Occurrence of FLT3 mutations in acute myeloid leukemia and comparison of different methodologies

Abstract

Fms-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of adult patients with acute myeloid leukemia (AML) and are clinically significant for prognosis and targeted therapy. Detection early is essential for treatment decisions. This study evaluated the performance of fragment analysis and next generation sequencing (NGS) in FLT3 mutations among adult patients with AML stratified by age. The cohort consisted of 959 patients diagnosed with AML between January 2020 and December 2024. FLT3 mutations were evaluated concurrently through fragment analysis and NGS, and all statistical analyses were implemented in R v4.4.1. Fragment analysis detected internal tandem duplication (ITD) in 28.68% of the patients (allelic ratio (AR): 0.02–13.9), while the positive rate was 30.55% detected by NGS (variant allele frequency (VAF): 1.01–95.7%). For FLT3-ITD, fragment analysis showed 93.86% positive percent agreement (PPA), 100% negative percent agreement (NPA), and 98.12% overall percent agreement (OPA) with NGS (kappa = 0.9550, p < 0.001). FLT3-ITD AR/(AR + 1) and FLT3-ITD VAF were strongly correlated (R = 0.8848, p < 0.0001). For the FLT3 tyrosine kinase domain (TKD), PPA was 70.27% with 100% NPA and 97.63% OPA. Most FLT3-ITD positive cases had one insertion (76.73%); median insertion length was 51 bps (range:6 bps-243 bps). Co-mutations were present in 91.27% of FLT3-ITD positive cases, most frequently NPM1 (48.36%) and DNMT3A (33.82%). The triple mutations FLT3-ITD / NPM1 / DNMT3A represented 26.91%. Patients with high FLT3-ITD AR (≥ 0.5) often have co-mutations of NPM1 (48.7%) and DNMT3A (38.0%), while those with low-AR (0.02–0.05) mainly involve mutations in the RAS pathway, notably NRAS (45.8%). Fragment analysis demonstrated high concordance with NGS for the detection of FLT3-ITD and provided a faster turnaround, which is advantageous for the rapid diagnosis of AML. However, fragment analysis show limited sensitivity to detect low-frequency FLT3 mutations, particularly FLT3-TKD, highlights the superiority of NGS as the preferred method of complete evaluation of FLT3-TKD. NGS enables the simultaneous assessment of co-mutations, with particular emphasis on the distinct co-mutation patterns associated with different AR. These two methodologies offer complementary clinical value.