J. Comp. Path. 2013, Vol. 149, 514e519 Available online at www.sciencedirect.com

www.elsevier.com/locate/jcpa

DISEASE IN WILDLIFE OR EXOTIC SPECIES

Primitive Neuroectodermal Tumour in a StripedDolphin (Stenella coeruleoalba) with Features ofEpendymoma and Neural Tube Differentiation

(Medulloepithelioma)

Cor

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J. L. Baily*, L. R. Morrison†, I. A. Patterson‡, C. Underwood*

and M. P. Dagleish*

*Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Near Edinburgh EH26 0PZ, †Veterinary

Pathology Unit, Royal (Dick) School of Veterinary Studies and Roslin Institute, Easter Bush Campus, University of

Edinburgh, Midlothian EH25 9RG and ‡Agri-Food and Biosciences Institute, Veterinary Sciences Division, Stoney Road,

Stormont, Belfast BT4 3SD, UK

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Summary

Primary brain tumours in cetaceans are rare with only four reported cases of intracranial tumours in the sci-entific literature. A juvenile female, striped dolphin live-stranded at Whitepark Bay, Co Antrim, NorthernIreland, UK, and died after an unsuccessful attempt at refloatation. Necropsy examination revealed a large,soft, non-encapsulated friable mass, which expanded and replaced the frontal lobes, corpus callosum andcaudate nucleus of the brain and extended into the lateral ventricles, displacing the thalamus caudally. Micro-scopically, this comprised moderately pleomorphic neoplastic cells arranged variably in dense monotonoussheets, irregular streams, ependymal rosettes, ‘ependymoblastomatous rosettes’ and multilayered to pseudos-tratified tubules. Liquefactive necrosis, palisading glial cells, haemorrhage and mineralization were alsoobserved. Immunohistochemically, the neoplastic cells expressed vimentin but not S100, glial fibrillary acidicprotein, cytokeratin, neuron-specific enolase or synaptophysin. Based on these findings a diagnosis of primitiveneuroectodermal tumour wasmade.Monitoring and recording such cases is crucial as neoplasia may be relatedto viral, carcinogenic or immunosuppressive chemical exposure and can ultimately contribute to assessing theocean health.

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Keywords: central nervous system; neoplasia; primitive neuroectodermal tumour; Stenella coeruleoalba

Primary brain tumours in cetaceans are rare withonly four reported cases: a cerebral lipoma in a hump-back whale (Megaptera novaeangliae) (Pilleri, 1966); acerebral neurofibroma in a fin whale (Balaenopteraphysalus) (Pilleri, 1968); a poorly differentiated carci-noma in the brainstem of a Beluga whale (Delphinap-

terus leucas) (Ridgway et al., 2002); and a microcysticmeningioma in a short-beaked common dolphin(Delphinus delphis) (Miclard et al., 2006). Additionally,Siebert et al. (2010) described intracranial metastasesof a gastric squamous cell carcinoma in a harbour

ondence to: J. L. Baily (e-mail: johanna.baily@moredun.ac.uk).

75/$ - see front matter

x.doi.org/10.1016/j.jcpa.2013.06.003

porpoise (Phocoena phocoena). The scarcity of reportsmay represent a genuine low incidence, lack of accessto cases due to death at sea, advanced autolysis ortechnical difficulties in examining the brain. Marinemammals are long-lived top level predators recog-nized as sentinels for ocean health (Bossart, 2011)and since neoplasia may be related to viral, carcino-genic or immunosuppressive chemical exposure, itis important to record and monitor these cases(Newman and Smith, 2006).

Striped dolphins (Stenella coeruleoalba) are social an-imals found throughout temperate and tropical wa-ters in dense schools of, on average, 100 animals.

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Fig. 1. Brain, sagittal section. A friable mass (delineated by ar-rows) expands and replaces the frontal lobes, corpus cal-losum and caudate nucleus and contains multiple foci ofmineralization (asterisk). CC, corpus callosum; CN,caudate nucleus; Th, thalamus; Ce, cerebellum; P, pons.

Fig. 2. Dense, monotonous sheets of poorly differentiated smallround cells with high nuclear to cytoplasmic ratio. HE.

Neuroectodermal Tumour in a Striped Dolphin 515

They are abundant globally and the most commondolphin in the Mediterranean Sea despite recentdeaths due to successive Morbillivirus epidemics,although some coastal populations have been greatlydepleted and perhaps extirpated (Reeves et al., 2002;Soto et al., 2011). Major threats to their populationsare bycatch and interaction with fisheries (Reeveset al., 2002). More recently, Toxoplasma gondii hasbeen associated with non-suppurative meningoen-cephalitis in striped dolphins in the Ligurian Sea(Di Guardo et al., 2010). Only two reports ofneoplasia in striped dolphins were listed in a reviewof marine mammal neoplasia (Newman and Smith,2006): a squamous cell carcinoma of the skin and acase of myelogenous leukaemia, although the latteroccurred in a bottlenose dolphin (Tursiops truncatus)according to the original reference (Cowan, 1994).

The purpose of this report is to present gross, histo-logical and immunohistochemical features of a prim-itive neuroectodermal tumour (PNET) in the centralnervous system (CNS) of a striped dolphin.

A juvenile female striped dolphin (S. coeruleoalba)(case No. 2010-001905) live-stranded at WhiteparkBay, CoAntrim, Northern Ireland, UK, and refloata-tion was attempted prior to death. The carcass wassubmitted to the Agri-Food and Biosciences Institute,Belfast, Northern Ireland, and a post-mortem exami-nation was performed 48 h after death. The carcassmeasured 162 cm from the tip of the beak to the tipof the fluke, and the girth measurement immediatelyrostral to the dorsal fin was 88 cm. Three blubberthicknesses were taken: dorsal midline immediatelyrostral to the leading edge of the dorsal fin and lateraland ventral midlines below the dorsal measurement.The animal was in good body condition denoted bya well-rounded carcass with a mean blubber thicknessof 15.5 mm. The weight of the carcass was unavai-lable.

Significant gross findings were confined to thebrain. The meninges were congested and a 6e7 cmdiameter, soft, friable mass expanded and replacedthe frontal lobes, corpus callosum and caudate nu-cleus and extended into the lateral ventricles, displac-ing the thalamus caudally. Themass wasmottled paletan and pink with multiple small to medium sized fociof haemorrhage and mineralization (Fig. 1). Grossfindings in other organs included overinflation ofthe lungs with rib indentations evident, a small num-ber of otoliths and a single focus of chronic gastric ul-ceration in the cardiac stomach and a moderateamount of digesta in the small intestine.

The whole brain and representative samples oflung, heart, liver and spleen were fixed in 10%neutral buffered formalin and processed routinely toparaffin wax. Sections (5 mm) were stained with hae-

matoxylin and eosin (HE). Selected sections were alsostained with periodic acideSchiff (PAS), Grocott’smethenamine silver (GMS), Perl’s Prussian blue(PPB) and phosphotungstic acid haematoxylin(PTAH) stains.

Microscopically, a discrete, occasionally infiltra-tive mass expanded and effaced the cerebral cortex,lateral ventricles and corpus callosum. Neoplasticcells were arranged variably in three main patterns:dense sheets (Fig. 2); irregular streams; and areascomposed of true ependymal rosettes (Fig. 3),although some areas also showed ‘ependymoblas-tomatous rosettes’ and distinct tubules. Ependymo-blastomatous rosettes were characterized by

Fig. 3. Intense labelling of neoplastic cells for vimentin. IHC.

516 J.L. Baily et al.

pseudostratified cells in a rosette pattern with a cen-tral lumen and frequent juxtaluminal mitoses, butdevoid of a limiting external membrane. Tubuleswere pseudostratified to multilayered, formed bycolumnar to cuboidal cells and reminiscent of theembryonic neural tube (Fig. 4). Cells were round,spindle shaped or tall columnar with moderateamounts of pale eosinophilic, wispy cytoplasm. Inareas of rosette formation, the cells had aperipherally-located nucleus and distinct eosino-philic cytoplasmic processes at the apical surface.Nuclei were medium sized, oval to elongated withcoarsely clumped chromatin and variably, one tofour, prominent nucleoli. Nuclear and cellular pleo-morphism was mild to marked. Mitotic activity washigh with an average of 10 mitoses per high-powerfield (400�). The mass was highly vascularized,but glomeruloid blood vessels were not observed.Large areas of liquefactive necrosis were present

Fig. 4. Pseudostratified tubules delineated by a GMS-positivebasement membrane. GMS.

within the mass, which were frequently borderedby palisading glial cells. Extensive areas of haemor-rhage, haemosiderin-laden macrophages, character-ized by golden brown pigment staining deepblue with PPB, and mineralization were alsofrequent within necrotic foci. Pseudorosettes wererare. Perivascular hyalinization was noted in areas.Neoplastic cells were negative for PTAH stain.Tubular structures were frequently delimited by aPAS-positive and GMS-positive basement mem-brane (Fig. 4). There was no evidence of metastasisin other organs examined. Heart and spleen werenormal. In the lung, mild multifocal interstitialpneumonia with intra-alveolar nematode wormswas present and in the liver there was mild, diffuselipidosis.

Immunohistochemistry (IHC) was performed onselected sections for cytokeratin (pan-CK; cloneMNF116, mouse monoclonal, Dako, Glostrup,Denmark [M0821], dilution 1 in 50), glial fibrillaryacidic protein (GFAP, rabbit polyclonal, Dako[Z0334], dilution 1 in 400), vimentin (vimentinNCL-VIM-V9, clone V9; mouse monoclonal; Novo-castra, Newcastle-upon-Tyne, UK, dilution 1 in 100),neuron-specific enolase (NSE, mouse monoclonal,Dako [M0873], dilution 1 in 600) and synaptophysin(clone SY36, mouse monoclonal, Dako [M0776],dilution 1 in 20) and ‘visualized’ with an Envision�kit (Dako) according to the manufacturer’s instruc-tions. Immunolabelling for S100 protein was per-formed using anti-S100 rabbit polyclonal antibody(Dako [Z0311], dilution 1 in 400) ‘visualized’ witha horseradish peroxidase (HRP)-conjugated goatanti-rabbit IgG antibody (Dako [P 0448], dilution 1in 50). Pan-CK immunolabelling required pretreat-ment with proteinase-K (ready-to-use, Dako) for10 min at room temperature. For NSE and synapto-physin, antigen retrieval involved microwave treat-ment at 110�C for 15 min in citrate buffer pH 6.0.Antigen retrieval for vimentin IHC was with vimen-tin high pH buffer H-3301 (Vector Laboratories,Peterborough, UK). For GFAP, antigen retrievalinvolved treatment with 0.05% trypsin and 0.05%chymotrypsin in a steamer for 10 min in Tris buffer(pH 7.8) at 37�C. Ovine brain, equine brain, equinecolon, ovine lung and canine tonsil were used as pos-itive control tissues, and internal controls includedstriped dolphin lung and brain. Negative control sec-tions were incubated with non-immune serum orisotype-matched mouse IgG in place of the primaryantibodies.

Immunohistochemical examination revealeddiffuse, strong, cytoplasmic immunolabelling of themajority (>95%) of neoplastic cells for vimentin(Fig. 3). Tumour cells failed to label for GFAP, but

Neuroectodermal Tumour in a Striped Dolphin 517

the stroma was strongly positive. Immunolabellingfor S100 protein showed a moderate signal in only asmall number of large, isolated macrophages withinthe mass, but no labelling of neoplastic cells. Immu-nolabelling for cytokeratin, NSE and synaptophysinwas negative. Positive and negative controlsconfirmed the specificity of the antibodies.

The gross and histological features of the mass sug-gested a poorly differentiated embryonal tumourarising from the lateral ventricles. The presence ofwell-differentiated multilayered and pseudostratifiedtubules, delineated by a PAS/GMS-positive basementmembrane, is consistentwith aPNETwith neural tubedifferentiation or medulloepithelioma. The main dif-ferential diagnoses for this tumour were ependymo-blastoma, medulloblastoma, anaplastic ependymomaand teratoma (Lantos et al., 2002). Medulloblastomawas excluded immediately based on the location out-with the cerebellum. Although the tumour had manyfeatures of ependymoblastoma, in particular the char-acteristic ‘ependymoblastomatous rosettes’, the pres-ence of neural tube-like structures excluded thisdiagnosis (Summers et al., 1995; Lantos et al., 2002).However, large areas of ependymal differentiation,including ‘ependymoblastomatous rosettes’, arecommonly found in medulloepitheliomas. Thisfinding is consistent with the highly primitive natureof this tumour and supports the argument thatmedulloepithelioma is a subset of the PNETs (Lantoset al., 2002; Adesina et al., 2010). The areas of markedcellular atypia, high mitotic activity and presence ofpalisading cells around necrotic foci were reminiscentof anaplastic ependymoma. Indeed, palisadingaround necrotic foci is not a recognized featureof PNETs, while it is a characteristic feature ofanaplastic ependymoma (Lantos et al., 2002). Howev-er, endothelial microvascular proliferation andpseudorosettes are also characteristic features ofanaplastic ependymoma (Lantos et al., 2002),but neither were present in this case. Furthermore,anaplastic features are a common finding inmedulloe-pitheliomas, further supporting the diagnosis(Summers et al., 1995; Lantos et al., 2002).

The classification of both human and animalembryonal tumours remains controversial due totheir anaplastic nature (Koestner and Higgins,2002) and constantly evolving nomenclature andgrouping. In the WHO International Histopathological

Classification of Tumors in Domestic Animals (1999),which reclassified CNS tumours based on the humanclassification at that time, animal embryonal tumourswere subdivided into PNETs, neuroblastomas andependymoblastomas (Koestner et al., 1999). PNETs,described as capable of differentiation alongneuronal, glial, ependymal and possibly mesen-

chymal cell lines, are further subdivided into thoseof cerebellar origin, known as medulloblastomas,and ‘PNETs excluding those of cerebellar origin’,otherwise indistinguishable from each other(Koestner et al., 1999). Shortly prior to this, however,Summers et al. (1995) used the term ‘medulloepithe-lioma’ for a primitive tumour derived from theneuroepithelium, but placed this entity under ‘miscel-laneous CNS tumours’, separately from medulloblas-tomas and PNETs.

The more recent human reclassification of embry-onal tumours by the WHO distinguishes three en-tities: medulloblastomas, located within thecerebellum, CNS PNETs and the atypical teratoidrhabdoid tumour. Within the CNS PNETs, CNSneuroblastoma, CNS ganglioneuroblastoma, medul-loepithelioma and ependymoblastoma are four recog-nized subdivisions defined by the presence of neuronalfeatures, neoplastic ganglion cells, features of theembryonal neural tube and ependymoblastomatousrosettes, respectively (Louis et al., 2007a,b). Thepresent tumour would therefore be termed PNETexcluding cerebellar origin in the veterinaryclassification or medulloepithelioma if adhering tothe human nomenclature.

CNS PNETs are rare, highly malignant tumours,found most frequently in young animals or in people<3 years of age, typically located supratentoriallyand classified as embryonal tumours. They arecomposed of poorly differentiated neuroepithelial cellsand show differentiation along neuronal, astrocytic,ependymal, muscular or melanocytic lines (Summerset al., 1995; Adesina et al., 2010). Occasional cases ofCNS PNETs occur in animals, with cases reportedin cattle (Lucas et al., 2003) and dogs (Headleyet al., 2009; Ide et al., 2010; Choi et al., 2012).

Medulloepitheliomas were described originally asthe most primitive of brain tumours (Bailey andCushing, 1926) and are thought to originate from apluripotent neuroepithelial stem cell progenitor.Their main feature is the presence of tubular struc-tures reminiscent of the embryonic neural tube andthey are regarded variably as a distinct entity or asanother form of differentiation within the PNETs(Adesina et al., 2010). It is important to note thatthese tumours should not be confused with the morewidely reported intraocular tumour, termed ‘teratoidmedulloepithelioma’, which, although sharing thesame name and histological features, carries a favour-able prognosis (Summers et al., 1995). There are noreports of CNS medulloepitheliomas in animals be-sides occasional cases of metastatic forms of ocularme-dulloepitheliomas (Aleksandersen et al., 2004).

In people, vimentin is present most consistently inmedulloepitheliomas, with inconsistent labelling of

518 J.L. Baily et al.

cytokeratin in both medulloepitheliomas andependymoblastomas (Adesina et al., 2010). In thecases recorded in animals, PNETs are reported asfrequently positive for vimentin and variably positivefor GFAP, NSE and cytokeratin (Lucas et al., 2003;Headley et al., 2009; Ide et al., 2010).

To the authors’ knowledge, this is the first detailedreport of neoplasia reported in a striped dolphin andonly the second case of intracranial neoplasia re-ported in any dolphin species. Specific tumour typesin this species may be of relevance with regard toexposure of marine animals to increasing concentra-tions of water-borne toxins and potential carcinogens,as well as exposure to potentially oncogenic virusesand radioactive nucleotides (Mossner andBallschmiter, 1997; Gulland et al., 2001). Improvingour understanding of disease in wild populations is akey step in any wildlife conservation strategy.

Acknowledgements

The authors wish to thank N. MacIntyre, R(D)SVS,for excellent technical assistance. J. Baily’s PhD stu-dentship is funded by the Moredun Research Insti-tute and the Royal Zoological Society of Scotland.This work was also funded by the Scottish Govern-ment.

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ccepted, June 14th, 2013

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