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.