title: The use of COLD-PCR, DHPLC and GeneScanning for the highly sensitive detection of c-KIT somatic mutations in canine mast cell tumours.
abstract: The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different 'driver' somatic mutations of c-KIT, together with the wild-type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild-type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50-20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5-1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.
authors: Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell'Emilia, Bologna, Italy.
authors: F
authors: F
authors: Gentilini
authors: Center for Applied Biomedical Research, S.Orsola-Malpighi University Hospital, Bologna, Italy.
authors: V
authors: V
authors: Mantovani
authors: Genefast Srl, Genefast Lab, Bazzano, Bologna, Italy.
authors: M E
authors: ME
authors: Turba
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000662:veterinary; D030541:Databases, Genetic; D004283:Dog Diseases / Q000175:diagnosis* / Q000235:genetics*; D004285:Dogs; D012515:Mast-Cell Sarcoma / Q000175:diagnosis / Q000235:genetics / Q000662:veterinary*; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011110:Polymorphism, Genetic; D011379:Prognosis; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D019009:Proto-Oncogene Proteins c-kit
keywords: COLD-PCR; DHPLC; GeneScanning; Mast cell tumours; c-KIT; somatic mutations