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.

Abstract: Background: Acute myeloid leukemias (AML) with rearrangements of core-binding factor (CBF) complex genes (CBF-AML), comprising t(8;21) and inv(16) subgroups, are considered as diseases with favorable outcome. Nevertheless, CBF-AML relapse rates remain high, with ~40% of patients (pts) relapsing after standard intensive chemotherapy. Aim: To dissect the biology of relapse in CBF-AML, we performed whole exome sequencing (WES) in a large cohort of 101 cases at the time of diagnosis and for 47 cases also at the time of relapse. Methods: All pts were treated either with standard chemotherapy or with standard chemotherapy and kinase inhibitor dasatinib within clinical trials of the German-Austrian AML Study Group (AMLSG). Using the Nextera Rapid Capture Exome kit (Illumina) we performed WES of paired diagnostic (dx), remission and relapse samples of 47 pts, namely 21 pts with t(8;21) and 26 pts with inv(16). RNAseq was performed in 18 of these pts using the Ribo Zero RNA-sequencing kit (Illumina). To better define genomic signatures related to CBF-AML relapse, we included WES data previously published by our group (Faber et al. Nat Genet 2016). This set comprised dx samples of 8 t(8;21) and 10 inv(16) pts who relapsed as well as a control group of 20 t(8;21) and 16 inv(16) CBF-AML pts, who did not experience relapse. Results: For the new cohort, WES sequencing of 47 pts was performed with a mean coverage of 127-fold. In t(8;21), we identified a median of 3.5 mutations exclusively present at dx (range: 0-8), 11.6 mutations persistent from dx to relapse (range: 4-19), and 4.0 mutations gained at relapse (range: 2-7). For the inv(16) subgroup a median of 2.0 mutations were dx specific (0-7), 6.0 mutations persisted during tumor evolution (3-26) and 2.5 were gained at relapse (0-9). As previously reported, the spectrum of genes affected by mutations showed little overlap between t(8;21) and inv(16), except for commonly affected 'signaling' genes such as KIT, RAS, FLT3 and epigenetic players such as TET2. In total, in t(8;21) we identified 94 relapse-specific mutations or mutations displaying a strong increase in variant allele frequency (VAF) at relapse, and 63 of such relapse-specific alterations in inv(16) pts. In addition to the previously reported RUNX1 and cohesin complex gene mutations showing an increase in VAF at relapse, we found recurrent novel relapse-specific mutations in LAMC3, which occurred exclusively in the t(8;21) subgroup affecting 9% of pts. In inv(16), recurrent mutations in the tumor suppressor gene WT1 occurred in 12% of pts, either acquired at relapse or already present at dx as a minor subclone. Remarkably, mutations in relapsed t(8;21) pts often affected genes involved in PI3K-AKT and in cell cycle regulation pathways. In the inv(16) relapse group, in addition to dysregulation of the MAPK signaling pathway, we found several non-recurrent mutations in genes involved in ribosomal RNA metabolism, like in PRNAD1. Conclusion: Our WES sequencing results already provide first insights into the molecular composition and mechanisms underlying relapse in CBF-AML which often affect pathways linked to proliferation, such as PI3K-AKT and MAPK signaling. While we are currently validating additional hits, updated results will be provided at the ASH meeting, which will also address combinatorial mutation patterns underlying chemotherapy resistance in t(8;21) and inv(16) positive AML. Disclosures Götze: Celgene: Research Funding. Fiedler:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria; ARIAD/Incyte: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding; Daiichi Sankyo: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Abbvie: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees. Thol:Celgene: 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; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Heuser:PriME Oncology: Honoraria; Abbvie: Consultancy; Stemline Therapeutics: Consultancy; Karyopharm: Research Funding; Roche: Research Funding; Bayer: Consultancy, Research Funding; Amgen: Research Funding; BerGenBio ASA: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; Astellas: Research Funding. Ganser:Novartis: Consultancy; Celgene: Consultancy. Paschka:Agios Pharmaceuticals: Consultancy, Speakers Bureau; Astex Pharmaceuticals: Consultancy; Astellas Pharma: Consultancy, Speakers Bureau; Celgene: Consultancy, Other: Travel, accommodations or expenses; Jazz Pharmaceuticals: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Otsuka: Consultancy; Pfizer: Consultancy, Speakers Bureau; Sunesis Pharmaceuticals: Consultancy; AbbVie: Other: Travel, accommodation or expenses, Speakers Bureau; Amgen: Other; Janssen Oncology: Other; BerGenBio ASA: Research Funding. Döhner:GEMoaB: Consultancy, Honoraria; AROG: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; 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; Roche: Consultancy, Honoraria; Pfizer: Research Funding; Oxford Biomedicals: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; Sunesis: Research Funding. Döhner:Novartis: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Daiichi Sankyo: Honoraria; Abbvie: Consultancy; Sunesis Pharmaceuticals: Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy; Astellas Pharma: Consultancy; Amgen: Consultancy, Research Funding; Agios: Consultancy; Roche: Consultancy; Arog: Research Funding. Bullinger:Amgen: 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; 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; Gilead: 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; Hexal: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; 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; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Abstract: Abstract 824 Background: Although acute myeloid leukemia (AML) with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) [hereafter referred to as inv(16)] is considered as a favorable AML subset, 30-40% of the patients (pts) are not cured by the current treatment strategies. Various genetic markers have been assessed to identify pts who are at high risk to fail therapy. However, the results among the different studies were not fully consistent. Methods: We studied diagnostic bone marrow and/or blood specimens from 179 adult AML pts with inv(16) for secondary chromosome abnormalities and gene mutations in FLT3 [internal tandem duplications (ITD) and tyrosine kinase domain mutations (TKD)], KIT, JAK2 (V617F) and in N-and K-RAS. All pts were treated on one of 7 prospective protocols of the German-Austrian AML Study Group (AMLSG) including anthracycline- and cytarabine-based induction therapy, and consolidation therapy incorporating higher doses of cytarabine in various settings, but also autologous and allogeneic 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: At least one gene mutation was found in 84% of the pts with 21% of the pts having multiple mutations. Pts with gene mutations ≥2 showed no significant differences in WBC, incidence of trisomy 22 and age. Mutations were most frequent in RAS (53%), followed by mutations in KIT (37%) and FLT3 (17%) [FLT3-TKD (15%) and FLT3-ITD (4%)] genes. No mutations were detected in JAK2. Concurrent mutations of KIT and RAS in the same leukemia sample were less likely to occur (P=0.003) than expected based on their frequencies as single markers; no other significant interactions between the gene mutations were observed. Median age of the cohort was 41 years (yrs; range, 18-74 yrs), and median white blood count (WBC) was 38.3 × 109/l (range, 1.1 to 294.9 × 109/l). Median follow-up for survival according to Korn was 4.9 yrs [95%-confidence interval (CI), 4.3-6.0 yrs] . While one multivariable model was constructed using the mutations in KIT, FLT3 and RAS as covariates (model I), the other included the number of mutated genes (≥2 vs 〈 2; model II) as single variable. Additional covariates included in the multivariable models were age, log10(WBC) and presence of trisomy 22. Model I revealed mutated KIT [Hazard ratio (HR), 1.85, P=0.04] and mutated FLT3 (HR, 2.03, P=0.04) as adverse factors for RFS, and mutated FLT3 (HR, 2.39, P=0.03) and older age (HR for 10 yrs change, 1.62, P=0.003) as significant factors for shorter OS. Model II revealed number of mutated genes ≥2 (HR, 2.38, P=0.004) and higher WBC (HR for change of one unit on log10 scale, 1.94, P=0.03) as adverse factors, and trisomy 22 (HR, 0.46, P=0.05) as favorable factor for RFS. In addition, number of mutated genes ≥2 (HR, 1.99, P=0.04) and older age (HR for 10 yrs change, 1.57, P=0.004) predicted for a shorter OS. Additional univariable analyses also revealed that pts with KIT mutations had a worse RFS compared with KIT wild-type (P=0.03; 5-yr RFS rates, 42% vs 60%), and that pts with FLT3 mutations had in trend a shorter OS compared with FLT3 wild-type pts (P=0.08; 5-yr OS rates, 58% vs 70%). Importantly, in univariable analysis, pts with number of mutations ≥2 had a worse RFS (P=0.009; 5-yr RFS rates, 36% vs 58%), but their OS was not significantly shorter (P=0.16; 5-yr OS rates, 58% vs 71%) than in pts with number of mutations 〈 2. However, once pts with trisomy 22 were excluded, the group with number of mutations ≥2 had a worse OS (P=0.02) than the group with number of mutations 〈 2. Conclusions: In this large cohort of genetically well defined inv(16)-positive AML we show that KIT and FLT3 mutations are independent factors for RFS and OS, respectively. Even more important, we demonstrate for the first time that harboring ≥2 secondary molecular lesions is an independent predictor for a worse RFS and OS. Thus, comprehensive genetic characterization may improve outcome prediction for AML with inv(16). Pts with multiple mutations may be candidates for a more aggressive treatment in combination with therapies targeting the mutated tyrosine kinases and GTPase proteins. Disclosures: No relevant conflicts of interest to declare.

Abstract: MRD assessment in t(8;21) AML allows identification of patients at high relapse risk at defined time points during treatment and follow-up. MRD− after treatment is the most favorable factor for relapse risk and survival, and serial MRD analyses define cutoffs predicting relapse.