Histological, immunohistochemical and molecular genetic characterisation of canine digital melanomas and mast cell tumours Canine digital melanomas are largely unexplored at the molecular genetic level. In canine cutaneous mast cell tumours, it is known that mutations in the c-KIT gene have prognostic and therapeutic relevance – however, no data existed on whether these relationships also apply to canine digital mast cell tumours. The two studies in the present work therefore focused on the molecular genetic characterisation of canine digital melanomas and mast cell tumours. In addition, these two digital tumours were evaluated histologically. For the digital mast cell tumours, immunohistochemical methods were additionally applied and their statistical relationship to the histological parameters and the c-KIT mutation status was analysed in order to better assess the dignity of this digital tumour. To do so, 86 canine digital melanomas (2014 to 2021; study 1) and 68 canine digital mast cell tumours (2014 to 2022; study 2) were collected from routine submissions to Laboklin GmbH & Co. KG and the corresponding formalin-fixed and paraffin-embedded tumour blocks were used. Remarkably, in the 86 canine digital melanomas of study 1, RAS mutations were identified in 40.7% of the cases. Mutations were distributed between the two exons KRAS and NRAS that were analysed: KRAS exon 2, codons 12 and 13 (22/86 = 25.6%); KRAS exon 3, codon 61 (2/55 = 3.6%); NRAS exon 2, codons 12 and 13 (2/83 = 2.4%); and NRAS exon 3, codon 61 (9/86 = 10.5%). The presence of RAS mutations was not statistically related to histological malignancy criteria or survival. However, the high proportion of RAS mutations and especially KRAS as a RAS isoform frequently affected by mutations differed significantly from what is known from the literature for human melanoma and canine oral melanoma. The finding that RAS mutations are common in canine digital melanoma may be of clinical and therapeutic relevance. Moreover, in contrast to human melanoma, in canine digital melanomas, there were neither mutations found in c-KIT exon 11 nor the canine BRAF V595E variant, which is equivalent to the BRAF V600E variant frequently detected in human melanomas. The copy number of KITLG varied between four and six – KITLG may be involved as a germline mutation in the development of digital melanomas in dogs with dark or black coats, but, so far, this is only a hypothesis due to a lack of control data. KITLG could therefore be a research target to influence the development of canine digital melanomas through breeding. The histological grading of the 68 canine digital mast cell tumours according to Patnaik et al. (1984) resulted in grade I in 22.1% of cases, grade II in 67.6% and grade III in 10.3%. In the histological grading according to Kiupel et al. (2011), the well-differentiated low-grade tumours were found in the majority of cases (86.8%), while the remaining mast cell tumours (13.2%) were poorly differentiated high-grade tumours. If the digital mast cell tumours had merely been assessed histologically, most of them would initially have been considered prognostically favourable. However, 58.8% of the digital mast cell tumours expressed an aberrant KIT pattern and 52.3% had increased Ki-67 antigen expression. Although KIT and Ki-67 showed no statistical relationship, there was statistical significance between KIT and the Patnaik system (p = 0.027) as well as between Ki-67 and the Kiupel system (p = 0.03). The two immunohistochemical parameters were also significantly associated (KIT: p = 0.018; Ki-67: p = 0.006) with internal tandem duplications (ITD) in c-KIT exon 11, which could be detected in 12.7% of cases. In the present study, French bulldogs, which are more likely to develop well-differentiated cutaneous mast cell tumours, were more frequently affected by the poorly differentiated forms at the digit and by c-KIT mutations in exon 11. Canine digital mast cell tumours may have an increased malignant potential, which could be of particular clinical relevance in French bulldogs. In conclusion, the differential consideration of the digit as an anatomical site for canine melanomas and mast cell tumours is important, as molecular genetic differences in particular could be demonstrated. This could be an impetus for future studies that further investigate these tumours and thereby include the factor of tumour site in their analyses.weiterlesen