Abstract
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.
Highlights
Occurring canine mammary gland tumors (CMTs) are the most common tumor type in intact female dogs [1, 2]
We investigated HER2 mRNA levels by assessing c-erbB2 expression in CMTs using RNA in situ hybridization (RNA-ISH) with a new quantitative assay method in retrospective formalin-fixed paraffin-embedded (FFPE) CMTs samples
The histological subtypes of CMTs were classified as simple adenoma (n = 4), complex adenoma (n = 2), simple carcinoma (n = 26), and complex carcinoma (n = 16)
Summary
Occurring canine mammary gland tumors (CMTs) are the most common tumor type in intact female dogs [1, 2]. CMTs in dogs share many epidemiological, biological, and clinical features with human breast cancer including their biological behavior and histologic features [3]. The few actively used prognostic factors for CMTs include histopathological classification and histologic grading, which have been modified to model the criteria for human breast cancer [4,5,6]. Further studies are required to provide a basis for treatments including chemotherapy for CMTs. In humans, breast cancer exhibits well-established intrinsic subtypes (luminal A, luminal B, HER2-enriched, and basal-like), facilitating accurate diagnosis and effective treatment [9]. Studies of HER2 expression in CMTs using IHC with an FDAapproved anti-HER2 polyclonal antibody (A0485, Dako, Glostrup, Denmark) revealed differences in the expression patterns and non-specific cytoplasmic staining patterns in accordance with the criteria for human breast cancer [18, 19]
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