基于高通量测序的血浆游离DNA甲基化组分析新方法及其在肿瘤诊断与监测中的应用

Aberrant methylation of CpG islands as cancer biomarkers have been discovered in plasma cell-free DNA for nearly twenty years. However, there is still a lack of omics analysis methods that are characterized as both high-sensitive (can detect targets of low abundance and proportion) and high through-put (can detect a large number of targets at the same time). In our previous work, we established a novel DNA methylome analysis method based on high-throughput sequencing: Methylated CpG Tandoms Amplification and Sequencing (MCTA-Seq). The results showed that MCTA-Seq is able to detect thousands of methylated CpG islands at high sensitivity of 0.1% to 1%. Further studies showed that MCTA-Seq was able to detect more than two thousands of methylated CpG islands from the plasma cell-free DNAs extracted from the peripheral blood of the hepatocellular carcinoma patients, including several hundreds of cancer-specific methylated CpG islands which were not detected in the peripheral blood of normal persons. Based on these previous studies, we apply for funding for 1) systematically optimizing MCTA-Seq; and 2) applying MCTA-Seq to analyze clinical samples of hepatocellular carcinoma and colorectal cancer patients for cancer detection and monitoring. These studies are aimed to accomplish MCTA-Seq as a high-sensitive, high through-put, low-cost and easy-operate DNA methylome analysis method for plasma cell-free DNA that can finally be applied in clinical cancer detection and monitoring.

血浆游离DNA中的异常甲基化CpG岛作为肿瘤生物标记物已有近二十年研究历史，但目前仍然缺乏同时具备高灵敏度（能检测含量和比例极少的靶目标）和高通量（能同时检测大量靶目标）特征的组学分析方法。在前期工作中，我们建立了一种基于高通量测序的DNA甲基化组分析新方法：甲基化CpG短串联扩增与测序法（MCTA-Seq）。结果显示，MCTA-Seq具有高灵敏（0.1~1%）地同时检测数千个甲基化CpG岛的能力。进一步，MCTA-Seq能从肝癌患者血浆游离DNA中检测到两千多个甲基化CpG岛，其中数百个为肿瘤特异性的、而在正常人外周血中检测不到的异常甲基化CpG岛。据此，我们拟：1）系统优化MCTA-Seq，并2）开展肝细胞癌和结直肠癌诊断和监测临床应用研究。以上研究旨在将MCTA-Seq建成一种高灵敏、高通量、低成本和操作简便的血浆游离DNA甲基化组分析方法，为其最终应用于临床肿瘤诊断与监测提供基础。

本项目发明了一种新颖的肿瘤无创早期检测新技术——甲基化CpG短串联扩增与测序（Methylated CpG tandom amplification and sequencing, MCTA-Seq；Wen, et al., Cell Research, 2015；已被授予国内与国际专利权）。该技术巧妙地采用CpG短串联引物对甲基化CpG岛进行富集性扩增与测序，在一次反应中同时检测近万个甲基化CpG岛，检测下限可低至1~2个细胞的基因组DNA。我们采用MCTA-Seq共分析了377份血浆样本，包括肝细胞癌患者（n = 36）、肝硬化患者（n = 17）、结直肠癌患者（n = 147）及正常个体（n = 172），以及137份组织样本。MCTA-Seq技术检测肝细胞癌的灵敏度为94%，特异度为89%；检测早期结直肠癌的灵敏度为74%，特异度为90%。该技术能够有效地在外周血中鉴别早期结直肠癌与早期肝细胞癌患者。综上所述，本项目研究结果表明MCTA-Seq是一种高效、简便的循环游离DNA甲基化组高通量测序新技术，其应用于肝细胞癌、结直肠癌以及其他人类肿瘤的无创早期诊断与监测具有广阔前景。