基于甲基化组、代谢组联合的大肠癌精准诊断生物标志物筛选的研究

Colorectal cancer is one of the most prevalent malignancies worldwide. The early stage diagnosis of colorectal cancer could obtain five-year survival rate in 97% patients by surgical remove and chemoradiotherapy. However, more than 80% patients have been diagnosed with mid-late stage cancer, which severely affects the prognosis. The leading reason is the diagnostic capability of current population screening and auxiliary diagnostic methods is unsatisfactory. Therefore, a non-invasive, early accurate diagnostic method is urgently needed. Plasma circulating tumor DNA (ctDNA) methylation is stable and with strong specificity; metabolites are sensitive and change quickly. These two kinds of biomarkers are expected to be the best combination with precise diagnostic capability. Therefore, in this study, we will primarily use Whole-Genome Bisulfite Sequencing (WGBS) to screen the potential ctDNA methylation of colorectal cancer. By integrating with the data of metabolome of colorectal cancer we have finished before, the pathway interaction network will be built; also, we will systematically elaborate the pathophysiological mechanism of colorectal cancer and further screen out ctDNA methylation biomarkers accurately. Then, in a two-stage case-control study, the reliable ctDNA methylation biomarkers will be confirmed. Simultaneously, we will assess the diagnostic values of individual biomarker or different combinations of biomarkers; analyze the association between biomarkers and pathological features of tumor. Based on the above results, we will establish the panel of biomarkers with the best diagnostic value and the panels of personalized classification of cancer, which will provide evidence for the early, precise, and non-invasive diagnosis of colorectal cancer.

大肠癌是世界范围内高发的恶性肿瘤。早期大肠癌通过手术放化疗5年生存率可达97%，但80%以上的患者确诊时已是中晚期，严重影响预后。主要原因是现有的筛检及辅助检查的诊断效果不理想。因此，迫切需要一种非侵入的早期精准诊断手段。血浆循环肿瘤DNA（ctDNA）甲基化稳定、特异性强；代谢产物敏感、变化迅速，这两类标志物可望构成大肠癌诊断的最佳组合。本课题将首先基于全基因组甲基化测序技术筛选潜在的大肠癌ctDNA甲基化位点，结合课题组已完成的大肠癌血浆代谢组学数据，整合甲基化组代谢组信息构建大肠癌互作通路网络，全面阐述病理学机制，准确筛选与大肠癌发病相关的标志物。然后，通过两阶段病例对照研究，确定可靠的ctDNA甲基化标志物；评价ctDNA甲基化和代谢标志物单独及联合应用的诊断学价值，分析各标志物与肿瘤病理特征的关系，构建大肠癌最佳诊断标志物组合及个体化分型组合，为大肠癌的早期精准无创诊断提供依据。

大肠癌是世界范围内高发的恶性肿瘤。早期筛查和诊断是提高大肠癌患者预后的重要手段。因此，结合甲基化组、代谢组多组学数据，筛选稳定、特异的大肠癌标志物验证评价；细化大肠癌的临床诊断是本研究的主要内容。.研究基于配对组织的全基因组甲基化测序（WGBS）发现10166个差异性甲基化区域（DMR）；转录组测序发现：癌组织中有350个基因上调，1528个基因下调；其中708个差异表达基因（DEGs基因）与DMR甲基化模式呈负相关。大肠癌患者血浆cfDNA的WGBS测序发现1251个甲基化基因在配对组织的DMR中。cfDNA的基因组功能元件的平均甲基化水平分布与癌组织一致。将cfDNA甲基化位点所对应的432个高甲基化DMR与白细胞WGBS对应的3933个高甲基化DMR比对。最终筛选出132个DMR为ctDNA甲基化所独有的大肠癌标志物。基于病例对照人群进行检测，3个ctDNA甲基化标志物 (DAB1, ST8SIA5, STK32B) 在区分癌及非癌组织，病例及对照血浆样本时都达到较好的诊断学效能（AUCs > 0.7）。此外，研究发现差异甲基化基因CRYAB（P=0.012/0.017）、AKAP12（P=0.022）、TNS1（P=0.039/0.055）、HSPB6（P=0.0018）的表达水平与结直肠癌患者的预后存在显著性关联。.研究在血浆中共发现147种大肠癌代谢物，在组织中发现93种，在系统综述中发现635种。经多步骤筛选后，由L-Tryptophan, 13-OxoODE, IDP, Glycochenodeoxycholate, and LysoPC(16:0)组成的标记物组合在中国人群中表现出良好的诊断性能、重要的生物学意义和肿瘤组织的特异性。四种代谢物L-Tryptophan, Linoleic acid, Glycocholic acid, and LysoPC(16:0)在多人群中可检测。靶标定量检测验证了四种标记物L-Tryptophon, Linoleic Acid, LysoPC(14:0), LysoPC(18:0)组合的诊断学价值（0.728, 0.681-0.775）。此外，基于组织的代谢分型分析结果发现，差异分型的患者具有不同的癌变依赖代谢机制和预后。整合甲基化及代谢标志物将对大肠癌患者的个体化治疗和预后预测具有重要意义。