Clinical and analytical accuracy of a 523 gene panel next-generation sequencing (NGS) assay on formalin-fixed paraffin-embedded (FFPE) solid tumour samples

Abstract Background We developed a tumor-only NGS assay (TruSight assay), a 523 gene panel covering 1.94 megabases (Mb), that detects small DNA variants, provides a microsatellite instability (MSI) score, and estimates tumor mutational burden (TMB) from FFPE tissue DNA. We demonstrate the assay’s ability to detect a variety of biomarkers compared to various orthogonal methods across a range of solid tumor tissues. We also demonstrate accuracy in determining TMB status, using a clinically based cutoff, in non-small cell lung cancer (NSCLC) FFPE samples compared to tumor-normal whole exome sequencing (WES). Methods The study used ∼400 paired tumor-normal FFPE samples from 7 tumor types, including 240 NSCLC. Tumor samples were tested using the verified assay workflow including software analysis. All tumor and normal FFPE were tested with internal WES (with an independent analysis pipeline) and most with a commercial MSI PCR kit (Promega). The TMB score threshold, in mutations (mut)/Mb, was established for the TruSight assay and WES using 119 independent NSCLC FFPE samples and correlated with an external published cutoff. Results Across different tumor types, there was high concordance in small DNA variant calling (>95% positive percent agreement [PPA], >99% negative percent agreement [NPA]) between the TruSight assay and WES. For MSI, >95% PPA and 100% NPA were achieved between the TruSight assay and MSI-PCR. TMB scores from the TruSight assay showed good correlation with WES TMB scores (R2 >0.95 on linear regression). Among the NSCLC samples, TMB scores (range 0 – 184.6 mut/Mb) determined by the TruSight assay correlated well with WES (R2 0.94). Using cutoffs of 12.3 and 8.3 mut/Mb (TruSight assay and WES, respectively) to assign TMB High and Low statuses, overall percent agreement (OPA) was 85%. Conclusions The tumor-only TruSight assay showed high accuracy in detecting biomarkers across a range of solid tumors. The assay showed a good correlation in TMB score and agreement in TMB status with tumor-normal WES in a collection of NSCLC samples. Legal entity responsible for the study The authors. Funding Illumina. Disclosure I. Deras: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. T. Du: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Zhao: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. N. Haseley: Full / Part-time employment: Illumina. A. Yazdanparast: Full / Part-time employment: Illumina. T. Jiang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. A. Mentzer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. A. Purdy: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. B. Crain: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Echegaray: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. D. Lee: Full / Part-time employment: Illumina. J. Lee: Full / Part-time employment: Illumina. J. Silhavy: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. K. O’Brien: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Vijayaraghavan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Garcia: Full / Part-time employment: Illumina. R. Haigis: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. T. Pawlowski: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. J. Dockter: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina.