Hepatitis B Virus-Associated Hepatocellular Carcinoma

Introduction

Liver cancer poses a significant global health challenge, with approximately 905,677 new cases reported in 2020, ranking it seventh among all cancers. In the same year, 830,180 deaths were attributed to liver cancer, making it the third leading cause of cancer-related mortality [1]. The incidence of liver cancer is the fifth most common among men and the ninth most common among women worldwide [2]. The majority of liver cancer cases (83%) were diagnosed in less developed nations. Hepatocellular carcinoma (HCC) constitutes the predominant form of primary liver cancer, accounting for 75-90% of cases, while intrahepatic cholangiocarcinoma (ICC) represents a significant portion of other cancer subtypes.

Figure 1: An overview of the pathogenesis of infection‐induced hepatocellular carcinoma (ref)

Recognized risk factors for HCC include chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV), exposure to dietary aflatoxin, fatty liver disease, alcohol-induced cirrhosis, obesity, smoking, diabetes, and iron overload [3].

Notably, the prevalence of HCC is increasing in the US and Western Europe. Most HCC patients exhibit underlying cirrhosis, primarily stemming from historical infections with HBV or HCV [4].

Hepatitis B virus (HBV) is a viral infection that attacks the liver, causing both acute and chronic diseases. It represents a major global health problem and is the most serious type of viral hepatitis. A highly effective vaccine is available to prevent hepatitis B infection. According to WHO recommendations in 1998, 190 countries worldwide had incorporated hepatitis B vaccination into their national childhood immunization programs by 2022 [5, 9] Even with available treatment options to lower the risk of hepatocellular carcinoma (HCC) in those with chronic HBV infection, a significant number of individuals worldwide remain unaware of their hepatitis B status [7].

Epidemiology of HBV

The incidence of HCC according to geographical area and aetiology (ref)

China bears the world's largest burden of HBV infection and is expected to be a major contributor to the global elimination of hepatitis B by 2030. Over the past three decades, HBV infection in China has transitioned from a highly endemic to an intermediate endemic area. The weighted prevalence of hepatitis B surface antigen (HBsAg) adjusted for people aged 1–59 years declined from 9.8% in 1992 to 7.2% in 2006. Among people aged 1–29 years, the weighted HBsAg prevalence decreased from 10.1% to 5.5% during 1992–2006 and from 5.5% to 2.6% during 2006–2014. In 2019, it is estimated that there are about 70 million HBsAg carriers, indicating a prevalence of 5–6% [8].

A review of published data from 161 countries reported between 1965 and 2013 provides a global estimate of HBsAg prevalence at 3.61%, with the highest rates in Africa (8.83%) and the Western Pacific regions (5.26%). In 2016, an updated estimate indicated that the total global prevalence of hepatitis B virus (HBV) infection increased to 3.9% (95% confidence interval, 3.4 to 4.6%), corresponding to 292 million people worldwide. However, only approximately 29 million individuals (10%) were diagnosed with HBV infection, and of those eligible for treatment, only 4.8 million (5%) had actually been treated [10].

Morphology of HBV

HBV is a double-stranded DNA virus, belonging to the Hepadnaviridae family (genus Orthohepadnavirus), a family of small hepatotropic DNA viruses that cause acute and chronic liver disease. The virus has a partially double-stranded DNA genome, present in the form of relaxed circular DNA (rcDNA [7]), enclosed within an icosahedral nucleocapsid, also known as the core particle. The nucleocapsid is surrounded by an envelope, also called the outer lipoprotein coat [11].

The structure of HBV virus (ref)

The life cycle of HBV

The HBV life cycle is unique in that the genomic DNA (relaxed-circular partially double-stranded DNA: rcDNA) is converted to a molecular template DNA (covalently closed circular DNA: cccDNA) to amplify a viral RNA intermediate, which is then reverse-transcribed back to viral DNA. The highly stable characteristics of cccDNA result in chronic infection and a poor rate of cure.

HBV entry

Hepatitis B virus (HBV) entry into hepatocytes involves several steps. Initially, the virus attaches to the cell surface through non-specific binding to heparan sulfate proteoglycans, like glypican 5, followed by specific and high-affinity interaction with its recepto. Epidermal growth factor receptor (EGFR) is implicated in triggering internalization, interacting directly with NTCP. NTCP oligomerization affects its ability to mediate viral internalization. The virus is internalized via endocytosis, leading to vesicle-mediated fusion of the viral envelope with the cell membrane. The nucleocapsid in the cytoplasm is directed to the nucleus along microtubules, and importin-dependent mechanisms facilitate nuclear entry through the nuclear pore complex.

cccDNA formation/maintenance

In the nucleus, HBV genomic DNA undergoes modification to form covalently closed circular DNA (cccDNA). A portion of incoming HBV DNA integrates into the host genome within a week after infection, contributing to HBs production and potential immune tolerance in HBV-related pathogenesis.

HBV transcription

cccDNA serves as a template for transcription, yielding four viral RNA lengths (3.5, 2.4, 2.1, and 0.7 kb). Transcription is governed by distinct promoters (preS1, preS2, core, and X) and two enhancers (Enhancer I and Enhancer II). Host RNA polymerase II machinery mediates this process.

Schematic representation of the regulation of HBV transcription

Epigenetic modification

cccDNA, existing as a minichromosome, associates with viral proteins and host factors. Histone modifications, particularly high levels of active markers (H3K4me3, H3K27ac, H3K122ac) and low levels of repression markers (H3K27ac, H3K9me2), govern cccDNA transcriptional activity. Histone modification enzymes, including acetyltransferases, deacetylases, methyltransferases, and demethylases, are recruited to regulate viral transcription on the cccDNA minichromosome. Interferon (IFN) targets these modifications, inducing hypoacetylation and recruitment of corepressors, leading to transcriptional silencing both in cell culture and mouse models.

Transcription factors

Cellular transcription factors recruited to cccDNA promoter/enhancer regions govern transcriptional activity. Liver-specific nuclear receptors, including HNF3, HNF4, RXRα, PPARα, and FXR, play key roles in HBV transcription. Additional factors that activate transcription include C/EBP, NF1, SP1, CREB, and LRH-1, while HNF6, PROX1, p53, and ZHX2 are reported suppressors of pgRNA transcription.

Role of HBx in HBV transcription

Multiple biological activities of HBx (ref)

HBx is crucial for HBV replication post-infection and plays multifunctional roles. It associates with the cccDNA minichromosome, influencing the kinetics of H3 acetylation. HBx modulates chromatin-modifying enzymes (p300, HDAC, SIRT1) to control the epigenetic status of cccDNA-associated histones, impacting acetylation, methylation, and phosphorylation. In the absence of HBx, cccDNA undergoes transcriptional silencing with decreased H3 acetylation, H3K4me3, and increased H3K9me2/3, leading to chromatin condensation. HBx expression reverses this silencing, restoring H3K4me3 and disrupting HP1 recruitment on cccDNA, thereby regulating HBV transcription.

Overall view of HBV infection (ref)

Mechanism of HBV mediated Oncogenesis

HBV can promote HCC through various mechanisms. There is a substantial amount of data describing the multiple pathways involved in this process, including the accumulation of genetic damage due to immune-mediated hepatic inflammation, the induction of oxidative stress, and virus-specific mechanisms involving the viral proteins HBx and HBs. This article specifically focuses on the immune and inflammatory factors caused by HBV [16].

In acute HBV infection, NK cells and NKT cells play crucial early roles, followed by a robust response from CD4+ and CD8+ T cells. B cells produce protective antibodies (anti-HBs, anti-HBe, anti-HBc) that clear HBV antigens, preventing reinfection. In chronic infection, stages include the "immune-tolerant" phase with high HBV-DNA replication, the "immune-active" phase causing liver injuries and inflammation, the "immune-inactive" phase with low HBV replication, and the "immune-reactive" phase leading to fibrosis, cirrhosis, and HCC. The late stage involves "immune exhaustion" [17].

HBV infection relates to the magnitude and quantity of anti-viral immune response

Chronic inflammation significantly contributes to cancer development, particularly in the liver, where repeated cycles of inflammation-induced apoptosis and hepatocyte regeneration elevate the risk of hepatocarcinogenesis. T cell dysfunction, cytokine production, and inflammation-induced alterations in signaling pathways are pivotal in hepatocellular carcinoma (HCC) development. During inflammation, the interplay between STAT3 and NF-κB is crucial, regulating communication between cancer and inflammatory cells. NF-κB and STAT3 inhibit apoptosis-based tumor surveillance, fostering immune escape, and promoting tumor angiogenesis and invasiveness. HBV infection induces NF-κB activation, facilitating immune evasion and HCC development. Additionally, STAT3 activation by interleukin-6, the IL-6 cytokine family, and IL-22 further supports HCC development.

Surveillance

High-risk populations for HCC include individuals with chronic HBV/HCV infection, cirrhosis, alcohol abuse, non-alcoholic steatohepatitis, or a family history of HCC, particularly among men over 40. The aMAP score in Chinese guidelines identifies high-risk individuals (aMAP >60, with a 1.6–4% annual HCC incidence). Surveillance in China relies on liver ultrasonography (US) plus alpha-fetoprotein (AFP) every 6 months for cost-effectiveness. In Japan, high-risk groups undergo liver US plus AFP, desgamma-carboxy prothrombin, and AFP-L3 testing every 6 months, with extremely high-risk groups (HBV/HCV-related cirrhosis) checked every 3–4 months. Challenges in China include limited testing items, surveillance frequency, and government support, affecting early HCC diagnosis rates. Integrating community resources into hospitals and exploring new blood-based biomarkers aim to address these issues, especially for AFP-negative patients [18].

Diagnosis

HCC diagnosis in patients with cirrhosis or chronic hepatitis B/C relies on typical imaging features like arterial phase hyper-enhancement (APHE) with a washout appearance. Multiphasic dynamic CT, dynamic MRI, and gadolinium-ethoxybenzyl diethylenetriamine pentaacetic acid-enhanced MRI (EOB-MRI) are used. EOB-MRI and contrast-enhanced ultrasound (CEUS) have been integrated since 2017 to enhance diagnostic sensitivity [18].

Prevention Vaccination

The HBV vaccine is the primary defense against chronic HBV infection and its complications. While global routine infant immunization coverage reached 84% in 2017, additional efforts are required to extend this coverage in various countries and establish national strategies for high-risk populations. Promising results from therapeutic vaccines in murine models and human trials suggest their potential, with ongoing multicenter clinical studies needed for validation. Combining therapeutic vaccines with other treatments, particularly those enhancing T-cell responsiveness, appears to enhance efficacy.

Anti-Hepatitis B Virus Vaccination

The hepatitis B vaccine, introduced in 1982 and widely available since 2000, is highly effective protection against chronic HBV infection and its complications. Three doses provide immunity for at least 20 years. Routine infant immunization is recommended by the WHO, with global coverage estimated at 84% for the third dose in 2017. This widespread vaccination has significantly reduced HBV prevalence and its socioeconomic impact in industrialized countries.

Vaccination for people at higher risk for contracting hepatitis B virus infection

The CDC emphasizes blood screening and anti-HBV vaccination for all prisoners lacking proof of vaccination completion or serological immunity. While vaccination has been recommended for prisoners since 1982, few countries implement it. In Italy, vaccination for prisoners is recommended at months 0, 1, 2, and 6. Homeless individuals follow an accelerated HBV immunization schedule (0, 7, 21 days) with a 12-month booster, showing higher completion and seroconversion rates than traditional programs.

In HIV-infected populations, vaccination against HBV is highly recommended, with double-dose rescue vaccination and regular anti-HBs antibody titers monitoring for optimal immunization [19].

Therapeutics and Treatment of HBV-induced HCC

Acute liver infection marked by necroinflammation involves transaminase level measures indicating innate immunity effectiveness in viral clearance. Studies emphasize cytotoxic T cells (CTLs) as crucial for HBV clearance. Adaptive immunity, through anti-HB antibodies, prevents hepatocyte attachment. Elevated HBsAg and HBeAb levels may hinder antigen-presenting cell function, reducing T cells. Diapedesis of viral proteins to the liver triggers proinflammatory cytokine production, contributing to immunopathogenesis. Untreated long-term HBV infection leads to chronic liver diseases like cirrhosis and HCC.

The replication cycle of HBV and sites of action of NAs (ref)

Diverse treatments target various HBV infection phases. Antivirals inhibit viral replication, preventing chronic hepatitis development based on viral load and HBV DNA presence. HCC treatments include nucleotide analogs (NAs) and PEGylated interferon-alpha (PegIFN-α). Host risk factors for HCC encompass age (>40), alcohol consumption, male gender, immunosuppressive drug use, and HIV infection.

Tenofovir, a Nucleotide Analog (NA), inhibits HBV reverse transcription by outcompeting natural nucleotides. Requiring only two phosphorylation steps, TDF competes for the HBV polymerase active site, reducing HBV DNA copies in LAM-resistant cases. Compared to entecavir (ETV), TDF shows superior efficacy, with a 16% lower mortality rate and longer recurrence-free survival.

The major signaling pathway through which IFN produces its inhibitory effect on HBV (ref)

PegIFN and NAs are key HBV treatments, each with distinct pros and cons. PegIFN offers finite treatment duration, high seroconversion rates, and resistance avoidance. However, it can increase morbidity and mortality. NAs have fewer side effects, some are safe in pregnancy, and drugs like TDF and ETV show minimal resistance. NAs' drawback lies in lower seroconversion rates, leading to prolonged treatment duration and unclear viral clearance timelines [20].

Conclusions

Persistent HBV infection and immune-mediated damage pose significant risk factors for hepatocellular carcinoma (HCC) development. Multiple genetic and molecular pathways contribute to HBV-related HCC, with ongoing research. Current HBV treatments reduce but don't eliminate HCC risk. HDV coinfection heightens HCC risk, but new treatments show promise. Etiologic therapy, such as antivirals for HBV, significantly improves outcomes in liver cirrhosis. Maintaining liver function is crucial for reducing HCC risk and enhancing overall outcomes. Hepatologists play a central role in managing HCC, while certain cases benefit from a multidisciplinary approach involving surgeons, infectivologists, radiologists, and oncologists.

Click to view enlarged poster.

    

Click to read full paper.

Impact Statement

My project focuses on hepatitis B virus-associated hepatocellular carcinoma, covering its epidemiology, morphology, clinical attributes, and future perspectives. etc. Throughout the program, I learned fundamental knowledge about the immune system and cancer, which played a great role later as I dived deeper into the topics. Weekly meetings with my mentor have been instrumental in keeping me on track and addressing problems during the process. I value the one-on-one assistance from my mentor, which has promoted my efficiency. Moreover, the poster section drove my creativity and learned a new way of making a review paper clearer. During the program, I learned how to read and dig into literature to look for information, and properly cite them. In terms of my project, I acquired a more general view of HBV, a virus that could be easily spread and nearly impossible to cure. This experience fosters my passion for diving deeper into life science and research.

References

1. Sung, Hyuna et al. “Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries.” CA: a cancer journal for clinicians vol. 71,3 (2021): 209-249. doi:10.3322/caac.21660

2. Liver cancer statistics, https://www.wcrf.org/cancer-trends/liver-cancer-statistics/

3. Wong, M., Jiang, J., Goggins, W. et al. International incidence and mortality trends of liver cancer: a global profile. Sci Rep 7, 45846 (2017). https://doi.org/10.1038/srep45846

4. De Flora, S.C.E.; Bonanni, P.; Ferro, A.; Vitale, F. Vaccines and Cancer Prevention/Screening Working Groups of the Italian Society of Hygiene, Preventive Medicine and Public Health (SItI). Incidence of infection-associated cancers in Italy and prevention strategies. Epidemiol. Prev. 2015, 39, 14–20.

5. Pattyn J, Hendrickx G, Vorsters A, Van Damme P. Hepatitis B Vaccines. J Infect Dis. 2021 Sep 30;224(12 Suppl 2):S343-S351. doi: 10.1093/infdis/jiaa668. PMID: 34590138; PMCID: PMC8482019.

6. Rizzo, G.E.M.; Cabibbo, G.; Craxì, A. Hepatitis B Virus-Associated Hepatocellular Carcinoma. Viruses 2022, 14, 986. https://doi.org/10.3390/v14050986

7. Changing epidemiology of liver disease in Asia: Dual infection of HBV and HCV https://onlinelibrary.wiley.com/doi/full/10.1111/liv.15040

8. Nguyen, Mindie H., et al. “Hepatitis B virus: Advances in prevention, diagnosis, and therapy.” Clinical Microbiology Reviews, vol. 33, no. 2, 2020, https://doi.org/10.1128/cmr.00046-19 .

9. Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking https://journals.asm.org/doi/full/10.1128/jvi.02741-06

10. Nakopoulou, L et al. “Expression of HBsAg and HBcAg in liver tissue: correlation with disease activity.” Histology and histopathology vol. 7,3 (1992): 493-9.

11. Senko Tsukuda, Koichi Watashi, Hepatitis B virus biology and life cycle, Antiviral Research, Volume 182, 2020, 104925, ISSN 0166-3542, https://doi.org/10.1016/j.antiviral.2020.104925 .

12. Neuveut, Christine & Wei, Yu & Buendia, Marie-Annick. (2010). Neuveut C, Wei Y, Buendia MAMechanisms of HBV-related hepatocarcinogenesis. J Hepatol 52: 594-604. Journal of hepatology. 52. 594-604. 10.1016/j.jhep.2009.10.033.

13. Tarocchi M, Polvani S, Marroncini G, Galli A. Molecular mechanism of hepatitis B virus-induced hepatocarcinogenesis. World J Gastroenterol. 2014 Sep 7;20(33):11630-40. doi: 10.3748/wjg.v20.i33.11630. PMID: 25206269; PMCID: PMC4155355.

14. Chen Y, Tian Z. HBV-Induced Immune Imbalance in the Development of HCC. Front Immunol. 2019 Aug 27;10:2048. doi: 10.3389/fimmu.2019.02048. PMID: 31507621; PMCID: PMC6718466.

15. Xie D, Shi J, Zhou J, Fan J, Gao Q. Clinical practice guidelines and real-life practice in hepatocellular carcinoma: A Chinese perspective. Clin Mol Hepatol. 2023 Apr;29(2):206-216. doi: 10.3350/cmh.2022.0402. Epub 2022 Dec 22. PMID: 36545708; PMCID: PMC10121293.

16. Stasi, Cristina, et al. “Hepatitis B vaccination and immunotherapies: An update.” Clinical and Experimental Vaccine Research, vol. 9, no. 1, 2020, p. 1, https://doi.org/10.7774/cevr.2020.9.1.1 .

17. Fung, James Yan‐Yue et al. “Nucleoside/nucleotide analogues in the treatment of chronic hepatitis B.” The Journal of antimicrobial chemotherapy 66 12 (2011): 2715-25 .

18. Li, Qirong, et al. “Interferon and interferon-stimulated genes in HBV treatment.” Frontiers in Immunology, vol. 13, 2022, https://doi.org/10.3389/fimmu.2022.1034968 .

_By: Weiran Yu_

The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of Elio Academy.