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Allogeneic hematopoietic cell transplantation (HCT) offers the highest chance for cure in patients with adverse-risk acute myeloid leukemia (AML) when performed in first remission (CR1). In contrast, patients in CR1 with favorable risk do not seem to benefit from allogeneic HCT due to the inherent risk of transplant-related mortality. Donor vs. no donor comparisons as well as prospective matched-pair analyses have suggested that allogeneic HCT performed in intermediate-risk AML may provide a higher probability of overall survival or relapse-free survival in patients ≤ 60 years of age with an acceptable risk for transplant-related mortality. On the other hand, many intermediate-risk patients relapsing after conventional chemotherapy may be successfully salvaged by allogeneic HCT. The role of allogeneic HCT in cytogenetically defined intermediate-risk AML patients in CR1 was addressed by a prospective randomized trial performed in 16 centers in Germany. Key inclusion criteria were: AML with intermediate-risk cytogenetics, first CR or CRi after conventional induction therapy, age of 18-60 years, and availability of an HLA-matched sibling or unrelated donor. For unrelated donors, a 9 out of 10 HLA allelic match was acceptable except for patients with an NPM1 mutation, for whom full 10/10 allele matching was required. Randomization was stratified according to age (< 40 vs. 40-60), NPM1/FLT3, and CEBP-alpha mutational status and unrelated vs. related donor availability. Endpoints included overall-survival as primary outcome and relapse-free survival (RFS), cumulative incidence of relapse, treatment-related mortality, and quality of life measured according to the short form (36) health status. From 2010 - 2018, 143 patients in CR1 were randomized into Arm A (n=76, allogeneic HCT) and Arm B (n=67, conventional consolidation and allo-HCT only in case of relapse). In July 2018, the trial was stopped prematurely due to slow accrual (143 out of 356 pts. randomized). Median age of the trial cohort was 51 years (range, 19-60), with 42% exhibiting an NPM1 and 25% a FLT3 mutation. A normal karyotype was reported in 84% of the included patients. All mentioned characteristics did not differ between both treatment arms. Sibling donors were available for 44 (31% of patients), matched unrelated donors for 99 (69%) patients. According to the intent-to-treat analysis, the probability of survival at 2 years was 71% (95% CI 60-81%) and 84% (95% CI 73-92%) in Arm A (Transplant) and Arm B (conventional consolidation), respectively (p=0.120, Figure 1A). RFS after allogeneic HCT was 69% (95% CI 57-80%) compared to 41% (95% CI 29-54%) after conventional consolidation (p=0.001, Figure 1B). Primary allogeneic HCT reduced the cumulative incidence of relapse at 2 years from 57% [95%-CI 46-71%] after conventional consolidation to 20% [95%-CI 13-31%] after HCT (p SF (36) scoring suggested a trend towards a lower physical functioning throughout the first 3 months after randomization in the primary HCT arm. No significant differences in vitality, mental health, social and emotional functioning could be documented between both treatment arms. In summary, the results of this first prospective randomized trial did not show that allogeneic HCT performed immediately after achievement of CR1 in patients with cytogenetically defined intermediate-risk AML ≤ 60 years of age conveys an overall survival advantage. However, allogeneic HCT in CR1 significantly reduced the relapse risk and was not associated with relevant impairments in quality of life. Although the limited statistical power of the trial does not allow definitive conclusions, delayed allogeneic transplantation seems to be a potential treatment algorithm in CR1 intermediate-risk AML with an available donor. Figure 1 Figure 1. Disclosures Schliemann: Jazz Pharmaceuticals: Consultancy, Research Funding; Roche: Consultancy; Philogen S.p.A.: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; AstraZeneca: Consultancy; Boehringer-Ingelheim: Research Funding; Novartis: Consultancy; Abbvie: Consultancy, Other: travel grants; Astellas: Consultancy; BMS: Consultancy, Other: travel grants. Schetelig: Roche: Honoraria, Other: lecture fees; Novartis: Honoraria, Other: lecture fees; BMS: Honoraria, Other: lecture fees; Abbvie: Honoraria, Other: lecture fees; AstraZeneca: Honoraria, Other: lecture fees; Gilead: Honoraria, Other: lecture fees; Janssen: Honoraria, Other: lecture fees . Glass: Riemser: Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau; Kite: Consultancy; BMS: Consultancy; Novartis: Consultancy; Helios Klinik Berlin-Buch: Current Employment. Platzbecker: Janssen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Geron: Honoraria; AbbVie: Honoraria. Burchert: Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding; Pfizer: Honoraria; Incyte: Honoraria; Gilead: Honoraria; BMS: Honoraria. Haenel: Jazz: Consultancy, Honoraria; GSK: Consultancy; Bayer Vital: Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria. Mueller: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; CTI: Membership on an entity's Board of Directors or advisory committees; Gentium: Other: Travel Support; Gilead: Other: Travel Support; Janssen: Other: Travel Support; Novartis: Other: Travel Support; Pfizer: Other: Travel Support; Sanofi: Other: Travel Support. Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Stelljes: Novartis: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Medac: Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Aim: Dynamic assessment of trends over time in patient- and transplant-specific characteristics and outcomes for patients undergoing 1st allogeneic haematopoietic cell transplant (allo-HCT) for Myelofibrosis (MF). Methods and Results: A total of 4142 MF patients were analysed who underwent allo-HCT between 1995-2018 (24-year period) across 278 centres based on data reported to the European Society for Blood and Marrow Transplantation. For analysis, 4 cohorts were considered based on year of allo-HCT: 60 years accounted for 8.7% of adults undergoing allo-HCT whereas for 2015-2018 this was 47%. Over time, increasing number of patients with a Karnofsky performance status (KPS) Conclusions: Despite a marked increase over this 24-year period in recipient age, RIC regimen utilisation and use of both URD and MMRD, this comprehensive analysis demonstrates stable OS and EFS rates. However, rates of GVHD have decreased over time, in particular extensive cGVHD. Further work is required to improve both the considerable NRM and relapse rates which remain significant. Disclosures McLornan: JAZZ PHARMA: Honoraria, Speakers Bureau; CELGENE: Honoraria, Speakers Bureau; NOVARTIS: Honoraria, Speakers Bureau. Platzbecker:Amgen: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Geron: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Chevallier:Incyte Corporation: Honoraria. Martínez-Lopez:Altum, Hosea: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Vivia Biotech: Honoraria; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Incyte: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding. Yakoub-Agha:Celgene: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria; Gilead/Kite: Honoraria, Other: travel support; Janssen: Honoraria.

Introduction: The most common cause of treatment failure after allogeneic hematopoetic stem cell transplantation (aHSCT) is relapse. The combination of venetoclax and the hypomethylating agents (HMA) azacitidine (AZA) or decitabine (DAC) have shown promising efficacy in elderly patients with AML. We here present clinical data on 32 patients, who were treated with an HMA/venetoclax combination therapy (HMAClax) for relapse of a myeloid malignancy after aHSCT, collected retrospectively from 11 German centers. Results: Sixteen patients (50%) were male, median age was 54 years (30.8-71.5). Diagnoses at aHSCT were 25 AML (17 primary, 8 emerging from MDS, CMML or OMF), 5 MDS, 1 CMML and one atypical CML. Twenty six patients were treated for relapse after their 1st and 6 after their 2nd aHSCT. Only 9 patients were in CR at aHSCT. The majority received a graft of a matched unrelated donor (21), 4 from an HLA-identical sibling and 7 from a haploidentical relative. Conditioning was myeloablative in 15 and RIC in 17patients. Median time from aHSCT to last relapse was 5.7ms (1.1-67.8). Five patients had molecular (MR) and 23 had hematologic relapses (HR), 4 patients had extramedullary manifestations 3 with concurrent HR and 1 with MR. Twenty-one patients were treated for 1st and 5 for 2nd relapse after 1st aHSCT. Four patients were treated for 1st and 2 for 2nd relapse after 2nd aHSCT. HMAClax was first line therapy for relapse in 8, 2nd line in 22, 3rd line 1 and 4th line in 1 patient. In 21 patients relapse had been refractory to HMA (+/- DLI, +/- lenalidomide). Median time from relapse to HMAClax was 1.8 ms (0.3-42.9). Twelve patients received AZA and 19 DAC with venetoclax. One patient was switched from AZAClax to DAClax because of rising MRD after 6 cycles and back to AZAClax after another 7 cycles. Six patients received DLI. Median number of cycles was 2 (1-15). Six patients are still on therapy. In total 75 cycles were given. Three patients had non-fatal tumor lysis syndrome. All but one patient had grade 3/4 neutropenia and 25 patients (78%) had grade 3/4 thrombocytopenia. Hospital admission for grade 3/4 infections was necessary in 23 patients (72%), 5 of these infections (22%) were fatal. Overall response rate was 43% (12/28, 2 CR MRD-, 4 CR, 2 CRi, 3 PR, 1 MLFS). Two patients died of infection before first response evaluation and in another 2 response has not been evaluated yet. ORR for patients who received first line HMAClax was 80% (4/5) and 35% (8/23) for salvage treatment. Three of 5 patients with MR reached CR, 2 received HMAClax first line. Time to best response was 1.2ms (0.7-3.8). Six patients lost best response after 1ms (0.4-3.3.) 2 underwent second transplant in remission, 4 have ongoing responses (0.4, 0.7, 3.1 and 8.8ms at last follow up). On July 25th 2019, median follow up was 3.3 ms (0.9-17.3), 20 patients (63%) had died and 12 were alive. Six were continuing HMAClax. One patient developed cGvHD and 4 underwent second aHSCT (2 in remission). Estimated median overall survival was 3.7ms (CI 2.9-4.7). Four responders are continuing treatment with HMAClax. Patients, who responded had an estimated OS of 11.1ms (2 underwent second aHSCT in remission). Median survival of patients with HMAClax first line therapy was 5.8ms and of patients with HMAClax salvage therapy 3.7ms. Conclusion: For patients relapsing after aHSCT, venetoclax plus AZA or DAC seems to be an effective, but also highly hematotoxic therapy. Responses occurred fast and were more frequently seen during 1st line treatment for relapse. Duration of response was short, especially in patients receiving HMAClax as 2nd, 3rd or 4th line therapy. Therefore HMAClax should be explored as 1st line therapy for relapse after aHSCT in combination with DLI or as a bridge to 2nd transplant. Disclosures Schuler: Celgene: Other: travel grants; Novartis: Honoraria, Other: travel grants; Alexion: Other: travel grants. Bug:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Hexal: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Other: travel grants; Celgene Neovii: Other: travel grant. Crysandt:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgem: Other: travel grant; celgene: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant. Jost:Jazz Pharmaceuticals: Honoraria; Sanofi: Honoraria; Gilead: Other: travel grants; Daiichi: Honoraria. Kaivers:Jazz Pharmaceuticals: Other: Travel Support. Mueller:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; CTI Life Sciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Financing of Scientific Research; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Gentium: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Jazz: Honoraria; Pharmaceuticals: Honoraria; Neovii: Honoraria; Novartis: Honoraria; Sanofi: Honoraria. Rautenberg:Jazz Pharmaceuticals: Other: Travel Support; Celgene: Honoraria, Other: Travel Support. Wolff:Takeda: Honoraria; Mallinckrodt: Honoraria; Novartis: Honoraria; Neovi: Honoraria. Schroeder:Celgene Corporation: Consultancy, Honoraria, Research Funding. Off Label Use: Venetoclax was used in combination with azacitidine or decitabine. The combination is not approved in the EU so far.. Kobbe:Pfizer: Honoraria, Other: Travel support; Abbvie: Honoraria, Other: Travel support; Biotest: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Jazz: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support, Research Funding; Takeda: Honoraria, Other: Travel support; Amgen: Honoraria, Other: Travel support, Research Funding; Neovii: Honoraria, Other: Travel support; Medac: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support. OffLabel Disclosure: Venetoclax was used in combination with azacitidine or decitabine. The combination is not approved in the EU so far.

Introduction The only curative treatment approach for patients with myelodysplastic syndromes (MDS) is allogeneic hematopoietic stem cell transplantation (HSCT), but disease relapse after transplantation is a major concern. Predictors for disease outcome after HSCT are limited. However, unfavorable cytogenetic abnormalities have been shown to serve as predictors for MDS-relapse after transplantation. Similar to the data available in MDS-patients not undergoing HSCT (Schanz et al. J Clin Oncol 2012), there is evidence that the novel 5-group cytogenetic classification has a better predictive value for outcome after HSCT than standard IPSS cytogenetics (Deeg et al. Blood 2012). The aim of this large multicentric, international study was to retrospectively determine the impact of the new 5-group cytogenetic MDS classification on outcome after HSCT. Patients and Methods Patients were selected from the EBMT database who had received HSCT for the treatment of MDS between 1982 and 2010 and for whom sufficient cytogenetic information was available. In total, 903 patients were included into the study. At time of HSCT, 97 (10.7%) patients had untreated MDS, 218 (24.1%) patients had advanced MDS or AML evolving from MDS in complete remission, and 227 (25.1%) patients were not in remission after treatment (in 12.3% information on stage of the disease was not available). Median time between diagnosis and transplant was 6.6 months (range 0.2-359.3). Matched related donor HSCT was performed in 574 patients (63.6%), and matched unrelated donor HSCT in 329 patients (36.4%). Bone marrow (35.4%) or peripheral blood (64.6%) served as stem cell graft. Myeloablative preparative regimens were used in 582 patients (64.5%), and a non-myeloablative regimen was given to 320 patients (35.4%). Impact of cytogenetic classification was analyzed in uni- and multivariate models regarding overall survival (OS) and relapse free survival (RFS) after HSCT. Predictive performance of the 2 classifications was compared by means of the cross-validated log partial likelihood. Results Estimated 5-year RFS and OS were 32% and 36% respectively. According to the 5-group cytogenetic classification 19 (2.1%) patients had very good risk cytogenetics, 204 (22.6%) normal risk cytogenetics, 438 (48.5%) intermediate risk cytogenetics, 178 (19.7%) poor risk cytogenetics, and 64 (7.1%) very poor risk cytogenetics. Good, intermediate, and poor risk cytogenetics according to IPSS were found in 192 (38.0%), 500 (40.2%), and 211 (23.7%) patients, respectively. In univariate analysis 5-group cytogenetic information was found to be strongly associated with OS and RFS (OS: log-rank test P

To assess the impact of spleen status on engraftment and early morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT), we analyzed 9,683 myeloablative allograft recipients from 1990 to 2006; 472 had prior splenectomy (SP), 300 splenic irradiation (SI), 1,471 with splenomegaly (SM), and 7,440 with normal spleen (NS). Median times to neutrophil and platelet engraftment were 15 vs. 18 days and 22 vs. 24 days for the SP and NS groups, respectively (p5.7x106/kg improved platelet engraftment at day+28. After adjusting variables by Cox regression, the incidence of graft-versus-host disease (GVHD) and overall survival were not different among groups. Splenomegaly is associated with delayed engraftment while splenectomy prior to HCT facilitates early engraftment without impact on survival.

Allogeneic stem cell transplantation (SCT) is the most effective treatment option for many hematologic malignancies, but graft-versus-host disease (GVHD) remains a major cause of treatment failure. Along with well-established risk factors for transplantation outcomes, recent single-center studies have identified a birth order effect in HLA-identical sibling SCT, with lower rates of acute and chronic GVHD and improved overall survival when the donor is younger than the recipient. One hypothesized mechanism for this effect is microchimerism due to fetomaternal and transmaternal sibling cell trafficking during pregnancy as the donor is exposed to recipient antigens in utero. The aim of the present study was to validate previously reported single-center data in a large, multicenter cohort provided by the Center for International Blood and Marrow Transplantation. All adult and pediatric patients (n = 11,365) with a hematologic malignancy who underwent allogeneic SCT with a graft from an HLA-identical sibling donor between 1990 and 2007 were included. When donors were younger than recipients, there was a significantly lower rate of acute GVHD grade II-IV and chronic GVHD in children, as well as a lower rate of chronic GVHD in adolescents. However, the hypothesized overall positive effect of lower relapse and better survival when donors are younger than recipients was not observed. Our data suggest that if otherwise equally matched, a graft from a younger sibling may be superior to a graft from an older sibling for children and adolescents undergoing SCT.

An alternative reduced-toxicity conditioning regimen for allogeneic transplantation, based on treosulfan and fludarabine, has recently been identified. The safety and efficacy of this new conditioning regimen has been investigated prospectively in patients with AML. A total number of 75 patients with AML in CR were treated with 3 × 14 g/m(2) treosulfan and 5 × 30 mg/m(2) fludarabine, followed by matched sibling or unrelated SCT. Patients were evaluated for engraftment, adverse events, GVHD, and for non-relapse mortality, relapse incidence, overall and disease-free survival (DFS). All patients showed primary engraftment of neutrophils after a median of 20 days. Non-hematological adverse events grade III-IV in severity included mainly infections (59%) and gastrointestinal symptoms (7%). Acute GVHD grade II-IV occurred in 21% and extensive chronic GVHD occurred in 16% of the patients. After a median follow-up of 715 days, the 2-year overall and DFS estimates were 61% and 55%, respectively. The 2-year incidences of relapse and non-relapse mortality reached 34% and 11%, respectively. In summary, our data confirm promising safety and efficacy of the treosulfan-based conditioning therapy in AML patients, ClinicalTrials.gov Identifier: NCT01063660.

Prognosis of patients (pts.) with refractory or relapsed acute myeloid leukaemia (AML) or advanced myelodysplasia (MDS) is poor. Preliminary reports suggest that induction of aplasia by a standard AML regimen followed by reduced intensity conditioned stem cell transplantation (RIC-SCT) in aplasia may be an effective strategy in this situation. A survey was conducted by the German Cooperative Transplant Study Group (GCTSG) to investigate the outcome after such an approach. In April 2006, GCTSG centers were asked if they had performed RIC-SCT in aplasia. Centers with positive response received a questionnaire including details on patient and donor characteristics, disease status, induction chemotherapy, transplant procedure and outcome. Data on 44 pts. (24 male, 20 female; median age 49y, range 19 – 66) with high-risk MDS (n=9), relapsed (n=16) or primary refractory AML (n=19) were reported by 3 centers. Pts. received aplasia-inducing chemotherapy with the following regimens: idarubicine / fludarabine / ara-C (n=16), daunorubicine / ara-C (n=15), fludarabine / ara-C / amsacrine (n=5), or other ara-C-based schedules (n=8). Subsequent transplant conditioning therapy consisted of melphalan (150 mg/m2) plus fludarabine (150mg/m2) in n=28, TBI (total body irradiation, 8Gy) plus fludarabine (150mg/m2) in n=11, and TBI (4Gy) plus cyclophosphamide (80 – 120 mg/kg) in n=5 pts. and was commenced after a median of 15 days (8 – 40) from the start of the preceding chemotherapy. Allogeneic peripheral blood (n=41) or bone marrow (n=3) stem cells from sibling (n=16), matched unrelated donors (n=18) or mismatched donors (n=10) were infused thereafter. Graft versus host disease (GVHD) prophylaxis was performed with cyclosporin A monotherapy (n=33) or by combination of a calcineurine inhibitor and methotrexate (n=11), n=31 received additional anti-thymocyte globuline. 43 pts. achieved stable engraftment and 41 showed > 90% donor chimerism after 28 – 90 days. Grade 2–3 acute GVHD occurred in 34%, whereas limited or extensive chronic GVHD was seen in 26% of the evaluable pts. The 100-day mortality rate was 24%: 5 pts. succumbed to treatment related complications, 4 to disease relapse. The median follow up is 183 days (19–1453). Actuarial survival rates estimated by Kaplan-Meier analysis are 56% and 30% at 6 and 12 months, whilst actuarial progression free survival rates are 42% and 30% at 6 and 12 months, respectively. This multi-center retrospective analysis suggests feasibility of the approach combining leukemia burden reduction by standard AML induction chemotherapy followed by RIC-SCT performed in aplasia. Treatment-related complications were comparable to those of a conventional transplant setting, thus disease control was achievable in a substantial proportion of patients with poor prognosis AML and MDS. Prospective studies are needed to assess whether durable remissions are achievable in patients with these otherwise fatal conditions.

Introduction:The role of autologous stem cell transplantation (ASCT) in patients with marginal zone lymphomas (MZL) is not fully elucidated. The aim of the present study was to determine the outcome of patients undergoing ASCT for relapsed/refractory MZL, and define prognostic factors affecting that outcome. Methods: Eligible for this study were patients with nodal, extra-nodal (MALT) or splenic MZL , aged ≥18 ears, who underwent a first ASCT between July 1994 and February 2013, and reported to the European Society for Blood and Marrow Transplantation (EBMT) registry, and/or the Fondazione Italiana Linfomi (FIL) and the Gruppo Italiano Trapianto di Midollo Osseo (GITMO) networks. Patients with a history of MZL transformation were excluded. Review of written diagnostic reports was mandatory for inclusion. Log rank tests were used to assess the impact of baseline characteristics on overall survival (OS) and event-free survival (EFS). In multivariate analysis, the effect of prognostic factors was evaluated using Cox regression models. Cumulative incidence of relapse (IR) and cumulative incidence of non-relapse mortality (NRM) were estimated with a competing-risk approach. Risk factors for IR and NRM were estimated by Pepe & Mori test or by Fine & Gray model. Results: The study included 199 patients, 111 patients (56%) with MALT lymphoma, 55 patients with nodal MZL (28%) and 33 patients (16%) with splenic MZL.. Median age at transplantation was 56 years (range, 25-71 years). Median time from diagnosis to ASCT was 2 years (0.1-28.0). Median number of prior therapies was 1 , (range 1-8) , including rituximab in 74%. 96% were transplanted with chemosensitive disease; 70 (37%) in CR1/PR1 ,113(59%) in CR/PR >1 and 7 in SD (4%) Median calendar year of ASCT was 2006, with 17,1% of transplants being performed before 2001. Total body irradiation-based high-dose regimen was used in 16 patients (8%), whilst 92% of the patients received high-dose chemotherapy only. Median follow-up was 4.1 years (0.1-19.1). Five-year cumulative incidence of relapse/progression (IR) and non-relapse mortality (NRM) were 38% (95%CI 30-45%) and 9 %( 95%CI 6-14%) respectively. Five-year event-free survival (EFS) and overall survival (OS) were 53% (95%CI 45-61%) and 73% (95%CI 65-79%), respectively. Multivariate analysis revealed age >65 years to be associated with shorter EFS and a shorter OS (HR=8.6, 95%CI 2.8-26.2, p Additionally, MZL predicted a shorter OS than MALT (HR=3.2, 95%CI 1.2-8.7, p=0.023). Notably, rituximab had no statistically significant effect on transplant outcome.Risk of secondary malignancies approached 6.8%. Conclusions: ASCT is a feasible and effective procedure when offered to MZL patients younger than 65 years, even in those previously exposed to rituximab. Disclosures Zaja: MedImmune: Research Funding.

Purpose The role of unrelated allogeneic stem-cell transplantation in acute lymphoblastic leukemia (ALL) patients is still not clear, and only limited data are available from the literature. We analyzed factors affecting clinical outcome of ALL patients receiving a related or unrelated stem-cell graft from matched donors. Patients and Methods The total study population was 264 adult patients receiving a myeloablative allogeneic stem-cell transplant for ALL at nine bone marrow transplantation centers between 1990 and 2002. Of these, 221 patients receiving a matched related or unrelated graft were analyzed. One hundred forty-eight patients received transplantation in complete remission; 62 patients were in relapse; and 11 patients were refractory to chemotherapy before transplant. Fifty percent of patients received bone marrow, and 50% received peripheral blood stem cell from a human leukocyte antigen–identical related (n = 103), or matched unrelated (n = 118) donor. Results Disease-free survival (DFS) at 5 years was 28%, with 76 patients (34%) still alive (2.2 to 103 months post-transplantation), and 145 deceased (65 relapses, transplant-related mortality, 45%). We observed an advantage regarding DFS in favor of patients receiving transplantation during their first complete remission (CR) in comparison with patients receiving transplantation in or after second CR (P = .014) or who relapsed (P < .001). We observed a clear trend toward improved survival in favor of B-lineage ALL patients compared with T-lineage ALL patients (P = .052), and Philadelphia chromosome–positive patients had no poorer outcome than Philadelphia chromosome–negative patients. Total-body irradiation–based conditioning improved DFS in comparison with busulfan (P = .041). Conclusion Myeloablative matched related or matched unrelated allogeneic hematopoietic stem-cell transplantation in ALL patients should be performed in first CR.

Patients with chromosome 5 abnormalities and high-risk myelodysplastic syndromes or acute myeloid leukemia have a poor outcome. We hypothesized that increasing doses of lenalidomide may benefit this group of patients by inhibiting the tumor clone, as assessed by fluorescence in situ hybridization for del(5q31).Twenty-eight patients at diagnosis or with relapsed disease and not eligible for standard therapy (16 with acute myeloid leukemia, 12 with intermediate-risk 2 or high-risk myelodysplastic syndrome) were enrolled in this prospective phase II multicenter trial and treated with lenalidomide up to 30 mg daily for 16 weeks. Three patients had isolated del(5q), six had del(5q) plus one additional aberration, 14 had del(5q) and a complex karyotype, four had monosomy 5, and one had del(5q) identified by fluorescence in situ hybridization only.Major and minor cytogenetic responses, assessed by fluorescence in situ hybridization, were achieved in 5/26 (19%) and 2/26 (8%) patients, respectively, who received one or more dose of lenalidomide, while two patients achieved only a bone marrow response. Nine of all 26 patients (35%) and nine of the ten who completed the 16 weeks of trial responded to treatment. Using the International Working Group criteria for acute myeloid leukemia and myelodysplastic syndrome the overall response rate in treated patients with acute myeloid leukemia was 20% (3/15), while that for patients with myelodysplastic syndrome was 36% (4/11). Seven patients stopped therapy due to progressive disease and nine because of complications, most of which were disease-related. Response rates were similar in patients with isolated del(5q) and in those with additional aberrations. Interestingly, patients with TP53 mutations responded less well than those without mutations (2/13 versus 5/9, respectively; P=0.047). No responses were observed among 11 cases with deleterious TP53 mutations.Our data support a role for higher doses of lenalidomide in poor prognosis patients with myelodysplastic syndrome and acute myeloid leukemia with deletion 5q. (Clinicaltrials.gov identifier NCT00761449).

Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal growth and accumulation of tissue mast cells (MC) in various organ systems, including bone marrow (BM). Indolent and advanced forms of SM have been described. Whereas patients with ISM have a normal or near normal life-expectancy, patients with advanced SM, including those suffering from mast cell leukemia (MCL) have a poor prognosis. In these patients, neoplastic MC are usually resistant against conventional drugs and various targeted drugs. In rapidly progressive aggressive SM (ASM) and MCL, polychemotherapy followed by allogeneic hematopoietic stem cell transplantation (alloHCT) has been proposed. However, outcome of alloHCT in advanced SM is unknown, and it also remains uncertain whether clinically relevant graft-versus-SM (GVSM) effects may occur in these patients, as only sporadic case reports have been published. We performed a retrospective multi-center analysis to evaluate the outcome of alloHCT in patients with advanced SM. Fifty-four advanced SM patients receiving SCT in 32 transplantation centers in Europe and America were identified between 1990 and 2013. The median patient age was 45 years. Donors were: HLA identical siblings (31), unrelated donors (URD) (15), umbilical cord blood donors (UCB) (2), and haploidentical donors (1). In 5 patients, stem cell source was not defined (5). Thirty-four patients received myeloablative conditioning (MAC) and 18 received reduced intensity conditioning regimens (RIC). In 2 patients, conditioning regimen was not specified. Indications for alloHCT were SM with an associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) (n=32), MCL (n=13, including one with MCL-AHNMD), 8 with ASM and 1 with myelomastocytic leukemia (MML). The most prevalent AHNMD was acute myeloid leukemia (AML, n=16). With follow-up of 35-6180 (median 365) days, SM responses (defined as ≥50% decrease in BM mast cells ± decrease in serum tryptase ± regression of other organ manifestations) were observed in 39 patients (72%), including complete responses (CR) documented in 12 patients (22%). Eleven patients had stable disease, whereas 4 patients (7%) progressed immediately after alloHCT (primary resistance). In addition, 10 patients progressed (5 of them within 100 days) after an initial response. Progression was most frequently seen in MCL patients (n=6, 50%). In the AHNMD group, only 8 patients relapsed/progressed (25%). The overall survival (OS) and SM progression-free survival (PFS) at 1 year were 63% and 50% for all patients, 77% and 68% for SM-AHNMD, 63% and 50% for ASM, and 25% and 17% for MCL, respectively. The strongest predictive variable associated with inferior survival was a diagnosis of MCL. Other factors associated with poor outcome were: Karnofsky performance status ≤70%, ≥2 SM regimens given before alloHCT (e.g., steroids, cladribine, chemotherapy, tyrosine kinase inhibitor), donor source (alternative donors-UCB and haploidentical compared to sibling or URD), SM progression within the first 100 days, normal cytogenetics (compared to t(8;21) (q22;q22), and RIC (compared to MAC). The following variables were not associated with poor outcome: patient and donor age, recipient-donor sex match status, graft source (BM vs. peripheral stem cells), BM mast cell percentage at time of alloHCT, and CR status of AML or SM response at time of alloHCT. This largest multi-center analysis of results in advanced SM provides evidence for clinical efficacy of alloHCT, presumably because of a GVSM effect of alloHCT (achieving CR, and response to donor-lymphocyte infusions and RIC alloHCT). However, responses varied among different SM categories: while patients with SM-AHNMD enjoyed excellent outcomes, the OS for MCL patients in general, was poor. Nevertheless it is remarkable that 3 of 13 patients with MCL – an otherwise fatal disease with a median survival of Disclosures: Vercellotti: Sangart Inc.: Research Funding; Seattle Genetics: Research Funding. Akin:Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding.

Abstract 1915 Bone marrow (BM) has been associated with a decreased rate of Graft-versus-Host-Disease (GvHD) when compared to peripheral blood stem cells (PBSC) in the context of allogeneic stem cell transplantation (allo-SCT). It has been reasoned that this may relate in part to the differential quantitative and qualitative immunologic composition of both stem cell sources. It has, for example, been noted that BM contains a higher percentage of CD8+ T-cells, natural killer (NK) cells and recently also more regulatory T-cells than PBSC. It is further known that the total nucleated cell concentration (TNC) correlates negatively with the BM harvest volume, which could argue for an increasing dilution of the transplant with peripheral blood (pB) during the BM harvest. How this impacts on the different immunological compartments and how the BM at different time points of the harvest procedure compares to PBSC products has not been determined yet. During routine BM harvest procedures (n = 24) we subjected a part of the aspirate drawn at the beginning, after withdrawing half of the prescribed volume and at the end of the harvest procedure to detailed immunophenotyping using multicolor flowcytometry including intracellular staining for FoxP3 and IL17. The PBSC products of an age matched group (n = 20) were analyzed in parallel with the same flowcytometry protocols and used as a comparison. During the BM harvest the median TNC dropped rapidly from 53.2 Gpt/l in the beginning to 17.0 Gpt/l after half the volume had been collected and finally reached 6.2 Gpt/l at the end of the procedure. As expected, the first BM specimen contained a higher concentration of NK- (6.7 vs. 4.6 %, p = 0.001) and B-cells (14.1 vs. 10.2 %, p < 0.001) but less T-helper-(Th)-cells (36.0 vs. 53.7 %, p < 0.001) than pB. After collection half of the prescribed volume the NK-cell concentration had already dropped to 4.5 %, which was not significantly different from pB (p = 0.412) anymore. The B-cell concentration, however, remained at levels comparable to the first specimen (14.5 %) and significantly differed from pB (p < 0.001). The last BM specimen drawn still had a higher B-cell concentration than pB (10.9 vs. 10.2 %), although this was not statistically significant (p = 0.265). Th-cells demonstrated a steady increase in concentration during the harvest with a median concentration of 43.4 and 47.0 % in the middle and at the end of the procedure. Both the halfway value and the end of harvest concentration were significantly different from pB (p < 0.001 and p = 0.004, respectively). An overview on the NK-, B- and Th-cell-concentrations at the different time points analyzed can be found in Figure 1. Within the Th compartment we were unable to see a clear trend for Treg which had a concentration of 7.6 % in the beginning, 7.0% halfway and 7.1 % after all BM had been collected. None of these concentrations was significantly different from that found in pB (7.6 %). The same applied to Th17 cells which made up 0.51 % of Th in the beginning, 0.59 % in the middle and 0.50 % at the end of the procedure and 0.84 % in the pB. Moreover there was no significant difference between the first BM specimen and PBSC products with respect to Treg (7.6 vs. 6.7 %, p = 0.181) or Th17 (0.51 vs. 0.61 %, p = 0.100). In contrast, the NK- and Th-cell concentration in PBSC products was significantly higher than in the first BM specimen (NK: 10.7 % vs. 6.7 %, p =0.005; Th: 40.3 % vs. 36.0 %, p =0.038). We conclude that the composition of immune cells within the BM changes significantly during the harvest procedure probably due to an increasing dilution with peripheral blood. These changes variably affect different compartments and may have an impact on post-transplant immunological function and complications. Therefore BM harvest volumes may need to be considered when comparing BM and PBSC with respect to clinical outcomes. In contrast to previous reports, we found no indication that BM, no matter at which time point analyzed, contained a higher concentration of Treg when compared to PBSC. Disclosures: No relevant conflicts of interest to declare.

s of the 11th International Symposium on Myelodysplastic Syndromes (MDS) 18 May 2011 21 May 2011 Oral Presentations / Leukemia Research 35 (2011) S14–S26 S25 66 Allogeneic stem-cell transplantation in chronic myelomonocytic leukemia (CMML): Factors affecting transplantation outcome. A study of the CLWP of the EBMT A. Symeonidis, A. van Bietsen, G. Mufti, J. Finke, D. Beelen, M. Bornhaeuser, H. Greinix, J.P. Jouet, L. Volin, R. Schwerdtfeger, T. de Witte, N. Kroeger. Hematology Division, Dept of Internal Medicine, University of Patras Medical School, Patras, Greece; Dept of Medical Statistics and Bioinformatics, Chronic Leukemias Working Party of the EBMT, Leiden, The Netherlands; Dept of Hematological Medicine, GKT School of Medicine, London, UK; Dept of Medicine, Hematology, Oncology, University of Freiburg, Freiburg, Dept of Bone Marrow Transplantation, University Hospital of Essen, Essen, Medizinische Klinik und Polyklinik I, Universitaetsklinikum Dresden, Dresden, Germany; Klinik fuer Innere Medizin I, Medizinische Universitaet Wien, Vienna, Austria; Service des Maladies du Sang, Hopital Claude Huriez, Lille Cedex, Lille, France; Division of Hematology, Dept of Medicine, Helsinki University Central Hospital, Helsinki, Finland; KMT Zentrum, Deutsche Klinik fuer Diagnostik, Wiesbaden, Germany; Dept of Hematology, Radboud University Nijmegen, Nijmegen, The Netherlands; Bone Marrow Transplantation Centre, University Hospital Eppendorf, Hamburg, Germany According to WHO classification CMML is characterized, first, as dysplastic or proliferative and second, as type-1 or type-2. Results of allogeneic stem-cell transplantation (allo-SCT) in CMML are usually pooled together with other MDS. We retrospectively analyzed transplantation outcome in 489 patients with CMML from the EBMT database (females 160, males 329, median age 52.7 years, range 19.5–75.4 years). Among 145 patients, 67 had CMML-dysplastic and 78 CMML-proliferative, whereas among 214 patients with known subtype, 87 had type-1 (40.6%), 32 had type-2 (15%) and 95 had evolved to AML (44.4%). Conditioning was standard in 249 (55.7%) and reduced-intensity in 198 (44.3%). Donors were HLA-identical siblings (245~50.1%), other related (23~4.7%) or unrelated ones (221~45.2%). Disease status at allo-SCT was complete remission (CR) in 108 patients (22.1%), no-CR in 331 (67.7%) and unknown in 50 (10.2%). Stem cell source was bone marrow in 125 (25.6%) and peripheral blood (PBSC) in 365 (74.4%). Engraftment was successful in 432/460 patients (93.9%) and unknown in 29. Grades 0–1 acute GVHD was reported in 291/448 evaluable patients (65%), grades 2–4 in 149 (33.3%) and was unknown in 8. Chronic GVHD was reported in 102/197 patients (limited 49, extensive 53). At the time of this analysis 224 patients were alive (45.8%), of whom 198 (40.5%) were disease-free and 120 (24.5%) had relapsed. Among 265 deaths, 95 were disease-related (35.8%), 145 transplant-related (54.7%) and 25 other cause-related (9.4%). The probability of nonrelapse mortality at 4 years was 35% and it was lower in female to female allo-SCT (p =0.069). Patients with abnormal cytogenetics had higher risk for relapse (p = 0.017) and for death after relapse (p = 0.03). Patients transplanted in CR had lower probability for non-relapse death (p = 0.006) and longer RFS and OS (p =0.010 and p=0.018 respectively). Kaplan–Meier estimates for OS of the whole group was 46.7 months (95% Confidence Interval 38.1– 55.2 months). Kaplan-Meier estimates for RFS was longer in PBSC recipients (p = 0.006). In multivariate analysis the only significant prognostic factor for OS was disease status at SCT (CR vs noCR, p = 0.010). Analysis also included interval from diagnosis to transplant, dysplastic vs proliferative and type-1 vs type-2 disease, intensity of the conditioning, donor matching, cytogenetics, stemcell source, T-cell depletion, administration of total body irradiation, grading of acute and chronic GVHD and the year of allo-SCT. Allo-SCT remains a promising treatment option for patients with CMML and patients should preferably be referred to allo-SCT after achieving the best possible remission status. 67 Response to ESA treatment in patients with MDS: Determination of a predictive score, from a retrospective analysis of 669 patients A. Symeonidis, P. Zikos, A. Galanopoulos, I. Kotsianidis, A. Kouraklis, E. Terpos, M. Protopapa, H. Papadaki, V. Lambropoulou, A. Aktypi, P. Bakarakos, S. Michalopoulou, A. Anastasiadis, E. Michalis, N. Zoumbos. Hematology Division, Dept of Internal Medicine, University of Patras Medical School, Hematology, St Andreas Hospital of Patras, Patras, Dept of Hematology, G. Gennimatas General Hospital of Athens, Athinai, Dept of Hematology, Democritus Thrace University, Alexandroupolis, Dept of Medical Therapeutics, National and Kapodistrian University of Athens, Athinai, Dept of Hematology, General Hospital of Serres, Serres, Dept of Hematology, University of Crete School of Medicine, Heraklion, Olympion Hospital of Patras, Patras, Greece To investigate factors predicting for a favorable response to treatment with ESA in patients with MDS, 669 patients with a complete follow-up, treated for at least 12 weeks, were retrospectively analyzed. Patients (441 males, 228 females, median age 74 years) were classified as RA (n =257), RARS (n =119), RAEB (n =210), CMML (n =71) and unclassified MDS (n =12). IPSS [available in 618 patients (92.4%)] was Low in 232 (37.5%), Int-1 in 249 (40.3%), Int-2 in 98 (15.9%) and High in 39 patients (6.3%). Age, gender, baseline hemoglobin and platelet count, serum Epo, Response to Anemia Index (RAI), defined as the Hb× log(Epo) component, WHO classification, IPSS, WPSS, cytogenetics, marrow blasts, marrow cellularity, previous transfusions and time from diagnosis to treatment were tested as predictors. At 12 weeks 136 patients (20.3%) achieved a complete (CR) and 150 (22.4%) a partial response (PR). Overall, 212 patients (31.7%) achieved CR and 123 PR [18.4%, overall response (OR) 50.1%]. OR was 66% in FAB-RA, 51.3% in FAB-RARS, 33.6% in RAEB, 46.5% in CMML, 77.6% in WHORA, 66.2% in WHO-RARS, 60.6% in CMML-D, 58.2% in RCMD, 48.9% in del-5q, 43.3% in RAEB-1, 32.6% in RCMD-RS, 31.7% in CMML-P, and 20.9% in RAEB-2. In multivariate analysis WPSS was the most important predictor, with a probability of CR at 12 weeks 59.6%, 37.2%, 34.4%, 9% and 0% for the very low, low, intermediate, high and very high risk group respectively (p 21 vs 26.5% for those with Epo > 200). Other factors affecting response were RAI (p < 0.001), interval from diagnosis to treatment (p< 0.001), bone marrow cellularity (p< 0.001), previous transfusions (p < 0.001), hemoglobin (p < 0.001), platelet count (p < 0.01), serum Epo (p < 0.01) and cytogenetic group (p < 0.02). By using the 4 main factors (WPSS, RAI, marrow cellularity and interval from diagnosis to treatment) we have created a score, which could predict 85%, 47%, 35%, 15% and 0% probability for response in patients exhibiting 1, 2, 3, 4 and 5 of these factors. Therefore, WPSS, RAI, marrow cellularity and interval from diagnosis to treatment constitute a score, highly predictive for response to ESA treatment in patients with MDS. 68 ASXL1 is frequently mutated and associated with a negative prognosis in MDS F. Thol, I. Friesen, F. Damm, H. Yun, E.M. Weissinger, J. Krauter, K. Wagner, A. Chaturvedi, A. Sharma, M. Wichmann, G. Gohring, C. Schumann, G. Bug, O. Ottmann, W.-K. Hofmann, B. Schlegelberger, M. Heuser, A. Ganser. Medizinische Hochschule Hannover, Hannover, University Hospital Mannheim, Mannheim, University of Frankfurt, Frankfurt/Main, Germany Myelodysplastic syndrome (MDS) is a heterogeneous disease of the hematopoetic stem cell caused by mutations, deregulated gene expression and epigenetic modifications of genes leading

Abstract 3720 Background: Dendritic cells (DCs) are professional antigen-presenting cells which display an extraordinary capacity to induce, sustain and regulate T cell responses. Recently, 6-sulfo LacNAc+ (slan) DCs (formerly termed M-DC8+ DCs) have been described as a major subpopulation of proinflammatory human blood DCs which are principal producers of tumor necrosis factor-alpha and interleukin-12. In addition, it has been demonstrated that slanDCs efficiently induce antigen-specific CD4+ and CD8+ T cells and direct the polarization of naïve CD4+ T lymphocytes into Th1 cells. In the present study, we investigated the reconstitution kinetics of slanDCs after allogeneic stem cell transplantation (aSCT) in comparision to CD1c+ myeloid DCs and plasmacytoid DCs representing two additional major human blood DC subsets. Material and Methods: The frequency of slanDCs, CD1c+ myeloid DCs and plasmacytoid DCs in the peripheral blood was quantified by flow cytometry in 70 patients following aSCT at different time points in early engraftment ( Results: (1) Early engraftment (30 days post transplantation): Interestingly, in the late phase post transplantation, the frequency of slanDCs steadily increases and these DCs represent the most abundant DC subpopulation in the second and third month post transplantation. The frequency of CD1c+ myeloid DCs and plasmacytoid DCs remains unchanged. Again, the majority of slanDCs show a mature phenotype in contrast to CD1c+ myeloid DCs and plasmacytoid DCs. Conclusion: Whereas the early engraftment phase after aSCT is dominated by CD1c+ myeloid DCs and plasmacytoid DCs, slanDCs represent the most abundant DC subset in the late engraftment phase. Furthermore, in both engraftment phases the majority of slanDCs display a mature phenotype in contrast to CD1c+ myeloid DCs and plasmacytoid DCs. Current studies are focused on functional assays and the role of individual DC populations in acute graft-versus-host disease and graft-versus-leukemia responses in the early and late phase following aSCT. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 4866 Introduction The first pandemic of the 21st century was caused by the novel influenza A (H1N1) virus, now known as pandemic H1N1-09 virus. Its first wave reached Germany in autumn 2009. Though for the German public health sector this pandemic was a challenge, a state of national emergency could not be recognized. A popular current believe is that the threat by this pandemic had been overestimated by national and international health care experts. Here, we report our experience with the pandemic H1N1-09 virus in hematologic patients when the first wave hit Germany between December 2009 and February 2010. Methods Viral diagnostic for all patients was performed in a central virologic laboratory. All patients with at least one sample tested positive by PCR for H1N1-09 between October 2009 and March 2010 were included into the analysis. Samples were obtained by nasal wash, nasopharyngeal swab or broncho-alveolary lavage. The medical charts of all patients with H1N1-09 infection were reviewed systematically. Results 15 patients with underlying hematologic diseases (10 male and 5 female) with a median age of 52 years (range 18–70 years) were tested positive for H1N1-09 virus by PCR in our department. Notably, in 12 Patients H1N1-09 was nosocomially acquired after a median of 16 days hospitalization (range 6 – 42 days). 13 patients (87%) got infected between December 2 and December 29. At the time of infection all patients were immunocompromised with 11 patients being cytopenic after chemotherapy and four patients after allogeneic hematopoietic cell transplantation (HCT) among the latter two patients were in aplasia after allogeneic HCT (day +5 and day +11 after HCT). CT scan was performed in 8 patients at the time of diagnosis. Seven patients presented with signs of atypical pneumonia on CT scan. Ground glass opacity, consolidation, airway wall thickening, airway dilatation, pleural effusion and lymphadenopathy were common findings. In 10 patients viral clearance was monitored by RT-PCR. The median duration of viral shedding was 10 days (range 4 – 41 days). Prolonged viral persistence was associated with severe lung injury. All patients received Oseltamivir as first-line therapy, except three patients, who died prior to the confirmation of the diagnosis by RT-PCR. Simultaneously broad spectrum antibiotics and antimycotics were administered. 5 patients (33%) with respiratory failure needed invasive mechanical ventilation (MV) at the time of the H1N1-09 infection. Three out of these patients died. Within a follow up of 6 months eight patients have died. Six patients (40%) have died from the infection. Among these three patients died from fulminant pulmonary failure whereas three patients died several weeks after H1N1-09 infection from subsequent respiratory or multiorgan failure. The impact on indirect mortality cannot yet be fully assessed, since in some patients the infection caused significant delay of anti-leukemic therapy and acquired comorbidities resulted in dose-reductions of chemotherapy. Conclusions In contrast to largely mild infections in the healthy German population pandemic H1N1-09 pneumonia represented a life-threatening infection for hematologic patients associated with a high mortality due to acute respiratory failure, late pulmonary complications and delay of antitumor treatment. One alarming finding was the frequency of nosocomial infections. This observation points to the possibility of transmission of the virus from patient to patient, visitors to patient or even from medical staff to patient. With this observation in our institution patients admitted to hospital were put under quarantine before they were allowed to be accommodated in a double room in the hematologic unit. Transmission of the virus from asymptomatic staff or visitors to patient is another major concern. The suspected vaccination rate of medical staff in Germany was less than 20%. Especially, when asymptomatic or mild H1N1 infections occur - as it was the case in Germany – the medical staff and visitors could become important vectors of infection. The most effective measure against this threat is active immunization of visitors prior to patient contact and medical staff in hematologic units. Disclosures: No relevant conflicts of interest to declare.

Abstract 3328 Poster Board III-216 Introduction Relapse is a major cause of treatment failure after alloSCT against acute leukaemia, and no standard treatment has been established in this challenging situation. The introduction of reduced conditioning regimens, and the broader availability of alternative donors have increased the possibilities to perform a second alloSCT as salvage treatment, using different preparative regimen and/or different stem cell donors. Methods To evaluate the role of a second alloSCT (tx2) for the treatment of relapse after first alloSCT (tx1), we performed a nationwide retrospective analysis based on the German registry for stem cell transplantation (DRST). Datasets were completed by the reporting centres on request, following a specifically designed questionnaire. Results 212 patients (69% AML, 31% ALL), from 23 centres were included. Median age at tx1 was 37y. Donor at tx1 were HLA identical siblings (41%), matched unrelated (39%), mismatched family or unrelated (17%) or syngeneic donors (3%). Conditioning intensity at tx1 was standard (SIC, 62%), intermediate (intC, 25%) or reduced (RIC, 13%). Median remission after tx1 was 7 months, median time from relapse to tx2 was 74d. At tx2, patients were aplastic (4%), in CR (20%) or showed active disease (76%). In 59%, the same donor was used for tx1 and tx2, whereas a different donor was chosen in 41%. Conditioning at tx1/tx2 were SIC/SIC (14%), intC/intC (10%), (RIC/RIC (10%), less intensive at tx2 (mostly intC or RIC after SIC, 58%), or more intensive at tx2 (SIC after RIC or intC, 8%). Following tx2, CR was achieved in 56% of patients, out of which 81% relapsed again. Hence, leukemia was the most frequent cause of death. With a median FU of 23 months after tx2, median OS after tx2 is 117d. In a univariate analysis (log rank), OS after tx2 depended on stage at tx1 (CR vs. active disease, p12m (31%), p Conclusion Survival of acute leukemia after second allogeneic SCT is determined by the duration of remission after tx1. Using an alternative donor for tx2 did not improve the results in our series. Further analysis is required to evaluate the role of RIC regimen for tx2. Disclosures No relevant conflicts of interest to declare.

Abstract 2307 Poster Board II-284 Objectives: T-cell depletion (TCD) is controversial in patients with chronic lymphocytic leukemia (CLL). While TCD is a powerful tool to prevent graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) concern is raised about an increased incidence of relapse. The aim of this retrospective analysis was to study the impact of in vivo TCD in patients with CLL registered with the EBMT database. Patients and Methods: Patients with CLL who received allogeneic HCT from a matched sibling (SIB) or unrelated donor (UD) and cyclosporine-based GVHD prophylaxis between 2001 and 2008 were eligible. Patients who received ex vivo T-cell depleted grafts were excluded. The outcome of three major groups of patients was compared: Patients who were transplanted without TCD, those who received anti-thymocyte globulin (ATG) and patients who received alemtuzumab (CAM). Baseline and follow-up data were downloaded from the EBMT database. Results: 413 patients were eligible. 73% of patients had SIB donors and 27% matched UD. Reduced intensity conditioning regimens were applied for the majority of patients (82%). No TCD was used in 234 patients, while 100 patients received CAM and 79 patients ATG. The median follow-up after HCT was 22 months (1 to 92 months). Univariate comparisons of GVHD and the rates of DLI are reported for patients with matched sibling donors only. After SIB-HCT the cumulative incidence of acute GVHD II-IV was 7% with CAM, 22% with ATG and 34% without TCD (gray test, p=0.0001). Subsequently, 28% of patients with CAM received prophylactic donor lymphocyte infusions (DLI) compared to 5% of patients with ATG and 9% without TCD (p=0.03). DLI for any reason was given to 45% of patients with CAM, 24% with ATG and 15% without TCD (p=0.009). As a result of late-onset GVHD the incidence of chronic GVHD after TCD with CAM increased from 20% at 1 year to 60% at 4 years after SIB-HCT. At 4 years after HCT comparable cumulative incidences of chronic GVHD were observed (70% without TCD, 59% after ATG and 60% after CAM; p=0.1). For the whole cohort of patients 4-year overall survival (OS) and progression-free survival (PFS) were 57% (95% CI, 50% to 64%) and 44% (95% CI, 37% to 51%). At 4 years the cumulative incidence of relapse was 28% (95% CI, 17% to 39%) and the incidence of non-relapse mortality was 28% (95% CI 18% to 38%). In multivariate Cox regression analysis of PFS when GVHD prophylaxis without TCD was used as reference category the adjusted hazard ratio for TCD with CAM was 1.4 (95% CI, 0.9 to 2.2, p=0.2) and for TCD with ATG 1.4 (95% CI, 0.9 to 2.3, p=0.1). For relapse incidence the adjusted hazard ratio for TCD with CAM was 1.4 (95% CI, 0.7 to 2.6, p=0.3) and for TCD with ATG 1.0 (95% CI, 0.5 to 2.1, p=0.9) and for non-relapse mortality the adjusted hazard ratio for TCD with CAM was 1.4 (95% CI, 0.8 to 2.8, p=0.3) and for TCD with ATG 1.9 (95% CI, 1.0 to 3.5, p=0.06). Conclusion: In patients with CLL the combination of in vivo TCD and DLI appears to result in comparable rates of chronic GVHD and PFS compared to T-cell replete HCT. Disclosures: Schetelig: Bayer Schering: Research Funding.

Relapse of hematological malignancy remains a major complication after allogeneic stem cell transplantation. This is especially true for patients receiving minimal or reduced-intensity conditioning therapy. Analysis of donor chimerism (DC) is an important diagnostic tool to assess the risk of relapse after allogeneic stem cell transplantation, especially in patients lacking a specific marker suitable for monitoring of minimal residual disease. The sensitivity of a standard PCR assay amplifying short-tandem-repeat sequences can be improved significantly by investigating sorted CD34+ peripheral blood cells. We prospectively compared the serial analysis of DC in selected CD34+ cells and unmanipulated whole blood (WB) within a randomised study in 131 patients with CD34+ hematological malignancies (AML, ALL and MDS) surviving more than 100 days after transplantation. The primary end-point was the association between decreasing CD34+ DC and haematological relapse. Whenever a decreasing CD34+ or WB DC was confirmed and no signs of active GvHD were present, a rapid taper of CsA or tracolimus (50% every 5–7 days) was suggested. If no GvHD occurred within 14 days after the stop of CsA or tacolimus, patients were scheduled to receive donor lymphocyte infusions (DLI) in incremental doses. The cumulative incidence of relapse was significantly increased in patients with decreasing or incomplete CD34+ DC (62% vs. 38%, p=0.01). This was associated with a lower probability of overall survival (20% vs. 39%, p=0.03). The interval between the decrease in CD34+ DC and hematological relapse was 35 days (range 0–567) in the study group compared to only 8 days (range 0–63) in the control group monitored by WB DC analysis (p=0.05). The median time between a decrease in CD34+ DC and WB DC was 14 days (range, 0 to 445). Patients receiving preemptive therapy triggered by decreasing CD34+ DC had a significantly higher probability of disease-free survival compared to cases monitored and treated according to WB DC (19% vs. 0%, p=0.009). Multivariate analysis revealed age, disease-risk and decreasing CD34+ DC as independent risk-factors for overall survival in the study group. In summary, we could demonstrate that the quantification of DC in CD34+ selected cells is a sensitive method to predict relapse and survival after allogeneic SCT. Although this technology opens a window for preemptive therapy, new treatment approaches have to be employed to improve the overall outcome of patients with recurrence of residual disease after allogeneic stem cell transplantation.

Graft versus host disease is a major cause of morbidity and mortality after allogeneic stem cell transplantation from unrelated donors. Strategies using intensified GvHD prophylaxis including T cell depletion did not result in better outcome due to increased risks of infection and relapse. The use of ATG in the conditioning regimen for in vivo-Tcell depletion for GVHD prophylaxis has been reported by several groups but not been tested in a large prospective randomized trial. Here we report on results from the first large prospective, randomized, multicenter, open-label, phase III trial comparing standard GvHD prophylaxis with cyclosporine A (CyA) and short course methotrexate (Mtx) days +1, +3, +6, +11 (15/10/10/10 mg/m2) with or without 3×20mg/kg ATG-Fresenius (ATG-F) after a median follow-up time of two years. Between 2003 and 2007, 201 patients, median age 40 (range 18–60) years with AML (n=101), MDS (n=10), ALL (n=70), CML (n=17), OMF (n=3) in early (1st CR or MDS-RA, n=107), or advanced status of disease (all other, n=94), were transplanted from HLA-A and -B (2 digit), DRB1, DQB1 (4 digit) identical unrelated donors after highdose myeloablative conditioning with marrow (n=37) or PBSC (n=164) grafts. Median follow up time was 732.5 (25%-quartile 604, 75%-quartile 1097) days. For treatment comparisons with regard to the occurrence of aGvHD grade III-IV or death within 100 days post Tx, logistic regression adjusted for status of disease, source of stem cells, and center was used. For treatment comparisons with regard to time-to-event variables, cumulative incidence rates considering relapse and death as competing events were estimated, and Cox regression modelling the event-specific hazard rates and adjusting for status of disease and source of stem cells was used. Engraftment with WBC > 1000/μl was achieved in 97% in the ATG-F group after median 26 days, and in 95% in the control group after median 19 days (p The addition of ATG-F to standard CyA/Mtx prophylaxis results in decreased incidence of acute and chronic GvHD without increase of relapse or TRM rates. This is the first randomized trial answering the long-standing question regarding the beneficial effect of additional ATG-F to a standard GvHD prophylaxis. A reduction of GvHD without compromising survival could be demonstrated.

Background : Peripheral blood progenitor cells (PBPCs) are routinely applied worldwide for allogeneic related and unrelated stem cell transplantation. Factors influencing the mobilisation of PBPC’s in healthy donors are poorly understood. The database of the Apheresis Centre at the University of Dresden was evaluated to identify predictors of PBPC mobilisation. Methods : 4050 PBPC collections (3928 first donations, 122 second donations) of healthy unrelated donors between 1/1996 and 1/2008 were carried out according to a standardized protocol and prospectively documented in a database. Peripheral blood CD 34 counts were performed before each leukapheresis. CD 34 yield of every PBPC product was calculated in absolute numbers and per kg body weight of the recipient. All parameters are presented as median and range. Mann-Whitney test was performed for discrete variables. Correlation analysis by Pearson was applied for continuous variables. Multivariate regression analysis was carried out to detect factors independently predicting mobilization efficacy. Results : The range of peripheral CD 34+ haematopoietic stem cells obtained on day 5 was 8.7–285/μl (median 67.5/μl) in male and 6–282/μl, (median 51/μl) in female donors. The median CD 34 yield from the first leukapheresis was 5.88 ×108. Inadequate CD34-yields (< 2 × 106/kg) were obtained only in 18 donors (0.45%). Peripheral CD 34 count at day 5 is significantly correlated with (positive linear regression): BMI, baseline platelet count, male sex, G-CSF application (split dose), baseline leukocyte count. Peripheral CD 34 count at day 5 correlates negatively with: female sex, single dose G-CSF application, regular alcohol consumption and regular smoking status. Linear regression analyses revealed no significant influence of donor age. Multivariate correlation analysis included sex, BMI, baseline platelets, G-CSF-application, baseline leukocytes, age and smoking. This model explained 21.2 % of the variabilityA strong correlation exists between peripheral CD 34-counts at day 5 and apheresis yield (70% of the variability explained). Conclusions : A dose of 7.5 μg/kg/d lenograstim proved to be safe and effective in a large cohort of unrelated donors for mobilizing sufficient haematopoietic progenitor cells for allogeneic transplantation. Our analysis of 4050 donations revealed significant, but very weak correlations of the peripheral CD 34-counts with donor characteristics and life style parameters. No single parameter derived from donor baseline investigation reliably predicts CD 34 yield. Further evaluation of healthy donors including comprehensive genomic linkage analysis is necessary to elucidate the variable efficiency of PBPC mobilization

In the German Multicenter ALL studies (GMALL) patients aged >55 years with high risk (B-lineage ALL with WBC at diagnosis >30000, late CR, t (4; 11), complex aberrant karyotype or prae-T or mature T-ALL) or very high risk (Ph+/BCR-ABL+) ALL are increasingly candidates for allogeneic stem cell transplantation (allogeneic SCT with a HLA identical sibling donor, MRD or a matched unrelated donor, MUD) or autologous SCT. Here, we report on 31 elderly patients transplanted within the GMALL studies 06/99 and 07/03. Median age of the patients was 61 years (56–65). 22 patients belonged to the very high risk group (VHR), 8 patients to the high risk group (HR) and 1 patient from the standard risk group (SR) was transplanted because of detection of minimal residual disease.17/31 patients were transplanted from a matched unrelated donor, 9/31 patients from a HLA identical sibling donor and 5/31 patients underwent autologous SCT. Conditioning regimens for MRD SCT were myeloablative (MAC) in 6 patients (TBI 12 Gy and chemotherapy n=2, radioimmunotherapy + chemotherapy n=2, chemotherapy only n=2) and 3 patients received reduced intensity conditioning (RIC).Conditioning regimens for MUD SCT changed over time with an increasing number of RIC in the study 07/03. In total, 7/17 patients received MAC (TBI 12 Gy and chemotherapy n=5, chemotherapy only n=2) and 10/17 patients received RIC. Conditioning regimens in autologous SCT were myeloablative (MAC) in 5/5 patients. Results: After allogeneic MRD SCT 4/9 patients (44%) are alive in CCR (d+ 24, d+ 611, d+ 1721, d+ 2321), 3/9 patients died due to leukemia, 2/9 due to transplant related mortality (TRM). After allogeneic MUD SCT 8/17 patients (46%) are alive in CCR (from d+ 165 to d+ 2176). 1 further patient is alive after re- SCT for treatment of relapse. 7/17 patients died due to TRM and 1 patient died due to relapse. After autologous SCT 2/5 patients are alive in CCR (d+ 1703, d+ 1731), 3/5 died due to relapse. Risk factors for TRM: In allo SCT and MAC 8/13 patients died due to TRM in contrast to 1/13 patients with RIC. In auto SCT none of the patients died due to TRM. Risk factors for relapse: In allogeneic MRD SCT 3/9 patients died due to relapse and 2/17 patients relapsed after MUD SCT. Due to the small number of patients, no difference between MAC and RIC could be found. In autologous SCT 3/5 patients died due to relapse. In conclusion: The study shows that allo MRD but also MUD SCT is very effective in a selected population of elderly ALL patients. Since the survival of elderly patients with chemotherapy only is about 25%, more patients should be encouraged to have a MRD or MUD SCT.

BACKGROUND: The toxicity of commonly used myeloablative regimens is the main factor limiting applicability and restricting upper age limit for allogeneic hematopoietic stem cell transplantation (alloHSCT). However, reduced intensity conditioning (RIC) protocols are associated with high risk of relapse. Previous dose-escalation trials revealed high-dose treosulfan-based conditioning an alternative treatment with myeloablative as well as antileukemic potential, accompanied by low non-hematological toxicity. The goal of this multicenter phase II trial was to evaluate safety and efficacy of treosulfan/fludarabine conditioning in AML patients. PATIENTS AND METHODS: Seventy-five patients (median age 45 years, range 19–59) with AML in 1st (80%), 2nd (17%), or 3rd (3%) complete remission were treated with alloHSCT from either matched related (MRD, 40%) or unrelated donors (MUD, 60%). Preparative regimen consisted of treosulfan 14 g/m2/day on days −6 to −4, fludarabine 30 mg/m2/day on days −6 to −2, and in case of MUD-HSCT additionally ATG (Fresenius) 10 mg/kg/day on days −4 to −2. Graft-vs.-host disease (GvHD) prophylaxis consisted of cyclosporine and short-course methotrexate. 25% of the patients had an increased risk of toxicity for standard myeloablative conditioning regimens because of higher age or co-morbidities. RESULTS: All patients engrafted after a period of absolute neutropenia with the median time to ANC >0.5 G/L and PLT >50 G/L of 21 (13–39) days and 19 (11–48) days, respectively. The cumulative incidence of complete donor chimerism was 72% on day +28 and 92% on day +100. Most frequent CTC grade 3 or 4 complications until day 28 after transplantation were febrile neutropenia (28%), infection in neutropenia (16%), and mucositis (5%). None of the patients experienced severe hepatic toxicity, including VOD. After a median follow up of 457 days (98–988), the probabilities of overall (OS) and disease-free survival (DFS) at 2 years equaled 67% resp. 58% (MRD-HSCT) and 58% resp. 45% (MUD-HSCT, p=NS). The 2-year cumulative incidence of relapse was 34% for MRD-HSCT and 37% for MUD-HSCT, whereas non-relapse mortality was 8% and 17%, respectively. Outcome was not significantly influenced by disease status (CR1 vs. ≥CR2) or age. CONCLUSIONS: Conditioning therapy based on treosulfan at a dose of 3x14 g/m2 in combination with fludarabine is very well tolerated, resulting in a low NRM. The regimen demonstrated reliable efficacy in AML patients with confirmed first or subsequent remission. Further randomized studies are warranted to verify advantageous survival results.

The major limitation of allogeneic stem cell transplantatiopn (SCT) in patients (pts) with myelofibrosis is the high treatment related mortality. We performed a prospective multicenter trial of a dose-reduced conditioning regimen, consisting of busulfan (10 mg/kg orally or 8mg/kg i.v), fludarabine (180 mg/m2) and anti-thymocyte globulin (ATG Fresenius: 30–60 mg/kg) followed by allogeneic SCT in pts with myelofibrosis. From 2002 to 2006, 104 pts with a median age of 55 years (range: 32–68) andlow risk with constitutional symptoms (18%) or intermediate risk (n= 58%) and high risk (n=19%) were included. Cytogenetic abnormalities and JAK2 mutation were noted in 22% and 48%, respectively. Bone marrow histology showed advanced fibrosis (MF 2 and 3) in all pts. All but 3 pts received peripheral blood stem cells as graft source either from related (n=33) or unrelated donor (n=71). All but one (1%) pts showed leukocyte and platelet engraftment after a median of 18 and 21 days, respectively. The median duration of leukocyte aplasia was 9 days (range: 3–21). Acute GvHD grade II to IV occurred in 19% and severe aGvHD III/IV in 7%, while chronic GvHD was seen in 32% of the pts. Non-relapse mortality at 1 year was 19% (95% CI: 11–27%) and significantly lower for pts younger than 50 years of age (0% vs 27%, p=0.004) and for pts with low risk vs intermediate/high risk disease (0% vs 27%, p= 0.02). The cumulative incidence of relapse at 3 years was 29% (95%CI: 15–43%). The 3 year overall (OS) and event-free survival (EFS) was 70% (95% CI 60–80%) and 55% (95% CI 42–68%). Significant factors for improved 3 year OS and EFS were age less than 50 years (92% vs 62%, p=0.003 and 79% vs 46%, p= 0,004) and low vs intermediate/high risk (100% vs 62%, p=0.01 and 72% vs 48%, p= 0.02), while no impact on survival was seen for cytogenetic abnormalities, JAK2 mutation status and donor (related vs unrelated). These prospective multicenter study show excellent outcome of an busulafan/fludarabine based reduced conditioning regimen followed by allogeneic SCT in pts with myelofibrosis.

Infections with herpesvirus and adenovirus is a major cause of morbidity and mortality in patients undergoing stem cell transplantation (SCT). In those patients, the frequency of herpesvirus and adenovirus co-infections as well as their contribution to the infectious burden remain so far unclear. In this study, we have postulated that co-infections with herpesviruses and adenoviruses are frequent in the 100 days following SCT, contributing significantly to the infectious burden. To address this issue, 615 blood samples collected from patients undergoing SCT (n=35) were retrospectively tested by quantitative real-time PCR for the presence of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus. Infections with a single virus were detected in 46.1% (283/615) of the samples, whereas co-infections with two or three viruses were detected in 9.9% (61/615) and 2.1% (13/615) of the samples, respectively. Interestingly, about 50% of the patients (17/35) displayed a co-infection with two or more viruses, with [CMV+HHV6 A/B], and [EBV + HHV6 A/B +adenovirus] being most frequently detected, respectively. Comparison of viral loads indicated that the infectious burden increases proportionally to the number of viruses detected. Moreover, a DNA-microarray allowing simultaneous detection of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus was developed. The assay displayed a high diagnostic accuracy, being an attractive device for routine assessment of the infectious burden in immunocompromised patients after SCT.

Animal models of acute graft versus host disease (GVHD) suggested the critical role of host antigen-presenting cells (APC) in the pathogenesis of acute GVHD (Shlomchik et al Science285:412, 1999). Depletion of host B cells reduced the incidence of acute GVHD (Schultz et al BMT16:289, 1995). We hypothesized that B-cell depletion by prior rituximab (RTX) therapy in lymphoma pts might result in reduced incidence of acute GVHD (Ratanatharathorn et al Blood96:391a, 2000). We now analyzed the outcomes of allogeneic PBSCT in 435 B-cell lymphoma pts reported to CIBMTR from 1999 to 2004. Pts who had prior therapy with anti-CD52 or anti-T-cell antibody or recipients of conditioning regimens containing any of these antibodies were excluded. All patients received unmodified PBSC grafts. Pts were considered to have prior RTX therapy if they received RTX within 6 months of transplantation. There were 256 pts who did not receive RTX within 6 months of transplantation (No-RTX group) and 179 pts received RTX within 6 months of transplantation (RTX group). In RTX group, most pts (67%) received the last dose of RTX within 3 months of transplantation and 54% received at least two doses of RTX. Baseline characteristics of the two groups were similar except for significantly more unrelated donor (32% vs 19%, P=0.002)), tacrolimus use (47% vs 26%, P Clinical outcomes Relative risk (95% CI) P value Grade II-IV acute GVHD 0.72 (0.53–0.97) 0.03 Grade III-IV acute GVHD 0.55 (0.34–0.91) 0.02 Chronic GVHD 0.89 (0.67–1.18) 0.41 TRM 0.68 (0.46–1.0) 0.05 Progression or relapse 0.7 (0.42–1.19) 0.19 Progression-free survival 0.68 (0.50–0.92) 0.01 Overall survival 0.63 (0.46–0.86) 0.004

We analyzed the prognostic impact of the most frequent genetic abnormalities detected by fluorescence-in situ-hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) in 101 patients with multiple myeloma, who underwent allogeneic stem cell transplantation after reduced melphalan/fludarabine-based conditioning from related (n=34) and unrelated (n=67) donors. The abnormalities were del(13q14) [62%], t(11;14)(q13;q32) [11%], t(4;14)(p16.3;q32) [16%], CMYC-gains (8q24) [17%], del(17p13.1) [16%], t(14;16)(q32;q23) [4%], whereas none of the patients had t(6;14)(p25;q32). Translocation t(4;14), CMYC-gains and del 17p were frequently associated with del(13q14): 64%, 80% and 92%, respectively. The complete remission (CR) rate was 45% for all patients. Patients with del(17p13) achieved fewer complete remission than others (7% vs. 56%; p=0.001), while no difference was seen for t(4;14) and other abnormalities. Univariate analysis revealed higher relapse rates for age > 50 years (p=0.002), del(13q14) (p=0.006) and del(17p13) (p=0.003). Patients with translocation t (4;14) had a similar four year event-free survival than others (50 vs 45%). In a multivariate analysis, only del(13q14) [HR: 2.34, p=0.03] and del(17p13) [HR: 2.24; p=0.04] influenced the risk of relapse, while for event-free survival, only age [HR 2.8; p=0.01] and del(17p13) [HR: 2.05; p=0.03] retained the prognostic value. These data seem to indicate that some adverse cytogenetic risk factors such as t(4;14) can be overcome by allogeneic stem cell transplantation, probably due to the graft versus myeloma effect. Del(17p13.1) is a significant factor for a lower chance of complete remissions and shorter event free survival following allogeneic stem cell transplantation. The presented data will have implications for risk-adapted strategies in the future.

Gemtuzumab ozogamicin (GO) has been associated with an increased risk of liver sinusoidal obstruction syndrome (SOS) when applied within three months before or after allogeneic stem cell transplantation. In addition, excess of liver toxicity has been observed when GO was used directly before conventional intensive preparative regimens. We hypothesized that GO might be safe and effective as part of a fludarabine-based reduced-intensity conditioning regimen. 30 patients relapsing after conventional induction chemotherapy (n=17) or after previous transplantation (autologous n=3; allogeneic n=10) have been included in a prospective phase I/II trial. Per protocol the preparative regimen contained 6 mg/sqm and 3 mg/sqm GO on day-21 and day-14 followed by fludarabine 120 mg/sqm, 200 or 800 cGy total-body irradiation (TBI; depending on age and pretreatment) and stem cell infusion during GO-induced aplasia. Five patients who had previously undergone TBI received melphalan 140 mg/sqm. GvHD prophylaxis was performed with tacrolimus (starting day-1) and mycophenolate mofetil (1.5g BID from day 0). Four patients with progressive disease after GO did not proceed to conditioning therapy and went off study. A reduction of marrow blast counts after GO monotherapy was achieved in 15/30 patients (50%). In 11 cases without response to GO, an additional salvage regimen containing anthracyclines and high-dose cytarabine was administered before conditioning therapy and transplantation. 26 patients received G-CSF mobilized peripheral blood progenitor cells from matched-sibling (n=6) or unrelated donors (n=20). Primary engraftment was observed in all cases. With a median follow-up of 20 months (range 6–55) only one patient experienced sinusoidal obstruction syndrome which was reversible after treatment with defibrotide. Grade II-IV acute GvHD occurred in 15 patients (54%). Nine patients are alive in complete remission. Reasons for death in the other patients were relapse/progression (n=10) and GvHD/Infection (n=7). The probability of overall and disease-free survival at two years for patients having received a transplant is 38% and 32%, respectively. Blast reduction after GO was associated with a superior disease-free survival (48%, p=0.1). These data suggest that GO can be integrated into reduced-intensity conditioning therapy before allogeneic stem cell transplantation in patients with relapsed AML. Refractory patients with no response to GO monotherapy have a low chance of cure and should probably receive other combination therapies.

No standard treatment is available for relapse of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic stem cell transplantation (alloSCT). Efficacy of donor lymphocyte transfusion (DLT) was limited, at least in part, by the rapid pace of the disease, overwhelming any allogeneic immune response. However, induction chemotherapy before DLT was complicated by severe toxicity. Based on a pilot trial conducted in Munich, a prospective, multicenter phase II trial for relapsed AML and MDS after alloSCT was initiated in 2000 by the German transplant cooperative group. The study evaluated the sequence of low-dose(ld)AraC for leukemic control (intensive chemotherapy for progressive disease only), transfusion of donor PBSC without immunosuppression as adoptive immunotherapy, and systemic application of GM-CSF. GM-CSF was included, since in combination with other cytokines, it has been able to improve the antigene presentation capacity of myeloid blasts in vitro. Between 2000 and 2006, 41 patients with hematological relapse of AML or MDS >3 months after alloSCT were included. Median age was 47y, 50% had an unrelated donor. Median remission after SCT was 223d (93–1614), median percentage of BM blasts at relapse was 40%. Median follow up was 28 months. Control of leukemic proliferation by ldAraC was achieved in 61%, allowing outpatient care in the majority of these patients. 39% required intensive chemotherapy. Three patients died from infections before donor cell transfusion (DCT), one patient was not transfused due to progressive leukemia. Median time from relapse to DCT was 52 days. 25/34 evaluable patients were found to be free of blasts in BM at d35 after PBSC and were considered initial responders. In an intent-to-treat analysis, overall survival (OS) of the entire cohort at 1 and 2 years from relapse was 41% and 32%. Among initial responders, 1y- and 2y-OS was 68% and 49%. A remission >6months after alloSCT, and control of leukemia by ldAraC prior to DCT were associated with better outcome. At last follow up, 8 patiens were in continous CR, 2 were alive with second relapse, 18 had died from leukemia, and 13 had died in remission or aplasia. In conclusion, ldAraC seems to be effective for initial control of leukemic proliferation in relapsed AML and MDS after alloSCT. A longer duration of post-transplant remission and response to ldAraC may identify patients who will benefit from adoptive immunotherapy. On an intend-to-treat-basis, the overall results of our trial compare favorably to other published strategies; nevertheless, outcome is still unsatisfying and warrants further investigation.

Deletions of chromosome bands 13q14 and 17p13 (P53) as well as t(4;14)(p16;q32) or overrepresentation of CMYC are known to be adverse prognostic factors for time to progression (TTP) and overall survival (OS) in myeloma patients receiving conventional or high-dose chemotherapy followed by autologous stem cell transplantation. We investigated the impact of these chromosomal abnormalities as detected by fluorescence in situ hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) on outcome after dose-reduced melphalan/fludarabin based allogeneic stem cell transplantation (SCT) in 64 patients (pts) with multiple myeloma (MM). Median age was 52 years (34 – 67 ys.), 38 patients were male, 26 female. 45 pts received stem cell graft from an unrelated donor and 19 pts from HLA-identical sibling. Deletion of 13q14 was found in 35 out of 64 pts (55%), P53 deletion in 7 out of 64 pts (11%), t(4;14)(p16;q32) in 11 out of 56 pts (20%) and trisomy of CMYC in 12 out of 55 pts (22%). The following strong correlations among the genetic changes have been seen: six out of seven pts with P53 deletion also showed deletion in chromosome band 13q14 (86%), which was also found in nine out of eleven pts with t(4;14) (82% del 13) and nine out of twelve pts with CMYC trisomy (75% del 13). The estimated event-free (EFS) and overall survival (OS) at four years for the entire study population was 48% and 57%, respectively. No significant difference of the estimated event-free survival at 3 years was seen for pts with del 13 (42% vs 54%, p=0,4), with P53 deletion (43% vs 49%, p=0,27) and for pts with t(4;14) (46% vs 62%, p=0,69) or CMYC overrepresentation (37% vs 50%, p=0,35). Comparison of pts showing both del 13 and P53 deletion, del 13 and t(4;14), del 13 and trisomy of CMYC or t(4;14) and CMYC trisomy with the rest of the study population did not show a significantly reduced EFS either. Even the comparison of pts with any chromosomal abnormalities and pts without any genetic changes detected by FISH did not show a significant difference in EFS (48% vs 50%, p=0,8). These preliminary data suggest that the negative prognostic impact of chromosomal abnormalities such as deletion of 13q14, P53 deletion, t(4;14) and overrepresentation of CMYC may be overcome by allogeneic SCT probably due to the graft versus myeloma effect.

Purpose: To analyse hematopoietic engraftment after allogeneic stem cell transplantation (SCT) in patients with chronic lymphocytic leukemia (CLL) who had received Campath prior to SCT. Patients and Methods: 40 patients with relapsed CLL were included into the analysis. All patients received Busulphan 8 mg/kg and Fludarabine 150 mg/m2 as conditioning regimen. The first 20 patients (Bu/Flu/Cam) received a cumulative dose of 75 mg Campath on days -9 to -5 and Cyclosporine (CSA) as GVHD-prophylaxis. The second 20 patients (preCam/Bu/Flu) received a conventional treatment (3 × 30 mg weekly) with Campath prior to SCT. Transplantation was scheduled 14 days from the last dose of Campath and GVHD-prophylaxis consisted of CSA and Methotrexate. The two groups did not differ in age, median number of prior chemotherapy regimens, prior fludarabine treatment or donor type. The observation of late secondary graft failure during the first phase of the study led to the analysis of lineage-specific engraftment (CD4+T-cells, CD8+T-cells, NK-cells and Neutrophils) and of Campath levels with an ELISA at the day of transplant. Complete donor chimerism was defined as more than 95% donor-DNA in a specimen. Results: Campath levels are available for 22 patients. The median antibody level was 307 ng/mL (range, 41 to 1820 ng/mL) after Bu/Flu/Cam and 62 ng/mL (detection limit to 490 ng/mL) after preCam/Bu/Flu. All patients reached neutrophil engraftment (>0.5/nl) after a median of 18 days (range, 14 to 27). The cumulative incidence of complete leukocyte chimerism at day +100 was 75% after Bu/Flu/Cam and 78% after preCam/Bu/Flu (p=0.858). The cumulative incidences of CD4+ and CD8+ T-cell donor chimerism at day +100 were 45% after Bu/Flu/Cam and 70% after preCam/Bu/Flu for both T-cell subsets (CD4+subset, p=0.481; CD8+subset, p=0.398). Of note, the Campath-level at the day of transplantation discriminated better between patients with prompt versus impaired T-cell engraftment. The incidences of complete CD4+ and CD8+ T-cell chimerism at day +100 were 14% and 29% in patients with Campath levels >250 ng/mL compared to 82% and 82% in patients with antibody levels 250 ng/mL compared to 0 out of 17 patients with Campath levels Conclusions: T-cell chimerism should be analysed in patients who have received Campath recent to SCT. The level of Campath at the day of transplant may be an important factor for delayed T-cell engraftment. Figure Figure

Seventy-one patients with acute myeloid leukemia (AML), most of them (63/71) considered ineligible for conventional allogeneic hematopoietic stem cell transplantation (HSCT), were enrolled into a phase II study on reduced intensity myeloablative conditioning with fractionated 8 Gy total body irradiation (TBI) and fludarabine (120 mg/m2) (Blood. 2005 Nov 1;106(9):3314–21). Patients received mobilized peripheral blood stem cells (n=68) or bone marrow (n=3) from siblings (n=39) or unrelated donors (n=32). HLA-typing was performed for HLA-A, -B, -Cw (serological matching or intermediate resolution DNA typing), DRB1 and DQB1 (high resolution DNA typing). Three patients had unrelated donors with an allele mismatch in HLA DRB1 (2 with an additional mismatch in HLA Cw) and 7 patients were transplanted from unrelated donors with an antigen mismatch in HLA Cw. Thirty-six patients were transplanted in complete remission (CR) and 35 with untreated or refractory disease (non-CR). Median patient age was 51 years (range, 20–66). Sustained engraftment was attained in all evaluable patients. With a median follow-up of now 41.3 months (range, 20.4–70.4) in surviving patients, probabilities of overall survival for patients transplanted in CR and non-CR were 80% (95% CI, 66 to 94%) and 17% (95% CI, 5 – 29%) at 4 years, respectively. Relapse-free survival rates were 57% (95% CI, 39 – 75%) and 14% (95% CI, 2 – 26%). Of the 35 evaluable patients transplanted in CR, 10 patients suffered a relapse between days 68 and 868 after transplantation (cumulative incidence 29%). Five patients with late relapse (>1 year after transplantation) achieved a subsequent CR after conventional chemotherapy, blood stem cell boost and treatment with granulocyte-macrophage colony-stimulating factor, lasting 2000+, 1841+, 909+, 847+ and 480 days, respectively. Depending on donor type, relapse-free survival was similar in patients transplanted from unrelated or sibling donors. Overall survival in patients transplanted in complete remission from unrelated vs. sibling donors was 84% (95% CI, 73 – 95%) vs. 77% (95% CI, 68 – 86%). The cumulative incidence of non-relapse mortality (NRM) in CR patients was 11% at 4 years and beyond (3 patients deceased before day 100 and 1 patient 25 months after transplantation), but amounted to 37% at 4 years in non-CR patients. Nine of the 33 surviving patients (27%) have actually active chronic GvHD (5 limited and 4 extensive disease). This update confirms that allogeneic HSCT from related or unrelated donors with 8 Gy TBI/fludarabine conditioning is feasible with low NRM and preserved long-term antileukemic activity in AML patients in first or later CR.

Herpesviruses and adenovirus are an important cause of end-organ disease (EOD) and are associated with graft versus host disease (GVHD) in patients undergoing allogeneic stem cell transplantation (SCT). Detection and monitoring of those viruses is routinely performed by simplex quantitative real-time PCR. Since more than one herpes- or adenovirus are reported to be present shortly after transplantation, detection of all possible (sub-)clinical viral infections is still time-consuming and cost-intense. We therefore designed a DNA-microarray for the simultaneous detection of herpes-simplex-virus-1 and -2 (HSV-1/2), cytomegalovirus (CMV), varicella-zoster-virus (VZV), Epstein-Barr-virus (EBV), human herpesvirus-6 (HHV-6) and adenovirus. The DNA-microarray was validated in comparison to simplex quantitative PCR routinely used in our diagnostic laboratory. We retrospectively screened 608 samples of 35 patients undergoing allogeneic SCT on a regular basis until day 100 post-transplantation. The detection limit of the DNA-microarray is 10 genome equivalents (GE)/ml. We detected one or more viruses in 367 samples (60,2%). Of the positive samples, CMV was detected in 272 (74,1%), EBV in 103 (28,1%), HHV-6 in 81 (22,1%), adenovirus in 35 (9,5%), HSV-1/2 in 21 (5,7%) and VZV in 14 samples (5,2%), respectively. Of the samples containing two viruses (n=62), we detected CMV/EBV in 19 (30,6%), CMV/HHV-6 in 27 (43,6%) and HHV-6/EBV in 7 (11,3%) samples, respectively. CMV/EBV/HHV-6 was detected in 4 (36,4%) of the samples containing 3 viruses (n=11). Results of the DNA-microarray showed good overall agreement in comparison to the simplex quantitative PCR. We conclude that the DNA-microarray is a highly sensitive and specific method for the simultaneous detection of up to six viruses in a single sample. By screening and monitoring SCT-patients for viruses known to be associated with EOD or GVHD we herewith introduce an innovative molecular technique that can simultaneously detect multiple viral infections on a large scale. The assay is currently used to determine the impact of multi-virus detection on clinical end-points like GVHD and infectious complications in patients undergoing allogeneic stem cell transplantation.

New immunosuppressive compounds are currently investigated to reduce the rate of lethal Graft-versus-Host disease (GvHD) after allogeneic hematopoietic cell transplantation without adding relevant toxicity. Although positive reports on the efficacy of Mycophenolate Mofetil (MMF) in patients with acute and chronic GvHD are available, there is only limited data on the optimal prophylactic dosing schedule in patients at high risk for GvHD. Since very low trough-levels of MPA are measured in recipients of allogeneic stem cell transplants, we performed a prospective phase I/II trial targeting daily MMF doses according to MPA AUC levels determined at several time-points after transplantation. Twenty-nine patients (18 male, 11 female) with a median age of 53 years were included. The indication for allogeneic transplantation from matched sibling (n=7) and unrelated donors (9/10 alleles matched as minimum requirement; n=22) was high-risk AML/MDS (n=19) or relapsed lymphoma/multiple myeloma (n=10). Conditioning therapy included 30 mg/m2 Fludarabine on days −9 to −6 (4 x 20 mg/m2) combined with intravenous Busulfan 3.45 mg/kg from day −5 to day −2 (4 x 3.45 mg/kg i.v.). Tacrolimus was given orally starting on day −1 at 0.03 mg/kg in order to achieve trough blood levels of 5–10 ng/ml. MMF was started on day 1 at 1500 mg intravenously every 12 hours. AUC measurements of mycophenolic acid (MPA) and its metabolite MPAG by HPLC were scheduled on day 3, 8, 14, 21 and 28 after transplantation. The MMF dose was modified in order to achieve an AUC of 35–60 μg/m*h. MMF was tapered from day 56 on, if possible The dose of MMF had to be increased in 15/29 (52%) patients to 1750–2500 mg every 12 hours on day 4. No patient required a reduction of dosing on day 4. With the respective dose adjustments 52% and 80% of patients reached the AUC target on day 8 and 14, respectively. There was no direct association between dose level and extramedullary toxicity. Early grade 3–4 gastrointestinal toxicity occurred in 4 patients and lead to a reduction of MMF back to 1000 mg every 12 hours. Trilineage engraftment and complete donor chimerism was observed in all patients included. Only one out seven patients with a matched related donor experienced acute GvHD > grade II. The respective proportion of grade III–IV acute GvHD in the unrelated setting was 7/20 (33%). The rate of viral (CMV) and fungal infections was not increased compared to historical controls using standard antimicrobial prophylaxis. With a median follow-up of 18 months 15 (52%) patients are alive and 14 are in complete remission. Reasons for death were relapse (n=5; 17%), pneumonia/sepsis (n=2; 7%), GvHD (n=5; 17%) and organ failure (n=2; 7%). So far, 8 out of 24 evaluable patients (33%) suffer from limited (n=3) or extensive (n=5) chronic GvHD. A retrospective analysis revealed a significant correlation between Cmax levels and the AUC for MPA. The respective target Cmax was shown to be 16 μg/ml. Given the high-risk patient population and the high proportion of unrelated donors (70%) the clinical results observed with the combination of tacrolimus and MMF as prophylactic regimen are encouraging. The regimen has to be optimised for recipients of unrelated transplants. MMF doses of up to 2500 mg every 12 hours can be infused early after stem cell transplantation without an increased risk of toxicity. A simplified MMF targeting strategy based on MPA Cmax levels seems to be warranted in future trials.

Haploidentical hematopoietic cell transplantation (HHCT) has been made feasible with the use of megadoses of CD34+ peripheral blood stem cells. Despite this progress, HHCT is still complicated by high treatment related toxicity, slow engraftment and immune reconstitution. A new regimen using graft CD3/CD19 depletion with anti-CD3- and anti-CD19-coated microbeads on a CliniMACS device and dose reduced conditioning may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, natural killer-, dendritic- and graft-facilitating-cells. As dose reduced conditioning fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (5 mg/day, day -5 to +14) without posttransplant immunosuppression was used. So far 9 consecutive adult patients have been transplanted with this regimen. Median age of the patients was 37 (range, 27–58) years and the diagnoses were AML (n=3), ALL (n=3), NHL (n=2) and multiple myeloma (n=1). All patients were high risk with relapse after preceding hematopoietic cell transplantation. The CD3/CD19 depleted haploidentical grafts contained a median of 8 x 10E6 (range, 5.2–17x10E6) CD34+ cells/kg, 6x10E7 (range, 0.02–8.6 x10E7) CD56+ cells/kg and 2x10E4 (range, 0.006–8.2x10E4) CD3+ T-cells/kg. 8 of 9 patients had a donor-recipient KIR-mismatch. The conditioning regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis and nausea. In 4/9 patients reversible peripheral neuropathy grade 2–3 occurred, combined with multifocal leucencephalopathy in one patient. Subsequently the dose of fludarabine was reduced to 150 mg/m2. Engraftment was rapid with median time to neutrophils >500/μL of 13 (range, 10–17) days, platelets >20000/μL of 11 (range, 8–14) days and full donor chimerism after two weeks in all patients. All evaluable patients had fast immune reconstitution with T-cells >100/μL by day +100. Five cases of grade 2 skin GVHD rapidly responded to steroids. Three patients with controlled fungal pneumonia at time of transplant had resolution of infiltrates with neutrophil reconstitution and antifungal therapy. Treatment related mortality in the first 100 days was low with one death due to idiopathic pneumonia syndrome. Three patients died after day 100, one each due to relapse, systemic adenoviral infection and CMV infection. Overall survival is 5/9 patients with a median follow-up of 406 days (range, 44–661). In conclusion, this regimen seems to be promising in adult high risk patients lacking a suitable donor and a prospective phase I/II study is ongoing.

Understanding the mechanisms underlying the reconstitution of the hematopoietic system after blood stem cell transplantation is of crucial importance for improving therapeutic modalities. In order to address these issues, we have recently established a novel co-culture system consisting of immuno-isolated human CD34+ hematopoietic stem and progenitor cells (HSPCs) growing on primary human mesenchymal stem cells (MSCs) as feeder layer in the presence of relevant cytokines. Here we have investigated i) the morphological modification of HSPCs induced by their interaction with MSCs; ii) the sub-cellular localization of the stem cell markers CD34 and CD133 (prominin-1) as well as other cell surface molecules potentially involved in such intercellular interaction, and iii) the intracellular pathway(s) responsible for the migration as well as the interaction of HSPCs with MSCs. The scanning electron microscopy analysis revealed that the adherent HSPCs display various morphologies; they are either round with, in some cases, the appearance of a microvillar pole or exhibit several distinct types of plasma membrane protrusions such as lamellipodium, filopodium and magnupodium. We could also observe very long and thin plasma membrane processes between adjacent HSPCs suggesting the formation of nanotubes, which have been implicated in intercellular communication. As previously reported (Giebel et al., 2004, Blood, 104:2332), the highly motile HSPCs acquire a polarized cell shape with the formation of a uropod at the rear pole and a leading edge at the front pole. The immunofluorescence analyses revealed that CD34 is randomly distributed over the entire surface of HSPCs, irrespective of their morphological shape, whereas CD133 shows various specific sub-cellular localizations, which are depending on the state of the cell. In migrating cells, CD133 is concentrated in the uropod at the rear pole, which contrasts with the enriched localization of the adhesion receptor β2 integrin in the leading edge. In HSPCs harboring other types of plasma membrane protrusions, which are in close contact with the feeder cell layer, CD133 is concentrated therein. In round adhesive cells, CD133 is located in one pole of the cell suggesting that the HSPCs are highly polarized even in the absence of plasma membrane outgrowths. Interestingly, the formation of uropod and microvillus-like structures is highly reduced in presence of Y-27632 inhibitor suggesting that the Rho GTPase/ROCK pathway is somehow involved in the polarization of HSPCs. Time lapse videomicroscopy revealed that the retraction of filopodia is also affected in Y-27632-treated HSPCs as well as their migration. Taken together, these data provide new insights regarding the molecular cell biology of HSPCs, and increase our understanding of cellular events associated with mobilization and engraftment in the context of blood stem cell transplantation.

Deletion of chromosome band 17p13 (P53) is known to be an adverse prognostic factor for time to progression (TTP) and overall survival (OS) in myeloma patients receiving conventional or high-dose chemotherapy. In this retrospective multicenter study, we investigated the impact of P53 deletion as detected by fluorescence in situ hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) on outcome after allogeneic blood stem cell transplantation (SCT) in 50 patients (pts) with advanced or relapsed multiple myeloma (MM). Median age was 51 years (34 – 67 ys.), 32 patients were male, 18 female. Thirty pts received a stem cell graft from an unrelated donor, and 20 pts from a HLA-identical sibling. P53 gene deletion was found in 5 out of 49 pts (10,2 %). Deletion 13 detected by cIg-FISH was found in 20 out of 47 pts (42%). There was a strong correlation between P53 deletion and deletion 13: four out of the five patients with P53 deletion also showed deletion in chromosome band 13q14. The estimated event-free (EFS) and overall survival (OS) at three years for the entire study population was 43% and 65%, respectively. For patients with del 13 only a trend for a worse 3 year EFS was seen (38% vs 42%, p=0.2), while pts with P53 deletion had a significant reduced EFS (0% vs 46%, p=0.0001). Three out of five pts with P53 deletion relapsed very early after allogeneic SCT (day 81, 108 and 287 respectively). These data suggest that P53 is a risk factor for patients with MM treated with allogeneic SCT which can not be overcome by this treatment strategy.

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment option for most high-risk patients with acute myeloid leukemia (AML). However, many high-risk patients regenerate with blasts or relapse early after induction therapy. Thus, consolidation with allogeneic HSCT in first CR is often not possible. Performing allogeneic HSCT within induction therapy can circumvent these problems and may furthermore reduce cumulative toxicity in high risk patients. Therefore, the prospective randomized treatment trial AML2003 for patients Within the first 8 months 107 AML patients with a median age of 48 (17–60) years were included in the study. Fast search diagnostics was complete within a median of 15 (range 5–31) days after arrival of the bone marrow samples for all patients. 57/107 patients were randomized into the intensified treatment arms. Out of these 25 (44%) patients with high risk characteristics have been identified. A suitable related or unrelated donor was found for 22 (88%) of those high-risk patients. Nine of those high risk patients with a donor (41%) received early allogeneic stem cell transplantation in aplasia after the first (n=4) or the second (n=5) induction therapy course on an intend to treat basis within the protocol. Three were transplanted with stem cells of related and six of unrelated donors. The preparative regimen consisted of melphalan 150mg/m2 and fludarabine 150mg/m2. So far no treatment associated death had to be recognized. These encouraging preliminary results show that fast risk-profiling and early donor-search is feasible in a large multi-center study. This leads to a significant proportion of early allogeneic stem cell transplants in aplasia after induction therapy within the group of high risk AML patients, which may improve the disastrous prognosis of this group of patients in the future.

Both number and origin of dendritic cells (DC) in the blood have been associated with acute graft-versus-host disease (aGvHD), graft-verus-leukemia effect, relapse and graft failure after human allogeneic haematopoietic cell transplantation (aHCT). Aim of this study was the systematic simultaneous investigation of skin and blood DC subtypes, the analysis of their host/recipient origin after HCT and the correlation of DC kinetics with treatment, outcome and incidence of aGVD and its treatment in 31 recipients of allogeneic HCT. Epidermal shave biopsies and peripheral blood samples were collected from patients pre and at defined time points following aHCT, DC from skin and blood isolated and examined by FACS and quantitative PCR for Short Tandem Repeat markers. A significant reduction in number of skin and blood DC subtypes was observed already before start of conditioning chemotherapy in patients compared to healthy volunteers. A majority of donor derived epidermal Langerhans Cells (median 95%, range 53–100%) and also dermal DC (median 94%, range 39–100%) was found in all examined patients even early after transplant independent from chemotherapy regimen, graft, occurrence of skin GvHD or time point after transplantation. Numbers of both skin DC subsets remained low for months post HCT. Reconstitution kinetics of CD 11c+ preDC1 and CD 123+ preDC2 blood DC were similar to each other and pre transplant numbers were reached again around day +112 post transplant. Recipients of grafts containing higher T cell numbers had lower preDC1 counts on day +28. PreDC2 reconstitution was negatively affected by steroid treatment. Patients with aGvHD tended to have more preDC1 on day +28 and lower numbers of both preDC subsets on day 56 post transplant. Residual host blood DC were rare at all time points (median

Objectives: In patients with acute myeloid leukemia and high-risk cytogenetic aberrations or minor response to the first cycle of induction chemotherapy (IC) the probability of achieving a sustained complete remission is low. Thus early treatment intensification may be warranted in order to achieve long-term disease control. We performed a prospective trial to evaluate whether reduced-intensity conditioning followed by allogeneic hematopoietic stem cell transplantation (HSCT) from related or unrelated donors can be performed during the aplastic phase of IC in patients with poor-risk AML. Methods: Seventeen patients (n=17) aged between 17 and 63 years (median 45) with acute myeloid leukemia and high-risk cytogenetic aberrations (n=14, complex, inv3 or t(3;3), t(3;5), −7 or del 7q, +8) or more than 10 % marrow blasts on day 15 after the first cycle of IC (n=3) were included so far. During aplasia a median of thirteen days (range 7–35) after the first (n=8) or second (n=9) cycle of IC patients received 5 x 30 mg/m2 fludarabine i.v. combined with either 8 mg/kg busulfan p.o. (n=4) or 150 mg/m2 melphalan iv. (n=13) followed by allogeneic G-CSF mobilized peripheral blood stem cells (PBSC, n=16) or bone marrow (n=1) from related (n=7) or unrelated (n=10) donors. Nine out of seventeen patients were not in complete remission before conditioning therapy was started. Patients with unrelated grafts received antithymocyte globulin (4 x 10 mg/kg ATG Fresenius). GvHD prophylaxis was performed with cyclosporine A (CSP). Results: All patients engrafted (ANC > 0.5 Gpt/l on day 11, range 8–19, platelets > 50 Gpt/l day 15, range 11–32) and went into remission. Acute GvHD grade II-IV occurred in 8 patients and extensive chronic GvHD was documented in 5 patients with a follow-up of > 100 days. Two patients died while being in remission from infectious complications associated with acute (n=1) or chronic (n=1) GvHD and two patients died during relapse eight and twelve months after PBSC. With a median follow-up of 15 months (range 1–65) thirteen out of seventeen patients (76 %) are alive and in remission. Conclusion: Early allogeneic HSCT as part of primary induction therapy seems to be an effective strategy in AML patients with either poor risk karyotype or minor response to the first induction cycle.

Short-term administration of rhG-CSF is a safe and effective procedure for mobilization of allogeneic PBPC in healthy related and unrelated donors. Pegfilgrastim (Neulasta™, Amgen Inc., Thousand Oaks, USA) is a covalent conjugate of filgrastim and polyethylene glycol with an increased elimination half-life due to decreased serum clearance. In cancer patients a single injection of pegfilgrastim has been shown to be equivalent to daily filgrastim in enhancing neutrophil recovery after chemotherapy. However there are no data concerning the efficacy of pegfilgrastim in mobilization of allogeneic PBPC. We report on 20 allogeneic PBPC donors (11 related, 9 unrelated, 12 male, 8 female), who have undergone a mobilization treatment with pegfilgrastim. The median age was 43 (range 28–74) years. Donors received a single subcutaneous injection of 2 vials (12 mg) pegfilgrastim resulting in a median dose of 161 μg/kg body weight (range 126–187). Monitoring included complete blood cell counts, number of CD34+ cells in peripheral blood (PB-CD34), cytokine related side effects and in five donors filgrastim pharmacokinetics. PBPC were collected by large-volume leukaphereses (4x blood volume) using Cobe Spectra 7.0 (Gambro BCT Inc.). Pegfilgrastim induced a rapid increase of WBC and ANC, which raised in median from 6.10 x 10^9/L at baseline to 34.41 x 10^9/L on day 2 and from 4.03 x 10^9/L to 28,05 x 10^9/L. Peak values of PB-CD34+ cells was found consistently on day 5 (median 76/μl, range 10–385). On day 6 the PB-CD34+ cell count was less than on day 5, and also less than on day 4 (day 6: median 38.5/μl, range 9–180; day 4: median 61.5/μl, range 4–135). In 19/20 donors the apheresis started on day 5. In a 74 years old family donor additional filgrastim at a dose of 10 μg/kg was administered on day 4 and 5 due to a PB-CD34+ cell count of 4/μl on day 4. The median apheresis yield was 8.23 x 10^6 CD34+ cells/kg donor weight (range 2.40–29.32), corresponding to a median of 9.5 x 10^6 CD34+ cells/kg recipient (range 3.22–39.71). The target cell dose (≥ 4.0 x 10^6 CD34+cells/kg recipient) was reached with one procedure in 15 (75 %) donors. Apheresis products contained in median 3.8 x 10^8 CD3+ T-cells. Mobilization and harvesting were well tolerated except for moderate bone pain which occurred in 19/20 donors. The mean values (± SD) for peak plasma concentration of pegfilgrastim (cmax), time to reach the maximum plasma level (tmax) and elimination half-life (t1/2) were 500 (± 417) ng/ml, 33 (±13) hours and 23 (± 9) hours, respectively. Among 14 recipients, who are evaluable for engraftment study at present, one graft failure occurred in a patient who received a haploidentical graft from her sibling donor. Pegfilgrastim is able to mobilize a sufficient number of CD34+ PBPC for stem cell collection in allogeneic donors. Schedule for mobilization and apheresis is very similar to conventional, non-pegylated rhG-CSFs. However, the PB-CD34+ cells were found to be higher on day 4 than on day 6. Pegfilgrastim is well tolerated and side effects are similar to Filgrastim. The most important benefit for the donor is the simplified once-per-mobilization dosing. Further evaluation with respect to graft function and incidence of graft-versus-host-disease is ongoing.

Gemtuzumab ozogamicin (GO) has been successfully used in older patients with relapsed CD33+ acute myeloid leukemia. Retrospective analyses suggest that the use of GO within a few months before or after hematopoietic stem cell transplantation (HCT) is associated with a increased risk for sinusoidal obstruction syndrome (SOS) or veno-occlusive liver disease (VOD). Ongoing studies investigate the use of GO in the induction and post-remission therapy of AML patients. We hypothesized that GO might be safe and effective as part of a reduced intensity conditioning regimen containing fludarabine and total-body irradiation (TBI) Fifteen patients relapsing after conventional induction chemotherapy (n=9) or after previous transplantation (autologous n=3; allogeneic n=3) were included so far. The last twelve patients were treated within an ongoing phase II trial. The preparative regimen contained 6 mg/m2 and 3 mg/m2 GO on day −21 and day −14 before allogeneic transplantation. Patients who responded to GO treatment and were below the age of 60 (n=8) received fludarabine 120 mg/m2 and 800 cGy TBI (n=6) during GO-induced aplasia followed by allogeneic HCT. Patients older than 60 years or those with relapsing AML These data suggest, that GO can be combined with a TBI-containing, fludarabine-based conditioning regimen before allogeneic HCT in patients with relapsed AML. No increased incidence of VOD has to be expected even in recipients who had undergone previous conditioning therapy and transplantation. Nevertheless, additional chemotherapy during aplasia before allogeneic transplantation is required in 60–70% of patients who do not respond to GO. For responding patients the combined regimen is associated with minimal toxicity and a considerable chance for long-term cure.