Abstract 1665: Development of standardized assay and software systems to deliver concordant results across sequencing platforms
Authors: Chowbina Sudhir , Cerqueira Gustavo , Sweeney Deacon , Shewale Shantanu , Hernandez James , Papp Eniko , Sausen Mark , Thompson John , Riley David , Angiuoli Sam 
Published: August 2019

Abstract
Introduction The role of NGS testing to inform clinical decisions has greatly expanded, now including biomarkers such as microsatellite instability (MSI) and Tumor Mutational Burden (TMB) for immune therapies. As oncology adopts widespread clinical use of NGS, standardization becomes essential. PGDx is developing IVD NGS assays optimized for these complex and comprehensive tumor profiling applications. Here, we describe the analytical performance for detection of various analytes using the PGDx elioTM tissue hybrid capture-based assay on the Ion Torrent S5 NGS platform. Notably, standardized and systematic design of wet chemistry and bioinformatics algorithms in combination yields high-performing results, regardless of sequencing platforms.
Method The comprehensive PGDx assay covers the coding regions of &gt;500 genes, complex structural alterations, and genomic signatures associated with solid cancers. Specifically, single-base substitutions (SBS), amplifications, fusions, MSI and TMB are analyzed. A custom bioinformatics pipeline was developed to accommodate single-end Ion Torrent reads with analyte-specific algorithms. PGDx’s VariantDx software was used to detect variants with allele frequencies &gt; 2%. MSI, amplifications and rearrangements were identified using proprietary methods and algorithms. TMB was calculated from non-synonymous somatic variants across the panel, excluding common germline alterations. TMB was normalized to the targeted region to achieve a mutation density of mutations per megabase. To determine analytical accuracy, we compared TMB values from the PGDx assay against a validated whole-exome sequencing (WES) method for 93 lung cancer patients.
Results TMB results demonstrated a Spearman correlation coefficient of &gt;0.90 compared to whole-exome TMB. MSI and amplifications were detected with 100% analytical accuracy, compared to orthogonal Illumina-based methods (n=19). SBS achieved &gt;94% agreement compared to samples sequenced with Illumina-based methods, covering the same &gt;500 gene panel. ROS1 and RET rearrangements were detected in 100% of 20 cell-line replicates obtained from Horizon®.
Conclusion Collectively, these data indicate the systematic co-development of a hybrid capture-based NGS assay with bioinformatics software is practical on the Ion Torrent S5 NGS platform. PGDx elio comprehensive tissue assays may be utilized for detection of SBSs, amplifications, fusions, MSI and TMB on the Ion Torrent S5 NGS platform with robust correlation to Illumina-based results and gold-standard WES.
Citation Format: Sudhir Chowbina, Gustavo Cerqueira, Deacon Sweeney, Shantanu Shewale, James Hernandez, Eniko Papp, Mark Sausen, John Thompson, David Riley, Sam Angiuoli. Development of standardized assay and software systems to deliver concordant results across sequencing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1665.