||KIT is a receptor tyrosine kinase present on the surface of hematopoietic progenitor cells. Binding of the KIT ligand results in receptor dimerization and activation of cell signaling pathways leading to a proliferative response. A kinase domain activating mutation, D816V, is common in mast cell disease, gastrointestinal stromal tumors (GIST) and core binding factor (CBF) AML. This mutation results in constitutive activation of KIT and confers resistance to tyrosine kinase inhibitors such as imatinib and a poor prognosis in the case of CBF-AML. Thus, testing for D816V mutations in KIT has utility in diagnosis and treatment of a variety of malignancies.
In this assay, KIT mutation D816V (Asp816Val) is detected by allele-specific PCR using two sets of primers in two separate PCR reactions. The first set of primers amplifies exon 17 and creates a 184 bp amplicon which serves as a control fragment for both wild type and mutant D816V. The second set of primers targets the D816V mutation, forming a 90 bp amplicon only when the D816V mutation is present. The two PCR products are mixed and analyzed on the ABI 3100 genetic analyzer. The presense of both 184 bp and 90 bp amplicons indicates the sample is positive for the D816V mutation. When no mutation is present, only a single, 184 bp amplicon is seen.