Original Article

DOI: 10.1111/vco.12106

A comparison of clinical, magnetic resonance imaging and pathological findings in dogs with gliomatosis cerebri, focusing on cases with minimal magnetic resonance imaging changes‡ R. T. Bentley1 , G. N. Burcham2 , H. G. Heng1 , J. M. Levine3 , R. Longshore4 , S. Carrera-Justiz5 , S. Cameron6† , K. Kopf7 and M. A. Miller2 1

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA 2 Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA 3 Department of Small Animal Clinical Sciences, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA 4 Gulf Coast Veterinary Neurology and Neurosurgery, Houston, TX, USA 5 VCA West Los Angeles Animal Hospital, Los Angeles, CA, USA 6 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA 7 Department of Small Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

Abstract

Keywords brain, canine, glioma, MRI, tumour

The primary study objective was to determine whether clinical examination and magnetic resonance imaging (MRI) can underestimate canine gliomatosis cerebri (GC); we also investigated immunohistochemical features. Seven dogs with GC were studied; four recruited specifically because of minimal MRI changes. Neuroanatomic localization and the distribution of MRI, gross and sub-gross lesions were compared with the actual histological distribution of neoplastic cells. In six cases, clinical examination predicted focal disease and MRI demonstrated a single lesion or appeared normal. Neoplastic cells infiltrated many regions deemed normal by clinical examination and MRI, and were Olig2-positive and glial fibrillary acid protein-negative. Four dogs had concurrent gliomas. GC is a differential diagnosis for dogs with focal neurological deficits and a normal MRI or a focal MRI lesion. Canine GC is probably mainly oligodendrocytic. Type II GC, a solid glioma accompanying diffuse central nervous system neoplastic infiltration, occurs in dogs as in people.

Introduction Correspondence address: R. T. Bentley Department of Veterinary Clinical Sciences College of Veterinary Medicine Purdue University 625 Harrison Street West Lafayette IN 47907, USA e-mail: [email protected]

Canine gliomatosis cerebri (GC) is a central nervous system (CNS) neoplasia that is not well-reported. Magnetic resonance imaging (MRI) † Present address: San Mateo Emergency Pet Care and Veterinary Specialists, San Mateo, CA, USA ‡ Part of this study was presented in abstract form at the Second Bi-Annual Veterinary Neurosurgical Society Symposium, Portland, OR, USA, August 2013.

© 2014 John Wiley & Sons Ltd

findings have only been reported for a small number of cases and the correlation between the regions which appear abnormal on MRI and the true histological distribution of the disease has not been studied. The starting point of this study was our observation of some of the dogs included here, in which limited MRI lesions supporting a focal disease process were detected, but widespread disease was found histologically. On the basis of a focal clinical neuroanatomic localization combined

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with focal MRI changes, GC had not been a differential diagnosis prior to necropsy for these cases; glioma and other classically focal tumours had been suspected. Reports of canine GC generally give the impression of widespread disease, with frequent involvement of multiple divisions of the CNS.1 – 5 All six dogs in one study had involvement of at least two of the following: telencephalon, diencephalon, brainstem, cerebellum or spinal cord.1 Four dogs in other reports all had disease of multiple brain divisions.2 – 5 Just two dogs with limited disease have been reported. In one, a small focus spanned the caudal brainstem and cranial cervical spinal cord; in another, neoplastic cells were restricted to the spinal cord at the level of the 13th thoracic to 2nd lumbar vertebrae.6 In GC, neoplastic cells insinuate among normal structures with minimal damage to neurons and axons; cells reminiscent of astrocytes as well as oligodendrocytes, cells of transitional appearance and unclassified cells are present.7 In dogs, it is mainly differentiated from solid gliomas, such as astrocytoma and oligodendroglioma, by the relative preservation of tissue architecture.8 The current World Health Organization definition of human GC9 includes infiltration of multiple cerebral lobes, with requisite forebrain involvement, while recognizing the frequent cases in which other areas of the CNS are also affected.10,11 In type I GC, there is diffuse infiltration of tumour cells into the CNS, whereas in type II GC, a mass lesion is present in addition to this infiltrative disease.11 – 12 Contrastingly, the veterinary definition of GC8 is applied more broadly to widespread and diffusely infiltrating glial neoplasms, and does not require involvement of any specific location(s), nor does it include the type I and II subdivisions. Dogs with mass lesions in addition to infiltrative disease have nonetheless been recognized.1,4,5 The histogenesis of canine GC remains unclear. Glial fibrillary acid protein (GFAP)-positive cells within lesions have been reported in one dog, but it was unclear as to whether these represented neoplastic cells or reactive astrocytes.2 In multiple other reported cases, GFAP-positive neoplastic cells were not detected.1,3 – 5 In one of these, 80% of neoplastic cells were positive

for Oligodendrocyte transcription factor 2 (Olig2), and all GFAP-positive cells resembled non-neoplastic astrocytes, leading to the diagnosis of oligodendroglial GC.5 Because Olig2 immunohistochemistry has not been reported from other dogs with GC so far, whether canine GC is usually oligodendroglial in origin, or astrocytic as in humans,10,11 is unknown. Our primary goal was to investigate whether clinical examination and MRI can underestimate the true topographical extent of GC according to histology; consequently we recruited a sub-population of dogs that may not be representative of all canine cases of GC. A secondary goal was to investigate the cell of origin of canine GC, by performing Olig2 immunohistochemistry, in addition to conventional histology and GFAP immunohistochemistry.

Materials and methods Case selection The inclusion criteria were client-owned dogs with a histological diagnosis of GC using the veterinary definition by Koestner et al.8 Banked paraffin blocks and an MRI of the appropriate region(s) of the CNS including at least multiplanar T1-weighted and T2-weighted imaging were also required. All GC cases in the pathology archives of Purdue University College of Veterinary Medicine for which an MRI was available were used. Further cases were recruited by contacting veterinary neurologists in outside institutions and requesting cases with minimal MRI lesions.

Medical records review Signalment, duration of clinical signs, findings on neurological examination and clinical neuroanatomic localization, and results of cerebrospinal fluid analysis were noted. The date of MRI and date of euthanasia were recorded.

Evaluation of MRI lesions The MRIs were jointly evaluated by a boardcertified neurologist (R. T. B.) and a board-certified radiologist (H. G. H.) and a consensus opinion reached. The MRI field strength and image quality was recorded for each case.

© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12106

Canine gliomatosis cerebri 3

Each MRI lesion was first described either as a mass lesion or as a T2-weighted hyperintensity of the parenchyma without mass effect, based on features such as displacement of adjacent structures, midline shift and ventricular distortion. Lesions were then characterized according to routine criteria for canine brain tumours13 – 15 and classifications reported as significant in the MRI of dogs with gliomas.16,17 Contrast enhancement, when available, was subjectively characterized as none-mild or as moderate-marked, with regards to the most markedly enhancing portion of the lesion.16 The T1-weighted and T2-weighted intensity were compared with normal grey matter, with regards to the largest fraction of the lesion if the lesion displayed multiple intensities.16 Ventricular distortion was characterized as absent or present16 ; mild asymmetry of the lateral ventricles alone was not considered abnormal.18 Peri-lesional T2-hyperintensity17 was characterized as either none-mild or as moderate-extensive16 ; the term ‘peri-lesional T2-hyperintensity’ was used rather than peri-lesional oedema due to difficulty in identifying the border between neoplastic tissue and adjacent oedema. Surface contact was characterized as absent or present17 and any apparent contact of T2-hyperintensity with the meninges was considered surface contact. If any portion of a lesion involved the internal capsule, thalamus16 or brainstem, that lesion was considered deep. Absence or presence of cystic structures16 was recorded.

Gross and sub-gross pathology, histopathology and immunohistochemistry Necropsy reports were reviewed for descriptions of gross lesions. For sub-gross evaluation, H&E, GFAP and Olig2 sections were digitized by two board-certified pathologists (G. N. B. and M. A. M.) and evaluated at low power to mimic evaluation of gross specimens with the naked eye. Haematoxylin and eosin-stained sections were produced by routine methods. Immunohistochemistry (GFAP and Olig2) was performed on an automated stainer (IntelliPATH FLX, Biocare Medical, Concord, CA, USA). Briefly, for GFAP immunohistochemistry sections were first treated with 3% hydrogen

peroxide in phosphate-buffered saline for 5 min to inactivate endogenous peroxidases. A blocking reagent (Background Punisher, Biocare Medical) was applied for 10 min to block non-specific reactions. A rabbit polyclonal antibody against bovine GFAP (Z0334, Dako, Carpinteria, CA, USA) diluted 1:1000 was incubated without antigen retrieval for 30 min, and detected with a biotin-free mouse-on-canine horseradish peroxidase polymer method (PromARK, Biocare Medical) using betazoid diaminobenzidine (Betazoid DAB Chromogen Kit, Biocare Medical). Normal astrocytes in non-neoplastic brain tissue were used as an internal positive control. The use of this anti-bovine antibody for canine brain tumours is well accepted.19 For Olig2 immunohistochemistry, pretreatment consisted of heat-induced epitope retrieval performed by placing sections in a pretreatment reagent (Borg Decloaker, Biocare Medical) (pH, 9.0) at 125 ∘ C and 20–25 psi (138–172 kPa) for 30 s. All other treatments were at room temperature. To inactivate endogenous peroxidases, sections were treated with 3% hydrogen peroxide in phosphate-buffered saline for 5 min. A blocking reagent (Background Punisher, Biocare Medical) was applied for 10 min to block non-specific reactions. Sections were then incubated with the primary antibody, rabbit polyclonal antibody to recombinant mouse Olig2 (AB9610, EMD Millipore, Billerica, MA, USA), diluted 1:500, for 1 h 30 min. The use of this anti-murine antibody for canine brain tumours has been reported.19 After three washes with tris-buffered saline, sections were incubated for 40 min with a rabbit-on-canine horseradish peroxidase-labelled polymer detection system (PromARK, Biocare Medical). After another three washes with tris-buffered saline, betazoid diaminobenzidine (Betazoid DAB Chromogen Kit, Biocare Medical) was used as a chromogen. Sections were counterstained with Mayer’s haematoxylin. For in-house validation of Olig2 immunohistochemistry, control tissues included normal canine cerebrum, two canine oligodendrogliomas, one canine astrocytoma and one canine cerebral histiocytic sarcoma. Haematoxylin and eosin, GFAP and Olig2 sections were evaluated microscopically (G. N. B. and M. A. M.) and a consensus opinion

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reached. For immunohistochemistry, the percentage of positive cells was semi-quantitatively estimated by reviewing entire cross-section(s) of the lesion.

Comparison of clinical neuroanatomic localization, MRI lesions, gross to sub-gross pathology, and histopathology The results of neuroanatomic localization, MRI, gross to sub-gross pathology, and histopathology including immunohistochemistry were used to compare the apparent distribution of neoplastic disease; histopathology was considered the gold standard. At each diagnostic evaluation (neuroanatomic localization, MRI, gross pathology, sub-gross pathology, histopathology) the lesion was described as focal or multifocal. To allow direct comparison with clinical examination findings, lesions involving only one of three standard divisions of the brain (forebrain, brainstem and cerebellum) or one of the four functional divisions of the spinal

cord (C1–C5, C6–T2, T3–L3 and L4–S3) were classified as focal. A single mass involving the optic chiasm and pituitary gland was also described as focal. For each of MRI, gross to sub-gross pathology, and histopathology, a comprehensive list of specific structures detectably involved by the disease process was recorded and the lesion lateralization was described as unilateral, bilateral, or a single lesion crossing the midline.

Results Cases Seven cases met the inclusion criteria. Three were identified in the Purdue University pathology archives, with one case being provided by each of four supporting institutions. These were three male (two castrated) and four female (three spayed) dogs of 1–13 years of age (median, 4 years). Included were two Boxer dogs, three other brachycephalic dogs (Boston Terrier, English Bulldog, Bull Mastiff) and two large mix-breed dogs. The duration of

Table 1. Clinical neurolocalization, magnetic resonance imaging lesions and gross pathological lesions in seven cases of

canine gliomatosis cerebri, compared with the gold standard of histology Clinical neurolocalization

MRI lesions

Gross to sub-gross lesions

Case 1 Unilateral forebrain

Unilateral thalamus

Unilateral thalamus

Case 2 Optic chiasm, hypothalamic-pituitary axis

Optic chiasm, pituitary

Optic chiasm, pituitary

Case 3 Bilateral L4–S3 Case 4 Bilateral brainstem

None detected None detected

Bilateral spinal cord None detected

Case 5 Unilateral brainstem

Unilateral thalamus, brainstem Unilateral cerebrum

Unilateral brainstem

Case 6 Bilateral (post-ictal) forebrain Case 7 Bilateral forebrain, brainstem/cerebellum, C6–T2

a

Unilateral cerebrum

Unilateral brainstema Bilateral cerebrum, thalamus, brainstema

Cervical syringomyelia was also present.

© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12106

Histological lesions (gold standard) GC: bilateral thalamus, cerebrum Astrocytoma: unilateral thalamus GC: bilateral thalamus, cerebrum, hippocampus, brainstem, cerebellum Oligodendroglioma: optic chiasm, pituitary GC: bilateral spinal cord GC: bilateral brainstem, cerebellum and thalamus (lesser extent) Oligodendroglioma: crossing midline of brainstem GC: bilateral brainstem, cerebellum GC: bilateral cerebrum, hippocampus, thalamus GC: bilateral cerebrum, hippocampus, thalamus, brainstema Oligodendroglioma: crossing midline of brainstem

Canine gliomatosis cerebri 5

clinical signs prior to examination ranged from 2 to 90 days (median, 28 days). Cerebrospinal fluid was analysed in four dogs. The protein concentration and nucleated cell counts were within the normal range for that laboratory for each case. Minimal cytological abnormalities were detected, with a single neutrophil and single eosinophil in one case and vacuolated macrophages in two others. In case 6, post-mortem MRI was performed immediately after euthanasia; IV contrast was not administered. Cases 2 and 7 were euthanized and submitted for necropsy on the same day as MRI. The other four dogs were euthanized 6–19 weeks after the MRI. Detailed neuroanatomic localization, MRI and histological descriptions are presented after the following comparison of distribution of neoplastic lesions by each of the different diagnostic modalities.

Comparison of the apparent distribution of GC according to each of clinical neuroanatomic localization, MRI lesions, gross and sub-gross pathology, and histopathology In most cases, a focal lesion was suggested by neuroanatomic localization, MRI and gross necropsy examination, but significantly more widespread and bilateral neoplasia was detected on histological examination (Tables 1 and 2). According to neuroanatomic localization, six cases had focal lesions and only case 7 was deemed multifocal. This compares to MRI where six cases had focal or no lesions, and case 7 had multifocal lesions. Grossly, all seven cases had a focal lesion or no lesions. In contrast, multifocal histological lesions were present in four cases. Although they underestimated the topography of histological lesions, there was much agreement between MRI and gross and sub-gross examination findings. In case 1 a thalamic mass was evident on both MRI and gross exam; this abutted what was considered to be perilesional T2-hyperintensity of the adjacent cerebrum by MRI, but was actually intensely cellular GC on histological exam. Similarly, in case 2, perilesional T2-hyperintensity affected the prosencephalon overlying an extra-axial skull base mass, matching

well with an area of discoloration noted grossly. In cases 3–6, MRI and gross and sub-gross findings were very similar. Case 7 differed, as myriad MRI lesions were detected with topography that matched well with the histological findings, but only a focal brainstem lesion was present on gross examination. Regarding the lesion laterality, neoplastic cells were histologically present bilaterally in the parenchyma of the CNS of every case. This included two cases in which an MRI had revealed no lesion, three cases in which there had been a purely unilateral lesion on MRI, and case 2 in which MRI revealed an extra-axial mass with no robust evidence of intra-axial neoplasia. Compared with the gold standard of histology, neuroanatomic localization failed to predict the full lesion location and laterality in cases 1, 2, 4 and 5. MRI failed to predict lesion location and laterality in cases 1–6. In all seven cases, lesions were detected histologically in area(s) that appeared grossly normal.

Clinical neuroanatomic localization Four cases had straightforward focal neuroanatomic localizations, whereas three cases warrant further description. In case 2, a focal neurolocalization including the optic chiasm and hypothalamic-pituitary axis was given, based on bilateral blindness, mydriasis and absent pupillary light reflexes with concurrent severe polyuria-polydipsia. Case 6 was presented following a seizure, and had bilateral neurological deficits referable to the forebrain and the post-ictal state. In case 7, a multifocal localization including the forebrain, brainstem and/or cerebellum, and C6–T2 was given; findings included cortical blindness with intact pupillary light reflexes, head tilt and vestibular quality ataxia, and thoracic limb hyporeflexia.

MRI characteristics of GC lesions Pre-contrast T1-weighted and T2-weighted images were acquired in all cases, and T2-weighted fluid attenuation inversion recovery (n = 5), T2* -weighted gradient echo (n = 5), and proton density (n = 2) images were also obtained.

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Table 2. Detectable distribution of neoplastic disease for seven cases of canine gliomatosis cerebri

Cerebral grey matter Cerebral white matter Hippocampus Thalamus Optic chiasm and pituitary gland Midbrain Pons Medulla Cerebellum Spinal grey and white matter

MRI

Gross pathology

Sub-gross pathology

Histopathology

2 unilateral, 2 bilateral 1 unilateral, 2 bilateral 2 unilateral, 1 bilateral 1 unilateral, 2 bilateral 1 crossing midline 1 unilateral, 2 bilateral 2 bilateral 2 bilateral 0 0

1 unilateral 0 0 1 unilateral 1 crossing midline 1 unilateral 2 unilateral 2 unilateral 0 0

2 unilateral 2 unilateral 1 unilateral 1 unilateral 1 crossing midline 1 unilateral 2 unilateral 2 unilateral 0 1 bilateral

4 bilateral 4 bilateral 3 bilateral 5 bilateral 1 crossing midline 4 bilateral 4 bilateral 3 bilateral 3 bilateral 1 bilateral

T1-weighted images were acquired post-contrast in all dogs except case 6. A short tau inversion recovery sequence was performed on case 3 (localized to L4–S3). The thoracic to sacral vertebral column was imaged in case 3, the brain and cervical vertebral column were imaged in case 7, and the brain alone was imaged in the other cases. Images were acquired in transverse, sagittal and dorsal planes. Field strengths were 0.2–1.5 T. Image quality was adequate for all cases. The MRI revealed a single lesion or no lesion in every dog except case 7 (Table 3). Case 1 (1.5 T field strength) had an intra-axial thalamic mass lesion with adjacent cerebral T2-hyperintensity (Fig. 1). Case 2 (1.5 T) was considered to have an extra-axial mass, on the floor of the rostral fossa effacing the optic chiasm and pituitary gland, with a limited area of adjacent perilesional T2-hyperintensity in the overlying diencephalon and cerebrum (Fig. 2). This extra-axial mass was the only lesion to show moderate to marked contrast enhancement; all lesions were none to minimally contrast-enhancing in the other five dogs administered contrast. In cases 3 (1.5 T) and 4 (0.2 T), no lesion was detected by MRI (Fig. 3). In case 5 (0.3 T), only a faintly discernible unilateral area of T2hyperintense parenchyma in the region of the midbrain was noted, with no detectable abnormalities on pre- and post-contrast T1-weighted images. In case 6 T2-hyperintensity and T1-hypointensity of the left pyriform lobe were both highly perceptible (Fig. 4). Only in case 7 (1.5 T) were multifocal lesions detected (Fig. 5). These were the regions of parenchymal T2-hyperintensity, noted bilaterally

Table 3. Magnetic resonance imaging characteristics of

seven canine cases of gliomatosis cerebri

Magnetic resonance imaging criteria Lesions present Single mass lesion Single T2-hyperintensea parenchymal lesion Multiple T2-hyperintensea parenchymal lesions No lesions detected Lesion characteristics Surface contact of lesion(s) Deep lesion(s) involving the internal capsule, diencephalon or brainstem Ventricular distortion Cystic structures T1-weighted mainly hypointense lesion(s)a T1-weighted mainly isointense lesion(s)a No T1-weighted lesionsa T2-weighted mainly hyperintense lesionsa No T2-weighted lesionsa Moderate to extensive peri-lesional T2-hyperintensitya Moderate to marked contrast enhancementb

Number of dogs 2 2 1 2 5 4 2 0 3 1 3 5 2 2 1

a T1-weighted

and T2-weighted signals were compared with normal grey matter. b IV contrast was administered to six of seven dogs.

and asymmetrically in the cerebral grey and white matter and the thalamus, with an additional bilateral region of parenchymal T2-hyperintensity of the midbrain, pons and rostral medulla. There was also cervical syringomyelia (non-enhancing T1-weighted hypointense dorsal spinal cord lesion >2 mm in diameter,20 T2-hyperintense with no signal on T2-weighted fluid attenuation inversion recovery images).

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Figure 1. Thalamic astrocytoma with oligodendroglial GC in a dog (case 1). By MRI, the lesion was apparently unilateral, a T2-hyperintense mass lesion and associated cerebral hyperintensity [(A) T2-weighted transverse image] that displayed minimal contrast enhancement [(B) T1-weighted transverse post-contrast image, arrow]. The sub-gross image [(C) H&E] includes the region where the pyriform and temporal lobes were most hypercellular (asterisks) and the rostral aspect of the astrocytoma (arrows). The cytoplasm of the neoplastic cells of the thalamic astrocytoma was GFAP-positive [(D) GFAP immunohistochemistry with haematoxylin counterstain]. Gliomatosis cerebri lesions [(E) H&E with Olig2 immunohistochemistry insert] were found bilaterally, characterized by neoplastic infiltrates with Olig2-positive nuclei that did not displace neurons (arrows depict neurons).

Differentiating T2-hyperintensity of the neoplastic lesion itself from surrounding T2-hyperintense parenchyma was difficult, even when post-contrast images were also taken into account. Characteristics such as apparent parenchymal oedema adjacent to the extra-axial mass in case 2 were considered, however, the peri-lesional T2-hyperintensity adjacent to the mass in case 1 proved to be an area of intense GC upon histology. In case 7, in which GC tracking along cerebral white matter was later confirmed histologically, it was considered impossible to differentiate so-called perilesional T2-hyperintensity from the lesions themselves.

Although mass effect and ventricular distortion were not common, three cases had extensive lesions with surface contact supportive of sub-pial involvement, peri-ventricular lesions (lateral ventricles), and lesions following the white matter pathways.

Histopathological and immunohistochemical features In all seven cases, GC lesions included diffuse and bilateral infiltration of neoplastic cells with relative preservation of the normal architecture. Peri-ventricular and sub-pial involvement were

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Figure 2. Discrete optic chiasm and pituitary gland oligodendroglioma with associated oligodendroglial GC in a dog (case 2). On MRI, a markedly contrast-enhancing, extra-axial mass lesion [(A) arrows, T1-weighted transverse post-contrast image] displayed moderate bilateral perilesional T2-hyperintensity [(B) arrows, T2-weighted fluid attenuation inversion recovery transverse image]. Grossly (C), a mass precluding identification of the optic chiasm (asterisk) was associated with a small region of parenchymal discoloration (arrow), but the remainder of the brain was normal. Microscopically, GC lesions consisting of Olig-2 positive neoplastic cells were found bilaterally in the cerebral hemispheres [(D) Olig2 immunohistochemistry], hippocampus, thalamus, brainstem and cerebellum; note the hypercellularity (asterisk) in the caudal colliculus subjacent to the cerebellum [(E) H&E].

both common, and lesions both involved grey matter areas and coursed down white matter tracts. There was an additional solid neoplastic mass with little to no identifiable neuroparenchyma (discrete glioma) in four cases. Accordingly, three cases of type I GC and four cases of type II GC were diagnosed, when applying recognized terminology for the human disease. The Olig2 immunohistochemistry was specific, with no reactivity in neurons, endothelial cells, or normal, reactive or neoplastic astrocytes in any of the cases or control tissues. Normal oligodendrocytes were Olig2-positive in all cases and control tissues. In control tissues, the two oligodendrogliomas displayed neoplastic cells with Olig2-positive nuclei, whereas the neoplastic cells in the astrocytoma and the cerebral histiocytic sarcoma were Olig2-negative. In every case, the GC lesions consisted of severe hypercellularity characterized by Olig2-positive, GFAP-negative cells. These cells displayed neoplastic morphology on H&E and immunohistochemistry, such as nuclear atypia (enlarged nuclei, anisokaryosis, mitotic figures), aiding

differentiation from satellitosis. They were differentiated from reactive or neoplastic astrocytes by both different morphology and completely opposite reactivity with GFAP and Olig2 immunohistochemistry. In six cases, the percentage of cells expressing Olig2 ranged from 50 to 90%. In case 5, 25–50% of the neoplastic cells were Olig2-positive. On GFAP immunohistochemistry, all GC lesions contained a variable but low number of positive cells, interpreted as reactive astrocytes, and all cells with neoplastic morphology were GFAP-negative. Microscopic morphology and immunohistochemistry in the four accompanying discrete gliomas varied. Three displayed H&E morphology consistent with oligodendrogliomas and had Olig2-positive, GFAP-negative neoplastic cells. The mass in case 1 was morphologically consistent with astrocytoma and neoplastic cells were GFAP-positive and entirely Olig2-negative.

Discussion Our cases indicate that GC should be considered in dogs with neurological disease and a single, focal

© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12106

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Figure 3. Lumbar spinal cord GC in a dog (case 3). No lesions were detected by MRI [(A) normal T2-weighted mid-sagittal image]. Sub-grossly [(B) H&E], the spinal cord was unilaterally swollen (asterisk) and bilaterally hypercellular. The nuclei of the neoplastic cells were stained positively for Olig2 [(C) lumbar grey matter]; note that neurons (short arrow) and astrocytes (long arrows) were negative for Olig2.

lesion on MRI, regardless of whether a parenchymal lesion or a mass is detected. This conclusion is supported by multiple reports, in which a solitary region of parenchymal T2-hyperintensity1,6 or a single mass lesion1,4 was described. The differential diagnosis for this MRI appearance should include a discrete glioma or other neoplasm; history, diffusion weighted imaging and cerebrospinal fluid analysis may help in evaluating the likelihood of other entities such as cerebrovascular accident or inflammatory disease.21,22 GC should also be in the differential diagnosis for certain dogs with progressive neurological disease in the face of a normal MRI. Two dogs with histologically confirmed GC and a normal MRI have been reported.1,6 Normal MRI findings in dogs with CNS disease have not been well-studied, beside certain diseases such as cerebellar atrophy23 and idiopathic and cryptogenic epilepsy.24 A normal MRI was reported for one of three dogs with multifocal oligodendroglioma25 and one cat with intracranial lymphoma,26 as well as six dogs with inflammatory cerebrospinal fluid27 and one dog thought to be suffering from transient ischaemic attacks.28 Although not reported previously, GC should also be in the differential diagnosis for apparently

extra-axial mass lesions in the region of the optic chiasm or pituitary gland. Other tumours that have been reported to have a similar appearance on MRI in dogs and cats include meningioma,13,29 pituitary carcinoma,13,29 granular cell tumour30 and metastatic pancreatic carcinoma,31 while craniopharyngioma32 and germ cell tumours33 also occur in the sellar region. An additional detail to note is that our cases displayed little contrast enhancement; enhancement was only considered moderate-marked in the apparently extra-axial mass, and was categorized as none-mild for all other cases administered contrast. Similarly, in all previous MRI reports of canine GC, no enhancement was detected.1,3,6,34 Other canine brain tumours that have been reported to lack contrast enhancement include medulloblastoma,34 ependymoma13 and a minority of discrete gliomas,13,15,34 especially low-grade gliomas.16,17 On the basis of the immunohistochemistry results in this study and previous reports, canine GC currently appears to be mainly oligodendroglial in origin. In one previous case report, 80% of the neoplastic cells expressed Olig2, while all GFAP-positive cells resembled normal or reactive astrocytes.5 This fits very well with our cases in which typically 50–90% of neoplastic cells were positive for Olig2, and all neoplastic cells were negative for GFAP. In previous studies, neoplastic cells in canine GC lesions have been negative for GFAP,1,3,4,6 CD181,3 and other leucocytic markers1,2,4 – 6 providing little evidence for astrocytic, leucocytic or microglial origin. In one case report, many GFAP-positive cells were observed, but it was considered unclear whether these cells resulted from reactive astrogliosis or belonged to the population of tumour cells, and Olig2 was not utilized.2 In the largest previous study of canine GC, all neoplastic cells were considered negative for GFAP, and all GFAP-positive cells were considered to be reactive astrocytes.1 The same conclusion was reached about the GFAP-positive cells in our cases. In contrast, 30 of 31 canine astrocytomas displayed GFAP-labelling, with unequivocal positive staining of neoplastic cells in 84%.35 Olig2 immunohistochemistry is an emerging but previously validated tool in canine brain tumours.19 It should be noted that astrocytes and

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Figure 4. Forebrain GC in a dog (case 6). A unilateral T2-hyperintense pyriform lobe lesion (arrow) was detected by MRI [(A) T2-weighted fluid attenuation inversion recovery transverse image). Oligodendroglial GC was present in the pyriform lobe [(B) H&E], and bilaterally in the cerebral hemispheres [(C) H&E], hippocampus and thalamus.

oligodendrocytes share a common precursor, and although normal and reactive astrocytes are Olig2-negative, Olig2-positive neoplastic cells have been detected in astrocytic tumours of dogs,19 cats19 and people.36 Critically, all such astrocytic tumours were also GFAP-positive.19,36 Olig2 was positive in nine canine oligodendrogliomas, eight of which were negative for GFAP and one of which was graded between positive and negative.19 In our control tissues, oligodendroglioma was positive for Olig2 but negative for GFAP. While human astrocytomas and oligoastrocytomas can contain Olig2-positive cells, the percentage of positive cells (primarily 11–50%) is lower than in oligodendrogliomas (85–96%).37 Consequently, Olig2 positive tumours can be of astrocytic origin, but typically they are also GFAP-positive19,36 and display astrocytic morphology.36,37 This supports the conclusion that our cases are probably oligodendroglial in origin. We recommend that canine GC be referred to as type I or type II, based on whether a discrete glioma accompanies the diffuse neoplastic infiltration. This builds upon the work of previous authors who have noted a mass lesion in addition to diffuse GC.1,4 This recommendation is not based on differing prognoses between various manifestations of GC in dogs, but on observed variations in morphological manifestations including MRI features, and could lead to more clarity in future studies. In planning therapy, it may be important to recognize that apparently focal lesions may be accompanied by widespread and distant microscopic disease. The classification of veterinary brain tumours has not been updated since 1999.8 Given the investigations

by others since then,1 the emerging trend towards an oligodendroglial over-representation,5 multiple reports of dogs with no cerebral hemisphere disease3,4,6 and the recognition of mass lesions in addition to diffuse infiltration,1,4 the veterinary definition of GC may need updating. Given that the term GC may be taken to imply cerebral involvement, either the name or the definition of this condition may need adjusting, and terms such as gliomatosis cerebelli4 or GC with only spinal cord involvement6 may need formal recognition as a variant of canine GC. Lack of a consistent MRI protocol and the use of different field strengths were both weaknesses of this study. However, it appears that MRI findings match well with gross findings, regardless of whether 0.2–1.5 T devices are used. It is possible that the MRI lesion in case 5 would have been less vague had a higher field strength been used, but image quality was acceptable for all cases. It is important to note that we particularly pursued additional dogs with limited MRI changes, and as such this population of dogs was not necessarily representative of dogs with GC. The seventh case, with multifocal neurological deficits and numerous MRI lesions, may be more reflective of the typical GC case. There was variation in the time period between MRI and submission for necropsy. However, this does not appear to impact most of our conclusions. Regarding the conclusion that MRI can considerably underestimate the extent of the disease process, there were two dogs (cases 2 and 6) euthanized the same day as the MRI in which microscopic neoplastic infiltration significantly extended beyond the

© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12106

Canine gliomatosis cerebri 11

Figure 5. Multifocal GC accompanied by a brainstem oligodendroglioma in a dog (case 7). T2-weighted fluid attenuation inversion recovery images [(A) level of the diencephalon, (B) level of the mesencephalon] revealed bilateral and asymmetric hyperintense prosencephalic lesions (arrows), and a single intra-axial lesion crossing the midline from the midbrain to the rostral medulla (asterisk). Grossly, only the brainstem lesion (arrow) was evident [(C) medulla]. This brainstem lesion was a discrete oligodendroglioma [(D) Olig2 immunohistochemistry]. Multifocal oligodendroglial GC lesions were also observed throughout the cerebrum, thalamus, brainstem and cerebellum. These were characterized by severe hypercellularity with infiltration of Olig2-positive cells with neoplastic morphology such as nuclear atypia including karyomegaly [(E) Olig2 immunohistochemistry of cerebral hemisphere with H&E inset]. While some of these cells were present in satellite position (arrow, normal neuron), the majority were scattered throughout the neuropil.

location of the MRI lesions. One of these cases showed the greatest disconnect between MRI and the true scope of the disease, with MRI revealing an extra-axial mass and no clear intra-axial neoplasia, and neoplastic cells histologically present bilaterally in the cerebrum, thalamus, brainstem and cerebellum. These cases highlight that the significantly increased distribution of disease on histological examination compared with MRI was not necessarily a result of time passing between MRI and euthanasia. On the basis of these cases, GC should be in the differential diagnosis for neurological dogs with a single, focal MRI lesion, and for certain dogs

with progressive CNS disease and a normal MRI. Clinical examination and MRI can considerably underestimate the microscopic distribution of GC. Canine GC currently seems to be a neoplasm that is primarily of oligodendroglial origin. Finally, type II GC, a discrete glioma accompanying diffuse infiltration of neoplastic cells, occurs in dogs as it does in people.

Acknowledgements The authors thank John Edwards, Texas A&M University; Gayle Johnson, University of Missouri; Jeanine Peters-Kennedy, Cornell University;

© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12106

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and David Caudell, Virginia-Maryland Regional College of Veterinary Medicine for post-mortem diagnosis and for contributing histological specimens. We would also like to thank the veterinary radiology services of Texas A&M University, Cornell University and the Virginia-Maryland Regional College for providing MRIs. Dee DuSold, Purdue University, performed the immunohistochemistry.

Conflict of interest

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11.

12.

This work was not supported by any grants. There are no conflicts of interest to report. 13.

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© 2014 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, doi: 10.1111/vco.12106

A comparison of clinical, magnetic resonance imaging and pathological findings in dogs with gliomatosis cerebri, focusing on cases with minimal magnetic resonance imaging changes(‡).

The primary study objective was to determine whether clinical examination and magnetic resonance imaging (MRI) can underestimate canine gliomatosis ce...
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