Ryan Edward Mills

Oncogenic viral infections are major contributors to cancer development worldwide. Tumor-associated viruses such as human papillomavirus (HPV), hepatitis B virus (HBV), Epstein–Barr virus (EBV), and Merkel cell polyomavirus (MCPyV) can promote malignant transformation through diverse mechanisms, including persistent viral gene expression, chronic inflammation, and, in some cases, integration of viral DNA into the host genome. Among these, HPV is one of the most clinically important DNA tumor viruses and is a major driver of cancers of the cervix, anus, penis, vagina, vulva, and oropharynx, collectively accounting for over 400,000 deaths annually ( 1 ). In infected cells, HPV can persist as episomal DNA or integrate into the host genome. Importantly, HPV integration plays an important role in tumorigenesis and often generates complex viral–host genomic rearrangements that are difficult to resolve using conventional short-read sequencing approaches. Long-read sequencing technologies offer new opportunities to reconstruct these intricate integration structures, but the performance of existing assembly strategies remains incompletely evaluated. In this study, we systematically review sequencing platforms and their application for detecting structural variants and evaluate long-read assembly tools for reconstructing HPV integration structures. Using three synthetic Oxford Nanopore DNA sequencing datasets representing different levels of integration complexity together with the UMSCC47 cell line as an authentic long-read sequencing dataset, we assess whether structural-variant detection methods and genome assembly can accurately identify complex integration structures, particularly under conditions of high copy number and structural rearrangement. Our results provide practical guidance for selecting sequencing technologies and computational approaches for viral integration detection and structural resolution, enabling a more comprehensive understanding of virus-driven genome remodeling in cancer.

Relative read depth across the MCPyV genome in each VP-MCC tumor. Left column shows MCPyV genome position (RefSeq NC_010277).

Summary of definitive treatment modalities for MCC patients in our cohort, stratified by stage. Data presented as n (%). a Including sentinel lymph node biopsy ± completion lymphadenectomy of regional lymphatic basin.

Summary of NGS data by individual tumor.

Summary of all MCPyV SNV calls in VP-MCC tumors.

Summary of all somatic genomic alterations identified in our MCC cohort.

List of human genes included in our targeted NGS panel.

Summary of all MCPyV integration events in VP-MCC tumors.

Primary tumor histopathologic parameters for patients with MCC treated with surgery. Data presented as n (%) or median (range).

Merkel cell carcinoma (MCC) is an aggressive disease with poor survival outcomes and increasing incidence. There is a clear and present need for enhanced understanding of cellular mechanisms of tumorigenesis, validation of robust genetic signatures predictive of aggressive disease, and novel informatics tools to simplify analysis of Merkel cell polyomavirus (MCPyV)–host genome interactions. Genomic DNA was harvested from 54 MCC tumors for exome sequencing and in-depth genetic profiling of a 226-gene panel. We further developed a robust informatics package (MCPyViewer) optimized for MCPyV integration site analysis with graphical output to simplify usability for end users. Finally, we assessed the prognostic impact of specific genetic signatures on MCC-specific survival in our cohort. Our study included 54 patients (n = 44 MCPyV positive), 11 (20.4%) of whom had died of MCC at last follow-up. Human genes altered at high frequency included LRP1B (n = 10, 18.5%), FAT1 (n = 9, 16.7%), KMT2D (n = 9, 16.7%), and RB1 (n = 7, 13.0%). In 36 of 44 (81.8%) MCPyV-positive tumors, we identified viral integration into the human genome with a median of two events per tumor. In six tumors, MCPyV integrated into Catalogue of Somatic Mutations in Cancer tier 1 or tier 2 cancer-related human genes.Implications:A combined genomics score incorporating tumor mutational burden and copy-number variation was strongly prognostic of MCC-specific survival controlling for lymph node metastases and tumor MCPyV status; thus, our study adds critical understanding to prognostic markers and tumorigenic mechanisms in MCC.