Background: Integration of HBV DNA into the human genome could progressively contribute to hepatocarcinogenesis. Both intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC) are known to be associated with HBV infection. However, the integration of HBV and mechanism of HBV-induced carcinogenesis in ICC and CHC remains unclear.

Methods: 41 patients with ICC and 20 patients with CHC were recruited in the study. We conducted HIVID analysis on these 61 samples to identify HBV integration sites in both the tumor tissues and adjacent non-tumor liver tissues. To further explore the effect of HBV integration on gene alteration, we selected paired tumors and adjacent non-tumor liver tissues from 3 ICC and 4 CHC patients for RNA-seq and WGS.

Results: We detected 493 HBV integration sites in ICC patients, of which 417 were from tumor samples and 76 were from non-tumor samples. And 246 HBV integration sites were detected in CHC patients, of which 156 were located in the genome of tumor samples and 90 were in non-tumor samples. Recurrent HBV integration events were detected in ICC including TERT, ZMAT4, MET, ANKFN1, PLXNB2, and in CHC like TERT, ALKBH5. Together with our established data of HBV-infected hepatocellular carcinoma, we found that HBV preferentially integrates into the specific regions which may affect the gene expression and regulation in cells and involved in carcinogenesis. We further performed genomic and transcriptomic sequencing of three ICC and four CHC patients, and found that HBV fragments could integrate near some important oncogene like TERT, causing large-scale genome variations on nearby genomic sequences, and at the same time changing the expression level of the oncogenes.

Conclusion: Comparative analysis demonstrates numerous newly discovered mutational events in ICC and CHC resulting from HBV insertions in the host genome. Our study provides an in-depth biological and clinical insights into HBV-induced ICC and CHC.