title: [High-throughput screening method of KRAS mutations at codons 12 and 13 in formalin-fixed paraffin-embedded tissue specimens of metastatic colorectal cancer].
abstract: Clinical studies overseas using the therapeutic anti-EGFR monoclonal antibodies, cetuximab or panitumumab against metastatic colorectal cancer(mCRC), have revealed KRAS mutations as a negative predictive marker of response. Accordingly, the Ministry of Health, Labour and Welfare in Japan approved medical reimbursement of the KRAS mutation test in April 2010. Anti-EGFR monoclonal antibody therapies are now used as first-line treatment for patients with mCRC. To advance the simple high-throughput KRAS mutation test, we established a high-throughput screening system for detecting KRAS mutations utilizing Luminex(xMAP)technology(the fluorescent bead-based multiplex analyte profiling method), in combination with the polymerase chain reaction-reverse sequence-specific oligonucleotide method. Here we evaluated the basic performance of our system and confirmed its high specificity and reproducibility in detecting KRAS mutations at codons 12 and 13 in both plasmid DNAs carrying mutant KRAS genes and formalin-fixed paraffin-embedded tissues from mCRC patients. We demonstrated the KRAS mutation status in paraffin-embedded tissues of mCRC and confirmed that the results were comparable to those of the direct sequencing method. Our high-throughput method has an advantage in simultaneous analysis of multiple mutations in one well of 96-well PCR plates, and will advance the KRAS mutation test in clinical laboratories.
authors.affiliation: Medical & Biological Laboratories Co., Ltd., Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Fukushima
authors.forename: Shoko
authors.initials: S
authors.lastname: Yanaka
authors.forename: Kazumi
authors.initials: K
authors.lastname: Murakami
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Abe
authors.forename: Takuya
authors.initials: T
authors.lastname: Koshizaka
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Hara
authors.forename: Chihiro
authors.initials: C
authors.lastname: Samejima
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Kishi
authors.forename: Makoto
authors.initials: M
authors.lastname: Kaneda
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshino
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003062:Codon / Q000235:genetics*; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009154:Mutation; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D016612:Paraffin Embedding / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins