表皮生长因子受体基因突变检测方法改进及初步临床验证

目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。

Clinical Evaluation of An Improved Method for Mutation Detection ofEpidermal Growth Factor Receptor

Objective:To develop a highly sensitive assay of Fluorescence Polymerase Chain Reaction(PCR) and assess its abilityto detect EGFR mutations in non-small cell lung cancer (NSCLC) by comparison with direct sequencing and amplification refractorymutation system (ARMS).Methods:From June 2013 to August 2015, one hundred and forty-one formalin-fixed paraffin-embeddedsamples of resected NSCLC were collected in Hunan Cancer Hospital. Direct sequencing, amplification refractory mutation system(fluorescence PCR) and the novel improved PCR assay were used to detect epidermal growth factor receptor (EGFR) mutationsseparately. The differences of these methods were then further analyzed and compared.Results:The detection success rates of all threemethods were 100 %. The consistent rate (completely same points and same mutation types) of the novel assay and direct sequencing was75.9 %(107/141). Among 96 samples with EGFR mutations detected via direct sequencing, the same mutations were detected in 92samples via the novel assay (95.8 %). However, in the other 45 samples without mutations tested by direct sequencing, 23 samples(51.1%) were found to be EGFR mutation-positive using the novel assay, and there were significant differences between these two methods(x2=40.745, P<0.05). Compared with direct sequencing, the sensitivity and specificity of the novel assay were 95.8 % and 48.9 % ,respectively, and the positive predictive value and negative predictive value were 80.0 %, and 84.6 %, respectively, with the accuracy of80.9 %. When compared with ARMS as the gold standard, the consistent rate of these two methods reached 84.4 %(119/141), with a highconsistence (Kappa=0.749, P<0.05). And the sensitivity and specificity of the novel assay were 94.1% and 84.6 %, respectively,Conclusion:The improved method of EGFR mutation detection has shown higher sensitivity and accuracy than direct sequencing, andthe results are highly consistent with ARMS.