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Updated: Jul 7, 2026
Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib
Published on: June 26, 2019
Peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based detection test for
Hitoshi Miyazawa1, Tomoaki Tanaka, Yoshiaki Nagai
1Department of Respiratory Medicine, Saitama Medical University, 38 Morohongo, Moroyama-machi, Saitama 350-0495, Japan.
Abstract:
Mutations in the epidermal growth factor receptor (EGFR) are observed in a fraction of non-small-cell lung cancers (NSCLS). EGFR mutation-positive NSCLS responds to gefitinib. Secondary T790M mutation confers gefitinib resistance to NSCLS. A detection test for the T790M mutation was designed based on the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method. The specificity and sensitivity of the test were both greater than 0.99. The test revealed that only a small population of the PC-13 cells carried the T790M mutation. The test also revealed that the T790M mutation was found in none of 151 NSCLC specimens obtained before gefitinib treatment, whereas it was found in four of four specimens obtained from NSCLS that had become refractory to gefitinib. In one patient in whom the L858R-positive EGFR allele was amplified to multiple copies, an L858R-T790M double-mutant allele emerged during the gefitinib therapy. This allele was expressed highly. The T790M mutation detection test based on the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method is sensitive and specific, and is applicable to clinical practice. It detects T790M-positive cells in the course of gefitinib treatment, and thus will help to devise therapies effective for T790M-positive NSCLS.
Insights
A new test accurately detects the T790M mutation, which causes resistance to gefitinib in non-small-cell lung cancer (NSCLC). This T790M mutation emerges during treatment, guiding future therapies for NSCLC patients.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Epidermal growth factor receptor (EGFR) mutations are implicated in non-small-cell lung cancer (NSCLC).
- Gefitinib is an effective treatment for EGFR mutation-positive NSCLC.
- Acquired resistance to gefitinib often arises due to the secondary T790M mutation.
Purpose of the Study:
- To develop and validate a sensitive and specific detection test for the T790M mutation.
- To assess the prevalence of the T790M mutation in NSCLC before and after gefitinib treatment.
- To investigate the emergence of the T790M mutation during gefitinib therapy.
Main Methods:
- Design of a detection test utilizing the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method.
- Evaluation of the test's specificity and sensitivity.
- Analysis of T790M mutation status in NSCLC cell lines and patient specimens before and after gefitinib treatment.
Main Results:
- The T790M detection test demonstrated high specificity and sensitivity (>0.99).
- The T790M mutation was absent in pre-treatment NSCLC specimens but present in all post-treatment refractory specimens.
- Emergence of a highly expressed L858R-T790M double-mutant allele was observed during gefitinib therapy in one patient.
Conclusions:
- The peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method provides a sensitive and specific test for T790M mutations.
- This test is suitable for clinical application, aiding in the management of gefitinib-resistant NSCLC.
- Early detection of T790M mutations can inform the development of targeted therapies for NSCLC.
