Canine Mammary Neoplasia Research Articles

Serum Extracellular Vesicles Cargo Approach in Bitches with Mammary Tumors.

This study investigated serum extracellular vesicles (EVs) in bitches with mammary neoplasms, in order to understand their size, shape, and concentration, as well as their association with tumor malignancy. Thirty bitches were categorized into control (n = 10), mammary tumor grades I and II (GI, n = 13), and grade III (GII, n = 7). Serum was separated from blood collected during mastectomy, and EVs were isolated using size exclusion chromatography. The analysis revealed no significant differences in EV concentrations among groups, with similar concentrations for control, GI, and GII. Ninety-one proteins were identified in EV-enriched samples, with six showing varied abundance across groups. Notably, keratin 18 was highly abundant in GI, while sushi domain-containing protein, EvC ciliary subunit 2, and the joining chain of multimeric IgM and IgA were increased in GII. Additionally, protocadherin 17 and albumin were upregulated in both GI and GII. ROC curves identified potential biomarkers for differentiating tumor grades. Enrichment pathway analysis revealed AFP gene upregulation in the GI. Mass spectrometry proteomics data were deposited in Mendeley Data. The study provides valuable insights into serum EV characterization in bitches, suggesting keratin 18 and protocadherin 17 as potential biomarkers for canine mammary neoplasia, with implications for future diagnostic and therapeutic strategies.

Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers.

Canine Mammary Neoplasia Induces Variations in the Peripheral Blood Levels of CD20, CD45RA, and CD99.

The idea of using tumour biomarkers as diagnostic tools is progressively increasing. Of these, serum biomarkers are of particular interest, as they can provide rapid results. In the present study, serum samples from 26 bitches diagnosed with mammary tumours, plus 4 healthy bitches, were obtained. The samples were analysed using CD antibody microarrays targeting 90 CD surface markers and 56 cytokines/chemokines. A total of five CD proteins, namely CD20, CD45RA, CD53, CD59, and CD99, were selected and further analysed, utilizing immunoblotting techniques to validate the microarray results. CD45RA showed a significantly lower abundance in the serum samples from the bitches carrying mammary neoplasia in comparison to the healthy animals. Regarding CD99, the serum samples from the neoplastic bitches showed it in a significantly higher abundance than those from the healthy patients. Finally, CD20 showed a significantly higher abundance in bitches carrying a malignant mammary tumour in comparison to healthy patients, but no differential expression between malignant and benign tumours was observed. According to these results, both CD99 and CD45RA are indicators of mammary tumour presence, but without distinguishing between malignant and benign.

Associations Between Dog Breed and Clinical Features of Mammary Epithelial Neoplasia in Bitches: an Epidemiological Study of Submissions to a Single Diagnostic Pathology Centre Between 2008–2021

Mammary cancer is one of the most common neoplasms of dogs, primarily bitches. While studies have been carried out identifying differing risk of mammary neoplasia in different dog breeds, few studies have reported associations between dog breeds and clinical features such as number of neoplastic lesions found in an individual case or the likelihood of lesions being benign or malignant. Such epidemiological studies are essential as a foundation for exploring potential genetic drivers of mammary tumour behaviour. Here, we have examined associations between breed, age and neuter status and the odds of a diagnosis of a mammary epithelial-origin neoplastic lesion (as opposed to any other histopathological diagnosis from a biopsied lesion) as well as the odds of a bitch presenting with either a single mammary lesion or multiple lesions, and the odds that those lesions are benign or malignant. The study population consisted of 129,258 samples from bitches, including 13,401 mammary epithelial neoplasms, submitted for histological assessment to a single histopathology laboratory between 2008 and 2021.In multivariable analysis, breed, age and neuter status were all significantly associated with the odds of a diagnosis of a mammary epithelial-origin neoplastic lesion. Smaller breeds were more likely to receive such a diagnosis. In cases diagnosed with a mammary epithelial neoplasm, these three factors were also significantly associated with the odds of diagnosis with a malignant lesion and of diagnosis with multiple lesions. Notably, while neutered animals were less likely to have a mammary epithelial neoplasm diagnosed, and were less likely to have multiple neoplasms, they were more likely to have malignant disease. Exploration of the patterns of risk of developing malignant disease, or multiple lesions, across individual breeds showed no breed with increased odds of both outcomes. Breeds with altered odds compared to the Crossbreed baseline were either at increased risk of malignant disease and decreased risk of multiple lesions, or vice versa, or they were at significantly altered odds of one outcome with no change in the other outcome. Our analysis supports the hypothesis that age, neuter status and intrinsic biological and genetic factors all combine to influence the biological heterogeneity of canine mammary neoplasia.

PIK3CA is recurrently mutated in canine mammary tumors, similarly to in human mammary neoplasia

Biological features of neoplastic disease affecting mammary gland tissue are shared between canines and humans. Research performed in either species has translational value and early phase clinical trials performed in canines with spontaneous disease could be informative for human trials. The purpose of this study was to investigate the somatic genetic aberrations occurring in canine mammary neoplasia by exome capture and next generation sequencing. Based on 55 tumor-normal pairs we identified the PIK3CA gene as the most commonly mutated gene in canine mammary tumors, with 25% of samples carrying mutations in this gene. A recurrent missense mutation was identified, p.H1047R, which is homologous to the human PIK3CA hotspot mutation found in different types of breast neoplasia. Mutations homologous to other known human mutation hotspots such as the PIK3CA p.E545K and the KRAS p.G12V/D were also identified. We identified copy number aberrations affecting important tumor suppressor and oncogenic pathways including deletions affecting the PTEN tumor suppressor gene. We suggest that activation of the KRAS or PIK3CA oncogenes or loss of the PTEN suppressor gene may be important for mammary tumor development in dogs. This data endorses the conservation of cancer across species and the validity of studying cancer in non-human species.

Consensus Regarding the Diagnosis, Prognosis and Treatment of Canine and Feline Mammary Tumors - 2019

The aim of this paper is to discuss and update criteria that can guide the diagnosis, prognosis and treatment of canine and feline mammary neoplasms. It was elaborated during the IV Mammary Pathology Meeting: Diagnosis, Prognosis and Treatment of the Canine and Feline Mammary Neoplasia, held on April 29th and 30th, 2019 in Belo Horizonte – MG, Brazil, sponsored by the Laboratory of Comparative Pathology of the Federal University of Minas Gerais (UFMG), with the support of the Brazilian Association of Veterinary Pathology (ABPV) and Brazilian Association of Veterinary Oncology (ABROVET). Academics from several regions of Brazil were present and contributed to this work.

Estrogen receptors genotypes and canine mammary neoplasia

BackgroundEstrogens are essential for the development and proper function of several hormone-dependent organs. There are, however, several lines of evidence associating estrogens with mammary carcinogenesis. A marked individual genetic variability concerning estrogens biosynthesis, metabolism and mechanism of action was recognized and associated with human breast cancer susceptibility, clinical features and progression. Although some genetic variations in canine ESR1 gene were reported, their influence in clinicopathological features and progression of canine mammary tumors has not been fully evaluated. This study aims to assess the influence of SNPs in ESR1 gene (rs397512133, rs397510462, rs851327560, rs397510612, rs852887655, rs852684753 and rs852398698) in canine mammary tumors characteristics and progression. A group of 155 non-neutered bitches with mammary tumors was included in the study. Follow-up information was assessed 24 months after surgery.ResultsGenetic profiles associated with a later onset of mammary tumors and less aggressive clinicopathological features, namely smaller tumor size (≤ 3 cm) with extensive tubular differentiation and low canine-adapted prognostic index (vet-NPI), were identified in this study.ConclusionsOur data suggest that the ESR1 genetic profile may help on the decision regarding the selection of individual tailored preventive measures against canine mammary tumors development, such as early neutering.

Transcriptomic profile reveals molecular events associated to focal adhesion and invasion in canine mammary gland tumour cell lines.

The prevalence of cancer in animals has increased significantly over the years. Mammary tumours are the most common neoplasia in dogs, in which around 50% are presented in the malignant form. Hence, the development and characterization of in vitro models for the study of canine tumours are important for the improvement of cancer diagnosis and treatment. Thus, the aim of this study was to characterize cell lines derived from canine mammary gland neoplasias which could be further used for basic and applied oncology research. Samples of canine mammary carcinomas were taken for cell culture and 2 cell lines were established and characterized in terms of cell morphology, tumourigenicity and global gene expression. Both cell lines presented spindle-shape morphology and shown common malignant features as in vitro invasion potential and expression of epithelial and mesenchymal proteins. Also, we found gene expression patterns between the 2 cell cultures in comparison to the normal mammary gland tissue. Cells from M25 culture showed a higher invasion and in vivo tumourigenic potential, associated to the overexpression of genes involved in focal adhesion and extracellular matrix communication, such as FN1, ITGA8 and THBS2. The phenotypic characterization of these cells along with their global gene expression profile potentially determine new therapeutic targets for mammary tumours.

Immunohistochemical Expression of P-glycoprotein and Breast Cancer Resistance Protein in Canine Mammary Hyperplasia, Neoplasia and Supporting Stroma

Immunohistochemical Expression of P-glycoprotein and Breast Cancer Resistance Protein in Canine Mammary Hyperplasia, Neoplasia and Supporting Stroma

Immunohistochemical Expression of P-glycoprotein and Breast Cancer Resistance Protein in Canine Mammary Hyperplasia, Neoplasia and Supporting Stroma

The ability of a tumour to become simultaneously resistant to different drugs is known as multidrug resistance and is often due to the expression of ATP-dependent binding cassette transporters (ABC-transporters) such as P-glycoprotein (PGP) and breast cancer resistance protein (BCRP). In this study, the expression of PGP and BCRP was determined in the components of hyperplastic and neoplastic canine mammary glands, including the supporting stroma. The variation of expression of these molecules in carcinomas was evaluated between lesions of different histological stage and grade of malignancy. Samples included 47 hyperplastic tissues and 10 benign and 46 malignant neoplasms. Tumours were classified into histological subtype, histological stage and grade. Immunohistochemical evaluation of PGP and BCRP expression showed that both markers are potentially expressed by epithelial cells, myoepithelial cells in complex tumours and mesenchymal cells in mixed tumours, but expression of both proteins was significantly higher in malignant epithelial cells versus hyperplastic epithelium or the epithelium of benign tumours. BCRP showed significantly higher expression in epithelial cells of simple carcinomas versus those of complex and mixed carcinomas. Grade II and III carcinomas had higher epithelial PGP expression than grade I tumours. The positivity of stromal fibroblasts was higher in histological stage II versus I carcinomas, and in histological grade II versus I carcinomas. Malignant and invasive tumours were more likely to express PGP and/or BCRP in luminal and stromal components and evaluation of these markers could provide valuable information for the identification of tumours characterized by an aggressive and chemoresistant phenotype.

Absence of Canine Papillomavirus Sequences in Canine Mammary Tumours

Human papillomaviruses (PVs) are found in human breast cancer tissue; however, it remains controversial as to whether these viruses play a role in the aetiology of this tumour. There has been minimal study of whether PVs are found in normal or abnormal mammary glands of animals. The present study investigated whether a PV sequence could be found in the mammary glands of 33 female dogs by rolling circle amplification and polymerase chain reaction. No PV DNA was found in normal or neoplastic canine mammary tissues, suggesting that canine PVs are probably not involved in the pathogenesis of canine mammary neoplasia.

Short interspersed CAN SINE elements as prognostic markers in canine mammary neoplasia

The genome of mammals is characterized by a large number of non-LTR retrotransposons, and among them, the CAN SINEs are characteristics of the canine species. Small amounts of DNA freely circulate in normal blood serum and high amounts are found in human patients with cancer, characterizing it as a candidate tumor-biomarker. The aim of this study was to estimate, through its absolute expression, the number of copies of CAN SINE sequences present in free circulating DNA of female dogs with mammary cancer, in order to correlate with the clinical and pathological characteristics and the follow-up period. The copy number of CAN SINE sequences was estimated by qPCR in 28 female dogs with mammary neoplasia. The univariate analysis showed an increased number of copies in female dogs with mammary tumor in female dogs >10 years old (p=0.02) and tumor time >18 months (p<0.05). The Kaplan-Meier test demonstrated a negative correlation between an increased number of copies and survival time (p=0.03). High amounts of CAN SINE fragments can be good markers for the detection of tumor DNA in blood and may characterize it as a marker of poor prognosis, being related to female dogs with shorter survival times. This estimate can be used as a prognostic marker in non-invasive breast cancer research and is useful in predicting tumor progression and patient monitoring.

Analysis of a New Histological and Molecular-Based Classification of Canine Mammary Neoplasia

Canine mammary tumors (CMTs) are morphologically and biologically heterogeneous, prompting several attempts to classify such tumors on the basis of their histopathological characteristics. Recently, molecular-based analysis methods borrowed from human breast cancer research have also been applied to the classification of CMTs. In this study, canine mammary neoplasms (n = 648) occurring in Korea from 2008 to 2011 were analyzed according to the histological classification and grading system proposed by Goldschmidt et al. Furthermore, randomly selected mammary carcinomas (n = 159) were classified according to the molecular subtype using immunohistochemical characteristics. Canine mammary neoplasia accounted for 52.6% (648/1250) of the tumors in female dogs, and 51.7% (340/648) of these were malignant. All of the carcinoma-anaplastic subtypes were grade III tumors (5/5, 100%), while most of the carcinoma-tubular subtypes (15/18, 83.3%) and carcinoma arising in a complex adenoma/mixed-tumor subtype (115/135, 85.2%) were grade I tumors. Tumor cell invasion into lymphatic vessels was most common in the comedocarcinoma, carcinoma-anaplastic, and inflammatory carcinoma subtypes. The most frequently occurring molecular subtype (70/159, 44%) was luminal A. However, the basal-like subtype was the most malignant and was frequently associated with grade III tumors and lymphatic invasion. The carcinoma-solid subtypes were also often of the basal-like subtype. Reclassification of CMTs using the newly proposed histopathological classification system and molecular subtyping could aid in determining the prognosis and the most suitable anticancer treatment for each case.

Detection of cancer stem cells by immunohistochemistry of canine mammary neoplasias

Background Canine mammary neoplasias are very common and, in Brazil, 70% are considered malignant. Cancer stem cells (CSCs) are able to self-renew and have abilities to form metastases. In canine mammary tumors these CSCs were isolated by flow cytometry by means of the surface markers CD44+/CD24-. The aims of this study was detect these CSCs by immunohistochemical reactions and correlate them with degrees of canine mammary neoplasias.

Use of CD10 as a marker of canine mammary myoepithelial cells

CD10 is an important cell marker in the diagnosis of acute lymphoblastic leukaemia and of breast myoepithelial (ME) cells in humans. The objective of this study was to assess the value of CD10 as a marker of canine ME cells using immunohistochemistry on routinely processed normal, dysplastic and neoplastic mammary tissue. Five different CD10 positive cell types were identified on the basis of cell morphology, pattern of immunoreactivity, and on the co-expression of additional cell lineage-specific markers. Type 1 cells were typical fusiform cells with a ME cell phenotype (calponin- and cytokeratin [CK] 14-positive, CK8/18-negative). Type 2 cells were typical or atypical polyhedral cells with a luminal epithelial (LE) cell phenotype (calponin- and CK14-negative, CK8/18-positive). Type 3 cells had a type 1 phenotype with variable morphology, and type 4 were atypical neoplastic cells with a mixed ME/LE phenotype. Type 5 cells were typical fusiform cells with a stromal phenotype. Type 1 cells were considered normal ME cells and were found in all sample types; type 2 cells were considered normal or neoplastic LE cells and were also found in all sample types; types 3 and 4 cells were restricted to tumour samples and to malignant tumours, respectively, and type 5 cells were found in all sample types, although predominantly in neoplastic tissue. The findings indicate that the CD10 antigen is a sensitive (although not specific) marker of canine ME cells in normal, dysplastic and neoplastic mammary tissue. Differences in the distribution and staining intensity of CD10-positive cells suggest a number of potential roles for this protein in the pathogenesis of canine mammary neoplasia.

Interleukin-8 as a prognostic serum marker in canine mammary gland neoplasias

Mammary gland tumors in female dogs are an excellent model for the clinic-pathological, diagnostic and prognostic investigation of mammary neoplasias. Prognostic and predictive markers are effective in research and routine diagnosis. Interleukins play a fundamental role in cancer, with a particular function in tumor growth, invasion and metastatic potential. Interleukin-8 (IL-8) is known to possess tumorigenic and pro-angiogenic properties, and its overexpression is seen in a number of human tumors. IL-8 serum levels were determined and correlated with the clinic-pathological features and clinical evolution of mammary gland neoplasias in female dogs. IL-8 was measured by an immunoenzymatic assay in 30 female dogs with mammary neoplasias within a 12 month follow-up and in 50 control animals. The correlation between IL-8 concentration and clinical parameters was investigated. A statistically significant difference in the IL-8 serum levels was found in tumor-bearing dogs compared to the controls. In addition, when the individual parameters were evaluated, IL-8 content showed a positive correlation with the tumor progression, lymph node involvement, recurrence and death. Single and multivariate analyses showed associations between tumor recurrence, metastasis, high clinical staging and high IL-8, and also with the death risk. This was also consistent with the high IL-8 content in dogs showing tumor recurrence and metastasis. IL-8 superexpression has been detected in a number of human tumors, usually associated with a poor prognostic. Besides promoting angiogenesis, IL-8 is strongly related with the metastatic phenotype of mammary tumor cells. High IL-8 concentration was found in mammary gland cancer patients with advanced disease stages. Our results show that IL-8 can be used as a non-invasive prognostic marker for mammary gland cancer, and can be useful for the prediction of disease progression and recurrence in dogs with mammary neoplasias. The increased level of this cytokine acts as an independent prognostic marker of survival and the identification of animals with the poor prognostic.

Possible Role of Snail Expression as a Prognostic Factor in Canine Mammary Neoplasia

Epithelial cells adhere tightly to each other by cell-to-cell adhesion and through the basement membrane barrier to prohibit movement. In carcinomas, neoplastic epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype during the epithelial-mesenchymal transition (EMT) for invasion and metastasis. The aim of this study was to identify Snail expression and examine the role of Snail protein in canine mammary tumour progression. Immunohistochemical expression of Snail, E-cadherin, oestrogen receptor, human epidermal growth factor receptor-2, cytokeratin 14 and p63 was analyzed in 54 samples of canine mammary epithelial tumours (11 adenomas and 43 carcinomas). Expression of mRNA encoding Snail was evaluated in seven samples (one normal mammary gland, two adenomas and four carcinomas) by reverse transcriptase-polymerase chain reaction. Snail mRNA was detected in all samples. Snail expression correlated significantly with histological type, grade and lymphatic invasion. However, there was no association between Snail expression and molecular subtype and between Snail expression and that of E-cadherin. Snail, a hallmark of EMT, might play an important role in invasion and metastasis of canine mammary carcinomas.

Histological and Immunohistochemical Identification of Atypical Ductal Mammary Hyperplasia as a Preneoplastic Marker in Dogs

This study describes and evaluates the morphological and molecular relationship between canine mammary ductal hyperplasias with atypia and canine mammary neoplasias. Ductal hyperplasia was identified in association with malignant neoplasia in 56 of the 115 cases (48,8%), and although ductal hyperplasia without atypia was the type most frequently noted in the cases, most examples of hyperplasia with atypia were associated with mammary tumors. Estrogen receptor, E-cadherin, and cytokeratins 1, 5, 10 and 14 (CK34bE12) expression was quite lower than in normal mammary tissue, and HER2 overexpression was absent in all proliferative cells of ductal hyperplasia. The Ki-67 expression, epidermal growth factor receptor and progesterone receptor expression appeared higher in those hyperplastic lesions analyzed than in normal mammary glands. These findings suggest that canine mammary atypical hyperplasia may play an important role in the process of malignant neoplastic transformation, with molecular alterations that are similar to precursor lesions reported in humans.

Interleukin-8 expression associated with canine mammary tumors

The use of prognostic markers for mammary cancer is important for routine diagnosis and research. Interleukin-8 (IL-8) is a chemotactic cytokine, produced by several cell types in response to inflammation. The expression, regulation and function of IL-8 in dogs are little known. Recent studies have associated angiogenesis and inflammatory processes with tumor malignancy. We investigated a possible correlation between IL-8 expression and mammary tumor prognosis in female dogs. IL-8 expression was measured in 50 dogs with mammary neoplasia by immunohistochemistry and real-time PCR. Immunohistochemical staining was done with anti-IL-8 antibodies and PCR amplifications were performed in a 7500 Fast Real-Time PCR system. Gene expression stability was analyzed by the geNorm software. Quantitative real-time PCR showed that IL-8 expression decreased in malignant mammary cells compared to normal mammary tissue, while weak immunostaining was associated with a diagnosis of carcinoma. Complementing earlier studies on IL-8 expression in several types of cancer, including mammary cancer, we conclude that IL-8 has potential for use as a prognostic marker for canine mammary neoplasia.

Expression of the plasma membrane markers aquaporin 1 (AQP1), glucose transporter 1 (GLUT1) and Na, K-ATPase in canine mammary glands and mammary tumours

This study investigated the expression of the plasma membrane markers aquaporin 1 (AQP1), glucose transporter 1 (GLUT1) and the alpha1 subunit of Na, K-ATPase in normal canine mammary glands and in benign and malignant mammary tumours, using immunohistochemistry and semi-quantitative histomorphometry. AQP1 immunoreactivity was absent from the majority of specimens studied. GLUT1 immunoreactivity was observed in normal mammary tissue and particularly in the epithelial and mesenchymal cells of benign, and in the epithelial cells of malignant tumours, respectively. Na, K-ATPase immunoreactivity was present in normal and neoplastic mammary epithelium and was significantly increased in the epithelium of both benign and malignant tumours. These results suggest that GLUT1 is more highly expressed in neoplastic epithelium and mesenchyme and that Na, K-ATPase is more highly expressed in neoplastic mammary epithelium. In consequence, these membrane proteins may have potential as diagnostic and prognostic biomarkers of canine mammary neoplasia.