ALBERT

Description: The presence of MRD in patients with acute myeloid leukemia (AML) who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) confer a negative prognostic impact by increasing the risk of relapse. However, the ability to detect these mutations in remission bone marrow specimens is hampered by the limited sensitivity of conventional polymerase chain reaction (PCR)-based assays, which detect approximately only 1 of every 100 (1%) mutant cells. To address this problem, we have developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations. Using isolated genomic DNA from bone marrow aspirates or whole-blood samples, PCR primers flanking exons 14 and 15 of the FLT3 gene were designed and highly diverse NGS libraries were generated. These libraries were then sequenced by Illumina's sequencing-by-synthesis method. The bioinformatics approach we used identifies unique FLT3-ITD mutations of varying length along with wild type sequences and calculates a mutant allelic frequency. The assay was validated using clinical samples to assess accuracy and reproducibility. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% but was capable of detecting FLT3-ITD mutations at a level as low as 0.003%. We next validated the assay using clinical samples from patients with FLT3-ITD AML. The negative prognostic impact of FLT3-ITD mutations can be mitigated in part when an FLT3 inhibitor is administered in combination with induction chemotherapy, as demonstrated in CALGB10603/RATIFY (N Engl J Med. 2017;377:454). It was reported in this study that patients treated with an FLT3 inhibitor combined with chemotherapy followed by allogeneic transplant in first remission had better overall survival than their counterparts in the control arm. One hypothesis for this outcome is that the FLT3-inhibitor-treated patients had a lower leukemic burden prior to transplant. As a pilot test of this concept, we used our MRD assay on a series of bone marrow aspirate samples collected from 10 patients with newly diagnosed FLT3-ITD AML. The patients were selected to be as uniform as possible. All patients had intermediate-risk karyotype, a detectable FLT3-ITD mutation by conventional PCR, and mutated NPM1. All patients received cytarabine-based intensive induction and achieved morphologic first remission with a single course of chemotherapy. Finally, all patients underwent allogeneic transplant in first remission. The sample analyzed for MRD was the first collected after remission induction, 5-8 weeks after the start of therapy. The investigators performing the MRD assay were blinded to the clinical data. Four patients received chemotherapy alone, while 6 were treated with chemotherapy (7+3) plus an FLT3 inhibitor. In all patients' remission samples, the MRD assay identified the FLT3-ITD mutation that precisely matched the one observed in the original diagnostic specimen. This demonstrates the sensitivity of the assay (all samples had a detectable mutation), and the unique length of each patient's mutation confers a degree of specificity not achievable with MRD detection methods that focus on other AML-associated mutations. Supporting our hypothesis was the observation that patients treated with FLT3 inhibitors had MRD levels lower than those in patients treated with chemotherapy alone (Figure). Our results help establish the role of NGS-based MRD assays for the clinical management of FLT3-ITD AML. This assay could be used to define the depth of remission, identify persistent disease, and help guide decision making in the use of FLT3 inhibitors as continuation therapy. This study provides validation of the clinical utility of our MRD assay, which will be used to analyze the remission samples from patients in the ongoing phase 3, randomized, double-blind, placebo-controlled QuANTUM-First clinical trial, in which patients with newly diagnosed FLT3-ITD AML are randomized to receive either the highly potent and selective FLT3 inhibitor quizartinib or placebo in combination with chemotherapy, followed by single-agent quizartinib as continuation therapy. Disclosures Shi: Novartis: Employment, Equity Ownership; Daiichi Sankyo: Other: Provide clinical trial testing services. Chang:Daiichi Sankyo: Employment. Laing:Novartis: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Berisha:Daiichi Sankyo: Employment. Adams:Johns Hopkins University: Employment. Ding:Navigate BP: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Nakamaru:Daiichi Sankyo: Employment. Lameh:Navigate BioPharma Inc,: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Pollner:Navigate BioPharma Inc.: Employment. Kobayashi:Daiichi Sankyo: Employment.

Description: Background: Quizartinib, a once-daily, oral, highly potent and selective FLT3 inhibitor, demonstrated a significant improvement in survival vs SC in FLT3-ITD-positive R/R AML in the global, randomized, phase 3 QuANTUM-R study (Cortes et al. Lancet Oncol, 2019; NCT02039726). Patients with R/R FLT3-ITD-positive AML were randomized 2:1 to receive single agent quizartinib or investigator's choice of pre-selected SC. We investigated the effects of baseline co-mutations and FLT3-ITD VAF on overall survival (OS) and response (composite complete remission [CRc]) to quizartinib and SC in QuANTUM-R. Methods: We analyzed 37 recurrently mutated genes in AML in baseline bone marrow samples from 304 patients (82.8% of ITT population [N = 367; quizartinib, n = 245; SC, n = 122]) with R/R FLT3-ITD-positive AML using next-generation sequencing and a customized Archer® Core Myeloid panel. Positive mutation status was defined as ≥ 1 mutation detected in the gene region using a VAF cutoff of 2.7%. FLT3-ITD VAF was measured separately by the Navigate BioPharma FLT3 Mutation Assay (polymerase chain reaction-based, VAF cutoff of 3%). Low and high FLT3-ITD VAF were defined as ≤25% and 〉25%, respectively. Results: In addition to FLT3-ITD, 5 key co-mutations were detected: DNMT3Amut (n = 182/304 [59.9%]), NPM1mut (n = 168/304 [55.3%]), TET2mut (n = 98/304 [32.2%]), IDH1/2mut (n = 49/304 [16.1%]) and CEBPAmut (n = 46/304 [15.1%]). Median OS was numerically longer with quizartinib vs SC in patients with DNMT3Amut, TET2mut, IDH1/2mut and NPM1mut, but not CEBPAmut (Table). CRc rates were numerically higher with quizartinib vs SC for each of the 5 key baseline co-mutations. For single gene mutations, the longest median OS was seen in patients with CEBPAmut treated with quizartinib or SC (37 and 37.6 weeks, respectively). As the majority of NPM1mut patients were also DNMT3Amut (138/168, 82%), we examined various permutations of these two mutations. Patients with NPM1wt/DNMT3Amut had significantly longer median OS with quizartinib vs SC (39.3 vs 19.6 weeks, respectively; HR, 0.239; P = 0.003 [Table]) while NPM1mut/DNMT3Amut patients had lower and similar median OS between the 2 arms (23.6 vs 23.4 weeks, respectively). Quizartinib treatment showed significantly longer median OS vs SC in patients with high FLT3-ITD VAF (23.9 vs 17 weeks respectively; HR, 0.689, P = 0.0148), while the median OS in patients with low FLT3-ITD VAF was similar (34.1 vs 26.6 weeks, respectively; HR, 0.857, P = 0.535). Conclusions: This is the first evaluation of the effect of baseline co-mutations on clinical outcomes in a large trial of R/R AML patients with FLT3-ITD mutations treated with quizartinib. Key co-mutations identified in this analysis were found to potentially impact treatment response and OS with quizartinib, relative to SC. Despite relatively low CRc rates in patients with IDH1/2mut, this group-as well as those with NPM1wt-derived the greatest OS benefit from quizartinib compared with SC on QuANTUM-R. CEBPA mutations were associated with high CRc rates and relatively long median OS, regardless of treatment arm. Patients with NPM1mut had a higher CRc rate with quizartinib vs SC, but this did not translate into longer survival on either arm compared with NPM1wt. A high allelic burden of FLT3-ITD at the time of salvage therapy was associated with relatively poorer median OS; quizartinib significantly improved survival of patients with high FLT3-ITD VAF relative to SC. Although these results require confirmation in an independent dataset, the modulatory effects of baseline co-mutations on treatment response and OS with quizartinib appear to differ from other FLT3 inhibitors. Our results indicate that a subset of R/R AML patients may particularly derive clinical benefit from quizartinib. Table Disclosures Perl: Daiichi Sankyo: Consultancy, Honoraria, Other, Research Funding; Arog: Consultancy, Other: Non-financial support included travel costs for advisory board meetings.; AbbVie: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Actinium Pharmaceuticals: Consultancy, Honoraria, Other: Clinical Advisory Board member, Research Funding; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Non-financial support included travel costs for advisory board meetings.; Jazz: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; NewLink Genetics: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Takeda: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Bayer: Research Funding; BioMed Valley Discoveries: Research Funding; FujiFilm: Research Funding; Novartis: Honoraria, Other: Advisory board, Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of the data., Research Funding; Astellas: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of trial data., Research Funding. Cortes:Astellas Pharma: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; BiolineRx: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Sun Pharma: Research Funding; Immunogen: Consultancy, Honoraria, Research Funding. Ganguly:Kite Pharma: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Seattle Genetics: Speakers Bureau; Daiichi Sankyo: Research Funding. Khaled:Omeros: Consultancy; Alexion: Consultancy, Speakers Bureau; Daiichi Sankyo: Other: Travel support. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; BMS: Research Funding. Martinelli:Novartis: Consultancy, Other: trial grant; Roche: Consultancy, Other: trial grant; Janssen: Consultancy, Other: trial grant; Incyte: Consultancy, Other: trial grant; Pfizer: Consultancy, Other: trial grant; Amgen: Consultancy, Other: trial grant; Celgene: Consultancy, Honoraria, Other: trial grant; Ariad: Consultancy, Other: trial grant; Abbvie: Consultancy, Honoraria, Other: trial grant; Daiichi Sankyo: Consultancy, Honoraria. Russell:Pfizer Inc: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria, Speakers Bureau; DSI: Consultancy, Honoraria, Speakers Bureau. Chang:Daiichi Sankyo: Employment. Mires:Daiichi Sankyo: Employment. Kato:Daiichi Sankyo, Inc.: Employment; Celgene: Employment, Equity Ownership. Zhang:Daiichi Sankyo: Employment. Korkhov:Precision for Medicine, Inc.: Employment; Daiichi Sankyo: Consultancy. Wang:Precision for Medicine, Inc.: Employment; Daiichi Sankyo: Consultancy. Günnel:Precision for Medicine, Inc.: Employment; Daiichi Sankyo: Consultancy. Sumi:Daiichi Sankyo, Inc.: Employment. Isoyama:Daiichi Sankyo Co, Ltd: Employment. Lesegretain:Daiichi-Sankyo Inc.: Employment, Equity Ownership. Berisha:Daiichi Sankyo: Employment. Dos Santos:Daiichi Sankyo: Employment. Levis:Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria.