数字PCR检测非小细胞肺癌患者血浆EGFR基因T790M突变平台的建立

Establishment of a digital PCR platform for detection of EGFR T790M mutation in plasma circulating DNA from NSCLC patients

目的建立数字PCR（dPCR）检测非小细胞肺癌（NSCLC）患者表皮生长因子受体（EGFR）基因T790M突变平台，并评估其基本性能和临床价值。方法方法学建立。收集2014年1月至2015年10月于复旦大学附属中山医院就诊的10例经EGFR—TKI治疗后发生耐药的NSCLC患者。设计EGFR基因T790M突变及野生型的引物及特异性探针，建立dPCR检测EGFR基因T790M突变平台，根据白配不同浓度标准品质粒验证该检测平台空白限、灵敏度和线性以建立最适报告和复检流程。采用dPCR和扩增受阻突变系统（ARMS）检测患者肺癌组织和血浆标本，卡方检验比较两种方法检测不同标本类型的EGFR基因T790M突变一致性，Pearson相关分析dPCR检测肺癌组织和血浆突变分子丰度的相关性。结果dPCR检测平台空白限为10拷贝，功能灵敏度为0．01％，且在0．01％～100％范围内线性良好（Y=1．226X-3．984，R^2=0．999）。dPCR和ARMS检测肺癌组织EGFR基因T790M突变一致性较高（kappa=0．80），而dPCR检测血浆突变阳性率高于ARMS（50％VS20％，P〈0．05）Pearson相关分析发现dPCR检测肺癌组织和血浆突变分子丰度高度相关（R=0．923，P〈0．05）。结论建立了高灵敏、绝对定量的dPCR检测EGFR基因T790M突变平台，其联合血浆etDNA能真正实现“液体活检”，对指导耐药后患者个体化治疗有重要价值。

Objective Digital PCR （dPCR） was established to detect plasma epidermal growth factor receptor （EGFR） T790M mutation of non-small cell lung cancer （NSCLC） patients and was evaluated in terms of analytical performance and clinical application significance. Methods The specific primers and probes for EGFR T790M mutation and wildtype were designed to establish dPCR platform. Limit of blank, sensitivity and linearity of dPCR were evaluated by the detection of plasmids with different concentrations to set up optimal reporting system and reanalyzing process. The mutation of EGFR T790M in plasma and tissue samples from 10 patients with advanced NSCLC resistant to EGFR-TKI therapy who were enrolled in Zhongshan Hospital Fudan University from January 2014 to October 2015 were analyzed by dPCR and amplification refractory （ARMS） , respectively. The consistency was evaluated between dPCR and ARMS by Chi-square test. The correlation of T790M abundance detected by dPCR between plasma and tissue samples was also analyzed by Peasrson correlation analysis. Results Limit of blank and sensitivity of dPCR was 10 copies and 0. 01% , respectively, dPCR was evaluated as linear in the range of 0. 01% - 100% （ Y = 1. 226X -3.984 ,R^2 = 0. 999 ）. The consistency between dPCR and ARMS of tissue samples was good （kappa = 0. 80）, while the positive rates of plasma T790M detected by dPCR was significantly higher than ARMS （50% vs 20% ,P 〈 0. 05）. It was found that T790M abundance detected by dPCR was highly correlated between lung cancer tissue and plasma （ R = 0. 923, P 〈 0. 05 ） using Pearson correlation analysis. Conclusions A new method of dPCR with high sensitivity and absolute quantification is established for the detection of EGFR T790M mutation in plasma from advanced NSCLC patients, which brings tumor liquid biopsy into real It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in patients with advanced NSCLC resistant to EGFR- TKI.