Abstract 2229: Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas

Abstract

Background: Human papillomavirus (HPV) infections are the principal cause of cervical cancers, subsets of anogenital and head and neck cancers (HNC). During persistent infection, viral DNA integration into the host genome may occur, which is suggested to affect carcinogenesis. One of the most critical limitations of currently used HPV integration detection techniques (PCR-based or NGS-based) is their application to FFPE material, because of DNA fragmentation. The aim of this study was to assess HPV integration in HPV-positive HNSCC cell lines and FFPE tissue comparing the new Targeted Locus Amplification (TLA) technology with previously used PCR technology (APOT/DIPS). Methods: Seven HPV-positive cell lines and FFPE material of 10 HPV-positive HNSCC were used for HPV integration detection by TLA, a proximity ligation-based next-generation sequencing technique. Crosslinked DNA is digested with restriction enzymes, and re-ligated into chimeric DNA molecules. For cell lines, a PCR based HPV16 target enrichment is performed, and for FFPE material a capture-based target enrichment is performed for HPV16 and HPV18 sequences, both followed by Illumina sequencing. Results: TLA was able to sequence up to 100 kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with APOT/DIPS PCR data and the literature. In the FFPE tissue samples, TLA identified integrated HPV in 6 out of 10 tumors, with simple and complex integration patterns. In general, TLA confirmed PCR data and detected additional integration sites. Conclusion: TLA provides the opportunity for reliable and robust detection of HPV integration in HNSCC cell lines and FFPE tissue. This new sequencing technology could be a useful tool for further research on HPV integration in disease and patient outcome and eventually in clinical diagnostics. Citation Format: Imke Demers, Harini Balaji, Harma Feitsma, Irinia Sergeeva, Joost Swennenhuis, Nora Wuerdemann, Steffen Wagner, Bernd Kremer, Christian Huebbers, Jens Peter Klussmann, Ernst-Jan Speel. Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2229.