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. 2022 Mar;71(3):616-626.
doi: 10.1136/gutjnl-2020-323153. Epub 2021 Feb 9.

Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma

Camille Péneau  1   2 , Sandrine Imbeaud  1   2 , Tiziana La Bella  1   2 , Theo Z Hirsch  1   2 , Stefano Caruso  1   2 , Julien Calderaro  3 , Valerie Paradis  4   5 , Jean-Frederic Blanc  6   7   8 , Eric Letouzé  1   2 , Jean-Charles Nault  1   2   9 , Giuliana Amaddeo  10 , Jessica Zucman-Rossi  11   2   12
Affiliations

Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma

Camille Péneau et al. Gut. 2022 Mar.

Abstract

Objective: Infection by HBV is the main risk factor for hepatocellular carcinoma (HCC) worldwide. HBV directly drives carcinogenesis through integrations in the human genome. This study aimed to precisely characterise HBV integrations, in relation with viral and host genomics and clinical features.

Design: A novel pipeline was set up to perform viral capture on tumours and non-tumour liver tissues from a French cohort of 177 patients mainly of European and African origins. Clonality of each integration event was determined with the localisation, orientation and content of the integrated sequence. In three selected tumours, complex integrations were reconstructed using long-read sequencing or Bionano whole genome mapping.

Results: Replicating HBV DNA was more frequently detected in non-tumour tissues and associated with a higher number of non-clonal integrations. In HCC, clonal selection of HBV integrations was related to two different mechanisms involved in carcinogenesis. First, integration of viral enhancer nearby a cancer-driver gene may lead to a strong overexpression of oncogenes. Second, we identified frequent chromosome rearrangements at HBV integration sites leading to cancer-driver genes (TERT, TP53, MYC) alterations at distance. Moreover, HBV integrations have direct clinical implications as HCC with a high number of insertions develop in young patients and have a poor prognosis.

Conclusion: Deep characterisation of HBV integrations in liver tissues highlights new HBV-associated driver mechanisms involved in hepatocarcinogenesis. HBV integrations have multiple direct oncogenic consequences that remain an important challenge for the follow-up of HBV-infected patients.

Keywords: cancer genetics; hepatitis B; hepatocellular carcinoma.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
HBV integrations in non-tumour tissues are associated with viral replication and more frequent in large highly expressed genes. (A) Repartition of clonality between all HBV integration events detected in viral capture (n=8809). (B) Coding genes with or without HBV integrations according to the gene length and the median gene expression in non-tumour liver tissues. Genes with recurrent clonal or subclonal HBV integrations are annotated. (C) Number of HBV integrations identified in non-tumour samples (n=142) according to the presence of episomal HBV DNA and replicative HBV DNA (Jonckheere’s trend test). (D) Correlations between HBV copy number/cell in 170 non-tumour liver tissues assessed by viral capture and clinical or molecular features. Positivity for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), HBV DNA (by PCR) in the patients’ serum and duration of antiviral treatments were obtained from clinical data. Wilcoxon signed-rank, Kruskal-Wallis or Pearson’s correlation statistical tests were applied with respect to the type of variable. P values were adjusted for multiple testing using the Benjamini-Hochberg method (false discovery rate). AFB1, aflatoxin-B1; BCP, basal core promoter; FPKM, fragments per kilobase of exons per million reads; PC, PreCore; RT, reverse transcriptase.
Figure 2
Figure 2
Tumours and non-tumour tissues have different HBV integration profiles and structures of HBV sequences. (A) Pan-genomic view of genomic locations of all HBV integration breakpoints in tumours (up) or non-tumour tissues (down) in 347 HBV-positive samples (non-tumour liver, n=170, and tumour samples, n=177). A line corresponds to a 1M-bin region. (B) Proportion of non-tumour tissues and tumours harbouring only unique integrations, at least one subclonal integration or at least one clonal integration (χ2 test). (C) Correlation between the HBV copy number per cell and the number of HBV integration breakpoints (Pearson’s correlation). (D) Number of HBV integration breakpoints per sample (Wilcoxon signed-rank test). (E) Localisation of HBV integration breakpoints along the HBV genome in tumours and non-tumour samples according to the orientation of the integrated sequence. (F) Number of structural variants in HBV genome per sample (Wilcoxon signed-rank test). (G) Proportion of non-tumour and tumour samples containing replicative HBV DNA, episomal non-replicative HBV DNA or no HBV episomal form (χ2 test). centr, centromeric; SV, structural variant; telo, telomeric.
Figure 3
Figure 3
HBV integrations induce chromosomal rearrangements and distant driver oncogenic alterations. (A) Proportion of HBV integration breakpoints (n=1436) associated with a copy number alteration (CNA) in 121 HBV-positive tumours (χ2 test). (B) Pan-genomic view of the number of HBV integration breakpoints according to their association with a CNA, split by chromosome arms. The three genomic regions containing the higher number of HBV integrations associated with CNA are annotated. Fisher’s exact tests were performed to compare the number of integrations with or without CNA and p-values were adjusted for multiple testing. (C) Translocation-like event in tumour #1733T: HBV integration is associated with a focal gain on chr5p and a large deletion on chr17p. (D) Duplication-inverted-like event in tumour #1597T, reconstructed with long-read sequencing: HBV integration is associated with a focal amplification including TERT.
Figure 4
Figure 4
HBV integrations in the TERT promoter induce a strong activation of TERT promoting hepatocellular carcinoma (HCC) development. (A) HBV copy number/cell in 177 tumours from the capture series according to their number of clonal HBV integration breakpoints (Jonckheere’s trend test). (B) Integrated HBV sequences located in the TERT promoter in 48 tumours harbouring clonal HBV integrations at this locus. The position of the breakpoints, of the viral enhancer regions and the orientation of the integrated sequences are annotated along the HBV genome. mRNA expression (-ddCT) from qRT-PCR data are represented above. (C) The impact of HBV integration in the TERT promoter was evaluated using promoter luciferase assays in Huh7 liver cell lines. Constructs of TERT promoter containing different HBV-integrated sequences with or without scrambled enhancer regions were compared with the WT promoter and to the TERT promoter with mutations at the −124 or −146 hotspots. Error bars correspond to SD of three independent transfections for each plasmid (Student’s t-test). (D) Molecular profile of 121 HBV-positive tumours with alterations in 14 HCC-associated genes (HBV integration, SV/CNA or mutation), according to clinical and other molecular features. mRNA expression of TERT, EPCAM and MKI67 were obtained from RNA-seq. AFB1, aflatoxin B1; Enh, enhancer; CNA, copy number alteration; SV, structural variant; WT, wild type; ns, not significant.
Figure 5
Figure 5
Integrative analysis reveals that a high number of HBV integrations is associated with poor survival of patients. (A) Mosaic plot: association of HBV infection with patient, tumour and viral characteristics in a series of 265 HCC (next-generation sequencing series) and association of geographic origin, aflatoxin B1 exposure, HDV infection, hepatitis B surface antigen (HBsAg) negativity or number of HBV integrations with patient, tumour and viral characteristics in a series of 177 HBV-positive HCC (capture series). Blue and red circles indicate negative and positive associations, respectively. Colour intensities represent different levels of statistical significance. Statistical analysis was performed using χ2 test, Wilcoxon signed-rank test or Pearson’s correlation with respect to the type of variable. (B) Kaplan-Meier curves for 5-year overall survival from 119 patients after curative R0 resection. (C) Multivariate Cox regression model for overall survival analysis. AA, aristolochic acid. *p<0.05; **p<0.01.
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References

    1. World Health Organization . Global hepatitis report, 2017; 2017.
    1. Thomas DL. Global elimination of chronic hepatitis. N Engl J Med 2019;380:2041–50. 10.1056/NEJMra1810477 - DOI - PubMed
    1. Bray F, Ferlay J, Soerjomataram I, et al. . Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394–424. 10.3322/caac.21492 - DOI - PubMed
    1. Kew MC. Hepatocellular carcinoma: epidemiology and risk factors. J Hepatocell Carcinoma 2014;1:115. 10.2147/JHC.S44381 - DOI - PMC - PubMed
    1. Papatheodoridis GV, Chan HL-Y, Hansen BE, et al. . Risk of hepatocellular carcinoma in chronic hepatitis B: assessment and modification with current antiviral therapy. J Hepatol 2015;62:956–67. 10.1016/j.jhep.2015.01.002 - DOI - PubMed

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