Canine Clitoral Carcinoma: A Clinical, Cytologic, Histopathologic, Immunohistochemical, and Ultrastructural Study.

Vaginal and vulvar tumors are uncommon in dogs. Knowledge of canine primary clitoral neoplasia is restricted to a few case reports, and only carcinomas have been reported. Cytologic and histologic features reported in the literature seem to overlap with those of canine apocrine gland anal sac adenocarcinoma (AGASA). Clinical features also recall those of canine AGASA, such as locoregional metastases and hypercalcemia of malignancy (HM). In this study, 6 cases of primary canine clitoral carcinomas (CCCs), with and without HM, were investigated by means of cytology, histopathology, electron microscopy, and immunohistochemistry for neuroendocrine markers including chromogranin A (CGA), synaptophysin (SYN), neuron-specific enolase (NSE), and S-100. In all 6 tumors, cytologic findings were consistent with malignant epithelial neoplasia of apocrine gland origin. The tumors examined were classified into 3 different histological patterns representing different degrees of differentiation: tubular, solid, and rosette type. Both CGA and SYN were mildly expressed in 2 of 6 tumors, while NSE was consistently expressed in all 6 cases. None of the tumors were S-100 positive. Transmission electron microscopy revealed electron-dense cytoplasmic granules compatible with neuroendocrine granules in all 6 cases. CCCs presented clinicopathologic features resembling AGASAs with neuroendocrine characteristics, and 2 of 6 neoplasms were considered as carcinomas with neuroendocrine differentiation and were positive for 3 neuroendocrine markers. CCCs can often present with HM, and long-term outcome is likely poor. Our study concludes that CCC seems to be a rare tumor, but it might be underestimated because of the overlapping features with AGASA. Further studies should aim to define the true incidence of this disease.

Monoclonal antibodies raised to the human FOXP3 protein can be used effectively for detecting Foxp3(+) T cells in other mammalian species.

A population of primarily CD4(+)CD25(+) regulatory T cells (Tregs), that have a critical role in maintaining the balance between tolerance and immunity, have been identified through their ability to provide protection against autoimmune disease. There is considerable interest in further exploring the role that Tregs play in autoimmune disease, cancer, and in regulating the immune response to pathogens. Currently the best single marker for labelling Tregs is the forkhead transcription factor FOXP3. Consistent with its essential functional role, sequence alignment showed that the FOXP3 protein is highly conserved across mammalian species. Lymphoid tissues were analysed for nuclear Foxp3 protein expression by immunohistochemistry to evaluate the utility of monoclonal antibodies raised to the human FOXP3 protein for labelling Foxp3(+) Tregs in other mammalian species. The T-cell specificity of those anti-FOXP3 antibodies that gave the most effective staining on each species was confirmed by double labelling with FOXP3 and CD3. Antibodies 236A/E7 and 206D/B1 showed least reactivity with other species, while 259D/C7 commonly exhibited non-specific nuclear staining of non-human lymphoid tissues. Antibodies 86D/D6, 150D/E4 and 157B/F4 are recommended as those which are most effective for labelling Foxp3(+) Tregs in studies utilising animal models.

Vincristine overdose in a cat: clinical management, use of calcium folinate, and pathological lesions.

A 6-year-old female neutered Burmese cat received a 10 times overdose (5mg/m(2)) of vincristine, administered in error. Supportive therapy, including administration of calcium folinate, was instigated within 8h. Despite treatment, the patient exhibited deterioration in renal and respiratory function and died 72 h after overdose. Necropsy was performed within 24h of death. Gross examination revealed pulmonary oedema and a pale brown liver with a prominent lobular pattern. Histological examination revealed marked apoptosis and necrosis of the bone marrow myeloid series, and mild to moderate apoptosis and necrosis of the erythroid and megakaryocyte series. Multifocal necrosis of the renal tubules, hepatocytes, and small intestinal crypt epithelium was also observed. Use of calcium folinate as a rescue therapy following vincristine overdose in humans has been previously documented. If treatment is to be successful in cases of vincristine overdose in cats, then a more complete understanding of the pathogenesis of vincristine toxicity in companion animal species is required.

Evaluation of variants of melanoma-associated antigen genes and mRNA transcripts in melanomas of dogs.

OBJECTIVE-To characterize variability in melanoma-associated antigen (MAA) genes and gene expression in melanomas of dogs. ANIMALS-18 dogs with malignant melanomas and 8 healthy control dogs. PROCEDURES-cDNA was prepared from malignant melanoma biopsy specimens and from pigmented oral mucocutaneous tissues of healthy control dogs. Genomic DNA was extracted from poorly pigmented melanomas. A PCR assay was performed by use of Melan-A, SILV, or tyrosinase-specific primers. RESULTS-Splice variants of Melan-A and SILV were identified in malignant melanomas and also in healthy pigmented tissues, whereas a tyrosinase splice variant was detected in melanoma tissues only. A short interspersed nuclear element (SINE) insertion mutation was identified in the SILV gene in 1 of 10 poorly pigmented melanomas. Six novel exonic single nucleotide polymorphisms (SNPs; 3 synonymous and 3 nonsynonymous) were detected in the tyrosinase gene, and 1 nonsynonymous exonic SNP was detected in the SILV gene. CONCLUSIONS AND CLINICAL RELEVANCE-Variants of MAA mRNA were detected in malignant melanoma tissues of dogs. The importance of MAA alternative transcripts expressed in melanomas and normal pigmented tissues was unclear, but they may have represented a means of regulating melanin synthesis. The tyrosinase splice variant was detected only in melanomas and could potentially be a tumor-specific target for immunotherapy. A SILV SINE insertion mutation was identified in a melanoma from a Great Dane, a breed known to carry this mutation (associated with merle coat color). The nonsynonymous SNPs detected in tyrosinase and SILV transcripts did not appear to affect tumor pigmentation.

Expression of cyclooxygenase-2 by equine ocular and adnexal squamous cell carcinomas.

OBJECTIVE: To investigate whether cyclooxygenase-2 (COX-2) is expressed by equine ocular and adnexal squamous cell carcinomas (SCC). METHODS: Forty-three samples of histologically confirmed cases of ocular SCC or carcinoma in situ (CIS) from 34 horses presented to the Animal Health Trust between 1992 and 2004 were subjected to a standard, two-layered, indirect immunohistochemical method using a rabbit polyclonal antihuman COX-2 antibody. Ten formalin-fixed, paraffin-embedded tissue samples taken from recognized predilection sites for SCC, from the grossly normal eyes of 10 horses euthanized for reasons unrelated to this study, were used as negative controls. Samples of equine fetal kidney were used as positive controls. Following immunolabeling, the number of normal and neoplastic epithelial cells exhibiting positive COX-2 expression was recorded along with staining intensity and distribution. RESULTS: Of 43 tumors, 34 were defined as first presentation tumors. When compared with control tissue, in which 0% (0/10) of samples expressed COX-2, significantly more of these samples with SCC (58.6%, 17/29: P = 0.002), CIS (60%, 3/5: P = 0.022) or either tumor type (58.8%, 20/34: P = 0.001) exhibited positive cytoplasmic and perinuclear immunohistochemical staining for COX-2. Of the samples exhibiting positive immunohistochemical staining, only 10% (2/20) showed staining in 2%-10% of neoplastic cells, while 90% (18/20) showed staining in 1% of neoplastic cells. About 70% (14/20) of those positively immunolabeled samples exhibited an intensity of staining greater than or equal to the staining exhibited by the equine fetal kidney positive control. CONCLUSION: Neoplastic tissue from both equine ocular SCC and CIS exhibit COX-2 expression at significantly higher levels than normal control ocular tissue. However, the percentage of cells expressing positive immunohistochemical staining is consistently low. On the basis of this study, it is unlikely that anti-COX-2 therapy would be of benefit in the treatment of equine ocular and adnexal SCC.

Canine mast cell tumors: correlation of apoptosis and proliferation markers with prognosis.

The Patnaik histologic grading system is commonly used to predict the behavior of cutaneous mast cell tumors (MCTs) in dogs, but it is less useful for grade 2 MCTs because they exhibit considerable variation in biological behavior. In this retrospective study, immunohistochemical staining for Ki-67, proliferating cell nuclear antigen (PCNA), and survivin and a standardized argyrophilic staining of nucleolar organizer regions (AgNOR) protocol were performed on 121 archived paraffin-embedded specimens of canine cutaneous MCTs, for which clinical follow-up data were available. Cox regression models indicated that the Ki-67 score (hazard ratio, 1.92; P < .001) and mean AgNOR score (hazard ratio, 2.57; P < .001) were significantly associated with Patnaik grade and survival time. A binary Ki-67 variable (cutoff point Ki-67 score = 1.8) was a significant predictor of survival for dogs with grade 2 MCTs. The estimated 1-, 2-, and 3-year survival probabilities for dogs with grade 2 MCTs and Ki-67 scores less than 1.8 were 0.92, 0.86, and 0.77, respectively (SEs, 0.08, 0.14, and 0.23, respectively; median not estimable). The corresponding survival probabilities for dogs with grade 2 MCTs and Ki-67 scores higher than 1.8 were 0.43, 0.21, and 0.21, respectively (SEs, 0.19, 0.18, and 0.18, respectively; median survival time, 395 days). No significant association was identified between survival and survivin score or PCNA score. This study shows that both mean AgNOR score and Ki-67 score are prognostic markers for canine MCTs. The Ki-67 score can be used to divide Patnaik grade 2 MCTs into 2 groups with markedly different expected survival times.

Vascular endothelial growth factor expression in canine intracranial meningiomas and association with patient survival.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a regulator of angiogenesis and vascular permeability. In human patients with meningiomas, increased VEGF expression is predictive of postsurgical recurrence. The objectives of this study were to evaluate VEGF expression in canine intracranial meningiomas and to determine whether an association between VEGF expression and patient survival existed. METHODOLOGY: Tumor tissue from 17 dogs with histologically confirmed intracranial meningiomas was obtained surgically. All dogs then were treated with radiotherapy. Immunohistochemistry was performed on 5-microm sections of paraffin-embedded tumor tissue with rabbit anti-human VEGF polyclonal antibody. The extent, intensity, and distribution of VEGF staining for each section were assessed with light microscopy by means of a semiquantitative scale. Survival was analyzed by the Kaplan-Meier procedure. Survival rates among groups were compared by log-rank tests with the significance set at P < or = .05. FINDINGS: VEGF expression was detected in all tumors, with >50% of cells staining positively in tissues from 15/17 dogs. Shorter survival times were associated with greater VEGF expression (P = .01). CONCLUSIONS: VEGF expression can be measured in canine intracranial meningiomas and may be associated with poor outcome. SIGNIFICANCE: The extent of VEGF expression in canine intracranial meningiomas may be used as a prognostic marker and suggests a potential future target for therapy.

Myeloma-related disorders in cats commonly present as extramedullary neoplasms in contrast to myeloma in human patients: 24 cases with clinical follow-up.

BACKGROUND: Myeloma-related disorders (MRD) are rare neoplasms of plasma cells. Published case reports describe a diversity of clinical presentations with confusing terminology and diagnostic criteria as a consequence of the assumption that MRD in cats are analogous to those in dogs or humans. OBJECTIVE: The aim of the study was to describe clinical, clinicopathologic and imaging findings, response to treatment, survival and possible associations with other diseases or vaccination in a large case series. A priori hypotheses were that cats with MRD commonly present with extramedullary involvement and uncommonly have radiographic bone lesions, in contrast to human patients. ANIMALS: Twenty-four cats with MRD confirmed by cytology or histopathology and immunohistochemistry. METHOD: A multicenter retrospective study was performed. RESULTS: Two types of clinical presentation were observed. The first group (n = 17) had neoplasia involving abdominal organs, bone marrow, or both. All developed systemic clinical signs and paraproteinemia. Five of 7 cats that received chemotherapy improved clinically or had decreased serum globulin concentration (median survival, 12.3 months; range, 8.5-22 months). The second group comprised 7 cats with skin masses, 2 of which were paraproteinemic and developed rapidly worsening systemic signs. In cats without systemic signs, excision of the skin masses appeared to be associated with prolonged survival (up to 2.4 years). Cats with MRD commonly presented with extramedullary involvement (67%), versus humans with MRD (5%) (P < .001), and uncommonly presented with radiographic bone lesions (8%) versus humans with MRD (80%) (P < .001). CONCLUSIONS: Radiographic bone lesions are uncommon in cats with MRD and extramedullary presentation is common, relative to human myeloma.

Association of argyrophilic nucleolar organizing regions, Ki-67, and proliferating cell nuclear antigen scores with histologic grade and survival in dogs with soft tissue sarcomas: 60 cases (1996-2002).

OBJECTIVE: To determine whether argyrophilic nucleolar organizing regions (AgNORs), Ki-67, and proliferating cell nuclear antigen (PCNA) scores were associated with histologic grade and survival in dogs with soft tissue sarcomas (STSs). DESIGN: Retrospective study. ANIMALS: 60 dogs with STSs. PROCEDURE: Medical records were examined and histologic specimens were reviewed. Tissue specimens obtained from archival materials were used to prepare sections for histologic staining for AgNOR and immunohistochemical staining for Ki-67 and PCNA labeling. Follow-up monitoring was obtained by reevaluation or telephone conversations with referring veterinarians or owners. RESULTS: 27 (45%) STSs were grade 1, 23 (38%) were grade 2, and 10 (17%) were grade 3. The mean and median AgNOR, Ki-67, and PCNA scores were determined, and significant positive associations among AgNOR and Ki-67 scores with histologic grade and mitotic score were detected. Fifty-four dogs had adequate follow-up examinations and were included in survival analysis and evaluation of prognostic factors. Overall median survival time was > 1,306 days. Twelve of 54 (22%) dogs died of tumor-related causes. Metastatic disease developed in 8 of 54 (15%) dogs. Results of univariate analysis indicated that increased mitotic score, increased AgNOR score, increased Ki-67 score, incomplete surgical margins, noncurative intent surgery, Ki-67 score greater than the median Ki-67 score, and AgNOR score greater than the median AgNOR score were prognostic factors for decreased survival time. Results of multivariate analysis indicated that increased AgNOR score was the only prognostic factor for decreased survival time. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that AgNORs and possibly Ki-67 should be routinely evaluated with histologic grading for STSs in dogs.

Extra-nodal subcutaneous Hodgkin's-like lymphoma and subsequent regression in a cat.

Hodgkin's-like lymphoma is a slow growing neoplasm, usually affecting the lymph nodes of the head and neck, which has been sporadically described in veterinary patients. This report describes the clinical and histopathological features in a 9-year-old male neutered Siamese cat that presented with a 6 week history of mid-dorsocranial swelling. Immunohistochemistry demonstrated positive staining for CD79a, paired box protein and B lymphocyte antigen-36, with variable, weak-to-moderate cytoplasmic staining for human leukocyte antigen-DR and CD18, and negative staining for antimacrophage antibody. The diagnosis based on incisional biopsy was Hodgkin's-like lymphoma; however, no evidence of neoplasia was found following wide surgical excision. This case report demonstrates two unreported items of note: the novel extranodal site of Hodgkin's-like lymphoma in a cat and tumour regression following initial biopsy. It is hypothesised that the surgical trauma of biopsying the lesion or the introduction of foreign material may have caused the regression of the neoplastic cells through induction of an anti-tumour immune or inflammatory response.

Cryptococcal pyelonephritis in a dog.

A 5-year-old castrated male Golden Retriever was evaluated for polyuria, polydipsia, and progressive regurgitation thought to be a result of bacterial pyelonephritis and megaesophagus. Bacteriologic culture of urine failed to yield clinically relevant growth, and results of a urine sediment examination were normal. With time, intention tremors and progressive neurologic dysfunction were also observed. At necropsy, a diagnosis of cryptococcal disease was confirmed histologically and immunohistochemically. Findings in the dog of this report were indicative of nephrogenic diabetes insipidus with polyuria and polydipsia caused by cryptococcal pyelonephritis. Neurologic manifestations of systemic cryptococcus infection included megaesophagus, esophageal hypomotility, and regurgitation attributed to localization of cryptococcal organisms in the brain stem in the region of the dorsal motor nucleus of the vagus nerve. To the authors' knowledge, this is the first report of polyuria secondary to cryptococcal pyelonephritis.

Ivermectin toxicosis after topical administration in dog-faced fruit bats (Cynopterus brachyotis).

Forty dog-faced fruit bats (Cynopterus brachyotis) were administered 1 drop of 1% ivermectin topically as part of a routine physical exam and deworming program, and 11 developed sudden generalized paresis. Six of the bats recovered within 24-48 hr, and the remaining 5 were presented recumbent and weak to the University of Florida Veterinary Medical Teaching Hospital. Ivermectin toxicosis was suspected, and the admitted bats died or were euthanatized within 3-5 days of the development of clinical signs despite supportive care. Three of the dead bats had renal tubular necrosis.

Unilateral squamous cell carcinoma of the renal pelvis with hydronephrosis in a cat.

A 4-year-old female neutered domestic shorthair cat was presented for evaluation of gradual onset of lethargy and anorexia. Physical examination revealed moderate abdominal distension. Investigations performed included complete blood count, serum biochemistry, urinalysis, pyelocentesis, abdominal fluid analysis, abdominal ultrasonography and exploratory celiotomy. Nephrectomy was performed on the hydronephrotic kidney and a sample of the omentum was also taken, as it was grossly abnormal. No other abnormalities were found in the remainder of the abdominal organs. Findings were consistent with unilateral hydronephrosis and squamous cell carcinoma of the renal pelvis with abdominal carcinomatosis. The patient was given supportive treatment while the results of the biopsies from the renal tissue and the omentum were pending. The patient deteriorated a short time after surgical intervention and was euthanased. This is the first report of a squamous cell carcinoma arising from the renal pelvis in a cat. A comparison with the disease presentation in humans is also discussed.

Total serum bilirubin as a negative prognostic factor in idiopathic canine chronic hepatitis.

Total serum bilirubin (TBIL) is used as a prognostic factor in chronic hepatitis (CH) in human beings. To date, the authors are unaware of any studies looking at the value of TBIL as a prognostic factor in idiopathic canine CH. The objective of the current study was to assess if TBIL is a negative prognostic factor in idiopathic canine CH, and to identify other prognostic factors. Thirty-nine dogs with histologically confirmed idiopathic CH admitted to 2 referral centers between 1999 and 2010 were included in the study. Patients with concurrent diseases that could affect TBIL or the survival time were excluded. Total serum bilirubin was measured prior to liver biopsy, and CH was diagnosed according to standardized histological criteria. Survival time was calculated from time of diagnosis to time of death or euthanasia. Cox proportional hazard analysis was performed to identify prognostic factors. The mean survival time for the 39 dogs included in the analysis was 197 days (1-2,677), and the mean total serum bilirubin was 11 µmol/l (2-265). Total serum bilirubin was statistically significantly associated with survival (odds ratio = 1.082, P = 0.047) as were weight (odds ratio = 1.028, P = 0.028) and the presence of ascites (odds ratio = 6.758, P = 0.013). The current study demonstrates that TBIL could be used as an additional prognostic factor in canine CH.

Comparison of a chlorambucil-prednisolone combination with an azathioprine-prednisolone combination for treatment of chronic enteropathy with concurrent protein-losing enteropathy in dogs: 27 cases (2007-2010).

OBJECTIVE: To compare treatment protocols for chronic enteropathy and concurrent protein-losing enteropathy that used prednisolone in conjunction with either azathioprine or chlorambucil in dogs. DESIGN: Retrospective case series. ANIMALS: 27 dogs. PROCEDURES: All dogs had hypoalbuminemia (serum albumin concentration, < 18.0 g/L) and chronic enteropathy as diagnosed via complete gastrointestinal tract investigations including intestinal biopsy. Dogs received either an azathioprine-prednisolone combination (group A; n = 13) or a chlorambucil-prednisolone combination (group C; 14). Response to treatment was assessed by evaluation of body weight gain, serum albumin concentration, and duration of primary treatment. RESULTS: No significant pretreatment differences were detected between groups for any baseline variable (signalment and weight), clinicopathologic variable (albumin, cobalamin, and folate concentrations), or histopathologic findings. After treatment, serum albumin concentration and weight gain were significantly greater in group C. Median survival time for group A dogs was 30 days (95% confidence interval, 15 to 45 days) and was not reached for group C dogs. Duration of primary treatment was positively associated with the histopathologic presence of mild lacteal dilatation and use of a chlorambucil-prednisolone combination. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that a chlorambucil-prednisolone protocol is more efficacious for treatment of chronic enteropathy and concurrent protein-losing enteropathy, compared with an azathioprine-prednisolone combination. Given these findings, a prospective randomized clinical trial is warranted.

Clinical features, survival times and COX-1 and COX-2 expression in cats with transitional cell carcinoma of the urinary bladder treated with meloxicam.

Records of 11 cats with transitional cell carcinoma of the urinary bladder, which had been treated with meloxicam, were reviewed for signalment, duration of clinical signs prior to diagnosis, results of diagnostic imaging, whether or not concurrent surgery was performed and survival. Immunohistochemical expression of cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) was assessed in the tumours of seven cats. Tumour location varied greatly. The cats had a mean age of 13 years. Three cats had a previous diagnosis of feline idiopathic cystitis of up to 2008 days duration. Ten of the cats showed clinical improvement (reduction of haematuria and/or dysuria), with a mean survival time (MST) of 311 days (range 10-1064); 1-year survival of 50%. All seven bladders assessed for COX staining were COX-1 positive and five were COX-2 positive. The MST for the COX-2-positive cats was 123 days, the MST for the COX-2-negative cases was 375 days.

Dogs really are man's best friend--canine genomics has applications in veterinary and human medicine!

In 2003, the US National Human Genome Research Institute (NHGRI) agreed to fund a project to sequence the entire genome of a boxer dog named Tasha. Although the USA is a country of dog lovers, with approximately 38 million households owning one or more dogs, why did one of the National Institutes of Health countenance the use of 30 m dollars for such a purpose? The answer is that the NHGRI recognised the value of the dog as an unrivalled model for the study of human disease. In this paper, the reasons why the dog is such a good model are examined. Examples of where the study of disease in dogs is increasing the understanding of the genetic basis of human disease, of the development of improved diagnostic assays and of the evaluation of clinical therapies are provided.

Cerebral phaeohyphomycosis caused by Cladosporium spp. in two domestic shorthair cats.

Two domestic shorthair cats presented for clinical signs related to multifocal central nervous system dysfunction. Both cats had signs of vestibular system involvement and anisocoria, and one had generalized seizure activity. Cerebrospinal fluid analysis revealed a neutrophilic pleocytosis with protein elevation in one cat and pyogranulomatous inflammation in the second. Electroencephalography and brain-stem auditory-evoked potentials in the first cat confirmed cerebral cortical and brain-stem involvement. Euthanasia was performed in both cats, and postmortem diagnoses of phaeohyphomycosis secondary to Cladosporium spp. were made based on histopathology and fungal culture in both cats.