基于血浆游离DNA含量和完整性特征的基因突变检测及其在非小细胞肺癌的应用

For non-small cell lung cancer (NSCLC), targeting therapy mainly lies on detection of genetic alterations, which nowadays are generally detected in biopsy samples. However, it is very difficult to obtain the biopsy samples that also cannot reflect the heterogeneity of tumor, as well as the single detection unlikely indicates the coming potential genetic alterations. Cell-free DNA (cfDNA) in plasma of cancer patients, mainly deriving from apoptosis and necrosis of cancer cells, is able to be served as a tool to detect genetic alterations, which circumvents the difficulties associated with lack of biopsy samples. Nevertheless, there appears to be an obstacle to clinical application that less generation or excessive degradation of cfDNA also leads to negative results. To sweep this obstacle, we have developed DNA-extraction-free technique for the determination of plasma total cfDNA and DNA integrity, supposing a new path to clinical application. In this subject, we have studied the coincidence for alterations of NSCLC targeting therapy related genes in paired biopsy DNA and plasma cfDNA, which is subsequently analyzed correlation with plasma total ctDNA and DNA integrity index in order to normalize conditions and criterions for determination of cfDNA genetic alterations. In addition, we have also revealed the mechanisms of genetic alterations and provided the evidences to overcome secondary drug resistance in the process of NSCLC targeting therapy.

非小细胞肺癌（NSCLC）的靶向治疗依赖于基因突变检测，目前较多采用癌组织来检测，存在取材难、局部取材不能反映肿瘤异质性和一次取材不能反映后期的基因突变变化等缺点。血浆游离DNA（cfDNA）在癌症患者主要来自于癌细胞的凋亡和坏死，可作为基因突变检测的样本，恰能弥补组织作为检材的不足，但也存在突变阴性结果不能判定是真阴性还是因cfDNA中来源于癌细胞的过少或过度降解的烦恼，一直阻碍其临床应用。为解决此问题，我们创立了免DNA提取的血浆cfDNA总量和DNA完整性测定技术，并提出基于血浆cfDNA含量和完整性可有效解决该问题的假设。本题通过比较以组织和血浆为对象检测NSCLC靶向治疗相关基因突变的吻合情况，分析后者与血浆ctDNA含量和完整性指数的关系，确定适合以血浆DNA为突变检测样本的条件和检测规范，并用该技术研究NSCLC靶向治疗过程中突变的变化规律，为克服靶向治疗继发性耐药提供依据。

非小细胞肺癌（NSCLC）的靶向治疗依赖于基因突变检测，目前较多采用癌组织来检测，存在取材难、局部取材不能反映肿瘤异质性和一次取材不能反映后期的基因突变变化等缺点。血浆游离 DNA（cfDNA）在癌症患者主要来自于癌细胞的凋亡和坏死，可作为基因突变检测的样本，恰能弥补组织作为检材的不足，但也存在突变阴性结果不能判定是真阴性还是因 cfDNA中来源于癌细胞的过少或过度降解的烦恼，一直阻碍其临床应用。为解决此问题，我们创立了免DNA提取的血浆cfDNA总量和DNA完整性测定技术，并提出基于血浆cfDNA含量和完整性可有效解决该问题的假设。本课题通过比较以组织和血浆为对象检测NSCLC靶向治疗相关基因突变的吻合情况，分析后者与血浆ctDNA含量和完整性指数的关系，确定适合以血浆 DNA突变检测样本的条件和检测规范，并用该技术研究 NSCLC 靶向治疗过程中突变的变化规律。本研究建立了一种免DNA提取cfDNA定量测定技术并申报发明专利2项；建立了基于超敏PNA-PCR的技术检测血浆相关基因突变；证实cfDNA含量是确保血浆检材用于突变检测可靠性的决定因素，可以基于血浆游离DNA含量建立血液DNA基因突变检测质量控制体系。在该课题的资助下，我们还进一步在其它肿瘤，如大肠癌等进行了研究。研究已发表论文6篇，其中SCI收录3篇，核心期刊3篇。另有2篇待发表。