title: High resolution melting analysis of KRAS, BRAF and PIK3CA in KRAS exon 2 wild-type metastatic colorectal cancer.
abstract: BACKGROUND
  KRAS is an EGFR effector in the RAS/RAF/ERK cascade that is mutated in about 40% of metastatic colorectal cancer (mCRC). Activating mutations in codons 12 and 13 of the KRAS gene are the only established negative predictors of response to anti-EGFR therapy and patients whose tumors harbor such mutations are not candidates for therapy. However, 40 to 60% of wild-type cases do not respond to anti-EGFR therapy, suggesting the involvement of other genes that act downstream of EGFR in the RAS-RAF-MAPK and PI3K-AKT pathways or activating KRAS mutations at other locations of the gene.
  
  
  METHODS
  DNA was obtained from a consecutive series of 201 mCRC cases (FFPE tissue), wild-type for KRAS exon 2 (codons 12 and 13). Mutational analysis of KRAS (exons 3 and 4), BRAF (exons 11 and 15), and PIK3CA (exons 9 and 20) was performed by high resolution melting (HRM) and positive cases were then sequenced.
  
  
  RESULTS
  One mutation was present in 23.4% (47/201) of the cases and 3.0% additional cases (6/201) had two concomitant mutations. A total of 53 cases showed 59 mutations, with the following distribution: 44.1% (26/59) in KRAS (13 in exon 3 and 13 in exon 4), 18.6% (11/59) in BRAF (two in exon 11 and nine in exon 15) and 37.3% (22/59) in PIK3CA (16 in exon 9 and six in exon 20). In total, 26.4% (53/201) of the cases had at least one mutation and the remaining 73.6% (148/201) were wild-type for all regions studied. Five of the mutations we report, four in KRAS and one in BRAF, have not previously been described in CRC. BRAF and PIK3CA mutations were more frequent in the colon than in the sigmoid or rectum: 20.8% vs. 1.6% vs. 0.0% (P=0.000) for BRAF and 23.4% vs. 12.1% vs. 5.4% (P=0.011) for PIK3CA mutations.
  
  
  CONCLUSIONS
  About one fourth of mCRC cases wild-type for KRAS codons 12 and 13 present other mutations either in KRAS, BRAF, or PIK3CA, many of which may explain the lack of response to anti-EGFR therapy observed in a significant proportion of these patients.
authors.affiliation: Departments of Genetics, Portuguese Oncology Institute, Porto, Portugal.
authors.forename: Joana G
authors.initials: JG
authors.lastname: Guedes
authors.forename: Isabel
authors.initials: I
authors.lastname: Veiga
authors.forename: Patrícia
authors.initials: P
authors.lastname: Rocha
authors.forename: Pedro
authors.initials: P
authors.lastname: Pinto
authors.forename: Carla
authors.initials: C
authors.lastname: Pinto
authors.forename: Manuela
authors.initials: M
authors.lastname: Pinheiro
authors.forename: Ana
authors.initials: A
authors.lastname: Peixoto
authors.forename: Maria
authors.initials: M
authors.lastname: Fragoso
authors.forename: Ana
authors.initials: A
authors.lastname: Raimundo
authors.forename: Paula
authors.initials: P
authors.lastname: Ferreira
authors.forename: Manuela
authors.initials: M
authors.lastname: Machado
authors.forename: Nuno
authors.initials: N
authors.lastname: Sousa
authors.forename: Paula
authors.initials: P
authors.lastname: Lopes
authors.forename: António
authors.initials: A
authors.lastname: Araújo
authors.forename: Joana
authors.initials: J
authors.lastname: Macedo
authors.forename: Fernando
authors.initials: F
authors.lastname: Alves
authors.forename: Camila
authors.initials: C
authors.lastname: Coutinho
authors.forename: Rui
authors.initials: R
authors.lastname: Henrique
authors.forename: Lúcio L
authors.initials: LL
authors.lastname: Santos
authors.forename: Manuel R
authors.initials: MR
authors.lastname: Teixeira
mesh_terms: D000328:Adult; D001483:Base Sequence; D058534:Class I Phosphatidylinositol 3-Kinases; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D059645:Mutation Rate; D009362:Neoplasm Metastasis; D021141:Nucleic Acid Amplification Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D044366:Transition Temperature; D016896:Treatment Outcome; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
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