Investigating HBV DNA integration and its role in genomic instability through HBx expression

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Hi everyone! My name is Dong and I’m a PhD student under Thomas Tu. I’ve been working on understanding the consequences of HBV X protein (HBx) expression from HBV DNA integration, and its role in genomic instability.

More than 90% of HBV induced Hepatocellular Carcinomas (HCCs) contain multiple HBV integrations in contrast to 1 in 10,000 of the surrounding hepatocytes. This suggests a strong association between virus integration and HCC development, but we don’t know exactly how integrations cause cancer over decades of infection.

I’m interested in how HBx (the smallest HBV protein) is involved. It is usually really important for virus replication, but it can also affect DNA stability in the host cell.

When HBV DNA integrates into the host genome (as opposed to making cccDNA), bits of the virus DNA get chopped off. This means the integrated HBV cannot produce infectious virus and represent a replicative dead-end. It also means some bits of HBx are cut off, so it may not work or it works differently.

My job is to see if the HBx derived from integrations is functional and could contribute to cancer.

We have developed novel molecular tools to isolate, manipulate, and characterize cells containing HBV integrations. We generated over 100 cell clones with de novo integrations.

Under our model, we found integration junctions found that HBx regions required for DNA destabilisation were present in over 70% of integrations. We found that HBx expression from the integrations averaged ~4-fold lower than expression from cccDNA. We also found that at least some of the integrations were functional and that cells with existing integrations were 2-12 fold more susceptible to integrations after a new HBV infection (consistent with DNA instability).

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Figure 1: HBV integration promoting a self-amplifying process through HBx expression

So now we’re developing a new model of understanding liver cancer: we think that genomic instability is driven by virus integrations and that this instability leads to more integrations, promoting a self-amplifying process (Figure 1). The resultant exponential increasing genomic instability then fuels accelerated acquisition of cancer driver mutations.

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Cool project @Dodo!

John.

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How can we prevent this hbv integration from causing cancer, this is very very scary

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Hi@chigoziekingsley5454, thank you for the question.

Although it is difficult to clear integrations that are established early, preventing new infections (e.g., by reducing viral load or entry inhibition) at the appropriate points during chronic infection could limit new integrations. These therapies could also act to limit inflammation, preventing ongoing clonal expansion and amplification of existing integrations.

The emerging technologies like CRISPR(mutating HBx) and RNA interference (transcriptionally silencing of HBx) are being explored for specifically target HBV DNA integration. Studies have shown effects on HBx-dependent replication and to some extent DNA damage. However, it is still unclear whether these could induce a reduction of HBV-associated liver cancer, independent of their antiviral effects.

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Thanks for the response sir, another question people who are inactive and always have there viral loads below 2000ul/ml, are they still at risk of developing liver cancer

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Hi Kingsley,

Inactive carriers with low viral DNA levels are generally at lower risk of liver cancer compared to active carriers. However, the risk of liver cancer is not completely absent.

A long-term follow-up study has shown that a small percentage of inactive carriers can develop liver cancer over time; the inactive carriers of HBV (seronegative for HBeAg, serum levels of HBV DNA <10,000 copies/mL, and normal liver enzymes) had 5-fold greater risk for HCC than controls (HBsAg-negative) (Chen JD, et al. Carriers of inactive hepatitis B virus are still at risk for hepatocellular carcinoma and liver-related death. 2010).

The risk of liver cancer in HBV infection is influenced by several known factors including viral load, age, family history of liver cancer and lifestyle. There are also potentially unknown factors, and research is ongoing to better understand them and develop strategies to reduce the risk. As I previously introduced my research, we suspect that integrations may play some role in promoting cancer.

Dong

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Congratulations @Dodo for placing in the top 8 of the 2024 Research Showcase :tada:.

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Also, don’t forget to ask our researchers any questions on their thread. Authors of the top 4 questions are awarded a $100 USD gift card each.

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Hi Dong, this is very interesting information and I thank you for researching and also explaining in a way that a lay person can attempt to understand. You mentioned that people with HBV whose viral load was below 10,000 copies still have a 5 fold greater risk of developing HCC than the controls. I was wondering if you have statistics for what those numbers are for the controls, that is what is the lifetime risk of developing HCC for someone who doesn’t have HCC? Thanks, Karin

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Hi @Karin, thank you for the question.

The data was cited from a sub-cohort analysis of REVEAL-HBV Study.(1) It’s reported HCC risk persists among individuals with lower levels of HBV-DNA, In REVEAL, even those with undetectable DNA(<300 copies/mL) had an HCC incidence of 108/100,000 person-years, underscoring the point that while HCC risk is reduced in the lower HBV-DNA tiers.(2) Please see the information below you may find helpful.

Cohorts:
Carriers of inactive hepatitis B virus (n = 1932): Participants were seropositive for HBsAg and seronegative for HBeAg and anti-HCV, had a normal serum ALT level (<45 U/L) and a serum HBV DNA level <10,000 copies/mL (~2000 IU/mL) at study entry ; did not have cirrhosis or HCC.

Controls (n = 18,137): Participants who were seronegative for HBsAg and anti-HCV, and matched by their clinical features of liver, ie, a normal serum ALT level without liver cirrhosis or HCC at study entry.

Results:
Numbers of newly developed HCC were 16 for the inactive HBV carrier group and 35 for the control group. The incidence rate of HCC per 100,000 person-years was 64 for the inactive HBV carriers and 15 for the controls, showing an incidence rate ratio of 4.4 with a 95% CI of 3.2−5.9. Among inactive HBV carriers, there were 6 incident cases of HCC for those with undetectable serum HBV DNA levels (n = 837), resulting in an incidence rate of 56 per 100,000 person-years. For the inactive HBV carriers with serum HBV DNA levels of 300−10,000 copies/mL (n = 1095), 10 developed HCC with an incidence rate of 71 per 100,000 person-years (crude incidence rate ratio = 1.3; 95% CI: 0.8−2.1 compared with inactive HBV carriers with undetectable serum HBV DNA level).

The multivariate-adjusted hazard ratios for carriers of inactive HBV, compared to controls, were 4.6 (95% confidence interval: 2.5-8.3) for HCC and 2.1 (95% confidence interval: 1.1-4.1) for liver-related death. Older age and alcohol drinking habits were independent predictors of risk for carriers of inactive HBV to develop hepatocellular carcinoma.(The multivariate-adjusted hazard ratios for risk predictors were derived from Cox regression models.)

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[Table 2] shows the HRa with 95% CI for risk predictors of HCC included in the Cox regression models. The risk of developing hepatocellular carcinoma was much higher in inactive HBV carriers than in controls (HRa = 4.6; 95% CI: 2.5−8.3). The other significant risk predictors included older age, a high-normal baseline serum ALT level, and an alcohol drinking habit. Among inactive HBV carriers, older age and an alcohol drinking habit were the 2 important predictors that remained significant. Additionally, the risk of hepatocellular carcinoma was higher in those with baseline serum HBV DNA levels of 300−10,000 copies/mL than those with undetectable serum HBV DNA levels, but this difference was not statistically significant (HRa = 1.6; 95% CI: 0.6−4.5). For inactive HBV carriers with undetectable baseline serum HBV DNA, an alcohol drinking habit was the only significant risk predictor in the analysis for hepatocellular carcinoma (HRa = 6.9; 95% CI: 1.1−41.9).

Some information about REVEAL-HBV Study: A community-based study conducted in Taiwan, with mean follow-up of 13.1 years. A total of 23,820 of 89,293 invited residents living in 7 townships in Taiwan participated in the prospective cohort study and provided informed consents in 1991−1992. Among the participants, 18,541 were seronegative for both HBsAg and anti-HCV. A total of 3931 participants who were HBsAg-seropositive, anti−HCV-seronegative, and free from hepatocellular carcinoma at study entry were invited for follow-up every 6 to 12 months with abdominal ultrasonography and serological tests until June 30, 2004.(2)

Reference:

  1. Chen JD, Yang HI, Iloeje UH, You SL, Lu SN, Wang LY, Su J, Sun CA, Liaw YF, Chen CJ; Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer in HBV (REVEAL-HBV) Study Group. Carriers of inactive hepatitis B virus are still at risk for hepatocellular carcinoma and liver-related death. Gastroenterology. 2010 May;138(5):1747-54.

  2. Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular carcinomaacross a biological gradient of serum hepatitis B virus DNA level.JAMA. 2006;295(1):65-73

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Hi and thank you so much for your prompt and detailed response to my question. This is very helpful and interesting.

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Hello all,

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Ask away.

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Hi everyone,

@Dodo has just published a review article in relation to their research. Viruses | Free Full-Text | Hepatitis B Viral Protein HBx: Roles in Viral Replication and Hepatocarcinogenesis (mdpi.com)
It’s an interesting read on HBV and the potential therapeutics to inhibit it.

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