Geographical and molecular disparity of HBV integration: Implications for hepatocarcinogenesis and clinical outcomes in HCC

Chronic HBV infection is responsible for approximately 50% of HCC cases worldwide.1 Extensive research into the molecular mechanisms of HBV-induced hepatocarcinogenesis has highlighted not only the role of chronic liver inflammation but also the integration of HBV into the human genome. This integration disrupts tumor suppressors or activates proto-oncogenes, which enhances cell survival and contributes to HCC development. Recent next-generation sequencing studies have revealed that HBV integration occurs not only in cancer-related genes, such as TERT and KMT2B, but also induces chromosomal rearrangements, suggesting a variety of mechanisms involved in HBV integration-driven hepatocarcinogenesis.2 Meanwhile, we have recently proposed a novel therapeutic strategy that targets vulnerabilities caused by HBV integration in HCC.3 To develop effective treatments for HBV-associated HCC, a deeper understanding of HBV integration-induced hepatocarcinogenesis is essential. In this issue of Hepatology, Lyu et al4 explored the disparity of HBV integration across different levels, including geographical cohorts, individual patients, and chromosomal locations. They first identified significant geographical differences in HBV integration events between Asian and Western countries. In Asian cohorts, HBV integration events were more frequent in HCC compared to nontumor liver (NTL) tissue, whereas the opposite was observed in western cohorts. Next, they examined the chromosomal distribution of HBV integration and found that these events were enriched in telomere and centromere (T&C) regions in HCC but not in NTL. This enrichment dynamically shifted from neutral regions (N peaks) to T&C regions during the malignant transformation from NTL to HCC. Since the integrity of telomeres and centromeres is crucial for maintaining genomic stability,5 the authors showed that HBV integration in these regions contributes to increased genomic instability, as evidenced by heightened DNA damage. The authors further investigated the co-occurrence of HBV integration events and observed a high concordance of integrations at specific gene pairs, particularly in the T&C regions. This co-existence was associated with poor survival outcomes. On a gene-specific level, key genes such as TERT, KMT2B, PLEKHG4B, LOC441666, and MTRNR2L1 were frequently integrated in HCC, with TERT integrations occurring upstream of the gene. As previously reported, integration near the TERT promoter harnesses the HBV EnhI region to recruit host transcription factors, such as ELF4, to activate TERT transcription.6 The study also demonstrated that the orientation and proximity of the TERT promoter and HBV EnhI are critical for TERT activation. Finally, the authors introduced the concept of “clonal disparity scores,” which reflect clonal HBV integration events across multiple tumors within an individual. Patients with higher clonal disparity scores had significantly worse survival outcomes. These scores were linked to integrations in the T&C regions and TP53 mutations, both of which promote genomic instability and malignant potential. While previous studies have reported HBV integration sites and mechanisms of TERT activation,6–8 this paper provides several new insights, including the geographical disparity of integration events, chromosomal landscape, co-existence of integration events, and clonal HBV integration across multiple tumors. These findings have important clinical implications, although there are still unanswered questions and limitations. The observed geographical disparity in HBV integration events between Asian and western cohorts is intriguing. Typically, the clonal enrichment of integration events in malignant cells leads to decreased diversity, with fewer integration events in HCC compared to NTL. However, the opposite pattern was observed in Asian cohorts. The authors suggest that this discrepancy may be due to differences in HBV transmission patterns—vertical transmission at birth in Asian populations and horizontal transmission in adulthood in western populations. Long-term infection may allow for greater expansion of HBV-integrated clones with a survival advantage. Indeed, the clonality value (the extent of clonal insertions) was higher in both NTL and HCC in the Asian cohort compared to the western cohort. Another possibility is that distinct mechanisms of hepatocarcinogenesis may operate between the 2 regions, with Asian HCC developing through the accumulation of weaker driver events, while western HCC may arise rapidly from fewer, stronger drivers. Further investigation is needed to clarify this geographical disparity. The oncogenic significance of co-occurring HBV-integrated gene pairs remains uncertain. The study found recurrent pairs, such as TERT and PLEKHG4B, and patients with these co-occurrences had worse clinical outcomes. However, the dual knockdown of TERT and PLEKHG4B did not demonstrate any additive or synergistic antitumor effect, leaving the role of cointegration unclear. It is possible that the co-occurrence of HBV integration in the T&C regions, rather than specific gene functions, induces chromosomal instability that promotes hepatocarcinogenesis. A limitation of this study is the lack of experimental validation for other gene pairs, which should be addressed in future studies. Regarding the clonal disparity score, although it was proposed as a potential biomarker for the aggressiveness of HBV-HCC, it was only significant in the Asian cohort. This raises questions about its broader utility, which may relate to the aforementioned geographical differences. The underlying reason for this discrepancy requires further clarification. These findings also underscore the importance of considering geographical differences in the study of HBV biology and biomarkers. Another interesting observation was the frequent integration of HBV into the FN1 gene, but only in NTL. This recurrent integration event, previously reported,7,8 suggests its validity. However, its clinical significance remains unclear. Given that this event is absent in HCC, HBV integration into FN1 may play a protective role against HCC, warranting further research. In summary, this study provides a comprehensive analysis of HBV integration disparities in HCC across geography, chromosomes, and genes, advancing our understanding of the molecular mechanisms underlying HBV-associated hepatocarcinogenesis.