基于drop-off ddPCR方法检测急性髓系白血病C-KIT基因N822位点突变及其临床应用  

Drop-off ddPCR-based method for detecting mutations in C-KIT gene N822 locus and its clinical application in acute myeloid leukemia

作　　者：李婷 金晔 袁倩 姚冬明[2,3] 向鹤麟 肖高飞[2,3] 于迪 冷加燕[1] 林江[2,3] 钱军[1,2,3] LI Ting;JIN Ye;YUAN Qian;YAO Dongming;XIANG Helin;XIAO Gaofei;YU Di;LENG Jiayan;LIN Jiang;QIAN Jun(Department of Hematology,Affiliated People′s Hospital of Jiangsu University,Zhenjiang Jiangsu 212002,China;Laboratory Center,Affiliated People′s Hospital of Jiangsu University,Zhenjiang Jiangsu 212002,China;Zhenjiang Clinical Research Center of Hematology,Affiliated People′s Hospital of Jiangsu University,Zhenjiang Jiangsu 212002,China)

机构地区：[1]江苏大学附属人民医院血液科,江苏镇江212002 [2]江苏大学附属人民医院中心实验室,江苏镇江212002 [3]江苏大学附属人民医院镇江市血液病临床医学研究中心,江苏镇江212002

出　　处：《江苏大学学报（医学版）》2024年第2期151-155,160,共6页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(82270179,81970118);镇江市血液病临床医学研究中心项目(SS2018009);镇江市社会发展项目(SH2022026,SH2021052)。

摘　　要：目的:建立急性髓系白血病(acute myeloid leukemia, AML)患者C-KIT基因N822位点突变的drop-off微滴式数字PCR(droplet digital PCR,ddPCR)定量检测方法,并评价其临床应用价值。方法:针对C-KIT基因第17外显子设计一对引物及探针,优化drop-off ddPCR反应条件及体系,评价该方法的特异性、灵敏度、重复性,使用所建立的方法对140例已行Sanger测序的AML初诊患者骨髓标本进行检测,并用二代测序(next generation sequencing, NGS)验证结果;用drop-off ddPCR对3例阳性患者化疗后C-KIT突变频率进行动态监测。结果:drop-off ddPCR检测C-KIT基因N822位点突变的最适退火温度为54℃,空白检测限为1.62拷贝数/μL,最低检测下限为10.12拷贝数/μL,线性良好。140例AML初诊患者样本中Sanger测序检出2例阳性(1.4%),而ddPCR共检出突变7例(5.0%),突变频率为0.29%~7.41%;进一步应用常规NGS方法对ddPCR阳性样本进行验证,共检出阳性3例(2.1%),等位基因频率为1.26%~8.00%。动态监测3例阳性患者C-KIT突变频率,结果显示治疗达完全缓解时C-KIT突变频率明显下降甚至降低至0。结论:本研究建立了检测C-KIT基因N822位点突变的drop-off ddPCR技术,具有良好的方法学检测性能,其灵敏度高于Sanger测序和NGS,有望用于阳性患者缓解后的可检测残留疾病监测及治疗指导。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 drop-off 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.

关 键 词：drop-off微滴式数字PCR C-KIT基因 基因突变 微小残留病 急性髓系白血病 预后 

分 类 号：R733.71[医药卫生—肿瘤]