Oral Sessions and Working Parties

ORAL SESSIONS AND WORKING PARTIES

Working Party Session Acute Leukaemia transplantation outcome. We, therefore, performed a retrospective registry-based study comparing outcomes after HLA matched sibling allo-HSCT in AML pts in CR given iv Bu/Cy (n=795) with 101 those given Cy/TBI (n=864). Engraftment rate was 98% and 99% Introduction and update on the EBMT Acute Leukaemia in the iv Bu/Cy and Cy/TBI groups, respectively. Acute GVHD II-IV Working Party activities was signifi cantly lower in the iv Bu/Cy compared to the TBI/Cy M. Mohty, M. Labopin, S. Giebel on behalf of the ALWP of EBMT group (P<0.0001). Similarly, chronic GVHD was signifi cantly lower in the iv Bu/Cy compared to the Cy/TBI group (p=0.003). Cumula- Transplant activity for acute leukemia continues to increase tive incidence of 2-years NRM was 12+1% in iv Bu/Cy and 15±2% worldwide. In the ALWP registry more than 85000 transplant pro- in Cy/TBI groups (p=0.14) and 2-years RI were 26±3% and 21±1%, cedures for AML and ALL (auto and allo-HSCT) were registered respectively (p=0.012). LFS rates were 61±2% after iv Bu/Cy and thus far. The ALWP objectives are: 64±2% after Cy/TBI (p=0.27). In multivariable analysis adjusting (i) to organize high level accredited educational activities perti- for diff erences between both groups, pts given iv Bu/Cy had lower nent to acute leukemia (latest symposiums: Nantes in 2008, Barce- acute and chronic GVHD, higher RI and a trend for a lower NRM. lona in 2009, Milan in 2010, Warsaw in 2011, and Milan in 2012); At the end, LFS was not statistically diff erent between the two (ii) to design and support prospective clinical trials in the fi eld of conditioning regimens. acute leukemia across member centres (the pan-european elderly In summary, in this retrospective study, fi nal outcomes after mye- AML randomized trial is currently recruiting patients: ClinicalTri- loablative conditioning regimen using iv Bu/Cy were not statisti- als.gov Identifi er: NCT00766779); cally diff erent of Cy/TBI. Similar results were observed in AML pts (iii) to generate high quality retrospective studies addressing transplanted not in remission and/or from unrelated donors. As diff erent issues related to acute leukemia management and full-dose myeloablative TBI is associated with serious late com- therapy; plications after allo-HSCT, our results question the absolute need (iv) to increase within the EBMT registry the quality of data perti- for high dose TBI, and may suggest that non-TBI based regimens nent to HSCT for acute leukemia; and should be preferred, due to possibly better post allo-HSCT quality (v) to generate guidelines pertinent to the management of acute of life. leukemia. Currently, the ALWP activities are organized and structured within 6 subcommittees (SC) focused on specifi c fi elds of interest: autol- 103 ogous HSCT SC, Immunotherapy SC, Alternative donors SC, RIC Haplo donors for all: are we there SC, Molecular markers SC, and the Developing centers SC. F. Ciceri, N.-C. Gorin, M. Mohty behalf of alternative donors The ALWP is currently chaired by M. Mohty (France) and the sec- Subcommittee of the ALWP of EBMT retary is S. Giebel (Poland) and includes representatives/members from most EBMT centres/countries, with expertise in both auto Despite improvements in World Marrow Donor Registries and and allogeneic HSCT for AML and ALL. The ALWP meets twice a Cord-Blood Banks networks, the curative potential of allogeneic year to discuss ongoing studies and new study proposals and hematopoietic stem cell transplantation (HSCT) is accessible to review manuscript preparation. All EBMT members are encour- approximately 50% of candidates patients. Conversely, almost all aged to submit study proposals (registry-based studies) to the patients who are candidate to transplantation have a 50% HLA- ALWP. After a quick feasibility assessment performed by the ALWP compatible (haploidentical) family donor promptly available. The offi ce, projects will be discussed during the ALWP winter (usually major limitation of this transplant modality is the high risk of severe October or November) and spring (during the EBMT annual meet- Graft-versus-Host Disease (GvHD), due to alloreactions mediated ing) business meetings. by donor T cells recognizing the mismatched HLA haplotype. The profound T cell depletion provided by ex vivo cell selection proved eff ective in preventing GvHD in this context and leukemia- 102 free survival obtained in patients with high-risk acute leukemia in High dose TBI for conditioning prior to allo-HSCT: any role remission were comparable in some studies to outcomes obtained left? from MUD and Umbilical Cord Blood (UCB) HSCT. Cell-based strat- A. Nagler, M. Labopin, M. Mohty on behalf of the RIC Subcommittee egies aimed at improving post-transplantation immune recovery of the ALWP of EBMT showed a benefi cial impact in abating infectious mortality but in patients transplanted with advanced disease the high incidence Allo-HSCT is the treatment of choice for high and intermediate of leukemia relapse still translated into a dismal clinical outcome. risk AML. The traditional preparative myeloablative regimens Recently, three main novel regimens of pharmacological immune include Cyclophosphamide (Cy) combined with total body irra- suppression have been tested in clinical trials, to allow infusion of diation (TBI) or the combination of Busulfan (Bu) and Cy (Bu/Cy unmanipulated bone marrow (BM) or peripheral blood (PB) graft: protocol). The original goals of the Bu/Cy protocol were to reduce 1- the use of high-dose post-transplantation cyclophosphamide, toxicity, improve outcome and provide an alternative for patients enabled the infusion of T cells harvested from the BM of haploi- (pts) who received prior radiation who would not be suitable dentical donors; 2- a rapamycin-based, calcineurin-inhibitor-free candidates for TBI. Several retrospective registry-based studies as prophylaxis of Graft-versus-Host Disease (GvHD) allowed for safe well as randomized studies compared the two types of prepara- infusion of high numbers of donor T cells from mobilized PB graft; tive regimens for allo-HSCT in pts with AML with some confl ict- 3- the G-CSF primed, unmanipulated BM with post-transplant ing results concerning outcome and toxicity. However, it can be combination of 5 drugs with diff erent points of attack: antithymo- hypothesized that using intravenous (iv) formulation of Bu, may cyte globulin, cyclosporine, methotrexate, mycophenolate mofetil reduce the risk of oral Bu-induced toxicities and thus improve and basiliximab, an anti-CD25 monoclonal antibody.

S6 In all of these unmanipulated graft platforms, myeloablative con- occurring within the favorable genotype mutated NPM1 in the ditioning followed by haploidentical BM or PB was associated absence of FLT3-ITD is currently unclear, and the same is true with encouraging outcome in terms of engraftment, GVHD, TRM, for FLT3 tyrosine kinase domain mutations on the genetic back- and relapse. Comparative, non-randomized studies in patients ground of an inv(16) [CBFB-MYH11], as well as DNMT3A mutations with high risk acute leukemias will prospectively compare T-cell in the context of normal karyotype AML in the absence of the repleted haplo HSCT with standard matched related or unrelated favorable genotypes mutated NPM1 in the absence of FLT3-ITD or donor transplantation. biallelic CEBPA mutations. By using quantitative RT-PCR (RQ-PCR), molecular markers such as the fusion genes PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11 104 and MLL-MLLT3 as well as NPM1 mutations are used to moni- The way for personalised care for cancer and leukaemia: tor molecular response to induction therapy as well as minimal the French National Cancer Institute (INCa) model residual disease (MRD) during consolidation and follow-up. This A. Buzyn (Paris, FR) information opens the possibility to modify the treatment strategy of individual patients at selected time points (after induction, The treatment of cancer is evolving quickly challenging our after consolidation, during follow-up) e.g. to proceed to alloge- organisations, the formations of health professionals to ensure neic hematopoietic stem cell transplantation. optimal up to date and secured care, and our capacity to provide In conclusion the integration of molecular markers in AML is indis- innovative therapies for all patients in a constrained budget. pensable at diagnosis, during treatment and at relapse to defi ne The consciousness of these challenges has led France to launch a the best available treatment option for an individual patient at cancer control plan in 2009, in which one of the objectives was to each time point. adapt the French health community to this new era, called personalised medicine, considering that cancer care is becoming more and more specialised and genetically driven. The issue was i) to 106 ensure the equitable access to molecular biology analysis for any Any role for transplant in refractory AML? tumour sample of French patients; ii) to ensure equitable access M. Mohty (Paris, FR) to innovative treatments. For that the French national cancer institute (INCa) had organised twenty-eight platforms of molecular In AML, primary treatment failure is defi ned by failure to achieve genetics all over the French territory, responsible for the molecu- a complete remission (CR) after two courses of induction chemo- lar testing of any genetic abnormality if this detection is useful for therapy or by early relapse within 6 months from fi rst CR. For these a clinical purpose such as prognosis or access to a target therapy. patients, overall survival (OS) at 1 year is less than 10%. In the light Up to now 17 tests are performed free of charge, and more than of these poor results with conventional therapy, salvage alloge- 65 000 patients had their tumour been tested in 2012 on the plat- neic stem cell transplantation (allo-SCT) may represent a potential forms. In the fi eld of leukaemia, bcr-abl, IgH/TCR are performed therapeutic tool for the treatment of refractory AML. However, in at a routine level but this list is very quickly evolving. To ensure a the context of standard myeloablative allo-SCT, results are limited fair access to innovation and bring high value on the way to per- in terms of feasibility in the larger patients’ population who is in sonalised medicine, the next step, programmed in 2013, will be to need, and by especially high non-relapse mortality (NRM). In the implement next generation sequencing (NGS) on the platforms recent years, the sequential FLAMSA strategy (Fludarabine, inter- in the aim of performing larger and quicker evaluation of a large mediate dose Ara-C, Amsacrine followed by low dose Total body number of genetic abnormalities. Acute leukaemia’s, with their irradiation (TBI), Cyclophosphamide, and Anti-thymocyte Globu- large panel of prognostic markers driving treatment stratifi cation, lin) developed by the Munich group, proved to be a promising should clearly benefi t from these techniques. The second objec- approach. Since the initial FLAMSA publications, several “FLAMSA- tive is to ensure access to innovative target therapies for a major- like” approaches (e.g. replacement of Fludarabine by Clofarabine ity of patients. For that INCa has organised and funded 16 early and replacement of TBI by I.V. Busulfan) have been used and phase clinical centres (CLIP2), able to perform phase I/II clinical developed. This approach is becoming increasingly used in this trials, with conventions with the pharmaceutical industry. These yet incurable group of patients. Here, we will discuss the role of 2 initiatives, their evolution in 2013 and their practical benefi t for strategies using early RIC allo-SCT in combination with sequen- leukaemia patients will be presented at this session. tial preparative regimens for allo-SCT, followed by active immune modulation and prophylactic infusion of donor lymphocytes (DLI) in patients with primary treatment failure/refractory AML. 105 Molecular markers of AML: an update R.F. Schlenk, H. Döhner, K. Döhner (Ulm, DE)

Genetic heterogeneity is one hallmark of acute myeloid leukemia Working Party Session Lymphoma (AML). Disease classifi cation, prognostication and prediction are based on the cytogenetic but even more and more important on the molecular profi le of the disease. As promising molecu- Jian-Jian-Luan Award larly targeted therapeutics becoming available fast pre-therapeutic molecular screening at diagnoses including at least the fusion genes PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11 as 109 well as mutations in the genes NPM1, FLT3, and CEBPA should Post-transplantation cyclophosphamide is safe and eff ective be performed. However, molecular screening should not only be to prevent immunological reaction after unmanipulated hap- restricted to the time point of diagnosis but also be performed at loidentical bone marrow transplantation following nonmy- relapse based on exciting new data of clonal evolution and out- eloablative conditioning for advanced lymphomas growth of small pre-therapeutic malignant subclones at relapse. L. Castagna (1), S. Bramanti (2), S. Fürst (1), L. Giordano (2), In addition, specifi c molecular characteristics of distinct markers R. Crocchiolo (1), B. Sarina (2), E. Mauro (2), L. Morabito (2), seem to add prognostic value such as biallelic versus monoal- R. Bouabdallah (1), M. Balzarotti (2), A. Anastasia (2), E. Todisco (2), lelic mutations of the CEBPA gene, mutant to wild-type ratio and C. Carlo Stella (2), E. Brusamolino (2), D. Blaise (1), A. Santoro (2) insertion site of FLT3 internal tandem duplications as well as sub- (1)Institut Paoli-Calmettes (Marseille, FR); (2)Humanitas Cancer types of isocitrate dehydrogenase gene mutations (IDH1-R132, Center (Milan, IT) IDH2-R140, IDH2-R172). Furthermore, the molecular signature of the disease rather than single gene mutations have to be taken Several studies have demonstrated the feasibility of allogeneic into account. Exemplarily, the prognostic value of IDH mutations stem cell transplantation with RIC/NMA, in advanced lymphomas.

S7 Only a fraction of patients had a HLA-identical sibling or matched unrelated donor (MUD) and alternative non identical donors such as cord blood or haploidentical related donors could expand the accessibility to transplantation for all patients. The aims of this study were to confi rm the reproducibility of haploidentical transplantation in terms of toxicity and effi cacy in a cohort of lymphoma patients treated in two institutions. Patients and methods. From April 2009, 47 lymphoma patients without related or unrelated HLA identical donors were included in 2 institutions because of relapse after high dose chemotherapy or refractoriness to conventional salvage chemotherapy in an auto-allo program. All patients received conditioning regimen consisting of cyclophosphamide (Cy), fl udarabine (F), and low dose TBI (2 Gy). GVHD prophylaxis was post-infusion Cy (day +3 and 4) and tacrolimus/cyclosporine and MMF. T cell replete bone marrow or peripheral blood was infused at d 0. Prophylaxis against molds, bacteria, and CMV was used. Results. The median follow-up was 10 months (3-42). Two patients developed donor specifi c antibody related graft failure. The median time to ANC > 0.5 and platelets > 20 000 was 20 (range 15-32) and 27 days (range 20-46). CMV reactivation was 26%, with non fatal 2 CMV-related diseases. EBV reactivation was 23% without EBV-related disease. BK virus-related cystitis was observed in 22% of patients. Invasive fungal infection incidence was 6%. The incidence of grade 2-4 aGVHD was 26% and only 2 patients had grade IV. The median time to aGVHD was 56 days (range 39-133). The incidence of moderate cGVHD was 4% and the Working Party Session Aplastic Anaemia median time to cGVHD was 167 days. The 1-year OS, PFS, and NRM were 70% and 62%, and 15%. Infections were the main cause of death. The relapse incidence was 13%, the median time to relapse 114 was 4.4 months (range 1.1-8.3). The median time to discharge Allogeneic haematopoietic stem cell transplantation in from hospital was 28 days (range 19-84). In univariate analysis, Fanconi Anaemia: the EBMT experience the disease status before transplantation aff ected the outcome. R. Peff ault de la Tour (Paris, FR) Conclusions. This study confi rms the feasibility of T cell replete haploidentical transplantation with post-infusion cyclophos- Although allogeneic hematopoietic stem cell transplantation phamide. Furthermore, the effi cacy was strongly suggested in a (HSCT) remains the only curative treatment for patients with Fan- cohort of advanced lymphomas patients. Infectious complica- coni Anemia (FA), published series mostly refer to single center tions were frequently and were the fi rst cause of mortality. experience with limited numbers of patients. We analyzed results in 795 FA patients who underwent fi rst HSCT between May 1972 and January 2010. With a six-year median follow-up, overall survival (OS) was 49% at 20 years (95%CI 38-65). For the 509 patients alive one year post-HSCT, a worse OS rate was observed in patients transplanted before the year 2000 (Hazard ratio - HR: 2.40, p=0.028), between 10 and 20 years of age (HR: 1.89, p=0.016), and who received peripheral blood (PB) as source of stem cells (HR: 2.43, p=0.046). Chronic GvHD and secondary malignancy were also deleterious: (HR 3.10, p<0.0001 and HR 22, p<0.0001, respectively). Risk factors for secondary malignancies included age at HSCT (between age 10 and 20, HR 2.70, P=0.002; after age 20, HR 3.62, P=0.031), clonal evolution (HR 4.44, p=0.004), use of PB (HR 2.97, p=0.016) and chronic GvHD (HR 3.03, p=0.0004). Changes in transplant protocols have signifi cantly improved the outcome of FA patients, who should be transplanted at a young age with bone marrow as the source of stem cells.

116 Unrelated donor HSCT for acquired severe aplastic anaemia: report from the EBMT Severe Aplastic Anaemia Working Party A. Kulasekararaj, A. Bacigalupo, H. Schrezenmeier, A. Tichelli, A.M. Risitano, M. Aljurf, B. Hochsmann, R. Peff ault de la Tour, E. Korthof, A. Rovo, S. Samarasinghe, S. Dufour, R. Oneto, G. Socié, J. Marsh, J. Passweg on behalf of th the EBMT Severe Aplastic Anaemia Working Party

Introduction: Acquired severe aplastic anaemia (AA) is a potentially fatal bone marrow failure syndrome (BMFS) and patients without a matched sibling donor who fail to respond to immunosuppressive therapy (IST) are off ered bone marrow transplantation from a suitable alternate donor. The outcome of unrelated donor (UD) transplants for AA patients has improved in the last decade, prompting even consideration of upfront UD transplants in children who do not have a sibling donor.

S8 Design and methods: We examined the EBMT database for from EBMT showed total response of 40% at 6 months (37% PR and patients (n=1209) with acquired aplastic anaemia undergoing UD 3% CR), and in a retrospective comparison with age and disease transplants either from matched or mismatched unrelated donors, severity matched patients from the EBMT database of patients over a period of 12 years (2000-2011).Only fi rst transplant proce- treated with horse ATG (Lymphoglobuline), similar late response to dures done for AA were included. We analysed the risks of graft horse ATG but inferior overall and transplant free survival with rab- failure, graft versus host disease, and also examined the overall bit ATG. A third prospective phase II study from Cleveland Clinic, survival in diff erent age groups, time periods, stem cell source and USA showed response of 45% at 6 months with rabbit ATG com- also in relation to time to transplantation. pared to 58% among historical controls treated with horse ATG Results: The median age was 21 years (range 1-74 years), of whom (ATGAM), p=0.44. Among retrospective studies, all showed better 48% (n=582) were <20 years, 34% (n=410) between 20-40 years response with horse ATG, apart from three studies, one from Japan and 17% (n=207) more than 40 years. The median time from and one from South Korea both showing similar response, and a diagnosis to transplantation was 397 days (IQR, 225-1023 days), third from Spain showing lower short term response with rabbit with 46% (n=561) and 54% (n=648) less than 12 months or more ATG but similar long term response to horse ATG. than 12 months from diagnosis to transplantation respectively. Since these prospective studies, horse ATG (ATGAM) has become The stem cell source was bone marrow (BM) in 59% (n=714) and available more widely outside the USA. The EBMT SAAWP made peripheral blood (PB) in 41% (n=495). Sixty per cent (n=791) of some proposals to help guide haematologists in the choice of ATG transplants were performed on or after 2006 whilst the rest (40%, to use, and proposed that horse ATG (ATGAM) is the fi rst choice of n=418) was in the period 2000-2005. ATG if it is available. The SAAWP noted that there may be ethnic Graft failure occurred in 8% (n=103) with primary non-engraft- diff erences, for example, that may result in diff erent responses, as ment in 7% (n=88) and late graft failure in 1% (n=15). The median shown in the retrospective studies from East Asia. time to neutrophil recovery was 18 days (range, 2-89 days).Acute The purpose of this part of the SAAWP session is to (1) summarise GvHD was observed in 40%, with grade II-IV in 10% of patients. results from a large study using horse ATG (ATGAM) from the There was no signifi cant diff erence in engraftment kinetics French group (2) discuss the rationale for a prospective randomise between patients with a shorter disease duration (<1year) com- study from the Asia Pacifi c group comparing standard dose with pared with disease duration >1 year pre-transplant. Engraftment reduced dose of rabbit ATG (3) review the updated results of the was signifi cantly faster and more patients engrafted with PB as Spanish study comparing horse with rabbit ATG and (4) sum- compared to BM (p<0.0001). Chronic GvHD was seen in 17%, lim- marise current practice recommendations for continued fi rst line ited in 10% and extensive in 7%.The cumulative incidence of day choice of horse ATG as IST for aplastic anaemia. 100 acute grade II-IV GvHD was 13±2% and the cumulative incidence of cGvHD at 5 years is 22±2%. The cumulative incidence of cGvHD at one year after transplantation with BM is 17±3% and 119 with PB is 20±4% (p=0.22). The Spanish experience of rabbit and horse ATG The 10-year probability of overall survival (OS) was 68±4%, with C. Vallejo on behalf of the BM failure Spanish Study Group no statistically signifi cant diff erence in the probability of 5 year (Pethema-GETH) OS between the two time periods (5 year OS, 2000-2005 68±5% vs 2006-2011 72±4%, p=0.07). The time from diagnosis to transplant Aplastic anemia (AA) is a life-threatening haematological disease, was a determinant of outcome with the 10-year probability of OS that was fi rst described by Paul Ehrlich in 1888. Currently, with a being 74±4% for patients transplanted within a year of diagnosis, prompt and proper management, AA is curable in a high propor- compared with 63±6% for patients transplanted after a year of tion of cases. The pathophysiology of acquired AA is based, gen- diagnosis (p=0.04). erally, on the immune destruction of the hematopoietic tissue. Conclusions: This large retrospective EBMT data analysis demon- Bone marrow transplantation (BMT) from HLA-identical sibling, strates that UD transplants done more than a year after diagnosis and immunosuppressive therapy (IST) are, nowadays, the most of acquired AA adversely impacts long term survival. Therefore, important fi rst-choice therapeutic approaches. early referral for transplantation should be advisable for patients When an HLA-id sibling is available, BMT is considered to be the failing IST who have a suitable alternate donor. fi rst-choice treatment for: 1) young pts (<40 years old) with newly diagnosed severe AA (SAA) or very severe (vSAA), and 2) children with non-SAA and transfusion (PRBC or platelet) dependence or 117 repeated or severe infections. Probability of cure with this thera- Treatment of aplastic anaemia with Antithymocyte globu- peutic approach ranges between 70 and 90 %. Favourable pre- lin (ATG): current situation regarding horse vs rabbit ATG; dictors are younger age, conditioning without TBI, and shorter a report from the EBMT Severe Aplastic Anaemia Working interval from diagnosis to transplant. Advantages of BMT include Party lower rates of relapse and clonal disorders (MDS, AML, PNH). Dis- J. Marsh, A. Bacigalupo, H. Schrezenmeier, A. Tichelli, A.M. Risitano, advantages are the lack of an HLA-id sibling in more than 70% of J. Passweg, M. Aljurf, B. Hochsmann, R. Peff ault de la Tour, E. Korthof, cases, and the development of extensive chronic GVHD in around A. Rovo, S. Samarasinghe, A. Kulasekararaj, C. Dufour, R. Oneto, 30% of the patients. G. Socié on behalf of th the EBMT Severe Aplastic Anaemia Working Party IST is considered to be the fi rst-line therapeutic option for: 1) older patients (>40 years old) with SAA or SAA, 2) younger patients with- ATG is a polyclonal IgG antibody preparation obtained by immun- out an HLA-id sibling, and 3) adults with non-SAA with transfusion ising horses or rabbits with human thymocytes. Clinical trials of dependence or infections. For diff erent reasons, IST is the most horse ATG have shown effi cacy in around 70% of patients. The frequently employed approach in AA patients. Median time to horse preparation has been the preferred source for the fi rst course response is 120 days or even higher. Response rates range between of immunosuppressive therapy (IST), and rabbit ATG was reserved 40 and 80% (half of them being CR). Robust responders have excel- for a second course following failure to respond or relapse after lent long-term survival, similar to that following BMT. Favourable horse ATG. Horse ATG (Lymphoglobuline) was withdrawn from the predictors are younger age, shorter interval diagnosistreatment, market in 2007 and replaced by rabbit ATG (thymoglobuline). Dur- and the employ of the combination of ATG and cyclosporine A. ing this time, another horse ATG preparation (ATGAM) continued Relapse and clonal evolution rates after IST are higher than after to be made and used almost exclusively in the USA. BMT. Little is known on the equivalence between the two kinds The withdrawal of Lymphoglobulin horse ATG prompted three pro- of ATG (horse ATG versus rabbit ATG), and on the role of a 2nd IST spective clinical studies comparing rabbit ATG as fi rst line IST with course in patients failing to respond to a fi rst course. Several papers horse ATG. A prospective randomised study from NIH, USA showed have been published comparing hATG and rATG in AA patients. inferior response (37% vs 68%) and overall survival (76% vs 96%) Some of them have showed inferiority of rATG (like Scheinberg in patients receiving rabbit ATG compared with horse ATG, ATGAM, et al; NEJM 2012), while others have found not diff erences between respectively. A prospective phase I/II pilot study using rabbit ATG both products (like Afable et al; Haematologica 2011).

S9 Retrospective analysis of the Spanish series includes 227 AA in establishing immune competence. Here, we investigated the patients treated with IST in 33 centers during the last decade. impact of circulating PDC measured at day +100 on clinical out- Median age was 45 years old (range: 1-84). 51.2% were male. Neu- come after allogeneic stem cell transplantation (allo-SCT). trophil counts were < 500/mcL in 55.8 % (SAA), and <200/mcL 79 consecutive patients were included in the analysis. Median in 27.9 % (vSAA). The vast majority of patients (98.2 %) had trans- age was 54 (range, 25-71) y. and there were 42 males (53%). fusion dependence. The majority of the IST courses included 44 patients (56%) were treated for myeloid malignancies, 33 cyclosporine A plus Thymoglobulin (TG) (2,5-4 mg/kg/day/x patients (42%) for lymphoid malignancies and 2 patients (2%) 5 days) (72.7%), or Lymphoglobulin (LG) (15mg/kg/day/x 5 days) for aplastic anemia. Stem cell source was peripheral blood in (20.6%). CR was considered when Hgb > 12 g/dL, RAN >1500/mcL, 55 patients (70%), bone marrow in 10 patients (12%) and cord and platelets > 100000/mcL were reached. PR when Hgb > 8 g/dL, blood in 14 patients (18%). Donors were HLA-identical siblings RAN >500/mcL, and platelets > 20000/mcL were reached. The rest in 37 patients (47%), matched unrelated in 25 patients (32%) and of the situations, as well as AA-related death, were considered NR. mismatched unrelated in 17 patients (21%). A myeloablative con- Early mortality (< 4 months) was 11,3%; it was due to infections ditioning was used in 9 patients (11%) and a reduced-intensity (62.5%) and bleeding (37.5%); the majority of patients who died conditioning in 70 patients (89%). BDCA2+ PDC were identifi ed in were non-responders (78.8%) or partial responders (18.2%). Late the blood at day +100 by staining PBMC for surface markers and mortality was 24.3 % (some of them after second line therapy with intracellular cytokines. HSCT). Median follow-up was 45 months (4-138), and overall sur- Patients were categorized by a median proportion of PDC into vival at last follow-up 64.4 %. Analysis of pts treated with TG or LG high PDC goup or low PDC group. The baseline characteristics of who received a single line therapy included: 195 pts at day +90, these groups were comparable. Overall, the cumulative incidence 141 pts at day +180, and 111 pts at day +365. Overall response in of grade II-IV acute GVHD was 36% (95%CI, 25-48%) and the those pts was: A) day +90: 54.5 % (LG), and 46.7 % (TG) (p <0.05); cumulative incidence of extensive chronic GVHD was 37% (95%CI, B) day +180: 73.5 % (LG), and 69.2 % (TG) (p =ns); C) Day +365: 25-49%). The incidence of grade II-IV acute GVHD was signifi - 86.7 % (LG), and 85.2 % (TG) (p =ns). cantly higher in the low PDC group (62% vs 18%, p=0.0003). On a functional level, patients with grade 0–I acute GVHD secreted signifi cantly higher amounts of IFN-α than patients with grade II–IV acute GVHD (p=0.002), probably highlighting the deleterious impact of corticosteroids on PDC function. With a median follow-up of 21 (range, 8-29) months for the 61 surviving patients, overall survival at 2 years was 74% (95%CI, 64-86%), signifi cantly better in the high PDC group (86% vs 55%, p=0.007). In the multivariate analysis, a high PDC count measured at day 100 after allo-SCT stayed an independent predictor of an improved overall survival(p=0.02; RR=3.41; 95% CI, 1.19-9.78). Based on these data, we plan to validate these fi ndings on another independant series and follow the kinetics of PDC recovery after allo-SCT. Nevertheless, we can already reasonably envision that monitoring of PDC after allo-SCT may represent a useful marker for patients’ management, and may have a signifi cant impact on the probability of a favorable outcome of allo-SCT.

Fifty-three pts received a second IST course. 77.4% of them were NR and 22.6% PR to the fi rst course. The second IST was based in TG in the majority of cases (77.6%). Overall response to second IST course was: A) day +90: 34.2 %; B) day +180: 44.7 %; C) day +365: 52.8 %; D) best response: 57.9% (CR: 23.7%). The conclusions of our study are: 1) short-term (< day +180) overall response of Thymoglobulin was lower than that of Lymphoglob- uline, but long-term (> day +180) overall response was similar for both kinds of ATG. According to this data, for some reason, Thy- moglobulin might need more time to reach the best response after an IST course. 2) A considerable proportion of patients who failed to respond to a fi rst IST course might reach response after a second IST course.

Tracking and expediting immune reconstitution post-transplantation O140 Early reconstitution of T-cell immunity to CMV after HLA- haploidentical haematopoietic stem cell transplantation is a O139 strong surrogate biomarker for lower non-relapse mortality Impact of plasmacytoid dendritic cell recovery on outcome rates after allogeneic stem cell transplantation M. Noviello, A. Forcina, M.T. Lupo Stanghellini, M. Carabba, Z. Peric, X. Cahu, F. Malard, E. Brissot, A. Clavert, P. Chevallier, A. Assanelli, M.R. Carbone, Z. Magnani, C. Corti, M. Bernardi, T. Guillaume, J. Delaunay, P. Moreau, M. Gregoire, B. Gaugler, M. Mohty J. Peccatori, F. Ciceri, C. Bonini, A. Bondanza CHU de Nantes (Nantes, FR) San Raff aele Scientifi c Institute (Milan, IT)

Plasmacytoid dendritic cells (PDC), the type I IFN secreting cells, Introduction: T-cell depleted HLA-haploidentical hematopoi- play a major role in linking innate and adaptive immunity, and etic stem cell transplantation (haplo-HSCT) is a readily available

S10 therapeutic option for patients with high-risk hematological In order to identify the Th17 cell, biopsies were tested for expres- malignancies who lack an HLA-compatible donor. Unfortunately, sion of CD161 and CCR6, and RORγt, the key transcription factor the profound state of immune incompetence associates with high for the diff erentiation of Th17 cells. Signifi cantly higher numbers non-relapse mortality (NRM) rates, mainly due to opportunistic of RORγt+ CD161+ and CCR6+ cells were counted in the skin of infections. Diff erent strategies have been developed to speed-up patients with aGVHD (p=0.001, p<0.0001 and p=0.01 for RORγt, immune reconstitution after haplo-HSCT. The development of CD161 and CCR6 expression respectively). Given the role of PDCs these strategies is however limited by the current lack of validated in triggering Th17-related cytokines, we determine the propor- T-cell biomarkers predictive of clinical events. tion of PDCs in the skin biopsies. This analysis showed a signifi - Aim: To analyze the immune reconstitution following haplo-HSCT cant increase of BDCA2+ PDCs in the skin of patients with aGVHD in order to fi nd early surrogate biomarkers of NRM compared with skin of patients without aGVHD (p=0.03). We Results: We prospectively studied T-cell immune reconstitution in observed a strong expression of the type I IFN-inducible protein 89 pts treated with haplo-HSCT from day 30 until day 360 post Mx1 in the skin of patients with aGVHD compared with skin of transplantation. Eighteen patients (20%) were given suicide gene- patients without aGVHD, refl ecting the high production of type I modifi ed DLI, while 71 patients (80%) received an unmanipulated IFN by the BDCA2+ PDCs. We show that Th17 cells and PDC graft followed by rapamycin. The incidence of grade III-IV acute infi ltrate skin biopsies from patients with aGVHD, suggesting a GVHD and chronic extensive GVHD were 12% and 28% respec- potential new pathophysiological link between PDCs and Th17 tively. T-cell counts recovery was accelerated: at day 90, median response in the context of skin aGVHD. These data raise the pros- CD3+ cells were 378 per microL (0-2817), CD4+ 127 (0-804), CD8+ pect of future innovative approaches to optimize immunosup- 173 (0-1922). There was a progressive normalization of both pression regimens for the treatment of acute GVHD by targeting memory diff erentiation phenotype and TCR spectratyping com- PDCs and the Th17 response. plexity score. Nevertheless, none of these biomarkers performed enough to be considered for surrogating lower NRM rates. In this series at high risk for CMV reactivation (CMV serostatus: H+/D+ O142 68%, H+/D- 27%), the event was observed in 46 pts (52%) and Reconstitution of regulatory T-cell subpopulations after CMV disease in 8 pts (9%), all treated according to guidelines. By allogeneic haematopoietic stem cell transplantation and using Receiver Operating Characteristics (ROC) curve analysis of graft-versus-host-disease CMV-specifi c IFN-γ ELISPOT results, we found that cut-off values A. Xhaard, H. Moins, M. Busson, M. Robin, P. Ribaud, N. Dhedin, of 1000 spots/mL allowed to discriminate with high specifi city S. Abbes, M. Carmagnat, A. Toubert, G. Socie, R. Peff ault de Latour (>95%) pts that did not reactivate the virus. Strikingly, while in pts Hopital St-Louis (Paris, FR) with <1000 spots/mL, the 2-yrs NRM rate was 32%, in those with >1000 spots/mL, this was 0% (P<0.05). Interestingly, NRM rates in No study yet analyzed naive (nTreg) and memory (mTreg) regu- pts that achieved or not a CD4+ cell value of 200 per mL were not latory T cells reconstitution in a longitudinal large cohort of signifi cantly diff erent (21% vs 30%, P=0.8). patients (pts) after allogeneic hematopoietic stem cell trans- Conclusions: These indicate that the early reconstitution of T-cell plantation (HSCT). We prospectively analyzed in our center 185 immunity to CMV after haplo-HSCT is a strong surrogate biomarker consecutive pts (median age: 41 years). Fresh whole blood sam- for lower NRM rates. Moreover, they warrant the investigation of ples obtained 3, 6, 12 and 24 months (M) post-HSCT were ana- a CMV-specifi c IFN-γ ELISPOT cut-off value of 1000 spots/mL as a lyzed by flow cytometry to quantify CD4 T cells (naive, activated, predictive biomarker in larger, multicenter series. central memory (CM) and effector memory (EM)) and Treg (CD4+ CD25+ CD127neg/lo), including nTreg (CD45RA+) and mTreg (CD45RAneg). Presented results (median values of circulating O141 cells) are statistically significant (p=≤0.01). HSCT was performed Plasmacytoid dendritic cells (PDC) and Th17 immune for a malignant disease in 92% after a reduced-intensity condi- response contribution in skin acute graft-versus-host disease tioning regimen (RIC) in 51% and from a matched-related donor (GVHD) (MRD) in 56%. The source of stem cells was peripheral blood F. Malard (1), C. Bossard (1), P. Chevalier (1), T. Guillaume (1), (PBSC), bone marrow (BM) and cord blood (CB) in 65%, 28% and J. Delaunay (1), E. Brissot (1), J.F. Mosnier (1), B. Gaugler (2), 7%, respectively. Graft-versus-host-disease (GVHD) occurring M. Mohty (3) before day 100 was defined as acute and chronic thereafter. Total (1)CHU de Nantes (Nantes, FR); (2)INSERM UMR645 (Besançon, FR); Treg (tTreg), nTreg and mTreg increased from 13, 1.8 and 10.7/μL (3)Hopital Saint Antoine (Paris, FR) at 3M to 44, 4.8 and 33/μL at 24M, remaining inferior to healthy controls (66, 24 and 42/μL). The CD4/Treg ratio was stable (12.6 Previous studies established an important role of Th1 cells in and 11.6) at 3 and 24M, while the nCD4/nTreg ratio increased aGVHD pathophysiology after allo-SCT. The role of proinfl am- from 17.4 at 3M to 42.7 at 24M, showing a larger expansion of matory Th17 cells in aGVHD has been demonstrated in mouse naive CD4 T cells than nTreg (Figure) and a larger expansion of models. However, their contribution in human skin aGVHD mTreg than memory CD4 T cells. At 3M, tTreg, nTreg and mTreg is unclear. The same, the role of PDC (the professional type I were significantly higher in PBSC recipients than in those who IFN-secreting cells), which play an important role in triggering received BM and CB grafts. Pts transplanted after a RIC regimen Th17-related cytokines is not established in aGVHD. This report had significantly more tTreg and mTreg than pts transplanted investigated the role of Th17 cells and PDC in skin biopsies taken after a standard regimen. Pts transplanted for an aplastic ane- from patients with or without aGVHD. The cohort included 38 mia had significantly fewer nTreg than pts transplanted for a patients who underwent allo-SCT for diff erent hematological malignant disease. At 6M, tTreg, nTreg and mTreg were signifi- malignancies, with a median age of 52 (range, 17-70) y. The stem cantly higher in pts transplanted from an MRD compared with cell source was PBSCs in 27 cases, CB in 6 cases and BM in 5 cases. pts transplanted from an unrelated donor. At 12 and 24M, pts 11 patients received transplant from a MRD, and 27 patients from ≤15 years had significantly more nTreg than older pts. In pts a MUD. A RIC regimen was used in the majority of cases (n=29). with previous acute GVHD, tTreg and mTreg were significantly Immunohistochemistry was performed on deparaffi nized tis- lower at 3 and 6M compared with pts without. Absolute num- sues sections. A quantitative evaluation of antigens expression bers of tTreg, nTreg and mTreg at 3, 6 and 12M post-HSCT, as well was performed by counting the number of positive cells in the as the ratios of Treg to activated, EM and CM CD4 T cells did not biopsy. In this cohort, based on standard pathology criteria, 29 predict a later episode of chronic GVHD up to 2 years post-HSCT. patients had a histologically proven skin aGVHD, biopsies were We believe these data are of particular interest regarding the taken before initiation of systemic corticosteroid therapy. The increasing number of Treg interventional studies in the context remaining 9 patients did not have histological signs of aGVHD. of HSCT.

S11 This study shows how Treg-based haploidentical HSCT is associated with quite low TRM and prevents GvHD while favouring immune reconstitution.

O144 Haematopoietic myeloid progenitor cells mobilized by G-CSF can inhibit GVHD M. D’Aveni (1), J. Rossignol (1), T. Coman (1), H. Trebeden-Nègre (2), R. Belhocine (3), M. Mohty (3), F. Zavala (1), O. Hermine (1), M.-T. Rubio (3) (1)Hôpital Necker (Paris, FR); (2)Hôpital Pitié-Salpêtrière (Paris, FR); (3)Hôpital Saint-Antoine (Paris, FR)

Despite the infusion of higher T cell doses in G-CSF mobilized peripheral blood stem cells (PBSC) as compared to bone marrow grafts, the incidence of acute graft versus host disease (aGVHD) is not increased. Mobilized CD34- myeloid derived suppressive cells (MDSCs), capable of suppressing alloreactive T-cell responses in vitro, have been recently associated with a lower risk of aGVHD. We describe here a novel subtype of murine and human mobilized hematopoïetic myeloid progenitor cells (HPCs) with features of pro-MDSCs. Methods: In the C57BL6 mouse, G-CSF mobilized pro-MDSCs were collected in the spleen as Lin- Sca1high cKithigh CD34- CD115+ CD11b+ Ly6C+ cells. Human pro-MDSCs were isolated from G-CSF-mobilized PBSC as Lin- CD34+ CD33high CD11blow CD14+ cells. In vitro, pro-MDSC functions were analyzed by co-culture Biology of graft-versus-host disease with T cells activated either by anti-CD28 and CD3 mAbs or allogeneic dendritic cells. In vivo, the eff ect of pro-MDSCs was assess either in the murine O143 C57BL6 (H-2b) (2x106 splenic T cells + 5x106 T depleted bone Adoptive immunotherapy with Tregs and Tcons rebuilds post marrow cells ± 0.5x106 HPCs) into lethally irradiated BALB/c (H-2d) transplant immunity and is associated with low TRM in recipients or by injecting 2x105 human HPCs with 2.5x106 human HLA-haploidentical transplantation PBMC into 2Gy irradiated Nod/SCID/γ c-/- mice. M. Di Ianni, F. Falzetti, A. Carotti, A. Terenzi, A. Pierini, M.S. Massei, Results: In vitro, both murine and human pro-MDSCs could inhibit L. Ruggeri, A. Velardi, M.F. Martelli the proliferation and induced the apoptosis of activated T cells. University of Perugia (Perugia, IT) The inhibition of T cell activation required IFN-γ produced by activated T-cells and the production of NO by pro-MDSCs in response In HLA-haploidentical stem cell transplantation we showed adop- to IFN-γ. NO suppressed T-cell functions through impaired tive immunotherapy with naturally occurring T regulatory cells responses to IL2, inhibition of JAK3 and STAT5 functions and (nTregs) followed by conventional T cells (Tcons) prevented acute induction of apoptosis. and chronic graft-versus-host-disease (GvHD), favoured lymphoid In vivo, the administration of murine pro-MDSCs signifi cantly reconstitution and immunity against pathogens (Di Ianni et al., reduced the development of clinical or histological GVHD signs as Blood 2011). The major drawback of that study was the extra- compared to allografted mice without HPCs. Murine pro-MDSCs haematological toxicity of the conditioning regimen in heavily could migrate to site of allo-priming and induced the apoptosis of pre-treated patients. allogeneic T cells. Apoptotic T cells subsequently triggered phago- In order to reduce transplant-related mortality (TRM) in the pres- cytes to engulf T cell debris and produce high levels of cytokines, ent new protocol 19 patients with high risk acute leukaemia (14 which in turn led to the induction of CD4+ CD25+ Foxp3+ regula- AML and 5ALL) received a conditioning regimen including TBI (8 tory T cells and, ultimately, immune tolerance. Gy on single fraction on day –10), thiotepa (4 mg/kg/day on days Human pro-MDSCs could protect all xeno-grafted Nod/SCID/γ c-/- –10 and -9), fl udarabine (40 mg/sqm/day from days –10 to –6 ) mice from GVHD mortality as compared to 100% GVHD lethality in and, instead of cyclophosphamide of the previous study, alemtu- controlled xeno-grafted mice without HPCs. zumab (20mg/sqm; day-21) or ATG (4-6mg/kg; day -21). The graft Conclusion: We describe a new subtype of HPCs that can regu- included: on day -4 2x106/kg nTregs (FoxP3+ cells 92% ± 8 SD), late alloreactive T cell activation in vitro and in vivo with potential on day 0 a megadose of CD34+ cells (mean 10.5x106/kg ±3.1 SD) therapeutical application in allogeneic haematopoietic stem cell and Tcons (1x106/kg). No post-transplant GvHD prophylaxis was transplantation. given. 19/19 patients achieved a rapid and sustained full donor- type engraftment. 4/19 (21%) valuable patients developed ≥ grade II acute GvHD. Three of these patients responded promptly to short course of immunosuppressive therapy. At present they O145 are alive without any sign of cGvHD. The incidence of TRM is 17% Purifi ed CD4 T-cell infusion can result in graft-versus- (3/19). As expected, extra-haematological toxicity was mild. CD4+ leukaemia without GVHD by recognition of broadly expressed and CD8+ peripheral blood counts reached 200/μL on days 48 minor histocompatibility antigens restricted by HLA class-II (range 27-111) and 37 (range 27-147) respectively. We observed P. van Balen, A.M. van Bergen, I. Jedema, S.A.P. van Luxemburg-Heijs, a rapid development of a wide T-cell repertoire and high fre- J.C. Harskamp, H.M. van Egmond, S.A.J. Veld, M. Griffi oen, W.A.F. quencies of specifi c CD4+ and CD8+ clones for opportunistic Marijt, C.J.M. Halkes, J.H.F. Falkenburg pathogens. Treg immunotherapy did not compromise post-trans- Leiden University Medical Center (Leiden, NL) plant generation of donor-vs-recipient alloreactive natural killer (NK) cell repertoires which was faster than controls and with an Donor lymphocyte infusion (DLI) after allogeneic stem cell transplan- enhanced alloreactivity against KIR-ligand mismatched targets. tation (alloSCT) can mediate Graft-versus-Leukemia (GVL) reactivity, Only 2 high risk patients have relapsed to date and at a mean frequently at the cost of Graft-versus-Host-Disease (GVHD). Donor follow-up of 1.2 years (range 0.05-2.6) 62% of the patients are alive CD8 T-cells recognizing HLA class-I restricted minor histocompatibil- and leukemia-free. ity antigens (MiHAs) that are broadly expressed on recipient tissues

S12 cause GVL and GVHD. Since expression of HLA class-II molecules Methods: We compared human B-lymphoblastoid cell line (LCL) under non-infl ammatory conditions is predominantly restricted to tumour rejection mediated by CB and PB CD3+ T-cells in vivo in a hematopoietic cells, donor CD4 T-cells may selectively recognize NOD-SCID γ knockout model. Mice were inoculated with 5×106 F- recipient hematopoietic cells resulting in GVL without GVHD. Luc labelled LCL sc to develop a human B cell lymphoma. To evalu- In a clinical trial we treat patients after T-cell depleted alloSCT ate anti-tumour activity, 5×106 HLA-mismatched CB or PB CD3+ from HLA-identical sibling donors with 106/kg purifi ed donor CD4 T-cells were injected iv after 2 days. Tumour growth was analysed T-cells. To characterize immune responses, in vivo activated T-cells using the Xenogen-IVIS system. are clonally isolated using fl owcytometric cell sorting at the time Results: In 2 consecutive experiments with diff erent T cell donors, of clinical response, expanded and tested for alloreactivity on we observed that control mice receiving LCL without T-cells (n=6) patient and donor derived target cells using IFN-γ ELISA. We char- and mice receiving LCL followed by PB T cells (n=10) had tumour acterized in detail the immune response in a patient transplanted growth exceeding 10 mm between 20-22 days after tumour inoc- for CML in blastic phase. After engraftment, recipient hematopoi- ulation and hence were sacrifi ced. In contrast, 9/10 mice receiving esis reoccurred varying from 5% of patient myelopoiesis to 90% of CB T-cells showed slower tumour growth followed by complete CD8 T-cells. After treatment with CD4 DLI, within 19 weeks conver- resolution (Fig.1). This resulted in signifi cantly improved survival sion to full donor chimerism was observed in all hematopoietic of mice treated with CB T-cells (p< 0.0003). Importantly, no signs cell lineages in total absence of GVHD. of xenogeneic GVHD were seen in mice receiving CB T-cells. Following cell sorting, no alloreactive CD8, but many alloreactive To study the immunological basis of these diff erential anti-tumour CD4 clones were found. Blocking experiments identifi ed HLA-DR eff ects, in subsequent experiment mice in both the groups were as restriction molecule for recognition. Using whole genome asso- sacrifi ced on day+24 at the start of tumour regression. Tumour ciation scanning, we identifi ed 4 MiHAs, including 3 novel MiHAs, infi ltrating lymphocytes (TIL) were isolated and perforin expres- as their target molecules. Synthesis and analysis of patient and sion was performed. Intracellular cytokine staining (IFN-y, TNF-a donor derived allelic peptide variants confi rmed the novel MiHAs and IL-4) was also performed after a 6 hour challenge with LCLs as LB-KHNYN-1K, LB-CTSB-1G and LB-ZDHHC13-1K. Cytotoxicity in vitro. CD4/CD8 ratios in TIL were signifi cantly higher in mice analysis of the MiHA specifi c T-cells illustrated that these CD4 receiving PB T cells (median of 0.32) than in animals receiving CB T-cells killed 80-97% of patient derived hematopoietic cells within T cells (median 0.12, p<0.0001), despite a higher initial CD4/CD8 48 hours. Gene expression profi les illustrated that the genes ratio in the CB group. Perforin expression in CB CD4 and CD8 T cells encoding the MiHAs were transcribed in several tissues including was signifi cantly higher than PB CD4 and CD8 T cells (p<0.0003). GVHD target tissues. This illustrates that the hematopoietic We observed signifi cantly higher TNF-a (p<0.0001) and lower IL-4 specifi city of the immune response was mainly defi ned by responses (p<0.01) in tumour infi ltrating CB T cells compared to restricted expression of HLA-DR on hematopoietic cells. PB T cells. No diff erence in IFN-y secretion was observed between In conclusion, infusion of donor CD4 T-cells after HLA- identical the 2 groups. alloSCT can result in GVL without GVHD by mediating an HLA Conclusion: These fi ndings demonstrate that in this model, allo- class-II restricted immune response against patient hematopoiesis. reactive CB T-cells may mediate more potent GVL eff ects than PB T-cells without causing xenogeneic GVHD. This GVL eff ect appears to be CD8 mediated and may involve TNF-a. O146 Cord blood lymphocytes mediate enhanced graft-versus- leukaemia eff ects compared with peripheral blood in a O147 xenogeneic mouse model CD24-Siglec-G interaction plays an important role in reduc- P. Hiwarkar (1), W. Qasim (1), I. Ricciardelli (1), P. Amrolia (2), P. Veys (2) ing experimental graft-versus-host disease (GVHD) (1)Institute of Child Health (London, GB); (2)Great Ormond Street T. Toubai (1), R. Evers (1), Y. Sun (1), I. Tawara (1), C. Liu (2), H. Tamaki Hospital (London, GB) (1), N. Mathewson (1), P. Zheng (1), Y. Liu (1), P. Reddy (1) (1)University of Michigan Cancer Center (Ann Arbor, US); (2)University Objective: The use of HLA-mismatched unrelated cord blood as of Florida (Gainesville, US) a stem cell source reduces relapse rate after transplantation for acute leukaemia. To model this graft-versus-leukaemia eff ect Members of the sialic acid binding Ig–like lectin-G (Siglec-G) is an (GVL) and dissect the immunological basis for this, we have com- immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM- pared the ability of cord blood (CB) vs peripheral blood (PB) T-cells like regions in its intracellular domain that negatively regulates to mediate GVL eff ects in a xenogeneic mouse model. immune activation induced by non-infectious damage associated [O146]

S13 molecules (DAMPs). But the role of negative regulators of DAMP T cells (generated by CD3/CD28 activation of human peripheral associated immune activation, such as Siglecs, in regulating allo- blood T cells and transduction with an MP71 retroviral vector car- reactivity is not known. We therefore utilized well defi ned clinically rying codon-optimized murinized HMMR-specifi c TCR). relevant murine models of allogeneic BMT to test the hypothesis Results: Co-cultures of HMMR-redirected T cells with human that defi ciency of Siglec-G in the hosts will increase GVHD. B6 wild CD34+ HSC from HLA-A2+, but not HLA-A2- donors, led to strong type (WT) and Siglec-G-/- animals were lethally irradiated (13Gy) reduction of colony forming units. HSC-containing bone marrow and transplanted on day 0 with 5x106 bone marrow and 3x106 of transgenic mice that were pre-cultured with HMMR-redirected splenic CD90+T cells from either WT-B6 or MHC mismatched T cells before transplantation failed to reconstitute hematopoiesis BALB/c donors. The Siglec-G-/- animals showed signifi cantly in irradiated mice; the animals died at the same time as the non- worse survival than the allo-WT animals (p<0.01) with an increase transplanted controls. However, mixed co-cultures of A2+ and donor T cell expansion and serum levels of pro-infl ammatory A2- HSC revealed no cross-killing of A2- HSC. Cross-recognition cytokines after BMT. Furthermore, consistent with the increased was not seen with any of 100 common self-peptides. Reactivity of levels of DAMPs, Siglec-G-/- animals showed higher GVHD only HMMR-redirected T cells against a panel of allogenic LCLs showed after 13Gy radiation but not after 8Gy conditioning. We next eval- exclusive recognition of LCL expressing the HLA-*02:01, 02:07 and uated whether this was because of Siglec-G defi ciency only on the 02:09 subtypes; which express the same peptide binding groove. radiosensitive host APCs. To this end we generated [B6 B6Ly5.2] This information is mandatory for designing exclusion criteria of and [Siglec-G-/- B6Ly5.2] BM chimeras and utilized them as recipi- donors, whereas patients with all three HLA-A2 subtypes could be ents. The allogeneic [Siglec-G-/- B6Ly5.2] animals demonstrated included in the clinical trial. signifi cantly worse survival than the [B6 B6Ly5.2] animals. These Conclusions: Adoptive T cell transfer of HMMR-redirected T cells data collectively demonstrate that Siglec-G expression only on for HLA-A2-mismatched stem cell transplantation promises two the host radiosensitive APCs is critical for protection from GVHD. clinical benefi ts: Firstly, targeted residual autologous HLA-A2 Because responses to non-infectious DAMPs are regulated by HSCs lead to faster donor chimerism and secondly the elimination Siglec-G-CD24 interaction we next hypothesized that enhanced of residual HLA-A2+ leukemic cells may allow curative treatment. CD24-Siglec-G interaction would mitigate GVHD. We fi rst characterized stimulation of allogeneic T cell responses by Siglec-G-/- APCs. We utilized CD24-/- and WT BALB/c T cells as responders in O149 an MLR with B6 and Siglec-G-/-stimulators. We found that Siglec- Over-expression of CXCR4 targets donor T-cells to tumour G-/- APCs expanded the CD24-/- T cells more than WT-B6 APCs. We niches and enhances graft-versus-lymphoma next tested in vivo whether enhanced CD24-Siglec-G interaction B.J.M Carpenter, S. Ghorashain, E. Nicholson, J. Griffi n, M. Ahmadi, would mitigate GVHD. We utilized a novel CD24 fusion protein L. Zhang, B. Flutter, A. Holler, H. Stauss, E. Morris, R. Chakraverty (day-1, 100μg/mouse) and found that it decreased GVHD mortal- UCL (London, GB) ity only in the WT but not in the Siglec-G-/- animals. Together our data demonstrate a critical role for CD24-Siglec-G interactions in The CXCR4-CXCL12 chemokine axis forms a critical component of regulating GVHD and suggest that administration of the novel many tumour niches, promoting tumour survival, metastasis and CD24 fusion protein may be an innovative strategy to mitigate recruitment of regulatory cell populations that block anti-tumour GVHD. immunity. In this study, we have tested the hypothesis that targeting therapeutic T cells to CXCR4-dependent niches will improve GVL following allogeneic BMT. Murine T cells were transduced with a retroviral vector containing CXCR4-GFP reporter (TCXCR4) or GFP reporter alone (TControl). To test homing functions in vivo, we tested the ability of cells to home Engineering cellular therapies to the bone marrow (BM), a site where stromal cells produce large amounts of CXCL12. In competitive assays, TCXCR4 CD8+ cells demonstrated a 2-fold greater capacity than controls to home to O148 the BM by 24h after transfer to sub-lethally-irradiated recipients. HMMR-redirected adoptive T-cell therapy for treatment In order to image the precise location of T cells in relation to the of refractory acute myeloid leukaemia in context of CXCL12-rich endosteal BM niche, we performed multi-photon mismatched stem cell transplantation microscopy through the frontal bones of sub-lethally irradiated A. Wieczorek (1), V. Marin (2), S. Raff egerst (2), S. Wilde (2), B. Mosetter (2), osteoblast (collagen 1-α-GFP) reporter recipients. At 24h, fl uores- S. Spranger (2), B. Frankenberg (2), M. Leisegang (3), W. Uckert (3), cently labelled TCXCR4 CD8+ cells were closer than TControl CD8+ L. Uharek (1), D. Schendel (1) cells to endosteal osteoblasts (13 μm vs. 17 μm p=0.007). The com- (1)Charité (Berlin, DE); (2)Helmholtz Zentrum (Munich, DE); petitive advantage for TCXCR4 CD8+ cells in the BM increased to (3)Max-Delbrück-Center for Molecular Medicine (Berlin, DE) over 15-fold after 2 weeks and such cells acquired higher levels of expression of CD44, a memory T cell marker involved in homing Background: The hyaluronic acid-mediated motility receptor and retention within the BM. In further experiments to test recall (RHAMM/HMMR) is an interesting antigen for adoptive T cell immunity to model antigen, TCR-transgenic CD8+ populations transfer. This is due to two observations: fi rstly, high expression were transduced with CXCR4 or control vector. In memory recall of RHAMM/HMMR mRNA in AML patients is correlated with poor experiments, CXCR4-transduced CD8+ T cells showed increased prognosis and secondly, the existence of immune responses frequencies of cells with a CD44high IL-7Rα+ IFN-γhigh memory against HMMR is linked to better clinical outcome. We previously phenotype. To determine how CXCR4-targeting would infl uence used dendritic cell priming to obtain allo-restricted HMMR-spe- GVL, we transferred B6 TCXCR4 or TControl cells and B6 BM to cifi c T cells restricted by HLA-A2. The redirected T cells recognized irradiated BALB/c recipients bearing A20 tumour administered HLA-A2+ HMMR+ tumor cells well in vitro. Leukemia outgrowth either by intra-bone or subcutaneous injection. Compared to was strongly retarded in vivo in humanized xenograft mice. We donor TControl, donor TCXCR4 increased GVL responses against now present the safety profi le of HMMR-redirected T cells. A20 at both sites. Of note, improved GVL mediated by donor Methods: We assessed the impact of HMMR-redirected T cells TCXCR4 cells was observed without increasing graft-versus-host on normal human stem cells (HSC) in vitro using a colony form- disease. ing unit assay (CFU) and in vivo using HLA-A2-transgenic mice. Over-expression of CXCR4 in therapeutic T cells increases their Cross-reactivity of HMMR-redirected T cells for other peptides and engraftment in BM and the induction of GVL. HLA allotypes (30 LCL cell lines with common HLA allotypes) was tested using mouse B3Z cells and HMMR-specifi c TCR-transgenic

S14 O150 TCR chain, with the generation of “single-edited” T cells, or both α Eff ectively targeting sensitive and resistant Burkitt and β chains, yielding “complete-edited” lymphocytes. By avoid- lymphoma by anti-CD20 chimeric antigen receptor (CAR) ing competition for surface expression between exogenous and modifi ed expanded natural killer (NK) cells combined with a endogenous TCR α and β chains, and by abrogating the risk of histone deacetylase inhibitor, romidepsin inappropriate TCR pairing, the TCR editing approach permanently Y. Chu, A. Yahr, J. Ayello, L. Lo, J. Katz, M. Cairo overcomes the major limitations of TCR gene transfer. TCR edited T New York Medical College (Valhalla, US) cells targeting WT1 recognized leukemic targets without mediating GvHD (Provasi, Genovese et al., Nat. Med. 2012). To promote Background: The outcome for patients with Burkitt lymphoma an eff ective and persistent therapeutic eff ect, and to facilitate its (BL) has improved signifi cantly but for patients who relapse, the clinical application, we established a scalable protocol of TCR sin- prognosis is dismal due to chemo-immunotherapy resistance gle editing, applicable within a single round of T cell stimulation. (Cairo et al, JCO, 2012). Rituximab (Rx) resistant BL cell lines pro- This approach completely and permanently abrogates expres- vided a good model to study monoclonal antibody resistance and sion of the endogenous TCR repertoire of donor T cells, which is design novel therapies (Czuczman et al, CCR, 2008). Romidepsin, responsible of GvHD. a histone deacetylase inhibitor, enhances NKG2D ligands expres- In addition, we validated the TCR editing approach on NY-ESO-1, sion (Satwani/Cairo, ASBMT 2009). Natural Killer (NK) cells play an antigen expressed by solid tumors and hematological malig- an important role in tumor eradication post allogeneic stem cell nancies. We observed similar high levels of TCR expression, mea- transplantation (Ruggeri, Science 2002). sured by NY-ESO-1 specifi c dextramer binding, in single edited Objective: We investigated 1) functional activities of anti-CD20 and transferred T cells (RFI 73 vs 66). Single edited cells effi ciently CAR modifi ed PBNK following mRNA nucleofection against CD20+ recognized and killed NY-ESO-1+ myeloma cell lines, without BL and 2) the combined eff ect of CAR+ PBNK cells with Romidep- aberrant response to NY-ESO-1- targets, and displayed improved sin against CD20+BL. tumor specifi city than unedited TCR-transferred cells in co-colture Methods: PBMC were expanded with inactivated K562-mbIL15- assays (99% of elimination of target cells in both single edited and 41BBL (geMK) cells and purifi ed. Anti-CD20-4-1BB-CD3ζ mRNA transferred cells; 0% vs 45.9% of elimination of unspecifi c targets, (CAR mRNA) was produced in vitro and nucleofected into exPBNK. respectively). Most importantly, the new editing protocol, can be CAR expression was detected by fl ow cytometry. exPBNK cyto- applied to a newly described memory T cell subset (TSCM), char- toxicity was assessed by europium release assay. Raji, Raji-2R and acterized by self-renewing, stem cell-like abilities (Cieri, Blood Raji-4RH (provided by Matthew Barth, MD) cells were treated with 2012). These results suggest that the TCR gene editing approach 10ng/ml Romidepsin, provided by Celgene. Cell viability, MICA/B can mediate Graft versus Tumor and overcome the risk of GvHD in expression, cell cycle were analyzed by fl ow cytometry. Romidep- patients with hematological malignancies undergoing allo-HSCT. sin treated BL cells were used for CAR PBNK mediated cytotoxicity assays. Results: The geMK expanded PBNK (exPBNK) were selected with more than 96% purity. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs until day 6 after CAR mRNA nucleofection. exPBNK in vitro cytotoxicity was signifi cantly enhanced by elec- CLL, MPD, MDS & Myeloma troporated CAR mRNA against CD20+ Raji, Raji-2R and Raji-4RH (p<0.001). Romidepsin induced signifi cant cytotoxicity at day 2 in BL cells, P<0.001, and cell cycle arrest in Rx resistant BL cells. O152 MICA/B expression was signifi cantly increased in Raji, Raji-2R DIPSS-plus improves outcome prediction after allogeneic and Raji-4RH (p<0.001) after romidepsin treatment. CAR PBNK in haematopoietic stem cell transplantation (HSCT) for vitro cytotoxicity was signifi cantly enhanced against romidepsin myelofi brosis treated BL (p<0.02) and Raji-4RH (p<0.001) compared to untreated M. Ditschkowski, A.H. Elmaagacli, N.K. Steckel, T. Gromke, R. Trenschel, targets. M. Koldehoff , D.W. Beelen Conclusion: Anti-CD20 CAR expression in exPBNK cells results in University Hospital of Essen (Essen, DE) signifi cant and specifi c in vitro cytotoxicity against sensitive and resistant CD20+ BL. CAR PBNK signifi cantly enhanced cytotoxicity Primary or secondary myelofi brosis (MF)is a chronic myeloprolif- against romidepsin treated BL and Rx resistant BL. Future direc- erative stem cell disorder exclusively curable by allogeneic HSCT. tions include examining the combination eff ect of Romidepsin 76 patients (pts) (40 male, 36 female; median age at transplant= and CAR PBNK against primary CD20+ BL in vitro and in xenograft 51 years) with primary (n=47), post-polycythemic (n=12) or post- mice. thrombocythemic (n=17) MF underwent allo-HSCT after myeloablative conditioning containing fractionated total body irradiation (TBI) (n=45), chemotherapy regimen (n=26) or reduced intensity O151 conditioning (n=5). Donors were HLA-identical (n=27) or mis- Towards clinical translation of TCR gene editing to mediate matched (n=3) siblings and matched (n=33) or mismatched unre- graft-versus-tumour in the absence of graft-versus-host lated (n=13). Transplants consisted of unmanipulated peripheral disease blood stem cells (n=68), bone marrow (n=6) or highly purifi ed S. Mastaglio (1), P. Genovese (1), Z. Magnani (1), E. Provasi (1), CD34+ cells (n=2). GVHD-prophylaxis was performed with CSA A. Lombardo (1), A. Reik (2), A. Brambilla (1), F. Ciceri (1), C. Bordignon + MTX (n=46), 17 pts. received anti-thymocyte-globulin (ATG) or (1), M. Holmes (2), P. Gregory (2), L. Naldini (1), C. Bonini (1) alemtuzumab (n=13). Dynamic international prognostic scoring (1)San Raff aele Scientifi c Institute (Milan, IT); (2)Sangamo BioSciences system (DIPSS) and DIPSS plus scores were generated for each pt. Inc. (Richmond, US) prior to HSCT. (SM and PG equally contributed to this work). The median follow-up was assessed 69 months among surviving pts. 1-year TRM was 22%, primary graft-failure occurred in 4 pts The genetic addition of a tumor specifi c receptor into T cells (5%) and relapse was observed in 17% (median 6 months post- yielded already substantial clinical results in patients aff ected transplant). 5-year overall survival (OS) was 55%, 5-year relapse- by hematological malignancies. To completely substitute T cell free survival (RFS) 53% with a median RFS time of 79 months specifi city, we recently developed the TCR gene editing approach, (95% CI: 14-143). Histories of pharmacological pre-treatment and based on the combination of: i. Somatic knockout of the endog- the post-transplant occurrence of chronic GvHD were signifi cant enous TCR genes (achieved by transient exposure to α and β chain independent unfavourable risk factors for post-transplant survival specifi c Zinc Finger Nucleases), and ii. Introduction of a tumor- in multivariate analysis. Using the DIPSS risk stratifi cation, low risk specifi c TCR by lentiviral vectors. With this approach, TCR gene patients showed signifi cantly improved overall (p=0.014, HR 1.4) transfer can follow the knockout of either a single endogenous and relapse-free (p=0.02, HR 1.3) survival compared to the other

S15 patient risk groups. Stratifi cation by DIPSS demonstrated 5-year OS of 77%, 48%, 50%, 50% for low, intermediate-1, intermediate-2 and high risk group. The additional inclusion of thrombocytopenia, abnormal karyotype and transfusion need (DIPSS plus) prior to transplantation resulted in a predicted 5-year OS of 100%, 55%, 54% and 44% for low, int-1, int-2 and high risk group revealing also signifi cantly higher OS for low vs. high risk (p=0.023). Mean survival was 108, 114, 67 and 36 months for low (95% CI: 92-123), int-1 (95% CI: 72-157), int-2 (95% CI: 48-87) and high risk (95% CI: 16-57). Our data point out that risk stratifi cation is more accurate by using DIPSS plus score. Furthermore, by DIPSS plus classifi cation the benefi cial and life prolonging eff ect of allogeneic transplantation becomes apparent for all risk groups.

O153 The eff ect of non-myeloablative conditioning (NMA) versus reduced-intensity conditioning (RIC) in chronic lymphocytic leukaemia (CLL) undergoing allogeneic haematopoietic cell transplantation (allo-HCT): a retrospective EBMT analysis N.S. Andersen, L. Vindeløv, M. Karas, M. Gramatzki, M. Machaczka, A. Vitek, M. Stadler, P. Dreger, E. Faber, E. Montserrat, G. Socie, M. Michallet, C. Moreno, D. Niederwieser, M. Bornhäuser, A. Henseler, L. Wreede, M. Gelder, N. Kröger, J. Schetelig on behalf of the CLL subcommitee, Chronic Malignancies Working Party

Objectives: NMA and RIC alloHCT are treatment options in very high risk CLL. Relapse after transplant is of major concern and the optimal conditioning regimen has not yet been defi ned. The objective of this analysis was to compare NMA to RIC with respect to survival outcomes. Patients and Methods: Patients with CLL who received a fi rst alloHCT from a HLA-identical sibling (SIB) or unrelated donor between 2000 and 2011 and whose baseline and outcome data have been upgraded in the new EBMT - CLL data quality initia- tive were analyzed. NMA was defi ned as a single dose of 2 Gray Total Body Irradiation with or without fl udarabine (FLU). RIC was defi ned as a combination of FLU and alkylators at non-myeloablative doses (oral busulphan under 10 mg/kg, intravenous busilvex under 8 mg/kg, melphalan under 150 mg/kg, or cyclophosphamide). The two groups of patients were compared in univariate comparisons and analysed in a multivariate Cox regression model with adjustment for age, cytogenetic risk categories, purine-ana- logue-sensitivity, remission status at HCT, and donor type. Results: 443 patients were included. The median follow-up after HCT was 3.5 years. The median age was 55 years (range, 25 to 74 years). The two cohorts did not diff er signifi cantly with respect to age (p=.2). However, in the NMA cohort more patients were transplanted in CR (20% vs 9%, p<.001) and exposed purine-ana- logue sensitive disease (59% vs 39%, p=.001), deletion 17p was less frequently observed (13% vs 21%, p=.003), and more patients received a SIB graft (51% vs 33%, p=.001). For the whole cohort O154 of patients the 5-year overall survival (OS), progression-free sur- Therapeutic management of patients relapsing after vival (PFS), cumulative incidence of relapse (CIR) and non-relapse allogeneic haematopoietic stem cell transplantation for mortality (NRM) were 48%, 37%, 30% and 33% (Fig.: OS by type of myelodysplastic syndrome: a large-scale study on behalf of conditioning treatment). After NMA, the respective probabilities the Société Française de Greff e de Moelle et de Thérapie for 5-year OS, PFS, CIR and NRM were 50%, 35%, 35% and 31% and Cellulaire (SFGM-TC) after RIC 47%, 38%, 28% and 34% . Between the two conditioning R. Guièze, G. Damaj, M. Robin, M. Mohty, M. Michallet, R. Tabrizi, regimens, none of these probabilities were signifi cantly diff erent Y. Beguin, D. Blaise, D. Roosweil, C.-E. Bulabois, F. Legrand, A. Huynh, over time. In multivariate analysis the eff ect of NMA compared to J. Cornillon, N. Contentin, F. Suarez, B. Lioure, N. Maillard, L. Clément, RIC on overall survival with a hazard ratio of 1.02 (95%-CI, 0.7 to G. Guillerm, M.-T. Rubio, F. Garnier, I. Yakoub-Agha for the Société 1.4; p=.9) and on PFS with a hazard ratio of 1.08 (95%-CI, 0.8 to 1.5; Française de Greff e de Moelle et de Thérapie Cellulaire (SFGM-TC) p=.6). A refi ned analysis on an extended data fi le will be presented at the meeting. Background: Treatment of patients (pts) with myelodysplastic Conclusion: After correction for known risk factors NMA versus RIC syndrome (MDS) relapsing after allo-SCT remains disappointing alloHCT resulted in similar rates of survival in CLL patients, and and prognostic factors for outcome are still unclear. Several thera- future studies should also address improved immunological dis- peutic approaches are off ered: palliative and supportive care, ease control. intensive chemotherapy (IC), demethylating agents (DMA) and immunotherapy with either donor lymphocyte infusion (DLI) or second allo-SCT. We propose to identify predictive factors for outcome and to investigate the impact of diff erent treatment groups on survival.

S16 Methods: We report on 145 consecutive MDS pts who relapsed ASCT2 compared with 10 months (95% CI 9, 12) for C-weekly, rep- after allo-SCT between Jul 1999 and Mar 2011 in 19 French and resenting a hazard ratio of 0.3 (HR 95% CI 0.18, 0.49; Cox regres- Belgian centers. Pts who received graft from either cord blood or sion analysis p<0.0001). Response to re-induction therapy had a mismatched donor were excluded. Since our study was not ran- signifi cant impact on TTP (HR 3.72, 95% CI 1.55, 8.89; p=0.0032) domized, we used a propensity score to adjust P values for pts whereas the time to progression from the fi rst ASCT had a margin- who received immunotherapy and those who did not. ally signifi cant eff ect on TTP (HR 1.74, 95%CI 1.01, 3.00; p=0.048). Results: At diagnosis, 64 pts presented RA/RARS/RCMD, 29 RAEB1 Overall, 7 (8%) in the ASCT2 and 8 (10%) in the C-weekly arms have and 52 RAEB2 (high-risk IPSS for 80 pts). At transplant, 80 pts pre- died with PD accounting for 4 deaths in each cohort. Currently, sented a progressive disease. Pts received either MAC (n=41) or there is no impact of the post-randomization intervention on OS RIC (n=104) conditioning followed by BM (n=43) or PBSC (n=102) (p=0.654), though interpretation is limited by the current short fol- from sibling (n=95) or HLA-allele-matched unrelated (10/10) low-up and C-weekly patients who received a second ASCT after (n=50) donor. Median age at relapse was 57 y (19-70). Post-trans- reaching the primary end-point. The fi rst analysis demonstrates a plant relapse occurred after a median time of 6 months [1-100]. At clear advantage in terms of durability of response when a ASCT2 relapse 59% of pts had more than 10% of marrow blasts. Multivari- consolidates re-induction therapy with a bortezomib-containing ate analysis performed with propensity score matching revealed regimen in myeloma patients at fi rst relapse. The impact on overall detrimental impact of the following factors on 2-year OS: early survival and quality of life remains to be clarifi ed. relapse within 6 mo after transplant (HR=1.57 [95% IC=1.03-2.42, p=0.01], high WBC count >10 G/L (HR=4.63 [95% IC=2.55-8.42, p<0.001]), low platelets count <50 G/L (HR=2.07 [95% IC=1.37- O156 3.13, p=0.001]), and the absence of immunotherapy (HR=2.38 Reduced-intensity conditioned allogeneic stem cell [95% IC=1.54-3.67, p<0.001]). transplantation for multiple myeloma relapsing or progress- Conclusion: Salvage immunotherapy (DLI or second allo-SCT) ing after autologous transplantation : a study by the seems to provide the best results and should, whenever possible, European Group for Blood and Marrow Transplantation be off ered to pts with MDS relapsing after allo-SCT especially Chronic Malignancies Working Party those with low tumor burden and delayed relapse. Our results H.W. Auner, R. Szydlo, A. van Biezen, S. Iacobelli, G. Gahrton, N. Milpied, emphasize the need of prospective protocols combining cytore- L. Volin, J. Janssen, S. Nguyen Quoc, M. Michallet, H. Schoemans, J. ductive treatments and immunotherapy since pts who received El Cheikh, E. Petersen, F. Guilhot, S. Schoenland, L. Ahlberg, C. Morris, cytoreductive treatment alone (IC or DMA) had less satisfactory L. Garderet, T. de Witte, N. Kröger for the Chronic Malignancies outcome. Working Party of the European Group for Blood and Marrow Transplantation

O155 The role of of reduced intensity-conditioned allogeneic stem cell A second autologous stem cell transplant induces superior transplantation (RIC allo-SCT) in patients with multiple myeloma response durability following bortezomib-containing re- (MM) relapsing or progressing after autologous (auto)-SCT is not induction therapy for relapsed multiple myeloma: results well defi ned. We therefore performed a study of 413 MM patients from the BSBMT/UKMF myeloma x (intensive) trial reported to the EBMT for undergoing a RIC allo-SCT between G. Cook (1), C. Williams (2), A. Szubert (3), K. Yong (4), J. Cavet (5), 1999 and 2008. All patients had undergone at least one prior H. Hunter (6), J. Bird (7), S. Bell (3), S. O’Connor (8), J. Cavenagh (9), auto-SCT, and patients had at least one documented progres- J. Snowden (10), C. Parrish (1), J. Ashcroft (11), J. Brown (3), sion/relapse after an auto-SCT, and no other SCT performed C. Morris (12) between the most recent documented relapse/progression and (1)St James’s Institute of Oncology (Leeds, GB); (2)Nottingham the RIC allo-SCT. Median age at the time of RIC allo-SCT was 54.1 Univeristy Hospital (Nottingham, GB); (3)CTRU (Leeds, GB); (4)UCLH years. Median time from the fi rst auto-SCT to the RIC allo-SCT was (London, GB); (5)Christie Hospital Trust (Manchester, GB); (6)Derriford 2.6 years. The majority of patients had undergone one (55%) or Hospital (Plymouth, GB); (7)United Bristol Hospitals Trust (Bristol, GB); two (37%) prior auto-SCTs. More than half of the stem cell donors (8)HMDS (Leeds, GB); (9)St Barts Hospital (London, GB); (10)Royal were siblings (58%), over a third were unrelated (38%). Patient Hallamshire Hospitals Trust (Sheffi eld, GB); (11)Mid-Yorks Trust or donor CMV serostatus was positive (hereafter referred to as (Wakefi eld, GB); (12)Queens University (Belfast, GB) “CMV seropositivity”) in 74% and negative for both patient and donor (“CMV seronegativity”) in 26% of evaluable patients. Acute ASCT in MM is standard fi rst line therapy however, evidence for GvHD grade II-IV developed in 33% of all evaluable patients. Lim- its use in salvage therapy is lacking. The aim of this multi-centre ited and extensive chronic GvHD were documented in 21.7% and phase III trial was to evaluate the durability of response of a sec- 26.7% of all evaluable patients, respectively. Best responses were ond ASCT(2) compared with a less intensive consolidation, after >PR in 36.5%, PR in 25.1%, and less than PR in 15.3% of evaluable a bortezomib-containing re-induction strategy. Eligible patients patients. Median OS from RIC allo-SCT for all patients was 24.7 with MM relapsing after a prior ASCT were enrolled. All patients months (95% CI, 18.7-30.7). In univariate analysis, patient/donor with documented PD were re-induced with PAD (Bortezomib CMV serostatus, age at RIC allo-SCT, number of prior auto-SCT, (1.3mg/m2 iv D1, 4, 8 & 11), Doxorubicin (9mg/m2/day iv D1-4) and remission status at RIC-allo SCT were associated with OS. In & Dexamethasone (40mg/day PO D1-4: additionally D8-14 & D15- multivariate analysis, CMV seronegativity and one (compared to 18 on cycle 1 only) delivered in 4-6 21-day cycles before being ≥ 2) prior auto-SCT remained associated with better OS, with age randomized 1:1 to receive either a ASCT2 (Melphalan 200mg/m2 almost reaching signifi cance. When adjusted for both age and iv) or low dose consolidation with weekly cyclophosphamide number of previous auto-SCTs, CMV seronegativity remained 400mg/m2 PO (C-weekly). Response assessment (by IMW criteria) associated with better survival (p=0.014). Median PFS from RIC was analyzed after re-induction and 100 days post-randomization. allo-SCT for all patients was 9.6 months (95% CI, 7.3-11.8). CMV The primary endpoint was Time-to-progression (TTP). The Data seronegativity and patient/donor gender mismatch were associ- and Safety Monitoring Board have reviewed the interim analysis ated with better PFS in uni- and multivariate analysis. Cumulative results and recommended closure and early release of results. 286 NRM was 10.5% at 3 months and 21.5% at 1 year. CMV serone- patients were entered into the study and 187 randomized from gativity and time from fi rst auto-SCT to RIC allo-SCT < 2.6 years April 2008 to October 2012: ASCT2 n=86, C-weekly n=81. Median were associated with lower NRM in uni- and multivariate analysis. age was 61 (range 38,75) with 74.9% of patients relapsing more The results of this study demonstrate that RIC allo-SCT can be an than 24 months from fi rst ASCT. ORR to re-induction therapy was eff ective salvage treatment in a subgroup of MM patients relaps- 94%. At the time of analysis, 79 of 187 patients have progressed, ing/progressing after auto-SCT, and identify patient and donor 32 in the ASCT2 (37%) and 47 in the C-weekly (58%) cohorts. CMV seronegativity as the key prognostic factor for transplant Median time-to-progression was 19 months (95% CI 16, 25) for outcome.

S17 O157 Prospective analysis of prognostic pre-transplant factors in myelodysplastic syndromes primarily treated by allogeneic haematopoietic stem cell transplantation: a study on behalf of the MDS subcommittee of the Chronic Malignancies Working Party of the EBM E. Cremers, L. de Wreede, E. Knödler, J. Finke, M. Ditschkowski, M. Robin, J. Maertens, L. Brinch, L. Volin, R. Martino, G. Kobbe, T. de Witte, N. Kröger on behalf of the Chronic Malignancies Working Party

Background: allogeneic hematopoietic stem cell transplantation (SCT) is the most potent curative modality in myelodysplastic syndromes (MDS). Non-relapse mortality (NRM) is the major cause of death after SCT. Number of red blood cell (RBC) transfusions prior to SCT, iron overload and comorbidity are recognized causes of NRM. In this large prospective analysis performed by the Chronic Myeloid Malignancies Party (CMWP) of the EBMT prognostic pre-transplant factors in MDS are analyzed. Objectives: to evaluate in this large prospective observational study the impact of specifi c disease modalities, number and history of transfusions, iron toxicity parameters, comorbidity, infections and non-infectious complications on the occurrence on non-relapse mortality and overall survival, in patients receiving an upfront allogeneic SCT for MDS after standard conditioning. Results: The CMWP has been collecting data of 200 adult patients O158 with cytologically proven MDS, according to the WHO-classifi ca- Higher probability of survival in complete remission tion. All patients received upfront allogeneic stem cell transplan- following tandem autologous/reduced-intensity allogeneic tation after myeloablative or reduced intensity. Here we present transplantation compared to autologous transplantation data of 125 patients transplanted between January 2009 and alone in the NMAM2000 multiple myeloma trial as estimated December 2012. Data were collected at diagnosis, transplanta- by multistate models tion, 6 weeks, 100 days, 1 year and 2 years after SCT. Table 1 illus- S. Iacobelli, L. De Wreede, B. Björkstrand, U. Hegenbart, A. Gruber, trates the disease and treatment modalities of the patients at time H. Greinix, L. Volin, F. Narni, A.M. Carella, M. Beksac, A. Bosi, G. Milone, of transplantation. P. Corradini, S. Schönland, K. Friberg, A. van Biezen, H. Goldschmidt, 58 Percent of the patients had a lower risk MDS according to IPSS. T. de Witte, C. Morris, D. Niederwieser, L. Garderet, N. Kröger, Seventy-nine percent of the patients had a high iron load score, G. Gahrton for the EBMT CMWP Plasmacell disorder Subcommittee including a ferritin level >1000 ng/ml. 76 Percent of the patients had received RBC transfusions (median:7 units, range: 1-61). Sixty Objectives: Complete remission (CR) following autologous or allo- percent of the patients had no additional comorbidities at SCT. geneic transplantation has previously been shown to be important Twelve patients have died during the fi rst 3 months of follow-up for both progression free survival (PFS) and overall survival (OS). leading to an OS of 90%, a relapse-free survival (RFS) of 85% and In the NMAM2000 study we have previously shown that patients NRM of 9%. OS, NRM, relapse incidence (RI) and, (RFS) were 74%, with myeloma treated with the autologous/ reduced intensity 18%, 21% and, 62% respectively 12 months after SCT. The mean conditioning (auto/RICallo) transplant regimen have superior CR, estimate overall survival (OS) is 27 months (CI 17.9-22.0 months). PFS and OS compared to patients treated with autologous (single Twenty-seven patients developed aGvHD >grade 1. or double) transplantation. Using the data from this prospective Conclusion: this large observational study is the fi rst prospective study, now with median 96 months follow up, we explore the role study which analyses the infl uence of several important prognos- of CR in relation to the transplant strategy and survival. tic factors including transfusional and iron load in MDS treaded Patients and Methods: Data from 346 patients enrolled (age 31- by allogeneic SCT. More elaborate data on the described pre- 69 years) of whom108 were allocated to the auto/RICallo arm transplant factors and early treatment outcome will be available according to availability of a matched sibling donor were utilised. in February 2013. The course of the disease was analysed in a multi-state model, using the standard non-parametric Nelson-Aalen estimator for transition intensities. Results: The CR rate at 96 months was 50% (95%CI 42%-61%) and 43% (95%CI 38%-50%) in the auto/RICallo and auto arms respectively, Auto/RICallo was superior in terms of predicted probability of survival in CR compared to auto, both comparing groups according to the treatment allocated at randomisation (28.8% vs 11.4% at 60 months, p=0.0004) and in an analysis according to the actual administration of the second transplant (25.6% vs 9.6%, p=0.008). CR achieved after auto/RICallo produced longer PFS than CR achieved after auto. Conclusions: The better outcome of patients in CR following auto/ RICallo indicates a better quality (depth) of CR after this treatment modality. This in turn may be due to the previously documented graft versus myeloma eff ect.

S18 Acute GvHD myeloablative (Cyclo/TBI) SCT for CML (fi rst chronic phase) in whom donor DNA was available. SC source was an HLA-identical sibling and 10/10 HLA-matched unrelated donor in 59% and 36% respectively. Mismatched donors were excluded. GvHD prophy- O159 laxis was Cyclosporine and Methotrexate. Recipients of unrelated Anti-CD26 monoclonal antibody for the treatment of steroid- SCs also received in-vivo T-cell depletion with Alemtuzumab. refractory acute graft-versus-host disease (SR-GvHD): a 65% (88/142) developed aGvHD, 31%, 45%, and 22% developing report of two prospective studies grades I,II,and III-IV respectively. On univariate analysis, the pres- A. Bacigalupo (1), C. Di Grazia (1), T. Lamparelli (1), F. Gualandi (1), ence of both KIR2DL2 and KIR2DS2 in the donor genotype was A. Ibatici (1), S. Bregante (1), M.T. van Lint (1), A.M. Raiola (1), associated with signifi cantly increased risk of severe (II-IV) aGvHD A. Dominietto (1), R. Varaldo (1), S. Geroldi (1), E. Tedone (1), S. Luchetti (P=0.013 and P=0.028. This association was especially signifi cant (1), F. Bertolotti (1), M. Montagna (2), M. Regazzi (2) in recipients of grafts from KIR2DL2+ and DS2+ sibling donors, (1)IRCCS Azienda Ospedaliera Universitaria San Martino – IST 58% and 57% recipients developing grade II-IV compared to 23% (Genoa, IT); (2)IRCCS Policlinico San Matteo (Pavia, IT) and 26% with KIR2DS2- and DL2– donors (P=0.002 and P=0.005). However in recipients of a T-deplete SCT (unrelated donor)eff ect Background: Inhibition of CD26 impairs T cells migration across of KIR2DL2/DS2 was no longer signifi cant (P=1.0 and P=1.0). Multi- the endothelial barrier (Trends in Immunology 2008; 29:295). A variate analysis including known aGvHD risk factors, revealed that murine anti-CD26 , IgG2B monoclonal antibody, produced by a donor KIR2DS2/2DL2 status remained an independent predictor of P3X63 hybridoma , has been made available for clinical use (BEGE- GvHD severity in sibling SCT (P=0.005 and P=0.005). KIR genotype DINA®, ADIENNE, Bergamo-Italy) and we have tested the antibody did not predict for chronic GvHD or relapse. KIR-HLA mismatch for pharmacokinetics, tolerance and effi cacy in two phase I/II clini- had no eff ect on GvHD. T-cells express KIR molecules. We com- cal trials . pared the production of pro-infl ammatory cytokines implicated Aim of the study: The primary end point of Study1 (EUDRACT in aGvHD between KIR2DS2/DL2 expressing and non-expressing 2007-005809-21) was safety and effi cacy of anti-CD26 in SR- T-cells of healthy volunteers. T-cell stimulation plus intracellular GvHD. The primary end point of the dose fi nding study, Study2, cytokine staining revealed the percentage of cells expressing IFN-γ, (EUDRACT 2012-001353-19), was the proportion of circulating TNF-α, MIP1-α, and MIP1-β was signifi cantly higher in KIR2DS2/ CD3+CD26+ T cells ,stained with an anti-mouse IgG2B. DL2 expressing compared to non-expressing cells; 48.76% v Patients and methods. In Study1, BEGEDINA® was given at the dose 24.65% (P<0.0001), 55.3% v 35.2% (P=0.0001), 60.39% v 20.26% of 2 mg/day i.v. for 5 days. In Study2 three cohort of 3 patients (P<0.0001), and 47.13% v 19.34 (P=0.03) respectively. Conversely, each, received 2mg/m2/day ,or 3 mg/m2/day, or 4.5 mg/m2/day for IL-2 production (important for T-reg development) was greatest 5 days. Baseline GvHD severity was grade II (n=1), grade III (n=17) in KIR2DS2/DL2-ve cells 28.63% v 20.71% (P=0.003).These data and grade IV (n=2). Pharmakokinetic studies with an ELISA assay suggest KIR2DS2/DL2 genotype is associated with increased risk were performed on serum samples on day 1 through day 5 of of severe aGvHD possibly due to pro-GvHD cytokine skewing of treatment. KIR2DS2/DL2+ve T-cells. This implicates donor T-cell KIR expres- Pharmacokinetics: In Study1, the median serum concentration of sion as an important predictor of GvHD regardless of KIR-ligand BEGEDINA® after the 5th dose , was 33 ng/ml, and the median pro- (mis)matching. portion of CD3+CD26+ T cells stained in vivo, 25%. In Study 2, the median serum concentration after the 5th dose was 22 ng/ml (with 2 mg/m2); 52 ng/ml (with 3 mg/m2) ; 600 ng/ml (with 4.5 mg/m2). O161 The proportion of CD3+CD26+ cells stained with anti-mouse Circulating mir-500a/ mir-532 clusters as potential IgG2B – on day 5- was respectively 41%, 58%, 70%. biomarkers and regulatory hub for acute graft-versus-host Clinical results: Twenty adults were enrolled in these 2 studies. disease following allogeneic haematopoietic stem cell The tolerance of BEGEDINA® infusions was excellent. There were transplantation 8 complete responders (40%) and 10 partial responders (50%). M. Lv (1), X.S Zhao (1), W. Jun (2), X.Y Zhao (1), D.H. Liu (1), L.P. Xu (1), Two patients were non responders (10%). Non relapse mortality K.Y Liu (1), X.J. Huang (1) occurred in 5/20 patients (25%) and was due to GvHD in 3 and (1)Peking University People’s Hospital (Beijing, CN); (2)Medical Center infections in 2. Relapse related death occurred in 5 patients (25%). of Peking University & Hong Kong Science and Technology University The projected actuarial 2 year survival is 42%, which compares (Shenzhen, CN) favourably with our experience of patients with grade III-IV SR- acute GvHD. Objectives: Acute graft-versus-host-disease(aGvHD) remains Conclusions: Pharmacokinetic studies show direct correlation a major obstacle to a more favorable therapeutic outcome of dose administered , serum concentrations and proportion of for patients receiving allogeneic hematopoietic stem cell circulating T cells stained with the antibody. Clinical responses transplantation(allo-HSCT). Both diagnostic tools and novel suggest promising effi cacy of BEGEDINA® in grade III-IV SR-GvHD. therapeutic targets for aGvHD are urgently needed. Very recently, A prospective randomized trial ,comparing BEGEDINA® with con- circulating microRNAs(miRNAs)have been found as promising ventional second line treatment ,is programmed. biomarkers and regulators in other disease models. This study is to screen targeted miRNAs and potential co-regulatory networks which might play important roles in aGvHD. O160 Methods: MicroRNA arrays (Exiqon miRCURY LNA) were employed Donor KIR2DS2/DL2 genotype status is associated with an to screen diff erentially expressed miRNAs (>0.5 log10) in increased incidence of severe acute graft-versus-host paired plasma samples from four patients with biopsy-proven disease following myeloablative haematopoeitic stem cell aGvHD(aGvHD+)and after complete response(aGvHD-), the transplantation quantitative real-time polymerase chain reaction (qRT-PCR) was I.H Gabriel, R. Sergeant, R. Szydlo, J.F Apperley, A. Alsuliman, applied for preliminary validation. Then the candidate miRNAs A. Khoder, J. Goldman, E. Kanfer, H. de Lavallade, D. Miljokovic, were further validated by qRT-PCR in 81 patients receiving allo- A. Rahemtulla, J. Pavlu, L. Foroni, D. Marin, K. Rezvani HSCT for systemic monitoring or symptomatic follow-up after Imperial College (London, GB) aGvHD occurrence. The most promising candidate miRNAs were introduced into co-regulatory network analysis with aGvHD- Killer Immunoglobulin-like receptor (KIR) repertoire is associated related genes and transcriptional factors. with outcome following allogeneic stem cell transplant (SCT). Results: It was found in microRNA arrays that 16 kinds of miRNAs Data regarding KIR and acute graft versus host disease (aGvHD) were up-regulated and 15 kinds were down-regulated remain confl icting. We investigated the impact of donor KIR geno- comparing aGvHD±, in which 7 up-regulated (let-7a, mir-532-5p, type on the risk of aGvHD in 142 consecutive patients receiving mir-192, mir-199a-5p, mir-500a, mir-28-3p and mir-200c) and 11

S19 down-regulated miRNAs (mir-223, mir-150, let-7i, mir-199a-3p, Since JAK1/2 inhibitor INC424 interrupts JAK1/2 mediated cyto- mir-423-5p, let-7d, mir-181a, mir-30a, mir-326, mir-629mir-130a) kine responses, we hypothesized that INC424 might suppress were confi rmed by qRT-PCR respectively. In further valida- cytokine driven infl ammatory tissue damage in GvHD. tion of miRNAs in cohort, only mir-500a(p=0.032) and mir- We created an allogeneic cell culture system, using 105 naïve 532-5p (p=0.016) showed signifi cantly diff erent between BALB/c CD4+ CD62L high T cells that were co-cultured with 2,5x aGvHD±groups(Figure-1),and were associated with higher risk of 104 CD11c+ C57BL/6 (B6) bone marrow derived dendritic cells aGvHD. As mir-500a and mir-532-5p belong to the same miRNA (DC). DC were either stimulated with 20 ng LPS at day -1 prior to clusters at GRCh37.p5 of chromosome X, the whole clusters were the co-culture or plated without stimulus. The JAK1/2 inhibitor incorporated to the co-regulatory network, and interleukin 4/10 Ruxolitinib (INCB018424), dissolved in DMSO at diff erent concen- receptor, glucocorticoid receptor(NR3C1), TGF-β activated kinase 1 trations (1:103 to 1:104) was added to the cultures at day -1, fol- (TAB3),etc. were found as hubs of feed forward loop in aGvHD lowed by 105 naïve T cells at day 0. regulation. The up-regulated clusters may strengthen the pro- After fi ve days of culture, supernatants were taken and analyzed infl ammatory signals by Interleukin-18 pathway, while reduce the by cytometric bead array (CBA). The cells were stained for fl ow sensitivity of glucocorticoid and endogenous suppressive cyto- cytometry analysis. kines, such as TGF-β,IL-10, etc. (Figure-2). For our in vivo studies, we used a major mismatch GvHD model Conclusion: Our study suggest that circulating mir-500a/ mir-532 (C57BL/6 donor; BALB/c H2d recipient mice). 5x106 T cell depleted are potential plasma biomarker for aGvHD, which may also serve donor bone marrow cells and 5x105 CD4+ and CD8+ T cells (in a as a targets for novel aGvHD therapy in future. 1:1 ratio) were used. Three diff erent cohorts of recipient mice were irradiated with 8 Gy and received cells in the aforementioned numbers: 1. T cell depleted bone marrow (T depl BM) alone, 2. T depl BM along with T cells and 3. T depl BM with T cells and 30 mg/kg INCB018424 administered as an oral gavage, twice daily, day 0 through 25. Mice were monitored for signs of GvHD for 25 days. According to our ICC data, INCB018424 blocked IFN-γ and IL-17 production of CD4+ T cells. CBA data revealed impaired production of IFN-γ, IL-17, IL-2, IL-6 and IL-4 in the presence of INCB018424 in a dose dependent manner. At the same time, regulatory CD4+ FOXP3+ T cells were induced at low concentrations, especially in the presence of non-LPS treated DC. In vivo, no signs of GvHD were seen in the control population (only T depl BM). Animals that had received both T depl BM and T cells developed GvHD. However, INCB018424 treated animals displayed improved survival and lower GvHD scores. The inhibition of JAK/STAT signaling using INCB18424 leads to suppression of proinfl ammatory cytokines and promotes regulatory T cells in an allogeneic in vitro setting. Also in vivo, INCB18424 treated mice have a better clinical outcome. Thus, Jak inhibitors are likely to improve treatment of GvHD after allo-HSCT.

O163 Graft-versus-host disease rates and survival after cord blood transplantation for acute leukaemia: a comparison of outcomes in the Japanese versus the U.S. populations Y. Kuwatsuka (1), Y. Atsuta (2), M. Horowitz (1), J. Inagaki (3), J. Kanda (4), K. Kato (5), K. Koh (6), M. Zhang (1), M. Eapen (1) (1)Medical College of Wisconsin (Milwaukee, US); (2)Nagoya University Graduate School of Medicine (Nagoya, JP); (3)National Kyusyu Cancer Center (Fukuoka, JP); (4)Saitama Medical Center Jichi Medical University (Saitama, JP); (5)Japanese Red Cross Nagoya First Hospital (Nagoya, JP); (6)Saitama Children’s Medical Center (Saitama, JP)

An earlier report that compared graft-versus-host disease (GVHD) and survival rates between Caucasians and Japanese children with leukemia after HLA-identical sibling bone marrow transplantation, observed higher acute and chronic GVHD in Caucasians (Blood 2005, 105:1408). There are no reports that have addressed whether racial diff erences are associated with GVHD rates after umbilical cord blood transplant (UCBT). To answer this question we analyzed data from the Center for International Blood and O162 Marrow Transplant Research and the Japan Society for Hemato- JAK1/2 inhibition leads to reduction of proinfl ammatory poietic Cell Transplantation; US Caucasians and Japanese patients cytokines and promotion of regulatory T-cells in an alloge- who received single unit UCBT for AML (n=181) or ALL (n=336) neic setting between 2000-2009 were eligible. Patients were 16 years or S. Spoerl (1), B. Michael (1), M. Schmickl (1), C. Peschel (1), J. Duyster younger, in fi rst (n=249) or subsequent (n=268) complete remis- (2), H. Poeck (1), R. Zeiser (2), N. von Bubnoff (2) sion at transplantation, received myeloablative regimen. The US (1)Technische Universität München (Munich, DE); (2) Universitätsklinik population was grouped based on whether in vivo T-cell depletion Freiburg (Freiburg, DE) was included in their transplant strategy or not. None of the Japa- nese patients received in vivo T-depletion. The three treatment Graft-versus-host-disease (GvHD) constitutes a major drawback of groups that were compared is as follows: Japanese (n=257), and allogeneic hematopoietic stem cell transplantation (allo-HSCT). In US Caucasians who received in vivo T-cell depletion (n=168) and GvHD, tissue damage is mediated by proinfl ammatory cytokines. who did not (n=92). Median ages of the Japanese and Caucasians

S20 who received in vivo T-cell depletion was 5 years; the remaining Our data show that application of proteomic peptide patterns Caucasians were slightly older, 8 years. Numbers of patients who allowed preemptive, accurate and investigator-independent received 4/6 or 5/6 HLA matched grafts were 210 (Japan), 126 diagnosis of severe aGvHD. (US T-depleted) and 75 (US non-T depleted). The corresponding median total pre-cryopreserved cells were 5.1, 7.4 and 5.7x107/kg. In multivariate analysis, there were no signifi cant diff erences in O165 acute grade 2-4 or grade 3-4 GVHD risk. However, compared to MiR-146a-5p and its targets in graft-versus-host disease the Japanese, chronic GVHD was signifi cantly higher in US Cauca- S. Atarod, W. Cope, J. Norden, M. Collin, X. Wang, A. Dickinson sians who did not receive in vivo T-cell depletion (RR 2.16, p<0.001) Newcastle University (Newcastle upon Tyne, GB) but not for those who received in vivo T-cell depletion (RR 1.43, p=0.08). Compared to the Japanese population, TRM was higher Introduction: Graft-versus-host disease (GVHD) is a fatal complica- in US Caucasians who received in vivo T-cell depletion (RR 1.81, tion of allogeneic haematopoietic stem cell transplantation (allo- p=0.01). However, higher chronic GVHD and TRM did not result HSCT) and is classically diagnosed as acute or chronic. There is in higher overall mortality rates for the US population. The 5-year no biomarker available clinically to predict the development and probabilities of overall survival adjusted for patient CMV serosta- severity of GVHD. MicroRNAs (miRs) are regulatory RNAs (19-22 tus and disease risk were not diff erent between the Japanese nucleotides) which have been shown to function in both innate (61%) vs. US Caucasian receiving T-depletion (51%, p=0.09) or non and adaptive immunity. The function of miR-155 has recently been T-depletion (57%, p=0.59). These data suggest acute GVHD rates implicated in GVHD pathogenesis. In addition, miR-146a-5p has after UCBT are not diff erent in Caucasian and Japanese children. been shown to play a crucial role in psoriasis. Thus, we studied miR- 146a-5p expression and its validated targets; TNF receptor associated factor 6 (TRAF6), interleukin-1 receptor-associated kinase O164 (IRAK1), signal transducer and activator of transcription 1- α (STAT1- Urinary proteomic peptide profi ling allows preemptive α) and interferon regulatory factor 5 (IRF5) in a Newcastle cohort of diagnosis of acute graft-versus-host-disease after allogeneic HSCT recipients with or without GVHD to examine whether miR- stem cell transplantation 146a-5p could be used as an early indicator of GVHD. E.M. Weissinger (1), J. Metzger (2), C. Dobbelstein (1), D. Wolff (3), Methods: Whole blood was collected from allo-HSCT patients M. Schleuning (4), Z. Kuzmina (5), L. Hambach (1), H. Greinix (5), (n=60) at various time-points pre- and post-transplant (Day -7 A.M. Dickinson (6), W. Mullen (7), M. Collin (6), M. Morgan (1), up to 12 months) in PreAnalytiX PAXgene tubes. Total RNA was I. Tchebotarenko (1), E. Dammann (1), S. Ehrlich (1), H. Diedrich (1), extracted and used to perform qRT-PCR for miR-146a-5p and its M. Stadler (1), M. Eder (1), E. Holler (3), H. Mischak (2), J. Krauter (1), targets. The Mann-Whitney U test was used to determine the sta- A. Ganser (1) tistical signifi cance and Receiver Operating Characteristic (ROC) (1)Hannover Medical School (Hannover, DE); (2)mosaiques- curves were generated to evaluate the sensitivity and specifi city diagnostics GmbH (Hannover, DE); (3)University of Regensburg of miR-146a-5p as a diagnostic biomarker for acute GVHD. (Regensburg, DE); (4)Deutsche Klinik für Diagnostik (Wiesbaden, Results: Our analysis showed that miR-146a-5p expression was sig- DE); (5)Medical University of Vienna (Vienna, AT); (6)University of nifi cantly down-regulated in II-IV acute GVHD patients at 28 days Newcastle (Newcastle, GB); (7)University of Glasgow (Glasgow, GB) post-transplant (p=0.003) compared to 0-I acute GVHD patients. The area under the curve (AUC= 0.84) for miR-146a-5p at 28 days Allogeneic hematopoietic stem cell transplantation (HSCT) is the post-transplant was highly signifi cant (p= 0.003) when 0-I acute only curative treatment for hematologic malignancies in adults, GVHD (n= 18) patient results were compared to patients with II-IV but is compromised by severe acute graft-versus-host disease acute GVHD (n= 10) (Figure 1). TRAF6 (p= 0.016 at 28 days) and (aGvHD). Prediction of severe aGvHD as early as possible is there- STAT1-α (p= 0.04, 3 months) were also signifi cantly down-regu- fore crucial. lated in the II-IV acute GVHD patient cohort. IRAK1 and IRF5 expres- A urinary proteome-based classifi er developed for aGvHD predic- sion remained unaltered regardless of GVHD severity. Expression tion has previously been described. Here we report on a multicen- of miR-146a-5p and its targets were not altered in chronic GVHD tre prospective validation of this peptide-classifi er in additional patients compared to the no chronic GVHD cohort. 463 patients. The majority of patients included were transplanted Conclusion: Our results suggest that miR-146a-5p could be an early for hematologic malignancies (n=453). At the time of transplan- diagnostic biomarker of acute GVHD severity. An investigation into tation, 50% (n=231) were not in CR. The majority of patients the validated targets at the protein level is currently on going. were transplanted from matched donors (n=370; 80%), while 93 (20%) received stem cells from mismatched donors. Acute GvHD prophylaxis consisted of cyclosporine A (CsA) and methotrexate (MTX; n=209; 45%) or mycophenolate mofetil (MMF, n=209); or tacrolimus in combination with others (n=34); ex vivo CD34- enrichment (TCD; T-cell-depletion) without additional GvHD-prophylaxis (n=7), or no GvHD-prophylaxis for other reasons (n=4). The leukemic cell burden in patients with malignant disease was reduced by myeloablative conditioning regimens (n=121; 26%) or reduced intensity conditioning protocols (RIC; n=342; 74%) with less toxicities. Immunosuppressive antibodies were added in 292 (61%) patients. Between 2005 and 2010, samples were collected prospectively from patients undergoing allo-HSCT in fi ve transplant centers. Samples (n=1106) were analyzed using capillary electrophoresis coupled on-line to mass spectrometry (CE-MS). The proteome-based classifi er, aGvHD MS17, led to correct classifi cation of patients with aGvHD grade III or IV 14 days prior to any clinical signs with a sensitivity of 71.2% and a specifi city of 77.8% (AUC 0.83). Multivariate regression analysis showed that aGvHD MS17 positivity was the highest predictive parameter for aGvHD development (P<0.0001). The classifi er consists of 17 peptides derived from albumin, β 2-microglobulin, CD99, fi bronectin, collagen α1 and 2, indicating infl ammation, activation of T-cells, and changes in the extracellular matrix as early signs of organ damage.

S21 Paediatric issues 1 myeloablative regimens. The median time of occurrence of AIHA, ES, and ITP from HSCT was 5.2, 9.2 and 40.2 months, respectively. 64% of AHD recipients failed steroid treatment and required additional immunosuppressive therapies.15 children were treated with O166 rituximab (RTX) administered as fi rst (n=1) or second line therapy Autoimmune haematological diseases after allogeneic (n=14) for AIHA (n=8), ES (n=4), and ITP (n=3), respectively. 87% haematopoietic stem cell transplantation in children. of patients receiving RTX (8 AIHA, 3 ES, 2 ITP) obtained complete Italian multicentre experience remission 60 days later (range 30-180), the remaining 2 died from M. Faraci, M. Pillon, A. Rovelli, M. Menconi, M. Ripaldi, F. Fagioli, severe intractable ES and relapse of malignancy. 46%, 40% and 20% M. Rabusin, O. Ziino, M. Zecca, F. Locatelli, A. Prete on behalf of the of patients aff ected by AIHA, ES and ITP, respectively, had viral infec- Italian Association of Pediatric Hematology and Oncology (AIEOP) tions before or during AHDs; 54% of these viral events were CMV reactivations. In our series,4 patients (9%)(1 AIHA,1 ES,2 ITP) died Introduction: Autoimmune hematological diseases (AHDs) may 87 days after AHD diagnosis (range 22-199) as a direct or indirect occur after allogeneic hematopoietic stem cell transplantation consequence of autoimmune disorders. (HSCT), but there are currently no large pediatric series reporting Conclusions: This observational retrospective study confi rmed these complications. that AHDs are possible complications of allogeneic HSCTs per- Material and Methods: The study involved all consecutive alloge- formed in children; non malignant diseases, AD-HSCTs and neic HSCT recipients reported to the Italian Association of Pediat- myeloablative regimens represent the most frequently observed ric Hematology and Oncology (AIEOP) - HSCT Registry and treated features in these patients; RTX represents the treatment of choice between 1998 and December 2011. Nine of 15 AIEOP-SCT centers for children who develop AHDs after transplant. (60%) agreed to participate in this study and collected a specifi c questionnaire including information on clinical features, laboratory characteristics, therapies and outcome of children with AHDs. O167 Results: Of the 1,569 allogeneic patients transplanted during the Osteopetrosis: a heterogeneous group of diseases requiring study period, a total of 33 children (2.1%) with AHDs were identifi ed. individualized therapeutic strategies - results of the The median follow-up for AHD patients was 38.5 months (range 2.5- osteopetrosis registry on behalf of ESID and EBMT 163.6). Fifteen out of 33 pts developed autoimmune hemolytic ane- A.S. Schulz, D. Moshous, C. Steward, M. Hoenig, C. Schuetz, mia (AIHA) (45%),10 had immune thrombocytopenia (ITP) (30%), C. Sobacchi, A. Fischer, K.-M. Debatin, A. Villa on behalf of the 5 had Evans’ Syndrome (ES) (15%), 2 had pure red cell aplasia (PRCA) Inborn Error Working Party (6%), and 1 had immune neutropenia (IN) (3%). 66.7% of these children were aff ected by non malignant diseases,78.8% received Osteopetroses (OP) are rare genetic diseases characterized by alternative donor (AD) HSCTs, and 72.7% were conditioned with increased bone density due to mutations in at least 7 diff erent

[O167]

S22 genes. Hematopoietic cell transplantation (HCT) is a curative centers between 06.2010 and 02.2012. The conditioning regimen approach for severe infantile and intermediate forms of OP. contained fl udarabine (150 mg/m2), treosulfan (42 g/m2; 36 g/m2 if A central OP registry has been developed in order to highlight <12kg), alemtuzumab (1.0 mg/kg for unrelated donors; 0.3 mg/kg clinical presentations and possible genotype/phenotype corre- for matched related donors (MRD)), and thiotepa (10 mg/kg; lations and to identify the best treatment option for individual 7 mg/kg if <12 kg). Depending on center preference, thiotepa was patients. omitted in patients in remission. Data has been collated on 244 patients from Europe, the Middle Results: The cohort included 4 FHL3 (familial HLH 3), 2 FHL4, East and Southern America from 1980 until now (analyzed retro- 6 FHL5, 1 XLP1 (x-linked lymphoproliferative syndrome 1), 2 XIAP spectively in 180 pts, prospectively in 50 pts, not in registry due defi ciency, and 2 undefi ned degranulation defects; median age to missing consent 30 pts). Of 187 patients with complete data 3.4 y at HSCT. Donors were HLA-mismatched (9/10) in 7 patients, sets, 155 pts (83%) have been transplanted; 99 transplanted pts matched unrelated in 5, matched related in 4, and haploidenti- (64%) and 18 non-transplanted pts (56%) are alive. The probability cal in 1. Both, overall and disease-free survival were 100% with of overall survival in evaluable transplanted pts according to caus- a follow-up of 6 to 20 months. Two patients required secondary ative gene and donor source is depicted in fi gure 1 and 2 respec- HSCT; one due to rejection of an HLA-haploidentical graft, the tively. Data analysis highlights the following: other one due to secondary failure of a mismatched graft. Ten • Genotype/ phenotype correlation is highly variable: “classical” recipients developed a donor chimerism of <95%. Five patients infantile OP was mainly seen in pts with TCIRG1, but also with received donor lymphocyte infusions (DLI) (one with a single SNX10 and some CLCN7 mutations; pts with OSTM1 and some CD34+ stem cell boost), with the lowest donor chimerism at with CLCN7 mutations have severe neurodegenerative disease; 24 – 70%, achieving a stable chimerism at 20 – 100%. Interest- intermediate forms with highly variable organ involvement are ingly, all 5 had a mismatched donor and 4/5 had not received thio- found, particularly in pts with CLCN7 and the rare RANK and tepa. Only one patient in the thiotepa cohort (n=12) required DLI. RANKL mutations. One patient experienced a 3° GvHD after DLI; one VOD occurred in • HCT results are promising in pts transplanted at an early age the haploidentical HSCT. Despite slow T-cell reconstitution, only 2 (<10 months) and/or with less advanced disease and/or from severe viral infections occurred, one infl uenza A pneumonitis and an HLA-matched donor; results in HLA haploidentical HCT one EBV reactivation requiring rituximab. improved with a busulfan, fludarabine and thiotepa based Conclusion: A combination of fl udarabine, treosulfan, alemtu- conditioning according to the latest EBMT/ESID guidelines, zumab, and thiotepa is an eff ective conditioning regimen with but are associated with a high risk of rejection and/or non- low toxicity in HLH patients. Thiotepa should be included to engraftment in advanced disease; HCT has no positive effect reduce the rate of autologous reconstitution, in particular with on neurodegeneration even if performed before the onset of mismatched donors. neurological symptoms. • Long term survivors of HCT are handicapped due to visual impairment, short stature and/or neurological sequelae; severe O169 neurological sequelae, e.g. developmental delay and autism, are Cognitive and psychosocial outcomes in childhood survivors rare. Quality of life is acceptable to parents in most cases. of haemophagocytic lymphohistiocytosis following haema- In summary these data highlight the importance of avoiding HCT topoietic stem cell transplantation in those with CNS neurodegeneration. Multidisciplinary follow up J. Jackson (1), P. Titman (2), S. Butler (1), K. Bond (2), P. Veys (2), A. Rao after HCT is crucial to treat long term sequelae. New or improved (2), R. Chiesa (2), A. Leiper (2), L. Riley (2), K. Gilmour (2), P. Amrolia treatment strategies are needed in distinct subgroups: those with (2), K. Rao (2) osteoclast independent RANKL mutations, neurodegenerative (1)University College (London, GB); (2)Great Ormond Street Hospital and/or advanced disease. NHS Trust (London, GB)

In our study, we present for the first time, a detailed assessment O168 of the cognitive and psychosocial outcome following HSCT for Fludarabin, treosulfan, thiotepa, and alemtuzumab as HLH. We have compared outcomes to a control sibling group and conditioning regimen for children with haemophagocytic our results are based on multiple informants including teacher lymphohistiocytosis: low toxicity and eff ective disease and parental reports. Our study investigated the cognitive and control psychosocial outcomes in childhood survivors of haemophago- K. Lehmberg (1), M. Albert (2), K. Beutel (3), B. Gruhn (4), R. Meisel (5), cytic lymphohistiocytosis (HLH) following haematopoietic stem A. Schulz (6), D. Stachel (7), R. Beier (8), T. Vraetz (9), W. Woessmann cell transplantation (HSCT). 21 children were assessed on stan- (10), G. Janka (1), I. Müller (1) dardised measures of cognitive and psychosocial functioning, (1)University Medical Center Eppendorf (Hamburg, DE); (2)University and compared with an unaffected sibling control group (n =14). Hospital - Ludwig-Maximilian University (Munich, DE); (3)University Parent and teacher reports were obtained to provide informa- Hospital - Westfalian Wilhelms University (Münster, DE); (4)University tion about the child’s education and psychosocial functioning. Hospital - Friedrich Schiller University (Jena, DE); (5)University The average full scale IQ for the patient cohort was 81 (95% Hospital - Heinreich Heine University (Düsseldorf, DE); (6)University confidence intervals 72-90), significantly lower than both the Hospital (Ulm, DE); (7)University Hospital - Friedrich Alexander population average of 100 (p=0.001) and the average for the University (Erlangen, DE); (8)Hannover Medical School (Hannover, unaffected sibling control group (99.2, p=0.002). 56% of school DE); (9)University Hospital (Freiburg, DE); (10)University Hospital - aged children were receiving additional support at school, the Justus Liebig University (Giessen, DE) majority needing high levels of support. These children also experienced greater psychosocial difficulties, particularly in Background: Children with hereditary forms of haemophagocytic social functioning. Lower socioeconomic status was associated lymphohistiocytosis (HLH) can be cured by haematopoietic stem with poorer cognitive outcomes, but age at transplant, time cell transplantion (HSCT). The high incidence of veno-occlusive from diagnosis to transplant, type of conditioning and mixed disease (VOD) and transplant related mortality after busulphan- chimerism were not. 10/21 (48%) of children had evidence of based myeloablative regimens could be reduced with melphalan- neurological involvement at diagnosis but surprisingly this was based reduced intensity conditioning (RIC), however at the cost of not significantly associated with adverse neurological outcome high rates of mixed chimerism. We hypothesised that treosulfan and some children who did not have any apparent neurologi- might be more myelosuppressive than melphalan and still pro- cal involvement at diagnosis had severe learning difficulties at duce less adverse eff ects than busulphan. follow up. In summary, childhood survivors of HLH are at risk of Objective and methods: To retrospectively determine rates of developing long-term cognitive difficulties and associated psy- survival, engraftment, donor chimerism, reactivation, and seri- chosocial difficulties. Based on our findings of significant cogni- ous adverse events after HSCT in 17 HLH patients in 9 German tive impairments in over half of our patients, we could speculate

S23 that the incidence of CNS involvement at diagnosis is probably O171 higher than currently recognised. Another unanswered question Outcome of children with ALL in second CR transplanted is the timing of the ‘first CNS hit’ in HLH.These findings add to from an unrelated donor has signifi cantly improved over the ongoing controversy regarding the timing and appropriate- time and is favourably infl uenced by the occurrence of grade ness of HSCT in asymptomatic siblings with genetically proven I-II acute GVHD and limited chronic GVHD HLH. Prospective and systematic long-term follow up of these D. Pagliara (1), M. Zecca (2), F. Fagioli (3), E. Lanino (4), A. Balduzzi (5), patients is essential for early identification and effective man- C. Messina (6), C. Favre (7), F. Porta (8), M. Ripaldi (9), F. Moretta (1), agement of these problems. A. Pession (10), A. Prete (10), F. Locatelli (1) (1)IRCCS Bambino Gesù Children’s Hospital (Rome, IT); (2)Fondazione IRCCS Policlinico “San Matteo” (Pavia, IT); (3)Ospedale S. Anna O170 (Turin, IT); (4)IRCCS G. Gaslini (Genoa, IT); (5)Fondazione MBBM, Outcomes after combined umbilical cord blood and bone University of Milan-Bicocca (Milan, IT); (6)University of Padua marrow haematopoietic stem cell transplantation from (Padua, IT); (7)Ospedale S. Chiara (Pisa, IT); (8)Spedali Civili (Brescia, the same HLA-identical sibling donor for children with IT); (9)SSD TMO, Pausilipon Hospital (Naples, IT); (10)Ospedale non-malignant and malignant diseases S. Orsola (Bologna, IT) L. Tucunduva, F. Volt, R. Cunha, A. Ruggeri, A. Yesilipek, S. Aksoylar, Y. Bertrand, F. Fagioli, C. Addari, J. de la Fuente, M. Zecca, M. Caniglia, Introduction: Allogeneic HSCT from an unrelated volunteer is F. Locatelli, T. Güngör, V. Rocha, E. Gluckman on behalf of Eurocord largely employed to treat children with ALL in second CR. We analyzed the outcome of patients transplanted in Centers belonging A family directed cord blood unit (CB) can be used for an HLA to the Associazione Italiana di Ematologia ed Oncologia Pediatrica identical sibling diagnosed with a disease that can be cured (AIEOP) between 1995 and 2009. by hematopoietic stem cell transplantation (HSCT). The CB can Methods: Two hundred thirty-eight patients (pts) with ALL in sec- be collected and stored regardless of its cellular content. An ond CR after a bone marrow (BM) (93%) or isolated extramedullary option to enhance the cell dose available for transplantation is recurrence (7%) were included in the study; 156 were males and to harvest bone marrow (BM) cells from the same sibling donor 82 females, median age at transplantation being 9 years (3-18). and infuse them in addition to the CB. There are few published BM and peripheral blood were the stem cell source in 212 and 26 reports on patients (pts) receiving CB combined with BM from pts, respectively. A myeloablative, TBI-containing regimen was HLA identical sibling and all include a low number of patients. used in 214 pts, while the remaining 24 were given chemotherapy- Therefore, we studied 148 children who received a combined based preparation. A combination of cyclosporine A, short-term graft from 1994 to 2011 in EBMT centers. The most common MTX and ATG was employed in 72% of pts for GVHD prophylaxis. indication of the BM harvest was a CB with a low cell count. In view of the Berlin-Frankfurt-Munster (BFM) classifi cation of fi rst Median age at transplantation was 7 years (1-20) and 77% of leukemia recurrence, 54%, 21% and 25% of pts were assigned to pts (n=115) were transplanted for non-malignant diseases (71%, the S2, S3 and S4 groups, respectively. n=81, for hemoglobinopathies). Acute leukemia (n=26) was Results: The 6-year disease-free survival (DFS) was 50%, while the the most frequent malignant diagnosis and all, but one patient 6-year cumulative incidence of transplantation-related mortality with this diagnosis, were transplanted in remission (CR). Most (TRM) and leukemia recurrence were 25% and 24%, respectively. pts received a myeloablative conditioning regimen, busulfan The 6-year DFS of children transplanted in the 3 time periods based in 87% of cases. Donors had a median age of 1.5 years (1995-1999, 2000-2004, 2005-2009) were 36%, 46% and 60%, (0-8) at BM harvest. The median infused total nucleated cell respectively (p=0.01). This improvement was due to a reduction of dose (TNC) was 2.0x107/kg (0.2-11.30) for CBU and 24.0x107/kg TRM, which in the 3 time periods was 43%, 31% and 14%, respec- (2.5-180) for BM, resulting in a total median infused TNC of 26.0 tively (p=0.0002). The 209 pts with B-cell precursor ALL had a sig- x107/kg (4.2-187.5). GVHD prophylaxis consisted in ciclosporin nifi cantly better outcome in comparison to patients with T-cell with (n=51) or without methotrexate( n=59). Median follow-up ALL, DFS being 52% and 38%, respectively (p=0.02). The DFS of was 36 months. Cumulative incidence (CI) of 60-day neutrophil pts who had grade I-II acute GVHD was 64%, while that of pts with recovery was 95% with a median time of 18 days. CI of acute either absent or grade III or grade IV acute GVHD was 39%, 38% GVHD II-IV was 13% (n=17 grade II, n=8 grade III, n=3 Grade IV). and 6%, respectively. Pts with limited chronic GVHD had a better CI of chronic GVHD was 10% (n=15) with only 3 pts presenting DFS as compared to those with either extensive or absent chronic extensive involvement. Three-year CI of mortality was 4%. Thir- GVHD (81%, 61% and 50%, respectively; p=0.05). Both grade I-II teen pts died; from which 4 had non-malignant diseases and acute GVHD and limited chronic GVHD protected from leukemia died of disease recurrence (n=2) or rejection (n=2), and 9 had recurrence. The DFS of pts assigned to the S2, S3 and S4 groups malignant diseases and died of relapse (n=6), GVHD (n=2) or was 66%, 39% and 31%, respectively (p=0.0001). All the variables unknown cause (n=1). Three-year disease free survival was 93% infl uencing DFS in univariate analysis remained signifi cant in mul- for pts with non-malignant and 67% for those with malignant tivariate analysis. diseases. In conclusion, incidences of acute and chronic GVHD Conclusions: The outcome of children with 2nd CR ALL has sig- are low after combined CB with BM HSCT from an HLA identi- nifi cantly improved over time, due to a reduction of TRM. Limited cal sibling donor. DFS is excellent for patients with malignant severity acute and chronic GVHD protect from leukemia recur- and non-malignant disorders. When the related CB has low TNC rence. BFM classifi cation of 1st relapse predicts outcome. (<2x107/kg malignant; <4x107/kg non-malignant) and the need for transplant is not immediate, harvesting BM of the same donor in a later date should be considered. O172 Impaired exercise capacity is related to reduced pulmonary function in long term follow-up after allogeneic haematopoietic stem cell transplantation in childhood S. Mathiesen, H.H. Uhlving, F. Buchvald, KG. Nielsen, K. Müller National University Hospital Rigshospitalet (Copenhagen, DK)

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) may lead to chronic pulmonary impairment and limited exercise tolerance. Our aim was to determine exercise capacity by peak oxygen uptake (VO2peak) at long term follow up after HSCT in children and to investigate associations between exercise capacity and

S24 pulmonary function tests (PFT), acute and chronic graft vs. host Results: Sixty-six subjects with a mean (range) age of 14.3 (7-24) disease (GvHD) and transplantation baseline data. years were included. The mean (range) follow up period was 7.5 Methods: We performed a cross-sectional follow up study in Dan- (3-10) years. Transplantation baseline data are shown in table 1. ish children and young adults years after allogeneic HSCT. Exercise Fifty-nine subjects (89%) met the criteria of a peak exercise test capacity (VO2peak (ml/min/kg)) was measured by a cycle ergom- (exhaustion, heart rate >185 bpm and respiratory exchange ratio eter test. PFT included spirometry, diff usion capacity of the lung >1.0). Twenty-fi ve percent of those had signifi cantly reduced val- for carbon monoxide (DLco) and bodyplethysmography. Abnor- ues of VO2peak (Table 2). mality was defi ned as z-score < -1.96. Chronic GvHD was assessed PFT revealed decreased values of forced expiratory volume in according to National Institutes of Health’s criteria. fi rst second (FEV1), forced vital capacity (FVC), total lung capacity

S25 (TLC), DLco and DLco adjusted for alveolar volume (DLco/VA) Donors issues compared to healthy subjects, but were signifi cantly abnormal in 10-30% of the patients (Table 2). Univariate analysis demonstrated signifi cant associations between VO2peak and FEV1, FVC, TLC, DLco and DLco/VA, respectively, but O174 no association to acute or chronic GvHD or any of the baseline Equivalent outcome between older siblings and unrelated parameters: Recipient age at HSCT, years from HSCT, donor age donors after reduced intensity allogeneic haematopoietic and type, diagnosis and conditioning by total body irradiation, stem cell transplantation for patients older than 50 years cyclophosphamide and busulfan. with acute myeloid leukaemia in fi rst complete remission Multiple linear regression confi rmed a signifi cant association R. Peff ault de Latour, M. Labopin, J. Cornelissen, L. Vindelov, D. Blaise, between VO2peak and FEV1 (p<0.001), DLco/VA (p<0.001) and N. Milpied, A. Huyn, C. Craddock, N. Russel, V. Koza, B. Lioure, busulfan conditioning (in 29/33 patients given intravenously) H. Schouten, D. Caballero, J. Cahn, N. Fegueux, M. Mohty on behalf (p=0.042). of the Acute Leukemia Working Party of EBMT Conclusion: Excercise capacity is impaired years after HSCT. We found a signifi cant association between VO2peak and pulmo- The requirement for an “older” matched sibling donor (MSD) when nary function parameters. The cause of reduced exercise capacity a healthy younger matched 8/8 unrelated donor (MUD) is available may be multifactorial, but the use of busulfan could be a risk fac- is still controversial for patients (pts) older than 50 years with AML tor. Our results emphasize the need for increased focus on exer- in CR1 transplanted after a RIC regimen. From 01/2000 to 12/2010, cise capacity as an important parameter in post HSCT patients. this retrospective multi-center study included 1102 pts with AML in CR1 who received PBSC after a RIC regimen, either from MSD (n=854) or from 8/8 HLA MUD (n=248). Conditioning regimen was O173 fl udarabine-based (95%) and GvHD prophylaxis consisted of CSA Costs and cost-eff ectiveness of allogeneic stem cell trans- + MMF (42%). Three groups of pts were defi ned: pts who were plantation in children are predictable transplanted from a MUD (MUD group), pts who received their S. Matthes-Martin (1), U. Poetschger (2), R. Barr (3), M. Martin (4), graft from a sibling aged less than 60 years old (MSD less than 60 H. Boztug (1), T. Klingebiel (5), A. Attarbaschi (1), W. Eibler (1), group) and pts who were transplanted from a sibling aged of 60 G. Mann (1) years old or more (MSD 60 or more group). Pts characteristics were (1)St Anna Children´s Hospital (Vienna, AT); (2)Children´s Cancer similar between the 3 groups for gender, disease distribution and Research Institute (Vienna, AT); (3)McMaster University (Hamilton, cytogenetic risks (MRC classifi cation). Recipients were younger in CA); (4)Imperial College (London, GB); (5)University Children´s the MSD less than 60 group. In the MUD group, pts were trans- Hospital (Frankfurt, DE) planted more recently, from a younger donor, with a longer time interval between diagnosis and HSCT and the conditioning regi- Stem cell transplantation (SCT), a well established and poten- men included more ATG. The median FU was 24 months (range, tially curative approach in several malignant and non-malignant 2-122) for MUD, 42 months (range, 1.5-140) for MSD less than 60 diseases, is considered to be cost intensive. In contrast to well and 33 months (range, 1-129) for MSD 60 or more. The cumula- defi ned costs per treatment course for expensive novel cancer tive incidence (CI) of acute GvHD was 28% in MUD versus 20% therapies, determining the costs of SCT is diffi cult. This is mainly and 17% in MSD less than 60 and MSD 60 or more, respectively due to the complexity of SCT in which various departments (trans- (p=0.006) while the CI of chronic GvHD at 2 years was about 50% plant ward, intensive care, outpatient clinic, radiation clinic, cell in all group (p=0.93). CI of treatment related mortality (TRM) and collection and processing unit) are involved. Moreover paediatric relapse as well as leukemia free survival (LFS) and overall survival SCT patients are an extremely heterogeneous group with vary- (OS) were not diff erent at 2 years between the 3 groups (Table1). ing toxic or infectious pre-transplant morbidity. Unpredictable In multivariate analysis, TRM was decreased in pts who received post-transplant complications, and the fact that SCT patients ATG in the conditioning regimen (HR: 0.62; p=0.03). A longer inter- need medical care during the fi rst year after transplant compli- val between AML diagnosis and CR1 (>median) was the only fac- cate the cost analysis. In the context of the actual discussions tor associated with relapse (HR: 1.35; p=0.02). Two independent on cost thresholds for cancer therapies we evaluated 1) factors factors were associated with worse LFS: a longer delay between which are known prior to SCT and which might correlate with the AML diagnosis and CR1 (HR: 1.34; p=0.01) and CMV donor sero- costs, making the costs of individual SCTs predictable and 2) the positivity (HR: 1.28; p=0.04). For pts alive after 2 years post HSCT costs of paediatric SCT per life year gained. The overall costs of (n=454), a signifi cant higher rate of TRM was found in pts trans- paediatric SCT including donor search, graft acquisition, recipient planted from MUD compared with the 2 other groups (Table 2). work-up and treatment during the fi rst year after transplant in a Outcomes using MUD, MSD less than 60 or MSD 60 or more were cohort of 141 consecutive children transplanted in a single insti- equivalent, supporting the recommendation to consider MSD for tution were calculated. Costs were correlated with patient and HSCT even after 60 years of age prior to younger 8/8 MUD for pts transplant characteristics and a risk score for transplant related with AML older than 50 years in CR1 after a RIC regimen. mortality (TRM). Cost-eff ectiveness was calculated as overall costs per surviving patient. Gained life years were extrapolated from the overall survival and the costs per expected gained life year were calculated. Overall median costs were 137,771 Euro with a wide range from 26,897 to 605,396. Costs increased signifi cantly with age, the use of donors other than matched siblings and with advanced disease. There was a strong correlation of costs with a simple TRM risk-score: the median total costs in Euro were 89,550 (score 0), 127,349 (score 1), 157,296 (score 2) and 266,108 (score 3) (p < 0.001). Cost eff ectiveness decreased with the TRM risk-score: the costs per survivor increased from 93,209 Euro (score 0) to a maximum of 1,215,436 Euro (score 3). Costs of paediatric SCT vary substantially, however the combination of the three variables age, disease and donor is predictive for costs and cost-eff ectiveness. Costs per life year gained are within the broadly accepted range in other cancer therapies even in the most cost intensive transplants.

S26 O175 Discrepancy analysis of microsatellite loci as a proxy for ances- O176 tral diff erentiation between donors and recipients: correlation Race/ethnicity is associated with donation experiences of between high scores and poorer overall survival in children US-based HSC donors undergoing matched unrelated donor transplantation for ALL G. Switzer (1), J. Bruce (1), L. Myaskovsky (1), D. Confer (2), J. Harvey (1), A. Green (1), S. Groves (2), J. Cornish (3), J. Moppett (3), L. Abress (2), A. DiMartini (1), D. Harrington (1), S. Ohngemach (1), M. Cummins (3), L. Keen (1), S. Culliford (1), A. Poles (1), P. Breslin (3), M.A. Dew (1) Y. Li (1), C. Steward (3) (1)University of Pittsburgh (Pittsburgh, US); (2)National Marrow (1)British Bone Marrow Registry, NHS Blood and Transplant (Bristol, Donor Program (Minneapolis, US) GB); (1)NHS Blood and Transplant (Bristol, GB); (2)Bristol University (Bristol, GB); (3)Bristol Royal Hospital for Children (Bristol, GB) Rationale: Previous research has indicated that there may be dif- ferences in unrelated hematopoietic stem cell (HSC) donor deci- Objective: Registries collect data on the racial origin of donor/ sion-making and experiences by racial/ethnic group. recipient(D/R)pairs so as to determine whether racial mismatch However, donor experiences have not been systematically or lon- impacts transplant outcome. However, the place of a person’s birth gitudinally examined by race/ethnicity. Our goal was to select a and their skin colour are poor predictors of their true genetic racial diverse sample of US-based donors and to examine characteristics origin. For example, American Latinos are a mix of European, African and experiences of these donors pre- and post-donation. Meth- and Native American ancestries; in the well studied Puerto Rican ods: We conducted telephone interviews with a random sample population, genetic markers of ancestral European origin vary from NMDP donors (N=522) stratifi ed by race/ethnicity at pre-dona- 30-79% and those of African origin from 7-50%. Multiple anthropo- tion, and 1 and 12 months post-donation. Measures included logical studies have used STR allelic length variability to calculate demographics, culturally-related variables (e.g., medical mistrust, genetic distance (GD). This has been shown to correlate in ances- religious objections to donation), psychosocial variables (e.g., tral populations (including Europeans) with genetic diff erentiation emotional distress, self-esteem) and donation-related variables in protein polymorphisms, which could potentially include those (e.g., ambivalence about donation, donation-related concerns). with eff ects on transplant outcome. We wished to test if STR length Analyses examined diff erences in these variables at the three tim- variability between D/R correlated with transplant outcome. points by racial/ethnic group. Method: It is thought that all humans derive from African ances- Results: At pre-donation, all minorities were more likely than tors and that short tandem repeat (STR) microsatellite allele whites (WH) to have religious objections to donation (F=18.45; lengths have drifted with GD. We hypothesised that a donor/ p<.001), to mistrust the medical system (F=4.05; p<.01), to mistrust recipient (D/R) weighted average allele size diff erence (WAASD) how HSCs would be allocated (F=5.66; p<.01), and to have been score - calculated from 15 highly informative STR’s (Promega, discouraged from donating (F=10.58; p<.05). In addition, Asian Powerplex 16TM), might provide a better readout of ancestral dif- Pacifi c Islanders (API) specifi cally were more likely than all other ferentiation, and hence potentially be correlated with transplant groups to be ambivalent about donation (F=4.31; p<.01) and to outcome.This score was derived for a previously reported popu- have medical concerns (F=6.18; p<.001). At 1 month post-dona- lation of 61 children who underwent transplantation for acute tion, API reported lower self-esteem (F=4.55; p<.01) and mastery lymphoblastic leukaemia during 2004-10. (F=6.76; p<.001), poorer physical health (F=3.10; p<.05), and more Results: The WAASD values obtained from HLA high resolu- concern about their overall health as a result of donation than did tion matched unrelated donor (MUD) transplant pairs formed a other groups (F=4.22; p<.01). Minorities overall reported more numeric continuum from 0.8 to 2.2, compared to 0 to 1.4 for sib- donation-related symptoms than did WH. At 12 months post- ling D/R pairs. There was increasing trend of signifi cance when donation, API reported poorer overall health (F=4.23; p<.01), less correlating post-transplant outcome with increasing WAASD dis- satisfaction with the donation decision (F=3.52; p<.05), more con- crepancy score for MUD pairs. This reached signifi cance at 1.8; 2 cern about their health as a result of donation (F=8.54; p<.001), and year OS was 77% in patients with a score of <1.8 (n=27) compared were less likely to report feeling back to normal following dona- to 43% in those with a score of >1.8 (n=14, p=0.02). Small num- tion (16.35; p<.001). Conclusions: These fi ndings have important bers prevent analysis of the impact of WAASD in sibling D/R pairs. implications for the management of donors, particularly donors Conclusion: STR size diff erence (averaged over 15 STR loci) may be belonging to ethnic minority groups and especially API donors. marker for multiple genetic diff erences between D/R pairs which Given that individuals who donate become ambassadors for HSC impact transplant outcome. This approach requires further confi r- donation in their communities it is critical to identify donors who mation in larger patient groups but has the potential to improve are at-risk for less positive donation outcomes and to intervene to MUD donor selection where multiple donors are available. ensure positive donation experiences.

S27 can be jeopardized by the inability to collect suffi cient number of O177 progenitors. Several studies were conducted to better understand Biosimilar fi lgrastim mobilizes haematopoietic stem cells in this variability in stem cell mobilization in healthy individuals. healthy volunteer donors with expected effi ciency and Factors such as age, sex, ethnic origin as well as the actual dose typical acute adverse eff ects: interim results of a post- of rhG-CSF have been shown to infl uence CD34+ mobilization. authorization safety study In addition, several studies suggest that associations exist with P.S.A. Becker (1), S. Brauninger (1), H. Bialleck (1), B. Luxembourg (1), donor genetic polymorphisms, consistent with pre-clinical obser- M. Schulz (1), M. Wiesneth (2), P. Reinhardt (2), J. Mytilineos (2), vations in strains of mice with diff erent genetic backgrounds. C. Seidl (1), O. Geisenberger (3), A. Schwebig (3), H. Schrezenmeier (2), Methods: We took advantage of access to DNA samples obtained E. Seifried (1), H. Bonig (1) from 225 related donors who were informed and consented to (1)German Red Cross Blood Service Baden-Württemberg-Hessen the study, and who were homogeneously treated, monitored and (Frankfurt, DE); (2)German Red Cross Blood Service Baden- collected at a single institution over a 14 year period of time, to Württemberg-Hessen (Ulm, DE); (3)Sandoz (Holzkirchen, DE) look at Single Nucleotide Polymorphisms (SNPs) that have been identifi ed in the coding or non coding sequences of biologically Introduction: Recommendations on healthy donor stem cell relevant genes, including G-CSF and its receptor, CXCL12 and its mobilization indicate skepticism about safety/effi cacy of biosimi- receptor, VLA4 and VCAM-1, CD44, CD34 and kit ligand. lar G-CSF. The biosimilar G-CSF Zarzio (Germany: Filgrastim Hexal) Results: Circulating CD34+ cell counts ranged from 6.7/mcL was approved by the European Medicines Agency (EMA) based on through 237.6/mcL. Polymorphisms were detected using a Snap- evidence of high physico-chemical similarity and comparable PK/ Shot multiplex PCR technique (Genoscreen, Lille, France). Diff er- PD vs. Neupogen in healthy volunteers and safety in neutropenic ent approaches were used for statistical studies: fi rst, we used cancer patients. Approval was granted for all approved indications ANOVA to compare circulating CD34+ counts with the three dif- of Neupogen including stem cell mobilization. In agreement with ferent classes for each studied SNP; second, the distribution of EMA a post-authorization safety study (PASS) is being conducted. SNPs was compared in the 25% group of donors with the lowest Methods: This PASS will enroll 200 consenting donors of the Ger- CD34+ counts and in the 25% group of donors with the highest man stem cell donor registry DSSD. Donor selection, evaluation, CD34+ counts; third, the distribution of SNPs was compared in treatment, apheresis collection and follow-up are performed “poor-mobilizers” (< 20 CD34+ cells/mcL) and in “good-mobilizers” according to governing laws and established SOP. Mobilization (> 20/mcL). In all analyses, the same SNP in VLA-4 was associated with standard-dose G CSF is followed by one or two large-volume with more effi cient mobilization and the same SNP for VCAM-1 aphereses. 10 year active donor follow-up (FU) is performed with was associated with less effi cient mobilization. Interestingly, we interim medical history/laboratory tests. The PASS was approved were unable to demonstrate statistically signifi cant associations by the local Ethics Committee. with SNPs in other studied genes. Results: 69/70 donors completed mobilization and apheresis Conclusion: This study confi rms the importance of the VLA4/ (1 cancelled transplantation), 38% and 21% completed 1- and VCAM-1 axis in progenitor cell mobilization. Further studies are 6-month FU. Donor population was Caucasian, 2/3 male, mean necessary to understand the link between SNPs and VCAM-1 or age 36 years (Range: 19-58), average weight, height and BMI. VLA4 expression and function Adverse events (AEs) during mobilization were frequent but tolerable and in agreement with known toxicities of G-CSF. 90% of donors reported pain (mostly bone). Circulating CD34+ cells after O179 the 9th dose of G-CSF were 111/μL (mean; range: 34-284/μL). G-CSF impairs the antiviral immune response of T-cells in Mean leukocyte count (WBC) under G-CSF was 48.8 G/L, with 78% stem cell donors: implications for the selection of CTL donors neutrophils. After one (93%) or two (7%) aphereses, 68/69 donors C. Bunse (1), S. Borchers (1), P. Varanasi (1), S. Tischer (1), C. Figueiredo achieved the target CD34+ cell dose of 5x10E6/kg of the recipi- (1), L. Goudeva (1), U. Kalinke (2), U. Köhl (1), B. Maecker-Kolhoff (1), ent (mean 9.2x10E6/kg, range 4.5-19.9x10E6/kg). Aphereses were A. Ganser (1), E. Mischak-Weissinger (1), R. Blasczyk (1), B. Eiz-Vesper (1) well tolerated. One possibly related SAE (hospitalization for chest (1)Hannover Medical School (Hannover, DE); (2)Twincore (Hannover, wall pain) completely resolved without medication. WBC, alkaline DE) phosphatase and LDH were elevated at the time of apheresis; all had normalized by the fi rst FU. Adoptive transfer of antiviral T cells has become a powerful and Discussion: Changes in WBC/biochemistry and acute toxicity of promising tool to complement impaired immune reconstitution in Zarzio were consistent with reported data, both in frequency and patients after allogeneic hematopoietic stem cell transplantation severity. Mobilization effi ciency for CD34+ cells was very good. FU (HSCT). HSCT patients are at risk of experiencing recurrent reactiva- surveillance will determine if long-term safety of Zarzio will also tion of persisting viruses like Cytomegalovirus (CMV) and Epstein- not diff er from Neupogen. Barr virus (EBV) or lytic viruses like Adenovirus (ADV). If available, Conclusion: Thus far, this PASS did not yield any unexpected seropositive stem cell donors may serve as a source for antiviral results which would question safety or biological similarity of T cells. Recently, expansion of cytotoxic T-lymphocytes(CTL) from Zarzio for healthy donor stem cell mobilization. G-CSF mobilized stem cell grafts has been suggested as a source for antiviral CTL. There is, however, growing evidence that G-CSF mobilization has negative eff ects on T-cell function. O178 Here, we analysed the eff ects of G-CSF on antiviral T cells in vitro SNP analysis of DNA obtained from 225 related donors for and in vivo in 87 mobilized stem cell donors and 25 unmobilized HSCT reveals the relation between CD34+ cell mobilization controls. The number of CMV-, EBV- and ADV-CTL were assessed intensity in response to rhG-CSF treatment and genetic using HLA-matched virus-specifi c multimers (A*01:01, A*02:01, polymorphisms in the VLA4 and VCAM-1 genes A*24:02, B*07:02, B*08:01, B*35:01). Functional activity was tested C. Fournel (1), A.M. Imbert (1), F. Bondi (2), C. Lemarie (1), B. Calmels (1), by IFN-γ ELISPOT assay after stimulation with overlapping peptide C. Chabannon (1) pools (CMV: pp65, IE-1; EBV: EBNA1, LMP2A, BZLF1; ADV5: hexon). (1)Institut Paoli-Calmettes (Marseilles, FR); (2)Inserm CBT-510 Interestingly, the absolute number of antiviral CTL detected after (Marseilles, FR) G-CSF stimulation and compared to unmobilized samples was not infl uenced by G-CSF, but function of T cells in response to all Objectives: Peripheral blood progenitor cells are nowadays the viral antigens was signifi cantly impaired as expressed by a mean preferred source of stem cells for allogeneic transplantation, from reduction in IFN-γ secretion of 73.9% (pp65 80.4%, IE-1 79.07%, related and unrelated donors. Donors display large inter-individual EBNA1 76.6%, LMP2A 57.8%, BZLF1 75.8%). To underline these variability in terms of response to rhG-CSF used for mobilization results in vitro stimulation assays were performed in presence or and increase in circulating CD34+ cell counts, and some behave absence of G-CSF using single HLA-restricted peptides. Expansion as « poor-mobilizers »; in this situation, recipient engraftment rates for G-CSF-cultured T cells did not diff er from untreated cells

S28 (e.g. A*02:01 CMVpp65: 2.6 vs 3.0 fold increase, B*08:01 EBVBZLF1: using HBsAg+ donors for HSCT are less supported by clinical data. 20.9 vs 20.3 fold increase), but IFN-γ secretion was reduced by To address this, we performed a case-control retrospective study 39.5% (mean all tested peptides). to investigate outcomes of patients who received an HBsAg+ Our study shows that during G-CSF mobilization, the functional transplant compared with those who received an HBsAg- trans- activity of antiviral memory T cells is impaired, indicating that plant. From 2005 to 2010, 10 HBsAg- recipients received HSCT from even stem cell donors may not be the best source of T cells. HBsAg+ donors were included as an observation group, while 30 Taken together, we suggest that (1) CTL for adoptive transfer in HBsAg- recipients received from HBsAg- donors were defi ned as a patients at high risk for viral reactivation (e.g. haploidentical T-cell control group. The anti-hepatitis B virus (HBV) therapy consisted of depleted HSCT) should be harvested prior to G-CSF mobilization lamivudine or entecavir for HBsAg+ donors. Stem cell harvest and in order to avoid an adoptive transfer of functionally impaired infusion were performed until donor’s serum HBV-DNA reached antiviral CTL and (2) third party donors should be considered in undetectable. Hepatitis B immunoglobulin were administered to patients at need of antiviral CTL early after HSCT. recipients within 24 hours, 1 month and 2 months after stem cell infusion. As shown in Figure 1, the transplant-related outcomes and liver complications were not statistically signifi cant between O180 the 2 groups (p>0.05), which implied the use of allografts from HBsAg- recipients had comparable outcomes and acquired HBsAg+ donors was safe. HBV-specifi c humoral immunity after haematopoietic stem HBV transmission may occur in HSCT from HBsAg+ donors. Unex- cell transplantation from HBsAg+ donors pectedly, none of the 10 HBsAg- recipients in observation group Y.X. Hu, Y. Luo, J. Shi, Y. Tan, W. Zheng, Z. Cai, H. Huang developed HBsAg+. Instead, 9 of the 10 recipients acquired HBsAb The First Affi liated Hospital,School of Medicine, Zhejiang University (Figure 1). This fi nding inspired us to further identify the origin (Hangzhou, CN) of HBsAb. Peripheral blood mononuclear cells (PBMCs) from 4 attainable recipients with HBsAb acquisition post-transplanta- Exclusion of HBsAg+ donors limits the application of hematopoi- tion were cultured with mitogens, CpG ODN-2006, and S. aureus, etic stem cell transplantation (HSCT). However, precise risks of Cowan to expand memory B cells. On day 6 HBsAb-secreting B cells were isolated by fl ow cytometry and short tandem repeat (STR) was performed. Consequently, STR showed 2 recipients (6 and 5 years post-transplantation) acquired donor-derived HBsAb, 1 recipient (31 months post-transplantation) acquired recipient-derived HBsAb while 1 recipient (17 months post- transplantation) acquired mixed donor and recipient- derived HBsAb (Figure 2). STR of PBMCs from all 4 recipients showed full donor’s chimerism. These results implied both recipient’s humoral immunity before transplantation and the microenvironment after transplantation might play crucial roles in humoral immune re-constitution via HSCT. In all, our data support that allo-HSCT from HBsAg+ donors is prac- ticable. Moreover, such transplantation mode provides unique disease models to study the contribution of microenvironment and to establish new therapeutic strategies for HBV clearance.

S29 Lymphocyte biology & Immunogenetics were stained with carboxyfl uorescein succinimidyl ester (CFSE) to evaluate their proliferation by fl ow cytometry. Results: Eff ects of fresh Tregs on the proliferation of diff erent lymphoma cell lines ranged from help to suppression. Tregs expanded O181 for 2-3 weeks with rapamycin suppressed proliferation of 10/12 In vitro generation of antigen-specifi c T-cells from CD34+ lymphoma cell lines tested, and of polyclonally activated autolo- haematopoietic progenitor cells: a new and promising gous or allogeneic conventional T cells. Tregs restimulated poly- immunotherapeutic strategy clonally or by allo-DC in the presence of rapamycin proliferated for S. Snauwaert, S. Van Coppernolle, G. Verstichel, G. Goetgeluk, Y. Van 6-9 weeks, maintained regulatory function, maintained demethyl- Caeneghem, S. Vanhee, I. Velghe, J. Plum, T. Kerre, B. Vandekerckhove ation of FOXP3 intronic Treg-specifi c demethylation region (TSDR), Ghent University (Ghent, BE) and constantly expressed FOXP3. Tregs expanded without rapamycin proliferated for a shorter period of time, did not develop Introduction: Transfer of high-affi nity antigen-specifi c T-cell regulatory function equivalent to that of Tregs expanded with receptor (TCR) genes into polyclonal peripheral blood T-cells is an rapamycin, and sometimes even helped lymphoma to prolifer- attractive immunotherapeutic strategy against malignancies and ate. Regulation of lymphoma cell proliferation by Tregs depended viruses. However, inappropriate crosspairing between introduced on both cell-to-cell contact and soluble factors (Fig. 1). Expanded and endogenous TCR chains can result in suboptimal activity and Tregs secreted IL-10 and IFN-γ, and expressed CTLA-4, CD39, CD73, unpredicted, potentially harmful antigen-specifi cities. Effi cient in GARP, TIGIT, GITR, and granzyme A, but not granzyme B or perfo- vitro generation of antigen-specifi c T-cells from CD34+ hemato- rin. Sorted CD127(lo) and CD127(hi) subsets of CD4+CD25+T cells poietic progenitor cells (HPCs) may eliminate these restrictions, after expansion with rapamycin similarly controlled lymphoma based on the hypothesis that early introduction of rearranged proliferation (Fig. 2), although FOXP3 expression was lower in the TCRα and TCRβ chains might result in allelic exclusion of the Tregs expanded from CD4+CD25+CD127(hi)T cells. endogenous TCRα and/or TCRβ locus. We and others have previ- Conclusions: Treg function directed against lymphoma B cells ously shown that HPCs commit to the T-cell lineage and become depends on Treg state of activation as well as on the Treg activa- CD4+CD8+ double positive (DP) precursors when cultured on tion pathway. Tregs expanded with rapamycin could be useful in OP9-DL1 stromal cells. designing a model of adoptive therapy post-autologous hema- Results: CD34+ HPCs from human postnatal thymus were retrovi- topoietic transplantation (HCT) to eradicate residual lymphoma rally transduced to express the TCRα and TCRβ chains of human and to counteract graft-versus-host disease as well as lymphoma leucocyte antigen (HLA)-A2 restricted TCRs recognizing epitopes regrowth after allogeneic HCT. of cytomegalovirus (CMV pp65) or Wilms’ tumor 1 (WT1). Diff erentiation in transduced cultures was studied. We confi rmed earlier reports showing that terminal maturation of TCR-transduced DP cells to mature CD8 single positive (SP) cells occurs, albeit at low effi ciency. We hypothesized that the observed maturation involved selection by TCR binding to HLA class I /peptide com- plexes present in culture. Therefore, we added the respective agonist peptide to the cultures. This induced rapid phenotypi- cal maturation to CD27+CD1- of the majority of TCRαβ+ DP cells. Antigen presentation by HLA-A2+ dendritic cells, HLA-A2+ tumor cell lines, and even cross-presentation by HLA-A2+ T-cell progenitors, but not by HLA-A2 negative cells, induced this maturation process. The mature cells consist of CD8αβ, CD8αα SP and CD4-CD8- cells. These T-cells expanded upon culture with phytohemagglutinin and interleukin-2 on irradiated feeders, indicating functionality. Upon activation, specifi c killing of T2 cells loaded with agonist peptide, was observed. In vitro generated T-cells were labeled brighter with the specifi c tetramers than TCR-transduced peripheral blood T-cells. TCR spectratyping revealed major inhibition of endogenous TCRα and TCRβ gene rearrangements. Conclusion: In vitro generation of functional antigen-specifi c T-cells from CD34+ HPCs is a promising new immunotherapeutic strategy.

O182 Ex vivo expanded human regulatory T-cells suppress B-cell lymphoma proliferation M.A. Grygorowicz, M. Biernacka, M. Bujko, E. Nowak, M. Omiotek, G. Rymkiewicz, J. Walewski, S. Markowicz Maria Sklodowska-Curie Memorial Institute and Oncology Centre (Warsaw, PL)

Objectives: Evaluation of interactions between T regulatory cells (Tregs), including ex vivo expanded Tregs, and lymphoma B cells. Methods: CD4+CD25+T cells were obtained from peripheral blood of healthy donors. Tregs were expanded from CD4+CD25+T cells in the presence of allogeneic monocyte-derived dendritic cells (allo- DC) or anti-CD3/28/2 Ab-coated beads, with IL-2 (10 or 50 U/ml, respectively), with or without rapamycin (100 ng/ml). Established cell lines of follicular lymphoma, diff used large B cell lymphoma, Burkitt lymphoma, plasma cell myeloma, mantle cell lymphoma, and Hodgkin lymphoma were co-cultured in the absence of rapamycin with fresh or ex vivo expanded Tregs. Lymphoma cells

S30 O183 in response to CD3/CD28 stimulation in vitro, while they showed Early development of immunity to CMV following haemato- signifi cant expansion in patients rendered lymphocytopenic after poietic stem cell transplantation is associated with graft- chemotherapy. Multi-drug effl uxing CD4+CD161+ T-cells were versus-leukaemia eff ect enriched within the viral-specifi c Th1 repertoire and persisted V. Guérin-El Khourouj (1), R. Porcher (2), D. Jorge Cordeiro (1), after chemotherapy in vivo. The high ABCB1-mediated drug effl ux S. Leveillé (1), J. Le Goff (2), B. Pédron (1), A. Baruchel (1), J.-H. Dalle (1), capacity of CD4+CD161+ memory T-cells facilitated their resis- G. Sterkers (1) tance to daunorubicin in vitro and, this resistance was abrogated (1)Robert Debré Hospital (Paris, FR); (2)Saint Louis Hospital by competitive ABCB1 inhibitors. Finally, we demonstrated that (Paris, FR) following infl uenza vaccination, the proportion of infl uenza-specifi c CD4+ T-cells that co-expressed CD161 was signifi cantly higher Relapse of disease remains the most common cause of failure at 2 years, compared to 4 weeks post-vaccination, suggesting that in children undergoing hematopoietic-stem cell transplantation CD161 is a marker for long-lived antigen-specifi c memory T-cells. (HSCT) for acute leukaemia. A favourable impact, on graft-versus These fi ndings suggest that CD4+CD161+ T-cells with rapid effl ux leukaemia eff ect (GVL), from recipient CMV-seropositivity before capacity contribute to the maintenance of viral-specifi c memory HSCT and from CMV-reactivations in the recipient after HSCT has T-cells. These data could have signifi cant implications for the been suggested. The potential role, in this process, for the immune development of novel immunotherapeutic approaches. response triggered by CMV has not been directly addressed so far. 108 children (median age 8 years) were included at HSCT fol- O185 lowing myeloablative conditioning for primary acute leukae- Human telomerase reverse transcriptase-specifi c T-cell mia (lymphoblastic 54%, myeloblastic 42%, biphenotypic 4%). receptor gene transfer redirects T cells to display an eff ective HSCT were from HLA-matched related (41%) or unrelated (44%) antitumour reactivity against adult T-cell leukaemia donors. 15% patients received cord blood units. CMV- DNAemia H. Fujiwara (1), Y. Miyazaki (1), F. Ochi (1), H. Asai (1), T. Ishida (2), was programmed weekly for at least 3 months post-HSCT. When S. Okamoto (3), J. Mineno (3), K. Kuzushima (4), H. Shiku (5), PCR showed ≥1000 copies/ml, patients received ganciclovir as M. Yasukawa (1) pre-emptive therapy. Immunity to CMV was evaluated sequen- (1)Ehime University (Toon, JP); (2)Nagoya City University (Nagoya, tially since the fi rst month post-HSCT using 3H-Thymidine incor- JP); (3)Takara Bio Inc. (Otsu, JP); (4)Aichi Cancer Center (Nagoya, JP); poration assay (T-cell proliferation) and intracytoplasmic cytokine (5)Mie University (Tsu, JP) accumulation assay (IFN-γ secretion). Median follow-up from transplant was 40 months. 57% of patients Purpose: Adult T-cell leukemia (ATL) still has a quite poor progno- were CMV-seropositive before HSCT. Cumulative incidence of sis, thus to develop a more eff ective treatment is urgently needed. recipients with CMV-DNAemia at day 120 was 31% (median time Graft-vs.-ATL eff ect observed in some cases successfully treated to onset: 26 days). Cumulative incidence of recipients with immu- with allo-HSCT suggests the eff ectiveness of cellular immune- nity to CMV (T-cell proliferation and/or IFN-γ secretion assays) at mediated approach. On the other hand, some laboratory fi ndings 1 year was 38% (median time to onset: 2 months). As expected, suggest the overexpression of human telomerase reverse tran- recipient CMV seropositivity represented a major factor contrib- scriptase (hTERT) in ATL tumor cells. In this study, we examined uting to DNAemia (p<0.0001) and to immunity (p<0.001) occur- the feasibility of hTERT-targeting redirected T-cell based immuno- rence as well. The 2 years cumulative incidence of relapse was therapy in order to explore a novel cellular immunotherapy for 26% among the 89 recipients free of relapse at day +120 and ATL. evaluated for immunity before that time, the 2 years cumulative Methods: mRNA and protein expression of hTERT in ATL tumor incidence of relapse was 15%. Multivariable analysis revealed a cells was examined using QRT-PCR and Western blotting. HLA- diff erent risk of leukemic relapse according to immunity to CMV A*2402-resticted hTERT461-469 nonamer epitope (VYGFVRACL) and CMV-reactivation (p=0.039), with the lowest risk in recipients specifi c CTL in peripheral blood was assessed by tetramer assay. with early (before day 120) immunity to CMV but no CMV-reac- HLA-A*2402-resticted hTERT461-469-specifi c TCR α/β genes tivation and the highest risk in recipients with early (before day obtained from K3-1, the epitope-specifi c CTL clone were inserted 120) CMV-reactivation but no immunity to CMV (HR (0.06, 95%CI into a second-generation 2A peptide-based siTCR vector encod- 0.01–0.69, p=0.024). ing siRNAs for endogenous TCR genes (hTERT-siTCR vector). In conclusion, early development of immunity to CMV rather than Using this vector, CD8+ T cells were gene-modifi ed and used as early viremia had a favourable impact on GVL in this pediatric eff ectors (hTERT-siTCR/CD8+). Anti-ATL tumor activity and safety series. against normal cells were assessed using standard 51Cr release assay, time-lapse assay, and xenografted mouse model with bio- luminescence assay. O184 Results: hTERT was overexpressed in ATL tumor cells, but not in Stem-like characteristics in a subset of CD161-expressing normal cells. hTERT461-469-specifi c CTL precursors in periph- human memory CD4+ T-cells may facilitate their survival eral blood were variably detected in HLA-A*2402+, but not after chemotherapy HLA-A*2402- ATL patients or HLA-A*2402+ healthy individu- A. Alsuliman (1), A. Khoder (1), K. Stringaris (1), N. Cooper (1), als. hTERT-siTCR/CD8+ displayed HLA-A*2402-restricted and S. Takuya (1), H. de Lavallade (1), A. Sarvaria (1), S. Mielke (2), D. Marin hTERT461-469-specifi c cytocidal activity against ATL tumor cells (1), K. Rezvani (3) but not normal cells including hematological progenitors. hTERT- (1)Imperial College London (London, GB); (2)University of Würzburg siTCR/CD8+ generated from heavily pretreated HLA-A*2402+ ATL (Würzburg, DE); (3)MD Anderson Cancer Centre (Houston, US) patients also lysed autologous ATL tumor cells according to each expression level of hTERT mRNA. Finally, using xenografted NOG The establishment of long-lived pathogen-specifi c T-cells is a fun- mouse models, therapeutically infused hTERT-siTCR/CD8+, but damental property of the adaptive immune response. However, not non-gene-modifi ed CD8+ from the identical donor success- the mechanisms for long-term persistence of antigen-specifi c fully suppressed growth of ATL tumor cells in vivo. CD4+ T-cells are not well-defi ned. Here we identify a subset of Summary: Here we for the fi rst time demonstrated that hTERT memory CD4+ T-cells capable of effl uxing cellular toxins through can be a promising therapeutic target antigen for anti-ATL cellu- the multidrug effl ux protein ABCB1. Drug-effl uxing CD4+ T-cells lar immunotherapy. Furthermore, our experimental observations were CD161+CD95+CD45RA-CD127hiCD28+CD25int and had support the development of a novel hTERT-targeting redirected T remarkable phenotypic similarity to the recently described CD8+ cell-based adoptive immunotherapy for ATL patients, especially stem-like memory T-cells. CD4+CD161+ T-cells proliferated poorly those for whom suitable allo-HSCT donors are lacking.

S31 O186 induction dendritic cells with low IL-12p40 release, that drive the Heparanase rs4693608 SNP correlates with allogeneic stem generation of induced FoxP3+ T cells. We have identifi ed single cell transplantation outcomes, including the response to nucleotide polymorphisms (SNP) in the exons of the gene encod- conditioning, engraftment and the risk of graft-versus-host ing IL-7Ra, including rs1494558C/T and rs1494555A/G, and found disease that the minor alleles are associated with acute graft versus host O. Ostrovsky (1), A. Shimoni (1), P. Gidelevich (1), Y. Margulis (1), disease (aGvHD) and mortality in patients undergoing SCT. We A. Apel (1), M. Mayorov (1), A. Shteingauz (2), N. Ilan (2), I. Vlodavsky hypothesized that the generation of regulatory T cells in SCT (2), A. Nagler (1) patients is infl uenced by IL-7R genotypes of the donor. (1)Chaim Sheba Medical Center (Ramat Gan, IL); (2)Technion Materials and Methods: Blood CD4+T cells were isolated using (Haifa, IL) immunomagnetic and mRNA profi les related to regulatory functions (FoxP3, CD25 and CTLA-4) were measured by Taqman Introduction: Heparanase, endo-β-glucuronidase that specifi cally semi quantitative RT-PCR. mRNA profi les were expressed as δ-Ct cleaves the saccharide chains of heparan sulfate proteoglycans, using CD4 as a reference gene. IL-7R SNPs were determined by is involved in the process of infl ammation and release of heparan a sequence specifi c PCR system. Two study populations were sulfate-bound chemokines, cytokines and bioactive angiogenic included: 1) 155 healthy individuals and 2) 92 SCT patients. The factors that are main players in the development of graft vs. host latter were investigated one year after the transplant, and T cell disease (GVHD). Our previous study revealed a highly signifi cant phenotypes were analyzed for associations with donor IL-7R gen- correlation of HPSE gene SNPs rs4693608 and rs4364254 with the otypes. risk of GVHD. Results: In SCT, rs1494555 genotypes of the donor were signifi - Patients and methods: Analysis of heparanase expression before cantly associated with CTLA-4 mRNA levels in CD4+ T cells (AA and after conditioning was performed in 198 patients. Correlation DCt: 1.2 (-4.0;3.9) (median, ranges) (n=46), AG/GG: 2.0 (-0.8;3.6) between time to neutrophils and platelets recovery and rs4693608 p= 0.012). For FoxP3 a borderline signifi cant association was seen was analyzed in 449 recipient-donor pairs. Statistic analysis was (AA: 1.0 (0.8;1.5), AG/GG: 1.2 (-0.4;3.9), p= 0.075), while this SNP performed using the NCSS software. was unassociated with CD25 mRNA levels (p=0.21). A similar pat- Results: Pre-transplantation conditioning was found to up-regu- tern was observed for the closely linked SNP rs1494558. No sig- late HPSE gene expression (p<10-7). Expression of heparanase nifi cant associations were found in healthy donors. correlated with rs4693608 both before and after conditioning. Conclusions: These data indicate that the genotypes previously RQ (relative quantifi cation) of AA carriers before conditioning was associated with aGvHD, tend toward reduce levels of regulatory 3.7 (1.2-12), while RQ of GG possessors was 1.2 (0.4-4.1), p=0.04. T cells. That points toward a biological mechanism which may After conditioning the correlation remained signifi cant (RQ of AA contribute or explain the previously found clinical associations.In possessors was 47.5 (15.6-141.5), while RQ of GG carriers was 10.6 contrast, IL-7R genotypes are not associated with the level of reg- (2.9-39.9), p=0.02). Moreover, a positive correlation was observed ulatory T cells in healthy adults, suggesting a diff erential role of between recipient and donor rs4693608 discrepancy and the time genetic variations in IL-7R, depending on whether the lymphocyte to neutrophil and platelet recovery. The mean time to neutrophil population is in a steady state or in a regenerative phase. and platelet recovery in the N1 group (relatively high heparanase expression in patients and low in the donors) was 13 and 16 days after transplantation, respectively. In the N2 group (the same HPSE gene expression between recipients and donors) and the N3 group (relatively low heparanase expression level in the patients Graft processing and manipulation and high in the donors) it was 14 and 18 days after transplantation, respectively (p=0.003 for neutrophils and p=0.03 for platelets). The rs4693608 also aff ected HPSE gene expression in lipopolysac- O191 charide (LPS) treated mononuclear cells (MNCs) from peripheral NiCord® expanded haematopoietic progenitor cells are blood (PB) and cord blood (CB). Possessors of the AA genotype capable of outcompeting the unmanipulated cord blood unit exhibited up-regulation of heparanase with high ratio in the LPS following myeloablative dual umbilical cord blood treated MNCs, while individuals with genotype GG showed down- transplantation regulation or no eff ect on HPSE gene expression. M Horwitz (1), P Stiff (2), N. Chao (1), D. Rizzieri (1), G. Long (1), K. Conclusions: The study emphasizes the importance of rs4693608 Sullivan (1), C. Gasparetto (1), J. Chute (1), A. Morris (1), C. McDonald SNP for HPSE gene expression in activated MNCs, indicating a role (1), S. Wease (3), D. Snyder (4), H. Shoham (4), E. Galamidi Cohen (4), in allogeneic stem cell transplantation including post condition- E. Friend (4), E. Landau (4), J. Kurtzberg (1), P. Peled (4) ing, engraftment and GVHD. (1)Duke University (Durham, US); (2)Loyola University Medical Center (Maywood, US); (3)EMMES Corp. (Rockville, US); (4)Gamida Cell Ltd. (Jerusalem, IL) O187 Interleukin-7 receptor polymorphisms: impact on regulatory Factors predicting for the dominant unit following dual UCB trans- T-cells in healthy individuals and patients undergoing plantation have not been fi rmly established. Heretofore, human myeloablative allogeneic stem cell transplantation (SCT) transplantation of ex vivo expanded UCB grafts are outcompeted Z. Shamim (1+2), T. Frisch (2), L.P. Ryder (2), H. O. Madsen (2), by the unmanipulated unit. A pilot study of myeloablative dual C. Heilmann (3), N. Jacobsen (4), K. Müller (5) UCB transplantation using the NiCord ex vivo expansion technol- (1)Dept. of Paediatrics and Adolescent Medicine, 4072 and Institute ogy has completed accrual. The NiCord UCB graft consisted of an of Infl ammation Research, 7541 (Copenhagen, DK); (2)Tissue Typing expanded CD133+ and an unexpanded CD133- T-cell fraction. Laboratory, 7631 (Copenhagen, DK); (3)Paediatric Department HPCs were expanded for 21 days in media containing cytokines (Copenhagen, DK); (4)Haematologic Department (Copenhagen, DK); supplemented with nicotinamide (NAM), reported to delay dif- (5)Paediatric Department and Institute of Infl ammation Research, ferentiation and enhance homing and engraftment. All patients 7541 (Copenhagen, DK) were conditioned with TBI (1350cGy), fl udarabine 160mg/m2 ±cyclophosphamide 120mg/kg (n=2). GvHD prophylaxis con- Objective: Interleukin-7 (IL-7) is a cytokine essential for T cell sisted of tacrolimus and MMF. Eleven patients (med. age 45; range development in the thymus and maintenance of the peripheral 21-61) with high-risk malignancies received NiCord and an unma- T cell population. The IL-7 receptor (IL-7Ra) is highly expressed nipulated graft (Table). Both units were comparably HLA-matched in most naive T-cells, but is expressed at low levels in FoxP3+ with the recipient; 4/6 (n=7), 5/6 (n=3) or 6/6 (n=1) vs. 4/6 (n=8), T regulatory cells. The CD127 chain is shared with the receptor of 5/6 HLA (n=3), respectively. However, the unmanipulated unit Thymus Stromal LymphoPoietin, a cytokine with a key role in the contained a larger pre-cryopreserved cell dose (3 x 10 7/kg [range development of natural FoxP3+ T cells in the thymus and in the 1.9-3.9] vs. 2.5 x 10 7/kg [range 1.7-3.8]). After expansion (CD133+

S32 fraction), NiCord contained a median TNC and CD34+ cell dose together with 100 ml of heparinized peripheral blood upon writ- of 2.7 x 10 7/kg (1.0-6.4) and 3.5 x 10 6/kg (0.9-18.3), respectively. ten informed consent. Tumors were dissociated to a single-cell The NiCord T-cell dose was substantially smaller than the unma- suspension and cultured in order to obtain tumor cell line for nipulated graft. every patient. Dendritic cells (DCs) were generated from previ- Results: Eight patients engrafted with NiCord (one of which is ously separated PBMCs, using a positive selection of CD14+ cells, a mixed donor chimera) and two with the unmanipulated graft cultured in presence of IL-4 and rhGM-CSF. Anti-tumor CTLs were (Table). One patient experienced primary graft failure. The elicited using DCs as antigen-presenting cells, autologous apop- median time to neutrophil engraftment was 10.5 (7-18) days for totic tumor cells as source of antigens, and CD-8+ enriched eff ec- those engrafting with NiCord. Four patients experienced grade I/ tors, with weekly stimulation. To evaluate the cytotoxic activity of II acute GvHD. There were no cases of Grade III/IV acute GvHD. No CTLs, IFN-γ secretion was assessed by ELISPOT. safety concerns were raised. The estimated 100-day treatment- Results: Tumor cell lines and dendritic cells were successfully related mortality is 10%. With a median follow-up of 9 months, obtained from 19 out of 61 patients. To date ELISPOT was per- the progression-free and overall survival are both 90%. formed for 6 patients: strong IFN-γ secretion was detected at the Conclusion: NiCord expanded HPC’s are capable of out-compet- third, fourth and fi fth stimulations for one patient and at the sec- ing those from the unmanipulated unit and predominate in the ond for another patient, whereas weakly secretion was detected majority of patients. NiCord expanded HPC’s reduce the time at the second and third stimulations of three patients. T-cells from to hematopoietic recovery and are capable of long term (>22 one patient did not react to the stimulations. months) neutrophil and T-cell engraftment. Stem cell transplan- Conclusions: generation of tumor-specifi c cytotoxic T lympho- tation using NiCord is feasible, and may provide a potent cord cytes suitable for ACT immunotherapy is feasible from peripheral blood graft enabling transplantation of a single expanded unit, blood in patients with colorectal cancer. without co-infusion of unmanipulated cells. Sponsored by Umberto Veronesi Foundation and ISS/ACC.

O193 IV plerixafor combined with G-CSF is highly eff ective for stem cell mobilization in lymphoma patients undergoing autologous stem cell collection A. Cashen, M. Rettig, F. Gao, T. Reineck, C. Abboud, K. Stockerl- Goldstein, R. Vij, G. Uy, P. Westervelt, J. DiPersio Washington University School of Medicine (Saint Louis, US)

Background: Plerixafor, combined with the standard G-CSF regimen and administered as a subcutaneous (sc) injection of 0.24 mg/kg, improves autologous stem cell collection in pts. with non- Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Intrave- nous (IV) administration of plerixafor may result in a faster rise and higher peak in the peripheral CD34+ cell count, allowing administration of plerixafor the same day as pheresis and improving stem cell collection. Methods: The primary objectives of this Phase I/II study were to determine the maximum tolerated dose of IV plerixafor, up to 0.40 mg/kg combined with G-CSF, and the effi cacy of IV plerixafor + G- CSF to mobilize > 2 x 106 CD34+ cells/kg from pts. with lymphoma. Pts. started mobilization with G-CSF (10 ug/kg SC daily) for 4 days before and on each day of pheresis (up to 4 pheresis days). IV plerixafor was given over 30 min. 4 hrs. before each pheresis. O192 Results: 61 pts. (median age, 53; 37 NHL/24 HL) were enrolled. Generation of tumour-specifi c cytotoxic T-lymphocytes from In Phase I, 25 pts. were treated with IV plerixafor at escalating peripheral blood of colorectal cancer patients for adoptive doses (0.16 mg/kg, 0.24 mg/kg, 0.32 mg/kg, and 0.40 mg/kg). t-cell transfer One dose-limiting toxicity (grade 2 chest pain) was observed at S. Carluccio (1), S. Delbue (2), P. Ferrante (1), M. Bregni (3) 0.32 mg/kg. In Phase II, an additional 36 pts. were treated at 0.40 (1)University of Milan (Milan, IT); (2)Ettore Sansavini Health Science mg/kg. No grade 3/4 toxicities attributed to plerixafor occurred Foundation (Lugo, IT); (3)Ospedale di Circolo (Busto Arsizio, IT) in any pt. treated at 0.40 mg/kg. Overall, 59 of 61 pts. (97%, 80% CI 92-99%) met the goal collection of > 2.0 x 106 CD34+ cells/ Background: Colorectal cancer is the third most common cancer kg, and 47 of 61 pts. (77%, 80% CI 69-84%) collected > 5.0 x 106 worldwide (9% of all cancer incidence), and the fourth most com- CD34+ cells/kg, in a median 2 days of pheresis. 45 of 61 pts. (74%, mon cause of death in the developed Western countries. Although 80% CI 65-81%) collected > 2.0 x 106 CD34+ cells/kg in 1 day of a clear association between tumor-infi ltrating T cells and clinical pheresis. In transplanted pts., there has been no delay in engraft- outcome has been documented in colorectal carcinoma, active ment. Analysis of CD34+ hematopoietic stem and progenitor and adoptive immunotherapy do not play an important role in cells (HSPCs) revealed that plerixafor preferentially mobilized the treatment of mCRC. CD34dimCD45RA+CD123hi plasmacytoid dendritic cell precur- Adoptive T-cell transfer (ACT) using autologous tumor-infi ltrat- sors. Flow cytometric analyses showed that the CD34+ subsets ing lymphocytes (TIL), grown ex-vivo and then infused into the preferentially mobilized by plerixafor expressed high levels of cell cancer patient, has emerged as an eff ective treatment for patients surface CXCR4. with metastatic melanoma and other solid tumors, such as naso- Conclusions: Plerixafor IV, at doses up to 0.40 mg/kg, is well-toler- pharyngeal carcinoma and soft-tissue sarcoma. However, in ated and eff ective when added to G-CSF for the mobilization of vitro studies performed on bulk TIL cultures purifi ed from mCRC stem cells from pts. with lymphoma. The percentage of patients patients demonstrated contrasting preliminary results on the lytic successfully mobilized with IV plerixafor (97%) compares very activity against autologous cancer cells. favorably to the outcomes reported in Phase II and III studies that Methods: 61 patients aff ected by colorectal carcinoma were used sc dosing. Our data suggest that G-CSF and plerixafor mobi- enrolled in the study. Tumor biopsies were obtained at surgery, lize distinct subsets of human CD34+ HSPCs.

S33 O194 laboratory variables have been reported so far. Eurocord and Net- Characterization of haploidentical stem cell grafts after cord have launched a retrospective analysis aimed at assessing negative depletion of B-cells and α/β+ T cells whether the major variables associated with the banking process G. Li Pira, F. Landi, P. Filippini, A. Bertaina, S. Ceccarelli, G. Del Principe, had an impact on transplantation outcome. D. Pagliara, K. Girardi, M. Romano, R. Pinto, F. Locatelli, S. Rutella Methods: With the aim of analyzing the banking procedures, we IRCCS Bambino Gesù Children’s Hospital (Rome, IT) selected an homogenous population of patients who 1: received a single CBT with a TNC > 3.0x10e7/kg at freezing, 2: had Acute Introduction: Allogeneic hematopoietic stem cell transplantation Leukemia in remission, 3: were transplanted in EBMT Centers (HSCT) from a HLA-haploidentical relative is a suitable option after 1997, 4: had availability of clinical follow-up, and 5: bank for patients lacking a compatible donor. We have developed a could be identifi ed. A specifi c questionnaire was circulated to the novel method of ex vivo T/B-cell depletion based on the selec- banks, aimed to assess the variables associated to banking of the tive elimination of α/β+ T cells through labeling with a biotinyl- selected cord blood units (CBU), from processing to shipment. In ated anti-TCR-α/β Ab, followed by incubation with anti-biotin and particular, CBU volume reduction (VR) technologies (analyzed as anti-CD19 Ab conjugated to paramagnetic beads (Miltenyi Biotec, below or above 30mL after processing) and cells viability assess- Germany). Here, we report the results of graft manipulation using ment in thawed sample were investigated. Questionnaires were this methodology. answered by 38/48 Banks worldwide and 677 patients met the Methods: Twenty-two children entered the study, 16 with hema- eligibility criteria. tological malignancies and 6 with non-malignant disorders. Cell Results: Table 1 reports patient, disease and transplant characteris- therapy products contained up to 60x109 white blood cells (WBC). tics. VR was performed in 399 out of 677 patients and its extension Graft aliquots were used to enumerate residual α/β+ T cells and (≤/>30 mL) did not infl uence any clinical endpoints. Therefore, all B cells, as well as other immune eff ector cells (type 1 and 2 DC multivariate analyses were performed including VR (yes/no). Tech- precursors, NK cells, invariant NKT cells, classical CD14+CD16- and nologies for viability assessment on a thawed sample were diff er- non-classical CD14+CD16+ monocytes). ent across the banks (32% Trypan-Blue, 33% Acridine Orange, 34% Results: Median recovery of CD34+ HSC and median number of 7-ADD) and were not analyzed for outcomes. Neutrophil recovery infused CD34+ HSC were 99.3% (range 55.4-100) and 14.7x106/ was 87% for patients given either a VR-CBU or an unmanipulated kg (range 7.9-37), respectively. The graft contained a median CB. NRM at 100 days and at two years were 14% and 29% for of 9.9x106 CD3+ T cells/kg (range 3.9-16) and 0.08x106 B cells/ patients given a VR CBU, and 19% and 31% for those without VR, kg (range 0.002-0.32). The log-depletion of α/β+ T cells was 4.1 respectively (p=ns). VR was not associated with any outcome dif- (range 3.33-4.96); the median number of transplanted α/β+ T cells ferences in a multivariate analysis which included CB age, year of was 42x103/kg (range 3-100.9). Patients received 28.5x106 CD56+ CB collection, year of Tx, patient age, diagnosis, status at Tx, Nr of NK cells/kg (range 13.6-192.0) and 9.1x106 γ/δ+ T cells/kg (range mismatches, cells dose, CMV status. 3.7-106.0). The grafts were also enriched in DC1 and DC2 (0.11% Conclusions: In this Registry study, manipulation of the CBUs of BDCA-1+ cells, range 0.02-0.43; 0.61% of BDCA-3+ cells, range aimed at volume reduction was not shown to infl uence the clini- 0.02-1.17); 0.66% of BDCA-2+ cells, range 0.25-1.28, and 0.28% cal outcome, indicating a satisfactory validation of the associated of BDCA-4+ cells, range 0.02-1.16), as well as in non-classical technologies across the banks. Cells viability assessment meth- CD14+CD16+ monocytes (10.0%, range 4.7-17.5), which may pre- odology varied among banks. Further eff orts to standardize the dict a reduced incidence of GVHD after allogeneic HSCT. All pts quality controls before CBU release are needed. rapidly engrafted, with the median time to reach 500 neutrophils and 50,000 platelets per μl of blood being 12 days (range 10-16) and 13 days (range 12-18), respectively. Only 2 pts developed skin grade I/II acute GVHD, while no pt had visceral acute GVHD. With a median follow-up of 6 months (range 2-8), no pt died of transplant-related complications. Conclusions: The results of immunomagnetic removal of α/β+ T cells and B cells were robust and reproducible. The grafts were enriched in CD34+ HSC, as well as in immune eff ector cells implicated leukemia and GVHD control, such as γ/δ+ T cells, NK cells, DC1, DC2 and non-classical monocytes.

O195 Eff ect of volume reduction of cord blood units before storage on transplantation outcomes: a retrospective analysis of Eurocord-EBMT and Netcord R. Saccardi (1), L. Tucunduva (2), A. Ruggeri (2), I. Ionescu (3), G. Koegler (4), S. Querol (5), L. Lecchi (6), F. Pouthier (7), H. Bittencourt (2), C. Kenzey (2), E. Gluckman (2), M. Labopin (8), E. Baudoux (9), V. Rocha (2) (1)Careggi University Hospital (Florence, IT); (2)Eurocord (Paris, FR); (3)Agence de la Biomédecine-Eurocord registry (Paris, FR); (4)Jose Carreras Cord Blood Bank, Medical Center, University of Düsseldorf (Düsseldorf, DE); (5)Barcelona Cord Blood Bank (Barcelona, ES); (6)Milan Cord Blood Bank (Milan, IT); (7)Besançon Cord Blood Bank of the Establisement Français du Sang (Besançon, FR); (8)Hospital Saint Antoine (Paris, FR); (9)Liège Cord Blood Bank, University of Liège (Liège, BE)

Background: Cord blood banking process is subjected to a wide range of procedures variability, despite international standards aimed at standardizing the process. Only single bank analyses of

S34 Working Party Session Chronic Patients and Methods: From 2001 to 2005 the study enrolled 357 myeloma patients up to the age of 69. Patients with an HLA-iden- Malignancies tical sibling were allocated to autoRICallo (n=108) and patients without a matched sibling donor to auto (n=249). Single (n=145) or tandem (n=104) auto were optional. Conditioning for auto 196 was melphalan 200 mg/m2, and for RICallo total body irradiation Second allogeneic haematopoietic stem cell transplantation 2 Gy plus fl udarabine 30 mg/m2/day x 3. Median follow-up time for relapse of malignant disease: retrospective analysis of was 96 months as compared to previously reported 61 months. outcome and predictive factors Primary endpoint was progression-free survival (PFS). Secondary T. Ruutu, L. de Wreede, A. van Biezen, R. Brand, M. Mohty, P. Dreger, endpoints were overall survival (OS), non-relapse mortality (NRM) C. Peters, L. Garderet, S. Schönland, A. Gratwohl, D. Niederwieser, and relapse/progression rate (RL). T. de Witte, N. Kröger on behalf of the European Group for Blood and Results: PFS was 22% at 96 months with autoRICallo, which was Marrow Transplantation (EBMT) signifi cantly better than 12% with auto (p=0.027). OS at this time was 49% with autoRICallo as compared to 36% with auto A signifi cant proportion of patients treated with allogeneic stem (p=0.030). The relapse/progression rate at 96 months was 60% cell transplantation (SCT) for a malignant disease suff er from a with auto/RICallo as compared to 82% with auto (p=0.0001). relapse after the transplantation. The best treatment approach in Non-relapse mortality at 36 months was 13% after autoRICallo this situation, including the role of SCT, is often uncertain. In a compared to 3% with auto (p=0.0004). In patients with the del13 retrospective analysis, all second allogeneic SCTs carried out for abnormality PFS with RICallo was 21% at 96 months as compared a relapse of malignant disease after the fi rst transplantation at to 5% with auto (p=0.026), and OS was 47% with auto/RICallo ver- the EBMT centres between 1994 and 2009 and reported to the sus 31% with auto at this time (p= 0.154). Survival from progres- EBMT registry (n =2632) were analysed for outcome and predic- sion was signifi cantly better with auto/RICalla, i.e. 48% versus 26% tive factors. The age of the patients was 1- 73 (median 36) years. at 60 months from progression (p= 0.019). The diagnoses were primary AML 948, ALL 590, MDS/CMML/sec- Conclusions: Long-term outcome in patients with multiple ondary leukaemia 406, CML 303, lymphoma 123, myeloma 80, myeloma was better with autoRICallo as compared to auto or and other 182. Thirty-one per cent of the patients had low disease auto/auto only. There was a tendency to overcome the poor burden, 52% had an advanced disease. In the second transplan- prognostic impact of the del13 abnormality seen with auto alone. tation 58% of the donors were HLA-identical siblings, 10% other Follow up longer than 5 years is necessary for correct interpreta- related and 31% unrelated. In 73% of the transplantations the tion of the value of auto/RICallo in multiple myeloma. donor was the same as in the fi rst transplantation. The conditioning was myeloablative in 52% and of reduced intensity in 39% of the transplantations (data missing 9%). The graft was PB stem 198 cells in 79%, BM in 20 %, and cord blood in 1%. All outcome data Use of second generation tyrosine kinase inhibitors prior are calculated from the second SCT. At 5 years, the overall survival to allogeneic stem cell transplantation in chronic myeloid was 20%, the cumulative incidence of relapse 45%, and that of leukaemia non-relapse death 40%. Fifteen per cent of the patients remained E. Olavarria, M. Schleuning, Y. Chalandon, F. Onida, A. Radujkovic, relapse-free until 5 years. Patients with CML had a clearly better M. Robin, N. Kröger on behalf of the European Group for Blood and survival than patients with other diseases. In multivariate analy- Marrow Transplantation (EBMT) sis, factors signifi cantly associated with better survival were low disease burden, longer remission duration after fi rst transplanta- Allogeneic stem cell transplantation (SCT) remains a treatment tion, longer interval between fi rst and second transplantation (> option for patients with chronic myeloid leukaemia (CML) who fail 1 vs. < 1 year), younger age, absence of grade II-IV acute GvHD tyrosine kinase inhibitors (TKI). However, nowadays most patients or chronic GvHD after the fi rst transplantation, and later year of referred for transplantation would have received prior therapy not transplantation. The EBMT risk score at the second SCT predicted only with Imatinib but with a second generation TKI such as Nilo- highly signifi cantly the outcome. Using the same donor as in the tinib or Dasatinib before allogeneic SCT. While prior exposure to fi rst transplantation vs. another donor had no predictive value Imatinib before SCT seems to have no adverse impact on the out- for survival. Sibling donor was a favourable predictive factor. The come of allogeneic SCT, there is no reliable information regarding intensity of conditioning in the second transplantation had no the impact of prior use of second generation TKI. signifi cant impact on the survival. The source of the graft (BM vs. We have undertaken a retrospective study looking a the use of PB) did not aff ect the outcome. In conclusion, second allogeneic second generation TKI prior to allogeneic SCT and its potential transplantation off ers a reasonable option especially for younger impact on the usual outcomes post SCT such as relapse incidence patients with a time interval of > 1 year from the fi rst transplanta- (RI), non-relapse mortality (NRM), overall (OS) and relapse-free tion and a low disease burden. survival (RFS). A total of 56 patients were studied. 36 were male (64%) and 20 female. The median age was 44 years (21-66), while 21 patients (37%) were older than 50 years. The median interval 197 between diagnosis and SCT (Dx-SCT) was 24 months (2-144), with Tandem autologous/reduced-intensity allogeneic stem 18 patients (32%) having a Dx-SCT of <12months and 21 patients cell transplantation versus autologous transplantation in (37%) of >36 months. multiple myeloma - update of the EBMT-NMAM2000 study at At the time of transplantation 15 patients (27%) were in fi rst 96 months median follow-up chronic phase (CP1), 20 patients (36%) in second or third CP G. Gahrton, S. Iacobelli, B. Björkstrand, U. Hegenbart, A. Gruber, (CP>1), 10 patients (18%) in accelerated phase (AP) and 11 H. Greinix, L. Volin, F. Narni, A. Carella, M. Beksac, A. Bosi, G. Milone, patients (20%) in blast crisis (BC). All patients received Imatinib as P. Corradini, S. Schönland, K. Friberg, A. van Biezen, H. Goldschmidt, fi rst line therapy, while 31 patients (55%) received Dasatinib and T. de Witte, C. Morris, D. Niederwieser, L. Garderet, N. Kröger for the 11 patients (20%) received Nilotinib as second line therapy prior EBMT CMWP Multiple Myeloma Subcommittee to allogeneic SCT. 14 patients (25%) received both drugs sequen- tially. The best response to the second line TKI (either Dasatinib Objectives: Long-term follow up of prospective studies compar- or Nilotinib) was major molecular remission (MMR) in 6 patients ing allogeneic transplantation to autologous transplantation in (11%), major cytogenetic (partial + complete) remission (MCR) in multiple myeloma are few and controversial. This is an update at 14 patients (25%), complete haematological remission only (CHR) 96 months median follow-up of the EBMT NMAM2000 myeloma in 14 patients (25%) and there was no detectable response (NR) in study comparing tandem autologous/reduced intensity condi- 19 patients (34%). tioning allogeneic transplantation (auto/RICallo) to autologous SCT donors were HLA-identical siblings in 17 cases (30%) and unre- transplantation - single(auto) or tandem(auto/auto). lated donors (URD) in 36 cases (64%). There were 3 mismatched

S35 family members and 9/36 URD were also HLA mismatched. In 8 Patients & Methods: Major eligibility criteria were 17p- CLL in patients there was an unfavourable gender mismatch (female remission, age below 70 years, availability of an HLA-compatible donor into male patient). The source of the stem cells was umbili- related or unrelated donor with up to one HLA-mismatch. The cal cord blood in 2 of the URD while it was bone marrow in 8 cases primary objective is to determine early progression-free survival and peripheral blood in the remaining 46 cases (82%). at one year after HCT. Here, we present fi rst results of this non- The median overall survival for the entire cohort was 11 months interventional study. (1-102). The estimated overall survival was 64% at 12 months Results: Forty-one patients from ten transplant centres have been and 34% at 3 years. Relapse occurred in 12 patients (cumulative recruited into this study by September 17, 2012. Clinical data on 35 incidence 15% at 12 months and 23% at 3 years) with a median patients are evaluable (9 females and 26 males). The median age time to relapse of 8 months (1-87). Overall 21 patients (37%) have was 59 years (range, 27 to 70 years). Twelve patients were trans- died following allogeneic SCT with an estimated NRM of 33% at planted in fi rst remission. The median number of pre-treatments 12 months and 41% at 3 years. Causes of death included relapse/ was 2 (range, 1 to 6) and included alemtuzumab in 28 patients. disease progression in 8 patients (29%), GVHD in 5 patients (18%), The remission status at HCT was assessed as partial remission in infections in 3 patients (11%), acute renal failure in 2 patients, 25 patients and as complete remission in 9 patients. The median multi-organ failure in 2 patients and VOD in 1 case. In total 28 time between diagnosis and HCT was 35 months (range, 4 to 209 patients (50%) remain alive and relapse-free with an estimated months) and the median time between fi rst treatment of CLL and RFS of 51% at 12 months and 37% at 3 years. Acute GVHD grade HCT was 10 months (range, 2 to 180 months). II-IV developed in 29 patients (52%) while chronic GVHD was diag- Myeloablative conditioning was administered in two patients, nosed in 24 patients (43%) of which 13 cases were limited and fludarabine-based reduced-intensity conditioning in 22 11 cases extensive cGVHD. patients and non-myeloablative conditioning based on 2 Gray There were no signifi cant diff erences between patients receiving TBI in eleven patients. Donors were HLA-identical siblings in Dasatinib, Nilotinib or both with regards to RI, NRM, OS and RFS. 9 patients and HLA-compatible unrelated volunteers in 26 However, patients treated with Nilotinib had an excellent 88% patients. Ten patients received ATG. Twelve patients experi- overall survival. As expected, patients in CP1 had the best OS (87% enced grade II to IV graft-versus host disease. By December 15, at 12 months). However, patients in CP>1 and AP enjoyed an OS 2012 twenty-seven patients were alive and eight patients have of 75% and 68% respectively. RFS at 12 months was 58%, 64%, died. Cause of death was relapse in three patients, graft-versus 46% and 22% for patients in CP1, CP>1, AP and BC respectively. host disease in four patients and infection in one patient. The Although only one of the 10 patients who achieved CCR or MMR median observation time for living patients was 13 months relapsed, there was no correlation between best response to TKI (range, 3 to 23 months). therapy and allogeneic SCT outcomes. Conclusions: In the light of advanced disease status and mostly In conclusion, patients with CML who fail Imatinib and subse- elderly patients we present favourable early results for this high- quently receive a second generation TKI that undergo allogeneic risk group of patients with 17p- CLL. Longer follow-up is needed. SCT have a reasonable outcome especially if the transplant is In order to determine the outcome in subgroups defi ned by remis- performed in chronic phase. There seems that the use of second sion, age and donor type recruitment into an extension study is generation TKI prior to allogeneic SCT has no impact on trans- ongoing. Interested centres are invited to contact the EBMT Data plant outcomes. Therefore, for CML patients in blastic transfor- Offi ce in Leiden ([email protected]). mation after Imatinib in whom an allogeneic is indicated, the use of a second generation TKI in order to achieve a second chronic phase seems a reasonable approach. Once a second CP has been 200 obtained, there seems to be no advantage in achieving a deeper Impact of graft-versus-host disease on relapse after alloge- cytogenetic or molecular remission. The CMWP of EBMT is cur- neic stem cell transplantation: an EBMT megafi le study rently doing a prospective non-interventional study of the use of M. Stern (1), L. de Wreede (2), R. Brand (2), A. van Biezen (2), P. Dreger second generation TKI prior to allogeneic SCT in CML patients and (3), M. Mohty (4), T. de Witte (5), N. Kröger (6), T. Ruutu (7) since 2010 near 300 patients have been entered. A recruitment (1)University Hospital Basel (Basel, CH); (2)Leiden University Medical update will be presented. Center (Leiden, NL); (3)University of Heidelberg (Heidelberg, DE); (4)CHU (Nantes, FR); (5)Radboud University Nijmegen Medical Center (Nijmegen, NL); (6)University Medical Center Hamburg Eppendorf 199 (Hamburg, DE); (7)Helsinki University Central Hospital (Helsinki, FI) Allogeneic HSCT in patients with 17p- CLL: fi rst results of a non-interventional prospective study Background: After allogeneic HSCT, graft-versus-host disease J. Schetelig (1), J. Hoek (2), S. Stilgenbauer (3), M. Bornhäuser (1), (GvHD) occurs through recognition of minor or major histocom- N. Andersen (4), C. Fox (5), S. Lenhoff (6), D. Bunjes (3), L. Volin (7), patibility mismatches by donor derived T lymphocytes. The same A. Shimoni (8), W. Schroyens (9), M. v. Gelder (10), A. van Biezen (2), mechanism also operates in the elimination of residual malig- L. de Wreede (11), T. De Witte (12), N. Kröger (13), P. Dreger (14) nant cells (the graft-versus-tumor or GvT eff ect). Earlier studies (1)University Hospital (Dresden, DE); (2)EBMT Data Offi ce (Leiden, have already shown reduced relapse risks for patients develop- NL); (3)Ulm University (Ulm, DE); (4)Rigshospitalitet (Copenhagen, ing GvHD (Weiden et al, NEJM 1979; Horowitz et al, Blood 1990). DK); (5)Nottingham City Hospital (Nottingham, GB); (6)University In particular, a large study in CML patients (Gratwohl et al, Blood Hospital (Lund, SE); (7)Helsinki University Central Hospital (Helsinki, 2002) showed that increasing grades of acute and chronic GvHD FI); (8)Chaim Sheba Medical Center (Tel-Hashomer, IL); (9)Antwerp are associated with a proportional decrease in relapse risk. Inci- University Hospital (Antwerp, BE); (10)University Medical Center dence and severity of acute and chronic GvHD might therefore be (Maastricht, NL); (11)Leiden University Medical Center (Leiden, NL); used as surrogate markers for GvT eff ects. (12)Radboud University Nijmegen Medical Center (Nijmegen, NL); Transplant procedures have changed signifi cantly since these (13)University Hamburg Eppendorf (Hamburg, DE); (14)University publications. Increased use of unrelated donors, peripheral blood Hospital (Heidelberg, DE) as stem cell source, and the introduction of reduced intensity conditioning regimens might aff ect the relationship between GvHD Introduction: 17p-/TP53-mutated CLL (17p- CLL) represents and GvT. Furthermore, previous studies have only analyzed trans- approximately 5 to 15% of newly diagnosed CLL. According to plants for AML, ALL and CML, the prevailing transplant indications current guidelines allogeneic hematopoietic cell transplantation at the time. Today, many patients receive transplants for MDS, (HCT) is recommended in 17p- CLL as part of the fi rst- or sec- plasma cell disorders (PCD) or lymphoma. We hypothesized that ond-line treatment. We performed a non-interventional study in comparing the eff ect of GvHD on relapse incidence might provide order to evaluate the outcome of allogeneic HCT in early stage a useful surrogate marker for the susceptibility of diff erent dis- 17p-CLL. eases to allo-immune eff ects.

S36 Methods: We studied 48,111 fi rst allogeneic transplants carried Discussion: These data confi rm earlier observations of a potent out and reported to EBMT between 1998 and 2007. The impact of GvT eff ect associated with GvHD. While GvHD and GvT are signifi - GvHD on relapse risk was assessed by including acute and chronic cantly associated in all diseases, the strength of this association GvHD as time-dependent covariates in Cox models for cause-spe- strongly diff ers between disease entities (strongest correlation in cifi c hazards adjusted for patient age, year of transplant, donor CML, ALL and MPN; weakest correlation in AML and PCD). A poor type, stem cell source, and type of conditioning regimen. correlation might point to either insensitivity of a particular dis- Results: Diseases were CML (N=7,711), AML (14,539), ALL (6,802), ease to GvT eff ects, to GvT eff ects operating in the absence of and MDS (5,447), Philadelphia-negative MPN (N=1’511), lymphopro- independent from GvHD, or to a signifi cant fraction of patients liferative disease (LPD, N=8,231), or plasma cell disorders (PCD, already cured before allogeneic SCT. N=3,870). Donors were HLA identical family donor (N=28,030), and HLA-identical unrelated donors (N=14,422) or mismatched donors (N=5,659). Stem cell source was bone marrow (N=13,273), peripheral blood (N=34,022), or cord blood (N=816). Condition- ing intensity was myeloablative (N=28,843), reduced intensity (15,889) or unknown (N=3,379). Incidence of grade I-IV acute GvHD was 49%, that of grade II-IV acute GvHD 30%. Limited chronic GvHD was diagnosed in 17% and extensive chronic GvHD in 20% of patients. Incidence of disease relapse was 22%, 28%, and 31% at 1, 2, and 4 years respectively. As shown previously, development of GvHD was associated with a reduced risk of relapse in our data. In CML, a clear reduction of relapse risk occurred with hazard ratios declining proportionally to severity of both acute and chronic GvHD (Figure 1). The protective eff ect of severe acute (grade III-IV) GvHD was similar to that of extensive chronic GvHD, whereas the protective eff ect of mild acute (grade I-II) GvHD was comparable to that of limited chronic GvHD. ALL and Philadelphia-negative MPN were almost equally sensitive to GvHD as CML, whereas MDS and LPD showed intermediate sensitivity (Figure 1). Acute and limited chronic GvHD were only associated with modest reductions in relapse risk in AML and PCD. The limited sensitivity of PCD to allo-immune eff ects was also evident in Kaplan-Meier curves of disease-free survival where - in contrast to other diseases - no plateau developed during follow-up (Figure 2, lower panel). Similarly, hazard rates of disease relapse failed to drop to values near zero in patients with PCD (Figure 2, upper panel). Interestingly, despite a comparatively poor association of GvHD and relapse in AML patients, a plateau in the survival curve occurred and hazard rates dropped in parallel to other diseases, suggesting that curative GvT eff ects operating independently of GvHD might occur in this disease.

S37 201 autologous HSCT and no patient developed HC. Of 313 patients Reduced versus standard conditioning in MDS/sAML: undergoing an allogeneic HSCT (HLA sib. n=140, MUD n=71, preliminary results of a prospective, randomized Phase III UCB n=28, Haplo n=74) 45 (14%) developed HC, which was of trial of Chronic Malignancies Working Party of the EBMT grade ≥II in 34 patients ( grade: II n=10, III n=21, IV n=3). All (RICMAC-Trial) these patients refractory to conventional therapy for HC were N. Kröger, R. Brand, D. Niederwieser, U. Platzbecker, K. Hubel, treated with FG. During cystoscopy bladder distension was T. Weber, M. Stelljes, M. Robin, B. Afanasiev, D. Heim, F. Onida, maintained at a constant pressure of 12 mmHg by a carbon P. Dreger, P. Massimo, S. Guidi, L. Volin, M. Gramatzki, W. Bethge, dioxide insufflator and FG was diffusely sprayed on bleeding X. Poire, G. Kobbe, R. Uddin, M. van Os, T. de Witte on behalf of the and raw surfaces of bladder mucosa by an endoscopic applica- Chronic Malignancies Working Party of the EBMT (RICMAC-Trial) tor. The response was evaluated at 10, 30 and 60 days from first FG application. Retrospective studies in MDS/sAML suggest that reducing the Results: The number of FG application was 1 in 21 patients, 2 in 10 intensity of the conditioning regimen prior to allogeneic stem cell and 3 in 3 with a median FG volume of 10.8 ml (range, 6.3-16). The transplantation reduces the risk of non-relapse mortality but is pelvic pain disappeared within the fi rst 24 hours from FG applica- associated with a higher risk of relapse, but prospective random- tion in all patients and the complete remission, defi ned as regres- ized studies for MDS are lacking so far. sion of all symptoms and absence of haematuria, evaluated at 10, Within the CMWP of EBMT we performed a prospective random- 30 and 60 days was achieved in 18%, 61% and 83% of patients, ized trial comparing a reduced intensity conditioning regimen respectively. The response was independent from platelets recov- (Busulfan 8 mg/kg and Fludarabine 150 mg/m2) and a standard ery and BK viruria and its treatment. myeloablative regimen (Busulfan 16 mg/kg and Cyclophospha- Conclusions: FG therapy is an eff ective, feasible, and reproducible mide 120 mg/kg) in patients with MDS or sAML (< 20% basts). procedure to treat grade ≥ II refractory HC. Between May 2004 and December 2012 129 patients were included from 18 centers and 7 nations. Accrual was closed in December 2012. Major inclusion criteria were: MDS (according 257 to FAB: RA, RARS, RAEB, RAEB-t), CMML and sAML, blasts less ECIL guidelines for bacterial resistance in the haematology than 20%, matched related or unrelated donor (1 mismatch was ward allowed), age between 18 and 60 (for unrelated) and 18 - 65 (for C. Cordonnier, D. Averbuch, M. Mikulska, C. Orasch, D. Engelhard, HLA-identical sibling. The primary study aim was therapy related C. Viscoli, I.C. Gyssens, W. Kern, G. Klyasova, O. Marchetti, mortality at 1 year. The study hypothesis was that RIC transplan- D.M. Livermore, M. Akova for the ECIL Group tation reduces the 1 year TRM to 20% from 40% in the standard conditioning regimen. The median age of the patient was 51.4 Bacterial resistance is a growing problem in many hematology years. The patients were well distributed in both arms regarding wards in Europe. Moreover, hematology patients receive multiples age, gender, IPSS risk profi le, number of blasts at transplantation, courses of antibacterials and have a high risk of collateral damage, related vs. unrelated donor, mismatched donor. arising via selection of multiresistant bacteria, or superinfection The preliminary analysis is based on all patients and the incidence by C. diffi cile and fungi. This may greatly impact on the survival curves on NRM, relapse incidence, disease free and overall sur- of SCT recipients. vival will be presented. The ECIL 4 meeting, held in September 2011, gathered experts Since the fi nal analysis of this trial requires all patients to have from the EBMT, the EORTC, the ELN, the ICHS and the ESCMID, to a minimal follow up of 1 year in order to compare the primary address this issue. The ECIL group reviewed the bacterial epide- endpoint NRM between both arms at 1 year, the current prelimi- miology at European hematology centers from published data nary analysis will focus on a description of estimates based on the (2005-2011) and from a questionnaire sent to 123 ECIL partici- currently available follow-up information. pants in July 2011. The questionnaire found a reduction in the Gram-positive:Gram-negative ratio compared with published data, and lower resistance rates for all bacteria, including P. aeruginosa. Median rates of ESBL producers, aminoglycoside-resistant bacteria, and carbapenem-resistant P. aeruginosa, were in the ranges 15-24%, 5-14%, and 5-14%, respectively, with higher rates Working Party Session Infectious in South/East Europe than North/West. Diseases Because adequate antibacterials are critical to the outcome of infection in immunocompromised patients, ECIL proposed two strategies in febrile neutropenia: (1) an ‘escalation’ approach, 256 avoiding empirical carbapenems and combinations, for patients Fibrin glue for treatment of severe haemorrhagic cystitis without particular risk factors; (2) a ‘de-escalation’ approach, with following allogeneic haematopoietic stem cell transplantation initial broad-spectrum antibiotics or combinations for patients M.C. Tirindelli, G. Flammia, F. Sergi, R. Cerretti, L. Cudillo, A. Picardi, with known prior colonization or infection with resistant patho- G. De Angelis, P. Bove, MG. Cefalo, E. Cerchiara, L. Altomare, G. Allori, gens, complicated presentation at onset of febrile neutropenia, A. Lanti, G. Avvisati, W. Arcese on behalf of the Rome Transplant or in centers with high rates of resistance. In all cases the anti- Network bacterial regimen should be re-assessed and narrowed if possible at 48-72h, based on laboratory results and the patient’s clinical Background: Patients undergoing hematopoietic stem cell trans- condition. Discontinuation of antibiotics from 72h should be plant (HSCT) are particularly exposed to the risk of developing considered in patients with fever of unknown origin who have haemorrhagic cystitis (HC), which is characterized by symptoms been hemodynamically stable since presentation and afebrile for ranging from macroscopic haematuria to renal failure. HC signifi - >48h. cantly aff ects quality of life and in some cases becomes intracta- As well as de-escalation, the principles of antimicrobial stew- ble leading to patient death. Its therapeutic management has not ardship should be implemented in all hematology wards, with been established. In this prospective study, we used Fibrin Glue equal priority to good infection control. These include (a) local (FG), an haemostatic agent derived from human plasma, to treat surveillance of antibiotic resistance, antibiotic consumption 34 patients with refractory post-transplant HC. and patient outcomes; (b) development and regular updat- Materials and methods: Between January 2006 and October ing of infection-management algorithms (c) swift report- 2012, 1116 (249 children and 867 adults) underwent an HSCT at ing of microbiological results and (d) optimization of dosing the Rome Transplant Network. Among adults, 554 received an regimens.

S38 Haemopoietic stem cell and the niche tested for their ability to inhibit ConA-activated splenocytes and also modulate macrophage activity in vitro and in vivo . Results: We observed that the ability to suppress T-cell proliferation in vitro was impaired in Dicer-/- MSC. Furthermore, Dicer-/- O284 MSC could not mediate the switch of activated macrophages into Candidate primary stroma stem/progenitor cells in normal a regulatory-like profi le in vitro. We also assessed the impact of human bone marrow are Lin-/CD45-/CD271+ and express the Dicer1 deletion on the ability of MSC to modulate the course low/negative levels of PDGFR-α of acute infl ammation in a mouse model of thioglycollate-induced H. Li (1), R. Ghazanfari (1), N. Ditzel (2), M. Kassem (2), S. Scheding (1) peritonitis. Although our results show that MSC administration (1)Stem Cell Center (Lund, SE); (2)University of Southern Denmark leads to a marked reduction of the infl ammatory response in vivo, (Odense, DK) once depleted from mature miRNAs, Dicer-/- MSC did not reduce the total number of cells mobilized into the peritoneal cavity, and Objectives: Human bone marrow (BM) contains a rare popula- the phenotype of the peritoneal cells was more similar to the phe- tion of non-hematopoietic mesenchymal stem cells (MSC), which notype of untreated mice, with higher number of infl ammatory play an important role in supporting, maintaining and controlling macrophages, instead of a reduced number of immature mono- hematopoiesis. We and others have shown that primary BM-MSC cytes. Interestingly, the administration of Dicer-/- MSC led to an were highly enriched in lin-/CD45-/CD271+ cells as refl ected by increase accumulation of neutrophils. high CFU-F frequencies (ca. 1 in 20). However, even this enriched Conclusions: Our results suggest that miRNAs have a strong population is likely to contain a considerable fraction of non-CFUs. impact on the immunomodulatory activity of MSC. Additional Therefore, this study aimed to identify novel MSC markers to more studies are warranted to dissect the eff ect of specifi c miRNAs on precisely defi ne the candidate stromal stem cell population. the regulatory function of MSC. Methods: Human lin-/CD45- BM cells were sorted based on CD271 expression and comparative gene expression profi ling was performed. Protein expression of candidate surface marker genes was O286 validated by fl ow cytometry. Candidate primary MSC populations Bone marrow mesenchymal stem cell characterization in as defi ned by the novel markers were FACS sorted, and assayed for severe multiple sclerosis patients treated with autologous MSC properties in vitro and in vivo at single cell and bulk level. haematopoietic stem cell transplantation Results: Twenty eight of the 215 genes that were up-regulated B. Mazzanti (1), A. Aldinucci (2), E. Bonechi (2), M. Boieri (1), A. Galante in the lin-/CD45-/CD271+ subset compared to CD271- cells cor- (2), A. Repice (2), M. Di Gioia (1), V. Carrai (1), I. Donnini (1), R. Saccardi related to surface markers. Several of the markers identifi ed by (1), C. Ballerini (2) this approach had been described previously (e.g., CD140b and (1)Careggi University Hospital (Florence, IT); (2)University of Florence CD106). However, the majority represented novel MSC markers, (Florence, IT) including molecules such as CD151, CD81, and CD140a. FACS analysis of these markers on lin-/CD45-/CD271+ cells revealed Introduction: Autologous hematopoietic stem cells transplanta- two staining patterns, i.e. marker expression either correlated tion (AHSCT) has been considered as a new novel therapy for the directly with CD271 expression and did thus not enable to fur- treatment of multiple sclerosis (MS) patients who are resistant to ther enrich for CFU-F (e.g. CD151), or the novel maker was only conventional treatments, but so far no data are available on bone expressed on a fraction of the lin-/CD45-/CD271+ cells. The latter marrow (BM) of MS patients before and after AHSCT. was the case for CD140a (PDGFR α), allowing to clearly identify a We therefore evaluated the BM microenvironment before and population of lin-/CD45-/CD271+/CD140a low/- cells which were after transplantation focusing on the characteristics of mesenchy- very highly enriched for CFU-F (24.15 ± 4.51 CFU-Fs per 100 plated mal stem cells (MSCs) isolated from MS patients. In MS patients cells, n=6). Primary CD140a low/neg MSC showed considerably we showed that IP10 production upon LPS stimulation is signifi - higher expression of ALPL, PPARg, and ACAN as well as Oct4, Sox2 cantly increased comparing with normal donors (HD) and we gain and Nanog compared to CD140a+ cells, and lin-/CD45-/CD271+/ insights on this observation investigating cell signalling pathways CD140a low/- cells co-expressed typical primary MSC markers and involved in IP10 production and in the modulation of MSCs prop- gave rise to typical cultured MSC (expression of standard surface erties. In the present work we tested AHSCT eff ects on MS MSCs markers, in-vitro diff erentiation capacity). Moreover, lin-/CD45- production of IP-10 and related signaling cascade. /CD271+/CD140a low/- derived stromal cells formed bone, adi- Material and methods: MSC isolated from 6 MS patients before pocytes and hematopoietic stroma when transplanted s.c. into and 4.5 (range 3-6) years after AHSCT were compared in terms of NOD-SCID mice. colony forming effi ciency, growth capacity, diff erentiation poten- Conclusion: Lin-/CD45-/CD271+/CD140a low/- BM cells represent tial, immunophenotype, immunomodulation on T cell proliferative a close to pure population of the candidate human primary mes- response, toll like receptor (TLR) expression, cytokine production enchymal stem/progenitors. and cell signalling pathways. Results: Morphological analysis of BM biopsies revealed a normal microenvironment in BM of MS patients both before and after O285 transplantation. In addition, no diff erences were found in colony microRNA regulates mesenchymal stromal cell-mediated forming effi ciency, as well as in growth potential. Immunopheno- anti-infl ammatory activity in vitro and in vivo types of MSC pre and post AHSCT are comparable and also is dif- M.B. Coelho, L.M. Lopes, B. Graham, M. Merkenschlager, F. Dazzi ferentiation potential. Imperial College London (London, GB) MS MSCs show signifi cant (p<0.003) increased production of IP10, a T cell recall chemokine, and increased phosphorylation of p38, Objectives: Mesenchymal stromal cells (MSC) exert an immuno- CREB, JNK, and STAT1. All tests were performed after TLR4 stimula- suppressive eff ect on the immune system and can switch acti- tion. Interestingly, the basal level of activation for all these factors vated macrophages into a regulatory-like profi le. Recently, several was higher in MS MSCs, except for STAT1. At the same time, MSC reports have shown that microRNAs (miRNAs) are a crucial regula- immunomodulatory action was not altered. This was preliminary tor of the immune system. Here we hypothesize that MSC modu- confi rmed in post AHSCT MS MSCs. late cellular immune response through mechanisms controlled by Conclusion: Despite in almost all patients (5 out of 6) no further dis- miRNAs. ease activity has been detected by MRI at a median follow-up of 4,5 Materials and methods: To test this hypothesis we derived MSC years (range 3 – 6) post-transplantation the increased production from the bone marrow of conditional Dicer1 knockout (Dicer-/-) of IP10 in MS MSCs seems to persist after AHSCT. This is in accor- mice. Dicer1 is an RNAIII endonuclease essential for miRNA biogen- dance with an altered basal activation state and within increased esis and RNA processing. Hence, upon genomic deletion of Dicer1 phosphorylation after TLR4 stimulation of factors forming signal- cells will be unable to produce mature miRNAs. Dicer-/- MSC were ling cascade responsible of IP10 production and secretion.

S39 O287 transplantation (HSCT). It can be prevented completely, but at Study of bone metabolism and angiogenesis markers the expense of other complications, rejection, relapse or delayed following high dose chemotherapy/autologous stem cell immune reconstitution. No optimal prevention or treatment transplantation method is defi ned. This is refl ected by the enormous heterogene- G. Boutsikas, E. Terpos, A. Papatheodorou, A. Meletiou, V. Telonis, ity in approaches in Europe. To advance our knowledge and defi ne K. Petevi, A. Kanellopoulos, P. Flevari, G. Gainaru, A. Koutsi, optimal approaches, correct information on the value of the diff er- L. Papageorgiou, A. Zannou, P. Tsirkinidis, Z. Galani, M. Dimou, ent strategies and methods in GvHD prevention and treatment is E. Lalou, M. Nikolopoulou, P. Panayiotidis, M.-C. Kyrtsonis, G. Pangalis, essential. Comparative retrospective analyses fail in this regard. In J. Meletis, T. Vassilakopoulos, M. Angelopoulou order to change this situation and to form a basis for forthcoming National and Kapodistrian University of Athens (Athens, GR) studies, an EBMT-ELN (European LeukemiaNet) working group has developed in a Delphi-like approach recommendations for pro- Objectives: Autologous Hematopoietic Stem Cell Transplantation phylaxis and treatment of GvHD. As a fi rst step, the present rec- (HSCT) is a classic example of bone marrow microenvironment ommendations apply for allogeneic HSCT using an HLA-matched and HSC interaction. The role of bone tissue both in mobiliza- sibling or unrelated donor in adult patients with standard risk tion and engraftment of HSC has not been adequately studied in malignant disease. A questionnaire including 60 items of the humans. The study of bone metabolism and angiogenesis mark- prophylaxis and treatment of GvHD was produced. Each item pre- ers during HSCT is the subject of the present paper. METHODS: 13 sented a proposal and a choice for approval or disapproval, in the patients (median age 37.5 years, males 54%, Hodgkin lymphoma latter case asking for comments and proposals for an alternative 46%, non-Hodgkin’s lymphoma 46%, multiple myeloma 8%) were formulation. The responses were returned to the coordinator of studied. Serum from each patient was collected before HDT (d-8 the process. If a suffi cient consensus was not reached regarding for lymphomas, d-4 for multiple myeloma), at the end of HDT (d- an item, it was reformulated according to the feedback and circu- 1), after the administration of HSC (d+1), at the nadir of myelo- lated among the participants for approval or disapproval. In some toxicity (d+5) and at engraftment (d+10). The following markers cases two or three alternatives were off ered. Approval by more were studied with ELISA: 1. Osteoclast regulators (sRANKL, OPG), than two thirds of the participants was regarded as provisional 2. Osteoblast inhibitors (dickkopf 1/DKK1), 3. Bone resorption consensus in each cycle. Altogether three rounds were needed markers (CTX, TRACP-5b), 4. Osteoblastic activity markers (bALP, after which a provisional consensus was regarded as having been OC), 5. Angiogenetic cytokines (Ang1, Ang2). reached. A report of the provisional consensus recommendations Results: Bone resorption decreases during autologous HSCT, with discussion was produced, and the fi nal consensus is based as shown by the decrease of TRACP-5b between d-8 and d+10 on the acceptance of the document by all participants. The panel (p=0.013) and the decrease of CTX between d-1 and d+10 (p=0.023). proposes these guidelines to be adopted as the routine standard Similarly, bone remodelling declines, as indicated by the decrease at transplantation centers and used as comparator in systematic of bALP and OC between d-8 and d+10 (p=0.015 and p=0.004 studies evaluating the advantages and disadvantages of practices respectively). DKK1 decreases between d-8 and d+10 (p<0.001). diff ering from these recommendations. Thereby the strategies sRANKL shows no signifi cant diff erences, while OPG increases and procedures could be developed based on scientifi c documen- (p=0.001) between d-8 and d+5 and then decreases between tation, the heterogeneity in GVHD management policies could be d+5 and d+10 (p=0.023). Conversely, the ratio sRANKL/OPG, as reduced, optimal procedures identifi ed, and hopefully the out- an osteoclastic activation marker, decreases (p=0.034) between come of allogeneic transplantations improved. d-8 and d+5 and increases between d+5 and d+10 (p=0.022). The changes of Ang1 and Ang2 lead to a progressive reduction of the ratio Ang1/Ang2 (p<0.001 between d-8 and d+10), suggesting destabilization of vessels. Furthermore, low TRACP-5b levels at d-8 and d-1 correlate signifi cantly with faster platelet engraft- Acute leukaemias ment (p=0.015, p<0.001 respectively), while high levels of DKK1 at d-8 and d-1 are associated with earlier neutrophil engraftment (p=0.009, p=0.046 respectively). On the contrary the number of O314 CD34+ cells/kg of body weight infused was not associated with Anti-CD19 BiTE® blinatumomab induces high complete neutrophil and platelet engraftment in this group of patients. remission rates in adult patients with relapsed/refractory Conclusion: Bone metabolism is clearly altered during autolo- B-precursor acute lymphoblastic leukaemia (ALL) including gous HSCT, while destabilization of the vascular endothelial cells relapse after stem cell transplantation (SCT) is evident. Moreover, some markers, such as TRACP-5b and DKK1 M. Topp (1), N. Goekbuget (2), G. Zugmaier (3), A. Viardot (4), might predict engraftment, even more strongly than the number M. Stelljes (5), S. Neumann (6), HA Horst (6), A. Reichle (7), R. Marks (8), of CD34+ cells infused. C. Faul (9), M. Brüggemann (6), M. Ritgen (6), P. Klappers (3), N. Mergen (3), M.-E. Goebeler (1), H. Einsele (1), D. Hoelzer (2), R. Bargou (5) (1)University Würzburg (Würzburg, DE); (2)University Frankfurt (Frankfurt, DE); (3)Amgen Research Munich GmbH (Munich, DE); (4)University Ulm (Ulm, DE); (5)University Münster (Münster, DE); Working Party Session Complications (6)University Schleswig-Holstein (Kiel, DE); (7)University Regensburg and Quality of Life (Regensburg, DE); (8)University Freiburg (Freiburg, DE); (9)University Tübingen (Tübingen, DE)

296 Relapsed/refractory ALL in adults has a dismal prognosis with only Prophylaxis and treatment of graft-versus-host disease: 35-40% complete remissions (CR) and a median overall survival EBMT – ELN working group recommendations for a (OS) of 4-6 months. In relapse after SCT, CR rates and survival are standardized practice even poorer. Blinatumomab, a bispecifi c T-cell engaging (BiTE®) T. Ruutu, A. Gratwohl, T. de Witte, J. Apperley, A. Bacigalupo, F. Dazzi, antibody that directs cytotoxic T-cells to CD19 expressing target P. Dreger, R.F. Duarte, J. Finke, L. Garderet, H.T. Greinix, E. Holler, cells (Topp et al, JCO, 2011), was tested in an exploratory phase II N. Kröger, A. Lawitschka, M. Mohty, A. Nagler, J. Passweg, O. Ringdén, trial in relapsed/refractory B-precursor ALL including relapse after G. Socié, J. Sierra, J. Stankevich, A. Sureda, W. Wiktor-Jedrzejczak, SCT. The primary endpoint was hematological CR or CR with par- A. Madrigal, D. Niederwieser on behalf of the EBMT-ELN working group tial hematological recovery (CRh*) within 2 cycles. Secondary endpoints included OS and safety. Blinatumomab was administered Graft-versus-Host Disease (GvHD) remains the major impediment by continuous i.v. infusion for 28 days followed by a 14-day inter- to broader application of allogeneic hematopoietic stem cell val. Responding patients could then receive 3 additional cycles or

S40 [O314]

proceed to SCT. Three dosing regimens were explored (Table 1). 36 possessed a CenB/B gene motif (p=0.025). Strikingly, presence patients were treated; 25 (69%) achieved CR/CRh*; ten out of 25 of a CenB/B motif in the donor showed a signifi cantly increased CR patients (28%) had a CRh*; 22 out of 25 (88%) responders even Non Relapse Mortality (NRM) rate (p=0.001). A trend towards achieved a molecular CR. The median OS for all 36 treated patients lower relapse risk was found. Presence of KIR2DL2, KIR2DS2 and is 9.0 months with a median follow-up time for living patients of KIR2DS3, all of which are specifi c for the group B haplotype, also 10.7 months. For patients with CR/CRh*, the median OS is 14.1 showed protection against relapse but an increased risk of NRM. months. Cytokine release syndrome (CRS) and CNS events were In summary, in our cohort of patients with AML undergoing reported as medically important events. CRS syndrome could be T-cell depleted, myeloablative, unrelated HSCT we also observed an either prevented or treated by adapting a dexamethasone regi- eff ect of donor KIR repertoire on clinical outcome. This study adds men. Six patients developed CNS events, leading to treatment to the knowledge on the effi cacy of NK cell alloreactivity in diff erent interruption; they were fully reversible. One patient stopped treat- HSCT cohorts. The contrasting results between our T-cell depleted ment due to fungal infection leading to death. No specifi c toxici- and the T-cell replete cohort illustrate the signifi cance of T-cells on ties were observed in patients with relapse after SCT. the biological NK cell eff ect after unrelated HSCT for AML. Based on safety and effi cacy considerations, 5 μg/m2/day in week 1 and 15 μg/m2/day for the remaining treatment was selected as fi nal dosing regimen (cohorts 2a and 3). This regimen produced O316 exceptionally high hematological and molecular remission rates. In vivo T-cell depletion with ATG or alemtuzumab prevents Adverse events were manageable, particularly if compared to GVHD without aff ecting leukaemia-free-survival in AML other intensive regimens used in this treatment situation. patients in CR1 given PBSC from HLA-identical sibling after chemotherapy-based RIC. A survey from the Acute Leukemia Working Party of the EBMT O315 F. Baron, M. Labopin, D. Blaise, D. Caballero, S. Vigouroux, The infl uence of donor KIR repertoire on the clinical outcome C. Craddock, M. Attal, P. Jindra, H. Goker, G. Socie, P. Chevallier, after unrelated, T-cell depleted HSCT for patients with AML P. Browne, A. Sandstedt, P. Duarte, A. Nagler, M. Mohty on behalf of J. Schellekens (1), B.E. Shaw (1), N.P. Mayor (1), L. Cooke (1), J. Perry the Acute Leukemia Working Party of the European group for Blood (2), R.M. Pearce (2), M. Wilson (2), K.E. Kirkland (2), M.N. Potter (3), and Marrow Transplantation N.H. Russell (4), A. Pagliuca (5), J.A. Madrigal (1), S.G.E. Marsh (1) (1)Anthony Nolan Research Institute (London, GB); (2)British Society A recent study from the CIBMTR has suggested that in-vivo T-cell of Blood and Marrow Transplantation data registry (London, GB); depletion increases relapse risk and decreases disease-free sur- (3)Royal Marsden Hospital (Surrey, GB); (4)Nottingham University vival in patients given grafts after chemotherapy-based RIC regi- Hospital (Nottingham, GB); (5)King’s College Hospital (London, GB) mens for various hematological malignancies (Soiff er et al., Blood 117:6963-70, 2011). This prompted us to perform a survey assess- Natural Killer (NK) cell function is determined by the net eff ect of ing the impact of in-vivo T-cell depletion on transplantation out- signalling through several receptor families, including Killer cell comes in an homogeneous cohort of 1250 adult patients with de Immunoglobulin-like Receptors (KIR). The impact of NK cells on novo AML in fi rst CR given PBSC from HLA-identical siblings after clinical outcome after Haematopoietic Stem Cell Transplanta- chemotherapy-based RIC between 2000 and 2011. 554 patients tion (HSCT) has been contradictory. One of the reasons for the did not receive any in-vivo T cell depletion (control patients), disparity in results is that many reports are based on a hetero- while ATG and alemtuzumab were given in 444 and 252 patients, geneous cohort with regards to underlying disease. Cooley et al respectively. The proportion of patients with poor-risk cytogenet- (Blood 2009 and 2010) have reported an improved clinical out- ics or FLT3-ITD was higher in ATG patients (23%) than in control come for patients who underwent T-cell replete, unrelated HSCT (17%) or alemtuzumab (18%) patients (P=0.04). RIC consisted of for Acute Myeloid Leukaemia (AML) when they received a graft fl udarabine plus busulfan in 49% of control patients, 85% of ATG from a donor with a group B KIR haplotype or centromeric B/B patients, and 15% of alemtuzumab patients, while 24% of control (CenB/B) KIR gene motif respectively. The aim of our study is to patients, 6% of ATG patients, and 82% of alemtuzumab patients identify whether there is a signifi cant survival advantage of using received a fl udarabine plus melpalan based RIC. The incidence of KIR group B haplotype donors for AML patients undergoing T-cell grade II-IV acute GVHD was 21.4, 17.6 and 10.2% in control, ATG, depleted, unrelated HSCT using transplant protocols in use in and alemtuzumab patients, respectively. With a median follow-up the United Kingdom. Each patient and donor was genotyped for of 28 months (range, 1-138 months), 3-year incidence of chronic all KIR genes. KIR haplotypes were predicted from the genotyp- GVHD and 3-year LFS were 57±2% and 51±2%, respectively, in ing results and analysed in the context of other known patient control patients, 39±3% and 51±3%, respectively, in ATG patients, and donor variables. T-cell depletion was carried out in vivo by and 37±3% and 52±3%, respectively, in alemtuzumab patients. In infusion of Alemtuzumab. This is a joint study between Anthony multivariate analysis, the use of ATG and the use of alemtuzumab Nolan and the British Society of Blood and Marrow Transplanta- were each associated with a lower risk of chronic GVHD (HR=0.66, tion Clinical Trial Committee. In our T-cell depleted, myeloablative P<0.001 and HR=0.55, P<0.001, respectively) and a lower risk of cohort (N=141) a signifi cantly lower overall survival (OS) and dis- extensive chronic GVHD (HR=0.48, P<0.001 and HR=0.23, P<0.001, ease-free survival (DFS) was observed for patients who received a respectively), but a similar risk of relapse (P=0.4 and P=0.8, respec- graft from a donor with a KIR Bx genotype (p=0.041 and p=0.049 tively), and of nonrelapse mortality (P=0.5 and P=0.7, respec- respectively). In addition, worse OS was observed when the donor tively), and similar LFS (P=0.7 and P=0.5, respectively) and OS

S41 (P=0.7 and P=0.3, respectively). In conclusion, these data suggest O318 that a certain amount of in-vivo T-cell depletion with ATG or alem- Outcomes of unrelated cord blood transplant for adults with tuzumab can be safely used in the conditioning of AML patients acute lymphoblastic leukaemia: a survey conducted by in fi rst CR given PBSC after chemotherapy-based RIC. Prospective Eurocord and the acute leukemia working party of EBMT studies are needed to determine the optimal dose and schedule L. Tucunduva (1), A. Ruggeri (1), G. Sanz (2), S. Furst (3), B. Rio (4), of administration of these agents in that setting. G. Socié (5), W. Arcese (6), M. Michallet (7), I. Yakoub-Agha (8), J. Cornelissen (9), J. Sanz (2), P. Montesinos (2), D. Purtill (1), E. Gluckman (1), M. Mohty (10), V. Rocha (1) O317 (1)Eurocord (Paris, FR); (2)Hospital Universitario La Fe (Valencia, ES); Risk factors and outcomes after umbilical cord blood (3)Institut Paoli Calmettes (Marseille, FR); (4)Hôtel-Dieu Assistance transplant for adults with Philadelphia positive acute Publique-Hôpitaux de Paris (Paris, FR); (5)Hôpital Saint-Louis (Paris, lymphoblastic leukaemia and impact of minimal residual FR); (6)University of Rome Tor Vergata (Rome, IT); (7)Edouard Herriot disease before transplant Hospital (Lyon, FR); (8)Hopital Claude Huriez (Lille, FR); (9)Erasmus L. Tucunduva (1), A. Ruggeri (1), G. Sanz (2), S. Furst (3), J. Cornelissen MC/Daniel Den Hoed (Rotterdam, NL); (10)Hospital Saint Antoine (4), W. Linkesch (5), L. Mannone (6), JM Ribera (7), P. Montesinos (8), (Paris, FR) E. Gluckman (1), M. Mohty (9), V. Rocha (1) (1)Eurocord (Paris, FR); (2)Hospital Univ. La Fe (Valencia, ES); Few studies on outcomes after umbilical cord blood transplant (3)Institut Paoli Calmettes (Marseille, FR); (4)Erasmus MC/Daniel (UCBT) for adult ALL have been reported. We performed a ret- Den Hoed (Rotterdam, NL); (5)Medical University Graz (Graz, AT); rospective survey of UCBT for adult ALL in Europe. From 2000 to (6)Hôpital de l`ARCHET (Nice, FR); (7)Hospital Universitari Germans 2011, 421 adult patients (pts) underwent UCBT for ALL. Median Trias i Pujol (Barcelona, ES); (8)Hospital Universitario La Fe (Valencia, age at UCBT was 32 years (18-76) and 59% of pts (n=247) were ES); (9)Hospital Saint Antoine (Paris, FR) older than 35 years. At UCBT 46% (n=195) were in fi rst complete remission (CR1), 32% (n=136) in CR2 and 22% (n=90) of pts The presence of chromosome Philadelphia (Ph) is one of the main were not in remission. At diagnosis, cytogenetic abnormalities prognostic factors in acute lymphoblastic leukemia (ALL). Only 34 were present in 229 out of 314 pts with available information. cases of umbilical cord blood transplant (UCBT) for adult Ph+ALL Pts were classifi ed as very high risk (n=129), poor risk (n=14) have been reported. We analyzed 129 patients (pts) who under- and standard risk (n=171). By this classifi cation, the frequency of went a UCBT for adult Ph+ALL from 2000 to 2011, of which 114 very high risk group was 69% (n=89) in CR1, 18% (n=23) in CR2 were transplanted in remission (CR). Median age at UCBT was 38 and 13% (n=17) in those not in remission. Double CBT (dUCBT) years and 79% of pts (n=90) were in fi rst CR. Median time from was performed in 173 pts (41%) and median TNC at freezing was diagnosis to UCBT was 7 months. We had information on the use 3.5x107/kg for single and 4.7x107/kg for dUCBT. Most pts received of tyrosine kinase inhibitor (TKI) for 84 pts. Most of them (n=73) CBU with one (24%, n=103) or two (55%, n=231) HLA disparities. A were treated with at least one TKI before UCBT for a median time myeloablative conditioning (MAC) was given to 314 pts (75%) and of 5 months (1-54) and a median interval of 12 days between 103 (25%) received a reduced intensity conditioning (RIC). Median drug withdrawal and UCB infusion. Information regarding MRD follow-up was 27 months. Cumulative incidence (CI) of 60-day before UCBT was available for 65 pts. The majority of MRD testing neutrophil recovery was 78%. CI of acute and chronic GVHD was was done by RT-PCR and MRD was positive in 34 and negative in 33% and 26%, respectively. Two-year CI of non-relapse mortality 31 pts. (NRM) was 42%. In multivariate analysis, age<35 years (HR: 0.5, Seventy-three pts (65%) received a myeloablative conditioning p=0.001), remission at UCBT (HR: 0.4, p<0.0001) and use of RIC (HR: (MAC) and 40 (35%) a reduced intensity conditioning (RIC). Dou- 0.36, p<0.0001) were associated with decreased NRM. In patients ble CBT was performed in 43% (n=49) and median total nucleated transplanted after RIC, the use of ATG as part of conditioning regi- cell dose infused was 3.0x107/kg. Most pts received CBU with one men was associated with increased NRM (HR: 3.3, p=0.003). Two- (32%, n=36) or two (46%, n=52) HLA mismatches. Median follow- year relapse incidence (RI) was 28% and factors associated with up was 30 months. decreased RI were use of MAC (HR 0.5, p=0.0004) and CR at trans- Cumulative incidence (CI) of 60-day neutrophil recovery was plant (HR: 0.4, p<0.0001). Two-year leukemia-free survival (LFS) was 77±4% and infused CD34 cells >1.2 x107/kg was related to 39% for pts in CR1 (n=195), 31% for CR2 (n=136) and 8% for pts not improved engraftment. CI of acute and chronic GVHD was 34±4% in CR (n=90). Cytogenetic risk group did not impact the outcomes. and 32±4%, respectively. There was no diff erence in the CI of In multivariate analysis 3 factors were associated with improved acute GVHD for pts receiving single and double grafts. Two-year LFS age <35 years (HR: 0.8, p=0.04), CR at UCBT (HR: 0.3, p<0.0001) CI of non-relapse mortality (NRM) was 35±5%. In multivariate and not using ATG in conditioning regimen (HR: 0.7, p=0.009). analysis, factors associated with higher NRM were age>42 years For pts receiving RIC only, acute GVHD as time-dependent (HR 3.3 p=0.0003) and use of MAC (HR 4.0 p=0.0009). Two-year variable was associated with improved LFS (HR 2.7 p=0.003). relapse incidence (RI) was 28±4% and use of MAC was associ- UCBT is an option to treat high risk adult ALL. Pts transplanted ated with decreased RI (HR 0.5 p=0.03). Two-year leukemia-free with advanced disease have a dismal prognosis. For those in CR, survival (LFS) was 36±5%. Factors associated with improved LFS the conditioning regimen, including the use of ATG, has an impact were age<42 years at UCBT (HR 0.41 p=0.003) use of RIC (HR 0.5 on outcomes. p=0.004). We evaluated the impact of MRD before UCBT (n=65). TKI before UCBT was administered to 58 pts in this group. MRD positivity was associated with increased RI (26% for MRD+ vs 13% for MRD- Aplastic anaemia & Autoimmune disorders p=0.04) and a trend for decreased LFS (38% for MDR+ vs 48% for MDR- p=0.06). Results after UCBT for Ph+ALL are encouraging. Rise in use of TKI O319 before transplant allows for molecular response, which seems Preclinical development of novel C3-targeted complement to be associated with decreased RI and probably increased LFS. inhibitors for paroxysmal nocturnal haemoglobinuria (PNH) Further data on MRD before UCBT in this population are being A. Risitano (1), P. Ricci (1), C. Pascariello (1), M. Raia (1), C.Q. Schmidt collected. (2), Y. Li (2), E.S. Reis (2), M. Sica (3), L. Del Vecchio (1), F. Pane (1), D. Ricklin (2), R. Notaro (3), J.D. Lambris (2) (1)Federico II University of Naples (Naples, IT); (2)University of Pennsylvania (Philadelphia, US); (3)Istituto Toscano Tumori (Florence, IT)

Objectives: PNH erythrocytes suff er from uncontrolled complement activation and subsequent lysis because of the lack of the

S42 complement regulators CD55 and CD59. The anti-C5 monoclonal higher in hATG160 group: 69% (95% CI 58-79%) versus 51% (95% antibody has proven eff ective in controlling intravascular hemo- CI 40-61%) in hATG100 group (P=0.023). As a result more patients lysis in vivo, but we have demonstrated that persistent upstream in hATG100 group received salvage ATG-based IST (30% vs 18%, activation of the C3 may limit hematological benefi t during eculi- P=0.091) and allogeneic HSCT (16% vs 5%, P=0.041). In univari- zumab treatment because of residual C3-mediated extravascular ate and multivariate analyses using logistic regression model hemolysis. Thus, upstream inhibition of the complement cascade both PNH clone presence (OR 3.20, 95% CI 1.51-6.76; P=0.002) and seems an appropriate strategy to improve the results of current baseline absolute reticulocyte count >30x109/L (OR 2.95, 95% CI anti-complement treatment. 1.15-7.55; P=0.024) independently predicted a higher response Methods: We used an in vitro model to evaluate two novel classes rate. The cumulative incidence of relapse and clonal evolution did complement inhibitors that both act at the level of C3 convertases. not diff er signifi cantly between the two groups. Median follow-up Erythrocytes from PNH patients were washed and incubated in of 134 living patients was 48 months (range, 4-167). The probabil- ABO-matched sera and exposed to pH-lowering to activate the ity of both 5-y and 10-y OS were 79.8% and 86.9% in hATG100 and alternative pathway, both in absence and presence of diff erent hATG160 groups respectively (P=0.498). complement inhibitors. Hemolysis and C3 activation/deposition Conclusions: The hATG (ATGAM) total dose of 160 mg/kg ver- were assessed by fl ow cytometry. sus 100 mg/kg provides better hematologic response rate and Results: AMY-101 and its pegylated derivative AMY-105 are ana- less need for additional IST and salvage allogeneic HSCT in AA logs of the peptidic inhibitor compstatin, which binds to C3 and patients. Compared dosing regimens have similar results in OS its activated fragment C3b, preventing the initiation, amplifi ca- due to second line treatment effi cacy in non-responders. tion and terminal damage of the complement cascade via all its major pathways (classical, alternative, and mannose/lectin). Mini- factor H (FH) is an engineered 43kDa protein that combines an increased affi nity for the opsonins C3b, iC3b and C3d with the regulatory activities of FH (convertase decay acceleration and cofactor activities), resulting in a potent and selective inhibition of the complement alternative pathway (classical and the mannose/lectin pathways remain preserved). AMY-101 and AMY-105 demonstrated a dose-dependent inhibition of hemolysis, with full inhibition at 6 μM. Similarly, mini-FH also showed a dose- dependent inhibition of hemolysis, with full inhibition at 0.1 μM (whereas both full-length FH and FR SCR1-4 were only partially eff ective). Both compstatin analogs and mini-FH completely prevented surface deposition of C3 fragments on PNH erythrocytes. Conclusion: We demonstrate that a C3-targeted complement inhibition effi ciently prevents hemolysis and C3-opsonization of PNH erythrocytes in vitro, likely preventing both intravascular and C3-mediated extravascular hemolysis in vivo. This eff ect may be obtained using either broad or pathway-specifi c inhibitors, pro- viding the rationale for translational plans aiming to assess their safety and effi cacy in PNH.

O320 The horse ATG (ATGAM) total dose of 160 mg/kg as compared with 100 mg/kg is superior in achieving haematologic response in aplastic anaemia patients A. Kulagin (1), M. Ivanova (1), I. Golubovskaya (1), E. Babenko (1), I. Kruchkova (2), N. Pronkina (2), S. Bondarenko (1), V. Vavilov (1), N. Stancheva (1), I. Lisukov (1), V. Kozlov (2), B. Afanasyev (1) (1)St. Petersburg Pavlov State Medical University (St. Petersburg, RU); (2)Institute of Clinical Immunology SB RAMS (Novosibirsk, RU)

Background: Combined immunosupression with horse antithymocyte globuline (hATG) and cyclosporine A (CsA) is a treatment of choice for patients with acquired aplastic anemia (AA) who are not candidate for allogeneneic HSCT. Despite its long history of clinical use current dosing regimens of hATG in AA remain more empirical than based on formal dose-fi nding trials. Here we report results of retrospective analysis of two dosing regimens of hATG for the fi rst-line IST in AA. Methods: We analyzed the response rate and survival in 155 patients (87 M and 68 F, median age 20, 1-66) with moderate (MAA 42), severe (SAA 66) and very severe (VSAA 47) AA who were treated with hATG (ATGAM, Pfi zer) and CsA (5 mg/kg/d) between O321 January 1998 and June 2012. There were two ATGAM dose regi- Excellent outcome of haploidentical haematopoietic stem mens: 20 mg/kg for 5 days (hATG100, n=81) and 40 mg/kg for cell transplantation in children and adolescents with 4 days (hATG160, n=74). Primary and secondary end-points of the acquired severe aplastic anaemia study were hematological response according criteria of B.Camitta H. Im (1), K. Koh (1), E. Choi (1), J. Seo (1), S. Jang (2), S. Kwon (2), (2000) and overall survival (OS) respectively. C. Park (2) Results: No signifi cant diff erences in gender, median age, base- (1)Asan Medical Center Children’s Hospital (Seoul, KR); (2)Asan line blood counts, disease severity, PNH and HLA DRB1*15 positiv- Medical Center (Seoul, KR) ity or interval from diagnosis and IST were observed between the two groups. A total of 92 patients (59 %) achieved at least partial The transplantation outcome for patients with severe aplastic ane- response after fi rst line IST. The response rate was signifi cantly mia (SAA), including that of transplants using matched unrelated

S43 donors, has improved markedly. However, the management of (iv cyclophosphamide 4gm/M2 followed by fi lgrastim 10μ/kg/d children or adolescents with acquired SAA that lack a suitable before randomisation to immediate (1 month) or delayed (13 related or unrelated donor is still a challenge. months) immunoablation and HSCT. Conditioning was with iv We evaluated the outcome of children and adolescents with cyclophosphamide 50mg/kg/day for 4 days, anti-thymocyte acquired SAA who received haploidentical hematopoietic stem globulin 2.5mg/kg/day and methylprednisolone 1mg/kg on days cell transplantation (HHCT) comprising in vitro T cell-depleted 3–5 before an unselected graft of 3-8x106/kg CD34+ stem cells peripheral blood stem cells. Twelve patients with acquired SAA was infused. Symptoms (Crohn’s disease activity index, CDAI), received a total of 15 HHCTs comprising in vitro CD3-depleted endoscopy (Simple Endoscopic Score, SES) and quality of Life are grafts between July, 2009, and July, 2012. All 12 patients received compared 1 year after mobilisation alone or after HSCT. The trial a conditioning regimen comprising fl udarabine, cyclophospha- tests whether HSCT can achieve sustained remission (defi ned as mide, and rabbit ATG, and 6 of them received additional low-dose no immunosuppressive drugs and a CDAI <150 over 3 months TBI (LD-TBI, 400 cGy). Calcineurin inhibitors and MMF were used before the 1 year assessment, with endoscopy/imaging showing to prevent GVHD. no evidence of active CD). The median age at the time of transplantation was 13.5 years Results: As of Nov 2012, data are available on 30/45 patients. In (range, 3.8-21.7 years). Most of the patients were heavily trans- 14 undergoing mobilisation and HSCT the CDAI (Remission = fused, and nine of the 12 patients received IST prior to HHCT. The score <150) fell by 162 (median, IQR 0-190), from 322 (253-386) to median time from diagnosis to HHCT was 69 months (range, 7-135 161 (73-246). Following mobilisation alone (n=16) the fall was 82 months). (41-137) from 351 (313-435) to 298 (220-370). The aggregate SES Among the 12 patients, 11 achieved neutrophil engraftment at (normal = 0) was 16.0 (10.0-25.0) before and 3.0 (1.8-12.0) after a median of 10 days (range, 9-13 days) after HHCT. One patient HSCT, a fall of 7.0 (7.0-14.0). For mobilisation alone, there was no failed to achieve primary engraftment, and two experienced change in the SES (IQR 5.0 rise to 12.0 fall) with a median score graft rejection soon after engraftment. All three patients who of 12.1 (6.5-14.0) before and 6.5 (3.0 to10.3) at 1 year. There have experienced early graft failure (GF) received a second HHCT, and been 62 SAEs in 19 patients randomised to early transplantation achieved sustained engraftment. Thus, the fi nal engraftment rate and 58 in 18 patients randomised to delayed transplantation. was 100%. None of the patients that received the TBI-containing One patient died following HSCT. Results concerning all but one regimen experienced GF. Acute graft versus host disease (aGVHD) patient with 1 year primary assessment data by February 2013 was assessed in nine patients, excluding the three with early GF. will be presented. Three of the nine patients developed aGVHD (two ≥ grade II and Conclusions: Immunoablation and HSCT appears to be an eff ec- one with grade III). All 12 patients survived and were transfusion- tive treatment for patients with Crohn’s Disease that may substan- independent at a median follow-up of 14.3 months (range, 4.1- tially reduce endoscopic evidence of disease, but carries a high 40.7 months). chance of adverse events. Final results will allow a rational evalua- Hematopoietic stem cell transplantation from haploidentical fam- tion of the eff ectiveness and safety of HSCT. ily donors with in vitro CD3 T cell depletion is a reasonable therapeutic option for children/adolescents with acquired SAA. We plan a future trial using a uniform protocol, which will help to solve the O323 problems associated with HHCT, and provide a valuable platform Immunoablation and haematopoietic stem cell transplanta- for the further development of HHCT as a therapy for SAA. tion in early type 1 diabetes E. Snarski (1), K. Halaburda (1), A. Milczarczyk (2), T. Torosian (1), M. Paluszewska (1), E. Urbanowska (1), M. Król (1), K. Jedynasty (2), O322 E. Franek (2), W. Wiktor Jedrzejczak (1) Clinical and endoscopic improvement following haemopoi- (1)Medical University of Warsaw (Warsaw, PL); (2)Central Hospital, etic stem cell transplantation on behalf of the Autoimmune Ministry of Interior Aff airs and Administration (Warsaw, PL) Diseases Working Party of the EBMT C. Hawkey (1), M. Allez (2), S. Ardizzone (3), E. Clark (4), M. Clark (5), Background: Since 2008 we have performed autologous hemato- J.F. Colombel (6), S. Danese (7), D. Farge-Bancel (2), M. Labopin (8), poietic stem cell transplantation in patients with early diabetes J. Lindsay (9), A. Norman (5), F. Onida (10), E. Ricart (11), G. Rogler type 1. The treatment led to remission of diabetes in majority of (12), M. Rovira (11), N. Russell (5), J. Satsangi (13), S. Travis (14), the transplanted patients. We report here follow up data of the A. Tyndall (15), S. Vermeire (16) 24 patients transplanted at our institution. (1) University of Nottingham (Nottingham, GB); (2)Hopital Saint-Louis Methods: The mobilization protocol was based on cyclophospha- (Paris, FR); (3)L. Sacco University Hospital (Milan, IT); (4)European mide and G-CSF and all patients mobilized well. The cyclophospha- Group for Blood and Marrow Transplantation (London, GB); mide (50 mg/kg/day on days -5,-4,-3, -2 prior to transplantation) (5)University Hospitals NHS Trust (Nottingham, GB); (6)HopitalHuriez with antithymocyte globulin (Thymoglobulin - of 0.5 mg/kg/day Lille (Lille, FR); (7)Istituto Clinico Humanitas (Milan, IT); (8)European given on day -5, and 1.0 mg/kg/day given on days -4,-3, -2 and -1) Group for Blood and Marrow Transplantation (Paris, FR); (9)Barts were used as conditioning prior to stem cell infusion. & the London School of Medicine (London, GB); (10)University of Results: The mean time of observation as of December 2012 Milan (Milan, IT); (11)Hospital Clinic Barcelona (Barcelona, ES); was 31 months (range 9 - 55 months). The independence of (12)University Hospital of Zurich (Zurich, CH); (13)University of exogenous insulin after the transplantation was achieved in 20 Edinburgh (Edinburgh, GB); (14)John Radcliff e Hospital (Oxford, GB); out of 23 patients. The median time without exogenous insulin (15)University Hospital (Basel, CH); (16)University Hospitals Leuven for all patients was 20 months (range 9 – 55 months). Thirteen (Leuven, BE) out of 23 (56%) remain in remission of diabetes (exogenous insulin free) with median follow up of 27 months (range 9 – 55 Background: The Autologous Stem Cell Transplantation Interna- months). The treatment lead to significant reduction of HbA1c tional Crohn’s Disease (ASTIC) Trial is a randomised controlled trial and good glycemic control in patients. One of the transplanted co-sponsored by EBMT (Autoimmune Diseases) and ECCO and patients died during neutropenia due to the sepsis and its supported by the Broad Foundation that investigates haemopoi- complications. etic stem cell transplantation (HSCT) in Crohn’s disease (CD). The Conclusion: The HSCT leads to remission of early diabetes in primary endpoint is measured at one year and all but one patient almost all patients with over 56% of patients remaining insulin- will have reached this endpoint by Feb 2013. free over 27 months after transplantation, with some patients Methods: Patients with impaired quality of life and active CD, remaining insulin free over 55 months. However, due to the non- despite >3 immunosuppressive agents all received mobilisation zero mortality concerns remain about safety of the treatment.

S44 Chronic GvHD validation cohort that included 180 cGVHD+ patients and 33 controls from the Fred Hutchinson Cancer Research Center (FHCRC). In the FHCRC set, median levels were signifi cantly diff erent between the cGVHD+ and cGVHD- groups (Figure 1). In addition, MIG lev- O324 els above the median of the entire FHCRC cohort were more likely MIG: a plasma chronic GVHD biomarker that is diagnostic at to have cGVHD (83% vs. 62%, p=0.003). In the UM cohort, median onset and associated with disease severity levels of MIG were higher in patients with NIH Mild/Moderate and C. Kitko (1), D. Couriel (1), J. Levine (1), P. Martin (2), M. Flowers (2), Severe cGVHD compared to cGVHD- (p=0.01 and p<0.001, respec- J. Hansen (2), L. Chang (1), T. Braun (1), D. Fox (1), M. Conlon (1), tively). In the FHCRC set, a statistically signifi cant diff erence B. Fiema (1), R. Morgan (1), A. Gomez (1), P. Pongtornpipat (1), between the cGVHD- and severe cGVHD groups was seen, but not K. Lamiman (1), B. Storer (2), J. Ferrara (1), S. Lee (2), S. Paczesny (1) the mild/moderate cGVHD vs cGVHD- groups (Figure 2), suggest- (1)University of Michigan (Ann Arbor, US); (2)Fred Hutchinson Cancer ing that the diff erence between cGVHD- and cGVHD+ groups may Research Center (Seattle, US) be driven by patients with more severe cGVHD at onset. In conclusion, we have performed discovery and validation analysis of Chronic GVHD (cGVHD) remains a major contributor to long-term cGVHD biomarkers and found that MIG, a known Th1 chemokine, morbidity and mortality in survivors of allogeneic hematopoietic distinguished those patients with and without cGVHD in indepen- cell transplant (HCT). Previous cGVHD biomarker identifi cation dent cohorts from two institutions. has yielded variable results. We compared University of Michi- gan (UM) plasma samples from onset of 17 patients with de novo cGVHD that was poorly responsive to initial therapy to 18 patients O325 without acute or chronic GVHD at similar time points post-HCT, Biomarker profi les of organ involvement in chronic graft- using quantitative microarrays dotted with 130 antibodies that versus-host disease and their modulation during targeted a diverse group of proteins including Th1, Th2 cytokines extracorporeal photopheresis treatment and chemokines. Nine proteins (BAFF, CCL1, CEACAM1, elafi n, R. Whittle, H. Denney, T. Maher, M. Foster, R. Goodgrove, C. Swift, GDF15, IL2Ra, MIG, CCL15, CCL19) showed diff erences between L. Robertson, F. Hammerton, J. Mayo, A. Alfred, P.C. Taylor the groups with p< 0.1. Using a customized ELISA, CD13 concen- Rotherham General Hospital (Rotherham, GB) trations were also signifi cantly diff erent between cGVHD+ and cGVHD- groups (p=0.003). Next, we analyzed plasma samples Introduction: A hallmark of chronic graft-versus-host disease from a validation set of 109 patients (45 de novo cGVHD and 64 (cGVHD) is eff ector T cell migration into target organs in response cGVHD-) at onset of cGVHD or at similar matched time points to infl ammatory stimuli. The role of specifi c chemokine & che- post-HCT. The median levels of fi ve proteins (BAFF, CD13, elafi n, mokine receptors in T cell recruitment to specifi c tissue sites is IL2Ra, and MIG) were signifi cantly diff erent between the cGVHD+ emerging. We present data that elevated serum elafi n, an epitheli- and cGVHD- groups. The composite of the area under the receiver ally expressed potent anti-protease generated in response to pro- operating characteristic curve (AUC) of the 5 proteins was 0.84 infl ammatory cytokines, is associated with the cutaneous form of whereas MIG alone had an AUC of 0.83. Thus, further analysis was cGVHD. Also we provide data connecting particular chemokine limited to MIG (monokine induced by interferon γ), also known profi les with cGVHD organ involvement and their correlation with as CXCL9. We confi rmed MIG as a cGVHD biomarker in a second organ response during Extracorporeal photopheresis (ECP).

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S45 Methods: We retrospectively evaluated 40 steroid-refractory O326 cGVHD adult patients undergoing ECP. Treatment schedule was Pre-transplantation risk factors for the development of 2-weekly dual treatments performed for an initial 3m, then sclerotic chronic GvHD after allogeneic HSCT: A multicentre monthly paired treatments. We developed a multiplex capture retrospective study from the Société Française de Greff e de assay with electrochemiluminescence-based detection to detect Moelle et de Thérapie Cellulaire (SFGM-TC) a panel of biomarkers including 15 chemokines and elafi n. These M. Detrait (1), R. Peff ault de la tour (2), I. Yacoub-Agha (3), were measured in sera prior to ECP and at 1, 3, 6, & 12m of therapy R. Crocchiolo (4), S. Vigouroux (5), J. Bay (6), P. Chevalier (7), M. Sobh (1), and compared with healthy controls( n=24). S. Morisset (1), N. Raus (8), F. Barraco (1), H. Labussière (1), R. Tabrizi Results: Chemokines CXCL8,9,10,11,16 and CCL3,17,27 were sig- (5), L. Magro (3), M. Mohty (7), N. Milpied (5), D. Blaise (4), G. Socié (2), nifi cantly amplifi ed in cGVHD patients. CXCL9,10, and 11 dem- M. Michallet (1) onstrated co-expression in cGVHD. Cutaneous cGVHD patients (1)Centre Hospitalier Lyon-Sud (Pierre Benite, FR); (2)Hôpital demonstrated 2.2 fold higher serum elafi n than patients without Saint-Louis (Paris, FR); (3)CHR Lille (Lille, FR); (4)IPC (Marseille, skin symptoms (Mean 96ng/ml vs 43ng/ml, p≤0.05) and particu- FR); (5)Hôpital du Haut Levêque (Pessac, FR); (6)Hôpital Estaing larly elevated CCL22 (p≤0.05). Patients initiating ECP with skin (Clermont-Ferrand, FR); (7)CHU de Nantes (Nantes, FR); (8)SFGM-TC scores >50 had higher mean serum elafi n than patients with (Lyon, FR) milder skin disease (84 vs 63ng/ml) and higher CXCL10 (820 vs 396pg/ml, p≤0.05). Patients with hepatic cGVHD (n=15) (NIH Sclerotic cGvHD is one of the most severe complications after score>1) or gut cGVHD (n=9) (NIH>0) exhibited signifi cantly allo-HSCT. Risk factors associated with this complication remain higher CXCL16, a chemokine reportedly associated with T cell not very well defi ned. Recently, TBI, PBSC, female recipient, and ingress to the liver and gut, than patients without liver (n=25) or prior severe skin aGvHD were identifi ed as factors associated with gut disease (n=31), (p≤0.05). Liver cGVHD patients responding to an increased risk while patients with HLA-mismatched donors ECP had signifi cantly lower CXCL16 & lower CXCL10 levels than and ABO major mismatch seem to have a better outcome. In non-responding patients (p≤0.05). After 1 month of ECP, a below the aim to validate the previous factors in France, we have con- median serum elafi n was associated with subsequent successful ducted a retrospective multicentric analysis, in 705 consecutive steroid taper of at least 50% by 3m (15/16 vs 6/13 patients with patients between 2005 and 2010 who received systemic therapy high 1m elafi n, p=0.01) and with superior skin disease improve- for cGvHD after a fi rst allo-HSCT. Analyses to determine pre-trans- ment after 6 & 12m compared to patients with high 1m elafi n (80 plantation risk factors included as variables: patient and donor vs 47% responders at 6m, 100% vs 64% at 12m, p≤0.05). age, kind of donor, HLA matching, ABO matching, sex-matching, Conclusions: Our data indicates that elafi n is elevated in the cuta- CMV status, diagnosis, stem cell source, gender, GvHD prophylaxis neous form of cGVHD in addition to the reported association with and antithymocyte globulin (ATG) in the conditioning regimen. All cutaneous acute GVHD and that serum measurement may have patients had hematological malignancies with a median age of some predictive therapeutic biomarker utility in cGVHD although 48 years (3-70), 366 patients (52%) had a matched related donor, further prospective studies are required. Our data adds to the evi- 222 patients (31.5%) had a matched unrelated donor, 53 patients dence that a number chemokines associated with eff ector T cell (7.5%) had a HLA- 9/10 unrelated donor and 64 (9%) had cord tissue entry are amplifi ed in cGVHD. Our fi ndings suggest that blood. The stem cell source was bone marrow for 181 patients, organ improvement generated by ECP is associated with modu- PBSC for 460 patients and cord blood for 64 patients. The median lations of particular chemokines associated with T cell migration time from allo-HSCT to chronic GvHD was 6.8 (1.8-41.5) months; into those target organs. 134 patients (19%) presented sclerotic features at initial diagnosis or after the onset of chronic GvHD. The cumulative incidence of sclerotic cGvHD was 18% (95%CI, 16.6-19.6) at 3 years after onset

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S46 of chronic GvHD (Figure). In univariate analysis, we found more TGF-B (≤10 pg/ml; n=36) were 3 times more likely to relapse (CI sclerotic cGvHD in patients transplanted with PBSC and when 17% vs 5%, p=0.05) and 4 times more likely to experience NRM the association of CsA and MMF as GvHD prophylaxis was used (23% vs 5%, p=0.01) than patients with high TGF-B (> 10 pg/ml; (p=0.0018). Cord Blood (p=0.0021) and ATG in conditioning were n=76). These increases resulted in lower progression free sur- protective (p=0.0016). In multivariate analysis (Table), factors vival (PFS 60% vs 89%, p=0.001) and overall survival (OS 72% vs associated with an increased risk of sclerotic cGvHD were young 92%, p=0.01). Low TGF-B was also signifi cantly correlated with patient age, multiple myeloma and PBSC as stem cells source. ATG the following features at onset: low performance status (p=0.01), in conditioning regimen and cord blood were associated with a platelet < 100K (p=0.01), and NIH lung 2/3 (p=0.01). However, the lower risk. The overall survival at 6 years is 50% and 65% for cGvHD impact of low TGF-B on OS and PFS was independent of each of patients without sclerotic form and sclerotic cGvHD patients these factors in bivariate analysis. There was no correlation with respectively (p=0.000148) (Figure). This form of cGvHD is not a TGF-B levels and other characteristics such as patient age, diag- negative factor on survival in this study. Immunologic reconstitu- nosis, high risk disease at HCT, donor type, overall NIH score or tion and immunologic mechanisms that account for the increased progressive onset cGVHD. Patients in the high TGF-B group and and decreased risk of sclerosis warrant future investigations. high IL-13 group (>200 pg/ml) were more likely to later develop sclerotic skin (n=20) (TGF-B 80% vs 65%, p=0.1; IL-13 85% vs 62%, p=0.04). Those with low levels of MMP2 (≤ 5000 pg/ml), an O327 antifi brotic metalloproteinase, were more likely to develop scle- Extracorporeal photopheresis induces neutrophilic rotic cGVHD (50% vs 21%, p=0.007). These results suggest ECM myeloid-derived suppressor cells in patients with turnover is active at the time of cGVHD onset, even prior to the graft-versus-host disease development of clinically apparent sclerosis. Further investigation D. Hartl, N. Rieber, I. Wecker, D. Neri, I. Schäfer, W. Bethge, of factors that impact the balance and transition between infl am- R. Handgretinger, O. Amon mation and fi brosis may provide a better understanding of the University of Tübingen (Tübingen, DE) pathophysiology and new biomarker opportunities for cGVHD.

Extracorporeal photopheresis (ECP) is a leukapheresis based immunomodulatory therapy, shown to be effi cient in a variety of T-cell mediated disorders, including graft-versus host disease (GvHD). The underlying immunological mechanism(s) by which Lymphoma ECP downregulates T cells responses, however, are incompletely defi ned. We hypothesized that ECP induces a myeloid cell population that dampens T cell responses. Myeloid-derived suppres- O329 sor cells (MDSCs) represent an innate immune cell population Autologous and allogeneic stem cell transplantation for characterized by their capacity to suppress T cell immunity. We diff use large B-cell lymphoma in the last decade. An analysis found that ECP treatment induced a distinct subpopulation of from the Lymphoma Working Party of the EBMT neutrophilic MDSCs (nMDSCs; low density CD66b+MHCII-IL-4R S. Robinson, A. Boumendil, H. Finel, R. Foa, I. Avivi, C. Thieblemont, α + cells) in GvHD patients (n=23). Isolated nMDSCs signifi cantly C. Craddock, M. Gilleece, S. Lenhoff , K. Orchard, U. Schanz, suppressed cytokine secretion and polyclonal proliferation of J. Cornelissen, A. Sureda, H. Schouten, P. Dreger on behalf of the both CD4 and CD8 T cell subsets in a dose-dependent manner. Lymphoma Working Party The eff ect of ECP on the induction of nMDSCs in peripheral blood was maintained over several weeks, but varied between individual Introduction: The last two decades have seen substantial changes patients. Increase in nMDSCs correlated with clinical response to in the therapy of diff use large B cell lymphoma (DLBCL) raising ECP (GvHD grading) in the majority of patients. The ECP-derived questions regarding the optimal treatment paradigm for relapsed mechanisms driving nMDSC accumulation remain to be solved. and refractory disease. We therefore fi rstly reviewed the outcome This study suggests that the induction of nMDSCs may represent of standard salvage therapy with an autologous stem cell trans- an underlying mechanisms of the immune modulatory mode of plant (autoSCT) over the last two decades. Secondly we have ana- action of the ECP treatment in GvHD patients. lysed the most recent outcomes of both autoSCT and allogeneic SCT (alloSCT) in patients with chemosensitive relapse or chemorefractory disease. O328 Methods: 5908 patients were identifi ed on the EBMT database Biomarkers of extracellular matrix remodelling in chronic satisfying the following criteria: - diagnosis DLBCL, diagnosis GVHD between 1992 and 2010, age 18-65, fi rst SCT procedure, chemo- C. Kitko (1), R. Saliba (2), S. Choi (1), P. Reddy (1), S. Goldstein (1), sensitive relapse or refractory disease. J. Magenau (1), A. Pawarode (1), M. Kennel (1), J. Levine (1), E. White Outcome of AutoSCT 1992-2002 vs 2002-10:- We identifi ed (1), D. Couriel (1) 1417 patients undergoing an autoSCT between 1992 and 2002 (1)University of Michigan (Ann Arbor, US); (2)MD Anderson Cancer and 4282 patients transplanted between 2002 and 2010. The Center (Houston, US) two groups were well balanced in pretransplant characteristics. Patients transplanted after 2002 had a signifi cantly lower NRM Chronic GVHD (cGVHD) is the major long-term complication of and relapse rate and a superior DFS and OS. In a multivariate allogeneic transplantation (HCT). It evolves from immune-medi- analysis transplantation before 2002, chemorefractory disease, ated infl ammation to fi brogenesis, resulting in end organ damage age >50 and time from diagnosis to SCT<1 year predicted for a such as sclerotic skin. Assessment of biomarkers of extracellu- signifi cantly worse DFS and OS. lar matrix (ECM) remodeling may help elucidate the biology of Outcomes of SCT for chemosensitive relapse 2002-2010:- The 4- cGVHD and contribute to outcome prediction. We conducted a year NRM rates were: - 6.4% for autoSCT , 20% for reduced intensity discovery experiment in 112 adult patients (median age 52.5 y, (RIC) alloSCT and 23.9% for myeloablative (MA) alloSCT respec- range 18-68) with new onset cGVHD. Median time to onset of tively. The 4-year RR was 49% for autoSCT, 44.5% for RICalloSCT cGVHD was 165 d (range 37-547) post-HCT. Plasma samples were and 39.4% for MA alloSCT (ns). The 4 year DFS was 45.5%, 37% and obtained at the time of cGVHD onset, biomarkers were assayed 37% for autoSCT, RICalloSCT and MA alloSCT respectively. The 4 simultaneously using a lab-derived (non-commercial) Luminex year OS was 55%, 57% and 42% for autologous SCT, RIC alloSCT bead-based assay, and levels of each analyte that provided the and MA alloSCT respectively. The DFS and OS were signifi cantly best discrimination with survival were determined. Levels of 3 worse for patients undergoing a MA alloSCT. profi brotic proteins (CCL2, IGF-1, IL-13Ra) signifi cantly correlated Outcomes of SCT for refractory disease 2002-2010:- The 4-year with TGF-B levels. Of these, TGF-B provided the strongest associa- NRM rates were:- 7.3% for autoSCT, 15.4% for RIC alloSCT and tion with outcomes 1 y post-onset of cGVHD. Patients with low 19.2% for MA alloSCT respectively. MA alloSCT was associated

S47 with a higher NRM. The 4-year RR was 73% for autoSCT, 81.5% for Achievement of CR before alloSCT (p=0.05 HR=0.5), but not donor RICalloSCT and 61.3% for MA alloSCT. The 4 year DFS was 20.7%, source, T-cell depletion or conditioning intensity aff ected OS after 3.9% and 20.7% for autoSCT, RICalloSCT and MA alloSCT respec- alloSCT. tively. The 4 year OS was 28%, 8.6% and 17.9% for autologous SCT, Conclusions: Patients with MCL who relapse within one year RIC alloSCT and MA alloSCT respectively. after autoSCT have an extremely dismal outcome even with Conclusions: The outcomes of autoSCT for DLBCL have improved alloSCT. In contrast, about half of the patients who have MCL in the last decade. For patients with chemosensitive relapse the recurrence beyond one year after autoSCT and can undergo outcomes following a RICalloSCT are comparable to those follow- salvage alloSCT enjoy long-term survival. A 2nd autoSCT does ing an autoSCT. not appear to be a promising option in patients with MCL failing a 1st autoSCT.

O330 Outcome and prognostic factors in patients with mantle cell O331 lymphoma relapsing after autologous stem cell Risk factors for Epstein Barr virus-related post-transplant transplantation: a retrospective study of the EBMT lymphoproliferative disease after allogeneic haematopoietic S. Dietrich, H. Finel, A. Boumendil, I. Avivi, L. Volin, J. Cornelissen, stem cell transplantation J. Walewski, C. Schmid, T. de Witte, H. Schouten, M. Kaufmann, M. Uhlin, H. Wikell, M. Sundin, O. Blennow, M. Mauerer, O. Ringden, C. Sebban, M. Trneny, G. Kobbe, P. Guardiola, J. Schetelig, J. Winiarski, P. Ljungman, M. Remberger, J. Mattsson M. Pfreundschuh, J. Diez-Martin, D. Bordessoule, S. Robinson, Karolinska University Hospital Huddinge (Huddinge, SE) P. Dreger on behalf of the EBMT Lymphoma Working Party Epstein-Barr virus associated post-transplant lymphoproliferative Background: It is little known about outcome of MCL recurrence disease (EBV-PTLD) is a severe complication after allogeneic hema- after autoSCT. We therefore conducted a retrospective analysis of topoietic stem cell transplantation (SCT). In immune- competent patients with MCL who failed autoSCT using the EBMT database. individuals EBV activity is strictly regulated by specifi c T cells. In Eligiblibility criteria were: age > 18 years and MCL relapse after the immune compromised milieu created by SCT, the pathogen autoSCT between 2000 and 2010. control is lost with viral reactivation and viremia as a possible Results: Median age at autoSCT of 360 included patients was 57 result. Several risk factors associated with PTLD after SCT. It is years (range: 37 to 77), 67% had undergone autoSCT as part of particularly common with EBV related complications when diff er- 1st-line therapy; 67% and 50% had documented exposure to ent in vivo or ex vivo T cell depletion strategies have been used rituximab (RTX) and high-dose ara-C (HA) before autoSCT; and 8% for graft versus host-disease (GVHD) prevention and/or following had refractory disease at autoSCT. Only 21 relapses (6%) occurred reduced intensity conditioning (RIC). This is a refl ection of the beyond 5 years after autoSCT. impaired T-cell mediated EBV-directed immunity and the pres- With a median observation time of 40 months, median OS after ence of residual recipient B-cells. relapse of the whole study group was 19 months. By univariate In total 1021 patients who underwent SCT at Karolinska University analysis, a long (>12mo) interval between autoSCT and relapse Hospital in Huddinge, Stockholm, between 1996 and 2011 were (p<0.001; HR 0.26; Figure 1A), 1st-line autoSCT (p=0.004; HR 0.7) included in this retrospective analysis of risk factors and clinical refractory disease (p<0.005, HR 1.9) and more recent year of relapse outcome for PTLD. (p<0.002, HR per year 0.9) signifi cantly infl uenced OS from relapse, PTLD was diagnosed in 40 (4%) of the 1021 patients transplanted whereas age, gender, RTX and HA exposure did not. By multivari- between 1996 and 2011. The median time of PTLD onset was 71 ate analysis refractory disease at autoSCT (p<0.02, HR=1.9), remis- (38-1199) days post SCT. sion duration after autoSCT (p<0.001 HR per 3 months 0.88) and Signifi cant risk factors identifi ed by multivariate analysis were; calendar year of relapse (p=0.02, HR per year 0.9) were confi rmed HLA-mismatch (p<0,001), serological EBV mismatch Recipient- to be predictors for OS. In addition, HA exposure prior autoSCT /Donor+ (p<0,001), use of reduced intensity conditioning (RIC) adversely aff ected OS from relapse (p=0.04, HR 1.4). (p=0,002), acute Graft vs. Host Disease grade II to IV (p=0,006), A high rate of chemotherapy refractory patients (41%) were splenectomy prior to SCT (p=0,008) and infusion of mesenchymal observed after fi rst relapse therapy. 80 patients (22%) received an stromal cells (MSC) (0=0,015). The risk of EBV-PTLD has increased allogeneic SCT (alloSCT), whereas only 7 patients (2%) received a in more recent years, from less than 2% before 1998 to more than second autoSCT after relapse. Survival after relapse for patients 6% after 2011. Additionally, we show that long term survival of who received a second autoSCT was poor with one long-term sur- patients with PTLD is poor despite initial successful treatment. vivor. AlloSCT performed for late relapse (>12mo) after autoSCT Three-year survival among the 40 patients with EBV-PTLD was was associated with superior OS compared to patients who 20% as opposed to 62% among patients without PTLD (p<0.001). received an allograft upon a shorter remission duration after The study identifi es patients at risk for PTLD after SCT in need of autoSCT (5-year OS from alloSCT 50% vs 0%; p=0.001; Figure 1B). preemptive measures.

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S48 O332 Patients and Methods: We reviewed the impact of twenty-one dif- ‘Plerixafor on demand’ in association with chemotherapy ferent prognostic / predictive factors prior to salvage chemother- and G-CSF halves rate of mobilization failure in lymphoma apy and integrated post-salvage chemotherapy FDG-PET results and in multiple myeloma: preliminary results of a multicen- in these HL patients to develop a “prognostic factors score-based- tre prospective study model” for post HDC ASCT outcome. Fine and Gray method for G. Milone (1), M. Martino (2), P. Scalzulli (3), G. Tripepi (4), M. Camuglia competing risk analysis and regression model was used to assess (1), G. Avola (1), S. Leotta (1), A. Cupri (1), S. Lingenti (1), F. Iacona the risk associated with diff erent factors on outcome. (1), A. Spadaro (1), S. Mercurio (1), E. Spina (1), E. Schinocca (1), Results: From 2004 to 2011, one hundred and forty-one patients V. Di Martina (1) had FDG-PET after salvage chemotherapy /prior to HDC ASCT; (1)Ospedale Ferrarotto (Catania, IT); (2)O. Riuniti di Reggio C. (Reggio male 55%, female 45%, relapsed 43%, refractory 57%, median age C, IT); (3)Ospedale Padre Pio (San Giovanni Rotondo, IT); (4)IBIM-CNR at ASCT 25.5 years, median follow-up 33 months. Etoposide + sol- (Reggio C, IT) umedrol + high dose Ara-C and cisplatinum (ESHAP) was used as salvage chemotherapy in 94% patients. Multivariate analysis iden- Background: Failure in PBSC harvest interests 7-10% of Mul- tifi ed HL International Prognostic Score (HL-IPS) of >3 at relapse tiple Myeloma (MM) cases and 20-30% of Lymphoma cases. “On (P=0.001) (Hazard ratio (HR) 3.7, (1.6-8.3)) and post salvage positive demand” Plerixafor (PLX) in association with “G-CSF” has been FDG-PET (P =0.011) (HR 3.4, (CI 1.3-8.9)) with higher hazard rate of reported (Micallef 2012, Horwitz 2012). However, no prospec- disease-specifi c death (model P=0.0001). Cumulative incidence tive data are available on “PLX on demand” in association with of disease-specifi c death for patients with 0, 1, 2 risk factors was “chemotherapy and G-CSF”. We have previously published on an 7%, 29% and 52% respectively (P=0.00003). For disease-specifi c algorithm for use of PLX “on demand” in association with CTX and event (persistent, progressive or relapsed disease), mediastinal G-CSF (Blood Transfusion 2012). involvement (P=0.024) (HR 2.7, (1.14-6.5)), B symptoms (P=0.027) Object: We present preliminary results of the fi rst phase II, prospec- (HR 2.1, (1.09-4.2)) and positive post-salvage FDG-PET (P=0.001) tive study in which PLX was employed “on demand” associated to Chemotherapy and G-CSF. PLX was administered according to our algorithm, in patients receiving HD-CTX or DHAP and G-CSF. Methods: Seventy-three patients were registered, 68 were evaluated for treatment effi cacy; 5 patients did not receive PLX in spite of having criteria for its use. Evaluated patients had a mean age of 54 y, 41 were male and 27 female, 52 were aff ected with MM and 16 with Lymphoma, 53 were mobilized using HD-CTX and 15 using DHAP. PLX was administered if, on day +13 after chemotherapy, CD34+ cell count in PB was below 10x10e6/l, or if CD34+ count was between 10 and 20 x10e6/l and fi rst apheresis yielded a CD34+ < 1x10e6/kg. Results: Overall success rate of CD34+ mobilization (defi ned as a CD34+ count in PB > 20x106/l) was 95.5%. Mobilization failure was registered in only 3.9% of MM and in 6.3% of Lymphoma. Overall success rate in harvesting a CD 34+ > 2x10e6/kg was 94% (MM: 96.1%; Lymphoma: 86.6%). Negative predictive value of the algorithm used was 100%, in fact, 61/61 patients were correctly predicted to reach a successful mobilization and harvest without requiring PLX. Five patients were predicted to not reach successful mobilization. However, due to drug shortage, these patients did not receive PLX. Only one of the fi ve (20%) reached successful mobilization, thus confi rming the high positive predictive value of the algorithm (80%). Seven patients were predicted as not reaching successful mobilization according to algorithm and were treated with PLX, 4/7 (57%) had a successful harvest. Overall the rate of PLX indication according to the algorithm was 16.4% (MM 6.5%, Lymphoma 30%). Conclusion: Compared to chemotherapy + G-CSF, on demand use of PLX in association with chemotherapy and G-CSF is able to halve the mobilization and harvest failure rate: failure of harvest in MM patients is reduced from 7-10% to 3.5% and in Lymphoma patients from 20-25% to 13.4%.

O333 Pre-transplant FDG-PET scan response and other prognostic factors based survival model in patients with relapsed and refractory Hodgkin’s lymphoma is useful to predict outcome after high dose chemotherapy and autologous stem cell transplant S. Akhtar, A. Al-Sugair, M. Abouzied, Y. AlKadhi, M. Abdelsalam, M. Dingle, H. Soudy, A. Darwish, A. Eltigani, T. Elhassan, M. Nabil-Ahmed, I. Maghfoor King Faisal Specialist Hosp. & Res. Ctr (Riyadh, SA)

Background: 18F-fl uorodeoxyglucose positron emission tomography (FDG-PET) documented response after salvage chemotherapy has been reported to impact survival in patients with HL undergoing HDC ASCT. Positive FDG-PET after salvage chemotherapy / prior to HDC ASCT has been reported with inferior post HDC ASCT outcome.

S49 (HR 3.3, (1.7-6.7)) were signifi cant (model P≤0.00001). Cumulative O335 incidence of disease-specifi c event with 0,1,2,3 risk factors was HLA haploidentical stem cell transplantation after removal 8%, 31%, 50% and 75% respectively (P=0.0000006). Table shows of α/β+ T-lymphocytes and B-lymphocytes: a new transplant the confi dence interval (CI) with upper and lower limits (UL) (LL) option for children with life-threatening, non-malignant and P=value. disorders lacking a HLA-identical donor Conclusion: Patients with relapsed or refractory HL, HL-IPS of >3 A. Bertaina (1), C. Messina (2), R. Masetti (3), M. Romano (1), and positive post salvage FDG-PET carries higher hazard rate of S. Rutella (1), A. Bauquet (1), D. Pagliara (1), M. Gazzola (2), D. Pende disease-specifi c death. Patients with mediastinal involvement, (4), S. Ceccarelli (1), M. Tumino (2), L.P. Brescia (1), G. Li Pira (1), with B symptoms and positive post-salvage FDG-PET carries M.E. Bernardo (1), G.M. Milano (1), G. Palumbo (1), M. Falco (5), higher hazard rate of disease-specifi c event. Patients with higher L. Moretta (5), F. Locatelli (1) scores have higher risk of treatment failure. Our FDG-PET based (1)IRCCS Bambino Gesù Children’s Hospital (Rome, IT); (2)University of model is signifi cant and patients with higher scores are potential Padua (Padua, IT); (3)Ospedale S. Orsola (Bologna, IT); (4)IRCCS AOU candidate for newer therapies along with HDC ASCT. San Martino-IST (Genoa, IT); (5)IRCCS G. Gaslini Institute (Genoa, IT)

To improve survival and immune recovery of patients with life- threatening non-malignant disorders given HLA haploidentical HCST, we developed a novel method of ex vivo T/B-cell depletion Genetic disorders based on elimination of α/β+ T cells and CD19+ B cells through a new selection strategy (Miltenyi Biotec, Germany). Here we report the results of 16 patients (pts) given this type of allograft. Eight O334 pts were males and 8 females, median age at HCST being 3 years Absence of natural killer cells in severe combined immuno- and 9 months (range 0.3-28.7). Five pts had SCID, three Fanconi defi ciency aff ords a permissive environment for donor T-cell anaemia (FA), 3 severe aplastic anaemia (SAA) and 1 each WAS, engraftment following non-conditioned allogeneic SCT IPEX, congenital amegakaryocytic thrombocytopenia (CAMT), A. Hassan (1), P. Lee (2), D. Moreira (3), P. Maggina (3), M. Slatter (3), HLH and thalassemia with autoimmune hemolytic anemia. All pts Z. Nademi (3), A. Jones (1), C. Cale (1), K. Rao (1), R. Chiesa (1), were transplanted from 1 of the 2 parents (8 from the mother and P. Amrolia (2), B Gaspar (2), E Davies (2), P. Veys (1), A. Gennery (3), 8 from the father), the median number of CD34+ cells, α/β CD3+ W. Qasim (2) cells and B cells being 19.4x106/kg, 48x103/kg and 4.2x105/kg. (1)Great Ormond Street Hospital (London, GB); (2)Institute of child The original conditioning regimen consisted of treosulphan and health, UCL (London, GB); (3)RVI (Newcastle Upon Tyne, GB) fl udarabine (FLU) in 7 (5 SCID, 1 IPEX, 1 WAS, 1 HLH and 1 CAMT), FLU and cyclophosphamide (CY) in 5 (3 FA and 2 SAA), thiotepa Introduction: Severe combined immunodefi ciencies (SCID) pre- (TT), CY and total nodal irradiation in 1 (SAA) and busulphan, senting in early infancy can be cured by allo-SCT. In many cases FLU and TT in 1 (thalassemia). All pts received ATG Fresenius (3 absence of eff ective host immunity obviates the need for pre- mg/kg/day) on days -5 through -3 before allografting; rituximab conditioning & donor T cell chimerism can be readily achieved. (200 mg/m2) was administered on day -1 to prevent EBV-related Whilst most infants with SCID have absent or greatly reduced PTLD. No pt received pharmacological GvHD prophylaxis. All pts T cell immunity & defective B cell immunity, natural killer cell func- but 3 engrafted, the median time to reach neutrophil and platelet tion may be preserved. We considered how this might infl uence recovery being 13 days (8-19) and 11 days (7-40), respectively. The donor T cell engraftment, reconstitution & long-term outcomes 3 pts (2 with SAA and 1 with thalassemia) who had primary graft after non-conditioned SCT. failure were successfully re-transplanted, 1 from the same parent Methods: Results for children with SCID who underwent non- and 2 from the other parent. Grade I/II skin acute GVHD and lim- conditioned allo-SCT between 1990-2010 at two tertiary referral ited skin chronic GVHD occurred in 4 and 1 pt, respectively, while centres were retrospectively analysed. Data was available for 75 no pt had visceral acute GVHD. Two pts (1 with SAA and 1 with children (50 male) with a median age at transplant of 3 months CAMT) died from respiratory failure secondary to CMV and adeno- (range one week - 17 months), & median follow-up 3119 days virus infection at 60 and 80 days after HSCT. No pt had EBV-related (range 592-7102). Detailed analysis of T & B cell immune recon- PTLD. With a median FU of 321 days (158-664), 14/16 pts are alive stitution and donor chimerism was compared between the two and disease-free (Fig. 1). Early T-cell recovery was sustained by γ/δ phenotypes. The NK- group comprised mainly SCID-X1, JAK3 defi - T cells, while, after 45 days from the allograft, α/β T cells predomi- ciency and data were pooled with ADA defi cient SCID patients, nated. These results indicate that the infusion of B-cell and α/β+ some of whom had received ERT prior to SCT. The NK+ group was T-cell-depleted hematopoietic progenitors from an HLA-haploi- heterogonous and included RAG1/2, Artemis, RMRP mutations dentical parent is an eff ective option for children with life-threat- and a number of molecularily undefi ned conditions. ening either congenital or acquired non-malignant disorders. Results: Overall survival was 80%, & was strongly infl uenced by donor selection, with 90% of matched sibling/family donor transplants surviving, compared to 60% of MUD or haploidentical procedures. Survival was not signifi cantly greater in infants transplanted <3 months of age (83%) compared to older children (77%). Infants with absent NK cells (n=51) were more likely to survive than NK+ recipients (n=24) (86% v 67%, p<0.05). Long- term recovery of CD4 T cell immunity was notably higher in NK- compared to NK+ forms of SCID with a mean of (996x10e6/ml v 465x10e6/ml, P<0.01). Donor T cells chimerism was above 90% for NK- disorders (and ADA SCID) but 68% in the NK+ group. In addition, when measured, thymic output as quantifi ed by TREC analysis was greater in NK- patients. Importantly, 32% of children with NK+ disorders underwent 2nd procedures, compared to 8% of children in the NK- group (p<0.005). Conclusion: Our data suggest that NK- SCID disorders are permissive for donor T cell (and committed T cell progenitor) engraftment without pre-conditioning, whereas an intact NK compartment impedes donor cell engraftment. This study will help inform decisions when determining which children with SCID should undergo conditioned procedures.

S50 O336 Conclusions: The results of this study about HSCT in children with Reticular dysgenesis: international survey on clinical presen- MIOP show this is an eff ective treatment modality that should be tation, transplantation and outcome consider urgent for saving vision and hearing. An umbilical cord M. Hoenig, C. Lagresle-Peyrou, U. Pannicke, M. Cowan, P. Stepensky, blood and haploidentical related donors, however, should be H. Al-Mousa, A. Al-Ghonaium, D. Al-Zahrani, A. Gennery, M. Slatter, used with caution for patients with MIOP. B. Gaspar, K. Oshima, K. Imai, H. Yabe, L. Noroski, N. Wulff raat, K-M. Debatin, A. Schulz, A. Fischer, W. Friedrich, K. Schwarz, M. Cavazzana-Calvo on behalf of the EBMT Working Party Inborn Errors O338 Haematopoietic stem cell transplantation (HSCT) in adult Introduction: Reticular Dysgenesis is defi ned by the combination patients with homozygous thalassemia of SCID, agranulocytosis and sensorineuronal deafness on the S. Santarone (1), P. Bavaro (1), P. Olioso (1), F. Papola (2), P. Accorsi (1), basis of mutations in Adenylate kinase 2 (AK2). S. Angelini (1), P. Di Bartolomeo (1) Objective and Methods: Data sets on clinical presentation, trans- (1)Ospedale Civile (Pescara, IT); (2)Ospedale San Salvatore plantation and outcome from 27 pts were collected from centers (L’Aquila, IT) in Europe, USA, Saudi Arabia, Kuwait, Japan and Israel. Results: Age at presentation was <1 week in 21/27 pts. One patient HSCT is an established therapy in children and adolescents with died prior to transplantation (sepsis). Mean age at transplantation thalassemia major (TM). In this study we report the outcome of was 3.4 months (0.5 to 11 months). Grafts originated from haploi- HSCT in 25 adult patients with TM who were transplanted either dentical donors in 17/26 pts, from matched family donors in 4/26; from HLA-identical sibling (n=23) or matched unrelated donor in 5/26 pts unrelated cord blood was used. Mean follow up for all (MUD) (n=2) between May 1989 and May 2011. Table 1 shows pts alive (17/27) is 6.7 years (0.2 to 23.3 years). Causes for early the clinical characteristics of patients and donors. Conditioning death (< 12 months; n=6) after transplantation were infections, regimen consisted of oral Busulfan (14 mg/Kg) and Cyclophos- toxicity or GvHD. Causes of death beyond 12 months (n=4) were phamide (200 mg/Kg) for 23 patients and Thiotepa (8 mg/Kg), all associated with neutropenia due to prolonged non or merely Treosulfan (42 g/m2) and Fludarabine 160 mg/m2 for the 2 patients partial engraftment, including two pts who developed MDS after transplanted from MUD. For graft-versus-host disease (GvHD) prolonged G-CSF stimulation and died after retransplantation. In prophylaxis all patients received Cyclosporine and short course contrast to this association of mixed chimerism with neutropenia, Methotrexate. The median number of nucleated marrow cells was two patients are stable long term survivors (10.6 and 23.3 yrs) with 4,1 x108/kg (1.9-7.6). Engraftment of 100% donor cells was docu- mixed chimerism but without neutropenia. Transplantation with- mented in 24 patients. The median time to achieve 0.5 x 109/L out conditioning (n=7) failed in all but one attempt. Conditioning neutrophils and 50 x 109/L platelets was day 16 (10-37) and day regimens leading to stable complete chimerism were myeloabla- 26 (12-43) respectively. The cumulative incidence (CI) of neutro- tive in most but not all cases. phil engraftment was 96+0.03%. Primary graft failure occurred in Conclusion: In comparison to other SCID entities considerable diff er- 1 patient. This patient showed thalassemia reconstitution after ences become evident. Even though the mechanism of posttrans- second HSCT from the same donor and is now living with transfu- plant neutropenia is not understood in detail, this phenomenon sion therapy. The CI of grade II-IV acute GvHD was 12+0.11%. Acute did not occur in patients with complete donor chimerism, which GvHD occurred in 10 patients and was grade I in 7 and grade II in therefore should be targeted in order to cure the disease. 3. Chronic GvHD occurred in 4 patients (2 limited, 2 extensive) and was resolved in all cases. The 5-yr CI of overall chronic GvHD (limited + extensive) was 14+0.09%. In the fi rst 12 months after HSCT, O337 8 patients developed CMV reactivation (CI 32±0.27%), 6 bacterial Treatment of malignant infantile osteopetrosis with haematopoietic stem cell transplantation A. Hamidieh, M. Behfar, A. Hosseini, L. Sharifi Aliabadi, S. Basirpanah, A. Ghavamzadeh Tehran University of Medical Sciences (Tehran, IR)

Objective: Malignant infantile osteopetrosis (MIOP) is a rare inherited disorder caused by osteoclast failure. Children with MIOP have life-threatening complications as a consequence of their disease, including hearing loss, blindness and bone marrow failure. At this time, the only potentially curative treatment option for MIOP is a successful hematopoietic stem cell transplant (HSCT). The aim of this study is to analyze the results of transplantation in these patients. Methods: This analysis identifi ed 20 MIOP patients with a median age of 20 months (range: 5-52) who received HSCT between 2007 and 2012. 12 patients were male. Patients underwent transplantation from HLA-identical sibling donors (n=7), fully matched other related donors (n=8), HLA-haploidentical related donor (n=2), unrelated partially matched cord blood donor (n=2) and unrelated full matched donor (n=1). All patients received busulfan and cyclophosphamide with or without ATG for conditioning regimen. The median numbers of MNC and CD34 injected were 6.71×108 cell/kg, 3.27×106 cell/kg, respectively. Cyclosporine with or without methotrexate were used as Graft-versus-host disease (GvHD) prophylaxis regimen. Results: Except one, patients engrafted successfully. The median times for neutrophil and platelet recovery were 16 and 24 days, respectively. With median follow-up of 16 months, 16 patients are still alive and all of them are disease free. Three patients who had grad III-IV acute GvHD had a favorable response to therapy. No patient developed chronic GvHD. 3 patients (1 fully matched other related donors, 1 haploidentical donor and 1 unrelated partially matched cord blood donor) died because of sepsis and ICH.

S51 septicemia (CI 24±0.32%), 9 hemorrhagic cystitis (CI 36±0.27%), O340 4 central nervous system complications (CI 16±0.17%), 1 invasive Favourable outcome of children with high-risk AML treated aspergillosis (CI 4±0.17%) and 2 veno-occlusive disease of the liver with either autologous or allogeneic haematopoietic stem (CI 8±0.07%). There was only one case of transplant-related death cell transplantation in the AIEOP LAM 2002/01 protocol due to idiopathic pneumonitis on day 41. The 5-yr CI of transplant- R. Masetti (1), R. Rondelli (1), M. Zecca (2), F. Fagioli (3), A. Rovelli (4), related mortality (TRM) was 4+0.25%. As of December 2012, 24 A. Mastronuzzi (5), C. Messina (6), E. Lanino (7), C. Favre (8), F. Porta patients are living and 23 of them are cured after a median fol- (9), M. Ripaldi (10), A. Pession (1), A. Prete (1), F. Locatelli (11) low-up of 16.8 years (1.5-25). The 10-yr Kaplan Meyer probability (1)Sant’Orsola-Malpighi Hospital (Bologna, IT); (2)IRCCS S.Matteo of overall survival and thalassemia-free survival was 96+3.9% and (Pavia, IT); (3)Regina Margherita Hospital (Turin, IT); (4)S.Gerardo 92+5.4% respectively. All surviving patients have resumed normal Hospital (Monza, IT); (5)IRCCS Bambino Gesù Children’s Hospital life activities and full-time work. Seven pregnancies were recorded (Rome, IT); (6)University of Padua (Padua, IT); (7)IRCCS G.Gaslini in 2 female patients and in partner of 1 male patient and resulted (Genoa, IT); (8)S.Chiara Hospital (Pisa, IT); (9)Spedali Civili (Brescia, in 8 normal babies. This study demonstrates that HSCT can cure IT); (10)Santobono-Pausillipon Hospital (Naples, IT); (11)IRCCS the majority of adult patients with TM with a very low TRM. Bambino Gesù Children’s Hospital (Rome, IT)

Introduction: HSCT is one of the most widely used post-remis- sional therapy in pediatric patients (pts) with AML in fi rst complete Paediatric issues 2 remission (CR), being able to off er a high chance of cure. Although the role of autologous (AUTO) HSCT remains controversial, the effi cacy of allogeneic (ALLO) HSCT from matched family (MFD) O339 and unrelated donor (MUD) has been confi rmed in high-risk (HR) The role of stem cell transplantation in paediatric patients with patients. We here report the results of the AIEOP AML 2002/01 chronic myeloid leukaemia in the era of tyrosine kinase inhibitors Protocol, which was designed to evaluate whether a large use of M. Suttorp, J.T. Tauer, N. von Neuhoff , C. Nowasz, J. Bradtke, A. Teigler- HSCT in HR pts was able to increase the 5-year event free survival Schlegel, C. Thiede & all participants of the study group CML-PAED (EFS) above 50%. Materials and Methods: In the AIEOP LAM 2002 Protocol pts with Background: In chronic myeloid leukaemia (CML) the very good core binding leukemia reaching CR after the fi rst induction course outcome of up-front treatment by tyrosine kinase inhibitors (TKIs) were Standard Risk (SR), whereas all the others were HR. After 2 like imatinib (IM), dasatinib (DA), or nilotinib (NI) has resulted in courses of standard induction therapy, HR pts in CR received 2 postponing stem cell transplantation (SCT) as 2nd or 3rd line high-dose cytarabine-based consolidation courses and under- therapy. This also holds true in the small group of paediatric CML went either ALLO or AUTO HSCT depending on the availability of a patients (pts). IM has been licensed since 2003 for pts <18 years, MFD. Infants, pts with either AML-M7, a complex karyotype (CK) or while for this age group the role of DA or NI is evaluated in ongoing FLT3-ITD positive, were eligible to receive HSCT from alternative trials. Since 2007 pts <18 years old with newly diagnosed CML were donors. A myeloablative conditioning regimen combining Busul- registered prospectively into the ongoing trial CML-PAED-II off er- fan (4mg/Kg/day for 4 days), Cyclophosphamide (60mg/Kg/day ing a standardized therapy with imatinib (300 mg/sqm in chronic for 2 days) and Melphalan (140mg/m2) was employed for both phase (CP); 400 mg/sqm in accelerated phase (AP); 500 mg/sqm in AUTO- and ALLO-HSCT. Pts given AUTO-HSCT had in vitro marrow blast crisis (BC) followed by SCT) under regular monitoring over a purging with mafosfamide. minimum time period of 2 years. Primary goal was to monitor the Results: Two hundred and thirty-fi ve HR pts received HSCT (102 response rates (haematological, cytogenetic, molecular), the side AUTO, 133 ALLO) in fi rst CR. Fifty-eight (25%), 45 (19%), and 30 (13%) eff ects, and in case of treatment failure (intolerance or resistance) received a MFD, MUD, and partially MUD (PMUD) respectively. The to switch to either 2nd line TKI treatment or SCT on an individual 100-day transplantation-related mortality was 2% and 0.7% for basis. We here report on the course of CML and decisions taken at AUTO and ALLO respectively. The 5-year cumulative incidence (CI) defi ned checkpoints and / or based on failure of TKI treatment. of relapse after AUTO- and ALLO-HSCT was 28% and 17% respec- Results: As of Nov. 2012 with a median recruitment rate of 20 pts tively (p= 0.043) (Figure 1). The 5-year EFS and overall survival (OS) annually, 130 pts have been enrolled (55 fem., 75 male; mean age: for pts given AUTO-HSCT was 63% and 76% respectively, while it 10.8 yrs; range: 1 – 17 yrs). Stage of disease was CML-CP (n=120), was 73% and 74% for pts given ALLO-HSCT (71% and 72%, 80% CML-AP (n=6), and CML-BC (n=4; each 2 myeloid and lymphoid). and 85%, 73% and 70% for MFD, MUD, and PMUD respectively) Out of the total cohort, 104 pts (80%) still are on IM (see Figure). (Table 1 and Figure 1). With a median follow-up of 57 months (3- All 4 pts in CML-BC achieved remission and were transplanted 130), the OS, EFS, and disease-free survival of HR patients enrolled within 6 mo. after diagnosis. Also 1 pt in CML-CP with a sibling in the protocol were 63%, 53% and 63% respectively. donor opted for SCT after 1 year of treatment although respond- Conclusions: A wide use of HSCT in HR pts enrolled in the LAM ing optimal to IM treatment. 21/126 pts (17%) in CML-CP expe- 2002/01 protocol resulted into an EFS above 50%. While the 100- rienced failure; in this situation 4/21 were transplanted from day CI of TRM was low in both pts given AUTO and ALLO-HSCT, the matched unrelated (MUD) or sibling donors and 17 switched to CI of leukemia relapse was higher in the former ones. A particularly DA. Because of insuffi cient response (n=4) or because a MUD was good outcome was observed in pts transplanted from an UD. identifi ed (n=6), in the remaining 10 pts DA was followed by SCT. 2/19 pts (11%) died after SCT, one from SCT-related complications and the other one with CML-BC from relapse. 2/7 pts on DA (29%) had to be switched to NI because of insuffi cient response. Conclusion: Although a MUD can be identifi ed for approx. 70% of pts, only a minority opted for SCT as 2nd line treatment. Side eff ects of IM are well tolerated by the majority of paediatric pts while the role of 2nd generation TKI still has to be clarifi ed in this age group.

S52 [O340]

O341 O342 Allogeneic stem cell transplantation in infants below one and Allogeneic haematopoietic stem cell transplantation (HSCT) two years old with acute leukaemia: results of a retrospec- for juvenile myelomonocytic leukaemia (JMML) in France: tive study in EBMT centres from 2000 to 2010, on behalf of a retrospective study of Société Française de Greff e de EBMT-PDWP Moelle et de Thérapie Cellulaire M. Fahd, J. Dalle, M. Zecca, Y. Bertrand, A. Toren, G. Dini, M. Diaz, D. Meyran (1), R. Porcher (2), N. Raus (3), M. Strullu (4), M. Ouache (1), C. Diaz De Heredia, F. Rialland, G. Michel, A. Seraihy, J. Cornish, K. Yakouben (1), C. Galambrun (5), B. Neven (6), P. Lutz (7), H. Cavé A. Salmon, C. Peters on behalf of the paediatric diseases working party (1), J-H. Dalle (1) (1)Hopital Robert Debré (Paris, FR); (2)Hopital Saint Louis (Paris, FR); Infant acute leukemia (AL) is a rare disease accounting for about (3)Hopital Edouard Herriot (Lyon, FR); (4)CHU Nantes (Nantes, FR); 4% of childhood leukemias. The main diagnoses are ALL and AML (5)Hopital La Timone (Marseille, FR); (6)Hopital Necker (Paris, FR); but there are some rare cases of AL of ambiguous lineage (ALAL). (7)CHRU Strasbourg (Strasbourg, FR) The outcome of older children has improved over decades to 85% OS for ALL, and 60% for AML, whereas infants below 2 y-o Juvenile myelomonocytic leukemia (JMML) is a rare and aggres- still have a worse prognosis due to very aggressive disease and a sive childhood haematological malignant disorder. Allogeneic higher TRM when the therapy includes HSCT. HSCT is the only proven curative therapy. This study reports more Treatments of infant AL includes intensive chemotherapy fol- than 20 years (1986-2011) of French experience in HSCT for chil- lowed in many cases by allogeneic HSCT for patients in CR, given dren with JMML. the overall poor prognosis and high relapse rates. However, HSCT Outcome of 107 HSCT (91 children) performed between March may be accompanied by severe acute toxicity in these very young 1986 and November 2011 in 18 French centres was retrospectively and fragile patients, and also by early relapse. Inevitably, patients studied. Overall Survival (OS), Non Relapse Mortality (NRM), Graft who survive may be expected to suff er from signifi cant long-term Versus Host Disease (GVHD) and relapse cumulative incidences sequelae. were analyzed per patient from the fi rst transplantation. Multi- We report the outcome of infant with AL below one (n=210) and variate analyses were performed to assess risk factors. Statistical two year-old (n=605) who underwent allogenic HSCT in EBMT analyses were carried out according to guidelines of European centers from 2000 to 2010. We examined the characteristics of group for Blood and Marrow Transplantation (EBMT). patients (age, sex, disease status at HSCT), stem cell source and Ninety-one children (58 males), median age 1.4 yrs (range 0.0-15,7 HLA match, conditioning regimen (myeloablative vs reduced- yrs) with JMML underwent 107 allogeneic HSCT (14 second graft intensity ; use of radiotherapy), GvHD prophylaxis, engraftment, and a third one). The median follow-up was 47 months (1-166). At toxicity, relapse rate, GvHD, death, OS and EFS. 72 months, OS was 59% (95% CI: 47-74). Cumulative incidence of Stem cell sources were 1. For patients below 2 years: bone marrow aGVHD was 48% (95% CI: 37-58). The 6-year cumulative incidence for 292 (48,3%) ; peripheral blood for 146 (24,1%) and cord blood of relapse and NRM was 34% (95% CI: 23-44) and 21% (95% CI: 13- for 159 (26,3%); 2. for patients below 1 year: 101 (48,1%), 45 (21,4%) 32), respectively. The median delay of relapse was 80 days (range and 58 (27,6%) respectively. Graft failure occurred in 3,8% and 4, 15-1096). In multivariate analysis, age at HSCT older than 2 years, 8%, and secondary graft failure in 0,7% and 0, 5% respectively. female donor to male recipient sex-mismatch, matched unrelated Neutrophil recovery was at a median of 17 days [3-97] in both or mismatched non cord donor, total body irradiation and no populations. Grade III/IV aGvHD occurred in 8,1% and 2,5% of pts. serotherapy in conditioning regimen predicted poorer outcomes. 5 year-OS is 55% and 58% for infants below 2 and 1 year-old, Age at HSCT older than 2 years was an increased risk of relapse respectively. 236/605 (39%) infants below 2 years-old and 79/210 whereas Busulfan/Cyclophosphamide/Melphalan conditioning (37.6%) below 1 year-old died from either TRM (10.6 and 12.4%, regimen was a decreased risk. Serotherapy was associated with a respectively) or relapse (25.3 and 37.6%, respectively). decreased risk of NRM. Age at HSCT older than 2 years and male This retrospective study demonstrates that more than half of recipient were associated with higher probability of dying of NRM patients presenting with infant acute leukaemia can be cured causes. by chemotherapy and allogeneic HSCT. However, the results still Our results show that allogeneic HSCT may cure approximately remain sub-optimal and new strategies should be developed 60% of patients with JMML and are similar to best results pub- through international co-operation leading to better disease lished by other groups. Relapse represents the main cause of control pre-transplantation. There should be better defi nition of treatment failure and a second HSCT should be proposed. Novel optimal SCT timing, donor selection, conditioning regime, GVHD strategies to decrease the risk of relapse are needed. prophylaxis and supportive care in this specifi c context of fragile patients with aggressive disease.

S53 O343 grafts (CB). Complete HLA matches were defi ned as 10/10 (or 6/6 Impact of serotherapy on the outcome after stem cell in case of CB). Patients were grouped for analysis according to transplantation in children: ATG versus Campath-1H whether conditioning included ATG (n=169) or C1H (n=41). C.M. Jol-van der Zijde, L. Willemsen, H. Putter, A.C. Lankester, Results: Children treated with ATG showed a signifi cantly better R.G.M. Bredius, M.J.D van Tol survival than the children treated with C1H (p=0.016, Log Rank Leiden University Medical Centre (Leiden, NL) test, Figure 1a.). Multivariate Cox regression analysis confi rmed this and showed a better survival in the ATG group compared with Objective: The outcome after allogeneic haematological stem the C1H group (HR=2.80; p=0.001). Time to engraftment (fi rst day cell transplantation (HSCT) is strongly aff ected by the kinetics neutrophils >0.5×109/L; HR=0.62; p=0.020) and the recovery of of reconstitution of the immune system. Serotherapy with anti- T-cells (fi rst day >100/uL; HR=0.36; p<0.001; Figure 1b) and thymocyte globulins (ATG) or Campath-1H (alemtuzumab, C1H) is NK-cells (fi rst day > 50/uL; HR=0.55; p=0.003) was signifi cantly often used as part of the conditioning regimen to prevent graft faster in the ATG group; no signifi cant diff erence was found for rejection and acute graft versus host disease (aGvHD). This study the recovery of B-cells. compared the eff ects of ATG and C1H on various outcome param- Compared to the ATG group, children treated with C1H had sig- eters after HSCT in children treated in a single SCT unit. nifi cantly more disseminated adenovirus infections (log DNA load Patients: The study cohort consisted of 210 children, median age in serum at least 2x >3.0; HR=4.79; p=0.004) and less EBV infec- 8.1 years (range 0.2-19.0), who underwent HSCT for malignant and tions (HR=0.20; p=0.009); no signifi cant diff erence was found for non-malignant haematological disorders in our centre between CMV infections. There was no signifi cant diff erence in occurrence January 2003 and May 2012. The median follow-up period was 4 of aGvHD (p=0.380) nor severe aGvHD (grade II, III or IV, p=0.400). years, 137 patients received bone marrow grafts (BM), 46 patients In a multivariate regression analysis all these outcomes were cor- peripheral blood stem cells (PBSC) and 27 patients cord blood rected for diagnose, grafttype, HLA match and age. Conclusion: Our results show that children treated with ATG as part of the conditioning regimen have a better overall survival and a faster T-cell and NK-cell reconstitution after HSCT than children treated with C1H.

Infectious diseases

O362 Donor CMV status infl uence on the outcome of allogeneic stem cell transplantation (HSCT) P. Ljungman, R. Brand, R. de la Camara, C. Cordonnier, H. Einsele, J. Styczynski, K. Ward, S. Cesaro for the Infectious Diseases Working Party of the EBMT

A previous study from the IDWP of the EBMT showed that CMV seropositive (pos) patients undergoing unrelated donor HSCT had increased non-relapse mortality (NRM) and decreased survival if they were grafted from a CMV seronegative (neg) donor. This fi nding has been controversial and we therefore decided to revisit the question with a larger number of patients and including new factors such as conditioning intensity. Patients were selected from the EBMT database who had received an allogeneic HSCT from 1992-2008 and for whom both the donor and recipient CMV antibody status were known. Patients receiving cord blood grafts were excluded. 54660 patients were identifi ed and included in the study; 32320 CMV pos and 22340 CMV neg patients. The diff erent donor categories (sibling, mismatched family, unrelated) were analyzed separately. Cox multivariate models were fi tted to estimate the eff ect of donor serological status (pos. vs neg) on outcome both in CMV pos and CMV neg patients adjusted for year of HSCT, donor and patient gender, recipient age, stem cell source, diagnosis, use of alemtuzumab or ATG, country, and conditioning intensity (RIC vs. MAC). Seronegative patients receiving grafts from CMV pos unrelated donors had a decreased overall survival (OS; HR 1.13; p<.01); relapse free survival (RFS; HR 1.10; p<.01) and increased non- relapse mortality (NRM; HR 1.13; p<.01) while no signifi cant eff ect was seen on relapse incidence (RI; HR 1.06). There were no signifi cant eff ects in patients receiving HLA-identical or mis-matched family donors. Seropositive patients receiving grafts from CMV pos unrelated donors had improved OS (HR 0.91; p<.01), RFS (HR 0.94; p<.05), and decreased NRM (HR 0.87; p<.01) if they had received MAC. These patients were less likely to die from viral infections than patients receiving grafts from CMV neg donors (p=.005). No eff ect of donor serostatus was seen in patients receiving unrelated donor grafts after RIC. There were no signifi cant eff ects in patients receiving HLA-identical or mis-matched family donors. A separate analysis regarding RI was performed only in AML patients in fi rst CR. No eff ect of donor serostatus on RI was found in either CMV pos or neg patients.

S54 In this analysis we confi rm the negative eff ect on OS, RFS, and NRM hematological diseases between January, 2006 and December, if a CMV seropositive unrelated donor is selected for a CMV sero- 2010 in Japan were included in this study. Data were collected negative patient. For a CMV seropositive patient our data support and analyzed retrospectively using the Transplant Registry the choice of a CMV seropositive unrelated donor if the patient is Unifi ed Management Program (TRUMP) of Japan Society for planned to receive a myeloablative conditioning regimen. Hematopoietic Cell Transplantation. Results: The 2-year cumulative incidence of IFI after HSCT was 11.2% (696 recipients), including 289 invasive candidiasis (IC) O363 (2.1%) and 953 invasive aspergillosis (IA) (7.1%). Among the 262 Role of BK virus-specifi c immunity in BKV-related Candida isolates from IC patients, the most common species was haemorrhagic cystitis after paediatric allogeneic HSCT C. parapsillosis (n = 77, 29.4%) and non-albicans Candida spp. L. Rubert, G. Quartuccio, I. Giudo, A. Gurrado, A. Pellerano, L. Calafi ore, were more commonly isolated than C. albicans (n = 32, 12.2%). F. Baldanti, T. Mina, C. Cugno, S. Basso, M. Zecca, P. Comoli Median onset of IFI was 70 (1 - 1162) days after HSCT. Risk factors Fondazione IRCCS Policlinico S. Matteo (Pavia, IT) which were signifi cantly associated with the incidence of IFI were adult, male, advanced disease status, CBT, HLA-mismatched HSCT, Hemorrhagic cystitis (HC) is a cause of morbidity and even mortal- more than one number of HSCT, reduced intensity conditioning, ity in patients receiving hematopoietic stem cell transplantation poor performance status at HSCT, active infectious disease at (HSCT). Polyomavirus BK (BKV) has emerged as the main infec- HSCT and chronic graft-versus-host disease. The 2-year cumu- tious cause of HC. It is controversial whether BKV-associated HC is lative incidence of IFI among patients who developed chronic mainly caused by virus-mediated cytopathic damage, or rather by graft-versus-host disease was 12.7% compared with 9.8% among an infl ammatory reaction to the virus. patients without chronic graft-versus-host disease (p<0.0001). Thus, the aim of our study was to investigate the role of BKV- The recipients who developed IFI after allogeneic HSCT showed directed cellular immunity in BKV-related HC. BKV specifi c immu- signifi cantly poor overall survival (29.7% vs 52.3% at 2 years, nity was evaluated prospectively in a cohort of 57 pediatric p<0.0001) due to high TRM (47.5% vs 22.2% at 2 years, p<0.0001). recipients of HSCT from an HLA-matched unrelated (MUD, n=31) Among them, the overall survival rate was signifi cantly lower for or haploidentical family (Haplo, n=26) donor. We measured by patients with IC than patients with IA (25.9% vs 31.2% at 2 years, interferon-γ (IFN-g) ELISPOT assay the frequency of BKV VP1 and p<0.0001). Median follow-up of surviving patients was 2.2 years. large T antigen-specifi c peripheral blood mononuclear cells. The The 60-day survival rate ranged from 52% for patients with IC to response to CMV pp65 and PHA was also evaluated. 70% for patients with IA. In our cohort, 26 pts (45%) developed BK viruria at a median time Conclusions: IFI occurred frequently and recipients who of 22 days after HSCT, with 19 progressing to HC. Overall, patients developed IFI after HSCT, especially IC showed poor overall who developed BK viruria showed a status of cellular immune survival due to high TRM, even now. defi ciency (response to PHA and to BKV signifi cantly lower than that observed in pts BKV-negative). There was a trend towards HC progression for recipients of a haplo-HSCT vs MUD (46% vs 22%, O365 p=0.06), whereas no correlation was found with TBI-based con- Gene polymorphism profi le may predict invasive aspergillosis ditioning. The parameter that showed the strongest correlation development in patients with acute leukaemias undergoing with HC development was the presence of T-cell immunity to BKV allogeneic stem cell transplantation LT antigen (p<0.01), whereas no correlation was found with the L. Gil, M. Wojtaszewska, A. Mol, D. Poplawski, K. Lewandowski, presence of response to BKV VP1 antigen, or CMV pp65 control M. Komarnicki antigen. In patients with HC, resolution of symptoms and clear- University oF Medical Sciences (Poznan, PL) ance of BKV in plasma and/or urine correlated with emergence of BKV-specifi c cellular immunity (response to BKV at HC and at reso- Invasive aspergillosis (IA) remains a major cause of morbidity and lution: median SFU/105 LT 0 vs 38; VP1 0 vs 34). Our data suggest mortality in pts undergoing alloSCT. The association between that failure of BKV immune surveillance due to a low frequency single nucleotide gene polymorphism (SNP) of components of of BKV-specifi c T cells favours BKV replication in the injured uro- the innate immune system and the risk for IA development has thelium, and may promote progression to HC. These data support been reported recently. Objective: analysis of molecular risk for the view that BKV-associated HC is not an immune reconstitution- IA development in pts with acute leukemias undergoing alloSCT driven pathology. based on gene polymorphism studies. Material and methods: Gene-candidates (TLR4, DECT1, IFNG, TNFR2) were based on published data and our previous research. The primer for TLR4 SNP was O364 designed using Lasergene Primer Select. IFNG, TNFR2 and DECT1 Invasive fungal infections after allogeneic haematopoietic polymorphisms were screened with published primers. PCR con- stem cell transplantation in Japan ditions were common to all reactions. Gene polymorphisms were K. Oshima (1), S. Taniguchi (2), S. Kurosawa (3), H. Ogawa (4), analyzed in a cohort of 90 pts with primary or secondary AML K. Ohashi (5), T. Eto (6), H. Sakamaki (5), H. Yabe (7), Y. Morishima (8), (64) or ALL (26) and their sibling (36) or unrelated (54) donors. Pts K. Kato (9), R. Suzuki (10), T. Fukuda (3) were conditioned with myeloablative (54) or RIC (36) regimen and (1)St. Luke’s International Hospital (Tokyo, JP); (2)Toranomon grafted with 3.5 (0.8-5.2)x106/kg of CD34+ cells. Standard defi ni- Hospital (Tokyo, JP); (3)National Cancer Center Hospital (Tokyo, JP); tions for neutropenic, bacterial, fungal and viral infections were (4)Hyogo College of Medicine (Hyogo, JP); (5)Tokyo Metropolitan used. Results: neutrophil recovery occurred in all pts at a median Cancer and Infectious Diseases Center Komagome Hospital (Tokyo, 22 (range; 13-36) days with complete donor chimerism by day +30. JP); (6)Hamanomachi Hospital (Fukuoka, JP); (7)Tokai University Infectious complications included neutropenic fever in 81 (90%), School of Medicine (Isehara, JP); (8)Aichi Cancer Center Research CMV infection in 39 (43.3%) and documented IA in 22 (24.4%) Institute (Nagoya, JP); (9)Japanese Red Cross Nagoya First Hospital pts (proven-4 and probable-18). Acute GVHD 2-4 grade was seen (Nagoya, JP); (10)Nagoya University Graduate School of Medicine in 26 (29%), while chronic extensive in 9 (10%) pts. Among pre- (Nagoya, JP) transplant factors (donor/recipient gene polymorphisms, age, sex, disease stage) the only signifi cant factor for development of Purpose: Invasive fungal infection (IFI) is one of the leading documented IA, by multivariate logistic regression analysis, was causes of transplant-related mortality (TRM) and its incidence in donor DECT1 SNP (p=0.016, HR 5.7). Among transplant-related allogeneic hematopoietic stem cell transplantation (HSCT) factors (dose and source of stem cells, intensity of conditioning, recipients varies among the reports. neutropenia duration, GVHD) signifi cant for IA development was Patients & Methods: Thirteen thousands fi ve hundred twenty-two CMV infection (p=0.021, HR=5.3). Over a median follow-up of 26.5 patients (7,848 males and 5,674 females) with a median age of (range; 2.5-77.5) months, 65 (72.2%) pts were alive with median 40-year-old (range 0-88) who underwent allogeneic HSCT for survival 27.5 (95%CI=5.0-78.9) months. Among 25 (20%) deaths,

S55 8 were related to IA. None of analyzed factors was signifi cant for seropositive third-party donors is a veritable option, particularly death from infections. No factors predicting the outcome of IA for infections following allogeneic cord-blood stem-cell trans- pts were identifi ed. Conclusion: Our data suggest that the donor plantation (SCT) or SCT from virus-seronegative donors. DECT1 SNP may be used as a prognostic factor for IA develop- To gain more insight into virus-specifi c memory T-cell pools in ment in acute leukemia pts after alloSCT. SNP profi le, performed healthy subjects and to identify the most effi cient antigens for with easy and cheap assay, may guide antifungal prophylaxis and adoptive immunotherapy, we assessed the frequencies of CMV, preemptive treatment in this group of patients. EBV and ADV-specifi c memory T cells in 204 HLA-typed healthy donors using peptides and peptide pools available in GMP quality. For each virus, we identifi ed at least 124 (61%) potential CTL O366 donors with highly signifi cant diff erences in frequencies of T cells Cytomegalovirus antiviral drug resistance and disease in against six viral antigens of CMV, EBV and ADV. Antigen-specifi c T patients receiving pre-emptive antiviral treatment after cells were detected in 100% of CMV-seropositive donors (n=124), haematopoietic stem cell transplantation 73% of EBV-seropositive donors (n=195) and 73% of ADV-sero- E. Shmueli, R. Or, M.Y. Shapira, I.B. Resnick, O. Kaplan, D.G. Wolf positive donors (n=196). Among the tested antigens frequen- Hadassah University Hospital (Jerusalem, IL) cies for CMV pp65 and EBV BZLF1 peptide pool were highest. Overall frequencies of peptide-specifi c T cells detected by T-cell Objectives: Preemptive antiviral therapy has reduced the occur- receptor staining were lower than those of the corresponding rence of early cytomegalovirus (CMV) disease after hematopoietic peptide pools. Short-term in vitro peptide stimulation assays stem cell transplantation (HSCT), however the development of revealed that, in the case of ADV and EBV, a donor response to antiviral drug resistance and late disease is increasingly recog- a certain peptide may not be determined with prior stimulation. nized. In this study we determined the rate and clinical impact of A modifi ed granzyme B ELISpot was established and used to detect antiviral drug-resistance, as well as the associated risk factors in T-cell specifi city and alloreactivity. Moreover, confi rmatory testing HSCT recipients receiving preemptive treatment. for CMV serology using western blot technique revealed 19/143 Methods: The study included 561 patients undergoing HSCT at (13%) false-positive results, possibly impacting future analysis the Hadassah University Hospital over a 5-year period. All patients and selection of potential stem cell and T-cell donors. were prospectively monitored for CMV viral load in peripheral blood, and received preemptive antiviral treatment. Prospec- tively-obtained specimens of patients with persistent/recurrent O368 infection or increasing viral load were subjected to analysis of CMX001 is a potential treatment for Adenovirus infection: drug-resistance mutations in the CMV UL97 and UL54 genes. preliminary antiviral activity results from an open-label, Results: CMV infection was detected in 62/206 (30.1%), 176/263 expanded access study of CMX001 for the treatment of (66.9%), 26/45 (57.8%), and 69/102 (66.7%) patients undergoing serious or life-threatening diseases caused by double- autologous, full-matched-, mismatched-, and haploidentical allo- stranded DNA viruses geneic HSCT, respectively. Importantly, drug-resistance mutations M. Grimley (1), G. Papanicolaou (2), V. Prasad (3), L. Dropulic (4), were exclusively identifi ed in haploidentical HSCT recipients. The H. Mommeja-Marin (5), T. Brundage (5), G. Chittick (5), D. Margolskee (5) rate of drug resistance among haploidentical HSCT recipients with (1)Cincinnati Children’s Hospital Medical Center (Cincinnati, US); CMV infection was high (10/69; 14.5%). Drug-resistance appeared (2)Memorial Sloan-Kettering Cancer Center (New York, US); (3)Duke after prolonged antiviral treatment (median 68d; range 21-330d), University Medical Center (Durham, US); (4)National Institutes of and was associated with higher maximal viral load (P<0.001). Health Medical Center (Bethesda, US); (5)Chimerix, Inc. (Durham, US) Six of 10 patients with drug-resistance mutations developed CMV disease. Overall, haploidentical HSCT recipients were sig- Background: In immunocompromised patients (pts), adenoviruses nifi cantly more vulnerable to the development of CMV disease (AdV) are an important cause of morbidity/mortality. Hemato- than all other HSCT recipients with CMV infection (8/69; vs 1/264; poietic cell transplant (HCT) pts are at especially high risk; infec- P <0.0001). Disease developed at 29-379d post transplantation tion associated with viremia is more severe, often disseminated (median 120d), and was associated with 87.5% mortality. Identi- and, once established, often rapidly fatal without treatment (tx). fi ed risk factors for CMV disease in haploidentical HSCT recipients No antiviral drugs are currently approved for AdV infections, which included drug resistance and higher maximal viral load. are typically managed by supportive care. A virologic response to Conclusion: Despite the overall low rate of antiviral drug resis- tx with intravenous (IV) cidofovir (CDV) has been associated with tance and disease among the total cohort of HSCT recipients, clinical improvement; failure to achieve a ≥1 log10 decrease in our fi ndings reveal a high rate of CMV antiviral drug resistance viral load (VL) during fi rst 2 weeks is associated with poorer pt out- and severe disease in haploidentical HSCT recipients receiving comes. However, IV CDV use is limited by signifi cant side eff ects. preemptive antiviral treatment. These remaining complications CMX001 is an orally bioavailable, broad spectrum, lipid acyclic argue for the potential advantage of a prophylactic rather than nucleoside phosphonate converted intracellularly into the active preemptive treatment approach in high-risk HSCT recipients, and antiviral, CDV diphosphate. We describe here preliminary antivi- highlight the need for new better-tolerable anti-CMV drugs. ral activity data in a subset of 57 AdV infected pts treated with CMX001 in an open label, expanded access study (CMX001 350; ClinicalTrials.gov identifi er: NCT01143181). O367 Methods: Pts received CMX001 twice weekly (BIW), typically 100 mg Adoptive T-cell immunotherapy from third-party donors: BIW for adults/adolescents or 2 mg/kg BIW for pediatrics characterization of donors and set up of a T-cell donor registry (≤12 yrs). AdV VL was measured at baseline (BL), regularly during C. Sukdolak, S. Tischer, D. Dieks, C. Figueiredo, L. Goudeva, H. Heuft, the tx period and 1- and 4 wks post-tx. M. Verboom, S. Immenschuh, S. Borchers, E. Mischak-Weissinger, Results: Most pts were male (61.4%), white (70.2%) and had R. Blasczyk, B. Maecker-Kolhoff , B. Eiz-Vesper received a HCT (84.2%). Median (range) age was 17 (0-68) yrs. Hannover Medical School (Hannover, DE) Median (range) duration of CMX001 tx was 12 (1 64) doses over 7 (1-43) wks. Thirty (30/57, 52.6%) pts survived; 10 (17.5%) died Human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and from AdV associated conditions and 17 (29.8%) from other causes. adenovirus (ADV) infection and reactivation are frequent and Overall, AdV-associated mortality was 37.5% (6/16) for pts with severe complications of hematopoietic stem cell transplantation disseminated disease, 11.5% (3/26) for localized disease, and (HSCT) and solid organ transplantation in immunocompromised 6.7% (1/15) for pts with BL viremia (1/15). Most (43/57) pts had recipients. Adoptive immunotherapy with virus-specifi c cyto- detectable viremia at BL and ≥1 post-BL assessments and were toxic T eff ector cells (CTLs) can eff ectively reconstitute antiviral evaluable for virologic response. Median (range) VL at BL was immunity without causing acute toxicity or increasing the risk of 4.82 (2.30-9.11) log10 c/mL. Thirty-four pts (79.1%) had ≥1 log10 GvHD. Treatment with allogeneic virus-specifi c CTLs from healthy decrease from BL or to below limit of detection (=100 c/mL) at the

S56 end of tx. The median (Q1, Q3) decrease was 1.57 (0.58, 2.64) log10 worldwide, few reports have studied health related quality of c/mL. Pts achieving a ≥1 log10 decrease (including in sputum or life (HRQoL) in the long term. This report investigates HRQoL, stool) had overall better outcomes, with 8.6% (3/35) AdV-associ- clinical and sociodemographic data, with a median follow-up of ated deaths (all within the fi rst 3 weeks of tx) vs 27.3% (6/22) for more than 20 years, in a large cohort of transplanted thalassemia pts with <1 log10 drop. patients. Conclusions: CMX001 appears to be a promising therapeutic Methods: Data on 130 surviving ex-thalassemia patients, trans- option for the tx of AdV infections. planted in the 1980s and 90s, were retrieved from the Sardinian public health demographics database. After a brief interview, patients were sent 2 questionnaires on HRQoL: (SF36) and Functional Assessment of Cancer Therapy–Bone Marrow Trans- Early and late complications plant (FACT-BMT). Sociodemographic data on the healthy Sar- dinian population were obtained from the National Institute of Statistics. Enrollment started in July 2011 and ended in O369 September 2012. Cytokine captured T-cell therapy for adenoviraemia in Results: 109/130 responded to the survey (83.8%, median age children following allogeneic stem cell transplantation 34, range 21-48). Median age at HSCT was 12 years (range 1-36) W. Qasim, K. Gilmour, S. Derniame, H. Zhan, W. Ip, R. Chiesa, K. Rao, with 75% of patients belonging to Pesaro risk classes 2-3. Median P. Hiwarkar, P. Amrolia, P. Veys, H. Gaspar follow up was 22.8 years (range 11.7- 30.3). All patients received Institute of child health, UCL (London, GB) myeloablation with oral busulfan and i.v. cyclophosphamide. Acute and chronic GVHD were registered in 35.8% and 18.3% of Introduction: Viral reactivation following allogeneic SCT can cause the patients, respectively. Within 5 years of HSCT, almost 50% of serious morbidity and mortality. In a recent survey at our centre, the patients reported a series of complications. 74.3% reported at virus related deaths accounted for 15% of overall mortality in pae- least one disease, with a higher incidence for liver, metabolic or diatric SCT, and Adenovirus reactivation was particularly prob- endocrine disorders. Two patients underwent organ transplanta- lematic. Children undergoing unrelated donor or mismatched tion (kidney, liver) and 4 reported a secondary malignancy. Most procedures and receiving serotherapy were highlighted as being patients (74.4%) had either resumed or completed their studies at highest risk of virus reactivation, and this was directly associ- after HSCT, with results comparable to the healthy population ated with delayed T cell reconstitution. Here we report adoptive (Figure 1). Surprisingly, employment status was considerably therapy using donor T cells with ADV specifi city identifi ed and higher among ex-thalassemia patients (77.1%) in comparison isolated by interferon-cytokine capture technology. with a healthy population aged 20-45 years (59.9%). 44.9% of Methods: Following hexon antigen stimulation, ADV specifi c patients lived with a spouse/partner. Overall 23 gave birth to 23 T cells that responded and secreted interferon γ (IFN-g) were healthy babies after spontaneous pregnancies. The resulting birth enriched with a bispecifi c antibody using the Miltenyi CliniMacs rate (4.7%) was only slightly lower than the total rate (7.9%) reg- magnetic bead cell separation system. istered in Sardinia in 2011. SF36 physical functioning (PF) scale Results: Five children received ADV-T cells following allo-SCT for showed good scores, comparable to those obtained from a sex- inherited immune disorders (n=3), autoimmune disease (n=1) or age matched healthy population. Lower scores on general health leukaemia (n=1). In vivo T cell depletion (Alemtuzumab) was given scales were signifi cantly associated with acute or chronic GVHD to four as part of their routine conditioning regimen and CD34 stem (p=0.03). cell enrichment with T cell depletion was used in the fi fth child. Conclusions: Our data provide robust evidence that ex thalas- All children developed adenovirus reactivation in blood within 28 semia patients achieve good HRQoL in the long term after HSCT days after SCT and had received Cidofovir and immunoglobulin with return to normal life style. replacement therapy. Peak viral loads ranged from 5.6x10e4/ml to 11x10e6/ml. Three children received captured T cells from their original stem cell donor and two received third-party haploidentical parental T cells. Flow cytomtery detected interferon–g secretion responses of between 0.13-3.3% which could be enhanced to between 19-64% by selection, yielding doses of 10e4 cells/kg and 10e5 cells/kg. ADV cell therapy was administered on average 80 days after the original stem cell graft, and in 4 children a single dose of freshly selected cells at a dose of 10e4/kg was given. ADV specifi c T cell responses were detectable in these children, with rapid resolution of viraemia in three. One child developed signifi cant skin and liver GVHD, thought to be mediated by bystander activation of alloreactive donor T cells from the original graft. The fi fth child received a single dose of 10e5 cells/kg from a mismatched family donor but died without evidence of antiviral reconstitution. Conclusion: This pilot study demonstrates the feasibility of gen- erating donor derived ADV specifi c T cells using cytokine capture, and shows the potential of cell therapy in resolving viral reactivation after allo-SCT.

O370 Quality of life in thalassemia patients twenty years after transplantation: long term outcome and return to normal lifestyle G. Caocci (1), F. Effi cace (2), A. Vacca (1), E. Piras (1), M. Sanna (3), G. Spanu (3), R. Littera (1), G. Lucarelli (4), G. La Nasa (1) (1)R. Binaghi Hospital (Cagliari, IT); (2)Italian Group for Adult Hematologic Diseases (GIMEMA) Data Center (Rome, IT); (3)University of Cagliari (Cagliari, IT); (4)University of Rome “Tor Vergata” (Rome, IT)

Objectives: Although more than 3000 thalassemia patients have received hematopoietic stem cell transplantation (HSCT)

S57 O371 O372 Prognostic and diagnostic value of endothelial damage Risk factors for thrombotic microangiopathy in allogeneic markers for veno-occlusive disease and transplant-related haematopoietic stem cell recipients receiving graft-versus- mortality after allogeneic stem cell transplantation host disease prophylaxis with tacrolimus plus methotrexate I.S. Moiseev, S.V. Lapin, E.A. Surkova, M. Lerner, E.V. Babenko, A. Sipol, or sirolimus V.N. Vavilov, B.V. Afanasyev J. Labrador (1), L. López-Corral (2), O. López-Godino (2), L. Vázquez Pavlov State Medical University (Saint-Petersburg, RU) (2), M. Cabrero-Calvo (2), R. Pérez-López (2), E. Pérez-López (2), C. Guerrero (1), I. Alberca (2), J.A. Pérez-Simón (3), J.F. San Miguel (2), The aim of this study was to evaluate vascular endothelial growth J.R. González-Porras (2), M.D. Caballero (2) factor (VEGF) A and C, number of circulating endothelial cell (1)Centro de Investigación del Cáncer, IBMC/CSIC-USAL (Salamanca, (CEC) in patients undergoing allogeneic stem cell transplanta- ES); (2)Hospital Universitario de Salamanca (Salamanca, ES); (3)CSIC tion (alloSCT). The relationship between VEGF levels, number of (Seville, ES) CEC and occurrence of veno-occlusive disease (VOD) and transplant-related mortality (TRM) was analyzed. The study included 91 Background and aims: Transplantation-associated thrombotic patients. 73% had acute leukemia, 10% chronic myeloid leukemia, microangiopathy (TA-TMA) is an uncommon but feared complica- 8% myelodysplastic syndrome and 9% other hematologic malig- tion of allogeneic hematopoietic stem cell transplant (HSCT) due nancies. Median age was 38 years (range 15-60), median ECOG to its high mortality rate. Although endothelial injury represents and modifi ed EBMT risk score were 1 (range 0-3) and 4 (range the fi nal common pathway of disease, the exact pathophysiology 1-6) respectively. 30% were grafted from related and 70% from of TA-TMA remains unclear, which could explain that this disor- unrelated donor. 24% received myeloablative conditioning, 76%- der responds poorly to conventional treatments for thrombotic reduced intensity conditioning. VEGF was measured by ELISA, thrombocytopenic purpura. The increasing use of tacrolimus plus CEC were measured by fl ow cytometry (CD45-CD146+CD31+). sirolimus (TAC/SIR) as graft versus host disease (GVHD) prophy- The sampling points were before conditioning, on day 0, upon laxis has been associated with an increased incidence of TA-TMA. engraftment and in case of VOD. However, in a recent phase II multicenter prospective trial (2007- VOD was diagnosed in 15% of patients, severe VOD- in 10%. VEGF 006416-32 trial by GEL-TAMO/GETH) conducted by our group, no A level was signifi cantly higher in VOD than in non-VOD patients diff erences were observed in the incidence of TA-TMA when TAC/ at two time points: on day 0 (p=0.027) and on the day of VOD SIR was compared with patients included in a prior prospective (p<0.01). VEGF C levels were not diff erent between VOD and non- trial with Cyclosporine-Mycophenolate. So, the increased risk of VOD patients at all time points (p>0.05). Overall, 1 year TRM was TA-TMA due to tacrolimus and sirolimus combination it is currently found signifi cantly lower in patients with VEGF A below level of not well defi ned. Our aim was to identify the incidence of TA-TMA quantifi cation of ELISA at day 0 (p=0.047) (fi g.1). Also there was in allo-HSCT recipients who received TAC/SIR GVHD prophylaxis a trend to better event-free survival in this group (59% vs 37%, compared with tacrolimus plus methotrexate (TAC/MTX). Other p=0.078). In the multivariate analysis, that included disease status, objectives were to determine the risk factors, including the role of age, and previous SCT, low VEGF A after conditioning was inde- immunosuppressive toxic levels in the development of TA-TMA. pendently predictive of lower TRM (HR=0.32, 95% CI 0.10-0.98, Methods: We retrospectively analyzed data from 102 allogeneic p=0.046). HSCT recipients who consecutively received TAC/SIR (n = 68) or Evaluating number of CEC showed that it was signifi cantly higher TAC/MTX ± ATG (n = 34) for GVHD prophylaxis. upon development of VOD than in non-VOD patients on day 0 and Results: With a median follow-up of 451 days (range, 28 – 1946 upon engraftment (148±77 vs 56±46 and 40±28 cell/ml, p=0.007 days), no signifi cant diff erences were observed in the incidence of and p=0.001 respectively). When a cut-of value of 100 cells/ml was TA-TMA when TAC/SIR was compared with TAC/MTX ± ATG (7.4% selected, sensitivity and specifi city of this method for diagnosis of vs 8.8%, p = 0.8). Only grade III – IV acute GVHD, previous HSCT and VOD were 75% and 94%. serum levels of tacrolimus > 25 ng/mL were associated with an In conclusion: endothelial damage markers could be used to sup- increased risk of TA-TMA, and we identifi ed 3 diff erent risk groups port the diagnosis of VOD. Also elevated due to cellular-derived with these variables (fi gure 1). Patients developing TA-TMA have secretion and release from extracellular matrix VEGF A after condi- a signifi cantly poorer survival when compared with those without tioning could represent the severity of tissue damage. This param- TA-TMA (p < 0.001). However, when TA-TMA was included in the eter could be used to predict TRM and survival. multivariate model, it did not remain an independent prognostic factor (p = 0.595). Conclusions: The combination of TAC/SIR does not appear to pose a higher risk of TA-TMA compared with TAC/MTX ± ATG for GVHD prophylaxis. Grade III – IV acute GVHD was the strongest determinant of TA-TMA and mortality.

S58 O373 O374 A prospective randomized trial on erythropoietin therapy Disease characteristics and new clinical approaches in following allogeneic haematopoietic cell transplantation bronchiolitis obliterans (BO) after allogeneic haematopoietic A. Jaspers, F. Baron, E. Willems, K. Hafraoui, G. Vanstraelen, C. Bonnet, stem cell transplantation (HSCT) Y. Beguin M. Ditschkowski, D.W. Beelen, T. Gromke, R. Trenschel, N.K. Steckel, M. CHU of Liège (Liège, BE) Koldehoff , A.H. Elmaagacli University Hospital of Essen (Essen, DE) Introduction: Based on the impairment of erythropoietin production observed following allogeneic hematopoietic cell trans- Bronchiolitis obliterans (BO) after HSCT is a terrifying progres- plantation (HCT), we previously reported in a phase-2 trial that sive non-infectious pulmonary complication leading to increased recombinant human erythropoietin (rhEPO) therapy was very morbidity and mortality. effi cient when started one month after transplantation. We also Among 982 long-term (>100 days) surviving patients (pts) after demonstrated that anemia after non-myeloabalative (NM) HCT HSCT between 1/2000 and 10/2010, 68 were diagnosed BO was less sensitive to rhEPO therapy, than after conventional allo- according to NIH criteria. The median onset of BO was 18 months geneic HCT. This prompted us to confi rm these fi ndings in a pro- post-transplant, 5-year cumulative incidence was 5.8% and 5-year spective randomized trial. mortality 41%. Concomitant chronic GvHD (p<0.001), transplan- Patients and methods: 131 patients were randomized (1:1) tation of peripheral stem cells (p=0.019) and ABO incompatibil- between no treatment (arm 1) or erythropoietin β (Neorecor- ity (p=0.006) were identifi ed as risk factors associated with BO. mon®) weekly at the dose of 500 U/kg/week (arm 2). Once the Among 982 long-term survivors ABO bidirectional incompatible target Hb (13 g/dL) has been attained, the dose of rhEPO was HSCT was assessed in 82 cases (8%), minor in 214 (22%), major in reduced by half, while it was withheld when Hb was ≥ 14 g/dL. 208 (21%) and identical in 477 (47%). Compared with ABO identi- Cohort A included 42 patients on day 28 after myeloablative cal HSCT BO incidence was higher for major (p=0.033) and minor HCT, cohort B 39 patients on day 28 after NMHCT, and cohort (p=0.048) but not bidirectional incompatibility. Blood quantita- C 50 patients on day 0 of NMHCT. Primary endpoints included tive measures of hypoxia-inducible factor 1 (HIF-1a) expression proportion of complete correctors (i.e. patients reaching Hb were higher in BO compared to healthy controls (p=0.02) and ≥ 13 g/dL) and median time to achieve Hb correction in each GVHD pts without lung involvement (p=0.007). Serum analysis of arm. heat shock protein 27 (HSP27) revealed higher values for BO than Results: The proportion of complete correctors before day non-BO (175 vs. 78, p=0.046). The mean exhaled nitric oxide con- 126 post-transplant was 0% in group 1A vs 52.4% in group 2A centrations (FeNO) were lower in BO than in HSCT pts without BO (p=0.0008), 0% in group 1B vs 69.5% in group 2B (p=0.0001) and (14 ppb ± 0.9 vs. 20 ppb ± 2.1) or controls (18 ppb ± 1.8, p<0.001). 19.1% in group 1C vs 70.2% in group 2C (p=0.0002). Median time Negative correlation was seen for FeNO level and HIF1a expres- to achieve Hb ≥ 13 g/dL was not reached in group 1B vs 49 days sion (r=0.02), positive correlation was ascertained for FeNO and in group 2B; 363 and 59 days in groups 1A and 1B respectively HSP27 (r=0.034). Fibrosis indicating serum procollagen-III-peptide and 363 and 87 days in groups 3A and 3B respectively (figure 1). levels were not diff erent in BO vs. non-BO [0.43 ± 0.05 vs. 0.6 ± Hb evolution in each group is shown in figure 2. 71 patients 0.06 E/ ml (mean ± SEM)]. (47/62 in control groups and 24/57 in treated groups, p=0.0003) Application of NO donating glyceroltrinitrate spray led to rapid required red blood cell transfusions. The difference was most FeNO increase in 60% of tested BO pts. (n=20) but only in 27% pronounced in cohort B. There was no difference in rates of healthy controls (n=12, p=0.05). Additive BO medication with the thrombo-embolic events or other complications between the NO donator molsidomin resulted in a subjective improvement 2 arms. of exertional dyspnoea in 4 of 12 treated BO pts after median In conclusion, this is the fi rst trial to demonstrate that EPO 3 months treatment. Pulmonary function tests demonstrated therapy hastens erythroid recovery and decreases transfusion good consolidation for vital capacity (VC), forced expiratory vol- requirements when started 1 month after allogeneic HCT. There ume in one second (FEV1) and mean expiratory fl ow at 25% VC was no benefi t to start rhEPO earlier after nonmyeloablative (MEF25) by additive molsidomin. Mean baseline level for VC, FEV1, HCT. MEF25 were 46%, 28%, 16% vs. 48%, 30%, 19% after therapy. Our data demonstrate that BO is characterized by a variety of pathologically modifi ed parameters. HIF1a and NO are probably involved in the regulatory response to hypoxia which could be the source for new treatment strategies.

[O373]

S59 O375 Conditioning therapies Second solid cancers in patients treated with allogeneic haematopoietic cell transplantation using reduced intensity/non-myeloablative conditioning regimens: a Center for O376 International Blood and Marrow Transplant Research study In vivo T-cell depletion with antithymocyte globulin or O. Ringden (1), R. Brazauskas (2), Z. Wang (2), C. Duncan (3), alemtuzumab for unrelated donor stem cell transplantation D. Jacobsohn (4), J. Mattsson (1), M. Sorror (5), N. Majhail (6) with reduced intensity conditioning: results of a multicentre (1)Karolinska University Hospital (Stockholm, SE); (2)Center for randomized phase II clinical trial (the Global study) from the International Blood and Marrow Transplant Research (Milwaukee, GITMO US); (3)Dana-Farber Cancer Institute (Boston, US); (4)Children’s A. Algarotti (1), C. Micò (1), P. Corradini (2), M. Falda (3), National Medical Center (Washington, US); (5)Fred Hutchinson E.P. Alessandrino (4), R. Fanin (5), E. Tagliaferri (6), L. Castagna (7), Cancer Research Center (Seattle, US); (6)National Marrow Donor N. Mordini (8), S. Sica (9), S. Santarone (10), A. Levis (11), A. Iori (12), Program (Minneapolis, US) S. Pollichieni (13), N. Sacchi (13), M. Scarano (14), A. Masciulli (14), R. Marchioli (14), A. Bacigalupo (15), A. Bosi (16), A. Rambaldi (1) Hematopoietic cell transplant (HCT) survivors are at risk for second (1)Ospedale Papa Giovanni XXIII (Bergamo, IT); (2)Istituto Nazionale solid cancers. Non-myeloablative (NMC) and reduced-intensity dei Tumori (Milan, IT); (3)Ospedale San Giovanni Battista (Turin, conditioning (RIC) has allowed HCT as treatment for patients who IT); (4)Policlinico San Matteo (Pavia, IT); (5)A.O. Università di Udine are otherwise ineligible for HCT using myeloablative conditioning (Udine, IT); (6)Ospedale Maggiore Policlinico (Milan, IT); (7)Istituto (MAC) because of advanced age or co-morbidities. In patients with Clinico Humanitas (Milan, IT); (8)A.O. Santa Croce e Carle (Cuneo, leukemia receiving chemotherapy, less chemotherapy is associ- IT); (9)Policlinico Gemelli (Rome, IT); (10)Ospedale Civile (Pescara, ated with lower risk of second cancers. It is therefore of importance IT); (11)A.O. SS Antonio e Biagio e C. Arrigo (Alessandria, IT); to analyze the risk of second solid cancers in recipients treated (12)Università La Sapienza (Rome, IT); (13)Ospedale Galliera (Genoa, with RIC/NMC compared to MAC. Patients treated with HCT for IT); (14)Istituto Mario Negri Sud (S. Maria Imbaro, IT); (15)Ospedale acute/chronic leukemia and lymphoma from 1995-2006 and San Martino (Genoa, IT); (16)Ospedale di Careggi (Florence, IT) reported to CIBMTR were studied (RIC/NMC=4,274, MAC=18,810 patients). Median followup for RIC/NMC and MAC cohorts was 6 Background and aim: We recently reported the clinical outcome and 7.6 years. RIC/NMC recipients were older, had longer interval of high risk patients undergoing allogeneic stem cell transplan- from diagnosis to HCT, less often received TBI, more often received tation (HSCT) from unrelated donors (UD) after a reduced inten- PBSC and more often had received a previous autograft. Median sity conditioning (RIC) based on Antithymocyte Globulin (ATG) or age was 53 years in the RIC/NMC and 34 years in the MAC group. Alemtuzumab (Alem) in vivo T cell depletion for GVHD prophy- Analyses were stratifi ed by diagnosis (leukemia and lymphoma). laxis (Rambaldi et al.: Leukemia 2012). Since both these programs Among patients with leukemia, cumulative incidence of second proved to be well tolerated and reasonably eff ective we compared solid cancers (excluding non-melanoma skin cancers) at 10 years them in a prospective randomized Phase II clinical trial. after HCT was 3.6% (95% CI, 2.4-5.0) in the RIC/NMC group and Study design and Patients: From March 2005 to September 2008, 2.3% (2.0-2.6) in the MAC group. The corresponding incidences for 245 patients were registered into this study at time of UD search patients with lymphoma were 3.0% (1.8-4.5) and 2.8% (1.8-3.9), activation. All of them were unfi t for conventional transplants due respectively. Table shows the standardized incidence ratios (SIR) to age or advanced disease. 112 patients underwent HSCT (median for solid cancers compared to age-, gender- and race-matched age 58 years, range 17-67) and were randomized to program A, (Mel- general populations. RIC/NMC recipients with leukemia had sig- phalan 30 mg/m2, Fludarabine 90 mg/m2, TBI 200cGy and Alem 80 nifi cantly higher risks than the general population for cancers mg) (n= 58) or program B, (Thiotepa 10 mg/Kg, Cyclophosphamide of the lip (SIR 14, P=0.02), other oropharynx (47, P≤0.001), bone 100 mg/Kg, Melphalan 30 mg/m2 and ATG 7,5 mg/Kg) (n= 54). (24, P<0.01), soft tissue (13, P<0.001) and vulva (19, P=0.01) and Results: The median time of neutrophil and platelet engraftment skin melanoma (3, P=0.02). RIC/NMC recipients with lymphoma was similar in the 2 study arms (18 vs. 16 days and 18 vs. 18 days, had signifi cantly higher risks of cancers of other oropharynx (67, respectively) but graft failure and graft rejection occurred most P<0.001), and skin melanoma (4, P=0.03). Multivariable analyses likely in arm A (9 vs. 1, p 0.036). At day +100 the incidence of acute are underway to compare the risks of solid cancers among RIC/ GVHD (> grade II) was slightly higher in the ATG arm (37% vs. 26%, NMC recipients with MAC recipients, while taking into account p 0.224) with a higher incidence of late occurring acute GVHD in age, exposure to TBI, and other disease and treatment related fac- the Alem arm, most likely because of DLI. The incidence of chronic tors. Our preliminary results show that the cumulative incidence GVHD was similar (40% and 41%). The immunologic reconstitution of second solid cancers among RIC/NMC recipients continues to was slower in arm A but no signifi cant diff erence was observed in increase over time, similar to what is seen among MAC recipients. the incidence of infections. With a median follow up of 15 months However, their risks are comparable to age- and gender- matched (range 1-39), at 2 years, the non-relapse mortality (36% vs. 35%) and general population, unlike MAC recipients who have signifi cantly the relapse rate (34% vs. 25%, p 0.294) do not diff er in the 2 arms. higher risks. Similarly, no signifi cant diff erence was observed in terms of event free survival (29% vs 41%, p 0.295) and overall survival (OS) (36% vs 52%, p 0,206) (Figure 1). A signifi cant decrease of the risk of death was associated with the incidence of chronic GVHD (p <0.001). Conclusion: A better engraftment and immune reconstitution as well as a trend for a lower relapse rate was observed in patients receiving an ATG based RIC. Chronic GVHD confi rms its protective role on the risk of death confi rming that an accurate dosing of

[O375]

S60 O378 Fludarabine, IV busulfan and antithymocyte globulins- based reduced-toxicity conditioning regimen (FB3) prior to allogeneic stem cell transplantation (allo-SCT): results of a multicentre prospective trial M. Mohty (1), D. Blaise (2), M. Labopin (1), N. Milpied (3), S. Furst (2), R. Tabrizi (3), T. Guillaume (4), S. Vigouroux (3), J. El-Cheik (2), J. Delaunay (4), S. Le Gouill (4), P. Moreau (4), P. Chevallier (4) (1)Hopital Saint Antoine (Paris, FR); (2)Institut Paoli-Calmettes (Marseille, FR); (3)CHU de Bordeaux (Bordeaux, FR); (4)CHU de Nantes (Nantes, FR)

We report the results of a phase 2 prospective multicenter trial (NCT00841724) aiming to assess a reduced-toxicity conditioning (RTC) regimen based on IV Busulfan 130mg/m2/d (single daily infusion over 3 hours) for 3 days (total dose equivalent to 9.6 mg/Kg), Fludarabine 30mg/m2/d for 5 days, and Thymoglobuline* in vivo T cell depleting agents is crucial for the long term clinical 2.5mg/Kg/d for 2 days (“FB3” regimen) in patients not eligible for a outcome after HSCT. standard myeloablative conditioning regimen. GVHD prophylaxis included CsA alone for a family donor, and CsA+MMF in case of MUD (10/10 or 9/10). O377 80 patients were included (47 males, 59%). Median age was 53 Impact of thiotepa-busulfan-fl udarabine versus (range, 25-64) y. Diagnoses included: ALL/AML/biphenotypic cyclophosphamide-based myeloablative conditioning acute leukemia (n=5; n=26; n=1; 40%); MDS/MPS (n=6; 8%); CLL regimen on outcomes after single unrelated cord blood (n=3; 4%); NHL and HD (n=24; 30%); and myeloma (n=15; 18%). 39 transplantation for adults with haematological malignancies patients (48%) were in CR1, 14 patients (18%) in CR2/CR3, and 27 according to disease status patients (34%) in more advanced phases. The donor was an HLA- H. Bittencourt, A. Ruggeri, G. Sanz, S. Nabhan, J. Sanz, W. Arcese, identical sibling donor in 28 cases (35%) and a MUD in 52 cases A. Sirvent, I. Ionescu, E. Gluckman, V. Rocha on behalf of Eurocord (65%). The stem cell source was PBSC in 77 cases (96%). At time of inclusion, the Karnofsky score was 70%, 80%, 90%, and 100% Fludarabine (FLU) has been increasingly used substituting cyclo- in 4 (5%), 11 (14%), 28 (35%), and 37 (46%) patients, respectively. phosphamide (CY) in myeloablative conditioning (MAC) regimens. 54 patients (68%) had at least one comorbidity, while 26 patients In single unrelated cord blood transplantation (UCBT), a particu- (32%) did not have any. lar conditioning, associating thiotepa, busulfan (Bu) and FLU (TBF) ANC>500/μL was achieved at a median of 15 (range, 10-23) days, showed a better outcome in patients with hematological malig- and 98% of patients had platelets >50.000/μL by day +60 (of nancies (Sanz BMT 2012). To compare TBF MAC regimen with the whom none was below 20.000/μL at any time point). The cumu- classical CY-based MAC regimen (associated with Bu or TBI), we lative incidences (CInc) of grade 2-4 and grade 3-4 acute GVHD retrospectively analyzed 229 patients who underwent sUCBT for were 32% (95%CI, 21-43%) and 9% (95%CI, 4-18%), respectively. acute leukemia(AL)/myelodysplasia/myeloproliferative disorder The CInc of relapse/disease progression (RI) at one year was 26% from 2000 to 2008. One hundred and seventy (74%) patients had (95%CI, 17-36%), while the CInc of NRM was 10% (95%CI, 5-18%) AL. According to IBMTR classifi cation, 64% of patients had early- or at one year. The KM estimates of OS and PFS at one year were 71% intermediate-risk (named early thereafter) disease stage. Median (95%CI, 61-81%) and 64% (95%CI, 53-74%), respectively. In terms age was 33 years, median follow-up, 44 months and 40% UCBT of prognostic factors, patient age, diagnosis, donor type, gender, had a 0-1 HLA mismatch. Median infused TNC was 2.5x10.7/kg. presence or absence of comorbidities and the HCT comorbidities GVHD prophylaxis consisted of CSA±steroids in 78% of pts and index, did not have any statistically signifi cant impact on NRM, RI, 91% pts received ATG. PFS and OS. Only a Karnofsky score <90% at time of allo-SCT had a Two groups of MAC were analysed: CY-based MAC (n=158; 69%), negative impact on OS (47% vs. 77%; p=0.008), while only disease and the TBF regimen (n=71; 31%). Patients were analyzed sepa- status (CR1 vs. other) impacted RI (p=0.02). rately as early or advanced stage since there was an interaction This prospective trial validated the effi cacy of a so-called RTC between conditioning and stage. TNC and CD34 cells infused did regimen which could overcome the deleterious impact of comor- not diff er between groups. bidities. Such FB3 regimen appears to be safe, and exhibits an effi - For early stage, TBF group presented a predominance of AL, cient disease control both in myeloid and lymphoid malignancies, less frequent ABO major incompatibility and UCBT has been warranting further investigations as part of phase 3 trials. performed more recently. Cumulative incidence (CI) of neutrophil/platelet recovery and non-relapse mortality (NRM) was 96% vs 82% (P=0.0006), 70% vs 50% (P=0.01) and 37% vs 48% O379 (P=0.04) for TBF and Cy-based MAC, respectively. CI of grade Comparison of myeloablative intravenous busulfan with 2-4 aGVHD, and relapse were similar between groups. Dis- cyclophosphamide (BuCy) or fl udarabine (BuFlu) prior to ease-free survival (DFS) and overall survival (OS) was 49% vs. haematopoietic cell transplantation (HCT) for myeloid 37% (P=0.06) and 52% vs. 38% (P=0.05) for patients receiving malignancies TBF and Cy-based MAC, respectively. In multivariate analysis, M. Pasquini (1), J. Le-Rademacher (2), K. Kato (3), X. Zhu (1), TBF was associated with better neutrophil recovery (HR: 1.91; P. McCarthy (4), V. Ho (5), K. Cooke (6), B. Armstrong (3), A. Smith (3), P=0.007) and OS (HR: 0.55; P=0.03) for patients in early stage J. Rizzo (1), J. Burkart (1), C. Brededson (7) of the disease. (1)Medical College of Wisconsin, CIBMTR (Milwaukee, US); (2)Medical For advanced stage, TBF group patients were older and UCBT has College of Wisconsin, Institute for Health and Society (Milwaukee, US); also been transplanted more recently. CI of relapse was higher for (3)Otsuka Pharmaceutical Development and Commercialization, patients receiving TBF regimen (35% vs. 14% - P=0.05). Neutro- Inc. (Princeton, US); (4)Roswell Park Cancer Institute (Buff alo, US); phil/platelet recovery, NRM, grade 2-4 aGVHD, DFS and OS were (5)Dana Farber Cancer Institute (Boston, US); (6)Ireland Cancer similar between groups in univariate analysis. There were no dif- Center, University Hospitals Case Medical Center (Cleveland, US); ferences on outcomes between the two groups in multivariate (7)University of Ottawa, (Ottawa, CA) analysis. These fi ndings support the use of TBF MAC regimen before single BuFlu is a well tolerated conditioning regimen and in the past UCBT in patients with hematological malignancies in early stage. decade became the second most commonly used myeloablative

S61 regimen in the U.S. Yet, comparisons to more traditional regi- bone marrow transplantation from sibling donor (n=133, 7.6%), mens, such as BuCy with intravenous (IV) Bu are lacking. The CIB- peripheral blood stem cell transplantation from sibling donor MTR conducted a prospective cohort study from March 2009 to (n=313, 18.0%), bone marrow transplantation from unrelated February 2011, comparing IVBU to total body irradiation contain- donor (n=504, 28.9%) and cord blood transplantation from unre- ing regimens. The current analyses focus on the non-irradiation lated donor (n=627, 36.0%). RIC regimens included Flu+Bu-based cohort, investigating diff erences in outcomes between BuFlu and (FB) (n=481, 33.8%), Flu+ivBu-based (FIB) (n=349, 24.5%) and BuCy. 1,025 patients (BuFlu, N=424, BuCy, N=601) from predomi- Flu+melphalan-based (FLP) (n=431, 30.3%) regimens. Three- nantly North American centers who received HCT from related or years overall survival and event-free survival of HSCT recipients unrelated donors with myeloablative conditioning and calcineu- with FIB regimens was signifi cantly higher (42.1%, 37.6%) than rin inhibitor (CNI) based graft-versus-host disease (GVHD) pro- those with FB (36.6%, 32.6%) and FLP (33.8%, 30.1%) (p=0.0004, phylaxis for treatment of myeloid malignancies. The BuFlu cohort 0.029). Cumulative incidence of relapse (RI) was similar among diff ered signifi cantly from BuCy by having older patients (median recipients with these three regimens (FB; 38.6%, FIB; 43.1%, FLP; age, 49 y vs. 43 y), more use of peripheral blood (90% vs. 68%), 38.9%, p=0.197), but cumulative incidence of transplant-related more unrelated donors (53% vs. 43%), once daily Bu administra- mortality (TRM) with FB and FLP was signifi cantly higher than FIB tion (78% vs. 16%), CNI plus mycophenolate mofetil (29% vs. 7%) (FB; 27.6%, FIB; 18.9%, FLP; 30.1%, p=0.00004). In the multivariate and use of anti-thymocyte globulin (ATG, 36% vs. 19%) compared analysis, FIB regimen had a signifi cantly higher risk of RI than FLP to BuCy. The distribution of performance score, comorbidity index (hazard ratio 1.43, 95% confi dence interval 1.09-1.89, p=0.011), (HCT CI), disease (AML 70%, MDS 20% and CML 10%) and disease and a signifi cantly lower risk of TRM (0.60, 0.41-0.87, p=0.007). status, dose of Bu and HLA mismatched donors were similar. After These results indicate that FIB can reduce the risk of TRM and may adjusting for diff erences in the cohorts, 2-year probabilities for improve the survival compared with FLP in allogeneic HSCT for overall survival were 56% (95% confi dence interval [CI], 51-62%) recipients with AML. and 57% (95% CI, 53-62%, p=0.79) for BuFlu and BuCy, respectively. Corresponding probabilities for progression-free survival were 50% (95% CI, 44-55%) and 47% (95% CI, 43-52%, p=0.55). O381 Two-year cumulative incidences for transplant-related mortality GSTA2 S112T polymorphism predicts survival, early TRM and (TRM), disease relapse and hepatic veno-occlusive (VOD) were hyperbilirubinemia after an allo-transplant prepared with 18%, 33% and 5% for both cohorts, respectively. Multivariate busulfan: in vitro model attempting to explain a statistical analysis was adjusted for age, race, HCT CI, use of ATG, graft source clinical association and donor type (table). This contemporary conditioning regimen F. Bonifazi (1), G. Storci (2), G. Bandini (1), M. Ferioli (1), E. Marasco comparison demonstrates comparable outcomes with BuFlu and (2), E. Dan (1), E. Zani (2), F. Albani (2), S. Bertoni (2), A. Bontadini BuCy when utilizing IV Bu. (2), B. Sinigaglia (1), S. De Carolis (2), S. Rizzi (1), M.R. Motta (1), P. Garagnani (2), V. Mantovani (2), M. Bonafè (2) (1)University of Bologna, S. Orsola-Malpighi Hospital (Bologna, IT); (2)University of Bologna (Bologna, IT); (2)S. Orsola-Malpighi Hospital (Bologna, IT)

Busulfan metabolism depends on liver glutathione (GSH) availability. Hepatic GSH is depleted as consequence of the enzymatic conjugation to exogenous and endogenous molecules by Gluta- thione Transferases (GST). GSTA2 S112T polymorphism represents a common (20% of serine homozygotes) aminoacidic substitution in the Italian population. Here, 40 polymorphisms at 27 candidate O380 genes were analysed by high throughput mass array Sequenom Clinical outcomes of allogeneic haematopoietic stem cell TM platform on 185 consecutive patients (median age 41 yrs) who transplantation with intravenous busulfan-based reduced- received busulfan-based conditioned allogeneic HSCT for hae- intensity conditioning for acute myeloid leukaemia: a matological malignancies at Institute of Hematology “Seràgnoli” nationwide retrospective study from the adult AML working from 2004 to 2009. GSTA2 S112T serine homozygotes showed group of JSHCT reduced OS (RR=2.388, 95% CI: 1.407-4.052), increased 100 days T. Yamashita, S. Taniguchi, T. Fukuda, H. Ogawa, Y. Morishima, TRM (RR=5.185, 95% CI: 1.971-13.64) and increased TRM at any T. Nagamura-Inoue, H. Sakamaki, Y. Atsuta, A. Takami on behalf of time (RR=4.912, 95% CI: 2.083-11.583) compared to threonine car- the Adult AML Working Group of the Japan Society for Hematopoietic riers. Toxicity was the main cause of death in the group of GSTA2 Cell Transplantation S112T serine homozygotes. Multivariate analysis revealed that GSTA2 S112T serine homozy- In allogeneic hematopoietic stem cell transplantation (HSCT) for gotes show increased systemic busulfan exposure (1214.36+570.06 recipients with acute myeloid leukemia (AML), fl udarabine (Flu)- vs 838.10+282.40 microMol x min, p=0.003), after adjusting for based regimens with the addition of Bu or melphalan (L-PAM) age, intensity of conditioning, sex mismatch, type of donor and have been developed as reduced-intensity conditioning (RIC) phase at transplant. These patients also disclosed increased post- regimens for the last decade. But recently, intravenous Bu (ivBu) transplant serum bilirubin levels (3.280+0.422 vs 1.874 +0.197 has been developed and Flu+ivBu has become one of the com- mg/dL, p=0.004). Respect to this issue, we found that the in vitro mon RIC regimens. In Japan, ivBu was introduced in 2006. In this exposure of the human hepatoma cell line (hep-G2) to busulfan, study, we evaluated the clinical outcomes of allogeneic HSCT for though does not elicit cell death, ignites pro-infl ammatory acti- AML, especially focusing on ivBu-based RIC regimens. vation, documented by the increase of NF-κB activity and IL-8 The study population included HSCT recipients reported to the mRNA expression. This up-regulation is paralleled by the down- Japan Society for Hematopoietic Cell Transplantation. From this regulation of a series of genes involved in bilirubin metabolism, database, we extracted the data of adult patients with AML who e.g. constitutive androstane receptor (CAR), ABCC2, SLCO1B1 and received fi rst allogeneic HSCT between 2001 and 2010. There SLCO1B3. The phenomenon is amplifi ed in GSTA2 knocked down were 7,027 recipients selected according to this criterion. And, we hep-G2 cells. Our data describes for the fi rst time a group of allo- excluded 33 (0.47%) cases from the study because of missing key transplants who show a genetic based association among poorer variables. Then, we divided these cases into two groups, that is, survival, higher mortality and busulfan exposure, and impaired the myeloablative conditioning group and the RIC group. bilirubin metabolism. Our patho-physiological model proposes In the RIC group, a total of 1,743 recipients were evaluated in that this association is due to busulfan-elicited pro-infl ammatory this study. Median age at transplant was 58 years (range, 16-82), hit in the liver which constitutes a marker of transplant frailty in and 37.2% (n=648) were female. Types of transplant included patients receiving busulfan as conditioning drug.

S62 O382 Alternative donor transplantation Favourable long-term disease-free survival following allogeneic stem cell transplantation in advanced stage mycosis fungoides and Sézary syndrome after non- O383 myeloablative conditioning with pentostatin and TBI200 Unmanipulated haploidentical transplants compared with F. Onida (1), G. Saporiti (1), E. Tagliaferri (1), C. Annaloro (1), L. Corti other alternative donors and matched sibling grafts: a single (1), S. Alberti Violetti (1), F. Grifoni (1), C. Olivares (1), G. Mometto (1), Center experience in 488 patients A. Cortelezzi (1), E. Berti (2) A. Bacigalupo, A. Dominietto, C. Di Grazia, T. Lamparelli, F. Gualandi, (1)Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico A. Ibatici, S. Bregante, M.T. van Lint, R. Varaldo, M. Gobbi, A.M. Carella, - University of Milan (Milan, IT); (2)Fondazione IRCCS Ca’ Granda A.M. Raiola Ospedale Maggiore Policlinico - University of Milano-Bicocca IRCCS Azienda Ospedaliera Universitaria San Martino – IST (Genoa, IT) (Milan, IT) Background: There are currently at least 4 possible donor types for Advanced tumor-stage mycosis fungoides (MF) and Sézary syn- an allogeneic transplant: HLA identical siblings (SIBS), unrelated drome (SS) are CTCL characterized by very poor prognosis, mainly donors (UD) , unrelated cord blood (UCB) and HLA haploidentical due to chemoresistance and rapid disease progression. Allogeneic family donors (HAPLO). haematopoietic stem cell transplantation (allo-HSCT) has been Aim of the study: The aim of the present study was to compare shown to be very eff ective in achieving long lasting complete disease free survival (DFS) of HAPLO transplants, , with SIBS , UD remission, possibly leading to cure in selected patients. However, and UCB grafts, in a single Center. high transplant-related mortality (TRM) limit its feasibility in the Patients: We therefore compared 92 HAPLO, with 176 SIBS, 291 UD vast majority of patients with CTCL. Reduced-intensity (RIC) or and 105 UCB grafts. All 488 patients had hematologic malignan- non myeloablative (NMA) conditioning regimens have been dem- cies , receiving a fi rst allogeneic transplant (SCT) in a single Center, onstrated to decrease TRM, allowing gradual establishment of full between jan-2006 and feb-2012. Patients were stratifi ed for dis- donor chimerism and graft-versus-lymphoma eff ect. ease phase as early (fi rst or second remission) (n=265) or advanced In our Institution, following a preliminary experience with a RIC (beyond second remission) (n=223). The diagnosis was acute leu- regimen including Fludarabine/CTX and TBI200 in 3 patients kemia (n=275), lymphoma (n=79), myelofi brosis (n=51), myelodis- (since 9/2000 to 4/2001), 18 patients underwent allo-HSCT after a plastic syndrome (n=94), other hematologic malignancies (n=19). pentostatin/TBI200 NMA regimen, between 09/2002 and 10/2012. The conditioning regimen was classifi ed as myeloablative (MAC) Graft-versus-Host Disease (GvHD) prophylaxis included CsA and (n=337) or reduced intensity (RIC) (n=151) . Graft vs host disease MMF (+ ATG in MUD transplants). (GvHD) prophylaxis was as follows: HAPLO transplants received At the time of transplant, all patients - 11 males and 7 females with unmanipulated marrow and post transplant cyclophosphamide a median age of 52 years (range 27-66) - had stage III/IV refractory (PT-CY) with cyclosporine (CsA) and mycophenolate (MMF), SIBS MF (n=13) or SS (n=5). Median time from diagnosis to HSCT was received CsA and methotrexate (MTX), UDs received CsA +MTX + 42 months (range 13-252). Donors were HLA-identical sibling in 13 antithymocyte globulin (ATG) and UCB received CsA+MMF+ATG. patients and HLA-matched unrelated (MUD) in 5. Source of stem The 4 groups were comparable for disease phase (p=0.2), age cells was peripheral blood in all patients but one, who received a (p=0.2), and conditioning regimen intensity (p=0.4), with the single umbilical cord blood unit. exception of UCB who received more frequently a MAC regimen. Full donor chimerism was obtained in 16/17 evaluable patients, in Results: Acute GvHD grade II-IV , was less frequent in UCB and a median time of 2 months (range 1-12). Acute GvHD occurred in HAPLOs (14%) as compared to SIBS and UDS (30%) (p=0.0003). 8 patients (4 grade I-II, 3 grade III and 1 grade IV), whereas chronic Chronic GvHD was less frequent in HAPLOs ((12% vs 24% of other GvHD was observed in 6 (extensive in 3). 6 patients died, 1 in com- donor types). plete remission (CR) for aGvHD and 5 with progressive disease. The cumulative incidence (CI) of transplant related mortality (TRM) Overall, clinical CR was obtained in 13 patients. With a median fol- was signifi cantly lower in SIBS and HAPLOS (17%) as compared low-up of 84 months (range 2-122), 12 patients are currently alive to UDs and UCB (34%) (p=0.0002). The CI of relapse was compa- with an estimated DFS at 10 years of 65% (fi g.1). Worth mention- rable in all 4 groups (26-36%, p=0.2). The 3 year actuarial DFS ,of ing, TRM at 2 years was only 12%. Of note, patients transplanted all patients including early and advanced disease, is respectively for SS are all alive and disease-free (regardless their disease status 41% , 39%, 36%, 43% (logrank, p=0.6) . In multivariate Cox analy- at allo-HSCT), whereas all patients who died in progression had sis, advanced disease was the only negative predictor of disease chemoresistant MF at time of transplant. free survival (RR 2.2; p<0.0001). In conclusion, allo-HSCT after a pentostatin/TBI200 NMA regi- In conclusion, we fi nd that unmanipulated HAPLO transplants, men is feasible and represents a highly eff ective strategy of cure with PT-CY, have outcome comparable to UD and MSD grafts, in in patients with advanced stage refractory MF/SS, with a key patients with hematologic malignancies. Advanced disease is the role of immunomediated graft-versus-lymphoma in maintaining only adverse factor for disease free survival . remissions.

S63 O384 sUCBT and dUCBT in CR1, in age, diagnosis, weight, CMV status, Changing trends of umbilical cord blood transplants for cytogenetics risk and number of HLA incompatibilities. However haematological malignancies in patients older than 50 years: dUCBT were performed more recently (2009 vs 2008), the time a Eurocord -CIBMTR study from CR1 to UCBT was longer and more frequently transplanted A. Ruggeri (1), M. Eapen (2), L. Tucunduva (1), F. Volt (1), M. Horowitz with CY+FLU+TBI2Gy (87% vs 68%), lower ATG use (21% vs 35%). (3), V. Rocha (1), D. Weisdorf (3), E. Gluckman (1) Also, dUCBT recipients received higher TNC dose (4x107/kg vs (1)Eurocord (Paris, FR); (2)CIBMTR (Milwaukee, US); (3)CIBMTR 3.1x107/kg). Median follow-up was 23 months. (Milwaukee, US) Cumulative incidence (CI) of 60 days neutrophil recovery was 82±3% and 76±2% after dUCBT after sUCBT, respectively (p=0.86) The number of unrelated cord blood transplant (UCBT) in adults and full donor chimerism at day 100 was similar in both groups. has increased with the use of two cord blood units (CBU) when Day 100, CI of acute GVHD was 35% in both groups, with a trend a single CBU does not contain adequate number of cells and of increased incidence of grades III-IV after sUCBT, but increased with the use of reduced-intensity conditioning regimens (RIC) to incidence of grade II aGVHD after dUCBT (28%) compared to 17% decrease toxicity. Using data from CIBMTR and Eurocord we stud- after sUCBT (p=0.05). CI of chronic GvHD at 2ywas 21±4% after ied indications of UCBT and survival in 1529 patients (pts) older dUCBT and 12±5% after sUCBT (p=0.15). Two years CI of non than 50-year (y) transplanted for hematological malignancies relapse mortality (NRM) after dUCBT was 28±4% and 30±6% after from 2005 to 2011. sUCBT (p=0.87). CI of 2y relapse was 21±4% after dUCBT and 848 pts>50y were transplanted in the US (CIBMTR data) and 681 38±2% after sUCBT (p=0.03). In an adjusted multivariate analysis, in EBMT centers (Eurocord data). Most were transplanted after dUCBT was associated with lower relapse incidence than sUCBT 2008 with a steady increment over time and 60% were aged (HR=0.74, p=0.01). There was an improved 2-y LFS after dUCBT 50-59y. In both registries the most frequent indication was acute (51±5% vs32±3%; p=0.03), confi rmed in a multivariate analysis myeloid leukemia(AML) (50%) followed by myelodysplastic (HR=0.64, p=0.04). syndrome(MDS) and non-Hodgkin Lymphoma(NHL). In Eurocord For patients transplanted in CR2 (n=148), there were no statisti- myeloma (MM) was the indication for UCBT in 15% of pts where cally diff erences of outcomes between dUCBT (n=93) and sUCBT as in the US this accounted for a single case. In both registries (n=55). At 2y LFS was 40±6% and 48±3% after dUCBT and sUCBT, most pts with AML (80%) were transplanted in CR1 or CR2. RIC respectively (p=0.32). regimens were used for 76% transplants in Eurocord and 49% of In this study for patients in CR1, neutrophil recovery, GVHD and transplants in US for pts in the range 50-59y. Among older pts RIC NRM were not diff erent after RIC-dUCBT or RIC-sUCBT, however regimens accounted for 93% in Eurocord and 69% in US. GVH pro- dUCBT recipients had decreased relapse and improved LFS. For phylaxis also varied by region; cyclosporine and prednisone was patients transplanted in CR2, there was no benefi t of using dUCBT the predominant regimen in Eurocord and tacrolimus and MMF in when compared to sUCBT. the US. Further, double UCBT were more in the US 71% compared to 61% in Eurocord. Probability of 2-y overall survival (OS) was similar in both cohorts. In CIBMTR 2-y OS for AML in CR1 was 41%, O386 30% in CR2 and 15% in advanced disease in pts aged 50-59y, and HOVON 106: a phase II study to assess engraftment and was 22%, 29%, and 16% in pts >60y, respectively. In Eurocord it engraftment kinetics after double cord blood transplantation was 33% in CR1, 48% in CR2, 10% in advanced disease if aged 50- preceded by a reduced-intensity conditioning regimen 59y, compared to 58% in CR1, 29% in CR2 and 15% in advanced J.AE Somers (1), B. van der Holt (2), K. Sintnicolaas (2), M. Oudshoorn disease in >60y old. (3), E. Braakman (4), E.J Petersen (5), W.A.F. Marijt (6), C. Huisman (7), For other malignancies, in Eurocord for age 50-59y, 2-y OS was M.E Groenendijk-Sijnke (2), A. Brand (3), J.J Cornelissen (4) 57% in MM, 52% in ALL, 49% in NHL, 47% in CLL and 25% in MDS. (1)Erasmus Medical Center Rotterdam / Sanquin Blood Supply In CIBMTR it was 54% in ALL, 48% in MDS, 41% in NHL and 40% (Rotterdam, NL); (2)HOVON Data Center (Rotterdam, NL); (2)Sanquin in CLL. Blood Supply (Rotterdam, NL); (3)Europdonor Foundation/Leiden Despite diff erences in transplant conditioning regimens and University Medical Center (Leiden, NL); (4)Erasmus Medical Center GVHD prophylaxis, survival rates were comparable across regions. Rotterdam (Rotterdam, NL); (5)University Medical Center Utrecht These results show similar trends in both registries collecting (Utrecht, NL); (6)Leiden University Medical Center (Leiden, NL); mostly European and US data. Although not a comparative study, (7)Academic Medical Center (Amsterdam, NL) this survey shows that use of UCBT is a good alternative source of stem cells in pts older than 50y and can yield encouraging survival Background: Umbilical cord blood transplantation (UCBT) is a in pts with hematologic malignancies. Further studies may defi ne potential curative treatment for patients (pts) in need for an allo- the best indications for UCB versus unrelated donor for transplan- geneic stem cell transplantation but lacking a matched unrelated tation in pts lacking an available family donor. donor. Single UCBT has been associated with a relatively high rate of primary graft failure and retarded or insuffi cient recovery of all hematopoietic lineages. The introduction of double UCBT O385 has resulted in enhanced engraftment, but the mechanism of Comparison between single and double cord blood improved engraftment and recovery is still incompletely under- transplantations in adults with acute leukaemia receiving stood, as only one UCB unit ultimately survives. In this multicentre reduced intensity conditioning regimen phase II study we evaluated engraftment, chimerism and trans- V. Rocha, M. Labopin, A. Ruggeri, D. Blaise, B. Rio, J. Cornelissen, plant outcomes in patients receiving a double UCBT preceded by N. Milpied, L. Tucunduva, A. Nagler, M. Mohty, E. Gluckman on behalf a 4 Gy TBI based conditioning regimen. of Eurocord and Acute Leukemia Working Party of EBMT Methods: Pts with high-risk hematological diseases received a double UCBT, preceded by a conditioning regimen of cyclofos- Double cord blood unit (dUCBT) is increasingly used over single famide 60 mg/kg, fl udarabine 160 mg/m2 and TBI 2x2 Gy. The CB unit (sUCBT) after reduced intensity conditioning regimen primary endpoint was the proportion of patients with primary (RIC). There is an increased relapse incidence (RI) after RIC-HSCT; graft failure. Secondary endpoints were the time to hematopoi- therefore we hypothesized that RI may be lower and leukemia-free etic recovery, overall survival (OS), progression-free survival (PFS), survival (LFS) higher after dUCBT than sUCBT, due to increased and non-relapse mortality (NRM). graft-versus leukemia. We analyzed 360 adults with ALL (n= 77) Results: Fifty-three patients with AML (n=30), ALL (n=10), CML or AML (n=238) in CR1 (n=212) or CR2 (n=148) transplanted (n=2), NHL/ CLL (n=7) or (V)SAA (n=4) from 6 transplant centers with a sUCBT (n=131) or dUCBT (n=229) after RIC. Only patients received a double UCBT. The median age was 51 (range: 20-65) transplanted from 2005-2011 with grafts containing more than years. The TNC of infused UCBs was 2.7 x106/kg (range: 1.3-5.2). 2.5x107/kg total nucleated cells (TNC) were included. There were 58%, 38% and 4% of CBUs were 4/6, 5/6 and 6/6 matched with the no statistical diff erences between the group of patients receiving recipient, respectively. Primary graft failure occurred in 1 patient.

S64 Engraftment was observed in 96% of pts. Seven pts died before O388 day +60 (relapse n=4; NRM n=3). The cumulative incidence (CI) Impact of graft-versus-host disease prophylaxis on of neutrophil recovery at day +60 was 85% with a median time outcomes after myeloablative single-unit umbilical cord to recovery of 35 (range: 15-100) days; single donor hematopoi- blood transplantation (sUCBT) esis was observed in 87% at that time point. Chimerism studies J Sanz, A. Picardi, V. Potter, C. Ferrá, C. Martín, D. Valcarcel, C. Nozzoli, showed early (day +18) single UCB predominance, indicating J.C. Hernández-Boluda, A. Rambaldi, A. Verdeguer, M.J. Pascual, rapid loss of the non-sustaining unit. Graft predominance was D. Serrano, B. Bartolozzi, A. Algarotti, A. Pagliuca, M.A. Sanz, studied in a subset of patients in more detail, showing that early W. Arcesse, G.F. Sanz on behalf of GETH and GITMO groups CD4+ T-cell chimerism predicted for ultimate graft predominance. With a median follow up of 13 months (range: 3-37), OS and PFS sUCBT from unrelated donors using busulfan, thiotepa, fl uda- at 1 year were 66% and 54%, respectively. The CI of relapse and rabine and ATG as myeloablative conditioning regimen (UCBT- NRM at 1 year were 25% and 22%, respectively. GETH-2005 protocol) has previously demonstrated high rates of Conclusion: Double UCBT preceded by a 4 Gy based condition- engraftment with fast neutrophil recovery and high anti-tumor ing regimen results in a high engraftment rate with low treatment activity in patients with high-risk hematologic malignancies. We related mortality, but only a single UCB ensures longterm hema- designed a new protocol substituting the long-term steroids ther- topoiesis in the majority of patients. apy administered for graft-versus-host disease (GvHD) prophylaxis by a short course of mycophenolate mofetil (MMF) together with a slight dose reduction of ATG (UCBT-GETH/GITMO-2007 pro- O387 tocol). From June 2007 and July 2012, 145 consecutive patients Outcome after 9/10 mismatched unrelated donor or cord entered the study and results were compared with 88 patients blood cells allogeneic stem cell transplantation (allo-SCT) in in the UCBT-GETH-2005 cohort. In the UCBT-GETH/GITMO-2007 the setting of reduced-intensity conditioning (RIC) protocol 55% of the patients were males with a median age of F. Malard (1), S. Furst (2), M. Loirat (1), P. Chevalier (1), J. El-Cheikh (2), 32 years (range, 1–57). Most patients had acute leukemia (42% T. Guillaume (1), J. Delaunay (1), S. Legouill (1), P. Moreau (1), D. Blaise AML and 37% ALL) and 41% were transplanted in an early stage (2), M. Mohty (3) of the disease. Donor–recipient HLA match was 4/6, 5/6 and 6/6 (1)CHU de Nantes (Nantes, FR); (2)Institut Paoli-Calmettes (Marseille, in 73%, 17% and 10% of the cases, respectively. The median TNC FR); (3)Hôpital Saint Antoine (Paris, FR) and CD34+ cell doses infused were 3.2 ×107/kg (range, 0.8–12.4) and 1.5×105/kg (range, 0.1–11.7), respectively. The cumulative Unrelated umbilical cord blood cells (UCB) have emerged as an incidence (CI) of myeloid engraftment at 60 days was 88% at a alternative stem cell source for allo-SCT in patients who lack a median time of 23 days. The CI of acute GVHD grades II–IV and matched-related or unrelated donor (MUD). Several studies found chronic extensive GVHD was 28% and 15%, respectively. The CI a similar outcome between HLA 4-6/6 matched UCB and HLA 8/8 of non-relapse mortality (NRM) at 100 days, 180 days, and 4 years or 7/8 MUD, mainly in the setting of MAC. However more common was 15%, 22%, and 35%, respectively. The 4-year CI of relapse was practice in many centres is to search for 10/10 or 9/10 MUD. Thus 22%, while disease-free survival (DFS) was 61%, 27%, and 19% for far, no study focussed on the comparison of outcome of patients patients transplanted in early, intermediate, and advanced stages who received double UCB versus 9/10 mismatched donors. This of the disease, respectively. When comparing the results obtained analysis assessed outcome after allo-SCT using double UCB or in two historical cohorts, patient, disease and graft characteris- 9/10 mismatched donors in the setting of RIC. We included 152 tics were similar in both groups except for a higher proportion consecutive patients treated for hematological malignancies in of HLA disparity in the UCBT-GETH/GITMO-2007 protocol. Overall 2 centers adopting similar transplant procedures. The median multivariable analysis showed delayed neutrophil recovery (RR age was 53 years (range, 16-69). Diagnoses included 59 AML, 21 0.58; 95% CI 0.43-0.77; P = 0.0002) after UCBT-GETH/GITMO-2007 MDS/MPN, 42 NHL, 5 HD, 18 ALL and 7 Myelomas. Conditioning protocol while acute and chronic GvHD, NRM, relapse and DFS regimen consisted of fl udarabine, cyclophosphamide and low rates were not signifi cantly diff erent in the two groups. This study dose TBI for 71% of patients, fl udarabine and busulfan for 23% confi rms that sUCBT from unrelated donors using busulfan-based of patients; and other regimens in 6% of patients. 33% received myeloablative conditioning is a valuable strategy for patients with ATG. The donor was double UCB in 110 cases (“dUCB” group) and hematological malignancies. Long-term DFS can be achieved in 9/10 mismatched unrelated in 42 cases (“9/10” group). Both cen- a substantial number of patients, especially if transplanted in an ters adopted the same strategy for donor choice: in the absence early stage of the disease. Introduction of MMF as GvHD prophy- of MRD or 10/10 MUD, 9/10 donors were searched. UCB cells were laxis had a deleterious eff ect on myeloid engraftment and should used if no 9/10 donor could be identifi ed within a reasonable time probably be avoided. frame (2-3 months after search initiation). With a median follow- up of 30.3 months (range, 6-72.4), the estimate of OS and PFS at 2 years were comparable between both groups 52% (95% CI, O389 42-61%) versus 48% (95% CI, 32-62%)(P=0.55) and 43% (95% CI, Single cord blood transplantation combined with an HLA 34-52%) versus 38% (95% CI, 23-53%)(P=0.55) in the dUCB group mismatched third party donor for high-risk haematological versus the 9/10 group. The cumulative incidence of NRM and patients and HIV infection relapse were comparable between both groups 26% versus 24% J. Kuball (1), M. Kwon (2), A. Wensing (1), P. Ellerbroek (1), P. Balsalobre (P=0.95) and 34% versus 38% (P=0.63) in the dUCB group versus (2), D. Serrano (2), J. Gayoso (2), K. van Besien (3), L. Petz (4), J.L. Diez- the 9/10 group (P=0.95). Grade 3-4 acute GVHD and extensive Martin (2) chronic GVHD incidences were 20% versus 21% (P=0.83), and 6% (1)UMCU (Utrecht, NL); (2)Gregorio Marañon G. U. Hospital (Madrid, versus 21% (P=0.02), in the dUCB group versus the 9/10 group, ES); (3)Cornell University (New York, US); (4)StemCyte International respectively. In multivariable analysis including the most impor- Cord Blood Center (Covina, US) tant parameters associated with outcome (patient’s age at transplant, patient’s sex, diagnosis, disease status at transplant, use of Background: The safety and usefulness of unrelated umbilical ATG, GVHD prophylaxis), the stem cell source (dUCB versus 9/10) cord blood (CB) stem cell transplantation (SCT) in patients candi- did not have any signifi cant impact on OS (HR=0.92 (95% CI, 0.41- dates for allogeneic SCT and HIV infection is unknown. Single CB 2.08); P=0.86). These data suggest that dUCB is likely an alterna- with the co-infusion of CD34+ cells from a third party HLA-mis- tive graft source compared to 9/10 mismatched unrelated donors matched donor (TPD), known as dual SCT, has shown to reduce in the setting of RIC allo-SCT. the period of post-transplant neutropenia and related early complications associated with single CB transplantation. This platform could potentially reduce the risk of early infections in this particular group of patients. On the other hand, the use of a cell source homozygous for the CCR5 δ32 allele mutation, which confers

S65 high resistance against HIV-1 acquisition, is of particular interest to CD146-CCR5-T cells (mean±SEM: 91.8±3.6 % vs. 15.1±4.6 %, in HIV+ patients. p<0.001). They also expressed a higher level of the activation Methods and Results: We report here on the fi rst two patients marker ICOS (p=0.01). Interestingly, although regulatory T cells with HIV infection and high-risk haematological malignancies (Tregs) were decreased in GI-GVHD patients, CD146+CCR5+Tregs who underwent dual SCT in two diff erent centers: (Case 1) a 53 were increased, suggesting a loss of regulatory function and that year-old male patient with poor risk MDS; (Case 2) a 34 year-old targeting this population with specifi c drugs may not be harm- male with Burkitt lymphoma in CR2. Both CB transplants were per- ful. In addition, we observed that CD146+CCR5+T cells circulate formed using the dual platform with the co-infusion of mobilized before clinical symptoms of GI-GVHD. As CD146 and CCR5 on and selected CD34+ cells from a TPD. Conditioning regimen were T cells are traffi cking markers, we verifi ed the expression of their myeloablative in both cases. Case 1 had the additional potential receptors in GI biopsies. CD146 expression on GI vessels was sig- benefi t from the use of a CB with the homozygous CCR5δ32 muta- nifi cantly higher (p=0.01) (Figure 2) and epithelial CCL14 trended tion. Both cases achieved neutrophil and platelet engraftment to be higher in GI-GVHD compared to non-GVHD enteritis. similarly to previous dual SCT performed in HIV negative patients, We conclude that CD146+CCR5+T cell frequency is a cellular bio- as well as full CB chimerism. However, case 1 developed beside marker of GI-GVHD with prognostic and predictive value. Mea- a severe lung infection, relapse of the underlying malignancy surement of this novel double positive T cell population may allow 2 months after SCT and died consequently. Case 2 presented one for early identifi cation of patients at risk for subsequent GI-GVHD, episode of bacterial sepsis with good response to antibiotics, and and is drug-targetable. one episode of CMV reactivation controlled with valgancyclovir. Six months after SCT, this patient is alive and in CR. Extensive analyses of viral and immunological compartments were performed in both patients. Conclusions: CB SCT is feasible in patients eligible for allogeneic SCT and HIV infection. The dual strategy with the co-infusion of auxiliary cells from a TPD seems to off er a fast neutrophil engraftment similarly to HIV negative patients. Therefore, this strategy should be considered in patients with HIV infection and indication for allogeneic SCT, whenever CB is the source of choice and especially if a CCR5 mutated unit is available. However, a harmoni- zation of transplanted patients is urgently required. To guide this process, an international consortium has been founded.

Biology of GvHD

O390 A novel CD4+CD146+CCR5+ T-cell population is a biomarker of intestinal graft-versus-host disease A. Gomez (1), S. G. Hammer (1), T. Braun (1), S. Hanash (2), J.L.M. Ferrara (1), J.K. Greenson (1), H. Wang (2), Q. Zhang (3), M. Vander Lugt (4), P. Pongtornpipat (1), K. Lamiman (1), M. Colon (1), L. Chang (1), S.W. Choi (1), J.E. Levine (1), A. Harris (1), D.R. Couriel (1), P. Reddy (1), S. Paczesny (5) (1)University of Michigan (Ann Arbor, US); (2)MD. Anderson (Houston, US); (3)Fred Hutchinson Cancer Research Center (Seattle, US); (4)Children’s Hospital of Pittsburgh (Pittsburgh, US); (5)Indiana University (Indianapolis, US)

Graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract is a major limitation of allogeneic hematopoietic stem cell transplantation (HSCT). We performed proteomic analysis to identify biomarkers using plasma taken 14 days prior to clinical manifestations of GI-GVHD. We selected candidates that were increased at least 1.5 fold in plasma taken from GI-GVHD patients compared to HSCT patients without GVHD. The chemokine motif ligand 14 (CCL14) and CD146 were the two lead candidates. CCL14 binds to the chemokine receptor CCR5 on T cells. CD146 is expressed O391 by activated T cells and endothelial cells. We analyzed peripheral Epithelial cytoprotection sustains ectopic expression of blood cells from 214 HSCT patients (71 GI-GVHD, 48 No GVHD, 33 tissue-restricted antigens in the thymus medulla during non-GVHD enteritis, 22 skin fi rst GVHD, 40 isolated skin GVHD) at murine acute graft-versus-host disease onset of symptoms. The frequency of CD146+CCR5+T cells was S. Dertschnig (1), C. Bucher (1), R. Ivanek (1), M. Hauri-Hohl (2), signifi cantly increased in GI-GVHD patients compared to patients G. Holländer (3), W. Krenger (1) without GVHD (p<0.001), or non-GVHD enteritis (p<0.001), or with (1)University of Basel (Basel, CH); (2)Benaroya Research Institute isolated skin GVHD (p=0.007) but not with skin fi rst and then GI- (Seattle, US); (3)University of Oxford (Oxford, GB) GVHD (p=0.28) (Figure 1). We then classifi ed patients into low and high risk groups according to the median CD146+CCR5+ frequency The development of acute graft-versus-host disease (aGVHD) pre- in GI-GVHD patients (2.3%). A high frequency of CD146+CCR5+T disposes to chronic GVHD whose autoimmune manifestations are cells predicted higher 6-month non-relapse mortality in patients integral components of disease. It remains uncertain, however, who eventually developed GI-GVHD (42% vs. 20%, p=0.02). We then whether and how autoimmunity is linked to antecedent alloim- further characterized this population. CD146+CCR5+T cells had a munity. A hallmark of murine aGVHD is the de novo generation primarily eff ector memory phenotype (CD45RA-CCR7-) compared of autoreactive T cells from donor HSC, suggesting a defect in

S66 thymic central tolerance. Essential for clonal deletion is the ecto- arguing against a local immunosuppressive eff ect by Tregs. Dif- pic expression of a full scope of tissue-restricted peripheral self- ferently from circulating T cells, however, BM T cells expressed antigens (TRAs) which is a distinct property of mature medullary the immunosuppressive receptor VEGFR-2. Peripheral blood T thymic epithelial cells (mTEChigh). Since the thymus epithelium cells co-cultured with VEGF-producing BM-resident stromal cells is a target of donor T-cell alloimmunity we hypothesized that thy- promptly up-regulated VEGFR-2, resulting in a dose-dependent mic aGVHD interfered with the mTEChigh capacity to sustain TRA suppression of proliferation in response to polyclonal activa- diversity. We found that reductions in mTEChigh compartment tors or allostimulation. Interestingly, VEGFR antagonists, such as sizes were universal manifestations of thymic aGVHD in murine sorafenib and sunitib, but not the control TKI dasatinib, partially models of haploidentical and fully MHC-disparate transplanta- reverted the antiproliferative eff ects of stromal cells. tion. Contraction of the total mTEChigh pool corresponded to a Conclusions: While protecting from GVHD, rapamycin-induced progressive decrease in the subset which expresses autoimmune Tregs do not traffi c to the BM, suggesting the full preservation of regulator (Aire), a key regulator of TRA expression. When testing the GVL eff ect. The potential immunosuppressive eff ect of BM- entire residual mTEChigh cell pools from individual recipients for resident stromal cells merits further investigation, as targeted global gene expression we found that aGVHD altered expression intervention may increase the GVL eff ect after HLA-haploindenti- of ubiquitous genes (Ub) and TRAs. Among the genes that were cal HSCT. repressed by greater than three-fold in relation to mice without aGVHD, TRAs were present at much higher frequencies than Ub, however (67%, O/E ratio > 1). We concluded that contraction in TRA diversity during aGVHD was due to non-uniform purging of TRAs secondary to a reduction in Aire+mTEChigh cells. The most substantially repressed TRAs were enriched for genes specifi c for known target tissues of chronic GVHD. Peritransplant administration of fi broblast growth factor-7 (Fgf7), a TEC cytoprotective agent, maintained a stable pool of Aire+mTEChigh at ~3000 cells/ mouse in mice with aGVHD. Rescue of mTEChigh numbers was due to enhanced proliferation of cells within the total mTEC compartment. In parallel, fewer TRAs were repressed by 7 weeks after transplantation. Hence, enhanced heterogeneity of the mTEChigh cell pool segregated with increased diversity of the TRA transcrip- tome in mice with aGVHD. Taken together, our data may provide a mechanism for how autoimmunity develops in the context of aGVHD. Moreover, approaches for epithelial cytoprotection may prove to prevent the emergence of thymus-dependent autoreactive T cells.

O392 O393 In vivo rapamycin-induced Tregs do not traffi c to the bone The expansion capacity of CD4- iNKT cells contained in marrow: preservation of the GVL eff ect after T-cell replete the graft highly predicts the occurrence of human acute HLA-haploidentical HSCT graft-versus-host disease F. Gullotta (1), J. Peccatori (1), A. Forcina (1), M. Carbone (1), M.T Rubio (1), M. Bouillié (2), N. Bouazza (3), T. Coman (2), H. Trebeden- M. Noviello (1), M.T. Lupo-Stanghellini (1), M. Battaglia (1), Negre (4), S. Lapusan (1), D. Sibon (2), A. Marçais (2), R. Belhocine M. Bernardo (2), F. Locatelli (2), F. Ciceri (1), C. Bonini (1), (1), C. Bompoint (1), E. Recchia (1), S. NGuyen (4), A. Brignier (2), A. Bondanza (1) M. Cavazzana-Calvo (2), F. Suarez (2), F. Norol (4), M. Mohty (1), (1)Ospedale San Raff aele Scientifi c Institute (Milan, IT); (2)Ospedale M. Leite De Moraes (2), S. Urien (3), O. Hermine (2) Pediatrico Bambin Gesù (Rome, IT) (1)Saint Antoine Hospital (Paris, FR); (2)Necker Hospital (Paris, FR); (3)Cochin Hospital (Paris, FR); (4)Pitié Salpétrière Hospital (Paris, FR) Introduction: we have recently completed a Phase I/II study of T-cell replete HSCT from an HLA-haploidentical donor in 113 pts Introduction: Invariant NKT (iNKT) cells represent a subtype of with hematological malignancies after treosulfan-based condi- immunoregulatory T lymphocytes. We have previously reported tioning, followed by GVHD prophylaxis with rapamycin (TrRaMM, that post-transplant iNKT reconstitution can predict the occur- Eudract Nr. 2007-005477-54). Incidences of grade III-IV acute rence of acute GvHD (aGvHD) and overall survival after human GVHD and chronic extensive GVHD were 21% and 35%, respec- allogeneic haematopoietic stem cell transplantation (HSCT) tively. Rapamycin treatment was associated with the expansion of (Rubio, Blood 2012). Here, we analyzed the impact of the graft circulating CD4+/CD25+/FoxP3+/IL-7Rα- Tregs (at day 30, median content in iNKT cells as well as of the expansion capacity of iNKT 6.5% range 0.2-37.2, P<0.01 compared with healthy and cyclo- cell subtypes on the occurrence of aGvHD. sporine-treated controls), which were suppressive ex vivo. Impor- Patients and Methods: 61 adult patients allografted for diff erent tantly, peripheral blood Tregs frequencies correlated with GVHD haematological malignancies (70% AML) entered this three cen- severity (Graph 1). One-year PFS was 44% in pts with early disease tre retrospective study. In all patients, the graft proportions and (n=18) and 30% in pts with advanced disease (n=95). absolute doses of total, CD4- and CD4+ iNKT, CD4+, CD8+, γ-δ+ Aim: A long-standing question is whether Tregs-based strategies T cells and naïve/ memory regulatory T cells were analyzed by fl ow may interfere with the GVL eff ect. We thus aimed at verifying if cytometry. An ex-vivo expansion of CD4+ and CD4- iNKT cells in rapamycin-induced Tregs traffi c to the BM and suppress eff ector the presence of IL-2 and α-GalCer for 15 days could be performed T cells locally. In parallel, we studied the immunosuppressive in 41 patients. Results were correlated to the occurrence of grade eff ect of BM-resident stromal cells. II to IV aGvHD (observed in 30% of the patients). Results: Despite peripheral Tregs expansion, the BM of rapamycin- Results: In univariate analysis, among the diff erent explored lym- treated pts was depleted of Tregs (at day 30, BM Tregs frequencies: phocyte subsets and functional analyses, only the absolute dose median 0.3% range 0.0-2.2, P<0.01 compared with PB Tregs). High- of CD4- iNKT cells/kg, the total iNKT/T and CD4- iNKT/T ratios and level CXCR4 expression on circulating Tregs suggested a specifi c the expansion capacity of CD4- iNKT cells were signifi cantly and antimigratory eff ect of rapamycin, rather than a failure to express inversely associated with the occurrence of grade II-IV aGVHD the addressin. On the contrary, the BM was heavily infi ltrated by (p=0.03, p= 0.005, p=0.003 and p=0.0005, respectively). In mul- CD45RA-/CD62L- eff ector memory CD8+ T cells. The resulting eff ec- tivariate analysis, the expansion capacity of the CD4- iNKT cells tor/Tregs ratio was strongly in favor of eff ector T cells (Graph 2), contained in the graft was an independent predictive factor of

S67 the occurrence of grade II-IV aGvHD (OR=0.44, 95% CI: 0.21-0.89, O395 p=0.021) as well as the use of a an unrelated donor (p=0.026). Blocking VIP-signalling after allogeneic bone marrow Using ROC curves, we found that the expansion factor of the transplantation (allo-BMT) enhances the cytotoxic graft- CD4- iNKT cells contained in the graft was the best predictive fac- versus-leukaemia activity of donor T-cells tor for the occurrence of grade II-IV aGvHD (AUC=0.82). All grafts E. Waller, H.D. Yun, J.M. Li included, the sensitivity of the test was 96% and the specifi city of Winship Cancer Institute (Atlanta, US) 70%. In PBSC grafts (n=24 with 7 grade II-IV aGVHD), the sensitivity and specifi city were of 100%. Background: Relapse following allogeneic bone marrow transplan- Conclusion: These results confi rm the hypothesis of an immuno- tation (allo-BMT) represents a failure of the graft-versus-leukemia regulatory role of CD4- iNKT contained in HSC grafts in controlling response, and tolerance of donor T-cells against residual cancer. aGvHD. In addition, we provide evidence that ex vivo expansion We have recently demonstrated that infl ammation up-regulates of CD4- iNKT cells might be used as a predictive test for the occur- the PD-1/PD-L1 signaling pathway that is associated with toler- rence of aGvHD. Our fi ndings should be confi rmed in a larger ance and anergy of T-cells. Vasoactive intestinal peptide (VIP) has number of donors and provide a rationale to decipher mechanism potent immune-suppressive activity and generates tolerogenic of action of iNKT CD4- cells and to explore their potential thera- dendritic cells (DC) that block graft-versus-host disease (GvHD) peutical benefi t. and suppress Th1-mediated cellular immunity in mouse models of BMT. We have published that transplanting VIP-knockout (VIP-KO) donor cells dramatically decreases PD-1 expression on O394 activated CD8 T-cells and increases cellular antiviral immunity Ikaros defi ciency in host haematopoietic professional leading to increased survival following murine cytomegalovirus APCs and leukaemic cells separates GVL from GVHD in a (mCMV) infection in C57BL/6 mice (Journal of Immunology 2011, calreticulin-dependent manner after experimental 187:1057). allogeneic BMT Methods: We used the B6-B10BR murine MHC-mismatched allo- T. Toubai (1), Y. Sun (1), N. Mathewson (1), H. Tamaki (1), C. Liu (2), BMT model to test whether blocking VIP-signaling could enhance P. Reddy (1) the GvL eff ect of donor T-cells. Murine allogeneic transplant recip- (1)University of Michigan Cancer Center (Ann Arbor, US); (2)University ients of wild type (WT) BM plus T-cell allografts were treated with of Florida (Gainesville, US) daily s.c. injections of VIPhyb, a peptide VIP antagonist, PBS (controls), or transplanted with donor cells from VIP-KO mice. Tumor Background: GVL eff ect following allogeneic BMT is critical for burden was monitored using a luciferase+ T-cell lymphoma cell its curative potential. However, GVL is tightly linked to GVHD. line administered by i.v. injection the day before transplant. Sur- Amongst hematological malignancies acute lymphoblastic leuke- vival and GvHD were monitored and ex vivo cytolytic activity mia (ALL) is most resistant to GVL,the reasons for which remain against tumor and allogeneic splenocytes was tested. poorly understood. Studies by our collaborators (Nature- 2007. Results: Tumor-free survival was dramatically enhanced in B6-B10. NEJM -2009. Leukemia- 2010) have identifi ed alterations in Ikaros BR transplant recipients treated with one week of VIPhyb injec- (Ik) transcription factor as the major marker for poor outcomes tions (80% 100-day survival) and B10.BR recipients of WT BM and in ALL. We have also shown that professional host hematopoietic splenocytes from C57BL/6 VIP-KO donor (35% 100-day survival) antigen presenting cells (APCs) are necessary for induction of compared with no survivors among PBS-treated recipients of WT robust GVL (Nature Medicine-2005). However, whether Ik plays grafts. GvHD was not increased by VIPhyb-treatment, and recipi- a role in host APC function and GVL responses is not known. In ents of VIP-KO BM plus splenocytes did not have more GvHD than this study we utilized multiple, clinically relevant murine models recipients of equivalent numbers of cells from WT donors. Ex vivo of allogeneic BMT to explore whether Ik expression in hemato- tumor-specifi c cytolytic activity, but not cytolytic activity against poietic APC and/ or in leukemic cells is critical for regulating GVL normal recipient-type or third party splenocytes, was signifi cantly and GVHD. enhanced in splenocytes harvested from VIPhyb treated recipi- Methods and results: We fi rst generated [Ik-/-B6-B6] (Ikaros defi - ents engrafted with WT cells. ciency is limited to professional host hematopoietic APCs) and Conclusion: Blocking VIP signaling enhanced GvL activity of donor the control [Wt-B6-B6] chimeras. They were utilized as recipients T-cells without increasing GvHD. The ability of VIP antagonist- in allogeneic-MHC matched minor disparate (C3H.SW) or MHC treatment to down-regulate PD-1/PD-L1 signaling and enhance disparate (BALB/c) models. GVL was tested against MBL2 tumors adaptive cellular immunity is a novel approach to treat and pre- (non-ALL ), p185BCR-ABL1 Arf (Wt-ALL ) and p185BCR-ABL1 Arf- vent relapse in allo-BMT. IKZF1 (Ik mutant ALL- similar to the clinically relevant Ik6 mutation) leukemias that were syngeneic to the host. The [Ik-/-B6-B6], animals demonstrated more severe GVHD than [Wt-B6-B6] ani- O396 mals. However, they showed reduced leukemia clearance regard- Detection of male DNA in CD34+ and CD34- cells derived less of all three types of leukemia demonstrating that absence of from G-CSF-exposed female donors Ik in the host hematopoietic APCs enhanced GVHD but reduced A.G.S. van Halteren (1), P. Rosier (1), B. Kemps-Mols (1), C. Vervat (1), GVL. The loss of GVL was even more pronounced when both the J. Pool (1), F. Calkoen (1), J. Zwaginga (1), M. Fechter (2), M.J.D. van leukemia and the host APCs, were defi cient in Ikaros. Mechanis- Tol (1) tic studies with tetramer analyses demonstrated reduced tumor (1)Leiden University Medical Center (Leiden, NL); (2)Europdonor specifi c immunodominant (gag+) antigen responses in the [Ik-/- Foundation (Leiden, NL) B6-B6] group. The Ik mutant leukemia cells demonstrated reduced expression of calreticulin that has been shown to impair sensitiv- Introduction: Feto- and trans-maternal exchange of male hemato- ity to immune mediated killing of tumors. Rescue of calreticulin poietic cells may lead to engraftment of male cells in both parous expression enhance the apoptosis of Ik defi cient leukemia to allo- and nulliparous women. This so called male microchimerism (Mc) T cell cytotoxicity. may persist for decades. Male DNA is frequently detected in CD34- Conclusions: Together our data demonstrate that absence of enriched peripheral blood stem cells (PBSC) collected from female Ikaros in, both host hematopoietic APCs and leukemia, enhances donors. Whether PBSC grafts also contain long-lived male CD34+ GVL resistance through downregulation of calreticulin despite progenitor cells is unclear. worsening of GVHD. Therefore strategies that overcome the Methods: We set out to analyze in more detail the chimeric cell impact of Ik defi ciency might enhance GVL without aggravating types which end up in the PBSC grafts prepared from n=42 G-CSF GVHD. treated donors with a well documented pregnancy and family history. The presence of male Mc in either whole blood cells or purifi ed cell types was quantifi ed by a real time PCR assay specifi c for a multicopy gene locus on the Y chromosome (DYS1). This sensitive

S68 assay reliably detects the presence of 1 male cell in 10E5 female O398 cells. Genetically modifi ed EBV-specifi c cytotoxic T-lymphocytes Results: In 9 out of 38 (24%) female donors male Mc was detected induce regression of autologous EBV-induced lymphopro- in total blood cells collected at the start of apheresis. The male liferation despite immunosuppression in xenografted NSG cells detected in these women were likely derived from diff er- mice: a novel strategy for EBV-associated post-transplant ent sources including older brothers of the donor’s mother or of lymphoproliferative disease the donor herself as well as male off spring. Separation by fl ow- I. Ricciardelli (1), J. Brewin (1), M. Blundell (1), M. Pule (2), P.J Amrolia (1) cytometric sorting revealed that male Mc was present both in (1)Institute of Child Health (London, GB); (2)Cancer Institute myeloid (CD45+ CD33+ CD14+) cells and non-myeloid (CD45+ (London, GB) CD33- CD14-) cells. To verify that the CD33- fraction may also contain male progenitor cells, CD34+ cells were isolated by magnetic Objective: EBV associated lymphoproliferative disease (PTLD) bead-guided separation (purity 90-96%) from the PBSC grafts of remains a major cause of morbidity/mortality after stem cell (SCT) n=9 chimeric donors. After culturing these cells for minimally 14 or solid organ (SOT) transplant. Adoptive immunotherapy with EBV- days in a standard CFU assay, male DNA could be detected in 3 specifi c cytotoxic lymphocytes (CTLs) therapy, whilst highly eff ec- out of 9 (33%) pooled colonies (erythroid, granulocytic and mono- tive in the SCT setting, have been less successful after SOT where cytic colonies). continued immunosuppression therapy is necessary to prevent Conclusion: Our study shows that the PBSC grafts of G-CSF rejection. Our group has previously genetically engineered EBV-CTL exposed women may comprise small quantities of male chimeric to enable them to function in the presence of the calcineurin inhibi- cells with a variable life span. Importantly, PBSC contamination tor Tacrolimus (FK506) through retroviral transfer of a calcineurin B by male cells is not confi ned to women who have given birth to mutant (CNB30). Here we examined the ability of FK506-resistant sons. Whether such ‘third party’ alloantigen-presenting male cells EBV-CTLs to control the tumour progression in the presence of actually engraft in the recipients of these grafts and whether such immunosuppression in vivo using a xenogenic mouse model. engraftment is associated with an increased risk of Graft-versus- Methods: NOD/SCID/IL2rγnull (NSG) mice were inoculated subcu- Host Disease is currently under investigation. taneously with 5×106 EBV-transformed lymphoblastoid B cell lines (LCL) labelled with F-Luc to develop human EBV+ B cell lymphoma. To evaluate in vivo antitumor activity, 5×106 CTLs retrovirally transduced with CNB30 and 5×106 eGFP control CTLs were injected intravenously after 7 days in the presence/absence of ip FK506. Cellular and gene therapies Tumour growth was analysed using the Xenogen-IVIS system. Results: Adoptive transfer of autologous CNB30 transduced CTLs induced EBV lymphoma regression in the presence of FK506, as O397 assessed both by IVIS (Figure 1) and tumour size, whereas FK506 Γ-δ T-cells elicited by CMV-reactivation after allo-SCT cross- treated mice receiving eGFP control CTLs had tumour progres- recognize CMV and leukaemia sion (p < 0.05). CNB30-CTL mice treated with FK506 survived W. Scheper (1), S. van Dorp (1), S. Kersting (1), F. Pietersma (1), signifi cantly longer (p<0.0001) than eGFP-CTLs treated animals. C. Lindemans (1), S. Hol (1), S. Heijhuurs (1), Z. Sebestyen (1), CNB30 transduced EBV CTLs persisted longer and expanded more C. Gründer (1), V. Marcu-Malina (1), A. Marchant (2), C. Donner (2), (p<0.0001) than eGFP-CTLs in peripheral blood of mice treated B. Plachter (3), D. Vermijlen (2), D. van Baarle (1), J. Kuball (1) with FK506 demonstrating a selective growth advantage and (1)UMC Utrecht (Utrecht, NL); (2)Université Libre de Bruxelles enrichment of CTLs resistant to immunosuppression (Figure 2). (Brussels, BE); (3)UMC of the Johannes Gutenberg-University Immunohistochemical staining showed that adoptively trans- (Mainz, DE) ferred CTLs home to the tumour and an increase of tumour infi l- trating T cells in CNB30-CTL compared with eGFP-CTLs treated Human cytomegalovirus (CMV) infections and relapse of disease mice in the presence of FK506 was observed. remain major problems after allogeneic stem cell transplanta- Conclusions: Our results indicate that CNB30 modifi ed EBV-CTL tion (allo-SCT), in particular in combination with CMV-negative can induce regression of EBV-associated tumours in vivo in the donors or cordblood transplantations. Recent data suggest a face of ongoing immunosuppression. Clinical application of this paradoxical association between CMV-reactivation after allo-SCT novel approach may enhance the effi cacy of adoptive transfer of and reduced leukemic relapse. Given the potential of V{δ}2-nega- EBV-CTL in SOT patients developing PTLD without the need for tive {γ}{δ}T-cells to recognize CMV-infected cells and tumor cells, reduction in immunosuppressive therapy. the molecular biology of distinct {γ}{δ}T-cell-subsets expanding during CMV-reactivation after allo-SCT was investigated. V{δ}2- negative {γ}{δ}T-cell expansions after CMV-reactivation were observed not only with conventional but also cordblood donors. Expanded {γ}{δ}T-cells were capable of recognizing both CMV- infected cells as well as primary leukemic blasts. CMV- and leukemia-reactivity were restricted to the same clonal population, whereas other V{δ}2-negative T-cells interact with dendritic cells (DCs). Cloned V{δ}1-TCRs mediated leukemia-reactivity and DC- interactions, but surprisingly not CMV-reactivity. Interestingly, CD8{α}{α} expression appeared to be a signature of {γ}{δ}T-cells after CMV exposure. However, functionally CD8{α}{α} was primarily important in combination with selected leukemia-reactive V{δ}1-TCRs, demonstrating for the fi rst time a co-stimulatory role of CD8{α}{α} for distinct γ δTCRs. Based on these observations, we advocate the exploration of adoptive transfer of unmodifi ed V{δ}2-negative {γ}{δ}T-cells after allo-SCT to tackle CMV-reactivation and residual leukemic blasts, as well as application of leukemia-reactive V{δ}1-TCR-engineered T-cells as alternative therapeutic tools.

S69 O399 from high viral titers (140.000 viral copies/ml PB) at day +66 after Direct tumour recognition and helper function of CD4+ T cells aSCT accompanied by high fever, night sweats, weight loss and modifi ed to express a CD19-specifi c CAR in vitro and in a liver enzyme elevation. Adoptive T-cell transfer was performed at preclinical lymphoma model day +105 at a dose of 1.0 Mio cells/kg. Here we present data using M. Hudecek (1), D. Sommermeyer (2), M.T. Lupo-Stanghellini (3), a TCRβ deep sequencing approach before and after (+21, +64 and P.L. Kosasih (2), H. Einsele (1), M.C. Jensen (4), S.R. Riddell (2) +232) transfer of EBV specifi c T-cells in combination with an exten- (1)University of Würzburg (Wuerzburg, DE); (2)Fred Hutchinson sive immunomonitoring by fl ow cytometry and HLA multimere Cancer Research Center (Seattle, US); (3)San Raff aele Scientifi c analysis. Prior to T-cell transfer the patients CD8+ compartment Institute (Milan, IT); (4)Seattle Children’s Research Institute comprised only EPL reactive T-cells in low numbers (20 cells/μl (Seattle, US) PB). In contrast, after peptide stimulation, the T-cell product contained high percentages of EPL (25.4%), QAK (0.97%), RAK (35.7%), Recent clinical trials of adoptive immunotherapy employing T cells YPL (0.17%), and HPV (3.65%) specifi c T-cells. After adoptive trans- modifi ed to express CD19-specifi c chimeric antigen receptors fer we observed an up to 400% increase of RAK and EPL specifi c T- (CARs) have demonstrated the potential to induce durable remis- cells over 10 days. The TCR deep sequencing data confi rmed that sions of advanced B-cell tumors in some patients, and antitumor predominantly CD8+ T-cells were transferred into to the patient, responses correlate with CAR T cell persistence and proliferation since the T-cell product TCR β rearrangements detected in the in vivo. Current clinical protocols have focused on using bulk patient exclusively belonged to CD8+ T-cells. Additionally, analy- PBMC that contain various proportions of the diff erent functional sis of the TCR β repertoire displayed that peptide stimulation of subsets of CD8+ and CD4+ T cells for CAR modifi cation. This PBMC resulted in selection of distinct TCR β rearrangements in the strategy allows poor control over the cellular composition of the T-cell product. These TCR sequences could in part be attributed fi nal CAR T cell product, and may in part explain the marked dif- to public EBV specifi c TCR sequences. Furthermore, when CD8+ ferences in cell persistence, toxicity, and effi cacy observed in the T-cells of the patient were analysed, we detected that TCR β rear- clinic. However, designing cell products of more uniform compo- rangements present in the T-cell product were present even long sition requires analysis of the role of individual subsets alone and term in the patient. Moreover, we were able to specify the per- in combination. centage of every clonotype in our samples at defi ned time points. Here, we derived CD19-CAR T cell lines from purifi ed CD8+ cen- This suggests that expansion of these TCRs resulted in control of tral memory (CM) T cells of healthy donors and from purifi ed CD4 EBV. These data show, that only limited numbers of TCRs emerge naïve (N), CM, and eff ector memory (EM) subsets and character- during the peptide stimulation and that these TCRs in part expand ized tumor recognition and the ability of CD4+ CAR T cells to pro- further in vivo. A subset of very few TCRs can be attributed to the vide “help” to CD8+ CAR CTL. CD4+ CAR T cells had specifi c but control of EBV reactivation. weak cytolytic activity against CD19+ tumors including primary CLL, Raji lymphoma, and K562 cells transfected with CD19. Mul- tiplex cytokine analysis detected high-level production of IFN-γ, O401 IL-2 and TNF-α from N and CM CD4+ CAR T cells, and CFSE staining T-cells co-expressing a CD44v6-specifi c CAR and a suicide showed signifi cantly higher proliferation after stimulation with gene enable the safe eradication of leukaemia and myeloma tumor compared to CD8+ CAR CTL. Co-culture experiments dem- M. Casucci (1), B. Nicolis di Robilant (1), L. Falcone (1), B. Camisa onstrated that the addition of CAR-transduced but not untrans- (1), P. Genovese (1), B. Gentner (1), M. Bernardi (1), M. Marcatti (1), duced CD4+ T cells resulted in maximum proliferation of CD8+ C. Bordignon (1), B. Savoldo (2), F. Ciceri (1), L. Naldini (1), G. Dotti (2), CAR CTL in vitro. C. Bonini (1), A. Bondanza (1) We confi rmed a benefi cial and synergistic eff ect of individual CD4- (1)San Raff aele Scientifi c Institute (Milan, IT); (2)Center for Cell and helper T cell subsets on the anti-tumor effi cacy of CD8+ CD19-CAR Gene Therapy (Houston, US) CTL in a mouse model of systemic lymphoma (NSG/Raji-ffl uc). In this model, addition of a non-curative dose of CD4+ CAR T cells to Introduction: The recent successes of T cells modifi ed with chime- a non-curative dose of CD8+ CD19-CAR CTL (0.5x106) resulted in ric antigen receptors (CARs) in B-cell tumors heralds a new frontier a more than 10-fold increase in absolute CD8+ T cell numbers in in cancer gene therapy. Targeted antigens are however limited the peripheral blood compared to mice treated with CD8+ CAR to B-cell lineage markers. The variant isoform 6 of the adhesion CTL alone, followed by complete tumor eradication and long- receptor CD44 (CD44v6) is widely expressed on acute myeloid term tumor free survival of all mice (n=6). These results show that leukemia (AML) and multiple myeloma (MM) cells and is crucially analyses of cell-intrinsic qualities can inform the rational design of involved in their interaction with the bone marrow niche. uniform cell products containing both tumor-specifi c CD8+ and Aim: Safe and eff ective targeting of AML and MM with T cells mod- CD4+ T cells, which may improve the predictability of response ifi ed to co-express a CD44v6-specifi c CAR and a suicide gene. and toxicity of cancer immunotherapy. Results: Knocking-down CD44v6 expression by short-hairpin RNA interference completely abated the ability of AML and MM cells to engraft in NSG mice. Moreover, primary AML cells co-cultured O400 with BM-derived mesenchymal stromal cells (MSCs) signifi cantly Deep insights after adoptive transfer of donor-derived EBV- up-regulated CD44v6 expression and acquired resistance to cell specifi c T-cells after matched unrelated allogeneic stem cell death induced by daunorubicin. Importantly, pre-incubation with transplantation a CD44v6-blocking mAb reverted the chemoresistant phenotype. R. Gary (1), J. Ritter (2), V. Seitz (2), M. Aigner (1), S. Standar (1), We therefore constructed a CAR by cloning the scFv of a human- S. Moi (1), H. Bruns (1), S. Völkl (1), M. Wurdack (1), W. Rösler (1), ized CD44v6 mAb in a CD28/TCR ζ chain backbone and expressed A. Mackensen (1), A. Moosmann (3), M. Hummel (2), A. Gerbitz (1) it with the Herpes simplex virus thymidine kinase (TK) suicide (1)University of Erlangen (Erlangen, DE); (2)Charite Berlin (Berlin, DE); gene by means of a LV carrying a PGK bi-directional promoter. (3)Helmholtz Center Munich (Munich, DE) After LV transduction, T cells acquired potent cytotoxic activity against autologous AML and MM cells, and selective sensitivity to Tracking of T-cell development using T-cell receptor (TCR) deep cell death induced by the prodrug ganciclovir. Importantly, add- sequencing allows for monitoring on a single cell level. It opens ing MSCs did not interfere with eff ector functions. Once infused new ways to follow up T-cell reconstitution in patients after allo- into NSG mice, CD44v6.CAR-trasnduced T cells persisted long- geneic stem cell transplantation (aSCT). We recently developed a term and completely eradicated previously engrafted autologous GMP conform manufacturing protocol to generate donor derived leukemic cells. Antitumor effi cacy was dependent on transduc- EBV specifi c T-cells by peptide stimulation. The product was used tion with CD3/CD28-beads and culture with IL-7/IL-15, according in a patient with EBV negative T-cell lymphoma after matched to a protocol that preserves the expression of the self-renewal unrelated aSCT. EBV-peptides used for stimulation matched MHC marker IL-7 receptor and of the BM addressin CXCR4. Interest- B0801 (QAK, RAK) and B3501 (YPL, EPL, HPV). The patient suff ered ingly, CD44v6.CAR-transduced T cells were not cytotoxic against

S70 CD34+CD38- hematopoietic stem cells, but recognized mature Conclusion: These results suggest that chimerism analysis using monocytes. Since TK is immunogenic, we evaluated the human indel-qPCR is a useful tool to early detect and prevent relapse inducible caspase 9 (iC9) as alternative suicide gene. Diff erently after allo-SCT for AL. form TK, iC9 allowed the ablation of CD44v6.CAR-trasnduced T cells even in the absence of proliferation and with a much faster kinetic. O403 Conclusions: Targeting CD44v6 with CAR-transduced T cells has High-sensitivity haematopoietic chimerism by qPCR for the potential to cure AML and MM. Co-expressing a suicide gene relapse prediction and specifi c identifi cation of HLA loss is an eff ective tool for rescuing off -tumor recognition and toxicity. leukaemic variants L. Vago (1), B. Mazzi (1), L. Chiesa (1), R. Greco (1), L. Crucitti (1), V. Tettamanzi (1), C. Pultrone (1), E. Toff oletti (1), C. Toff alori (1), O402 P. Crivello (1), M. T. Lupo Stanghellini (1), M. Bernardi (1), C. Corti (1), Prediction of relapse and impact of immunomodulation J. Peccatori (1), D. Bost (2), F. Ciceri (1), K. Fleischhauer (1) based on quantitative RT-PCR of insertion/deletion (1)San Raff aele Scientifi c Institute (Milan, IT); (2)Celera Genomics polymorphism for chimerism analysis in acute leukaemia (Alameda, US) N. Jacque (1), S. NGuyen (1), J. Golmard (1), A. Garnier (1), M. Uzunov (1), J. Vernant (1), D. Bories (2), N. Dhédin (3) Background: In the last decades, allogeneic Hematopoietic Stem (1)Hôpital de la Pitié Salpétrière (Paris, FR); (2)Laboratoire Cell Transplantation (HSCT) has greatly improved the outcome d’hématologie (Créteil, FR); (3)Hôpital Saint Louis (Paris, FR) of hematologic malignancies. Still, post-transplantation disease recurrence remains a major issue, and therapeutic strategies Objectives: Detection of increasing mixed chimerism (IMC) using against relapsed disease remain to date unsatisfactory. Thus inter- standard PCR is correlated with relapse after allogeneic stem cell est has risen in how to anticipate the detection of residual malig- transplantation (allo-SCT) for acute lymphoblastic and myeloid nant cells by more sensitive techniques, such as those based on leukemias (AL); however, it is not known whether using a very quantitative Polymerase Chain Reaction (qPCR). Moreover, our sensitive method such as quantitative real time PCR of insertion/ group has recently described novel variants of leukemia post- deletion polymorphism (indel-qPCR) would be of benefi t. Here, transplantation relapse, characterized by genomic loss of the we studied whether this technique is useful for predicting relapse mismatched HLA (Vago et al, N Eng J Med, 2009), that are not after transplant for AL, and whether an immunomodulation (IM) detected by routine diagnostic assays and that should be treated (ie withdrawal of immunosuppressive drugs (IS) and/or donor diff erently from “classical” relapses, warranting the development lymphocyte infusion (DLI)) based on the detection of IMC would of novel specifi c assays. improve outcome. Methods: We prospectively validated a commercial hematopoi- Methods: We retrospectively include patients transplanted for AL, etic chimerism assay based on qPCR (AlleleSEQR® Chimerism excluding those without at least 2 analyses of chimerism. Eighty- Assay, Celera Genomics) in follow-up bone marrow samples har- fi ve patients (58 myeloblastic and 27 lymphoblastic AL) who vested from 55 patients transplanted in our centers, comparing it received 89 transplants were studied (69% in complete remission with standard Short Tandem Repeat (STR) chimerism analysis. To (CR) 1, 20% in CR2, 11% in progressive disease). Median age was discriminate between “classical” and “HLA loss” relapses, we devel- 41 years (18-67). Conditioning regimen was myeloablative in 59 oped chimerism assays targeted to specifi c HLA-A allele groups, cases, the source of stem cell was bone marrow in 43 patients, based on the same qPCR technology and targeted to the same peripheral blood in 41 and cord blood in 5. An IMC was defi ned sensitivity of the commercial assay. by an increasing of patient’s DNA rate of 1 log when it was under Results: qPCR chimerism displayed higher sensitivity in detec- 0.1% and an increasing of at least 0.1% beyond this threshold. tion of residual host cells as compared to STR: of the total 199 Results: The 3-year overall survival (OS) was 57.4% (95% CI: 48.1- samples tested, 52 exhibit a host chimerism above 0.5% and 33 66.7%), with a median follow-up of 30 months for the survivors above 1%, whereas 27 were positive in STR. qPCR chimerism (range 4-86). Twenty-one patients relapsed, leading to a 3-year could predict impending relapse with a sensitivity of 57% and incidence of relapse of 28.9%. Among the 57 transplants pre- a specificity of 43% for a 0.5% host threshold, and with a sen- sented IMC, 21 relapsed, whereas none of the 32 patients with sitivity of 29% and a specificity of 54% for a 1% host threshold, stable or decreasing chimerism relapsed. By multivariate analysis, comparing favourably with STR (sensitivity 17%, specificity 54%). the detection of IMC and the disease status at transplant were We setup 7 qPCR assays able to specifically detect 12 different strongly associated with relapse (p=0.0002; HR: 27.6, 95%CI: 3- allele groups of the HLA-A locus, with a combined frequency of 246 and p=0.0037; HR: 3.4 95%CI: 1.4-8.2, respectively). After the 81 % in the Caucasian population, and to discriminate HLA loss detection of one IMC (n=57), an IM was performed in 27 patients, from classical relapses in chimeric samples. The assays are cur- consisted in withdrawal of IS drugs for all, and followed by DLI in 2. rently being used on an exploratory basis in our Bone Marrow For these 27 patients, the 1 and 3-year relapse rate was 15.7%, ver- Transplantation Unit. sus 57.6% in the 30 other patients (p=0.0007). After IM, 6 patients Conclusions: Hematopoietic chimerism detection by qPCR appears developed graft versus host disease (GVHD), including the 2 who promising, displaying a higher sensitivity and a superimposable received DLI. Four of them died. Altogether, the 3-year overall sur- specifi city as compared to techniques currently in use, and allow- vival was 68.3% after IM versus 42.6% without any immunological ing the simultaneous detection of disease variants characterized intervention (p=0.02). by genomic HLA loss.

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