早期发现食管鳞状细胞癌（ESCC）可以大大改善患者的预后。异常的细胞外游离DNA（cfDNA）甲基化特征是检测ESCC的一种有前途的工具。然而，基于细胞外DNA甲基化的可用标志物仍然不足。本研究旨在确定ESCC特异性的cfDNA甲基化标志物，并评估其在早期检测ESCC中的诊断性能。我们对24例ESCC组织及其正常邻近组织进行了全基因组亚硫酸盐测序（WGBS）。基于WGBS数据，我们确定了21,469,837个合格的CpG位点。通过整合多个甲基化数据集，我们确定了几个有前途的ESCC特异性细胞外DNA甲基化标志物。最后，我们基于甲基化的KCNA3和OTOP2开发了一个双标志物组合板，并在我们的训练和验证队列中评估其性能。基于KCNA3和OTOP2构建的ESCC诊断模型在训练队列中具有0.91的AUC值（95％CI：0.85-0.95），并具有84.91％的最佳敏感性和94.32％的最佳特异性。在独立的验证队列中，AUC为0.88（95％CI：0.83-0.92），敏感性为81.5％，特异性为92.9％。对于I-II期ESCC，模型的敏感性为78.4％，略低于III-IV期ESCC模型的敏感性（85.7％）。基于cfDNA的双目标板显示出了优异的ESCC检测性能，并可能成为筛查ESCC的替代策略。 版权所有 © 2023 中华医学会，Wolters Kluwer, Inc. 根据CC-BY-NC-ND许可协议制作。

Early detection of esophageal squamous cell carcinoma (ESCC) can considerably improve the prognosis of patients. Aberrant cell-free DNA (cfDNA) methylation signatures are a promising tool for detecting ESCC. However, available markers based on cell-free DNA methylation are still inadequate. This study aimed to identify ESCC-specific cfDNA methylation markers and evaluate the diagnostic performance in the early detection of ESCC.We performed whole-genome bisulfite sequencing (WGBS) for 24 ESCC tissues and their normal adjacent tissues. Based on the WGBS data, we identified 21,469,837 eligible CpG sites (CpGs). By integrating several methylation datasets, we identified several promising ESCC-specific cell-free DNA methylation markers. Finally, we developed a dual-marker panel based on methylated KCNA3 and OTOP2, and then, we evaluated its performance in our training and validation cohorts.The ESCC diagnostic model constructed based on KCNA3 and OTOP2 had an AUC of 0.91 [95% CI: 0.85-0.95], and an optimal sensitivity and specificity of 84.91% and 94.32%, respectively, in the training cohort. In the independent validation cohort, the AUC was 0.88 [95% CI: 0.83-0.92], along with an optimal sensitivity of 81.5% and specificity of 92.9%. The model sensitivity for stage I-II ESCC was 78.4%, which was slightly lower than the sensitivity of the model (85.7%) for stage III-IV ESCC.The dual-target panel based on cfDNA showed excellent performance for detecting ESCC and might be an alternative strategy for screening ESCC.Copyright © 2023 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.