A novel, minimally invasive diagnostic test for KIT exon 11 internal tandem duplications in canine cutaneous mast cell tumours II: proof-of-principle applications

Background The clinical management of canine cutaneous mast cell tumour (cMCT) presents challenges. Treatment decisions are often informed by histopathological and molecular analysis of excisional biopsy samples, however, these may not always be available. Furthermore, current gold standard assays for the most informative of molecular biomarkers, KIT receptor tyrosine kinase exon 11 internal tandem duplications (ITDs), are not quantitative and cannot offer insights into tumour heterogeneity. Here, we have tested the performance and detection limits of our qPCR-based assay for KIT exon 11 ITDs, the minA assay, on a new cohort of canine cMCT formalin-fixed paraffin-embedded (FFPE) tissue. As a proof-of-principle, we have also assessed its suitability for use with fine needle aspirates (FNAs) and ‘liquid biopsy’ (blood/plasma samples). Results The minA assay performed with 100% sensitivity and specificity on sixteen (nine wild type, seven with ITDs) archival FFPE samples, consistent with our previous results. The ITD sequence could be diluted down to 4% of the overall sample and still be detected in a quantitative manner, providing each reaction contained at least 5ng of sample DNA. The minA assay was demonstrated to work in FNA cytology samples and an ITD was detected in a lymph node FNA containing metastatic tumour cells. The minA assay was also able to detect the exon 11 ITD in cell-free DNA isolated from plasma of dogs with cMCTs. However, while some plasma samples gave a strong signal, other cases gave weak or equivocal signals, or, in one known ITD-positive case, no signal at all. Conclusions The minA assay is a rapid, sensitive, specific, quantitative approach to determining KIT exon 11 status in canine cMCT. It is suitable for use in formalin-fixed material and fine needle aspirates, making it ideal for situations where excision biopsy of a mass is not possible. However, for liquid biopsy, better standardisation of sample collection and longitudinal studies are required to understand the clinical significance of detecting (or not detecting) an ITD. This assay represents a significant advance in the management of canine cMCT as well as establishing a new tool to address tumour heterogeneity and response to therapy.