<scp>PD</scp>‐<scp>L1</scp>, <scp>PD</scp>‐1, and <scp>CTLA</scp>‐4 <scp>mRNA</scp> In Situ Expression by Canine Oral Melanoma Cells and Immune Cells of the Tumour Microenvironment

Canine oral melanoma (OM) exhibits poor prognosis and limited treatment options. The success of immune checkpoint inhibitors (ICIs) in human melanoma has driven interest in similar therapeutic approaches in the dog, although the immunosuppressive mechanisms adopted by canine OM remain unclear. This study aimed to evaluate the expression of the immune checkpoints PD-1/PD-L1 and CTLA-4 by RNAscope in situ hybridization (ISH) in canine OM, to investigate their expression pattern and explore their potential role in melanoma progression. Twenty-four formalin-fixed, paraffin-embedded canine OM were included in the study. PD-L1 expression by tumour cells was detected in 100% melanomas (score 1-3), especially at the host-tumour interface. PD-1 and CTLA-4 expression by tumour cells was detected in 13/24 (54%, score 1-2) and 18/24 (75%, score 1) melanomas, respectively. Dual ISH-immunohistochemistry with Melanoma Triple Cocktail, CD3, CD20 and Iba1 demonstrated the expression of tested immune checkpoints in neoplastic and immune cells. Notably, PD-1 and CTLA-4 were predominantly expressed by tumour-infiltrating T lymphocytes, while PD-L1 was primarily expressed by tumour-associated macrophages. PD-1 expression in neoplastic cells was significantly correlated with mitotic count (p < 0.05), while no associations were found between immune checkpoint expression and disease-free interval or overall survival. Whole tumour PD-L1 and PD-1 expression, assessed by image analysis, correlated to PD-L1 scores in neoplastic cells and the grade of tumour-infiltrating lymphocytes, respectively. Collectively, PD-L1, PD-1 and CTLA-4 likely contribute to immunosuppression in canine OM. Further studies are warranted to investigate whether ISH can serve as a biomarker for selecting patients suitable for ICI treatment.