Temporal and structural patterns of hepatitis B virus integrations in hepatocellular carcinoma

Abstract

Chronic infection of hepatitis B virus (HBV) is the major cause of hepatocellular carcinoma (HCC). Notably, 90% of HBV-positive HCC cases exhibit detectable HBV integrations, hinting at the potential early entanglement of these viral integrations in tumorigenesis and their subsequent oncogenic implications. Nevertheless, the precise chronology of integration events during HCC tumorigenesis, alongside their sequential structural patterns, has remained elusive thus far. In this study, we applied whole-genome sequencing to multiple biopsies extracted from six HBV-positive HCC cases. Through this approach, we identified point mutations and viral integrations, offering a blueprint for the intricate tumor phylogeny of these samples. The emergent narrative paints a rich tapestry of diverse evolutionary trajectories characterizing the analyzed tumors. We uncovered oncogenic integration events in some samples that appear to happen before and during the initiation stage of tumor development based on their locations in reconstituted trajectories. Furthermore, we conducted additional long-read sequencing of selected samples and unveiled integration-bridged chromosome rearrangements and tandem repeats of the HBV sequence within integrations. In summary, this study revealed premalignant oncogenic and sequential complex integrations and highlighted the contributions of HBV integrations to HCC development and genome instability.

Keywords: chromosomal translocation; clonal evolution; hepatocellular carcinoma; intratumor heterogeneity; multiregion sequencing; viral integration.

Figure 1.. The genomic landscape of analyzed HCC tumor biopsies.

(A) The number of total mutations, (B) the number of non-silent mutations, (C) the nucleotide base substitution frequencies, (D) the COSMIC mutation signature compositions, and (E) the copy number variations in individual biopsies.

Figure 2.. HBV sequence and integrations identified in analyzed tumor and tumor adjacent biopsies.

(A) The distribution of sequencing reads on the HBV genome in four selected tumor biopsies. (B) A summary of the location and allele frequency of the identified HBV integrations in all tumor and tumor adjacent biopsies. (C) The allele frequency of integrations shared by different numbers of tumor biopsies. No integration was observed in three out of five biopsies for any of the tumors analyzed in this study.

Figure 3.. Phylogenetic trees of six analyzed HCC tumors.

Selected oncogenic mutations, tumor genes affected by copy number alterations, and integrations were mapped to trunks and branches, as indicated.

Figure 4.. HBV integration-bridged chromosomal recombination identified by long-reads sequencing.

(A) The number and percentage of integrations with single or both human-virus breakpoints identified by short-reads sequencing. BP: breakpoint. (B) The location of integrations identified by short-reads and long-reads sequencing in two biopsies. Lines represent integration-bridged chromosomal translocation or inversions. (C) Structure of the four integration-bridged chromosomal translocation identified by long-reads sequencing. The lines represent single PacBio reads. (D) Mapping of supporting short-reads to the integration-bridged chromosomal translocation between chr7 and chr9 in Patient 6 Biopsy 5. (E) Mapping of supporting short-reads to the integration-bridged chromosomal inversion of chr17 and chr9 in Patient 2 Biopsy 3. (F) Log R ratio showing copy number alterations around breakpoints showing in (D). (G) Structure of the four complicated integrations identified by long-reads sequencing. The lines represent single PacBio reads.