title: KRAS gene mutation in a series of unselected colorectal carcinoma patients with prognostic morphological correlations: a pyrosequencing method improved by nested PCR.
abstract: BACKGROUND
  Inhibition of EGFR is a strategy for treating metastatic colorectal cancer (CRC) patients. KRAS sequencing is mandatory for selecting wild-type tumor patients who might benefit from this treatment. DNA from formalin-fixed paraffin-embedded (FFPE) tissues is commonly used for routine clinical detection of mutations, and its amplification succeeds only when all preanalytical histological processes have been controlled. In cases that are not properly processed, the DNA results can be poor, with low peak pyrosequencing findings. We designed and tested a pair of forward and reverse primers for a nested PCR method, followed by pyrosequencing, in a single Latin American institution series of 422 unselected CRC patients, correlating KRAS mutations with pathological and clinical data.
  
  
  METHODS
  Patient DNA samples from tumors were obtained by scraping or laser microdissection of cells from FFPE tissue and extracted using a commercial kit. DNA was first amplified by PCR using 2 primers that we designed; then, nested PCR was performed with the amplicon from the preamplification PCR using the KRAS PyroMark™ Q96 V2.0 kit (Qiagen). Pathological data were retrieved from pathology reports.
  
  
  RESULTS
  KRAS mutation was observed in 33% of 421 cases. Codon 12 was mutated in 76% of cases versus codon 13 in 24%. Right-sided CRCs harbored more KRAS mutations than left-sided tumors, as did tumors that presented with perineural invasion.
  
  
  CONCLUSIONS
  Our findings in this Latin American population are consistent with the literature regarding the frequency of KRAS mutations in CRC, their distribution between codons 12 and 13, and type of nucleotide substitution. By combining nested PCR and pyrosequencing, we achieved a high rate of conclusive results in testing KRAS mutations in CRC samples - a method that can be used as an ancillary test for failed assays by conventional PCR.
authors.affiliation: Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil. Electronic address: maripetaccia@gmail.com.
authors.forename: Mariana Petaccia
authors.initials: MP
authors.lastname: de Macêdo
authors.affiliation: Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil.
authors.forename: Fernanda Machado
authors.initials: FM
authors.lastname: de Melo
authors.affiliation: Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil.
authors.forename: Bianca Cristina Garcia
authors.initials: BC
authors.lastname: Lisboa
authors.affiliation: Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil.
authors.forename: Louise D Brot
authors.initials: LD
authors.lastname: Andrade
authors.affiliation: Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil.
authors.forename: Maria Dirlei Ferreira
authors.initials: MD
authors.lastname: de Souza Begnami
authors.affiliation: Surgical Oncology Department, A.C. Camargo Cancer Center, Brazil.
authors.forename: Samuel Aguiar
authors.initials: SA
authors.lastname: Junior
authors.affiliation: Abdominal Surgery Department, A.C. Camargo Cancer Center, Brazil.
authors.forename: Heber Salvador de Castro
authors.initials: HS
authors.lastname: Ribeiro
authors.affiliation: Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil.
authors.forename: Fernando Augusto
authors.initials: FA
authors.lastname: Soares
authors.affiliation: Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Genomics and Molecular Biology, CIPE/ A.C. Camargo Cancer Center, Brazil.
authors.forename: Dirce Maria
authors.initials: DM
authors.lastname: Carraro
authors.affiliation: Diagnostic Molecular Pathology Laboratory, Anatomic Pathology Department, A.C. Camargo Cancer Center, Brazil; Laboratory of Investigative Pathology, CIPE/A.C. Camargo Cancer Center, Brazil.
authors.forename: Isabela Werneck
authors.initials: IW
authors.lastname: da Cunha
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002277:Carcinoma / Q000175:diagnosis / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D017422:Sequence Analysis, DNA / Q000379:methods*; D018631:ras Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: Colorectal cancer; KRAS gene mutation; Molecular diagnostics; Molecular pathology; Pyrosequencing