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 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.     

Overexpression of HER-2 in feline invasive mammary carcinomas: an immunohistochemical survey and evaluation of its prognostic potential

The role of c-erbB-2 protooncogene status in feline invasive mammary carcinomas (FMCs) was assessed through the HER-2 receptor immunohistochemical expression. The HER-2 overexpression was then correlated with some relevant histologic parameters and with the clinical course of the disease during a 2-year follow-up. Forty-seven FMCs from surgically treated queens were considered. Tumors were classified according to the WHO criteria and stromal or lymphatic invasion (or both) and histologic grading were recorded. The immunohistochemical staining was performed on paraffin sections and a well-defined scoring system based upon numbers of HER-2 receptors expressed on the cell surface was applied according to standard guidelines. Overall survival (OS) distributions were generated with the Kaplan-Meier method. HER-2 overexpression was detected in 28 of the 47 carcinomas (59.6%). This parameter was demonstrated to be significantly correlated with the shorter OS (P = 0.02). However, the HER-2 overexpression did not show significant correlation with histologic type, tumor grading, or presence of lymphatic invasion. Furthermore, the HER-2 overexpression appeared with a higher percentage in FMCs than what is reported in canine or human mammary carcinomas. The significant correlation with a shorter OS suggests a possible role of HER-2 as an additional marker of malignancy in FMCs and as a reliable prognostic indicator. As in the human oncology practice, the identification of the FMCs that overexpress HER-2 may also promote new therapeutic strategies.     

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.     

Expression and significance of PTEN and VEGF in canine mammary gland tumours

To investigate the relationship between the expression of the PTEN (phosphatase and tensin homolog deleted on chromosometen) and VEGF (vascular endothelial growth factor) and the clinicopathological features in canine mammary gland tumours, the expression levels of PTEN and VEGF protein were assessed in 50 cases of canine mammary gland tumours tissues and 4 cases of normal mammary gland tissues with using immunohistochemical method. The over-expression rate of PTEN protein was 100% in normal and well-differentiated mammary gland tissues and 67% in breast cancer cases respectively with a significant difference between the two groups (P<0.01). Expression of PTEN was not related to age and tumour size, but closely correlated to lymph node metastasis (P<0.01). The over-expression rate of VEGF protein was 33.3% in normal mammary gland tissues, and 78% in canine mammary gland tumours with a significant difference between the two groups (P<0.01).Expression of VEGF was not related to age or tumour size, but closely correlated with lymph node metastasis and clinical stage (P<0.05).Therefore the combination detection of PTEN and VEGF could serve as an important index to estimate the biological behavior and prognosis of canine mammary gland tumours. Reduced expression of PTEN might be involved in carcinogenesis and progression of canine breast cancer by up-regulating the VEGF expression to enhance angiogenesis.     

Prognostic impact of neoadjuvant aglepristone treatment in clinicopathological parameters of progesterone receptor‐positive canine mammary carcinomas

Neoadjuvant treatment of canine mammary carcinomas with the progesterone receptor (PR) antagonist aglepristone has a PR expression‐related inhibiting effect on proliferation index (PI). The aim of this study was to evaluate the effect of the treatment in the disease‐free period (DFP) and overall survival (OS) of canine mammary carcinomas. Fifty female dogs with mammary carcinomas were treated with aglepristone (n = 34) or oil vehicle (n = 16) before surgery (day 15). PR expression and PI were analysed by immunohistochemistry in samples taken at days 1 and 15. Epidemiological and clinicopathological data were assessed. DFP and OS data were retrieved every 4–6 months for at least 24 months after surgery. Aglepristone treatment increased DFP of animals bearing PR+ tumours with size smaller than 3 cm, complex and mixed tumours, with histologic grades I and II, and with PI ≤ 10%. Although further studies are necessary, current evidence points to treatment with aglepristone as useful for the management of canine mammary tumours.     

VEGFR‐2 expression in malignant tumours of the canine mammary gland: a prospective survival study

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is the main receptor activated by vascular endothelial growth factor ‐A (VEGF‐A) to promote tumour angiogenesis. Its clinical prognostic value has not been studied in canine mammary tumours (CMTs). Dogs with mammary cancer were enrolled in a survival study and the immunohistochemical expressions of VEGFR‐2 and VEGF‐A were analysed and associated with clinicopathological features. VEGFR‐2 expression was associated with VEGF immunoreactivity in cancer cells, supporting the presence of an autocrine loop that may be involved in CMTs growth and survival. VEGFR‐2 was also expressed by endothelial cells from tumour vasculature and positively associated with stromal matrix metalloproteinase‐9 (MMP‐9), suggesting the existence of a link between endothelial cells activation and up‐regulation of matrix degrading proteins. Carcinosarcomas exhibited high VEGFR‐2 expression suggesting that it may be one of the activated molecular pathways in this aggressive histological type and that VEGFR‐2 inhibitors may constitute a potential treatment to improve the prognosis of these patients. Both VEGF and VEGFR‐2 immunoreactivities were independent of patients' overall survival (OS) and disease‐free survival (DFS).     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

Comparison of steroid receptor expression in normal, dysplastic, and neoplastic canine and feline mammary tissues

Steroid receptor expression was assessed by immunohistochemistry in neoplastic, hyperplastic/dysplastic, and normal mammary tissue samples removed from 68 queens and 47 bitches, using monoclonal antibodies against human oestrogen-alpha (ER) and progesterone receptors (PR). Mammary lesions were classified according to World Health Organization (WHO) criteria, and all animals with invasive carcinomas were clinically followed for 2 years. Stromal and/or lymphatic invasion and histological grading were also recorded. In both species, ER expression was significantly higher in healthy tissues, hyperplastic/dysplastic lesions, and benign tumours than in carcinomas. The loss of ER expression was more marked in feline than in canine carcinomas. In queens, PR expression increased in dysplastic lesions and "in situ" carcinomas and decreased in invasive carcinomas, even if parts of these tumours were still PR-positive. In bitches no significant variation in PR expression was observed between normal tissue, dysplasias, and benign neoplasms, but was significantly lower in carcinomas. In both species ER and PR expression in invasive carcinomas did not correlate either with histological parameters or overall survival time. This study demonstrates several differences in steroid hormone dependency between the two species. The percentage of PR-positive feline carcinomas suggests a possible role of progesterone in promoting early tumour cell growth in queens. The low percentage of ER-positive invasive carcinomas further demonstrated the aggressive phenotype and behaviour of feline mammary tumours.     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas

Calponin is a 34‐kDa smooth muscle‐specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin–biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin‐positive cells were identified: (1) Type 1: cytokeratin‐14‐positive pre‐existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin‐14‐positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin‐14‐positive atypical cells indistinguishable from non‐reactive atypical cells, which should have never been detected in haematoxylin and eosin‐stained sections and (4) Type 4: cytokeratin‐14‐negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin‐negative and cytokeratin‐14‐positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high‐molecular‐weight cytokeratins in luminal epithelial‐type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.     

Expression of the glutamine metabolism‐related proteins glutaminase 1 and glutamate dehydrogenase in canine mammary tumours

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumour growth and glutamine metabolism. Because of their similarities, canine mammary carcinomas are useful for studying human breast cancer. Accordingly, we investigated the correlations between the expression of glutamine metabolism‐related proteins and the pathological features of canine mammary tumours. We performed immunohistochemical and western blot analysis of 39 mammary tumour tissues. In immunohistochemical analysis, the expression of glutaminase 1 (GLS1) in the epithelial region increased according to the histological grade (P < .005). In the stromal region, complex‐type tumours displayed significantly higher GLS1 intensity than simple‐type tumours. However, glutamate dehydrogenase expression did not show the same tendencies as GLS1. The western blot results were consistent with the immunohistochemical findings. These results suggest that the expression of GLS1 is correlates with clinicopathological factors in canine mammary tumours and shows a similar pattern to human breast cancer.     

The expression of sialyl Lewis X in canine and feline mammary gland tumors

The expression of sialyl Lewis X (sLe(x)) in 93 canine and 15 feline mammary gland tumors (MGT) obtained by surgical resection at Veterinary Medical Center, the University of Tokyo was examined by immunohistochemistry. Their clinicopathological features and prognosis were also reviewed. Approximately 60% of MGT tissues showed sLe(x) positive expressions, while all normal mammary gland tissues were negative. However, its expression was not correlated with clinicopathological features and prognosis significantly. This study suggests that sLe(x) may be a tumor-associated antigen in canine and feline MGTs.     

High COX‐2 expression is associated with increased angiogenesis,proliferation and tumoural inflammatory infiltrate in canine malignant mammary tumours: a multivariate survival study

COX‐2 expression affects mammary tumourigenesis by promoting angiogenesis and cell proliferation, encouraging metastatic spread and tumour‐associated inflammation. Samples of canine mammary tumours (n = 109) were submitted to immunohistochemistry to detect COX‐2, CD31, VEGF, Ki‐67, CD3 and MAC387 expression. Concurrent high expression of COX‐2/CD31, COX‐2/VEGF, COX‐2/Ki‐67, COX‐2/CD3 and COX‐2/MAC was associated with elevated grade of malignancy, presence of intravascular emboli and presence of lymph node metastasis. Tumours with high COX‐2 (P < 0.001) and tumours with concurrent expression of high COX‐2 and high CD31 (P = 0.008); high VEGF (P < 0.001); high Ki‐67 (P < 0.001); high CD3+ T‐lymphocytes (P = 0.002) and elevated MAC387 macrophages (P = 0.024) were associated with shorter overall survival (OS) time. Interestingly the groups with high COX‐2/CD31 and high COX‐2/VEGF retained their significance after multivariate analysis arising as independent predictors of OS. Present data highlight the importance of COX‐2 in canine mammary tumourigenesis.     

Immunohistochemical analysis of cyclin A, cyclin D1 and P53 in mammary tumors, squamous cell carcinomas and basal cell tumors of dogs and cats

The involvement of cyclin A, cyclin D1 and p53 proteins in canine and feline tumorigenesis was analyzed immunohistochemically. In the present study, a total of 176 cases were examined, among which there were 108 canine cases (75 mammary lesions, 16 squamous cell carcinomas and 17 basal cell tumors) and 68 feline cases (43 mammary lesions, 20 squamous cell carcinomas and 5 basal cell tumors). Speckled nuclear staining for cyclin A was observed in 19/38 (50%) canine malignant mammary tumors and 18/37 (48.6%) feline mammary carcinomas, while this was not seen in benign mammary tumors of either dogs or cats. Marked intense nuclear cyclin A staining was seen in 7/16 (43.8%) canine squamous cell carcinomas and 18/20 (90.0%) feline squamous cell carcinomas. Only 3/17 (17.6%) canine basal cell tumors showed slight and scattered staining for cyclin A. Expression of cyclin D1 was very rare in both canine and feline tumors. Nuclear staining of p53 was found in 7/37 (18.9%) feline mammary carcinomas. Intense immunoreactivity for p53 was found in 6/16 (37.5%) canine squamous cell carcinomas and 8/20 (40%) feline squamous cell carcinomas. These results suggest that cyclin A may have a role in the proliferation of canine malignant mammary tumors, feline mammary carcinomas and squamous cell carcinomas of dogs and cats, and p53 may associate with the tumorigenesis of feline mammary carcinomas and squamous cell carcinomas of dogs and cats.     

MIB-1 labeling index in feline dysplastic and neoplastic mammary lesions and its relationship with postsurgical prognosis

Samples from feline normal, dysplastic, and neoplastic mammary tissues were used to investigate the usefulness of MIB-1 labeling index (MIB-1 I) as a prognostic indicator. Forty-eight queens bearing invasive carcinomas were included in a 2-year follow-up study. Mammary lesions were classified according to the World Health Organization system, and invasive carcinomas were further graded on the basis of the degree of tubule formation, the degree of nuclear and cellular pleomorphism, and mitotic count. Additional sections were immunostained using MIB-1 antibody, and MIB-1 I was expressed as a percentage of positive nuclei. In normal mammary gland tissues, the mean MIB-1 I was <1%. A low proliferation rate was found in all mammary adenosis and in situ carcinomas, and the highest rates were observed in feline mammary hypertrophy and invasive carcinomas. Twenty-one (43.7%) of the queens bearing invasive carcinomas were still alive at the end of the trial, and 27 (56.2%) had died. The MIB-1 I was not significantly correlated with clinical outcome, age, histologic type, or grading of the tumors, but a borderline correlation was observed with invasion of lymphatic vessels. Univariate analysis showed that high MIB-1 I was also not associated with decreased overall survival, whereas the grading system of the tumors had high predictive value (P = 0.0040) for postsurgery survival. The lack of correlation between MIB-1 I and postsurgery survival suggests that this marker alone is not sufficient to determine a correct prognosis in feline mammary carcinomas, even if it is a useful proliferation marker.     

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk‐3)

Reduced expression in immortalized cells (REIC/Dkk‐3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk‐3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk‐3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk‐3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk‐3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk‐3 represents a potential molecular target for the development of therapies against feline mammary cancers.     

Amplification of the cyclin A gene in canine and feline mammary tumors

DNAs from 33 canine mammary tumors and 8 feline mammary carcinomas were examined by Southern blot analysis to clarify genomic abnormalities of the cyclin A gene. Amplification of cyclin A was detected in 27.3% (9/33) of canine mammary tumors and 87.5% (7/8) of feline mammary carcinomas. It was suggested that amplification of cyclin A do not correlate directly with the tumorigenesis of canine mammary tumors, because there was no significant difference of incidence of cyclin A amplification between the benign and malignant tumors. In feline mammary carcinomas, the high frequency of cyclin A amplification raised the possibility that the amplification lead to the protein overexpression and play an important role in the tumorigenesis.     

Association of tumour‐infiltrating regulatory T cells with adverse outcomes in dogs with malignant tumours

Regulatory T cells (Tregs) infiltrate into a variety of tumour tissues and associate with poor prognosis in humans. However, data on association of Treg infiltration with prognosis is limited in canine tumours. The purpose of this study was to examine the number of tumour‐infiltrating Tregs and its association with overall survival (OS) in dogs with malignant tumours. The following 168 canine tumours were included: 37 oral malignant melanomas (OMMs); 14 oral squamous cell carcinomas (OSCCs); 16 pulmonary adenocarcinomas (PAs); 37 mammary carcinomas (MCs); 36 mast cell tumours (MCTs) and 28 hepatocellular carcinomas (HCCs). Normal tissues were obtained from 8 healthy dogs as controls. The number of forkhead box P3 (Foxp3)‐positive Tregs in intratumoral and peritumoral areas was investigated by immunohistochemistry. OS was compared between high and low Treg groups. The number of intratumoral and peritumoral Foxp3‐positive Tregs was significantly higher in OMM, OSCC, PA and MC compared with each normal tissue. There were few Foxp3‐positive Tregs in MCT and HCC. With intratumoral Tregs, the OS in the high Treg group was significantly shorter than that in the low Treg group in OMM, OSCC and PA. With peritumoral Tregs, there was no significant difference for OS between the 2 groups in each tumour type. These results suggest that Tregs infiltrate into a variety of canine tumours and the abundance of Tregs are associated with poor prognosis in some solid tumour types.