Abstract 3114: Analytical validation of Illumina's TruSight Oncology 500 ctDNA assay
Authors: Verhein Kirsten C. , Hariani Gunjan , Hastings Stephanie B. , Hurban Patrick 
Published: September 2020

Abstract
While tissue biopsies and imaging techniques remain the current standard of care, minimally invasive liquid biopsy tests are emerging as compelling complements in the clinical management of cancer patients. The ability to accurately detect low frequency somatic mutations, tumor mutational burden (TMB) and microsatellite instability (MSI) biomarkers from a single plasma sample using a broad-based NGS panel is of high value for biomarker discovery, detection and monitoring. Herein we report the results of our analytical validation of Illumina's TruSight Oncology 500 (TSO500) ctDNA assay which employs a hybrid-capture approach for target enrichment of 523 clinically-relevant cancer genes with unique molecular indices (UMIs) to enable detection of low frequency variants, CNVs, DNA fusions, TMB and MSI analysis.
To characterize performance of small variant detection (SNVs and indels) and TMB analysis using the TSO500 ctDNA assay, we utilized various contrived reference standards with &gt;1,500 unique variants below 5% variant allele frequency (VAF) and over 50 uncharacterized diseased and 20 healthy plasma samples. Cell-free DNA (cfDNA) isolates from healthy donors were used to evaluate the false positive (FP) rate as well as to evaluate potential impacts of blood collection tube (BCT) type on variant calling. For the tube type comparison, we procured 5 matched sets of healthy donor blood in both EDTA and Streck cfDNA BCTs. Where possible, unexpected somatic variants detected in healthy donor samples were orthogonally confirmed using droplet digital PCR (ddPCR). Additionally, to evaluate robustness of input on variant detection and TMB precision, cfDNA isolates from late stage cancer patients were tested at varying input amounts (10, 20, 30ng) in the TSO500 ctDNA assay.
Analytical sensitivity, specificity and positive predictive value (PPV) were evaluated using reference standards. We observed &gt;99% sensitivity for detecting SNVs and &gt;98% for indels at or above 0.5% VAF. Additionally, we observed very high specificity and PPV (&gt;99%), with a false positive rate of &lt; 2 FP per 1.3 Mb down to 0.2% VAF. Further support for high specificity was observed in healthy donor samples where 25/26 apparent false positive somatic variants (0.2-0.7% VAF) were confirmed using ddPCR. Robustness of input studies revealed comparable coverage depth and uniformity can be achieved with 20 - 30 ng of cfDNA. Precision of variant detection and TMB will be reported for a subset of the diseased plasma sample cohort. In conclusion, the TSO500 ctDNA assay enables robust detection of low frequency variants and other clinically relevant biomarkers in EDTA and Streck derived plasma samples with at least 20ng of cfDNA input.
Citation Format: Kirsten C. Verhein, Gunjan Hariani, Stephanie B. Hastings, Patrick Hurban. Analytical validation of Illumina's TruSight Oncology 500 ctDNA assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3114.