Immunohistochemical Characterisation of Mismatch Repair Proteins in Feline Cancers: A Pilot Study Using Validated Cross-Reactive Antibodies.

DNA mismatch repair (MMR) deficiency is a clinically important biomarker in human oncology, yet its relevance in feline neoplasia remains poorly understood due to limited characterisation and the absence of validated reagents. In this study, we established a practical immunohistochemistry (IHC) approach for evaluating feline MMR proteins by confirming the cross-reactivity of anti-human MLH1, MSH2 and MSH6 antibodies using CRISPR/Cas9-engineered feline tumour cell lines lacking each target gene. Using these validated antibodies, we conducted a pilot screening to assess MMR protein expression in 83 feline tumours and observed variable reductions or losses of MLH1, MSH2 and MSH6 across several tumour types. Notably, MLH1 expression was lost in all melanoma cases (6/6, 100%) and in most osteosarcomas (14/15, 93%), indicating that MMR perturbations may be particularly relevant in these malignancies. These findings provide the first analytically validated framework for assessing MMR proteins in feline oncology and suggest that MMR dysregulation may be more widespread than previously recognised. Although the clinical sample size is limited, this initial pilot screening maps the preliminary landscape of feline MMR protein expression. Further large-scale validation studies incorporating microsatellite instability testing, methylation analysis and genomic profiling are warranted to clarify the biological significance of these alterations.