title: A new microarray substrate for ultra-sensitive genotyping of KRAS and BRAF gene variants in colorectal cancer.
abstract: Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAF(V600E) mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.
authors.affiliation: San Raffaele Scientific Institute, Genomic Unit for the Diagnosis of Human Pathologies, Center for Translational Genomics and Bioinformatics, Milan, Italy. galbiati.silvia@hsr.it
authors.forename: Silvia
authors.initials: S
authors.lastname: Galbiati
authors.forename: Francesco
authors.initials: F
authors.lastname: Damin
authors.forename: Pamela
authors.initials: P
authors.lastname: Pinzani
authors.forename: Irene
authors.initials: I
authors.lastname: Mancini
authors.forename: Serena
authors.initials: S
authors.lastname: Vinci
authors.forename: Marcella
authors.initials: M
authors.lastname: Chiari
authors.forename: Claudio
authors.initials: C
authors.lastname: Orlando
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
mesh_terms: D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
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