Definition and selection of stage-specific differentially methylated region (DMR) during hepatocellular carcinoma (HCC) development. (A) Chromosomal views of the genome-wide methylation statuses of chronic hepatitis B infection(CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) and non-small cell lung cancer(NSCLC). Black bars represent hypermethylation relative to HC. DNA hypermethylation occurs as early as the CHB stage and becomes more extensive as HCC development progresses. NSCLC, an alternative non-liver malignancy included in this study to test effects of different organs on cfDNAm. (B) Venn diagram of the hyper-differentially methylated genes (DMGs) that occurred in the CHB, LC and HCC stages. The diagram was used to further analyze the relationships among these three groups. (C) Gene mappings based on DMG information. To expand upon the identification of genes that are affected during the early, middle and late stages in the DMRs, genes that were located proximal to and involved in the hypermethylation events, such as “CGI only”, “both CGI and shore” or “shore only”, were examined. Here, “shore” is defined as a region that flanks traditional CGIs (up to 2 kb in distance). (D) Venn diagram depicting the comparison of the hyper-DMGs that occurred in HCC versus NSCLC. In total, 405 hyper-DMGs are shared by HCC and NSCLC, indicating the general homogeneities of the different malignancies, such as HCC and NSCLC in this case.