用于肺癌液体活检的新型微液滴数字多重PCR方法的研究

The liquid biopsy technique measures circulating tumor DNA (ctDNA) and has the potential to facilitate early cancer diagnosis, personalized treatment and post-surgery evaluation of cancer patients. It has the advantages of being minimally invasive, cost saving and offering real-time detection; it thus may change the conventional cancer diagnosis and treatment methods. However, liquid biopsy is now still in the stage of concept. Currently, there is no reliable non-invasive ctDNA detection technique nor related commercial products available. Droplet digital PCR (ddPCR) is the most sensitive among the methods having single molecule detection ability. Herein we propose a novel proprietary multi-plex digital PCR detection method, which integrates addressable, quantum dots-encoded microbeads with our previously developed ddPCR technique. We will use this novel multi-plex ddPCR method to study EGFR, which is a therapeutic target of non-small cell lung cancer (NSCLC). We will perform parallel detection mutations simultaneously and quantification of low-abundance mutations at the single-molecule level, and then evaluate the application of this method in liquid biopsy using peripheral blood from NSCLC patients. This study will accelerate the application of lung biopsy in diagnostics and have a significant impact in clinic practice.

基于循环肿瘤DNA（ctDNA）的“液体活检”为肿瘤的早期诊断、个体化治疗及术后评估提供了一种微创、经济、实时的检测方法，具有重要的应用价值，可能将颠覆肿瘤的诊疗模式。但迄今尚没有一项可靠的无创肿瘤液体活检技术和产品。微液滴数字PCR（聚合酶链式反应）是目前检测DNA灵敏度最高的新方法，本项目拟在团队成功研制微液滴数字PCR检测系统的基础上，首次将量子点编码微球阵列寻址技术与自主研发的微滴数字PCR技术相整合，开发具有独立知识产权的，使用方便的新型多重数字PCR单分子检测方法；基于此新型微液滴数字PCR方法，针对非小细胞肺癌靶向治疗密切相关的基因EGFR多个基因突变进行并行检测和单分子定量检测，并用肺癌病人外周血样本进行液体活检评估研究。本项目有望提供一种可靠的EGFR多靶标超高灵敏度检测方法，极大促进肺癌液体活检在临床检验中的应用，具有非常重要的临床价值。

基于外周血肿瘤循环DNA（ctDNA）的液体活检技术有望在非小细胞肺癌靶向治疗的分子分型和耐药检测中发挥至关重要的作用。针对这一重大需求，本项目开发了基于微液滴数字PCR的肺癌主要驱动基因EGFR的检测试剂。通过自主知识产权的数字PCR平台和专利的封闭探针法建立和优化了适用于临床的EGFR液体活检试剂，为了进一步验证该试剂的临床适用性，组织了多中心临床测试，同时和罗氏液体活检诊断试剂cobas EGFR对比测试，总体结果表明，基于数字PCR的EGFR液体活检方法比目前性能较好的罗氏COBAS EGFR产品灵敏度高，且特异性好，能保证准确度，是临床方法的补充和更佳选择。.本项目针对肿瘤液体活检的重大需求，在数字PCR技术和相关检测方法上均有重要成果，所开发的微液滴数字PCR平台提供了一种高灵敏和防污染的自动化检测装置，所建立的新的基于数字PCR的EGFR液体活检试剂灵敏度高，且特异性好，是临床实验方法的有益补充和更佳选择。