Ultrasensitive Detection of FLT3-ITD Mutations via Primer Competition Enhanced Mutant Accumulation

Abstract

The in-frame internal tandem duplication of the FLT-3 gene (FLT3-ITD), a prevalent genetic aberration, significantly contributes to treatment failure and poor prognosis in acute myeloid leukemia (AML). A robust and cost-effective assay for minimal residual disease (MRD) detection in FLT3-ITD⁺ AML is crucial for guiding therapeutic decisions. However, current MRD monitoring methodologies for FLT3-ITD⁺ patients are limited by sensitivity and adaptability, particularly for dynamically quantifying complex and heterogeneous FLT3-ITD mutations. In this study, we developed a primer competition enhanced mutation accumulation (PCEMA) technique designed to selectively enrich FLT3-ITD in the context of abundant wild-type alleles. By integrating the PCEMA with capillary electrophoresis, we significantly improved the discrimination between mutant and wild-type genes, increasing the minimum detectable sensitivity to 0.001%, comparable to next-generation sequencing. The competitive amplification between ITD-specific and universal primers facilitated the selective enrichment of mutant alleles, enabling highly sensitive and specific real-time FLT3-ITD mutation monitoring. We thoroughly evaluated the analytical performance and adoptability of the PCEMA technique in conjunction with quantitative fluorescent PCR (qPCEMA). Our results demonstrated that qPCEMA quantitatively differentiates FLT3-ITD with a mutation frequency below 0.1%, offering an effective, rapid, and reliable method for long-term FLT3-ITD monitoring in clinical AML patients. The PCEMA technique, characterized by its robustness, sensitivity, specificity, timeliness, and adoptability, presents a promising alternative for clinical FLT3-ITD mutation detection. It is anticipated to provide significant technical support for timely diagnosis, prognosis assessment, drug evaluation, and personalized treatment of AML patients, with substantial potential for clinical application.