Abstract 246: Evaluation of a comprehensive genomic profiling assay for characterization of biomarkers in FFPE tissue
Authors: Li Xinyan , Frady Lauren , Feltz Juliana , Shaban Hiba , Sims Jennifer 
Published: April 2023

Next generation sequencing (NGS)-based comprehensive genomic profiling has emerged as a powerful tool for the large-scale detection of genomic changes. Illumina's TruSight Oncology 500 (TSO500) Comprehensive Assay employs a hybrid-capture approach for target enrichment of 523 clinically relevant cancer genes to enable detection of small variants (SNVs, insertions, and deletions), CNV (copy number variations), TMB (tumor mutational burden), and MSI (microsatellite instability) in DNA and detection of fusions and splice variants in RNA. Briefly, purified DNA and RNA were sheared to the desired length by sonication or heat mediated fragmentation. Fragmented DNA and cDNA then underwent end repair and adapter incorporation, followed by target sequence capture, amplification, and purification using a bead-based approach. The enriched libraries were then pooled and sequenced on the Illumina NovaSeq 6000. Sequencing data (≥ 80M paired-end reads for DNA and ≥ 16 M paired-end reads for RNA) was analyzed using Illumina Primary Analysis pipeline and custom scripts developed for validation data analysis.To characterize the analytical performance of variant detection using the TSO500 Comprehensive Assay following guidelines established by Clinical Laboratory Improvement Amendments of 1988 (CLIA) and College of American Pathologists (CAP), we utilized various commercially available reference standards and over 59 formalin-fixed, paraffin-embedded (FFPE) samples, characterized by selected comparator testing methods. The comparator methods were as follows: for DNA small variants, the QCCP (Q2 Solutions Comprehensive Cancer Panel) NGS assay; for CNV, the NanoString nCounter Cancer Copy Number Assay; and for RNA fusions and splice variants, the TruSeq RNA Exome assay. Variant calls for orthogonal assays were manually curated during review by the Medical Director at Q2 Solutions. Analytical sensitivity, accuracy, precision, specificity, and positive predictive value (PPV) were evaluated using both reference standards and clinical FFPE samples. We observed 95% sensitivity, &gt; 99.99% accuracy and specificity as well as &gt; 99.5% PPV for detecting small DNA variants with variant allele frequency (VAF) above 4.5%. The intra-run and inter-run precision for SNV, insertions/deletions were 99.54% and 99.52%, respectively. Additionally, we observed &gt; 98% accuracy for CNV. The sensitivity, accuracy, specificity, and PPV for fusion and splice variant detection were 94.64%, 99.93%, 100%, and 100%, respectively. Robustness of input studies revealed comparable coverage depth and uniformity can be achieved with varying DNA and RNA input (30, 40, and 60 ng). In conclusion, the TSO500 Comprehensive Assay enables robust detection of variants and other clinically relevant biomarkers in FFPE samples.

Citation Format: Xinyan Li, Lauren Frady, Juliana Feltz, Hiba Shaban, Jennifer Sims. Evaluation of a comprehensive genomic profiling assay for characterization of biomarkers in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 246.