Peripheral markers of oxidative stress in Labrador retrievers with copper-associated hepatitis.

OBJECTIVES: To evaluate biomarkers of oxidative stress in dogs with copper-associated hepatitis (CAH) as compared with healthy controls, and to evaluate if these markers correlate with hepatic copper concentrations and hepatic histopathologic features. MATERIALS AND METHODS: Prospective study. Plasma reactive metabolite concentrations, plasma antioxidant potential, and plasma and urine isoprostane concentrations were determined in Labrador retrievers with copper-associated hepatitis (n=9) as well as in breed- and sex-matched (n=9) and age- and sex-matched (n=9) healthy control populations. Possible correlations between markers of oxidative stress and hepatic histopathological features also were investigated. RESULTS: Reactive metabolites (median, range) were over twofold greater in dogs with copper-associated hepatitis (87.2 RFU/µL, 60.9 to 185.6 RFU/µL) as compared to breed- and sex-matched (38.2 RFU/µL, 22.4 to 116.8 RFU/µL) and age- and sex-matched controls (32.0 RFU/µL, 18.5 to 127.4 RFU/µL). Antioxidant potential was decreased in copper-associated hepatitis dogs (6.5 TE/µL, 5.1 to 7.7 TE/µL) as compared to breed- and sex-matched controls (8.2 TE/µL, 5.3 to 11.8 TE/µL). Both reactive metabolite concentrations and the reactive metabolite to antioxidant potential ratio were positively correlated with hepatic copper concentrations. Plasma and urine isoprostanes were variable and not significantly different between populations. CLINICAL SIGNIFICANCE: Labrador retrievers with copper-associated hepatitis have altered oxidant status. Plasma reactive metabolite concentrations and the reactive metabolite to antioxidant potential ratio could be useful biomarkers. However, neither plasma nor urine isoprostanes were useful biomarkers for copper-associated hepatitis.

Pericardial Effusion in a Dog with Pericardial Hemangiosarcoma.

An adult Jack Russel terrier dog presented for evaluation of large-volume peritoneal and pleural effusion. Echocardiography revealed scant pericardial effusion and abnormally thickened pericardium. Electrocardiography revealed complete atrioventricular block with junctional and ventricular escape beats and occasional ventricular premature complexes. Computed tomography of the thorax confirmed diffuse abnormal thickening of the pericardium, and a tentative diagnosis of constrictive-effusive pericarditis was made. The dog underwent subtotal pericardiectomy to remove the parietal pericardium and permanent epicardial pacemaker implantation to manage bradycardia. Based on pericardial histopathology and immunohistochemistry, a diagnosis of pericardial hemangiosarcoma was made. Systemic chemotherapy was initiated with doxorubicin 1 month after surgery. Despite initial improvement with chemotherapy, the dog was euthanized 4 months after surgery because of development of recurrent pleural effusion. To the author's knowledge, this is the first case report in dogs to describe isolated pericardial location of hemangiosarcoma resulting in constrictive-effusive pericarditis.

Equine Odontoclastic Tooth Resorption and Hypercementosis: Histopathologic Features.

Equine odontoclastic tooth resorption and hypercementosis (EOTRH) is a painful progressive condition of older horses that involves multiple teeth, including canines and incisors. EOTRH is uncommonly recognized by veterinary pathologists and in some cases may be misdiagnosed as cementoblastoma. The cause is unknown. The goals of this study were to describe the histopathologic features of EOTRH in 17 affected horses from the United States and to increase awareness of this condition. Samples ranged from affected tooth to the entire rostral mandible and maxilla. Affected teeth exhibited cemental hyperplasia and lysis. The marked proliferation of cementum in severe cases caused bulbous enlargement of the intra-alveolar portions of affected teeth. Several teeth contained necrotic debris, bacteria, and plant material in the regions of cemental lysis. All horses exhibited dentinal lysis in at least affected tooth, and several contained necrotic debris in these regions. Endodontic disease was often present with inflammation, lysis, necrotic debris, fibrosis, and/or a thin rim of atubular mineralized tissue in the pulp cavity. Periodontal disease was a common feature that was primarily characterized by moderate lymphoplasmacytic inflammation. Resorption with secondary hypercementosis appears to begin on the external surface of the teeth rather than within the pulp cavity. Distinguishing EOTRH from other diseases requires a complete history that includes the number and location of affected teeth, a gross description of regional hard/soft tissue health, and radiographic findings.

Beyond H&E: integration of nucleic acid-based analyses into diagnostic pathology.

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

A retrospective analysis of the efficacy of Oncept vaccine for the adjunct treatment of canine oral malignant melanoma.

Oral malignant melanoma (OMM) in the dog is often locally aggressive with a high metastatic potential and there are few treatment options that have been demonstrated to improve outcome of this disease. The purpose of this study was to determine whether adjunctive treatment with the Oncept melanoma vaccine affected the outcome of dogs with OMM that had achieved loco-regional cancer control. Medical records from 45 dogs that presented to the Animal Cancer and Imaging Center were reviewed, including 30 dogs with stage II and III disease. Dogs that received the vaccine did not achieve a greater progression-free survival, disease-free interval or median survival time than dogs that did not receive the vaccine.

Acquired proximal renal tubular dysfunction in 9 Labrador Retrievers with copper-associated hepatitis (2006-2012).

BACKGROUND: Copper-associated hepatitis (CAH) has been well described in Labrador Retrievers. However, the association of CAH with proximal renal tubular dysfunction in this breed has not been characterized. OBJECTIVES: To report clinical features, hepatic and renal histopathologic findings, tissue copper concentrations, and outcome of Labradors with CAH and proximal renal tubular disease. ANIMALS: Nine Labrador Retrievers with renal glucosuria and biopsy-confirmed CAH. METHODS: Clinical, clinicopathologic, and light microscopic findings were retrospectively reviewed. Rhodanine staining or atomic emission spectroscopy was performed on all hepatic samples and available renal tissue (4 dogs) to assess copper concentrations. RESULTS: Eight dogs had a history of polyuria and polydipsia, and all dogs had increased serum bilirubin concentrations. Five dogs had hyperchloremic metabolic acidosis. Three dogs with acidemia had paradoxical alkalinuria. All renal specimens had increased copper concentrations. Renal tubular vacuolization, degeneration, and regeneration were observed on light microscopy. Four dogs died within 10 days of diagnosis. One dog survived 2 months; 4 dogs survived more than 1 year. In long-term survivors, including 2 that did not undergo immediate copper chelation, resolution of renal tubular dysfunction occurred within weeks to months. CONCLUSIONS AND CLINICAL IMPORTANCE: Labrador Retrievers with CAH can develop clinical and laboratory evidence of renal tubular dysfunction in association with increased renal copper concentrations. Given the rarity of renal tubular disorders, detection of renal glucosuria and increased ALT activity in a Labrador Retriever is suggestive of CAH. Although renal tubular dysfunction may indicate advanced disease, successful long-term outcome is possible with a variety of therapies.

Prognostic markers for canine melanocytic neoplasms: a comparative review of the literature and goals for future investigation.

Many studies have evaluated various prognostic markers for canine melanocytic neoplasms either as primary or secondary goals; however, design, methodology, and statistical validation vary widely across these studies. The goal of this article was to evaluate and compare published canine melanocytic neoplasm studies in relation to the principals established in the Recommended Guidelines for the Conduct and Evaluation of Prognostic Studies in Veterinary Oncology. Based on this evaluation, we determined which parameters currently have the most statistically supported validity for prognostic use in canine melanocytic neoplasia. This information can also be used as part of evidence-based prospective evaluations of treatment regimens. Additionally, we highlight areas in which the current data are incomplete and that warrant further evaluation. This article represents an initiative of the American College of Veterinary Pathologists' Oncology Committee and has been reviewed and endorsed by the World Small Animal Veterinary Association.

Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior.

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples.

Differentiating between inflammatory bowel disease (IBD) and small intestinal lymphoma in cats is often difficult, especially when only endoscopic biopsy specimens are available for evaluation. However, a correct diagnosis is imperative for proper treatment and prognosis. A retrospective study was performed using surgical and endoscopic intestinal biopsy specimens from 63 cats with a history of chronic diarrhea or vomiting or weight loss. A diagnosis of lymphoma or inflammation was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone, HE-stained sections plus results of immunohistochemical labeling (IHC) for CD3e and CD79a, and HE staining, immunophenotyping, and polymerase chain reaction (PCR) results for B and/or T cell clonality. In addition, various histomorphologic parameters were evaluated for significant differences between lymphoma and IBD using Fisher's exact test. The sensitivity and specificity of each parameter in the diagnosis of lymphoma were also determined. Results of Bayesian statistical analysis demonstrated that combining histologic evaluation of small intestinal biopsy specimens with immunophenotyping and analysis of clonality of lymphoid infiltrates results in more accurate differentiation of neoplastic versus inflammatory lymphocytes. Important histologic features that differentiated intestinal lymphoma from IBD included lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. Based on the results of this study, a stepwise diagnostic algorithm that first uses histologic assessment, followed by immunophenotyping and then PCR to determine clonality of the lymphocytes, was developed to more accurately differentiate between intestinal lymphoma and IBD.

Immunohistochemical diagnosis of canine oral amelanotic melanocytic neoplasms.

Definitive diagnosis of canine oral melanocytic neoplasms is often difficult because of variability in pigmentation and cellular pleomorphism. These neoplasms can resemble carcinomas, sarcomas, and round cell neoplasms, which differ in prognosis and treatment. A variety of immunohistochemical antibodies have been used for diagnosis of melanocytic neoplasms in humans and dogs; however, sensitivity and specificity of many markers have not been determined in amelanotic melanocytic neoplasms in dogs. The authors investigated a comprehensive panel of immunohistochemical markers in 49 canine oral amelanotic melanocytic neoplasms--namely, Melan-A, PNL2, HMB-45, microphthalmia transcription factor (MiTF), S-100, tyrosine hydroxylase, tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2), and CD34. Ten well-differentiated cutaneous soft tissue spindle cell sarcomas were negative controls. Melan-A, PNL2, TRP-1, and TRP-2 were highly sensitive and 100% specific for the diagnosis of canine oral amelanotic melanocytic neoplasms. S-100 and MiTF showed high sensitivity but were less specific; that is, they also labeled a proportion of the soft tissue spindle cell sarcomas. HMB-45, tyrosinase, and tyrosine hydroxylase were 100% specific but had low sensitivities. CD34 did not label any of the melanocytic neoplasms but did label 80% of the soft tissue spindle cell sarcomas. A cost-effective and efficient immunodiagnostic cocktail containing antibodies against PNL2, Melan-A, TRP-1, and TRP-2 was created that had 100% specificity and 93.9% sensitivity in identifying canine oral amelanotic melanocytic neoplasms. The spindloid variant was the variant with the lowest sensitivity to the cocktail. The likelihood of correctly diagnosing canine oral amelanotic melanocytic neoplasms was dramatically higher when biopsy samples contained ample overlying and adjacent epithelium.

Prognostic evaluation of Ki67 threshold value in canine oral melanoma.

Oral melanoma is a common canine cancer with a historically poor prognosis. Recent evidence suggests that a subset of cases may have a more favorable outcome, defined as long-term survival in the absence of intervention other than initial surgery. Traditional histological parameters have had prognostic significance in some studies but not in others, potentially due to interobserver variation. We evaluated the prognostic utility of Ki67 immunohistochemistry in a group of 79 canine oral melanomas using a technique easily applied in a veterinary diagnostic laboratory. A threshold Ki67 value of >19.5 had a sensitivity and specificity of 87.1% and 85.4%, respectively, at predicting death or euthanasia due to melanoma by 1 year postdiagnosis. Threshold values for classical histological parameters were also identified for most cases and were >4 (>30%; sensitivity = 83.9%, specificity = 86.0%) for the nuclear atypia score and >4/10 hpfs (sensitivity = 90.3%, specificity = 84.4%) for the mitotic index. In this study, the percentages correctly classified with respect to death by 1 year postdiagnosis were comparable for Ki67 (86.1%, 68/79), the nuclear atypia score (86.3%, 63/73), and the mitotic index (86.8%, 66/76). High pigmentation (>50%) had a high negative predictive value of 90.9% (18/20), but overall, only 61.0% (47/77) of cases could be correctly classified by this parameter. Based on these results, we recommend a panel of prognostic parameters, including the nuclear atypia score, the mitotic index, Ki67, and pigmentation quantification to more accurately predict the likely outcome of canine oral melanomas.

Recommended guidelines for submission, trimming, margin evaluation, and reporting of tumor biopsy specimens in veterinary surgical pathology.

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Recommended guidelines for the conduct and evaluation of prognostic studies in veterinary oncology.

There is an increasing need for more accurate prognostic and predictive markers in veterinary oncology because of an increasing number of treatment options, the increased financial costs associated with treatment, and the emotional stress experienced by owners in association with the disease and its treatment. Numerous studies have evaluated potential prognostic and predictive markers for veterinary neoplastic diseases, but there are no established guidelines or standards for the conduct and reporting of prognostic studies in veterinary medicine. This lack of standardization has made the evaluation and comparison of studies difficult. Most important, translating these results to clinical applications is problematic. To address this issue, the American College of Veterinary Pathologists' Oncology Committee organized an initiative to establish guidelines for the conduct and reporting of prognostic studies in veterinary oncology. The goal of this initiative is to increase the quality and standardization of veterinary prognostic studies to facilitate independent evaluation, validation, comparison, and implementation of study results. This article represents a consensus statement on the conduct and reporting of prognostic studies in veterinary oncology from veterinary pathologists and oncologists from around the world. These guidelines should be considered a recommendation based on the current state of knowledge in the field, and they will need to be continually reevaluated and revised as the field of veterinary oncology continues to progress. As mentioned, these guidelines were developed through an initiative of the American College of Veterinary Pathologists' Oncology Committee, and they have been reviewed and endorsed by the World Small Animal Veterinary Association.

Detection of group C rotavirus in juvenile ferrets (Mustela putorius furo) with diarrhea by reverse transcription polymerase chain reaction: sequencing and analysis of the complete coding region of the VP6 gene.

Nine juvenile ferrets (Mustela putorius furo) with a history of diarrhea were severely dehydrated and had distended abdomens and thin-walled small intestines that contained gas and fluid. Histologically, small intestines exhibited acute superficial atrophic enteritis. Transmission electron microscopy of the small intestine showed rotavirus-like particles within apical vacuoles. Reverse transcription polymerase chain reaction (RT-PCR) was negative for group A rotavirus. A group C rotavirus-specific RT-PCR assay was developed using consensus primers designed from the alignment of VP6 gene sequences of porcine, bovine, and human strains. A 182-bp product of the VP6 gene was sequenced and showed significant similarity to group C rotavirus VP6 sequences. This strain was designated "Ferret Rota C-MSU." The entire coding sequence of VP6 was determined and compared with other rotaviruses. Ferret Rota C-MSU virus was found to be most closely related to Shintoku group C rotavirus. This is the first definitive identification of a group C rotavirus in ferrets, based upon RT-PCR, sequencing, and genetic analysis.