High-grade astrocytoma in a sheep: clinical,pathology,and genetic investigations

Abstract

Primary neoplastic diseases of the CNS in livestock are rare compared to companion animals. Here we describe the clinical, pathologic, and genetic findings in an adult sheep with a high-grade cerebral astrocytoma. Clinically, apathy, ataxia, bilateral amaurosis, and horizontal resting nystagmus were observed. The signs were suggestive of a space-occupying lesion affecting the vestibular and ascending reticular activating systems. The CSF had high total protein, hypercellularity, and pleocytosis. Macroscopically, a friable, gray-pink, ovoid mass (6 × 3 × 4 cm) was observed within most of the right parietal, occipital, and temporal cerebral subcortical areas, extended into the thalamus, hippocampus, and the cerebral peduncles, and reached the ipsilateral choroid plexus and the lateral ventricles. Histologically, the well-delimited, unencapsulated lesion consisted of proliferating neoplastic spindloid-to-polygonal cells mainly arranged in streams and bundles supported by minimal fibrous stroma, and random areas of coagulative necrosis and hemorrhages without pseudopalisading in the center of the mass. Immunohistochemistry revealed strong immunolabeling of spindle cells for S100, vimentin, and glial fibrillary acidic protein. Immunolabeling for neuron-specific enolase was restricted to trapped neurons; oligodendrocyte transcription factor 2 (OLIG2), pan-cytokeratins, and SOX10 were immunonegative. TP53 gene sequencing of the neoplasm did not demonstrate any point mutations involving exons 2–11.

Keywords

brain high-grade astrocytoma neuropathology sheep

Primary neoplastic diseases of the CNS in livestock are rare compared to dogs and cats. In a survey of 891 tumors recorded in sheep in the United Kingdom, only 5 (0.56%) were tumors of the CNS, including 2 gliomas, which occurred in lambs up to 9-mo-old, but no further description was given.⁹ Astrocytomas are common primary intracranial tumors that have been reported in dogs, cats, cattle, horses, goats, wildlife, and, on 2 occasions, in sheep.^{5
–7,11,13,15} In humans, mutations of the p53 tumor suppressor gene (TP53) may contribute to oncogenesis, including for brain tumors, and have been reported frequently in astrocytic tumors. Such mutations are absent in autologous healthy tissue.^15,23

Here we describe the clinical, pathologic, and genetic findings in an adult sheep with a high-grade cerebral astrocytoma. To our knowledge, this tumor has not been reported previously in sheep.

A 7-y-old Finn Dorset ewe in very poor body condition and with neurologic signs, which were first noticed 35 d previously, was submitted to the Animal and Plant Health Agency (APHA) Weybridge, in Addlestone (UK) for further investigation. The ewe was dull, with low head carriage, no obvious head tilt, with a tendency to close the eyelids, and reluctance to walk (Suppl. Video 1). Heart and respiratory rate and rectal temperature were all within normal limits. The sheep had a bilaterally absent menace response and pupillary light reflex, and horizontal resting nystagmus with the fast component to the left that did not change direction with different head positions. Ophthalmoscopic examination did not reveal any abnormalities. During the assessment of vision, the gait was compromised by reluctance to walk. If pushed, the ewe had disequilibrium with a tendency to stand with increased extensor tone on the left, and mild leaning to the right, resulting in less effort needed to push the sheep to the right than to the left. Lifting of the head resulted in restless behavior with wagging of the tail. No response was elicited to scratching of the back and blindfolding.

The clinical signs were suggestive of a brain lesion affecting the ascending reticular activating system (dullness), the visual pathways (absent menace response and light reflex), and the vestibular system (nystagmus). The direction of nystagmus away from the site of the lesion and the decreased muscle tone based on the site of the lesion were consistent with a right-sided location of a lesion.²⁰ Possible diagnoses for progressive chronic neurologic disease with lateralization included intracranial abscesses or tumors, parasitic cysts of Coenurus cerebralis, internal otitis, atypical scrapie, chronic borna disease, visna, aberrant intracranial larva of Oestrus ovis, old trauma, and mild hydrocephalus.^1,14

Immediately after euthanasia, carried out with quinalbarbitone–cinchocaine (Somulose; Dechra) administered intravenously, CSF was collected from the atlanto-occipital cistern. Total protein was measured (Micro-BCA protein assay kit; Thermo Scientific) according to the manufacturer’s instructions, using a bovine serum albumin standard curve to calculate protein concentration. Total WBC count was performed using a hemocytometer. The CSF was colorless but slightly gelatinous. Total protein values and cell count were high (3.80 g protein/L [RI: <0.30 g/L]; 0.105 × 10⁹ WBC/L [RI: <0.003 × 10⁹ WBC/L]).¹⁷ A Giemsa-stained cytocentrifuged sample had pleocytosis with lymphocytes predominating (~90%) followed by monocytes and eosinophils (~10%). Slight blood contamination (up to 5 RBCs per visual field at 10× magnification) was also noted.

At postmortem examination, both right temporal and parietal bones were markedly thinned (0.2 mm). The volume of the right hemisphere was increased markedly, with flattening of the gyri. In parasagittal sections, a friable, gray-pink, focally extensive ovoid mass (6 × 3 × 4 cm) was observed within most of the dorsoventral width of the caudal half of the right hemisphere (Fig. 1A, 1B). The mass was well-delimited dorso-rostrally and laterally, was poorly delimited ventrally, and compressed the surrounding tissues. The mass expanded within the parietal, temporal, and occipital cerebral subcortical areas (mainly involving the white matter), and extended into the thalamus, hippocampus, and cerebral peduncle, reaching the ipsilateral choroid plexus and the lateral ventricles.

Figure 1.

High-grade cerebral astrocytoma in a sheep. A. Well-delimited, gray-pink mass affecting the right parietal and temporal cerebral subcortical areas. Note the cerebral gyri flattening. Inset: cross-section of the mass. B. Parasagittal section of the relatively well-demarcated ovoid formalin-fixed mass (6 × 3 × 4 cm), within most of the right parietal, occipital, and temporal cerebral subcortical areas.

The brain was fixed in 10% neutral-buffered formalin and processed routinely; 4-µm sections were stained with H&E for histologic examination. Microscopically, a well-delimited, unencapsulated lesion formed by proliferating neoplastic cells was observed. Cells were arranged mainly in streams or bundles and occasionally in a rosette pattern. Both gray and white matter were affected. Most of the cells were spindloid-to-polygonal, with abundant eosinophilic, occasionally vacuolated, cytoplasm, and round-to-oval euchromatic nuclei with one nucleolus (Fig. 2A). Rarely, binucleate or multinucleate cells were observed. Severe pleomorphism, anisokaryosis, anisocytosis, and low mitotic count (1 mitosis in 2.37 mm², 40× objective, 10× ocular, FN 22 mm) were seen. Mild-to-moderate multifocal lymphocytic infiltrates were noted in the ventral areas of the mass, along with random areas of coagulative necrosis and hemorrhages in the center of the mass (Fig. 2B). No palisading cells were observed surrounding the areas of necrosis.

Figure 2.

High-grade cerebral astrocytoma in a sheep. A. Streams and bundles of proliferating spindloid-to-polygonal neoplastic cells with abundant eosinophilic, occasionally vacuolated, cytoplasm, and round-to-oval euchromatic nuclei with one nucleolus; rare binucleate or multinucleate cells. H&E. B. Area of coagulative necrosis and hemorrhage in the center of the mass. H&E. C. Strong diffuse cytoplasmatic vimentin immunolabeling of the spindle cells (>90%). D. Strong cytoplasmatic GFAP immunostaining of the spindle cells (>75%).

Sections were prepared for immunostaining and labeled with S100, vimentin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), oligodendrocyte transcription factor 2 (OLIG2), pan-cytokeratins, and SOX10 (Table 1). Used as positive controls were sheep skin (pan-cytokeratins, SOX10, vimentin) and sheep brain (GFAP, S100, NSE, OLIG2). The omission of a primary antibody and the use of tissue samples that did not express the biomarker served as negative controls.

Table 1.

Details of immunohistochemical stains performed on sections of a high-grade astrocytoma in a sheep.

Antibody	Host	Dilution	Antigen retrieval; incubation time/temperature	Source
GFAP	Rabbit	1:10,000	High TRS (pH 9); 25 min at RT	Dako
NSE	Mouse	1:2,368	Citrate buffer (pH 6); 30 min at RT	Dako
OLIG2	Rabbit	1:400	Low TRS (pH 6.1); 25–30 min at 97°C	Proteintech
Pan-cytokeratins	Mouse	1:200	Low TRS (pH 6.1); 25–30 min at 97°C	Dako
S100	Mouse	1:100	High TRS (pH 9); 25 min at 37°C	Abcam
SOX10	Mouse	1:200	High TRS (pH 9); 25–30 min at 97°C	GenomeMe
Vimentin	Mouse	1:200	Citrate buffer (pH 6); 20 min at RT	Dako

GFAP = glial fibrillary acidic protein; NSE = neuron-specific enolase; OLIG2 = oligodendrocyte transcription factor 2; RT = room temperature; TRS = target retrieval solution.

Immunohistochemistry (IHC) revealed strong immunolabeling of spindle cells for S100 (>90%), vimentin (>90%; Fig. 2C), and GFAP (>75%; Fig. 2D). Immunostaining for NSE was restricted to trapped neurons. Immunolabeling for OLIG2, pan-cytokeratins, and SOX10 were diffusely negative. Statutory prion tests were negative for scrapie.

Serial paraffin-embedded sections and formalin-fixed samples of the tumor and from the surrounding histologically healthy brain were submitted to the Istituto Zooprofilattico Sperimentale della Sardegna “G. Pegreffi” (Sassari, Italy) for genetic investigations. Genomic DNA was extracted from 5-µm sections (QIAamp DNA FFPE tissue kit; Qiagen), after addition of xylene and ethanol (96–100%) and incubation at room temperature until all residual ethanol had evaporated. Incubation at 90°C was performed to partially reverse formaldehyde modification of nucleic acids. Exons 2–11 (DNA-binding domain) of TP53 were amplified by PCR using specific primers (Table 2). Finally, the TP53 exon sequences were detected by DNA sequencing on both strands of the PCR products (BigDye Terminator v.1.1 cycle sequencing kit; Life Technology). Consensus sequences from the tumor mass and healthy tissue were assembled (BioEdit v.7.2.5),¹⁹ and the sequences of the single exons in the tumor were compared with corresponding exons of healthy tissue and with the NCBI database reference (NM_001009403.1) by using ClustalW alignment (http://www.clustal.org/clustal2/). TP53 gene sequencing did not reveal point mutations in exons 2–11.

Table 2.

PCR primers used for ovine TP53 exon detection.

Exon target	Product size, bp	Sequence
2	241	F: 5′-CCCCCAGCAGATCCTCA-3′
		R: 5′-CCTCCAGACCTCCACTCA-3′
3	253	F: 5′-TGTGAGTGGAGGTCTGGA-3′
		R: 5′-AGCTCGGAGGACTACAGA-3′
4	198	F: 5′-CCGGGCCTTCTGATGCA-3′
		R: 5′-GGAGGGGACAAAGGACGA-3′
	244	F: 5′-CAGAGCCTCCTGCCCAA-3′
		R: 5′-GGCAAAGGCTGAAGTGGA-3′
5	260	F: 5′-ACAGGACCCGGCTTCCA-3′
		R: 5′-CGCTCATGGTGGGGACA-3′
	252	F: 5′-GTTCTGCCAGCTGGCCAA-3′
		R: 5′-GGCCAGACCTGAGAGCAA-3′
6	216	F: 5′-AGAGCCCCCAATCCCCTA-3′
		R: 5′-TCCCACCTAGGGTGGTCA-3′
7	216	F: 5′-AGGCTCGTGGAGGCTGA-3′
		R: 5′-GAGGGAAGCTAGCGTGGA-3′
8	265	F: 5′-GGGACCTGGTTCACAGGA-3′
		R: 5′-ATTGGGGGCAGAACTGCA-3′
9	291	F: 5′-GTGCAGTTCTGCCCCCAA-3′
		R: 5′-CTGCCGTCTATGGGGTCA-3′
10	264	F: 5′-CCTGTATTGGCAGGTGGA-3′
		R: 5′-TACGTGTGTGTGTGTGCA-3′
11	215	F: 5′-TGCCTCAGGCCCTTCGA-3′
		R: 5′-GGGTGGGGATGTCAACCA-3′

Based on the above histopathologic findings, a diagnosis of astrocytoma, specifically a fibrillary astrocytoma subtype of a diffuse astrocytoma grade II,¹⁰ was made. Astrocytomas are neuroepithelial cell tumors and are considered common cerebral neoplasms both in humans and animals.⁶ They are more prevalent in the cerebrum compared to the spinal cord and cerebellum.⁸ Gross appearance and histologic classification vary largely based on the degree of malignancy.^5,10,21 According to the grading used for canine gliomas,¹² which considers the simultaneous or nonsimultaneous presence of necrosis, microvascular proliferation (with or without pseudopalisading), mitosis, and universal features of malignancy to differentiate astrocytoma into low- or high-grade, the neoplasm in our case was classified as a high-grade astrocytoma. Immunohistochemical labeling is similar for all astrocytomas, with GFAP, S100, and vimentin being consistently positive,⁸ although negative vimentin staining of an astrocytoma has been reported in the spinal cord of a hedgehog.⁸

Although immunolabeling for GFAP could be influenced by formalin fixation, it was one of the most successful antibodies used in this panel. Despite vimentin, S100, and pan-cytokeratins being less specific than GFAP, as they are expressed in multiple tissues and non-glial neoplasms, they were used to complement the panel and detect or dismiss other possible tumor components in our case.¹³ Similarly, positive NSE immunolabeling can be detected in astrocytomas and, rarely, in other gliomas.²² The negative immunostaining for OLIG2 ruled out oligodendroglioma as a differential diagnosis; the negative pan-cytokeratin and SOX10 immunolabeling excluded potential histogenesis from transformed Schwann cells or meningothelial cells. However, for diagnostic accuracy, it is important to note the importance of the morphologic appearance rather than the use of special stains and IHC despite their fundamental investigative role, at least in canine and feline CNS tumors.² Finally, our case had mitotic activity similar to astrocytomas in other species.^15,18

In our case, the high CSF total protein concentration and the high WBC count were considered results of an immune-mediated response to the large areas of necrosis within the neoplasm.³ However, the WBC count observed in cats with CNS neoplasms is not always abnormal.¹⁶

The development of astrocytomas, as for other neoplasms, is multifactorial. In humans, TP53 mutations were among the first variations identified in astrocytic tumors²³; exonic mutations in dog brain tumors were found in 3.4²³–35%¹⁸ of the samples investigated. TP53 exonic mutations have also been described in a sheep glioblastoma,¹⁵ but data are not available for brain tumors in other species. We did not find any gene mutations in our case. Preliminary data in humans⁴ described an increase in overall survival in human patients with TP53-mutant compared to TP53-wild type (not mutated) glioblastomas. Therefore, a mutation is not excluded in our case given the extent and aggressiveness of the neoplasm.

To our knowledge, using the routine scientific research platforms (Google, PubMed, Scopus, CAB Direct, and Web of Science) for the search terms “astrocytoma”, “sheep”, “ovine”, “ewe”, only 2 astrocytomas have been described in sheep. Specifically, a protoplasmic astrocytoma⁷ and a glioblastoma,¹⁵ classified as low- and high-grade astrocytomas, respectively. However, for the latter there was an additional oligodendrogliomal component.¹⁵ We did not find a sole high-grade astrocytoma reported in sheep in the literature.

Footnotes

Acknowledgements

Our case was identified as part of the TSE surveillance program, funded by the UK Department of Environment, Food and Rural Affairs (TS5300). We are grateful to former and present staff in the Pathology Department (Animal and Plant Health Agency [APHA] Weybridge) for their contribution to the investigation of this case. We thank Mr. L. Thorne (Virology Department, APHA Weybridge) for performing the CSF analysis; the Veterinary Pathology Diagnostic Service, School of Veterinary Science (University of Liverpool, Leahurst) for their technical assistance with some IHC staining; and Dr. A. Schock (Pathology Department, APHA Lasswade) for review of the manuscript.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Sebastian A. Mignacca

Supplemental material

Supplemental material for this article is available online. Supplemental Video 1. Examination of the sheep prior to euthanasia. The sheep stands with low head carriage and is reluctant to walk. It tends to stand with the left hind leg more abducted than the right, which makes it more difficult pushing it toward the left than toward the right. Menace response is absent; the palpebral reflex is present. A light source directed to the left eye does not elicit a pupillary light reflex. Horizonal nystagmus with the fast component to the left is evident.

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