Secreted Frizzled-Related Protein 2 Expression in Canine Thyroid Tumors
Deitz K., Formanek M., Metivier K., et al.
The purpose of this study was two-fold: 1) to determine differentially expressed genes in canine thyroid tumor samples when compared to normal thyroid tissue using microarray analysis and 2) to use immunohistochemistry (IHC) to conﬁrm differential protein expression of one gene that was greater than 10-fold up-regulated via microarray analysis. For gene expression, dogs were prospectively enrolled with informed owner consent if they had a likely diagnosis of follicular thyroid carcinoma (FTC). The affected dogs then underwent a biopsy and a 1 cm3 piece of tissue was saved in RNAlater for further analysis. Microarray analysis (5 FTC and 4 histologically normal thyroid glands) was performed using the commercially available Affymetrix GeneChips Canine Genome 2.0 Array. Microarray data were imported into and analyzed using Genedata Expressionist Analysts. A standard t-test was conducted to compare, for each probe set, the mean expression for the FTC group with that of the normal thyroid using a false discovery rate of q o 0.052. A probe set was considered to be differentially expressed if it had a fold change of 2 or greater, a q-value o 0.05 and had valid expression values for all samples. There were 610 differentially expressed probe sets corresponding to 208 unique genes with characterized proteins. Eight genes had a fold change greater than 10. One of these genes was secreted frizzled-related protein 2 (SFRP2), which was greater than 10 times up-regulated in all tumor samples versus normal thyroid, and was chosen for further analysis (IHC). For IHC, twenty archived samples of thyroid neoplasia were chosen (including 5 samples from gene expression studies) along with 4 normal samples (also from gene expression studies) and analyzed with IHC using goat anti-SFRP2 antibody. All samples were given two separate scores for distribution and intensity of staining. Thyroid tumor samples had signiﬁcantly higher distribution and intensity scores (p-value o 0.01) than the normal samples, which lacked immunoreactivity for SFRP2. Results of this study indicate that SFRP2 is up-regulated in canine FTCs versus normal thyroid tissue at both the gene and protein level and may play a role in the pathogenesis of this tumor type.