多重数字PCR平台检测cfDNA突变在NSCLC患者中的应用评估

Evaluation for application of multiplex dPCR platform in detection of cfDNA mutations of NSCLC patients

目的基于多重数字PCR(dPCR)技术检测非小细胞肺癌(NSCLC)患者血浆游离DNA(cfDNA)中表皮生长因子受体(EGFR)基因突变,评估其检测性能及临床应用价值。方法选取不同浓度EGFR-19Del、L858R、T790M突变的质粒样本作为研究对象,对基于多重dPCR的新平台进行性能验证(包括检测精密度、灵敏度、空白限与线性范围)。收集2019年6月至2020年11月复旦大学附属中山医院呼吸科确诊的49例NSCLC患者标本,采用多重dPCR和Super-ARMS方法分别检测cfDNA标本中EGFR基因突变水平,使用卡方检验比较两组间的差异,并进一步比较检测结果的一致性。结果多重dPCR平台检测L858R、19Del、T790M 3种常见突变,在10%、5%、1%的检测精密度均达到厂商声明的CV<15%;对于3种常见突变位点在不同检测限(0.5%、0.1%、0.05%)的检测均报告阳性。L858R、19Del、T790M突变的检测空白限分别为0.0382、0.000和0.053。T790M、19Del、L858R突变百分比在0.05%~10%(0.05%、0.1%、0.5%、1%、10%)时,实测值与预期百分比的线性回归方程中的R 2值分别为0.9977,0.9954和0.9984,P<0.01,相关性显著。49例临床样本中,基于多重dPCR与Super-ARMS技术检测S768I、G719X、19Del、T790M、20ins、L858R突变的方法总符合率分别为100%、100%、100%、97.96%、93.62%和89.80%。两方法检测L858R(χ^(2)=0.425,P>0.05)、T790M(χ^(2)=0.089,P>0.05)、19Del(χ^(2)=0,P>0.05)、20ins(χ^(2)=0.211,P>0.05)结果差异均无统计学意义。结论多重dPCR新平台检测cfDNA EGFR基因常见突变的性能较好,在评估突变丰度中具有优势,可为肿瘤精准治疗提供实验依据。

Objective To validate the new digital PCR(dPCR)-based assay platform for the detection of plasma-free DNA EGFR mutations in the patients with non-small cell lung cancer(NSCLC),and confirm its performance and clinical evaluation for future clinical application.Methods The samples with different concentrations of 19Del,L858R and T790M mutation provided by the reagent manufacturer were selected for the performance verification of a new platform based on multiple digital PCR,including precision,sensitivity,limit of blank and linearity test.The samples of 49 patients with NSCLC diagnosed in the Pneumology Department of Zhongshan Hospital,Fudan University from June 2019 to November 2020 were collected and analyzed by multiple dPCR and Super-ARMS.Chi-square test was used to compare the differences and the consistency of test results between the two groups.Results The accuracy of dPCR platform was good for the coefficient of variation among the three abundances of the three mutations(L858R,19Del,T790M)was less than 15%,which is in the line of the manufacturer′s statement.Positive results were reported based on the three common mutations at different predicted detection limits(0.5%,0.1%and 0.05%).The limits of blank for T790M,20ins and L858R mutations were 0.0382,0.000 and 0.053.The linearity was good in the range of 0.05%to 10%(0.05%,0.1%,0.5%,1%and 10%).The R 2 in the linear regression equation of the measured value withthe expected percentage were 0.9977,0.9954 and 0.9984,respectively(P<0.01),indicating significant correlation.For the results of 49 clinical samples,the overall agreements between the new multiplex dPCR platform and the Super-ARMS platform in the detections for S768I,G719X,19Del,T790M,20ins and L858R mutations were 100%,100%,100%,97.96%,93.62%and 89.80%,respectively.The Chi-square test showed that no significant difference was found between the results of L858R(χ^(2)=0.425,P>0.05),T790M(χ^(2)=0.089,P>0.05),19Del(χ^(2)=0,P>0.05)and 20ins(χ^(2)=0.211,P>0.05).Conclusion The new multiplex dagital PCR platform showed good performance in detecting common mutations of EGFR in free DNA,which may be tentatively considered as an advantageous method for detecting mutation abundance,thus could provide laboratory basis for supporting tumor precision therapy.