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. 2023 Jul 4;28(1):15.
doi: 10.1007/s10911-023-09543-z.

SFRP1 Expression is Inversely Associated With Metastasis Formation in Canine Mammary Tumours

Judith Seitz  1 , Alan Bilsland  2 , Chloé Puget  3 , Ian Baasner  3 , Robert Klopfleisch  3 , Torsten Stein  4
Affiliations

SFRP1 Expression is Inversely Associated With Metastasis Formation in Canine Mammary Tumours

Judith Seitz et al. J Mammary Gland Biol Neoplasia. .

Abstract

Background: Canine 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.

Method: A 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.

Results: While the 18-gene signature as a whole did not have any prognostic power, a subset of three RNAs: Col13a1, Spock2, and Sfrp1, 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-antagonist Sfrp1 showed 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).

Conclusion: The 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.

Keywords: Canine Mammary Tumours; Metastasis; RNA Signature; SFRP1.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Complete separation of the 27 cases of canine mammary cancer based on the sum of expressions of Col13a1, Spock2, and Sfrp1 mRNAs. A Lymph node positive cases (y-axis = 1) and negative cases (y-axis = 0) as defined by [19] were completely separated by the weighted sum of Col13a1, Spock2, and Sfrp1 expression: -2.04 × Col13a1 – 0.968 × Sfrp1 – 1.27 × Spock2. These coefficients are scaled from those observed in standard logistic regression at the maximum iteration. As discussed in the main text and Table 1, Firth’s method of penalised logistic regression was subsequently employed to generate finite coefficient estimates. Group separation of tumours with (LNpos) and without metastases (LNneg), as defined in [19] with an alternative score using these coefficients is also shown (right panel) (B). C Kaplan–Meier survival analysis using RNA sequencing data from the Kim et al. [25] dataset of 158 CMTs showed that patients within the lowest expression quartile (Q1) for the three mRNAs had a significantly lower overall survival (p < 0.005)
Fig. 2
Fig. 2
Comparison of RT-qPCR results for Col13a1, Spock2, and Sfrp1 using total RNA isolated from FFPE-material from metastatic and non-metastatic CMTs, and morphologically normal canine mammary tissue. Box and whisker plots showing the mRNA abundance for Col13a1, Spock2, and Sfrp1 as % of Rps19 expression in CMTs with (metastasis, n = 17) and without (no metastasis; n = 16) tumour spread, as well as in morphologically normal mammary tissue (morph. normal; n = 5). Boxes define the interquartile range, with the median (horizontal line) and average expression (X) shown. Dots show individual outliers. Only Sfrp1 showed a significant difference in abundance levels between CMTs with and without metastases in a Mann–Whitney U Test analysis (p = 0.001)
Fig. 3
Fig. 3
SFRP1 protein expression (IHC) in CMTs with and without metastasis. A Immunohistochemistry for SFRP1 in six randomly chosen examples of CMTs with (M1-6) and without (N1-6) metastasis compared to the mRNA expression levels (B) shown as % of Rps19 expression (bars show standard deviation of replicates) of the same FFPE-blocks showing good correlation between protein and mRNA abundances. Bars represent 200 µm
Fig. 4
Fig. 4
β-catenin protein expression (IHC) in CMTs with and without metastasis. A Immunohistochemistry for β-catenin in the same six examples of CMTs with (M1-6) and without (N1-6) metastasis as shown in Fig. 2. Membrane staining did correlate with metastasis status, but not with SFRP1 expression. Bars represent 200 µm
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