Abstract: Abstract 1600 Poster Board I-626 Background The V-ets erythroblastosis virus E26 (ETS) oncogene family is one of the largest families of transcription factors. ETS transcription factors are characterized by two major functional domains, a transcription domain and an evolutionarily highly conserved DNA-binding domain, also known as ETS domain that mediates binding to purine-rich DNA sequences. Most ETS family proteins are nuclear targets for activation of the Ras-MAP kinase signalling pathway. Therefore, they play a significant role in regulating cellular functions such as cell growth, apoptosis, development and differentiation. ETS transcription factors have been implicated in leukemia by chromosomal rearrangement, and more commonly by gene amplification and/or overexpression. Moreover, overexpression of ERG was shown to be an adverse predictor for clinical outcome in AML with normal cytogenetics (CN). In our recent study on complex karyotype AML, array-CGH (comparative genomic hybridization) analysis identified small genomic amplifications affecting ERG/ETS2 in 21q22 and ETS1/FLI1 in 11q23 in about 10% of the cases. Correlation with global gene expression profiling showed that ERG and ETS2 as well as ETS1 and FLI1 were overexpressed in these cases. Aims: To evaluate expression levels of ERG, ETS2, ETS1 and FLI1 in a large cohort of younger (16 to 60 years of age) adult CN-AML patients (pts) and their impact on clinical outcome. Methods The expression of ERG, ETS2, ETS1 and FLI1 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay in 343 CN-AML pts who were entered on 3 AMLSG treatment protocols (AMLHD93, AML HD98-A, AMLSG 07-04). ERG, ETS2, ETS1, and FLI1 were dichotomized into two major groups according to their expression levels. The upper quartile was chosen as the cut point and the set of patients with gene expression above were defined as Q4 group. Univariable as well as multivariable regression models were used to evaluate the influence of ERG, ETS2, ETS1 and FLI1 on induction success, event-free, relapse-free and overall survival. Multivariable analyses were stratified for AMLSG treatment protocols. Results Partial correlation analysis revealed positive correlations of expression levels between ETS2 and ERG (ρ=0.45) being the strongest, followed by ERG and FLI1 (ρ=0.4), as well as ETS1 and FLI1 (ρ=0.31). Correlation of ERG, ETS2, ETS1 and FLI1 with white blood count (WBC) revealed a significant association between high gene expression (Q4) and elevated WBC (ERG, p=0.004; ETS2, p=0.002, FLI1 p 〈 0.001), whereas high expression of ETS1 was associated with a significantly lower WBC (p 〈 0.001). Univariable as well as multivariable analyses on induction success revealed high ETS2 as an unfavourable marker (OR, 0.29, p=0.01). In univariable analysis, there was a significantly inferior relapse-free survival (RFS) and overall survival (OS) for high ERG (p=.01; p=.06, respectively) and high ETS2 (p=.002; p=.03, respectively) that was even more pronounced when both ERG Q4 and ETS2 Q4 (ERG Q4 ∩ ETS2 Q4) (p 〈 0.001; p=.001, respectively) were included as one variable and compared with the rest. In multivariable analysis for the endpoints event-free survival (EFS), RFS and OS, a significant effect was found for RFS for ERG Q4 ∩ ETS2 Q4 (p=.002); the only significant variables that consistently appeared in the model were NPM1mut, FLT3-ITDpos and WBC. In subgroup analysis for the genotypes CEBPAmut, NPM1mut/FLT3-ITDneg, and all others (NPM1mut/FLT3-ITDpos, NPM1wt/FLT3-ITDpos, NPM1wt/FLT3-ITDneg) according to the hierarchical model, ERG Q4 was associated with an inferior EFS (p=.04) and OS (p=.03) in the favorable CEBPAmut genotype and became even more significant for the variable ERG Q4 ∩ ETS2 Q4 (EFS, p=.007, RFS, p=.002; OS, p=.06, respectively). For the NPM1mut/FLT3-ITDneg subgroup, again ERG Q4 ∩ ETS2 Q4 was associated with an adverse RFS (p=.04), but not with OS (p=0.07). Conclusions In our study on a large cohort of homogenously treated CN-AML patients, ERG and ETS2 expression were highly correlated. Overexpression of both genes had a significant impact on clinical outcome of CN-AML patients. Moreover, adverse effects of high ERG and high ETS2 expression on prognosis were also shown for the genetic AML subgroups CEBPAmut and NPM1mut/FLT3-ITDneg. Disclosures No relevant conflicts of interest to declare.

Abstract: Background Overall survival (OS) in acute myeloid leukemia (AML) treated with intensive chemotherapy has improved over the last 20 year especially in younger adults (18-60 years) but still remains poor in older patients ( 〉 60 years) (Döhner et al. Blood 2010). The German-Austrian AMLSG performed controlled prospective treatment trials since 1993 starting with a risk-adapted approach (phase I, 1993-1997), followed by randomized and risk-adapted treatment strategies based on cytogenetic risk groups (phase II, 1997-2002); since 2003 addition of differentiating agents and HiDAC inhibitors to intensive induction therapy was evaluated (phase III, 2003-2007). Of note, until 2007 younger and older patients ( 〉 60 years) were treated in separate protocols with significantly lower dosages of chemotherapy in older patients. Starting from 2008, risk-adapted therapies were replaced successively by a genotype-adapted approach and the artificial age cut-off at 60 years was abandoned (phase IV, 2008-2012). Aims To evaluate the outcome of adult AML patients within the different time periods. Methods The study included 4705 intensively treated adults (younger, n=3546; older, n=1159) with newly diagnosed AML enrolled on 11 AMLSG treatment trials between 1993 and 2012. Patients with acute promyelocytic leukemia were excluded. All patients received intensive induction and consolidation therapy. Analyzed outcome variables were first complete remission rates (CR1), relapse-free survival (RFS), survival after relapse (SAR) and OS. Analyses were performed according to age groups (18-60 vs. 〉 60 yrs). In younger patients comparisons were performed for the 4 treatment phases (I-IV), whereas for older patients analyses were restricted to phase II-IV. Results In younger patients CR rates did not improve over time (1993-2013) and varied between 72% and 77% (p=0.12), whereas early and hypoplastic (ED/HD) death rates significantly declined from 10% to 5% (p=0.0001). In older patients CR rates significantly improved over time from 44% to 50% between 1998 and 2007 to 67% after 2008 (p 〈 0.0001); ED/HD rates gradually declined from 12% to 8%, but the difference was not statistically significant (p=0.17). The proportion of younger patients receiving an allogeneic hematopoietic stem cell transplantation (alloHSCT) increased from 30% (15% in CR1) in phase I to 58% (29% in CR1) in phase III and remained there in phase IV with 53% (26% CR1), whereas the proportion of patients receiving an autologous HSCT constantly decreased from maximally 16% (15% in CR1) in phase II to 0.4% (0.2% in CR1) in phase IV; the proportion of older patients receiving an alloHSCT steadily increased from 4% (2% CR1) in phase II to 21% (12% CR1) in phase IV; autoHSCT was rarely performed. OS at 4 years in both age groups significantly improved (p 〈 0.0001, each) from 41% to 56% and from 10% to 23% in younger and older patients, respectively. This beneficial effect on OS over time in younger patients was due to a better RFS (p=0.01) and SAR (p 〈 0.0001), whereas in older patients no improvement in RFS (p=0.20) and only in trend for SAR (p=0.07) was noted. In cytogenetically high-risk patients, OS in younger (p=0.001) and in older (p=0.007) patients got better; in older patients mainly driven by increase in CR rates (p=0.001) and in younger patients by an improvement in RFS (p=0.02) and SAR (p=0.05). Nearly the same pattern was identified for cytogenetically intermediate risk patients with a better OS in younger (p 〈 0.0001) and older patients (p=0.01) due to higher CR rates in older patients (p 〈 0.0001), no improvement in RFS in both age groups and a significantly better SAR in younger patients (p=0.0002). In contrast, in low risk patients improvement in OS was only present in older patients (p=0.02), due to a better RFS in older patients (p=0.02) but without any progress in younger patients. Furthermore we performed two subgroup analyses in intermediate risk patients. In the subgroup of patients characterized by the genotype NPM1-mut/FLT3-ITDneg a significant better OS was present only in younger patients (p=0.03); in FLT3-ITD positive AML a better OS was seen in younger patients (p 〈 0.0001) due to a better RFS (p=0.05) and SAR (p=0.01). Conclusions Based on the German-Austrian AMLSG experience the prognosis in younger and older AML patients has improved over time. In older patients this is mainly a result of higher CR rates and in younger patients of better RFS and SAR. Disclosures: Schlenk: Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Ambit: Honoraria. Off Label Use: Pomalidomide in Myelofibrosis. Greil:Novartis: Honoraria, Research Funding.

Abstract: Background: Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in roughly 25% of younger adult patients (pts) with acute myeloid leukemia (AML). The multi-targeted kinase inhibitor midostaurin combined with intensive chemotherapy has shown activity against AML with FLT3 mutations. However, toxicity and potential drug-drug interactions with strong CYP3A4 inhibitors such as posaconazole may necessitate dose reduction. Aims: To evaluate the impact of age and midostaurin dose-adaptation after intensive induction chemotherapy on response and outcome in AML with FLT3-ITD within the AMLSG 16-10 trial (NCT01477606). Methods: The study included adult pts (age 18-70 years (yrs)) with newly diagnosed FLT3-ITD positive AML enrolled in the ongoing single-arm phase-II AMLSG 16-10 trial. Pts with acute promyelocytic leukemia were not eligible. The presence of FLT3-ITD was analyzed within our diagnostic study AMLSG-BiO (NCT01252485) by Genescan-based DNA fragment-length analysis. Induction therapy consisted of daunorubicin (60 mg/m², d1-3) and cytarabine (200 mg/m², continuously, d1-7); midostaurin 50 mg bid was applied from day 8 until 48h before start of the next treatment cycle. A second cycle was allowed in case of partial remission (PR). For consolidation therapy, pts proceeded to allogeneic hematopoietic-cell transplantation (HCT) as first priority; if alloHCT was not feasible, pts received three cycles of age-adapted high-dose cytarabine (HDAC) in combination with midostaurin starting on day 6. In all pts one-year maintenance therapy with midostaurin was intended. The first patient entered the study in June 2012 and in April 2014, after recruitment of n=147 pts, the study was amended including a sample size increase to 284 pts and a dose reduction to 12.5% of the initial dose of midostaurin in case of co-medication with strong CYP3A4 inhibitors (e.g. posaconazole). This report focuses on age and the comparison between the first (n=147) and the second cohort (n=137) of the study in terms midostaurin dose-adaptation. Results: Patient characteristics were as follows: median age 54 yrs (range, 18-70; younger, 68% 〈 60 yrs; older, 32% ≥ 60 yrs); median white cell count 44.7G/l (range 1.1-1543 G/l); karyotype, n=161 normal, n=16 high-risk according to ELN recommendations; mutated NPM1 n=174 (59%). Data on response to first induction therapy were available in 277 pts; complete remission (CR) including CR with incomplete hematological recovery (CRi) 60%, PR 20%, refractory disease (RD) 15%, and death 5%. A second induction cycle was given in 54 pts. Overall response (CR/CRi) after induction therapy was 76% (76%, younger; 76%, older) and death 6% (4%, younger; 10% older). The dose of midostaurin during first induction therapy was reduced in 53% and 71% of patients in cohort-1 and cohort-2, respectively. Reasons for dose reduction were in 58% and 49% toxicity, and in 9% and 23% co-medication in cohort-1 and cohort-2, respectively. No difference in response to induction therapy was noted between cohorts (p=0.81). Median follow-up was 18 months. Overall 146 pts received an alloHCT, 128 in first CR (n=94 younger, n=34 older; n=92 from a matched unrelated and n=36 from a matched related donor). In pts receiving an alloHCT within the protocol in median two chemotherapy cycles were applied before transplant (range 1-4). The cumulative incidence of relapse (CIR) and death after transplant were 13% (SE 3.2%) and 16% (SE 3.5%) without differences (p=0.97, p=0.41, respectively) between younger and older patients. So far maintenance therapy was started in 86 pts, 61 pts after alloHCT and 25 pts after HDAC. Fifty-five adverse events 3°/4° were reported being attributed to midostaurin; cytopenias after alloHCT were the most frequent (29%). CIR in patients starting maintenance therapy was 20% one year after start of maintenance without difference between alloHCT and HiDAC (p=0.99). In addition, no difference in CIR was identified in patients after consolidation with alloHCT or HDAC according to dose reduction of midostaurin during first induction therapy (p=0.43, p=0.98, respectively). Median overall survival was 25 months (younger, 26 months; older 23 months; p=0.15). Conclusions: The addition of midostaurin to intensive induction therapy and as maintenance after alloHCT or HDAC is feasible and effective without an impact of age and dose adaptation on outcome. Disclosures Schlenk: Amgen: Research Funding; Pfizer: Honoraria, Research Funding. Fiedler:GSO: Other: Travel; Pfizer: Research Funding; Kolltan: Research Funding; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding; Gilead: Other: Travel; Ariad/Incyte: Consultancy; Novartis: Consultancy; Teva: Other: Travel. Lübbert:Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid. Greil:Janssen-Cilag: Honoraria; Genentech: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Merck: Honoraria; AstraZeneca: Honoraria; Boehringer-Ingelheim: Honoraria; GSK: Research Funding; Ratiopharm: Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Sanofi Aventis: Honoraria; Eisai: Honoraria; Amgen: Honoraria, Research Funding. Greiner:BMS: Research Funding. Paschka:ASTEX Pharmaceuticals: Consultancy; Novartis: Consultancy; Medupdate GmbH: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer Pharma GmbH: Honoraria; Celgene: Honoraria. Heuser:Bayer Pharma AG: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Honoraria; Pfizer: Research Funding; BerGenBio: Research Funding; Tetralogic: Research Funding.

Abstract: Background: Measurable residual disease (MRD), as determined by quantitation of Nucleophosmin 1-mutated (NPM1mut) transcript levels (TL), provides significant prognostic information independent of other risk factors in patients (pts) with acute myeloid leukemia (AML). This is also addressed by the 2017 European LeukemiaNet (ELN) risk stratification system, which recommends taking into account results from MRD monitoring when selecting the appropriate post-remission therapy. Furthermore, MRD monitoring provides a powerful tool to evaluate treatment effects within clinical trials investigating novel therapies. Aims: To determine the impact of the anti-CD33 immunotoxin Gemtuzumab-Ozogamicin (GO) on kinetics of NPM1mut TL in pts with newly diagnosed NPM1mut AML [18 to 82 years (yrs), median age 58 yrs] enrolled in our randomized Phase III AMLSG 09-09 study (NCT00893399). In this study GO was randomized (1:1) to standard chemotherapy plus ATRA. Patients and Methods: In total, 588 evaluable pts were enrolled in the clinical AMLSG 09-09 study. Standard treatment comprised two cycles of induction therapy with A-ICE (ATRA, idarubicin, cytarabine, etoposide; arm A) followed by three consolidation cycles of high-dose cytarabine (n=371, 63%) or allogeneic hematopoietic cell transplantation (n=42, 8%). In the investigational arm (arm B), GO (3 mg/m²) was given at d1 of each induction and in the first consolidation cycle. 296 pts were randomized to arm A and 292 pts to arm B. For this correlative study, outcome analysis was restricted to the clinical endpoint cumulative incidence of relapse (CIR) due to study protocol requirements allowing overall survival analysis to be performed only two years after the last pt had been enrolled. MRD monitoring was performed in a total 503 pts for whom at least one bone marrow (BM) sample was available using RQ-PCR technique; the median follow-up (FU) of the 503 pts was 2.8 yrs. NPM1mut TL (ratio of NPM1mut/ABL1 transcripts x 104) were determined by RQ-PCR (sensitivity 10-5 to 10-6). Results: In total, 3711 BM samples were analyzed (at diagnosis, n=415; during treatment, n=1765; during FU, n=1531). Both study arms were well balanced with regard to pts characteristics and pretreatment NPM1mut TL. First, we evaluated the impact of GO on kinetics of NPM1mut TL during treatment. After the first induction cycle, median NPM1mut TL were significantly lower in the investigational arm (p=.001) and this was true for all subsequent treatment cycles [after induction II (p=.008), consolidation I (p 〈 .001), consolidation II (p=.006), consolidation III (p=.009)]. Next, we evaluated treatment effects on NPM1mut TL after two cycles of induction therapy in pts in complete remission (CR, n=378). At this time point, there was no difference in the proportion of pts achieving RQ-PCR negativity (RQ-PCRneg) [arm A 15% (28/192), vs arm B 17% (32/186); p=.57] between the two treatment arms. However, treatment according to investigational arm B with GO was associated with a significantly lower CIR rate (CIR at 4 yrs: arm B 29% vs arm A 45%, p=.02). In multivariate analysis randomization to arm B revealed as an independent prognostic factor for remission duration (HR 0.63, p=.018). At the end of treatment (EOT, n=288 pts in CR) the proportion of pts achieving RQ-PCRneg was significantly higher (55% vs 41%; p=.02) in the investigational arm; pts treated in arm B had a significantly lower CIR rate compared to arm A (CIR at 4 yrs: arm B 29% vs arm A 45%, p=.04). Conclusion: In our randomized Phase III AMLSG 09-09 study, the addition of GO to intensive chemotherapy plus ATRA was associated with a significantly better reduction of NPM1mut TL after each treatment cycle. This better clearance translated into a significantly lower CIR in the investigational arm with GO. Disclosures Paschka: Otsuka: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Jazz: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Travel support; Janssen: Other: Travel support; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astex: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Takeda: Other: Travel support. Krönke:Celgene: Honoraria. Fiedler:Amgen: Other: support for meetíng attendance; GSO: Other: support for meeting attendance; Teva: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Pfizer: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Other: support for meeting attendance. Schroeder:Celgene: Consultancy, Honoraria, Research Funding. Lübbert:Janssen: Honoraria, Research Funding; TEVA: Other: Study drug; Cheplapharm: Other: Study drug; Celgene: Other: Travel Support. Götze:JAZZ Pharmaceuticals: Honoraria; Novartis: Honoraria; Takeda: Honoraria, Other: Travel aid ASH 2017; Celgene: Honoraria, Research Funding. Schleicher:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Investigator; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Ipsen: Membership on an entity's Board of Directors or advisory committees; Eissai: Other: Investigator; Astra Zeneca: Other: Investigator; Pfizer: Speakers Bureau; Janssen: Speakers Bureau; Celgene: Speakers Bureau. Schlenk:Pfizer: Research Funding, Speakers Bureau. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Döhner:Amgen: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Abstract: Background: FLT3-ITD occurs in ~25% of adult AML patients (pts) and is associated with poor prognosis. MRD monitoring is of high prognostic relevance, but restricted to certain AML subtypes. FLT3-ITD represents an attractive target for MRD monitoring in particular in pts treated with a tyrosine kinase inhibitor. FLT3-ITD MRD monitoring is hampered by the broad heterogeneity of ITD length and insertion site (IS). NGS may overcome these limitations offering the opportunity for MRD monitoring in FLT3-ITD+ AML. Aims: To validate our recently established NGS-based FLT3-ITD MRD assay in a defined cohort of FLT3-ITD+ AML pts treated within the AMLSG16-10 trial (NCT01477606) combining intensive chemotherapy with midostaurin followed by midostaurin maintenance and to evaluate the prognostic impact of FLT3-ITD MRD monitoring. Methods: Using FLT3-ITD paired-end NGS (Illumina MiSeq) with a variant allele frequency (VAF) sensitivity of 10-4-10-5 (Blätte et al., Leukemia 2019), 227 bone marrow (BM) and 17 peripheral blood samples from 61 FLT3-ITD+ AML pts were analyzed at diagnosis (Dx), after two cycles of chemotherapy (Cy2), at the end of treatment (EOT), and during 3-6 months follow-up (FU). All pts achieved complete remission (CR) after Cy2. Allogeneic hematopoietic cell transplantation in first CR was performed in 40 (66%) pts. Mutational status for NPM1 and DNMT3A was available for all pts (NPM1mut, n=48; DNMT3Amut, n=33; NPM1mut/DNMT3Amut, n=31), and NPM1mut MRD data for 41 pts. Results: At Dx we identified 191 ITDs; median length was 45 nucleotides (range, 9-194) and median VAF 0.279% (range, 0.006-90.21). Of the 191 ITDs, 133 (70%) located in the juxtamembrane domain (JMD) and 58 (30%) in the tyrosine kinase domain-1 (TKD1). There was no correlation of VAF with length or IS, whereas ITD size correlated with IS: the more C-terminal the IS, the longer the ITD (Rho=0.51; p & lt;.001). Total ITD VAF per pt was in median 34.3% (range, 0.007-90.21) and correlated positively with white blood cell count (WBC, Rho=0.314; p=.021) and lactate dehydrogenase serum level (LDH, Rho=0.274; p=.04), and inversely with the number of ITDs (Rho=-0.265; p=.04). Most pts (67%) exhibited & gt;1 ITD at Dx (median 2; range, 1-16). Categorizing pts according to IS as JMDsole (46%), JMD/TKD1 (34%), and TKD1sole (20%) revealed that JMD/TKD1 pts exhibited more ITD subclones (p & lt;.001) and a lower total VAF at Dx (p=.03). There were no correlations with any other clinical or genetic features. Pts' total ITD VAF significantly decreased after Cy2 and at EOT (median log10 reduction: 4.4 and 4.7; p & lt;.001, each), and MRD negativity (MRD-) was achieved in 67% and 87% of pts, respectively. According to subgroups, pts with JMDsole or TKD1sole showed deeper MRD reduction compared to JMD/TKD1 pts after Cy2 (4.6 vs 4.7 vs 3.7 log10; p=.06) and at EOT (4.8 vs 4.8 vs 4.0 log10; p=.02) but this did not result in a significant difference in achievement of MRD-. Concurrent NPM1mut was of favorable impact for log10 VAF reduction (median, 4.7 for DNMT3Amut/NPM1mut vs 4.6 for NPM1mut vs 2.8 others; p=.003) and MRD- (77 vs 76 vs 31%; p=.01) after Cy2, but exerted no impact at EOT. Correlating NPM1mut and FLT3-ITD MRD course revealed a positive correlation after Cy2 (Rho=0.327; p=.03), but not at EOT (Rho=0.250; p=.10), likely due to the higher sensitivity of the real-time quantitative PCR-based NPM1mut MRD assay. Median follow-up was 3.4 years (95% CI, 2.6-4.6). Survival analyses with respect to cumulative incidence of relapse (CIR; n=60) and overall survival (OS; n=61) revealed significantly lower CIR for total VAF at Dx & gt;34.3% (p=.03), a VAF reduction & gt;4.7 log10 (MR4.7) at EOT (p=.001), and for MRD- pts at EOT (p=.001). There was no impact on OS. In preliminary exploratory Cox regression (n=48), including BM blasts, WBC, LDH, age, and NPM1mut as covariables, MRD- at EOT was the only consistent favorable variable for CIR (HR, 0.1; p=.001) and OS (HR, 0.27; p=.03). During FU, 5/8 (63%) MRD+ pts at EOT became MRD- and 4/53 (8%) MRD- pts converted to MRD+ resulting in consecutive relapse in 2 pts. Conclusion: In this first cohort of FLT3-ITD+ AML pts treated with intensive chemotherapy and midostaurin in the prospective AMLSG16-10 trial we could demonstrate that FLT3-ITD NGS-based MRD monitoring is feasible and represents a promising tool to evaluate therapy response and identification of pts at a higher risk of relapse. Further analysis of the study cohort is ongoing. Disclosures Kapp-Schwoerer: Jazz Pharmaceuticals: Honoraria, Research Funding. Paschka:Sunesis Pharmaceuticals: Consultancy; BerGenBio ASA: Research Funding; Novartis: Consultancy, Speakers Bureau; Otsuka: Consultancy; Pfizer: Consultancy, Speakers Bureau; Astellas Pharma: Consultancy, Speakers Bureau; Celgene: Consultancy, Other: Travel, accommodations or expenses; Astex Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy, Speakers Bureau; Agios Pharmaceuticals: Consultancy, Speakers Bureau; Amgen: Other; Janssen Oncology: Other; AbbVie: Other: Travel, accommodation or expenses, Speakers Bureau. Fiedler:Ariad/Incyte: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accomodations; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: support in medical writing; Daiichi Sankyo Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accomodations; Morphosys: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: support in medical writing; Servier: Honoraria, Other; BerGenBio ASA: Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accomodations, support in medical writing, Research Funding; Gilead: Honoraria. Salih:Novartis: Consultancy; Pfizer: Consultancy; Philogen: Consultancy; Medigene: Consultancy; Synimmune: Consultancy, Research Funding. Salwender:Bristol-Myers Squibb/Celgene: Honoraria; Janssen-Cilag: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Oncopeptides: Honoraria; Sanofi: Honoraria; GlaxoSmithKline: Honoraria; AbbVie: Honoraria. Götze:Celgene: Research Funding. Luebbert:Janssen: Research Funding. Schlenk:PharmaMar: Research Funding; 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; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. 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:Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Karyopharm: Research Funding; Abbvie: Consultancy; PriME Oncology: Honoraria; Amgen: Research Funding; Astellas: Research Funding; Roche: Research Funding; Stemline Therapeutics: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; BerGenBio ASA: Research Funding; Bayer: Consultancy, Research Funding. Ganser:Novartis: Consultancy; Celgene: 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. Bullinger:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Döhner:Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria; Sunesis Pharmaceuticals: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding; Roche: Consultancy; Astex Pharmaceuticals: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Agios: Consultancy; Abbvie: Consultancy.

Abstract: Background: Deletion of the long arm of chromosome 9, del(9q), is a recurrent genomic abnormality, which occurs at a frequency of ~2% in AML. Interestingly, deletions of 9q are mainly found in t(8;21)-positive AML, as well as in AML with NPM1 (NPM1mut) or CEBPA (CEBPAmut) gene mutation, thereby suggesting that del(9q) can act as cooperating event in these prognostically favorable AML subgroups. Aims: In order to dissect the biology of AML with del(9q), we comprehensively characterized a large cohort of 9q21 deleted cases (n=45) at the molecular level. Methods: We performed SNP 6.0 microarray analysis to delineate the minimally deleted region on 9q, and we analyzed gene expression in selected cases to determine whether 9q21 deletions are displaying a characteristic expression pattern. Potential candidate genes were further studied by shRNA based knock-down experiments in cell line models. Finally, we performed whole exome sequencing (WES) of paired diagnostic and remission samples from n=20 del(9q) patients with NPM1mut (n=7), NPM1wt/CEBPAmut (n=7), and t(8;21) (n=6) to identify additional aberrations cooperating with 9q loss in leukemogenesis. Results: By SNP microarray analysis, we could confirm a minimally deleted region (MDR) on 9q21 encompassing seven genes (GKAP1, KIF27, C9orf64, HNRNPK, RMI1, SLC28A3, NTRK2). By targeted resequencing in n=50 non-9q deleted cases, we detected a mutation in HNRNPK, which was recently confirmed to be recurrently mutated by The Cancer Genome Atlas (TCGA) project. These findings point to HNRNPK as the most important candidate gene of the MDR. HNRNPK encodes for a ubiquitously expressed heterogeneous nuclear ribonucleoprotein (hnRNP), which influences pre-mRNA processing and other aspects of mRNA metabolism, and it is thought to play a role during cell cycle progression. To further evaluate the biology underlying 9q deleted/HNRNPK haploinsufficient cases, gene expression data were generated by microarray technology comparing NPM1mut cases with and without del(9q) (n=11 vs n=119, respectively). These analyses showed deregulated expression of genes involved in splicing and mRNA processing, and there was an overlap with gene expression changes following shRNA-mediated HNRNPK knock-down in AML cell lines, which also suggested a growth advantage for haploinsufficient cells. While these data further support that HNRNPK might play a cooperating role in AML, we were eager to see whether there are additional mutations commonly linked to del(9q). By WES, we detected on average 7.8 somatic protein altering point mutations per sample (missense and nonsense SNVs) and 2.5 frameshift insertions or deletions affecting genes known to play a role in AML as well as genes not yet linked to AML. In accordance with the general mutational spectrum of t(8;21), NPM1 or CEBPA mutant AML, we identified mutations in known epigenetic regulators such as ASXL1, ASXL2, TET2 or DNMT3A, but we also could find novel somatic mutations in additional genes involved in the regulation of the chromatin structure such as BRD3 or BRWD3. Furthermore, we identified mutations in genes associated with mRNA processing and RNA splicing,as well as mutations affecting the RAS- signaling pathway and DNA repair mechanisms. Conclusions: While ongoing analyses are likely to identify additional gene mutations in del(9q) AML, first results suggest HNRNPK haploinsufficiency as a potential "driver" mutation playing a role in the pathomechanism of 9q deleted AML. A better understanding of the HNRNPK function in normal hematopoietic cells as well as leukemia cells without del(9q), and studying the impact of HNRNPK mutations in AML might enable novel therapeutic approaches for del(9q)/HNRNPKmut AML. These authors contributed equally to the work: AD and SRC as well as KD and LB. Supported by: FP7 NGS-PTL project, and DFG SFB 1074 B3 project. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 825 Background: Acute myeloid leukemia (AML) with t(8;21)(q22;q22) is considered as a prognostically favorable subgroup of AML. However, outcome is heterogeneous and almost half of adult patients (pts) cannot be cured by current treatment. Candidate molecular markers have been assessed in an effort to predict outcome in AML with t(8;21) at the time of diagnosis and to potentially guide the development of genotype-specific approaches. In most, but not all studies, KIT mutations were associated with adverse prognosis in AML with t(8;21). However, no larger study has elucidated the independent prognostic impact of various gene mutations in a comprehensive molecular analysis. Methods: Bone marrow and/or blood specimens from 146 adult pts diagnosed with de novo (n=137), therapy-related (n=5) or unknown history (n=4) t(8;21) AML were studied for the presence of additional chromosome abnormalities and for mutations in FLT3 [internal tandem duplications (ITD) and tyrosine kinase domain mutations (TKD)], N-/K-RAS, KIT and JAK2 (V617F) genes. All pts were treated on one of 7 prospective protocols of the German-Austrian AML Study Group (AMLSG). For induction pts received anthracycline-and cytarabine-based therapy regimens; pts achieving a complete remission (CR) were assigned to postremission therapy incorporating higher doses of cytarabine in various settings or to autologous stem cell transplantation. Multivariable analyses were performed to assess the prognostic value of gene mutations on relapse-free (RFS) and overall survival (OS) and were stratified for treatment protocols. Results: Mutations were identified in 56% of the pts with the highest frequency observed in KIT (30%), followed by mutations in RAS (21%), FLT3 (13%) [ITD (9.5%) and TKD (3.5%)] and JAK2 (3.5%) genes. When correlating gene mutations with clinical features, pts with RAS mutations had a higher WBC (P=0.003) and a lower frequency of the most common secondary chromosome abnormality represented by the loss of a sex chromosome (LOS; P=0.03) when compared to pts with wild-type RAS; for the other genes studied no differences in pretreatment characteristics were observed. The median age of the study cohort at diagnosis was 46 years (yrs; range, 17-73 yrs), and the median white blood count (WBC) was 8.7 × 109/l (range, 0.9-152 × 109/l). Median follow-up for survival according to Korn was 3.4 yrs [95%-confidence interval (CI), 2.6.-5.2 yrs] . The CR rate in the entire study cohort was 89% and none of the gene mutations impacted as single marker on the CR rate. In univariable and multivariable analyses, only FLT3 mutations significantly affected relapse-free survival (RFS) and overall survival (OS). No significant difference in RFS and OS was observed with respect to the mutational status of KIT, RAS and JAK2 genes. In univariable analyses, pts with FLT3 mutations relapsed more frequently (P=0.03; 3-yr RFS rates, 22% vs 58%) and had a shorter survival time (P=0.006; 3-yr OS rates, 26% vs 63%) than those without FLT3 mutations. Multivariable analyses revealed the mutational status of FLT3 as independent prognostic variable for RFS and OS. Age was a significant risk factor for OS. Additional variables that were also included in multivariable models were mutational status of KIT and RAS, log10(WBC), and presence of LOS. Pts harboring FLT3 mutations relapsed more frequently (HR, 3.20, P=0.01) than pts with FLT3 wild-type. In addition, the risk of death in pts with FLT3 mutations was more than four times higher (HR, 4.24, P=0.004) than in pts lacking these mutations. Conclusions: In conclusion, we show here in a large group of adult AML pts with t(8;21) that the presence of activating FLT3 mutations independently predicts for poor outcome within this favorable subset of AML. Thus, adults with t(8;21)-positive AML and FLT3 mutations require alternative treatment strategies. Our data support the rationale of evaluating FLT3 tyrosine kinase inhibitors in these pts. Disclosures: No relevant conflicts of interest to declare.

Abstract: Background: Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1) resulting in the RUNX1-RUNX1T1 gene fusion is considered favorable in the 2017 genetic risk stratification by the European LeukemiaNet (ELN). After intensive chemotherapy most patients (pts) achieve complete remission (CR), but relapse occurs in about 50% and is associated with poor prognosis. In this AML subgroup monitoring of measurable residual disease (MRD) has been shown to identify pts at higher risk of relapse. Aims: To assess the prognostic impact of MRD monitoring in bone marrow (BM) and peripheral blood (PB) in a large cohort of 155 clinically well-annotated t(8;21)-AML pts enrolled in one of six AMLSG treatment trials. Methods: RT-qPCR was used to quantify RUNX1-RUNX1T1 transcript levels (TL) reported as normalized RUNX1-RUNX1T1 values per 106 transcripts of the housekeeping gene B2M. Samples were analyzed in triplicate, the sensitivity was up to 10-6. Results: While pretreatment RUNX1-RUNX1T1 TL did not impact prognosis, both reduction of RUNX1-RUNX1T1 TL and achievement of MRD negativity (MRDneg) at end of treatment (EOT) were of significant prognostic importance in BM as well as in PB: MR2.5 ( 〉 2.5 log reduction) after treatment cycle 1 and MR3.0 after cycle 2 were significantly associated with a reduced relapse risk (MR2.5, BM: P=.034; PB: P=.008 and MR3.0, BM: P=.028; PB: P=.036, respectively). After completion of therapy, MRDneg was an independent favorable prognostic factor for cumulative incidence of relapse (CIR) (4-year CIR BM: 17% vs 36%, P=.021; PB: 23% vs 55%; P=.001) and overall survival (OS) (4-year OS rate BM: 93% vs 70%, P=.007; PB: 87% vs 47%; P 〈 .0001). Moreover, maximally selected Gray´s statistic defined specific MRD cut-offs at EOT associated with a lower relapse risk: 〈 83 RUNX1-RUNX1T1 TL in BM and 〈 5 in PB predicted for superior 4-year CIR (BM: 18% vs 61%; P 〈 .0001; PB: 23% vs 65%; P 〈 .0001). During follow-up serial MRD analyses allowed prediction of relapse in 77% of pts exceeding an arbitrary cut-off of 150 RUNX1-RUNX1T1 TL in BM and in 84% of pts with 〉 50 TL in PB, respectively. KIT mutation observed in 28% of pts predicted for lower CR rate and inferior outcome, but its prognostic impact was outweighed by RUNX1-RUNX1T1 TL during treatment. To determine whether PB could provide similar prognostic information as BM, we compared 680 paired samples (diagnosis, n=125; after cycle 1, n=80; after cycle 2, n=86; at EOT, n=78; during follow-up, n=311). At diagnosis RUNX1-RUNX1T1 TL tended to be slightly higher in BM than in PB (P=.072), but were significantly higher after cycle 1 (P=.008), cycle 2 (P 〈 .001), at EOT (P=.002), and during follow-up (P 〈 .001). RUNX1-RUNX1T1 TL in BM and PB correlated well (r=.87; P 〈 .0001) with on average 1-log lower values in PB. However, 2.5%, 26.7%, 26.9%, and 24.8% of all pairs were discrepant (BMpos/PBneg or BMneg/PBpos) after cycle 1, cycle 2, at EOT, and during follow-up. Of 104 PBneg samples obtained during treatment, 46 (44%) were still BMpos. In the post-treatment period, this fraction decreased to 28% (77 BMpos/276 PBneg pairs) (P=.003). Of note, RUNX1-RUNX1T1 TL in all but four of the 77 (5.2%) BMpos samples were below the cut-off of 150 TL. Virtually all relapses occurred within one year after EOT with a very short latency from molecular to morphologic relapse strongly suggesting to perform MRD assessment at short intervals during this period. Based on our data we refined the practical guidelines for MRD assessment in RUNX1-RUNX1T1-positive AML: i) along with the current ELN MRD recommendations, BM and PB should be analyzed after each treatment cycle; ii) during the follow-up period, in particular the first year after EOT, MRD monitoring in PB should be performed monthly; in pts with TL 〉 50 in PB, increase of MRD TL 〉 1-log, and/or conversion from MRDneg to MRDpos a complementary BM samples should be analyzed timely. Summary: RUNX1-RUNX1T1 MRD monitoring allows for the discrimination of pts at high and low risk of relapse. MRDneg in both BM and PB after completion of therapy was the most valuable independent favorable prognostic factor for relapse risk and OS. During follow-up, serial MRD analyses allowed the definition of cut-offs predicting relapse. Moreover, considering that virtually all relapses occurred within the first year after EOT with a very short latency from molecular to morphologic relapse MRD assessment in PB at shorter intervals during this period is indispensable. Disclosures Weber: Celgene Corporation: Research Funding. Schroeder:Celgene Corporation: Consultancy, Honoraria, Research Funding. Götze:AbbVie: Membership on an entity's Board of Directors or advisory committees. Fiedler:Amgen, Pfizer, Abbvie: Other: Support in medical writing; Amgen, Pfizer, Novartis, Jazz Pharmaceuticals, Ariad/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Amgen, Jazz Pharmaceuticals, Daiichi Sanchyo Oncology, Servier: Other: Support for meeting attendance. Greil:Gilead: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; MSD: Consultancy, Honoraria, Other: Travel/accomodation expenses, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Sandoz: Honoraria. Krauter:Pfizer: Honoraria. Bullinger:Amgen: Honoraria; Astellas: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Hexal: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria; Menarini: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Abbvie: Honoraria; Bayer: Other: Financing of scientific research; Sanofi: Honoraria; Seattle Genetics: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria. Paschka:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Other: Travel expenses, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Travel expenses; Otsuka: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Travel expenses; Janssen: Other: Travel expenses; Abbvie: Other: Travel expenses; Sunesis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Astex: Membership on an entity's Board of Directors or advisory committees, Travel expenses; Astellas: Membership on an entity's Board of Directors or advisory committees. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; Celgene, Novartis, Sunesis: Honoraria, Research Funding; AROG, Bristol Myers Squibb, Pfizer: Research Funding. Döhner:Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria; Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria.

Abstract: Activating mutations in the receptor tyrosine kinase FLT3 occur in roughly 30% of acute myeloid leukemia (AML) patients (pts), implicating FLT3 as a potential target for kinase inhibitor therapy. The multi-targeted kinase inhibitor midostaurin (PKC412) shows potent activity against FLT3 as a single agent but also in combination with intensive chemotherapy. Besides its mere presence, the allelic ratio as well as ITD insertion site within the FLT3 gene had been reported as prognostic factors in FLT3-ITD positive AML. Furthermore, pharmacokinetic analyses revealed clinically important interactions between potent CYP3A4 inhibitors, such as azoles, and midostaurin. Aims To evaluate the pharmacodynamic activity of midostaurin measured as inhibition of the degree of phosphorylated FLT3 (pFLT3) in correlation to co-medication and outcome data. Methods The study includes intensively treated adults (age 18-70 years) with newly diagnosed FLT3-ITD positive AML enrolled in the ongoing single-arm phase-II AMLSG 16-10 trial (NCT: NCT01477606). Pts with acute promyelocytic leukemia are not eligible. The presence of FLT3-ITD is analyzed by Genescan-based fragment-length analysis (allelic ratio 〉 0.05 required to be FLT3-ITD positive). Induction therapy consists of daunorubicin (60 mg/m², d1-3) and cytarabine (200 mg/m², continuously, d1-7); midostaurin 50 mg twice daily is applied from day 8 onwards until 48h before start of the next treatment cycle. For consolidation therapy, pts proceed to allogeneic hematopoietic stem cell transplantation (HSCT) as first priority; if allogeneic HSCT is not possible pts receive three cycles of age-adapted high-dose cytarabine in combination with midostaurin from day 6 onwards. In all pts maintenance therapy for one year is intended. A total sample size of n=142 is planned to show an improvement in event-free survival from 25% after 2 years to 37.5%. Plasma inhibitory activity assay (PIA) for pFLT3 is performed as previously described (Levis MJ, et al. Blood 2006; 108:3477-83). For PIA, measured time points include day 15 of induction therapy, the end of each treatment cycle and every three months during maintenance therapy. Results To date, 72 pts (median age, 54.5 years; range, 29-69 years) have been included and PIA was performed so far in 37 pts during induction therapy. Median pFLT3 inhibition after one week of midostaurin intake measured on day 15 of cycle 1 (C1D15) was 57.5% (range, 14.2-93.7%) with 2 of 31 pts showing inhibition 〉 85%. At the end of the first induction cycle (C1end), median inhibition was 60.3% (range, 0-99.8%); here, 6 of 37 pts had an inhibition 〉 85%. Co-medication with azoles was present in 7 of 23 pts at C1D15 and 13 of 28 pts at C1end. There was no significant difference in pFLT3 inhibition either on C1D15 (p=0.79) or at C1end (p=0.70) between pts on (median pFLT3 inhibition: 52.5%) or off (median pFLT3 inhibition 57.5%) azoles. Response data were available in 56 pts: complete remission (CR) was achieved in 78.5%; rates of early death and refractory disease (RD) were 9% and 12.5%, respectively. In first analyses, there was no difference in pFLT3 inhibition in pts achieving CR (n=30) as compared to those with RD (n=3; p=0.99). In contrast to our previously published data from three historical trials without a FLT3 inhibitor which showed that high allelic ratio was associated with low CR rates (Kayser S, et al. Blood 2009;114:2386-92), in the current trial CR rates remained high (81.5%) despite of a high allelic ratio above the median ( 〉 0.58). In addition, we did not see a negative prognostic impact of ITD insertion site within the tyrosine kinase domain of the FLT3 gene (p=0.99). Analyses are currently ongoing, measurement of FLT3 ligand levels and evaluation of pharmacokinetics of midostaurin are also intended. Conclusions The addition of 50 mg midostaurin twice daily to intensive induction therapy resulted in a moderate pFLT3 inhibition during induction therapy. Nonetheless, CR rates are promising, especially in pts with unfavorable FLT3-ITD characteristics. Concomitant azoles do not appear to significantly influence pFLT3 inhibitory activity of midostaurin. Disclosures: Levis: Ambit Biosciences: Consultancy. Schlenk:Ambit: Honoraria; Chugai: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Amgen: Research Funding.

Abstract: Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1-mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TLs) by quantitative reverse-transcription polymerase chain reaction and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TLs and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase 3 AMLSG 09-09 trial. A total of 3733 bone marrow (BM) samples and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥ 3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after 2 treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity was consistently revealed to be a poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TLs were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly greater proportion of patients achieving MRD negativity at EOT (56% vs 41%; P = .01). The better reduction in NPM1mut TLs after 2 treatment cycles in MRD positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR, 29.3% vs 45.7%, P = .009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction in NPM1mut TLs across all treatment cycles, leading to a significantly lower relapse rate.