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Spontaneously occurring canine mammary gland tumors share many features with human breast cancer, including biological behavior and histologic features. Compared to transgenic murine model, canine models have advantages including naturally occurring models of human diseases and cancer. In humans, breast cancer is divided into molecular subtypes based on ER, PR, and  HER2  expression. In contrast with humans, few studies have evaluated these subtypes in canine mammary gland tumors, including expression of  HER2 .  HER2  expression in canine mammary tissues has been further complicated by controversy regarding the antibody’s specificity. This study aimed to investigate  c-erbB2  mRNA expression in retrospective formalin-fixed paraffin embedded samples, using RNA  in situ  hybridization with a novel quantitative assay and to compare this method with immunohistochemistry. Using 48 canine mammary tumor samples and 14 non-neoplastic canine mammary tissues, RNA  in situ  hybridization was performed with RNAscope ®  using a canine-specific target gene probe ( ERBB2 ), and quantitative measurement was performed using the housekeeping gene ( POLR2A ) to calculate the target gene/housekeeping gene ratio. The ratio of  ERBB2/POLR2A  was quantified using open-source image analysis programs and compared with the immunohistochemistry results. A significant correlation was observed between the  HER2  immunohistochemistry score and  ERBB2/POLR2A  RNA  in situ  hybridization ( P  < 0.001). When the  HER2  immunohistochemistry score was 3+, significantly higher expression of  HER2  mRNA was observed by RNA  in situ  hybridization. Interestingly,  HER2  mRNA was also observed in non-neoplastic mammary tissues by RNA  in situ  hybridization. This assay potentially facilitates the reliable quantification of mRNA expression levels in retrospective formalin-fixed paraffin-embedded samples. Further studies are required to elucidate the role of  HER2  in canine mammary gland tumors and to implement clinical trials in dogs.

plos one

Quantitative analysis of HER2 mRNA expression by RNA in situ hybridization in canine mammary gland tumors: Comparison with immunohistochemistry analysis