Abstract 417: Comparative analysis of clinically validated NGS-based assays reveals high concordance across short variants
Authors: Dewal Ninad P. , Skoletsky Joel , Yip Wai-Ki , Patriquin Caitlin , He Yuting , Ross Jeffrey S. , Miller Vincent A. , Stephens Philip J. , Burns Christine , Vietz Christine , Li Yali , Pritchard Colin , Sun James X. 
Published: August 2018

Abstract
Background: Traditional FDA-approved assays employing PCR, FISH, or IHC report presence or absence of single markers for therapeutic guidance. Comprehensive Genomic Profiling (CGP) via hybrid-capture NGS-based assays, however, can simultaneously interrogate hundreds of markers to offer a broader picture for clinical actionability. To support the reliability of using hybrid-capture NGS as a clinical platform, we present a comparative analysis on the variant calls detected between two clinically validated assays - FoundationOne CDx (F1CDx), which is the first FDA-approved NGS-based platform that indicates personalized therapies; and a clinically-validated NGS tumor panel assay, performed in an experienced CLIA-certified and CAP-accredited lab in the academic setting. The assays capture 324 and 262 genes, respectively.
Methods: DNA from 188 clinical cases across an array of tumor types was selected to be sequenced by both assays, performed at the respective labs. For F1CDx, CGP was performed using a hybrid-capture, adaptor ligation-based NGS assay to a mean coverage depth of over 500X. Short variants - base substitutions, small insertions and small deletions - were identified and curated. A similar procedure was followed for the academic lab. Resulting variant calls were compared at FMI.
Results: Across the 157 genes that overlap between the assays, 550 clinically reportable short variants that met filtering criteria were identified. Of these, 491 were called by both, 15 were unique to the academic assay, and 44 to F1CDx. This resulted in a bi-directional agreement of ~94% (97.0% with the academic NGS assay as the reference method, and 91.0% using F1CDx as the reference method). The bi-directional agreement for base substitutions alone is (97.4% (discordances due to low allele fraction (AF)), 92.5%, respectively), while for indels in non-homopolymer regions is (96.1%, 89.7%, respectively). Negative agreement between the assays was greater than 99.8%.
Conclusions: Overall concordance among short variant calls between the two NGS assays is high, comparable to reproducibility results at low AF in qPCR. A further advantage over traditional assays is that variant calling is mostly automated but with some curation, whereas with use of IHC or FISH, the call is wholly dependent on pathologic review and interpretation. This supports the ability of robustly validated hybrid-capture based NGS to subsume sequential testing using other technologies commonly employed in oncology in the clinical diagnostics space.
Citation Format: Ninad P. Dewal, Joel Skoletsky, Wai-Ki Yip, Caitlin Patriquin, Yuting He, Jeffrey S. Ross, Vincent A. Miller, Philip J. Stephens, Christine Burns, Christine Vietz, Yali Li, Colin Pritchard, James X. Sun. Comparative analysis of clinically validated NGS-based assays reveals high concordance across short variants [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 417.