Hepatocellular carcinoma (HCC), the most prevalent form of
liver cancer, is growing in incidence but treatment options remain limited, particularly for late stage disease. As
liver cirrhosis is the principal risk state for HCC development, markers to detect early HCC within this patient population are urgently needed. Perturbation of epigenetic marks, such as DNA methylation (5mC), is a hallmark of human
cancers, including HCC. Identification of regions with consistently altered 5mC levels in circulating
cell free DNA (
cfDNA) during progression from
cirrhosis to HCC could therefore serve as markers for development of minimally-invasive screens of early HCC diagnosis and surveillance. Methods: To discover DNA methylation derived
biomarkers of HCC in the background of
liver cirrhosis, we profiled genome-wide 5mC landscapes in patient
cfDNA using the Infinium HumanMethylation450k BeadChip Array. We further linked these findings to primary tissue data available from TCGA and other public sources. Using biological and statistical frameworks, we selected CpGs that robustly differentiated
cirrhosis from HCC in primary tissue and
cfDNA followed by validation in an additional independent cohort. Results: We identified CpGs that segregate patients with
cirrhosis, from patients with HCC within a cirrhotic liver background, through genome-wide analysis of
cfDNA 5mC landscapes. Lasso regression analysis pinpointed a panel of probes in our discovery cohort that were validated in two independent datasets. A panel of five CpGs (cg04645914, cg06215569, cg23663760, cg13781744, and cg07610777) yielded area under the receiver operating characteristic (AUROC) curves of 0.9525, 0.9714, and 0.9528 in
cfDNA discovery and tissue validation cohorts 1 and 2, respectively. Validation of a 5-marker panel created from combining hypermethylated and hypomethylated CpGs in an independent
cfDNA set by
bisulfite pyrosequencing yielded an AUROC of 0.956, compared to the discovery AUROC of 0.996. Conclusion: Our finding that 5mC markers derived from primary tissue did not perform well in
cfDNA, compared to those identified directly from
cfDNA, reveals potential advantages of starting with
cfDNA to discover high performing markers for liquid biopsy development.