[show abstract][hide abstract] ABSTRACT: BACKGROUND: Although several histopathological and clinical features of canine mammary gland tumours have been widely studied from a prognostic standpoint, considerable variations in tumour individual biologic behaviour difficult the definition of accurate prognostic factors. It has been suggested that the malignant behaviour of tumours is the end result of several alterations in cellular physiology that culminate in tumour growth and spread. Accordingly, the aim of this study was to determine, using a multivariable model, the independent prognostic value of several immunohistochemically detected tumour-associated molecules, such as MMP-9 and uPA in stromal cells and Ki-67, TIMP-2 and VEGF in cancer cells. RESULTS: Eighty-five female dogs affected by spontaneous malignant mammary neoplasias were followed up for a 2-year post-operative period. In univariate analysis, tumour characteristics such as size, mode of growth, regional lymph node metastases, tumour cell MIB-1 LI and MMP-9 and uPA expressions in tumour-adjacent fibroblasts, were associated with both survival and disease-free intervals. Histological type and grade were related with overall survival while VEGF and TIMP-2 were not significantly associated with none of the outcome parameters. In multivariable analysis, only a MIB-1 labelling index higher than 40% and a stromal expression of MMP-9 higher than 50% retained significant relationships with poor overall and disease-free survival. CONCLUSIONS: The results of this study indicate that MMP-9 and Ki-67 are independent prognostic markers of canine malignant mammary tumours. Furthermore, the high stromal expressions of uPA and MMP-9 in aggressive tumours suggest that these molecules are potential therapeutic targets in the post-operative treatment of canine mammary cancer.
BMC Veterinary Research 01/2013; 9(1):1. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: To immunohistochemically evaluate matrix metalloproteinase (MMP)-9 expression in benign and malignant mammary gland tumors (MMTs) in dogs and relate expression to prognostic factors and patient outcome.
118 female dogs with naturally occurring mammary gland tumors and 8 dogs without mammary gland tumors.
24 benign mammary gland tumors and 94 MMTs (1/affected dog) were obtained during surgical treatment; control mammary gland tissue samples were collected from unaffected dogs after euthanasia for reasons unrelated to the study. Tumors were evaluated for proliferation, invasive growth, histologic grade, and metastatic capacity; expression of MMP-9 was determined immunohistochemically, and its relationship with clinical and histologic findings was investigated. For dogs with MMTs, follow-up continued for 2 years; data were used to compute overall survival time and disease-free interval and construct survival curves.
MMTs had significantly higher MMP-9 expression in stromal cells and in neo-plastic cells than did the benign neoplasms. Stromal MMP-9 expression was also higher in highly proliferative tumors and in tumors with invasive growth, high histologic grade, and metastatic capacity. Furthermore, tumors from patients with shorter overall survival times and disease-free intervals had higher expression of MMP-9 in stromal cells.
In dogs with MMTs, level of MMP-9 expression by stromal cells was related to factors of poor prognosis and shorter overall survival times and disease-free intervals. These results suggested that MMP-9 produced by tumor-adjacent stromal cells contributed to MMT progression in female dogs and that assessment of MMP-9 expression may be a valuable prognostic factor.
American Journal of Veterinary Research 05/2012; 73(5):689-97. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bracken (Pteridium aquilinum) has long been known to cause cancer in farm and laboratory animals. Ptaquiloside, a norsesquiterpene glycoside found in bracken, is considered its main carcinogenic toxin and is capable of inducing tumours in a variety of organ systems, but especially in the urinary bladder, depending on the animal species, the administration route employed and the duration of exposure. In the present study, 12 male CD-1 mice were intraperitoneally administered with 0.5 mg ptaquiloside weekly for 15 weeks, followed by 15 weeks without any treatment. Twelve animals used as controls were administered the vehicle solution (phosphate buffered saline). Two exposed animals died during the experimental work. On necropsy, blood and tissue samples (brain, eyes, thymus, heart, lungs, liver, digestive system, spleen, bladder, kidney, adrenal gland, urinary bladder, sexual accessory glands, testes, muscle, skin and femur) were collected for histological analysis. Leukograms were prepared from blood smears and total WBC counts obtained with a Neubauer chamber. Flow cytometry was used to assess blood T-(CD3(+)) and B-(CD19(+))-lymphocytes, medullary granulocytic (CD11b(+)/Ly-6G(-), CD11b(+)/Ly-6G(+)) and lymphocytic (CD19(+)/IgM(-), CD19+/IgM(+)) populations and thymic lymphoid (CD4(+), CD8(+), CD4(+)/CD8(+)) populations. Lymphoproliferative lesions were analysed immunohistochemically using antibodies against CD45R and CD3. All of the 10 surviving mice developed a lymphoproliferative malignancy. Lymphoproliferative disease was characterized by multifocal B-(CD45(+)/CD3(-))-lymphocytic renal (10/10 animals) and hepatic (2/10 animals) invasion, splenic white pulp hyperplasia (10/10) together with a significant increase in circulating B-(CD19(+))-lymphocytes and the appearance of circulating dysplastic lymphoid cells. Eight out of 10 ptaquiloside-exposed animals developed urothelial dysplasia (six low-grade dysplasia and two high-grade dysplasia). No lesions were detected in control mice. These results show that ptaquiloside is capable of inducing malignant transformation in mice and provide an in-depth characterisation of lymphoproliferative lesions. Furthermore, the urinary bladder is shown to be a target organ for this toxin in mice as well as in other animal species.
[show abstract][hide abstract] ABSTRACT: Urokinase plasminogen activator (uPA) has been associated with aggressive behaviour and poor prognosis in human breast cancer, but there is no information on its expression in canine mammary tumours (CMT). uPA immunohistochemical expression was studied in 119 CMT (24 benign, 95 malignant) to investigate its relationship with clinical and histopathological parameters. Dogs with malignant mammary tumours (MMT) underwent a 2-year follow-up evaluation. MMT expressed significantly more uPA than benign tumours. In MMT, high uPA stromal expression was significantly associated with larger tumour size, high Ki-67 expression, invasive growth, high histological grade, regional lymph node metastases, development of distant metastases, and lower overall survival (OS) and disease-free survival (DFS). On the basis of these results, uPA could be considered a useful prognostic factor in dogs with MMT.
The Veterinary Journal 07/2011; 189(1):43-8. · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Canine mammary tumours (CMTs) are a very heterogeneous group of neoplasms with variable prognosis. Their aggressiveness is mainly due to their ability to invade locally and to metastasize. The degradation of extracellular matrix components is an important determinant of the invasive phenotype. The aims of this study were to analyse by immunohistochemistry and double immunofluorescence the expression of metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in eight normal canine mammary glands and 118 CMTs (24 benign, 94 malignant) and to investigate relationships with metastatic disease and survival. MMP-2 and TIMP-2 expression was higher in malignant tumours than in normal canine mammary tissue and benign tumours. The main difference between benign and malignant CMTs was the pattern of expression of both molecules: benign tumours presented TIMP-2 and MMP-2 immunoreactivity in the myoepithelial cells lining the basement membrane of tubuloalveolar structures, while malignant tumours showed mainly diffuse expression in neoplastic cells. In malignant tumours, increased TIMP-2 expression was significantly associated with the development of distant metastases, lower overall survival and lower disease-free survival. MMP-2 expression was not significantly associated to any of these parameters. These results suggest that the immunohistochemical expression of TIMP-2 is a useful prognostic factor in CMTs.
The Veterinary Journal 01/2011; 190(3):396-402. · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: The histopathological and clinical aspects of canine mammary tumours (CMTs) have been widely studied, but the variation in the biological behaviour of these neoplasms hampers the identification of prognostic factors. Sustained angiogenesis has been suggested to be one of the most important factors underlying tumour growth and invasion. This process involves the action of several growth factors including vascular endothelial growth factor (VEGF). The present study characterizes the relationship between immunohistochemical expression of VEGF and gross (e.g. size and tissue fixation) and microscopical (e.g. type, growth, necrosis, lymphoid infiltration, lymph node metastasis, histological grade and proliferation index) features of CMTs. Forty-eight benign and 64 malignant CMTs were evaluated. Statistical analysis failed to show a significant relationship between VEGF expression and the pathological features, suggesting that VEGF expression occurs in both benign and malignant tumours and is independent of histological type, proliferation, tissue invasion or local metastatic capacity.
Journal of comparative pathology 11/2010; 143(4):268-75. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Caveolin-1 (Cav-1) is a major structural protein of caveolae, plasma membrane invaginations related to several cellular processes including regulation of signal transduction. In recent years there has been some controversy regarding the distribution of Cav-1 in normal and neoplastic mammary cell types, which may be attributed to different scoring systems adopted in different studies. The present study compares Cav-1 immunoexpression in normal (n=17) and neoplastic (n=79) canine mammary tissues assessed by two different scoring methods (previously reported by others with conflicting results) and associates Cav-1 expression with metastasis and overall survival (OS). Results obtained with both scoring methods were similar, revealing absence of immunoreactivity in normal luminal epithelium and in benign neoplasms and clearly associating Cav-1 expression with malignant transformation. The data suggest that Cav-1 expression is associated with highly malignant subtypes of mammary tumours (i.e. basal-like carcinoma), invasion and metastasis, thus supporting the hypothesis that it may play a major role in the epithelial-mesenchymal transition process. Furthermore, one of the scoring systems employed associated Cav-1 expression with unfavourable prognosis in canine mammary carcinomas, showing a strong correlation between Cav-1-positive carcinomas and shorter OS.
Journal of comparative pathology 02/2010; 143(2-3):87-93. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Caveolins are the major structural proteins of caveolae and play a role in tumour surveillance. The expression of caveolin-1 (Cav-1) was investigated immunohistochemically in samples of normal canine mammary tissue (n=5), benign (n=23) and malignant (n=45) mammary tumours, tumour emboli (n=10) and metastatic lesions (n=10). Cav-1 was not expressed by normal luminal epithelium, but was consistently expressed by normal myoepithelial cells. There was no epithelial expression of Cav-1 by the benign mammary lesions, but the molecule was detected in 35.6% of the malignant lesions, 80% of the tumour emboli and in 40% of the metastatic lesions. These findings link increased expression of Cav-1 to neoplastic transformation and suggest that Cav-1 expression is associated with more malignant canine mammary tumours and their vascular invasion and regional lymph node metastasis.
Journal of comparative pathology 02/2010; 143(1):39-44. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Catechol-O-methyltransferase (COMT) is an important enzyme involved in inactivation of catechol estrogens, which are metabolites with carcinogenic properties. Some investigations in human breast cancer associate a genetic polymorphism in the COMT gene (COMT val158met) with an increased risk and poor clinical progression of the disease. In dogs, there are 2 recognized single nucleotide polymorphisms in the COMT gene (COMTG216A and COMTG482A); however, their influence on the outcome of mammary neoplasms has never been investigated. The purpose of this study is to investigate the influence of COMT in the clinical progression of canine mammary tumors, namely in recurrence, metastasis and survival by testing 2 SNPs (G216A and G482A), and 2 genotypes of the COMT gene. A case series was conducted analyzing genomic DNA samples by polymerase chain reaction-restriction fragment length polymorphism from 80 bitches with mammary tumors. Animals were submitted to an active follow-up study for a period of 24 months after surgery. We observed that bitches carrying both genetic variations simultaneously are more likely to develop recurrence of mammary lesions. Our results demonstrate a possible role for COMT genotypes in the outcome of mammary neoplasms in the dog. Identifying a genetic factor predictive of recurrence may be useful in selecting the most effective surgical approach for canine mammary neoplasms.
[show abstract][hide abstract] ABSTRACT: COX-2 expression was examined immunohistochemically in samples of normal canine mammary tissue (n=22) and benign (n=36) and malignant (n=45) mammary tumours including metastases (n=12). COX-2 was constitutively expressed in normal mammary tissue with membranous apical labelling of glandular epithelium, suggesting a role for this molecule in normal mammary physiology. By contrast, in neoplastic lesions and in adjacent non-neoplastic mammary tissue COX-2 was expressed in the cytoplasm of epithelial cells, suggesting that internalization of the molecule is associated with oncogenesis. Marked expression of COX-2 was observed in 8.3% of benign neoplasms and in 42.2% of malignant neoplasms, mainly in poorly differentiated areas. The majority of metastatic lesions (58.3%) exhibited strong COX-2 labelling and in almost all cases (83.3%) the labelling intensity was similar or stronger to that of the primary neoplasm. This finding is consistent with the hypothesis that COX-2 metabolites are important promoters of angiogenesis and invasiveness and therefore contribute to metastatic spread.
Journal of comparative pathology 03/2009; 140(4):247-53. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Modifications in cell surface glycosylation affecting cell adhesion are common characteristics of transformed cells. This study characterizes the N-glycosylation profile of E-cadherin in models of canine mammary gland adenoma and carcinoma evaluating the importance of these glycosylation modifications in the malignant phenotype. Our results show that the pattern of E-cadherin N-glycosylation in mammary carcinoma is characterized by highly branched N-glycans, increase in sialylation and an expression of few high mannose structures. Detailed mass spectrometry analysis demonstrated a new N-glycosylation site containing a potential complex type N-glycan in E-cadherin from a mammary carcinoma cell line. Our study demonstrates the importance of E-cadherin N-glycans in the process of tumor development and in the transformation to the malignant phenotype.
Biochemical and Biophysical Research Communications 02/2009; 379(4):1091-6. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Catechol-O-methyltransferase (COMT) is an important enzyme participating in inactivation of carcinogenic oestrogen metabolites. In humans there is a single nucleotide polymorphism in COMT gene (COMT val158met) that has been associated with an increased risk for developing breast cancer. In dogs, there is a single nucleotide polymorphism in COMT gene (G482A), but its relation with mammary carcinogenesis has never been investigated. The aim of this study was to focus on the evaluation of such polymorphism as a risk factor for the development of mammary tumors in bitches and on the analysis of its relationship with some clinicopathologic features (dog's age and weight, number and histologic type of the lesions, lymph node metastasis) of canine mammary neoplasms. A case-control study was conducted analyzing 90 bitches with mammary tumors and 84 bitches without evidence of neoplastic disease. The COMT G482A polymorphism was analyzed by PCR-RFLP. We found a protective effect of the polymorphism in age of onset of mammary tumors, although we could not establish a significant association between COMT genotype and other clinicopathologic parameters nor with mammary tumor risk overall. Animals carrying the variant allele have a threefold likelihood of developing mammary tumors after 9 years of age in comparison with noncarriers. The Kaplan-Meier method revealed significant differences in the waiting time for onset of malignant disease for A allele carrier (12.46 years) and noncarrier (11.13 years) animals. This investigation constitutes the first case-control study designed to assess the relationship between polymorphic genes and mammary tumor risk in dogs. Our results point to the combined effect of COMT genotype with other genetic and/or environmental risk factors as important key factors for mammary tumor etiopathogenesis.
[show abstract][hide abstract] ABSTRACT: Recent studies of canine malignant mammary tumours suggest that reduction of E-cadherin and/or beta-catenin correlates with invasive behaviour and lymph node metastasis. The aims of this study were to examine the interrelationships between the expression of E-cadherin and beta-catenin, and the relationship between the expression of E-cadherin and/or beta-catenin and the mode of growth and metastatic capacity of canine malignant mammary tumours. 90 spontaneous malignant tumours and local and regional lymph nodes were studied. A significant relationship was evidenced between membranous expression of E-cadherin and beta-catenin (p=0.0027), but not between E-cadherin and cytoplasmic beta-catenin. Only E-cadherin as a separate factor was significantly related to tumour invasion (p=0.0072) and lymph node metastasis (p=0.0001). Neither membranous nor cytoplasmic beta-catenin expression was significantly related to either of these phenomena.
[show abstract][hide abstract] ABSTRACT: Cutaneous mast cell tumours are one of the most common neoplasms in dogs and show a highly variable biologic behaviour. Several prognosis tools have been proposed for canine mast cell tumours, including histological grading and cell proliferation markers. CD117 is a receptor tyrosine kinase thought to play a key role in human and canine mast cell neoplasms. Normal (membrane-associated) and aberrant (cytoplasmic, focal or diffuse) CD117 immunoexpression patterns have been identified in canine mast cell tumours. Cytoplasmic CD117 expression has been found to correlate with higher histological grade and with a worsened post-surgical prognosis. This study addresses the role of CD117 in canine mast cell tumours by studying the correlations between CD117 immunoexpression patterns, two proliferation markers (Ki67 and AgNORs) histological grade, and several other pathological variables.
Highly significant (p < 0,001) correlations were found between CD117 immunostaining patterns and histological grade, cell proliferation markers (Ki67, AgNORs) and tumoral necrosis. Highly significant (p < 0,001) correlations were also established between the two cellular proliferation markers and histological grade, tumour necrosis and epidermal ulceration. A significant correlation (p = 0.035) was observed between CD117 expression patterns and epidermal ulceration. No differences were observed between focal and diffuse cytoplasmic CD117 staining patterns concerning any of the variables studied.
These findings highlight the key role of CD117 in the biopathology of canine MCTs and confirm the relationship between aberrant CD117 expression and increased cell proliferation and higher histological grade. Further studies are needed to unravel the cellular mechanisms underlying focal and diffuse cytoplasmic CD117 staining patterns, and their respective biopathologic relevance.
BMC Veterinary Research 02/2007; 3:19. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sialyl Lewis x (sLex) antigen is a carbohydrate antigen that is considered not only a marker for cancer but also implicated functionally in the malignant behaviour of cancer cells. Overexpression of sLex is associated with enhanced progression and metastases of many types of cancer including those of the mammary gland. Canine mammary tumours can invade and give rise to metastases via either lymphatic or blood vessels.E-Cadherin is specifically involved in epithelial cell-to-cell adhesion. In cancer, E-Cadherin underexpression is one of the alterations that characterizes the invasive phenotype and is considered an invasion/tumour suppressor gene. Partial or complete loss of E-Cadherin expression correlates with poor prognosis in canine malignant mammary cancer. The aim of this study was to analyse the sLex expression in canine malignant mammary tumours and to evaluate if the presence of sLex correlates with the expression of E-Cadherin and with clinicopathological features.
Fifty-three cases of canine mammary carcinomas were analysed immunohistochemically using monoclonal antibodies against sLex (IgM) and E-Cadherin (IgG). The clinicopathological data were then assessed to determine whether there was a correlation with sLex tumour expression. Double labelled immunofluorescence staining was performed to analyse the combined expression of sLex and E-Cadherin.
sLex expression was consistently demonstrated in all cases of canine mammary carcinomas with different levels of expression. We found a significant relationship between the levels of sLex expression and the presence of lymph node metastases. We also demonstrated that when E-Cadherin expression was increased sLex was reduced and vice-versa. The combined analysis of both adhesion molecules revealed an inverse relationship.
In the present study we demonstrate the importance of sLex in the malignant phenotype of canine malignant mammary tumours. Our results support the use of sLex as a prognostic tumour marker in canine mammary carcinomas. Furthermore, we showed that sLex and E-Cadherin expression were inversely correlated. Future studies are warranted to clarify the molecular mechanism underlying the relation between sLex and E-Cadherin in canine mammary carcinoma cells which represents an important comparative model to woman breast cancer.
[show abstract][hide abstract] ABSTRACT: Abstract
Sialyl Lewis x (sLe<sup>x</sup>) antigen is a carbohydrate antigen that is considered not only a marker for cancer but also implicated functionally in the malignant behaviour of cancer cells. Overexpression of sLe<sup>x </sup>is associated with enhanced progression and metastases of many types of cancer including those of the mammary gland. Canine mammary tumours can invade and give rise to metastases via either lymphatic or blood vessels.
E-Cadherin is specifically involved in epithelial cell-to-cell adhesion. In cancer, E-Cadherin underexpression is one of the alterations that characterizes the invasive phenotype and is considered an invasion/tumour suppressor gene. Partial or complete loss of E-Cadherin expression correlates with poor prognosis in canine malignant mammary cancer.
The aim of this study was to analyse the sLe<sup>x </sup>expression in canine malignant mammary tumours and to evaluate if the presence of sLe<sup>x </sup>correlates with the expression of E-Cadherin and with clinicopathological features.
Fifty-three cases of canine mammary carcinomas were analysed immunohistochemically using monoclonal antibodies against sLe<sup>x </sup>(IgM) and E-Cadherin (IgG). The clinicopathological data were then assessed to determine whether there was a correlation with sLe<sup>x </sup>tumour expression. Double labelled immunofluorescence staining was performed to analyse the combined expression of sLe<sup>x </sup>and E-Cadherin.
sLe<sup>x </sup>expression was consistently demonstrated in all cases of canine mammary carcinomas with different levels of expression. We found a significant relationship between the levels of sLe<sup>x </sup>expression and the presence of lymph node metastases. We also demonstrated that when E-Cadherin expression was increased sLe<sup>x </sup>was reduced and vice-versa. The combined analysis of both adhesion molecules revealed an inverse relationship.
In the present study we demonstrate the importance of sLe<sup>x </sup>in the malignant phenotype of canine malignant mammary tumours. Our results support the use of sLe<sup>x </sup>as a prognostic tumour marker in canine mammary carcinomas. Furthermore, we showed that sLe<sup>x </sup>and E-Cadherin expression were inversely correlated. Future studies are warranted to clarify the molecular mechanism underlying the relation between sLe<sup>x </sup>and E-Cadherin in canine mammary carcinoma cells which represents an important comparative model to woman breast cancer.
[show abstract][hide abstract] ABSTRACT: The relationship between MIB-1 labelling indices (LI), as detected by immunohistochemical methods, and other clinico-pathological characteristics was studied in a series of 77 malignant mammary tumours surgically removed from 47 female dogs. The immunostaining was assessed on the basis of the estimated percentage of positive cells in the areas of highest labelling. Multivariate logistic regression demonstrated no influence of breed, age, previous pregnancies, previous progestin administration, histological type or location of the tumour on MIB-1 LI. MIB-1 LI was significantly related to the size of the tumour, necrosis, invasive growth and histological grade, but not with ulceration, lymph node metastasis, skin fixation or E-cadherin expression. The significant relationship between MIB-1 LI and other known factors of poor prognosis suggests that a high LI may have prognostic value in canine malignant mammary tumours.
Anticancer research 26(3A):1821-6. · 1.71 Impact Factor