新型循环游离DNA甲基化组测序及分析方法在上皮性卵巢癌诊断与监测中的应用

Circulating cell-free DNA (ccfDNA) has emerged as a promising approach for non-invasive diagnosis. Our group developed a methylated CpG tandems amplification and sequencing (MCTA-Seq) method which was potentially used to diagnose epithelial ovarian cancer. Firstly, MCTA-Seq will be optimized by analyzing the fully methylated molecules instead of the methylation level of CpG islands which gives MCTA-Seq higher detecting sensitivity. Primer with CGGCGCGG CpG tandem will be added to primer B for detecting methylation level of BRCA2 and MyD88 promoter regions. Secondly, MCTA-Seq will be applied to detect the epithelial ovarian cancer (EOC) specific hypermethylated regions and establish the EOC classifier. Further, we will identify markers for discriminating EOC, hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) and establish a classifier which could distinguish 3 types of cancer. Then , we will monitor the dynamic changes of methylation in ccfDNA and white blood cell during the treatment. In conclusion, our research provides a novel method and new insights into the ccfDNA sources and non-invasive detection of epithelial ovarian cancer.

近年来，循环游离DNA检测作为一种极具前景的人类疾病无创检测手段被广泛研究。我与合作者建立了一种新型的循环游离DNA甲基化组检测技术—甲基化CpG短串联扩增与测序技术（MCTA-Seq）。基于以上技术（Wen，Cell Research, 2015），我们计划探究MCTA-Seq用于上皮性卵巢癌无创性检测的应用价值。第一方面，我们将对MCTA-seq进行优化：采用单碱基甲基化模式的分析策略，提高MCTA-Seq对肿瘤特异高甲基化位点的检测灵敏度；在引物种加入CGGCGCGG等CpG短串联序列用于检测卵巢癌相关重要基因甲基化水平。第二方面，我们将MCTA-Seq应用于检测临床样本：鉴定卵巢癌特异甲基化位点；建立卵巢癌血浆游离DNA甲基化组诊断模式；结合已有数据，开发基于肝癌、结直肠癌、卵巢癌异常甲基化位点的诊断模式；监测上皮性卵巢癌患者治疗前后血液中DNA甲基化变化。

本项目采用一种新型的循环游离DNA甲基化组检测技术（MCTA-Seq），分析了155例样本，包括卵巢癌患者血浆（n=75）,非癌妇科疾病患者血浆（n=6），健康女性血浆（n=41），卵巢癌组织（n=23），正常卵巢癌组织（n=10）。另外，下载数据库中的105例健康女性样本对甲基化标志物进行初筛。采用机器学习算法建立卵巢癌无创DNA甲基化分类器，对I、II、III、IV期卵巢癌检测，AUC分别为0.825, 0.975, 1.00, 0.995。综上所述，本项目研究结果表明MCTA-Seq在卵巢癌无创早期诊断与监测中具有广阔的临床应用前景。