A new rapid method for detecting epidermal growth factor receptor mutations in non-small cell lung cancer

Takata,Miyako; Chikumi,Hiroki; Matsunami,Keiji; Kodani,Masahiro; Sakamoto,Tomohiro; Hashimoto,Kazuhiro; Nakamoto,Masaki; Okada,Kensaku; Kitaura,Tsuyoshi; Matsumoto,Shingo; Kurai,Jun; Yamasaki,Akira; Igishi,Tadashi; Burioka,Naoto; Shimizu,Eiji

doi:10.3892/or.2015.3716

A new rapid method for detecting epidermal growth factor receptor mutations in non-small cell lung cancer

Authors:
- Miyako Takata
- Hiroki Chikumi
- Keiji Matsunami
- Masahiro Kodani
- Tomohiro Sakamoto
- Kazuhiro Hashimoto
- Masaki Nakamoto
- Kensaku Okada
- Tsuyoshi Kitaura
- Shingo Matsumoto
- Jun Kurai
- Akira Yamasaki
- Tadashi Igishi
- Naoto Burioka
- Eiji Shimizu
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Affiliations: Division of Medical Oncology and Molecular Respirology, Department of Multidisciplinary Internal Medicine, Tottori University, Yonago-shi, Tottori-ken, Japan, Trust Medical Co., Ltd., Hyogo, Japan, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, Chiba, Japan
Published online on: January 14, 2015 https://doi.org/10.3892/or.2015.3716
Pages: 1040-1048
Copyright: © Takata et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Mutations in the epidermal growth factor receptor (EGFR) gene are associated with a favorable clinical response to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib in non-small cell lung cancer (NSCLC). We present here, a new method for the rapid detection of the two most common EGFR mutations (delE746-A750 and L858R) from clinical samples. The methodology involves the combination of newly designed mutation-specific primers and a novel real-time PCR machine with an innovative thermo-control mechanism that enables ultrarapid PCR. We evaluated this method using a cell mixture composed of various ratios of lung cancer cells harboring mutated or wild-type EGFR, lung cancer tissues obtained by surgery, and a cytology sample obtained by bronchoscopy from a lung cancer patient. In the cell mixture analysis, our method detected 0.1% of cells with delE746-A750 and 1% of cells with L858R among cells with wild-type EGFR. In 143 lung cancer tissues, the result of this assay was concordant with those of direct sequencing in 138 samples. The five samples with discordant results were tested using a PCR-Invader assay and the result matched those of our method at 100%. We also successfully detected EGFR mutations in the lavage obtained from a lung cancer patient. The turnaround time for this method was <10 min, and all steps could be accomplished in <50 min after sample collection. Thus, our novel PCR method offers a rapid, simple, and less expensive test for EGFR mutations and can be applied as a point-of-care diagnostic test.

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1	Ferlay J, Parkin DM and Steliarova-Foucher E: Estimates of cancer incidence and mortality in Europe in 2008. Eur J Cancer. 46:765–781. 2010. View Article : Google Scholar : PubMed/NCBI
2	Azzoli CG, Baker S Jr, Temin S, et al: American Society of Clinical Oncology Clinical Practice Guideline update on chemotherapy for stage IV non-small-cell lung cancer. J Clin Oncol. 27:6251–6266. 2009. View Article : Google Scholar : PubMed/NCBI
3	Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 350:2129–2139. 2004. View Article : Google Scholar : PubMed/NCBI
4	Maemondo M, Inoue A, Kobayashi K, et al: Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 362:2380–2388. 2010. View Article : Google Scholar : PubMed/NCBI
5	Mitsudomi T, Morita S, Yatabe Y, et al: Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 11:121–128. 2010. View Article : Google Scholar
6	Mok TS, Wu YL, Thongprasert S, et al: Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 361:947–957. 2009. View Article : Google Scholar : PubMed/NCBI
7	Rosell R, Carcereny E, Gervais R, et al: Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 13:239–246. 2012. View Article : Google Scholar : PubMed/NCBI
8	Zhou C, Wu YL, Chen G, et al: Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol. 12:735–742. 2011. View Article : Google Scholar : PubMed/NCBI
9	Han JY, Park K, Kim SW, et al: First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung. J Clin Oncol. 30:1122–1128. 2012. View Article : Google Scholar : PubMed/NCBI
10	Paez JG, Janne PA, Lee JC, et al: EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304:1497–1500. 2004. View Article : Google Scholar : PubMed/NCBI
11	Pao W, Miller V, Zakowski M, et al: EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA. 101:13306–13311. 2004. View Article : Google Scholar
12	Shigematsu H, Lin L, Takahashi T, et al: Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 97:339–346. 2005. View Article : Google Scholar : PubMed/NCBI
13	Han SW, Kim TY, Hwang PG, et al: Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol. 23:2493–2501. 2005. View Article : Google Scholar : PubMed/NCBI
14	Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T and Mitsudomi T: Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res. 64:8919–8923. 2004. View Article : Google Scholar : PubMed/NCBI
15	Pao W and Ladanyi M: Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method. Clin Cancer Res. 13:4954–4955. 2007. View Article : Google Scholar : PubMed/NCBI
16	Mitsudomi T and Yatabe Y: Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci. 98:1817–1824. 2007. View Article : Google Scholar : PubMed/NCBI
17	Hall JG, Eis PS, Law SM, et al: Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc Natl Acad Sci USA. 97:8272–8277. 2000. View Article : Google Scholar : PubMed/NCBI
18	Naoki K, Soejima K, Okamoto H, et al: The PCR-invader method (structure-specific 5′ nuclease-based method), a sensitive method for detecting EGFR gene mutations in lung cancer specimens; comparison with direct sequencing. Int J Clin Oncol. 16:335–344. 2011. View Article : Google Scholar : PubMed/NCBI
19	Nagai Y, Miyazawa H, Huqun, et al: Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res. 65:7276–7282. 2005. View Article : Google Scholar : PubMed/NCBI
20	Yatabe Y, Hida T, Horio Y, Kosaka T, Takahashi T and Mitsudomi T: A rapid, sensitive assay to detect EGFR mutation in small biopsy specimens from lung cancer. J Mol Diagn. 8:335–341. 2006. View Article : Google Scholar : PubMed/NCBI
21	Kimura H, Kasahara K, Kawaishi M, et al: Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer. Clin Cancer Res. 12:3915–3921. 2006. View Article : Google Scholar : PubMed/NCBI
22	Takata M, Chikumi H, Miyake N, et al: Lack of AKT activation in lung cancer cells with EGFR mutation is a novel marker of cetuximab sensitivity. Cancer Biol Ther. 13:369–378. 2012. View Article : Google Scholar : PubMed/NCBI
23	Fujimoto T, Konagaya M, Enomoto M, et al: Novel high-speed real-time PCR method (Hyper-PCR): results from its application to adenovirus diagnosis. Jpn J Infect Dis. 63:31–35. 2010.PubMed/NCBI
24	Barbau-Piednoir E, Botteldoorn N, Yde M, Mahillon J and Roosens NH: Development and validation of qualitative SYBR^®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes. Appl Microbiol Biotechnol. 97:4021–4037. 2013. View Article : Google Scholar :
25	Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 25:169–193. 2000. View Article : Google Scholar : PubMed/NCBI
26	Newton CR, Graham A, Heptinstall LE, et al: Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17:2503–2516. 1989. View Article : Google Scholar : PubMed/NCBI
27	Newton CR, Kalsheker N, Graham A, et al: Diagnosis of α₁-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products. Nucleic Acids Res. 16:8233–8243. 1988. View Article : Google Scholar : PubMed/NCBI
28	Ellison G, Zhu G, Moulis A, Dearden S, Speake G and McCormack R: EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples. J Clin Pathol. 66:79–89. 2013. View Article : Google Scholar :
29	Goto K, Satouchi M, Ishii G, et al: An evaluation study of EGFR mutation tests utilized for non-small-cell lung cancer in the diagnostic setting. Ann Oncol. 23:2914–2919. 2012. View Article : Google Scholar : PubMed/NCBI
30	Kim YH, Yang I, Bae YS and Park SR: Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. Biotechniques. 44:495–496. 498500passim. 2008. View Article : Google Scholar : PubMed/NCBI
31	Nitsche A: Oligonucleotide design for in-house real-time PCR applications in microbiology. Real-Τime PCR in Microbiology. Mackay IM: Caister Academic Press; Norfolk: pp. 41–69. 2007