Abstract 1346: Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay
Authors: Hastings Stephanie B. , Hariani Gunjan , Weigman Victor J. , Jiang Tingting , Zhao Chen , Raja Rajiv , Pawlowski Traci , Brohawn Philip , Hurban Patrick 
Published: August 2019

Abstract
Checkpoint inhibitor (CPI) therapy demonstrates a remarkable clinical benefit in many cancer types. However, the ability to successfully select patients who will benefit from CPIs is still limited. Tumor mutational burden (TMB), a measure of the number of somatic mutations per coding area of tumor genome, is a putative biomarker of response showing great promise in CPI and immunotherapy combination trials. The ability to measure TMB from tumor biopsies or plasma samples will be important for clinical adoption of this biomarker. Herein we report on initial performance evaluation of Illumina’s TruSight™ Oncology 500 gene (TSO500) NGS assay for the analysis of TMB in FFPE tissue and plasma cell-free (cf)DNA specimens. Illumina’s TSO500 assay employs hybrid-capture based approach for target enrichment coupled with unique molecular indices to enable low frequency variant detection of single nucleotide variants and indels. This comprehensive cancer panel interrogates relevant cancer biomarkers in &gt;500 cancer genes (~2 Mb) from as little as 40 ng of FFPE DNA or 30 ng of cfDNA. In addition to variant calls, Illumina’s analysis pipeline reports a TMB score and microsatellite instability (MSI) status. Results obtained with the TSO500 TMB assay were compared to our validated whole exome sequencing (WES) TMB assessment for FFPE tissue specimens, with and without matched normal. As previously reported, the WES TMB tumor-only pipeline uses somatic variant classifications determined using a random forest model to generate TMB scores from analysis of tumor FFPE specimens. TSO500 TMB and MSI statues were evaluated in a set of 50 tumor FFPE specimens obtained from late-stage (III+) colorectal and bladder cancer patients. TSO500 TMB results correlated well with the WES-derived TMB results, although some differences were observed in the magnitude of high TMB scores. TSO500 TMB levels correlated strongly with the MSI status, with 100% of MSI-high tumors demonstrating high TMB scores (&gt; 20), and greater than 85% of MSI-low tumors characterized as TMB-low (&lt;20%). Additionally, correlations between tissue and blood-based TMB assessments in matched FFPE and plasma specimens are reported. Overall, our results demonstrate a strong correlation between TSO500 panel-based and whole exome sequencing-based TMB results in tissue and report comparisons to TMB calling in liquid biopsies. Our data further confirm that tumor mutational load is strongly connected to DNA damage repair deficiency in colorectal and bladder cancer patients.
Citation Format: Stephanie B. Hastings, Gunjan Hariani, Victor J. Weigman, Tingting Jiang, Chen Zhao, Rajiv Raja, Traci Pawlowski, Philip Brohawn, Patrick Hurban. Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay [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 1346.