American Association for Cancer Research, Cancer Research, 2023
DOI: 10.1158/0008-5472.can-22-3030
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Abstract HPV16 is the most oncogenic type of human papillomavirus (HPV). Integration of HPV into the human genome is an important mechanism of carcinogenesis but is absent in at least 30% of HPV16+ tumors. We applied long-read whole-genome sequencing (WGS) to cervical cancer cell lines and tumors to characterize HPV16 carcinogenesis in the absence of integration. Large tandem arrays of full-length and unique truncated viral genomes integrated into multiple chromosomes were identified in two HPV16+ cell lines. The dispersion of characteristic viral variants to multiple integrations sites indicated that viral deletions formed as extrachromosomal DNA (a phenomenon we termed HPV superspreading). In addition, an HPV16+ cell line was identified with unintegrated (episomal) DNA that had tandem arrays of full-length, truncated, and rearranged HPV16 genomes (multimer episomes). Cytogenetic analysis of this cell line showed intense extrachromosomal HPV staining, including structures resembling double-minute chromosomes. Whole genome sequencing of HPV16+ cervical tumor samples from Latin America revealed that 11/20 tumors with only episomal HPV (EP) had intact monomer episomes. The remaining nine EP tumors had multimer and rearranged HPV genomes. The majority (80%) of HPV rearrangements and deletions disrupted the E1 and E2 genes, and EP tumors overexpressed the E6 and E7 viral oncogenes, a similar profile to tumors with HPV integration. Tumors with putative multimer HPV integrations displayed HPV multimers and concatemers of human and viral sequences. This data uncovered a novel mechanism for HPV16 to cause cancer without integration through aberrant episomal replication to form rearranged, mutated, and multimer episomes.