Comparison of Direct Sequencing, PNA Clamping-Real Time Polymerase Chain Reaction, and Pyrosequencing Methods for the Detection of EGFR Mutations in Non-small Cell Lung Carcinoma and the Correlation with Clinical Responses to EGFR Tyrosine Kinase Inhibitor Treatment
2
조회
0
다운로드
13
인용
0
참고문헌
키워드
초록
Background: The aims of this study were to evaluate the abilities of direct sequencing (DS), peptide nucleic acid (PNA) clamping, and pyrosequencing methods to detect epidermal growth factor receptor (EGFR) mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) samples and to correlate EGFR mutational status as determined by each method with the clinical response to EGFR tyrosine kinase inhibitors (TKIs). Methods: Sixty-one NSCLC patients treated with EGFR TKIs were identified to investigate somatic mutations in the EGFR gene (exons 18-21). Results: Mutations in the EGFR gene were detected in 38 of the 61 patients (62%) by DS, 35 (57%) by PNA clamping and 37 (61%) by pyrosequencing. A total of 44 mutations (72%) were found by at least one of the three methods, and the concordances among the results were relatively high (82-85%; kappa coefficient, 0.713 to 0.736). There were 15 discordant cases (25%) among the three different methods. Conclusions: All three EGFR mutation tests had good concordance rates (over 82%) for FFPE samples. These results suggest that if the DNA quality and enrichment of tumor cells are assured, then DS, PNA clamping, and pyrosequencing are appropriate methods for the detection of EGFR mutations.
Abstract
Background: The aims of this study were to evaluate the abilities of direct sequencing (DS), peptide nucleic acid (PNA) clamping, and pyrosequencing methods to detect epidermal growth factor receptor (EGFR) mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) samples and to correlate EGFR mutational status as determined by each method with the clinical response to EGFR tyrosine kinase inhibitors (TKIs). Methods: Sixty-one NSCLC patients treated with EGFR TKIs were identified to investigate somatic mutations in the EGFR gene (exons 18-21). Results: Mutations in the EGFR gene were detected in 38 of the 61 patients (62%) by DS, 35 (57%) by PNA clamping and 37 (61%) by pyrosequencing. A total of 44 mutations (72%) were found by at least one of the three methods, and the concordances among the results were relatively high (82-85%; kappa coefficient, 0.713 to 0.736). There were 15 discordant cases (25%) among the three different methods. Conclusions: All three EGFR mutation tests had good concordance rates (over 82%) for FFPE samples. These results suggest that if the DNA quality and enrichment of tumor cells are assured, then DS, PNA clamping, and pyrosequencing are appropriate methods for the detection of EGFR mutations.
참고문헌 (27)
- Paez JG, "EGFR mutations in lung cancer : correlation with clinical response to gefitinib therapy", Science, 304, pp.1497~1500 (2004)
- Lynch TJ, "Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib", N Engl J Med, 350, pp.2129~2139 (2004)
- Penzel R, "EGFR mutation detection in NSCLC: assessment of diagnostic application and recommendations of the German Panel for Mutation Testing in NSCLC", Virchows Arch, 458, pp.95~98 (2011)
- Helland Å, "EGFR gene alterations in a Norwegian cohort of lung cancer patients selected for surgery", J Thorac Oncol, 6, pp.947~950 (2011)
- Pirker R, "Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop", J Thorac Oncol, 5, pp.1706~1713 (2010)
- Eberhard DA, "Biomarkers of response to epidermal growth factor receptor inhibitors in Non-Small-Cell Lung Cancer Working Group: standardization for use in the clinical trial setting", J Clin Oncol, 26, pp.983~994 (2008)
- Kim HJ, "Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer", Lung Cancer, 75, pp.321~325 (2012)
- 김희정, "EGFR 돌연변이 검출에 있어 PNA-Mediated Real-Time PCR Clamping과 직접 염기서열 분석법의 비교 분석", Tuberculosis and Respiratory Diseases, 70(1), pp.21~27 (2011), 대한결핵및호흡기학회
- 이계영, "비소세포폐암에서 PNA-Mediated PCR Clamping을 이용한 EGFR 돌연변이 분석법", Tuberculosis and Respiratory Diseases, 69(4), pp.271~278 (2010), 대한결핵및호흡기학회
- Ronaghi M, "A sequencing method based on real-time pyrophosphate", Science, 281, pp.363~365 (1998)
- Takano T, "Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer", J Clin Oncol, 23, pp.6829~6837 (2005)
- Dufort S, "Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC", J Exp Clin Cancer Res, 30, pp.57~ (2011)
- Pao W, "Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method", Clin Cancer Res, 13, pp.4954~4955 (2007)
- Travis WD, "World Health Organization classification of tumours: pathology and genetics of tumours of the lung, pleura, thymus and heart" (2004), IARC Press
- Goldstraw P, "The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours", J Thorac Oncol, 2, pp.706~714 (2007)
- Yoo SB, "Epidermal growth factor receptor mutation and p53 overexpression during the multistage progression of small adenocarcinoma of the lung", J Thorac Oncol, 5, pp.964~969 (2010)
- Sun PL, "High incidence of EGFR mutations in Korean men smokers with no intratumoral heterogeneity of lung adenocarcinomas: correlation with histologic subtypes, EGFR", TTF-1 expressions, and clinical features, 2012, pp.7, 323~330
- Therasse P, "New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada", J Natl Cancer Inst, 92, pp.205~216 (2000)
- Han HS, "Detection of EGFR mutation status in lung adenocarcinoma specimens with different proportions of tumor cells using two methods of differential sensitivity", J Thorac Oncol, 7, pp.355~364 (2012)
- Choi JJ, "Peptide nucleic acid-based array for detecting and genotyping human papillomaviruses", J Clin Microbiol, 47, pp.1785~1790 (2009)
- 서안나, "Comparison of the DNA Preservation in Neutral-Buffered Formalin Fixed Paraffin-Embedded Tissue and in Non-Buffered Formalin Fixed Paraffin-Embedded Tissue", Journal of Pathology and Translational Medicine, 45(6), pp.549~556 (2011), 대한병리학회
- Greer CE, "Sample preparation and PCR amplification from paraffin-embedded tissues", PCR Methods Appl, 3, pp.113~122 (1994)
- Liu D, "A useful protocol for analyses of mutations of the epidermal growth factor receptor gene", Oncol Rep, 15, pp.1503~1505 (2006)
- Goto K, "An evaluation study of EGFR mutation tests utilized for non-small-cell lung cancer in the diagnostic setting", Ann Oncol, 23, pp.2914~2919 (2012)
- Ahmadian A, "Pyrosequencing: history, biochemistry and future", Clin Chim Acta, 363, pp.83~94 (2006)
- Mok TS, "Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma", N Engl J Med, 361, pp.947~957 (2009)
- Mitsudomi T, "Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence", J Clin Oncol, 23, pp.2513~2520 (2005)