Felisbina L. Queiroga

Role of steroid hormones and prolactin in canine mammary cancer

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.

The role of Cox-2 expression in the prognosis of dogs with malignant mammary tumours.

Immunohistochemical detection of Cyclooxygenase (Cox)-1 and -2 enzymes in canine mammary tumours (CMT) has recently been described. However, the prognostic value of their expression needs to be established. The aim of this study was to investigate Cox (-1 and -2) prognostic value in malignant CMT by evaluating its correlation with clinicopathological parameters (tumour size, histological type, necrosis, lymph node metastasis) and with Disease Free Survival (DFS) and Overall Survival (OS). Twenty seven female dogs with malignant tumours were included. Cox-2 expression was associated with lymph node metastasis at surgery time, development of distant metastasis during follow-up (p=0.038), DFS (p=0.03) and OS (p=0.04). Multivariate survival analysis showed that Cox-2 did not retain its significance as an independent prognostic factor. For Cox-1 expression, no statistically significant association was observed. Present study suggests the usefulness of testing Cox-2 specific inhibitors as part of an adjuvant therapy in female dogs with malignant mammary neoplasias.

COX-2 over-expression correlates with VEGF and tumour angiogenesis in canine mammary cancer

This study was designed to investigate the possible roles of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in canine mammary cancer angiogenesis. Immunohistochemistry was performed on 70 tumours (28 benign and 42 malignant) in order to detect COX-2 and VEGF expression. Microvessel density (MVD) was determined by CD31 immunolabelling to assess tumour angiogenesis. There was a significantly higher expression of COX-2 (P<0.001), VEGF (P<0.001) and MVD (P<0.001) in malignant compared to benign tumours. In the malignant group, the MVD of COX-2 positive tumours was significantly higher than that of COX-2 negative tumours (P=0.026). A similar association was observed for VEGF (P<0.001) positive tumours. The results from this study suggested that over-expression of COX-2 and VEGF may contribute to increased angiogenesis and aggression in malignant tumours.

COX-1 and COX-2 expression in canine cutaneous, oral and ocular melanocytic tumours.

In order to evaluate the potential value of non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of canine malignant melanoma, expression of cyclooxygenase (COX)-1 and COX-2 was determined in 20 cutaneous, nine oral and two ocular malignant melanomas, and in nine cutaneous melanocytomas. Almost all tumours expressed COX-1, but COX-2 expression was restricted to the malignant tumours being found in 11 of the 20 cutaneous malignant melanomas, all oral malignant melanomas and in one of two ocular malignant melanomas. COX-1 expression did not differ significantly between benign and malignant skin lesions, but COX-2 expression was significantly greater in cutaneous malignant melanoma compared with melanocytoma (P=0.047). COX-2 labelling was particularly intense in the more highly malignant oral tumours. The results of the study suggest that NSAIDs, particularly COX-2 inhibitors, may be useful in the treatment of canine malignant melanoma.

Crosstalk between GH/IGF-I axis and steroid hormones (progesterone, 17β-estradiol) in canine mammary tumours

Growth hormone (GH), insulin-like growth factor I (IGF-I), progesterone (P4) and 17beta-estradiol (17-E2) concentrations have been studied in 84 mammary tumours (44 dysplasias and benign tumours and 40 malignant neoplasias) from 33 female dogs. Thirteen normal mammary glands from 80 healthy female dogs were also analysed as controls. GH concentrations were determined in mammary homogenates by radio-immunoassay. IGF-I, P4 and 17-E2 tissue levels were determined by enzyme-immunoassay (EIA) techniques. The potential correlations between GH/IGF-I concentrations and P4 and 17-E2 mammary tissue levels were investigated. Tissue GH (p<0.01) and IGF-I concentrations (p<0.01) were significantly higher in malignant tumours than in benign neoplasms. Likewise, malignant tumours were the mammary lesions that displayed the highest P4 and 17-E2 tissue levels. Strong correlations between GH/IGF-I (n=84; r=0.436; p<0.001), P4/GH (n=84; r=0.562; p<0.001) and 17-E2/IGF-I (n=84; r=0.638; p<0.001) were observed in tumoral tissue homogenates. Our study provides evidence that P4 might increase autocrine GH production which might directly stimulate local or systemic IGF-I secretion. Additionally, the IGF-I effect might be influenced by local levels of 17-E2. These results suggest that all these hormones and factors might act as local growth factors stimulating the development and/or maintenance of canine mammary tumours in an autocrine/paracrine manner.

Inflammation and cancer: till death tears them apart.

Advances in biotechnology have enabled the collection of an immeasurable amount of information from genomic, transcriptomic, metabolomic and proteomic studies of tumours within their microenvironments. The dissection of cytokine and chemokine networks has provided new clues to the interactions between cancer cells and their surrounding inflammatory landscape. To bridge the gap between chronic inflammation and cancer, dynamic participants in the tumour microenvironment have been identified, including tumour-associated macrophages (TAMs) and regulatory T cells (Tregs). Both of these cell types are notable for their ability to cause immunosuppressive conditions and support the evasion of tumour immune surveillance. It is clear now that the tumour-promoting inflammatory environment has to be included as one of the major cancer hallmarks. This review explores the recent advances in the understanding of cancer-related inflammation and how this is being applied to comparative oncology studies in humans and domestic species, such as the dog.

Serum and intratumoural GH and IGF-I concentrations: prognostic factors in the outcome of canine mammary cancer.

The biological implication of the growth hormone/insulin like growth factor-I (GH/IGF-I) axis in canine mammary tumours (CMT) has been recently demonstrated, however its clinical and prognostic implications are unknown. Our aim was to investigate its prognostic significance. Hormonal determinations were done by enzyme immunoassays techniques validated for canine species in serum and tumour tissue from 32 bitches with CMT and in serum and normal mammary tissue from 10 controls. Serum and tissular GH and IGF-I concentrations were significantly higher in the case of malignant tumour compared with benign and controls. GH and IGF-I elevated concentrations were significantly associated with tumour relapse and/or metastases during follow-up and in dogs with reduced survival times; however these parameters were not independent prognostic factors in multivariate analysis. This association demonstrates a link between high serum and intratumoural GH and IGF-I concentrations and a worse prognosis and opens the possibility to new anticancer endocrine therapies in dogs.

A Role for T-Lymphocytes in Human Breast Cancer and in Canine Mammary Tumors

Chronic inflammation in the tumor microenvironment has a prominent role in carcinogenesis and benefits the proliferation and survival of malignant cells, promoting angiogenesis and metastasis. Mammary tumors are frequently infiltrated by a heterogeneous population of immune cells where T-lymphocytes have a great importance. Interestingly, similar inflammatory cell infiltrates, cytokine and chemokine expression in humans and canine mammary tumors were recently described. However, in both species, despite all the scientific evidences that appoint for a significant role of T-lymphocytes, a definitive conclusion concerning the effectiveness of T-cell dependent immune mechanisms has not been achieved yet. In the present review, we describe similarities between human breast cancer and canine mammary tumors regarding tumor T-lymphocyte infiltration, such as relationship of TILs and mammary tumors malignancy, association of ratio CD4+/ CD8+ T-cells with low survival rates, promotion of tumor progression by Th2 cells actions, and association of great amounts of Treg cells with poor prognostic factors. This apparent parallelism together with the fact that dogs develop spontaneous tumors in the context of a natural immune system highlight the dog as a possible useful biological model for studies in human breast cancer immunology.

EGFR and microvessel density in canine malignant mammary tumours.

The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor which has been shown to have an important role in human breast cancer. Its role appears to be associated with increased angiogenesis and metastasis. In order to clarify its role in canine mammary tumours (CMT), 61 malignant neoplasms were studied by using immunohistochemistry, comparing expression of EGFR, microvessel density (MVD) by CD31 immunolabelling and characteristics of tumour aggressiveness. High EGFR immunoexpression was statistically significantly associated with tumour size, tumour necrosis, mitotic grade, histological grade of malignancy and clinical stage. High CD31 immunoreactivity was statistically significantly associated with tubule formation, histological grade of malignancy and clinical stage. A positive correlation between EGFR and CD31 immunoexpression (r = 0.843; P < 0.001) was also observed. Results suggest that an over-expression of EGFR may contribute to increased angiogenesis and aggression in malignant CMT, presenting the possibility of using EGFR inhibitors in the context of metastatic disease treatment.

Prognostic value of tumour-associated macrophages in canine mammary tumours

Tumour-associated macrophages (TAMs) have already been associated in human breast cancer to a poor prognosis. As a part of a tumoural microenvironment, TAMs have an important contribution influencing neoplastic progression. Hitherto, in canine mammary tumours (CMT) the prognostic value of TAMs has not been reported. In this study, MAC387 immunohistochemical expression was evaluated in 59 CMTs (20 benign and 39 malignant). The TAM value was significantly higher in malignant than benign CMT (P = 0.011). In malignant CMT, TAMs were associated with skin ulceration (P = 0.022), histological type (P = 0.044), nuclear grade (P = 0.031) and tubular differentiation (P = 0.042). The survival analysis revealed a significant association between tumours with higher levels of TAMs and the decrease in overall survival (P = 0.030). TAMs have proven to have a prognostic value. These findings suggest the future possibility of using TAMs as a novel therapeutic target in CMT.