Rapid and Sensitive Detection ofKRASMutation 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

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, PC-qPCR 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.

Comparison of PNA clamping and direct sequencing for detecting KRAS mutations in matched tumour tissue, cell block, pleural effusion and serum from patients with malignant pleural effusion

“…Peptide nucleic acid (PNA) is a synthetic nucleic acid that binds to its complementary DNA or RNA sequence . The characteristics of PNA are: (i) higher stability of the PNA/DNA complex compared with the corresponding DNA/DNA complex; (ii) higher specificity of PNA binding to DNA; and (iii) inefficiency of PNA as a primer for DNA polymerases. PNA clamping has the advantages of higher sensitivity, speed, simplicity and lower cost compared with the standard direct sequencing, although it cannot be used to detect new mutations.…”

Detection ofBRAFV600EMutations in Papillary Thyroid Carcinomas by Peptide Nucleic Acid Clamp Real-Time PCR: A Comparison with Direct Sequencing

“…It is necessary to apply a more sensitive and specific method for the detection of BRAF V600E mutations in PTC for diagnosis and treatment. The peptide nucleic acid clamp real-time PCR (PNAcqPCR) procedure has been developed for enhanced amplification,20 and has successively detected KRAS mutations in colorectal cancers with specificity and sensitivity 21. In this study, we analyzed 200 PTCs for the presence of the BRAF V600E mutation by the two methods to evaluate their efficiency for BRAF V600E mutant detection, and the results of BRAF V600E in PTC were analyzed with clincopathologic parameters.…”

KRAS mutation testing in colorectal cancer as an example of the pathologist’s role in personalized targeted therapy: a practical approach

“…In addition, this method detected more occult metastases in lymph nodes than standard HE analysis [117]. In another study [98], the PNAClamp test detected mutations in 1% of the mutant cells, while direct sequencing barely found mutations in 20-50% of the mutant cells. The PNA-Clamp test is a very sensitive method for detecting mutants in a very small amount of DNA (optimal range: 10-25 ng of total DNA), and it is suitable for KRAS mutation testing in small biopsy specimens (Fig.…”

“…The PNA-Clamp test is a very sensitive method for detecting mutants in a very small amount of DNA (optimal range: 10-25 ng of total DNA), and it is suitable for KRAS mutation testing in small biopsy specimens (Fig. 2) [88,98,118].…”