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Background TP53 mutations are associated with adverse outcome of AML treated with cytarabine-based regimens. Interestingly, DNA-hypomethylating agents (HMAs) induce a higher response rate in TP53-mutated (MUT) compared to TP53 wildtype (WT) AML (Welch et al., N. Engl. J. Med. 2016, Döhner et al., Leukemia 2018). We conducted a randomized phase II trial (NCT00867672, 2x2 factorial design) asking whether the in vitro cooperativity of DAC with VPA or ATRA translates into clinical benefit. While VPA added to DAC affected neither objective response rate (ORR) nor overall survival (OS), ATRA significantly improved ORR and OS, without added toxicity (Lübbert et al., J. Clin. Oncol. 2020). Preclinical data suggest that HMAs and ATRA have cooperative effects also in TP53 MUT AML. We therefore performed a post-hoc analysis to determine the predictive impact of TP53 status. Patients and Methods Key inclusion criteria: newly diagnosed AML pts >60 yr (non-M3) unfit for induction, ECOG performance status (PS) 0-2. Treatment: DAC 20 mg/m 2 day 1-5 (arms A/B/C/D), VPA p.o. from day 6 (arms B/D), ATRA p.o. day 6-28 (arms C/D) of each 28-day course. For TP53 mutation analyses, the Illumina TruSight Myeloid Sequencing Panel was used for library preparation and an Illumina MiSeq device for sequencing. Key endpoints: ORR (CR/CRi/PR, ELN 2010 criteria) and OS. Original sample size calculation of a total of 200 patients (pts) was based on the primary endpoint ORR (Lübbert et al., Haematologica 2012). ORR was analyzed with logistic regression, OS with Cox regression. Odds ratios (OR) for the effect on ORR and hazard ratios (HR) for the effect on death with 95% confidence intervals (CI) are presented in the genetic subgroups TP53 MUT and TP53 WT including tests for interactions (TFI) between treatment and TP53. These are post-hoc exploratory analyses, hence p-values have to be interpreted in a descriptive sense. Results Between 12/2011 and 2/2015, 200 pts were randomized and treated. Information on TP53 status was available for 168 of 200 pts (84%); 155 of them (92%) had died at last follow-up (June 2021). 61% of pts were aged >75 years (range 61-92), ECOG PS 0/1/2: 19/62/19% (a single pt had a PS of 3); 53% had an HCT-CI >3, 19% WBC >30.000/µl, 30% adverse genetics (ELN 2010), 51% an antecedent hematologic disorder. TP53 aberrations were detected in 42 pts (25%), with 1 (n=27) or 2 mutations (n=12, median variant allele frequency 44%, range, 1.3-99%) in 39 pts, and TP53 deletions in 3 additional pts. The 42 pts with TP53 MUT showed a higher ORR (23.8%) than the 126 pts with TP53 WT (ORR 15.1%), with an OR of 2.04 (95% CI 0.83-4.98), p=0.12. OS (irrespective of treatment) in the TP53 MUT v WT pts was not different (HR, adjusted for treatment: 1.14 [95% CI 0.78-1.66], p=0.51; Fig. A). In both genetic groups, the addition of ATRA had a favorable effect on ORR (ATRA v no ATRA in TP53 MUT: 31.3% v 19.2%, OR 1.91 [95% CI 0.45-8.03]; ATRA v no ATRA in TP53 WT: 18.8% v 10.5%, OR 1.98 [95% CI 0.70-5.61]), TFI p=0.97 (Fig. B). A positive effect of ATRA on OS in both genetic groups was reflected by a median OS of 6.0 v 4.5 months (ATRA v no ATRA in TP53 MUT: HR 0.75 [95% CI 0.38-1.48]), and a median OS of 8.9 v 4.7 months (ATRA v no ATRA in TP53 WT: HR 0.58 [95% CI 0.39-0.86], all results adjusted for VPA, ECOG, HCT-CI, sLDH, Hb), TFI p=0.49 (Fig. C). VPA did not affect ORR in either of the 2 genetic groups (VPA v no VPA in TP53 MUT: 21.7% v 26.3%, OR 0.76 [95% CI 0.18-3.21]; VPA v no VPA in TP53 WT: 16.7% v 13.3%, OR 1.34 [95% CI 0.5-3.61]), TFI p=0.53. The impact of VPA on OS differed between TP53 MUT pts (VPA v no VPA: median OS of 4.2 v 5.3 months, HR 1.31 [95% CI 0.69-2.48], and TP53 WT pts (VPA v no VPA: median OS of 8.4 v 4.8 months, HR 0.67 [95% CI 0.46-0.99], all results adjusted for ATRA, ECOG, HCT-CI, sLDH, Hb; TFI p=0.08, Fig. C). Conclusions Our results confirm the reported higher response rate to DAC in pts with TP53 MUT compared to TP53 WT; the addition of ATRA led to a higher ORR. Improved OS with ATRA was observed particularly in TP53 WT pts. In contrast, VPA did not affect the ORR in either genetic group; TP53 WT pts may benefit from VPA regarding OS. Our exploratory post-hoc results need confirmation in other trials, e.g. in the DECIDER-2 study (adding ATRA or placebo to the recently approved dual treatment of a HMA combined with venetoclax). Cooperative effects of HMAs and retinoids deserve a deeper mechanistic understanding, which may have implications not only for AML but also for other malignancies with impaired TP53. Figure 1 Figure 1. Disclosures Becker: BMS: Honoraria; Pierre Fabre Pharma: Honoraria; Servier: Honoraria; MSD: Honoraria; Novartis: Honoraria. Crysandt: Incyte: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees. Thol: Abbvie: Honoraria; Pfizer: Honoraria; Astellas: Honoraria; Novartis: Honoraria; BMS/Celgene: Honoraria, Research Funding; Jazz: Honoraria. Heuser: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; Astellas: Research Funding. Goetze: Abbvie: Other: Advisory Board; BMS/Celgene: Other: Advisory Board, Research Funding. Schlenk: Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Abbvie: Honoraria; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Salih: Synimmune GmbH: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria; BMS: Honoraria. Schittenhelm: Takeda: Other: advisory board; Astellas: Other: advisory board; BMS: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Müller-Tidow: Pfizer: Research Funding; Janssen: Consultancy, Research Funding; Bioline: Research Funding. Germing: Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding. Giagounidis: Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Wäsch: Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; BMS/Celgene: Consultancy; Gilead: Consultancy. Döhner: Jazz Roche: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Other: Advisory Board; Agios and Astex: Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Honoraria, Other: Advisory Board; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Ganser: Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria. Döhner: Astellas: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Oxford Biomedicals: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Helsinn: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Berlin-Chemie: Consultancy, Honoraria; Astex: Consultancy, Honoraria; Agios: Consultancy, Honoraria, Research Funding; Ulm University Hospital: Current Employment; Jazz: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; GEMoaB: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding. Hackanson: Roche: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria; MSD: Consultancy, Honoraria. Lübbert: Cheplapharm: Other: study drug (ATRA); TEVA: Other: study drug (valproic acid); Janssen: Consultancy, Other: study drug (decitabine), Research Funding; Syros: Consultancy, Honoraria; Aristopharm: Other: study drug ; Imago BioSciences: Consultancy, Other: study support with study drug; AbbVie: Consultancy, Honoraria; Astex: Consultancy, Honoraria. OffLabel Disclosure: ATRA, in non-M3 AML valproic acid, in non-M3 AML

Background: Monitoring of measurable residual disease (MRD) in patients (pts) with advanced myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) who achieve complete remission (CR) can predict hematological relapse. Recently published data from the first cohort of the RELAZA2-trial have shown that pre-emptive therapy with azacitidine (AZA) can prevent or substantially delay an overt relapse in MRD-positive pts with MDS or AML (Platzbecker et al. Lancet Oncol. 2018). Aims: To evaluate outcome of the entire patient cohort of the RELAZA2-trial and determine whether MRD-guided pre-emptive AZA treatment could prevent relapse in molecularly defined cohorts. Methods: Between 12/2011 and 07/2018 380 pts with advanced MDS or AML, who had achieved CR after conventional chemotherapy or allogeneic hematopoietic stem-cell transplantation (allo-HCT) were prospectively screened for MRD in monthly intervals either in bone marrow (BM) or peripheral blood (PB). A total of 94 pts (AML, n=83; MDS, n=11) became MRD positive during 24 months from baseline by either quantitative PCR (qPCR) or analysis of CD34+ donor-chimerism and entered the treatment phase. Preemptive MRD-triggered treatment consisted of AZA 75 mg/m2 per day subcutaneously on days 1-7 of a 29-day cycle for up to 24 cycles. After six cycles, MRD status was reassessed and pts with MRD negativity were eligible for treatment de-escalation. Primary endpoint was relapse-free survival (RFS) six months after start of pre-emptive treatment. For mutational analysis next generation sequencing (NGS) with a panel of 54 genes was performed (Illumina Trusight Myeloid). Results: Median age was 60 yrs (range: 22-80 yrs); 52 (55%) of the pts were female. Prior therapy consisted of chemotherapy in 42 (45%) and allo-HCT in 52 (55%) of the pts. Cytogenetics could be analyzed in 93 (99%) of the 94 pts. Risk categorization according to ELN 2017 was favorable in 30 (37%), intermediate in 31 (38%) and adverse in 21 (26%) of the AML pts, respectively. Type of MDS was advanced in all 11 pts and all were previously transplanted. Fifty-two (61%) of 85 pts with available NPM1 status were positive. NGS on 64 (68%) pts with available DNA at the time of first diagnosis revealed additional mutations in DNMT3A (n=25), TET2 (n=15), FLT3-ITD (n=12), IDH1 (n=9), FLT3-TKD (n=8), ASXL1, NRAS, TP53 (n=7, each), IDH2 (n=6), PTPN11, WT1 (n=5, each), GATA2, U2AF1 (n=4, each), CBL (n=3), CEBPA, CSFR3, CUX1, EZH2, KIT, RAD21, RUNX1, SF3B, STAG2, ZRSR2 (n=2, each), and KRAS (n=1). MRD data were correlated with outcome in 45 pts for NPM1, in 3 for RUNX1-RUNX1T1, whereas CD34-donor-chimerism was analyzed in 39 pts (missing, n=7). There was a significant faster and deeper decline of MRD in PB as compared to BM (P=0.03). The same held true with regard to the increase of donor-chimerism, which was achieved faster in PB as compared to BM (P=0.05). Secondary molecular abnormalities (MAs) had no impact on MRD response as measured by qPCR, which was also true if MAs were categorized functionally. Similarly, additional chromosomal abnormalities had no impact on MRD response in both MRD methods. However, in pts with measurement of donor-chimerism ASXL1 mutations were a negative factor for MRD response (P Conclusions: AZA as a pre-emptive therapy was effective in delaying hematological relapse of advanced MDS or AML pts, regardless of the underlying genetic signature. Based on these encouraging results, intensifying treatment with AZA in combination with pembrolizumab is currently investigated as MRD-guided treatment in NPM1 positive AML (PEMAZA; ClinicalTrials.gov Identifier: NCT03769532). Disclosures Wolf: Celgene: Honoraria, Research Funding. Bug:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hexal: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Eurocept: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz: Honoraria; Neovii: Other: Travel; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Sanofi: Other: Travel. Götze:Celgene: Research Funding. Stelljes:Amgen: Consultancy, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Subklewe:Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Seattle Genetics: Research Funding; Morphosys: Research Funding; Janssen: Consultancy; AMGEN: Consultancy, Honoraria, Research Funding; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding. Haenel:Amgen, Novartis, Roche, Celgene, Takeda, Bayer: Honoraria. Rollig:Amgen, Astellas, BMS, Daiichi Sankyo, Janssen, Roche: Consultancy; Abbvie, Novartis, Pfizer: Consultancy, Research Funding. Müller-Tidow:Pfizer: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Geron: Consultancy, Honoraria. Thiede:AgenDix GmbH: Other: Co-owner and CEO. OffLabel Disclosure: Off-label: treatment with azacitidine to prevent or substantially delay an overt relapse in MRD-positive patients with MDS or AML

Background: DNA-hypomethylating agents are providing a very well-accepted backbone for non-intensive combination treatment of AML/MDS patients (pts), and an in vivo synergism has been demonstrated for the azacitidine+venetoclax combination in the VIALE-A trial (DiNardo et al., EHA 2020). The DAC+ATRA combination also resulted in an improved response rate and survival compared to DAC without ATRA (DECIDER trial, Lübbert et al., J. Clin. Oncol. 2020), also in pts with prior hematologic disorder (mostly MDS); no benefit was seen when valproic acid (VPA) was added to DAC (2x2 factorial design). In a previous study, we had investigated the outcome of elderly pts with oligoblastic AML (i.e. with 20-30% bone marrow blasts, defined as MDS RAEBt according to the French-American-British classification) treated with either DAC or best supportive care within the EORTC 06011 phase III trial (Becker et al., Ann. Hematol. 2015), observing a median overall survival (OS) of 8.0 months (mths) in DAC-treated RAEBt pts. We now hypothesized that the outcome of pts with oligoblastic AML may be improved by the addition of ATRA to DAC. Therefore, in the present exploratory subgroup analysis, pts from the DECIDER cohort with 20-30% bone marrow blasts were analyzed for clinical outcome. Patients and Methods: Key inclusion criteria: newly diagnosed pts >60 years (yr), unfit for induction with non-M3 AML (WHO, de novo or after antecedent hematologic disorder [AHD], therapy-associated [t]AML), ECOG performance status (PS) 0-2. Treatment: DAC 20 mg/m2 day 1-5 (treatment arms A/B/C/D), ATRA p.o. day 6-28 (arms C/D), VPA p.o. continuously from day 6 (arms B/D), of each 28-day course (repeated until relapse/progression, prohibitive toxicity, withdrawal or death). Key endpoints: objective response rate (ORR): CR/CRi/PR, overall (OS) and event-free survival (EFS). Sample size calculation was based on the primary endpoint ORR, assuming an ORR of 25% in arm A (Lübbert et al., Haematologica 2012). For a power of 80% (test in this phase II study at 1-sided alpha=0.1) for an increase of ORR to 40% with ATRA or VPA, 176 pts were necessary, planned sample size 200. Between 12/2011 and 2/2015, 200 pts were randomized and treated. Efficacy analyses were performed in the intention-to-treat (ITT) population. ATRA was investigated by comparing arms C+D vs arms A+B, VPA by comparing arms B+D vs arms A+C, ORR was analyzed with logistic regression estimating odds ratios (OR), OS/EFS with Cox regression estimating hazard ratios (HR), each with 95% confidence intervals (CI), and presented with descriptive two-sided p values of the tests of no treatment effect. Central hematopathologic review (blinded as to treatment arms) was conducted by an independent morphologist. Results: In 56/200 pts of the DECIDER cohort, bone marrow blasts were 20-30% (median, 25%). The number of pts in the randomized arms were: 13 in arm A, 21 in arm B, 9 in arm C, 13 in arm D. Baseline pt characteristics were as follows: male 77%, median age: 75 yr (range 61-88), median WBC: 3400/µl (range 500-52,600), adverse genetics (ELN 2010) present in 25%, ECOG 2 in 13%, comorbidities (HCT-CI) ≥ 3 in 48%, AHD in 68%, tAML in 11% (only slight random imbalances across randomized treatment arms). A median of 5 DAC courses were administered (per arm: 2/5/11/4). Six pts attained a CR, 7 pts a CRi, and 1 pt a PR, resulting in an ORR of 25% (arm A: 7.7%, arm B: 28.6%, arm C: 33.3%, arm D: 30.8%, respectively). Effect on ORR of ATRA vs no ATRA (31.8 vs 20.6%): OR 1.85, CI [0.54,6.37], p=0.33; and of VPA vs no VPA (29.4 vs 18.2%): OR 1.93, CI [0.51,7.24], p=0.33. With 40 deaths out of 56 pts, median OS was 9.5 mths (arm A: 7.6 mths, arm B: 8.9 mths, arm C: 37.2 mths, arm D: 11.2 mths, respectively). Effect on OS of ATRA vs no ATRA (12.5 vs 7.6 mths median OS): HR 0.47, CI [0.24,0.94], p=0.032 (after adjustment for PS, HCT-CI, WBC, LDH, genetic risk: HR 0.42, CI [0.19,0.90], p=0.025); and of VPA vs no VPA (10.0 vs 8.4 mths median OS): HR 0.99, CI [0.51,1.92], p=0.98: A comparable benefit on EFS of ATRA vs no ATRA (but not VPA vs no VPA) was observed. Conclusion: In elderly pts with oligoblastic AML ineligible for induction chemotherapy, the addition of ATRA, but not VPA, to DAC resulted in a clinically meaningful survival benefit; OS of pts receiving DAC without ATRA was very similar to that observed in a previous study. It is tempting to speculate that the combination of an HMA with a retinoid such as ATRA may also be active in MDS pts with excess of blasts. Disclosures Jost: JAZZ: Other: travel support; Roche: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: travel support. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Heuser:Amgen: Research Funding; Bayer: Consultancy, Research Funding; BerGenBio ASA: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Stemline Therapeutics: Consultancy; Janssen: Consultancy; PriME Oncology: Honoraria; Karyopharm: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy; Astellas: Research Funding; Roche: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Götze:Celgene: Research Funding. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Roche: Research Funding; AstraZeneca: Research Funding; PharmaMar: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Döhner:Sunesis Pharmaceuticals: Research Funding; Abbvie: Consultancy; Agios: Consultancy; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Arog: Research Funding; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy; Janssen: Consultancy, Honoraria; Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria; Novartis: Honoraria, Research Funding. Salih:Synimmune: Consultancy, Research Funding; Philogen: Consultancy; Medigene: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Schittenhelm:Pfizer: Consultancy; Astellas: Consultancy. Mueller-Tidow:Jose-Carreras-Siftung: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; BMBF: Research Funding; Deutsche Krebshilfe: Research Funding; Deutsche Forschungsgemeinschaft: Research Funding; Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer AG: Research Funding. Brugger:MorphoSys: Current Employment. Bug:Jazz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Hexal: Membership on an entity's Board of Directors or advisory committees; 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; Eurocept: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Sanofi: Other: Travel; Neovii: Other: Travel. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Ganser:Celgene: Consultancy; Novartis: Consultancy. Döhner:AstraZeneca: Consultancy, Honoraria; Sunesis: Research Funding; Roche: Consultancy, Honoraria; Pfizer: Research Funding; Oxford Biomedicals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Helsinn: Consultancy, Honoraria; Jazz: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Astex: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; AROG: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; GEMoaB: Consultancy, Honoraria. OffLabel Disclosure: ATRA is approved for APL treatment but not for non-APL AML

Background: In the context of a haploidentical stem cell transplantation (Haplo-SCT) platform with post transplantation cyclophosphamide (PT-Cy) for acute myeloid leukemia (AML) patients, the optimal conditioning regimen remains unknown. A non-myeloablative conditioning (NMAC) regimen (cyclophosphamide + fludarabine + 2Gy TBI [CyFluTBI]) was initially reported by the Johns Hopkins group as a safe approach in this setting, notably to treat patients of advanced age and/or with comorbid conditions. However, relapse incidence after NMAC Haplo-SCT remains high in AML where it can reach 45%. Alternatively, a reduced intensity conditioning (RIC) regimen containing an antileukemic drug combination like thiotepa and reduced-dose busulfan in addition to fludarabine (TBF) may decrease AML relapse. However, this anticipated benefit may be counterbalanced by a higher incidence of toxicity, graft-versus-host disease (GVHD) and non-relapse mortality (NRM). To date, no study comparing TBF vs. CyFluTBI has been published in complete remission (CR) AML. We performed this retrospective comparison on behalf of the Acute Leukemia Working Party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT). Methods: We retrospectively analyzed 398 patients from the EBMT registry database with the following inclusion criteria: 1) adult patient in CR1 or CR2 AML; 2) T-replete Haplo-SCT with PT-Cy; 3) no in vivo depletion using antithymocyte globulin or alemtuzumab; and 4) receiving either TBF RIC (equivalent of 2-day iv busulfan dose) or CyFluTBI NMAC regimen. We compared separately TBF vs. CyFluTBI in patients younger (n=170, 82 TBF vs. 88 CyFluTBI) and older (n=228, 141 TBF vs. 87 CyFluTBI) than 60 years. Results: In patients younger than 60 years, the 2-year cumulative incidence of relapse (CIR) was significantly lower in the TBF group compared with the CyFluTBI group (TBF vs. CyFluTBI: 14% vs. 43%, p In patients older than 60 years, univariate analysis did not show any significant difference in outcome according to the type of conditioning regimen (2-year NRM: TBF vs. CyFluTBI: 33% vs. 25%, p=0.23; 2-year CIR: TBF vs. CyFluTBI: 23% vs. 28%, p=0.20; 2-year LFS: TBF vs. CyFluTBI: 44% vs. 47%, p=0.96). Multivariate analysis showed a significant reduction in the risk of NRM after CyFluTBI (HR 0.5, 95%CI [0.2-0.9], p=0.04), while a non-significant increase in the risk of relapse was observed (HR 1.9, 95%CI [0.8-4.2], p=0.13). Finally, there was no significant difference in LFS (HR 0.9, 95%CI [0.5-1.5], p=0.67) and OS (HR 0.9, 95%CI [0.5-1.5], p=0.67). Conclusion: Our study suggests that in CR AML patients aged younger than 60 years, the use of TBF RIC provides better outcomes than NMAC CyFluTBI due to lower incidence of relapse, without significant increase in the risk of NRM. Conversely, it seems that older patients do not benefit from such conditioning intensification, due to a significantly higher risk of NRM after TBF RIC. Thus, in CR AML patients who will not receive a truly myeloablative regimen prior to PT-Cy Haplo-SCT, age could be used for determining the conditioning intensity from the wide variety of reduced toxicity conditioning regimens. Beyond the patient age, further prospective trials should assess patient-based parameters that may be useful for a fine tuning of conditioning intensity in a more individualized approach. Disclosures Labopin: Jazz Pharmaceuticals: Honoraria. Blaise:Jazz Pharmaceuticals: Honoraria. Bug:Sanofi: Other: Travel; Neovii: Other: Travel; Jazz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Hexal: Membership on an entity's Board of Directors or advisory committees; 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; Eurocept: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel. Mohty:Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Acute myeloid leukemia (AML) is driven by niche-derived and cell-autonomous stimuli. Although many cell-autonomous disease drivers are known, niche-dependent signaling in the context of the genetic disease heterogeneity has been difficult to investigate. Here, we analyzed the role of Bruton tyrosine kinase (BTK) in AML. BTK was frequently expressed, and its inhibition strongly impaired the proliferation and survival of AML cells also in the presence of bone marrow stroma. By interactome analysis, (phospho)proteomics, and transcriptome sequencing, we characterized BTK signaling networks. We show that BTK-dependent signaling is highly context dependent. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)–positive AML, BTK mediates FLT3-ITD–dependent Myc and STAT5 activation, and combined targeting of FLT3-ITD and BTK showed additive effects. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)–negative AML, BTK couples Toll-like receptor 9 (TLR9) activation to nuclear factor κΒ and STAT5. Both BTK-dependent transcriptional programs were relevant for cell cycle progression and apoptosis regulation. Thus, we identify context-dependent oncogenic driver events that may guide subtype-specific treatment strategies and, for the first time, point to a role of TLR9 in AML. Clinical evaluation of BTK inhibitors in AML seems warranted.

Introduction: Complex aberrant karyotypes (CK, ≥3 cytogenetic aberrations, CA) are associated with an unfavorable prognosis and an increased AML transformation rate in MDS. However, even MDS with CK (CK-MDS) are heterogeneous in terms of genetic profile and prognosis. Recently, we demonstrated that a high number of CA as well as mutations in TP53 (TP53mut) are associated with increased risk in CK-MDS (Haase et al, 2019). However, as there is a strong association between CK-MDS and TP53mut, it is still a matter of debate whether the karyotype and TP53mut are prognostically independent genetic markers. Furthermore, loss of heterozygosity (LOH) of 17p13 (TP53LOH), due to loss of genetic material or to copy number neutral LOH (CN-LOH), is also associated with a poor prognosis. We here aimed to characterize TP53mut andTP53LOH in CK-MDS and to elucidate the impact of cytogenetics, TP53mut and TP53LOH on the outcome of CK-MDS. Methods: We included 178 pts with MDS (N=138), CMML (N=5) and secondary AML after MDS (AML with myelodysplasia-related changes, N=35), all with CK. The median precentage of bone marrow (bm) blasts was 11% (range: 0-90%). The median age was 72 yrs (range: 30-95 yrs). The male:female ratio was 1.23:1. The number of CA was determined by banding analysis in all cases. The karyotype was confirmed by multicolor FISH in 134 cases. TP53LOH was verified by FISH analysis of the TP53 locus in 17p13 (146 analyses) and/or molecular karyotyping (MK, 41 analyses). In 144 cases further FISH probes in addition to TP53 were used. TP53mut was identified by NGS (54 cases) or Sanger sequencing (124 cases). Follow-up data for survival analyses were available for 127 pts with MDS and oligoblastic AML with less than 30% bm blasts. Results: The median number of CA was 7 (range: 3-46), 98/178 pts (55%) showed a TP53mut (median VAF: 34%, range: 8-93%) and 64/178 (36%) a TP53LOH (median FISH clone size: 65%, range: 6-99%), including 9 pts with a CN-LOH in 17p13. The CN-LOH was either identified by MK (5/41 pts (12%) where MK was available showed a CN-LOH, 4/5 with TP53mut) or by NGS (4/54 pts (7%) where NGS was available showed a VAF >70% and normal TP53-FISH). In total, a TP53mut and/or a TP53LOH was identified in 116/178 pts (65%). Overall survival (OS) did not significantly differ between CK-MDS with TP53mut only, TP53LOH only, and TP53mut+TP53LOH (Fig.1). Therefore, we merged TP53mut and TP53LOH to TP53altered in all further analyses. Regarding the cytogenetic characterization of pts with TP53altered, the number of CA was significantly higher in pts with TP53altered than in pts with normal TP53 (median 9 CA (range: 3-46) vs 5 CA (range: 3-24), P The number of CA as well as the TP53 status contributed significantly to OS (Fig.2). The presence of anemia (Hb Conclusions: The presence of ≥5 CA is associated with reduced OS in CK-MDS. A TP53mut as well as a TP53LOH both further segregate outcome. The impact of the clone size of TP53mut and TP53LOH on survival is currently being evaluated. Our data imply that the TP53 status (TP53mut and/or TP53LOH) and the complexity of the karyotype are independent prognostic markers. Based on the presence of anemia, the TP53 status (TP53mut and/or TP53LOH), and the number of CA, the individual risk of CK-MDS can be estimated more accurately. This will allow to better tailor treatment decisions for individual pts with CA. Funding (FS): 2017 SGR 288-GRC Disclosures Germing: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding. Kaivers:Jazz Pharmaceuticals: Other: Travel Support. Kröger:Celgene: Honoraria, Research Funding; DKMS: Research Funding; JAZZ: Honoraria; Medac: Honoraria; Neovii: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Riemser: Research Funding; Sanofi-Aventis: Research Funding. Hertenstein:RS Media: Research Funding. Döhner:Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bug:Hexal: Membership on an entity's Board of Directors or advisory committees; Celgene Neovii: Other: travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Sanofi: Other: travel grants; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees. Nickelsen:Roche Pharma AG: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant; Janssen: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Introduction: Posttransplantation cyclophosphamide (PTCy) based T cell-replete haploidentical (haplo) hematopoietic stem cell transplantation (HSCT) is a valid option for patients with indication for allogeneic HSCT without a human leucocyte antigen (HLA) matched donor. However, selection criteria to determine the optimal among several available haplo donors are still a matter of debate. Especially, the impact of killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen (HLA) incompatibilities (inc) in the setting of PTCy T cell-replete haplo HSCT is unclear. PTCy has been reported to eliminate most mature donor NK cells infused with the graft, including single KIR+ NK cells, thereby blunting NK cell alloreactivity in this setting (Russo et al., Blood 2018). Willem et al. (J Immunol 2019) reported (i) a significant loss of KIR2DL2/3+ NK cells at day +30 in patients with inhibitory KIR/HLA incompatibility (inc.) suggesting that PTCy might target responsive KIR NK cells and (ii) a correlation of genetic KIR2DL/HLA inc. with less relapse, but more graft-versus-host-disease (GvHD). Similarly, NK alloreactivity defined as KIR receptor-ligand mismatch or group B KIR haplotype with the presence of KIR2DS2 has been correlated with improved survival (Salomon et al., BBMT 2018). Aims of our study were to evaluate the impact of (i) HLA/KIR inc, (ii) donor KIR genotype and (iii) HLA-DP mismatch status on survival and incidence of relapse, acute and chronic GvHD in our homogeneously treated, independent patient cohort. Patients and methods: We retrospectively analyzed the outcome of 51 consecutively transplanted patients (AML/MDS (n=28/5), ALL (n=9), HD (n=2), NHL (n=5), CML (n=1), PMF (n=1)) receiving a PTCy based T cell-replete haplo HSCT between 01/2011-12/2018. All patients received a myeloablative conditioning regimen (fludarabine/total body irradiation (FTBI) or thiotepa/busulfan/fludarabine (TBF)) with unmanipulated bone marrow (98%) as the preferred graft (median CD34+ cells: 3.02 x 106/kg (range, 1.50-6.90) and median CD3+ T cells: 3.54 x 107/kg (range 1.52-43.74)). GvHD prophylaxis with ciclosporin A started on day 0, mycophenolate-mofetil on day +1, PTCy was applied on day +3 and +5. Results: Patient, donor and transplant characteristics as detailed in table 1 were well balanced between the inh. KIR/HLA inc. group (n=29) vs. no inh. KIR/HLA inc. group (n=22) with the exception of the median donor age (41.7 (range, 23.4-73.7) vs. 33.6 years (range, 19.0-56.2), resp. All patients engrafted. At day +28 (range, 20-29; n=26) CD3+ cells were 88.5/nL (range, 3-665), CD3+CD4+ cells 22.5/nL (range, 0-277.0), CD3+CD8+ cells 117.0/nL (range, 7-478), CD19+ cells 1.0/nL (range, 0-12), CD56bright cells 74.4/nL (range11.1-93.4), CD56dim cells 25.5/nL (range, 6.4-88.9) measured by flow cytometry and without differences between the inh. KIR/HLA inc. group vs. no inh. KIR/HLA inc. group. Cytomegalovirus (CMV) reactivation occurred in 73.3% of patients at risk and median time of occurrence was 32 days (range, 12-97) without difference between groups. Median follow-up for surviving patients was 26.1 months (range, 2.8-92.8) and we found no significant differences in 2-year overall survival (OS; 65.3±10.3 vs. 89.6±7.0, p=0.311), 2-year relapse-free survival (RFS; 66.0±9.4 vs 77.8±10.2, p=0.235), GvHD- and relapse-free survival (GRFS; 48.4±9.8 vs 60.5±12.0, p=0.182) as well as cumulative incidence (CI) of relapse (23.3% vs 16.2%, p= 0.283), acute GvHD grade 2-4 (27.6% vs 31.8, p=0.563), moderate-severe chronic GvHD (22.2% vs. 9.9%, p=0.227) and NRM (16.3% vs 5.3%, p=0.283) between the inh. KIR/HLA inc. group vs. no inh. KIR/HLA inc. group. This was also the case for donor KIR genotype AA vs AB (n=46; 2-y OS: 74.9±13.0% vs. 73.0±9.9%, p=0.844; 2-y RFS: 60.0±14.8% vs 74.5±8.4%, p=0.645) and HLA-DP-identical/permissive mismatch (MM) vs non permissive MM (n=45; 2-y OS: 70.7±10.0% vs 72.7±13.4%, p=0.945; 2-y RFS: 73.2±8.2% vs 63.6.0±14.5%, p=0.798) Conclusion: Our outcome data support the hypothesis of PTCy eliminating mature donor NK cells infused with the graft and thereby reducing the impact of alloreactivity in this setting. However, our patient number is quite small and the findings need to be validated in larger cohorts and preferably prospective studies. Disclosures Lindner: Celegene, Sanofi, Neovii: Honoraria, Research Funding. Berg:Riemser Pharma GmbH: Consultancy, Honoraria; Incyte, Abbvie, Astellas, Alexion and Celgene: Other: travel support. Bug:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene Neovii: Other: travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria, 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; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Sanofi: Other: travel grants. Schwaeble:Uniqure BV: Research Funding. Ullrich:CellGenix: Honoraria, Research Funding; Novartis: Research Funding.

Background: Measurable residual disease (MRD) can identify patients (pts) with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) in complete hematological remission (CR) at high risk of relapse even after allogeneic hematopoietic stem cell transplantation (HSCT). We have recently shown in 53 pts treated within the first cohort of the RELAZA2 trial that pre-emptive therapy with azacitidine (AZA) at the time of MRD-positivity (MRDpos) can successfully prevent imminent hematological relapse (Platzbecker et al. Lancet Oncol. 2018). We now report on the results of the second cohort of 41 pts undergoing MRD-guided treatment in the RELAZA2 trial (ClinicalTrials.gov NCT01462578) by the Study Alliance Leukemia (SAL). Methods: Between 2015 and 2018, 166 MDS/AML pts were screened and centrally monitored for MRD in bone marrow or peripheral blood at monthly intervals for a period of 2 years prospectively in 9 centers in Germany. Of these 166, 41 pts with either advanced MDS (n=6) or AML (n=35) in CR after either conventional chemotherapy only (n=13) or consecutive allogeneic HSCT (n=28) developed MRD above a threshold defining imminent hematological relapse. Still being in morphological CR, these pts pre-emptively received 6 cycles of AZA (75mg/m2, s.c. days 1-7), which was followed by a risk-adapted AZA-maintenance therapy based on MRD-response for up to 18 additional months. Pts developing a hematological relapse went off study. MRD was detected by either the quantification of NPM1 mutation level (n=19), leukemia-specific fusion genes DEK-NUP214 (n=1) or RUNX1/RUNX1T1 (n=2) or a sensitive donor chimerism analysis of sorted CD34(+)/CD117(+) peripheral blood cells (n=28) in pts undergoing allogeneic HSCT. Here, we report the analysis of the primary endpoint of the 41 pts in the second cohort as well as the data for the entire 94 pts who entered the treatment phase of the RELAZA-2 study. Results: At a median of 110 days (range 28-476) after start of screening, 41 (25%) out of 166 prospectively screened pts became MRDpos as defined by either a decrease of CD34(+)/CD117(+) donor chimerism to 1% (NPM1 n=18) while being still in hematological CR. All of these MRDpos pts started AZA-based pre-emptive treatment to prevent imminent hematological relapse. Six months after start of MRD-guided therapy, 25 out of 41 pts were still in CR (61%, 95%-CI 45-76%, p Conclusion: These multicenter prospective data provide further strong evidence that continuous MRD monitoring is feasible and can identify MDS/AML pts at high risk of hematological relapse. Pre-emptive MRD-guided therapy with AZA is an effective treatment to prevent or at least substantially delay hematologic relapse in these pts. Disclosures Platzbecker: Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Wolf:Celgene: Honoraria, Research Funding; Abbvie: Honoraria. Krämer:Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding. Bug:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria, 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; 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. Götze:AbbVie: Membership on an entity's Board of Directors or advisory committees. Stelljes:Novartis: Honoraria; Amgen: Honoraria; Jazz Pharmaceuticals: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; MDS: Consultancy. Subklewe:AMGEN: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Janssen: Consultancy; Roche: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Morphosys: Research Funding. Hänel:Celgene: Other: advisory board; Novartis: Honoraria; Takeda: Other: advisory board; Amgen: Honoraria; Roche: Honoraria. Dührsen:Gilead: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Takeda: Consultancy, Honoraria; Teva: Honoraria; Celgene: Research Funding; Roche: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Janssen: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; CPT: Consultancy, Honoraria. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Thiede:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; AgenDix GmbH: Employment, Equity Ownership.

Spleen tyrosine kinase (Syk) induces cell survival and proliferation in a high proportion of acute myeloid leukemia (AML) blasts, but the underlying molecular events of Syk signaling have not been investigated. Proteomic techniques have allowed us to identify the multiprotein complex that is nucleated by constitutively active Syk in AML cells. This complex differs from the B-lymphoid Syk interactome with respect to several proteins, especially the integrin receptor Mac-1, the Fc-γ receptor I (FcγRI), and the transcription factors STAT3 and STAT5. We show in several AML cell line models that tonic signals derived from the Fc-γ chain lead to Syk-dependent activation of STAT3 and STAT5, which in turn induces cell survival and proliferation. Moreover, stimulation of Mac-1 or FcγRI intensifies the constitutive Syk-mediated STAT3/5 activation in AML cells, a scenario likely to take place in the bone marrow niche. In accordance with these findings, we observed that β2 integrins, including Mac-1, trigger proliferation of AML cells in an AML cell/stroma coculture model. Taken together, we identified an oncogenic integrin/Syk/STAT3/5 signaling axis that might serve as a therapeutic target of AML in the future.

The European LeukemiaNet classification combines a heterogeneous group of aberrations as adverse-risk abnormalities. Our goal was to investigate the outcomes associated with distinct high-risk chromosomal abnormalities in acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). We performed a retrospective cohort analysis in patients with high-risk AML who received first, HLA-compatible, allogeneic HSCT between January 2005 and December 2008. Data from 236 patients with a median age of 55 years were included. Because complex karyotype (CK), −5/5q−, and abnl(17p) are overlapping categories, a hierarchical classification system based on the presence or absence of abnl(17p) and −5/5q− was developed. Patients with abnl(17p) had a 2-year event-free survival (EFS) of 11% (95% confidence interval [CI], 0%-25%), patients with −5/5q− but no abnl(17p) a 2-year EFS of 29% (95% CI, 14%-44%), and patients with adverse-risk AML but neither of the 2 marker lesions a 2-year EFS of 49% (95% CI, 39%-59%). Notably, complex and monosomal karyotypes lost their prognostic value when these marker lesions were excluded. In conclusion, hierarchical classification of adverse-risk karyotypes by 2 marker lesions, abnl(17p) and −5/5q−, is effective in prognostication of the outcome of allogeneic HSCT in AML.

Introduction In older patients (pts), host factors such as functional deficits, comorbidities and other age-related factors are increasingly recognized as predictors for outcome of leukemia treatment. Thus, prospective clinical trials increasingly implement functional studies (often termed geriatric assessment) in their pretreatment diagnostic workup. In older, fit AML pts receiving standard chemotherapy, Klepin et al. demonstrated a predictive value of this approach for overall survival (OS) in a single-center study (Blood 2013). We also developed a Frailty Score, assessed at 3 study centers and predicting OS of elderly AML/MDS pts receiving either hypomethylating agents (n=66) or sole best supportive care (n=35; Deschler et al., Haematologica 2013). To validate this score, which is composed of performance status (PS), activities of daily living (ADL) and fatigue, we prospectively assessed these parameters in the DECIDER trial (AMLSG 14-09, NCT00867672). Methods In the DECIDER trial, 200 non-fit AML pts aged >60 years (yr) were randomized between four treatment arms with either decitabine (DAC) alone, or DAC plus all-trans retinoid acid (ATRA) or DAC plus valproic acid (VPA) or DAC plus ATRA and VPA. We assessed PS via ECOG, ADL via Barthel index and fatigue via the EORTC QLQ-C30 questionnaire, which were available for 200, 175 and 156 pts, respectively. Pts with missing ADL and/or fatigue assessments tended to have a lower PS than those with complete data, which may explain at least in part why the assessments could not be performed. The Frailty Score was calculated using ECOG PS as a substitute for the Karnofsky Index (KI, as applied when establishing this score), with ECOG 0-1 taken as corresponding to KI ≥80, and ECOG 2-3 to KI Results For 141 pts with complete data in all 3 parameters building the Frailty Score, median follow-up time was 28 months, and 122 pts had died. The median age of pts at AML diagnosis was 76 yr (range 61-90) and 96 pts (68.1%) were males. Median WBC was 3.100/µl (interquartile range [IQR] 1.400 to 11.500/µl), median platelet count 49.000/µl (IQR 30.000 to 98.000/µl), median hemoglobin (Hb) 9.2g/dl (IQR 8.3 to 10.1g/dl), with median peripheral blasts of 48% (IQR 25 to 76%) and median LDH 287 U/l (IQR 203 to 459 U/l). 32.6% of the pts had adverse genetic risk according to ELN 2010 criteria. 56.7% had relevant comorbidities (HCT-CI ≥3). The majority of pts had a reduced PS (21.3% ECOG 0, 62.4% ECOG 1 and 16.3% ECOG 2-3). When assessing ADL, 30.5% had limitations (ADL 1, ADL Conclusions This previously described pt fitness assessment to calculate the Frailty Score could now be implemented in a multicenter academic trial setting. In our independent, large AML cohort, the score was able to separate pts into groups with different OS, underlining the emerging roles of ADL and fatigue as predictors of outcome, in addition to the already well-established role of PS. A second prospective validation study, in AML pts aged ≥ 60 yr receiving standard "7+3" induction therapy or 10-day DAC (EORTC AML21 "inDACtion vs. induction" trial, NCT02172872), is ongoing. Disclosures Schlenk: Pfizer: Research Funding, Speakers Bureau. Salih:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Bug:Janssen: Other: Travel Grant; Celgene: Honoraria; Jazz Pharmaceuticals: Other: Travel Grant; Amgen: Honoraria; Novartis Pharma: Honoraria, Research Funding; Neovii: Other: Travel Grant; Astellas Pharma: Other: Travel Grant. Germing:Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Götze:Takeda: Honoraria, Other: Travel aid ASH 2017; Celgene: Honoraria, Research Funding; JAZZ Pharmaceuticals: Honoraria; Novartis: Honoraria. Scholl:Deutsche Krebshilfe: Research Funding; Novartis: Other: Travel support; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Döhner:Celator: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Pfizer: Research Funding; Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding. Lubbert:Teva: Other: Study drug; Celgene: Other: Travel Grant; Janssen: Honoraria, Research Funding.

Introduction: After an allogeneic stem cell transplantation (SCT), analysis of donor chimerism (DC) is routinely used to monitor engraftment. In patients with myeloid malignancies, loss of a complete donor chimerism (CC) may indicate graft failure, but more often imminent leukemic relapse. Especially in patients without a valid marker for minimal residual disease (MRD), chimerism analysis may prompt reduction of immunosuppression or therapeutic interventions such as donor lymphocyte infusions (DLI) or hypomethylating agents (HMA). We retrospectively analyzed DC data and outcomes of 255 consecutive patients (pts) transplanted for an acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) at our center. Aims of our study were to evaluate the impact of (i) a falling DC, (ii) the first chimerism-guided intervention, and (iii) the application of DLI on survival and incidence of acute and chronic (a/c) GvHD. Patients and Methods: 255 pts that received a first SCT between 2005 and 2016 were monitored regularly (approx. every two weeks from day +14 to +100, then monthly) for DC using a validated, CE-labeled multiplex-STR PCR at a single laboratory (AgenDix GmbH, Dresden, Germany). CC was defined as ≥99% and mixed chimerism (MC) as 245 pts (median age 53 years (range 19-73), 136 male) with AML (n=222) or MDS (n=23) achieved a CC within 60 days post SCT and were eligible for our analysis, 10 pts were excluded due to refractory disease (n=9) or early death (n=1). 101 out of 222 AML pts (45%) had intermediate (int)-2 or adverse cytogenetics according to ELN guidelines, and 10 out of 23 MDS pts (43%) had IPSS int-2 or high risk. 121 pts (49%) were transplanted in first complete remission (CR), 107 (44%) with active disease. For SCT, 96 pts (39%) had received myeloablative (MAC) and 149 (61%) reduced intensity conditioning regimens (RIC). Donors were HLA-matched siblings (MRD, n= 60) or unrelated donors (MUD, n=149), mismatched related (MMRD, n= 1) or unrelated donors (MMUD, n=27), or haploidentical family members (n=8). Results: A MC was detected in 95 pts (39%) at a median of 104 (range, 28-1764) days post SCT, of whom 18 pts (32%) had aGVHD G2-4. Pts with MC had received RIC significantly more often compared to pts with continued CC (69% vs 55%, p=0.046), the two groups did not differ regarding high risk cytogenetics/IPSS and remission status at SCT. MC prompted reduction of immunosuppressive therapy (IST, n=35), DLI (n=7), HMA (n=16), DLI+HMA (n=7), chemotherapy and/or 2ndSCT (n=7), small molecules (n=10) or best supportive care (BSC, n=13) as deemed appropriate by the treating physician. Median OS and GFRS were significantly better for pts with CC (OS not reached; GFRS 46 months (mths)) compared to pts with MC (OS 15.7 mths; Hazard ratio (HR) 0.25, 95%-CI 0.17-0.37, p In the whole cohort, 46 pts (19%) received a median of 2 DLIs (median dose 0.5x106CD3+cells/kg). PDLIs were administered to 33 pts (72%) and tDLIs to 13 pts with relapsed disease (28%). The pDLI group had a 3-year survival of 82.9% and did not reach median OS, compared to 24.6% 3-year survival and 22 mths median OS in the tDLI group. Median GRFS was 91.4 vs 6.6 mths for the pDLI and tDLI group, respectively. No pt developed aGVHD G2-4 after DLI administration, 1 pt (8%) in the tDLI and 4 pts (12%) in the pDLI group developed cGVHD requiring systemic IST. Conclusion Occurrence of MC seems predictive of an inferior outcome, but early intervention such as careful reduction of IST if feasible or administration of DLI with or without HMA may effectively prolong OS and GRFS. Administration of pDLI after discontinuation of IST starting with low doses is safe and results in low rates of cGvHD. Disclosures Lang: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding. Toenges:Bayer: Research Funding. Schetelig:Sanofi: Consultancy, Research Funding; Roche: Honoraria; Abbvie: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding. Serve:Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Bug:Astellas Pharma: Other: Travel Grant; Novartis Pharma: Honoraria, Research Funding; Neovii: Other: Travel Grant; Jazz Pharmaceuticals: Other: Travel Grant; Janssen: Other: Travel Grant; Celgene: Honoraria; Amgen: Honoraria.

In adult Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL), minimal residual disease (MRD) after stem cell transplantation (SCT) is associated with a relapse probability exceeding 90%. Starting imatinib in the setting of MRD may decrease this high relapse rate. In this prospective multicenter study, 27 Ph+ ALL patients received imatinib upon detection of MRD after SCT. Bcr-abl transcripts became undetectable in 14 (52%) of 27 patients, after a median of 1.5 months (0.9-3.7 months) (earlyCRmol). All patients who achieved an earlyCRmol remained in remission for the duration of imatinib treatment; 3 patients relapsed after imatinib was discontinued. Failure to achieve polymerase chain reaction (PCR) negativity shortly after starting imatinib predicted relapse, which occurred in 12 (92%) of 13 patients after a median of 3 months. Disease-free survival (DFS) in earlyCRmol patients is 91% ± 9% and 54% ± 21% after 12 and 24 months, respectively, compared with 8% ± 7% after 12 months in patients remaining MRD+ (P < .001). In conclusion, approximately half of patients with Ph+ ALL receiving imatinib for MRD positivity after SCT experience prolonged DFS, which can be anticipated by the rapid achievement of a molecular complete remission (CR). Continued detection of bcr-abl transcripts after 2 to 3 months on imatinib identifies patients who will ultimately experience relapse and in whom additional or alternative antileukemic treatment should be initiated.

We have previously reported on the efficacy of the JAK1/2 inhibitor ruxolitinib in corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) in 95 patients (pts) (Leukemia 2015;29(10):2062-8). To assess long-term follow-up results, we collected data from the same pts treated in 19 centers in Europe and the US. Pts were classified as SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). Median numbers of pre-ruxolitinib GVHD treatment lines were 3 (1-7) and 3 (1-10) for SR-aGVHD and SR-cGvHD, respectively. The median follow-up was 19 and 24 months for aGVHD and cGVHD, respectively. The 1-year overall survival (OS) from was 62.4% (CI: 49.4%-75.4%) and 92.7% (CI: 84.7%-100%) for SR-aGVHD and SR-cGvHD, respectively. The estimated median OS (50% death) was 18 months for aGVHD and not reached for cGVHD patients. The median duration of ruxolitinib treatment was 5 and 10 months for patients with SR-aGVHD and SR-cGVHD, respectively reflecting the different biology of the diseases. At follow-up, 22/54 (41%) of SR-aGVHD patients and 10/41 (24%) of SR-cGVHD patients have an ongoing response and are free of any immunosuppression. GVHD relapse or progression after achieved PR/CR was observed in 14/45 (31%) and 13/36 (36%) patients with SR-aGVHD and SR-cGVHD, respectively. Response to re-treatment with Ruxolitinib or any immunosupressive therapy was seen in 11/14 (78%) and 11/13 (86%) patients with SR-aGVHD and SR-cGVHD, respectively. Cytopenia (any grade) and CMV-reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. These findings extend our previous report by showing that patients with SR-aGVHD and SR-cGVHD may benefit long-term from ruxolitinib treatment with an OS that is relatively high for steroid-refractory GVHD. GVHD-relapse or GVHD-progression rates were moderate and more than 75% of the relapse/progression patients responded to re-treatment with ruxolitinib or other immunosuppression. Disclosures Meyer: Stanford University: Patents & Royalties. Marks:Pfizer: Honoraria. Lübbert:Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding. Scheid:Novartis: Other: funding outside this work; Celgene: Other: funding outside this work; Janssen: Other: funding outside this work. Kobbe:Celgene: Honoraria, Other: travel support, Research Funding; Jansen: Honoraria, Other: travel support. Negrin:Stanford University: Patents & Royalties. Brune:Meda Pharma: Consultancy. Mielke:JAZZ Pharma: Speakers Bureau; Novartis: Consultancy; MSD: Consultancy, Other: Travel grants; Gilead: Other: Travel grants; Celgene: Other: Travel grants, Speakers Bureau. Kuball:Gadeta B.V,: Membership on an entity's Board of Directors or advisory committees. Kröger:Sanofi: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Peschel:MophoSys: Honoraria. von Bubnoff:BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding.

In patients with relapsed or refractory (r/r) Acute Myeloid Leukemia (AML), allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is considered to be the only treatment providing long-term disease control for fit patients. The BRIDGE trial studied the safety and efficacy of a clofarabine-based salvage therapy prior to HSCT in patients with r/r AML. Here, we report the long-term follow up of this Phase II, multi-center, Intent-To-Transplant study and the impact of comorbidity on outcome. Eighty-four patients with a median age of 61 years (range 40 - 75) were enrolled. Patients were scheduled for at least one cycle of salvage therapy with CLARA (clofarabine 30 mg/m2 and cytarabine 1 g/m2, days 1-5). Chemo-responsive patients with a donor received HSCT after first CLARA. In the event of a prolonged donor search, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine 30 mg/m2, day -6 to -3, and melphalan 140 mg/m2 on day -2. The ECOG score, hematopoietic cell transplantation-specific comorbidity index (HCT-CI) and Cumulative Illness Rating scale (CIRS) were obtained at study enrolment as well as prior to HSCT. Sixty-seven percent of the patients received HSCT within the trial. After a median follow up of 40months (95% CI, 38-49 months), the estimated 4-year OS (Figure 1) for all enrolled patients was 38% (95% CI, 28-50%) and Disease-Free Survival for transplanted patients was48% (95% CI, 36-64%). The CIR at four years was 30% (95% CI, 17-43%) and the NRM 22% (95% CI, 10-33%).Those patients who received an allogeneic HSCT within the trial had a median HCT-CI at the time of study enrollment of 1 (range, 0 - 6) compared to a median of 2 (range, 0 - 6) for those who did not proceed to allogeneic HSCT (p = .17). Corresponding figures for the CIRS were a median of 2 (range, 0 - 9) compared to 4 (range, 0 - 8) (p = .09). The median ECOG score was 1 (range, 0 - 3) in both groups. Compared to the time point of study enrollment, both the HCT-CI as well as the CIRS increased to a median of 2 (observed range of score, 0 - 7) and a median of 4 (observed range of score, 0 - 12), respectively, at the time of start of the conditioning regimen. This was almost exclusively due to an increase in infectious complications (Figure 2). Inmultivariate analysis, both the baseline HCT-CI and the ECOG score had a statistically significant impact with a HR of 1.22 (p = .025) and 1.72 (p = .001), respectively, on OS. Using a clofarabine-based salvage therapy combined with early allogeneic HSCT we were able to achieve good long-term results for patients with r/r AML. In this cohort, both the HCT-CI and the ECOG score gave prognostic information on OS, showing feasibility of comorbidity evaluation at the time of diagnose of r/r AML. Figure 1 OS of all enrolled patients Figure 1. OS of all enrolled patients Figure 2 Changes of the HCT-CI at baseline to HSCT Figure 2. Changes of the HCT-CI at baseline to HSCT Disclosures Middeke: Sanofi: Honoraria. Rösler:Janssen: Consultancy, Other: Travel/Accommodation/Expenses. Thiede:AgenDix: Employment, Other: Ownership. Schetelig:Sanofi: Honoraria.

Background: During the last years, based on its efficacy and favourable toxicity profile the hypomethylating agent (HMA) Azacitidine (Aza) has proven to be a valuable treatment option for patients with AML or MDS who relapse after allo-SCT. In contrast to Aza, reports on the use of Decitabine (DAC), the second HMA approved in Europe for the treatment of AML, as salvage therapy for relapse after allo-SCT are scarce covering a total of 9 patients so far. This prompted us to perform a retrospective survey in order to gather more experience on the use of DAC after allo-SCT. Patients and Methods: Retrieving information from the EBMT Med-A form and a study-specific questionnaire we were able to analyze data of 36 patients (median age 56 years, range 21-72 years) from 6 German transplant centers who had received at least one cycle of DAC for the treatment of relapse of AML (n=29) or MDS (n=7) after allo-SCT. Median time to haematological (n=34) or molecular (n=2) relapse was 370 days (range 43-2623 days). Results: Overall, DAC was the first treatment for relapse in 16 pts (44%), whereas 20 pts (56%) had previously received one (n=14), two (n=2) or three (n=4) lines of salvage therapy for relapse after allo-SCT. This included 16 pts treated with Aza, 3 pts with intensive chemotherapy and 2 pts with radiation. Five pts had received a second allo-SCT and 9 pts donor lymphocyte infusions (DLI) before DAC therapy. Patients received a median of 2 DAC cycles (range, 1-10) with 24 pts (67%) treated with the approved dose of 20 mg/m2 for 5 days and 12 pts (33%) treated with 20 mg/m2 for 10 days based on the local policy of the individual transplant center. In addition to DAC, DLI (median number of DLI =1, range: 1-5) were administered to 22 pts (61%). Following treatment with DAC +/- DLI the median survival was 5 months (range 1 - 40 months). Six pts achieved a complete remission (CR, 17%) and 3 pts achieved a partial remission (PR, 8%) leading to an overall response rate of 25%. Median time to documentation of CR was 157 days (range: 47-255 days) and 4 DAC cycles (range: 1-8 cycles). Of 6 patients achieving CR after DAC, 3 had received DAC as first salvage therapy and 3 had previously received Aza, including 2 pts not responding to Aza and 1 patient switched to DAC therapy due to Aza intolerability. With a median follow-up of 12 months (range: 5-40 months), 3 of 6 patients remain in ongoing remission for 4, 23, and 33 months respectively without any further antileukemic therapy, while the other 3 patients died in remission due to infectious complications after second transplant. The 2-year overall survival rate of the entire group as calculated from the start of DAC therapy was 9%. Incidence and severity of acute GvHD (overall: 19%, grade I: 3%, grade II: 8%, grade III: 5%, grade IV: 0%, missing: 3%) and chronic GvHD (overall: 6%, limited 6%, extensive 0%) were low and mild. Conclusion: Our analysis shows, that also the second HMA DAC exerts relevant clinical efficacy in patients with AML or MDS relapsing after allo-SCT and can induce durable remissions in individual pts. Given the heterogeneity of our patient group and the limitations of a retrospective analysis this asks for confirmation in a prospective trial. Disclosures Platzbecker: Onconova, Teva, Celgene, Janssen, Novartis, Amgen: Honoraria, Research Funding. Kobbe:Jansen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support, Research Funding.

Background. Haploidentical bone marrow transplantation (HAPLO-BMT) with post-transplant cyclophosphamide (PT-CY) is being increasingly used in patients with acute myeloid leukemia (AML) who lack a suitable HLA-matched donor. The standard Baltimore regimen calls for PT-CY 50 mg/kg on days +3 and +4, with a calcineurin inhibitor and mycophenolate (MMF) starting on day +5 after transplant. Aim of the study. We have modified the original Baltimore regimen (BBMT 2013; 19:117), and are now reporting a multicenter retrospective analysis of HAPLO-BMT in 142 patients with AML. GvHD prophylaxis. All patients received a uniform GvHD prophylaxis , consisting of cyclosporine (CsA) starting on day 0, mycophenolate (MMF) starting on day +1, and PT-CY 50 mg/kg , on days +3 and +5. Patients and conditioning regimen. All patients received umanipulated haploidentical marrow between year 2010 and 2016. Clinical characteristics included: 73 males and 69 females, median age of 50 years (17-74); low ELN risk group (3%) intermediate risk (34%) and high risk (63%); FLT3-ITD positivity (22%) ; first complete remission (CR1) (46%) , second CR (CR2) (21%) and active disease (33%). The median dose of TNC collected and infused was 3,1x108/kg (range 0,8-6,5). All patients received a myeloablative regimen: either thiotepa (10 mg/kg), busulfan (3.2 mg/kgx3), fludarabine (50 mg/m^2x3) (TBF) in 114 patients (median age 55 years), or full dose TBI in 28 patients (median age 37 years). Busulfan was capped at 2 days in patients over 60 years. The median follow up for surviving patients is 532 days (100-1893) Results. 133 patients (94%) engrafted; the median interval to a neutrophil count of 0,5x10^9/L was day 18 (range 13-56). The 100 day cumulative incidence (CI) of grade II-IV and III-IV aGVHD was 17% and 3%. Chronic GVHD was observed in 65 patients with a cumulative incidence of moderate and severe cGVHD of 16% at 3 years. The cumulative incidence of transplant related mortality (TRM) at 5 years is 18%; the CI of relapse related death (RRD) is 32%. Causes of death were relapse (n=26), infections (n=14), graft failure (n=3), multi-organ failure (n=2) chronic GvHD (n=2) , and interstitial pneumonia (n=2). Patients in CR1, CR2, or with active disease, have an actuarial 5 year overall survival (OS) of 79%, 67% and 22%, respectively (p In multivariate Cox analysis, active disease at transplant is the only negative predictor of survival. Despite older age of patients given TBF (almost 20 year difference), survival is comparable to the TBI regimen: the OS of 22 patients aged 60 and over, receiving TBF, grafted in CR1 or CR2, is 82%. Center effect. There was no significant Center effect and the actuarial 5 year OS for CR1+CR2 patients grafted in Genova (n=68) or outside Genova (n=27) was identical (75%). Conclusion . This study shows that our modified PT-CY regimen, with CyA given before PT-CY, and one day rest between the two CY doses, can be successfully applied in a multicenter setting of unmanipulated HAPLO-BMT for AML. For CR1,CR2 patients the outcome is excellent in terms of TRM, RRD and survival, particularly with the TBF conditioning, also in patients over 60. Relapse remains a problem in patients with active disease, as seen with any conventional transplant platform, and may require post-transplant interventions. The incidence of severe acute and chronic GvHD is very low, with marrow as the only stem cell source for all patients. Disclosures Bug: Novartis: Honoraria, Research Funding; Nord Medica: Consultancy; Celgene: Honoraria, Other: Travel Grant; Janssen: Other: Travel Grant; Astellas: Other: Travel Grant; Teva Oncology: Other: Travel Grant. Angelucci:Novartis oncology, celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: Allogeneic hematopoietic cell transplantation is a potentially curative therapy for patients with hematological malignancies. However a fraction of patients will develop corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) which both cause a high mortality and impaired quality of life. Pre-clinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib by modification of T cells and dendritic cells. Methods: In this retrospective analysis, 19 stem cell transplant centers in Europe and the United States reported clinical outcome data from 95 patients who had received ruxolitinib as salvage-therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGvHD (1-10). The median follow-up times were 26.5 (3-106) for SR-aGVHD and 22.4 (3-135) weeks for SR-cGVHD-patients. Results: The ORR was 81.5% (44/54) in SR-aGVHD including 25 CRs (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). The median time to response was 1.5 (1-11) and 3 (1-25) weeks after initiation of ruxolitinib treatment in SR-aGVHD and SR-cGVHD, respectively. Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3%-90.7%,95% CI) and 97.4% (92.3%-100%,95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and CMV reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Relapse of the underlying malignancy occurred in 9.3% (5/54) and 2.4% (1/41) of the patients with SR-aGVHD or SR-cGVHD, respectively. Conclusion: Ruxolitinib constitutes a promising new treatment option for SR-aGVHD and SR-cGVHD. Its activity in SR-aGVHD and SR-cGVHD should be validated in a prospective trials in both, SR-aGvHD and cGvHD. Disclosures Bertz: GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scheid:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding.

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the detection of mutations in genes like DNA methyltransferase 3A (DNMT3A) and has recently been described to occur in healthy people and to predispose them to myeloid malignancies. DNMT3A is frequently mutated in acute myeloid leukemia (AML) and mutations have been detected in CD3 positive T-cells of some AML patients. In these patients DNMT3A mutations are early events that are likely to arise from CHIP. It is unknown how a history (hx) of CHIP influences the characteristics of AML patients and their response to therapy. We studied this question on the basis of a large cohort of DNMT3A mutated AML patients. Patients and Methods: 171 DNMT3A mutated AML patients (aged 18-87 years) were included in our study. 127 patients were treated intensively in trials of the AMLSHG and AMLSG. 34 patients received non-intensive therapy and for 10 patients the therapy is unknown. 148 patients carried a mutation at arginine R882. At the time of diagnosis and relapse samples were further sequenced for 54 genes involved in leukemia with next generation sequencing (NGS) on the Illumina platform. Library preparation of diagnostic samples was performed with the TruSight Myeloid sequencing panel (Illumina). T-cells (CD3+ CD11b- CD14- CD33-) were purified by flow cytometry from AML samples at the time of diagnosis. DNMT3A mutational analysis of T-cell samples and of mononuclear cells during remission or at relapse was performed also with ultra-deep sequencing using customized DNMT3A NGS primers. Presence of a DNMT3A mutation in sorted T cell populations was used as an indicator of a hx of CHIP. Results: A total of 40 patients (23%) were found to have the DNMT3A mutation in mononuclear cells and T-cells (hx of CHIP), while 131 patients (77%) had a DNMT3A mutation in mononuclear cells, but not T-cells (control cohort). Comparing these two patient cohorts revealed that significantly more patients in the hx of CHIP cohort had secondary AML (p=0.009), were older (p=0.005) and less likely to receive intensive treatment (p=0.047) while other clinical parameters did not significantly differ. Analysing the mutational profile of 54 genes revealed that the number of mutations per patient between these 2 groups was similar (median 5 vs 4 mutations, p=0.39). Patients with a hx of CHIP were significantly more likely to harbour mutations in TET2 (p=0.006), RUNX1 (p=0.004), SF3B1 (p=0.049), U2AF1 (p=0.015) but less likely to be NPM1 mutated (p=0.005). There was no significant difference in the allelic burden of DNMT3A in the CHIP hx (mean 43.6) vs control group (mean 44.5). The mean variant allele frequencies of DNMT3A, RUNX1 and NPM1 were highest (44, 45 and 43 respectively) as compared to other mutated genes like IDH1, IDH2 and FLT3 (32, 37 and 34). In relapse samples (n=11), the identical DNMT3A mutation could always be identified. However, patients with a hx of CHIP (n=2) had comparable allelic frequencies compared to diagnosis of mutated DNMT3A ( 10% difference), while 7 out of 9 patients in the control group had a change in the allelic frequency at the time of relapse (mostly reduction). In all remission samples DNMT3A mutations could be identified with ultra-deep NGS but with variable allelic frequencies (0.13-50.01% in the control group, 0.25-70.14% in the hx of CHIP group). In the cohort of patients with intensive therapy there was no difference in CR rates between hx of CHIP and control groups (82 vs 90%, p=0.31). Overall survival (OS) was not influenced by a hx of CHIP (whole cohort: HR 1.09; 95%CI 0.67-1.79; P=.73; intensively treated cohort: HR 0.72; 95%CI 0.34-1.51; P=.38). Relapse-free survival (RFS) was also not different in the hx of CHIP vs the control group (HR 1.06; 95%CI 0.58-1.93; P=.85; intensively treated cohort only HR 0.91; 95%CI 0.46-1.78; P=.78). However, when looking at the influence of allogeneic stem cell transplantations (HSCT) on outcome in intensively treated patients, patients with a hx of CHIP showed abenefit from HSCT (HR 0.082; 95%CI 0.009-0.75; P= 0.027 Figure 1A) as compared to the control group (HR 0.68; 95%CI 0.39-1.21; P= 0.19, Figure 1B). Conclusion: AML patients with a hx of CHIP, as defined by mutated DNMT3A in T-cells, show a distinct clinical and molecular profile and may benefit from HSCT. Figure 1A. Figure 1A. Figure 1B. Figure 1B. Disclosures Bug: TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Fiedler:Pfizer, Amgen, Kolltan: Research Funding; Teva, Amgen, Astellas: Other: Travel Grant; Karyopharm: Research Funding. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Teva: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.

Introduction The extent of systemic iron overload (SIO), quantified by magnetic resonance imaging (MRI), has been associated with adverse outcome in some studies in MDS and AML patients undergoing allogeneic stem cell transplantation (allo-SCT), whereas others were unable to demonstrate a significant impact. It has been hypothesized that the release of reactive iron species such as labile plasma iron (LPI) during the transplant procedure mediates iron-associated cellular toxicity by catalyzing the generation of oxygen radicals and fostering the growth of microbial agents. The association between SIO, the occurrence of LPI and the outcome after allo-SCT has not been prospectively studied so far. Patients, Material and Methods This was a Geman-Austrian prospective multicenter observational trial in 133 patients with AML or MDS undergoing allo-SCT between 2013 and 2015 (NCT01746147). Inclusion criteria were either having a ferritin above 500 ng/ml or having received more than 10 red blood cell concentrates. Liver iron content (LIC) was determined by MRI prior to and on day +100 and day +360 after allo-SCT. Enhanced labile plasma iron (eLPI) was measured using the Ferros eLPI Kit (Afferix) prior to, during and after conditioning and an eLPI above 0.4 was defined as positive. Results At the time of analysis 21 MDS and 90 AML patients were evaluable for LIC. The median age of the cohort was 61 years (range: 21 to 75 years) and the majority (80.2 %) received reduced intensity conditioning regimens. Median LIC prior to conditioning was 110 µmol/g and 45.9 % had a LIC above the pre-specified threshold of 125 µmol/g (7 mg/g) indicating SIO. A LIC >=125 µmol/g was associated with a significantly increased cumulative incidence (CI) of early (day +100) NRM (19.8 % vs. 6.8 % p = 0.034), thus confirming our previous observations (Wemke et al. ClinCancRes 2012). Prior to the initiation of the conditioning regimen positive eLPI levels were found in 26 of 109 evaluable patients. A significant correlation between LIC and pre-conditioning eLPI (Pearson's correlation coefficient: 0.470; p < 0.001) was noted. In fact, the median LIC in patients with a pre-conditioning eLPI > 0.4 was 190 µmol compared to 100 µmol/g in patients below this threshold (p < 0.001). Mean eLPI levels increased continuously during the course of the conditioning regimen and then gradually decreased starting on day +7, while most patients had negative eLPI levels by day +100 after allo-SCT (Figure 1). The presence of an eLPI above 0.4 prior to the initiation of the conditioning regimen was strongly associated with an increased early NRM (CI at day +100: 34.6 % vs. 6.0 % p < 0.001, Figure 2) and this association was confirmed in a multivariate analysis incorporating other factors known to predict for NRM (HR 7.0; 95% confidence interval: 2.076 to 23.91; p = 0.002). Of note, patients remaining LPI positive at day +14 also had a significantly increased NRM (19.0 % vs. 4.9 % p = 0.025), which also held true, when the analysis was restricted to patients being LPI negative prior to conditioning (12.5 % vs. 0.0 % p = 0.013). Patients having an eLPI above 0.4 prior to conditioning had a slightly higher CI of bacterial infections during the course of transplant (CI at day +100: 88.5 % vs. 83.3 %, p = 0.023). There was no association between a positive pre-conditioning eLPI and the occurrence of acute graft versus host disease of grade 2 or higher (CI: 39.1 % vs. 38.6 %). Conclusions The results of the prospective ALLIVE trial confirm recent single center observations that SIO prior to allo-SCT is associated with an increased mortality in AML and MDS patients. Given the fact that a positive eLPI prior to the initiation of the conditioning regimen and the persistence of positive eLPI levels after transplantation are strongly predictive for adverse outcome, it is reasonable to believe that reactive iron species are the key pathogenetic mediators in this context. Therefore, clinical trials assessing therapeutic interventions e.g. by peri-transplant iron chelation are warranted. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Wermke: Boehringer: Research Funding; Novartis: Research Funding. Bug:Celgene, Novartis: Research Funding; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; TEVA Oncology, Astellas: Other: Travel Grant. Theurl:Gilead Science: Research Funding. Platzbecker:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Boehringer: Research Funding.

Introduction Deletion of chromosome 5q (del(5q)) defines a distinct clinical subtype of myelodysplastic syndromes (MDS) and qualifies patients to specific treatment with Lenalidomide (LEN). Therefore, detection and monitoring of this deletion is an important element in routine clinical diagnostics for determining molecular response. Current methodologies for performing these analyses consist of cytogenetics, fluorescence in situ hybridization (FISH) or microarrays. All of these methods have downsides due to the high demands to the input material, i.e. viable cells or necessity for large amounts of high quality genomic DNA (gDNA). To perform quantitative assessment of cytogenetic lesions in low quantity or residual material we here present the establishment of a PCR-based assay for interrogation of del(5q) in MDS, based on the allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Methods Genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of n=86 MDS del(5q) patients. 49 non-del(5q) MDS patients were used as controls. Serial chronological BM samples (n=95) following treatment with LEN from n=40 del(5q) patients, who were enrolled in the LEMON-5 trial from the German MDS study group, were analysed. Using 10ng DNA, 12 fluorochrome-labelled PCR amplicons of STR loci located between chromosomal bands 5q21 and 5q31 were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis and allele size quantification of heterozygous STR loci was performed. Finally, the degree of skewing in the allelic ratios of all informative STR markers was averaged and translated into an allelic burden of del(5q). Results Paired quantitative correlation of clone sizes using STR-PCR and interphase FISH was carried out in n=34 samples and revealed highly concordant results with r²=0.924. The diagnostic accuracy of the PCR assay was evaluated by receiver operating characteristic (ROC) analysis and revealed an area under the curve of 0.989 (sensitivity and specificity of 0.977 and 0.948, respectively). Prior to treatment with LEN, clone sizes as determined by STR-PCR were heterogeneous (mean: 57%, range: 11-91%). During follow-up analysis, while cytogenetic analyses failed (e.g. metaphase failure) in 7/40 (18%) cases, our STR-PCR assay successfully generated estimates of del(5q) cell burden in all available samples. Upon LEN treatment, n=12 patients achieved major cytogenetic remission (absence of del(5q)-positive metaphases). The mean clone size carrying del(5q) determined by STR-PCR in that group was 7% (range 3 - 10%) and significantly increased compared with n=15 patients who reached minor cytogenetic response (defined as 50% reduced aberrant metaphases, mean 13%, range 5 - 39%, p=0.025). Intriguingly none of n=6 patients without cytogenetic response achieved a del(5q) clone size of less than 35% as determined by STR-PCR (mean 46%, range 35 - 66%), highlighting the correlation of PCR based follow-up analysis with currently used cytogenetic methods for response evaluation. Finally in n=93 matched PB and BM samples a correlation of del(5q)-frequency in BM versus PB showed r²=0.81. Moreover, in 96% of samples in which the BM still showed clone sizes >10%, we reliably detected del(5q) in corresponding PB cells with a robust sensitivity of 5% deleted cells. Discussion We present a highly adaptable tool for precise measurement of large chromosomal deletions, requiring only minute amounts of genomic DNA. It shows a very good quantitative correlation with established methods and good diagnostic accuracy. Most importantly, this PCR based assay does not require dividing cells so it can be performed from PB, which shows a sufficient correlation with clone sizes in BM and rarely involves the risk of underrepresentation of del(5q)-clones in PB, possibly allowing the use of PB as a regular specimen for clone size monitoring. Thus, especially in the context of serially monitored patients this assay represents an alternative method for less invasive tracking of cytogenetically aberrant clones. Disclosures Platzbecker: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Boehringer: Research Funding. Schlenk:Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background: Relapse is the most frequent cause of failure after alloSCT for acute leukemia (AL). Unlike in CML, infusion of donor lymphocytes (DLI) is of limited efficacy in overt hematological relapse. Hence, it may be preferable to give DLI in complete hematological remission (CHR) after alloSCT, to exploit the allogeneic graft-versus-leukemia (GvL) effect either as maintenance in high risk patients (prophylactic DLI, proDLI), or as early intervention to prevent hematological recurrence in case of decreasing donor chimerism or minimal residual disease (preemptive DLI, preDLI). However, no systematic analysis of this strategy is available so far, neither concerning the optimal way of application, nor with respect to safety and clinical efficacy. Here, the Acute Leukemia Working Party of the EBMT presents results from a registry-based survey on 343 patients with AML (n=266) or ALL (n=77), who received DLI in CHR after alloSCT. Patients: Median age was 48y, 64% of patients had received alloSCT from a matched sibling, 36% from an 8/8 matched unrelated donor. Disease status at time of alloSCT was CR1/CR2/advanced in 68%/14%/17% of cases, respectively. Patients had received standard/reduced intensity conditioning in 53%/47% of cases. Before alloSCT, 55% had received in vivo T-cell depletion (TCD), 16% ex vivo TCD, 10% in vivo plus ex vivo, and 19% no TCD. Reasons for preDLI were persisting mixed or decreasing donor cells chimerism (n=167, 49%) and persisting or recurrent minimal residual disease (MRD; n=32, 9%). ProDLI without any sign of leukemia was given to 144 patients (42%) with high risk disease Results: Median follow up from DLI1 was 6.5 years (range, 1.1-14.5). Median interval from alloSCT to first DLI (DLI1) was 180 days (range, 15-1178). Patients received a median of 2 infusions with the median CD3+ cell dose at DLI1 being 1x106/kg (range, 0.1-163). Reasons to discontinue DLI were: number of planned infusions reached (56%), GvHD (17%), disease progression (13%), and documented improvement of donor chimerism (6%). At 5y from DLI1, cumulative incidence of leukemia relapse was 43% and 28% in patients receiving preDLI for MRD and mixed chimerism, and 28% among recipients of proDLI given for maintenance in high risk disease. The corresponding 5y OS rates were 55%, 66% and 64%, 5y-LFS rates were 52%, 57% and 58%. Efficacy of preDLI could be directly demonstrated by decreasing MRD in 71% (15/21) and by improvement of donor chimerism in 68% (110/163) of informative patients. Furthermore, hematologic improvement was observed in 13 patients following proDLI. Cumulative incidences of acute GvHD grade II-IV and chronic GvHD after DLI were 13% and 32%, respectively. A multivariate model identified a history of aGvHD ≥grade II after alloSCT (p=0.009, HR 2.1, 95% CI 1.2-3.7), an interval from alloSCT to DLI 1 x 106/kg at DLI1 (p=0.024, HR 1.011, 95% CI 1.001-1.021) as risk factors for induction of GvHD after DLI in CHR. One hundred and thirty three patients (39%) had died at last follow-up, with relapse still being the most frequent cause of death (n=87). Sixteen patients (5% of the entire cohort) died from DLI-induced GvHD, and 29 patients died from other courses. In summary, in this large cohort of patients receiving DLI for AL in CHR after alloSCT, efficacy of preemptive DLI cells could be demonstrated in 69% of patients. About half of patients with MRD, and >70% of patients with mixed chimerism did not experience hematological relapse during a follow up period of >5 years, suggesting a clinically meaningful effect of preDLI in AL. GvHD was the most devastating complication, leading to death in 5% of patients. The identification of risk factors for GvHD may influence the selection of candidates for prophylactic and preemptive DLI in general, and may help to refine the use of DLI in this context with respect to cell dose and timing. Disclosures Schmid: Neovii: Consultancy; Janssen Cilag: Other: Travel grand. Bug:Celgene, Novartis: Research Funding; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; TEVA Oncology, Astellas: Other: Travel Grant. Tischer:Sanofi-Aventis: Other: advisory board. Esteve:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Background: Leukemic relapse and graft-versus-host disease (GvHD) remain major obstacles after an allogeneic stem cell transplantation (HSCT). Panobinostat is a potent inhibitor of class I, II and IV deacetylases and has shown antileukemic as well as immunomodulatory activity. The hypothesis of our phase I/II PANOBEST trial was that panobinostat can effectively prevent relapse in patients (pts) with high-risk (HR) myeloid diseases while simultaneously reducing GvHD. We aimed to determine dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) of panobinostat in adult pts with HR acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) in complete hematologic remission (CR) after a reduced-intensity HSCT. Secondary objectives were evaluation of safety and tolerability of panobinostat, and overall (OS) and disease-free survival (DFS) at 1 and 2 years after HSCT. Methods: In two sequential schedules, panobinostat was administered orally thrice weekly, (TIW), either every week (A) or every other week (B). In schedule A, panobinostat was started at a dose of 10 mg TIW and escalated to 30 mg TIW using a 3+3 design; in schedule B, panobinostat was given at doses of 20-40 mg TIW. Panobinostat was initiated between day +60 and +150 after HSCT and given for up to 1 year. Eligibility criteria included: ANC ≥ 1,000/μL, platelets ≥ 75,000/μL, adequate organ function and no severe GvHD. DLT was defined as prolonged G4 hematologic toxicity or any non-hematologic toxicity ≥ G3 unrelated to disease progression or intercurrent illness within 28 days of the first panobinostat dose. Results: 42 pts (37 AML, 5 MDS) were enrolled, with a median age of 52 years (range, 21-71). Cytogenetics were classified as low (n=6), intermediate-1/2 (n=20) or adverse risk (n=16) according to ELN criteria. Panobinostat was started a median of 98 days (range, 60-147) after HSCT from a matched related (n=9), matched unrelated (n=24), mismatched unrelated (n=6) or haploidentical donor (n=3). The majority of patients (n=28, 67%) were transplanted with active disease (bone marrow blasts 0-80%, median 21%, 1 pt. with extramedullary AML), 9 in CR1 (21%) and 5 in CR2 (12%). Patient and transplant characteristics were equally distributed between schedules A and B. Of 42 pts, 22 (54%) have completed one year of panobinostat, 1 remains on treatment and 19 (46%) discontinued prematurely after a median of 70 days (range, 12-342) due to adverse events (AEs) (n=10), relapse (n=6), patient decision (n=2) or prohibited comedication (n=1). To date, 24 out of 42 patients experienced panobinostat-related grade 3/4 AEs (schedule A: n=14, 67%; schedule B: n=10, 48%). The most common AEs were hematologic toxicity (G3: 14 pts, 33%; G4: 2 pts, 5%), constitutional (G3: 7 pts, 17%) and gastrointestinal symptoms (G3: 5 pts, 12%). Neurological AE and pain (G3, 2 pts each, 5%) as well as metabolic/laboratory alterations (G3: 3 pts., 7%) and renal toxicity (G3, 1 pt, 5%) were also reported. AEs were fully and rapidly reversible after interrupting panobinostat (n=24); 14 patients needed dose adjustment and no study-related deaths occurred. The RP2D was 20 mg TIW in arm A and 30 mg TIW every other week in arm B based on 5 DLTs: fatigue G3 at 20 mg, colitis and nausea/emesis G3 each at 30 mg (arm A), diarrhea and headache G3 at 40 mg each (arm B). Prophylactic or preemptive donor lymphocyte infusions (median 2, range, 1-6) were administered to 10 pts (42%, median 1.5x106 CD3+ cells/kg) in arm A and 8 pts (33%, median 0.5x106 CD3+ cells/kg) in arm B. Cumulative incidence (CI) of moderate (n=8) or severe (n=2) chronic GvHD was 24±0.4% at one year after HSCT and did not differ between both arms. There was no evidence of impaired immune reconstitution. To date, median OS and DFS have not been reached after a median follow up of 22 months (range, 6-57). At two years after HSCT, 5 patients have died from relapse (n=3), sepsis (n=1) or sudden death (n=1 at 3.5 months after study discontinuation) and CI of relapse was 21±0.5%, resulting in probabilities for 2-year OS and DFS of 88±5% and 74±7%, resp. Discussion: Panobinostat maintenance following HSCT for high-risk AML or MDS is feasible with a RP2D dose of 20 mg TIW weekly or 30 mg TIW every other week and associated with a low relapse rate. This provides a rationale for a prospective randomized trial of panobinostat as post-transplant intervention. Disclosures Bug: NordMedica: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Other: Travel grants, Research Funding; Novartis Oncology: Honoraria, Other: Travel grants, Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Astellas: Other: Travel grant; TEVA Oncology: Other: Travel grant; Gilead: Honoraria. Off Label Use: Panobinostat has not been approved for maintenance therapy after an allogeneic stem cell transplantation in ANL and MDS patients . Burchert:Bristol Myers Squibb: Honoraria. Bader:Neovii: Other: Institutional grants; Medac: Other: Institutional grants; Amgen: Consultancy; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy; Riemser: Other: Institutional grants. Ottmann:Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction: Treatment of acute myeloid leukemia (AML) in elderly patients remains challenging. Low-dose DNA hypomethylating agents are a therapeutic option in myelodysplastic syndromes and AML. However, the mechanism of action of hypomethylating agents and the role of induction of DNA hypomethylation in the clinical response is still unclear. To unravel the in vivoeffects of sequential cycles of decitabine, we set out to characterize methylomes of leukemic blasts, T cells (presumably not part of the malignant clone) and granulocytes before and during treatment of AML patients enrolled in the randomized phase II DECIDER clinical trial (NCT00867672). We developed a statistical model for longitudinal data analysis to identify the strongest hypomethylation response. Methods: Peripheral blood mononuclear cells (PBMC) from AML patients were collected before and during therapy (i.v. 20 mg/m2 decitabine for 5 days, with or without subsequent oral drug add-on). Leukemic blasts and T-cells were isolated using automatic magnetic sorting of cells (autoMACS) labelled with anti-human CD34, CD117 and CD3 MACS microbeads (Miltenyi Biotec), respectively. Granulocytes were isolated using dextran sedimentation. Cell type specific genome-wide DNA methylation profiles were obtained using Infinium Human Methylation 450 BeadChip arrays. Data were analyzed using R packages RnBeads applying beta mixture quantile dilation for normalization (Teschendorff et al. Bioinformatics, 29:189–196, 2013) and a modified version of NHMMfdr for multiple testing. Results: Peripheral blood blasts (median purity: 92%) were isolated from 20 patients, and T cells (median purity: 94%) from 26 patients before treatment and on days 4 and/or 8 and 15 of treatment cycle 1. From 10 patients, blasts and T cells were also collected during and/or after cycle 2. In total, until now 127 methylomes (46 blasts, 47 T cells, 34 granulocytes) were generated and used for mathematical modelling. Since the trial is still recruiting, genome-wide methylation was interpreted blinded to all clinical data including drug add-on (ATRA, valproic acid). First, the methylation dynamics of each individual CpG site described by a specified summary statistics were identified. Then, inter-probe distance and CpG annotation were incorporated to explain the dependence structure between CpG sites. In order to control the false discovery rate (FDR), we adapted a method proposed for differential DNA methylation (Kuan & Chiang, Biometrics 68: 774–783, 2012). The summary statistics for each CpG site were modelled to follow a non–homogeneous hidden Markov model. Statistical testing was validated by simulations revealing a very high discriminative power for affected CpGs even with very low methylation dynamics. Applying the model to blasts and T cells, extensive differences in the in vivomethylation changes became apparent. In blasts, 13% of CpG (59,920 CpGs of total 460,343 CpGs) showed significant DNA hypomethylation (Δβ>0.1, FDR Conclusions: Our mathematical model revealed significant DNA hypomethylation by day 8, with striking remethylation by day 15 from start of decitabine treatment in AML blasts in vivo. Most of the hypomethylated CpGs resided in non-promoter regions. In contrast, T-cells were much less affected, which might be due to the low cell division rate and the fact that they are non-malignant cells. This model will hopefully allow determination whether the effects of decitabine are targeted or random, by including sequential samples from later treatment cycles. Unblinding of the patients' clinical data will reveal potential biomarkers of response to epigenetic therapy. Disclosures Lübbert: Ratiopharm: received study drug valproic acid, received study drug valproic acid Other; Johnson&Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees, received study drug decitabine Other.

Background: In relapsed or refractory acute myeloid leukemia (AML), long-term disease-free survival may only be achieved with allogeneic hematopoietic stem cell transplantation (HSCT). Within the BRIDGE Trial, the safety and efficacy of a clofarabine salvage therapy as a bridge to HSCT was studied. Here, we report long-term survival data and the impact of donor availability at the time of study enrollment. The BRIDGE trial (NCT 01295307) was a phase II, multicenter, intent-to-transplant study. Patients and Methods: Between March 2011 and May 2013, 84 patients with relapsed or refractory AML older than 40 years were enrolled. Patients were scheduled for at least one cycle of induction therapy with CLARA (clofarabine 30 mg/m2 and cytarabine 1 g/m2, days 1-5). Patients with a donor received HSCT in aplasia after first CLARA. In case of a prolonged donor search, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine 30 mg/m2, day -6 to -3, and melphalan 140 mg/m2 on day -2. In patients with partially matched unrelated donors, ATG (Genzyme) at a cumulative dose of 4.5 mg/kg was recommended. GvHD prophylaxis consisted of CsA and mycophenolate mofetil. Results: Forty-four patients suffered from relapsed AML and 40 patients had refractory disease. The median patient age was 61 years (range 40 – 75). According to the current ELN risk stratification 17% of pts were classified as favorable risk, 35% as intermediate I, 17% as intermediate II and 20% as adverse risk. The overall response rate assessed at day 15 after start of CLARA was 80% (defined as at least a marked reduction in BM blasts or BM cellularity and absence of blasts in the peripheral blood) with 31% of patients having less than 5% BM blasts at that time. Seventeen patients did not respond to CLARA, and were subsequently treated off-study. Due to early death, three patients were not evaluable for treatment response. Overall, 66% of the patients received HSCT within the trial. Donors were HLA-identical siblings in eight cases (14%), HLA-compatible unrelated donors in 30 cases (55%) and unrelated donors with one mismatch in 17 cases (31%). Treatment success was defined as complete remission (CR), CR with incomplete recovery (CRi) or CRchim (BM donor chimerism >95% and absolute neutrophil count >0.5/nL) on day 35 after HSCT. Treatment success was achieved in 61% of the patients. With a median follow up of 25 months, the OS for all enrolled patients at two years was 42% (95% CI, 32% to 54%). (Figure 1) The Leukemia-free survival at two years for those 51 patients who achieved the primary endpoint was 52% (95% CI, 40% to 69%). (Figure 2) At the time of enrollment, 14% of patients had a related donor and 33% had an unrelated donor available. In 46% of the patients, donor search was initiated at the time of enrollment. For 7% of patients, donor search was unsuccessful prior to enrollment and reinitiated. The OS at 2 years for patients with a related or an unrelated donor available was 75% (95% CI, 54% to 100%) and 47% (95% CI, 31% to 71%), respectively, while it was 29% (95% CI, 18% to 48%) for patients for whom donor search was initiated at time of enrollment (p = .09). Conclusions: Salvage therapy with CLARA, and subsequent conditioning with clofarabine and melphalan prior to allogeneic HSCT, provides good anti-leukemic activity in patients with relapsed or refractory AML. Fast unrelated donor search and work up, with conditioning in aplasia allowed a high rate of successful HSCTs. The leukemia-free survival for this group of elderly, high risk AML patients is very promising. Figure 1 Figure 1. Overall survival for all patients, n=84 Figure 2 Figure 2. Leukemia-free survival for all patients with primary treatment success, n=51 Disclosures Middeke: Genzyme: Speakers Bureau. Off Label Use: Clofarabine for AML. Schetelig:Genzyme: Research Funding; DKMS German Bone Marrow Donor Center: Employment.

Background Two prospective studies and small retrospective series have demonstrated feasibility and clinical efficacy of Azacitidine (Aza) as salvage therapy for relapse of AML or MDS after allo-HSCT. As a consequence, Aza has become a clinically relevant treatment alternative in this setting. Still, due to the heterogeneity and limited number of patients reported so far predictive factors for response and long-term survival are unknown. Patients and Methods We analyzed data of 115 pts (median age 50 years, range 21-72 years) with AML (n=90), MDS (n=23) or myeloproliferative syndrome (MPS n=2), who had experienced relapse (hematological n=101; molecular n=14) after a median of 181 days (range: 19-3349 days) after allo-HSCT. Patients were treated with Aza ± DLI at 11 German transplant centers and in 109 of them (95%) Aza was the first treatment of relapse. Patients received a median of 3 courses Aza (range 1-14) and 78 patients (68%) received DLI (median number of DLI = 2, range: 1-6). Thirty patients were treated within a prospective phase-II trial (NCT-00795548). Their results have been published previously but were updated for this analysis. Results Following this treatment, 34 pts achieved CR (29%) and 8 patients achieved PR (7%) resulting in an overall response rate of 36%. Median time to CR was 84 days (range: 26-430 days) corresponding to 3 Aza cycles (range: 1-8 cycles). Of 6 patients with an extramedullary relapse, 3 patients achieved CR. The 2-year OS rate was 34%. With a median follow-up of 17 months (range 1-82) for survivors, 22 pts (65%) are in ongoing CR for a median of 16 months (range 2-48) without further treatment, while 12 pts relapsed again after a median of 11 months (range 2-48). Bone marrow blast count (< 20% vs. ≥20%, CR rate 47% vs. 12% p=0.0001) and type of relapse (molecular vs. hematological, CR rate 71% vs. 24%, p=0.0007) were identified as predictive factors for the achievement of CR. No correlation was found for any karyotype abnormalities, time interval from allo-HSCT to relapse, the presence of blasts in the peripheral blood and the diagnosis of MDS vs de-novo AML. Incidence and severity of acute GvHD (overall: 25%, grade I: 10%, grad II: 6%, grade III: 7%, grade IV: 2%) and chronic GvHD (overall 23%, limited 19%, extensive 4%) were low and mild. Conclusion This analysis shows that the combination Aza and DLI is a valuable treatment option in the challenging situation of relapse after allo-HSCT. The association of disease burden with the likelihood to respond emphasizes the need for stringent disease monitoring and early intervention. Disclosures: Schroeder: Celgene: Financial travel support Other, Honoraria. Bug:Celgene: Honoraria, Travel grants Other. Platzbecker:Celgene: Honoraria. Kobbe:Celgene: Research Funding.

Despite aggressive chemotherapy and early allogeneic transplantation, acute myeloid leukemia (AML) frequently relapses, so that over-all disease-free survival remains below 50%. Strategies to overcome the chemoresistance of relapse-initiating residual AML blasts are, therefore, warranted. Evidence has been provided that AML cells are sheltered from the insult of chemotherapeutic agents by interacting with bone marrow stroma. Integrin alpha4beta1 (VLA4) mediates adhesion of hematopoietic cells to bone marrow stroma cells and extracellular matrix and has been implicated in cell adhesion-mediated chemotherapy resistance. Based on the evidence thereof provided for ALL blasts, VLA4 is here proposed as a therapeutic target for refractory AML. For this purpose, VLA4 was functionally blocked in vitro and in vivo on patient-derived AML cells using an anti-functional humanized VLA4 antibody, Natalizumab (NZM). VLA4-positive (>90%) patient-derived (primary) AML cells were plated on immobilized human VCAM1 or human stromal cell line HS-5 and treated with control (IgG4) or Natalizumab (NZM) for 2 days. NZM de-adhered 94.0%±7.6 AML cells from its counter receptor VCAM-1, yet only 31.3%±13.8 from HS-5, indicating that stroma cells offer ligands for a wider panel of adhesion receptors besides VLA4. We tested also whether VLA4 blockade is beneficial against AML when combined with chemotherapy. For this purpose, primary AML cells were incubated with NZM and incubated on uncoated tissue culture plates or HS-5 stromal layers in the presence or absence of Ara-C (1µM) for two days. AML cells showed higher viability under Ara-C therapy when incubated with HS-5 cells compared to controls, indicating the chemoprotective effect of the stromal layer. The viability of the AML cells treated with combined Ara-C and NZM was similar to the controls, indicating that HS-5-mediated chemo-protection was completely abrogated by NZM. Significantly more AML cells treated with Ara-C+NZM stained AnnexinV+/7AAD- than after Ara-C+control Ig4 treatment (44.4%±5.6 vs. 29.8%±4.8, p=0.03) indicating increased apoptosis of AML cells. On its own, NZM did not induce apoptosis. Next, we tested NZM as a single agent in our NOD/SCIDIL2Rγ deficient (NSG) xenograft model of primary AML. Luciferase-labeled AML cells were intrafemorally injected into NSG mice (1x105 cells / mouse). NZM (5mg/kg) was given intraperitoneally once per week for 4 weeks. NZM-treated animals survived significantly longer than control Ig-treated animals (Median Survival Time, MST=107 days vs. MST=76 days; *p=0.008 by Log-rank Test.To determine effects of NZM on leukemia cell burden/distribution in different organs, primary AML cells were injected into NSG mice and allowed to engraft for 3 days, subsequently treated with a single dose of NZM or Ig control. 72 hours later, AML cell burden in femurs and spleens of NZM-treated animals was significantly decreased compared to control treated mice, however AML cells were not increased into the peripheral blood, so that whether leukemia cells were selectively killed ormobilized and then retained in non-hematopoietic organs remains to be determined. Further studies addressing molecular mechanisms of increased apoptosis after combined VLA4 blockade and chemotherapy are ongoing. Our data suggest that the paradigm of leukemia cell targeting by VLA4 blockade, previously demonstrated by us for ALL, can also be applied to AML. Disclosures: No relevant conflicts of interest to declare.

Activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling plays a role in cell proliferation, survival, and drug resistance in solid tumors and hematologic malignancies including chronic myeloid leukemia (CML), B-cell precursor acute lymphoblastic leukemia (BCP-ALL), T-ALL and acute myeloid leukemia (AML). The investigational compound BEZ235 is a potent dual pan-class I PI3K and mTOR complex C1 and C2 inhibitor and an attractive agent for relapsed or refractory leukemias. Primary objectives of this phase I study were determination of the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) in pts. with advanced acute leukemia. Secondary objectives included assessment of pharmacokinetics (PK), pharmakodynamic (PD) parameters and preliminary evidence of anti-leukemic activity. Inclusion criteria included age > 18years, relapsed or refractory AML, ALL or CML-BP considered ineligible for intensive or established treatment. Pts. with a fasting blood glucose >160mg/dl or an HbA1c >8% were excluded. The starting dose of BEZ235 was 400 mg twice daily (BID), administered orally during 28d cycles. Dose escalation was based on a “rolling-six”design, followed by an expansion phase at the RP2D. PK analyses were performed on days 1 and 15 by HPLC and fluorescence detection, PD analysis included assessment of phosphorylation of AKT, S6 and 4EBP1 by Western blotting (WB) and flow cytometry. The presence of PI3KCA, AKT or PTEN mutations was evaluated by direct sequencing of exons with known mutation hotspots. All pts. gave informed written consent, the study was approved by the Ethics Committee of the University of Frankfurt. 22 pts. (13m, 9f), median age 62.5 years (range 29-82), were enrolled. Types of leukemia were AML (n=11), BCP-ALL (n=9), T-ALL (n=1) and CML in myeloid blast phase (CML-BP, n=1). 6 pts. were in first and 9 pts. in second or later relapse, 7 refractory or in refractory relapse, 7 pts. had extramedullary leukemia, 14 pts. previously received an allogeneic stem cell transplant (SCT). Six pts. were evaluated at the starting dose of BEZ235 (400 mg BID). No DLTs were observed, but BEZ235-related AEs (stomatitis and GI toxicity grades 1-3) necessitated treatment interruptions in 3 of 6 pts. 400 mg BID was considered not tolerable for prolonged administration and 16 pts. were subsequently treated at dose level -1 (300 mg BID). The most frequent non-hematologic AEs were gastrointestinal primarily of grades 1 and 2 with diarrhea (n=20), nausea/vomiting (n=18/6), stomatitis/mucositis (n=20), decreased appetite (n=14), fatigue (n=10) and hyperglycemia (n=21). Grade 3/4 AEs included sepsis (n=6), pneumonia (n=4), diarrhea (n=3), hyperglycemia (n=2), mucositis and fatigue (2 each). No patient started at dose level -1 was dose-reduced and none discontinued BEZ235 because of toxicity, 300 mg BID was selected as the RP2D. Clinical responses were observed in 4 of 22 pts. (3/10 ALL): one pat. with pro-B ALL achieved a complete hematologic and molecular remission with full donor chimerism, ongoing after 11 cycles of BEZ235. Hematologic improvement was observed in two pts. with BCP-ALL (1 Ph+, 1 Ph neg) and stable disease of 4 mos. duration in an AML patient. Nineteen of 22 pts. discontinued because of disease progression, median time to progression was 28 days (5d-112d). PK analysis revealed substantial interpatient variability of peak and trough levels at steady state, with no clear dose-dependency. All three responders in whom PK data are already available had low steady state trough levels below 100 ng/ml. No activating mutations of PIK3CA, AKT or PTEN were identified in any of the 22 pts. Phospho-flow and WB analysis provided no evidence of PI3K pathway activation, even in responding pts. In conclusion, the RP2D for BEZ235 was determined to be 300 mg BID, without formal definition of DLTs and an MTD. Single-agent anti-leukemic efficacy was most pronounced in ALL, with an overall response rate of 30% and a sustained molecular remission in one patient. Results of PK analysis and assessment of PD markers associated with PI3K signaling did not correlate with response. The PI3K pathway appears to be a “driver pathway” in only a small minority of pts. with ALL or AML, but more comprehensive genomic analysis may identify a subset of patients likely to benefit from treatment with dual PI3K-mTOR inhibitors. Disclosures: Ottmann: Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background In relapsed or refractory acute myeloid leukemia (AML) long-term disease-free survival may only be achieved with allogeneic stem cell transplantation (HSCT). However, only about 40% of patients (pts) with relapsed AML receive HSCT. A number of factors contribute to this low rate, among them, a moderate activity of currently available salvage regimens and accumulating toxicity of chemotherapy. Clofarabine is considered to have a favorable risk-benefit ratio in this indication and has been successfully used in conditioning regimens. Our goal was to study the safety and efficacy of a clofarabine salvage therapy as a bridge to HSCT. Here, we report the results of the BRIDGE trial (NCT 01295307), a phase II, multicenter, intent-to-transplant study. Patients and Methods Between March 2011 and May 2013, 84 pts with relapsed or refractory AML older than 40 years were enrolled. Pts were scheduled for at least one cycle of induction therapy with CLARA (clofarabine 30 mg/m2 and cytarabine 1 g/m2 days 1-5). Pts with a donor received HSCT in aplasia after first CLARA. In case of a prolonged donor search HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine 30 mg/m2 day -6 to -3 and melphalan 140 mg/m2 on day -2. In pts with partially matched unrelated donors ATG (Genzyme) at a cumulative dose of 4.5 mg/kg was recommended. GvHD prophylaxis consisted of CsA and mycophenolate mofetil. Results Median age was 61 years (range 40 – 75). Forty-four pts suffered from relapsed AML and 40 pts had refractory disease. According to the current ELN risk stratification 17% of pts were classified as favorable risk, 35% as interm. I, 17% as interm. II and 20% as adverse risk. Complex and monosomal karyotypes were present in only 12% and 10% of pts, respectively. FLT3, NPM1 and CEPBA mutations were found in 16%, 24%, and 4% of the pts. The mean value of the HCT-CI score was 1.6 (range 0 - 7) at the time of study enrollment and 2.3 (range 0 - 7) at the time of conditioning. The overall response rate assessed at day 15 after start of CLARA was 80% (46% good response defined as less than 10% blast in the bone marrow (BM) and 33% moderate response with at least a marked reduction in BM blasts or BM cellularity and absence of blast in the peripheral blood). Seventeen pts did not respond to CLARA and were subsequently treated off study. Due to early death, three pts were not evaluable for treatment response. Overall, 66% of the pts received HSCT within the trial. Donors were HLA-identical siblings in eight pts (14%), HLA-compatible unrelated donors in 30 pts (55%) and unrelated donors with one mismatch in 17 pts (31%). Treatment success defined as complete remission, CR with incomplete recovery or >95% BM donor chimerism and an absolute neutrophil count >0.5 /nL on day 35 after HSCT was achieved in 62% of the pts. Disease-free survival (DFS) is shown in Figure 1. With a median follow up of 16 months the OS for all enrolled patients at one year is 51% (95% CI, 39% to 63%). At the time of enrollment, 14% had a related donor and 33% had an unrelated donor. In 46% of the pts donor search was initiated at the time of enrollment. For 7% of pts donor search was not successful. Time from study entry to HSCT was remarkably low with a median of 33 days (range 19 – 116 days). Of note, time interval did not differ between related and unrelated donors (Figure 2). Day 30 and day 100 mortality, which covered salvage therapy and HSCT, was 9% and 27%, respectively. Six out of seven pts who died within the first 30 days hat refractory AML and thus entered the trial already with a history of long-lasting neutropenia. Liver toxicity was the most frequent adverse event. Fifty percent of the pts had transiently elevated liver enzymes CTCAE grade III considered to be related to clofarabine. Twenty-one patients developed CTCAE grade III – IV sepsis throughout the study treatment. GvHD grade II – IV and III-IV until day 100 after HSCT occurred in 36% and 21% of the pts, respectively. Conclusions This intent-to transplant study allows for a realistic estimate for the outcome of elderly pts with relapsed or refractory AML. We demonstrate a high rate of leukemia-control by CLARA. Fast unrelated donor search and work up and conditioning with clofarabine and melphalan in aplasia allowed for a high rate of successful HSCTs. While the long-term results require longer follow-up the overall results are promising. Disclosures: Middeke: Genzyme: Speakers Bureau. Schetelig:Genzyme: Research Funding. Off Label Use: Clofarabine, not approved for AML.

Inhibitors of class I/II histone deacetylases (HDACi) possess anti-leukemic activity and have been reported to modulate the function of immune effector cells. Thus, they could provide specific clinical benefit in the allogeneic hematopoietic stem cell transplantation (HSCT) setting. Panobinostat (PAN) is a potent, orally available pan-HDACi reported to either suppress or stimulate regulatory T cells (T reg), depending on the administered dose (Shen L & Pili R, OncoImmunology 1;7:948, 2012). The feasibility and efficacy of PAN treatment following HSCT for patients (pts) with high risk acute myeloid leukemia (AML) has not been established. We report clinical and translational results of the dose-escalation phase of the PANOBEST study with PAN as post-HSCT maintenance. Primary objectives were, based on dose-limiting toxicity (DLT), determining the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) of PAN in patients with high risk AML or myelodysplastic syndromes (MDS) in complete remission (CR) after reduced-intensity conditioning HSCT. Secondary objectives were the evaluation of safety, tolerability and immunoregulatory properties of PAN, and overall (OS) and disease-free survival (DFS) of treated patients. PAN was started at 10 mg p.o. three times a week (TIW) and escalated to 20 and 30 mg TIW using a 3+3 design. Treatment was initiated 60-150 days after HSCT and continued for up to one year. Eligibility criteria included: recovery of peripheral blood counts, adequate organ function and no severe graft versus host disease (GvHD). All pts gave written informed consent. DLT was defined as prolonged grade 4 hematologic or grade 3/4 non-hematologic toxicity within 28 days of the first PAN dose. Immunophenoytyping of lymphocyte subsets was performed pre-treatment and on days 3, 8, 30, 90, 180 and 360. 12 pts (11 AML, 1 MDS), median age of 52 years (21-62) were enrolled. PAN was started within a median of 73 days (60-126) after HSCT, which was performed with active disease (n=11) or in CR2 (n=1). The RP2D was determined to be 20 mg TIW based on one DLT (fatigue grade 3) at 20 mg and two DLTs (nausea/emesis and colitis grade 3 each) at 30 mg. Grade 2-4 adverse events (AEs) were reported in 10 out of the 12 pts (83%). Grade 3/4 AEs included hematologic toxicity (50% of pts), laboratory alterations (33%), gastrointestinal symptoms (25%), fatigue, pulmonary infection (17% each), sepsis, herpes stomatitis, diabetes, syncope, deep vein thrombosis and pulmonary embolism (8% each). Toxicity was reversible and required at least one PAN dose reduction in 3 pts. Acute GvHD grade 2 (1 pt) and 3 (2 pts) was responsive to steroids in 2 pts or salvage therapy in 1 pt. Four pts developed mild (n=3) or moderate (n=1) chronic GvHD. To date, 5 pts have completed one year of PAN and 2 pts remain on treatment (days 238, 290). Five pts discontinued treatment prematurely after 10-217 days due to grade 3 toxicities (n=4) or AML relapse (n=1). With a median follow up of 579 days (129-911), 11/12 pts are alive and 10/12 in continuous CR after HSCT. Seven pts received prophylactic donor lymphocyte infusions (DLIs, 1-5 doses of 0.1-20x106 CD3+ cells/kg). Immunophenotyping revealed no impact of PAN on absolute T reg numbers (9 pts), but a significantly reduced proportion of CD4+CD25++CD127dim/- T reg to CD3+CD4+ T helper (Th) cells by day 8 after 3 doses of PAN (mean±SEM: 14.6±2.6 vs. 9.6±1.2%, p value of t test =0.03). While Treg/Th proportion continuously decreased in pts with ongoing CR, it again increased after PAN discontinuation or remained stable under PAN treatment in both relapsing patients. Outcome of the study population was compared with a historical cohort of 29 consecutive pts with active AML transplanted in Frankfurt in 2000-2009. Both cohorts were similar in age, gender, disease stage or BM blasts at time of HSCT, donor type and use of DLIs. In a landmark analysis including all pts who were in CR and without severe GvHD on day 73 after HSCT, probabilities for DFS and OS at 18 months after HSCT were 83±11% vs. 55±9% (p=0.145, log-rank test) and 92±8% vs.66±9% (p=0.085) in the PANOBEST vs. historical cohort (Fig 1). PAN is well tolerated after HSCT at a RP2D of 20 mg TIW. Comparison with a historical control cohort of pts transplanted with active AML shows a low relapse rate, which appears to be associated with a PAN-induced modulation of the T reg/Th proportion. Disclosures: Bug: Novartis: Honoraria, Travel grants, support for the clinical study Other. Ottmann:Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Purpose Patients with acute myeloid leukemia (AML) and abnormalities (abnl) of the short arm of chromosome 17 (17p) are considered to be at high risk of treatment failure after conventional chemotherapy. Small studies have suggested that this abnormality may portend a poor prognosis even after allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to assess the prognostic role of abnl(17p) in a larger cohort of patients with AML undergoing allogeneic HSCT, and to analyse the impact of disease status, conditioning regimen, and type of abnormality. Here, we present data on the outcome of 201 patients with abnl(17p) AML transplanted since 2000. Patients and Methods We performed a retrospective cohort analysis based on study-registries from two AML groups, HOVON and SAL, and transplant-registries of the Fred Hutchinson Cancer Research Center (FHCRC) and the German Cooperative Transplant Study Group (GCTSG). Inclusion criteria were AML diagnosed according to the current WHO criteria with 17p abnormalities and a first HSCT between January, 1, 2000 and January, 1, 2011. Overall survival (OS), event-free survival (EFS), cumulative incidence of relapse (CIR) and non-relapse-mortality (NRM) after HSCT are reported for the whole cohortand for patients receiving HSCT in first complete remission (CR1). We tested for center effects (FHCRC, HOVON, SAL, GCTSG) in a multivariate Cox regression model. Results Data from 201 patients with full information on the karyotype were analysed. The median age was 54 years with a range from 2 years to 75 years. Five patients were younger than 18 years. Sixty-one percent of the patients suffered from de novo AML, while 26% had secondary AML and 11% therapy-related myeloid neoplasm. Complex and monosomal karyotypes were present in 90% and 77% of patients, respectively. Eighty-four patients (42%) were in CR1 at the time of HSCT. Seventy patients (35%) were treated with standard myeloablative conditioning (MAC) regimens while the remaining patients received reduced intensity conditioning (RIC). Donors were matched siblings in 34%, matched unrelated donors in 43% and partially matched or mismatched unrelated donors in 18% of the patients. Eight patients (4%) had a haploidentical donor. At the time of analysis 30 patients were alive with a median follow-up of 30 months (range 1 to 121 months). At three years, the probabilities of OS and EFS were 15% (95% CI, 10% to 20%) and 12% (95% CI, 7% to 16%), respectively, whereas the CIR was 49%. For patients transplanted in CR1 the probability of OS at three years was 22% (95% CI, 13% to 32%) compared to 9% (95% CI, 3% to 15%) for those with advanced disease (p= The incidence of grade II to IV acute GvHD up to day 100 was 32% while grade III to IV occurred in 11% of the patients. Due to the high frequency of competing events (death or relapse before onset of GvHD) the cumulative incidence for chronic GvHD at one year was very low with 8%. Conclusion Patients with abnl(17p) AML have a poor outcome after HSCT. The observation of better outcome in patients with less advanced disease stages and without MK argues against primary resistance to allogeneic immune effects of 17p abnormalities. While transplantation in CR1 may still be considered the treatment of choice due to the lack of more promising alternatives, novel strategies to prevent relapse are highly warranted for this group of patients. For patients with abnl(17p) AML in more advanced stages experimental approaches should be considered. Disclosures: No relevant conflicts of interest to declare.

Introduction Epigenetic therapies with azanucleoside DNA hypomethylating agents, alone or in combination with histone deacetylase inhibitors (HDACi), show clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly when given at non-cytotoxic doses. They are able to reactivate epigenetically silenced genes including, among others, a number of highly immunogenic proteins dubbed Cancer/testis antigens (CTAs), predominantly the CTAs located on the X chromosome. We have previously shown that decitabine can induce expression of several CTAs, including MAGEB2 and NY-ESO-1, in myeloid cells in vitro and thereby trigger an immune response (Almstedt et al., Leuk. Res. 2010). Induction of a CTA-specific cytotoxic T cell response in vivo was reported also in AML patients treated with azacitidine and sodium valproate (VPA) and correlated with clinical response (Goodyear et al., Blood 2010). To the best of our knowledge, no data have yet been reported on the effect of combination treatment with decitabine and panobinostat or sodium valproate (VPA) on CTA reactivation in myeloid leukemia. Aim We hypothesized that by combining decitabine with HDACi we could further enhance expression of CTAs in myeloid leukemia cells and thereby boost recognition of the malignant cells by the cytotoxic T lymphocytes. Methods The myeloid cell lines U937 and Kasumi-1 were treated with decitabine alone or in combination with the HDACi VPA or panobinostat applied at non-toxic concentrations (>80% cell viability). Expression of CTAs was analyzed by RT-qPCR and Western blot after 48 hours of HDACi treatment. DNA methylation of NY-ESO-1 and MAGEB2 promoter regions was quantified by pyrosequencing. Bone marrow mononuclear cells from 19 AML patients (treated with or without VPA as add-on to decitabine in the ongoing randomized phase II DECIDER clinical trial, NCT00867672) were collected before and on day 15 of treatment, in some patients also after 2 treatment cycles. CTA mRNA expression and promoter DNA methylation were quantified as described above. Results VPA or panobinostat alone did not induce MAGEB2 or NY-ESO-1 expression in vitro. However the pretreatment of cells with decitabine prior to addition of either HDACi resulted in a synergistic dose-dependent reactivation of MAGEB2 and NY-ESO-1 on the mRNA level (confirmed for the latter on the protein level). Pyrosequencing analysis of the heavily methylated NY-ESO-1 and MAGEB2 promoters revealed, as expected, no methylation changes upon HDACi treatment, but a dose-dependent hypomethylation upon decitabine. In recently initiated in vivo studies (DECIDER trial), until now cells from 19 AML patients receiving epigenetic treatment were sequentially analyzed. Induction of MAGEB2 mRNA was observed in 9 patients (from absent to a median of 0.002 relative to GAPDH, range 0.0004-0.043), with concomitant DNA hypomethylation of the MAGEB2 promoter from median 83% pretreatment methylation (range 63%-90%) to 63% posttreatment (range 44%-74%). In 5 patients modest hypomethylation without changes in MAGEB2 expression was observed (from median pretreatment values of 89% [72%-92%] to 82% [58%-87%] posttreatment). Another 5 patients disclosed neither hypomethylation nor reexpression of MAGEB2 (results as yet blinded to treatment arm and clinical response). Conclusions Combined epigenetic treatment with the hypomethylating agent decitabine and the HDACi VPA or panobinostat synergistically induced a dose-dependent reactivation of the CTAs MAGEB2 and NY-ESO-1 in vitro, accompanied by promoter hypomethylation. First translational results of the DECIDER AML trial also indicate in vivo effects of the epigenetic treatment on CTA induction. The unmasking of CTAs to the immune system by epigenetically active drugs can increase anti-tumor immune responses, and thus has clear implications for future clinical trials combining epigenetic therapy and specific immunotherapy in myeloid neoplasia. Disclosures: No relevant conflicts of interest to declare.

Abstract 411 Background: LDAC is an established treatment option for patients (pts) with AML considered ineligible for intensive remission induction treatment. However, the outlook for pts who receive LDAC remains unsatisfactory, and novel therapeutic strategies are needed to improve clinical outcome in these pts. Plk1 plays a key role in mitosis and cell cycle progression and is an attractive target for novel therapeutic approaches in cancer. Volasertib (V) is a first-in-class, selective and potent cell cycle kinase inhibitor that induces mitotic arrest and apoptosis by targeting Plks. The phase I part of this trial determined the maximum tolerated dose of V in combination with LDAC (V + LDAC) to be 350 mg and demonstrated antileukemic activity of V and V + LDAC in pts with relapsed/refractory AML ineligible for intensive therapy (Bug et al, ASH 2010 and 2011). Here we present preliminary phase II data for the randomized comparison of V + LDAC vs LDAC in pts with newly diagnosed AML ineligible for intensive treatment. Methods: In the phase II part of this open-label study, eligible pts were randomized to receive V (350 mg 1-hr intravenously, days 1, 15 Q4W) + LDAC (20 mg bid subcutaneously, days 1–10 Q4W), or LDAC alone until progression/relapse or intolerance. The primary endpoint was objective response (complete remission [CR] or CR with incomplete blood count recovery [CRi]); secondary endpoints included event-free survival (EFS), overall survival (OS), safety and pharmacokinetics (PK). Results: 87 pts were treated with V + LDAC (n=42) or LDAC (n=45). Pt characteristics (V + LDAC/LDAC) were largely balanced: median age, 75/76 yrs; secondary AML, 40.5%/64.4%; adverse cytogenetic group, 35.7%/33.3%. At time of analysis (February 22 2012) 15 pts were still on treatment (12 with V + LDAC). Pts received a median (range) of 2 (1–12) cycles of V + LDAC and 2 (1–11) cycles with LDAC. A significantly greater proportion of pts who received V + LDAC achieved a CR or CRi compared with pts who received LDAC (31.0% vs 11.1%; odds ratio 3.59 [95% CI: 1.15, 11.18]; P = 0.0277), with a median (range) time to remission of 71 (29–158) days and 69 (34–125) days, respectively. Remissions with V + LDAC were observed across genetic groups, including pts with adverse cytogenetics. A trend for longer median EFS was observed for pts who received V + LDAC compared with those who received LDAC (HR 0.61 [95% CI: 0.35, 1.05]; P = 0.0725; Figure). Follow-up for OS was ongoing at the time of this analysis. Among pts achieving CR/CRi, only 2 had experienced recurrence or death at the time of analysis (1 in each arm after a remission duration of 57 [V + LDAC] or 67 [LDAC] days). For all other pts ongoing in remission, the remission duration was censored after 53–407 days (LDAC + V) or 32–282 days (LDAC), consistent with prolonged duration of remission in some pts. The most frequent all grade adverse events (AEs) in the V + LDAC arm were febrile neutropenia (50%), constipation (45.2%), nausea (40.5%) and anemia (40.5%). In the LDAC arm, the most common all grade AEs were nausea (33.3%), anemia (28.9%), pyrexia (28.9%), and constipation, asthenia and diarrhea (26.7% each). More pts who received V + LDAC experienced ≥ grade 3 AEs than those who received LDAC (95.2% vs 68.9%), particularly for blood and lymphatic system disorders (81.0% vs 44.4%), gastrointestinal disorders (21.4% vs 6.7%), and infections and infestations (45.2% vs 22.2%). The early death rates (V + LDAC/LDAC) at 30, 60 and 90 days were comparable between the two treatment arms: 30 days, 9.5%/8.9%; 60 days, 21.5%/17.8%; 90 days, 28.9%/33.4% (rates calculated using Kaplan-Meier method). PK analyses demonstrated that V is a moderate clearance drug with multi-compartmental PK behavior, a large volume of distribution and a long terminal half-life. Preliminary data suggest no drug-drug interactions following combination of V with LDAC. Conclusions: These preliminary phase II data demonstrate a significantly improved CR/CRi rate and a trend for EFS benefit with V + LDAC compared with LDAC alone in pts with newly diagnosed AML ineligible for intensive treatment. An increased frequency of AEs was observed with the addition of V, which was expected given its myelosuppressive mechanism of action; available data do not suggest increased early mortality for V + LDAC vs LDAC. A confirmatory phase III trial is needed to determine the clinical benefit of V + LDAC in pts with AML ineligible for intensive treatment. Disclosures: Off Label Use: Volasertib is an investigational agent. Fiedler:Pfizer, Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Bug:Boehringer Ingelheim: Honoraria, Membership on an entity's Board of Directors or advisory committees. Müller-Tidow:Boehringer Ingelheim: Research Funding. Voss:Boehringer Ingelheim: Employment. Taube:Boehringer Ingelheim: Employment. Fritsch:Boehringer-Ingelheim: Employment. Döhner:Celgene, Amgen, Ambit, Astellas, Lilly: Consultancy.

Abstract 485 Transfusional iron overload (TIO) contributes considerably to treatment-related morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Data on post-HCT treatment of secondary hemochromatosis are limited. The results of an open-label, single arm, multi-center trial evaluating the efficacy and safety of the iron chelator deferasirox (DEX) in recipients of HCT with TIO are presented. Patients and methods: The study was conducted in 6 German centers. The primary objective was to evaluate if DEX could provide clinically acceptable chelation in a target pool of 76 adult patients (pts) with TIO 3–12 months after related/unrelated HCT irrespective of conditioning regimens. TIO was defined as a serum ferritin (SF) ≥1000ng/ml without active inflammation (CRP 20 units of red blood cell transfusions (RBC) or 100mL/kg of prepacked red blood cells. Main exclusion criteria were ANC 5xULN, and serum creatinine (Cr) >1.5xULN. DEX 30 minutes prior to lunch was started with 10 mg/kg/d over 52 weeks or until SF 33% of baseline (BL) or was >1.5xULN. DEX had to be interrupted if any toxicity > grade 2 according to the NCI common toxicity criteria (CTC) occurred. An independent Data Safety Monitoring Board reviewed safety data on an ongoing basis. Surrogate markers monitored were SF, serum transferrin and transferrin saturation (TS). Results: After a median of 168 days after HCT, 47 (62%) males and 29 (38%) females [median age 56 y] were included. Cyclosporine was taken by 54 (71%) pts. Median number of RBC prior to study entry was 37 units. Post-HCT, 14 (18.4%) pts received transfusions. Post-HCT HFE genotype was mutated in 27/67(40%) pts (heterozygote-H63D,n=13; heterozygote-C282Y,n=7; heterozygote for other mutations, n=6; homozygote-H63D,n=1). At BL, median SF and TS were 2045ng/ml and 56% respectively at a median CRP of 2.7 mg/l. SF correlated with TS (r=0.5, pgrade 2 were 1.6%, 2.3%, 5.5%, and 2.8% respectively. Main non-hematologic adverse events (AEs) were gastrointestinal (GIT) (nausea, vomiting, diarrhea) (16.9%); infections (15.1%); skin (rash, erythema) (4.4%). Cr values above BL were observed in 41 (54%) pts. At EOS, median Cr was above BL by a median of 10 μmol/l. AEs were mostly mild to moderate, transient, and dose-related. On study, a median of 2 drug dosage adjustments/interruptions were made mainly for Cr elevation (27%), GIT-AEs (15%), TA elevation (10.4%), and skin AEs (3.8%). Study drug was permanently discontinued because of GIT-AEs in 3 (4%), skin AEs in 3 (4%), TA in 2 (2.6%), and Cr in 2 (2.6%) pts. Conclusions: A highly significant reduction in serum ferritin could be induced with deferasirox after allogeneic HCT with an acceptable safety profile. Stable trough serum cyclosporine levels could be maintained over time irrespective of the daily dose of deferasirox. Our results indicate that a daily dose of 7.5–10mg/kg is effective, and well tolerated in a complex cohort with TIO after allogeneic HCT. Disclosures: Al-Ali: Novartis: Honoraria. Lieder:Novartis Germany: Employment. Albrecht:Novartis Germany: Employment. Leismann:Novartis Germany: Employment. Bug:Novartis Germany: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant Other. Kroeger:Novartis Germany: Honoraria, Research Funding. Platzbecker:Novartis Germany: Advisory Board Other, Honoraria.

Abstract 1964 Introduction: Treatment with azacitidine (Aza) and donor lymphocyte infusions (DLI) can induce sustained remissions in some patients (pts) with AML or MDS relapsing after allogeneic stem cell transplantation (allo-SCT). Meanwhile incidence and severity of GvHD seems to be relatively low when compared to historical data using DLI alone. As a potential mechanism murine models have suggested that Aza upregulates the transcription factor FoxP3 thereby expanding CD4+ regulatory T cells (Tregs). This has also been recently shown in 17 AML pts receiving Aza maintenance therapy following allo-SCT (Goodyear et al., 2012). Patients and Methods: To confirm and expand this knowledge we monitored CD4+CD25+FoxP3+ Tregs and lymphocyte subsets (CD3+; CD3+/CD4+; CD3+/CD8+; CD3−/CD56+; CD20+) by flow cytometry in 46 pts during salvage therapy with Aza (up to 8 cycles either 100 mg/m2/day d1-5 or 75 mg/m2/d d1-7) and DLI (envisaged on day 34/90/146) for relapse following allo-SCT. PB samples were obtained prior treatment (d0), after the 1st (d6), 2nd (d34), 4th (d90) and 6th cycle (d146). To assure a serial measurement only pts who had received at least 4 Aza cycles were eligible. Thereby 13 pts could be included, while 33 pts were excluded as a consequence of early drop-of resulting from progression or death (n=21), or due to missing samples (n=12). Results: Relapse of AML (n=8) or MDS (n=5) occurred in median 446 d (range:19–1688) following allo-SCT in these 13 pts. They received a median of 6 Aza cycles (range: 4–8). DLI were administered in all patients with a median number of 2 DLI per patient (range:1–4) resulting in a median total T cell dose of 5.0×106CD3+ cells/kg per patient (range:1–119). A CR rate of 62% (n=8) was observed in these 13 pts being overestimated in comparison to a CR rate of 33% (n=15) in the whole group due to positive selection of pts. Prior to relapse 6 pts (46%) had suffered from aGvHD (Io 1 pt, IIo 1 pt, IIIo 3 pts, IVo 1 pt) and 2 pts (15%) from cGvHD (limited 1 pt, extensive 1 pt). At the beginning of Aza treatment 3 pts were still on immunosuppresion which could be tapered in all cases without GvHD flare. Following treatment with Aza aGvHD was observed in 5 pts (overall 38%, Io 3 pts, IIIo2 pts) in median 129 d (range: 20 – 253) following the 1st DLI, while cGvHD developed in 6 pts (overall 46%, limited 5 pts, extensive 1 pt). In concordance with this rather mild presentation of GvHD, a 1.5-fold increase of Tregs was observed after 4 Aza cycles (d0: 8.23/μl vs. d90: 13.26/μl, p=0.0479). By grouping the pts on the basis of the median time to relapse (day 446), we observed a 3.2-fold increase of the absolute number (d0: 4.7/μl vs. d90: 14.8/μl, p=0.031) as well as an 1.9-fold increase of the frequency of Tregs (d0: 6.7% vs. d90: 12.9% of CD3+CD4+ cells, p=0.06) during treatment with Aza in the group of patients who relapsed early. On the other hand, in those patients who relapsed late the absolute number of Tregs (d0: 12.2/μl vs. d90: 11.9/μl, n. s.) was already higher and remained together with the Treg frequency (d0: 4.7% vs. d90 3.9%, n. s.) unchanged during treatment. Of interest, in those patients with early relapse only 1 pt developed aGvHD (Io), in contrast to 4 pts with aGvHD in those with late relapse. No significant changes were observed with regard to other lymphocyte subpopulations. Conclusions: We here demonstrate an intra-individual Treg expansion, which might be induced by Aza and may explain the low rate and mild presentation of GvHD observed following the combination of Aza and DLI. In line with the data of Goodyear et al. Aza-induced expansion of Tregs seems to be restricted to patients relapsing early after allo-SCT where the Treg repertoirs is still immature. Disclosures: Schroeder: Celgene: Travel support Other. Platzbecker:Celgene: Honoraria, Research Funding, Speakers Bureau. Bug:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, Travel support Other. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kröger:Celgene: Research Funding. Kobbe:Celgene: Research Funding.

Abstract 3816 Background Conventional chromosome banding (CCB) analyses of bone marrow (bm) metaphases represent the gold standard of cytogenetic diagnostics in myelodysplastic syndromes (MDS), but they are not suitable for frequent follow-up analyses. Most aberrations can also be detected by fluorescence in situ hybridisation (FISH), and they are provable in CD34+ cells from peripheral blood (pb). In our prospective multicenter German diagnostic study “Screening and genetic monitoring of patients with MDS under different treatment modalities by cytogenetic analyses of circulating CD34+cells” (ClinicalTrails.gov NCT01355913) we followed MDS pts by sequential FISH analyses. Methods CD34+ pb cells were enriched by immunomagnetic cell sorting (MACS®) and analysed by FISH using a “Superpanel” (D7/CEP7, EGR1, CEP8, CEP XY, D20, TP53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p36/1q25, CSF1R, all Abbott® Products) at initial screening, every 12 months during follow-up and in case of suspected disease progression and a “Standardpanel” (EGR1, D7/CEP7, CEP8, TP53, D20, TEL/AML1, CEP XY, plus -if necessary- another informative probe) every 2 months in the 1st and every 3 months in the 2nd and 3rd year. If bm aspirate was available, additional CCB and FISH analysis of CD34+ and native bm cells were performed. Cut-off values for each FISH probe were evaluated in our lab. Cytogenetics, bm morphology, clinical course and therapies were documented in a database. All pts gave their written informed consent. The study was approved by all local ethic committees. Results After 3 years of study time, 361 patients (25 AZALE (University of Dresden), 110 LEMON5 (University of Duesseldorf), 226 CD34+FISH) have been included in the study, resulting in a total number of 19,516 FISH analyses: Median age, gender distribution and MDS subtypes were typical for the disease, median follow-up at the time of analysis was 8.2 (1–36) months. Chromosomal aberrations could be detected by FISH of CD34+ pb cells in 71.5% of pts (55% of CD34+FISH-cohort, 99% of LEMON5-trial pts, 100% of AZALE-trial pts). FISH and CCB were highly correlated: p Discussion Our interim results demonstrate that FISH analysis of circulating CD34+ pb cells provides relevant cytogenetic informations. It is a reliable novel method for screening and cytogenetic monitoring of MDS pts during the course of disease and under different therapies, and helps in cases where a bm biopsy is not possible or not successful. Disclosures: Braulke: Celgene: This study was supported by Celgene. Other. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:Celgene: Honoraria, travel support, advisory board Other; Novartis: Honoraria, travel support, advisory board, travel support, advisory board Other; Boehringer Ingelheim: Honoraria, travel support, advisory board, travel support, advisory board Other. Schafhausen:Novartis: Honoraria, travel support Other; BMS: Honoraria, travel support, travel support Other; Roche: Honoraria, travel support, travel support Other; Celgene: Honoraria, travel support, travel support Other; Alexion: Honoraria, travel support Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 921 This 3+3 dose escalation phase I trial by the German MDS study group aimed to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of sequential azacitidine (AZA) 75 mg/m2 for 5 days followed by 14 days of up to 25 mg lenalidomide (LEN) in patients with higher-risk myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) and del(5q) cytogenetic abnormalities. Twenty patients (median age 69 years) were enrolled, including 16 (80%) with complex cytogenetic abnormalities and 65% of patients harboring at least one TP53 mutation. Nine patients (45%) had no prior treatment while relapse or progression to single agent azacitidine or lenalidomide treatment (30%) and allogeneic HSCT (15%) were the most common regimens among previously treated patients. Three of 6 patients treated with LEN at the 25 mg dose level experienced DLTs, with 20 mg subsequently identified as MTD. All eligible patients experienced treatment-related Grade 3/4 thrombocytopenia and neutropenia. Hematologic responses occurred in 4 of 9 (44%) previously untreated patients with complex karyotypes, including 3 with a TP53 mutation and were preceded by a significant decrease of del(5q) CD34+ progenitor cells in the blood during cycle 1. In fact, compared with baseline, the percentage of peripheral blood CD34+ cells with del(5q) clone was decreased in the hematologic responders after 1 cycle of azacitidine followed by lenalidomide therapy (P = 0.001) (Figure 1A). In contrast, the percentage of peripheral blood CD34+ cells with del(5q) remained unchanged in hematologic non-responders (Figure 1B). By applying TP53 mutation directed deep-sequencing technology we observed a concomitant early reduction and disappearance of minimal residual disease in MDS patients achieving a complete cytogenetic response. In one patient, a reemergence of the TP53 clone was observed later despite continuation of the treatment but by consolidation (lower dose and every 8 weeks compared to 4 weeks with induction) and it preceded the hematologic and cytogenetic relapse by several months. In conclusion, in a population of higher-risk MDS/AML patients with del(5q) that included a high proportion of patients with complex karyotypes and mutations of TP53, the sequential combination of azacitidine and lenalidomide was shown to be a feasible and potentially effective treatment strategy, even in those patients with TP53 -mutated clones. We additionally observed a correlation between the percentage of peripheral CD34+ cells with del(5q) and hematologic response, suggesting that monitoring of this cell population may be a surrogate marker of response in this setting. Our results encourage an application of sequential azacitidine and lenalidomide as first line therapy for higher-risk MDS patients in future trials. (A) HEMATOLOGIC RESPONDERS (B) HEMATOLOGIC NON-RESPONDERS (A) HEMATOLOGIC RESPONDERS (B) HEMATOLOGIC NON-RESPONDERS Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kuendgen:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 1402 Over 20 different fusion partner genes of NUP98 have been reported in hematological malignancies with the majority of NUP98 fusions occurring in acute myeloid leukemia (AML). Recently, a specific fusion of NUP98 with Nuclear receptor-binding SET domain protein 1 (NSD1) has been analyzed in a large cohort of pediatric and adult AML patients. In this report, 16.1% of pediatric AML samples and 2.3% of adult cytogentically normal (CN)-AML cases were found to be NUP98/NSD1-positive. NUP98/NSD1-positive patients had an adverse outcome. The aim of our study was to investigate the frequency, clinical features and the prognostic impact of NUP98/NSD1 in another large, uniformily treated adult AML cohort, and in patients with myelodysplastic syndromes, which frequently precede overt leukemia. We studied 504 younger AML patients ( Analysis in the AML cohort was also performed for mutations in FLT3-ITD, NPM1, DNMT3A, IDH1, IDH2. Additional mutation analyses were performed in the subgroup of CN-AML for CEBPA, MLL-PTD, WT1 and WT1 SNP rs16754, NRAS. The NUP98/NSD1 fusion was identified by a nested RT-PCR using patient-derived cDNA. cDNA from a patient with proven NUP98-NSD1 fusion was used as a positive control. NUP98/NSD1 fusions were identified in 7 out of 504 younger adult AML patients (1.4%) while the NUP98/NSD1 fusion was not found in any of the 193 MDS patients. In the AML cohort, NUP98/NSD1 positive patients showed a higher number of peripheral blood blasts (P=.002), while other clinical characteristics such as FAB-subtype, type of AML, hemoglobin levels, white cell count or platelet count did not significantly differ between patients with or without the NUP98/NSD1 fusion. The majority of NUP98/NSD1 positive patients (5 out of 7) showed a normal karyotype while one patient was found to have a del(9) and another patient an inv(3)(q21q26) with monosomy 7. We identified an association between FLT3-ITD and NUP98/NSD1 fusion in our cohort (P=.007, 5 patients with NUP98/NSD1 also had a FLT3-ITD). This finding confirms the data by Hollink et al. and suggests a possible functional link between FLT3-ITD and the NUP98/NSD1 fusion in leukemogenesis. NUP98/NSD1 fusions were not associated with other mutations like those suggested in epigenetic regulation (DNMT3A, IDH1 and IDH2). In CN-AML, we also looked for an association between BAALC, ERG, EVI1, MN1, MLL5 and WT1 expression but did not find a significant difference between the expression of these genes and NUP98/NSD1 fusion genes. Due to the low frequency of the aberration outcome analysis was performed for explorative purposes. Considering the whole AML cohort we did not detect a significant difference for OS and for RFS between NUP98/NSD1 positive and negative patients (OS, HR 1.6; 95%CI 0.66–3.88; P=.3; RFS, HR 2.33; 95%CI 0.74–7.30; P=.147). However, NUP98/NSD1 positive patients had significantly lower complete remission (CR) rates (43 vs. 77 percent, P=.037). When considering only patients with CN-AML the NUP98/NSD1 positive patients (n=5) had no significantly different OS and RFS (OS, HR 2.08; 95%CI 0.77–5.64; P=.15; RFS, HR 2.88; 95%CI 0.71–11.71; P=.14). Again, a significantly lower CR rate was observed in the NUP98/NSD1 positive patients compared to NUP98/NSD1 negative patients with CN-AML (40 vs. 80 percent, P=.03). Because of the association between FLT3-ITD and NUP98/NSD1 we also investigated the prognostic impact in the subgroup of CN-AML patients also positive for FLT3-ITD. Again, OS and RFS did not differ between NUP98/NSD1 positive and negative patients (OS, HR 1.31; 95%CI 0.47–3.64; P=.61; RFS, HR 2.04; 95%CI 0.49–8.57; P=.33). For this subgroup a trend towards a lower CR rate was observed for NUP98/NSD1 positive FLT3-ITD positive CN-AML patients (40 vs 75 percent, P=.08). In summary, our analysis confirms the presence but low incidence of the NUP98/NSD1 fusion gene in adult AML patients and the strong association with FLT3-ITD. The specific association of NUP98/NSD1 with FLT3-ITD mutations warrants clinical investigation with FLT3 inhibitors in these patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4926 Introduction: Chromosomal banding analysis (CBA) of bone marrow metaphases is the gold standard to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). We aim to comprehensively detect and follow chromosomal abnormalities during the course of the disease without the need of repeated bone marrow biopsies. In ongoing studies we attempt to achieve this goal by performing serial fluorescence in situ hybridization (FISH) analysis on CD34+ peripheral blood cells (PBC). The aim of this pilot study was to establish SNP-array-analysis (SNP-A) on CD34+ PBC to complement genetic analysis on peripheral blood by identifying chromosomal abnormalities not detectable by FISH and/or CBA. Methods: We immunomagnetically enriched CD34+ PBC of 20 patients (pts) with MDS (16 pts), suspected MDS (1 pts) and secondary acute myeloid leukemia (sAML, 3 pts). SNP-A was performed with arrays from Affymetrix (3x SNP 6. 0, 4x Cyto 2. 7, 13x CytoScanHD). Fresh or frozen CD34+ PBC of 10 pts and in methanol/acetic acid fixed CD34+ PBC of 9 patients were successfully processed. One whole genome amplified sample was included. CBA and FISH-A was done for all patients. Results: By CBA, 3 pts had no chromosomal abnormalities, 8 pts had one abnormality, 6 pts had 2–4 abnormalities and 3 pts had more than 6 abnormalities. By SNP-A on CD34+ PBC, additional abnormalities could be revealed in 13/20 pts. In two pts they were also confirmed by FISH-A. Most of them were micro-deletions not detectable by CBA. In addition, SNP-A revealed uniparental disomies (UPD) in 5/20 pts. Of the 3 pts with no detectable abnormalities in CBA, one had a micro-deletion in 4q24 (TET2). The other two had an insufficient number of metaphases. One of them showed a highly complex karyotype by FISH-A and SNP-A on CD34+ PBC. The other one had suspected MDS and did not show any abnormalities by SNP-A. The 17 pts with ≤ 6 abnormalities in CBA showed 55 abnormalities by CBA, FISH-A and SNP-A altogether. 34/55 (62%) abnormalities could be detected by SNP-A and/or FISH-A, but not by CBA. 24/55 (44%) abnormalities could only be detected by SNP-A. 4/55 (7%) of abnormalities were structural abnormalities or small clones and were only detected by CBA. Serial analysis indicated clonal evolution: A patient with 16 abnormalities detectable by CBA and additional three by FISH and SNP-A developed two further micro-deletions (del(2)(q31q32), del(4)(q24q26)) within four months. When a MDS patient with a known 20q-deletion (isolated by CBA and FISH) progressed to AML 25 months after first diagnosis we detected 3 micro-deletions by SNP-A of peripheral blood (0. 98 Mb on 4q, 1. 31 Mb on 12q, 2. 55 Mb on 12q) thus resulting in 4 cytogenetic alterations fulfilling the criteria of complex and prognostically unfavorable abnormalities. Conclusions: Recently it was shown that abnormalities detectable by SNP-A, but not by CBA, could worsen prognosis of MDS patients. We succeeded in detecting these additional abnormalities without the need of bone marrow biopsies out of peripheral blood. Nevertheless, by parallel FISH and SNP-A of CD34+ PBC, most abnormalities detectable by CBA of bone marrow metaphases could be detected. Comprehensive genetic analysis at close intervals thus is possible without the need of bone marrow biopsies to study clonal evolution. The information gained could be used for therapy decisions, to improve prognostication and to unravel genetic evolutionary steps towards acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 1316 In acute myeloid leukemia (AML), translocations and the resulting fusion proteins (FPs) such as PML/RAR, AML1/ETO and DEK/CAN represent the leukemia initiating event. t(6;9)(DEK/CAN)-positive AML is classified as a separate clinical entity, because of its early onset and poor prognosis. We recently have shown that DEK/CAN is a leukemia-inducing oncogene, which targets a very small subpopulation of primitive hematopoietic stem cells (HSC) for leukemic transformation. Like other FPs, DEK/CAN also interferes with the epigenetic regulation of transcription by modifying key processes of chromatin modeling such as histone acetylation and methylation as well as DNA methylation. In the DEK/CAN fusion protein, all the chromatin binding domains of DEK are conserved and we recently showed that DEK/CAN is associated to chromatin and strongly interferes with chromatin modeling by inhibiting the decondensation of chromatin and accessibility to transcription. As a “Class 1 mutation”, the oncogenic internal tandem duplication (ITD) of the receptor tyrosine kinase Flt3 (Flt3-ITD) is found in 88% of the t(6;9)-positive AML-patients, which otherwise is present in about 30% of other AML cases. The simultaneous presence of Flt3-ITD and DEK/CAN in AML is correlated with a high WBC and significantly lower rates of complete remission. Aim of the study was to determine the effect of Flt3-ITD on the DEK/CAN-induced leukemic phenotype. Therefore we expressed Flt3-ITD and DEK/CAN from a single vector as p2A fusion protein in order to obtain an equimolar expression of the two proteins. We investigated the capacity to mediate factor-independent growth of the single factors and in combination in the myeloid progenitor cell line 32D upon IL-3 withdrawal. The leukemic phenotype was studied in primary Sca1+/Lin- murine hematopoietic stem and progenitor cells (mHSPC) retrovirally transduced with DEK/CAN, FLT3-ITD and FLT3-ITD-p2a-DEK/CAN. These cells were tested for their differentiation potential in liquid culture, for their serial replating capacity in semi-solid medium, their stem cell capacity in colony-forming unit spleen - day12 (CFU-S12) assays, and their potential to induce leukemia in sublethally irradiated recipients. Here we show that I.) Flt3-ITD mediated factor-independent growth alone and in presence of DEK/CAN, but the onset of factor-independent growth was delayed by DEK/CAN; II.) FLT3-ITD did not influence the differentiation potential of DEK/CAN-positive HPSCs; III.) Flt3-ITD increased the colony-number of DEK/CAN-positive, but not the overall serial replating efficiency of DEK/CAN-positive HPSCs; IV.) FLT3-ITD accelerated and increased efficiency of leukemia induction by DEK/CAN in vivo, without modifying either the morphological or the immunological phenotype of DEK/CAN-induced leukemia. Finally we investigated whether FLT3-ITD influences the known capacity of histone-deacetylase (HDAC) inhibitors (HDACi) to revert the leukemogenic potential of DEK/CAN. Therefore we employed a xenograft model based on the patient derived FKH-1 cell line expressing both FLT3-ITD and DEK/CAN. We found that exposure to the HDACi Dacinostat prevented the leukemia-induction in this model. Taken together these findings strongly suggest that DEK/CAN drives the transformation of immature HPSCs which is supported by the presence FLT3-ITD regarding proliferation, without strong effects on the leukemic phenotype induced by DEK/CAN. Disclosures: Bug: Novartis Oncology: Honoraria, Travel grants Other.

Abstract 656 Background: Relapse after allo-SCT is a major cause of treatment failure in patients (pts) with myeloid malignancies and is associated with a poor prognosis. As therapeutic options are limited, treatment of these pts is challenging. Indeed, there is a need for novel strategies which ideally target the leukemic clone and direct the immune system towards an enhanced GvL effect without aggravating GvHD. The hypomethylating agent Aza might provide these properties, and results from retrospective studies investigating Aza+/−DLI in pts with AML/MDS relapsing after allo-SCT were encouraging. Design/Methods: We here report the final results from a prospective single-arm EBMT multicenter phase II trial which aimed to investigate the efficacy and safety of a combination of Aza and DLI as 1st salvage therapy in pts with AML or MDS with hematological relapse after allo-SCT. Treatment schedule contained up to 8 cycles Aza (100 mg/m2/d d1-5, every 28 d) followed by DLI with increasing dosages (1-5×106–1-5×108cells/kg) after every 2ndAza cycle. Results: A total of 30 pts (19 f/11 m, median age 56 years, range 29–71) from 6 German transplant centres were included between January 2009 and May 2010. The majority of pts (n=28, 93%) suffered from AML, while 2 pts (8%) had MDS or MDS/MPN, respectively. At transplant, 16 pts (53%) had active disease (6 induction failure, 7 relapse I/II, 3 untreated) and 14 pts (47%) were in remission (12 CR1, 2 CR2). Following standard-dose (n=4, 13%) or dose-reduced conditioning (n=26, 87%), 10 pts (33%) received a graft from MSD and 20 pts (67%) from MUD. PBSC were used in 29 pts (97%), while 1 pt (3%) received BM. Acute GvHD occurred in 13 pts (46%) and 4 pts (13%) had chronic GvHD prior inclusion. None of the pts had active GvHD at the time of relapse, but 6 pts were still on immunosuppressive therapy. All pts had hematological relapse (median BM blasts: 34%, median chimerism: 67%) at a median time of 160 days (range 19–1699) following allo-SCT. A median of 3 courses Aza (range 1–8) were administered, and 22 of 30 pts (73%) received DLI (median: 1, range: 1–4, median CD3 dose 5×106/kg/DLI, range: 1–100×106). Overall response rate was 47%. Seven pts (23%) achieved CR or CRi, 2 pts (7%) PR, and 5 pts (17%) had stable disease (SD). Median time and median number of Aza cycles to best response were 79 days (range 28–299) and 3 cycles (range 1–8) respectively. Of the 7 pts who achieved a CR, 5 pts continue in CR for a median of 605 days (range 307–763) without any additional treatment, while 1 pt relapsed after 490 days and 1 pt died from GvHD. By July 2011 median follow-up of surviving pts is 645 days (range 564–857) and 5 of 30 pts (17%) are currently alive. Twelve pts have died due to progressive disease (PD), while 7 pts died during (n=3, 2 infection, 1 bleeding) or after the end of therapy (n= 4, 1 GvHD, 2 infection, 1 bleeding). All 5 pts who underwent 2nd allo-SCT died. Median overall survival (OS) of all pts is 117 days (95% CI 66–168 days). Patients with CR/CRi had a significant longer OS than pts not reaching CR/CRi (not reached vs. 83 days, p Conclusion: Aza and DLI as first salvage therapy is a safe and active approach for pts with AML or MDS who relapse after allo-SCT, and induces durable remissions in a subgroup of pts. Further research to better define target patient groups and combination partners for AZA+DLI is needed. Disclosures: Schroeder: Celgene: Financial travel support. Bug:Celgene: Lecture fees, Membership on an entity's Board of Directors or advisory committees. Luft:Celgene: Research Funding. Kobbe:Celgene: Financial travel support, Research Funding.

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 1734 Musashi (MSI) represents an evolutionarily conserved family of RNA-binding proteins. MSI2 is the predominant form in hematopoetic stem cells (HSC). Recently, overexpression of MSI2 in a mouse model was found to increase cell cycle progression and to cooperate with BCR-ABL1 to induce aggressive leukemia. The aim of this study was to analyze the prognostic importance of MSI2 expression in myelodysplastic syndrome (MDS) and in acute myeloid leukemia (AML). Diagnostic bone marrow or peripheral blood samples were analyzed from 454 adult patients (aged 17–60 years) with de novo (n=406) or secondary AML (n=48) with French-American-British (FAB] classification M0-M2, or M4-M7, who were entered into the multicenter treatment trials AML SHG 0199 or AML SHG 0295. Additionally, we analyzed a cohort of 148 patients (aged 36–92 years) with MDS with low to high IPSS scores. Real-time reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed using patient-derived cDNA and ABL as an endogenous control. MSI-2 expression levels were quantified using the TaqMan Gene Expression Assay (Applied Biosystems, Assay ID MSI2: Hs01592567_m1). To define the MSI2 expression status patient cohorts were divided into four quartiles (Q) according to levels of MSI2/ABL expression and subsequently dichotomized into two groups including the three lower Qs (Q1, Q2, and Q3) and the upper Q (Q4) of MSI2/ABL values. Patients of the quartile with highest MSI2 expression levels were classified as high MSI2 expressing patients. In MDS, MSI2 high versus low expression was independent of transfusions (P=.55), cytogenetic risk (P=.6), and ASXL1 mutations (P=.99), but was associated with a higher blast percentage (p=.034). In AML, higher MSI2 expression correlated with increased peripheral blood blasts (P=.036), and inversely correlated with the favourable cytogenetic risk group defined by inv(16) and t(8;21) according to Medical Research Council criteria (P Next, we evaluated the effect of MSI2 expression levels on patient outcome in MDS and AML. In MDS, high MSI2 expression predicted an increased likelihood of and more rapid progression to AML (HR 2.95, 95%CI 1.55–5.64, P=.001), but MSI2 expression levels did not affect overall survival (OS, HR 1.34, 95%CI 0.81–2.23, P=.25). In multivariate analysis when adjusting for karyotype, transfusion dependence, percentage of bone marrow blasts, ASXL1 frameshift mutation status and IDH1 mutation status, high MSI2 expression remained an independent prognostic factor for AML transformation in MDS (HR 2.33, 95%CI 1.16–4.67, P=.018). In AML, OS was shorter in AML patients with higher MSI2 expression as compared to low expression (HR 1.48, 95%CI 1.13–1.95, P=.005). However, relapse free survival (RFS) and complete remission (CR) rates were not influenced by high versus low MSI2 expression (RFS, HR 1.27, 95%CI.92–1.76, P=.15; CR, P=.39). In multivariate analysis, when considering MSI2 high versus low expression, DNMT3A mutations, age, platelet count, secondary versus de novo AML, cytogenetic risk group, NPM1/FLT3-ITD high vs. low risk group, and MLL5 expression levels, MSI2 expression did not remain an independent prognostic marker for OS (HR 1.31, 95%CI.98–1.76, P=.07). In patients with cytogenetically normal AML (CN-AML), MSI2 also presented a negative prognostic marker for OS (HR 1.59, 95%CI 1.08–2.33, P=.019) but did not influence RFS (HR 1.10, 95%CI 0.69–1.76, P=.68) and CR (P=.39). In multivariate analyses for CN-AML, when considering age, platelet count, DNMT3A mutations, NPM1FLT3-ITD high vs. low risk group, CEBPA mutation status and WT1 SNPrs16754, MSI2 did not independently predict OS (HR 144, 95%CI 0.94–2.19, P=.096). In summary, our data suggest that MSI2 expression might contribute to the transformation from MDS to AML. Whether MSI2 expression is dysregulated through other associated prognostic markers, or whether it is an independent event in AML pathogenesis remains to be determined. However, it may represent a valuable therapeutic target both in MDS and AML patients. Disclosures: Ottmann: Novartis Corporation: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Hofmann:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 1549 Background: The prognosis of patients (pts) with relapsed or refractory (rel/ref) AML who are considered unlikely to benefit from or tolerate intensive salvage treatment is unfavorable and novel treatment strategies are needed. Repeated cycles of LD-Ara-C are a therapeutic option for palliative treatment; however, the outlook for these pts remains unsatisfactory. Plks are critical in cellular division and mitotic progression and Plk1 is overexpressed in many cancers including AML. Volasertib is a first in class, selective and potent cell cycle kinase inhibitor that induces mitotic arrest and apoptosis by targeting Plk. In phase I/II trials in pts with solid tumors, volasertib demonstrated a favorable safety profile and encouraging antitumor activity. Here, we present updated results from the phase I part of an ongoing phase I/II study of volasertib in combination with LD-Ara-C or as monotherapy in AML pts considered ineligible for intensive salvage treatment. Material and Methods: This study follows a two-stage design. The phase I part, reported here, investigates the maximum tolerated dose (MTD) of volasertib as a 1-hr intravenous infusion on days 1 and 15 Q4W as monotherapy or in combination with fixed dose LD-Ara-C 20 mg bid subcutaneously on days 1–10 Q4W in pts with rel/ref AML. Dose escalation follows a 3+3 design with de-escalation. Blood samples for pharmacokinetic (PK) analyses were taken in cycles 1 and 2 and concentrations of volasertib and LD-Ara-C were determined. Results: In the monotherapy arm, increasing volasertib doses (150, 200, 350, 400, 450 mg) were evaluated in 29 pts (median age: 71 yrs [range 26–84]). Drug-related adverse events (AEs) were reported in 8 pts (27.6 %). Most frequent drug-related AEs (>5%) were anemia in 3 pts (10.3%), and thrombocytopenia, epistaxis, and nausea in 2 pts each (6.9%). Grade 3/4 drug-related AEs included thrombocytopenia (2 cases), anemia, diarrhea, mucositis, neutropenia, and pneumonia (1 case each); there was 1 fatal (grade 5) drug-related AE (fungal pneumonia). Of the drug-related AEs, the following were dose-limiting toxicities (DLTs) per protocol: grade 4 pneumonia and fatal fungal pneumonia (n=1, at 150 mg), and grade 3 mucositis (n=1, at 400 mg). Monotherapy dose escalation is ongoing; pts have received volasertib doses of 500 mg without having reached the MTD. Preliminary best response data indicated minor antileukemic activity at low doses (150 and 200 mg); with 4/13 pts achieving no change as best response, mostly of short duration (median number of cycles initiated: 1 [range 1–5]). At higher monotherapy doses (≥350 mg), antileukemic activity was observed with 4/16 pts achieving a complete remission with incomplete blood count recovery (CRi) and 5/16 having temporarily stable blood values as best response. In the combination arm, volasertib doses of 150–400 mg were investigated. The MTD for volasertib in combination with LD-Ara-C was 350 mg (Bug et al ASH 2010). Seven out of 32 pts treated with volasertib + LD-Ara-C achieved a complete remission (CR or CRi). In responding patients, a median number of 6 treatment cycles was initiated (range 3–13) and a preliminary analysis revealed a median overall survival of 551 days (range 165–595). PK analysis showed that volasertib is a moderate clearance drug with multi-compartmental PK behavior with a large volume of distribution (>4000 L) and a long terminal half-life (∼111 hrs). No drug interaction after co-administration of LD-Ara-C was observed. Conclusions: The phase I part of the study determined the MTD of volasertib in combination with LD-Ara-C to be 350 mg; the MTD of volasertib monotherapy has not yet been determined. Volasertib was well tolerated in this heavily pretreated AML pt population at doses above the recommended phase II volasertib dose used in pts with solid tumors. Most of the reported higher grade drug-related AEs were due to the myelosuppressive effect of volasertib. Preliminary results from the phase I trial show antileukemic activity of volasertib as monotherapy and in combination with LD-Ara-C. These results indicate Plk to be a potential new target for AML treatment and warrant proceeding with further clinical investigation of volasertib in AML pts. Disclosures: Bug: Novartis Pharma GmbH: Consultancy, Honoraria; Celgene GmbH: Consultancy, Honoraria. Off Label Use: Volasertib is an investigational agent. Müller-Tidow:Boehringer Ingelheim: Research Funding. Krug:Boehringer Ingelheim: Research Funding. Voss:Boehringer Ingelheim: Employment. Taube:Boehringer Ingelheim: Employment. Fritsch:Boehringer Ingelheim: Employment. Garin-Chesa:Boehringer Ingelheim: Employment. Ottmann:Boehringer Ingelheim: Consultancy. Döhner:Celgene, Clavis: Membership on an entity's Board of Directors or advisory committees.

Abstract 1294 Background: Patients with AML or MDS who relapse after allogeneic HSCT have a poor prognosis and therapeutic options are limited. The DNA hypomethylating agent Aza has significant activity in patients (pts) with AML and MDS and retrospective analyses have recently shown encouraging results with the use of Aza +/− DLI in patients with AML and MDS, who relapsed after allogeneic HSCT (Czibere et al., 2010; Luebbert et al., 2010). In line with these clinical observations preclinical data suggest that Aza enhances a Graft-versus-Leukemia (GvL) effect while mitigating Graft-versus-Host Disease (GvHD). Design/Methods: To evaluate the activity and safety of Aza in combination with DLI as first salvage therapy in pts with AML or MDS relapsing after HSCT, we conducted a prospective, multicenter, single-arm phase-II trial. Pts were allowed to receive up to 8 cycles Aza (100 mg/m2/d d1-5, every 28 days) and 3 DLI with increasing dosages (1-5×106 – 1–5×108 cells/kg) after every 2nd Aza treatment cycle. Additional DLI were permitted. Results: Between January 2009 and May 2010, 30 pts from 6 German transplant centres were included into this trial. So far, 25 pts (15 female/10 male) were evaluable and are presented in this analysis: Of these, 23 (92%) suffered from AML (15 de novo/8 secondary following MDS), 1(4%) from a MDS (RAEB-1) and 1 (4%) from a myelodysplastic/myeloproliferative syndrome (MDS/MPS, CMML-1). Median age was 54 years (range 29–71). Conditioning was myeloablative in 24 pts (96%) and non-myeloablative in 1 patient (4%). Eight pts (35%) received a graft from a matched sibling donor, while 15 (65%) were transplanted with a matched unrelated donor (2 pts missing data). Peripheral blood stem cells (PBSC) were used in 24 pts (96%; 1 pt missing data). At the time of transplant 6 pts (24%) had primary induction failure, another 6 (24%) suffered from first or secondary relapse, 10 pts (40%) were in first or second complete remission (CR), while 3 pts (12%) were untreated. With regard to their molecular and genetic characteristics at diagnosis, 21 pts belonged to an adverse (9 pts) or intermediate (12 pts) group, whereas 2 pts were diagnosed with a favourable genetic phenotype (2 pts not performed). Prior to relapse 9 (36%) and 3 (12%) pts had episodes of acute GvHD and/or chronic GvHD, respectively. Relapse occurred in all pts after a median of 160 days (range 19–1199) following HSCT (median BM blasts: 34%, range 5–100%, median chimerism: 63% range-1-100%). At the time of relapse, karyotype was evaluable in 13 of 25 pts (52%). Of these 13 pts, 4 pts had a normal karyotype, while 9 had chromosomal aberrations including 6 pts with a complex karyotype. Patients received a median of 3 courses Aza (range 1–8) and 18 of 25 pts (72%) received DLI (median: 1, range: 1–4, median CD3 dose 5×106/kg/DLI, range: 1–207×106). Following treatment, overall response rate was 64% with 5 pts (20%) achieving a CR or CRi, 3 (12%) a partial remission (PR) and 8 (32%) a stable disease (SD). Median response duration was 266 days. Acute GvHD occurred in 6 pts (24%) (2 skin/6 liver/ 2 gut) after a median of 65 days (range 19–179) following the first DLI, while chronic GvHD was observed in 3 pts (12 %, all limited). Hematotoxicity (grade III-IV) was observed in 64% of all evaluated patients. Common adverse events were gastrointestinal side effects as well as infections. After a median follow-up of 100 days (range 25–485) 15 of 25 pts (60%) are currently alive. Median overall survival of all pts is 184 days (range 87–281). All pts, who achieved a CR/CRi, remained in ongoing remission for a median time of 229 days. Achieving a CR (CR: not reached vs. no CR: 117 days, p .008) or any type of response (CR/CRi, PR or SD) to the combination of Aza and DLI (any response: not reached vs. no response: 79 days, p .0001) were associated with a significantly longer overall survival. Conclusion: The combination of Aza and DLI as salvage treatment for patients with AML or MDS who relapse after allogeneic HSCT seems to be safe and shows significant anti-leukemic activity. Response, including CR rates, so far match those from retrospective analyses. Data presented in this interim-analysis suggest that salvage therapy with Aza and DLI is of substantial therapeutic benefit in these challenging patients. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bug:Celgene: Honoraria. Luft:Celgene: Research Funding. Fenk:Celgene: Research Funding. Kobbe:Celgene: Research Funding.

Abstract 3316 Background: Patients with refractory or relapsed AML have a poor prognosis and new treatments are needed for this patient population. While younger AML patients might benefit from intensive salvage treatments, a substantial number of elderly patients are considered ineligible for intensive treatment approaches. For these patients, repeated cycles of low-dose cytarabine (LD-Ara-C) are an accepted therapeutic option for palliative treatment. The serine/threonine kinase Polo-like kinase 1 (Plk1) controls several key steps in mitosis. BI 6727 is a first in class, highly selective and potent cell cycle kinase inhibitor targeting Plk1, and has demonstrated antiproliferative activity in multiple cell lines and animal models. Targeting Plk1 with BI 6727 results in cell cycle arrest in prometaphase (referred to as polo arrest) leading to eventual apoptosis. In a phase I dose escalation trial in patients with advanced solid tumors a favorable safety profile and encouraging antitumor activity was reported. BI 6727 has demonstrated a long terminal half life of 111 hours and a high volume of distribution suggesting excellent tissue distribution in patients. Here, we present preliminary results from the Phase I part of an ongoing Phase I/II study of BI 6727 in combination with LD-Ara-C in patients with relapsed or refractory AML considered ineligible for intensive treatment. Methods: This study follows a two stage design: the maximum tolerated dose (MTD) of BI 6727 in combination with fixed dose LD-Ara-C was evaluated in the Phase I dose escalation part of the trial following a 3+3 design with de-escalation. In a second ongoing treatment schedule the MTD of single agent BI 6727 is investigated, the MTD of single agent BI 6727 has not been reached yet. In the planned randomized Phase II part of the study, efficacy of BI 6727 plus LD-Ara-C will be compared to LD-Ara-C alone. BI 6727 was administered as a one hour intravenous infusion on days 1+15 every 28 days in combination with fixed dose LD-Ara-C (20 mg bid s.c). The BI 6727 starting dose was based on the MTD previously determined in solid tumor patients. Patients with no progression after the first cycle were allowed to continue treatment. Results: Patient characteristics were as follows: median age was 71 years (range 40 – 81); ECOG performance score 0: 9 pts; 1: 17 pts; 2: 5 pts. Increasing BI 6727 doses in combination with LD-Ara-C were evaluated in 31 patients (21 males, 10 females). Safety: Drug related adverse events (AEs) were reported in 17 of the 31 patients. The most frequent AEs reported (>5%) were: anemia and febrile neutropenia (each 9.7%), infections (pneumonia), decreased appetite and headache (each 6.5%). Dose-limiting toxicities (DLTs) were reported in 4 patients treated with BI 6727 + LD-Ara-C. DLTs as rated per protocol were: pneumonia, mucositis, hypersensitivity/allergic reaction and myocardial infarction. Based on the preliminary reports on DLTs the MTD for BI 6727 in combination with LD-Ara-C was determined. Preliminary response data of 28 patients with relapsed/refractory AML treated at different BI 6727 doses in combination with LD-Ara-C are available: 5 patients achieved a CRi or CR, 2 patients achieved a PR. Six patients had temporarily stable blood values (“no change” as best response). 10 patients suffered from progression during or at the end of the 1st treatment cycle, and 5 patients were ineligible for response assessment. An update of the phase I part of this trial with further details on patient/disease characteristics, safety and efficacy of BI 6727 in combination with LD-Ara-C will be reported at the meeting. Conclusion: Preliminary results indicate that BI 6727 in combination with LD-Ara-C is well tolerated in patients with relapsed/refractory AML ineligible for intensive treatment. The MTD of BI 6727 in combination with LD-Ara-C was determined. BI 6727 in combination with LD-Ara-C showed first signs of clinical activity in AML patients. Safety and efficacy of BI 6727 + LD-Ara-C will be further explored in the phase II part of the trial. Disclosures: Off Label Use: LD-Ara-C in combination with BI 6727 for treatment of patients with relapsed refractory AML ineligible for intensive treatment. Fleischer:Boehringer Ingelheim Pharma GmbH & Co KG: Employment. Taube:Boehringer Ingelheim Pharma GmbH & Co KG: Employment.

Abstract 4000 Lenalidomide has shown single agent activity in patients with MDS (Myelodysplastic Syndromes) and a del(5q) cytogenetic abnormality. Further, studies with the DNA methyltransferase inhibitor 5-azacytidine (5-aza) have been conducted in high-risk MDS (IPSS INT-2 or HIGH) and patients with acute myeloid leukemia (AML) resulting in considerable responses with a low rate of extramedullary toxicity compared to conventional induction chemotherapy (IC). Given the poor outcome of high-risk MDS and AML patients with chromosome 5 abnormalities, there is a significant clinical need to perform studies with new regimens in this patient population. We report first results of an ongoing phase I clinical trial evaluating the maximum tolerated dose (MTD) of lenalidomide in combination with 5-aza in patients with either high-risk MDS, refractory/relapsed AML or de novo AML not eligible for conventional IC with chromosome 5 abnormalities including monosomy 5 or del(5q). Given the mechanism of action of both drugs and also in contrast to a recent study in non-del(5q) MDS patients, a sequential approach was chosen. In fact, induction therapy consisted of 5-aza (75mg/m2 days 1–5) followed by increasing doses of lenalidomide (starting with 10mg p.o., days 6–19). In patients achieving a complete remission this was followed by a combined maintenance therapy every 8 weeks until disease progression. To determine the MTD, a standard “3+3” design was used. The dose limiting toxicity (DLT) is determined during the first cycle only and is defined as either inability to deliver the full dosing schedule of lenalidomide due to any ≥ Grade 3 non-hematologic toxicity or absence of hematological recovery after completing the 1st cycle despite complete marrow blast clearance or treatment delay of ≥ 4 weeks as a result of unresolved grade 4 non-hematological toxicity. Of 8 patients currently enrolled, median age was 67 years (range, 45 to 74 years), interval from primary MDS or AML diagnosis was 9 months (range, 1 to 100 months). IPSS categories were INT-2 (n = 1) and HIGH (n = 3) whereas 4 patients were included with advanced AML. It is of note, that all but two patients had a complex karyotype including a del(5q) abnormality. Prior treatment included IC (n=1), IC plus allogeneic HSCT (n=3) and/or single agent 5-aza (n=3) while 4 patients had received supportive care only prior to study entry. A median of 2 induction cycles were administered. During the first cycle of cohort I (10mg lenalidomide) and cohort II (15mg lenalidomide) grades 3 to 4 non-hematologic toxicities included febrile neutropenia (n = 3), enterocolitis (n = 1) and pneumonia (n=3) whereas therapy-induced grade 3–4 neutropenia or thrombocytopenia occurred in four and five patients, respectively. The MTD has not been reached yet. One patient (12.5%) with AML showed rapid progression while receiving the 1st cycle. Out of the remaining seven patients, one (12.5%) achieved a marrow CR together with a partial cytogenetic remission, and six patients (75%) had stable disease. Interestingly, two out of these achieved a partial cytogenetic remission. These preliminary data of an ongoing phase I trial demonstrate the safety and the potential of a combination of 5-aza and lenalidomide in patients with advanced MDS or AML and a del(5q). Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kuendgen:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hofmann:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.