A. Gama

Identification of molecular phenotypes in canine mammary carcinomas with clinical implications: application of the human classification

Similarly to humans, canine mammary cancer represents a heterogeneous group in terms of morphology and biological behaviour. In the present study, we evaluated a series of canine mammary carcinomas based on a new human classification, initially based on gene expression profiling analysis. Similarly to human breast cancer, by using an immunohistochemistry surrogate panel based on five molecular markers [estrogen receptor, human epidermal growth factor receptor 2 (HER2), cytokeratin 5, p63 and P-cadherin], we were able to classify canine mammary carcinomas into four different subtypes: luminal A [estrogen receptor (ER)+/HER2−; 44.8%], luminal B (ER+/HER2+; 13.5%), basal (ER−/HER2− and a basal marker positive; 29.2%) and HER2 overexpressing tumours (ER−/HER2+; 8.3%). Luminal A-type tumours were characterised by lower grade and proliferation rate, whereas basal-type tumours were mostly high grade, high proliferative and positive for cytokeratin 5, p63 and P-cadherin. In addition, as in humans, basal subtype was significantly associated with shorter disease-free and overall survival rates, and we propose canine mammary carcinomas as a suitable natural model for the study of this particular subset of human carcinomas.

p63: A Novel Myoepithelial Cell Marker in Canine Mammary Tissues

Several immunohistochemical markers have been used to demonstrate the presence of myoepithelial cells in order to determine their role in the histogenesis of mammary tumors. p63, a recently characterized p53 homologue, is consistently expressed in myoepithelial cells of the human breast; however, no assessment of its immunoreactivity has been reported so far in canine mammary tissues. We investigated p63 immunohistochemical expression, as a novel myoepithelial cell nuclear marker, in 81 samples of normal (n = 2), hyperplastic (n = 11), and neoplastic (n = 68) canine mammary tissues. Myoepithelial phenotype was confirmed by using complementary monoclonal antibodies: alpha-smooth muscle actin, cytokeratin 14, cytokeratin AE1/AE3, and vimentin. p63 expression was observed in 91.4% (74/81) of the samples evaluated. Normal mammary glands, mammary hyperplasias, and benign tumors showed 100% immunoreactivity, with p63 expression restricted to myoepithelial cell nuclei. In general, benign mixed tumors showed a basal cell compartment immunoreactive to p63, with a gradual decrease of its expression during myoepithelial transformation. p63 expression was found in 72% of malignant tumors, allowing myoepithelial or basal cell identification in spindle-cell carcinomas (2/2), tubulopapillary carcinomas (8/9), solid carcinomas (7/10), and carcinosarcomas (1/3). The osteosarcoma analyzed was p63 negative. In our series, stromal components were consistently nonreactive to p63. In conclusion, the present study reveals p63 as a sensitive and highly specific marker of myoepithelial cells in canine mammary tissues, and the authors suggest p63 as an additional marker for defining myoepithelial histogenesis.

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.

P-Cadherin Expression in Canine Mammary Tissues

P-cadherin is a classical cadherin expressed by myoepithelial cells in mammary tissue. Its expression in human breast cancer has been associated with aggressive tumour behaviour. To analyse the possible role of P-cadherin in canine mammary carcinogenesis, its expression was examined immunohistochemically in 82 samples of normal (n=2), hyperplastic (n=11) and neoplastic (n=69) canine mammary tissues. In normal and hyperplastic canine mammary glands, P-cadherin was restricted to myoepithelial cells, usually at sites of cell-to-cell contact. In tumour tissues, however, P-cadherin expression was observed in both epithelial and myoepithelial cells, with a cytoplasmic pattern of cellular distribution. Aberrant epithelial P-cadherin immunolabelling was found in 19/44 (43%) benign tumours and in 16/25 (64%) malignant tumours (P<0.001). In malignant tumours, a significant correlation between P-cadherin expression intensity and histological type was observed (P<0.05).

Expression and prognostic significance of CK19 in canine malignant mammary tumours.

Expression of the luminal cytokeratin CK19 was examined in 102 canine mammary carcinomas by immunohistochemical analysis and associated with known expression of oestrogen receptor (ER), basal/myoepithelial cell markers, proliferation and survival. Negative staining for CK19 was present in 23.5% of tumours and was associated with histological type, grade, invasiveness, Ki67 index and expression of basal/myoepithelial cell markers. Positive staining for CK19 was associated with ER expression. Reduced expression of CK19 was associated with both shorter overall and disease-free survival rates; however, CK19 was not an independent prognostic factor in multivariate analysis. Reduced expression of CK19 in canine mammary carcinomas is related to an aggressive phenotype and may play a role in tumour progression.

The effects of repeated oral gavage on the health of male CD-1 mice

Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.

Ultrasonographic, thermographic and histologic evaluation of MNU-induced mammary tumors in female Sprague-Dawley rats

BACKGROUND As the worldwide breast cancer burden increases, non-invasive tools, such as ultrasonography and thermography are being increasingly sought after. N-methyl-N-nitrosourea-induced rat mammary tumors are important tools to investigate the usefulness of such imaging techniques. OBJECTIVE This study aimed to integrate both ultrasonographic and thermographic approaches to the vascularization and the superficial temperature of chemically-induced rat mammary tumors. MATERIALS AND METHODS Twenty-five female Sprague-Dawley rats were divided into two groups: group I (intraperitoneally administered with N-methyl-N-nitrosourea) and group II (control group). Thirty-five weeks after the administration of the carcinogen, mammary tumors were evaluated using Power Doppler, B Flow and Contrast-enhanced ultrasound, thermography and histology analyses. RESULTS Group I animals showed an average of 2.5 mammary tumors per animal, mostly papillary and cribriform non-invasive carcinomas. B Flow detected higher counts of colour pixels than Power Doppler. Contrast-enhanced ultrasound analysis showed a centripetal enhancement order of contrast agent and clear margins. Maximum tumor temperature and thermal amplitude determined by thermography were significantly correlated with tumor volume and with color pixel density, determined by Power Doppler. CONCLUSION B Flow was more sensitive than Power Doppler in detecting tumor vessels, but Power Doppler correlates with thermographic data concerning superficial temperature and may reflect tumor angiogenesis.

Cadherin cell adhesion system in canine mammary cancer: a review.

Cadherin-catenin adhesion complexes play important roles by providing cell-cell adhesion and communication in different organ systems. Abnormal expression of cadherin adhesion molecules constitutes a common phenomenon in canine mammary cancer and has been frequently implicated in tumour progression. This paper summarizes the current knowledge on cadherin/catenin adhesion molecules (E-cadherin, β-catenin, and P-cadherin) in canine mammary cancer, focusing on the putative biological functions and clinical significance of these molecules in this disease. This paper highlights the need for further research studies in this setting in order to elucidate the role of these adhesion molecules during tumour progression and metastasis.

P‐cadherin expression in canine lactating mammary gland

To the Editor: We read with interest the article recently published by Peralta Soler et al. in Journal of Cellular Biochemistry on the expression of the P-cadherin soluble fragment in human milk. In this article, the authors reported that a high concentration (100–400mg/ml) of a soluble 80-kDa fragment of P-cadherin adhesion protein was present in human milk. They evaluated the pattern of P-cadherin expression in normal lactating mammary tissue, through immunohistochemistry, immunoprecipitation, and Western blotting, using a monoclonal antibody to the extracellular domain of P-cadherin (BD Transduction Laboratories, clone 56) [Peralta Soler et al., 2002]. In lactating tissue, P-cadherin appears as a protein secreted by epithelial cells, rather than a cell–cell adhesion protein, while in the non-lactating mammary gland, P-cadherin is restricted to myoepithelial cells, and is present at sites of cell–cell contact [Peralta Soler et al., 2002]. Recently, we have found a similar distribution of P-cadherin while studying its expression in canine mammary tumors. P-cadherin expression has been described as a marker of aggressiveness and poor outcome in human breast cancer [Soler et al., 1999; Gamallo et al., 2001; Paredes et al., 2002] and, since there are no studies on P-cadherin expression in canine mammary tissues hitherto described, we sought to evaluate the immunoexpression of this adhesion molecule in this animal model. The study of the biology of spontaneous mammary tumors in the dog, which is one of the most prevalent neoplasms in this domestic animal, may be of interest for the clinical management of human breast cancer [Mottolese et al., 1994]. Most importantly, the study of canine mammary tumors may serve as an ideal model to evaluate P-cadherin expression, owing to the high frequency of tumors showing features of a myoepithelial/basal cell differentiation/histogenesis. Immunohistochemistry and evaluation of P-cadherin expression in canine mammary gland tissue was performed as described in our recent study [Paredes et al., 2002]. To the best of our knowledge, up to now, there are no specific antibodies against canine P-cadherin; thus we used the anti-human P-cadherin antibody from Transduction Laboratories (clone 56, 1:50 dilution). To optimize the immunohistochemistry technique to canine mammary tissue, heat-induced antigen retrieval was carried out using an EDTA buffer (Lab Vision Corporation, Fremont), pH 8.0, in a boiling bath, during 20 min. Our findings largely corresponded to the previous observations in human breast tissue, by Peralta Soler et al., 2002, reinforcing their results. In normal non-lactating canine mammary glands, P-cadherin was restricted to the myoepithelial cells (Fig. 1A). In canine lactating, pseudo-lactating mammary tissue, and in areas with pseudolactional hyperplasia of breast lobules and ducts, P-cadherin was found in secretory luminal epithelial cells and milk secretion (Fig. 1B). Its expression in luminal epithelial cells was strong and cytoplasmic, similar to that of a secreted protein, which suggests that P-cadherin is not functioning as an adhesion molecule, and could be involved in canine breast tissue remodelling, since it only appears in luminal epithelial cells during the lactation period. On the other hand, milk secretion P-cadherin may derive from proteolytic cleavage of the

Long-term exercise training as a modulator of mammary cancer vascularization

BACKGROUND Breast cancer remains a leading cause of death by cancer worldwide. It is commonly accepted that angiogenesis and the expression of angiogenic factors such as vascular endothelial growth factor-A (VEGF-A) is associated with the increased risk of metastasis and poor patient outcome. OBJECTIVE This work aimed to evaluate the effects of long-term exercise training on the growth and vascularization of mammary tumors in a rat model. MATERIALS AND METHODS Fifty female Sprague-Dawley rats were divided into four groups: two N-methyl-N-nitrosourea (MNU)-exposed groups (exercised and sedentary) and two control groups (exercised and sedentary). MNU was administered once, intraperitoneally at 7 weeks-old. Animals were then exercised on a treadmill for 35 weeks. Mammary tumors were evaluated using thermography, ultrasonography [Power Doppler (PDI), B Flow and contrast-enhanced ultrasound (CEUS)], and immunohistochemistry (VEGF-A). RESULTS Both, MNU sedentary and exercised groups showed 100% of tumor incidence, but exercised animals showed less tumors with an increased latency period. Exercise training also enhanced VEGF-A immunoexpression and vascularization (microvessel density, MVD) (p<0.05), and reduced histological aggressiveness. Ultrasound and thermal imaging analysis confirmed the enhanced vascularization of tumors on exercised animals. CONCLUSION Long-term exercise training increased VEGF-A expression, leading to enhanced tumor vascularization and reduced tumor burden, multiplicity and histological aggressiveness.