Abstract: Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are among the most frequent alterations in acute myeloid leukemia (AML) and can be found in ∼20% of patients at diagnosis. Among 4930 patients (median age, 56 years; interquartile range, 45-66) with newly diagnosed, intensively treated AML, we identified IDH1 mutations in 423 (8.6%) and IDH2 mutations in 575 (11.7%). Overall, there were no differences in response rates or survival for patients with mutations in IDH1 or IDH2 compared with patients without mutated IDH1/2. However, distinct clinical and comutational phenotypes of the most common subtypes of IDH1/2 mutations could be associated with differences in outcome. IDH1-R132C was associated with increased age, lower white blood cell (WBC) count, less frequent comutation of NPM1 and FLT3 internal tandem mutation (ITD) as well as with lower rate of complete remission and a trend toward reduced overall survival (OS) compared with other IDH1 mutation variants and wild-type (WT) IDH1/2. In our analysis, IDH2-R172K was associated with significantly lower WBC count, more karyotype abnormalities, and less frequent comutations of NPM1 and/or FLT3-ITD. Among patients within the European LeukemiaNet 2017 intermediate- and adverse-risk groups, relapse-free survival and OS were significantly better for those with IDH2-R172K compared with WT IDH, providing evidence that AML with IDH2-R172K could be a distinct entity with a specific comutation pattern and favorable outcome. In summary, the presented data from a large cohort of patients with IDH1/2 mutated AML indicate novel and clinically relevant findings for the most common IDH mutation subtypes.

Abstract: Introduction: Cytogenetic testing is routinely performed in newly diagnosed acute myeloid leukemia (AML) for risk stratification. Elaborate risk classifications based on karyotyping are provided by both the European Leukemia Net (ELN) and the Medical Research Council (MRC). Complex aberrant, monosomal and abnl(17p) karyotypes confer a poor prognosis. In cytogenetic studies, chromosome aberrations that cannot be identified due to gross rearrangement, thereby preventing the allocation to a specific chromosome, are designated as "Marker Chromosomes" (MC). The significance of MC as prognostic factor for AML has remained elusive so far. In this study we have assessed frequency, cytogenetic characteristics and prognostic impact of MC as well as their underlying biological origin. Given the gross structural chromosomal damage inherent to MC we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. Patients and Methods: Patients recruited intwo large consecutive, prospective, randomized, multicenter clinical trials for newly diagnosed non-M3 AML patients from the German Study Alliance Leukemia (SAL) were analyzed (AML96, NCT00180115; AML2003, NCT00180102). All karyotypes were retrospectively screened for MC. For the detection of chromothripsis array-CGH was used. For each sample 50 ng of DNA were hybridized to an Affymetrix® CytoScan HD Oligo/SNP-array and scanned with the Affymetrix GeneChip® Scanner 3000 7G. Chromothripsis was defined according to the criteria of Rausch et al., which require at least 10 switches in segmental copy number involving two or three distinct copy number states on a single chromosome. Results: MC were detectable in 165/1026 (16.1%) of aberrant non-CBF karyotype cases. Adverse-risk karyotypes displayed a higher frequency of MC (40.3% in complex aberrant, 26.5% in adverse-risk as defined by MRC criteria and 41.2% in abnl(17p) karyotypes, p 〈 .001 each). MC were associated with a poorer prognosis compared to other non-CBF aberrant karyotypes as well as with lower remission rates (CR+CRi; 36.0% vs. 55.8% in AML96 ≤60 years, p=0.01; 14.3% vs. 44.1% in AML2003, p 〈 0.001), inferior event-free survival (2.24 vs. 6.54 months, p 〈 0.001; 3.45 vs. 8.03 months, p 〈 0.001) and overall survival (5.72 vs. 11.87 months, p 〈 0.001; 8.68 vs. 20.78, p=0.01). In multivariate analysis with co-variables age, prior MDS, therapy-related AML and adverse-risk cytogenetics according to MRC criteria, MC independently predicted poor prognosis in AML96 ≤60 years but not in AML2003 with its higher allogeneic transplantation rate. As detected by array-CGH, in about one third of MC karyotypes (18/49, 36.7%, including 3 cases with 8 or 9 copy number switches) MC had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of complex aberrant karyotypes without MC (1/34) (p 〈 0.001). Chromothripsis in MC karyotypes typically involved one single chromosome (n=11), with two or three chromosomes affected in 5 and 2 patients, respectively. There was no predilection for a particular chromosome. MC karyotypes positive for chromothripsis were characterized by a particularly high degree of karyotype complexity as compared to those that were negative for chromothripsis (complex aberrant 100% vs. 64.5% p 〈 0.01; abnl(17p) 50.0% vs. 16.1%, p=0.01). In 12/18 (66.7%) cases, at least one of the chromothriptic chromosomes was reported as loss in the karyotype formula, suggesting that the grouping of a chromothriptic chromosome as a marker is paralleled by a putative loss of the affected chromosome. The chromothripsis positive MC karyotype subgroup had a particularly dismal prognosis with a combined CR+CRi rate of 2/16 vs. 10/31 (p=0.14). It also displayed inferior event-free and overall survival, though statistical significance was not reached for either endpoint, likely due to the already poor prognosis of the entire MC positive group. Conclusion: This is the first study showing that MC are a frequent finding predominantly in adverse-risk AML and associated with particularly poor prognosis. Our data provide evidence that a substantial portion of MC arise from chromothripsis. Disclosures Thiede: AgenDix: Employment, Other: Ownership.

Abstract: Thalidomide (Thal) is a drug with antiangiogenic, anti-inflammatory, and immunomodulatory properties that was found to inhibit the production of tumor necrosis factor-α (TNF-α) in vitro. We studied single nucleotide polymorphisms at positions −308 and −238 of the TNF-α gene promoter and measured the corresponding TNF-α cytokine levels in 81 patients (pts) with refractory and relapsed multiple myeloma (MM) who were treated with Thal. In myeloma pts carrying the TNF-238A allele (n = 8), we found a correlation with higher pretreatment TNF-α levels in peripheral blood (P = .047). After Thal administration, this TNF-238A group had a prolonged 12-month progression-free and overall survival of 86% and 100% versus 44% and 84% (P = .003 andP = .07) in pts with the TNF-238G allele, respectively. These findings suggest that regulatory polymorphisms of the TNF-α gene can affect TNF-α production and predict the outcome after Thal therapy, particularly in those MM pts who are genetically defined as “high producers” of TNF-α.

Abstract: Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.

Abstract: Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are frequently found in patients with acute myeloid leukemia (AML) and several other tumors. Mutant IDH1 produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, and leads to a block in differentiation to promote tumorigenesis. Methods: We developed a novel, highly active oral pan-IDH1 inhibitor, BAY-1436032, for clinical evaluation. Its inhibitory potency was evaluated in primary human AML cells in vitro for the five major IDH1R132 mutation types and in two patient derived AML xenograft (PDX) models in vivo, in which BAY-1436032 cleared leukemic blasts in peripheral blood and prolonged survival by induction of differentiation and inhibition of leukemia stem cell self-renewal. Results: R-2HG production by mutant IDH1 was effectively inhibited in patient derived AML cells with all reported IDH1R132 mutations ex vivo by BAY-1436032 with an IC50 between 3 to 16 nM. AML cells cultured ex vivo showed morphologic differentiation and marked upregulation of the myeloid differentiation markers CD14 and CD15. For in vivo experiments, human AML cells from two patients were transplanted into sublethally irradiated NSG mice. After stable engraftment at 17 (PDX1) or 90 (PDX2) days post transplantation, mice were treated with BAY-1436032 orally every day at a dose of 150 mg/kg or vehicle for 100-150 days (n=10 per group). The R/S-2HG ratio in serum was reduced to near normal levels by BAY-1436032. Leukemic cell counts in peripheral blood constantly increased in control mice, while leukemic cells declined from day 30 of BAY-1436032 treatment onwards with morphologic and immunophenotypic evidence of differentiation (Figure). Importantly, all BAY-1436032 treated PDX1 mice survived until the end of treatment at 150 days. In contrast, vehicle-treated mice died with a median latency of 91 days (range 70-95, P 〈 .001). In an independent second model (PDX2) 6 of 10 BAY-1436032 treated mice survived until the end of treatment at day 100 with a median of 15% leukemic cells in peripheral blood, while all vehicle-treated mice suffered from high leukemic burden and died from leukemia with a median survival of 62 days (P=.014). Early mortality was increased with 4 mice dying in the BAY-1436032 group reminiscent of clinical differentiation syndrome in AML patients treated with the IDH1 inhibitor AG-120. To assess the effect of BAY-1436032 on leukemic stem cell self-renewal we treated PDX1 mice with 150 mg/kg BAY-1436032 or vehicle for 4 weeks and performed a limiting dilution transplantation experiment in secondary recipient mice. LSC frequency was 100-fold lower in BAY-1436032 treated compared to control mice. Gene expression profiling showed that stemness associated genes were downregulated, while genes associated with myeloid differentiation like PU.1 and CEBPA were upregulated upon treatment with BAY-1436032. In addition, cell cycle progression was slowed and E2F transcription factors concomitantly inhibited. In accordance with gene expression profiling results, methylation of the PU.1 promoter decreased, while E2F1 promoter methylation increased upon treatment with BAY-1436032. Finally, histone trimethylation levels at residues H3K4, H3K9, H3K27, and H3K36 decreased in both IDH1R132C and IDH1R132H mutant AML cells but not in IDH1 wildtype cells upon BAY-1436032 treatment. Conclusion: In summary, the novel oral pan-mutant IDH1 inhibitor BAY-1436032 is active against all IDH1R132 mutation types and shows strong anti-leukemic activity in two independent AML PDX mouse models. Clinical development is ongoing with a first in man study with BAY-1436032 in IDH1 mutant solid tumors. * M. Heuser and L. Herbst contributed equally to this article #A. Krämer and A. Chaturvedi share senior authorship Figure Human leukemic cells in peripheral blood of mice treated with BAY-1436032. ** P 〈 .001, ns, not significant. Figure. Human leukemic cells in peripheral blood of mice treated with BAY-1436032. ** P 〈 .001, ns, not significant. Disclosures Heuser: Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Research Funding. Pusch:German Cancer Research Center: Patents & Royalties: WO2013/127997A1. Kaulfuss:Bayer Pharma AG: Employment. Panknin:Bayer Pharma AG: Employment. Zimmermann:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210 . Toschi:Bayer Pharma AG: Employment. Neuhaus:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210. Haegebarth:Bayer Pharma AG: Employment, Equity Ownership. Rehwinkel:Bayer Pharma AG: Employment, Equity Ownership, Patents & Royalties: WO2015/121210. Hess-Stumpp:Bayer Pharma AG: Employment. Bauser:Bayer Pharma AG: Employment. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. von Deimling:German Cancer Research Center: Patents & Royalties: IDH1R132H mutant specific antibody H09; BRAF V600E mutant specific antibody VE1; BAY-1436032 patent.

Abstract: Background In newly diagnosed acute myeloid leukemia (AML), the general recommendation is to start treatment immediately after the diagnosis has been made. This paradigm is based both on the observation that untreated acute leukemia has a poor prognosis and on retrospective analyses demonstrating a shorter survival in younger AML patients (pts) in whom treatment was delayed by more than 5 days (Sekeres et al., 2009). A more recent single-center analysis came to a different conclusion, showing no prognostic effect for the time from diagnosis to treatment (TDT; Bertoli et al., 2013). We explored the relationship between TDT and prognosis on a large set of real-world data from the AML registry of the Study Alliance Leukemia (SAL) and compared it to the published cohorts. Methods The SAL runs a transregional AML registry in 46 treatment centers across Germany (NCT03188874). All registered patients with an intensive induction treatment, a minimum follow-up time of 12 months and no acute promyelocytic leukemia were selected (n=2,200). Treatment start was defined by the first day of cytarabine, whereas single agent hydroxyurea (HU) was labeled as pretreatment. We analyzed the influence of TDT on complete remission (CR), early death (ED) and overall survival (OS) in univariable analyses for each day of treatment delay, in groups of 0-5, 6-10, 11-15 and & gt;15 days of TDT, and by using the restricted cubic spline (RCS) method for data modelling. In order to adjust for the influence of established prognostic variables on the outcomes, we used multivariable regression models and propensity score weighting. The influence of HU pretreatment on outcomes was investigated by introducing an interaction term between TDT and the presence of HU pretreatment. Results The median age was 59 years (y) (IQR 50-68), the proportion of pts with favorable, intermediate and adverse genetic risk according to ELN was 27%, 53%, and 20%; & gt;95% of pts received induction treatment with standard 7+3. HU pretreatment was administered in 4% of pts. The median TDT was 3 days (IQR 2-6). Descriptive statistics after grouping of pts showed the highest median age and the lowest proportion of NPM1 mutated and favorable risk in the TDT group 11-15. Of all pts, 79% achieved a CR/CRi; unadjusted CR rates for the patient groups with TDT of 0-5, 6-10, 11-15 and & gt;15 days were 80%, 77%, 74% and 76%, respectively (p=0.317). In multivariable analysis accounting for the influence of ELN risk, age, WBC, LDH, de novo versus secondary AML and ECOG, the OR for each additional day of TDT was 0.99 (95%-CI, 0.97-1.00; p=0.124). Four percent of pts died within the first 30 days from treatment start. The respective rates in the four TDT categories were 4.0%, 3.8%, 5.1% and 4.1% (p=0.960). In multivariable analysis, the OR for TDT was 1.01 (95%-CI, 0.98-1.05; p=0.549). After a median follow-up of 40 months, the 2-y OS of all pts was 51%. The unadjusted 2-y OS rates stratified by TDT of 0-5, 6-10, 11-15, & gt;15 days were 52, 49, 46, and 51% (see Table 1 and Figure 1). The hazard ratio (HR) for each day of treatment delay was 1.00 (95%-CI; 0.99-1.01; p=0.317). In multivariable Cox regression analysis, the HR for TDT as continuous variable was 1.00 (95%-CI, 0.99-1.01; p=0.689). When OS was analyzed separately stratified for age ≤60 and & gt;60 ys and for high versus lower initial WBC defined by a threshold of 50 x 109/L, no significant differences between TDT groups were observed. Multivariable models using TDT as a grouped variable or with RCS did not provide evidence for a significant influence of TDT on outcomes. Propensity score matching of pts in the four TDT groups did not reveal an influence on outcomes. The use of HU was not associated with CR, ED nor OS. Conclusion Our study on 2,200 newly diagnosed registry pts receiving consistent intensive induction with standard-dose cytarabine plus daunorubicin (7+3) suggests that TDT is not related to response or survival, neither in younger nor in older pts. Despite multivariable analyses, a bias towards longer TDT intervals in pts judged to be clinically stable by the treating physician cannot be excluded entirely. As treatment stratification in intensive first-line treatment of AML evolves, the TDT data suggests that it may be a safe and reasonable approach to wait for genetic and other laboratory test results in order to assign clinically stable pts to the best available treatment option before the start of intensive treatment. Disclosures Krämer: Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; BMS: Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hänel:Roche: Honoraria; Amgen: Honoraria; Celgene: Other: advisory board; Novartis: Honoraria; Takeda: Other: advisory board. Jost:Daiichi: Honoraria; Sanofi: Honoraria; Gilead: Other: travel grants; Jazz Pharmaceuticals: Honoraria. Brümmendorf:Merck: Consultancy; Janssen: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment; Ariad: Consultancy. Krause:Siemens: Research Funding; Takeda: Honoraria; MSD: Honoraria; Gilead: Other: travel; Celgene Corporation: Other: Travel. Scholl:Novartis: Other: Project funding; Pfizer: Other: Advisory boards; Gilead: Other: Project funding; Daiichi Sankyo: Other: Advisory boards; AbbVie: Other: Advisory boards. Hochhaus:Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding. Kiani:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Middeke:Sanofi: Research Funding, Speakers Bureau; Roche: Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Gilead: Consultancy; Janssen: Consultancy, Speakers Bureau; MSD: Consultancy. Thiede:AgenDix GmbH: Employment, Equity Ownership; Novartis: Research Funding, Speakers Bureau; Bayer: Research Funding; Daiichi-Sankyo: Speakers Bureau. Stoelzel:JAZZ Pharmaceuticals: Consultancy; Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding. Platzbecker:Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

Abstract: ATR-Seckel syndrome and ataxia-teleangiectasia are autosomal recessive disorders associated with hematologic malignancies. They share the feature of genetic instability and are caused by defects in the DNA damage response: underlying causes are a hypomorphic ATR mutation and inactivating ATM mutations, respectively. A common substrate of ATM and ATR in the DNA damage response is the nuclear checkpoint kinase Chk1, which we have recently shown to localize to interphase centrosomes and thereby negatively regulate entry into mitosis by preventing premature activation of cyclin B-Cdk1 (Nat Cell Biol6: 884–891, 2004). Here, we demonstrate that DNA damage by ultraviolet radiation or hydroxyurea treatment enhances the centrosomal localization of Chk1. In fibroblasts derived from ATR-Seckel or ataxia-teleangiectasia patients, this DNA damage-dependent centrosomal accumulation of Chk1 was rather more pronounced in comparison with control fibroblasts. Accordingly, this cellular reaction is not dependent on ATR or ATM. These results indicate that centrosomal accumulation of Chk1 might contribute a novel regulatory mechanism to the DNA damage checkpoint. Our study might help to further dissect the contributions of ATR and ATM to the DNA damage response, and to better understand their role in the development of hematologic malignancies.

Abstract: Introduction Plasma cell disorders (PCD) are clonal outgrowths of pre-malignant or malignant plasma cells (PC), characterized by extensive chromosomal aberrations. Centrosome aberrations (CA) were identified to be a major driver of chromosomal instability in cancer. However, their origin, incidence, and composition in patient-derived tumor cells is only poorly understood. Moreover, while most studies on CA in primary tissues rely on immunostaining against centrosomal proteins at low resolution, systematic analysis of structural aberrations on an ultrastructural level is lacking. Here, we use a multi-modality approach integrating high-throughput volume electron microscopy (EM) and immunofluorescence (IF) imaging, expression profiling, and clinical data to enhance our understanding of CA evolution in primary cancer cells, using the PCD spectrum as a paradigm for malignant progression. Methods Consenting patients enrolled in the study were either healthy donors or diagnosed with PCD (monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SM), overt multiple myeloma (MM), or plasma cell leukemia (PL)) or other B-cell malignancies (B-cell acute lymphoblastic leukemia (B-ALL) and B-cell chronic lymphocytic leukemia (B-CLL)). Bone marrow aspirates (except B-ALL/B-CLL) were sorted for CD138 + PCs and prepared for both IF and electron tomography (ET) assessment. For ET, slot grids were loaded with 5 consecutive, 200 nm thick sections of epoxy resin-embedded cells. High-throughput screening by transmission EM was performed on the central section to identify cells containing centrosomes (Schorb et al., Nature Methods 2019). Serial-section ET was performed after semi-automated targeting of centrosomes on adjacent sections. Tomograms were reconstructed to produce volumes of at least 1 µm in Z for each centrosome. At least 30 completely featured centrioles were evaluated per case. An online repository of all acquired ET data will be made publicly available for interactive visualization upon conclusion of the study. For IF-based analysis, cells were fixed and stained for nuclei and the centrosomal proteins centrin and pericentrin. Results EM screening of 42,876 cells yielded 1873 completely featured centrioles in 1297 CD138 + PCs from 21 PCD patients and eight healthy donors. Both ET and IF revealed no increased frequency of supernumerary centrosomes in normal and primary patient PCs as compared to healthy cells of B-lymphatic origin. In contrast, ET revealed frequent centriole over-elongation over 500 nm, structural aberrations, and decoration of centrioles with supernumerary appendages (Fig. 1). Unexpectedly, in healthy individuals, centriole over-elongation was most pronounced and increased with age to a maximum of 75.0 % of cells. In PCD, over-elongation decreased in frequency from the early disease stages MGUS and SM, via overt MM to PL, where it was fully absent. Similarly, the amount of additional structural aberrations correlated with centriole length and decreased from healthy donors via MGUS, SM, and MM to PL, where they were absent as well. In line with these findings, gene expression profiling revealed significantly elevated mRNA levels of centriole elongation activators in healthy CD138 + PCs as compared to malignant PCs from MGUS and MM. MM patients with & gt; 20 % over-elongated centrioles showed significantly better progression-free (p & lt;0.001) and overall survival (p & lt;0.001; PFS and OS, respectively) as compared to those with fewer over-elongated centrioles. Interestingly, neither numerical nor structural CA were detected in B-ALL or B-CLL samples. Conclusions Our data imply that centrioles lengthen with individual cellular age in healthy donor PCs. In vitro, over-elongated centrioles were shown to perturb mitotic spindle symmetry and to contribute to multipolar spindle formation previously (Marteil et al., Nature Communications 2018). Centriole over-elongation and subsequent structural CA in long-lived, quiescent PCs might hence offer a possibility for chromosomal instability development in early disease stages if these cells re-enter the cell cycle. Within increasingly advanced PCD, structural CA became less frequent, indicating an inverse relationship between centriole length and PC proliferation rate. Accordingly, in MM patients, a low rate or absence of over-elongated centrioles was associated with poor PFS and OS. Figure 1 Figure 1. Disclosures Weinhold: Sanofi: Honoraria. Goldschmidt: MSD: Research Funding; GSK: Honoraria; Incyte: Research Funding; Adaptive Biotechnology: Consultancy; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Johns Hopkins University: Other: Grant; Molecular Partners: Research Funding; Mundipharma: Research Funding; Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Novartis: Honoraria, Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Takeda: Consultancy, Research Funding. Müller-Tidow: Pfizer: Research Funding; Bioline: Research Funding; Janssen: Consultancy, Research Funding. Raab: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dreger: BMS: Consultancy; Riemser: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Bluebird Bio: Consultancy; AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy; Gilead Sciences: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Hegenbart: Janssen: Consultancy, Research Funding; Akcea: Honoraria; Pfizer: Consultancy, Honoraria; Prothena: Research Funding; Alnylam: Honoraria. Schönland: Sanofi: Research Funding; Pfizer: Honoraria; Janssen: Honoraria, Other: Travel grants, Research Funding; Takeda: Honoraria, Other: Travel grants; Prothena: Honoraria, Other: Travel grants. Krämer: F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Other: Honoraria to Institution, Travel/Accomodation/expenses; Bayer: Other: Honoraria to Institution, Research Funding; AbbVie: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Other: Travel/Accomodation/Expenses; Merck: Research Funding; Celgene: Other: Travel/Accomodation/Expenses; Bristol Myers Squibb: Consultancy.

Abstract: Treatment of elderly patients with AML remains challenging. While increasing doses of induction and consolidation chemotherapy have failed to improve outcome, efforts to decrease relapse rates using the graft-versus-leukemia effect have shown promising results in phase II studies. In the present analysis of the prospective OSHO 2004 study we evaluated the effect of post-induction hematopoietic cell transplantation (HCT) in comparison to conventional consolidation chemotherapy (CT) on outcome in elderly patients with AML. The OSHO 2004 study is part of the German intergroup study. Upon achieving complete remission (CR) after induction, patients were assigned to CT or HCT depending on the availability of a matched related or unrelated donor. Unrelated, single antigen mismatched donors were accepted in high risk situations. By April 2014 from 817 eligible patients, 505 entered CR (62%) after one or two induction therapies. From the 452 patients who received consolidation in CR 1, 31 patients (7%) relapsed and 10 (2%) died of complications during consolidation. No further therapy for medical reasons was given to 73 patients, 206 patients received second consolidation with cytarabine (0.5 g/m2 i.v. bid d1, 3, 5) plus mitoxantrone (10 mg/m² d1-2) and 132 patients underwent HCT. Most frequent conditioning regimens for HCT were low dose TBI (83%) and treosulfan/fludarabine (12%). Most of the patients received HCT from unrelated (80%) donors and the majority received grafts from HLA-identical (78%) donors. Our analysis was restricted to the 315 patients 〈 75 years receiving either CT or HCT. Probabilities for overall survival (OS) and leukemia free survival (LFS) were estimated according to the Kaplan-Meier method and differences tested by the log-rank test. Relapse incidence (RI) and non relapse mortality (NRM) were described by estimating the cumulative incidence and testing the differences using the Gray's test. Multivariate Cox regression models and competing risks regression models were used to identify independent prognostic variables for outcomes. The median age was 67 (60-74) and 65 (60-74) years in the CT and the HCT groups (p 〈 0.0005), respectively. There were no differences between CT and HCT regarding gender, AML type (de novo, secondary or therapy related) and FLT3 mutation status. However more patients with mutated NPM1 were observed in the CT as compared to the HCT group (39% vs 28%; p=0.07) and more patients entered into remission after one induction in the CT as compared to the HCT group (89% vs. 81%; p=0.05). Low risk cytogenetics and normal karyotype were present more frequently in the CT than in the HCT arm (p 〈 0.0005). The interval from CR to CT was 50 days and from CR to HCT 72 days (p 〈 0.0005). Patients receiving related or unrelated matched/mismatched HCT had superior LFS than those receiving CT (32±5% vs. 13±4% at 8 years, respectively; p 〈 0.0005). The difference was more distinct when only those patients with matched related or unrelated donors were compared to those receiving CT (36±6% vs. 13±4% at 8 years; p 〈 0.0005). Similar figures were obtained for overall survival [OS, 35±5% matched/mismatched HCT vs. 24±4% for CT (p=0.18) and 41±6% for matched HCT patients vs. 24±4% for CT (p=0.09)]. RI was lower after HCT (40±5%) than after CT (79±5%; p 〈 0.0001). In contrast, NRM was higher in HCT patients (28±7%) than in CT patients (9±11%; p 〈 0.0001). Subpopulation analyses identified no difference in LFS and OS between matched related versus unrelated HCT. The difference in LFS between HCT and CT was highest in patients with normal karyotype, high risk cytogenetics and patients with non-monosomal karyotyp. Prognostic factors for LFS, OS, RI and NRM were analyzed in a multivariate analysis. Significant prognostic factors for LFS were cytogenetic risk (p=0.04), HCT (p=0.01) and FLT3 mutation status (p=0.07). OS was determined by cytogenetics p 〈 0.01) with a trend for lower age (p=0.07) and HCT (p=0.14). Prognostic factors for RI were cytogenetics (p 〈 0.0006), FLT3 mutation status (p 〈 0.03) and HCT (p 〈 0.0005). NRM was influenced by HCT (p=0.002). Conclusions: HCT from related or unrelated donors improved LFS and OS in patients with AML over the age of 60 years and in particular in those with high risk cytogenetics or normal karyotype disease. The LFS of over 30% after 8 years achieved by HCT represents a marked improvement in the prognosis of patients with AML aged 60-75 years in CR1. Disclosures Al-Ali: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Wolf:Bayer: Honoraria; Geo Pharma: Honoraria. Hochhaus:ARIAD Pharmaceuticals, Inc.: Research Funding. Maschmeyer:Celgene: Consultancy.

Abstract: Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: 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; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.