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Estrogen receptors (ERs) are located in both healthy and neoplastic tissues. The type of estrogen receptor expressed varies depending on its location, tumor type, and species. Estrogen action is mediated by binding to ER and activating the transcriptional and signaling processes that result in the control of gene expression. There are two main types of estrogen receptors: ER alpha (ERα) and ER beta (ERβ). Both receptors are functionally different, they may act antagonistically and are distributed in different tissues but their structure is similar – as they are composed of 5 different domains: A/B, C, D, E, and F. The signaling pathway and hence regulation of the gene expression by ERs is a complex and multifactorial process that involves both genomic and nongenomic actions. In the human reproductive tract, both ERα and β are present, with predominant expression of ERβ, while there are no satisfactory data distinguishing the type of ERs expressed in the canine reproductive tract. In mammary gland neoplasia, a decreased or lacking ERα expression in humans is associated with a poorer prognosis. This is similar to dogs, where higher ERα expression intensity was noted in benign tumors than in carcinomas. In human hematopoietic malignancies, ERβ is a predominant receptor. Selective and non-selective ERβ agonists have an antiproliferative and pro-apoptotic effect on human lymphoma cell lines and may be effective in the therapy of ERβ positive lymphomas and leukemias. In canine lymphoma tissues, none or only marginal expression of ERs was detected over the decades. Considering available data, we conducted preliminary studies proving that, in contrast to humans, the dominant ER expressed in canine hematopoietic tumors is ERα. [ABSTRACT FROM AUTHOR]

Programmed death ligand 1 (PD-L1) is an immune checkpoint molecule that plays a crucial role in regulating antitumor immune responses. Canine mammary carcinomas (CMCs) are common tumors of dogs. Despite extensive studies on the heterogeneity of CMCs, there is still a lack of effective precision therapies for the treatment of CMCs. In this study, we aimed to investigate the correlation between PD-L1 mRNA and protein expression in CMCs and explore its association with histopathological grade and molecular markers, including the estrogen receptor, epidermal growth factor receptor 2, and cytokeratin 5/6 (CK5/6). Formalin-fixed paraffin-embedded samples were evaluated for PD-L1 mRNA expression using RNA in situ hybridization and PD-L1 protein expression using immunohistochemistry. We observed no substantial correlation between PD-L1 mRNA and protein expression in CMCs; however, PD-L1 mRNA levels were significantly higher in grade 3 than in grade 1 tumors (P =.001). In addition, we observed a positive correlation between PD-L1 protein expression and CK5/6 expression in CMCs (P =.032). These findings suggest that PD-L1 expression in CMCs is heterogeneous and may be regulated post-transcriptionally. Further studies are needed to explore the prognostic and therapeutic implications of PD-L1 expression in different molecular subtypes of CMCs and their potential as predictive biomarkers for immunotherapy. [ABSTRACT FROM AUTHOR]

Mammary tumour is the most common type of tumour in dogs, especially in unneutered female dogs. Homoharringtonine (HHT) is a natural alkaloid that can be used to treat various types of human tumour. However, the inhibitory effect and mechanism of HHT on canine mammary carcinomas (CMC) remain unclear. This study aimed to evaluate the inhibitory effect of HHT on CMC in vitro and determine its underlying molecular mechanism. The effects of HHT on the cytotoxicity of CMC U27 cells were evaluated by the cell counting kit‐8, wound healing, and Transwell assays. HHT‐induced apoptosis of U27 cells was detected by JC‐1 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Moreover, the gene expression of B‐cell lymphoma‐2 (Bcl‐2) and Bcl‐2 associated X protein (Bax) were analysed using quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR), and the protein expression of protein kinase B/mammalian target of rapamycin (AKT/mTOR) and mitochondrial apoptosis proteins were determined by western blotting. Furthermore, mammary tumour‐bearing mouse models were established using 4T1 cells to evaluate the therapeutic effect of HHT. It was found that HHT could significantly down‐regulated the protein expression of p‐AKT, p‐mTOR, and Bcl‐2, and up‐regulated the protein expression of P53, Bax, cleaved caspase‐3, and cleaved caspase‐9. In addition, HHT significantly suppressed both tumour volume and mass in mammary tumour mice. In conclusion, HHT damages CMC cells by inhibiting the AKT/mTOR signalling pathway and inducing mitochondrial apoptosis. Such findings lay a theoretical foundation for the clinical treatment of CMC and provide more options for clinical medication. [ABSTRACT FROM AUTHOR]

Purpose: Mammary gland tumors are the most common neoplastic diseases in elderly female dogs, about 50% of which are considered to be malignant. Canine mammary tumors are similar to human breast cancers in many respects, so canine mammary tumors are frequently studied alongside human breast cancer. This article mentioned KI-67, HER-2, COX-2, BRCA1, BRCA2, P53, CA15-3, MicroRNA, Top2α and so on. All these markers are expected to have an important role in the clinic. Methods: Existing markers of canine mammary carcinoma are reviewed, and the expression of each marker and its diagnostic role for this tumor are described in detail. Results: This article introduced several effective markers of canine mammary tumors, among them, antigen KI-67 (KI-67), human epidermal growth factor receptor 2 (HER-2), cyclooxygenase 2 (COX-2) are promising and can be detected in both serum and tissue samples. Breast cancer caused by mutations in the breast cancer 1 gene (BRCA1) and breast cancer 2 gene (BRCA2) is also a hot topic of research. In addition to the above symbols, tumor protein p53 (p53), cancer antigen15-3 (CA15-3), MicroRNA (miRNA), topoisomerase πα (Top2α), proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR) and E-cadherin will also be involved in this paper. We will also mention Mammaglobin, which has been rarely reported so far. [ABSTRACT FROM AUTHOR]

Spontaneous canine mammary carcinomas (CMCs) have been widely considered a good research model for human breast cancers, which brings much attention to CMCs. In recent years, the oncolytic effect of Newcastle disease virus (NDV) on cancer cells has been widely studied, but its effect on CMCs is still unclear. This study aims to investigate the oncolytic effect of NDV LaSota strain on canine mammary carcinoma cell line (CMT‐U27) in vivo and in vitro. The in vitro cytotoxicity and immunocytochemistry experiments showed that NDV selectively replicated in CMT‐U27 cells, and inhibited cell proliferation and migration but not in MDCK cells. KEGG analysis of transcriptome sequencing indicated the importance of the TNFα and NF‐κB signalling pathways in the anti‐tumour effect of NDV. Subsequently, the significantly increased expression of TNFα, p65, phospho‐p65, caspase‐8, caspase‐3 and cleaved‐PARP proteins in the NDV group suggested that NDV induced CMT‐U27 cells apoptosis by activating the caspase‐8/caspase‐3 pathway and the TNFα/NF‐κB signalling pathway. Nude mice tumour‐bearing experiments showed that NDV could significantly decrease the growth rate of CMC in vivo. In conclusion, our study demonstrates the effective oncolytic effects of NDV on CMT‐U27 cells in vivo and in vitro, and suggests NDV as a promising candidate for oncolytic therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Canine mammary carcinomas (CMC) are associated with poor clinical outcomes and high mortality. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and tumor progression in many solid tumors, including mammary carcinomas. The goal of this work was to establish the therapeutic value of MB02 monoclonal antibody biosimilar to bevacizumab that targets VEGF in CMC. For this purpose, first, we were able to predict in silico that bevacizumab was able to recognize and bind canine VEGF. This was confirmed in vitro using an ELISA-based assay. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These results show a high potential for MB02 as a promising therapeutic agent for the management of CMC. Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC. [ABSTRACT FROM AUTHOR]

Enhancer of zeste homologue 2 (EZH2) is the human homologue of Drosophila zeste gene enhancer. The aim of this study was to determine the expression of EZH2 in canine mammary carcinomas (CMCs) and its relationship with clinicopathological features.

Simple Summary: Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor whose homonymous gene is an important oncogenic driver in 10–20% of invasive breast cancer. The role of HER2 in canine mammary carcinoma is controversial; therefore, this study aimed to investigate the protein expression and cytogenetic alterations of HER2 and their correlation with the proliferative fraction, estrogen receptor, and other clinicopathological parameters in canine mammary carcinoma. The HER2 gene was amplified in a subgroup of canine mammary carcinomas, and the HER2 gene copy number was correlated with HER2 protein overexpression but not with the tumor's biological behavior. Surprisingly, a possible translocation of HER2/CRYBA1 was found. This is the first study in the canine species that found cytogenetic alterations with fluorescence in situ hybridization in mammary carcinoma. Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical–pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical–pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns. [ABSTRACT FROM AUTHOR]

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.

Cancer-associated stroma (CAS) profoundly influences progression of tumors including mammary carcinoma (mCA). Canine simple mCA represent relevant models of human mCA, notably also with respect to CAS. While transcriptomic changes in CAS of mCA are well described, it remains unclear to what extent these translate to the protein level. Therefore, we sought to gain insight into the proteomic changes in CAS and compare them with transcriptomic changes in the same tissue. To this end, we analyzed CAS and matched normal stroma using laser-capture microdissection (LCM) and LC-MS/MS in a cohort of 14 formalin-fixed paraffin embedded (FFPE) canine mCAs that we had previously characterized using LCM-RNAseq. Our results reveal clear differences in protein abundance between CAS and normal stroma, which are characterized by changes in the extracellular matrix, the cytoskeleton, and cytokines such as TNF. The proteomics- and RNAseq-based analyses of LCM-FFPE show a substantial degree of correlation, especially for the most deregulated targets and a comparable activation of pathways. Finally, we validate transcriptomic upregulation of LTBP2, IGFBP2, COL6A5, POSTN, FN1, COL4A1, COL12A1, PLOD2, COL4A2, and IGFBP7 in CAS on the protein level and demonstrate their adverse prognostic value for human breast cancer. Given the relevance of canine mCA as a model for the human disease, our analysis substantiates these targets as disease-promoting stromal components with implications for breast cancer in both species.

Canine mammary gland tumors (CMTs) are one of the most prevalent cancers in dogs and a good model for human breast cancer (BC), however gene expression analysis of CMTs is scarce. Although divergence of genes expression has been found in BC of different human races, no such research of different dog's breeds has been done. The purpose of this study was to investigate expression of the VEGF-B, VEGF-A, FLT-1, KDR, ERBB2, EGFR, GRB2, RAC1, CDH1 and HYAL-1 genes of canine mammary carcinomas, compare the expression levels with clinicopathological parameters and analyze expression disparities between different breeds. Carcinomas and adjacent tissues were collected from female dogs to perform routine histopathology, immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). We found that VEGF-B and EGFR genes were overexpressed in the mammary gland carcinomas compared to adjacent tissue. VEGF-B gene expression had associations with different parameters (tumor size, grade, and absence of metastasis). Furthermore, differences in VEGF-B, FLT1, ERBB2, GRB2, RAC1, CDH1 and HYAL-1 genes expression have been found in different breed dogs (German Shepherd, Yorkshire Terrier) and mixed-breed dogs indicating that a dog's breed could determine a molecular difference, outcome of cancer and should be accounted as a confounding factor in the future gene expression research. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical–pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical–pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns.

Background: In the last two decades, canine mammary cancer has played an important role in human breast cancer research. In many cases, there are various similarities between the biological and clinical features of canine breast cancer and female breast cancer. Clinical studies and evaluation of prognostic parameters of canine mammary cancer can increase confidence in generalizing results for human cancers. This study was performed in the direction of comparative oncology. Methods: Clinical-pathological data from invasive type of canine mammary carcinoma were collected from clinical records and pathology reports. The parameters of age, tumor laterality, tumor size, lymph node status and tumor grade were recorded and the relationships between the parameters were evaluated using linear regression analysis. Results: 97 patients were included in the study and the mean age was 10.06 ± 2.73 years. 51% of the left mammary glands were involved and pT2 tumor size was the most common. Lymph nodes were involved in 27% of patients and 43% of tumors were grade I. Statistical analysis showed no statistical relationship between tumor size and laterality with other clinico-pathological features. However, there was a statistically significant relationship between tumor size and tumor grade and the condition of lymph nodes, so that with increasing tumor size, tumor grade increased and the likelihood of lymph node involvement increased. Conclusions: The results of this study are very similar to breast cancer in women and show that canine mammary carcinoma is a suitable model in comparative oncology research. Dogs live shorter than humans, so researchers can get the results of treatment and survival rate assessments faster in clinical trials. By observing ethical principles, dogs with breast cancer may replace phase I and II of human clinical trials in some types of cancer in the near future.

Canine mammary carcinoma (CMC) is the most common type of neoplasm in intact female dogs. While a previous study in Western countries validated the 2011 classification as an independent prognostic indicator in CMC, its role in CMC prognostication in Asian countries such as Korea remains unclear. In the present study, we estimate the survival rates in CMC types defined by the 2011 classification, elucidate the prognostic significance of the histological subtype and grade and that of the lymphatic invasion status in CMC, and validate the 2011 classification as an independent prognostic indicator in a large cohort of CMCs (excluding cases of multicentric CMCs). A total of 155 CMC cases retrieved from archived formalin-fixed, paraffin-embedded tissues, along with 2-year follow-up data, were retrospectively analysed. A significant association was found between the histological subtype of the 2011 classification and the tumour-specific survival. Carcinosarcoma, adenosquamous carcinoma and anaplastic carcinoma subtypes were associated with the poorest prognosis. Dogs with comedocarcinoma and solid carcinoma followed a disease course that was more aggressive than that observed in dogs with a carcinoma arising in a benign mixed tumour. Moreover, age, histological grade and lymphatic invasion status significantly correlated with tumour-specific survival in univariate analysis. In multivariate analysis, histological subtype, age and lymphatic invasion status remained independent prognostic factors for CMC. [Display omitted] [ABSTRACT FROM AUTHOR]

Cancer-associated stroma (CAS) profoundly influences progression of tumors including mammary carcinoma (mCA). Canine simple mCA represent relevant models of human mCA, notably also with respect to CAS. While transcriptomic changes in CAS of mCA are well described, it remains unclear to what extent these translate to the protein level. Therefore, we sought to gain insight into the proteomic changes in CAS and compare them with transcriptomic changes in the same tissue. To this end, we analyzed CAS and matched normal stroma using laser-capture microdissection (LCM) and LC-MS/MS in a cohort of 14 formalin-fixed paraffin embedded (FFPE) canine mCAs that we had previously characterized using LCM-RNAseq. Our results reveal clear differences in protein abundance between CAS and normal stroma, which are characterized by changes in the extracellular matrix, the cytoskeleton, and cytokines such as TNF. The proteomics- and RNAseq-based analyses of LCM-FFPE show a substantial degree of correlation, especially for the most deregulated targets and a comparable activation of pathways. Finally, we validate transcriptomic upregulation of LTBP2, IGFBP2, COL6A5, POSTN, FN1, COL4A1, COL12A1, PLOD2, COL4A2, and IGFBP7 in CAS on the protein level and demonstrate their adverse prognostic value for human breast cancer. Given the relevance of canine mCA as a model for the human disease, our analysis substantiates these targets as disease-promoting stromal components with implications for breast cancer in both species. [ABSTRACT FROM AUTHOR]

Canine mammary carcinoma represents a model for the study of human breast cancer, although the prognostic value of various clinical, histological and immunohistochemical parameters has shown contradictory results. A prospective study, through a 4‐year follow‐up, was performed in 77 patients with mammary carcinoma to analyse the association between histological diagnosis, grade of malignancy, peritumoral and vascular invasion. We have also performed immunohistochemistry for the expression of oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and cyclooxygenase‐2 (COX‐2) that define human biomarkers of disease progression and treatment response. An association between histological diagnosis and clinical stage was observed with a high proportion of complex carcinoma classified as stage I. There was a higher proportion of ER+/PR+/HER2− tumours in stage I. In contrast, triple‐negative tumours (ER−/PR−/HER2−) were found mainly in advanced clinical stages and were associated with vascular and peritumoral invasion. The tumours included in group VII (carcinosarcoma/adenosquamous carcinoma/other special types of carcinoma) had a higher expression of COX‐2. The univariate analysis showed that those patients with complex carcinoma had the lowest incidence of metastases and the highest probability of survival. In contrast, a high proportion of patients with anaplastic/inflammatory carcinoma developed metastases and showed the lowest probability of survival. In addition, the estimated survival time was shorter for those patients with triple‐negative tumours and those with high COX‐2 expression. However, in the multivariate analysis, only the peritumoral invasion maintained its prognostic significance. In conclusion, in our study anaplastic/inflammatory carcinomas had the worst prognosis with a high proportion of triple‐negative tumours in this category. [ABSTRACT FROM AUTHOR]

Background: In the last two decades, canine mammary cancer has played an essential role in human breast cancer research. There are various similarities between biological and clinical features of canine breast cancer and female breast cancer in many cases. Clinical studies and evaluation of prognostic factors in canine mammary cancer can increase reliability in generalizing results to human cancers. This study was performed in the direction of comparative oncology. Methods: We collected clinicopathological data of an invasive type of canine mammary carcinoma from clinical records and pathology reports. Age, tumor laterality, tumor size, lymph node status, and tumor grade were recorded, and the relationships between the parameters were evaluated using linear regression analysis. Results: Ninety-seven patients were included in the study, and the mean age was 10.06 ± 2.73 years. The left mammary gland was involved in 51% of cases, and pT2 was the most common tumor size. Lymph nodes were involved in 27% of patients, and 43% of tumors were grade I. Statistical analysis showed no relationship between tumor size and laterality with other clinicopathological features. However, there was a statistically significant relationship between tumor size and tumor grade, and lymph node status. As the tumor size increased, tumor grade and the risk of lymph node involvement raised. Conclusion: Similar results of this study to breast cancer in women show that canine mammary carcinoma is a suitable model in comparative oncology research. Dogs live shorter than humans so that researchers can get the results of treatment and perform survival rate assessments faster in clinical trials. By considering ethical principles, dogs with breast cancer may replace phases I and II of human clinical trials in some cancer types soon. [ABSTRACT FROM AUTHOR]

Canine mammary carcinomas are common tumours in female dogs and histopathological examination has an important role in identifying whether they are benign or malignant. The latest and most commonly used histological grading system was established by Peña et al. (2013) and is based on the extent of tubule formation, nuclear pleomorphism and number of mitoses. Before the establishment of this grading system, tumour size and classical histological indicators of malignancy such as lymphovascular invasion, infiltration into surrounding tissue, necrosis and presence of a micropapillary pattern were important predictors of biological behaviour. However, the system of Peña et al. does not consider tumour size or these histological features. Clarifying the association of these features and histological grade, especially in grade II and III carcinomas, is important. In this study, we confirmed that the system of Peña et al. is effective for predicting biological behaviour and that evaluation of histological features of malignancy reinforced histological grade, as determined by the system of Peña et al. , especially in grade II carcinomas. [ABSTRACT FROM AUTHOR]

Canine mammary gland tumors (CMTs) are one of the most prevalent cancers in dogs and a good model for human breast cancer (BC), however gene expression analysis of CMTs is scarce. Although divergence of genes expression has been found in BC of different human races, no such research of different dog’s breeds has been done. The purpose of this study was to investigate expression of the VEGF-B, VEGF-A, FLT-1, KDR, ERBB2, EGFR, GRB2, RAC1, CDH1 and HYAL-1 genes of canine mammary carcinomas, compare the expression levels with clinicopathological parameters and analyze expression disparities between different breeds. Carcinomas and adjacent tissues were collected from female dogs to perform routine histopathology, immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). We found that VEGF-B and EGFR genes were overexpressed in the mammary gland carcinomas compared to adjacent tissue. VEGF-B gene expression had associations with different parameters (tumor size, grade, and absence of metastasis). Furthermore, differences in VEGF-B, FLT1, ERBB2, GRB2, RAC1, CDH1 and HYAL-1 genes expression have been found in different breed dogs (German Shepherd, Yorkshire Terrier) and mixed-breed dogs indicating that a dog’s breed could determine a molecular difference, outcome of cancer and should be accounted as a confounding factor in the future gene expression research.

Spontaneous canine simple mammary tumors (CMTs) are often viewed as models of human breast cancer. Cancer-associated stroma (CAS) is central for initiation and progression of human cancer, and is likely to play a key role in canine tumors as well. Until recently, however, canine CAS in general, and in CMT in particular, lacked detailed characterization and it remained unclear how canine and human CAS compare. This void in knowledge regarding canine CAS and the resulting lack of unbiased cross-species analysis of molecular homologies and differences undermined the validity of the canine model for human disease. To assess stromal reprogramming in canine breast tumors, we have recently established a protocol to specifically isolate and analyze CAS and matched normal stroma from archival, formalin-fixed paraffin embedded (FFPE) clinical tumor samples using laser-capture microdissection followed by next-generation RNA-sequencing. Using this approach, we have analyzed stromal reprogramming in both malignant canine mammary carcinomas (mCAs) as well as benign canine mammary adenomas in a series of studies. Our results demonstrate strong stromal reprogramming in CMTs and identify high-grade molecular homology between human and canine CAS. Here, I aim to give a short background on the value of comparative oncology in general, and spontaneous CMT in particular. This will be followed by a concise review of the current knowledge of stromal reprogramming in both malignant canine mCA and benign adenoma. Finally, I will conclude with insights on highly conserved aspects of stromal reprogramming between CMT and human breast cancer that accentuate the relevance of CAS in CMT as a model for the human disease.

Spontaneous canine simple mammary carcinomas (mCA) are often viewed as models of human mCA. Cancer-associated stroma (CAS) is central for initiation and progression of human cancer, and is likely to play a key role in canine tumours as well. However, canine CAS lacks characterisation and it remains unclear how canine and human CAS compare. Formalin-fixed paraffin embedded (FFPE) tissue constitutes a valuable resource of patient material, but chemical crosslinking has largely precluded its analysis by next-generation RNA sequencing (RNAseq). We have recently established a protocol to isolate CAS and normal stroma from archival FFPE tumours using laser-capture microdissection followed by RNAseq. Using this approach, we have analysed stroma from 15 canine mCA. Our data reveal strong reprogramming of canine CAS. We demonstrate a high-grade molecular homology between canine and human CAS, and show that enrichment of upregulated canine CAS genes strongly correlates with the enrichment of an independently derived human stromal signature in the TCGA breast tumour dataset. Relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. Finally, we establish the prognostic potential of the canine CAS signature in human samples, emphasising the relevance of studying canine CAS as a model of the human disease. In conclusion, we provide a proof-of-principle to analyse specific subsections of FFPE tissue by RNAseq, and compare stromal gene expression between human and canine mCA to reveal molecular drivers in CAS supporting tumour growth and malignancy.

P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) expression are frequently related to multidrug resistance (MDR) in neoplastic cells. Canine inflammatory and grade III noninflammatory mammary carcinomas (IMC and non-IMC) are aggressive tumors that could benefit from chemotherapy. This study describes the immunohistochemical detection of P-gp and BCRP in 20 IMCs and 18 non-IMCs from dogs that had not received chemotherapy. Our aim was to determine if P-gp and BCRP expression was related to the "inflammatory" phenotype, to establish a basis for future studies analyzing the response to chemotherapy in dogs with highly malignant mammary cancer. Immunolabeling was primarily membranous for P-gp with a more intense labeling in emboli, and immunolabeling was membranous and cytoplasmic for BCRP. P-gp was expressed in 17 of 20 (85%) IMCs compared to 7 of 18 (39%) non-IMCs (P = 0.006). BCRP was expressed within emboli in 15 of 19 (79%) emboli in IMC, 12 of 15 (80%) primary IMCs, and 12 of 18 (67%) non-IMCs, without statistically significant differences (P >.05). All IMCs and 67% of non-IMCs expressed at least 1 of the 2 transporters, and 63% (12/19) of IMCs and 39% (7/18) of non-IMCs expressed both P-gp and BCRP. P-gp and BCRP evaluation might help select patients for chemotherapy. P-gp, expressed in a significantly higher percentage of IMCs vs non-IMCs, might play a specific role in the chemoresistance of IMC. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are major actors in multidrug resistance (MDR) phenomenon in both human and canine mammary carcinomas (CMCs). The aim of this study was to investigate an association between the intrinsic expression of P-gp and BCRP compared to the immunophenotypes and outcome in CMCs. Fifty CMCs were evaluated at immunohistochemistry (IHC) for P-gp, BCRP, Estrogen receptor alpha (ER), Progesterone receptors (PR), Human Epidermal Growth Factor Receptor type 2 (HER2), basal cytokeratins 5/6 (CK5/6), Epidermal Growth Factor Receptor 1 (EGFR), and Ki67 proliferation index. P-gp and BCRP positive cases were, respectively, 52% and 74.5%, with a significantly higher expression of BCRP than P-gp. Five immunophenotypes were defined in 37 out of 50 CMCs: 9 (24.3%) Luminal A, 5 (13.5%) Luminal B, 9 (24.3%) HER2 overexpressing, 9 (24.3%) Triple-negative basal-like, and 5 (13.5%) Triple-negative non-basal-like. In all CMCs at least one marker was expressed. Follow-up data were available for 25 animals. The average cancer-specific survival was 739 ± 444 days. A number of CMCs bear a high expression of P-gp and BCRP but no significant association was found between their expression and the immunophenotypes, Ki67 index, the histological grade, and tumor-related death.

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The incidence of canine mammary carcinoma varies with age, breed, and spay status, being among the main tumors appearing in intact female dogs. Thirty-six canine mammary carcinoma patients received injections of canine interferon-β (cIFN-β) and HSV-thymidine kinase/ganciclovir (HSV-tk/GCV) carrying lipoplexes, into the tumor bed, immediately after surgery. Next, they started periodic subcutaneous injections of lipoplexes carrying a human granulocyte-macrophage colony stimulating factor and interleukin-2mixedwith allogeneicmammary carcinoma extracts. This combined strategy was safe and well tolerated. In addition, only two out of 26 patients treated with complete surgery developed a local relapse, and 0 out of 29 stage II and III patients displayed distant metastases, suggesting both local and systemic antitumor activities. The most encouraging result was the long survival times: 22 > 1 year (where 13 > 2 and 4 > 3 years), while maintaining a good quality of life. The preliminary results in five patients presenting with local disease, an additional HSV-tk/GCV plus cIFN-β gene treatment induced local antitumor activity, evidenced by four objective responses (one complete, three partial) and one stable disease. This successful outcome supports further studies to validate this approach not only for canine veterinary patients, but also for translation to human patients. [ABSTRACT FROM AUTHOR]

Cyclooxygenase (COX)-2 expression is positively correlated with malignant features in canine mammary carcinomas. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and may therefore possess anticancer effects. Meloxicam is an NSAID that is widely used in human and veterinary medicine. High concentrations of meloxicam have been reported to be antitumorigenic in vitro; however, the effect of meloxicam at concentrations that are equivalent to those that can be obtained in vivo remains unknown. In the current study, the in vitro effects of low-dose meloxicam (0.25 µg/ml) on CF41.Mg canine mammary carcinoma cells were evaluated. The effects on cell proliferation, apoptosis, cell migration and invasion, in addition to the expression of different molecules associated with tumor invasiveness were analyzed. No effect on cell viability and apoptosis were observed. However, cell migration and invasion were significantly reduced following treatment with meloxicam. MMP-2 expression and activity were similarly reduced, explaining the impaired cell invasion. In addition, β-catenin expression was downregulated, while its phosphorylation increased. These results indicate that 0.25 μg/ml meloxicam reduces cell migration and invasion, in part through modulating MMP-2 and β-catenin expression. Additional studies are required to elucidate the mechanism associated with the anti-invasive effect of meloxicam on CF41.Mg cells. The results of the present study suggest that meloxicam has a potential adjunctive therapeutic application, which could be useful in controlling the invasion and metastasis of canine mammary carcinomas. [ABSTRACT FROM AUTHOR]

Salinomycin (SAL), a polyether ionophore antibiotic, has been demonstrated to selectively kill cancer stem cells (CSCs) in various types of human tumor. The aim of the present study was to investigate the effects of SAL on canine mammary CSCs. CSCs in canine mammary carcinoma cell lines (CMT7364 and CIPp) were identified using a sphere formation assay and flow cytometry. The chemoresistance, invasive potential and expression of stem cell‑associated proteins of these spheres was then analyzed. This demonstrated that the spheres exhibited characteristics of CSCs, including a cluster of differentiation (CD)44+/CD24‑/low phenotype, upregulation of Wnt/β‑catenin signaling pathway‑associated proteins and chemoresistance. The viability of the spheres was decreased in a concentration‑ and time‑dependent manner following treatment with SAL, and the spheres did not exhibit increased resistance to SAL compared with their parental cells. In addition, exposure to SAL inhibited sphere‑formation and invasive potential in canine mammary CSCs in a dose‑dependent manner. Furthermore, SAL decreased the CD44+/CD24‑/low population and downregulated the expression of Wnt/β‑catenin signaling‑associated proteins (β‑catenin, Cyclin D1 and octamer‑binding transcription factor 4) in the spheres. In conclusion, the present study demonstrated that SAL is an effective inhibitor of canine mammary CSCs in vitro, indicating that SAL is a promising chemotherapeutic agent for the treatment of canine mammary carcinoma. [ABSTRACT FROM AUTHOR]

Mammary tumors are the most common neoplasm in female dogs and are considered important health problems. In this study we aimed to investigate Transforming Growth Factor Beta-1 (TGF-β1) expression in canine mammary carcinomas (CMCs) by immunohistochemistry and its relationships with histologic grading, survival rate, and recurrence. Tissues were selected from 35 cases of CMCs and were included in a 2-year follow-up study. Immunohistochemistry for detection of TGF-β1 protein was carried out using Avidin-Biotin Complex (ABC) method, and the immunostained slides were scored according to the distribution and intensity of positive reactions. Results indicated that TGF β-1 expression increased significantly in CMCs in comparison to canine normal mammary gland ( P < 0.05), 6/35 (17.14%) of tumor samples showed weak immunoreactivity, 15/35 (42.85%) moderate, and 14/35 (40%) showed strong immunoreactivity for TGF-β1. It is also revealed that TGF β-1 increased expression is in significant relationships with higher histologic grades and shorter survival rate after 2 years but not with tumor recurrence. [ABSTRACT FROM AUTHOR]

Synthesis of structurally diverse derivatives of aconitine-type diterpenoid alkaloids and their anti-proliferative effects on canine breast cancer cells. [Display omitted] • A series of structurally diverse aconitine-type diterpenoid alkaloid derivatives were synthesized. • Three derivatives exhibited good proliferation inhibitory against canine breast cancer cells. • The importance of the A-ring, 8-long chain ester, 13-hydroxyl, 14‑phenyl ring and N -ethyl substitution were revealed. • The best compound, aconitine linoleate (25) was able to block the cell cycle of canine breast cancer cells in the G0/G1 phase by inducing apoptosis. As one of the most common malignancies in female dogs, no drugs have been developed specifically for the treatment of canine mammary carcinoma. In our previous study, a series of diterpenoid alkaloids derivatives were synthesized and exhibited good anti-proliferative activity in vitro against both normal and adriamycin-resistant human breast cancer cells lines. In this study, a series of structurally diverse aconitine-type alkaloids derivatives were also synthesized basing on the minimal modification principle, by modifying on A-ring, C-ring, D-ring, N -atom or salt formation on aconitine skeleton. Their anti-proliferative effects and mechanism on canine mammary cancer cells were investigated, exhibiting the importance of the substitution at A ring, the long chain ester at the C8, the hydroxyl group at the C13, the phenyl ring at the C14 and the N -ethyl group, while the methoxy group at the C1 and C16 showed little effect on the activity. The results of the proliferation, apoptosis and ultrastructure tests of the treated canine mammary carcinoma cells referred that the representative compound, aconitine linoleate (25) could block the cell cycle of canine mammary carcinoma cells in the G0/G1 phase, and exhibit the anti-proliferative effect by inducing apoptosis. [ABSTRACT FROM AUTHOR]

The incidence of canine mammary carcinoma varies with age, breed, and spay status, being among the main tumors appearing in intact female dogs. Thirty-six canine mammary carcinoma patients received injections of canine interferon-β (cIFN-β) and HSV-thymidine kinase/ganciclovir (HSV-tk/GCV) carrying lipoplexes, into the tumor bed, immediately after surgery. Next, they started periodic subcutaneous injections of lipoplexes carrying a human granulocyte-macrophage colony stimulating factor and interleukin-2 mixed with allogeneic mammary carcinoma extracts. This combined strategy was safe and well tolerated. In addition, only two out of 26 patients treated with complete surgery developed a local relapse, and 0 out of 29 stage II and III patients displayed distant metastases, suggesting both local and systemic antitumor activities. The most encouraging result was the long survival times: 22 > 1 year (where 13 > 2 and 4 > 3 years), while maintaining a good quality of life. The preliminary results in five patients presenting with local disease, an additional HSV-tk/GCV plus cIFN-β gene treatment induced local antitumor activity, evidenced by four objective responses (one complete, three partial) and one stable disease. This successful outcome supports further studies to validate this approach not only for canine veterinary patients, but also for translation to human patients.

The CD44+/CD24– phenotype identifies cancer stem cell (CSC) properties in canine mammary carcinoma (MC); however, the histopathological features associated with this phenotype remain to be elucidated. Here, we determined whether the CD44+/CD24– phenotype was associated with hormonal receptor (HR; estrogen receptor [ER] and/or progesterone receptor [PR]) status and/or triple (ER, PR, and human epithelial growth factor receptor 2)–negative (TN) subtype; conventional histological evaluation was also performed. We found that, as single markers, both CD44+ and CD24+ were associated with less aggressive histological types, low grade, and a non-TN subtype; both markers were associated with HR positivity. On the other hand, a CD44+/CD24– phenotype was associated with higher grade of carcinoma. Therefore, our results suggest that immunohistochemical phenotyping for CD44/CD24 is useful for the evaluation of tumor behavior as well as CSC-like properties in canine MCs. [ABSTRACT FROM AUTHOR]

Background: Dogs spontaneously develop invasive mammary carcinoma with a high prevalence of the triple-negative (TN) subtype (lack of ER-Estrogen Receptor and PR-Progesterone Receptor expression, lack of HER2-Human Epidermal Growth Factor Receptor 2 overexpression), making this animal model relevant for investigating new therapeutic pathways. Insulin-like growth factor Type-1 receptor (IGF1R) is frequently overexpressed in primary human breast cancers, with a growing role in the TN phenotype. The purpose of this study was to investigate the Dog as a candidate model for IGF1R-overexpressing mammary carcinoma.Methods: 150 bitches with canine mammary carcinoma (CMC) and a known 2-year follow-up were retrospectively included. IGF1R expression was assessed by immunohistochemistry (IHC) using a similar scoring system as for HER2 in breast cancer. The prognostic value of the IGF1R expression was assessed in terms of overall and specific survival as well as disease-free interval (DFI).Results: 47 CMC (31 %) were classified as luminal and 103 (69 %) as triple-negative (TN-CMC). 41 % of CMC overexpressed IGF1R (IHC score 3+) of which 76 % were TN-CMC and 62 % grade III. IGF1R overexpression was associated with aggressive features including lymphovascular invasion, histological grade III, low ER expression and the TN phenotype. Univariate and multivariate analyses revealed that IGF1R overexpression was associated with shorter overall and specific survivals and shorter DFI in TN-CMC.Conclusions: IGF1R overexpression is common and related to a poor outcome in canine invasive mammary carcinoma, particularly in the triple negative subtype, as in human breast cancer. Preclinical studies using the Dog as a spontaneous animal model could be considered to investigate new therapies targeting IGF1R in triple-negative breast cancer. [ABSTRACT FROM AUTHOR]