Abstract
Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.
Highlights
IHC is considered the method of choice for analyzing hormone receptor status in human breast cancer [1], studies are prone to variability due to variation in antibody clones and assay interpretation [2]
The aim of the present study was to investigate a multiplex branched DNA (bDNA) assay for analyzing hormone receptor gene expression in canine mammary tissue comparing fresh frozen and FFPE samples
This might be due to small sample numbers considering that in a recent study, ESR1/progesterone receptor (PGR) in malignant mammary tumors was found to be significantly reduced in 30 neutered females compared to 49 intact females with malignant mammary tumors [21]
Summary
IHC is considered the method of choice for analyzing hormone receptor status in human breast cancer [1], studies are prone to variability due to variation in antibody clones and assay interpretation [2]. Quantitative real-time PCR (qRT-PCR) is a common method for gene expression analysis, it is limited by its low multiplex capacity [6]. A multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. In comparison to qRT-PCR, the bDNA assay amplifies the signal instead of the target gene [8]. The simple assay format with decreasing pipetting errors is a major advantage of the bDNA assay in comparison to qRT-PCR [9]. Its sensitivity allows small quantities of specimens to be analyzed
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