Development and Clinical Validation of OncCNV

Abstract

Large-scale tumor molecular profiling has enabled the discovery of diagnostic, prognostic, and therapeutic biomarkers, and expanded the clinical utility of alterations such as gene amplifications (GAMPs), homozygous deletions (HMZ-Dels), and biallelic inactivation (BI) of tumor suppressor genes. Comprehensive clinical detection of these events is essential for optimal patient management. Illumina's TruSight Oncology 500 (TSO500) kit detects multiple biomarkers, including GAMPs for select genes, but does not assess HMZ-Del or BI events. To address this gap, OncCNV, a genome-wide copy number analysis and visualization pipeline that integrates both on-target and off-target probe data from TSO500 sequencing, was developed. Performance optimization evaluated copy number calling tools, on-target and off-target probe selection strategies, off-target bin sizes, and panel-of-normal configurations. Clinical validation was conducted using 132 unique solid tumors characterized by a clinically validated microarray assay. OncCNV showed >96% positive percentage agreement, >99% negative percentage agreement, and >99% accuracy at the assay's established limit of detection (40 ng DNA at 40% tumor content). Sensitivity for HMZ-Del and BI detection decreased to 62%–70% at tumor content of 20%–39% in in silico dilution experiments; however, intrarun, interrun, and interanalyst precision remained >99%. OncCNV extends the analytical capabilities of TSO500 by enabling robust and precise detection of GAMP, HMZ-Del, and BI events, enhancing solid tumor comprehensive molecular profiling.