Whole exome HBV DNA integration is independent of the intrahepatic HBV reservoir in HBeAg-negative chronic hepatitis B

Abstract

ObjectiveThe involvement of HBV DNA integration in promoting hepatocarcinogenesis and the extent to which the intrahepatic HBV reservoir modulates liver disease progression remains poorly understood. We examined the intrahepatic HBV reservoir, the occurrence of HBV DNA integration and its impact on the hepatocyte transcriptome in hepatitis B ‘e’ antigen (HBeAg)-negative chronic hepatitis B (CHB).DesignLiver tissue from 84 HBeAg-negative patients with CHB with low (n=12), moderate (n=25) and high (n=47) serum HBV DNA was analysed. Covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) were evaluated by quantitative PCR, whole exome and transcriptome sequencing was performed by Illumina, and the burden of HBV DNA integrations was evaluated by digital droplet PCR.ResultsPatients with low and moderate serum HBV DNA displayed comparable intrahepatic cccDNA and pgRNA, significantly lower than in patients with high HBV DNA, while hepatitis B core-related antigen correlated strongly with the intrahepatic HBV reservoir, reflecting cccDNA quantity. Whole exome integration was detected in a significant number of patients (55.6%, 14.3% and 25% in high, moderate and low viraemic patients, respectively), at a frequency ranging from 0.5 to 157 integrations/1000 hepatocytes. Hepatitis B surface antigen >5000 IU/mL predicted integration within the exome and these integrations localised in genes involved in hepatocarcinogenesis, regulation of lipid/drug metabolism and antiviral/inflammatory responses. Transcript levels of specific genes, including the proto-oncogene hRAS, were higher in patients with HBV DNA integration, supporting an underlying oncogenic risk in patients with low-level to moderate-level viraemia.ConclusionsHBV DNA integration occurs across all HBeAg-negative patients with CHB, including those with a limited HBV reservoir; localising in genes involved in carcinogenesis and altering the hepatocyte transcriptome.

The Impact of Nucleos(t)ide Analogs on Hepatitis B Virus DNA Integration: Hype or Reality?

“…Accordingly, Hsu et al 8 also demonstrated that the number of expressed viral integrations strongly correlated with levels of serum biomarkers associated with active viral replication and transcription (such as circulating levels of HBV DNA, HBV RNA, hepatitis B surface antigen, and hepatitis B corerelated antigen) in all of the samples at baseline. The findings of this investigation complement those of earlier studies 11,12 and further demonstrate that active HBV replication and HBV integration are linked processes.…”

Innate and Adaptive Immunopathogeneses in Viral Hepatitis; Crucial Determinants of Hepatocellular Carcinoma

“…In line with this, we have also shown evidence of HBV-DNA integration and clonal hepatocyte expansion in these patients considered immune-tolerant, indicating that hepatocarcinogenesis could be underway even in the early stages of HBV [ 157 ]. Moreover, in those subjects deemed as having immune control, evidence of HBV-DNA integration is again present, with the upregulation of genes involved in carcinogenesis [ 158 ]. The link between immunity and HBV DNA integration, however, remains tenuous, but with the use of novel technologies (e.g., tissue CyTOF and spatial transcriptomics) we can come to understand more about this relationship in the future.…”

HBV-Integration Studies in the Clinic: Role in the Natural History of Infection

“…As highlighted previously, the HBeAg-negative phase of chronic HBV infection may be associated with different virologic/clinical outcomes including HBeAg-negative chronic infection (serum HBV DNA < 2 × 10 3 IU/mL and normal ALT) and HBeAg-negative chronic hepatitis (moderate/high serum HBV DNA levels and elevated ALT) characterized by a higher risk of disease progression, the development of cirrhosis, and HCC [15]. A very recent study, analyzing, by whole-exome sequencing, HBV integration in liver tissue from 84 HBeAg-negative patients with low, moderate, and high serum HBV DNA, has shown that HBV integration occurs across all HBeAg-negative patients with CHB, including little-viremic patients with limited hepatic HBV reservoirs, and that by AUROC (Area Under the Receiver Operating Characteristics), HBsAg > 5 × 10 3 IU/mL identified the occurrence of HBV integration in the whole exome with the best diagnostic accuracy (86.5%) [65]. The preservation of HBsAg production independently of HBV replication leads us to hypothesize its role in viral persistence.…”