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Published on: August 11, 2017
Detection of low-level EGFR T790M mutation in lung cancer tissues
Ji Eun Oh1, Chang Hyeok An, Nam Jin Yoo
1Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Abstract:
Epidermal growth factor receptor (EGFR) gene mutation status is critical to predicting responsiveness to EGFR tyrosine kinase inhibitor (TKI) therapies in non-small cell lung cancer (NSCLC) patients. However, a vast majority of the patients experience recurrence of the cancers by a secondary mutation of EGFR (T790M). Earlier studies suggested evidence that subclones bearing EGFR T790M mutation pre-exist in NSCLCs even prior to the therapies. However, to date, the status of T790M mutation in primary NSCLC is largely known. In this study, we developed an assay using peptide nucleic acid (PNA)-clamping PCR for detection of low-level EGFR T790M mutation. We found that the assay showed the highest sensitivity (0.01% mutation detection) in the clamping condition. We analyzed 147 NSCLC tissues [70 adenocarcinomas (AD), 62 squamous cell carcinomas (SQ), 12 large cell carcinomas (LC), and three adenosquamous carcinomas] that had not been exposed to the TKI therapies, and found 12 (8.2%; 12/147) EGFR T790M mutation in eight AD (11.4%), three SQ (4.8%), and one LC (8.3%) by the PNA-clamping PCR. However, this mutation was not detected by conventional DNA sequencing. Our data indicate that EGFR T790M exists in pretreatment NSCLC at low levels irrespective of histologic types. This study provides a basis for developing an applicable protocol for detecting low-level EGFR T790M mutation in primary NSCLC, which might contribute to predicting recurrence of the tumor in response to the TKI therapies.
Insights
The EGFR T790M mutation, often causing cancer recurrence in non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs), can exist at low levels before therapy. A new PNA-clamping PCR assay detects this pre-existing mutation in primary NSCLC.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Epidermal growth factor receptor (EGFR) mutations are key predictors of tyrosine kinase inhibitor (TKI) response in non-small cell lung cancer (NSCLC).
- Cancer recurrence in NSCLC patients treated with EGFR TKIs is frequently associated with the development of a secondary EGFR T790M mutation.
- Evidence suggests that EGFR T790M-bearing subclones may pre-exist in NSCLC prior to TKI therapy, but their prevalence in primary tumors remains largely uncharacterized.
Purpose of the Study:
- To develop a sensitive assay for detecting low-level EGFR T790M mutations in pre-treatment NSCLC.
- To investigate the presence and prevalence of EGFR T790M mutations in primary NSCLC tissues before TKI therapy.
Main Methods:
- Development of a peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR) assay.
- Optimization of the PNA-clamping PCR assay for maximum sensitivity in detecting low-frequency mutations.
- Analysis of 147 primary NSCLC tissue samples (adenocarcinomas, squamous cell carcinomas, large cell carcinomas, adenosquamous carcinomas) using the developed PNA-clamping PCR assay and conventional DNA sequencing.
Main Results:
- The PNA-clamping PCR assay achieved a high sensitivity of 0.01% for mutation detection under optimal clamping conditions.
- EGFR T790M mutations were detected in 8.2% (12/147) of pre-treatment NSCLC samples.
- The detected EGFR T790M mutations were present at low levels and were not identified by conventional DNA sequencing, occurring across different NSCLC histologic subtypes.
Conclusions:
- EGFR T790M mutations exist at low levels in primary NSCLC, irrespective of histologic type, before exposure to TKI therapy.
- The PNA-clamping PCR assay is a sensitive method for detecting low-level EGFR T790M mutations in pre-treatment NSCLC.
- Detection of pre-existing EGFR T790M mutations may aid in predicting tumor recurrence in NSCLC patients undergoing TKI therapy.
