Prognostic impact of neoadjuvant aglepristone treatment in clinicopathological parameters of progesterone receptor-positive canine mammary carcinomas.

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.

Isolation, purification, culture and characterisation of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system.

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.

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.

Widespread epithelioid angiosarcoma with ventricular wall involvement in a dog.

This paper reports on a canine angiosarcoma, presenting as an "undifferentiated metastasizing tumor". A 14-year-old female Cocker Spaniel was referred to the University of Extremadura Veterinary Clinic for clinical examination after suffering rapid deterioration, with chronic cough, anorexia and cachexia. One week after clinical examination, the dog died of right congestive heart failure and ventricular arrhythmia. Blood counts revealed lymphopenia and platelet depletion. The biochemistry profile was within normal limits, except for a drop in blood urea nitrogen. Cytological evaluation of liver and spleen biopsies revealed clustered anaplastic cells that lacked convincing tissue differentiation. Major findings at necropsy were enlarged spleen and multiple, beige to dark-red nodules ranging from 0.5 to 3 cm in diameter in the heart, lung, liver and spleen. At histological examination, multiple nests of anaplastic epithelioid cells were found in sections from all affected organs. Immunohistochemistry revealed widespread expression of CD31 and Factor VIII-related antigen. The neoplastic cells were negative for CD 18. The diagnosis of epithelioid angiosarcoma, localized in the myocardium, lung, liver and spleen was made. The primary site of the neoplasm could not be determined.

Aglepristone decreases proliferation in progesterone receptor-positive canine mammary carcinomas.

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.

Myoepithelial cell layer integrity in canine mammary carcinoma.

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.

Hypomyelination in three Weimaraner dogs.

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.

Morphological and immunohistochemical characterization of spontaneous mammary gland tumors in the guinea pig (Cavia porcellus).

Ten spontaneous mammary gland tumors affecting guinea pigs (GP) were analyzed histologically and immunohistochemically. Histologically, 3 were benign (2 simple adenomas and 1 benign mixed tumor) and 7 were malignant (1 simple solid carcinoma and 6 simple tubulopapillary carcinomas). Immunohistochemical data revealed the glandular immunoprofile of all the tumors and suggested their ductal origin on the basis of cytokeratin 20 expression. Interestingly, cytokeratin 7 was detected in basal/myoepithelial cells. Further, all tumors were positive for type alpha estrogen and progesterone receptors, suggesting a role for steroid hormones in the development of these neoplasias in GP. This article describes the morphological and immunohistochemical features of the normal mammary gland and spontaneous mammary gland tumors in GP.

Presumed primary uveal melanoma with brain extension in a dog.

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.

A retrospective analysis of radiation therapy for the treatment of feline vaccine-associated sarcoma.

We retrospectively evaluated predictive prognostic factors in 73 cats with vaccine-associated sarcoma given postsurgical curative (n = 46, most with clean margins) or coarse fractionated radiotherapy (n = 27, most with either macroscopic disease or dirty margins). The former animals displayed a median survival of 43 months and a median progression free interval (PFI) of 37 months, the latter reached a median survival of 24 months and a median PFI of 10 months. In cats undergoing coarse fractionated therapy, factors predictive of a better outcome included lack of visible mass (n = 10) as opposed to macroscopic disease (n = 17, survival: 30 versus 7 months, P = 0.025; PFI: 20 versus 4 months, P = 0.01), adjuvant chemotherapy for gross disease (n = 5/17, survival: 29 versus 5 months, P = 0.04) and a smaller number of surgeries preceding radiation therapy (coeff = 0.41, P = 0.03). The Ki67 index was not predictive for survival. We concluded that postsurgical curative and coarse fractionated radiotherapy are effective legitimate options for managing vaccine-associated sarcomas.

Expression of steroid receptors and calponin in a cervical leiomyoma in a young pig.

Thickening of the uterine cervix and bilateral ovarian cystic change was identified in a 6-month-old pig during routine abattoir inspection. Microscopically, the cervical lesion comprised a non-encapsulated mass of densely packed, large and monomorphic spindle cells within the myometrium. Immunohistochemically, the majority of these neoplastic cells expressed the cytoplasmic terminal smooth muscle differentiation marker calponin, the nuclear oestrogen receptor alpha and the progesterone receptor. The ovarian cysts were classified as follicular cysts. A diagnosis of leiomyoma of the uterine cervix with bilateral ovarian follicular cysts was made. The expression of calponin as a marker of smooth muscle differentiation in tumours of the genital tract of the pig has not previously been reported. The expression of steroid hormone receptors suggests a role for steroid hormones derived from the ovarian follicular cysts in tumourigenesis.

Bilateral retiform Sertoli-Leydig cell tumour in a bitch. Alpha-inhibin and epithelial membrane antigen as useful tools for differential diagnosis.

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.

Spontaneous trichoepithelioma in a laboratory mouse: gross, microscopic and immunohistochemical findings.

A spontaneous trichoepithelioma occurred in a Swiss OF1 outbred, four-month-old, intact, nulliparous female mouse from a breeding colony. At necropsy, the tumour was a single, well-delineated mass measuring 4.2 cm in major diameter, located in the thoracic region and had an intact haired surface. The regional lymph nodes were not enlarged and no other abnormalities were found. Microscopically, it was composed of a random admixture of budding epithelial islands and cystic structures variable in size. The epithelial islands were composed of basaloid cells. The cystic structures were lined by squamous epithelium with or without a granular cell layer and contained lamellar or amorphous keratin, as well as wide areas of matrical keratinization (ghost cells) with or without a peripheral layer of basaloid cells and calcified contents. Mitotic activity of basaloid cells was moderate to high, but nuclear or mitotic atypia were not observed. High and low molecular weight cytokeratins, profilaggrin and involucrin expression were observed in the tumour. The immunohistochemical profile of this rare type of tumour of the skin of mice, which includes a first-time description of involucrin expression, confirms the histological evidence of differentiation towards more than one segment of follicular epithelium.

Expression of maspin in mammary gland tumors of the dog.

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.

Cyclic changes of the ovarian surface epithelium in the rat.

The ovarian surface epithelium (OSE) plays pivotal roles during ovulation and postovulatory wound repair. In this paper we describe the proliferative activity of the OSE through the estrous cycle in adult cycling rats, by immunohistochemical detection of DNA-incorporated bromodeoxyuridine (BrdU). Immunohistochemical detection of estrogen receptor alpha (ERalpha) and progesterone receptor was also performed. The cycle of the OSE consists of a proliferative phase (that lasts for two consecutive estrous cycles) and a quiescent phase of variable duration. Cyclic changes in the OSE were related to the underlying ovarian structure. OSE areas covering growing follicles entered into the proliferative phase during the transition from proestrus to estrus, with the appearance of fast-growing class 1 follicles, destined to ovulate at the end of the current estrous cycle. A labeling index (after pulse-labeling BrdU treatment) of about 7% was maintained throughout the estrous cycle in parallel to follicle growth. Cumulative BrdU-labeling (after daily BrdU treatment) indicated that about 1/3 of the total OSE cell proliferation was related to follicle growth. Following ovulation, OSE cells covering newly-formed corpora lutea showed a labeling index of about 50% that decreased through metestrus and diestrus (about 13% and 3%, respectively), returning to basal levels by proestrus. Cumulative BrdU-labeling indicated that about 2/3 of the total proliferative activity was related to ovulation repair/luteinization. The remaining OSE covering ovarian stroma or structurally regressing corpora lutea of previous cycles showed negligible BrdU labeling. The equivalent proliferative activity found in the OSE covering newly-formed corpora lutea in indomethacin-treated rats lacking rupture of the OSE at the apex, demonstrated that ovulation-triggered proliferation was not dependent on the loss of integrity of the OSE at the ovulation site. OSE cells expressed ERalpha throughout the cycle, but no differential expression was found between proliferating and quiescent OSE areas. On the contrary, OSE cells did not express PR at any time of the cycle. These data indicate the existence of a cycle of the OSE, related to the cyclic changes in the underlying ovarian structure and strongly suggest that the proliferative activity of the OSE is regulated by local microenvironmental rather than by systemic factors.

Immunohistochemical expression of estrogen receptor beta in normal and tumoral canine mammary glands.

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.

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.

Lipid-rich carcinomas of the mammary gland in seven dogs: clinicopathologic and immunohistochemical features.

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.

Oncogene HER-2 in canine mammary gland carcinomas: an immunohistochemical and chromogenic in situ hybridization study.

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.