title: A comprehensive procedural approach to genotyping KRAS and BRAF from paraffin embedded tissues for diagnostic purposes.
abstract: BACKGROUND
  Mutations in the Kirsten Ras 1 (KRAS) and V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) genes may be predictive of response to drugs directly linked to the Epidermal Growth Factor Receptor (EGFR) signaling pathway.
  
  
  METHODS
  A total of 230 samples from patients with metastatic colorectal cancer were analyzed for KRAS exon 1 and 2 and for BRAF exon 15 mutations. DNA from paraffin-embedded tumor sections was analyzed using microdissection, direct sequencing analysis and allelic separation by cloning.
  
  
  RESULTS
  KRAS mutations were present in 44.3% of the tumor samples. The mutation frequency at hot-spot codons of exon 1 was 84.2%, whereas non-canonical variants had a frequency of 11.8%. Approximately 4% of the cases exhibited concomitant variations. BRAF mutations were present in 3.9% of the tumor samples.
  
  
  CONCLUSIONS
  Our experience suggests that sequential microdissection, direct sequencing and allelic separation by cloning may improve the approach to mutational analysis of KRAS and BRAF in patients with colorectal cancer.
authors: Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele Pisana, via della Pisana 235, 00163 Rome, Italy. raffaele.palmirotta@sanraffaele.it
authors: Raffaele
authors: R
authors: Palmirotta
authors: Giorgia
authors: G
authors: Ludovici
authors: Maria Laura
authors: ML
authors: De Marchis
authors: Barbara
authors: B
authors: Leone
authors: Vincenzo
authors: V
authors: Formica
authors: Giuseppe Maria
authors: GM
authors: Ettorre
authors: Francesco
authors: F
authors: Cavaliere
authors: David
authors: D
authors: Della-Morte
authors: Patrizia
authors: P
authors: Ferroni
authors: Mario
authors: M
authors: Roselli
authors: Fiorella
authors: F
authors: Guadagni
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics; D017931:DNA Primers; D005260:Female; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
chemical_list: D017931:DNA Primers; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf