微滴式数字PCR检测急性髓系白血病NPM1基因突变及其临床应用价值

Detection of NPM1 Mutation in Acute Myeloid Leukemia by Droplet Digital PCR and Its Clinical Application Value

目的:建立微滴式数字PCR(ddPCR)检测急性髓系白血病(AML)患者NPM1基因A型突变的方法,评价该方法的特异性、灵敏度及其临床应用价值。方法:使用NPM1突变型及野生型质粒对该方法进行性能验证;ddPCR及Sanger测序法检测87例初诊AML患者骨髓标本,用二代测序验证;ddPCR动态监测5例NPM1 A型突变患者化疗后突变负荷水平。结果:检测NPM1基因A型突变的ddPCR方法,空白限(LOB)为1.1拷贝/微升,最低检测下限(LOD)为2.43拷贝/微升,且线性良好;ddPCR及Sanger测序法检测87例初诊标本NPM1突变结果完全一致,阳性17例(19.5%);二代测序验证12例突变阳性样本,结果显示,NPM1 A型突变8例、D型突变2例、罕见型突变2例。5例患者NPM1突变水平动态监测结果显示,化疗达完全缓解时NPM1突变水平明显下降甚至接近于0,但复发时再次明显增高。结论:本研究建立了一个检测NPM1突变的ddPCR方法,具有良好的灵敏度和重复性,可用于AML初诊患者的NPM1突变筛选和阳性患者的缓解后微小残留病监测以指导治疗。

Objective:To establish the droplet digital PCR(ddPCR)assay for the detection of NPM1 type A mutation in patients with acute myeloid leukemia(AML),and to evaluate its specificity,sensitivity and its value in clinical application.Methods:NPM1 mutant and wild-type plasmids were used to verify the performance of ddPCR.Both ddPCR and Sanger sequencing were used to detect the bone marrow samples of 87 AML patients,which were confirmed by next generation sequencing(NGS).Moreover,NPM1 mutation burden was dynamically monitored in five patients by ddPCR.Results:The limit of blank(LOB)of ddPCR established for NPM1 mutation detection was 1.1 copies/μl,and the limit of detection(LOD)was 2.43 copies/μl,which had good linearity.Among the 87 newly diagnosed AML patients,ddPCR identified seventeen cases positive for NPM1 mutation(19.5%),which was consistent with Sanger sequencing.NGS confirmed 12 positive cases,including 8 of type A mutations,2 of type D mutations,and 2 of rare type mutations.The results of dynamic monitoring of NPM1 mutation burden in 5 patients showed that the NPM1 mutation burden decreased obviously even close to 0,when patients achieve complete remission after chemotherapy.However,the mutation burden was increased again at the time of relapse.Conclusion:In this study,we established a ddPCR method for detection of NPM1 mutation with good sensitivity and repeatability,which can be used for screening NPM1 mutation in newly diagnosed AML patients and for minimal residual disease monitoring after remission in positive AML patients to guide treatment.