Structure and Transcription of Integrated HPV DNA in Vulvar Carcinomas

HPV infections are associated with a fraction of vulvar cancer. Through hybridization capture and DNA sequencing, HPV16 DNA was detected in five of thirteen vulvar cancers, and it was integrated into human DNA in three of the five. The insertions were in introns of the human NCKAP1, C5orf67, and LRP1B genes, each in the opposite transcriptional orientation as the human genes. Two of the three integrations (NCKAP1 and C5orf67) were flanked by short direct repeats in the human DNA, consistent with the possibility of HPV DNA insertions at sites of abortive LINE retrotransposon DNA incisions. Long-range DNA sequencing showed that the insertion in C5orf67 was present as either a 36 kbp human-HPV hetero-catemer, extrachromosomal, circular DNA or a 36 kbp tandem repeat integrated into the human genome, with definition of the human circularization/concatemerization junction at single nucleotide resolution. The integrated viral DNAs all retained an intact segment including the upstream regulatory region and the adjacent viral E6 and E7 oncogenes. RNA sequencing revealed that the only HPV genes consistently transcribed from the integrated viral DNAs were E7 and the E6*I spliced form of E6. The other two HPV DNA+ tumors had coinfections, one with HPV6 plus HPV16, the other with HPV53 plus HPV62, but no evidence for integration of any of those viral DNAs was detected. HPV-positive and HPV-negative vulvar cancers exhibited contrasting global gene expression patterns among human genes, with a number of those genes demonstrating concordance with previously observed differential expression profiles between HPV-positive and HPV-negative tumors in other types of human cancer. In addition, a substantial fraction of differentially expressed genes involved immune system function. Thus, HPV DNA integration and transcription in vulvar cancers parallel those observed in other HPV-positive cancers, underscoring a significant correspondence and shared biological characteristics This study emphasizes the power of hybridization capture coupled with DNA and RNA sequencing approaches to identify a broad spectrum of HPV types, determine human genome integration status of viral DNAs, elucidate structures of integrated and hetero-catemer DNAs, and detect meaningful insights about viral and human gene transcription in HPV-associated cancers.