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Updated: Jul 9, 2025
Author Spotlight: Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
Clinical implications of PNA‑sequencing as a complementary test for EGFR mutation analysis in human lung cancer
Yi-Lin Chen1,2,3, Chien-Chung Lin4, Yu-Ting Yu5
1Molecular Diagnostics Laboratory, Department of Pathology, National Cheng Kung University Hospital, Tainan 704, Taiwan, R.O.C.
Abstract:
Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line regimen for the treatment of non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, false-negative results are occasionally observed, even with FDA-approved molecular tests. Such examples in have been reported in our pilot study showing a slightly upward-shifted amplification curve using commercial reverse transcription-quantitative (RT-q)PCR. Verification using peptide nucleic acid (PNA) clamping-sequencing, which has a sensitivity of ~0.1%, may allow better prediction of which patients will benefit from EGFR-TKI therapy. To confirm this hypothesis, samples were prospectively collected from 1,783 lung cancer cases diagnosed in National Cheng Kung University Hospital between 2012-2018. An independent lung cancer cohort of 1,944 cases was also recruited from other hospitals. The clinical significance of mutant-enriched PCR with PNA-sequencing was analyzed and patient outcomes were followed. A total of 17 of 34 cases (50%) were found to harbor EGFR mutations by PNA-sequencing. A total of 22 cases were discovered in the independent lung cancer cohort, and 14 of these (63.6%) cases had EGFR mutations. TKIs were administered to 14 of the 17 mutation-positive patients, and a partial response was observed in 4 cases and stable disease in 10 cases. Patients with EGFR mutations receiving a TKI regimen had a longer overall survival (OS) (median: 40.0 vs. 10.0 months) compared with those without treatment. The difference in OS was not significant. Based on the results of the present study, combining RT-qPCR with PNA-sequencing may be a practical supplementary technology in a clinical molecular laboratory for a subset of lung cancer patients in selection of EGFR TKI therapy.
Insights
False-negative EGFR mutation results in non-small cell lung cancer (NSCLC) can be reduced by combining RT-qPCR with PNA-sequencing. This approach improves the selection of patients who will benefit from epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are standard first-line treatment for non-small cell lung cancer (NSCLC) with EGFR mutations.
- Current FDA-approved molecular tests can yield false-negative results, potentially excluding eligible patients from effective therapy.
- Pilot studies indicated subtle amplification curve shifts in RT-qPCR for some EGFR mutations.
Purpose of the Study:
- To evaluate the clinical utility of peptide nucleic acid (PNA) clamping-sequencing as a supplementary diagnostic tool for EGFR mutations in NSCLC.
- To determine if this combined approach improves the identification of patients who can benefit from EGFR-TKI treatment.
- To analyze the impact of EGFR mutations detected by PNA-sequencing on patient outcomes and overall survival.
Main Methods:
- Prospective collection of 1,783 lung cancer cases from National Cheng Kung University Hospital (2012-2018).
- Recruitment of an independent cohort of 1,944 lung cancer cases from other hospitals.
- Analysis of mutant-enriched PCR with PNA-sequencing for EGFR mutations and follow-up of patient outcomes.
Main Results:
- PNA-sequencing identified EGFR mutations in 50% (17/34) of cases from the primary cohort and 63.6% (14/22) from the independent cohort.
- Among 17 mutation-positive patients receiving TKIs, 4 showed partial response and 10 had stable disease.
- Patients with EGFR mutations treated with TKIs demonstrated longer overall survival (median 40.0 months) compared to untreated patients (median 10.0 months), though this difference was not statistically significant.
Conclusions:
- Combining reverse transcription-quantitative PCR (RT-qPCR) with PNA-sequencing offers a practical supplementary method for clinical molecular laboratories.
- This approach can enhance the accurate selection of non-small cell lung cancer patients for EGFR TKI therapy.
- Improved detection of EGFR mutations may lead to better patient stratification and potentially improved treatment outcomes.
