Background : Human papillomavirus (HPV) is generally recognized as the main cause of cervical precancerous and cancerous lesions. In infected cells, the HPV episomal form is generally maintained; however, in high-grade cervical lesions and carcinomas, HPV is likely to integrate the host cell genome. Furthermore, HPV integration is frequently associated with partial or complete loss of the E1 and E2 genes, which regulate the activity of viral oncoproteins E6 and E7 involved in the regulation of the cellular cycle. The HPV integration event lead to an overexpression and constitutive activity of E6 and E7 and therefore contribute to the progression of low-grade lesions towards more severe lesions and, ultimately, carcinomas. In this study, using a double-capture system followed by high-throughput sequencing, we determined : i) the HPV genotype(s) present in liquid-based cervical (LBC) smears performed on a Gabonese urban population, ii) the HPV integration status, iii) as well as possible integration site(s).
Methods: The women participating in this study were recruited during a previous multi-center cross-sectional study carried out at two hospitals in Libreville, Gabon. The main inclusion criteria were based on cytology and the presence of the HPV16 genotype detected using molecular assays. A double HPV-specific capture was performed to guarantee an optimal HPV-enrichment and to maximize the fraction of HPV reads in bioinformatics analyses. Illumina Sequencing was conducted using a Miseq benchtop sequencer. The integration sites were defined as zones of chimeric reads corresponding to human and viral sequences within a concatenated or single read. The process of finding the chimeric reads was based on several successive mappings against human and viral genomes.
Results: The rate of HPV integration in the host genome varied with cytological grade: 85.7% (6/7), 71.4% (5/7), 66.7% (2/3) 60% (3/5) and 30.8% (4/13) for carcinomas, HSIL, ASCH, LSIL and ASCUS, respectively. For high cytological grades (carcinomas and HSIL), genotypes HPV16 and 18 represented a total of 92.9% of samples (13/14). The HPV16 genotype was mainly found in high-grade lesions in 71.4% of samples regardless of cytological grade. Minority genotypes (HPV33, 51, 58 and 59) were found in LSIL samples, except HPV59 which was identified in one HSIL sample. Among all the HPV genotypes identified after double capture, 10 genotypes (HPV30, 35, 39, 44, 45, 53, 56, 59, 74 and 82) were detected only in episomal form; no virus-human chimeric sequences were detected after mapping them on reference sequences.
Conclusion: Our study revealed that the degree of HPV integration varies with the cytological cervical grade. The integration event might be a potential clinical prognostic biomarker for the prediction of the progression of neoplastic lesions. However more samples from early stage lesions need to be included in further study to support this.
Bringing sequence to the clinic (i.e., diagnostics, cancer, inherited disorders)
