Hypomethylation in HBV integration regions aids non-invasive surveillance to hepatocellular carcinoma by low-pass genome-wide bisulfite sequencing

BMC Med. 2020 Aug 3;18(1):200. doi: 10.1186/s12916-020-01667-x.


Background: Circulating cell-free DNA (cfDNA) methylation has been demonstrated to be a promising approach for non-invasive cancer diagnosis. However, the high cost of whole genome bisulfite sequencing (WGBS) hinders the clinical implementation of a methylation-based cfDNA early detection biomarker. We proposed a novel strategy in low-pass WGBS (~ 5 million reads) to detect methylation changes in circulating cell-free DNA (cfDNA) from patients with liver diseases and hepatocellular carcinoma (HCC).

Methods: The effective small sequencing depth were determined by 5 pilot cfDNA samples with relative high-depth WGBS. CfDNA of 51 patients with hepatitis, cirrhosis, and HCC were conducted using low-pass WGBS. The strategy was validated in an independent WGBS cohort of 32 healthy individuals and 26 early-stage HCC patients. Fifteen paired tumor tissue and buffy coat samples were used to characterize the methylation of hepatitis B virus (HBV) integration regions and genome distribution of cfDNA.

Results: A significant enrichment of cfDNA in intergenic and repeat regions, especially in previously reported HBV integration sites were observed, as a feature of cfDNA and the bias of cfDNA release. Methylation profiles nearby HBV integration sites were a better indicator for hypomethylation of tumor genome comparing to Alu and LINE (long interspersed nuclear element) repeats, and were able to facilitate the cfDNA-based HCC prediction. Hypomethylation nearby HBV integration sites (5 kb flanking) was detected in HCC patients, but not in patients with hepatitis and cirrhosis (MethylHBV5k, median:0.61 vs 0.72, P = 0.0003). Methylation levels of integration sites certain candidate regions exhibited an area under the receiver operation curve (AUC) value > 0.85 to discriminate HCC from non-HCC samples. The validation cohort achieved the prediction performance with an AUC of 0.954.

Conclusions: Hypomethylation around viral integration sites aids low-pass cfDNA WGBS to serve as a non-invasive approach for early HCC detection, and inspire future efforts on tumor surveillance for oncovirus with integration activity.

Keywords: Cell-free DNA; DNA methylation; HBV integration; Hepatocellular carcinoma; Low-pass WGBS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Hepatocellular / genetics*
  • Cell-Free Nucleic Acids / genetics*
  • Cohort Studies
  • DNA Methylation / genetics*
  • Female
  • Genomics / methods*
  • Hepatitis B virus / pathogenicity*
  • Humans
  • Liver Neoplasms / genetics*
  • Male
  • Pilot Projects
  • Sulfites / metabolism*


  • Cell-Free Nucleic Acids
  • Sulfites
  • hydrogen sulfite