The Adverse Effect of Myelodysplasia-Related Mutations in De Novo Acute Myeloid Leukemia Is Associated with Higher Variant Allelic Frequency (VAF): A Proposal for a Numeric Cutoff for Variant Allelic Frequency

Mutations in myelodysplasia-related genes ASXL1, BCOR, EHZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 (MR^mut) are class-defining alterations in acute myeloid leukemia (AML) in the latest World Health Organization (WHO, with the exception of RUNX1) and International Consensus Classification (ICC) schemes and indicate adverse-risk per European Leukemia Net (ELN) 2022 guidelines, unless occurring in favorable-risk AML. The lower limit of detection (LOD) for most clinical-grade next generation sequencing (NGS) is ~1-2% variant allelic frequency (VAF); however, data on the prognostic significance of MR^mut relative to VAF is lacking. No guidelines that provide a numeric cutoff for VAF are available for utilizing these mutations to classify AML. Our aim was to define a clinically meaningful VAF cutoff for MR mutations in de novo AML.

We included patients (pts) with de novo, untreated AML with MR^mut treated at our institution between 2017 to 2022. We excluded pts with acute promyelocytic leukemia, core binding factor AML, and other ELN adverse-risk categories (adverse karyotype and/or TP53 mutation). NGS at baseline was performed using a panel of 81 genes recurrently mutated in myeloid neoplasms (LOD ~1-2%). We performed a best fit classifier on individual MR^mut to determine optimal VAF cutoffs using event-free survival (EFS) and overall survival (OS) as endpoints. VAF for MR^mut involving X-linked genes was adjusted for men.

Pts (n=145) included 92 (63%) men and 53 (37%) women with a median age of 68 years (range, 58-75). 85 (59%) pts had diploid karyotype AML, 51 (35%) had other intermediate-risk non-diploid karyotype AML, and 9(6%) had insufficient metaphases for analysis. 34 (23%) pts had ELN favorable-risk and 111(77%) had ENL adverse-risk AML. Pts were treated with low-intensity+venetoclax (VEN) (72/50%), low-intensity (26/18%), high-intensity (22/15%), and high-intensity+VEN (12/17%) regimens.

Median follow-up was 26.9 months (range, 13.9 - 40.8). 72 (49%) pts had 1 MR^mut; the remaining had 2-4 concurrent MR^mut. MR^mut had the following frequencies and VAFs: ASXL1 (28%; median VAF: 26%, [range 2-48%]); BCOR (18%; median VAF 37% [3-100%]); EHZH2 (9%; median VAF 7% [1-55%]), RUNX1 (32%; median VAF 47% [1-96%]), SF3B1 (9%; median VAF: 38% [1-55%]), SRSF2 (46%; median VAF: 37% [1-76%]), STAG2 (20%; median VAF: 44% [15-98%]), U2AF1 (12%; median VAF: 33% [2-43%]) and ZRSR2 (2%; median VAF: 94% [93-95%]). 34 (23%) pts had ELN favorable-risk AML including 28 (23%) with mutated NPM1 without FLT3-ITD and 6 (4%) with CEBPA bZIP mutations, respectively; in 12/34 pts NPM1 or CEBPA bZIP mutations were subclonal compared with clonal MR mutation(s). A summary of baseline genetic and treatment data is provided in Fig 1A.

The overall composite remission rate (CR+CRi) was 88% (64% and 24%, respectively) Median OS was 30.3 months and estimated 12/24 month OS were 77%/60%. The median EFS was 24.6 months and estimated 12/24 month EFS were 65%/51%. Pts with ELN favorable-risk AML with MR^mut had longer OS and EFS compared with the adverse-risk group (median OS 34.9 vs 27.5 months, median EFS 34.9 vs 22.9 months) but the difference was not significant (p=0.60 and 0.30, respectively). The VAF cutoff for MR^mut associated with EFS and OS, respectively, was: ASXL1: 21%/21%, BCOR 25%/25% , EHZH2 15%/15%, RUNX1 25%/27%, SF3B1 31%/31%, SRSF2 32%/35%, STAG2 40%/21%, U2AF1 44%/44% and ZRSR2 47/47%.There was no significant difference in OS or EFS between pts with 1 MR^mut vs those involving ≥2 MR genes. Pts treated with high-intensity, high intensity+ VEN and low intensity+VEN regimens had longer OS (Fig 1B) and EFS compared with pts treated with low intensity regimens without VEN (OS: not reached, 31.7 months, 31.4 months vs 8.9 months, respectively, p<0.001; EFS: not reached, 31.7 months, 29.6 months vs 3.7 months, respectively, p<0.001).

Our data suggest that the poor prognostic effect of MR^mut in de novo AML is associated with higher VAF and not a binary presence or absence of MR^mut. The minimum VAFs associated with worse outcome for each MR^mut ranged from 15-47%. Additional analysis to create predictive models is in progress to determine the optimal VAF cutoff when using MR^mut to assign AML to the WHO/ICC MR categories and to ELN adverse-risk group. Importantly, our results suggest that pts with MR^mut and non-adverse karyotype have significantly better outcomes when treated with high-intensity and/or venetoclax-based treatment regimens.

Loghavi:Amgen: Research Funding; GLG: Consultancy; QualWorld: Consultancy; PeerView: Honoraria; Astellas: Research Funding; Abbvie: Consultancy, Current equity holder in publicly-traded company. Kanagal-Shamanna:Amgen: Consultancy; Novartis: Consultancy; Aptitude Health: Speakers Bureau; Physicians Education Resource: Speakers Bureau. Kadia:cyclacel: Research Funding; Novartis: Consultancy; Astex: Honoraria; Regeneron: Research Funding; Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Servier: Consultancy; PinotBio: Consultancy; Glycomimetics: Research Funding; Iterion: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; JAZZ: Consultancy, Research Funding; Delta-Fly: Research Funding; cellenkos: Research Funding; AstraZeneca: Research Funding; Astellas: Research Funding; Genfleet: Research Funding; Ascentage: Research Funding; Agios: Consultancy; Abbvie: Consultancy, Research Funding. Daver:Agios, Celgene, SOBI and STAR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos and Jazz Pharmaceuticals: Other: Data monitoring committee member; Karyopham Therapeutics and Newave Pharmaceutical: Research Funding; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Novartis, Jazz, Amgen, Servier, Karyopharm, Trovagene, Trillium, Syndax, Gilead, Pfizer, Bristol Myers Squibb, Kite, Actinium, Arog, Immunogen, Arcellx, and Shattuck: Consultancy, Other: Advisory Role; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Gilead, Immunogen, Pfizer, Bristol Myers Squibb, Trovagene, Servier, Novimmune, Incyte, Hanmi, Fate, Amgen, Kite, Novartis, Astex, KAHR, Shattuck, Sobi, Glycomimetics, Trillium: Research Funding. Takahashi:Celgene/BMS: Consultancy; GSK: Consultancy; Agios: Consultancy; Ostuka Pharmaceuticals: Honoraria; Novartis: Consultancy; Symbio Pharmaceuticals: Consultancy; Mission Bio: Honoraria; Illumina: Honoraria. Garcia-Manero:AbbVie: Honoraria, Research Funding; Gilead Sciences: Research Funding; Curis: Honoraria, Research Funding; Aprea: Honoraria; BMS: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Acceleron Pharma: Consultancy; Novartis: Honoraria, Research Funding; Astex: Consultancy, Honoraria, Research Funding. Kantarjian:Jazz Pharmaceuticals: Research Funding; NOVA Research: Honoraria; KAHR Medical Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding; Ipsen Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; ImmunoGen: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Astellas Health: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ascentage: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Takeda: Honoraria. Ravandi:Astex/Taiho: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Biomea Fusion, Inc.: Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Consultancy; Prelude: Research Funding; Xencor: Research Funding; Amgen: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; Syos: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding. Short:Stemline Therapeutics: Research Funding; Novartis: Consultancy; AstraZeneca: Consultancy; Amgen: Consultancy, Honoraria; Takeda Oncology: Consultancy, Research Funding; Pfizer: Consultancy; Astellas: Research Funding. DiNardo:ImmuneOnc: Honoraria, Research Funding; LOXO: Research Funding; Cleave: Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Forma: Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Kura: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Research Funding; Astex: Research Funding; Novartis: Honoraria; Foghorn: Honoraria, Research Funding; Takeda: Honoraria; Jazz: Honoraria; GenMab: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Honoraria; Astellas: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria.