p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer‐related signalling pathways. In this context, the deregulated expression of p62 – Sequestosome1 (p62/SQSTM1) – a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62‐based medicine. In veterinary oncology the role of p62 in cancer‐related pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 non‐neoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62‐negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour‐rejection antigen for an anti‐cancer immunotherapy.     

Prognostic value of tumour‐associated macrophages in canine mammary tumours

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

Decorin concentrations in canine normal and neoplastic mammary gland tissues

In the present study, the concentration of decorin in canine normal and neoplastic mammary gland tissues was examined to understand the potential role of decorin in development and progression of canine mammary tumours. The homogenates of 48 mammary gland tumours (10 benign and 38 malignant) and 10 samples of normal canine mammary gland tissue were used in the study. The presence and quantification of decorin was examined in the homogenates using Western blot and specific canine ELISA. Western blotting confirmed the presence of decorin both in the normal mammary gland tissues and in the mammary gland tumours. The concentration of decorin was significantly higher (p < .05) in the benign tumours and non-metastatic malignant tumours than in the normal mammary gland. The concentration of decorin was significantly lower (p < .05) in the malignant tumours with metastasis to regional lymph nodes compared with benign tumours and non-metastatic malignant tumours. No significant differences were found in the level of decorin between the benign and the non-metastatic malignant tumours. Both the histological type of malignant tumours and the histological grade did not significantly affect the concentration of decorin. These findings suggest that neoplastic transformation in the canine mammary gland leads to increase in the decorin protein synthesis. The reducing decorin concentration in canine malignant mammary tumours appears to facilitate the metastatic spread of these tumours.     

Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas

Calponin is a 34‐kDa smooth muscle‐specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin–biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin‐positive cells were identified: (1) Type 1: cytokeratin‐14‐positive pre‐existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin‐14‐positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin‐14‐positive atypical cells indistinguishable from non‐reactive atypical cells, which should have never been detected in haematoxylin and eosin‐stained sections and (4) Type 4: cytokeratin‐14‐negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin‐negative and cytokeratin‐14‐positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high‐molecular‐weight cytokeratins in luminal epithelial‐type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.     

Mast cells in canine mammary gland tumour: number, distribution and EPOR positivity

Erythropoietin (EPO)-mediated mitogenic and anti-apoptotic effects involve all the cells expressing functional receptors for EPO (EPOR), as demonstrated by in vitro and in vivo studies. EPO shows pleiotropic effects and acts as an endogenous mediator of adaptive tissue response to metabolic stress protecting tissues from different injuries. Recently, the EPO/EPOR complex has been identified in several neoplastic cell lines and solid tumours. In this study, the authors investigated the mast cells (MCs) number, distribution and their immunoreactivity for EPOR in normal, dysplastic and neoplastic canine mammary gland. The results showed that MCs were more numerous in displastic glands compared with normal and neoplastic glands. As far as the EPOR immunoreactivity is concerned, we did not observe MCs reaction on cancer, in contrast with previously published data where epithelium of neoplastic gland showed an increase in EPOR expression along with the neoplastic progression. Overall, our results might be suggestive for MCs role in oncogenesis and offer new insight regarding to the expression of EPOR in mammary gland cancer in dog.     

Plasma free amino acid profiles of canine mammary gland tumors

The purpose of this study was to elucidate the relationship between plasma free amino acid (PFAA) levels and the clinical stages of mammary gland tumors (MGT) in dogs. PFAA levels in canines with malignant mammary tumors were decreased compared to those of healthy animals. The levels of aspartate and ornithine, in the dogs with tumor metastasis were significantly decreased when compared to those of dogs that did not have metastases. Results of this study indicate that PFAA levels could be a risk factor or biomarker for canine MGT metastasis.     

Photodynamic detection of canine mammary gland tumours after oral administration of 5‐aminolevulinic acid

5‐Aminolevulinic acid (5‐ALA) is widely used in photodynamic detection (PDD) and therapy. We evaluated the pharmacokinetics of 5‐ALA‐induced porphyrins and its effectiveness in PDD in dogs with mammary gland tumours (MGTs) following oral administration. Healthy dogs and those with MGTs (nine each) were orally administered 4 mg kg^?1 5‐ALA. Protoporphyrin IX (PpIX) was not detected in the plasma of healthy dogs but it peaked in dogs with MGT at 2 h after 5‐ALA administration. In the PDD study, 16 dogs with MGT were orally administered 40 mg kg^?1 5‐ALA, and MGT but not normal tissue showed red fluorescence after 2–4 h. Photon counts were 6635–63 890 and 59–4011 (median, 19 943 and 919) for MGT and non‐tumour tissues, respectively. Cell density strongly correlated with PpIX photon counts of MGT tissue of the dogs (R = 0.743, P = 0.0009). We suggest that 5‐ALA‐PDD might be an effective diagnostic tool for MGTs.     

Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.     

Microtomographic characterization of calcifications in canine mammary tumours

The present work describes the microtomographic characterization of macro‐ and microcalcifications present in excised canine mammary glands. In human breast cancer, microcalcifications are highly relevant for diagnosis and prognosis, often being the sole element determining biopsy. Canine mammary tumours are considered a model for human breast cancer, but the morphological features of calcifications had still to be studied in this species. The objective of this research is to contribute to the characterization of the mineralization features of the canine mammary gland. In the present study, the excised mammary glands of 33 bitches underwent fluoroscopic examination. In 30 of the samples, the presence of calcification was suspected, and multiple biopsies were taken of these areas. Biopsy fragments underwent microtomographic scanning. Microcalcifications were found in non‐neoplastic glandular tissue, benign and malign lesions, as it is known to happen in humans. Qualitative evaluation regarding morphology of the imaged calcifications showed similarities to breast cancer findings, based on the BI‐RADS 2013 classification, such as pleomorphism and shape. No differences in the quantitative morphological parameters of volume, surface, surface/volume, SMI and structure thickness were found when macrocalcifications were considered. However, although significant differences existed in these parameters between microcalcifications from malignant canine mammary tumours and the two other groups, none were found between non‐neoplastic and benign tumours. Findings further support the use of this spontaneous animal model for the study of human breast cancer, considering how clinically relevant microcalcifications are in humans.     

Molecular phenotyping of malignant canine mammary tumours: Detection of high-risk group and its relationship with clinicomolecular characteristics

Canine mammary gland tumours (CMTs) constitute the most common cancer in female dogs and comprise approximately 50% of all canine cancers. With the advent of high-throughput technologies such as microarray and next-generation sequencing, the molecular phenotyping (classification) of various cancers has been extensively developed. The present study used a canine RNA-sequencing dataset, namely GSE119810, to classify 113 malignant CMTs and 64 matched normal samples via an unsupervised hierarchical algorithm with a view to evaluating the association between the resulting subtypes (clusters) (n = 4) and clinical and molecular characteristics. Finally, a molecular classifier was developed, and it detected 1 high-risk molecular subtype in the training dataset (GSE119810) and 2 independent validation datasets (GSE20718 and GSE22516). Our results revealed four molecular subtypes (C2–C5) in malignant CMTs. Furthermore, the normal samples constituted a distinct group in the clustering analysis. Marked significant associations were observed between the molecular subtypes (especially C5) and clinical/molecular features, including positive lymphatic invasion, high tumour grades, histopathology diagnoses, short survival and high TP53 mutation rates (ps <.05). The high-risk subtype (C5) was further characterized through the development of a cell cycle-based gene signature, which comprised 37 proliferation-related genes according to the support vector machine algorithm. This signature identified the high-risk group in both training and validation datasets (ps <.001). In the validation analysis, our potential classifier robustly predicted patients with positive lymphatic invasion, metastases and short survival.     

Expression of E‐cadherin in canine anal sac gland carcinoma and its association with survival*

The objective of this study was to determine whether an association could be demonstrated between survival and the expression of the adhesion molecule E‐cadherin by the neoplastic cells in a group of dogs with anal sac gland carcinomas (ASGCs). Archived formalin‐fixed, paraffin wax‐embedded primary tumour specimens were obtained for 36 cases of canine ASGC with known clinical management and survival data. Immunohistochemical methods were used to evaluate E‐cadherin expression by the neoplastic cells and data were evaluated for an association between E‐cadherin expression and survival. On univariate analysis, the median survival time for cases with tumours expressing E‐cadherin in more than 75% of cells was significantly greater than that for cases with tumours expressing E‐cadherin in fewer than 75% of cells (1168 versus 448 days, P = 0.0246). Both E‐cadherin expression and presence or absence of distant metastases were significantly associated with survival on multivariate analysis. This study demonstrates that expression of E‐cadherin at the cytoplasmic membrane in canine ASGCs is variable and potentially predictive of survival.     

Prognostic value of regional lymph node status in canine mammary carcinomas

In this study, we have determined the prognostic value of the presence of the micrometastases and metastases greater than 2 mm in the regional lymph nodes for bitches with mammary carcinomas. The study involved 51 dogs diagnosed with a single malignant epithelial tumour in the 4th or 5th mammary gland. All animals underwent regional mastectomy; the 4th and 5th mammary glands were removed together with the inguinal lymph node. The lymph nodes were examined immunohistochemically using the anti-cytokeratin antibody, clone AE1/AE3. The bitches were followed up every 6 months for 2 years after surgery to determine the disease-free survival (DFS) and overall survival (OS). The Kaplan-Meier analysis showed a statistically significant difference in DFS and OS only between the group of bitches without metastases and the group with lymph node metastases greater than 2 mm. No significant differences between these two groups versus bitches with lymph node micrometastases were found.     

Expression of the glutamine metabolism‐related proteins glutaminase 1 and glutamate dehydrogenase in canine mammary tumours

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumour growth and glutamine metabolism. Because of their similarities, canine mammary carcinomas are useful for studying human breast cancer. Accordingly, we investigated the correlations between the expression of glutamine metabolism‐related proteins and the pathological features of canine mammary tumours. We performed immunohistochemical and western blot analysis of 39 mammary tumour tissues. In immunohistochemical analysis, the expression of glutaminase 1 (GLS1) in the epithelial region increased according to the histological grade (P < .005). In the stromal region, complex‐type tumours displayed significantly higher GLS1 intensity than simple‐type tumours. However, glutamate dehydrogenase expression did not show the same tendencies as GLS1. The western blot results were consistent with the immunohistochemical findings. These results suggest that the expression of GLS1 is correlates with clinicopathological factors in canine mammary tumours and shows a similar pattern to human breast cancer.     

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.     

The relationship between tumour size and expression of prognostic markers in benign and malignant canine mammary tumours

Tumour size is considered one of the most important determinants of clinical staging in cancer patients. The aim of this study was to assess the value of tumour size as an indicator of the differentiation of mammary neoplasias in female dogs. The tumour, nodes metastates (TNM) system, based on primary lesion size, the extent of its dissemination to regional lymph nodes and the presence or absence of distant metastases, was applied to 120 female dogs diagnosed with mammary neoplasias. Paraffin blocks from 38 cases were selected and studied by immunohistochemical staining for prognostic and predictive markers of breast cancer. The Kaplan–Meier survival curve was estimated for 110 female dogs. Larger tumours (T3) were mostly malignant and showed lower expression of progesterone receptor and higher expression of cellular proliferation markers. Global survival time was shorter in female dogs with large tumour masses. This study highlights the importance of tumour size as a prognostic indicator of mammary neoplasias in female dogs.     

Tumour‐associated macrophages are associated with vascular endothelial growth factor expression in canine mammary tumours

Tumour‐associated macrophages (TAMs) have been implicated in carcinogenesis including an important role in angiogenesis. In this study, we describe the relationship between TAMs and angiogenesis in canine mammary tumours (CMT). Formalin‐fixed paraffin‐embedded CMT samples [(n = 128: malignant (n = 97) and benign (n = 31)] were submitted to immunohistochemical staining to detect MAC387, vascular endothelial growth factor VEGF and CD31 expression. A statistical analysis was carried out to assess possible associations with clinicopathological variables and biological markers of tumour angiogenesis. TAMs, detected by MAC387 expression, were significantly associated with malignant CMT (P < 0.001) and VEGF positive tumours (P = 0.002) and also associated with VEGF expression within malignant CMT (P = 0.043). Associations with clinicopathological variables were found between TAMs and the presence of infiltrative growth (P = 0.031), low tubule formation (P = 0.040) and lymph node metastasis (P = 0.016). The results support the hypothesis that TAMs influence angiogenesis in CMT suggesting TAMs may represent a therapeutic target in this disease.