title: Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR.
abstract: Patients with metastatic colorectal carcinoma (mCRC) carrying activating             mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth             factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing             of mCRC patients is mandatory in the clinical setting for the choice of the most             appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR)             is a novel modification of the conventional PCR method that selectively amplifies             minority alleles from a mixture of wild-type and mutant sequences irrespective             of the mutation type or position within the sequence. In this study, we compared             the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the             real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions             of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS             status, we found that Fast COLD-PCR is more sensitive than the conventional PCR             method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations.             The detection of G>C transversions was not improved by either Fast COLD-PCR             or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen             52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples             with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen             and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed             by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were             all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen             or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among             16 samples with <30% tumor cells (12.5%), whereas Therascreen and Fast COLD-PCR             identified 6 mutants (37.5%). These data suggest that Fast COLD-PCR has a higher             clinical sensitivity as compared with conventional PCR in detecting G>C to             A>T changes in the KRAS gene, which represent >90% of the mutations of this             oncogene in CRC.
authors.affiliation: Pharmacogenomic Laboratory, CROM - Centro Ricerche Oncologiche                     di Mercogliano, Avellino, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Carotenuto
authors.forename: Cristin
authors.initials: C
authors.lastname: Roma
authors.forename: Salvatore
authors.initials: S
authors.lastname: Cozzolino
authors.forename: Francesca
authors.initials: F
authors.lastname: Fenizia
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Rachiglio
authors.forename: Fabiana
authors.initials: F
authors.lastname: Tatangelo
authors.forename: Alessia
authors.initials: A
authors.lastname: Iannaccone
authors.forename: Luigi
authors.initials: L
authors.lastname: Baron
authors.forename: Gerardo
authors.initials: G
authors.lastname: Botti
authors.forename: Nicola
authors.initials: N
authors.lastname: Normanno
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D044366:Transition Temperature; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins