Rapid Evaluation of Mutant Exon-11 in c-kit in a Recurrent MCT Case Using CD117 Immunocytofluorescence, FACS-Cell Sorting, and PCR

A 13-year-old, poodle-mixed, male dog was referred to the oncology unit in our faculty’s small animal teaching hospital with the problem of rapid recurrent MCT. The owner and the veterinarian would like to use a tyrosine kinase inhibitor (TKI) for the dog. Therefore, fine-needle aspiration (FNA) was performed to collect the MCT cells and these cells were submitted to our laboratory for the detection of internal-tandem-duplicated (ITD) mutation of exon-11 in c-kit, prior to the treatment. The aim of this paper is to demonstrate the use of combinatorial protocol for the rapid evaluation of ITD mutation in MCT cells harvested by FNA. However, there was no ITD-mutant exon-11 that had been observed in this case. 1. Introduction Canine cutaneous mast cell tumors (MCT) are the second most skin tumors found in dogs. The incidence is probably medium to high in some breeds, such as boxer, bull dog, pug, poodle, labrador retriever, and golden retriever. In general, all MCT patients required an aggressive diagnosis and therapy because the progression of disease is very rapid [1]. Among obscured tumorigenesis of MCT, however, there has been much information from various studies showing that the mutation of exon-11, called internal-tandem-duplication (ITD), of proto-oncogene, c-kit, is involved in MCT formation. Principally, c-kit is a protein-coding gene responsible for KIT (CD117) formation and it is usually expressed in many cell species, such as normal mast cells, melanocytes, and Purkinje cells including MCT cells [2]. KIT is a member of the receptor-tyrosine-kinases class III (RTKs class III). It consists of three functioning domains, extracellular (ectodomain), transmembrane (TM), and intracellular domains, respectively [3]. The intracellular domain is further separated into two subsidiary portions; juxtamembrane encoded by exon-11 and kinase domains encoded by the remaining exons. The function of KIT is triggered when the ectodomain binds to the specific ligand, stem cell factor (SCF), followed by KIT dimerization and cross-autophosphorylation to tyrosine residues on the dimerized KITs. The consequence is to activate the downstream-signaling cascades, which are responsible for the growth and proliferation as well as antiapoptosis of mast cells. In case of MCT, ITD mutation of exon-11 results in the abnormally autonomous KIT dimerization without any specific ligand binding. This mechanism leads to uncontrollable autophosphorylation followed by MCT formation. Nevertheless, this type of mutation also affects MCT therapy when a tyrosine kinase inhibitor (TKI),