Takayuki Ishige1, Sakae Itoga2, Kenichi Sato3, Kouichi Kitamura4, Motoi Nishimura4, Setsu Sawai4, Kazuyuki Matsushita4, Kazufumi Suzuki5, Satoshi Ota6, Hideaki Miyauchi5, Hisahiro Matsubara5, Yukio Nakatani6, Fumio Nomura4. 1. Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan. Electronic address: ishige.t@chiba-u.jp. 2. Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan. 3. Department of Medical Technology and Sciences, International University of Health and Welfare, Fukuoka, Japan. 4. Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan. 5. Department of Frontier Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan. 6. Department of Diagnostic Pathology, Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Pathology, Chiba University Hospital, Chiba, Japan.
Abstract
OBJECTIVES: Recent studies have demonstrated that, in advanced colorectal carcinoma (CRC) patients, extended RAS (in KRAS exons 2-4 and NRAS exons 2-4) and BRAF mutations are negative predictors for anti-EGFR treatment efficacy and negative prognostic factor, respectively. Thus, high-throughput and cost-effective methods for identification of the mutation status are required. DESIGN AND METHODS: We developed a PCR-high-resolution melting (HRM)-based method for screening extended RAS and BRAF mutations, and relative frequency of mutations in formalin-fixed paraffin-embedded samples of CRC was analyzed. RESULTS: Among 93 CRC samples, 29 harbored mutations in KRAS exon 2, and 9 harbored mutations in BRAF exon 15. Analysis of 55 KRAS exon 2 and BRAF exon 15 wild-type CRC samples identified the following mutations: 1/55 in exon 3 and 2/55 in exon 4 of KRAS; 1/55 in exon 2, 3/55 in exon 3, and 0/55 in exon 4 of NRAS. CONCLUSIONS: Our PCR-HRM method will enable rapid determination of the extended RAS and BRAF mutation status prior to anti-EGFR treatment in the clinical setting.
OBJECTIVES: Recent studies have demonstrated that, in advanced colorectal carcinoma (CRC) patients, extended RAS (in KRAS exons 2-4 and NRAS exons 2-4) and BRAF mutations are negative predictors for anti-EGFR treatment efficacy and negative prognostic factor, respectively. Thus, high-throughput and cost-effective methods for identification of the mutation status are required. DESIGN AND METHODS: We developed a PCR-high-resolution melting (HRM)-based method for screening extended RAS and BRAF mutations, and relative frequency of mutations in formalin-fixed paraffin-embedded samples of CRC was analyzed. RESULTS: Among 93 CRC samples, 29 harbored mutations in KRAS exon 2, and 9 harbored mutations in BRAF exon 15. Analysis of 55 KRAS exon 2 and BRAF exon 15 wild-type CRC samples identified the following mutations: 1/55 in exon 3 and 2/55 in exon 4 of KRAS; 1/55 in exon 2, 3/55 in exon 3, and 0/55 in exon 4 of NRAS. CONCLUSIONS: Our PCR-HRM method will enable rapid determination of the extended RAS and BRAF mutation status prior to anti-EGFR treatment in the clinical setting.