title: KRAS and BRAF mutations in 203 esophageal squamous cell carcinomas: pyrosequencing technology and literature review.
abstract: BACKGROUND
  Epidermal growth factor receptor (EGFR) signaling is one of the most promising targets for molecular-targeted therapies in esophageal squamous cell carcinoma (ESCC). Thus, the molecular diagnosis of KRAS and BRAF mutations is clinically important in therapeutic decision making. However, the frequency of KRAS and BRAF mutations in ESCCs remains inconclusive because of the limited sample sizes of previous studies (all N ≤ 80). Pyrosequencing is a nonelectrophoretic nucleotide extension sequencing technology that can be used for mutation testing.
  
  
  METHODS
  The frequency of KRAS and BRAF mutations was examined using a nonbiased database of 203 resected ESCCs and a high-throughput pyrosequencing assay.
  
  
  RESULTS
  The validity of the KRAS pyrosequencing method was initially demonstrated by detection of all 4 types of KRAS mutations [c.35G>T (codon 12 GGT>GTT), c.35G>A (codon 12 GGT>GAT), c.34G>T (codon 12 GGT>TGT), c.38G>A mutation (codon 13 GGC>GAC)], which had been previously diagnosed using Scorpion-ARMS technology, in 9 colon cancer tissues (9 of 9; 100 %). Similar results were demonstrated for BRAF mutational status in 3 colon cancer cell lines (HCT116, Colo201, and HT29), which were validated by Sanger dideoxy sequencing. Subsequently, the KRAS mutation was found to be extremely rare (1 of 203; 0.5 %), and the BRAF mutation was absent (0 of 203; 0 %), in the dataset of 203 ESCCs.
  
  
  CONCLUSIONS
  These results suggest that KRAS and BRAF mutations play a limited role in the development of ESCC and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in ESCC.
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Shigaki
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Baba
authors.forename: Masayuki
authors.initials: M
authors.lastname: Watanabe
authors.forename: Keisuke
authors.initials: K
authors.lastname: Miyake
authors.forename: Asuka
authors.initials: A
authors.lastname: Murata
authors.forename: Shiro
authors.initials: S
authors.lastname: Iwagami
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Ishimoto
authors.forename: Masaaki
authors.initials: M
authors.lastname: Iwatsuki
authors.forename: Naoya
authors.initials: N
authors.lastname: Yoshida
authors.forename: Hideo
authors.initials: H
authors.lastname: Baba
mesh_terms: D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics* / Q000556:secondary; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology; D004252:DNA Mutational Analysis; D004938:Esophageal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D005260:Female; D005500:Follow-Up Studies; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008207:Lymphatic Metastasis; D008297:Male; D058990:Molecular Targeted Therapy; D009154:Mutation / Q000235:genetics*; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012196:Review Literature as Topic; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000235:genetics*
chemical_list: D000970:Antineoplastic Agents; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins