E-cadherin and beta-catenin reduction influence invasion but not proliferation and survival in canine malignant mammary tumors

E-Cadherin and beta-catenin are known for their role in tumor invasion, but both proteins also exert an influence on tumor proliferation. This study, performed on canine mammary tumors, aimed to analyze the influence of E-cadherin (E-cad) and beta-catenin (beta-cat), immunohistochemically assessed singly and in combination (E-cad/beta-cat), on survival and their relationship with several proliferation indices (AgNOR index, MIB1 index, mitotic index). Immunohistochemistry was carried out on 60 formalin-fixed, paraffin wax-embedded specimens of canine mammary malignancies. The labeling was defined as preserved when prevalent on cell membranes of more than 75% of cells and reduced in other forms of expression (i.e., membranous less than 75%, cytoplasmic, and negative). E-cad, beta-cat, and E-cad/beta-cat were preserved respectively in 22, 12, and 11 out of 60 cases. Immunohistochemical expression of the two proteins in the same tumors was significantly correlated (P = 0.0001; R = 0.57). Survival analysis revealed no difference in outcome comparing the preserved versus reduced cases (E-cad, P = 0.31; beta-cat, P = 0.29; E-cad/beta-cat P = 0.36). Grouping cases for histologic invasiveness, the expression of E-cad or beta-cat and E-cad/beta-cat showed a progressive reduction that paralleled an increase in invasiveness from noninfiltrating to stage-II tumors (E-cad, P < 0.001; beta-cat, P < 0.05; E-cad/beta-cat, P < 0.05). No significant difference was obtained comparing mitotic index, MIB 1 index, and AgNOR index by analysis of variance between the cases grouped for preserved or reduced E-cad, beta-cat, and E-cad/beta-cat variables. In conclusion, reduced expression of E-cad, beta-cat, or E-cad/beta-cat was significantly associated with the progression from noninfiltrating to highly infiltrating tumors but not with proliferation or survival.     

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.     

Alpha basic crystallin expression in canine mammary tumors

The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (αB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were αB-c positive but myoepithelial cells were negative. In benign or simple type malignant tumors, αB-c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative. In benign or complex type malign tumors, positive staining was predominantly found in the cytoplasm of epithelial cells. Immunoreactivity of αB-c was also observed in neoplastic myoepithelial cells. Statistically, the number of cells immunolabeled with αB-c was found to be significantly different among tissues from normal canine mammary glands, benign lesions, and malignant tumors (p < 0.05). αB-c immunoreactivity was higher in malignant tumors than the control mammary tissues (p < 0.001). Data obtained in the current study revealed a strong association between high expression levels of αB-c and primary mammary gland tumors in canines.     

Expression of maspin in mammary gland tumors of the dog

Maspin is a serine protease inhibitor that inhibits tumor invasion and metastasis in human breast cancer and is consistently expressed by mammary myoepithelial cells (MECs). To analyze the value of maspin as a marker of the MEC layer of the normal and tumoral canine mammary gland, the immunohistochemical expression of maspin was studied in formalin-fixed tissues from 55 benign and malignant tumors (40 tumors also contained the surrounding normal mammary gland) using a commercially available monoclonal antibody. Periacinar and periductal MECs of all 40 normal mammary glands were stained by the anti-human maspin monoclonal antibody, and immunoreactivity was observed in the nucleus and cytoplasm of these cells. In addition, maspin was found in 53 (98%) of the tumors studied, reacting with the MECs in 100% of benign tumors and 93% of malignant tumors and to the epithelial cells of 16% of benign and 73% of malignant tumors. In the MEC compartment, immunoreactivity was observed in the cytoplasm of hypertrophic MECs, fusiform MECs, stellate MECs, rounded (myoepithelial) cells, and chondroblasts. In the epithelial cell compartment, immunoreactivity was observed in the cytoplasm of cells with and without squamous differentiation. Stromal myofibroblasts were unreactive. Maspin appears to be a very sensitive marker of the normal and neoplastic myoepithelium that, contrary to smooth muscle differentiation markers, does not stain stromal myofibroblasts. In addition, a subset of neoplastic epithelial cells reacted with the maspin antibody. The relationship between maspin expression in different cellular compartments of canine mammary carcinomas and the biologic aggressiveness of the disease remains to be elucidated.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Expression of connexins 26 and 43 in canine hyperplastic and neoplastic mammary glands

Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis; reported studies in rodent and human mammary glands, which normally express connexins 26 and 43, confirm these alterations in malignancies. Mammary neoplasms represent the second most frequent neoplasm in dogs, and since there are no reports on the study of connexins in canine mammary glands, the present study investigated the expression of connexins 26 and 43 in normal, hyperplastic, and neoplastic mammary glands of this species, to verify if altered patterns of connexin staining are related to higher cell proliferation and malignant phenotypes. A total of 4 normal, 8 hyperplastic mammary glands, 9 benign, and 51 malignant mammary gland neoplasms were submitted for the immunostaining of connexins 26 and 43, E-cadherin, and proliferating cell nuclear antigen (PCNA). Normal, hyperplastic, and benign neoplastic mammary glands showed a punctate pattern for connexin 26 and 43 staining and an intercellular E-cadherin staining. Malignant neoplasms, especially the most aggressive cases with high cell proliferation rates, presented either fewer gap junction spots on the cell membranes or increased cytoplasmic immunostaining. Malignant tumors also expressed a less intense immunostaining of E-cadherin; the expression of this adhesion molecule is important for the transportation of connexins to cell membranes and in forming communicating gap junctions. Deficient expression of E-cadherin could be related to the aberrant connexin localization and may contribute to the malignant phenotype. In conclusion, the expression and distribution of connexins and E-cadherin are inversely correlated to cell proliferation in malignant mammary neoplasms of dogs and may well be related to their more aggressive histologic type and biologic behavior.     

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.     

Immunolocalization of the smooth muscle-specific protein calponin in complex and mixed tumors of the mammary gland of the dog: assessment of the morphogenetic role of the myoepithelium

The immunohistochemical expression of the smooth muscle-specific protein calponin was studied to assess the contribution of myoepithelial cells to the histogenesis of spindle cells of complex and mixed tumors of the mammary gland of the dog and the origin of cartilage and bone in mixed tumors. Formalin-fixed tissues from 55 benign and malignant tumors (49 also containing surrounding normal mammary gland) were evaluated. Periacinar and periductal myoepithelial cells of all the 49 normal mammary glands were diffusely stained by the anti-human calponin monoclonal antibody. Calponin was found in 53 (98%) of the tumors studied, reacting with the myoepithelium-like cells of 86% of benign tumors and their remnants in 85% of malignant tumors. Five different types of calponin-immunoreactive myoepithelial cells were identified: hypertrophic myoepithelial cells. fusiform cells, stellate myoepithelial cells, rounded (myoepithelial) cells, and chondroblasts. Differences in staining intensity and staining pattern among these five types of cells suggested a transition of myoepithelial cells to chondroblasts. Stromal myofibroblasts also showed calponin immunoreactivity, but they did not react with a cytokeratin 14 monoclonal antibody, which recognizes myoepithelial cells in mammary gland. Calponin appears to be a very sensitive marker of normal and neoplastic myoepithelium in the canine mammary gland, and its identification in different cell types of complex and mixed tumors of the mammary gland of the dog suggests a major histogenetic role for myoepithelial cells.     

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.     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

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.     

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.     

Immunohistochemical expression of Epidermal Growth Factor Receptor (EGFR) in canine mammary tissues

Epidermal Growth Factor Receptor (EGFR) has been extensively studied in human breast cancer; however, systematic studies of EGFR protein expression in canine mammary gland tumours are lacking. Therefore, we evaluated its immunohistochemical expression in a series of 136 canine mammary tumours and representative areas of adjacent normal and hyperplastic mammary tissue and investigated a possible correlation between EGFR overexpression and several clinicopathological parameters and survival. In normal and hyperplastic canine mammary glands, EGFR expression was consistently observed in myoepithelial cells, with luminal cells usually negative. In tumour tissues, EGFR overexpression was found in 9 benign (19.6%) and 38 malignant (42.2%) lesions, with EGFR positivity significantly related with malignancy. Besides animal age and tumour size, there were no significant associations between other clinicopathological parameters and EGFR overexpression. On survival analysis, tumours with EGFR overexpression showed a reduced disease-free and overall survival; however these associations failed to reach statistically significant levels.     

Computerized morphometry of mean nuclear diameter and nuclear roundness in canine mammary gland tumors on cytologic smears

BACKGROUND: The use of computer-based image analysis systems in veterinary oncology has increased. Computerized morphometry is a part of image analysis that describes geometric figures of cellular structures in any dimension. Most investigators have performed morphometric analysis on histologic specimens. Computer-assisted nuclear cytomorphometry can provide important preoperative information on neoplastic lesions in animals. OBJECTIVES: The aim of this study was to define whether the morphometric parameters of mean nuclear diameter and nuclear roundness could be used to differentiate benign from malignant canine mammary gland tumors on cytologic specimens. METHODS: Mean nuclear diameter and nuclear roundness were determined by computer-assisted morphometry of epithelial cells in Hemacolor-stained cytologic smears from normal canine mammary gland (n = 7) and from canine mammary adenomas (n = 8), tubulopapillary carcinomas (n = 9), and solid carcinomas (n = 6). Data were analyzed by the Mann-Whitney U test. RESULTS: Significant differences (P <.001) were found in mean nuclear diameter and nuclear roundness among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for nuclear roundness between tubulopapillary carcinomas and solid carcinomas). CONCLUSIONS: The morphometric parameters of mean nuclear diameter and nuclear roundness can be used in the preoperative differentiation of benign from malignant canine mammary gland tumors.     

Immunohistochemical expression of estrogen receptor beta in normal and tumoral canine mammary glands

To date, two isoforms of estrogen receptors (ER) have been identified, cloned, and characterized from several species, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). Although the presence of ERalpha has been demonstrated in normal and tumoral canine mammary tissues, the issue of ERbeta expression has not been addressed in the dog. In this study, we have analyzed the expression of ERbeta in formalin-fixed, paraffin-embedded tissue samples of nonaltered mammary gland, 30 malignant (six complex carcinoma, 12 simple carcinoma, three carcinosarcoma, and nine carcinoma or sarcoma in benign tumor), and five benign (one fibroadenoma, one complex papilloma, one complex adenoma, and two benign mixed tumors) mammary tumors of the dog by using a polyclonal ERbeta antibody and the avidin-biotin-peroxidase complex immunohistochemical technique. Our results show that high numbers of normal ductal and acinar epithelium and approximately one third of canine mammary tumors express ERbeta. This expression was higher in benign than in malignant tumors. Furthermore, expression was higher in complex and mixed histologic subtypes of malignant tumors when compared with simple subtypes.     

The expression of p63 and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms

Cytokeratin 5 and p63 have been described as basal and myoepithelial cell markers in human breast. Mixed tumors of the canine mammary gland have been associated with a myoepithelial origin. Cytokeratin 5 expression has not been evaluated in these tumors. We investigated the relation between cytokeratin 5 and p63 double-immunohistochemical expression in 23 mixed tumors of the canine mammary gland (10 benign mixed tumors and 13 carcinomas arising from benign mixed tumors) and their origin. Cytokeratin 5 and p63 co-expression was observed in myoepithelial cells of benign mixed tumors, as well as in squamous differentiation of carcinoma arising from benign mixed tumors. Though a few interstitial spindle cells of the mesenchymal components expressed both p63 and cytokeratin 5, the basal epithelial cells were labeled only by cytokeratin 5. The co-expression of p63 and cytokeratin 5 in myoepithelial cells and squamous differentiation suggest that, like in human breast, cytokeratin 5 can also be considered a myoepithelial- and squamous-cell differentiating marker in canine tumors. The presence of some interstitial spindle cells stained for p63 and cytokeratin 5 might be associated with a myoepithelial origin of the mesenchymal component of mixed tumors of the canine mammary gland. Moreover, contrary to p63, basal epithelial cells were labeled by cytokeratin 5, indicating that cytokeratin 5 may not represent an exclusive myoepithelial cell marker but also a basal epithelial cell marker in canine mixed tumors. According to these data, basal epithelial cells may be related to the origin of the epithelial component of mixed tumors of the canine mammary gland.     

Expression of HER-2 and nuclear localization of HER-3 protein in canine mammary tumors: histopathological and immunohistochemical study

HER-2 and HER-3 are transmembrane receptor proteins that are considered to be important but poorly understood biomarkers in canine tumors. In this study, the expression and the localization of HER-2 and HER-3 were evaluated immunohistochemically in canine mammary tumors (n = 64; 12 benign, 52 malignant).HER-2 overexpression was identified in 2/12 (16.7%) benign and in 18/51 (35.3%) malignant cases. HER-3 was expressed in a non-nuclear localization in 11/12 (91.7%) benign and 18/52 (34.6%) malignant tumors. In contrast, HER-3 was expressed in the nucleus of neoplastic cells in 0/12 (0%) benign and 22/52 (42.3%) malignant tumors. Nuclear HER-3 expression was higher in neoplastic epithelial cells compared to myoepithelial cells, and positively correlated with high histological grade and lymphatic vessel invasion. These results suggest that nuclear HER-3 expression is significantly associated with tumor progression and metastasis and may serve as a useful prognostic biomarker in canine malignant mammary tumors.