Development of Human Papillomavirus Integration Analysis Technologies for Human Papillomavirus-Associated Cancer Research

Abstract

Viruses associated with human cancers, known as "tumor viruses," can induce cellular transformation or immortalization, representing a crucial step in cancer initiation. In past decades, the association between viruses and cancer has been a prominent focus in cancer research. Many tumor viruses, such as human papillomavirus (HPV) and hepatitis B virus (HBV), possess the capability to integrate their genomic DNA or RNA into the target host cell, whereas others, like hepatitis C virus (HCV), rarely integrate into the host genome. Recent studies propose that virus integration may introduce additional oncogenic mechanisms. For instance, in cervical and head and neck cancer, HPV integration directly influences cancer-related gene expression, leading to the generation of hybrid viral-host fusion transcripts. Therefore, detecting viral integration sites in the host genome is crucial for further understanding their oncogenic mechanisms in cancer development. HPV is a well-established driver of malignant transformation in various cancers. However, the impact of HPV integration into the human genome remains largely unresolved due to sample size limitations and existing informatics challenges in identifying viral-host breakpoints from low-read-coverage sequencing data, especially in the presence of complex structural variations around fusion points. In response to these challenges, we developed SearcHPV, a novel method using targeted capture sequencing (TCS) to identify and assemble HPV integration sites in the genome. Our analysis of three HPV+ models demonstrated that SearcHPV detected HPV-host integration sites with higher sensitivity and specificity than two other commonly used methods. Additionally, we validated the junction assembly of SearcHPV, aiding in the accurate identification of viral-host junction breakpoint sequences. Our findings indicated that viral integration occurs through diverse DNA repair mechanisms, including microhomology-mediated repair, etc. We expanded our study to 291 head and neck squamous cell carcinoma (HNSCC) patients, employing TCS, RNA-Seq, and nanopore sequencing. We devised a novel approach to locally resolve complex HPV integrations using nanopore sequencing. Using statistical models, we labeled complex structures as "Type2", characterized by multiple integrations clustered with high copy numbers, and less complex integrations as "Type1." We revealed that Type2 events exhibited significantly more non-canonical splicing sites and were more likely to be transcribed, suggesting a complex transcription pattern. Additionally, RNA expression levels of oncogenes around Type2 events were significantly higher than Type1, indicating potential differences in oncogenic mechanism alterations induced by different types of integrations. In a subset of 78 patients with recurrent or metastatic samples, we explored the heterogeneity of HPV integration structures, revealing unique HPV integrations and varying copy numbers in different tumor sites of the same patients. Using nanopore sequencing on one cell line with primary and recurrent samples, we uncovered potential clonal selections of HPV integrations during tumor progression. Our findings emphasize the heterogeneous and complex nature of HPV integration associated with genome rearrangement, potentially contributing to distinct tumorigenic consequences. We broadened our methodology to other viral-associated cancers and investigated 48 Mucoepidermoid carcinoma (MEC) patients with both TCS and RNA-Seq. We detected one patient with HPV integrated into 13 host genes and exhibiting high expression of HPV16 oncogenes E6 and E7. The genetic mechanisms of host genome integration were found to be similar to our previous findings in HNSCC. This study provided insights into the role of HPV in tumorigenesis of MEC.