Detecting gene copy number alterations by comprehensive genomic profiling: a comparative study in a real-world series of FFPE tumor samples

Copy number alterations (CNAs) play a fundamental role in cancer development and constitute a potential tool for tailored treatments. The CNAs recognition in formalin fixed paraffin embedded (FFPE) material, to date, relies on fluorescence in situ hybridization, but the introduction of large-scale next-generation sequencing (NGS) has dramatically improved their discovery at genome-wide level. The detection of CNAs by NGS in FFPE material is, nonetheless, a complex issue, which still requires validation studies. Herein, the CNAs detection by a widely used NGS assay (Oncomine Comprehensive Assay plus®, OCA+) were evaluated in 14 FFPE samples mirroring diagnostic daily practice and compared to a whole-genome assay. OCA+, a targeted DNA panel, showed lower CNAs detection sensitivity and equal specificity for gains and losses. According to proprietary software pipeline, OCA+ accurately identify gains characterized by CN ≥5,2. A much less robust threshold (CN ≤1.18) was identified that maximized the difference between true and false positive losses. Orthogonal FISH tests validated seven CNAs characterized by CN gain ≥6 or complete loss. Considering the CNAs growing significance in precision medicine, our findings further prompt towards a robust validation of NGS detection in FFPE materials. The current molecular diagnostic procedure in our laboratories relies on the amplicon-based Oncomine Comprehensive Assay plus® (OCA+) targeted gene panel (Thermofisher), which beyond the identification of SNVs, tumor mutational burden and microsatellite instability also allows the detection of genes with gain, loss and loss of heterozygosity (LOH) in shorter time than other commercially available target gene panels, compliant with the turn-around-time and the costs for NGS analyses required by the National and Regional healthcare systems. Considering the potential use of NGS as a method for CNAs discovery, the assessment of its accuracy in FFPE material obtained in daily practice conditions must be considered a milestone. To this end, OCA+ performance was investigated in 14 samples from diagnostic routine and results were compared to those obtained using the Whole Genome Array (WGA) OncoScan® (OCS) [8], which has been already successfully validated for CNAs detection in FFPE samples [9, 10]. Potentially “druggable” alterations were investigated by fluorescence in situ hybridization (FISH).