Plasma Cell-Free DNA Methylomes for Hepatocellular Carcinoma Detection and Monitoring after Liver Resection or Transplantation

Hepatocellular carcinoma (HCC) is a highly lethal malignancy with frequent recurrence after curative-intent surgery. Current detection and monitoring methods rely heavily on imaging, and genomic strategies are limited by access to tumor tissue and the challenges of mutational heterogeneity. Therefore, a cost-effective noninvasive approach with sufficient sensitivity, specificity, and high accuracy is needed to enhance HCC management. In this study, we evaluated the clinical potential of cfMeDIP-seq for HCC detection and postoperative monitoring. A total of 236 cfDNA samples were collected at surgery (b-HCC, n=89) and during follow-up (f-HCC, n=112) from 89 HCC patients undergoing liver transplantation (n=57) or resection (n=32), along with 35 healthy controls (CTL). cfMeDIP-seq was performed, followed by machine learning to develop an HCC-specific classifier in a discovery cohort (52 b-HCC vs. 35 CTL), which was subsequently tested in a validation cohort of 37 patients. An HCC methylation score (HMS) was assigned to reflect the probability of a sample containing HCC-derived cfDNA, and relationships between HMS and clinical variables were assessed. The classifier identified HCC with 97% sensitivity and 99% specificity in the discovery cohort and 97% accuracy in the validation cohort. Baseline HMS >0.9 was associated with higher recurrence risk (HR 3.43, 95% CI 1.30-9.06, p=0.013), and HMS decreased by 3-44% (median 17%) within 13 weeks post-surgery. HMS trajectories diverged for recurrent and non-recurrent patients, with an increase in HMS indicating clinical recurrence, and the HMS was independent of other clinicopathologic variables. These findings demonstrate that tumor-agnostic cfDNA methylomes can accurately detect HCC and predict recurrence after liver resection or transplantation, suggesting that this approach may have important implications for HCC diagnosis, treatment, and monitoring. ### Competing Interest Statement Gonzalo Sapisochin discloses consultancy for Astra-Zeneca, Roche, Novartis, Evidera, Natera, Integra and HepaRegeniX. Gonzalo Sapisochin has received financial compensation for talks for Roche, AstraZeneca, Chiesi, and Integra. Gonzalo Sapisochin has received a grant from Roche. Gonzalo Sapisochin has research collaborations with Astra-Zeneca, Roche, Natera and Stryker. ### Funding Statement This study was conducted with support from the Canadian Institutes of Health Research (CIHR Project grant #152982) and the UHN Foundation (Lillian MacKenzie Fund). Trevor J. Pugh holds the Canada Research Chair in Translational Genomics and is supported by a Senior Investigator Award from the Ontario Institute for Cancer Research and the Gattuso-Slaight Personalized Cancer Medicine Fund, and the Ontario Institute for Cancer Research Genomics Program, and the Canada Foundation for Innovation, Leaders Opportunity Fund (CFI #32383 and #38401), and the Ontario Ministry of Research and Innovation, Ontario Research Fund Small Infrastructure Program. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the University Health Network Research Ethics Board (REB#08-0697, REB#17-5925). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Raw sequencing data for all 203 HCC samples and 23 healthy controls are deposited at the European Genome-phenome Archive (EGA) under the accession number EGAS50000000450. Raw sequencing files for another 12 healthy normal controls can be requested at https://www.ontariohealthstudy.ca/for-researchers/data-access-formsand-templates. Processed intermediate data and objects used to replicate the results in this study are available at https://doi.org/10.5281/zenodo.11251606. Code scripts and markdown instruction to reproduce the figures and analyses are available at https://github.com/pughlab/HCC_cfMeDIP.