Prognostic impact of variant allele frequency in intensively treated patients with NPM1-mutated AML: a PETHEMA study

NPM1-mutated acute myeloid leukemia (AML) is genetically well defined, but clinical outcomes remain heterogeneous, suggesting that quantitative clonal features may refine current risk stratification. We analyzed 688 intensively treated NPM1-mutated (NPM1mut) AML integrating variant allele frequency (VAF), mutation order, and clonal architecture inferred by PyClone and ClonEvol. Comutations were present in 97% of patients (median, 3 per case), dominated by DNMT3A (49%), FLT3-ITD (46%), TET2 (22%), and IDH2 (20%). Prognostic modeling of NPM1 VAF identified an optimal cutoff of 31.44%, defining NPM1high and NPM1low groups. NPM1low correlated with splicing-related alterations and independently predicted inferior overall survival (hazard ratio [HR], 1.46; P = .037) and relapse-free survival (HR, 1.40; P = .036). Gene-specific VAF analyses revealed divergent effects across partners: high DNMT3A, FLT3-OTHER (non-ITD/TKD), KRAS, and PTPN11 burdens were adverse, whereas high IDH2 VAF was protective. Combined models showed that patients with NPM1high and favorable comutation VAFs had the best outcomes, whereas dual unfavorable burdens conferred the poorest survival. Mutation ordering inferred from VAFs positioned NPM1 after epigenetic and splicing lesions but before signaling and transcription-factor mutations. Noncanonical orders, such as early FLT3-OTHER/TKD or WT1 before NPM1, significantly stratified outcomes. Clonal reconstruction revealed predominantly linear evolutionary trajectories (84.3%), with increased mutational burden and clonal diversity associating with inferior survival. Notably, intraclonal colocalization of NPM1 with IDH1 or TET2 was associated with improved outcomes, whereas colocalization with WT1 predicted dismal prognosis. These results demonstrate that quantitative and structural dimensions of clonality refine the biological and prognostic landscape of NPM1-mutated AML beyond mutational status alone.