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

• Published in: Cancer control 2023-01, Vol.30, p.10732748231167257-10732748231167257
• Main Authors: Bontoux, Christophe, Guyard, Alice, Lupo, Audrey, Diallo, Karim, Rebah, Khedidja, Hercent, Agathe, Guenzi, Edouard, Cros, Jérome, Lamoril, Jérome, Leroy, Karen, Theou-Anton, Nathalie
• Format: Article
• Language: English
• Subjects: c-Met protein; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - genetics; Copy number; DNA Copy Number Variations; ErbB-2 protein; Fibroblast growth factor receptor 1; Fibroblast growth factor receptor 2; Fibroblast growth factor receptors; Fluorescence in situ hybridization; Formaldehyde; Gene amplification; High-Throughput Nucleotide Sequencing; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Lung; Lung cancer; Lung carcinoma; Next-generation sequencing; Oncogenes; Paraffin Embedding; Polymerase Chain Reaction; Reproducibility of Results; Retrospective Studies; Technical Note
• ISSN: 1073-2748; 1526-2359
• DOI: 10.1177/10732748231167257

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.