Abstract 4054: Efficient identification of integration sites of human papilloma virus (HPV) genome in HPV-related head and neck squamous cell carcinoma cell lines with next-generation sequencing-based novel approach
Authors: Hatano Takashi , Sano Daisuke , Takahashi Hideaki , Hyakusoku Hiroshi , Isono Yasuhiro , Shimada Shoko , Sawakuma Kae , Watanabe Yoshiyuki , Yamamoto Hiroyuki , Itoh Fumio , Myers Jeffrey N. , Oridate Nobuhiko 
Published: July 2016

Abstract
Background: The incidence of human papillomavirus (HPV)-related oropharyngeal cancers (HPV-OPC) is increasing remarkably these days, in contrast to other head and neck cancers with use of tobacco and/or alcohol as major risks. While the detail mechanism of HPV integration on the host genome for the development of cervical cancer has been well characterized, less is known about the interactions between HPV integration and the human genome in HPV-OPC. The purpose of this study is to reveal genome-wide maps of HPV integration loci in HPV-related head and neck squamous cell carcinoma (HNSCC) cell lines. Methods: Three HPV 16-related HNSCC cell lines (UPCI:SCC090; tongue base, UPCI:SCC152; hypopharyngeal recurrence of a tongue base cancer, UPCI:SCC154; tongue) were prepared for this study. To detect integrated HPV sequences in the genome of these 3 HPV-related HNSCC cell lines, we used technique of target enrichment for next-generation sequencing analysis A total 427 of custom RNA probes were designed to specifically target the HPV genome for this study. After capture and amplification, the templates were sequenced using next-generation sequencer (NGS). Reads that aligned perfectly to the human or HPV 16 genome were removed. Reads aligned partially to human genome and HPV 16 genome were reserved. The integration sites, exact joint position of human and HPV 16 sequence, were then efficiently detected from the reserved reads. PCR and direct sequencing were used to verify the detected HPV 16 integration sites. Results: The average read length was 414.18 bp. The average read quality was 31.30, corresponding to &gt;99.9% accuracy. The HPV integrants mapped to chromosomes 2q23, 3p12, 6p21, and 9q22 in UPCI:SCC090, chromosomes 2q23, 3p12, 9q22, and 9q31 in UPCI:SCC152, and chromosome 21q21 in UPCI:SCC154 were detected. Conclusion: In the present study, we identified novel integration sites in HPV-related HNSCC cell lines with our unique approach with deep and high-throughput sequencing which enabled enrichment of viral fragments for sequencing using custom-made probes based on the sequence of the HPV genome, without conducting unnecessary sequencing of regions where the HPV genome is not integrated.
Citation Format: Takashi Hatano, Daisuke Sano, Hideaki Takahashi, Hiroshi Hyakusoku, Yasuhiro Isono, Shoko Shimada, Kae Sawakuma, Yoshiyuki Watanabe, Hiroyuki Yamamoto, Fumio Itoh, Jeffrey N. Myers, Nobuhiko Oridate. Efficient identification of integration sites of human papilloma virus (HPV) genome in HPV-related head and neck squamous cell carcinoma cell lines with next-generation sequencing-based novel approach. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4054.