Purpose: Detection of colorectal carcinomas (CRC) at a time when there are more treatment options is associated with better outcomes. This prospective case-control study assessed the 5-hydroxymethylcytosine (5hmC) biomarkers in circulating cell-free DNA (cfDNA) for early detection of CRC and advanced adenomas (AA) Experimental Design: Plasma cfDNA samples from 2,576 study participants from the multi-center METHOD-2 study (NCT03676075) were collected, comprising patients with newly diagnosed CRC (n=1,074), AA (n=356), other solid tumors (n=80), and non-CRC/AA controls (n=1,066), followed by genome-wide 5hmC profiling using the 5hmC-Seal technique and the next-generation sequencing (NGS). A weighted diagnostic model for CRC (stage I-III) and AA was developed using the elastic net regularization in a discovery set and validated in independent samples. Results: Distribution of 5hmC in cfDNA reflected gene regulatory relevance and tissue of origin. Besides being confirmed in internal validation, a 96-gene model achieved an area under the curve (AUC) of 90.7% for distinguishing stage I-III CRC from controls in 321 samples from multiple centers for external validation, regardless of primary location or mutation status. This model also showed cancer-type specificity as well as high capacity for distinguishing AA from controls with an AUC of 78.6%. Functionally, differential 5hmC features associated with CRC and AA demonstrated relevance to CRC biology, including pathways such as calcium and MAPK signaling. Conclusions: Genome-wide mapping of 5hmC in cfDNA shows the promise as a highly sensitive and specific non-invasive blood test to be integrated in screening programs for improving early detection of CRC and high-risk AA.

中文翻译：

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基于 5-羟甲基胞嘧啶的非侵入性模型，用于早期检测结直肠癌和晚期腺瘤：METHOD-2 研究


目的：在有更多治疗选择时检测结直肠癌 (CRC) 与更好的结果相关。这项前瞻性病例对照研究评估了循环游离 DNA (cfDNA) 中的 5-羟甲基胞嘧啶 (5hmC) 生物标志物，用于早期检测 CRC 和晚期腺瘤 (AA) 实验设计：来自多中心 2,576 名研究参与者的血浆 cfDNA 样本收集了 METHOD-2 研究 (NCT03676075)，其中包括新诊断的 CRC 患者 (n=1,074)、AA (n=356)、其他实体瘤 (n=80) 和非 CRC/AA 对照患者 (n=1,066) ，然后使用 5hmC-Seal 技术和下一代测序 (NGS) 进行全基因组 5hmC 分析。使用发现集中的弹性网络正则化开发了 CRC（I-III 期）和 AA 的加权诊断模型，并在独立样本中进行了验证。结果：cfDNA 中 5hmC 的分布反映了基因调控相关性和组织来源。除了在内部验证中得到证实外，96 基因模型在来自多个外部验证中心的 321 个样本中区分 I-III 期 CRC 和对照的曲线下面积 (AUC) 达到了 90.7%，无论原发位置或突变状态如何。该模型还显示出癌症类型特异性以及区分 AA 和对照的高能力，AUC 为 78.6%。在功能上，与 CRC 和 AA 相关的差异 5hmC 特征证明与 CRC 生物学相关，包括钙和 MAPK 信号传导等途径。结论：cfDNA 中 5hmC 的全基因组图谱显示出作为一种高度敏感和特异的非侵入性血液检测的前景，可集成到筛查计划中，以改善 CRC 和高风险 AA 的早期检测。