Histomorphological and immunohistochemical study of angiogenesis and angiogenic factors in the ovary of the mare

Cyclical ovaries of 18 mares were examined histologically and immunohistochemically for vascular endothelial growth factor A and B (VEGF A; VEGF B), angiopoietin1 and 2 (Ang1; Ang2), vascular endothelial growth factor receptor 1 and 2 (VEGF-R1; VEGF-R2), angiopoietin receptor (Tie2) and von Willebrand factor. The most intensive coexpression of the examined factors and receptors was detected in the periovulatory period, when a distinctive ovarian angiogenesis takes place, being essential for tertiary follicle maturation and for the endocrine function of the Corpus luteum. Based on the immunohistochemical results, VEGF A, Ang2, VEGF-R2 and Tie2 in particular seem to play a significant role on angiogenesis during follicular and luteal development in the mare, while Ang1 supports vessel stabilisation. The findings of luteal regression and follicular atresia showed that, in the absence of VEGF A, Ang2 and its receptor Tie2 contribute substantially to vessel regression and therefore to luteolysis and follicular atresia.     

Immunohistochemical diagnosis of canine ovarian epithelial and granulosa cell tumors.

In humans and canines, the morphology of granulosa cell tumors is extremely variable and causes diagnostic difficulties. In human pathology, immunohistochemistry has been widely used for the diagnosis of granulosa cell tumors, whereas, limited studies are present in canine species. The aim of this study was to investigate the expression of cytokeratins, vimentin, and inhibin-alpha in canine normal ovaries, epithelial ovarian tumors, and granulosa cell tumors to establish an immunohistochemical panel for the differential diagnosis of ovarian tumors. Formalin-fixed, paraffin-embedded tissue sections from 4 normal ovaries, 8 granulosa cell tumors, and 6 epithelial ovarian tumors (2 adenomas and 4 adenocarcinomas) sections were obtained and stained with hematoxylin and eosin and immunohistochemically for cytokeratin AE1/AE3, cytokeratin 7, vimentin, and inhibin-alpha. In normal ovaries, cytokeratin 7, cytokeratin AE1/AE3, and vimentin were expressed in the surface epithelium. Granulosa cells were negative for cytokeratin 7 and displayed variable expression of vimentin, cytokeratin AE1/AE3, and inhibin-alpha toward follicular maturation. Granulosa cell tumors were negative for cytokeratin 7 and positive for inhibin-alpha. Conversely, ovarian epithelial cells tumors were positive for cytokeratin 7 and negative for inhibin-alpha. Both granulosa and epithelial cell tumors displayed variable expression of vimentin. Cytokeratin AE1/AE3 was expressed by all epithelial-derived tumors and 6 of 8 granulosa cell tumors. The results of this study suggest that useful immunohistochemical markers to distinguish epithelial ovarian tumors from granulosa cell tumors are cytokeratin 7 and inhibin-alpha.     

Two atypical cases of granulosa cell tumours in Belgian Blue heifers

This paper describes two atypical cases of granulosa cell tumours (GCTs) in Belgian Blue heifers. In the first case, an 18-month-old heifer died after ovariectomy, and a granulosa-theca cell tumour was diagnosed post-mortem. The second heifer (31 months of age) was near term and was successfully treated surgically for a malignant granulosa cell tumour. Unfortunately, no further reproductive data are available, as the heifer was sold for slaughter. This is the first report describing GCTs in Belgian Blue beef cattle.     

Immunohistochemical study of hormonal receptors and cell proliferation in normal canine mammary glands and spontaneous mammary tumours

The expression of hormone receptors and their relationship to cell proliferation in six samples of normal canine mammary tissue, and 11 benign and 10 malignant mammary neoplasms from female dogs were assessed by immunohistochemistry in formalin-fixed paraffin-embedded samples, by using monoclonal antibodies against progesterone and oestrogen receptors, and nuclear antigen Ki-67 (MIB-1). Malignant tumours negative for progesterone receptors proliferated at higher rates than progesterone receptor-positive tumours, suggesting that the progression towards malignancy in spontaneous mammary tumours is accompanied by a decrease in hormonal steroid dependency. Only one malignant tumour was positive for oestrogen receptors.     

Treatment of haemoperitoneum secondary to ruptured granulosa cell tumours in two mares

This report describes two cases of successful surgical management of granulosa cell tumours (GCT) in mares presenting with haemoperitoneum (HP). Controlled abdominal drainage was initially attempted in Case 1 but was not successful. A ventral midline exploratory laparotomy allowed removal of a haemorrhaging 13 kg GCT. The mare made a full recovery and returned to normal work as a driving pony 11 months post-operatively. In Case 2 controlled abdominal drainage was followed by standing left flank laparoscopic visualisation of the bleeding ovary and transection of the ovarian pedicle by electrocautery. The GCT was then removed via a ventral midline incision due to its large size. Haemoperitoneum can be associated with GCTs and in some cases is severe enough to prompt emergency treatment. Stabilisation of the patient and removal of the haemorrhaging GCT can lead to a successful outcome.     

Review of seven cases of granulosa cell tumour of the equine ovary

This paper reviews the equine granulosa cell tumour (GCT) and describes the clinicopathological features, treatment and outcome in seven cases of GCT in mares. Mares were presented with unilateral ovarian enlargement during the 2007 to 2010 breeding seasons. The mean (sd) age of the mares was 11.7 (5.96) years. Three mares were multiparous barren, three were nulliparous and one was primigravida. Behaviour at presentation was 57 per cent anoestrus, 28 per cent with stallion-like behaviour and 14 per cent with persistent oestrus. All mares had unilateral ovarian enlargement. Six non-pregnant mares had a small and inactive contralateral ovary; the pregnant mare had a single small corpus luteum on the contralateral ovary and was at three-and-a-half months' gestation. Enlarged ovaries measured 7 cm to an estimated 30 cm in diameter. 28 per cent had a multicystic ultrasound appearance, 57 per cent were dense structures and 14 per cent were of mixed appearance. Mean concentrations of progesterone were <1 ng/ml, oestrone sulphate 3.06 (2.32) ng/ml and testosterone 0.58 (0.64) nmol/l in non-pregnant mares. Inhibin was elevated in all non-pregnant cases at 7.6 (12.45) ng/ml. The pregnant mare had concentrations of progesterone 2.5 ng/ml, oestrone sulphate 81.0 ng/ml, testosterone 1.9 nmol/l and inhibin 1.31 ng/ml. Mares demonstrating stallion-like behaviour had a significantly higher (P<0.001) testosterone concentration (1.85 [0.07] nmol/l) than those that did not (0.34 [0.26] nmol/l). Three mares underwent unilateral ovariectomy and resumed cyclic ovarian activity within nine months of surgery.     

Immunohistochemical study of the expression of E-cadherin in canine mammary tumours

To investigate the possible role of E-cadherin in canine mammary tumours 20 benign and 40 malignant tumours were evaluated by immunohistochemistry in formalin-fixed paraffin wax-embedded samples. In all the benign tumours, E-cadherin was strongly expressed at the intercellular borders of epithelial cells, but it was less strongly expressed in 17 (43 per cent) of the malignant tumours. Furthermore, poorly differentiated carcinomas were less immunoreactive for E-cadherin than moderately and well differentiated carcinomas.     

Hemoperitoneum caused by rupture of a juvenile granulosa cell tumor in an equine neonate

A neonatal foal was examined because of apparent abdominal pain and distention, anemia, and hemoperitoneum. Exploratory laparotomy was performed, and a large spherical mass, which had ruptured, was found in the area of the left ovary. Left salpingo-oophorectomy was performed. The mass was determined to be a juvenile granulosa cell tumor.     

Immunohistochemical expression of HER - 2 and Ki - 67 in granulosa cell tumor in bitches

Granulosa cell tumour, an ovarian neoplasm of stromal origin, is an important tumour related to oestrogenic dominance syndrome and cystic endometrial hyperplasia–pyometra complex. In order to analyse ovarian tumour´s malignant potential, immunohistochemical markers can be used, such as anti-HER2 and anti-Ki-67. The aim of this study was to evaluate the expression of immunohistochemical markers HER-2 and Ki-67 in granulosa cell tumour from bitches´ ovaries. In HER-2 immunomarker analysis using the HercepTest^® method, most tumours were classified as 2+ (moderate labelling). Concerning Ki-67 immunomarker, only one case was described as having a high proliferative index. An association was found between immunostained cell percentage by anti-HER-2 antibodies and high pleomorphism, represented by the pattern of follicular/trabecular tumour arrangement. There was no correlation between anti-Ki-67 and anti-HER-2 antibody immunostaining intensities, probably due to only one case with a high Ki-67 index. With an effective protocol for HER-2 and Ki-67 immunohistochemical identification in granulosa cell tumours in bitches, it was possible to characterize this neoplasm proliferation profile.     

Immunohistochemical expression of Mdm2 and p53 in canine cutaneous mast cell tumours

The objective of this study was to evaluate by immunohistochemical means nuclear reactivity for Mdm2 and p53 proteins in 71 canine cutaneous mast cell tumours. Detectable reactivity for Mdm2 was observed in 17 of 23 grade I tumours, 19 of 27 grade II tumours, and 14 of 21 grade III tumours, the grading method used was that by Patnaik et al. [Vet. Pathol., vol. 21, 1984, p. 469]. Increased reactivity for Mdm2 was detected in grade III tumours compared with grade I tumours. In contrast to Mdm2, detectable reactivity for p53 was observed in 17 tumours. Of 39 cases with moderate or marked Mdm2, 34 showed mild or no detectable p53, although only five showed moderate or marked p53. The results suggest that Mdm2 overexpression plays a crucial role in canine mast cell tumorigenesis and is consistent with the histologic grade, and its expression may be induced without p53 overexpression.     

Immunohistochemical and ultrastructural investigation of granular cell tumours in dog, cat, and horse.

Six canine, one feline and one equine granular cell tumours (GCTs) were investigated electron microscopically and immunohistochemically. The tumours were tested for reactivity with monoclonal antibodies against vimentin and desmin and with polyclonal antibodies against cytokeratin, S-100 protein, glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE). All GCTs were characterized by their PAS positive cytoplasmic granules in light microscopy, which in electron microscopy appeared as lysosome-like granules. In each case two canine GCTs were stained by the antibody against cytokeratin, vimentin and S-100 protein. Cells of the equine GCT showed reactivity with the antiserum against S-100 protein. In the feline GCT no reactivity with any of the antibodies tested was observed. These differences of the immunohistochemical reactions of GCTs suggest a nonuniform histogenesis of GCTs in domestic animals. The reactivity of the tumour cells with the antiserum against NSE is discussed.     

Immunohistochemical study of the local humoral immune system of the equine respiratory mucosa

An indirect immunoperoxidase technique was used to demonstrate both free immunoglobulin and immunoglobulin-containing plasma cells of IgG, IgA, and IgM classes in the mucosa of the equine respiratory tract. IgA-producing plasma cells predominated in the upper airways, whereas IgG-producing cells predominated in the lower respiratory tract. IgM-secreting cells were uncommon, but present in their highest numbers in the nasopharynx. Plasma cells specific for all of the immunoglobulin classes were identified in the surface epithelium, lamina propria connective tissue, glandular tissue and organised lymphoid tissue. The surface epithelium stained strongly for free IgA and IgM, but weakly for IgG, whereas glandular cells and the lamina propria connective tissue stained most strongly for IgG.     

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.     

Immunohistochemical evaluation of prostaglandin E2 and vascular endothelial growth factor in canine cutaneous mast cell tumours

Vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE-2) appear to play a critical role in tumour neovascularization. In this study, we have investigated the expression of VEGF and PGE-2 in 53 canine cutaneous mast cell tumours (MCTs). Immunohistochemistry of tissue sections revealed that VEGF and PGE-2 were expressed in all mast cell tumours studied. When the expression patterns of VEGF and PGE-2 were compared with tumour grade according to Patnaik criteria, the only significant correlation observed was between PGE-2 staining intensity and tumour pathological grade, with grade II and III tumours having higher PGE-2 staining, both in intensity and percentage of cells stained, than grade I tumours ( P < 0.05).     

Microvascularization and angiogenic activity of equine corpora lutea throughout the estrous cycle

Corpus luteum growth and endocrine function are closely dependent on the formation of new capillaries. The objectives of this study were to evaluate (i) tissue growth and microvascular development in the equine cyclic luteal structures; (ii) in vitro angiogenic activity of luteal tissues in response to luteotrophic (LH, PGE₂) and luteolytic (PGF₂) hormones and (iii) to relate data to luteal endocrinological function. Our results show that microvascular density was increased in the early and mid luteal phase, followed by a fall in the late luteal phase and a further decrease in the corpus albicans. Hyperplasia of luteal tissue increased until the mid luteal phase and it was followed by tissue regression. Luteal explants were cultured with no hormone added, or with PGF₂, LH, PGE₂, LH + PGE₂ or LH + PGF₂. Media conditioned by equine luteal tissue from different stages of the luteal phase were able to stimulate mitogenesis of bovine aortic endothelial cells (BAEC), suggesting the presence of angiogenic activity. No difference was observed among luteal structures on their mitogenic capacity, for any treatment used. Nevertheless, Late-CL conditioned-media with PGF₂ showed a significant decrease in BAEC proliferation (p < 0.05) and LH + PGF₂ a tendency to reduce mitogenesis. Thus, prostaglandin F₂ may play a role on vascular regression of the CL during the late luteal phase in the mare. These data suggest that luteal angiogenesis and vascular regression in the mare are coordinated with the development of non-vascular tissue and might be regulated by many different factors.     

Immunohistochemical characteristics of canine aortic and carotid body tumours

In an immunohistochemical study of 25 canine chemodectomas, 17 tumours were stained with antisera to neurone specific enolase and the same number were stained for synaptophysin; a single tumour was stained for S100. Staining for Ki-67 occurred in 18 cases; the Ki-67-labelling index and the intensity of immunostaining was increased in more pleomorphic and malignant tumours, as assessed on histological grounds. Immunohistochemistry did not aid in recognition of less well-differentiated tumours.     

Tumour angiogenesis,anti‐angiogenic therapy and chemotherapeutic resistance

In order for a tumour to continue to grow and disseminate, it must acquire a new blood supply. Neovascularisation can be enacted by a number of different mechanisms. This dependence of tumour progression on an augmented vascular supply has been exploited by the development of anti‐angiogenic drugs, which are designed to inhibit new blood vessel formation or disrupt existing tumour‐associated vasculature, both leading to ischaemic–hypoxic tumour cell death. However, the clinical benefits of these therapeutic approaches are frequently variable and often transient, the neoplasm sometimes being able to use other neovascularisation mechanisms to maintain its blood supply and thus evade the current anti‐angiogenic therapy. Tumours may also develop a more malignant phenotype following this treatment. Clinical outcomes may be improved by simultaneously inhibiting different angiogenic pathways, abetted by more effective drug delivery regimens such as metronomic chemotherapy and the concurrent use of other antitumour modalities.