Abstract / Summary
Abstract 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. Here, we have tested our new qPCR-based assay for KIT exon 11 ITDs, the minA assay, on a cohort of canine cMCT formalin-fixed paraffin-embedded (FFPE) tissue. We have also assessed its suitability for use with fine needle aspirates (FNAs) and ‘liquid biopsy’ (blood/plasma samples). Results DNA was extracted from nineteen archival FFPE samples. DNA extraction failed in one sample. Two additional samples consistently failed to give interpretable PCR results. Of the remaining sixteen, the minA assay correctly identified nine samples as wild type, seven with ITDs (confirmed by exon 11 sequencing; 100% sensitivity and specificity of interpretable samples). The ITD sequence could be diluted 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 tested in seven FNA cytology samples; in one of these (from a lymph node containing metastatic tumour cells) an ITD was detected. The minA assay was also able to detect the exon 11 ITD in cell-free DNA isolated from plasma of dogs with cMCTs (nine samples from eight animals). 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.