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Whole Exome HBV DNA Integration Is Independent of the Intrahepatic HBV Reservoir in Hbeag-Negative Chronic Hepatitis B

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Whole Exome HBV DNA Integration Is Independent of the Intrahepatic HBV Reservoir in Hbeag-Negative Chronic Hepatitis B Hepatology Original research Gut: first published as 10.1136/gutjnl-2020-323300 on 21 December 2020. Downloaded from Whole exome HBV DNA integration is independent of the intrahepatic HBV reservoir in HBeAg- negative chronic hepatitis B Valentina Svicher,1 Romina Salpini,1 Lorenzo Piermatteo,1 Luca Carioti,1 Arianna Battisti,1,2 Luna Colagrossi,1,3 Rossana Scutari,1 Matteo Surdo,4 Valeria Cacciafesta,4 Andrea Nuccitelli,4 Navjyot Hansi,2 Francesca Ceccherini Silberstein,1 Carlo Federico Perno,5 Upkar S Gill,2 Patrick T F Kennedy 2 ► Additional material is ABSTRACT published online only. To view, Objective The involvement of HBV DNA integration in Significance of this study please visit the journal online promoting hepatocarcinogenesis and the extent to which (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2020- 323300). the intrahepatic HBV reservoir modulates liver disease progression remains poorly understood. We examined ► Hepatitis B ‘e’ antigen (HBeAg)- negative phase For numbered affiliations see of HBV infection is associated with a wide end of article. the intrahepatic HBV reservoir, the occurrence of HBV DNA integration and its impact on the hepatocyte disease spectrum, ranging from quiescent low viraemic disease to chronic HBeAg- negative Correspondence to transcriptome in hepatitis B ’e’ antigen (HBeAg)-negative Dr Patrick T F Kennedy, Barts chronic hepatitis B (CHB). hepatitis, with a high risk of evolution to Liver Centre, Immunobiology, Design Liver tissue from 84 HBeAg-negative patients cirrhosis and hepatocellular carcinoma. Blizard Institute, Barts and The with CHB with low (n=12), moderate (n=25) and high ► Hepatitis B surface antigen (HBsAg) and London School of Medicine (n=47) serum HBV DNA was analysed. Covalently closed hepatitis B core- related antigen (HBcrAg) & Dentistry, Queen Mary are increasingly used as surrogates of the University of London, Newark circular DNA (cccDNA), pregenomic RNA (pgRNA) Street, London, E1 2AT, UK; were evaluated by quantitative PCR, whole exome and intrahepatic HBV reservoir. p. kennedy@ qmul. ac. uk transcriptome sequencing was performed by Illumina, ► HBV DNA integration is believed to play a key and the burden of HBV DNA integrations was evaluated role in hepatocarcinogenesis. VS and RS contributed equally. by digital droplet PCR. What are the new findings? http://gut.bmj.com/ Results Patients with low and moderate serum HBV Received 11 October 2020 ► HBV DNA integration in the whole exome is Revised 2 November 2020 DNA displayed comparable intrahepatic cccDNA and Accepted 16 November 2020 detected in a significant proportion of HBeAg- pgRNA, significantly lower than in patients with high negative patients, including low viraemic HBV DNA, while hepatitis B core-related antigen patients with a limited HBV reservoir. correlated strongly with the intrahepatic HBV reservoir, ► HBV DNA integrations occur in regions crucial reflecting cccDNA quantity. Whole exome integration for human gene expression and involve genes on September 29, 2021 by guest. Protected copyright. was detected in a significant number of patients (55.6%, regulating cell proliferation, carcinogenesis in 14.3% and 25% in high, moderate and low viraemic addition to antiviral immunity and hepatocyte patients, respectively), at a frequency ranging from 0.5 to metabolism. 157 integrations/1000 hepatocytes. Hepatitis B surface ► High levels of HBsAg (>5000 IU/mL) can predict antigen >5000 IU/mL predicted integration within HBV DNA integration events in HBeAg- negative the exome and these integrations localised in genes patients. involved in hepatocarcinogenesis, regulation of lipid/ drug metabolism and antiviral/inflammatory responses. How might it impact on clinical practice in the Transcript levels of specific genes, including the proto- foreseeable future? oncogene hRAS, were higher in patients with HBV DNA ► The demonstration of HBV DNA integration in integration, supporting an underlying oncogenic risk in patients with chronic hepatitis B perceived as patients with low-level to moderate-level viraemia. low risk mandates a re- evaluation of treatment © Author(s) (or their Conclusions HBV DNA integration occurs across candidacy. employer(s)) 2020. Re- use permitted under CC BY- NC. No all HBeAg-negative patients with CHB, including ► Future studies will have to dissect the role of commercial re- use. See rights those with a limited HBV reservoir; localising in genes HBV DNA integration in hepatocarcinogenesis and permissions. Published involved in carcinogenesis and altering the hepatocyte and how this can be mitigated against. by BMJ. transcriptome. ► Novel HBV therapies should target both To cite: Svicher V, Salpini R, integrated HBV DNA in addition to covalently Piermatteo L, et al. Gut Epub closed circular DNA to enhance treatment ahead of print: [please outcomes and reduce hepatocellular carcinoma include Day Month Year]. INTRODUCTION risk. doi:10.1136/ Chronic hepatitis B (CHB) virus infection is a gutjnl-2020-323300 dynamic disease reflecting the balance between the Svicher V, et al. Gut 2020;0:1–12. doi:10.1136/gutjnl-2020-323300 1 Hepatology virus itself and the host immune response. In CHB, the hepa- MATERIALS AND METHODS Gut: first published as 10.1136/gutjnl-2020-323300 on 21 December 2020. Downloaded from titis B ‘e’ antigen (HBeAg)- negative phase is associated with a Study population spectrum of clinical outcomes spanning from HBeAg- negative Eighty- four HBeAg- negative patients with CHB were recruited chronic infection (serum HBV DNA <2000 IU/mL and normal from Viral Hepatitis clinics at The Royal London Hospital (Barts alanine aminotransferase (ALT)) to HBeAg-negative chronic Health NHS Trust) UK from 2013 to 2016. All patients under- hepatitis characterised by a higher risk of disease progression, went a liver biopsy and tissue surplus to diagnostics was used the development of cirrhosis and hepatocellular carcinoma for experimental assays as described. Matched serum samples (HCC).1 were also collected (online supplemental figure S1 depicts the There remains a paucity of data on the extent and productivity patient cohort and virological analyses that were performed). All of the intrahepatic HBV reservoir (in terms of covalently closed patients with CHB were treatment- naïve and monitored for ≥2 circular DNA (cccDNA), intrahepatic total HBV DNA (itHBV years with virological and biochemical parameters for disease DNA) and pregenomic RNA (pgRNA)) in the HBeAg- negative stratification prior to tissue sampling. Patients were HBV mono- phase of CHB. Unravelling this issue is critical since the intra- infected; and co- infection with HCV, HIV and hepatitis Delta hepatic HBV reservoir plays a pivotal role in modulating liver virus was excluded. Patients were stratified according to serum disease progression.2 3 Elucidating the accuracy of novel non- HBV DNA levels (calculated as the mean value obtained by invasive HBV biomarkers in predicting the intrahepatic HBV consecutive measurements during a 2-year period of monitoring before undergoing liver biopsy): reservoir also remains an unmet clinical need.4 5 HBV replica- Group 1: patients with serum HBV DNA persistently tion can promote HBV DNA integration into the genome of the ► <2000 IU/mL; low viraemia (n=12). human hepatocyte, considered a key mechanistic step underlying ► Group 2: patients with serum HBV DNA between 2000 and HBV- mediated carcinogenesis, even in the absence of necroin- 20 000 IU/mL; moderate viraemia (n=25). flammation.6–8 Indeed, HBV DNA integration can compromise ► Group 3: patients with serum HBV DNA persistently cell genome stability and modify the expression of genes regu- >20 000 IU/mL; high viraemia (n=47). lating cell cycle/proliferation, predisposing the hepatocytes to pre- neoplastic transformation.7 DNA and RNA isolation from liver biopsy tissue HBV DNA integration has been detected in the early phases of Total intrahepatic DNA and RNA were isolated from patients’ CHB virus infection.9 Importantly, a recent study from our group liver tissues by using AllPrep DNA/RNA Mini Kit (QIAGEN, demonstrated high rates of HBV DNA integration along with Hilden, Germany) (procedure described in online supplemental clonal hepatocyte expansion in patients with HBeAg- positive material (SM)). chronic infection, highlighting a potential carcinogenetic risk in these patients, often considered to be in a benign disease phase, devoid of disease progression.9 A lower rate of HBV DNA inte- Quantification of intrahepatic HBV markers: total HBV DNA, gration has been observed in HBeAg- negative patients with cccDNA and pgRNA CHB.10 Although controversial, it has been hypothesised that, Intrahepatic DNA was used to perform itHBV DNA and cccDNA quantification as described in SM. pgRNA levels were deter- during the immune clearance phase and HBeAg seroconversion, http://gut.bmj.com/ the development of a strong immune response may favour the mined for 41/84 liver biopsies with an available RNA sample, by selection of those hepatocytes in which HBV DNA integrations applying an in-house digital droplet (dd)-PCR assay (described in confer a survival advantage, thus potentially contributing to liver SM). itHBV DNA, cccDNA and pgRNA values were normalised cancer development.8 11 12 to cell number, according to the quantification obtained by The occurrence and localisation of HBV DNA integration in Albumin- based ddPCR copy number assay: Alb, Human (Bio- Rad, Pleasanton, California, USA) and, thus reported as itHBV
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