Immunohistochemical expression of protein p53 in neoplasms of the mammary gland in bitches

The aim of the study was to investigate the presence of protein p53 in correlation with other tumor traits: histological type, tumor grade and proliferative activity. Material for the investigation comprised mammary gland tumours collected from dogs, the patients of veterinary clinics, during surgical procedures, and archival samples. Alltogether 21 adenomas, 31 complex carcinomas, 35 simple carcinomas and 12 solid carcinomas were qualified for further investigation. No protein p53 expression was found in adenomas. Cancers show positive reaction in 32.5%. The highest percent of p53 positive neoplasms was observed in solid carcinomas and neoplasms with the highest degree of histological malignancy. The smallest number showing this expression was observed in adenomas and the highest was characteristic for solid carcinomas. Considering the tumour grading, it was found that an increase in neoplasm malignancy was positively correlated with the number of the cells showing the expression of protein p53. The differences were statistically significant. Statistically significant positive correlations were observed between the proliferative activity and protein p53 expression. Higher accumulation of protein p53 in more malignant neoplasms suggests that mutations of protein p53 can be responsible for higher proliferation in neoplasms with advanced progression of malignancy.     

Immunohistochemical evaluation of expression of heat shock proteins HSP70 and HSP90 in mammary gland neoplasms in bitches

Heat shock proteins have essential roles in a number of pathophysiologic conditions including carcinogenesis and represent a group of novel molecular markers in cancer management. The aim of this study was to investigate heat shock protein expression in correlation with other neoplasm traits such as: histological type, differentiation grade, proliferative activity, estrogenic receptor expression, and cyclooxygenase-2 and p53 proteins. Material for the investigation comprised 133 tumors of the mammary gland collected from bitches. In total 14 adenomas, 66 complex carcinomas, 47 simple carcinomas and 6 solid carcinomas were collected. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Expression of heat shock protein 70 was observed in all types of evaluated neoplasms. A higher average number of cells undergoing expression of heat shock protein 70, which was statistically insignificant, was established in complex and simple cancers and in cancers with the 1st and the 2nd degree of histological malignancy. Expression of heat shock protein 90 was observed in all studied neoplasms; it was very insignificant in adenomas, compared to cancers, and the highest expression was established in the solid cancers, as well as in cancers with the 2nd degree of histological malignancy. This high expression of heat shock protein 90 was correlated with proliferative activity. The results suggest that heat shock protein 90 is involved in canine mammary gland carcinogenesis. The results also suggest that heat shock protein 90 may be a prognostic factor, but this requires detailed clinical confirmation.     

Dysregulation of beta-catenin is common in canine sporadic colorectal tumors.

Human colorectal tumorigenesis is often initiated by APC (adenomatous polyposis coli) or beta-catenin (CTNNB1) mutations, which result in dysregulation of beta-catenin expression, followed by alterations in E-cadherin and/or p53. We examined 32 canine intestinal tumors for expression and intracellular distribution of beta-catenin, E-cadherin, and p53 using immunohistochemistry. beta-Catenin in normal mucosal epithelial cells was restricted to lateral cell membranes, but 13/13 (100%) colorectal adenomas had intense cytoplasmic and/or nuclear reactivity. Three of six (50%) colorectal carcinomas, 2/13 (15%) small intestinal carcinomas, and dysplastic cells in 1/2 focal hyperplastic lesions in the small intestine had a similar pattern of staining; remaining tumors had normal membranous beta-catenin reactivity. There was a correlation (P = 0.007) between abnormal beta-catenin and E-cadherin staining with 11/13 (85%) colorectal adenomas, 3/6 (50%) colorectal carcinomas, and 3/13 (23%) small intestinal carcinomas showing decreased membranous reactivity compared with normal mucosal epithelium. E-cadherin staining was reduced more often in adenomas than in carcinomas (P = 0.04). There were two patterns of nuclear p53 staining: > 60% of nuclei in 2/26 (8%) carcinomas (one colorectal, one small intestinal) were strongly labeled, whereas three colorectal adenomas and one small intestinal carcinoma had fainter staining in 10-20% of cells. Dysregulation of beta-catenin appears to be as important in canine colorectal tumorigenesis as it is in the human disease and could be due to analogous mutations. Malignant progression in canine intestinal tumors does not appear to be dependent on loss of E-cadherin or beta-catenin expression or strongly associated with overexpression of nuclear CMI antibody-reactivity p53.     

Expression of E-cadherin in normal, hyperplastic and neoplastic feline mammary tissue

This paper describes an immunohistochemical study of the expression of E-cadherin in four samples of normal, eight samples of hyperplastic and 19 samples of neoplastic feline mammary tissue. In the normal tissues, the luminal epithelial cells showed a strong pattern of staining for E-cadherin at the cell-cell boundaries, whereas the myoepithelium showed no immunoreactivity. In the hyperplastic tissues and the five benign neoplasms, there were disturbances in the expression of E-cadherin in the luminal epithelium, in the form of the coexistence of membranous and cytoplasmic staining, together with immunoreactivity in a small percentage of myoepithelial cells. In 11 of 14 carcinomas, there was a reduction or absence of E-cadherin expression and abnormalities in the pattern of immunostaining; these changes were more pronounced in cribriform and solid carcinomas.     

Immunohistochemical analysis of c-yes and c-erbB-2 oncogene products and p53 tumor suppressor protein in canine mammary tumors

In order to evaluate the involvement of c-yes and c-erbB-2 oncogene products, and p53 tumor suppressor protein in canine mammary neoplastic lesions, sections of archived paraffin-embedded samples of 79 mammary tumors were analyzed immunohistochemically using antibodies against human c-yes p62 and c-erbB-2 products and p53. These 79 tumors were divided into 2 groups: 32 benign (2 adenosis, 7 simple adenomas, 14 complex adenomas, and 9 benign mixed mammary tumors) and 47 malignant tumors (26 simple adenocarcinomas, 7 complex adenocarcinomas, 5 solid carcinomas, 2 sclerosing carcinomas, 6 malignant mixed mammary tumors, and 1 malignant myoepithelioma). As a result of immunostaining, 40.6% (13/32) of the benign tumors and 21.3% (10/47) of the malignant tumors expressed the c-Yes oncogene product, ErbB-2 expression was detected in 50% (16/32) of the benign tumors and in 19.1% (9/47) of the malignant tumors. P53 expression was detected in 16% (4/25) of the benign tumors and in 30.6% (11/36) of the malignant tumors. Co-expression of c-Yes and ErbB-2, ErbB-2 and p53, and all 3 products was detected in 6, 1 and 7 tumors, respectively.     

Insulin receptor is expressed in normal canine mammary gland and benign adenomas but decreased in metastatic canine mammary carcinomas similar to human breast cancer

Insulin receptor (INSR) or insulin-like growth factor (IGF) signalling is speculated to be involved in mammary tumour development. Expression levels of members of the insulin receptor family (INSR, IGF1R, IGF2R, GHR) and their ligands IGF1and IGF2 were quantified in macro- and microdissected tissue samples of normal canine mammary gland, adenomas, carcinomas and their lymph node metastases to evaluate their potential impact on the carcinogenesis of canine mammary tumours. Normal mammary gland and adenomas had strong INSR expression, while carcinomas and metastases had significantly decreased expression. No differences were observed for IGF1R expression. IGF1, IGF2 and GHR mRNA expressions were strongly decreased in adenomas, carcinomas and metastases. INSR and IGF1R are therefore expressed in normal gland and adenomas and an increased stimulus by their ligands may be a proliferative stimulus in those tissues. However, decreased INSR expression carcinomas and their metastases render questionable its impact at late stages of carcinogenesis.     

The expression of intermediate filaments in canine mammary glands and their tumors

Monoclonal antibodies specific for different types of intermediate filaments (cytokeratin, vimentin, desmin and neurofilaments) were used to study the histogenesis of canine mammary glands and 57 canine mammary tumors by immunocytochemistry. The intra- and interlobular duct epithelium, acinar, and intralobular myoepithelial cells stained positively for cytokeratin. Peripheral ductal and acinar cells, as well as interstitial cells, stained positively for vimentin. A similar staining pattern was seen in adenomas, complex adenomas, benign mixed tumors, ductular carcinomas, and one myoepithelioma-like tumor. Additionally, cytokeratin positive cells were scattered interstitially in one single adenoma, most complex adenomas, some benign mixed tumors, complex carcinomas, and in the malignant mixed tumors. All stromal cells stained positively for vimentin. The fibrosarcomas were positive only for vimentin, while the following expressed both desmin and cytokeratin: epithelial-like cells in one adenoma, three complex adenomas, the myoepithelioma-like tumor, the single comedo carcinoma, two complex carcinomas, the single lobular carcinoma, one malignant mixed tumor, and three osteosarcomas. Epithelial-like cells in one adenoma, six complex adenomas, two benign mixed tumors, two complex carcinomas, the lobular carcinoma, and the malignant schwannoma stained for neurofilaments. Three tumors, one adenoma, one complex adenoma, and the lobular carcinoma expressed both desmin and neurofilaments in addition to cytokeratin and vimentin. The results show the expression of different types of intermediate filaments and indicate that there might be a stem cell origin in most of the canine mammary tumors.     

E-cadherin and β-catenin expression in canine colorectal adenocarcinoma

E-cadherin and its associated cytoplasmic proteins, including β-catenin, have been examined as potential oncogenic markers due to the significant correlation between tumour dedifferentiation and the invasive capacity of epithelial tumours. The purpose of this study was to evaluate the expression of E-cadherin and β-catenin in canine colorectal cancer using immunohistochemistry and to examine the relationship between this expression and various clinicopathological variables. The expression pattern of E-cadherin and β-catenin was investigated in 44 colorectal canine carcinomas. In the intestinal mucosa of noncancerous areas, epithelial cells demonstrated equally strong membranous expression of E-cadherin and β-catenin localised to the cell–cell junctions. Reduced expression of E-cadherin and β-catenin was demonstrated in 75% and 81.8% of the colorectal carcinoma cases, respectively. The down-regulation of both E-cadherin and β-catenin was correlated with decreased differentiation and increased tumour grade. In addition, the expression of β-catenin was correlated with tumour size. These results suggest that dysfunction of the E-cadherin–catenin complex starts in the early stages of carcinogenesis and that the disruption of the tissue architecture is progressively associated with the invasion of the tumour.     

Loss of p27 expression in canine mammary tumors and their metastases

The p27 gene is a member of the cyclin-dependent kinase inhibitors, which arrest G1- to S-phase transition of the cell cycle. We have previously shown a significant reduction of p27 mRNA expression level in laser-microdissected mammary carcinomas and their lymph node metastases when compared to non-neoplastic mammary gland of the same dog. Here, p27 expression was analyzed on the protein level in non-neoplastic mammary gland, primary mammary carcinomas, their lymph node metastases and intravascular tumor cells of 49 dogs, adenomas of 49 dogs and non-neoplastic mammary gland of 98 dogs by immunohistochemistry. A significantly (p ? 0.05) decreased percentage of p27 positive tissue samples was found when normal gland was compared with adenomas, carcinomas and lymph node metastases. Specifically, 91% of normal gland epithelium displayed nuclear p27 expression. In contrast, only 22% of the adenomas, 20% of carcinomas, 12% of lymph node metastases and 32% of intravascular tumor cells had p27 reactivity. Cell cycle control by p27 is therefore lost in the majority of canine mammary tumors. The lack of significant differences between benign and malignant mammary tumors indicates that decreased p27 expression is an early step in carcinogenesis of canine mammary tumors and hinders the use of p27 as a marker of malignancy for this tumor type.     

Indicators of malignancy of canine adrenocortical tumors: histopathology and proliferation index

Tumors of the adrenal cortex account for 10-20% of the naturally occurring Cushing's syndrome diagnosed in dogs. Differentiating between adrenocortical adenoma and carcinomas is often difficult. The purposes of this study were to determine which histopathologic criteria can be used as markers for malignancy in canine adrenocortical tumors and the relevance of the proliferation marker, Ki-67, for differentiation between cortical adenomas and carcinomas. Twenty-six adrenocortical carcinomas, 23 adenomas, and 11 normal adrenal glands were examined. Morphologic criteria significantly associated with adrenocortical carcinomas included a size larger than 2 cm in diameter, peripheral fibrosis, capsular invasion, trabecular growth pattern, hemorrhage, necrosis, and single-cell necrosis, whereas hematopoiesis, fibrin thombi, and cytoplasmic vacuolation were significantly associated with adrenocortical adenomas. The mean (+/- SD) proliferation index, measured by immunohistochemistry for the Ki-67 antigen, was 9.3 +/- 6.3% in carcinomas, 0.76 +/- 0.83% in adenomas, and 0.58 +/- 0.57% in normal adrenal glands. The Ki-67 proliferation index was significantly higher in carcinomas compared with adenomas and normal adrenal glands. A threshold value of the proliferation index of 2.4% reliably separated carcinomas from adenomas. Based on these results, it appears that thorough evaluation of morphologic features combined with immunohistochemical assessment of the proliferation index is extremely useful for differentiating between adrenocortical adenomas and carcinomas in dogs.     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.     

Expression of matrix metalloproteinase 2 (MMP-2), E-cadherin and Ki-67 in metastatic and non-metastatic canine mammary carcinomas

Background

The aim of the study was to demonstrate the immunohistochemical expression of proteins that affect the metastatic potential of a tumour, including matrix metalloproteinase 2 (MMP-2) and E-cadherin. Another objective was to determine their correlation with the expression of the Ki-67 antigen in metastasizing and non-metastasizing mammary carcinomas in female dogs. The study was conducted on 32 canine mammary carcinomas (12 metastatic and 20 non-metastatic), classified as simple tubular and tubulopapillary carcinomas. Immunohistochemistry was performed to evaluate the expression of MMP-2, E-cadherin and Ki-67 antigen.

Results

MMP-2 was expressed in 85 % of the non-metastatic tumours and in all the metastatic tumours, while E-cadherin was expressed in 85 % of the non-metastatic tumours and in 66 % of the metastatic tumours. The Ki-67 antigen was expressed in 65 % of the non-metastatic tumours and in 91 % of the metastatic tumours. The mean Ki-67 expression was slightly higher in tumours that had metastasized (1.5?±?0.90 vs 1.1?±?0.94; p?=?0.22). A similar relationship was found in terms of the intensity of the MMP-2 expression (2.9?±?1.9 vs 2.7?±?2.4; p?=?0.50). A decrease in the expression of E-cadherin (2.8?±?2.5) was found in metastatic tumours compared to the expression in non-metastatic tumours (3.2?±?2.3). However, these differences were not statistically significant (p?=?0.63).

Conclusion

We did not show significant differences in MMP-2, E-cadherin and Ki-67 expression between metastatic and non-metastatic tumours due to low number of cases studied, however further experiments are necessary to assess the role of these antigens in the process of canine mammary tumours metastasis.

     

Immunohistochemical Characterization of a Renal Nephroblastoma in a Trp53-mutant and Prolyl Isomerase 1-deficient Mouse

A nephroblastoma is a tumor arising from metanephric blastema occurring in childhood. Among laboratory rodents, nephroblastoma has been frequently reported in rats, but it remains exceedingly rare in mice. The present work describes a nephroblastoma in a young mouse homozygous for the specific Trp53 R172H point mutation coupled with targeted deletion of the Pin1 gene. The affected kidney was effaced by a biphasic tumor with an epithelial component arranged in tubules surrounded by nests of blastemal cells. Immunohistochemically, the neoplasm was diffusely positive for Wilms’ tumor antigen. The epithelial component expressed markers of renal tubular differentiation including wide-spectrum cytokeratin, E-cadherin and folate-binding protein. Furthermore, the neoplasm exhibited a high proliferative index and diffuse nucleocytoplasmic β-catenin expression. Based on histological and immunohistochemical features, a diagnosis of nephroblastoma potentially associated with Trp53 loss and oncogenic β-catenin activation has been proposed.     

Cyclooxygenase-2 expression in canine mammary tumors

Mammary tumors are the most common neoplasms in female dogs. Induction of cyclooxygenase-2 (COX-2) has been implicated in various cancers in humans. However, expression of COX-2 has not been investigated in canine mammary tumors. Normal mammary gland (n = 4), simple or complex adenomas (n = 63), and simple or complex adenocarcinomas (n = 84) were studied by immunohistochemistry. Results showed that COX-2 was not expressed in the normal gland but was detected in 24% of adenomas and in 56% of adenocarcinomas (P < 0.001). The incidence of COX-2 expression and the intensity of the COX-2 signal were higher in adenocarcinomas than in adenomas (P < 0.001). These results demonstrate for the first time that COX-2 is induced in a proportion of canine mammary tumors and that COX-2 expression is more frequent and more intense in malignant than in benign tumors, suggesting a potential role for COX-2 in canine mammary tumorigenesis.     

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.     

Localization of connexin 32 in spontaneous liver lesions of mice

We examined the localization of connexin 32 (Cx32), a component of gap junctions, in 24-month-old male B6C3F1 mice with spontaneously occurring hepatocellular altered foci or tumors. Immunohistochemically, Cx32-staining intensity in cell-to-cell membranes of altered hepatocytes was decreased in eosinophilic foci and increased in basophilic foci as compared to those in intact hepatocytes. These alterations were enhanced in adenomas and carcinomas with both eosinophilic and basophilic cytoplasm. In cell membranes facing on the sinusoidal portions, the intensities increased in all lesions. Image analyses confirmed that the spot areas of Cx32 were decreased in eosinophilic foci, but increased in basophilic foci, adenomas and carcinomas. These results demonstrate that Cx32 shows different expression in different types of hepatic lesions.     

Expression of cell adhesion molecules in canine choroid plexus tumors

Choroid plexus tumor (CPT) is a primary intracranial neoplasm of the choroid plexus epithelium in the central nervous system. In the current World Health Organization classification, CPT is classified into two categories; choroid plexus papilloma (CPP) and carcinoma (CPC). In the present study, we investigated immunohistochemical expressions of N-cadherin, E-cadherin and β-catenin in 5 canine CPT cases (1 disseminated CPC, 2 CPCs and 2 CPPs). One CPP case was positive for N-cadherin and β-catenin, but negative for E-cadherin. The disseminated CPC case was positive for E-cadherin and β-catenin, but negative for N-cadherin. The other cases were positive for the three molecules examined. These results suggest that loss of the N-cadherin expression might associate with the spreading of CPC cells.     

Relationship between cell proliferation and tenascin-C expression in canine gastrointestinal tumours and normal mucosa

Tenascin-C is an extracellular matrix glycoprotein that has been implicated in cell proliferation and adhesion by in vitro experiments. Its expression is known to be increased in canine and human gastrointestinal tumours. The aim of this study was to investigate the possible relationship between cell proliferation and tenascin expression in these tumours. In tissue sections of normal stomach, small intestine and colon, and gastrointestinal epithelial tumours, the monoclonal antibody Ki-67, which is directed against a proliferation-associated nuclear antigen, was used to identify proliferating cells. Serial sections were also stained for tenascin. Serial sections stained for tenascin and Ki-67 were compared to determine whether there is a correlation between tenascin expression and tumour cell proliferation. In the normal gastric mucosa, Ki-67 positive cells were confined to the neck region and in the normal small intestinal mucosa positive cells were confined to the lower parts of the crypts. In adenomas and carcinomas, the frequency of positive cells was increased at the edges of adenomas and invasive tumour margins of carcinomas and there was inter- and intra-tumoural heterogeneity. Carcinomas with lymphatic invasion showed a high Ki-67-index. There was no relation between cell proliferation and tenascin expression in both normal tissues and tumours studied. The absence of a correlation between tenascin and Ki-67 expression suggests that the main function of tenascin in both normal tissues and tumours of the canine gastrointestinal tract is antiadhesion rather than proliferation.     

Expression of claudin‐5 in hepatoid gland biopsies

Claudins are integral membrane proteins involved in the structure of the tight junctions found in epithelial and endothelial cells. This study evaluated the expression of claudin‐5 in 67 hyperplastic and neoplastic lesions of canine hepatoid glands. These included normal hepatoid glands (n = 10), nodular hyperplasia (n = 10), adenomas (n = 12), epitheliomas (n = 15), differentiated carcinomas (n = 15) and anaplastic carcinomas (n = 15). There was intense lateral membrane expression of claudin‐5 on epithelial cells from normal hepatoid glands, nodular hyperplasia and adenomas, but expression was weaker in hepatoid gland epitheliomas. Basal reserve cells from normal glands, nodular hyperplasia, adenomas and epitheliomas never expressed claudin‐5. There was membrane‐bound immunoreactivity for claudin‐5 in selected areas of the epitheliomas where the cells exhibited typical hepatoid features. The weak expression of claudin‐5 molecule in epitheliomas may nevertheless lead to cellular disorientation, detachment and invasion. Claudin‐5 expression seemed to be helpful in distinguishing poorly differentiated carcinomas, differentiated carcinomas and epitheliomas of the hepatoid glands. Increased claudin‐5 expression by invasive anaplastic carcinomas may facilitate invasion and metastasis through the activation of matrix metalloproteinases.