Targeting internal tandem duplications in the FLT3 gene in canine mast cell tumors and a comment to the method

Mast cell tumours (MCTs) are among the most common and clinically significant cutaneous neoplasms in dogs. While their pathogenesis is not yet fully understood, internal tandem duplications (ITDs) in genes encoding receptor tyrosine kinases KIT and FLT3 have been reported as frequent genetic abnormalities. In this study, we analysed 52 canine MCT samples for the presence of ITDs in exons 14 and 15 of the FLT3 gene using PCR with newly designed primers (P1 primers). No ITDs or point mutations were detected by gel electrophoresis and Sanger sequencing, respectively. Subsequently, we reanalysed 15 of these samples using the P2 primers, i.e. the primers previously used in the published study that reported FLT3 ITDs at a high frequency in canine MCTs. Both P1 and P2 primer sets target the same FLT3 region. Using the P2 primers, two additional PCR bands were observed in 66.7 % of the samples; however, these products corresponded to regions within the SRGAP2 and SLC2A9 genes as revealed using Sanger sequencing. These findings highlight the importance of rigorous methodological validation, even when employing previously published procedures, to ensure accuracy in both research and diagnostic settings. Besides, they suggest that ITDs in exons 14 and 15 of the FLT3 gene are not common in MCTs, at least in European dogs.