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.

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. Variant calls from comparator testing methods 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, > 99.99% accuracy and specificity as well as > 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 > 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). Interfering substances such as ethanol, xylene, and paraffin had no to minimal impact to assay performance in terms of quality control metrics or variant calling capability. In conclusion, the TSO500 Comprehensive Assay enables robust detection of variants and other clinically relevant biomarkers in FFPE samples.

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