Diagnostic immunohistochemistry of primary and secondary central nervous system neoplasms of dogs and cats.

The diagnosis of primary and secondary CNS neoplasms of dogs and cats relies on histologic examination of autopsy or biopsy samples. In addition, many neoplasms must be further characterized by immunohistochemistry (IHC) for a more refined diagnosis in specific cases. Given the many investigations assessing the diagnostic and prognostic IHC profile of CNS neoplasms in the veterinary literature, it may be difficult for the diagnostic pathologist or pathology trainee to narrow the list of reliable diagnostic IHCs when facing a challenging case. Here we compile a comprehensive list of the most diagnostically relevant immunomarkers that should be utilized for the diagnostic support or confirmation of the most common primary and secondary CNS neoplasms of dogs and cats.

Primary and secondary leptomeningeal gliomatosis in dogs.

Leptomeningeal gliomatosis (LG) is characterized by extensive dissemination of neoplastic glial cells in the subarachnoid space either without an intraparenchymal glioma (primary LG or PLG) or secondary to an intraparenchymal glioma (secondary LG or SLG). Given the low frequency of LG in human and veterinary medicine, specific diagnostic criteria are lacking. Here, we describe 14 cases of canine LG that were retrospectively identified from 6 academic institutions. The mean age of affected dogs was 7.3 years and over 90% of patients were brachycephalic. Clinical signs were variable and progressive. Relevant magnetic resonance image findings in 7/14 dogs included meningeal enhancement of affected areas and/or intraparenchymal masses. All affected dogs were euthanized because of the poor prognosis. Gross changes were reported in 12/14 cases and consisted mainly of gelatinous leptomeningeal thickening in the brain (6/12 cases) or spinal cord (2/12 cases) and 1 or multiple, gelatinous, gray to red intraparenchymal masses in the brain (6/12 cases). Histologically, all leptomeningeal neoplasms and intraparenchymal gliomas were morphologically consistent with oligodendrogliomas. Widespread nuclear immunolabeling for OLIG2 was observed in all neoplasms. The absence of an intraparenchymal glioma was consistent with PLG in 3 cases. The remaining 11 cases were diagnosed as SLG.

Glioma with cribriform plate involvement in 6 dogs.

Distinct patterns of local infiltration are a common feature of canine oligodendroglioma and astrocytoma, and typically involve the surrounding neuroparenchyma, ventricles, or leptomeninges. Infiltration of adjacent extraneural sites is rare and has not been well documented in veterinary medicine. Here we describe 6 canine gliomas with cribriform plate involvement (compression or infiltration) and caudal nasal invasion confirmed by neuroimaging, autopsy, and/or histology. All affected dogs were adults (9-12-y-old), and 3 were brachycephalic. Clinical signs were associated with the brain tumor, with no respiratory signs reported. Magnetic resonance imaging in 2 patients revealed a rostral intraparenchymal telencephalic mass with extension into the cribriform plate. All dogs were euthanized. Gross changes consisted of poorly demarcated, white or pale-yellow, soft, and, in oligodendrogliomas, gelatinous, intraparenchymal masses that expanded the rostral portions of the telencephalon and adhered firmly to the ethmoid bone and cribriform plate. Gliomas were classified as high-grade oligodendrogliomas (4 cases) and high-grade astrocytomas (2 cases) based on histology and immunohistochemistry for OLIG2 and GFAP. In all cases, there was evidence of cribriform plate invasion and, in one case, additional invasion of the caudal nasal cavity.

Rare diagnosis of intraosseous lipoma in the scapula of a 15-year-old domestic shorthair cat.

Case summary: A 15-year-old male castrated domestic shorthair cat originally presented to its primary care veterinarian with a chief complaint of a multi-month history of decreased appetite, weight loss and mild weight-bearing lameness. On physical examination, over the right scapula there was mild-to-moderate muscle wasting and a palpable firm, bony mass measuring approximately 3.5 cm × 3 cm. A complete blood count, chemistry panel, urinalysis, urine culture and baseline thyroxine were clinically unremarkable. Further diagnostics included CT, which revealed a large, expansile, irregularly mineralized mass centered over the caudoventral scapula at the site of attachment of the infraspinatus muscle. Following wide surgical excision by means of complete scapulectomy the patient regained function of the limb and has been free of disease since. The clinical institution's pathology service examined the resected scapula with associated mass and diagnosed an intraosseous lipoma. Relevance and novel information: Intraosseous lipoma is a rare form of bone neoplasia that has only been reported once in the small animal veterinary literature. Histopathology, clinical signs and radiographic changes were consistent with what is described in human literature. It is hypothesized that these tumors occur because of invasively growing adipose tissue within the medullary canal following trauma. Considering the rarity of primary bone tumors in cats, intraosseous lipomas should be considered as a differential diagnosis in future cases with similar signs and history.

Feline eosinophilic sclerosing fibroplasia - a characteristic inflammatory response in sites beyond the gastrointestinal tract: case report and proposed nomenclature.

Case summary: An adult male neutered Russian Blue cat presented for a right-sided nasal mass with bilateral retropharyngeal and right mandibular lymphadenomegaly. Medial retropharyngeal lymph node excision with nasal mass biopsy revealed eosinophilic sclerosing lymphadenitis and eosinophilic and lymphoplasmacytic rhinitis, respectively. Bacterial culture of the lymph node grew Pseudomonas aeruginosa, and treatment with pradofloxacin was started. Despite initial improvement, clinical signs recurred after 9 months, and fine-needle aspirates of the right mandibular and left medial retropharyngeal lymph nodes showed eosinophilic and mastocytic infiltration. Bacterial culture of the left medial retropharyngeal lymph node grew P aeruginosa, and treatment with anti-inflammatory doses of prednisolone and, later, marbofloxacin was instituted. Relevance and novel information: This report describes a case of feline eosinophilic sclerosing lymphadenitis diagnosed outside of the abdominal cavity and is the first case reported to be associated with P aeruginosa. Feline eosinophilic sclerosing lymphadenitis should be considered as a differential for lymphadenopathy occurring in areas other than the abdominal cavity. Feline eosinophilic sclerosing lymphadenitis may develop in cats due to a species-specific inflammatory response to chronic bacterial and fungal infections.

High-field MRI using standard pulse sequences has moderate to substantial interobserver agreement and good accuracy for differentiation between intracranial extra-axial histiocytic sarcoma and meningioma in dogs.

Intracranial extra-axial histiocytic sarcoma shares common MRI features with meningioma. As histiocytic sarcoma carries a generally worse prognosis than meningioma, the ability to differentiate between these two neoplasms is of clinical value. The aim of this retrospective diagnostic accuracy and observer agreement study was to evaluate the accuracy and reliability of high-field MRI to differentiate between these two tumors, using standard pulse sequences and published MRI features. A total of 51 dogs were included (26 meningiomas and 25 histiocytic sarcomas). Magnetic resonance imaging examinations were independently assessed by three experienced board-certified radiologists, evaluating 18 imaging features. They were asked to assign each case to one of three categories (meningioma, histiocytic sarcoma, and undetermined). Agreement for the MRI diagnosis across all three reviewers was moderate (κ 0.54) while paired interobserver agreement ranged from moderate to substantial (κ 0.58-0.74) with percent agreement ranging between 86.1% and 87.7%. Overall, the probability of correctly diagnosing meningioma in a dog with this tumor ranged between 79.2% and 94.4%, and the probability of correctly diagnosing histiocytic sarcoma in a dog with this tumor ranged between 76.0% and 92.3%. The overall probability to diagnose the correct tumor, irrespective of type, ranged between 79.2% and 89.7%. Histiocytic sarcomas tended to have more extensive edema and more often had combined perilesional and distant meningeal enhancement affecting both pachy- and leptomeninges, while for meningiomas, meningeal enhancement tended to more commonly be perilesional and pachymeningeal. Imaging features that seemed more useful to make a correct diagnosis included "location/type of meningeal enhancement," "osseous changes in the adjacent neurocranium," "cystic changes," and "herniation severity."

Meningeal B Cell Clusters Correlate with Submeningeal Pathology in a Natural Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an idiopathic demyelinating disease in which meningeal inflammation correlates with accelerated disease progression. The study of meningeal inflammation in MS has been limited because of constrained access to MS brain/spinal cord specimens and the lack of experimental models recapitulating progressive MS. Unlike induced models, a spontaneously occurring model would offer a unique opportunity to understand MS immunopathogenesis and provide a compelling framework for translational research. We propose granulomatous meningoencephalomyelitis (GME) as a natural model to study neuropathological aspects of MS. GME is an idiopathic, progressive neuroinflammatory disease of young dogs with a female bias. In the GME cases examined in this study, the meninges displayed focal and disseminated leptomeningeal enhancement on magnetic resonance imaging, which correlated with heavy leptomeningeal lymphocytic infiltration. These leptomeningeal infiltrates resembled tertiary lymphoid organs containing large B cell clusters that included few proliferating Ki67+ cells, plasma cells, follicular dendritic/reticular cells, and germinal center B cell-like cells. These B cell collections were confined in a specialized network of collagen fibers associated with the expression of the lympho-organogenic chemokines CXCL13 and CCL21. Although neuroparenchymal perivascular infiltrates contained B cells, they lacked the immune signature of aggregates in the meningeal compartment. Finally, meningeal B cell accumulation correlated significantly with cortical demyelination reflecting neuropathological similarities to MS. Hence, during chronic neuroinflammation, the meningeal microenvironment sustains B cell accumulation that is accompanied by underlying neuroparenchymal injury, indicating GME as a novel, naturally occurring model to study compartmentalized neuroinflammation and the associated pathology thought to contribute to progressive MS.

Outcome, prognostic factors and histological characterization of canine gastrointestinal sarcomas.

Canine gastrointestinal sarcomas, a group of tumours that includes leiomyosarcomas (LMSAs), gastrointestinal stromal tumours (GISTs) and other rarer sarcomas, comprise about 10-30% of all gastrointestinal tumours. This study aims to characterize the histologic characteristics and clinical behaviour in order to identify prognostic factors predictive of outcome. A single institution database search for surgically treated gastrointestinal sarcomas yielded 47 cases with adequate tissue remaining for histologic analysis and 42 cases available for analysis of clinical outcome. Tumours were then prospectively evaluated for mitotic count, necrosis, haemorrhage and inflammation, as well as categorized via immunohistochemical (IHC) staining for smooth muscle actin, c-KIT and DOG-1. IHC analysis defined 32 tumours as GISTs, 14 as LMSAs and one as a sarcoma not otherwise specified. For both GISTs and LMSAs, the overall median survival time (MST) is 1024 days (range 31-1456), which did not differ statistically between tumour types (p = .92). The overall metastatic rate of GISTs in this study was 32.1% (n = 9) which was not significantly different to that of LMSAs at 15.3% (n = 2, p = .45). In multivariate analysis, mitotic count under 9 in GIST patients and complete surgical excision in all tumour types correlated with improved MST. For patients with GISTs, the intensity of c-KIT staining also correlated positively with survival, with an MST of 250 days in cases with weak staining and an MST of 1418 days in cases with moderate or strong c-KIT staining (p = .005).

The hedgehog pathway suppresses neuropathogenesis in CD4 T cell-driven inflammation.

The concerted actions of the CNS and the immune system are essential to coordinating the outcome of neuroinflammatory responses. Yet, the precise mechanisms involved in this crosstalk and their contribution to the pathophysiology of neuroinflammatory diseases largely elude us. Here, we show that the CNS-endogenous hedgehog pathway, a signal triggered as part of the host response during the inflammatory phase of multiple sclerosis and experimental autoimmune encephalomyelitis, attenuates the pathogenicity of human and mouse effector CD4 T cells by regulating their production of inflammatory cytokines. Using a murine genetic model, in which the hedgehog signalling is compromised in CD4 T cells, we show that the hedgehog pathway acts on CD4 T cells to suppress the pathogenic hallmarks of autoimmune neuroinflammation, including demyelination and axonal damage, and thus mitigates the development of experimental autoimmune encephalomyelitis. Impairment of hedgehog signalling in CD4 T cells exacerbates brain-brainstem-cerebellum inflammation and leads to the development of atypical disease. Moreover, we present evidence that hedgehog signalling regulates the pathogenic profile of CD4 T cells by limiting their production of the inflammatory cytokines granulocyte-macrophage colony-stimulating factor and interferon-γ and by antagonizing their inflammatory program at the transcriptome level. Likewise, hedgehog signalling attenuates the inflammatory phenotype of human CD4 memory T cells. From a therapeutic point of view, our study underlines the potential of harnessing the hedgehog pathway to counteract ongoing excessive CNS inflammation, as systemic administration of a hedgehog agonist after disease onset effectively halts disease progression and significantly reduces neuroinflammation and the underlying neuropathology. We thus unveil a previously unrecognized role for the hedgehog pathway in regulating pathogenic inflammation within the CNS and propose to exploit its ability to modulate this neuroimmune network as a strategy to limit the progression of ongoing neuroinflammation.

Metastatic Mixed Germ Cell Tumour with Embryonal Carcinoma and Choriocarcinoma in a Female Eurasian Harvest Mouse (Micromys minutus).

Mixed germ cell tumours occur rarely in veterinary species. This report describes a case of metastatic mixed germ cell tumour in a female Eurasian harvest mouse (Micromys minutus). The tumour was extensive in one ovary and the uterus, and was characterized by two distinct tumour cell populations with features typical of embryonal carcinoma (EC) and choriocarcinoma (CC). Metastases of CC to the lungs and liver were observed. The exact origin of the CC was unclear, but the possibility of a non-gestational CC is favoured, given the context of a mixed germ cell tumour and lack of p53 expression. EC diagnosis was confirmed by immunohistochemical labelling of CD30 and lack of immunoreactivity for c-Kit. In addition, membranous ß-catenin expression was present in the EC component, indicating an inactive Wnt/ß-catenin pathway, which is required for the maintenance of pluripotency.

Clinical outcomes, ultrastructure and immunohistochemical features of canine high-grade olfactory neuroblastoma.

Olfactory neuroblastoma (ONB) is a rare intranasal neoplasm in both dogs and humans. Similar clinical presentation and overlapping histologic and immunohistochemical features of ONB with other intranasal neoplasms can make diagnosis and treatment of intranasal neoplasia challenging. Furthermore, in part because of their rarity, there is a lack of reporting on therapeutic regimen for these neoplasms. In humans, initial debulking surgery is usually followed by radiation therapy. Here we report on the histologic, immunohistochemical, and ultrastructural characteristics of canine ONB and report on the clinical progression of cases treated with radiation therapy. In all nine canine ONB examined here, neoplastic cells were arranged in a lobular manner amidst a prominent neurofibrillary matrix and had features consistent with Grade III (high grade) ONB. The neoplastic cells demonstrated positive immunohistochemical staining for TuJ-1, a Class III beta-tubulin neuronal cytoskeletal protein, and variable staining for other markers, including chromogranin, synaptophysin, AE1/AE3 and MAP2. The longest surviving case was treated with a regimen similar to that used in humans, consisting of debulking surgery followed by definitive radiation therapy. Our study found that TuJ-1 is a useful marker for ONB and that radiation therapy, even in cases of advanced disease, may result in prolonged survival.

Neuropathology of Spontaneous Hypertensive Encephalopathy in Cats.

Pathologic features of 12 cats with naturally acquired systemic hypertension and concomitant hypertensive encephalopathy were analyzed. All cats demonstrated acute onset of signs localized to the forebrain and/or brainstem, including stupor, coma, and seizures. All cats had systemic hypertension, ranging from 160 to 300 mm Hg. Gross lesions were identified in 4 of 12 cases, including caudal herniation of the cerebrum and cerebellum, sometimes with compression of the rostral colliculus and medulla. Histologically, all cases featured bilaterally symmetrical edema of the cerebral white matter. Associated vascular lesions, especially arteriolar hyalinosis, were also observed. Concurrent lesions were chronic tubulointerstitial nephritis (11/12 cases), adenomatous hyperplasia of the thyroid gland (4 cases), hypertensive choroidal arteriopathy (6 cases), and left ventricular hypertrophy (5 cases). This study demonstrates that the typical histologic manifestation of spontaneous hypertensive encephalopathy in cats is bilaterally symmetrical edema of the subcortical cerebral white matter.

Pathology and Epidemiology of Oxalate Nephrosis in Cheetahs.

To investigate cases of acute oxalate nephrosis without evidence of ethylene glycol exposure, archived data and tissues from cheetahs ( Acinonyx jubatus) from North America ( n = 297), southern Africa ( n = 257), and France ( n = 40) were evaluated. Renal and gastrointestinal tract lesions were characterized in a subset of animals with ( n = 100) and without ( n = 165) oxalate crystals at death. Crystals were confirmed as calcium oxalate by Raman spectroscopy in 45 of 47 cheetahs tested. Crystals were present in cheetahs from 3.7 months to 15.9 years old. Cheetahs younger than 1.5 years were less likely to have oxalates than older cheetahs ( P = .034), but young cheetahs with oxalates had more oxalate crystals than older cheetahs ( P < .001). Cheetahs with oxalate crystals were more likely to have renal amyloidosis, interstitial nephritis, or colitis and less likely to have glomerular loop thickening or gastritis than those without oxalates. Crystal number was positively associated with renal tubular necrosis ( P ≤ .001), regeneration ( P = .015), and casts ( P ≤ .001) but inversely associated with glomerulosclerosis, renal amyloidosis, and interstitial nephritis. Crystal number was unrelated to the presence or absence of colitis and was lower in southern African than American and European animals ( P = .01). This study found no evidence that coexisting chronic renal disease (amyloidosis, interstitial nephritis, or glomerulosclerosis), veno-occlusive disease, gastritis, or enterocolitis contributed significantly to oxalate nephrosis. Oxalate-related renal disease should be considered as a potential cause of acute renal failure, especially in young captive cheetahs. The role of location, diet, stress, and genetic predisposition in the pathogenesis of oxalate nephrosis in cheetahs warrants further study.

Identification of a Second Raccoon-Associated Polyomavirus.

Raccoon polyomavirus 1 (RacPyV1) is the suspected cause of an outbreak of fatal brain tumors among raccoons (Procyon lotor) in the western United States. Spleen samples from Georgia raccoons were screened for polyomaviruses. Although RacPyV1 was not detected, a previously unknown polyomavirus, which we designate RacPyV2, was identified and sequenced.

BRD4 is associated with raccoon polyomavirus genome and mediates viral gene transcription and maintenance of a stem cell state in neuroglial tumour cells.

Polyomavirus infection often results in persistence of the viral genome with little or no virion production. However, infection of certain cell types can result in high viral gene transcription and either cytolysis or neoplastic transformation. While infection by polyomavirus is common in humans and many animals, major questions regarding viral persistence of most polyomaviruses remain unanswered. Specifically, identification of target cells for viral infection and the mechanisms polyomaviruses employ to maintain viral genomes within cells are important not only in ascribing causality to polyomaviruses in disease, but in understanding specific mechanisms by which they cause disease. Here, we characterize the cell of origin in raccoon polyomavirus (RacPyV)-associated neuroglial brain tumours as a neural stem cell. Moreover, we identify an association between the viral genome and the host cell bromodomain protein, BRD4, which is involved in numerous cellular functions, including cell cycle progression, differentiation of stem cells, tethering of persistent DNA viruses, and regulation of viral and host-cell gene transcription. We demonstrate that inhibition of BRD4 by the small molecule inhibitors (+)-JQ1 and IBET-151 (GSK1210151A) results in reduced RacPyV genome within cells in vitro, as well as significant reduction of viral gene transcripts LT and VP1, highlighting its importance in both maintenance of the viral genome and in driving oncogenic transformation by RacPyV. This work implicates BRD4 as a central protein involved in RacPyV neuroglial tumour cell proliferation and in the maintenance of a stem cell state.

Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV) for downstream use in virus characterization.

Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA), and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV) [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus.

Polyomavirus and Naturally Occuring Neuroglial Tumors in Raccoons (Procyon Lotor).

Polyomavirus (PyV) infections are widespread in human populations and, although generally associated with silent persistence, rarely cause severe disease. Among diseases convincingly associated with natural PyV infections of humans, there are remarkably different tissue tropisms and outcomes, including progressive multifocal leukoencephalopathy, transient or progressive nephropathy, and cancer. The variable character and unpredictable outcomes of infection attest to large gaps in our basic understanding of PyV biology. In particular, the rich history of research demonstrating the oncogenic potential of PyVs in laboratory animals begs the question of why cancer is not more often associated with infection. Raccoon polyomavirus (RacPyV), discovered in 2010, is consistently identified in neuroglial tumors in free-ranging raccoons in the western United States. Exposure to RacPyV is widespread, and RacPyV is detected in tissues of raccoons without tumors. Studying the relationship of RacPyV with its natural host is a unique opportunity to uncover cogent cellular targets and protein interactions between the virus and its host. Our hypothesis is that RacPyV, as an intact episome, alters cellular pathways within neural progenitor cells and drives oncogenesis.

The raccoon polyomavirus genome and tumor antigen transcription are stable and abundant in neuroglial tumors.

UNLABELLED: Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by in situ hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer. IMPORTANCE: The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor tissue or in unaffected raccoons. Unlike other oncogenic polyomaviruses, RacPyV was episomal, not integrated, in these tumors. To determine the stability of the viral genome and sustained transcription of the oncogenic tumor antigen genes, we cultured primary raccoon tumor cells and passaged them in mice, confirming the nonintegrated state of the virus and the maintenance of viral gene transcription throughout. RacPyV provides a naturally occurring and tractable model for a novel mechanism of polyomavirus-mediated oncogenesis.