A novel, minimally invasive diagnostic test for KIT exon 11 internal tandem duplications in canine cutaneous mast cell tumours I: Assay development

Background In canine cutaneous mast cell tumours (cMCTs), Internal Tandem Duplications (ITDs) of exon 11 of the KIT receptor tyrosine kinase are associated with higher histological grade and greater risk of metastasis. They also predict response to tyrosine kinase inhibitors (TKIs). Assessment of KIT mutation status provides important information when identifying candidate patients for TKI therapy. Current KIT ITD assays based on semi-quantitative PCR and electrophoretic separation of PCR products lack sensitivity and are not easily comparable between samples. They are limited to analysis of biopsy material from tumours and cannot be used for monitoring of residual disease during routine clinical follow-up, for instance using fine needle aspirates or blood tests. Here, we describe a novel, sensitive, quantitative qPCR assay based on melting curve analysis with potential for use in a wide range of samples during initial clinical assessment/staging and also routine follow up of cMCT patients. Results We identified a minimal region (‘min’ region) of KIT exon 11 commonly amplified in cMCT and designed qPCR primers against that region to produce a small product (44bp) from a wild-type (WT) target. However, an ITD including the min region duplicates primer annealing sites, meaning that additional, larger (>90bp) PCR products are generated. The presence of an ITD-derived product can be distinguished from the WT product by examining peaks on the qPCR melt curves (the WT product produces one peak, the ITD product produces a second distinct peak). The assay was tested on fifteen FFPE cMCT samples with known KIT exon 11 status (five WT, ten with an ITD) and on MDCK normal canine epithelial cell DNA. Considering each individual replicate carried out on all samples, the analytical specificity of the assay was 94% and the sensitivity was 100%. Conclusions We have developed a novel, rapid, qPCR-based assay for the presence of KIT exon 11 ITDs in canine cutaneous Mast Cell Tumours. The assay uses PCR-product melt curve analysis to detect the duplications with high sensitivity and specificity. The assay has the potential for use in a wide range of cMCT samples, including non-surgical samples such as blood or fine needle aspirates.