Studies on histogenesis of mixed tumours of the mammary gland in bitches

The aim of the study was the immunohistochemical assessment of the reactivity of mixed tumours in bitches with special regard to myoepithelial cells and cartilage. The materials for study were 20 mixed tumours collected during surgery or autopsy. Paraffin-embedded sections were routinely stained with hematoxylin and eosin and HID method was also used. Immunohistochemical assays were made using the AB-Complex method with the use of monoclonal antibodies against: cytokeratin MNF-116, cytokeratin 19, vimentin, actin and S-100 protein. On the basis of the reactions, 5 types of myoepithelial origin cells transformed during the formation of cartilage cells were diagnosed.     

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.     

Epithelial‐to‐mesenchymal transition: immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours

Background –  Epithelial‐to‐mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of β‐catenin and gain of mesenchymal markers. Hyposthesis/Objectives –  Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. Methods –  Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti‐vimentin, ‐cytokeratin, ‐survivin and ‐Hsp72 antibodies. A semi‐quantitative method was used to analyse the results, as follows: 0 to <5%; ≥5 to <10%; ≥10 to <25%; and ≥25% of positive cells. Immunofluorescence was performed to investigate survivin–vimentin and survivin–Hsp72 colocalization in selected SCCs. Results –  In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin–survivin colocalization. Conclusions and clinical importance –  A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested.     

Alkaline phosphatase reaction of canine mammary mixed tumours: a light and electron microscopic study

Alkaline phosphatase (ALK-P) reactions at the light and electron microscopic levels were performed on eight canine mammary mixed tumours. In the morphologically normal gland next to the tumours, the myoepithelial cells and the stromal tissues near the acinar basement membranes both showed a positive ALK-P reaction by light microscopy. At the electron microscopic level, those parts of the myoepithelial cell membrane that were in contact with other cells--myoepithelial or lumenal epithelial--showed an ALK-P-positive reaction, as did the adjacent stromal tissue. The characteristic tumour cells, at sites of early proliferation, were ALK-P-positive, while in the mucoid and chondroid areas of the mixed tumours they were largely ALK-P-negative by light microscopy. By electron microscopy, those neoplastic cells which were in contact with other cells typically showed a positive ALK-P reaction, while those cells which were in contact with mucoid or chondroid matrix were largely negative. Although the cytoplasmic filaments of the neoplastic cells were 10 nm thick, and those of normal myoepithelial cells were 6 to 8 nm thick, the observations reported provide further evidence for the view that the essential cells of the canine mammary mixed tumour are derived from myoepithelial cells.     

Histologic and immunohistochemical characterization of thymic epithelial tumours in the dog

Thymic epithelial tumour (TET) histologic subclassification has not been well described in the veterinary literature as it has in humans. The objective of this study was to identify and describe TET subtypes in dogs and to determine the utility of immunohistochemistry (IHC) in differentiating these subtypes. Samples were reviewed and classified according to a modified World Health Organization (WHO) criteria for human tumours of thymic origin. Signallment, presenting signs, treatment and survival data was collected from medical records. Histologic review confirmed the same subtypes as described in humans. Presence of high stage disease, pleomorphism, mitotic figures and capsular invasion was more common in atypical thymomas and thymic carcinomas than in thymomas. IHC was performed for GLUT‐1, CD5, CD117 and CK8/18; however, this was not useful in classifying the tumours.     

Canine mammary mixed tumours: immunohistochemical expressions of EGFR and HER‐2

Background Benign mixed tumours (BMTs) are frequently found in the mammary glands of female dogs, but the factors determining malignant transformation in these tumours are unknown. Objective To evaluate the expression of the oncoproteins, human epidermal growth factor receptor 2 (HER‐2) and epidermal growth factor receptor (EGFR), in 46 carcinomas in BMTs (CBMTs) and to verify their possible association with the malignancy of the tumours. Methods Immunohistochemical expression was analysed in benign and malignant components separately, and then compared with 74 cases of BMTs. Results Among the CBMTs, positivity for HER‐2 was found in the benign histological component of 4.3% (2/46), in the malignant epithelial non‐invasive component of 14.8% (4/27) and in the malignant invasive epithelial component of 13.6% (6/44) of cases. Two of the 24 (8.3%) BMTs were positive for HER‐2. There was no relationship between HER‐2 and the tumour components. There was no significant difference between BMTs and CBMTs. Positivity for EGFR was found in the benign component of 17.4% (8/46) of the CBMTs, in the malignant epithelial non‐invasive component of 40.7% (11/27%) and in the invasive epithelial malignant component of 45.4% (20/44). EGFR positivity was significantly associated with the invasive component of CBMTs. Conclusion EGFR may contribute to malignant epithelial transformation of BMTs. In contrast, HER‐2 overexpression may not be associated with the acquisition of a malignant epithelial phenotype.     

Plate and collagen-gel cultures of normal canine mammary epithelial cells.

Plate and collagen gel (floating or embedded) cultures were made to study the cell morphology and growth characteristics of canine mammary epithelial cells in vitro. Hiratsuka's method was satisfactory for the separation of the epithelial cells from the fibroblasts in explants of canine mammary tissue. In the plate culture method, epithelial cells migrated out from the explants to form colonies. The characteristic features of epithelial colonies in this culture system were polygonal cells situated in the centre, elliptical cells in the outer region and large spindle-shaped cells, thought to be myoepithelial cells, at the periphery. These epithelial cells showed a pavement-like appearance and were positive for antikeratin antibody. On floating gels, the cells changed their shape dramatically from flattened to rounded when the gel was released from the glass dish. Cells embedded in collagen gels grew to form duct-like structures.     

An assessment of the clonality of the components of canine mixed mammary tumours by mitochondrial DNA analysis

The aim of this study was to investigate if mutations in the mitochondrial DNA (mtDNA) D-loop fragment control region of canine mammary mixed tumours could be used as clonal markers that identified the cell population of origin. Ten benign mixed mammary tumours and nine carcinomas arising from benign mixed tumours were microdissected and DNA from epithelial and mesenchymal tumour cells and from normal mammary tissue was examined for sequence variations in a fragment of the hypervariable control region.Identical sequence variants in both the epithelial and mesenchymal components (as well as in the corresponding normal tissue) were found in 80% of the benign mixed tumours and in 89% of the carcinomas arising from benign mixed tumours suggesting a shared clonal origin. The distinctive sequence alterations identified in the epithelial and mesenchymal components of 15.8% of all 19 tumours examined, suggests the possibility that a minority of mammary tumours are polyclonal in origin or that early clonal divergence occurs. Increased mutation within the mtDNA D-loop fragment of mixed tumour components was not observed.     

Isolation and characterization of primary canine lens epithelial cells

OBJECTIVE: The purpose of this study was to characterize the proliferation and differentiation of primary canine lens epithelial cells (LEC) under standard culture conditions. PROCEDURE: Canine LEC were isolated by mechanical dissection of the canine globe and enzymatic digestion of the lens capsule from fresh lenses. Isolated capsules and cell suspensions were seeded in laminin-coated culture flasks. Canine LEC proliferated and formed monolayers, which could be passaged and maintained for approximately 2 weeks. Cells were characterized morphologically and cell lysates examined for expression of protein markers of epithelial origin and differentiation. RESULTS: Canine LEC exhibit morphologic characteristics of epithelial cells when cultured on laminin/lysine coated flasks. Expression of epithelial cell marker, cytokeratin 5, was highest at passage 1 and diminished with increasing passage number. Expression of gamma-crystallin, a protein found only in differentiated lens fiber cells, increased at passage 6. A laminin/lysine-coated surface supported optimal proliferation of canine LEC. Both an initial seeding density of 1 x 10(5) cells/cm(2) and culture in Dulbecco's modified essential media (DMEM) supplemented with 10% FBS supported a doubling time of less than 48 h in canine LEC. CONCLUSION: This study demonstrates that primary canine LEC retain the characteristics of lens epithelial cells prior to passage 6 under the described culture conditions and represent a suitable in vitro model for investigating lens physiology and cataractogenesis.     

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.     

Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III β tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.     

Effect of cyclooxygenase inhibitors in a xenograft model of canine mammary tumours

Inhibition of cyclooxygenase-2 (COX-2) represents a possible avenue for the prevention and/or treatment of some cancers. Our goal was to compare the effect of a selective inhibitor of COX-2, deracoxib, and a COX-1 and -2 inhibitor, piroxicam, on the growth of canine mammary tumours in a murine model. CMT-9 was used to induce xenografts in nude mice. Mice were treated with piroxicam (0.6 mg kg(-1)), deracoxib (6 mg kg(-1)) or a control solution. Tumour volumes between 0 and 24 days post-treatment showed no significant difference between all groups. A second series of experiments was performed with a higher dose of piroxicam (0.9 mg kg(-1)). Tumour volumes between 14 and 21 days post-treatment were significantly smaller in piroxicam-treated mice compared with controls. These results demonstrate that COX inhibition reduced the growth of canine mammary cancer xenografts in mice, suggesting that COX inhibitors could have a positive effect in dogs.     

Effect of recombinant feline interferon-ω alone and in combination with chemotherapeutic agents on putative tumour-initiating cells and daughter cells derived from canine and feline mammary tumours

Interferons (IFNs) are naturally produced cytokines with multiple important biological functions. The activity of recombinant feline IFN-ω (rFeIFN-ω) alone and in combination with chemotherapeutic drugs was tested on canine and feline mammary carcinoma cell lines (REM134 and CAT-MT) and putative tumour-initiating cells (TIC) derived from these cell lines by sphere assay. Viability was measured by chemoluminescence and a one-way analysis of variance and Student's t -tests were used for statistical analysis. rFeIFN-ω showed in vitro antitumour activity on feline and canine mammary carcinoma cells and putative TICs with a dose-dependent and target cell-specific action. Putative TICs were more resistant to the action of rFeIFN-ω compared with daughter REM134 and CAT-MT cells. REM134 cells and TICs were more sensitive to rFeIFN-ω compared with the feline counterparts. An additive effect was noticed between rFeIFN-ω and conventional anticancer drugs, in particular following co-culture of cells with anthracycline drugs. The results suggest that rFeIFN-ω warrants further investigation as a therapeutic adjunct in feline and canine mammary tumours.     

Prognostic studies of canine and feline mammary tumours: the need for standardized procedures

For several years, veterinary oncologists have been struggling with the prognosis of mammary tumours in dogs and cats. Translation of tumour characteristics into prognostic information is an invaluable tool for the use of the most appropriate therapies, as well as for planning innovative therapeutic trials. Moreover, canine and feline spontaneous mammary gland tumours are good models for the study of human breast cancer. Collecting and interpreting information regarding the prognosis of canine and feline mammary tumours is difficult due to the fact that different methods have been applied to study various components and characteristics. This review identifies some of the challenges of prognostic studies of spontaneous canine and feline mammary tumours and suggests standardized procedures to overcome these challenges and facilitate reproducibility and assessment of results.     

Immunohistochemical evaluation of MMP-2 and TIMP-2 in canine mammary tumours: a survival study

Canine mammary tumours (CMTs) are a very heterogeneous group of neoplasms with variable prognosis. Their aggressiveness is mainly due to their ability to invade locally and to metastasize. The degradation of extracellular matrix components is an important determinant of the invasive phenotype. The aims of this study were to analyse by immunohistochemistry and double immunofluorescence the expression of metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in eight normal canine mammary glands and 118 CMTs (24 benign, 94 malignant) and to investigate relationships with metastatic disease and survival.MMP-2 and TIMP-2 expression was higher in malignant tumours than in normal canine mammary tissue and benign tumours. The main difference between benign and malignant CMTs was the pattern of expression of both molecules: benign tumours presented TIMP-2 and MMP-2 immunoreactivity in the myoepithelial cells lining the basement membrane of tubuloalveolar structures, while malignant tumours showed mainly diffuse expression in neoplastic cells. In malignant tumours, increased TIMP-2 expression was significantly associated with the development of distant metastases, lower overall survival and lower disease-free survival. MMP-2 expression was not significantly associated to any of these parameters. These results suggest that the immunohistochemical expression of TIMP-2 is a useful prognostic factor in CMTs.     

Fine-needle aspiration biopsy of canine mammary gland tumours: a comparison between cytology and histopathology

In the current study, a total of 90 mammary neoplasms obtained from 55 female dogs were used to determine the accuracy of fine-needle aspiration cytology in the diagnosis of canine mammary tumours and to investigate the feasibility of this technique for the differentiation of simple tumours from complex or mixed tumours. Three aspirations were performed on each mammary gland mass using a 22-gauge needle attached to a 5-ml syringe before the mammary glands were surgically excised and submitted for histopathological examination. Twenty-five (27.7%) of 90 samples were classified as insufficient/inadequate for diagnosis. Of the remaining 65 samples, six (9.2%) were benign, 51 (78.5%) were malignant tumours and 8 (12.3%) were suspicious. Histopathological examination of the 90 specimens revealed five (5.6%) benign, 84 (93.3%) malignant and one (1.1%) non-neoplastic lesion. The diagnostic accuracy, sensitivity and specificity of cytologic examination for diagnosing malignancy were 96.5%, 96.2% and 100%, respectively. However, when inadequate (n = 25) and suspicious (n = 8) samples were included, the diagnostic accuracy and sensitivity decreased to 63.3% and 60.7%, respectively, but no change was observed in the specificity. Furthermore, it was not possible to differentiate simple tumours from complex and mixed tumours because spindle cells were seen in both 28% of the simple tumours and 39.3% of the complex or mix tumours. In conclusion, we believe that fine-needle aspiration cytology of canine mammary tumours is a valuable diagnostic tool, although our results indicated lower accuracy when inadequate samples were taken into consideration.