Hepatitis B Virus DNA Integration Drives Carcinogenesis and Provides a New Biomarker for HBV-related HCC

Abstract

Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection. However, HBV DNA is present in the genome of approximately 90% of HBV-related HCCs and is the most common somatic mutation. Whole genome sequencing of liver tissues from chronic hepatitis B patients showed integration occurring at random positions in human chromosomes; however, in the genomes of HBV-related HCC patients, there are integration hotspots. Both the enrichment of the HBV-integration proportion in HCC and the emergence of integration hotspots suggested a strong positive selection of HBV-integrated hepatocytes to progress to HCC. The activation of HBV integration hotspot genes, such as telomerase (TERT) or histone methyltransferase (MLL4/KMT2B), resembles insertional mutagenesis by oncogenic animal retroviruses. These candidate oncogenic genes might shed new light on HBV-related HCC biology and become targets for new cancer therapies. Finally, the HBV integrations in individual HCC contain unique sequences at the junctions, such as virus-host chimera DNA (vh-DNA) presumably being a signature molecule for individual HCC. HBV integration may thus provide a new cell-free tumor DNA biomarker to monitor residual HCC after curative therapies or to track the development of de novo HCC. Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection. However, HBV DNA is present in the genome of approximately 90% of HBV-related HCCs and is the most common somatic mutation. Whole genome sequencing of liver tissues from chronic hepatitis B patients showed integration occurring at random positions in human chromosomes; however, in the genomes of HBV-related HCC patients, there are integration hotspots. Both the enrichment of the HBV-integration proportion in HCC and the emergence of integration hotspots suggested a strong positive selection of HBV-integrated hepatocytes to progress to HCC. The activation of HBV integration hotspot genes, such as telomerase (TERT) or histone methyltransferase (MLL4/KMT2B), resembles insertional mutagenesis by oncogenic animal retroviruses. These candidate oncogenic genes might shed new light on HBV-related HCC biology and become targets for new cancer therapies. Finally, the HBV integrations in individual HCC contain unique sequences at the junctions, such as virus-host chimera DNA (vh-DNA) presumably being a signature molecule for individual HCC. HBV integration may thus provide a new cell-free tumor DNA biomarker to monitor residual HCC after curative therapies or to track the development of de novo HCC. SummaryMechanistic studies of HBV integration-induced insertional mutagenesis help develop precise HCC therapy. HBV integration provides a unique junctional DNA biomarker valuable for monitoring residual HCC after therapy or the development of de novo recurrence. Mechanistic studies of HBV integration-induced insertional mutagenesis help develop precise HCC therapy. HBV integration provides a unique junctional DNA biomarker valuable for monitoring residual HCC after therapy or the development of de novo recurrence. Hepatocellular carcinoma (HCC) ranks 6th among the 10 leading human cancers worldwide.1Sung H. Ferlay J. Siegel R.L. et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin. 2021; 71: 209-249Crossref PubMed Scopus (23614) Google Scholar This disease burden is especially heavy in Asia and Africa, largely because of the high prevalence of chronic hepatitis B virus (HBV) infection (CHB), ranging from 3% to 15% among the local populations. Overall, CHB is present in 50%–60% of all HCC cases worldwide.2de Martel C. Georges D. Bray F. et al.Global burden of cancer attributable to infections in 2018: a worldwide incidence analysis.Lancet Glob Health. 2020; 8: e180-e190Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar To control CHB, universal HBV vaccination programs have been implemented in most countries since 1986, resulting in a sharp decline in HBV carriers among children and young adults from 10% to 0.5%, as in the example of Taiwan.3Ni Y.H. Huang L.M. Chang M.H. et al.Two decades of universal hepatitis B vaccination in taiwan: impact and implication for future strategies.Gastroenterology. 2007; 132: 1287-1293Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar,4Ni Y.H. Chang M.H. Huang L.M. et al.Hepatitis B virus infection in children and adolescents in a hyperendemic area: 15 years after mass hepatitis B vaccination.Ann Intern Med. 2001; 135: 796-800Crossref PubMed Scopus (308) Google Scholar This is accompanied by a reduction in HCC in children and teenagers, confirming the carcinogenic effect of CHB.5Chang M.H. Chen C.J. Lai M.S. et al.Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children: Taiwan Childhood Hepatoma Study Group.N Engl J Med. 1997; 336: 1855-1859Crossref PubMed Scopus (1586) Google Scholar For CHB patients, active antiviral therapies such as nucleos(t)ide analog (NA) reverse transcriptase inhibitors have been widely adopted since 2000. Effective control of viral replication results in much less hepatitis activity, improving the clinical outcomes of CHB and reducing the death rate from cirrhosis, hepatic failure, and HCC to 50% of historic levels. These combined efforts have led to a significant decline in HBV-related end-stage liver diseases and death, as witnessed in the last decade.6Choi J. Han S. Kim N. et al.Increasing burden of liver cancer despite extensive use of antiviral agents in a hepatitis B virus-endemic population.Hepatology. 2017; 66: 1454-1463Crossref PubMed Scopus (73) Google Scholar Despite this impressive progress in controlling liver cirrhosis and hepatic failure resulting from the use of long-term NA therapies, the absolute cases of HBV-related HCC were actually only modestly reduced, largely because of an accumulation of surviving CHB patients with advanced liver fibrosis or cirrhosis who still carry a high risk of HCC. Therefore, the major challenge for CHB patients in the NA therapy era is to decrease development of HCC. CHB has been shown to increase HCC risk by approximately 10- to 30-fold compared with the risk in the non-HBV population.7Huang Y.T. Jen C.L. Yang H.I. et al.Lifetime risk and sex difference of hepatocellular carcinoma among patients with chronic hepatitis B and C.J Clin Oncol. 2011; 29: 3643-3650Crossref PubMed Scopus (155) Google Scholar The lifelong risk for HCC in CHB patients is estimated to be approximately 30% in male carriers and 10% in female carriers. Both direct and indirect mechanisms are generally assumed to contribute to carcinogenesis. Chronic viral hepatitis creates a microenvironment in the liver for cycles of hepatocyte death and regeneration, favoring the growth of hepatocytes with proliferation advantages, eventually leading to cancer. The direct mechanisms include transforming viral proteins, such as the well-known HBx or deleted Pre-S,8Choi M.S. Kim D.Y. Lee D.H. et al.Clinical significance of pre-S mutations in patients with genotype C hepatitis B virus infection.J Viral Hepat. 2007; 14: 161-168Crossref PubMed Scopus (66) Google Scholar, 9Lin C.L. Liu C.H. Chen W. et al.Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma.J Gastroenterol Hepatol. 2007; 22: 1098-1103Crossref PubMed Scopus (76) Google Scholar, 10Kim C.M. Koike K. Saito I. et al.HBx gene of hepatitis B virus induces liver cancer in transgenic mice.Nature. 1991; 351: 317-320Crossref PubMed Scopus (1049) Google Scholar and even more important is HBV DNA integration in the genomes of HBV-related HCC cells. This review will focus on the importance of HBV DNA integration as the driving force in HCC carcinogenesis, with implications for targeted therapies and even as a novel liquid biopsy cancer biomarker. Similar to most human cancers, HCC is considered a disease caused by accumulated somatic mutations in the chromosomes of hepatocytes. Comprehensive whole genome sequencing (WGS) of HCC identified some well-known somatic mutations of host genes. Among them, the TERT promoter -124G>A or -146G>A point mutations were the most frequently identified, which are detected in up to 60% of non-HBV HCCs and approximately 30% of HBV-related HCCs; others are TP53, CTNNB1, AXIN, and ARID1/2.11Totoki Y. K. et of hepatocellular carcinoma PubMed Scopus Google J. S. et of hepatocellular carcinoma are to gene mutations and Hepatol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar However, common somatic mutations in the or advanced of HCC. the of cirrhosis, the of sequencing common somatic mutations even TERT promoter point at positions et mutations and in and human PubMed Scopus (155) Google Scholar Therefore, the common mutations in or advanced HCC are more the to cancer development in with the presumably occurring and in HCC which may for the hepatocytes to have been identified or However, the most common in HBV-related HCC is HBV DNA integration This in the the of HCC chromosomes with an HBV C. 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