Adenosquamous Carcinoma of the Upper Eyelid in a Dog.

A 12-year-old, male mongrel dog was presented for a 6-month history of a progressive eyelid mass of the right upper eyelid. The dog's medical history reported long-term bilateral topical application of 0.03% tacrolimus ophthalmic ointment for third eyelid plasmoma and treatment cycles with systemic corticosteroids for a long-standing atopic dermatitis. Complete physical and ophthalmologic examination of the dog as well as complete blood count, serum biochemical analysis, urinalysis, thoracic radiographs, and abdominal ultrasonography were performed. The mass involved the skin and the margin of the upper eyelid, which was ulcerated. Conjunctival hyperemia, and the thickening and partial depigmentation of the third eyelid due to plasmoma were present. The plasmoma was observed bilaterally. Histological examination of the eyelid mass showed an invasive proliferation of malignant epithelial cells with intermixing of both adenocarcinomatous and malignant squamous cell components. An eyelid adenosquamous carcinoma was diagnosed. To the best of the authors' knowledge, this is the first report on an adenosquamous carcinoma of the eyelid in a dog. Immunosuppression has been found to be a significant clinical risk factor for cutaneous adenosquamous carcinoma in humans and was considered a possible risk factor for this dog.

Correction to: Fixation of polyvinylidene fluoride (PVDF) mesh with cyanoacrylate-derived glues in a rat experimental model: histopathologic immunohistochemical and morphometric study.

Unfortunately, during the revision of the manuscript the title has been incorrectly published.

Fixation of polyvinylidene fluoride (PVDF) mesh with cyanoacrylate-derived glues in a rat experimental model: histopathologic immunohistochemical and morphometric study.

PURPOSE: The use of cyanoacrylate (CA)-based tissue adhesives for mesh fixation in abdominal hernia repair is increasing due to the fast action and bond strength of these glues. The aim of the present study was to assess tissue changes induced by different CA glues used for mesh fixation in an animal model. METHODS: Parietal defects were induced in the abdominal wall of 60 rats and repaired by polyvinylidene fluoride (PVDF) mesh fixation using different CA glues. At 1, 7, 15, and 30 days post-surgery, macroscopic and histopathological studies were performed to evaluate mesh adhesion, the presence of complications and the tissue response. RESULTS: All meshes were successfully fixed without signs of inflammatory reaction, displacement or detachment. In areas where CA adhesives were applied, the acute tissue response was limited and transient. At 7 days post-surgery, collagen fibril production around prosthetic materials was observed, and collagen maturation was achieved at 30 days post-surgery. Good mesh incorporation was detected with all three glues, but the application of Glubran-2 was associated with an early macrophagic response and the early production and maturation of collagen fibrils. CONCLUSIONS: Our study confirmed that CA tissue adhesives induced the good incorporation of prosthetic mesh within host tissue with a low incidence of complications and reduced acute tissue reaction. At 30 days post-surgery no signs of mesh disinsertion or migration were observed, the prosthetic mesh adhesion was due to the presence of a dense mature connective tissue rich in type I collagen fibres.

Overexpression of HER-2 via immunohistochemistry in canine urinary bladder transitional cell carcinoma - A marker of malignancy and possible therapeutic target.

Transitional cell carcinoma (TCC) is the most commonly diagnosed neoplasm in the urinary bladder. Distant metastases to the regional lymph nodes, lungs, abdominal organs or bones are noted in up to 50% of dogs at time of death. Surgical excision is often not practical as TCC typically involve the trigone of the bladder and/or occurs multifocally throughout the bladder with field cancerization. Therapeutic approaches are very challenging and the requirement to evaluate alternative therapeutic protocols that may prolong survival times in dogs bearing these tumours is compelling. We assessed the immunohistochemical expression of HER-2 in 23 cases of canine TCCs of the urinary bladder and compare it with non-neoplastic urothelium in order to evaluate a rationale for targeted therapies and gene-based vaccines. HER-2 positivity was recorded in 13/23 (56%) neoplastic lesions. The receptor was significantly overexpressed in neoplastic than in non-neoplastic samples (P = .015). According to our preliminary results, it would be of interest to further evaluate the role of HER-2 in canine TCCs as a marker of malignancy and a therapeutic target for cancer vaccine and antibodies. Moreover, the significantly different overexpression of HER-2 in TCCs than in non-neoplastic urothelium further supports to investigate its role in the progression toward malignancy of non-neoplastic lesions.

Urinary capillariosis in six dogs from Italy.

Canine urinary capillariosis is caused by the nematode Pearsonema plica. P. plica infection is seldomly detected in clinical practice mainly due to diagnostic limitations. This report describes six cases of urinary capillariosis in dogs from Italy. Recurrent cystitis was observed in one dog, whereas another patient was affected by glomerular amyloidosis. In the remaining animals, the infection was considered an incidental finding. Immature eggs of the parasite were observed with urine sediment examination in 3/6 patients. Increased awareness of the potential pathogenic role of P. plica and clinical disease presentation could help identify infected animals.

Conjunctival myxoma in a dog: clinical and histopathological features.

CASE REPORT: A 9-year-old male Bull Terrier was presented for a 3-month history of a progressive, non-painful conjunctival mass of the right eye. The mass was exophytic and located in the temporal bulbar conjunctiva. No adhesion to the sclera was detected. A presumptive clinical diagnosis of a conjunctival neoplasia was made. Complete physical and ophthalmological examinations of the dog, as well as complete blood count, serum biochemical analysis, urinalysis, thoracic radiography, echocardiography and abdominal ultrasonography, were performed. The mass was surgically removed and a double freeze-thaw cycle of cryotherapy was performed locally. Histological examination of the removed tissue showed a well-delineated, non-encapsulated mass composed of spindle cells in loose myxomatous stroma. No nuclear atypia was observed in the tumour cells. A positive Alcian blue stain confirmed the mucin origin of the stroma. Tumour cells stained positive on immunohistochemistry for vimentin and negative for cytokeratins. A diagnosis was made of conjunctival myxoma. No evidence of local recurrence or distant metastases was identified during the 24-month follow-up period. CONCLUSION: To the best of the authors' knowledge, this is the first report on a conjunctival myxoma in dogs.

Correlation Between Cyclo-oxygenase-2 and Vascular Endothelial Growth Factor Expression in Canine and Feline Squamous Cell Carcinomas.

Overexpression of cyclo-oxygenase (COX)-2 is involved in tumour growth and spread by modulating the production of angiogenic factors such as vascular endothelial growth factor (VEGF). Expression of COX-2 and VEGF was investigated immunohistochemically in 51 canine and feline cutaneous and non-cutaneous squamous cell carcinomas (SCCs) and the correlation between expression of these molecules and clinicopathological variables was evaluated. COX-2 and VEGF expression was not observed in normal skin keratinocytes. COX-2 overexpression occurred in 53% and 61% of the canine and feline SCCs, respectively. The expression of both markers was higher in cutaneous compared with non-cutaneous SCCs. In both species COX-2 and VEGF expression was correlated with the progression of the disease, but not with the presence of lymphatic invasion, tumour grading or tumour classification in the cutaneous tumours. Further study will be required to understand the role of the COX-2 pathway in angiogenesis in SCC.

Immunohistochemical Labelling for Cyclo-oxygenase-2: Does the Positive Control Guarantee Standardized Results?

Since the identification of cyclo-oxygenase-2 as a potentially important therapeutic target in veterinary oncology, numerous studies on its expression have been conducted. Unfortunately, results have been heterogeneous and conclusions are difficult to draw. We tested the ability of a defined positive control to guarantee reproducibility of results among different laboratories. Valid positive controls were defined by positivity of the renal macula densa without background labelling. Fifteen colorectal tumours and 15 oral squamous cell carcinomas were labelled immunohistochemically by six European laboratories. Slides were evaluated in blinded fashion for percentage of positive cells and labelling intensity by three pathologists, and results were analyzed statistically for reproducibility and inter-reader variability. Macula densa positivity was an insufficiently sensitive control to guarantee reproducible results for percentage of positive cells and labelling intensity. Inter-reader variability was proven statistically, making the case for image analysis or other automated quantitative evaluation techniques.

COX-2, mPGES-1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours.

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E2 (PGE2 ) tumour-promoting activity has been confirmed and its production is controlled by Cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). Evidence suggests that mPGES-1 and COX-2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX-2, mPGES-1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non-neoplastic tissues. COX-2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES-1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX-2, EP2 receptor and mPGES-1 expression was significantly higher in carcinomas than in non-neoplastic tissues and adenomas. COX-2, mPGES-1 and EP2 receptor expression was strongly associated. These findings support a role of the COX-2/PGE2 pathway in the pathogenesis of these tumours.

Validation of tissue microarray for molecular profiling of canine and feline mammary tumours.

Tissue microarray (TMA) is a high-throughput method adopted for simultaneous molecular profiling of tissue samples from large patient cohorts. The aim of this study was to validate the TMA method for the molecular classification of canine and feline mammary tumours. Twelve samples, five feline and five canine mammary tumours and two canine haemangiosarcomas, were collected. TMA construction was based on Kononen's method of extracting a cylindrical core of paraffin wax-embedded 'donor' tissue and inserting it into a 'recipient' wax block. Seven consecutive sections from each tissue array block were subjected to immunohistochemistry (IHC) using primary antibodies specific for oestrogen receptor (OR), progesterone receptor (PR), c-erbB-2, cytokeratin (CK) 5/6, CK14, CK19 and p63. The same panel of antibodies was applied to the full sections from all cases. Comparison between full sections and TMA scores revealed different results depending on the antibodies. Labelling for OR, PR, CK19 and p63 showed total concordance, c-erbB2 (score +2, +3) was concordant in nine out of ten cases, CK5/6 and CK14 in eight out of ten cases. The TMA platform preserves the molecular profile of canine and feline mammary tumour markers, representing a useful tool for rapid and cost-effective analysis for the first phenotypic screening using OR, PR and c-erbB2 antibodies. Basal cytokeratin, used for triple negative identification, shows a multifocal 'niche' expression pattern, for which IHC of the full section or multiple core array is recommended.

Immunohistochemical expression of cyclooxygenase-2 in normal, hyperplastic and neoplastic canine lymphoid tissues.

There is much interest in the potential use of selective inhibitors of cyclooxygenase (COX)-2 in combination with other cancer therapeutics. COX-2 is a key enzyme in prostaglandin synthesis and has been implicated in the pathogenesis of numerous canine and feline malignancies. There are few data on the potential role of COX-2 in the pathogenesis of canine lymphoma. The present study examined COX-2 expression in normal, hyperplastic and neoplastic canine lymphoid tissues. Immunohistochemical expression was evaluated in 12 samples of non-pathologically enlarged normal lymph nodes, 24 samples of hyperplastic lymph node and 44 samples of lymphoma (22 B-cell and 22 T-cell lymphomas). The labelling was scored semiquantitatively and a score of +2 or +3 was interpreted as overexpression. In hyperplastic lymph nodes only a few macrophages were COX-2-positive while six of the 44 lymphomas (13.6%; three B- and three T-cell lymphomas) overexpressed COX-2. These data provide a rationale for further investigation of COX-2 expression in canine lymphoma for prognostic, chemopreventive and chemotherapeutic purposes.

Molecular phenotype of primary mammary tumours and distant metastases in female dogs and cats.

Distant metastases represent a major step in the progression and fatal outcome of canine and feline mammary carcinomas. Recent studies have characterized the molecular phenotypes of mammary tumours and provided information on molecules that may allow targeted therapy in sites from which the tumours may not readily be surgically resected. Molecular phenotypes were determined immunohistochemically in three feline and two canine cases of mammary neoplasia, each presenting with multiple distant metastases. These tumours and their metastases often overexpressed the c-erbB-2 phenotype. A basal-like phenotype was found in the distant metastases from two cases. These findings suggest that canine and feline mammary tumours with distant metastases may be amenable to novel targeted therapies.

Molecular phenotype in mammary tumours of queens: correlation between primary tumour and lymph node metastasis.

The molecular characterization of mammary tumours represents a new stage in the development of effective predictive models and targeted therapies. The aim of this study was to evaluate the relationship between the molecular phenotype of a primary feline mammary tumour and that of a related lymph node metastasis. Twenty-one mammary tumour samples and their lymph node metastases were selected and evaluated immunohistochemically for expression of oestrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (c-erbB-2), cytokeratin 5/6, cytokeratin 14, cytokeratin 19 and protein 63. Mammary tumours were classified into five subtypes: luminal A, luminal B, c-erbB-2 overexpressing, basal-like and normal-like, based on an algorithm applied in both human and veterinary medicine. Concordance between the primary tumour and its lymph node metastasis was detected in 12 of 21 cases (57.1%). In the remaining nine cases (42.9%) there was discordance in the molecular profile at the two sites. Therefore, the tumour molecular profile must be evaluated in both sites in order to obtain definitive identification of the tumour profile (or profiles) and to plan an appropriate therapy.

Immunohistochemical expression of COX-2, mPGES and EP2 receptor in normal and reactive canine bone and in canine osteosarcoma.

Accumulating evidence suggests that cyclooxygenase (COX)-2 is involved in the pathogenesis of human and canine osteosarcoma. The aim of this study was to investigate the expression of COX-2 in normal, reactive and neoplastic canine bone and the events downstream to COX-2 that lead to prostaglandin E(2) (PGE(2)) production. COX-2, microsomal PGE(2) synthase-1 (mPGES-1) and the PGE(2) receptor (EP2) were assessed by immunohistochemistry in 12 samples of normal bone, 14 cases of fracture callus and 27 appendicular osteosarcomas. No immunoreactivity to COX-2, mPGES-1 or EP2 receptor was observed in normal bone. Fifty percent of reactive bone samples expressed COX-2 and 57% expressed mPGES-1 and EP2 receptor, although with weak labelling intensity. Ninety-three percent of osteosarcomas expressed COX-2, while mPGES-1 was expressed by 85% and EP2 receptor by 89% of the tumours. The data confirm that COX-2 is expressed at high level in osteosarcoma and support the use of COX-2 inhibitors to improve the response to chemotherapy. The possibility of blocking the EP2 or the selective inhibition of mPGES-1, rather than COX-2 activity, might decrease the incidence of adverse effects that occur due to the inhibition of prostanoids other than PGE(2).

Canine invasive lobular carcinoma of the mammary gland: morphological and immunohistochemical characterizations of three cases.

Invasive lobular carcinoma (ILC) represents 15% of invasive human breast tumours. This report describes the morphological and immunohistochemical features of three canine mammary tumours comparable with human ILC. These tumours were composed of a non-delimited proliferation of discrete cells infiltrating fibrous connective tissue. Multifocal in-situ carcinoma associated with invasive lesions was present. Invasive tumour cells and in-situ lesions expressed cytokeratin and CK34betaE12, but not E-cadherin. Based on these morphological and immunohistochemical characteristics, the tumours were classified as canine ILC.

Expression of vascular endothelial growth factor in canine inflammatory and non-inflammatory mammary carcinoma.

Inflammatory mammary carcinoma (IMC) is the most aggressive type of mammary tumour in the dog and has been proposed as a model for human inflammatory breast cancer. The aim of this study was to investigate angiogenesis in canine IMC by immunohistochemical assessment of the expression of vascular endothelial growth factor (VEGF). Tissues from 19 cases of IMC were compared with tissues from 27 cases of invasive mammary carcinoma without inflammation (non-IMC). Immunohistochemical expression of oestrogen receptor (ER), progesterone receptor (PR) and HER-2 receptor was also assessed. VEGF was strongly expressed in all IMCs and the percentage of VEGF-immunoreactive tumour cells was significantly higher in IMC than in non-IMC (P=0.02). There was no difference in HER-2 receptor expression between IMC and non-IMC, and no IMC expressed ER or PR. These results suggest that VEGF may contribute to the high angiogenic phenotype of canine IMC and that this expression may underlie the tendency towards local and systemic metastasis of these tumours.

Reduced PTEN protein expression and its prognostic implications in canine and feline mammary tumors.

Phosphatase and tensin homolog (PTEN) belongs to the group of gatekeeper tumor suppressor genes and is involved in multiple mechanisms leading to cellular defense against neoplastic transformation and progression. Twenty-four dogs and 17 cats were submitted to a 2-year follow-up study, and clinicopathologic features were recorded and compared with immunohistochemical PTEN staining. PTEN-negative status occurred in 33% of canine and 76% of feline mammary carcinomas. In canine mammary carcinomas, there was a significant (P < .05) correlation between loss of PTEN protein expression and simple carcinoma histotype, lymphatic vessel invasion, lymph node metastases, distant organ metastases, tumor dedifferentiation, tumor recurrence, and shorter overall survival. In feline mammary tumors, a significant correlation between loss of PTEN protein expression and lymphatic vessel invasion was found. Loss of PTEN expression could be a useful prognostic marker in canine mammary carcinomas.

Steroid hormone receptors in normal, dysplastic and neoplastic feline mammary tissues and their prognostic significance.

The expression of oestrogen-alpha and progesterone receptors was determined in 13 normal, 21 dysplastic and 53 neoplastic feline mammary tissues. Expression of the receptors was correlated with cell proliferation, as assessed by the MIB-1 immunolabelling index, and with the clinical course of the disease. The expression of oestrogen receptors was significantly higher in healthy tissues and in adenosis than in neoplastic lesions, and the levels of progesterone receptors increased in fibroadenomatous changes and in "in situ" carcinomas but decreased in invasive carcinomas. The oestrogen and progesterone receptor status of the invasive carcinomas did not correlate either with the histological parameters or with the overall survival of the cats, although the oestrogen receptor-negative tumours had a poor prognosis. Oestrogen receptor-positive neoplasms had a significantly lower MIB-1 immunolabelling index than oestrogen receptor-negative neoplasms.

COX-2 expression in canine and feline invasive mammary carcinomas: correlation with clinicopathological features and prognostic molecular markers.

Cyclooxygenase (COX)-2 is an inducible enzyme linked to tumor growth and angiogenesis. Its expression occurs in a wide range of preneoplastic and neoplastic conditions in humans, including colon and breast carcinomas. We evaluated the role of COX-2 as a mediator of angiogenesis in feline and canine invasive carcinomas (IMCs) and its role as a prognostic indicator. COX-2 expression was assessed in neoplastic samples and healthy mammary glands by immunohistochemistry, and related to the following clinicopathological parameters: age, tumor size, histologic type, tumor grading, vessel invasion, estrogen (ER) and progesterone receptor (PR) status, Ki-67, HER-2 overexpression, microvessel density (MVD), VEGF expression and overall survival (OS). In both species, COX-2 immunoreactivity was not observed in healthy tissues, whereas 96% of feline and 100% of canine invasive carcinomas scored positive. In queens, COX-2 overexpression was significantly correlated to ER-negative status (p=0.04) and to increased PR (p=0.038) expression, and angiogenesis assessed by VEGF expression (p=0.002). In bitches an increased COX-2 expression was significantly correlated to HER-2 overexpression (p=0.013) and to tumor dedifferentiation (p=0.03). In both species increased levels of COX-2 were correlated to poorer prognosis (p=0.03 in dogs and p=0.002 in cats). COX-2 is expressed in mammary tissues during tumorigenesis and its expression is associated with a poorer prognosis in bitches and queens. The correlation of COX-2 expression and angiogenesis provides support for a potential role of COX-2 inhibitors for the prevention and the treatment of feline IMCs via their anti-angiogenic properties. In the canine species, moreover, COX-2 may be important for mediating HER-2 induced mammary tumors.

The role of vascular endothelial growth factor and its receptor Flk-1/KDR in promoting tumour angiogenesis in feline and canine mammary carcinomas: a preliminary study of autocrine and paracrine loops.

Vascular Endothelial Growth Factor (VEGF) and its receptor KDR are involved in the regulation of angiogenesis and are up-regulated in a number of tumours in humans and in particular, breast cancer. We therefore evaluated the prognostic potential of the angiogenetic process in feline and canine mammary carcinomas by the immunohistochemical assessment of VEGF expression and micro vessel density (MVD) quantification and examined the interplay between VEGF and KDR. These variables were related to some relevant clinicopathological parameters and to overall survival (OS). VEGF and KDR expression were evaluated in epithelial, stromal and endothelial compartments in order to identify autocrine and/or paracrine loops. In dogs an increased VEGF expression did not show any statistical correlation with the clinicopathological parameters examined and was not correlated to a poorer prognosis. MVD was found to be significantly correlated to the histologic type (P=0.04), tumour grading (P=0.02), and to the OS (P=0.01). In cats VEGF expression was significantly correlated to tumor grading (P=0.01) and OS (P=0.03), while no significant associations were found between MVD and the other parameters. VEGF and KDR were found to be detected on the epithelial, and/or endothelial and/or stromal cells of the carcinomas in both species, suggesting indications for some possible autocrine and paracrine loops. Our results encourage further studies on the possible prognostic role of VEGF and MVD in canine and feline mammary tumours and on the role of growth factors and their receptors in promoting tumour proliferation and an "angiogenetic shift". The VEGF/KDR system may play a role in malignant transformation and tumor progression.