Detection of Nine Oncogenes Amplification in Lung and Colorectal Cancer Formalin-Fixed Paraffin-Embedded Tissue Samples using Combined Next-Generation Sequencing-Based Script and Digital Droplet Polymerase Chain Reaction

Introduction Gene copy number variations have theranostic impact and require reliable methods for their identification. We aimed to evaluate the reliability of combined next-generation sequencing (NGS) and digital droplet PCR (ddPCR) method for gene amplification evaluation. Methods We conducted a retrospective multicentric observational study. MET/ERBB2 amplifications were assessed in patients with lung or colorectal carcinoma (cohort A), from 2016 to 2020, by fluorescence in situ hybridization (FISH)/immunohistochemistry (IHC), NGS and ddPCR. NGS-based script and ddPCR were then used to detect amplifications of 7 additional oncogenes ( EGFR, KRAS, BRAF, FGFR1, FGFR2, FGFR3, PIK3CA) in a cohort of patients (cohort B). Results 55 patients (9 control, 25 ERBB2- amplified and 21 MET- amplified) out of 3779 patients tested were included in cohort A. Correlation coefficient between NGS-based script and FISH/IHC results were .88 for MET (P < .001) and .89 (P < .001) for ERBB2 . Using a threshold ratio of 1.56 with the NGS-based script, the sensitivity was 100% for both genes and the specificity 69% for MET and 90% for ERBB2 , respectively . With an alternative 1.76 threshold, sensitivity was 94% for MET and 96% for ERBB2 , while specificity was 85% for MET and 90% for ERBB2 . Correlation coefficient between FISH and ddPCR ratio was .90 for MET and .88 for ERBB2 . In both cohorts, NGS-based script and ddPCR results were significantly correlated regarding all genes (P < .001). Conclusion Combined NGS-based script and ddPCR method is reliable and easily feasible for the detection of gene amplifications, providing useful data for guided therapy in cancer