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: Extramedullary manifestations (EM) are rare in acute myeloid leukemia (AML) and their impact on clinical outcomes is controversially discussed. Methods We retrospectively analyzed a large multi-center cohort of 1583 newly diagnosed AML patients, of whom 225 (14.21%) had EM. Results AML patients with EM presented with significantly higher counts of white blood cells ( p   〈  0.0001), peripheral blood blasts ( p   〈  0.0001), bone marrow blasts ( p  = 0.019), and LDH ( p   〈  0.0001). Regarding molecular genetics, EM AML was associated with mutations of NPM1 (OR: 1.66, p   〈  0.001), FLT3 -ITD (OR: 1.72, p   〈  0.001) and PTPN11 (OR: 2.46, p   〈  0.001). With regard to clinical outcomes, EM AML patients were less likely to achieve complete remissions (OR: 0.62, p  = 0.004), and had a higher early death rate (OR: 2.23, p  = 0.003). Multivariable analysis revealed EM as an independent risk factor for reduced overall survival (hazard ratio [HR]: 1.43, p   〈  0.001), however, for patients who received allogeneic hematopoietic cell transplantation (HCT) survival did not differ. For patients bearing EM AML, multivariable analysis unveiled mutated TP53 and IKZF1 as independent risk factors for reduced event-free (HR: 4.45, p   〈  0.001, and HR: 2.05, p  = 0.044, respectively) and overall survival (HR: 2.48, p  = 0.026, and HR: 2.63, p  = 0.008, respectively). Conclusion Our analysis represents one of the largest cohorts of EM AML and establishes key molecular markers linked to EM, providing new evidence that EM is associated with adverse risk in AML and may warrant allogeneic HCT in eligible patients with EM.

Abstract: In fit patients with newly diagnosed acute myeloid leukemia (AML), immediate treatment start is recommended due to the poor prognosis of untreated acute leukemia. We explored the relationship between time from diagnosis to treatment start (TDT) and prognosis in a large real-world data set from the German Study Alliance Leukemia–Acute Myeloid Leukemia (SAL-AML) registry. All registered non–acute promyelocytic leukemia patients with intensive induction treatment and a minimum 12 months of follow-up were selected (n = 2263). We analyzed influence of TDT on remission, early death, and overall survival (OS) in univariable analyses for each day of treatment delay, in groups of 0 to 5, 6 to 10, 11 to 15, and & gt;15 days of TDT, adjusted for influence of established prognostic variables on outcomes. Median TDT was 3 days (interquartile range, 2-7). Unadjusted 2-year OS rates, stratified by TDT of 0 to 5, 6 to 10, 11 to 15, and & gt;15 days, were 51%, 48%, 44%, and 50% (P = .211). In multivariable Cox regression analysis accounting for established prognostic variables, the TDT hazard ratio as a continuous variable was 1.00 (P = .617). In OS analyses, separately stratified for age ≤60 and & gt;60 years and for high vs lower initial white blood cell count, no significant differences between TDT groups were observed. Our study suggests that TDT is not related to survival. As stratification in intensive first-line AML treatment evolves, TDT data suggest that it may be a feasible approach to wait for genetic and other laboratory test results so that clinically stable patients are assigned the best available treatment option. This trial was registered at www.clinicaltrials.gov as #NCT03188874.

Abstract: 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 & lt;0.001). At hematological relapse, only 1 (2%) of 45 pts with NPM1 measurement was not congruently MRD positive. Six months after start of MRD-guided therapy, 56 (60%) of 94 pts were still in CR while a total of 38 pts (40%) developed a hematological relapse after median of 3 AZA cycles. 38 (40%) pts responded with either a decline of MRD below a predefined threshold (increasing donor-chimerism to ≥80% or PCR MRD & lt;1%), while a stabilization in the absence of relapse was achieved in 18 (19%) pts. Overall response rate was not statistically different between pts with (63%) or without (55%) antecedent allo-HCT (P=0.5). RFS rate at 6 months was 60% (56/94 pts). With a median follow-up of 9 months after start of MRD-guided pre-emptive treatment 12-months overall and progression-free survival rates were 94% and 44%, respectively. 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

Abstract: Tandem-duplication mutations of the UBTF gene ( UBTF -TDs) coding for the upstream binding transcription factor have recently been described in pediatric patients with acute myeloid leukemia (AML) and were found to be associated with particular genetics (trisomy 8 (+8), FLT3 -internal tandem duplications ( FLT3 -ITD), WT1 -mutations) and inferior outcome. Due to limited knowledge on UBTF- TDs in adult AML, we screened 4247 newly diagnosed adult AML and higher-risk myelodysplastic syndrome (MDS) patients using high-resolution fragment analysis. UBTF -TDs were overall rare ( n  = 52/4247; 1.2%), but significantly enriched in younger patients (median age 41 years) and associated with MDS-related morphology as well as significantly lower hemoglobin and platelet levels. Patients with UBTF -TDs had significantly higher rates of +8 (34% vs. 9%), WT1 (52% vs. 7%) and FLT3 -ITD (50% vs. 20.8%) co-mutations, whereas UBTF -TDs were mutually exclusive with several class-defining lesions such as mutant NPM1 , in-frame CEBPA bZIP mutations as well as t(8;21). Based on the high-variant allele frequency found and the fact that all relapsed patients analyzed ( n  = 5) retained the UBTF -TD mutation, UBTF -TDs represent early clonal events and are stable over the disease course. In univariate analysis, UBTF -TDs did not represent a significant factor for overall or relapse-free survival in the entire cohort. However, in patients under 50 years of age, who represent the majority of UBTF -mutant patients, UBTF -TDs were an independent prognostic factor for inferior event-free (EFS), relapse-free (RFS) and overall survival (OS), which was confirmed by multivariable analyses including established risk factors such as age and ELN2022 genetic risk groups (EFS [HR: 2.20; 95% CI 1.52–3.17, p   〈  0.001], RFS [HR: 1.59; 95% CI 1.02–2.46, p  = 0.039] and OS [HR: 1.64; 95% CI 1.08–2.49, p  = 0.020]). In summary, UBTF -TDs appear to represent a novel class-defining lesion not only in pediatric AML but also younger adults and are associated with myelodysplasia and inferior outcome in these patients.

Abstract: Acute myeloid leukemia (AML) is characterized by recurrent genetic events. The BCL6 corepressor (BCOR) and its homolog, the BCL6 corepressor-like 1 (BCORL1), have been reported to be rare but recurrent mutations in AML. Previously, smaller studies have reported conflicting results regarding impacts on outcomes. Here, we retrospectively analyzed a large cohort of 1529 patients with newly diagnosed and intensively treated AML. BCOR and BCORL1 mutations were found in 71 (4.6%) and 53 patients (3.5%), respectively. Frequently co-mutated genes were DNTM3A, TET2 and RUNX1. Mutated BCORL1 and loss-of-function mutations of BCOR were significantly more common in the ELN2017 intermediate-risk group. Patients harboring loss-of-function mutations of BCOR had a significantly reduced median event-free survival (HR = 1.464 (95%-Confidence Interval (CI): 1.005–2.134), p = 0.047), relapse-free survival (HR = 1.904 (95%-CI: 1.163–3.117), p = 0.01), and trend for reduced overall survival (HR = 1.495 (95%-CI: 0.990–2.258), p = 0.056) in multivariable analysis. Our study establishes a novel role for loss-of-function mutations of BCOR regarding risk stratification in AML, which may influence treatment allocation.

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.

Abstract: Background: Relapse of disease remains the major cause of treatment failure in patients with acute myeloid leukemia (AML) or advanced myelodysplastic syndrome (MDS), even after allogeneic hematopoietic stem cell transplantation (HSCT). Treatment of relapsed AML or MDS is difficult, especially after HSCT, and long-term prognosis of patients suffering from relapse is dismal. One approach to overcome this problem is to use sensitive molecular diagnostic strategies to detect recurring disease already at the level of minimal residual disease (MRD), thus avoiding the development of overt hematologic relapse by treatment of patients at the stage of molecular relapse. We have recently implemented preemptive treatment with the demethylating drug 5-Azacitidine (AZA) in patients with molecular evidence of recurrent disease in a prospective Phase II study (RELAZA). In this study, 80% of the patients showed responses, with reduction of MRD and prolonged leukemia free survival, 20% of patients even showed molecular clearance of their leukemia and long-term disease free survival. More recently, results from several groups studying demethylating agents in MDS or AML suggested that patients with mutations in genes involved in epigenetic DNA-modification, such as TET2, DNMT3A or IDH1 or IDH2 might be more responsive to treatment with these drugs. Since we observed varying clinical response in the patients treated preemptively with AZA for molecular evidence of recurrent disease, we correlated the clinical response in these patients with the presence of mutations in epigenetic regulator genes in order to identify potential predictors of response. Patients and Methods: A cohort of 44 patients (23 f/21 m), median age 55.6 years (range 21-75 years), in hematological remission with AML (N=40) or MDS (N=4) were given AZA to treat molecular relapse defined by mutant NPM1 (N=23) or CD34+ chimerism (N=21). Patients were monitored post allogeneic HSCT (N=26) or standard chemotherapy (N=18). The cohort received a median of 5 cycles of AZA (ranging from 1-18 cycles). DNA taken at first diagnosis was analyzed using amplicon based resequencing on a MiSeq next generation sequencing system for the following genes, either analyzing the complete coding region (EZH1, EZH2, DNMT3A, TET1 and TET2) or hot-spot regions (ASXL1, ASXL2, IDH1, IDH2). First diagnosis samples were unavailable for 4 patients. In these, DNA from sorted CD34+ cells taken at the time of molecular relapse was used as a substitute. Results: Amplicon sequencing revealed mutations in one or more genes in 25/44 patients (56.8%). With 15 mutations (34%), DNMT3A was the most frequently mutated gene, the majority of the alterations (9; 60%) were located in exon 23. Mutations in TET2 were found in 8 patients, IDH1 was mutated twice, ASXL2, EZH2 and TET1 were mutated once each. In 20 of the 44 patients (45.5%), no mutations in the investigated genes were found. A comparison of primary response to AZA-treatment (defined as stabilization or decrease of the MRD-marker) between patients with and without mutations revealed no significant difference (79.2 vs 66.6%; P=.48). Likewise, the rate of hematologic relapse was comparable in both cohorts (54% vs. 56%). However, a more detailed look at the patients with mutations revealed differences. The highest initial response rate was observed in patients with DNMT3A mutations (87%), whereas patients with isolated TET2 mutations were less likely to respond (50%). Also, the rate of hematologic relapse was highest in patients with TET2-mutations (75%) compared to patients with DNMT3A-mutations alone (41.6%). In support of a role of TET2-mutations in mediating resistance, an analysis of matched diagnosis and relapse samples in three patients indicated persistence of TET2-loss of function mutations in one patient as well as an acquisition of a second mutant TET2- allele or a switch to a loss-of function-mutation in two patients, indicating that a clonal evolution favoring a subclone with an inactivating TET2-allele under treatment with AZA occurred. Conclusions: Our data confirm that mutations in epigenetic regulator genes are common in patients with AML. Although based on small numbers, these preliminary data do not support that mutations in these genes are associated per se with an improved response to treatment with AZA, but might indicate a differential effect of certain alterations, i.e. DNMT3A-mutations or mutations of TET2. Disclosures Middeke: Genzyme: Speakers Bureau. Thiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

Abstract: Abstract 489 Systemic iron overload (SIO) can be frequently encountered in AML and MDS patients (pts) and predominately occurs as a consequence of recurrent red blood cell transfusions (RBC). SIO has been associated with an increased risk for infectious complications and acute graft-versus-host disease (aGvHD) as well as with excessive early non-relapse mortality (NRM) after allogeneic stem cell transplantation (allo-SCT). However, most of these studies relied on surrogate markers like ferritin and transfusion burden, which might be interference prone in this patient population. Consequently, the prognostic impact of these parameters is under considerable debate and a variety of different thresholds for risk stratification have been proposed. Studies using improved and objective assessments of SIO like magnetic resonance imaging (MRI) are rare and limited in patient number. We aimed at determining prospectively the influence of SIO as quantified by MRI on post-transplant outcome in a large cohort of AML and MDS patients undergoing allo-SCT. From 2009 on, MRI-based assessment of liver iron content (LIC) according to the methods described by Gandon (Lancet 2004) and Rose (Eur J Haematol 2006) was routinely performed prior to conditioning in all AML and MDS patients at risk for SIO undergoing allo-SCT at our center. Further, serological parameters of SIO were determined at the same time. Post-transplant outcome including the occurrence and extent of GVHD as well as NRM were correlated with variables of SIO. Categorial variables were assessed using Fisher's excact test, while competing events statistics was used to compare cumulative incidences of NRM and aGvHD. Correlations are reported as Spearmans rank correlation coefficient (r). Over a period of 30 months 59 AML- and 22 MDS patients with a median age of 57 years were prospectively screened with liver MRI. Median LIC was 140 μmol/g (range: 40 – 350 μmol/g). Ferritin was elevated in all patients with a median of 2204 ng/ml (range: 305 – 45049 ng/ml) and patients had received a median of 24 units of packed red blood cells (RBC, range: 4 – 127). There was a strong positive correlation between transfusion burden and LIC (r = 0.702, p 〈 0.001) as well as between ferritin and LIC (r = 0.594, p 〈 0.001). A threshold of 20 or more RBC, which is widely accepted as a good marker for SIO, was found to predict an elevated LIC ( 〉 =125 μmol/l) with a sensitivity and specificity of 70.0 % and 81.8 %, respectively. Contrasting, the commonly used criterion of ferritin above 1000 ng/ml albeit, being very sensitive (95.5%), provided only very poor specificity (27.0%). Increasing the ferritin threshold to 2500 ng/ml, a level known to correlate with increased NRM, lead to increased specificity 81.1% at the price of moderately reduced sensitivity (63.6%). None of the three SIO parameters was associated with an increased risk of aGvHD or infections after allo-SCT, while preexisting aspergillosis was more common in iron overloaded patients (LIC 〉 = 125 μmol/g: 33.3% vs. 0.0 %, p 〈 0.001; RBC 〉 =20: 27.1 vs. 3.4%, p = 0.013; Ferritin 〉 = 1000 ng/ml: 21.2% vs. 0.0 %, p = 0.199). A moderate correlation between LIC (r = 0.494, p 〈 0.001) as well as transfusion burden (r = 0.478, p 〈 0.001) and the time between diagnosis and allo-SCT was noted, while ferritin was not associated with that parameter. Interestingly, both transfusion burden (r = 0.248, p = 0.026) and ferritin (r = 0.329, p = 0.003) but not LIC showed a weak but significant correlation with hematopoietic transplantation comorbidity scores. Regarding NRM we were able to show only a modest trend for transfusion burden of 20 or more RBC as a predictor for an adverse prognosis (100 day CI of NRM: 26.2% vs. 7.7%, p = 0.080). Furthermore, ferritin above 2500 ng/ml (CI: 26.0 vs. 13.4%; p = 0.231) did not correlate with NRM. In contrast, an LIC of 125 μmol/g or more, which is known to be associated with organ toxicity in thalassemia patients, predicted for a significantly increased risk of NRM (CI: 30.8 % vs. 6.3%; p = 0.016). Multivariate analysis confirmed LIC but not transfusion burden or ferritin as an independent risk factor for an increased NRM (HR 1.007 for every 1 μmol/g increase, p = 0.022). We conclude that systemic iron overload is an independent negative prognostic factor for post-transplant outcome in AML and MDS patients but definition of SIO should be based on reliable parameters like MRI- measured LIC. Disclosures: Wermke: Novartis: Research Funding. Platzbecker:Novartis: Research Funding.