Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing
Targeted therapies and, more precisely, <i>EGFR</i> tyrosine kinase inhibitors (TKIs) have been a major improvement in the therapeutic management of <i>EGFR</i>-mutated non-small-cell lung cancers (NSCLCs). Earlier administration of these TKIs throughout tumor progression is...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-07-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/10/8/527 |
| _version_ | 1797560970294329344 |
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| author | Amandine Billaud Veronique Verriele Jonathan Dauvé Louise-Marie Chevalier Alain Morel |
| author_facet | Amandine Billaud Veronique Verriele Jonathan Dauvé Louise-Marie Chevalier Alain Morel |
| author_sort | Amandine Billaud |
| collection | DOAJ |
| description | Targeted therapies and, more precisely, <i>EGFR</i> tyrosine kinase inhibitors (TKIs) have been a major improvement in the therapeutic management of <i>EGFR</i>-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 <i>EGFR</i>. 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 <i>EGFR</i> 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. |
| first_indexed | 2024-03-10T18:07:51Z |
| format | Article |
| id | doaj.art-24d585ecb5f94e1db54e5a95d438559e |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-10T18:07:51Z |
| publishDate | 2020-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-24d585ecb5f94e1db54e5a95d438559e2023-11-20T08:20:57ZengMDPI AGDiagnostics2075-44182020-07-0110852710.3390/diagnostics10080527Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and PyrosequencingAmandine Billaud0Veronique Verriele1Jonathan Dauvé2Louise-Marie Chevalier3Alain Morel4Université d’Angers, Inserm, CRCINA, F-49000 Angers, FranceInstitut de Cancérologie de l’Ouest Nantes-Angers, 49000 Angers, FranceInstitut de Cancérologie de l’Ouest Nantes-Angers, 49000 Angers, FranceUniversité d’Angers, Inserm, CRCINA, F-49000 Angers, FranceUniversité d’Angers, Inserm, CRCINA, F-49000 Angers, FranceTargeted therapies and, more precisely, <i>EGFR</i> tyrosine kinase inhibitors (TKIs) have been a major improvement in the therapeutic management of <i>EGFR</i>-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 <i>EGFR</i>. 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 <i>EGFR</i> 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.https://www.mdpi.com/2075-4418/10/8/527peptide nucleic acidPNApreamplificationnon-small-cell lung cancerNSCLCEGFR |
| spellingShingle | Amandine Billaud Veronique Verriele Jonathan Dauvé Louise-Marie Chevalier Alain Morel Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing Diagnostics peptide nucleic acid PNA preamplification non-small-cell lung cancer NSCLC EGFR |
| title | Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing |
| title_full | Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing |
| title_fullStr | Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing |
| title_full_unstemmed | Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing |
| title_short | Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing |
| title_sort | non small cell lung cancer sensitive detection of the p thr790met egfr alteration by preamplification before pna mediated pcr clamping and pyrosequencing |
| topic | peptide nucleic acid PNA preamplification non-small-cell lung cancer NSCLC EGFR |
| url | https://www.mdpi.com/2075-4418/10/8/527 |
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