title: Simultaneous identification of 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit.
abstract: BACKGROUND
  Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction.
  
  
  METHODS
  Tumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status.
  
  
  RESULTS
  In total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1-9.2), 2.7 months (1.2-4.2), and 1.6 months (1.5-1.7); median OS was 13.8 months (9.2-18.4), 8.2 months (5.7-10.7), and 6.3 months (1.3-11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11-0.44; P < 0.0001; OS: 95% CI, 0.15-0.61; P < 0.0001).
  
  
  CONCLUSIONS
  Our newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA detected in Asian patients were not predictive of clinical benefits from cetuximab treatment, similar to the result obtained in European studies.
authors.affiliation: Department of Gastroenterology and Gastrointestinal Oncology, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. tyoshino@east.ncc.go.jp.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Bando
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshino
authors.forename: Eiji
authors.initials: E
authors.lastname: Shinozaki
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nishina
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yamazaki
authors.forename: Kensei
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yuki
authors.forename: Shinya
authors.initials: S
authors.lastname: Kajiura
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujii
authors.forename: Takeharu
authors.initials: T
authors.lastname: Yamanaka
authors.forename: Katsuya
authors.initials: K
authors.lastname: Tsuchihara
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ohtsu
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D044466:Asian People / Q000235:genetics; D058534:Class I Phosphatidylinositol 3-Kinases; D003062:Codon; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics* / Q000401:mortality; D005260:Female; D006801:Humans; D007564:Japan; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation; D009362:Neoplasm Metastasis; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D016896:Treatment Outcome
chemical_list: D058534:Class I Phosphatidylinositol 3-Kinases; D003062:Codon; D019869:Phosphatidylinositol 3-Kinases; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D011933:Reagent Kits, Diagnostic; C484760:PIK3CA protein, human