Drop-off ddPCR-based method for detecting mutations in C-KIT gene N822 locus and its clinical application in acute myeloid leukemia
LI Ting1, JIN Ye1, YUAN Qian2,3, YAO Dongming2,3, XIANG Helin1, XIAO Gaofei2,3, YU Di1, LENG Jiayan1, LIN Jiang2,3, QIAN Jun1,2,3
(1. Department of Hematology, 2. Laboratory Center, 3. Zhenjiang Clinical Research Center of Hematology, Affiliated People′s Hospital of Jiangsu University, Zhenjiang Jiangsu 212002, China)
Abstract： Objective： To develop a quantitative drop-off ddPCR technique for identifying of mutations at the C-KIT gene′s N822 locus in patients with acute myeloid leukemia (AML), and assess the methodological performance and its clinical value. Methods： A pair of primers and probes were designed for the exon 17 of C-KIT gene. The system and reaction conditions of drop-off ddPCR were optimized. The method′s specificity, sensitivity, and repeatability were assessed. The established method was used to test bone marrow samples from 140 newly diagnosed AML patients previously Sanger sequenced, and the results were further confirmed by next generation sequencing (NGS). In addition, the KIT-N822 mutation frequency was monitored dynamically in 3 patients by dropoff ddPCR. Results： The optimal annealing temperature of drop-off ddPCR for the KIT-N822 mutation was 54 ℃. With good linearity, the limit of detection was 10.12 copies/μL, and the limit of blank was 1.62 copies/μL. Among 140 AML samples, Sanger sequencing detected only 2 positive cases (1.43%), however, ddPCR identified 7 positive (5.00%) with mutation frequency of 0.29%~7.41%, among which NGS found only 3 positive cases (2.1%) with variant allele frequencies ranging from 1.26% to 8.00%. The results showed that the C-KIT mutation frequency decreased significantly or even to 0 when the treatment reached complete remission. Conclusion： The drop-off ddPCR method for detecting KIT-N822 mutations was developed with better methodological performance and higher sensitivity than both Sanger sequencing and NGS. It maybe employed for monitoring measurable residual disease and guiding treatment following remission in positive patients.
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