Metallothionein expression in benign and malignant canine mammary gland tumours

The presence of metallothioneins (MTs) were demonstrated immunohistochemically using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in canine mammary tumours. In a semiquantitative analysis MT expression in the tumour cells was observed in 54/54 cases of benign and 32/40 malignant mammary neoplasms. A statistically significant difference at the level of P<0.01 was observed for MT expression between benign and malign mammary tumours in terms of immunoreactivity score. It is concluded that immunohistochemically demonstrated MT expression is significantly associated with benign canine mammary tumours.     

Immunohistochemical detection of P-glycoprotein (clone C494) in canine mammary gland tumours

Elevated levels of P-glycoprotein have been reported in multidrug-resistant tumours in both humans and dogs. In the present study, we investigated the expression of P-glycoprotein in 57 canine mammary gland tumours, 10 mammary gland hyperplasia and seven normal mammary glands by immunohistochemistry. Tissue sections were incubated with an anti-Pgp monoclonal antibody and visualized with En Vision-DAB polymer. Normal and hyperplastic mammary tissues were negative or showed slight cytoplasmic immunoreactivity. Neoplastic cells in benign mammary tumours showed diffuse cytoplasmic staining, in contrast to malignant tumours that showed mainly a membranous staining pattern for Pgp (C494). We observed statistically significant differences among all the different groups of tissues analysed except for benign tumours versus hyperplasia (P = 0.221). Receiver-operating characteristic analysis showed that the best cut-off point to differentiate the threshold to differentiate negative from positive tissue samples was 18.40% of immunostained cells. These results provide a first indication that routine evaluation of Pgp expression in canine mammary gland tumours, taking into consideration a cut-off point for positivity, may be useful for selecting cases for chemotherapy.     

Estrogen receptors in canine mammary gland tumours

The aim of the present study was to establish the relationship between estrogen receptor expression in neoplastic parenchymal cells and the type of canine mammary gland tumour. The research material included mammary gland tumours obtained from 66 dogs. Paraffin sections were stained with hematoxylin-eosin and immunohistochemically with monoclonal antibodies and LSAB/Peroxidase/Universal Kit. The estrogen receptor expression was observed in nuclei of neoplastic cells in about 59% of the cases and in the cytoplasm in about 89% of the cases. In about 20% of the cases the expression in the nuclei and in the cytoplasm was extremely weak. No correlation was found between the expression of estrogen receptors and the value of mitotic indexes in the neoplasms investigated.     

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.     

Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.     

Expression of bone morphogenetic protein-6 (BMP-6) in myoepithelial cells in canine mammary gland tumors.

Seventy-three mammary tumors and three mammary tissue specimens were examined to elucidate the expression of bone morphogenetic protein (BMP)-6 in the myoepithelial cells of canine mammary gland tumors. Morphologically, the myoepithelial cells were classified into four types: resting and proliferating cells inside the basement membrane, and spindle- and star-shaped cells proliferating in the outer area of the basement membrane. The characteristics of these myoepithelial cells were confirmed by immunohistochemistry using antibodies raised against keratin, cytokeratin 19, alpha-smooth muscle actin, and vimentin. In simple adenoma, a small number of resting myoepithelial cells was immunopositive for BMP-6. In complex adenomas and benign mixed tumors, all types of myoepithelial cells, depending in some cases on their specific location within the tumor, were immunopositive for BMP-6, but almost all of the tubular epithelial cells were immunonegative. Foci consisting of a proliferation of BMP-6-positive star- and spindle-shaped cells had mucinous stroma with marked hyaline and chondroid changes. In contrast, the foci with BMP-6-negative spindle- and star-shaped cells tended to have mucinous stroma without chondroid change. Several types of mesenchymal cells including chondrocytes, osteoblasts, and fibroblastlike cells in the mixed tumors, showed an intense immunopositive reaction for the BMP-6 antibody, and were located close to the ectopic cartilage and bone matrix. No significant immunoreactivity for BMP-6 was observed in most of the malignant mammary tumors; only one malignant mixed tumor was examined. All of these findings indicate that BMP-6 expression in myoepithelial cells may increase in complex adenomas and benign mixed tumors in canine mammary glands, and that BMP-6 expression is most intense in the vicinity of chondroid matrix in these tumors.     

Expression of bone morphogenetic protein-6 and bone morphogenetic protein receptors in myoepithelial cells of canine mammary gland tumors

To study the ectopic chondrogenesis in canine mammary mixed tumors, the expression of bone morphogenetic protein-6 (BMP-6) and specific BMP receptors (BMPRs), BMPR-IA, BMPR-IB, and BMPR-II, was examined using immunohistochemical and immunoblot analysis in 39 canine mammary gland tumors. Immunohistochemically, BMP-6 and all three types of BMPRs were coexpressed in the myoepithelial cells and chondrocytes in six of eight benign mixed tumors. In complex adenomas, myoepithelial cells showed an expression pattern of BMP-6, BMPR-IA, and BMPR-II similar to those in benign mixed tumors, whereas immunoreactivity for BMPR-IB was very mild. The myoepithelial cells proliferating within the basement membrane showed more intense immunoreactivity for BMP-6 and all BMPRs as compared with those proliferating in the interstitial areas. Western blotting analysis revealed immunopositive bands at 40-45 kDa for BMP-6 in the samples from simple and complex adenomas and benign mixed tumors. The BMPR-IB-specific bands at 45 kDa were most detected in benign mixed tumors. Because among BMPRs, BMPR-IB is thought to be the major receptor for BMP-6 for primary chondrogenesis, these findings suggest that the expression of BMP and its receptors on the myoepithelial cells might play a role in the ectopic cartilage formation in canine mammary gland tumors, especially in benign mixed tumors.     

Expression and significance of PTEN and VEGF in canine mammary gland tumours

To investigate the relationship between the expression of the PTEN (phosphatase and tensin homolog deleted on chromosometen) and VEGF (vascular endothelial growth factor) and the clinicopathological features in canine mammary gland tumours, the expression levels of PTEN and VEGF protein were assessed in 50 cases of canine mammary gland tumours tissues and 4 cases of normal mammary gland tissues with using immunohistochemical method. The over-expression rate of PTEN protein was 100% in normal and well-differentiated mammary gland tissues and 67% in breast cancer cases respectively with a significant difference between the two groups (P<0.01). Expression of PTEN was not related to age and tumour size, but closely correlated to lymph node metastasis (P<0.01). The over-expression rate of VEGF protein was 33.3% in normal mammary gland tissues, and 78% in canine mammary gland tumours with a significant difference between the two groups (P<0.01).Expression of VEGF was not related to age or tumour size, but closely correlated with lymph node metastasis and clinical stage (P<0.05).Therefore the combination detection of PTEN and VEGF could serve as an important index to estimate the biological behavior and prognosis of canine mammary gland tumours. Reduced expression of PTEN might be involved in carcinogenesis and progression of canine breast cancer by up-regulating the VEGF expression to enhance angiogenesis.     

Use of CD10 as a marker of canine mammary myoepithelial cells

CD10 is an important cell marker in the diagnosis of acute lymphoblastic leukaemia and of breast myoepithelial (ME) cells in humans. The objective of this study was to assess the value of CD10 as a marker of canine ME cells using immunohistochemistry on routinely processed normal, dysplastic and neoplastic mammary tissue. Five different CD10 positive cell types were identified on the basis of cell morphology, pattern of immunoreactivity, and on the co-expression of additional cell lineage-specific markers.Type 1 cells were typical fusiform cells with a ME cell phenotype (calponin- and cytokeratin [CK] 14-positive, CK8/18-negative). Type 2 cells were typical or atypical polyhedral cells with a luminal epithelial (LE) cell phenotype (calponin- and CK14-negative, CK8/18-positive). Type 3 cells had a type 1 phenotype with variable morphology, and type 4 were atypical neoplastic cells with a mixed ME/LE phenotype. Type 5 cells were typical fusiform cells with a stromal phenotype.Type 1 cells were considered normal ME cells and were found in all sample types; type 2 cells were considered normal or neoplastic LE cells and were also found in all sample types; types 3 and 4 cells were restricted to tumour samples and to malignant tumours, respectively, and type 5 cells were found in all sample types, although predominantly in neoplastic tissue. The findings indicate that the CD10 antigen is a sensitive (although not specific) marker of canine ME cells in normal, dysplastic and neoplastic mammary tissue. Differences in the distribution and staining intensity of CD10-positive cells suggest a number of potential roles for this protein in the pathogenesis of canine mammary neoplasia.     

Xenobiotic-metabolizing enzymes in canine mammary tumours

Mammary tumours are the most common neoplasms in female dogs. The present study was designed to evaluate the relationship between different clinical stages with activities of phase I and phase II carcinogen-metabolizing enzymes in canine mammary tumours. The levels of cytochrome P450 and cytochrome b5 and the activities of glutathione S-transferase (GST), gamma-glutamyl transpeptidase (GGT), DT-diaphorase (DTD) and NADPH diaphorase in tumour tissues of 25 bitches was estimated. Enhanced levels of cytochrome P450 and b5 and phase II enzyme activities were observed in tumour tissues compared to the corresponding uninvolved adjacent tissues. The magnitude of the changes in phase I and phase II enzyme status was, however, more pronounced in stages I and II compared to stages III and IV. The results suggest that the balance between phase I carcinogen activation and phase II detoxification systems may play an important role in canine mammary tumour development.     

VEGFR‐2 expression in malignant tumours of the canine mammary gland: a prospective survival study

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is the main receptor activated by vascular endothelial growth factor ‐A (VEGF‐A) to promote tumour angiogenesis. Its clinical prognostic value has not been studied in canine mammary tumours (CMTs). Dogs with mammary cancer were enrolled in a survival study and the immunohistochemical expressions of VEGFR‐2 and VEGF‐A were analysed and associated with clinicopathological features. VEGFR‐2 expression was associated with VEGF immunoreactivity in cancer cells, supporting the presence of an autocrine loop that may be involved in CMTs growth and survival. VEGFR‐2 was also expressed by endothelial cells from tumour vasculature and positively associated with stromal matrix metalloproteinase‐9 (MMP‐9), suggesting the existence of a link between endothelial cells activation and up‐regulation of matrix degrading proteins. Carcinosarcomas exhibited high VEGFR‐2 expression suggesting that it may be one of the activated molecular pathways in this aggressive histological type and that VEGFR‐2 inhibitors may constitute a potential treatment to improve the prognosis of these patients. Both VEGF and VEGFR‐2 immunoreactivities were independent of patients' overall survival (OS) and disease‐free survival (DFS).     

Immunohistochemical study of the expression of E-cadherin in canine mammary tumours

To investigate the possible role of E-cadherin in canine mammary tumours 20 benign and 40 malignant tumours were evaluated by immunohistochemistry in formalin-fixed paraffin wax-embedded samples. In all the benign tumours, E-cadherin was strongly expressed at the intercellular borders of epithelial cells, but it was less strongly expressed in 17 (43 per cent) of the malignant tumours. Furthermore, poorly differentiated carcinomas were less immunoreactive for E-cadherin than moderately and well differentiated carcinomas.     

Expression of class II beta-tubulin by proliferative myoepithelial cells in canine mammary mixed tumors

Benign mammary mixed tumors in dogs resemble human salivary pleomorphic adenomas with regard to their histogenesis, including the occurrence of cartilaginous or bony metaplasia as well as the expression pattern of cytoskeletal proteins in proliferative myoepithelial cells. Recently, a monoclonal antibody specific for class II beta-tubulin has been developed. The epitope it recognizes was determined to be the heptapeptide Glu-Glu-Glu-Glu-Gly-Glu-Asp, which is the common sequence found among the canine, rat, mouse, and human class II beta-tubulin-specific regions. We carried out immunohistochemical studies on mammary mixed tumors obtained from three female dogs using this the monoclonal antibody. The antibody to class II beta-tubulin reacted intensely with proliferative myoepithelial cells in canine mammary mixed tumors, whereas staining was barely detectable in normal myoepithelial cells surrounding alveoli and alveolar ducts within the tumor and adjacent normal tissue. Proliferative myoepithelial cells also expressed vimentin, but alpha-smooth muscle actin (alphaSMA) staining was barely detectable. Immunoblot analysis showed that class II beta-tubulin and vimentin were expressed in myoepithelial cell lines prepared from the three mammary mixed tumors. On the other hand, only one cell line, which was negative for alphaSMA, produced cartilage-specific type II collagen. These results suggest that class II beta-tubulin could be a new molecular marker of proliferating myoepithelial cells in canine mammary mixed tumors and that differential expression of cytoskeletal components is associated with cartilaginous metaplasia of proliferative myoepithelial cells in mixed mammary tumors.     

Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas

Calponin is a 34‐kDa smooth muscle‐specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin–biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin‐positive cells were identified: (1) Type 1: cytokeratin‐14‐positive pre‐existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin‐14‐positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin‐14‐positive atypical cells indistinguishable from non‐reactive atypical cells, which should have never been detected in haematoxylin and eosin‐stained sections and (4) Type 4: cytokeratin‐14‐negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin‐negative and cytokeratin‐14‐positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high‐molecular‐weight cytokeratins in luminal epithelial‐type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.     

Oxidative stress parameters in bitches with mammary gland tumours

The aim of the present study was to describe some of the oxidative stress parameters in bitches suffering from spontaneously occurring mammary gland tumours. The experiment involved 28 bitches which had mammary gland tumours removed surgically (15 bitches with malignant tumour and 13 with benign tumour) as well as 10 clinically healthy bitches. The activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were determined in haemolysates of erythrocytes derived from the animals. The concentrations of thiobarbituric acid reactive substances (TBARS), as well as -SH groups, were determined in blood plasma. GSH-Px activity was significantly higher in the malignant tumour group than in healthy animals. SOD activity was significantly higher in animals with tumours compared with the control group. Activities of both enzymes were higher in animals with malignant tumours than in benign groups, but the differences were not statistically significant. The concentrations of TBARS and -SH groups were similar in all examined groups. The increase of antioxidative enzyme activities in these animals may suggest the activation of antioxidative defence mechanisms in mammary gland carcinogenesis. Moreover, it might indicate the participation of oxidative stress in malignancies. Further experiments involving more animals, with more frequent sample collection and the use of other oxidative stress markers are necessary.