Segmental Copy Number Variant Detection Using an Amplicon-Based Next-Generation Sequencing Panel for Integrated...

Next-generation sequencing is a first-tier test in molecular oncology, capable of detecting sequence variants and copy number variants (CNVs). Although most amplicon-based gene panels are not designed to detect segmental chromosomal CNVs, they can still be inferred. This study describes a custom analysis for CNV detection using the amplicon-based Oncomine Comprehensive Assay version 3, for critical glioma biomarker 1p/19q codeletion, +7/-10, EGFR amplification, and CDKN2A/B homozygous deletion. The segmental CNV calling algorithm leverages vendor-normalized gene-level CNV data with intersample normalization to control regions. Assay performance was assessed across three cohorts: a retrospective discovery cohort (N = 52) for threshold establishment, a prospective validation cohort (N = 39) for threshold evaluation against parallel fluorescence in situ hybridization (FISH) testing, and a prospective clinical cohort (N = 53) to assess workflow impact. Positive thresholds were defined to achieve 100% specificity, whereas permissive thresholds triggered reflex FISH to ensure 100% sensitivity. Samples with <30% tumor cellularity were excluded. Threshold evaluation in the validation cohort demonstrated 100% concordance with FISH across all biomarkers. Across glioma subtypes, Oncomine Comprehensive Assay version 3 enabled integrated classification in most cases, particularly in oligodendroglioma and glioblastoma, by simultaneously assessing sequence variants and CNVs. Implementation in the prospective clinical cohort reduced required FISH studies by 90%, significantly shortened turnaround times, and decreased molecular testing costs.