Abstract 3417: Detection and analysis of oncovirus integration sites in FFPE-derived human tumor samples using hybrid capture and massively parallel sequencing
Authors: Slevin Michael K. , Powers Winslow T. , Burns Robert T. , Wollison Bruce M. , Coleman Haley A. , Paskavitz Amanda L. , Nag Anwesha , Manning Danielle K. , Garcia Elizabeth , Ducar Matthew D. , Thorner Aaron R. , MacConaill Laura E. 
Published: August 2018

Viral genome integration into the host's genetic material can provide cells with selective advantages that make critical contributions to unregulated growth, cell division, and genomic instability. Current clinical tests include in situ hybridization, immunohistochemical, and real-time PCR assays that are unable to identify viral integration points in relation to oncongenic somatic mutations. This precludes a complete picture of how such mutations work together to promote, maintain, and disperse tumor progression. Precision cancer medicine employs genomic technologies (e.g., massively parallel sequencing) for high-throughput genomic profiling to molecularly define patient tumors, allowing identification of clinically actionable mutations. We created a custom probe set for targeted hybrid capture enrichment of several oncoviruses and have tested it in combination with our clinical tumor DNA profiling probe set, OncoPanel v3.

Selected viruses were chosen due to their serving as causative cancer agents that disrupt tumor suppressors, facilitate genomic instability, and increase oncogene expression. The full viral genomes of hepatitis B virus and high-risk human papilloma virus (HPV) strains, 16, 18, 33, and 45 were targeted. Additionally, regions coding for E6 and E7 oncoproteins were targeted for several of the low-risk HPV strains, as was the LANA region of Kaposi's sarcoma-associated virus.

FFPE-derived human tumor samples of known infection status were fragmented to 250 bp and converted into Illumina libraries. Pooled libraries underwent hybrid capture with custom, Agilent-designed OncoPanel and viral probes, and resulting captures were sequenced on an Illumina HiSeq2500 sequencer.

SvABA (Structural variation and indel analysis by assembly) was used to perform de novo sequence assembly on soft-clipped and discordant aligned reads, and viral integration sites were identified by realigning contigs to the host genome to obtain breakpoint coordinates. Reduction of false positives was facilitated using dual-matched sample barcodes (IDT), which virtually eliminate barcode cross-talk, allowing for confident detection of low allele frequency events. Furthermore, inclusion of unique molecular identifiers (UMI) permitted discrimination of PCR duplicates and errors.

Our results demonstrate high concordance with clinical samples of known infection status and provide viral integration break point locations in association with genomic tumor mutations. Viral integration sites are often associated with deletions or amplifications of the host's flanking genomic regions. Furthermore, viral integration may disrupt or enhance expression of tumor suppressors and oncogenes, respectively. Coupled with tumor molecular profiling, this information will better inform patient treatment.

Citation Format: Michael K. Slevin, Winslow T. Powers, Robert T. Burns, Bruce M. Wollison, Haley A. Coleman, Amanda L. Paskavitz, Anwesha Nag, Danielle K. Manning, Elizabeth Garcia, Matthew D. Ducar, Aaron R. Thorner, Laura E. MacConaill. Detection and analysis of oncovirus integration sites in FFPE-derived human tumor samples using hybrid capture and massively parallel sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3417.