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

Christophe Bontoux; Alice Guyard; Audrey Lupo; Karim Diallo; Khedidja Rebah; Agathe Hercent; Edouard Guenzi; Jérome Cros; Jérome Lamoril; Karen Leroy; Nathalie Theou-Anton

doi:10.1177/10732748231167257

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

Cancer Control. 2023 Jan-Dec:30:10732748231167257. doi: 10.1177/10732748231167257.

Authors

Affiliations

¹ Laboratory of Clinical and Experimental Pathology, Centre Hospitalier Universitaire de Nice, Université Côte d'Azur, Hospital-Integrated Biobank, Hôpital Pasteur, Nice, France.
² Department of Genetics, University Hospital Bichat-Claude Bernard, AP-HP, Paris University, Paris, France.
³ Department of Pathology, University Hospital Bichat-Claude Bernard, AP-HP, University Paris-Diderot, Paris, France.
⁴ Department of Pathology, Hôpitaux Universitaire Paris Centre, Hôpital Cochin, AP-HP, Université de Paris, Paris, France.
⁵ Department of Pathology, University Hospital Beaujon, AP-HP, University Paris-Diderot, Paris, France.

Abstract

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.

Keywords: amplification; bioinformatics; colorectal cancer; copy number; digital droplet PCR; genes; lung cancer; sequencing data.

Publication types

Observational Study

MeSH terms

Colorectal Neoplasms* / genetics
DNA Copy Number Variations*
Formaldehyde
High-Throughput Nucleotide Sequencing
Humans
In Situ Hybridization, Fluorescence
Lung
Oncogenes
Paraffin Embedding
Polymerase Chain Reaction
Reproducibility of Results
Retrospective Studies

Substances

Formaldehyde