Suprasellar Differentiated Germ Cell Tumor in a Male Dog

Brief communications

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18. White ME, Pennock PW, Seiler IU: 1978, Atlanto-axial subluxation in five young cattle. Can Vet J 19:79-82. 19. Whitwell KE: 1978, Craniovertebral malformations in an Arab foal. Equine Vet J 10:125-126.

20. Wilson WD, Hughes SJ, Ghoshal NG, et al.: 1985, Occipitoatlantoaxial malformation in two non-Arabian horses. J Am Vet Med Assoc 187:36-40.

J Vet Diagn Invest 5:462-467 (1993)

Suprasellar differentiated germ cell tumor in a male dog Abraham Nyska, Alon Harmelin, Gad Baneth, Boris Yakobson, Per1 Shmuel, Uri Orgad, Trevor Waner Germ cell tumors arise as the result of neoplastic changes that occur during embryonic development.12 In humans, germ cell tumors are classified into germinomas and differentiated germ cell tumors. The germinoma is regarded as a pure cell line.4 The differentiated tumors are further subclassified into embryonic (embryonal carcinoma and mature or immature teratoma) and extraembryonic (choriocarcinoma and yolk sac or endodermal sinus tumor) types. Each tumor represents the malignant correlate of a normal stage of embryonic development.4 Valentine11 reassessed the terminology regarding canine germ cell tumors in the intracranial site. He reclassified previously diagnosed craniopharyngiomas as germ cell tumors and emphasized the need to study additional canine tumors to better understand the classification. This article describes the morphologic and immunohistochemical characteristics of a differentiated embryonal germ cell tumor (germinoma mixed with teratomatous elements) in a male miniature Doberman Pinscher dog. A 3-year-old male miniature Doberman Pinscher dog was referred to the Koret Veterinary Hospital with a history of complete blindness that had been preceded by difficulties in identifying distant objects. Two weeks previously, the dog had developed exophthalmus, conjunctivitis, and polyuria. The physical examination revealed bilateral mydriasis, lack of menace reflex, and absence of direct or consensual pupillary light reflex. Ophthalmoscopy did not demonstrate any pathology. The dog showed disorientation, proprioception deficits, severe ataxia, and circling. Hematology and blood chemistry profiles were within the normal range. Urine specific gravity was 1.010 (hypostenuria). The dog was euthanized at the owner’s request. At necropsy, a 10- x 10- x 6-mm dark red irregular firm mass was found in the hypothalamic area dorsal to the sella turcica (Fig. 1). The tumor was compressing the hypothalamus, optic chiasma, and pituitary gland. Tissues were fixed in 10% buffered formalin saline, embedded in paraffin, sectioned at 4 µm, and stained with he-

matoxylin and eosin (HE), periodic acid-Schiff (PAS), and Masson’s trichrome stain. Immunohistochemical staining for α-fetoprotein (AFP),a S-100,a neuron-specific enolase (NSE),a keratinb and vimentinb were performed on selected slides using the avidin-biotin peroxidase complex (ABC) technique.5 Positive controls were fetal liver (AFP), peripheral nerve (S-100) brain (WE), skin (keratin), and small intestine (vimentin). Immunohistochemical reactions were graded as negative (-), slight (+), moderate (+ +), and marked (+ + +). Histologic examination showed that the tumor grew by expansion with short invading neoplastic tongues penetrating the adjacent brain. The unencapsulated neoplasm was characterized by a complex mixture of various cell types and growth patterns. The predominant component, involving about 75% of the tumor mass, was composed of lobular islands of noncohesive medium-sized pleomorphic neoplastic germinal type cells with scanty acidophilic cytoplasm (Fig. 2). The islands were separated by collagenous septa of various widths. The collagen composition was confirmed by Masson’s trichrome stain and by vimentin-positive reaction (Fig. 3, Table 1). The nuclei of the germinal cells either were

From the Department of Pathology, Kimron Veterinary Institute, Beit Dagan 50250, Israel (Nyska, Yakobson, Orgad), the Koret School of Veterinary Medicine, Hebrew University, Jerusalem, Israel (Harmelin, Baneth), and Life Science Research, PO Box 139, Ness Ziona 70451, Israel (Waner). Received for publication August 10, 1992.

Figure 1. Subgross photograph, transverse section of the brain of a dog at the thalamic area. Germ cell tumor is mixed with teratomatous elements and is not encapsulated. Note the hypothalamic compression. The arrow indicates the third ventricle. Some areas are composed of lobular islands of germinal cells (A) or a mixture of germinal, mesenchymal, and epithelial cells (B).


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Figure 2. Histologic appearance of germinal cell islands in brain tumor of a dog. Note the uniform cell population separated by collagenous septa. Masson’s trichrome.

vesicular with marginated chromatin and a central prominent nucleolus or were small and homogeneously hyperchromatic. Cells arranged in true rosettes with scanty acidophilic material in the center, reminiscent of immature nervous tissue, were rare. Mitotic figures were frequently noted. The cytoplasm of the germinal cells had slight to moderate diffuse positive reaction to AFP antibodies. A second type of neoplastic tissue (approximately 25% of the entire tumor mass) was located near the midline adjacent to the brain and was contiguous with the main body of the tumor. Germinal, mesenchymal, and epithelial tissue components were intermingled (Figs. 4, 5). The germinal tissue

layered structures with desquamating cell debris and inflammatory cells in their center. A few irregular cysts of various sizes lined by single to multilayered squamous epithelium were also seen (Fig. 6). The cysts contained PAS-positive proteinaceous fluid, cell debris, erythrocytes, and a few polymorphonuclear cells. The tumor tissue was immunohistochemically negative for NSE and S-100. Staining for keratin antigens aided in discriminating between germinal and differentiated squamous cells; the squamous cells showed a marked diffuse cytoplas-

cells tended to be arranged perpendicular to the surrounding basement membrane. The mesenchymal tissue was composed of rarefied bony trabeculae and irregular fibrous tissue. The bony tissue was mostly unmineralized, with only a few basophilic calcified foci. Masson’s trichrome stain was used to differentiate between osteoid and squamous epithelial elements; the osteoid tissue stained green. The epithelial elements contained squamoid cells arising from the germ tissue. The more mature cells were rich in acidophilic cytoplasm. In some areas the squamous cells were arranged in multiTable 1. Results of immunohistochemical staining* of different elements of the germ cell tumor of a dog.

Figure 3. Brain tumor of a dog, immunohistochemical staining for vimentin. Note positive reaction (arrow) confined to the fibrous collagenous septa. ABC method, Mayer’s hematoxylin counterstain.

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Figure 4. Brain tumor of a dog. Note the transition zone between the germinal cell islands (left), the woven bone (center), and cysts lined by squamous epithelium (upper, right). Masson’s trichrome.

mic staining, whereas the germinal cells were negative (Table 1; Figs. 7, 8). The adjacent neural tissue underwent compression as a result of the expanding growth of the tumor. This compression was evident from the spongy appearance of the brain tissue around the tumor, the frequent hemorrhage, and the appearance of polymorphonuclear cells. The pituitary gland and the optic chiasma were not available for histologic examination. Based on the morphologic and immunohistochemical characteristics, this suprasellar tumor was diagnosed as a differentiated embryonal germ cell tumor with predominant

Figure 5.

trichrome.

germinomatous characteristics and some teratomatous elements. The reason for development of germ cell tumors in the intracranial site is unknown; however, the most widely accepted theory relates to the misplacement of germ cells.7 These cells arise from the yolk sac endoderm and migrate widely in the embryo before localizing in the gonadal ridge.1,7 Sometimes, primitive cells wander and develop in the cranial cavity of the embryo. These cells survive the physiologic dissolution during the development of the embryo and later may progress into germ cell tumors. The germ cell tumor in all its sites, gonadal or otherwise,

Brain tumor of a dog. Note the transition zone between the germinal cell layer (upper) and woven bone (lower). Masson’s


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Figure 6. Irregular epidermoid cysts and ducts in brain tumor of a dog. Note the multilayered desquamating squamous epithelium and the thick fibrous external wall. Masson’s trichrome.

is primarily a midline neoplasm.1 Intracranial localization has been described mostly in the parapineal, hypothalamic, and basal areas. The clinical signs in this dog were similar to those in humans4 and previously described in dogs.6,9 Diabetes insipidus has been documented in 41% of cases of central nervous germinomas, visual field defects in 33%, pyramidal tract signs in 14%, and ataxia in 9% of cases. Although not confirmed, the polyuria in this dog was probably a sign of diabetes insipidus. Intracranial germinomas tend to cause a higher frequency of neurologic signs in dogs than in humans, possibly because of anatomical differences of the skull and brain between dogs and humans. Germ cell tumors are rare in dogs. 11 The classification of the tumor is problematic and has led many investigators to misdiagnose the growth as a craniopharyngioma. Valentine11 based his classification on human literature and summarized the criteria for the diagnosis of germ cell tumors: 1) location; 2) the presence of several distinct cell types, 1 population that resembles seminoma or dysgerminoma and others that suggest teratomatous differentiation (secretory glandular and squamous elements); and 3) positive staining for AFP. Based on these criteria, the mass in this dog was diagnosed as a germ cell tumor. The presence of teratomatous tissue (bone and cysts lined by squamous epithelium) allows for the further subclassification of the tumor as a differentiated embryonic germ cell tumor. One of the tumors reclassified by Valentine was a craniopharyngioma in a dog.3 This tumor was reclassified as a germ cell tumor on the basis of the lack of cells resembling ameloblastoma, which is considered to be a diagnostic feature of craniopharyngioma in humans. We agree with this approach but would further subclassify the tumor as differentiated and of embryonic origin. This tumor strongly resembles the growth described in the present report, which is the second report of osseous tissue in a canine suprasellar germ cell tumor. The tumor in this report was stained for AFP because AFP

has been used as a marker in human and canine germ cell tumors.11 In the present case, AFP and keratin immunohistochemical staining was useful for distinguishing germinal. cells from mature squamous cells. Horowitz organized the germ cell tumors according to their increasing malignant potential.4 The tumor described in the, present report was considered of low malignancy because of the short invading tumor extensions and the differentiated areas. This assessment is in agreement with the classification of Horowitz,4 who stated that teratomas tend to be the least malignant, and choriocarcinomas are the most malignant. Differential diagnosis for germ cell tumor also includes intracranial teratoma and intracranial meningioma. Intracranial teratomas also occur in intracranial midline locations in the hypothalamus, suprasellar areas, and pineal gland in dogs. A prominent feature of the intracranial teratomas is the presence of a mixture of differentiated cells of more than 1 embryologic stem cell derivative in various proportions.7 This feature was not observed in the germ cell tumor described in this report. Although the intracranial meningioma may be found in the basal location of the brain, the histologic appearance allows for its differentiation from other brain tumors.10 The most common microscopic appearance is that of whorls of elongated crescentic cell profiles; bone and cartilage elements are only rarely found.2 Immunohistochemical staining of intracranial meningioma in dogs has been positive for vimentin and negative for cytokeratin,10 in contrast to the marked positivity in neoplastic islands described in the germ cell tumor in the present case. However, the cytokeratin reaction appears to be variable; in other reports, a small number of intracranial meningiomas have been found positive for cytokeratin-s Forty percent of the 10 dogs described by Valentine” belonged to the Doberman Pinscher breed. The present case also involved a Doberman Pinscher, albeit the miniature

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Figures 7,8. Brain tumor of a dog, immunohistochemical staining for keratin. Note positive reaction of mature squamous cells arising adjacent to the negative staining germinal cells. ABC method, Mayer’s hematoxylin counterstain.

variation, and provides further evidence of a predilection of

this breed to this type of tumor. The male : female ratio for the 10 dogs with germ cell tumors previously described is 1.57: 1, which is similar to the ratio in humans (2.24: 1).4 The diagnosis of this differentiated embryonal germ cell tumor in a male miniature Doberman Pinscher was based

on the morphologic and immunohistochemical characteristics. A more complete categorization for this type of tumor, as used in human pathology and outlined here, should be adopted. The publication of more reports on this type of tumor in dogs will allow for better understanding these germ -cell neoplasms. Acknowledgement. We acknowledge the technical assistance of Mrs. N. Shehat.

Sources and manufacturers a. Dako Corp., Carpinteria, CA. b. Biogenex Laboratories, San Ramon, CA.

References 1. Cordy DR: 1984, Intracranial germinoma in a dog. Vet Pathol 21:357-358. 2. Cordy DR: 1990, Tumors of the nervous system and eye. In: Tumors of domestic animals, ed. Moulton JE, 3rd ed., pp. 640665. University of California Press, Berkeley, CA. 3. Hawkins IU, Diters RW, McGrath JT: 1985, Craniopharyngioma in a dog. J Comp Pathol 95:469474. 4. Horowitz MB, Hall WA: 1991, Central nervous system germinoma. A review. Arch Neural 48:652-657.

Brief communications 5. Hsu SM, Raine L, Farger H: 1981, Use of avidin-biotin-peroxidase-complex (ABC) in immunoperoxidase techniques: a comparison between the ABC and unlabeled antibody (PAP) procedure. J Histochem Cytochem 29:577-580. 6. Neer TM, Reavis DU: 1983, Craniopharyngioma and associated central diabetes insipidus and hypothyroidism in a dog. J Am Vet Med Assoc 182:519-520. 7. Patnaik AK, Nafe LA: 1980, Intracranial teratocarcinoma in a dog. Vet Pathol 17:764-769. 8. Ribas JL, Carpenter JL, Mena H, et al.: 1991, Central system meningioma in the dog: a review of 50 cases. J Neuropathol Exp Neurol 50:373.

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9. Saunders LZ, Richard CG: 1952, Craniopharyngioma in a dog with apparent adiposogenital syndrome and diabetes insipidus. Cornell Vet 42:490-495. 10. Schulman FY, Carpenter JL, Ribas JL, Brum DE: 1992, Cystic papillary meningioma in the sella turcica of a dog. J Am Vet Med Assoc 200:67-69. 11. Valentine BA, Summers BA, de Lahunta A, et al.: 1988, Suprasellar germ cell tumors in the dog: a report of five cases and review of the literature. Acta Neuropathol 76:94-100. 12. Walsh JW: 1985, Suprasellar germinomas. In: Neurosurgery, ed. Wilkins WH, Rengachary SS, pp. 921-925. McGraw-Hill, New York, NY.

J Vet Diagn Invest 5:467-471 (1993)

Meningoencephalitis in mink associated with a Sarcocystis neurona-like organism J. P. Dubey, Olaf R. Hedstrom Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. are the 3 major protozoans that cause encephalomyelitis in animals. 1,6 This is the first report of Sarcocystis-associated meningoencephalitis in mink. Two l-month-old female Mahogany mink (Mustela vison) were submitted live to the Oregon State University Veterinary Diagnostic Laboratory, Corvallis, for diagnosis. These kits came from a farm of approximately 15,500 mink. Affected kits had just been weaned and started on solid feed (turkey and fish parts). The owner described the kits as weak and ataxic, and he thought that they were starving to death even though they had access to adequate feed. Prior to necropsy, the 2 submitted mink were weak and comatose. At the time of submission, 50 kits had died over a 2-week period. Once a litter was affected, eventually the majority of the remaining litter died. The deaths were randomly distributed throughout the farm. The owner also reported an increased number of abortions and fetal loss during this kindling season. Virgin breeding mink were vaccinated at breeding and with yearly boosters of a 4-way commercially available vaccine for mink distemper, botulism, mink viral enteritis, and hemorrhagic Pseudomas aeruginosa pneumonia. The submitted mink were euthanized and necropsied. Grossly, there was a focal region of light yellow discoloration with indistinct boundaries in the right frontal cerebral cortex of 1 mink. The lumens of stomach and intestines of both mink were empty. Specimens of the heart, brain, kidneys, urinary bladder, liver, thymus, stomach, small and large intestines, lymph nodes, and lungs from both mink were fixed in 10% buffered formalin. Paraffin-embedded 5-6-µm-thick sections were

From the Zoonotic Diseases Laboratory, Livestock and Poultry Sciences Institute, BARC-East, ARS, USDA, Beltsville, MD 207052350 (Dubey), and Oregon State University, Veterinary Diagnostic Laboratory, PO Box 429, Corvallis, OR 97339-0429 (Hedstrom). Received for publication November 27, 1992.

initially stained with hematoxylin and eosin (HE) and evaluated microscopically. Selected specimens of the brain were embedded in methacrylate, and 2-3-µm-thick sections were stained with HE and periodic acid-Schiff (PAS). Paraffinembedded sections were retrospectively stained with antisera to T. gondii, N. caninum, and Sarcocystis cruzi as reported previously.* For ultrastructural examination, a 4-mm area of brain lesions from a paraffin block was routinely processed for transmission electron microscopy (TEM). Microscopically, in the cerebral cortex there were focally extensive areas of severe necrosis and inflammation that often involved an entire gyrus. The sheets of inflammatory leukocytes consisted of primarily neutrophils with fewer macrophages and lymphocytes. In adjacent regions and scattered throughout the remaining brain parenchyma, there was multifocal abscessation, angiocentric accumulations of leukocytes, vasculitis, meningitis, and gliosis (Fig. 1). Numerous protozoa seen in 6-µm HE-stained sections appeared to be structurally similar to T. gondii (Fig. 1B). A closer examination of plastic-embedded 1-3-µm sections revealed immature and mature schizonts (Fig. 1D). The structure of the protozoa as seen in 1-µm toluidine blue-stained sections and by TEM are shown in Figs. 2-4. Only asexual stages were seen. The protozoa divided by endopolygeny, a divisional process similar to that of Sarcocystis schizonts.6 Schizonts were located in the cytoplasm of neurons without a parasitophorous vacuole. The earliest merozoite ready to differentiate into a schizont had a centrally located nucleus (Fig. 2A). The earliest schizont (5 x 7 µm) contained a 4-5-µm-diameter nucleus with a prominent nucleolus (Fig. 2B). In later schizonts, the nucleus was lobed (Figs. 2C, 3). The parasite nucleus was at times wrapped around the host cell nucleus. The nucleus in immature schizonts was highly irregular, and the nucleolus disappeared when the nucleus became multilobed (Fig. 2D). Merozoites were formed internally as well as peripherally (Fig. 2E), and the merozoite formation was not synchronous. Sometimes 2 schizonts were seen within the same host cell,