Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing

Amandine Billaud; Veronique Verriele; Jonathan Dauvé; Louise-Marie Chevalier; Alain Morel

doi:10.3390/diagnostics10080527

Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing

Diagnostics (Basel). 2020 Jul 29;10(8):527. doi: 10.3390/diagnostics10080527.

Authors

Amandine Billaud^{1

2}, Veronique Verriele², Jonathan Dauvé², Louise-Marie Chevalier^{1

2}, Alain Morel^{1

2}

Affiliations

¹ Université d'Angers, Inserm, CRCINA, F-49000 Angers, France.
² Institut de Cancérologie de l'Ouest Nantes-Angers, 49000 Angers, France.

Abstract

Targeted therapies and, more precisely, EGFR tyrosine kinase inhibitors (TKIs) have been a major improvement in the therapeutic management of EGFR-mutated non-small-cell lung cancers (NSCLCs). Earlier administration of these TKIs throughout tumor progression is imperative to improve patient outcomes. Consequently, studies have focused on refining the characterization of biomarkers, especially concerning the resistance mutation p.Thr790Met of EGFR. Herein, we developed peptide nucleic acid (PNA)-mediated PCR clamping followed by pyrosequencing, favoring enrichment of the mutated fraction. A preamplification step was first added to increase the amplifiable DNA fraction. Throughout the application of our method on DNA extracted from FFPE samples of 46 patients with NSCLC who had relapsed under first-generation EGFR TKI, we evaluated a sensitivity of 93.3% and a specificity of 100%. All 19 patients who were positive for the p.Thr790Met mutation with NGS were also found to be positive with our protocol. The only discordant case was a sample with no mutation detected with NGS, but which was positive with PNA. This protocol allows for the detection of the p.Thr790Met mutation with a sensitivity of 0.5% which will permit earlier detection and an improvement of therapeutic management.

Keywords: EGFR; NSCLC; PNA; T790M; biomarkers; detection sensitivity; non-small-cell lung cancer; p.Thr790Met; peptide nucleic acid; preamplification.