A multi-centre, prospective trial of a methylation-based liquid biopsy for early detection of liver cancer in high-risk populations

Abstract

Background and aims: Existing imaging and serum-marker assays miss many early liver cancers, especially in high-risk chronic liver disease carriers. We aimed to create a highly accurate, non-invasive, methylation-based liquid biopsy for early detection.

Methods: We used a comprehensive, multi-platform, multi-cohort strategy for marker discovery, starting with methylation profiling of hepatocellular carcinoma samples from TCGA and in-house cohorts. From 30 initial candidates, nine highly liver-specific methylation markers were shortlisted, and three optimal cfDNA markers (RNF135, CHFR, PAX5) were selected to develop a robust diagnostic model, tuned in a training set (N = 280) and locked in an internal testing set (N = 124). The model was then validated in a prospective, large-scale trial conducted at four geographically distinct Chinese centres.

Results: The clinical trial included 1097 participants from two groups, (i) a diagnosing group (N = 646) that prospectively enrolled individuals without prior diagnostic results and represented a real-world high-risk population, and (ii) a diagnosed group recruited after pathology confirmation. Overall, the model achieved 94.43% (95% confidence interval, 92.12–96.09%) sensitivity and 95.16% (92.78–96.78%) specificity for liver cancer, with stage-I sensitivity of 93.10% (89.78–95.40%). Within the diagnosing group, overall sensitivity was 93.99% (91.28–95.90%), and for the 267 stage-I cases, it reached 92.88% (89.15–95.39%). As for specificity, it remained high across confounders: 92.78% (85.84–96.46%) in cirrhosis, 91.74% (85.46–95.45%) in other-cancer interference samples. Besides, the model outperformed the traditional liver cancer biomarker AFP and showed changes in methylation signals before and after surgery, suggesting a possible role in perioperative monitoring. Each centre independently reported sensitivities and specificities exceeding 90%, demonstrating robust geographic performance.

Conclusions: Using a systematic marker-discovery pipeline and a multi-centre prospective cohort, we developed a methylation-based liquid biopsy that reliably detects early liver cancer in high-risk populations.

Clinical trial number: Chictr.org identifier: ChiCTR2400092883.

Key points:

Keywords

cfDNA / early detection / liquid biopsy / liver cancer diagnosis / methylation marker / non-invasive diagnosis

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RIGHTS & PERMISSIONS

2026 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.