본문 바로가기
KERD
논문KCI 등재

Rapid and Sensitive Detection of KRAS Mutation by Peptide Nucleic Acid-based Real-time PCR Clamping: A Comparison with Direct Sequencing between Fresh Tissue and Formalin-fixed and Paraffin Embedded Tissue of Colorectal Cancer

2

조회

0

다운로드

1

인용

0

참고문헌

주제분류병리학

초록

Background: Rapid and sensitive detection of KRAS mutation is needed to maximize the bene­fits for patients who are being treated with monoclonal antibodies to target the epidermal growth factor receptor in colorectal cancer. The aim of this study is to evaluate the efficacy of the peptide nucleic acid clamp real-time PCR (PCqPCR) as compared to that of direct sequencing (DS) be­tween using fresh colorectal cancer tissue and the matched formalin-fixed and paraffin-embed­ded (FFPE) colorectal cancer tissue. Methods: The efficacy of PCqPCR was evaluated and com­pared with that of DS using fresh tissue and matched FFPE tissue from 30 cases of colorectal cancer. Results: PCqPCR is more sensitive than DS for detecting KRAS mutation. PCqPCR de­tected 1% of mutants in 1 ng DNA. PCqPCR detected mutation in 1% of mutant cells, while DS barely detected, by manual reading, that in 20-50% of mutant cells. In the clinical samples, PC­qPCR detected KRAS mutation in 60.0% while DS detected KRAS mutation in 53.3% of the colorectal cancers. The two methods showed a 100% concordance rate for detecting KRAS muta­tion between the fresh tissue and FFPE tissue. Conclusions: The PCqPCR method is efficiently applicable for the detection of KRAS mutation in a clinical setting.

Abstract

Background: Rapid and sensitive detection of KRAS mutation is needed to maximize the bene­fits for patients who are being treated with monoclonal antibodies to target the epidermal growth factor receptor in colorectal cancer. The aim of this study is to evaluate the efficacy of the peptide nucleic acid clamp real-time PCR (PCqPCR) as compared to that of direct sequencing (DS) be­tween using fresh colorectal cancer tissue and the matched formalin-fixed and paraffin-embed­ded (FFPE) colorectal cancer tissue. Methods: The efficacy of PCqPCR was evaluated and com­pared with that of DS using fresh tissue and matched FFPE tissue from 30 cases of colorectal cancer. Results: PCqPCR is more sensitive than DS for detecting KRAS mutation. PCqPCR de­tected 1% of mutants in 1 ng DNA. PCqPCR detected mutation in 1% of mutant cells, while DS barely detected, by manual reading, that in 20-50% of mutant cells. In the clinical samples, PC­qPCR detected KRAS mutation in 60.0% while DS detected KRAS mutation in 53.3% of the colorectal cancers. The two methods showed a 100% concordance rate for detecting KRAS muta­tion between the fresh tissue and FFPE tissue. Conclusions: The PCqPCR method is efficiently applicable for the detection of KRAS mutation in a clinical setting.

참고문헌 (28)

  1. Jemal A, "Cancer statistics, 2008", CA Cancer J Clin, 58, pp.71~96 (2008)
  2. Fong Y, "Liver resection for colorectal metastases", J Clin Oncol, 15, pp.938~946 (1997)
  3. Karapetis CS, "K-ras mutations and benefit from cetuximab in advanced colorectal cancer", N Engl J Med, 359, pp.1757~1765 (2008)
  4. Freeman DJ, "Association of K-ras mutational status and clinical outcomes in patients with metastatic colorectal cancer receiving panitumumab alone", Clin Colorectal Cancer, 7, pp.184~190 (2008)
  5. Lyon E, "LightCycler technology in molecular diagnostics", J Mol Diagn, 11, pp.93~101 (2009)
  6. Demers DB, "Enhanced PCR amplification of VNTR locus D1S80 using peptide nucleic acid (PNA)", Nucleic Acids Res, 23, pp.3050~3055 (1995)
  7. Kyger EM, "Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping", Anal Biochem, 260, pp.142~148 (1998)
  8. Sun X, "Detection of tumor mutations in the presence of excess amounts of normal DNA", Nat Biotechnol, 20, pp.186~189 (2002)
  9. Thiede C, "Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping", Nucleic Acids Res, 24, pp.983~984 (1996)
  10. Taback B, "Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes", Int J Cancer, 111, pp.409~414 (2004)
  11. Masuda N, "Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples", Nucleic Acids Res, 27, pp.4436~4443 (1999)
  12. Park YN, "Detection of hepatitis C virus RNA using ligation-dependent polymerase chain reaction in formalin-fixed, paraffin-embedded liver tissues", Am J Pathol, 149, pp.1485~1491 (1996)
  13. Monzon FA, "The role of KRAS mutation testing in the management of patients with metastatic colorectal cancer", Arch Pathol Lab Med, 133, pp.1600~1606 (2009)
  14. Egholm M, "PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules", Nature, 365, pp.566~568 (1993)
  15. Orum H, "Single base pair mutation analysis by PNA directed PCR clamping", Nucleic Acids Res, 21, pp.5332~5336 (1993)
  16. Angulo B, "A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations: a morphology-based approach in colorectal carcinoma", J Mol Diagn, 12, pp.292~299 (2010)
  17. Tsiatis AC, "Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications", J Mol Diagn, 12, pp.425~432 (2010)
  18. Dufort S, "Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues", Anal Biochem, 391, pp.166~168 (2009)
  19. Kobunai T, "The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology: a comparison between direct sequencing and real-time PCR", Biochem Biophys Res Commun, 395, pp.158~162 (2010)
  20. Oh JE, "Detection of low-level KRAS mutations using PNA-mediated asymmetric PCR clamping and melting curve analysis with unlabeled probes", J Mol Diagn, 12, pp.418~424 (2010)
  21. Su YH, "Detection of mutated K-ras DNA in urine, plasma, and serum of patients with colorectal carcinoma or adenomatous polyps", Ann N Y Acad Sci, 1137, pp.197~206 (2008)
  22. Di Fiore F, "Clinical interest of KRAS mutation detection in blood for anti-EGFR therapies in metastatic colorectal cancer", Br J Cancer, 99, pp.551~552 (2008)
  23. Oliveira C, "Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status", Hum Mol Genet, 13, pp.2303~2311 (2004)
  24. Edkins S, "Recurrent KRAS codon 146 mutations in human colorectal cancer", Cancer Biol Ther, 5, pp.928~932 (2006)
  25. Allegra CJ, "American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy", J Clin Oncol, 27, pp.2091~2096 (2009)
  26. Cerottini JP, "The type of K-ras mutation determines prognosis in colorectal cancer", Am J Surg, 175, pp.198~202 (1998)
  27. Span M, "A detailed analysis of K-ras point mutations in relation to tumor progression and survival in colorectal cancer patients", Int J Cancer, 69, pp.241~245 (1996)
  28. Scicchitano MS, "Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples", J Histochem Cytochem, 54, pp.1229~1237 (2006)

관련 논문AI추천

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay오우진, 이성학, 이아원, 이연수, 정은선, 최영진, Arthur Minwoo Chung대한병리학회 · Journal of Pathology and Translational Medicine2017 · 제51권 제1호KRAS Mutation Detection in Non-small Cell Lung Cancer Using a Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping Method and Comparative Validation with Next-Generation Sequencing권미정, 이보람, 이보인, 최윤라, 한강민, 한정호대한병리학회 · Journal of Pathology and Translational Medicine2014 · 제48권 제2호Comparison 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 Treatment김지은, 김효진, 이현주, 정진행, Pingli Sun, Xianhua Xu, Yan Jin대한병리학회 · Journal of Pathology and Translational Medicine2013 · 제47권 제1호Peptide Nucleic Acid Clamping Versus Direct Sequencing for the Detection of EGFR Gene Mutation in Patients with Non-small Cell Lung Cancer김규식, 김영철, 박철규, 신홍준, 오인재, 윤성훈, 장진선, 최유덕, 최하영대한암학회 · Cancer Research and Treatment2015 · 제47권 제4호Can Serum be Used for Analyzing the KRASMutation Status in Patients with Advanced Colorectal Cancer?김승태, 김열홍, 성재숙, 장원진, 최윤지, Lihua Jin대한암학회 · Cancer Research and Treatment2015 · 제47권 제4호Peptide Nucleic Acid Clamping and Direct Sequencing in the Detection of Oncogenic Alterations in Lung Cancer: Systematic Review and Meta-Analysis송재욱, 이종후연세대학교의과대학 · Yonsei Medical Journal2018 · 제59권 제2호

이 간행물의 다른 논문 (16건)