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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.

Background: Acute myeloid leukemia (AML) is characterized by a high relapse rate, indicating insufficient clearance of leukemia-initiating cells. Depending on genetic risk stratification, consolidating chemotherapy proves to significantly reduce the risk of relapse. In particular, in younger AML patients higher dosage of cytarabine appears to improve long-term outcome, while there is no apparent benefit of multiagent combination, compared to cytarabine monotherapy. However, to this end the optimal dosage of single agent cytarabine in consolidation therapy after 7+3 remission induction remains elusive. Methods: Here, we retrospectively assessed the impact of different dosages of cytarabine consolidation on outcome in a large real-world data set from the German Study Alliance Leukemia-Acute Myeloid Leukemia (SAL-AML) registry. Patients below 65 years of age, registered between April 2005 and September 2020 with non-acute promyelocytic leukemia, who attained complete remission after intensive induction and received at least one consolidation cycle with intermediate (IDAC) or high dose cytarabine (HiDAC) were selected. To account for differences in patient and disease characteristics between both groups, the average treatment effect was estimated by propensity score weighting. Results: 642 patients received HiDAC consolidation with a median dosage of 5794.88 (IQR, 4745.48-5971.56) mg/m 2/d with a median number of 3 cycles (IQR, 2-3), whereas 178 patients received IDAC consolidation with 1946.16 (IQR, 1869.51-2469.15) mg/m 2/d with a median of 2 cycles (IQR, 1-3). IDAC-treated patients showed in average a higher age (median (IQR) 58.5 (49-62) years vs. 50 (41-56) years) and more comorbidities with 43.8% having an HCT-CI score of 2-4, compared to 22.3% among HiDAC-treated patients. Alongside, significantly more secondary (5.1% vs. 3.1%) and therapy-related (12.4% vs. 4.1%) AML as well as more adverse (14.5% vs. 6.5%) and less favorable (40.6% vs. 56%) genetic risk features according to ELN 2017 risk classification were found among IDAC-treated patients. After propensity score weighting for differences in patient and disease characteristics, overall survival after 5 years was comparable between HiDAC-treated (71.1 %) and IDAC-treated (67.7%) patients. Moreover, no significant differences in relapse-free survival were observed after 5 years (47.4 vs. 45.2%). Notably, more patients treated with IDAC received allogeneic stem cell transplantation in first remission (37.6 vs. 19.8%) while significantly more HiDAC-treated patients underwent allogeneic stem cell transplantation in relapse (30.8 vs. 20.2%). Censoring for allogeneic stem cell transplantation in first remission revealed no significant survival difference with regard to cytarabine dosage. Considering only ELN favorable risk AML patients, there was no difference in 5-years overall (80.5% vs. 83.9%) nor relapse-free (57.7% vs. 56.8%) survival. Of note, significantly more patients treated with HiDAC suffered from ≥3 CTCAE infectious complications (56.7 vs. 44.1%), which was more striking in patients above 50 years of age. The rate of other ≥3 CTCAE non-hematological toxicities and secondary malignancies was comparable in both treatment groups. Conclusion: This retrospective analysis suggests no significant benefit of high dose cytarabine compared to intermediate dosages in consolidation for AML patients under 65 years of age, independent of ELN risk group. Disclosures Krause: Siemens: Research Funding; Takeda: Honoraria; Pfizer: Honoraria; art-tempi: Honoraria; Kosmas: Honoraria; Gilead: Other: travel support; Abbvie: Other: travel support. Schliemann: Philogen S.p.A.: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Other: travel grants; Astellas: Consultancy; AstraZeneca: Consultancy; Boehringer-Ingelheim: Research Funding; BMS: Consultancy, Other: travel grants; Jazz Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy; Roche: Consultancy; Pfizer: Consultancy. Haenel: Jazz: Consultancy, Honoraria; GSK: Consultancy; Bayer Vital: Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria. Brummendorf: Takepart Media: Honoraria; Repeat Diagnostics: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Patents & Royalties, Research Funding; Janssen: Honoraria; Bristol Myers: Research Funding. Fransecky: Abbvie: Honoraria, Research Funding; Medac: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Takeda: Honoraria. Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding. Held: MSD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Roche: Research Funding; Acortech Biopharma: Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Platzbecker: Janssen: Honoraria; Celgene/BMS: Honoraria; AbbVie: Honoraria; Geron: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Baldus: Amgen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Jazz: Honoraria. Mueller-Tidow: Janssen Cilag: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bioline: Consultancy, Research Funding.

Achievement of complete remission (CR) signifies a crucial milestone in the therapy of acute myeloid leukemia (AML) while refractory disease is associated with dismal outcomes. Hence, accurately identifying patients at risk is essential to tailor treatment concepts individually to disease biology. Machine Learning (ML) is a branch of computer science that can process large data sets for a plethora of purposes. The underlying mechanism does not necessarily begin with a manually drafted hypothesis model. Rather the ML algorithms can detect patterns in pre-processed data and derive abstract information. We used ML to predict CR and 2-year overall survival (OS) in a large multi-center cohort of 1383 AML patients who received intensive induction therapy using clinical, laboratory, cytogenetic and molecular genetic data. To enable a customizable and reusable technological approach and achieve optimal results, we designed a data-driven platform with an embedded, automated ML pipeline integrating state-of-the-art software technology for data management and ML models. The platform consists of five scalable modules for data import and modelling, data transformation, model refinement, machine learning algorithms, feature support and performance feedback that are executed in an iterative manner to approach step-wisely the optimal configuration. To reduce dimensionality and the the risk of overfitting, dynamic feature selection was used, i.e. features were selected according to their support by feature selection algorithms. To be included in an ML model, a feature had to pass a pre-determined threshold of overall predictive power determined by summing the normalized scores of the feature selection algorithms. Features below the threshold were automatically excluded from the ML models for the respective iteration. In that way, features of high redundancy or low entropy were automatically filtered out. Our classification algorithms were completely agnostic of pre-existing risk classifications and autonomously selected predictive features both including established markers of favorable or adverse risk as well as identifying markers of so-far controversial relevance. De novo AML, extramedullary AML, double-mutated (dm) CEBPA, mutations of CEBPA-bZIP, NPM1, FLT3-ITD, ASXL1, RUNX1, SF3B1, IKZF1, TP53, U2AF1, t(8;21), inv(16)/t(16;16), del5/del5q, del17, normal or complex karyotypes, age and hemoglobin at initial diagnosis were statistically significant markers predictive of CR while t(8;21), del5/del5q, inv(16)/t(16;16), del17, dm CEBPA, CEBPA-bZIP, NPM1, FLT3-ITD , DNMT3A, SF3B1, U2AF1, TP53, age, white blood cell count, peripheral blast count, serum LDH and Hb at initial diagnosis as well as extramedullary manifestations were predictive for 2-year OS. For prediction of CR and 2-year OS, AUROCs ranged between 0.77 - 0.86 and 0.63 - 0.74, respectively. We provide a method to automatically select predictive features from different data types, cope with gaps and redundancies, apply and optimize different ML models, and evaluate optimal configurations in a scalable and reusable ML platform. In a proof-of-concept manner, our algorithms utilize both established markers of favorable or adverse risk and also provide further evidence for the roles of U2AF1, IKZF1, SF3B1, DNMT3A and bZIP mutations of CEBPA in AML risk prediction. Our study serves as a fundament for prospective validation and data-driven ML-guided risk assessment in AML at initial diagnosis for the individual patient. Image caption: Patient features were automatically selected by machine learning to predict complete remission (CR) and 2-year overall survival (OS) after intensive induction therapy. Based on a continuous feature support metric with a predefined cut-off of 0.5 (determined by optimal classification performance), 27 and 25 features were automatically selected for prediction of CR (A) and 2-year OS (C), respectively. For each of these features predicted by machine learning, odds ratios and 95% confidence intervals (CI) were calculated for CR (B) and 2 year OS (D). BMB: bone marrow blast count; FLT3h/low: FLT3-ITD ratio, h=high>0.5; Hb: hemoglobin; karyotype, c: complex aberrant karyotype (≥ 3 aberrations); karyotype, n: normal karyotype (no aberrations); LDH: lactate dehydrogenase; PBB: peripheral blood blast count; PLT: platelet count; WBC: white blood cell count. Figure 1 Figure 1. Disclosures 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 . Platzbecker: Janssen: Honoraria; Celgene/BMS: Honoraria; AbbVie: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Geron: Honoraria. Müller-Tidow: Pfizer: Research Funding; Janssen: Consultancy, Research Funding; Bioline: Research Funding. Baldus: Celgene/BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria; Jazz: Honoraria. Krause: Siemens: Research Funding; Takeda: Honoraria; Pfizer: Honoraria; art-tempi: Honoraria; Kosmas: Honoraria; Gilead: Other: travel support; Abbvie: Other: travel support. Haenel: Bayer Vital: Honoraria; Jazz: Consultancy, Honoraria; GSK: Consultancy; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria. Schliemann: Philogen S.p.A.: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Other: travel grants; Astellas: Consultancy; AstraZeneca: Consultancy; Boehringer-Ingelheim: Research Funding; BMS: Consultancy, Other: travel grants; Jazz Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy; Roche: Consultancy; Pfizer: Consultancy. Middeke: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Jazz: Consultancy; Astellas: Consultancy, Honoraria; Sanofi: Honoraria, Research Funding; Novartis: Consultancy; Gilead: Consultancy; Glycostem: Consultancy; UCB: Honoraria.

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.

Identification of the origins of acute myeloid leukemia (AML) stem cells has been a holy grail for a better understanding of the developmental biology of the disease and the design of effective treatments. Studies using primary AML samples on patient-derived xenograft models identified AML stem cells in multiple different CD34/CD38 cellular fractions, which shared similar immunophenotypes of hematopoietic stem/progenitor cells including hematopoietic stem cells (HSCs), lymphoid-primed multipotent progenitors (LMPPs) and granulocyte-macrophage progenitors (GMPs). However inference of cells-of-origin based on the retrospective approach has major limitations as the reported HSC/LMPP/GMP-like AML stem cells are phenotypically different from their normal counterparts; and AML stem cells identified in late developmental stages do not necessarily maintain the same immunophenotypes of the disease initiating (pre-leukemic) cells, which can retain a relatively normal differentiation potential, and only their descendants acquire additional events becoming AML stem cells. While prospective disease modelling using mouse cells has provided unique insights into the potential origins of AML stem cells, human and mouse cells have different transformation requirements, distinct telomere biology, and a significant degree divergence of transcriptional regulation, chromatin state and gene regulatory networks that can profoundly affect their transformation potential and associated cancer biology. Therefore we reason that deconstruction of AML stem cell hierarchy from primary human samples followed by reconstruction of the corresponding human disease using candidate cell populations will give novel insights into this issue. Given that AML is a highly heterogeneous disease, our study initially focused on genetically well-defined MLL-rearranged AML frequently found in both infant and adult leukemia. By analyzing primary human AML patient samples, the current study unexpectedly revealed that AML stem cells driven by MLL fusions almost exclusively resided in immunophenotypically mature CD34-/loCD38+ compartments, as demonstrated by in vitro long-term culture initiating cells and in vivo limiting dilution xeno-transplantation assays into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using highly purified populations of hematopoietic stem/progenitor cells from normal human umbilical cord blood (UCB) to reconstruct of the human disease, we found that only human HSCs and common myeloid progenitors (CMPs), but not developmentally branched LMPPs and GMPs, could be disease initiating cells, transformed by MLL-fusions and induced leukemia in vivo, suggesting a key difference between human and mouse leukemia. As revealed by RNA-sequencing, LMPPs and GMPs failed to activate stem cell transcriptional programs and genes essential for MLL-leukemia including MEIS1 and HMGA2 in the early phase of transformation. MLL-fusions transformed HSCs and CMPs were immunophenotypically indistinguishable to leukemic cells of human MLL-AML patients, and also had an enrichment of AML stem cells in CD34-/loCD38+ compartment. Using machine learning, a specific gene signature could stratify patients into HSCs- and CMPs-derived AML, in which HSCs-derived AML showed a significantly poorer prognosis. To further investigate the biology of HSCs/CMPs-derived MLL-AML related to treatment responses, we subjected HSC/CMP MLL-AML leukemia with chemotherapeutic drugs and inhibitor of Bromodomain and Extra-Terminal motif (BET) currently used in AML trial. Interestingly, CMP-derived MLL-AML was treatment sensitive and PDX models transplanted with CMP-like AML samples could be largely cured by chemotherapy or BET inhibitor targeted therapy. In contrast, human HSCs-derived AML was highly resistant to the treatments. Strikingly, shRNA-mediated knockdown or pharmacological inhibition by fidaxomicin targeting ATP-binding cassette (ABC) transporters, ABCC3 that is highly expressed in HSCs-derived MLL-AML could re-sensitize the cells to the current chemotherapy. Together, the current study not only for the first time functionally identifies the origin of human MLL-AML stem cells, but also provides a new actionable venue for overcoming stem cells-associated treatment resistance by repositioning an anti-diarrhea drug, fidaxomicin currently available in the clinics. Disclosures Mufti: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cellectis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Clinical translation of chimeric antigen receptor (CAR) T cell therapy in myeloid malignancies is progressing slowly compared to its success in treatment of B cell malignancies. Clinical experiences with CAR T cell therapies against the currently investigated tumor-associated antigens (TAA) (e.g. CD33, CD123 and FMS-like tyrosine kinase 3 (FLT3)) were discouraging and severe side effects occurred (cytokine release syndrome, neurotoxicity and myeloid aplasia) (Hoffmann et al. Journal of Clinical Medicine 2019). Probably targeting a single TAA is insufficient to treat high risk myeloid malignancies with CAR T cell therapies. Therefore, combined targeting of two or even more TAAs seems to be a promising approach. In order to implement such a multiple tumor targeting strategy, we developed a modular CAR T cell system termed UniCAR. The system consists of a universal CAR (UniCAR) directed against the La peptide epitope E5B9 combined with single-chain variable fragment (scFv) -based target modules (TM). In contrast to conventional CARs, anti-tumor activity of UniCAR T cells is only turned on in the presence of the TMs. Thus, this approach will allow UniCAR T cell control due to the short half-life of the TM and therefore has a favorable safety profile. Furthermore, different TMs against several TAAs can be administered both sequentially or in parallel to increase the anti-tumor efficacy or face disease relapse due to antigen escape mechanisms. In the field of myeloid malignancies our group developed retargeting strategies against the TAAs CD33 and CD123 (Cartellieri et al. Blood Cancer Journal 2016). In addition, we have developed a new TM for the UniCAR system that is directed against the TAA FLT3. FLT3 is highly expressed on acute myeloid leukemia (AML) cells and also present on CD123low AML samples (Riccioni et al. British Journal of Haematology 2011). The novel FLT3 TM was constructed by fusion of the variable domain of the heavy and the light chain of the murine anti-FLT3 monoclonal antibody (4G8) to the E5B9 UniCAR epitope. In light of a potential clinical application, we in parallel generated a humanized FLT3 TM to further decrease its immunogenicity. Both FLT3 TMs were tested in vitro against different AML cell lines, by using flow cytometry based killing assays as described elsewhere (Fasslrinner et al. British Journal of Haematology 2019). The functionality of the FLT3 TMs in vitro was highly effective. Both FLT3 TMs were able to redirect UniCAR T cells for AML cell lysis already in the picomolar range and were moreover comparable effective than the previously developed CD123 TM. Thus, humanization of the FLT3 TM did not lead to a decrease in anti-tumor efficacy. In summary, we could show that both the novel murine FLT3 TM and the humanized counterpart redirected UniCAR T cells and induced highly effective elimination of AML cells in vitro. Thus, the flexible application of the FLT3-based UniCAR system seems to be a promising tool for cell-based AML therapy alone or even in combination with other AML-specific TMs (e.g. CD33, CD123). Disclosures Koristka: Intellia Therapeutics: Employment. Jung:Synimmune: Other: shareholder interest. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

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

The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Introduction The involvement of the bone marrow microenvironment (BMME) into disease progression and therapeutic response of myelodysplastic syndromes (MDS) is indisputable. Hereby, mesenchymal stromal cells (MSCs) play an important role for both the support of the leukemic clone and the remaining healthy hematopoietic stem and progenitor cells (HSPCs). The extracellular matrix (ECM) secreted by MSCs regulates stem cell fate through the modulation of cytokine and growth factor delivery and may also be targeted by clinically available drugs such as luspatercept, a novel recombinant fusion protein containing modified extracellular domain of activin receptor IIB. Luspatercept is a first-in-class erythroid maturation agent with promising results in lower-risk MDS patients with red blood cell transfusion dependency. Aim To shed light on the largely unknown composition and function of the MSC-derived ECM, we have characterized ECM from MDS patients vs. healthy controls and elucidate how luspatercept may modulate their functional characteristics. Methods Bone marrow-derived MSCs from patients with lower-risk MDS and age-matched healthy donors (HD) were treated with RAP-536, a murine homologue of luspatercept harboring the same activin receptor IIB domain. MSCs of three patients were treated with RAP-536 and RNA sequencing was carried out. Gene expression and pathway analyses were performed using the Reactome tool (https://reactome.org). Candidate genes were validated by quantitative real-time PCR (qPCR). For the generation of ECM, MSCs were seeded on poly-octadecene-alt-maleic anhydride and human fibronectin coated glass slides in the presence or absence of RAP-536. To yield cell-free ECM structures, cultures were decellularized at day 10 and analyzed by scanning electron microscopy (SEM), sulfated glycosaminoglycan (GAG), fibronectin and collagen staining as well as GAG quantification (Blyscan assay). Moreover, purified HD CD34+ HSPCs were cultured on ECM scaffolds for 6 and 9 days, respectively. Subsequently, expansion of adherent and supernatant cells was determined and the phenotype was analyzed by flow cytometry. Results RNA sequencing of MDS MSCs after six days of RAP-536 treatment revealed a total of 58 significantly regulated genes, thereof 24 up- and 34 down-regulated genes. Gene enrichment and pathway analyses revealed a striking involvement in ECM organization, collagen biosynthesis and formation. Moreover, integrin cell surface interaction genes showed significantly differential expression. Focusing on collagens as important ECM components, we identified Col7A1 and Col4A2 to be down-regulated. Indeed, both collagen mRNAs were significantly decreased by 46% and 25%, respectively, in MSCs after RAP-536 treatment compared to untreated controls. SEM characterization and immunofluorescence staining of the ECM showed a more compact fiber network produced by MDS MSCs. Moreover, MDS ECM contained higher levels of collagen and GAGs. Blyscan assay confirmed the latter observation, showing significantly higher sulfated GAG concentrations in MDS ECM. Interestingly, trapping of TGFβ superfamily ligands, such as GDF-11, by RAP-536 clearly reduced Col4 staining intensity in MDS MSC ECM. Structural and compositional ECM differences had functional impact on the expansion of HSPCs cultured on the matrices. Significant higher total cell numbers were detected on healthy ECM (18.3-fold vs. 12.1-fold expansion, *p< 0.05) but not on MDS ECM (12.9-fold) after 9 days of culture. The number of adherent cells increased 8.5-fold on healthy and 4.3-fold on MDS ECM and could be further increased after RAP-536 treatment of MSCs. Using flow cytometry, we found a 3.1-fold increased proportion of CD90+ HSPCs in the adherent fraction on healthy but only 1.8-fold on MDS MSC ECM. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be significantly higher expressed in the adherent HSPC fraction. RAP-536 treatment resulted in up to 20% higher expression of both CD90 and integrin subunits. Summary We demonstrate an association between induced collagen abundance and reduced hematopoietic support in ECM derived from MDS MSCs and conclude that compact MDS ECM structure induced by TGFβ superfamily members may alter the cytokine environment for HSPCs. Consequently, TGFβ ligand trapping by RAP-536/luspatercept leads to ECM re-organization and thus an improved hematopoietic support. Disclosures Stoelzel: Shire: Consultancy, Other: Travel funding; Neovii: Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

The clinical efficacy of CAR T cell therapies has been widely recognized, particularly in the treatment of hematologic malignancies. Nevertheless, CAR T cells also have the capability to elicit undesired effects such as on-target/off-tumor recognition and cytokine release syndrome. To increase clinical safety of CAR T cell therapy, a novel modular universal CAR platform termed UniCAR was developed by our group. In the UniCAR system, antigen-binding specificity and signaling features are two distinct moieties, in which the antigen specificity is provided by targeting modules (TMs) to redirect UniCAR T cells in an individualized time- and target-dependent manner. In this way, UniCAR T-cells acquire killing potential only in the presence of a tumour-specific TM. Given the reduced size of such molecules, they are rapidly eliminated and therefore, need to be continuously infused. Thus, possible side effects and activation of UniCAR T cells can be easily monitored and controlled by TM dosing. During the onset of therapy, tumor burden and the risk for severe side effects are high and regulation of CAR T cell activity is particularly important at this stage. For this reason, TMs with extended half-life may play an important role by improving eradication of residual tumor cells in late phases of treatment and further expedite clinical application. In this line of thought, a set of novel short-lived and longer-lasting TMs directed against several tumor-associated antigens was developed. Short-lived TMs are composed of a tumor-specific binding moiety fused to the La peptide epitope (E5B9) which is recognized by UniCAR T cells. In order to generate extended half-life TMs, these two components are fused via an Fc domain derived from the human IgG4 molecule. In vitro and in vivo assays have shown that both short-lived and longer-lasting TMs efficiently redirect UniCAR T cells to cancer cells in a highly target-specific manner, thereby promoting the secretion of pro-inflammatory cytokines and tumor cell lysis. Further assays using PET-imaging, demonstrated that all TM formats specifically enriched at the tumor site presenting either short or prolonged serum half-lives. From a clinical point of view, after the initial reduction of tumor burden promoted by the small TMs, IgG4-based TMs could be subsequently administrated allowing a more convenient and personalized treatment of the patients avoiding the continuous infusion of the short-lived TMs. Furthermore, the specific accumulation of such IgG4-based TMs at the tumor site sets these molecules as attractive candidates for in vivo imaging and endoradiotherapy. Taken together, combination of switchable UniCAR T cells and TMs with different sizes, specificities and half-lives represent a flexible and individualized approach at different stages of cancer treatment. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Hematological malignancies are successfully treated with chimeric antigen receptor (CAR) armed T cells. Despite the clinical success, CAR T cell therapy struggles still with some problems including the selection of tumor escape variants and on-target, off-tumor side reactions as well as massive cytokine release and uncontrollability of CAR T cell activity in the patients. In order to enable controllability of CAR T cells and to avoid unspecific side effects, we established a novel switchable, split and adaptable CAR platform technology, termed RevCAR system. The novel RevCARs lack the single chain variable fragment (scFv) commonly used as extracellular domain in conventional CARs. Instead of the scFv, RevCARs contain only a small peptide epitope as extracellular portion. This design reduces the CAR size, avoids unspecific antigen binding and prevents antigen independent tonic signaling caused by scFv dimerization. As RevCAR T cells do not recognize anything, they are per se inert. Only in the presence of a corresponding bispecific antibody based target module (RevTM) they can be specifically redirected to tumor cells. Therefor RevTMs consist of two scFvs. One recognizes the RevCAR peptide epitope and the other one simultaneously binds to a tumor associated antigen (TAA). By dosing of the RevTM, which has a very short half-life, the reactivity of RevCAR T cells can be switched on and off reversibly. Another advantage is that the RevCAR system can be flexibly adapted to any tumor antigen simply by exchanging the RevTM. Furthermore, the small RevCAR size is favorable for inserting more than one RevCAR in the same T cell thus facilitating the mode of gated targeting which is a highly attractive approach to minimize the risk for on-target, off-tumor toxicities against healthy tissues and to increase tumor specificity of conventional CAR T cells. For 'AND' gate targeting via the RevCAR system, two different RevCARs were constructed and expressed simultaneously in the same T cell. The two RevCARs differed with respect to the extracellular peptide epitope and the intracellular signaling domain. Moreover, the respective transmembrane domain was selected to isolate the respective RevCAR signal. The first RevCAR is designed to transmit the activation signal, the second RevCAR to deliver a costimulatory signal. For efficient RevCAR T cell activation, both RevCARs must be engaged via their respective RevTM which on the one hand binds to one of the two RevCAR epitopes and on the other hand to one of two TAAs expressed on the same target cell. Here, we present two RevCAR/RevTM systems for retargeting of AML cells as well as solid tumor cells including via gated targeting. In summary, we show proof of concept for a novel switchable RevCAR system that can be used for retargeting of AML cells as well as solid tumors. The novel modular RevCAR platform is characterized by small size, lacks unwanted tonic signaling effects, allows the control of RevCAR T cell activity, enables gated targeting strategies, and can be adapted to any tumor antigen and tumor type. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Mutations of the key myeloid transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) are found in 5-10% of patients with acute myeloid leukemia (AML). Two mutational clusters exist, in the aminoterminal transcription activation domains (TAD1 or 2) and in the basic leucine zipper domain (bZIP) located at the carboxyterminal-part of the protein. Biallelic mutations (biCEBPA) have been found to be associated with improved outcome and are now included as an independent entity in the WHO-classification. In contrast, monoallelic CEBPA-mutations (moCEBPA) do not appear to provide prognostic information. We characterized a large cohort of AML patients for CEBPA mutations and further analyzed the mutational spectrum of mono- and biallelic CEBPA-mutant AML patients to better understand potential differences in the biology of these groups. Patients and Methods: Patients (including all age groups) analyzed had a newly diagnosed AML and were registered in clinical protocols of the Study Alliance Leukemia (SAL)(AML96, AML2003 or AML60+, SORAML) or the SAL-register. Screening for CEBPA mutations was done using PCR and capillary electrophoresis. All identified CEBPA mutations were confirmed using conventional Sanger sequencing and the samples were further analyzed using next generation sequencing (Trusight Myeloid Panel, Illumina) for the presence of associated alterations. Results: In the 4578 patients analyzed, 228 (5%) with CEBPA-mutations were identified. An initial analysis revealed substantial clinical differences between the different mutation subtypes. Patients with biCEBPA (n=111) were significantly younger (median age 46 yrs) than wt-CEBPA patients (median 57 yrs; p Disclosures Middeke: Sanofi: Honoraria. Platzbecker:Janssen-Cilag: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Thiede:AgenDix: Employment, Other: Ownership.

Introduction The identification of compounds which increase the number but also keep or enhance the activity of hematopoietic stem and progenitor cells (HSPCs) could improve the clinical outcome after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). So far, most attempts to increase HSPC numbers ex vivo have been unsuccessful because of either inadequate cell numbers and/or loss of engraftment capacity and HSPC quality during expansion. Executing drug discovery screens in vertebrate systems is generally expensive, technically challenging and time consuming. Therefore, the zebrafish represents a versatile vertebrate model allowing HSPC regulation and development studies during embryogenesis and adulthood. Methods We used a semi-automated chemical screen to identify modulators of HSPC activity by transgenic (cmyb:EGFP) zebrafish embryos. Verification of identified histone deacetylase (HDAC) inhibitor candidates was carried out in vitro using human CD34+ HSPCs which were isolated from apharesis samples of healthy donors after mobilization with G-CSF by anti-CD34 coupled magnetic beads. The influence of HDAC inhibitors on HSPC phenotype, gene expression pattern as well as adhesion and migration capacity was analyzed after 5 days of treatment either in single or in co-culture with bone marrow-derived mesenchymal stromal cells (MSCs). Results The HDAC inhibitors valproic acid (VPA), resminostat and entinostat were shown to significantly amplify the number of hematopoietic precursors in a chemical in vivo zebrafish embryo screen (Arulmozhivarman et al. 2016). Treatment of human CD34+ HSPCs with these compounds in vitro resulted in a significantly increased percentage of CD34+CD90+ cells up to 60% compared to controls which showed only 2% of double positive cells as well as in 3-fold higher CD34+ and about 12-fold higher CD34+CD90+ absolute cell numbers. CD34 is a well-known surface marker for human immature HSPCs and in combination with CD90 it defines a potentially pluripotent subpopulation. In a co-culture setting, we found that VPA treated cells showed 2 to 3-fold higher attachment capacity on MSCs compared to the control cells. This finding led us to quantify the adhesive capacity of cells using static adhesion assay and atomic force microscopy based single-cell force spectroscopy (AFM-SCFS). Interestingly, detachment forces of VPA treated HSPCs were 3 times increased on MSCs compared to control cells and a similar phenotype was observed by static adhesion assay. Accordingly, the chemokine-mediated migration of VPA treated HSPCs towards SDF-1/CXCL12 was inhibited. To reveal underlying downstream molecules and mechanisms mediating the modified cellular characteristics, a whole genome expression array was carried out for HSPCs treated with VPA in comparison to untreated controls. Amongst a panel of regulated genes, the melanoma cell adhesion molecule (MCAM/CD146), Notch 3 and its downstream effector Hes-1 as well as the SDF-1 receptor CXCR-4 were found to be significantly changed. Whereas the decreased expression of CXCR4 correlates with the inhibited migration potential of VPA-treated HSPCs and Notch-3/Hes-1 have a known role in normal and malignant hematopoiesis (Gu et al. 2016), the induced expression of MCAM on HSPCs was not described so far. The result was confirmed by flow cytometry which revealed a 40% MCAM-positive cell population when treated with VPA, whereas the control showed only negative cells. Additionally, significant higher transcript levels were detected for MCAM by quantitative real-time PCR in VPA expanded cells. Recently, we described a role of MCAM in MSCs for the hematopoietic support (Stopp et al. 2013). The inducible expression in HSPCs may reflect homotypic interactions which preserve a more immature subpopulation with high stem cell activity. Conclusion We describe for the first time the ability of the HDAC inhibitors VPA, resminostat and entinostat to efficiently expand CD34+ HSPCs ex vivo especially supporting a CD34+CD90+ subpopulation with potentially high stem cell activity. Moreover, a potential role of MCAM in this context may offer new perspectives of the HSPC expansion ex vivo for the improvement of HSCT. Disclosures No relevant conflicts of interest to declare.

While adoptive transfer of virus antigen specific T cells has shown to be effective in therapy of resistant recurrent viremia which is frequently associated with the lack of protective immunity following hematopoietic stem cell transplantation, the transfer of leukemia associated antigen specific (LAA) T cells is less implemented and appears to depend on factors that hamper a successful translation into the clinic. Among them are low frequencies and low antigen affinity of LAA specific T cells which currently mandate laborious in vitro expansion protocols. Moreover, screening of healthy individuals with regard to the presence of LAA specific T cells revealed contradictory results. Since we failed to detect LAA specific T cells in healthy donors using single peptide specificities to known LAA epitopes coupled to MHC Streptamers, here we asked if the use of peptide mixes comprising 15mers overlapping by 11 amino acids and spanning the entire LAA protein could elicit in vitro T cell responses in healthy donors, otherwise undetectable by single peptide staining. A cohort of 48 HLA A*0201 healthy individuals was screened using intracellular cytokine staining (ICS) after stimulation with tumor specific peptide mixes representing well known LAAs (WT1, PRAME, NY-ESO, Survivin and p53). While distinct T-helper cell responses were not observed in either of the specimen tested, cytotoxic T lymphocytes could be elicited and measured after incubation with peptide mixes for 5 hours and subsequent CD8+ IFNγ+ staining in 12 out of 48 healthy subjects. Only one individual displayed specifies against multiple antigens (WT1:0,1%; PRAME:0,5%; NY-ESO:0,1%; p53:0.06%), while the remaining responses were directed to one single antigen per individual. Most prevalent and highest T cell frequencies were found against PRAME in 5 out of all screened subjects (mean 0.4±0.3%; max. 0.8%), followed by WT1 in 4 (mean 0.07±0.03%; max. 0.1%) and NY-ESO in 3 individuals (mean 0,07±0,04%; max. 0,1%); one showed CD8 T cells specific against Survivin (0,03%) and 2 individuals had CD8 frequencies specific against p53 (0,05±0,01; max. 0,06%), respectively. The calculated limit of detection (LOD) for the enumeration of LAA specific T cells was 0,02%. In contrary, testing LAA positive individuals with according MHC Streptamers presenting single peptides of previously described epitopes showed no frequencies exceeding LOD. Further analysis showed LAA specific CD8+ IFNγ+ T cells exhibit mainly a less differentiated phenotype (CD45RA+, CCR7+/-, TNFα+, IL-2+/-) and could be immune-magnetically isolated to purities of 94.5±0.7% using a PRAME-specific IFN-γ capture assay yielding 1*104 antigen specific T cells out of 4*107 PBMCs. Simultaneous enrichment of helper T cells to a purity of 73.0±7.6% proofed their existence, despite no CD4+ response could be detected via ICS in the first place. The cytotoxic potential of the cell product was confirmed in an Europium assay using T2 cells loaded with PRAME peptide mix. The specific lysis accounted to 19.3% at an E:T ratio of 1:1 after 90 minutes of co-incubation. In conclusion, using LAA specific peptide mixes in combination with ICS we were able to show a relatively high prevalence of LAA specific T cells, especially for PRAME, in healthy donors. These LAA specific T cells can be enriched without the need of in vitro expansion culturing ex vivo using the IFN-γ capture assay with regard to achieving a functional LAA specific T cell product for adoptive T cell transfer. Furthermore, a less differentiated phenotype exhibited by a large proportion of LAA specific T cells might contribute to their long term survival in a patient after transplantation. Disclosures No relevant conflicts of interest to declare.

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.

Background The aim of this study was to evaluate whether HPC counts measured with the hematology analyzer can predict CD34 + levels in peripheral blood and in the apheresis product, as detected by standard flow cytometry. The main focus was the evaluation of HPC counts in poor mobilizers. Methods Progenitor cell quantification was performed measuring HPC counts provided by the Sysmex XE-2100 hematology analyzer and CD34 + counts obtained in parallel by flow cytometry. Peripheral blood of patients who had received chemotherapy and G-CSF (142 measurements) and healthy donors mobilized with G-CSF alone (106 measurements) was investigated. HPC counts in peripheral blood were also correlated with apheresis yield. Results HPC counts were significantly higher than CD34 + counts (3.5 fold in patients and 1.7 fold in healthy donors, p=0.0015). Our data indicate that HPC counts ≤10/μL in pretreated patients predict a low probability of adequate CD34 + counts in peripheral blood and yields 6 /kg in subsequent aphereses. Furthermore, repetitive low HPC enumerations in an individual were followed by insufficient CD34 + counts in peripheral blood or aphereses in 81% of investigations. In healthy donors low HPC counts (≤10/μL; 12/106 measurements) did not exclusively predict low CD34 + counts (median 23/μL). Discussion HPC counts can be used to schedule the start of CD34 + measurements (threshold>10 HPC/μL) in patients mobilized after chemotherapy for autologous donation. Thus, expensive and time-consuming CD34 + enumerations can perhaps be minimized. HPC measurements cannot completely replace flow cytometric CD34 + enumeration. In particular, healthy stem-cell donors should be monitored with both methods to exclude false negative HPC measurements.

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.

Background ABCB1 (=MDR1, multidrug resistance protein 1) single nucleotide polymorphisms (SNPs) were shown to have a significant impact on therapy outcome in patients with acute myeloid leukemia (AML). Furthermore, an independent significant impact on treatment response and patient survival of SNPs in the genes for ABCC4 (MRP4), ABCC5 (MRP5) and ABCC11 (MRP8) related SNPs has also been demonstrated. In contrast, therapeutic strategies trying to modulate the anthracycline efflux of these transporters have failed in most clinical trials so far. Recently, higher dosages of daunorubicin used during induction chemotherapy have been associated with a better outcome in certain subgroups of AML patients. Hence, in times of individual diagnostic genetic analyses available as point-of-care diagnostics, the goal of this study was to further investigate whether SNPs in ABC-transporter genes, which are responsible for anthracycline efflux, have an independent impact on treatment outcome. Patients and Methods DNA samples were obtained from bone marrow aspirates of 160 Caucasian patients with newly diagnosed AML as part of the prospective AML2003 trial (NCT00180102). The cohort solely consisted of patients with a normal karyotype, based on conventional G-banding, minimizing false results in case of gain or loss of chromosomal material. All patients received double induction chemotherapy with daunorubicin and cytarabine. After DNA extraction, quantitative real time PCR was performed, using a total of 49 SNP assays investigating SNPs of seven different ABC genes. The identification of the corresponding SNPs was performed in an in silico analysis using the NIH dbSNP database and HapMap while statistical univariate and multivariate analyses were performed using SPSS. Results We detected three ABCC1 (MRP1) SNPs: rs129081 (CACCCC[C/G]ACTCCA), rs212090 (TTACTG[A/T]TCCCAC), and rs212091 (ACCTTA[A/G]AGAACA) with a significant influence on disease-free survival (DFS) or overall survival (OS), respectively. Patients carrying the homozygous rs129081 GG-SNP had a significant longer 5-year OS and 5-year DFS compared to the homozygous wildtype CC and heterozygous CG patients (OS: 68% [GG] vs. 40% [CC] vs. 64%, [CG], p=.035; DFS: 64% vs. 35% vs. 50%, p=.01). SNP rs212090 revealed a statistically significant difference in DFS when comparing homozygous alleles TT and AA (wildtype), 40% vs. 68%, p=.021. SNP rs212091 showed a significant difference concerning OS, with homozygous SNP GG leading to worse OS (0% vs. wildtype AA 64% vs. heterozygous AG 59%, p=.006). Again, there was a significant difference in DFS between both homozygous alleles AA (wildtype) and GG (55% vs. 0%, p=.018). Furthermore, there were no significant differences of standard clinical and laboratory baseline characteristics, FLT3-ITD mutation, or NPM1-mutation status, or chemotherapeutic toxicities. In order to exclude false positive findings of SNPs conferred as a result of leukemic transformation, we obtained saliva germline DNA from patients in complete remission who were treated by chemoconsolidation and performed a confirmatory analysis with the investigated SNPs, including rs129081, rs212090, and rs212091. Here, all SNPs were shown to be expressed in germline DNA in remission and bone marrow samples at diagnosis alike. The multivariate models for rs129081, rs212090 (TT), rs212091(AG), and rs212091(AA) revealed significances of p=.024, p=.029, p=.042, and p=.017 respectively for DFS but not for OS (except for rs212091[AA]). After adjustment for a false discovery rate of 5% still a trend towards the association of the SNPs and DFS could be seen. Therefore, more research is necessary to strengthen this evidence. Conclusion In this study we found a significant influence of rs129081, rs212090, and rs212091 SNPs (ABCC1, MRP1) on survival in AML in univariate analyses. Interestingly, these polymorphisms were not associated with other AML specific characteristics at diagnosis and were shown to be expressed in germline DNA and AML DNA alike. Hence, we suggest a prognostic effect of these SNPs which might be responsible for differential anthracycline susceptibility. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 2350 CXCL12 is a chemokine known to be critical for the regulation of the interaction between hematopoietic stem cells (HSCs) and their niche in the bone marrow, e.g. mesenchymal stem cells (MSCs). MicroRNAs (miRNAs) are post-transcriptional regulators recently shown to mediate a variety of cellular processes in the bone marrow niche. However, identification of specific miRNAs and their regulatory role in the crosstalk between HSCs and MSCs are still poorly understood. From a library of 470 miRNAs, 26 miRNAs were shown to downregulate the levels of CXCL12 in the supernatant of the human MSC line SCP-1. Eight of them (miR-23, 130b, 135, 200b, 200c, 216, 222, 602) were chosen for further investigation according to their significant interaction with the 3'UTR of CXCL12 as determined by luciferase assay. Among them, miR-23a,130 and 222 were expressed in 46 human primary MSCs, whereas the other miRs show negligible expression in resting MSCs. However, we observed, that MSCs that underwent adipogenic and osteogenic differentiation showed strongly decreased CXCL12 protein values early (day 5) and at later stages (day 14). The later drop in CXCL12 expression was clearly associated with an increased expression of miR-23a and miR-200. We furthermore tested a subset of stimuli (proinflammatory cytokines, cytotoxic drugs, chemokines) for their ability to modulate the described miRNAs. Amongst them, exclusively the application of transforming growth factor ß1 (TGF-ß1), resulted in the induction of miR-23a and at the same time reduction of CXCL12. The effect was counteracted by transfection of anti-miR-23 molecules. Taken together, we have shown for the first time that CXCL12-targeting miRNAs (in particular miR23a) have a significant potential to regulate the properties of the stem cell niche. Moreover, miR-23 is implicated in the signalling pathway of TGF-ß1 in human MSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 3048 Acute graft-versus-host disease (aGvHD) of the gastrointestinal (GI) tract is still a major clinical challenge after allogeneic stem cell transplantation. Patients with steroid-refractory disease have a poor prognosis. Pentostatin, an inhibitor of adenosine deaminase, has shown efficacy as salvage therapy in steroid-refractory aGvHD of the GI tract in small single center studies. Here we report on the experience with pentostatin in severe steroid-refractory aGvHD of the GI tract at seven German transplant centers. PATIENTS: A total number of 123 patients who had been treated with pentostatin due to intestinal steroid-refractory aGvHD between 2000 and 2011 were retrospectively analyzed. Steroid-refractory aGvHD was defined as progression or no improvement of diarrhea despite treatment with prednisolone (≥ 2mg/kg/d) for ≥ 3 days. Pentostatin was infused at a dose of 1mg/m2 for 3 consecutive days. In patients with impaired renal function the dose of pentostatin was reduced. Patients received 1–4 cycles. Steroids and calcineurin inhibitors (CNI) were continued. Response after therapy with pentostatin was classified as complete (CR, no ongoing symptoms of GvHD), very good partial (VGPR, residual symptoms only) or no response (NR). 50 females and 73 males with a median age of 50 (range: 19–70) years were included. The underlying diseases were AML (n=71), ALL (n=15), CML/MPS (n=6), lymphoma (n=12), MDS (n=10), and multiple myeloma (n=9). 85 patients received reduced intensity and 38 myeloablative conditioning. Patients had been transplanted from matched related (n=38), matched unrelated (n=53) or mismatched donors (n=32). All patients suffered from severe steroid-refractory intestinal aGvHD overall grade III (n=59) or IV (n=64). Patients received pentostatin as first line salvage (n=109) or beyond first line salvage therapy (n=14). Results: 52 patients (43%) responded after salvage therapy with pentostatin. 39 patients (32%) achieved CR, 13 patients (11%) VGPR. Median survival was 104 days; 2-year and long term survival rates were 26 and 19% with a median follow up of 45 months. Among 109 patients who received pentostatin as first line salvage therapy 49 (45%) responded (37 × CR [34%] and 12 × VGPR [11%]). Median survival, 2-year and long term survival were essentially the same as in the total cohort of patients. After the first infusion of pentostatin clinical improvement occurred within a median of 14 (range: 1–58) days. 71 patients (57%) did not respond. Responding patients had a significantly (p Conclusions: The outcome after salvage therapy of III/IV° steroid-refractory intestinal aGvHD with pentostatin is at least within the range as reported for other salvage approaches. In this critical clinical situation pentostatin has some superior characteristics: a sustainable effect, moderate toxicity, easy application and cost-effectiveness. Moreover, this analysis suggests that the outcome of steroid-refractory aGvHD cannot be improved by the application of more than one immunosuppressive salvage drug in addition to steroids and CNI or by second line salvage approaches. Disclosures: Klein: Hospira: Honoraria, Research Funding. Off Label Use: pentostatin is not licensed for use in acute GvHD.

Abstract 3060 Background: Chromosome 5 abnormalities in patients (pts) with acute myeloid leukemia (AML) or advanced myelodysplastic syndrome (MDS) are mostly associated with a poor outcome. Although allogeneic hematopoietic stem cell transplantation (HCT) is the treatment approach with the highest curative potential, early relapse rates following HCT are still considerably high. Since therapeutic options in these pts are often limited, the prevention of relapse represents a major challenge. Lenalidomide (LEN) has been successfully used in MDS pts with del(5q) cytogenetic abnormalities. Besides a direct anti-proliferative effect on del(5q) progenitors, its immunmodulatory function might also enhance T-or NK cell mediated GVL effects. Therefore it could be an effective maintenance drug to prevent relapse after allogeneic HCT in pts with MDS or AML and del(5q) abnormalities. Methods: We report results of a prospective multicenter phase II clinical trial evaluating the efficacy to prevent relapse as well as safety of LEN maintenance following HCT in pts with MDS or AML and cytogenetic abnormalities including del(5q). Only pts achieving a complete hematological remission (CR) after HCT were eligible. In the absence of toxicity or relapse pts could receive up to 12 cycles of LEN maintenance at a dose of 10 mg/day orally, for 21 days, with 7 days rest (28 day cycle). Results: Ten pts with either MDS (n=1, RAEB-1) or AML (n=9) with a median age of 65 years (range 40–72) were included. The disease status prior to allogeneic HCT were CR in 4 pts, relapse/refractory disease in 3 pts, unknown in 2 pts while the MDS pt had not received any prior therapy. A complex aberrant karyotype including del(5q) was documented in 5 pts. Two pts had an additional cytogenetic abnormality besides del(5q) and 3 pts displayed single del(5q). While three pts underwent allogeneic HCT from a matched sibling donor, four pts were transplanted from a matched unrelated and three from a mismatched unrelated donor (single allele or antigen mismatch). Conditioning was of reduced intensity and was followed by the infusion of peripheral blood stem cells in all pts. LEN maintenance therapy was started after complete hematopoietic recovery with documented complete remission at a median of 2.5 months (range 2–4 months) following HCT. After a median of 4 cycles 8 of 10 pts (80%) had to discontinue LEN treatment due to relapse (n=4), development of severe GvHD grade 2–4 (n=2) or other adverse events (n=2). Altogether, 6 of 10 pts (60%) developed severe acute GvHD grade 3–4 within the first 2 cycles of LEN maintenance. All pts were still under systemic immunosuppression at the time of GvHD appearance. Other common adverse events were gastrointestinal side effects (nausea) and myelotoxicity. Reversible neutropenia grade 3/4 was documented in 3 of 10 (30%) of the pts while thrombocytopenia grade 3/4 occurred in 4 of 10 (40%) of them. During the treatment course specific T cell responses against different tumor/leukemia-associated antigens (TAAs/LAAs) using ELISpot analysis for Interferon alpha and granzyme B were measured. Sufficient T cells before and during LEN treatment were only available in one pt, who showed an increase of specific T cell responses against the TAAs/LAAs. With a median follow-up of 254 days (range 32–677) from the start of LEN maintenance, 5 of 10 pts (50%) are currently alive with four pts in continuous CR since the time of HCT. The study was stopped prematurely because of suspected induction of GVHD by LEN. Conclusions: Early LEN maintenance to prevent relapse following HCT in pts with MDS or AML and del(5q) may be associated with the induction of severe acute GVHD. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy. Germing:Celgene: Consultancy, Research Funding. Bornhaeuser:Celgene: Honoraria. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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 3486 Delayed myeloid engraftment after HCT is a risk for increased morbidity and mortality, especially in patients with splenomegaly (SM) at time of transplant. Time to engraftment and overall survival after HCT have not been well analyzed in patients with prior splenectomy (SP) or splenic irradiation (SI), when compared to patients with normal spleens (NS) or with SM. A total of 9,683 recipients with myeloproliferative diseases and/or myelodysplasia who were reported to CIBMTR after receiving a myeloablative allogeneic HCT between 1990 to 2006 were compared according to the spleen status at transplant: 472 SP; 300 SI; 1,471 SM and 7,440 NS. Recipients of cord blood grafts were excluded. The median age was 39 years for all groups, the SP group had a higher proportion of patients with Karnofsky performance score OR d21 WBC1 (95% CI2) p-value OR d28 Platelet3 (95% CI) p-value RR4 Overall Mortality (95% CI) p-value Normal spleen (NS) 1 - 1 - 1 - Splenectomy (SP) 2.25 (1.76–2.89) Disclosures: Maziarz: Millenium: Speakers Bureau; Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 2154 Background: Myeloid Sarcoma (MS) is defined as an extramedullary mass composed of myeloid blasts occurring at an anatomical site other than the bone marrow. Furthermore, the term extramedullary manifestation (EM) is applied if it accompanies overt acute myeloid leukemia (AML) and represents non-effacing tissue infiltration. EM is reported to correspond often to the skin but can affect almost every site of the body. The prognosis of MS or EM has been discussed controversially in the past. EM at diagnosis of AML is generally thought to be a rare event. However, data defining the prevalence of EM at diagnosis of AML and its prognostic value are missing. The aim of this analysis was to provide data for estimating the prevalence of EM at diagnosis of AML and to determine its relevance by including clinical and laboratory data from patients being treated in the prospective AML96 trial of the Study Alliance Leukemia (SAL) study group. Patients and Methods: A total of 326 patients with AML (age 17 – 83 years) and EM were treated within the AML96 trial with a median follow up of 8.8 years (95% CI, 8.4 to 9.3 years). All patients received double induction chemotherapy. Consolidation therapy contained high-dose cytosine arabinoside and for patients ≤ 60 years of age the option of autologous or allogeneic hematopoietic stem cell transplantation (HSCT). Logistic regression analyses were used to identify prognostic variables for CR rates. The method of Kaplan-Meier was used to estimate OS and EFS. Confidence interval (CI) estimation for the survival curves was based on the cumulative hazard function using the Greenwood's formula for the SE estimation. Survival distributions were compared using the log rank test. Results: 17% of the AML patients entered into the AML96 trial were diagnosed with EM. In 313 of the 326 patients (96%) EM was evident at diagnosis. The majority of patients with EM were diagnosed with de novo AML (84%, n=273), whereas gingival infiltration (51%, n=166) displayed the main EM of AML with CNS involvement being less common (4%, n=14). The majority of patients had a cytogenetic intermediate risk profile (71%, n=221) with a total of 172 patients (56%) harboring a normal karyotype. Patients with EM had a statistically significant lower median CD34-positivity of bone marrow blasts, higher percentage of FAB subtypes M4 and M5, higher WBC counts and LDH at diagnosis and higher percentage of NPM1 mutations compared to those patients without EM (all p Conclusions: This analysis represents the largest study so far investigating the impact of EM AML. Patients with EM AML have distinct differences from AML patients without EM regarding their clinical and molecular characteristics at diagnosis. However these differences do not translate into differences in response to induction chemotherapy. Compared to patients without EM, survival analysis revealed differences according to the NPM1/FLT3-ITD mutation status which is also described for patients without EM AML. However, the prognosis for patients with EM who harbor a mutated NPM1 the prognosis at relapse seems to be dismal. Disclosures: No relevant conflicts of interest to declare.

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.

Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became clear that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD34 purified HSC expansion and differentiation demonstrating a promoting impact of MSC on adherent HSC in comparison to non adherent HSC in terms of phenotype, migration capacity and clonogenicity. Performing phase contrast microscopy and confocal microscopy we are able to distinguish HSC which are located on the surface of a MSC monolayer (phase-bright cells) and HSC which are covered by MSC monolayer (phase-dim cells). Both HSC fractions and the non-adherent cells were isolated separately by performing serial washing steps. All three fractions were analyzed at fixed time points during the first week of co-culture in term of cell cycle progression, proliferation, maturation and cell division accompanied differentiation. First we performed propidium iodide (PI) staining for cell cycle analysis revealing that the phase-bright cells contained the highest percentage of G2 cells in comparison to the non adherent cells and the phase-dim cells; 13.9 ±1.0% vs 1.3 ±1.2% vs 2.7 ±2.0%, p

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.

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.

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

Hypoxia increases IL-8 secretion of mesenchymal stroma cells affecting migratory capacity in an autocrine manner Manja Wobus, Katrin Müller, Gerhard Ehninger, Martin Bornhäuser Department of Hematology/Oncology, University Hospital Dresden, Fetscherstr. 74, 01307 Dresden, Germany Adult bone marrow-derived stem cells represent an important source of cells for regeneration and repair of a number of damaged tissues. Mesenchymal stromal cells (MSCs) give rise to the cellular components of the bone marrow microenvironment and support expansion and differentiation of hematopoeitic stem cells in the respective niche. Low oxygen tension is thought to be an integral component of the endosteal niche microenvironment. When used for therapeutic purposes, MSCs cultured in standard conditions must adapt from 21% oxygen to less than 1% oxygen in the ischemic tissue. Understanding the mechanisms by which the production of cytokines and growth factors by MSCs is regulated may represent an important way to optimize their beneficial paracrine and autocrine effects. Human primary MSCs were incubated under normoxic (20% O2) and hypoxic (0.5% O2) conditions over five days and the pattern of released cytokines in the cell culture supernatants was compared using a human cytokine array (R & D Systems). Amongst others, we found upregulated IL-8 levels under hypoxic conditions leading us to further investigation of IL-8 expression in MSCs and its role for in-vitro migration. As expected, IL-8 mRNA levels were significantly higher in hypoxic MSCs. The result of the cytokine array was confirmed by examination of secreted IL-8 in the cell culture supernatant by ELISA (PeproTech). The migration capacity was investigated in a 24-well transwell chamber assay with 8 μm pore size. Using recombinant IL-8 as a chemoattractant in the lower chamber, we detected an almost twofold enhanced MSC migration rate after 24 hours under hypoxic conditions. As a different approach to investigate the migratory capacity, we used an in-vitro scratch assay. A wound was applied to a MSC monolayer in absence or presence of IL-8 which clearly enhanced the migration of MSCs into the wound area after 24 hours. In summary, IL-8 secretion by human primary MSCs is clearly increased under hypoxic conditions. IL-8 in turn seems to be a chemotactic factor for MSCs and enhances their migratory capacity in an autocrine manner.

MicroRNAs (miRNAs) can regulate hematopoietic stem/progenitor cells (HSPC) by modulation of intrinsic cell components such as transcription factors and receptors. In addition, miRNAs could play a role in the microenvironment were HSPC host and consequently affect HSPC via extrinsic factors, which to our knowledge is an hypothesis that has not been tested. Since the chemokine stromal derived factor 1alpha (SDF-1alpha) is essential for both homing and retention of HSPC in the bone marrow, we tested if miRNAs expressed by human bone marrow-derived mesenchymal stem cells (MSC; a key source of SDF-1alpha), could potentially inhibit SDF-1alpha expression. In deed, using luciferase reporter-systems we show specific binding of miR-23a to the 3′UTR of SDF-1alpha. Consequently, transfection of MSCs with a precursor miR-23a (pre-miR-23a) leads to a 30% reduction of SDF-1alpha at both mRNA and protein levels. In contrast, inhibition of endogenous miR-23a with anti-sense oligonucleotides (anti-miR-23a), leads to a significant increase of SDF-1alpha also at both mRNA (30%) and protein (10%) levels as compared to controls (scramble pre-/anti-miR). As a result, migration of CD34+ HSPC in transwell assays is strongly affected upon overexpression or inhibition of miR-23a in MSCs (with pre-miR-23a 35% less migration; with anti-miR-23a 20% more migration, as compared to respective controls). Interestingly, transforming growth factor beta 1 (TGF-beta1) inhibits SDF-1alpha expression in MSCs in a concentration dependent manner, while miR-23a is increased under same experimental settings. Even more, silencing endogenous miR-23a significantly reduces the effect of TGF-beta1 on SDF-1alpha mRNA and protein levels, suggesting that at least in part TGF-beta1 inhibits SDF-1alpha expression via increasing miR-23a levels. This is to our knowledge the first established connection between miRNA biology and HSPC-niche related factors.

Objectives: Chronic myeloid leukemia (CML) is effectively treated by Imatinib (IM) via inhibition of the BCR-ABL tyrosine kinase. However, also related tyrosine kinases like abl, c-Kit, PDGF-R, and c-FMS are blocked by IM. As shown in adult humans and mice, abl-controlled protein folding as part of the endoplasmatic stress response in heart myoblasts as well as bone “remodeling” depending on PDGF-R and c-FMS is impaired under imatinib exposure (Dewar AL et al 2005, Kerkelä R et al 2006, Fitter S et al 2008). The influence of IM on the growing heart and skeleton of immature animals has not been studied so far. With respect to treatment of pediatric CML we report alterations in these organs of juvenile mice chronically exposed to IM during the growth period. Methods: From the age of 4–14 weeks (w) [development milestones of mice: weaning 3 w; puberty 7 w; epiphysial lines closure 18 w] C3H/Neu male and female wild-type mice were chronically exposed to IM via the drinking water at concentrations of 500 mg/l (group A), 750 mg/l (group B), and 1000mg/l (group C). Femur length and overall skeletal development was analysed by whole body X-ray analysis using a mammography device. Bone metabolic activity was assessed by total body Na18F PET and CT after 5w and 10w of exposure using dedicated small animal tomographs. Bone mineral density and microstructure of tibiae were analysed by pQCT and microCT (resolution 12.5μ m) while the number of osteoclasts and resorption lacunae in femora and vertebrae was assessed by histomorphometry. Plasma concentration of IM, osteocalcin, and activity of the tartrate resistant acid phosphatase (TRAP5b) was also determined. The heart was examined histologically and ultrastructurally by electron microscopy. Results: IM was tolerated well and mean uptake of 80 mg/kg/d 110 mg/kg/d and 150 mg/kg/d resulted in serum levels of 60–674 ng/ml, 36–242 ng/ml and 51–534 ng/ml, respectively. Body weight gain was delayed in groups B and C until the age of 8 w while no change in overall growth, development and behaviour was observed at 14 w. At higher doses of IM and at younger age there was a non-significant trend to a reduction in femur length. Heart morphological examination exhibited an increased number (p Conclusion: In juvenile mice, a chronic exposure of IM resulted in toxic damage of the cardiomyocytes at higher dose rates. However, these alterations do not necessarily imply also a functional impairment which can only be studied in vivo. In the skeleton, IM reduced the number of osteoclasts and resorption lacunae in long bones but not in vertebrae. IM showed an antiresorptive effect in cancellous bone and increased cortical thickness and trabecular number by inhibiting the expansion of the marrow cavity. The effects were more pronounced in male mice and at younger age.

Introduction: 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 upfront allogeneic HSCT for remission induction as part of induction therapy has the potential to circumvent these problems and might furthermore reduce cumulative toxicity in high-risk patients. Therefore, in 2003 we started a prospective multicenter randomized trial that investigates both the feasibility and efficacy of upfront allogeneic stem cell transplantation for remission induction in high-risk AML patients. Methods: The AML2003 study compares in a randomized fashion an intensified treatment approach using upfront allogeneic transplantation in high risk patients as part of the induction therapy (IT) during marrow aplasia achieved by DA (daunorubicin 60 mg/ m2 – day 3–5; cytarabine 100 mg/m2 – day1–7) to a “conventional” treatment strategy, which allows for allogeneic transplantation only in patients achieving remission after two induction courses (DA). To do this, rapid analysis of cytogenetics, FLT3 status and HLA-DNA-typing of the patient and possible family donors is of utmost importance. This “fast search diagnostics” together with routine analyses of morphology and immunophenotyping is accomplished centrally in all enclosed patients. The dose-reduced preparative regimen for upfront allogeneic stem cell transplantation within induction therapy consisted of melphalan 150mg/m2 and fludarabine 150mg/m2. Results: Until the last update we recruited 679 patients Conclusions: These preliminary results show that rapid risk profiling and fast donor-search is feasible in a large multi-center study. This leads to a significant proportion of upfront allogeneic stem cell transplants as part of the induction therapy within the group of high-risk AML patients, which may improve the disastrous prognosis of this group of patients in the future.

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.

FLT3-mutations are among the most common abnormalities in adult acute myeloid leukemia. These mutations induce constitutive activation of the receptor and downstream signaling pathways, including RAS/ERK, PI3K and STAT5-signalling. The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with inferior prognosis. We have recently demonstrated by microarray analysis that leukemic samples of patients with FLT3-ITD mutations have significantly upregulated expression levels of Autotaxin (ATX). The ATX protein acts as a secreted lysophospholipase D (lysoPLD) through generating lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). LPA has several important functions in cell migration and proliferation and acts via G-protein coupled receptors. It has been shown that ATX represents an aberrantly expressed motility and growth factor in a variety of cancer cells. However data on ATX in myeloid leukemias and especially in AML are missing. To study more deeply the role of ATX in leukemogenesis, we screened a series of human leukemic model cell lines for ATX expression. Using retroviral transduction, two alternatively spliced transcripts of ATX were expressed in several human leukemic cell lines without detectable levels of endogenous ATX. To investigate the ATX function and expression in normal haematopoiesis we used CD34+ human progenitor cells. The expression of ATX transcripts was confirmed at both mRNA and protein levels by RT-PCR and Western blotting, respectively. Transwell migration assays in the presence of LPC or LPA were performed to study effects of ATX on cell motility. Proliferation and clonogenic potential were investigated using MTT and colony forming assays. Moreover, we examined the effect of the FLT3 inhibitor N-benzoylstaurosporine (PKC412) on ATX expression and ATX mediated migration in MV4-11 cells. High ATX expression was found primarily in malignant cells. Western blot analysis showed that detectable levels of ATX are secreted into medium within 2 hours. In normal cells, highest ATX mRNA expression was found in purified CD34 cells, but could also be found at lower levels in T-cells. Stable overexpression of FLT3-ITD in OCI-AML3 cells induced an increased ATX expression (up to 6 fold). Vice versa, inhibition of FLT3-ITD by sublethal doses of PKC412 in MV4-11 cells resulted in a significant reduction of ATX expression down to 10% of the initial expression level. Furthermore, PKC412 treatment resulted in a complete loss of LPC or LPA induced specific migratory capacity in MV4-11 cells. The transduction of ATX increased the colony-forming capacity by 75% and significantly increased the short term proliferation. LPA increased chemotaxis in human leukemic cell lines and human CD34+ progenitors in a dose dependent manner and induced significantly higher migratory rates by at least 50%. LPC induced chemotaxis by 80–200% only in cells with high expression of endogenous or exogenous ATX, demonstrating the autocrine activity of ATX. Ongoing studies on the mechanism of ATX expression showed that ionomycin, an activator of the Ca2+–calcineurin–NFAT signalling pathway, upregulated ATX mRNA in several leukemic cell lines as well as in human primary progenitors up to 5-fold, which could be completely blocked by cyclosporine A treatment, indicating an involvement of NFAT in the regulation of ATX. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration of hematopoietic stem cells and its deregulation may contribute to the pathogenesis of AML, especially in patients with FLT3-ITD mutations.

Notch, expressed on hematopoietic progenitors plays a crucial role in hematopoiesis. Mesenchymal stem cells (MSC) express both, Notch and its ligand Jagged and are known to support self renewal of hematopoietic progenitors via cell-cell contact and cytokine secretion. The Jagged/Notch signaling pathway has been implicated in the differentiation process of MSC, however it is not completely understood and current observations are contradictory. In order to analyze the effect of Notch signaling on human MSC differentiation we constructed lentiviral vectors that contained either the GFP-marker gene, hJagged1 IRES GFP, hNotch1 intracellular domain (NICD) IRES GFP or a gene fusion between dominant negative Mastermind1 (MAML1dn - inhibitor of Notch signaling) and the Cherry reporter gene. Primary hMSC that were obtained from bone marrow of 3 different donors were transduced with respective lentivirus vectors to greater than 98%. After exposure to adipogenic and osteogenic differentiation stimuli hMSC differentiation was quantified by Alizarin red or oil red staining, alkaline phosphatase (AP) activity and expression levels of adipogenic or osteogenic markers by Real-time PCR. Jagged1 transduced hMSC demonstrated enhanced calcium phosphate deposits and enhanced AP activity and expression levels in osteogenic differentiation medium, while adipogenic differentiation was strongly inhibited as quantified by oil red staining and low mRNA expression of genes upregulated during adipogenic differentiation (pprY, Fabp4). Similarly, overexpression of NICD induced strong and rapid osteogenic differentiation while inhibiting adipogenic differentiation and reducing cell viability. Moreover, NICD overexpression upregulates the expression of endogenous Jagged1 up to 5-fold. Inhibition of Notch signaling via overexpression of MAML1dn partially blocked the effect of hJagged1 and NICD in co-transduction experiments. In another approach MSC samples obtained from 20 donors with various osteogenic differentiation potential as measured by AP activity were analyzed for Notch1 and Jagged1 expression. While there was no correlation between AP activity and Notch1 levels we observed a significant positive correlation for AP activity and Jagged1 expression. In summary, our data strongly suggest that increased Jagged/Notch signaling enhances the osteogenic differentiation of hMSC while inhibiting their adipogenic fate.

NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a potential curative therapy to patients with advanced NHL. RIC HCT induces potent graft-versus-lymphoma effects with best results in patients with low tumor burden at time of HCT. Combined use of radioimmunotherapy (RIT) with RIC may increase anti-lymphoma activity of RIC while HCT provides rescue from hematologic toxicity of RIT. This may allow dose escalation of RIT. A multicenter phase I/II study of allogeneic HCT combining RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20) with two RIC regimens for treatment of patients with NHL has been initiated. Patients with indolent NHL (Arm A) receive RIT with Y90-CD20 (0.4 mCi/kg) on day −14 combined with RIC using fludarabine (30 mg/m^2 day −4 to−2) and 2 Gy TBI (day 0). Patients with aggressive NHL (Arm B) receive an escalated dose of Y90-CD20 (0,6–0,8 mCi/kg) on day −14 combined with RIC using fludarabine (30 mg/m^2 day −8 to−4), melphalan (140 mg/m^2 day −3) and campath (20–30 mg day −3 to−2). For postgrafting immunosuppression either CSA/MMF (Arm A) or CSA alone (Arm B) is used. To date, 31 patients have been enrolled (Arm A=23, Arm B=8). Diagnoses in Arm A were FL (n=12), MCL (n=6), CLL (n=4) and Immunocytoma (n=1). Diagnoses in Arm B were DLBCL (n=6), blastoid MCL (n=1) and transformed CLL (n=1). Median age was 55 (range, 34–67) years. PBSC grafts were either from matched related (n=10) or matched unrelated donors (n=21). All patients were high risk with refractory disease or relapse after preceding HCT. Disease status after salvage therapy at time of HCT was in Arm A: CR=1, PR=18, SD=4 and in Arm B: PR=8. No additional toxicity due to RIT was observed. Engraftment was rapid and sustained with no graft rejections. In Arm A median time to >500 granulocytes/μL was 13 (range, 0–69) days and to >20000 platelets/μL 3 (range, 0–69) days (in 11 patients platelets never dropped 500 granulocytes/μL was 17 (range, 9–23) days and to >20000 platelets/μL 11 (range, 8–29) days. TRM in the first 100 days was 3%, overall 19%. Incidence of grade II-IV GVHD in Arm A was 35% (II=3, III=4, IV=1) and in Arm B 25% (II=2). Best disease response observed was in Arm A: CR=18, PR=5 and in Arm B: CR=3, PR=5. To date, 16/23 patients in Arm A and 6/8 patients in Arm B are alive with a median follow-up of 271 (range, 20–390) days, resulting in a Kaplan-Meier 1 year survival estimate of 65% in Arm A and 62% in Arm B. Causes of death were infection=5, GVHD=1, relapse=1 in Arm A and relapse=2 in Arm B. A combination of RIT with RIC is feasible with no additional toxicity due to RIT and stable engraftment in all patients. Preliminary response data suggest that this strategy may improve early post-transplant disease control, but long-term disease-free survival remains to be determined.

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.