To permit the large-scale study of HPV genome variability and precancer/cancer, we developed low cost, high-throughput next-generation sequencing (NGS) HPV whole-genome methods. We used our viral whole-genome sequencing assay to investigate HPV16’s unique epidemiology and cervical carcinogenicity among 4,626 HPV16-infected women in the NCI-KPNC PaP Cohort and SUCCEED study.
We have developed a PCR based next-generation sequencing (NGS) assay using the Thermo Fisher Life Sciences’ Ion Torrent Proton, custom Ion Ampliseq panels and an analytic pipeline to whole-genome sequence HPV16. We have validated NGS variant calls by comparing with Sanger and Illumina based sequence data. We have further designed a NGS assay to sequence the entire genome of the 13 high-risk HPV types concurrently.
Using European sublineage A1 as reference in this mainly White population, we assessed HPV16 genetic variation (from lineage to SNP level) with worst histologic outcome, including: CIN2 (n=1,284), CIN3 (n=1,395), AIS (n=103), SCC (n=187), adenocarcinoma (n=61), controls (n=1,596, ≤CIN1).
A4 (Asian) sublineage was associated with an increased risk of cancer, specifically adenocarcinoma (OR 9.8, 95% CI 2.0-47.7). The non-European lineage B (African-1) conferred significantly lower risk of CIN3 (OR 0.5, 95% CI 0.3-0.9) while lineage C (African-2) yielded increased risk (OR 2.1, 95%CI 1.1-3.9). D2/D3 sublineages were strongly associated with an increased risk of CIN3+, particularly D2 (OR 7.6, 95% CI 3.0-19.5). D2 had the strongest increased risk of glandular lesions, AIS (OR 29.2, 95% CI 8.9-95.5) and adenocarcinomas (OR 137.3, 95% CI 37.2-506.9). At the SNP level, we have identified 2,679 variable positions, with 67 individual European SNPs and 122 non-European SNPs significantly associated with CIN3+. These data allowed us to determine that controls have a significant burden of rare variants. Deep sequencing has also revealed HPV16 variant lineage co-infections in 24.4% of women. HPV16 variant lineage co-infections were linked to multi-HPV-type infections, and lower CIN3+ risk. We detected several hundred different HPV16 isolates in this population.
NGS HPV genome sequencing has enabled the sequencing of thousands of HPV16-containing specimens from epidemiologic studies for the evaluation of the genetic basis of HPV carcinogenicity. HPV16 actually represents hundreds of co-transmitted viral isolates, providing finer detail for epidemiologic study of viral acquisition and persistence/clearance/re-appearance. Viral genetic variation at the variant lineage and SNP levels strongly influences HPV16 carcinogenicity and histologic outcome and helps explain HPV16’s unique properties.