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

Haikun Zhang, Peiling Dong, Shicheng Guo, Chengcheng Tao, Wei Chen, Wenmin Zhao, Jiakang Wang, Ramsey Cheung, Augusto Villanueva, Jian Fan, Huiguo Ding, Steven J. Schrodi, Dake Zhang, Changqing Zeng

Research output: Contribution to journalArticlepeer-review

25 Scopus citations


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.

Original languageEnglish
Article number200
JournalBMC Medicine
Issue number1
StatePublished - 3 Aug 2020


  • Cell-free DNA
  • DNA methylation
  • HBV integration
  • Hepatocellular carcinoma
  • Low-pass WGBS


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