Abstract 4699: Analytical validation of solid tumor fusion gene detection in a comprehensive NGS-based clinical cancer genomic test
Authors: Yelensky Roman , Donahue Amy , Otto Geoff , Nahas Michelle , He Jie , Juhn Frank , Downing Sean , Frampton Garrett M. , Chmielecki Juliann , Ross Jeffrey S. , Zakowski Maureen , Ladanyi Marc , Miller Vincent A. , Stephens Philip J. , Lipson Doron 
Published: May 2015

Abstract
Background: As the number of clinically actionable cancer genes grows and the size of most diagnostic biopsies decreases, next-generation sequencing (NGS) becomes increasingly attractive as a diagnostic tool, as it can detect all classes of genomic alterations in all cancer genes in a single test. However, for NGS to achieve its full utility in the clinic, robust analytical validation and performance comparisons against established detection methodologies are required for each class of targetable genomic alteration.
Methods: Previously, we reported on the development and validation of an NGS-based diagnostic test for accurate detection of clinically-relevant genomic alterations across all exons of 287 cancer genes in routine FFPE specimens (Frampton, et. al, Nat. Biotech. Oct 2013). Here, we present systematic validation of fusion gene detection in the test, enabled by hybrid-selection and deep sequencing of commonly rearranged introns in 19 (or, in an alternative version, 31) genes. We created reference samples reflecting key determinants of detection accuracy for gene fusions, including extent of stromal admixture and variety of partner genes: We obtained 5 solid tumor fusion-bearing cell-lines (2 ALK, 1 RET, 1 ROS1, 1 TMPRSS2) and mixed these into 23 variably sized pools, such that each fusion was represented at 100%, 50%, 33%, 25%, and 20% simulated cellular fraction at least once. Gene fusions were called if a well-mapped cluster of 5 chimeric reads or greater was observed in a targeted intron. We then verified that the observed performance translated to FFPE clinical samples by examining test concordance in 43 tumors (20+/23-) characterized for ALK rearrangement by FISH. Finally, we evaluated the test broadly by assessing detection prevalence of the three most common gene fusions (ALK, ROS1, RET) in 724 lung adenocarcinoma FFPE patient samples, including needle biopsies.
Results: Of 32 tested gene fusion instances in the 28 cell-lines samples, all were successfully detected (sensitivity 100%, 95% CI 89%-100%), with no false positive calls. Robust performance translated to FFPE: of the 20 ALK FISH+ specimens, 18 were unequivocally (+) by NGS, with remaining 2 specimens showing sub-threshold evidence of the events. 22/23 FISH- specimens were NGS-, with the remaining specimen an apparent false (-) by FISH. Clinical lung adenocarcinoma samples showed 5% ALK, 3% RET, and 2% ROS1 rearrangement rate respectively, in line with published data.
Conclusions: We present rigorous validation and performance benchmarks for efficient targeted fusion gene detection for solid tumors in an NGS-based test for use in clinical oncology. Given the ability of NGS to detect a much broader range of genomic alterations than currently available technologies, particularly on limited tissue, this type of testing can be a direct component of patient care and potentially expand targeted treatment options.
Citation Format: Roman Yelensky, Amy Donahue, Geoff Otto, Michelle Nahas, Jie He, Frank Juhn, Sean Downing, Garrett M. Frampton, Juliann Chmielecki, Jeffrey S. Ross, Maureen Zakowski, Marc Ladanyi, Vincent A. Miller, Philip J. Stephens, Doron Lipson. Analytical validation of solid tumor fusion gene detection in a comprehensive NGS-based clinical cancer genomic test. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2014-4699