晚期非小细胞肺癌血浆小片段DNA中EGFR基因突变检测率的研究

Epidermal growth factor receptor (EGFR) mutation is strongly associated with the therapeutic effect of tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Nevertheless, tumor tissue that needed for mutation analysis is frequently unavailable, particularly from patients with refractory NSCLC. Plasma samples, which often contain circulating free DNA derived from tumor tissues, have been used as surrogate tumor tissues for detecting genetic alterations. Previous studies have demonstrated that tumor-free DNA, as evidenced by the presence of a specific mutation, is mostly degraded and has a median size of 150-250 bp. The ssDNA concentration correlated with enhancing tumor volume and grade gliomas. Most importantly, we were able to detect the IDH1R132H mutation in the ssDNA from patients with mutated glioma. IDH1R132H identification in the ssDNA has a strong diagnostic value in patients not amenable to biopsy. Therefore, in this study, we will analyze patients with NSCLC for ssDNA and correlations between the ssDNA concentration and clinical/pathologic parameters, to determine the potential clinical implications of ssDNA. We also analyze EGFR mutations by using plasma samples and matched tumor tissues to determine the utility of plasma as a surrogate tissue for EGFR mutation analysis. We try to resolve the problems in clinical practice such as relatively lower mutation rate and poor clinical correlation by optimizing the extraction procedure with ssDNA, using coamplification at lower temperature-PCR (COLD-PCR) to enrich mutant DNA in a background of wild-type DNA, using ARMS method with sensitivity to single DNA molecule. Plasma-based EGFR mutation analysis will be feasible and has value in predicting tumor response in pathologically confirmed NSCLC patients not amenable to biopsy.

明确病理诊断却无充足组织标本的中晚期非小细胞肺癌患者亟需寻找一种肿瘤组织标本替代物，检测EGFR基因的突变状态以指导个体化治疗。外周血是组织标本替代物研究的热点。前期研究发现肿瘤来源、含特异性突变的游离循环DNA断裂为小片段DNA，小片段DNA浓度与脑胶质瘤分级及大小呈正相关，且在小片段DNA中检测到与肿瘤细胞一致的基因突变，显示其诊断价值。本研究探索小片段DNA在非小细胞肺癌中的临床应用价值，比较癌症患者与正常人体内小片段DNA含量的差别以及它与临床病理间的关系，并深入研究肿瘤组织及配对外周血中EGFR突变阳性率异质性原因。通过优化小片段DNA提取方法，采用COLD-PCR技术富集EGFR突变体，应用敏感的EGFR检测方法以及规范标本获取时间等方式，提高二者EGFR突变阳性一致率，为明确病理诊断却无充足组织标本的中晚期NSCLC患者找到方便、经济、可行的组织标本替代物，实现个体化治疗。

背景：表皮生长因子受体（EGFR）突变分析已成为指导肺癌治疗决策的常用方法。血浆中循环肿瘤DNA（ctDNA）分子突变检测可作为无法获取足够肿瘤组织的替代检测方法，以及重复活检监测治疗反应时使用。.目的：开发一个具有时效性和成本效益的可以准确检测ctDNA中 EGFR突变的检测方法，并可以提示临床药物疗效和耐药发生。.设计：分析评估了一组经典的表皮生长因子受体突变的参考材料。以最近研发的泊松分布为基础的方法来了解检测灵敏度。对I期至IV期患者224对血浆和匹配的组织进行EGFR突变检测一致率检测及临床评价。然后对390个连续的血浆样品的EGFR突变率与以前报道的亚洲人口患病率进行了比较。.结果：我们的研究结果表明，EGFR荧光定量聚合酶链反应（PCR）检测有10份限制突变检测，最低检测拷贝数可以扩展到一位数的水平。I-IV期肺癌检测的敏感性为53.3%，III-IV中晚期阶段的敏感性81.4%，具有较高的特异性为100%。临床观察显示两组人群中，IV期总阳性率分别为32.5%和41.4%，后者与先前报道的EGFR突变在亚洲人群中的一致。.结论：我们的研究结果支持在血液ctDNA中使用超灵敏qPCR检测EGFR突变的临床应用。