title: Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.
abstract: Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10(-5)). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.
authors.affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Sakai
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Tsurutani
authors.affiliation: Department of Biostatistics, Yokohama City University, Yokohama, Kanagawa, Japan.
authors.forename: Takeharu
authors.initials: T
authors.lastname: Yamanaka
authors.affiliation: Department of Pathology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Yoneshige
authors.affiliation: Department of Pathology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Ito
authors.affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Togashi
authors.affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Marco A
authors.initials: MA
authors.lastname: De Velasco
authors.affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Terashima
authors.affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Fujita
authors.affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Shuta
authors.initials: S
authors.lastname: Tomida
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Nakagawa
authors.affiliation: Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Nishio
mesh_terms: D045744:Cell Line, Tumor; D003106:Colon / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D004247:DNA / Q000097:blood / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D020558:GTP Phosphohydrolases / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012007:Rectum / Q000378:metabolism / Q000473:pathology
chemical_list: C117307:KRAS protein, human; D008565:Membrane Proteins; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)