Squamous Papillomas of the Conjunctiva in Dogs: A Condition Not Associated With Papillomavirus Infection.

Papillomas of the conjunctival surface in people can be of viral or nonviral origin and are found in high association with human papillomavirus. Canine conjunctival papillomas are seldom described, and published accounts have mostly been associated with canine oral papillomavirus infection. Here, we describe conjunctival squamous papillomas that do not express papillomavirus proteins and compare them with papillomavirus-associated conjunctival papillomas. Conjunctival squamous papillomas presented a distinct histopathologic profile and lacked the cytopathic effects seen in viral papillomas. They appeared as exophytic, papilliferous, pedunculated lesions with delicate fronds and angular terminal margins. Squamous papillomas presented with a delicate fibrovascular core and were associated both clinically and grossly with a feeder vessel. Pigmentation was variable within the epithelium and stroma of these lesions, and inflammatory infiltrates were characteristically minimal. Conjunctival squamous papillomas resembled squamous papillomas of the skin; however, they lacked significant hyperkeratosis. Compared with conjunctival viral papillomas, these masses occurred in older dogs and were smaller and solitary. Furthermore, polymerase chain reaction and immunohistochemistry failed to demonstrate papillomavirus genetic material and antigens in conjunctival squamous papillomas. Both viral and nonviral conjunctival papillomas were considered benign.

When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry--the red, brown, and blue technique.

Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.

Immunohistochemical expression of melanocytic antigen PNL2, Melan A, S100, and PGP 9.5 in equine melanocytic neoplasms.

The immunoreactivity of PNL2, Melan A, and protein gene product (PGP) 9.5 was compared with that of S100 protein in 50 formalin-fixed, paraffin-embedded equine melanocytic neoplasms. PNL2, PGP 9.5, and S100 protein were detected in all 50 neoplasms; none expressed Melan A. PNL2 was not expressed in 62 nonmelanocytic tumors (equine sarcoids, schwannomas, carcinomas, sarcomas, endocrine tumors, sex-cord stromal tumors, germ cell tumors, and leukocytic tumors) or in normal tissues other than epidermis. In summary, antibody PNL2 is a sensitive marker of equine melanocytic neoplasms and is more specific than S100 protein or PGP 9.5. In contrast, the monoclonal antibody to Melan A did not react with any of the equine melanomas.

Immunohistochemical evaluation of the effects of paraffin section storage on biomarker stability.

Environmental stresses can alter immunoreactivity of biomarkers in stored tissue sections. The effect of temperature and lighting on 49 cellular or microbial antigens was evaluated in 4 serial paraffin sections, cut 12 months, 10 months, 8 months, 5 months, 3 months, 1 month, 3 days, and 1 day before immunohistochemistry. Slides were stored at room temperature (RT) in the dark, at 4°C in the dark, at RT under fluorescent light, or at RT with windowpane exposure to sunlight. Immunohistochemistry was performed simultaneously in an automated immunostainer. Immunoreactivity was compared with that in the corresponding 1-day-old section and scored as 4 (<10% reduction), 3 (10%-25% reduction), 2 (26%-60% reduction), 1(>60% reduction), or 0 (no reactivity). Any loss of immunoreactivity was proportional to the tissue section age and was least in sections stored in the dark. Immunoreactivity was only completely lost in light-exposed sections and as early as 1 month for CD45. Other markers with complete loss of immunoreactivity were bovine viral diarrhea virus, CD18 (only with fluorescent light), CD31, CD68, canine parvovirus, chromogranins, and thyroid transcription factor-1. Markers with complete loss after light exposure also had reduced immunoreactivity when stored in the dark, as early as day 3. Eight markers (Bartonella spp, CD11d, high molecular weight cytokeratins, feline coronavirus, GATA-4, insulin, p63, progesterone receptor) had minimal decrease in immunoreactivity, regardless of treatment. In conclusion, light-induced antigen decay (tissue section aging) is antigen dependent and could explain unexpectedly weak or negative immunohistochemical reactions in stored paraffin sections.

Association between cigarette smoke exposure and urinary bladder cancer in Scottish terriers in a cohort study.

Canine urothelial carcinoma (UC) initially responds favorably to treatment, but is ultimately lethal in most cases. Research to identify modifiable risk factors to prevent the cancer is essential. The high breed-associated risk for UC, e.g. 20-fold higher in Scottish terriers, can facilitate this research. The objective was to identify environmental and host factors associated with UC in a cohort of Scottish terriers. Information was obtained through dog owner questionnaires for 120 Scottish terriers ≥ 6 years old participating in a bladder cancer screening study, with comparisons made between dogs that did or did not develop UC during the 3 years of screening. Univariable models were constructed, and variables with P < 0.20 were included when building the multivariable model, and then removed using a backward stepwise procedure. P < 0.05 was considered statistically significant. Urine cotinine concentrations were measured by liquid chromatography-mass spectrometry to further investigate potential cigarette smoke exposure. Biopsy-confirmed UC which was found in 32 of 120 dogs, was significantly associated with the dogs living in a household with cigarette smokers (odds ratio [OR], 6.34; 95 % confidence intervals [CI], 1.16-34.69; P = 0.033), living within a mile of a marsh or wetland (OR, 21.23; 95 % CI, 3.64-123.69; P = 0.001), and history of previous bladder infections (OR, 3.87; 95 % CI, 1.0-14.98; P = 0.050). UC was diagnosed in 18 of 51 dogs (35.3 %) with quantifiable cotinine concentrations, and six of 40 dogs (15.0 %) without quantifiable cotinine concentrations in their urine (P = 0.0165). In conclusion, the main modifiable risk factor for UC in this cohort of dogs was exposure to second-hand tobacco smoke.

Comparison of CD34, CD31, and factor VIII-related antigen immunohistochemical expression in feline vascular neoplasms and CD34 expression in feline nonvascular neoplasms.

The diagnosis of vascular neoplasms is often facilitated by the use of immunohistochemical markers such as factor VIII-related antigen, CD31, and CD34. However, the relative sensitivity and specificity of these markers have not been compared in cat vascular neoplasms. In this study, these 3 immunohistochemical markers were evaluated in 61 endothelial neoplasms (50 hemangiosarcomas and 11 hemangiomas) in 59 cats. All neoplasms were labeled by all 3 markers. CD34 had the highest average immunolabeling intensity in neoplastic endothelial cells. CD31 had the lowest average background labeling, followed by CD34 and factor VIII-related antigen, respectively. CD34 expression was also examined in 130 nonvascular neoplasms of cats; 14 of 62 epithelial neoplasms, 39 of 43 mesenchymal neoplasms, 8 of 23 leukocytic neoplasms, and 2 of 2 melanomas were positive. Given the broad expression of CD34 in mesenchymal neoplasms, this marker has limited diagnostic relevance for vascular neoplasms of cats.

Immunohistochemical identification of canine melanocytic neoplasms with antibodies to melanocytic antigen PNL2 and tyrosinase: comparison with Melan A.

The immunoreactivity of PNL2 and antityrosinase in formalin-fixed, paraffin-embedded canine melanocytic neoplasms (n = 101) was compared with that of Melan A. Of the 113 samples overall, 106 were positive for PNL2, 101 for Melan A, and 90 for tyrosinase. Six melanomas that were positive for PNL2 were negative for Melan A; 1 melanoma that was negative for PNL2 was positive for Melan A. Eighty tumors were positive for all 3 markers; 111 reacted with at least 1 the 3 antibodies. Decalcification with formic acid for up to 1 week did not affect immunoreactivity of any of the markers; however, decalcification with HCl for 1 day or 1 week notably decreased or completely abrogated immunoreactivity for Melan A and PNL2. There was only minor loss of immunoreactivity for tyrosinase in tissues decalcified with HCl for 1 week. Prolonged fixation (up to 2 months) did not affect PNL2 or tyrosinase immunoreactivity; however, Melan A immunoreactivity was reduced after 1 month of fixation. PNL2 was not expressed in 120 nonmelanocytic tumors (carcinomas, sarcomas, steroid-producing tumors, and leukocytic tumors). In summary, antibody PNL2 is slightly more sensitive than Melan A and more sensitive than tyrosinase in the identification of canine melanocytic neoplasms. Furthermore, PNL2 does not appear to cross-react with nonmelanocytic neoplasms. PNL2 is resistant to prolonged fixation but sensitive to strong decalcification. Results indicate that PNL2 is an excellent marker in the identification of canine melanomas and that the sensitivity is close to 100% when used in conjunction with Melan A and tyrosinase.

Immunohistochemical expression of E-cadherin does not distinguish canine cutaneous histiocytoma from other canine round cell tumors.

Immunohistochemistry for E-cadherin (ECAD) has been used to distinguish canine cutaneous histiocytoma from other leukocytic neoplasms ("round cell tumors"). To determine the specificity of this test, 5 types of canine cutaneous round cell tumors were evaluated for immunohistochemical expression of ECAD. Tumors of all 5 types had variable cytoplasmic, plasma membrane, and/or paranuclear ECAD expression: All 13 cutaneous histiocytomas were ECAD+; all but 1 of 14 mast cell tumors expressed ECAD; 10 of 12 epitheliotropic lymphomas reacted with E-cadherin antibody; of 72 plasmacytomas, 54 were ECAD+; and 5 of 5 histiocytic sarcomas were positive. Conclusions based on these results include the following: First, immunoreactivity for ECAD is not limited to leukocytes of cutaneous histiocytoma; second, antibody to ECAD also labels neoplastic cells in most mast cell tumors, plasmacytomas, cutaneous histiocytic sarcomas, and epitheliotropic lymphomas; third, although most histiocytomas have membranous ECAD expression, the immunoreactivity varies among round cell tumors and is frequently concurrent in different cellular compartments; fourth, the distinctively paranuclear ECAD expression pattern in epitheliotropic lymphomas might distinguish them from other round cell tumors; and, fifth, ECAD should be used with other markers (eg, MUM1 for plasmacytomas, KIT for mast cell tumors, CD3 and CD79a for lymphomas) to distinguish among canine round cell tumors.

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.

Systemic sarcocystosis in a striped skunk (Mephitis mephitis).

A striped skunk with neurological signs was euthanized and examined via necropsy. Histologically, protozoa were found in multiple tissues. Protozoal schizonts measured 15 to 25 mum in diameter and contained 4 to 6 mum crescent-shaped merozoites. Protozoa were associated with necrosis and inflammation in the lung, brain, liver, and nasal epithelium. Immunohistochemistry labeled protozoa strongly positive for Sarcocystis neurona. Polymerase chain reaction-amplified products from the protozoan were 99.6% identical to the corresponding portion of the nuclear small subunit ribosomal RNA gene of S neurona. S neurona origin was further confirmed by amplifying a 451-base pair DNA fragment from the skunk lung, which differed by just 2 or 3 base pairs from the small subunit ribosomal RNA gene of S neurona. Striped skunks act as intermediate and aberrant hosts for S neurona; however, S neurona has rarely been found in extraneural tissues in any species, and systemic sarcocystosis has not been reported in skunks. Additionally, canine distemper virus infection was confirmed with histopathology and immunohistochemistry. Concurrent canine distemper suggests that immunosuppression may have played a role in S neurona infection in this skunk.

Immunohistochemical detection of CD34, E-cadherin, claudin-1, glucose transporter 1, laminin, and protein gene product 9.5 in 28 canine and 8 feline meningiomas.

The variation in histologic pattern of meningiomas can make their diagnosis challenging. The immunohistochemical profile of 28 canine and 8 feline meningiomas was examined. Tumor types included anaplastic (6 dogs), angiomatoid (1 cat), fibroblastic (3 dogs, 1 cat), meningothelial (1 dog), microcystic (2 dogs), myxoid (3 dogs), psammomatous (4 cats), and transitional (13 dogs, 2 cats). The authors compared the expression of novel markers (CD34, E-cadherin, claudin-1, glucose transporter 1 [GLUT-1], laminin, and protein gene product [PGP] 9.5) with published markers (cytokeratins, glial fibrillary acidic protein [GFAP], progesterone receptor, S100, and vimentin). Neoplastic cells were immunohistochemically positive for vimentin in 100% of the meningiomas; CD34, 94%; GLUT-1, 86%; E-cadherin, 81%; S100, 75%; laminin, 72%; claudin-1, 60%; PGP 9.5, 55%; progesterone receptor, 44%; pancytokeratins, 39%; cytokeratins 8/18, 17%, and GFAP in 9%. Ki67 index did not correlate well with mitotic index. Based on these results and those in the human literature, immunohistochemistry for vimentin, CD34, and E-cadherin is proposed to support a diagnosis of meningioma. Immunohistochemistry for claudin-1, albeit of only moderate to low sensitivity in canine and feline meningiomas, may help to distinguish meningioma from some mesenchymal neoplasms involving the brain and associated structures, such as schwannomas, which in humans express claudin-1 poorly or not at all. Further studies with CD34, E-cadherin, and claudin-1 in canine and feline tumors that may mimic meningiomas are needed to determine the adequacy of this approach.

Immunohistochemical evaluation of GATA-4 in canine testicular tumors.

GATA-4 is a transcription factor expressed in Sertoli cells and less commonly in Leydig (interstitial) cells but not germ cells in adult human beings, cattle, pigs, and mice. We examined GATA-4 in 76 formalin-fixed, paraffin-embedded canine testicular tumors, including 21 Sertoli cell tumors (SCT), 28 Leydig (interstitial) cell tumors (LCT), 24 seminomas (GCT), and 3 mixed germ cell sex cord-stromal tumors (MGSCT). Our hypothesis was that immunohistochemistry for GATA-4 could discriminate between germ cell and sex cord-stromal tumors of the canine testis. SCTs (21/21) had strong diffuse nuclear and weak and inconsistent cytoplasmic staining. LCTs (27/28) also had strong diffuse nuclear staining and much weaker and granular cytoplasmic staining. GCTs were negative for this marker. Sex cord-stromal cells of MGSCT were also positive. These results indicate that GATA-4 is mainly expressed in sex cord-stromal tumors and not in germ cell tumors of the canine testis.

Pheochromocytoma in six New World primates.

Six New World primates, including 2 golden lion tamarins (Leontopithecus rosalia), 2 cotton-top tamarins (Saguinus o. oedipus), 1 black howler monkey (Alouatta caraya), and 1 black-handed spider monkey (Ateles g. geoffroyi), were diagnosed with unilateral (4/6) or bilateral (1/6) adrenal or extra-adrenal (1/6) pheochromocytoma by light microscopy and immunohistochemical staining for chromogranin A. Overt invasive behavior or metastases were not observed in any primate, and thus these neoplasms were considered benign. All primates either died spontaneously (4/6) or were euthanatized (2/6) as a result of concurrent malignant neoplasia, infection, renal disease, or a combination of several disease processes. Although we did not determine whether these pheochromocytomas were functional, all 6 primates had myocardial fibrosis, and some had arteriosclerosis.

Cerebellar abiotrophy in an alpaca (Lama pacos).

Cerebellar abiotrophy, a premature degeneration of cerebellar neurons, has been described in most domestic animals. Affected animals typically present with progressive neurologic signs after a variable period of postnatal normalcy. This report describes the clinical, histopathologic, and immunohistochemical (IHC) findings of cerebellar abiotrophy in an alpaca. The alpaca developed intention tremors, hypermetria, and a wide-based stance at 1.5 years of age. Histologic lesions, confined to the cerebellar vermis, included marked absence of Purkinje cells, decreased granule cells, narrowing of the molecular layer, and thinning of white matter tracts consistent with abiotrophy. Increased cell processes in the molecular layer immunolabeled for glial fibrillary acidic protein, whereas immunoreactivity for neurofilament was reduced in the molecular layer and cerebellar folia white matter. To the investigators' knowledge, this is the first report of cerebellar abiotrophy in a camelid and the first documentation of IHC findings associated with this condition.

Sequencing of the von Hippel-Lindau gene in canine renal carcinoma.

BACKGROUND: Similarities in human and canine renal cell carcinoma (RCC) epidemiology and biologic behavior suggest that molecular mechanisms of tumorigenesis may be similar in both species. Approximately 75% of RCC in people are of the clear cell subtype, up to 85% of which are associated with mutation of the von Hippel-Lindau (VHL) gene. The canine VHL coding deoxyribonucleic acid (DNA) shares 90% identity with the human VHL gene. OBJECTIVE: To determine whether or not RCC in dogs are associated with VHL mutations, and if so determine the prevalence, type, and location of these mutations. ANIMALS: Thirteen dogs with RCC, 2 dogs with primary renal sarcomas, and 10 dogs without neoplastic kidney disease. METHODS: DNA was extracted from paraffin-embedded RCC tissue; DNA extracts from paraffin-embedded and snap-frozen nonneoplastic canine kidneys and canine whole blood were used as negative controls. Polymerase chain reaction and sequencing of the 3 VHL exons was performed, and results compared with the accessioned canine sequence. RESULTS: All VHL exons were amplified from 9 of 13 canine RCC samples, both renal sarcomas, 8 of 10 nonneoplastic kidney samples, and canine whole blood; only exon 2 could be amplified from 2 RCC samples. Mutations were not identified in any exons. A maximal prevalence of 33.6% for VHL mutations in canine RCC was determined. CONCLUSION AND CLINICAL IMPORTANCE: Although similarities between canine and human RCC merit further investigation of the dog as a model for some subtypes of renal tumors, the lower prevalence of VHL mutations suggests that oncogenesis in these 2 species differs.

Cluster of cases of malignant schwannoma in cattle.

Between 1998 and 2001, several cases of ataxia and paresis followed by recumbency and death were reported in cows from different farms in a restricted area of the Argentinian Patagonia. Five cases of this cluster were studied and a diagnosis of malignant schwannoma was established. Electron microscopy (em) of tumour samples from three of the animals revealed intracytoplasmic or interstitial structures resembling retroviral particles. Attempts to isolate a viral agent from the tumours were unsuccessful but the epidemiological data and the em findings suggest a viral aetiology.

Retrospective analysis of factors affecting clinical outcome following CHOP-based chemotherapy in dogs with primary nodal diffuse large B-cell lymphoma.

Numerous factors are known to affect the prognosis of dogs with chemotherapy-treated lymphomas. However, prognostic factors for dogs with specific subtypes of lymphoma are less clearly defined. The objective of this study was to identify prognostic factors for dogs receiving CHOP-based chemotherapy for primary nodal diffuse large B-cell lymphoma (DLBCL). Medical records of dogs treated for DLBCL at the Purdue Veterinary Teaching Hospital (PUVTH) from 2006 to 2016 were reviewed. Factors potentially related to prognosis were analysed using multivariable statistical methods. Ninety-eight dogs were included in the study. Best overall response to chemotherapy was complete remission in 80 dogs (81.6%) and partial remission in 18 dogs (18.4%). Median progression-free survival (PFS) for the entire population was 252 days (range 19-1068). Factors significantly associated with achieving partial (rather than complete) remission following CHOP included presence of thrombocytopenia at diagnosis (OR 6.88; 95% CI 1.98-23.93; P = .002), baseline serum globulin concentration (OR 2.63; 95% CI 1.03-6.75; P = .044), and age at diagnosis (OR 1.36; 95% CI 1.08-1.71; P = .009). Factors significantly associated with PFS in the lowest quartile (≤93 days) included presence of thrombocytopenia at diagnosis (OR 8.72; 95% CI 1.54-49.33; P = .014), age at diagnosis (OR 1.47; 95% CI 1.12-1.94; P = .005), and baseline neutrophil count (OR 1.18; 95% CI 1.02-1.37; P = .025). Presence of thrombocytopenia, greater age, higher neutrophil count, and higher serum globulin concentration all may be associated with a particularly poor outcome in dogs receiving CHOP-based chemotherapy for DLBCL.

Immunohistochemical Profile of 20 Feline Renal Cell Carcinomas.

Renal cell carcinoma (RCC) is uncommon in cats, but makes up the majority of epithelial neoplasms in the kidney. The immunohistochemical profile of 20 feline RCCs (13 tubular carcinomas, four tubulopapillary carcinomas, one papillary carcinoma and two anaplastic carcinomas) was evaluated. Primary antibodies used were specific for Pax8, KIT, CD10, cytokeratins and vimentin. A polymer-based immunoperoxidase procedure was used. Nineteen tumours (95%) expressed Pax8; 12 (60%), KIT; 15 (75%), CD10; 20 (100%), cytokeratins; and 19 (95%), vimentin. Nuclear Pax8 immunoreactivity was readily apparent, but variation in labelling intensity was present within a given section. KIT reactivity was diffuse, cytoplasmic and relatively homogeneous. CD10 immunoreactivity was predominantly membranous along the apical border of tubular epithelial cells and was less commonly cytoplasmic. CD10 immunoreactivity was less intense in areas with papillary differentiation and absent in solid areas. Cytoplasmic cytokeratin expression was strong in 18 tumours and weak in two; the papillary portion of one tumour had distinct submembranous expression. Vimentin immunoreactivity, which ranged from diffuse to focal, was difficult to evaluate due to strong stromal immunoreactivity and its patchy expression in phenotypically similar neoplastic cells. Fewer non-renal tumours were positive for Pax8 than for CD10. Considering overall sensitivity and specificity, Pax8 appears to be a valuable marker for distinguishing feline tumours arising in the kidney from other neoplasms.