基于双重PNA钳制反应的循环肿瘤DNA突变检测新方法及其在肿瘤靶向治疗中的应用评价

Targeted therapy in cancer is based on the detection of mutant DNA in tumor tissues that are sometimes difficult to obtain. Since circulating tumor DNA (ctDNA) contains genetic information of tumors in which mutant DNA can be detected, ctDNA could be an alternative to tumor tissues. However, the fact that detection results are inconsistent between ctDNA and tissue DNA by the existing methods due to their lower sensitivity, limits the clinical application of ctDNA. To establish a highly sensitive method for detecting mutant in ctDNA, a single-tube nested PCR were developed by our group though the control of primers’ Tm values and thermal cycling temperature. Although the single-tube nested PCR has a high amplification efficiency, it is still insufficient for detecting mutant in ctDNA because of the interference of the large amount of coexisting wid-type DNA. To further address the interference of wild-type DNA, we aim to establish a novel method that combines PNA-clamping reaction with single-tube nested PCR, whose specificity is expected to achieve 1 mutant molecule in 1000 wild type molecules (0.1%). In this method, the outer primers as well as the inner primers, which are used to amplify wild-type DNA, will be dually clamped by a wild-type PNA probe, but the primers used to amplify mutant DNA will not be affected for the mismatch between PNA probe and mutant templates. By this means, the amplification of wild-type DNA is inhibited, and the mutant in ctDNA can be detected with a high sensitivity and specificity. By exploring the correlation of ctDNA and tumor tissue DNA, we are able to establish a novel tumor-targeted medication platform oriented by ctDNA, providing a specific and non-invasive new approach for personalized treatment.

肿瘤靶向治疗是基于肿瘤组织突变检测结果，但肿瘤组织有时难以获得。循环肿瘤DNA（ctDNA）含有肿瘤的遗传信息，可代替肿瘤组织进行突变检测。但现有突变检测方法灵敏度低的缺点导致了ctDNA与肿瘤组织检测结果有差异，限制其临床应用。为建立高灵敏ctDNA突变检测方法，课题组先利用引物Tm值差异和热循环温度控制，创建了单管巢式PCR法，该法具有很高扩增效率，但仍难克服ctDNA中大量野生型DNA的干扰。为此，本课题拟进一步将肽核酸钳制反应与单管巢式PCR相融合，建立可检测0.1%突变的双重PNA钳制反应介导的单管巢式PCR法。其原理是利用肽核酸探针对巢式外引物、内引物扩增的双重钳制作用，选择性抑制野生型DNA富集突变型DNA，进而实现对ctDNA突变的高灵敏、高特异检测。最后，本课题拟以非小细胞肺癌为例，利用该方法阐明ctDNA代替肿瘤组织的可行性，为肿瘤个体化靶向治疗提供特异、无创的新途径。

肿瘤靶向治疗是基于肿瘤组织突变检测结果，但肿瘤组织有时难以获得。循环肿瘤DNA（ctDNA）含有肿瘤的遗传信息，可代替肿瘤组织进行突变检测。但现有突变检测方法灵敏度低的缺点导致了ctDNA与肿瘤组织检测结果有差异，限制其临床应用。本研究拟开发一种新型的基于单管巢式PCR和双重PNA钳制反应的ctDNA检测技术，并利用该技术评价非小细胞肺癌（NSCLC）患者血浆ctDNA代替肿瘤组织的可行性，为基于血浆ctDNA的肿瘤个体化靶向治疗提供理论依据。结果显示，本研究建立的基于单管巢式PCR和双重PNA钳制反应的ctDNA检测技术在10的0次方~10的6次方之间具有线性，最低检测限为1拷贝的DNA突变，且能实现对低至0.1‰的极微量基因突变的检测。利用本研究建立的ctDNA检测技术与数字化PCR同时对100例组织样本的EGFR L858R位点进行了检测，本研究技术与数字化PCR结果完全一致，表明了本研究建立的方法和数字化PCR具有相同的准确性。进一步的组织和血浆的一致性分析结果显示，在中国人群中，NSCLC患者血浆ctDNA与肿瘤组织的一致性为79%。且该一致性与肿瘤分期具有显著的相关性。血浆和组织一致性最高的是IV期NSCLC患者，两者一致性为100%。因此，要用血浆来代替肿瘤组织进行基因突变检测，选择IV期NSCLC患者能得到最佳的检测效果。总之，本研究建立了一种新型的血浆ctDNA基因突变检测技术，为临床血浆中极微量血浆ctDNA基因突变检测提供了一种高灵敏、可定量、简便快速和低成本的新技术；该技术可用于因不适合手术或术后复发与转移等无法获得肿瘤组织样本的IV期NSCLC患者提供了一种简单、特异、无创地获取基因突变信息的新途径。将该技术应用于临床，可为基于血浆ctDNA的非小细胞肺癌患者靶向药物个体化治疗方案提供技术支持。