidity (N=10;30%)) and/or poor risk leukemic period. After the second imatinib cycle, the BCR-ABL features (n=24;65%; poor Cytogenetics (N=13,35%); failure of transcript rapidly decreased to an undetectable status in most first induction (n=10,27%); secondary leukemia (n=4;11%), cases. Finally, all patients underwent HSCT in a favourable high WBC count (n=5;14%) or partial remission (n=1,3%)) status and are alive in a leukemia free status in the range of were included. After CR1, all patients received a low dose 3~42 months after HSCT. Thus, it should be considered to cytarabine consolidation chemotherapy followed one month undergo allogeneic HSCT, considering the poor prognosis of later by one course of high dose cytarabine (24 g/m²) and Ph+ AML and the possibility of resistance to imatinib as in anthracycline (HIDAC). Pts were then scheduled to receive CML and Ph+ ALL. RIC ASCT (fluda.,180 mg/m²; Bu, 8 mg/kg; Thymoglobulin, Conclusion: The present study is the first report that 2.5 to 10 mg/kg) followed with BMT (n=10;28%) or PBSCT successfully incorporates imatinib into standard chemotherapy (n=26;72%). However after treating the first pts, it appears as an alternative for bridging to HSCT in Ph+ AML. As a first- that this schedule was not associated with prohibitive toxicity. line imatinib interim therapy, it appears to be a useful strategy Thus, all following pts were proposed to receive one month to bridge the time to HSCT for patients with Ph+ AML. Further after HIDAC, one course of melphalan (140 mg/m²) (HDMEL) follow-up with a sizable population is needed to define the with auto-SCT that preceded the RIC ASCT. Overall, 21 pts impact of imatinib on the long-term outcome of transplantation received HIDAC only and 16 HIDAC and HDMEL. Median FU for AML. is 3 y. (16-70 m.). 15 pts experienced aGVHD (Grade 1:7;Grade 2:4;Grade 3-4:4). The CI of grade 2-4 aGVHD was 22% (9-35). 33 pts (90%) were evaluable for cGVHD: 10 and P420 14 pts presented a limited and extensive form respectively. Conditioning with 8-Gy total body irradiation for The CI of cGVHD was 65 % (50-80). Three deaths were allogeneic haematopoietic stem cell transplantation in attributed to non-relapse causes (aGVHD:1;cGVHD:2;CI of acute myeloid leukaemia non-relapse death (NRD):8% (95%CI: 0-17). In all, 8 pts A. Ferrant, E. Van Den Neste, M.-C. Vekemans, X. Poiré, S. relapsed at 5 m. (2-19) (CI:22%;(95%CI,9-35). Relapse was Costantini, F. Richard, V. Gregoire associated with the abence of cGVHD (cGVHD:4% (4-12), no Cliniques Universitaires Saint-Luc (Brussels, B) cGVHD 44% (12-76), P=0.02), and at a lesser extent with the intensity of prior consolidation chemotherapy (HIDAC:33% Seventy-eight patients with acute myeloid leukemia (AML) (13-53); HIDAC+AUTO;6% (0-19%), P=0.06). 26 pts are still received a single dose of 8-Gy total body irradiation (TBI) and alive in CR1 for an overall survival and leukemia-free survival cyclophosphamide for conditioning. By reducing the dose of (LFS) probability estimates at 4 years of 67 % (95%CI, 49- TBI, the intention was to minimize toxicity while keeping 81%) and 68% (95%CI, 50-81%) respectively. When antileukemic activity. restricting the analysis to the 33 pts evaluable for cGVHD, Patients and methods: TBI was delivered over 6 h, with a cGVHD was the only independent risk factor positively mean dose rate of 2.2 cGy/min. The patients received bone influencing LFS (cGVHD:83% (59-74); no cGVHD (56% (27- marrow (n=51) or mobilized peripheral blood stem cells (n=27) 81), P=0.03). We conclude that RIC ASCT preceded by from siblings (n=67) or from an alternative donor (n=11). appropriate prior intensive chemotherapy may offer a Forty-nine patients had a transplant in first remission (CR), 15 relatively low NRD while exerting a sustained leukemia control in second or later remission, and 14 had untreated relapse or even in high risk patients. refractory disease. Ten patients had a good prognosis karyotype, 40 had a normal diploid karyotype, and 25 had poor prognosis cytogenetics. No mitoses could be obtained in 3 patients.

S59 Results: With a median follow-up of 10.7 years in surviving BMT). So far, this trial seems to be tolerable in terms of patients, probabilities of overall survival (OS) for patients who toxicities, during induction and post remission therapies. received a transplant in first CR, second/later CR, or non-CR, Among IPG, auto PBPCT arm had tendency of superior were 75%, 45%, and 12% respectively.The cumulative median survival in terms of OS and LFS without statistical relapse incidences (RI) were 24%, 27% and 93% respectively. differences(HDAC:8m, auto-PBPCT:12m, allograft:8m). This Relapse-free survival (RFS) rates were 68%, 35%, and 7% intention to treat trial, which started in Jan, 2000, has been respectively. The cumulative incidence of nonrelapse mortality going on until now in order to have better statistical power for (NRM) was 8% for the whole patient group. the subset analysis. Cumulative incidence of aGvHD II-IV was 21%. For extensive cGvHD, the cumulative incidence was 9%. Only status at transplantation predicted for OS, for RFS, for P422 RI, and for NRM (logrank, p<0.001 for all). A second Impact of allogeneic HSC transplantation in normal malignancy occurred in 7 patients (skin 3, breast 1, soft palate karyotype adult acute myeloid leukaemia and correlations 1, myelodysplastic syndrome 1, prostate 1), with a cumulative with multidrug resistance protein expression incidence of 7%. All of these patients are alive. D. Damiani (1), M. Tiribelli (1), A. Geromin (1), A. Michelutti Conclusions: Outcome data indicate that allogeneic HSCT (1), D. Russo (2), C. Filì (2), R. Fanin (1) with 8-Gy conditioning is feasible with low NRM and preserved (1)Division of Hematology and BMT (Udine, I); (2)Unit of antileukemic activity in AML patients in first or later CR. The Blood Diseases and C.T. (Brescia, I) incidence of second malignancies remains high. Background: Patients with acute myeloid leukemia (AML) and normal karyotype represent about 40-50% of all AML cases. P421 Despite being included in an “intermediate” risk group, they The intention to treat analysis of the different post- display an heterogeneous clinical course. Thus, post-induction remission therapy modalities in AML patients with the therapy, including the role of allogeneic hematopoietic stem Intermediate Risk Group based on cytogenetics cells transplantation (HSCT) is still under debate in these Y.C. Mun (1), S.M. Lee (1), S.H. Park (2), E.K. Cho (2), J.H. patients. Multidrug resistance (MDR) proteins over-expression Lee (2), D.Y. Jo (3), I.H. Kim (4), S.S. Yoon (4), S.Y. Park (4), is associated with worse prognosis in acute , and S.M. Bang (4), Y.J. Min (5), J.J. Seo (5), M.H. Lee (6), C.S. we have found that P-glycoprotein (PGP) positivity is related Kim (6), Y.D. Joo (7), S.Y. Chung (8), D.Y. Oh (8), D.Y. Jang with a reduced overall survival (OS) also in cytogenetically (9), K.H. Lee (10), M.S. Hyun (10), H.S. Song (11), H.S. Kim normal (CN) AML patients. (11), K.Y. Kwon (11), H.C. Kwon (12), H.J. Kim (12), Y.H. Objectives: We analyzed the outcome of allogeneic HSCT in Park (13), M.J. Ahn (13), J.S. Ahn (13), K.T. Park (13), S.H. 51 patients with CN AML, correlating the outcome of Bae (14), H.M. Ryoo (14), H.J. Yoon (15), B.S. Kim (16), T. transplant with PGP expression at diagnosis. We also Ghim (17), C.M. Seong (1) compared the survival in the transplanted patients with 98 (1)Ewha Womans University (Seoul, KOR); (2)Gachon cases of CN AML who did not received allogeneic HSCT. Medical School (Inchon, KOR); (3)Chungnam University (Dae- Methods: Fifty-one CN AML patients who were considered at Jeon, KOR); (4)Seoul National University (Seoul, KOR); high risk and had an HLA identical donor underwent to (5)University of Ulsan (Ulsan, KOR); (6)Inha University allogeneic HSCT at our Centre. High risk was defined (Inchon, KOR); (7)Inje University (Pusan, KOR); (8)Pochon according to initial clinical presentation (WBC >30x109/l, PGP CHA University (Pochon, KOR); (9)Hallym University (Seoul, MFI >6) or on the basis of a poor response to induction KOR); (10)Yeungnam University (Daegu, KOR); therapy. PGP was over-expressed in 12 patients (24%). (11)Keimyung University (Daegu, KOR); (12)Dong-A Median age was 45 years (range: 16-69) and median time University (Pusan, KOR); (13)Sungkyunkwan University from diagnosis to HSCT was 7 months (range: 1-135). (Masan, KOR); (14)Catholic University of Daegu (Daegu, Twenty-eight patients (55%) had a sibling donor, 23 an KOR); (15)Kyung-Hee University (Seoul, KOR); (16)Seoul unrelated one. HSC source was bone marrow in 30 cases, Veterans Hospital (Seoul, KOR); (17)Korea Cancer Center peripheral blood in 21. Twenty-eight transplants were Hospital (Goyang, KOR) performed in first (n=23) or subsequent (n=5) remission. Conditioning regimen was myeloablative in 40 cases (78%), The result of cytogenetics is one of the most important non-myeloablative in 11. prognostic factors on the prognosis of AML. HDAC, auto Results: Transplant related mortality was 22% (11 cases). PBPCT and allogeneic BMT after post remission therapy were Acute GvHD (n=3) and infections (n=5) were the principal given as last consolidation therapy in Intermediate Risk Group causes of mortality. Median OS was 41 months; survival was (IPG) based on cytogenetics using MRC definition. We similar in the 12 PGP+ patients (38 months) and in the 39 studied complete remission, relapse, toxic death, DFS and PGP- cases (43 months) (p=0.50). The OS of the transplanted OS. Inclusion criteria were age<65, PS<3 with reasonable patients was significantly longer than in 98 CN AML patients functions, cardiac ejection fraction>50%, (33 PGP+ and 59 PGP-) who did not received HSCT (41 vs bilirubin<2.0mg/dl, creatinine<2mg/dl in de novo AML, 12 months, p<0.001). The advantage of transplantation was secondary AML and RAEB-T. The aims of this prospective evident both in the PGP+ (38 vs 6 months, p=0.005) and in intention to treat analysis was to compare the CR, recovery the PGP- (43 vs 12 months, p=0.007) cases. kinetics, DFS and OS in IPG based on cytogenetics with Conclusions: Allogeneic HSCT is an effective therapy in CN different consolidation treatment modalities. AML, especially in cases with high risk, such as over- Three plus seven(Idarubicin 12mg/m², D1-D3; Ara-C expression of PGP. Transplant overcomes the impact of PGP 100mg/m², D1-D7) were given to de novo AML, secondary on survival and may be therefore pursued in patients who AML and RAEB-T. Three times of post remission therapy display this high risk feature at diagnosis. including HDAC, or auto PBPCT followed by two times of post remission therapy was given to IPG. If HLA-identical sibling was available, then allo BMT was tried after 1st post- P423 remission therapy. Retrospective evaluation of allogeneic stem cell 194 patients were enrolled. Median follow-up was transplantation in elderly patients with acute myeloid 32.5months. Among them, 92.3% was de novo AML. Over all leukaemia compared with conventional chemotherapy remission rate after 1st induction(3+7) was 69.9%. Relapse H. Wolschke, T. Zabelina, W. Struss, N. Kröger, C. rate after induction was 40.5%. Grade 3 or 4 toxicity were Bokemeyer, A. Zander found in 26.1% during induction therapy. Median days for University Hamburg Eppendorf (Hamburg, D) ANC >500/µl and >20k/µl during induction were 22 days and 21 days respectively. Toxicities profiles including The median age of patients with AML, above all with abnormal mucositis hepatic, cardiac and bleeding episodes are similar karyotyp is > 50 years. The Question of our retrospective on 3 different therapy modalities(HDAC, auto PBPCT and allo evaluation in the university hospital of Hamburg was: profit

S60 elderly patients from an allogeneic stem cell transplantation situ hybridization) and RT-PCR. However, each method has after a reduced conditioning regimen compared with a limitations i.e. low sensitivity in karyotyping, uncertainty of conventional chemotherapy? which molecular probe to be used in FISH or RT PCR Patients and methods: 87 pts. with age > 55 years (41 pts. methods. Multiplex RT-PCR (MRT-PCR)TM allows with normal/good risk karyotyp) were treated with a simultaneous detection of 29 fusion genes, more than 80 conventional chemotherapy. 44 pts. underwent SCT after a breakpoints and splice variant. Our aim was to demonstrate reduced intensity conditioning (14 pts. with identical sibling whether a MRT-PCR system might be successfully used to donor, 30 pts. with unrelated donor). 19 pts. of this group with screen a large number of patients with acute leukemia and normal/good karyotyp. compare the result with that of chromosome studies. Results: 5 years overall survival in the SCT-group was 47%, in Design and method: Frozen bone marrow cells from patients, the chemotherapy group 17% (p=0.01). 5 years OS in patients who were diagnosed with acute leukemia at Ajou university with normal and good risk karyotyp was 58% in the SCT group hospital between September 1994 and May 2006, were used versus 20% in chemotherapy patients (p =0.07). In for MRT-PCR. In all samples with a known conventional transplanted patients with 1.complete remission was the 5 cytogenetic results, we performed MRT-PCR and compared years OS 61%, in the chemotherapy group 26%. with conventional cytogenetic study regarding the Conclusions: Our data demonstrate that patients with acute concordance rate and analyzed discordant cases regarding myeloid leukemia, who receive an allogeneic stem cell their types. transplantation have a better outcome, compared to standard Results: 132 samples (68 male and 64 female patients) were chemotherapy. SCT after reduced- intensity chemotherapy analyzed, and there were 99 AML, 25 ALL, 5 biphenotypic produce a long-term survival. Rate of relapse and refractory is leukemia, 2 Juvenile myelomonocytic leukemia, and 1 patient higher in the group of standard treatment. Elderly patients with of Hairy cell leukemia. We successfully obtained the mRNA acute myeloid leukemia profit from allogeneic haematopoetic from all frozen samples. In 30 cases, we identified gene cell transplantation after reduced-intensity conditioning. abnormalities with chromosome studies and most of them (24/30), the same abnormalities were detected with MRT-PCR method. P424 In 102 patients with normal karyotype by cytogenetic Feasibility of allogeneic stem cell transplantation in technique, we identified 30 translocations of clinical adults with acute lymphoblastic leukaemia in first relapse significance by MRT-PCR method. In these 30 discordant E. Tavernier-Tardy, J.M. Boiron, F. Huguet, K. Bradstock, N. cases, there were 8 cases with t(8;21), 5 cases with t(15;17), Vey, T. Kovacsovics, A. Delannoy, N. Fegueux, P. Fenaux, A. 4 cases with t(9;22), 4 cases with t(9;11), 2 cases with Stamatoullas-Bastard, O. Tournilhac, A. Buzyn, O. Reman, C. t(11;19) and 7 others [t(9;9), inv(16), t(10;11), t(1;19), t(4;11), Charrin, C. Boucheix, J. Gabert, V. Lheritier, J.P. Vernant, H. t(11,21), TAL 1d (40kb deletion)] Dombret, X. Thomas on behalf the GET LALA Group, The In 99 AML patients, We found MRT-PCR was comparable to Swiss Group for Clinical Cancer Research SAKK and the chromosome study for prognosis (MRT-PCR; p=0.013, Australasian Leukaemia and Lymphoma Group chromosome study; p=0.042) Conclusion: There were 72.7% concordance between In the LALA-94 trial, 54% (421/771) of adults with acute cytogenetic technique and MRT-PCR. Furthermore clinically lymphoblastic leukemia (ALL) experienced a first relapse. 355 significant translocations were detected by MRT-PCR in 30 of patients were given re-induction therapy with various 102 normal karyotype patients, indicating improved sensitivity chemotherapy regimens, 3 with autologous stem cell with MRT-PCR. Further investigations are needed to ascertain transplantation (SCT) from cells harvested in first complete the usefulness of MRT-PCR as the screening tool of leukemic remission (CR), 1 with donor lymphocyte infusions (DLI), and genetic abnormalities. 14 with geno- or pheno-identical allogeneic SCT. 187/421 patients (44%) achieved CR. The median disease-free survival (DFS) was 5.2 months with an estimated 5-year DFS P426 at 12%. After chemotherapy, patients in CR with a donor were Factors influencing the collection of peripheral blood systematically assigned to allogeneic SCT (61 patients). stem cells in acute myeloid leukaemia in first complete Allogeneic SCT was also proposed to refractory patients with remission a donor (24 patients). 111 patients had an identical sibling F. Ferrara, S. Palmieri, M. Annunziata, M. De Simone, F. donor identified during first line therapy and 9 at the time of Pollio, P. Correale, F.P. Tambaro, P. Morabito, C. Lopardo, A. relapse. Geno-identical allogeneic SCT was performed in 55 Viola, G. Mele patients, and 3 patients received DLI. 44 transplantations Cardarelli Hospital (Naples, I) were performed from an unrelated donor (of which 4 from cord blood). A higher survival rate was observed when SCT was Background: While several studies have investigated factors performed in second CR as compared to SCT performed at influencing rates and quantity of peripheral blood stem cell the time of relapse (p = 0.02) or after failure of chemotherapy (PBSC) mobilization in patients with non myeloid (p = 0.005). 3-year survival tended to be better with a pheno- malignancies, there are very few reports on low number of than with a geno-identical donor (31% vs 21%). Relapse patients concerning the efficiency of PBSC mobilization in incidence was 41% vs 64% at 3 years, while toxicity-related patients with acute myeloid leukemia (AML). We analyzed the mortality was 46% vs 37% at 1 year. Difficulties to achieve CR effects of different potentially influential variables on and to organize allogeneic SCT combined with a definitive mobilization of CD34 positive (CD34+) from a series of 160 advantage for SCT in terms of survival, questions on the consecutive patients with AML in first complete remission opportunity to transplant earlier in the evolution of adult ALL. (CR1). Patients and methods: Data were collected from a cohort of 160 consecutive patients with AML in CR1. The median age P425 was 52 years (14-78). There were 134 patients with de novo Application of multiplex reverse-transcriptase polymerase AML (84%) and 26 patients (26%) with secondary AML (s- chain reaction for identification of leukaemia-associated AML). According to MRC criteria, cytogenetic findings at gene abnormalities diagnosis were classified as favourable in 11%, intermediate H.W. Lee, J.I. Park, S.Y. Kang, J.S. Park, J.H. Jang, J.H. in 69%, and adverse in 20% of patients. Patients up to 60 Choi, H.C. Kim years (n=116, 72%) received anthracycline based induction Ajou University School of Medicine (Suwon, KOR) and consolidation, while those aged more than 60 (n=44, 28%) were treated in induction/consolidation with Puopose: The best prognostic predictor for acute leukemia is fludarabine/ARA-C given as continuous sequential infusion. known to be the findings of genetic abnormalities in leukemic The following variables were analyzed: age > or < 60 years, cells. Methods for detecting the genetic abnormalities include de novo vs. s-AML, anthracycline vs. fludarabine based chromosomal studies for karyotyping, FISH(Fluorescence in treatment, cytogenetics at diagnosis, presence of FLT3

S61 mutations (either ITD or D835 mutation), WBC at diagnosis (more or less than 50x106/l), number of courses (i.e. 1 vs 2) needed for achievement of CR. The above parameters were correlated to successful mobilization (defined as collection of > 2x106/l). Results: Overall, 136 (85%) patients had a successful mobilization of CD34+ cells (median 7,86x106/kg, range 2,6- 152,6) obtained with a median of 2 (range 1-4). Either univariate or multivariate analysis failed to demonstrate significant influence for any parameter which were considered into the study. In particular no effect was observed as median number of CD34+ cell collection, median number of apheresis, peak of SC in PB and median number of CD34+ cells collected per single apheresis were concerned. In addition, none of the above parameters was significantly related to hematopoietic reconstitution after autologous stem cell transplantation in terms of WBC and platelet recovery. Conclusion: We conclude that successful mobilization in AML is unpredictable by using the variables analyzed in our study. In particular, elderly age per se does not represent an unfavourable factor for mobilization.

P427 ID-Ara-C, Idarubicine and mylotarg Gemtuzumab ozogamicin as salvage treatment in advanced AML patients L. Cardarelli (1), S. Capria (1), S.M. Trisolini (1), G. Cimino P428 (1), D. Diverio (1), A. Guarini (1), M. Mancini (1), G. Martinelli High-dose melphalan in relapsed AML: update of an (2), R. Foà (1), G. Meloni (1) ongoing phase II study (1)Università "La Sapienza" (Rome, I); (2)Istituto Seragnoli H. Martin, G. Bug, J. Atta, S. Mousset, S.A.L. Klein, D. (Bologna, I) Hoelzer Clinical Hospital J.W. Goethe-University (Frankfurt, D) The management of relapsed and refractory acute myeloid leukemia (AML) patients (pts) remains a major problem. Background: Long-term survival in relapsed AML has been Whenever a second complete remission (CR) is attained, the reported to range between 10-15%. We recently introduced median duration of the second relapse-free interval (RFI) is high-dose melphalan and autologous PBSC to salvage generally considerably shorter than that of the first RFI at least patients with relapsed AML (Bug et al. Ann Hematol 2005; in pts not eligible for transplant procedures. The aims of our 84:748-54). study were to evaluate the feasibility and efficacy, of a salvage Objectives: (1) To improve second CR rate in relapsed AML; chemotherapy showed in Tab 1. Pts achieving CR were (2) to increase the proportion of relapsed AML patients scheduled to receive a consolidation course with the same achieving a consolidation with an allogeneic transplant; (3) to drug and schedule, followed by an allogenic transplantation. improve long-term survival in patients with relapsed AML. At our center 13 pts were treated according to this protocol; 11 Methods: AML patients in first relapse received salvage of the 13 pts were in first relapse, while 2 were refractory to therapy with 200 mg/m² melphalan and autologous PBSC, first line chemotherapy. The main characteristics of the pts are which had been cryopreserved in early CR1. Subsequently an shown in the table 2. The overall response rate was 85% (7 allogeneic donor was searched and a consolidating allogeneic CR, 2 CRp and 2 PR). Two pts, refractory to first line transplant scheduled within 2 – 3 months. chemotherapy, showed no response and died. Results: Eighteen consecutive patients with AML in first Extrahematological toxicity was mild: only 1 pt suffered from relapse who had cryopreserved autologous PBSC are grade 3 oral mucositis. No pt presented alopecia. Recovery evaluable. The median age was 48 (range 29-61) years. was evaluable in 9/13 pts. The median time required to attain Sixteen of 18 pts. (89%) achieved a second CR and 16/18 an absolute neutrophil count in excess of 0.5 x 109/L was 14 pts. (89%) achieved a consolidating allogeneic transplant in days (range 8-62). A sustained platelet count exceeding 50 x CR2 (n=14) or PR2 (n=2) from a related (n=3) or unrelated 109/L was reached after a median of 31 days (range 19-62). (n=13) donor within a median of 2,4 (1,9 - 4,5) months after Eight pts had fever (3 documented infections, 3 FUO and 2 HD-Mel. Treatment-related mortality was 0/18 after HD-Mel pneumonias). Empirical antifungal therapy was administered and 4/16 (25%) after the subsequent allograft; 4 patients died to 2 pts because of persistence of fever. Of the 9 patients in due to relapse and 10 patients are alive in CR with a follow-up second CR, 1 relapsed after consolidation and died, 1 of median 20 (range 6 – 66) months and a projected long-term underwent a sibling transplant and is alive in second CR 3 survival of 50%. months after SCT, 1 pt received an haploidentical transplant Conclusions: Salvage therapy using HD-Mel in relapsed AML and is alive in second CR, 1 pt, relapsed after a sibling is superior to any other reported salvage regimen, however transplant in first CR, is alive in third CR after an more patients need to be studied to confirm these results. extramedullary relapse, 1 pt is currently undergoing an allogenic transplant, 1 died after an unrelated donor transplant of transplant-related mortality, 2 pt died while waiting for an allogenic transplant, 1 due to CNS relapse and the other of hematological relapse and 1 pt developed an important aplasia after consolidation treatment. Two pts who achieved a PR relapsed rapidly, after consolidation therapy, and died. Despite the relatively small cohort of pts, the regimen utilized seems promising and feasible allowing to proceed to a SCT transplant in the majority of AML pts with relapsed or resistant disease. A longer follow-up and a prospective multicenter study is necessary to conclusively validate our data.

S62 P429 Methods: The material of the study - bone marrow from 79 Allogeneic haematopoietic stem cell transplantation after children with initial diagnosed ALL, 37 children with initial first relapse of acute myeloid leukaemia: a retrospective diagnosed AML and 7 controls (leukocytes peripheral blood analysis on behalf of the Northern Italy Leukaemia Group from healthy donors). We studied the quantities of several (NILG) experience proteins cyclin-dependent kinases (cdk2 and cdk4) and I. Cavattoni (1), E. Morello (1), M. Casini (1), T. Intermesoli cyclins (cyclin D, cyclin E), E2F1, pRb, caspase 3 and 8, Bclx, (2), E. Oldani (2), E. Borlenghi (3), G. Rossi (3), C. Minotto Bax, p53 by Western blotting. The rate of proliferating cells (4), E. Terruzzi (5), E. Pogliani (5), E. Audisio (6), F. Marmont was analyzed by flow cytometry, using nuclear cell (6), A. Rambaldi (2), R. Bassan (2), S. Cortelazzo (1) proliferation-associated (Ki67). The level of (1)Bolzano Central Hospital (Bolzano, I); (2)Ospedali Riuniti spontaneous apoptosis was estimated by flowcytometry as (Bergamo, I); (3)Spedali Civili (Brescia, I); (4)Ospedale Civile the number of cells after propidium iodide and annexin V (Noale, I); (5)Chair of Hematology (Monza, I); (6)Chair of staining. Hematology (Turin, I) Results: The levels of expression of cyclin D, cyclin E, cdk2, cdk4 and level spontaneous apoptosis was much higher of AML patients in first relapse have a poor prognosis and initial B-ALL in children in comparison with T-ALL, pro-B-ALL alloHSCT is the sole curative option. However, only a few pts and controls(P<0.05). The positive rates of proliferation achieve a long term survival after this procedure. Therefore, activity cell (Ki67) was much higher of initial T-ALL in children adequate prognostic criteria are required for selecting pts who in comparison with B-ALL. The levels of expression of cdk2, can benefit from this approach. cdk4 and level spontaneous apoptosis in complete remission The aim of this report was to assess prognostic factors ALL was higher than in relapse samples (p<0.05). The Ki67 affecting OS in a cohort of 42 AML pts who received alloHSCT and the levels of expression of Bax in cell in relapse was as salvage treatment following a risk-oriented frontline higher than in complete remission ALL samples (p<0.05). The chemotherapy. probability of 5-year DFS in patients with high levels of cyclin Forty-two out of 145 AML pts in first relapse after frontline D, cyclin E, cdk2, cdk4 and level spontaneous apoptosis was chemotherapy (NILG-AML01-00) received allograft as salvage 99% versus 77%, 76%, 82%, 81% and 79% in patients with therapy. They all expressed adverse prognostic factors such low levels of these proteins (p<0.05). The probability of 5-year as age >45yr (n=27) and high risk FAB subtypes M0,M6,M7, DFS in patients with low levels of Ki67 was 93% versus 61% biphenotypic leukemia or MDS-associated AML (n=9). Many in patients with high levels of these proteins (p<0.05). were defined at high risk at diagnosis (n=28), or had a relapse Conclusions: Expression rates of cyclin D, cyclin E, cdk2, free interval (RFI) <6 months (n=11). At time of transplantation cdk4, Ki67 and the level spontaneous apoptosis have clinical 10 did not received reinduction therapy (BM blasts <15%), and prognostic significance in childhood leukemia. The initial 27(64%) were in CR2 and 5(12%) in PR. The donor was high levels of cyclin D, E, cdk2, cdk4 and the level matched related for 19(45%), matched unrelated for 15(36%) spontaneous apoptosis in leukemic cells are associated with and haploidentical for 8 pts(19%). Stem cell source was PB in good prognosis of 5 years disease-free survival. The initial low 27, BM in 10 and CB in 5 pts. Thirty-one pts received levels of Ki67 in leukemic cells are associated with good myeloablative conditioning (MA), and 11(26%) a reduced prognosis of 5 years disease-free survival. intensity one (RIC). Univariate and multivariate analyses were performed to evaluate the correlation of several diagnosis/relapse/transplant-related risk factors with OS. P431 After a median follow up of 10 mos. (range 3-49) the projected Monitoring of cardiotoxicity in acute leukaemia patients 3-yr OS was 17% and 14 pts(33%) were in CCR2. Seventeen treated with anthracycline-based chemotherapy and pts relapsed at a median time of 6 mos. from transplant, haematopoietic cell transplantation 14/31(45%) conditioned with a MA and 3/11(27%) with a RIC. J.M. Horacek (1), L. Jebavy (1), M. Tichy (1), R. Pudil (2), P. Among the 15 pts transplanted with active disease, 12(80%) Zak (2), J. Maly (2) achieved CR2 after alloHSCT and 4(26%) are in CCR2. (1)School of Military Health Sciences (Hradec Kralove, CZ); Nineteen pts developed acute or chronic GVHD. The pts who (2)Charles University Hospital (Hradec Kralove, CZ) received a MA regimen died of treatment related toxicity (12%). Univariate analysis showed high risk AML, RFI <6 mo, Aim: Assessment of acute and chronic cardiotoxicity in ECOG-performance status (PS) >0 and unfavorable karyotype patients treated for acute leukemia with biochemical markers at diagnosis to be predictive for a poor outcome (p<0.05). By – N-terminal pro brain natriuretic peptide (NT-proBNP), Cox multivariate analysis only short RFI and PS maintained cardiac troponin T (cTnT), creatine kinase MB (CK-MB mass); an independent prognostic value (p=0.033 and 0.034, echocardiography (ECHO) and electrocardiography (ECG). respectively). Patients and methods: Twenty six adult acute leukemia Our report suggests that salvage allograft should be offered patients (mean age 46.2±12.4 years, 15 males) treated with preferentially to pts with a RFI >6 mos. and very good PS. RIC 2–6 cycles of anthracycline-based chemotherapy (CT), seems to be as effective and less toxic than MA conditioning followed by myeloablative preparative regimen (PR) and regimen. Prospective studies including a large number of hematopoietic cell transplantation (HCT) were studied. allotransplanted cases are required for confirming the role of Cardiac evaluation was performed at the baseline (before CT), RIC in AML salvage strategies. after first CT (cumulative ANT dose 136.3±28.3 mg/m²), after last CT (cumulative ANT dose 464.4.3±117.5 mg/m²) and circa 6 months after completion of CT (6 Mo after CT). P430 Results: The results are summarized in the Table. Significance of expression of cell cycle proteins and Six months after CT, NT-proBNP concentrations correlated parameters of apoptotic proteins in leukaemic cells in with systolic and diastolic LV dysfunction on ECHO – children with acute leukaemia (r=0,514; p<0.01) and (r=0,587; p<0.01). Decreased QRS T. Astrelina, E. Osipova, E. Vladimirskaya voltage on ECG correlated with systolic and diastolic LV Research Institute of Pediatric Hematology (Moscow, RUS) dysfunction on ECHO – (r=0.660; p<0.001) and (r=0,592; p<0.01). Objectives: The investigation of the cell cycle proteins and Conclusions: Our results demonstrate acute and chronic check points of apoptotic program may occur very useful in cardiotoxicity of anthracyclines in combination with PR and understanding the mechanisms of cell resistance to HCT. Clinical manifestation of cardiotoxicity in terms of chemotherapy the biological basis for the therapy of failure developed in 2 (7.7 %) patients. In asymptomatic ontological diseases. patients, abnormal cardiac findings (NT-proBNP elevations, Aims: to estimate the expression of several proteins involved diastolic LV dysfunction, QTc prolongation) represent in the cell cycle and the regulation of cell proliferation, subclinical cardiotoxicity, which indicates a risk for apoptosis in leukemic áells in cases in childhood ALL. development of heart failure and malignant ventricular arrhythmias. In regard of late anthracycline cardiotoxicity,

S63 further cardiology follow-up is warranted in all acute leukemia leukemia (AML). Several studies tried to transfer these survivors. positive experiences onto elderly patients. Due to extensive Supported by Research Project MO 0FVZ 0000 503. toxicity and therapy-related mortality those strategies failed to show any benefit for elderly patients (R.J. Mayer et al, NEJM, 1994,331(14):896; R.M. Stone et al, Blood,2001,98(3):548). Autologous and allogenic transplantation after high dose Busulfan (BU) have been shown to be very safe in patients (pts.) with chronic myeloid leukemia (E. Olavaria et al, BJH,2000,108:769). BU is an effective drug also in acute myeloid leukemia. Therefore we performed a pilot study in 18 elderly pts. with AML. Median age was 63 years (range 58- 67). High dose regimen consisted in oral BU 4x1mg/kg x 4 days followed by autologous stem cell (SC)transplantation three days later. SCs were harvested after two or three cycles of induction/consolidation chemotherapy with G-CSF support. P432 A median of 4.8 (1.1-12.3) CD34/kg progenitor cells were 5-azacitidine in combination with donor lymphocyte collected. Median duration to reach leucocytes >1000/µl and infusions for the treatment of patients with MDS or AML >20.000/µl was 12 and 15 days, respectively. relapsing after allogeneic stem cell transplantation We reported in 2003 that this approach is safe without any A. Czibere, T. Graef, I. Bruns, F. Zohren, U. Germing, R. toxic death (Bone Marrow Transpl ,31,Suppl.1,#640). Now we Haas, G. Kobbe report the follow-up of these 18 patients (pts.)with a median of University of Duesseldorf (Duesseldorf, D) 53,5 months for the surviving pts.. Seven out of 18 pts. (39%) and 6 out of 11 pts. with standard risk AML (54%), Purpose: Therapeutic options for patients with high-risk respectively, are still disease-free. In contrast, 6 out of 7 pts. myelodysplastic syndrome (MDS) or acute myelogenous with high-risk cytogenetics or secondary AML relapsed within leukemia (AML) who relapse after allogeneic stem cell a median of 12 months (3-49). All pts. in complete remission transplantation (SCT) are limited and prognosis is dismal. If have an excellent performance status. applicable, transfusion of donor lymphocytes (DLI) with or Conclusion: High dose BU with autologous SC support as without chemotherapy is the current standard therapy. But, in consolidation therapy is promising in elderly standard risk contrast to chronic myelogenous leukemia (CML), response AML pts. and should be compared prospectively with dose rates after sole DLI in patients with relapsing MDS or AML reduced allogeneic transplantation. Relapse free survival and after allogeneic SCT are poor. Patients & treatment: In an quality of life should be the objectives. intent-to-treat approach we treated 6 patients with high risk MDS or AML who relapsed after allogeneic SCT with 5-Aza plus DLI. Patients' median age was 47.5 years (range 32-71 P434 years). Before allogeneic SCT 4 patients had active disease, High-dose chemotherapy and autologous peripheral and 2 were in complete remission (CR). Two had family blood stem cell transplantation in HIV + patients with donors, 4 had unrelated donors. Median time for relapse after acute myeloid leukaemia receiving highly active SCT was day +99 (range day +84 to day +300). Once relapse antiretroviral therapy was diagnosed patients received 100mg/m² 5-Aza for five A. Re (1), C. Cattaneo (1), S. Casari (1), C. Almici (1), M. days via subcutaneous injection in two to four weeks intervals. Capucci (1), M. Micheletti (1), E. Borlenghi (1), C. Schiantarelli If practical, patients received 1x106 CD3+ cells/kg bodyweight (2), R. Verardi (1), P. Ferremi (1), G. Rossi (1) following the first course of 5-Aza, and in the absence of graft- (1)Spedali Civili di Brescia (Brescia, I); (2)Ospedale Niguarda versus host disease, this was followed by additional 5x106 (Milan, I) CD3+ cells/kg bodyweight after 3 months. Results: Five out of 6 patients responded to treatment with 5- Myelodysplastic changes are known complication of HIV, Aza and DLI. Three patients achieved a complete remission while acute myeloid leukaemia (AML) is slightly increased (CR), two a partial remission (PR) and one patient died early during HIV infection and only few cases have been reported. due to progressive disease. Two patients developed extensive Long-lasting CR are rare and optimal treatment has not been graft-versus host disease (GvHD), while, so far, four patients defined. We report our experience with of high dose did not show any signs of GvHD. Two of these three patients chemotherapy (CT) and peripheral blood stem cell (PBSC) achieving initial CR are alive, one in CR, one in PR, while one transplantation in 3 HIV + pts with AML receiving HAART. patient died due to CNS disease. One of the patients Patient 1. 45-year-old HIV+ on HAART with AML M4 (46XY) achieving a PR died due to progressive disease, and the other in Oct 2001; CD4:284/mmc. After 2 induction courses died after a second allogeneic SCT with progressive disease. (ida+ara-C;3+7), CR was obtained. After consolidation he Median survival of all patients was 125 days (range 39-397 received ara-C 1g/mq bid x4d (“A8”) + G-CSF 10 mcg/Kg with days). successful PBSC collection (CD34+ 4.5x106/Kg, CFU-GM 5.6, Conclusion: Overall, induction of CR after treatment with 5- BFU-E 2.2 and CFU-GEMM 0.2x105/Kg). In Jun 2002 Aza, and consolidation of CR via DLI followed the 5-Aza received high dose CT (THIO 10 mg/Kg + CY 50 mg/Kg x 2) treatment is promising. with stem cell rescue + G-CSF 5mcg/Kg from d1. No toxicities were seen and engrafment was prompt (N>500 d10; plt>20.000 d12). CR was confirmed. In July 2004 he P433 developed anemia and leuco-monocytosis and was diagnosed High-dose busulfan post-remission therapy in the elderly with CMML (complex cariotype). In Sep 2005 died by with acute myeloid leukaemia supported by autologous Pseudomonas sepsis. stem cell transplantation: long-term follow-up Patient 2. 33–year-old patient with AML M1 (47XY,+8) and H. Wandt (1), K. Schäfer-Eckart (1), W. E. Aulitzky (2), M. HIV detection in May 2004. CD4 count was 132/mmc. He was Bornhäuser (3), A. D. Ho (4), J. Kaesberger (5), N. (6), started on HAART and received induction therapy with N. Schmitz (7), M. Wilhelm (1) doxo+ara-C+VP-16 (3+7+5) and cytogenetic CR was (1)Klinikum Nürnberg Nord (Nuremberg, D); (2)RBK-Stuttgart achieved. After consolidation he received “A8” + G-CSF 10 (Stuttgart, D); (3)Uniklinikum Dresden (Dresden, D); mcg/Kg and PBSC collection (CD34+ 6.2 x 106/Kg, CFU-GM (4)University Heidelberg (Heidelberg, D); (5)Diakonie-Stuttgart 15.9, BFU-E 15.2, CFU-GEMM 1.6x105/Kg). In Dec 2004 he (Stuttgart, D); (6)Klinikum Cottbus (Cottbus, D); (7)St.Georg received high dose CT (THIO 10 mg/Kg + CY 50 mg/Kgx2) Hamburg (Hamburg, D) with PBSC rescue. No toxicities were seen and engrafment was prompt (N>500 d11;plt>20.000 d13). In Feb 2005 a BM Intensive consolidation stragegies proofed to be safe and biopsy showed CR and light myelodisplastic features (normal effective in patients below 60 years with acute myeloid

S64 cariotype). In Sep 2005 he developed acute cholecystitis, P436 refused treatment and died. Busulfan-melphalan regimen in autologous stem cell Patient 3. A 53-year-old HIV+ pt was diagnosed with AML transplantation for adult patients with acute myeloid M4Eo (46,XY,inv16) in Jun 2006. CD4 was 2048. In 2004 he leukaemia was treated with doxorubicin and HAART for visceral KS. M. Tozzi (1), G. Marotta (1), S. Sammassimo (1), P. Galieni HHV8 was negative by PCR on peripheral blood. He received (2), S. Falciani (2), C. Bigazzi (2), G. Buonfrate (1), F. Lauria ida+ara-C+VP-16 (3+7+5) and after consolidation obtained (1) cytogenetic CR. Then received “A8”+G-CSF 10 mcg/Kg but (1)Azienda Ospedaliera Universitaria Senese (Siena, I); mobilization failed. In Nov 2006 a BM biopsy showed CR. He (2)Ospedale Ascoli Piceno (Ascoli Piceno, I) is now receiving consolidation with high dose Ara-C. Intensive CT with HAART is feasible and allow to achieve CR Autologous stem cell transplantation (ASCT) improves the in HIV+ patients with AML; high dose CT with PBSC rescue survival of patients with acute myeloid leukemia (AML). In the as consolidation seems feasible and safe. However, caution is past, high-dose regimens utilized in ASCT were derived from needed and additional studies are required to confirm allogeneic setting. Since these regimens (TBI-Cy, BU-Cy, BU- feasibility and explore long-term outcome. VP-Cy) provide both cytoreduction and immunosuppression to facilitate the allogeneic engraftment, we evaluated the efficacy of a regimen that theorically gives the maximum therapy to P435 eradicate the disease. A modified beac-conditioning protocol: a safe and Between April 1997 and May 2006, 29 AML patients in effective therapy for high-risk ALL Complete Remission (CR) (16 females and 13 males; median O. Krieger, H. Kasparu, J. König, S. Machherndl-Spandl, O. age 52 years, range 17-70 with 9 patients > 60 years; 9 Zach, D. Lutz patients had FAB M1, 9 FAB M2, 8 FAB M4 and 3 had a Elisabethinen Hospital (Linz, A) secondary AML) underwent ASCT. Conditioning regimen consisted of 4d Busulphan (4 mg/ Kg from day -5 to -2) Stem cellt ransplantation is included in most treatment followed by Melphalan (140 mg/m²) for 1d (day -1); BU-MEL strategies for high-risk ALL. Conventional conditioning doses were reduced (3 mg/Kg for 4d and 120 mg/m², schedules (eg.TBI+cyclophosphamide and others) do have a respectively) in patients > 60 years. Unpurged peripheral substantial toxicity which has to to be regarded seriously. blood stem cells in 24 (83%) patients were used, while 5 Therefore we used a 7 days lasting conditioning regimen (17%) patients reinfused bone marrow receiving respectively a consisting of BCNU (300mg/m²), Etoposide (800mg/m²), median of 3.9 x 106/Kg CD34+ cells (range 0.96-11.5) and ARA-C (3g/m²), Cyclophosphamide (120mg/kg) and 2- 1.45 x 108/Kg nucleated cells (range 0.29-2.6). All patients Chlorodeoxyadenosine (24mg/m²) without TBI for these achieved full hematological recovery. Median number of days patients. For UD-transplantation ATG was added for two days. to neutrophil count of 0.5 x 109/l and platelet count of 20 x Patients: So far, 12 adult patients (6 male, 6 female) with a 109/l was 14 (range 12-22) and 18.5 (range 13-50) with no Ph+ pos. ALL (8), Pre- T-ALL (2), Pro-B ALL (1) in first CR significant differences between the two groups <60 years and and 1 relapsed cALL, with a median age of 41 years (19-53) >60 years. Transplant related mortality was 3.5% with 1 were treated. In 6 patients a HLA ID Allo-PBPCT was patient died for septicemia. As major extra-hematological performed, 1 HLA ID Allo-BMT, 1 family one mismatched- treatment-related toxicity 21 (73%) patients developed a PBPCT and 3 fully matched UD Allo-PBPCT. One 51 year old mucositis episode which was severe (grade III-IV WHO) in 15 woman received an autologous transplant. All Allo-Tx patients of them. There were 16 documented bacterial infections, while received a standard GVHD prophylaxis with cyclosporine and 8 patients had fever of unknown origin. After a median follow- short course MTX. G-CSF was administered in 10/12 patients up for surviving patients of 30 months from ASCT, 22 patients from day +5 to PMN >1,0 G/l. (74%) are alive and 21 (70%) are in continous CR. In Results: All patients reconstituted with a donor granulopoiesis particular, analysis of two groups <60 years and >60 years within 11 days (PMN > 0,5 G/l, range 10-20), and showed OS and DFS values respectively of 85% vs 50% and thrombopoiesis within 15 days (plt>50 G/l, range 12-29). 79% vs 46%. Chimerism at day 28 was over 95 % donor hematopoiesis by In conclusion, despite the reduced number of patients and the quantitative PCR in all Allo-transplanted patients. Non short follow-up, our results demonstrated the feasibility of BU- hematologic side effects were usually mild (mucositis I-II in 10 MEL regimen as conditioning treatment for AML patients who pat., nausea/emesis grade I-II: 11, enteritis grade II in 2 pat.), will undergo ASCT also in elderly and secondarily the efficacy severe side effects rare (mucositis grade IV: 1 pat., 1 emesis of this schedule as evidenced by the high number of continous grade III). All infectious complications were treated CR in patients <60 years. It remains to evaluate the exact role successfully (3 FUO, 3 urogenital inf., 1 gastro.intest.inf., 1 of this approach in patients >60 years, where the acceptable pneumonia, 1 invasive aspergillosis and 1 septicaemia). One toxicity is not associated with encouraging results. patient with transient renal failure during acute GVHD-phase (day 38) required passager dialysis. Within the first 100 days 2 patients were treated for a CMV reactivation. Acute GVHD grade 0-I developed in 4 pat. and GVHD grade II-IV in 6 pat. Chronic leukaemia No early TRM occurred. 8/12 patients are alive (7 complete haematologic remissions, 1 Ph+ALL with relapsed disease) for 1-82 months (median 9 months). 3 patients died of relapse (4, 10, 16 months after P437 Tx) and only one TRM occurred 10 months after Tx (extensive Dasatinib, nilotinib and imatinib inhibit CD8+ effector cGVHD+CMV enteritis). T-cells as well as CD4+CD25hi regulatory T-cells with Conclusion: According to these first experiences, the modified different potency BEAC conditioning protocol is safe with a very low TRM rate J. Chen (1), A. Schmitt (1), F. Fei (1), Y.Z. Yu (1), B. Chen (2), and similarly efficient to other commonly used conditioning S. von Harsdorf (1), M. Ringhoffer (1), D. Bunjes (1), M. regimen in high risk ALL-patients. Schmitt (1) (1)University Clinic Ulm (Ulm, D); (2)Southeast University Medical School (Nanjing, RC)

Background: For patients at relapse or with residual disease after allogeneic stem cell transplantation (allo-PBSCT), we have the therapeutical option to administer one of the tyrosine kinase inhibitors (TKIs) imatinib (Glivec), nilotinib (Tasigna; AMN107) and dasatinib (Sprycel; BMS-354825) which will inhibit the proliferation of CML progenitor cells, but might also

S65 hamper the graft-versus-leukaemia (GVL) effect considered to The Goodness of Fit of the models was assessed by the be crucial for the eradication of the disease. Moreover, CD8+ resulting Kaplan-Meier curves which showed clinically relevant T cells specific for cytomegalovirus (CMVpp65) might be differences between the good, intermediate and poor impaired. prognostic groups. The worst prognostic scores always Objectives: We wondered if TKIs might afflict anti- included the absence of ATA/ACC in the donor. Relapse was leukemic/viral CD8+ T cells, as well as CD4+CD25hi T cells associated with clinical factors; absence of female to male regulating the GVL. transplants and presence of T cell depletion but no significant Methods: Imatinib, dasatinib and nilotinib were added at association was found in this cohort with genetic factors. concentrations of 0-20 µM, 0-4 µM and 0-50 nM respectively GvHD incidence was associated with clinical factors and the to proliferation assays of CD4+CD25+ and CD8+ T cells. The genetic factors, absence of recipient IL10 ATA/ACC; more influence of TKIs on apoptosis was measured by bromo- severe GvHD (grades II-IV) was associated with absence of deoxyuridine/ annexin V. Mixed lymphocyte peptide cultures IL-4 (allele T) and IL-6 GG i.e. potential lack of a Th2 (MLPCs) were performed with peptides derived from a) response. This study suggests that distinct high risk patterns influenza matrix protein (IMP) as a recall antigen, b) CMVpp65 of NHP of patients and donors can be defined and influences as a viral antigen and c) the receptor for hyaluronic acid survival which may be associated with an increased risk of mediated motility (RHAMM-R3) as a leukaemia-associated GvHD without the benefit of GvL. Data add to the growing list antigen. CD8+ T cells from these MLPCs from healthy donors of situations (e.g. age, sex, multiparity of the donor) where an and patients with CML after allo-PSCT by tetramer unrelated donor may be preferred compared to a high risk staining/multi-color flow cytometry and enzyme linked sibling donor; NHPs should be integrated into donor selection immunosorbent spot (ELISPOT) assays, as well as algorithms. CD4+CD25hi Tcells after 3 days of culture with anti-CD3 and anti-CD28. Results: Proliferation of CD8+ T cells and regulatory T cells P439 was significantly inhibited by TKIs without increase of T cell Monitoring BCR-ABL transcripts after allogeneic apoptosis. The release of interferon gamma and granzyme B haematopoiteic stem cell transplant for patients with by CD8+ HLA-A2/tetramer+CD45RA+ CD27-CD28-CCR7- CML: is peripheral blood equivalent to bone marrow? effector T cells specific for IMP, CMVpp65 and RHAMM-R3 A. Dominietto, G. Cirmena, A. Garuti, A.M. Raiola, A. Ibatici, was inhibited by all TKIs in a dose-dependent fashion and M. van Lint, F. Patrone, F. Frassoni, A. Bacigalupo correlating to the time of TKI exposure. The inhibition was S.Martino's Hospital (Genoa, I) reversible after removal of the drugs from the MLPC. The proliferation and function of CD4+CD25hiFoxP3+GITR+ Background: Molecular monitoring of the BCR-ABL transcripts TGFß1+CD69+CD152+ regulatory T cells were also in patients with chronic myeloid leukemia (CML) using significantly inhibited by TKIs in a dose-related fashion. The quantitative real-time polymerase chain reaction (QRT-PCR) potency of T cell inhibition was imatinib:nilotinib:dasatinib = provides important information about the leukemia cell mass 1:2:40 in therapeutical serum respectively culture medium and the response to therapy. After allogeneic hematopoietic levels (2 µM:1 µM: 25 nM). stem cell transplants (HSCT) patients with persistently positive Conclusion: When administering TKIs to patients after allo- levels of BCR-ABL transcript have a molecular relapse and PBSCT, the inhibition of both CD8+ and CD4+CD25hi T cells some of these patients progress to develop a cytogenetic or must be taken into consideration with respect to GVL and anti- hematologic relapse. viral T cell response. Objectives: To test whether molecular detection of BCR-ABL transcripts is comparable using peripheral blood (PB) and bone marrow (BM) aspirate samples after allogeneic HSCT. P438 Patients and Methods: BCR-ABL transcripts were monitored A distinct pattern of non-HLA polymorphisms predicts an the interval of time 2003 – 2005 in 118 patients who received increased risk for GvHD without benefit of GvL in HLA an allogeneic HSCT in chronic or advanced disease phase. matched sibling transplants for chronic myeloid BCR-ABL transcripts were evaluated concomitantly in 200 leukaemia blood samples and 200 marrow samples (total 400 A. M. Dickinson, K. Pearce, J. Norden, A. Neylon, E. Holler, V. determinations) using real time PCR (QRT-PCR) assay to Rocha, E. Gluckman, H.J. Kolb, I. Hromadnikova, P. analyze BCR-ABL gene rearrangement (p210 b2a2 and Sedlacek, D. Niederwieser, R. Brand, T. Ruutu, J. Apperley, b3a2). A complete molecular response (CMR) both for PB and E. Goulmy, E. Olavarria, T. de Witte, A. Gratwohl on behalf of BM we defined has undetectable levels of p210 and Major Eurobank and The Chronic Leukaemia Working Party of the molecular response (MMR) was defined when p210/ABL EBMT ratios where <0.02%. Results: 135 double BM+PB determinations (67%) proved Non-HLA polymorphisms (NHP) influence GVHD BCR-ABL negative; 61 double determinations (31%) were incidence,severity and outcome of allogeneic haematopoietic classified as "persistently positive" both in peripheral blood stem cell transplants (HSCT). Their effects on GvHD vs GvL, and in blood marrow; 4 determinations (2%) were discordant. remains to be defined. A cohort of 291 CML HLA matched Therefore 196/200 BCR-ABL determinations in peripheral sibling transplants with known clinical risk factors as defined blood and bone marrow (98 %) were concordant (Spearman by the EBMT risk score, were typed for NHPs (IL-1Ra, IL-4, correlation coefficient r=0.871; 95% CI 0.832 - 0.902). QRT- IL-6, IL-10, IFN-g, TNF-a, TNFR 11), steroid hormone PCR assay’s sensibility and specificity were respectively receptors (VDR and ER-a) and NOD2/CARD15 mutations. 93.5% and 98.5%; positive and negative predictive values TNFRII-196 allele R; IL-10 ATA/ACC; IL-1 Ra (allele 2) and IL- were respectively 96.7% and 97.1%. 4–T were significantly associated with survival using Conclusions. This study shows that BCR-ABL QRT PCR univariate analysis. Two clinical Cox proportional hazards monitoring of CML patients after allogeneic HSCT with models were generated and used as a basis for further peripheral blood cells is concordant with bone marrow cells in development: (i) using the EBMT risk score as a single 98% of cases, and thus may be used to monitor the disease. variable on an ordinal scale or (ii) using the individual clinical This may be relevant for patients, especially when quality of factors of the EBMT risk score as categorical variables. After life issues are discussed together with the need for post- step-wise variable selection using the significant genetic transplant monitoring. factors, the resulting multivariate models indicated that absence of TNFRII-196 R, i.e. down regulation of TNF in the recipient, absence of IL-10 ATA/ACC, i.e. intermediate IL-10 production in the donor and presence of IL-1Ra (allele 2) i.e. down regulation of IL-1 in the donor were associated with poor outcome. The addition of these genetic variables significantly improved the model which contained the EBMT risk score.

S66 P440 Phenotypic detection of MRD was performed by 4-color and, After allogeneic stem cell transplantation in patients with more recently, by 6-color FC (CD19-gated cells in chronic lymphocytic leukaemia the level of campath at kappa/lambda/CD45/CD5/CD19/CD20, CD43/CD79b/CD45/ the day of transplant may be associated with delayed T- CD5/CD19/CD20 combinations and cell engraftment CD81/CD22/CD5/CD38/CD19/CD20 combinations) with J. Schetelig, C. Thiede, M. Bornhäuser, B. Mohr, U. sensitivity of 1:104. For RQ-PCR, clonal IgH-PCR products Oelschlägel, G. Geissler, R. Schwerdtfeger, H. Baurmann, K. were directly sequenced using ABI310 automated DNA Klima, C. Cox, W. Siegert, G. Ehninger on behalf of the sequencer. Individual ASO primers were designed to match German Cooperative Transplant Study Group the hypervariable CDR3 region of the sequenced IgH-gene. RQ-PCR with TaqMan probe was carried out in a real-time Purpose: To analyse the effect of persisting Campath levels at thermal cycler. Sensitivity for RQ-PCR was mainly 1:105. the day of transplant on T-cell engraftment after allogeneic Results: A total of 178 BM samples were taken for MRD stem cell transplantation (SCT) in patients with chronic analysis, and 86 of them were analyzed in parallel by FC and lymphocytic leukemia (CLL). RQ-PCR. ASO primer was successfully designed for all Patients and methods: 40 patients with relapsed CLL received cases. FC and RQ-PCR were concordant in 78% of the Busulphan 8 mg/kg and Fludarabine 150 mg/m2 as analyses. FC did not detect MRD in PCR-negative cases. RQ- conditioning regimen. The first twenty patients received 75 mg PCR detected MRD despite FC negativity in 22% of the Campath as part of the conditioning regimen. The second analyses. cohort of twenty patients received Campath as a conventional ASCT resulted in CR in 20/23 (87%) of the patients. By FC, cytoreductive treatment with the last application scheduled 14 8/23 (35%) of autotransplanted patients became MRD days before SCT. The median age was 54 years (range, 35 to negative, but only 2/16 (12.5%) PCR negative. Median OS 65 years) and the median number of prior chemotherapy was 57 months. regimens was 4. The observation of late secondary graft All alloSCT patients achieved CR, and 3/8 (37%) became failure in the first cohort of patients led to the analysis of PCR-negative (sensitivity of RQ-PCR <0.003, <0.001 and lineage-specific engraftment and Campath levels on the day <0.002, respectively), 2 of them having non-myeloablative of transplant. conditioning. TRM rates for allo and auto SCTs were 10% and Results: Data on Campath levels and lineage-specific 0, resp. chimerism are available for 24 patients. The median Campath Conclusion: CLL clones identified by sequenced IgH-gene are level at the day of SCT was 110 ng/mL (range, <31.25 to stable, and thus, RQ-PCR is suitable for detecting MRD in 1820). After establishing the association of the Campath level CLL. FC was less sensitive, but as specific as RQ-PCR, giving with the time to complete (>95% donor chimerism) T-cell no false positive results. After ASCT, 12.5% of patients with engraftment in a cox regression model (p=0.036) an arbitrary unmutated CLL achieved a molecular response by RQ-PCR, cut-off level of 250 ng/mL Campath was defined. 7 patients even when transplanted up-front. These patients may need had Campath levels above this treshold, while 17 patients had alternative treatment modalities. RQ-PCR provides a rapid lower plasma levels. The cumulative incidences of complete tool for judgement of the efficacy of new treatments. CD4+ and CD8+ T-cell chimerism at day +100 were 14% and 29% in patients with Campath levels >250 ng/mL compared to 82% both in patients with levels <250 ng/mL (CD4- P442 subset, p=0.006; CD8-subset, p=0.017). Other potential Comparative analysis of BCR/ABL mRNA transcript factors like the type of study treatment, the CD34-count and clearance using quantitative PCR after allogeneic stem CD3-count of the graft or the donor type did not have a cell transplantation between imatinib-resistant and significant impact on the kinetics of T-cell engraftment. imatinib-naïve/responsive/intolerant patients with chronic Despite the use of donor lymphocytes to convert incomplete myeloid leukaemia T-cell engraftment the cumulative incidence of progression at D. Kim, H. Messner, M. Minden, V. Gupta, J. Kuruvilla, J. 1-year was 48% in patients with Campath levels > 250 ng/mL Lipton compared to 12% in patients with levels <250 ng/mL (gray- Princess Margaret Hospital (Toronto, CAN) test, p=0.043). Conclusions: A level > 250 ng/mL of Campath at the day of The introduction of imatinib mesylate (IM) in the treatment of SCT may be a risk factor for delayed T-cell engraftment in chronic myeloid leukemia (CML) improved patients’ survival patients who receive reduced intensity conditioning with and resulted in current trend of avoiding up-front allogeneic Busulphan and Fludarabine. Whether delayed T-cell stem cell transplantation (SCT) in CML patients although engraftment leads to an increase incidence of progression allogeneic SCT is the only known curative treatment option for deserves further investigation. CML. The current recommendation for allogeneic SCT is for the cases resistant or intolerant to IM. The clearance kinetics of BCR/ABL transcript after allogeneic SCT, especially in IM P441 resistant case is still to be elucidated. Flow cytometry and RQ-PCR in assessment of MRD after The current study investigated the kinetics of BCR/ABL mRNA stem cell transplantation in CLL transcript reduction after allogeneic SCT, and compared M. Itälä (1), A-R. Huhtinen (1), V. Juvonen (1), V. Kairisto (1), between IM naïve/responsive and resistant groups. T-T. Pelliniemi (1), T-L. Penttilä (1), A. Rauhala (2), A. Since 2002, 16 CML cases were transplanted at PMH, Tienhaara (1), K. Remes (1) Toronto, ON, Canada (median age 40 years (19-60 years), 6 (1)Turku University Central Hospital (Turku, FIN); (2)Vaasa male and 10 female, 15 myeloablative TBI-based and 1 Central Hospital (Vaasa, FIN) nonmyeloablative conditioning, 10 sibling and 6 unrelated transplantation). Eight patients were IM naïve (n=4), intolerant Purpose: To define phenotypic and molecular response rate (n=2) or responsive case (n=2; Group A), while 8 patients after autologous (ASCT) and allogeneic SCT in poor-risk CLL, were IM resistant case (no molecular response, n=7 and loss and to compare two methods for MRD assessment, 4- 6-color of response n=1; Group B) including T315I (n=1), F359V flow cytometry (FC) and allele specific RQ-PCR. (n=1) and A459K mutation (n=1). No difference was noted Patients and methods: MRD was evaluated in 33 patients with between 2 groups in terms of age, disease stage at CLL who underwent SCT (auto n=23, allo n=10). A mutational presentation and at the time of transplantation. The prior dose status was available for 14 patients (10 autotransplanted) and of IM before SCT in group B was 800mg/day (n=7) and in 13 as unmutated. ASCT with BEAC/BEAM (n=16) or 600mg/day (n=1). CY+TBI (n=7) conditioning was performed, up-front in 70% of The reduction of BCR/ABL mRNA transcript below 5.0 log patients. In alloSCTs, 50% of the patients received reduction was achieved within median 2 months in group A, myeloablative and 50% non-myeloablative reduced intensity while within 9 months in group B (p=0.007). The serial conditioning. measures of BCR/ABL mRNA using quantitative PCR showed significant delay of clearance of BCR/ABL mRNA in group B

S67 at 4 months (4.34 vs 2.25 log reduction, p=0.002) and 6 and the absence of a final plateau (Fig1). The percentage of months (4.41 vs 2.47 log reduction, p=0.003) compared to long-term survivors for alloT was 34% (Fig 2) showing rather group A. However, after 9 months after SCT, no difference of good adequacy. The study of the impact of usual prognosis BCR/ABL mRNA transcript levels was noted between 2 factors (age, time diagnosis-transplant, sex match, HLA groups. Overall survival rate was similar between 2 groups match, CMV status, type of conditioning, BM or PBSC, ABO (p=0.435) with median follow-up of 22 months among compatibility and disease status before transplantation) on the survivors. percentage of long-term survivors showed that only the status The CML cases resistant to IM seemed to have delayed of disease at transplant had a significant impact: (CR vs SD or clearance of BCR/ABL mRNA transcript after allogeneic SCT. PD, HR: 0.11 [0.02-0.5] p=0.01 and PR vs SD or PD, HR: Further study including large number of patients will be 0.30 [0.09-0.96] p=0.04). This study pointed out the possibility necessary to reach a clear conclusion on this issue. The of curing B-CLL patients who responded to conventional GVHD titration up to 9 months after allogeneic SCT for IM chemotherapy with allogeneic transplantation. resistant case will be a reasonable approach to induce more graft-versus-leukemia effect, and to achieve in-depth remission in IM resistant CML patients.

P443 Allogeneic haematopoietic stem cell transplantation cures CLL: a retrospective analysis from the SFGM-TC registry M. Michallet (1), Q.-H. Le (2), N. Raus (2), M. Sobh(2), J.-P. Vernant (1), F. Nicolini (2), L. Sutton (1), J.-L. Harousseau (3), P. Colombat (4), E. Deconninck (1), B. Cazin (5), D. Blaise (1) (1)SFGM-TC (Paris, F); (2)Hôpital Edouard Herriot (Lyon, F); P444 (3)Hôpital Hôtel-Dieu (Nantes, F); (4)CHU (Tours, F); The impact of pretransplant imatinib use in allogeneic (5)Hôpital Claude Huriez (Lille, F) haematopoietic cell transplantation for CML: a single-centre comparative analysis This retrospective study concerned 471 B-CLL patients A. Ugur Bilgin, E.A. Soydan, P. Topcuoglu, Y. Günaydan, A. registered in the SFGM-TC registry from 1984 to 2005 : 313 Uysal, N. Konuk, M. Beksaç, H. Akan, G. Gürman, M. Ozcan, patients underwent autologous transplantation, 138 females Ö. Arslan, T. Demirer, O. Ilhan, M. Arat (F) and 175 males (M) with a median age of 54 years, 236 Ankara University School of Medicine (Ankara, TR) received peripheral blood stem cell (PBSC) and 77 bone marrow (BM); 158 patients underwent allogeneic Introduction: Imatinib has become the standard primary transplantation, 78 F and 80 M, median age = 49 years, 77 approach for the treatment of a newly diagnosed chronic PBSC, 81 BM from 17 related and 141 unrelated donors. phase myeloid leukemia (CML) patient, recently. Complete Before conditioning, 302 autoT and 143 alloT were evaluated cytogenetic response is achieved on the majority of patients for disease status: 100 and 26 patients were in CR, 170 and under treatment, but the disease remains in molecular level 55 in PR, 4 and 13 in stable disease, 28 and 49 in progressive and after discontinuation of the drug, relapses occur disease for autoT and alloT respectively. Among alloT inevitably. Allogeneic hematopoietic stem cell transplantation patients, 73 received reduced intensity conditioning and 85 (AHSCT) is still the only curative treatment modality. In standard conditioning (72 Cyt+TBI, 33 Fluda+TBI, 23 imatinib era centers are referring only the patients, who has Fluda+Bu+ATG, 8 Cyt+Bu and 21 other). Before autoT the imatinib resistance or patients with high relapse risk to conditioning consisted of 224 Cyt+TBI, 45 BEAM and 44 transplant centers. Secondly, the period from diagnosis to other. After alloT, 71 patients developed an acute GVHD > transplantation is an important nominator. We have performed grade II and 60 developed a chronic GVHD. The non-relapse a case-matched control analysis in our CML (n=173) cohort. mortality at 1 year was 29%. With a mean follow-up of 28 Patients and methods: CML patients who received imatinib months for autoT and 40 months for alloT, the probabilities of before transplantation (n=20), were matched retrospectively 3-year, 5-year and 8-year overall survival were 80%, 66%, according to EBMT (Gratwohl) score with imatinib naive ones 45.5% after autoT and 52%, 48% and 35% after alloT (n=40) in 1:2 ratio. The median age at transplant in the respectively. An analysis aimed to determine the percentage imatinib group was 39 (19-57), gender F/M: 10/10, disease of long-term survivors, or patients focused on the final plateau status 1st CP:13, 2nd CP: 4, AP: 2, BP: 1. The median of survival curves was performed on alloT and autoT groups. imatinib dose that was used until transplantation was 200 gr, A mixture model, gfcure with Splus statistical package and imatinib therapy was discontinued at a median of 17.5 (7- determined the percentages of long-term survivors and its 210) days prior to transplantation. Only one of 20 patients was adequacy was verified graphically. The percentage of long- in molecular remission before transplantation. The median term survivors for the autoT group was 1.2%, with a mean age of 40 patients in imatinib naïve historical control group survival length for uncured population of 160 months, both curves were close and consequently show good adequacy

S68 was 34 years (14-53), gender F/M: 14/26, and disease status significant impact of the EBMT score on EFS (HR=1.28 (0.07- 1st CP:32, 2nd CP:2, AP: 3, BP: 3. 3.13), p=0.0017), and on OS (HR=1.36 (0.08-3.71), Results: The pretransplant variables were homogenous within p=0.0002). OS was negatively influenced by the pre- groups with the exception of the time from diagnosis to transplant disease status (HR=2.07 (0.29-2.47), p=0.01) and transplant as expected. There was no significant difference in HU (HR=2.19 (0.31-2.47), p=0.01, and positively influenced by engrafment kinetics between the two groups. The cytogenetic the sex mismatch (HR=0.55 (0.29-2.02), p=0.04). A mixture response, transplantation related mortality, the incidence of model, gfcure with Splus statistical method determined the acute and chronic GVHD were not significantly different in two percentages of long-term survivors and its adequacy was groups . Moreover, no significant difference was observed till verified graphically. The percentage of long-term survivors for now in 2-year’s disease free survival and overall survival the whole cohort was 41.3% [figure1], with a deep impact of estimations (Table). the EBMT score (HR 1.98 (1.41-2.79), p<0.01). This study Conclusion: Our single center retrospective case-matched points out the importance of the EBMT score in determining analysis is in concordance with several recent reports. We the risks of transplant in CML, even at very long-term (>20 observed that imatinib use prior to AHSCT does not negatively years). affect the early transplant related outcome and our 2 year’s survival analysis. We have to wait for long term analysis of imatinib receiving transplant recipients for emergence of unwanted complications like late relapses.

P446 Immune reconstitution after allogeneic stem cell transplantation (aSCT) in patients with B-CLL correlates with the administration of campath U. Oelschlägel (1), C. Thiede (1), M. Bornhäuser (1), G. Geissler (2), H. Baurmann (3), G. Ehninger (1), J. Schetelig P445 (1) Allogeneic stem cell transplantation for chronic (1)University Hospital Dresden (Dresden, D); (2)Hospital myelogenous leukaemia: is it still applicable? Lessons Chemnitz (Chemnitz, D); (3)German Clinic of Diagnostics from long-term survivors (Wiesbaden, D) M. Michallet (1), F. Maloisel (2), Q. Lê (1), B. Lioure (3), A. Thiébaut (1), S. Ducastelle (1), N. Raus (1), F. Nicolini (1) Objectives: In a first cohort of patients with B-CLL in an (1)Edouard Herriot Hospital (Lyon, F); (2)Hôpital Civil ongoing study on allogeneic stem cell transplantation (aSCT) (Strasbourg, F); (3)Hôpital Hautepierre (Strasbourg, F) after reduced intensity conditioning with Fludarabine and Busulfan a cumulative dose of 75 mg Campath was given We analysed 207 allogeneic stem cell transplantations for between day –9 and day –5 (Bu/Flu/Cam) plus Cyclosporine chronic myelogenous leukaemia (CML) (121 M and 86 F, as GVHD-prophylaxis. Because of considerable infectious median age=36) performed between 1981 and 2005. The complications in this cohort of patients, the mode of Campath median interval diagnosis-transplant was 12 Mo (2 Mo-24 treatment was changed. The second cohort of patients years). Before transplant, 136 pts received hydroxyurea (HU) received Campath as pre-conditioning therapy for one month alone, 62 IFN-a (with 26+AraC) and only 9 received imatinib. with the last dose scheduled for day –14 (preCam/Bu/Flu), EBMT scores were 0 (n=3), 1 (n=49), 2 (n=64), 3 (n=54), 4 and Methotrexate was added to CSA. The aim of our (n=25), 5 (n=9), 6 (n=3). Stem cell sources were PBSC investigation was to compare the reconstitution of T cell (n=16), BM (n=187), and BM+PBSC (n=4), from 182 HLA counts as surrogate marker of the immune competence. identical related and 25 unrelated donors. Seven pts received Patients and Methods: In 38 patients (12 after Bu/Flu/Cam reduced intensity conditioning regimen, 200 received standard and 26 after preCam/Bu/Flu) a number of overall 398 four- conditioning (125 +TBI and 75 -TBI). Of the 203 evaluated pts, color flow cytometric measurements were performed to follow the majority was transplanted in chronic phase (n=163), 19 in the reconstitution of CD3/CD4/CD8 and CD56 absolute counts accelerated phase and 20 in blast crisis. After transplant, 197 (per µl) after aSCT (at least at days 30/100/180/365). engrafted, 103 pts developed an aGVHD •grade II (51 grades Chimerism analysis of lymphocyte subpopulations were III+IV) and 63 developed a cGVHD (29 limited/34 extensive). performed, too. The Campath level at the day of transplant Forty-nine pts relapsed: 17 hematological, 7 cytogenetic, 1 was analysed with an ELISA for both cohorts. molecular and 24 unspecified. At latest follow-up, 90 pts were Results: Immune reconstitution after preCam/Bu/Flu was alive, 18 with a limited and 12 with an extensive cGVHD, 117 significantly faster than after Bu/Flu/Cam. Early T cell pts died, 114 of whom the causes were known with 99 non- reconstitution at day 30 was significantly better after relapse causes (35 GVHD, 3 second malignancies and 51 preCam/Bu/Flu - median CD3 absolute counts: 259 vs. 79/µl, other causes), 12 from disease progression and 3 from p=0.026; CD4: 112 vs. 28/µl, p=0.011 and CD8: 129 vs. 39/µl, unrelated causes. Median follow-up was 144 months, the 3- p=0.038. At day 100 CD4 counts remained significantly higher year and 5-year overall survival (OS) and event-free survival (224 vs. 77/µl, p=0.014). Normal T cell counts were achieved (EFS) were 42% and 32.6%, 30% and 25.3% respectively. A significantly earlier after preCam/Bu/Flu: day 87 vs. day 269 multivariate analysis (age, pre-transplant status, EBMT score, (p=0.007) after aSCT. NK cells at day 100 and even at day graft, sex matching, HLA matching, conditioning, pre- 365 were significantly lower after preCam/Bu/Flu – 135 vs. transplant treatment, TBI, interval diagnosis-transplant) 403/µl, p=0.003 and 124 vs. 282/µl, p=0.001. The CD4 count showed a significant negative impact of the pre-transplant is known to be a critical value in the resistance to infections. disease status on EFS (HR=2.24 (0.28-2.87), p=0.004), a Therefore, we analysed the time to achieve an absolute donor

S69 CD4 count of 100/µl and 200/µl after aSCT, respectively. After quantitative high throughput assay. JAK2 mutation detection preCam/Bu/Flu these thresholds were obtained significantly was carried out using a chip-based matrix-assisted laser earlier: 100/µl – at day 63 vs. day 213, p=0.005; 200/µl – at desorption-time-of-flight mass spectrometer (Sequenom, San day 65 vs. day 332, p=0.004. Of note, we were able to Diego, CA) with a specific primer extension assay designed to correlate the time to achieve 100/µl CD4 cells with the detect the JAK2 V617F mutation. The level of mutation was Campath level at day 0. A Campath level • 250ng/ml was calculated by the following formula: % T allele = (AUC T associated with a delay in CD4 reconstitution (p=0.005). allele) x 100 / (AUC T allele +AUC G allele). T allele Conclusion: Surrogate markers for T-cell immune proportions were compared with routine methods for reconstitution can be correlated with the mode of Campath chimerism detection. administration and the antibody level at day 0. Thirteen pts underwent alloSCT in the study period (1.2001- 6.2006), mean age 59 (34-72) years, 8 males and 5 females. Their diagnoses included idiopathic myelofibrosis -5, post P447 polycythemia myelofibrosis -2, post essential Progenitor cell mobilization and collection in patients thrombocythemia myelofibrosis -1 and sAML -5. JAK2 V617F with chronic lymphocytic leukaemia: a nation-wide was found in 7/13 (54%) pts, their clinical characteristics are analysis presented in Table 1. All but one pt had more than 50% of the E. Jantunen (1), M. Itälä (2), T. Siitonen (3), T. Kuittinen (1), J. mutated allele (mean T allele 74%, range 16-98%), Heiskanen (4), E. Koivunen (5), E. Juvonen (4), R. suggesting the existence of a homozygous clone for this Silvennoinen (5), T. Nousiainen (1), P. Koistinen (3), L. Volin mutation in most pts at the time of alloSCT. Compared to pts (4), K. Remes (2) without JAK2 V617F, more pts with JAK2 V617F underwent (1)Kuopio University Hospital (Kuopio, FIN); (2)Turku alloSCT due to sAML (4/7 vs. 1/6) and more have undergone University Central Hospital (Turku, FIN); (3)Oulu University splenectomy (4/7 vs. 1/6) (ns). Disease duration (227 vs. 126 Hospital (Oulu, FIN); (4)Helsinki University Central Hospital months) and median survival (863 vs. 871 days) was the (Helsinki, FIN); (5)Tampere University Hospital (Tampere, same in both groups. After alloSCT, level of the mutant T FIN) allele decreased from baseline in 4 patients and remained unchanged in one. There was a very high correlation between Autologous stem cell transplantation is a treatment option in the decrease in the mutant T allele and the decrease in host patients with chronic lymphocytic leukaemia (CLL). Previous cells as detected by FISH (n=1) and microsatelites (n=3) reports suggest that a significant proportion of patients are (r=0.97, p<0.001). difficult to mobilise. In 1995-2005 altogether 128 patients with In summary, in our cohort pts with JAK V617F MPD and CLL (86 males, 42 females, median age 58 years) received clinical indication for alloSCT were mostly homozygous for the mobilisation therapy and were considered for progenitor cell mutation and they seemed to have a more aggressive clinical collection in five Finnish university hospitals. The median time behavior than pts without the mutation. The good correlation from diagnosis to mobilisation was 26 months (3-220). Eighty- between JAK2 V617F and other methods for chimerism eight patients (69 %) had received only one treatment line. detection in the post alloSCT period suggests that our novel The most common mobilisation regimen was intermediate- sequenom based assay may be used for follow up in pts with dose cyclophosphamide (CY) (4 g/m²) plus G-CSF (89 MPD undergoing alloSCT. patients, 71 %). At least 2 x 106/kg CD34+ cells were collected after the 1st mobilisation attempt in 83 patients (65 %), whereas 45 patients (35 %) experienced a mobilisation failure according to this definition. No differences were observed between these groups in regard to gender, age, time from diagnosis to mobilisation, number of treatment lines, number of fludarabine courses, time from the last chemotherapy to mobilisation, disease status or amount of marrow infiltration at the time of mobilisation. Patients who failed to achieve collection of at least 2 x 106/kg CD34+ cells had lower median platelet counts at the time of mobilisation (146 vs. 162 x 109/l, P=0.019). Altogether 30 % of the patients who failed to achieve this goal had platelet counts < 100 x 109/l compared to only 4 % of those with successful collections (P=0.001). Fourteen patients received a re-mobilisation course, which was successful in seven patients (50 %). To conclude, progenitor cell mobilisation is a problem in many patients with CLL. An adequate marrow function including platelet counts > 100 x 109/l seems to be an important factor in terms of successful progenitor cell collection. P449 Treatment of relapse after allogeneic stem cell transplantation in CML patients: imatinib only versus P448 imatinib&DLI High throughput sequenom-based assay for monitoring S. Civriz Bozdag, E.A. Soydan, P. Topcuoglu, A. Ugur Bilgin, JAK2 V617F post-allogeneic stem cell transplantation for S.M. Bakanay, H. Koc, M. Beksac, O. Ilhan, M. Ozcan, G. classic myeloproliferative disorders Gurman, M. Arat M. Koren-Michowitz, A. Nagler, A. Vivante, L. Trakhtenbrot , Ankara University School of Medicine (Ankara, TR) R. Loewenthal, G. Rechavi, N. Amariglio, A. Shimoni , Y. Cohen Donor lymphocyte infusion (DLI) is the gold standard for Chaim Sheba Medical Center (Ramat Gan, IL) relapse after alloHSCT. The role of single agent or combined application of imatinib,which is highly effective for induction of Allogeneic stem cell transplantation (Allo SCT) is currently the molecular remission in CML patients, in post alloHSCT only curative treatment in classic myeloproliferative disorders relapse is still an open question. In this single center study, we (MPD) and is often performed in advanced disease stages. retrospectively analyzed molecular response and graft JAK2 V617F mutation is thought to play a causative role in the functions of relapsed CML patients after transplantation, who pathogenesis of MPD. There is limited data on patients (pts) received imatinib only (n=10), Imatinib+DLI (n=9). The median with JAK2 V617F undergoing alloSCT and on the course of age of patients in the imatinib group (group 1) was 29 (range, this mutation in the post transplantation period. 19-54) , F/M:3/7. Nine patients were in CP1 and one was in To investigate the course of JAK2 V617F mutation in pts CP2 at the time of transplantation. In the combination group undergoing alloSCT, we developed a sensitive and (group 2), the median age was 41 years (20-54) , F/M:3/6. In

S70 group 2 the status of patients at transplantation was CP1: 4, from 0,4 to 12 years (median: 4,4 years) post HSCT for the AP: 3 and BP:1. Three patients received reduced intensity and level of T cell receptor excision circles (TREC) and T cell the remaining myeloablative conditioning. Molecular remission subsets in blood. Five and 26 patients received myeloablative (MR) was defined as; complete negativity of RQ-PCR (<3 log). or nonmyeloablative conditioning, respectively and were The great majority of the patients achieving MR proved to be transplanted from 17 sibling or 14 unrelated donors. in complete chimeric (CC) status. After 6 months of follow-up; TREC numbers were determined by quantitative PCR (qPCR) 9 of 10 patients treated with only imatinib (90%) achieved MR and T cell subsets CD4+CD27+CD45RO, CD4+CD27- at median 3(2-5)months. Only one patient remained refractory CD45RO+, CD4+CCR7+ and CD4+CCR7- were described by to treatment and died because of pneumocystis carinii flow cytometry. Sixteen patients (51,6%) were TREC positive pneumonia. Two patients relapsed after 6 months, received (mean number 559,98, from 1,30 to 4412,00). DLI, one of them died of DLI induced GVHD, the other one TREC positive patients were not significantly younger than obtained MR. After 12 months of follow-up, 3 of 7 patients TREC- patients (28 vs 37 years) and not significantly more relapsed and 2 had remission after addition of DLI to imatinib. recent post transplant. At the end of 12 months in group 1 only four patients TREC positive patients differ from their TREC negative remained in sustained MR with Imatinib only (40%) and three counter partners with respect to: after combination with DLI (70%). In group 2, 4of 9 patients I. lower frequency of cGvHD (3/16 vs 8/15, p=0,044) were treated with upfront DLI plus imatinib and 5 of 9 patients II. higher proportion of CD4+CCR7+ cells (5,07±0,75% vs with imatinib followed by DLI up to 6 months. After 6 months 2,67±0,33%, p=0,02) of follow up; 5 patients remained refractory but 4 patients III. higher proportion of CD4+CD27+CD45RO- (3,67±0,61% achieved MR (44%). At the end of 12 months; 3 of the 4 vs 2,45±0,55%, p=0,089) patients were still in molecular remission (33%). Two patients IV. lower proportion of CD4+CD27-CD45RO+ cells population died of progressive disease, 3 patients remained refractory (5,38±0,87%, vs 9,09±1,67% p=0,093) (recurrent relapse) and one patient experienced molecular V. the presence of post transplant chimerism was complete in relapse. Our single center experience has shown that MR and all TREC positive but mixed in 3 out of 15 TREC negative pts. CC status is achievable after imatinib in relapsed CML VI. post transplant relapse which was seen only in TREC patients after alloHSCT but the response is not durable. DLI negative patients synergized with imatinib for induction of MR but the We conclude that the presence of TREC is affected by cGvHD cumulative high risk CML patients in group 2 caused to low and older age at transplant and favoured the presence in remission rates. DLI only should be the standard approach in blood of naive and central memory cells. TREC negativity CML patients with molecular relapse. constitute a risk of mixed chimerism and relapse. Supported by FP6 AlloStem project

P450 Complement-dependent and complement-independent cytotoxicity of polyclonal antithymocyte globulins in P452 chronic lymphocytic leukaemia Mobilization of peripheral blood stem cells in poor risk F. Ayuk, A. Zander, N. Atassi, G. Schuch, C. Bokemeyer, B. CLL patients after • 2 lines of treatment and previously Fehse, N. Kröger underwent to therapy with chlorambucil University Medical Center (Hamburg, D) G. Console (1), M. Martino (1), G. Irrera (1), G. Messina (1), G. Pucci (1), I. Callea (1), A. Condemi (1), A. Dattola (1), A. Polyclonal antithymocyte globulins (ATG) are used in Pontari (1), G. Pratico (1), T. Moscato (1), E. Massara (1), F. allogeneic stem cell transplantation mainly due to their anti-T Gatto (1), D. Marcuccio (1), V. Callea (1), C. Stelitano (1), E. cell activity. ATG contains targeting Quartarone (2), S. Neri (3), P. Iacopino (1) expressed on various hematopoietic cells. Because of the (1)Azienda Ospedaliera Bianchi- Melacrino- Morelli (Reggio known effects of ATG on B cells, we assessed anti-CLL Calabria, I); (2)University of Messina (Messina, I); (3)Azienda activity of two commercially available ATGs at clinically Ospedaliera Papardo (Messina, I) relevant concentrations (10 – 100µg/ml) compared to Alemtuzumab (1- 10µg/ml) in CLL samples from 16 patients. Autologous stem cell transplantation is performed in an Cytotoxicity was determined by staining with 7-amino- increasing number of Chronic Lymphocytic Leukaemia (CLL) actinomycin D (7-AAD) and flow cytometry. ATG-Fresenius patients (pts). There are contradictory data about the impact (ATG-F) and Thymoglobulin at 100µg/ml induced a mean of of more lines of chemotherapy and previous treatment with 30.2% and 53.7% apoptosis. Alemtuzumab did not induce Chlorambucil (CLB) on stem cell harvest. From September complement-independent cytotoxicity. Addition of complement 2004 to February 2005 we treated 10 poor-risk CLL pts, (7 Rai strongly enhanced the cytotoxic effect of both ATG stage II, Binet B, 2 Rai I Binet B, 1 Rai III Binet C, 7 ZAP 70 preparations. At 100µg/ml mean complement-dependent positive, 3 ZAP 70 negative, 7 CD38+, 3 CD 38-), median age cytotoxicity (CDC) was 68.5% for ATG-F, 82.4% for 56,4 years (range 34-65), 6 male, 4 female, the median follow Thymoglobulin and 61% for Alemtuzumab (10µg/ml). CDC up to diagnosis was 4,5 years (range 1-8 years).The pts was mediated by reactive oxygen species (ROS). Both ATGs underwent previously to 2 lines (7),3 lines (1 ) or 4 lines (2) of enhanced the effect of Fludarabine. Absorption on primary T chemotherapy;all previously underwent treatment with CLB, cells revealed that the anti-CLL effect of ATG was in part due median time 4,8 months,(range 1-9 ). The treatment of to antibodies targeting antigens not expressed on primary T mobilization was a intermediate-dose (ID) of Ara-C, 800 cells. We conclude that ATG is an effective agent against CLL mg/m² every 12 h for six doses + G-CSF 10 mcg/Kg The in vitro. status at time of mobilization was: 9 pts in partial remission (PR), 1 pts in complete remission (CR). The target yield was > 5.0x106 CD34+ cells/kg. All pts mobilized peripheral stem P451 cells. A median of 401 cells/mcl was count of peripheral Associated with the presence of naive T-cells in blood in CD34+ cells (range 35-732). A median of 13,49.x106 CD34+ CML patients post HSCT and the lower incidence of cells/kg was collected per pts (range 5,1-26,5). The median cGvHD and relapse time of the first collection was 14,9 days (range 11-21).The D. Drabczak-Skrzypek (1), B. Wysoczanska (1), E. Jaskula median number of apheresis to obtain the target was 2,4( (1), M. Sedzimirska (2), D. Dlubek (1), A. Lange (2) range 1-3) The study of minimal residue disease (mrd) (1)L.Hirszfeld Institute (Wroclaw, PL); (2)Lower Silesian demonstrated median infiltration on collected product of 8% Center for Cellular Transplantation (Wroclaw, PL) (range 1-21) Only 3 pts received ( 2 for pts),while platelet support was needed in 7 pts,. 1 pts in CML offers long lasting remission which experienced mucositis W.H.O. grade 2, 3 patients had fever largely depends on the immunological surveillance employing W.H.O. 2, In conclusion, stem cells collection is feasible in alloreactivity. Therefore we studied a group of 31 patients CLL pts treated previously with more lines of chemotherapy

S71 and underwent to treatment with CLB. The toxicity was Myelodysplasia manageable The mechanisms of CLB on stem cell harvest must be further investigated. P454 P453 Effect of cytogenetics classification according to IPSS on Distinct immunoglobulin variable heavy chain genes as the outcome of allogeneic HSCT from HLA-identical possible early prognostic indicator for allogeneic stem siblings in patients with MDS or secondary AML: a cell transplantation in patients with chronic lymphocytic retrospective analysis from the EBMT-CLWP leukaemia F. Onida, R. Brand, A. van Biezen, F. Frassoni, D. Beelen, J. L. Cevreska, I. Panovska-Stavridis, M. Ivanovski, A. Finke, P. von dem Borne , M. Boogaerts, T. Ruutu, N. Milpied, Stojanovik, B. Georgievski P. Alessandrino, N. Kröger, T. de Witte on behalf of the University Clinical Center Skopje (Skopje, MK) EBMT-CLWP

In spite of the treatment progress for chronic lymphocytic Cytogenetics (CG) represent one of the most important leukemia (CLL) in the last few years, the disease remains prognostic factors for pts with MDS, being therefore required incurable. Although, allogeneic stem cell transplantation by the IPSS to predict life-expectancy. However, the impact of (alloSCT) is still considered as experimental approach for CLL CG on the outcome of pts undergoing allo-HSCT for MDS is and is associated with a number of risks to a number of still unclear. We aimed to retrospectively assess the impact of progressive CLLs is being offered. CG on overall survival (OS), relapse-free survival (RFS), The mutational status of the immunoglobulin variable heavy relapse probability (REL), and TRM in pts with MDS/sAML chain (VH) genes is a major prognostic indicator for the undergoing allo-HSCT from HLA-identical siblings. clinical course and outcome of the CLL patients. More Data from 1506 pts with MDS/sAML who underwent a first recently, additional prognostic categories have been identified allograft from HLA-identical siblings from 1981 to 2006 by recognizing disease subsets which utilize unique VH reported to EBMT were assessed. The following covariates genes. Examples include the V3-21, V1-69 and V3-72 genes were included: CG (good- vs standard- vs high-risk according which are invariably associated with progressive and stable to the IPSS); stage at HSCT (untreated vs treated in CR vs disease, respectively. treated not in CR); FAB classification (RA/RARS vs The aim of our study was to investigate the possibility to RAEB/CMML vs RAEB-t/sAML); age; time from dx to HSCT identify early after diagnosis suitable CLL candidates for (<5 vs 5 to 8 vs > 8 months); calendar year in which HSCT alloSCT among CLL subsets that utilize unique VH genes. was performed; type of conditioning (standard vs RIC); source Methods: The study group consisted of 106 consecutive CLLs of stem cells (BM vs PB). A complete information dataset was that had been diagnosed at our Institution prior to 2000, available in 839 pts. 18% of pts had RA or RARS, 29% had according to standard morphologic and immunophenotypic RAEB or CMML, and 53% had RAEB-t or sAML. Age was >50 criteria. VH gene family usage and mutational status were years in 24%. CG classified 56% of pts as good-, 23% as obtained by direct sequencing of RT-PCR amplified RNA intermediate-, and 21% as high-risk. At the time of HSCT, samples. Correlations between the different CLL subsets were 39% were untreated; among treated pts, 52% were in CR. A made using standard statistical tests. RIC regimen was administered to 15%. Source of stem cell Our results showed that 61 (57,6 %) patients utilize mutated was PB in 43%. VH genes. The most frequently rearranged VH gene in our By univariate analysis, subdivision of pts in the IPSS risk- CLLs was V1-69 gene, in all 25 cases (23.6%) with unmutated categories for CG associated with significantly different OS (at sequence. We compared the overall survival (OS) of the V1- 60 mos, alive pts in good-, interm.-, and high-risk groups were 69 subgroup against all the other patients. The two groups 47%, 40% and 31%, respectively), RFS (alive pts at 60 mos were comparable regarding the sex, age, total tumor mass 43%, 40% and 23%, respectively), relapse probability (34%, (TTM) score and Rai stage. The VH1-69 group has median 35% and 57% at 60 mos, respectively) and TRM (34%, 40% OS of 56.7 months and all others patients have median OS of and 43% at 60 mos, respectively). 125. 8,(p<0.0001). Further analyzes of the survival between By multivariate COX, CG and age were the variables VH1-69 cases and all other unmutated VH genes showed no associated with all the outcomes. FAB classification together differences between the two subgroups regarding the age, with stage at HSCT associated with OS, RFS, and REL; gender, TTM score, Rai stage and OS. No other unique VH conditioning intensity associated both with TRM and REL. gene, utilized in our study group, had frequency important of Concerning REL, the interval between diagnosis and HSCT analyzing. was also significant, whereas the source of stem cells Our data do not support the thesis that patients expressing associated only with OS, with the hazard associated with PB V1-69 gene form a distinct subgroup of CLL patients, but our being higher in comparison to BM. results confirmed that CLL with unmutated VH gene sequence In summary, this study showed that CG have strong has poorer OS. We suggest that all younger patients that prognostic impact on outcome of pts undergoing allo-HSCT utilize unmutated VH genes should be consider as candidates from HLA-identical siblings for MDS/sAML and should be for early alloSCT, immediately after the first complete taken into consideration when selecting candidates for this remission, if HLA identical donor is available. treatment strategy. Detailed analyses will be presented.

P455 Retrospective comparison of transplant from matched unrelated donor or identical sibling in adult MDS: evidence for a graft-versus-myelodysplasia effect in unrelated donor transplant E. Alessandrino, A. Bacigalupo, M. Falda, G. Lambertenghi Deliliers, W. Arcese, F. Benedetti, F. Ciceri, P. Di Bartolomeo, G. La Nasa, A. Rambaldi, P. Pioltelli, P. De Fabritiis, P. Marenco, C. Pascutto, G. Bandini, R. Fanin, A. Bosi for GITMO

In this retrospective multicenter study, we report the outcome of 231 patients with myelodysplastic syndrome who underwent transplantation with a human leukocyte antigen identical sibling donor (group A: 160 pts) or with an unrelated well-matched volunteer donor (group B: 71 pts.). All patients

S72 received a myeloablative conditioning regimen. Those who P457 transplanted from an HLA unrelated donor received, in Monitoring graft-versus myelofibrosis-effect with highly addition, ATG at the dose of 7.5 mg – 10 mg/Kg recipient sensitive real-time PCR of V617F-JAK2-mutation after body weight. The median age of patients was 42.7 years allogeneic stem cell transplantation in patients with (range 17.3 – 66 years); 126 were males and 105 females. myelofibrosis The age and sex distribution were comparable in the two N. Kröger (1), A. Badbaran (1), E. Holler (2), J. Hahn (2), G. groups, as well as the disease status at transplant. The overall Kobbe (3), M. Bornhäuser (4), A. Reiter (5), T. Zabelina (1), A. survival (OS) was not significantly different in the two groups Zander (1), B. Fehse (1) (5-year survival for group A: 38.5%; group B: 37% log-rank (1)University Hospital (Hamburg, D); (2)University Hospital test: p= 0.17). The prevalence of acute GvHD was similar in (Regensburg, D); (3)University Hospital (Duesseldorf, D); the two groups: 41% in group A and 37.5% in group B. The (4)University Hospital (Dresden, D); (5)University Hospital relapse rate was significantly increased (log-rank test: p=0.02) (Mannheim, D) in patients receiving an HLA identical sibling transplant (5-year cumulative probability of relapse = 46.6%) compared to The V617F-JAK2-mutation occurs in about 50 % of patients patients with an unrelated well-matched donor (20.6%). The 5- with myelofibrosis and is a reliable marker to monitor residual year non-relapse mortality rate was lower in sibling transplants disease after allogeneic stem cell transplantation. The (37.1% vs. 46.8%, log-rank test: p=0.03). Although the OS is establishment of valid complete remission criteria for similar in the two groups, the lower relapse rate in group B myelofibrosis especially after allogeneic stem cell suggests a stronger graft versus myelodysplasia effect in this transplantation remains a major issue. We developed a new, subgroup of patients. In this view, the use of a reduced highly sensitive real-time PCR to monitor and quantify V617F- intensity regimen in patients undergoing transplantation with JAK2-positive cells after dose-reduced allogeneic stem cell an unrelated donor might lead to an improvement in the transplantation. The mutated differs from wild-type JAK2 allele outcome by just one nucleotide exchange (G à T) leading to the valine phenylalanine (V à F) transition. Using PrimerExpress® we designed a TaqMan® PCR where the reverse primer (RP) P456 terminates at the (3';) nucleotide corresponding to this point Equal effectiveness of haematopoietic stem cell mutation. Thus, this reverse primer should bind with higher tranplantation for patients with therapy-related affinity to the mutated than to the wild-type allele. To increase myelodysplastic syndrome or acute myeloid leukaemia the specificity while conserving optimal sensitivity of the MRD- induced by prior curative cytotoxic therapy specific PCR we generated a set of primers shortened each R. Trenschel, M. Hlinka, N.C. Steckel, L. Kordelas, S. Pluntke, time by one nucleotide at their 5' end. In parallel, all those M. Ditschkowski, H. Ottinger, M. Koldehoff, A. Elmaagacli, C. shortened primers were designed to contain an additional Schulte, D. Beelen mutation at the third to last 3' position. This allowed us to University Hospital of Essen (Essen, D) identify the reverse primer combining high specificity with so far not reported sensitivity (0.01 %). After 22 allogeneic stem Therapy-related myelodysplasia (t-MDS) and acute leukemia cell transplantation procedures in 21 JAK2-positive patients (t-AML) are reported to imply a worse prognosis as compared with myelofibrosis, 78 % became PCR-negative. In 15 out of to de novo MDS and s-AML. Between 04/94 and 10/06, 147 17 patients (88 %), JAK2 remained negative after a median patients (median age 47 years) with MDS received allogeneic follow-up of 20 months. JAK2-negativity was achieved after a HSCT in our center including 11 patients with t-MDS and 24 median of 89 days post allograft (range, 19 - 750 days). A with t-AML. Treatment related disease was induced by prior significant inverse correlation was seen for JAK2-positivity and chemotherapy for NHL (n=11), HD (n=9), gynaecologic donor cell chimerism (r: - 0.91, p < 0.001). Four of five malignancies (n=8), testicular cancer (n=4), endocrine patients who never achieved JAK2-negativity fulfilled during malignancies (n=2), ALL (n=1). The outcome of HSCT in the entire follow-up all criteria for complete remission recently patients with de novo and therapy-related diseases were proposed by the Internationaal Working Group. Suggesting a compared. The effects of prior cytotoxic exposure, major role for JAK2 measurement to determine depths of cytogenetics, IPSS, donor type, and disease stage were remission. In one case, residual JAK2 positive cells could be appraised to determine influences on overall survival, disease eliminated by donor lymphocyte infusion, supporting the graft free survival, relapse, and non relapse mortality (OS, DFS, versus myelofibrosis effect. In conclusion, allogeneic stem cell REL, and NRM). We found no difference in OS, EFS, REL, transplantation after dose-reduced conditioning induces high NRM for t-MDS/t-AML compared to MDS/s-AML. Using the rates of molecular remission in JAK2-positive myelofibrosis Kaplan Meyer method OS was 37% and 36% (Log Rank patients and quantification. V617F-Jak2 mutation by real-time p=0,45), EFS was 36% and 37% (Log Rank p=0,34), relapse PCR allows detecting minimal residual disease to guide occurred in 25% and 20% (Log Rank p=0,47), NRM in 49% adoptive immunotherapy. and 47% (Log Rank p=0,53) for MDS/s-AML and t-MDS/t- AML after a follow-up of 10 years, respectively. Subgroup analysis for MDS and t-MDS as well as for s-AML and t-AML P458 generated similar results. Cox Regression was performed to A limited impact of chemotherapy when not followed by identify disease related risk factors. Only advanced disease stem cell transplantation on prolonged survival in stage was identified to worsen outcome: REL, OS, and EFS patients with myelodysplastic syndrome (p=0.02). Advanced disease state is associated with an M. Markova, J. Cermak, A. Vitek, M. Lukasova, P. Cetkovsky inferior OS (48% vs. 24%), EFS (46% vs. 22%), and an Institute of Haematology (Prague, CZ) increased relapse ratio (39% vs. 13%). Alternative donor and matched sibling donor grafts had similar results. In this The influence of different clinical and laboratory parameters analysis no influence of prior cytotoxic exposure, cytogenetic and various therapeutic approaches on the survival was abnormalities according to the IPSS classification, as well as retrospectively studied in a group of 142 adult patients with IPSS Score for MDS at diagnosis on above mentioned advanced forms of primary myelodysplastic syndrome (MDS): endpoints was detectable. RAEB with more than 10% blasts or RAEB-T. Median survival, In conclusion, HSCT for cytotoxic induced and de novo estimated 1 year overall (OS1y), disease free (DFS1y), 3 year MDS/s-AML is equally effective in this long term follow-up overall (OS3y) and disease free (DFS3y) survival, and relaps analysis. risk (RR3y) of patients stratified according to different therapy are shown in the table.

S73 total, 5415 post-transplant samples were analyzed. Recipient genotype discrimination was possible in 96% with a mean number of 2.5 (1 to 7) markers. Sensitivity of the method was ” 1E-3 in 83.3% of the assays. It was possible to very accurately detect autologous signals from 0 to 0.5% [confidence interval (CI) 95% ± 0.2], from 0.5 to 1% (CI 95% ± 0.4), from 1 to 2% (CI 95% ± 0.6) and from 2 to 5% (CI 95% ± Allogeneic stem cell transplantation (SCT) regardless to the 1.2). Reproducibility of the quantified signals was independent number of bone marrow blasts at the time of conditioning, was from the amount of DNA. the most significant parameter affecting survival in univariate To our knowledge, this is the first report on a SP-chimerism analysis (P=0.00001). However, achievement of complete system allowing to perform routine chimerism analyses for (CR) or partial (PR) remission after chemotherapy had a virtually all patients with high sensitivity, excellent significant impact on survival, whether followed by SCT or not reproducibility and accuracy. Quantification of chimerism is (P=0.0001). A multivariate analysis revealed SCT as the most reliably possible with marker-specific standard dilution series important independent variable determining survival in the from volunteers. We provide unequivocal data on CIs 95% of whole group of patients (P=0.00001), SCT performed in autologous signals for measuring changes of autologous CR/PR was a significant variable in patients younger than 55 signals in consecutive samples as statistically significant or years (P=0.04). In non-transplanted patients, only not. This is an important prerequisite for reliable assessment achievement of CR or PR had a significant impact on survival of the clinical impact of dynamic developments of chimerism. (P=0.001). The system allows conducting prospective studies to evaluate Despite a significant impact on survival compared to single whether SP-RQ-PCR is superior to STR-PCR in terms of agent therapy or supportive care (P=0.005), combination clinical impact of chimerism. chemotherapy not followed by SCT had only minimal effect on prolonged survival not different from low-dose chemotherapy (P=0.4) even in patients who achieved CR or PR (estimated 3 P460 years survival was only 4,3 %). On the other hand, Allogeneic stem cell transplantation in children and achievement of CR or PR prior SCT was a significant factor adolescents with high-risk alveolar rhabdomyosarcoma affecting long-term survival in patients younger than 55 years P. Bader, J. Soerensen, A. Jarisch, G. Weber, H. Kreyenberg, (estimated 3 years survival for SCT in CR/PR was 75% R. Esser, A. Willasch, E. Rettinger, U. Koehl, S. Kuci, T. compared to only 36% for those with more than 5% bone Klingebiel marrow blasts prior SCT). Our data support the usefulness of University Children's Hospital (Frankfurt, D) combination chemotherapy followed by allogeneic SCT as an optimal treatment regimen for younger patients with advanced Rhabdomyosarcoma (RMA) is a rare and aggressive disease MDS. of childhood and adolescence. According to the German CWS 96 Study, patients above the age of ten with bone and or bone marrow metastasis and an alveolar histology do not have a realistic chance to survive their disease (2 years EFS: 0%). Therefore, we were evaluating whether allogeneic Paediatric issues might be an option to improve these patients’ prognosis. For effective allogeneic stem cell transplantation an important prerequisite is a cellular target on the malignant cells. Such a P459 target can be the Wilmstumour Gen 1 Peptide (WT1). We Characterisation of chimerism by sequence could show that RMA tumours and RMA cell lines express polymorphism systems for quantitative real time WT1 as high as AML cells. As this tumour entity also polymerase chain reaction - high informativity and expresses HLA- class 1 antigens on the cell surface, alveolar sensitivity as well as excellent reproducibility and RMA patients fulfil substantial requirements to be good precision of measurement candidates for allo-SCT. A.M. Willasch (1), G. Schneider (2), B.S. Reincke (1), N. Based on these findings, we started a pilot trial in which 9 Shayegi (1), H. Kreyenberg (1), S. Kuci (1), G. Weber (1), B. patients were included. All patients have had an alveolar van der Reijden (3), D. Niethammer (4), T. Klingebiel (1), P. subtype (n=9). Median age was 17.5 years (range: 5.0 to Bader (1) 27.2). Patients were treated in CR 1 (n=4), CR2 (n=1) or PR (1)J. W. Goethe University Children's Hospital (n=4). As patients were heavily pre-treated with chemo- and (Frankfurt/Main, D); (2) J. W. Goethe-University (Frankfurt, D); radiotherapy and two of them have had already high dose (3)Radboud University Medical Centre (Nijmegen, NL); (4)E. chemotherapy, a reduced conditioning regimen was chosen. Karls University Children's Hospital (Tuebingen, D) This consisted of fludarabine 150mg/m², thiotepa 10 mg/kg, melphalan 140 mg/m² and 10 days of OKT3. All patients Characterization of increasing mixed chimerism offers the received a CD3/CD19 depleted peripheral stem cell grafts possibility to identify patients with impending relapse. Several from either an HLA haploidentical family donor (n=8) or from a prospective trials showed, that pre-emptive immunotherapy HLA identical sibling (n=1). The median number of stem cells can principally prevent relapse in these high risk patients. was 7.7x106CD34 pos cell/kg with a substantial number of However, these studies were not able to identify ongoing NK, monocytes and dendritic cells. All patients showed rapid relapse in all patients. Possible reasons might be the and sustained engraftment with an ANC >1000 on day 12 moderate sensitivity (1E-2) of the applied methods as well as (range: 9-16) and more than 20.000 Thrombocytes on day 16 the fact that relapse can develop within days. Short tandem (range: 12-19). GVHD grade 0-I occurred in 6 patients, grade repeat PCR (STR-PCR) is the gold standard for II in 2 patients and grade III in one patient. All patients characterization of chimerism. However, due to technical responded to transplantation and achieved complete reasons this method shows a limited sensitivity of 1E-2. Our remission (n=9; 100%) on day 60 post transplant. goal was to establish a chimerism analysis method with high Consecutively, three patients relapsed; one patient died of informativity, sensitivity, reproducibility and accuracy. TRM and one due to non-TRM reasons. From the relapsing The improvement of sensitivity is achievable by a quantitative patients 1 could be rescued by additional treatment. At PCR (RQ-PCR) approach with sequence polymorphisms present 5/9 patients are alive and in complete remission with a (SPs) as targets. SPs represent the most frequent genetic median survival time of 246 days (68 to 374 days). In variation. Most of the SPs are biallelic and differ only in one conclusion, allogeneic stem cell transplantation seems to be a single nucleotide. In this study, a panel of 29 selected markers promising approach in the treatment of patients with high risk was evaluated in 317 patients with leukemia and MDS, who RMA who otherwise did not have a realistic chance to survive. received allo-SCT. Marker specific dilutions from volunteers’ DNA served as standard for quantification of chimerism. In

S74 P461 difference was observed in the probability of extensive cGvHD Allogeneic stem cell transplantation for leukocyte (0.33 vs 0.2 in the mismatched vs matched groups; p=NS). adhesion deficiency: 20-year single-centre experience Cox model disclosed no correlation between the number of W. Qasim, G. Davies, B. Gaspar, C. Cale, P. Amrolia, K. Rao, grafted CD34+ cells/kg or CD3+ cells/kg and the incidence of P. Veys severe acute or chronic GvHD and no influence of other Great Ormond Street Hospital (London, UK) factors incl. stem cell source, sex mismatch or ATG brand. Pts in the mismatched group were given slightly higher numbers Leukocyte adhesion deficiency (LAD) is caused by defective of CD3+ cells/kg (4.9 vs 3.0 x 108 in the matched group) and expression of the beta-2 integrin, CD18, and can result in life similar numbers of CD34+ cells/kg (median 8.1 vs 8.9 x 106 in threatening infections in the first few years of life. The CD18 the matched group). Despite a higher incidence of severe molecule forms part of the lymphocyte function associated aGvHD in the mismatched group, probability of 5-year survival antigen-1 (LFA-1) when associated with CD11a, Mac-1 with was slightly better in that group (pS = 0.61 vs pS = 0.52 in the CD11b and p150-95 with CD11c. These molecular complexes matched group, p=NS). are essential for effective migration and homing of immune In conclusion, there is no correlation between the numbers of cells, including neutrophils, dendritic cells and T lymphocytes. grafted CD34+ cells/kg and CD3+ cells/kg and the incidence Allogeneic haematopoietic stem cell transplantation (SCT) of severe GvHD in the UD setting in children. It is safe and offers the possibility of curative therapy for LAD, however feasible to perform allogeneic transplants from mismatched conventional myeloablative conditioning may not be sufficient UD, provided a detailed selection of > 1 HLA-allele- to result in complete donor chimaerism (Thomas et al Blood; mismatched donors is performed. It is not necessary to limit 86: 1629-35), and agents such as Etoposide may be required the number of transplanted CD34+ cells to 6-8 x 106/kg, which in addition to Busulphan and Cyclophosphamide. This may had been previously suggested by studies performed in lead to significant toxicity and a mortality rate of over 25% was Europe or USA in adults. Further studies are warranted to reported in this European study that treated 14 patients. An compare results of unmanipulated and TCD-mismatched UD alternative strategy might be to use a more or haploidentical transplants. immunosuppressive but less myeloablative conditioning regimen. We report our experience in transplanting LAD deficiency over a 20 year period: P463 The first 3 children all succumbed after myeloablative SCT. Retrospective analysis of stem cell transplantations for Five subsequent children received matched unrelated donor T severe combined immunodeficiency in Japan cell replete bone marrow grafts following conditioning with K. Imai (1), H. Kanegane (2), S. Kumaki (3), T. Ariga (4), S. Melphalan (140/m²), Fludarabine (150/m²) and Alemtuzamab Tsuchiya (3), T. Morio (5), Y. Tsuji (1), K. Agematsu (6), H. (1mg/kg). All had successful multi-lineage reconstitution with Takada (7), S. Nonoyama (1), T. Miyawaki (2) full donor chimaerism in four patients and 94% in the fifth. (1)National Defense Medical College (Saitama, JP); Complete immunological reconstitution was confirmed by (2)University of Toyama (Toyama, JP); (3)Tohoku University mitogen proliferation assays, and detection of normal antibody (Sendai, JP); (4)Hokkaido University (Sapporo, JP); (5)Tokyo responses in the four patients vaccinated to date. Normal Medical and Dental University (Tokyo, JP); (6)Shinshu levels of CD18 expression were confirmed by flow cytometry. University (Matsumoto, JP); (7)Kyushu University (Fukuoka, All patients are alive and well, and remain free of infective JP) complications. Our experience highlights the benefits of reduced intensity We analyzed retrospectively the stem cell transplantations conditioning for LAD and emphasises the importance of (SCTs) for severe combined immunodeficiency (SCID) in treating patients early after diagnosis and before serious Japan between 1981 and 2005. 143 cases were diagnosed as infective complications develop. SCID and 100 cases were reported to be transplanted. Of these 72 times of SCTs of 68 cases were analyzed. B+ SCID patients were 44 cases, B- SCID patients were 15 P462 cases and other types including Omenn syndrome were 9 High numbers of grafted CD34+ and CD3+ cells/kg have cases. 13 cases were transplanted below 3 months of age. no negative impact on the incidence of severe GvHD and Bone marrow (BM) from HLA matched related donor (MRD) survival in a large series of children undergoing including matched sibling donor (MSD), BM from HLA unmanipulated allogeneic HCT from matched or matched unrelated donor (UR-BM), BM from mismatch related mismatched unrelated donors donor (mMRD), and cord blood (CB) were transplanted in 16, K. Kalwak, J. Owoc-Lempach, A. Dyla, E. Gorczynska, M. 4, 29 and 19 cases, respectively. 43 cases were transplanted Ussowicz, D. Turkiewicz, M. Slociak, D. Wójcik, A. Chybicka without conditioning. Myeloablative conditioning (MAC) and Wroclaw Medical University (Wroclaw, PL) reduced intensity conditioning (RIC) were performed in 12 and 13 cases respectively. Death until 40 days after SCT was The aim of the study was to analyze the correlation between observed in 12 cases. 9 cases were dead during 40 days and the number of grafted CD34+ and CD3+ cells/kg and the 1 year after SCT. 5 cases were dead after 1 year of SCTs. incidence of severe GvHD in children undergoing allogeneic Early deaths were mostly related to pre-SCT pulmonary HCT from HLA-matched or mismatched unrelated donors infections especially due to cytomegalovirus. Late deaths (UD). were related to insufficient engraftment of lymphocytes. No 115 pts (median age 11.4; ALL n=41, AML n=22, CML n=21, significant difference of prognosis was observed among the MDS/JMML n=10, NHL/HD n=4, SAA/FA n=13, SCID/WAS types of SCID. Overall survival (OS) rate for patients receiving n=4) underwent unmanipulated allogeneic BMT (n=34) or UR-CB (4years-OS: 79.0%) was comparable to those of PBSCT (n=81) from UD between 1999 and 2005. There were MRD-BM (5 years-OS: 87.5%) and significantly superior to 88 matched- and 27 mismatched donor-recipient pairs, those of UR-BM mMRD (5 years-OS: 50.0%) and mMRD (5 respectively (acc. to BFM). Among 88 matched pairs, 51 were years-OS: 50.7%). Deaths after 1 year were only seen in 10/10 HLA-allele matched, whereas in another 37 cases 9/10 mMRD-BMT. alleles were matched. In the mismatched group there were 22 Among the patients receiving UR-CB, all seven patients with pairs with a 8/10 allele-match and 5 pairs with a 7/10 allele- RIC are not dependent on intravenous immunoglobulin (IVIG) match, respectively. No single DRB1* mismatch was substitution. All patients receiving MSD-BM without accepted. For in-vivo TCD pts were given either median 30 conditioning are not dependent on IVIG. mg/kg ATG-Fresenius (94 pts) or 10 mg/kg Thymoglobuline Our results indicate that transplantation using UR-CB is as (21 pts). effective as that using MSD. Thus in Japan UR-CBT is A distinct correlation between the degree of HLA-match and recommended as a donor source for the patients without the risk of grades III-IV aGvHD was found (p=0.04; Cox MRD. In the case of UR-CB, RIC is recommended to improve univariate analysis). Probability of aGvHD was 0.34 and 0.17 the engraftment of B cells. in the mismatched/matched groups, respectively. No

S75 P465 The outcome of children with constitutional single cytopenia post allogeneic stem cell transplantation from matched related donor: a single-centre experience A. Al-Ahmari, A. Radwan, M. Ayas, A. Al-Jefri, A. Al Seraihy, M. Al-Mahr, S. Rifai, H. El-Solh King Faisal Specialist Hospital (Riyadh, SA)

Severe congenital cytopenia (SCN) and Diamond-Blackfan anemia (DBA) are constitutional bone marrow disorders that present with a specific single lineage cytopenia Allogeneic stem cell transplantation (SCT) is the treatment of choice for SCN patients who failed G-CSF treatment or developed myelodysplasia or leukemia and, for DBA patients who required high doses or failed corticosteroid therapy. We report on 15 (median age of 5 years, range of 0.4 to 12.2) constitutional single cytopenia patients (5 SCN and 10 DBA) who were transplanted between 1993 and 2006.The reason P464 for transplantation in all cases is failure of medical treatment. Bone marrow transplantation in children with acute None has developed myelodysplasia or leukemia. All patients leukaemia and Down syndrome had received HLA-identical graft from sibling in14 patients and B. Meissner (1), A. Borkhardt (2), D. Dilloo (3), D. Fuchs (4), from mother in the remaining patient. They were conditioned W. Friedrich (5), R. Handgretinger (6), C. Peters (7), A. with standard doses of busulfan and cyclophosphamide Schrauder (8), F.R. Schuster (2), J. Vormoor (9), B. Maecker (BU/CY) regimen. ATG was added to the regimen only for (1), K.W. Sykora (1), F. Zintl (4), M. Sauer (1) heavily transfused patients (6 patients). Graft versus host (1)Medical School Hannover (Hannover, D); (2)Dr von disease (GvHD) prophylaxis was cyclosporine (CSA) only in 2 Haunersches Kinderspital (Munich, D); (3)Heinrich-Heine- patients or CSA and short course of methotrexate (MTX) in University (Duesseldorf, D); (4)Friedrich-Schiller-University the remaining patients. Myeloid engraftment was achieved in (Jena, D); (5)University of Ulm (Ulm, D); (6)Children's all patients. Platelets failed to engraft in one DBA patient and University Hospital (Tuebingen, D); (7)St Anna Children's another one died early prior to platelet engraftment. Two DBA Hospital (Vienna, A); (8)University Hospital Schleswig- patients rejected their grafts 1 and 9 months post transplant. Holstein (Kiel, D); (9)Children's University Hospital (Muenster, One continued to be transfusion dependant and the other one D) failed the second and third transplants and subsequently died of disseminated candidiasis 10 months post the first Background: Intensive chemotherapy in children with Down transplant. Two other patients (DBA and SCN) succumbed to syndrome (DS) has been associated with a higher risk of pulmonary complications 1and12 months post transplantation, treatment related toxicity and infection-associated respectively. Acute GvHD was seen in one patient and chronic complications. Reports on bone marrow transplantation in gut GvHD in another. children with DS are limited. The overall survival (OS) and event-free survival for the whole Methods: Histories of 11 children with acute leukaemia and group were 86.2% and 75.4 % respectively. DS who underwent bone marrow transplantation (BMT) in In conclusion, allogeneic stem cell transplant from a match- eight German/Austrian BMT centers between 1996 and 2006 related donor for children with constitutional single cytopenia were analysed retrospectively. Indications for transplantation disorders is a curative and safe procedure. were acute lymphoblastic leukaemia (ALL) in 8 cases (2 x first complete remission (CR1, 4 x CR2, and 2 x CR3) and acute myeloid leukaemia (AML) in 3 cases (2 x CR2 and 1 x partial response after first relapse). Five children received a graft from an HLA-matched sibling, four from an HLA-matched unrelated donor, one from an HLA-matched mother, and one from an haploidentical mother. Conditioning regimens included total body irradiation (TBI) in seven children. A reduced intensity (RIC) protocol containing 2 Gy TBI and fludarabin was given to two patients, another five received a myeloablative regimen with 12 Gy TBI either combined with cyclophosphamide or etoposid. Treosulfan or busulfan were used in the remaining four children. Results: 4/11 (36%) patients are alive at 8, 12, 39, and 63 months after BMT. Three of them were treated with 12 Gy TBI, one received busulfan instead. Main cause of treatment failure was relapse. All relapsed patients (5/11) died 3, 7.5, 8.5, 13, and 20 months after BMT, respectively. 2 children died of transplant related complications, one from severe exfoliative dermatitis and sepsis after a treosulfan-containing regimen, the other from GVHD-related infections after RIC. Unusual regimen related toxicity (RRT) was reported in 3/11 (27%) patients, including exfoliative dermatitis, sepsis, pancreatitis, renal insufficiency, pulmonary edema, and severe stomatitis. Acute GvHD of the skin was observed in 10/11 patients. Conclusion: In our small group of patients relapse was the major cause of death rather than RRT. A fully myeloablative conditioning regimen was used in all 4 surviving patients. The incidence of skin GVHD seems to be high.

S76 P466 Allogeneic stem cell transplantation for JMML: P467 single-centre experience Allogeneic stem cell transplantation for acquired severe O. Mosleh, M. Ayas, H. El-Solh, A. Al-Jefri, A. Al Seraihy, S. aplastic anaemia in paediatric patients in the Czech Rifai, M. Al -Mahr, A. Al-Ahmari Republic – clinical outcomes: retrospective study King Faisal Specialist Hospital (Riyadh, SA) (1991 – 2006) E. Pindurova (1), P. Sedlacek (1), R. Formankova (1), P. Juvenile myelomonocytic leukemia (JMML) is a rare childhood Keslova (1), K. Zdrahalova (1), M. Sukova (1), V. Vavra (1), P. leukemia for which the allogeneic stem cell transplantation Smisek (1), P. Kobylka (2), M. Dobrovolna (2), M. Vrana (2), (SCT) is the only curative therapeutic option. Eleven L. Kupkova (3), B. Malinova (1), J. Stary (1) consecutive patients (age 0.7-6.9 years) with JMML (1)University Hospital Motol (Prague, CZ); (2)Institute of underwent allogeneic SCT at our institution between 1995 and Hematology and (Prague, CZ); (3)Czech 2005.The median time to transplant is 6 (range: 3-14) months. Bone Marrow Donor Registry (Prague, CZ) All patients but one were pretreated with chemotherapy and 3 patients were splenectomised prior to transplant. At the time Allogeneic stem cell transplantation (SCT) is a standard part of conditioning all patients were having stable disease. The of a treatment algorithm in childhood acquired severe aplastic grafts were related marrows in 9 patients (HLA-identical anemia (SAA), either as a first option in case when HLA relative n= 8 and one-antigen mismatched mother n=1) or matched sibling (MSD) is available or as an alternative given mismatched unrelated cord in 2 patients. to the patients who failed previous immunosuppressive Six patients were conditioned with busulfan and therapy (IST). In a summarizing report, we analyze our 16- cyclophosphamide (BU/CY) regimen and the remaining five year (I/1991 – X/2006) experience with allo-SCT for SAA in patients were conditioned with busulfan, cyclophosphamide children all over the Czech Republic. At the only pediatric and Etoposide (BU/CY/VP-16) in standard dosages. GVHD transplant centre providing allo-SCT in our country so far 34 prophylaxis consisted of cyclosporine and short-course of patients (17 male, 17 female) of median age 13 years (range methotrexate in 7 patients or steroid in 2 patients. 2 - 18) underwent SCT for associated SAA (n=7), All patients but one engrafted at a median time of 25 days idiopathic SAA(n=26) or MSAA/MDS-RC (n=1). 25 children (range: 15- 34) post stem cell infusion. Grade 1 acute skin (73.5%) underwent SCT using MSD (2-27 years, median 14). GVHD was observed in one patient only and veno-occlussive 9 of them (1991-1994) received prior SCT corticoids and/or disease (VOD) in 3 patients. anabolics, 16 underwent SCT as the treatment of choice. The Three out of the eleven patients relapsed at a median time of interval between diagnosis and transplantation ranged from 15 42 days post transplant and subsequently died of progressive to 1250 days (median 32). Bone marrow (BM) was used as a disease. One engrafted patients lost his graft at 245 days post stem cell source in all but 4 (16%) patients, who received transplant and had autologous marrow recovery and remained PBSC (combined with BM in one). 84% of patients were in remission for 8 years post transplant. One patient failed to conditioned with ALG/ATG and cyclophosphamide (CYC). engraft and died of severe VOD and sepsis. All patients who With standard MTX/CsA prophylaxis, acute GVHD grade II-IV received VP-16 as a part of their conditioning regimen developed in 12.5% and extensive chronic GVHD in 14 % of continued to be long-term survivors. them. 9 patients, who failed previous even repeated courses In summary, allogeneic SCT has dramatically improved the of IST (ALG or ATG/CsA), received grafts from unrelated JMML survival. Adding VP-16 to the conditioning regimen donors (UD; n=8) or HLA identical parent (n=1). The interval might have a favorable outcome however this finding merits a between diagnosis and transplantation ranged from 212 to prospective evaluation in a larger cohort of patients. 1713 days (median 360). 8 patients were conditioned with fractionated total body irradiation (TBI) (6-14.4 Gy) and CYC (120mg/kg), 1 patient received single dose TBI (200cGy), fludarabine and CYC. All received ATG. Acute graft rejection occurred in the latter patient, who afterwards received a second graft from the same donor. Acute GVHD grade II-IV was observed in 66%, chronic GVHD occured so far in 28% of patients in risk. At a median follow up of 68 months (2-191) the overall survival of the whole cohort is 94%, with transplant- related mortality of 6% (mucor and aGVHD gr.III-IV were the primary causes of death in two patients). Our experience is proving the benefits of allo-SCT in children suffering from SAA, current conditioning regimen used in MSD SCT is satisfactory. We are now focusing on decreasing the risk of later side effects by reducing TBI in recipients of grafts from UD. Partly supported by grants VZ FNM 00064203, IGA CR NR 7984

S77 P468 cardiac or gastrointestinal symptoms. >89% of pts had no CD34+ haematopoietic progenitor cell selection of bone prior fungal infection. marrow grafts for autologous transplantation in paediatric Statistical analysis of demographic characteristics to predict patients who would be at risk for abnormal CT findings identified a K. Kasow, L. Sims-Poston, P. Eldridge, G. Hale trend towards significance only in the auto cohort. Males were St. Jude Children's Research Hospital (Memphis, USA) more likely to have abnormal abdominal findings than females (P=0.053), and the other race group was less likely to have Objectives: CD34+-selection of hematopoietic grafts for abnormal sinus findings than Caucasians (P=0.038). For patients undergoing autologous hematopoietic stem cell those with abnormal CT findings, the existence of a transplantation (HSCT) is frequently used to obtain a tumor- relationship between findings being 1) primary disease and free graft. The majority of published experience is with non-infectious related (DR) and 2) infectious related and not peripheral blood stem cell (PBSC) products; only scant related to the primary disease (DU) was investigated. For allo information has been published on bone marrow grafts. We pts with abnormal chest/abdominal CTs, only diagnosis at reviewed our experience using CD34+-selection of bone enrollment showed a trend towards significant difference marrow grafts in children undergoing autologous bone marrow between those with DR findings and those with DU findings transplantation (BMT). (P=0.046, chest; P=0.029, abdomen). For auto pts, age at Methods: After obtaining institutional approval, we performed HSCT showed a trend towards significant difference between a retrospective review of the medical records of patients who those with DR chest abnormalities and DU findings (P=0.055) underwent autologous stem cell collection at St. Jude. From while diagnosis was significant for abdominal abnormalities January 1, 1999, to December 31, 2003, 373 patients (P=0.018). underwent autologous HSCT; 133 received marrow grafts, Conclusion: Based on these findings, routine CT imaging of 232 received PBSC grafts, and 8 received a combination. the chest and abdomen may not be warranted in Seventeen patients, 15 with neuroblastoma and 2 with non- asymptomatic pts; this change would decrease patients’ Hodgkin lymphoma, underwent bone marrow harvests for radiation exposure. Sinus imaging, which was frequently CD34+-selection of their stem cell grafts. Grafts were abnormal, may be justified to help plan post-HSCT supportive analyzed for residual tumor when appropriate. All grafts were care. included in the processing analysis. Results: Seventeen patients had 20 grafts processed on the Isolex 300i Magnetic Cell Selection System® device, and 16 P470 received 19 CD34 purified grafts. Four patients were not Fludarabine, cyclophosphamide plus thymoglobulin included in the engraftment analysis: a) one did not receive conditioning regimen for unrelated bone marrow the collected product, b) one received a tandem product, and transplantation in paediatric severe aplastic anaemia: c) two received products that were composed of 2 or 3 phase II prospective multicentre study in Korea combined purified products. Following selection, marrow H.J. Kang, H.Y. Shin, J.E. Park, B. Cho, H.K. Kim, Y.H. Lee, grafts contained a median of 1.4 x 106 CD34+ cells/kg (range, K.H. Yoo, K.W. Sung, H.H. Koo, H.S. Ahn on behalf of The 0.09 - 8.3 x 106/kg) and a median of 0.014 x108 TNC cells/kg Korean Society of Pediatric Hematology-Oncology (range, 0.001 - 0.09 x 108/kg). The median CD34% recovery was 30.9% (range, 9.3-57.1%), with the median CD34 purity Anti-thymocyte globulin (ATG) has been used in severe being 95.5% (range, 62.2%-98.8%). All patients engrafted. aplastic anemia (SAA) as a part of the conditioning regimen. The median time to ANC • 500/mm³ was 19 days (range, 12 – Among the many kinds of ATG preparations, thymoglobulin 35 days), and to platelet recovery was 28 days (range 18 - 37 had been found to be more effective in preventing graft- days). No patient died from transplant-related complications. versus-host disease (GVHD) and rejection of organ Conclusion: Our study demonstrates that CD34+-selection of transplants. After the promising result of the pilot study (Bone marrow grafts is feasible and these grafts are able to Marrow Transplant. 2004. 34; 939), phase II prospective multi- successfully reconstitute hematopoiesis in patients center clinical trial was performed with fludarabine, undergoing autologous BMT. To our knowledge, this is the cyclophosphamide plus thymoglobulin conditioning regimen to first series describing CD34+-selection involving bone marrow allow good engraftment in unrelated bone marrow grafts. transplantation (BMT) for SAA and preliminary results are presented here. Ten patients underwent BMT with cyclophosphamide (50 mg/kg once daily i.v. on days -9, -8, -7 P469 & -6), fludarabine (30 mg/m² once daily i.v. on days -5, -4, -3 & Clinical utility of screening chest, abdomen, and sinus -2) and thymoglobulin (2.5 mg/kg once daily i.v. on days -3, -2 computed tomography prior to haematopoietic stem cell & -1) from HLA matched unrelated donors. GVHD prophylaxis transplantation: a single-centre experience regimen was composed of cyclosporine, methotrexate (15 K. Kasow, J. Krueger, J. Okuma, D. Srivastava, G. Hale mg/m² i.v. on day 1, 10 mg/m² i.v. on days 3 and 6) and St. Jude Children's Research Hospital (Memphis, USA) thymoglobulin (1.25 mg/kg once daily i.v. on days 7, 9 and 11). The median infused cell dose of nucleated cells and Objective: Prior to hematopoietic stem cell transplantation CD34 positive cells were 3.3x108/kg (1.6-33.9x108/kg) and (HSCT), all patients (pts) underwent CT chest, abdomen, and 2.9x106/kg (1.5-14.7x106/kg), respectively. The median sinus to screen for infections. However, it is unclear whether number of days required for ANC of more than 0.5x109/l and this extensive imaging evaluation is warranted. 1.0x109/l were 16.5 days (10-35 days) and 19 days (11-40 Methods: Medical records of HSCT pts at St. Jude from Jan.1, days), respectively. Complete donor type hematologic 2004 to Dec. 31, 2005 were reviewed. Of 198 pts identified, recovery was achieved in all patients. Five patient developed 11 were excluded for tandem HSCTs, no pre-HSCT scans grade II-III acute GVHD resolved after treatment. Three performed, or greater than 2 months between imaging and patients died about 2 months after BMT owing to the HSCT; 187 were included in the final analysis. coagulopathy, pneumonia, and cardiac infarction, respectively. Results: 131 pts received allogeneic (allo) grafts and 56 All the other 7 patients are still alive with median 136 days. autologous (auto) grafts; females compromised 51 allo and 25 Fludarabine, cyclophosphamide plus thymoglobulin auto pts. The mean age at HSCT was 10.6 years (allo) and conditioning allows for the promising result of good 10 years (auto). 73 allo and 39 auto pts were Caucasian. engraftment, although serious events occurred in some Peripheral blood stem cell (60%) or bone marrow (40%) grafts patients. More patients will be enrolled in this study to detect were infused. Of the allo pts, 48.1% received related improvement of engraftment and survival rate than historical mismatched grafts and 30.5% received HLA-matched control in literatures. unrelated grafts. Clinical symptoms and physical examination were reviewed in the medical record to correlate with imaging findings; >85% of the allo and >80% of auto pts did not have sinus, pulmonary,

S78 P471 CR1=4, CR2=5), NHL (n=1) acute (n=5; CR1=1, CR2=4) and Therapy of haematological relapse of acute leukaemia chronic myeloic leukemias (n=3), MDS (n=3) and Wiskott- following allogeneic stem cell transplantation in children Aldrich (n=1). Median age was 10 years (0.5-18). The donors R. Formankova, P. Sedlacek, P. Keslova, L. Sramkova, J. were matched for HLA-A/B (medium resolution typing) and Trka, E. Fronkova, K. Muzikova, E. Mejstrikova, O. Hrusak, H. DRB1/DQB1 (high resolution). A short course of MTX (2-3x10 Zizkova, J. Stary on behalf of CPH and CLIP mg/m²) and CSA (2-3 mg/kg, adjusted to blood levels) until day 100 were given. 21/22 patients had primary engraftment Relapse of leukemia remains the major cause of treatment with a median time to ANC >500 of 18 days (12-60). No G- failure in allogeneic stem cell transplantation (SCT). Between CSF was given. One patient rejected his graft and was I/1994 and VII/2006 a total of 120 children with acute leukemia successfully retransplanted from another MUD. Platelet (ALL - 79, AML - 36 and acute hybrid leukemia; AHL - 5) were recovery was fast (median time to reach independence from transplanted at our unit. The median age at first SCT was 9,4 substitution: 20 days). Mean numbers of CD3+, CD3+CD4+ years (ranged 1,5 to 20,5). Donors were HLA-identical siblings and CD3+CD8+ on day 180 (365) were 259/µl (868/µl), 100/µl (48), unrelated donors (63), matched family donors (3) or (400/µl) and 146/µl (456/µl). 19/22 patients had GvHD grade mismatched family donors (6). Eight grafts were T-cell 0-I (86%), 1 and 2 patients had grade II and III, (5%, 10%) depleted. EFS of this cohort is 57%, 18% died due to respectively. Chronic GvHD occurred in 2 patients (10%). transplant related mortality (TRM), 13% died in a 15/22 patients are alive with a median follow up of 3 years consequence of leukemia relapse. Post-transplant relapse (1.5-5). 3 year EFS was 64% (all patients), 50% (ALL/NHL) following first allogeneic SCT was documented in 34 (28%) and 73% (myeloic leukemias /MDS). Relapse probability at 2 patients (23 ALL, 11 AML) 34 to 1091 days (median 191) after years was 25%, probability of TRM at 2 years was 15%. SCT. Intensive therapy of hematological relapse was initiated Causes of death were relapse (n=4) and infections (n=3). in 18 (53%) patients (11 ALL, 7 AML). Chemotherapy (CHT) Thus, stable and favourable survival rates with a low was aplied to 10 patients (6 ALL, 4 AML), CHT combined with incidence of GvHD were achieved. The method allows to donor lymphocyte infusion in 4 ALL patients, gemtuzumab administer clearly defined T cell numbers independent from ozogamicin in 2 AML patients and dasatinib in one patient with the size of the grafts. This may be of advantage in particular in Ph+ ALL. Additional local radiotherapy was applied in 2 small children and if the donor does not accept a bone marrow patients with ocular and testicular relapse respectively, 1 harvest: all available stem cells can be infused without the patient underwent second SCT without previous CHT. Mixed limitation of unacceptable high T cell numbers. chimerism was documented in peripheral blood samples using PCR of VNTR in 16 of 18 patients at the start of post- transplant relapse treatment. After the first line treatment 3 P473 patients (ALL) died due to infectious complications and 5 Haplo-identical transplantation following ''FLAMSA-RIC'' patients (2 ALL, 3 AML) due to disease progression/further conditioning in children with refractory acute leukaemia relapse. One patient treated for isolated testicular relapse of F.R. Schuster (1), M. Führer (1), S. Behrendt (1), J. Hauer (1), ALL is alive in further complete remission 13 months after J. Tischer (2), G. Ledderose (2), M. Sauer (3), T. Feuchtinger diagnosis of post-transplant relapse and was not re- (4), M. Hammer (5), D. Prevalsek (2), I. Bigalke (6), C. Schmid transplanted. Nine patients underwent second allogeneic SCT (7), R. Buhmann (6), H.J. Kolb (2), A. Borkhardt (8) using the same (8) or other (1) donor in hematological (1)Dr. von Haunersches Kinderspital (Munich, D); (2)Klinikum remission 10,5-39 months (median 16) after the first SCT, all Großhadern (Munich, D); (3)Hannover Medical University of them again achieved complete donor chimerism before (Hannover, D); (4)Children's Hospital (Tuebingen, D); second transplantation. Three of 9 patients died due to TRM (5)Charité (Berlin, D); (6)GSF Haematologikum (Munich, D); (1) or further relapse of leukemia (2). Six patients (2 ALL, 4 (7)Klinikum Augsburg (Augsburg, D); (8)Oncology and AML) are alive and well in continuous complete remission 4 to Immunology (Duesseldorf, D) 113 months (median 22.5) after second SCT. In both surviving ALL patients minimal residual disease (MRD) was detectable Objectives: Although survival rates of children with acute prior to first SCT and they achieved MRD negativity before lymphoblastic leukemia (ALL) or acute myeloid leukemia second transplantation. Intensive chemotherapy and re- (AML) increase in the last decades, the subgroup of patients transplantation might be considered in patients in a good with refractory acute leukemia (RAL) still have a very bad clinical status. outcome. Allogeneic stem cell transplantation (SCT) remains Partially supported by VZ FNM MZ 000 64203 the therapy with the highest potential for cure. However, the rapid course of RAL often allows only limited time to identify an human leukocyte antigen (HLA)-matched donor. P472 Haploidentical family members are readily available. Based on Favourable long-term survival and low incidence of GvHD the encouraging data obtained from adults in the HLA- after transplantation of positive selected stem cells with identical setting (Schmid et al., Blood 2006) we adapted the T-cell add-backs from unrelated donors in children FLAMSA-RIC protocol for haploidentical SCT in children with P. Lang (1), J. Greil (2), P. Bader (3), U. Mura (1), M. Schumm RAL. (1), T. Feuchtinger (1), M. Pfeiffer (1), R. Handgretinger (1) Methods: Five children (age 1.5-15 years) with RAL (2x ALL, (1)University Children's Hospital (Tuebingen, D); (2)University 3x AML) received a SCT from their haplo-identical mothers. Children's Hospital (Heidelberg, D); (3)University Children's The three AML patients showed blasts in the bone marrow Hospital (Frankfurt, D) (BM) at the time of SCT, one ALL patient was in aplasia, one was in cytological remission. Conditioning consisted of high There is still a significant risk of GvHD in transplantation of dose cytosine arabinoside, amsacrine and fludarabine peripheral stem cells from matched unrelated donors, despite (FLAMSA), followed by total body irradiation with 4 Gy (age the use of ATG and pharmacological prophylaxis. Especially >2 years) or half doses of i.v. busulfan (<2 years), followed by in the case of young pediatric patients and adult donors, grafts cyclophosphamide and anti-thymocyte globulin. The recipients with high numbers of stem cells but also with extreme receive unmanipulated BM on day 0 and CD6-depleted amounts of T cells can be obtained. peripheral blood stem cells at day 6. Cyclosporine A and We present a current update of a clinical study with T cell methotrexate were given in standard doses for graft versus reduced grafts in 22 patients, which offer both large stem cell host disease (GvHD) prophylaxis. numbers and clearly defined amounts of T cells. Positive Results: Conditioning was well tolerated. All patients showed selection of peripheral stem cells was done with CD34 or stable engraftment (d+19 - +38). Four of five showed a full CD133 coated magnetic microbeads and the CliniMACS™ donor chimerism on d+30 in the BM, in three of them full device. Afterwards, an unselected aliquot containing chimerism still remains. Three patients developed acute 10x106/kg T cells was added to the purified stem cells and GvHD I-II, two patients suffered from extended chronic GvHD. infused on day 0, resulting in a median T cell depletion of 2 All episodes of GvHD respond to immunsuppressive log. The diagnoses were: acute lymphatic leukemia (n=9; treatment. Further complications consisted of Adenovirus and

S79 Epstein Barr Virus reactivation, which were successfully versus host disease (GvHD) prophylaxis in all cases. treated with antiviral drugs and specific donor T-cells. Four of Neutrophil engraftment was seen at a mean of 15 days (range the five patients are still alive (d+114 - +395), one of these 11-20 days) and platelet engraftment at a mean of 49 days relapsed on day +243. The fifth patient died on day +116 after (range 12-60 days). 3 episodes of primary graft rejection relapse in the central nervous system. occurred in 2 patients. Both these patients engrafted Conclusion: Haploidentical transplantation with “FLAMSA- successfully after a haploidentical parent was used. 3 patients RIC” conditioning may offer a therapeutic option in children showed complete chimerism (CC) at day 28 and this has suffering from RAL. In all five children complete engraftments persisted. 2 patients that showed increasing mixed chimerism and moderate toxicity were observed. An effective graft versus (i-MC) were treated with escalating doses of donor leukemia reaction can be postulated, as in three of five cases lymphocyte infusions (DLI) with fludarabine. This resulted in an ongoing hematological remission can be observed. CC in one patient and stable mixed chimerism in the other. 4 patients developed grade 2 acute skin GvHD (2 post-DLI), 1 patient developed chronic skin GvHD. Clinical improvement P474 or stabilisation was seen in 4 patients. Disease progression Outcome of child recipients of blood and marrow occurred in 1 patient in spite of CC. There was no transplant transplantation requiring intensive care related mortality. 1 patient developed acute immune A. Faggyas, E. Ujhelyi, G. Krivan thrombocytopenic purpura which needed immunoglobulin Szt. László Hospital (Budapest, HUN) therapy. Hypercalcemia needing pamidronate therapy was seen in 1 patient. Both have resolved. We have shown that Introduction and methods: Incidence, chacteristics and the use of alternate stem cell donors, especially haploidentical outcome of stem cell transplant (SCT) pediatric patients parents, in the absence of a fully matched sibling donor, is a requiring intensive care was studied retrospectively in a safe, readily available and effective transplant option that is teaching hospital for infectious diseases. likely to improve the outcome in children with malignant Results: Pediatric stem cell transplantation was performed in osteopetrosis. 182 children in our institution between 1992 and 2006. Several patients required more than one SCT (n=192). From the 182 pediatric patients with SCT, 40 patients (22%) P476 had 54 admissions to the pediatric intensive care unit (PICU). Single dose of peg-filgrastim in children with poor-risk Mean age of the 24 boys and 16 girls was 85 ± 64 (median= malignant tumours receiving chemotherapy. 66) months. Mean PRISM-score on admission was 7±6 Enhancement of neutrophils recovery and peripheral (median= 6). Overall mortality was 21/40 (52,5 %). The most blood progenitor cells mobilization frequent reasons for PICU admission were pulmonary S. Dallorso (1), E. Castagnola (1), T. Emanueli (1), L. Scarso complications 30/54 (55,5%), hemodinamic failure 4/54 (1), M. Iannachino (1), R. Haupt (1), F. Fagioli (2), M. Miano (7,5%), neurologic disease 8/54 (15%) and other 12/54 (22%). (1) Distribution according to days from SCT to admission in PICU (1)IRCCS Gaslini (Genoa, I); (2)Regina Margherita Children's was: 10/54 (0-30 days), 14/54 (31-100 days), 30/54 (>100 Hospital (Turin, I) days). Mechanical ventilation (MV) was required in 27 of the 54 Objectives: to evaluate efficacy of peg-filgrastim (PEG), admissions (50 %). Mortality in patients requiring MV and not administrated after chemotherapy, in enhancing neutrophils requiring MV was 19/25 (76%) and 2/15 (13 %), respectively. recovery and peripheral blood progenitor cells (PBPC) Acut respiratory distress syndrome (ARDS) was diagnosed in mobilization, in children suffering from malignant tumours. 14 patients with a mortality rate 11/14 (78%). Twenty-seven Methods: From August 2005 to October 2006 25 children patients had multiorgan failure (MOF) involving 3 or more received at least 1 dose of PEG and were assessable. organs. Sepsis was proven in 20/54 cases (37% of all Diagnosis were Neuroblastoma (10), bone (8), brain (3) and admissions) with a high mortality rate (80%). Distribution of 20 renal tumours (2), non-Hodgkin lymphoma and patients according to graft versus host disease (GVHD) was Hepatoblastoma. Inclusion criteria were: age < 18 years, 9/20 Grad III-IV and 11/20 Grad I-II. diagnosis of high-risk solid tumour or non-Hodgkin lymphoma Discussion: Mortality rate was high in patients with sepsis, without marrow involvement, myelosuppressive treatment, ARDS, MOF and in mechanically ventilated children. Our Karnofsky status > 50 and adequate renal, hepatic and results indicate that in case of life-threatening complications, cardiac function. A single subcutaneous injection of PEG, at requirement for PICU support is indicated in SCT children, dose of 100 mcg/kg, was given 3 days after the end of because the overall survival was 46% and 24% in ventilated chemotherapy. Primary endpoint: duration of grade 4 patients. neutropenia (absolute neutrophils count (ANC) < 0.5 x 109/L). Secondary endpoints: incidence and characteristics of febrile neutropenia (FN), myeloid recovery (ANC > 0.5 x 109/L), P475 characteristics of PBPC mobilization (days from CD34 + cells Use of alternate stem cell sources for allografting in raise until their fall <20 CD34+ cells/microL (threshold level for malignant infantile osteopetrosis: single-centre collection) and of PBPC harvest (CD34+ cells x experience 106/kg/apheresis). Chemotherapy before PEG was: Cyclo and L. Karnik, G. Kochethu, M. Velangi, S. Lawson, P. Darbyshire Etoposide alone or in combination, IVADO Birmingham Children's Hospital (Birmingham, UK) (Ifo+VCR+Doxo+Act-D), Ifo+ Carbo+ Etoposide, Topotecan+ VCR+ Doxo. Malignant infantile osteopetrosis (MIOP) is a lethal disorder of Results: All patients experienced grade 4 neutropenia, lasting osteoclast dysfunction. Haematopoietic stem transplantation is a median of 5 days. Only 2 patients complained about mild the only curative treatment currently available. The best bone pain responsive to paracetamol. Myeloid recovery results are seen with a fully matched sibling donor; however occurred at a median of 6 days (range 4-9) from PEG the use of alternate donors is being explored. Early administration. Threshold CD34+ cells level for PBPC allografting is essential in order to minimise permanent collection was reached in 14 out of 20 children in whom CD34 neurosensory deficit. We studied 5 children with MIOP who was monitored (median value 147/microL, range 20-421) and underwent 8 allograft procedures in our unit in 3 years. The lasted for a median of 6 days (range 4-9). 12 children, mean age was 18 months (2-53 months). Donors were candidate to autologous stem cell transplantation (SCT) were matched sibling (n=1), unrelated cord (n=2) and haploidentical able to collect more than 4 x 106/kg CD 34+ cells. In 10 cases parent (n=5). The mean CD34 dose was 8.4X106/kg and a only one apheresis was performed. dose of 10X106/kg was achieved with all haploidentical grafts. Conclusions: PEG can be safely administered in children, Common conditioning regimes were used. Haploidentical myeloid recovery is similar to that seen with filgrastim, PBPC grafts were T cell depleted and patients received serotherapy mobilization was possible in 70% of attempts. Failures were (ALG in 4 and OKT3 in 1). Cyclosporine was used for graft observed in patients older than 10 years, heavily pre-treated

S80 or with previous spinal radiotherapy. A collection window Cell Factor (SCF) S/C at 20 µg/kg/d with GCSF (5 or 10 wider than that usually observed with filgrastim was often µg/kg/d) used in Onco-Pediatric patients in this special observed. A prospective randomized comparative study is context. then needed and it is in progress at our Institutions. Patients: Between 11/2000 and 08/2006, 32 children, median age=14,2 years [range: 1,1-22,2], with high risk solid tumor (neuroblastoma=8, ewing-PNET=6, rhabdomyosarcoma=4, P477 medulloblastoma=3, nephroblastoma=2, osteosarcoma=2, High rate of stable allogeneic engraftment in heavily others solid tumors=3) or lymphoma (Hodgkin Disease=3, pretransfused children with SAA after conditioning with Burkitt type=1), with a mean of 2[1-3] mobilization failure were treosulfan, cyclophosphamide and rabbit ATG treated with SCF+GCSF in steady state (30 mobilizations) or J. Owoc-Lempach, K. Kalwak, E. Gorczynska, M. Slociak, D. after chemotherapy (4 mobilizations). 2 pts were twice Wójcik, A. Dyla, D. Turkiewicz, A. Chybicka mobilized with this association. Median number of prior Wroclaw Medical University (Wroclaw, PL) chemotherapy cycles was 7 [4-26]. The SCF was administered during hospitalization after a medical treatment The aim of the study was to analyze outcome in heavily combining Cetirizine, Ranitidine and Salbutamol. pretransfused children with SAA undergoing allogeneic HSCT Results: No serious anaphylactic effect due to SCF was after conditioning containing treosulfan, cyclophosphamide observed. After a mean delay of 4 [3-7] days for the pts and ATG in comparison with pts conditioned without mobilized in steady state and 15 days for the pts mobilized by treosulfan. CT, 44 were performed among 23/32 pts with a Twelve pts (Group 1) with median age 10.8 years (range 4.5- mean of 2 [1-4] leukapheresis by pt. Harvest allows 2,83 [0,5- 17.8), median interval between dgn and HSCT 705 days 7,27] x 106 CD34+/kg and 29,41 [6,2-114] x 104 CFUGM/kg (range 75-4968) underwent allogeneic HSCT from unrelated per child. The correlation CD34+/CFUGM (10,3) was adequat. donors (UD, 10 pts) or from matched sibling donors (MSD, 2 The number of circulating CD34+ cells is higher at d5 [22(9- pts) after conditioning regimen consisted of TREO 30g/m², CY 40)/µl] for the pts collected in steady state. 14/32 pts had a 120 mg/kg and ATG-Fresenius 30mg/kg. All pts but one PSC collection > 2,5 x 106 CD34+/kg. Other hematopoietic received an immunosuppressive treatment before HSCT and stem cell collection was necessary in 9 cases (bone all were transfusion-dependent. Source of SC was PB in 9 pts marrow=4). 21/32 pts received at least 1 [1-3] intensive with median of CD 34+ cells 13.7 x10(6)/kg and BM in 3 pts treatment with autologous PSC alone or with bone marrow with median of CD 34+ cells 2.3 x106/kg. transplantation. Thirteen pts (Group 2) with median age 12.8 years (range 1.6- Conclusion: With non toxicity, the combination of SCF+GCSF 20), with shorter median interval between dgn and HSCT 173 is efficient to collect PSC in 72% of poor mobilizer onco- days (range 30-1338) underwent allogeneic HSCT from UD (4 pediatric pts, allowing a collection of more than 2,5 x 106 pts), MSD (8 pts) and from HLA-mismatched relative (1pt) CD34+/kg among 44% of them. after conditioning regimen consisting of : CY 120 and ATG (9 pts), CY 120, FLU 120, ATG or Campath (3pts) and BU 16, FLU 120, CY 120 and ATG (1pt). All pts but six received an P479 immunosuppressive treatment before HSCT and all were HbF output following cord blood allogeneic transplant transfusion-dependent. Source of SC was PB (4 pts) with mirrors HbF output following birth and is evidence for median of CD 34+ cells 5.3 x106/kg and BM (9 pts) with intrinsic stem cell programming median of CD 34+ cells 1.3 x106/kg. J. Ruell, T. Carr, C. Hutchinson, H. Church, M. Gharib, J. In the Group 1 no graft failure occurred. Engraftment with Wraith, R. Wynn ANC (>500/ul) and platelet (>50 000/mcl) occurred at a Royal Manchester Children's Hospital (Manchester, UK) median of 16 days (range 14-25) and 21 days (range 12-37), respectively. Two pts died before engraftment because of Following cord engraftment we noted that during preparation sepsis and cerebral edema, respectively. With a median of a white cell pellet, acid resistant red cells were found to be follow-up of 522 days (range 48-841) 8/12 pts survived with present. Using the principle of the Kleihauer test which relies complete chimerism (CC) in 5 cases or mixed chimerism (MC) on the acid resistance of foetal cells we postulated that this in 3 cases. Two further pts died due to TTP and cerebral represented Hb F red cells within the peripheral blood. In this haemorrhage, respectively. In the Group 2 all pts but one paper we document the Hb F output of engrafted cord blood engrafted with neutrophil and platelet recovery occurring at a transplant recipients. median of 16 days (range 11-24) and 32 days (range 14-960), Hb F levels were determined by flow cytometry in 4 patients respectively. With a median follow-up of 1837 days (range undergoing cord transplantation, minimum follow up time was 813-2949) 8/13 pts survived with CC in 1 case or MC in 7 6 months. These 4 patients have been transplanted for cases. 2 pts required a second HSCT from the same donor metabolic disorders, unlike the majority of case reports because of graft rejection, 2 pts relapsed. 5 pts died due to reporting Hb F levels have been based on patients TRM. transplanted for red cell disorders. Our data confirm that conditioning with TREO, CY and rabbit The results show mean levels of Hb F at 3 months of 50%, ATG in heavily pretransfused children with SAA undergoing and at 6 months levels of 24%. Peak levels occurred at 116 PBSCT or BMT from UD or MSD is associated with high rate days post transplant. 3 patients achieved full chimeric status, of stable allogeneic engraftment. and achieved HbF levels of 50, 43, and 83 at 3 months, whilst the patient who achieved chimerism of 40%, HbF levels at 3 months of 22%. P478 Our results show as one may predict a similar pattern of Hb F Mobilization of haematopoietic stem cell by stem cell levels comparable to a newborn where Hb F levels of 60-90% factor + GCSF in poor mobilizer onco-paediatric patients at birth fall to adult levels at 6 months. Hb F levels are likely to P. Brault (1), D. Valteau-Couanet (2), D. Orbach (1), P. correlate with engraftment. This data provides evidence for an Voultoury (3), J. Michon (1), C. Boccaccio (3), O. Hartmann intrinsic stem cell pre-programming of the Hb, rather than an (2) environmental trigger for the Hb switching mechanism. (1)Institut Curie (Paris, F); (2)Institut Gustave Roussy (Villejuif, F); (3)EFS Ile de France site IGR (Villejuif, F)

The classical schema of Peripheral Stem Cell (PSC) mobilization [chemotherapy (CT)+ GCSF or GCSF in steady state] are efficient to collect frequently a sufficient number of CD34+ cells to administer high dose chemotherapy (CT). However there is a low fraction of patients (pts) who are “poor mobilizer”. We report the results of the combination of Stem

S81 were considered high risk if they had one of the following risk factors: tumor volume > 200 ml, a poor prognosis tumor site (pelvis, chest wall, vertebra) or metastasis. treated with high dose TT (750 mg/m²), CPM (60 mg/kg), VP16 (600 mg/m²) (ETC conditioning regimen) followed by autologous stem-cell- rescue. Median age at diagnosis and at transplantation was 6,9 yr (range 1-13 yr) and 7,6 yr (range 1,6-13,7 yr) respectively. Primary tumour was localised in extremities (6), pelvis (3), paravetebral (3), chest wall (3), abdomen (1), and skull (1). Metastatic disease was detected in 6 pts: lung metastasis alone in 4 cases; lung, bone marrow infiltration and brain localisation in one and diffuse bone marrow infiltration in another one. Before HDC all pts received 7 courses of induction chemotherapy based on Ifosfamide, VP16, P480 Carboplatin, CPM, Adriamicine, Vincristine. was Impact of colony forming units-granulocyte macrophage performed in 12 pts. Complete remission (CR) was achieved on engraftment following unrelated cord blood in 10 pts and partial response (PR) in 7 pts. Three of 6 transplantation in paediatric recipients metastatic pts were in CR after conventional chemotherapy K.H. Yoo, S.H. Lee, H.J. Kim, K.W. Sung, H.L. Jung, H.K. and surgery. Park, H.A. Kim, H.H. Koo Results: Toxicity, evaluated according to Bearman Samsung Medical Center (Seoul, KOR) classification, was mainly haematological. Median absolute neutrophil count recovery > 500/mm³ was at day 10+ (range The number of total nucleated cells (TNC) and CD34+ cells 6-27). Median platelet recovery > 50000/mm³ was at day 19 + has been a commonly used standard to predict engraftment (range 9-114). Oral mucositis was reported in 11 pts: grade II after cord blood transplantation (CBT). Although colony in 9 and grade III in 2. Diarrhoea grade I was reported in 1 pt forming units-granulocyte macrophage (CFU-GM) is a and grade II in 2 pts. No HDC related death occurred. At a representative for clonogenic potential, no study has clearly median overall survival from diagnosis of 43 months (16-104 documented the clinical impact of post-thaw CFU-GM on months) 11 pts are alive in CR, 1 pt is alive in PR and 5 pts engraftment in human subjects after CBT. To investigate the are died for recurrence after HDC. Relapsed occurred at a clinical relevance of CFU-GM in CBT for pediatric patients, we median of 7,5 months (range 3,4-11) from HDC. Kaplan Meier performed CFU-GM assay using post-thaw cord blood 5 yr OS and PFS was 65% in all population and respectively samples along with the counts of TNC, CD34+ cells, and Kaplan-Meier 5yr OS was 78% in non-metastatic pts and 45% CD3+ cells. A total of 71 cord blood units prepared for 53 in metastatic pts. recipients (double unit transplant in 18 patients) were Conclusions: preliminary evaluation shows feasibility of HDC evaluated after thawing. At least 4/6 matched units were with ETC conditioning regimen in the high risk ESFT pts selected for transplantation. Except for 4 patients with marrow treatment with promising results in term of survival and failure such as aplastic or Fanconi anemia, patients were acceptable toxicity. Further study warrants to evaluate conditioned with a myeloablative regimen. The infused cell response rate of ETC HDC. dose per kg of recipient weight was as follows: CFU-GM 1.64±1.83x105, TNC 5.15±2.50x107, CD34+ 1.93±1.52x105, and CD3+ 1.06±0.96x107. Among the 4 parameters of cell dose, CFU-GM and CD34+ counts were significantly lower in those who failed to show donor-derived hematopoiesis (P=0.028 and 0.005, respectively). CFU-GM, TNC, and CD34+ counts were well correlated to engraftment of donor- derived neutrophils (P=0.004, 0.037 and 0.004, respectively), whereas only CFU-GM showed a significant correlation to platelet engraftment (r=-0.385, P=0.024). In double unit transplants, CFU-GM count was the only significant variable predicting engraftment of predominating unit among the cell dose parameters (P=0.006). We conclude that post-thaw CFU-GM is a reliable predictor of predominating unit as well as of rapid engraftment after CBT. It would be beneficial to perform CFU-GM assay for several candidate cord blood units in advance if the cord could provide small aliquots of cryopreserved samples before CBT to avoid selecting the unit that might possess a low clonogenic potential.

P481 High-dose chemotherapy with thiotepa, cyclophosphamide and etoposide in high-risk Ewing sarcoma family tumours paediatric patients: single-centre experience I. Ilari, M.A. De Ioris, R. Pessolano, A. Castellano, A. Serra, A. Jenkner, L. De Sio, R. Cozza, P. Fidani, C. De Laurentis, A. Donfrancesco Ospedale Pediatrico Bambino Gesù IRCCS (Rome, I)

Objective: to asses efficacy and safety of high dose chemotherapy (HDC) with Thiotepa (TT), Cyclophosphamide (CPM) and Etopside (VP16) followed by hematopoietic stem- cell-rescue in high risk Ewing Sarcoma Family Tumors (ESFT) paediatric patients. Methods: from November 1998 to March 2006, 17 pts ( M/F 7/10) with high risk ESFT were included in our study. Patients

S82 P482 Conclusions: In this small series, patients with IMC had a Increased mixed chimerism in stem cell transplanted similar incidence of relapse, GvHD and graft rejection children is not correlated to delayed immuno- compared to non IMC patients. The only cell line with isolated reconstitution IMC was the T cell lineage. In patients transplanted for M. Umaerus (1), A. Fasth (2), K. Mellgren (2), C. Allard (1), J. malignant diseases IMC was not always a predictor of Abrahamsson (2) relapse. (1)Gothenburg University (Gothenburg, S); (2)The Queen Silvia Children's Hospital (Gothenburg, S) P484 Aim of the study: To determine the correlation between Results of second stem cell transplantation in children - increased mixed chimerism (IMC) in T-, B- and myeloid (M) report from a paediatric bone marrow transplantation cells and immuno-reconstitution after paediatric stem cell centre transplantation (SCT). B. Wojcik, K. Drabko, M. Choma, A. Zaucha-Prazmo, J.R. Material and Methods: Forty-six consecutive children with Kowalczyk malignant and non-malignant disorders underwent 51 SCT Children's University Hospital (Lublin, PL) during 2000 – 2006. The patients were followed for 24 months after SCT. Chimerism was analyzed by PCR analysis of short The second stem cell transplantation is burdened with high tandem repeats on separated T, B and myeloid cells. IMC was risk complication, however it is the only treatment option for defined as an increase of recipient DNA >10% above the some patients. lowest value after SCT in any cell-line at >2 consecutive Patients: From1999 to 2006 in our BMT unit 162 children have samples. undergone hematopoietic stem cell transplantation (HSCT): Lymphocyte subsets were analysed by flow cytometry and the 83- autologous (auto SCT) and 79- allogeneic (alloSCT). In 2 proliferative response of lymphocytes after PHA or ConA patients after auto SCT second autologous procedure was stimulation was measured. T cell excision circles (TREC) were performed. 8 children were transplanted twice from allogeneic determined by PCR. All patients were investigated before donors (3-sibling, 5-unrelated) and in all those patients in both transplantation and at 3, 6, 9, 12, 18 and 24 months after. procedures the donors were the same. The overall number of Results: IMC was present in 11 SCT. The regeneration of patients subjected retransplatation in our Unit was 10 (6,1%), lymphocyte subsets (CD3+, CD3+4+, CD3+8+, CD4+45RA+, aged from 5 months to 18 years (median 8,5 years).Among CD4+45RO+ , CD8+45RA+, CD8+45RO+ and CD19+) was those children 5 suffered from malignancies (AML-1, sAML-1, not significantly different in patients with or without IMC. ALL-1, NHL-2) while 5 had nonmalignant diseases (SAA-3, Response to PHA or ConA and level of TREC were also S.Omenn-1, S. Wiscott Aldrich-1). Indication for second SCT similar in both groups. After 24 months 50% of patients still in the group with malignancies was relapse, and in group of had subnormal levels of CD4+ T lymphocytes. patients with nonmalignant diseases - was rejection or graft Conclusion: In this single centre material the presence of IMC failure. The interval between first and second transplantation did not correlate with delayed immuno-reconstitution. This range from 1 to 29 months (median 12,5 months). At the finding needs to be substantiated in a larger material. moment of second SCT 4 children with malignancies were in Furthermore, two years after SCT, half of the patients still had CR (3 in CR2, 1 in CR3), and one child was in partial T cell dysfunction. remission (PR). Conditioning before second SCT was myeloablative in 3 procedures, reduce intensivity (RIC) in 5 and 2 children (SAA) received ATG only as preparative P483 regime. Increased mixed chimerism in stem cell transplanted Results: 9/10 patients engrafted. 4/5 children retransplanted children and its correlation to relapse, rejection and graft- due to relapse are alive and are still in remission, with versus-host disease observation time from 6 to 74 months (median 30 months). C. Allard (1), A. Fasth (2), J. Abrahamsson (2), M. Umaerus Among 5 children with nonmalignant diseases 4 are alive: 3 (1), K. Mellgren (2) with complete hematopoietic recovery and 1 with twolineage (1)University of Gothenburg (Gothenburg, S); (2)The Queen hematopoetic recovery. The most serious complications were: Silvia Children's Hospital (Gothenburg, S) posttransplant limfoproliferative disease (PLTD- 2 patients), and GvHD (III0 -1, IV0-1). The other transplantation-related Aim of the study: The aim of the present study was to complications (infection, organ toxicity) were mild. 2 children determine the presence of increased mixed chimerism (IMC) died (20%): 1 due to GvHD IV0 and 1 due to PLTD. in T-, B- and myeloid (M) cells after paediatric stem cell Conclusions: Our results suggest, that second SCT may be transplantation (SCT) and its correlation to diagnosis, considered as therapeutic option for patients with relapse or presence of graft-versus-host disease (GvHD) and relapse or graft failure. The choice of preparative regimen should be rejection of the graft. made individually, depending on the patient condition, Material and methods: Forty-six consecutive children with diseases status and previous treatment. Transplant- related malignant and non-malignant disorders underwent 51 SCT toxicity and mortality in our group of patients are acceptable, during 2000 – 2006. The patients were followed for 24 months which may be granted nonmyeloablative conditioning in 70% after SCT. Chimerism was analyzed by PCR analysis of short of the all procedures. Long term observation is needed for tandem repeats on separated T, B and myeloid cells IMC was better evaluation of second SCT results. defined as an increase of recipient DNA >10% above the lowest value after SCT in any cell-line at >2 consecutive samples. P485 Results: IMC was observed in 11 patients, transplanted for The presence of more than one 2DS4 activatory KIR acute lymphatic leukaemia (5/15), immunodeficiency (5/9) or genes in donors and donor recipient C1 and C2 thalassemia (1/6). IMC of T-cells alone was observed in 3 incompatibility favor complete chimerism after HSCT in patients. IMC of B- and M- cells but not T-cells was observed immunodeficiency children in 5 patients and 3 patients presented IMC in all 3 cell-lines. K. Koscinska (1), B. Wysoczanska (2), A. Lange (1) In the 11 transplants with IMC, 2/5 patients with malignant (1)Lower Silesian Center for Cellular Transplantation disease relapsed and 2/6 with non-malignant disease rejected (Wroclaw, PL); (2)L.Hirszfeld Institute (Wroclaw, PL) the graft. In the 40 transplants with no IMC, 7/30 patients with malignant disease relapsed and 2/10 with non-malignant Twenty five immunodeficiency children SCID (13) and non- disease rejected. One patient with > 85% recipient DNA in all SCID (12) were transplanted in our BMT unit. Five, 7 and 13 3 cell-lines is still alive and without relapse 18 months after children were transplanted from sibling, haploidentical and presentation of IMC. GvHD was observed in 6/11 of patients unrelated donors, respectively. In 3, 10 and 12 cases the with ICM and in 18/40 of transplantations without IMC. transplantation procedure resulted in autologous reconstitution (AR), mixed chimerism (MC) and complete chimerism (CC),

S83 respectively. In haploidentical transplants CD3+ cells were compared to 2004, and the joint discussion between the three only partially removed with the use of counter- flow centrifugal units of common indicators led to advances in our QMS. elutriation. T cells were also present (add-back) in one out of 2 Conclusions: The good grounding of ISO9001 QMS, the joint transplant materials obtained by positive selection of CD34+ efforts of the integrated team towards JACIE implementation, cells. and the involvement of all personnel have been fundamental Chimerism post transplant (detected with the use of for the improvement of clinical activity of our Transplantation informative gene STR alleles detection) was analyzed in Programme in the context of JACIE Accreditation. association with the presence of KIR ligands and KIR genes (Olerup-SSP Kit) in donors and recipients. HLA-C typing was performed by PCR-SSP at the high resolution level. HLA-C P487 specificities were divided into C1 and C2 subgroups according Incidence and outcome of posterior reversible the published literature data. Association between the level of encephalopathy syndrome in paediatric patients following chimerism and the presence of C1 and C2 specificities, and allogeneic haematopoietic stem cell transplantation number of the presence of activatory KIR genes in donor was A.A. Hussein, A. Mansour, A. Kamona, R. Rihani, F. Abdul- analyzed. It was found that: Rahman, T. Ensirat, A.H. Hussain, M. Sarhan (i) Complete chimerism was more frequent in patients King Hussein Cancer Center (Amman, JOR) transplanted with donors carrying more than one activatory KIR genes (9/11 vs 4/10, p=0,09) Purpose: Posterior Reversible Encephalopathy (ii) The presence of more than one activatory KIR genes in Syndrome(PRES)has never been studied in Jordan;here we donors and/or a lack in recipient C1 or C2 specificity present reviewed the incidence,predisposing factors,radiological in a donor favored occurrence of complete chimerism after features and clinical and radiological outcome of PRES in HSCT (11/12 vs 4/10, p=0,015). pediatric patients who underwent allogenic Hematopoietic Conclusions: mixed chimerism post HSCT was more frequent Stem Cell Transplantation (HSCT) from matched sibling in children receiving transplant from donors having only one donors. 2DS4 gene. Therefore the activation of activatory KIR favors Patients and Methods: Between January 2003 and October graft vs host reactivity. The hypothesis that NK cells play a 2006,104 pediatric patients received allogenic HSCT at role in this reactivity was further supported by the finding that KHCC.Nine patients(8.6%)developed clinical and radiological a lack of C1 or C2 specificity in recipients which were present features consistent with in the donors also favored complete chimerism. Therefore, if Pres. Results: The median age of the affected patients was 7 donors had more than one 2DS4 gene and/or at the recipient years,6 were males & 3 females. Tow third of the patients had site there was a lack of the donor C1 or C2 specificity the non-malignant diseases;Thalassemia(n=3),Aplastic complete chimerism could be frequently reached. Anemia(n=1),Fanconi Anemia(n=1)and Sickle Cell Anemia (n=1),while one third had malignant disorders;Acute Leukemia (n=2)and MDS/AML (n=1).Eight patients received P486 myeloablative conditioning while one patient received non- JACIE accreditation programme: first experience of an myeloablative conditioning prior to HSCT. PRES occurred at a Italian paediatric centre median of 91 days(range, 23 -114 days).All patients I. Ferrero, E. Vassallo, L. Perugini, E. Madon, F. Fagioli developed visual disturbances, head ache,seizure and Regina Margherita Children Hospital (Turin, I) elevation of blood pressure.All patients were receiving Calcineurines at the time PRES occurred(8 patients received Objectives: To collect, process and transplant haematopoietic cyclosporine and one patient received FK-506).Also all stem cells (HSC) there are standardized procedures. These patients were receiving steroid at more than 1mg/kg for GvHD require specific haematological and laboratory knowledge and at the time of PRES.MRI showed abnormalities in all patients firm quality controls applied to all processes from cell including occipital lobe involvement in all patients,the parietal manipulation up to patient infusion, in order to guarantee both lobe was involved in 7 patients,temporal lobe in 6 safety and clinical efficacy. Therefore, quality assurance is a patients,frontal lobe in 4 patients,periventricular in 2 high interest topic at all levels of HSC transplantation. patients,brain stem in 1 patient and cerebullar lobes in1 The implementation of a Quality Management System (QMS), patient.Eight(89%)patients had changes that were according to ISO9001 and JACIE Standards arises from the symmetrical and about half of the cases demonstrated necessity to optimise the quality our Paediatric HSC variable degree of enhancement.Complete resolution of Transplantation Programme provides in a general context of radiological findings was observed in 7 out of 8 patients within improving health care quality, and to reach a high level of a median period of 39 days.After a median follow up of 16 practice in accordance with agreed standards of excellence. months(2.5-20 months),all patients survived except the patient The aim of this study was to evaluate the impact of our QMS who had partial radiological remission;experienced relapse of on the quality of the clinical activities one year on from PRES after 5.5 months with deterioration of neurological implementation. status that led to death. Methods: In order to assess the QMS of our Paediatric Conclusion: PRES is a relatively frequent complication of autologous and allogeneic HSC Transplantation Programme, pediatric allogenic HSCT.It appears that the incidence of we established an integrated team from the Clinical and the PRES following HSCT for non-malignant disorder is high. Processing Units (under the HSC Transplantation Unit), and Concomitant administration the Aphaeresis Unit of Service, of steroid and calcineurines seems to be an important risk coordinated by the Programme Director. Each unit had factor for the development of PRES. Patients with non- previously followed independent approaches to draw up complete radiological remission should be followed up for the Quality Manuals (QMs) according to ISO9001 standards. In possibility of relapse. addition to those QMs, in order to comply with JACIE Standards, we organized detailed Standard Operating Procedure manuals, containing written policies and procedures addressing collection, processing and clinical activities. All personnel had been involved in common training meetings and in the drawing up of the documents. The impact of quality parameters on HSC transplantation was assessed by evaluating process indicators. Results: Our integrated Transplantation Programme applied for JACIE accreditation in February 2005 and, after the December 2005 on-site inspection, obtained JACIE Accreditation in August 2006. An analysis of 2005 outcome indicators revealed an improvement of process performance

S84 P488 estimate for relapse after BMT was 38%. Actually, TRM Busulfan for paediatric bone marrow transplantation: seems to be lower than 5-10 years before. If MRD is not which doses in infants less than 9 kg of body weight? available, MUD or CB should be considered for paediatrics N. Bleyzac, V. Bertholle, C. Galambrun, V. Mialou, T. Basset, patients with high risk ALL. B. Kitio, P. Sahakian, Y. Bertrand Debrousse Hospital (Lyon, F) P490 Although IV form of busulfan seems to allow to achieve a safe Infectious complications in children with increased risk of and efficient systemic exposure, young children less than 9 kg conventional regimen toxicity conditioned for allogeneic might present over or under dosage with the recommended haematopoietic stem cell transplantation with reduced regimen (i.e. 1 mg/kg/dose), both with oral or IV form. To toxicity treosulfan-based preparative regimen – the Polish evaluate which dosage regimen was necessary to achieve paediatric experience target AUC, 19 patients, weighting between 3.9 and 8.5 kg G. Grund (1), M. Leda (1), E. Gorczynska (2), B. Wójcik (3), K. and receiving busulfan (IV : 4, oral : 15) with BU Bayesian Kalwak (2), K. Drabko (3), A. Chybicka (2), J.R. Kowalczyk individualization of dosage regimens were enrolled. Mean total (3), J. Wachowiak (1) dose was 15.3 mg/kg but with very large range (8,7 – 24,5 (1)Department of Pediatric Oncology, Hematology and mg/kg). Thirty seven percent of patients received a higher Transplantology (Poznañ, PL); (2)Department of Pediatric dose than 16 mg/kg while 53 % received a lower one. Bone Marrow Transplantation, Oncology and Hematology Incidence of VOD was 26.3%. Patients with VOD received (Wroclaw, PL); (3)Department of Pediatric Hematology and doses of busulfan varying from 10.2 to 24.5 mg/kg and AUC Oncology (Lublin, PL) values were all in the target range (971 to 1164 µM.min). Incidence of mixed engraftment <95% donor was 42%. Mean As demonstrated previously in children not eligible for AUC reached in patients with complete engraftment was not conventional myeloablative conditioning, the treosulfan different from those of patients with mixed chimerism. (TREO) based preparative regimen for allogeneic HSCT (allo- Significant linear relationship was found between the HSCT) demonstrates almost exclusively mucosal toxicity, estimated dose per 6 hour dosing interval necessary to reach along with sufficient myeloablative and antileukemic effects a target AUC value of 4 µg.h.ml-1 and body weight (p< 0.001). (Wachowiak et al., 2005). However, until now infectious The optimal doses of busulfan per 6 hour dosing interval complications in this group of patients were not analysed. should be: 0.8 mg/kg for BW between 4 and 6 kg, 0.9 mg/kg Between 2000-2005 total of 51 children, including 42 with for BW between 6 and 8 kg, and 1.0 mg/kg for BW around 9 hematological malignancies and 9 with congenital disorders kg. underwent allo-HSCT after conditioning with TREO-based regimen. Out of 42 patients (pts) with usually advanced hematological malignances 19 obtained HSCT from MSD, and P489 23 from MUD. Among 9 pts with congenital diseases 5 were Haematopoietic stem cell transplantation in children with transplanted from MSD, and 4 from MUD. As preparative acute lymphoblastic leukaemia in second or subsequent regimen TREO (3x10-14 g/m²) was given i.v. in various complete remission: a study from Société Française de combination with other cytostatics according to diagnosis, risk Greffe de Moelle, COOPRALL 97 factors of regimen related toxicity and/or regimen used for first A. Salmon (1), A. Babin-Boilletot (2) HSCT. Prior MUD-HSCT and in all pts with congenital (1)CHU Nancy Hôpital d'Enfants France (Vandoeuvre les disorders ATG (n=27) or Campath (n=4) was given. GvHD Nancy, F); (2)CHU Haute Pierre (Strasbourg, F) prophylaxis usually consisted of cyclosporine CsA+MTX (n=24) or CsA (n=18). Prior HSCT in 16 (31.4%) pts infections In acute lymphoblastic leukaemia (ALL), disease free survival with high risk of reactivation or progression occurred. One (DFS) is quite low for children after a relapse. These patients child was conditioned and transplanted in course of should be considered as candidates for allogeneic bone pneumonia. By day +100 in 42 pts with malignancy 38 marrow transplantation (BMT). In France, children with ALL infections were documented, including 22 viral (52%), 10 relapse between April 1997 and November 2002 were bacterial (24%), and 5 fungal (12%). FUO was observed in 5 included in Cooprall protocol, 173 children in group G2 (early (12%) pts. One (2.4%) early infection-related death occurred extramedullar relapse in ALL high risk, isolated marrow in patient, who after MUD-HSCT developed ADV infection with relapse or combined in ALL HR, in T ALL, and isolated multiorgan failure (day +66). In addition 2 (5%) pts died late marrow relapse or combined in B ALL six months after the after MUD-HSCT in course of chronic GvHD due to fungal end of treatment). Once the second remission obtained and infection (day +634 and +865). By day +100 among 9 pts with the avaibility of a donor, the allograft was performed.128 B congenital disorders 8 (89%) demonstrated 13 episodes of ALL, 40 T ALL, 5 ALL (no clearly defined) were included. The infection, including 6 viral (46%), 6 bacterial (46%), and one average time between the first complete remission and the fungal (8%). In pts transplanted for congenital disorders relapse is 21.9 months (1.8 months-7.3 years). Second or neither early nor late infection-related death occured. All-in, subsequent complete remission was obtained for 130 children out of 51 pts studied 3 (5.9%) died in result of infectious (75%). 5 of them received an autograft, all died (4 relapse, 1 complication. Conclusions: Profile, incidence and timing of toxicity); 3 lost sight of the fact. For 30 children, allogeneic infections observed in children conditioned for allo-HSCT with BMT was not possible (early relapse or/and no donor TREO-based regimen were comparable to those observed available). 92 children received an allogeneic BMT: 33 HLA after conventional regimens. Despite of high risk of transplant matched related donor (MRD), 2 HLA mismatched related relate complications the rate of fatal infections in studied donor (MMRD) and 57 matched unrelated donor (MUD) group of children was low, both in early and late post HSCT including 21 cord blood (CB), 33 bone marrow (BM), 3 period. peripheric stem cell (CSP). The 92 conditioning regimens Supported by grant 2PO5E04829 were myeloablativ: total body irradiation-aracytine-melphalan was the most used. Graft versus host disease (GVHD) prophylaxis varied over years and teams: methotrexate and cyclosporine A was the most frequent (n=38). The cumulative incidence of transplant related mortality (TRM) at J100 was 3% for MRD BMT; 14% for MUD CB transplantation; 19% for MUD (BM and CSP). Relapse occurred in 31 patients (9 MRD, 9 CB, 13 BMT). The 2 patients (MMRD) are still alive without GVHD. Acute GVHD (d II) and chronic GHVD developed respectively in 46.6 %, 21% for MRD BMT, 31.5%, 9% for MUD CB transplantation and 63.3%, 2.7% for MUD BMT. Overall survival was 40%. DFS was 41%. The cumulative

S85 P491 patients presented grade I GVHD and 2 grade II. None of the Risk of engraftment failure and veno-occlusive disease in patients developed chronic GVHD. Seven patients are alive children who underwent allogeneic haematopoietic stem with complete donor chimerism and normal a-L-iduronidase cell transplantation conditioned with a regimen levels or levels comparable to those of the donor. containing oral busulfan compared to total body- Visceromegalies present in 4 patients remitted. irradiation Cardiomyopathy present in 3 patients improved. Skeletal Y. Kawano, K. Tabuchi, S. Kato for the HSCT Committee of abnormalities failed to improve in all cases. Neurologic Japanese Society of Pediatric Hematology development in patients transplanted before the age of 2 was normal. In patients transplanted between the ages of 2 and 3, An oral preparation of busulfan (BU) has been widely used in neurologic damage did not progress. Finally, two patients with Japan as a part of a conditioning regimen for allogeneic a moderate phenotype transplanted at the ages of 5 and 10, hematopoietic stem cell transplantation (HSCT) before the respectively, did not later develop severe neurologic damage. quite recent introduction of an intravenous preparation of BU. Patients with Hurler Syndrome undergoing HCT have To characterize the potential advantages/disadvantages of prolonged survival and, if it is performed early , do not present this medication in pediatric patients, we retrospectively neurologic deterioration. UCB has proved to be a valid source analyzed the database of the Japan Pediatric HSCT Registry of hematopoietic progenitors for transplant in these patients to compare outcomes after HSCT with those following a regimen containing oral BU (16 mg/kg) and total body- irradiation TBI (=>12Gy), particularly focusing on engraftment P493 and the occurrence of veno-occlusive disease (VOD). Immune reconstitution after bone marrow transplantation Between Jan 2000 and Dec 2004, 195 children (122 male and in children with malignant and non-malignant diseases 73 female) were treated with a BU-containing regimen. Their S. Aytac, S. Yalcin, O. Kucukbayrak, M. Cetin, M. Tuncer, I. diseases were ALL (n=41), AML (51), MDS (30), CML (12), Tezcan, D. Uckan and others (31). The median age of the patients was 5 years Hacettepe University (Ankara, TR) (range, 3 mo-15y). As a control group, 622 children (median age, 8 years; range, 8mo-15y) who underwent HSCT with a Objective: Study reports show that immune reconstitution TBI-containing regimen were also analyzed. Their diseases takes longer time in malignant diseases when compared to were ALL (n=385), AML (130), CML (21), MDS (37), and non-malignant disorders and immune deficiencies after bone others (49). Patients who received any fludarabine- or other marrow transplantation. We aimed to determine immune iradiation-containing regimens were excluded from the study. reconstitution on the basis of lymphocyte subpopulations of As a result, in HLA-matched allogeneic transplantation, the our patients and retrospectively studied them in a group of 73 graft engraftment rate was 94.3% and 95.8%, in BU- and TBI- children after bone marrow transplantation. containing regimens, respectively, and this difference was not Method: Absolute CD4, CD8, CD4/CD8, CD3, CD19, statistically significant. In HLA-mismatched transplantation, CD3+NK, NK, CD3/DR, HLA-DR subsets of 73 patients were there was no significant difference (p=0.1459) between BU- evaluated at +1, +3 and +6 months of transplantation. The (79.6%) and TBI- containing regimens (87.3%). For the underlying disease was a malignant disease in 27, non- analysis of VOD, 141 cases with the BU-regimen and 444 with malignant in 32 and an immune deficiency disorder in 14 the TBI regimen were eligible. The incidence of VOD was patients. The latter group of patients were excluded from the significantly higher in BU- (10.6%) than in TBI-regimen (4.7 %, study, as they were not given conditioning regimen because of p=0.018), and this was due to the high rate (23.5%) of VOD in their underlying disease. In the study group (n=59) there were mismatched cases with BU-regimen. There was no significant 34 boys and 25 girls, ages ranging from 6 months to 20 years difference in the day +100 survival rates (72.2 % and 70.4%, (median 7). BuCy or CyTBI (with or without etoposide, respectively). melphalan or ATG) was the conditioning regimen in 49 In conclusion, conditioning with an oral BU-containing regimen patients, while 10 patients were treated with reduced intensity does not seem to carry a significant risk of engraftment failure therapy. The donors were HLA 6/6 matched family member in or the occurrence of VOD due to variable plasma 55 patients and 1-3 antigen mismatched related donor in 4. concentrations of the drug compared to a TBI-regimen in HLA- The mean engrafment days for WBC and platelets were matched transplantation, but such associations were 22,95±18,17 and 29,40±16,18 days. confirmed in mismatched transplantation. This may suggest Results: There were no significant differences in the absolute that oral BU should not be used in this clinical setting. CD4, CD8, CD3, CD19, CD3+NK, NK, CD3/DR, HLA-DR levels or CD4/8 ratio after bone marrow transplantation between malignant and non-malignant patients` groups during P492 the follow-up period (p>0,05). In addition, the relapse rate Haematopoietic cell transplantation for children with among patients with malignant diseases did not show any Hurler syndrome correlation with absolute numbers of lymphocyte C. Díaz de Heredia, T. Olivé, J. Ortega, D. Vizmanos, J. subpopulations or the CD4/CD8 ratios. Sánchez de Toledo Conclusion: In this pediatric group of patients the kinetics of Hospital Infantil Vall d'Hebon (Barcelona, E) immune reconstitution was comparable between those with malignant or non-malignant disease. In previous studies the Hurler Syndrome, an inborn error of metabolism due to delayed reconstitution in malignant diseases had been deficiency of a-L-iduronidase enzyme, produces accumulation attributed to older age of patients in that group. Thus, our of heparin and dermatan sulphates, causing findings with comparable data may suggest a role for better hepatosplenomegaly, skeletal abnormalities, cardiomyopathy preservation of thymic reserve in the pediatric age patients. and progressive neurologic deterioration, generally leading to death before the age of 8. HCT can improve the clinical course. Eight patients with Hurler Syndrome (4 boys/ 4 girls), P494 who received a HCT, median age at diagnosis 10m (6m-5y) Neurological complications in paediatric patients with severe phenotype in 6 and moderate in 2 are presented. receiving haematopoietic stem cell transplantation, a Three received enzyme replacement therapy prior to single-centre experience transplant. Median age at transplant was 26m (17m-10a). V. Mialou, J.M. André, C. Galambrun, E. Doré, N. Bleyzac, Y. Four patients received an HLA-identical sibling bone marrow Bertrand donor transplant (ISDT) and 4 an unrelated donor transplant Hopital Debrousse (Lyon, F) (UDT) (2 BM/2 UCB). Conditioning regimen consisted of BU 16-20 mg/kg and CY 200 mg/kg in ISDT and BU+CY+ATG or Objectives: the aim of this study was to evaluate the TNI 6 Gy in UDT. GVHD prophylaxis in BMT consisted of frequency and type of neurological complications after CyA+MTX and, in UCBT CyA+prednisone. All patients allogenic Haematopoietic Stem Cell Transplantation (HSCT). engrafted except one who died from disease progression. Five

S86 Methods: We performed a retrospective analysis of 147 conditioning. The median follow up for surviving patients is 5 consecutive children admitted in our institution for HSCT years (range 1-12 years) between January 2000 and January 2006. Prophylaxis of Results: The actuarial 10 year survival is 40%, (60% vs 30% GVHD consisted of cyclosporine for all patients (with in CR1 or >CR1 disease). The cumulative incidence (CI) of monitoring of ciclosporine levels) and anti-thymocyte globulins transplant related mortality (TRM) at 10 years is 29% (18% vs for unrelated donors. 36% for CR1 or >CR1 disease); TRM for CR1 patients grafted Results: One hundred and forty seven transplantation were from identical siblings (n=94) is 12%. Acute GvHD grade III-IV performed, 92 for malignancy and 55 for other diseases. was seen in 6% of sibling HSCT and 12% of alternative donor Donors were of unrelated origin for 82 patients, familial HLA grafts. CI of relapse related death (RRD) at 10 years was 27% identical for 59 and haploidentical for 6. Thirteen children (18% vs 32% in CR1 or >CR1). In multivariate analysis on (8.8%) developed severe neurological events after a median survival and TRM, significant predictors were disease phase of 42 days (range 4 to 310 days) from HSCT. Those events and intensity of the conditioning (RR 1.66 for intensified includes: convulsions related to cyclosporine A (CsA) regimens). These two variables (disease phase and neurotoxicity (n=3) or to other aetiology (n=2), intracranial conditioning intensity) were also significant predictors of TRM. hypertension related to CsA (n=2), viral infection of central Conclusions: This 12 year study, shows that THIO based nervous system (n=3), irradiation or chemotherapy related regimens produce encouraging long term survival, with low toxic leucoencephalopathy (n=2) and early cerebral leukaemia incidence of GvHD and TRM, when used in combination with relapse (n=1). CY or FLU alone. The addition of MEL or TBI reduces RRD, HSCT was performed for malignant disease in 7 patients, but increases significantly TRM and does not improve survival myelodysplasia, severe aplastic anaemia or Fanconi anemia Disease phase remains a major predictor of outcome. in 4 and sickle cell disease in 2. Median age at HSCT was older in patients with neurological complications (11.9 years) than in the other group of patients P496 who received an HCST at the same period (5.5 years). Long-term follow-up of reduced-intensity allogeneic Allogenic HSCT with unrelated donor was performed in 55% transplantation in patients older than 50 years of cases in both groups. TBI containing regimen was R. Tabrizi, K. Bouabdallah, M. Sauvezie, A. Pigneux, T. administered in 38% of patients that developed neurological Leguay, J.M Boiron, G. Marit, C. Foucaud, M.S Dilhuydy, F. symptoms and 34% in the other group (p=ns). All patients Bijou, C. Melot, V. Frossard, N. Milpied received CsA as GVHD prophylaxis. The incidence of GVHD CHU Bordeaux (Bordeaux, F) was the same in both groups. No patient died because of neurological complications. We have evaluated the outcome of RIC in 128 adult pts with Conclusion: Incidence of severe neurological complications malignant disease (AML: 54, MM: 31, NHL: 14, MDS: 8, ALL: was low in our experience as compared to literature data and 6, CML: 4, CLL: 3, MPD:3, MDS/MPD:2 and Solid tumor: 2) didn’t lead to death of the patients in any cases. We found no between October 1997 and August 2006. factors that could increase the risk of such complications (no There were 87 female, median age was 56 yo (50-65). Pts influence of the type of HSCT, GVHD, conditioning regimen). had received a median of 2 line of treatment before RIC (0-5). Forty-four had a previous autologous transplant. At time of RIC, 32 patients were refractory or in relapse while 57 were in CR (Ist: 33; 2d or more : 24). The donor was an id sib for 93 Reduced-intensity transplants and a MUD in 35 (with 1allelic mismatch in 6). Overall 81 had ATG as part of cond reg. GVHD prophylaxis consisted of CSA or CSA-MTX in 109 pts, 19 had CSA-MMF. With a median FU of surviving pts of 30 m (3-110) the survival P495 (OS) and EFS are 45% and 39% at 3y and 33% and 32% at Thiotepa-based conditioning regimens for allogeneic 5y respectively. Sixty-nine pts died with (50% relapse and stem cell transplants: long-term outcome of 374 patients 50% toxicity).The probability of TRM at 100 d and 3 y are 10% with a median age of 48, grafted from related or unrelated and 31% respectively. The 5y probability of relapse is 49%. donors These figures are strictly superimposable for pts with Myeloid A. Bacigalupo, F. Gualandi, D. Occhini, S. Bregante, T. or Lymphoid disease. Lamparelli, A.M. Raiola, A. Ibatici, C. Di Grazia, A. Dominietto, The factors affecting significantly (p<0.05) the 5y OS and EFS B. Bruno, M.T. van Lint, F. Frassoni were: disease status at time of RIC (OS:40% CR vs 26%; S.Martino's Hospital (Genoa, I) EFS: 40% in CR, 30% in CP and 18% in progressive disease), an id sib as donor (OS:64% vs 25%; EFS: 35% vs 30%), Background: Following the experienced of the Perugia group, having an aGVHD grade 1 or 2 ( OS: 43% vs 10% if aGVHD who first introduced thiotepa (THIO) in allogeneic stem cell grade 3-4; EFS: 40% vs 15%), and a CGVHD limited or transplantsd (HSCT), we have developed several conditioning extensive (OS: 50% vs 23% if no CGVHD; EFS: 50% vs regimens, including THIO, cyclophopshamide (CY), 24%). The factors that affected the risk of relapse was the Fludarabine (FLU), melfalan (MEL) and TBI mainly for patients occurence of an acute and or chronic GVHD. The TRM was above the age 45. significantly increased in pts with previous autologous Aim of the study: assess the outcome of patients undergoing transplant, an aGVHD. EFS and TRM is affected in pts with an allogeneic HSCT with a THIO based conditioning regimen. previous autologous transplant. Patients: 374 patients were allografted with a THIO based Conclusion: The extended follow-up in this series of pts older regimen, between 1994 and 2005, from HLA identical siblings than 50 years, identifies a high risk of disease relapse and (n=221) or family partially mismatched (n=67) or unrelated TRM but provides evidence that RIC allografts can produce (n=86) donors. Median patient age was 48 years (range16- sustained DFS in a significant number of patients with .67). The stem cell source was unmanipulated in all cases, controlled disease. The best results are achieved in patients in either bone marrow (n=276) or peripheral blood (n=98). The CR. conditioniong regimens were classified as reduced intensity (n=177) (THIO+CY or THIO+FLU) or intensified (n=197) (THIO +CY with MEL or TBI 200r). The disease was in 1stCR (n=221) or more advanced phase (n=153). Diagnosis were as follows chronic myeloproliferative disease (n=123), acute leukemia (n=120), myelodisplasia (n=46), other (n=85, including lymphoma and myeloma). All, patients received cyclosporin methotrexate GvHD prophylaxis. Alternative donor transplants received additional anti-thymocyte globulin in the

S87 P498 Reduced-intensity allogeneic stem cell transplantation is an effective salvage strategy for relapsed/refractory peripheral T-cell non-Hodgkin lymphomas P. Corradini , A. Dodero, F. Narni, R. Fanin, P. Matteucci, F. Ciceri, R. Scime', L. Farina, C. Carniti, A. Rambaldi, F. Bonifazi, P. Di Bartolomeo, A. Olivieri, F. Benedetti, G. Console, E. Angelucci, A.M.Gianni , C. Tarella on behalf of GITMO

Clinical outcome of most of patients with PTCLs is usually poor with the exception of ALK-positive anaplastic large cell lymphomas (ALCL). In fact, the majority of patients experience relapse either after conventional chemotherapy or autologous stem cell transplantation (SCT). In 2004, we have demonstrated that allogeneic SCT (allo-SCT) is an attractive salvage strategy. Therefore, we extended our previous observations to 32 patients. All patients received several courses of debulkying chemotherapy followed by allo-SCT with a RIC regimen. Ten patients (31%) received T-cell depletion with alemtuzumab. Patients’ median age was 42 P497 years (range, 15-64). Histologic PTCL subtypes included: Mixed chimerism after allogeneic haematopoïetic stem unspecified (n=14), ALCL (n=8; n=5 ALK neg, n=3 ALK pos), cell transplantation following reduced-intensity AILD (n=4), intestinal (n=3), lymphoblastic (n=2), panniculitis- conditioning in patients in complete remission like T-cell lymphoma (n=1). Twenty-seven pts (84%) received F. Nicolini (1), R. Meyer (2), W. Herr (2), I. Mollet (3), V. transplant from HLA-identical sibling donor, 3 from Dubois (3), M. Michallet (1), C. Helg (4), J. Passweg (4), Y. haploidentical donor and 2 from unrelated donor. The median Chalandon (4), E. Roosnek (4) time from diagnosis to allo-SCT was 16 months (range, 6- (1)Edouard Herriot Hospital (Lyon, F); (2)Johannes Gutenberg 100). Seven-teen patients (53%) had failed a previous auto Hospital (Mainz, D); (3)HLA laboratory EFS Rhone Alpes and 22 pts (68%) had received more than 2 lines of therapies. (Lyon, F); (4)Geneva University Hospital (Geneva, CH) Disease status at time of allograft was CR in 9 cases, PR in 14 cases, and PD in 8 cases, and untested in one case. At Reduced-intensity conditioning (RIC) is increasingly used for median follow-up of 30 months (range, 6-86), 22 (69%) were patients not considered candidates for conventional alive (n= 16 in CR, n= 2 PR, n=4 PD) and 10 died (n=7 Hematopoietic Stem Cell Transplantation (HSCT). Long- disease, n=3 toxicity). The estimated 5 year OS and PFS lasting mixed hematopoiesis has been considered a hallmark projections were 62% (95% CI, 42-82%) and 53% (95% CI, of RIC in the first clinical trials but this may no longer be the 35-71%), respectively. After allo-SCT, the disease case for the RIC applied to date. Here we report the relapsed/progressed in 14 patients (44%) at median time of 5 occurrence of mixed chimerism (MC) analyzed by PCR- months (range, 2-12): 7 died of disease and 7 are still alive (4 amplification of short tandem repeats in granulocytes and T responded to chemotherapy and/or DLI). Patients receiving cells purified using magnetic MicroBeads and an allo-SCT more than 1 year after diagnosis have better PFS autoMACS Separator from 24 patients after HSCT following 4 and OS (PFS: 57% versus 42%, P=0.5; OS: 69% versus 44%, different Fludarabine (Flu) based protocols. Twenty-four adults P=0.4). We did not observe a significant difference in PFS and transplanted for MM with good partial responses (7), for ALL OS between patients with specific versus unspecified variants (2), AML (8), CML (1), NHL (2) or HD (2) in complete (PFS: 57% versus 46%, P=0.9; OS: 73% versus 55%, P=0.8). remission (CR) or for treated MDS (2) were enrolled in the Based on these findings, allo-SCT is an effective salvage study. All patients engrafted successfully. Blood from patients strategy for PTCL. Due to the limited number of patients, it is in CR were tested at d+28, d+60, d+90 and for approximately difficult to evaluate which subtype respond better. half of patients also at d+180. The following 4 RIC based protocols have been used: A) Flu: 30 mg/m² (Flu30) d-4 to d-2 + TBI 2 Grays at d0 (n=4), B) Flu30 d-6 to d-2 + Busulfan (Bu) P499 4mg/kg d-5 to d-4 + ATG 2.5 mg/kg d-4 (n=9), C) Flu30 d-7 to Reduced-intensity conditioning for patients with non- d-3 + Melphalan 140mg/m² on d-2 + Alemtuzumab (Campath- SCID primary immune deficiency disorders undergoing 1H) 20mg d-8 to d-4 (n=5) and D) same as RIC B but patients allogeneic stem cell transplantation. The experience of a received Alemtuzumab T-cell depleted grafts with an add back specialist hospital research centre, Saudi Arabia of 130x106 T cells/kg at d+1 (n=6). GVHD prophylaxis M. Ayas, A. Al-Seraihi, A. Al-Ghoneim, H. Al-Mousa, A. Al- consisted mostly of MMF d+1 to d+28 in combination with Jefri, S. Al-Mohsen, M. Al-Mahr, S. Rifai, H. Al-Dhekri, N. El- CSA tapered during the second and third month. After RIC B, Kum, H. El-Solh C or D, all granulocytes at d+28 were of donor origin King Faisal Specialist Hospital (Riyadh, SA) indicating that hematopoiesis of the recipient had ceased. By contrast, traces of recipient granulocytes (max 15%) limited to Background: The only curative modality for patients (pts) with the first d+28 sample were detected in patients after RIC A. In primary immune deficiency disorders (PID) is allogeneic stem patients who had received Alemtuzumab in vivo (RIC C), T sell transplantation (ASCT). Over the years, the results of cell MC occurred occasionally, scattered over 30% of the ASCT in pts with severe combined immune deficiency (SCID) samples, but half of patients after RIC B and all patients after have improved but pts with non-SCID PID are still a RIC A or RIC D had T cells of mixed origin that, once present challenging group since they require conditioning before SCT at d+28, persisted through all the samples tested. T cell MC and the results with myeloablative regimens have generally was most prominent in patients receiving ex vivo T cell been disappointing with the major causes of death being depleted grafts (RIC D). We conclude that recipient infections and toxicity. We report here 12 pts with non-SCID hematopoiesis reflected by MC in lineages other than T cells PID who were transplanted using a non-myeloablative is rare in patients after Flu-based RIC and virtually absent conditioning regimen. when Flu is used together with Bu or Me. By contrast, T cell Patients and methods: From May 2005 to July 2006, 12 pts MC is frequent and its extent is subjective to differences in the with confirmed PID underwent ASCT at our institution; 7 pts T cell depletion methods applied. had Bare Lymphocyte syndrome, 2 pts had Wiskott Aldrich syndrome (WAS), 1 patient (pt) had chronic granulomatous disease (CGD), one had Omenn syndrome, and one had CD40 ligand deficiency. Upon admission for SCT, none of the

S88 pts had evidence of active infections or significant organ P501 dysfunction. The stem cell source was bone marrow from Anti-D alloimmunisation after RhD-mismatched allogeneic matched related donors in 10 pts and partially matched stem cell transplantation following reduced-intensity unrelated cord blood in 2 pts; the CD34 dose for pts with conditioning matched ASCT was 6 x 106/kg (range, 3.9-12 x 106/kg). The N. Worel, H. Greinix, G. Körmöczi, M. Mitterbauer, A. conditioning was with fludarabin 30 mg/m²/dose x 5 days, Schulenburg, A. Rosenmayr, P. Kalhs, P. Höcker melphalan 140 mg/m²/dose for one dose and rabbit anti- Medical University (Vienna, A) thymocyte globulin (ATG) 5 mg/kg for 2 doses pre-SCT and 2 doses post SCT; the CGD pt received Campath 0.2 Anti-D alloimmunization develops in up to 20% of RhD- mg/kg/dose for 5 doses instead of ATG. CSA was given for negative patients on chemotherapy following exposure to RhD GVHD prophylaxis at the standard doses; cord blood antigen, but is reported to be rare in recipients of transplant pts received steroids in addition. haematopoietic stem cell transplants (HSCT), especially Results: The regimen was overall well tolerated, one pt died following myeloablative conditioning. The objective of our before engraftment at 55 days due to respiratory failure. Ten study was to evaluate the incidence of anti-D alloimmunization pts engrafted with a median time to an absolute neutrophil after D-mismatched HSCT following reduced-intensity count (ANC) of • 0.5 x 109/L of 12.5 days, and a median time conditioning (RIC). to a platelet count of • 20 x 109/L of 24 days; chimerism From 112 consecutive patients receiving RIC-HSCT between (assessed by short tandem repeats, STR) ranged from 24% to April 1999 and March 2006, 26 patients had a D-mismatched 100% in the lymphoid line. Acute GVHD grade • 2 developed donor. Twelve RhD-positive patients had a RhD-negative in 5 pts (40%), all responded to steroids therapy and are in donor, 14 RhD-negative patients received a RhD-positive remission now. Eleven pts remain alive; the one year overall graft. RIC consisted of fludarabine (3x30mg/m²) and 2 Gy total survival and event free survival are 91% and 81% body irradiation (TBI) or polychemotherapy containing respectively. All engrafted pts are well now with no major amsacrine (4x100mg/m²), fludarabine (3x30mg/m²), infections and the 2 pts with WAS have normal platelet cytarabine (4x2g/m²), cyclophosphamide (2x40-60mg/kg), counts. ATG (3x10-20mg/kg) and 4 Gy TBI. For GvHD prophylaxis Conclusions: The results of non-myeloablative conditioning in cyclosporin A and mycophenolate mofetil were given. After ASCT of pts with non-SCID PID are encouraging. Longer HSCT, red blood cell support consisted of donor Rh-type follow up is needed to determine the durability of engraftment. RBCs. After a median follow-up of 30 months, 17 patients with a D- mismatch donor are alive. Two RhD-negative patients died P500 within 10 days after HSCT and were not evaluable for anti-D Conventional versus reduced-intensity conditioning alloimmunization. Eight patients developed acute GvHD haematopoietic stem cell transplantation in AML between days 7 and 52 and 11 patients chronic GvHD J.A. Perez , M.J. Arcos, J.E. Ritchmon, C. Encinas , I.F. between days 75 and 274 after transplantation, respectively. Graciani, M.D. Caballero, L. Vazquez, C. Canizo , F. Sanchez RhD-positive patients with RhD-negative donors received a Guijo, J.F. San Miguel median of 11 (range, 0-92) RhD-negative RBC units during a Hospital Clinico de Salamanca (Salamanca, E) median of 229 days (range, 0-38 mos.) after HSCT. RhD- negative patients with RhD-positive donors were transfused Objectives: Results comparing conventional (C-HSCT) versus with a median of 11 (range, 0-44) RhD-positive RBC units reduced conditioning (RIC-HSCT), specially regarding de long during a median of 60 days (range, 0-54 mos). None of the term risk of relapse, are still lacking. In the current study we 24 evaluable patients developed an anti-D antibody after a have analysed our experience on RIC vs C-HSCT in a series median serological follow-up of 290 days (range, 14 days to 4 of 58 AML patients. years) after transplantation. There was no difference in Patients and Methods: 58 high risk AML patients undergoing transfusion requirements between D-mismatched vs. D- C-HSCT (n=31) or RIC-HSCT (n=26) from January 1996 to matched patients. May 2006 were analyzed. Median age was 34 years (14 to 58) Reduced-intensity conditioning containing fludarabine and/or for C-HSCT and 53 years (39 to 68) for RIC-HSCT (p<0.001). ATG effectively prevented anti-D formation in both RhD- 84% vs 78% of patients were in 1st or 2nd CR and 9.7% vs negative recipients of a RhD-positive graft and RhD-positive 11% were in progression /relapse at transplant in C-HSCT vs recipients of a RhD-negative graft in our group of patients. To RIC-HCST. 71% and 55% of patients undergoing C-HSCT vs avoid transfusion related anti-D formation after D-mismatched RIC-HSCT had a pre-transplant Charlson score of 0-1 HSCT, donor Rh-type RBCs should be given from the day of (p=0.22). Three and 11% of patients had received a previous stem cell graft infusion. transplantation, respectively (p= 0.11). 10% vs 19% of patients received HSCT from an unrelated donor among those undergoing C-HSCT vs RIC-HSCT, respectively (p=0.27). P502 Finally, regarding source of progenitor cells, 80% vs 100% of In patients with aplastic anaemia, conditioning with patients undergoing C-HSCT vs RIC-HSCT received reduced-intensity followed by infusion of T-cell depleted peripheral blood progenitor cells (p=0.01). grafts is associated with limited transplant related Results: 93% C-HSCT vs 81.5% RIC-HSCT of patients morbidity and universal survival reached CR after the transplant (p= 0.17). Regarding relapse N. Novitzky (1), V. Thomas (2), C. du Toit (2), A. McDonald (2) rate 26% vs 15% of patients relapsed after transplantation, (1)University of Cape Town (Cape Town, ZA); (2)Groote respectively (p= 0.24). The incidences of grades 2-4 aGVHD Schuur Hospital (Cape Town, ZA) were 57% and 31% in C-HSCT vs RIC-HSCT patients, respectively (p= 0.13), while the incidence of extensive Introduction: The survival of patients receiving allogeneic stem cGVHD was 45% vs 60% (p=0.05). Upon analysing only cell transplantation (SCT) for aplastic anaemia (AA) has patients receiving HSCT from a matched related donor the improved significantly, but many patients develop extensive incidence was 46% and 49% for C-HSCT vs RIC-HSCT, graft vs. host disease (GvHD), with the associated morbidity respectively (p=0.16). Transplant related mortality was 17% vs and mortality. T-cell depletion of grafts decreases the 29% among patients receiving C-HSCT vs RIC-HSCT, incidence and severity of GvHD but may result in greater risk respectively (p=0.47).With a median follow up of 36 months of graft failure. among alive patients, event free survival projected at 7 years Patients and methods: Between 2004 and 2006, 16 patients was 49% vs 57% among patients receiving C-HSCT vs RIC- with AA were conditioned with fludarabine 30 mg/m² for 5 HSCT (p=0.73) consecutive days (150 mg/m²) followed by cyclophosphamide Conclusion: In spite of a higher median age, patients (CFM) 120 mg/kg. In addition 2 (one with Fanconi's anaemia) undergoing RIC-HSCT displayed similar TMR response and received busulfan 8 mg/kg instead of CFM. HLA identical relapse rates as well as similar incidence of GVHD as sibling donors were mobilised with filgrastim (5-10 ug/kg) for 5 compared to those receiving conventional conditioning. days. Donor cells were quantitated and the CD34+ cell

S89 number determined. PBPC grafts were then incubated "in the in patients with advanced hematological malignancies shows bag" with alumtuzumab (1 mg/109 mononuclear cells) at 20°C encouraging rates of GvHD control without significant increase for 30 minutes. In addition, patients received cyclosporin from in toxicity. day -1 and continued for 90 days. End points were OS, DFS, incidence and severity of GvHD. Results: Median age was 21.5 (7-49) years and 4/15 was P504 females. Median CD34+ cell infused was 3.93 x 106/kg, Radioimmunotherapy with yttrium-90-ibritumomab containing median of 22.2 x 14/kg CFU-GM. The median "ex tiuxetan as part of a reduced-intensity conditioning vivo" alumtuzumab dose employed was 10 mg (range 5-10 regimen for allogeneic haematopoietic cell mg). There was universal engraftment and leukocytes transplantation in patients with advanced reached 0.5 x 109/L at a median of 12 days (8-17) post low/intermediate grade non-Hodgkin lymphoma transplantation. Extra haematological toxicity of the W.A. Bethge (1), M. Bornhäuser (2), T. Lange (3), V. Vucinic conditioning was limited to grade I-II intestinal side effects. (3), L. Uharek (4), M. Stelljes (5), M. Stadler (6), D. Bunjes (7), Eight individuals did not develop pyrexia during the C. Faul (1), L. Kanz (1) neutropenic period. No patient developed GvHD. Two (1)University of Tuebingen (Tuebingen, D); (2)University of subjects had CMV reactivation (pp65+) and responded to Dresden (Dresden, D); (3)University of Leipzig (Leipzig, D); therapy with gancyclovir. Another two developed reactivation (4)University of Berlin (Berlin, D); (5)University of Muenster reactivation of BK virus and haemorrhagic cystitis. One patient (Muenster, D); (6)University of Hannover (Hannover, D); developed delayed graft failure 6 months post SCT and (7)University of Ulm (Ulm, D) recovered following a second T- cell depleted transplant with similar conditioning. Donor-recipient sex mismatch was Allogeneic hematopoietic cell transplantation (HCT) is the only present in 6 instances and post transplant bone marrow potential curative therapy for patients with low/intermediate karyotype analysis showed that haematopoiesis was of grade non-hodgkin lymphoma (NHL). High treatment related donor’s origin in each case. All patients survive at a median of mortality (TRM) and toxicity using conventional high dose 651 days (range 60-1353) with normal blood parameters. conditioning regimens excluded most patients with Conclusion: Purine analogues in combination with alkylators low/intermediate grade NHL from this treatment modality are a sufficiently intense immunosuppressive conditioning to given their intense pretreatment, frequent comorbidities and allow successful engraftment of T-cell depleted PBPC grafts in older age. Reduced intensity conditioning (RIC) regimens AA. Alumtuzumab "in the bag" led to excellent GvHD allow successful allogeneic HCT even in patients with prophylaxis, resulting in minimal morbidity of the procedure. contraindications for conventional HCT. RIC HCT can induce potent graft-versus-lymphoma (GVL) effects with promising results even in advanced patients with NHL. Results appear to be best in patients with low tumor burden at the time of HCT. P503 Combined use of radioimmunotherapy (RIT) with RIC may A combination of tacrolimus and mycofenolat mofetil increase anti-lymphoma activity of the conditioning and after non-myeloablative unrelated matched donor improve engraftment without increased toxicity to non-target allogeneic blood stem cell transplantation organs. We initiated a multicenter phase I/II study combining F. Zohren, R. Fenk, T. Graef, A. Czibere, H. Pape, N. Safaian, RIT using yttrium-90-ibritumomab tiuxetan on day -14 with M. Kondakci, U. Germing, R. Haas, G. Kobbe fludarabine (30 mg/m2 day -4 to-2) and 2 Gy TBI (day 0) Heinrich Heine University Duesseldorf (Duesseldorf, D) followed by an HLA-matched allogeneic peripheral blood stem cell (PBSC) graft on day 0 for treatment of patients with In this prospective study we evaluated a combination of advanced lgNHL. For postgrafting immunosuppression the tacrolimus (FK506) and mycophenolate mofetil (MMF) as patients receive mycophenolate mofetil and cyclosporine. So graft-versus-host disease (GVHD) prophylaxis following far, 14 patients have been enrolled. Diagnoses were follicular nonmyeloablative peripheral blood stem-cell transplantation lymphoma (n=7), mantle cell lymphoma (n=4) and chronic (PBSCT) from matched unrelated donors (MUD). lymphatic leukemia (n=3). Median age was 56 (range, 47-67) Forty-four patients (median age, 52 years), suffering as well years. Patients were “high risk” with refractory disease or from advanced hematological malignancies as from additional relapse after preceding HCT. Nine patients are evaluable for comorbidities, unable to undergo conventional myeloablative engraftment. Their PBSC grafts from either matched related conditioning because of unacceptable treatment related risks, (n=1) or matched unrelated donors (n=8) contained a median received fludarabine (30mg/m², day –4 to day –2) and total- of 5.6x 106 (range, 2.6-14.7x106) CD34+cells/kg. The regimen body irridation (TBI, 2Gy, day 0), directly followed by an was very well tolerated with no additional toxicity attributable infusion of peripheral blood stem cells from matched unrelated to RIT. Engraftment was rapid and sustained with median time donors. As GvHD prophylaxis a combination of oral to >500 granulocytes/µL of 17 (range, 0-25) days and 2 Tacrolimus at 0,15mg/kg twice daily (from day –1) and oral patients with a nadir <20000 platelets/µL for 1 or 11 days, MMF at 15mg/kg twice daily (from day 0) was applied. MMF respectively. No TRM in the first 100 days was observed. was tapered from day +28 to day +50 in patients without Incidence of grade II-IV GVHD to date was 33% with grade occurrence of any acute GVHD symptoms. Tacrolimus was II=1 and III=2. Median follow-up is still short with 99 (range, tapered from day +70 to day +100 in patients suffering from 49-154) days, therefore definite evaluation for tumor response aggressive malignancies (n=29), and from day +100 in those and graft-versus-host disease is still pending. In conclusion, a with indolent malignancies (n=15). Regimen related toxicities combination of RIT with RIC is feasible with no additional were mild. Fourty-two (95%) patients had engraftment of toxicity due to RIT and stable engraftment in all patients. donor cells and 39 Patients (89%) achieved a complete donor chimerism after PBSCT. Two patients experienced non-fatal early graft-rejection. The incidences of grades II-IV and III-IV P505 acute GvHD were 56% (n = 25) and 4,5% (n = 2), Pharmacokinetics of everolimus in combination with CSA respectively. First symptoms of acute GvHD were diagnosed in a canine transplantation model at a median of day +75 (range 12-99). Thirty-two patients S. Lange, S. Rathsack, C. Adam, R. Wacke, S. Altmann, D. were evaluable for chronic GvHD. Of these, 18 patients (56%) Wolff, B. Drewelow, M. Freund, C. Junghanss developed cGvHD, 10 (31%) showed limited and 8 (25%) University of Rostock (Rostock, D) experienced extensive disease. Thirteen Patients (29%) died from relapse or progressive Background: Everolimus (RAD) is an inhibitor of m-TOR disease. Treatment related deaths from GvHD and associated (mammalian target of rapamycin) and has been shown to complications occurred in 12 Patients (27%). After a median prevent graft rejection in kidney, heart, and lung follow-up of 653 days (range, 31 – 2054 days), 19 patients transplantation. Combination of RAD and cyclosporine A (43%) are alive. Median overall survival was 474 days. The (CSA) resulted in synergistic immunosuppressive activity and use of tacrolimus/MMF after MUD non-myeloablative PBSCT allowed CSA dose reduction leading to an improved safety

S90 profile. No data are available about RAD pharmacokinetics, investigated as possible risk factors. A lower dose of CD4 safety and efficacy in hematopoietic stem cell transplantation graft cells was significantly associated with the presence of (HSCT). Here, we analysed pharmacokinetics of RAD and CMV infection and CMV disease (p=0.008). Patients RAD/CSA in dogs to determine the dosing scheme for diagnosed with CMV infection were treated with either subsequent nonmyeloablative HSCT. ganciclovir, valganciclovir or foscarnet plus IVIG. In our study, Methods: Healthy dogs were given different doses of RAD the reactivation of CMV has not been identified as a statistical (BID, PO) alone or combined with 15 mg/kg CSA BID PO for 5 significant factor affecting the outcome in terms of OS and days. At day 5 blood samples were collected before and 0.5, TRM. The OS was strongly associated with advanced disease 1, 1.5, 2, 3, 4, 6, and 12 h after the morning dose. RAD status at HCT and aGvHD (p<0.05). Patients undergoing RIC- concentrations were analysed by a fluorescence-polarisation- HCT are at risk for CMV-infection or disease especially those immunoassay. Initial RAD dose of 1.5 mg BID was adapted in with low dose of CD4 graft cells. Close monitoring for CMV- subsequent experiments to reach targeted trough level of 3-8 load is of clinical importance throughout immunosuppression ng/ml, as recommended in solid . period. Hematotoxicity, blood lipids, and creatinine were monitored. Results: At a dose of 1.5 mg RAD alone (n = 4) the median trough level was 6.2 (range 4.8-13.1) ng/ml, cmax was 16.2 P507 (12.6-27.1) ng/ml and the corresponding AUC amounted to Influence of graft composition on the incidence of acute 117 (96-197) ng/ml*h. However, 1.5 mg RAD/CSA (n = 2) and chronic graft-versus-host disease after non- caused an increase in median trough level to 28.7 (24.1-33.4) myeloablative allogeneic peripheral blood stem cell ng/ml, in cmax to 77.2 (76.7-77.6) ng/ml, and in AUC to 643 transplantation (579-707) ng/ml*h. After RAD dose reduction to 0.5 mg (n = 2) P. Tsirigotis, R. Or, M.Y. Shapira, M. Bitan, S. Samuel, Y. and 0.25 mg (n = 2) trough levels were 14.5 (14.1-14.9) ng/ml Polyansky, A. Ackerstein, B. Gesundheit, S. Slavin, I.B. and 6.6 (5.5-7.7) ng/ml, cmax were 28.7 (22.1-27.5) ng/ml and Resnick 11.7 (9.4-14.0), and AUC were 190 (159-220) ng/ml*h and 97 Hadassah Hebrew University (Jerusalem, IL) (85-110) ng/ml*h, respectively. Trough concentrations and cmax correlated with AUC (r = 0.927 and 0.966, respectively; Introduction: The influence of graft composition on the p < 0.01 both). CSA blood concentrations were unaffected by outcome of allogeneic non-myeloablative stem cell different RAD doses. At 0.25 and 0.5 mg RAD/CSA and 1.5 transplantation (NST) using mobilized peripheral blood stem mg RAD alone hematologic parameters, blood lipids, and cells (PBSC) remains controversial. In this study, we analyzed creatinine values remained within normal limits. Increased the impact of CD34 and CD3 cell doses on the incidence of toxicity (severe diarrhea and hypercholesterolemia) was acute and chronic graft-versus-host disease (GVHD) after observed at 1.5 mg RAD/CSA. PBSC using a standard NST regimen. Conclusion: RAD and CSA show a linear correlation in dogs. Patients and methods: A total of 103 consecutive patients with Application of 0.25 mg RAD in combination with 15 mg/kg various malignancies were included in this study. Preparative CSA BID leads to trough levels of 3-8 ng/ml in healthy animals conditioning consisted of a combination of fludarabine 180 and is well tolerated. This regimen is currently tested in a mg/m², oral busulfan 8 mg/kg or intravenous busulfex 6.4 nonmyeloablative canine HSCT model (data will be mg/kg, and anti-thymocyte globulin (ATG, Fresenius) 40 presented). mg/kg. Results: Data analysis did not reveal any association between CD34 and CD3 cell dose and the incidence of acute or chronic P506 GVHD (table). Spearman’s rank correlation of the number of Low CD4 graft cell dose is associated with CD34 and CD3 in the stem cell inoculum, grade of acute and cytomegalovirus infection and disease in allografted severity of chronic GVHD did not show any significant patients conditioned with reduced-intensity regimen association either (‘p’ range from 0.062 to 0.926). M. Papathanasiou, I. Batsis, I. Zorbas, P. Kaloyannidis, C. Conclusion: Following NST, neither the dose of CD34 nor the Vadikolia, C. Smias, D. Sotiropoulos, G. Dourvas, I. Sakellari, number of CD3 T cells in the stem cell inoculum influenced A. Fassas, A. Anagnostopoulos the incidence or severity of acute or chronic GVHD. The G.Papanicolaou (Thessaloniki, GR) incidence of GVHD is probably dependent on the degree of alloreactivity between the donor and the recipient, which may Cytomegalovirus (CMV) infection remains a severe vary between each donor-recipient pair, thus suggesting that a complication even in patients undergoing reduced intensity personalized approach may be mandatory to control GVHD in regimen allogeneic transplantation (RIC-HCT). We recipients of T cell repleted allografts. retrospectively investigated the incidence and risk factors for Table: Influence of infused CD34+ cell dose on the incidence CMV infection and CMV disease and their effect on OS and of GVHD (compared groups with cell dose higher or lower TRM following RIC-HCT in 37 patients aged 50(15-63) years than median, and higher than 75 percentile versus lower than old. Thirty-five were allografted from siblings and 2 from 25 percentile). matched unrelated donors. Median follow-up time was 16 months. According to donor/recipient (D/R) CMV-seropositivity status, 32 patients were evaluated as high risk for CMV infection (D/R: +/+), 3 low risk (D/R: -/-) and 2 intermediate risk. All the conditioning regimens were Fludarabine-based (33 reduced intensity, 4 non myeloablative). ATG was applied in 19 patients. The median graft cell-dose administered was CD34: 5.6x106/kg, CD3: 4.8x108/kg, CD4: 2.75x108/kg. All patients received antiviral prophylaxis with valacyclovir plus multipotent immunoglobulin (IVIG). CMV infection was evaluated by serological and molecular methods (real-time PCR) weekly during the first trimester and on clinical P508 indications afterwards. CMV infection was defined as the Nucleic acid based drugs interfere with nucleoside presence of at least one PCR-positive sample (serum, gastric analogues in cancer therapy juice, BAL, CSF) plus clinical manifestations. In a total, 548 R. Buhmann, T. Yang, I. Steinmann, H.-J. Kolb samples were examined with real-time PCR; seven patients GSF (Munich, D) were diagnosed with CMV infection, 3 with CMV disease while on immunosuppression. At a median time of 6 months the Background: In the past years, an increasing number of estimated incidence of CMV infection was 37% and of CMV nucleic acid based drugs (e.g. antisense oligonucleotides, disease 13%. In a multivariate analysis, age, CMV-status, aptamers, ribozymes, RNA interference, defibrotide) have ATG, aGvHD, cGvHD, CD34, CD3, CD4 graft cell-dose were been identified and already tested in clinical trials. But until

S91 now, no information is available whether these treatment HR 0.44 (0.28-0.7) p=0.002), conditioning regimen (use of approaches might interfere with chemically and structurally ATG; HR 1.86 (1.08-3.2) p=0.025) and the occurrence of related drugs, e.g. nucleoside analogues (NA) used in cancer acute (HR 1.54 (1.17-2.01); p=0.001) and severe GVHD (HR or in antiviral therapy. 2.36 (1.38-3.05) p=0.0006; 82% of patients with grade 3-4 Objective: In the current survey we investigated, whether acute GVHD were transfused) were the parameters addition of single-stranded (ss) polydeoxyribonucleotides significantly associated with platelets transfusion needs in (PDRN) could interfere with the cytotoxic effects of NAs (e.g. multivariate analysis. In this cohort, 145 pts could be fludarabine, cytarabine) on lymphocytes or myeloid blasts in assessed for platelets recovery at day +100: among them, 99 vitro. (68%) had a platelet count >99 G/L. Univariate analysis found Methods: For this purpose, T-lymphocytes or CD33 positive a significant impact of acute GVHD (p=0.0001) and platelet myeloid blasts (purity >95%) were labelled with [1µM] count prior to conditioning (p=0.012) but only acute GVHD carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA- (HR 5.52 (2.48-12.25); p=0.001) was associated with a SE) and incubated with different concentrations of fludarabine delayed platelet recovery in a multivariate model. No impacts or cytarabine (200µM, 20µM und 2µM). Cellular proliferation of pathology, GVHD prophylaxis regimen or CD34+ cell dose was induced by addition of CD3/CD28 Dynabeads for the T- were demonstrated. cells, or a cytokine cocktail containing 50ng/mL of SCF, Overall, these observations show a significantly lower rate of 50ng/mL of IL-3, 200ng/mL of GM-CSF, 100ng/mL of G-CSF, platelets transfusions and a quicker kinetic of platelets 2 U/mL of EPO, 0.47g/L of human transferrin, and 5×10- recovery after RIC allo-SCT and point out the effect of acute 5mmol/L of 2-ME for the myeloid blasts. Defibrotide and GVHD. random PDRNs of different length (20-80b) and concentrations were added immediately or with a delay of 24, 48 or 72 hours to the assay. After 5 days, quantitative CFDA distribution was measured by flow cytometry to assess the cellular proliferation of the T cells and the myeloid blasts. Moreover, cell viability was measured by trypan blue exclusion. Each experiment was performed at least three times. Results: PDRNs of different length and composition could antagonize the cytotoxic effects of NAs (fludarabine, cytarabine) in both, T-cells and myeloid blasts. Thereby, the antagonistic effects of PDRNs revealed to be concentration- and time dependent. Moreover interference with aequimolar concentrations of PDRNs could be detected even with a delay of 48 hours. Of importance, the concentration of NAs used in these experiments, referred to concentrations applied in standard clinical treatment protocols. Conclusion: Treatment approaches using nucleic acid based drugs might be critical in context with NAs like fludarabine and cytarabine and diminish the efficacy of these drugs. Further P510 experiments are necessary to elucidate the precise Treosulfan/fludarabine as transplant conditioning mechanism of these effects. regimen for patients with high-risk haematologic malignancies and co-morbidity D. Baronciani (1), A. Rambaldi (2), A.P. Iori (3), F. Pilo (1), C. P509 Mico (2), S. Deplano (1), D. Dessi (1), C. Depau (1), E. Kinetics of platelets recovery and transfusion needs after Angelucci (1) reduced-intensity conditioning allogeneic peripheral (1)Ospedale A. Businco (Cagliari, I); (2)Ospedali Riuniti blood stem cell transplantation (Bergamo, I); (3)Universita' La Sapienza (Rome, I) T. Prebet, M. Mohty, P. Ladaique, C. Chabannon, C. Faucher, S. Furst, H. De Lavallade, J. El Cheikh, M. Ferrando, P. Viens, Treosulfan has recently demonstrated antileukaemic and J.A. Gastaut, D. Blaise immunosuppressive activity in pre-allogeneic HSCT in high Institute Paoli-Calmettes (Marseille, F) risk hematologic patients. Therefore we started a Treosulfan– based conditioning with the aim to find a less toxic, safer and Few data are currently available regarding platelets efficient treatment for patients with high risk malignancies, non transfusion needs and the kinetics and predictive factors for fitting age and comorbidity criteria with standard conditioning platelets recovery after educed-intensity conditioning (RIC) regimens. Evaluations of TRM, OS, DFS, and relapse were allogeneic peripheral blood stem cell transplantation (allo- secondary objectives. Since July 2005 to October 2006 , 31 SCT). In this study, we analyzed the profile of platelets patients (16 males, 15 females ) entered this study. Mean recovery and transfusion needs in the first 100 days after age was 42 years (range 17-65). Underlying diseases were: sibling PBSC RIC in a single institution series of 166 Acute Leukemias(8 ALL, 8 AML), 3 CML, 3 MM, 6 MDS, 1 consecutive transplantations. Patients and graft hystiocitic sarcoma, 1 HD, 1 Myelofibrosis). All patients were characteristics were: age 49 y. (range: 18-70), diagnoses: 66 heavily pretreated; only 13/31 were in CR at the moment of myeloid malignancies (40%), 64 lymphoid malignancies transplant; mean Hematopoietic cell transplant-comorbidity (39%), and 36 metastatic solid tumors (21%). 112 pts (67%) index (HCT-CI ) was 2 according to the Seattle criteria. received an ATG-based RIC, while 54 pts (33%) received a Conditioning consisted of Treosulfan 12-14 gr/m2 for 3 days in low dose irradiation-based RIC. 75 pts (45%) developed grade combination with Fludarabine 30 mg/m2 for 5 days. 2-4 acute GVHD. Platelets recovery (>20 G/L) was observed Cyclosporine plus short MTX were used as GVHD at a median of 9 days (range: 0-99). The kinetics profile of prophylaxis; anti-Thymocyte globulin was used in patients platelets recovery is shown in the figure below. In the whole receiving PBSCs and MUD transplants. Seventeen patients study population, the nadir was observed around day +7 after received HSCs from HLA identical siblings and 14 from match allo-SCT, and a plateau was reached about day +35. Filtered unrelated donors. Source of stem cells were bone marrow in 9 and irradiated donor apheresis platelets were used and patients and peripheral blood stem cells in 22 patients. patients needed a median of 1 unit (range: 0-53). In this Twenty-nine patients (93%) engrafted; mean time to series, 83 pts (50%) did not require any platelets transfusion neutrophil >500 x109/L was 16 days (range 10-26), to platelets during the follow-up period (median follow-up: 442 days) and >20000x109/L was 16 days (range 10- 24). One patient did not 83 patients (50%) received at least one transfusion of platelets engraft (a sAML patient with previous thyroid cancer and I 131 (54 were not transfused beyond day +100 after allo-SCT). radiation);one patient was not evaluable for early death at day Platelets count prior to RIC allo-SCT (median count 144 G/L; +9. Four patients experienced GI toxicity (two grade II, two

S92 grade I), one patient had grade II liver toxicity and five grade P512 I, one patient had grade I renal toxicity. Six patients presented Fludarabine and melphalan conditioning with tacrolimus aGvHD (four grade I, two grade II ). One patient had Cr- as GvHD prophylaxis is effective reduced-intensity GVHD and it was limited. Twenty patients are alive: KM combination regimen for allogeneic stem cell transplant probabilities of survival at 3 and 15 months were 81%(95%CI recipients compared to conventional regimen 67-96%)and 48%(95%CI 24-72%), respectively. Fithteen out O. Imataki, H. Ohnishi, A. Kitanaka, Y. Kubota, T. Tanaka, T. of 31 patients are in complete remission with a median follow- Ishida up of 8 months (range 1-15 months ). Eleven patients died: 8 Kagawa University (Kagawa, JP) for recurrent disease, 3 for TRM. This preliminary study underlines that Treosulfan-Fludarabine Purpose: Controlling regimen-related toxicities and GVHD was can be safely associated in pre-allogeneic conditioning critical issues for allogeneic stem cell transplantation (SCT) regimen even in high risk patients. Longer follow up is recipients with increasing age. Among various RIST regimens, necessary to evaluate the anti-tumor capacity of this regimen. a combination of fludarabine and melphalan (FM) has limited transplantation-related mortality (TRM) by using an appropriate immunosuppressant. We retrospectively P511 compared an efficacy of RIST with conventional SCT. Ibritumomab tiuxetan (Zevalin®) combined with reduced- Methods: Eighty-two consecutive patients who underwent intensity conditioning and allogeneic stem cell allogeneic SCT in our hospital from October 1998 to July 2006 transplantation in patients with chemo-refractory non- for hematological malignancies were enrolled in this study. Hodgkin lymphoma RIST was performed since August 2000 in our hospital. A. Shimoni, T. Zwas, Y. Oksman, I. Hardan, N. Shem-Tov, R. Preparation for RIST was consisted with 25 mg/m2 fludarabine Yerushalmi, A. Avigdor, I. Ben-Bassat, A. Nagler on days -6 to -2 and melphalan 70 mg/m2 on days -3 to -2. Chaim Sheba Medical Center (Tel-Hashomer, IL) Melphalan dose was modified according to the patients’ age, condition and disease status as following; (1) reduction to 40 Allogeneic SCT is an effective therapy in lymphoma by mg/m2 for the high ages older than 50 years or recipients with providing both intensive chemotherapy and graft-versus- organ dysfunction, (2) escalation to 90 mg/m2 for the non- lymphoma effect. Thus, allogeneic SCT is associated with remission cases before transplantation. GVHD prophylaxis for reduced incidence of relapse compared to autologous SCT. RIST was tacrolimus alone or occasionally incorporated with However, myeloablative allogeneic SCT is associated with short term methotrexate. Conventional regimen was with high high risk for non-relapse mortality (NRM) in heavily pretreated dose cyclophosphamide and total body irradiation (12 Gy) or lymphoma patients (pts). Reduced-intensity conditioning (RIC) busulfan and high dose cyclophosphamide. Cord blood stem reduces NRM rate, but is associated with high-risk for relapse cell transplantation case was excluded. in active disease. This study was designed to explore the Results: Among 82 patients, 42 cases received conventional safety and outcome following inclusion of Zevalin with RIC SCT and 40 did RIST. The median age was 43 years (range, regimens prior to allogeneic SCT in pts with refractory 16-71 years) in both, 35 years for conventional SCT and 51 lymphoma with an attempt to augment anti-lymphoma effect of years for RIST. Stem cell source was 26 cases of bone RIC while maintaining reduced NRM. The study included 12 marrow (BM) and 16 of peripheral blood stem cell (PBSC) in pts, median age 54 years (range, 37-62), having SCT from an conventional SCT and 21 of BM and 19 of PBSC in RIST. HLA matched sibling (n=7) or matched unrelated donor (n=5). HLA full-matched donor was available in 69% (29/42) cases in Histology was diffuse large cell (n=3), transformed low grade conventional SCT and 75% (30/40) in RIST. In both groups, 4 (n=7) or mantle cell lymphoma (n=2). The median number of patients developed engraftment failure and the residues prior therapies was 4 (range, 2-6) and 4 pts have previously achieved to engraftment on median day 14. The probability of failed an autologous SCT. All pts had active disease by PET- non-relapse mortality (NRM) was 17% (7/42) in conventional CT at SCT and 3 pts also had extensive marrow involvement. SCT and 8% (3/40) in RIST. Grade II to IV GVHD occurred in Rituximab 250 mg/m² followed by Zevalin 0.4 mCi/kg were 38% in conventional SCT and 28% in RIST. Grade III to IV given on day -14 and fludarabine combined with iv busulfan GVHD was represented in 17% in conventional SCT and 18% (n= 6) or melphalan (n=6) was started on day -6. All pts in RIST. Overall survival observed in 1035 days of median engrafted, a median of 14 days after SCT (10-22). Nine pts follow-up time was equally 43%. are evaluable for response; 5 achieved CR, 4 achieved PR, 1 Conclusion: RIST with FM incorporated tacrolimus exploited died early and 2 are too early for evaluation. With a median the low TRM incidence and had a moderate risk of GVHD in follow-up of 9 months (range, 1-27), 6 pts are alive. The Japanese patients. It is suggestive that our regimen might estimated 1-year PFS is 34%. Only 2 pts relapsed with a exert a potential benefit of graft-versus-tumor effect in RIST cumulative incidence of 20%. One pt died early after SCT of comparative to conventional SCT. multi-organ toxicity. Two pts died of acute GVHD and one of chronic GVHD. The cumulative incidence of acute GVHD grade III-IV was 60% and the rate of NRM was 46%. The P513 inclusion of Zevalin in RIC allogeneic SCT is feasible with no Morbidity and mortality with non-myeloablative compared impairment in engraftment and relatively low organ toxicity in to myeloablative conditioning before stem cell heavily pretreated pts with refractory lymphoma. The low transplantation incidence of relapse suggests an augmented anti-lymphoma Y. Brychtova (1), M. Doubek (1), M. Krejci (1), J. Muzik (2), M. effect. NRM was relatively high due to increased incidence of Navratil (1), J. Mayer (1), J. Vorlicek (1) severe acute GVHD. This is probably related to the planned (1)University Hospital Brno (Brno, CZ); (2)Masaryk University early withdrawal of immunesuppression. Zevalin in (Brno, CZ) combination with allogeneic SCT merits further study. Better results may be achieved in pts earlier in disease course and Background: Nonmyeloablative regimens for allogeneic with longer duration of immune suppression. hematopoietic cell transplantation (HCT) have been developed for patients ineligible for myeloablative conditioning to reduce the regimen-related toxicities (RRT) and non- relapse mortality (NRM). Methods: In this retrospective analysis describing our results after the first HCT in patients with AML and CML after reduced intensity conditioning (RIC), we compare RRT and NRM with group of patients after myeloablative conditioning (MC). The RIC and MC groups were balanced regarding the diagnosis and disease stage. The RIC patients, however, significantly differed from MC patients in median age (51y vs. 37 y). The RIC consisted of fludarabine (30 mg/m2/d; 5 days), busulfan

S93 (total dose 8-12 mg/kg), and ATG Fresenius (10 mg/kg/d; 4 0,6y (0,3-11.8). The stage was classified as I in 7%, II 47%, days), post-transplant immunosuppression was mostly III 37% and IV 10% at time of dg. The reason for HDT was an cyclosporine alone. The conventional regimen was busulfan adujvant approach in pts with high relapse risk (• LN involved) (total dose 14-16 mg/kg) + cyclophosphamide (120 mg/kg) in 57% pts, neoadjuvant setting in inflammatory BC in 5% pts, followed by post-transplant cyclosporine and methotrexate. In primary metastatic disease in 10% and both groups, PBSC from related donors were mostly used as relaps/progression/resistance after/to previous therapy in a stem cell source. RRT were graded using Seattle toxicity 28% pts. Tandem HDT approach was planned in 18 pts criteria. (30%). Peripheral blood progenitor cells was used in all pts, Results: The RIC vs. MC group had a median follow up additional BM in two procedures. The majority of HDT 1019.5 vs. 1049 days, respectively. None or mild RRT was regimens for single transplant approach (n 42) was more frequent and severe RRT less frequent in RIC group in represented by CTCb (54%) and ICE (24%). The first HDT in comparison with MC group (Tab 1). Sixteen percent and 17 % tandem program consisted out of melphalan and paclitaxel in of patients in the RIC and MC group did not achieve a 67% and melphalan alone in 28% of cases. The 2nd HDT was complete chimerism, usually for early disease progression, or performed in 17 pts (one pt did not proceed to 2nd HDT due to graft rejection. Median time to achievement a complete disease progression) and the most frequent used regimen chimerism was 83.5 vs. 82 days for RIC and MC, respectively. was CTCb (88%). The median follow-up is 7 years. Concerning aGvHD, the incidence and severity was less in the Results: All patients engrafted. There were observed 39 RIC group (Tab. 1). Nine/16 AML patients (56%) in RIC group events during the follow up, disease progression or relapse in relapsed and 4 of them died (44%), whereas 9/20 AML 36 (60%) pts and treatment related mortality in 3 pts (5%). All patients (45%) relapsed and 7 of them died (78%) in MC together 35 (58%) deaths was observed. The 5y probability of group. Twenty/22 (91%) RIC CML patients are in molecular progression free survival for adjuvant th, primary metastatic remission (MR) versus 20/25 (80%) CML patients from MC and relapsed/resistant pts resp. was as follows: 59%, 33% group. Mortality from any cause was 18% vs. 36%, NRM was and 12% resp. (p<0.005). The relapse risk at 5y was 39%, 5% vs. 19%, early TRM was 2.5% (for disease progression) 60% and 81% resp. (p<0.005). Overall survival at 5y was vs. 17%, late TRM was 5% vs. 2% for RIC and MC patients, 61%, 33% and 18% resp. (p<0.005). No secondary respectively. malignancy has been observed. Conclusion: Reduced intensity conditioning containing Conclusion: The longterm outcome of pts who underwent the fludarabine, busulfan, and ATG Fresenius is very well HDT with ASCT for BC is presented with the significant tolerated regimen, with lower RRT, NRM, early and late TRM difference among the distinct subgroups. Nowadays there is than conventional myeloablative regimen. This regimen can not possible to consider the HDT for pts eligible for biological completely replace standard conditioning in patients with therapy based on the hormonal receptors positivity and/or myeloid malignancies. HER-2 positivity and new chemotherapeutic drugs. The patients who are negative in all three biological markers can be however eligible for prospective trials. The inclusion criteria and possible trial design will be disscused. Supported by grant IGA MZ CR 8223-3

P515 Treatment of germ cell cancer patients who relapse after high-dose chemotherapy and autologous bone marrow transplantation T. Sarosiek, P. Rzepecki, C. Szczylik Institute of Health Services (Warsaw, PL)

Patients and methods: Between May 1999 and December 2005 44 patients with chemoresistant or relapsed germ cell tumors (GCT) have been treated in our centre with high dose chemotherapy and autologous stem cell transplantation (HSCT). HSCT was in average third line of systemic therapy in our patients. From the 44 transplanted patients 17 had relapse of disease, amongst them 14 had marker positive relapse, or tumor that displayed germ cell histology in obtained tissue specimens. Other 3 had secondary neoplasm (or malignancies that evolved from residual teratoma) – AML, Solid tumours fibrosarcoma and neuroendocrinal carcinoma. Of the 17 patients with relapse / secondary neoplasm, 15 received further treatment. This treatment consisted of surgery alone in P514 2 patients, chemotherapy alone in 2, radiotherapy alone in 1, High-dose therapy with autologous stem cell rescue for combined surgery + chemotherapy in 5, chemotherapy + breast cancer patients - long-term (7 years) follow-up surgery +radiotherapy in 3 and chemotherapy + radiotherapy M. Trneny (1), J. Abrahamova (2), L. Petruzelka (1), B. in 1 patient. Systemic therapy consisted of Vackova (1), V. Stahalova (3), J. Nepomucka (2), Z. Donatova paclitaxel/gemcitabine combinations in GCT patients, and (2), P. Vitek (3), M. Trnkova (4) disease specific regimens in patients with secondary (1)Charles University General Hospital (Prague, CZ); neoplasms. (2)Thomayer Memorial Hospital (Prague, CZ); (3)Institute of Results: Median survival from the moment of relapse was 95 Oncology (Prague, CZ); (4)Czech National SCT Registry days (3 months) in all patients, and 178 days (6 months) in the (Prague, CZ) group of 14 patients who received further treatment. In patients who received no further treatment median survival The HDT with ASCT for breast cancer (BC) was frequently from the moment of relapse was only 25 days. Three patients used in nineties. Randomized trials however failed to prove are still alive with survival times 37,4; 24, 3 and 6,2 months any significant outcome benefit over control arms and the from the moment of relapse. In all of those three patients number of transplants has declined to zero. We present the relapse was confined to a single metastatic site, they received long term follow up of our patients treated in those days. multimodal treatment (chemotherapy + surgery or Patients: There were performed 77 transplant procedures radiotherapy) and all of them achieved CR in the course of from Sept 1994 till Oct 2001 in 60 patients. The median age at palliative therapy. ASCT was 47.5 (21-61), median interval from dg to ASCT was

S94 Conclusions: Further chemotherapy in germ cell cancer Median age at SCT was 3.1 years (range, 1.3-5.6 years). Pts patients who relapse after a HSCT is controversial. However were all treated with 5 cycles of induction chemotherapy our results show that even such heavily pretreated patients (adriamycin, cisplatin, ifosfamide and etoposide); resection of may benefit from further palliative treatment. Best chances for the primary tumor was performed after the 4th or 5th cycle, long survival have those who experience relapse confined to and radiation therapy was given to any gross residual tumor one metastatic site and receive combined treatment that and/or metastatic site remaining positive on the MIBG scan consists of surgery or radiotherapy + systemic therapy. before SCT. Bone marrow harvest was done after the 3rd or 4th cycle, purging was achieved by the CD34 positive selection method. Median CD 34 count was 3.82 x 106/kg P516 (range, 1.2-11.4 106/kg). The conditioning was with Beneficial effect of autologous peripheral blood stem cell carboplatin, melphalan and etoposide. Stage IV pts (11) transplantation for high-risk rhabdomyosarcoma in received total body irradiation (TBI); and those with stage III childhood disease (4) did not. Cis Retinoic acid was given orally for 6 C.J. Lyu (1), N.K. Kim (1), S.C. Won (1), W.M. Kim (), J.E. months post transplant. Park (3), H.J. Jung (3) Results: The regimen was overall well tolerated, all patients (1)Yonsei University, College of Medicine (Seoul, KOR); developed mucositis grade IV. All pts engrafted with a median ()Yonsei University, Wonju College of Medicine (Wonju, KOR); time to an absolute neutrophil count (ANC) of • 0.5 x 109/L of (3)Ajou University (Suwon, KOR) 15 days (range, 11-48 days), and a median time to a platelet count of • 20 x 109/L of 46 days (range, 24-134 days). CMV Background: Since 1970's Intergroup Rhabdomyosarcoma infection (detected by CMV antigenemia test) developed in 5 Study (IRS) committee has conducted various treatment pts (33%). Two deaths occurred; one was at 49 days post strategies for pediatric soft tissue sarcoma patients. Recently, SCT due to sepsis and the other was late due to refractory high dose chemotherapy followed by autologous peripheral CMV infection. The overall event free survival is 86.7 % at 26 blood stem cell transplantation (HDC/PBSCT) has been tried months post SCT. for these high-risk patients by some institutes, but the results Conclusions: Our results are encouraging, although longer seem controversial, until these days. follow up is necessary. CMV infection may be a problem in Patients and Methods: This study was performed based on this population when purging with CD34 + positive selection the retrospective review of medical records of patients with method, likely due to the delayed immune reconstitution after RMS in a single institute. Thirty-one patients who had been SCT. diagnosed as RMS and treated in Severance Hospital, Seoul, Korea from 1982 were enrolled in this study. All of these patients were classified as high-risk RMS (stage III & IV). Results: Median follow up period for all patients were 3.1 Autoimmune diseases years. The 3-year event free survival (EFS) for patients who underwent HDC/PBSCT and only chemotherapy were 43 ± 16% and 20 ± 8%, respectively (P=0.11). But, patients who respond well to conventional induction chemotherapy P518 (complete response or very good partial response) showed 3- Autologous haematopoietic stem cell transplantation is year EFS as 80 ± 16%, while 23 ± 9% for HDC/PBSCT and more effective in relapsing/remitting multiple sclerosis chemotherapy group, respectively (P=0.04). Especially, forms patients with preoperative (TNM) stage IV presented 43 ± R. Saccardi (1), B. Allione (2), A. Bonini (3), A. Bosi (1), E. 16% for HDC/PBSCT group and 0% for chemotherapy group Capello (4), F. Ciceri (5), P. di Bartolomeo (6), M. Di Gioia (1), (P=0.002). A. Donelli (7), I. Donnini (1), S. Guidi (1), F. Gualandi (4), A. Conclusion: High dose therapy with autologous stem cell Guerrasio (8), G. La Nasa (9), G. Longo (1), F. Pagliai (1), F. transplantation seems to achieve prolonged remissions in Papineschi (10), G. Mancardi (4) pediatric high risk RMS. It must be considered in patients who (1)Careggi Hospital (Florence, I); (2)Alessandria Hospital are in VGPR or CR following conventional-dose therapy to do (Alessandria, I); (3)Azienda Ospedaliera ASMN (Reggio HDT/PBSCT for pediatric high risk RMS, especially TNM Emilia, I); (4)San Martino Hospital (Genoa, I); (5)San Raffaele stage IV. Hospital (Milan, I); (6)Osepdale Civile (Pescara, I); (7)Univeristy Hospital (Modena, I); (8)Ospedale San Luigi Orbassano (Turin, I); (9)R. Binaghi Hospital (Cagliari, I); P517 (10)Univeristy Hospital (Pisa, I) Megadose chemotherapy followed by autologous stem cell transplantation in patients with high-risk Introduction: Autologous HSCT has been increasingly neuroblastoma, the experience of a specialist hospital, employed for the treatment of severe progressive Multiple Saudi Arabia Sclerosis (MS). Actively inflammatory phases of the disease S. Rifai, M. Ayas, I. Al-Fawaz, Z. Habib, S. Al-Sheneifi, A. Al- are believed to be the ideal target of the procedure. We Seraihi, A. Al-Jefri, Y. Khafaga, A. Al-Ahmari, M. Al-Mahr, H. analyzed the Italian GITMO-Neuro database in order to EL-Solh evaluate different outcome according to this variable. King Faisal Specialist Hospital (Riyadh, SA) Patients and methods: Forty-six MS patients, aged between 16 and 52 years (median 36 years), 30 females and 16 males, were reported to our database. All of them were mobilized Background: Neuroblastoma has a highly variable clinical 2 outcome, ranging from spontaneous regression to rapidly fatal with Cyclophosphamide 4 g/m and G-CSF; unmanipulated disease. High risk features such as advanced stage, age of PBSC were infused after BEAM conditioning regimen and more than 365 days, unfavorable histology, MYCN ATG administration. Disease phase were 27 Secondary amplification, 1p deletion and/or 17q+ and 11q LOH are Progressive (SP), 16 Relapsing Remitting (RR), 3 of them usually associated with poor outcome. Historically, high risk classified as malignant diseases of Marburg type, 1 Primary Neuroblastoma patients (pts) have had very dismal long term Progressive (PP) and 2 Optical neuromyelitis. survival probabilities, but it has been shown lately that Results: All the patients engrafted but one (RR) who died 27 megadose chemotherapy followed by autologous stem cell days after HSCT for a septic shock. Data are shown only rescue (SCT) may significantly improve the outcome. We about SP and RR forms. EDSS disability scale modification report here our results in 15 such pts. according to baseline is available in 40 patients (14 RR and Patients and methods: From October 2003 until June 2005, 15 26 SP), and is reported in the following graphic. Median pts (5 females and 10 males) with high risk neuroblastoma follow-up time in the two groups is 22,5 (1-96) and 21,5 (6-84) were referred to King Faisal Specialist Hospital & research months respectively. Chi square analysis showed a center (KFSHRC) for treatment, the diagnosis was confirmed statistically significant difference (p=0,008). and the high risk features were ascertained at KFSHRC.

S95 Conclusions: Our data show that, in a retrospective analysis, P520 the neurological outcome is better in RR/RP forms, confirming Autologous stem cell transplantation for severe systemic that HSCT, performed following BEAM+ATG regimen, is sclerosis: update on the ASTIS-trial effective in slowing down inflammatory activity of rapidly J. van Laar (1), D. Farge (2), C. Bocelli-Tyndall (3), J. Sont progressing patients. (1), R. Saccardi (4), I. Miniati (4), M. Matucci-Cerinic (4), K. Machold (5), S. Arfi (6), D. Adoue (7), F. Sarrot-Reynauld (8), J. Constans (9), P. Roblot (10), I. Queré (11), J. Durand (12), J. Sibilia (13), A. Himsel (14), J. Kaltwasser (14), H. Peter (15), I. Kötter (16), S. Kleinert (17), H. Tony (17), A. Fassas (18), A. Voskuyl (19), M. Vonk (20), F. van den Hoogen (20), P. Villiger (21), S. Weiner (22), S. Dass (23), P. Emery (23), A. Gratwohl (3), A. Tyndall (3) (1)Leiden University Medical Center (Leiden, NL); (2)Hopital St Louis (Paris, F); (3)University Hospital Basel (Basel, CH); (4)University of Florence (Florence, I); (5)Allgemeines Krankenhaus (Vienna, A); (6)CHU La Meynard (Fort de France, F); (7)Hopital Purpan (Toulouse, F); (8)CHU de Grenoble (Grenoble, F); (9)Hopital St André (Bordeaux, F); (10)Hopital St Bernard (Poitiers, F); (11)CHU de Montpellier (Montpellier, F); (12)Hopital de la Conception (Marseille, F); (13)Hopital de Hautepierre (France, F); (14)JW Goethe University (Frankfurt, D); (15)Albert-Ludwigs University (Freiburg, D); (16)Medical University (Tuebingen, D); P519 (17)Medical University (Wuerzburg, D); (18)Papanikolaou Autologous stem cell transplantation in Crohn's disease Hospital (Thessaloniki, GR); (19)Free University Medical C. Annaloro (1), F. Onida (1), P. Usardi (1), A. Della Volpe (1), Center (Amsterdam, NL); (20)Radboud University Medical A. Cassinotti (2), G. Bianchi Porro (2), G. Lambertenghi Center (Nijmegen, NL); (21)Inselspital (Berne, CH); Deliliers (1) (22)Medical University Herne-Bochum (Herne-Bochum, D); (1)Ospedale Maggiore di Milano IRCCS (Milan, I); (2)Polo (23)Leeds General Infirmary (Leeds, UK) Universitario L. Sacco (Milan, I) Background: High dose immunosuppressive therapy (HDIT) The relatively few but encouraging reports concerning and hematopoietic stem cell transplantation (HSCT) is a novel autologous hematopoietic stem cell transplantation (AHSCT) treatment for patients with severe systemic sclerosis (SSc). in patients with Crohn's disease (CD) describe the use of Previous studies showed durable responses in two thirds of selected CD34 cells. We autografted four CD patients (three patients up to 3 yrs after HSCT (1). This treatment modality is males aged 26, 28 and 45 years, and one female aged 35 now further investigated through the ASTIS-trial (autologous years) who had a CD activity index of >200 and were stem cell transplantation international scleroderma trial), a refractory to at least four lines of conventional therapy prospective, controlled, randomized trial to compare safety including salicylates, corticosteroids, azathioprine and and efficacy of HDIT + HSCT versus monthly i.v. infliximab. Disease activity manifested as relapsing peri-rectal cyclophosphamide in SSc patients at risk of major organ abscesses in all four patients, two of whom were also affected failure or early mortality. by refractory intestinal bleeding requiring regular transfusions. Objectives: To evaluate whether HDIT + HSCT is superior Unselected PBSCs were collected after mobilisation with CTX over conventional treatment in terms of safety and efficacy in 1.5 g/m² and G-CSF 10 mg/kg. The conditioning regimen SSc patients, and to assess potential predictive factors of included CTX 50 mg/kg on days -5 to -2, and rabbit anti- response. thymocyte globulin 2.5 mg/kg on days -4 to -2. No growth Methods: SSc patients with early active diffuse disease with or factors were administered after PBSC infusion. All of the without major organ involvement are eligible. SSc patients patients developed febrile episodes, but none required randomized to the transplant arm undergo mobilization with empirical antifungal therapy; one patient also experienced BK- cyclophosphamide 2x2 g/m², conditioning with positive hemorrhagic cystitis, which resolved after intravenous cyclophosphamide 200 mg/kg, rbATG 7.5 mg/kg, followed by hyperhydration. The patients reached unsupported platelet reinfusion of CD34+ selected autologous HSCT. Those counts of 50,000/l eight or nine days after AHSCT, and randomized to the control arm are treated with 12x monthly neutrophil counts of 500/l after respectively 10, 11, 12 and 13 i.v. bolus cyclophosphamide 750 mg/m². The primary endpoint days. They were all discharged after peripheral hematopoietic is event-free survival, defined as survival until death or recovery, with the remission of baseline CD symptoms and development of major organ failure during 2 years follow-up. without developing any further signs of disease activity. Results: Eighty SSc patients have been enrolled in 20 Intestinal bleeding recurred in one patient one month after European centers per October 2006: 34 male, 46 female, AHSCT, whereas no abscesses were observed during the 12- mean age 43 yrs, mean disease duration 1,8 yr, mean VC month follow-up; the other patients are disease free three, 81%, mean DLCO 59%. Thirty-eight patients were seven and eight months after AHSCT. CD is a randomized to the transplant arm, 42 to the control arm. No pathogenetically self-standing immune-mediated disorder, unexpected toxicities have yet been observed in either arm which enabled us to evaluate the feasibility of AHSCT using with a median follow-up of 23 months (range 1-54). One unselected PBSC after a conventional conditioning regimen. fatality in the transplant arm was categorised as probably Transplantation allowed our patients with end-stage CD at treatment-related. least a transient remission, with an acceptable rate of Conclusion: The lower than expected TRM (2.6% for HSCT) procedure-related complications and prompt peripheral and unexpected toxicities with 80 SSc patients randomized recovery. As in the case of all preliminary reports, it is not underscores the feasibility of the ongoing ASTIS-trial. clear whether the single early relapse reflects the worst References: van Laar JM, Farge D, Tyndall A, on behalf of the prognosis of the patient, or if the disease-free survival of the EBMT/EULAR Scleroderma Study Group. The ASTIS-trial, others simply reflects the short period of follow-up; however, hope on the horizon. Ann Rheum Dis 2005;64:1515. the results suggest that AHSCT with unselected PBSCs may find a reasonable place in at least in some subsets of patients with advanced CD.

S96 P521 Methods: Male BXSB (H-2b) mice (10 weeks old) were Cyclophosphamide/ATG autograft for chronic received 20x106 BALB/c (H-2d) BM cells. 3 Gy TBI on day -1 inflammatory demyelinating polyradiculoneuropathy and anti-CD40LmAb (2mg) on day 0 to BMT was given. Skin R. Weinkove (1), M. Mahdi-Rogers (2), R.A. Hughes (2), M.A. grafting (donor BALB/c; third party C3H) was performed one Kazmi (1) day after BMT to assess tolerance. Chimerism in peripheral (1)Guy's and St Thomas' NHS Foundation Trust (London, blood was followed by FCM analysis. Mice were sacrificed at UK); (2)King's College London School of Medicine, Guy's 16 weeks post-BMT. Anti-dsDNA antibody IgG (ANA) titer was Hospital (London, UK) measured by ELISA. Peripheral blood counting was performed by autoanalyzer. Microscopic structural changes in Objectives: Chronic inflammatory demyelinating were investigated. polyradiculoneuropathy (CIDP) is a progressive or relapsing Results: 3 Gy TBI on day -1 with anti-CD40LmAb and BMT on neurological disorder characterised by symmetrical weakness day 0 allowed induction of high levels of multi-lineage mixed and sensory impairment, reduced deep tendon reflexes and chimerism (donor CD4+ T cell; 52.2±9.5 % and CD8+ T cell; specific electrophysiologic findings. We describe three 42.4±8.5 % at 14 wks). No clinical signs of GVHD were patients with progressive CIDP refractory to conventional shown. There was no significant difference in anti-dsDNA IgG treatments who were treated with cyclophosphamide and titer between chimeric mice (27.6±27.6 mg/ml) and BXSB antithymocyte globulin (ATG) autologous stem cell transplant. mice (31.1±27.6 mg/ml) (p=0.77). There was significant Methods: All immunosuppressives except steroids and difference in number of platelets between chimeric BXSB mice intravenous immune globulin (IvIg) were stopped prior to stem (50.4±12.9x104 /ml) and BXSB mice (26.0±18.5x104 /ml) cell mobilisation with cyclophosphamide 2g/m²/day for two (p<0.01). The monocytosis and destructive change of spleen days and filgrastim 10mcg/kg/day from day 6. Peripheral was significant in BXSB mice, while these changes were slight blood stem cells were harvested on days 12 to 13. Transplant in chimeric BXSB mice. conditioning consisted of cyclophosphamide 50mg/kg/day on Conclusion: Allogeneic mixed chimerism achieved by BMT days -6 to -3 inclusive followed by methylprednisolone with less toxic, non-lymphoablative conditioning regimen, 2mg/kg/day and rabbit ATG 2.5mg/kg/day on days -3 to -1 which induced donor-specific tolerance without GVHD inclusive. improved lymphoproliferative disorder and cured Results: Three patients with CIDP (two paraprotein- thrombocytopenia in BXSB lupus mice without associated) were treated; the median age was 40 years immunosuppressive therapy. (range 29 - 72). Patients had received a median of 7 prior immunosuppressive therapies; all had previously received steroids, , IvIg and rituximab. Stem cell P523 mobilisation and harvest was successful in all patients with a Autologous stem cell transplantation in progressive median yield of 16.7 x106/kg CD34 cells. Pre-transplant, multiple sclerosis – an interim comparative analysis of median Overall Neuropathy Limitation Scale (ONLS) score two different conditioning regimens was 7, median Medical Research Council (MRC) sum score A.A. Ribeiro (1), N. Harmeschlak (1), M. Rodrigues (1), J.M. was 46, and median 10 metre walk time was 11.5s (one Kutner (1), J.C. Voltarelli (2), J. Zanis Neto (3), F. Dulley (4), patient could not walk). A median of 5.2 x106/kg CD34 cells C.A. Souza (5), A.C. Vigorito (5), C. Leite Santana (6), W. were infused. All patients had grade 4 neutropenia; one Azevedo (7), J.C. Barros (8), M.C. Oliveira (2), D. Moraes (2), required prolonged intensive care support. All patients B. Simoes (2), A.B. Stracieri (2), F. Picioni (2) engrafted (median neutrophil engraftment 12 days; median (1)Hospital Israelita Albert Einstein (São Paulo, BR); platelet engraftment 17 days). At 6 - 12 months post- (2)Faculdade de Medicina da USP - Ribeirao Preto (Ribeirao transplant, median ONLS score was 7, median MRC sum Preto, BR); (3)Hospital de Clinicas (Curitiba, BR); score was 48 and median 10 metre walk time was 9.6s (one (4)University of Sao Paulo (São Paulo, BR); (5)University of patient could not walk). At last follow-up (median 12 months) Campinas (Campinas, BR); (6)Hospital Araújo Jorge (Goiania, all patients are alive. Two of three report significant BR); (7)Hospital Socor (Belo Horizonte, BR); (8)Santa Casa improvement in function; one reports worsening of function. (São Paulo, BR) Serial lymphocyte subset analysis shows a marked post- transplant fall in CD4 and CD8 counts, but increase in or Background: High-dose chemotherapy followed by autologous preservation of natural killer cell numbers in all patients. peripheral blood progenitor cells (PBPCs) infusion is being Conclusions: Cyclophosphamide/ATG autologous stem cell used as an alternative treatment for auto-immune diseases transplant may prove an effective treatment for refractory like systemic multiple sclerosis (SMS) refractory to CIDP, but is associated with significant toxicities. A immunossupressive therapy. However, the toxicity of this randomised study comparing autologous transplant with aggressive treatment is still a concern. We report our conventional immunosuppressive treatments is warranted. experience with 2 different high-dose conditioning regimens in patients with SMS, and their clinical efficacy and toxicity. Methods: We studied 41 patients (24 women, 17 men; median P522 age, 42 years: range, 27-53 years) with progressive SMS Allogeneic mixed chimerism cured autoimmune (median EDSS score= 6.5). All patients were mobilized with thrombocytopenia in BXSB lupus mice receiving donor cyclophophamide (2g/m2) and G-CSF . No CD34+ selection BMT with non-lymphoablative conditioning of low-dose was performed. Twenty one patients (51,2%) were treated TBI and anti-CD40L mAb with BEAM regimen plus equine antithymocyte globulin and E. Takeuchi, K. Kamata, M. Higashihara, Y. Takeuchi 20 patients were treated with cyclophosphamide (200mg/Kg; Kitasato University School of Medicine (Sagamihara, JP) Cy-200) plus rabbit antithymocyte globulin. Both groups were comparable in terms of age, sex and EDSS. Background: We have demonstrated that allogeneic BMT with Results: Post-transplant hematopoietic recovery was low-dose TBI and anti-CD40L mAb without lymphoablative successful in all patients and the median time to engraftment treatment achieved durable mixed chimerism and permanent was 9 days (range 7-10 days). Thirty-five patients had enough donor-specific tolerance. Also, allogeneic mixed chimerism follow-up time for evaluation. Three patients (all in the BEAM induced by anti-CD40L-containing regimen overcomes both group) died: sepsis, alveolar hemorrhage and conditioning allo- and auto-immunity in NOD mice. BXSB mice that regimen toxicity. In the Cy-200 group, 10 patients (10/14; possess features of lymphoproliferative disorder, 71,4%) responded to the treatment (the disease stabilized or splenomegaly, glomerulonephritis and thrombocytopenia is a improved) , and in the BEAM group, 12 patients (12/18; model of human SLE. Prolonged immunosuppessive therapy 66,6%) responded. Treatment failure, defined by an EDSS is required for treatment of autoimmune thrombocytopenia in increase, was seen in 10 patients (31,2%). No gadolinium human disease. We investigated whether allogeneic mixed enhanced lesion was seen on post-treatment magnetic chimerism induced by non-lymphoablative regimen cured resonance imaging in the 28 patients analyzed. The principal autoimmune thrombocytopenia in BXSB lupus mice. early toxicities were: fever during neutropenia (46%), infection

S97 (20%) and allergic reactions with antithymocite globulin was 66% at 100 months. PFS of MS patients was 89% at 100 (12,5%). The BEAM group presented more clinical months, but 23% for the others. This difference may be complications (71,4%) than the Cy-200 group (40%); p=0.04. interpreted as the effect of the combination of the more The main late events were also infectious, but 3 patients intensive conditioning regimen for the MS patients (BEAM) presented with deep venous thrombosis. These late adverse than for the others (Thiotepa-cyclophosphamide: TT) with effects occurred in both groups: 63,0% with BEAM and 35% possibly intrinsic disease characteristics. This 10.years with Cy-200 (p=0.08). experience confirms the view that autologous 5CT may Conclusions: More intensive immunosuppressive regimens, significantly alter the progression of SADS.Elegant studies like BEAM, did not seem to yield better results in terms of argue for a renewal of the T cell repertoire, but no equivalent clinical efficacy than Cy-200. Even though this initial studies have made in patients treated with high-dose conclusion need to be confirmed with longer follow-up to immunosuppression alone. observe disease progression and relapse, the greater [1] Marmont AM: Bone Marrow Transplant 2006;38:1061-1063 incidence of some serious side effects, does not favor this [2] Marmont AM at al Bone Marrow Transplanted intensive regimen. 2006;37:1061-1063 [3] Marmont AM et al: Biol Blood Marrrow Transplant 2006; [4] Marmont AM et al: Autoimmunity 2006;39:305-311 P524 Autologous stem cell transplantation for multiple sclerosis P526 V. Vereschagina, A. Totolyan, N. Bondarenko, E.I. Darskaya, Autologous haematopoietic stem cell transplantation in A. Skoromets, V. Afanasyev early severe systemic sclerosis St. Petersburg State Pavlov Medical University (St. G. Lambertenghi Deliliers (1), F. Onida (1), C. Annaloro (1), C. Petersburg, RUS) Vener (1), A. Della Volpe (1), W. Maglione (2), P. Usardi (1), D. P. Comina (2), R. Giordano (1), N. Del Papa (2) Background: During the last time hemotopoietic stem cell (1)Fondazione IRCCS Ospedale Maggiore (Milan, I); transplantation has become a prospective method of therapy (2)Ospedale G. Pini (Milan, I) in multiple sclerosis. Material: From 2000 till now in St-Petersburg State Medicine Systemic sclerosis (SSc) is an autoimmune disease University there 13 autologous stem cell transplantations characterized by progressive fibrosis that involves both skin (SCT) for multiple sclerosis (MS) were performed. 4 patients and visceral organs, progressively affecting quality of life and had secondary progressive MS, 5 patients had primary reducing life expectation. Autologous hematopoietic stem cell progressive MS and 4 patients had relapsing-remitting MS. All transplantation (AHSCT) has been proposed as a new the patients previously underwent conventional treatment. therapeutic strategy in patients refractory to conventional Median baseline expanded disability status scale (EDSS) was treatments. In our Institution 7 patients with early (< 4 years) 6,0 (4,5-6,5). active diffuse cutaneous SSc (M/F 1/6, median age 38 years, Methods: We used granulocyte colony-stimulating factor (G- range 21-59) were treated with a reduced intensity CSF) for mobilization. High-dose immunosuppressive therapy conditioning regimen followed by AHSCT. All the patients had included BEAM+ATG (antithymocyte globuline) in 10 patients a modified Rodnan skin score (mRSS) greater than 14. The and Fludarabine+Melphalane + ALG in 3 respectively. aims of the study were primarily the reduction of mRSS and of Regimen-related toxicities were mild. Period after SCT was the disease activity, and secondly the improvement of associated with transient worsening of neurological symptoms physician global assessment (PGA), health assessment in all patients: emphasis ataxia, paresis and in 2 cases acute questionnaire (HAQ) and visceral function tests. Skin score bladder dysfunction. Estimate EDSS was conducted at base- and visceral function evaluation were recorded 3 and 6 line, 1, 3, 6, 12 months and every 6 months after SCT; MRI months after transplantation. Hematopoietic stem cells were was conducted at base-line and every 6 months after SCT. mobilised using CTX (4g/m2) and filgrastim (10mcg/Kg/day). Results: We carefully observe 10 patients after SCT. With a After leukapheresis CD34+ cells were positively selected. median follow-up of 17,9 (range, 3 - 36) months. EDSS Conditioning regimen included i.v. CTX (50 mg/Kg on days -5 decreased in 2 patients, 1 patient has progressive and other through -2) and rabbit antithymocyte globulin (2.5 mg/Kg on patients are stable. MRI plaque of 6 patients had not changes days -3 through -1). The median number of reinfused CD34+ after SCT (in different terms). cells was 5.1 x 106/Kg (range 2-5.9). All of the patients Oligoclonal bands in cerebrospinal fluid decreased in 2 experienced at least one febrile episode during post-transplant patients across 9 months and 1 year after SCT. We detect neutropenia which completely resolved after hematopoietic negative correlation between effects of SCT and duration recovery. No grade III-IV extra-hematologic toxicity and disease/ degree EDSS. treatment related mortality were observed. Six months after Conclusion: Clinical response was observed in most patients AHSCT all patients showed a significant reduction in mRSS (9 of 10) after SCT. These findings confirm that SCT might be (-65%, p<0.001) and disease activity (-85%, p<0.0001). considered as an effective treatment for MS. Improvement of PGA and HAQ scores were documented, whereas no significant changes in the lung, heart and kidney function were observed. According to this preliminary P525 experience the prompt clinical improvement of skin Autologous haematopoietic blood and marrow stem cell involvement is significant. The lack of visceral improvement transplantation for severe autoimmune diseases. The may be due to the relatively short duration of follow up. We Genoa experience conclude that AHSCT is feasible and effective in selected A.M. Marmont, F. Gualandi, L. Grassia, M.T. Van lint, G.L. patients with early severe SSc. Mancardi, A. Uccelli, E. Capello, A. Bacigalupo Az. Osp.-Universitaria S. Martino (Genoa, I)

Between 1996 and 2006 35 patients with severe autoimmune diseases (SADs) were transplanted with autologous (34), syngeneic (1) and allogeneic (3) hematopoietic stem cell transplantation ( STC). The case histories of allo- transplanted patients have been published 1-4. Patients treated with autologous autologous STC included 21 with multiple sclerosis (MS),7 with systemic lupus erythematosus arthritis (RA), autoimmune thrombocytopenic purpura (AITP), Behçet’s disease (BD) and vasculitis. The overall survival (OS) was 97% at 150 months, and the progression-free survival (PFS)

S98 P527 diagnosed using Poser's criteria. MRI with contrast Efficacy and safety of autologous haematopoietic stem enhancement and EVP investigation were perfomed monthly cell transplantation in four patients affected by diffuse for a pretreatment period of 3-6 months and compared with cutaneous systemic sclerosis refractory to conventional serial MRI and EVP following 6 months and then once every 3 doses of cyclophosphamide months. I. Miniati (1), R. Saccardi (2), S. Guiducci (1), M.L. Conforti Results: Mobolization was successful in all cases, and a (1), G. Salvadorini (1), S. Guidi (2), L. Lombardini (2), C. median nuber of 7.4 x 10 x 6/kg CD34+ cells were collected. Nozzoli (2), A. Bosi (2), A. Tyndall (3), M. Matucci Cerinic (1) The median follow-up is now 18 months(range 4 to 36 (1)Division of Medicine I and Rheumatology (Florence, I); months). There no treatment-related mortality in our group of (2)Bone Marrow Transplantation Unit (Florence, I); patients. Lymphopenia was profound and prolonged enough (3)Department of Rheumatology (Basel, CH) but was not associated with serious late infections. Common side effects were mucositis (n = 9), allopecia (all), general Background: Autologous HSCT is indicated in rapidly fatigue (all), hypothermia (n=15), cystitis (n=2). Neurological progressive diffuse SSc. The rationale is to establish a toxicity: In the first week after engraftment, loss of dexterity resetting of autoimmune disease through the administration and general muscular weakness were noted in all patients. of an immunoablative regimen followed by reinfusion of Our study shows that maximum intensive immunosupressive autologous hematopoietic stem cells. The primary goal of this protocol is associated with serious non-neurological therapy is to improve quality of life, induce a remission of the coplications. The number of contrast-enhancing lesions disease and increase survival. Monthly pulsed decreased after mobilization with CY and finally dropped to cyclophosphamide is currently used for the conventional zero in 17 cases after conditioning regimen. Clinically, patients treatment of SSc. improved slightly or remained stable. EVP data start Objectives: to evaluate dose intensification of CYC in diffuse improved in 6-8 months after ASCT. cutanteous systemic sclerosis (dcSSc).patients resistant to Conclusion: ASCT is considered an important new promising conventional therapy. theraputic procedure for refractive autoimmune disease like Patients and methods: Three patients (1 male and 2 females) MS because of lower transplant-related mortality and greater with severe dcSSc were screened for HSCT and were found feasibility than allogenic transplantation. These results to be not eligible for the ASTIS prospective trial due to the demonstrate that the theraputic sequence "in vivo T cell cumulative dose of CYC previously administered. Prior to depletion-CY-GCSF-ASCT" has the capacity to suppress HSCT, patients were assessed with physical examination, MR-enhancing activity, improve EVP data, an effect that modified Rodnan Skin Score (mRSS), chest CT scan, sustained with time. ASCT cannot be recomended as pulmonary function test with DLCO, cardiac echocolordoppler standard therapy for severe and rapidly worsening MS. The and HAQ. A mobilization regimen with CYC 4g/m² and final impact of this procedure on MS course remains to be recombinant human granulocyte colony stimulating factor (rHu established. G-CSF), was used for all patients. The graft was manipulated through selection of CD 34 + cells and patients were conditioned with Cyc 200 mg/Kg/ and intravenous rbATG (Thymoglobulin) 7,5 mg/Kg. Graft-versus-host disease Results: All patients engrafted and were discharged from the BMT unit within the normal time range. They were followed up with periodical physical examination, routine laboratory tests and instrumental examinations according to rheumatological P529 practice. The median follow up time was 15 months (range 6- Autologous graft-versus-host disease after an 36). No opportunistic infections have been observed during 2 alemtuzumab containing conditioning regimen and years of follow up. No transplant related toxicity was reported. autologous stem cell transplantation in CLL: Skin score and HAQ improved significantly in all patients. immunological mechanisms and potential anti-leukaemia Lung function was stable. Mild-moderate pulmonary effect hypertension.was detected in 1 patient before HSCT and T. Zenz (1), M. Ritgen (2), G. Haerter (1), P. Dreger (3), T. improved after the procedure Barth (1), S. Böttcher (2), M. Kneba (2), D. Bunjes (1), M. Conclusions: SSc patients who are resistant to Cyc Hallek (4), H. Döhner (1), A. Mackensen (5), S. Stilgenbauer conventional doses may show a satisfactory clinical response (1) to autologous HSCT conditioned with high doses CYC and (1)University of Ulm (Ulm, D); (2)University of Kiel (Kiel, D); ATG. (3)University of Heidelberg (Heidelberg, D); (4)University of (Cologne, D); (5)University of Regensburg (Regensburg, D) P528 Autologous haemapoetic stem cell transplantation for A high incidence of auto-GvHD was observed after an severe multiple sclerosis. A single-centre experience alemtuzumab containing myeloablative conditioning regimen A. Fedulau (1), A. Uss (2), V. Zmazhinsky (2), N. Milanovich and autologous stem cell transplantation (SCT) in patients (3), A. Baida (4), L. Drazhina (4), P. Mitskevich (3), V. with CLL. We performed a detailed analysis of the defects of Smolnikova (3), N Soloviova (3) immune reconstitution in patients receiving alemtuzumab and (1)Belorussion State Medical University (Minsk, BLR); analysed the influence of auto-GvHD on minimal residual (2)Belorussion Postgraduate Medical Academy (Minsk, BLR); disease. We treated 27 patients with CLL (Binet B / C) with (3)National BMT Center (Minsk, BLR); (4)9th Hospital (Minsk, autologous SCT in two trials of the German CLL Study Group BLR) at the University of Ulm. In the CLL3 trial (n=11) autologous SCT was performed after standard conditioning (TBI/Cy). Objective: To determine in severe cases of MS the effect of Patients in the CLL3C trial (n=16) received additional ASCT on MRI, cognitive, motor, sensitive evoked potentials alemtuzumab before SCT. Blood samples were analysed for (EVP)data and T cell depletion to obtain information on intracellular cytokine production (IL2, INF-y). Immune clinical course and safety. reconstitution was assessed by immunophenotypic analysis of Methods: In cooperative study, from 01.2004 to 09.2006 18 different lymphocyte subsets (CD45RA, RO, CD27, CD62L, patients with rapidly evolving secondary progressive MS were CD28, CD4, CD25, FoxP3, CD152 (CTLA-4), CD69, GITR). T transplanted, after original conditioning regimen, with cell receptor repertoire was assessed by spectratyping. unmanipulated autologous peripheral blood SC mobilized with There were no skin rashes or auto-GvHD in the standard high-dose cyclophosphomide (CY), ATG and granulocyte- TBI/Cy group. In contrast, 12 of 16 patients (75%) receiving colony-stimulating factor (GCSF). During pretransplantation Alem/TBI/Cy developed a skin rash between 43 and 601 days phase in vivo T cell depletion with Leicladin was perfomed. after SCT. In 7 patients a clinical diagnosis of auto-GvHD was Patients were considered for inclusion if MS had been

S99 made. The median duration of GvHD was 517 days (range 60- P531 867). Mesenchymal stromal cells administered post- The reconstitution of CD4 and CD8 positive cells was severely transplantation, but not pre-transplantation, ameliorate delayed in the Alem/TBI/Cy group with a particular depletion murine graft-versus-host disease of CD8+ cells for up to 2 years. The CD4/CD8 ratio was M. Kambouris, B. Turner, K. Atkinson, A. Rice abnormally high in the Alem/TBI/Cy group. This increased Mater Medical Research Institute (South Brisbane, AUS) ratio was mainly caused by the extreme CD4/8 ratio imbalance in patients with GvHD. Interestingly, histology A novel means of immunosuppression in graft-versus-host showed a predominant invasion of the skin by CD4 positive T disease (GVHD) is the use of multipotent, mesenchymal lymphocytes. Molecular analysis revealed oligoclonal stromal cells (MSC). MSC have been shown to be non- expansion of T cells in the skin biopsy samples of patients immunogenic and actively suppress T cell function and with GvHD with similar Vß subfamilies (BV3, BV18)(n=3). ameliorate GVHD clinically. Despite this, there is a paucity of Detailed analysis of the immune reconstitution will be pre-clinical data and a lack of consensus as to the efficacy of presented. MSC. Our aim was to determine the optimal dose and time of The addition of alemtuzumab led to continuous MRD MSC administration for the treatment of murine GVHD. We negativity in 10/14 patients (71%) compared to 0/7 patients examined the effects of intraperitoneally injected, donor- receiving TBI/Cy (p=0.0039). Within the Alem/TBI/Cy group derived MSC in two murine models of haematopoietic stem continuous MRD negativity was observed in 6/6 patients with cell transplantation (HSCT). In the first and most severe auto-GvHD (100%) vs. 4/8 patients without auto-GvHD (50%; model, BALB/c (H-2d) mice were myeloablatively conditioned p=0.08) ). and transplanted with bone marrow and spleen from a major The current study demonstrates a remarkable incidence of a histocompatability complex (MHC) mismatched donor (UBI- GvHD-like syndrome attributable to the addition of GFP/Bl6 (H-2b)). MSC were injected either 4 hours prior to alemtuzumab to the conditioning regimen before autologous HSCT (0, 3x105 or 4x105 per mouse) or 24 hours post HSCT SCT in patients with CLL. Longer follow-up will show if the (0, 2x104, 2x105, 3x105 or 4x105 per mouse). Mice were GvHD-like syndrome will lead to prolonged MRD negativity. monitored for the onset and severity of GVHD. In MHC mismatched HSCT recipients, doses of 3x105 (p<0.01) and 4x105 (p<0.05) MSC infused post-transplantation showed P530 greater survival than controls, or recipients injected pre- Heparanase and stem cell transplantation: dual effects on transplantation. Death from GVHD in the post-transplant MSC the donor and the recipient cohort was delayed by up to 33 days (figure 1). In MSC M. Bitan (1), L. Weiss (1), S. Reich (1), M. Zeira (1), E. treated mice we observed no negative effects of MSC on Zcharia (1), I. Vlodavsky (2), S. Slavin (1) donor cell engraftment and less diarrhoea (indicative of (1)Hadassah-Hebrew University Medical Center (Jerusalem, reduced gut GVHD) was seen as compared to controls. In our IL); (2)Technion (Haifa, IL) second model mimicking that of sibling transplantation in the clinic, MHC matched, multiple minor mismatch model donor Heparanase is an endo-beta-D-glucuronidase that cleaves cells (UBI-GFP/Bl6 (H-2b)) were injected into BALB.B (H-2b) heparan sulfate (HS) saccharide chains. The enzyme mice that were conditioned and transplanted as above. MSC promotes cell adhesion, migration and invasion and plays a were injected either pre or post transplantation and monitored significant role in cancer metastasis and angiogenesis. We for onset and severity of GVHD. We showed a similar result to investigated the effect of heparanase in models of stem cell that seen in the full MHC mismatch model, with injection of transplantation, in order to evaluate its potential function in the MSC post transplantation showing increased (although not development and maturation of stem cells and hematopoietic significant) survival when compared to controls or mice progenitors. Applying a mouse model of bone marrow injected with MSC pre-transplantation (figure 2). Death by transplantation (BMT), we evaluated the effect of heparanase GVHD in these animals was delayed by up to 44 days. In on the engraftment process and the development of graft- conclusion, injecting MSC post-transplantation, not pre- versus-host disease (GVHD). It was found that white blood transplantation, has significant consequences on survival and cells (WBC) counts in the peripheral blood of the donor were elucidation of the mechanism by which these cells act may significantly higher in heparanase treated mice, reaching peak lead to wider application of HSCT. levels two days after administration. The of the heparanase treated mice were significantly bigger with less WBC content but with higher total number of stem cells (evaluated by CFU counts). Analysis of the recipient mice, demonstrated a better and faster engraftment in mice receiving cells from donors that were treated with heparanase. While control mice rapidly developed signs of GVHD (i.e., weight loss, hair loss, diarrhea) and died after 12 days, heparanase treated mice showed only a mild appearance of GVHD, and died only 27 days post transplantation, indicating a significant improvement in survival. Next, we applied a murine model of transgenic mice over-expressing heparanase as the recipients of BMT. Monitoring clinical parameters of GVHD, the transgenic mice showed 100% survival on day 40 post transplantation, compared to 50% survival on day 14 in the control group. In vitro and in vivo studies revealed that heparanase modified the function of T cells following transplantation. It inhibited the activation of T cells through modulation of their repertoire of cytokines indicated by a marked increase in the levels of Interleukin-4 (IL-4), IL-6 and IL-10, and a parallel decrease in IL-12, tumor necrosis factor(TNF)-alfa and interferon (IFN)-gamma. T cell activation was inhibited regardless of whether the cells were incubated with active or latent heparanase. Moreover, by using point mutated inactive enzyme, we have found that the documented cytokine shift was independent of heparanase enzymatic activity. Our study demonstrates, for the first time, a direct effect of heparanase on T-cell activation and cytokine profile, resulting in beneficial therapeutic outcome in vivo.

S100 P532 Peristence of host cells after reduced-intensity conditioning: a potential mechanism for delayed onset GvHD B. Turner, M. Kambouris, J. Lange, A. Burns, D. Hart, K. Atkinson, D. Munster, A. Rice Mater Medical Research Institute (South Brisbane, AUS)

Graft versus Host Disease (GVHD) is a major limitation to the widespread application of allogeneic haematopoietic stem cell transplantation (HSCT). Pre-transplant myeloablative conditioning (MAC) regimens controls malignancy, ablates host immune responses and facilitates haematopoietic reconstitution, but they are associated with significant treatment related mortality (TRM). Conditioning drives host dendritic cell (DC) and donor T-cell interactions resulting in GVHD. Reducing the intensity of the conditioning (RIC) regimen maintains anti-leukaemic activity and reduces TRM but the overall incidence of GVHD is unchanged. No clinically relevant in vivo murine models of RIC allogeneic HSCT exist, preventing studies into the mechanism of RIC-associated delayed-onset GVHD. We have developed two RIC HSCT P533 models (full MHC mismatched (UBI-GFP/BL6 [H-2d]-BALB/c Immature donor plasmacytoid dendritic cells attenuate [H-2b] and MHC matched, minor histocompatibility acute graft-versus-host disease by inhibition of TNF mismatched (UBI-GFP/BL6 [H-2d]-BALB.B [H-2d]) that result secretion in delayed onset GVHD, recapitulating that seen in the clinic. T. Banovic, K. MacDonald, A. Burman, E. Morris, R. Kuns, A. In the fully MHC mismatched HSCT model, whilst there was Don, H. Bofinger, G. Hill no difference in overall survival between RIC and MAC HSCT The Qld Institute of Medical Research (Brisbane, AUS) recipients, RIC HSCT recipients required sacrifice for lethal GVHD significantly later (d+23.11±2.88, n=9) than MAC The functional effects of plasmacytoid dendritic cells (pDC) on recipients (d+6.6±0.1, n=23; p<0.000001). In the MHC outcome after allogeneic stem cell transplantation remain matched, miHA mismatched HSCT model, more RIC HSCT unknown. We have studied this using novel pDC depleting recipients survived until d+90 than MAC recipients (p<0.001) reagents in well-defined murine models of allogeneic bone and those RIC recipients requiring sacrifice were taken marrow transplantation (BMT) and G-CSF mobilized stem cell significantly later (d+60±7.9, n=11) than their MAC transplantation (SCT). Surprisingly, donor pDC reconstituted counterparts (d+23.04±1 (n=24); p<0.000001; Fig 1). The very early after either allogeneic transplant, comprising up to development of these models has allowed the effect of RIC on 28% of the splenic cells 7 days after transplant before rapidly chimerism and DC chimerism and subsequent GVHD to be declining to undetectable levels 4 weeks later. In contrast, investigated. RIC activates and increases overall DC numbers reconstitution of pDC occurred more slowly but was sustained post-transplantation without triggering early GVHD. Delayed in T cell depleted allogeneic transplant recipients in which onset GVHD in these RIC HSCT recipients is characterized by graft-versus-host disease (GVHD) was absent. Specific the persistence of host DC and delayed emergence of depletion of donor pDC in bone marrow grafts by antibody activated donor DC (Fig 2). Significantly, excess activated (120G8) significantly increased GVHD (survival: 0% v 43%, host DC in the BM of RIC mice was the only difference P<0.01, 120G8 v control mAb), despite the rapid between RIC and MAC mice in the absence of donor cells. reconstitution of pDC after BMT. Additional sustained RIC does not alter levels of proinflammatory cytokines (TNFa depletion of pDC after allogeneic BMT prevented donor pDC and IL-1bƒwƒnsecreted during conditioning or early post reconstitution and further increased GVHD severity (clinical transplant. After HSCT this excess of activated host DC scores: 4.7 ± 0.1 vs. 3.8 ± 0.1, P<0.01, 120G8 vs control should be expected to cause earlier or more severe GVHD but mAb), confirming that bone marrow derived pDC indeed the reverse was true suggesting that the pathogenesis of RIC- promote transplant tolerance. Furthermore, immature associated delayed onset GVHD is different to that seen in the reconstituting donor pDCs suppressed donor CD4+ T cell MAC setting. proliferation and TNF secretion after BMT and induced a 3- fold expansion of Foxp3+ regulatory T cells (Treg, P<0.01) without alteration of Th1/Th2 differentiation. In the SCT model, depletion of pDC within splenic grafts throughout or after G- CSF mobilization had no effect on GVHD mortality and morbidity. In contrast, G-CSF driven reconstitution of immature pDC (low levels of CD40 and CD86) within grafts following depletion prior to G-CSF administration resulted in a 2-fold increase in the numbers of Treg (P<0.05) and significantly improved GVHD (survival: 56% v 0%, P<0.01). This was the result of reduced levels of TNF in the sera (222 ± 27.5 pg/ml v 461 ± 99.2 pg/ml, P<0.05) since the protective effects of these immature pDC were lost when TNF deficient grafts were transplanted. Thus, we demonstrate for the first time that donor pDC can attenuate GVHD, due to their ability to inhibit the CD4 dependent inflammatory arm of GVHD. However, this is requisite on pDC being immature, a constitutive property of marrow pDC which can be promoted in the periphery by G-CSF.

S101 P534 cells were then sorted to perform functional assays by IFN- Elevated numbers of immature/transitional CD21- B gamma ELISPOT (to check the inhibitory capacity in an lymphocytes identify chronic graft-versus-host disease allogeniec co-culture) and RQ-PCR for Foxp-3, TGF-beta and patients with severe immunodeficiency IL-10 mRNA quantification. ECP was accompanied by a H. Greinix, D. Pohlreich, U. Körmöczi, I. Lohmann, K. significant increase of circulating CD4-CD25+ cells after 6 Feldmann, M. Kouba, P. Kalhs, C. Zielinski, W. Pickl procedures, going from 10.6 to 28.3% of total CD4 (p<0.05), Medical University of Vienna (Vienna, A) with a simultaneous increase of GITR expression on CD4- CD25+ cells (from 15% to 36.6%, p<0.05). This increase was Introduction: Chronic graft-versus-host disease (cGVHD) is a sustained after 14 procedures. CD4-CD25 bright cells were major complication of allogeneic hematopoietic stem cell almost totally positive for CD62L, CD45RO and Fox-P3 transplantation (HSCT) and a leading cause of nonrelapse expression. Sorted CD4-CD25 bright cells were potently mortality resulting from profound immunodeficiency. Currently, inhibitory towards CD25-negative cells when matched with an biology-based markers both for diagnosis and monitoring allogeneic target (IFN-gamma secretion was reduced for disease activity of cGVHD as well as its functional >80%, as detected by ELISPOT) and such capacity was consequences on the immune system are lacking. dependent on cell-to-cell contact, as demonstrated by Methods: Within nine months 70 consecutive patients a transwell experiments. RT-PCR revealed a significant median of 47 (range, 4-150) months after HSCT were enrolled expression of Fox-p3 in CD4-CD25+ cells when compared to into the study, including 21 without any history of cGVHD and CD25-negative cells (p<0.01), but there was no expression of 49 with active or resolved cGVHD. Evaluations comprised TGF-beta and IL-10. All the patients presented an clinical parameters including cGVHD severity according to the improvement of GvHD, tapering off or discontinuing other NIH Consensus Development Project and treatment, serum immunosuppressive drugs, at a median follow-up of 9 months immunoglobulin (Ig) levels, and infections according to the after the first ECP. Even though the number of patients is Infection Module of Common Toxicity Criteria of the National limited, nevertheless our study shows a temporal correlation Cancer Institute. Peripheral blood (PB) leukocytes were between ECP and increased percentages of circulating T-reg analyzed by multiparameter flow cytometry after staining for cells, which are GITR, CD62L and Foxp3-positive, do not CD19+, staining for surface Ig and the B cell memory marker secrete inhibitory cytokines, but present a strong inhibitory CD27+ and staining for CD21, which is absent on function mediated by cell-to-cell contact. Future studies on a immature/transitional B lymphocytes. larger number of patients and a longer follow-up will ultimately Results: No significant differences in absolute B, T, and NK- demonstrate the real impact of T-reg cells in mediating the cell numbers between the groups with and without cGVHD clinical effect of ECP. were observed. Elevated numbers (>15%) of immature/transitional CD19+/CD21- B lymphocytes were significantly associated with occurrence of severe infections P536 (p=0.001). All patients with active cGVHD and elevated The degree of liver infiltration with Foxp3+ regulatory T- numbers (>15%) of CD19+/CD21- B lymphocytes had cells discriminates between viral hepatitis and hepatic experienced severe infections. In contrast, only 21% of GvHD in patients after allogeneic stem cell patients with active cGVHD and low immature/transitional B transplantation cell numbers had a history of severe infections (p<0.0001). K. Rieger (1), D. Wolff (2), C. Loddenkemper (1), A. Muessig The relative and absolute numbers of both non-class-switched (1), C. Gentilini (1), S. Ganepola (1), F. Prall (2), E. Thiel (1), and class-switched memory B cells were significantly lower in E. Berg (1), L. Uharek (1) patients with active cGVHD when compared to patients never (1)Charite Campus Benjamin Franklin (Berlin, D); experiencing cGVHD (p=0.003 and 0.001). Preliminary data (2)University of Rostock (Rostock, D) obtained from patients studied repeatedly support a trend towards normalization of B cell homeostasis in patients with Objectives: The diagnosis of hepatic GvHD is often decreasing cGVHD activity whereas ongoing active cGVHD complicated, even if bioptical material is present. Because of was associated with continuous B cell disturbance in PB. their pivotal role in the induction of tolerance, we have recently Conclusions: Perturbation of circulating B lymphocyte proposed to include the number of Foxp3+ cells as a novel compartments identifies cGVHD patients at risk for severe immunological marker to discriminate between infectious infections and may serve as novel biomarkers for monitoring causes of inflammation and GvHD in pts. with enteropathy cGVHD activity and its impact on the immune system. (Blood 107, 2006). We have now extended our investigations Supported by European Commission Grant QLK-CT-2002- to pts. with signs of severe liver disease after allogeneic SCT. 01936 Transeurope and by MCRTN-CT-2004-512253. In particular, we focussed on infiltrating FOXP3+ regulatory T cells in the portal field of the liver. Methods: A total of 90 paraffin-embedded tissue samples of P535 liver biopsies of 86 patients (17 of patients with suspected Immunological changes in CD4+CD25+ circulating GvHD, 22 of patients with suspected rejection after liver regulatory T cells (T-reg) after extracorporeal transplantation, 25 specimen of patients with hepatitis C and photochemotherapy: results of a prospective monitoring 26 with autoimmune hepatitis) were analysed by double of 10 patients affected by GvHD after allogeneic HSCT immunoenzymatic labeling. Serial sections were stained E. Biagi, G. Gaipa, V. Leoni, I. Di Biaso, G. Renoldi, V. Rossi, conventional HE, and immunhistologically for CD8 and P. Perseghin, A. Biondi FOXP3. San Gerardo Hospital (Monza, I) Results: All samples with histologically proven acute GvHD, liver graft rejection as well as hepatitis C, or autoimmune Extracorporeal photochemotherapy (ECP) has been hepatitis showed a significant increase of infiltrating T cells. As associated with clinical improvement in refractory acute or expected, the dominating lymphocyte subset was CD8+ and chronic GvHD. Immunological mechanisms of action of ECP was localized in the portal fields (PF) of the liver (e.g. mean 15 are still under investigation. We have evaluated the ± 3 CD8+ /PF in GvHD). Although the numbers of CD8+ immunological changes in frequency, immune-phenotype and effector T cells were similar in all four groups, patients with functionality of circulating T-reg cells in 10 patients undergoing GvHD and patients with liver rejection after liver allogeneic bone marrow transpantation, affected by acute transplantation had significantly reduced numbers of FOXP3+ (n=3) or chronic (n=7) refractory GvHD. Patients were affected T cells compared to hepatitis C or autoimmunhepatitis, by cutaneous GvHD, with the exception of 3 patients who also resulting in a low FOXP3/CD8 ratio. presented hepatic GvHD. Patients received between 14 and Conclusion: As in gastrointestinal GvHD, the number of 24 ECPs. T-reg were monitored by immune-phenotyping with regulatory T cells is significantly decreased in the portal fields CD3, CD4, CD25 and GITR antibodies. Further analysis of patients with hepatic GvHD. Evaluation of the FOXP3+ T included CD62L, CD45RO and intracytoplasmatic staining of cells may also be a helpful marker in the diagnosis of GvHD Foxp-3 on gated CD4-CD25 bright cells. T-reg CD25 bright vs infectious diseases of the liver. Future studies will show if

S102 adoptive immunotherapy with regulatory T cells will be able to ATG-G pts received the product from day (d)–5 to –2, the later adjust this deficiency and help to reduce the severity of ones from d–4 to –1, whereas ATG-F pts had ATG from d–3 hepatic GvHD. to –1. Most pts of cohort1 had a daily ATG-G dose of 2,5 compared to 15–20 mg/kg BW ATG-F in cohort 2. Results: At a md follow up of 938 d there was no significant P537 difference in engraftment, chimerism, disease status or overall Alemtuzumab as second-line therapy in acute survival (OS). Probability of OS for cohort1 was 63% and 69% graft-versus-host disease for cohort2 (p=0,25). There was a trend towards more M. Egger, H. Bertz, A. Rückert, R. Wäsch, J. Finke frequent (73 vs 57%) and earlier readmissions (md 158 vs 209 University Clinic Freiburg (Freiburg, D) d) in ATG-G pts. ATG-G pts had a md of 3 infectious episodes between d30 an 365 compared to 2 in ATG-F. Infections were A major cause of morbidity and mortality following allogeneic categorized as equally severe in both groups. Acute GvHD°II- hematopoietic stem cell transplantation (HSCT) is severely IV was seen in 64% of ATG-G compared to 43% of ATG-F pts acute graft-versus-host disease (aGvHD). Currently, there is (p=0,03). 76% of cohort1 and 67% of cohort2 developed no standard therapy for steroid-resistant aGvHD. Here we chronic GvHD (p=0,38) which was extended in 64 vs 39% report our experience with 14 pts suffering from steroid- (p=0,04) and lasted longer in cohort1 (md 296 vs 69 d, resistant gut aGvHD since late 2004 (13 pts de novo, 1 DLI- p=0,03). ATG-G pts had more liver (p=0,05) whereas ATG-F associated). Clinical diagnosis was histologically confirmed. pts had more skin (p=0,04) and gut cGvHD (p=0,04). Description: The patients' median age was 57y (22-68): Conclusion: Both treatments gave good results in matched diagnosis AML (n=8), MM (n=3) and ALL, MDS and and mismatched unrelated donor transplants without lymphoma (each n=1). All but two had advanced disease prior significant differences in overall and disease free survival. to transplant. Acute GvHD started at median day 55 (33-176) Progress in donor selection, immunosuppressive and anti- after PBSCT from MRD(n=1)/MUD(n=12), and 387 days infectious drugs and conditioning may work in favour of cohort following the first DLI, respectively. All 14/14 pts presented 2. Still at the given schedule a probably lower aequivalence with aGvHD of the gut (III n=7, IV n=7); additional dose of ATG-F prevented at least as much severe cGvHD as manifestations were the skin (6/14) and liver (1/14). Steroid ATG-G. Differences in type of cGvHD seem to emerge which resistance was defined as persistence of GvHD after 7d of need further evaluation. prednisolon 2 x 1 mg/kg/day. Methods: Campath 1H (Alemtuzumab) was given at a total dose of 10 mg once weekly with up to 4 (median 2) doses. 1 patient received 2 courses of pentostatin additionally. All P539 patients were treated with broad antibiotic, antiviral and mTOR inhibitors for treatment of severe chronic GvHD antifungal prophylaxis. following allogeneic stem cell transplantation Results: GvHD completely resolved in 13/14 pts as M. Schleuning, D. Judith, I. Burlakova, R. Taube, M. Heshmat, determined by clinical criteria. 12/14 pts were also evaluated H. Baurmann, R. Schwerdtfeger by colonoscopy and in 11/14 aGvHD grade 0 or I was German Diagnostic Clinic Foundation (Wiesbaden, D) confirmed by histology. Median duration to response from the initial dose of campath was 22 d (11-78). 12/14 pts could be Chronic graft-versus-host disease (cGVHD) remains a major discharged from hospital. cause of morbidity and mortality in hematopoietic transplant With a median follow-up period of 7.5 months (3-17) since the recipients. The mammalian target of rapamycin inhibitors onset of aGvHD, 8/14 pts are alive and well. In total 6 pts (mTOR-I) sirolimus and everolimus are immunosuppressants died: due to relapse (n=2) (which were chemotherapy- that have activity in the prevention and treatment of acute refractory pre-transplant), infection (n=3), and in the context of GVHD. Sirolimus in combination with calcineurininhibitors aGvHD and toxicity (n=1). (CNI) has also been shown to have activity in chronic GVHD, Conclusion: Low-dose Campath is a promising, highly-active however at the cost of considerable toxicity. Since mTOR-I therapeutic option in steroid-resistant acute gut GvHD. exert their action by blocking interleukin 2 receptor signaling Infections are of particular concern in this situation. Further and thus arresting the cells at G1 phase they are promising trials are warranted to determine ideal schedules and candidates for tolerance induction. In addition, they also have dosages. antiangiogenic and antiproliferative activity. We hypothesized that treatment of cGVHD with mTOR-I and without CNI would be more effective in inducing tolerance than the combination P538 of both and would have a lower toxicity profile. In this Retrospective comparison of two different antithymocyte retrospective analysis we report 27 patients (pts) with severe globulins as part of the conditioning for unrelated stem cGVHD treated with mTOR-I (everolimus n=18; sirolimus cell transplantation: emerging differences in clinical n=9). Two pts terminated treatment prematurely because of effects adverse events (1xsevere cough; 1xhypertriglyceridemia). Ten H. Baurmann, G. Hoppe, K. Davis, D. Judith, M. Schleuning, pts had de novo cGVHD, 5 progressive cGVHD developing R. Schwerdtfeger from acute GVHD and 10 had cGVHD following donor Deutsche Klinik für Diagnostik (Wiesbaden, D) lymphocyte transfusions. Organ involvement included skin (scleroderma) in 20, lungs in 11, mucous membranes in 9, Introduction: Antithymocyte globulin (ATG) is increasingly liver in 5, gut in 1 and eyes in 3 pts. Sixteen pts received incorporated into conditioning in unrelated stem cell mTOR-I in combination with steroids (n=16), one received a transplantation. Several brands of ATG with different antibody monotherapy and 8 pts received additional spectrum and potency are available. Little is known about their immunosuppressive drugs, no CNI however. To further reduce potentially different effects in this setting. We reported on the the risk of adverse events drug trough levels were monitored immune reconstitution of 108 patients (pts) with and the dosing was adjusted to low therapeutical levels (3-8 haematological malignancies after unrelated transplants and ng/ml). Nine pts had a complete response and 10 a partial conditioning with either rabbit ATG Genzyme response with an overall response rate of 70%. Steroids could (ThymoglobulinR, ATG-G) or ATG FreseniusR (ATG-F) (BMT be tapered and stopped in 10 out of 18 surviving pts. No 2004, 33, S3). Here we analyse their clinical outcome. difference was observed in everolimus- and sirolimus-treated Patients: 66 pts (cohort1) received ATG-G, the following 42 pts. Major adverse events possibly related to mTOR-I were pts (cohort2) ATG-F. There were no differences between the hyperlipidaemia and impaired wound healing, especially in pts groups regarding age, Karnofsky index, disease or risk status, with ulcerative lesions of skin or mucous membranes. None of CMV risk and match. 31% had • one class I Ag MM or one the pts developed thrombotic microangiopathy (TMA) and class II allel MM. Cohort2 had more primary AML (36 vs 17%), infectious complications were rare. Seven pts died, 4 of the reduced conditioning (47 vs 26%), MMF (+CSA) instead of non-responders either due to cGVHD (n=2) or infection (n=2) MTX (26 vs 5%) and slightly more PBSC (60 vs 42%). 71% of and 3 of the responders due to relapse of the underlying

S103 malignancy. The median follow-up for surviving pts is 15 stem cell transplantation, we applied recombinant human IL-7 months. Controlled trials comparing this approach with (rIL-7) in an experimental model of murine syngeneic bone alternative strategies are warranted. marrow transplantation (BMT) using in vitro incubated bone marrow cells (BMC), followed by in vivo administration of rIL- 7. Results showed a two-fold increase in BMC cellularity after P540 in vitro incubation of bone marrow cells with rIL-7, associated Activating killer immunoglobuline-like receptor with an enhanced reconstitution of BMC and thymocytes at 45 incompatibility affects outcome after allogeneic days post transplant. In addition to increased cellularity, mice haematopoietic stem cell transplantation treated with rIL-7 also showed enhanced proliferative S. Giebel (1), I. Nowak (2), T. Czerw (1), J. Wojnar (1), T. responses to mitogenic stimulation. Furthermore, riL-7 Kruzel (1), M. Krawczyk-Kulis (1), A. Karolczyk (1), M. significantly increased rearrangement activating gene (RAG-1) Markiewicz (1), I. Wylezol (1), P. Kusnierczyk (2), J. expression and promoted V beta 8 (D)j gene rearrangement of Holowiecki (1) the T cell receptor in the thymus. Treatment with rIL-7 induced (1)Silesian Medical University (Katowice, PL); (2)Institute of secretion of IL-2 by thymic cells in vitro. The finding that rIL-7 Immunology and Experimental Therapy (Wroclaw, PL) induces expansion of BMC, enhances differentiation and proliferation of immature lymphocytes, thus counteracting post Activating and inhibitory killer immunoglobulin-like receptors BMT immune deficiency, makes it a promising new agent for (KIRs) are expressed on NK cells and a subset of T cells. KIR clinical application for treatment of immune deficiency and for genotype is characterized by two main haplotypes: haplotype enhancing immunohematopoietic reconstitution of stem cell A including KIR2DS4 as the only activating KIR and haplotype allograft recipients. B with varying number of activating KIRs. The goal of this prospective study was to evaluate prognostic significance of KIR genotype, including the presence of P542 particular genes in the donor and the recipient as well as Association of TLR-4 mutations in host and donor cells incompatibility in respective loci. Mismatches in graft-vs.-host and the risk for acute GVHD after allogeneic (GVH) direction were considered if gene was present in the haematopoietic stem cell transplantation recipient and absent in the donor. Incompatibility in HVG T. Imado, T. Iwasaki, T. Kuroiwa, S. Kitano, S. Tsunemi, H. direction was recognized in opposite situation. Sano, H. Ogawa Seventy patients with hematological malignancies, aged 32.5 Hyogo College of Medicine (Nishinomiya, JP) (18-58)y, given transplant from an HLA-identical sibling (n=28) or matched unrelated donor (n=42) were included. The Lipopolysaccharide (LPS) has been implicated in the conditioning regimen was myeloablative and based on pathogenesis of acute graft-versus-host disease (GVHD). The chemotherapy alone (76%) or TBI (24%). GVHD prophylaxis toll-like receptor (TLR)-4 has been recently identified as a consisted of cyclosporin, methotrexate, and, in case of MUD- major receptor for LPS. It is possible that mutations of TLR-4 HSCT, pre-transplant ATG. have been associated with LPS hyporesponsiveness, thereby In a multivariate analysis the OS rate was negatively affected reducing the risk of acute GVHD in allogeneic hematopoietic by KIR2DS4 incompatibility (48% vs. 83%; RR=4.4; p=0.01), stem cell transplantation (HSCT). However, stimulation of and the presence of B haplotype in the donor (69% vs. 94%, TLRs may also initiates a signaling cascade for induction of RR=10.45; p=0.03), together with higher recipient age, various pro-inflammatory cytokines, leading to microbial unrelated donor, and blood group mismatch. Increasing eradication. Therefore, mutations of TLR-4 may increase risk number of activating KIR mismatches in HVG direction of microbial infection, thereby accelerating acute GVHD in (analyzed as continues variable) was associated with allogeneic HSCT recipients. Retrospective studies to decreased DFS (RR=3.45; p=0.008), increased NRM determine the frequency of TLR-4 mutations and their (RR=1.43, p=0.02), and the incidence of grade II-IV acute possible association with acute GVHD contradict each other. GVHD (RR=1.35; p=0.03). Any KIR2DS4 incompatibility In the present study, we examined the association of TLR-4 resulted in increased incidence of NRM (R=7.56, p=0.001). mutations in host and donor cells and the risk for acute GVHD KIR2DS4 mismatch in GVH direction was associated with after allogeneic HSCT. To examine the role of TLR-4 in host higher risk of grade II-IV acute GVHD (RR=3.74; p=0.04) and cells, we analyzed TLR-4 expression in the intestine by extensive chronic GVHD (R=2.38; p=0.03), whereas mismatch immunohistochemistry. Lethally irradiated C3H/HeN mice in HVG direction resulted in increased risk of CMV infection received allogeneic HSCT from C57BL/6 donors. TLR-4 (RR=4.67; p=0.02). Inhibitory KIR genotype was not found to expression in the intestinal epithelial cells was significantly influence any of the study end-points. increased after GVHD induction. Next, we used a mouse Conclusions: Increasing number of activating KIRs in the strain (C3H/HeJ) that has a single amino acid substitution in donor, in particular in the absence of their couterparts in the its TLR-4 as recipients in an allogeneic HSCT. Lethally recipient, seems to enhance GVH alloreactivity resulting in irradiated either C3H/HeN or C3H/HeJ hosts received HSCT impaired survival. Whether this effect is associated with the from C57BL/6 donors. C3H/HeN recipients receiving C57BL/6 activity of NK cells or KIR-bearing T cells requires further donors developed significantly less GVHD as measured by investigation. The role of KIR2DS4 appears diverse. Our mortality and intestinal histopathology compared with results suggest that it may serve as minor histocompatibility C3H/HeJ recipients receiving C57BL/6 donors. Next, we antigen when expressed in the recipient and not in the donor. examined the role of TLR-4 in donor cells. Lethally irradiated C57BL/6 hosts received allogeneic HSCT from either C3H/HeN or C3H/HeJ donors. Mice receiving C3H/HeJ P541 donors developed significantly less GVHD as measured by IL-7 is a potent cytokine for enhancement of mortality and intestinal histopathology compared with mice immunohaematopoietic reconstitution following bone receiving C3H/HeN donors. Taken together, these results marrow transplantation suggest that TLR-4 in host cells is crucial in the microbial A. Abdul-Hai (1), A. Ben-Yehuda (2), S. Slavin (1) eradication in the intestine and the protection of intestinal (1)Hadassah University Hospital (Jerusalem, IL); (2)Hadassah GVHD, while responsiveness of donor cells to LPS may be an School of Medicine (Jerusalem, IL) important risk factor for acute GVHD.

Interleukin-7 (IL-7) is a critical cytokine in early B and T cell development and thus may play an important role in facilitating impaired immune reconstitution resulting in increased risk of infectious complications post allogeneic stem cell transplantation. In order to study the role of IL-7 on thymopoiesis and peripheral T cell expansion and reconstitution from uncommitted stem cells in the setting of

S104 P543 activity in intestinal and hepatic GVHD and for distinguishing The graft content of donor T-cells expressing active GVHD from irreversible end organ damage. gamma/delta TCR+ and CD4+foxp3+ predicts the risk of Prospective studies are warranted to evaluate how CK18F acute graft-versus-host disease after transplantation of may assist in diagnosis, differentiation, prognosis, and allogeneic peripheral blood stem cells from unrelated treatment guidance of GVHD. donors U. Platzbecker, C. Pabst, C. Theuser, E. Ehninger, M. Bornhäuser P545 University Clinic Carl Carus (Dresden, D) Escalated-dose donor lymphocyte infusion depletion of alloreactive T-cells may limit infections and malignant Objectives: Recently, high numbers of regulatory T-cells within relapse without causing GvHD after haplotype the stem cell graft were described to be associated with less mismatched myeloablative stem cell transplantation graft versus host disease (GVHD) after related peripheral D.C. Roy, S. Cohen, L. Busque, D. Fish, T. Kiss, S. Lachance, blood stem cell transplantation (PBSCT). Studies in mice also G. Sauvageau, P. Caudrelier, J. Roy suggest a distinct role of gamma/delta TCR+ T-cells in Hop. Maisonneuve-Rosemont (Montreal, CAN) mediating GVHD. Therefore, the aim of this study was to define the yet unknown role of regulatory and gamma/delta Patients with very high risk hematologic malignancies who TCR+ T-cells in human PBSCT from unrelated donors. cannot find an HLA-matched related or unrelated donor can Methods: The frequency of both T-cell subsets within the graft benefit from haplo-mismatched transplantation. The latter is, was analyzed in 63 patients receiving matched unrelated however, complicated by frequent and severe infectious allogeneic PBSCT after a busulfan or TBI based intensive complications and disease relapse due to delayed immune conditioning. No ATG was used during conditioning and all reconstitution. We have previously reported that patients received unmanipulated PBSC on day 0 containing a photodynamic therapy (PDT) could selectively deplete donor median of 7.6 x106/kg CD34+ cells (range 3.4-17.9). All alloreactive populations while preserving lymphocytes for patients were given cyclosporine A and methothrexate as immune responses. We present results of an ongoing Phase I GVHD prophylaxis. The respective amounts of T-cell subsets clinical trial of haplo-mismatched allogeneic stem cell in the graft were quantified by flowcytometry and PCR and transplant (SCT) supplemented with donor lymphocyte further correlated with clinical outcome. infusions (DLIs) PDT depleted of host-reactive T cells. Nine Results: The grafts contained a median of 11.2 x106/kg high-risk patients with hematologic malignancies (5 AML CD4+foxp3+ and 9.8 x106/kg gamma/delta TCR+ T-cells, relapsed or refractory, 2 MDS, 1 NHL, 1 refractory CLL) respectively. Patients receiving more CD4+foxp3+ cells had a entered the trial (6 M, 3 F) and are evaluable for acute GVHD lower cumulative incidence of acute GVHD II-IV (44% vs. and immune reconstitution. Donor mononuclear cells (MNCs) 65%, p=0.03). Interestingly, in patients who received higher were incubated with recipient MNCs for 4 days, exposed to concentrations of donor gamma/delta TCR+ T-cells acute TH9402 PDT, stored frozen, and administered on day 30 after GVHD II-IV was more frequent (66% vs. 40%, p=0.02). In transplant at graded DLI dose levels: 1x104(1pt), 5x104(3pts), multivariate analysis only the graft concentration of 1.3x105(3pts) and 3.2x105(2pts) CD3+ cells/kg. Anti-host gamma/delta TCR+ T-cells (p=0.002) and a positive CMV- cytotoxic T lymphocyte precursors (CTLp) were depleted from status of the recipient (p=0.03) were significantly associated DLIs by approximately 1.5 logs, and flow cytometry showed with the occurrence of acute GVHD II-IV. greater than 90% elimination of activated T cells (CD4+CD25+ Conclusion: Graft composition of T-cell subsets seems to and CD8+CD25+) by TH9402 PDT. Median age at SCT was affect the outcome of patients receiving allogeneic PBSCT 55 years (range: 21-58). The myeloablative regimen consisted from unrelated donors. Therefore, selective manipulation or of TBI (1200 cGy), thiotepa (5 mg/kg) and fludarabine (40 add-back of particular subsets might be a promising strategy mg/m2/dayx5 days) followed by infusion of CD3 depleted HSC to reduce the incidence of GVHD. grafts. No GVHD prophylaxis was administered. All patients showed durable trilineage hematologic engraftment and complete donor chimerism. No patient developed acute GVHD P544 (grade II-IV), while 3 patients developed signs of chronic Serum cytokeratin-18 fragments as biomarker for GVHD. Five patients died: one (cohort 1) of a post-transplant hepato-intestinal GvHD activity lymphoproliferative disease, one (cohort 2) of relapsed AML, T. Luft (1), M. Conzelmann (1), E. Rodionova (2), M. Hess (2), and 3 (cohort 2 and 3) of infectious complications. Two pts U. Strohhäcker (2), A. Benner (2), M. Görner (3), U. recovered greater than 0.3x109 CD3+ and CD4+ cells/L at 2 Hegenbart (1), A. D. Ho (1), P. Dreger (1) and 5 months post-DLI, and 4 pts had >0.2x109 CD3+ cells/L (1)University of Heidelberg (Heidelberg, D); (2)DKFZ at 6 mo post-DLI. The overall disease-free-survival and (Heidelberg, D); (3)City Hospital (Bielefeld, D) survival are 44% at 1 year (median follow-up: 10.3 mo). Our results indicate that the post-transplant infusion of a PDT A biomarker for sensitive measurement of GVHD activity is treated DLI is feasible, does not induce acute GVHD, and may still lacking. Since apoptosis is the histopathological hallmark accelerate T cell reconstitution. This PDT strategy could in GVHD, we investigated whether the caspase-cleaved neo- represent an appealing alternative for patients in the higher epitope of cytokeratin 18 fragments (CK18F) released during age range who are at high risk for GVHD. apoptosis might be a serum marker for ongoing GVHD- induced target organ destruction. Serum CK18F kinetics was monitored by M30 antibody-based P546 ELISA in 51 patients who fulfilled histopathological and/or Extracorporeal photopheresis for the treatment of steroid clinical criteria diagnostic for GvHD. Both intestinal and refractory acute GvHD hepatic GVHD were associated with significant elevations of P. Perfetti (1), P. Carlier (1), P. Strada (1), F. Gualandi (1), CK18F levels over baseline (6.3±3.8 fold and 4.6±0.8 fold, M.T. Van Lint (1), T. Lamparelli (1), A.M. Raiola (1), C. Di respectively), whereas isolated skin GVHD was accompanied Grazia (1), S. Bregante (1), E. Pogliani (2), A. Bacigalupo (1) by only slight CK18F changes. Responses of hepato-intestinal (1)San Martino (Genoa, I); (2)Ospedale San Gerardo Monza GVHD to immunosuppressive therapy were mirrored by (Monza, I) CK18F decreases. The extent of CK18F increases correlated with serological severity of acute (r=0.96) and chronic (r=0.76) Extracorporeal photochemotherapy (ECP) is a therapeutic liver GVHD. Clinical conditions that might represent relevant approach currently used for the treatment of T-cell mediated differential diagnoses, such as non-complicated, infection- diseases, as T-cell-lymphomas, chronic Graft versus Host related diarrhea and toxic hyperbilirubinemia were not Disease (cGvHD), graft rejection and autoimmune diseases. associated with CK18F rises. In recent reports also acute GvHD (as an expression of In conclusion, CK18F monitoring provides the first marker for alloreactivity) has shown some response to ECP. From 1996 quantitative assessment of GVHD-associated apoptotic to 2006 we have been treating with ECP 23 patients with

S105 steroid-refractory acute GvHD, after receiving a stem cell (35,7%), hyperlipidemia in 4 ( 28,6%), LDH elevation in 3 transplantation from a full matched or 1-antigen mismatched (21,4%). Treatment had to be terminated due to adverse related or unrelated donor. Their median age was 41 (range event (TTP) in 5 pts (35,7%). CMV infection was observed in 18-66) and diagnoses were as follows: 5 chronic myeloid 3 with spontaneous resolvement in 2 pts. leukemia, 8 acute leukemia, 3 myelodysplastic syndrome, 2 Outcome: 7 pts are alive, 2 pts died due to GVHD, 5 due to multiple myeloma, 2 myelofibrosis, 3 aplastic anemia; 40% of infection. Response of GVHD: CR was achieved in 5 (38%), these patients was not transplanted in complete remission. PR in 2 (15,4%), no change in 2 and progression in 4 (30,8%). The median duration of ECP treatment was 6.6 months (range Conclusions: we have observed a high overall response of 0-32.8), and the median number of ECP cycles (1 cycle=2 pts with advanced cGVHD under treatment with RAD001, treatments) was 10 (range 1-25). After three months of treatment was well tolerated with a high compliance due to therapy 56% (13 of 23) of patients improved aGvHD application. TTP remains the major problem with an incidence manifestations (52% improved, 4% had a complete of approximately 35%. Our experience suggests that further resolution). An improvement was obtained in 80% (8 of 10) of prospective studies are warranted. patients with global grade II aGvHD, 71% (5 of 7) with grade III and none (0 of 6) with grade IV. 70% of patients with a skin disease responded to ECP treatment, 57% of those with liver P548 aGvHD and 65% with gut involvement (this series had no Low incidence of acute GvHD after adoptive DLI patients with grade +++ aGvHD of the gut). ECP treatment application post transplantation of highly purified CD34 started after a median time of 56 days (range 14-148) from positive cells aGvHD onset; in both groups of patients, treated before and A.H. Elmaagacli, R. Peceny, M. Koldehoff, N. Steckel, M. after 56 days, 54% respoded to ECP. Overall survival after a Ditschkowski, R. Trenschel, H. Ottinger, D. Beelen mean follow-up of 24 months (range 11-61) is 52% (12 of 23); University Hospital of Essen (Essen, D) seven patients (30%) died of uncontrolled aGvHD during ECP treatment, two patients (9%) died of infection, and two (9%) of The application of DLI for treatment of relapse is a widely disease relapse. Among ECP responding patients 100% used therapy option after allogeneic transplantation. But one progressed into a chronic GvHD (31% had a limited and 69% of the major complication of this therapy modality is the an extensive diesease); 70% of them achieved a complete occurrence of severe acute GVHD. Here we evaluated the resolution of cGvHD manifestations during follow-up, with only incidence of acute GVHD after adoptive application of DLI with 15% discontinuing immunosuppression after 26 to 30 months increasing T cell doses post transplantation of highly purified from stem cell transplantation (SCT). In 46% of patients CD34+ cells in patients transplanted from HLA identical immunosoppressive therapy could be tapered at a median siblings for various haematological diseases. DLI was applied time of 13 months from SCT(range 5-26). with a starting dose of CD3+ 3,3x 105 at day 90, thereafter In conclusion : this study confirms that ECP has a role in the DLI was administered at day 135 post transplant and in case management of steroid refractory acute GvHD. Responses of MRD or relapse. are obtained with time and one needs to be patient. We were 57 patients transplanted for CML (n=52), AML (n=2), ALL particularly impressed with responses in patients with gut (n=1) and multiple myeloma (n=2) received DLI afer GvHD. transplantation. 19% of the patients had a progressive disease stage at time point of transplantation and 81% a standard disease stage at time point of transplantation. P547 DLI was applied five times at median (range1-8) with Efficacy and tolerability of the novel mTOR antagonist increasing T cell doses. At median a T cell dose of 3,3 x106 Everolimus (RAD001) in treatment of patients with CD3+ cells/kg/BW were applied until a haematological or advanced chronic GcHD after allogeneic stem cell molecular response was achieved. transplantation After transplantation acute GVHD grade 1 was diagnosed in S. von Harsdorf (1), M. Schmitt (1), M. Ringhoffer (1), R. 15% of patients and acute GVHD grade 2 in 3% of patients. In Schlenk (1), H. Döhner (1), M. Wiesneth (2), D. Bunjes (1) none of the patients a GVHD grade 3-4 occurred, whereas (1)University Hospital (Ulm, D); (2)DRK Dept. of Transfusion 85% of the patients had no GVHD. After DLIs, acute GVHD Medicine (Ulm, D) grade 1 was diagnosed in 18% if patients and acute GVHD grade 2 in 14% of patients. Severe GVHD occurred in 12% of Objectives: pts with cGVHD not responding to steroids have a the patients, whereas no GVHD was seen in 56% of the poor prognosis. Everolimus, (RAD001, Certican) a new mTOR patients after DLI. Four of the seven patients who suffered antagonist, only known as immunosuppressant in organ from severe GVHD died. The estimate to develop a GVHD transplantation, has not been used in treatment of pts with grade2-4 after transplantation and DLI was only 27%. A GVHD after allo-SCT. Here we report our experience in a haematological disease reoccurrence were found in 14% only. retrospective analysis of 14 pts with hematologic malignancies The concept of transplantation if highly purified CD34+ cells after a median follow up of 17,54 months (range: 6,31-53,7 and adoptive DLI was associated with a low five-year estimate mo). for TRM and disease free survival of 80% for all patients and Pts and methods: 14 pts (AML 7, CML 2, CLL 2, NHL 1, MM 90% for CML-patients. 1) with a median age of 48,8 years underwent allo-SCT from April 2002 to December 2005. Myeloablative conditioning was used in 8, reduced-intensity conditioning in 6 pts. All pts P549 received peripheral stem cells, 11 from HLA-identical related The impact of histopathological subtype of cutaneous donors, 3 from matched unrelated donors. GVHD-prophylaxis chronic GvHD on alloHSCT outcome: lichenoid versus consisted of CsA/Mycophenolat (MPA) in 10 pts, scleradermoid CsA/Campath 100 mg in 4 pts. ATG as rejection prophylaxis E.A. Soydan, G. Cengiz, H.E. Sanli, P. Topcuoglu, A.U. Bilgin, was used in 4 pts. CMV-serostatus was positive in 9 pts., with S.M. Bakanay, O. Arslan, M. Ozcan, T. Demirer, H. Akan, O. seronegative donors in 4. Ilhan, M. Beksac, N. Konuk, A. Uysal, G. Gurman, M. Arat Results: acute GVHD occurred in 10/14 (71,4%), grade II-IV School of Medicine, Ankara University (Ankara, TR) in 6 (42,9). All pts developed cGVHD with limited disease in 3 (21,4%) and extensive disease in 11 (78,6%). At the time of Introduction and aim: Chronic graft versus host disease starting treatment with RAD001, all pts were on steroids and (cGVHD) is the most common late complication of allogeneic under second line therapy with tacrolimus/MPA in 4 (28,6%) hematopoetic cell transplantation (AHCT). The most or more than second line treatment in 10 (71,4%). The starting characteristic expression of cutaneous involvement in cGVHD dose of Certican was 2 x 0,75 mg/day orally until reaching includes lichenoid and/or sclerodermatous manifestations, plasma levels of 3-8 ug/l. Median treatment duration was pigment and mucosal changes. Lichenoid lesions usually 154,5 days (range: 4-585). Following adverse events occur in early cGVHD and clinically resemble idiopatic lichen occurred: TTP/HUS/haemolytic anemia (MAHA) in 5 pts planus. Patients with cutenous changes showing all the typical

S106 histological aspects of lichenoid GVHD seem to have a worse hematological malignancies (73%) or aplastic syndromes prognosis. Sclerodermoid lesions usually develop (27%). Median age was 29y(5-58) and median follow-up was progressively, long after acute GVHD in the site of previously 24mo(1-54). The donor was HLA identical in 43% and involved lichenoid lesions or on healthy skin. It is unrelated in 57%. Myeloablative regimen was used in 77%. As characterized by fibrosis and in severe forms patients mobility GVHD prophylaxis CsA+MTX was used in 26 patients, CsA and quality of life is affected. In this study we retrospectively alone in 14 and in 3 patients CsA+steroids. Bone Marrow cells analysed the demographic data and life expectancy of were used in 62%, PB in 25% and Cord Blood in 13%. patients with chronic skin GVHD according to type of their skin Treatment of aGVHD consisted of prednisolone 2mg/kg IV in involvement proven by skin biopsies. all patients associated to CsA. Once criteria of resistance Method and results: Allogeneic transplants performed in 428 were established, CsA was stopped and replaced by MMF patients between 1988-2006 in our center. Mean age 31.2 (5- and/or FK506. FK was not added if renal insufficiency was 64) years, M/F: 248/180, diagnosis were 144 CML, 148 AML, observed. Criteria of steroid-resistant aGVHD were A in 7 50 ALL, 22 SAA, 20 MDS, 11 MM, 6 lymphoma, 5 FAA and 22 patients; B in 26; C in 19, and D in 1 patient. Steroid resistant other diseases. 357 of the patients received ablative acute GVHD grade I was observed in 9 patients(17%); II in conditioning mostly busulphan based non-TBI regimens with 26(49%); III in 19(36%) and IV in 3(6%). MMF was used in 15 short term CSA and MTX GVHD prophylaxis. We observed patients(28%) and associated to FK506 in 38(72%). Median cGvHD in 160 out of 314 patients surviving after +100th day at time to start second line treatment was 16days (7-100). CR in postranplantation;114 (71.3%) had limited and 46 (28.7%) had skin was observed in 74% of the patients, 66% in gut and 75% extensive cGVHD. All donors were HLA identical siblings. in liver. However, overall CR was observed in 21 patients Stem cell source was periperal blood in 245 and bone marrow (40%), partial response in 5 (9%) and failure in 27 (51%). in 183 of the patients. In our patient cohort chronic GVHD Median time to obtain CR/ PR was 8 d(2-20). Cumulative incidence was 50.9 %. In the cGVHD group 32.5 % (n=52) of incidence of CR was 39%. Age, CMV status, source of cells, the patients had skin involvement. Distrubution of these 52 conditioning regimen, HLA incompatibility and MMF alone or patients according to histopatologic diagnosis were 61.5% associated to FK were not associated with CR. Overall lichenoid, 23.1% sclerodermoid and 15.4% were other survival was 35±7%; it was 74±10% for 21 patients achieving hystopathologic types. Patient characteristic were summarized CR; 5±4% for 27 non-responders and 1 out 5 patients with in the table. All patients received MP±CSA for initial treatment. PR. In conclusion, use of MMF and/or FK506 is an option to Mycophenolate mofetil was given to patients steroid-refractory treat steroid resistant GVHD but did not result in 1-year or dependent cases. ECP was applied to steroid refractory or survival that would justify to set-up a larger randomized study. intolerant progressive patients. Conclusion: Our 52 patients with pathologically proven and histologically subgrouped cutaneous cGVHD did not differ in P551 terms of long term transplant outcome. The usage of broad spectrum chemokine inhibition reduces the severity of idiopathic pneumonia syndrome after allogeneic stem cell transplantation G.C. Hildebrandt (1), S. Miklos (1), Y. Chang (1), G. Mueller (1), D.J. Grainger (2), E. Holler (1), R. Andreesen (1) (1)University of Regensburg Medical School (Regensburg, D); (2)University of Cambridge (Cambridge, UK)

Idiopathic pneumonia syndrome (IPS) is a major cause of mortality after allogeneic (allo) stem cell transplantation (SCT). IPS pathophysiology includes the secretion of inflammatory cyto- and chemokines along with the recruitment of donor T cells and monocytes/macrophages. We have previously shown, that inflammatory CC- and CXC- chemokines, increasingly expressed in the lungs after allo-SCT, play a critical role in this context, as abrogating the interaction of only one of these chemokines with its respective receptor was able to partially reduce pulmonary injury. The novel broad spectrum chemokine inhibitor (BSCI) BIM-58001 suppresses the in vitro- and in vivo-migration of leukocytes in response to several CC- and CXC-chemokines. We therefore P550 hypothesized, that after allo SCT the administration of BIM- Mycophenolate mofetil with or without tracolimus (FK506) 58001 inhibits chemokine-induced migration of donor effector as a second line treatment for steroid-resistant acute cells to the lung, thereby reducing injury and improving graft-versus-host disease. The experience of Saint Louis pulmonary function (PF). Lethally irradiated B6D2F1 mice Hospital were transplanted with either syngeneic (syn) B6D2F1 or allo G. Fisher, V. Rocha, M. Dos Santos, A. Devergie, M. Robin, C57BL/6 bone marrow and T cells. IPS progressively R. Peffault de Latour, P. Ribaud, C. Ferry, E. Gluckman , G. developed in allo recipients until week 6. Chemokine Socié expression of MCP-1, RANTES, Mip-1 alpha peaked around Hopital Saint Louis (Paris, F) day + 14, whereas Mig and IP-10 levels remained continuously elevated. Next, animals were treated with either We conducted a phase II trial to test the role of second line BIM-58001 or vehicle control (vc) from day 0 to day +14, treatment using mcophenolate mofetil (MMF) associated or analyzed on day +42 and histopathological changes were not to FK506 in steroid resistant aGVHD in 53 patients. Four correlated with PF. Compared to vc and BIM-58001 treated criteria of steroid-resistant aGVHD were defined: A) no syn controls, allo recipients treated with vc showed significant improvement of signs of skin GVHD after one week of damage to the lung with increased inflammatory cell infiltrates treatment with prednisolone 2mg/kg; B) signs of skin in the alveoli, interstitial spaces, around vessels and around progression or no response of visceral (gut or liver) GVHD 3 bronchial structures. These changes correlated with impaired days after treatment; C) visceral signs of progression 2 days PF (figure 1). In contrast, when treated with BIM-58001, allo after treatment; D) progression to organ stage 4, 1 day after recipients demonstrated a clear reduction in pulmonary treatment. Response of signs of aGVHD to MMF pathology along with PF improvement. Next, we assessed the (30mg/kg/day) or FK506 (0.05mg/kg/day) was defined as effect of BIM-58001 treatment later during IPS development complete remission (CR)(all organs); partial remission (PR) or by giving BIM-58001 or vc from day +15 until day +42. Again, failure (progression even in only one organ). HSCT were animals were analyzed on day +42 for histopathological performed between 1999 and 2004 for patients with changes and PF. Interestingly, this time no significant

S107 differences were seen between groups after allo-SCT, P553 suggesting that interrupting early chemokine signalling is more Pre-engraftment serum C-reactive protein value may efficient. In conclusion, our data show, that usage of BSCI predict acute graft-versus-host disease and non-relapse may provide a new tool in the prevention and treatment of mortality in patients undergoing allogeneic myeloablative IPS, and that early intervention is critical to hinder the cascade haematopoietic stem cell transplantation of events during IPS development. S. Fuji, S. Kim, S. Mori, T. Fukuda, R. Tanosaki, Y. Heike, K. Tobinai, Y. Takaue National Cancer Center Hospital (Tokyo, JP)

Background: In a mouse model, Ferrara et al. clearly showed that inflammatory cytokines play a primary role in the afferent and effector phases of acute graft-versus-host disease (aGVHD). In the human setting, C-reactive protein (CRP) is a simple and reliable marker of inflammation, represented by infectious diseases, when the production of inflammatory cytokines, including TNF-alpha and IL-6, is increased. In our routine transplant procedure, the CRP level is measured serially at least three times a week. In this study, the correlation between the pre-engraftment CRP value and clinical events was analyzed to test whether the CRP value could be used as a practical surrogate marker of inflammation that leads to post-transplant complications including aGVHD. P552 Methods: The data from a cohort of 118 consecutive adult Targeted mycophenolate mofetil for graft-versus-host patients with hematological malignancies who were treated disease prophylaxis after related and unrelated between January 2002 and July 2006 were reviewed haematopoietic cell transplantation retrospectively. Sixteen patients were excluded due to graft J. Freiberg-Richter, I. Haentschel, A. Jenke, P. Lorenz, A. failure, preexisting neutropenia and previous allogeneic Kiani, J. Schetelig, G. Ehninger, E. Schleyer, M. Bornhäuser transplant. The remaining 102 patients (median age, 36 y; 18- University Hospital Dresden (Dresden, D) 57 y) were categorized according to the maximum CRP value during neutropenia before engraftment. All patients received Purpose: We performed a prospective phase I/II trial targeting myeloablative conditioning regimens including TBI-based daily MMF doses according to MPA AUC levels. (n=49) and non-TBI-based (n=53) regimens. GVHD Patients and Methods: Twenty-nine patients (18 male, 11 prophylaxis included cyclosporine- (n=71) and tacrolimus- female) with a median age of 53 years were included. The based regimens (n=31). The "low CRP" group (CRP < 15 indication for allogeneic transplantation from matched sibling mg/dl) included 70 patients and the "high CRP" group (n=7) and unrelated donors (n=22) was high-risk AML/MDS (CRP>15 mg/dl) included 32 patients. The primary endpoint of (n=19) or relapsed lymphoma/multiple myeloma (n=10). this study was the occurrence of grade II-IV aGVHD. The Conditioning therapy included 30 mg/m² Fludarabine on days - secondary endpoints were overall survival (OS) and non- 9 to -6 combined with intravenous Busulfan 3.45 mg/kg from relapse mortality (NRM). day -5 to day -2. Tacrolimus was given orally starting on day - Results: Grade II-IV and grade III-IV aGVHD were both more 1 in order to achieve trough blood levels of 5-10 ng/ml. MMF frequent in the high CRP group than in the low CRP group was started on day 0 at 3 g intravenously bid. AUC (p<0.001 and p=0.047, respectively). A Cox proportional measurements of MPA and its metabolite MPAG by HPLC hazard model showed that a high CRP level and HLA- were scheduled on day 4, 7, and 11 after transplantation. The mismatch were associated with an increased risk of grade II- MMF dose was modified in order to achieve an AUC of 35-60 IV aGVHD. OS tended to be worse in the high CRP group µg/ml*h. than in the low CRP group (p=0.08). NRM was significantly Results: The dose of MMF had to be increased in 15/29 (52%) higher in the high CRP group than in the low CRP group patients to 3.5-5 g every 12 hours on day 5. No patient (p=0.004). A Cox proportional hazard model showed that a required a reduction of dosing on day 4. With the respective high CRP level and older age were associated with a high dose adjustments 36% and 80% of patients reached the AUC NRM rate. target on day 7 and 11, respectively. There was no direct Conclusion: Our results suggest that the pre-engraftment CRP association between dose level and extramedullary toxicity. value in humans reflects inflammation that could lead to the Early grade 3-4 gastrointestinal toxicity occurred in 4 patients development of aGVHD and subsequent NRM, as has been and lead to a reduction of MMF back to 2 g bid, although a proposed in a mouse model. direct association to the study drug was difficult to prove given the direct toxic effects of the preceding conditioning regimen. Trilineage engraftment and complete donor chimerism was observed in 28 out of 29 patients included. Only one out seven patients with a matched related donor experienced acute GvHD > grade II, which was due to non-compliance in the outpatient setting. The respective proportion of grade III-IV acute GvHD in the unrelated setting was 7/28 (25%). With a median follow-up of 18 months 15 (52%) patients are alive and 14 are in complete remission. So far, 8 out of 24 evaluable patients (33%) suffer from limited (n=3) or extensive (n=5) chronic GvHD. A retrospective analysis revealed a significant correlation between Cmax levels and the AUC for MPA (r=0.48; p<0.0001). The respective target Cmax was shown to be between 16 and 25 µg/ml. Conclusion: Given the high-risk patient population with 70% unrelated donors, the clinical results observed with the combination of tacrolimus and MMF as prophylactic regimen are encouraging. A simplified MMF targeting strategy based on MPA Cmax levels seems to be warranted in future trials.

S108 P555 CD3/CD19 depleted grafts for haplo-identical stem cell transplantation in children: un update P. Lang (1), I. Mueller (1), M. Schumm (1), M. Pfeiffer (1), T. Feuchtinger (1), J. Greil (2), P. Bader (3), R. Handgretinger (1) (1)University Children's Hospital (Tuebingen, D); (2)University Children's Hospital (Heidelberg, D); (3)University Children's Hospital (Frankfurt, D)

Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation. We present our preliminary results with a direct depletion procedure using antiCD3/antiCD19 coated magnetic microbeads and the CliniMACS device. Only patients with high risk malignancies (n=30, most of them had active disease or relapsed after previous trp.) or with nonmalignant diseases and high risk of graft failure (n=2) were included. The diagnoses were: AML/MDS/CML (n=16), ALL(n=6), relapsed Neuroblastoma / Ewing- / Rhabdomyosarcoma (n=8), SAA, PNH (n=2); Remission P554 status: NR=11, 2nd transplantation=10, CR/CP1=3, CR2-4=6. De-escalation of alemtuzumab in GvHD-prophylaxis is The patients received either TBI or Bu based standard feasible without increased acute GvHD morbidity or conditioning regimens (n=9) or a toxicity reduced protocol mortality in allogeneic sibling and matched unrelated (Flud, TT, Mel, OKT3, MMF, n=23). The grafts comprised a donor transplantation median number of 16x106/kg (7-41) stem cells as well as high H. Bertz, J. Akkad, M. Blos, U. Denz, M. Egger, C. Gruellich, amounts of NK-cells (137x106 (9-550)) with 49x103/kg (7-200) F. Kaul, R. Marks, A. Rueckert, R. Waesch, J. Finke residual CD3+ cells and <0.01% CD20+ cells. Primary Albert Ludwigs University Medical Center (Freiburg, D) engraftment occurred in 90% of patients (after reconditioning and second stem cell donation: 100%). Median time to reach The goal in GvHD prophylaxis is to find a combination of >500 neutrophiles/ul and independence from platelet immunosuppressive (IS) drugs, which preserve a high graft substitution was 11 and 9 days respectively. Recovery of versus malignancy (GvM)- effect with no, tolerable or curable CD3+ cells was favorable (d90: 320/ul, d180: 600/ul). Acute GvHD. Alemtuzumab (MabCampathTM) was introduced in GvHD grade 0-1 occurred in 72%, 25% had GvHD grade II combination with calcineurin antagonists for GvHD- and one patient had grade III. Limited chronic GvHD occurred prophylaxis in high-doses resulting in a high rate of primary in 4 patients. No transplant related mortality (TRM) and, in graft failure, posttransplant lymphoproliferative diseases particular, no lethal infections were observed. (PTLD), viral infections and relapse. 17/32 patients (53%) were alive with a median follow up of 0.6 Within a protocol we used low dose Alemtuzumab in years (0.2 - 2.9). Single cause of death was relapse. Disease combination with CSA starting with 2x20mg/d (day-2 and day - status was predictive: 9/12 patients with leukemias and active 1, group 40)(10/2002 - 06/2005), deescalating the dose to disease relapsed, whereas only 2/10 patients in CR relapsed. 2x10mg/d (day -2 and day -1, group 20) (07/2005 - 05 /2006) 4/8 patients with solid tumors are alive. and since June 2006 to 10mg/d on day -1(group 10) in HLA The toxicity-reduced conditioning regimen helped to avoid identical sibling or MUD alloTX. TRM, despite intensive pretreatment (including previous Patients: Overall, 174 consecutive pts. (group 40 n=74; group transplantation) of the patients. Conclusions: our preliminary 20 n= 65; group 10 n=35) received after a mainly fludarabine- results indicate that CD3/CD19 selected grafts can improve based reduced intensity conditioning (99%; 98%; 94%) a graft immune reconstitution and TRM. Engraftment rates similar to (PBSC in 99%) from a sibling (40%; 32%; 23%) or MUD (60%; that of patients with myeloablative standard conditioning 68%; 77%). Diagnosis were mainly AML/MDS (40%; 78%; regimens and positive selected haploidentical stem cells could 77%) or lymphoma/MM (51%; 23%; 14%). Remission at TX be achieved, although most patients of the study group was mainly advanced disease stage/ >CR1 (92%; 86%; 87%), received an intensity reduced regimen. However, the outcome also reflected by the fact that within each group 45%, 22% was poor in patients with active disease. Thus, further options and 37% received their second transplantation, respectively. have to be investigated to increase the potential antileukemic All pts. received standard care including aciclovir prophylaxis activity of donor derived effector cells. till day +100, PjP- prophylaxis starting day +25 and weekly CMV-PCR diagnostic procedures. Results: Primary graft failure was observed in one pt. each in P556 groups 40 and 10; both have been rescued with an additional Genetic risk factors for post allogeneic HSCT graft. complication among Japanese population The incidence of acute GvHD II-IV and III-IV was identical in M. Onizuka (1), M. Shimada (1), M. Kojima (1), Y. Sirasugi (1), each group (23% and 8% in group 40, 25% and 8% in group 20, 23% and 8% in group 10), respectively. M. Oki (1), Y. Ogawa (1), K. Miyamura (2), Y. Kodera (2), H. Until day +100 30 pts. died (17pts.; 7pts.; 6pts.). Comparing Inoko (1), K. Ando (1) (1)Tokai University School of Medicine (Kanagawa, JP); the groups causes of death were relapse (44%; 43%; 57%), non-viral-infection (17%; 29%; 0%), viral infection (17%; (2)Japanese Red Cross Nagoya First Hospital (Nagoya, JP) 0%;14%), aGvHD (6%; 0%; 0%) and others (17%; 29%; 29%). Despite recent advances in our understanding of the relation In group 40 (n=3) and group 20 (n=4) PTLDs was observed. between the genetic background and the risk of complication CMV reactivation was treated in up to 53% of the pts. at risk after allogeneic hematopoietic stem cell transplantation (recipient and/or donor with positive CMV-serology) (HSCT), it is difficult to apply it exceeding between races Conclusion: Dose deescalation of Alemtuzumab as part of our because patterns of genetic variation are different depending GvHD-prophylaxis from 40mg to 10mg absolutely, is feasible, on population. We investigated previously described post with low incidence of acute GvHD in all dose groups for MUD HSCT complication related genetic polymorphisms and as well as in sibling alloTX. compared the frequency of developing non-infectious pulmonary dysfunction (NIPD), acute graft versus host disease (aGVHD) and chronic GVHD (cGVHD) with that of allele frequency and clinical factors in Japanese population. We retrospectively analyzed the incidence of complication and donor/recipients polymorphisms of ACE, GSTM1,

S109 NOD2/CARD15, FCLR3, VDR, PAI1, CD14 and some and plasmacell leukemia (n=1). All donors were HLA identical cytokines and chemokine (TNFa, IL1a, IL6, IL10, RANTES) in at 1 haplotype and mismatched for 2 or 3 loci on the 126 Japanese patients who underwent HSCT from HLA- unshared haplotype. The conditioning regimen consisted of identical sibling donor. Clinical factors such as recipient age, Cytarabine 3 g/sqm/d for 3 days and Cyclophosphamide 45 gender mismatch, disease stage and conditioning regimen mg/Kg/d for 2 days combined with 10 Gy fractionated-total were included in the analysis. NIPD patients were high body irradiation (n=4), or Treosulfan 14 g/sqm/d for 3 days frequent rate of the genotype of ACE DD, IL6-634 GG, and (n=3) or i.v Busulfan 3,2 mg/Kg/d for 3 days (n=2) Because of TNFa-857 AA or CA (p = 0.0017, 0.048 and 0.048 graft failure following a double haploidentical T-cell depleted respectively). GSTM1 null genotype was significantly low or an autologous PBSC transplant, 2 patients were prepared frequency among sever aGVHD (grade III to IV) patients (p = with Fludarabine alone 40 mg/sqm/d for 3 days. As part of the 0.012). FCLR3 CC genotype was significantly low frequency conditioning regimen, all patients received Fresenius anti- among cGVHD patients (p = 0.0080). Although previous thymocyte globulin at 5 mg/Kg/d from day -4 to -1. As GvHD studies demonstrated strong evidence of the association prophylaxis, the patients received Cyclosporine at 1,5-3 between NOD2/CARD15 SNP13 and sever aGVHD, we could mg/Kg/day i.v. from day -7 to +28 and orally at 5 mg/Kg until not find the SNP among Japanese population. To decide day +365; Methotrexate 15 mg/sqm on day +1 and 10 mg/sqm NOD2/CARD15 haplotype in Japanese, we typed new three on day +3, +6 and +11; Mycophenolate mofetil 1 g/d from day SNPs based on international HapMap project. There was no +7 to +100; Basiliximab 20 mg i.v. on day 0 and +4. Donors statistical significance between Japanese NOD2/CARD15 were primed with Filgrastim at 3-4 microg/Kg/d from day -7 to haplotype and HSCT complication. -1. Bone marrow cells were infused fresh and unmanipulated. In conclusion, our studies revealed the association of genetic The median dose of nucleated cells, CD34+ and CD3+ cells background and post allogeneic HSCT complication. was 6,6x108/kg (1,01-10,8), 2,3x106/Kg (1,17-6,06) and Especially, FCLR3 is a new candidate cGVHD associated 30,8x106/Kg (16,9-66,6) respectively. Ten patients engrafted gene. However, this type of analysis should deeply consider with a median time of 21 days to reach >0.5 x109/L PMNs and the difference of population. 26 days to reach >25x109/L platelets. Six patients had no evidence of acute GvHD and 4 showed clinical manifestation of grade I. No patient had evidence of chronic GvHD. Two P557 patients (18%) died, 1 of septic shock on day +8 and 1 of Two steps to clinical multipotent mesenchymal stromal multi-organ failure on day 35. Nine out of 11 patients are now cells with human platelet lysate living and doing well in hematological remission after a K. Schallmoser, C. Malischnik, E. Rohde, A. Reinisch, K. median follow-up of 122 days (range, 35-479). The small Kashofer, G. Lanzer, W. Linkesch, D. Strunk number of patients described here does not permit any final Medical University Graz (Graz, A) conclusion. Nevertheless, the regimen of GVHD prophylaxis seems to be very effective in the setting of the unmanipulated Human multipotent mesenchymal stromal cells (MSC) are haploidentical transplant. The results are particularly promising candidates for a growing spectrum of regenerative encouraging and deserve further study with more patients and and immunomodulatory cellular therapies. Translation of longer follow-up. auspicious experimental results into clinical applications has been limited by the dependence of MSC propagation from fetal bovine serum (FBS). P559 We analyzed the capacity of human platelet lysate (HPL) to Successful therapy of steroid-refractory acute graft- replace FBS for clinical scale MSC propagation. versus-host disease with sequential alemtuzumab HPL could be efficiently produced from buffy coats. Multiplex R. Repp, A. Claviez, N. Schub, B. Gahn, A. Schrauder, M. analyses allowed delineating a distinct HPL growth factor Gramatzki profile. Using a previously established two-step clinical scale University of Kiel (Kiel, D) procedure, HPL was reproducibly more efficient than FBS in supporting MSC outgrowth. Using only 3 x 105 primary culture- After allogeneic stem cell transplantation severe acute graft- derived MSC we obtained mean 4.36 ± 0,51 x 108 MSC within versus-host disease (GvHD) is the major limitation of this a single secondary 11-13 day culture step. Although treatment modality. While in acute GvHD addition of steroids morphologically distinct, HPL-MSC and FBS-MSC did not to immunosuppressive prophylaxis lead to a response rate of differ significantly regarding immunophenotype, differentiation 30% to 70%, treatment options of steroid resistant acute potential in vitro and lack of tumorigenicity in nude mice in GvHD are limited and no accepted standard for salvage vivo. therapy exists. Alemtuzumab (Campath-1H) is a commercially Replacing FBS with HPL excludes bovine prion, viral and available humanized antibody targeting the CD52 antigen, zoonose contamination of the stem cell product. This new which is expressed on T-cells, B-cells and on some monocyte efficient FBS-free two-step-procedure for clinical scale MSC derived dendritic cells. For years, Alemtuzumab has been propagation will largely facilitate rational clinical testing of shown to be effective in GvHD prevention when used in MSC based therapies. Based on this protocol a clinical trial for conditioning regimes, however infectious complications did the treatment of steroid-refractory Graft versus Host Disease limit this approach when routinely applied. In order to evaluate with HPL-MSC has been initiated. efficacy and safety of sequential alemtuzumab for severe acute GvHD, ten patients with steroid-resistant acute GvHD of grade III or IV involving the gut or/and liver were treated in our P558 Division. The initial three patients, all with advanced grade IV Successful graft-versus-host disease prophylaxis in GvHD of the liver and already receiving cyclosporine, unmanipulated bone marrow transplantation from haplo- mycophenolate mofetil and high-dose steroids, were treated identical related donor for haematological malignancies with up to 80 mg of alemtuzumab followed by additional 40 mg S. Santarone (1), E. Di Bartolomeo (1), M. Postorino (2), L. CD52 antibody within the next four weeks. While dramatic Franceschini (2), W. Arcese (2), P. Di Bartolomeo (1) clinical responses were seen, such as a bilirubin level of 48 (1)Ospedale Civile (Pescara, I); (2)University Tor Vergata mg/dl returning to normal within 6 weeks, virus reactivation (Rome, I) and bacterial infections were limiting this approach. Although pronounced lymphocyte depletion seems inevitable for In this pilot study we investigated the feasibility and clinical efficacy, additional patients were treated with lower doses of value of an intensive regimen for graft-versus-host disease 20 to 30 mg Alemtuzumab, repeated approximately every two (GvHD) prophylaxis in unmanipulated bone marrow weeks, while concomitant immunosuppressive drugs could be transplantation from HLA-haploidentical related donor. Eleven tapered within 3 to 4 weeks. Acute GvHD improved in all patients with a median age of 35 years (12-59) were patients treated so far. A complete response of liver and gut transplanted for acute myeloid leukemia (n=8), acute GvHD was seen in two patients, an additional patient lymphoblastic leukemia (n=1), myelodisplastic syndrome (n=1) developed a vanishing bile duct syndrome but has no

S110 evidence of ongoing GvHD. Another two patients are still on patients (median age 41, range 15-68 years) between May treatment with major improvement of gut and liver-GVHD, 1996 and December 2005. Among these patients, 243 respectively. Thus, alemtuzumab given sequentially in (20.4%) developed hepatic graft-versus-host disease (GvHD) moderate doses shows promising activity in severe, steroid (124 pts had a hepatic GvHD of grade > II). We followed 25 refractory acute GvHD. However, monitoring for infectious patients (11 male, 14 female) in whom GvHD of the liver complications, particularly by reactivated viruses presented with marked elevation of serum aminotransferases, (CMV, adenovirus) is mandatory for months after treatment. clinically resembling acute hepatitis and auto/antibodies characteristics for AIH. The median age at transplant was 35.0 (range, 16-54) years. Onset of liver dysfunction was at 286 P560 days (range, 55-2766) after HSCT. Median peak serum was HLA-A, B, C allele mismatches have no major impact on 312 (range 105-1750) U/L for alanine aminotransferase, 629 the risk of chronic graft- versus-host disease (133-2410) U/L for gamma-glutamyl transferase and 1.74 (0.5- H.D. Ottinger, S. Ferencik, A. Elmaagacli, R. Trenschel, H. 23.4) mg/dl for bilirubin. The autoantibody profiles of AIH were Grosse-Wilde, D. Beelen 60% for anti-nuclear antibody, 44% for antibodies to liver- University Hospital of Essen (Essen, D) kidney microsomes, 24% for antibodies to smooth-muscle antigens, 28% for anti-mitochondrial antibody, 16% for Graft-versus-Host Disease (GvHD) is a frequent and often antibodies to actin, 8% for antibodies to nucleoli, and 4% for serious complication after allogeneic hematopoietic stem cell other autoantibodies. AIH had a higher prevalence in younger transplantation (HSCT). While the impact of HLA-A,B,C and in female patients. AIH occurred in 92% in patients, who mismatches at the antigen (i.e. low resolution or 2 digits) and were transplanted with G-CSF mobilized and peripherally the allelic (i.e. high resolution or 4 digits) level on acute GvHD collected stem cells (PSC), but in only 8% in patients with has been intensively studied, corresponding data for chronic bone marrow (BM) source (p<0.02), comparing all GvHD (cGvHD)are sparse. Thus, the impact of HLA-A,B,C transplanted patients (922 PSC, 267 BM). Stem cell grafts allele mismatches on cGvHD risk was studied in a cohort of from matched sibling donor or matched unrelated donor were 177 consecutive unrelated transplants by means of uni- and similar in the two groups. Acute GvHD of grade > II occurred multivariate statistical analysis. The entry critera of the study more frequently in the groups with AIH (15/25 vs. 489/1189, were as follows: Patient age > 15 years, underlying disease p<0.002), and chronic GvHD (11 limited, 14 extensive) was CML, AML or ALL, no graft manipulation (e.g. no T- cell ascertained in all AIH patients vs. 43.7% in all transplanted depletion), use of standard regimens for conditioning (total patients (p<0.0001). Three patients with AIH died from body irradiation 4 x 2.5 Gy plus cyclophosphamide) and pulmonary bleeding, chronic GvHD, and relapse, whereas 22 GvHD prophylaxis (CSA plus short course MTX), donor- patients with AIH are still alive (88%) at a median survival time recipient HLA-identity according to our pre-transplant typing of 2570 days. In conclusion, our evaluation confirms a strong routine (i.e. identity of HLA-A,B,C at the antigen and of association between G-CSF mobilized PSC, chronic GvHD DRB1,DQB1 at the allelic level). Retrospective sequence and the development of AIH. based typing was used to assess the HLA-A,B,C alleles of donors and recipients. Impact of allele mismatches on outcome was evaluated after stratification for nil, one and two P562 or more discrepancies. Enrolled into the analysis were n=138 Impact of the 14 bp deletion/insertion HLA-G gene patients surviving day 100 post transplant of whom n=90 polymorphism on the outcome of unrelated (65%) developed cGvHD (de novo/ secondary chronic haematopoietic stem cell transplantation in thalassaemia n=5/n=133, limited/extended (n=54/n=36) respectively. Cross patients table analysis/ Fisher`s exact test revealed a clear correlation G. La Nasa (1), R. Littera (1), S. Lai (1), F. Alba (1), M. of cGvHD with acute GvHD (p<0.001), but only a trend Mulargia (1), G. Caocci (1), A. Vacca (1), E. Piras (1), C. towards an increased risk of cGVHD incidence with increasing Giardini (2), F. Locatelli (3), C. Carcassi (4) number of HLA-A,B,C allele mismatches (p=0.08 with 0,1 and (1)Osp. R. Binaghi (Cagliari, I); (2)Osp. S. Salvatore (Pesaro, two or more mismatches being present in about 46%, 34% I); (3)Policlinico S. Matteo (Pavia, I); (4)Università di Cagliari and 19% of the cases. Kaplan-Meier analysis could also (Cagliari, I) confirm this trend (p=0.06). After correction for disease status, multivariate stepwise backward analysis excluded patient and Non classical HLA-G Class I molecules exert their donor age as well as stem cell source and sex mismatch from immunomodulatory function by interaction with certain the Cox model, leaving the number of allele mismatches as receptors on immune effector cells. These molecules are the only independent risk factor for cGvHD (RR: 1.2, p=0.2, encoded by a gene mapped on the short arm of Chromosome i.e. not significant). Thus (1) if present at all HLA-A,B,C allele 6, telomeric to the classical HLA-A, -B, and –C genes. HLA-G mismatches have only a very moderate biological impact on allelic variants may be also characterized by a 14 bp deletion- cGvHD risk and (2) donors with these type of mismatches insertion polymorphism located in the 3' UT region of the HLA- should not be excluded from donation in clinically urgent G gene. The presence of the 14 bp insertion is known to settings if no fully matched donor is available in time. generate an additional splice whereby 92 bases are removed from the start of exon 8. HLA-G mRNAs having the 92-base deletion are more stable than the complete mRNA forms, P561 suggesting that this region may be involved in the Incidence and outcome of auto/alloimmune hepatitis with mechanisms controlling post-transcriptional regulation of HLA- hepatic graft-versus-host disease after haematopoietic G molecule associated with allelic variants. stem cell transplantation We investigated the influence of the 14bp deletion/insertion M. Koldehoff (1), A.H. Elmaagacli (1), R. Klein (2), R. polymorphism on the outcome of allogeneic hematopoietic Trenschel (1), V. Heuer (1), M. Ditschkowski (1), C. Schulte stem cell transplantation (HSCT) in patients affected by (1), D. Beelen (1) thalassemia major. (1)University of Duisburg-Essen (Essen, D); (2)University of Forty-five thalassemia patients transplanted from an unrelated Tuebingen (Tuebingen, D) donor were enrolled in the study. The conditioning regimen was the same in all patients. All donor/recipient pairs were Auto/alloimmune hepatitis (AIH) is an inflammatory liver HLA class I and II complitely identical at molecular level. All disease characterized histological by a dense mononuclear donor/ recipient pairs were analyzed for the presence of the cell infiltrate in the portal tract and serological by the presence 14bp deletion/insertion polymorphism in the 3' untranslated of non-organ and liver-specific antibodies, high transaminases region of the HLA-G gene. and increased levels of IgG. The relation between allogeneic Out of 45 transplanted patients, 32 (71%) are thalassemia free hematopoietic stem cell transplantation (HSCT) and survivall, 7 rejected, 6 patients died. 12 (27%) developed auto/alloimmune disease is complex. To examine this grade II-IV acute GvHD. The frequencies of the 14bp deletion association, we retrospectively studied 1,189 allogeneic or insertion were 55% and 45% respectively, which was

S111 analogous to that found in other studies. Ninety percent of the P564 recipients had the same polymorphism as their donor. A comparison between 2 intra-arterial treatment protocols Patients that were homozygous or heterozygous for the 14bp for hepatic steroid resistant/dependent graft-versus-host deletion had a higher risk of developing GvHD compared to disease patients with the 14bp insertion (RR = 18.20; 95% CI 0.88 – M.Y. Shapira, A.I. Bloom, R. Or, I.B. Resnick, M. Aker, M. 375.17; p = 0.019). Bitan, I. Zilberman, S. Miron, L. Yoffe, A. Levovic, S. Slavin, A. The frequency of rejection was similar in the two groups of Verstandig examined patients. Hadassah – Hebrew University (Jerusalem, IL) The number of genetic factors capable of influencing the outcome of HSCT is steadily rising. Introduction: It has been previously shown by us, that intra- Careful evaluation of the KIR gene profile, the 14 bp arterial (IA) targeted steroid therapy may be useful even in polymorphism of the HLA-G gene and the interleukin 10 steroid refractory hepatic graft vs. host disease (GVHD). In the promotor in donor/recipient pairs should make it possible to 1st protocol, an intermediate dose steroid with low dose better assess the risk for GvHD and/or rejection and methotrexate was used (protocol I). Due to suspected toxicity consequently, to adjust immunosuppressive therapy, all of of methotrexate, it was omitted and substituted with high dose which will most certainly reflect positively on the outcome of steroids (protocol II). transplantation. Patients and methods: 7 patients with steroid resistant/dependent GVHD were treated with protocol I and 13 with protocol II at time of analysis. Serum bilirubin level was P563 followed for the evaluation of efficacy. Mice model of graft-versus-host disease based on Results: Median time from stem cell transplantation (SCT) to chemotherapy conditioning IA and from GVHD to IA tended to be longer in the patients B. Sadeghi (1), Z. Hassan (1), M. Abedi Valugerdi (2), M. treated with protocol II. No life threatening event was seen Hassan (1) with protocol II. Four out of 7 and 10/12 patients responded to (1)Karolinska Institute (Stockholm, S); (2)Stockholm University the treatment (protocol I and protocol II respectively – figure (Stockholm, S) 1). There was a trend toward faster time to response in the patients treated with protocol II (table 1). one out of 7 and 7/12 Background: Haematopoietic stem cell transplantation (HSCT) patients are long term survivors in the 2 protocols is used successfully for reconstitution of haemato- respectively. lymphopoiesis in the bone marrow and provides curative Conclusion: Intra-arterial catheter guided steroid therapy for therapy for a number of malignant and non-malignant steroid resistant/dependent GVHD with high dose steroids is diseases. Preconditioning of the recipient with a proper at least as good as intermediate dose steroids with preparative regimen is an essential prerequisite for HSCT. methotrexate and may be safer. Transplantation related complications such as graft versus host disease (GVHD), drug toxicity, veno-oclussive disease, hypothyroidism, cataract, pulmonary complication, and hemorrhagic cystitis are limiting factors for HSCT. At the present the majority of GVHD mice models are based on the use of radiotherapy as a conditioning regimen. However, these models can’t cover the variety of HSCT in clinical settings. Many patients are conditioned with chemotherapy, which may affect the occurrence and rate of transplantation related complications. Aims: To establish a mice model of GVHD using the chemotherapy as conditioning regimen. Methods: Female BALB/c and male C57BL/6 mice were used in the study. Group I-IV considered for allogeneic BMT and group V and VI considered for syngeneic BMT. Group I-IV included dose escalation of busulfan (Bu) and cyclophosphamide (Cy). Conditioning started at day -7 days using Bu 20 or 25 mg/kg/day for four days followed by Cy 100 or 150 mg/kg/day for two days in the allogeneic setting. Syngeneic control mice received Bu 20 or 25 mg/kg/day for four days followed by Cy 100 mg/kg/day for two days. Both Bu and Cy administrated as IP. GVHD was studied by histological analysis of skin, intestine and liver. The chimerism and engraftment were surveyed by FACS analysis. The plasma levels of cytokine were measured by Multiplex illumination assay. Results: Symptoms of acute GVHD started at day + 5 post transplantation. GVHD signs were found as histological changes in affected organs in all allogeneic transplanted mice but none of the syngeneic groups. Engraftment, H-2Kb (donor MHC class I) versus H-2Kd (recipient MHC class I) was established successfully in all groups and found from day +7. Mortality rate increased by increment of conditioning dose and survival was poorest in group II and IV (Bu 20-Cy 150 and Bu25-Cy150, mg/kg/day). Conclusion: We have established a new mouse model of GVHD using chemotherapy that is compatible of conditioning in human. This model can also be used to study the basic mechanisms underlying GVHD that might be caused by the effect of the conditioning regimen on different cell sub-populations.

S112 to the shorter immunosuppressive treatment, usually 2 months vs. 6 months after an unrelated transplantation.

P566 Retrospective study of extracorporeal photopheresis in acute and chronic steroid refractory graft-versus-host- disease: clinical results in accordance with the literature K. Bilger, A. Laplace, B. Lioure, A. Bohbot, V. Liu, P. Lutz, N. Entz Werle, N. Cojean, R. Herbrecht CHU de Hautepierre (Strasbourg, F)

The recent article by Couriel et al and the NIH consensus renew interest and motivation for the evaluation of extracorporeal photopheresis (ECP) in the management of chronic graft versus host disease (cGVHD. We report 22 cases of steroid refractory GVHD, including 2 patients with acute GVHD, treated by ECP between 1996 and 2006. Median age was 45 years (15-58), 7/22 patients were female. Twelve patients received myeloablative regimens and 10 reduced-intensity conditioning regimens. Only 3/22 patients had a matched unrelated donor. Stem cell source was bone marrow in 7 patients and peripherical blood in 15 patients. All patients received ECP using the Therakos system. Median time between transplantation and start of ECP was 482 days (51-2784). Of the 20 patients treated for extensive cGHVD, 13 presented a progressive and 7 a de novo form.The schedule of ECP was 2 procedures every 2 weeks for 3 months, 2 procedures per month for 6 months and 2 every 2 months for 1 year.All patients were evaluated by the same two physicians. The organs involved were the skin in all patients with a sclerodermal form in 6 cases, the mouth in 15 patients, the eyes in 8 patients, the liver in 5 patients and the lung in 2 patients. No gastrointestinal GVHD was initially observed. Patients received a median number of 32 procedures (10-44) in a median of 406 (61-876) days. The response rate by organ was 88% for oral mucosa, 80% for eyes, 73% for skin, 60% for liver; however 6 patients progressed with pulmonary GVHD. Using the criteria of Rubegni et al to evaluate the overall response, the ECP was P565 determinant in 3 patients (14%), good in 8 patients (36%) and Risk-factors for chronic GvHD after allogeneic ineffective in 11 patients (50%). The median follow up was 22 haematopoietic stem cell transplantation months (5-57),the overall survival was 51% at 3 years; the G. Afram (1), M. Remberger (2), P. Ljungman (1), O. Ringden survival for responders vs non-responders was 68% vs 38% (3), B. Omazic (3), J. Aschan (4), H. Hägglund (1) (p=0.047) Nine patients died, 2 from relapse. When we (1)Hematology Center (Stockholm, S); (2)Dept of Immunology examined patient characteristics and correlation with (Stockholm, S); (3)CAST (Stockholm, S); (4)Hematology response, we found no difference with respect to age,type of Center, CAST (Stockholm, S) conditioning regimen, source of cells,Akpek prognostic score and time of transplant to ECP. Only de novo onset cGVHD Among 372 consecutive recipients of hematopoietic stem cell correlated with response. transplantation, grafted between January 2000 and Thus, our results confirm the data of ECP in cGVHD in overall September 2005, 340 patients survived more than 3 months response and response rate by organ with large differences after HSCT and were included in the study. Both pediatric and in efficacy according to the type of organ involved, better adult patients were included. The median age was 38 (1-67) survival for responders. It is difficult, however, to find years. There were 206 males and 134 females. Among the predictive factors for response and to define time to response. donors 194 were unrelated and 146 related. Myeloablative conditioning was used in 190 and RIC in 150 patients. The stem cell sources were; PBSC in 226, bone marrow in 101 P567 and cord blood in 13. Most of the patients had a Myeloablative conditioning followed by T-cell depletion is haematological malignancy (n=252), but 45 had a non associated with low treatment-related mortality malignant disorder and 43 a solid tumor. A total of 98 (27%) N. Novitzky (1), V. Thomas (2), C. du Toit (2), A. McDonald (2) patients developed cGVHD, 79 (81%) mild, 14 (14%) (1)University of Cape Town (Cape Town, ZA); (2)Groote moderate and 5 (5%) severe. The 3-year overall survival in Schuur Hospital (Cape Town, ZA) patients without cGVHD was 58% and in patients with mild, moderate and severe it was 74%, 71% and 60% (p=0.02), Introduction: Classically the conditioning for transplantation respectively. We analyzed 18 potential risk factors for cGVHD. has been myeloablative, but this has been associated with In the multivariate analysis, a sibling donor (RH, 1.69, 95% CI, substantial transplant related mortality, particularly from 1.13-2.52; p=0.01), recipient age >40 years (RH, 1.58; CI GvHD. Reduced intensity conditioning is immunosuppressive 1.03-2.43; p=0.04) and year of HSCT (RH, 0.88; CI 0.78-0.99; and has lower treatment related mortality (TRM), but is p=0.04) were independent risk factors for all grades of associated with high rates of GvHD and disease recurrence. cGHVD. However, in the PBSC group the 3-year probability of To better decide on conditioning strategies, we prospectively cGVHD were 41% vs 24% in recipients of BM, but PBSC were studied the outcome of patients who received similar GvHD not a significant risk-factor in multivariate analysis; (RH, 1.62; prophylaxis after myeloablative conditioning. CI 0.97-2.71, p=0.06). Surprisingly, the risk for developing Patients & methods: Patients with haematological cGVHD at our center has decreased during the more recent malignancies, in remission or still responsive to years in spite of the increased use of PBSC. The reason for chemotherapy, who had an HLA identical sibling were offered the increased cGVHD risk in sibling transplants might be due T-cell depleted stem cell grafts. Conditioning was with ablative

S113 doses of either chemotherapy or total body radiotherapy. Group I: Incidence of aGVHD Gr. I, II, III, IV was 18%, 13%, Stem cells were mobilised with G-CSF (5-10ug/kg x 5) and 2% and 0%. 67% of the patients had no aGVHD. Extensive grafts were harvested by large volume apheresis. GvHD cGVHD have 4 pts. and limited cGVHD 3 pts. Two patiens prophylaxis was by “ex vivo” incubation of grafts with experienced graft failure with the need of retransplantation. 23 CAMPATH-1 H (1 mg/109 mononuclear cells) at 20oC, and pts. died (16 due to relapse, 1 due to graft failure, 2 due to therapeutic doses of cyclosporin until day 90. End points were GVHD and 4 due to infection). Seven of 7 (7/7) (100%) CML- rate of GvHD, toxicity, TRM, disease recurrence rate and I.CP patients, 8/9 (90%) MMM pts, 10/13 (77%) LG-NHL pts, overall survival (OS) time. and 6/13 (46%) AML pts. survive 4-66 months post HSCT. Results: 92 patients with median age of 44 (17-62) years were Group II: Incidence of aGVHD Gr. I, II, III, IV was 22%, 20%, enrolled. The diagnosis was acute leukaemia (ALL in 7) in CR 2% and 3%. 52% of the patients had no aGVHD. 28 patients in 39, myeloproliferative disorders in 16, lymphoproliferative died (3 due to GVHD, 10 due to relapse, 11 due to infection disease in 25, and multiple myeloma in 11. Median and 4 due to VOD). Probability of survival is 70% at 60 transplanted CD34+ cell number was 2.69 (1- 12.3) and the months for CML, 80% at 60 months for ALL and 30% at 40 median dose of CAMPATh-1H was 10 (range 7.5-45) mg. months for AML patients. Median time to engraftment was 11 days. At 6 months post Summary: ATG(Fresenius) given during conditioning regimen transplantation, 8 patients with CML received prophylaxis with reduces the risk of severe GVHD in fully matched as well as donor lymphocytes. Overall, 21 (23%) individuals died, while mismatched HSCT in the dose of 20 mg or 40 mg/kg b.w. treatment related mortality occurred in 17% (n= 16; VOD 3, respectively. infections in 8, pneumonitis in 2, lymphoma in 1 and GvHD in 2). GvHD (>grade 1) occurred in 8 patients, was controlled with further immunosuppression but led to death from P569 infections in 6. Disease recurrence was seen in 14, but 7 with Serum granzyme A and granzyme B levels are enhanced CML or myeloma responded to DLI. At a median follow up of in patients developing chronic GvHD after HLA-identical 688 days, 73% survive disease free. Cox analysis showed stem cell transplantation that occurrence of GvHD was associated with increased B. Kircher, P. Schumacher, D. Nachbaur mortality. Immunobiology and Stem Cell Laboratory (Innsbruck, A) Conclusions: Myeloablative conditioning is well tolerated in patients receiving T-cell depleted grafts and treatment related Complications after stem cell transplantation (SCT) are mortality of <20% can be expected with this strategy. This mediated by activated cytotoxic T lymphocytes and natural information is useful to more precisely select patients who killer cells. It has been shown perforin-positive lymphocytes would benefit most from reduced intensity conditioning infiltrate lesions of acute graft-versus-host disease (GvHD), schedules. however granzyme (Gr) production of these cells in patients after SCT has not been studied yet. Aim of this study was therefore to search for a possible P568 correlation between in vivo GrA and GrB levels and the ATG (Fresenius) 20 mg/kg b.w. + CSA is sufficient GvHD occurrence of complications after SCT. prophylactic regimen for fully matched related and GrA and GrB levels were determined by enzyme-linked unrelated HSCT, while 40 mg/kg b.w. of ATG + CSA and immunosorbant assays (ELISA) from a cohort of 86 HLA- MMF should be used for partially matched HSCT - a identical and HLA-mismatched SCT patients at various time single-centre experience points before and after SCT. Accordingly, patients were A. Vitek (1), P. Cetkovsky (1), M. Dobrovolná (1), E. Ivaskova divided into a high and a low Gr-producing group when Gr (2), P. Korinkova (1), P. Kovarova (1), L. Kupkova (2), M. levels exceeded or fell below median Gr levels at least at two Loudova (1), M. Markova (1), E. Matejkova (1), J. Navrátilova time points. (3), H. Pittrova (3), D. Pohlreich (1), J. Sajdova (1), D. Further evaluations were performed on a study cohort of 70 Sponerova (1), V. Valkova (1), M. Vrana (1), J. Vytiskova (1) HLA-identical, sibling or unrelated high-risk SCT recipients. A (1)UHKT (Prague, CZ); (2)IKEM (Prague, CZ); (3)CNMDR clear association between high GrA and GrB levels and the (Pilsen, CZ) occurrence of chronic GvHD was detected. The risk of developing this complication in the sub-cohort transplanted ATG, given during conditioning regimen, reduces the risk of with a myeloablative conditioning regimen was 74% for high severe aGVHD as well as of chronic GVHD in stem cell GrA and 86% for high GrB producers versus 43% for low GrA recipients. ATG-Fresenius(40 mg/kg b.w.) was originally and 46% for low GrB producers (p=0.089 and p=0.082, recommended for BuFluATG nonmyeloablative conditioning respectively). The same pattern was observed in the sibling regimen. We employed this dose of ATG in our GVHD sub-cohort producing high GrB levels (75% versus 45%; prophylactic strategy for MRD and MUD nonmyeloablative as p=0.043). In contrast, patients transplanted under reduced- well as for MUD myeloablative transplantations. First we were intensity conditions and releasing high GrA and GrB levels using ATG 40mg/kg b.w., later we reduced the dose to 20 displayed a decreased incidence of developing acute GvHD. mg/kg b.w., given as a single dose on D-1 in MRD as well as Nevertheless, high GrB producers of this sub-cohort showed a in MUD (10/10 HR matched) transplantations with CSA alone reduced survival (24% vs 51% (p=0.096)). Patients postransplant. In HSCT from mismatched donors we transplanted with an HLA-identical sibling and releasing high continued in administration of 40 mg ATG/kg b.w.( 20 mg/kg GrB levels tended to have a lower risk of relapse (24% vs b.w. infused D-2 and D–1), and we added MMF to CSA 50% (p=0.077)). Interestingly, no clear correlation was postransplant. Regimen with 20 mg/kg b.w. ATG in fully observed between Gr production levels and the occurrence of matched HSCT (Goup I) was used in 55 patients (12 CML, 13 cytomegalovirus infections. AML, 7 MDS, 7 MPS, 5 NHL, 7 CLL, 1 HD, 1 MM), 18 MRD In conclusion, measurement of Gr levels in serum of SCT and 37 MUD, 25 RIC and 30 myeloablative SCT. Regimen patients may be a simple and useful tool to identify patients with 40 mg/kg b.w. of ATG in mismatched HSCT (Group II) developing chronic GvHD already before or in the first week was used in 58 patients ( 19 CML, 13 AML, 11 ALL, 7 MDS, 2 after SCT. CLL, 2 MMM, 2 NHL, 1 MM and 1 HD). The most frequent mismatch in this cohort of the patients was class I. and II. combined mismatch (31%) followed by isolated C locus mismatch (26%). Multiple mismatches in class I were present in 22% and one single mismatch on A or B loci in 7% of the pts. 3% pts received graft with one and 8% with multiple mismatches in class II. HLA. 41 patiens had MMF+CSA and 17 had CSA alone postransplant. Results:

S114 P570 In unrelated transplants (149), both the patient and the donor Kinetics of dendritic cells reconstitution and co- were genotyped while in sibling transplants (99) only the stimulatory molecules expression after myeloablative patient was genotyped. allogeneic haematopoetic stem cell transplantation: Results: In unrelated transplants, 16 (11%) mismatches were implications for the development of acute graft-versus- found. Although an increase in the incidence of chronic GVHD host disease was found among patients receiving a graft with MIC-A R. Horvath, V. Budinsky, J. Kayserova, R. Formankova, J. mismatch, there were no significant differences in leukemia Stary, P. Sedlacek, J. Bartunkova, R. Spisek free survival (LFS), transplant related mortality (TRM) and Charles University (Prague, CZ) GVHD between patients with MIC-A match or mismatch. At the MIC-A allele level, no specific allele was found to be Allogeneic hematopoetic stem cell transplantation (allo-HSCT) associated with different clinical outcomes after HSCT. with myeloablative conditioning represents a unique Conclusion: MIC-A mismatch is a rare event in HLA matched opportunity to monitor the kinetics of reconstitution of HSCT. Neither the MIC-A mismatch nor the MIC-A alleles hematopoetic cells. Dendritic cells (DCs) are the most efficient seem to have a major effect on the clinical outcome after antigen presenting cells. Two distinct subsets of DCs have HSCT. been identified, myeloid (mDCs) and plasmacytoid (pDCs) that have different roles in the regulation of immunity. In immature state DCs express low levels of costimulatory P572 molecules and have very low capacity to stimulate antigen Donor-derived myofibroblasts are present in the specific T cells. After activation, DCs undergo a process conjunctiva after allo-SCT also in patients without graft- termed maturation characterized by an increased expression versus-host disease of costimulatory molecules and cytokine production. Mature D. Hallberg, P. Wernstedt, K. Stenberg, U. Stenevi, C. DCs migrate to lymphoid organs and activate antigen specific Hansson, M. Brune T cells. In this pilot study, we analyzed the kinetics of DCs Gothenburg University (Gothenburg, S) subsets reconstitution in 5 patients (age range: 5-18 years) undergoing unmanipulated allo-HSCT from unrelated donor. Objective: To assess the presence and quantity of donor Myeloid DCs were defined as lineage -, CD11c+, HLA-DR+ myofibroblasts in the conjunctiva of long-term follow-up and pDCs as lineage -, CD123+ and HLA-DR+. We further patients without symptomatic GvHD. evaluated activation status of circulating DCs by simultaneous Patients: Eight women, median age 44 (32-57) yrs, with CML monitoring of expression of costimulatory molecules CD80, (n=4), AML (n=3), or Mb Waldenström all allografted from CD83 and CD86. Peripheral blood DCs were monitored from male donors (3 siblings, 5 unrelated) using bone marrow (n=2) the earliest phase of hematopoetic reconstitution. Both mDCs or peripheral blood stem cells (n=6), after full (n=2) or reduced and pDCs appeared at earliest stages after engraftment and (n=6) conditioning. Samples were obtained a median of 58 relative numbers within white blood cells compartment peaked (12-93) months after transplantation. All pts were without between days 19-25 after HSCT. Their proportion then signs and symptoms of systemic or local GVHD. Two pts were gradually declined and steady-state absolute levels found in on prednisolone (5 mg) after previous GvHD. the control group were reached between days 80-180 after Methods: Conjunctival cells were sampled from pts by blotting BMT for mDCs and beyond day 180 for pDCs. Expression of the eye with a sterile filter membrane, lifting an imprint from costimulatory molecules, especially CD83 and CD86 the ocular surface. Cells thus obtained, were immediately transiently increased between days 15 and 35 and then went fixed and analysed on the filter using immunohistochemistry back to low steady state levels. Interestingly, patients who using the myofibroblast (MFB) marker alpha-SMA (Smooth developed acute graft-versus-host disease (aGVHD) had Muscle Actin). Donor cells were identified using sex- lower absolute numbers of circulating DCs before appearance chromosome specific fluorescence in situ hybridization (FISH). of clinical symptoms. Administration of glucocorticoids for the Results: Cells were typically arranged in a monolayer, treatment of aGVHD abrogated circulating pDCs and led to a predominated by recipient epithelial cells. Myofibroblasts rapid decrease of mDCs within 24h similarly to the findings in accounted for 16% (range 10-19) of all cells on the filter. Of systemic autoimmune diseases. In this pilot study the these cells 72% (23-93) were of donor origin. development of aGVHD was preceded by decreased numbers Discussion: Myofibroblasts are associated with wound-healing of circulating DCs though now we would like to confirm this and the formation of fibrosis. However, the patients in this hypothesis in larger series of patients. study did not show any signs of ocular disease and the Supported by 6th Framework project Allostem 503319, fraction of MFB present did not vary in spite of a wide range of MSM0021620812 and GAUK 52/2005. variables such as duration from transplant, diagnosis and stem cell source. The finding of MFB of both donor and recipient origin in allografted pts is intriguing and suggests that P571 MFB may have differentiated not only from progenitors or No association between MIC-A polymorphism and clinical stem cells in the graft, but also from remaining recipient cells. outcome after allogeneic HSCT The role of conjunctival donor and recipient MFB, as well as M. Uzunel, M. Remberger the differentiation route(s) of these cells after allo-SCT is Karolinska Institutet (Stockholm, S) currently under study.

Background: MHC class I chain related molecule A (MIC-A) shows homology with the classical HLA molecules. MIC-A is P573 polymorphic with more than 50 recognized alleles and is Intra-arterial treatment for steroid resistant/dependent described as a “marker of stress” since it is up-regulated on graft-versus-host disease epithelial cells at heat and virus infection. The presence of M.Y. Shapira, A.I. Bloom, R. Or, I.B. Resnick, M. Aker, M. MIC-A antibodies in patient sera has been associated with Bitan, I. Zilberman, S. Miron, L. Yoffe, A. Levovic, S. Slavin, A. rejection after organ transplantation. The significance of MIC- Verstandig A polymorphism in hematopoietic stem cell transplantation Hadassah – Hebrew University Medical Center (Jerusalem, (HSCT) has not been studied. IL) In this study we wanted to see the degree of MIC-A mismatch between patients and donors and to investigate if the MIC-A Introduction: Graft versus host disease (GVHD) still is the genotype was associated with clinical outcome after HSCT. major draw-back of allogeneic stem cell transplantation (SCT). Materials and Methods: For MIC-A genotyping, we performed In its resistant form, it carries high morbidity and mortality. The PCR amplification with allele-specific primers. A total of 248 mainstay of GVHD therapy is preventive and once it is leukemia patients were included in the study. All received developed the first line therapy is systematically high dose myeloablative conditioning and GVHD prophylaxis consisted steroids, and cyclosporine or tacrolimus. It has been shown by mainly of a combination of cyclosporine A and methotrexate.

S115 us and other groups, that intra-arterial targeted steroid therapy Overall survival is 94%, with median and maximum follow-up may be useful even in steroid refractory GVHD. of 22 and 67 months, respectively. Methods: A total 35 patients with 37 cases of steroid Conclusion: Pulse Cy has remarkably good efficacy and resistant/dependent gastrointestinal (GI) (n=16), hepatic toxicity profile, and the cost of the drug is negligible. According (n=15) or combined (n=6) GVHD, were included and given to our results, pulse Cy is extremely effective treatment of intra-arterial treatment, for one or more of the following steroid-refractory liver or liver/skin GvHD. arteries - the hepatic, gastro-duodenal, superior mesenteric, inferior mesenteric, internal iliacs. We defined GI partial response and complete response as the day that symptoms P575 decrease or resolved, respectively. Hepatic partial response Tacrolimus with high-dose dexamethasone pulses for and complete response were defined as the day that bilirubin chronic graft-versus-host disease management level began to decrease or decreased below 30% of initial M. Markova, A. Vitek, V. Valkova, D. Sponerova, P. Cetkovsky level, respectively. Institute of Haematology (Prague, CZ) Results: The procedure was safe with only 1 major complication (renal failure and pancytopenia). We found that We evaluate retrospectively 21 patients with chronic graft Intra-arterial catheter guided steroid therapy was associated versus host disease (c-GVHD). Except of four patients with with partial and complete remission among patients with matched sibling donors, all the other 17 donors were steroid resistant or dependent GI or Hepatic GVHD. Hepatic unrelated, 8 of them completely matched while 9 donors were partial response was observed in 14 (66.6%) patients among with acceptable mismatch. whom 7 (33.3%) reached complete response. GI partial C-GVHD was classified as extensive in 15 patients and as response was observed in 19 (86.4%) patients among whom limited disease in 6. In addition the severity of impairment was 12 (54.4%) reached complete response. An early graded according to internal scale to mild, moderate and administration of the local therapy, female gender, myeloid severe. basic disease, and a non-active status of the basic disease at Patients were treated with tacrolimus in doses achieving the day of transplantation were found related for predicting a therapeutic plasmatic level. Dexamethasone was added better response for the intra-arterial treatment. orally, 40 mg per day for four days. The next pulse was Conclusion: Intra-arterial catheter guided steroid therapy is designed according to clinical status, e.g. either due to safe and effective in steroid resistant or dependent GVHD. A recurrence or refracterity, not sooner than in a 3 weeks further research is warranted characterizing the patients interval. The median of pulse number was 3 (1-10). benefit most. The effect was evaluated according to scale: none, partial and substantial, and the results are shown in the table. Although the reccurence of c-GVHD symptoms was frequent, P574 9 patients (43%) finally achieved stable state with minimal or Surprisingly high efficacy and low toxicity of pulse none symptomatology, and with no need of other steroid cyclophosphamide in the treatment of corticosteroid- immunosupression. refractory liver GvHD Infectious complications were the major adverse event and M. Krejci, J. Mayer, M. Doubek they were the only cause of death (overall in 3 patients 14%). Faculty Hospital Brno (Brno, CZ) 1 patient died from sepsis and 2 from pneumonia. 2 other patients were complicated with bacterial pneumonia, but are Objectives: Corticosteroid-refractory GvHD is difficult to still alive, herpes zoster infection was diagnosed in 2 patients, manage, and is associated with high morbidity and mortality. viral parotitis in 1 patient and cytomegalovirus reactivation Based on our previous experience, we saw that liver GvHD without CMV disease was found in 2 patients. Muscular not associated with gut GvHD had very encouraging response weakness especially in legs was major complain in 5 patients rate to the treatment with pulse cyclophosphamide (Cy). Here (24%). None of the patients relapsed with original disease. we update our previous data and add further information Conclusion: Management of chronic GVHD with high dose based on larger group of patients and longer follow-up. dexamethasone pulses in addition to tacrolimus is effective, Methods: This is a retrospective study of 18 patients (pts) after multiple courses are necessary for severe or refractory forms, allogeneic PBSC transplantation: 10 pts had acute GvHD (2 infection is the major complication and cause of death. pts grade I, 5 pts grade II, 3 pts grade III), 3 pts had chronic extensive GvHD and 5 pts developed liver GvHD upon DLI. Three pts had only liver GvHD, 15 pts had GvHD with involvement of liver and/or oral mucosa, or skin. Furthermore, all pts had hepatitic variant of the liver GvHD (serum aminotransferase ALT or AST elevation above 5 times the upper normal limit). Classical elevation of bilirubin was observed in 11 pts. All pts were treated by cyclosporine A and steroids before pulse Cy. Pulse Cy was administered at a P576 median dose of 1g/m²; 12 pts received one pulse Cy, 6 pts Mucositis induced by myeloablative or reduced-intensity received two pulses of Cy for slow response. Complete conditioning is a risk factor for acute graft-versus-host response (CR) was defined as resolution of GvHD disease following matched sibling allogeneic stem cell manifestation, partial response (PR) as a decrease in organ transplantation stage by 1 or decrease in aminotransferase levels to values A. Ng, R. Hoyt, A. Grigg, A. Roberts, D. Curtis, J. Szer, D. under 50% of initial level. Patients with no response were Ritchie signed as NR. Royal Melbourne Hospital (Melbourne, AUS) Results: There were 45% CR (8/18), 33% PR (6/18) and 22% NR (4/18). However, in 4 pts with NR their clinical status Introduction: GVHD is a significant complication of allogeneic stabilized and they responded to another GvHD treatment. No SCT. Transplant models suggest a relationship between the death from GvHD were observed, however, one patient died degree of mucositis, systemic cytokine levels and acute from relapse of leukemia. Leukopenia and neutropenia WHO GVHD severity. Lower GVHD rates associated with RIC are grade 4 developed in 4 patients. When myelosuppression hypothesised to be due in part to lower rates of severe occurred, it was usually short-lived (2-5 days). Importantly, no mucositis, however, this hypothesis has not been influence of pulse Cy on chimerism and disease status was systematically assessed in clinical cohorts. observed. Eight infectious complications occurred in 6 of 18 We undertook a retrospective analysis to explore the pts (pneumonia, n=1; febrile neutropenia, n=1; CMV positivity, relationship between conditioning regimen intensity, mucositis n=4; BKV positivity, n=2), all of them resolved after therapy. severity and the incidence and severity of acute GVHD. No other significant toxicity after Cy pulse was observed. Methods: Consecutive patients undergoing histocompatible sibling allogeneic SCT between 1999 and 2004 were identified

S116 from the institutional database and classified according to P577 conditioning regimen, maximum mucositis severity by WHO Alefacept treatment for chronic extensive graft-versus- grading, and the incidence and maximum grade of acute host disease GVHD. Comparative statistical analysis was performed using M.Y. Shapira, I.B. Resnick, M. Bitan, P.D. Tsirigotis, M. Aker, two-sided Fisher’s exact test, unless otherwise stated. B. Gesundheit, L. Yoffe, I. Zilberman, S. Miron, A. Levovic, S. Results: 151 patients were identified: 54% male, median age Slavin, R. Or 45 years (range 17-66). Myeloablative conditioning was used Hadassah – Hebrew University Medical Center (Jerusalem, in 108 patients: Bu( 16mg/kg) - Cy (120mg/kg) (n=30), Cy IL) (120mg/kg)–TBI (12Gy) (n=30), TBI (13.2Gy)-etoposide (60mg/kg) (n=16) and Flu (125mg/m²)-Mel (120mg/m²) (n=32). Introduction: Alefacept (AMEVIVE®) is an High-dose Flu (125mg/m²)-Cy (60mg/kg) (n=23) and low-dose immunosuppressive dimeric fusion protein that consists of the Flu (125mg/m²)-Cy (1g/m²) (n=20) were RIC regimens. All extracellular CD2-binding portion of the human leukocyte patients received GVHD prophylaxis (cyclosporin and function antigen-3 (LFA-3) linked to the Fc (hinge, CH2 and methotrexate+/-prednisolone). CH3 domains) portion of human IgG1. We have recently The severity of mucositis varied by conditioning regimen (1 shown its effect in acute, steroid resistant/dependent GVHD. way ANOVA, p<0.0001), with TBI-etoposide associated with In this study, we describe the effect of alefacept treatment on greatest and Flu-Cy regimens associated with the least chronic extensive graft versus host disease (cGVHD). mucosal toxicity (Figure 1). Patients and methods: A total of 12 patients (13 cGVHD Acute GVHD occurred in 89 of 151 (59%); grade I (n=18), II episodes) were included in this study, 7 males and 5 females (n=51), III (n=14), IV (n=6) (Figure 2). The incidence of acute with median age 27 years (range 3–60 years). Basic disease grade II-IV GVHD was less with RIC (Flu-Cy) compared to was AML (n=5), ALL (n=3), NHL (n=1), hemophagocytosis myeloablative regimens (p=0.029), but no significant (n=1) and malignant melanoma (n=1). Seven patients were difference in incidence was detected between different transplanted from HLA-A, B, C and high resolution DR fully myeloablative regimens (p=0.33). Notably, the incidence of matched family members (siblings=6, father=1); 2 patients grade II-IV mucositis was significantly related to grade II-IV received stem cell graft from fully matched unrelated donor gastrointestinal tract (GIT) acute GVHD (p=0.0065). (MUD) non-reactive in mixed lymphocyte culture; 2 from Conclusion: The incidence and severity of mucositis varied mismatched family members and 1 from a matched unrelated according to conditioning regimen. The incidence of acute cord blood unit. All patients had an extensive cGVHD. grade II-IV GVHD was notably less in the RIC cohort, which Alefacept dose for children was 15mg given intramuscularly also had less grade 2-4 mucositis compared to myeloablative (IM) once weekly. The dose for adults was 30mg IM once regimens. This suggests that regimen-induced mucositis is a weekly. risk-factor for acute GVHD, especially GIT GVHD. Mucositis Results: a median of 9 (range 1-25 injections were given to severity, however, was not predictive of GVHD severity. the patients. Eight out of 12 patients (9/13 episodes) showed response. The median time to initial response was 2.25 weeks (range 1-8). The response was either marked (n=3), moderate (n=2) or minimal (n=4). One patient with pulmonary GVHD had a consistent improvement (figure 1). In 2 responding patients, the response was only temporary. Treatment complications included infection (n=3), pericarditis (n=1) and squamous cell carcinoma of the lip (n=1). All these events may be related to other drugs given simultaneously. Currently, 7/12 patients are alive all with stable or improved cGVHD. Five patients died due to GVHD progression. Conclusion: Alefacept is effective for the treatment of cGVHD, dose and treatment's time intervals should be explored.

P578 The effect of alefacept treatment on lymphoid subpopulations in patients with chronic extensive graft- versus-host disease A. Abdul Hai, I.B. Resnick, M. Bitan, P.D. Tsirigotis, M. Aker, B. Gesundheit, L. Yoffe, I. Zilberman, S. Miron, S. Slavin, R. Or, M.Y. Shapira Hadassah – Hebrew University Medical Center (Jerusalem, IL)

Introduction: Alefacept (AMEVIVE®) is an immunosuppressive dimeric fusion protein that consists of the extracellular CD2-binding portion of the human leukocyte function antigen-3 linked to the Fc portion of human IgG1. We have recently published preliminary data demonstrating its effect in acute graft versus host disease (GVHD). In this study,

S117 we followed the subpopulations in the peripheral blood while 19 received PBSC. Conditioning was chemotherapy lymphoid cells of patients treated with alefacept due to chronic (n=33) or TBI based regimen (n=2), while 2 patients received extensive GVHD (cGVHD). no conditioning. Donors were matched related (n=18), Patients and methods: seven patients were included in this matched unrelated (n=14) and mismatched (n=5). Incidence study, 5 males and 2 females with median age 23 years of and parameters influencing mortality, GVHD development (range 3–47 years). All were heavily pretreated with multiple and relapse of disease after retransplantation have been immunosuppressive agents due to cGVHD. Alefacept was analyzed. given 15mg given intramuscularly (IM) once weekly in children Results: The overall 5-year survival after retransplantation and 30mg IM once weekly in adults. Peripheral blood was was 15%, and transplantation-related mortality was 41%. analyzed throughout the treatment for the number and ratio of Incidence of acute GVHD, grades II-IV, was 62%, with PBSC CD2, CD3, CD4, CD8, CD20, CD45RA and CD45RO positive being a risk factor for development (p<0.05). The incidence of cells. chronic GVHD was 30%. The incidence of relapse after Results: informative data was acquired from 6 patients, In retransplantation was 46% and was not associated with general, there was a decrease of the percentage of total GVHD prophylaxis or GVHD status of the patient. lymphocytes, both B and T cells, in most patients under the Univariate analysis showed a better survival for patients with treatment of alefacept. A significant reduction of memory cells GVHD grades 0-II (19%) in comparison to grades III-IV (0%), (CD45RO+) as compared to naïve (CD45RA+) cells was seen p<0.05. Also, bone marrow as graft source and a female in all patients (figure 1). Additionally, we found In some donor to male recipient could be identified as risk factors for patients, a decrease of the percentage of the sub-types of T inferior survival after retransplantation (p<0.05). cells, including cytotoxic T cells or reduction of the percentage In multivariate analysis acute GVHD grades III - IV (p<0.001) of B cells. and a bone marrow graft (p=0.004) were significantly Conclusion: as reported in psoriatic patients treated with correlated to an inferior survival. Patients, having one of these alefacept, we found an increase in the percentage of naïve T factors (n=23) or both (n= 2) showed poorer 1-year survival cells following alefacept treatment in patients with cGVHD. (30% and 0%) compared to patients having none of the risk These results make alefacept a promising new agent for factors (n=12, 75%). Patients with acute GVHD grades III-IV clinical application for treatment of patients with cGVHD. (n=7) showed poorer survival (0%) compared to patients with marrow as graft source (n=16, 38%). Conclusions: Retransplantation is associated with poor survival and high incidence of moderate to severe acute GVHD. Although the size of the study limits conclusions, it is suggested to use PBSC as stem cell source and to prevent development of severe acute GVHD.

P580 Rituximab reduces the incidence of acute graft-versus- host disease in an unselected series of twenty-nine patients undergoing allogeneic stem cell transplantation at a single centre M. Christopeit, V. Schütte, L.P. Müller, M. Eikam, W. Grothe, T. Weber, A. Mütherig, S. Theurich, G. Behre BMT Section (Halle, D)

Recent evidence highlights an implication of B lymphocytes in the development and sustainment of chronic graft versus host disease (cGvH). The monoclonal anti-CD20-antibody rituximab has proven effective against steroid refractory cGvH. Data on the role of B lymphocytes in acute GvH (aGvH) is sparse. We report on a reduction of the incidence of aGvH in patients receiving rituximab as an element of conditioning as compared to patients conditioned without rituximab. All 11 patients receiving rituximab suffered from CD20 positive Non- Hodgkin-Lymphoma (NHL). All patients received cyclosporine and either mycophenolate or short course methotrexate immunosuppression. Only patients beyond day 25 are included in the analysis; as of november 23rd, 2006 median follow up is 178 days (range 29 to 448 days). Out of 9 patients receiving rituximab as an element of conditioning, 2 (18.2 %) P579 developed moderate aGvH. Out of 18 patients who did not Retransplantation is associated with severe GvHD and receive rituximab during conditioning, 13 (72.2 %) developed poor survival moderate to severe aGvH. 2 patients developed severe acute M. Okas, J. Mattsson, O. Ringdén, H. Hägglund, M. GvH after early cessation of immunosuppression and / or Remberger donor lymphocyte infusion for progressive disease. If removed Karolinska Institutet (Stockholm, S) from this analysis, 11 out of 16 patients (68.8 %) developed aGvH after conditioning without rituximab. Taken together, Background: Second allogeneic hematopoietic stem cell these data present a trend that in patients conditioned with transplantation (HSCT) has been previously reported as a rituximab acute GvH occurs less frequently. These are treatment option in cases of disease relapse or graft failure. In amongst the first data indicating that rituximab might suppress this retrospective study we present data resulting from the development of acute GvH. They are in accordance with retransplantations carried out at a single institution between work published before yet this is the first study systematically years 1985 and 2006. comparing two groups of patients. At present, no data Material and Methods: Thirty-seven patients with a exploring the influence of CD20 positive B lymphocytes or the haematological malignancy underwent a second HSCT due to effect of rituximab on graft-versus-tumor effects exist. It relapse (n=29) or graft failure (n=8). Median age was 19 years therefore remains to be investigated whether the suggested (range 2 – 60). The donor was changed for 6 patients. GVHD suppression of GvH by rituximab benefits overall survival by prophylaxis was introduced in 19 patients, while 18 received reducing transplantation related mortality or whether it is no prophylaxis. A bone marrow graft was used in 18 patients, associated with a higher relapse rate due to an abrogated

S118 graft versus tumor effect. Furthermore, it remains to be Haematopoietic Stem Cell Transplantation (AHSCT). Gastro- elucidated whether the administration of rituximab before intestinal (GI) aGVHD shows a progressive worsening of the transplantation or during the restoration of the allogeneic symptoms, but the prognosis may be improved by an early immune system exerts prophylactic effects against chronic diagnosis and timely treatment. Differential diagnoses include GvH and whether this influences both graft versus tumor intestinal infections and chemo/radiotherapy side-effects. effects and survival. Finally, we conclude that the addition of Conventional diagnostic procedures include the histologic rituximab to the treatment of severe steroid refractory acute assessment of the upper and lower GI tract, requiring invasive graft versus host reactions has to be tested in randomized endoscopic procedures in severely ill patients. trials. Aim Aim of the present study was to determine whether High Resolution Sonography (HRS) of the bowel may represent a useful non invasive procedure for the diagnosis and P581 assessment of aGVHD and whether HRS may be helpful for a Chronic graft-versus-host disease after allogeneic timely treatment of aGVHD. reduced-intensity conditioning haematopoietic stem cell Methods: From August 2004 to September 2006, 24 patients transplantations (15M, 45 yrs, range 18-58) underwent AHSCT (patients M. Michallet, Q. Le, N. Raus, M. Sobh, D. Revesz, A. received: 15/24 peripheral blood stem cell transplantation and Thiébaut, X. Thomas, Y. Chelgoum, S. Ducastelle, J. Troncy, 9/24 bone marrow transplantation). Indication for AHSCT was: M. Clark, F. Nicolini ALL in 3 pts, AML in 8 pts, CML in 2 pts, NHL in 6 pts, HL in 1 Hôpital Edouard Herriot (Lyon, F) pt, multiple mieloma in 3 and myelodisplastic syndrome in 1 patient. GI symptoms were observed in 18/24 patients, We studied the impact of demographic, pre and post- including anorexia, nausea, vomiting, watery diarrhoea, transplant parameters on chronic graft-versus-host disease intestinal bleeding. Symptoms onset began after a median of (GVHD) occurrences in 141 allogeneic reduced intensity 59 days (range 10-100) from AHSCT. Within 24 hours from conditioning transplant (RICT). There were 52 female and 89 the onset of GI symptoms, HRS was performed by using a 7 male recipients [median age: 51 yrs], 71 female and 70 male MHz linear and a 3.5 MHz convex array transducers. HRS donors [48.5 yrs]. There were 83 sex-mismatched pairs (59%) changes included: increased bowel wall thickness and small (M/F: 32, F/M: 51), 48 ABO incompatibilities (34 minor, 14 bowel and/or colon dilation. major) and 68% CMV seropositive pairs. Concerning Results: Among the 18 patients with GI symptoms, aGVHD transplant procedures, 33 patients received bone marrow and was confirmed by clinical assessment in 10 patients, by 105 peripheral blood stem cells with a median CD34+ cell histology in 6 patients. In 10/10 patients who developed number of 5x106 /kg (0.42-64) and a median number of CD3+ aGVHD after AHSCT, HRS revealed at least one site of cells of 197x106 /kg (0-761) and 3 cord blood cells from 121 involvement as defined by an increased bowel wall thickness. HLA identical related donors and 20 unrelated donors (17 HLA An increased small bowel wall thickness was found in 8/10 matched and 3 mismatched). For conditioning, 68 patients patients with aGVHD. In 8/10 aGVHD patients a thickening of received Fluda+Busulfan+ATG with doses varying from the colonic wall (either in ascending and/or distal colon) was 2.5mg/kg to 12.5 mg/kg, 39 Fluda+TBI (2 grays), 6 found. In 6/10 aGVHD patients both small and large bowel Fluda+Treosulfan+ATG and 28 other combinations. The appeared to be involved. GVHD prophylaxis consisted of methotrexate+Ciclosporine Conclusion: HRS is a feasible and non-invasive diagnostic (CsA) for 41 patients, CsA alone for 50, CsA+MMF for 39 and tool which may provide useful information for a prompt and other combinations for 11. The pre-transplant diagnosis was appropriate treatment of patients with suspected aGVHD. The 30 AML, 5 ALL, 13 CML and myeloproliferative disorders, 8 possible role of HRS for predicting the development of Hodgkin disease, 10 CLL, 16 MDS, 43 MM and 16 NHL; 40 aGVHD and for grading its severity warrants evaluation in were in CR (28%), 54 in PR (38%), 4 in stable disease and 43 prospective studies. in progressive disease (30%). The median interval between diagnosis and transplant was 20 months (5 - 300). We observed 10 graft failures, 51 patients (39.5%) developed P583 acute GVHD • II and 54 (42%) chronic GVHD (28 limited - 26 Pre-transplant low dose ATG as prophlylaxis for chronic extensive). At the last follow-up, 73 patients (52%) relapsed GvHD after peripheral blood stem cell myeloablative and 97 patients had died (55 from TRM causes and 42 from sibling transplants in AML: a single-centre experience relapse), 43 (30.5%) received DLI. The 3-year probability of D. Pastore, A. Mestice, P. Carluccio, T. Perrone, F. Gaudio, overall survival was 28.5% and TRM at 1 year and 3 years M. Leo, V. Liso, G. Specchia was 24% and 34% respectively. We analysed in multivariate University of Bari (Bari, I) analysis using a Cox proportional hazard model the relation between chronic GVHD occurrence and (1) demographic Chronic graft versus host disease (cGvHD) incidence and parameters: donor and recipient age, sex matching (2) pre severity are generally found to be greater after peripheral and post-transplant variables: HLA matching, number of blood stem cell transplantation (PBSCT) than after bone previous transplant, CMV status (D/R), ABO compatibility, marrow transplantation, and some studies increased mortality interval diagnosis-transplant, HSC source, CD34+ cell from cGvHD, particularly the extensive type. In vivo, T-cell number, CD3+ cell number, conditioning, ATG dose, status of depletion with antithymocyte globulin (ATG) is an effective disease, acute GVHD and DLI. We did not find any significant strategy for decreasing the incidence of cGvHD but may impact factor on chronic GVHD occurrence. Due to these include side effects such as high risk of infection and relapse. negative predictive findings and too few patients, it is vital to The main utilization of ATG has been as prophylaxis of GvHD do prospective trials in RICT focused on chronic GVHD in unrelated bone marrow transplantation but few data are prevention and/or early treatment. available on myeloablative HLA-identical sibling PBSCT. In our study we evaluated the incidence of infections, relapse and cGvHD in 28 patients with acute myeloid leukemia (AML) P582 in first complete remission after a peripheral stem cell High-resolution sonography of the bowel for timely myeloablative HLA-identical sibling transplant. diagnosis of intestinal acute graft-versus-host disease Conditioning regimen was oral busulfan (16 mg/Kg), after allogenic haematopoietic stem cell transplantation cyclophosphamide (120 mg/Kg) and ATG-Thymoglobuline (4 M. Postorino, E. Calabrese, L. Franceschini, G. Del Vecchio mg/Kg total dose given on days -2 and -1); the median dose of Blanco, R. Cerretti, L. Cudillo, A. Picardi, G. De Angelis, L. Di CD34+ cells and CD3+ cells infused was 4.6 x106/Kg (range Caprio, F. Pallone, W. Arcese 3.1-9) and 250 x106/Kg (range 84-442), respectively. GvHD Policlinico Tor Vergata (Rome, I) prophylaxis was Cyclosporin A (2 mg/Kg/Die) and short-term Methotrexate (15 mg/m² on day +1, 10 mg/m² on day +3, +6, Background: Acute Graft Versus Host Disease (aGVHD) is a +11). The median time to neutrophil (>0.5 x109/L) and platelet major complication during the first 100 days after Allogenic (>20 x109/L) engraftment was 15 (range 13-17) and 18 (range

S119 15-20) days, respectively. Acute GvHD grade I-II was P585 observed in 5/28 or 18%; no patients experienced aGvHD The role of ATG in reducing acute GvHD and primary graft grade III-IV. CMV antigenemia was monitored twice a week rejection in related and unrelated donor transplants for the first 100 days; 15/28 (54%) patients developed CMV Y. Zalyalov, B. Ganapiev, N. Ivanova, A. Alyanskiy, B. reactivation but no CMV disease occurred. As to Afanasyev immunological recovery, the median number of CD3+ and State I.Pavlov Medical University (St. Petersburg, RUS) CD4+ after 1, 3 and 6 months after transplantation was 690, 850, 930 and 125, 214, 256/microL, respectively; the median Myeloablative conditioning regimen followed by allo-HSCT number of CD8 and NK cells after 1, 3 and 6 months was 430, from HLA-identical donors has become a curative treatment 538, 653 and 216, 156, 230/microL, respectively. At a median option for patients with a number of hematologic observation time of 32 months (range 8-55) the overall cGvHD malignancies. Antithymocyte globulin (ATG) is used in the was 5/28 (18%) (limited 3/5 or 10% and extensive 2/5 or 8%); conditioning regimens for donor transplants to reduce the risk the relapse rate was 7/28 or 25%. of GvHD and rejection. Reduce intensity conditioning regimen Our data suggest that low dose of ATG is effective in (RIC) is important option for treatment patients > 50 yo and preventing acute and chronic GvHD without increase in heavy-pretreated. relapse; prospective randomized trials are needed to evaluate We analyzed the outcome of 125 high-risk pts with the role of ATG in AML undergoing allogeneic stem cell hematologic and nonhematologic malignancies receiving transplantation from HLA-identical sibling. myeloablative and RIC regimen for allo-SCT from HLA- identical donors with or without ATG. Since 2000 we have performed 125 allo-HSCT (88 (70%) with ATG and 37 (30%) - P584 without ATG) for the patients: AML (n=26), ALL (n=55), CML Extracorporeal photochemotherapy in treatment of acute (n=17), NHL (n=7), AA (n=7), HD (n=4), MDS (n=3), and chronic graft-versus-host disease osteomyelofibrosis (n=1), solid tumor (n=1). All patients were S. Sammassimo, G. Marotta, M. Tozzi, G. Buonfrate, D. stratified into two groups according to the risk of diseases Raspadori, F. Lauria recurrence: Azienda Ospedaliera Universitaria Senese (Siena, I) 1) Low risk • 2 complete remission/chronic phase (57 pts) 2) High risk >2 complete remission, no remission, partial Photopheresis (extracorporeal photochemotherapy, ECP) remission, progression diseases (64 pts) and the other characterized by exposure of peripheral blood mononuclear diseases: Kostmann's syndrome (n=1), hypereosinophilic cells to the photosensiting agent 8-methoxypsoralen and UV- syndrome (n=1), Krabbe disease (n=1), Wiskott-Aldrich A radiation, has been shown to be effective in treatment of syndrome (n=1). selected oncological and autoimmune diseases. Additionally, Dose of ATG was 60 mg/kg.b.w. (45-120). Myeloablative recent reports indicate that this therapy is promising also in conditioning regimen consisted mainly of Bu-Cy- 55 pts (44%), patients who develop Graft versus Host Disease (GvHD) after RIC-Flu-Cy- 70 pts (56%). The GvHD prophylaxis was: short allogeneic hematopoietic stem cell transplantation (HSCT) for term MTX and CsA. hematological malignancies. We present 19 patients (with a Results: The rate of GvHD grade 0-II in pts with ATG was median age of 48 years): 13 developed an acute GvHD 83% (67 pts), GvHD grade III-IV - 17% (14 pts). The rate of (aGvHD, 11 grade II, 1 grade III and 1 grade IV) and 6 a GvHD 0-II in group pts without ATG was 78% (22 pts) and chronic GvHD (cGvHD, 3 limited and 3 extensive) after GvHD III-IV- 22% (6 pts). In ATG group we observed primary (HSCT) and were referred to ECP. All patients were refractory rejection in 7 pts (8%). In the group without ATG primary or poor responders to steroid therapy. Seventeen patients rejection was higher (7 pts (19%)). Two pts without ATG died received a stem cell grafting from sibling donor and 2 from after engraftment because of severe infections without signs unrelated donors while 8 were treated with an ablative of acute GVHD. TMR in the group with ATG was 56% (49 conditioning and 11 with a nonmyeloablative regimen. The pts), without ATG - 67% (25 pts). OS within 6 years in pts with stem cell were collected from peripheral blood in 18 cases and ATG was 44%, without ATG - 33% (p=0.04). from bone marrow in 1 case. The majority of patients had skin Conclusion: The use of ATG in conditioning regimen reduces and liver involvement (only 3 patients with aGvHD had gut the incidence of acute GvHD grade III-IV, reduces risk primary manifestations). The ECP procedure was performed using the rejection and increases overall survival in patients after both UVAR XTS photopheresis system (Therakos Inc., West related and unrelated allo-HSCT without increased risk of Chester, PA, USA). Patients were treated on 2 consecutive relapse. days (one cycle) every week for two months, every two weeks for the following two months and then ECP was tapered on an individual basis. The median number of treatments was 21 P586 and no adverse events were observed. Of the 13 aGvHD Enteral budesonide in treatment of mild and moderate patients, 7 (54%) achieved a complete resolution (CP) of chronic GvHD GvHD manifestations, 2 (15%) a partial remission (PR) and 4 H. Andree, I. Hilgendorf, M. Leithäuser, C. Junghanss, C. no response. In the 6 cGvHD patients, 3 obtained a CR and Kahl, J. Casper, M. Freund, D. Wolff the other 3 a PR. The immunosuppressive therapy was University of Rostock (Rostock, D) discontinued in 32% of patients (all cases with grade II aGvHD or limited cGvHD) and reduced in 37%. Our findings suggest Introduction: Budesonide (BUD) is a highly potent steroid with that ECP is an effective adjunct therapy for both acute and a low bioavailability and has been used for topical treatment of chronic GvHD with cutaneous and liver involvement. In oral manifestations of GVHD as well as gastrointestinal addition, this beneficial effect is obtained without the GVHD. Here, we present the results of a retrospective complications typically encountered with immunosuppressive evaluation of the efficacy of BUD in treatment of mild or regimens used to control GvHD. However, in patients with moderate chronic GVHD (cGVHD). aGvHD grade IV or extensive cGvHD other therapeutic Methods: Eleven patients median age 52 years (41-65) after options are warrented. Randomized studies on a larger allogeneic hematopoietic stem cell transplantation for number of patients are needed to confirm the efficacy of ECP hematologic malignancies receiving single agent treatment in GvHD and its role in the treatment of this complications with BUD for cGVHD were evaluated for response. The stem after HSCT. cell source consisted of peripheral blood stem cells (n=10) or bone marrow (n=1). The donor types were matched unrelated donor (n=7), mismatched unrelated donor (n=2), and matched sibling donor (n=2). Six patients had prior acute GVHD grade 2-3, while the remaining 5 patients developed de novo cGVHD. Three patients had isolated gastrointestinal involvement of cGVHD and 8 patients had mild multi-organ involvement including gastrointestinal (n=8), skin (n=4), and

S120 liver (n=4) manifestations. Four patients received Cyclosporin (2) National Institutes of Health Consensus Development (CsA) at the time of onset of cGVHD, which was continued Project on Criteria for Clinical Trials in Chronic Graft-versus- during treatment with BUD. Diagnosis of cGVHD was Host Disease .BBMT 2005(1)- confirmed by histology (skin n=1, duodenum/stomach n=9) in 10 patients. Treatment consisted of BUD with an initial dose of 3x3mg orally. Results: Complete resolution of cGVHD was achieved in 6 Stem cell research patients, one patient had complete resolution of all clinical symptoms of cGVHD but persisting thrombocytopenia and two patients achieved partial remission (PR) of cGVHD. Two patients progressed on BUD and responded subsequently to P588 systemic steroids. Two patients relapsed with cGVHD after Chemotherapy induced mesenchymal stem cell damage withdrawal of BUD and 7 patients are currently on BUD. Ten in patients with haematological malignancies patients are currently alive with a median follow up of 168 K.C. Kemp (1), R. Morse (1), S. A Wexler (2), C. Cox (2), J.M. days after start of BUD. One patient with PR died due to Hows (1), C. Donaldson (1) invasive mycosis. Beside response of gastrointestinal (1)University of the West of England (Bristol, UK); (2)Royal involvement of cGVHD responses have been observed in liver United Hospital (Bath, UK) manifestations (n=2), lungs (n=1), skin (n=2) and eyes (n=2). Conclusion: BUD represents a treatment option in mild to High dose chemotherapy (HDC) used to treat haematological moderate cGVHD, which is well tolerated and associated with malignancy is known to damage the mature marrow stromal a high response rate in gastrointestinal manifestations of microenvironment. The extent that marrow MSCs are cGVHD. Furthermore systemic responses have been damaged by HDC is unknown. If damage to marrow MSCs is observed. The observed relapse of symptoms of cGVHD after substantial, cultured autologous or allogeneic MSCs are a withdrawal of BUD may be due to a dominating local effect on therapeutic possibility. We have compared the proliferative cGVHD and best treatment duration has yet to be determined. capacity and cluster of differentiation (CD) marker expression of marrow MSCs from patients with haematological malignancies who have received no treatment, low, or high P587 dose chemotherapy. Diagnostic value of two classifications of chronic graft- All MSC cultures were processed with minor modifications as versus-host disease and its therapeutic impact described by Wexler et al. 2003. Primary cultures established S. Saba, J. Malacalza, C. Mendez, C. Canepa from treated marrows displayed a mixed morphology UTMO - Hospital CUCAIBA (La Plata Buenos Aires, RA) compared to those from untreated marrows which grew as a typical densely packed fibroblast like monolayer. The Introduction: Chronic Graft- versus-Host Disease (cGVHD) is proliferation of marrow MSCs from patients who have received still one of the more important causes of Morbidity/Mortality HDC, n=25 was significantly reduced when compared to related to the allogeneic Stem Cell Transplant (aSCT).The MSCs from patients who had received no prior chemotherapy increase of aSCT in the few last years, with clear benefits, treatment, n=20, (p<0.01). MSCs from treated patients were requires to improve diagnoses and prognostic criteria of phenotypically similar to MSCs from untreated patients as cGVHD, in order to establish the suitable treatment, without follows; CD105+, CD166+, CD29+, CD44+, CD45neg, losing GVL effect. (p>0.84). However, the level of MSC CD44 expression after Purpose: to analyze, to re-classify and to compare two 2nd passage was significantly lower in patients who had internationally adopted classifications, in cGVHD, in order to received HDC than in untreated patients, (p<0.05). The improve future treatments with immunosuppressive drugs. frequency of colony-forming unit-fibroblasts (CFU-F) in the Patients and methods: 130 patients submitted to aSCT were second passage MSC population was determined using a evaluated in retrospective form, between June 1994/ April standard CFU-F assay. The number of CFU-F in each sample 2006.Evaluated patients: n=100 (100% donor HLA-matched was correlated to its MSC proliferation capacity at 2nd related siblings). Median age: 31.8(15-54),M/F: 71/29(r 2.4/1). passage. Although there was a significant reduction in MSC Diagnosis ( n ):CML:37, AML:19, AA:15, ALL:11, MDS:6, proliferation in the HDC group (p<0.05), there was no HL:5, NHL:3, MM:2, NPH:1, BFL:1.Conditioning regimen= significant difference in the number of CFU-F present in BUCY:78, CyGat: 9, FluMel:6, BuCyVP16:2, BEAM:2, culture from patients receiving HDC and patients who had FluBu:1, Flu-Ara-Ida:1, Mel200. A hundred patients were received no prior treatment, (p>0.30). There was a positive available for evaluation of cGVHD, diagnosed according to correlation between the number of CFU-F and the proliferative Seattle classification of limited / extensive (1), and then capacity in MSC cultures from patients who had received no compared according to criteria of the International prior chemotherapy (p<0.05), this was not evident in the HDC Consensus(IP)(2): mild/moderate/ severe. group, (p>0.64). Results: 47 patients developed cGVHD (1 ) ,one case limited, We conclude that marrow MSCs sustain prolonged injury due and 46 extensive. According IP 46 patients developed to recurrent courses of HDC. The reduction in proliferative cGVHD: mild: 2, moderate: 18, severe: 26. Median time to capacity and changes in adhesion molecule expression onset day +193 (61-494). All patients were treated by observed could disrupt vital interactions between MSC and systemic inmunosupresion (corticoids, alternate days protocol, HSC within the bone marrow microenvironment. thalidomide, ursodeoxycholic acid, mycophenolate mofetil, References: Wexler, S.A., Donaldson, C., Denning-Kendall, total lymphoid irradiation, and extracorporeal P., Rice, C., Bradley, B. & Hows, J.M. (2003). Br J Haematol, photoaphaeresis) 121, 368-74. Commentaries: According to the analyzed results, we conclude that: 1-Two patients should not have received aggressive P589 treatment, avoiding morbi/mortality. Protection by keratinocyte growth factor against the 2-International Consensus 2005 will help us to be less altered expression of CD44 on mesenchymal stem cells aggressive with the inmunosupresion in the cases of after chemotherapeutic insult involvement of eye and mouth (mild compromise) K.C. Kemp, R. Morse, K. Sanders, J.M. Hows, C. Donaldson 3-We also must consider Karnofsky performance status, University of the West of England (Bristol, UK) platelets count and diagnosis of the disease to decide the treatment. Bone-marrow stromal cells provide signals for differentiation, References: proliferation and self-renewal of haemopoietic stem cells (1) Chronic Graft-versus-Host in man. Am J Med, 1980, 69 (HSC) through direct cell-cell interactions and secretion of haemopoietic growth factors and cytokines.

S121 We have previously shown that the level of CD44 expression compare it with 486 publicly available microarray datasets on marrow mesenchymal stem cells from patients receiving from other mouse tissues and cell types. Principal component high dose chemotherapy (HDC) regimens is significantly analysis and hierarchical clustering identified a unique pattern reduced when compared to levels of expression from patients of gene expression capable of distinctively classifying MSCs who have received no prior HDC, (p<0.05). We have now from other tissues and cells. Analysis of absolute and relative examined the CD44 expression on marrow MSC from abundance of transcripts in all cell types revealed that the set haematologically normal donors, before and after in-vitro of transcripts uniquely expressed by MSCs is enriched in treatment with commonly used chemotherapeutic drugs transcription factors and in components of the Wnt signaling including cyclophosphamide with or without S9 liver extract, pathway. This analysis also identified a set of genes vincristine and chlorambucil. Drugs were added to the cultures specifically involved in the HSC niche. Interestingly, some of in therapeutic concentrations for 48 hours. In addition we these genes play a role in the maintenance of HSCs in a have investigated the use of keratinocyte growth factor (KGF) quiescent state supporting their survival but preventing them as a cyto-protective agent against chemotherapeutic damage from proliferating and differentiating. These findings provide to MSC in culture. novel and important insights on the mechanisms of T cell After in-vitro treatment of normal donor MSC, significant function regulation by MSCs and help to cement the rationale reductions in CD44 expression were observed with all drugs for their application in the treatment of autoimmune diseases. tested, (p<0.05). Decreased levels of CD44 expression on MSC were evident up to twelve weeks post in-vitro treatment with cyclophosphamide + S9 liver extract (figure 1). CD44 P591 protein expression on MSC was also examined in untreated Human mesenchymal stem cells promote survival of T- and cyclophosphamide + S9 in-vitro treated samples using cells in a quiescent state two-dimensional gel electrophoresis immuno-blots. The CD44 A. Uccelli (1), F. Benvenuto (1), E. Pedemonte (1), S. Ferrari expression pattern was altered after treatment, indicating that (1), E. Gerdoni (1), F. Gualandi (2), F. Frassoni (2), V. Pistoia exposure of MSC to cyclophosphamide may alter CD44 (3), G. Mancardi (1) structurally as well as quantitatively on the membrane surface (1)University of Genoa (Genoa, I); (2)San Martino Hospital of MSC. (Genoa, I); (3)IRCCS G. Gaslini (Genoa, I) The cyto-protective role of KGF to MSC was tested. KGF was added to MSC cultures 48hrs, 24hrs, and 4hrs prior to the in- Mesenchymal stem cells (MSC) are part of the bone marrow vitro treatment with chemotherapeutic agents. The addition of where they provide signals supporting survival and growth of KGF to MSC prior to treatment with cyclophosphamide + S9 bystander haematopoietic stem cells (HSC). MSC modulate liver extract resulted in significant preservation of CD44 also the immune response as they inhibit proliferation of expression, (p<0.05). lymphocytes. In order to investigate whether MSC can support We conclude that in-vitro treatment of normal donor MSCs survival of T cells we investigated MSC capacity of rescuing T with chemotherapeutic agents causes prolonged injury as lymphocytes from cell death induced by different mechanisms. previously shown in patient MSC exposed in-vivo to high We observed that MSC prolong survival of unstimulated T dose chemotherapy regimens. This study demonstrates that cells and apoptosis-prone thymocytes cultured under starving KGF protects normal donor MSC from in-vitro damage by condition. MSC rescued T cells from activation induced cell cyclophosphamide. It is possible that KGF given prior to HDC death (AICD) by downregulation of Fas receptor and Fas may protect the marrow microenvironment in the clinical ligand on T cell surface and inhibition of endogenous setting. proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl-2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a mild capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state providing a model of their activity inside the HSC niche.

P592 Implementation and results of a test dose programme for individualized dosing of once daily intravenous busulphan in combination with fludarabine in allogeneic P590 stem cell transplantation conditioning The molecular signature of therapeutic mesenchymal S.B. Kangarloo (1), F. Naveed (1), D. Quinlan (2), M. Geddes stem cells exposes the architecture of the haematopoietic (2), A. Chaudhry (2), D. Stewart (2), A.M. Magliocco (2), J. stem cell niche synapse Russell (2) A. Uccelli (1), E. Pedemonte (1), F. Benvenuto (1), S. (1)Tom Baker Cancer Centre (Calgary, CAN); (2)Tom Baker Casazza (1), G. Mancardi (1), J. Oskenberg (2), S. Baranzini Cancer Centre and University of Calgary (Calgary, CAN) (2) (1)University of Genoa (Genoa, I); (2)University of California Busulphan (Bu) given in myeloablative doses is a frequent (San Francisco, USA) component of cytotoxic preparative regimens for hematopoietic stem cell transplantation. Variations in the area The hematopoietic stem cell (HSC) niche of the bone marrow under the concentration/time curve (AUC) for Bu may result in is comprised of HSCs, osteoblasts, endothelial cells and a over/under dosing, which may increase the risk of toxicity or stromal component of non-hematopoietic multipotent cells of reduce efficacy. A pharmacokinetically guided test dose mesenchymal origin called mesenchymal stem cells (MSCs). strategy prior to administration of high dose Bu may be utilized MSCs are able to modulate T cell functions in vitro and, upon to achieve a specific target AUC. Previously we have shown in vivo administration, ameliorate experimental autoimmune that exposure to Bu as indicated by AUC above 6000 encephalomyelitis (EAE). However, the mechanism by which microM.min is associated with poorer clinical outcome. MSCs exert their effect is largely unknown. Here we analyze Objectives: Our aim was to avoid toxic levels of Bu exposure the global transcriptional profile of therapeutic MSCs and above an AUC of 6000 microM.min and to accurately and

S122 consistently adjust dose prior to the first day of intravenous P594 (IV) Bu treatment based on the kinetics of a reduced test dose Development of human natural killer cells from adult given a few days prior to the high dose treatment. haematopoietic stem cells in an in vitro system using Methods: Pharmacokinetic (PK) monitoring was conducted for human mesenchymal stem cells both the test dose and day one of high dose IV Bu. For the M. Pfeiffer, M. Wömpner, I. Müller, S. Viebahn, F. Gieseke, R. test dose 0.8 mg/kg of IV Bu was administered at a fixed Handgretinger, P. Lang infusion rate a minimum of two days before the first 3.2 mg/kg University of Tuebingen (Tuebingen, D) IV Bu dose. If the test dose predicted an AUC of 6000 microM.min or above the Bu dose for day one was reduced to Natural killer (Nk) cells have been shown to play an important target an AUC of 5500 microM.min. Bu concentrations in role in the graft versus leukaemia reaction especially after plasma were determined by UV-HPLC. All PK data were haploidentical transplantation. Leukaemia reactive Nk clones analyzed by non-compartmental analysis using WinNonlin could be observed only in the first few months after Version 5.0.1 software (Pharsight Corporation, Mountain transplantation ant this may be correlated to an increased View, CA, USA). number of KIR negative and KIR single positive Nk cells in Results: To date 53 patients have been monitored for Bu patients in this time period post transplantation. A better levels using this test dose strategy. The test dose has an understanding of Nk differentiation stages could help to excellent predictive performance for day one AUC of high decode the mechanisms underlying these observations. dose Bu. Bias as determined by the average percent error for Several in vitro systems for the in vitro generation of human day one AUC of Bu as predicted by the test dose was 2.08%, natural killer cells have been described, most using cord blood with a 95% confidence interval of +0.29% to +3.87%. The stem cells, murine stroma cell lines and different cytokines. range of percent error for the test dose predicted AUC for day We developed an in vitro system using autologous or one Bu was -9.06% to +21.34%, with 98% of the predictions allogeneic human mesenchymal stem cells as feeder layer falling below 20% error and 89% falling below 10% error. The and a cytokine combination previously shown to be optimal for test dose predicted that day one AUC for two of the patients Nk development on a murine fetal liver cell line (Miller et al would have exceeded the critical value of 6000 microM.min. Blood. 2001;98:705-713). Nk cells developed through different Bu dose was successfully reduced in these two patients to stages of maturation in regard to expression of CD117 and target an AUC of 5500 microM.min. CD94 which have been described in isolated Nk cells from Conclusions: This pharmacokinetically guided test dose human lymph nodes. We found a early expression of NKp44 strategy for monitoring IV Bu is highly accurate, consistent, and CD161, while expression of other NCRs, NKG2A and and reliable. Using this strategy toxic exposure level of Bu can NKG2D occurred later. These still KIR-negative, NKG2A be avoided; furthermore this strategy offers the promise of positive and NCR positive Nk cells showed production of targeted individualized dosing for IV Bu. interferon-gamma and a good cytolytic activity against K562. If such KIR-negative Nk cells also have a good activity against leukaemic blasts remains to be investigated. As we use P593 human mesenchymal stem cells and not a murine stroma cell Immune cells mimic endothelial progenitor colonies line our system would allow the generation of such KIR E. Rohde, C. Malischnik, K. Schallmoser, A. Reinisch, G. negative and cytolytic Nk cells according to GMP. Lanzer, W. Linkesch, C. Guelly, D. Strunk Medical University Graz (Graz, A) P595 Colony-forming units of endothelial progenitor cells (“CFU- Allogeneic haemopoietic stem cell transplantation after EC”) have been introduced as a powerful biological marker for conditioning with i.v. busulfan or i.v. thiotepa-containing vascular function and cumulative cardiovascular risk. The regimens level of “CFU-EC” has even been described to predict the A. Bacigalupo (1), M.T. van Lint (1), N. Bahlis (2), M.L. Savoie occurrence of cardiovascular events and death from (3), A.R. Turner (2), L. Larratt (4), A. Chaudhry (2), M. Geddes cardiovascular causes and may identify patients at increased (2), J. Storek (2), C. Brown (2), D. Quinlan (2), D. Stewart (2), cardiovascular risk. The precise mechanisms underlying J. Russell (2) “CFU-EC” formation and their cellular composition are (1)S.Martino's Hospital (Genoa, I); (2)Alberta Blood and BMT unknown. Program (Calgary, CAN); (3)Alberta Blood and BMT Program We performed detailed subtractive “CFU-EC” analyses in (Vancouver, CAN); (4)Alberta Blood and BMT Program blood samples of 18 healthy volunteers. The impact of various (Edmonton, CAN) blood cell types and kinetics of protein and gene expression were studied by cell sorting, flow cytometry, quantitative RT- Background: The most popular conditioning regimens without PCR and full genome microarray analyses. “CFU-EC”-derived total body irradiation (TBI) , is busulfan –cyclophosphamide soluble factors were measured by multiplex cytokine display (BU-CY). BU is now available in the intravenous formulation and functionally tested during endothelial network formation. and has been used in conjunction with fludarabine. Thiotepa “CFU-EC” contained more than 99% CD45+ nucleated (THIO) is a myeloablative agent, which has also been used in hematopoietic cells mainly comprising T cells and monocytes regimens with or without TBI. admixed with B and NK cells. Interestingly, purified T-cells Aim of the study: The aim of the present study was to plus monocytes were able to form “CFU-EC” clusters. The compare two different conditioning regimens , one based on complete lack of colony formation after depletion of T cells or i.v. BU (developed in Alberta Canada ) and one based on i.v. monocytes was contrasted by an unaffected “CFU-EC” THIO (developed in Genova Italy). formation after depletion of CD34+ progenitors. Gene Patients and regimens: The analysis is restricted to 104 expression profiling revealed an activation of various immune patients with leukemia , over the age of 40 allografted function-related biological processes but no changes in between 1999 and 2005, grafted from an HLA identical sibling. pathways assigned to angiogenesis. Soluble factors derived Patients transplanted in Alberta (n=57) received i.v. BU 3.2 from “CFU-EC” cultures supported the vascular regenerative mg/kg/day x4 with fludarabine 50 mg/m²x5 with low dose function of three different types of EC in vitro. antithymocyte globulin (ATG). Patients grafted in Genova Unravelling “CFU-EC” formation as a result of a functional (n=47) received thiotepa 10-15 mg/kg with cyclophosphamide crosstalk between T cells and monocytes shifts expectations 100-150 mg/kg and no ATG. GvHD prophylaxis was on vascular regenerative medicine. The data support a switch Cyclosporin Methotrexate for all patients. The two groups from a singular view on circulating endothelial progenitors were balanced for diagnosis (p=0.5) (AML (33,29) ALL (7,3) towards models that recognize the contribution of immune cell CML (17,15), and disease phase (p=0.1) (1st CR 44,30) ; BU functions during vascular regeneration. patients were younger (p=0.0003) (median age 47 vs 53) and received more frequently peripheral blood (PB) grafts (p=0.0001) (72% vs 15%). Median follow up for surviving patients was 4,6 years.

S123 Results Median day to 500 PMN in BU vs THIO patients was 16 vs 18 (p=0.2) respectively; acute GvHD grade II-III was 9% vs 32% (p=0.002) and chronic GvHD (minimal+limited+ extensive) 87% vs 81% (p=0.3). Cumulative incidence of transplant related mortality (TRM) was 11% vs 13% (p=0.7), cumulative incidence of relapse was 35% vs 46% (p=0.2) and actuarial 5 year survival was 64% in both arms (p=0.9) Conclusions: This study suggests that TRM, relapse and survival are comparable in leukemia patients over the age of 40, receiving conditioning regimens based on i.v. busulfan or i.v. thiotepa.; the lower incidence of acute GvHD in the BU patients is due to low dose ATG used in Alberta. Whether these regimens can be modified to reduce leukemia relapse, with low dose TBI or additional myeloablative agents, is being currently explored.

P596 Targeted and novel therapies for non-Hodgkin lymphoma: the role of radioimmunotherapy and haemopoietic stem cell transplantation V. Pavone (1), R. De Francesco (1), A. Mele (1), A. Rana (1), G. Greco (1), V. Frusciante (2), A. Varraso (2), C. Del Casale (1), F. Dicembrino (2), P. Tabacco (2), M. Caputo (1), A. Ostuni (1) (1)Hospital Cardinale G. Panico (Tricase, I); (2)Hospital Casa Sollievo della Sofferenza (San Giovanni Rotondo, I) P597 High-dose therapy followed by autologous hematopoietic The usage of mesenchymal stem cells for prophylaxis stem-cell transplantation is a main indication for relapsed or and treatment of GvHD in patients undergoing allo-HSCT high-risk first remission aggressive non-Hodgkin Lymphoma B. Afanasyev (1), D. Polintsev (2), L. Zubarovskaya (1), P. (nHL) and plays a role for relapsed follicular lymphoma. Kruglyakov (2), A. Golovacheva (1), Y. Stankevich (1), E. Preliminary studies suggest that Y-90 ibritumonab tiuxetan in Morozova (1), S. Alexeev (1), E. Darskaya (1), S. Bondarenko combination with high dose therapy and autologous stem (1), E. Babenko (1), O. Paina (1) transplantation is associated with high response rates, durable (1)St. Petersburg State Pavlov Medical University (St. remissions and acceptable toxicity. Petersburg, RUS); (2)Trans Technology LtD (St. Petersburg, We evaluated the combination of Y-90 ibritumonab tiuxetan at RUS) standard dose (0.4 mCi/Kg) plus high dose regimen (BEAM) in 9 patients with advanced stage nHL who failed to achieve Background: Within the bone marrow there exist subsets of complete remission (CR) after first line chemotherapy. nonhematopoietic cells referred to as fibroblast mesenchymal Methods: The treatment plan is shown in figure 1. PBSCs progenitor cells (A.Fridenstein,1968) or mesenchymal stem were collected after mobilization with DHAP and G-CSF. cells (MSCs). MSCs can suppress immune response by Patients’ characteristics are shown in table 1. multiple mechanisms. GVHD still remains the major Results: The median CD34+ cells infused was 7.92 x 106/Kg complication of allo-HSCT, especially MUD and haploidentical (range 3.2-21.6). The median time to platelet counts higher setting. The usage of MSCs for prophylaxis and treatment of than 20x109/L were 15 days (range, 9-28 days). The median severe GVHD (both acute and chronic) seems to be time to an absolute neutrophil count greater than 0.5x109/L reasonable (K.LeBlank,2004). were 10 days (range, 8-14). Median CD3+, CD4+, CD8+ and Patients and methods: From 10.2005 till 11.2006 7 patients CD56+ cells count at day + 30 were 580 xmille/mmc (70- (pts) received co-transplantation of HSC and MSCs for 2350), 110 xmille/mmc (30-540), 350 xmille/mmc (40-1630) prophylaxis of GVHD and 7 pts received isolated infusions of and 140 xmille/mmc (60-180), respectively. No grade IV MSCs for treatment of acute and chronic steroid-resistant mucositis was documented. Febrile neutropenia occurred in (aGVHD, chGVHD). Diagnosis of patients (pts): AML-4, ALL- 88% of cases. Median time onset and the duration of fever 5, MDS-1, CML-BC-2, NHL-2. Related allo-HSC was were day +2 (range, 1-4) and 4 days (range, 1-12). We performed in 6 pts, MUD–6 pts, haploidentical (haplo)–2 pts. observed 1 episode of FUO, 4 pneumonitis and 5 blood Seven patients received bone marrow (BM) and 5 pts PBSC stream infections, mainly by Gram+. One patient developed as the HSC source. The sources of HSC for patients with an atrial fibrillation. Median follow-up is 77 days (range, 36- haplo-HSCT were combination of primed BM and PBSC after 300). The overall response rate is 78% with CR in 56% of the separation by CliniMacs (“Miltenyi Biotec”). MSCs harvested patients. Median time to progression was 92 days (2 cases). from the BMof HLA-identical sibling-matched donors in a case One patient died for documented viral encephalitis at day +70. of related allo-HSCT and from the BM of HLA-haploidentical Conclusion: The use of RIT plus BEAM provides sufficient (third-party) donors in a case of unrelated allo-HSCT, results (CR 56%) with sustained engraftment, an acceptable processed by “Trans-Technology” Ltd company(license ¹ 99- extra-haematological toxicity and a rapid immunological 01-002224 from 14.07.2005) that include selection and recovery. The power of this program needs to be assessed in expansion for achieving a dose of 2.0x106 MSC/kg b.w. of a larger series of patients and in a multicenter setting. recipient. On day -1, pts are given MSCs (2.0 x 106/kg b.w.IV) 24 ours before infusion of donor’s HSC. Isolated infusion of MSCs is provided in a case of developing steroid-resistant aGVHD or chGVHD, 5pts received 1 MSC dose, 1pt -2 doses and 1pt-3 doses. Results: No toxicities were observed related to the infusion of MSC. Among the 14 pts, 10(71%) remain alive between 1 or 12 months after allo-HSCT, 4 pts died – 1 pt non-engraftment, 3 pts progression or relapse. In group with co-transplantations of MCSs-HSC engraftment was at D+13-29, no pts had severe aGVHD (0-I stage-6 pts, II-IV–0%).After isolated infusion of MSCs a partial response (PR) was observed in 1 pt, complete response in 3 pts with GVHD, III stage including

S124 2 pts with haplo-HSCT, PR in 1 pt with mild chGVHD. Overall extensive= 1). 8-year disease free survival were 73.17%. 8- response was 85%. year overall survival were 79.85%. Conclusion: MSCs infusion is well tolerated, safe, without Conclusion: According to this study, for an acceptable immediate infusion-related or late MSC-associated toxicities. outcome in Thalassemia class 3 We need better conditioning Randomized clinical trials are necessary for the estimation of and GVHD prophylaxis regimens to decrease therapeutic effect of MSCs in allo-HSCT pts. cardiopulmonary and liver complications. The results of blood and marrow transplantation showed that it is better than supportive therapy such as transfusion and desferal therapy. P598 Mesenchymal stem cells impair dendritic cell functions S. Chiesa (1), E. Traggiai (2), A. Uccelli (1) P600 (1)Universiity of Genoa (Genoa, I); (2)Gaslini Institute (Genoa, Side population (SP) cells are present in highly purified I) human CD34+ cells obtained from peripheral blood progenitor cells in patients with Hodgkin's lymphoma, but Several evidences demonstrate that murine mesenchymal the level of SP cells do not affect short-term engraftment stem cells (mMSC) modulate many immune functions of T, B, D. Josefsen, L. Forfang, A. Blystad, M. Dyrhaug, G. Kvalheim NK lymphocytes and dendritic cells (DC). Here we addressed Rikshospitalet-Radiumhospitalet Medical (Oslo, N) the effect of bone marrow derived mMSC on DC maturation, effector functions and T cells priming. In the presence of The expression of the CD34 antigen on hematopoietic stem mMSC at different concentrations, maturation of bone marrow cells (HSC) and immature hematopoietic progenitor cells precursors to DC was strongly impaired as depicted by an (HPC) are widely used to characterize a cell population which arrest of cell division and a significant decrease of CD11c also include cells with self-renewal potential. However, positive DC grown in the presence of mMSC as compared to previous findings suggest that early HPC might lack CD34 controls. After DC activation by LPS exposure, DC treated expression, further demonstrating the complexity of early with mMSC during maturation, showed a decreased hematopoiesis. A novel Hoechst 33342 dye efflux assay has expression of CD83 and an impaired secretion of IL-12. been developed which identifies a population of immature Similarly, mMSC-treated mature DC downregulated MHC HPC termed side population (SP) cells. In several previous class I and class II as well as CD80 and CD86 costimulatory studies the SP cells examined in mononuclear cell (MNC) molecules in comparison to controls. Finally, DC matured in fractions were shown to be predominantly CD34 negative. In the presence of mMSC showed an impaired capacity to prime this study we examined whether SP cells were present in CD4+ T cells obtained from the spleen of tetanus toxoid highly purified CD34+ (98-99%) cells from PBPC in patients immunized mice. Interestingly, a similar defect of effector with Hodgkin lymphomas. Furthermore, we examined whether functions was observed also when mature DC where exposed variations in the level of SP+ cells influenced short term to mMSC only during LPS activation. Overall, these findings engraftment. suggest that the immunoregulatory activity of MSC on T cell Highly enriched CD34+ cells were isolated from PBPC functions is the result also of a direct effect on DC and obtained from patients with Hodgkin lymphomas. therefore support MSC utilization for the modulation of To identify the SP+ cells, enriched CD34+ cells were stained abnormal immune responses. with Hoechst 33342 dye. Using flowcytometric techniques (FACStar+, Becton Dickinson, San Jose, CA) we were able to visualize the dye efflux in SP+ cells. The cells were P599 phenotyped using PE or FITC conjugated monoclonal Allogeneic blood and marrow transplantation in antibodies against CD34, CD38, CD133 and Thy-1. The thalassaemia major class 3: an experience in Iran percentage of SP+ cells in the CD34+ cell fraction varied. M. Iravani, K. Alimogaddam, F. Khatami, M. Jalili, L. Interestingly, we observed that 0,3-69% of the total CD34+ Nedaeifard, N. Babhadiashar, A. Mousavi, M. Jahani, B. cell population were SP+ cells. We obtained similar results in Bahar, A. Khodabandehlu, E. Baibordi, A. Gavamzadeh both freshly isolated as well as in thawed CD34+ cells. Except Hematology- Oncology & Bone Marrow (Tehran, IR) for a higher level of CD133+ cells in the SP+CD34+ cell fraction compared to the SP-CD34+ cell fraction, the Objective: Our aim for this study was to describe the outcome phenotypical profile of the two cell populations were of blood and marrow transplantation in patients with class 3 comparable with regard to these antigens. Thalassemia major. The frequency of LTC-IC was markedly increased in Methods: Since December 1992 till July 2006, fifty-two SP+CD34+ cells compared to SP-CD34+ cells, in line with patients with Thalassemia class 3 received blood and marrow previous reports. We did not observe significant differences in transplantation from their Human Leukocyte Antigen(HLA)- short term engraftment (ANC>05, Trombocytes>20) with identical siblings. Thirty-two patients received bone marrow regard to percentage of SP+ cells. Moreover, the number of and twenty patients received peripheral blood stem cell CD34 positive cells reinfused and CFU-C was not correlated transplantation. Conditioning regimen in 47 patients was to the number of SP positive cells, but number of CD34+ cells Cyclophosphamide 40 mg/kg/day (from day -5 to -2) and were correlated to CFU-C. Busulfan 3.5 mg/kg/day (from day -9 to -6) and in these In conclusion, Immature SP+ cells are present at variable patients Graft Versus Host Disease (GVHD) prophylaxis levels in CD34+ cells obtained from human PBPC. However, regimen was Cyclosporine.A (CY.A) 1.5mg/kg /day/IV (day - the level of SP cells was not correlated to the number of 3), then 3mg/kg/day IV (days +7, +11), then 12.5 CD34+ cells or CFU-C, and variation in number of SP cells did mg/kg/day/PO and Methotrexate 10mg/m² (day +1), 6mg/m² not influence time to short term engraftment. (days +3, +6). Conditioning regimen in 5 patients was Fludarabin 40 mg/m² (from -6 to -2) and Busulfan 4 mg/kg (from -5 to -2) and in this patients GVHD prophylaxis regimen was (CY.A) 3 mg/kg/day IV (days +3 and +7), then 12.5 mg/kg/day PO. Results: Median age at time of transplantation was 8.5 years (age range: 1-26), Male/Female: 21/31.Median time of absolute neutrophil count >0.5 x 109 /L was +14 and Median time of platelet recovery > 20 x 109 /L was +25. At present 42 out of 52 are alive and 10 patients died due to acute GVHD, chronic GVHD, rejection, veno-occlusive disease, infection and the others. Thirty-four patients (64%) developed acute GVHD (grade 1 = 13, grade 2 = 7, grade 3= 10, grade 4= 4). Three patients (5.8%) developed chronic GVHD (limited= 2,

S125 P601 retrospective study including all the patients submitted to Immune regulatory effect of human multipotent autologous stem cell transplant (ASCT) from June 1996 to mesenchymal stromal cells: role of TLR3/TLR3-ligand June 2006 in our center. We also evaluated other factors interaction on in vitro immunoglobulin secretion influencing engraftment like gender, age, diagnosis and R. Maccario (1), A. Moretta (1), M.A. Avanzini (1), A. Cometa CD34+ doses. (1), M. La Rocca (2), C. Dos Santos (1), M.E. Bernardo (1), P. Results: Engraftment data was only available in 129 of a total Gallina (1), M. Marconi (3), D. Montagna (1), P. Comoli (1), F. of 167 patients submitted to ASCT. Platelet and leukocyte Locatelli (1), A. Moretta (4) engraftment was delayed in all patients who received a (1)IRCCS Policlinico San Matteo (Pavia, I); (2)University Tor CD34+ ASCT dose lower than 3x106 per Kg (table 1). Vergata (Rome, I); (3)University of Pavia (Pavia, I); Analyzing the data we found that group O patients who (4)University of Genoa (Genoa, I) received a CD34+ ASCT dose above 5x106 per Kg presented a later engraftment than the non-group O patients (table 2). In vitro propagated multipotent mesenchymal stromal cells Conclusion: ABO group may play an important role in platelet (MSC) represent a promising tool for , and engraftment especially at higher CD34+ doses delaying cell therapy approaches in the setting of hematopoietic stem platelet recovery in Group O patients. cell transplantation. Several studies have documented relevant immune modulatory function of MSC. Toll-like receptors (TLR) are immune sensors of invading pathogens, which are distributed on cells of the immune system. Recently it has been demonstrated that also MSC express several TLR (TLR 1-6 and 9). Aim of the study was to investigate the effect of human MSC on immunoglobulin (Ig) secretion induced in vitro by TLR3/TLR3-ligand interaction. In preliminary experiments, we observed that addition of the TLR3-ligand Poly-IC (20 ug/ml) to MSC culture determined up-regulation of surface HLA-class I (MFI 204 in the presence vs. MFI 77 in the absence of Poly-IC) and a sharp increase in IL-6 production (17,5 ng/ml in the presence vs. 0.9 ng/ml in the absence of Poly-IC). Peripheral blood mononuclear cells (PBMC) of two healthy donors (Don) were then cultured in the presence or absence of Poly-IC and of autologous (auto) or allogeneic (allo) MSC (PBMC:MSC ratio, 40:1);10 ng/ml of rIL- 2 were added to culture medium. After 7-day culture, a higher % of activated B-lymphocytes (CD27bright on gated CD20+ cells) was observed in the presence rather than in the absence of MSC (Don 1: PBMC alone 12%, PBMC+Poly-IC 23%, PBMC+Poly-IC+MSC-auto 56%, PBMC+Poly-IC+MSC- Stem cell source allo 33%. Don 2: PBMC alone 2%, PBMC+Poly-IC 6%, PBMC+Poly-IC+MSC-auto 22%, PBMC+Poly-IC+MSC-allo 12%). Addition of allo-MSC caused more than 50% decrease P603 in cell recovery evaluated after 10-day culture, if compared A novel MDR-dependent pharmacological approach for with addition of Poly-IC or Poly-IC+MSC-auto. Evaluation of Ig ex-vivo expansion of haematopoietic stem cells from secretion (ng/ml) in 10-day culture supernatants demonstrated human umbilical cord blood an enhancing effect of both auto and allo MSC on IgG and H. Galski, I. Bar, A.J. Treves, A. Nagler IgM secretion for both Don, and on IgA secretion only for Don Chaim Sheba Medical Center (Tel Hashomer, IL) 1 (Table). Further experiments are in progress to confirm data obtained with TLR3-ligand and to investigate the role of other While umbilical cord blood (UCB) is an attractive source for TLR-ligands. transplantation, major disadvantage is its relative low number of hematopoietic stem cells (HSCs). A rational approach thus involves ex-vivo expansion of UCB-derived HSCs. However, the current expansion processes are laborious, expensive and also providing unwanted differentiated sub-populations. As it has been reported that the multidrug transporter MDR1 (ABCB1) gene product (Pgp) is over-expressed in various stem cells relatively to their differentiated progeny, we compared the expression level and activity of Pgp in UCB- derived CD133+ HSCs relatively to CD133- cells. Moreover, we reasoned that higher Pgp activity in these CD133+ HSCs will make them more resistant to cytostatic agents as P602 colchicine (COL) and thus its administration during the Influence of ABO type on platelet engraftment after expansion process could be applicable to their selection and autologous stem cell transplant enrichment. Towards this end, we isolated CD133+ HSCs C. Borges, D. Ferreira, J. Tomaz, L. Pais from UCB of various donors (n=6) by CD133- Hospitais da Universidade de Coimbra (Coimbra, P) immunomagnetic separation (MACS). Pgp-expression level was measured by flow cytometry using the Pgp-antibodies Background: Cell-surface carbohydrates, particularly those MRK-16, and its activity was measured by efflux assay of the containing fucose, play integral roles in hematopoietic cell Pgp-substrate Rh123. We further analyzed the CD133+/Pgp+ adhesion, growth, and differentiation. The H and H-active and the CD34+/CD38-/Pgp+ subsets during 8 weeks of antigens are strongly expressed on CD34+ and in standard procedure of their expansion in the absence or the megakaryocytic progenitors. The H-antigen seems to play an presence of COL. Analyses of freshly isolated CD133+ HSCs important roll in mediating stroma cell adhesion (fibroblasts indicated that the majority (>92%) of these HSCs express Pgp and extra cellular matrix proteins) and so improving on the cell surface. Moreover, the Pgp is functional as the megakaryocytic expansion and long-term preservation. cellular level of Rh123 was approx. 260-fold lower relatively to Objective and methods: The aim of our study was to find a CD133 negative cells and the Pgp modulator R-VRP inhibited possible relationship between the patient ABO group and the the efflux of Rh123. Analyses after ex-vivo expansion platelet as well as the leukocyte engraftment. We made a demonstrated a significant dose-dependent enrichment of

S126 CD133+ cell fraction by COL. At optimal COL dose (2.5 P605 ng/ml), the fold-enrichment of CD133+/CD34+/CD38- HSCs Quantitative monitoring of lineage-specific chimerism was 5.2 ± 2.3 higher than in its absence. At 8 weeks of after dual haematopoietic stem cell transplantation expansion the CD133+ cell number increased from 105 cells (umbilical cord blood with co-infusion of third party donor to 0.56±0.19 x109 and 1.60±0.41 x109 in the absence and CD34+ cells) presence of COL, respectively and thus the total yield of N. Sanchez-Hernandez, C. Manzano, D. Barroso, G. Iglesias, CD133+ HSCs after expansion in the presence of COL was P. Balsalobre, D. Serrano, R. Carrion, A. Gomez-Pineda, J. 2.9 ± 0.5 fold higher than in its absence. The long exposure of Diez-Martin, I. Buño CD133+ HSCs to COL at the expansion process did not affect Hosp. G.U. Gregorio Marañon (Madrid, E) their ability to form various hematopoietic colonies in semisolid culture after COL removal. In conclusion, we show that UCB- Background: Allogeneic umbilical cord blood (UCB) stem cell derived HSCs over-express a functional Pgp and this may be transplantation (SCT) shows some advantages (HLA beneficial for their expansion ex-vivo towards clinical matching requirements or availability) respect to SCT using transplantation. other sources of matched unrelated donor (MUD) stem cells. However, it is correlated with slower engraftment, increasing risk of infections and early mortality. It has been recently P604 shown that co-infusion of third party donor (TPD) CD34+ cells Study of cord blood derived mesenchymal stromal cells (dual SCT) is useful to speed up engraftment. and comparison with their bone marrow counterparts Objective: To evaluate the usefulness of lineage-specific C. Perdikogianni, H. Dimitriou, E. Stiakaki, G. Martimianaki, M. chimerism quantification in the management of this transplant Kalmanti setting. University of Crete (Heraklion Crete, GR) Patients and methods: 8 dual SCT (Tables 1, 2) in 7 patients (1 CML-BC, 2 AML-M2, 1 AML-M4, 1 ALL-Ph+, 1 biphen. Cord blood (CB) constitutes an easily accessible source of ALL, 1 NHL). Chimerism was analyzed by STR-PCR hematopoietic progenitors suitable for transplantation and (AmpFlSTR SGM Plus, Applied Biosystems; sensitivity 1%) recently its feasibility as a source of mesenchymal stromal and quantitative real-time PCR (qrt-PCR) of null alleles and cells (MSC) is assessed. The aim of this study was to insertion/deletion polymorphisms (Light Cycler, Roche; determine the optimal culture conditions for the expansion of sensitivity 0,01%). Peripheral blood (PB) and leukocyte CB MSC, to assess their functional and immunophenotypic lineages (T cells, CD3+, and myeloid cells, CD15+), isolated characteristics and to compare them with bone marrow (BM) by positive selection using automated immunomagnetic MSC from children. technology (AutoMACS, Miltenyi Biotec), were analyzed Eighteen CB samples from full term deliveries were analyzed weekly. Bone marrow (BM) was analyzed at days +30, +100, and the results were compared with MSC from 23 BM from +180 and +365). children with benign hematological disorders without BM Results: 4/8 cases showed recipient cells in PB after dual SCT involvement (ITP n=13, autoimmune neutropenia n=10). during a median period of 12 days (range 4-18 days). In three Culture conditions were initially based on the technique used patients no TPD cells were observed after dual SCT. 5/8 for the isolation of BM MSC and sequentially different culture cases showed initially TPD cells in PB which were conditions were tried in order to optimize MSC expansion. progressively replaced by UCB cells. UCB complete They included different cell concentrations, enrichment of the chimerism (UCB-CC, absence of recipient and TPD cells even culture medium with FGF-2, different concentrations of the in qrt-PCR assays) was acquired in a median of 22.5 days specially selected FCS, different culture surface area and (range 18-39). In 3/8 cases, recipient cells were found once pretreatment of the culture plate with FCS. CC had been acquired, which allowed early diagnosis of 1 BM derived mononuclear cells formed stroma that reached graft rejection and 2 relapses. One patient developed a confluency in 17±0.9 days. Cell doubling time was 3.6±0.15 ganglionar relapse in CC. T cells (CD3+) were mainly of UCB days at the first passage (P1) and remained the same until origin early after dual SCT and reached UCB-CC a median of P3. Following stroma formation, no hematopoietic markers 7 days (range 0-21) before PB. However, myeloid cells were expressed whereas CD105, CD29, CD44, CD146 were (CD15+) derived primarily from the TPD and reached CC detected at a percentage of > 85%. Histochemical and together with the PB. TPD cellularity favoured early molecular analysis showed that BM MSC had the capacity to engraftment (before UCB-CC took place) in 3 cases. differentiate into osteocytes, chondrocytes and adipocytes. Conclusions: Lineage-specific chimerism quantification Isolation of CB MSC was achieved in 25% of the samples allowed a close monitoring of the dynamics of engraftment of cultured under optimal conditions which were found to be the cells of both donors which is of key importance in this SCT higher initial concentration of cells (3x106 cells/cm²), the setting. Moreover, lineage-specific chimerism analysis was enrichment of the culture medium with FCS (20% compared to useful to diagnose one graft rejection and two out of three 10% of the BM protocol), the addition of 5ng/ml FGF-2 (1ng/ml relapses. in the BM protocol), the pretreatment of the culture surface with FCS and the initial volume of the sample (> 40 ml). MSC were morphologically similar to the ones derived from BM, but appeared late in culture requiring 34 days to reach confluency and 55 days from P1 to P2. Immunophenotypic analysis of P1 cells showed no expression of CD34 while CD45 ranged from 0-17.83% and CD105 ranged from 49-83%. CFU-F colonies were developed in one case. These findings suggest that CB cannot be regarded as a sufficient source of MSC for clinical use. Intense research should continue on the optimal culture conditions and further focus on the search of alternative sources.

S127 P606 CML, 2 CLL, 1 Waldenström, 3 follicular lymphoma, 1 Hodgkin Double cord blood transplantation for patients with high- disease, 3 myeloma, 1 PNH, 1 SAA. risk haematological malignancies: delayed immune Five pts received double cord units. Graft characteristics in the recovery and high incidence of infections 37 single unit UCBT were: number of nuclear cells and CD34 V. Rocha, A. Madureira, M. Robin, M. Carmagnat, J. collected: 3.63x107/kg (2.6-5,2) et 1.4x105/kg (0.5-6.5); HLA Fernandes, R. Peffault de Latour, C. Ferry, R. Traineau, P. compatibility 4/6 in 27 (in HLA class I: 17, class I plus class II: Ribaud, A. Devergie, C. Rabian, E. Gluckman , G. Socié 10). Hopital Saint Louis (Paris, F) Results: median day to obtain 0.5x109 ANC: D15±8 (0-39). Full chimerism was present in 29 pts, autologous We have performed a phase II study on 16 dCBT from 2004 to reconstitution in 2, early relapse in 6 pts (not yet evaluated in 2006. Ten patients had high risk malignant disorders (ALL=1, 5). Infections: 8 CMV reactivations; 3 bacteremias; 1 VRS AML+MDS=6, CML=3) and 6 high risk of rejection (SAA=4, pneumonia, 2 toxoplasmosis. Limbic encephalitis developed in PNH=1 and Fanconi Anemia=1). Among them, 4 received a 2 pts. Acute GVHD incidence: 29±7% (6 grade II, 1 grade III, 3 dCBT as a rescue of previous non-engrafted transplants (2 grade IV) and 11 pts/32 developed chronic GVHD. Four pts SAA, 1 AML and 1 CML). Analyses of T, B and NK cells died and EFS was 62±9%. Median follow-up: 10 mo. phenotype were performed once a month during the first 3 In conclusion, UCBT after RIC in adults is an alternative in months and every two months until 12 months. The median absence of related or unrelated donor and could be the future age was 21 years (11-42), the median weight 63 kg (30-90) for allogeneic hematopoietic stem cell transplantation. A and the median follow-up time was 6 months (3-18). national protocol (MINICORD) is proposed in France for AML Conditioning regimen varied according to disease pts. European extension should be discussed. (myeloablative) or second transplant (reduced intensity), all except two patients have received ATG. Graft-versus-host disease (GVHD) prophylaxis consisted in Cyclosporine A + P608 steroids in 12 patients and associated to MMF in 4. CD34+ cells recirculation and immunophenotyping after Results: 2 patients did not engraft (both with SAA), one patient autologous peripheral blood stem cell transplant for relapsed 8 days after dCBT, and 13 patients engrafted at a haematological malignancies median of 23 days (14-42). Chimerism available in 12 patients S. De Matteis, N. Piccirillo, F. Sorà, M. Tarnani, L. Laurenti, C. before day 100 showed both CB units in 7 patients. After day Rumi, G. Fancello, P. Chiusolo, G. Reddiconto, S. Sica, G. 100, in 9 evaluable patients, 3 patients had evidence of both Leone CB units engraftment. Grades II-III acute GVHD was observed Università Cattolica del Sacro Cuore (Rome, I) in 5 patients (grade II in 4 and grade III in 1) and chronic GVHD in 7 out of 12 at risk. During the first 100 days, 9 CMV Hemopoietic progenitor cells (HPC) can be identified by the infections were diagnosed; 4 HSV (resistant to acyclovir); 3 expression of the CD34 antigen. Previous studies showed that HHV6 infections; 3 EBV infections; 3 adenovirus disease, 4 HPC circulates in the peripheral blood during engraftment SRV infections, 3 septicemia, 3 fungal infections, 1 after peripheral blood stem cell transplantation (PBSCT). Thus disseminated toxoplasmosis. After day 100, we observed 4 we investigated the kinetics of appearance of CD34+ cells CMV infections, 1 CMV and 1 adenovirus disease, 3 HSV and after autologous PBSCT (aPBSCT), its correlation with 1 EBV infections. Severe lymphopenia was observed in all haematological engraftment and the immunophenotypic patients (median of 259mm³ at 3 months (n=14); 389 at 6 characteristics of the HPC. We studied the percentage of months (n=13) and 480 at 12 months (n=8). Median number CD34+ cell in peripheral blood every other day from day +2 of CD3/CD4 at 3, 6 and 12 months were: 8, 15 and 46 mm³ until patient discharge in 29 patients (pts) (table 1) treated with respectively; of NK cells 203, 249 and 115mm³, and of B cells aPBSCT for haematological malignancy. These cells were was 0, 0 and 110 mm³, respectively. At 6 months overall detectable starting from day +8 after transplantation. The peak survival was 58±14% and event-free survival was 52±14%. of the CD34+ cells was at day +12 (range 10-16) when the Six patients died, 2 of relapse and 4 from infections. Three out number of CD34+cells/mmc was 6.85 (range 0.6-116). of 4 patients have been rescued of previous non engraftment Statistical analysis showed a significant negative correlation and are alive and well (4 -18 months after dCBT). (r-0.44, p=0.022) between age of pts and peak of CD34+, In conclusion, despite the short follow-up dCBT seems to be while a significant positive correlation (r0.41, p=0.031) an option to treat patients with high risk diseases and without between age and day of CD34+ peak. There was a significant a suitable compatible HLA donor. High incidence of infections correlation between total number of CD34+cells/kg infused and delayed immune recovery are major problems after and percentage of CD34+ peak (r0.42, p=0.027). Furthermore dCBT. we observed a significant positive correlation between day of CD34+ peak and time to absolute lymphocyte count >0.5x109/l (r0.54, p=0.003) and to absolute neutrophil count P607 >0.5x109/l (r0.47, p=0.012). Subsequently we compared the Unrelated cord blood transplantation after reduced- immunophenotypic profiles of the HPC circulating after intensity conditioning in adults aPBSCT in 12 pts (table 1) on day +11 and+14 to profile of B. Rio, M. Renaud, S. Francois, P. Ceballos, C. Bulabois, M. the HPC mobilized in 3 pts using chemotherapy and growth Uzunov, L. Fouillard, D. Blaise, E. Gluckman, V. Rocha on factors administration. The CD34+ cell subset almost entirely behalf of the SFGM-TC; Eurocord expressed HLA-Dr, 50% of cells were CD38+, 74% were CD133+ on day 11 and 59% on day 14, only 47% on day 11 In absence of related or unrelated donor, cord blood is an and 19% on day 14 expressed CD33 (myeloid committed). alternative source of hematopoietic stem cells. We report here Thus the majority of HPC were pluripotent cells. Interestingly the preliminary results of unrelated cord blood transplantation the major difference between HPC circulating after aPBSCT (UCBT) after reduced intensity conditioning (RIC) in adults in and during mobilization was the expression of the CD117: France. 41% and 36% on day 11 and 14 after a PBSCT respectively Between October 2003 and April 2006, 42 patients (pts) and 4% only in mobilized HPC (figure 1). This result could received UCBT after RIC associating cyclophosphamide 50 clearly differentiate mobilized HPC from circulating HPC after mg/kg (D-6), fludarabine 40 mg/m²/d (D-6 to D-2) and total transplant. Thus the latter cells might be considered as body irradiation at 2 Gy. GVHD prophylaxis associated surrogate marker of bone marrow reserve. Furthermore they ciclosporine et mycophenolate mofetyl from D-3. G-CSF was seem to be more prone to interact with the hemopoietic niche started from D+1 until 5x109 WBC/L. thought the persistent high expression of CD117, adhesion Pts characteristics were: median age: 48±13 (16-69); weight: molecule. Further analysis of CD34+ subset are ongoing to 64±11 kg (45-90); recipient gender: 15 males/27 females; confirm these data and to clarify their significance. CMV seropositivity in 24 pts; major and minor ABO incompatibility in 18 and 9 respectively. Diagnosis: 3 ALL (1 CR1, 2 CR2), 21 AML (8 CR1; 12 CR2; 1 CR3), 4 MDS, 2

S128 P610 The role of the autologous stem cell rescue in the unrelated cord blood transplant A. Picardi (1), R. Cerretti (1), A.P. Iori (2), A. Spagnoli (1), I. Rana (3), M. Screnci (2), L. Cudillo (1), L. Cupelli (1), L. De Felice (1), G.P. Del Proposto (1), I. Rana (3), E. Iannella (2), A. Tendas (1), W. Arcese (1) (1)Tor Vergata University (Rome, I); (2)La Sapienza University (Rome, I); (3)OPBG (Rome, I)

Umbilical cord blood (UCB) represents a valid alternative haematopoietic stem cell source for pts who need an allogeneic stem cell transplant (SCT) but lack an HLA matched related donor. The widespread applicability of UCB has been limited by the low number of cells contained in a single CB unit, which can negatively affect the probability of hematopoieitc engraftment. The increased risk of graft failure after unrelated UCB SCT represents an indication to cryopreserve patient’s stem cells as rescue. Aim: In order to re-define whether the back-up collection continues to be mandatory for pts undergoing UCBT, we have reviewed the use of stem cell back-up in a large cohort of UCBT recipients for whom a stem cell rescue was prospectively planned. Methods: We have collected data from 4 SCT Units in Rome. The resulting database consisted of a cohort of 118 pts (81 children\37 adults), submitted to UCBT between June1995 and October 2006. There were 94 AL (32 AML/62 ALL), 10 MDS, 6 Lymphomas and 8 other diseases. Median age and body weight was 12 years (1-60) and 44 kg (6–100), P609 respectively. Median number of infused NCx107/kg, Use of autologous bone marrow harvests in patients CD34+x105/kg and CFU-GMx104/kg was 3.3, 2.3 and 1.5, undergoing volunteer unrelated donor stem cell respectively. According to serologic (class-I) or high resolution transplantation: a single-centre experience HLA-typing (class-II), 4 pts received an HLA matched UCB, P.S. Junagade, S. Simpson, A. Ho, S. Devereux, A. Pagliuca, which was different at 1 or 2 HLA loci in 51 and 59 pts, A. Mijovic, G.J. Mufti respectively. A stem cell rescue was provided from 116 out of (1)King's College Hospital (London, UK) 118 pts. Results: Overall, 103 pts engrafted (87%) within 100 days with The incidence of graft failure in patients undergoing volunteer a median time to achieve PMN>500 of 25 days (11-99), 11 pts unrelated donor(VUD) haematopoietic stem cell died of TRM. Four pts, all transplanted before 2002 and transplantation(HSCT) is 2-5%. As a precautionary measure, receiving NC < 2.5x107/kg, failed to engraft and infused the many centres perform autologous backup stem cell harvest autologous back-up at 38, 46, 48 and 50 days, respectively. prior to transplantation. Due to the potential risk of leukaemic An autologous reconstitution was observed on day +74 in 1 transformation with the use of G-CSF, many centres prefer to patient, while 2 pts died of acute GVHD and 1 of infection.This perform bone marrow harvests in patients with myeloid analysis has shown that the back-up was required in only malignancies. However, the cost of each BMH is 3.4% of pts and was successful in only 0.8% of all approximately £3000, and the procedure carries the inherent transplanted pts. risk of general anaesthesia, together with the need for Conclusions: At present, the significant improvement in the inpatient stay and possible blood transfusion post procedure. selection of both pts and cord blood units and the use of We retrospectively identified 53 patients with myeloid double UCB units have significantly reduce the risk of graft malignancies who received a back up BMH performed over a failure after UCBT. Moreover, reservation of a second UCB 4 year period, all of whom underwent VUD HSCT(6 unit or the use of an haploidentical related donor can be myeloablative,47 RIC). Median age of recipients was 52 offered to pts who need an urgent stem cell rescue following years(range:16-71). The median platelet count was UCBT failure. Therefore, the policy to provide pts of an 141.5(range:15-380), with median BM cellularity at harvest autologous stem cell rescue should not be considered was 35%(range:5-95%). The median CD34+ yield of mandatory for UCBT procedure. 0.65x106/kg(range:0.3-6.88). 65% patients required blood transfusion post procedure(median of 2 units) with a median admission of 2 nights. On analysis, there was no correlation P611 between age or platelet count and CD34+ yield. However, High recovery of haematopoetic stem cells using a closed patients with BM cellularity >25% had significantly improved apheresis system for erythrocyte depletion and volume median CD34+ yield compared with those <25% (0.99 vs reduction of AB0-incompatible bone marrow transplants 0.35, p=0.004). Stem cells were re-infused in only 2 P. Reinhardt, P. Schauwecker, K. Schwarz, E. Krug, B. patients(pancytopenia post-adenovirus infection=1, secondary Maccari, H. Schrezenmeier, M. Wiesneth autologous transplant=1). Both patients died from transplant- IKT Ulm (Ulm, D) related complications. Our data suggest very infrequent use of backup BMH even for Purpose: Bone marrow (BM) is an important source of VUD HSCT. In addition, the cell yield from these procedures is hematopoetic progenitor cells (HPC). For small volume often sub-optimal. As the procedure involves considerable recipients, AB0-incompatible transplants (Tx), cost, exposure to potential complications, blood transfusion cryopreservation or manipulation, the collected BM requires a and increasing demand on storage space, we propose that safe and effective red blood cell- (RBC) and volume-reduction back up bone marrow harvests should be considered on without significantloss of HPC. individual basis. Multi-centre collaboration may help to Methods: 40 BM-aspirates for transplantations requiring an identify uniform criteria to select patients undergoing BMH RBC- and vol.-reduction were analysed retrospectively. ACD- pre-transplant. A was added to the collected BM to 10% (vol/vol) and transferred to a BM processing set (Gambro, BCT) in the clean room. The BM was processed >4 times on a

S129 CobeSpectra cell separator using the respective BM-setting 10.2 - p= 0.7). GVHD prophylaxis was done in both groups (all Gambro, BCT) in essence following manufacturer with CSA plus MTX and median number of MTX doses was instructions. Enriched MNC fractions were usually collected not different in BM and PBSC groups (3.41 versus 3.48), sequentially in 2 bags. Vol.- and RBC-reduction, MNC-, CD34- Engraftment time to Neutrophils > 0.5 x 109 /L was and CD3-recovery as well as clonogenic assays (MethoCult, comparable in the two groups (respectively 18.3 in BM vs 18.0 StemCell Technologies) were assessed. in PBSC group,log rank p value = 0.4). A count of PLT > 50 x Results: 28 BM-Tx were AB0-incompatible, 27 Tx were for 10e9/L was reached faster in PBSC group (median 15.1 days) patients weighing <40kg (6-38 kg) and most transplants were than to BM group (median 18.2 days - Log rank p value = to be partially cryopreserved. The original BM vol. ranged 310- 0.08). 2228 ml with an average Hct of 29% i.e. 81-624 ml of RBC. In conclusion BM harvest can results in a CD34+ cells content Cytapheresis lasted 100-150 min. The BM-vol. was processed equivalent to the one regarded as optimum when using PBSC 4 to 16 times (average 8). The initial vol. was reduced in harvest and with a comparable myeloid engraftment time. average to 14% (final volume 61-359 ml) and a Hct of 6% (RBC 1-29 ml) in collection bag 1; in bag 2 a vol. of 34-124 ml (8%) and an average Hct of 5%. As expected the recovery of P613 NC was low (bag 1=34%, bag 2=6%), better for MNC (bag Unrelated cord blood transplantation in paediatric 1=55%, bag 2=10%)and very good for CD34 (bag 1=83%, patients, the experience of a specialist hospital and bag 2=13%) and CD3 (bag 1=86%, bag 2=14%). The research centre, Saudi Arabia recovery of BFU-E and CFU-GEMM in 20 assayed transplants M. Ayas, A. Al-Seraihi, A. Al-Jefri, A. Al-Ahmari, S. Rifai, A. Al- revealed no loss of HPC, but instead suggested an improved Ghoneim, S. Al-Mohsen, H. Al-Mousa, H. Al-Dhekri, Z. Al- regenerative potential in vitro of the volume- and RBC- Hasnan, A. Khairy, S. Markiz, I. Al-Hassan, H. El-Solh reduced transplants (recovery BFU-E median=133% p=0.28, King Faisal Specialist Hospital (Riyadh, SA) CFU-GEMM median=169% p=0.0009, all MethoCult). Conclusions: To maintain the Hct between 2-7 % in the Background: For patients (pts) lacking a suitable matched collected MNC-layer is technically challenging. Processing the related donor, unrelated cord blood transplantation (CBT) has initial vol. 4-6 times usually allows the recovery of >80% of emerged as a valid alternative with many advantages such as CD34 and CD3 cells (bag 1), additional proc. (bag 2) may be easy procurement, and acceptable outcome even with partial useful as back-up. Tests to clarify the surprisingly high HLA mismatches. We report here our experience at King recovery of CFU are currently in progress. Cytapheresis is an Faisal Specialist Hospital (KFSHRC) in 51 pts who underwent efficient and GMP-compatible technique to reduce RBC`s and partially matched unrelated CBT. vol. of BM-Tx even for AB0-incompatible Tx of infants or Patients and methods: From January 2003 till October 2006, further processing without major loss of HPC`s. we performed 54 CBT on 51 pts; second transplants (3 pts) are excluded from this analysis. Conditioning was according to the disease in addition to antithymocyte globulins (ATG); 2 pts P612 received reduced intensity conditioning. GvHD prophylaxis In normal donors bone marrow harvests can results in a was with cyclosporin and methylprednisolone. All pts received CD34+ contents and in a neutrophils engraftment time GCSF from day +1 until engraftment. equivalent to that obtained using PBSC mobilization and Results: Forty three pts engrafted (88%), median time to PBSC collections absolute neutrophil count (ANC) • 0.5 x 109/L was 17 days G. Milone, S. Coppoletta, K. Battiato, P. Murgano, V. Pinto, M. (range, 10-49 days), and median time to a platelet count of • Poidomani, S. Leotta, S. Mercurio, B. Farsaci, A. Strano, E. 20 x 109/L was 45 days (range, 18-149); chimerism, assessed Mauro, M.G. Camuglia, R. Giustolisi by short tandem repeats (STR), ranged from 24% to 100%. Ospedale Ferrarotto (Catania, I) Acute GvHD grade II-IV developed in 12 pts (23 %) and acute GvHD grade III-IV developed in 6 pts (12%). CMV infection Number of infused cells has an important effect on patient's developed in 26 pts (51%), 2 of them developed CMV disease survival. Patients receiving a number of CD34+ cells below (retinitis in one and pneumonitis in another). Twenty nine pts median have an inferior survival than those receiving a are currently alive (56.9 %), with a probability of overall and superior amount of progenitor cells. event-free survival at 46 months of 45.5% and 35.5 % Median CD34+ cells content of Bone Marrow harvests in respectively. A trend of improvement was noted in the results normal donors is usually reported to be between 2.4 and 2.7 x over the years; when calculated for pts transplanted in 2003- 106/CD34 cells and that of TNC (total nucleated cells) 2004 only, the one-year probability of overall and event free between to be 2.3 and 3.2 x 108/Kg. survival was 51.5% and 43.5% respectively versus a one-year In order to increase the median number of CD34 cells infused probability of overall and event free survival of 67.7% and we have optimized our bone marrow harvest techniques. 50.5% respectively for pts transplanted in 2005-2006. For pts 60 consecutive patients who underwent in our institution HSC with acute leukemia (11 pts), the one-year probability of Transplant from HLA identical sibling using BM or PBSC were overall and event-free survival was 40.4% and 36.4 % studied; 35 patients received BM harvests and 25 received respectively, and for pts with immune deficiency disorders (16 PBSC with a target infusion dose of 5 x106/Kg CD34+. The pts), the one-year probability of overall and event-free survival HSC source (BM or PBSC) was choosen at physician was 55% and 50% respectively. discretion on the basis of disease stage, donor sex and ABO- Conclusions: At KFSHRC, CBT offers a reasonable chance of incompatibility. Optimization of BM Harvests was obtained event free survival to pts who lack matched related donors using large and multiple-bore needle (11 gauges), small with acceptable results. CMV infection is a major cause of volume of aspiration (3-5 ml), performing 5-10 skin punctures morbidity in this population. The improvement noted in the for each side and allowing only to experienced physicians to survival of pts in the 2005-2006 era is probably due to a better perform the procedure. During this study our laboratory selection of pts as well as improved supportive care and participated to an external quality control (UKNEQAS) for prompt management of complications. CD34 enumeration obtaining results comprised between 25- 75 percentiles. Median recipient age was 38 years (range 15- 61). Median Volume of harvested BM (ml / Kg donor weight) was 17,8 (range 11-25). Median donor weight was 74.9 Kg in BM group and 71.2 Kg in PBSC group (t-test: p= 0.6). Median number of TNC in BM group was significantly lower than in PBSC group (4.7 x 10e8/Kg vs 5.9 x 10e8/Kg respectively – p = 0.001). To the contrary median number of CD34+ cells in BM group was 5.4 x 106/Kg recipient weight (range 2.4-17.8) and it was 5.4 x 106/Kg recipient weight also in PBSC group (range 3.4-

S130 P614 monoclonal antibodies enumerating each subpopulation as Low platelet count before growth factor administration is absolute cell count and proportion of the total lymphocytes. a significant predictor of poor autologous peripheral Results: median number of infused cells per kilogram of blood progenitor cell mobilization recipient are reported in the table. In multivariate analysis, A. Lopez de la Guia, A. Kerguelen Fuentes, M. Martin Salces, early disease status at transplant and a lower number of F. Hernández-Navarro, R. Arrieta CD19+ cells were associated with a better overall survival H.U. La Paz (Madrid, E) (OS). Risk of death was increased in patients receiving >0.5 x 108 CD19+ cells infused/kg (HR = 6.4, p = 0.01). The Previous studies have proposed combination of various incidence of grade II to IV acute GVHD was 36%. In factors that predict poor mobilization after GCSF stimulation. multivariate analysis the disease status and the number of However, there is no single clinical or laboratory test that CD19+ cells correlated significantly with the incidence of reliably correlates with PBPC mobilization potential before acute GVHD (p= 0.03). A trend for higher transplant related growth factor administration. Peripheral blood CD34+ cell mortality (TRM) was seen with a greater number of CD19+ count (PCD34) reliably correlates with PBPC mobilization and cells, but this correlation was not significant (p= 0.14). yield after growth factor administration. Disease-free-survival (DFS) was better for patients receiving a We have prospectively followed 130 autologous PBPC lower number of CD19+ and CD56+ cells (p=0.01 and p=0.04, transplant candidates, from the time of initial evaluation to respectively). Eleven of 36 patients at risk experienced transplant, to evaluate factors that predict poor autologous chronic GVHD, not influenced, however, by lymphocyte PBPC collection. subsets. The patients'median age was 42 (range 1-68).76 of the 130 Conclusions: a high cell dose of CD19+ lymphocyte increases patients were diagnosed of non-Hodgkin’s disease, 33 had the incidence of acute GVHD and TRM and is associated with Hodking’s disease and the remaining 21 had Multiple a reduced OS and DFS in patients undergoing PBSCT. This Myeloma. Between inclusion criterios were patients which data could confirm the role played by B cells in pathogenesis were mobilized with G-CSF a 10-20 mg/kg. Our evaluation of GVHD and could suggest new therapeutic options in showed platelet count prior to growth factor administration prevention and treatment of GVHD. significantly correlated with total CD34+ cell yield (spearman: r = 0.281,p< 0.05). In adition,daily platelet count during PBSC harvest correlated with CD34+ cell yield for the day (spearman: r = 0.267,p>0.05 in the first harvest, spearman: r = 0.636, p< 0.05 in the second harvest, spearman: r = 0.671, p< 0.05 in the third harvest , spearman: r = -1.000, p> 0.05 in the four harvest ). Using multiple linear regression model P616 (F=19.1,p <0.001, R2 0.64 ), we have determined type of Use of totally implantable catheter for peripheral blood disease (p=0.037) and platelet count (p=0.031), significantly stem cell apheresis correlate with total CD34+ cell yield but not patients´s age (p=0.7). Further analysis showed low baseline platelet count L. Carausu, G. Clapisson, I. Philip, H. Sebban, P. Marec- before growth factor administration prediction poor Berard mobilization. Using Receiver Operating Curve (ROC), we Centre Leon Berard (Lyon, F) have determined that platelet count of 258,000 ul was the best Purpose: The collection of peripheral blood stem cells (PBSC) cut/off collecting at least 2 million Kg CD34+ cells with by leukapheresis requires two venous accesses (VA), one for sensitivity of 54 % and specificity of 100% . Baseline platelet the inflow and one for the outflow. The use of a totally count is a sensitive indicator of autologus PBPC mobilization implantable venous access device (TIVAD) has never been and may be considereted prior to growth factor administration reported in this setting. to determinate optimal period to mobilization patients for Patients and methods: We retrospectively analyzed the use of autologous PBPC collection. TIVAD (systematically inserted at the time of diagnosis) for performing leukapheresis by means of a continuous flow- blood cell separator (Spectra-Gambro BCT, France). The P615 objective was to obtain 2x106 CD34+/kg per graft. The Influence of B-cell dose in granulocyte colony-stimulating second VA was either peripheral when possible or a factor-mobilized peripheral blood mononuclear cell grafts temporary central VA inserted before the first apheresis. The on outcome of HLA-identical sibling allogeneic peripheral study was conducted between 01-2000 and 06-2005 on 64 blood stem cell transplants patients (including 41 children) who required intensification for L. Cupelli (1), T. Dentamaro (1), A. Tendas (1), A. Bruno (1), I. the treatment of a solid tumor. de Meis (2), A. Picardi (1), L. Cudillo (1), M. Mirabile (1), P. de Results: Mean body weight was 26kg (range 8-91kg) for a Fabritiis (1) median age of 8.5 years (range 0.7-66years). 115 (1)Tor Vergata University (Rome, I); (2)S. Eugenio Hospital leukapheresis were performed with a mean of 1.79 (Rome, I) apheresis/patient. The second VA was cubital vein in 85 procedures and a temporary central VA in 30. The mean Background: more than 60% of patients with haematological duration of each apheresis was 3 hours (range 30-274mn). malignancies are submitted to allogeneic stem cell transplant The mean flow rate was 41.3ml/min (range 12-85ml/mn). The utilizing granulocyte colony-stimulating factor-mobilized mean collection rate was 59.2% for CD34+cells and 70% for peripheral blood stem cells from HLA identical sibling. The mononuclear cells. The total number of CD34+cells collected influence of the lymphocyte subsets in the apheretic product was 2.5x106/kg per apheresis (range 0.3-20x106/kg), and 5.9 on the outcome of transplant is not clear. x106/kg per patient (range 2.4–33x106/kg). Several Aim: the aim of this study was to analyze the influence of the complications occurred during hospitalization for lymphocyte subsets and the CD34+ cell dose on the outcome leukapheresis: 1 catheter-related sepsis (0.86%), 4 catheter of peripheral blood stem cell transplantation (PBSCT). occlusions (3.47%) and 8 cases of hemodynamic instability Methods: thirty-nine patients receiving PBSCT from HLA related to extracorporeal volume. Weight <10Kg is a risk identical sibling for haematological malignancies (16 acute factor of complication (p=0.0006). Only 2/115 procedures myeloid leukaemias, 3 acute lymphoblastic leukaemias, 7 were interrupted because of complication (1 obstruction of multiple myelomas, 6 lymphomas, 5 myelodisplastic peripheral catheter and 1 catheter-related sepsis). syndromes, 1 aplastic anemia and 1 chronic myeloproliferative Conclusion: TIVAD are effective and safe for PBSC collection disease) were analyzed. and is consistent qualitatively and quantitatively with published T cells (CD3+, CD4+ and CD8+), B cells (CD19+) NK cells reports. Placement of a second central VA (requiring general (CD56+/16+) and CD34+ cells were evaluated in the apheretic anaesthesia for children) could be avoided in a majority of product by immunophenotypic analysis using appropriate patients.

S131 P617 required platelet transfusions for severe thrombocytopenia; Comparison between TNC and CD34+ cell counts none required red cells. There were no treatment-related reported at the time of pre-cryopreservation of unrelated deaths. All patients achieved a peripheral blood CD34 count > cord blood units and those observed at the time of their 5 cells/uL and underwent apheresis. The median CD34- transplantation positive stem cell harvest was 4.4 x106/kg (range 1.92 – 7.16), V. Antonenas, K. Bradstock, P.J. Shaw, D.J. Gottlieb achieved over a median of 2 harvest days (range 1 – 2). Westmead Hospital (Sydney, AUS) Conclusion: Cytarabine-based stem cell mobilisation schedule is highly effective in patients who have previously failed Background: Engraftment after Unrelated Cord Blood (UCB) cyclophosphamide mobilisation, but may lead to hospital transplantation is highly dependent on total nucleated cell admission for febrile neutropenia or bleeding. Patients (TNC) count and CD34+ cell content. Typically TNC and receiving cytarabine-based stem cell mobilisation should be CD34+ counts provided to transplant centres are determined monitored closely and facilities for rapid admission and before freezing of CB units. No data are available to treatment should be available. determine whether thawed CB units actually contain the TNC and CD34+ cell numbers determined by collection centres. We compared cell numbers after CB units were thawed P619 immediately before infusion with those provided by CB Banks. Pharmacokinetics of AMD3100 in patients with Methods: 39 CB units were received at our laboratory for haematologic malignancies transplantation. All except 3 CB units were thawed without R. McFarland (1), D. Stewart (2), A. Cashen (3), J. DiPersio washing prior to infusion. Units had been stored in liquid (3), K. Badel (1), G. Calandra (1) nitrogen for a mean duration of 3.5 years (range 1-7 yrs) prior (1)AnorMed (Langley, CAN); (2)University of Calgary to infusion. TNC and CD34+ counts were determined (Calgary, CAN); (3)Washington University School of Medicine immediately post-thaw. (St. Louis, USA) Results: The mean recovery for TNC count for the 39 units analysed was 78% (range 54-108%). 14/39 CB units had TNC Background: AMD3100, a selective antagonist of CXCR4, recoveries < 70%. 11/39 CB units had <2x107 TNC/Kg mobilizes long-term repopulating HSCs with adequate homing according to post-thaw counts. Eight received a second cord and engraftment capacity. The aim of this study was to unit but 3 received a single unit (cell doses 1.5, 1.48 and 1.6 generate pharmacokinetic results for AMD3100 after a 240 x107 TNC/Kg respectively). The mean recovery for viable µg/kg subcutaneous injection (s.c.) in Multiple Myeloma (MM), CD34+ cell count was 67% (range 10-160%). 19/31 CB units Non-Hodgkin Lymphoma (NHL), or Hodgkin's Disease had viable CD34 recoveries < 70%. 29/39 CB units contained (HD)patients that had been pretreated with G-CSF. <2x105 viable CD34/Kg according to post-thaw counts. Methods: PK parameters were determined from samples Conclusions: There is a consistent reduction in TNC and obtained immediately prior to dosing, 0.25, 0.5, 1, 2, 4, 6 and CD34+ cell numbers observed by transplant centres post- 8 hours after the injection of the first dose of AMD3100, and thaw when compared with those reported before freezing. just prior to apheresis. Samples were available from 4 MM pts, This may result in infusion of cell numbers considered 3 NHL pts, and 5 HD pts. inadequate for successful CB transplantation. Correlation Results: Following s.c. administration at 240 µg/kg median between post-thaw TNC and CD34+ cell counts and maximum plasma concentrations of 1020 ng ml-1, 821 ng ml- transplant outcomes is required. Pending this, we recommend 1, and 872 ng ml-1 were observed at 0.15-1 h for MM, NHL, that all CB Banks provide TNC and CD34+ cell numbers on and HD patients respectively. Furthermore, MM, NHL, and HD thawed pilot vials to provide some guide to transplant patients had median elimination half lives of 4.68 h, 4.46 h, physicians at the time of unit selection. and 3.69 h and median area under the concentration-time curves of 3973 hr*ng ml-1, 2781 h*ng ml-1, and 3305 h*ng ml- 1 respectively. P618 Conclusions: The pharmacokinetics of AMD3100 in cancer Successful cytarabine stem cell mobilisation after failed patients pretreated with G-CSF was similar to results obtained cyclophosphamide mobilisation in healthy volunteers given AMD3100 alone (see figure). R. Weinkove, O. Keane, B.A. Abdulkareem, M.A. Kazmi Guy's and St Thomas' NHS Foundation Trust (London, UK)

Objectives: Failure of autologous peripheral blood stem cell mobilisation is associated with prior chemo- and radiotherapy. We retrospectively reviewed the outcomes of a cytarabine- based stem cell mobilisation schedule in heavily pre-treated patients who had previously failed cyclosphophamide-based mobilisation. Methods: Five patients who had previously failed peripheral blood stem cell mobilisation with intravenous cyclophosphamide 1.5 mg/m² (single dose) and lenograstim 5mcg/kg/day received cytarabine-based stem cell mobilisation. Patients received six doses of intravenous cytarabine 800 mg/m² 12-hourly (total dose 4.8 g/m² cytarabine); lenograstim 5 mcg/kg/day was commenced the day after chemotherapy (day 4). Peripheral blood CD34 count was estimated daily from day 14, and peripheral blood stem cell harvest performed if the CD34 count was > 5 cells/uL. Results: Five patients (4 with myeloma, 1 with P620 lymphoplasmacytic lymphoma) received cytarabine-based Clinical scale expansion of human cord blood-derived stem cell mobilisation. The median age was 56 years (range multipotent stem cells in a new FBS-free system 45 – 61). The patients had received a median of three (range A. Reinisch, C. Malischnik, E. Rohde, K. Schallmoser, V. 3 – 5) prior lines of chemotherapy, which included prior Bjelic-Radisic, G. Lanzer, W. Linkesch, D. Strunk melphalan autologous stem cell transplant in four out of five Medical University Graz (Graz, A) patients. The patients had failed a median of one (range 1 – 2) prior cyclophosphamide-based stem cell mobilisations. Two Umbilical cord blood (UCB) is an easily accessible source for patients required hospital admission for febrile neutropenia multipotent mesenchymal stromal cells (MSC) and is generally (one patient) or bleeding (one patient) following cytarabine- believed to provide MSC with a higher proliferative potential based stem cell mobilisation. Three out of five patients

S132 compared to adult bone marrow (BM). Limitations in cell platelet engraftment received platelet transfusions more often number and strict dependence of expansion procedures from than those with <21 days for platelet engraftment (median 9 v selected lots of fetal bovine serum (FBS) obviously hampered 2 transfusions, p <0.001). Statistical analysis was performed the progress of clinical applications with UCB-derived MSC. using two-tailed, non parametric Mann Whitney U-test. We analyzed isolation and proliferative potential of human The absolute number of CD34+/CD110+ cells /kg infused at UCB-MSC as compared to BM-MSC under optimized ex vivo time of transplantation appears to be an important factor culture conditions. We further investigated the impact of identifying patients at risk of delayed (>21 days) platelet human platelet lysate (HPL) as an alternative to replace fetal engraftment. Those with <6 x 104 are at particularly high risk bovine serum for clinical scale expansion of both types of of delayed (>21 days) platelet engraftment requiring multiple MSC. Progenitor cell function was determined by CFU-F transfusion after transplantation. assays and correlated to acquisition of cellular senescence over time. MSC functions were studied combined with multiplex cytokine display and immunophenotyping by flow P622 cytometry. Cryopreservation of allogeneic peripheral blood stem MSC could be reproducibly propagated from UCB with and cells does not influence the recovery of lymphocyte without FBS. MSC propagation was effective in 46% of UCB counts after allogeneic transplantation samples compared to 100% of BM samples. A UCB sample D. Kim, N. Jamal, R. Saragosa, J. Lipton, M. Minden, V. volume > 40mL correlated with a higher probability of Gupta, J. Kuruvilla, H. Messner successful outgrowth. Once established, the proliferation Margaret Hospital (Toronto, CAN) kinetics of UCB-MSC did not differ significantly from that of BM-MSC under optimized culture conditions resulting in a Over the last 3 years, the allogeneic BMT program at PMH mean maximum of 40 population doublings after only 10 conducted a prospective study using cryopreserved peripheral weeks. A clinical quantity of more than 1 billion MSC could be blood stem cells (PBSCs). A preliminary report (Kim et al, obtained from BM-MSC within shorter time compared to UCB- IBMTR 2007) showed an equivalent transplant outcome MSC due to significant differences in primary cell amounts. compared to fresh PBSCT in terms of overall survival, Vascular regenerative function in matrigel assays in vitro was mortality and graft-versus-host disease (GVHD). In the current more efficient in BM-MSC. Functional integrity of UCB-MSC study, we analyzed the impact of cryopreservation on the was further confirmed by testing their hematopoietic support recovery of absolute lymphocyte counts (ALCs) after and immune inhibitory properties. allogeneic PBSCT. We demonstrate for the first time that human MSC can be A total of 89 patients received cryopreserved PBSCT between obtained and propagated to a clinical quantity from UCB in a Sep 2003~Dec 2005. 106 patients transplanted with fresh completely FBS-free system although with lower efficiency PBSCT between Jun 2001~Aug 2003 served as historical compared to BM. Surprisingly, our data argue against a control. A gradual rise in absolute lymphocyte counts (ALC) generally superior proliferative potential of UCB-MSC. was observed after allogeneic PBSCT for both groups. The Additional functional differences indicate the need for further absolute numbers varied considerably between individual studies to select the appropriate cell source for defined patients. The overall pattern of lymphocyte recovery was therapeutic approaches. indistinguishable (p=0.926) for both groups. A differnce in overall survival (p= 0.921), non relapse mortality (p=0.835) and recurrence rate (p= 0.342) was not observed. However a P621 difference was seen when the outcome of patients with Absolute number of transplanted CD34+ cells expressing differing ALCs was examined separately. Cryopreserved and C-mpl (CD110) correlates with speed of platelet fresh PBSCT resulted in ALCs of >1.0x109/L on day 30 in 42 engraftment following autologous stem cell and 47% of patients (p=0.434).The overall survival of patients transplantation and their survival at distinct time points at 1 and 2 years with M. Sartor, V. Antonenas, F. Garvin, K. Bradstock, D. Gottlieb an ALC at day 30 of > 1.0x109/L was superior to that of Westmead Hospital (Sydney, AUS) patients with an ALC of <1.0x109/L. The observation was made for both cryopreserved (p=0.046) and fresh grafts Recovery of neutrophil numbers after peripheral blood stem (p=0.014). Based on the eqiuvalent outcome the data of both cell transplantation (PBSCT) is closely associated with graft patient groups (n=195) were pooled to facilitate further CD34+ cell dose. Predicting the speed of platelet recovery is subgroup analyses. The group with high ALC over 1.0x109/L more difficult but would be of value given that a significant showed 77% and 71% of 1- and 2-year(s) overall survival minority of patients experience delayed platelet recovery and rates, while those with low ALC less than 1.0x109/L at day 30 bleeding complications after transplantation. In this study we showed 58% and 48% of survival, respectively (p=0.004). The retrospectively analysed the graft composition of 41 patients 1-year non-relapse mortality was significantly different who underwent autologous transplantation, using peripheral favoring a high ALC group (14±4% vs 26±5%, p=0.011). The blood stem cells to assess the utility of CD110 (c-mpl) high ALC group seemed to have a trend towards a lower expression on CD34+ cells as a predictor of platelet incidence of recurrence compared to the group with a low ALC engraftment (ie. time to platelet count greater than 20 x 109/L recovery (9% vs22%) (p=0.078). for seven consecutive days without the need for platelet In conclusion, ALCs at day 30 can predict transplant transfusion). outcomes including overall survival and non-relapse mortality Absolute CD34+ cells and CD34 subsets expressing CD110 after allogeneic PBSCT. Cryopreservation does not seem to were enumerated using an in-house single platform viable influence lymphocyte recovery and transplant outcomes as CD34 flow cytometry assay (BMT, 2005). measured by overall survival, non-relapse mortality and Of the 41 patients 9 required at least 21 days for platelet relapse after allogeneic PBSCT. engraftment. These patients received a median graft dose of 5.5 x 104 CD34+CD110+ cells/kg compared with a median dose of 16.0 x 104 cells/kg received by patients who experienced platelet engraftment within 21 days of transplant (p=0.002). In contrast, there was no difference in the number of CD34+ cells/kg infused (4.6 v 3.0 x 106/kg for < versus >21 days to platelet engraftment respectively, p=0.07). There was a weak correlation between the absolute number of CD34+ cells and the number of CD34+CD110+ cells in the graft (r² = 0.48). Similarly there was no correlation between the percentage of CD34+ cells expressing c-mpl and the speed of platelet engraftment (8.1 v 5.8%) for > or < 21 days for platelet engraftment respectively, p=0.39). Patients with >21 days

S133 P624 Is there any benefit of secondary approaches for stem cell (auto-PBSC) collection in poor mobilising myeloma and lymphoma patients? O. Ilhan, P. Topcuoglu, E. Ayyildiz, M. Ozcan, E.A. Soydan, M. Ozcan, M. Arat, O. Arslan School of Medicine - Ankara University (Ankara, TR)

For successful engraftment an auto-PBSC graft should P623 contain adequate number of CD34+ cells of at least 2x106/kg. Transfusion requirements and costs during the acute Some factors related to patients such as advanced age, transplant episode for patients undergoing an autologous underlying disease, heavy pretreatment, radiotherapy prior to or allogeneic haematopoietic stem cell transplant mobilization etc. may play important role at poor mobilization B. Thomas, M. Lasebai, K. Hill, C. Hurlock, J. Newman, N. and collection. We aimed to retrospectively evaluate the McKeag, V. Kitchker, S. Churchill, D. Richardson, K. Orchard efficiency of subsequent attempts to mobilize and collect stem Southampton University Hospital (Southampton, UK) cells in poor mobilizing lymphoma and myeloma patients. Patients: Between 1999 and 2006 in 285 adult patients with Introduction: As part of the quality management programme of myeloma (n=99) or lymphoma (n=186) auto-PBSC the Wessex Blood and Marrow Transplantation service and in mobilization was done in our center. However, adequate stem response to national health strategies to minimise blood cell dose was not reached in 30 patients (10.5 %, F/M: 14/16). product usage, we reviewed cellular transfusion Median age was 44 years (range, 16-66). Their diagnoses requirements during the transplant episode for patients were 18 lymphoma (NHL:11; HL:7) and 12 myeloma. Data undergoing autologous and allogeneic Haematopoietic Stem were retrospectively collected from Apheresis Unit’s database. Cell Transplant (HSCT). Results: In 23 out of 30 patients a second mobilization Objective: To report the cellular blood product transfusion regimen was used for auto-PBSC collection. We evaluated requirements and costs for patients during the acute these poor mobilizing patients in two groups according the admission for autologous and allogeneic HSCT over a twelve success after subsequent mobilizing (Table-1). Group I: In 16 month period from 2005-06. patients (53.3 %) adequate CD34+ cell dose was reached Design: Retrospective observational study after the use of a second mobilization regimen Group II: In 7 Setting: Regional HSCT centre patients (23.3%) we failed to collect the minimum number of Patients: Autologous and Allogeneic HSCT recipients during CD34+ cells. We also analyzed the patients in the group I into their acute transplant episode. All autologous and 18/20 three different subgroups as follows: Group Ia (n=8): First allogeneic transplants were performed with peripheral blood mobilization regimen was unsuccessful for the collection, but stem cells. 2/20 allogeneic transplants were performed with adequate CD34+ cells was obtained after a second bone marrow alone. mobilization regimen; Group Ib (n=5): First mobilization Method: Review of clinical and laboratory data of the patients regimen has reached the target CD34+ cell dose, but a admitted for HSCT in the financial year April 05 – March 06 second mobilization regimen was used due to additional (inclusive). Use of plasma components was not assessed, as requirement of higher CD34+ cells, but adequate CD34+ cell these products were rarely used during the acute transplant was not obtained. In this group a second mobilization regimen phase. The cellular blood product requirements from the time was tried in 3 patients after high dose therapy. Group Ic (n=3): of admission up to time of discharge were recorded. The target CD34+ cell dose was achieved in total after Results: 2/40 autologous transplant patients received no blood subsequent attempts of mobilizations. products.15/40 autologous recipients and 2/8 allogeneic Conclusion: In over 90 % of our myeloma and lymphoma recipients received no red blood cells (RBC). The typical blood patient cohort our hemapheresis center was able to collect product cost (based on median number of products received) adequate CD34+ cells after first mobilization attempt. was autologous - €1450, RIC allogeneic - €3583 and full Secondary mobilization attempts could reach the required intensity allogeneic - €6944. target number of CD34+ cells in only about half of the patients Conclusions: Results show a clear difference in the median failing first mobilization. In the light of existing data an blood product requirements dependent on the type of experienced hemapheresis center is able to collect sufficient transplant. There was no difference in blood product autologous stem cells in the majority of the patients including requirements between BEAM and high dose Melphalan poor mobilizers. conditioning for the autologous recipients. The increased RBC requirements in 4/20 allograft recipients could be predicted from major ABO incompatibility or bone marrow as source of stem cells. The wide range of variability in platelet requirements in 5/20 of the allograft recipients was not predictable and depended on individual patient characteristics including cerebral haemorrhage, veno-occlusive disease and therapeutic anticoagulation. These results show that blood product costs are only a small proportion (approx. 5%) of the total cost of stem cell transplantation. The results of this study will be used as a standard of care for our future blood product usage to ensure this remains consistent.

S134 Stem cell donor

P626 Estimation of duration and success of unrelated stem cell donor searches, based on HLA-DRB1 allele and DRB1- DQB1 haplotype frequencies K. Hirv, K. Bloch, M. Fischer, H. Schrezenmeier, J. Mytilineos University of Ulm, DRK Ba-Wü/Hessen (Ulm, D)

In our facility more than 600 unrelated blood stem cell donor searches (UDS) are performed annually. The average searching time is approximately 70 days. However, this strongly varies depending on the patient’s HLA phenotype frequency. We have developed an algorithm, based on the HLA-DRB1 allele frequencies as well as on the DRB1-DQB1 linkage disequilibrium, which allows an estimation of the search complexity and determines the number of donors to be requested for confirmatory typing (CT). We hypothesized that P625 our algorithm would correlate with the UDS duration as well as Chimerism after multi-donor SCT: principles and with the number of HLA-identical donors found. paradigms for a quantitative analytic approach HLA-DRB1 allele frequencies were calculated based on a D. Kristt (1), B. Gesundheit (2), J. Stein (3), M.Y. Shapira (2), dataset of 65752 individuals published by the National Marrow R. Or (2), A. Amar (2), I. Yaniv (3), T. Klein (1) Donor Program. HLA-DRB1-DQB1 haplotype frequencies (1)Rabin MC (Petach Tikvah, IL); (2)Hadassah MC were retrieved from different literature sources. Information on (Jerusalem, IL); (3)Schneider's Children's MC (Petach Tikvah, HLA-DRB1 allele frequencies within two-digit defined allele IL) groups are of importance, since in most cases only a low resolution typing of donors is available at the time of UDS Multi-donor (MD) hematopoietic stem cell transplantation initiation. HLA-DRB1 alleles with a frequency of >80%, 20- (SCT) is increasingly used to expedite engraftment and 79% and <20% within their allele group were considered as improve outcome of SCT. Although chimerism (Chm) testing very frequent, frequent and rare, respectively. In addition, an is essential for following engraftment status, MD association between a particular HLA-DRB1 allele and a transplantation (MDT) may result in complete allelic sharing certain DQB1 allele of >80%, 20-79% and <20% was that precludes using standard methods for quantitative considered as very frequent, frequent and rare, respectively. monitoring. Consequently, the goals of this project were to: (1) The rarest allele or haplotype assignment defined the analyze theoretical MD Chm models for the relationship of probability of finding a fully compatible donor. For the patients alleles to their components; (2) formulate principles for with high, medium or low probability, 2-3, 4 or 5-6 potentially quantitative MD Chm testing, using STR-PCR, based on the matched unrelated donors were requested for CT at the theoretical analysis; (3) develop a practical approach for initiation of the UDS, respectively. routine monitoring of MD Chm. The project’s results are In a group of 128 patients, the UDS duration and the average represented by the following paradigm: number of identified donors was calculated. The average UDS STAGE 1. Identifying the chimeric components: When using duration was 29, 52 and 146 days in the groups with high, the typical three or four peaked chimeric locus as a model, the medium and low probability, respectively. In average 2,8 relative “quantity” of DNA in each of the alleles allows donors were found in the group with high, 1,8 donors in the distinguishing the following chimeric configurations: D1 (Donor group with medium and 0,8 donors in the group with low #1), D1+D2, D2, R (Recipient), R+D1, R+D2. We posit that probability. An HLA-DRB1-DQB1-identical donor was found the quantity of DNA in a shared allele will be a multiple of a for 100%, 81% and 42% of patients with high, medium or low non-shared allele. This permits numerically identifying the probability, respectively. “absence” of an expected signal representing D1, D2 and/or Established algorithm shows a significant correlation between R. Therefore, Stage 1 data also are a basis to establish 0% or UDS duration and search success rate and can be used for 100% Chm. the prediction of these search characteristics. A more precise STAGE 2. Defining a MD-informative locus: We found that the differentiation of the number of donors, to be requested for following criteria are required to utilize a locus for quantitation: CT, should allow further reduction of expenses in the UDS (1) a configuration whereby D1 and/or D2 have an allele not process without prolongation of the UDS duration. present in R prior to SCT; (2) D1 and D2 may share this allele. STAGE 3. Computing % Mixed Chm: We extended the usual locus D:R ratio statement to MD SCT as follows: %Donor P627 Chm = Sum-DNA MD / Sum-DNA (R+MD); where Second stem cell harvest from unrelated donors for MD=D1+D2. Due to allelic sharing, the DNA measurements patients with relapse or graft failure after first need “validation” that can be achieved using an expression for transplantation: patient age > 50 years and relapse of optimum performance: [R-1+ (D1-1) + (D2-1)] = [R-2 + (D1-2) malignant disease are associated with poor outcome + (D2-2)] (Leukemia 2006; 20, 1169). The algorithm’s 3- U. Platzbecker, S. Zapf, C. Theuser, A. Schmidt, C. Rutt, G. stages are required for “inter-current” MDTs. The stages also Ehninger, M. Bornhäuser are individually useful for analyzing aspects of serial single- University Clinic Carl Gustav Carus (Dresden, D) donor, and in-utero SCTs. In conclusion, this work represents the first algorithmic Objectives: Hematopoietic progenitor cells from unrelated quantitative approach for assessing MD chimerisms. The donors are increasingly used for allogeneic stem cell stages of the paradigm can be used in concert, or transplantation (SCT). A second donation of peripheral blood independently, to accommodate a range of MD situations. stem cells (PBSC) or bone marrow is widely accepted in case Using this algorithm, it becomes possible to routinely track the of graft failure of relapse. progressive changes in Chm status after a MDT. Assessing Methods: We retrospectively investigated the outcome of the latter is the main justification for performing “quantitative” seventy (n=70) patients with hematological malignancies Chm monitoring. (AML/MDS n=29, CML n=19, ALL n=17, other n=5) receiving a second SCT using PBSC (n=67) or bone marrow (n=3) from the same unrelated donors. Patients had a median age of 30 years (range 1–59) and the reason for a second donation of

S135 stem cells consisted of either relapse (n=37) or graft failure P629 (n=33). Whereas in 21 patients no conditioning was used in Donor follow-up after filgrastim mobilization for unrelated the remaining patients it was busulfan or TBI-based in the blood stem cell transplantation majority of cases. C. Guenther (1), U. Hahn (1), B. Englmann (1), V. Spitzer (1), Results: After a median follow-up of 33 months (range 7.9- H. Diem (2), H. Knabe (1) 76.2) after 2nd SCT a total of 55 patients died due to (1)Bayerische Stammzellbank (Gauting, D); (2)Würmtal-Labor treatment-related mortality (63%) or relapse (16%) resulting in (Gauting, D) a median overall survival (OS) for all patients of 3 months. The OS was significantly better in patients < 50 years (38 vs. Filgrastim is not only known to mobilize CD34+ progenitor 8 %, p=0.01) and in case of graft failure (37% vs. 7%, p=0.02). cells but acts also as a pleiotropic immune modulator. We Time interval between SCT, HLA-disparity, disease type started to conduct the systematic donor follow-up in March (AML/MDS vs. CML vs. ALL), infused CD34+ cells/kg or graft 2004 after Filgrastim mob. in healthy donors. The follow-up source at first transplant had no impact. focused on lab testing incl. lymphocyte subsets and the Conclusion: These data show the lack of efficacy of a second reports of adverse events i.e. infectious episodes or other infusion of HSC from the same donor especially in older symptoms. patients and in cases in which relapse of malignancy is the Mat. and meth.: 233 consecutive PBSC donors were tested at reason for second transplant. Given the currently unknown different time points (1, 3, 6 mths) after stem cell collection. long-term effects of a repeated exposure to G-CSF, the Lab. testing included blood count and diff. count, lymphocyte factors mentioned above should be kept in mind whenever a subsets (CD3,4,8,19, 16/56), LDH, Crea, aP, GPT, Uric acid. request for a second HSC donation is considered. All donors received questionnaires. In case of adverse events donors were contacted. Results: Overall 82% of donors responded at any time point. P628 Blood count: after 1 mth 88% of samples were normal; 9 Analysis of RIC allografted patients over 55: young donors had leukopenia (min 2.7 G), 2 thrombopenia and 7 low unrelated donors and older sibling donors have Hb. The diff. count showed neutropenia (6 donors), comparable outcomes eosinophilia or lymphocytosis. Lymph. subsets were abnormal P. Jindra, V. Koza, K. Steinerova, M. Karas in 36%, predominantly affecting NK- and B-cells. Lab. findings University Hospital Pilsen (Pilsen, CZ) were abnormal in 32% incl. elevated uric acid (13 donors), Crea. (14 donors), GPT, GOT and LDH. These changes were Backround: RIC allograft allows the SCT even in the elderly. demonstrable up to six mths in some donors tested or However their siblings are naturally older and the question is appeared de novo after mths. We did not expect long-lasting whether younger age of unrelated donors could compensate effects on leukocyte counts or lymph. subsets and renal for the higher immune incompatibility. The superiority of parameters changes. The significance of these findings is not younger donor age was shown in the setting of conventional clear. The donors reported some infectious episodes following HSCT; however for the RIC the data are limited. stem cell mobilization (n=5: bronchitis, atypical pneumonia, Method and Patients: Retrospective analysis of group of 62 herpes, influenza-like affection), some allergic episodes consecutive patients • 55 years of age (median 60 years, (exanthema, newly aquired allergic reaction to apitoxin) and range 55-68) allografted either with related (SIB group, n=32, autoimmune-like symptoms (n=8) as tendovaginitis, median age 60;55-68) or unrelated, 9-10/10 HLA allele-level fibromyalgia, arthralgia, new onset of thyreoiditis. We could matched donors (MUD group, n=30, median age 60;55-68). not establish a correlation between abnormal lab. testing and Groups were well balanced for disease characteristics. SIB the onset of adverse events due to the lack of samples tested has more CMV matched pairs (90vs 60%, p=0.007), more at that time point. female donors (53 vs. 23%,p=0.02) and longer follow-up (33 Conclusion: We demonstrated that some of the healthy mts; 5-56 vs. 16 mts; 5-40, p=0,03). As expected, the MUD donors show long-lasting alterations of the blood count, received significantly more CD 34+ (6.1 vs. 4.3 106/kg, differential count and lymphocyte subsets and renal p<0.0001) and T-cells (2.8 vs.2.6x108/kg, p<0.001). parameters after Filgrastim mobilization for peripheral blood Results: For all pts (median follow-up of 20 mts, 5-56), the stem cell collection. The significance, if any, remains to be 3year Kaplan-Meier OS and EFS probabilities were 32% and clarified. The reports of adverse events including infectious 29%, whereas overall NRM 39% (24/62) and relapse rate 45% episodes and signs of autoimmune disorder should be (19/48). After splitting to SIB vs. MUD the figures were as analyzed carefully. The data support the need for a systematic follows: number of surviving pts 11/32 (34%) vs. 15/30 (50%) donor follow-up. Data should be exchanged between Donor resulting in 3y OS probabilities for SIB and MUD of 29% and centres and evaluated in order to define possible side-effects 42% (p=0,85) whereas the corresponding figures for EFS in the long-term follow-up. were 22% and 34% (p=0,42). The relapse rates were significantly higher in the SIB - 54% vs. 23%, p=0,009; on the contrary there was a nonsignificant trend for lower 100-day P630 and 1-year NRM in the SIB (19% vs.27%, p=0,55 and 34% Acute severe pulmonary toxicity in a healthy stem cell vs.43%, p=0,61). Rejections (altogether 8/54 of evaluable donor during G-CSF mobilization pts), occurred more frequently in the SIB (20% vs. 8% in C. Guenther (1), U. Hahn (1), A. Fertl (2), K. Häußinger (2), H. MUD), again statistically nonsignificant (p=0,28).The Knabe (1) incidences of gr. II-IV aGVHD and cGVHD were also similar (1)Bayerische Stammzellbank (Gauting, D); (2)Asklepios- (SIBxMUD: 25% vs. 37%,p=0,41 and 50% vs. 59%, p=0,57). Klinik (Gauting, D) Conclusions: Our results clearly indicate that unrelated donors did not adversely affect RIC-SCT outcome in the patients We report severe pulmonary toxicity with hemoptysis, aged • 55. We even observed trend for better outcome for dyspnoe and hypoxemia in a healthy stem cell donor MUD, probably due to the better disease control. The mobilized with Filgrastim. Mob. had to be discontinued and intriguing question whether a younger allele-matched stem cell collection could´nt be performed. Physical unrelated donor is superior to an older HLA-identical sibling examination of the 38 yrs old male donor did not show any donor is thus arising and should be tested in larger patient’s pathological findings with normal functions of heart, liver, cohort. If confirmed, we should not be reluctant with MUD in kidney and lung (incl. chest-x-ray), no allergic asthma nor older pts and the priority of sibling donor would be challenged pulmonary disease but smoker. He received the standard when younger MUD will be available. dose of Filgrastim of 11 mg/kg (split in two doses) for 4 days. On day 3 of mob. the donor developed massive hemoptysis and cough, but no dyspnea or fever. He contacted the DC´s physician on duty. The chest x-ray performed in an emergency ambulance showed diffuse infiltrates in the middle and lower parts of the lung.The next day (day 4 of mob.) the

S136 donor had to be admitted to hospital with continuing hemoptysis, dyspnea and hypoxemia (O2 < 55 mmHg). Filgrastim was discontinued immediately. The donor received oxygen and clarithomycin as an underlying infection could not be ruled out. He gradually improved and could be discharged 4 days later with only very mild hemoptysis and a normalized blood gas status. The pulmonary CT-scan showed diffuse opaque infiltrates with accentuation of the lower parts. The broncho-alveolar lavage demonstrated diffuse hemorrhage. The histological analysis was normal. The donor had no excessive leukocytosis (44 G/l) or other abnormal lab. findings at the onset of lung toxicity. The donor recovered completely as shown in the control examination performed two mths later. P632 Fortunately an alternative fully matched unrelated donor could The advantage of prospective search prognosis in the be identified within 24 hrs in our registry. The bone marrow unrelated donor search was performed one day after the scheduled PBSC collection. A.M. van Walraven (1), M.B.A. Heemskerk (1), J. Lie (2), M. The patient, who was fully conditioned, did receive a stem cell Oudshoorn (2) graft in time and recovered. Conclusion: It is recommended to (1)Leiden University Medical Centre (Leiden, NL); monitor PBSC donors closely with respect to pulmonary (2)Europdonor Foundation (Leiden, NL) symptoms and other complications during Filgrastim mob. as not only pat. with malignant diseases and concomitant Time is a factor of major importance in the unrelated donor chemotherapy may experience ARDS-like toxicity but also search. Our group has shown that for most of our patients the healthy donors. Donors with pulmonary symptoms should be search time span, not the number of donors available, is the admitted to hospital immediately.This report demonstrates the major influencing factor determining the chance of reaching necessity of a 24h on duty physician (donor centre) who is transplantation for the majority of patients. Search facilitating experienced in PBSC mobilization and cytokine side-effects. tools such as the match programs of Bone Marrow Donors This is the first report of hemoptysis during Filgrastim Worldwide and the European Marrow Donors Information mobilization in a healthy donor. Fortunately the donor System make an efficient and fast unrelated donor search recovered but had to be hospitalized for some days. possible. The Europdonor Transplant Centre Coordination Unit has been offering prospective search prognosis (PSP) for patients that are not in direct need of transplantation (e.g. P631 patients that receive treatment such as Gleevec, ATG etc.) but Factors influencing CD34+ cell rate in the final allograft may need a transplantation in future. If the likelihood of finding product after GCSF-mediated mobilization in healthy an acceptable unrelated donor is poor, the transplant centre donors (TC) might consider to start an unrelated donors search (UDS) N. Piard, C. Le Berre, M. Balère, E. Marry, V. Lapierre on instead of awaiting the outcome of initial treatment. The behalf of the French Donor Center: Angers, Besançon, objective of this study was to validate the prognostic value of Bordeaux, Brest, Caen, Lille, Limoges, Lyon, Marseille, PSP. Montpellier, Nantes, Nice, Paris Saint Antoine, Paris Saint A group of 108 patients were enrolled in the PSP study. Five Louis, Paris Hotel Dieu, Paris Pitié Salpétrière, Créteil, patients were excluded due to preliminary death. All patients Poitiers, Rennes, Rouen. were typed for HLA-A, -B, -C, -DRB1 and –DQB1 on high resolution DNA. HLA haplotypes were confirmed where Aim: To analyse factors influencing the total CD34+ cells possible. The likelihood of finding a donor was determined contained in the allograft product after G-CSF-mediated based on the available donors and cord blood units in BMDW mobilization in healthy donor. and the chances that available donors are allele matched with Method: A retrospective analyze was performed on 535 the patient and matched for additional loci such as HLA-C and healthy donors mobilized with 10 mucrog/kg/d in 25 French -DQB1. In order to do this we used published data on centres from 2001 to 2005. The studied factors were: age, sex frequency of alleles in a given population and linkage and donor weight, recipient weight, number of daily G-CSF disequilibrium between the different loci. administrations, day of first aphaeresis, number of aphaeresis, In 53% of all cases, a 10/10 match was predicted; for 96% this circulating CD34+ cell rate before donation and number of prediction was correct. In 29% of all cases, a 9/10 match was treated blood mass. predicted as best donor; this was correct for 54%. However, Results: In multivariate analysis, as expected, the most for 34% a 10/10 match was identified. A poor prognosis was important factor correlated to CD34+ cells contained in the predicted for 18 % of all patients; for these patients a 9/10 final product, is circulating CD34+ cells before the first match was found in 27%, no donor in 33% and 40% resulted donation (p<0.0001). Moreover male donor (p=0.0003), donor in a suitable cord blood unit. These results show that PSP can weight (p=0.005), two daily G-CSF injection (p=0.03), number point out for which patient a 10/10 match is likely to be found, of treated blood mass (p=0.02) were associated with high but that the PSP underestimates the chance of finding CD34+ cells contained in the allograft product. Factors acceptable donors (10/10 or 9/10). influencing the CD34+ cell circulating rate before the first PSP is a practical tool to enhance the efficiency in UDS but donation were two daily injections of GCSF (p=0.0007), donor still needs some adjustments. It can help TC’s decide on age (p=0.04), and first aphaeresis performed at d5 several choices namely: cord blood or stem cell donor, start (p=0.0003). urgently or wait for the results of alternative therapy, and On a pragmatic level, table 1 resume different strategic which planning. procedures. Conclusion: Two daily injection of 5 mucrog/kg of G-CSF and first aphaeresis performed at J5 seems to be the best strategy to obtain the CD34+ cell count required for an allogeneic HSC graft containing at least 4, 6 or 8X106 CD34+ cells. Randomized study must be performed to confirm these findings.

S137 P633 had a myeloablative haemopoietic stem cell transplant Do transplantation outcomes differ according to donor (HSCT) and 220 received non-myeloablative HSCT. The after myeloablative conditioning with daily intravenous breakdown of patient numbers based on disease sub groups busulphan and fludarabine ± TBI with thymoglobulin? An are MDS 39, AML 38, CML 8, CLL 4, Myeloma 7, NHL 17, analysis involving 350 adult patients others 2. Second donations were requested for J. Russell (1), L. Kmet (2), A. Chaudhry (1), A. Balogh (1), L. 115/278(41.3%) patients (91 DLI only, 12 stem cells and DLI, Savoie (1), N. Bahlis (1), J. Storek (1), C. Brown (1), R. Turner 12 stem cells only from same donor, 2 stem cells from (3), L. Larratt (3), D. Quinlan (1), M. Geddes (1), D. Stewart different donor for a repeat allogeneic transplant,). 63/115 (1) patients had sibling allografts and 52/115 patients received a (1)Tom Baker Cancer Centre (Calgary, CAN); (2)Calgary VUD HSCT with 8/115(7%) patients receiving myeloablative Health Region (Calgary, CAN); (3)Cross Cancer Institute and 107/115(93%) non-myeloablative (Edmonton, CAN) conditioning.109/115(94.7%) patients had T- depleted allografts. Of the 24 patients who were retransplanted a Although results of hematopoietic stem cell transplants (SCT) different donor was used in 6 patients including 2 patients who for hematologic malignancy from donors other than had three allogeneic transplants. 24 patients had a second genotypically identical siblings (MRD) may be improving, stem cell donation within 8 months (1.5 – 48) median time historically they have been worse than those from MRD period post transplant. 91 patients required 368 DLI infusions particularly in low-risk (LR) disease. We have investigated over a period of 5 years with median time to first infusion whether this remains the case when all patients (pts) receive being 7(3-52) months. 2 second donation requests were fludarabine (Flu)/busulphan (Bu) based conditioning declined and one was delayed due to donor (FLUBUP) with Thymoglobulin (TG) included in the graft- unavailability.107/220(48.6%)of patients receiving non- versus-host disease (GVHD) prophylaxis. We treated 350 pts myeloablative allografts required second donation compared between 05/99 and 05/05 with Flu 50mg/m² x 5, intravenous to 8/58(13.7%) myeloablative HSCT. Our data demonstrates Bu (Busulfex, PDL Pharma) 3.2 mg/kg daily x 4 ± total body that upto half the patients receiving a non myeloablative irradiation (TBI) 200cGy x 2. Prophylaxis for GVHD was HSCT would be likely to need a second donation.In donor cyclosporine A, methotrexate and TG (Genzyme) 4.5 mg/kg counselling sessions, practicality of second donation is total dose. Patients were divided into four groups depending discussed briefly, but we do not provide donors with data such on donor – 205 MRD, 22 genotypically mismatched family as time to second donation and its frequency. We realise,due members (MMRD), 83 unrelated donors (UD) matched for to heterogeneity in conditioning protocols,nature of disease HLA-A, -B, C, DR & DQ (MUD) and 40 UD mismatched by 1-3 and status at time of transplant it is difficult to inform donors alleles/antigens (MMUD). The proportion of LR pts (acute about the prospect of second donation. We propose that bone leukemia CR1/2, CML CP1) was 42%, 55%, 58% and 33% in marrow registries should prospectively collect data on second the 4 groups respectively. Median age was 44 years (range donation requests made by individual transplant centres and 18-63). Five-year survival (OS) estimates for LR, high-risk and reliably inform future practice. combined groups were MRD 80%, 47%, 60%, MMRD 74%, 40%, 58%, MUD 75%, 39%, 57%, MMUD 53%, 37%, 38% rspectively. By Cox regression analysis MRD, MMRD and P635 MUD SCT had similar outcomes and were combined for a Collection of cord blood from siblings of children affected more robust analysis. The hazard ratio for OS for MMUD vs all with constitutional or acquired disorders: the Marseilles others was 2.03 (95% CI 1.31-3.16, p = 0.002). This was single-centre experience largely accounted for by a difference in OS of LR pts at 53% C. Chabannon (1), C. Lemarie (1), B. Calmels (1), C. for MMUD vs 77% for the other 3 groups (p = 0.03). After Malenfant (1), I. Malenfant (2), H. Chambost (2), D. Reviron adjusting for gender, risk group, patient age (continuous), (3), G. Michel (2) donor age (continuous), TBI, stem cell source, female donor (1)Institut Paoli-Calmettes (Marseilles, F); (2)Hôpitaux de to male recipient, CMV status and CD34+ cell dose Marseille (Marseilles, F); (3)Etablissement Francais du Sang (continuous) the hazard ratio was 1.69 (1.04-2.74) (p = 0.03). (Marseilles, F) The cumulative incidence of relapse mortality was similar across all 4 groups, the difference in outcome was mostly due We retrospectively reviewed ten years of a single-centre to transplant-related mortality (TRM). The cumulative activity, and 50 consecutive cord blood (CB) collections incidence of TRM at 3 years was 31.6% (17.5%-46.7%) for obtained at birth of a sibling from an affected child treated for MMUD vs 14.5% (10.8%-18.8%) for all others. We conclude a constitutional disorder or a malignant blood disease. CB that the FLUBUP protocol ± TBI with TG gives comparable OS collection was decided because the affected child was a for recipients of SCT from all donors apart from MMUD. As potential candidate for an allogeneic haematopoietic cell with other myeloablative protocols differences between donors transplantation and had a mother who was pregnant again; are more apparent in LR pts. Patients with LR disease should the decision was made following discussion between the be advised of the increased risk of SCT when the latter donors medical centre (Service d’Hématologie Pédiatrique, AP-HM) are the only ones available. and families. CB collection was organized by the cell therapy laboratory at the regional cancer research centre (Institut Paoli-Calmettes, IPC). The obstetrical team was contacted, P634 and the procedure to collect CB was explained in detail prior Impact of non-myeloablative conditioning on the to sending all necessary disposables. The mother was asked frequency of second donation: what should donors be to provide an informed consent regarding a directed donation. told? The 50 CB were collected at 17 different sites, none of them K. Ramasamy, M. Kenyon, Z. Lim, A. Ho, S. Devereux, A. participating in the constitution of an unrelated CB bank. The Pagliuca, G.J. Mufti average volume upon reception was 95 ± 37 mL, with 87.5 ± Kings College Hospital (London, UK) 47 x107 CD45+ cells and 3.2 ± 5.1 x106 CD34+ cells; thus 30/50 CB met the minimal volume (80mL) required to qualify Second donation in the form of bone marrow (BM), peripheral unrelated CB collection by the French Cord Blood Bank blood stem cell (PBSC) and donor lymphocytes (DLI) are Network (Réseau Français du Sang Placentaire, RFSP, requested either for improving engraftment or a repeat FranceCord) and 22/50 met the minimal progenitor cell allogeneic transplant. We performed retrospective analysis on content (>2x106 CD34+ cells). In many cases, part or all of second donations requested for allografts performed between the virological testing necessary to secure safety was missing, January 2001- November 2005 who have either received a as well as the demonstration of geno-identity or lack of geno- sibling or an unrelated donor transplant. Censor date for data identity between the affected patient and the neonate. Finally collection was 1-year post last analysed allograft. We have – since February 1997 – two affected children in this cohort performed 278 allografts in the 5-year period {113 sibling were transplanted, one with the CB collected at birth from his donors, 165 voluntary unrelated donors (VUD)}. 58 patients geno-identical sibling, the second one with a bone marrow

S138 collection from a sibling whose CB was cryopreserved. We P637 conclude that related CB collection can be organized, in case Follow-up of 49 MUD peripheral stem cell donors: no that the mother of an affected child is pregnant again, even haematologic malignancy, but 4 cases of arthralgia, 6 when the birth clinic is not routinely engaged in CB collection. cases of neurovegetative disturbances and 1 case of The use of related CB for allogeneic transplantation of siblings long-lasting neutropenia were observed after PBSC- affected with constitutional or acquired disorders will benefit donation from a better definition of national and international rules for B. Pelzmann (1), L. Kajkut (2), G. Leitner (2), N. Worel (2), H. validation, determination of HLA-compatibility and clinical Tüchler (1), P. Höcker (2), A. Rosenmayr (1,2) decisions. (1)Austrian Bone Marrow Donor Registry (Vienna, A); (2)University Hospital Vienna (Vienna, A)

P636 Between September 2000 and March 2006, 49 MUD Incidence of allo-reactive NK cells after allogeneic (matched unrelated) donors donated PBSC in our harvest haematopoietic stem cell transplantation centre. We now overlook the follow up of 49 donors between J. Hasenkamp (1), I. Missal (1), A. Borgerding (1), C.S. Falk 0,5 and 6 years after harvest. (2), T. Wommelsdorf (1), S. Dingeldein (1), B. Chapuy (1), G. After application of rhG-CSF, nucleated cell counts rose from Wulf (1), W. Jung (1), L. Trümper (1), B. Glass (1) 5,4 x 10 3 / ul (median value) to 46.1 (median value) before (1)Georg-August-University (Goettingen, D); (2)NCT - harvest. Nucleated cells returned to 4.5 (median value) one National Centre for Tumour Diseases (Heidelberg, D) year after donation. No particularities of blood counts were noted in the donors after one year. Under G-CSF, 39/49 Allo-reactivity of natural killer (NK) cells has been observed (79%) reported bone pain, 6/49 (12%) persons reported fever after haplo-mismatch hematopoietic stem cell transplantation and/ or flew like symptoms, 5/49 (10%) individuals had (HSCT) with killer cell immunoglobuline-like receptor (KIR)- fatigue, 4/49 (8%) had a head ache. During apheresis, the ligand mismatch in Graft-versus-Host (GvH) direction. The most prominent side effect was citrate toxicity, (15/49, 30%), occurrence of allo-reactive NK cells is correlated with a 16/49 donors (32%) had fatigue and 5/49 (10%) persons favorable influence on relapse rates, engraftment and reported to have nausea. incidence of GvH disease. We study prospectively the PBSC-donors underwent a health check 1, 7, 14 days and 1, incidence and impact of allo-reactive NK cells after HLA A, B, 3, 6 months after harvest and then annually. In the framework DR and DQ-matched HSCT on a functional level. Patients of these checks, donors were interviewed about their health (pts) receiving an allogeneic HSCT are consecutively situation. This was used as the basis for detailed donor follow registered and monitored repeatedly post HSCT. From 4/04 to up. After PBSC donation, 4 donors reported to have joint pain 8/06 107 pts entered the study. History, chimerism (PCR), and swelling of at least one joint. In addition, 6 donors immune-reconstitution (FACS) and frequencies of functional reported to have had neurovegetative disturbances after active and allo-reactive NK cells (Granzyme B ELISpot) are donation. In 2 cases, this was accompanied by a neurological analyzed in 22 pts so far. Pts suffered from AML (9), CML (3), problem (1 tinnitus, 1 paraesthesia of the hand) and in one ALL (2), NHL (6), MM (13q-) (1) and OMF (1). 15 pts received case with inflammation in two joints. Furthermore, a second myeloablative (8 x TBI/Cy, 7 x FBC12) and 7 pts received case of tinnitus was noted. Beyond that, 6 donors reported to reduced conditioning regimen FB8. GvHD-prophylaxis have had severe infections (1 tonsillitis, 2 pneumonias, 1 consisted of CsA or tacrolimus and short course MMF without candida-infection, 2 cases of influenza). One donor had the T cell depletion in 21/22 pts. In 5/22 pts we detected allo- excision of a polyp of the jejunum. One donor had a long reactive NK cells at d +128 (case #19), +56 (#8), +355 (#1), lasting phase of neutropenia of 2 years following PBSC +364 (#22) and +379 (#13). 5/22 pts (#1, 13, 19, 8, 22) were donation but has finally returned to normal. transplanted with KIR-ligand mismatch in GvH direction. In In conclusion, we now overlook the follow up of 49 PBSC two of these pts we detected allo-reactive NK cells at d +355 donations with more than 6o donor years after harvest. We (#1) and +379 (#13) in PB. In three pts (#9, 12, 21) NK allo- have not seen any haematopoietic malignancy in these reactivity remained absent despite KIR-ligand mismatch. donors, but report here 6 cases of neurovegetative 17/22 pts were transplanted in KIR-ligand match. In 3 of these disturbances, 4 cases of arthralgia and 1 case of long lasting pts we detected allo-reactive NK cells in PB at d +56 (#8), neutropenia in MUD donors after PBSC donation. +128 (#19), +364 (#22). In 14/17 pts transplanted in KIR- ligand match no allo-reactive NK cells occurred. In 3/22 pts (#4, 6, 12) NK cell activity was absent at all, correlated with P638 acute GvHD and use of glucosteroids. All other cases showed Is there a superiority of single-dose pegfilgrastim over at least once active NK cells in PB. After median observation daily conventional filgrastim in mobilization of allogeneic time of 258 d, 2 pts died due to relapse (#3, 12), 12 pts died in peripheral blood stem-cells? Results from a retrospective CR due to infection (8) or GvHD (4). None of the pts with NK comparative study cell allo-reactivity experienced relapse. We observed F. Kroschinsky (1), K. Hölig (1), K. Poppe-Thiede (2), M. previously unknown late occurrence of allo-reactive NK cells Blechschmidt (1), R. Ordemann (1), A. Haack (1), G. Rall (2), transplanted in KIR-ligand mismatch regardless of M. Bornhäuser (1), C. Rutt (2), G. Ehninger (1) myeloablative or reduced conditioning. Some cases (1)Dresden University Hospital (Dresden, D); (2)Cellex transplanted in KIR-ligand mismatch lacked allo-reactive NK Apheresis Center (Dresden, D); (2)German Bone Marrow cells after HSCT, which may explain heterogeneous results Donor Center (DKMS) (Tuebingen, D) from retrospective studies investigating the influence of KIR- ligand mismatch in HSCT. Recently we reported about feasibility and efficacy of single- dose pegfilgrastim (PEGFIL) in the mobilization of allogeneic peripheral blood stem cells (PBSC) [Haematologica 2005; 90: 1665-1671]. Now we present an update of the ongoing study and a comparison with a retrospective cohort of donors who received daily conventional Filgrastim (FIL) to mobilize allogeneic PBSC. Thirty-five donors (15 related, 20 unrelated, 23 male, 12 female, median age 40 years) were mobilised with PEGFIL. Donors received a single s.c. injection of 2 vials (12 mg) PEGFIL (Neulasta™, Amgen Inc., Thousand Oaks, USA) resulting in a median dose of 161 µg/kg body weight (range 126-203) on day 1. FIL (Neupogen™, Amgen Inc.) was used for mobilization in 64 donors (2 related, 62 unrelated, 44 male, 20 female, median age 31 years) at a dose of 10 µg/kg on

S139 days 1 to 5. Monitoring included blood cell counts, number of Donor was treated with diuretics and was discharged in a CD34+ cells in peripheral blood (PB-CD34) and cytokine markedly improved condition on the fifth day of hospitalization. related side effects. Leukaphereses (LPH) started on day 5. This case presents a very rare, albeit severe adverse event, - PBSC were collected by large-volume LPH (4x blood volume) capillary leak syndrome, in a healthy donor following using Cobe Spectra 7.0 (Gambro BCT Inc.). stimulation with G-CSF. This case has resulted in a revision of In all donors sufficient numbers of CD34+ cells for allografting our future handling of peripheral stem cell donors, stressing could be collected. PB-CD34+ count on day 5 was the importance of an increased awareness of these specific significantly higher after PEGFIL compared to FIL (72/µl vs. symptoms and a recommendation of following the body weight 53/µl, p=0.03). Whereas an LPH on day 6 had to be during and up to one week after stimulation with G-CSF. performed only in 6 donors (17%) after PEGFIL, a second procedure was needed in 56/64 (87%) donors who had received daily FIL. The harvest results of both cohorts are P640 presented in the table. Although CD34+ yields on day 5 (LPH- Means to an end – parents’ commitment to participation 1) were significantly higher after PEGFIL than after FIL the in mesenchymal stem cell donation total numbers of collected stem cells were not different. The S.M. van Walraven (1), M.E. Bernardo (2), A. Brand (1), H. worse yields on day 6 (LPH-2) in the PEGFIL cohort Roelofs (1), F. Locatelli (2), R.M. Egeler (1), W.E. Fibbe (1), correspond to the observation that PB-CD34+ cells were L.M. Ball (1) higher on day 4 than on day 6, which is in contrast to the (1)Leiden University Medical Centre (Leiden, NL); (2)IRCCS kinetics of CD34+ mobilization with conventional G-CSFs. The Policlinico, San Matteo (Pavia, I) incidence of bone pain and headache was similar in both cohorts. However, there were a significantly higher number of Introduction: Mesenchymal stem cells (MSC’s) can be used in donors who received PEGFIL who characterized the the experimental transplant setting for treatment of life symptoms as severe. threatening GvHD or to improve engraftment. Presently the In conclusion, there might be a superior mobilization efficacy European MSC consortium has three established multi center of 12 mg single-dose PEGFIL over daily 10µg/kg FIL. Due to a clinical protocols in use. MSC’s expanded ex vivo from donor faster CD34+ kinetics LPH should be started already on day bone marrow are of parental origin. Requests for multiple 4. However, the effects could be related to the much higher donations of various types of cell products can raise questions cytokine dose if PEGFIL is used. The possible advantages of about medical and ethical considerations. single-dose administration have to be discussed with regard to Aims: We reviewed donation data from two participating side effects and costs. centers, to establish donor commitment, donation and procedural related complications. Donors and methods: Parents of children undergoing haplo- identical PBSCT or UCBT were asked to donate 50 cc of bone marrow 4-5 weeks prior to HSCT. Parents of children with non-steroid responsive aGvHD (grade III-IV) were asked at the time of treatment failure. Bone marrow was aspirated either under general or local anesthesia. Haplo-identical PBSC donors received in addition 5 days of G-CSF immediately before stem cell harvest. Donors could also consent for additional MSC’s to be donated for research or use as 3rd party donations for other patients. Donors were cleared for donation by an independent physician. Results: Twenty-two (8 mothers, 14 fathers)of 23 parents asked, agreed to donate bone marrow for MSC expansion and all uses. Median age was 39 years (range 26-55). One parent whose child died before receiving MSC’s requested their use as 3rd party donations. Thirty patients (included 4 adults) were treated with MSC’s (10 haplo-identical PBSCT, 8 UCBT and 12 aGvHD). Only 19 patients (63%) received MSC’s from a P639 parent, the remaining treatments being 3rd party. All donors Capillary leak syndrome in a healthy donor following G- were considered suitable for PBSC or BM donation, although CSF stimulation excess weight, mild hypertension and depression were P. Andersen (1), S. Gertsen (2), K. Assing (1), E. Dickmeiss documented in a minority of donors. No adverse events were (1) documented after bone marrow donation, but one parent (1)University Hospital Copenhagen (Copenhagen, DK); suffered severe bone pain requiring opiate analgesia, during (2)Århus Hospital (Århus, DK) G-CSF administration. At 6 weeks follow up no adverse effects were reported. A 39 years old female peripheral stem cell donor was Conclusions: MSC donation either alone or in combination stimulated with G-CSF (16mg/kg/day) for five days in order to with stem cell mobilization can be achieved without major donate peripheral stem cells for allogeneic stem cell problems. Parental donor commitment is definitely related to transplantation. the needs of the child. However, donor altruism to include The donor underwent leukapheresis for two days. White blood other unknown patients was in this study without limitation. It cell count (WBC) was 42 x 109/L the first day of is therefore essential when introducing experimental leukapheresis. On the second day after the final treatments to establish a balance between donor’s leukapheresis, WBC and absolute neutrophile count (ANC) commitment and patient's needs. were 65 x 109/L and 56 x 109/L, respectively. During this period the donor had fluctuating periorbital oedema, headache, nausea and vomiting culminating in abdominal pain and dyspnoe. The donor experienced a weight gain of 6 kg, on day 6 after initiation of G-CSF therapy or 60 hours after the last injection of G-CSF, resulting in rehospitalization. At admission abnormal laboratory values were Hb 6,4mmol/L, WBC 17,8 x 109/L, ANC 10 x 109/L, platelets 111 x 109/L, elevated alkaline phospatase and alanine aminotransferase and hypoalbuminemia. Computer tomograhy scan of thorax and abdomen revealed pleural effusion and ascites. Additionally, the donor had slightly generalised oedema.

S140 P641 There was no statistically significant correlation (r=-0,09, Myeloablative transplantation from matched siblings with p>0,05; Student’s t-test) between transplanted CD34+ cell a daily intravenous busulphan/fludarabine regimen with number per kg patient’s body weight (BW) and day of thymoglobulin: analysis involving 200 patients indicates leucocyte recovery. However, the number of transplanted low transplant-related mortality in all but older patients CFU per kg BW correlated well (r=-0,29; p<0,0001) with with high-risk disease engraftment kinetics and seems to be a more reliable J. Russell (1), N. Bahlis (1), M. Geddes (1), A. Chaudhry (1), parameter (vs. CD34+ dose) for rapid haematological A. Balogh (1), L. Larratt (1), R. Turner (2), L. Savoie (1), J. recovery confirming importance of using functional assays Storek (1), D. Quinlan (1), C. Brown (1), D. Stewart (1) rather than phenotypical markers. (1)Tom Baker Cancer Centre (Calgary, CAN); (2)Cross Engraftment after both syngeneic (n=8) as well as autologous Cancer Institute (Edmonton, CAN) (n=67) was seen significantly earlier than after allogeneic (n=449) HSCT (day 9.63±1.19 and 11.54±1.65 vs. This analysis aims to identify recipients of a fludarabine 16.04±4.00; p<0.0001). The engraftment delay in autologous (Flu)/busulphan (Bu) (FLUBUP) regimen given prophylaxis for as compared to syngeneic HSCT is still significant (p<0,005) graft-versus-host disease (GVHD) incorporating pointing to an influence of primary disease and/or previous Thymoglobulin (TG) who may be at particular risk of chemotherapy cycles on the engraftment of autologous HSC. transplant-related mortality (TRM) and perhaps merit some When focussing on allogeneic HSCT there is a significant modification of the protocol. Two hundred stem cell transplant advantage of HLA-matched (n=305) in comparison to HLA- patients (pts) were treated between 05/99 and 07/05 with Flu mismatched (n=144) donors (15.32±4.01 vs. 17.01±4.40 days; 50mg/m² daily x 5 and intravenous Bu (Busulfex, PDL p<0,001). Also, engraftment was reached earlier with HSC Pharma) 3.2 mg/kg daily x 4. Forty-six acute leukaemia pts from related (n=148) as compared to unrelated (n=301) had additional total body irradiation 200cGy x 2. Graft-versus- donors (14.80±3.38 vs. 16.84±4.45 days; p<0,001). host disease (GVHD) prophylaxis was cyclosporine A, Importantly, there were no significant differences in methotrexate and Thymoglobulin (TG, Genzyme) 4.5 mg/kg transplanted CD34+ cell number and CFU per kg BW. Also, total dose. Cell source from matched sibling donors was all above differences hold true when bone marrow and mobilised blood cells in 172 and marrow in 28. Median age peripheral blood stem cell transplantations were analysed was 46 (range 18-65) and follow-up of survivors was 13-87 separately. months (median 42). For low-risk (LR, acute leukaemia In conclusion our data clearly support a crucial role of the CR1/CR2, CML CP1) pts projected 5 year TRM and overall HSCT type on the speed of leukocyte recovery independent of survival (OS) was 4% and 76% for those ” 45 years old (n = the HSC source (BM or PBSC) and transplanted cell numbers. 54) and 6% and 83% for those >45 (n = 31). For high-risk Thus genetic disparities seem to be the most important (HR) pts TRM was 6% vs 27% (18% at one year) (p = 0.04) predictor of delayed engraftment. and OS 64% vs 37% (p = 0.046) in younger (n = 40) and older (n = 75) pts respectively. To correct for imbalance in HR diagnoses we matched each of 17 younger HR (YHR) pts with P643 2 older HR (OHR) pts by diagnosis and details of stage and Allogeneic stem cell transplants: impact of mobilization thereafter for other risk factors. For the YHR and OHR pts regimen on graft composition respectively OS was 70% vs 37% (p = 0.02), TRM 0 vs 34% J. DiPersio (1), A. Smith (2), D. Sempek (2), A. Baker (2), S. (p = 0.02) and relapse 64% (48% at 3 years) vs 46% at 3 and Jiang (3), R. Vij (1), A. Cashen (1), P. Westerfelt (1) 5 years (p = ns). For a more robust analysis of the whole data (1)Washington University School of Medicine (St. Louis, USA); set OHR pts were compared with the other 3 groups (2)Barnes-Jewish Hospital (St. Louis, USA); (3)University of combined giving TRM of 27% vs 5% respectively (p= 0.0003). British Columbia (Vancouver, CAN) Incidence of acute GVHD grade II-IV, acute GVHD grade III-IV and chronic GVHD was 23% vs 10% (p = 0.02), 4% vs 2% (p Background: Transplantation of G-CSF-mobilized peripheral = ns) and 66% vs 41% respectively (p = 0.001). Nine of 14 blood cells is the current standard practice in allogeneic non-relapse deaths in the OHR group were related to GVHD transplantation. Alternative mobilization agents are available or its treatment compared with 3 of 6 in all others (p value for or under investigation. Different mobilization strategies may GHVD related death = 0.01). These data provide some affect graft composition, i.e., the relative contribution of support for our practice of using this regimen in all adults, lymphocyte subsets and NK cells, and ultimately impact regardless of age. The effect of age on TRM is only apparent immune reconstitution and transplant outcome. in HR pts and is not explained by heterogeneity in diagnoses. Methods: From December 1994 to July 2006, a total of 566 High-risk older pts experience more GVHD and more GVHD donors have undergone peripheral blood stem cell related death than others but TRM is no higher than reported mobilization and apheresis at the Siteman Cancer Center, with many non-myeloablative regimens. It is possible that Washington University School of Medicine. The database these pts might benefit from additional GVHD prophylaxis. registered 604 individual cases of mobilization and apheresis; 529 mobilization records contained full data on subset cell collection on apheresis Day 1. Retrospective analysis of this P642 large database allows the comparison of graft composition Engraftment after haematopoietic stem cell between various standard mobilization regimens, as well as transplantation: which predictors are most important? an investigational agent, AMD3100. M. Lioznov, R. Ikogho, B. Fehse, A. Zander Results: The age of donors ranged from 1 month to 78 years, University Medical Centre Eppendorf (Hamburg, D) with a median age of 45.5 years; 57% were male. Donors were mobilized with G-CSF (55%), G-CSF/GM (29%), GM Rapid engraftment is one of the most important preconditions (7%), or AMD3100 (3%). Median CD34+/kg collected was for successful haematopoietic stem cell transplantation 7.00 x 10E6; range 8.19E4 – 2.02E8. Median absolute CD34 (HSCT). Conditioning regimen, HSC dose and HLA disparities yield on apheresis Day 1 by mobilization regimen is shown in are parameters thought to be of crucial importance in this figure 1. Mobilization regimen affects graft composition. respect. In fact, more rapid engraftment after syngeneic vs. Compared to G-CSF, GM-containing mobilization regimens allogeneic transplantation of purified HSC was shown in the have signficantly reduced levels of absolute CD3+, CD4+, and murine system (Uchida et al, 1998). However, an analogous CD8+ T-lymphocytes (2.2 v 1.36 x 1E10, 1.47 v 0.99 x 1E10, influence of the HSCT type has not been systematically 5.4 v 2.55 x 1E9, resp.). In contrast, AMD3100 is associated addressed in human HSCT yet. with increased absolute levels of these subtypes (3.51, 2.4, We retrospectively analysed leukocyte engraftment (>1000/µl) and 9.49, resp.). Subset yields expressed as absolute subset in relation to HSC and CFU dose as well as donor types for cell/ absolute CD34 are summarized in Table 1. While the 524 haematological patients receiving HSC transplants after ratio's appear similar between G-CSF and GM- regimen, myeloablative full-conditioning. AMD1300-mobilized grafts have higher ratio's of all subsets analysed.

S141 Conclusions: As the number of available mobilizing agents Minimal residual disease continues to grow, the impact of these different agents on graft composition and transplant outcome (immune reconstitution, graft-versus-host, and graft-versus-leukemia) requires rigorous evaluation. This large database will be P645 further evaluated for pt outcome. WT1 expression as a marker of disease in patients with acute myeloid leukaemia undergoing allogeneic HSCT S. Pozzi, A. Dominietto, G. Piaggio, A. Ibatici, N. Sessarego, M.T. van Lint, A.M. Raiola, F. Frassoni, M.B. Pinazzi, F. Bertolotti, A. Bacigalupo S.Martino's Hospital (Genoa, I)

Background: relapse remains a significant problem in patients with acute leucemia undergoing an allogeneic HSCT. A marker of disease predicting hematologic relapse would be useful to monitor patients post-transplant. Aim of the study. To assess the role of WT1 expression in AML patients after allogeneic HSCT Methods: We studied 54 patients with AML who received an allogeneic HSCT between 2004 and 2006 in our Unit. WT1 mRNA levels of expression were normalized with respect to the number of Abl transcripts and expressed as WT1 copy numbers every 104 copies of ABL. Data obtained from the quantitative assessment of the WT1 transcript in 20 normal bone marrow controls showed values in the range between 3 and 180 WT1 copies /104 ABL. MRD was considered positive when WT1 copy numbers every 104 copies of ABL were greater to 180. Patients bone marrow cells were monitored for WT1 expression, and cytology, at day +30 and every month untile day +180. Escalating dose donor lymphocyte infusions (DLI) were given , when possible, in MRD+ patients. Results: WT1 was found positive (>180) in 12 patients (22%) at a median interval from HSCT of 120 days (range 38-180) and always negative in 42 patients (78%). Hematologic relapse was seen in 8/12 MRD+ patients (66%) and 9/42 (21%) MRD- patients (p=0.005). Of the 12 MRD+ patients 4 received DLI (MRD+DLI+) on the basis of their WT1 expression: 1 relapsed, 3 remain in remission with a negative WT1 expression. GvHD developed in 2/4 patients receiving DLI. Of the 8 patients MRD+ who did not receive DLI P644 (MRD+DLI-) 6 relapsed (p=0.1). There is a statistical Rhabdomyolysis in allogeneic peripheral blood stem cell difference in relapse for MRD- and MRD+DLI+ patients (n=46, donor 10 relapses) and MRD+DLI- patients (n=8, 6 relapses L. Kaynar, F. Altuntas, S. Kabukcu , I. Kocyigit , L. Akyol, M. )(p=0.005). Relapse related death in these 2 groups of Karakükcü, B. Eser , T. Patiroglu , A. Ünal, M. Çetin patients is 4/46 (10%) in MRD- or MRD+DLI+ and 3/6 (50%) Erciyes Medical School (Kayseri, TR) in MRD+DLI-. Conclusions: This study suggests that WT1 expression can be Granulocyte colony-stimulating factor (G-CSF) has been used as a marker of MRD in patients with AML , following an exclusively used in normal donors to mobilize circulating stem allogeneic HSCT: relapse is significantly more frequent in cells. G-CSF is well tolerated by healthy donors. Severe side MRD+ as compared to MRD- patients. In addition, infusion of effects requiring discontinuation of treatment G-CSF are donor lymphocytes in MRD+ patients may reduce their risk of infrequent. We report a case of a 6-year old allogeneic PBSC relapse and of relapse related death. donor observed to have rhabdomyolysis after G-CSF therapy. The donor received G-CSF (filgrastim, 10 µg/kg/day, 5 days, SQ) to mobilize PBSC. On the third and fourth days of filgrastim, the donor complained of slight bone pain; paracetamol was given at a 250 mg dose. One day later, donor suffered from bone pain, myalgia and vomiting. Laboratory analysis showed: CPK=1095 U/L (40–226), LDH= 312 U/L (100–190), AST= 85 U/L (0–40), uric acid= 8,5 mg/dL (2,6–8), Cr=0,5 mg/dL (0.6-1.3), BUN=10 mg/dL (6-20), K= 3,3 mmol/L (3,6–5,1), WBC= 39.800 /µL, Hb= 12,5 g/dL, Hct= 37,4% and platelet= 350.000 /µL. Urine examination showed a high myoglobin level (110 ng/mL). In this case, a diagnosis of rhabdomyolysis was made with clinic and laboratory findings. Intravenous hydration, potassium replacement and P646 alkalization of the urine by adding sodium bicarbonate were Detection of host chimerism by patient-specific HLA performed. Subsequently, donor’s muscular signs and alleles is a more sensitive marker than standard flow symptoms improved, and the laboratory examination results cytometry for relapse prediction of high-risk leukaemia returned to normal after three days. Stem cells were after T-cell depleted stem cell transplantation harvested from peripheral blood on fifth day of G-CSF T.D. Clerici, R. Crocchiolo, B. Mazzi, A. Palini, C. Tresoldi, J. therapy. In conclusion, G-CSF and/or paracetamol may cause Peccatori, M. Bernardi, P. Servida, C. Corti, M.T. Lupo rhabdomyolysis. Therefore, serum CPK levels should be Stanghellini, K. Fleischhauer, F. Ciceri followed carefully in an allogeneic PBSC harvest setting, San Raffaele Scientific Institute (Milan, I) especially in donors with myalgia. After T-cell depleted (TCD) hematopoietic stem cell transplantation (HSCT) detection of minimal residual disease (MRD) is a useful tool for early prediction of disease relapse

S142 because it allows the prompt intervention with preemptive family of IgH. Comparison of primary structures of VH-genes immunotherapy strategies. A molecular marker for MRD study exhibiting clonal rearrangements and of germinal VH-genes is available only for a small number of hematologic revealed somatic mutations in 100% of cases (homology level malignancies. Hematopoietic chimerism (HC) has been ranged from 72,4 to 96%). Molecular monitoring of treatment proposed as a surrogate marker for MRD. We have previously efficacy was performed on 11 patients received HDT followed developed a semiquantitative method for HC analysis based by autologous PBSCT. Partial remission was documented in 6 on studying patient and donor-specific HLA alleles by PCR cases, complete remission (CR) - in 5 of 11 patients (45.4%). and reverse sequence-specific oligonucleotides (HLA method) Three patients from those with CR showed molecular which was shown to have a higher sensitivity (1%) and a remission (MR) also. Overall follow-up period came to 12, 27 higher predictive power for disease relapse than PCR-STR, and 33 months in these patients and they still remain in MR. the standard method for HC study (BMT 2006;37,s1:Ab824). MRD persistence was observed in 2 patients, one of them Here, we report on an investigational study aimed at developed MM relapse after 11 months following second comparing study of HC by the HLA method with standard flow transplantation, while another patient has been remaining in cytometry analysis of tumor-specific cell surface markers on a hematological remission for 20 months. limited number of cells, for detection of MRD after allogeneic Conclusion: Current study provides evidences for correlation HSCT. of molecular status with clinical disease course. However, we We performed a monthly follow-up of 59 TCD-HSCT for high suggest hopefully that the treatment approach in MM patients risk acute leukemia from alternative donor (43 haplo, 15 MUD, will depend on MRD detection results in the nearest future. 1 UCB) for the state of HC and phenotype in bone marrow samples in parallel by HLA-method and by standard flow cytometry. The stage of disease at HSCT was complete P648 remission (44.1%) or advanced disease (55.9%). The median Evaluation of haematopoietic chimerism following follow-up with HC analysis and flow cytometry analysis was allogeneic peripheral blood stem cell transplantation with 139 days. Total number of relapses was 20 (18 hematologic, 1 amelogenin marker cytogenetic, 1 molecular). S.H. Ghaffari, B. Chahardouli, K Alimoghaddam, A. 11/20 (55%) relapses were predicted by the HLA method only; Gavamzadeh 5/20 (25%) relapses were predicted by the HLA method and Tehran University Medical Sciences (Tehran, IR) by standard flow cytometry; 4/20 relapses (20%) were not predicted. There were no relapses predicted by standard flow Introduction: Several techniques are available for detecting cytometry alone. In total, the HLA method predicted 16/20 mixed chimerism after bone marrow transplantation (BMT); relapses (80%), whereas standard flow cytometry predicted one example is STR-PCR. However, this technique requires 5/20 (25%) relapses. The median time from diagnosis to DNA sample from both donor and recipient before BMT for relapse was 52 (21-261) and 36 (14-57) days, respectively, by interpretation of the result. One approach to this is the sex the HLA method and standard flow cytometry. identification of both male and female cells in the cell mixtures We conclude that HC analysis based on the study of of patients after the sex-mismatched bone marrow mismatched HLA alleles after TCD-HSCT for high risk transplantation. In humans, the amelogenin gene is present leukemia is a sensitive tool for early prediction of disease on both the X and the Y chromosomes. However, there are relapse. In particular, this assay has a higher sensitivity than size differences in this gene between these chromosomes, standard flow cytometry. These data underline the usefulness which have been utilized for sexing in forensic casework and of recently developed flow cytometry assays which allow MRD prenatal identification. The aim of the present study was to detection at higher levels of sensitivity. evaluate the application of the amelogenin gene for assessment of chimerism in PBL and/or BM samples of patients who had received sex-mismatched BMT. P647 Methods: PCR techniques using a set of amelogenin primer Individual molecular monitoring of minimal residual alone or in combination with STR primers were first preformed disease after autotransplantation in patients with multiple on whole WBC and/or BMA samples, and in some cases on myeloma isolated T-cell and granulocytes subsets of 30 recipient V. Zherebtsova, V. Surin, V. Savchenko, L. Mendeleeva, O. patients. Then, between September 2000 and April 2006, Pokrovskaya, N. Turina, A. Misurin more than 1400 samples from 300 BMT patients suffering Russian Research Center for Hematology (Moscow, RUS) from different types of leukemia and non-malignant hematologic were evaluated for detection of chimerism after Efficacy of MM treatment is evaluated by morphological and the transplantation. immunochemical methods. Improvement of transplantation Result: About 51% of patients were received sex-mismatched strategies and novel therapies result in a better treatment BMT. In 98% of cases, amelogenin marker in combination outcome. This gives rise to the need for sensitive methods to with STR markers has been used without a single false- detect the minimal residual disease (MRD). Qualitative positive or negative results, as confirmed by clinical outcome. molecular monitoring using allele (patient)-specific In 5% of the cases, when there was no pre-BMT sample from oligonucleotide polymerase chain reaction (PCR) for the either donor or recipient, the applicability of this assay has immunoglobulin heave chain (IgH) is well established to detect become of crucial important to our treating physicians. clonotypic myeloma cells during and after therapy. The aim of Conclusion: The application of amelogenin marker alone or in study was monitoring of MRD in patients with MM after high- combination with STR system can be used for relative dose therapy (HDT) followed by autologous PBSCT and quantitative analysis of mixed chimerism and for observing thalidomide therapy. kinetics of engraftment in patients who have sex-mismatched Materials and methods: 41 patients with untreated MM were BMT. Comparison of values obtained with amelogenin PCR included in the trial. Molecular marker (clonally rearranged V- analysis showed an excellent correlation with the STR-PCR D-J regions) was revealed by reverse transcriptase results. polymerase chain reaction (RT-PCR) with family-specific primers (VHL, VHD). RNA was extracted from bone marrows samples. The patient-specific primers were generated after sequencing of amplified fragments. MRD was evaluated using nested-PCR on bone marrows DNA samples of MM patients. The first round of PCR was performed using family-specific primers, the second one - with patient-specific oligonucleotide. Method sensitivity equaled 10-3-10-4. Results: Clonal rearrangements of VH-genes were detected in 37 (90%) of 41 patients. More than half of patients with MM demonstrated VH3-family and about 25% of patients – VH1-

S143 Lymphoma P650 Prolonged survival in patients with diffuse large B-cell lymphoma and aaIPI Score 2-3 receiving front-line rituximab-supplemented high-dose chemotherapy with P649 multiple autologous haematopoietic stem cell support: a Rituximab markedly improves the efficacy of high-dose prospective multicentre study programmes with autograft for B-cell lymphoma: a C. Tarella (1), M. Zanni (1), M. Di Nicola (2), C. Patti (3), R. multicentre GITIL survey on 957 patients Calvi (4), A. Pescarollo (5), V. Zoli (6), D. Caracciolo (1), M. C. Tarella, M. Zanni, M. Magni, F. Benedetti, T. Barbui, M. Ladetto (1), M. Magni (2), L. Devizzi (2), R. Rosato (1), M. Boccadoro, C. Patti, F. Ciceri, A. Gallamini, S. Cortelazzo, I. Boccadoro (1), M. Bregni (5), P. Corradini (2), A. Gallamini Majolino, S. Mirto, P. Corradini, R. Passera, G. Pizzolo, A.M. (4), I. Majolino (6), S. Mirto (3), A.M. Gianni (2) Gianni, A. Rambaldi on behalf of GITIL (Gruppo Italiano (1)University of Turin (Turin, I); (2)I.N.T. (Milan, I); (3)AO Terapie Innovative nei Linfomi) Cervello (Palermo, I); (4)AO S. Croce & Carle (Cuneo, I); (5)HS Raffaele (Milan, I); (6)AO S. Camillo (Rome, I) Introduction: The outcome of B-cell lymphoma has definitely improved since the introduction of the anti-CD20 Rituximab, Introduction: The outcome of Diffuse Large B Cell Lymphoma which can be effectively combined into conventional (DLB-CL) patients has definitely improved since the chemotherapy regimens. Rituximab can also be added to introduction of the anti-CD20 Rituximab, which can be high-dose chemotherapy programs with autograft. However, effectively combined into conventional chemotherapy the clinical benefit of combining Rituximab and autograft- regimens Indeed, Rituximab-supplemented CHOP is now based programs has not been proved yet. This issue is considered the most effective treatment option for DLB-CL. addressed in the present study. However, despite the inclusion of Rituximab, there is still a Patients and Methods: Data were collected from 957 B-cell marked difference in the outcome between patients with lymphoma patients undergoing a high-dose sequential (HDS) adverse IPI prognostic factors and those without. Rituximab- chemotherapy program, at 10 Italian Centers associated to supplemented intensive programs with autograft might GITIL (Gruppo Italiano Terapie Innovative nei Linfomi). represent an effective alternative to R-CHOP in DLB-CL Although the HDS schedule has been introduced almost 20 patients with unfavorable clinical presentation. yrs. ago, most patients were treated in the last decade. They Aim of the study: To perform a multicenter, prospective study received either the original HDS scheme or the hd-Ara-C- evaluating applicability and efficacy of the combination of supplemented scheme developed for mantle-cell and diffuse Rituximab and high-dose (hd) sequential chemotherapy large cell lymphoma; overall, Rituximab was added to HDS delivered with multiple autologous peripheral blood progenitor (R+) in 483 patients, the remaining 474 received Rituximab- cell (PBPC) support (R-HDS-maps regimen) in previously- free HDS (R-). All patients entered the HDS-protocols due to untreated patients with DLB-CL and scores 2-3 of the age- high-risk prognostic features, their median age was 49 yrs. adjusted IPI (aaIPI) prognostic classification. Treatment (range 17-70); 403 patients (232 R+) had low-grade and 554 schedule. R-HDS-maps includes: i. 3 APO courses; ii. (251 R+) had intermediate/high grade B-cell lymphoma; HDS sequential administration of hd-cyclophosphamide (CY), hd- was delivered to 542 (259 R+) patients at diagnosis and to Ara-C, both supplemented with Rituximab, hd- 415 (224 R+) at first or subsequent disease recurrence. etoposide/Cisplatin, with PBPC harvests following hd-CY and Results: at a median follow-up of 5 yrs., the 5-yr Overall hd-Ara-C; iii. hd-Mitoxantrone + L-Pam + 2 further Rituximab Survival (OS) and Event-free Survival (EFS) projections were doses; iv. involved-field radiotherapy. PBPC rescue was 66% and 55%, respectively, with a significantly better outcome scheduled following Ara-C, etoposide/Cisplatin and for patients treated at diagnosis (5-yr OS: 72%, EFS: 61%) Mitoxantrone/L-Pam. compared to patients at relapse (5-yr OS: 56%, EFS: 45%). Results: Six hematological Centers affiliated to GITIL (Gruppo As shown in Table 1, Rituximab addition was associated with Italiano Terapie Innovative nei Linfomi) participated to the significant improvements; in particular, a significant prospective trial. Between 1999 and 2004, 112 consecutive improvement was observed for the EFS projection both in patients (74 score 2, 38 score 3) entered the study protocol patients at diagnosis and at disease recurrence; moreover, and received the R-HDS-maps regimen. There were 5 early the benefit of Rituximab was clear both in low-grade (5-yr and 2 late toxic deaths. Overall 90 patients (80%) reached CR EFS: 65% for R+ vs. 41% for R-) and intermediate/high-grade and, at a median follow-up of 48 mos., 87 (78%) patients are subtypes (5-yr EFS: 64% for R+ vs. 52% for R-). In the Cox alive, 82 (73%) in continuous CR, with 4-yr overall survival multivariate analysis, Rituximab addition confirmed its (OS) and event-free survival (EFS) projections of 76% (C.I. significant impact on the EFS. 68-85%) and 73% (C.I. 64-81%), respectively. Conclusions: the addition of Rituximab to high-dose programs Conclusion: The results of the prospective trial show that R- with autograft may improve response and long-term outcome HDS-maps is feasible at the multicenter level, with acceptable in high-risk B-cell lymphoma patients. toxicity, and leads to prolonged survival in a high proportion of patients. These observations give further support to the use of early-intensified chemotherapy regimens, possibly supplemented with Rituximab, in younger patients with high- risk DLB-CL.

P651 Impact of complete remission achievement on the survival of lymphoma patients undergoing high-dose sequential chemotherapy and autograft (HDS programme): a GITIL (Gruppo Italiano Terapie Innovative nei Linfomi) retrospective study on 1,266 patients C. Tarella, M. Zanni, S. Rambaldi, M. Magni, M. Sorio, R Passera, C. Patti, F. Ciceri, A. Gallamini, S. Cortelazzo, I. Majolino, S. Mirto, M. Boccadoro, P. Corradini, F. Benedetti, T. Barbui, A.M. Gianni on behalf of GITIL (Gruppo Italiano Terapie Innovative nei Linfomi)

Introduction: Several observations stressed the importance of achieving an adequate tumor shrinking as a preliminary step prior to high-dose therapy and autograft. This concept was considered while designing the high-dose sequential (HDS)

S144 chemotherapy program (Gianni & Bonadonna, 1989). Indeed, 9 achieved PR, 5 of whom converted to CR with additional the HDS approach is characterized by the combination of an radiation therapy to residual disease (overall 16/21 achieved early dose-intensification followed by a conclusive phase with CR), 1 pt did not respond. With a median follow-up of 17 high-dose therapy and autograft with peripheral blood months (range, 6-27), 16 pts are alive. The estimated 2-year progenitor cells (PBPC). The HDS program has been overall and progression-free survival were 67% (95% CI, 46- extensively used in the management of both non-Hodgkin s 87%) and 52% (95% CI, 31-72%). Seven pts relapsed or did (NHL) and Hodgkin s Lymphoma (HL). The outcome of a large not respond, all within 6 months of aSCT. The 2-year series of lymphoma patients treated with the HDS approach at cumulative relapse incidence is 31% (95% CI, 17-57%). There 10 Centers associated to GITIL is here reported. were no early infusion reactions associated with Zevalin. Two Patients and methods: Data were collected on 1,266 patients, pts died of multi-organ toxicities early after aSCT. One died who received either the original or slightly modified HDS from an infection and one of bronchiolitis obliterans, 4 and 5 regimen. There were 213 HL and 1,053 NHL patients (630 months after SCT, respectively. The day100 rate of non- aggressive, 423 low-grade); median age was 46 yrs, 57% relapse mortality is 9%. This rate can be expected in such were male. Overall, 671 (53%) patients received HDS as heavily pretreated pts and there was no additional toxicity salvage treatment whereas 595 (47%) had HDS front-line. related to Zevalin. In conclusion, inclusion of Zevalin in the Most patients were autografted with PBPC, few received BM conditioning regimen prior to aSCT is relatively safe and may cells (alone or with PBPC); 158 (12%) patients did not improve outcome in pts with refractory lymphoma. Although undergo autograft, due to toxicity, disease progression, or excess non-relapse mortality can not be ruled out, relapse poor harvests. rate was low resulting in improved PFS. Zevalin in Results: Overall, 1,013 (80%) patients reached Complete combination with aSCT merits further study in larger scale Remission (CR) following HDS program. Up to now, 93 (7%) randomized studies and standard risk pts with chemosensitive patients died for early/late toxicities, 328 (26%) died for disease may also benefit from this combination. lymphoma, 844 are known to be alive; at a median follow-up of 5 yrs, the 5-yr Overall Survival (OS) projection is 64%. As shown in Table 1, a markedly higher survival was observed in P653 patients achieving CR vs. those not in CR, including Partial High-dose 90yttrium ibritumomab tiuxetan (Zevalin®) with Remission, Stable or Progressive disease. On multivariate PBSC support in refractory-resistant NHL patients: Cox analysis, both status at HDS (diagnosis vs relapse) and preliminary results of a phase I/II study post-HDS response (CR vs. no CR) maintained a significant A. Vanazzi, P.F. Ferrucci, C. Grana, M. Cremonesi, M. Clerici, impact on the 5-yr OS. D. Radice, S. Papi, L. Calabrese, G. Paganelli, G. Martinelli Conclusions: i. the long-term outcome of lymphoma patients European Institute of Oncology (Milan, I) receiving the HDS program is definitely good in patients achieving CR; ii. given their poor outcome, early salvage We present feasibility and toxicity results of a phase I/II study treatment options, including allogeneic transplant, should be of HD-Zevalin with PBSC support in resistant-refractory NHL considered for those patients unable to reach CR following a pts. From 04/04 to 11/06, 20 pts were enrolled. Median age HDS treatment approach. was 66.5ys (21-75). 17/20 pts had advanced stage disease (III/IV) at diagnosis. 12 had DLBCL, 4 MCL, 2 FL, 1 transformed FL and 1 transformed MZL. Median number of prior therapies was 3 (1-6), including Rituximab, RT and HD- CT. Three Zevalin activity levels were fixed: 30, 45, 56 MBq/kg; 4 pts at the first, 4 at the second and 12 at the third level. One week before treatment all patients underwent dosimetry with Indium-111 (111In) ibritumomab-tiuxetan in order to assess organ doses and plan the activity level. ASC were reinfused 13 day after Zevalin. On d28 from reinfusion engraftment was considered to be delayed if ANC<1.0x109/L or PLT<20.0x109/L. P652 Results: Dosimetry showed acceptable radiation-absorbed Yttrium-90 - ibritumomab tiuxetan (Zevalin®) combined doses to normal organs in all cases. Median adsorbed doses with high-dose BEAM chemotherapy and autologous (mGy/MBq): 0.8±0.2 (RM), 3.0±1.7 (heart wall), 1.7±0.8 stem cell transplantation for chemo-refractory aggressive (lungs), 3.9±2.3 (liver), 2.5±1.6 (spleen), 2.2±1.1 (kidneys), non-Hodgkin lymphoma 3.0±1.0 (testes), 0.6±0.1 (total-body). Only 1 pt received 30Gy A. Shimoni, T. Zwas, Y. Oksman, I. Hardan, N. Shem-Tov, R. to the liver, without developing toxicity. Median activity of 90Y- Yerushalmi, A. Avigdor, I. Ben-Bassat, A. Nagler Zevalin delivered: 3.5 GBq, range 2.1 – 5. 55 GBq; 10 pts Chaim Sheba Medical Center (Tel-Hashomer, IL) received more than 3,7 GBq. All patients engrafted promptly after ASC transplantation. PLT and ANC-count nadirs were High-dose chemotherapy and autologous stem-cell reached 8 and 4 days after transplantation, with median transplantation (aSCT) have an established therapeutic role in values of 11x109/L PLT (4-35) and 0.01x109/L ANC (0.01- patients (pts) with chemosensitive relapse of aggressive 1.09). The time of nadir did not change as a function of 90Y- lymphoma. However, aSCT has only limited success when ibritumomab tiuxetan dose. Median time to engraftment was performed in refractory or progressive stage of the disease. 12 (0-22) and 19 (1-48) for PLT and ANC respectively. One The expected long-term progression-free survival (PFS) in this transient acute G3 liver toxicity was observed at the 3rd level setting is less than 20%. This study was designed to explore and 1 pt died 4 months after treatment due to HCV the safety and outcome following inclusion of Zevalin in the reactivation. 18/20 pts are evaluable for response: 7CR, 4PR, conditioning regimen given prior to aSCT in pts with refractory 1SD, 6PD. lymphoma. The study included 23 pts, median age 55 years Conclusion: Zevalin at myeloablative activity is feasible with (range, 35-66). Histology was diffuse large cell (n=15), PBSC support and it could be safely delivered also in elderly transformed low grade (n=7) and mantle cell lymphoma (n=1). pts. We suggest dosimetry in order to avoid unpredictable Disease status was primary refractory (n=11) or refractory toxicity and an activity of 45 MBq/kg in heavily pretreated pts. relapse (n=12), and 14 pts had bulky disease at aSCT. The Clinical efficacy and mild treatment-related toxicities suggest median number of prior therapies was 3 (range, 1-6). HD-Zevalin is an interesting modality treatment to be further Rituximab 250 mg/m² followed by Zevalin 0.4 mCi/kg were investigated as an alternative therapeutic option in ABMT given on day -14 and high-dose BEAM chemotherapy started setting. We are continuing our experience in order to verify on day -6. All pts engrafted, a median of 10 days after aSCT initial results. (9-22). Zevalin had no impact on engraftment kinetics. Twenty-one pts are evaluable for response; 11 achieved CR,

S145 P654 P655 Multicentre randomised GITMO-IIL trial in high-risk Efficacy of the allogeneic stem cell transplant against follicular lymphoma at diagnosis: rituximab- cutaneous T-cell lymphoma supplemented high-dose sequential chemotherapy is M. Postorino, M. Cantonetti, R. Cerretti, L. Cudillo, A. Picardi, superior to CHOP-R in molecular remissions rate, EFS L. Franceschini, G. De Angelis, G. Baliva, S. Faccia, D. Renzi, and PFS M. Rizzo, W. Arcese M. Ladetto, F. De Marco, F. Benedetti, U. Vitolo, C. Patti, A. Policlinico Tor Vergata (Rome, I) Rambaldi, A.M. Liberati, M. Musso, A. Pulsoni, A. Olivieri, E. Pavone, E. Pogliani, D. Rota Scalabrini, A. Gallamini, V. Background: Sezary syndrome (SS), tumor-stage Mycosis Callea, I. Ricca, M. Boccadoro, I. Majolino, P. Corradini, A.M. Fungoides (t-sMF) and other cutaneous T-cell Lymphomas Gianni, C. Tarella on behalf Of GITMO/IIL (CTCL) in advanced stage or refractory to front-line chemotherapy are generally considered incurable with This is an update of the GITMO-IIL trial comparing R-HDS and currently available treatments.In this context the role of the CHOP-R in high-risk FL <60 years. Curent median follow-up is allogeneic stem cell transplant (SCT) should be explored in now 36 months. Eligibility was based on age-adjusted IPI 2 order to investigate if an immunotherapeutic effect of “Graft (125 pts) or according to the Italian Lymphoma Intergroup versus Tumor” does exist against such dismal diseases. score 3 (11 pts). 136 pts were stratified according to histology Patients and Methods: From May 2004 to Agust 2006, 5 (grade I or II 101, grade III 35) and randomized (68 each). patients with a median age of 48 years (range, 28- Clinical features were: median age 50 yrs. (22-60), stage III-IV 62)underwent an allogeneic stem cell transplant for SS (n=2), 98%, elevated LDH 78%, bulky disease 66%, B symptoms t-sMF (n=1), Panniculitis (n=1)and CD30+ T-cell Lymphoma 47%, extranodal disease (other than BM) 45%, ECOG PS >1 (n=1). was The median time from diagnosis to SCT was 5 47%. R-HDS has been already described (Ladetto et al ASH years (range, 2-10) with a median number of 3 (range 2-4) 2005). The CHOP-R arm consisted of CHOP and Rituximab lines of given therapies . Four patients were grafted from an delivered sequentially as already published(Rambaldi et al HLA identical sibling, while the patient with CTCL received a Blood 2002). Cross-over was allowed in case of CHOP-R single, unrelated, 2 HLA antigen mismatched cord blood unit. failure. Centralized minimal residual disease (MRD) analysis The chemotherapy based conditioning regimens were with the bcl-2/IgH was planned on BM. Analysis was intention myeloablative in 2 patients with t-sMF and CTCL and non- to treat. Toxic deaths were 4 (2 in each arm); in addition 1 myeloablative in the 3 patients affected by SS or Panniculitis solid cancer and 2 MDS-ANLL occurred in the R-HDS arm Results: All patients engrafted with complete clinical and 1 solid cancer in the CHOP-R arm. CR rates were 59% remission. Furthermore, molecular disease remission and with CHOP-R and 85% with R-HDS (p<0.001). Progressions chimerism of full donor origin were detected at 60 days after were 28% with CHOP-R and 10% with R-HDS (p<0.025). At SCT. No patient developed acute GVHD, while extensive 36 months EFS and PFS for CHOP-R are 36% and 38% while chronic GVHD occurred in 2 patients. No other major for R-HDS are 66% and 72% (EFS: figure 1a). The better complication was observed. The relapse occurred in one outcome for R-HDS was seen both in grade I, II and grade III patient with SS at 270 days after transplant and a further pts. OS at 36 months was 83% in each arm. Cross-over from complete remission was achieved by 2 DLI infusions from the CHOP-R to R-HDS was chosen in 67% patients failing CHOP- original donor, after an induced cutaneous acuteGVHD. At a R with a CR rate of 73%. MRD analysis is available in 44% of median follow-up of 21 months (range, 4-31 ), all patients are pts. A stable molecular remission (MR) was achieved in 26% alive and in complete remission. of CHOP-R pts and 78% of R-HDS pts (p<0,001). A persistent Conclusion: Despite the small number of patients, our results MR was associated to an improved PFS (p<0,001) (figure 1b). suggest that allogeneic SCT seems a very effective therapy Interestingly, PFS of PCR+ and PCR- pts was similar, for different T-cell cutaneous lymphomas, which can be regardless of the treatment received: 3-years PFS for PCR+ particularly sensitive to its GvT immunotherapeutic activity . pts was 25% for CHOP-R and 32% for R-HDS, while 3-years PFS for PCR- pts was 67% with CHOP-R and 76% with R- HDS). This is the first randomized study with a significant P656 proportion of molecularly-studied pts in FL and compares for Allogeneic stem cell transplantation for lymphoma the first time an intensified versus a conventional schedule in V. Valkova, K. Benesova, A. Vitek, E. Faber, Z. Koristek, P. the Rituximab age. Conclusions are: a) R-HDS induces more Zak, M. Trneny for the Czech National SCTregistry CRs and ensures a better EFS and PFS compared to CHOP- R in this rare and aggressive population of pts. b) R-HDS Background: Allogeneic stem cell transplantation (allo-SCT) is induces more MRs; c) the similar outcome in PCR+ and PCR- considered to be a therapeutic option for poor risk patients pts, regardless of treatment received, indicates that the with lymphoma. We report a retrospective analysis of allo-SCT superior outcome of R-HDS is largely explained by the in patients with lymphoma from four centers in years 1999- superior rate of MRs achieved with the intensified treatment. 2006. Methods: We analysed 78 patients (pts), sex ratio M/F 49/29. Histological subtypes were: Hodgkin´s lymphoma (HL) n=18, diffuse large cell lymphoma (DLCL) n=16, follicular lymphoma (FL) n=17, mantle cell lymphoma (MCL) n=9, peripheral T-cell lymphoma n=8, and 10 other lymphomas. Median age was 45 years. Donors were identical siblings (IS) in 48 pts (62%) or unrelated volunteers (UD) in 30 (38%). Sixty (77%) pts received reduce-intensity conditioning (RIC), eighteen (23%) were treated with myeloablative conditioning (MC). Forty-one (53%) pts had previous autologous SCT. At time of allo-SCT, forty-eight (62%) pts had chemosensitive disease, twenty-four (31%) were chemoresistant (six were untested). Median interval from diagnosis to SCT was 25 months (2-198). Results: Acute graft-versus-host disease (aGVHD) occurred in 26 (33%) pts (17x grade 1-2, 9x grade 3-4). Of the 53 evaluable pts nineteen (36%) developed chronic GVHD. Overall 100-day and 1-year resp 3-years TRM rates were 27%,33% and 33% resp. (22%,30% and 30% for RIC, 39%, 44% and 44% for MC). Relapse incidence was 26%. With a median folow-up of 26 months (3-109) of living pts, actuarial overall survival (OS) at 3 years is 44%, event free survival (EFS) 42% and relaps rate 45%. The 3-years OS of pts with

S146 HL, DLCL, FCL, MCL and PTL is 65%, 23%, 53%, 32% and P658 88% resp (DLCL vs PTL, p=0,01 and PTL vs all others, Pre-transplant FDG-PET predicts survival in lymphoma p=0,01). patients undergoing high-dose sequential chemotherapy Pts with chemoresistant disease had significantly less followed by autologous stem cell transplantation outcome (3y OS 22% vs 56%,p=0,002). There was no R. Crocchiolo, D. Clerici, M. Tassara, A. Assanelli, P. Servida, correlation between GVHD and relapse rate. There was no M. Bernardi, J. Peccatori, F. Ciceri significant diference in 3y OS between pts treated with MC San Raffaele Scientific Institute (Milan, I) and RIC (54% vs 42%), between IS and UD (46% vs 48%), or between pts with vs without previous ASCT (35% vs 52%, Objectives: to evaluate the prognostic role on progression-free p=0,14). At time of last follow-up 41 pts are dead. survival (PFS) and overall survival (OS) of 18F-fluoro- Conclusion: Allogeneic SCT represents an effective deoxyglucose positron emission tomography (FDG-PET) therapeutic option for patients with poor prognosis lymphoma, performed before autologous stem cell transplantation (ASCT) however TRM as well as relapse risk are still high. RIC may in lymphoma patients who underwent high-dose sequential be an option for erderly patients but it´s not clear if offer the chemotherapy (HDS), supplemented or not with Rituximab, same effectivity as standard MC. Transplants from UD and IS followed by ASCT. had the same outcome. Methods: we retrospectively analyzed 47 lymphoma patients, Study was supported with grant of IGA N0 NR/8223-3 10 with Hodgkin’s disease (HD) and 37 with non-Hodgkin’s lymphoma (NHL), treated with HDS followed by ASCT, between February 1999 and March 2006 at our institution. P657 The median age was 45 years old (range: 18-69), and patients Autologous stem-cell transplantation in patients with were treated upfront (if they had an age-adjusted IPI • 2 at primary testicular lymphoma - a study of the Lymphoma diagnosis) or at relapse after conventional first-line Working Party of the EBMT chemotherapy; FDG-PET scans were all performed at our A. Symeonidis, C. Canals, G. Taghibour, G. Martinelli, D. institution within 46 days before ASCT. HDS scheme is a Blaise, J.L. Harousseau, P. Colombat, A. Rambaldi, A. sequence of different chemotherapeutic agents Goldstone, A. Sureda on behalf of the Lymphoma Working (cyclophosphamide, cytarabine, etoposide, cysplatine) Party of the EBMT administered at high doses at established intervals. The most common conditioning regimen was mitoxantrone and high- Primary testicular lymphoma represents ~1% of all dose melphalan. lymphomas and is characterized by an aggressive clinical Results: with a median follow-up of 31 months (range: 8-81), course and a tendency for additional extranodal involvement. no significant differences in PFS or OS were observed We analyzed the EBMT experience of 129 patients, who between PET-positive and PET-negative group (fig.1); the underwent autologous stem-cell transplantation (ASCT). median PFS and the OS for both groups were not reached. Median age at SCT was 50 years (range 2-74), 6 patients However, a significant difference was observed in patients were <18 years and 21 patients >60 years old. Histological who received HDS for relapsed disease (n=33) when we subtypes were diffuse large B-cell (63), Burkitt’s (8), T- compared OS with respect to FDG-PET status before ASCT lymphoblastic (7), B-lymphoblastic (5), follicular (7), other low- (fig.2). The 2-year OS was 90% vs 51% in PET-negative grade (6), Hodgkin’s (3), and various differently specified (30). (n=23) and PET-positive (n=10) group, respectively. The At diagnosis testicular involvement alone was found in 36 median OS was not reached in any of the 2 groups. patients (28%) and additional extranodal involvement in 41 Interestingly, all the 13 patients affected by lymphomas other (32%), most frequently bone marrow (24), central nervous than HD and diffuse-large-B-cell and Hodgkin’s lymphomas (7 system (10), lung (8), bones (5) and gastrointestinal tract (5). follicular lymphomas, 2 anaplastic T-cell lymphomas, 2 Elevated serum LDH was found in 35/87 patients (40%) and mantle-cell lymphomas and 2 MALT lymphomas) reached systemic symptoms in 31/96 (32%). Median time from FDG-PET negativity, probably due to the impact of dose- diagnosis to ASCT was 9 months (2.3-92). Disease status at intensity of the above-mentioned chemotherapeutic regimen ASCT was CR1 in 45 patients (31%), CR2 in 26 (20%), PR1 on the metabolic activity of these hystotypes. in 27 (21%), refractory relapse in 15 (12%) and others in 16 Conclusion: pre-transplant FDG-PET is predictive of survival patients. Stem-cell source was bone marrow in 16 patients, in relapsed patients affected by HD and NHL treated with HDS PBSC in 109 and both in 4. TBI was used for conditioning in followed by ASCT. Patients with positive pre-transplant FDG- 19 patients (15%). The regimens more frequently used were PET should be considered candidate to tandem autologous BEAM in 64, Cy-TBI in 12 and BEAC in 7. Seven patients died and allogeneic transplantation. from non-relapse mortality (NRM) and 59 progressed / relapsed post-transplant, with a 2-year cumulative incidence of 6% and 46%, respectively. After a median follow-up of 25 months (range 1-152), 74 patients are alive, 60 of them disease-free, with a KM probability of OS and PFS at 2 years of 58% and 48%. PFS at 2 years for patients transplanted in CR1 was 57%, for those transplanted in CR2 44%, in first PR 45% and in refractory relapse 46%. On multivariate analysis, disease status at ASCT (others vs CR1; p=0.04) and the presence of additional extranodal involvement at diagnosis (p=0.001) correlated with higher risk of relapse after ASCT. A trend to a lower risk of relapse (p=0.06) was observed for patients receiving TBI. Disease status at ASCT (other vs CR1; p=0.015) and the presence of additional extranodal involvement (p=0.008) predicted for a shorter PFS. In conclusion, the factor with a higher impact on the outcome after ASCT was the presence of other extranodal involvement. Better results are expected when the procedure is performed in first CR. Finally, TBI-containing regimens seem to be associated with a lower risk of relapse.

S147 P660 High-dose chemotherapy and autologous stem cell transplantation as first-line treatment without consolidating radiotherapy for primary CNS lymphoma G. Illerhaus, F. Müller, R. Marks, C. Ostertag, J. Finke University Hospital (Freiburg, D)

Purpose: Primary central nervous system lymphoma (PCNSL) have a dismal prognosis despite initial response to steroids and radiotherapy (RT). Addition of high-dose methotrexate (HD-MTX) to RT has improved the prognosis of patients (pts) with PCNSL. However, the majority of pts eventually relapse. High-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) with consolidating whole-brain radiation (WBRT) showed favourable results in a recent phase II trial with 30 pts. (Illerhaus et al., J Clin Oncol. 2006). However, the rate of leukoencephalopathy (5 of 30) was of concern. Here we present the results of a monocentric pilot study using early HDT and ASCT for PCNSL without obligatory WBRT to reduce neurotoxicity for patients (pts) under 65 yrs. P659 Patients and Methods: Chemotherapy treatment included 3 Long-term follow-up of a subgroup of peripheral T-cell sequential steps: 1) induction with up to 4 cycles of high-dose lymphomas unspecified treated with and without autoSCT systemic methotrexate (8 g/m²); 2) two cycles AraC (2x 3 V. Koza, K. Steinerova, D. Lysak, S. Vokurka, M. Karas g/m²) and thiotepa (40 mg/m²) followed by rG-CSF and stem Charles University Hospital (Pilsen, CZ) cell mobilization; 3) conditioning regimen with BCNU (400 mg/m²) and thiotepa (4x5 mg/kgBW) followed by ASCT. Introduction: Prognosis of peripheral T-cell lymphomas WBRT was restricted to incomplete responders after HDT + (PTCLs) is considered dismal not only after antracycline- ASCT. containing chemoterapy but also after autoSCT. Regarding Results: Eleven patients with PCNSL with a median age of 54 this clinical studies incorporating aloSCT as up-front therapy yrs (range 38-67) were enrolled. Objective response was 9 of have been drafted. But PTCLs are a heterogenous group of 11 (7 CR, 2 PR). Two refractory diseases were observed, one malignancies, experience with auto SCT is limited and after AraC/Thiotepa, the other after HDT and ASCT. Both pts controversial (particularly in up-front treatment), experience with PR after ASCT received WBRT resulting in a sustained with autoSCT in single histologic subtypes is missing at all. complete remission. During follow-up one patient died The aim of our study was to perform a retrospective analysis because of early systemic and cerebral relapse from CR 8 of peripheral T-cell lymphomas unspecified (PTCLu) as the months after ASCT. Treatment related mortality was 0%. With largest subgroup of PTCLs in terms of autoSCT performed in a median follow-up of 19 months (range: 2-44) 8 of 11 (73%) CR1 or after first relaps. patients are alive in CR and in good condition without clinical Methods: All T-NHL treated in years 96-05 were histologically signs of leukoencephalopathy. reviewed. Pts. with PTCLu and treated with/without autoSCT Conclusion: Sequential systemic application of maximum were selected. OS of all transplanted pts. and OS of pts. dosed cytostatic agents followed by HDT+ASCT without transplanted up-front and after first relaps were analysed. obligatory WBRT is highly effective as initial therapy for Results were compared with the group of pts. treated in the patients with PCNSL. HDT + ASCT was tolerated well, with same period without autoSCT and reasons for diversed little acute toxicity. A multicenter phase two study using this treatment were analysed. protocol will be implemented in January 2007. Results: Histological specimens of 54 pts. with T-NHL were reviewed. PTCLu has been confirmed in 27 cases. 16 pts (median age 46,5 years) underwent autoSCT (5x in CR1, 11x P661 after first relaps ). 11 pts. (median age 66 years) were not Long-term outcomes of high-dose etoposide and transplanted (4 because of death during induction, 4 because melphalan chemotherapy and autologous stem cell of fast and chemoresistant relaps, 2 because of high age). transplantation for non-Hodgkin lymphoma Median follow-up of both groups is 6 years. OS of all J. Kuruvilla, S. Zadeh, N. Franke, T. Nagy, R. Saragosa, M. transplanted pts. is 67%, OS of pts. transplanted in CR1 and Crump, A. Keating after relaps is 66% and 58%, p=0,5056. No pt. with relaps Princess Margaret Hospital (Toronto, CAN) after SCT survives. OS of the non-transplanted group is 9%, only one pt has been surviving for 10 months. Introduction: High dose chemotherapy (HDCT) and Conclusions: Despite the objection a cohort of any subgroup autologous stem cell transplantation (ASCT) are the standard of PTCLs must be small for a single center study and biased of care for relapsed or refractory aggressive histology non- by retrospective analysis we strongly believe: 1) OS of pts. Hodgkin’s lymphoma (NHL) and is an accepted therapy for with chemosensitive PTCLu transplanted in CR1 might be other types of NHL. There is no gold standard HDCT regimen much better, even excellent, compared to OS of pts. with T- although BEAM, CBV and CY-TBI are commonly employed. NHL in all. 2) AutoSCT seems to reach excellent outcome We have reviewed our experience of ASCT for NHL using a even in pts. with PTCLu after chemosensitive relaps. 3) Only high dose regimen of VP16/Melphalan. pts. with PTCLu chemoresistant/progressive to first line Methods: We retrospectively reviewed a computerized antracycline-containing chemotherapy have very dismal database and charts of 467 patients (pts) with NHL who prognosis and should be considered for innovative treatments underwent ASCT between Jan 1st 1986 and June 30th 2006. like alloSCT. This subgroup seems to consist of significantly Pts had disease that did not respond to or relapsed after initial older pts. and RIC protocols should be considered. anthracycline-based chemotherapy; a small number of pts with mantle cell lymphoma (MCL) or T-NK lymphoma underwent ASCT as consolidation of first line therapy. Responses have been retrospectively assessed using International Workshop Criteria. Pts typically received 2-3 cycles of platinum-based salvage therapy to assess chemotherapy sensitivity; responding pts proceeded to PBSC mobilization or bone marrow harvest and subsequent ASCT. The intensive therapy regimen typically consisted of high-dose

S148 VP16 60 mg/kg day –4 and melphalan 180 mg/m² day –3 with P663 stem cell infusion day 0 (TBI day –2 to 0 if used). Pts with Single versus tandem high-dose chemotherapy and bulky disease at relapse (> 5cm) received involved field autologous stem cell transplantation in non-Hodgkin radiation post-ASCT. lymphoma not achieving complete response or relapsing Results: 467 pts were identified with 57% male and 43% after induction chemotherapy female. The median age at ASCT was 49.8 (range 19 – 67). B. Sarina, E. Todisco, A. Anastasia, R. Mazza, A. Nozza, M. Disease status at ASCT was: CR/CRU 39.4%, PR 57.4%, SD Balzarotti, M. Magagnoli, I. Timofeeva, A. Santoro, L. 1.5% and unknown 1.5%. NHL histology was: DLBC 52.8%, Castagna Transformed 15.4%, follicular 12.4%, T-cell: 12.4% and other: Istituto Clinico Humanitas (Milan, I) 7%. Stem cell source was: peripheral blood 59.1%, bone marrow: 29.8% and mixed 11.1%. The high dose therapy Background: We retrospectively analyzed the survival and the regimen was VP16 60 mg/kg + melphalan 180 mg/m²: 77%, safety of single and tandem high-dose chemotherapy (HDC) VP16 60 mg/kg + melphalan 160 mg/m²+ 12 Gy TBI: 19% and with autologous stem-cell rescue in 46 patients with other: 4%. With a follow-up of up to 210 months post-ASCT aggressive non-Hodgkin lymphoma (NHL) who failed to (median 22 months), the overall survival of the entire cohort is achieve complete remission (CR) or relapsed after front-line 51.9% and the progression-free survival is 52.6%. Causes of chemotherapy. death were recurrent disease: 31%, treatment-related Patients and methods: Twenty eight patients underwent to a mortality (TRM): 7%, other non-relapse mortality: 6% and single autologous stem cell transplantation (s-HDC), 18 to unknown: 4%. tandem autologous stem cell transplantation (t-HDC). At the Conclusions: With an experience of over 450 transplants time of transplantation in s-HDC group, 50% of patients were employing VP16/melphalan based HDCT, long-term OS and in CR, 35% in partial remission (PR), 14% in progressive PFS are approximately 50% with an acceptable TRM. These disease (PD). In t-HDC group, 38% of patients were in CR, results demonstrate that VP16/melphalan is a regimen with 50%, in PR and 11% in PD. In s-HDC, 4 patients (14%) excellent long-term toxicity and provides excellent long-term received BEAM (BCNU 300 mg/m² day -6, VP-16 800 mg/m² disease control of NHL. and cytarabine 1600 mg/m² in 4 days, melphalan 140 mg/m² day -1); ten patients (36%) high dose melphalan (200 mg/m², at day –1); 14 (50%) received TM regimen (Thiotepa 600 P662 mg/m² day -3, PAM 160 mg/m² day -1). In t-HDC the first Reduced-intensity conditioning allogeneic stem cell course of HDC consisted of MEL200 and the second one of transplantation for patients with refractory or relapsed BEAM. Overall survival (OS) and freedom from progression non-Hodgkin lymphoma (FFP) were calculated with Kaplan–Meier method. H. De Lavallade, R. Bouabdallah, C. Faucher, S. Furst, J. El Results: The CR rate was 71% in s-HDC and 87% after t-HDC Cheikh, N. Vey, A.M. Stoppa, D. Sainty, C. Arnoulet, L. Xerri, (p= 0.6), meanwhile the PR rate was 11% s-HDC and 17% in P. Cassier, J.A. Gastaut, D. Blaise, M. Mohty t-HDT. With a median follow-up time of 30.5 (1-102) months, Institute Paoli-Calmettes (Marseille, F) the 3-year overall survival and freedom from progression in s- HDC 66% and 52%, and in t-HDT were 83% and 77 %, This study aimed to evaluate the role of Reduced intensity respectively. In both groups, no statistical differences were conditioning (RIC) allogeneic stem cell transplantation (allo- seen in hematological and extra hematological toxicity and SCT) for relapsed or refractory non-Hodgkin s lymphoma 100-day treatment-related mortality was null. (NHL). We report here our experience in 25 consecutive Conclusions: Tandem HDC is feasible and safe in poor patients transplanted in a single center for high grade (n=17) prognosis NHL. It seems to improve clinical results compared or follicular NHL (FL; n=8). In the high grade NHL group, to a standard approach with single HDC. Long-term follow-up median age was 46 (range, 24-63) years, and all 17 patients of a larger cohort of patients and a randomized study will be received 2 or more previous chemotherapy regimens prior to needed to confirm the overall efficacy of this strategy and to RIC allo-SCT. In addition, 12 patients (71%) had failed evaluate long-term toxicity. autologous SCT and 6 patients (35%) had chemoresistant disease at time of allo-SCT. Among the 8 patients transplanted for a heavily pretreated follicular NHL (FL), P665 median age was 52 (range, 34-59) years and median number Primary refractory Hodgkin’s lymphoma: outcome after of prior lines of therapy was 3 (range, 2-5), with 3 patients high-dose chemotherapy and autologous stem cell (38%) having chemoresistant diseases and 4 patients (50%) transplantation and impact of various prognostic factors relapsing after autologous SCT. on overall and event-free survival. A single institution Among the 17 patients with aggressive high grade NHL, we result of 66 patients compared the outcome of T-cell and B-cell aggressive NHL. S. Akhtar, I. Maghfoor, A. El Weshi, M. Abdelsalam, H. With a median follow-up of 15.4 (range, 3.4-65.2) months, the Hussaini, I. Janabi, M. Rahal cumulative incidence of non-relapse mortality was 6%, King Faisal Specialist Hospital and Research Center (Riyadh, (95%CI, 0.3%-31%) and the Kaplan-Meier estimate of SA) progression-free survival (PFS) was significantly higher in the T-cell as compared to the B-cell group (P= 0.03; 100% vs. Purpose: The outcome of high dose chemotherapy (HDC) and 40% at 3 years). autologous stem cell transplant (ASCT) in primary refractory In the FL group, the cumulative incidence of non-relapse Hodgkin’s lymphoma (PR-HL) is not as encouraging as in mortality was 25% (95%CI, 3%-65%). Six patients (75%) relapsed HL. The purpose of this study is to report our showed objective disease response with complete remission experience with HDC and ASCT in PR-HL. (CR) occurring concomitantly to graft-versus-host disease, Patients and Methods: From 1996 to May 2006, 66 including one CR after donor lymphocytes infusion. With a consecutive patients with HL who failed induction median follow-up of 19 (range, 7-85) months, 6 patients from chemotherapy i.e. partial response (PR), no response (NR), the FL group are still alive of whom 5 in CR. stable disease (SD), progressive disease (PD) or relapsing We conclude that a potent graft-vs.-lymphoma (GVL) may be within 3 months of finishing the planned treatment received achieved in FL patients, even those with chemoresistant salvage chemotherapy followed by BEAM as HDC. Patients disease or who have relapsed after autologous SCT. In the with progressive disease on salvage chemotherapy were not high grade NHL group, strategies aiming to enhance the GVL eligible for HDC ASCT. Impact of various prognostic factors effect (Rituximab-based RIC and/or Rituximab maintenance prior to the initiation of salvage chemotherapy on events free therapy) in the B cell subtype are still needed. However, RIC survival (EFS) and overall survival (OS ) was evaluated using allo-SCT is a feasible and promising strategy for aggressive multivariate regression analysis NHL, with particularly low toxicity, and T-cell aggressive NHL Results: Male 35 (53 %), female 31 (47 %), Median age at benefiting most from a potent GVL effect, likely overcoming diagnosis 21 years and at ASCT 22.75 years. Prior to salvage the poor prognosis usually associated with this phenotype. chemotherapy, stages I:II:III:IV were 2:21:14:29, bulky disease

S149 (•8 cm) in 18 (27 %), involvement of mediastinum in 50 (79 ASCT. 10 (22%) had no response or progressive disease post %), spleen 17 (26 %) and extranodal-site in 31 (47 %) HDC ASCT. 26 (41%) patients with PR after ESHAP achieved patients. 23 (35%) had short lived CR, and 16 (24 %) had CR as a result of HDC ASCT. As of September 2006, 27 prior XRT. 38 patients (58 %) had tissue confirmation at (43%) patients are in CR. 20 (32%) had relapsed, 31 ( 49%) relapse/progression. ESHAP was used as first line salvage in had died. For the whole group, median OS from diagnosis is 60 patients (92 %); median cycles given were 3. Disease 68 months and from ASCT is 43 months. EFS is 44% for the evaluation post ASCT showed overall response in 50 patients whole group. Median OS for 45 relapsed patients is 98 (77 %); CR 43 (65 %), PR 14 (21 %), NR / SD 3 (5%) and PD months (from diagnosis) and 50 months (from ASCT ) and for in 13 (20 %) patients. 2 patients had treatment related 18 primary refractory patients, 28 months and 12 months mortality. 24 out of 53 patients (45 %) with no CR prior to respectively. OS from diagnosis and HDC ASCT was better in ASCT achieved CR after ASCT. 12 (28 %) of 42 patients in favor of relapse vs primary refractory disease; P = 0.015 and CR post HDC ASCT relapsed. Median followup: from p = 0.027 respectively. diagnosis 36 months and from HDC and ASCT 18 months; Conclusion: Although we have 70% CR rate in this group, EFS is 38 %; 31 (47 %) remain in CR, 19 ( 29 %) died of 46% of total CR patients have relapsed and EFS is only 43%. disease and 14 ( 21 %) were alive with disease at last follow- Out come of primary refractory disease is even worse. Better up. treatment strategies are urgently needed for these groups of Conclusion: In patients with PR-HL, ESHAP + BEAM patients. combination results in 76 % response rate with 43 % CR rate. Despite this high responsiveness, almost quarter of patients with CR relapsed. 29 % patients died due to disease. At a median follow up of 18 months, EFS is only 38 %. We failed to identify impact of any prognostic factor on EFS and OS. Better understanding and treatment strategies are needed for this group of patients.

P667 The role of bone marrow graft in autologous stem cell transplantation in patients with non-Hodgkin lymphoma E. Juvonen (1), E. Elonen (1), L. Volin (1), A. Räsänen (2), A. Nihtinen (1), F. Ebeling (1), T. Ruutu (1) (1)Helsinki University Central Hospital (Helsinki, FIN); (2)Kotka Central Hospital (Kotka, FIN)

High dose chemotherapy and autologous stem cell transplantation is an established treatment for high risk or relapsed NHL. Peripheral blood stem cell (PBSC) graft has displaced bone marrow (BM) because of faster engraftment and benefits both in the harvest and handling of the graft. However, in heavily pretreated patients the mobilization may fail and BM may remain the only available source of graft. We P666 have analyzed the outcome of autologous transplantation in ESHAP as salvage followed by BEAM as high-dose NHL according to the type of the graft. 106 adult patients chemotherapy and autologous stem cell transplant for treated at the Helsinki University Hospital in 1991–2006 were relapsed or refractory diffuse large cell non-Hodgkin studied retrospectively. The graft was BM in 26 and PBSC in lymphoma. A single-centre experience of 63 patients 80 cases. Marrow graft was used in most cases because of S. Akhtar, A. Al-Zahrani, M. Rahal, A. El Weshi, S. mobilization failure (17/26) or heavy pretreatment predicting Bazarbashi, D. Ajarim, Y. Khafaga, I. Maghfoor poor mobilization. According to the WHO classification the King Faisal Specialist Hospital and Research Center (Riyadh, type of NHL was diffuse large B-cell in 40 patients, follicular in SA) 38 and transformed follicular in 5 patients, peripheral T-cell in 7 patients, anaplastic T-cell in 8 patients, and other in 8 Objective: To evaluate the outcome of patients treated with patients. Of the patients 54 were males and 52 females. At high dose chemotherapy and autologous stemcell transplant transplantation the median age was 48 years (range 20–65 (HDC ASCT) for relapsed or refractory diffuse large cell years). 40 patients were in CR1, 38 in PR1. In 7 patients the lymphoma (DLCL) at our institution. disease was primarily refractory. 21 patients were in •CR2 or Material and Methods: From 1996 to May 2006, 63 PR after relapse. Before the intensive treatment the median consecutive patients with DLCL who received either ESHAP number of CHOP-like treatments given was 10 (range 4–24) alone or ESHAP alternating with some other salvage in the BM group and 7 (3–16) in the PBSC group. The median chemotherapy regimen prior to ASCT were identified. number of mononuclear cells in the BM grafts was Responding patients received ESHAP for mobilization of 0.33x108/kg (range 0.11–1.4). In the PBSC grafts the median PBSC followed by BEAM as HDC. Refractory disease is number of CD34+ cells was 4.0x106/kg (0.46–13.2). The defined as patient who failed induction chemotherapy i.e. median engraftment time (neutrophils >0.5x109/l) in the BM partial response (PR), no response (NR), stable disease (SD) group was 18 days (9–93) and in the PBSC group 12 days (8– or progressive disease (PD). 33). The median follow-up of all patients was 55 months (0– Results: Males 36 , females 27, median age at diagnosis 35 175), in the BM group 95 months (0–175), and in the PBSC years ( 14 to 67 years) and at transplant 41 years ( 14 to 67 group 49 months (1–159). 84 patients (79%) were alive; 19 years). Stage at initial diagnosis I (13), II (16), III (14), IV (18). patients (73%) in the BM group and 65 (81%) in the PBSC Initial chemotherapy was CHOP in 50 patients (79%). (71%) group (p=0.53). At five years the cumulative progression free had prior CR / CRu, 17 (27%) patients had primary refractory survival was 0.81 in the BM group and 0.82 in the PBSC disease. 44 patients (70%) had 3 cycles and 10 (16%) had 4 group. 23 patients had died. The cause of death was relapse cycles of ESHAP. 18 (28%) had CR /CRu prior to HDC ASC. in 11 cases, treatment complication in 7, and other in 4 cases. Median follow up is 35 months from diagnosis and 18 months after HDC ASCT. 44 (70%) patients had CR / CRu after HDC

S150 Conclusion: In patients with mobilization failure, last years, the use of the drug has been restored due to the transplantation using a bone marrow graft is a good option ability of significantly increase the dose 10-22 fold, in and may give results comparable to those achieved with comparison to only 3-5 fold in most of other anti-tumor agents, PBSC graft. while rescue was done by reinfusion of cryopreserved autologous bone marrow or peripheral stem cells. Yet, there is no sufficient information about its administration in high doses P668 with or without other chemotherapeutic agents in lymphatic Long-term remission in a patient with refractory malignancies. hepatosplenic gamma-delta T-cell lymphoma treated with Methods: One day following inoculation of BALB/c mice with bortezomib and high-dose CHOP-like chemotherapy between 105 to 108 BCL1 leukemia cells (simulating various followed by autologous peripheral stem cell pre-transplant leukemia loads) each mice group received one transplantation of the "induction-like" irradiation and/or cytotoxic containing Z. Otrock, H. Hatoum, Z. Salem, A. Tawil, G. Zaatari, A. conditioning regimens simulating the common BMT induction Bazarbachi methods. Five to 11 days from the end of treatment, adoptive American University of Beirut (Beirut, LBN) transfer to untreated BALB/c mice was done in order to evaluate the effect of the induction treatment and the residual Hepatosplenic gammadelta T-cell lymphoma (HS GD TCL) is disease. Recipients were observed for the development of a distinct clinico-pathological entity among peripheral T-cell leukemia for at least 150 days. Splenomegaly and peripheral lymphomas (TCL) with an extremely poor prognosis. Recently, blood lymphocyte counts were monitored to confirm presence we have shown that the selective proteasome inhibitor of leukemia and to determine if leukemia was indeed the bortezomib (Btz) induces cell growth arrest and apoptosis in cause of death. malignant but not normal T cells supporting a potential Results: given alone, high dose TH did not change the time for therapeutic role for Btz in TCL (Nasr et al Oncogene appearance of leukemia. The cyclophosphamide (CY) 2005;24:419-30). containing regimens were the most effective ones. Combining We report a 33 year-old woman who presented with CY with TH showed borderline significance (p=0.067) when hepatosplenomegaly and pancytopenia. Bone marrow biopsy AT was done on day 5 as compared to the BU-TH was hypercellular with increased erythroblasts and combination. Additionally, in a different study, a synergistic dyserythropoiesis. Laparoscopic splenectomy revealed effect have been seen in the combination of TH-CY (none of expansion of the red pulp with dilatation of the sinusoids the animals developed leukemia) whereas 4/10 animals in the containing atypical lymphoid cells and abundant CY-TBI group developed leukemia (p=0.029, figure 1). hemophagocytosis. Liver biopsy revealed diffuse sinusoidal Conclusion: although TH did not show a significant effect dilatation containing the same atypical lymphoid cells which against BCL1 leukemia, its combination with CY is promising. were strongly + for CD2, CD3, CD7, CD56, TiA-1 and CD45RO while CD5 was negative. Molecular testing confirmed the rearrangement of the gammadelta chains of the T-cell receptor and the diagnosis of HS GD TCL. She received 4 cycles of platinum-cytarabine based therapy (ESHAP). Repeat liver biopsy showed minimal response. After informed consent, she received Btz (Velcade 1 mg/m²/day on days 1 and 5) with a modified high dose CHOP chemotherapy (ACVB) with G-CSF support every 2 weeks for 4 cycles. Because of potential additive peripheral neurotoxicity with Btz, vincristin was deleted from the first 2 cycles. This treatment was well tolerated; two cycles were complicated by febrile neutropenia and 2 cycles by transient grade 3 thrombocytopenia. Laparoscopic liver biopsy showed almost complete remission (CR) with a single small atypical lymphoid infiltrate. The patient then underwent stem cell mobilization with high dose cyclophosphamide+G-CSF followed by stem cell collection and cryopreservation. She then received BEAM conditioning regimen followed by autologous peripheral stem cell transplantation (APSCT). The post-transplant course was P670 relatively smooth with WBC and platelet engraftment at 9 and High-dose therapy with autologous transplantation for 6 days respectively. Three years after diagnosis and 26 relapsed and refractory Hodgkin’s lymphoma: a single- months after transplantation the patient is still in CR. These centre experience data demonstrate that the combination of Btz and high dose E. Cocorocchio, D. Laszlo, S. Steffanoni, A. Vanazzi, P. CHOP-like chemotherapy followed by APSCT is a feasible Bertazzoni, S. Bassi, F. Gigli, L. Calabrese, D. Radice, F. and potentially efficient way to treat HS GD TCL. The Peccatori, G. Martinelli remarkable efficacy of this combination in a patient with HS European Institute of Oncology (Milan, I) GD TCL stresses the need for further trials of this regimen in peripheral TCL. High-dose chemotherapy (HDT) with peripheral blood stem cell support can provide sustained remissions in pts with refractory/relapsed HL. We report a retrospective analysis of P669 71 consecutive HL (34 recurrent and 37 refractory) pts who The effect of high-dose thiotepa, alone or in combination were submitted to HDT between 1995 and 2006 in our with other chemotherapeutic agents on murine B-cell institution. The major part of pts had nodular sclerosis HL (63 leukaemia model simulating autologous stem cell pts). Stage at diagnosis was II in 45 pts and III-IV in 26 pts. transplantation ABVD, MOPP, hybrid regimens represented the first-line A. Abdul Hai, M.Y. Shapira, L. Weiss, S. Reich, S. Slavin therapy in 36 of pts while 35 pts received a chemo- Hadassah – Hebrew University (Jerusalem, IL) radiotherapy combination. Median age at transplantation time was 32 years (range18-56). Before HDT, pts had been treated Introduction: Thiotepa (TH), an ethylene amide, developed by with a median of two chemotherapy lines (range 1-4). Two Lederle Laboratories in 1952, possesses mechlorethamine- cycles of ESHAP regimen was the induction therapy. At the like alkylating activity and has been used clinically more than time of transplantation, 21 (29 %) pts were in CR, 37 (52%) in 40 years. It is utilized especially in breast cancer, PR, 8 (11%) had SD/PD and for 5 pts response data was not carcinomatous meningitis and for bladder carcinoma. In the

S151 available. All pts were conditioned with chemotherapy: 59 lymphoma. Monoclonal anti-CD20 therapy (Rituximab) has received BEAM and 13 pts Idarubicine (15mg/sqmx3d)/LPAM been available since November 2002 in Hungary. In vivo (180mg/sqm) combination. Radiotherapy was performed after purging with Rituximab is proven to prolong survival after transplantation in 13 pts. No treatment related mortality was hemopoetic stem cell transplantation. observed and engraftment was achieved in all pts. Two pts Methods: Main demographic data, the number of apheresis, developed ITP after 43 and 10.5 months from transplant and the cryopreserved stem cell count and the transplantation one patient developed an AML after 49 months from outcome were compared in stem cell mobilization regimens transplant. Fifty-two (73.2%) out of 71 pts achieved CR (27 of containing Rituximab (Group A) with regimens without recurrent and in 25 of refractory pts), 8/71 (12%) a PR and Rituximab (Group B). 11/71 (15%) a SD/PD. With a median follow-up of 23 months Results: Figure (range 2-112), 44/71 pts are in continuous CR, while 26/71 Conclusions: Addition of Rituximab to mobilization regimens progressed and twelve died because of their disease. Our has no adverse effects on stem cell harvesting (number of data support the activity of HDT in this subset of pts, mainly in apheresis procedures, CD34 + cell counts and time to the relapsed pts (DFS 70% in relapsed vs 54% in refractory engraftment). It is safe and produces durable remissions. pts). Refractory pts can also benefit of the salvage therapy although more efficacious alternative therapeutic options should be proposed; the role of non-myeloablative transplant procedure in pts with an identical sibling donor after HDT is under investigation.

P671 Cyclophosphamide or disease-specific chemotherapy for stem cell mobilization in patients with relapsed diffuse large B-cell lymphoma? E. Jantunen (1), S. Leppä (2), T. Kuittinen (1), L. Keskinen (3), O. Kuittinen (4), M. Mokka (5), T. Turpeenniemi-Hujanen (4), T. Nousiainen (1), K. Remes (5), T. Lehtinen (3), T. Wiklund (2) (1)Kuopio University Hospital (Kuopio, FIN); (2)Helsinki University Central Hospital (Helsinki, FIN); (3)Tampere University Hospital (Tampere, FIN); (4)Oulu University Hospital (Oulu, FIN); (5)Turku University Central Hospital Multiple myeloma (Turku, FIN)

Optimal strategy for progenitor cell mobilisation in patients P673 with relapsed diffuse large B-cell lymphoma (DLBCL) is Allogeneic stem cell transplantation in patients with high- unknown. We analysed efficiency and toxicity of mobilisation risk de novo or relapsed multiple myeloma. A single- therapy in this patient category. Between 1998 and 2005 centre experience altogether 88 patients with relapsed DLBCL (58 M, 30 F) with S.O. Schonland, S. Gerull, M. Goerner, T. Moehler, H. a median age of 53 yrs (23-69) received either Goldschmidt, A.D. Ho, P. Dreger, U. Hegenbart cyclophosphamide (CY) plus G-CSF (N=45) or disease- University of Heidelberg (Heidelberg, D) specific chemotherapy (CT) plus G-CSF (N=43) for mobilisation of progenitor cells in five Finnish centres. The Background: Allogeneic hematopoietic stem cell most common CT regimens were CHOP-like (N=12), VIG transplantation (allo-SCT) is currently being used as upfront or (N=10) and dexa-BEAM (N=8). At least 2 x 106/kg CD34+ salvage therapy for patients with multiple myeloma (MM). cells were collected after the first mobilisation attempt in 69 Methods: We performed a retrospective analysis of all patients patients (78 %). This goal was achieved in 82 % of patients in with MM (n=89) who underwent allo-SCT in our institution CY-group and in 72 % of patients mobilised with CT + G-CSF between 2000 and 2006. Indications for upfront allo-SCT were (P=NS). No differences were observed in the median number chromosome 13 deletion, beta-2-microglobulin >3 mg/l (n=27) of aphaeresis between these groups (2 vs. 2), but the median or primary progressive disease (n=4). Indications for allo-SCT number of CD34+ cells collected tended to be higher in as salvage treatment were relapse after auto-SCT (n=47) or patients mobilised with CY + G-CSF (5.0 vs. 3.4 x 106/kg, progressive MM after first-line therapy (n=11). In all upfront P=0.069). Neutropenic fever was more common in the CY- patients, allo-SCT was applied 3-6 months after an auto-SCT. groups (38 % vs. 14 %, P=0.03) and the in-hospital stay Conditioning with 2 Gy total body irradiation and fludarabine during aphaeresis and supportive care longer (5 vs 3 days, (90 mg/m²) was used in all but 6 patients who received P=004). Rituximab was used during relapse treatment in 26 % melphalan 140 mg/m² + fludarabine 90 mg/m². Of note, no of the patients but did not have effect on mobilisation patient had in-vivo or ex-vivo T cell depletion. Median age was efficiency. In this retrospective survey, no clear preferences 52 (range 29-69) years. Donors were related in 49 patients suggesting superiority of one approach over another was and unrelated in 40 patients. In 24 patients at least one observed. Thus local preferences and need for effective antigen or allele mismatch out of ten loci was present. Twelve disease control seem to be decisive in regard to optimal patients (13%) were in CR prior to allo-SCT. mobilisation strategy in this patient group. Since a significant Results: As best response, 32/81 patients (40%) achieved CR proportion of patients with relapsed DLBCL are difficult to after allo-SCT. After a median follow-up of 17 months (range mobilise with either strategies, novel agents for mobilisation 0,5 - 76), median event-free survival (EFS) and overall are needed. survival (OS) were 9 months and 39 months, respectively. Incidence of chronic GvHD was 79% (2/3 extensive). Treatment-related mortality (TRM) was 16%. Significant P672 factors for OS and EFS were achievement of CR after allo- Therapeutic impact of Rituximab® containing stem cell SCT (p<0.001) and the presence of chronic GvHD (p<0.01). mobilization regimens in diffuse large B-cell lymphoma Patients with upfront SCT showed a significant prolonged OS A. Szomor, M. David, T. Vidra, H. Losonczy (p=0.04), but EFS was not different. No significant differences University Pecs (Pecs, HUN) regarding OS and EFS were observed between related vs. unrelated donors and matched vs. mismatched donors. Objectives: Two hundred and fifteen patients underwent Conclusions: Disease recurrence or progression remains the autologous hemopoetic stem cell transplantation in our clinic. major cause of treatment failure after allo-SCT in high risk Fifty four of them (25%) had diffuse large B-cell (DLCL) MM. The favorable impact of chronic GVHD on disease

S152 control confirms the importance of the GvM effect. Further mobilization with Cyclophosphamide 5 g/m² and granulocyte concepts are necessary to increase CR rates, to prevent colony-stimulating factor and 1 course of melphalan 200 relapse or progression e.g. using maintenance strategies, and mg/m2 immediately prior to ASCT. To detect p53 deletions, a to reduce TRM after allo-SCT. Spectrum Red-labeled DNA probe (LSI p53 Vysis) specific for the p53 locus on 17p13.1 was combined with a Spectrum Green-labeled probe for the chromosome 17 alphasatellite- P674 DNA centromere. Allogeneic stem cell transplantation in multiple myeloma: Results: An interstitial p53 deletion identified by one red (p53) single-centre experience of 106 patients and 2 green (CEP17) signals was detected in 7 of 50 patients. H. Uotinen, L. Volin, E. Juvonen, A. Nihtinen, T. Ruutu The median percent of myeloma cells with an interstitial p53 Helsinki University Central Hospital (Helsinki, FIN) deletion was 47% (range, 28%-90%). A FISH analysis informative for t(4;14),13q status and t(11;14) was available At the Helsinki University Central Hospital 106 patients with for all cases. Patients with p53 deletions had significantly multiple myeloma (MM) have been treated with allogeneic higher serum calcium and creatinine levels, but there was no stem cell transplantation (SCT) since 1987. The conditioning significant correlation between p53 status and C-reactive was at first myeloablative (MA), but since 1999 reduced protein (CRP), albumin level and lytic bone lesions. The intensity conditioning (RIC) after autologous SCT has mostly overall response rates were similar in patients with and been used. MA conditioning has been considered only in without p53 deletions (65% versus 69%, respectively). young patients with aggressive disease. Of the patients 53 However, patients with p53 deletions had significantly shorter were male and 53 female. The median number of PFS (median,8.1 months) than patients without p53 deletions chemotherapy lines was 1 (range 1-7) before allogeneic SCT. (median, 26.1 months, OS was also significantly shorter In addition, prior autologous SCT had been performed to 55 (median, 13.7 months) for patients with a p53 deletion than for patients. The median time from diagnosis to allogeneic SCT patients without a deletion (median, 46.1 months). was 12 (4-168) months, and the time between autologous and Conclusion: p53 deletions in pts with multiple myeloma treated allogeneic SCT 6 (2-146) months. At the time of allogeneic with high-dose chemotherapy is an independent risk factor for SCT 13 patients were in CR, 73 in PR, 3 in MR, 4 had SD, both PFS and OS. and 13 showed progression. The median age at the time of allogeneic SCT was 49 (27–65) years. 90 patients had a sibling donor: 86 were HLA -identical, one 1 antigen P676 mismatch, and 3 syngeneic. 16 patients had an HLA-matched Reduced-intensity conditioning allogeneic stem cell unrelated donor. The conditioning was MA in 52 and RIC in 54 transplantation as salvage treatment for relapsing patients. The MA conditioning consisted of Cy/TBI in 37, multiple myeloma: a "donor" vs. "no donor" Cy/Bu in 4, HDMel/TBI in 3, and Treosulphan/Fld in 8 patients. comparison RIC was the Seattle protocol in 45, reduced Treosulphan/Fld H. De Lavallade, P. Ladaique, C. Faucher, S. Furst, J. El in 8, and Fld/Cy in 1 patient. 44 patients received a BM graft Cheikh, A.M. Stoppa, D. Coso, R. Bouabdallah, N. Vey, D. and 62 patients a PB graft. As GVHD prophylaxis, 27 patients Sainty, C. Chabannon, J.A. Gastaud, D. Blaise, M. Mohty were given Cya/Mtx, 28 Cya/Mtx/MP, 3 Mtx/MP, 45 Cya/MMF, Institut Paoli-Calmettes (Marseille, F) and 3 nothing (syngeneic). The median follow-up time from allogeneic SCT was 34 (1.6–206) months. Until 1995 MA The role of RIC allo-SCT in MM is still controversial. This allogeneic SCT was performed to 18 heavily treated patients single centre study aimed to evaluate RIC allo-SCT for and all but one of them have died. Among the 88 (34 MA and relapsed MM, using a genetic randomization through a donor 54 RIC) patients transplanted since 1995 the OS was 50% at vs. no donor comparison. Between 2002 and 2005, 32 61 months post SCT and there were no deaths after 61 patients with relapsed or refractory MM, and with an identified months. The cumulative incidence of acute GVHD grade 2-4 sibling, were referred to our centre for HLA typing. In all, 19 was 28%. The cumulative incidence of chronic GVHD was patients (59%; donor group) had an HLA-identical sibling 72%, 58% in the MA and 83% in the RIC patients (p=0.074). donor, while the remaining 13 pts (41%; no donor group) had There was no statistical difference in the incidence of acute or no HLA-identical sibling donor. There were no significant chronic GVHD by donor (72 siblings/16 unrelated). 100-day differences between these two groups that were comparable transplant related mortality was 4.5%. Of the 60 patients alive, as for demographic, disease and prognosis factors. Median the maximum response achieved so far is CR in 28, PR in 19 age was 54 (range, 37-65), and all pts had previously failed and 13 have progressive disease. The cause of death in the auto-SCT. Pts from the no donor group received salvage patients transplanted after 1995 was MM in 17, GVHD in 7, therapy including thal., bortezomib, dex., and/or additional and infection in 4 patients. In conclusion, in the present high dose chemotherapy. material early transplant related mortality is low and the Among the 19 pts from the donor group , 18 (95%) could survival is encouraging. proceed to allo-SCT. With a median overall FU of 36 m., 11 patients (85%;95%CI:54-98%) from the no donor group had disease progression despite salvage therapy, and only 6 of P675 them are still alive, of whom 5 (80%) in progressive disease at Adverse prognostic factor of p53 gene delection detected last FU. In contrast, only 5 pts (28%;P=0.001) from the donor by FISH in multiple myeloma following autologous stem group progressed after RIC allo-SCT. In the RIC allo-SCT cell transplantation group, 10 pts (56%;95%CI;33-79%) are still alive, with 4 pts V. Pitini, G. Altavilla, C. Arrigo, C. Naro, L. Siracusano, P. being in CR, and 5 in PR or VGPR. Only one patient is Sciarrone currently experiencing disease progression and receiving University of Messina (Messina, I) salvage therapy. Interestingly, 11 pts (61%;95%CI,39-83%) from the RIC allo-SCT group showed objective disease In multiple myeloma the frequency of p53 deletions detected response, usually concurrent to chronic GVHD. In all, 6 pts by fluorescence in situ hybridization (FISH) is reported to died from TRM for an overall incidence of TRM of 33% range from 9%to 34% and associated with poor survival. We (95%CI,11-55%) in this population of heavily pretreated and investigated the relevance of p53 deletions to the clinical relapsed or refractory MM population. In an intention-to-treat outcome of patients with multiple myeloma (MM) treated with analysis, the KM estimate of progression-free survival was high-dose chemotherapy and autologous stem cell significantly higher in the donor group as compared to the no transplantation (ASCT). donor group (P=0.01;46%vs.8% at 3 y.; Fig. below). In all, Material and Methods: Between January 1998 and December these results compare favorably with those achieved using 2003, 50 patients were diagnosed and treated for MM with other standard non-allo-SCT salvage therapies for relapsed high-dose chemotherapy followed by ASCT. All patients MM. Therefore, RIC allo-SCT from an HLA-identical sibling is received 4 cycles of the VAD regimen (Vincristine, a feasible and potential therapy that should be proposed for Adriamycin, and Dexamethasone) followed by stem cell refractory or relapsed MM, since a potent graft-vs.-MM effect

S153 can be induced despite heavy pretreatments, allowing for P678 significantly longer PFS. Also, the latter results are expected Salvage therapy with bortezomib for advanced multiple to be further improved with the systematic and early use of myeloma after reduced-intensity conditioning allogeneic maintenance therapies after RIC allo-SCT. stem cell transplantation J. El Cheikh, A.M. Stoppa, T. Aurran, J. Rey, R. Bouabdallah, N. Vey, D. Coso, V. Ivanov, A. Charbonnier, C. Faucher, J.M. Schiano de Collela, J.A. Gastaut, D. Blaise, M. Mohty Institut Paoli-Calmettes (Marseille, F)

Reduced intensity conditioning (RIC) allogeneic stem cell transplantation (allo-SCT) is currently under investigation for advanced multiple myeloma (MM) with promising results in terms of feasibility and toxicity. However, MM progression is still a matter of concern after such approaches. Bortezomib has demonstrated activity and safety in heavily pre-treated patients with relapsed and/or refractory MM. In addition, animal data suggested that bortezomib may have immunomodulatory effects impacting GVHD activity. This report describes the outcome of 12 MM pts who received bortezomib (1.0 or 1.3 mg/m2 IV bolus on days 1, 4, 8, and 11, every 21 days) as salvage therapy after progression following RIC allo-SCT. Median age of pts was 51 (27-58) y. and all patients have failed auto-SCT prior to RIC allo-SCT. Median duration between allo-SCT and bortezomib initiation was 17 (1-65) months. 7 pts failed thalidomide prior to bortezomib. Also, 5 patients failed to respond to DLI. At time of bortezomib P677 initiation, 9 pts (75%) were in progressive disease and 3 pts Features and risk factors of peripheral neuropathy during (25%) were in PR. None of the 12 pts had active GVHD, and treatment of advanced multiple myeloma with bortezomib all of them were off immunosuppressive therapy. Patients J. El Cheikh, A.M. Stoppa, S. Duran, T. Aurran, V. Ivanov, J. received a median of 8 (1-13) cycles of bortezomib (+dex. in 8 Rey, R. Bouabdallah, N. Vey, D. Coso, A. Charbonnier, C. pts). During bortezomib therapy, 2 pts experienced limited Faucher, J.M. Schiano de Collela, J. Camerlo, J.A. Gastaut, chronic GVHD that did not require systemic D. Blaise, M. Mohty immunosuppression. Among other toxicities, 3 pts Institute Paoli-Calmettes (Marseille, F) experienced exacerbation of prior peripheral neuropathy that was manageable through bortezomib dose reduction. Also, 4 The aim of this retrospective single centre study was to cases of mild (grade 1 or 2) thrombopenia were encountered. determine the frequency, characteristics, and reversibility of With a median FU of 7 (2-35) m. from bortezomib initiation PN from bortezomib treatment of 100 consecutive advanced and 48 (16-123) m. from RIC allo-SCT, 7 pts (58%) had an multiple myeloma (MM) patients treated with bortezomib. PN objective disease response (1 CR, 3 VGPR, 3 PR) and 1 was evaluated by investigator neurological examination at patient had a SD. 4 pts continued to progress (of whom 3 died baseline, during the study, and at last follow-up. Patients of their disease). characteristics at baseline prior to bortezomib initiation were In all, we conclude that bortezomib is an efficient salvage as follow: median age: 60 (range,27-77), prior history of therapy without induction of significant deleterious GVHD. diabetes: n=8 (8%), prior auto-SCT: n=76 (76%), prior Drug-related side effects were limited and manageable after treatment with thalidomide: n=75 (75%) with a median dose of dose reduction. Thus, it represents a valid option for MM pts 200 (range,50-600) mg for a median duration of 8 (range,1- progressing after RIC allo-SCT. These results provide the 61) months. Median duration between thalidomide framework for testing the earlier use of bortezomib in MM pts discontinuation and bortezomib initiation was 5 (range,0-43) undergoing RIC allo-SCT, especially those pts who do not m. Before treatment with bortezomib, 48 pts (48%) already develop alloimmune reactions and/or those pts who do not had some form of PN [grade 1,n=27(56%);grade achieve disease response early after allo-SCT. 2,n=16(33%); grade 3,n=5(11%)]. With a median FU of 8 (range, 0.1-32) months from bortezomib initiation, pts from this series received a median of 4 (range, 1-12) cycles of P679 bortezomib. Bortezomib-related emergent PN was observed in Role of PET-TAC in multiple myeloma staging at 38 pts (38%;95%CI,28-47%), with grade 1, 2, 3, and 4 PN diagnosis and in restaging after high-dose therapy occurring in 17 (45%), 15 (39%), 5 (13%) and 1 (3%) pts F. Patriarca (1), R. Mestroni (1), S. Buttignol (1), E. Englaro respectively. Median time to onset of bortezomib-related PN (2), F. Zaja (1), A Sperotto (1), A. Geromin (1), A. Candoni (1), was 53 (range, 11-182) days after bortezomib initiation. In O. Geatti (3), R. Fanin (1) most cases (n=30; 79%), pts had sensory symptoms, while 8 (1)Clinica Ematologica (Udine, I); (2)Istituto di Medicina pts (21%) experienced both sensory and motor symptoms. Nucleare (Udine, I); (3)Istituto di Medicina Nucleare (Udine, I) Bortezomib-related PN led to dose reduction or discontinuation in 18 pts. Of the 38 pts with bortezomib- Very little information is available regarding the diagnostic related PN, resolution to baseline or improvement occurred in utility of PET-CT in multiple myeloma (MM) patients. The 20 (53%) pts, at a median time of 3 (range, 1-8) m. objective of this study is to define the role PET-CT in In multivariate analysis, the total number of cycles of assessing the extent of active disease at the time of initial bortezomib (less or more than 4 cycles), and a prior history of presentation and in evaluating treatment response in MM. We treatment with thalidomide were the strongest parameters studied 25 previously untreated MM patients at diagnosis, significantly associated with an increased incidence of after induction therapy and 3 months after ASCT. The results bortezomib-related PN (P=0.03; OR=2.6;95%CI,1.1-6.1 and of PET-CT scans were compared with standard whole skeletal P=0.02;OR=3.9;95%CI, 1.2-12.6 respectively). The finding survey and whole spine Magnetic Resonance (MR) at that the development of bortezomib-related PN seems to be diagnosis and with clinical response to treatment at restaging. dependent of the patient history of prior neurotoxic therapy Thirteen patients were male, 12 female, median age was 59 with thalidomide, raises the question of the optimal sequence years (range 44-69), 14 had IgG, 9 IgA monoclonal protein and schedule of administration of these anti-MM effective and one had a Bence Jones MM. Two patients had stage IA drugs as single agents, but also in combination. MM, 4 had stage IIA and1 9 stage IIIA MM according with Durie and Salmon classification. The patients underwent induction therapy consisting of thalidomide plus

S154 dexamethasone or VAD for 4 months, then proceeded to These preliminary data demonstrate that the detection and the collection of PBSC mobilized by 4 g/m² Cyclophosphamide + follow-up of RQ-PCR chimerism status in purified CD138+ BM G-CSF and myeloablative treatment with 200 mg/m² cells after allo-SCT is feasible and provides specific early Melphalan. information on the kinetics of malignant PC and might be a At diagnosis PET-CT scans were negative in 10 patients useful tool to guide early therapeutic interventions after RIC (40%) and positive in 15 patients (60%). PET-CT scans and allo-SCT in order to improve results and curability of this skeletal radiographic findings were concordant in 16/25 disease. patients (64%). In 5/25 cases (20%) PET-CT scans detected more disease sites than skeletal survey or MR (all bone lesions). MR showed a focal pattern of bone marrow involvement of a few vertebras in 4 patients, who had both negative radiographic and PET-TAC findings. Twenty-one patients had a restaging after induction chemotherapy (3) or after ASCT (18). Out of 16 responsive patients, 9 had positive PET-CT scans at diagnosis: 5 showed a significant reduction of the number and the extension of the sites of abnormal uptake after therapy and 4 patients, who obtained a complete remission (negative serum immunofixation) after ASCT, achieved a negative PET-CT scan. We conclude that in our series of newly diagnosed MM PET- CT could identify additional sites of diseases to skeletal survey or MR in 5/25 (20%) of the patients. In other 4/15 patients (16%) whole spine MR could identify minimal involvement of single vertebras, that were not recognized by PET-CT and skeletal survey. Moreover, all the 9 patients with pre-treatment positive PET –CT scans showed a reduction (5) P681 or a disappearance (4) of the sites of abnormal uptake after Pharmacokinetics of AMD3100 in volunteers with renal therapy, that correlated with clinical response. impairment R. McFarland, R. Scarborough, S. Becker, A. Khong, K. Badel, G. Calandra P680 AnorMed (Langley, CAN) Interests of CD138+ plasma cells chimerism follow-up by real-time quantitative PCR for multiple myeloma patients Background: High-dose therapy with autologous stem cell after reduced-intensity conditioning regimen following transplantation improves survival in patients with myeloma allogeneic stem cell transplantation and lymphoma. AMD3100, a selective antagonist of CXCR4, C. Martin (1), F. Nicolini (2), I. Mollet (1), V. Ortiz-Corbella (2), mobilizes long-term repopulating hematopoietic stem cells C. Giannoli (1), N. Raus (2), A. Thiebaut (2), D. Revesz (2), V. with adequate homing and engraftment capacity. As renal Dubois (1), M. Michallet (2) impairment (RI) is common among myeloma pts, we (1)HLA laboratory (Lyon, F); (2)Hematology Department evaluated the pharmacokinetic characteristics of this (Lyon, F) investigational agent in volunteers with varying levels of RI. Methods: This open-label study examined the Multiple myeloma (MM) is a clonal disorder characterized by pharmacokinetics of single 240 ug/kg subcutaneous doses of the accumulation of malignant plasma cells (PC) in the bone AMD3100; pharmacokinetic parameters evaluated included marrow (BM) and the secretion of a paraprotein. This disease area under the concentration time curve from 0 to 24 hours can be cured by conventional allogeneic stem cell (AUC0-24), half-life (t1/2), renal clearance (CLr), and systemic transplantation (allo-SCT) with high transplant-related clearance (CLs). Results: Renal excretion was determined to mortality (TRM). Reduced intensity conditioning (RIC) be a major mechanism of clearance for AMD3100, with 60 % regimens followed by re-infusion of allogeneic stem cells have of the dose excreted unchanged in the urine of volunteers with been proposed to reduce the TRM and keep a graft-versus- normal renal function over 24 hours post administration. myeloma (GVM) effect. However, this technique is hampered Pharmacokinetic characteristics for volunteers with either no, by important rates of relapse and the monitoring of chimerism mild, or severe RI are summarized in Table 1. Compared to status is of help to modulate the immunosuppressive regimen volunteers with normal renal function (CLcr > 90 ml/min), and thus, the GVM effect by allogeneic effector cells. To date, AUC0-24 increased 40 % in volunteers with moderate RI STR chimerism status was followed-up in total peripheral (CLcr = 31-50 ml/min) and 37 % in those with severe RI blood (PB) or total BM cells after transplantation but these (CLCR < 31 ml/min). Half-life increased from 4.9 hours in methods failed to detect early relapses. In order to improve volunteers with normal renal function, to 11.6, and 17.3 hours the follow-up, we have used a BM CD138+ cell enrichment in those with moderate and severe RI respectively. Renal technique combined to real-time quantitative PCR (RQ-PCR) clearance of AMD3100 decreased from 40.3 ml/min in to assess the chimerism status of such cells after allo-SCT. volunteers with normal renal function, to 15.3 and 5.0 ml/min Fourty-two samples from 17 MM patients (pts) have been in those with moderate and severe RI respectively. analysed from 72 days to 4 years after conventional (1) or RIC Conclusions: The increase of T1/2 in pts with RI is consistent allo-SCT (16 pts). Samples were analysed from BM using a with renal excretion being a major clearance mechanism for CD138 cell purification technique (Miltenyi Biotec®). DNA was AMD3100. As the effect of RI on systemic exposure to extracted and RQ-PCR was performed as previously AMD3100 was < 50% increase in AUC, utilization of described by Alizadeh. Mixed Chimerism (MC) was defined as AMD3100 for stem cell mobilization at the 240 ug/kg dose in the presence of • 0.2% recipient cells (RC). The purity of MM pts with RI is feasible. CD138+ after enrichment was on average 19.6% (1.34-93.5) on all BM samples. For all 17 pts, at least once, while total BM cell chimerism was total donor (TD), CD138+ assessment detected RC (3.2-8.6%) at • 2 years post-allo SCT. Three pts exhibited increased CD138+ RC few weeks prior to clinical relapse (Figure 1). In other 3 pts, the impact of therapeutic measures (DLI, immunosuppressive modulation, bortezomib) was closely correlated to variations of CD138+ chimerism status. A stable CD138+ MC with constant levels of serum paraprotein demonstrated the absence of significant relapse in one patient.

S155 P682 P683 The assessment of CD138+ cells chimerism after Outcomes of autologous transplantation in multiple reduced-intensity conditioning allogeneic stem cell myeloma: an analysis of 408 patients from the Czech transplantation is able to identify early marrow relapse in Transplant Registry multiple myeloma patients M. Krejci (1), R. Hajek (1), B. Vackova (2), V. Maisnar (3), E. V. Ortiz-Corbella (1), F. Nicolini (1), C. Martin (2), S. Faber (4), E. Gregora (5), A. Vitek (6), A. Svobodnik (1), I. Ducastelle (1), I. Mollet (2), N. Raus (1), A. Thiébaut (1), C. Spicka (2), V. Scudla (4) Giannoli (2), D. Revesz (1), V. Dubois (2), M. Michallet (1) (1)Faculty Hospital Brno (Brno, CZ); (2)General Faculty (1)Edouard Herriot Hospital (Lyon, F); (2)Etablissement Hospital (Prague, CZ); (3)Faculty Hospital (Hradec Kralove, Français du Sang (Lyon, F) CZ); (4)Faculty Hospital (Olomouc, CZ); (5)Faculty Hospital KV (Prague, CZ); (6)IHBT (Prague, CZ) Allogeneic stem cell transplantation (Allo-SCT) for multiple myeloma (MM) is the only curative treatment to date. Objectives: Autologous stem cell transplantation (ASCT) has However, high relapse rates suggest that molecular an established role in the treatment of symptomatic multiple remissions are not complete. Thus, the follow-up of MRD after myeloma (MM). We report results of 408 patients (pts) with Allo-SCT is a major prognostic factor, particularly in the RIC MM after ASCT from the Czech Transplant Registry. The data setting. The careful monitoring of BM plasma cell (CD138+) from 6 transplant centers were analysed with aims to evaluate chimerism should allow us to detect patients (pts) at high-risk outcomes of ASCT and to detect significant prognostic of clinical relapse and lead to therapeutic intervention. In this parameters. study, we analysed the CD138+ chimerism kinetics by RQ- Methods: We have retrospectively evaluated 408 pts with MM PCR in 13 MM pts that underwent RIC Allo-SCT in our center undergoing ASCT between 1994 and 2005, median follow up recently, and determined its utility in predicting from ASCT is 33 months, range 1-117 months. Subgroup of relapse/progression over conventional follow-up parameters 44 pts was more than 10 years from ASCT. At diagnosis, 32 (Total BM and PB CD3+ chimerism, paraprotein analysis). pts (8%) had stage I according to Durie-Salmon (DS), 117 pts Mixed chimerism was defined as the persistence of • 0.2% of (29%) stage II and 259 pts (63%) stage III. The median age recipient cells. Median age was 51 (35-61) years with 10 M was 56 years. Most of pts (83%) were transplanted to one and 3 F. All pts were in stage III (9 A, 4 B) at diagnosis with 8 year from diagnosis. One transplant was performed at 229 pts IgG, 3 IgA, 2 light chains. Ten pts had • 2 lines and 3 one, (56%), 2 or more transplants at 179 pts (44%). prior to Allo-SCT; all had recieved autologous PBSCT (11, 1 Results: Following ASCT, 94 pts (23%) were in complete and 2, 2). At Allo-SCT 11 pts were in PR and 2 in CR. remission (CR). The median progression-free (PFS) and Conditionings were Flu-Bu-ATG for 8 and Flu-TBI for 5. Stem overall (OS) survivals were 27.5 and 62.3 months, cell source was PBSC for 12 and BM+PBSC for 1, GVHD respectively. prophylaxis consisted in 9 Ciclo alone, combined to MMF in 4. Significant prognostic parameters for poor survival were: Eight pts developed aGVHD (3 pts>II) and 6 a cGVHD (5 clinical stage III according to DS (p = 0.005), IgA type of extensive). Median follow-up was 21 (4-64) Mo. A the last monoclonal immunoglobulin (p = 0.003), renal impairment with follow-up 11 pts were alive with 8 in relapse. A median of 2.9 serum creatinine at diagnosis above 2mg/dl (p under 0.001), (range 2-6) BM samples were analysed/pt from 2 to 55 Mo. no achievement of CR after ASCT (p under 0.001). post-tranplant. After BM CD138+ magnetic purification, purity Correlation between PFS and OS was found, pts with PFS was 26.5 (4-93.5)%. In 3 pts, while total BM and PB CD3+ under 30 months had significantly shorter OS than others (p chimerism remained total donor, a decrease in CD138+ donor under 0.001). Patients with progression of MM to one year chimerism with time was observed and correlated with a BM after transplant had very poor prognosis, median PFS of this and (+extra-medullar in 2) relapse, whereas immunofixation subgroup was 7.3 months only and median OS was 21.6 remained persistently negative for 2 of them (Figure 1). In all months. Long-term outcomes of ASCT were evaluated in relapsed pts, median time prediction to clinical progression subgroup of 44 pts, who had been transplanted before 1997: was 3 (3-4) Mo. In 4 relapsed pts, a progressive CD138+ 12 pts (27%) are alive, only 4 pts (8%) are disease free. The increasing donor chimerism was correlated with a clinical status of disease before ASCT, age, number of transplants (1 response after post-transplant retreatment (all with versus 2), time from diagnosis to transplant did not Bortezomib). In 2 pts a Graft Versus MM effect was shown significantly affect OS after ASCT. with increasing CD138+ donor chimerism while Conclusion: In our group of pts the survival after ASCT immunosuppression was withdrawn. These preliminary data correlated with clinical response after transplant (CR versus suggest that the follow-up of BM CD138+ donor chimerism non CR), stage according to DS (III versus I+II), type of after RIC Allo-SCT is a useful tool to monitor disease paraprotein (IgA versus others) and renal impairment at response/resistance and drive quick therapeutic intervention, diagnosis. The most significant parameters for better survival and maybe improve the results of RIC Allo-SCT in MM. of MM pts after transplant are: no presence of renal impairment at diagnosis and achievement of CR after transplantation. The work was supported by grant IGA MZ CR NR/8223-3.

P684 Allogeneic stem cell transplantion in multiple myeloma A.M. Carella, M.M. Greco, L. Savino, N. Cascavilla Irccs "Casa Sollievo Della Sofferenza" (San Giovanni Rotondo, I)

We evaluated the feasibility, efficacy and toxicity of allogeneic stem cell transplantation for 29 (17 M, 12 F) newly diagnosed stage IIIA(n=24)-IIIB(n=5) multiple myeloma patients (pts) up to the age of 65.Twenty-three and 6 pts underwent a matched sibling donor allogeneic transplant after a nonmyeloablative or myeloablative regimens , respectively. Twenty-one pts were treated with autografting followed by reduced intensity conditioning allotransplantation. All these patients received HD Melphalan (200 mg/mq) followed by autologous PB-SCT. After a median of 90 days, the pts underwent RICT (Fludarabine + 2 Gy TBI). Acute GVhD prophylaxis consisted of MM and cyclosporine. Chimerism analysis was performed

S156 using STR-PCR and donor engraftment was evaluated at day (22 % of all the transplants); transplant-related mortality at day +15,+30,+45,+60,+90 on unfractionated BM cells. All pts +90 was 0. received a HLA identical donor mobilized PBSC and the graft Our preliminary data suggest that ASCT with early discharge contained a median of 3,3 x 10E6 (range 1-6,8) CD34+ is feasible in elderly patients with MM with a speed cells/kg body weight. After RICT, on day +15, 3 (13%) pts engraftment, very low transfusion requirement and negligible showed a complete donor chimerism; on day +90, 21 (90%) severe extrahaematological toxicity. showed a complete donor chimerism; two pts with mixed chimerism received a DLI on day +30 and one of these achieved full donor chimerism. Six pts received a conventional P686 hematopoietic SCT. All patients showed a complete donor Influence of glucocorticoid receptor polymorphism on chimerism at the time of engraftment. efficacy of VAD therapy in patients with multiple myeloma Results: For RICT pts grade I-II and III acute GVHD occurred N. Stepanova, V. Larionova, A. Voitovich, S. Moiseev in 5 (22%) and 1 (4%) pts respectively. Five patients (22%) State Pavlov Medical University (St. Petersburg, RUS) developed a mild and 4 (19%) an extensive chronic GVHD. After RICT 11 pts (48%) achieved CR and they are in CCR Polymorphisms and mutations glucocorticoid receptor gene at+66,+66,+60,+58,+,55,+29,+24,+13, +9 and +9 months; 1 may be associated with an altered sensitivity to patient in CR died for cardiopathy. Six pts (26%) are in PR. glucocorticoids in patients with lymphoid neoplasms. Six (26%) pts not in CR showed a progressive disease and 5 Efficacy of VAD therapy in dependency of polymorphisms (22%) of them died. With a median follow-up of 26 months, G+647C glucocorticoid receptor gene (2 intron) was 18 (78%) are alive. Instead the pts that received a evaluated in 38 patients with multiple myeloma II-III stages. myeloablative conditioning regimen, grade II-IV acute GVHD Patients received 4-6 courses of VAD therapy as first line occurred in 3 patient and one of these died for aGVHD. All pts therapy. Polymorphism glucocorticoid receptor gene was developed mild chronic GVHD. Two patients relapsed and evaluated by PCR-RFLP analysis. CC genotype was died. To date, 3 patients are alive and are in CCR at + 37 and revealed in 12 (38%), CG genotype – in 21 (55%), GG +13 and +12 months. genotype – in 5 (13%) patients with multiple myeloma. No patients died after RICT. Pancytopenia after RICT was Distribution of these polymorphisms in patients with multiple minimal and sustained allogeneic stem cell engraftment myeloma did not differ from distribution in population of St- occurred in 90% of patients. A good correlation between Petersburg. Efficacy of VAD therapy in patients with multiple GVHD, full chimerism and remission was found. myeloma was dependent on glucocorticoid receptor G+647C polymorphisms. Uneffectiveness of therapy (progression disease) was revealed in 4 (19%) patients with CG genotype, P685 in 4 (36%) patients with CC genotype and in 5 (100%) c GG Autologous stem cell transplantation with early discharge genotype. Complete remission (CR) was received in 5 (24%) in elderly patients with multiple myeloma patients with CG genotype. None of the patients with CC and M. Montanari, I. Scortechini, G. Gini, A. Poloni, M. Offidani, G. CG genotype reached CR. Not complete response (near CR, Mancini, S. Trappolini, R. Re, P. Leoni partial remission, minimal response, stabilization disease) was Ospedali Riuniti di Ancona (Ancona, I) received in 12 (57%) patients with CG genotype, 7 (64%) patients with CC genotype and in none of the patients with GG Multiple myeloma (MM) is a disease of the elderly with a genotype. 6 patients after VAD therapy received autologous median age at diagnosis of 70 years. Autologous stem cell peripheral stem cell transplantation (APSCT). 3 patients with transplantation (ASCT)is an effective standard treatment for CG genotype after APSCT stayed alive during 3 years. 3 MM patients and previous data showed that it is feasible also patients with other genotypes had relapse and died. in elderly patients. 13 patients with multiple myeloma after ineffective VAD A program of ASCT with early discharge in elderly patients therapy as second line therapy received treatment by velcade with MM was started in December 2001 at our department (bortezomib) 1,3 mg/m2 at 1, 4, 8, 11 days and dexametazone with the following inclusion criteria: age 60-75 years (y), 20 mg at 1,2, 4,5, 8,9, 11,12 days every over 10 days. performance status 0-1 (WHO), absence of severe Patients received 8 such courses. 6 (46%) patients reached comorbidities, availability of a caregiver, estimated current CR, 1(8%) patient – near CR, 2 (15%) – partial remission, 2 time to reach the transplant centre no more than 40 minutes (15%) – stable disease, 2 (15%) – progression disease. and good compliance to homing therapy. Efficacy of velcade was not dependent on G+647C The aim of our study was to evaluate the feasibility of this polymorphisms of glucocorticoid receptor gene (2 intron). approach. Preliminary results may indicate the greater efficacy of VAD Twenty-seven patients were enrolled: 17 male and 10 female therapy in patients with CG genotype, that should be taken with a median age of 65 y (range 60-74); 48% patients were into consideration in pretransplant preparing of patients with over 65 y and 11% over 70 y; all of them were analysed multiple myeloma. according to the intention criteria to perform an early discharge within 24 hours post stem cells reinfusion, with clinical and laboratory surveillance 3 times a week until the P687 complete haematological recovery. Autologous stem cell transplantation in elderly patients The conditioning regimen was high dose Melphalan (200 with multiple myeloma (• 65 years). The experience of one mg/mq) with previous infusion of Amifostine 750 mg; 13 centre patients transplanted until 2004 received the combination of A. Garcia Guiñón, A. Sureda, R. Martino, D. Valcárcel, J. granulocyte colony stimulating factor (G-CSF) 5 gamma/die Briones, J. Delgado, J.L. Piñana, B. Sánchez-Gonzalez, S. s.c. plus erytropoietin 10000 UI/die s.c. starting from day +1 Brunet, J. Sierra until the complete haematological reconstitution; the others Hospital Santa Creu i Sant Pau (Barcelona, E) received pegylated G-CSF 1 fl s.c. and darbepoietin 500 mcg 1 fl s.c. at the day +1. Objective: To analyze the tolerance and effectiveness of an The median of CD34+ x 106/kg cells reinfused was 6 (range 1- autologous stem cell transplantation procedure(ASCT) in 10.9) and a speed engraftment was observed in all patients patients(pts) with myeloma(MM) • 65 years. with a median of 10 days (range 8-15) for absolute neutrophil Patients and methods: From a total number of 191 ASCT for count >500/mcl and 12 days (range 9-27) for platelets > MM performed in our center from June’1991 to June’2006, 35 20000/mcl. The median of transfusion requirement was 0 and of them(18%) have been performed in pts • 65 years.There 1 for red cell and platelets units, respectively. are 22 males with a median age at diagnosis of 66(range,59– Severe mucositis (grade 3-4 WHO) was observed in only two 71) yrs and at ASCT of 68(range,65–72)yrs.Twenty pts(57%) patients (7%) and neutropenic fever (> 38°C) in four (14%) presented with stage III at diagnosis and 7 pts(20%) had renal with a consequent second hospitalization in these six patients function impairment.The induction therapy consisted of alternating cycles of VBCMP/VBAD in 31 pts(88%),2 pts

S157 received the VAD protocol(6%) and the remaining 2 pts(6%) hepatitis B virus reactivation. One patient had to stop therapy received alternating cycles of bortezomib plus because of grade III neuropathy and 1 patient because of dexametasone.Median time from diagnosis to ASCT was of grade IV thrombocytopenia. The response rate(RR) was 11 months.Seven pts(20%) were autografted in complete 66%(CR, n=2; PR,n=4) after a median number of 4 cycles of remission(CR) with IF-, 24 pts(68%) in partial remission(PR), BTZ. Median time to achieve the best response was 2 pts(6%) with minimal response and 2 pts(6%) with 4.5(range,1.8–11) mo. Two patients(22%) progressed under progressive disease. Conditioning regimen consisted of high- BTZ and 1 patient remained in SD. After a median follow-up of dose melphalan (200mg/m² iv) in 30 pts (86%),melphalan plus 6 mo after finishing therapy, the RR is 55%(CR,n=2; PR, n=3), oral busulfan in 3 pts (8%), mephalan plus TBI in 1 patient 3 patients present with PD and one patient has died from (3%) and BEAM in the remaining patient.The median number disease progression. of CD34+ cells x106/Kg infused was 4.2(range,1.7-7.4). G- Conclusion: BTZ is a feasible salvage therapy for MM pts CSF(5ug/kg/day,sc) was administered in all cases from day progressing or relapsing after a RIC-Allo procedure. Toxicity +7 after ASCT to neutrophil recovery. profile is acceptable and overall RR high taking into account Results: Thirty-four pts(97%) engrafted with a median time of the bad prognosis of the pts included in the analysis. neutrophil engraftment(>0.5x109/l) of 12 days(range,9- 18),platelet recovery was also fast and complete.One patient did not engraft due to early transplant related mortality P689 (TRM).Grade III gastrointestinal toxicity was observed in 3 Gamma-delta T-cell receptor expression estimation in (8%) pts and gIII/IV oral mucositis in 7(20%) pts. Febrile peripheral blood lymphocyte population in multiple neutropenia was present in 88% of the pts myeloma and lymphoma malignum patients treated analyzed(n=31).Two pts(6%) died from transplant-related megachemotherapy with autologous bone marrow causes before day +100 after ASCT.Late TRM(>100 days transplantation (auto-BMT) after ASCT) was 0%. Overall disease response at 3 months L. Usnarska-Zubkiewicz, E. Sowinska, K. Kuliczkowski after ASCT was 43% with 5 pts(14%) achieving a CR (with Wroclaw Medical University (Wroclaw, PL) negative IF) and 7 pts(20%) achieving a PR. Nine patients (26%) received a 2nd ASCT at a median time of 6(range,4–9) Background: Gamma-delta T lymphocytes (gd) appear a months from the first one.Early and late TRM after this 2nd immune response to acute lymphoblastic leukemia following autologous procedure was 0% in both instances.None of allogenic BMT, resultating in improved relapse-free survival these patients achieved a CR after the 2nd ASCT.2-years for those patients, who survive 100 days from transplant. overall survival for the whole series is 71%±7%. Activated gd T cells express antigens CD25+ or CD69+ on Conclusions: ASCT can be considered a therapeutic option in their surface. elderly pts with symptomatic MM.Early and late TRM as well Aim: Gamma-delta T-cell mean percentage (%) in peripheral as response rate after ASCT are similar to what is seen in blood in multiple myeloma (MM) and lymphoma malignum younger pts with this disease. (NHL) patients treated megachemotherapy (mega-chmt) with autologous bone marrow transplantation. (auto-BMT) was investigated. P688 Material and methods: 10 patients (pts): 6 MM and 4 NHL pts Bortezomib as salvage therapy for patients with multiple treated in Department of Hematology, Blood Neoplasms and myeloma relapsing after a reduced intensity allogeneic Bone Marrow Transplantation Wroclaw Medical University, stem cell transplantation Poland and 14 healthy controls were included into analysis. A. Garcia Guiñón (1), A. Sureda (1), J. Beltran (2), S.J. Bravo Among 6 MM pts 2 were in II, 3 in III stages, 1 pt was MM (3), J. Sierra (1) non-secretorius, according to Durie-Salomon classification. 4 (1)Hospital Santa Creu i Sant Pau (Barcelona, E); (2)Hospital pts were classifieled to A and 2 to B groups. Ammong 4 NHL de Basurto (Bilbao, E); (3)Hospital Dr.Negrín (Las Palmas pts 3 were in IVA and 1 pt in IVB stages according to Ann- Gran Canaria, E) Arbor classification. Blood samples were taken before and after mega-chmt + auto-BMT. gd T cells were estimated by Objectives: The tolerance and effectiveness of bortezomib flow-cytometry (FACS), using a fluorescence-activated cell (BTZ) as salvage therapy in patients (pts)with multiple sorter and monoclonal antibodies (MoAbs: TCRgamma1-FITC myeloma (MM) relapsing or progressing after a reduced (Becton-Dickinson), CD14-RPE, CD45-FITC, CD25-RPE, intensity allogeneic stem cell transplantation (RIC-Allo) was CD69-RPE (DAKO) and IgG1kappa-FITC and IgG1-RPE as analyzed in a group of 9 pts treated in 3 different Spanish negative controls. institutions [H.de la Santa Creu i Sant Pau(n=7), H.de Results: Mean % of total gd T lymphocytes in peripheral blood Basurto(n=1), H.Doctor Negrin(n=1)]. before mega-chmt+autoBMT was 1,72 of all MM and 3,63 of Patients and methods: There were 4 males with a median age all NHL pts. It was lower than in control group 5,63%. In MM at diagnosis of 51(range,43–63)yrs. Eight pts received a pts, after mega-chmt+auto-BMT,gd T cells mean % increased previous autologous stem cell transplantation (ASCT), 7 of to 3,73, compared before treatment, but was also lower than them as a part of a tandem procedure (ASCT+RIC-Allo), and in healthy volunteers. Similarly, after mega-chmt+auto-BMT in 1 patient received two prior ASCT. Median time between MM pts activated gd T CD25+ and gd T CD69+ cells mean % diagnosis and RIC-Allo was 15(range,12-69) mo. Three were higher than before transplantation (gd T CD25+: 0,36 vs pts(33%) were allografted in partial remission(PR), 2 0,06 and gd T CD69+: 0,85 vs 0,35). Instead in NHL pts after patients(22%) in stable disease(SD) and 4 patients(44%) in mega-chmt+auto-BMT, dg T cells mean % decreased to 1,13, progressive disease(PD). The conditioning regimen was iv compared before treatment 3,63 and was lower than in fludarabine 150 mg/m² plus melphalan 70mg/m². Graft-versus- healthy volunteers 5,63. After mega-chmt+auto-BMT in NHL host-disease prophylaxis consisted of ciclosporine A plus pts activated gd T CD69+ cells mean % were lower than mycofenolate mofetil. BTZ was administered alone as a 21- before treatment(0,16 vs 0,52), but gd t CD25+ cell mean% no day cycle (1.3 mg/m2 iv on days +1,+4,+8 and +11). One changed. All MM and NHL pts (100 %) stayed in complete patient was treated with the combination of bortezomib and remission (RC) 6 months after mega-chmt+autoBMT. dexametasone. Conclusions: This study shows a favorable trend, that in MM Results. BTZ was started 29(6-68) mo [median (range)] from pts the increase and in NHL pts the decrease of all gd T the RIC-Allo. Seven pts(77%) presented with PD before BTZ, lymphocytes and activated gd T cells subpopulations in 1 patient(11%) with PR and 1 patient(11%) with SD. The peripheral blood after mega-chmt+auto-BMT may be median number of cycles of BTZ was 4(range,2-8). Severe connected with normal immunological reconstruction after grade III/IV thrombocytopenia was observed in 1 patient(11%) transplantation and can indicate better prognosis, because all and grade IV neutropenia in another patient. Grade II of studies pts stayed in complete remission half a year after neuropathy was observed in 2 patients(22%) and grade III transplantation. Our studied patients group is small, so it neuropathy in 1 patient(11%). Grade II gastrointestinal toxicity needs a further examination. was observed in 2 patients(22%) and 1 patient(11%) had a

S158 P690 Infectious complications Autologous haematopoietic stem cell transplantation as an up-front therapy in stage III multiple myeloma: the Lebanese experience A. Ibrahim (1), Z. Otrock (2), A. Mugharbel (1), R. Mahfouz (2), Z. Salem (2), W. Shebbo (1), H. Hatoum (2), G. Nsouli (1), P691 A. Shamseddine (2), R. Jalloul (1), A. Taher (2), N. El-Saghir High incidence of EBV viraemia but low risk of post- (2), E. Baz (2), J. Makarem (2), A. El-Kinge (2), N. Droubi (1), transplantation lymphoproliferative disorder following T- N. Kreidieh (2), N. Yassine (1), T. Jisr (1), A. Bazarbachi (2) cell depleted stem cell tranplantation with CAMPATH-1H (1)Makassed Hospital (Beirut, LBN); (2)American University of M.N. Dimopoulou, S. Grace, M. Raza, P. Griffiths, P. Beirut (Beirut, LBN) Kottaridis, A.K. Fielding, S. Mackinnon, R. Chakraverty Royal Free Hospital (London, UK) Autologous stem cell transplantation (ASCT) has become the gold standard therapy for young patients (pts) with multiple The aim of this study was to evaluate the incidence of EBV myeloma (MM). We report the results of ASCT used as up- viraemia and PTLD following SCT with CAMPATH-1H T-cell front therapy in 71 pts with stage III (Durie-Salmon) MM depletion (TCD, in vivo or ‘in the bag’) in adult patients (pts). autotransplanted between March 1997 and October 2006 in We have previously shown that high doses of in vivo the two major BMT units in Lebanon. Median age was 49 CAMPATH-1H (100mg) are cleared from the circulation more years (29-70). There were 49 males and 22 females. Twelve slowly that CAMPATH-1H (20mg) ‘in the bag’. Weekly pts (17%) were in stage IIIB. IgG, IgA and light chain were the quantitative PCR for EBV DNA in peripheral blood was most frequent types (58%, 21% and 17% respectively). Sixty- performed in 37 pts transplanted over an 18 months. 19 six pts (93%) received VAD protocol (3-6 cycles) and 4 pts received a Cyclophosphamide-TBI-based conditioning (± (6%) received thalidomide + dexamethasone. Seventy pts Fludarabine) with CAMPATH-1H 20mg ‘in the bag’ and 18 received autologous peripheral stem cell transplantation received conditioning with Fludarabine, Melphalan and (APSCT). Cyclophosphamide (4-5g/m²) followed by G-CSF CAMPATH-1H in vivo (FMC, CAMPATH-1H doses 100mg (10µg/kg/day) were given for peripheral stem cell mobilization. n=14, 40mg n=2, 30mg n=1, 20mg n=1). EBV viraemia was The median number of CD34 + cells collected was 10.24 x defined as >200 copies/ml. Pts with a viral load >40000 106/kg (1.36-60). High dose chemotherapy consisted of copies/ml had CT scans, bone marrow and CSF examination Melphalan which was given at a dose of 200mg/m² in 60 pts to exclude PTLD and were treated with one dose of rituximab (85%); the others received Melphalan at a dose of 140mg/m². (375/m2). Median patient age was 44 (range 15-67). Fifty-nine pts (83%) underwent one ASCT and 12 pts (17%) Diagnoses were AML/MDS (21), Myelofibrosis (6), CLL (2), underwent tandem ASCT. The median number of CD34 + NHL (5), Hodgkin (3). Donors were matched/1 ag mismatched cells transplanted was 5.79 x 106/kg (1.36-20) per transplant related (18), matched unrelated (8) and mismatched unrelated for the pts who underwent APSCT. Bisphosphonates were (11). 94% of pts were EBV seropositive. EBV serostatus was given IV to all pts for about 2 years after ASCT. Response avaliable in 28/37 donors and 93% were positive. 38% of pts was assessed according to the EBMT criteria (Blade J. et al, reactivated EBV over a median follow up time of 6mo (range Br J Haematol, 1998. 102: 1115-1123). Disease status before 0.4-18). The incidence was 55.6% for FMC with in vivo ASCT was as follows: 8 pts (11%) were refractory, 4 pts (6%) CAMPATH-1H vs. 21% for myeloablative SCT with had minimal response, 54 pts (76%) had partial response and CAMPATH-1H ‘in the bag’ (p<0.05). Median time to EBV 5 pts (7%) were in complete remission (CR). Transplantation- reactivation post SCT was 70 days (range 21-180). 9/10 of related mortality rate was 1.4%. Twenty-one pts (30%) were in the pts who reactivated in the FMC group had received 100mg CR after ASCT. Thirty-six pts (51%) who relapsed or of CAMPATH-1H and 1 pt 20mg. At the time of EBV progressed after ASCT received thalidomide ± reactivation 13/14 pts were on immunosuppression, 3 had dexamethasone (30 pts), a second ASCT (4 pts) and Velcade chronic GVHD but none had acute GVHD. None of the pts (2 pts). The median overall survival (OS) and the median had symptoms or clinical findings related to EBV infection or progression-free survival (PFS) were 60 and 32 months PTLD. Median peak viral load was 14027 copies/ml (range respectively (Figure 1). In conclusion, the feasibility of APSCT 1000-2,352,829). 5/14 pts had a viral load > 40000 copies /ml for MM in Lebanon was acceptable with an OS and PFS rates and were fully staged with none of them showing evidence of comparable to the EBMT registry. PTLD, and subsequently treated with rituximab. Median duration of viraemia was 56 days (range 11-340). 4/9 non- treated pts remained intermittently positive on a low level for > 6 mo. All pts treated with Rituximab became EBV DNA negative rapidly (median 15 days). None of the pts studied developed PTLD. The incidence of EBV viraemia post-SCT is high following TCD with CAMPATH-1H, and significantly higher with in vivo CAMPATH-1H. Using a pre-emptive approach of rituximab for high levels of EBV viraemia, no patients developed PTLD.

P692 The hCMV-antigens pp65 and IE-1 are equally effective inducers of CMV-specific cytotoxic T-cell responses after allogeneic stem cell transplantation S. Ganepola (1), S. Fuhrmann (2), F. Kern (2), HD. Volk (2), E. Thiel (1), L. Uharek (1) (1)Charite Campus Benjamin Franklin (Berlin, D); (2)Charite Campus Mitte (Berlin, D)

Rapid reconstitution of cytotoxic T cell function is mandatory for the control of human cytomegalovirus (CMV) reactivation. Whereas tetramer staining is sufficient to determine the frequency of antigen specific T cells, the assessment of their cytotoxic potential requires functional assays. Here, we have used the CD107a degranulation assay along with 8-color flow- cytometry to determine ex-vivo T-cell degranulation after

S159 stimulation with CMV IE-1 and pp65 peptide pools in patients Patients diagnosed of tuberculosis were treated with 3 drugs after allogeneic stem cell transplantation (allo SCT). for nine months. The cases with NTM were treated with 4 Material and Methods: T cells from 6 healthy CMV exposed drugs for 12 and 18 months respectively. Four patients (2 m donors and 16 allotransplanted patients were stimulated with tuberculosis and 2 m. kansasii) are alive and disease free 10 peptide pools representing the pp65 (UL83) and IE-1 (UL123) months to 8 years after SCT. One patient with extended proteins. T-cell activation was determined by intracellular cGVHD was diagnosed of tuberculosis 584 days after staining for TNF-alpha, IFN-gamma, and IL-2. Activated T- transplantation, and died from infection 2 months after cells were assessed for CD107a expression as a measure of diagnosis. degranulation. Conclusions: Our incidence of MBI in pediatric allogeneic HSC Results: A specific CD8+ T cell response against pp65 was recipients is similar to the observed in other european observed in 6/6, against IE-1 in 5/6 healthy donors and in countries.We suggest that constant vigilance is required to 11/16 and 9/16 allotransplanted patients, respectively. Healthy early detect MBI in SCT recipients. donors presented slightly higher numbers of effector type (CD3+/CD8+/CD28-) T cells against pp65 (77% vs. 61%) and IE-1 (87% vs. 65%). However, there was no difference P694 between healthy donors and transplant recipients regarding Human metapneumovirus infection in haematopoietic the number of degranulating CD8 T-cells, neither in regards of stem cell transplant recipients from São Paulo, Brazil pp65 (78% vs. 75%) nor in regards of IE-1 (74% vs. 75%). R.R. Oliveira, A.F. Tateno, J.E. Levi, L.S. Vilas Boas, C.S. Also, there was no significant difference between healthy Pannuti, C.M. Machado donors and patients in terms of cytokine production. University of São Paulo (São Paulo, BR) Conclusion: We conclude, that pp65- as well as IE-1-specific T-cells are equally effective inducers of hCMV-specific T cell Introduction: Respiratory viruses can cause significant responses after allo SCT. Moreover there is no significant morbidity in immunocompromised hosts. Human difference between healthy donors and allotransplanted metapneumovirus (HMPV) has been increasingly associated patients concerning the percentage of specific effector T cells to lower respiratory tract infection in HSCT recipients, with and, most importantly, the ability of pp65 or IE-1 induced mortality rates up to 50% in some series. No data on the degranulation of CMV-specific T cells. According to our data, occurrence of HMPV infection in HSCT recipients has been the quality of CMV-specific T cell responses is not impaired in reported in Brazil. allotransplanted patients.In order to look into this matter Methods: HMPV was detected by PCR with primers further, the granule content will have to be examined. complementary to the N region of HMPV genome. To determine HMPV group, amplified products were sequenced (Big-Dye, Applied Biosystems) and analyzed using the P693 software SeqMan (DNA-Star, ABI). Mycobacterial infections in paediatrics stem cell Results: 251 nasal washes (NW) were taken from 155 transplant recipients in Spain. A GETMON survey symptomatic HSCT recipients from 2001 to 2003. The number A. Muñoz (1), M. G-Vicent (2), I. Badell (3), C. Diaz-Heredia of episodes of respiratory tract infection during follow-up (4), P. Gomez (5), M.S. Maldonado (1), E. Bureo (6), A. varied from 1 to 4, median 1.6 episodes per patient. Ten of the Martinez (7), J.M. P-Hurtado (8), A. Verdeguer (9), E. G- 155 patients (6.5%) acquired HMPV infection. The prevalence Valentin (10), J.M. Couselo (11) was 7% in 2001, 3.1% in 2002 and 10.6% in 2003. HMPV was (1)Hospital Ramón y Cajal (Madrid, E); (2)Hospital Niño Jesus detected during fall, winter and spring months. Eight of the 10 (Madrid, E); (3)Hospital San Pau (Barcelona, E); (4)Hospital HMPV positive samples were sequenced. Both HMPV group Valle Hebron (Barcelona, E); (5)Hospital Reina Sofia A and B circulated each year. In 2001, 2 of the 3 HMPV (Cordoba, E); (6)Hospital Valdecilla (Santander, E); detected (75%) belonged to group A; in 2002 group A and B (7)Hospital La Paz (Madrid, E); (8)Hospital Virgen Rocio circulated equally and in 2003, HMPV group A (75%) (Sevilla, E); (9)Hospital La Fe (Valencia, E); (10)Hospital predominated over group B. One patient died on day +16, Carlos Haya (Malaga, E); (11)Hospital Xeral (Santiago, E) eight days after HMPV infection was diagnosed. However, death could not be attributed to HMPV since influenza B co- Objectives: The incidence of Mycobacterial infections (MBI) in infection was detected in this case. Spanish population is high and an increase of nontuberculous Conclusions: Prevalence rates of HMPV infection in HSCT mycobacterial (NTM) has been observed during the last recipients living in Brazil were similar to those observed in decade. We studied the incidence, clinical presentation, northern hemisphere. The majority of HMPV infections were diagnosis and outcome of MBI in pediatrics stem cell diagnosed in late winter and spring. transplant (SCT) in our country. Financial support: FAPESP 05/01455-5 Methods: A research was performed in the eleven Spanish pediatrics centers in which SCT was applied from 1985 to 2005. All patients under 15 years of age who had received an P695 allogeneic (n=1073) or autologous (n=1539) were included. Molecular characterisation of respiratory syncytial virus Results: Five cases of MBI were diagnosed among the 1073 in haematopoietic stem cell transplant unit during two allogeneic SCT (0.47%). No cases of MBI were recorder in the outbreaks in consecutive year autologous SCT recipients. A.F. Machado, L.S. Vilas Boas, A.F. Tateno, M.A.M. Sallum, Underlyng disease was ALL in four patients and C.M. Machado hemophagocitic lymphohistiocytosis in 1.All recipients were University of São Paulo (Sao Paulo, BR) considerer not at risk for MBI at pre-SCT visit and had not been vaccinated with BCG. The 5 patients had been Introduction: Respiratory Syncytial Virus (RSV) is recognized transplanted from matched donors (3 related and 2 unrelated). as the leading cause of nosocomial respiratory infection The diagnosis was made at a median of 120 days after SCT among recipients of hematopoietic stem cell transplant (range 90 to 540 days). The mean time interval between the (HSCT) causing considerable morbidity and mortality among first symptoms attributable to MBI and diagnosis was 39 days. these patients. All patiens had a normal total neutrophil count at diagnosis of Objective: To characterize the way of transmission of RSV in MBI. HSCT Unit at Hospital das Clínicas, University of São Paulo Lung was the organ involved in the 5 patients. Diagnostic Medical School during RSV season in 2001 and 2002. procedure was bronchoalveolar lavage in 4 cases and open Methods: From April 2001 to October 2002, RSV was lung biopsy in one.Four patients evidenced microbiollogicaly identified in symptomatic HSCT recipients at Hospital das proven MBI; 2 had m. tuberculosis and two an atypical Clínicas. Patients admitted to the HSCT ward were under mycobacteria (m. kansasii). One patient was diagnosed by strict nosocomial control policies to avoid respiratory virus histological findings. transmission. Outpatients were assisted at the HSCT Day- Hospital where the implementation of such policies was

S160 limited. RNA extraction, cDNA synthesis and semi-nested P697 PCR with primers complementary to RSV genes G e F were Pre-emptive treatment for EBV reactivation after performed in samples that tested positive by IFA. PCR allogeneic haematopoietic stem cells transplantation: an products were analyzed by nucleotide sequencing of C- effective strategy terminal region of the gene G for typing (in group A or B) and I. Ahmad, C. Nguyen Van, J. Bennani, J. Kwan, M. Aoun, P. genotyping (Peret et al, 1998). Lewalle, N. Meuleman, P. Martiat, D. Bron Results: Of the seven strains analyzed from HSCT recipients Institut Jules Bordet (Brussels, B) during 2001, only two belonged to group B, the other five belonged to group A. Of these seven strains, three were Background: Epstein-Barr virus reactivation (EBV-R) occurs in identical and were from patients assisting the outpatient HSCT up to 50% of allogeneic haematopoietic stem cell transplant Unit. In 2002, of the nine strains analyzed, three belonged to (alloHSCT) patients (pts) and can lead to a posttransplant group A and the other six belonged to group B. Five strains lymphoproliferative disorder (PTLD) which is a rare (<5% were identical and were also from the outpatient setting HSCT) but lifethreatening complication. The main risk factor suggesting that nosocomial transmission in the Day-Hospital for PTLD is T-cell depletion but predictive markers for PTLD unit was more likely. Two strains isolated from 2001 were development after EBV-R are unknown. Preemptive therapy identical to two strains isolated from 2002. No hospitalized (PT) with anti-CD20 antibody rituximab (RTX) has shown patient acquired RSV infection within the Hematopoietic Stem efficacy in abrogating PTLD-related mortality, but indications Cell Transplant ward, indicating community-acquired infection. and duration of PT remain unknown. Conclusion: Multiple strains of RSV co-circulated in the Methods: From 08/2000 to 09/2006, pts undergoing alloHSCT Hematopoietic Stem Cell Transplant units (ward and Day- were monitored weekly for EBV-R by polymerase chain hospital) of Hospital das Clínicas between 2001 and 2002. reaction (PCR) on peripheral blood mononucleated cells. The Nosocomial transmission was more likely to occur at the criteria for PT with RTX were 2 consecutive rising viral loads HSCT Day-hospital as compared to the HSCT ward. Infection (VL) of • 10,000 copies/mL or a single VL • 40,000/mL. The control practices should be also implemented at Day-hospital number of weekly cycles of RTX (375 mg/m²) was tailored Units to avoid nosocomial transmission of RSV and other according to weekly PCR results. Disappearance of EBV VL respiratory viruses in outpatient units. and incidence of PTLD were prospectively evaluated. Results: 124 pts were transplanted during this period (71 identical sibling[s], 17 MUD[u], 36 haploidentical[h], 46 P696 nonmyeloablative[n]). EBV-R requiring PT occurred in 19 pts Prospective study of the dynamics of human (15.3%) -10s[14%], 4u[24%], 5h[14%], 4n[9%]. 12 patients papillomavirus infection in female HSCT recipients: (63%) had received ATG as part of the conditioning regimen preliminary results or for steroid-refractory acute graft-versus-host disease L.Q. Mauad (1), A.F. Tateno (2), J.E. Levi (2), M.A. Mauad (1), (aGVHD). Median time from transplant to EBV-R was 86 [29- M. Souza (1), V.R. Colturado (1), M.V. Ikoma (1), C.M. 304] days and median viral load was 42,748 [12,480 – Machado (1, 2) 268,118] copies/mL at initiation of RTX. PT was started after a (1)Amaral Carvalho Foundation (Jau, BR); (2)University of median of 6.5 days of PCR result [1-13]. Median number of São Paulo (São Paulo, BR) weekly cycles of PT was 3 [1-4]. 17 (89%) patients had sustained normal VL after PT. 1 pt presented a serious Introduction: Hematopoietic stem cell transplant (HSCT) adverse reaction to RTX that was discontinued. He developed recipients are at increased risk for the development of PTLD and died from aGVHD. 7 pts (37%) had a normal VL secondary neoplasias such as oral cavity cancer and uterine after a single cycle of RTX. In the 2 pts who received 4 cycles, cervix neoplasia. Particular Human Papillomavirus (HPV) one had responded to the first cycle and the other died from types are implicated in the genesis of cervical cancer. No aGVHD before PCR negativity. study has prospectively evaluated the occurrence of HPV Conclusion: Our observations confirm that PT with RTX is an infection, cytological alterations and the risk factors associated effective strategy to prevent PTLD in HSCT. All pts should be with progression to cervical cancer in HSCT recipients. monitored weekly for EBV-R until withdrawal of Methods: Women admitted to undergo hematopoietic stem immunosuppressive therapy. Increased VL should be treated cell transplantation at the BMT Unit of Amaral Carvalho with RTX and duration of therapy can be tailored weekly to Foundation in Jaú, SP, Brazil, were invited to participate in minimize costs and side effects. This attitude deserves to be this prospective study. Gynecological examination and further confirmed in prospective trials. cervical samples were taken pre-transplant and at regular intervals thereafter (+90, +180, +365). Information about the use immunosuppressive drugs, GVHD, CD4 and CD* counts, P698 sexual activity, menopause symptoms, etc, was prospectively Impact of posaconazole prophylaxis on colonisation and taken in each visit. HPV was detected by PCR and HPV azole susceptibility typing was performed by RFLP. O. Cornely (1), A. Ullmann (2), P. Chandrasekar (3), D. Results: 30 women were enrolled in the study so far. Median Winston (4), J. Perfect (5), V. Reddy (6), P. McNicholas (7), T. age was 32 years, ranging from 15 to 50 years old. Twenty- Brundage (7), D. Angulo (7), H. Patino (7), T. Black (7) two patients received an allogeneic graft (one mismatch, 21 (1)University of Cologne (Cologne, D); (2)J.-Gutenberg full match) and the remaining 8 were autologous transplants. University (Mainz, D); (3)Harper Hospital (Detroit, USA); Underlying diseases (UD) were AML in 10, CML in 8, MM in 4, (4)UCLA CA (Los Angeles, USA); (5)Duke University Hospital HD in 3, and other in 5 patients. The median time from (Durham, USA); (6)University of Florida (Gainesville, USA); diagnosis of UD to transplant was 222.5 (46 – 4497) days. (7)Schering-Plough Research Institute (Kenilworth, USA) HPV was detected in 6 patients (20%) pre-HSCT. HPV types were 51, 59 (in two patients), 33, 45 and 16. Types 16 and 45 Objective: Antifungal prophylaxis with azoles is effective in are considered of high risk for the development of neoplasia. decreasing the frequency of invasive fungal infections in high- Six of the 30 patients (20%)died during follow-up. The risk patients with hematologic malignancies including stem cell remaining 24 patients have been followed-up for a median of transplant (SCT) recipients. However, there is concern that 150 days. Five of the 6 patients with HPV positive cervical prophylaxis may increase the frequency of non-albicans samples pre-transplant remained so (with same HPV type) in Candida species and promote azole resistance. This study consecutive samples tested. evaluated Candida colonization and azole susceptibility during Conclusions: Prevalence of HPV infection pre transplant was 2 clinical trials comparing posaconazole (POS), fluconazole 20%. HPV (same types) tend to persist months after HSCT. In (FLU), and itraconazole (ITZ) for prophylaxis. the present series two patients were identified with HPV types Methods: Colonization and susceptibility were assessed in 2 considered of high risk for the development of neoplasia. large studies (N=600 and N=602) comparing POS vs. FLU or ITZ for prophylaxis in SCT recipients with graft-versus-host disease or patients with acute myelogenous leukemia or

S161 myelodysplastic syndrome undergoing induction clearance may carry serious risks, especially when used with chemotherapy. Samples from mouth, throat, urine, and stool haematotoxic comedication. Further, prospective studies on were collected. Breakthrough infections were matched to long-term safety of VGC in patients after allogeneic HSCT colonization data. seem warranted. Results: Some species transitions were noted upon comparing isolates at baseline and during days 29-42 of prophylaxis: C. albicans decreased from 31% (165/534) to 6% P700 (13/221) on POS, 28% (132/473) to 9% (16/174) on FLU; C. Human polyoma virus BK infections in haematopoietic glabrata increased from 6% (32/534) to 9% (19/221) on POS, stem cell transplant recipients 7% (32/473) to 8% (14/174) on FLU; C. krusei changed from C. Fehér (1), P. Molnár (1), I. Mihály (1), P. Reményi (2), A. 3% (17/534) to <1% (1/221) on POS, 3% (12/473) to 5% Barta (1), L. Gopcsa (1), É. Torbágyi (1), L. Lengyel (1), T. (8/174) on FLU. For subjects with the same species at Masszi (2), G. Kriván (1) baseline and end of treatment a > 4-fold minimum inhibitory (1)St. László Hospital (Budapest, HUN); (2)National Medical concentration increase in any azole was observed in: C. Centre (Budapest, HUN) albicans, 2/25 and 4/26 POS and FLU patients, respectively, and C. glabrata, 9/20 and 5/12 POS and FLU patients, Objectives: BK virus (BKV) infections are associated with late respectively. Breakthrough infections by colonizing Candida post-transplant hemorrhagic cystitis (HC) and represent a species occurred while on study drug in 2 POS (2 C. glabrata) significant morbidity after hematopoietic stem cell and 2 FLU (C. albicans and C. krusei) patients and 3 POS (2 transplantation (HSCT). We evaluated retrospectively the BKV C. glabrata and 1 C. krusei) and 4 FLU (4 C. glabrata) patients infections of a 3 year period (28/02/2003 - 28/02/2006) in our after removal of the study drug transplant unit. Conclusion: Species transition and decreased susceptibility to Patients and methods: A total of 269 consecutive HSCT cases azoles were infrequent. C. glabrata was the colonizing were retrospectively analysed. Virus shedding (VSh) had been organism most frequently associated with infections during monitored by detection of BKV-infected epithelial cells in the and after azole prophylaxis. urine using direct immunofluorescence method (DIF). Our objective was to find some (if any) correlation between certain variables (age, sex, conditioning regimen, relation between P699 donor and recipient /RDR/, graft-versus-host disease /GvHD/) Evaluation of efficacy and long-term safety of pre-emptive and VSh intensity (ratio of infected cells in urine sample: ICR). therapy with or without oral valganciclovir for CMV Relation between the above mentioned variables and the reactivation after allogeneic haematopoetic stem cell degree of HC was also analyzed. Furthermore, relation transplantation between ICR and the degree of HC was examined and, finally, M. Stadler, C. Barkam, E. Dammann, L. Hoy, S. Buchholz, B. we mapped and correlated the seasonal changes of VSh to Hertenstein, J. Krauter, M. Eder, A. Ganser the prevalence of HC. Hannover Medical School (Hannover, D) Results: The diagnosis of post-transplant BKV infection was established in 31/269 (11,5%) cases. Symptoms of HC were Background: Valganciclovir (VGC), an orally available prodrug present in 27 cases while in the remaining 4 cases only of ganciclovir (GCV), has been shown to effectively clear CMV symptomless VSh was registered. There were 116 DIF-tests viraemia in several smaller studies of patients treated pre- performed on the materials of our 31 cases. emptively for CMV reactivation after allogeneic Significant correlation (p”0.05) was found to the ratio of haematopoietic stem cell transplantation (HSCT), without positive DIF-results with respect to age (children>adults) and significant early toxicity observed. Since VGC obviates GvHD (GvHD>no GvHD). ICR and RDR were also hospitalisation, it is increasingly being adopted, although not significantly related (more intensive VSh in case of unrelated licensed, in allogeneic HSCT. donor). The degree of HC only correlated significantly with Patients and methods: We evaluated efficacy and long-term ICR. We also found two peaks on the trend line of the monthly safety of preemptive therapies for the first episode of CMV ratio of positive DIF-results (December/March) followed by reactivation in all consecutive patients allotransplanted at our corresponding peaks of the prevalence of HC institution between 1996 and 2006. Among 164 patients (February/May). treated for CMV viraemia, 76 received oral VGC (44 as first, Conclusions: Our results indicate that post-transplant BKV second, or third line therapy, and 32 for oral maintenance after shedding is more frequent in children and patients with GvHD; GCV, Foscarnet (FCN), or Cidofovir (CDF) treatment), while VSh is more intensive in patients with unrelated donors; HC is 88 were given GCV, FCN, or CDF, only. Despite the more severe in the case of high ICR values and, finally, VSh retrospective study design, both the VGC and the non-VGC and the prevalence of HC show corresponding seasonal group were comparable for demographic, disease- and patterns with peaks in the winter and in the spring. transplant-related data such as gender, diagnosis, and donor type. In accordance with historical trends in HSCT, VGC patients were generally older and more of them had received P701 reduced intensity conditioning, whereas less non-VGC Distribution of HIV-1 resistance-conferring polymorphic patients had been taking haematotoxic or nephrotoxic alleles SDF-1-3'A and CCR5-Delta32 in relation to EBV comedication. reactivation in patients after HSCT Results: In both groups, the rate of CMV clearance was K. Bogunia-Kubik (1), A. Gieryng (1), A. Kuzyk (1), E. Jaskula similar (VGC 74% vs. non-VGC 70%). As expected, (1), A. Lange (2) hospitalisation for pre-emptive therapy was significantly lower (1)L.Hirszfeld Institute (Wroclaw, PL); (2)L.Hirszfeld Institute / in VGC-treated patients. Conversely, severe neutropenias Lower Silesian Center for Cellular Transplantation (Wroclaw, (including two cases of agranulocytosis) occurred more PL) frequently in the VGC group (18% vs. 8% in the non-VGC group), especially in patients with VGC maintenance therapy Epstein-Barr virus (EBV) reactivation is a serious complication (22%), accounting for significantly increased subsequent re- affecting the recipients of allogeneic hematopoietic stem cell hospitalisation in the VGC group (mean, 7.7 days vs. 0.6 days transplants (HSCT). CC-chemokine receptor-5 (CCR5) and for non-VGC patients). There was no difference between the CXC receptor-4 (CXCR4) are known to be involved in groups in total hospitalisation requirement for both initial pre- perpetuation of HIV infection (as co-receptors for HIV entry). emptive therapy and later treatment of complications. The clinical course of this infection is favored by the presence Conclusions: In our retrospective evaluation, the initial of the 32-nucleotide deletion within the CCR5 gene (CCR5- advantage of oral VGC for pre-emptive therapy of CMV Delta32) and A at the -801 position within the 3’UTR of the reactivation after allogeneic HSCT was outbalanced by its stromal derived factor-1 (SDF-1) encoding gene. SDF-1 is a long-term suppressive effect on granulopoiesis. Our data CXCR4 ligand. The CCR5 deletion mutation results in a non- further indicate that oral VGC maintenance after CMV functional chemokine receptor. The effects of the SDF-1-3'A

S162 on HIV infection and AIDS progression have been attributed to points when pts lacked CMV DNA copies (0.30±0.06 vs the altered amount of SDF-1. 0.77±0.19%, p=0.008, U MW test). From the latter observation In the present study the CCR5-Delta32 polymorphism and the it was also apparent that proportion of CMV pentamers lower A/G substitution within the SDF-1 gene were analyzed in 76 than 0.30% in lymphocytes gate is highly predictive of CMV HSCT donor-recipient pairs and related with EBV load. Viral reactivation. load were assessed 2-3 months after transplantation in blood This study shows that higher risk of CMV reactivation cells employing a real-time PCR technique. The detection associates with: (i) MUD donor transplantation, (ii) the threshold for viral reactivation equaled 10 EBV-DNA presence of sever acute or extensive GvHD (iii) the use of copies/105 cells. EBV reactivation was apparent in 27 Campath for conditioning and more specifically with a low patients. The CCR5-Delta32 and SDF-1 (-801 G/A) proportion of CMV specific C8+ cells. The latter measurement polymorphisms were analyzed by PCR and PCR-RFLP may provide an argument to implement anti-CMV pre-emptive technique, respectively. treatment. Distributions of CCR5 and SDF-1 genotypes were similar in This work was supported by FP6 grant AlloStem. patients and donors. The higher number of EBV copies were detected in patients lacking CCR5-Delta32 deletion (0.42 vs 0.13, p=0.026) No association was found for the donor CCR5 P703 polymorphism. No significant correlation was observed IFNG 3/3 genotype may be associated with the higher between EBV reactivation and the presence of the SDF-1-3’A incidence of CMV reactivation in patients post HSCT allele either in recipients or donors of HSCT. E. Jaskula (1), A. Mlynarczewska (1), K. Bogunia-Kubik (1), D. In conclusion, the susceptibility for EBV reactivation appeared Duda (2), A. Lange (2) to be associated with the recipient CCR5-Delta32 allele, but (1)L.Hirszfeld Instute (Wroclaw, PL); (2)Lower Silesian Center not with the SDF-1 polymorphism. The latter one might be for Cellular Transplantation (Wroclaw, PL) explain by the lack of evidence for the contribution of the SDF- 1 (-801 A/G) polymorphism to the amount of the SDF-1 mRNA Cytomegalovirus (CMV) reactivation is a serious complication in EBV-transformed lymphoblastoid cells. These results affecting the recipients of allogeneic hematopoietic stem cell suggest (in opposite to HIV infection) the involvement of transplants (HSCT). IFN-gamma is an important antiviral CXCR4 independent mechanisms in EBV reactivation. factor and polymorphism within the IFNG gene is associated Supported by the Polish Ministry of Science and Higher with expression of this gene and protein production and also Education (grant No. 2 P05B 085 28)and the FP6 Project with the risk of acute and chronic graft-versus-host disease. AlloStem. Our former studies also showed that the presence of the IFNG 3/3 low producer genotype in the patients was associated with a lower susceptibility to Epstein-Barr virus (EBV) reactivation. P702 In the present study IFNG polymorphic features were related Severe CMV load post HSCT is inversely correlated with a with CMV load. proportion of CD8high+ Pro5 pentamer HLA- Sixty seven patients (30 HLA-matched sibling and 37 matched A*0201/NLVPMVATV (CMV pp65)+ cells and associates unrelated donor (MUD) transplant recipients) were analyzed with a risk of fatal complications for microsatellite polymorphism (CA)n within the first intron of E. Jaskula (1), D. Dlubek (1), D. Drabczak-Skrzypek (1), D. the IFNG gene by PCR-STR technique and for CMV DNA Duda (2), M. Sedzimirska (2), A. Lange (2) copies with the use of a real-time PCR. Number of CMV DNA (1)L.Hirszfeld Institute (Wroclaw, PL); (2)Lower Silesian copies in peripheral blood cells were determined in patients Center for Cellular Transplantation (Wroclaw, PL) within the observation period ranging from day 7 to 100 post HSCT with 5 measurements per patients in an average. Seventy two recipients receiving HSCT from sibling (SIB) A higher number of CMV copies were detected in patients (n=34) and unrelated (MUD) donors (n=38) were examined for having the IFNG 3/3 low producer genotype in comparison to a number of cytomegalovirus (CMV) DNA copies in blood the recipients with other IFNG genotypes (mean±sem: within the observation period ranging from day 7 to 3 yrs post 1044±405 vs 448±148 CMV copies/105 cells, p<0.001, Mann- HSCT with the use of a qPCR. Number of copies from 10 to Whitney U test (U MW test)). The association of IFNG 3/3 99 and that equal or higher than 100 per 105 cells were genotype with higher CMV load was also seen in patients recognized as positive and highly positive (severe load), transplanted from unrelated donors (1251±550 vs 681±335, respectively. p=0.003, U MW test) and from sibling donors (511±298 vs MUD transplanted pts had significantly higher number of CMV 70±36 CMV copies/105 cells, 0.038, Student’s T test). This copies than those receiving sibling transplantation observation was also valid when the patients were divided into (mean±sem: 914±243 vs 91±38 CMV DNA copies/105 cells, groups: (i) receiving reduced intensive conditioning regimen p=0.008, Mann-Whitney U test (U MW test)). Patients with (1229±475 vs 550±257 CMV copies/105 cells, p<0.001 U MW aGvHD grades • II and/or extensive cGvHD had higher test); (ii) receiving Campath (1124±641 vs 71±71, p=0.013, U number of CMV copies than those who lacked or had only MW test) or (iii) ATG (1079±550 vs 556±268, p=0.035, U MW grade I aGvHD or limited cGvHD (612±207 vs 451±161 CMV test). copies/105 cells, p=0.025, U MW test). In conclusion, the risk of CMV reactivation in patients post Only 2 out of 14 pts receiving Campath in comparison to 25 HSCT is associated with 3/3 IFNG low producer genotype. out of 43 pts on ATG and to 6 out of 15 pts not receiving anti- Supported by the FP6 project AlloStem and the Polish Ministry lymphocyte antibodies as a part of conditioning regimen had of Science and Higher Education (grant No. 2 P05B 085 28). aGvHD grades • II and/or extensive cGvHD (chi square=8.44, p<0.025). The use of Campath reduced the risk of aGvHD but was associated with a higher incidence of severe CMV load as compared to pts not receiving anti-lymphocyte antibodies (448±262 vs 25±20 CMV copies/105 cells, p=0.004, U MW test). The pts with a fatal outcome of HSCT had a higher frequency of severe CMV load as compared to the group of pts enjoying long term survival (12/20 vs 14/52, p=0.010, Fisher’s exact test). 18 pts were followed from day 11 to 2 yrs post transplant for a percentage of CD8high+ PRO5 pentamer HLA- A*0201/NLVPMVATV (CMV pp65)+ cells. It was found that proportion of this pentamer staining positive cells was significantly lower in pts post HSCT at the time of CMV reactivation as compared to staining results at observation

S163 P704 preventive strategies in specific subgroup of patients after RIC Features of long-term infectious complications following allo-SCT. The aim of this pilot study was to investigate the reduced-intensity conditioning allogeneic HLA-identical feasibility and tolerance of weekly prophylactic administration sibling transplantation of once-weekly high-dose (7.5 mg/kg; Ambisome®) L-AmB A. Etienne (1), M. Mohty (1), C. Faucher (1), S. Furst (1), J. El therapy in 21 adult patients receiving high dose steroids (2 Cheikh (1), C. Zandotti (2), P. Berger (1), N. Vey (1), A.M. mg/Kg/day) for acute GVHD therapy after RIC allo-SCT. The Stoppa (1), R. Bouabdallah (1), P. Viens (1), J.A. Gastaut (1), median age of recipients was 51 (range, 18-70) years. Seven D. Blaise (1) patients had a myeloid malignancy, whereas 11 patients were (1)Institut Paoli-Calmettes (Marseille, F); (2)Hopital La Timone diagnosed with lymphoid malignancies. The remaining 3 (Marseille, F) patients were treated for metastatic non-hematological malignancies. In addition to multiple lines of previous This prospective report describes infectious complications chemotherapy, 10 patients from this study had received prior occurring beyond 6 m. after allo-SCT, in 159 pts who received auto-SCT. A sustained ANC of more than 500/ìL, was reached reduced-intensity conditioning (RIC) allo-SCT from an HLA- at a median of 19 (range, 6-28) days. Per protocol, all patients identical sibling. Pts characteristics are as follow: median age: had acute GVHD (11 grade 2, 10 grade 3-4) occurring at a 50 (18-68)y. 68 pts (43%) had a myeloid malignancy, whereas median of 42 (range, 12-84) days after allo-SCT. Patients 66 (41%) had a lymphoid malignancy. 25 pts (16%) had a received a median of 4 (range, 1-8) infusions of L-AmB. metastatic non-hematological malignancies. 24 pts (15%) Seven patients (33%; 95%CI, 13-53%) discontinued taking the received bone marrow (BM), while 135 pts (85%) received study drug due to study drug-related adverse events, including PBSCs. In addition to fludarabine and busulfan, the RIC elevated serum creatinine (>1.5 times from baseline values; regimen included high dose ATG in 20 pts (13%) and low n=5), hypotension and pain (n=1), and chest pain and dose ATG in 95 (60%). 24 pts (15%) received fludarabine, arrhythmia (n=1). The overall frequency of infusion-related busulfan and TLI, while the remaining 24 pts (15%) received reactions was 29% (n=6; 95%CI, 10-48%), but these reactions fludarabine and low dose TBI. With a median FU of 19 were always transient and relieved by stopping the infusion or (range,6-90) m., 120 pts (75%) experienced at least one administering paracetamol. With a median follow-up of 377 infectious episode (total, 366) beyond the first 6 months after (range, 74-983) days, no deaths were attributed to fungal allo-SCT, at a median of 8 (range,6-34) m. 212 infectious infections, although the study was not powered to assess episodes (58%) required hospitalization (7% in the ICU) for a efficacy. This safety data provide support for a prospective median duration of 10 (1-91)d. 144 episodes (39%) could be efficacy study of this prophylaxis strategy, because this documented (bacterial,n=48;viral,n=78; fungal,n=18). approach may provide useful protection against fungal Documented infections were: gram negative bacteria (18%), infections, and may help further improving the outcome of RIC other bacteria (15%), CMV positive antigenemia (17%), HSV or nonmyeloablative allo-SCT. (19%), VZV (15%), other viruses (3%), aspergillus (6%), candida species (6%), other (1%). 76% of pts with an infection were under systemic immunosuppressive therapy for chronic P706 GVHD at time of infection. Moreover, 85 pts (71%) A prospective evaluation of sensitivity and specificity of experienced more than one infectious episode BK viruria and BK viraemia in paediatric patients given (median,2;range,1-12). In multivariate analysis, active or prior allogeneic haematopoietic stem cell transplantation with history of extensive chronic GVHD and the use of a BM graft haemorrhagic cystitis were the strongest factors significantly associated with an S. Cesaro (1), C. Facchin (2), G. Tridello (1), MA. Biasolo (3), increased risk of long term infections A. Calò (1), L. Barzon (3), E. Calore (1), M. Pillon (1), S. (P=0.0003;RR=2.04;95%CI,1.4-3.0; and Varotto (1), C. Mengoli (3), G. Palù (3), C. Messina (1) P=0.005;RR=2;95%CI,1.2-3.2 respectively), highlighting the (1)Pediatric Hematology Oncology (Padua, I); (2)Infectious raising concern about the deleterious impact of severe chronic Diseases of Pediatric Immunocompromised Host (Padua, I); GVHD occurring after RIC allo-SCT, but also the protective (3)Department of Histology, Microbiology and Medical effect of donor origin immunity based on graft content. In this Biotechnology (Padua, I) series, the overall long term TRM was 11% (n=18), of whom 12 deaths were attributed to chronic GVHD and its Hemorrhagic cystitis (HC) has been reported in 3.6%-68% of complications including infections, and 5 deaths solely patients given HSCT. Recently, its pathogenesis has been attributed to infections. In all, these results suggest that, associated to infection or reactivation of polioma virus, despite reduction in early toxicity associated with the use of especially BK virus. RIC regimens, long term debilitating chronic GVHD and its From September 2001 to March 2006, we analyzed corollary of continuous immunosuppression and subsequent prospectively 74 patients who underwent allo-HSCT to assess infections are still a matter of concern. Prospective efforts to the 100-day incidence of HC and its association with BK develop optimal antimicrobial preventive strategies are infection as determined by PCR on blood and urine. They needed. were 44 males and 30 females, with median age at transplant of 9.6 years (range 0.5-17); affected by malignant disease, 61 patients, or non-malignant disease, 13 patients. 16 of 74 P705 patients developed 17 episodes of HC within day +100. The High-dose weekly liposomal amphotericin B antifungal cumulative incidence of HC was 22% (C.I. 12-31). The median prophylaxis following reduced-intensity conditioning time from HSCT to HC was 35 days (range 6-56); and the allogeneic stem cell transplantation median duration of HC was 11 days (range 1-44). HC was J. El Cheikh, C. Faucher, S. Furst, S. Duran, P. Berger, N. classified as grade I, II, III, IV in 4, 8, 4, and 1 patients Vey, A.M. Stoppa, R. Bouabdallah, J.A. Gastaut, P. Viens, D. respectively. After a median f-up of 1.2 years, range 0.1-4, HC Blaise, M. Mohty patients, compared to non-HC patients, had a lower OS, 37% Institut Paoli-Calmettes (Marseille, F) vs 54%, p 0.009; and a lower EFS, 38% vs 57%, p 0.017; though the 100-day TRM resulted similar, 6% vs 5%, p 0.9. Although promising results have been reported in regards to BK virus on urine by PCR was detected in 14 of 16 HC engraftment and early transplant-related toxicities, graft- episodes investigated; whilst BK was found on blood by PCR versus-host disease (GVHD) and its corollary of long-term in 9 of 12 HC episodes tested. detrimental immunosuppressive therapy and opportunistic Weekly monitoring of all transplanted patients from January infections, remain a matter of concern after RIC allo-SCT. We 2005 to March 2006 showed a significant association between and others have previously shown that the use of high doses at least 2 consecutive positive PCR for BK on blood or urine of steroids for GVHD treatment represent a major risk factor and HC, p 0.003 and < 0.001, respectively. Moreover, the for long-term infections, with invasive aspergillosis (IA) being analysis of quantitative BK-PCR results by ROC curve the principal cause of infectious-related mortality, suggesting showed that a cut-off of 9 x 106 genomic copies/ml on urine that efforts are warranted to develop optimal antifungal had a sensitivity of 95% and a specificity of 90% for HC; whilst

S164 a cut-off of 1 x 103 genomic copies/ml on blood had a P708 sensitivity of 40% and a specificity of 93% for HC. P2X7 receptor polymorphism and clinical outcomes in In conclusion, BK viruria and BK viremia are significantly HLA-matched sibling allogeneic haematopoietic stem cell associated to HC in pediatric allo-HSCT patients; moreover, transplantation BK load on urine has an higher predictive value for HC than I. Kim, K.-H. Lee, K.-H. Kim, J.-W. Kim, J.-Y. Lee, D.-Y. Kim, BK load on blood. B.-S. Kim, S.-Y. Oh, S.-W. Han, S.-Y. Kim, E.-K. Park, S.-M. Bang, S.-S. Yoon, J.-S. Lee, S. Park, B.K.K. Kim Seoul National University Hospital (Seoul, KOR) P707 Posaconazole prophylaxis in haematopoietic stem cell The P2X7 receptor (P2X7R) is a key player in the processing transplant recipients and neutropenic patients: and release of IL-1. To evaluate whether the A1513C pharmacokinetics and safety analysis polymorphism of P2X7R is related to allogeneic stem cell J. Maertens (1), A. Langston (2), A. Ullmann (3), O. Cornely transplantation outcome, we performed association analysis (4), J. Lipton (5), D. Winston (6), J. Perfect (7), P. between this polymorphism and clinical outcome in patients Chandrasekar (8), T. Brundage (9), H. Patino (9), G. Krishna treated by HLA-matched sibling stem cell transplantation. (9) Patients (n=152) with a malignancy or aplastic anemia (1)University Hospital (Leuven, B); (2)Emory University underwent allogeneic stem cell transplantation at a single Hospital (Atlanta, USA); (3)J.-Gutenberg University (Mainz, institute. The frequencies of the A and C alleles in all 295 D); (4)University of Cologne (Cologne, D); (5)Princess study subjects were 72% and 28%, respectively. Patient Margaret Hospital (Toronto, CAN); (6)UCLA CA (Los Angeles, genotypes were; AA in 75, AC in 58, and CC in 12, and donor USA); (7)Duke University Hospital (Durham, USA); (8)Harper genotypes were AA in 74, AC in 70, and CC in 6. Overall Hospital (Detroit, USA); (9)Schering-Plough Research Institute survival was significantly shorter for recipients with the CC (Kenilworth, USA) genotype than for those with the AA or AC genotypes (92 days for 1513CC vs. 821 days for 1513AA or 1513AC, Objectives: Hematopoietic stem cell transplant (HSCT) P=.012), and for recipients from donors with the CC genotype recipients and neutropenic patients undergoing induction than for recipients from donors with the AA or AC genotypes chemotherapy for acute myelogenous leukemia (AML) or (63 days for 1513CC vs. 702 days for 1513AA or 1513AC, myelodysplastic syndrome (MDS) are at high risk for the P=.024). Multivariate analyses, which included sex, age, development of invasive fungal infections (IFI). In 2 large, transplant method (reduced intensity conditioning vs. comparative, clinical trials (N~1200), the azole posaconazole conventional conditioning), stem cell source, risk group, and (POS) was evaluated vs fluconazole (FLU) or itraconazole for P2X7R recipient and donor genotype, identified a high-risk prophylaxis in these high-risk patients. The objective of this group (hazard ratio 3.25, 95% confidence interval 1.83~5.77) analysis was to investigate the relationship between POS and a donor 1513CC genotype (hazard ratio 2.66, 95% pharmacokinetics and common adverse events identified in confidence interval 1.02~6.91) as risk factors of a shorter these trials. survival. Microbiologically documented bacteremia occurred in Methods: 301 HSCT recipients with graft-vs-host disease 66.7% of recipients with the CC donor genotype and in 17.6% (GVHD) (study 1) and 304 neutropenic AML or MDS (study 2) of recipients that received transplants of the AA or AC were randomized to receive oral POS 200 mg three times genotypes (P=0.014). We conclude that the A1513C daily. POS average and maximum plasma concentrations polymorphism in the P2X7R gene is related to the occurrence (Cav and Cmax) were determined and their relationship to of infections and survival after allogeneic stem cell adverse events was analyzed. transplantation. Thus, the determination of this polymorphism Results: POS plasma concentrations were available for 246 may be helpful for the optimal selection of patients and (study 1) and 215 (study 2) patients (table). Although the donors. mean Cav and Cmax differed between the 2 patient populations, the frequency of treatment-related adverse events (AEs) was similar. Disorders of the gastrointestinal P709 system were most commonly reported, including nausea (7%, Epstein-Barr virus associated lymphoproliferative 7%), vomiting (4%, 5%) and diarrhea (3%, 7%) for study 1, disorder following allogeneic transplantation for acquired study 2, respectively. POS plasma levels were not associated aplastic anaemia: the role of prior immunosuppressive with the incidence of AEs, including those involving the central therapy and intensity of conditioning nervous system, visual disturbances, and increases in liver S. Chakrabarti, H. Buyck, S. Ball, L. O'Regan, P. Rice, J. enzymes. Overall, the safety and tolerability of POS were Marsh similar to that of FLU. St George's Hospital (London, UK) Conclusion: POS prophylaxis was well tolerated in HSCT recipients with GVHD and neutropenic AML/MDS patients at EBV-LPD is a serious and often fatal post-transplant high risk for development of an IFI. No association was found complication which has a widely variable incidence depending between POS plasma concentrations and AEs commonly on the several factors such as donor and recipient sero- related to azole therapy. positivity, T cell depletion, use of serotherapy, steroids and GVHD. We reviewed the incidence of EBV-LPD in 89 patients (median age, 20 yrs, range 4-53) with acquired aplastic anaemia (AAA) receiving allogeneic transplantation from a related (n=69) or unrelated (n=20) donor between 1989 and 2006. 78 patients received conventional conditioning (CIC) with cyclophosphamide (200mg/kg) alongwith ATG or Campath, whilst 11 received a reduced intensity conditioning (RIC) with fludarabine, reduced dose cyclophosphamide and Campath or ATG. The overall incidence of EBV-LPD was 5 % (4/89) with no cases diagnosed in those receiving an allograft for malignant diseases (n=57) or constitutional bone marrow failure syndromes (n=30) during the same period. There was no impact of age, gender, donor status, CMV seropositivity, GVHD and graft cell dose on the occurrence of LPD. There was no influence of serotherapy used in conditioning either, with 1/19 patients receiving ATG and 3/63 receiving Campath developing LPD. However, prior use of ATG as immunosuppressive therapy had a strong influence on development of LPD with an incidence of 11.3 ±5.3% (4/40),

S165 compared to none in those not exposed to ATG before P711 transplantation (p=0.02). The incidence of LPD in those Weekly high-dose cidofovir is an effective treatment for receiving RIC was 18.2 ± 11% (2/11), compared to 2.9 ± 2% symptomatic BK virus-associated haemorrhagic cystitis (2/78) for those receiving CIC (p=0.02). Three patients after allogeneic stem cell transplantation survived with withdrawal of immunosuppression, DLI or DLI M. Michael, G. Kourti, I. Baltathakis, E. Grispou, M. Bouzani, and rituximab. The lymphoproliferation was identified as that M. Anagnostopoulou, I. Apostolidis, D. Karakasis, N. of host origin in all 3 patients evaluated. This is the first study Harhalakis, O. Paniara, E. Nikiforakis on the incidence of LPD in AAA who unlike those with Evangelismos Hospital (Athens, GR) malignant diseases are not exposed to cytotoxic therapy prior to transplant, but are often exposed to anti-T cell therapy. A proportion of cases of hemorrhagic cystitis(HC) after Prior treatment with ALG and nonmyeloablative conditioning allogeneic stem cell transplantation are associated with might play a role in preferential survival of residual B infection from BKV. Supportive treatment is not sufficient for lymphocytes of host origin, creating a unique environment for the relief of symptoms in many patients with BKV-associated host derived EBV-lymphoproliferation. Use of Campath at high HC. Cidofovir is an antiviral agent active against BKV but its doses might protect against EBV-LPD as previously reported, experience in BKV-associated HC is anecdotal and the but reduced dose Campath does not seem to have a similar optimal dose schedule of the drug has not been determined. effect, at least in this group of patients. In conclusion, patients We administered weekly high-dose cidofovir in 5 patients for with AAA who have received prior treatment with ATG are the treatment of 6 episodes of symptomatic BKV-associated likely to be at a higher risk of developing EBV-LPD, more so, HC, which were refractory to first-line therapy with hydration in association with RIC. and continuous bladder irrigation. One patient was treated twice with cidofovir for 2 temporally separated episodes of HC, combined with papillary necrosis and ureteritis respectively. P710 All 5 patients were males, aged 21-44 years and had received Toll-like receptor 2 signalling regulates oxidative burst of grafts from siblings (n=2), matched unrelated (n=1) or human neutrophils, but not cytokine response of haploidentical (n=1) donors. The median time from dendritic cells after contact with aspergillus fumigatus transplantation to diagnosis of HC was 61.5 (26-303)days. In M. Mezger, H. Einsele, J. Loeffler all cases the development of HC was associated with severe University Hospital (Wuerzburg, D) immunosuppression due to profound lymphopenia or treatment for GVHD. BKV-associated HC was confirmed by Introduction: Toll-like receptors (TLRs) play a crucial role for detection of BKV in the urine by real-time PCR (1.5 × 107-5.5 detecting conserved pathogen-associated molecular patterns × 109 copies/ml). In 2 episodes, the patients also developed comprising fungal structures. Activation of polymorphonuclear BKV viremia (5.0-9.9 × 10³ copies/ml). The median time from granulocytes (PMNs) as well as mobilization of antigen the manifestation of symptoms to initiation of cidofovir was presenting cells is important for successful clearance of fungal 12(range, 4-53) days. Cidofovir was started at a dose of 2.5 infections. Here, the role of TLR2 and TLR4 for activation of mg/kg/week intravenously over 1 hour. Subsequent doses of 5 PMNs and immature dendritic cells (iDCs) through Aspergillus mg/kg were given at weekly intervals for a total of 4-6 doses. fumigatus was analyzed. Oral probenecid and intravenous hydration were also Material and Methods: Generation of iDCs was achieved by administered. No patient developed renal toxicity. In 5 of the 6 cultivating monocytes in the presence of GM-CSF and IL-4. cases, clinical improvement of patients was observed with a PMNs were obtained from fresh blood. In blocking studies, median time to response of 5 (range, 3-8) days and complete cells were preincubated with anti-TLR2 and / or anti-TLR4 resolution of symptoms of HC was achieved in these 5 cases antibody before stimulation with Aspergillus fumigatus after 4 to 6 doses of cidofovir. BKV viral load was monitored germinating conidia. For RNA interference (RNAi) weekly in urine and blood during therapy. Clinical responses experiments, iDCs were transfected with short-interfering RNA did not correlate with a reduction in viral load in urine. (siRNA) via electroporation and gene expression was Concurrent CMV infection needed no further treatment. controlled by quantitative real-time PCR. In conclusion, treatment of BKV-associated HC with cidofovir Oxidative burst of PMNs was induced after contact with at the dose of 5 mg/kg/week seems to be safe and may result Aspergillus or TLR2 ligand zymosan. Release of oxygen in prompt and sustained relief of the debilitating symptoms of intermediates could be significantly reduced if PMNs were this condition. Additional manifestations of BKV-associated preincubated with anti-TLR2 antibody. disease (papillary necrosis and ureteritis) and concurrent CMV Results: In contrast to monocytes, cytokine secretion (IL-12 infection can be managed successfully with this dose and TNF-a) of iDCs was not altered if anti-TLR antibodies schedule. were used. To study the role of TLR2 and TLR4 more detailed, an RNAi system for iDCs was established to downregulate gene expression. Determination of siRNA P712 transfection rate revealed an efficiency of about 85% and Outbreak of adenovirus in paediatric transplant unit allowed significant downregulation of TLR2 and TLR4 (> coincided with increased incidence of adenovirus in 90%). In accordance to blocking studies, expression of IL-10, general population IL-12 and TNF-a was not reduced after TLR2 or TLR4 siRNA P. Hubacek (1), P. Sedlacek (1), L. Hornofova (1), D. transfection and stimulation with Aspergillus. Brynychova (2), K. Hyncicova (3), M. Zajac (3), B. Nagyova Conclusions: TLR2 plays an important role in the induction of (1), P. Keslova (3), R. Formankova (1), J. Stary (1) the oxidative burst of PMNs whereas cytokine release of iDCs (1)Charles University and Motol University (Prague, CZ); seems to be independent of TLR2 / TLR4 signalling. The (2)National Reference Laboratory on Viral Respiratory established siRNA system allows the detection of receptors Infections, National Institute of public Health (Prague, CZ); responsible for activation of iDCs. (3)Motol University Hospital (Prague, CZ)

Adenovirus (AdV) could cause serious life-threatening complications in recipients of allogeneic hematopoietic stem cells (HSCT). Surprisingly over a period of detailed surveillance (AdV study EU project - I/2003-IX/2005) we have in our unit only rarely detected AdV. This was in contrast to more frequent AdV infections reported by other involved European transplant centers. Using quantitative real-time PCR we have over that time screened for AdV DNA group A-C 1535 samples of whole blood and 8 obtained from BAL, stool and urine in 65 children early after allogeneic HSCT. Only 12 blood samples (4 patients) and 2 BAL samples (1 patient)

S166 were positive. But within one week in July 2006 we have specific T-cells under certain conditions. Thus monitoring of detected AdV infection in five children. In 4 of them AdV31 the persistence or development of virus-specific T-cells after was detected, AdV2 was documented in one patient. None of HSCT will lead to the detection of patients at risk for viral the children at the time of diagnosis showed clinical signs of reactivation and may allow early detection of patients profiting severe infection, mild diarrhea has appeared in two. In all from CMV-specific T-cell transfer. children we have detected significant viral load in blood (1.8x103 - 1.7x107/ml), and also in stool (2,6x104 - 4x1010/ml). In four with higher load we have started cidofovir (CDV) 1mg/kg/dose 3x weekly and hygiene-epidemiological (HE) regime of the transplant unit was even more restricted (restriction of contacts, gloves, etc.). After introduction of this regime no new infection with AdV was observed in next 4 months. We have documented the positive effect of CDV (prevention of further increase). Viral load in blood disappeared in three children, in two decreased. Reconstitution of immunity played crucial role in all cases. Two patients died 5.6 and 3.2 months after HSCT (severe GvHD in one, disseminated AdV infection and intracranial hemorrhage in one). In later patient samples from the autopsy were tested by PCR and we have detected AdV 31 in most of the organs. Because there was no change in type of transplants, supportive care, regime, etc. at our unit compare to previous period we assume that spreading of AdV on the unit reflected increased adenovirus infections observed in general population by National reference laboratory. At least in some of patients with AdV31 there is clear epidemiological connection and therefore it is obvious that the original source P714 in them was the same. We have documented that strict HE Artesunate as a potent anti-CMV agent in a patient after regime is the efficient way how to prevent further spread of mismatched haematopietic cell transplantation infection and CDV may be effective to control the infection at M.Y. Shapira (1), I.B. Resnick (1), T. Stamminger (2), T. least in some patients. Efferth (3), O. Kaplan (1), M. Marshall (2), D.G. Wolf (1) Supported by MSMT CR 002162081 and EU: QLK2-CT-2002- (1)Hadassah Hebrew University (Jerusalem, IL); (2)Institute 01432 for Clinical and Molecular Virology (Nuremberg, D); (3)German Cancer Research Center (Heidelberg, D) P713 Introduction: Human cytomegalovirus (CMV) has remained a Monitoring of virus-specific T-cells with virus specific major cause of morbidity and mortality following hematopoietic tetramers – implication for virus-specific T-cell therapy? stem cell transplantation (SCT). All antiviral drugs currently S. Borchers, B. Grabow, S. Luther, M. Heuer, A. Ganser, E.M. used for the treatment of CMV infection target the viral DNA Weissinger polymerase, and are often limited by bone marrow and renal Hannover Medical School (Hannover, D) toxicity, low oral bioavailability, and the development of antiviral drug resistance. Recently, the non-toxic anti-malaria Cytomegalovirus (CMV) reactivation contributes to morbidity drug artesunate, has been shown to be a highly effective and mortality after allogeneic hematopoietic stem cell inhibitor of CMV in vitro and in an experimental animal model. transplantation (HSCT). Risk factors for the reactivation of Here, we describe the first clinical use of artesunate in the CMV are the conditioning regimen and graft versus host SCT setting. disease (GvHD) prophylaxis, like T-cell depletion of the graft, Case report: A 12 years old boy, with X-linked the development and the treatment of acute or chronic GvHD. adrenoleukodystrophy underwent a haploidentical SCT from In addition, transplantation of a CMV-seropositive recipient his father with T cell depletion. Following this treatment, the with cells a CMV-seronegative donor comprises a risk factor patient developed CMV infection. He was treated with for recurrent reactivation of CMV. Since T-cells play an foscarnet because of severe myelosuppression precluding important role in the control of viral reactivation, we started to ganciclovir therapy with initial partial response. However his evaluate the number of CMV-specific T cells in our patient viral load subsequently increased during treatment. Foscarnet cohort after allogeneic HSCT. was discontinued. Treatment trials with cidofovir and To improve understanding of reactivation and restoration of ganciclovir, combined with two courses of high dose CMV-immunity, we established a FACS based assay for immunoglobulin were unsuccessful, and the CMV DNA load monitoring absolute numbers of CMV-reactive cytotoxic T- increased to 1.15 x106 copies/ml. At this point, all cells using MHC-I-Peptide-tetramers available for the major conventional anti-CMV drugs were discontinued, and oral HLA class I groups. The available tetramers allow monitoring treatment with artesunate (100 mg/d) was initiated on a of about 63% of our patients. The screening interval for CMV- compassionate basis. A dramatic decrease in viral load was epitope specific CD8+ T-cells was prior to HSCT and on days noted with 2-, 3-, and 4-log reduction in viral load within 10, +50, +80, +100, + 180 and +365 or weekly at the time of CMV 21, and 28 days of artesunate therapy, respectively. No side reactivation or increased immunosuppressive treatment due to effects were observed during a month of artesunate treatment. development of GvHD. To date we have enrolled 27 patients The patient had no rebound viremia during a 60 days follow in this screening. Of these, 12 CMV-seropositive recipients up after completion of therapy. were transplanted from CMV-seronegative donors. Trends of Conclusion: Artesunate proved to be a highly effective CMV-specifiv T-cell reconstitution after HSCT are currently inhibitor of CMV replication in a high risk immune suppressed available for 9 patients. patient following mismatched SCT, and should be considered All patients with more than 10 CMV-specific T-cells per in similar cases of clinically drug-resistant CMV infection. A microliter blood experienced no CMV reactivation. Two of clinical trial of preemptive artesunate treatment in patients these patients were transplanted from seronegative donors. In undergoing SCT is currently underway at our center. both patients CMV-reactive T-cells (total of 13 to 59 cells per IB Resnick and MY Shapira contributed equally to this report. µl) were detected around day +50 and seem to provide DG Wolf and M Marschall contributed equally as senior protection against CMV ( Figure 1). authors. Our data indicate that monitoring of CMV-specific T-cells is well suited to predict the risk of CMV-reactivation and/or protection against reactivation due to recipient/donor CMV-

S167 P715 Aim: To investigate the immunogenicity of the 7 valent Adenovirus infections following allogeneic HSCT. A pneumococcal conjugate vaccine Prevenar following single UK centre experience transplantation in patients with myeloma. L. Green, C. Bagot, S. Ramalingam, Z. Lim, M. Sudhanva, S. Design: We followed the UK DoH guidance for vaccination of Devereux, A. Ho, M. Zuckerman, A. Pagliuca, G.J. Mufti high risk children; 2 doses of Prevenar separated by a Kings College Hospital (London, UK) minimum of 2 months followed by a dose of the 23-valent PS vaccine after a further 2 months (Group 1). Patients who had Adenovirus(Adv) infections are a frequent cause of morbidity received the 23-valent vaccine after transplant were only and mortality following allogeneic HSCT. We retrospectively offered the vaccinations with Prevenar (Group 2). analysed the incidence, risk factors and outcome of Results: Data are available on 24 patients. Median age was symptomatic adenovirus infection among allogeneic HSCT 61 years (range 52-69). All patients had undergone an recipients at a single UK centre from Jan 2003 to May 2005. autologous transplant. 4 patients had received 2 prior All patients were screened twice weekly for a minimum of 6 autografts and 1 had undergone a tandem autograft followed months for the presence of Adv in the peripheral blood by by a RISCT. DNA-PCR. Median time from last treatment was 14 months (range 6-43). A total of 183 patients received an allogeneic HSCT during the 8 patients were receiving maintenance thalidomide. 1 patient study period, of which 13 patients(7%) developed Adv had low baseline IgM levels and 3 had low IgA. infection. The median age of recipients developing Adv was We utilised a serotype-specific IgG Bioplex assay for all 7 44 years(range:18-62). 12/13 patients(92%) received serotypes in Prevenar. WHO guidelines suggest levels of • alemtuzumab as part of their conditioning. 10 patients(76%) 0.35µg/ml are protective. We also assessed the proportion of received RIC. 7 patients(53%) received stem cells from a patients acheiving a 2 and 4 fold increase from baseline matched unrelated donor. The stem cell source was PBSC in levels. 8 patients(61%). The median time to initial Adv detection was Prior to vaccination the median number of serotypes with 40 days post-HSCT(range:11-160). Adv was isolated in urine protective antibody levels was 0 in Group 1 [n=20], (range 0- in 5/13 cases(38%), stool in 9/13(69%), peripheral blood in 2) and 1 in Group 2 [n=4] (range 0-3). 10/13(76%), nasal pharyngeal aspiration(NPA) in 8/13(61%) Following vaccination the median number of serotypes with and one patient had skin involvement. In 30%(4/13) Adv was protective antibody levels increased to 4 in both groups (range isolated in four sites(blood, urine, stool and NPA) and 0-7). 5 patients, (21%) achieved full protection. Antibody mortality rate in this group was 100%. The major presenting levels doubled in both groups for a median of 5.5 serotypes symptoms at the time of diagnosis were diarrhoea(9/13), (range 0-7)and quadrupled for a median of 3 serotypes (range haematuria(4/13). 7 patients(53%) had GvHD at time of 0-7). diagnosis. Severe lymphocytopenia(<0.3x109/L) at the time of Conclusions: The pneumococcal conjugate vaccine Prevenar first detection was noted in 80% of recipients, and 12/13 appears to be immunogenic for some individuals in this group. patients were in receipt of concurrent immunosuppressive Most patients did achieve an increase in baseline antibody therapy. levels, however, it did not provide cover in all cases to all 11/13 cases were treated with Cidofovir as first line treatment serotypes. Further studies are required to determine optimal and 2/11 patients subsequently received alternative treatment dosage and predictors of response prior to its introduction in with Forscanet(1), Ribavirin + adenovirus specific cytotoxic T- clinical practice. cell lymphocytes(1). The overall mortality rate was 76%(10/13) and the median time to death after initial Adv detection was 23.5 days. All 10 patients who died had 2 or more organ failure, and 8/10 had viral infections including CMV(n=7), P717 BK/JK virus(n=4),Others(n=2). The 3 surviving patients had AFLP epidemiological investigation of an outbreak of Adv detected in the peripheral blood(2), and via NPA(1), and Pseudomonas aeruginosa sepsis in stem cell none developed end-organ damage. transplantation patients In a cohort of patients predominately receiving alemtuzumab R. Fanci (1), B. Bartolozzi (1), P. Pecile (1), D. Cecconi (1), R. as part of the conditioning regimen, we report a 7% incidence Mannino (1), E. Casalone (2), A. Gordillo Leon (2), S. Sergi of Adv post-HSCT. Patients developing Adv were more likely (2), S. Guidi (1), P. Nicoletti (1), G. Mastromei (2), A. Bosi (1) to have concurrent GvHD and to be receiving (1)Careggi Hospital (Florence, I); (2)University of Florence immunosuppression. In addition prognosis was poorer in (Florence, I) those patients who had multiple organ damage or disseminated infection (isolating Adv from 3 or more sites). Pseudomonas aeruginosa represents one of the main causes of nosocomial infections in immunocompromised patients, responsible for high morbidity and mortality. From May to September 2006, in the Bone Marrow Unit of the P716 Careggi Hospital in Florence, Italy, 5 severe P. aeruginosa Evaluation of immune response to the pneumococcal bloodstream infections were diagnosed in patients undergoing conjugate vaccine (Prevenar®) following transplantation stem cell transplantation (3 autologous and 2 allogeneic in patients with myeloma transplants). All isolates resulted phenotipically similar (same H.M Greenfield (1), R. Borrow (2), R. Warrington (2), P. biochemical pattern, same pattern of sensitivity to antibiotics). Balmer (2), J. Cavet (3), J. Adams (1), K. Mutton (2), J.A. Liu But only three of the pts (2 pts with acute leukemia receiving Yin (1) allogeneic transplant and one pt with non Hodgkin Lymphoma (1)Manchester Royal Infirmary (Manchester, UK); (2)North receiving autologous transplantation) had shared the same West Regional Health Protection Agency (Manchester, UK); room in different periods. In the hypothesis of a possible (3)Christie Hospital (Manchester, UK) environmental source of the infection, samples for microbiological evaluation (phenotyping analysis and AFLP- Infection with Streptococcus pneumoniae is responsible for amplified fragment-length polymorphism) were collected from significant mortality in myeloma patients where the humoral different sources that had potential for cross-contamination response is defective. In addition, protective immunity is lost throughout the Bone Marrow transplantation Unit including the following stem cell transplant. Immunisation with electrolytic disinfectant used for medication of skin and pneumococcal polysaccharide (PS) vaccine is poorly mucosae (Irgasan) and facilities (5% Antisapril). A strain of P. efficacious in this group. Conjugation of PS antigens to a aeruginosa with the same phenotype of patients was isolated protein carrier renders vaccines more immunogenic via T-cell from the Irgasan soap. dependency. Impressive results in children lend themselves to To confirm the hypothesis of the clonal origin of the isolates evaluation in other high risk groups. and of the transmission of the infection through the soap, a molecular typing by AFLP was performed on the P. aeruginosa isolates from the 5 blood cultures and the soap.

S168 The AFLP analysis showed an internal similarity of 93% within least 6 weeks post-transplant. Allele-specific PCR was applied the samples of the three pts sharing the same room and the for matrix metalloproteinase-1 (MMP-1, 1G/2G) plasminogen soap. These data were highly suggestive of a single infection activator inhibitor-1 (PAI-1, 4G/5G), and MDR-1 (C3435T) source and strongly suggest an environmental transmission gene variants. Statistical evaluation was performed by means route. of non-parametric criteria. Results: 1. As expected, serological studies of patients and donors pretransplant have shown high prevalence of CMV- P718 seropositivity in the patient-donor pairs. The post-transplant Aciclovir versus ganciclovir plus foscarnet for CMV PCR-positive state (determined as >2 positive findings until prophylaxis in allogeneic haematopoietic stem cell d+100) was detected in 33%, 24%, and 26% for, resp., HSV, transplantation EBV and CMV. No gross correlations were observed between C. Nozzoli, B. Bartolozzi, S. Guidi, A. Orsi, A. Gozzini, L. incidence of the three viral pathogens in blood cells, and Lombardini, R. Saccardi, A. Vannucchi, A. Bosi source of transplant, age or sex of the patients. However, Careggi Hospital (Florence, I) EBV- and CMV-positivity in myeloablative transplants was observed more common than in HSCT with reduced Cytomegalovirus (CMV) infection is a major cause of morbidity conditioning. (P<0.005). CMV PCR-positivity was often and mortality in allogeneic bone marrow transplant recipients. associated with clinical infection (non-engraftment, pneumonia Risk factors are donor/recipient CMV status, type of transplant etc.). HSV reactivation correlated with appropriate lesions of and acute graft versus host disease (aGVHD). PCR-based skin and mucosal surfaces. Increased percentage of HSV CMV DNA detection assays are the most sensitive methods positivity was found with unrelated transplants (41% incidence for monitoring CMV reactivation. Currently prophylactic or pre- vs 19% in familial HSCT, P=0,015). Interestingly, the risk of emptive antiviral treatment, are used for prevention of CMV HSV reactivation was strongly associated with hyperactive disease but their role is still debated . 2G/2G allele of collagenase 1 (MMP-1), thus confirming the We analysed the impact of antiviral prophylaxis on CMV role of extracellular matrix degradation in viral invasion. reactivation in 191 consecutive patients treated with Conclusions: Higher intensity of conditioning regimen myelotoxic allogeneic haematopoietic stem cell transplant determines increases risk of herpesvirus reactivation post- (HSCT) in our Unit from January 1999 to October 2006. Two transplant. Moreover, some genetic factors may represent consecutive series of patients received different antiviral additional risk factors for HSV and, probably, CMV infections prophylaxis: 90 pts transplanted from January 1999 to post-transplant December 2001 received acyclovir i.v. 750mg three times per day from the start of conditioning to day +20 and then 400 mg twice a day by mouth until d +180; 101 pts from January 2002 P720 received gancyclovir 10mg/kg/day from start of conditioning to EBV reactivation in paediatric unrelated hamatopoietic day -2 and foscarnet 90mg/kg/day from d +4 to d + 20 and stem cell transplantation: a single-centre experience then acyclovir 400 mg twice a day by mouth until d +180. All J.M. André (1), V. Mialou (1), M.P. Goutagny (1), C. pts were monitored by quantitative-PCR on plasma and cells Galambrun (1), P. Morand (2), Y. Bertrand (1) once a week from d +21. We considered as CMV reactivation (1)Hopital Debrousse (Lyon, F); (2)Hopital A. Michallon a DNA load >104 copies/ml or an increase of 1x103 (Grenoble, F) copies/ml in two consecutive detections. The two series of pts were comparable for CMV serum status recipient (pos 68 vs Epstein Barr Virus (EBV) reactivation following unrelated 86; p0.22), donor type ( IS 51vs55, VUD 39 vs46; p0.46), donor (UDR) hematopoietic stem cell transplantation (HSCT) aGVHD incidence (27 vs 32 pts; p0.6) but not for stem cell is a common event. Use of monoclonal antibodies anti-CD 20 source (BM 45 vs 30, PB 41 vs 106; p0.016), according to the (Rituximab®) dramatically improved the management of EBV transplant era. Interestingly CMV reactivation was similar in reactivation. the two groups (29 pts vs 38 pts; p0.68). In univariate and Objectives: To evaluate the incidence of lymphoproliferative multivariate analysis CMV reactivation was significantly disease, EBV reactivation and effectiveness of preemptive correlated with the donor type (p0.001), stem cell source and curative therapy by Rituximab® in pediatric URD HSCT. (p0.037), CMV serum status (p0.002), aGVHD (p0.000) but Methods: We retrospectively analysed the cohort of children not with the type of prophylaxis (p0.68). who received an HSCT with an URD in our institution between Our results suggest that the introduction of ganciclovir plus January 2001 and September 2006. The EBV DNA load was foscarnet in CMV prophylaxis did not result in a significant measured weekly in whole blood by a quantitative real time improvement in CMV reactivation rate. PCR until the third month following HSCT. Viral genome detection was considered positive when DNA quantification was above 10 copies/mL. EBV disease was defined by P719 positive detection of viral DNA and clinical manifestations Incidence of herpesvirus activation in young HSCT including fever associated with cutaneous rash or a patients lymphoproliferative syndrome. All patients received A. Chukhlovin, M. Ovsyannikova, E. Semenova, O. antithymocytes globulines (ATG) in the conditioning regimen Pankratova , L. Zubarovskaya, B. Afanasyev and GVHD prophylaxis by ciclopsorine and no T-cell St. Petersburg State Pavlov Medical University (St. depletion. Petersburg, RUS) Results: Seventy-seven patients were eligible for the study among 134 children who received HSCT. Four patients were Objectives: Herpesviruses are known to reside in most healthy excluded because of early death (within 30 days after HSCT). humans, and undergo reactivation in immunocompromised The hematopoietic stem cell source was bone marrow for 53 hosts, e.g., following alloHSCT. Hence, the aim of our study patients, peripheral stem cells for 6 and cord blood for 14. was to assess efficiency of PCR monitoring for assessment of Thirty-three patients with seronegative donor and recipient viremia in various variants of HSCT. were not monitored, none had EBV disease. Among the 40 Patients and methods: The study included 100 young patients monitored patients, 27 had a quantifiable viral genome: 6 were (mean age 19 years) subjected to allo-HSCT, mostly for ALL not treated, with a median viral load of 4880 copies/ml (range (n=45), AML (n=21), and CML (n=12). Bone marrow and from 200 to 30000 copies/ml). None developed EBV disease. PBSC were transfused, resp., in 31% and 69% of cases. Fourteen patients were treated preemptively with a median Transplantation from matched unrelated donors was made in viral load of 11295 copies/mL (range from 1950 to 50000) and 63%. Reduced conditioning regimens were applied for 54% of among them none developed an EBV disease. Seven patients the patients. Virus-specific IgG and IgM antibodies were presented a rapid EBV disease without previous increasing detected by ELISA technique. Monitoring of HSV type 1 and 2, viral load ; they all received Rituximab® and one patient died CMV, and EBV DNA by means of PCR in blood and marrow from EBV and VRS pneumopathy. None of the patients keukocytes was carried out weekly before HSCT and for at

S169 treated, curatively or preemptively had severe adverse event P722 reliable to Rituximab®. Safety of cidofovir in high-risk paediatric patients Conclusion: This study suggests that: V. Bhadri (1), L. Lee-Horn (2), P.J. Shaw (1) - Rituximab® preemptive treatment is efficient and safe with a (1)The Children's Hospital (Sydney, AUS); (2)University of low average threshold value of more than 10000 copies/mL. Sydney (Sydney, AUS) The number of Rituximab® injections should be adapted to evolution of viral load. Purpose: Cidofovir (CDV) has activity in adenovirus (ADV) - Rituximab® curative treatment is efficient for patients with and cytomegalovirus (CMV) infections. It is licensed in EBV disease, without serious adverse effects. Australia for the treatment of CMV retinitis in AIDS patients over the age of 12 years, but is nephrotoxic and contra- indicated with the use of other concomitant nephrotoxic P721 agents. Licensed drug information recommends an aggressive CMV disease and reactivation in 91 paediatric unrelated hydration regimen. Despite this it is a valuable anti-viral agent, haematopoietic stem cell Transplantation: a single-centre particularly in the setting of blood and marrow transplantation experience (BMT). This study assessed the safety of CDV in a high-risk J.M. André (1), V. Mialou (1), M.P. Goutagny (1), C. paediatric population. Galambrun (1), N. Lévêque (2), F. Najioullah (2), N. Bleyzac Methods: 234 CDV infusions in 23 patients were studied. The (1), Y. Bertrand (1) CDV dose, renal function, other drugs given and adverse (1)Hopital Debrousse (Lyon, F); (2)Faculté Rockefeller (Lyon, events were obtained from the notes. F) Patients: were 5months - 19years (mean 6.6 years); male 17, female 6; 16 were post allogeneic BMT, 4 post autoBMT, 3 CMV disease is a major complication during HSCT procedure. other malignancies. The indication for CDV was adenovirus Monitoring of viral reactivation and preemptive therapy are isolate in 20; adenovirus prophylaxis as part of haplo-identical now generalised practices. However, the threshold value of BMT in 2, and in 1 for sepsis without an identified organism. CMV DNA quantification for starting preemptive treatment is CDV dose was 1mg/kg (117, 50%), 3mg/kg (70, 30%) and still a debated question. 5mg/kg (47, 20%). CDV was given with a regimen of oral Objectives: To analyse the incidence of CMV asymptomatic probenecid 40mg/kg at hour -3, 20ml/kg of intravenous 0.9% reactivation, CMV disease, and the results of curative and NaCl from hour -1 to 0, CDV infusion with 20ml/kg 0.9% NaCl preemptive therapy in paediatric unrelated HSCT. from hour 0 to +1, maintenance intravenous fluids from hour Methods: We retrospectively analysed the data from all +1 to +3, followed by oral probenecid 20mg/kg at hour +3 and patients receiving an HSCT from an unrelated donor between +9. All patients were receiving concomitant nephrotoxins: 17% January 2000 and September 2006. Viral DNA quantification receiving 1; 17% receiving 2; 22% receiving 3 and was monitored weekly until the third month following HSCT by 43%receiving 4 or more agents. quantitative PCR assay. Viral genome detection even if not Results:There was no significant change in serum creatinine quantifiable was considered positive. Cut-off for quantification for any doses or on overall analysis (paired T-Test, SPSS). was 500 copies/mL. CMV disease was defined by positive The only change in urea was a decrease, most likely reflecting detection of viral DNA and clinical manifestations including improved hydration with CDV. In the 19 patients with normal fever associated with cutaneous rash, pneumopathy, intestinal renal function pre-CDV, there was no evidence of or ocular symptoms. All patients received anti-thymocyte nephrotoxicity with CDV. In 4 (17%) with pre-existing renal globuline as part of conditioning regimen and GVHD impairment, there was no deterioration in renal function. prophylaxis by ciclopsorine . Symptomatically CDV was tolerated by 20 (87%) patients Results: One hundred and sixty two children were without adverse events. The only toxicity attributable to CDV transplanted during the study period, 91 patients were eligible therapy was 2 patients with pre-existing pneumonitis post- for the study. Four patients, without CMV disease, were not allogeneic BMT, where the fluid regimen precipitated included because of early death. respiratory deterioration secondary to fluid overload. The hematopoietic stem cell source was bone marrow for 66 Conclusions: CDV is safe and well-tolerated in this high-risk patients, peripheral stem cells for 7 and cord blood for 14. paediatric population, even with concomitant nephrotoxic Forty seven patients with negative donor and recipient agents. Given this, the amount of hydration fluids should be serostatus were not monitored and didn’t have a CMV reviewed, particularly in patients with pulmonary co-morbidity. disease. Forty patients were either seropositive for the CMV and/or had a positive donor. Seventeen of them always had a negative P723 viral load. Two patients had severe combined Low citrulline concentration after high-dose melphalan: a immunodeficiency revealed by a CMV disease previously to prerequisite of bacteraemia? HCST. Sixteen patients were initially CMV positive below 500 A.H.E. Herbers, J.P. Donnelly, N.Blijlevens copies/mL, among them, 5 were not treated, and 11 were University Medical Centre St. Radboud (Nijmegen, NL) subsequently treated because of an increasing viral load (median 2300 copy/ml). One patient developed a CMV Introduction: Myelo-ablative regimen-induced mucosal disease. Five patients were initially CMV positive > 500 damage of the intestines can be measured by citrulline. copy/ml (median 3600) and were all treated, 2/5 developed a Mucosal damage is a known risk factor of Gram-positive CMV disease despite preemptive therapy and one died from bacteraemia in transplant patients who develop fever during CMV. neutropenia. We were interested to see when the nadir of Conclusion: All but one patients preemptively treated citrulline was reached after high-dose melphalan (HDM) and recovered, with a low rate of CMV disease. There was no its relationship to the onset of neutropenic fever and severe adverse event reliable to antiviral therapy. All the bacteraemia. patients who reached a viral load of 500 copies/ml Methods: We evaluated 29 patients who underwent subsequently need treatment, so the threshold value of 500 consecutively an autologous haematopoeitic stem cell copies/mL for starting treatment is effective and safe. transplantation (HSCT) after HDM 100 mg/m² (day –3 and – 2). Plasma citrulline was collected before starting conditioning therapy and 3 times weekly thereafter until discharge. Blood cultures were taken at the onset of fever both peripherally and via each lumen of the catheter before starting empirical antimicrobial therapy. Results: The baseline mean citrulline concentration was 28 µmol/L, which is lower than the 35 µmol/L that is found for healthy individuals. The nadir of citrulline (mean 6.7 µmol/L) was rapidly reached on HSCT day +8. 20 patients developed

S170 fever during neutropenia, but bacteraemia was detected in to expand cytotoxic populations, or possibly caused immune only 10 cases (50%) The organisms cultured included neutropenia. Ferum-DFO complex may be used by fungi as Streptococcus mitis (in 5 patients) and coagulase-negative growth factor. Considering that Rituximab as well as DFO are Staphylococci (in 8 patients); in 5 patients the infections were crucial agents to manage serious complications after alloTx, polymicrobial. Patients with bacteraemia had citrulline values further studies are needed to define their possible involvement below 10 µmol/L (range 2-9). The mean (5.5 µmol/L) was in fungal infections. significantly lower (p= 0.02, 95% CI 0.8-8.4) than for patients without bacteraemia (10.2 µmol/L). No differences in the duration and extent of neutropenia, leukopenia or CRP levels P725 were found. Citrulline concentrations only increased gradually Toxoplasmosis after allogeneic stem cell transplantation: and were still significantly low at a mean of 12 µmol/L when a report of eleven cases patients were discharged (figure 1). S. Buchholz, H. Kamal, E. Dammann, T. Winkler, C. Conclusion: The occurrence of bacteremia coincides with very Koenecke, M. Stadler, H. Diedrich, J. Krauter, B. Hertenstein, low serum citrulline levels rather than the onset of A. Ganser, M. Eder neutropenia, supporting the consideration that mucosal barrier Hannover Medical School (Hannover, D) injury is a major risk factor for infectious complications of cytotoxic therapy. Introduction: Toxoplasma disease (TD) after SCT is a rare complication with high mortality even with specific therapy. TD usually results from reactivation of latent infection due to immunosuppression. We report on 11 cases with local or disseminated TD and retrospectively analyse patient characteristics, potential risk-factors, therapy, and outcome in our institution. Patients and methods: The incidence of proven, probable or possible TD (Martino et al. 2000) from 01/95 to 12/05 is 2.4/100 allogeneic SCT. TD-patients with AML (n=4), CML (n=5), myeloma (n=1), and PNH (n=1) were transplanted with BM (n=2) or PBSC (n=9) from MUD (n=4), MMUD (n=3) and MRD (n=4). Conditioning was TBI/Cy (n=5), Bu/Cy (n=1), FLAMSA/TBI/Cy (n=4), and Flu/Mel (n=1) with CSA/MTX (n=5), CSA/MMF (n=4), CSA/Pred (n=1), and T-cell depletion (n=1) as GvHD prophylaxis. 4 patients suffered from definitive TD with 3 diagnosed at autopsy (1 myocardial, 2 P724 disseminated). Two patients had probable TD based on Use of Rituximab®, Desferioxamine® and high levels of clinical evidence and positive PCR from peripheral blood serum ferritin may induce the development of invasive samples. In 3 cases clinical findings with cranial CT/MRI (n=2) fungal infections, in patients undergoing allogeneic bone suggested possible TD. TD manifestation was disseminated marrow transplantation (4), cerebral (2), myocardial (1), retinal (1) and BM (1). Two I. Sakellari, P. Kaloyannidis, D. Adamidou, D. Bartzoudis, I. patients were treated based on serologic findings only. 9/11 Batsis, E. Yannaki, C. Smias, D. Mallouri, V. Tzarou, A. TD patients were seropositive for T. gondii before SCT, for Kontodimou, E. Proya, P. Palladas, A. Anagnostopoulos, A. two patients pretransplant serology was not available. Median Fassas time to TD onset was 86 days (30–685) with 9 patients under G. Papanicolaou Hospital (Thessaloniki, GR) immunosuppression, two due to aGvHD and cGvHD. One patient was neutropenic and one had no immunosuppressive A rising incidence of invasive fungal infections (IFI) has been treatment. At diagnosis of TD none patient received reported in patients undergoing allogeneic bone marrow cotrimoxazole but 8 pentacarinat for Pneumocystis transplantation (alloTx) despite the use of primary antifungal prophylaxis. TD patients diagnosed at lifetime received prophylaxis. Introduction of novel strategies in alloTx field treatment with pyrimethamine (n=5), atovaquone (n=2) or demands the research of new risk factors predisposing to IFI. cotrimoxazole (n=1) in combination with sulfonamides and/or We retrospectively analyzed the incidence and risk factors for clindamycin. Five patients responded and are alive with a IFI in a group of 135 allografted patients during 2002-2006, median follow-up of 1.7 years (0.8-10.7). One patient aged 36(7-63) years. No patient received chemotherapy for responded with PCR negativity but died due to leukemic relapse post alloTx. Thirty patients received a reduced relapse, a second died because of TD and leukemic relapse, intensity regimen. Sibling donors were 107, unrelated and the third due to a combination of cerebral toxoplasmosis volunteers (VUD) 25, haploidenticals 3. Antithymocyte globulin and pneumocystis carinii pneumonia. (ATG) was administrated in 41 patients, in 38 as part of Conclusion: TD is a rare complication after allogeneic SCT preparative regimen and in 3 as therapy for refractory graft- with multiple clinical manifestations and significant mortality versus-host disease (GvHD). Twenty seven patients received even under specific therapy. TD diagnosis may be difficult and antiCD20 (Rituximab) as management for EBV overload requires imaging and PCR methods. Prophylaxis with (>1000 genomes/ml in 2 consecutive samples) and 2 for cotrimoxazole as early as possible in seropositive SCT refractory thrombotic thrombopenic purpura. In 99 patients patients should be considered. high levels of serum ferritin were detected and 35/99 received Desferioxamine (DFO) for coexisting haemochromatosis. Previously resolved IFI was reported in 15/135 patients. In a P726 median time of 5 months (0.5-28), 34/135 patients developed T-cell neogenesis after allogeneic stem cell IFI (probability 40% at 4.5 years). According to EORTC 6 transplantation cases were probable, and 28 possible. In univariate analysis J. Winkler (1), B. Spriewald (1), N. Schub (2), W. Rösler (1), IFI pre-Tx, refractory chronic GvHD, ferritin level >2000ng/ml, M. Gramatzki (2) alternative vs sibling donor and administration of ATG, DFO or (1)University Erlangen-Nuremberg (Erlangen, D); (2)University rituximab were proven significant risk factors (p<0.05). Kiel (Kiel, D) Appearance of acute GvHD showed tendency to significance (p:0.07). Multivariate analysis revealed as risk factors IFI pre- Impaired T cell reconstitution after allogeneic stem cell Tx (p:0.0004), ferritin>2000ng/ml (p:0.01), use of DFO transplantation (SCT) is a major cause of morbidity and (p:0.05), Rituximab administration (p:0.01), while advanced mortality. Previous studies of T-cell reconstitution after SCT disease (p:0.06), and acute GvHD (p:0.08) retained tendency determined newly generated T lymphocytes by T-cell receptor to significance. Rituximab probably induces development of excision circles (TREC) in addition to the proliferation of donor IFI through either suppression of B-cells, which are necessary “memory” T lymphocytes. The occurrence of TRECs seems to

S171 be associated with the restoration of the T-cell receptor repertoire and may lead to a lower incidence of infections after SCT. The aim of this study was to analyze the influence of T cell depletion by anti-thymocyte globulin on T cell neogenesis. Anti-thymocyte globulin (ATG) is established as part of the conditioning regimen in matched unrelated donor situation and in order to reduce graft versus host disease (GVHD). Patients who had received ATG (ATG-Fresenius mostly) showed significantly less CD3+ T cells in the first 6 months than patients without ATG (mean 4.3 ± 0.7 and mean 17.1 ± 3.1, p= 0.0005). T cell neogenesis in these patients was analyzed by determining CD4+/CD45RA+ positive T lymphocytes by flow cytometry and TREC-positive T cells by real time PCR. The percentage of CD45RA+ T-cells among CD4+ T-cells in patients who did not receive ATG did not change significantly 1 month after transplantation and thereafter. Elevated levels of TRECS were not found in these patients. In patients receiving ATG and myeloablative transplantation two groups of patients could be defined by their percentage of CD4+/CD45RA+ T- P728 cells and TRECs early after transplantation. A subpopulation Quantitive analysis of Epstein-Barr virus DNA load in of patients after ATG had elevated frequencies of bone marrow transplant recipients using real-time PCR CD4+/CD45RA+ T-cells and TRECs. When reduced intensity S.H. Ghaffari (1), S. Sabri (2), K. Alimoghaddam (1), M. conditioning (RIC) was performed with ATG application, SadeghiZadeh (2), B. Chahardouli (1), A. Ghavamzade (1) TREC-positive CD3 T cells were found elevated more (1)Tehran University Medical Sciences (Tehran, IR); frequently. (2)Tarbiat Modares University (Tehran, IR) In summary, T cell neogenesis can be monitored after SCT by measuring CD45RA+ T cells and/or TREC in T cells. Introduction: A major risk faced by bone-marrow and solid Indications for T cell neogenesis were only found in a organ transplant patients is the development of post- subfraction of transplanted patients with ATG treatment. transplant lymphoproliferative disease or post-transplant lymphoma (PTLD). Method: To measure the Epstein-Barr virus (EBV) virus load P727 in patients with stem cell transplantation (SCT), we developed Breakthrough invasive aspergillosis in allogeneic a highly sensitive and quantitative PCR assay using haematopoietic stem cell transplant recipients treated LightCycler. A plasmid containing a target sequence from the with caspofungin BALF5 gene of EBV was constructed as a positive control A. Madureira (1), A. Bergeron (1), C. Lacroix (1), M. Robin (1), template. After purification of the EBV clone and calculation of V. Rocha (1), R. Peffault de Latour (1), C. Ferry (1), A. copy number, the standard curve was constructed by using Devergie (1), J. Lapalu (1), E. Gluckman (1), G. Socié (1), M. serial dilution of the plasmid clone. Sequential blood samples Ghannoum (2), P. Ribaud (1) (275 samples) were collected from 35 patients as monthly (1)Hopital Saint Louis (Paris, F); (2)University Hospitals of intervals up to at least 6 months post-BMT. A TaqMan-based Cleveland (Cleveland, USA) real-time PCR assay was developed and standardized to quantify EBV DNA copy number in peripheral blood Background: Caspofunin (C) has been used at our centre for mononuclear cells (PBMNC) of SCT patients. empirical treatment of fever since January 2004. Results: The real-time PCR assay could detect from 2 to over Methods. We reviewed the clinical records of 103 SCT 107 copies of EBV DNA with a wide linear range. Viral DNA patients (pts) who received C since then, to the exclusion of could be detected in majority of PBMNC samples collected pts treated for a probable or definite IA.. Definitions of invasive from 35 patients during the follow up. The threshold for normal aspergillosis (IA) were made according to consensus criteria EBV viral load in our experiment was defined as 1x102 EBV (Ascioglu, 2002). genomes/ 2x105 PBMNC. In this study, no patient developed Results: We recorded 6 breakthrough (BT) IA (IA occurring > PTLD during the follow up post transplant; however, EBV copy 6 days (d) after C initiation), among which 1 proven and 5 number in 22 samples (3 patients) was higher than the probable infections. Aspergillus sp. was isolated in 4 pts: A. threshold value with symptoms like fever and pulmonary fumigatus (n = 2), A. ustus (n = 1), and A. nidulans (n = 1). nodes. The mean EBV DNA copy number in these patients The 100-day cumulative incidence post C initiation, with death was 104 copies per 2×105 PBMNC. as a competing factor, was 6% (95%CI: 1-11). Patient and IA Conclusion: These results suggest that the real-time PCR main characteristics, and susceptibility testing of the isolates assay is useful for quantification of EBV viral load and for are shown in table. Median age was 28 years. Median time to monitoring the virus reactivation in BMT patients. IA diagnosis was 93 days post transplant (range: 47 to 188). Median duration of C treatment before IA diagnosis was 32 days (range: 7 to 197). Susceptibility testing of breakthrough P729 Aspergillus isolates showed that 2 out of 4 isolates tested Development and evaluation of a real-time quantitative have high MEC to caspofungin. Voriconazole induced 2/6 CR. PCR for the detection of human cytomegalovirus DNA in However, all pts ultimately died a median of 5 weeks after IA bone marrow transplant recipients diagnosis. S.H. Ghaffari, N. Obeidi, K. Alimoghaddam, M. Dehghan, A. Conclusions: Whether this high incidence of BT IA in SCT pts Ghavamzadeh under C therapy represents a clinically meaningful limitation of Tehran University Medical Sciences (Tehran, IR) C in this population of pts, or is only a reflection of their poor immune status is unknown. Introduction: Cytomegalovirus (CMV) has been recognized as the most important viral pathogen in persons undergoing bone marrow transplantation (BMT). In this study, we present the development of a TaqMan-based real-time PCR assay to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Materials and Methods: A plasmid containing the target sequence from the pp65 region (UL83) of CMV was constructed as a positive control template. Serial dilutions of

S172 107 to 101 plasmids per assay were prepared. Sequential Conclusions: MBL levels are elevated for the first 6 weeks blood samples (415 specimens) were collected from 51 following aBMT in MBL wild type patients. TRM seems to be patients as weekly intervals until day 100 after transplantation. higher in profoundly MBL deficient patients. MBL deficiency is CMV DNA was quantified by RQ-PCR in parallel with the pp65 associated with infection, engraftment and GvHD, which may antigenemia assay in PBL samples. be related to the higher mortality. Results: The real-time PCR assay could detect from 10 to over 107 copies of CMV DNA with a wide linear range. Real- time PCR assay results correlated with those of the CMV P731 pp65 antigenemia assay (P < 0.01). CMV DNA was detected Impact of long-term low versus high-dose acyclovir by RQ-PCR in 13 patients a mean of 15 days prior to the prophylaxis on cytomegalovirus infection and survival appearance of antigenemia. About 76% of patients developed after allogeneic stem cell transplant: a single-centre, more than one episode of CMV replication. If copy number of retrospective study CMV was increased, preemptive therapy was initiated. The A.P. Iori, E. Iannella, G.F. Torelli, F. Milano, A. Capobianchi, administration of anti-CMV drugs led to a rapid decrease in B. Lucarelli, L. Malandruccolo, W. Barberi, E. Arleo, P. the numbers of viral copies and positive nuclei. After Martino, R. Foà, G. Gentile preemptive therapy, 16 days (7-21 days) needed the result of University Sapienza (Rome, I) RQ-PCR to become negative. Conclusion: The TaqMan real-time assay may be a useful High dose Acyclovir (ACV) has been shown to reduce the risk tool for rapid quantification of CMV infection and for of CMV infection and improved overall survival in stem cell monitoring of CMV reactivation in bone marrow transplant (SCT), in a european, multicentric, randomized transplantation recipients. RQ-PCR was more sensitive than study (Prentice,Lancet 1994). We retrospectively compared pp65 Antigenemia. After preemptive therapy, negative results two groups of patients who underwent an allogeneic SCT, of RQ-PCR were the best indicator for determining of both receiving i.v. ACV (15 mg/Kg/day) from day -7 to day +20 successful treatment. If copy number of CMV increased one after transplant followed by low-dose oral (200 mg x 4/day) log, CMV reactivation developed 1.22 fold. (group A) or high-dose oral (800 mg x 4/day) (group B) AVC for a period of 6 months. Aim of the study was to evaluate whether there was any difference in the rate of CMV infection, P730 CMV disease, adverse events, probability of survival and Role of mannose-binding lectin polymorphisms in transplant related mortality (TRM) between the two groups. complications after allogeneic bone marrow We analyzed 108 patients (60 in group A, 48 in group B) at transplantation risk of CMV infection (recipient and/or donor seropositive for O. Neth (1), O. Waschke (2), P. Das (2), T. Zabelina (2), F. CMV), managed between January 1999 and July 2006.CMV Ayuk (2), B. Fehse (2), N. Kröger (2), M. Lioznov (2), N. Klein infection was monitored by pp65 antigenemia assay. Pre- (1), A. Zander (2) emptive therapy with ganciclovir or foscarnet was used on the (1)Institute of Child Health and Great Ormond Street Hospital basis of pp65 antigenemia positivity. The two groups were (London, UK); (2)University Medical Centre Eppendorf comparable for age, sex, hematological disease, disease (Hamburg, D) phase at transplant, CMV serology, combination between donor and recipient at transplant, HLA compatibility, Introduction: Life-threatening complications such as graft- conditioning regimens and supportive therapy. No differences versus-host disease and infection remain major barriers to the were observed between the two groups considering the rate of success of allogenic bone marrow transplantation (aBMT). engraftment and acute and chronic GVHD. Cumulative A growing body of evidence indicates that genetic factors are incidence of CMV infection was 70% in group A and 60% in involved in an increased risk of infection.Genetic factors within group B; this difference was not statistically significant. CMV the innate immune system are increasingly being identified disease was observed in 2 patients in group A and in 1 patient and their role in a clinical setting is under investigation. in group B. Herpes simplex virus and varicella zoster virus Mannose-binding lectin (MBL) has emerged as an important infections were not observed in both groups during the first 6 innate host defense molecule.MBL binds a range of months after transplant. Observed adverse events that were pathogens independently of antibody and activates possibly related to the treatment were nausea and vomiting complement lysis and phagocytosis.It is a C-type serum lectin (55% group in A vs 66% in group B) and creatinine increase produced by the liver, and it responds as an acute-phase (>1.5mg/dl) (45% group in group A vs 56% in group B).The 1- reactant.MBL is a central player in the innate immune year cumulative incidence of TRM was 17% and 21% for response. groups A and B respectively; the difference was not Objectives: We investigated whether NBL gene statistically significant. The 1-year probability of survival by the polymorphisms causing low levels of MBL are associated with Kaplan-Meier method was 78% and 75% for group A and B the development of infection and GvHD in patients undergoing respectively. The 5-year probability of survival was similar aBMT. between the two groups (63% for group A vs 53% for group B; Methods: From 01/2001 to 10/2003 a total of 131 patients p=0.5). (age range 14 to 69 years, mean 43.5) were enrolled in a In conclusion we didn’t observe any difference in the combined retro-and prospective study. Frequency, causes of incidence of CMV infection, CMV disease, survival or TRM infections and episodes of GVHD were recorded, and MBL between the two groups. Larger, randomized studies are genotype of recipient and donor were determined by required to confirm these results. heteroduplex analysis and MBL phenotype by ELISAs. Serial MBL concentrations were also measured pre BMT, day 0-45 and day 46-7 post aBMT, and the results correlated with the P732 MBL genotype (A/A indicating wild type, A/O indicating Influence of pantoprazole on voriconazole plasma heterozygous for MBL structural- gene mutations and O/O concentrations indicating homozygous, for such mutations). W. J. Heinz, C. Kloeser, A. Helle, S. Scheuermann, A. Kuhn, Results: MBL concentrations increased between days 0 and C. Guhl, H. Einsele, H. Klinker 45 in all patients (A/A p<0,001) from pre-transplant levels. Medizinische Klinik and Poliklinik II (Wuerzburg, D) Between days 46-75 levels fell significantly (A/A p<0.0001, A/O p=0.005 and O/O p=0.005). There were no significant Objectives: Invasive fungal infections (IFI) are a life differences observed in frequency and causes of infections. threatening complication in patients with haematological However of the 8 patients with very low MBL levels malignancies, especially after allogenic stem cell (<400ng/ml) 5 died of transplant related mortality (TRM) (2 transplantation. As these patients often receive quite a patients from sepsis, 1 from infection and GVHD, 1 from number of different medications drug interactions are a toxicity and 1 from a new malignancy). This is twice the TRM relevant problem. Voriconazole is broadly used in treatment of seen in the group as a whole (62% vs 30%). IFI. Metabolized by cytochrome P450 (CYP) it shows in

S173 addition inhibiting and inducing activity for several isoenzymes complaints spontaneously. No changes were noticed on (2C9, 2C19 and 3A4). For the proton pump-inhibitor (PPI) electrocardiography. One patient (SC12001) developed omeprazole inhibition of several isoenzymes (e.g. 2C9, 2C10, elevated transaminase levels four days following HSCT, with 2D6, 3A4) could be demonstrated. This may cause reduced AST elevated 5 times (198 U/l) and ALT 7 times (336 U/l) the metabolization of voriconazole. On the other hand the direct ULN, meaning a grade 2 respectively 3 adverse event effect of increased gastric pH may reduce absorption of the according to the Common Terminology Criteria for Adverse azole. Indeed, for the comedication of omeprazole an Events (version 3.0). Since concomitant drug use including increased area under the curve of voriconazole has been fluconazole could also have caused these abnormalities, this demonstrated. Other PPIs have different affinity to CYP side effect was rated possibly related to the administration of enzymes; for pantoprazole e.g. a higher inhibitory activity on hLF1-11. Spontaneous and full recovery occurred not CYP 2C9 and 3A4 has been shown. Therefore we necessitating any interventions. Otherwise no adverse events investigated the plasma through concentrations (PC) of occurred, other than those expected for patients undergoing voriconazole depending on the comedication of pantoprazole. HSCT. Methods: A high performance liquid chromatography (HPLC) Conclusion: Administration of a single dose of 5 mg hLF1-11 was established to determine PC of voriconazole. Plasma was well tolerated and proved safe in patients receiving samples from haematological patients with oral voriconazole autologous HSCT following HDM. treatment have been collected and voriconazole concentrations determined. The results were analyzed in correlation to the comedication of pantoprazole. P734 Results: We analyzed 130 samples from 42 patients, 23 Usefulness of the systematic measurement of serum samples without comedication (group 1), and 107 from concentration of galactomannan antigen in patients with patients also receiving pantoprazole (group 2). PC showed a acute leukaemia or stem cell transplantation broad range; in group 1 37 have been below 200 ng/ ml and M. Batlle, J. Dominguez, N. Lloveras, A. Oriol, C. Ferrà, J.M. no above 10.000 ng/ml, in group 2 26 (25%) have been below Sancho, B. Xicoy, A. Flores, M. Jiménez, S. Piernas, S. Vives, 200 ng/ ml and 4 above 10.000 ng/ml 4. Average PC have A. Serrano, F. Vall-llovera, J.M. Ribera, E. Feliu been 1212 ng/ml (median 463, interquartiles Range (IQR) Hospital Germans Trias (Badalona, E) 62,5 – 1717,5 for group 1) and 1840 ng/ml (median 803, IQR 206,5 - 2199 for group2). In Mann-Whitney-U-Test the Objetive: To evaluate prospectively the usefulness of periodic difference was not significant. measurement of the serum concentrations of galactomannan Conclusion: In this retrospective analysis we could show that antigen in patients with acute leukemia (AL) or stem cell comedication of the PPI pantoprazole did not reduce the PC transplantation (SCT) to predict invasive fungal infections. of voriconazole. Similar to the data shown for omeprazole Patients and methods. All consecutive patients from one there is a tendency to higher plasma levels. This was not center on intensive treatment for AL or submitted to SCT significant, probably due to the broad range of plasma levels during two years (2004-2005) were evaluated prospectively. detected. As only few PC have been above 10.000 ng/ml an Serum galactomannan antigen levels were measured by increased toxicity seems not to be likely and no dose ELISA twice weekly from admission to discharge. Values over adjustment should be recommended so far. 0.5 ng/mL were considered positive for fungal infection suspicion. Results: A hundred and eighty-nine patients were recruited P733 (33 AML, 12 ALL, 40 allo-SCT and 54 auto-SCT). Out of 1,443 Safety and tolerability of human lactoferrin 1-11 (hLF1-11) determinations, 59 (4%) were positive, corresponding to 28 in patients receiving an autologous HSCT after episodes (26 patients,16 males, median age 47 years, range conditioning with high-dose melphalan (a phase I trial) 15-70). Diagnoses of these patients were AML 17 (60%, 8 W.J.F.M. van der Velden, N.Blijlevens, J.P. Donnelly, M. during induction treatment, 5 during consolidation and 4 Wulferink, B.M. Giannetti, M.P. Velders during allo SCT), ALL 3 (9%) (1 during induction and 1 allo- Radboud University Nijmegen Medical Center (Nijmegen, NL) SCT), 12 (43%) during allo-SCT and 1 during auto-SCT. In 13/28 episodes (46%) the subsequent galactomannan Background: Human lactoferrin is a natural defence protein measurement was negative and, consequently, were present in several body fluids and the secondary granulae of considered not evaluable and no further action was taken. In 9 polymorphonuclear neutrophils. Lactoferrin has pleiotropic out of these 13 episodes borderline values (0.5 to 1 ng/mL) functions including broad anti-microbial, anti-inflammatory and were found. In 15/28 (54%) the positivity persisted in the immune-modulating activity, properties of interest in the subsequent determination and were considered as having treatment of infectious and inflammatory complications in probable Aspergillus infection. In 4 of the probable infections haematopoietic stem cell transplantation (HSCT) recipients, in no focality could be identified and empirical treatment was which levels of lactoferrin are decreased. The peptide administered (voriconazol 2, liposomal amphotericin 1 and comprising the N-terminal 11 amino acids of the human caspofungin 1) up to negativization of the galactomannan lactoferrin protein, representing the first cationic domain level. Eleven patients presented focal or systemic infection (8 (hLF1-11), has been shown more effective in vitro and in vivo pulmonary, 3 invasive fungal infection - IFI). Treatments than the full length protein and other peptide fragments included voriconazol (4), liposomal amphotericin (2), thereof. Results from animal models representing systemic caspofungin (1) or associations (4) and the outcome was fungal and local bacterial infection have shown hLF1-11 to be fovorable in 6/11 (55%). Five patients died (3 of them with a a promising agent in HSCT management, in which confirmed IFI). Globally, a favorable evolution was observed in transplantation-related morbidity and mortality due to 10/15 (67%) episodes. infections remains high. Conclusion: Sequential measurements of galactomannan Aim: To determine the safety and tolerability of a single antigen allowed the detection and preemptive treatment of intravenous dose of 5 mg hLF1-11 given to autologous HSCT probable Aspergillus infections. This approach was mainly recipients following conditioning with high dose melphalan useful in patients with AML and in those submitted to allo- (HDM). SCT. Methods: Patients with multiple myeloma or Supported in part by the grant P/EF-05 of the José Carreras lymphoplasmacytoid lymphoma received one dosage of 5 mg Foundation. hLF1-11 administered intravenously at 1 ml/min in 20 minutes on the day of transplantation. Follow up was 28 days during which side effects, laboratory results and electrocardiograms were monitored. Results: As scheduled eight patients (N = 8) were included and received the full dose of study drug on day 0 of HSCT. No infusion reactions occurred and the study patients reported no

S174 P735 with/without ATG (40(64,5%) vs 15(57,7%)). Active CMV- Role of BK virus infections in haemorrhagic cystitis after infection considerably delayed the time of engraftment of allogeneic bone marrow transplantation white blood cells • 1,0x109 (D+21,9 vs 17,3 in pts without M. Ramzi, R. Yaghobi, E. Shakiba CMV, p=0,006), granulocytes • 0,5x109 (D+22,2 vs 17,7, Shiraz University of Medical Sciences (Shiraz, IR) p=0,01) and platelets •50x109 (D+27,5 vs 20,2, p=0,02). Conclusion: The rate of early CMV-infection after allo-HSCT is Background and objectives: Hemorrhagic cystitis (HC) is an higher in children ”15 y.o., in seropositive pts, especially in important cause of morbidity and mortality in Bone Marrow cases with D/R “-/+”, after allo-HSCT from MUD and Transplant (BMT) recipients. Its incidence has varied from 7- myeloablative conditioning. ATG didn’t influent on the rate of 70% in BMT patients. HC presentations are classified to early CMV-infection in our study. Active CMV-infection delays the and late onset and one of the causative agents of late onset time of engraftment. HC is viral infections. In this study we evaluated the role of BK polyoma virus (BKV) infections in late onset HC and the prevalence of this viral infection in BMT recipients were P737 determined. Usefulness of micafungin for prevention of invasive Material and methods: We studied 30 recipients and 24 fungal infection after minimized cord blood donors.Total number of 293 EDTA treated blood samples and transplantation 293 urine samples were collected from BMT recipients and S. Taniguchi, Y. Matsuhashi, S. Takagi, D. Kato, H. donors. One sample was collected before transplantation from Yamamoto, N. Matsuno, S. Seo, N. Uchida, K. Masuoka, A. BMT recipients and donors and also one sample per week for Yoneyama, A. Wake 100 days from BMT recipients. BKV- monoplex in house PCR Toranomon Hospital (Tokyo, JP) were used for determination of this virus prevalence in patients. Also data of this investigation were statistically Background: Therapeutic outcomes for hematological analyzed with version 12 of SPSS. diseases have recently been markedly improved due to the Results: Incidence of HC in BMT recipients with measurement introduction of hematopoietic stem cell transplantation of RBC in urine (grade of 0-4 HC) and with sonographic (HSCT), improvement of therapeutic regimens, and records of bladder bleeding were 53.3% and 36.7% advancement of supportive therapy. Although invasive fungal respectively. Significant correlations were found between BKV infection (IFI) is one of the major causes of morbidity and -PCR positive results and HC presentation. mortality in HSCT, the proper regimen of prophylaxis as well Conclusion: Due to significant correlations of HC clinical as treatment of IFI has not been clearly established. In this presentations with positive results of BKV-PCR test early study, we retrospectively surveyed the incidence of IFI after diagnosis and treatment of this viral infection have a critical minimized cord blood transplantation (CBT) performed at our role in prevention and management of HC disorder in BMT hospital to investigate the effectiveness of antifungal agents recipients. for prevention of IFI. Methods: A retrospective survey on the prevention of fungal infection was performed in 183 patients (AML 40, ML 32, MDS P736 25, ALL24, ATL 21, MDS+AML 12, CML 7, AA 4, MM 3, Cytomegalovirus infection after allogeneic haematopoetic others 15) who underwent minimized CBT (188 stem cell transplantation transplantations) at our hospital between March 2002 and M. Ovsjannikova, E. Semenova, N. Stancheva, N. December 2005. The presence or absence of IFI (fungal Zubarovskaya, T. Antonova, O. Pankratova, B. Afanasyev breakthrough) within 50 days after transplantation, mainly St. Petersburg State Pavlov Medical University (St. Aspergillus infection, was surveyed. Petersburg, RUS) Results: Invasive pulmonary aspergillosis (IPA) was observed after 3 transplantations in 3 patients. These patients had been Cytomegalovirus (CMV) is one of the main causes of infection continuously treated with fluconazole (FLCZ) before and after complications in patients after allogeneic haematopoetic stem transplantation, and IPA developed during the treatment (3/71, cell transplantation (allo-HSCT). The aim of the study was to incidence rate: 4%). These patients were treated with estimate the rate of early CMV-infection in transplant amphotericin B and/or micafungin (MCFG) after the onset, but recipients in depending on age, donor’s and receipient’s died of pulmonary aspergillosis. In contrast, no breakthrough serologic status (D/R), type of transplantation and preparative IFI occurred in a group (46 patients) in which FLCZ was regimen and it’s influence on engraftment. changed to MCFG immediately at the sign of infection such as Patients and methods: Data include 88 patients (pts) from 1,5 fever despite the administration of FLCZ before and after till 66 y.o. after allo-HSCT performed since Nov 2000 till May transplantation. 2005. Patients are devided into 4 age groups: ”10 y.o. (22), Conclusion: Although oral FLCZ is administered to prevent 11–15 y.o. (22), 16–21 y.o. (20) and >21 y.o. (24). The fungal infection before and after transplantation, this agent diagnoses: ALL (42), AML (19), CML (10), NHL (7), others has a weak antifungal activity against Aspergillus sp. A (10). 33 pts received allo-HSCT from matched related donors regimen to change FLCZ to MCFG immediately after a sign of (MRD) and 55 from matched unrelated donors (MUD). fungal infection appears is recommended not only for the Conditioning regimens were myeloablative in 43 and with prevention of fungal infection but also for the avoidance of reduced intencity (RIC) in 45 pts. Antithymocyte globuline adverse events caused by immunosuppressants because (ATG) was used in 62 pts. Serologic test for CMV before MCFG is less interactive with them. Moreover, a regimen transplant was “+” in 74 pts, “-“ in 14 pts. D/R was “+/+” in 44 switching FLCZ to MCFG promptly after transplantation cases, “-/+” - in 30, “+/-“ - in 8, “-/-“ - in 6. For monitoring of should be considered. CMV infection we used qualitative PCR in all pts and quantitative PCR in 21 pts. Results: Early CMV-infection developed in 55 (62,5%) pts, P738 more frequently in pts ”10 y.o. -17(77,3%) and 11–15 y.o. - Oral valganciclovir is an effective primary pre-emptive 17(77,3%) than in pts 16–21 y.o. –9(45,0%) and adults – therapy of cytomegalovirus disease in recipients of 12(50,0%), p=0,004. There was tendency to more frequent allogeneic stem cell transplant development of CMV-reactivation in seropositive pts vs A. Candoni, R. Mestroni, E. Simeone, M. Cerno, R. Fanin primary infection in seronegative ones (49(66,2%) vs University Hospital (Udine, I) 6(42,9%), p=0,089) with highest rate in D/R “-/+” cases (80%(24 pts), p=0,012). The rate of CMV-infection was higher Cytomegalovirus (CMV) infection is a common complication after allo-HSCT from MUD than MRD (38(69,1%) vs after allogeneic SCT. Valganciclovir hydrochloride (VALCYTE- 17(51,5%), p=0,07) and after myeloablative conditioning than VGC) is a pro-drug of ganciclovir, orally available, that has after RIC (33(76,7%) vs 22(48,9%), p=0,013). There was no been used in CMV infection in high-risk solid organ significant difference between rate of CMV-infeciton in groups transplants (donor positive, recipient negative).The primary

S175 aims of our study were the assessment of efficacy and safety P739 of VGC as pre-emptive therapy of CMV disease after Comparison of CMV antigenaemia and quantitative real- allogeneic BMT. In this abstract we are reporting the time PCR CMV-DNA assay in the detection of CMV preliminary results (first 10 patients)of this study. During a infection in patients after allogeneic haematopoietic stem five-month-period VGC was administered to 10 consecutive cell transplantation outpatients (pts) with a CMV infection which was diagnosed J. Gozdzik, A. Mordel, E. Lesko, A. Krasowska-Kwiecien, E. after a median time of 86 days (range 59-480) from transplant. Kacinska, A. Wedrychowicz, W. Czogala, O. Wiecha, S. There were 6 males and 4 females (myelofibrosis 2, leukemia Skoczen, M. Majka 4, myeloma 2, lymphoma 2). The median age was 55 years Jagiellonian University Medical College (Cracow, PL) (range 43-66); 7/10 pts underwent BMT from unrelated and 3/10 from related donors; 7/10 (70%) pts received a reduced Cytomegalovirus (CMV) disease remains an important cause intensity conditioning regimen (RIC); CMV prophylaxis of morbidity and mortality in patients undergoing HSCT. consisted in acyclovir in all cases. Pre-transplant CMV Developing reliable methods detecting CMV infection is serology showed that in 100 % of cases either recipient and/or important to identify patients at risk. The aim of this study was donor were positive (D+/R+=5/10, D-/R+=5/10. At the onset of to compare two methods: CMV pp65 antigen and quantitative CMV infection 9/10 (90%) pts have an acute or chronic graft real-time PCR (qRT-PCR) DNA-CMV assays. CMV antigen versus host disease for which were received therapy including was detected in peripheral blood leukocytes (PBL) by prednisone plus other immunosuppressive agents. The pp65 immunohistochemical staining using Clonab CMV Kit antigenemia assay were positive in all cases with a mean (Biotest). The test was considered positive when pp65 was number of positive nuclei of 21±35. The starting treatment detected in one or more leukocyte per 2.6x105 PBL. CMV- dosage of VGC was 900 mg twice a day and it was continued DNA loads in patients plasma was measured by TaqMan. until the CMV antigenemia and viremia (CMV-DNA in the PCR was considered positive when more than 1 copy of CMV pheripheral blood) became negative in two consecutive DNA per 1ml of plasma was detected. The evaluation was samples. performed in 3 consecutive years. Results: All 10 cases obtained a clearance of antigenemia In 2004 only antigenemia studies were performed. Assessing after a median of 8 days of VGC therapy (range 5-16 81 samples we obtained 27% positive, 56% negative and 6% days)[Figure 1]; viremia became negative in all cases. The doubtful results. In 11% of samples repetition of the study was median length of manteinance therapy with VGC (900 mg necessary because of technical reasons. daily) was 21 days (range 8-32). Only one patient developed a In 2005 160 samples were evaluated using antigenemia mild deterioration of renal function that required dose assay, 11 of which were also analysed by qRT-PCR. Using adjustment (VGC 450 mg daily); mild anemia was reported in both methods we obtained 14%/18% positive, 79%/82% 3/10 (30%) pts, neutropenia in 5/10 (50%) pts and negative and 2%/0% doubtful results, respectively. Among thrombocytopenia in 4/10 (40%)[Figure 2]. samples assessed by antigenemia assay 4% required Conclusions: repetitions. 1)Pre-emptive therapy with VGC after allogeneic SCT seems In 2006 83 samples were assessed with the use antigenemia to be safe and effective (with a rapid clearance of antigenemia assay and 131 samples by qRT-PCR method. The results and viremia. were as follows: 19%/9% positive, 52%/82% negative, 2)The simple once or bi-daily VGC oral regimen can improve 11%/0% doubtful results, respectively. A determination of the compliance of the pts. pp65 antigen had to be repeated in 18% cases, in 2% 3)Regular blood counts should be performed to early detect samples inhibition of PCR was observed. In 51 cases were cytopenia. evaluated with both methods. In this group 4% samples were 4)The optimal dose and duration of VGC therapy need to be doubtful with the antigenemia but positive with the qRT-PCR established. and 8% samples were positive in antigenaemia assay, while in the qRT-PCR they were negative. In 29% of cases immunohistochemical staining had to be repeated. Comparison of these two methods showed that positive results were found in 20% vs 15%, negative in 62% vs 82%, doubtful in 6% vs 0% and repetition necessity in 11% vs 1%, respectively. Reevaluation of the antigenemia study increased costs and delayed introduction of treatment. Moreover, positive and doubtful results in absence of infection caused exposure to risk of unnecessary treatment and increased costs. A time needed to obtain results was much shorter for qRT-PCR, which could be of great value in immunocompromised patients.

P740 Relapse of acute leukaemia or myelodysplastic syndrome after an allogeneic stem cell transplantation: increased risk for invasive mould infections A. Nihtinen, L. Volin, V.-J. Anttila, E. Juvonen, T. Ruutu Helsinki University Central Hospital (Helsinki, FIN)

Recipients of allogeneic stem cell transplantation (SCT) and patients with acute leukaemia are at high risk for invasive mould infections. Since little is known about the incidence of these infections in a setting of a relapse after an allogeneic SCT, a retrospective study was performed. Between January 1 2000 and December 31 2005, 220 adult patients (114 AML, 65 ALL, 39 MDS, 2 other) with a median age of 46 years (range 17 – 65) received an allogeneic SCT at the Helsinki University Central Hospital. At the time of transplantation, 119 patients were in first remission. The donor was an HLA- identical sibling in 123 cases and unrelated in 97. Of the grafts, 129 were PB and 91 BM. As conditioning, Cy+TBI was used for 181, Busulfan for 10 and Treosulfan for 22. Reduced

S176 intensity conditioning according to the Seattle protocol was bacilloscopy of the lesions was consistent with leprosy and given to 7 patients. ATG was given to 89 patients with an treatment with ofloxacin was initiated. The patient died of unrelated donor as a part of the conditioning. After the pneumonia on D+13 after AHSCT. Table 1 summarizes the transplantation, acute graft versus host disease (GVHD) grade characteristics of the six patients showing that the association 2-4 was seen in 22 patients, five of these patients later had a of leprosy and AHSCT may occur like de novo opportunistic relapse. Of the 92 patients with chronic GVHD (limited 48, mycobacterium infection, immune mediated reaction extensive 44), 10 relapsed. With a median follow up time of exacerbated by GVHD in a previously treated leprosy patient 690 days (range 6 – 2297), 74 (31 AML, 24 ALL, 17 MDS, 2 and simultaneous occurrence of leprosy and a potentially other) patients have relapsed. The median time from transplantable disease. Thus, leprosy must be considered in transplantation to relapse was 184 days (range 16-1621). The the differential diagnosis of skin and neurological lesions after relapse was treated with chemotherapy alone in 23, AHSCT and does not seen to be a contraindication for the chemotherapy + DLI in 32, DLI alone in one and palliative procedure. care in 18 patients. No systemic fungal prophylaxis was used. Invasive mould infection was diagnosed in eight of the 74 (10.8%) relapsed patients and three of the 146 patients without a relapse (2%) (p 0.008). In the group of relapsed patients, a proven Aspergillus infection was seen in four patients, Fusarium in one patient (blood culture) and Mucor in one patient (orbita). Two patients had a probable Aspergillus infection. All eight patients had received a combination of chemotherapy and DLI for their relapse and had developed P742 GVHD. Of the relapsed patients, 64 (86.5%) have died of the Emergence of Candida parapsilosis and Candida haematological malignancy. Of the eight patients with a mould guilliermondii bloodstream infections in allogeneic infection, one is alive 483 days after the relapse. In the non- haematopoetic stem cell transplant recipients receiving relapse group of patients, 111/146 are alive. In conclusion, the long-term caspofungin therapy incidence of invasive mould infections was significantly N. Kabbara (1), C. Lacroix (1), M. Robin (1), V. Rocha (1), R. increased in patients with acute leukaemia or MDS relapsing Peffault de Latour (1), A. Devergie (1), H. Esperou (1), E. after an allogeneic SCT and systemic prophylaxis should Gluckman (1), G. Socie (1), M. Ghanoum (2), P. Ribaud (1) therefore be considered for these patients. (1)Hôpital Saint Louis (Paris, F); (2)University Hospitals of Cleveland (Cleveland, USA)

P741 Between 07/2004 and 06/2006, 209 stem cell transplants Leprosy: report of six cases associated with allogeneic (SCT) were performed in our unit. Only 3 patients (pts) had haematopoietic stem cell transplantation candidemia [C. parapsilosis (Cp) n=2; C. guilliermondii (Cg) F. Pieroni (1), A.B. Stracieri (1), D. Moraes (1), E.J.A. Paton n=1)]. All were on Caspofungin (C) therapy. Pt 1 A 18 year-old (2), F.P. Saggioro (1), G. Barros (1), M.C. Oliveira (1), M. male with aplastic anemia received a 1st unrelated SCT in Coutinho (1), N.S. Castro (3), A.C. Vogoritto (4), P. Trabasso 01/2004 without engraftment (E). He later had a definite lung (4), C.A. Souza (4), M. Souza (5), M.A. Mauad (5), V.A.R. invasive aspergillosis (IA) treated with Voriconazole (V). He Colturato (5), B. Simões (6), N.T. Foss (6), J.C. Voltarelli (6) received a 2nd unrelated cord blood transplant in 05/2005. V (1)University Hospital of Ribeirão Preto (Ribeirão Preto, BR); was continued as 2ry prophylaxis. V was switched to C on day (2)Pio XII Foundation (Barretos, BR); (3)Santa Casa de (d) 7. On d 48, a blood culture (BC) was positive (+) for Cp. C Misericórdia (São Paulo, BR); (4)State University of Campinas was stopped [total treatment duration (TTD) = 47 d] and (Campinas, BR); (5)Amaral Carvalho Foundation (Jaú, BR); liposomal amphotericin B (LAB) was started. Due to the pt's (6)University of São Paulo (Ribeirão Preto, BR) poor condition, the central vein catheter (CVC) was not removed. BCs became negative on d 54. The pt died from Leprosy is an infectious disease with severe skin and multi-organ failure and no E on d 72. Skin and throat peripheral nerves lesions. We reported the association of colonization (Co) with Cp was documented from d 39. Pt 2 A leprosy and allogeneic hematopoietic stem cell transplantation 46-year old male with ALL received a genoidentical SCT in (AHSCT) on six patients. The first one developed 05/2005. Antifungal prophylaxis (AP) consisted of fluconazole erythematous plaques on the limbs without neurosensitive (F). C was empirically started on d 6. Granulocytes recovery symptoms on D+222. Biopsy showed lepromatous leprosy (GR) occurred on d 28. He presented a severe acute graft- (LL), treatment was rifampicine, clofazimine and dapsone versus-host disease (aGvHD) treated with increased (RCD), improvement occurred after 30 days. A type II reverse immunosuppression (IS), and a CMV infection. On d 58-60, 3 reaction was treated with prednisone plus thalidomide and BCs were + for Cp. C was stopped (TTD=50 d). LAB and F cleared leprosy lesions. The second case developed annular were initiated, and the CVC was replaced. The BCs became erythematous skin plaques on the face and limbs 174 days negative (-). However, the pt ultimately died from aGvHD and after AHSCT. Biopsy showed borderline lepromatous (BL) respiratory failure (RF) on d 86. Skin, GI and respiratory track leprosy, treatment with RCD improved the lesions after 30 (RT) Co with Cp was documented from d 33. Pt 3 A 32-year days. The third case presented erythematous painless old male with a lymphoblastic lymphoma received an plaques on the right shoulder 43 months after AHSCT. Biopsy unrelated SCT in 11/2005. AP consisted of F. GR occurred on showed borderline tuberculoid leprosy, monotherapy with d 21. He presented recurrent episodes of aGvHD treated with clofazimine did not clear lesions after six months. The fourth increased IS and developed several episodes of CMV case had a previous history of LL without lesions before the infections and bacteremias. The CVC was replaced on day AHSCT. On D+318, cutaneous cGVHD was associated with 61. AP was switched to C on d 95. On d 118, a BC was + for sharp pain and paresthesia in the upper and lower limbs. Cg. He developed a probable lung IA, concomitantly. C was Nerve biopsy showed tuberculoid leprosy. No treatment was stopped (TTD=26 d), and V was started. The BCs became -, given after AHSCT and currently he presents paresthesis in and IA resolved. He ultimately died from GvHD and RF on d the lower finger tips. The fifth case presented erythematous 197. GI and RT Co with Cp were documented between d 116 plaques on the trunk, abdomen, cripples and buttocks, after and d 123. Table 1 shows minimal inhibitory concentrations of one week of treatment with hydroxyurea for CML. Biopsy the first blood isolate. showed BL leprosy. Initial treatment with ofloxacin and In immunocompromised pts, the emergence of Candida dapsone was done with regression of lesions after 28 days. known to have a decreased susceptibility to C could become After D+229 atypical type I reverse reaction developed and an increasing problem. Persisting Co with such species may presented intermittent corticosteroid-dependent course. In the be an indication, in C treated pts, for switching to an last follow-up there was no evidence of leprosy. The sixth alternative antifungal drug. case had brownish plaques on the trunk, buttocks and limbs, with a diagnosis of aplastic anemia. Before AHSCT

S177 compatibilities; but other prognostic factors could be implicated. Recent findings indicate that the outcome of hematopoietic stem cell transplantation (HSCT) could be affected by NK cell alloreactivity via killer Ig-like receptors (KIRs). The purpose of this study was to determine the influence of donor-recipient HLA and KIR genotypes on the outcome of UCBT in 48 pediatric patients with hematological malignancies. Our results with donor-recipient pairs characterized by missing P743 donor HLA ligand in the recipient (HLA-C1, HLA-C2 or HLA- Diving suit: sterile, ambulatory, personal device for Bw4) for corresponding donor KIR, not only failed to show any protective and preventive isolation of nosocomial beneficial effect but also indicated trends for a deleterious invasive fungal infections in neutropenic patients effect on DFS (p=0.09), a higher relapse frequency (p=0.08) hospitalised in laminar air flow room who need a and a lower aGVHD rate (p=0.03). The latter finding contrasts disruption in isolation with those from previous reports on HSCT and could be due A. Thiebaut (1), M. Perraud (1), D. Lyonnet (1), S. Ozil (2) to an education of naïve cord blood NK cells after (1)Hôpital E. Herriot (Lyon, F); (2)Delta Protection (Lyon, F) transplantation. Furthermore, multivariate analysis showed that KIR3DS1 positive in donor is an independent predictor of Background: Patients (pts) with long neutropenia are at high disease relapse (p=0.03) whether in the presence or absence risk to develop poor prognosis invasive pulmonary of recipient KIR3DS1. aspergillosis (API). Pulmonary Computed Tomography Scans This is the first study to analyze the influence of HLA and KIR (CT scan) allow earlier diagnosis of API and improve genotypes in donors and recipients on the outcome of UCBT management. However, CT scans require disrupting isolation, in pediatrics patients. We observed no beneficial effect on which is potentially dangerous. We propose a diving suit to relapse frequency in patients lacking HLA ligand for donor preserve the preventive isolation. inhibitory KIR. This could be due to an educational effect of Material and Methods: The diving suit is personal, sterile, the naïve NK cells from the donor. The main finding was that ambulatory and transparent for monitoring, visual and verbal presence of KIR3DS1 in donor is an independent factor of contact. It has a self-contained ventilation station with a 12- disease relapse in UCBT. Our data suggest that HLA and KIR hour air supply. Air contamination is controlled with 2 HEPA genotypes are implicated in the outcome of UCBT and could filters. Fifteen pts treated for acute leukaemia tested it after be helpful for graft selection. These preliminary results will giving informed consent. require confirmation in larger retrospective multicenter trials as Results: The diving suit is validated for air contamination, well as in prospective trials. Further study will also be needed physiological (CO2), CT scan feasibility and pt acceptance. All to determine the biological relevance of these data and pts (5 male, 10 females) presented a severe neutropenia propose an algorithm of selection of CB associating the HLA (PNN < 0.5 G/l more than 10 days) at time of CT scan. Three and KIR genotypes. pts had already had a pulmonary CT scan before using the suit and 2 claimed to be claustrophobic. No pt described dyspnea (pain or discomfort). All pts felt reassured and agreed P745 to a new CT scan with this outfit. Two pts had another CT Haplo-identical allogeneic haematopoietic cell scan with the suit for suspicion of API. The diving suit was transplantation in adults after reduced-intensity very easy to manipulate and did not disrupt monitoring or conditioning with CD3/CD19-depleted grafts treatment administration. It was also compatible with Doppler W.A. Bethge (1), C. Faul (1), M. Bornhäuser (2), G. Stuhler and Nuclear Magnetic Resonance. (3), P. Lang (1), M. Stelljes (4), R. Handgretinger (1), L. Kanz For one pt with an anti-bacteriologic drug resistant fever, 3 (1) thoracic CT scans were performed (1 per week) without any (1)University of Tuebingen (Tuebingen, D); (2)University of sign of API. The 4th CT scan showed an API diagnosis with Dresden (Dresden, D); (3)University of Wuerzburg halo sign. This diagnosis was confirmed by histology after (Wuerzburg, D); (4)University of Muenster (Muenster, D) surgery. In this case, contamination was not possible during the first CT scan because of the diving suit. Haploidentical hematopoietic cell transplantation (HHCT) after Conclusion: Pulmonary CT scans very often need to be high dose conditioning with CD34-selected stem cells has performed in neutropenic pts to diagnosis API. Ambulatory, been complicated by high regimen related toxicities, slow personal protective clothing preserves isolation for engraftment and delayed immune reconstitution leading to immunocompromised pts during a CT scan. It can also be increased treatment related mortality (TRM). A new regimen proposed for medical staff protection when treating patients using reduced intensity conditioning (RIC) and graft with SARS or other highly contagious agents. CD3/CD19 depletion with anti-CD3 and anti-CD19 coated microbeads on a CliniMACS device may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, natural killer-, graft-facilitating- and Graft engineering dendritic-cells. RIC was performed with fludarabine (150-200 mg/m²), thiotepa (10 mg/kg), melphalan (120 mg/m²) and OKT-3 (5 mg/day, day -5 to +14) and no posttransplant P744 immunosuppression. Twenty seven patients (median age=41 KIR and HLA genotypes in unrelated cord blood (range, 21-59) years) have been transplanted with this transplantation regimen. Diagnosis were AML (n=15), ALL (n=7), NHL (n=2), C. Picard (1), C. Frassati (1), C. Galambrun (2), C. Curtillet MM (n=2) and CML (n=1). Patients were “high risk” with (2), C. Chabannon (3), I. Thuret (2), H. Chambost (2), D. refractory disease or relapse after preceding HCT. The Reviron (1), G. Michel (2) CD3/CD19 depleted haploidentical grafts contained a median (1)EFS Alpes Mediterranée (Marseille, F); (2)La Timone of 7.6 x 106 (range, 3.4-17x106) CD34+cells/kg, 5.4x104 Hospital (Marseille, F); (3)Institut Paoli-Calmettes (Marseille, (range, 0.006-44x104) CD3+T-cells/kg and 7. 5x107 (range, F) 0.02-37.3 x107) CD56+cells/kg. Donor-recipient KIR-ligand- mismatch was found in 17 of 27 patients. The regimen was Unrelated cord blood transplantation (UCBT) has been used well tolerated with maximum acute toxicity being grade 2-3 as an alternative treatment for hematological malignancies in mucositis. Because of severe neurotoxicity in 4 patients pediatric patients. Outcome of UCBT has been related to the treated with 200 mg/m² fludarabine, the dose was reduced to total nucleated cell dose and number of HLA-A, -B, -DR 150 mg/m² with no further cases of neuropathy thereafter.

S178 Engraftment was rapid with median time to >500 P747 granulocytes/µL of 13 (range, 10-21) days, >20000 Clinical outcome data using unseparated and volume- platelets/µL of 11 (range, 7-38) days and full donor chimerism reduction of cord blood units including an automated after 2-4 weeks in all patients. Incidence of grade II-IV GVHD system - the Duesseldorf experience was 48% with grade II=9, III=2 and IV=2. One patient, who L. Koerschgen (1), M. Aktas (1), F. Garnier (2), P. Verde (3), received the highest T cell dose, developed lethal grade IV P. Wernet (1), G. Kogler (1) GVHD. TRM in the first 100 days was 6/27 (22%) with deaths (1)ITZ (Duesseldorf, D); (2)Eurocord (Paris, F); due to idiopathic pneumonia syndrome (n=1), mucormycosis (3)Coordination Center for Clinical Trials (Duesseldorf, D) (n=1), pneumonia (n=3) or GVHD (n=1). Seven patients died after day 100, six due to relapse and one with systemic Standardization of unrelated CB banking was initiated in adenoviral infection. Overall survival is 14/27 patients (52%) Düsseldorf in 1992. To date a total of 10875 CB samples with with a median follow-up of 252 days (range, 18–1120). In a mean total NC count of 1.1x109 have been cryopreserved. conclusion, this regimen is promising in high risk patients 1940 CB units were stored unseparated and 8935 volume- lacking a suitable donor, and a prospective phase I/II study is reduced using HES. 7415 CB units were separated manually ongoing. and 1520 using the automated cell processing system Sepax (BIOSAFE) with the CS490 separation kit. The mean recovery of NC, WBC and MNC after manual volume reduction was P746 85±9%; 84±9% and 82±9.5% and for the automated sytem Retransplantation with stem cells from mismatched 85±9.4, 86±9.8 and 84.5±7.6%. Recovery of CD34+ and CFC related donors after graft failure in paediatric patients with both volume reduction (VR) methods was 95±5% and P. Lang (1), J. Greil (2), I. Mueller (1), P. Bader (3), T. 90±10%. However the consistency of the automated system Klingebiel (3), F. Heinzelmann (4), C. Belka (1), B. Kremens with regard to total processing time (n= 45 minutes), reduction (5), A. Reiter (6), P. Schlegel (7), R. Handgretinger (1) of time for training as well as the direct possibility to volume- (1)University Children's Hospital (Tuebingen, D); (2)University reduce to a defined volume (21 or 42ml) depending on the Children's Hospital (Heidelberg, D); (3)University Children's original size (cell count, total volume, HKT) of a CB unit were Hospital (Frankfurt, D); (4)Department of Radiooncology advantages after introducing this system into routine operation (Tuebingen, D); (5)University Children's Hospital (Essen, D); of the cord blood bank. The respective clinical outcome data (6)University Children's Hospital (Giessen, D); (7)University of both unseparated and volume-reduced single CB unit Children's Hospital (Wuerzburg, D) transplants were documented, also because of regulatory requirements. 364 CB units (197 for children and 153 for Graft failure is a rare but life-threatening complication after adults, 14 double CB units) have been delivered to transplant transplantation of hematopoietic stem cells. Treatment centers worldwide. The median age of these patients was 7 comprises immunoablative reconditioning regimens and a years (range 0.3-60) with a median weight of 26 kg (range 4- second stem cell donation from the same or from a different 116); they received a median cell dose of 4x107/kg BW. The donor as soon as possible to minimize the time of majority of patients were transplanted for malignancies (76%); pancytopenia and its sequelae. 19 % of patients had metabolic diseases and We report a cohort of 11 pediatric patients with leukemias immunodeficiencies and 4% AA. The cumulative incidence of (acute lymphatic n=4, acute or chronic myeloic / MDS n=4) neutrophil engraftment for both VR (n=55) and unseparated and severe aplastic anemia (n=3) who experienced graft CB units (n=88) in children was 83% at day 60 (p=0.126), the failure (nonengraftment,n=1; rejection, n=10) after TBI, overall survival rate at one (50%) and two years (43%) also busulphan or melphalan based myeloablative transplantation not statistically different (p= 0.596). The Kaplan-Meier from mismatched related donors (MMRD) (n=6) or after cord estimate of survival in adults at one year was 45 %, at two blood / matched unrelated donor (MUD) transplantation (n=5) years 38% and was statistically not significant for both VR between 2000 and 2005. In the latter the original donor was (n=27) and unseparated CB (n=29) units (p=0.43). In adults, not available a second time. Thus, all patients were re- the cumulative incidence of neutrophil engraftment at day 60 transplanted with CD34+ positive selected or CD3/CD19 was 82%. There were no differences between the two groups depleted stem cells from a second, haploidentical parental with regard to relapse rates (p= 0.170 children, p= 0.49 adults) donor (MACS method, Miltenyi Biotec). Median time span or aGVHD. Although the total number of patients analyzed is from diagnosis of graft rejection to second transplantation was small, these data show that an efficient volume reduction 9 days. The reconditioning regimens consisted of total method generates an identical outcome in this single center lymphoid irradiation or cyclophosphamide, thiotepa (5mg/kg), analysis. fludarabine (120 mg/m²) and ATG /OKT3. A median number of 25x10E6/kg of body weight stem cells with 95.000/kg residual T cells were infused. Mofetilmycophenolat was given P748 as GvHD prophylaxis, if residual T cells exceeded 25 000/kg Clean-room conditions for HPC processing have no bw. 7/10 evaluable patients had therapy resistant fever and impact on incidence of product contamination or on high levels of LDH (median of maximum values: 929 (347- clinical outcome 1422 IU/ml). Flow cytometry analyses revealed a M. Vasilcan, F. O'Brien, M. Sekhavat, S. Mackinnon, M.W. predominance of CD3+8+ lymphocytes and an almost Lowdell complete decrease of B and NK subsets. Sustained Royal Free & UCL Medical School (London, UK) engraftment was achieved in all patients (ANC>500/ l: 9 (11- 32) days). No GvHD > grade II was observed. T cell recovery In October 2006 the second technical annex to the EU was delayed, however no lethal viral infection occurred. Tissues & Cells Directive was published (2006/83/EC) which Severe organ toxicity was observed in 2 patients (BOOP, determined the appropriate conditions for “open” processing to hemorrhagic cystits) and moderate mucositis in 11 patients. be Grade A air in a Grade D background. Grade A air is 8/11 patients are disease free (median follow up 1.9 (1.0-6.9) achieved by processing within a class II microbiological years; 1 year EFS=73%). Causes of death were: BOOP (n=1), cabinet. The standard for grade D air is a maximum of infection (n=1); only one patient with refractory AML relapsed. 3500000 particles >0.5 microns in size per m³ of air and Thus, transplantation of stem cells from haploidentical donors 20000 >5.0 microns. This standard is below that already represents a realistic option to rescue patients with graft required or in use in some member states (e.g. Gemany and failure within a short time span and for whom a second UK) but is above those required by JACIE/FACT. It is donation of the original donor is not available. The use of a significantly below the standard required for manufacture of different donor may help to avoid a second rejection. somatic cell therapy pharmaceutical products for clinical trials. HPC transplants have been performed in many EU MS for over 25 years and the great majority have involved products which were procured and/or processed in “open” conditions in a grade A environment in a conventional laboratory which did

S179 not meet even grade D standards. We undertook a In summary, we have developed a novel lyophilization retrospective analysis of the frequency of bacterial technique maintaining the viability and functionality of CB contamination of HPC-A and HPC-M received and released HSCs. Using this technique the CB HSC have survived by our laboratory over the 25 years of HPC transplantation. All complete desiccation while maintaining their clonogenicity products received by the laboratory were tested for aerobic capabilities upon rehydration. In vivo NOD/SCID experiments and anaerobic contaminants at reception and post-processing. are ongoing. These results may be of significant clinical The data were compared with products received/released importance reducing current CB progenitor cell loss post post-2002. Pre 2002 data included 1648 allogeneic or cryopreservation. autologous HPC-M or HPC-A which were compared with 332 products produced in clean-room conditions. These frequencies of contamination were not significantly different to P750 those observed after relocation into clean-room facilities. 11 Processing of cord blood after thawing should be patients received an HPC product which was known to be different for volume-reduced units before freezing and microbially contaminated. No adverse events were reported non volume-reduced ones and all engrafted with no significant delay. C. Lemarie (1), B. Calmels (1), C. Picard (2), S. Furst (1), C. Conclusions: The chance of microbial contamination of an Curtillet (3), D. Blaise (1), G. Michel (3), C. Chabannon (1) HPC product is mostly determined by the harvesting (1)Institut Paoli-Calmettes (Marseille, F); (2)Etablissement procedure and not the laboratory processing. The incidence of Francais du Sang (Marseille, F); (3)Hopital Timone-Enfants laboratory-acquired microbial contamination is low and is (Marseille, F) affected by the degree of processing, cryopreservation being associated with a doubling of risk in the case of HPC-M. We Objectives: A major factor that affects cord blood (CB) can find no evidence of a reduction in this risk following the transplantation outcome is cell dose. Cell loss consecutive to relocation into clean-room laboratories. post thaw processing is reported to be unpredictable and sometimes significant. The goal of our study was to analyze a single centre experience of CB thawing for transplantation, in order to evaluate the influence of volume-reduction before freezing and post-thaw processing on cell recovery after thawing. Methods: Sixty-one unrelated CB were thawed at a unique cell therapy facility from December 2004 to November 2006. Thirty CB had been volume-reduced before freezing, and 31 had P749 been frozen without volume-reduction (whole CB). To obtain a Lyophilization of human umbilical cord blood may graft volume below 10ml/kg, CB were either washed after improve cell yield and reduce cell loss following thawing using a standard SOP based on P. Rubinstein cryopreservation technique (n=48; volume-reduced and whole CB), or diluted to A. Nagler (1), D. Natan (2), M. Mayerov (1), M. Binyamini (2), obtain a final DMSO concentration below 1% (n=13; volume- A. Arav (2) reduced CB). At the end of the post-thaw processing, the (1)Chaim Sheba Medical Center (Tel Hashomer, IL); (2)Core Total Nuclear Cells (TNC) count was measured with an Dynamics Inc. (Rechovot, IL) automatic counter, whereas the viable CD34+ cell count was measured by cytometry with the Stemkit® single platform Currently, most of the cord blood banks (CBB) cryopreserve assay. After infusion, potential immediate adverse events human umbilical cord blood (HUCB) units as a mononuclear were graded according to the CTC-NCI classification. cell (MNC) fraction using DMSO which results in 20-30% cell Results: At thawing, the median TNC recoveries were 84% loss. The units are usually stored in liquid nitrogen (LN) tanks. and 76% for volume-reduced and whole CB respectively. The Long term storage in LN is costly, requires means to avoid median CD34+ cell recoveries were identical in the 2 transient warming events and there is a risk (although small) subgroups (60%). Considering volume-reduced CB, the of cross contamination between stored samples. One way to median TNC recoveries were 74% and 85% respectively for avoid these problems is by lyophilization. Lyophilization is a CB that were washed (n=15) or diluted (n=13) after thawing. process in which ice crystals sublimate (form vapor without The median CD34+ recoveries were 48% and 80% going through the liquid phase) resulting in a dry, stable and respectively for washed (n=17) or diluted (n=10) CB. Patient low weight sample. We have developed a freezing apparatus immediate tolerance to infusion was reported for 43 named the Multi Thermal Gradient (MTG) device. This device transplantations. Two grade 2 adverse events occurred: one is based on directional freezing technology and enables the in the diluted CB subgroup (n=10) and one in the volume- precise control of ice crystal morphology during the freezing reduced CB subgroup washed after thawing (n=33). process, thus making the use of intracellular cryoprotectant Haematopoietic reconstitution and chimerism could not be agents (CPAs) redundant. HUCB units from consenting analyzed due to variability of multiple factors: patients adults mothers were split into 2 portions and a comparison between versus pediatrics, myeloablative versus reduced intensity fresh un-manipulated CB cells and cells that underwent conditioning regimens, single versus double CB lyophilization was performed. The MNC fraction was transplantations. separated on an F/H gradient. A freezing solution named IMT- Conclusion: We conclude that processing of CB after thawing 2 was added to the cell pellet and 2.5 ml of the cell has to be adapted to frozen unit characteristics. The initial suspension were put in a glass test tube and frozen using the washing SOP described by P. Rubinstein in 1998 concerned MTG-516 freezing apparatus. After freezing, samples were CB frozen without volume reduction. To optimize the cell placed in a commercial lyophilizer for 3.5 days. Rehydration recovery of volume-reduced CB after thawing, CB may simply was done by adding 2.4 ml of double distilled water (DDW) be diluted to reduce the DMSO concentration if the patient pre-warmed to 37ºC. After lyophilization and rehydration weight allows it. samples were evaluated for: viability, CFU-GM growth and CD34+ cell number. Viability was assayed by live/dead fluorescent stains using SYBR/PI. GM-CFU frequencies were assessed in semisolid cultures and CD34+ expression was assessed by FACS. Viability after lyophilization was 89.21±1.5% as compared to fresh CB cells. The number of CD34+ cells was 15,600 cells/ml before freezing and 17,450±150cells/ml after lyophilization. GM-CFU growth after lyophilization was reduced by only 30% as compared to the fresh un-manipulated sample.

S180 P751 low and high TNC viability groups, respectively. Median viable Immunosuppressive TLI-based reconditioning enables CD34+ cell recuperation after thawing and washing was 74% engraftment after graft rejection/graft failure in patients for the low TNC viability group, as compared to 77% for the with haematological malignant diseases remaining grafts. Both groups had an identical number of F. Heinzelmann (1), P. Lang (1), C. Faul (1), H. Ottinger (2), infused viable CD34+ cells (3.9x106/kg) and a similar R. Handgretinger (1), C. Belka (1) hematopoietic reconstitution: 12.0 days to reach a circulating (1)University of Tuebingen (Tuebingen, D); (2)University of neutrophil count of 0.5x109/L after infusion. Significant Essen (Essen, D) differences between these groups were only observed for the absolute number of granulocytes in the apheresis product Primary non-engraftment or early graft rejection represent a (13x109 for the low TNC viability group versus 7x109) and the rare but life-threatening complication that occassionally occurs occurrence of infusion-related adverse events (17% in the low after myeloablative conditioning followed by allogeneic TNC viability group versus 7%). hematopoetic stem cell transplantation (HSCT) with an Our data indicate that a low TNC viability in the infused HSC incidence below 5%. Management of early graft failure has graft does not compromise CD34+ cell recovery and been problematic as the risk of fatal infectious complications subsequent hematopoietic reconstitution. However, infusion of increases with the time of pancytopenia and retransplantation such grafts should be carefully monitored because of a higher preceded by a conventional myeloablative conditioning occurrence of infusion-associated complications. regimen is associated with high rates of cumulative organ toxicity such as pneumonitis and sinusoidal obstruction syndrome. Therefore we elected to prove an experimental P753 primarily immunosuppressive TLI-based reconditioning Clinical validation of a cGMP-compliant dry thawing regimen in order to obtain rapid engraftment paralleled by low device for haematopoietic stem cell grafts treatment toxicity. B. Calmels, C. Lemarié, C. Malenfant, C. Chabannon 14 patients (7 adults and 7 children) with a variety of Institut Paoli-Calmettes (Marseille, F) hematologic malignant diseases underwent TLI-based reconditioning regimen with 7 Gy single dose application plus Thawing and washing of hematopoietic stem cell (HSC) grafts OKT3/ antithymocyte globulin (ATG)/ fludarabine, followed by before infusion is for some transplant centers a routine an infusion of peripheral blood stem cells (PBSC) from procedure. Each year, more than 300 HSC graft are thawed at related/ unrelated donors. our institution, a procedure that still requires a waterbath. This Eleven of 14 recipients were evaluable for engraftment open system is operator-dependant and not convenient to following TLI-based reconditioning since three adults died handle in a cGMP facility, due to daily change of water. We (day 2/ 5/ 15) due to infectious complications. Remaining four thus evaluated an automated, closed thawing system, adults initially engrafted at a median of 13,5 days (range 11- CytoTherm CT-D2, for its ability to preserve HSC viability and 16 days) whereas six of seven children had sustained recuperation after thawing. engraftment with a median of 10 days (range 9-32 days). TLI- Previous pre-clinical experiments performed on 8 disqualified based reconditioning was well-tolerated with no severe toxicity apheresis products that were divided in 2 identical cryobags concerning the thyroid gland, lung, liver and bladder. Overall/ revealed comparable CD34+ cell recuperation and nucleated disease-free survival for the whole cohort was 42,8%. After a cell viability between our traditional waterbath-based median follow-up of 681 days, in children disease-free/ overall technique and the dry thawer. Based on these results, we survival are 85,7%/ 85,7%, respectively. decided to use this device for autologous HSC graft thawing. In conclusion, our results clearly demonstrate that in heavily We here report our data on the first 100 transplantations using pretreated patients with graft failure/ graft rejection after the CytoTherm CT-D2, as compared to 100 historical HSCT, TLI-based reconditioning regimens allow sustained procedures using a conventional waterbath. Measured water engraftment paralleled by minimal toxicity potentially leading temperature for each of the 2 systems was set to 40°C. to long-term survival. Average number of thawed bags was 2.0 for each condition, and numbers of CD34+ cells before cryopreservation were 5.4x106/kg for the waterbath group, and 5.0x106/kg for the P752 CytoTherm group. After thawing, all grafts were diluted 2-fold A low total nucleated cells viability after haematopoietic and supernatant was removed following a 15 min stem cell grafts thawing does not impair CD34+ cell centrifugation step. Average viable CD34+ cell recuperation recovery measured on the final product was comparable (85% versus B. Calmels, C. Lemarié, C. Chabannon 83% with the CytoTherm CT-D2). Total nucleated cell viability Institut Paoli-Calmettes (Marseille, F) was 63% when using the CytoTherm as compared to 59% when using the waterbath, as evaluated with the StemKit Autologous hematopoietic stem cell graft thawing and washing single platform assay. Time to reach a circulating neutrophil before infusion is systematically performed in some transplant count of 0.5x109/L after infusion was 12.0 days for both centers to limit DMSO-related complications and to groups of patients. standardize the thawing step. Total nucleated cells (TNC) CD34+ cell recuperation and total nucleated cell viability, viability and CD34+ cell recovery are considered and recorded measured on 100 thawing procedures, were equivalent to the as the main quality parameters for the thawing process. We traditional waterbath technique. Our data suggest that this report on a retrospective cohort of 370 hematopoietic stem closed, fully standardized automated system, drastically cell (HSC) grafts washed and assessed at a unique cell reduces water handling, potentially limits water-borne therapy facility, the biological and clinical factors associated contaminations and has to be preferred in cGMP facilities. with a low TNC viability in the thawed cell product. HSC bags were thawed with a traditional waterbath (n=165) or using a dry thawing device (n=205). The washing procedure P754 was performed on a standard centrifuge after dilution (n=210), Rapid and effective CD3-T-cell depletion using a novel or using a closed automated washing device, CytoMate magnetic cell sorting program to produce peripheral (n=160). CD34+ cell recuperation and TNC viability were blood stem cell products for haplo-identical measured on the infused cell product, using the StemKit transplantation in children and adults single platform assay that includes the 7-AAD viability dye. J.H. Dykes (1), J. Toporski (2), G. Juliusson (3), A. N. We defined the low TNC viability group as the first quartile of Bekassy (2), S. Lenhoff (4), A. Lindmark (4), S. Scheding (3) our population (n=90), that includes HSC grafts characterized (1)Blood Centre (Lund, S); (2)Paediatric Oncology (Lund, S); by a TNC viability lower than 55% (25th percentile). Median (3)Stem cell Centre (Lund, S); (4)Haematology (Lund, S) TNC viability was 50% for this group and 70% for the high TNC viability group (n=280). Median CD34+ cell content Background: Effective T-cell depletion is a prerequisite for before cryopreservation was 5.5x106/kg and 5.0x106/kg for the haploidentical stem cell transplantation. We investigated the

S181 performance of magnetic cell sorting (MACS)-based direct P756 large-scale T-cell depletion, which is an attractive alternative Post-thawed analysis of peripheral blood derived stem to standard stem cell enrichment procedures. cells – handle with care and let them breath Study design and methods: Peripheral blood stem cells A. Grunspan, Y. Kodman, Y. Sverdlov, A. Peles-Bortz, A. (PBSC) were harvested from 11 HLA-haploidentical family Davidson, I. Yaniv, J. Stein donors. T-cells labelled with anti-CD3 coated beads were Schneider Children's Medical Center (Petach Tikva, IL) depleted with the CliniMACS device using either the Depletion 2.1 (D2.1, n=12) or the novel D3.1 (n=11) program. If We sought to optimize conditions for post-thaw evaluation of indicated, additional positive CD34-selections were performed cryopreserved, G-CSF mobilized peripheral blood derived (n=6). Eleven patients received T-cell depleted grafts after stem cells (PBSC) from children with solid tumors, as part of reduced-intensity conditioning. our quality control program. Cells were frozen in 10% DMSO Results: The median log10 T-cell depletion was better with the and autologous plasma at concentrations of <150,000 D2.1 compared to the D3.1 program(log 3.6 vs. log 2.3, cells/ml in pilot tubes in tandem with bags containing the p<0.05) and could be further improved by introducing a PBSC units. Samples were thawed after a minimum of 24 blocking step (log 4.5 and log 3.4, respectively). D3.1 was hours in liquid nitrogen. Immediately after thawing at 38o, an superior to D2.1 (p<0.05) in median recovery of CD34+ cells equal volume of FACS buffer (PBS + 2% fetal calf serum) was (90 % vs. 78 %) and in median recovery of CD3 negative cells added. 150 ul of the sample was immediately analyzed for (87 % vs. 76 %). Total processing times were 1.7 to 5.3 hours total nucleated cell (TNC) count and differential, viability (D2.1) and 1.0 to 2.5 hours (D3.1). The transplanted grafts (trypan blue exclusion), and CD34+ cell count (IM aliquot), (directly T-cell depleted products ± CD34+ selected cells) and the remainder was washed (centrifugation 200g x 10 contained a median of 10.5×106/kg CD34+, 0.93×105/kg min, 4o), resuspended in FACS buffer, and then analyzed as CD3+, and 11.6×106/kg CD56+. Rapid engraftment was above (WASH aliquot). CFU-GM colony growth from both achieved in 10 patients. The incidence of acute GvHD was < aliquots was assessed in GM-CSF supplemented 0.9% 20% (grade I/II) and 0% (grade III/IV). methylcellulose. Mean TNC recovery (% of pre-freezing Conclusion: Both, the D2.1 and the novel D3.1 program values) was lower in WASH vs. IM aliquots (56% vs. 94%, enable effective, time-saving large-scale T-cell depletion. respectively (p<.0001), and mean viabilities of the samples Combining direct depletion techniques with standard CD34- were 75% and 79% (p=0.004). Mean number of granulocytes selection enables the composition of grafts optimized to the was similar in both aliquots. Mean CD34+ cell recovery was specific requirements of the patients. lower in WASH vs. IM aliquots (28% vs. 53%, p<0.0001). Mean CFU-GM colonies/1 x 105 cells were 12 vs. 76 in WASH and IM aliquots, respectively (p<.001). In a separate P755 experiment, IM cells were analyzed immediately or incubated Removal of dimethylsulfoxide prior to reinfusion of at 4o for 30 minutes before analysis. Incubation did not effect frozen-thawed leukaphereses products, does not TNC recovery, viability, or the number of CFU-GM/ 1 x 105 preclude engraftment cells, while mean CD34+ cell recovery rose to 71% vs. 53% in P. Ramos Oliva (1), M.L. Ramos Oliva (1), L. Larrea (2), E. unincubated samples(p=.012). It is likely that this increase in Roldan (1), J. Garcia-Larana (1), J. Perez-Oteyza (1) CD34+ cell recovery results from enhanced of (1)Hospital Ramon Y Cajal (Madrid, E); (2)Centro De antigenicity/presentation of the thawed CD34 molecule on the Transfusiones (Valencia, E) surface of incubated cells, rather than from increased expression of this protein on cells. Post-thaw evaluation of Reinfusion of cryopreserved and thawed haemopoietic cryopreserved G-CSF molibilized PBSC should be performed progenitors may induce adverse events such as nausea, on diluted but otherwise unmanipulated cells that have been vomiting and cardiac arrythmia, some of which have been incubated at 4o for at least 30 minutes. attributed to the presence of the cryoprotectant Dimethylsulfoxide (DMSO). On the other hand, extensive manipulation of thawed cells could reduce the number of P757 haemopoietic progenitors in the graft, and potentially Challenge testing of DMSO-resistant sterile filters jeopardize haematologic recovery. In order to avoid these incorporated in a closed bag system for cryopreservation problems, we have developed a semi-automated method for of cellular products removal of DMSO from thawed leukaphereses products using A. Humpe (1), B. K. Tischer (2), S. Schubert (2), P. Jansen the COBE 2991 cell processor. We have also analysed (1), C. Beck (1), I.-D. Adamzik (3), J.-H. Maas (4), A. Strate haematologic recovery after the reinfusion of washed cells in (4), M. Gramatzki (1), J. Riggert (4) haemopoietic stem cell transplant recipients. (1)Division of Stem Cell and Immunotherapy (Kiel, D); Methods: A total of 275 aphereses products from 91 (2)Department for Infection Medicine (Kiel, D); (3)Department consecutive patients undergoing transplantation at a single of Transfusion Medicine (Kiel, D); (4)Department of institution were processed. After thawing in a 37º C water Transfusion Medicine (Goettingen, D) bath, cell suspensions were 50% diluted in an hypertonic solution containing saline dextran, human albumin, and acid- Purpose: Processing of cellular therapeutics in an open citrate-dextrose (ACD-A). Cells were centrifuged and washed system according to GMP guidelines requires a cleanroom in the COBE 2991 cell processor. Quality controls included grade A with surrounding grade B conditions. A new closed cell counts, viability, CD34+ cell enumeration, microbiological bag system with an incorporated DMSO-resistant sterile filter cultures and haemopoietic progenitor cell assays (CFU-GM, (C3 Cell Freeze TM, CharterMedical) offers the opportunity to CFU-E) process cellular products outside of cleanroom facilities. To Results: There were no adverse events during cell test the capacity of the filter challenge experiments with manipulation. All microbiological controls were sterile. After bacteria were performed. washing, recovery of total nucleated cells was 76.4 %. Methods: 3 different batches (#06C10, #06D21, and #108763) Recovery of CD34+ cells was 143 %. Functional cultures of the systems were challenged with defined suspensions of showed 65 % and 71% recovery of CFU-GM and CFU-E, Bacillus subtilis strain ATCC 6051 or Staphylococcus respectively. Neutrophil engraftment occurred at a median of epidermidis strain ATCC 14990. 20ml of DMSO with a final 10 days (8-15), whereas platelet engraftment occurred at day load of 10E+03, 10E+04, 10E+05, or 10E+06 bacteria/ml and 12 (8-60). 20ml of NaCl 0.9% with a bacterial load of 10E+06 bacteria/ml Conclusions: our results demonstrate that washing thawed were applied to the sterile filter. Samples for evaluation of leukaphereses cell products prior to reinfusion, is safe and bacterial growth were taken before and after passage of the does not preclude haemopoietic reconstitution. filter. Bacterial titers were counted after overnight incubation at 37°C at ambient air and agar plates screened for cross- contaminations. In addition, in the S. epidermidis series samples before and after passage of the filter were tested by

S182 quantitative Real-Time PCR with Staphylococcus spec. P759 specific primers and an universal 16SrRNA TaqMan probe Analysis of the minor histocompatibility antigen HA-1- using a LightCycler 2.0 instrument (Roche) and the QuantiTec specific immune response after HA-1-mismatched Probe PCR Kit (Qiagen). haematopoietic cell transplantation Results: In the S. epidermidis series samples taken before M. Hudecek, H. Al-Ali, A. Reinhardt, E. Greb, D. Niederwieser, filtration revealed no bacterial growth when suspended in S. Tschiedel DMSO. Nevertheless, PCR analyses of the same series after University of Leipzig (Leipzig, D) filtration still revealed positive results. Challenging with B. subtilis, a more DMSO-resistant bacterium, revealed a filter Objective: Minor histocompatibility antigens are considered failure of lot # 108763 regardless of the applied medium responsible for both GvHD as well as GvT effects after HCT. (DMSO or NaCl 0.9%). The other filter lots tested successfully In the present analysis we investigated the endogenous HA-1- retained the inoculated bacteria. specific immune response in HLA-A*0201 HA-1 positive Conclusion: This study demonstrates that the reliability of patients after HA-1-mismatched HCT in conjunction with the closed systems for cryopreservation of cellular products is clinical course. dependent on a sufficient and effective quality control and Methods: A total of 9 HLA-A*0201 HA-1 positive patients with assurance system both on the side of the manufacturer of hematological malignancies received HCT from a HLA- sterile filters and on the side of the distributor of the whole matched HA-1-mismatched donor, 7 of them with reduced system. intensity conditioning (RIC). The appearence of HA-1-specific T-cells was monitored with HLA-A*0201/HA-1 Tetramers and the IFN-g Elispot Assay. From 2 patients T-cell lines were generated at different time points after engraftment. Graft-versus malignancy Results: Tetramer staining detected the emergence of significant numbers of HA-1-specific T-cells in 3 of the 9 patients, 2 of them with RIC and 1 after conventional HCT. During the first 3 months after HCT, frequencies of up to P758 0.18% HA-1-specific T-cells occurred and increased further Serotherapy with ATG and monoclonal antibodies with time. In the first RIC patient frequencies of maximum differentially influences frequency and function of natural 0.24% were detected and strong HA-1-specific reactions could killer cells after allogeneic stem cell transplantation be observed by Elispot after discontinuation of O. Penack, L. Fischer, A. Stroux, C. Gentilini, K. Rieger, A. immunosuppression at day 1000 post HCT. The second RIC Nogai, A. Muessig, S. Ganepola, E. Thiel, L. Uharek patient required continuous immunosuppression to control Charité University Hospital (Berlin, D) GvHD. After tapering CycA, increases in HA-1-specific T-cell frequency were associated with reduction of tumor cell burden Background: NK cells are important mediators of graft-versus- (increase of donor CD34+ chimerism and decrease in leukemia activity. Since ATG and monoclonal antibodies are leukemic host CD34+ cells). Contrary, augmented increasingly used for in vivo T cell depletion to prevent GvHD, immunosuppression was associated with lower HA-1-specific we investigated the effect of different types of serotherapy on T-cell numbers and reappearance of tumor cells. In this the frequency and function of NK cells in vitro and in vivo. patient the fluctuations in HA-1-specific T-cell frequency were Methods: Induction of apoptosis in vitro: NK cells and T cells not detected by Elispot. The third patient had a maximum of were separated from PBMCs by positive and negative 0.17% HA-1-specific T-cells 557 days post transplant while in selection of CD56 and CD3 (Micro Beads). Cells were molecular remission of the disease. T-cell lines were incubated with Thymoglobulin, Lymphoglobulin, ATG generated from PBMC of the 2 RIC patients and showed Fresenius, Alemtuzmab and OKT3 in concentrations ranging strong HA-1-specific reactions. Tetramer staining showed up from 0.001 µg/ml to 1000 µg/ml for 72 hours. Cell death and to 77.2% HA-1-specific CD8+ T-cells in the cell lines after 6 Apoptosis was determined by flow cytometry using the weeks of culture. propidium iodide and the Annexin V-FITC Detection Kit. NK Conclusions: HA-1-specific immune reactions are observed in cell activity in vivo: PBMCs of 34 patients at day +30 after vivo after HA-1-mismatched HCT. Reactivity to HA-1 allogeneic HSCT were coincubated for 3 hours with the NK increases during the first 3 months after HCT and after sensitive cell line HL60, NK cells were stained (CD56, CD3) discontinuation or reduction of immunosuppression. Tetramer and CD107a expression was assessed by flow cytometry. staining is more sensitive than the Elispot Assay in monitoring Results: In vitro: NK cells were affected differentially by the the HA-1-specific immune response. T-cell lines can be antibodies: OKT3 did not induce apoptosis; Lymphoglobulin established from PBMC of in vivo sensitized patients >10 µg/ml was needed to induce apoptosis in >50% of NK independent from the frequency of HA-1-specific T-cells. cells; in contrast <1 µg/ml Alemtuzumab, Thymoglogulin or ATG-Fresenius induced apoptosis in >50% of NK cells. T cells were effected equally by the antibodies: for induction of P760 apoptosis in >50% of T cells >10 µg/ml of all tested antibodies Favourable outcome after T-cell depleted stem cell were needed. In vivo: Fourteen pts received Thymoglobulin, transplantation combined with pre-emptive and ten pts Alemtuzumab and ten patients no T cell depletion. The therapeutic donor lymphocyte infusions in patients older median NK cell (T cell) count was: 250 /µl (630 /µl) in pts than 50 years without T cell depletion, 400 /µl (630 /µl) in pts with M. Hendriks, A. Schattenberg, F. Preijers, T. de Witte, N. Thymoglobulin and 100 /µl (100 /µl) in pts receiving Schaap Alemtuzumab (p<0.0005). The median percentage of Radboud University Nijmegen Medical Centre (Nijmegen, NL) degranulating NK cells was 4,4% without T cell depletion, 2,8% when Thymoglobulin was used and 0,8% when Allogeneic stem cell transplantation (SCT) with reduced Alemtuzumab was given (p<0.0005). intensity conditioning (RIC) regimens may be of benefit in Conclusion: The antibodies used for T cell depletion older patients. However, the probability of relapse may be differentially impact NK cells and T cells in vitro and in vivo. In higher compared to patients transplanted with standard vitro, we found that low concentrations of Thymoglobulin, conditioning regimens. In addition the incidence of chronic Alemtuzumab and ATG-Fresenius induced NK cell apoptosis, graft-versus-host-disease (GVHD) may be relatively high whereas high concentrations of Lymphoglobulin were needed which may have impact on quality of life and late mortality. to induce NK cell apoptosis. In patients receiving T cell In this study we evaluated outcome of partial T cell-depleted depletion with Alemtuzumab, NK cell activity and T cell counts allogeneic SCT together with pre-emptive and therapeutic were considerably reduced. In contrast, Thymoglobulin had donor lymphocyte infusions (DLI) in 71 patients older than 50 only moderate impact on the NK cell activity in the early phase years (median age, 55 years). Patients were transplanted for post allogeneic HSCT. acute myeloid leukaemia in first remission (n = 24), acute lymphoid leukaemia in first and second remission (n = 4),

S183 chronic myeloid leukaemia in first chronic phase (n = 12), P762 chronic lymphoid leukaemia (n = 4), myelodysplastic Ex vivo assessment of early reconstituted natural killer syndrome (n = 16) and non-Hodgkin’s lymphoma (n = 11). cells following HLA-matched haematopoietic stem cell The conditioning regimen consisted of cyclophosphamide 120 transplantation mg/kg body weight, idarubicine 42 mg/m² (in 64 patients) and G. Pittari (1), S. Wittnebel (1), S. Chouaib (2), A. Caignard (2), total body irradiation 9 Gy (TBI). In 10 patients busulphan J.H. Bourhis (1) replaced TBI. Donors were HLA-identical siblings. Stem cells (1)Institut Gustave-Roussy (Villejuif, F); (2)INSERM U753 were derived from either bone marrow (n = 53, fixed dose of T (Villejuif, F) lymphocytes: 0.7 x 106/kg) or from peripheral blood (n = 18, fixed T cell dose: 0.1 x 106/kg). Pre-emptive DLI was given to Objectives: Early reconstituted Natural Killer (NK) cells after patients if they developed acute GVHD ” grade 1 without the HLA-matched hematopoietic stem cell transplantation (HSCT) occurrence of chronic GVHD. The median follow-up was 4.4 might be involved in Graft-versus-Leukemia (GvL) years (median, 0.5-10.0). phenomena. To provide rationale for GvL-enhancing After SCT, acute GVHD • grade 2 occurred in 26% of immunotherapeutic strategies focused on such effectors, we patients. Extensive chronic GVHD occurred in 16% of performed an ex-vivo assessment of the functional status of patients. 17 patients received pre-emptive DLI. 17 out of 21 early reconstituted, resting NK cells following HSCT by a relapsed patients after SCT were treated with therapeutic DLI novel CD107a degranulation assay. and 9 of them responded. The two years probability of non- Methods: Blood samples at two different timepoints after relapse mortality was 29% in all patients. The 4 years HSCT – engraftment and day 60 – have been obtained from probabilities of relapse, current relapse (defined as persisting 12 patients undergoing HSCT for hematological malignancies. or recurrent relapse after DLI), survival, leukaemia-free No GvHD was present at the time of sampling. To induce the survival (LFS) and current LFS (defined as LFS after DLI) for degranulation of resting NK cells, whole peripheral blood all patients were 40% (molecular relapse included), 26%, mononuclear cells (PBMC) and K562 erythroblastoid cell line 59%, 40% and 50% respectively. were cocultured for 4h to 5h at a ratio of 10/1 respectively. We conclude that T cell-depleted SCT combined with pre- Cells were subsequently immunostained with anti-CD3, anti- emptive and therapeutic DLI is a feasible procedure in CD56, anti-CD107a fluorescence conjugated monoclonal patients older than 50 years. The low incidence of extensive antibodies and CD107a-positive NK cells were assessed by chronic GVHD (16%) and a plateau in the survival curve of multiparameter flow cytometry. For each sample, NK baseline these patients after median 2 years may exceed the results degranulation was used as negative control. The obtained by RIC transplantation strategies. degranulation potential of reconstituted NK cells was compared to the degranulation potential of NK cells obtained from healthy donors. P761 Concomitantly to the degranulation assay, an extensive Improved immunological reconstitution and amplified phenotypic profile – including natural cytotoxicity receptors graft-versus-leukaemia effects in a murine model of B cell (NCRs) 1-2-3, killer Ig-like receptors (KIRs) – 2DL1-2/3, leukaemia/lymphoma NKG2A/D, CD16 – of resting, unmanipulated NK cells has A. Abdul-Hai (1), L. Weiss (1), A. Ben-Yehuda (2), D. Ergas been performed in an attempt to correlate specific phenotypic (2), S. Reich (1), S. Slavin (1) patterns to the NK functional state. (1)Hadassah University Hospital (Jerusalem, IL); (2)Hadassah Results: School of Medicine (Jerusalem, IL) 1. At engraftment, early reconstituted, resting NK cells did not significantly differ from adult NK cells from healthy donors in Interleukin 7 (IL-7) plays a key role in maturation and function terms of CD107a expression after K562-mediated triggering. of both T and B cells. In this study, we investigate the 2. Patients could be divided in two groups according to the potential use of recombinant human IL-7 for facilitation of kinetic of expression of CD107a along reconstitution. In the graft-versus-leukemia (GVL) effects mediated by T cells first group (n=5), CD107a expression increased in the first two following transplantation in a murine model. GVL effects were months following HSCT, while in the second (n=7) it induced in (BALB/cxC57BL/6)F1 mice 24 hours after decreased. The differential expression (ExpressionDay60- conditioning with Cyclophosphamide by transplantation of ExpressionEngraftment) of NCR2 (NKp30) was strictly related C57BL/6 spleen cells activated or naïve with BCL1, followed to the one of CD107a (r value: 0,9214; p <0,01). by treatment of recipients with one or two cycles of IL-7 in Conclusions: Early reconstituted NK cells after HLA-matched vivo. Administration of IL-7 improved disease-free survival: HSCT display lytic potential and might reveal promising 67% of mice treated with IL-7 remained alive and disease free targets for immunotherapeutic strategies aiming at the >60 days, in comparison to 17% of the controls (p<0.05). enhancement of GvL effect. Their functional state may vary Similar results were obtained when C57BL/6 spleen cells over time and might be at least under the control of the sensitized against irradiated BCL1 cells in the presence of IL- up/down-regulation of NCR2. 7 were transplanted to F1 mice, followed by IL-7 treatment in vivo. Of the BALB/c mice that received spleen cells from F1 mice treated with IL-7 following transplantation of C57BL/6 P763 spleen cells sensitized with irradiated BCL1 in the presence of Natural killer cell dose and donor killer-cell IL-7, only 29% developed leukemia, as compared to 79% in immunoglobulin-like receptor genotype in HLA-identical the control group (p<0.05). Mice treated with IL-7 showed haematopoietic stem cell transplantation increased splenic and thymic cellularity and improved T cell- J. Clausen, D. Wolf, A. Petzer, E. Gunsilius, P. Schumacher, dependent proliferative responses compared to the controls B. Kircher, G. Gastl, D. Nachbaur (p<0.05). Based on the murine data, IL-7 may provide a novel Medical University Innsbruck (Innsbruck, A) tool to enhance immune reconstitution following transplantation of mismatched stem cells, as well as for Background. Natural killer (NK) cells are considered a relevant enhancement of GVL effects mediated by alloreactive component of the haematopoietic stem cell graft by promoting lymphocytes. engraftment and possibly contributing to a graft-versus- malignancy effect. The role of killer-cell immunoglobulin-like receptor (KIR) – ligand compatibility is still unclear, as is the impact of the NK cell dose of the graft. Methods. To investigate these issues in HLA-identical sibling peripheral blood stem cell transplantation (PBSCT), 43 consecutive transplants for haematological malignancies were retrospectively analyzed. Twenty-four patients underwent myeloablative conditioning and 19 patients received

S184 busulfan/fludarabine-based reduced intensity conditioning CTLs with lower leukemia-cytotoxic activity than in DC-T (RIC). training (38% vs.62%). Results. In patients with acute myelogenous leukaemia (AML) These data confirm that leukemia derived DC could train lytic or myelodysplastic syndrome (MDS) (n=18), regardless of T-cells in MLC and therefore qualify for immunotherapeutic conditioning type, no relapse occurred following transplants therapy to fight off residual leukaemia before or after SCT in meeting both, a high (above median) natural killer (NK) cell AML. count and missing KIR ligand, compared to all other (1st an 2nd authors contributed equally) AML/MDS patients (0% vs. 44%; p=0.049). Quantitative graft composition was found to have significant impact exclusively in RIC transplants. Here, a trend towards reduced relapse P765 incidence was found in patients receiving high numbers of NK Long-term IL 2 therapy after transplantation of T-cell cells (p=0.09). Overall survival (OS) was superior in recipients depleted stem cells from alternative donors in children of high T cell numbers (p=0.01). Non-relapse mortality (NRM) P. Lang (1), H. Teltschik (1), M. Pfeiffer (1), T. Feuchtinger (1), was significantly reduced in patients receiving high T cell E. Koscielniak (2), I. Müller (1), M. Ebinger (1), J. Greil (3), P. numbers (p=0.046). Multivariate analysis, including KIR-ligand Bader (4), T. Klingebiel (4), R. Handgretinger (1) matching, showed the probability of relapse to be significantly (1)University Children's Hospital (Tuebingen, D); (2)Olga decreased only in patients receiving high NK cell numbers Children's Hospital (Stuttgart, D); (3)University Children's (p=0.039). Hospital (Heidelberg, D); (4)University Children's Hospital Conclusion. These data suggest that both, the number of (Frankfurt, D) transplanted NK cells, and their KIR genotype in relation to the given HLA-type, play a role in the graft-versus-malignancy Relapse remains the major cause of death after allogeneic effect in HLA-identical PBSCT. Quantitative graft composition transplantation in children with high risk malignancies. T cell influenced outcome only in RIC transplants, while missing KIR mediated Graf versus host Disease (GvHD) has been shown ligand(s) improved the relapse incidence particularly in AML to be associated with Graft versus Leukemia (GvL) effects, but and MDS patients. also natural killer (NK) cells and other cells may exert cytotoxic activity. Thus, we evaluated the feasibility of low dose interleukin (IL) 2 therapy in patients with very high risk of P764 relapse after transplantation of completely T cell depleted A successful anti-leukemic cytotoxic reaction depends on stem cells from matched unrelated (n=2) and mismatched at least two partners: elucidation of the role of related donors (n=9) and analyzed the effects on NK cell (leukaemia-derived) dendritic cells and anti-leukaemia- mediated cytotoxicity. Diagnoses and remission status: directed T-cells AML/MDS, n=5 (NR1, 2, or second transplantation), ALL, n=2 A. Liepert (1), C. Grabrucker (1), A. Kremser (1), J. Loibl (1), (NR, CR3), JMML, n=1 (NR), Ewing sarcoma, n=2 (NR1, C. Schmid (1), R. Buhmann (1), T. Yang (1), T. Kroell (1), F. NR2). No posttransplant GvHD prophylaxis was given. IL2 (1 de Valle (2), W. Treder (2), H.J. Kolb (1), H.M. Schmetzer (1) Mio IU/square meter) was administered s.c. 3x/week for a (1)University Hospital of Munich (Munich, D); (2)Municipal median time of 73 days (15-250). 10 patients received IL 2 in Hospital (Oldenburg, D) order to prevent relapse, starting at day 41(13-77), one patient received IL2 after having relapsed, in combination with low For the mediation of a successful cytolytic reaction DC and dose chemotherapy. The regimen was well tolerated without specific T-cells play a central role. Here we present severe side effects (transient fever (n=4), fatigue (n=3), local characteristics of DC and T-cells that are necessary to inflammation (n=4), possible induction of GvHD II-IV (n=2)). mediate a blast lysis. Cytotoxicity assays against the standard cell line K562 were We generated DC with 3 methods in parallel (Lee, 2003), performed in 9 patients (2-4 experiments per patient, 1-2, 3-4, (Houtenbos, 2003), (Sato, 2003)). DC/ DCleu were quantified 4-8, and 12-24 weeks after start of IL2). In 8/9 patients a high by FACS. On average 27-32/25-34% DC in AML/MDS could and stable NK cell mediated specific lysis of K 562 was be generated with 45-52/39-52% mature (CD83+) and 31- observed (median lysis at E:T=20:1: 50%). In 5 patients, lysis 33/32-40% migratory (CCR7+) DC. 52-66/54-59% of those during IL2 therapy was compared with the NK activity before DC were leukemia-derived (DCleu); on average 43-48/43-49% IL2 administration: in 5/5 patients, NK activity was of blasts were convertible to DCleu.. substantially enhanced by in vivo IL2 injections (11% pre IL2 For cytotoxic analysis, mixed lymphocyte culture (MLC) were vs. 41, 47, 53 and 62% 1-2, 3-4, 4-8, and 12-24 weeks post set up with MNC- or DC-stimulated T-cells as effector cells IL2). 7 patients relapsed despite treatment between 41 and and their cytotoxic activity towards naive blast cells was 319 (median 100) days. 3/11 patients are disease free (4-5 quantified in 11 cases. In the DC-T training, a leukemia- years posttransplant). specific lytic activity (LA) was achieved in 6 cases while in 5 Conclusions: sustained, low dose IL2 administration is cases a stimulation of leukemic cells was seen (no-LA). feasible in children after transplantation from alternative Amounts of DC/DCleu or blast convertibility to DCleu, as well donors without severe side effects for several months. GvHD as T-cell-subpopulations were similar (CD4, CD8, CD45RA was induced only in two patients and a continuously high NK and RO, CCR7, viable cells) except for the proliferating T-cells activity could be achieved. However, the majority (63%) of our (49% in the LA fraction vs. 22% in the non-LA fraction). very high risk patients relapsed. Further studies are necessary Subdividing the group in LA-cases with high proliferation to evaluate the role of long-term administration of IL2. (>42%) and low proliferation (LA and non-LA), they presented with 70/19% CD4+, 53/19% CD45RO+, 6/39% non-viable cells while the other surface markers were expressed similarly P766 (CD8, CD45RA, CCR7). The DC characteristics of this group Double allogeneic haematopoietic stem cell showed 28/18 % DC, 62/49% DCleu, overall DCleu values of transplantation as a rescue therapy for high-risk 14/7% and blast convertibility of 44/28%. haematological malignancies MLC with MNC based T-cell training give rise to leukaemia- G. Stussi (1), J. Halter (1), A. Tichelli (1), S. Meyer-Monard specific CTL in 3 of 10 cases compared to 6 of 11 in the DC (1), C. Arber (1), D. Heim (1), J.R. Passweg (2), J.R. based T-cell training. However, after DC-training on average Rischewski (3), M. Paulussen (3), A. Gratwohl (1) 62% vs 37% of leukemic blasts after MNC-training were lysed. (1)University Hospital (Basel, CH); (2)University Hospital The T-cell subpopulations were similar in the LA and non-LA (Geneva, CH); (3)University Children's Hospital (Basel, CH) group except for the proliferating T-cells (56% vs. 15%). Summary and conclusion: Lytic activity in MLC with DC-T Treatment of patients with hematological malignancies and training is associated with high DC/DCleu counts and a good relapse or with primary refractory disease presents a blast convertibility to DCleu as well as high rates of therapeutic challenge. Results with direct allogeneic proliferating, CD4 and memory-T-cells. Some cases with hematopoietic stem cell transplantation (HSCT) are MNC-T training showed lytic activity however resulting in unsatisfactory and forced reinduction chemotherapy adds

S185 toxicity or can induce prolonged aplasia. Double allogeneic rejection, documented autologous reconstitution and survival HSCT (dHSCT) may improve the antileukemic effect by the of more than 3 months were identified in the data base among two conditioning regimens and add safety to the procedure by 10 887 patients. Fourteen patients with a sustained shortening the aplasia without GvHD by a first T-cell depleted autologous documented hematopoiesis and lasting remission HSCT. We followed this strategy over the last ten years in 25 were identified. None of these received a second allogeneic selected patients with advanced disease (19 male, 6 female, graft after the autologous recovery. For two cases a donor median age 37 years, range 5-70). Inclusion criteria were lymphocytes infusion was used. The characteristics of the informed consent and willingness of patients and donors as patients at transplantation were as followed: acute leukemia well as a sufficiently good general health status for such an (AL = 5 in CR), chronic myeloid leukemia (CML = 3, one in intensive treatment. 17 patients received dHSCT after a acute phase and 2 Phi+ in chronic phase), myelodysplastic previous HSCT (14 patients after one, 3 patients after 2 syndrome (MDS = 4), CLL (n = 1, progressive disease), previous HSCT) after a median of 308 days (range 99-2212) refractory myeloma (n =1); the conditioning regimen was and 8 patients as primary rescue therapy. The first dHSCT myeloablative in 11 cases and non myeloablative in 3 cases. was based on intensive chemotherapy or high-dose A transient engraftment was documented in 5 cases while no melphalan; the second T-replete HSCT was performed engraftment occurred in 9 cases. As of 02/06, only one patient immediately after recovery from the first HSCT (median 29 (CML in acute phase) died because of relapse, one is lost to days, 24-61) and was based on TBI±chemotherapy or BuCy follow up and 10 are alive and in CR. The range time of CR depending on prior irradiation or on BEAM-TBI in patients with after autologous reconstitution was 7,5 to 238 months (median HD. Donors were HLA-identical siblings for 22 patients, 102 months). The remission in the 2 CML (chronic phase) haploidentical donors for 2, and a syngeneic twin for 1 patient. patients after autologous reconstitution was not supported by Ten patients (40%) are still alive with a median follow-up of imatinib treatment. 390 days (27-3666). The 1-year probability of survival is 53% While one cannot exclude that a least a proportion of the 14 (95%-CI 33-74%). Five patients (20%) died of transplant- patients were cured prior to alloSCT and/or that some of the related mortality (2 infections, 2 VOD, 1 heart failure), 10 remission were in relation with the conditioning regimen, this patients (40%) died of relapse or progression despite this retrospective analysis data does suggest an efficient GvL intesified procedure. Only one patient experienced grade II effect can be observed without a sustained donor chimerism. acute GvHD in between the dHSCT, 8 patients after the The transient presence of donor T cells might indeed be second dHSCT (maximal grade II). In conclusion, dHSCT can sufficient to induce a powerful and long-lasting GvT effect. be performed with acceptable toxicity and TRM and may induce long-term remission in some patients. DHSCT might be further investigated as primary therapy for high risk or P768 refractory leukemias. Haplo-identical stem cell transplantation in children with high-risk neuroblastoma I. Dolgopolov, V. Boyarshinov, R. Pimenov, M. Yankelevich, N. Subbotina, G. Mentkevich Institute of Ped. Oncology/Hematology (Moscow, RUS)

Children with stage IV NB without CR after induction treatment have a very poor prognosis. We hypothetized that NK cells activated by KIR receptors disparity as well as T-cells will mediate graft vs NB effect after mismatched SCT transplant. Seven pts (4 M, 3 F) with stage IV NB with bone and/or BM involvement underwent G-CSF mobilized haploSCT from 3/6 HLA mismatched relatives (3 mothers, 4 fathers). Five pts were in PR (MIBG I123 positive bony lesions), one had PD, one pt with recurrent disease 8 mo after autologous SCT was in 2nd CR after resection of solitary CNS metastasis. The P767 median age of pair recipient/donor was 4.0 (2.5-9) and 33 (29- Anti-tumour effect of allogeneic haematopoietic 42) years, respectively. RIC regimen included Flu 180 mg/m², transplantation without sustained engraftment: myth or and ATG 40 mg/kg in combination with Bu 8 mg/kg (n=5) or reality? Treosulfan 30000 mg/m² (n=2). The median numbers of E. Daguindau (1), A. Buzyn (2), P. Chevalier (3), Z. Chir (4), CD34+ and CD3+ were 7.9 (2.9-35)x106/kg and 4.3 (2.7- C. Faucher (5), M. Kuentz (6), B. Lioure (7), M. Robin (8), P. 6.7)x108/kg. Cells were infused after incubation with Tiberghien (9), E. Deconinck (9) vincristine and methylprednisolone. GvHD prophylaxis (1)Hôpital Jean-Minjoz (Besançon, F); (2)Hôpital Necker consisted of 3 doses of MTX and cyclosporine A. Five pts (Paris, F); (3)Hôtel Dieu (Nantes, F); (4)French Society of developed GI and/or liver toxicity of stage>2. All pts engrafted Bone Marrow Transplantation and Cellular Therapy (Paris, F); with WBC>1.0x109/l on d11 (10-18), PLTs>20x109/l on d16 (5)Institut Paoli-Calmettes (Marseille, F); (6)Hôpital Henri (13-100). Full donor chimerism (CC) was achieved by d30 in Mondor (Créteil, F); (7)Hôpital Hautepierre (Srasbourg, F); all pts and remained stable for all period of the follow-up. One (8)Hôpital Saint-Louis (Paris, F); (9)University of Franche- pt did not develop any acute GvHD, 3 pts had gr. I GvHD, 1 pt Comté (Besançon, F) had gr. II GvHD, and 2 pts had gr. III at 100 days after SCT. Acute GvHD was treated with steroids, MMF, ATG, MTX or Graft versus tumor (GvT) mediated by the donor T cells is the cyclophosphamide. Three out of 4 long term survivors had most beneficial effect of allogeneic hematopoietic stem cell localized chronic GvHD. One pt died on d +50 from interstitial transplantation (alloSCT). Few cases suggest that sustained pneumonia and VOD. Four pts progressed 1, 5, 5, and 5 mos. allogeneic engraftment is not absolutely necessary to obtain a after graft. In all these pts immunosupression was stopped. long-lasting anti-tumor effect. Clinical remissions of GvHD reached gr. I in 1 and gr. II in 3 pts. Two pts died from hematological malignancies despite donor marrow cell DP in 2 and 12 mo. after SCT. Three pts are alive. One pt is in rejection have been reported after a myeloablative or non- CR 12 mo after SCT (clearance of MIBG I123 5 mo post- myeloablative conditioning regimens. In an attempt to SCT). Two pts with PD are alive with at 11 and 12 mo post- demonstrate a potential benefit stemming from transient post SCT. One of these pts is on low dose of MMF and received 5 SCT alloreactivity, we took advantage of the Société courses of cis-retinoid acid. This pt has a SD in bone and BM. Française de Greffe de Moëlle et Thérapie Cellulaire registry Another pt with brain metastasis relapsed in CNS 5 mos after to document patients with an efficient and lasting GvL effect SCT (5 metastases) and received 2 courses of CT and RT after alloSCT for malignant hematological diseases despite with a good response based on negative MIBG I123 scan. transient or absence of engraftment. Between June 1984 and We conclude that haploidentical PBSCT with RIC regimen in October 2005, 40 patients with no engraftment or graft NB pts with very poor prognosis is feasibleOur experience

S186 with using RIC in NB pts demonstrated evidence of graft vs NB effect and could be a promising approach to create a basis for immunotherapy.

P769 In haplo-identical transplants, administration of low-dose GM-CSF in patients who cannot benefit from NK alloreactivity, drastically reduces relapse rate at the price of an increased incidence of GvHD P. Lewalle, J. Kwan, J. Bennani, I. Ahmad, M. Aoun, D. Bron, R. Rouas, A. Delforge, P. Martiat Institut Bordet, ULB (Brussels, B)

We initiated a three-step phase I study trying improve transplant related mortality, relapse rate and immunity: first G- CSF + DLI, secondly GM-CSF + DLI, and next patient and disease adapted strategy (step 3). This paper reports the outcome of the 25 evaluable patients included in step 3 which consists of a patient adapted strategy: no more aspecific DLI Aplastic anaemia (only secondary prophylactic selective anti-CMV DLI) ; in myeloid disorders with NK allo-reactivity: no GF. In the other cases, GM-CSF (at a reduced total dose of 500 mcg) is given P771 over 5 days. The follow-up range of these 25 patients is Monoclonal T-cell expansions in paediatric patients with currently 40 to 3 months. Overall, TRM at day 100 was 4/25, aSAA suggests an autoimmune process located in the reflecting the good tolerance of the conditioning in a heavily bone marrow pre-treated population (median age : 38). NRR-mortality (7/25) B. Hubner (1), M. Führer (1), K. Dornmair (1), R. Hohlfeld (1), at one year remains acceptable in such a group of patients F.R. Schuster (1), A. Borkhardt (2) and was higher in the GM-CSF group (5 vs 2), mainly due to a (1)University of Munich (Munich, D); (2)University Duesseldorf higher incidence of aGVHD and immunosuppressive therapy. (Duesseldorf, D) More importantly, the overall relapse rate (6/25), equally balanced (3 vs 3) between the two group, strongly suggests a Acquired Severe Aplastic Anemia (aSAA) is a rare disease GVL effect induced by GM-CSF. The overall DFS is 13/25 characterized by pancytopenia and bone marrow with a median follow-up for patients at risk of 30 months, hypocellularity. There is good clinical and laboratory evidence which is rather good in this type of patients. We conclude that that a T- cell mediated immune attack against stem- and our current strategy drastically improves the relapse rate in progenitor cells located in bone marrow plays an important patients who do not benefit from NK allo-reactivity, at the role in the pathogenesis of aSAA. If aSAA is indeed mediated expense of a higher incidence of GVHD. by antigen-specific lymphocytes, putative autoimmune T- cell clones should be dominantly expanded in bone marrow compared to peripheral blood. Thus, we characterized the T- P770 cell receptor (TCR) repertoire in 3 newly diagnosed aSAA N-acetylcysteine does not inhibit the lymphokine patients by V beta- /J beta specific PCR- based spectratyping activated cells activity but reduces graft-versus- analysis and compared the results with 3 age- matched leukaemia effect in murine models healthy donors. In the bone marrow of aSAA patients we M.Y. Shapira, L. Weiss, S. Reich, M. Zeira, S. Slavin found an extremely skewed TCR- repertoire of CD4 and CD8 Hadassah University Hospital (Jerusalem, IL) positive lymphocytes compared to the corresponding repertoire in peripheral blood as well as in bone marrow and N-acetylcysteine (NAC) is a modulator of the glutathione blood of the healthy control group. In addition to the cellular content effects and a known antioxidant. In stem cell oligoclonal skewing we found even an unexpectedly high transplantation (SCT), NAC was suggested for prevention and number of monoclonal expansions in bone marrow. Molecular treatment of graft-vs.-host disease (GVHD), veno-occlusive analysis of 37 (CD4) and 55 (CD8) CDR3 regions of those T- disease (VOD) and idiopathic pneumonia syndrome (IPS). We cell clones did not reveal a) a preferential usage of specific V investigated wether it is safe to include NAC in the treatment beta families b) J beta- segments and c) uncommon features of patients during and following SCT by assessment of the in CDR3 sizes compared to V(D)J- rearranged sequences in possible effect of NAC on lymphokine activated cells (LAK) the ImMunoGeneTics database. These findings allow us to activity and graft-vs.-leukemia effect (GVL) in murine models. analyze putatively new autoimmune T- cell clones in detail After 10 days of either oral or intraperitoneal NAC treatment in and provide further evidence that SAA is mediated by an doses in the range of treatment in humans, the cytotoxic strong autoimmune T- cell response. activity of the LAK cells against Yac cells (H-2a, an NK sensitive tumor cell line) did not significantly differ from LAK activity generated from spleen cells obtained from untreated P772 controls. However, NAC slightly suppressed GVL in animals Favorable outcome of fludarabine and cyclophosphamide transplanted from C57BL/6 into (BALB/c x C57BL/6) F1 and as a bone marrow transplantation conditioning regimen inoculated with BCL1 leukemia (the appearance of BCL1 for patients with severe and very severe aplastic anaemia leukemia in 8/36 animals treated with NAC as compared to H. Al-Zahrani, F. Al-Mohareb, F. Al-Shareif, H. Al-Omar, A. Al- 0/20 in the transplantation control group, p=0.023, figure 1). In Shanqeeti, N. Chaudri, E. Sahovic, S. Zaidi, A. Saleh, K. Al- spite of this mild GVL suppression, no negative effect on Anazi, A. Abdulwahab, A. Al-Shahri, M. Morshed, A. Nassar, engraftment, as judged by achievement of donor chimerism, M. Aljurf was seen. King Faisal Specialist Hospital (Riyadh, SA) We conclude that though further clinical studies are warranted, the use of NAC in SCT seems relatively safe in Introduction: Bone marrow transplantation is widely accepted regard to the GVL effect. as the standard potentially curative treatment option for young patients with severe and very severe aplastic anemia. Ideal pre-transplantation conditioning therapy should secure adequate lympho/immunoablation necessary for adequate engraftment, preferably with the lowest possible risk of graft versus host disease (GVHD) in this non-neoplastic disease.

S187 Fludarabine is a potent lympho/immunoablative therapy, Conclusions: Immunosuppression by MSC of normal adults particularly if given with Cyclophosphamide. and aplastic anemia patients can be detected in direct cell-cell Study: Between August 2004 and October 2006, we have contact and in transwell experiments while no effect of MSC used the combination of Fludarabine (30 mg/m2 x 3 days) and supernatant was seen. The mechanism of Cyclophosphamide (50 mg/kg x 4 days) as conditioning immunosuppression ist not fully understood and further regimen for 29 (17 males) consecutive patients with severe investigations are needed. and very severe aplastic anemia. HLA-identical siblings marrow was used as the stem cell source in all patients. Median age of our patients was 21 years (14-36), median P774 dose of CD34 positive cells was 2.44 x 106 per kg of recipient Allogenic geno-identical haematopoietic stem cell weight (range 3.8-19 x 106). GVHD prophylaxis consisted of transplantation with procarbazine, cyclophosphamide CSA and short course of methotrexate (MTX), except for one and antithymocyte globulin conditioning in 69 patients patient who received MMF/MTX because he developed renal with acquired severe aplastic anaemia impairment early after transplantation. The regimen was well R. Ahmed-Nacer, R.M. Hamladji, M. Benakli, F. Mehdid, R. tolerated. Median time for achievement of ANC >500 and Belhadj, F. Kaci, N. Rahmoune, M. Baazizi platelets >20,000 was 21 and 23.5 days, respectively. Acute Pierre and Marie Curie Center (Alger, DZ) GVHD developed in 9 patients (31%), with grade I-II in 6 and grade III-IV in 3 patients. Chronic GVHD was observed in 4 Introduction: Allogenic hematopoietic stem cell transplantation patients (14%) which was limited in 3 and extensive in 1 (HSCT) from HLA-identical siblings is used successfully to patient. Four patients (14%) developed subsequent graft treat severe aplastic anaemia SAA), particularly in young failure. Four patients died early after transplantation, 1 patients. HSCT leads to complete hematopoietic recovery and secondary to graft failure, 2 with infection and 1due to cure of disease in 60 to 80% of patients treatment toxicity. At a median follow up of 12 months, 25 Materials and methods: During an 91 months period (from patients are alive, 22 are disease free (overall survival of May 1998 to November 2005)69 patients with SAA 86.2% and disease free survival of 76%). Detailed information (idiopathic:66,HPN:2,post hepatitis:1) received 72 allogenic about recipient pre-transplantation disease severity status, HSCT and 3 boosts from HLA-identical sibling donors; median transfusion status, pre-transplantation pulmonary fungal age 19 years (5 to 40), 36 patients under 20 years and 3 over infections and observed regimen related toxicity will be 30 years; sex ratio:1,65; median interval from diagnosis to presented at the meeting. allograft 12,8 months (1,5 to 114); all patients were transfused Conclusion: The combination Fludarabine and but 47 (68,1%) had received more than 20 transfusions before Cyclophosphamide is an effective and well tolerated allograft.Twenty four patients (43,8%) had previously conditioning regimen for severe aplastic anemia with immunosuppressive therapy failure. All patients received acceptable risk of GVHD and graft failure. Further prospective conditioning with procarbazine 37,5 mg/kg,Cy 200 mg/kg and studies are needed to compare the outcome of this regimen ATG (Fresenius) 40 mg/kg and GVHD prophylaxis with with other accepted standard conditioning regimens for methotrexate/cyclosporin (Seattle). Nine patients received transplantation in aplastic anemia. bone marrow transplant (within one boost) of median mononuclear cells 4,32x10 /kg (range 2-7) and 60 patients peripheral blood stem cell (within 2 boosts and 3 additional P773 transplant for 2 patients) of median CD34+cells 3,55x10 MSC from AA Patients exert a normal /kg(0,6-26). At September 2006 maximal follow up is 101 Immunosuppression in vitro months and minimal 11 months. V. Mailänder, E. Hennel, M. Flören, M. Rojewski, M. Results:The median time to engraftment was 20 days (10 to Wiesneth, H. Reichel, H. Schrezenmeier 32). Fifty two patients (75,4%) are alive with successful University of Ulm (Ulm, D) engraftment after median follow up 51 months (11 to 96 months). Four patients (5,8%) rejected graft and one patient is Purpose: Mesenchymal stem cells (MSC) are increasingly alive after successful second transplant.A late loss of stem used for therapy of GvHD or immunosuppression in cell transplant characterized by pancytopenia and successful autoimmune diseases. It was hypothesized that MSC from hematopoietic complete recovery after boost was observed in aplastic anemia patients are less immunosuppressive thus 2 patients. Acute GVHD accured in 16 patients (25,8%) with 6 allowing immunoreaction against autologous hematopoesis. grade II-IV and chronic GVHD in 20 patients (37,7%), While the immunosuppressive effect of healthy MSC can be extensive in 10. Seventeen patients (24,6%) died: 14 (20,2%) demonstrated in vitro and in vivo models as well as in clinical of TRM (infectious:7, GVHD: 4, cerebral hemorrhage: 1, studies there is uncertainty about the mechanism. cardiomyopathy: 6, metabolic disorder: 1) and 3 after graft Methods and Results: MSC from bone marrow were failure (4,3%). Chimerism data (PCR-STR) were available for characterized by FACS (CD9+, CD44+, CD105+, CD34-, 27 patients and all achieved 100% donor chimerism with CD45-) and differentiation assays. In a standard mixed median follow up 60,5 months (15,5 to 96 months). Actuarial lymphocyte reaction (MLR) MSC in a ratio of 1:1 to the overall survival is 75% at 8 years. responder cells clearly suppressed the MLR reaction (80- Conclusion:Our results confirm that HSTC is the choice 100% reduction of proliferation). A 1:10 ratio of treatment for aquired severe aplastic anaemia patients under MSC:responders was not effective in most experiments and at 30 years. a 1:100 ratio we never observed a suppressive effect. MSC from patients with aplastic anemia were studied for their immosuppressive potential. In all experiments (n=5) P775 suppression of proliferation was observed contrary to the Allogeneic stem cell transplantation for aplastic anaemia, results of other groups (Bacigalupo A et al.Exp Hematol. PNH and hypoplastic MDS – a single-centre experience 2005, 33:819). S. Buchholz, E. Dammann, C. Koenecke, M. Stadler, G. In experiments with MSC supernatant we did not find any Buesche, H. Kreipe, J. Krauter, B. Hertenstein, A. Ganser, M. suppressive effect. In contrast there was some effect when we Eder put the MSC in a transwell system (roughly 50% suppression Hannover Medical School (Hannover, D) of proliferation). Data in the literature on the mechanism are very controversial. Among others it was proposed that Introduction: Acquired bone marrow aplasia in adults may be prostaglandin mediates the effect. However by inhibition of due to rare diseases such as aplastic anemia (AA), prostaglandin synthesis with indomethacin and NS398 we paroxysmal nocturnal hemoglobinuria (PNH), and hypoplastic could not reduce the MLR-suppressive effect of MSC. myelodysplastic syndromes (MDS). Currently we are investigating whether depletion of tryptophan Patients and Methods: We report the outcome of allogeneic plays an important role. SCT for consecutive patients with AA (n=18), PNH (n=4, one aplastic patient), and hypoplastic MDS as defined by bone

S188 marrow histology (n=11). MDS patients were classified as RA Dexa scans have been performed in 19 patients following (n=2), RCMD (n=3), RAEB (n=5, 2 with myelofibrosis), and transplantation. Of these 9 showed reduced bone density (T unclassified MDS (n=1). Median age was 28.5 years (17-45) score <-1.0), 4 of whom had received steroids for treatment of for AA and PNH, and 46 years (33–64) for MDS patients. graft versus host disease. Renal function was impaired in 14 Karyotyp was normal in 21 cases with AA/PNH; 6 and 3 of 9 subjects, and ovarian/testicular failure identified in 3. evaluable MDS patients had unfavourable (monosomy 7) and Conclusion: Allogeneic transplantation is a successful intermediate cytogenetics, respectively. AA and PNH patients treatment modality for selected patients with severe aplastic (13 patients pretreated by conventional immunosuppressive anaemia. Complications of this treatment include graft versus therapy) were transplanted with BM (n=18) or PBSC (n=4) host disease, which is seen more commonly in those who from either matched (n=10), haploidentical (n=1) or syngeneic have achieved a 100% donor chimera profile, and recurrent (n=2) family donors or matched (n=7) and mismatched (n=2) pancytopenia, which is seen more frequently in those who unrelated donors. GvHD prophylaxis consisted of CSA/MTX have evidence of significant residual recipient (n=17), CSA/Pred (n=2), CSA and T-cell depletion (n=1) and haematopoiesis. Osteopenia and osteoporosis are also none for both syngeneic transplantations. MDS patients were complications of transplantation and are more frequent in transplanted with BM (n=5) or PBSC (n=6) from matched those who have received treatment with steroids. related (n=5) or unrelated (n=6) donors. Seven patients received CSA/MTX, three CSA/MMF, and one CSA/Pred for GvHD prophylaxis. Conditioning for the AA/PNH-group varied P777 depending on disease and donor type and mainly included Trial with anti TNF alpha in Fanconi anaemia: report of the cyclophsophamide/ATG (n=9) and first treated patients fludarabine/cyclophosphamide/ATG with or without low-dose J. Svahn (1), P. Ghezzi (2), T. Lanza (1), A. Corcione (1), M. body irradiation (n=6). In the MDS-group eight patients Mengozzi (2), M. Lanciotti (1), E. Castagnola (1), E. Ferretti received oral or iv busulfan/cyclophosphamide and three (1), I. Lorenzi (1), R. Tonelli (1), G. Casazza (3), V. Pistoia (1), FLAMSA/TBI/Cy (Schmid et al. JCO, 2005) for conditioning. C. Dufour (1) With a median follow-up of 39 months (range 4 – 132) for the (1)G.Gaslini Childrens' Hospital (Genoa, I); (2)Mario Negri AA/PNH group all but one PNH patient have had favourable Institute (Milan, I); (3)Santa Chiara Hospital (Pisa, I) outcomes (> 95% survival) with high performance status independent of donor type and pre-treatment. Acute and Anti TNF-a molecule Etanercept improves the growth in vitro chronic GvHD was observed in 35% and 21%. In contrast, of haematopoietic cells of Fanconi Anemia (FA) patients. A survival for MDS patients was only 33% with a median follow- clinical trial with 0.4mg/kg of Etanercept twice/week in FA up of 16 months (range 10-102). All deaths were due to patients with severe marrow hypocellularity (less than 25%), treatment related mortality. Acute and chronic GvHD occured no overt MDS or leukemia who lack a stem cell donor was in 50% and 59% of evaluable MDS patients, respectively. approved by Gaslini Institute Ethical Committee (EC). MN, boy Conclusion: Whereas allogeneic SCT for AA has proven very 9 years and GD, boy 7 years entered the trial 12 and 8 successful independent of donor type and pre-treatment months after diagnosis of FA, with deteriorating blood counts allogeneic SCT for patients with hypoplastic MDS results in and unavailability of stem cell donor. much lower overall survival due to high post-transplant Marrow apoptosis of MN after Etanercept start decreased morbidity and mortality. (annexin V+ cells) and so did intracellular TNF-a and IFN-g. Committed progenitor growth mildly increased after Etanercept start in both MN and GD. LTCIC were 0 in both P776 MN and GD prior to and after therapy. Etanercept neither Allogeneic transplantation in the treatment of severe improved nor worsened blood count and transfusion aplastic anaemia frequency in both patients. No relevant side effects occurred. K. Ryan The drug reached a plateau in peripheral serum of 0.4mg/ml St. James's Hospital (Dublin, IRL) in both patients after 2 months and a level of 0.12-0.27mg/ml in the marrow after 1 month. Aplastic Anaemia is a haematological disorder characterised TNF-a increased in peripheral serum to 140-270pg/ml and in by failure of development of maturing blood cell precursors marrow serum to 100-180pg/ml. Levels were 2500-5000-fold resulting in pancytopenia. Spontaneous marrow recovery is lower than those of serum Etanercept and 1500-3500-fold rare, and treatment options include immunosuppression and lower than those of marrow Etanercept. Bioassay showed that allogeneic bone marrow transplantation. We aimed to both serum and marrow TNF-a were biologically inactive. determine the outcome of patients treated with allogeneic MN was transplanted 100 days after start of Etanercept with bone marrow transplantation in our centre. an 8/10 antigen compatible donor identified 30 days earlier. Method: We identified all patients with severe aplastic GD is still on Etanercept, 5 months after start. No optimal anaemia treated by allogeneic bone marrow transplantation in stem cell donor is identified as yet. St. James’s Hospital. Their charts were reviewed to obtain These data show: demographic data, their conditioning regimens and establish 1. Etanercept was safe in the treated patients. their subsequent outcomes. End-points included chimerism 2. The drug reaches the marrow in biologically active status, full blood count and renal function at 1 year and also amounts. morbidity including renal impairment, graft versus host 3. The initial increase of TNF-a is not biologically active as it is disease, bone density abnormalities and endocrine neutralized by Etanercept as shown by the lack of activity in disturbance. the bioassay. Results: Between February 1987 and August 2005, 26 4. No relevant impact was seen on the blood count and allogeneic bone marrow transplants were carried out on transfusion requirement. patients with severe aplastic anaemia (15 male and 11 5. Marrow apoptosis, progenitor growth and cytokine burden female). The average age at transplantation was 18years were improved thus suggesting that the drug may have a role (median 17 years, range 12-31 years). Follow-up was for an in contrasting marrow failure progression of FA. average of 8 years (median follow-up 10 years, range 4-19 Points 4. and 5 can be explained by the too advanced stage of years). The most commonly used conditioning regimen was marrow failure that while precluding gross haematological Cyclophosphamide/ATG. Chimera analysis at one year post- improvements might have allowed subclinical changes. The transplant revealed complete donor chimera in 10 patients, 3 EC of Gaslini Institute, has now approved a protocol of whom had graft versus host disease. 11 patients had a amendment to enrol patients at an earlier stage of marrow mixed chimera profile and a further 2 displayed complete failure. This may enable to evaluate the impact of this recipient reconstitution. Persistent pancytopenia was seen in 3 experimental treatment also on blood and marrow cellularity. patients with a mixed chimeric profile, all of whom had a chimera of less than 95%.

S189 P778 anti-thymocyte globulin (ATG) and cyclosporine A (CSA). Two Stem cell transplantation in Fanconi anaemia. A single- pts. received unmanipulated BM from a 8 and 9/10 compatible centre experience donor (2,4 and 1,3 x 106 CD34 positive cells/kg BW) M.A. Sanna, M.C. Addari, M.G. Orofino, A. Piroddi, F. Rizzo, respectively, one patient received T-cell depleted peripheral M. Badiali, R. Galanello, F. Argiolu blood stem cells (PBSC) from a 10/10 matched donor (4,5 x Ospedale per le Microcitemie (Cagliari, I) 106 CD34, 10,4 x 104 CD3 pos. cells/kg BW). Conditioning regimen consisted of cyclophosphamide (Cy), Flud-Arabin and Fanconi anemia (FA) is a genetic disorder associated with serotherapy. The pt. with the T-depleted graft received 2 x 2.5 congenital abnormalities, progressive bone marrow failure and Gy total lymphoid irradiation (TLI) in addition. All three pts. increased risk of leukaemia and other cancers. rejected their graft and were successfully regrafted from the Haematopoietic stem cell transplantation (HSCT) is presently same donor after an intensive immunablative conditioning the best treatment for FA patients developing bone marrow based on Cy (2 x 60 mg/kg), Flud-Arabin and serotherapy with failure. Success for HSCT in patients with an HLA identical OKT3 ± ATG. The graft consisted of unmanipulated PBSC in sibling donor is 70%, whereas in those transplanted from an all three pts. (8.1, 8.7 and 11.6 CD34/kg BW). No significant unrelated donor figures are reported in the range of 29-43%, toxicity was seen after the 2nd transplant. All three pts. with graft rejection, GvHD and regimen related toxicity being showed prompt (range 10 – 13 days) and stable engraftment the main causes of failure. These results have limited the with complete donor chimerism. Two pts. developed mild ability to perform marrow transplantation other than with HLA acute GvHD (I-II) of the skin, and one pt. suffers from limited identical siblings for this disease. Recently a fludarabine (FLU) chronic GvHD. based cytoreductive regimen has been successfully used in T We conclude that in young pts. with SAA re-transplantation cell depleted haploidentical/mismatched transplant of FA from the same MUD after non-engraftment or early graft patients. failure can be performed successfully with unmanipulated Between June 1999 and September 2005, four patients with PBSC after an immunoablative conditioning regimen FA (three with bone marrow failure and one with MDS) containing Cy, Flud-Arabin and OKT3 alone or in combination underwent HSCT in Cagliari. Patients’ characteristics, with ATG. conditioning regimen and outcome are shown in Table 1. Conclusions. All three patients transplanted with reduced doses of Cyclophosphamide (CY), FLU and ATG (Thymoglobuline) engrafted promptly, achieved full Haemoglobinopathy haematopoietic chimerism (VNTR mean 13 days) and adequate immunological recovery. Neither patient developed toxicity, nor GvHD. UPN 77, transplanted with a more aggressive preparatory P780 regimen for the development of clonal disease (MDS), showed Allogeneic stem cell transplantation in patients with erythroderma and acute/chronic limited GvHD. thalassaemia: the Saudi experience Fludarabine-based regimen could also be proposed to cure A. Al-Jefri, M. Ayas, A. Al-Musa, M. Al-Mahr, M. Saleh, S. FA patients with clonal disease and without an HLA matched Rifai, R. Sabbah, A. Al-Seraihi, A. Al-Ahmari, A. Khariy, I. Al- donor, decreasing the long term side effects of other more Hassan, H. El-Solh toxic regimens. King Faisal Specialist Hospital (Riyadh, SA) Background: Allogeneic stem cell transplantation (STC) is curative in patients (pts) with Thalassemia major with outcome depending on age and disease status at SCT. We report here our experience. Patients and methods: from January 1998 to July 2006, 62 cases of SCT were performed on 59 pts with a- Thalassemia; 7 pts had hepatitis C with stable liver condition. Conditioning was with one of 2 regimens, regimen A (24 pts): Busulfan (BU), Cyclophosphamide (CY) and anti-thymocyte globulins, and regimen B (38 pts): BU/CY only. GvHD prophylaxis was with cyclosporine and methotrexate in both regimens; those undergoing second SCT were conditioned P779 with CY and total body irradiation (TBI). Median age at SCT Successful re-transplantation from the same donor after was 5 years (range, 1.5-13.9 years). Pts were classified rejection of a graft from an unrelated donor in children according to disease severity; mild (23 pts), moderate (25 pts) with severe aplastic anaemia and severe (12) based on hepatomegaly, regular transfusion M. Führer (1), A. Claviez (2), A. Schrauder (2), A. Borkhardt and chelation plus the level of ferritin. The source of stem cells (3) was unmanipulated marrows from HLA-matched related 6 (1)Children University Hospital (Munich, D); (2)Children donors; the median CD34+ cells was 7 x 10 /kg (range, 1.6- 6 University Hospital (Kiel, D); (3)Children University Hospital 17 x 10 /kg). Engraftment was assessed by VNTR/STR or (Duesseldorf, D) FISH. Results: All pts engrafted; median time to ANC of 0.5 x 109/l was 20 days (range, 11-29 days) and median time to self 9 Since the results of bone marrow transplantation (BMT) from sustained platelet count of 20 x 10 /l was 31 days (range, 16- matched unrelated donors (MUD) have improved in severe 68 days). Donor chimerism ranged from 59 % to 100 %. The 5 aplastic anemia (SAA), BMT from MUD has become an year probability of overall survival (OS) and event free survival accepted second line therapy for children not responding to (EFS) for all pts was 94% and 77% respectively. The 5-year the first course of immunosuppressive therapy (IST). probability of OS and EFS for pts with mild disease was 96% However, non-engraftment and early rejection are still and 83% respectively and for pts with moderate and severe important reasons for treatment failure and death. Because of disease the 5-year probability of OS and EFS was 89% and the high risk for lethal bacterial or fungal infections, immediate 73% respectively. All pts with hepatitis C had stable liver re-transplantation must be considered. In this situation the condition post SCT and remain alive; with only one graft conditioning regimen should be highly immunoablative to rejection. Pts who had second SCT remain alive and free of allow for engraftment but less toxic to prevent acute tissue disease at (271, 503, and 1449 days post SCT for the 3 pts. damage and consecutive increase of the risk for graft-versus The incidence of grade III-IV GvHD was 25% in pts with host disease (GVHD). severe disease, 20% in pts with moderate disease and 13% We report on three patients (pts.) who developed SAA at age for pts mild disease, graft rejection rate was 25% in pts with 7, 11 and 17 years and underwent MUD BMT 12, 94 and 120 severe disease and 13 % in pts with mild disease. Conclusion: months after 1 – 2 unsuccessful courses of combined IST with The outcome of allogeneic SCT at KFSHRC is satisfactory.

S190 SCT at a younger age with mild disease offers the best Inborn errors of metabolism outcome. More advanced disease is associated with a higher rate of rejection and severe GVHD. Conditioning with CY/ TBI appears adequate in pts undergoing second SCT. Patients with Hepatitis C and stable liver status have a favorable P782 outcome. Haematopoietic stem cell transplantation in patients with cartilage hair hypoplasia V. Bordon (1), A. Gennery (2), M. Slatter (2), E. Vandecruys P781 (1), T. Güngör (3) Unusual late rejection of the first graft seven years after (1)Ghent University Hospital (Ghent, B); (2)University of allogeneic transplant for thalassaemia successfully Newcastle upon Tyne (Newcastle upon Tyne, UK); treated with second BMT (3)University Children's Hospital (Zurich, CH) C. Giardini (1), G. Visani (1), M. Lucesole (1), G. Leopardi (1), G. Sparaventi (1), F. D'Adamo (1), G. Nicolini (1), B. Guiducci Cartilage Hair Hypoplasia (CHH) is a rare autosomal (1), S. Barulli (1), L. Malerba (1), A. Isidori (1), M. Manna (1), recessive disease, caused by mutations in the RMPR gene, M.G. Romeo (2) characterised by prenatal onset of growth failure with (1)Osp. S.Salvatore (Pesaro, I); (2)Pediatria Università di metaphyseal chrondrodysplasia and different grades of Catania (Catania, I) immunodeficiency. Haematopoietic Stem Cell Transplantation (HSCT) has been suggested for rare patients presenting with We report an unusual case of late rejection with autologous severe immunodeficiency. reconstitution after BMT for thalassemia. A three years old Here, we summarise the indications, complications, outcome female patient was submitted to allogeneic bone marrow and immune reconstitution in six patients from 3 centres after transplantation on February 1998 for Thalassemia major HSCT. See table for the details. (Class of risk 1) from her HLA identical brother. Conditioning All patients received Busulfan-based conditioning and replete regimen consisted of Busulfan 16 mg/kg and marrow. All achieved full donor chimerism. Cyclophosphamide 200 mg/kg. The post-transplant course Normal T and B cell numbers were achieved. T-cell function was uncomplicated, with only a grade 1 skin acute GvHD. with normalization of mitogen- and antigen-specific After hematological reconstitution, sexual chromosome lymphocyte stimulation was seen in all patients. marker has been repeatedly studied to follow an incomplete Immunoglobulin levels were normal in 2 patients, whereas 3 donor engraftment, with a persistent and increasing patients exhibit continuous absent production of IgA. Specific percentage of recipient cells documented on bone marrow. antibody production against protein and polysaccharide Despite this progressive decrement of donor cells percentage, antigens has normalized in all 5 surviving patients. the patient maintained a Hemoglobin level between 10 to 11 Autoimmune phenomena disappeared after successful HSCT. and she was transfusion-free and free of symptoms of the As yet, no secondary malignancy has been observed. genetic disease until late 2005, when the patient was again We conclude that HSCT treatment is successful at correcting referred to us for progressive anemization. At that time, the the immunodeficiency in patients with CHH. These patients donor chimerism was found to be only 5% and the patient may have immunological investigations that are reassuringly became again transfusion-dependent. normal, but experience infections that are more significant and On May 2006 the patient has been submitted to a second serious than the immunological function would suggest. transplant using the same HLA identical family donor. The Careful clinical evaluation is necessary, and in patients with conditioning regimen consisted on: THIOTHEPA 8 mg/kg, clinical evidence of immunodeficiency, HSCT should be TREOSULFAN 14 g/mq/day for 3 days, FLUDARABINE 40 performed before severe organ damage, severe autoimmune mg/mq/day for 3 days. The post-transplant course was again phenomena or malignancy occur to avoid-transplant related uncomplicated with no evidence of acute GvHD. The post- mortality. transplant chimerism study showed: Day +21: 100% donor cells; 6 months after BMT: 92% donor cells. The patient is continuing the post-transplant follow-up and Cyclosporine tapering. Conclusions: This is the latest rejection with autologous reconstitution ever documented after a transplant in Thalassemia. The progressive loss of the allogeneic graft is really unusual even in mixed chimeras, where the situation of mixed chimerism is generally considered to be “persistent” and stable after one year of post-transplant follow-up. Although the follow-up after the second BMT is still short, the conditioning regimen employed (without the use of Cyclophosphamide) leads to eradication of the thalassemic marrow without significant organ toxicity and is considered promising for further clinical investigation.

P783 Biochemical monitoring after haemopoietic stem cell transplant for Hurler syndrome and implications for funtional outcome after transplant in metabolic disease J. Ruell (1), H. Church (1), T. Carr (1), A. O'Meara (2), R. Wynn (1) (1)Royal Manchester Children's Hospital (Manchester, UK); (2)Our Lady's Hospital for Sick Children (Dublin, IRL)

Hurler Syndrome is corrected by allogeneic BMT by the action of donor enzyme on recipient tissue. In this paper we describe monitoring of 39 patients transplanted in 2 centres to determine donor chimerism, enzyme level and residual

S191 substrate – expressed as dermatan sulphate to chondroitin P785 sulphate ratio. We show that in fully engrafted recipients the Comparison of immune reconstitution following anti- enzyme level, expressed as µmol/g total protein/hr, post CD52 T-cell depleted haematopoietic stem cell transplant transplant is 24.2 from an unrelated donor and 10.2 from a versus CD34+ positive stem cell selection for SCID in a heterozygote family donor (P<0.0001). There is a tight single centre relationship between mean post-transplant enzyme level and M. Slatter (1), K. Brigham (2), D. Barge (2), A. Jackson (3), T. residual substrate – Spearman’s rank correlation coefficient Flood (1), A. Cant (1), M. Abinun (1), A. Gennery (1) (Rho) was -0.76 and -0.80 at 12 and 24 months respectively (1)Newcastle General Hospital (Newcastle upon Tyne, UK); (P<0.0001). We propose that these differences affect patient (2)Royal Victoria Infirmary (Newcastle upon Tyne, UK); outcome. As unrelated donor transplant outcomes improve (3)International Centre for Life (Newcastle upon Tyne, UK) and especially given the higher levels of donor cell engraftment following cord transplants our data might Introduction: Severe combined immunodeficiencies (SCID) influence donor selection. are primary T cell differentiation defects with/out defects in B and NK cells. Results from treatment by haematopoietic stem cell transplantation (HSCT) have improved over the past two P784 decades due to better diagnosis, stem cell sources, Haematopoietic stem cell transplantation for children with chemotherapy conditioning regimens and new methods for purine nucleoside phosphorylase deficiency is feasible detecting and treating viral and fungal infection. For many and successful by using low-toxicity conditioning patients without an HLA-identical relative or unrelated donor, regimens and alternative donors T cell depleted (TCD) haplo-identical family transplants have T. Güngör (1), C. Ehlert (2), U. Duffner (3), F. Scherer (1), J. been successful with survival rates of 80-90% over the last 5 Vormoor (2), C. Niemeyer (3), E. Boltshauser (1), R. Seger years. Methods to remove viable T cells from the graft include (1), P. Veys (4), G. Davies (4) addition of anti-CD52 antibodies and, more recently, CD34+ (1)University Children's Hospital (Zurich, CH); (2)University positive HSC-selection with magnetic bead-labelled Children's Hospital (Muenster, D); (3)University Children's antibodies. We have previously presented results of long-term Hospital (Freiburg, D); (4)Great Ormond Street Children's follow up of children who received anti-CD52 TCD marrow Hospital (London, UK) between 1987- 19981. We compared the quality of engraftment in these patients with more recent patients who The prognosis of children with purine nucleoside received CD34+ positive HSC-selected product. phosphorylase (PNP) deficiency after HSCT is poor because Methods: Survivors with SCID who received CD52 TCD or of a high incidence of transplant-related lethal complications CD34+ HSC-selected HSCT were reviewed. Chimerism, T and rejection. Five children (1-6 years) with proven PNP- and B cell subsets, and immunoglobulin level at last follow up, deficiency (with mild to moderate neurological symptoms), were examined. The non-parametric Wilcoxon rank sum and opportunistic viral infections, or autoimmune disease (AIHA, the Kruskal-Wallis one-way ANOVA analyses were used to ITP) received reduced toxicity (n=4) or myeloablative (n=1) determine differences between groups. conditioning regimens using alternative donors. In 3 of 5 Results: 19 patients received CD52-treated, 19 received patients, reduced intensity conditioning regimens comprised CD34-treated HSC. There were no statistically significant fludarabine (5 x 30 mg/qm), melphalan (140 mg/qm) and in differences in myeloid chimerism, T cell numbers, new thymic vivo T-cell depletion with rabbit ATG-Genzyme (4 x 5 mg/kg) emigrants, B cell or memory B cell numbers. There was no or Campath IH (5 x 0.2 mg/kg) followed by transplantation of significant difference in donor B cell chimerism between the maternal HLA-phenoidentical BM (pat. 1), haploidentical T-cell groups. The only significant difference was that more patients depleted (pat. 2) and paternal HLA phenoidentical PBSC (pat. who received CD52-treated HSC had IgG class-switched B 5). The second low-toxicity conditioning regimen used, cells (p=0.023) and discontinued IVIG (0.049) than CD34- consisted of intravenous busulfan (13.2 mg/kg), treated patients. More patients with common gamma chain cyclophosphamide (120 mg/kg), rabbit ATG-Fresenius (40 (CgC) or JAK3 SCID in the CD52-treated group had donor B mg/kg) and PBSC from a MUD donor (pat. 3). Full cells (4/5) than similar patients in the CD34-treated group myeloablative conditioning with busulfan (16 mg/kg), (1/10), although patient numbers were small (p=0.054). cyclophosphamide (200 mg/kg) together with in vitro T cell Conclusion: There appear to be no long-term differences in depletion of a MUD BM transplant using Campath 1M (with T immune reconstitution or chimerism between patients cell addback) and in vivo T-cell depletion using Campath IG receiving CD52- or CD34-treated HSC. Differences in B cell was used in patient 4. After timely hematopoietic engraftment function/IVIG requirement are likely due to uneven patient mix and completed T-cell reconstitution, persisting viral and between groups, with more CgC/JAK3 patients in the CD34- autoimmune diseases resolved. Because of decreasing donor treated group, most having dysfunctional recipient B cells. chimerism, patient 1 received a single DLI infusion; while 1. Gennery AR, et al. Cytotherapy. 2001;3:221-32. patient 2 required boosting with haploidentical stem cells (SCB) after a single dose of melphalan (140 mg/qm). All patients achieved full long-term donor chimerism except P786 patient 4 who shows stable mixed chimerism (23 % donor) 9 Comprehensive airway evaluation and management in years after MUD transplant. Patient 5 developed GVHD grade children with malignant infantile osteopetrosis III of the gut with good response to therapy. After a follow-up K. Kasow (1), R.M. Stocks (2), S. Kaste (1), R. Schoumacher period of 54-171 months, all patients are alive and well without (2), E. Horwitz (1) signs of chronic GHVD and with completed T- and B-cell (1)St. Jude Children's Research Hospital (Memphis, USA); immune reconstitution. PNP activity of red cells is normal in all (2)University of Tennessee Health Sciences (Memphis, USA) patients except in patient 4 with pre BMT levels in red cells but normal levels in leukocytes. Low toxicity conditioning Objective: Malignant infantile osteopetrosis (MIOP) is a rare, regimens using alternative donors are feasible in high-risk autosomal recessive inherited disorder caused by children with PNP-deficiency. Autologous reconstitution still dysfunctional osteoclasts, leading to thickened bony remains a complicating factor, however DLI or SCB therapy trabeculae, decreased bone marrow space, and eventually are appropriate to induce long-term sustained full donor bone marrow failure. Clinically, MIOP children have classic chimerism. Importantly, neurological PNP-related disease did facial features, such as macrocephaly, frontal bossing and not progress after HSCT. microagnathia, and a small thorax, all due to the lack of bone remodeling. These features may potentially place these children at significant risk for developing obstructive sleep apnea (OSA), chronic hypoxemia, and pulmonary hypertension. Therefore, to objectively document OSA and determine the extent, a comprehensive airway evaluation was performed with results impacting management.

S192 Methods: We retrospectively reviewed the medical records of FK506 and MTX, but MTX was quitted after the second 7 patients with MIOP treated at St. Jude. To evaluate for administration since it caused severe hepatic toxicity. OSA, 6 children underwent polysomnograms during the pre- Engraftment was documented on day +19, with an absolute hematopoietic stem cell transplantation (HSCT) evaluation; neutrophil count greater than 0.5 x 109 cells/L. Following one patient had the initial polysomnogram 3 years after HSCT. uneventful engraftment with just grade 1 acute GvHD, NEMO To determine the existence of a relationship between OSA protein expression was normalized and cytokine response and radiological imaging, initial chest radiographs were given was significantly improved. Lineage-specific genetic analysis a severity score by a single radiologist and bone mineral confirmed full donor chimerism. Ten months after UCBT, the density (BMD) was determined by QCT in 5 children and patient is in perfect performance status. This outcome shows resulted as a Z-score. that correction of immunodeficiency associated with NEMO Results: Pre-HSCT patients had a median respiratory mutation is possible by SCT with a reduced-intensity regimen disturbance index (RDI) of 17.51 (range, 6.67 -25.28; normal and UCB can be a suitable alternative source for affected 0-2), indicating OSA, and a median minimal oxygen saturation patients. of 79% (range, 68%-88%), indicating intermittent hypoxemia. For those who had BMD studies, the median Z-score was 27.3 (range, 22.4-32.6). Neither the chest radiograph score P788 nor the BMD correlated with the severity of the OSA Correction of toxic accumulation of thymidine and measured by the RDI. Four patients received tracheostomies deoxyuridine in a patient with mitochondrial either prior to or during the peri-HSCT period, and 2 neurogastrointestinal encephalopathy by allogeneic underwent tonsillectomy/ adenoidectomy pre-HSCT. Three haematopoietic stem cell transplantation surviving children underwent polysomnograms at least a year R. Elhasid (1), I. Zaidman (1), N. Levi (1), N. Chamaysi (2), M. post-HSCT and had a median RDI of 1.3 (range, 0.46-2.3), Ben Arush (1), J.M. Rowe (1), H. Mandel (1) indicating near to complete resolution of OSA. (1)Rambam Medical Center (Haifa, IL); (2)Clalit Health Conclusions: Using a quantitative approach, our study Services (Caffar Cana, IL) demonstrates that MIOP children have significant OSA and hypoxemia, which is unrelated to their BMD. Upon successful Background: Deficiency of thymidine phosphorylase (TP) HSCT, OSA resolves but these children may benefit from results in progressive fatal mitochondrial neurogastrointestinal aggressive airway management prior to HSCT. Severe OSA is encephalopathy (MNGIE) disease, clinically defined by a not a contraindication for HSCT in MIOP children, but gastrointestinal dysmotility, cachexia, peripheral neuropathy, aggressive airway evaluation and management pre-HSCT white-matter changes in brain MRI. The cause for this disease may reduce the risk of life-threatening pulmonary is believed to be the toxic accumulation of thymidine and complications known to occur during HSCT. deoxyuridine which generate imbalanced mitochondrial deoxyribonucleoside triphopahte pool, which in turn is responsible for the mitochondrial DNA (mtDNA) mutagenesis P787 and depletion. The appeal of TP deficiency as a candidate Correction of immunodeficiency associated with NEMO disease for curative hematopoietic stem cell transplantation mutation by unrelated cord blood transplantation using a (HSCT) arose from the clinical observation that infusions of reduced-intensity conditioning regimen platelets, which contain abundant functional TP, reduced C. Tono (1), Y. Takahashi (1), K. Terui (1), S. Sasaki (1), T. circulating levels of thymidine and deoxyuridine in 3 patients. Toki (1), N. Tachibana (2), T. Yoshioka (3), T. Nakahata (3), Methods and results: A 10-year old patient with MNGIE T. Morio (4), R. Nishikomori (3), E. Ito (1) disease underwent HSCT from her HLA identical mother. (1)Hirosaki University School of Medicine (Aomori, JP); Conditioning regimen included: busulphan 16mg/kg, (2)Aomori Central Hospital (Aomori, JP); (3)Kyoto University cyclophosphamide 200mg/kg, anti thymocyte globulin (ATG, (Kyoto, JP); (4)Tokyo Medical and Dental University (Tokyo, Fresenius) 25mg/kg, and fludarabine 200mg/kg. In order to JP) prevent graft-versus-host disease (GVHD), moderate T cell depletion, giving 105/kg donor T cells, was performed using X-linked recessive anhidrotic ectodermal dysplasia with positive selection of CD34 cells by immunomagnetic beads immunodeficiency (XL-EDA-ID) is a developmental and (Miltenyi Biotech). On day 0 she received 10.5 x 106/kg CD34 immunologic disorder caused by hypomorphic mutations in cells and 105/kg T cells. No post-transplant the nuclear factor-k B essential modulator (NEMO) gene. immunossuppression was given. Engraftment of leukocytes Affected boys are susceptible to infections with various occurred on day +8 post transplantation. Last platelet microorganisms, mostly pyogenic bacteria and mycobacteria. transfusion was given on day +9 post transplant. Concomitant Stem cell transplantation (SCT) is thought to be the only with engraftment, 12 days post transplantation, the curative treatment, but successful SCT has not yet been concentration of serum thymidine and deoxyuridine declined reported, one of the probable reasons was concern for markedly, and became undetectable 27 days following regimen related toxicity due to their increase in apoptosis. We transplantation. Six months later signs of cachexia here report the first successful treatment of the disorder by disappeared and she gained 9kg. Acute GVHD grade II SCT. The patient was a 3-year-old boy, born from mother with (eosinophilia, skin rash and mild diarrhea) occurred 40 days mild incontinentia pigmenti. He had frequent episodes of post transplantation and was treated with prednisone 2mg/kg bacterial enterocolitis and sepsis. Mutation analysis revealed with prompt response. Later she developed chronic GVHD NEMO mutation (1167 insC) in both the patient and his treated with cyclosporine, prednisone and photopheresis with mother. Flow cytometry analysis showed lower NEMO improvement. She is now 15 months post transplantation with expression in each PBMC lineage, and CD40L induced less stable GVHD and complete donor chimerism. up-regulation of CD23, CD54, CD86, and CD95 in the Conclusion: These results constitute a proof of concept that patient's cells. The patient did not have an HLA-matched restoration of TP activity by HSCT can ameliorate biochemical related or unrelated bone marrow donor. At 2.5 years of age, imbalances that cause the disease. The weight gain could he suffered severe pyloric stenosis following gastric ulcers indicate improvement in gastrointestinal function. Close and Candida glabrata sepsis. For this life threatening episode, clinical follow-up is needed to evaluate the long-term, multi- an unrelated cord blood transplantation (UCBT) was systemic efficacy of this treatment. performed at 3 years of age after written consent was obtained from his parents. Pretransplant conditioning consisted of fludarabin (30 mg/m²/d) from day -7 to day -3, melpharan (70 mg/m²/d) from day -6 to day -5, and rabbit anti- T-lymphocyte globulin (ATG) (2.5 mg/kg/d) from day -5 to day -1. The patient received 4.6 x 107 total nucleated cells/kg from a male donor. The recipient and the donor were one antigen mismatched by serology. GvHD-prophylaxis was planned with

S193 P789 MPS I Registry: baseline description of 10 years of experience with haematological stem cell transplantations in MPS I J.E. Wraith on behalf of the MPS I Registry European Board of Advisors

The MPS I Registry (www.mpsiregistry.com) is an observational, voluntary registry program initiated in October 2003, to track the clinical onset, symptoms and outcomes of patients with a confirmed diagnosis of Mucopolysaccharidosis type I (MPS I), regardless of disease severity or treatment status. As of June 2006, 491 MPS I patients were enrolled in the Registry with 110 (26%) reporting to ever having received Haematological Stem Cell Transplantation (HSCT) and 85 (77%) reported a date for HSCT within the selected time interval of 1995 to 2006. Of these 85 patients, 27 (32%) also received enzyme replacement therapy, and 22 patients with a mean time of 5 months prior to HSCT. Seventy four (87%) of the 85 patients reported a 1st transplant prior to age 2, with 23 (27%) reporting a transplant before the age of 1 and 51 (60%) reporting a transplant between age 1 and 2. The remaining 11(13%) patients were older than 2 at the time of transplant. The mean time from 1st enzyme replacement therapy infusion P790 to 1st transplant was 5 months with a median of 2 months, Effects of haematopoietic stem cell transplantation on ranging from 1 month to 2.7 years (the patient with a time of cardiac function of children with mucopolysaccharidosis 2.7 years did not receive HSCT until age 5). I-H The overall mean for age at 1st transplant was 1.5 years, with P. Corti (1), A. Rovelli (1), R. Parini (1), G. Sersale (1), F. a median of 1.3 years, ranging from 2 months to 5.4 years. Furlan (1), F. Santus (1), C. Uderzo (1), A. Balduzzi (1), P.L. The most frequently reported HSCT type was “Bone Marrow” Russo (1), V. Fesslova (2) with 52 (61%) patients and the “Cord Blood” type was (1)University Milan-Bicocca (Monza, I); (2)Policlinico San reported in 30 patients (36%). Donato (Milan, I) Fiftysix out of 85 patients reported an engraftment outcome; 36 (64%) reported a “Full Engraftment”, 8 (14%) reported a Cardiac involvement is common in mucopolysaccharidosis I-H “Failed Engraftment”, 6 (10%) reported a “Partial (MPS I-H). In untreated patients it contributes to the Engraftment”, and 6 (10%) reported “Unknown”. relentlessy lethal course of the disease. A first baseline overview was provided of the data on HSCT Eight children (1 M, 7 F), diagnosed with MPS I-H (median currently available for MPS I over the time interval of 1995 to age 1.2 yrs, range 0.5-1.5), were referred to our center for 2006. As HSCT data in the MPS I Registry accumulate, HCT. They underwent echocardiography at baseline and at potential trends may become visible. regular intervals thereafter. A scoring system that combined echocardiogram findings was used to determine cardiac severity scores (3-point scale for each cardiac anomaly: 1 mild, 2 moderate, 3 severe) at baseline and at latest evaluation. Cardiac involvement was present in 6 pts. before HCT (requiring standard cardiac medications in 4) with a cardiac severity score ranging from 1 to 3.5. The most common finding was involvement of the mitral valve (present in 6), often associated with aortic valve insufficiency (in 5), asymmetric septal hypertrophy or hypertrophic cardiomyopathy (in 3), pericardial effusion (in 2) and tricuspid valve involvement (in 1). Median age at HCT was 1.7 yrs (range 1.2-2.5); the 2 most recent cases received enzymatic replacement therapy from diagnosis to 1 month after HCT, when normal endogenous enzymatic activity was detected. Conditioning regimen was BuCy and ATG; fludarabine was added in 3 cases (2 of them at 2nd HCT). HCT was performed in all, but one, cases from an unrelated donor (URD). Two patients, after graft rejection (8 and 11 months after HCT), successfully underwent a 2nd URD-HCT (13 and 20 months after the first transplant). One patient died 1 year after HCT from a matched family donor, for immune complications during rejection. Normal enzymatic activity level and stable complete (in 6) or partial (in 1) chimerism have been periodically documented in the 7 survivors. The follow-up after HCT is too short in 2 patients for assessing HCT long-term impact on heart function; in the other 5 patient (median age 7.9 yrs, range 7.0-8.5; median post-HCT follow-up 6.2 yrs, range 5.4- 6.9), cardiac impairment continued to mildly progress over the first 2 years following HCT (median cardiac severity score 4.5, range 3.5-5.5), but then stabilised in 3 and even improved in 2 cases (median cardiac severity score 4.0, range 1.5-4.5); at the latest follow-up only 2 patients require cardiac medications.

S194 HCT probably modifies the expected natural progressive iduronidase or mannosidase activity, respectively The course of cardiac abnormalities in children with MPS I-H. preparative regimen consisted of busulfan, fludarabine and anti-thymocyte globuline. For MMFD and MUD transplantations, melphalan was added. P791 1 patient was transplanted with full bone marrow from an HLA- Role of haematopoietic stem cell transplantation in identical sibling donor, GVHD-prophylaxis was CSA+MTX. 3 children with life-threatening metabolic disorders patients were transplanted from their haploidentical mothers J. Motwani, S.E. Lawson, M. Velangi, P.J. Darbyshire and 5 from unrelated donors, all received CD-34+-selected Birmingham Children's Hospital, UK (Birmingham, UK) PBSC (CliniMACS, Miltenyi Biotech), mean CD34+ cell doses were 37.3 x 106/kg (range 10,0-54,8), residual CD3+ cell We present a single-centre experience of haemopoietic stem content was 3.8 x 104/kg (range 0,0-5.4). 7 patients had no cell transplantation (HSCT) in patients with metabolic post-transplant immunosuppression, 1 received MMF because disorders. of a residual CD3+ cell number of > 5 x 104/kg. 19 children with various metabolic disorders (osteopetrosis-8, Results: All children are alive 3 months to 5 years after SCT Adrenoleukodystrophy(ALD)-5, congenital erythropoietic and show donor-derived hematopoiesis. PMN > 0.5/nl was porphyria-1, Hurler’s syndrome-2, Mannosidosis-2, Glycogen seen after 15.3 days (range 10-21), platelet counts > 50/nl storage disease-1) received HSCT between 1991-2006(14 were reached after 16.5 days (range 9-25). The patient with between 2000-2006). There were 14 males and 5 females. MAN rejected and required re-transplantation and a T-cell Median age at HSCT was 18 months (range 2 months to 13 boost. No GvHD > grade I occurred, no toxicity > grade II was years). 6 had matched unrelated donor graft, 1 had seen in the MPS1H-patients, the MAN patient required haploidentical PBSC graft, which was TCell depleted (TCD) intensive care for a systemic inflammatory response during his before re-infusion & 12 received HSCT from matched family graft rejection. 1 MPS1H patient developed a subdural donors, parent-6, siblings-4, sibling cord-2. Most received hematoma on day 6 after SCT necessitating surgical removal. similar conditioning regimen: Fludarabine, Busulphan In 6 children, complete donor chimerism was achieved; 3 &cyclophosphamide .3 patients who had multiple HSCT patients have mixed chimerism. In 3 patients, donor- received slightly different conditioning regimen. lymphocyte infusions (DLI) were given to stabilize or convert None of our patients had any conditioning related toxicity. All mixed to full chimerism. engrafted within 2-3 weeks post-HSCT. 3 patients rejected Conclusion: SCT is the therapy of choice in children with their first HSCT between 1-8 weeks post engraftment. 2 had a MPS1H. This follow-up shows that a fludarabine based second successful transplant from the same donor. 1 patient conditioning regimen in combination with a CD34-selected had multiple HSCT, and presently is well 2 years post his graft is safe, non-toxic and effective in achieving stable successful HSCT. 1 patient who received haploidentical PBSC engraftment in MPS-1H. More patients are needed to answer from mother, became mixed chimaeric but became fully donor the question whether these results can be reproduced in chimaeric following use of fludarabine and DLI. Our observed MAN-patients. rejection rate is 15%. 15 alive patients are mainly/fully donor chimaeric and well after a median follow up of 4 years (range 5 months-15 yrs). 4 patients died (ALD-2, osteopetrosis-2) P793 within 6-16 weeks post transplant due to disease progression Successful haematopoietic stem cell transplantation with (ALD) and infection related complications. Mortality was reduced-intensity conditioning in a child with combined observed more frequently amongst the older patients. immuno-deficiency and multiple opportunistic infections Children who died were aged 3 months, 8, 10 and 13 years V. Mialou (1), J.M. André (1), C. Galambrun (1), K. Kébaïli (1), each.6 of 18 (33%) had grade 2-4 graft versus host disease N. Bleyzac (1), F. Najioullah (2), C. Malcus (3), V. Dubois (4), (GVHD) involving skin, gut, liver or a combination. Steroids Y. Bertrand (1) were used to treat GVHD in all patients, and in 2 patients (1)Hopital Debrousse (Lyon, F); (2)Faculté Rockefeller (Lyon, immunosuppressive therapy was changed from cyclosporin to F); (3)Hopital P. Wertheimer (Lyon, F); (4)Etablissement tacrolimus. All 15 patients alive are also disease free with Français du Sang (Lyon, F) resolution/improvement/stabilisation of preHSCT disease related symptoms including normalisation of blood counts, Haematopoietic stem cell transplant (HSCT) is the best reduced infections and progression in previous delayed therapy to cure children with combined immunodeficiency. development. However, previous opportunistic infection and organ Conclusion: Our single centre experience shows that HSCT dysfunction can be a barrier to the success of the transplant even with alternative donors, is successful in metabolic procedure with high infectious and toxic mortality. disorders . Early diagnosis and early transplantation with Methods: A 9-year-old boy presented in early childhood with specialists in metabolic disorders involved in decision making Hyper IgM syndrome without CD40 ligand deficiency. Despite will probably result with best outcomes. immunoglobulin substitution, continuous prophylactic and curative antibiotics, he presented with many complications consisting of bronchiectasis, severe cryptosporidiosis with P792 admission in intensive care unit for 15 days, asymptomatic T-cell depleted haematopoetic stem cell transplantation viral infection with CMV and EBV (increasing viral load) and for children with lysosomal storage diseases: a follow-up adenovirus gut infection and severe immunologic deficiency M. Sauer (1), L. Grigull (1), T. Lücke (1), A. Das (1), B. (with CD3+:1100/µl, CD4+: 200/µl and IgM: 1.02 g/l) Maecker (1), B. Meissner (1), K. Welte (1), D. Fuchs (2), M. There was no HLA identical familial donor and due to the poor Suttorp (3), K.W. Sykora (1) general condition we decided to peform an unrelated HSCT (1)Hannover Medical University (Hannover, D); (2)Friedrich- with a 4/6 HLA cord blood and reduced intensity conditioning Schiller-University (Jena, D); (3)Technical University Dresden with Fludarabine (150 mg/m²), melphalan (140 mg/m²) and (Dresden, D) Anti-thymocytes globulin (10 mg/Kg). Cyclosporine and corticosteroids were used as GVH prophylaxis. Allogenic stem cell transplantation (SCT) can provide a life- Anti-infectious prophylaxis consisted in a laminar airflow room, long correction of the enzymatic deficiency in lysosomal bactrim as pneumocystis prophylaxis, gut decontamination, storage diseases (LSD). Here, we report the follow-up on nine weekly Ig substitution, cidofovir to prevent CMV and children with LSD transplanted with a fludarabine based adenovirus infection and prophylactic anti-CD20 to prevent conditioning regimen that includes T-cell depletion for all MUD EBV lymphoproliferative disease. Cryptosporidiosis was and MMFD transplantations (Grigull et al. BMT 2005). treated with nitazoxanide and azithromycine. Patients: 8 patients had Hurler’s disease (MPS1H) and one Results: Haematologic recovery occured at day 30 with a alpha-Mannosidosis (MAN). By the time of SCT the children complete chimerism (100% donors cells) at day 32. There was were 10 months to 3.75 years old. MPS1H and MAN were no metablic or toxic complication and the HSCT was only diagnosed clinically and by reduced leukocyte alpha- complicated only by a CMV reactivation (increased viral load)

S195 which was treated by switch from cidofavir to foscarnet and Early side effects / Late effects and ganciclovir. Six months after HSCT this boy was alive and well, without quality of life any infectious disease. Treatment for cryptosporidiosis was withdrawn. Immune reconstitution status showed CD3+: 700/µl, CD4+: 150/µl and CD19+ B cell count at 250/µl) with a P795 100% donor chimerism. Clinical characteristics of early and late non-infectious Conclusion: The prophylactic use of anti-viral and anti-CD20 pulmonary complication following cord blood injection can avoid the risk of severe viral reactivation in a transplantation very immunocompromised child during a HSCT procedure A. Wake, N. Uchida, S. Takagi, D. Kato, H. Yamamoto, Y. with a RIC regimen and a cord blood transplant. Matsuhashi, S. Seo, N. Matsuno, K. Masuoka, S. Miyakoshi, A. Yoneyama, S. Taniguchi Toranomon Hospital (Tokyo, JP) P794 Long-term immune reconstitution analysis in MHC class II Objectives: Non-infectious pulmonary complications (NIPC) deficiency patients after haematopoietic stem cell are fatal disorders following allogeneic SCT. NIPCs includes transplantation idiopathic pneumonia syndrome (IPS), which occurs early C. Malcus (1), F. Poitevin-Later (1), V. Mialou (2), V. Dubois period, and late onset NIPCs (LONIPCs) such as bronchiolitis (3), G. Souillet (2), F. Touraine-Moulin (1), Y. Bertrand (2) obliterans (BO), cryptogenic organizing pneumonia (COP), (1)Hôpital Neurologique, GHE (Bron, F); (2)Hôpital Debrousse and interstitial pneumonia (IP). There has been little (Lyon, F); (3)EFS Gerland (Lyon, F) information on NIPCs following cord blood transplantation (CBT). We tried to characterize NIPCs after CBT by Objectives: MHC class II deficiency (MHCII-Def) is a severe estimating the incidence, types of NIPC, and prognosis, and to immunodeficiency characterized by deficient T helper cell- identify risk factors for them. dependent immunity. The curative treatment of this Methods: We reviewed medical records of 223 patients with immunodeficiency consists of haematopoietic stem cell hematological diseases who had received CBT between Jan. transplantation (HSCT). However, the success rate of early 2002 and Dec. 2005 at Toranomon Hospital, Tokyo, Japan. immunological recovery in this disease is poor. Moreover, it Median age was 55 years (17 E9). Most of them had has been suggested that T-cell immunity may be impaired diseases in advanced status (n=198). Median follow-up time later in life because of long-term graft failure or accelerated was 601 days (111-1544). Most of our pre-transplant loss of thymus output. To address this issue, we compared conditioning were reduced intensity consisted of fludarabine, immunological parameters of 2 MHCII-Def sibling patients (P1 melphalan and TBI 4Gy (n=189). Cyclosporine (CSP) (n=97) and P2) with those of their HLA-identical sibling donors (D1 or tacrolimus (Tac) alone (n=126) were used as GVHD and D2). One of the patients (P2) presented long-term prophylaxis. HLA disparities were as follows; 6/6 (n=6), 5/6 impaired immune reconstitution with autoimmune disease (n=47), 4/6 (n=174), and 3/6 (n=2). Diagnosis was made by (Evans’s syndrome). During the late follow-up care, P2 was Chest X-ray, computed tomography, broncho-alveolar lavage treated by anti-CD20. and pulmonary function test. Methods: P1 and P2 received respectively bone marrow and Results: The incidence of IPS was 20.6% (n=46), with median cord blood transplantation from HLA-identical sibling donors onset of 17 days post-transplant (2-57). Among IPS patients, D1 and D2, in 1998 and 1995 after a conditioning regimen 17 cases were accompanied by pleural effusion. LONIPCs consisting of Busulfan and cyclophosphamide. Eight and were observed in 13 cases (5.8%), including 3 cases of COP, eleven years after HSCT, chimerism (in P1 and P2), 8 cases of IP, and 2 cases of BO. Multivariate analysis qualitative and quantitative T receptor Vbeta family revealed 3 risk factors for NIPC; GVHD prophylaxis with CSP complementary-determining region 3 sequence mRNA over Tac (p=0.036, RR 1.65), grade III-IV acute GVHD over (TCRV-b CDR) analysis (TcLandscape®), lymphocyte grade 0-II (p=0.007, RR 1.84), and 2 and greater HLA subtypes (T, B, NK, CD45RA, CD31, CD45RO, DR, flow mismatch over less than 2 (p=0.046, RR 1.587). Overall cytometry FC500® Beckman-Coulter) and lymphocyte specific survival (OS) of the patients with IPS was 22.1 % at day100 proliferation were assessed in P1, P2, D1 and D2. despite high dose steroid therapy. Interestingly, day100 OS of Results: The chimerism analysis revealed that P1 cells were IPS with pleural effusion (PE) was significantly better than all of donor origin whereas in P2, cells were mixed those without PE (44.2% and 10.0%, p=0.003). Cases with recipient/donor origin (70% cells from donor and 8% CD3 cells LONIPCs responded favorably to low dose steroid therapy. from the recipient). P1 and P2 had quasi-normal standard Among LONIPC patients, those who had LONIPC later than lymphocyte subset values and normal MHCII expression but day 100 post-transplant (n=3), no one required long-term P2 had no B cell and his specific proliferative T cell response oxygen therapy or died directly from respiratory failure due to was not observed. In P2, TCRV-b CDR was more skewed the lung diseases. (6/26 altered V-b families vs. P1 2/26, D1 3/26 and D2 1/26) Conclusion: The incidence of IPS was relatively high, whereas and less up-regulated (1/26 V-b families vs. P1 5/26, D1 3/26 that of LONIPCs was low after reduced-intensity CBT. CBT and P2 6/26). Moreover recent thymic emigrant (RTE) CD4 may reduce the late pulmonary complication and improve the cells (CD31+CD45RA+CD4+) were deeply decreased in P2 quality of life in hematological patients. compared with P1, D1 and D2 RTE (2% vs. 17%, 41% and 31% respectively). Conclusion: Chimerism analysis showed different patterns of P796 engraftment in the two patients. Slight alterations of Growth hormone, IGF-I, IGFBP3 and growth after stem immunological parameters were found in both patients P1 and cell transplantation in children and adolescents P2 compared with sibling donors. But only, the very low P. Schwarze thymic lymphocyte output evaluated by RTE CD4+ cells could University Childrens Hospital (Tuebingen, D) account for the worse long term immune reconstitution observed in P2 patient. Objective: To study growth hormone status, IGF-I and IGFBP- 3 levels and growth before and after stem cell transplantation and to gain insight in possible interaction and regulation. Methods: 416 patients, 168 (40%) female and 248 (60%) male patients aged 8.4 years (median, range 0.1 to 22.6) were registered. According to a standardised late-effects protocol 416 patients were evaluated over a period of 5 years. Diagnosis were ALL (24%), AML (13%), neuroblastoma (10%), CML (7%), severe aplastic anaemia (6%), rhabdomyosarcoma (5%) und myelodysplastic syndrome

S196 (4%). Height (HT), weight (WT), pubertal status and IGF-I and survival was 91.5% (95% CI 87-96%). With a median follow- IGFBP-3 levels were assessed at each visit. A growth up of 33 mths hormone (GH) stimulation test (arginine test) was performed (range 3-83 mths), 2 year OS, TRM and RR were 62% (95% before SCT and when indicated after SCT. CI 56-70%), 17.5% (95% CI 11-24%) and 35.5% (95% CI Results: The median maximum GH peak (maxGH) in the 26.5-44.5%) respectively. The incidence of aGVHD •II was 25 arginine test before SCT was 8.8 ng/ml (0.4 - 44.7), N = 172, % (95% CI 4-39%). with 19 ” 4 ng/ml (group 1), 27% > 4 and <8 ng/ml (group 2) Conclusion: The results of this extended study suggest that and 54% • 8 ng/ml (group 3). maxGH was not correlated to defibrotide is an effective prophylaxis for VOD. IGF-I (SDS) and IGFBP-3 (SDS) levels, HT-SDS, difference between HT-SDS and target HT-SDS (delta THT), changes in HT-SDS (delta HT-SDS) during follow-up and prior P798 radiotherapy treatment. maxGH was negatively correlated to Bone marrow transplants out of the hospital/in hotel - BMI (SDS). IGF-I (SDS) and IGFBP-3 (SDS) levels were pos. experience with 108 patients correlated over the entire follow-up period. Increases in IGF-I C. Demosthenous, P. Statham, S. Rowley, A. Goldstone (delta IGF-I) within the first year pos. correlated with delta University College London Hospital (London, UK) IGF-I during further follow-up. Delta IGF-I and delta IGFBP-3 were pos. correlated during 3 years after SCT. IGF-I and Background: Since the onset of the ambulatory care (AC) IGFBP-3 were pos. correlated with HT-SDS over the entire initiative at University College London Hospitals (UCLH), over follow-up. Prior radiotherapy was not correlated to HT-SDS, 250 patients have received acute haematology treatments delta THT and IGF-I and IGFBP-3 levels. Delta HTSDS after previously delivered as in-patients. In order to maximise 2, 3, 4 and 5 years was correlated with radiotherapy given as efficiency in the AC facility and ensure robust governance all part of the SCT regimen. Taking HT-SDS before SCT into aspects of the service have been audited prospectively. account, HT-SDS after 1 to 5 years was correlated to the Aims: The primary aim of the AC service is to safely transfer presence or absence of radiotherapy for SCT. Delta HT-SDS the delivery of in-patient treatments to the ambulatory setting. was pos. correlated to delta WT-SDS over the entire follow-up Objectives: period. Delta HT-SDS after ½ and 1 year were pos. correlated • To define eligibility of patients for outpatient/ambulatory with delta HT-SDS during further follow-up. transplant Conclusions: Early increases after SCT in HT-SDS, IGF-I and • To identify and minimise risk factors when adapting inpatient IGFBP-3 may enable us to predict changes during later follow- transplant treatments to the ambulatory setting up. After SCT, IGF-I and IGFBP-3 seem to be in part • To determine reasons for and potential median day of differentially regulated. The growth inhibiting effect of SCT transfer to hospital for specific transplant protocols related radiotherapy may be detected from 2 years after SCT. Sample: Since December 2004 108 patients have received Results from arginine stimulation tests must be carefully either autologous or low intensity allogenic (LI) transplantation interpreted taking IGF-I/IGFBP-3 levels and growth into in AC; 50 (46%) a BEAM autograft, 29 (27%) a Melphalan account. autograft and 29 (27%) a LI allogenic transplant. Results/Discussion: Audit results show reasons for hospital transfer from AC fall into 7 categories: GI symptoms, febrile P797 neutropenia, non-neutropenic fever, fatigue, anxiety, drug Efficient prevention of veno-occlusive disease of the liver reactions and carer issues. Data allows the identification of with defibrotide after allogeneic haematopoietic stem cell median day of hospitalisation for transplant patients; day +3 transplantation. An extended study for BEAM autograft, day +4 for Melphalan autograft and day+4 Y. Chalandon, E. Roosnek, C. Helg, F. Verholen, S. Knipp, H. for LI allogenic transplant. Of the 108 transplant patients in AC Ozsahin, J. Passweg 13 (12%) have required out-of-hours hospital admissions due University Hospital of Geneva (Geneva, CH) to febrile neutropenia, 5(38%), GI symptoms,3 (23%), drug reactions, 3(23%), non-neutropenic infections, 1(8%) and Veno-occlusive disease of the liver (VOD) of the liver is anxiety 1(8%). Infection issues, including infectious seasonal reported to occur in 10-30% of patients after allogeneic outbreaks, continue to pose a risk and complicate hospital haematopoietic stem cell transplantation (HSCT), with admissions from AC. In order to reduce risk to all patients, mortality rates up to 50%. We previously reported a pilot study Unit policies have been amended and practice reviewed within of VOD prophylaxis with defibrotide (Prociclide Crinos, Como, the ongoing audit. Italy) showing a significantly lower rate of VOD as compared Conclusion: Our findings augment data presented at EMBT to historical controls. We now report the results of an 2006; for eligible patients, autograft and allograft extended study. transplantation remain suitable ambulatory treatments in the Between 2000 and 2006, 157 consecutive patients were hotel setting. A diverse patient-mix is necessary to optimise transplanted for hematological diseases. Median age was 43 y AC space and staff resources and robust out-of-hour policies, (range 5-61 y), M/F: 96/61; diagnosis was AML=42, ALL=19, risk management and infection control procedures are CML=28, MDS/MPS=24, lymphoma=22, MM=12, AA=7, solid fundamental in order to provide a safe AC service. Admission tumor=3. Donors were HLA-identical siblings (65%), unrelated to hospital for these transplant patients is ultimately inevitable. (32%) mismatched relatives (3%). Stem cell source was However, efficiencies in in-patient bed usage, cost savings peripheral blood (87%) or bone marrow (13%). The and improvements in patient satisfaction are advantageous. transplants were T-cell depleted by CAMPATH-1H in vitro, with (73%) or without (7%) T-cell add-back or left unmanipulated (20%). Conditioning was myeloablative with P799 CyTBI (53%), BuCy (10%) other myeloablative (7%) or non- The impact of disparity in short tandem repeats analysis myeloablative (30%) using Fludara/Bu/ATG. GVHD on the outcome of allogeneic haematopoietic cell prophylaxis was CSA with or without MTX or MMF. Nine pts transplantation from HLA-identical sibling donors received a second transplant for relapse or graft failure. E. Serbest, P. Topcuoglu, K. Dalva, E.A. Soydan, O. Arslan, Defibrotide was given intravenously 6 hourly starting prior to M. Ozcan, T. Demirer, H. Akan, N. Konuk, A. Uysal, O. Ilhan, conditioning up to day +21 posttransplant (dose range 20-30 G. Gurman, M. Beksac, M. Arat mg/kg) in addition to low dose heparin. The Baltimore criteria School of Medicine, Ankara University (Ankara, TR) were used to score VOD. Results: No VOD was observed after 165 transplants. STRs were widely used for monitoring of chimerism after allo- Defibrotide was well tolerated and no side effects were HCT. The effect of STR disparity on allo- HCT has been attributed to this medication. Median peak levels of bilirubin analyzed in a few studies. We aimed to evaluate the impact of were 21 umol/l (range 9-286 umol/l), aPTT: 34.5 sec (range STR disparity on allo-HCT outcome retrospectively in our 24-83 sec) and fibrinogen: 7.1 g/l (range 3.1-10 g/l). Day 100 single center cohort. Between Sep 2000 and May 2005, total 88 patients (F/M:45/43) underwent allo-HCT from an HLA-

S197 identical sibling donor (PB/BM: 71/17) Median age was 34 ys developed sMDS/AL, with a cumulative incidence of sMDS/AL (16-64). Multiplex PCR was performed to amplify 16 STR loci of 4.2% at 5 yrs. Median time of sMDS/AL occurrence was 2.2 (D3S1358, HUMvWA, D16S539,D2S1338, yrs since autograft. Several clinical parameters, including age, Amelogenin,D8S1179, D21S11,D18S51, D19S433, THO1, sex, histology, disease status, BM involvement, were FGA, D7S820, CSF1PO, D13S317, TPOX, D5S818) (ABI assessed for possible association with sMDS/AL occurrence; Prism 310). These loci were classified as full- and partial- however, none of them appeared to be of relevance. Patients matched, and full mismatched(mm) between donors and autografted with PBPC collected after hd-Ara-C had a higher recipients.Results:D2S1338 locus (full matched, 21.6%-n=19; risk of sMDS/AL development. On multivariate analysis this partial matched 50 %-n=44 and full mm 28.4 %-n=25) was the was the only parameter marginally associated with sMDS/AL most informative one, but CSF1PO (43.2 %, n=4; 52.2 %, occurrence. n=46 and 4.5%, n=38, respectively) was the least. The Conclusions: Overall, the incidence of sMDS/AL observed general incidences of acute(a) severe and chronic(c)GvHD following HDS is among the lowest reported so far in were 29.5% and 61.8%. Full mm in TPOX locus (p=0.04) led lymphoma patients treated with high-dose therapy and to more acute severe GvHD than in partial matched or full autograft. The autograft performed with large quantities of matched loci. However, full mm in D2S1338 locus significantly CD34+ve cells and the preferential use of TBI-free decreased the incidence of aGvHD in patients with partial conditioning regimens might have lowered the risk of matched and full matched loci (5.3%, 29.5% and 48%, sMDS/AL occurrence. In addition, the study suggests that the respectively p=0.009). However, more cGvHD was only seen type of graft employed in the autografting procedure may be in the patients with full matched D7S820 donors (p=0.04). In critical for sMDS/AL development. our cohort,2-year probability of disease free survival(DFS) and overall survival(OS) were 53.1% ±5.9 and 66.2 %±5.6, respectively in mean 46.5±3.8 moths of follow-up. Although P801 the full-mm in D8S1179 locus increased the relapse incidence Neurologic complication of cyclosporine in bone marrow (62.5% vs 33.3% vs 18.8%, p=0.04), there was a tendency to transplanted thalassaemic patients, a 10-year experience a decrease of DFS in patients with full-mm CSF1PO or partial M. Ramzi, A. Parhizkar, H. Haghighi matched D7S820 (p=0.06). Early transplant-related mortality Shiraz University of Medical Sciences (Shiraz, IR) increased in patients with D7S820 full-mm (p=0.02). This unfavorable effect was reflected on OS (p=0.002).Besides, Background: Cyclosporine neurotoxicity is an important patients with full-mm TPOX locus had a decreased probability complication of bone marrow transplantation (BMT), reported of OS. TPOX and D7S820 were also less informative as the most common neurologic complication of BMT. resembling CSF1PO.We were not able to show any Identification of risk factors can help physicians to predict the correlation of stem cell source, conditioning regimen, donor or probability of this complication, so prepare to detect and recipient age, and sex mm on early TRM, aGVHD, cGVHD, manage it at early stages and decrease complications. Goal of and OS rate. Conclusion:The presence of STR disparity could this study was determination of frequency, presentations and be associated with the development of complications and risk factors of neurotoxicity of cyclosporine in thalasemic unfavorable survival probability after allo-HCT from HLA- patients after allogenic BMT. identical siblings.This analysis approach should be repeated Methods: During 10 years 117 thalasemic patients underwent on partial matched and unrelated donor transplants. allogenic marrow transplantation. Conditioning chemotherapy regimen were Busulfan and cyclophosphamide ± antithymocyte globulin. The GVHD prophylaxis regimen was P800 cyclosporine and methyl prednisolone with or without Moderate risk of secondary myelodysplastic methotrexate. In order to find any risk factor many clinical syndrome/acute leukaemia occurrence following high- features were compared between patients with cyclosporine dose sequential chemotherapy and autograft: a GITIL neurotoxicity and ones without it. We used statistic methods. (Gruppo Italiano Terapie Innovative nei Linfomi) survey Results: Cyclosporine induced neurotoxicity was developed in on 1266 lymphoma patients 27(23.1%) of patients at an average time of 39.1 days post- M. Zanni , M. Magni, A. Rambaldi, F. Benedetti, R. Rosato, R. transplantation. It was responsible for 42.8% of all-causes of Passera, K. Patti, F. Ciceri, A. Gallamini, S. Cortellazzo, I. neurologic sequella in bone marrow transplanted thalasemic Majolino, S. Mirto, P. Corradini, M. Boccadoro, A. Andreini, T. patients. Headache was the most common manifestation of Barbui, A.M. Gianni, C. Tarella on behalf of GITIL cyclosporine neurotoxicity in these patients (74%). Other neurotoxic manifestations in order of frequency were: Introduction: The occurrence of secondary myelodysplastic Seizure(62.9%) ,Decrease-level of consciousness (55.6%), syndrome/acute leukemias (sMDS/AL) is a critical issue in the visual problems(33.3%). Patients with cyclosporine autograft setting, and represents a major cause of failure in neurotoxicity were significantly older than ones without it both non-Hodgkin s (NHL) and Hodgkin s Lymphoma (HL) (P=0.037). Blood pressure and serum creatinine level of potentially cured after high-dose chemotherapy. However, the patients with cyclosporine neurotoxicity at the time of reported cumulative incidences of sMDS/AL are quite various, presentation were significantly elevated with respect to ranging between 3 up to 24%. admission time (P=0.000, 0.009 in order) but BUN was not Aim of the study: To evaluate the frequency and risk factors of different (P=0.101). Analysis of risk factors showed that sMDS/AL in a large series of lymphoma patients, treated with degree of splenomegally, status of chelation therapy and high-dose sequential (HDS) regimen followed by Peripheral Pesaro risk classification group are significantly related to Blood Progenitor Cells (PBPC) autograft. Methods: During the cyclosporine neurotoxicity in univariate analysis(P=0.05, last 2 decades 1266 lymphoma patients were treated with 0.004, 0.012 in order). HDS at 10 Italian Centers associated to GITIL. All patients Conclusion: Cyclosporine induced neurotoxicity has a received either the original or somewhat modified HDS considerable incidence in bone marrow transplanted regimen with autograft; PBPC were usually collected after thalasemic patients (23.1%) causing significant morbidity. This hd.cyclophosphamide; PBPC harvested after a second study shows that irregular chelation, large splenomegally (4 mobilization procedure with hd-Ara-C were employed in a cm or above) or presence of splenectomy and Pesaro risk subgroup of patients. The series included 213 HL and 1053 classification group III significantly increase the risk of NHL patients (630 high-grade and 423 low-grade NHL). cyclosporine neurotoxicity. Median age was 46 yrs. Overall, 595 patients received HDS as first-line therapy, and 671 patients as salvage treatment. Most patients were autografted with PBPC (median CD34+ve cells: 8x106/kg) and only few patients received BM cells. TBI was employed only in 7% of patients. Results: At a median follow-up of 5 yrs, the 5-yr Overall Survival (OS) projection is 64%. Overall, 38 (3%) patients

S198 P802 P803 Marked telomere shortening in autografted patients could Outpatient-based autologous peripheral blood stem cell predict secondary myelodiyplastic symdromes transplantation for patients with multiple myeloma I. Ricca, A. Rocci, M. Ruella, C. Dellacasa, C. Lobetti Bodoni, S. Palmieri, M. Annunziata, G. Mele, C. Copia, F. Pollio, M.R. R. Francese, M. Compagno, M. Zanni, M. Ladetto, D. D'Amico, A. Viola, M. Alberti, D. Graziano, C. Falco, F. Caracciolo, D. Ferrero, M. Boccadoro, C. Tarella Ferrara University of Turin (Turin, I) Cardarelli Hospital (Naples, I)

Background: sMDS and AML may occur following autologous Background: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT). A retrospective analysis at stem cell transplantation (ASCT) represents standard practice our Institution showed a 5-year cumulative probability of for newly diagnosed patients with multiple myeloma (MM). developing sMDS/AML of 4.8% for lymphoma patients who Accordingly, there is a growing demand for ASCT resulting in underwent high-dose chemotherapy (HDS) and ASCT. The increased pressure on available hospital beds. The main molecular pathogenesis of sMDS/AML remains uncertain and advantages of at-home ASCT are the improvement in the we still lack a simple and wildly-applicable method to predict quality of life and the decrease in the number of days during the rising of sMDS/AML. We recently observed a marked which patients need hospitalization, with a consequent telomere shortening in hematopoietic cells of autografted reduction of costs, risk of nosocomial infections, and more lymphoma patients that persists at long-term. AIM In this rational utilization of beds. The aim of this study was to study, we assessed post-ASCT telomere dynamics in order to confirm the feasibility and safety of performing ASCT on an verify whether accelerated telomere loss in hematopoietic outpatient basis, according to an early discharge method. cells might underlie the pathophysiology of sMDS/AML. Patients and methods: A total of 107 cases out of 124 Patients and methods: We retrospectively analyzed telomere consecutively autograft procedures for 101 patients affected restriction fragments length (TRF-L)of BM cells from 35 long- by MM in partial or complete remission were selected to term survivors autografted lymphoma patients (22M/13F) and receive ASCT on an outpatient basis. In particular, after 51 healthy donors (31M/20F). Among the 35 autografted conditioning with high-dose melphalan and stem cell infusion, patients we studied also 5 patients who developped patients were programmed to go home and to be sMDS/AML (2 AML,2 clinical sMDS,1 persistent pancitopenia rehospitalized in the case of febrile neutropenia or other with cytogenetic abnormalities). Patient median age at follow- severe toxicities. The remaining 17 cases were excluded from up was 51 yrs (24-68 yrs) while donor median age was 53 yrs the program because of poor performance status (n=4), (18-82 yrs). The sMDS/AML patients do not differ from the BEAM conditioning (n=8), geographical distance and/or lack other autografted patients in terms of clinical features and no of caregiver (n=7), more than one reason being present in 2 differences were found in terms of CD34+ cells reinfused. cases. All asked patients accepted the outpatient-based Median follow-up from ASCT was 6 yrs (3.5-10 yrs). All procedure. patients were in CCR since autograft at the time of TRF-L Results: Out of 107 cases, 83 (78%) did spend the aplastic analysis. phase entirely at home following high-dose chemotherapy and Results: Patient TRF-L was compared to that of donors and a stem cell infusion. A second hospital admission was required significant telomere loss was observed in patients (Fig. panel in 24 patients (22%). Febrile neutropenia and severe A, squares and grey circles). Indeed, patient median TRF-L mucositis needing total parenteral nutrition were the most was 6739bp (4472-9004) while donor median TRF-L was frequent causes of hospitalization (45 and 38%, respectively). 7791bp (5836-11058) (p<0.005). Of note all the sMDS/AML However, there were no documented infections and either patients had a TRF-L shorter than the median TRF-L of fever or mucositis was easily resolved at the time of autografted patients (Fig. panel A, black circles). Thus, hematopoietic recovery in all patients. Neither patient’s nor comparing only age-matched autografted patients (12) and disease’s characteristics before ASCT were found to be healthy subjects (19), we found that of sMDS/AML patients related to need for re-hospitalization. Of note, percent of had TRF-L significantly shorter than both donors and second admission was 43% in the initial 30 patients and 14% autografted patients (median TRF-L 5738bp, range 4472- in the following 77 procedures (p:0.002). 6493, p<0.0005 and p<0.01, respectively) (Fig. panel B). Conclusion: We conclude that ASCT on an outpatient basis is Conclusion: A)patients with short telomeres seem to have a feasible and safe in patients with MM and more than 75% of higher risk of developing sMDS/AML suggesting that telomere patients are entirely manageable at home, provided that a loss may be relevant to the pathogenesis of sMDS; B) if these caregiver is available. A progressive increase in expertise results will be confirmed on larger and independent results in a significant reduction of hospitalization. populations, they will add further knowledge into the pathogenesis of sMDS. P804 Long-term psychosocial aspects and risk factors for severe infections, ocular, pulmonary, bone, thyroid and other complication after allogeneic haematopoietic stem cell transplantation for children with haematologic malignancies F. Christèle, G. Gemayel, V. Rocha, M. Labopin, H. Esperou, M. Robin, R. Peffault de Latour, P. Ribaud, A. Devergie, E. Gluckman, G. Socié Saint Louis Hospital ApHp (Paris, F)

Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective therapy for children with hematologic malignancies. However, many long term complications are observed with a longer follow-up. Few data is available analyzing psycho-social aspects and risk factors of various long term complications in a same cohort of children. We have reviewed the charts of 112 children younger than 16 years, transplanted for hematologic malignancies between 1985 and 2000 at Saint Louis Hospital, and who survived at least 1 year after transplant. Median age at transplant was 8 years (0.73- 15) and median follow-up from 1 year after transplantation was 8 years (0.3-16.5). Most children had acute leukemia (n=101,90%) were transplanted with an HLA-identical sibling

S199 donor (n=92). Total body irradiation (TBI) was used in 87 Results: Sustained engraftment was achieved in all pts at day patient as part of conditioning regimen and fractionated TBI 19 (range 9-47) for ANC > 0.5x109/L, and day 30 (range 16- (FTBI) was used since march 1994 in 37 patients. 111) for platelets > 50x109/L. Complete chimerism (> 99%) At 10 years, overall survival was 75±5%, transplant related was seen in 31/36, and 5/36 were mixed chimeras but had mortality (TRM) 18±4 and relapse 14±3%. Ten year mainly donor cells(> 85%). IVBu was well tolerated: no grade cumulative incidence of severe infections (mostly bacterial) (G) IV toxicity, G III (mainly stomatitis) occurred in 14 pts. was 31%±4 (n=33); it was 44±4% for cataract (n=48); 20±4% VOD occurred in 8% but none was severe. G I-II and III-IV for pulmonary dysfunction (n=20); 29±5% for osteoarticular acute GVHD rates were 50% and 6%, respectively.There was complications (n=27); 36±4% for hypothyroidism (n=36); one death (3%) at day+100, and the cumulative incidence (Ci) 11±3% for cardiac complications and 7±3% for secondary of TRM was 6% (hemorrhage =1, c-GVHD =1). After a median malignancies (n=8). The number of complications per patient follow-up of 36.7 months (range 3.9-49.1) the Ci of relapse, increased with time, from 1 at a median observation time of 73 EFS, and OS were: 12% ±11% (all among AML), 82% ±13, months to 3 at a median of 120 months. Chronic GVHD was and 85% ±12%, respectively. For pts with myeloid significantly associated with higher risk of pulmonary malignancies (mainly AML): Neither VOD nor TRM. EFS and dysfunction (p=0.02). In a multivariate analysis, risk of OS rates were 82% and 86%, respectively. infections were associated with older patients (>10 years at Conclusions: These favourable results indicate that IVBu transplant) (p=0.004)and positive CMV serology (p=0.01). dosing in IVBuCy regimen is an efficacious, myeloablative However, single dose TBI or only TBI was the most important conditioning with a reduced toxicity for children undergoing factor among other factors that increased the risk of cataract HSCT. (p<0.001), pulmonary (p=0.004), osteoarticular (p=0.04) and thyroid complications (p<0.0001). Concerning psychological health and social issues; half of the patients had psychological disturbance (depression, eating behavior disorder). Twelve patients achieved superior level studies and 69 had scholar difficulties. In conclusion with a longer follow-up in survivors of HSCT long term complications become an important issue. Development of less toxic conditioning regimen, avoid single dose TBI and probably better control of chronic GVHD may prevent late effects and improve quality of life of long term survivors.

P805 Clinical results of allogeneic haematopoietic stem cell transplantation following a new intravenous busulfan dosing strategy as part of BuCy conditioning regimen in children and adolescents: sustained engraftment and less toxic deaths H. Espérou (1), G. Vassal (2), B. Neven (3), F. Méchinaud (4), C. Galambrun (5), K. Yakouben (6), C. Paillard (7), P. Bordigoni (8), C. Levrault (9), H. Zouabi (9), G. Michel (10) (1)Hopital Saint Louis (Paris, F); (2)Inst Gustave Roussy (Villejuif, F); (3)Hopital Necker-Enfants Malades (Paris, F); (4)Hopital Hotel Dieu (Nantes, F); (5)Hopital Debrousse (Lyon, F); (6)Hopital Robert Debré (Paris, F); (7)Hopital Hotel Dieu (Clermont-Ferrand, F); (8)Hopital d'Enfants Brabois (Vandoeuvre Les Nancy, F); (9)Institut de Recherche Pierre Fabre (Boulogne, F); (10)Hopital la Timone (Marseille, F)

Introduction: In children and adolescents allogeneic (allo-) hematopoietic stem cell transplantation (HSCT) is a standard therapeutic option, but its use remains limited by the risk of transplant-related mortality (TRM). Busulfan (Bu) is often included in conditioning regimens but under- or overdosing may have a fatal outcome. We published that instead of an age-based dosing a body-weight- (BW) based calculation of IVBu can successfully target a therapeutic blood exposure without any dose adjustment (Vassal G et al BMT 2006). This approach was applied prospectively and we report here the final clinical results. Patients and Methods: 36 children (21 boys/15 girls); median age 7.5 y (range 0.3 to 17.2 y) and weight 27 kg (range 5.0 to 62.0). IVBu (Busilvex®) was given over 2 h q6h x 16 doses at: 1.0 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 0.95 mg/kg, and 0.8 mg/kg for patients(pts) with <9 kg, 9 to <16 kg, 16-23 kg, >23-34 kg, and >34 kg BW, respectively. Cy was administered at 50 mg/kg qdx4. Clonazepam was used as seizures prophylaxis. Graft-versus-host disease (GVHD) prophylaxis was given according to local practice. Indications for HSCT were: AML (n= 17: 14 CR1), ALL (n= 1), CML (n= 3), MDS (n= 1), SAA (n= 1); SCD (n= 7), thalassemia (n= 1), Wiskott-Aldrich (n= 3), LAD (n= 1), and CGD (n= 1). Pts received bone marrow (n= 35), and PBPC (n= 1) containing 6.4 x106 CD34+/kg (range 1.1-29) from matched related (28/36) or unrelated (5/36) donors.

S200 P806 (n=26)donors. Conditioning was with (n=1243) or without TBI Palifermin (Kepivance) in prevention of oral mucositis in (n=408). Primary disease risk was high in 714 & low in 937 patients with haematological diseases undergoing patients. TBI dose was <9.5 Gy (n=342), 10.5 Gy (n=613) or > allogeneic stem cell transplantation 10.5 Gy (n=282) delivered in single (n=1015) or multiple B. Nasilowska-Adamska (1), P. Rzepecki (2), A. fractions (n=222). Donor was sibling (n=900), MUD (n=135) or Tomaszewska (3), M. Bieniaszewska (4), A. Czyz (5), M. mismatched family member (n=53). 357 received cranial Markiewicz (6), J. Manko (7), K. Halaburda (1), A. Szczepinski radiation. Stem cell source was marrow (n=1312), PBSC (1), A. Dmoszynska (7), J. Holowiecki (6), M. Komarnicki (5), (n=320) or both (n=19). GVH prophylaxis was CyA alone A. Hellman (4), W. Jedrzejczak (3), K. Warzocha (1), B. (n=527), CyA with other agent (n=516) or other measures Marianska (1) (n=45). 136 patients received more than one transplant. 897 (1)Institute of Hematology (Warsaw, PL); (2)Military Medical patients (54.3%) survived for at least 1 yr. with a median Institute (Warsaw, PL); (3)Medical University (Warsaw, PL); follow-up of 27yr. 50 patients developed SMNs with the (4)Medical University (Gdansk, PL); (5)K. Marcinkowski incidence of 7.5% at 10 yr (95% CI: 4.9-10.1) & 11.5% at 15yr University of Medical Science (Poznan, PL); (6)Silesian (95%CI: 7.6-15.4). Site of SMN was brain (n=10), breast Medical University (Katowice, PL); (7)Medical University (n=6), cervix (n=3), GIT (n=2), lung (n=1), skin (n=9), sarcoma (Lublin, PL) (n=3), thyroid (n=1), oral cavity (n=4), MDS (n=6), CML (n=1), ovary (n=1) & non EBV related lymphoma (n=3). Risk factors Aims: Oral mucositis (OM) is a frequent complication of the in univariate analysis were chronic GVHD (5% vs. 10%, myeloablative therapy and allogeneic stem cell p=0.008, high risk disease (5% vs. 11%, p=0.0007), cranial transplantation.(alloSCT) which previously had no effective RT (4% vs. 18%, p=0.015) & ALL as primary diagnosis (4% treatment. vs. 10%, p=0.06). In multi-variate analysis advanced stage In this multi-center study we assessed the ability of palifermin disease (RR: 2.4, 95% CI: 1.3-4.2, p=0.004), Chr. GVHD (RR: (rHu-KGF1) to reduce the incidence, duration and severity of 2.5, 95% CI: 1.4-4.4, p=0.003) & cranial RT (RR: 2.3, 95% CI: OM induced by high-dose chemotherapy followed by alloSCT 1.2-4.4, p=0.01) were independent risk factors. Multi-variate in patients with hematological diseases. We also evaluated risk factors were different for site specific SMNs. Risk factors the requirement for analgesics and total parenteral nutrition for brain tumours were cranial RT (RR: 12.8, 95% CI:2-75.5, (TPN) administered because of OM. Moreover, the influence p=0.006) & age <16 yr at the time of transplant (RR:15.7, 95% of palifermin on the engraftment and length of hospitalization CI:1.9-130.6, p=0.01) & for epithelial malignancies Chr. GVHD after alloSCT was assessed in this study. (RR: 5.8, 95% CI: 2.2-15.5, p=0.0005), female sex (RR: 3.0, Methods and Results: Sixty two patients with hematological 95% CI:1.1-8.3, p=0.03) & advanced stage disease (RR: 3.6, diseases were enrolled to the study. 95% CI: 1.4-9.4, p=0.01). No independent risk factors could Thirty one of them (50%) received palifermin (60 be identified for skin or haematological malignancies. 25 microg/kg/day) for three consecutive days before and after patients have died due to SMN (49%) at a median of 31 conditioning therapy. Each reporting center submitted equal months. Survival was better in skin SMNs (85%) as compared number of patients to palifermin and control group. The to haematological SMNs (20%), brain tumours (30%) & patients in retrospective control group were similar to patients epithelial SMNs (56%)(p=0.02). This analysis shows that the in palifermin group in terms of sex, age, diagnosis, type of risk of developing SMN does not relate to the type of donor-related/unrelated, type of transplant- conditioning & increases with longer follow-up necessitating myeloablative/reduced intensity conditioning, conditioning prolonged observation in this group. regimens and risk of OM. OM was assessed daily after alloSCT according to the World Health Organization (WHO) scale. P808 The incidence of OM of WHO grade 1-4 was 69,5 percent in Sequential organ failure assessment predicts survival in palifermin group and 95,8 percent in control group and grade patients treated at the intensive care unit after allogeneic 3-4 was 16,6 percent in palifermin group and 66,6 percent in SCT control group. The median duration of OM grade 1-4 was 5,5 K. Gilli, M. Remberger, H. Hjelmqvist, O. Ringden, J. Mattsson days (range, 0 to 19) in palifermin group and 12 days (range, Karolinska University Hospital Huddinge (Stockholm, S) 0 to 32) in control group. As compared with control, palifermin was also associated with significant reductions in the use of Methods: We retrospectively analysed all allogeneic SCT analgesics ( 41vs.87,5 percent ), opioid analgesics ( 37,5 vs. patients treated at the intensive care unit (ICU) from January 83,3 percent) and TPN ( 25 vs. 70 percent). 1995 to December 2005 at Karolinska University-Hospital, No significant differences in the median duration of Huddinge. During this period 661 patients underwent hospitalization were observed between groups. Also allogeneic SCT at our centre. Ninety-one patients were palifermin did not appear to impair engraftment . admitted to the ICU, 4 patients were admitted twice. The The drug was generally well tolerated. Adverse events, mainly median age of the patients was 34 (1-67) years. Twenty-six rash, pruritus, erythema, generalized edema, mouth/tongue (29%) were children younger than 18 years. Diagnoses were: thickness and discoloration and taste alteration were mild to acute leukaemia (n=43), CML (n=17), solid tumors (n=7), moderate in severity and were transient. other malignancy (n=15) and non-malignant disease (n=9). Conclusions: Administration of palifermin significantly reduced Thirty-one donors were HLA-identical related, 46 MUD and 14 the incidence, severity and duration of OM and did not have patients received HLA-mismatched grafts. Conditioning was negative effect on engraftment in the patients with TBI-based (n=44), chemotherapy (n=28) or non- hematological diseases after alloSCT. It was generally well myeloablative(n=19). Sixty-three patients received ATG tolerated and safe. before SCT. GVHD prophylaxis was mainly a combination of cyclosporine A and methotrexate. The patients were monitored for respiration mode, need of vasopressor P807 treatment, Apache-score and SOFA-score. Patients younger Second malignant neoplasms after haematopoetic stem than 10 years were not analysed with Apache-score or SOFA- cell transplant score. S. Kulkarni, R. Saso, J. Brennan, M. Ethell, J. Treleaven, P. Results: Reasons for ICU-admission were: respiratory Kaczmarek, C. Horton, F. Saran, R. Powles, G. Morgan, M. insufficiency (n=39), septicemia (n=29), neurological Potter symptoms (n=11) or others (n=16). Fifty-three patients died in Royal Marsden Hospital (Sutton, UK) the ICU and further 14 within 1 month after discharge from the ICU. Median time from SCT to ICU admission was 69 (-24 – From 1973 to 2003, 1651 patients (age: 28yr., range: 1-74 yr.; 1572 days) and median stay at ICU was 4 (1-60) days. M: 982, F: 669) received SCT for haematological Median time from ICU-admission to death was 14 (0-612) malignancies (Ac. Leuk: 1246, Chr. Leuk: 182, other: 223) days. The survival after ICU admission day 100 was 22% and using allogeneic (n=1088), autologous (n=537) or syngeneic 16% at 1 year. Median SOFA-score was 10 (1-17). SOFA-

S201 score was predictive for survival after admission to the ICU. Irrespective of underlying diagnoses trabecular parameters fell Patients with SOFA-score <8 (n=24) showed 54% survival within the first 6 months after SCT. Afterwards, trabecular compared to 23% with SOFA-score 8-11 (n=24) and no parameters varied over time, however, none of the survival with SOFA-score >11 (n=23) (p<0.0001). No patient parameters went back to baseline values. Number of patients transplanted for a second time survived ICU treatment analysed over time was to small to find out causative clinical compared to 34% survival in patients receiving the first SCT parameters like chronic GvHD. Radiologic bone density was (p=0.003). Patients receiving TBI as conditioning treatment available for these patients and showed synchronous showed less survival compared to patients given only variations over time. chemotherapy (14% vs. 45%, p=0.02). Conclusions: Severe changes of trabecular bone mass and Conclusion: Long-time survival in SCT-patients treated at the structure develop early after allogeneic SCT and underscore ICU is relatively poor. The SOFA-score was predictive for the severeness of bone loss observed in these patients. Even prognosis in SCT-patients treated at the ICU. after prolonged follow-up, structural changes and diminished bone mass persist and indicate a long-term cause of morbidity in these patients.

P810 Vascular endothelial growth factor and activin-A serum levels following allogeneic haematopoietic stem cell transplantation D. Nachbaur, P. Schumacher, J. Auberger, J. Clausen, B. Kircher Innsbruck Medical University (Innsbruck, A)

Background: Liver toxicity is a frequent complication following allogeneic hematopoietic stem cell transplantation with a high mortality in its severe form. Marked elevations of coagulation parameters, adhesion molecules, and of pro inflammatory cytokines occur in patients with liver toxicity although the exact mechanisms of endothelial injury still remain elusive. Vascular endothelial growth factor and activin-A, a member of the transforming growth factor beta superfamily, are important for endothelial integrity and tissue repair. Methods: We therefore serially measured vascular endothelial growth factor and activin-A serum levels in seventy patients P809 following allogeneic stem cell transplantation. Severe alterations of bone mass and bone structure after Results: Vascular endothelial growth factor serum levels were allogeneic stem cell transplantation: an osteopathologic significantly decreased within the first two weeks after the analysis transplant, returned to pre transplant levels after G. Massenkeil (1), C. Fiene (2), O. Rosen (2), G. Delling (3), hematopoietic regeneration, and were inversely correlated R. Arnold (2) with total maximal serum bilirubin and with the number of days (1)Ruhr-University (Bochum, D); (2)Charité (Berlin, D); with serum bilirubin >2 mg/dL with day +20. Patients with high (3)University Hospital (Hamburg, D) vascular endothelial growth factor levels (> 151.2 pg/mL) on day +15 had a significantly faster hematopoietic recovery and Loss of bone density after SCT has been observed in a significantly better overall survival due to less transplant- radiologic analyses (Ebeling 1999, Schulte 2000, Massenkeil related mortality and a lower incidence of severe acute graft- 2001). We have studied pathologic bone mass alterations in versus-host disease. Even in a multivariate Cox model high bone marrow biopsies after SCT. vascular endothelial growth factor levels on day +15 were 67 patients after allogeneic SCT were included in this associated with a significantly lower risk for death. No analysis. Median age was 36 years (range 17-56), 35 M, 32 F. correlation was found between activin-A serum levels and liver Diagnoses: ALL 27 patients (pts.), AML 14, MDS 6, CML 20. toxicity, overall survival, or graft-versus-host disease. 38 pts. were in 1. CR or 1. cP, 29 were transplanted with Conclusion: Monitoring of vascular endothelial growth factor advanced disease. 64 received standard high-dose levels following allogeneic hematopoietic stem cell conditioning and 3 were transplanted after reduced intensity transplantation might help to identify patients with a very high conditioning. 42 received bone marrow and 25 mobilized risk for transplant-related complications including severe acute peripheral blood as stem cell source. 40 pts. were graft-versus-host disease. transplanted from HLA-identical related and 27 from unrelated donors. 31 bone marrow biopsies underwent Micro-CT (Scanco, P811 Zurich, CH) and were analysed for parameters of trabecular Lower body mass index is associated with the structure after 3-D reconstruction. appearance of microangiopathic haemolytic anaemia Results: Median bone volume/tissue volume (BV/TV), which following total body irradiation serves as a parameter of histopathologic bone mass was D. Mallouri, P. Kaloyannidis, K. Hatziioanou, I. Batsis, I. 13.7% before SCT (range 8.6-44.4), well below the Sakellari, E. Yannaki, A. Papalexandri, M. Ganidou, A. osteopathologic threshold for osteoporosis (appx. 18%). At Tsobanakou, V. Yannoulakos, D. Misailidou, A. Fassas, A. first follow-up 6-9 months after SCT, BV/TV had dropped to Anagnostopoulos 10.1% (5.9-24.6%) and at 1-2 years after SCT BV/TV was still G. Papanicolaou Hospital (Thessaloniki, GR) below the pre-SCT values at 12.2% (6.1-19.3%). Number of trabecules (Tb.N.) dropped from 1.63/mm (range Microangiopathic hemolytic anemia (MAHA) is a common 0.93-2.32/mm) to 1.34 (1.01-5.61) after 6-9 months and complication post-allogeneic transplantation (alloTx). Total slightly increased to 1.54 (1.26-1.88) after 1-2 years. body irradiation (TBI) as pathogenic factor of endothelium Trabecular thickness (Tb. Th.) fell from 0.20 mm before SCT damage has been associated with the appearance of MAHA. (normal Tb. Th. 0.20-0.30 mm) (range 0.16-0.49 mm) to a We retrospectively analyzed the incidence and the minimum of 0.16mm (0.14-0.25mm). predisposing factors of MAHA in 48 consecutive alloTx As a consequence, intertrabecular space (Tb.Sp., normal performed in 45 patients (29 male and 16 female) with a appx. 0.60mm) increased from 0.60mm (0.36-1.11mm) to 0.73 median age of 27(7-54) years diagnosed with lymphoid mm (0.23-1.04) after SCT. malignancies between 2000-2006. Three of them were

S202 transplanted twice because of relapsed disease. Disease significantly. In summary, we characterize for the first time status was: CR (24), relapse (12), and refractory (12). Sibling changes in breathing patterns plus PF during the course of donors were 40 and matched unrelated 8. ABO incompatibility acute LI after experimental allo SCT. Allo recipients show was marked in 11 alloTx. Twenty three patients received TBI- insufficient ventilation starting at 1 week after SCT due to based conditioning regimen, 25 non TBI-based. A linear combined restrictive and progressively obstructive changes in accelerator type 600C Varian was used and irradiation was the lung. In addition to post mortem obtained parameters of LI, delivered by a two oppose fields technique (anterior- PF testing provides an important in vivo tool to understand posterior), with the patient in a lateral decubidus. Body dose better the pathophysiology of this serious complication after was 14.4 Gy and lung dose 12 Gy, divided in 6 fragments over allo SCT. 3 days. Acute graft versus host disease (aGvHD) prophylaxis consisted of CSP/MTX (39) and Tacrolimus/MTX (9). MAHA staging was estimated according to LDH value and the percentage of schistocytes. Seventeen patients developed MAHA grade>2 with a cumulative incidence of 40%. TBI was found to be significant factor for MAHA gr>2 (p=0.02) in a univariate analysis. In multivariate analysis the presence of aGvHD, disease status, female sex and tacrolimus were significant factors for MAHA gr>2 (p<0.05). In order to identify possible factors enhancing the endothelium damage post TBI, age, immunosuppressive agents, aGvHD, disease status, ABO-incompatibility, infection, sex, donor type and body mass index (BMI) were multivariably analyzed in the separate group of patients conditioned with TBI-based regimen. BMI ”24 was found to be strongly associated with MAHA gr>2, with a 9-fold possibility of occurrence (p=0.0001). Significant adverse factors were also viral infection (p=0.01) and Tacrolimus (p=0.002). Adipose tissue affects the distribution of irradiation. Given as a fact that smaller endothelium surface exists in adipose tissue, possibly non-overweight patients receive higher irradiation dose in a larger endothelium surface. The current study indicates that further investigation is needed regarding the role of the peripheral vascular tissue and the techniques of compensation, in order to reduce the complications of TBI.

P813 P812 New myeloablative conditioning regimen with fludarabine Characterisation of breathing patterns and pulmonary and busulfan for allogeneic stem cell transplantation: function after allogeneic stem cell transplantation: comparison with BuCy2 lessons from a murine model S.K. Sohn (1), Y.S. Chae (1), J.G. Kim (1), Y.Y. Cho (1), J.H. S. Miklos, G. Müller, E. Holler, G.C. Hildebrandt Moon (1), H.J. Shin (2), J.S. Chung (2), G.J. Cho (2), D.H. University of Regensburg Medical School (Regensburg, D) Yang (3), J.J. Lee (3), Y.K. Kim (3), H.J. Kim (3) (1)Kyungpook National University Hospital (Daegu, KOR); Using rodent models of allogeneic (allo) stem cell (2)Pusan National University Hospital (Pusan, KOR); transplantation (SCT) is important to study transplant- (3)Chonnam National University Hospital (Gwangju, KOR) associated complications such as graft-versus-host disease and lung injury (LI). While changes in histopatholoy, Background: A regimen of busulfan and cyclophosphamide is bronchoalveolar lavage cellularity and cytokines have been standard therapy before transplantation of allogeneic stem the hallmark of experimental LI, pulmonary function (PF) tests cells in hematologic malignancies. We evaluated the have been only sporadically used to determine the extent of hypothesis that fludarabine can replace cyclophosphamide in disease. Here, we characterized breathing patterns and PF myeloablative allogeneic stem cell transplantation. after allo-SCT using a well established murine model. Lethally Methods: From September 1998 to November 2006, 95 irradiated B6D2F1 mice received bone marrow and T cells consecutive patients (male: n = 54; median age: 36, range 17- from either syngeneic (syn) or allo C57BL/6 mice. In vivo 54 years) with hematologic malignancies underwent breathing patterns (tidal volume (TV), respiratory rate (RR), allogeneic stem cell transplantation from HLA-identical donors minute ventilation (MV),inspiratory (Ti) and expiratory (Te) following busulfan+cyclophosphamide (BuCy, n = 55) or time , peak inspiratory (PIF) and peak expiratory flow (PEF) busulfan+fludarabine (BuFlu, n = 40). The patients were rates) were determined weekly for 6 weeks. On day +42, lung affected by acute myeloid leukemia (AML, n = 60), acute volumes (vital capacity (VC), forced VC (FVC), functional lymphoblastic leukemia (ALL, n = 12), chronic myeolgenous residual capacity (FRC), forced expiratory volume (FEV) at leukemia (CML, n = 10), myelodysplastic syndrome (MDS, n = 50ms and forced expiratory flow (FEF) at 25ms) were 6), non-Hodgkin's lymphoma (NHL, n = 5), and measured and animals were sacrificed for histopathologic hemophagocytic lymphohistiocytosis (n = 2). We evaluated analysis. Compared to syn and naive controls, allo recipients the efficacy of fludarabine in HLA-identical allogeneic stem showed a significant decrease in MV from week 1 until week 6 cell transplantation compared to cyclophosphamide. due to decrease in RR and TV along with dramatically Results: No significant differences in terms of baseline increasing Te (figure 1). PIF and PEF were also strongly characteristics, stem cell source, type of donor, GVHD reduced, whereas Ti remained normal. A decrease of MV prophylaxis, or infused cell dose were observed between the <45% was associated with increased mortality (p=0.041). two groups. However, a shorter duration of engraftment (P = Lung compliance was reduced as early as one week after 0.001), lower incidence of acute and chronic GVHD (P < SCT (86.8% of naive; p<0.01). Parallel to these changes in 0.001 and P = 0.003, respectively), and non-relapse mortality breathing patterns, allo recipients showed progressive (P = 0.039) were observed in BuFlu group. Furthermore, interstital, alveolar, peribronchial and periluminal inflammatory event-free survival and overall survival were significantly cell infiltrates. In addition, on day+42, animals after allo SCT higher in BuFlu group compared to BuCy group (P = 0.036 demonstrated clear evidence of obstructive changes in PF and P = 0.002, respectively). After adjustment for age, risk when compared to syn controls (FEV50: 58.1 vs. 70.4%, status of disease, GVHD prophylaxis and donor type, BuFlu p=0.04; FEF25: 49.9 vs. 74.3% , p=0.03; % = relative to naive; regimen was independent favorable risk factor for event-free figure 2). VC was also reduced after allo-SCT, although not

S203 survival (HR, 0.165; 95% CI, 0.045-0.062; P = 0.006) and diseases and symptoms were sent to 102 out of 106 patients overall survival (HR, 0.170; 0.037-0.768; P = 0.021). and the answers of 84 (84%) patients responded were Conclusion: From this retrospective analysis we concluded analyzed. that Fludarabine based myeloablative conditioning regimen is Results: The median age of patients at the time of transplant superior to standard BuCy regimen in HLA matched was 38 (range 15-54), and 80 (95%) underwent HCT for allogeneic stem cell transplantation. hematological malignancies. 11 were still on immunosuppressants. The most frequent self-reported health problem was eye problems (64%) followed by bone-skeletal P814 (38%) and skin problems (31%). Neurological, cardiovascular, Low-dose alemtuzumab is preferred in allogeneic stem gastrointestinal, endocrine, and oral problems were also cell transplantation setting with cytomegalovirus reported in about 20-25% of patients. In addition, long-term seropositive recipient and donor survivors reported problems regarding sexuality issues (42%) Y.S. Chae (1), S.K. Sohn (1), J.G. Kim (1), Y.Y. Cho (1), J.H. and financial problems (45%) The mean score of each SF-36 Moon (1), M.S. Hyun (2), K.Y. Kwon (3), D.H. Yang (4), J.J. dimensions of all long-term survivors was the same as that of Lee (4), Y.K. Kim (4), H.J. Kim (4) age- and sex-matched control; however, 25 out of 84 (29.7%) (1)Kyungpook National University Hospital (Daegu, KOR); had SF-36 dimensions which score was lower than that of (2)Yeungnam University (Daegu, KOR); (3)Keimyung control population. The median number of impaired SF-36 University Dongsan (Daegu, KOR); (4)Chonnam National dimensions per patient was 1.8 ranging from 1 to 8. Many of University Hospital (Gwangju, KOR) the self-reported health problems were significantly associated with low scores of SF-36 dimensions except for social Background: Previous studies demonstrated that in vivo functioning in univariate analysis, and dimensions administration of alemtuzumab (an anti-CD52 antibody) was representing physical aspects of QOL were affected more effective in decreasing the incidence of GVHD after allogeneic than those representing mental aspects of QOL. Multivariate stem cell transplantation (SCT). However, major concerns of analysis showed that oral (p=0.01), neuro-psychiatric serious infections have been raised because of its negative (p=0.03), musculoskeletal (p=0.01), endocrine (p=0.04), and impact on the host's immune recovery after transplantation. skin (p=0.006) problems remained as significant factors of Patients and methods: Thirty-five patients receiving allogeneic impairing QOL assessed by SF-36. In addition to those health SCT with alemtuzumab-containing conditioning regimen were problems, financial problems but not problems regarding classified to two groups (low dose, treated with <50mg of sexuality was associated with impaired SF-36 alemtuzumab; high dose group, with • 50mg) based on the dimensions(p=0.01). median dose of alemtuzumab (50mg), and were evaluated for Conclusion: Although indistinguishable in many aspects, long the incidence of CMV antigenemia or disease, clinical term survivors after HCT should be carefully monitored for outcomes and complications. health problems described above to improve their QOL. Results: Among thirty-five patients with hematologic malignancies, 17 (48.6%) were high risk. All recipients (9 BM, 26 PB) and donors were CMV-seropositive before P816 transplantation. The median dose of alemtuzumab was 50mg Prospective Oral Mucositis Audit (POMA): patterns of (range, 20-75). The median survival duration was 14.03 prophylaxis and treatment for oral mucositis months (range, 0.67-24.10) and 2 year survival rate was B. Quinn, N. Blijlevens, S. McCann, P. Bacon, R. Cinnéide, M. 47.0%. The probability of non-relapse mortality at 100 days Schwenkglenks, R. Stone on behalf of the EBMT Mucositis and 2 year was 13.6% and 29.7%, respectively. The Advisory Group cumulative incidence of grade II-IV acute GVHD at day 100 was 24.7%. The overall incidence of chronic GVHD was Objectives: In addition to the primary goal of estimating oral 20.8%, including an 8.3% incidence of extensive chronic mucositis (OM) incidence and duration, the POMA study GVHD. The cumulative incidence of CMV antigenemia was aimed to assess patterns of OM prophylaxis and treatment in 70.2% (n = 24). There were no statistical differences in European practice. engraftment (P = 0.573), acute and chronic GVHD (P = 0.359 Methods: Patients (pts.) with multiple myeloma (MM) or non- and P = 0.335, respectively), relapse (P = 0.849), mortality (P Hodgkin lymphoma (NHL), receiving high dose melphalan or = 0.229), CMV antigenemia (P = 0.227) or overall survival (P BEAM followed by autologous stem-cell transplantation, were = 0.510) between the two groups. prospectively observed until 30 days post transplant or Conclusion: Our study has shown that alemtuzumab was hospital discharge. OM assessments used the WHO scale. effective for GVHD prevention but caused a high incidence of Sites were asked to report OM-related resource use. OM CMV antigenemia regardless of its dose. Thus a low dose onset was used to distinguish prophylaxis from treatment. alemtuzumab (<50mg) seems to be preferable in allogeneic Results: Of 197 evaluable pts. from 25 centres across 13 SCT setting with CMV seropositive recipient and donor both. countries, 109 (55%) had MM and 88 (45%) had NHL. The incidence of ulcerative OM (WHO scale grade 2-4) was 64% and the incidence of severe OM (WHO scale grade 3-4) was P815 44%. Reported frequencies of prophylaxis use (% of all pts.) Self-reported health problems and quality of life in long- were: mouthwash 78%; antifungals 54%; antivirals 44%; term survivors of allogeneic haematopoietic stem cell antibiotics 36%; other 20%; total parenteral nutrition (TPN) transplantation 11%; opioids 8%. Multivariate regression results indicated that S. Okamoto, T. Mori, K. Kiuchi, H. Kaneko, M. Kudo, Y. Ikeda, some prophylactic agents were associated with a reduction in S. Kondo the incidence of severe OM (mouthwash: odds ratio [OR] 0.4, Keio University School of Medicine (Tokyo, JP) 95% confidence interval [CI] 0.1-1.1; antifungals: OR 0.3, CI 0.1-0.6) or the duration of severe OM (TPN: difference of 1.7 Background and Purpose: Much of the research on HCT days, CI 0.6-2.9 days; antibiotics: difference of 3.5 days, CI survivors has mainly focused on major medical events rather 2.4-4.7 days). After the onset of OM, frequencies of treatment than daily health issues and QOL, and systemic information use of agents (% of pts. with any OM) were: mouthwash 84%; has not been available in routine monitoring and management antifungals 70%; antibiotics 55%; antivirals 50%; opioids 47%; of health care needs after 5 years for this population. The aim TPN 37%; other 27%. Treatment use of TPN, opioids and of this study is to clarify health problems and health-related antibiotics increased with grade of OM experienced (p for QOL for 5 years< survivors after HCT. trend ” 0.018; Figure 1). Overall mean (standard deviation) Patients and Methods: Between 1983 and 2001, 204 patients days of use in those who had the respective agents were: with hematologic diseases underwent allogeneic HCT at Keio mouthwash, 19 (6); antivirals, 18 (7); antifungals, 16 (7); BMT Program. 106 are currently alive and disease-free for 5 < antibiotics, 14 (6); TPN, 11 (5); other, 11 (8); opioids, 8 (5). years. Medical Outcome Survey-Short Form 36 (SF-36) Country- and site-specific results were heterogeneous without questionnaires for QOL and self-reported questionnaire for 85 clear patterns being visible.

S204 Conclusion: This study confirmed OM-related medical resource use to be substantial, even if an element of over- reporting due to competing or overlapping reasons of medication use may be present in the data. Some prophylactic agents appeared to be effective in reducing severe OM incidence and duration. Additional results showed a trend toward an increase in resource use with increasing grade of OM experienced.

P818 The impact of treatment-related distress on cognitive functions in candidates for allogeneic stem cell transplantation F. Schulz-Kindermann, L. Schirmer, A. Mehnert, A. Scherwath, A. Zander, U. Koch University Medical Center Hamburg (Hamburg, D) P817 Microsatellite instability in second primary neoplasms Purpose: In the course of high-dose chemotherapy after childhood cancer neuropsychological complications are observed in a significant K.-K. Kober (1), P. Lang (1), J. Greil (2), R. Kandolf (3), R. portion of patients. Remarkably, studies show that cognitive Handgretinger (1), M. Ebinger (1) dysfunctions may be already present before the onset of (1)University Children’s Hospital (Tuebingen, D); (2)University therapy. One reason for this observation could be the Children’s Hospital (Heidelberg, D); (3)Institute of Molecular influence of distress related to the specific treatment Pathology (Tuebingen, D) modalities at admission for HSCT. The aim of the present study was to evaluate the specific effects of treatment Objectives: A defective DNA mismatch repair (MMR) system modalities and treatment-related psychological distress on increases the risk of developing a malignant neoplasm. The cognitive functions before the onset of HSCT. MMR defective mutator phenotype is characterized by Method: Patients scheduled for high-dose therapy and microsatellite instability (MSI). MSI is seen in patients with allogeneic stem-cell transplantation were admitted to an colon carcinoma and associated tumours, in not related ongoing prospective multicenter study. They were assessed tumours a form of MSI called ‘elevated microsatellite with a standardized neuropsychological test battery, alterations of selected tetranucleotide repeats’ (EMAST) is measuring cognitive performance in the domains of memory, observed. In childhood malignant neoplasms a higher rate of learning and attention. Furthermore subjects filled out the predisposing factors has to be expected because of the early Cancer Treatment Distress Scale (CTXD; Syrjala 2005), a onset. Indeed MSI is found in a relevant percentage in self-report questionnaire with 7 subscales related to specific childhood malignancies. Furthermore, individuals suffering features of HSCT. form second primary neoplasms (SPN) are supposed to have Results: Baseline data of 60 patients from one study center an increased load of genetic risk factors. Several publications will be presented. Cognitive impairments were found in a describe a clearly increased rate of MSI in SPN (Gafanovich, subgroup of patients with percentages of impairments ranging Cancer 1999; Casorelli, DNA Repair 2003). To date it is not from 5% to 20%, being most pronounced in the verbal possible to answer the question whether a) the preceding learning domain. Subdimensions of treatment-related distress chemotherapy is inducing MSI and thereby increasing the risk were significantly, but moderately (r=-.26 to -.35) related with of a second neoplasm, or b) the individuals with MSI-positive several areas of cognitive performance. tumours represent a high risk group with primary mutator Conclusion: Regarding cognitive performance phenotype, cumulating two hazardous factors: the basal neuropsychological deficits can be observed in a subgroup of increased risk of developing a neoplasm and the increased patients before the onset of HSCT. The influence of treatment vulnerability to the mutagenicity of the applied chemotherapy. related specific distress should be considered especially in the Patients and methods: MSI is analysed by PCR and fragment situation of admission for HSCT. length analysis. Because of the sensitive methodology Acknowledgements: The study was funded by the Deutsche samples of poor DNA quality can be included, e.g. paraffin José Carreras Leukämie-Stiftung. embedded tissue or bone-marrow smears. Furthermore an approach has been established to evaluate the MSI status without the need of reference tissue (as peripheral blood) to P819 minimize the logistic efforts. From all individuals with SPN Liver dysfunction in long-term survivors of stem cell registered at the German Childhood Cancer Registry (GCCR) transplantation in Mainz (< 400 patients), samples of 172 patients are R. Nathwani, E. Olavarria, E. Kanfer, A. Rahemtulla, D. Marin, collected. J. Todd, J. Apperley, N. Salooja Results: Table 1 shows the results of a pilot study including 47 Hammersmith Hospital (London, UK) samples of SPN and 33 samples of independent primary neoplasms. The incidence of liver dysfunction in asymptomatic long term Conclusions: MSI is more frequent in SPNs than in primary survivors of SCT is unknown. Recognised complications of neoplasms. The predominant form of MSI is EMAST in both SCT which could contribute to abnormal liver function tests groups. If DNA is available, the corresponding primary (LFTs) include GVHD, viral hepatitis and iron overload. We malignancy of the patients with SPN will be evaluated to have evaluated 50 consecutive patients in a dedicated follow- answer the question mentioned above whether MSI is a up clinic for apparently well patients a minimum of 9 years primary underlying defect or a secondary treatment induced post SCT. All patients had the following tests: AST, ALT, phenomenon. The influence of the applied therapy, especially bilirubin, ALP, GGT, ferritin, lipids, hepatitis A,B and C. In of certain high risk elements, e.g. stem cell transplantation patients with abnormal LFTs repeat samples were taken and and administration of alkylating drugs, will be studied. additional tests included liver autoantibodies (smooth muscle and anti-mitochondrial), and abdominal ultrasound (US). Three patients had liver biopsies.

S205 A total of 50 patients (27 male), median age 46y (range 24-71) Only one pt developed transient hyperbilirubinemia and Grade were evaluated a median of 16 (range 9-27) years post SCT. II VOD. There was no increased risk of infections. Acute 40 patients had an underlying diagnosis of CML and the GVHD was observed in only 4 pts (Grade II-3, III-1), while 4 source of stem cells was syngeneic (n=2), allogeneic (n=34) developed chronic GVHD (limited-2, extensive-2 -1 after or unrelated (n=14). stopping immunosuppression). With a median followup of 11 Only 15 (30%) patients had LFTs which were in the normal (2-13) months, ten pts are alive, 9 in CR, while 3 died of range. 35/50 (70%) patients had at least one persistently disease progression. We may conclude that in pts undergoing abnormal LFT and 21/50 (42%) patients had at least one SCT following Dasatinib there is no evidence that Dasatinib parameter greater than twice the upper limit of the normal adversely affect post SCT outcome as no increased transplant range. related organ toxicities, non-engraftment, GVHD or infections Of 7 patients whose transaminases were more than double were observed. Larger studies are indicated to confirm these the normal range (± elevated gamma GT), two patients had preliminary results. hepatits C, one had biopsy proven GVHD and four had a presumptive diagnosis of fatty liver based on US appearance and evidence of dyslipidaemia (cholesterol>6mmol/l, P821 HDL/cholesterol ratio >5). One of these four patients had Permanent telomere erosion after serial autologous stem received a liver transplant 10 years previously for liver GVHD cell transplantation Of 14 patients whose gamma GT was more than double the T. Widmann, H. Kneer, J. Schubert , M. Pfreundschuh normal range (mean 162iu/l range 62-539) without University Hospital Homburg (Homburg, D) significantly elevated transaminases (<2x normal), two had liver biopsies confirming diagnoses of iron overload and Introduction: Hematopoietic stem cells (=HSCs) are known to GVHD. Of the remaining 12 patients, 8/12 had dyslipidaemia have robust mechanisms to overcome short term proliferative and 2/12 had elevated ferritins (>1000ug/l). Of the 8 patients stress during infections, chemotherapy or even single stem with dyslipidaemia, liver US data were available for 6. Three cell transplantation procedures. Here we have studied had an appearance suggestive of fatty change and 1 had telomere length, the ability to mobilize HSCs and proliferative mildly increased echogenicity. Two US scans were normal. Of responses of lymphocytes from Aggressive NHL (=aNHL) the 4 patients with a cholesterol less than 6 mmol/l, two patients and one seminoma patient after repetitive cycles of patients had alcohol consumption in excess of 21 units per high dose chemotherapy. week but there were no additional risk factors for liver disease Methods: 6 previously untreated aNHL patients with an age (ferritin normal, liver autoantibodies negative) adjusted International Prognostic Index (= IPI, IPI • 2) were Long term survivors of SCT have a high prevalence of liver treated in the Phase III MegaCHOEP trial (one course dysfunction of which the commonest cause is hepatic hiCHOEP und 3 courses MegaCHOEP + Rituximab and steatosis associated with dyslipidaemia. autologous stem cell support, including stem cell apheresis before each cycle of high dose chemotherapy). Also one patient diagnosed with seminoma (induction cheomotherapy: P820 cisplatin, Etoposid und ifosphamid for stem cell mobilisation Prior treatment with Dasatinib does not increase and 3 repetitive cycles of high dose chemotherapy with transplant related toxicity in chronic myelogenous Carboplatin and Etoposid, including stem cell support from the leukaemia and Ph+ acute lymphoblastic leukaemia first apheresis procedure) was studied. Telomere length M. Leiba (1), A. Shimoni (1), F. Guilhot (2), G. Martineau (2), analyses by FlowFish were done before and after repetitive M. Renaud (2), S. Francois (2), I. Hardan (1), M. Schleuning cycles of chemotherapy and at a follow up date. Furthermore (3), A. Nagler (1) the amount of mobilized CD34+ HSC in apheresis products (1)Chaim Sheba Medical Center (Tel Hashomer, IL); (2)CHU after each cycle of chemotherapy was determined in n=17 La Miletrie (Poitiers, F); (3)Stiftung Deutsche Klinik for aNHL patients. Flow-cytometric analysis of proliferative Diagnostik (Wiesbaden, D) responses from CD3+ lymphocytes was quantified (n=4). Results: Telomere length decrease significantly in after three Dasatinib is a dual SRC/ABL TK inhibitor being used in autologous transplantations in granulocytes (p<0.048) but not advanced Chronic Myelogynous Leukemia (CML) or refractory in lymphocytes (p=0.20) from aNHL patients. Even follow up Ph+ Acute Lymphoblastic Leukemia (ALL). Most of these pts samples showed that telomere shortening could not be will eventually undergo SCT, raising the question of whether compensated by replicative reserve of HSCs. In contrast, pre-SCT Dasatinib therapy will increase transplant related telomere length was stable in one seminoma patient. complications and/or adversely affect outcome. We report Furthermore, lymphocyte proliferation decreased alongside thirteen pts: CML–11 (CP1-4, low sokal score–3, high sokal with telomere shortening in the lymphocyte compartment, but score–1, CP2-2, AP-3, BC-2) and Ph+ ALL –2 who received was not statistically significant (n=4, decrease from an Dasatinib prior to alloSCT (n=12) or autoSCT (n=1). Donors average of 27.0% to 16.0%, p=0.3). Parallel to telomere were matched unrelated–7, matched siblings–4 or mismatch shortening, the percentage of mobilized CD34+ HSC per total related donor (haploSCT) –1. Seven were male and 6 female leukocytes during leukapheresis decreased significantly with with a median age of 45 (16-56) years. First line therapies each consecutive cycle of apheresis/ stem cell transplantation included Hydroxyurea or Interferon followed by Imatinib for the in patients with aNHL (average 6,18% during 1st apheresis CP CML pts and chemotherapy followed by Imatinib for the procedure to 1,49% for the 3rd apheresis procedure, advanced CML and Ph+ ALL pts. All pts subsequently p=0.006). received Dasatinib 70mg x 2/day due to resistance to Imatinib, Conclusion: Our study demonstrates that repetitive resulting in complete hematological response in 11 pts., as autografting causes permanent telomere erosion in aNHL well as complete (n=6) or partial cytogenetic response (CyR) patients granulocytes and results in significant reduction of the (n=2) prior to SCT. Three pts did not achieve CyR prior to HSCs mobilized to the peripheral blood. SCT. The pts were conditioned with either a myeloablative protocol (n=8) or a reduced intensity protocol (n=5). GVHD prophylaxis consisted of CSA and MTX (n=11) or complete T- P822 cell depletion (n=1). Pts received a mobilized peripheral blood Abnormal liver enzymes 2 years after haematopoietic stem cell graft (n=12) or BM graft (n=1) with 8.7 (5–24) x 106 stem cell transplantation in children: prevalence and risk CD34+ cells/kg. The pt that underwent autoSCT was factors successfully mobilized (2 apheresis cycles yielding 5.7 x 106 D. Bresters, L.T. Muller, P.F. Egberts, R. Wolterbeek, I. van CD34+/kg). Dasatinib was stopped 6 days before mobilization. Gils, A. Lankester, R. Bredius, J.J. Schweizer All pts successfully engrafted reaching ANC > 0.5x109/L on LUMC (Leiden, NL) day +17 (11-22) and PLT >20x109/L on day +17.5 (11-32). Chimerism was 99.4–100%. Transplant related toxicities were Several studies have shown that the prevalence of minimal. Only 4 pts developed severe (>Grade III) mucositis. persistently or recurrently elevated liver enzymes late after

S206 haematopoetic stem cell transplantation (SCT) varies from 40 cyclophosphamide in 17 pts, treosulphan and fludarabine to 58%. Aetiology includes viral hepatitis, iron overload, auto- (n=10), cyclophospamide alone (n=4), total body irridation with immune hepatitis and/or chronic graft versus host disease cyclophosphamide (n=5), treosulphan and cyclophosphamide (cGvHD) in a majority of these patients, but in some aetiology (n=3), busuphan and fludarabine in 1 patient and others remains unclear. regimens in remaining 4 patients. GVHD prophylaxis Aim of the study: was to establish the prevalence and clinical consisted of cyclosporine (CsA) and methotrexate (Mtx) in 39 relevance of elevated liver enzymes in children surviving long pts, Mtx and prednisone in 7 pts and CsA alone in 3 patients. term after SCT in a country with a low prevalence of HBV and The median number of transplanted mononuclear cells was: HCV among blood donors. 4.74x108/kg (range 0.1-26.4) including CD34+ cells- Methods: A single centre retrospective study was performed, 3.02x106/kg (range 0.9-21.7). Granulocyte engraftment in which AST and ALT values from before SCT until 2 years >0.5x109/L occurred after median of 21 days (7-32) and after SCT were collected in all children transplanted between platelet exceeded >50x109/L after median of 23.5 day (12- 1980 and 2002. Patient characteristics such as age, sex, 109). Acute GVHD developed in 23 patients (52%). Six diagnosis, type of transplant and conditioning regimen and patients (13%) transplanted with major and bi-directional post-transplant complications involving the liver (veno- ABO-incompatibility developed PRCA. The long-term occlusive disease, acute GvHD, viral reactivation) were treatment using different strategies (plasmapheresis, EPO, collected. We applied the Chi-square test and univariate CsA off, donor lymphocyte infusion, rituximab) resulted in logistic regression analysis to analyse possible risk factors for erythroid recovery in 5 out of 6 patients after median of 13 abnormal liver enzymes 2 years after SCT. months (3-16). Median follow-up of PRCA group is 37 Results: 290 of 455 patients who underwent SCT were alive 2 months(16-51).Median RBCs transfusion was 10.1 litres (2.2- years after SCT. In survivors median age at SCT was 7.1 15.9). years and the majority were transplanted for a haematological Conclusions: PRCA after ABO-incompatible alloSCT requires malignancy and with bone marrow of a matched sibling donor. long-lasting treatment, but majority of patients achieve In 59% of patients conditioning included TBI. AST and ALT complete erythroid recovery. were assessed at 2 years after SCT in 210 patients and values were above normal in 97 (46%). In 22 patients (10.5%) AST and/or ALT value was at least twice the upper limit of normal. In 15 of these 22 patients aetiology could be studied P824 and was consistent with iron overload in 7, including one with Total parenteral nutrition for patients undergoing severe haemochromatosis, portal hypertension and autologous stem cell transplantation: comparison of oesophagus varices, cGvHD in 4 (1 died) and unknown standard, glutamine alone and glutamine plus fish-oil N-3 aetiology in 4 patients. None of these 15 patients had chronic fatty acid enriched regimens viral hepatitis. Risk factors for elevated liver enzymes were M. Postorino, L. Cudillo, M. Lupattelli, A. Picardi, L. also studied and will be presented. Franceschini, R. Cerretti, C. Ditto, G. De angelis, L. Di caprio, Conclusions: The prevalence of abnormal liver enzymes in our W. Arcese centre is not different from the prevalence reported in previous Policlinico Tor Vergata (Rome, I) studies. Like previous studies, ours showed that all but 2 of 97 patients with abnormal liver enzymes were asymptomatic. Background: Glutamine (GLN), usually defined a “non Aetiology was different from that reported in other studies essential” amino acid, is one of the most important substrate however, i.e. no chronic viral hepatitis was found. Longer for ammonia genesis in the gut and for acid-base homeostasis follow-up is needed to establish the clinical relevance of regulation in kidney. Moreover, it plays an important role in the elevated liver enzymes late after SCT. glutathione’s synthesis and in promoting the growth of fibroblasts, lymphocytes and enterocytes. On the other hand, the fish-oil-derived n 3 fatty acids (n-3FA) have been referred P823 to interfere with inflammatory and immune response by Resistant pure red cell aplasia following major and bi- regulating prostaglandins, Tromboxane A2 and leukotrienes directional ABO incompatible allogeneic stem cell synthesis. transplantation – recovery of donor-derived Patients and Methods: From 2000 to 2002, 29 patients erythropoiesis after long-term treatment using different undergoing autologous SCT for malignant hematological therapeutic strategies diseases entered the study comparing standard TPN regimen G. Helbig, B. Stella-Holowiecka, J. Wojnar, M. Krawczyk, S. (Group A: n = 16) with a TPN regimen containing GLN Kyrcz-Krzemien, M. Sadus-Wojciechowska, M. Markiewicz, M. (Dipeptiven, Fresenius) at dose of 0.22g/kg/day (Group B: n = Kopera, I. Wylezol, J. Holowiecki 13) The patients in the 2 groups were comparable for age, Silesian Medical University (Katowice, PL) diagnosis, pre-transplant therapy and pre-transplant conditioning regimens. Isonitrogeneous and isocaloric TPN Background: Pure red cell aplasia (PRCA) following ABO- was started on day +1 after SCT and discontinued when mismatched allogeneic stem cell transplantation (AlloSCT) patients recovered oral intake for 50% of nutritional support. results from the presence of recipient-derived, residual No difference in terms of gastro-intestinal toxicity and general lymphocytes B or plasma cells, which can produce patient outcome was observed between group A and B. isohemagglutinins against donor erythroid cells. From 2003 to 2005, 16 patients with similar characteristics Material and methods: Between 1993-2006, 548 patients received TPN containing GLN at dosage 0.22 g/K/day plus underwent alloSCT for malignant and non-malignant fish-oil n-3 fatty acid ( n-3FA Omegaven, Fresenius) at dosage haematologic disorders. In 44 (8%) we revealed major and of 1.5ml/kg.These patients (Group C) were retrospectively major-minor ABO blood group incompatibility between donor compared with the other two groups (A and B) of patients for a and recipient. Bone marrow grafts were major ABO number of variables: length of hospitalization, number days on incompatible in 30 patients, there was bi-directional mismatch TPN, duration of febrile neutropenia, number days on in remaining 14 patients. antibiotic therapy, use of antifungal drugs, days to PMN Results: Stem cell donors were HLA identical siblings in 26 engraftment, grade of mucositis, diarrhea and hemesis. patients, 18 recipients received transplant from unrelated Results: The use of GLN -EPA enriched TPN (group C) was donors. The source of stem cells were: bone marrow (BM) in significantly associated with lower length of hospitalization (p 30 patients, peripheral blood (PB) in 9 pts and BM+PB in 5 =0.001), lower duration of febrile neutropenia (p=0.006) cases. 21 patients received transplant for acute myeloid reduced number of days on antibiotic therapy (p=0.008), and leukemia, 10 for acute lymphoblastic leukemia, 7 for chronic on TPN (p=0.004), reduced use of antifungal drugs and lower myeloid leukemia, 2 for severe aplastic anaemia, 1 for incidence and severity of mucositis. No difference was paroxysmal nocturnal haemoglobinuria, 1 for myelodysplastic observed for number of days to PMN engraftment. syndrome, 1 for Hodgkin lymphoma and 1 for multiple Conclusion: Our retrospective study strongly suggests that a myeloma. Conditioning regimens consisted of busulphan and number of clinical outcomes can be improved in patients

S207 undergoing an autologous transplant by the use of GLN-n3FA contacted by the first author at a median time of 12,5 years enriched TPN and provides the clinical base for designing a after transplant ( range: 10-23,8y). All of them agreed to prospective randomized trial. answer a set of questionnaires regarding QOL. Gender: 137M/75F. Median age at transplant: 21y(3-51y) Median age at evaluation : 34,8y (18-64y). All pts received bone marrow P825 from related (208) or unrelated (4) donors. 18 pts received a Keratinocyte growth factor reduces mucosal damage and 2nd transplant. Malignant diseases: 42,5%. Non malignant consecutively the need for intravenous antibiotic diseases:57,5%. 212 adults recruited at a regional blood bank treatment following autologous and allogeneic were used as controls.Median age:34,4y. haematopoietic stemcell transplantation Gender:103M/109F(p<0,001). W. Grothe, M. Bauer, A. Rupprecht, M. Christopeit, L. P. Results: Quality of life as well as overall health was Mueller, A. Muetherig, S. Theurich, T. Weber, G. Behre significantly better in pts than in the control group (p< 0,001). University of Halle-Wittenberg (Halle, D) They also had a better functional well being when compared to controls (p<0,001). Even though most pts came from a low Background: Oral mucositis (OM) and intestinal mucositis (IM) socio-economic background there was no significant following autologous and allogeneic hematopoietic stem-cell difference between social classes or academic level. Pts had transplantation (HSCT) result in significant morbidity. more fatigue, more sexual dysfunction (especially women) Treatment with keratinocyte growth factor (KGF) reduces the and more cognitive deficits when compared to controls but extent of OM and IM. The extent of IM is difficult to determine. these symptoms did not change what they felt about their Citrulline, an amino acid mainly synthesized in the small overall health status. No statistical difference was found intestine, serves as a marker of small intestine injury. Aim of between both groups regarding mood disorders (depression the present study was to determine the extent of OM and IM or anxiety) or family and social well being. No difference was before and after the introduction of KGF in the treatment of found between pts having GVHD or not. 53pts transplanted OM and IM. below the age of 13 remember very little about the procedure Methods: Since 05/2006 the extent of OM and IM in but confirm the importance of family support at that time. autologous and allogeneic HSCT was evaluated Conclusions: This study shows that despite living in a prospectively. Beside routine clinical and laboratory values developing country and coming from a low social economic including the daily oral mucositis score (DMS) and daily gut background, pts were able to reach similar and sometimes score (DGS) the plasma citrulline level was determined in better indices related to QOL when compared to normal health week 1 (W1), week 2 (W2) and week 3 (W3) following HSCT. controls. Sexual dysfunction, fatigue and cognitive deficits Moreover the dosage of therapeutic intravenous (i.v.) should be followed closely after transplant in order to improve antibiotic treatment with meropeneme, QOL in surviving pts. piperacilline/combactam and teicoplanin in addition to antibiotic prophylaxis was noted. Since 09/2006 KGF (60µg/kg body weight per day, for 3 days before conditioning therapy P827 respectively after HSCT) was applied to all patients (pt) Incidence of second malignant neoplasms after undergoing HSCT in our institution. Pt with oropharyngeal conditioning with total body irradiation before bone radiotherapy were excluded from analysis. marrow transplantation Results: Until 11/2006 20 pt (2f/18m) were evaluated. 15 of Z. Elsayed, M.P. Sunyach, H. Ghesquiere, C. Sebban, P. these pt underwent autologous and 5 allogenic HSCT. 5 of the Biron, C. Carrie autologous and 3 of the allogenic transplanted pt received Centre Léon Bérard (Lyon, F) KGF. In pt with KGF clinically significant OM and moderate to severe IM were seen less often (W1: 1/8 pt vs. 3/12 pt and Purpose: To determine the risk of second malignant W2: 1/8 pt vs. 2/12 pt without KGF). CRP (in mg/l) was lower neoplasms in patients who had total body irradiation (TBI) as in the group of pt with KGF (W2: average 69.9 (min 9.4 – max a part of conditioning regimen before bone marrow 174.2) vs. 109.9 (min 6.0 – max 259.8) in pt without KGF). All transplantation (BMT). these differences were statistically non significant. Citrulline Patients and methods: Between 1980 and 1998 500 patients serum levels (in µmol/l) were significantly higher in pt underwent intensive chemotherapy withTBI (8-12 Gy in 4-6 receiving KGF (W2: average 24 vs. 15.2 in pt without KGF (p fractions/2-3 days in 90% of patients). Patients were included = 0.002)). The need for therapeutic i.v. antibiotics was lower in a program of allogeneic(22.4% ) and autologous (77.2% ) for pt receiving KGF (average of 5.43 days vs. 13.23 days for BMT The majority of cases were Non Hodgkin's lymphoma pt without KGF (p = 0.037)). Subgroup analysis confirmed the (NHL) (233 patients), neuroblastoma (92 cases ) and acute described tendencies in autologous HSCT. lymphatic leukemia(87 cases) .Age of the patients ranged Summary: Even in this small number of pt a positive effect of from 1-70 years .All cases that developed second KGF on oral and intestinal mucositis was detectable. The malignancies were recorded and the incidence was compared reduced need for therapeutic i.v. antibiotics seen as a to that in the general population. tendency in this study should be re-evaluated in a larger group Results: Median Follow up from BMT was 8.5 years (range 5- of pt to prove the cost effectiveness of KGF treatment. 21 years). Twenty second malignancies were observed (18 solid tumors and 2 acute mylogenous leukemia).The most frequent malignant diseases were neoplasms of the thyroid P826 (4patints ) and lung (3 cases ) One of the patients developed Quality of life 10 or more years after bone marrow 2 second malignancies (skin and parotid).There were 11 transplantation: an analysis of 212 patients transplanted males and 8 females with a median age of 35 (range 2-64) at in a single centre the time of BMT.Median time to develop a second malignancy M. Doro, C. Bonfim, M. Bitencourt, C. de Medeiros, J. Zanis was 5 years (range 1-17).The initial diagnosis was NHL in 9 Neto, I. Kafka, E. Cesario, J. Pelaez, R. Pasquini patients (47.4%), 15 patients (78.9%) had autologous BMT Federal University of Parana (Curitiba, BR) .Second malignancy was the cause of death in 13 patients. Patients’ characteristics and type of conditioning Although we find in the literature a great number of recent chemotherapy regimen used were investigated for their ability publications, there is still limited data regarding quality of to predict the development of second cancer and none was life(QOL) for pts transplanted in developing countries. Low found to be significant. income, low level of education and sanitary measures will Conclusion: Long follow up for patients who received TBI directly affect the life of pts transplanted in these countries before BMT demonstrates an increased incidence of second and QOL should be analyzed within this scenario. malignant neoplasms which can be an important cause of Material and methods: 312 pts, 18 years or older and death in long-term survivors of BMT. This incidence continues surviving more than 10 years after transplant were identified in to rise and prolonged follow up is extremely important. our registry. 100 pts were lost to follow-up and 212pts were

S208 P828 to the Seattle criteria and classified retrospectively as mild, Relationship between the mucositis seen after the moderate and severe. allogeneic haematopoietic stem cell transplantation and Results: Out of 133 allogeneic transplants, we detected 22 the human leucocyte antigen types cases of VOD with an overall incidence of 16%. The general G. Kabacam (1), E. Akcaglayan Soydan (2), P. Topcuoglu (2), characteristics of this patients are showed in Table 1. There K. Dalva (2), P. Ozdemir (3), M. Ozcan (2) were 12 male and 10 female. 2 patients had undertaken (1)Ataturk Research and Education Hospital (Ankara, TR); double autologous transplants. The underlying disease and (2)Ankara University Faculty of Medicine (Ankara, TR); other clinical characteristics are described in Table 1. (3)Hacettepe University Faculty of Medicine (Ankara, TR) Pretransplant elevated serum AST concentration was described in 3 patients (13%, median ALT 101 UI/L, range 70 Oral mucositis (OM) is an important complication of allogeneic to 136). The VOD prophylaxis was low molecular weight hematopoietic stem cell transplantation, that leads to severe heparin (LMWH) in 15 (54%) patients, ursodeoxycholic acid 1 morbidity and mortality, characterised by immunological (4,5%) or both in 6 (36%) patients. Use of specific antibiotics mechanisms in its pathogenesis. In the literature, diseases that have been associated with VOD was as follows: acyclovir like Behçet’s Disease and Systemic Lupus Erythematosus, (81%), vancomycin (18%) and amphotericin (13%). 18 with similar inflammatory ulcerative oral lesions, are found to patients required diuretics and other supportive therapy and 4 have pathogenetic relationships with HLA system. patients (18%) were treated specifically with defibrotide. VOD Objectives: Aim of this study was to define, if there is any was graded as mild in 4 (18%), moderate in 15 (68%) and relationship between oral mucositis seen in posttransplant severe in 2 (9%) patients. There was only one death setting and the HLA system. attributable to VOD and multiorgan failure. Material and methods: Study was conducted retrospectively in Conclusion: The overall incidence of VOD in our series was 202 patients of a single transplant center, who were 16%, similar to that described by other groups. We did not find transplanted in previous 5 years. To increase the statistical any specific correlation, probably due to the low number of power of the results, corrected p values according to events. New prophylactic and treatment approaches are Bonferroni method were used in determination of significance. required to decrease the incidence, morbidity and mortality of Results: Post-transplant mucositis occured in 160 (79,6 %) of this complication. the patients, which was mild in 31,2 %, and severe in 68,5 % of them. Duration of the mucositis was 12,5 (±7) days. As an indicator of severity, need for total parenteral nutrition and/or narcotic analgesic treatment was seen in 42,7 % of the cases. HLA-DRB11 antigen was detected to be a strong protective factor against the development of mucositis and the severity of mucositis (OR: 0,33, p=0,006). HLA-A24 (OR: 0,30, p=0,02), B53 (OR: 0,02, p=0,02), and DRB15 (OR:0,31, p=0,04) were protective against mucositis. A24 was also a decreasing factor for severity of the mucositis (Trends test: P830 0,014). All of the cases expressing HLA-A33 and DRB16 Transplantation from related and unrelated donors in phenotype developed mucositis, so these antigens are elderly patients shows equal toxicity and survival thought to be risk factors for OM. Mucositis severity was M.Y. Shapira, P.D. Tsirigotis, I.B. Resnick, M. Bitan, S. higher in cases with HLA B41 antigen (OR: 26.3, p=0,02). Samuel, S. Elad, B. Gesundheit, R. Or, S. Slavin HLA-A30 (OR: 8,23, p=:0,03), A68 (OR:46.5, p=0,02), B8 Hadassah University Hospital (Jerusalem, IL) (OR:3,77, p=:0,03), and B15 (OR: 21.6, p=0,008) expressing patients are susceptible for severe mucositis that needs Most of the hematological malignancies where allogeneic therapeutic intervention. Patients with HLA-C3 phenotype had stem cell transplantation (SCT) is a therapeutic option occur in lower risk for severe mucositis requiring therapeutic elderly patients. Additionally, in this age group, the probability intervention (OR:0,24, p=0,04). of finding a matched sibling donor decreases due to mortality Conclusion: Some specific HLA groups are related to and co-morbidities of patient s siblings which may exclude development of post-transplant mucositis. This shows the role them from donation. In such case, SCT from an unrelated of immune system in the pathogenesis of oral mucositis so in donor (UD) can offer a therapeutic solution for elderly patients the future, patients expressing these HLA antigens should be in need although such a procedure is considered more evaluated accordingly about the risk of developing mucositis hazardous than SCT from a matched sibling (mostly due to in the clinics. procedure and GVHD related morbidity and mortality). We present our data on transplantation, in 68 elderly (age • 55 years) patients that underwent SCT from a matched sibling P829 (n=51) or UD (n=17) mostly using fludarabine-based regimen Veno-occlusive disease of the liver: incidence and clinical (table 1). outcome in 133 allogenic haematopoietic stem cell Median recipient age was 58.3y in the sibling group and 60.8y recipients from a single institution in the UD group (NS) (range 55-69.5y and 55-75y, V. Pérez Andreu, J.B. Nieto, F. Iniesta, M. Blanquer, M.J. respectively). With a median follow-up of 40 months of the López, M.J. Martínez, J.M. Moraleda survivors after sibling SCT, the 1-year OS was 32%. Similarly, Hospital Morales Meseguer (Murcia, E) in the UD group the 1-year OS was 35% (survivors median follow-up 48.5m) (NS). Transplant related mortality occurred in Objectives: Veno-occlusive disease of the liver (VOD) is a 22/49 (45%) and 9/17 (53%) of the patients transplanted from serious complication following hematopoietic stem cell sibling or UD respectively (NS). Occurrence of acute GVHD transplantation (HSCT) with broad ranges of incidence was noted in 26/43 and 7/11 (figure 1) evaluable patients variability among institutions. Our goal was to determine the transplanted from matched sibling or UD respectively (NS). incidence, severity, and outcome of VOD in a uniform series Diagnosis of chronic GVHD was made in 50% (14/28) and of patients that underwent allogeneic transplantation in the 63% (5/8) of the patients in the sibling and UD group last ten years at our hospital. correspondingly. GVHD related mortality occurred in 11 and 2 Methods: We reviewed the clinical charts of all consecutive patients, respectively. patients that underwent allogeneic HSCT from January 1996 We conclude that using fludarabine based regimen, allogeneic to January 2006. Data collection included variables known to SCT from an UD is equally effective as in patients undergoing affect VOD: baseline haematological disease and phase, SCT from matched family member. Age per-se should not be conditioning regimen, VOD prophylaxis, preexisting liver currently used as an exclusion criterion for either initiation of disease, use of specific antibiotics during HSCT and source of donor search or SCT from UD. haematopoietic cells. VOD was diagnosed clinically according

S209 grade and no evidence of apoptotic changes. Platelets thrombi were rarely observed. Conclusion: i-TAM could be defined pathologically as ischemic enterocolitis with microangiopathy and non-apoptotic enteropathy. i-TAM was high frequent HSCT complication than gut-GVHD. Reference to specific findings is need for differential diagnosis between i-TAM and gut-GVHD.

P832 Comparing efficacy and toxicity of conditioning regimen “treosulfane + cyclophosphomide” versus “busulfane + cyclophosphomide” in allogeneic haematopoietic stem cell transplantation N. Stancheva, E. Semenova, N. Zubarovskaya, O. Paina, E. Morozova, S. Bondarenko, E. Darskaya, L. Zubarovskaya, B. Afanasyev St. Petersburg State Pavlov Medical University (St. Petersburg, RUS)

Myeloablative conditioning regimens have severe organotoxicity with different complications. The goal of many investigations is to find the medicine with sufficient myeloablative effect and lower organotoxicity. One of candidates with such options is treosulfan (“Medac”). The aim of our study: to estimate efficacy and toxicity of two conditioning regimens in allogeneic haematopoietic stem cell transplantation (allo-HSCT): treosulfan (TREO) + cyclophosphamid (CY) (1-st group) and busulfan (BU) + CY (2-nd group). Patients and methods: First group includes 10 patients (pts), follow-up is from Feb, 2004 till Nov, 2006: 6 boys, 4 girls by age from 1 till 10 y.o. (mediana –7.1y.o.), ALL-9 pts (relapse- P831 3 pts, 1 complete remission (CR) –1 pts, 2 CR –5 pts), AML Histopathology of intestinal type of stem cell in 2 CR -1 pt. Conditioning regimen included TREO (10 g/ m² transplantation associated microangiopathy IV -1 pt, 12 g/m² IV -3 pts, 14g/m² IV-6 pts) administrated at M. Ito, M. Fujino, T. Ohba, Y. Kuwatsuka, K. Miyamura, Y. day -6,-5,-4 and CY (60mg/kg IV) given at day -3,-2. HSC Kodera donors were 2 matched related donor (MRD), 8 matched Japanese Red Cross Nagoya 1st. Hospital (Nagoya, JP) unrelated donor (MUD). Follow –up 2-nd group pts was from November 2000 till Aims: Hematopoietic stem cell transplant (HSCT) -associated January 2006 and consisted of 36 children (19 boys, 17 girls), microangiopathy (TAM) is one of the important complication. age corresponded to 1-st group (mediana –10.6 y.o.). AML –6 We have previously reported the intestinal type of TAM (i- pts (1CR –3 pts, 2 CR-2 pts, relapse –2 pts), ALL –25 pts (1 TAM), which was clinically similar to gut GVHD, worse CR-1 pt, 2 CR-12 pts, 3 CR -7pts, prognosis and might be associated with immunosuppressant. relapse- 5 pts), relapse of NHL –1 pts, CML -3 pts (1 chronic Gut GVHD is well documented pathologically as apoptotic phase (CP)-2 pts, 2 CP -1pt), Kostmann’s syndrome –1 pt. enterocolitis. However, i-TAM has not been well described HSC donors were 10 donor MRD, 26 MUD. Conditioning pathologically. Aims of this study are to establish the regimen was BUCY (BU 4 mg/kg, orally at day -7,-6,-5,-4 and histological criteria of i-TAM distinguishing from gut-GVHD CY 60mg/kg IV at day -3,-2). and to show the frequency of i-TAM. In both group prophylaxis of acute graft-versus-host-disease Cases and pathological examinations: We have reviewed 74 (aGvHD) included standard CsA dose and short course of cases of biopsy materials under colonoscopy from our SCT MTX. Unrelated allo-HSCT pts received ATG (“Pfizer”) 20 case file (1998-2006). Non-GVHD gut complication cases mg/kg b.w. at days –3,-2,-1. were selected by following eligibility, without evidence of acute Results: 1-st group vs 2-st group: 2-year overall survival (OS) skin GVHD, no evidence of CMV infection, biopsied within 3 60% vs 40%, respectively. Common toxicity criteria (CTC) days after clinical symptoms and without additional III/IV: hepatic toxicity 30% vs 33.5 %, VOD 0% vs 2.7%, immunosuppressive therapy. All cases were histologically mucositis 20% vs 69.4%, severe neurology symptoms 0% vs examined focusing on the characteristic findings; 1. apoptotic 8.3%, aGvHD 30 % vs 25%, chronic GvHD 44.8% vs 47%, enterocolitis with intraepithelial lymphocytes infiltration (gut- engraftment at day +17 vs +14, primary non engraftment 0% GVHD), 2. microangiopathy of capillary and small vessels, 3. vs 11.1%, early transplant-related mortality 20% vs 25%, cryptogenic epithelial cells degeneration without apoptosis, 4. respectively. crypt ghost with wedge shaped ischemic changes, 5. residual Conclusion: Treosulfan - based conditioning regimen is well neuroendocrine cells nest, 6. platelet thrombi, 7. hemorrhage tolerable, safe, and efficient. In contrast with BU+CY this (2-7 were i-TAM), 8. CMV inclusion cells. preparative regimen has lower toxicity and can be used in Results: 5 cases were fulfilling the eligibility and also clinical heavy-pretreated children having severe complications after TAM criteria. 4 of them were showed i-TAM as ischemic colitis previous chemotherapy. with microangiopathy, non-apoptotic enterocolitis and 1 of CMV colitis, respectively. These 4 cases were showed microangiopathy, endothelial cells degeneration, wall thickening with myxofibromatous degeneration and decreased vascular myocyte. Cryptogenic degeneration without apoptosis was observed in all. Platelet thrombi were observed in 1 case. Residual neuroendocrine cells nests were observed in 2 cases. 74 cases were histologically subdivided into 8 of gut-GVHD, 10 of gut-GVHD and i-TAM, 33 of i-TAM, 8 of CMV enterocolitis and 16 of others by according histological findings. i-TAM cases had each 2-5 findings with various

S210 P833 None of these pts showed any signs of or the Long-term outcome of allogeneic haematopoietic cell presence of anti-erythrocytes antibodies before conditioning. transplant recipients with relapsed or secondary ATG (Fresenius) was given either at the dose 10 mg/kg/day malignancies after prior autologous haematopoietic cell for 4 consecutive days or 20 mg/kg/day for 3 consecutive transplantation days. Eight of 10 pts (80%) showed elevated bilirubin and A. Czyz (1), W. Knopinska-Posluszny (2), L. Gil (1), J.M. LDH levels, all pts (100%) showed elevated free plasma Zaucha (2), A. Lojko (1), A. Hellmann (2), M. Komarnicki (1) hemoglobin and decrease in haptoglobin. Nine of 10 pts (1)University of Medical Sciences (Poznan, PL); (2)Medical (90%) showed decrease in hemoglobin level more than 20g/L University School of Gdansk (Gdansk, PL) after the 2nd dose of ATG. Platelet count decreased rapidly in all pts (100%) during the administration of ATG. Number of The allogeneic hematopoietic cell transplantation (alloHCT) in substituted packed red blood cells units ranged from 2 to 8 patients with relapsed or secondary hematological units and number of units ranged from 2 to malignancies occurring after autologous hematopoietic cell 5 during 5 days from the start of the ATG. Screening papain- transplantation (autoHCT) is the potential curative salvage tests gave unspecific positivity, but both the indirect and direct treatment option. However, cumulative toxicity of chemo- and antiglobulin tests were negative. We performed 5 tests to radiotherapy remains the main cause of early mortality. We detect the reactivity of serum with erythrocytes incubated with evaluated long-term outcome of alloHCT following autoHCT ATG and the reactivity of serum in the presence of ATG. We after median 24 (5-73) months in 14 pts, median age 38 (27- compared them with negative controls. Both tests with 53) years with relapsed or secondary hematological erythrocytes incubated with ATG and tests with ATG malignancies. The indication for alloHCT was relapse of AML administration to serum-erythrocytes reaction were positive for (4 pts), CML (3 pts) or lymphoma (5 pts: 2-HD, 1-NHL, 2-MM) hemolysis. and secondary AML after lymphoma treatment in 2 pts. Conclusion: ATG causes hemolysis laboratory and also Different conditioning regimen were used: fludarabine-based clinically. The action of ATG is doubled – in immunocomplex reduced intensity regimen in 9 pts, TBI/Cy in 2 pts, and reaction and the adsorbtion on pts erythrocytes but the direct Treo/Cy, BuCy2, and BEAM respectively in 3 other pts. The antiglobulin test was never positive. We can only speculate patients received infusion of filgastrim-mobilized PBSC (11 that the antibody reaction passed but the antibody is unstable pts), bone marrow cells (1 patient) or cord blood (1 patient) a can be detected only in enzymatic tests. ATG administration from HLA-matched sibling (11 pts), HLA-mismatched sibling is linked with potentially clinically serious hemolysis requiring (1 patient) or unrelated HLA-matched donor (1 pts). GVHD therapy and with unspecific immunohematological reactions prophylaxis consisted of CsA and a short course of MTX in 10 for hemolysis. pts or CsA and MMF in 4 pts. ATG was added in 5 pts. Two patients developed non-fatal early complication of vascular origin (SOS- 1, capillary leak syndrome-1). The incidence of P835 acute GVHD grade II-IV was 29% (4/14 pts). Chronic GvHD Secondary failure of platelet recovery after allogenic occurred in 7/12 (58%) pts surviving day 100, extensive in 6 haematopoietic stem cell transplantion cases. With a median follow-up of 20 (2-123) months 6/14 pts T. Maksud, B. Padhye, A. Bakshi, K. Prabhash, N. Karanth, P. died, 1 in first 100 days after alloHCT due to multiorgan- Parikh dysfunction syndrome and 5 after 100 days (GVHD-2, Tata Memorial Hospital (Mumbai, IND) relapse-1, pulmonary hemorrhage- 1, HUS-1). OS and PFS were 45% (95% CI 14-76) and 47% (95% CI 17-78) Objective: Late persistent decline in platelet count after respectively at 5 years estimated with the Kaplan-Meier primary engraftment of platelets not due to relapse, called method. The cumulative incidence of NRM was 46% (95% CI secondary failure of platelet recovery, (SFPR) is a significant 24-90). problem after allogenic hematopoietic stem cell Conclusions: Our findings on a limited number of pts transplantation (HSCT). The reported incidence in literature is confirmed that alloHCT may offer a sustained remission in approximately 20%. We analyzed the incidence, clinical high proportion of poor prognosis patients with relapsed or features, risk factors and outcome of SFPR in Indian patients secondary hematological malignancies after autoHCT with undergoing allogenic hematopoietic stem cell transplantation. acceptable early toxicity, but still high NRM. Reduced intensity Methods: We retrospectively analyzed the incidence and or low-toxicity conditioning regimen should be considered to outcome of SFPR in Indian patients undergoing allogenic reduce the rate of side effects. High incidence of chronic HSCT. SFPR was defined as decline in platelet count below GVHD remains the main cause of morbidity. It suggests that 20,000/mm3 for 7 consecutives days or requiring platelet new GVHD prophylaxis strategy should be studied in further transfusion after achieving sustained platelet count over clinical trials for heavily pretreated patients with advanced 50,000/mm3 without transfusion for 7 consecutive days after hematological malignancies. hematopoietic stem cell transplantation.(HSCT). Results: The study population consisted of 210 consecutives patients who underwent allogenic stem cell transplantation at P834 our centre between 1983-Aug 2006. Majority of these patients Haemolysis accompanying the administration of ATG were males (81%) and most common indication for transplant (Fresenius) in conditioning regimens – prospective was Chronic Myeloid Leukemia (81%). 21/210 (10%) monitoring, mechanism and clinical importance developed SFPR. Median time of onset of SFPR after HSCT M. Navratil, A. Pejchalova, Z. Koristek, M. Tomiska, E. was 65 days (34-254). 13 patients (61.9%) had associated Tesarova, J. Mayer neutropenia. Immediate post transplant complications in these Masaryk University Hospital (Brno, CZ) group were acute graft versus host disease (GVHD) followed by sepsis and venoocclusive disease. 3/21 patients who Introduction and Methods: Several side effects of ATG are developed SFPR were CMV positive and 2/21 developed described. However, ATG-caused acute hemolysis has not antiplatelet antibodies. The most common factors associated been systematically studied. In context with our retrospective with SFPR were GVHD, CMV DNA positivity and data presented on EBMT 2006 meeting, we designed a infection.15/21(71%) died following SFPR, median survival prospective study protocol of monitoring the patient with ATG after HSCT being 150 days (49-810). administration during the period January – October 2006. The Conclusion: Secondary failure of platelet recovery is a patients (pts) were immunohematologicaly tested before, significant problem after allogenic hematopoietic stem cell during, and after the ATG administration. Standard laboratory transplantation and is associated with high mortality. markers of hemolysis and the clinical status of the patient Thrombocytopenia leads to utilization of substantial financial were also monitored. resources also. Further studies are needed to understand the Results: Ten pts were eligible (Bu+Flu+ATG, n=3; pathogenesis of this entity so that effective preventive Bu+Cy+ATG, n=1; FLAMSA+Cy+ATG, n=4), transplanted strategies can be planned. mostly for myeloid malignancies, and aplastic anemia, n=2.

S211 P836 biocompatible and biodegradable and not associated with Efficacy and safety of palifermin in a large cohort of inflammation, foreign body reaction, tissue necrosis or patients with haematological malignancies undergoing extensive fibrosis. Reabsorption of fibrin clot is achieved autologous haematopoietic stem cell transplantation during normal wound healing within few days after application L. Cavagna (1), G. Rossi (2), G. Saglio (3), C. Pilatrino (3), M. and its activity for tissue repair process is well known. We Petti (4), A. Mengarelli (4), G. Meloni (5), S. Capria (5), G. aimed at evaluating the efficacy of fibrin glue, obtained from Visani (6), A. Isidori (6), E. Benincasa (1) , in the treatment of two female patients, (1)Amgen INC (Milan, I); (2)Ospedali Civili (Brescia, I); aged 46 and 41 years, who developed a severe HC following (3)Ospedale San Luigi (Orbassano, I); (4)INT (Rome, I); an unrelated cord blood and an haploidentical HSCT, (5)Università La Sapienza (Rome, I); (6)Ospedale San respectively. Both patients were prepared with an intensive, Salvatore (Pesaro, I) chemotherapy-based, myeloablative conditioning regimen. The first symptoms of HC occurred at 60 and 42 days after Oral mucositis (OM) is an acute, severe and often dose- transplant. Both patients received all the sequence of the limiting toxicity in patients undergoing Hematological stem cell conventional therapy, including hyperhydration, bladder transplantation (HSCT). OM significantly affects functional irrigation, anti viral therapy against isolated cytomegalovirus, status and patient's quality of life; however, until recently, no polyomavirus and BK virus, supportive therapy and massive effective therapy was available for this condition. Palifermin platelet and red blood cell concentrates. Nevertheless, HC (Kepivance®) is a recombinant human keratinocyte growth was unresponsive with persistence of gross hematuria and factor that stimulates proliferation, differentiation, and clot formation. Using a specially designed single-use migration of epithelial cells. Palifermin has been shown to endoscopic catheter (Vivostat Endoscopic Application decrease the incidence and duration of severe OM in patients System), after a carbon dioxide insufflation fibrin sealant was with hematologic malignancies receiving myelotoxic therapy applied on large bleeding surfaces of the vesical mucosa and HSCT (Spielberger NEJM 2004). The safety and efficacy through a cystoscope. Due to the preformed nozzle, the spray of palifermin have not been established in patients with non- tip could easily be manipulated in many directions and fibrin hematologic malignancies. sealant applied to all the row surfaces. After 10 days, We report the results of an Expanded Access Program complete regression of HC symptoms was achieved in both conducted in Italy from July 2005 through October 2006. After patients, who remain well and free of transfusions. Fibrin Glue obtaining ethics committee approval, 29 centers administered seems to be an effective approach to the treatment of palifermin 60 mcg/kg/day intravenously on 3 consecutive days refractory HC and its early usage in the course of this before the conditioning regimen and on 3 consecutive days complication should be considered. immediately after the day of stem cell infusion. Data from 140 patients (76 men, 64 women; median age 51.5 years, range 18-72) were analyzed. The most common diagnoses were P838 multiple myeloma (MM) (n=55) and non-Hodgkin lymphoma Palifermin as oral mucositis prophylaxis in autologous (NHL) (n=56). Conditioning therapy and supportive care were transplant patients after myeloablative therapy administered according to standard institutional practice (high- N. Miranda (1), F. Trigo (2), P. Pimentel (3), A.B. Sousa (4), I. dose melphalan for MM and the BEAM regimen for NHL). OM Sousa (5), J.F. Lacerda (6) was evaluated daily for 28 days after transplantation or until (1)Instituto Português de Oncologia de Francisco Gentil severe OM resolution using the World Health Organization (Lisbon, P); (2)Hospital de São João (Porto, P); (3)Instituto (WHO) oral-toxicity scale. The incidence of severe OM (Grade Português de Oncologia de Francisco Gentil (Porto, P); 3-4) was 12.7 % and the incidence of ulcerative OM was (4)HCapuchos-CHL-ZC (Lisbon, P); (5)Hospitais da 31.3%. The mean duration was 2.1 ± 3.8 days (CI,1.4-2.7) in Universidade de Coimbra (Coimbra, P); (6)Hospital de Santa patients with OM Grade 0-4, 8.6 ± 3.6 days (CI, 6.8-10.4) in Maria (Lisbon, P) patients with OM Grade 3-4, and 6.6 ± 4.0 (CI, 5.4–7.8) in patients with OM Grade 2-4. Adverse events due to Palifermin Oral mucositis (OM) is a complication of intensive chemo and (rash, pruritus, erythema, mouth and tongue disorders, and radiotherapy with no effective treatment. Palifermin (PAL) is a taste alteration) were mild to moderate in severity and were recombinant keratinocyte growth factor that decreases the transient. The frequency of these adverse events was incidence and duration of OM in patients with hematologic consistent with those observed in clinical trials. In conclusion, malignancies receiving myeloablative chemotherapy and we observed that treatment with palifermin reduces the hematopoietic stem cell transplantation (SCT). The primary incidence and duration of OM if compared with historical objective of this study was to evaluate the incidence, duration data(Sonis Cancer 2004), offering effective OM prophylaxis in and severity of OM. The secondary objective was to evaluate patients with hematologic malignancies undergoing HSCT. the safety of PAL in these patients. Observational, retrospective and multicenter study in patients with hematologic malignancies who received PAL in the P837 setting of autologous SCT. Fifty-two PAL treated patients were Successful fibrin glue application for refractory included, 54% male, mean age 47±12 years old. The haemorrhagic cystitis following cord blood and haplo- diagnosis were Multiple Myeloma (MM) (N=15), Hodgkin’s identical haematopoietic stem cell transplantation Disease (N=11), non-Hodgkin’s Lymphoma (N=16), Acute M.C. Tirindelli (1), G. Flammia (1), E. Cerchiara (1), F. Sergi Leukemia (AL) (N=7) and others (N=3). The majority of (1), R. Cerretti (2), A. Picardi (2), M. Postorino (2), G. Avvisati patients were conditioned with BEAM/BEAM-like regimens (1), W. Arcese (2) (52%) and high dose melfalan (31%). World Health (1)University Campus Bio-Medico (Rome, I); (2)University Tor Organization (WHO) Oral Toxicity Scale was considered to Vergata (Rome, I) score OM. Fifty-four percent of the enrolled patients showed OM, 50% Hemorrhagic cystitis (HC) is considered a major cause of male, mean age 46±12 years old. OM appeared after 16±7 morbidity following hematopoietic stem cell transplantation days of conditioning regimen, median WHO score of 1 for 8±6 (HSCT) and its overall incidence is approximately 10%. days (mean). Forty-eight percent of all patients needed opioid Symptoms range from microscopic hematuria to painful, analgesics, median WHO score of 1 for 8±2 days (mean). massive hematuria, urinary obstruction due to clots formation Sixty-nine percent of the patients tolerated solid food (Grade and renal failure. Conventional therapies including pain relief, 0-2), 21% liquids only (Grade 3) and 9% nothing per os hyperhydration, bladder irrigation and antiviral drugs result (Grade 4). Seven patients received parenteral nutrition (6 due often ineffective. Invasive intervention such as fulguration, to OM), median WHO score of 1 for 12±9 days (mean). OM vescical air embolism and partial or total cystectomy affected mostly patients with MM (N=11) and AL (N=6). employed in life-threatening conditions are associated with Bleeding was referred by 6 patients, 15 mentioned tongue high morbidity and mortality. Fibrin glue is an hemostatic scalloping and 16 reported saliva alterations. The most agent derived from plasma coagulation proteins. It is frequent ulcerations were on the cheeks (57%), right ventral

S212 and lateral tongue (27%) and soft palate (25%). Left cheek, P840 right cheek and soft palate were the most reported as Cord blood transplantation in adult patients for erythema sites (75%, 64% and 57%, respectively). Adverse recurrence after allogeneic transplantation events were reported in 40 patients. Rash (N=36), pruritus S Seo (1), N. Uchida (1), K. Masuoka (1), H. Yamamoto (1), (N=29), weight increase (N=18) and edema (N=8) were S. Takagi (1), D. Kato (1), Y. Matsuhashi (1), N. Matsuno (1), probably related to PAL and were transient. Diarrhoea (N=34) A. Wake (1), S. Miyakoshi (2), S. Taniguchi (1) was interpreted as a result of the conditioning regimen. (1)Toranomon Hospital (Tokyo, JP); (2)Tokyo Metropolitan Although the mean duration of OM was 8 days, most of the Geriatric Hospital (Tokyo, JP) patients reported a mild grade of OM according to WHO. Palifermin was easy to use and the adverse events were Backgrounds: For more than 10 years, umbilical cord blood easily manageable. Further studies are needed to access the has become an alternative stem cell source for the patients full benefits of Palifermin. with hematological malignancies requiring allogeneic stem cell transplantation. Cord blood transplantation (CBT) can be performed more quickly than other stem cell transplantation, P839 since cord blood units are preserved in the deep freeze and The role of pre-transplant therapy in the incidence of even three HLA mismatched donors are acceptable. secondary malignancies after a PBSCT for Considering these advantage, we examined the feasibility of lymphoproliferative disorders CBT including reduced-intensity regimens for adult relapsed L. Laurenti, P. Chiusolo, F. Sora', N. Piccirillo, G. Farina, M. patients after allogeneic transplantation. Tarnani, S. Sica, G. Leone Patients/methods: We reviewed medical records of 32 Universita' Cattolica del Sacro Cuore Roma (Rome, I) patients who received CBT at Toranomon Hospital between November 2003 and October 2006. Median age of the Many reports indicate different incidence of secondary patients was 36 years old (range, 18-66). Underlying diseases myelodysplastic syndrome (sMDS)/acute myeloid leukemia were acute leukemia (n=25), chronic myelogenous leukemia (sAML) and solid tumor in patients receiving autologous (n=1), myelodysplastic syndrome (n=2) and lymphoma (n=4). transplantation for lymphoprolipherative disorders (LPD). We The stem cell source of the first transplantation were bone conducted a retrospective study in a selected group of 142 marrow from sibling donor (n=3), bone marrow from unrelated patients out of 323 undergone to autologous transplantation donor (n=10), peripheral blood stem cell from sibling donor for LPD between January 1988 and December 2004. The risk (n=7) and unrelated cord blood (n=12). Conditioning regimens factor was evaluated with univariata and multivariata analysis comprised fludarabine 125-180 mg/m² in several combination including: gender, sex, age, diagnosis, radiotherapy and with melphalan 80-140 mg/m², Busulfan 8-16mg/kg and total chemotherapy before conditioning regimen, disease status at body irradiation (4-8 Gy). Graft-versus-host disease (GVHD) PBSCT, type of harvest. Out of 142 patients included in the prophylaxis was cyclosporine alone (n=4) or tacrolimus alone study, 3 (2.1%) (2.48% at 5 years) developed sMDS/AML at 6, (n=28). Median number of total nucleated cells and CD34+ 15 and 41 months after PBSCT respectively for NHL, CLL and cells was 2.56x107 cells/kg (1.91-5.94) and 0.86x105 cells/kg NHL. One patient died 6 months after diagnosis of sAML, two (0.57-1.77), respectively. HLA disparities were 5/6 (n=3), 4/6 patients are alive after allogeneic stem cell transplantion at 7 (n=26) and 3/6 (n=3). and 35 months respectively after diagnosis of sMDS/AML. Results: Median observation period after the second At univariate analysis diagnosis and use of drugs were the transplantation was 78 days (range, 9-485). Overall survival only variables significantly associated with the development of for 1 year was 25% and 18 patients were died of disease sAML/MDS, with higher incidence rate in patients with CLL (p progression (n=7) and infection (n=11). Six of 18 dead = 0.045), and in patient who received fludarabine (p = 0.0005) patients showed engraftment failure. No grade IV toxicities and monoclonal antibody (p = 0.012). At multivariate analysis, (NCI-CTC Ver.3.0) were observed. Patients with stronger the variables associated to sAML/MDS were fludarabine and conditioning regimen (such as Flu 180mg/m² + Mel 140mg/m²) disease status at PBSCT (PD respect to CR). presented higher survival rate compared to the patients with Out of 142 patients considered, 3 patients (2.1%) developed a reduced-intensity regimen (64% vs 23%). The stage of the solid neoplasia, one affected by HD and two with LNH. The disease in the second transplantation, the duration between patients who developed secondary solid neoplasia (pancreas, two transplantations and HLA disparities did not influence the colon and lung) 24, 46, 127 months respectively after outcome. autologous transplant died at median time of 4 months (range Discussion: We demonstrated that CBT could be an available 3-33 months) from second diagnosis. At univariate analysis and feasible treatment for the relapsed patients after stem cell we found that radiotherapy (p = 0.021), and monoclonal transplantation. Moreover, the death rate from regimen related antibodies (p = 0.028) were significantly associated with the toxicities are acceptable even in the patients with an development of secondary solid neoplasia. Multivariate advanced disease. analysis confirmed the results of univariata analysis. The low incidence of sAML/MDS after ASCT could be abscribed to the stringent criteria lead to establish the real P841 incidence of sMDS/AML due to prior chemo-radiotherapy Outcome of intensive care unit patients following excluding: the use of chemo-radiotherapy or biological allogeneic haematologic stem cell transplantation response modifiers after autotransplant for progression compared to solid organ transplantation recipients disease requiring treatment, cytogenetical alteration before R. Benz, L. Bircher, M. Fischler, G. Stüssi, M. Maggiorini, U. conditioning regimen, low risk MDS during the first year after Schanz, J.D. Seebach aPBSCT. The absence of TBI as conditioning regimen could University Hospital Zurich (Zurich, CH) explain the low icidence of secondary solid tumor (expecially skin and bladder neoplasia). Objectives: Patients admitted to intensive care units (ICU) following allogeneic hematopoietic stem cell transplantation (HSCT) have one of the highest mortality rates compared to other ICU patients. Several parameters including need for mechanical ventilation, number of organ dysfunctions and time from transplantation to ICU admission have been related to outcome. Here, we analyzed the clinical parameters of this poor risk patient group in comparison to recipients of allogeneic solid organ transplantation (SOT) admitted to ICU. Methods: A retrospective single-center analysis was performed on all patients admitted to the medical ICU between 2000 and 2004 who had received an allogeneic HSCT or SOT previously. Primarily, overall survival, causes of

S213 death, and quality of life at 6 mo were evaluated. In addition, Comments: In this retrospective study we have confirmed that causes leading to ICU admission, time from transplantation to adult patients needing mechanical ventilator treatment for ICU, time from ICU admission to death, number of organ more than 24 hours and having signs of liver and/or renal failures and treatment were analyzed. failure and needing vasopressor treatment after allogeneic Results: Out of 131 allogeneic HSCT performed 15 patients stem cell transplantation still have a very poor prognosis. (11%) were admitted to the ICU 16 times with a 6 mo ICU Based on these and previously published results the survival rate of 20% (3/15). The 6 month survival rate after continuation of intensive care for more than 4 days, if there transplantation was 20% in the ICU group compared to 95% are no signs of improvement, may be questionable, and must (110/116) of the non-ICU group. The three surviving HSCT be reconsidered on a daily basis. patients were admitted to ICU 13.7 mo after transplantation, compared to 2.7 months in the 12 patients who died. In the latter death occurred 15 d (0-36 d) following ICU admission. In P843 the same time period a total of 161 SOT patients were A 3-day short course of Palifermin significantly reduces admitted to the ICU (27% [average of 120 SOT/year]), with a 6 toxicity and need for supportive care during high-dose mo ICU survival rate of 72.2% (114/161). Time from therapy and autologous blood stem cell transplantation in transplantation to ICU admission was 80.5 mo in the solid patients with multiple myeloma organ group compared to 4.8 mo after HSCT. Time from ICU G. Kobbe, N. Hieronimus, T. Graef, I. Bruns, A. Czibere, F. admission to death in the SOT group was 9 d (1-530 d). A Zohren, N. Safaian, U. Steidl, U. Germing, R. Haas, R. Fenk detailed analysis of the causes leading to ICU admission, Heinrich Heine University (Duesseldorf, D) number of organ failures, and required ICU treatment will be presented as well as causes of death. Background: A 6-day course of Palifermin, a human Conclusion: The ICU admission rate of our HSCT patients recombinant keratinocyte growth factor has been shown to was low compared to previously published data with a similar reduce mucositis and need for supportive care after a very 6 mo survival rate. In contrast, SOT recipients admitted to the intensive, TBI containing high-dose conditioning regimen. We ICU had a much higher survival rate, and a much longer time here combined a short-course of Palifermin (3 days) before between transplantation and ICU admission. These high-dose Melphalan (HDM) and a single dose of differences reflect the different pattern of risk factors Pegfilgrastim after stem cell infusion to reduce toxicity in associated with HSCT and SOT, respectively, and will be patients with multiple myeloma (MM). discussed in the light of the detailed analyses of the events Patients and methods: So far 18 patients (the Pal-Peg group, leading to ICU admission. 9 male, 9 female, median age 59, range 45-73) with MM myeloma received 3 daily injections of Palifermin (60ug/kg/d) before HDT. At least 24 hours after the last Palifermin dose P842 patients were treated with HDM (median dose 200mg/m², Very low long-term survival in adult patients needing range 100-200) followed by ASCT of a median of 3.5 mechanical ventilator treatment for more than four days CD34+cells/kg (range 1.9-18.8). Pegfilgrastim (6mg) was after allogeneic stem cell transplantation given one day after blood stem cell infusion. This group of L.J.C. Brinch (1), S. Lenhof (2), C. Malm (3), J. Mattson (4), A. patients was compared to a previous cohort of 21 patients (the Wahlin (5) Peg-only-group, 9 male, 12 female, median age 57, range 39- (1)Rikshospitalet National Hospital (Oslo, N); (2)University 69) with multiple myeloma who had received no Palifermin. In Hospital (Lund, S); (3)University Hospital (Linköping, S); these patients a median of 4.5 CD34+cells/kg (range 2 -12) (4)Karolinska Hospital Huddinge (Huddinge, S); (5)Umeå and a single dose of 6mg Pegfilgrastim were given after HDM. University Hospital (Umeå, S) Patients in the Pal-Peg group had more advanced disease. Results: Time to hematopoietic reconstitution was not Introduction: Patients with respiratory failure needing significantly different in both groups with a median time to a mechanical ventilator treatment for more than 24 hours after WBC >1x109/l of 11 days (range 9-13) in the Pal-Peg group allogeneic stem cell transplantation have a poor prognosis. and 10 days (median, range 9-14) in the Peg-only group. The This is especially the case in patients with bilirubin >68 the same was true for platelet reconstitution. Due to a mmol/L and /or creatinin >177 mmol/L during the first 3-4 days markedly reduced incidence and severity of mucositis in the of ventilator treatment, vasopressor treatment for more than 4 Pal-Peg group the duration of hospitalisation was significantly hours per 24 hours in the first 3-4 days, and/or lung injury with shorter (median 17 days, range 13-23 vs 21 days, range 15- a need for inspiratory oxygen concentration of more that 60% 34, p<0.05). In addition the need for iv antibiotics (median 0 or a PEEP of more than 5 cm H2O 24 to 72 hours after start of days, range 0-9 vs 4 days, range 0-21, p<0.05), morphine ventilator treatment. Two large retrospective studies from the (median 0 days, range 0-12 vs 4 days, range 0-21) parenteral 90-ties have described more than 95% death rate in such nutrition (median 0 days, range 0-12 vs 6 days, 0-16, p<0.05) patients, and more that 80% in patients not having the and erythrocyte transfusions (median 0, range 0-4 vs 2, range mentioned high risk features. 0-14, p<0.05) was significantly reduced. Aims of study: To investigate whether these results might Conclusion: The combined use of a 3-day short course of have improved during the last 5 years and with the purpose of Palifermin before HDM and a single dose of Pegfilgrastim developing guidelines for intensive care of patients needing after stem cell transplantation significantly reduces toxicity in mechanical ventilator treatment, we undertook a retrospective patients with multiple myeloma and improves patient survey of Nordic centres performing allogeneic stem cell convenience. Our data clearly favours the use of Palifermin to transplantation. reduce toxicity of HDM in patients with MM. However, the Methods: A questionnaire was sent to 10 Nordic transplant need for the full 6 day course seems at least questionable. centres asking for the total number of patients transplanted during 2001 until 31.12.2005. We asked how many patients were treated with a mechanical ventilator and how many P844 patients with the above mentioned characteristics were Nutrition and cancer: an epidemiological survey in discharged alive from the hospital and how many were still France. Focus in haematology alive after one year. M. Michallet (1), C. Beauvillain de Montreuil (2), F. Results: 5 of 10 centres returned the questionnaire. In those Goldwasser (3), E. Lemarié (4), N. Raus (1), M. Sobh (1), C. centres a total of 711 patients were transplanted. 56 were Chambrier (1), F. Nicolini (1), X. Hébuterne (5) treated in the intensive care unit with mechanical ventilation (1)Hôpital Edouard Herriot (Lyon, F); (2)Hôpital Hôtel-Dieu for more than 24 hours. 11 of these patients were discharged (Nantes, F); (3)Hôpital Cochin (Paris, F); (4)Hôpital from the intensive care unit, 8 patients could be discharged Bretonneau (Tours, F); (5)Hôpital L'Archet 2 (Nice, F) from the transplant hospital, of whom 2 were still on ventilator treatment. An epidemiological study was performed in France in 2005, Only 4 patients were alive more that 1 year after transplant. with an overview of the nutritional status and nutritional care of

S214 non selected cancer patients (pts). This study involved 2068 receive BMMC or placebo implantation plus conventional pts (879 female, 1189 male) and 86 clinical departments, CABG, serial samples of peripheral blood mononuclear cells, including hematology departments with 419 pts either serum and plasma were collected before and after surgery. hospitalized (n=391) or ambulatory (n=28). Height, current Results: No procedure related side effects, especially no and usual body weight were assessed and body mass index significant arrhythmogenic effects of BMMC transplantation (BMI) was calculated. Malnutrition was defined when have been observed so far. Two patients died due to non- BMI<18.5 (18-74 yrs) or BMI<21 (• 75 yrs) and/or when pts transplantation related cause after some months from the presented weight loss • 10% from the beginning of the procedure cardiac related. A first group of 16 patients, with 6 disease. On the 2068 pts, nutritional status was available for and 12 months follow-up, showed a significant improvement 1903 pts (377 pts in hematology with 73 autologous both in LVEF and in LV volumes (P<0.01). Flow cytometric Hematopoietic Stem Cells Transplant (HSCT) pts and 37 analysis of PBMC of all patients showed that, after allogeneic HSCT pts). Overall, the prevalence of malnutrition intervention, absolute number/ml of HPC co-expressing was 39%. Among the 377 pts presenting a hematological CD34+/CD133+ was statistically higher at + 24 h. as malignancy, 34% were severely malnourished (36% lymphoid compared with the baseline value (P<0.005). Similarly, an versus 30% myeloid malignancies). The prevalence of early and transient increase of mature EPC co-expressing malnutrition was particularly high for ALL pts (48%; n=31), B- CD34+/CD31+, peaked already at + 24 h. (P<0.0001). A and T-NHLs (39%; n=108), multiple myeloma (36%; n=69), significant higher number of EPC co-expressing and for CLL (44%; n=16). In-pts were significantly more CD34+/CD117+ and CD34+/ VEGFR-2+ peaked at + 24 h affected by malnutrition than out-pts (35% versus 15%). and +7 days (P<0.003, P<0.001, respectively). Both Multivariate analysis (logistic regression) conducted on the circulating HPC and EPC returned to baseline levels within 30 whole population revealed mainly 3 anti-tumoral treatments days, after surgical intervention. Serum VEGF-2 level was significantly associated with malnutrition: previous over-secreted at +7 days (23,25 vs 570,3 + SD 473 pg/mL, radiotherapy, allogeneic graft and chemotherapy. Indeed, P<0.0001), returning to baseline values at + 30 days. whatever the tumor localization, age and tumoral staging, The plasma SDF-1 alpha level was significantly appreciable radiotherapy increased by 1.53 (OR) the probability of being within 24 hours post-surgery (400,32 vs 889 + SD 250 pg/mL, malnourished (CI:1.21-1.93; p=0.0004) whereas previous P<0.003), being higher at +30 days (400,32 vs 1420 + SD allogeneic HSCT or chemotherapy multiplied the same pg/mL, 744, P<0.0001). Interestingly, in a first group of malnutrition risk by, respectively, 3.97 (1.12-14.09; p=0.033) patients with 6 and 12months-follow-up, the systemic homing and 1.40 (1.05-1.89; p=0.023). Furthermore, age and tumoral factor production showed a significant value than those found staging were also significant factors associated with at baseline (p<0.001, p<0.02, respectively).We found a strong malnutrition (OR between 1.01 and 2.97; p<0.05). Considering correlation between secretion fashion of SDF-1 alpha and the global population of cancer pts, near 1 over 2 pts (47.3%) CD62E/CD11b expression on PBMC (p<0.0001). did not receive any nutritional support. For the hematology Of interest, a small group of patients showed no difference of population, only 44.5% of the malnourished pts received circulating CD34+ cell subsets and cytokines secretion before nutritional support. The high prevalence of malnutrition in and after intervention onco-hematology (34%) impair the dose-intensity of chemo- Conclusion: From our experience certain interim conclusions radiotherapy, prolong intervals between chemotherapy can be drawn. One, adjunctive bone marrow-derived cell courses, alter the quality of life of pts and may favor infectious implantation at the time of bypass surgery appears to be complications, a factor that could be even more determining feasible and safe. The second conclusion is that both cell and when considering HSCT pts. This underlines the absolute placebo infusion in infarcted patients could regulate requirement for optimal nutrition care in these pts. endothelial progenitor cell mobilization and angiogenic/homing factors which might act to recruit circulating stem cells to the ischemic heart. Of interest, only few cases showed no substantial modification of biological parameters. However, we Cellular and Gene therapies are not able to provide any definitive conclusion on the association between biological results and clinical outcome since this study is still blinded. P845 Progenitor cell trafficking in patients with infarcted P846 myocardium undergoing autologous bone marrow High-dose chemotherapy +autologous stem cell mononuclear cell injection. Interim analysis of a double- transplantation in multiple sclerosis patients: follow-up blind randomised phase II clinical trial results of a prospective multicentre study M. Cuzzola (1), G. Irrera (1), A. Pontari (1), I. Callea (1), G. J. Shevchenko (1), A. Novik (1), A. Kuznetcov (1), B. Pucci (1), G. Martinelli (2), M. Braccio (2), C. Nesta (2), A. Afanasiev (2), I. Lisukov (3), O. Rykavicin (1), A. Myasnikov Condemi (1), A. Dattola (1), E. Spiniello (1), P.E. Puddu (1), (4), T. Ionova (2), S. Shamanski (1), A. Kulagin (3), O. D. Sergi (1), F. Benedetto (1), M. Cassese (2), P. Iacopino (1) Malysheva (3), N. Baziy (1), V. Melnichenko (1), D. Fedorenko (1)Azienda Ospedaliera Bianchi-Melacrino-Morelli (Reggio (1), A. Zdorov (4), A. Kishtovich (2), R. Ivanov (1), J. Fedotov Calabria, I); (2)Sant’Anna Hospital (Catanzaro, I) (2), G. Gorodokin (5) (1)Russian Coop. Group for Cellular Therapy (Moscow, RUS); Aim of the study: The primary aim of our double-blind (2)Russian Coop. Group for Cellular Therapy (St. Petersburg, randomized phase-II clinical trial was to assess therapeutic RUS); (3)Russian Coop. Group for Cellular Therapy effectiveness of intramyocardial bone marrow mononuclear (Novosibirsk, RUS); (4)Russian Coop. Group for Cellular cells (BMMC) injections in patients with ischemic Therapy (Petrozavodsk, RUS); (5)New Jersey Center for cardiomyopathy undergoing coronary artery bypass grafting Quality of Life and Health Outcome Research (New Jersey, (CABG). Herein we present an analysis of the procedure USA) feasibility and safety together with the longitudinal biological study regarding (a) the possible therapeutic mobilization Treatment outcomes in MS patients after HDCT+ASCT are process of both peripheral hematopoietic progenitor cells under-reported. We aimed to study clinical treatment response (HPC) and early-late endothelial CD34+ cells ( EPC), and (b) and quality of life (QoL) treatment response in MS patients the modification of humoral factors potentially related to HPC after early, conventional and salvage HDCT +ASCT. mobilization and mechanism of tissue repair. Forty patients with MS (secondary progressive – 19 patients, Patients and Methods: Between March 2004 and October primary progressive –10, progressive-relapsing – 1, relapsing- 2006, a total of 37 patients who had suffered from acute remitting – 10) from 6 medical centers were included in this transmural myocardial infarction less than 6 months before study – (mean age - 32.0, range: 17-51; male/female – 16/24). admission, with left ventricular ejection fraction (LVEF) less Seven patients underwent early HDCT +ASCT, 30 patients – 35, were recruited. The patients were randomized 1:1 to conventional HDCT+ASCT and 3 patients – salvage HDCT

S215 +ASCT. Median EDSS at base-line was 6.0 (range 1.5 – 8.0). Taken together, mRNA based nucleofection is a powerful, The median follow-up duration was 18 months (range 6 – 84 highly efficient and non-toxic approach for transient labelling months). Neurological and QoL evaluation was provided at of human progenitor cells or, via transfection of selective baseline, at discharge, at 3, 6, 9, 12 months, and every 6 proteins, for transient manipulation of stem cell function. It months thereafter following HDCT+ASCT. FACT-BMT and may thus be useful to transiently manipulate stem cell FAMS were used for QoL evaluation. characteristics and combine principles of and All 27 patients with the follow-up longer than 1 year, included tissue engineering. in the analysis, experienced a clinical stabilization or improvement. More than half of them improved: 6 patients showed significant improvement in EDSS (by more than 1.0 P848 point), 4 patients improved by 1.0 point, and 5 patients - by May we predict a “pure” graft versus leukaemia effect 0.5 points on EDSS. Twelve patients achieved stabilization. after donor lymphocyte infusions for chronic myeloid Two patients deteriorated to a worse score after 18 months of leukaemia relapsing after allogeneic stem cell transplant? stabilization; 2 other patients progressed after 12 and 30 C. Guglielmi, S. Bergantini, S. Iacobelli, A. van Biezen, E. months of improvement, respectively. All the patients with Olavarria, A. Gratwohl, A. Schattenberg, L. Verdonck, D. clinical stabilization and improvement had negative MRI Niederwieser, T. de Witte on behalf of the Chronic Leukaemia scans. Working Party of the EBMT Out of 21 patients included in the analysis of QoL response 19 exhibited improved QoL 6 months post-transplantation. At Donor lymphocyte infusions (DLI) have radically changed the one year after HDCT+ASCT 1 patient exhibited a maximal prognosis of patients (pts) relapsing after allogeneic QoL response; 3 patients – good QoL response; 7 patients – hematopoietic stem cell transplant (SCT) for chronic myeloid moderate QoL response and 8 patients – minimal QoL leukemia (CML). Major obstacles to success with DLI are response. At 2.5 years post-transplantation 3 more patients represented by leukemia resistance and by secondary GvHD had a maximal QoL response. Further QoL improvement was (GvHD2). The best result of is when a patient treated with DLI observed at longer follow-up. achieve a durable molecular remission (MR) without In conclusion, clinical response was observed in 100% of MS experiencing GvHD2. It is unclear which factors may predict patients after early, conventional and salvage HDCT+ASCT. for such a favourable outcome when CML pts are treated with The majority of patients with clinical response had a good or DLI. moderate QoL response. Further studies should be done to We retrospectively identified 500 pts, treated with DLI for CML investigate the clinical and patient-reported outcomes in MS relapse (16% molecular, 30% cytogenetic, 42% patients receiving early, conventional and salvage haematological chronic, 12% hematological accelerated) at 68 transplantation to better define treatment success. EBMT centres before 2004: 73% had an HLA-identical sibling donor, 27%unrelated. Single (58%) or multiple (42%) infusions were given. P847 Results: 340 pts(68%)achieved MR in a median of 7.5 Highly efficient mRNA- and cDNA-based transient gene months, 44% had GvHD2 in a median of 3 months, 16 pts delivery into human progenitor cells recurred at a median of 19 months. Actuarial probability of J. Greiner (1), J. Torzewski (1), P. Ponsaerts (2), M. Rojewski being alive in MR without GvHD2 was 29% and 27% at 5 and (1), D. Kronawitter (1), H. Schrezenmeier (1), V. Hombach (1), 10 years, respectively. H. Döhner (1), M. Schmitt (1), M. Wiesneth (1), O. We studied the prognostic effect of following factors: patient Zimmermann (1), J.M. Wiehe (1) age at DLI, donor type, donor sex, sex mismatch with the (1)University of Ulm (Ulm, D); (2)University of Anwerp donor, phase at SCT, stem cell source, T-depletion, total body (Antwerp, B) irradiation in the conditioning regimen, GvHD prior to DLI, interval from SCT to DLI, type of relapse. Multivariate analysis The method of gene transfer into progenitor cells is critical as with a Cox model adjusted for the period of DLI (<1998 vs viral vector transduction involves the risk of tumor induction by >1997), showed that chronic GvHD after transplant and prior non-specific genomic integration. Non-viral transfection to relapse (hazard ratio [HR]: 1.5, 95CI: 1.2-1.9, p<0.001), an systems often fail due to low transfection efficiency. However, interval from SCT to DLI <1 year (HR: 1.7, 95CI: 1.3-2.2, gene transfer into human CD34+ hematopoietic progenitor p<0.001), and hematological relapse (HR: 1.6, 95CI: 1.2-2.0, (HPC) and mesenchymal stem cells (MSC) is an essential tool p<0.001), were adverse features. 94 pts (20%), 222 (48%), for in vitro- and in vivo-applications and therapeutic strategies 133 (28%), and 17 (4%) had 0, 1, 2, and 3 adverse features, such as tissue engineering and gene therapy. We recently respectively. Survival in remission without experiencing reported an transient genetic labelling of human CD34+ HPC GvHD2 at 5 years improved from 14%, 30%, to 56% in pts with deltaLNGFR-plasmid-DNA for in vivo application: with 2-3, 1, and 0 adverse features, respectively. Transient transfection was efficient for both, CD34+ HSC We conclude that: [a] pure GvL effect (ie. durable remission (41%±2.0% ) and leukemia cell lines (55%±4.9%) using the without GvHD2) was observed in more than 25% of pts method of nucleofection. Moreover, mature myeloid cells treated with DLI for CML relapsing after allogeneic SCT; [b] (CD66b+) derived from transfected human CD34+ HPC and occurrence of chronic GvHD prior to relapse, the interval from leukemia cells maintained deltaLNGFR expression at a high SCT to DLI, and the type of relapse are the main factors percentage (70% ± 1.6% and 58% ± 2% respectively). associated with the chance of a pure GvL effect; [c] pts In this work, we investigated labelling of CD34+ HPC with treated with DLI beyond 1 year from SCT for a mRNA. Human CD34+ HPC and human MSC were molecular/cytogenetic relapse that was not preceded by transfected with in vitro-transcribed mRNA for deltaLNGFR, a chronic GvHD have more than 50% chance of exploiting the marker gene approved for human in vivo-application, using pure GvL effect. nucleofection. EGFP was used as a control. 24h after nucleofection, FACS-analysis showed a higher transfection efficiency compared to plasmid transfected CD34+ HPC and P849 MSC: A high transfection frequency was found for mRNA- Cytochrome P450 2C19 loss of function polymorphism is transfected HPCs using deltaLNGFR (82.4±9.7%) and EGFP associated with an increased TRM in patients who (88.7±2.6%). We found also a high transfection rate for MSC underwent allogeneic transplantation using the marker genes deltaLNGFR (92.4±3.6%) and EGFP A.H. Elmaagacli, M. Koldehoff, N.K Steckel, M. Ditschkowski, (83.3±4.1%). Cell viability was not affected by mRNA- R. Trenschel, H. Ottinger, D. Beelen transfection. Moreover, differentiation assays of deltaLNGFR- University Hospital of Essen (Essen, D) selected MSC after transfection, showed that differentiation of MSC into mesenchymal cells like chondrocytes, adipocytes Background: The polymorphic expression of drug- and osteoblasts was not affected by mRNA nucleofection. metabolizing enzymes is one of the major factors which cause individual variability in a drug metabolism and thereby in

S216 pharmacologic and toxicologic responses. Previous studies on P851 genetic polymorphisms of CYP 2C19 showed that individuals Propagation of human mesenchymal stem cells under could be classified into the following groups: poor metabolizer defined serum-free culture conditions (PMs), intermediate metabolizers (IMs), and extensive J.C. Brune (1), A. Tormin (1), D. Melzer (1), Å. Borg (1), S. metabolizers (EMs). Patients receiving an allogeneic Scheding (2) transplant are faced to use a huge number of different kind of (1)Lund Stem Cell Center (Lund, S); (2)Lund University drugs, i.e. for immunesuppression, chemotherapeutical Hospital (Lund, S) agents, or anti-infectious medication. Never before the influence of genetic polymorphisms of CYP 2C19 on patients Background: Human MSC are multipotent cells that are who underwent allogeneic transplantation, was evaluated. promising candidates for clinical cell therapy approaches such Methods: Here we genotyped in a retrospective study 290 as GvHD treatment and cell replacement strategies. However, patients for CYP 2C19 expression who underwent allogeneic the culture of MSC requires fetal calf serum which is transplantation for various diseases and analyzed their considered a high-risk ancilliary product and, therefore, should outcome. Genotyping of CYP 2C19 was performed by real- be avoided. Therefore, we aimed to develop a serum-free time PCR. culture system that allows to effectively propoagate MSC in- Results: 201 patients (72,5%) were genotyped as EMs, 71 vitro. patients (24,5%) as IMs and nine patients (3,1%) as PMs. Materials and methods: MSC were generated from human From the donors 208 donors (71,7%) were EMs and had a bone marrow using standard methods. Kryopreserved MSC homozygous wild-type gene, 68 donors (23,4%) were were thawed and allowed to attach in complete MSC medium genotyped as IMs and had a heterozygous genotype, and 14 for three days (1000 MSC/well). Thereafter, cells were donors (4,8%) were typed as PMs with a homozygous gene washed and cultured in either standard MSC medium or mutation. Calculated genotype frequencies did not differ from serum-free CellGro culture medium ± cytokines. Functional that reported earlier by other studies for caucasiens. Five-year tests including differentiation assays, LTC-IC assays as well estimate for TRM was highest in PM-patients with 50% +18,6 as gene-expression profiling were performed. % compared to EM patients (25,1 % + 3,7 % [p<0.018]) and Results: Without cytokine stimulation, MSC numbers IM patients 22,7 % + 5,6 % [p<0.042]), whereas the five-year decreased over time under serum-free conditions (2% ± estimate for relapse or overall survival were not statistically 0.05% of serum controls after 4 weeks culture). Among the different between the groups. No differences for five-year cytokines and cytokine doses tested, was the combination of estimates for TRM, relapse rate, or OS were seen in patients PDGF-BB (100 ng/mL) plus bFGF (1.0 ng/mL) both necessary with either EM-, IM-or PM donors. No statistic differences and sufficient to amplify MSC to levels comparable to were found in the incidence of acute GVHD grade 2-4 within standard serum-containing cultures (33 ± 11.3-fold and 32 ± the study groups. 9.6-fold, respectively, after 4 weeks culture). The addition of Conclusions: These results suggests that patients with loss-of- SCF and IL-3 markedly inhibited MSC growth, an effect that function polymorphism of CYP450 2C19 do have an increased was even more pronounced when additional cytokines such TRM after transplantation. Genotyping for CYP450 2C19 as IL-6, Flt3-ligand, thrombopoietin, and IL-1 were added. might help to identify patients with higher risk for TRM. PDGF + FGF-stimulated serum-free cultured MSC showed a typical spindle-shaped morphology and expressed MSC surface markers (CD73/CD90/MHC-Ipos, P850 CD14/CD34/CD45neg). Moreover, these MSC effectively Interleukin 7 engineered human stromal cells enhance supported hematopoiesis when used as feeder cells in immune reconstitution in NOD/SCID mice standard long-term culture initiating cell assays and they were P. Sportoletti, B. Del Papa, L. Moretti, A. Terenzi, M. De capable of differentiating into osteoblasts, adipocytes and Ioanni, L. Fontana, F. Falzetti, M. Di Ianni, B. Falini, A. Tabilio chondrocytes. Gene-expression profile comparison of serum- University of Perugia (Perugia, I) free MSC vs. MSC grown in standard serum-containing culture revealed a total of 486 differentially expressed genes. Impaired immune reconstitution after allogeneic bone marrow Conclusion: Taken together, our data demonstrate that human transplantation (allo-BMT) remains a major obstacle to its MSC can be propagated under defined serum-free GMP clinical application. Enhancing thymus-dependent immune conditions at efficiencies matching standard serum-containing reconstitution is the most efficient way to improve post culture by supplementing PDGF-BB and bFGF. transplant immune recovery. Key players in human T cell development are bone marrow stromal cells (MSCs) and cytokines such as interleukin-7 (IL-7) which modulate the P852 immature thymocytes differentiation. Human MSCs can be Production of soluble HLA-G antigens is involved in the engineered to produce IL-7. They home to bone marrow, inhibition of the lymphoproliferative response by human spleen and thymus (Di Ianni et al., Hum Gene Ther 16:752; mesenchymal stromal cells from different sources 2005). In the present study we co-transplanted 5x105 human F. Lanza (1), D. Campioni (1), S. Moretti (1), L. Ferrari (1), L. IL-7 engineered MSCs (hMSC-IL-7) producing 1000pg/ml Melchiorri (2), R. Rizzo (2), G. Bagnara (3), O. Baricordi (2) together with 1x106 human CD34+ stem cells into sublethally (1)Section of Hematology (Ferrara, I); (2)Section of Medical irradiated NOD/SCID mice. We investigated the effects on Genetics (Ferrara, I); (3)Section of Histology (Bologna, I) immune reconstitution in bone marrow, spleen and thymus by staining with anti-CD3 monoclonal antibody and by measuring Clinical and experimental data have demonstrated an T-cell receptor excision circles (TRECs) levels by real time immunoregulatory function of bone-marrow BM-MSCs, which PCR. Immunohistological analysis detected human CD3+ could contribute to the decreased incidence of graft-versus- cells in the thymus and spleen of NOD/SCID mice co- host disease following allogeneic stem cell transplantation. transplanted with human CD34+ cells and hMSC-IL7 cells. Soluble factors produced by BM-MSCs were suggested to TREC levels were significantly increased in thymus, bone play a role in the inhibition of mixed lymphocyte reaction marrow and spleen (17894±1634 copies/ìg DNA; 161±18 (MLR) response. Human leukocyte antigen G (HLA-G) is non- copies/ìg DNA; 2106±90 copies/ìg DNA respectively). In mice classic MHC class I molecule characterized by a limited transplanted with CD34 cells alone, no human CD3 cells were polymorphism and a splicing mechanism that regulates detected in any organ. No trecs were found. In conclusion membrane and soluble isoforms (sHLA-G). Since IL-10 up- hMSC-IL-7 cells could restore thymopoiesis of allo-BMT modulates sHLA-G production and increased IL-10 levels recipient by reconstructing thymic microenvironment and were reported in MLR/MSCs culture supernatants, we delivering an effective concentration of IL-7 directly into the investigated sHLA-G production by MSCs obtained from thymus. Thus, the use of hMSC-IL-7 cells may be different sources (BM, dental pulp, amniotic membrane, therapeutically useful for enhancing immune reconstitution chorion, skin). The results showed the presence of detectable after allo-BMT. sHLA-G molecules in the supernatants of either MLR/MSCs or MSC/phytoemoagglutinin (PHA) stimulated cultures but not in

S217 controls. RT-PCR analysis confirmed the presence of HLA-G the effects of human recombinant IL-7 (rIL-7), human m-RNA in MLR/MSCs cultures. The extent of sHLA-G mesenchymal cells (hMSC) and hMSC engineered with the IL- production, as well as MSC lymphoproliferative response was 7 gene (hMSC/IL-7) on regulatory T cells expressing a dependent on MSC aging (MSCs culture passages: T1-T10) memory (CD4/CD25/CD45RO) or naive (CD4/CD25/CD45RA) and MSC source. The sHLA-G production is also positively phenotype. T cells from healthy donors were enriched by correlated with the in vitro MSCs immunosuppressive immnuselection to provide populations of CD45RA+ cells (95 capacities. In situ immunocytochemical analysis demonstrated % ± 2.9) and CD45RO+ cells (97 % ± 0.25). Enriched naive that both (activated) MSCs and CD14+ monocytes could and memory cells were cultured in presence rIL-7 (100 ng/ml), produce sHLA-G. Overall these data suggest a functional role hMSC (ratio 5:1) or hMSC/IL-7 (ratio 5:1). After 7 days’ culture for HLA-G antigens in mediating inhibition of the allogeneic in the naive T cell population the T reg starting fraction of 0.05 response. % ± 0.01 of CD4/CD25 positive cells, did not change in the presence of rIL-7 while it rose to 0.2 % ± 0.14 in presence of human MSC and reached 1.67 % ± 0.6 in presence of IL-7 P853 engineered human MSC. In the memory T cell population the Pregnancy outcome following haematopoetic stem cell T reg starting fraction of 0.3 % ± 0.05 of CD4/CD25 positive transplantation cells, did not change in presence of rIL-7 while it rose 1.5 % ± S. Kulkarni, M. Ethell, R. Saso, J. Brennan, P. Kaczmarek, C. 0.9 in the presence of human MSC and more interestingly Horton, F. Saran, R. Powles, G. Morgan, M. Potter reached 11.55 % ± 7.5 in the presence of human IL-7 Royal Marsden Hospital (Sutton, UK) engineered-MSC. In the naive T reg starting fraction 3 % ± 1.2 expressed CD127 which was down-regulated to 0.96 % ± 0.5 Between 1973 and 2003, 747 patients (M: 430, F:317; age: in the presence of rIL-7, to 0.29 % ± 0.2 with human MSC 29yr, range:1-65) who survived at least 1 yr. post SCT and and to 0.37 % ± 0.02 with human IL-7engineered-MSC. were 15 to 65 years of age (range:15-71) at the last follow-up Memory T reg cells expressed CD127 in 15% ± 1.2 of the (LFU) were analysed to assess the outcome of pregnancies. starting fraction which was down-regulated to 1.2 % ± 0.45 in At the time of SCT, 138 patients were pre-pubertal (M: 79, F: the presence of rIL-7, to 1.32 % ± 0.34 with hMSC and to 59, p=0.8). Transplants were allogeneic (n=429, median age: 4.01% ± 0.74 in presence of hMSC/IL-7. FoxP3 expression 28yr), autologous (n=304, median age: 32) or syngeneic was measured by real time quantitative PCR in sort-purified (n=14, median age: 37) performed for Ac. Leukaemia (n=583), subsets of peripheral blood, identified by staining with a Chr. Leukaemia (n=82) or other diseases (n=82).Conditioning combination of CD4, CD25, CD45RA or CD45RO. In naïve T included TBI in 561 patients (single fraction:478, fractionated: reg FoxP3 expression was increased 1.15 fold in the presence 83) and 168 patients also received cranial radiation. Stem cell of hMSC and 2.7 fold in presence of hMSC/IL-7. In memory T source was marrow (n=573), PBSC (n=166) or both (n=8). reg FoxP3 expression was increased 1.14 fold in the presence GVHD prophylaxis was CyA alone (n=188), CyA with another of hMSC and 2.67 fold in presence of hMSC/IL-7. Given the agent (n=227) or other measures (n=14). The overall survival different responses to IL-7/engineered and non engineered was 65% at 10yr (median FU: 7yr). 27 patients (10 females stromal cells, it appears that different regulatory systems may and 17 partners of male patients) had 42 pregnancies and underlie different functions within the T reg sub-populations. sired 45 children (4 twin pregnancies). The probability of conception was 4% at 10yr. Pregnancies occurred in 17/429 allografts (3.96%; 6/186 females; 11/243 males), 8/304 P855 autograft patients (2.63%; 4/123 females, 4/181 males) and Thymoglobulin suppresses the immunomodulatory effect 2/14 syngeneic transplants (14.3%; 0/8 females, 2/6 males). of human mesenchymal stem cells Amongst 10 female patients 3 had assisted conception. S. Tschiedel, C. Pfrepper, A. Reinhardt, K. Bartsch, D. Amongst 17 partners of male patients, 6 had assisted, 10 had Niederwieser, N. Basara natural and 1 had assisted and natural conception. In uni- University of Leipzig (Leipzig, D) variate analysis chances of pregnancy were higher with age at SCT> 15yr.(5% vs. 1% at 10 yr., p<0.001), non-TBI Objectives: Mesenchymal stem cells from human bone conditioning (9% vs. 2%, p=0.001), age 20-45 yr.at LFU (5% marrow (hMSC) reduce the incidence and severity of graft- vs. 2%, p=0.005) and use of PBSC (8% vs. 5%, p<0.001). versus-host disease (GvHD) in clinical phase I studies, thus Use of PBSC and non-TBI conditioning had significant indicating the immunomodulatory function of hMSC. correlation explaining the association. Multi-variate analysis Moreover, several studies have been initiated regarding the identified non-TBI conditioning (RR: 3.7, 95% CI: 1.7-8.3, use of hMSCs in the prophylaxis of GvHD. Since p=0.001), age at transplant>15 yr. (RR: 11.6, 95% CI: 1.6- thymoglobulin has being routinely used for the GvHD- 86.3, p=0.02) and age of 20-45 at LFU (RR: 5.53, 95% CI: prophylaxis in unrelated- and within the studies in related 1.9-16.1, p=0.002) as independent predictors.Two donor transplants we examined for the first time the in vitro pregnancies resulted in miscarriages leading to 3 foetal effect of thymoglobulin (0-200ug/ml) on immunomodulatory deaths (2/42, 5%; 3/45, 7%). Four babies were born and differentiation function of hMSCs (0.1-10% prematurely and required admission to neonatal unit and 2 endconcentration). In addition, we examined if the secretion of died after 6 weeks. None had congenital malformations. IL-6, IL-8, IP-10, RANTES and MCP-1 by hMSCs has been Currently, 40 children are alive and well; 26 after natural influenced by Thymoglobulin. conception and 14 after assisted fertilization. In conclusion, Methods and Results: Thymoglobulin binds to hMSCs and even though the chances of conception are lower in patients down regulates the HLA-I class expression. The expression of undergoing transplant pregnancy outcomes following natural CD54, CD105, CD73 and CD90 on hMSCs has not been and assisted conception are likely to be similar. influenced by thymoglobulin. hMSCs significantly reduced the proliferation of lymphocytes tested in mixed lymphocyte culture (MLC) in a dose dependent manner. However, P854 thymoglobulin caused increased proliferation of lymphocytes Response of naive and memory Treg cells to IL-7 compared to MLC with untreated hMSC (0.01) (Fig. 1). engineered stromal cells M. Di Ianni, B. Del Papa, L. Moretti, P. Sportoletti, E. Bonifacio, D. Cecchini, M. De Ioanni, F. Falzetti, A. Tabilio University of Perugia (Perugia, I)

T regulatory (T reg) cells constitute 1-2% of peripheral blood cells in adults and function prevalently as immunosuppressors. 70% of T reg are memory/effector cells with a CD45RO+ phenotype; the others have a naive T cell phenotype (CD45RA+). In the present study, we investigated

S218 identical:3;median age 10 years( 6 to 13); sex ratio:1; median interval from diagnosis to transplant 38 months (2 to 300); 12 patients (54,5%) had received more than 20 transfusions before allograft. Two conditioning regimens was used: BU-Cy protocol with busulfan 6mg/kg and cyclophosphamide 40mg/kg for 9 patients (40,9%) and FLU-Cy-ATG protocol with fludarabin 150mg/m, Cy 20mg/kg and anti thymocyte globulin (Fresenius) 40mg/kg for 13 patients(59,1%). GVHD prophylaxis associated cyclosporin and methotrexate 5mg/m(J1-J2-J3). Two patients received bone marrow transplant and 20 peripheral blood stem cell. At September The secretion of IL-6, IL-8, IP-10, RANTES and MCP-1 by 2006 maximal follow up is 97 months and minimal 4 months. hMSCs has been significantly inhibited by Thymoglobulin (50- Results: The median time to engraftment was 13 days (10 to 200ug/ml, p<0.001). The differentiation capacity of hMSCs 19). Eleven patients (50%) are alive with successful into adipocytes has not been influenced by Thymoglobulin, engrafment after median follow up 58 months (3 to 91), 2/9 however the differentiation of the hMSCs from passage 11 patients (22,2%) had BU-Cy conditioning and 9/13(69,2%) pre-treated with Thymoglobulin was more completed in FLU-CY-ATG. Late graft failure (J60 to J511) was observed in comparison to passage 2. 4 patients (19%). Acute GVHD accured in 11 patients (52,4%) Conclusion: Our data have shown for the first time that with 4 grade II-IV and extensive chronic GVHD in 2 patients Thymoglobulin suppress the immunomodulatory effect of (12,5%). Eleven patients died (50%): 6 (27,3%) by transplant hMSCs in vitro without influencing their differentiation related mortality (TRM): grade II/IV acute GVHD:4,e xtensive capacity. This finding offers new insight into the interactions chronique GVHD:1, hemorrhage:1 and 6 patients by another between hMSCs and Thymoglobulin, suggesting that in vivo causes: graft rejection:3, secondary neoplasia:1 and effect of hMSCs in GvHD could be altered. unknown:1. Actuarial overall survival (OS) is 36% at 7,5 years. TRM was higher in BU-Cy conditioning group (77,7%) than FLU-CY-ATG group (30,6%) and OS is 52% at 7,5 years in P856 FLU-Cy-ATG group. Tissue repair using allogeneic mesenchymal stem cells Conclusion: FLU-Cy-ATG conditioning regimen appears better for haemorrhagic cystitis, pneumomediastinum and than BU-Cy. A best outcome can be reached by precoce perforated colon diagnosis and by reduction of interval from diagnosis to O. Ringden, M. Uzunel, B. Sundberg, L. Lonnies, S. Nava, J. allograft. Gustafsson, L. Henningsohn, K. Le Blanc Karolinska Institutet (Stockholm, S) P858 Mesenchymal stem cells (MSC) can differentiate to several Regional intrathecal infusions of allogeneic donor mesenchymal tissues and have been suggested to be used lymphocytes in patients with brain tumours: a phase I for tissue repair. We explored the possibility to use MSC to trial heal chemoirradiation-induced tissue toxicity. I. Dolgopolov (1), M. Budarin (1), G. Tchkadia (2), R. Pimenov Ten consecutive patients who were treated with MSC due to (1), M. Yankelevich (1), A. Popa (1), G. Mentkevich (1) tissue toxicity following allogeneic hematopoietic stem cell (1)Institute of Ped. Oncology/Hematology (Moscow, RUS); transplantation (ASCT) were included. Median age was 48 (2)Institute of Experimental Diagnostics and Therapy of (13-64) years. Seven had severe hemorrhagic cystitis grades Cancer (Moscow, RUS) 2-5, two had pneumomediastinum and one had perforated colon diverticulitis and peritonitis. MSC were from HLA- In light of the published clinical evidence that “graft vs. brain mismatched (n=11), from HLA-haploidentical donors (n=3) or tumor” effect exists in the settings of ch. GVHD after HLA-identical siblings (n=2). MSC were given i.v. and median allogeneic bone marrow transplant in a pt with recurrent cell dose was 1.0 (range 0.7-2) x 106/kg. ependymoma we hypothesized that repeat regional infusions Two patients with severe hemorrhagic cystitis had decreased of allogeneic lymphocytes across blood brain barrier may transfusion requirements after MSC infusion, but died of multi- potentially provide antitumor effect in pts with brain tumors. organ failure. In one of them, MSC donor DNA was Five pts (3 M, 2 F) with recurrent/progressive anaplastic demonstrated in the urinary bladder. In five patients, severe ependymoma (AE) (n=4) and glioblastoma (GB) (n=1) were hemorrhagic cystitis cleared after MSC infusion. In two enrolled into phase I trial of regional i.t. and/or intratumoral patients, pneumomediastinum disappeared after MSC infusion of allogeneic donor lymphocytes. The mean age of infusions. A patient with steroid-resistant graft-versus-host pts was 9.8 years (2-18). The relatives (mother – 3, sister – 1, disease of the gut experienced perforated diverticulitis and grandmother – 1) served as lymphocyte donors. HLA peritonitis that was reversed twice by MSC. matching was 3/6 in 4 and 1/6 antigen in 1 pt. PBSCs were To conclude, MSC is a novel therapy for therapy-induced harvested after G-CSF stimulation. A median of 4.2 x 106 tissue toxicity that deserves further evaluation. (1.6-8.1) CD34+/dose was infused i.t. or into a tumor cavity every 2 weeks. In 2 pts the intervals between infusions were shortened to the next level (every week). All pts also received P857 CT (at least initially) and one pt received autologous dendritic Allogenic haematopoietic stem cell transplantation for 22 vaccine in parallel with allogeneic cells infusions. A total of 51 patients with Fanconi anaemia performed in 93-month regional allogeneic cell infusions (range 2 to 14) were given to period 5 pts. No complication neither CSF abnormalities were R. Ahmed-Nacer, M. Saidi, F. Harieche, M. Benakli, F. observed. One pt with AE died of DP 3 mo after the first Mehdid, R. Belhadj, N. Rahmoune, M. Baazizi, R.M. Hamladji infusion. Four pts are alive with SD and still receiving Pierre and Marie Curie Center (Alger, DZ) treatment 1 to 9 mo from the beginning of the trial. We were able to evaluate isolated effect of regional intraCNS infusions Introduction: Fanconi anaemia( FA) is an autosomal recessive of allogeneic donor lymphocytes in only one pt. This 18 yo disease which evolves frequently to bone marrow failure and male with 3rd recurrence of GB demonstrated gradual clinical acute myeloid leukemia. Allogenic hematopoietic stem cell improvement of strength, ambulation and seizures started (HSTC) is the treatment of choice for patients with FA, it approximately 1 week after the first allogeneic cell infusion. leads to normal complete hematopoietic recovery and This pt was not on any steroids or other treatment. MRI 3 prevention of acute leukemia. months after the first alloinjection revealed SD with some Materials and methods: From June 1998 to March 2006 (93 improvement of contrast enhancement around tumor resection months period) 22 patients with FA underwent allogenic HSCT site; however autologous dendritic cell vaccination was also (from HLA-identical sibling donors:19 and pheno started in this pt 2 mo prior to the date of the scan.

S219 In conclusion, regional repeat intraCNS infusions of allogeneic P860 donor lymphocytes in pts with brain tumors were feasible and Early consolidation with bortezomib, thalidomide and well tolerated. The questions of intraCNS allogeneic cells dexamethasone on molecularly-detectable disease in MM clearance and traffic into the tumor/brain remained patients in CR or VGPR following ASCT: uncommon unanswered. Both cytotoxic T-cells and NK cells can achievement of molecular remission despite evidence of potentially provide an antitumor effect. Further laboratory and tumour load reduction by real time PCR clinical exploration of allogeneic donor immunocompetent M. Ladetto (1), M. Astolfi (1), L. Santo (1), I. Avonto (1), F. cells vs brain tumor effect is warranted. Cavallo (1), G. Pagliano (1), P. Pregno (2), M. Grasso (3), A.M. Liberati (4), T. Caravita (5), F. Pisani (5), T. Guglielmelli (2), E. Genuardi (1), R. Crivelli (1), C. Cristiano (1), D. Drandi P859 (1), S. Ferrero (1), M. Boccadoro (1), A. Palumbo (1) Analysis of the revascularization capacity of monocytes (1)University of Turin (Turin, I); (2)Italian Multiple Myeloma obtained from peripheral blood Network, GIMEMA (Turin, I); (3)Italian Multiple Myeloma N. López-Holgado (1), M. Alberca (1), F. Sánchez-Guijo (1), J. Network, GIMEMA (Cuneo, I); (4)Italian Multiple Myeloma Almeida (2), J. Rivas (3), J.M. López-Novoa (3), C. Garcia (1), Network, GIMEMA (Perugia, I); (5)Italian Multiple Myeloma J.F. Pérez-Fontán (1), B. Blanco (1), L.I. Sánchez-Abarca (1), Network, GIMEMA (Rome, I) S. Tabera (1), J.A. Pérez-Simón (1), J.F. San Miguel (1), C. del Cañizo (1) Background: ASCT is unable to induce molecular remission (1)University Hospital of Salamanca (Salamanca, E); (MR) in MM as opposed to allogeneic transplantation. It is (2)Cancer Research Center of Salamanca (Salamanca, E); unknown if new non-chemotherapeutic agents following ASCT (3)University of Salamanca (Salamanca, E) might ensure further cytoreduction allowing patients to enter MR. In this study we assessed this hypothesis by Cells from the monocyte-macrophage system can be involved consolidating with Bortezomib, Thalidomide and in the revascularization process and even it has been Dexamethsone (VTD) patients achieving CR or VGPR suggested that endothelial progenitors can derive from these following an ASCT-containing regimen. cells. Patients and methods: Patients were eligible if they had: 1) a The aim of this study was to assess the “in vitro” differentiation molecular marker based on the IgH rearrangment; 2) a ability of monocytes into endothelial cells and their angiogenic documented CR or VGPR following ASCT. The VDT had to be properties in a murine model of hind limb ischaemia. started within 6 months from ASCT. Each cycle included: a) Monocytes were obtained from buffy-coats of blood donations Bortezomib 1.6 mg/m² as an IV injection once weekly (on days (n=10) by adherence. They were cultured in the presence of 1, 8, 15, 22) followed by a 13-day rest period (days 23-35); b) VEGF (10ng/ml), FGF (2ng/ml) and IGF (1ng/ml). Thalidomide at the initial dose of 50 mg/day PO once daily, Immunophenotypic analysis were performed before and 26 with increments of 50 mg every 7 days to acceptable days after initiating the culture using the following monoclonal tolerance (maximum 200 mg); c) Dexamethasone 20 mg/day Antibodies (McAb): CD13, CD14, CD15, CD16, CD31, CD33, PO once daily, on days 1 to 4, 8 to 11 and 15 to 18 followed CD34, CD45, CD64, CD105, CD133, KDR, Lysozyme,Ve- by a 17-day rest period (days 19-35) A total of 4 cycles were cadherine and vWF. ”In vivo” studies were performed in a delivered. Bone marrow samples for molecular analysis were mouse model of ischaemia. Acute hind limb ischaemia was taken at study entry, after two and four courses, and then at induced after ligation and excision of the left femoral artery in three months intervals. Minimal residual disease (MRD) has 6 swiss-nu/nu 6-8 weeks old mice. Revascularization was been evaluated using clone-specific primers by nested PCR measured by two approaches: blood flow using a laser and real time PCR as published elsewhere (Voena et al, Doppler device (moor LAB server) and by assessing capillary Leukemia 1997 and Ladetto et al Biol Bone Marrow Transpl density (number vessels/um²). Two groups of mousse were 2000). used for the study: one receiving 105 monocytes (n=6) and Results: 21 Patients have entered the study so far. One was other in which only a saline solution was injected (control already PCR negative after ASCT, one is not evaluable due to group) (n=6). Blood flow was measured the day before artery early toxicity. 10 patients have been evaluated only after two ligation (basal) and on days +1, +7, +14 and +28. On that day, courses and eleven also at the end of the program. For seven mice were sacrified and capillaries counted in both vastus patients also follow-up samples at three and six months are muscles (ischaemic and non ischaemic) by using available. Nested PCR was always PCR positive with the immunochemistry with anti-CD31McAb.The Visilog program exception of three patients. One had a PCR-negative sample was used in order to obtain the number of capillaries/sample. after 50% of consolidation therapy but reverted to PCR Monocytes purity after adherence was 87% (75-92). These positivity at the end of treatment and during follow-up. One cells expressed the following markers: CD13, CD31, CD33, patient is PCR-negative after two courses but we do not have CD45, CD64, lysozyme CD15, CD105, CD16, Ve-cadherine subsequent follow-up samples. Only one patient achieved but were negative for CD34, CD133 and vWF. 1.3% of cells PCR negativity following two VTD courses and mantained MR expressed the KDR antigen. After 28 days in culture all during follow-up. Real time PCR is available in six patients. In hematopoietic and myeloid specific markers showed a four a reduction of tumor burden was observed following VTD decreased expression when compared to that of the basal treatment (median reduction 0.77 log). population. Regarding CD14 expression, two cell Discussion: Consolidation with VTD is active on MRD as subpopulations could be observed: one positive (69%) and documented by real time PCR. However in most patients, one negative (31%) and both of them expressed the KDR VTD seems unable to reduce the tumor load below the antigen. sensitivity threshold of PCR. Blood flow increased after ischaemia in both groups of animals at day +7, +14 and +28 but always recovery was faster in mice receiving monocytes. This difference was P861 statistically at day +14. Risk factors for late infections after allogeneic The number of capillaries was significantly higher in mice haematopoietic stem cell transplantation from a matched receiving monocytes. In no case we observed a monocytes related donor incorporated into a mouse vessels. M. Robin, R. Porcher, R. De Castro Araujo, R. Peffault de Latour, A. Devergie, V. Rocha, J. Larghero, H. Espérou, L. Adès, E. Gluckman, P. Ribaud, J.Y. Mary, G. Socié Hôpital Saint Louis (Paris, F)

After allogeneic hematopoietic stem cell transplantation (HSCT), early infections represent a major cause of morbidity and mortality but little information has been previously reported on late infections. Late infections incidence and risk

S220 factors were retrospectively determined in 196 long-term GVM effects in man, as observed in preclinical animal models, survivors after HLA matched related HSCT. Patients must be determined in a prospective clinical trial. transplanted for aplastic anemia, chronic myeloid leukemia *Igor B. Resnick and Michael Y. Shapira contributed equally to (CML) and acute myeloblastic leukemia (AML) were included. this study. Median follow-up was 8 years. 30 patients died beyond the first year, causes of death were relapse (n=10) and infections (n=19, associated with GVHD in 16 patients). Late severe P863 bacterial (LSB) and fungal infections occurred in 30 and 8 Imbalance in NK cell subsets reconstitution after HLA- patients, yielding to an 8-year cumulative incidence of 15 matched haematopoietic stem cell transplantation (95%CI: 10-20) and 4% (95%CI: 1-6), respectively. The N. Dulphy (1), P. Haas (1), M. Busson (1), S. Belhadj (2), P. majority of viral infections were hepatitis C and VZV (8-year Loiseau (3), C. Rabian (3), D. Charron (3), A. Janin (2), G. cumulative incidence: 10 (95%CI: 5-14) and 27% (95%CI: 20- Socié (4), A. Toubert (1) 34) , respectively). 3 risk factors for LSB have been identified (1)INSERM U662 (Paris, F); (2)INSERM U728 (Paris, F); in multiple Cox analysis: CMV status (positive recipient and (3)Service d'Immuno-Histocompatibilité (Paris, F); (4)Service negative donor) [HR: 2.5, 95%CI: 1.1-5.9, p=0.033], irradiation d'Hématologie-Greffe de Moelle (Paris, F) based conditioning regimen [HR: 3.1, 95%CI: 1.2-7.8, p=0.016] and chronic extensive GVHD [HR: 2.9, 95%CI: 1.3- NK cells constitute a main actor of the anti-tumour immune 6.9, p=0.013]. Chronic extensive GVHD was the only risk response. They are divided in two sub-populations according factor for non-HCV viral infections in patients transplanted for to the expression of CD56. CD56brigth NK cells are AML or CML [HR: 2.7, 95%CI: 1.4-5.1, p=0.002]. After HSCT, specialized in inflammatory cytokines production whereas patients remain at high risk of infections even late after CD56dim cells are mainly cytotoxic. transplantation, in particular with the above risk factors, and We investigated the reconstitution of both sub-populations in required a prolonged follow-up. 43 HLA-matched non T-cell depleted hematopoietic stem cell donor-recipient pairs using flow cytometry-based analysis. The study was performed on samples before and at 3, 6 and P862 12 months after graft. We confirmed the early expansion of Irradiated lymphokine-activated killer cells: a safe tool for NK cells as compared to T-cells. However this expansion is immunotherapy after haplo-identical stem cell observed only for CD56bright cells, resulting in the transplantation modification of the ratio between the NK sub-populations. This M.Y. Shapira, I.B. Resnick, R. Or, S. Morecki, Y. Gelfand, O. perturbation is observed for at least one year after graft. Eizik, M. Aker, M. Bitan, I. Zilberman, B. Gesundheit, S. Recipient CD56brigth NK cells show an overexpression of the Samuel, L. Dray, A. Ackerstein, S. Slavin activating receptors NKG2D and NKp46 and the inhibitory Hadassah Hebrew University (Jerusalem, IL) receptor NKG2A as compared to donor CD56brigth populations. Interestingly the intra-cellular perforin is also Objectives: The therapeutic effect of haplo-identical stem cell overexpressed in recipient CD56brigth NK cells and this transplantation (HSCT) in malignancies and non-malignant overexpression correlates with the occurrence of acute Graft- diseases depends on reactivity of the graft against versus-Host disease. We confirmed the capacity of recipient undesirable target cells of host origin. The mandatory T cell CD56bright NK cells to degranulate perforin and to expose depletion (TCD) across MHC as the only approach for CD107a on the cell-surface during lysis of target cells. Our successful prevention of graft-versus-host disease (GVHD) results would suggest an impact of NK cell sub-populations on can cause graft failure, delayed immune reconstitution and the induction of GvH reaction in HLA-matched hematopoietic lack of graft-vs.-malignancy effects. Our goal was to induce stem cell transplanted patients. GVM effects following HSCT while avoiding GVHD. Scientific background. GVHD is induced by alloreactive donor derived T lymphocytes. Previous investigations in murine P864 models suggest that neither naïve nor rIL-2 activated NK cells Photodynamic purging of alloreactive T-cells for adoptive can cause GVHD. immunotherapy after haplo-identical stem cell Patients and Methods: Treatment with irradiated lymphokine- transplantation activated killer cells (ILAK): was performed in 7 patients after K. Perruccio, F. Topini, A. Tosti, A. Carotti, T. Aloisi, F. T cell depleted haploidentical HSCT from related donors. All Aversa, M.F. Martelli, A. Velardi patients had high risk diseases: 3 ALL, 3 AML and 1 X-ALD. University of Perugia (Perugia, I) Total 17 procedures were performed from 1 to 4 procedures per patient; interval between infusions was 6 to 68 (median After haploidentical stem cell transplantation immune recovery 17) days in each session. Donor cells were incubated in the is inevitably slow and infectious related mortality is about 30- presence of rIL-2 for 96 hours, irradiated with 15 to 30 Gy and 40%. Immune reconstitution could be improved by infusing infused in doses from 0.7 to 6.2 (median 2.3) x107 per kg. In donor T cells, but the obstacle is Graft-versus-Host Disease. vitro cytotoxicity was controlled for each cell sample. In a mixed lymphocyte reaction, alloantigen-stimulated T cells Results: Four out of 7 patients developed symptoms of acute uptake 4,5-dibromorhodamine methyl ester (TH9402), a GVHD: 1 grade I, 2 grade III and 1 grade III-IV. In all but one compound that is structurally similar to rhodamine. TH9402 case acute symptoms were reversible with conventional preferentially localizes in mitochondria and when exposed to treatment. In one patient we saw no symptoms of GvHD with 500-600 nm wave-length visible light delivered through the transition of mixed to 100% donor chimerism. In the last TheraluxTM device (Celmed, Montreal, Canada) it becomes patient effect was difficult to estimate as immediately after the highly cytotoxic through oxidative damage. This study first iLAK infusion relapse of his ALL developed which investigated a range of parameters, and combinations thereof, required appropriate treatment with chemotherapy. In one with the aim of achieving optimal T cell allodepletion and case after 3 infusions of iLAK, an episode of acute GVHD preservation of pathogen-specific responses. We report on 9 transformed to chronic gastrointestinal GVHD. Overall survival clinical scale dry-runs which reproducibly yielded the following in this high risk group is 2 out of 7, all five deceased patients results. PBMC were stimulated with haploidentical irradiated suffered relapse of their leukemia, and mortality was not (20 Gy) PBMC in a MLR. Cells were then incubated with associated with iLAK infusion. Both surviving patients are free TH9402 and exposed to light delivered through the of any undesired consequences of iLAK infusion with no TheraluxTM device. Optimal conditions for T cell allodepletion chronic GVHD. emerged as: Conclusion: iLAK can be a safe, inexpensive tool for cellular 1) duration of mixed lymphocyte reaction: 24 hours; immunotherapy of residual disease while avoiding severe 2) responder cell concentration: 3-5 x 106/ml; GVHD after HSCT. The exact dose of ionizing radiation 3) TH9402 concentration: 5 ìM; required for complete prevention of GVHD while retaining 4) quantity of internalized TH9402, as measured by mean fluorescence intensity (MFI): 20,000-25,000 MFI;

S221 5) energy delivered: 0.1 J/cm2. P866 Only under these conditions were the frequencies (by limiting Loss of ex vivo Valpha24+ natural killer cell expansion dilution analyses) of alloantigen-specific T cells maximally efficacy in cancer patient reduced, i.e., 3,583 ± 1,909 (mean ± SD) times, when O. Imataki (1), Y. Heike (2), K. Oda (3), K. Shirakawa (3), H. compared with non TH9402-treated cells. Pathogen-specific Ishida (3), D. Hashimoto (3), Y. Takaue (2) responses to pathogen antigens such as CMV, Adenovirus, V- (1)Kagawa University (Kagawa, JP); (2)National Cancer Z, Aspergillus, Candida, Toxoplasma were retained, although Center Hospital (Tokyo, JP); (3)Saitama Medical School with a 63 ± 22 times reduction in frequency. This remarkable (Saitama, JP) drop in frequency of alloreactive T cells is expected to allow safe infusion of relatively large numbers of T cells across Background: Vaplha24+ natural killer T (NKT) cells are histocompatibility barriers for adoptive transfer of donor primary effectors of innate immune systems, and also play an immunity. Consequently, a clinical trial is being designed to important role in effectively stimulating and regulating adaptive incorporate infusion of photo-allodepleted donor T cells after immune responses. Human NKT cells, which is very small haploidentical stem cell transplantation with the aim of population in peripheral blood mononuclear cells (PBMC), decreasing infection-related mortality. effectively proliferated by using a specific ligand aplha- galactosylseramide (alpha-GalCer), while in cancer patients, this proliferation is suppressed. P865 Object: We analyzed characteristics of PBMC taken from Isolation of monocytes from leukapheresis products for cancer patients to identify the factors that contributed to ex large-scale GMP-grade generation of CMV-specific T-cell vivo expansion of Vaplha2+ NKT cells and compared to those lines by means of an automated elutriation device from healthy volunteers. (Elutra®) Methods: PBMC were collected from healthy volunteers P. Perseghin (1), G. D'Amico (2), E. Dander (2), M. Dassi (1), (n=10) and colon cancer patients (n=17). PBMC were cultured G. Gaipa (2), E. Biagi (2), A. Biondi (2) in AIM-V medium supplemented with 5% auto-plasma, 100 (1)Ospedale San Gerardo (Monza, I); (2)University Milan ng/mL aplha-GalCer and 100 U/mL recombinant human IL-2, Bicocca (Monza, I) which was supplied every 3 days. On day 12, expansion efficacy of Vaplha2+ NKT cells was calculated. To evaluate Background: Dendritic cells (DC) act as antigen presentig the contribution of CD14+ cells, we sorted CD14+ cells from cells (APC) in immune responses against malignant cells healthy donors and co-cultured them with a CD14- negative and/or viral or fungal pathogens. CD14+ monocytes have fraction including NKT cell precursor from cancer patients. been so far isolated by means of plastic adherence Results: Although the proportions of Vaplha24+ NKT cells in techniques or by using immunomagnetic methods. Here we peripheral blood before culture were comparable between investigated the effectiveness of a newly released device healthy donors and cancer patients (0.02±0.036 vs (Elutra®) in the separation of monocytes obtained from 0.02±0.026 %, P=0.49), the number of cultured Vaplha24+ mononuclear cell (MNC) collections and the large-scale NKT cells derived from cancer patients was less than that in production of CMV-specific T-cell lines. healthy donors with marginal significance (472±886 vs Study design and methods: Six MNC collections obtained by 7.85±11.2, x 10²/mL, P=0.06). There were no significant using a continuuos flow cell separator (COBE Spectra) were recovery of NKT cell expansion observed, in culture with processed with the Elutra system. The monocyte-enriched healthy donors' plasma (1.01±1.11 patient auto-plasma vs fraction was differentiated into DCs by sequential addition of 1.26±1.82 healthy donors plasma, x 10²/mL, P=0.78), nor in GM-CSF, IL-4, IFN-gamma, IFN-alpha, IL1-beta, TNF-alpha culture with healthy donors' CD14+ cell (1.05±0.70 patient and poly (I:C). DC were pulsed with a pool of 47 MHC class I CD14+ cells vs 26.4±29.3 healthy donor CD14+ cells, x and II binding CMV peptides derived from different CMV 10²/mL, P=0.09). Specific surface markers (CD94, CD69, proteins (pp65, pp50, pp150 and IE1) for simultaneous CD161, NKG2A and NKG2D) on NKT cells between healthy expansion of CD8 and CD4 T-cell lines. Lymphocytes were donors and cancer patients before culture were not then stimulated with mature autologous CMV peptide pulsed- significantly different. DC for 3 rounds of weekly stimulation. Conclusions: Previous our data demonstrated that the Results: MNC collections contained a median of 10.03 x 109 expression of CD1d molecules on CD14+ cells was lower in total nucleated cell count, with a median 78.5 % lymphocytes cancer patients than that in healthy donors. However, CD14+ and 15 % monocytes content. Elutriation procedure took a cell obtained from healthy donor could not overcome the mean of 59 ± 4 minutes. After elutriation mean monocyte yield reduced expansion of NKT cells in cancer patients. Those was 0.89 ± 0.65 x 109, with a 51.0 ± 31.6 % recovery and a results might suggest that Vaplha24+ NKT cell in cancer 51.1 ± 35.4 % purity. We observed a significant correlation patient lose a potentiality to expand, in other mechanisms when basal monocyte content was related to the post- related to common NK cell activation mechanisms. elutriation recovery (P< 0.0116). In fact, when the basal product contained more than 1.5 x 109 monocytes we observed both an higher purity (81v s 45 %) and recovery (74 P867 vs 37 %) when compared with products with lower monocyte Expression patterns of oncogenes and tumor suppressor contents. At the end of the differentiation process >60% of genes in mesenchymal stromal cells derived from bone plated monocytes were differentiated into DCs and a robust marrow of children enrichment in CMV antigen-specific T cells was observed in D. Choumerianou, E. Linardakis, H. Dimitriou, E. Stiakaki, C. all samples (average percentage of pentamer+ CD8+ Perdikogianni, G. Martimianaki, M. Kalmanti cells=35% compared to the initial value = 2%) University of Crete (Heraklion Crete, GR) Conclusion: Our findings might be helpful in appropriately tailoring MNC collection from healthy donors, in order to Recent studies have shown that mesenchymal stromal cells maximize the efficiency of the elutriation system and to (MSCs) can become targets of neoplastic transformation subsequentely obtain an optimal monocyte-enriched yield for leading to cells with malignant potential. We studied the further DC generation together with lymphocyte-enriched expression profile of oncogenes and tumor suppressor genes ancillary units to be stimulated by CMV-pulsed DC. Moreover, (TSG), known to play an important role in the malignant the elutration procedure proved to be more cost effective than transformation of cells. We assessed their expression levels in immunomagnetic methods. bone marrow (BM) mononuclear cells (MNCs) and compared them to the respective ones in MSCs as well as their levels in different MSC passages (P). MNCs and MSCs were isolated from BM of children diagnosed with idiopathic thrombocytopenic purpura (ITP, n=8), autoimmune neutropenia (n=5), solid tumors without BM involvement (n=6) and B cell acute lymphoblastic leukaemia

S222 (ALL) at diagnosis (n=5). MSCs were cultured for 6 passages. survival. The proposed discrepancy index appears feasible to RNA was isolated from MNCs as well as from MSCs at quantify differences between donor and recipient NK cell passages 2 and 6. RT-PCR was performed for p53, p16, Rb phenotype pattern. and H-Ras genes. Expression of GAPDH housekeeping gene was used as internal control and HUVEC cells as normal control for adherent cells. In all groups studied, MSCs at P2 expressed higher levels of Cytokines p53, Rb and H-Ras and lower levels of p16 in comparison to MNCs from the same patients. More specifically, statistical significant differences between MNCs and MSCs at P2 were shown on the expression levels of p53, Rb and H-Ras, in the P869 ITP group. Similar expression patterns were found in Positron emission tomography identifies a differential neutropenia apart from p53 and in solid tumor samples with pattern of bone marrow FDG uptake in "poor" and "good" the exception of H-Ras. However, there were no differences in peripheral stem cell mobilizers expression levels between passages P2 and P6 and between G. Marcacci, C. Caracò, G. D'Arena, L. Aloj, G. Corazzelli, F. P2 and HUVEC. In ALL samples though, expression levels Frigeri, S. Lastoria, A. Pinto were similar between MNCs and MSCs at P2. National Cancer Institute, Fond. Pascale (Naples, I) These results indicate that despite the differences in oncogene and TSG expression levels between MNCs and Objectives: The use of prophylactic G-CSF is associated to MSCs, the profile of the same genes does not change during the increase of bone marrow (BM) fluorodeoxyglucose (FDG) cultivation of MSCs up to passage 6. This finding, combined uptake as detected by Positron Emission Tomography (PET). with the similar expression levels between MSCs and HUVEC In contrast, no data is available as to changes in BM FDG- cells, suggests that MSCs may not be transformed for a uptake during peripheral blood stem cell (PBSC) mobilization. number of serial passages. This study was aimed at investigating patterns of BM FDG uptake during mobilization as quantified by Standardized Uptake Value (SUV) determinations. We also evaluated P868 whether PET scanning may turn of value for identifying good Reconstitution of NK cell phenotype after allogeneic and poor mobilizers. To our knowledge, this is the first PET- haematopoietic stem cell transplantation based study in this setting. S. Giebel (1), J. Dziaczkowska (1), T. Czerw (1), T. Kruzel (1), Methods: Nine patients(pts)(M/F=6/3), median age 41 yrs (r J. Wojnar (1), M. Krawczyk-Kulis (1), M. Markiewicz (1), I. 33-60), with relapsed lymphoma (NHL/HD=8/1) without BM Wylezol (1), A. Holowiecka-Goral (1), P. Kusnierczyk (2), J. involvement, were accrued after informed consent. Baseline Holowiecki (1) PET was obtained at relapse, before salvage therapy and any (1)Silesian Medical University (Katowice, PL); (2)Institute of CSF administration. After salvage regimes, pts were mobilized Immunology and Experimental Therapy (Wroclaw, PL) by VRL/CTX or idARA-C; G-CSF (10 ug/kg/day) was given from day +4 through apheresis. PET scans were obtained on NK cells are considered to play a role in anti-infectious and day +9 or +10 (after nadir with a WBC>1000/ul). SUVmax and anti-leukemic reactions. Their phenotypic maturation is average (avg) were measured (whole lumbar spine and gradual and is expected to eventually resemble the donor-type bilateral iliac regions) and compared to SUV of the same BM pattern. The goal of this study was to assess factors regions at baseline PET. The aim was to calculate a BM influencing NK cell maturation and association of this process specific D-SUV (mobilizing vs steady-state D-SUV) for each with clinical outcome. single patient. Results: Five pts mobilized PBSC (median CD34 peak 42.8/ul, Seventy adult patients with hematological malignancies given 6 alloHSCT from sibling (n=28) or unrelated (n=42) donor were r 27.9-280.5/ul; median CD34 in the harvest 3.3 x 10 /Kg, r included. NK cell phenotype was studied in the donor before 2.1-12.5) while 4 pts were poor mobilizers (median CD34 HSCT and in the recipient on days +28, +56, +100 and +180, peak 10.9/ul, r 7.5-14.1/ul). In the group of good mobilizers, using antibodies specific for KIR2DL1/DS1, KIR2DL2-3/DS2, apheresis was performed at CD34 peak (day +11,+13), with a KIR3DL1/DS1, and NKG2A. Individual pattern was created for median of 1 apheresis/pt (r 1-2). Unexpectedly, effective each donor based on frequencies of NK cells expressing mobilization was associated with a low BM uptake of FDG: respective receptors together with mean fluorescence median BM D-SUVmax and D-SUVavg of 3.0 (r 1.0-3.8) and intensity (MFI). To quantify differences between donor and 3.3 (r 0.9–3.9), respectively. In contrast, poor mobilizers recipient after alloHSCT, we developed a new discrepancy displayed a median D-SUVmax and D-SUVavg of 5.7 (r 2.4– index (DI) based on sum of differences defined as distances in 12.8) and 6.9 (r 4.1–14.2), respectively. coordinate system constructed by frequencies and MFI axes. Conclusions: While BM FDG uptake usually increases upon DI equaled 8.0 (3.2-15.1) on day +28 and decreased to 7.4 CSF administration, our results suggest that PBSC (2.6-15.0) on day +56 (p=0.003, vs. d. +28), 6.3 (2.3-15.0) on mobilization may be associated with a complex metabolic day +100 (p=0.001, vs. d. +28), and 6.2 (1.8-11.4) on day pattern of BM as detected by PET. We documented that, 48 to +180 (p=0.0005, vs. d. +28). At all time-points NK cells had 72 hrs before CD34 peak, poor mobilizers display a higher BM increased expression of NKG2A compared to the donors. On FDG uptake (D-SUV>4) as compared to good mobilizers (D- days +28, +56, and +100 DI values correlated negatively with SUV<4). These preliminary results indicate that BM PET may the number of CD3+, CD3+CD4+, and CD3+CD8+ cells, and represent a new tool for early identification of poor mobilizers were increased for patients receiving steroids for the allowing a timely modification of the mobilization strategy to treatment of acute GVHD. A positive correlation was found possibly rescue the procedure. between the DI and a maximum dose of methylpredisolone but not with the grade of acute GVHD. On day +180 DI was increased for patients receiving CsA (p=0.04) which, however, P870 did not depend on the presence of chronic GVHD. TNF receptor type II 196M/R genotype of the donor is The OS rate at 18 months was higher for patients with DI <9 associated with survival after sibling allogeneic stem cell vs. >9 on day +28 (83% vs. 64%, p=0.04). The difference transplantation resulted from NRM, which was associated with late fatal J. Nersting, C. Heilmann, J. Andersen, L. Ryder, K. Rieneck, infections (10% vs. 32%, respectively, p=0.02). H. Baekgaard, N. Jacobsen , K. Müller Conclusions: The pattern of NK cell phenotypic reconstitution Rigshospitalet (Copenhagen, DK) after alloHSCT generally tends to recapitulate the donor-type. The process is negatively affected by the use of steroids and TNF is a cytokine that plays a central role in the inflammation cyclosporin, and seems to be parallel to reconstitution of T cascade. A single nucleotide polymorphism in the TNF type II cells. In turns, delayed maturation of NK cells may contribute receptor (TNFRII) gene, codon 196, results in substitution of to impaired anti-infectious defense translating into decreased arginine (R allele) for methionine (M allele). In transfected cell

S223 lines the 196R allele transduces TNF signalling more found a higher incidence in patients with TNF beta and alfa effectively than 196M. The 196R allele has been associated homozygous variant(p=0.0004, p=0.02) and IL10 -1082 with systemic lupus erythematosus and with the severity of heterozygous variant (p=0.005); in addition we observed that rheumatoid arthritis and acute GvHD (aGvHD). We the patients with advanced status disease had a lower investigated the impact of donor and recipient TNFR II 196 survival( p=0.002) . These data suggest that the determination genotypes on survival in Danish SCT patients. of functional genotypes in the cytokine pathways may have Patients and methods: TNFR II 196 genotypes were clinical utility for risk assessment, counseling and treatment determined in amplified genomic DNA by using restriction planning before trasplantation. enzymes and agarose gel electrophoresis. Transplants with either matched sibling donors (n=159) or matched unrelated donors (MUD) (n=102) were included by the availability of DNA. The indications for SCT were ALL (n=58), CML (n=59), AML (n=63), MDS (n=15), other types of haematologic malignancy (n=28) and non-malignant disorders (n=38). The logrank test was applied. Results: In sibling transplants the 196 RR genotype of the donor was associated with decreased overall survival (p=0,041). A selective analysis of treatment related mortality showed the same pattern (p= 0,015). There was no significant association between TNFR II 196 genotypes and the frequency or grade of aGvHD, and TNFR II 196 genotypes were unrelated to the risk of relapse of leukaemia. Carriage of the R allele by the recipient was not associated with survival or with aGvHD. In MUD transplants we did not observe any association between TNFR II 196 genotypes and outcome. Conclusion: Our data suggest that the TNFR II 196R allele of the donor is associated with treatment related mortality in sibling donor SCT, and points to TNF as a mediator in the P872 pathogenesis of treatment related complications. Selection of Donor genotype for the IL-10 -1064 polymorphism donors based on TNFR II 196 typing, in addition to HLA- influences the development of chronic GvHD, in a typing, may help to reduce treatment related mortality in SCT. chimerism mediated fashion, after HLA-identical allogeneic haematopoietic stem cell transplantation C. Manzano, N. Sanchez-Hernandez, D. Barroso, P. P871 Balsalobre, M. Gonzalez-Rivera, D. Serrano, R. Carrion, A. Role of non-HLA genetic polymorphisms after Gomez-Pineda, J.L. Diez-Martin, I. Buño haemopoietic stem cell transplantation Hosp. G.U. Gregorio Marañon (Madrid, E) G. Farina, G. Reddiconto, A. Fiorini, S. Giammarco, D. De Ritiis, S. Marietti, M. Palladino, L. Laurenti, G. Leone, P. Background: Polymorphisms in regulatory sequences of Chiusolo, S. Sica cytokine genes are known to influence their expression and, Catholic University S. Cuore (Rome, I) therefore, the intensity of the immune response. Some of such polymorphisms have been associated with the outcome of HSCT may be associated with a variety of complications such allogeneic stem cell transplantation (SCT). as GVHD and major non infectious transplant-related Objective: To evaluate the association between donor (D) and complications (TRC). Although HLA compatibility has the recipient (R) genotype for the IL-10 gene -1082 SNP (single central role in selecting donors and determining transplant nucleotide polymorphism) and -1064 STR (short tandem outcome, the sequencing of the human genome has revealed repeat) polymorphism with the dynamics of chimerism and the numerous non HLA encoded single nucleotide polymorphisms development of complications after SCT. whose significance in allogenic HSCT could be relevant. Patients and methods: 24 HLA-matched conventional SCT. IL- Due to these polymorphisms patients may be predisposed to 10 genotypes were determined in an -1082 SNP allele-specific releasing high levels of certain cytokines pre and post PCR (A vs G) including the -1064 STR in the product which is trasplant during the “cytokine storm” and therefore be more revealed by capillary electrophoresis. Results were analyzed predisposed and/or protected from GVHD and mortality. In our using Fisher's exact test due to the reduced sample size. study we examined the association of single nucleotide Results: The frequency of alloreactive genotypes (AA or A) for polymorphisms at position 677 and 1298 in the MTHFR gene, the SNP was 37,5% AA and 79,2% A in the D and 33,3 AA at –308 in the TNF alfa gene and – 252 in the TNF beta gene, and 95,8% A in the R . No association was observed between at –1082 and –592 in the IL10 gene, at –238 in the IL10 the homozygous or heterozygous presence in the D or R of receptor gene and at 908, 702, 1007 in the NOD2/CARD15 the alloreactive allele for the SNP genotype and the dynamics gene, on incidence of GVHD (grade II-IV) and mortality in a of chimerism or complications post-SCT. Alleles 4 to 10 for the group of 35 patients submitted to allogenic HSCT from 2004 in STR genotype were found in contrast to previous reports our institute and in their donors respectively. Variant (alleles 7-16). The frequency of alloreactive alleles genotypes were determined using PCR/restriction fragment (homozygous, +/+, or heterozygous, +), with greater number length polymorphisms, except for variant 1007 and 702 of dinucleotide repeats (alleles 8-10 in the present series) for determined using allelic specific PCR. Table 1 presents the STR was 8,3% +/+ and 62,5% + in the D and 8,3% +/+ patients’ characteristics. Kaplan Meyer incidence curves , with and 50% + in the R. The presence of alloreactive alleles differences compared by the Log-Rank test, was used to (heterozygous) in the donor was significantly associated with assess the association between polymorphisms and clinical the development of extense chronic GVHD (EcGVHD, Table covariates (age, disease status, regimen conditioning, gender 1). In patients transplanted from such donors, the incidence of compatibility, HLA mismatches) and GVHD development and mixed chimerism (MC) in peripheral blood during the first mortality. The level of significance was performed to p<0.05. month post-SCT was lower although without statistical Our results showed that global incidence of GVHD( grade III- significance (Table 1). The higher alloreactivity of these IV) was 25.7%. In univariate analysis we found an association donors favors the establishment of complete chimerism and statistically significant between development of GVHD and therefore, increases the incidence of EcGVHD. TNF beta homozygous variant recipient (p: 0.02) and TNF alfa Conclusions: The present study shows an association homozygous variant donor (p:0.001). We did not observe between the presence in the donor of alloreactive alleles for significant differences between genotypic variants and clinical the IL-10 -1064 STR polymorphism and the development of characteristics versus acute GVHD. Regarding mortality we EcGVHD, in a chimerism mediated fashion, after HLA-

S224 identical SCT. The analysis of a larger number of patients as well as further cytokines will eventually allow to confirm these observations and to establish this type of studies as a means for an improved management of transplanted patients.

P873 Prognostic significance of NOD2/CARD15 variants in haematopoietic stem cell transplantation G. Reddiconto, S. Giammarco, G. Farina, A. Fiorini, S. Marietti, M. Palladino, M. Tarnani, G. Leone, P. Chiusolo, S. Sica Università Cattolica S. Cuore (Rome, I)

The inflammatory response following the activation of imflammatory cells by bacterial compounds is a further important player in the induction of severe complications following HSCT. Regulatory issues The NOD2/CARD15 protein has been described as an intracellular sensor to muramyl dipeptide mediating consecutive nuclear factor-kB activation and belongs to a P874 large family of protein involved in intracellular pathogen JACIE accreditation in our centre 18 months later, what is recognition. Its expression is restricted to intestinal epithelial new? cells and cells of monocyte/macrophage lineage. Three A. Thiébaut, C. Bréchard, J. Noyel, S. Petiot, M. Saint-Sorny, common single nucleotide polymorphisms in the leucine –rich M. Chatain, M. Clark, M. Michallet repeats domain R702W, G908R and L1007finsC, might be Hospital E. Herriot (Lyon, F) associated with an abnormal inflammatory response against normal bacterial flora by diminishing NFkB response. Introduction: After 2 years of work, our haematopoietic stem These variants are associated with a higher incidence of acute cell (HSC) transplant programme was accredited by JACIE for GVHD and transplant related mortality after allogeneic SCT autologous and allogeneic transplantation in adult patients, due to transplant related toxicity and sepsis. collection of bone marrow and peripheral blood progenitor and Our purpose was to evaluate the prevalence of these variant therapeutic cells and cell processing. This accreditation is polymorphisms in both subsets of patients submitted to auto effective from June 2005 to June 2008. The purpose of this (56 pts) and alloSCT (35 pts) and their influence on mortality abstract is to describe the follow-up of this quality in the autoSCT group and GVHD and mortality in the alloSCT management plan. respectively. Patient’s characteristics are described in Table 1. Results: In 2005, 51 allogeneic and 40 autologous HSC Variant genotype 908 was determined using PCR/restriction transplantations were performed in our department. Two fragment length polymorphisms , while variant 1007 and 702 quality management reviews were done the first year: doctors, was determined using allelic specific PCR. Kaplan Meyer nurses, nurse supervisors, and secretaries were present. The incidence curves , with differences compared by the Log-Rank first decision was to modify the operation schedule with test, was used to assess the association between fewer.meetings. One area to improve is the collection of non- polymorphisms and clinical covariates (age, disease status, conformity where failure to transmit information was the most regimen conditioning, gender compatibility, HLA mismatches) frequent. Only 8 cases were collected in 2005, which seems and GVHD development and mortality. The level of erroneous with respect to the number of transplants per year. significance was performed to p<0.05. In the subgroup of pts To involve the nurses in the quality management, an annual submitted to autoSCT we did not find any correlation between nurse report review with criteria proposed by nurses was NOD2/CARD15 polymorphisms and overall survival, proposed. On the contrary, clinical report review, bone marrow transplant related toxicity and incidence of sepsis. (p=ns). In collection review, and follow-up indicators are successful. the subgroup of patients submitted to alloSCT no association However, annual review procedures are long and difficult to was found between the same polymorphisms and the maintain. For allogeneic transplantation, 38 standard incidence of GVHD and mortality. operating procedures (SOP) were written, six of which need to This is the first study regarding the influence of be modified for example “Choice of cell source” and “Choice NOD2/CARD15 in a autoSCT set of patients and we did not of conditioning regimen”. We have integrated cord blood demonstrate any correlation with transplant related transplantation, not performed here before accreditation, and complications. In the allogeneic subgroup we were not able to reduced conditioning regimens. Two SOP for autologous demonstrate any correlation with mortality and incidence of transplantation and 9 SOP for both allo and auto transplants GVHD probably due to the low number of patients and the must be changed according to new comparative studies and dishomogeneity of conditioning regimens. new drugs. Sixteen register documents have to be modified or removed. The review procedure is tedious, requires working groups and above all demands heavy secretarial support. The main difficulty is the loss of our JACIE secretarial support immediately after our accreditation in June 2005. Conclusion: JACIE accreditation is an interesting challenge, however the follow up of the programme is difficult due to lack of time and resources. For other teams, we suggest paying careful attention to the feasibility of their programme.

S225 Tolerance and rejection transduced cells within the injected cell-suspension. Thus GFP/OVA expressing cells were successfully deleted. In control vector transduced cells GFP expression was present in the developing tumours. When splenic T-cells were isolated P876 from mice that rejected OVA expressing lymphoma we found In vitro CD4+ T-cell expansion: induction of regulatory T- up to 0.6% T-cells specific for SIINFEKL, a peptide deriving cells with sirolimus and CD3/CD28 dynabeads from OVA. When lymphoma cells were analysed for MHC G. Borelli (1), T. Aarvak (2), A. Brunsvig (1), A. Rasmussen class I and II expression before injection we found that MHC (1), G. Kvalheim (1) expression was very low compared to wild type B-cells of (1)Rikshospitalet-Radiumhospitalet Medical (Oslo, N); C57BL/6 mice. After injection of control vector transduced (2)Dynal-Invitrogen (Oslo, N) cells we observed an up to 8fold increase in MHC class I and up to 20fold induction of MHC class II expression on Background: T regulatory cells (Treg) are a subset of T lymphoma cells. By contrast, upregulation of MHC class I and lymphocytes defined by CD4+CD25+ markers and high II was much more pronounced in OVA/GFP expressing FOXP3 expression. They play a key role in self-reactivity and lymphoma with up to 18 fold induction of MHC class I and up alloreactivity control. In allogenic hematopoietic stem cell to 100fold induction of MHC class II. GFP negative transplantation (HSCT), these cells could be potential useful in lymphomas from different mice were explanted and reducing graft versus host disease without impairing graft recultured. MHC class I and II levels dropped to values versus tumor effect. observed before injection within 14 days. Induction of MHC Objective: To optimize the in vitro T- cell expansion conditions class I and II expression could be partly restored by treatment for clinical grade production of Treg cells. with murine IFNg (100U/ml) up to 2fold for MHC class I and 12 Material and methods: CD4+ cells were obtained by positive fold for MHC class II respectively.The results indicate strong and negative immunomagnetic selection from peripheral blood tumour editing by infiltrating host T-cells. derived lymphocytes. CD4+ cells were stimulated with anti CD3/CD28-coated Dynabeads at a bead/cell ratio of 3:1. The cells were cultured with X-VIVO20 media, autologous plasma, P878 IL2, IL4 and with and without Sirolimus. At day 12 cells were Serum crossmatch analysis is not predictive for rejection washed and depleted of CD3/CD28 Dynabeads. After 24 h re- or acute GvHD after allogeneic stem cell transplantation incubation with CD3/CD28 Dynabeads, cytokine secretion J. Mattsson (1), A. Nordlander (1), M. Remberger (1), J. was analyzed in the supernatant by BioPlex. Cell phenotype Holgersson (2), D. Hauzenberger (2) and FOXP3 expression was evaluated by flow cytometry and (1)Centre for Allogeneic Stem Cell Transplantation suppressive capacitive was measured using standard (Stockholm, S); (2)Clinical Immunology (Stockholm, S) proliferation assay. Results: 80% of CD4+ cells cultured with Sirolimus expressed Background: Evaluation of patient and donor sera using CD25 and secreted low levels of both Th1 and Th2 cytokines. cytotoxic crossmatch analysis prior to allogeneic SCT is a In contrast, only 20% of CD4+ cells cultured without Sirolimus routine procedure in many centers. However, the predictive expressed CD25 and secreted high level of Th1 cytokines. value of cytotoxic crossmatch analysis prior to SCT is unclear. Culture conditions either with or without Sirolimus resulted in Methods: We retrospectively analysed the clinical impact of the same number of FOXP3+ cells. However, cells cultured cytotoxic T- and B-cell crossmatches before SCT performed at with Sirolimus showed strong suppressive capacity and could Karolinska University Hospital, Huddinge between January suppress CD4+CD25- T cell proliferation by 80% even at 1:32 2000 and June 2005. Crossmatch analysis was performed in ratio of CD4+CD25-:CD4+CD25+ cells. patients receiving grafts from unrelated donors. During this Conclusions: Sirolimus induces generation of Treg cells with a period 257 patients received stem cells from unrelated donors. strong inhibitory power over CD4+ CD25- cells proliferation. Crossmatching was performed in 157 patients. Median age of Key words: T cells, T regulatory cells, sirolimus, hematopoietic the patients was 35 (0-67) years. Diagnoses were: acute stem cells transplantation. leukemia (n=58), CML (n=27), other hematological malignancies (n=31), solid tumors (n=22) and non-malignant disease (n=19). Fifty-four patients received bone marrow and P877 103 patients were grafted with PBSC. Fully matched grafts Rejection of syngeneic high-grade B-cell-lymphoma by a (6/6) were given to 130 patients. Twenty-six patients received single minor antigen in a murine model an allele mismatched graft and one patient received a graft M. Sukumar (1), A. Wilke (1), E. Jäger (1), J. Mautner (2), H.- with one major antigen mismatch. Thirty-three patients J. Kolb (1), J. Ellward (3), G. Bornkamm (3), A. Gerbitz (1) received TBI based conditioning regimen, 50 were given (1)University of Munich LMU (Munich, D); (2)Technical BuCy, 53 patients received non-myeloablative and 21 patients University (Munich, D); (3)GSF (Munich, D) received other conditioning protocols. All patients received ATG before SCT. Most patients received a combination Syngeneic rejection of high grade lymphoma is hampered by cyclosporine and methotrexate as GVHD prophylaxis. several mechanisms including fast growth of the lymphoma, Results: Ninety patients are still alive. Acute GVHD grade I secretion of anti-inflammatory cytokines and low expression of was diagnosed in 41 (26%) patients and grades II-IV in 50 MHC molecules. To better understand the immunological (32%). Rejection was diagnosed using chimerism analysis in mechanisms behind lymphoma rejection we have developed a 15 patients (10%). In 11 of these patients cytotoxic murine model that allows us to investigate anti lymphoma crossmatching was performed before SCT. One of 11 patients immunity directed against a single minor antigen. Using a with rejection was positive in T-cell crossmatching in HVG- C57BL/6 c-myc transgenic mouse lymphoma model, we direction and 4/11 in B-cell crossmatching. No correlation was established primary lymphoma cell lines that were transduced found between cytotoxic crossmatching and GVHD. Only two by an MSV based retroviral vector containing chicken patients with GVHD grades II-IV had a positive T-cell ovalbumin (OVA) and IRES-GFP. Control cell lines were crossmatch before SCT. Concerning rejection, T-cell transduced with an IRES-GFP containing vector. When cells crossmatching showed a sensitivity of 9% and a specificity of were subcutaneously injected (0.1 Mio) into syngeneic 97% in the present study. For acute GVHD T-cell C57BL/6 GFP transgenic mice that are tolerant to GFP, we crossmatching showed a sensitivity of 3% and a specificity of could show that introduction of OVA markedly slowed down 98%. A positive crossmatch before SCT had no effect on tumour growth relative to control vector transduced cells. Mice survival in this study as compared to patients with a negative receiving control vector transduced cells developed lethal crossmatch. lymphoma within 23 days whereas OVA transduced Conclusion: Cytotoxic T- and/or B-cell crossmatching before lymphoma progressed slowly after 28 days. This delay was SCT is not predictive for either rejection or GVHD. We accompanied by a loss of GFP expression within the therefore currently evaluate novel crossmatch tests prior to developing lymphoma, indicating a selection for non- SCT with better predictive value at our center.

S226 P879 transplantation. These were of the IgG+IgM class and titres GFP serves as a minor antigen in a murine model of high- increased in the post-transplantation period and were present grade B-cell lymphoma before any signs of clinical rejection. We demonstrate that E. Jäger (1), M. Sukumar (1), A. Wilke (1), J. Mautner (2), H.- purified IgG fractions from patients with rejections but not J. Kolb (1), G. Bornkamm (3), A. Gerbitz (1) controls, lysed donor CD34+/VEGFR-2+ stem cells and (1)University of Munich LMU (Munich, D); (2)Technical significantly decreased the ability of these cells to form University (Munich, D); (3)GSF (Munich, D) hematopoietic and endothelial colonies and tube-formation in matrigels. Retroviral vectors containing green fluorescence protein We will also present data on how antibody mediated rejection (GFP) as a reporter gene are widely used. Whether the of HSC may be avoided. A 2-year-old girl with expression of GFP from retroviral constructs enhances hemophagocytic lymphohistiocytosis (HLH) was transplanted immunogenicity of transduced cells is presently under debate. in April 2005. The patient rejected the graft verified with By using a transgenic mouse lymphoma model, where the chimerism analysis. She was re-transplanted in September human proto-oncogene c-myc is driven by parts of the 2006. We will show data on how plasmapheresis, rituximab immunoglobulin lambda locus representing a t(8;22) and development of acute GVHD grade III saved the translocation as found in Burkitt´s lymphoma, we developed a transplant. The patient is today in good clinical condition and model to test whether GFP expressed in lymphoma cells can complete donor chimera. serve as a minor antigen. C57BL/6 lambda-hu-c-myc We conclude that donor-specific antibodies to transgenic mice were crossbred with C57BL/6UBC-GFP CD34+/VEGFR-2+HSC may significantly contribute to the transgenic mice, giving rise to a “green” lymphoma within 200 rejection process after HSCT. days post birth. These cells exhibit two potential minor antigens in WT C57BL/6 mice: GFP and the human c-myc gene. Peptide prediction of GFP in the context of MHC H2Kb P881 and H2Db revealed several octamers and nonamers as GvHD prophylaxis with sirolimus in combination with possibly immunogenic. Primary cell lines from GFP transgenic mycophenolate mofetil in allogeneic stem cell lymphoma were established and cells were injected after transplantation for high-risk leukaemia patients irradiation into wild type C57BL6 mice. After booster injection M. Schleuning, D. Judith, A. Bertelsmann, Z. Jedlickova, M. after 4-6 mice a strong antibody response towards GFP was Heshmat, H. Baurmann, R. Schwerdtfeger detectable in the serum as measured by ELISA. Similar German Diagnostic Clinic Foundation (Wiesbaden, D) results were observed when GFP protein was used for vaccination. In order to compare immunogenicity of GFP Sirolimus, an immunosuppressant structurally related to the negative and GFP positive primary cell lines we retrovirally calcineurininhibitor (CNI) tacrolimus, exerts its action in a later transduced GFP negative lymphoma cells with an MSV based stage of T-cell activation by inhibiting the mammalian target of IRES-GFP containing vector. After cell sorting, GFP- rapamycin (mTOR) and thus blocking the interleukin 2 transduced lymphoma cells displayed up to 98% GFP receptor signaling. In addition to its immunosuppressive expression. This phenotype was maintained in culture over effects sirolimus also has potent antineoplastic activity. several weeks. When retrovirally transduced GFP positive Sirolimus has been used successfully in solid organ lymphoma cells were subcutaneously injected into wild type transplantation and in combination with CNI for graft versus C57BL/6 mice (0.1 Mio. cells), lymphoma development was host disease (GVHD) prophylaxis in hematopoietic stem cell slowed down compared to C57BL/6 UBC-GFP transgenic transplantation (SCT). We hypothesized that replacing CNI hosts, which are tolerant towards GFP. C57BL/6UBC-GFP with sirolimus would preserve effective prophylaxis of GVHD transgenic mice developed lethal systemic lymphoma within while enhancing the antileukemic potential of allogeneic SCT 20 days whereas wild type C57BL/6 mice displayed at a low toxicity profile. Within the FLAMSA-RIC protocol for lymphoma progression only after 28 days. Explanted high risk leukemia we enrolled 5 high risk patients (pts) in a lymphomas from wild type C57BL/6 hosts displayed reduced pilot trial to test the efficacy of sirolimus in combination with GFP expression and were partially GFP negative, indicating a MMF without CNI in allowing engraftment and preventing selection for low GFP expression within the injected GVHD after allogeneic SCT from HLA-matched related (n=2) lymphoma cell suspension. These data indicate that GFP can and unrelated donors (n=3). All but one pt also received ATG serve as a minor antigen since rejection of lymphoma in during conditioning. The underlying diagnoses were refractory C57BL/6 UBC-GFP transgenic mice could not be observed. T-ALL with complex karyotype (n=1), refractory AML with flt3- ITD (n=3), CML in refractory myeloid blast crisis (n=1). One pt died in aplasia on d +6 from aspergillosis and pulmonary P880 bleeding. The remaining pts engrafted after a median of 19 Novel antibodies to the donor stem cell population days. Grade I GVHD occurred in 3 pts and was limited to the CD34+/VEGFR-2+ are associated with rejection after skin. One patient developed signs of chronic GVHD after haematopoietic stem cell transplantation cessation of immunosuppression which improved after A. Nordlander (1), J. Mattsson (1), B. Sundberg (2), S. resumption of sirolimus treatment. Despite the one death from Sumitran-Holgersson (3) aspergillosis peritransplantation toxicity was mild. One pt (1)Centre for allogeneic stem cell transplantation (Stockholm, suffered from HHV6-associated erosive gastritis, which S); (2)Clinical Immunology (Stockholm, S); (3)Transplantation necessitated laser coagulation and her condition improved Surgery (Stockholm, S) only after terminating sirolimus at day +48. None of the pts developed thrombotic microangiopathy or sinusoidal Reconstitution of hematopoiesis after hematopoietic stem cell obstruction syndrome. At a median follow-up of 118 days after transplantation (HSCT) occurs via a reservoir of donor CD34+ transplantation all 4 pts are alive and in complete remission. HSC. CD34+/VEGFR-2+ is a primitive, quiescent Although the follow-up is rather short sirolimus in combination subpopulation of HSC known to generate hematopoietic or with MMF seems to be a promising alternative to CNI-based endothelial progeny in vitro and in vivo. Here, we regimens for GVHD prophylaxis after allogeneic SCT. hypothesized that donor-specific antibodies to the Engraftment was ensured, although it might be delayed to CD34+/VEGFR-2+ stem cell population may be associated some extent. Transplant-related toxicity was modest, as was with rejections after HSCT. acute GVHD. So far no relapse occurred in this high-risk Ten patients without and eleven with rejections after HSCT population. In conclusion, sirolimus-based GVHD prophylactic were included. We provide novel evidence that significantly regimens deserve further investigation. higher numbers of patients with rejections 8/11 (73 %), while 0/10 in the controls had antibodies against donor CD34+/VEGF-R2+ cells, but not CD34-/VEGFR-2- cells. Importantly, in seven transplantations, antibodies against donor CD34+/VEGF-R2+ cells were detected already prior to

S227 Non-haematopoietic tissue repair defecation and asthenia. Clinical examination showed necrosis of the cervix confirmed by MRI. In spite of local treatment necrosis progressed. Based on limited animal data and after obtaining ethics P882 approval the patient was treated on a 2-step experimental The expression of VEGF after the injection of BM cells is HSC protocol. First, G-CSF was used at 1 mth after diagnosis related to the neovascularisation in hind-limb ischaemic to “mobilize” HSC to the necrotic tissue (5 mg/kg sc bid for 5 mice days). CD34+ counts increased to 105/ml on d5 (undetectable J.-A. Kim (1), J.-Y. Kwak (2), S. Lee (1), J. Hong (1) on d0 and d11). Second, because of continued progression of (1)The Catholic University (Seoul, KOR); (2)Conbuk National necrosis at 2 mths, 110 ml of bone marrow (harvested by University (Jeonju, KOR) sternal and anterior iliac puncture) was injected peri-lesionally (4 injections) after isolation by Ficoll-hypaque density gradient Backgrounds: Improved neovascularization is an important centrifugation. Total CD34+ counts were 6.4x106 (0.1x106/kg), therapeutic goal after myocardial infarction and limb ischemia. CFU-GM 1.1x106 (1.8x104/kg). Half of the HSC were given In the recent years, increasing evidence suggests that bone- immediately (the other half was cryopreserved for a later use). marrow derived circulating cells home the sites of ischemia There were no complications associated with the injections. and contribute to the formation of new blood. The direct On d21 after HSC injection progression of the necrosis injection of bone marrow cells (BMCs) in the ischemic sites stopped with revascularization at the edges, improvement in nevertheless contributes to neovascularization though the odor, defecation and weakness. Through the necrosed cervix, mechanism is unknown. remaining intrauterine necrotic tissue was removed. As of last Aims: This study focuses on the angiogenic roles of BMCs in observation the necrotic process has been stopped. C57BL/6 hind-limb ischemic mice. We examined whether the These results suggest that peri-lesional injection of HSC for BMCs might induce the angiogenesis in a mouse model of radionecrosis is feasible and may possibly contribute to tissue hind limb ischemia and evaluated the expression of related regeneration. molecules. Future studies are planed to confirm those encouraging Methods: BMCs were obtained from C57BL/6. Unilateral hind results and to try to understand the mechanisms involved. limb ischemia was surgically induced by femoral artery ligation in C57BL/6 mice (control group; n=4), autologous BM-MNCs (transplantation group; n=6, 1.58 ± 0.3 x107 cells/animal) P884 were transplanted into the ischemic limbs after 10 days. The Long-term magnetic resonance study of intracoronary capillary/muscle ratios were evaluated and VEGF, eNOS, infusion of bone marrow stem cells in acute myocardial SDF-1, CXCR-4 and MMP-9 assayed in tissue homogenates infarct patients using western blotting. M. Blanquer (1), M.J. Antolinos (1), D.A. Pascual (2), P. Bleda Results: The capillary/ muscle ratio was elevated continuously (1), F. Iniesta (1), F. de Arriba (1), M. Valdés (2), J.M. in BMCs transplantation group. 4 weeks later transplantation Moraleda (1) group had a higher capillary/ muscle ratio (1.07 ± 0.03) than (1)Hospital Morales Meseguer (Murcia, E); (2)Hospital Virgen control (0.62 ± 0.12, p<0.05). VEGF was not expressed after de la Arrixaca (Murcia, E) femoral artery dissection but it was expressed only 2 days after BMCs were injected in the ischemic sites. The Aim: In vitro studies have shown the potential of bone marrow expression of SDF-1 was detected in the ischemic sites on stem cells (BMSC) to transdifferentiate into myocardial cells. day 2 and day 3 after femoral artery dissection, and 7 days Preclinical studies have shown the potential of these cells to after BMCs injection. CXCR-4 was up-regulated ameliorate the outcome of Acute Myocardial Infarct animal transplantation group more than control and normalized in 14 models. Our goal was to assess the feasibility, security and days. efficacy of the intracoronarial injection of BMSC. Conclusions: The direct local transplantation of autologous Methods: We recruited 13 consecutive patients, age 49 ± 10, BMCs augments the neovascularization in the ischemic 12 of them male, who fulfilled the following criteria: successful tissues. And the expression of VEGF after the injection of reperfusion by primary angioplasty in the first 12 h of the AMI BMCs induces the expression of SDF-1 and mobilizes the (TIMI flow 3, residual stenosis <30%), at least 2 non viable CXCR-4 cells in ischemic sites. adjacent segments by gadolinium magnetic resonance (GMR) and age 18 to 75. Patients were given conventional postAMI treatment with beta-blockers, statins, ACEI and double P883 antiaggregation. 60 mL of bone marrow were extracted Bone marrow derived haematopoietic stem cells for the 9,9±3,4 days post-AMI. Ficoll gradient was performed treatment of radionecrosis of the cervix uteri: a case obtaining 340±140 x106 mononuclear cells with a median of report 6,6x106 CD34+ cells that were resuspended in 20 mL of Y. Popowski, J-F. Lambert, J. Passweg, T. Zilli, A-T. Vlastos, autologous plasma and immediately infused by coronary H. Bonnefoi, Y. Chalandon catheterism. Efficacy of the procedure was evaluated one year University Hospital of Geneva (Geneva, CH) later by GMR measuring necrotic mass (NM), left ventricle ejection fraction (LVEF) and telediastolic (TDV) and Hematopoetic stem cells (HSC) are investigated for their telesystolic volume (TSV). regenerative potential through transdifferentiation (stem cell Results: There were no adverse events related to the plasticity), cell fusion, or cytokine mediated effects. Animal procedure during the time of the study. No significant studies have shown G-CSF to improve organ damage e.g. in difference was observed between the basal and one year myocardial infarction or after irradiation, possibly by localizing GMR results: NM (gr) 17,16±10,83 vs 18,24±12,18 (p=0,38,) HSC to damaged tissues. Local injection of HSC is LVEF(%) 42,26±7,02 vs 41,27±7,6 (p=0,53), TDV (mL) investigated in many different settings. 192,55±44,7 vs 195,13±61,3 (p=0,95), TSV (mL) 112,9±38,7 We here describe a patient treated successfully with bone vs 120,56±47,11 (p=0,95). marrow derived HSC injected locally for radionecrosis after Conclusions: Intracoronary infusion of BMSC is a feasible and radiation therapy to the cervix. secure procedure. In our study we observed no significant A 36 years old female, diagnosed with epidermoid carcinoma improvement on the necrosis mass or in the left ventricle of the cervix (FIGO IIB N1) with bilateral extended iliac lymph function parameters. These results are in consonance with the node involvement was treated by pelvic radiotherapy (45Gy recently published randomized studies in which no benefit was (pelvis) + 10Gy (parametria and cervix) + 12Gy (iliac lymph observed among the patients with mild ventricular disfunction. nodes) + 21Gy (cervical brachytherapy associated with radio- sensitizing cisplatin) over 2 mths. One mth after treatment, she complained of abdominal and groin pain, malodorous vaginal discharge, left leg proximal weakness, painful

S228 P885 Lack of transdifferentiation of umbilical cord stem cells in a central nervous system dysmyelinating model M. Blanquer, F.M. Ruiz, P. Bleda, F. Iniesta, V. Vicente, S. Martínez, J.M. Moraleda Hospital Morales Meseguer (Murcia, E)

Background and Objectives: No effective treatment is available for demyelinizating neurodegenerative diseases. Our goal was to asses the ability of umbilical cord blood stem cells to regenerate a demyelinizating injury. Methods: A demyelinizating injury in the right parietal hemisphere was performed by intracerebral infusion of LaLysolecitin to 59 mice of 4 to 8 weeks of age. Fresh umbilical cord blood mononuclear cells (MNC) were infused in the opposite hemisphere. These cells were previously labelled with CellTracker Orange (n=22) or with Qtracker 605 (n=8). Non labelled cells were infused in 12 mice. Control mice were infused with saline (n=8), the supernatant of the last wash of the labelling with either CellTracker (n=5) or Qtracker’s (n=2). No infusion was performed in 2 mice. The animals were sacrificed 1 (n=8), 7 (n=8), 15 (n=19) or 30 (n=24) days after the infusion. Immunofluorescence with anti-Human Nuclei, anti-HLA ABC and anti-CD45 was done to check the human origin of the cells, with anti-Doublecortin and anti-Nestin to assess their differentiation into neural stem cells, with anti-bIII- tubulin to check their differentiation into neurons, anti-NG2 and anti-AA3 for oligodendrocytes and anti-GFAP for astrocytes. Results: A median of 250.000 mononuclear cells with 755 CD34+ and 531 CD133+ cells were infused. One day after the infusion, CD45, HLA ABC, Human nuclei and CellTracker labelled immunopositive cells were observed in the infusion place and in the corpus callosum, crossing to the opposite hemisphere in coincidence with the presence of hemorrhage. Mice brains with survival >1 week showed the presence of cells with a brownish, autofluorescent, Sudan black positive material in the same locations where the hemorrhage had been localized. Some of these cells were positive for CellTracker and QTracker. We did not see any human antigens´ positive cells in these brains and colabelling of CellTracker or QTracker positive cells with the brain cells antigens studied was not observed. Conclusions: Human cord blood cells survive in the adult mice at least for 1 day, and migrate towards the injured area. After one week the human cells disappear and are replaced by cells containing a hemofucsin-like material. Some of these cells contain CellTracker or QTracker suggesting that they are mice phagocytic cells and that the human cells have suffered an immunological rejection. Immunosupression of the animals is necessary to obtain stable human grafts.

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