Abstract: We conducted a single-arm, phase 2 trial (German-Austrian Acute Myeloid Leukemia Study Group [AMLSG] 16-10) to evaluate midostaurin with intensive chemotherapy followed by allogeneic hematopoietic-cell transplantation (HCT) and a 1-year midosta urin maintenance therapy in adult patients with acute myeloid leukemia (AML) and fms-related tyrosine kinase 3 (FLT3) internal tandem duplication (ITD). Patients 18 to 70 years of age with newly diagnosed FLT3-ITD-positive AML were eligible. Primary and key secondary endpoints were event-free survival (EFS) and overall survival (OS). Results were compared with a historical cohort of 415 patients treated on 5 prior AMLSG trials; statistical analysis was performed using a double-robust adjustment with propensity score weighting and covariate adjustment. Results were also compared with patients (18-59 years) treated on the placebo arm of the Cancer and Leukemia Group B (CALGB) 10603/RATIFY trial. The trial accrued 440 patients (18-60 years, n = 312; 61-70 years, n = 128). In multivariate analysis, EFS was significantly in favor of patients treated within the AMLSG 16-10 trial compared with the AMLSG control (hazard ratio [HR] , 0.55; P & lt; .001); both in younger (HR, 0.59; P & lt; .001) and older patients (HR, 0.42; P & lt; .001). Multivariate analysis also showed a significant beneficial effect on OS compared with the AMLSG control (HR, 0.57; P & lt; .001) as well as to the CALGB 10603/RATIFY trial (HR, 0.71; P = .005). The treatment effect of midostaurin remained significant in sensitivity analysis including allogeneic HCT as a time-dependent covariate. Addition of midostaurin to chemotherapy was safe in younger and older patients. In comparison with historical controls, the addition of midostaurin to intensive therapy led to a significant improvement in outcome in younger and older patients with AML and FLT3-ITD. This trial is registered at clinicaltrialsregistry.eu as Eudra-CT number 2011-003168-63 and at clinicaltrials.gov as NCT01477606.

Abstract: In the international randomized phase 3 RATIFY (Randomized AML Trial In FLT3 in patients less than 60 Years old) trial, the multikinase inhibitor midostaurin significantly improved overall and event-free survival in patients 18 to 59 years of age with FLT3-mutated acute myeloid leukemia (AML). However, only 59% of patients in the midostaurin arm achieved protocol-specified complete remission (CR), and almost half of patients achieving CR relapsed. To explore underlying mechanisms of resistance, we studied patterns of clonal evolution in patients with FLT3-internal tandem duplications (ITD)-positive AML who were entered in the RATIFY or German-Austrian Acute Myeloid Leukemia Study Group 16-10 trial and received treatment with midostaurin. To this end, paired samples from 54 patients obtained at time of diagnosis and at time of either relapsed or refractory disease were analyzed using conventional Genescan-based testing for FLT3-ITD and whole exome sequencing. At the time of disease resistance or progression, almost half of the patients (46%) became FLT3-ITD negative but acquired mutations in signaling pathways (eg, MAPK), thereby providing a new proliferative advantage. In cases with FLT3-ITD persistence, the selection of resistant ITD clones was found in 11% as potential drivers of disease. In 32% of cases, no FLT3-ITD mutational change was observed, suggesting either resistance mechanisms bypassing FLT3 inhibition or loss of midostaurin inhibitory activity because of inadequate drug levels. In summary, our study provides novel insights into the clonal evolution and resistance mechanisms of FLT3-ITD–mutated AML under treatment with midostaurin in combination with intensive chemotherapy.

Abstract: High CD33 expression in acute myeloid leukemia (AML) with mutated NPM1 provides a rationale for the evaluation of gemtuzumab ozogamicin (GO) in this AML entity. We conducted a randomized trial to evaluate GO in combination with intensive induction and consolidation therapy in NPM1-mutated AML. PATIENTS AND METHODS Between May 2010 and September 2017, patients ≥ 18 years old and considered eligible for intensive therapy were randomly assigned up front for induction therapy with idarubicin, cytarabine, etoposide, and all- trans-retinoic acid with or without GO. The early ( P = .02) primary end point of event-free survival (EFS) was evaluated 6 months after completion of patient recruitment. RESULTS Five hundred eighty-eight patients were randomly assigned (standard arm, n = 296; GO arm, n = 292). EFS in the GO arm was not significantly different compared with that in the standard arm (hazard ratio, 0.83; 95% CI, 0.65 to 1.04; P = .10). The early death rate during induction therapy was 10.3% in the GO arm and 5.7% in the standard arm ( P = .05). Causes of death in both arms were mainly infections. The cumulative incidence of relapse (CIR) in patients achieving a complete remission (CR) or CR with incomplete hematologic recovery (CRi) was significantly reduced in the GO arm compared with the standard arm ( P = .005), with no difference in the cumulative incidence of death ( P = .80). Subgroup analysis revealed a significant beneficial effect of GO in female, younger (≤ 70 years), and FLT3 internal tandem duplication–negative patients with respect to EFS and CIR. CONCLUSION The trial did not meet its early primary end point of EFS, mainly as a result of a higher early death rate in the GO arm. However, in patients achieving CR/CRi after induction therapy, significantly fewer relapses occurred in the GO compared with the standard arm.

Abstract: Background:Despite recent advances in identifying novel molecular targets in AML patients, intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (HSCT) still remains a cornerstone of AML therapy. However, outcome of HSCT depends on the availability of a donor and the donor type. Prior studies comparing HSCT from HLA-matched related donors (MRD) with matched unrelated donors (MUD), demonstrated conflicting results with regards to outcome. These conflicting results might be attributed to the genetic heterogeneity of AML. Aims:To analyze outcome with respect to donor type of 952 AML patients who received HSCT in first complete remission (CR) and were treated within prospective AMLSG trials. Methods:Within the AMLSG trials conducted between 1993 and 2013, of a total of 4991 patients (excluding acute promyelocytic leukemia), 3408 (2744 younger ( 〈 61 years old), 664 older (≥61 years old)) patients achieved a first CR after intensive double induction therapy. Of these, 867 (31%) younger and 85 (13%) older patients received HSCT in first CR. Distributions of donor types were 511 matched related donors (MRD), 435 matched unrelated donors (MUD) and 6 haplo-identical donors. The latter were grouped together with MUD. Results:Distributions of donor type over time are illustrated in table 1 indicating two clear trends with increasing numbers of MUD transplants and increasing median age in MUD- and MRD-transplants in recent years. There was no significant difference in overall survival, cumulative incidence of relapse (CIR) and death (CID) all estimated at 4 years according to the three time periods for MRD (p=0.56, p=0.15, p=0.10, respectively) and MUD (p=0.27, p=0.20, p=0.88, respectively). Table 1 Time period 1993-2002 2003-2007 2008-2013 Total no. 1036 1102 1270 MRD  No. 186 (18%) 182 (17%) 143 (11%)  Median age 42.7yrs 46.0yrs 51yrs  4-yr-OS (95%-CI) 59% (53-67) 66% (59-73) 61% (53-72)  4-yr-CIR (SE) 21% (3%) 25% (3%) 29% (4%)  4-yr-CID (SE) 25% (3%) 15% (3%) 18% (3%) MUD  No. 42 (4%) 131 (12%) 268 (21%)  Median age 41.1yrs 47.9yrs 50.6yrs  4-yr-OS (95%-CI) 52% (39-70) 46% (38-58) 54% (47-61)  4-yr-CIR (SE) 21% (3%) 25% (3%) 29% (4%)  4-yr-CID (SE) 25% (3%) 15% (3%) 18% (3%) Table 2 ELN risk category low inter-1 inter-2 high Total no. 867 711 433 318 MRD  No. 78 (9%) 122 (17%) 66 (15%) 57 (18%)  4-yr-OS (95%-CI) 84% (76-93) 50% (51-69) 53% (41-67) 57% (44-72)  4-yr-CIR (SE) 7% (3%) 24% (4%) 35% (6%) 49% (7%)  4-yr-CID (SE) 13% (4%) 23% (4%) 23% (6%) 12% (4%) MUD  No. 21 (2%) 139 (20%) 76 (18%) 109 (36%) 4-yr-OS (95%-CI) 69% (52-93) 58 (49-68) 52% (41 67) 35% (26-46)  4-yr-CIR (SE) 0% 28% (4%) 32% (6%) 44% (5%)  4-yr-CID (SE) 31% (11%) 20% (4%) 17% (5%) 28% (4%) There were no differences in stratified survival analyses for time period between MRD and MUD-transplants in the low, intermediate-1 and intermediate-2 risk groups with respect to OS (p=0.12, p=0.86, p=0.98), CIR (p=0.28, p=0.54, p=0.94) and CID (p=0.09, p=0.57, p=0.39). In the high risk group, OS was significantly superior after MRD-transplant compared to MUD-transplant (p=0.02), but without significant differences in CIR (p=0.74) and CID (p=0.08). Equivalent efficacy could also be shown in a subgroup analyses focusing on all FLT3-ITD positive patients (MRD, n=103, MRD, n=147) for OS (p=0.71), CIR (p=0.53) and CID (p=0.69). Conclusions: Our results based on prospective interventional studies support the perception that MUD-transplants are equal to MRD-transplants in patients with AML in first CR. Only within the ELN high risk group, patients with MRD-transplants showed superior OS but without differences in CIR and CID as compared to MUD-transplants. Disclosures Kobbe: Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other. Götze:Celgene Corp, Novartis Pharma: Honoraria. Fiedler:TEVA: Travel reimbursement for meeting attendance Other. Petzer:Celgene: Honoraria, unrestricted grant Other. Lübbert:Cephalon / TEVA: Travel support Other. Greil:Bristol-Myers-Squibb: Consultancy, Honoraria; Cephalon: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Amgen: Honoraria, Research Funding; Eisai: Honoraria; Mundipharma: Honoraria, Research Funding; Merck: Honoraria; Janssen-Cilag: Honoraria; Genentech: Honoraria, Research Funding; Novartis: Honoraria; Astra-Zeneca: Honoraria; Boehringer-Ingelheim: Honoraria; Pfizer: Honoraria, Research Funding; Roche: Honoraria; Sanofi Aventis: Honoraria; GSK: Research Funding; Ratiopharm: Research Funding. Döhner:Novartis: Research Funding. Döhner:TEVA: Research Funding.

Abstract: Background: Mutations of the NPM1 gene are one of the most frequent genetic aberrations in adult AML. AML with mutated NPM1 is categorized as a disease entity according the WHO-2016 classification and clinically associated with female sex, high white blood cells at diagnosis, normal karyotype and high CD33 antigen expression. We recently showed that patients with NPM1-mutated AML benefit from all-trans retinoic acid (ATRA) as adjunct to intensive induction therapy (Ann Hematol. 2016; 95:1931-1942; Haematologica. 2009;94:54-60). Based on the regular high CD33 expression in AML with mutated NPM1 we hypothesized that gemtuzumab ozogamicin (GO) added to intensive therapy with ATRA may further improve clinical outcome in AML with mutated NPM1. Aim: To evaluate GO in combination with intensive induction and consolidation therapy and ATRA in NPM1 mutated AML within the randomized AMLSG 09-09 trial (NCT00893399) Methods: Between May 2010 and September 2017, patients ≥18 years of age and considered eligible for intensive therapy were randomized up-front for open-label treatment with GO. Induction therapy consisted of two cycles of A-ICE (idarubicin 12mg/m² iv, day 1,3,5 [in induction II and for patients 〉 60 years reduced to d 1, 3]; cytarabine 100mg/m² continuous iv, day 1 to 7; etoposide 100mg/m² iv, day 1-3 [in induction II and for patients 〉 60 years reduced to d 1, 3]; ATRA 45 mg/m²/day po on days 6-8 and 15mg/m² days 9-21, +/- GO 3mg/m² iv day 1). Consolidation therapy consisted of 3 cycles of high-dose cytarabine (HiDAC; 3g/m² [reduced to 1g/m² in patients 〉 60 years] bid, days 1-3; Pegfilgrastim 6mg sc, day 10; ATRA 15 mg/m²/day po, days 4-21; +/- GO 3mg/m² on day 1 [first consolidation only] ). The primary endpoints of the study were event-free survival (EFS) as early endpoint tested 6 months and overall survival (OS) tested 4 years after study completion with sequential testing according the fallback procedure described by Wiens (Statistics 2003;2:211-215). This report focusses on the early EFS endpoint. Further secondary endpoints were response to induction therapy, cumulative incidence of relapse (CIR) and cumulative incidence of death (CID). Results: In total 588 patients were evaluable for analysis (n=296, standard-arm; n=292 GO-arm). Median age was 58.7 years (range, 18.4-82.3 years), ECOG performance status was 0 in 34.1% and 1 in 55.1%, and FLT3-ITD was present in 16.8% of the patients, with baseline characteristics well balanced between the two arms. After first induction therapy death rates were significantly higher in the GO-arm (7.5%) (p=0.02) compared to the standard-arm (3.4%); in both study-arms causes of death were mainly infections. Following induction therapy complete remission (CR) and CR with incomplete count recovery (CRi) were 88.5% and 85.3% (p=0.28), refractory disease (RD) 6.1% and 5.1% (p=0.72), death 5.4% and 9.6% (p=0.06) in the standard- and GO-arm, respectively. Due to prolonged thrombocytopenia after second induction therapy in the GO-arm, the protocol was amended in that GO was omitted in second induction and first consolidation cycles, if prolonged cytopenias were observed during first induction therapy. The study treatment was completed in 197 and 171 patients (p=0.11), allogeneic hematopoietic cell transplantation in first CR was performed in 18 and 21 patients (p=0.51) in the standard- and GO-arm, respectively. Median follow-up was 2.6 years (95%-CI, 2.4-3.1 years). Two- and 4-year EFS were 53% (95%-CI, 48-60%) and 58% (95%-CI, 52%-64%), and 44% (95%-CI, 38-52%) and 52% (95%-CI, 46%-59%) in the standard- and GO-arm, respectively. According to the pre-specified significance level of 0.025, EFS in the GO-arm was not different to that in the standard-arm (p=0.21). In patients achieving CR/CRi after induction therapy, CIR was significantly reduced in the GO-arm compared to the standard-arm (p=0.018), whereas no difference in CID was noted between both arms (p=0.89). Conclusion: The addition of GO to intensive induction therapy with ICE plus ATRA was associated with a higher death rate. In patients achieving a CR/CRi after induction therapy significantly less relapses occurred in the GO- compared to the standard-arm. Disclosures Schlenk: Pfizer: Research Funding, Speakers Bureau. Paschka:Astex: 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; Otsuka: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Jazz: Speakers Bureau; Amgen: Other: Travel support; Janssen: Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Travel support. Fiedler:Amgen: Other: support for meetíng attendance; Gilead: Other: support for meeting attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; 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; GSO: Other: support for meeting attendance; Teva: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Daiichi Sankyo: Other: support for meeting attendance. Lübbert:Cheplapharm: Other: Study drug; Celgene: Other: Travel Support; Janssen: Honoraria, Research Funding; TEVA: Other: Study drug. Götze:Novartis: Honoraria; Takeda: Honoraria, Other: Travel aid ASH 2017; JAZZ Pharmaceuticals: Honoraria; 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. Greil:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Honoraria, Research Funding; Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sandoz: Honoraria, Research Funding; Amgen: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heuser:Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Astellas: Research Funding; BergenBio: Research Funding; Karyopharm: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Döhner:Agios: Consultancy, Honoraria; Pfizer: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Jazz: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Seattle Genetics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

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: Background: CBF-AML is defined by recurrent genetic abnormalities which encompass t(8;21)(q22;q22), inv(16)(p13.1q22) or less frequently t(16;16)(p13.1;q22). Most frequent secondary chromosome aberrations in t(8;21) AML are del(9q) or loss of a sex chromosome, and in inv(16)/t(16;16) AML trisomy 22 or trisomy 8. At the molecular level mutations involving KIT, FLT3, or NRAS were identified as recurrent lesions in CBF-AML. However, the underlying genetic alterations which might trigger relapse in CBF-AML are not well delineated. Thus, the aim of our study was to characterize the clonal architecture of relapsed CBF-AML. Methods: We performed mutational profiling (KIT, FLT3-ITD, FLT3-TKD, NRAS, ASXL1) in paired samples obtained at diagnosis and at relapse from 66 adults with CBF-AML [inv(16), n=43; t(8;21), n=23] who all were treated within the AMLSG studies. Results: In inv(16) AML, the following mutation pattern was identified at diagnosis: KIT 13/40 (33%; exon 8, n=6; exon 17, n=5; exon 8+17, n=1; exon 11, n=1; missing data, n=3), NRAS 18/43 (42%), FLT3-TKD 4/43 (9%); none of the pts harboured FLT3-ITD or ASXL1 mutations. At the time of relapse, there was a shift in the mutation pattern in 26 pts (60%): KIT mutations (exon 8, n=5; exon 17, n=2; exon 8+17, n=1) were lost in 8 pts and 1 pt acquired an exon 17 KIT mutation; similarly, 15 pts lost and 1 pt gained NRAS mutation, respectively. Of note, all FLT3-TKD mutations were lost at the time of relapse, and only one pt gained a FLT3-ITD mutation. Based on these findings we calculated the stability in inv(16) AML for KIT, NRAS and FLT3-TKD mutations as 38%, 17%, and 0%, respectively. AML with t(8;21) presented a different diagnostic mutation profile: KIT 9/23 (39%; exon 17, n=8; exon 11, n=1), FLT3 -ITD 3/23 (13%), NRAS 2/23 (9%), and ASXL1 1/23 (4%); there were no FLT3-TKD mutations. At the time of relapse, the mutation pattern changed in 9 pts (39%); KIT mutations were lost in 4 pts (exon 17, n=3; exon 11, n=1), but acquired in 2 pts with both of them located in exon 17; only 1 pt lost the NRAS mutation. FLT3-ITD was lost in 2 and gained in 3 pts. There was no change in the ASXL1 mutation status. Thus, the stability for KIT, NRAS, FLT3-ITD and ASXL1 mutations in t(8;21) AML was calculated as 56%, 50%, 33% and 100%, respectively. Of note, mutations affecting the KIT and NRAS gene were almost mutually exclusive; there were only 3 pts with concurrent KIT and NRAS mutations at diagnosis [inv(16), n=2; t(8;21), n=1] . Conclusion: CBF-AML cases display a high degree of molecular heterogeneity with shift of the mutation pattern at relapse in both CBF-AML subtypes. The frequent loss of KIT and NRAS mutations at relapse suggests that there might be other important secondary lesions driving relapse. Ongoing high-resolution genome-wide profiling will further unravel the clonal hierarchy and genomic landscape in CBF-AML. Disclosures Götze: Novartis: Honoraria; Celgene Corp.: Honoraria. Greil:Celgene: Consultancy; Ratiopharm: Research Funding; Sanofi Aventis: Honoraria; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Astra-Zeneca: Honoraria; GSK: Research Funding; Novartis: Honoraria; Genentech: Honoraria, Research Funding; Janssen-Cilag: Honoraria; Merck: Honoraria; Mundipharma: Honoraria, Research Funding; Eisai: Honoraria; Amgen: Honoraria, Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria; AOP Orphan: Research Funding; Roche, Celgene: Honoraria, Research Funding. Schlenk:Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding.

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.