Abstract 2991: Detection of gene rearrangements using OncoPanel: a targeted next-generation sequencing assay
Authors: Garcia Elizabeth P. , Ligon Azra H. , Abo Ryan P. , Dal Cin Paola S. , Weremowicz Stanislawa , Shivdasani Priyanka , Davineni Phani K. , Zepf Dimity L. , Ducar Matthew D. , Van Hummelen Paul , Jia Yonghui , Kuo Frank C. , Sholl Lynette M. , MacConaill Laura E. , Lindeman Neal I. 
Published: August 2015

Abstract
Identification of structural gene rearrangements is vital for cancer patients as these events can provide definitive diagnoses, prognostic value, and influence the course of treatment. While FISH, karyotype analysis and aCGH array have traditionally been used to identify and confirm the presence of structural variants, the advent of next generation sequencing has enabled genetic testing including detection of multiple structural variants (SVs) from genomic DNA. To this end, we have developed and validated Oncopanel, a cancer-specific targeted next generation sequencing (NGS) assay designed to detect SNVs, indels, and copy number alterations across 300 genes, and 35 clinically actionable or informative SVs. Each rearrangement was specifically targeted by baiting genomic locations frequently identified to contain breakpoints reported in the literature and publicly available databases. Using BreaKmer, an internally developed SV detection tool (Nucleic Acids Res. 2014 Nov 26, doi: 10.1093/nar/gku1211), rearrangements, including the exact breakpoint coordinates and the genes involved in or adjacent to the breakpoint(s), were identified. Here we examine the utility of Oncopanel using genomic DNA to identify structural variants.
We report the results of 3,291 cancer patients tested in our personalized cancer medicine program (Profile), a joint venture between Dana-Farber Cancer Institute, Brigham and Women's Hospital, and Boston Children's Hospital. As compared to conventional cytogenetics, FISH analysis, and molecular detection by PCR methods, Oncopanel's overall sensitivity and specificity for SVs was 81.4% and 100%, respectively. Most discordant results were identified in (1) tumors with SVs involving the IGH enhancer regions (60% of discordant results), or (2) in samples with &lt; 20% tumor (25% of discordant results). Several SVs involving the IGH enhancer regions were missed likely due to lack of Oncopanel coverage. Oncopanel was designed to target a finite sequence pool, but due to IGH enhancer region's large size (1.2Mb), only a small portion of this region was specifically interrogated. Inclusion of all possible IGH enhancer sequences would have hampered the effectiveness of SNV, indel and copy number alteration detection for other cancer critical genes. Discrepant Oncopanel and cytogenetic results were also observed in samples with low tumor purity likely due to masking of variant sequences by stromal contamination.
In conclusion, we find that Oncopanel has utility to detect structural variants with a sensitivity of 92%, barring detection of rearrangements involving IGH, and a specificity of 100%. Based on the baiting strategy, detection of many rearrangements can also be interrogated in parallel with SNV, indel and CNV detection resulting in reduced sample input requirements and the inclusion of precise information regarding the breakpoints and the class of rearrangement identified.
Citation Format: Elizabeth P. Garcia, Azra H. Ligon, Ryan P. Abo, Paola S. Dal Cin, Stanislawa Weremowicz, Priyanka Shivdasani, Phani K. Davineni, Dimity L. Zepf, Matthew D. Ducar, Paul Van Hummelen, Yonghui Jia, Frank C. Kuo, Lynette M. Sholl, Laura E. MacConaill, Neal I. Lindeman. Detection of gene rearrangements using OncoPanel: a targeted next-generation sequencing assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2015-2991