Background

Human papillomaviruses (HPV) cause most cervical carcinomas and substantial fractions of oropharyngeal, anal, vulvar, penile, and vaginal carcinomas. During infection, HPV DNA initially replicates as circular episomes. However, it becomes integrated into the host genome in most HPV-induced cancers. Following integration, HPV is most commonly found as a subgenomic segment that includes the non-coding regulatory region (URR) and viral oncogenes E6 and E7. In tumors, HPV DNA can exist as extrachromosomal DNA (ecDNA) circles, chimeric HPV-human tandem repeats (heterocatemers), or both. The presence of ecDNA is associated with poor patient prognosis across cancer types. However, distinguishing whether particular amplified DNAs in tumors are ecDNAs and/or intrachromosomal heterocatemers and determining the extent of genomic rearrangement surrounding them, present technical challenges. Characterizing HPV-related integration structures accurately and completely is essential for advancing our understanding of HPV-driven tumorigenesis and the mechanisms behind viral integration.

Methods

We applied a battery of genomic analysis techniques at various size-scales to the HPV16-positive human oropharyngeal squamous cell carcinoma cell line UM-SCC-47. These techniques included a custom hybridization capture assay coupled with short-read Illumina sequencing (HC+SEQ), Nanopore long-read DNA and RNA sequencing, Bionano optical genome mapping, and single-cell fluorescence in situ hybridization (FISH).

Results

HC+SEQ and long-read DNA sequencing defined the structure of the integrated HPV16 DNA, determined the human-virus DNA junctions, and mapped a repeated 23.5 kb HPV-human heterocatemer at single-base-pair resolution. RNA-seq revealed that these heterocatamers could produce spliced, polyadenylated virus-human fusion transcripts alongside high viral oncogene expression. Optical genome mapping further showed that the 23.5 kb heterocatemer occurred in tandem arrays of various lengths, ranging from one unit to more than 27 tandem units. These large-scale arrays (up to 700 kb) were further rearranged with adjacent human DNA in even larger-scale structures. Additionally, single-cell FISH analysis revealed that every cell had intrachromosomal HPV16 associated with human DNA, with a subset of cells also containing HPV16 ecDNA.

Conclusion

These findings highlight extensive genomic instability, complexity, and heterogeneity of HPV16-human DNA structures in this cell line, including previously undescribed higher-order organizational patterns. They also underscore the value of combining large-scale analyses to resolve complex genomic rearrangements and emphasize the importance of FISH for distinguishing intrachromosomal from extrachromosomal DNA structures. Ultimately, understanding how integrated HPV DNA higher-order structures contribute to tumorigenesis could lead to patient stratification based on individual risk of progression or inform the development of targeted therapeutic strategies.