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.

Dongjun Jeong; Yujun Jeong; Jonghyun Lee; Moo Jun Baek; Yongbae Kim; Ji Hye Lee; Hyun Deuk Cho; Mee Hye Oh; Chang Jin Kim

doi:10.4132/KoreanJPathol.2011.45.2.151

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Original Article 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.: Dongjun Jeong, Yujun Jeong, Jonghyun Lee, Moo Jun Baek, Yongbae Kim, Ji Hye Lee, Hyun Deuk Cho, Mee Hye Oh, Chang Jin Kim; Journal of Pathology and Translational Medicine 2011;45(2):151-159
DOI: https://doi.org/10.4132/KoreanJPathol.2011.45.2.151

¹Department of Pathology, Soonchunhyang University College of Medicine, Cheonan, Korea. cjkim@sch.ac.kr
²Seoul High School, Seoul, Korea.
³Department of Surgery, Soonchunhyang University College of Medicine, Cheonan, Korea.
⁴Department of Preventive Medicine, Soonchunhyang University College of Medicine, Cheonan, Korea.

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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.

Keywords: Sequencing;DNA;Peptide nucleic acids;Polymerase chain reaction;Colorectal neoplasms;KRAS gene;

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