Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV‐associated head and neck squamous cell carcinoma cell lines

Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV‐associated head and neck squamous cell carcinoma (HPV‐HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next‐generation sequencing using a target‐enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16‐related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele‐specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV‐HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus‐associated carcinogenesis of HPV‐HNSCC.