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

Chen,Yi-Lin; Lin,Chien-Chung; Yu,Yu-Ting; Chen,Wan-Li; Yang,Shu-Ching; Huang,Wenya; Su,Wu-Chou; Chow,Nan-Haw; Ho,Chung-Liang

doi:10.3892/ol.2023.14126

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

Authors:
- Yi-Lin Chen
- Chien-Chung Lin
- Yu-Ting Yu
- Wan-Li Chen
- Shu-Ching Yang
- Wenya Huang
- Wu-Chou Su
- Nan-Haw Chow
- Chung-Liang Ho
View Affiliations

Affiliations: Molecular Diagnostics Laboratory, Department of Pathology, National Cheng Kung University Hospital, Tainan 704, Taiwan, R.O.C., Department of Internal Medicine, National Cheng Kung University Hospital, Tainan 704, Taiwan, R.O.C., Department of Pathology, National Cheng Kung University Hospital, Tainan 704, Taiwan, R.O.C.
Published online on: November 2, 2023 https://doi.org/10.3892/ol.2023.14126
Article Number: 539
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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.

View Figures

View References

1	Midha A, Dearden S and McCormack R: EGFR mutation incidence in non-small-cell lung cancer of adenocarcinoma histology: A systematic review and global map by ethnicity (mutMapII). Am J Cancer Res. 5:2892–2911. 2015.PubMed/NCBI
2	Hsu PC, Jablons DM, Yang CT and You L: Epidermal growth factor receptor (EGFR) pathway, yes-associated protein (YAP) and the regulation of programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC). Int J Mol Sci. 20:38212019. View Article : Google Scholar : PubMed/NCBI
3	He J, Huang Z, Han L, Gong Y and Xie C: Mechanisms and management of 3rd-generation EGFR-TKI resistance in advanced non-small cell lung cancer (Review). Int J Oncol. 59:902021. View Article : Google Scholar : PubMed/NCBI
4	Gelatti ACZ, Drilon A and Santini FC: Optimizing the sequencing of tyrosine kinase inhibitors (TKIs) in epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC). Lung Cancer. 137:113–122. 2019. View Article : Google Scholar : PubMed/NCBI
5	Guo Y, Song J, Wang Y, Huang L, Sun L, Zhao J, Zhang S, Jing W, Ma J and Han C: Concurrent genetic alterations and other biomarkers predict treatment efficacy of EGFR-TKIs in EGFR-mutant non-small cell lung cancer: A review. Front Oncol. 10:6109232020. View Article : Google Scholar : PubMed/NCBI
6	Vallée A, Le Loupp AG and Denis MG: Efficiency of the Therascreen^® RGQ PCR kit for the detection of EGFR mutations in non-small cell lung carcinomas. Clin Chim Acta. 429:8–11. 2014. View Article : Google Scholar : PubMed/NCBI
7	Thiede C, Bayerdörffer E, Blasczyk R, Wittig B and Neubauer A: Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping. Nucleic Acids Res. 24:983–984. 1996. View Article : Google Scholar : PubMed/NCBI
8	Sano R, Takahashi Y, Nakajima T, Yoshii M, Kubo R, Takahashi K, Kominato Y, Takeshita H, Yasuda T, Tsuneyama H, et al: ABO chimerism with a minor allele detected by the peptide nucleic acid-mediated polymerase chain reaction clamping method. Blood Transfus. 12:431–434. 2014.PubMed/NCBI
9	Chen YL, Lee CT, Lu CC, Yang SC, Chen WL, Lee YC, Yang CH, Peng SL, Su WC, Chow NH and Ho CL: Epidermal growth factor receptor mutation and anaplastic lymphoma kinase gene fusion: Detection in malignant pleural effusion by RNA or PNA analysis. PLoS One. 11:e01581252016. View Article : Google Scholar : PubMed/NCBI
10	Chen YL, Lu CC, Yang SC, Su WP, Lin YL, Chen WL, Huang W, Su WC, Chow NH and Ho CL: Verification of wild-type EGFR status in non-small cell lung carcinomas using a mutant-enriched PCR on selected cases. J Mol Diagn. 16:486–494. 2014. View Article : Google Scholar : PubMed/NCBI
11	Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, et al: New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 45:228–247. 2009. View Article : Google Scholar : PubMed/NCBI
12	Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y, et al: Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 361:947–957. 2009. View Article : Google Scholar : PubMed/NCBI
13	Rotow J and Bivona TG: Understanding and targeting resistance mechanisms in NSCLC. Nat Rev Cancer. 17:637–658. 2017. View Article : Google Scholar : PubMed/NCBI
14	Yeo WL, Riely GJ, Yeap BY, Lau MW, Warner JL, Bodio K, Huberman MS, Kris MG, Tenen DG, Pao W, et al: Erlotinib at a dose of 25 mg daily for non-small cell lung cancers with EGFR mutations. J Thorac Oncol. 5:1048–1053. 2010. View Article : Google Scholar : PubMed/NCBI
15	Tan CS, Gilligan D and Pacey S: Treatment approaches for EGFR-inhibitor-resistant patients with non-small-cell lung cancer. Lancet Oncol. 16:e447–e459. 2015. View Article : Google Scholar : PubMed/NCBI
16	Ricciardi S, Tomao S and de Marinis F: Pemetrexed as first-line therapy for non-squamous non-small cell lung cancer. Ther Clin Risk Manag. 5:781–787. 2009.PubMed/NCBI
17	Okamoto I, Aoe K, Kato T, Hosomi Y, Yokoyama A, Imamura F, Kiura K, Hirashima T, Nishio M, Nogami N, et al: Pemetrexed and carboplatin followed by pemetrexed maintenance therapy in chemo-naive patients with advanced nonsquamous non-small-cell lung cancer. Invest New Drugs. 31:1275–1282. 2013. View Article : Google Scholar : PubMed/NCBI
18	Jeon SH, Kim HW, Kim BN, Kang N, Yeo CD, Park CK, Kim YK, Lee YH, Kim TJ, Lee KY, et al: Comparison of PNA clamping-assisted fluorescence melting curve analysis and PNA clamping in detecting EGFR mutations in matched tumor tissue, cell block, pleural effusion and blood of lung cancer patients with malignant pleural effusion. In Vivo. 33:595–603. 2019. View Article : Google Scholar : PubMed/NCBI
19	Fouz MF and Appella DH: PNA clamping in nucleic acid amplification protocols to detect single nucleotide mutations related to cancer. Molecules. 25:7862020. View Article : Google Scholar : PubMed/NCBI
20	Billaud A, Verriele V, Dauvé J, Chevalier LM and Morel A: Non-small-cell lung cancer-sensitive detection of the p.Thr790Met EGFR alteration by preamplification before PNA-mediated PCR clamping and pyrosequencing. Diagnostics (Basel). 10:5272020. View Article : Google Scholar : PubMed/NCBI
21	Zhang S, Chen Z, Huang C, Ding C, Li C, Chen J, Zhao J and Miao L: Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp. Analyst. 144:1718–1724. 2019. View Article : Google Scholar : PubMed/NCBI
22	Song JU and Lee J: Peptide nucleic acid clamping and direct sequencing in the detection of oncogenic alterations in lung cancer: Systematic review and meta-analysis. Yonsei Med J. 59:211–218. 2018. View Article : Google Scholar : PubMed/NCBI
23	Orum H: PCR clamping. Curr Issues Mol Biol. 2:27–30. 2000.PubMed/NCBI
24	Zhang T, Wan B, Zhao Y, Li C, Liu H, Lv T, Zhan P and Song Y: Treatment of uncommon EGFR mutations in non-small cell lung cancer: New evidence and treatment. Transl Lung Cancer Res. 8:302–316. 2019. View Article : Google Scholar : PubMed/NCBI