Development and multicenter validation of a cfDNA methylation-based PCR assay (myESCAP) with machine-learning prediction for non-invasive detection of esophageal cancer.

Authors

Xin Wang, Zhen Wang, Ling Qi, Shuang Zhou, Weixin Liu, Xuefeng Zhang, Hui Wang, Qiye He, Xinyu Mei, Yong Li

Published in

Journal of advanced research. Oct 30, 2026. Epub Oct 30, 2026.

Abstract

Esophageal cancer (EC) remains a significant public health burden in China. Current diagnostics modalities such as endoscopy and imaging are invasive, poorly accessible, or insensitive for early-stage cases. DNA methylation alterations in circulating free DNA (cfDNA) are promising for non-invasive cancer detection.
We aimed to develop and validate a methylation PCR-based assay for EC detection using EC-specific DNA methylation targets. Here, we report myESCAP, a PCR-based assay that amplifies and quantifies levels of EC-specific methylation in cfDNA METHODS: We conducted a multi-center, retrospective case-control study with 489 EC cases and 555 non-malignant controls (benign esophageal/gastric diseases or asymptomatic) to develop myESCAP, a methylation-specific PCR assay containing 11 EC-specific methylation markers from our previous genome-wide methylation screening, selected for strong discriminative performance and PCR compatibility. cfDNA was collected, bisulfite-converted, and tested by myESCAP, and results were analyzed by six modeling strategies (generalized linear model, k-nearest neighbors, C5.0 decision tree, gradient boosting machine, random forest, support vector machine) to train EC-prediction models using 347 EC cases and 349 non-malignant controls, and validated in 142 EC cases and 206 controls.
All myESCAP markers demonstrated significant differential methylation between ECs and non-malignant controls, with adjusted p values between 5.8×10⁻³ and 3.1×10⁻⁸⁸. The six models showed robust performance in the training cohort (average AUC = 0.835, average sensitivity = 76.13%, average specificity = 90.88%). They maintained performance in the validation cohort (average AUC = 0.823, average sensitivity = 75.59%, average specificity = 89%). Specifically, for stage I cases, myESCAP had an average sensitivity of 47.2%, highlighting the challenge of early detection with limited circulating tumor DNA and the need for further optimization.
myESCAP accurately differentiated EC from non-malignant controls. The six models collectively confirm myESCAP's robustness and clinical potential as a cost-effective, non-invasive diagnostic tool for EC.

PMID:
42962916
Bibliographic data and abstract were imported from PubMed on 31 Oct 2026.

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