KIT amplitude-based multiplex droplet digital polymerase chain reaction outperforms direct sequencing for sensitive KIT D816 genotyping in core binding factor acute myeloid leukemia.

Abstract

Introduction: The prognosis of acute myeloid leukemia is stratified by genetic abnormalities; however, the detection sensitivity varies by method. The KIT D816 mutation is frequently found in core binding factor leukemia and is associated with a poor prognosis. In this study, we aimed to investigate the performance of multiplex droplet digital polymerase chain reaction (ddPCR) for detecting KIT D816 mutations and propose the practical mutation analysis method for clinical laboratory testing.

Methods: We evaluated the detection limit of ddPCR using mixed probes for HEX-labeled wild-type and FAM-labeled mutations (D816V, D816Y, and D816H) by analyzing plasmid mixtures containing these sequences. We compared the frequency of KIT mutations detected by direct sequencing and ddPCR in 20 patients with core binding factor leukemia.

Results: Multiplex ddPCR successfully discriminated between mutation types based on plot positions on a 2-dimensional map, with a detection limit of 0.1%. The frequency of D816 mutations was 42.5% using ddPCR and 20% using direct sequencing. Most patients with KIT D816 mutation require hematopoietic stem cell transplantation for chimeric gene clearance.

Discussion: Amplitude-based multiplex ddPCR efficiently provides sensitive and accurate genotyping of KIT D816 and has potential applications for other genetic abnormalities.