基于PASS技术的EGFR基因突变比例检测方法的建立与评价

The establishment and evaluation of the method for EGFR gene mutation proportion detection based on PASS technology

目的近期临床研究发现EGFR-TKI并非对所有EGFR基因激活突变的NSCLC患者都有效,提示EGFR-TKI的治疗效果也可能与NSCLC患者中EGFR基因突变比例相关。本研究基于平行等位基因特异性测序(Parallel Allele-Specific Sequencing,PASS)方法,建立一个稳定可靠的EGFR突变基因比例检测平台。方法构建野生型和突变型EGFR基因重组质粒,建立EGFR基因突变比例检测的PASS平台,从灵敏度、精密度以及准确度对PASS检测平台进行性能评价。结果建立的PASS平台可用于EGFR基因点突变和缺失突变的检测,能够检测到的最小基因拷贝数为1,能够稳定检测到的最小基因拷贝数为10,在野生型基因中最低能检测出0.01%的突变基因。线性回归分析显示,检测到的突变型/野生型比例与预期的突变型/野生型比例呈良好的线性相关(R_(2)=0.9962)。结论该方法的建立能够对EGFR基因突变比例进行灵敏、准确测定,这对其在临床中的应用奠定了良好的技术基础,对于评价EGFR基因突变比例与EGFR-TKI靶向治疗NSCLC患者疗效的关系也具有重要意义。

Objective Recently,clinical researches found that EGFR-TKI is not effective in all NSCLC patients with activating mutations in the EGFR gene,which suggests that the therapeutic effect of EGFR-TKI may also be affected by the proportion of mutant EGFR gene in NSCLC patients.In this study,we established a reliable platform for detecting the proportion of mutant EGFR gene,based on Parallel Allele-Specific Sequencing(PASS)method.Methods Construct recombinant plasmid containing wild and mutant EGFR gene and establish PASS platform for detecting the proportion of mutant EGFR gene.Meanwhile evaluate the performance of PASS detection platform in terms of sensitivity,precision and accuracy.Results The results show that this technology platform can be applied to detect both point mutation and deletion mutation of EGFR gene.The minimum number of gene copies that can be detected by this technology is one,and the minimum number of gene copies that can be stably detected is 10.Particularly,PASS platform can detect 0.01%of the mutated genes from wild type genes.Linear regression analysis revealed a good linear correlation(R_(2)=0.9962)between detected and expected mutant/wild-type ratios.Conclusion The establishment of this method achieved the sensitive and accurate determination of the proportion of mutant EGFR genes,which had laid a good technical foundation for its clinical application and was of great significance for evaluating the relationship between the proportion of mutant EGFR gene and the efficacy of EGFR-TKI targeted therapy in NSCLC patients.