基于目标序列捕获测序法的肺腺癌循环肿瘤DNA基因检测与组织标本的对比研究

Detection of EGFR and ALK gene mutations in patients with lung adenocarcinoma is more and more common in routine clinical practice. But there are challenges ahead. Sometimes it is difficult to obtain the tumor tissue, and the heterogeneity of tumor may also affect the result..This prospective study is to evaluate the feasibility of the gene detection of peripheral blood circulating tumor DNA in Ⅱ-ⅢA stage lung adenocarcinoma. We utilized targeted sequencing with a next-generation sequencing (NGS) platform to detect driver mutations in matched tumor tissue DNA(fine-needle aspiration, surgical tumor tissue) and plasma ctDNA samples with matched white blood cell (WBC) DNA as a control. We also quantify the concentration of ctDNA before surgery and in a series of times after surgery by digital PCR to assess the accuracy of blood biopsy and the time of ctDNA degradation after surgery..Most previous studies are lack of prospective clinical data and aim at advanced lung cancer. This is the first study to compare gene detection between these three types of specimens in patients with early stage lung adenocarcinoma. With previous research in patients’ inrollment, acquisition of specimen, improvement of gene detection method and preliminary comparison of blood and tissue sample, this study is practicable and creative, and will provide reliable data in selecting optimal procedures of gene mutations detection of lung cancer patients..

肺腺癌患者进行驱动基因的检测日趋成为临床常规。然而肿瘤组织标本作基因检测存在标本获取困难以及肿瘤异质性等问题，影响了检测结果的可靠性。. 本研究拟前瞻性的同期收集Ⅱ-ⅢA期肺腺癌的穿刺组织学小标本、术后大体标本以及外周血标本，通过目标序列捕获测序法进行血循环肿瘤DNA（ctDNA）的基因检测，并对比三种标本的检测结果，评估对可手术的早期和局部进展期肺癌患者进行外周血ctDNA基因检测的可行性。研究还将对ctDNA的浓度通过数字PCR方法进行定量检测。对比术前术后ctDNA水平的变化，以及术后多时间点的差异，进而从定量的角度评价对接受手术的肺癌患者进行外周血ctDNA检测的准确性和最佳时间。. 本研究是首个针对可手术的早期肺腺癌患者进行外周血ctDNA检测的研究。课题组前期已先后完善了入组人群筛选，检测标本获取，检测方法的改良，以及初步的定性对比，研究有充分的可行性及创新性。

肺癌患者进行基因检测已成为临床常规，但组织标本存在获取困难和异质性等难题。循环肿瘤DNA（ctDNA）是坏死的肿瘤细胞所脱落分离出来的小DNA片段，理论上可提供肿瘤的全景视图并且取材方便。但既往多应用于对晚期癌症患者。本课题率先研究了循环肿瘤DNA在早期肺癌中检测的可行性，并探索了实际应用价值。.研究首先通过对76例早期肺癌患者的肿瘤组织和血液ctDNA进行基于二代测序技术的目标序列捕获法基因检测。结果显示，68.4% 患者的ctDNA与组织tDNA检测结果一致，ctDNA对早期肺癌的诊断敏感性高于传统肿瘤标志物（63.2% vs 49.3%）。证实了早期肺癌血ctDNA检测的可行性。.在此基础上，进一步开展了前瞻性的ctDNA代谢动力学研究（DYNAMIC）。通过围手术期多时间点采血并对检测到的变异等位基因频率进行计算，发现肿瘤R0切除术后血浆ctDNA浓度呈快速下降趋势，A、B、C、D时间点突变等位基因平均丰度分别为2.72%、2.11%、1.14%、0.17%，根据指数模型计算ctDNA中位半衰期仅为35分钟。随后进行长期随访，并对术后1天，术后3天，1月进行ctDNA检测。结合影像学检查发现，术后3天ctDNA阳性的患者较阴性的患者，无瘤生存期和总生存期均更短，说明ctDNA可用于检测术后动态监测。.DYNAMIC是国际第一项精准评估ctDNA围手术期动态变化的前瞻性研究，探索了ctDNA术后监测的最佳时间点。同时也是亚洲第一项应用ctDNA 监测肺癌术后微小病灶残余的研究，为非小细胞肺癌的临床决策及患者的精准治疗提供了宝贵的参考依据。