Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma.

Yangxing Zhao,Zhenfeng Zhang,Shicheng Guo,Feng Xue,Xiaoyu Zhou,Yinghua He,Hongyu Zhang,Junfeng Geng,Bijun Qiu,Jinfeng Sun,Qiang Xia,Wei Wang,Jun Gu,Jian Yu,Ning Tang

doi:10.1186/1868-7083-6-30

Abstract

BackgroundAn important model of hepatocellular carcinoma (HCC) that has been described in southeast Asia includes the transition from chronic hepatitis B infection (CHB) to liver cirrhosis (LC) and, finally, to HCC. The genome-wide methylation profiling of plasma cell-free DNA (cfDNA) has not previously been used to assess HCC development. Using MethylCap-seq, we analyzed the genome-wide cfDNA methylation profiles by separately pooling healthy control (HC), CHB, LC and HCC samples and independently validating the library data for the tissue DNA and cfDNA by MSP, qMSP and Multiplex-BSP-seq.ResultsThe dynamic features of cfDNA methylation coincided with the natural course of HCC development. Data mining revealed the presence of 240, 272 and 286 differentially methylated genes (DMGs) corresponding to the early, middle and late stages of HCC progression, respectively. The validation of the DNA and cfDNA results in independent tissues identified three DMGs, including ZNF300, SLC22A20 and SHISA7, with the potential for distinguishing between CHB and LC as well as between LC and HCC. The area under the curve (AUC) ranged from 0.65 to 0.80, and the odds ratio (OR) values ranged from 5.18 to 14.2.ConclusionsOur data revealed highly dynamic cfDNA methylation profiles in support of HBV-related HCC development. We have identified a panel of DMGs that are predictive for the early, middle and late stages of HCC development, and these are potential markers for the early detection of HCC as well as the screening of high-risk populations.Electronic supplementary materialThe online version of this article (doi:10.1186/1868-7083-6-30) contains supplementary material, which is available to authorized users.

Highlights

An important model of hepatocellular carcinoma (HCC) that has been described in southeast Asia includes the transition from chronic hepatitis B infection (CHB) to liver cirrhosis (LC) and, to HCC
Association of plasma cell-free DNA methylation disturbances with the progressive stages of hepatocellular carcinoma development We used hierarchical clustering to trace the progression of methylation disturbances during the carcinogenic process for the healthy control (HC), CHB, LC and HCC samples
We estimated the relationships between the aberrant methylation patterns in the various stages of HCC development by the pairwise overlap of the peak bases from each stage, which revealed the similarities between HC and CHB as well as between LC and HCC (Additional file 2: Table S5)

Summary

Introduction

An important model of hepatocellular carcinoma (HCC) that has been described in southeast Asia includes the transition from chronic hepatitis B infection (CHB) to liver cirrhosis (LC) and, to HCC. The highest incidences are observed in eastern Asia, where a model of multi-stage carcinogenesis that includes progression from chronic HBV infection (CHB) to HBV-related liver cirrhosis (LC) and, to HBV-related HCC (HBV-HCC) has been suggested by both epidemiologic data and laboratory investigations [3,4]. Previous reports have demonstrated that HBV products, including the HBx protein, may manipulate DNMT1 to inactivate some TSGs, such as CDH1, by the dysregulating methylation or by aberrantly methylating miRNAs to influence downstream targets, such as DNMT3A, leading to further disturbances in methylation [9]

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