Mixed Glioma (Oligoastrocytoma) in the Brain of an African Hedgehog (Atelerix albiventris)

J. Comp. Path. 2014, Vol. 151, 420e424

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DISEASE IN WILDLIFE OR EXOTIC SPECIES

Mixed Glioma (Oligoastrocytoma) in the Brain of an African Hedgehog (Atelerix albiventris) S. S. Benneter*, B. A. Summers†, W. J. Schulz-Schaeffer‡, W. H€ artigx, J. Mollidork and S. Sch€ oniger* *Institute of Pathology, Faculty of Veterinary Medicine, University of Leipzig, An denTierkliniken 33, 04103 Leipzig, Germany, † The Royal Veterinary College, Department of Pathology and Pathogen Biology, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK, ‡ Department of Neuropathology, University Medical Center G€ottingen, Georg-August University of G€ottingen, Robert-Koch-Str. 40, 37075 G€ottingen, x Paul-Flechsig-Institute for Brain Research, University of Leipzig, Jahnallee 59, 04109 Leipzig and k Tierarztpraxis Dr. J. Mollidor, Niehler Str. 68, 50733 K€oln, Germany

Summary This report describes an oligoastrocytoma in the brain of a 3.5-year-old female pet African hedgehog (Atelerix albiventris) that showed progressive central nervous system signs for 6 months. Microscopical examination of the brain revealed a widely infiltrative, deep-seated glioma within the white matter of the cerebral hemispheres, basal nuclei, hippocampus, thalamus, midbrain, pons and the medulla of the cerebellum with extension of neoplastic cells into the cerebral cortex and overlying leptomeninges. Morphological features of the neoplastic cells, together with variable immunohistochemical expression of glial fibrillary acidic protein, Olig-2 and Nogo-A, indicated the presence of intermingled astrocytic and oligodendroglial tumour cells with an astrocytic component of approximately 40% consistent with an oligoastrocytoma. The distribution of the tumour is consistent with gliomatosis cerebri. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: African hedgehog; brain tumour; immunohistochemistry; oligoastrocytoma

Gliomas in man and domestic animals are subclassified according to their histopathological features. Astrocytic and oligodendrocytic tumours are most frequent (Koestner et al., 1999; Koestner and Higgins, 2002; Burger and Scheithauer, 2007; Louis et al., 2007). Diffuse infiltrating astrocytic tumours include low-grade astrocytoma, anaplastic astrocytoma and the most aggressive variant, glioblastoma multiforme. For subclassification the following features are evaluated: hypercellularity, cellular pleomorphism, nuclear atypia, mitotic activity, glomeruloid vascular or endothelial proliferation and presence of necrotic areas (Koestner et al., 1999; Koestner and Higgins, 2002; Burger and Scheithauer, 2007; Louis et al., 2007). Diffuse and anaplastic astrocytomas lack glomeruloid vascular or endothelial proliferation and/or Correspondence to: S. Sch€oniger (e-mail: Sandra.Schoeniger@vetmed. uni-leipzig.de). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.07.002

necrotic areas. These findings together with marked nuclear atypia are diagnostic for glioblastoma multiforme (Koestner et al., 1999; Koestner and Higgins, 2002; Burger and Scheithauer, 2007; Louis et al., 2007). Oligodendrogliomas are composed of neoplastic oligodendroglial cells. In formalin-fixed paraffin wax-embedded tissue sections the cells commonly show perinuclear shrinkage artefacts in the form of clear halos and are intermingled with branching capillaries. They can occur as relatively benign tumours or as anaplastic variants (Koestner et al., 1999; Koestner and Higgins, 2002; Burger and Scheithauer, 2007; Louis et al., 2007). Oligoastrocytomas are composite tumours formed of neoplastic astrocytes and oligodendrocytes. These two cell populations may be intermingled (diffuse variant) or located in separate clusters (biphasic or compact variant) (Koestner et al., 1999; Louis et al., 2007). In human medicine, whether or not the glioma is an astrocytoma, oligodendroglioma or Ó 2014 Elsevier Ltd. All rights reserved.

Oligoastrocytoma in an African Hedgehog

oligoastrocytoma has prognostic and therapeutic significance (Louis et al., 2007). The most common tumours reported in the African hedgehog (Atelerix albiventris) are mammary gland carcinomas, lymphomas and oral squamous cell carcinomas (Raymond and Garner, 2001; Heatley et al., 2005). Primary tumours of the central nervous system (CNS) appear to be rare; reported cases include astrocytomas located in the cerebellum (n ¼ 2), hippocampus (n ¼ 1), brainstem (n ¼ 4), medulla oblongata and cervical spinal cord (n ¼ 1) or spinal cord (n ¼ 1) as well as a cerebral microglioma (Gibson et al., 2008; Garner et al., 2010; Nakata et al., 2011). The present case report describes an oligoastrocytoma with extensive spread in the brain of an adult African hedgehog. A 3.5-year-old female African hedgehog showed clinical signs of neurological disease that progressed over 6 months. Initially, manege movement (walking in circles to the right) was noted, but the hedgehog could be encouraged to walk in a straight line with assistance. Later, the animal was unable to walk without falling to the left. The animal was humanely destroyed because of the poor prognosis and submitted for necropsy examination. The animal was in a good body condition. There was multifocal mild to moderate acute pulmonary alveolar emphysema and oedema. All other organs including the brain and spinal cord showed no significant gross changes. The brain, spinal cord, brachial plexus, sciatic nerves, skeletal muscle of the thoracic and pelvic limbs, as well as additional selected tissue samples, were fixed in 10% neutral buffered formalin and processed routinely. Microscopically, there was a widely infiltrative glioma within the white matter of the cerebellum, midbrain, thalamus, basal nuclei and both cerebral hemispheres. Neoplastic cells expanded the corpus callosum, infiltrated the cerebral cortex, extended along the white matter tracts into the hippocampus and pons, formed perivascular cuffs and spread via the cerebral leptomeninges. They integrated into and expanded the parenchyma without formation of a discrete tumour mass, although areas with a dense accumulation of tumour cells were observed. In areas with a higher tumour cell density, the parenchyma showed microvacuolation (Fig. 1). Two distinct tumour cell populations were discerned. On the one hand, neoplastic cells had an oligodendroglial morphology with round or oval hyperchromatic nuclei surrounded by clear halos; some of these cells were clustered. On the other hand, there were tumour cells with larger, cigarshaped or irregularly-shaped vesicular nuclei, indicating an astrocytic component (Fig. 1), of which a few were gemistocytic astrocytes; such cells were occa-

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Fig. 1. Oligoastrocytoma, cerebrum. Mixed neoplastic glial cells are located predominately within the white matter (black dot), which fills the figure, except for the grey matter (lower left; asterisk) where neoplastic cells are located perivascularly (arrowhead). The white matter shows multifocal mild microvacuolation (thin black arrow). Neoplastic oligodendrocytes (thick black arrows) are intermingled with other neoplastic cells indicative of an astrocytic population (thick white arrows). HE. Bar, 50 mm.

sionally binucleate. Scattered tumour cells with nuclear features of oligodendrocytes, but an eccentrically located nucleus and a moderate amount of eosinophilic cytoplasm, were observed. These cells were consistent with minigemistocytes, which are regarded as a variant of neoplastic oligodendrocytes. There was an average of 10 mitotic figures in 10 high-power (40 objective) fields; mitotic figures were observed in both neoplastic cell populations. A Gomori’s reticulin stain revealed no basal lamina delineating the tumour cells. Intralesional blood vessels were proliferating, but did not show an endothelial or a glomeruloid vascular proliferation and no necrotic areas were observed. Additional findings were marked splenic extramedullary haemopoiesis and multifocal mild interstitial lymphoplasmacytic infiltrates within the renal parenchyma. Tumour cells were further characterized by immunohistochemistry (IHC) using a biotinestreptavidin technique (for evaluation of expression of vimentin, Nogo-A and glial fibrillary acidic protein [GFAP]) or the peroxidaseeantiperoxidase method (for evaluation of Olig-2 and GFAP expression) with 3, 30 diaminobenzidine as chromogen. Approximately 40% of the tumour cells expressed GFAP (antibody from Dako, Glostrup, Denmark, 1 in 1,000 dilution). The GFAP labelling was of varying intensity (mild, moderate or strong) with most immunolabelled cells having branching fibrillary processes (resembling fibrillary astrocytes), while a few had morphological features consistent with those of gemistocytes (Fig. 2). A similar cell population (interpreted as astrocytes) expressed

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vimentin (clone Vim34, Dako; 1 in 300 dilution; protease antigen retrieval). Other tumour cells, which often formed small clusters and had round or oval hyperchromatic nuclei, reacted with antibody specific for isoform A of the neuritic outgrowth inhibitor (NogoA; clone 11C7; 1 in 200 dilution; antigen retrieval by heating in citrate buffer; kindly donated by Professor M. E. Schwab, Brain Research Institute, University of Zurich and Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland), indicating an oligodendrocytic origin, together with the

presence of shrinkage artefacts (Fig. 3). Approximately 60% of the tumour cells showed nuclear expression of the oligodendrocyte transcription factor Olig-2 (Millipore, Schwalbach, Germany; 1 in 500 dilution; Fig. 4). Double immunofluorescence labelling was performed by concomitant incubation with guinea pig anti-GFAP (Synaptic Systems, G€ottingen, Germany; 1 in 400 dilution) and rabbit anti-Olig-2 (1 in 100 dilution) followed by applying a mixture of Cy3tagged donkey anti-guinea pig immunoglobulin (Ig) G and Cy2-conjugated donkey anti-rabbit IgG (both from Dianova, Hamburg, Germany; 20 mg/ ml). Labelling was visualized with an Axioplan 2 microscope (Zeiss, G€ottingen, Germany) equipped with a structured light confocal system (OptiGrid, Qioptiq, Fairport, New York, USA) and a digital camera (ORCA-ER, Hamamatsu, Hamamatsu City, Japan). Tumour cells expressed either GFAP or Olig-2. Cells co-expressing both antigens were not observed. In the hedgehog glioma, GFAP/vimentin on the one hand and Nogo-A on the other hand were expressed in tumour cells resembling fibrillary astrocytes and oligodendrocytes, respectively. These findings are consistent with an intermingled (diffuse) oligoastrocytoma with an astrocytic component of 40%. For the diagnosis of an oligoastrocytoma, either neoplastic lineage (astrocytes or oligodendrocytes) must constitute at least 25% of the tumour (Mork et al., 1986; Koestner et al., 1999). GFAP is an intermediate filament of astrocytes, but can also be expressed in minigemistocytes of oligodendrogliomas (Louis et al., 2007). Vimentin expression in astrocytes occurs during CNS development and is frequently

Fig. 3. Oligoastrocytoma, cerebral white matter. Immunolabelling for Nogo-A is observed in approximately 40% of the tumour cells. Nogo-A immunolabelled neoplastic cells show an oligodendroglia-like morphology and are often clustered (arrowheads). IHC. Bar, 45 mm.

Fig. 4. Oligoastrocytoma, cerebral white matter. Immunolabelling for the transcription factor Olig-2 is detected in approximately 60% of the neoplastic glial cells (arrowheads); some of these form small clusters. IHC. Bar, 50 mm.

Fig. 2. Oligoastrocytoma, cerebral white matter. Approximately 40% of the neoplastic cells express GFAP (black arrows) and have numerous slender cell processes. One GFAPimmunolabelled tumour cell is binucleate (arrowhead). Typical of neoplastic astrocytes, GFAP labelling of tumour cells is heterogeneous, weaker in some cells (white arrows) than others. IHC. Bar, 20 mm.

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observed in fibrillary astrocytomas (Louis et al., 2007). Nogo-A is a marker of oligodendrocytes (Kuhlmann et al., 2007). Moderate to strong NogoA immunoreactivity is detectable within the majority of human oligodendrogliomas, but only in some glioblastomas and anaplastic astrocytomas (Marucci et al., 2012). In the adult human brain, Olig-2 is detected in oligodendrocytes as well as in NG2-glia (Nishiyama et al., 2005; Ligon et al., 2006). In human glial tumours, Olig-2 can be found in oligodendrogliomas and oligoastrocytomas, but also in approximately 80% of astrocytomas (Mokhtari et al., 2005; Ligon et al., 2006). In these cases, oligodendrogliomas contained significantly higher numbers of Olig-2 immunopositive cells than astrocytomas (Mokhtari et al., 2005). In the hedgehog glioma, nuclear Olig-2 immunolabelling was noted in more cells than were positive for Nogo-A and was always associated with an absence of expression of GFAP. Similarly, human anaplastic oligodendrogliomas contain higher numbers of Olig-2 immunopositive cells than those expressing Nogo-A (Jung et al., 2011). The presence of significant numbers of reactive astrocytes within the tumour was ruled out, since astrocytic cells lack a hypertrophic cytoplasm and show nuclear irregularities as well as a variable intensity of GFAP expression. Glioblastoma multiforme with an oligodendrocytic component was excluded, since endothelial or a glomeruloid vascular proliferation and/or necrosis were absent (Louis et al., 2007). Due to a few hypercellular areas, the observed mitotic activity and the infiltration of the leptomeninges, this hedgehog astrocytoma had features of an anaplastic oligoastrocytoma (Koestner et al., 1999; Louis et al., 2007). In the veterinary literature, oligoastrocytomas are rarely described; case reports exist in dogs (Koestner and Higgins, 2002; Walmsley et al., 2009) and a hooded crane (Ide et al., 2009). The growth pattern of this hedgehog oligoastrocytoma was consistent with ‘gliomatosis cerebri’. This designation is used for human gliomas (astrocytic, oligodendroglial or mixed) with the contiguous presence of neoplastic cells within at least three cerebral lobes (Louis et al., 2007). In veterinary medicine, ‘gliomatosis cerebri’ is defined as diffuse glioma that widely involves the brain and spinal cord (Koestner et al., 1999). It has been diagnosed in a few dogs (Koestner et al., 1999; Porter et al., 2003; Gruber et al., 2006; Gal an et al., 2010; Plattner et al., 2012) and a goat (Braun et al., 2005). Neoplastic glial cells were either astrocytic (Braun et al., 2005), oligodendroglial (Gal an et al., 2010) or could not be definitively identified histologically or immunohistochemically (Porter et al., 2003; Plattner et al., 2012).

Acknowledgement The authors are grateful to Dr. Kacza, Institute of Anatomy, Faculty of Veterinary Medicine, University of Leipzig, for providing his expertise in using the Axioplan 2 microscope.

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April 28th, 2014 ½ Received, Accepted, July 7th, 2014