Investigating Cox-2 and EGFR as Biomarkers in Canine Oral Squamous Cell Carcinoma: Implications for Diagnosis and Therapy.

Oral squamous cell carcinoma (OSCC) is a common and highly aggressive dog tumor known for its local invasiveness and metastatic potential. Understanding the molecular mechanisms driving the development and progression of OSCC is crucial for improving diagnostic and therapeutic strategies. Additionally, spontaneous oral squamous cell carcinomas in dogs are an excellent model for studying human counterparts. In this study, we aimed to investigate the significance of two key molecular components, Cox-2 and EGFR, in canine OSCC. We examined 34 tumor sections from various dog breeds to assess the immunoexpression of Cox-2 and EGFR. Our findings revealed that Cox-2 was highly expressed in 70.6% of cases, while EGFR overexpression was observed in 44.1%. Cox-2 overexpression showed association with histological grade of malignancy (HGM) (p = 0.006) and EGFR with vascular invasion (p = 0.006). COX-2 and EGFR concurrent expression was associated with HGM (p = 0.002), as well as with the presence of vascular invasion (p = 0.002). These data suggest that Cox-2 and EGFR could be promising biomarkers and potential therapeutic targets, opening avenues for developing novel treatment strategies for dogs affected by OSCC. Further studies are warranted to delve deeper into these findings and translate them into clinical practice.

Immunohistochemical Expression Levels of Epidermal Growth Factor Receptor, Cyclooxygenase-2, and Ki-67 in Canine Cutaneous Squamous Cell Carcinomas.

Squamous cell carcinoma (SCC) stands as the second most prevalent skin cancer in dogs, primarily attributed to UV radiation exposure. Affected areas typically include regions with sparse hair and pale or depigmented skin. The significance of spontaneous canine cutaneous SCC as a model for its human counterpart is underscored by its resemblance. This study assesses the expression of key markers-Epidermal Growth Factor Receptor (EGFR), Cyclooxygenase-2 (Cox-2), and Ki-67-in canine cutaneous SCC. Our objective is to investigate the association between their expression levels and classical clinicopathological parameters, unraveling the intricate relationships among these molecular markers. In our retrospective analysis of 37 cases, EGFR overexpression manifested in 43.2% of cases, while Cox-2 exhibited overexpression in 97.3%. The EGFR, Cox-2 overexpression, and Ki-67 proliferation indices, estimated through immunohistochemistry, displayed a significant association with the histological grade, but only EGFR labeling is associated with the presence of lymphovascular emboli. The Ki-67 labeling index expression exhibited an association with EGFR and Cox-2. These findings propose that EGFR, Cox-2, and Ki-67 hold promise as valuable markers in canine SCC. EGFR, Cox-2, and Ki-67 may serve as indicators of disease progression, offering insights into the malignancy of a lesion. The implications extend to the potential therapeutic targeting of EGFR and Cox-2 in managing canine SCC. Further exploration of these insights is warranted due to their translational relevance and the development of targeted interventions in the context of canine SCC.

Dietary omega-3 fatty acids in the management of feline periodontal disease: What is the evidence?

Periodontal disease is an oral disorder with high prevalence in cats from 2 years of age, resulting from an inflammatory response against bacterial plaque. Treatment depends on the stage of the disease and may include dental scaling, local application of perioceutics, tissue regeneration and/or even tooth extraction and periodontal surgery. As multimodal therapy is often required, new strategies have been developed to improve the therapeutic response in these patients. Adjuvant use of omega-3 fatty acids has been reported in humans with periodontal disease, but the current evidence of its effect in companion animals, especially cats, is still considered to be scarce and conflicting. This review describes the state of the art regarding feline periodontal disease and seeks to clarify the potential effect of omega-3 fatty acids on its clinical management in light of the evidence available in the current literature.

The Dog as a Model to Study the Tumor Microenvironment.

Cancer is a complex and dynamic disease with an outcome that depends on a strict crosstalk between tumor cells and other components in tumor microenvironment, namely, tumor-infiltrating immune cells, fibroblasts, cancer stem cells, adipocytes, and endothelial cells. Within the tumor microenvironment, macrophages and T-lymphocytes appear to be key effectors during the several steps of tumor initiation and progression. Tumor cells, through the release of a plethora of signaling molecules, can induce immune tolerance, by avoiding immune surveillance, and inhibit immune cells cytotoxic functions. Furthermore, as the tumor grows, tumor microenvironment reveals a series of dysfunctional conditions that potentiate a polarization of harmful humoral Th2 and Th17, an upregulation of Treg cells, and a differentiation of macrophages into the M2 subtype, which contribute to the activation of several signaling pathways involving important tissue biomarkers (COX-2, EGFR, VEGF) implicated in cancer aggressiveness and poor clinical outcomes. In order to maintain the tumor growth, cancer cells acquire several adaptations such as neovascularization and metabolic reprogramming. An extensive intracellular production of lactate and protons is observed in tumor cells as a result of their high glycolytic metabolism. This contributes not only for the microenvironment pH alteration but also to shape the immune response that ultimately impairs immune cells capabilities and effector functions.In this chapter, the complexity of tumor microenvironment, with special focus on macrophages, T-lymphocytes, and the impact of lactate efflux, was reviewed, always trying to demonstrate the strong similarities between data from studies of humans and dogs, a widely proposed model for comparative oncology studies.

Clinicopathological significance of caspase-3 and Ki-67 expression in canine mammary gland tumours.

UNLABELLED: Fifty canine mammary gland tumours (CMGT) (18 benign and 32 malignant) were studied by immunohistochemical detection of active caspase-3 and Ki-67 antigens in order to determine their association with several clinicopathological parameters. The percentage of caspase-3 positive cells was significantly higher in benign tumours as compared to their malignant counterparts (P ≤ 0.001). In the group of malignant tumours there was no significant association between active caspase-3 and the clinicopathological variables considered. The percentage of Ki- 67 positive cells was significantly higher in malignant tumours compared to the benign ones (P ≤ 0.001). In the group of malignant tumours, Ki-67 expression showed a statistically significant association with tumour size (P = 0.025), histological type (P = 0.010), mitotic grade (P ≤ 0.001), nuclear grade (P = 0.025), differentiation grade (P = 0.004), histological grade of malignancy (P = 0.002), and presence of metastases in regional lymph nodes (P = 0.025). Furthermore, this study revealed a negative correlation between the percentages of active caspase-3 and Ki-67 (r = -0.39; P = 0.04). Thus, our results suggest a loss of balance between cell death and cell division in CMGT. KEY WORDS: Apoptosis, caspase-3, Ki.

Serum Biochemistry Parameters of the Endangered Miranda's Donkey Breed: Reference Intervals and the Influence of Gender and Age.

Miranda's donkey is an endangered, autochthone breed from Northern Portugal. Understanding the physiological and pathological conditions of Miranda's donkey is crucial for the conservation of the breed. Our study aimed to establish reference intervals (RIs) for blood biochemistry parameters and to evaluate the influence of gender and age on these parameters. Blood samples from 75 clinically healthy animals were analyzed for 21 blood biochemistry parameters using Respons® 920 and Start® 4-Diagnostica-Stago. RIs were calculated according to the ASVCP guidelines, utilizing Reference Advisor software V. 2.1 and the statistical program SPSS version 29 to analyze the effects of gender and age. Significant gender-related differences (p < 0.05) were observed in cholesterol, chlorine, creatinine (CREA), glucose (GLU), sodium, and triglycerides (with higher values in females) and in aspartate aminotransferase, creatine phosphokinase (CK), gamma-glutamyl transferase, potassium, magnesium (Mg), and urea (with higher values in males). Age-related differences (p < 0.05) were noted for alkaline phosphatase, CK, fibrinogen, GLU, and phosphorus (higher in young animals) and for globulin, Mg, and total protein (higher in adults). The RIs described here are invaluable for assessing and monitoring the health status of individual animals and herds. Our study highlights the importance of considering gender and age in the interpretation of biochemical parameters, providing critical insights for the conservation and management of this endangered breed.

Stage Migration in Canine Multicentric Lymphoma: Impact of Diagnostic Techniques on Assessing Disease Extent.

BACKGROUND/AIM: Stage migration, a phenomenon triggered by technological advancements allowing more sensitive tumor spread detection, results in alterations in the distribution of cancer stages within a population. Canine multicentric lymphoma is staged I to V based on the affected anatomic site(s) and substage a or b depending on the presence of tumor-related clinical signs. The primary objective of this study was to assess the influence of various diagnostic techniques on staging accuracy and determine whether multiple staging methods lead to significant stage migration, impacting the reliability of disease stage assignments. MATERIALS AND METHODS: Dogs cytologically diagnosed with multicentric lymphoma were staged using four different staging methods (A-D): A (physical examination, hemogram, blood smear), B (A plus thoracic X-ray, abdominal ultrasound), C (B plus liver and spleen cytology) and D (C plus bone marrow cytology). RESULTS: Twenty-three dogs were enrolled: 16 females (70%) and seven males (30%). Regarding immunophenotype, 21 dogs (91.3%) were B-cell and two dogs (8.7%) were T-cell. Stage migration was observed between all staging methods. Between A and B, 12 animals migrated from stage III to stage IV. Between B and C, four animals migrated, three to a higher stage (stage III to IV) and one to a lower stage (stage IV to III). Between C and D, one animal migrated from stage IV to V. The differences between staging methods A and B were statistically significant (p≤0.001). CONCLUSION: Stage migration in canine multicentric lymphoma depends on the diagnostic methods used and reinforces the need to use standardized staging methods to avoid it.

Immunohistochemical Expression and Prognostic Value of COX-2 and Alpha-Smooth Muscle Actin-positive Cancer-associated Fibroblasts in Feline Mammary Cancer.

BACKGROUND: Cyclo-oxygenase-2 (COX-2) and cancer associated fibroblasts (CAFs) play an important role in the development and progression of tumor malignancy in humans and animals, showing that both can influence the tumor microenvironment. However, the impact of these two markers in feline mammary carcinogenesis has not yet been addressed. MATERIALS AND METHODS: In the present study, the clinicopathological significance of COX-2 immunoexpression and alpha-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs) was determined and correlated with disease-free and overall survival of 50 felines with malignant mammary tumors. RESULTS: COX-2 overexpression was positively associated with mitotic index (p=0.031), degree of malignancy (p≤0.001), lymph node metastasis (p≤0.001), vascular invasion (p=0.002), disease recurrence (p=0.019) and distant metastasis (p=0.036). α-SMA-positive CAFs were associated with mitotic index (p=0.004), lymph node metastasis (p=0.027), vascular invasion (p=0.05), disease recurrence (p≤0.001) and distant metastasis (p≤0.001). Additionally, both markers were correlated with disease-free and overall survival, emerging as predictors of poor prognosis. CONCLUSION: Our results indicate for the first time that the presence of two markers, COX-2 and α-SMA, is associated with carcinogenesis and worse prognosis in feline mammary cancer and that α-SMA-positive CAFs have a role in feline mammary tumorigenesis, cancer development, and clinical outcome.

Corrigendum: Melanoma of the dog and cat: consensus and guidelines.

[This corrects the article DOI: 10.3389/fvets.2024.1359426.].

Melanoma of the dog and cat: consensus and guidelines.

Melanoma of the dog and cat poses a clinical challenge to veterinary practitioners across the globe. As knowledge evolves, so too do clinical practices. However, there remain uncertainties and controversies. There is value for the veterinary community at large in the generation of a contemporary wide-ranging guideline document. The aim of this project was therefore to assimilate the available published knowledge into a single accessible referenced resource and to provide expert clinical guidance to support professional colleagues as they navigate current melanoma challenges and controversies. Melanocytic tumors are common in dogs but rare in cats. The history and clinical signs relate to the anatomic site of the melanoma. Oral and subungual malignant melanomas are the most common malignant types in dogs. While many melanocytic tumors are heavily pigmented, making diagnosis relatively straightforward, melanin pigmentation is variable. A validated clinical stage scheme has been defined for canine oral melanoma. For all other locations and for feline melanoma, TNM-based staging applies. Certain histological characteristics have been shown to bear prognostic significance and can thus prove instructive in clinical decision making. Surgical resection using wide margins is currently the mainstay of therapy for the local control of melanomas, regardless of primary location. Radiotherapy forms an integral part of the management of canine oral melanomas, both as a primary and an adjuvant therapy. Adjuvant immunotherapy or chemotherapy is offered to patients at high risk of developing distant metastasis. Location is the major prognostic factor, although it is not completely predictive of local invasiveness and metastatic potential. There are no specific guidelines regarding referral considerations for dogs with melanoma, as this is likely based on a multitude of factors. The ultimate goal is to provide the best options for patients to extend quality of life and survival, either within the primary care or referral hospital setting.

The role of COX expression in the prognostication of overall survival of canine and feline cancer: A systematic review.

Cyclooxygenase (COX) isoforms-1 and -2 have been extensively investigated in cancer. Although COX-2 is the isoform most studied and has been described in several malignancies associated with histologic criteria of malignancy and worse prognosis, COX-1 has also been linked to some forms of cancer. With the present review our aim was to summarize the current state of knowledge and clarify if and in which type of tumours COX-1 and/or COX-2 expression have real prognostic implications. We searched PubMed database for prognostic studies using predefined inclusion criteria in order to ascertain the prognostic value of COX-1 and COX-2 in malignant neoplasia in dogs and cats. Eighteen studies were analysed. COX-2 was shown to be a negative prognostic factor in canine and feline mammary tumours, canine mast cell tumour, canine melanoma, canine osteosarcoma and canine renal cell carcinoma. COX-1 showed a negative prognostic value in feline oral squamous cell carcinoma (SCC). We found high heterogeneity among studies regarding COX immunohistochemical evaluation methodology even in the same type of neoplasia pointing out the need for its standardization at least by tumour type. The available data support the use of COX-2 as a prognostic factor in canine (mammary carcinoma, mast cell tumour, melanoma, osteosarcoma and renal carcinoma) and feline (mammary carcinoma) cancers. For COX-1, its use is advised in feline oral SCC.

Adjuvant doxorubicin vs metronomic cyclophosphamide and meloxicam vs surgery alone for cats with mammary carcinomas: A retrospective study of 137 cases.

This study aims to evaluate the efficacy and side effects of low dose cyclophosphamide chemotherapy plus meloxicam as an adjuvant treatment, compared with high dose doxorubicin or surgery alone in cats with mammary carcinoma. Medical records of 228 female cats treated for mammary carcinoma between 2008 and 2018, were reviewed in eight veterinary institutions. Only cats with complete tumour staging and radical mastectomy were included in the study. One hundred and thirty-seven cats were divided into three treatment groups: group 1 (n = 80) cats treated with surgery, group 2 (n = 34) cats that had surgery and adjuvant treatment with doxorubicin, and group 3 (n = 23) cats with surgery and adjuvant treatment with low dose metronomic cyclophosphamide and meloxicam. The study endpoints were disease free interval (DFI) and overall survival (OS). Toxicity was evaluated according to the VCOG-CTCAE criteria. The median DFI was 270, 226 and 372 days in groups 1, 2 and 3, respectively. The median OS was 338 (group 1), 421 (group 2) and 430 (group 3) days. The differences between groups were not significant (DFI P = .280 and OS P = .186). Toxicity was observed in 52.9% (n = 18) of cats in group 2 and 39.1% (n = 9) of cats in group 3, with mild to moderate intensity. Differences were not significant (P = .306). In conclusion, adjuvant chemotherapy treatment did not improve survival and the overall benefit remains unproven. Randomized prospective trials are necessary to clarify the effectiveness of adjuvant chemotherapy treatment for feline mammary carcinomas.

Epithelioid hemangiosarcomas of the bovine urinary bladder: a histologic, immunohistochemical, and ultrastructural examination of four tumors.

Epithelioid hemangiosarcoma is a specific variant of hemangiosarcoma that has recently been recognized in domestic animals. These malignant vascular neoplasms histologically resemble, and may be mistaken for, carcinomas. Four epithelioid hemangiosarcomas in the urinary bladders of 4 cows with severe enzootic hematuria are described in the current study. Grossly, the vesicular mucosa of the urinary bladder of each cow contained a single red elevated nodule. Histologically, each neoplasm was composed of short strands, cords, or nests of epithelioid, round, or slightly spindle-shaped endothelial cells that formed small vascular structures. Neoplastic cells were immunohistochemically positive for factor VIII-related antigen and vimentin, and were negative for cytokeratin and desmin. Ultrastructurally, the neoplastic cells often contained cytoplasmic intermediate filaments, a prominent granular endoplasmic reticulum, a Golgi complex, mitochondria, marked pinocytotic activity, and rare Weibel-Palade bodies. These neoplasms were diagnosed as epithelioid hemangiosarcomas based on their histologic, immunohistochemical, and ultrastructural features. The present report widens the spectrum of mesenchymal tumors of the bovine urinary bladder and aids in the characterization of these vascular neoplasms.

Prognostic factors for cats with squamous cell carcinoma of the nasal planum following high-dose rate brachytherapy.

OBJECTIVES: The aim of the study was to investigate retrospectively the prognostic impact of variables such as sex, neuter status, breed, age, number of lesions, location and size of the tumour, tumour extension beyond the nasal planum, ulceration and lymph node status, among others, in a population of cats treated with high-dose rate brachytherapy. METHODS: This study reviews the outcome of 58 cats with cytologically and/or histologically confirmed squamous cell carcinoma of the nasal planum, treated at the Clinic Alliance (Bordeaux, France) with high-dose rate brachytherapy from 2010-2016. The total radiation dose delivered was 30 Gy, administered in two different schedules: five fractions of 6 Gy for a period of 4 days (Tuesday-Friday) or four fractions of 7.5 Gy for a period of 3 days (Tuesday- Thursday). Data were collected from cats' clinical records. RESULTS: Complete response was achieved in 72% (n = 36) of the cats, partial response in 24% (n = 13) and 2% (n = 1) did not respond. Median progression-free survival and overall survival times were 316 and 835 days, respectively. CONCLUSIONS AND RELEVANCE: Results indicated that sex (P = 0.045), extension of the tumour from the nasal planum to the upper lip (P = 0.015), tumour size (P = 0.015; P = 0.001), the existence of a previous treatment (P = 0.043) and the tumour response to high-dose rate brachytherapy (P = 0.038; P <0.001) are prognostic factors for cats with squamous cell carcinoma of the nasal planum following high-dose rate brachytherapy.

Assessing the Interleukin 35 Immunoexpression in Malignant Canine Mammary Tumors: Association With Clinicopathological Parameters and Prognosis.

BACKGROUND/AIM: IL-35 has a prominent immunosuppressive role and its overexpression has been reported in human breast cancer. However, the impact of IL-35 in canine mammary carcinogenesis has not been addressed yet. The present study determined the clinicopathological significance of IL-35 immunoexpression and its correlation with overall survival (OS) in 72 malignant canine mammary tumor (CMT) patients. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded malignant CMT samples (n=72) were submitted to immunohistochemical staining to detect IL-35 expression. Survival curves were obtained by the Kaplan-Meier method and the log-rank test was used for the survival estimates. Cox proportional hazard model for multivariate analysis was also performed. RESULTS: IL-35 overexpression was associated with: skin ulceration, tumor necrosis, mitotic index, nuclear pleomorphism, tumor differentiation, histological grade of malignancy (HGM), neoplastic intravascular emboli and lymph node metastasis. Additionally, IL-35 was also correlated with a worse overall survival in multivariate analysis, arising as an independent predictor of poor prognosis. CONCLUSION: IL-35 is associated with carcinogenesis and worse prognosis of CMT.

Preservation of wild bird species in northern Portugal - Effects of anthropogenic pressures in wild bird populations (2008-2017).

In this study we aim to obtain a holistic view over the consequences of human-induced threats to the wild bird populations in the study area, based on data collected from a Wildlife Rehabilitation Centre (WRC) through the method of Partial Least Squares-Path Modelling (PLS-PM). The study area comprised 76 rural and urban municipalities located in northern Portugal. Within PLS-PM the threats ("anthropogenic pressures") are termed exogenous latent variables, while the final environmental consequence (wild bird mortality) is termed endogenous latent variable. Latent variables are concepts assessed by numerical parameters. The PLS-PM results identified as most significant pressures, the number of small and medium companies in the municipalities, both in traumatic and non-traumatic deaths. Although the pattern of weights is similar regardless of the general cause of death, traumatic causes seem to play a more prominent role given the larger weights in the relevant specific causes (number of companies). The high coefficients of determination (R2 > 0,8) reveal that the variance of wild bird mortality is largely explained by the variance of the pressures, which indicates a cause-effect relationship between the independent (pressures) and dependent (mortality) variables. The ample coverage of northern Portugal with a huge dataset suggests that this cause-effect relationship is typical from this region. The use of a sophisticated statistical method PLS-PM and its incorporation into a Geographic Information System (GIS) revealed to be an important tool for analysing wildlife impacts of environmental and human factors. The results obtained with this model indicate that there is a substantial negative impact of human activity on wild bird mortality.

Crosstalk between GH/IGF-I axis and steroid hormones (progesterone, 17beta-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.

Bidirectional Regulation of COX-2 Expression Between Cancer Cells and Macrophages.

BACKGROUND/AIM: Our aim was to investigate the crosstalk between tumor and immune cells (M2 macrophages) and its effects on cyclo-oxygenase-2 (COX2) regulation in canine mammary tumors (CMT). MATERIALS AND METHODS: Sh1b CMT cells and human BT474 mammary or HT29 colon cancer cells were co-cultured with canine peripheral blood mononuclear cells (PBMCs) or with macrophage-like differentiated THP1 monocytes (dTHP1). Intracellular COX2 expression by PBMCs, dTHP1 and cancer cells was evaluated by flow cytometry. RESULTS: Co-culturing of Sh1b and canine PBMCs induced COX2 overexpression in CMT cells. In turn, COX2 expression by PBMCs, mostly CD68+ macrophages, was attenuated by co-culture with Sh1b (p=0.0001). In accordance, co-culture with dTHP1 prompted intracellular production of COX2 in both Sh1b CMT cells and HT29 human colon cancer cells and reduced production of COX2 in BT474 human mammary cancer cells. The intracellular COX2 expression from dTHP1 decreased when treated with conditioned medium from cultured Sh1b and HT29 cancer cells. CONCLUSION: Bidirectional COX2 regulation between cancer and monocytes/macrophages might shape a tolerogenic tumor microenvironment in CMT.

Investigating associations of cyclooxygenase-2 expression with angiogenesis, proliferation, macrophage and T-lymphocyte infiltration in canine melanocytic tumours.

Cyclooxygenase-2 (COX-2) is known to be involved in tumour progression and has been suggested as a therapeutic target in many human and animal malignancies. A number of different pathways subjacent to cancer hallmarks are considered to be involved in COX-2-mediated tumour progression, although these are still largely undefined. Our aim is to investigate associations between COX-2 expression and angiogenesis, proliferation and the inflammatory microenvironment in canine melanocytic tumours. Understanding the involvement of COX-2 with cancer hallmarks might enable us to adapt therapeutic strategies for canine melanomas, an aggressive and often lethal malignancy with value in comparative oncology. Immunohistochemical staining of COX-2, Ki-67 (proliferation index), vascular endothelial growth factor (VEGF), factor VIII (microvessel density), CD3 (lymphocytes) and MAC387 (macrophages) was performed in 51 melanocytic tumours (31 malignant melanomas, 20 melanocytomas). Statistical associations between COX-2 and the other parameters detected were analysed. In melanocytic tumours (n=51), both COX-2 labelling extension and intensity showed a statistically significant association with angiogenesis by factor VIII, VEGF, Ki-67, CD3+ T lymphocytes and MAC387. Within malignant melanomas, COX-2 expression has shown significant associations with microvessel density (factor VIII), lymphocyte and macrophage infiltration and, considering all melanocytic tumours, COX-2 was also associated with VEGF intensity and Ki-67 cell proliferation. Our results point to a role for COX-2 in angiogenesis and in the establishment of an inflammatory microenvironment, favourable to melanoma tumour progression. Further mechanistic studies are warranted to dissect molecular pathways in which COX-2 is involved. Present evidence suggests that COX-2 inhibitors might be useful as an adjuvant treatment to hinder canine melanoma progression.

Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors.

The activity of regulatory T cells (Tregs) is closely associated with the expression of FoxP3 transcription factor. FoxP3 regulatory T cells (FoxP3Treg) have immunosuppressive properties and can work for prevention of harmful autoimmune responses, however can also interfere with beneficial anti-tumor immunity. In human breast cancer these cells play a crucial role in tumor progression. In canine mammary tumors (CMT) this topic is not well-documented. This study included 80 malignant CMT and studied, by immunohistochemistry, the intratumoral FoxP3 expression together with microvessel density (MVD), vascular endothelial growth factor (VEGF) and several clinicopathological characteristics. Abundant FoxP3Treg cells were associated with tumor necrosis (p=0.001), high mitotic grade (p<0.001), more marked nuclear polymorphism (p=0.001), poor differentiation of tumors (p<0.001), high histological grade of malignancy (HGM) (p<0.001), presence of neoplastic intravascular emboli (p<0.001) and presence of lymph node metastasis (p<0.001). Intratumoral FoxP3 was correlated with MVD (r=0.827; p<0.001) and associated with VEGF (p=0.001). Additionally tumors with abundant FoxP3Treg cells were associated with shorter overall survival (OS) time in univariate and multivariate analysis (p<0.001 Kaplan-Meier curves and 7.97 hazard ratio, p<0.001 Cox proportional hazard model). Results suggest that Treg cells play a role in CMT progression and may contribute to increased angiogenesis and aggression in these tumors. The association of intratumoral FoxP3 expression with shorter OS in multivariate analysis suggests the usefulness of Treg cells as an independent prognostic marker.