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Journal of Veterinary Diagnostic Investigation Vol. 18 Issue 5, 499-503
Copyright © 2006 by the American Association of Veterinary Laboratory Diagnosticians
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Brief Communication

Malignant nonteratoid ocular medulloepithelioma in a llama (Llama glama)

Sandra Schoeniger1, Ludvik R. Donner and William G. Van Alstine

Correspondence: 1Corresponding Author: Sandra Schoeniger, The Royal Veterinary College, Department of Pathology and Infectious Diseases, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK


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A 6-year-old female llama presented with buphthalmos of its right eye owing to the presence of an intraocular mass. The affected globe was enucleated and submitted for microscopic examination. The intraocular mass was diagnosed as malignant medulloepithelioma. Within the following months, the llama developed soft tissue masses, which completely filled the right orbital cavity and expanded the cranial portion of the right mandibular bone, and enlarged mandibular lymph nodes. Euthanasia was elected 30 months after the initial diagnosis. The carcass was submitted for postmortem examination, which revealed the presence of medulloepithelioma metastases within the right orbit, mandible, mandibular lymph nodes, lungs, liver, and mesenteric and sublumbar lymph nodes. The primary intraocular tumor and its metastases were composed of neoplastic undifferentiated neuroepithelial cells, which formed tubules, Flexner-Wintersteiner and Homer Wright rosettes, and rare solid sheets. Electron microscopy showed that tumor cells were connected by desmosome-like junctions and contained rare intracytoplasmic basal bodies. Neoplastic cells were positive for vimentin, nestin, microtubule-associated protein 1B, S-100 protein, and glial fibrillary acidic protein (GFAP). To the best of the authors' knowledge, this is the first report of a malignant nonteratoid ocular medulloepithelioma with distant metastases in a llama and of the ultrastructural and extended immunohistochemical characterization of a nonteratoid medulloepithelioma in this species.

Key Words: Electron microscopy • immunohistochemistry • medulloepithelioma • nonteratoid

Medulloepitheliomas are rare primitive neuroepithelial tumors.13 In animals, most intraocular medulloepitheliomas are described in horses,7,14,16,17 and only rare case reports exist in dogs,2,18 cats,12 and cockatiels.3,15 They are classified as nonteratoid and teratoid and can be benign or malignant.6,10,13,16 Nonteratoid ocular medulloepitheliomas are composed only of undifferentiated neuroepithelium, while teratoid ocular medulloepitheliomas contain additional heteroplastic components such as cartilage or brain tissue.10,13,16 This report describes a nonteratoid ocular medulloepithelioma in a llama with widespread metastasis to regional lymph nodes and internal organs.

A 6-year-old female llama (Llama glama) was presented to the referring veterinarian with buphthalmos and excessive tearing of its right eye owing to the presence of an intraocular mass. The globe was enucleated and submitted for microscopic examination. One month after enucleation, the right orbit was swollen with a firm mass. Radiation treatment (cobalt) at this time dramatically reduced the orbital mass. However, a palpable mass returned within 2 months following radiation treatment. Six months after enucleation, the llama developed swelling of head and neck lymph nodes and masses within the rostral portion of the mandible. At this time, euthanasia was elected and a complete necropsy was performed. Time between detection of the intraocular mass and euthanasia was approximately 30 months.

The enucleated globe measured 4.5 x 5 x 5 cm and contained a firm to friable, lobulated, white-reddish mottled mass. The mass was connected to the posterior-lateral wall of the globe, replaced vitreous and lens, filled anterior and posterior chambers, and protruded approximately 0.15 cm through the ruptured cornea (Fig. 1A).


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  		Figure 1 A, Right globe: The medulloepithelioma filled anterior and posterior chambers, replaced vitreous and lens, and protruded through the ruptured cornea. B, Right lateral thoracic and abdominal cavities: The lungs (arrowheads) and liver (arrows) contained multiple variably sized medulloepithelioma metastases.

 
At necropsy, the right orbit was completely filled with a lobulated mass, which caused outward bulging of the overlying sutured eyelids, infiltrated and filled the right maxillary sinus and right nasal cavity, and caused compression of nasal turbinates. The cranial portion of the right mandibular bone was expanded and replaced by masses resulting in superficial gingival ulceration with loss of right canine and lateral incisor teeth and left deviation of intermediate and medial right incisor teeth. Mandibular and retropharyngeal lymph nodes were bilaterally enlarged measuring 7 x 3 x 3 cm owing to expansion by lobulated masses. Pulmonary and hepatic parenchyma contained multiple gray nodules measuring up to 7 cm in diameter (Fig. 1B). Mesenteric and sublumbar lymph nodes were enlarged with similar masses measuring up to 9 x 5 x 4 cm. Masses within all affected tissues had similar gross features as the primary intraocular tumor. They were expansile and infiltrative, multilobulated, and composed of firm to friable, white-reddish mottled tissue. Additional macroscopic lesions included ascites and ulcers within the third gastric compartment.

Tissue samples from the globe and visceral organs were fixed in 10% neutral-buffered formalin, routinely processed, and embedded in paraffin. Histologic sections (4 µm) were prepared from paraffin-embedded tissues and stained with hematoxylin and eosin (HE) and periodic acid–Schiff stain (PAS). Under microscopic examination, the ocular mass was identified as a medulloepithelioma, and masses within the right orbit, maxillary sinus, nasal cavity, lymph nodes, mandible, lungs, and liver as medulloepithelioma metastases. The primary and metastatic tumors had similar microscopic features. They were composed of densely-packed, undifferentiated, neoplastic, neuroepithelial cells. Tumor cells were arranged in tubules, sheets, and true rosettes (numerous Flexner-Wintersteiner rosettes and scattered Homer Wright rosettes) separated by fine septa of fibrovascular stroma (Fig. 2). Tubular structures predominated within the tumor and were lined by pseudostratified, neoplastic, primitive neuroepithelium. Flexner-Wintersteiner rosettes were lined by pseudostratified columnar neoplastic cells surrounding a clear central lumen, which occasionally contained PAS-positive material. Arrangement of pseudostratified neoplastic cells around a central accumulation of fibrillary eosinophilic tangles was consistent with Homer Wright rosette formation. Basal portions of tubules and rosettes were supported by a thin PAS-positive membrane. Neoplastic cells forming tubules and rosettes were cuboidal to columnar containing a single vesicular to hyperchromatic nucleus and a moderate amount of cytoplasm. Tumor cells forming sheets were polygonal to ovoid with a single polygonal hyperchromatic nucleus and a small amount of pale eosinophilic cytoplasm. Neoplastic cells had mild anisocytosis, anisokaryosis, and cellular pleomorphism and a high mitotic rate of 2 to 10 mitotic figures per high dry magnification field (Fig. 2B). In the enucleated globe, tumor cells invaded cornea, conjunctiva, and sclera and replaced retina, choroid, ciliary body, iris, and lens. Neoplastic cells were admixed with small remnants of choroid and retinal pigment epithelium. The intraocular tumor contained small areas of hemorrhage, which were located mainly perivascular within the stroma septa and extended between neoplastic cells. Tumor emboli plugged pulmonary vessels, hepatic sinusoids, and sinuses of affected lymph nodes. Within affected organs, neoplastic cells multifocally infiltrated and replaced the parenchyma.


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  		Figure 2 Light microscopy. A, Tumors contained numerous tubules and Flexner-Wintersteiner rosettes, which were lined by columnar pseudostratified neoplastic neuroepithelium. B, Neoplastic neuroepithelial cells had a high mitotic rate (arrows) and formed rare solid nests (circle) and scattered Homer Wright rosettes (asterisk). HE stain.

 
Transmission electron microscopy was performed on 60- to 90-nm thick sections of plastic embedded neoplastic tissue, which had been fixed in 10% nonbuffered formalin, postfixed in 1% osmium tetroxide, and stained with uranyl-acetate and lead citrate. Rosette-forming neoplastic cells were connected at their apical poles by desmosome-like junctions, contained rare intracytoplasmic basal bodies within their apical cytoplasm, and had basally located nuclei (Fig. 3). Neoplastic cells within solid clusters had few intercellular desmosome-like junctions and rare intracytoplasmic basal bodies. Tumor cells contained scattered organelles (e.g., mitochondria, endoplasmic reticulum, ribosomes).


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  		Figure 3 Electron microscopy. Tubules and rosettes were lined by pseudostratified polarized cuboidal to columnar neoplastic neuroepithelial cells, which contained intracytoplasmic basal bodies within their apical cytoplasm (arrowheads, inlet A) and were connected at their apical poles by desmosome-like junctions (arrows, inlet B).

 
Immunohistochemistry was performed on paraffin-embedded, formalin-fixed sections (4 µm) of llama medulloepithelioma tissue (primary intraocular medulloepithelioma, pulmonary, and hepatic metastases), llama cerebellum (control), and human control tissue using the EnVision DAB systema. Monoclonal antibodies for detection of the following antigens were used: vimentina, an intermediate filament (IF) of mesenchymal cells and neuronal and glial cell precursor cells; nestinb, an IF present in neuronal and glial cell precursor cells; {alpha}-internexinb, an IF of neurons appearing early during neurogenesis; neurofilaments (NFs)b, IFs of neurons; microtubule-associated protein 1B (MAP1B, formerly MAP5)c, a microtubule-associated protein, which appears early during neurogenesis; glial fibrillary acidic protein (GFAP)b, an IF of mature astrocytes; S-100 proteinb, a calcium binding protein present in multiple cell types including glial cells and melanocytes; and retinoblastoma protein (Rb protein, clone1F8)d, a tumor suppressor protein, which is often downregulated or absent in ocular retinoblastomas in humans owing to mutation in its gene.

Positive immunohistochemical results are shown in Table 1. Neoplastic neuroepithelial cells were vimentin, nestin, MAP1B, GFAP, and S-100 protein positive. Immunolabeling for vimentin, MAP1B, and GFAP was located in the cytoplasm. Nestin immunostaining was observed mainly in the cytoplasm, but also repeatedly in the nucleus. Neurons within the llama cerebellum displayed intracytoplasmic labeling for {alpha}-internexin, NFs, and MAP1B. Rare granular cell neurons showed weak intranuclear nestin immunostaining. Primary and metastatic tumors and the cerebellum of the llama were negative for detection of Rb protein, whereas epithelial and mesenchymal cells in human control tissue had positive immunostaining.


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  		Table 1 Immunohistochemical staining of medulloepithelioma and control tissue.

 
The diagnosis of intraocular medulloepithelioma with metastases to regional lymph nodes and visceral organs was based on typical microscopic findings together with electron-microscopic and immunohistologic features of neoplastic cells. To the authors' knowledge, this is the first case report describing a malignant nonteratoid medulloepithelioma with widespread visceral metastasis in a llama.

In llamas, only 2 previous reports of ocular medulloepitheliomas exist.9,10 While one of the cases was a malignant intraocular teratoid medulloepithelioma,10 the other reported case was not further characterized.9

Main differentials for intraocular neuroectodermal tumors are medulloepithelioma and retinoblastoma. Retinoblastomas are embryonal tumors arising from neuroepithelial cells of the developing retina. Intraocular medulloepitheliomas are derived from the undifferentiated medullary epithelium lining the optic cup, which forms retina, iris, and choroid. They may show partial differentiation toward 1 or several of the derivates of inner- and outer-medullary epithelium including photoreceptor-, neuronal- and glial-cell differentiation.13 Commonly, intraocular medulloepitheliomas arise from the ciliary body because of its delayed development and more rarely from papilla of the optic nerve6,7 In the present case, the tumor filled the entire globe, and thus the area of origin could not be determined.

Microscopic features of the presented tumor were diagnostic for medulloepithelioma and similar to those described for the neoplastic neuroepithelial cells in the malignant teratoid medulloepithelioma of the llama.10 Presence of columnar, often pseudostratified, neuroepithelium arranged in papillary and tubular formations is considered the hallmark of medulloepithelioma.4,13 In contrast to medulloepitheliomas, retinoblastomas are mainly composed of sheets of undifferentiated neuroepithelial cells.1,8,13 Recently, an ocular retinoblastoma was reported in a llama.8 Shared microscopic features of both tumor types include presence of Flexner-Wintersteiner and/or Homer Wright rosettes.1,8,10,13 However, rosettes in medulloepitheliomas are usually larger than in retinoblastomas.13

In the present case, electron-microscopic features of the llama medulloepithelioma were similar to those previously described for medulloepitheliomas.5,13,14 Typical ultrastructural features of neuroepithelial cells forming rosettes and tubules are straight lateral cellular membranes and apical desmosome-like junctions,5,13 and the presence of basal bodies (blepharoplasts) or cilia can be observed.13,14

Results of immunohistochemistry showed the tumor was composed of neoplastic primitive neuroepithelial cells (vimentin and/or nestin immunopositive cells) and tumor cells with neuronal (MA1B immunopositive) and glial (GFAP and/or S-100 positive) differentiation. Vimentin and nestin are intermediate filaments located in the cytoplasm of neuronal and glial cell progenitor cells.11 During neuronal development, vimentin and nestin are successively replaced by {alpha}-internexin and neurofilaments.11 The reason for the repeatedly observed mild intranuclear nestin staining of the neoplastic neuroepithelial cells and the granular cell neurons of the llama cerebellum is uncertain. Microtubule-associated proteins (MAPs) are components of the neuronal cytoskeleton and MAP1B (formerly MAP5) is expressed early during neurogenesis. Immunoreactivity for GFAP and S-100 protein indicates partial glial cell differentiation.

Immunohistochemical studies on medulloepitheliomas in animals are scarce.2,3,10 Previously reported medulloepitheliomas contained undifferentiated neuroepithelial cells2 or neoplastic cells with neuronal3,10 and glial cell differentiation.3 The anti-Rb antibody failed to react with llama tissue, whereas the human control tissue had a positive reaction. Lack of interspecies cross-reactivity may have been responsible for absence of immunohistochemical staining in llama tissue.

The number of reports of intraocular neoplasms including medulloepitheliomas in llamas is very limited.8–10 The present communication shows that ocular medulloepitheliomas in llamas can be highly malignant causing extensive metastases in regional lymph nodes and visceral organs.


   Acknowledgments
 
The authors are very grateful to Drs. Leon Thacker and Steven Hooser (School of Veterinary Medicine, Purdue University, West Lafayette, IN) and to Dr. Dirk Werling (Department of Pathology and Infectious Disease, Royal Veterinary College, London, UK) for critical review of the manuscript. We thank Dr. Jose Ramos-Vara (School of Veterinary Medicine, Purdue University) for helpful advice.


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From the Department of Veterinary Pathobiology and Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1175 (Schoeniger, Van Alstine), and the Department of Pathology, Memorial Hospital, Texas A&M University College of Medicine, Temple, TX 76508 (Donner). Back

a. EnVision DAB system, Dako Corp., Carpinteria, CA. Back

b. Nestin, {alpha}-internexin, neurofilaments, glial fibrillary acidic protein (GFAP), and S-100 protein, Chemicon International, Temecula, CA. Back

c. Microtubule-associated protein 1B (MAP1B), Sigma, St. Louis, MO. Back

d. Retinoblastoma protein, Neomarkers, Lab Vision Corp., Fremont, CA. Back

e. Novocastra, Vector Laboratories, Burlingame, CA. Back


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