HBV Genome-Enriched Single Cell Sequencing Revealed Heterogeneity in HBV-Driven HCC

Abstract

\n Background: HBV-HCC is heterogeneous and frequently contains multifocal tumors. To interrogate heterogeneity of HBV-HCC, we developed a HBV genome enriched single cell sequencing (HGE-scSeq) procedure and a computational method to identify HBV integration sites and infer DNA copy number variations (CNVs). Results: We performed HGE-scSeq on 269 cells from 4 tumor sites and 2 tumor thrombi of a HBV-HCC patient. HBV integrations were identified in 142 out of 269 (53%) cells sequenced, and were enriched in two HBV integration hot spots chr1:34,397,059 (CSMD2) and chr8:118,557,327 (MED30/EXT1). There were also 162 rare integration sites. HBV integration sites were enriched in DNA fragile sites and sequences around HBV integration sites were enriched for microhomologous sequences between human and HBV genomes. Cells were grouped into 4 clonal groups based on CNVs. The HBV integration heterogeneity was associated with single cell’s CNVs. All of 269 cells carried chromosome 1q amplification, a recurrent feature of HCC tumors, suggesting that 1q amplification occurred before HBV integration events in this case study. Further, we performed simulation studies to demonstrate that the sequential events (HBV infecting transformed cells) could result in the observed phenotype with biologically reasonable parameters. Conclusion: Our HGE-scSeq data reveals high heterogeneity of HCC tumor cells in terms of both HBV integrations and CNVs.