MTC 03 II-01The role of HPV genome sequencing

12. Genomics
L. Mirabello 1, Y. Meredith 2, N. Wentzensen 1, M. Cullen 2, J. Boland 2, Y. Kai 1, Z. Chen 3, Q. Yang 2, B. Zhu 1, J. Mitchell 2, D. Roberson 2, S. Bass 2, L. Burdett 2, Y. Xiao 1, S. Wagner 2, T. Raine-Bennett 4, T. Lorey 4, P.E. Castle 5, R. Burk 5, M. Schiffman 1.
1Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, MD (United States), 2Cancer Genomics Research Laboratory, Leidos Biomedical Research, Inc., Frederick, MD (United States), 3Department of Microbiology, The Chinese University of Hong Kong, Hong Kong (Hong Kong), 4Regional Laboratory and Women’s Health Research Institute, Division of Research, Kaiser Permanente Northern California, Oakland CA (United States), 5Department of Epidemiology and Population Health, at Albert Einstein College of Medicine, Bronx, NY (United States)

Background / Objectives

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.


Methods

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).


Results

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.


Conclusion

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.


References