Evaluation of angiogenesis in canine mammary tumors by quantitative platelet endothelial cell adhesion molecule immunohistochemistry

Angiogenesis was evaluated by immunohistochemistry for platelet endothelial cell adhesion molecule (CD31) in a series of benign and malignant canine mammary neoplasms. Computer image analysis was used to measure the intratumoral microvessel density (number of microvessels per square millimeter) and the area and perimeter of immunolabeled vascular structures. A higher intratumoral microvessel density and lower values for area and perimeter were found in malignant neoplasms compared with benign tumors and among the malignant tumors, in less differentiated phenotypes and in more anaplastic histological types (solid and squamous carcinomas), most of which had metastasized. These findings are consistent with an increase in angiogenesis in multistage neoplasia. Therefore, in more anaplastic malignant neoplasms, numerous but small and often malformed new vessels can be identified. The correlation of angiogenesis data with differentiation grade and histological type of mammary neoplasms is consistent with the findings in human medicine and demonstrates that angiogenesis can also have a prognostic value in veterinary medicine.     

The Prognostic Significance of Angiogenesis in Canine Mammary Tumors

The purpose of the study was to determine if neovascularization, a measure of angiogenesis, is correlated with metastasis of mammary tumors in dogs. Forty-six paraffin-embedded tissue blocks of benign and malignant canine mammary tumors obtained from 42 clinical cases at the Iowa State University Veterinary Teaching Hospital were retrieved from the archives of the Department of Veterinary Pathology. Of the dogs with malignant tumors, cases with and without lymph node metastasis were chosen. Neovascularization was quantified by light microscopy on formalin-fixed, paraffin-embedded sections of canine mammary tumors using an avidin biotin immunoperoxidase assay for factor VIII-related antigen. Mean microvessel counts for each group were statistically evaluated using analysis of variance. The mean number of microvessels was highest in the malignant tumors of dogs with lymph node metastasis (44). This number was significantly different from the mean number of microvessels in the benign tumors (28; P = .03) and a trend occurred toward higher microvessel counts in malignant tumors with lymph node metastasis versus malignant tumors of dogs without metastasis (32; P = .1). No significant difference was found between the number of microvessels found in malignant tumors without metastasis versus benign tumors. The trend toward higher microvessel counts in mammary tumors that have metastasized supports the premise that angiogenesis may be an independent and significant prognostic indicator in dogs with malignant mammary tumors, as it is in women with breast cancer.     

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.     

Correlation of vascular endothelial growth factor expression to overall survival in feline invasive mammary carcinomas

Samples from feline invasive mammary carcinomas (FMCs) were used to determine the prognostic significance of the immunohistochemical expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD). Forty-eight queens bearing FMCs were included in a 2-year follow-up study. Mammary tumors were classified according to the World Health Organization system and graded on the basis of histologic criteria. Tumor sections were immunostained using anti-VEGF and anti-von Willebrand factor (vWf) antibodies. VEGF expression was quantified on the basis of the percentage of positive cells. MVD of vWf-positive microvessels was determined by both mean microvessel counts and highest microvessel counts. Normal mammary gland tissues showed an inconspicuous VEGF staining. In FMCs the proportion of VEGF-positive cells was significantly higher in papillary and solid carcinomas than in tubular and papillary cystic tumors. An increased number of cells expressing VEGF was also observed in poorly differentiated FMCS. Sixteen (33.3%) of the queens bearing invasive carcinomas were still alive at the end of the 2-year follow-up period, and 32 (66.7%) had died. The VEGF expression was significantly correlated with the clinical outcome, but no correlation was observed with the invasion of lymphatic vessels. A correlation between the higher percentage of VEGF-positive cells and the unfavorable prognosis was demonstrated by the estimation of curves for overall survival (P = 0.03). Univariate analysis showed that MVD did not correlate with the overall survival. The results of our study demonstrated that VEGF expression, although not associated with increased angiogenesis, is a prognostic indicator in feline mammary tumors. In contrast, there is no support for a role of neovascularization as an indicator of survivability.     

Tumour‐associated macrophages are associated with vascular endothelial growth factor expression in canine mammary tumours

Tumour‐associated macrophages (TAMs) have been implicated in carcinogenesis including an important role in angiogenesis. In this study, we describe the relationship between TAMs and angiogenesis in canine mammary tumours (CMT). Formalin‐fixed paraffin‐embedded CMT samples [(n = 128: malignant (n = 97) and benign (n = 31)] were submitted to immunohistochemical staining to detect MAC387, vascular endothelial growth factor VEGF and CD31 expression. A statistical analysis was carried out to assess possible associations with clinicopathological variables and biological markers of tumour angiogenesis. TAMs, detected by MAC387 expression, were significantly associated with malignant CMT (P < 0.001) and VEGF positive tumours (P = 0.002) and also associated with VEGF expression within malignant CMT (P = 0.043). Associations with clinicopathological variables were found between TAMs and the presence of infiltrative growth (P = 0.031), low tubule formation (P = 0.040) and lymph node metastasis (P = 0.016). The results support the hypothesis that TAMs influence angiogenesis in CMT suggesting TAMs may represent a therapeutic target in this disease.     

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

An epidemiologic study of canine multiple primary neoplasma involving the female and male reproductive systems

The significance of canine multiple primary neoplasms involving the reproductive system and one or more additional anatomic sites was evaluated using data from the Alameda and Contra Costa County Animal Tumor Registry. Among the registry's data base of 35 015 histologically confirmed tumors, both sequential and simultaneous occurrences of histologically distinct benign and malignant tumors were identified. Retrospective analysis demostrated that there was a decreasing risk gradient for multiple tumors of the reproductive systems. The intact female had the highest risk followed in order by the spayed female and all male dogs. The incidence-based analyses also indicated a strong predilection for development of multiple tumors within both the female and the male reproductive systems. A four- to five-fold increase in observed numbers of mammary tumors and other histologically distinct reproductive tumors was found. Benign and malignant mammary tumors were strongly associated in their occurrence, and dogs with benign mammary neoplasms had a three-fold increased risk for subsequent development of histologically different malignant mammary tumors. These quantitative results were consistent with observations in man for malignancies and provide additional evidence that multiple primary malignant and benign neoplasms of the female and male reproductive systems are biologically associated.     

RADIOGRAPHIC CHANGES ASSOCIATED WITH DIGITAL, METACARPAL AND METATARSAL TUMORS, AND PODODERMATITIS IN THE DOG

Fifty-two of digital, metacarpal or metatarsal canine tumors and 48 pododermatitis lesions were reviewed retrospectively. The tumors were divided into 42 malignant neoplasms, 6 benign neoplasms and 4 non-neoplastic tumor-like growths. The distribution and radiographic changes of the lesions associated with the tumors and inflammation were compared. Pododermatitis and benign processes could not be radiographically differentiated from malignant neoplasms of the foot; however, lesions exhibiting osteolysis were more likely (p < 0.05) to be associated with malignant neoplasms.     

Clinical significance of circulating vascular endothelial growth factor in dogs with mammary gland tumors

Increase in circulating vascular endothelial growth factor (VEGF) is suggested as a prognostic indicator in human patients with malignant tumors. The purpose of this study was to evaluate the clinical significance of circulating VEGF in dogs with mammary gland tumors (MGT). Both plasma and serum VEGF were significantly higher in dogs with MGT when compared with those in the healthy dogs. In dogs with MGT, the plasma and serum VEGF of the malignant group increased significantly compared with those of the benign group. Additionally, there was a significant difference between the plasma and serum VEGF in the groups with postoperative metastasis and no metastasis. Circulating VEGF is expected to be clinically available for the determination of prognosis in canine MGT.     

Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1-3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P<0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.     

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.     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in canine simple mammary gland adenocarcinomas

The expression of 5 markers associated with angiogenesis, proliferation, and apoptosis was studied in 26 canine simple mammary gland adenocarcinomas (SMGAs). The adenocarcinomas were graded histologically, and tissue sections were immunohistochemically stained for the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), intra-tumor microvessel density, and tumor proliferation (PI) using antibodies against VEGF, VEGFR-2, von Willebrand factor, and Ki-67 antigen, respectively. Apoptotic indices (AI) were determined by an apoptosis assay. Markers VEGF and VEGFR-2 were detected in 96% and 100% of SMGAs, respectively. A high correlation between histologic grade and PI (r = 0.73), a moderate correlation between VEGF and histologic grade (r = 0.33), and between VEGF and PI (r = 0.42) were found. There was a significant difference in median PI among the 3 histologic grade groups (r < 0.05). Vascular endothelial growth factor may stimulate tumor cell proliferation through an autocrine loop, since VEGF and VEGFR-2 were expressed in most tumors.     

Mast cells in canine cutaneous hemangioma,hemangiosarcoma and mammary tumors

Mast cell count (MCC) in 45 dogs with cutaneous hemangioma (HA, n = 12), hemangiosarcoma (HSA, n = 12), mammary adenoma (AD, n = 9) and mammary adenocarcinoma (AC, n = 12) was made using Toluidine blue stained sections. Antibodies against endothelial cell markers, Factor VIII and VEGF were used to visualize and determine the hot spot micro-vessel density (MVD). Total MCC and MCC along the invasive edges were significantly higher (p < 0.001) in canine mammary AC than in AD. The total MCC did not significantly differ (p > 0.05), in HSAs (8.6 ± 3.3) than in HAs (5.5 ± 2.8). There is a positive correlation (r = 0.14) between the hot spot MCC and MVD in mammary AC, although not significant (p = 0.3172), indicating that mast cells are associated with angiogenesis in canine mammary AC. This study suggests that mast cells may play an important role in neovascularization of canine cutaneous vascular and mammary neoplasms. Detailed studies encompassing correlation of MCC and MVD with clinical outcomes and prognosis in these neoplasms are recommended.     

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours*

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.     

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

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

Metallothionein expression in benign and malignant canine mammary gland tumours

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

The role of vascular endothelial growth factor and its receptor Flk-1/KDR in promoting tumour angiogenesis in feline and canine mammary carcinomas: a preliminary study of autocrine and paracrine loops

Vascular Endothelial Growth Factor (VEGF) and its receptor KDR are involved in the regulation of angiogenesis and are up-regulated in a number of tumours in humans and in particular, breast cancer. We therefore evaluated the prognostic potential of the angiogenetic process in feline and canine mammary carcinomas by the immunohistochemical assessment of VEGF expression and micro vessel density (MVD) quantification and examined the interplay between VEGF and KDR. These variables were related to some relevant clinicopathological parameters and to overall survival (OS). VEGF and KDR expression were evaluated in epithelial, stromal and endothelial compartments in order to identify autocrine and/or paracrine loops. In dogs an increased VEGF expression did not show any statistical correlation with the clinicopathological parameters examined and was not correlated to a poorer prognosis. MVD was found to be significantly correlated to the histologic type (P=0.04), tumour grading (P=0.02), and to the OS (P=0.01). In cats VEGF expression was significantly correlated to tumor grading (P=0.01) and OS (P=0.03), while no significant associations were found between MVD and the other parameters. VEGF and KDR were found to be detected on the epithelial, and/or endothelial and/or stromal cells of the carcinomas in both species, suggesting indications for some possible autocrine and paracrine loops. Our results encourage further studies on the possible prognostic role of VEGF and MVD in canine and feline mammary tumours and on the role of growth factors and their receptors in promoting tumour proliferation and an "angiogenetic shift". The VEGF/KDR system may play a role in malignant transformation and tumor progression.     

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.