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Transforming growth factor-β (TGFβ) and FoxP3 regulatory T cells (Treg) are involved in human breast carcinogenesis. This topic is not well documented in canine mammary tumors (CMT). In this work, the tumoral TGFβ expression was assessed by immunohistochemistry in 67 malignant CMT and its correlation to previously determined FoxP3, VEGF, and CD31 markers and other clinicopathologic parameters was evaluated. The high levels of TGFβ were statistically significantly associated with skin ulceration, tumor necrosis, high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastases. The observed levels of TGFβ were positively correlated with intratumoral FoxP3 (strong correlation), VEGF (weak correlation) and CD31 (moderate correlation). Tumors that presented a concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31 markers were statistically significantly associated with parameters of tumor malignancy (high HGM, presence of vascular emboli and nodal metastasis). Additionally, shorter overall survival (OS) time was statistically significantly associated with tumors with an abundant TGFβ expression and with concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31. The presence of lymph node metastasis increased 11 times the risk of disease-related death, arising as an independent predictor of poor prognosis in the multivariable analysis. In conclusion, TGFβ and Treg cells seem involved in tumor progression emerging as potential therapeutic targets for future immunotherapy studies.

Canine cutaneous histiocytoma (CCH) represents a significant proportion of dog skin tumours, often manifesting as the most common neoplastic skin condition in young animals. Predominantly affecting dogs under four, these tumours appear primarily as solitary lesions that may regress spontaneously. This study, conducted over five years at the University of Trás-os-Montes e Alto Douro, involved a detailed histopathological and ultrastructural examination of 93 CCH cases. Histologically, these tumours showed distinct patterns of lymphoid infiltration, which contributed to their classification into four groups based on the inflammatory response and histological architecture. Most tumours displayed signs of epidermal invasion and frequent mitotic figures, with necrosis present in over half of the cases. Ultrastructurally, the neoplastic cells were characterised by pleomorphism, abundant organelles, and adherens-type junctions. This study offers significant insights into the pathophysiology and morphological characteristics of CCH, underscoring the importance of detailed histological and ultrastructural analysis in accurately diagnosing and understanding this common canine tumour.

Background: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC., Results: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside., Conclusion: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© 2021 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Background/aim: Canine Cutaneous Histiocytoma (CCH) is a Langerhans' cells benign tumour that undergoes spontaneous regression. The aim of the present study was to investigate the role of angiogenesis, a key step for tumour development, in CCH regression., Materials and Methods: 50 CCH samples were classified into 4 histological groups according to a regression scale, and evaluated for expression of vascular endothelial factor-A (VEGF-A) and its receptor VEGFR-2 as well as microvessel density (MVD)., Results: Tumours during early stages of the regressive process had a lower MVD compared to later stages, while CCH tumoural cells showed a limited production of VEGF, but higher levels of VEGFR-2. On the contrary, tumours in advanced phases of regression showed a higher number of neovessels, probably associated with the inflammatory state and the healing process., Conclusion: Our results suggest that angiogenesis may be compromised at early stages of histiocytoma development and this may be a determinant of regression in this tumour., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Inflammatory breast cancer (IBC) in humans is the most aggressive form of mammary gland cancer and shares clinical, pathologic, and molecular patterns of disease with canine inflammatory mammary carcinoma (CIMC). Despite the use of multimodal therapeutic approaches, including targeted therapies, the prognosis for IBC/CIMC remains poor. The aim of this review is to critically analyze IBC and CIMC in terms of biology and clinical features. While rodent cancer models have formed the basis of our understanding of cancer biology, the translation of this knowledge into improved outcomes has been limited. However, it is possible that a comparative "one health" approach to research, using a natural canine model of the disease, may help advance our knowledge on the biology of the disease. This will translate into better clinical outcomes for both species. We propose that CIMC has the potential to be a useful model for developing and testing novel therapies for IBC. Further, this strategy could significantly improve and accelerate the design and establishment of new clinical trials to identify novel and improved therapies for this devastating disease in a more predictable way., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Canine inflammatory mammary cancer (IMC) and its human counterpart, inflammatory breast cancer, are extremely aggressive types of cancer. Our aim was to characterize immunohistochemical expression of C-C chemokine receptor 2, colony stimulating factor 1 receptor and metalloproteinase-9 in canine IMC versus non-IMC and to analyze associations with clinicopathological variables., Materials and Methods: Immunohistochemical staining of CCR2, CSF1R and MMP9 was performed in a series of 25 IMC and 15 non-IMC tumors., Results: No differences in the expression of these biomarkers between IMC and non-IMC were observed. Distinct nuclear subcellular expression of CCR2 was observed in IMC (p<0.001). For IMC, higher CCR2 expression was associated with increased nuclear grade (p=0.037), and higher neoplastic MMP9 expression was associated with fewer mitoses (p=0.022), higher nuclear grade (p=0.047) and increased CSF1R expression (p=0.025)., Conclusion: Expression of CCR2, CSF1R and MMP9 in canine IMC could contribute to increased nuclear pleomorphism, but the biological mechanisms involved warrant further investigation., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

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.

In this study 80 malignant CMT were submitted to immunohistochemical detection of CD3, c-kit, VEGF, and CD31, together with clinicopathological parameters of tumor aggressiveness. CD3+ T-cells and c-kit overexpression revealed a positive correlation with VEGF (r = 0.503, P < 0.0001; r = 0.284, P = 0.023 for CD3 and c-kit, resp.) and CD31 (r = 0.654, P < 0.0001; r = 0.365, P = 0.003 for CD3 and c-kit, resp.). A significant association (P = 0.039) and a positive correlation (r = 0.263, P = 0.039) between CD3 and c-kit were also observed. High CD3/VEGF, c-kit/VEGF, and CD3/c-kit tumors were associated with elevated grade of malignancy (P < 0.0001 for all groups), presence of intravascular emboli (P < 0.0001 for CD3/VEGF and CD3/c-kit; P = 0.002 for c-kit/VEGF), and presence of lymph node metastasis (P < 0.0001 for all groups). Tumors with high CD3/VEGF (P = 0.006), c-kit/VEGF (P < 0.0001), and CD3/c-kit (P = 0.002) were associated with poor prognosis. Interestingly high c-kit/VEGF tumors retained their significance by multivariate analysis arising as independent prognostic factor.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background/aim: Canine cutaneous histiocytoma (CCH) is a tumour that undergoes spontaneous regression. The aim of this study was to establish a possible relationship between regression of CCH and tumoural cell proliferation and apoptosis., Materials and Methods: Immunostaining with Ki-67 antigen and the terminal deoxytransferase (TdT) deoxyuridine-5'-triphosphated (dUTP) nick-end labelling (TUNEL) method were performed on 93 specimens of CCH, grouped into four histological groups., Results: The proliferative index evaluated with Ki-67 antigen expression was on average 23.56 ± 7.91%. The apoptotic index determined by the TUNEL method was on average 39.37 ± 5.87%. Neither the proliferative nor apoptotic index differed between histological groups. Moreover, the proliferative and apoptotic indices did not correlate significantly. However, apoptotic activity was higher than proliferative activity in almost all tumours., Conclusion: A reduction of proliferation or an increase of apoptosis does not appear to justify regression of CCH. However, our results suggest that an imbalance between cell proliferation and apoptotic cell death plays a significant role in spontaneous regression of CCH.

In order to investigate the immune mechanisms involved in regression of canine cutaneous histicytoma (CCH), major histocompatibility complex (MHC) class-II immuno-expression and the number of T- and B-lymphocytes and macrophages were analyzed in 93 cases of CCH. MHC class-II was also studied in 16 cases of CCH by immunoelectron microscopy. All tumors expressed MHC class-II, and two major staining patterns were identified: focal juxtanuclear cytoplasmic staining and rim-like staining along the cell periphery. The MHC class-II labelling pattern and T- and B-lymphocyte infiltrates were associated with tumor regression. In regressing lesions, MHC class-II molecules shift to the cell surface and an increase of both T- and B-lymphocytes were noted. The increasing expression of MHC class-II molecules on the cell surface could be a significant factor for the onset and progression of tumour regression.

Mast cell tumours (MCTs) are among the most common cutaneous neoplasms in dogs and have a highly variable clinical behaviour. Cyclooxygenase (Cox) catalyzes the rate-limiting step in prostanoid biosynthesis and has recently gained attention as a prognostic factor and therapeutic target in human and animal oncology. In order to evaluate the potential value of non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of canine MCTs, expression of Cox-2 was determined in 49 such tumours (14 of grade I, nine of grade II and 22 of grade III). Cox-2 was expressed by 86% of the tumours studied. The percentage of labelled cells ranged from isolated positive cells throughout the tumour (n=8) to localized foci of labelled cells (n=3) or diffuse labelling of >50% of the cells (n=31). The intensity of Cox-2 labelling ranged from weak (n=4) to moderate (n=16) and strong (n=22) and was greatest at the advancing margin of the tumour. The intensity of Cox-2 labelling was significantly different between the three histological groups (P=0.018). However, no significant differences were noted for the percentage of Cox-2 positive cells (P=0.122) and for the immunoreactivity score (P=0.348) between the histological grades. The results of this study suggest that NSAIDs, particularly Cox-2 inhibitors, may be of value in the treatment of canine MCTs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In recent years, considerable progress has been made in understanding the role of the immune system in tumour progression. However, in canine mammary tumours (CMT), the prognostic value of T-lymphocytes is not established. The aims of the present study were to characterize T-lymphocytic infiltrate in 57 canine mammary tumours (21 benign and 36 malign), by immunohistochemical detection of CD3 antigen, and to determine its association with several clinicopathological parameters and overall survival. CD3+ positive cells were counted in 10 high-power fields within the tumour (i.e. The tumour-infiltrating T-lymphocytes, TIL), in the peripheral area of the tumour and in the adnexal non-tumoural mammary gland. CD3(+) TILs were significantly more frequent in benign than in malignant tumours (p<0.001). Conversely, peripheral CD3(+) TILs were significantly more frequent in malignant than in benign neoplasias (p<0.001). For CD3(+) T-lymphocytes in the adnexal non-tumoural mammary gland, there was no statistical difference in their frequency between benign and malignant tumours. On survival analysis, there was a tendency towards an association of a higher number of CD3(+) TILs and a shorter overall survival (p=0.08). Interestingly for CD3(+) T-lymphocytes in the adnexal non-tumoural mammary gland, a statistically significant relationship was observed, with a higher number of lymphocytes conferring a reduced overall survival (p=0.045). Further studies will be required to better understand the biological implications of the current findings.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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., (Copyright 2009 Elsevier Ltd. All rights reserved.)