Clinical implications of PNA‑sequencing as a complementary test for EGFR mutation analysis in human lung cancer

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.