title: KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancer.
abstract: Treatment of colon carcinoma with the anti-epidermal growth factor receptor antibody Cetuximab is reported to be ineffective in KRAS-mutant tumors. Mutation testing techniques have therefore become an urgent concern. We have compared three methods for detecting KRAS mutations in 59 cases of colon carcinoma: 1) high resolution melting, 2) the amplification refractory mutation system using a bifunctional self-probing primer (ARMS/Scorpion, ARMS/S), and 3) direct sequencing. We also evaluated the effects of the methods of sectioning and coring of paraffin blocks to obtain tumor DNA on assay sensitivity and specificity. The most sensitive and specific combination of block sampling and mutational analysis was ARMS/S performed on DNA derived from 1-mm paraffin cores. This combination of tissue sampling and testing method detected KRAS mutations in 46% of colon tumors. Four samples were positive by ARMS/S, but initially negative by direct sequencing. Cloned DNA samples were retested by direct sequencing, and in all four cases KRAS mutations were identified in the DNA. In six cases, high resolution melting abnormalities could not be confirmed as specific mutations either by ARMS/S or direct sequencing. We conclude that coring of the paraffin blocks and testing by ARMS/S is a sensitive, specific, and efficient method for KRAS testing.
authors.affiliation: Department of Pathology, University of Colorado Denver, Fitzsimons Campus, 12801 E. 17 Ave., Aurora, CO 80045, USA. wilbur.franklin@uchsc.edu
authors.forename: Wilbur A
authors.initials: WA
authors.lastname: Franklin
authors.forename: Jerry
authors.initials: J
authors.lastname: Haney
authors.forename: Michio
authors.initials: M
authors.lastname: Sugita
authors.forename: Lynne
authors.initials: L
authors.lastname: Bemis
authors.forename: Antonio
authors.initials: A
authors.lastname: Jimeno
authors.forename: Wells A
authors.initials: WA
authors.lastname: Messersmith
mesh_terms: D002277:Carcinoma / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D005557:Formaldehyde; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins