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The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in canine mammary tumours. The studied material included 10 benign and 32 malignant mammary tumours from female dogs treated with mastectomy. Primary mouse monoclonal antibodies directed against p63 protein clone 4A4 and calponin clone CALP were used in single- and doublestain system of immunohistochemical reaction. The investigations have shown that majority of myoepithelial cells in benign tumours and carcinomas in situ exhibited strong positive labelling for both markers. In other malignant tumours strong immunoreactivity was observed in resting myoepithelial cells (MECs) and hypertrophic myoepithelial cells (HMECs), while the immunoreactivity in spindle-stellate myoepithelial cells (SMECs) and rounded myoepithelial cells (RMECs) was moderate. The granular-diffuse nuclear expression of p63 protein was observed only in myoepithelial cells. In terms of calponin, diffuse cytoplasmic expression was noted not only in myoepithelial cell but also in some stromal fibroblasts and vascular smooth muscle cells. The epithelial cells did not exhibit specific expression of the investigated markers. The obtained results indicate that p63 is a sensitive and more specific marker of myoepithelial cells in canine mammary tumours compared with calponin. These findings suggest that the immunohistochemical analysis peformed with the use of p63 can be a valuable complement of routine histological examinations of canine mammary tumours facilitating identification of tumours with myoepithelial component., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Canine mammary tumours (CMTs) are one of the most common malignancies in bitches. Platelet-derived growth factor receptor (PDGFR) α and β, vascular endothelial growth factor receptor-2 (VEGFR-2) and CD117 are tyrosine kinase receptors involved in several tumours and represent suitable targets for specific therapy with toceranib phosphate. The purpose of this study was to evaluate the expression of these receptors in the pathogenesis and progression of CMTs. PDGFRα, PDGFRβ, VEGFR-2 and CD117 were expressed in 46/83 (55.4 per cent), 33/83 (39.8 per cent), 46/83 (55.4 per cent) and 32/83 (38.5 per cent) of CMTs, respectively. Immunohistochemical results showed a statistically significant loss of PDGFRα and PDGFRβ expression in simple carcinomas compared with complex/mixed carcinomas. Protein expression by western blot revealed specific bands corresponding to PDGFRα and VEGFR-2 in 3/7 and in 1/7 cell lines. Moreover, in vitro treatment showed that toceranib phosphate weakly reduced cell proliferation in one canine mammary cell line. Before considering TKR inhibitors for possible therapeutic approaches, actually further studies are necessary to evaluate the effect of these drugs on CMTs in vivo., Competing Interests: Competing interests: None declared., (© British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The receptor activator of nuclear factor-kB ( RANK) gene is found in both human and murine mammary epithelial cells and in human cancer cell lines. We analyzed RANK expression in normal and proliferative canine mammary tissue samples ( n = 47) and cell lines ( n = 10), and identified its expression in epithelial cell populations. The correlation of RANK protein with clinicopathologic parameters was also studied. A double immunohistochemical method using RANK and p63 antibodies was applied to 33 tissue samples to analyze RANK protein expression and its possible co-expression with p63 protein, the latter used to identify myoepithelial (ME) cells (p63-positive) or luminal epithelial (LE) cells (p63-negative). RANK protein expression was found in ~75% of the tissue samples analyzed, at a similar level in all of the histologic types studied: dysplasias (4 of 4, 100%), malignant tumors (13 of 17, 76%), normal glands (12 of 17, 70%), and benign tumors (6 of 9, 67%). ME and LE cells expressed RANK protein at a similar level. A higher level of RANK protein expression was found in older animals (≥10 y, p = 0.027). Quantitative RT-PCR was applied to 6 ME (1 normal and 5 neoplastic) and 4 LE (1 normal and 3 neoplastic) primary cell lines. The RANK gene was found at similar expression levels in all canine mammary ME and LE cell lines studied. We found RANK expression in normal, dysplastic, and neoplastic canine mammary tissues and cell lines, in both ME and LE cell populations.

Neoadjuvant treatment of canine mammary carcinomas with the progesterone receptor (PR) antagonist aglepristone has a PR expression-related inhibiting effect on proliferation index (PI). The aim of this study was to evaluate the effect of the treatment in the disease-free period (DFP) and overall survival (OS) of canine mammary carcinomas. Fifty female dogs with mammary carcinomas were treated with aglepristone (n = 34) or oil vehicle (n = 16) before surgery (day 15). PR expression and PI were analysed by immunohistochemistry in samples taken at days 1 and 15. Epidemiological and clinicopathological data were assessed. DFP and OS data were retrieved every 4-6 months for at least 24 months after surgery. Aglepristone treatment increased DFP of animals bearing PR+ tumours with size smaller than 3 cm, complex and mixed tumours, with histologic grades I and II, and with PI ≤ 10%. Although further studies are necessary, current evidence points to treatment with aglepristone as useful for the management of canine mammary tumours., (© 2016 John Wiley & Sons Ltd.)

Mammary tumours are the most common neoplasms of female dogs. Compared to mammary tumours of humans and cats, myoepithelial (ME) cell involvement is common in canine mammary tumours (CMT) of any subtype. Since ME cell involvement in CMT influences both histogenetic tumour classification and prognosis, correct identification of ME cells is important. This review describes immunohistochemical methods for identification of canine mammary ME cells used in vivo. In addition, phenotypic and genotypic methods to isolate ME cells for in vitro studies to analyse tumour-suppressor protein production and gene expression are discussed. The contribution of ME cells to both histogenetic classifications and the prognosis of CMT is compared with other species and the potential use of ME cells as a method to identify carcinoma in situ is discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Progesterone receptors play a key role in the development of canine mammary tumours, and recent research has focussed on their possible value as therapeutic targets using antiprogestins. Cloning and sequencing of the progesterone receptor gene has shown that the receptor has two isoforms, A and B, transcribed from a single gene. Experimental studies in human breast cancer suggest that the differential expression of progesterone receptor isoforms has implications for hormone therapy responsiveness. This study examined the effects of the antiprogestin aglepristone on cell proliferation and mRNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n = 22) or vehicle (n = 5) twice before surgery., Results: Formalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor and both isoforms by RT-qPCR and Ki67 antigen labelling. Both total progesterone receptor and isoform A mRNA expression levels decreased after treatment with aglepristone. Furthermore, a significant decrease in the proliferation index (percentage of Ki67-labelled cells) was observed in progesterone-receptor positive and isoform-A positive tumours in aglepristone-treated dogs., Conclusions: These findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A.

Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors., (© The Author(s) 2013.)

Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A 9-year-old male rottweiler was presented with abdominal distension, ascites and respiratory distress and marked bulging in the perineal region. At necropsy examination the animal had profuse ascites and hydropericardium and a multinodular mass in the right auricle of the heart infiltrating the epicardium and pericardium and metastasizing to the caudal lobe of the left lung. Microscopically and immunohistochemically the tumour was composed of neoplastic cells with muscular, cartilaginous and adipose differentiation. A diagnosis of malignant mesenchymoma with leiomyosarcomatous (≈ 50%), rhabdomyosarcomatous (≈ 30%), chondrosarcomatous (25%) and liposarcomatous (5%) components was made. Metastatic malignant mesenchymoma has not been reported previously at this site in the dog., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

14-3-3 σ protein is a negative cell cycle regulator, with both reduced and elevated levels associated with cancer in humans. This study assessed the expression of this protein in canine mammary tissues using immunohistochemistry and Western blotting. 14-3-3 σ was detected in 97% of the mammary tissue samples examined and was found in both myoepithelial (MECs) and epithelial (ECs) cells. Expression levels were elevated and reduced in neoplastic ECs and MECs, respectively (P<0.001). Intense expression of 14-3-3 σ was detected in neoplastic ECs infiltrating blood vessels and lymph nodes and suggests a possible role for this protein in the malignant transformation of mammary neoplasms. Moreover, double immunostaining for 14-3-3 σ and the MEC-specific marker p63, confirmed that 14-3-3 σ is a highly sensitive marker of MECs since all p63-positive cells were also positive for 14-3-3 σ. However, this protein is not exclusive to MECs as ECs also labelled positively., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine whether the myoepithelial (ME) cell marker calponin could be used to analyze the integrity of the ME cell layer as a means of identifying canine mammary carcinoma in situ. Tissue from 74 canine mammary lesions was evaluated (two dysplasia, eight benign tumours and 64 carcinomas including one carcinoma in situ). The 63 carcinomas included examples of histological grade 1 (n=32), grade 2 (n=23) and grade 3 (n=8). Expression of calponin was determined by immunohistochemistry. The percentage of proliferating cells surrounded by a single layer of calponin-positive cells formed the basis of classification as type I (≥ 90%), type II (70-90%) and type III (≤ 70%). Expression of Ki67 was used to determine the proliferation index (PI). The malignant tumours comprised of an approximately equal mixture of type I, II and III lesions. The two examples of dysplasia, the carcinoma in situ and two thirds of the benign tumours were classified as type I lesions. Some overlap in the level of calponin expression was observed between benign and malignant tumours. Positive correlations between the degree of calponin expression and the type of lesion (i.e. benign versus malignant; R=+0.3, P=0.08) and the histological grade of malignancy (R=+0.54, P=0.000001) were found. A negative correlation between the degree of calponin expression and PI (R=+0.027, P=0.016) was found. The ME cell marker calponin may be used as an aid in the identification of canine carcinoma in situ, but the study of the ME cell layer integrity is not definitive for the diagnosis of malignancy in canine mammary tumours., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Progesterone receptor (PR) antagonist aglepristone (RU534) has been used successfully for pregnancy termination and therapy of pyometra, vaginal tumors, and mammary hyperplasia in bitches and queens. All of these conditions share with canine mammary carcinomas the expression of PR., Objectives: To study the effect of RU534 on proliferation and apoptosis in canine mammary carcinomas in relation to PR expression., Animals: Twenty-seven nonspayed bitches with mammary carcinomas were treated with either 2 doses of 20 mg/kg RU534 (n = 22, RU534-treated group) or oil placebo (n = 5, control group) on days 1 and 8., Methods: Tumor samples were collected before (day 1) and after (day 15) treatment for immunohistochemistry. PR expression, proliferation index (PI), and apoptotic index (AI) were determined using antibodies against PR, Ki67, and cleaved lamin A/C antigens, respectively. The effect of treatment on these parameters was analyzed., Results: Differential expression of PR between day 1 (59.1% PR-positive tumors) and day 15 (36.4% PR-positive tumors) was observed in RU534-treated tumors exclusively. After RU534 treatment, mean PI was significantly decreased in PR-positive but unchanged in PR-negative RU534-treated tumors. A reduction of ≥20% in PI was found in 61.5% of RU534-treated tumors with PR expression. Conversely, no effect on AI was observed after RU534 treatment., Conclusions and Clinical Importance: Neoadjuvant RU534 treatment had PR expression-related inhibiting effects on proliferation of canine mammary carcinoma cells., (Copyright © 2011 by the American College of Veterinary Internal Medicine.)

Gliomatosis cerebri (GC) is a rare, diffusely infiltrating, glial cell tumour of neuroepithelial origin. This report describes a case of oligodendroglial GC in a 6-year-old male Poodle with central nervous system symptoms. Computed tomography revealed anomalous parenchyma density and ventricular asymmetry. Cerebrospinal fluid showed elevated protein (30 mg dL(-1)) and nucleated cell count (20 µL(-1)). Presumptive diagnosis of necrotizing meningoencephalitis was made. Because of rapid deterioration of the general condition of the animal, the dog was euthanized. Histologically there was an infiltration of round or ovoid neoplastic cells in the white matter of the left cerebral hemisphere and in leptomeninges. Immunohistochemistry showed that 80% of the neoplastic cells expressed Olig2 and some 50% expressed glial fibrilary acidic protein. On the basis of clinical, histological and immunohistochemical features, a diagnosis of oligodendoglial GC was done. This case represents the first report of a case of oligodendroglial GC in the canid., (© 2010 Blackwell Publishing Ltd.)

Hypomyelination syndrome of the Weimaraner dog is a disease characterised by a reduction or absence of myelin in the axons of the central nervous system (CNS) exclusively. The objective of this study was to analyse the cause of this deficiency of myelin. Tissue samples of the CNS of three Weimaraner dogs with neurological signs were fixed in 10% formalin and embedded in paraffin wax, and histochemical, immunohistochemical and ultrastructural studies were performed. Histochemical staining with haematoxylin and eosin and Kluver-Barrera techniques showed generalised pallor in the peripheral areas of the ventral and lateral funiculi of the spinal cord. Immunohistochemical analysis showed a weak expression of both proteolipid protein (PLP) and myelin basic protein (MBP) and a marked decrease of Olig2(+) cells in the demyelinated areas. The immunohistochemical findings suggested a myelination or remyelination failure because of the smaller population of oligodendrocytes. However, PLP gene mutations may also be the cause of the decrease of PLP expression as described in other species., (© 2010 British Small Animal Veterinary Association.)

A 13-year-old, female, mixed, cocker spaniel was examined for a unilateral exophthalmia and protruding mass in episcleral region of the right eye. Mode B ocular ultrasonography revealed a mass extended intraocular from anterior chamber to posterior pole without evidence of extraocular extension. A presumptive diagnosis of melanocytic tumour was made. A complete blood count and chemistry and thoracic radiographs did not show any abnormal changes. The recommended treatment was enucleation, and melanocytic nature of the tumour was confirmed by immunohistochemistry. Three months after surgery, the animal showed a status epilepticus refractory to treatment. Computed tomographic examination of the brain revealed changes compatible with a tumour. Cerebrospinal fluid analysis was normal. Because of the poor clinical prognosis, the owners elected to have the dog euthanased.

Sertoli-Leydig cell tumours with a retiform pattern similar to the pattern of the rete testis are a subtype of sex cord-stromal tumours recognized in the human WHO histological classification of ovarian tumours but not in the equivalent classification for domestic animals. The morphology of the tumour may be confused with that of the more common ovarian epithelial tumours. The gross, microscopical and immunohistochemical features of a canine retiform Sertoli-Leydig cell tumour and its comparison with the human counterpart are presented in this report. Both ovaries were enlarged and cystic. Microscopically, the tumour was cystic with tubulopapillary growth characterized by narrow, elongated branching tubules. Immunohistochemically, the tumour cells expressed alpha-inhibin, while epithelial membrane antigen was not detected, indicating a sex cord-stromal origin of the tumour. Additionally, the tumour cells expressed cytokeratin and vimentin in addition to oestrogen receptor alpha and progesterone receptor.

A 12-year-old, entire, nulliparous crossbreed female dog was presented with a history of vulval bleeding, bulging of the perineum and faecal tenesmus. A firm, non-painful perineal mass, measuring 9.11x5.4 cm, with erythema was detected. Abdominal radiography showed compression and elevation of the rectal ampulla. A dose of 10 mg/kg aglepristone was administered subcutaneously on days 1, 2, 8, 15, 28 and 35. An incision biopsy was taken on day 15 and immunohistochemical analysis showed that the majority of neoplastic cells expressed progesterone receptors. Both the cutaneous erythema and the faecal tenesmus had resolved by day 28. A 50 per cent reduction in size was observed by day 60 (surgical excision). This study shows that benign tumours of the vagina of the dog that contain progesterone receptors can be reduced in size in a palliative or neoadjuvant setting using the progesterone receptor antagonist aglepristone.

The expression of oestrogen receptor-alpha (ERalpha) and progesterone receptor (PR) was examined in 32 canine genital tract tumours diagnosed as smooth muscle tumours (benign or malignant, pure or mixed). The immunohistochemical expression of calponin was used to assess the smooth muscle differentiation of the tumours. Nineteen human uterine leiomyomas were also examined. Calponin expression was detected in 89.3% of canine and 100% of human genital tract tumours diagnosed as leiomyomas, as well as in the majority of other tumours examined (canine or human, genital or extragenital, benign or malignant) with the exception of canine negative control tumours (cutaneous fibroma and hepatoid gland adenoma). ERalpha was found in 56.3% of canine and 52.6% of human leiomyomas, while PR was found in 84.4% of canine and 94.7% of human tumours. These results indicate that calponin is a good marker for differentiating neoplasia of the canine genital system of uncertain origin, as in human patients. They also show that canine tumours with smooth muscle differentiation of the genital tract of the bitch express steroid hormone receptors, a finding that opens up the possibility of hormone therapy.

Maspin is a serine protease inhibitor that inhibits tumor invasion and metastasis in human breast cancer and is consistently expressed by mammary myoepithelial cells (MECs). To analyze the value of maspin as a marker of the MEC layer of the normal and tumoral canine mammary gland, the immunohistochemical expression of maspin was studied in formalin-fixed tissues from 55 benign and malignant tumors (40 tumors also contained the surrounding normal mammary gland) using a commercially available monoclonal antibody. Periacinar and periductal MECs of all 40 normal mammary glands were stained by the anti-human maspin monoclonal antibody, and immunoreactivity was observed in the nucleus and cytoplasm of these cells. In addition, maspin was found in 53 (98%) of the tumors studied, reacting with the MECs in 100% of benign tumors and 93% of malignant tumors and to the epithelial cells of 16% of benign and 73% of malignant tumors. In the MEC compartment, immunoreactivity was observed in the cytoplasm of hypertrophic MECs, fusiform MECs, stellate MECs, rounded (myoepithelial) cells, and chondroblasts. In the epithelial cell compartment, immunoreactivity was observed in the cytoplasm of cells with and without squamous differentiation. Stromal myofibroblasts were unreactive. Maspin appears to be a very sensitive marker of the normal and neoplastic myoepithelium that, contrary to smooth muscle differentiation markers, does not stain stromal myofibroblasts. In addition, a subset of neoplastic epithelial cells reacted with the maspin antibody. The relationship between maspin expression in different cellular compartments of canine mammary carcinomas and the biologic aggressiveness of the disease remains to be elucidated.

The immunohistochemically determined estrogen receptor (ER) alpha (ERalpha) and progesterone receptor (PR) status, as well as recognized, well-accepted prognostic indicators and host factors were prospectively analyzed in 84 cases of primary canine mammary carcinoma for their effect on disease-free period (recurrence free, metastasis free, or combined) (DFP) after an observation period of 18 months. The presence of one or both receptors, as well as tumor size, lymph node status, histologic grading, intravascular growth, and necrosis, were of prognostic value for DFP. In multivariate analysis, only tumor size and histologic grading proved to be independent prognosticators. None of the host factors analyzed were of prognostic value for DFP. ERalpha, PR, or both were detected in 173 out of 228 tumors: 70 ERalpha and PR; 5 ERalpha only; 98 PR only. Statistically significant differences regarding the presence of one or both receptors were observed between benign and malignant tumors and between complex, mixed, and simple histologic subtypes of benign and malignant tumors. In the group of malignant tumors (n=155), the presence of one or both receptors was more frequent in tumors smaller than 3 cm, without lymph node metastasis, with tubulopapillary rather than solid patterns of growth among simple carcinomas, of histologic grades I and II, without both intravascular growth and necrosis, and with lymphocyte cell infiltrates. The most frequent groups of hormone receptors-positive tumors were the ERalpha-positive and PR-positive group among benign and the ERalpha-negative and PR-positive group among malignant tumors.

To date, two isoforms of estrogen receptors (ER) have been identified, cloned, and characterized from several species, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). Although the presence of ERalpha has been demonstrated in normal and tumoral canine mammary tissues, the issue of ERbeta expression has not been addressed in the dog. In this study, we have analyzed the expression of ERbeta in formalin-fixed, paraffin-embedded tissue samples of nonaltered mammary gland, 30 malignant (six complex carcinoma, 12 simple carcinoma, three carcinosarcoma, and nine carcinoma or sarcoma in benign tumor), and five benign (one fibroadenoma, one complex papilloma, one complex adenoma, and two benign mixed tumors) mammary tumors of the dog by using a polyclonal ERbeta antibody and the avidin-biotin-peroxidase complex immunohistochemical technique. Our results show that high numbers of normal ductal and acinar epithelium and approximately one third of canine mammary tumors express ERbeta. This expression was higher in benign than in malignant tumors. Furthermore, expression was higher in complex and mixed histologic subtypes of malignant tumors when compared with simple subtypes.

Lipid-rich carcinomas occurred in seven female dogs. Affected dogs were purebred (all but one), intact (all but one), and between 4 and 13 years of age. Five of them had a history of parity, one had pseudopregnancy, and none had received contraceptive steroids. The tumors were single (five cases) or multiple (two cases) well-circumscribed masses of different sizes (varying from 1 to 6 cm in diameter), composed of solid nests and cords of tumor cells separated by a moderate amount of stroma. The tumor cells contained either multiple and small or large and solitary vacuoles that pushed the nucleus to the periphery of the cell (signet-ring cell). A glandular epithelial immunophenotype (cytokeratins 5 and 8 and 8 and 18) was observed in the majority of tumor cells. All tumors lacked both estrogen and progesterone receptors, and five out of seven tumors gave rise to local recurrence and proximal or distant metastases or both.

Immunohistochemical (IHC) HER-2/neu protein overexpression was found in 17.6% of canine mammary gland carcinomas, a percentage similar to that observed in human breast carcinoma, but there was no gene amplification by chromogenic in situ hybridization (CISH). Canine mammary carcinoma would be a suitable natural model of that subset of human breast carcinomas with HER-2 protein overexpression without gene amplification.

The immunohistochemical expression of the smooth muscle-specific protein calponin was studied to assess the contribution of myoepithelial cells to the histogenesis of spindle cells of complex and mixed tumors of the mammary gland of the dog and the origin of cartilage and bone in mixed tumors. Formalin-fixed tissues from 55 benign and malignant tumors (49 also containing surrounding normal mammary gland) were evaluated. Periacinar and periductal myoepithelial cells of all the 49 normal mammary glands were diffusely stained by the anti-human calponin monoclonal antibody. Calponin was found in 53 (98%) of the tumors studied, reacting with the myoepithelium-like cells of 86% of benign tumors and their remnants in 85% of malignant tumors. Five different types of calponin-immunoreactive myoepithelial cells were identified: hypertrophic myoepithelial cells. fusiform cells, stellate myoepithelial cells, rounded (myoepithelial) cells, and chondroblasts. Differences in staining intensity and staining pattern among these five types of cells suggested a transition of myoepithelial cells to chondroblasts. Stromal myofibroblasts also showed calponin immunoreactivity, but they did not react with a cytokeratin 14 monoclonal antibody, which recognizes myoepithelial cells in mammary gland. Calponin appears to be a very sensitive marker of normal and neoplastic myoepithelium in the canine mammary gland, and its identification in different cell types of complex and mixed tumors of the mammary gland of the dog suggests a major histogenetic role for myoepithelial cells.

Myxomas of the joints are extremely rare in domestic animals, only four cases, all in dogs, having been reported previously. This paper describes a myxoma originating from the synovium of the right radiocarpal joint of a mature female Dobermann pinscher with right front limb lameness. The tumour was excised surgically and no recurrence was detected during a 2-year follow-up period. Immunohistochemically, the tumour cells reacted with antibodies to vimentin and S100 protein but not with antibodies to cytokeratins (high and low molecular weight) or human callus keratin., (Copyright Harcourt Publishers Ltd.)

A cutaneous melanocytoma-acanthoma in a 2-year-old female German Shepherd Dog was characterized by the presence of two populations of neoplastic cells: epithelial and melanocytic. The epithelial component consisted of nests of well-differentiated stratified squamous epithelium closely associated with neoplastic melanocytes. The epithelial cells immunoreacted with both monoclonal and polyclonal anti-cytokeratin antibodies, and immunoreaction to S-100 protein and vimentin was observed in the melanocytic cells. This rare pigmented skin neoplasm of the dog apparently has a benign behavior.

A five-year-old crossbred dog was referred with rapidly growing masses over its penis and right popliteal lymph node. The dog had severe blepharospasm, congestion of episcleral vessels and rubeosis iridis of the left eye. A presumptive diagnosis of transmissible venereal tumour (TVT) and iridocyclitis was made based on the results of fine needle aspiration. Chemotherapy with vincristine and prednisolone was initiated and after four months the dog made a complete recovery. However, the dog subsequently relapsed, showing miosis, blepharospasm and a well defined mass within the anterior chamber of the left eye. In addition, the dog exhibited generalised 'grand mal' type seizures. Computed tomographic (CT) examination of the brain revealed two distinct masses in the left frontal lobe. Because of the poor prognosis, the owners elected to have the dog euthanased. On histopathology, metastases of TVT in the left eye and left cerebral hemisphere were found, showing no specific staining for CD3, immunoglobulin (Ig) G, IgM and lambda light chains. It was therefore concluded that the tumour growth was progressive, and that there was an absence of local humoral immune response against TVT in this case.

Forty-seven feline and 60 canine epithelial tumors were studied to test the coordinate expression of cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) using commercially available monoclonal antibodies and an avidin-biotin immunoperoxidase staining technique. Previously, the distribution of both cytokeratins was examined in normal tissues from 4 cats and 4 dogs. The pattern of distribution of CK 7 in normal tissues was similar, with minor differences, to that described in humans, whereas the reactivity pattern of CK 20 in cats and dogs was wider than that in humans. The subset of tumors strongly expressing CK 7 and CK 20 included pancreatic adenocarcinomas (100%), transitional cell carcinomas (75%), and endometrial carcinomas (67%) in the cat. None of the canine tumors had this immunophenotype. Feline (50%) and canine (56%) mammary gland carcinomas and canine cholangiocarcinomas (67%) were the only tumors presenting the CK 7 +/CK 20- immunophenotype, whereas the CK 7-/CK 20+ immunophenotype included thyroid carcinomas (100%), intestinal adenocarcinomas (60%), bronchioloalveolar carcinomas (50%), and renal carcinomas (50%) in the cat and intestinal adenocarcinomas (56%), gastric adenocarcinomas (50%), and ovarian carcinomas (50%) in the dog. The CK 7-/CK 20- immunophenotype included the rest of the analyzed tumors. The immunohistochemical evaluation of coordinate expression of both CK 7 and CK 20 in feline and canine carcinomas using monoclonal antibodies provides important information that can help to discriminate among carcinomas from different primary sites and could be particularly helpful in the determination of the primary site of origin of carcinomas presenting as metastatic disease.

The presence of apoptotic cell death was evaluated in routinely processed tissue samples of 39 neoplasms of the skin and subcutaneous tissues of the dog using the method of terminal deoxynucleotidyl transferase (T d T) mediated deoxyuridine-5'-triphosphate (d UTP)-biotin nick end labelling (TUNEL). The degree of apoptosis was related to the frequency of mitosis, an index of cell proliferation. The correlation between the apoptotic index (AI), the percentage of positive cells after randomly enumerating 1000 cells and the mitotic count (MC), the number of mitotic figures in 10 fields at a magnification of 400 times was assessed by the Spearman non-parametric correlation test. TUNEL signals were observed in all types of tumours as brown products detected in non-pyknotic nuclei, in non-identifiable rounded structures (so-called apoptotic bodies) and occasionally in the cytoplasm, either singly or in combination. An inverse relationship between AI and MC was observed in benign tumours, while no correlation was found between AI and MC in either malignant or locally invasive tumours. Among benign tumours, intracutaneous cornifying epithelioma, fibroma, haemangioma and Schwannoma had high AI and low MC, while histiocytomas had low AI and high MC and pilomatrixomas low AI and MC. All malignant tumours had low AI and high MC, except for fibrosarcomas, which had high AI and MC. Finally, higher heterogeneity was observed among locally invasive tumours, as they had high AI and low MC (squamous cell carcinomas), and low AI with either low MC (haemangiopericytomas) or high MC (basal cell tumours). The classification of the tumours according to their AI (>15.8% high and <15.8% low) and MC (>9 high, <9 low) did not reflect the clinical behaviour of some tumour types., (Copyright 1999 W.B. Saunders Company Ltd.)

The presence and distribution of S100 protein (alpha and beta subunits), cytokeratin polypeptides, glial fibrillary acidic protein, neurofilaments, vimentin, neuron specific enolase, synaptophysin, HLA class II DR antigen, and pituitary hormones (prolactin, adrenocorticotropic hormone and human chorionic gonadotrophin) in stellate cells were studied immunohistochemically in four normal canine pituitary glands, five canine pituitary adenomas, two canine pituitary carcinomas and two equine pituitary adenomas (with surrounding normal glandular tissue). Stellate cells of the pars distalis and pars intermedia of canine and equine adenohypophyses showed a strong reaction with antibodies against S100 protein subunits alpha and beta. They also reacted with antibody against high and low molecular weight cytokeratins, but not with those against other intermediate filament proteins, neuroendocrine markers, the HLA-class II DR antigen or the pituitary hormones. Other populations of cells expressing both subunits of the S100 protein were polygonal cells of the pars distalis of the adenohypophysis (horse) and marginal epithelial cells of the pars intermedia of the adenohypophysis (dog and horse). Some pituitary tumours had S100-immunoreactive cells with a distribution of alpha and beta subunits that differed between the two species. Some canine tumours (one adenoma and one carcinoma) expressed only the alpha subunit, but both of the equine adenomas expressed alpha and beta protein subunits. Some of the S100-immunoreactive tumour cells reacted with RCK-102 (cytokeratins 5+8) antibody in the dog but not in the horse. The results suggested that canine and equine stellate cells of the adenohypophysis are more closely related to epithelial than to glial cells, as is the case in cattle, sheep and goats but not human beings or mice. No subpopulation of cells of bone marrow origin could be identified among canine stellate cells, as they lack MHC class II antigen. The results also suggested that the presence of S100-immunoreactive cells is more striking in canine and equine tumours than in human tumours.

The collective immunohistochemical expression of human lysozyme, human alpha-1-antitrypsin, human CD3 antigen, calf vimentin, human keratins, human lambda light chains,canine immunoglobulins IgG, IgM, and bovine protein S-100 has been analyzed on formalin-fixed, paraffin-embedded tissue sections of 25 spontaneous canine transmissible venereal tumors (CTVT) from both genital and extragenital locations using the avidin-biotin-peroxidase complex technique. Lysozyme immunoreactivity was detected in 10/25 CTVT, alpha-1-antitrypsin in 14/25 CTVT, and vimentin in 25/25 CTVT. All CTVT cells were negative to keratins 5 + 8 of the Moll catalogue (RCK-102), S-100 protein, lambda light-chain immunoglobulins, IgG, IgM, and CD3 antigen. The intratumoral T-and B-lymphocyte infiltrate was differentiated using CD3 antigen, lambda light-chain immunoglobulins, IgG, and IgM, and this technique could be useful to evaluate the regressive or progressive growth stage of venereal tumors. Our findings support the hypothesis of a histiocytic immunophenotype for CTVT, and these staining techniques could be used in the differential diagnosis with lymphomas.

The immunohistochemical expression of the oncofetal proteins alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), and the intermediate filament proteins keratin and vimentin was analysed in 18 canine liver carcinomas. All the tumours other than hepatocellular carcinomas, with the exception of one poorly differentiated carcinoma, were AFP negative, and only cholangiocarcinomas and mixed (hepatocellular and cholangiocellular) carcinomas were CEA positive. All the histological types of tumours expressed high and low molecular weight keratins, and keratin and vimentin were both expressed in three tumours (one moderately differentiated hepatocellular carcinoma, one mixed carcinoma and one poorly differentiated carcinoma). The findings demonstrate the use of immunohistochemical staining methods for analysing the expression of some tumour markers in routinely processed tissue samples of canine liver carcinomas, and suggest that some of the tumour markers are correlated with histological types of tumour.

A tumour in an 11-year-old male crossbred Fox Terrier, showing the clinical and pathological features of epithelioid sarcoma in man is reported. Clinical aspects, location, peculiar pattern of spread and evolution, together with histopathological and immunohistological findings of this rare tumour simulating a granuloma are described. Controversial histogenesis is discussed.