Genome Instability Precedes Viral Integration in Human Papilloma Virus Transformed Tonsillar Keratinocytes

Approximately 70% of oropharyngeal squamous carcinomas (OPSCC) are associated with human papillomavirus (HPV). While HPV-positive (HPV+) OPSCC responds better to standard therapies and patients with HPV+ tumor generally have better outcomes than those with HPV-negative (HPV-) tumors, a subset of HPV+ patients do have poor outcomes. Our previous work suggested that tumors with integrated virus exhibit significantly greater genome wide genomic instability than those with only episomal viral genomes and patients with HPV+ OPSCC with episomal viral genomes had better outcomes. To explore the causal relation between viral integration and genomic instability, we have examined the time course of viral integration and genetic instability in tonsillar keratinocytes transformed with HPV16. HPV-infected human tonsil keratinocyte cell lines were continuously passaged and every fifth passage some cells were retained for genomic analysis. Whole genome sequencing and optical genomic mapping confirmed that virus integrated in five of six cell lines while remaining episomal in the sixth. In all lines genome instability occurred during early passages, but essentially ceased following viral integration but continued to occur later passages in the episomal line. To test tumorigenicity of the cell lines, cells were injected subcutaneously into the flanks of nude mice. A cell line with the integrated virus induced tumors following injection in the nude mouse while that with the episomal virus did not. We conclude that genomic instability is not the result of viral integration but likely promotes integration. Moreover, those transformants with episomal virus appear to be less tumorigenic than those with integrated virus.