Double Trouble: Composite Mutations in Driver Genes in Acute Myeloid Leukemia (AML)
Authors: Shahin Shadma , Achrekar Aarti Ramesh , Ugale Priyanka Rajesh , Joshi Swapnali , Terse Vishram , Shetty Dhanlaxmi Lalit , Jindal Nishant , Tembhare Prashant , Mirgh Sumeet , Shetty Alok , Gokarn Anant , Punatar Sachin , Nayak Lingaraj , Jain Hasmukh , Sengar Manju , Khattry Navin , Bagal Bhausaheb , Rajpal Sweta , Chatterjee Gaurav , Subramanian PG , Gujral Sumeet , Patkar Nikhil 
Published: December 2024

Introduction: Composite mutations (CM) occurring in driver genes are a putative mechanism that drives oncogenesis and cancer progression. It is possible that CM result in mutant proteins with novel functions. Recent pan-cancer evaluation has shown CM to be a relatively common event that may occur selectively in specific oncogenes and tumor suppressor genes (Saito et al. Nature 2020); however, data on AML is largely lacking. Here, we describe the frequency, patterns and clinical significance of CM in de novo AML.

Methods: A total of 506 patients of adult AML (≥18 years) were accrued over 10 years (2012-2023) from a single center. Diagnostic samples were sequenced using a 50-gene myeloid panel (till 2020) based on single molecule molecular inversion probes and subsequently using a 135-gene hybrid capture-based panel. Based on cytogenetic and genomic information, cases were risk stratified as per 2022 European LeukemiaNet (ELN) recommendations. Patients were uniformly treated with “3 + 7” induction. MRD was evaluated at post-induction (PI) timepoint using 10-colour or 16-colour flow cytometry (MFC-MRD). CM was described as two or more distinct somatic non-synonymous mutations in a driver gene from a diagnostic AML sample. Survival analysis was done in comparison to cases having single mutation (SM) or wildtype (WT) genes. Correlation of PI MRD and CM was performed using odds ratio. The prognostic relevance of CM on overall survival (OS) and relapse-free survival (RFS) was computed using the Kaplan-Meier method and compared using log-rank test for time-to-event analyses. Multivariable Cox proportional hazard model was used for survival endpoints.

Results: The median age of the entire cohort was 35.0 years (M:F, 1.6:1) with a median follow-up of 29.5 months. The median OS was 72 months (95% CI 35.5 to 87.3), and the median RFS was 52.3 months (95% CI 35.2 to 87.9). We describe here that incidence of CM is relatively common in AML. More than a quarter of AML cases (n=134, 26.5%) harbored CM. Median age of patients with CM was 33.0 years (M:F, 1.48:1). The median OS was 32.8 months (95% CI 19.7 to 42.2), and the median RFS was 40.3 months (95% CI 24.6 to 52.3). CM involved signalling pathway (28.6%), cell survival (28.6%), epigenetic modifier (19%), cohesin complex (9.5%) as well as DNA repair (9.5%) genes. In order of descending frequency, cases with CM included FLT3 (5.92%), CEBPA (5.53%), NRAS (3.95%), KIT (3.16%), TET2 (2.76%), and WT1 (2.17%). There was enrichment of CM in FLT3 and TET2 in NPM1 mutated AML; KIT and ASXL2 in AML with RUNX1::RUNX1T1; and WT1 and GATA2 in AML with mutated CEBPA. PI MRD positivity was evident in 44.8% (n=60) of CM cases and 46% (n=233) in the entire cohort. CM did not predict for PI MRD positivity (p=0.79). Of the total cases (n=506), patients were stratified as ELN22 favorable (n=260), intermediate (n=169), and adverse risk (n=77). ELN22 adverse risk patients had inferior OS [HR 3.2 (95% CI 2.0 to 5.0) p&lt;0.0001] and RFS [HR 2.7 (95% CI 1.4 to 5.5) p=0.0002] as well as those classified as intermediate risk had inferior OS [HR 1.7 (95% CI 1.2 to 2.4)] and RFS [HR 1.3 (95% CI 0.9 to 1.9)] when compared to favorable risk category. On further ELN22 risk stratification on the basis of CM, patients classified as favorable risk with CM (n=72) had inferior RFS [HR 2.28 (95% CI 1.1 to 4.7); p=0.038] as compared to WT cases. Gene wise risk stratification in favorable risk group revealed that CM of WT1 had significantly inferior RFS [HR 6.76; p=0.01]; FLT3 CM had worse RFS [HR 7.3; p=0.005] and OS [HR 9.7; p=0.0005] to wildtype cases. CM did not impact outcomes in ELN22-intermediate risk category (n=41). ELN22-adverse risk patients with CM (n=21) had inferior OS [HR 3.5 (95% CI 1.3 to 9.2); p=0.013] in comparison to WT cases with the presence of TET2 CM in particular having worse OS [HR 9.03; p=0.026]. On multivariate analysis of our cohort, ELN adverse risk group [HR 1.8 (95% CI 1.5 to 2.19); p&lt;0.0001] and CM were independent predictors of worse OS [HR 1.3 (95% CI 1.03 to 1.65); p=0.027].

Conclusion: We demonstrate the prognostic utility of composite mutations in AML. Composite mutations in FLT3 and WT1 had inferior outcome in ELN22 favorable risk AML, whereas TET2 composite mutation was associated with inferior outcome in ELN22 adverse risk AML. Incorporation of CM is warranted for improved risk stratification of these patients.