Program: Oral and Poster Abstracts
Session: 617. Acute Myeloid Leukemia: Biology, Cytogenetics, and Molecular Markers in Diagnosis and Prognosis: Poster I
The novel fms-like tyrosine kinase 3 (FLT3)/AXL inhibitor, gilteritinib, showed robust antileukemic activity at doses ≥80 mg/day in FLT3 mutation-positive (FLT3mut+) patients with R/R AML in the CHRYSALIS Phase 1/2 study (NCT02014558). We analyzed variant allele frequency (VAF) in a CHRYSALIS study FLT3mut+ R/R AML subpopulation with baseline FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations using two different assays: capillary electrophoresis (CE) and next-generation sequencing (NGS). We assessed analytical concordance and survival outcomes at two FLT3 loci: ITD and D835 mutations in the TKD.
Assays were performed on bone marrow and blood samples. The CE (LeukoStrat® CDx) FLT3 mutation assay detected both FLT3-ITD and D835/I836 FLT3-TKD mutations; these loci were amplified using polymerase chain reaction. Amplicon size differences distinguished samples with FLT3-ITD mutations from those with wild-type FLT3. Undigested amplicons following restriction enzyme digestion identified the presence of FLT3-TKD mutations. The capture based NGS assay targeted potential mutation loci in all FLT3 exons. Whole genome libraries were generated and hybridized with a custom probe to capture target fragments, which were sequenced on an Illumina® MiSeq platform. The CE FLT3 signal ratio outputs were converted to estimated VAF values for comparison with NGS outputs. For FLT3-ITD analysis, both assays were optimized to detect large ITD mutations. Median overall survival (OS) was determined for patients who received ≥80 mg/day gilteritinib, specifically among those with FLT3-ITD with/without concomitant TKD mutations, and those with TKD mutations only. Median VAF values were calculated; patient VAFs were stratified according to whether they were greater than/equal to, or less than the median value. Lastly, OS of patients with a FLT3-ITD mutation detected by NGS was stratified using a ≥5% VAF cut point.
Bone marrow and blood samples from 241 patients were analyzed. The two assays demonstrated strong concordance for both qualitative (mutation detection) and quantitative (VAF) measures at both FLT3-ITD and FLT3-TKD loci. Using a VAF threshold of ≥0.05 and the most prominent ITD in each sample, FLT3-ITD detection by CE or NGS showed a strong concordance in 98.8% (238 of 241) of samples (155 and 156 positives by CE and NGS, respectively). All 3 discordant samples had VAFs that were near (±0.02) the 0.05 VAF threshold and within the 95% CI values for both assays. Strong concordance for FLT3-ITD detection was maintained when multiple ITDs were observed within a sample; a strong linear correlation between both assays was observed for mutation frequencies (R2=0.987) (Figure 1). At a VAF threshold of 0.05, FLT3-TKD mutation concordance was observed in 98.3% (236/240) of samples (34 and 30 positives by CE and NGS, respectively). The 4 discordant samples were near (±0.04) the 0.05 VAF threshold and there was a strong a linear correlation between observed mutation frequencies (R2=0.906).
At a VAF threshold of ≥5% for FLT3 mutation positivity, there was no apparent difference in OS in the ≥80 mg/day gilteritinib subgroup among those with FLT3-ITD with/without concomitant TKD mutations who had VAF values that were greater than/equal to or less than the median values (Figure 2A and 2B), regardless of the assay used. Patients who had a TKD mutation only with VAF values greater than/equal to the median value had longer OS than patients with VAF values less than the median value. Patients who had a TKD mutation only with VAF values greater than/equal to the median value had OS durations similar to those in patients with ITD mutations. By NGS, patients with a FLT3-ITD VAF <5% had similar OS duration as patients with a FLT3-ITD VAF ≥5% (Figure 3).
We observed high concordance using both CE and NGS methods to measure FLT3 mutational burden in patients with R/R AML who received gilteritinib in the CHRYSALIS study. Analytical concordance between the 2 assays was high and survival outcomes with stratification based on FLT3 mutational burden were similar with both assays. Our results suggest that FLT3-ITDmut+ R/R AML patients may benefit from gilteritinib therapy, regardless of FLT3 mutational burden. In patients with FLT3-TKD mutations only, a higher mutational burden may be associated with gilteritinib benefit; this finding will be further explored in future studies.
Disclosures: Smith: Plexxikon Inc.: Research Funding; Astellas Pharma: Research Funding. Perl: Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Asana Biosciences: Other: Scientific advisory board; Astellas: Consultancy; Novartis: Other: Advisory Board; Seattle Genetics: Other: Advisory board; Daiichi Sankyo: Consultancy; Arog Pharmaceuticals: Consultancy. Altman: Syros: Consultancy; BMS: Consultancy; Celgene: Consultancy; Astellas: Consultancy; Novartis: Consultancy; Janssen Pharmaceuticals: Consultancy; Ceplene: Consultancy; NCCN: Other: Educational speaker; ASH: Other: Educational speaker; Hemedicus: Other: Advisor to CME Company. Carson: Invivoscribe, Inc.: Employment. Cortes: Sun Pharma: Research Funding; Teva: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Stenzel: Invivoscribe, Inc.: Employment. Hill: Ligacept, LLC: Equity Ownership; Patent: Patents & Royalties: WO2013163419A1 - pending; Patent: Patents & Royalties: US7862995B2 - Issued; Astellas Global Pharma Development: Employment. Lu: Astellas Pharma Global Development: Employment. Bahceci: Astellas Pharma Global Development: Employment. Levis: Daiichi Sankyo, Inc.: Consultancy, Honoraria; FujiFilm: Research Funding; Astellas Pharma Us: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.
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