The association between the activating mutations of K-ras gene and poor clinical response to current targeted therapies against epidermal growth factor receptor (EGFR ) such as cetuximab (Erbitux®) and panitumumab (Vectibix®) in colorectal cancer (CRC) has been well documented. Therefore detection of K-ras mutation status will guide effective therapeutical options for patients with CRC. Here we developed a new method for the rapid and reliable detection of K-ras mutations by using a combination of the modified mutation-specific ARMS primers, peptide nucleic acid (PNA) clamping and taqman-MGB probes, named ARMS-PM system. In the ARMS-PM system, the wild-type K-ras DNA is completely blocked by PNA at a higher annealing temperature, and the mutant gene is selectively amplified by the modified ARMS primers and the amplicon extending signals are collected by the taqman-MGB probe at the same time. The ARMS-PM system can detect approximately 10 copies of the mutant gene stably in somatic cells at the background of 10ng genomic DNA. In addition, the premixed reaction was introduced into this system to allow the assay to be completed in only one step of sample loading, which minimizes the potential contamination significantly. By using this assay, we analyzed the K-ras mutations in 1014 Chinese CRC and 205 Chinese lung cancer FFPE samples in comparison with Sanger sequencing. Our results showed that the positive rates were 38.6% and 10.2% 38.0% in CRC and lung cancer samples respectively, which were higher than that measured by Sanger sequencing (38.0% and 7.1%). The coincidence rates of the two methods were 97.3% for the positive samples and 97.7 for the negative samples. The total coincidence rate was 97.8%. In conclusion, our study provides a basis for the detection of somatic mutations in K-ras in a routine clinical setting. The ARMS-PM assay allows the detection of the activating K-ras mutations in FFPE samples with better sensitivity and feasibility than the current assays used in clinic. <!–EndFragment–> Citation Format: Wei-Tao Duan, Xiujuan Wang, Lihong Hu, Feng Zhu, Dehua Yu. A highly sensitive and reliable methodology (ARMS-PM system) for detection of K-ras mutations in FFPE samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1508. doi:10.1158/1538-7445.AM2014-1508
Background The most common activating B-raf mutations (T1799A point mutation) lead to a substitution of valine (V) to glutamic acid (E) at the position 600 of the amino acid sequence. This oncogenic mutation in the B-raf gene constitutively activates the MAPK signaling pathway which results in increased proliferation and inhibition of apoptosis. B-raf V600E mutation was described in approximately 8% of all solid tumors (43% melanomas, 39-67% papillary thyroid carcinomas and 12% colorectal adenocarcinomas). Currently, the gold standard to identify B-raf mutations is sequencing of the tumor DNA. However this method is not practical for routine clinical detection due high risk of contamination and low sensitivity. Recent development of therapeutic intervention using B-raf inhibitors requires an accurate and rapid detection system for B-raf mutations. Methods Here we developed a new methodology for the rapid and reliable detection of B-raf (V600) mutations by combination of the modified real-time PCR primers, peptide nucleic acid (PNA) clamping and MGB probe, named of PM-PCR method. In this assay, the mutant gene is selectively amplified by the modified PCR primers and the amplicon extending signals are collected by the taqman-MGB probe in a real-time fashion, while the amplification of wild-type B-raf DNA is completely blocked by the specifically designed PNA with a higher annealing temperature. The assay procedure includes only one step of sample loading to avoid potential contamination. The PM-PCR method can detect 3-10 copies of the mutant gene stably in somatic cells with background of a total of 10ng wild-type genomic DNA. Tumor samples from 97 patients with malignant melanoma (N=20) and papillary thyroid carcinoma (N=78) were evaluated for the B-raf V600mutation. Genomic DNA was extracted from FFPE tissue sections and analyzed by PCR amplification followed by Sanger sequencing for comparison. Results Among the 97 tested samples, 74% (72/97) of them were positive for B-raf V600 mutation detected by our ARMS-PM assay, while 60.8% (59/97) were detected positive by Sanger sequencing. All the 25 negative cases detected by our assay were confirmed by Sanger sequencing with a concordance rate 100% (25/25). The overall concordance between our assay and Sanger sequencing was (87%) . Conclusion We have developed a new method for detection of B-raf V600 mutation by combination of the modified real-time PCR primers, PNA and MGB probes (PM-PCR method). The new assay demonstrates significantly improved sensitivity and feasibility for clinical application. <!–EndFragment–> Citation Format: Xiujuan Wang, Hui Lan, Qi-Lin Chen, Wei-Tao Duan, Shanshan Zhao, Dehua Yu. A highly sensitive, repaid and reliable assay for detection of human B-raf (V600) mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1511. doi:10.1158/1538-7445.AM2014-1511
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