目前尚未开发出一种有效的基于血液的结直肠癌（CRC）诊断方法。免疫细胞的分子变化在肿瘤起初阶段就会发生，为基于免疫细胞特征的早期癌症诊断提供了理论依据。因此，我们旨在开发一种基于外周血单个核细胞（PBMCs）的有效检测方法，以提高CRC的诊断水平。通过微阵列、测序和目标亚硫酸盐测序对来自CRC患者和健康对照组的PBMCs进行全基因组甲基化分析，发现了适用于CRC诊断的五个DNA甲基化标记物，特别是早期CRC（eCRC）。建立了一种单管多甲基化特异定量PCR检测方法（multi-msqPCR），可同时检测五个甲基化标记物，可以量化含有0.1% PBMC DNA的样本，且其辨别性能优于单分子检测。随后，基于甲基化标记物和多甲基化特异定量PCR方法构建了一种CRC诊断模型（CDM），在eCRC的诊断上达到了高准确度（AUC=0.91；敏感性=81.18%；特异性=89.39%），相较于CEA（AUC=0.79）有所提高。该CDM还可对晚期腺瘤案例进行高度区分（AUC=0.85；敏感性=63.04%）。随访数据还证明了该CDM能够在目前使用的诊断方法之前提前2年识别CRC潜在风险。总之，本研究构建的基于PBMC来源的DNA甲基化标记物和多甲基化特异定量PCR方法的策略是一种有前景且易于实施的eCRC诊断方法。

An effective blood-based method for the diagnosis of colorectal cancer (CRC) has not yet been developed. Molecular alterations of immune cells occur early in tumorigenesis, providing the theoretical underpinning for early cancer diagnosis based on immune cell profiling. Therefore, we aimed to develop an effective detection method based on peripheral blood mononuclear cells (PBMCs) to improve the diagnosis of CRC. Analysis of the genome-wide methylation landscape of PBMCs from CRC patients and healthy controls by microarray, pyrosequencing, and targeted bisulfite sequencing revealed five DNA methylation markers for CRC diagnosis, especially early-stage CRC (eCRC). A single-tube multiple methylation-specific quantitative PCR assay (multi-msqPCR) for simultaneous detection of five methylation markers was established, which allowed quantitative analysis of samples with as little as 0.1% PBMC DNA and had better discriminative performance than single-molecule detection. Then, a CRC diagnostic model (CDM) based on methylation markers and the multi-msqPCR method was constructed that achieved high accuracy for eCRC (AUC=0.91; sensitivity=81.18%; specificity=89.39%), which was improved compared to CEA (AUC=0.79). The CDM also enabled a high degree of discrimination for advanced adenoma (AA) cases (AUC=0.85, sensitivity=63.04%). Follow-up data also demonstrated that the CDM could identify CRC potential up to 2 years before currently used diagnostic methods. In conclusion, the approach constructed in this study based on PBMC-derived DNA methylation markers and a multi-msqPCR method is a promising and easily implementable diagnostic method for eCRC.