title: Cycleave polymerase chain reaction method is practically applicable for V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)/V-raf murine sarcoma viral oncogene homolog B1 (BRAF) genotyping in colorectal cancer.
abstract: Activating V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and V-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene mutations are important predictive markers for antiepidermal growth factor receptor chemotherapy in colorectal cancer (CRC). However, a rapid and accurate assay for KRAS/BRAF mutation detection from routine pathological specimens is lacking in clinical practice. We applied the cycleave polymerase chain reaction (PCR) method to routine KRAS/BRAF genotyping of CRC patients at our institution from 2001 to 2009. The accuracy of cycleave PCR genotyping was shown by the high concordance with reverse transcriptase-PCR-coupled direct sequencing. KRAS gene mutations were analyzed successfully from small biopsy or cytology specimens. Although some surgical specimens could not be evaluated by cycleave PCR, corresponding biopsy specimens could be used instead. This PCR failure observed for some biopsy specimens may have been a result of the use of formalin fixation, as overfixation of surgical specimens by formalin impaired PCR amplification. In conclusion, cycleave PCR is practically applicable to KRAS/BRAF genotyping using small amounts of biopsied tumor cells. Care must be taken in the selection of pathological specimens for KRAS/BRAF testing.
authors.affiliation: Department of Clinical Oncology, Aichi Cancer Center Hospital, Kanokoden, Chikusa-ku, Nagoya, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Yokota
authors.forename: Noriko
authors.initials: N
authors.lastname: Shibata
authors.forename: Takashi
authors.initials: T
authors.lastname: Ura
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takahari
authors.forename: Kohei
authors.initials: K
authors.lastname: Shitara
authors.forename: Kei
authors.initials: K
authors.lastname: Muro
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Yatabe
mesh_terms: D000818:Animals; D015179:Colorectal Neoplasms / Q000235:genetics*; D017931:DNA Primers; D005784:Gene Amplification; D011905:Genes, ras / Q000235:genetics*; D005838:Genotype; D006801:Humans; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012513:Sarcoma, Experimental / Q000235:genetics*; D018631:ras Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; C482120:Braf protein, mouse; C482123:Braf protein, rat; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins