BackgroundCanine mammary tumours (CMTs) are the most frequent tumours in intact female dogs and show strong similarities with human breast cancer. In contrast to the human disease there are no standardised diagnostic or prognostic biomarkers available to guide treatment. We recently identified a prognostic 18-gene RNA signature that could stratify human breast cancer patients into groups with significantly different risk of distant metastasis formation. Here, we assessed whether expression patterns of these RNAs were also associated with canine tumour progression.MethodA sequential forward feature selection process was performed on a previously published microarray dataset of 27 CMTs with and without lymph node (LN) metastases to identify RNAs with significantly differential expression to identify prognostic genes within the 18-gene signature. Using an independent set of 33 newly identified archival CMTs, we compared expression of the identified prognostic subset on RNA and protein basis using RT-qPCR and immunohistochemistry on FFPE-tissue sections.ResultsWhile the 18-gene signature as a whole did not have any prognostic power, a subset of three RNAs:Col13a1,Spock2, andSfrp1, together completely separated CMTs with and without LN metastasis in the microarray set. However, in the new independent set assessed by RT-qPCR, only the Wnt-antagonistSfrp1showed significantly increased mRNA abundance in CMTs without LN metastases on its own (p= 0.013) in logistic regression analysis. This correlated with stronger SFRP1 protein staining intensity of the myoepithelium and/or stroma (p< 0.001). SFRP1 staining, as well as β-catenin membrane staining, was significantly associated with negative LN status (p= 0.010 and 0.014 respectively). However, SFRP1 did not correlate with β-catenin membrane staining (p= 0.14).ConclusionThe study identified SFRP1 as a potential biomarker for metastasis formation in CMTs, but lack of SFRP1 was not associated with reduced membrane-localisation of β-catenin in CMTs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10911-023-09543-z.