Equine Adrenocortical Carcinoma with Hypercalcemia
A. S. FIX AND L. D. MILLER
This report describes histological and ultrastructural char- acteristics of an equine adrenocortical carcinoma (ACC) as- sociated with hypercalcemia. Reports of ACC are relatively rare in the veterinary literature, and most describe occur- rences in dogs and cattle.1,3,4 Ultrastructural characteristics of these tumors have been described in humans10 but not in animals. Two previous case reports of equine adrenal cortical tumors, one adenoma and one carcinoma,6,9 include light microscopic findings but lack ultrastructural evaluation. Hy- percalcemia associated with non-parathyroid neoplasia is a well-documented entity in animals3,4 but has rarely been re- ported in Equidae; one in a horse with lymphosarcoma5 and another with gastric squamous cell carcinoma.8
A 14-year-old quarter horse stallion presented with a his- tory of chronic weight loss, inappetance, and vague abdom- inal pain. Clinical signs at admission were lethargy, stiffness, scrotal edema, and pulmonary rales. Pertinent laboratory data included elevated white count (total white blood cells 16,200; neutrophils 14,094), hypercalcemia (15.4 mg/dl; nor- mal 9.0-13.0 mg/dl), and hypophosphatemia (1.1 mg/dl; nor- mal 3.1-7.0 mg/dl). A large mass was detected in the area of the left kidney by rectal palpation. At laparotomy there was an extensive perirenal mass joined to adjacent abdom- inal viscera by fibrous adhesions. Biopsy diagnosis by frozen section was undifferentiated carcinoma. The horse was eu- thanatized.
At necropsy there was an irregular mass, approximately 20 cm in diameter and 40 cm in length, firmly attached to and compressing the anterior pole of the left kidney. The mass was multilobulated, very firm, and white to yellow-tan. The tumor had invaded the adjacent caudal vena cava and aorta, but not the adjacent renal capsule. The left adrenal gland was completely replaced by the neoplasm, and the right adrenal gland was firmly attached to it. Fibrous adhesions connected much of the abdominal viscera and there was diffuse fibrinous peritonitis. Many metastatic nodules were evident throughout the liver, spleen, and lung. These nodules were 1-6 cm in diameter, firm, and yellow-tan. Enlarged mesenteric, mediastinal, and tracheobronchial lymph nodes contained similar tissue. Presumptive diagnosis was neopla- sia with metastasis, probably of adrenal gland origin.
Tissues were fixed in 10% neutral buffered formalin, pro- cessed by routine paraffin technique, and sectioned at 5 um. Stains included hematoxylin and eosin (HE), periodic acid- Schiff, Lewis and Lobban, alcian blue, toluidine blue, oil red O, and Giemsa. Gross chromaffin reaction on fresh tumor tissue was negative. For ultrastructural evaluation, formalin- fixed tissues were immersed in 3% glutaraldehyde in a 0.1 M cacodylate buffer at pH 7.3, and post-fixed in 1% osmium tetroxide in the same buffer. After acetone dehydration, tis- sues were infiltrated and embedded in epoxy resin (EMbed 812, Electron Microscope Sciences, Fort Washington, PA),
sectioned, and stained with 2% methanolic uranyl acetate and Reynolds’ lead citrate.
Histologically, the primary neoplasm consisted of sheets, cords, and nests of epithelial cells separated by connective tissue bands of varying thickness (Fig. 1). Cells were generally oval to polygonal although some were elongated and fusi- form. Nuclear size varied considerably. Neoplastic cells gen- erally had vesicular, round to oblong nuclei with prominent nucleoli, and finely granular, sometimes vacuolated, eosin- ophilic cytoplasm. Mitotic figures were generally few in num- ber. Frequently, homogeneous, lightly basophilic material separated individual tumor cells or was pooled centrally within a large nest of cells. Other features included multiple foci of necrosis, hemorrhage, and suppurative inflammation. Some regions of the tumor were highly vascular. All metastatic sites contained cells morphologically similar to those found in the primary mass. In the lung, liver, and spleen there were many poorly encapsulated discrete nodules, however, much of the liver and enlarged lymph nodes were diffusely infiltrated by neoplastic cells. Invasion into blood vessels was common. Neoplastic cells resembled those of the zona fasciculata in some areas but undifferentiated carcinoma in others. Nega- tive gross chromaffin reaction tentatively ruled out pheo- chromocytoma, however, ultrastructural evaluation was nec- essary to confirm ACC.
Special stains were employed to determine the nature of the pale, homogeneous material throughout this neoplasm. Periodic acid-Schiff and alcian blue stains were negative for neutral and acidic mucopolysaccharides, respectively. In ad- dition, significant characteristics were not revealed by metachromatic stains. The Lewis and Lobban method for sterol identification was positive for androgenic sterols. Oil red O stain demonstrated sporadic, variably-sized intracyto- plasmic lipid vacuoles irregularly throughout the neoplasm.
Ultrastructurally, neoplastic cells from primary and met- astatic sites were similar. Adjacent cells were joined by nu- merous desmosome-like cell junctions that lacked tonofila- ments (Fig. 2). Many cells contained a prominent Golgi apparatus, well-developed rough endoplasmic reticulum, and moderately developed smooth endoplasmic reticulum (Fig. 3). Some cells also contained lysosomes. Nuclei were fre- quently indented and folded. No membrane-limited, elec- tron-dense granules were found.
This equine ACC was anaplastic, had widespread metas- tasis, and therefore was more aggressive than the equine ACC in a previous report.9 Histologic descriptions of ACC in dogs4,7 resemble our equine case. The homogeneous basophilic ma- terial present in this tumor was incompletely characterized. The presence of androgens in parts of this amorphous ma- terial may represent sterol production by neoplastic cells of the zona reticularis. In contrast to the sporadic lipid vacuoles found in our case, moderate amounts of intracellular lipid were reported in the previous equine ACC.9
D
3
Ultrastructural characteristics of this tumor were similar to ACC in humans, as cell membrane junctional complexes were reported in each of 22 human cases.1º Electron-dense, membrane-limited secretory granules characteristic of pheochromocytomas3 were not found. Desmosome-like cell junctions and lack of secretory granules are helpful in dif- ferentiating adrenal tumors of medullary origin from those of cortical origin. Cytoplasmic glycogen, irregular microvilli, and dense matrical mitochondrial granules, reported in hu- man ACC,10 were not found in our specimen.
Functional ACC are frequently associated with clinical signs and atrophy of the contralateral adrenal cortex.3,4 Pituitary- mediated negative feedback control via ACTH suppression, resulting in atrophy of the remaining adrenal gland, is strong- ly suggestive of tumor hyperfunction.7 Evidence for func- tional ACC is well-documented in dogs4,7 but tentative in the previously reported equine case.9 In our case, clinical signs of hyperadrenocorticism were absent and the contralateral adrenal cortex was not atrophic.
The cause for hypercalcemia and hypophosphatemia in this case is unknown, however, it is likely associated with the neoplastic process. These abnormalities could be found
with primary hyperparathyroidism, pseudohyperparathy- roidism, or primary renal disease.2 Other causes for hyper- calcemia, including vitamin D toxicosis and exposure to cal- cinogenic plants, were excluded based on the history. In addition, these substances usually produce hyperphospha- temia with severe soft tissue mineralization.4 Primary renal disease was excluded by normal blood urea nitrogen values (21 mg/dl) and normal renal histology. Primary hyperpara- thyroidism, although not recognized in the horse,2 cannot be excluded since the parathyroid glands were not found at nec- ropsy. Parathyroid atrophy could account for this and would be anticipated in pseudohyperparathyroidism as a result of suppression by hypercalcemia secondary to a tumor-pro- duced factor that mimics parathyroid hormone.4,8 However, normal equine parathyroid glands are widely separated and often difficult to locate.2 Other tumor-related mechanisms for hypercalcemia include extra-renal production of vitamin D, skeletal metastasis, and tumor production of osteoclast- activating factor or prostaglandin E2.2 C-cell hyperplasia is expected in response to chronic hypercalcemia,4 but was not evident in our case. The only reports of hypercalcemia and suspected pseudohyperparathyroidism in the horse are men-
tioned above and occurred in association with lympho- sarcoma5 and gastric squamous cell carcinoma.8
Acknowledgements
The authors thank Dr. E. Reinertson and Dr. M. Gaul for providing case material, Dr. A. Ledet for consultation, and Jane Fagerland, Kay Pierce, and Sally Pyle for technical as- sistance.
References
1 Appelby EC: Tumours of the adrenal gland and para- ganglia. Bull WHO 53:227-235, 1976
2 Brewer BD: Disorders of equine calcium metabolism. Compend Contin Educ Pract Vet 4:244-251, 1982
3 Capen CC: Tumors of the endocrine glands. In: Tumors in Domestic Animals, ed. Moulton JE, 2nd ed., pp. 385- 392. University of California Press, Berkeley, 1978
4 Capen CC: The endocrine glands. In: Pathology of Do-
mestic Animals, ed. Jubb KVF, Kennedy PC, and Palmer N, 3rd ed., pp. 237-303. Academic Press, Orlando, 1985
5 Esplin DG, Taylor JL: Hypercalcemia in a horse with lymphosarcoma. J Am Vet Med Assoc 170:180-182, 1977
6 Evans LH, Hubben K, Siegel ET: Clinico-pathologic conference. J Am Vet Med Assoc 152:1778-1786, 1968
7 Kelly DF, Siegel ET, Berg P: The adrenal gland in dogs with hyperadrenocorticalism. Vet Pathol 8:385-400, 1971
8 Meuten DJ, Price SM, Seiler RM, Krook L: Gastric carcinoma with pseudohyperparathyroidism in a horse. Cornell Vet 68:179-195, 1978
9 Raker CW, Fegley H: Clinico-pathologic conference. J Am Vet Med Assoc 147:848-860, 1965
10 Silva EG, Mackay B, Samaan NA, Hickey RC: Adre- nocortical carcinomas: an ultrastructural study of 22 cases. Ultrastruct Pathol 3:1-7, 1982
Request reprints from Dr. Andrew S. Fix, Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (USA).
Vet. Pathol. 24:192-194 (1987)
Cerebral Phaeohyphomycosis in Two Dogs and a Cat
D. L. DILLEHAY, J. L. RIBAS, J. C. NEWTON, JR., AND R. P. KWAPIEN
Phaeohyphomycosis is a mycotic infection of man and animals caused by opportunistic, dematiaceous (pigmented) fungi. The fungi grow in tissue as darkly pigmented, septate hyphae with occasional branching.2 Various pigmented fungi have been isolated from superficial, cutaneous, subcuta- neous, and systemic lesions. 1,2,6 Cerebral phaeohyphomycosis is a rare, fatal infection of man and animals and almost always caused by Cladosporium bantianum.2,8 C. bantianum is considered synonymous with C. trichoides since the fungi are antigenically alike.4 We found only two reports of cerebral phaeohyphomycosis in animals.5,8 This report describes the clinical and pathological findings of cerebral phaeohypho- mycosis in two dogs and a cat.
Case 1. A 4-month-old female pit bulldog had a 3 day history of seizures and a temperature of 105.0 F. The dog had not received any vaccinations and had been healthy. Because of neurologic abnormalities, it was euthanized.
Case 2. A 6-month-old, male Dachshund mixed breed dog had a 3 week history of neck stiffness and pain upon manipulation. It had not received vaccinations. Treatment with antibiotics and steroids resulted in temporary clinical improvement. Cervical radiographs were normal. Analysis of cerebrospinal fluid showed white blood cells, 26,400/cu mm; protein, 298 mg/dl; and glucose, 80 mg/dl. Cultures of cerebrospinal fluid were negative. The dog had several grand mal seizures and was euthanized.
Case 3. A 6-year-old male castrated Persian cat had a 3 week history of intermittent ataxia, lethargy, and occasional episodes of circling to the left with right-sided hemiparesis.
Antibiotics and steroids resulted in temporary improvement. Chest and cranial radiographs were normal. Electroenceph- alograms suggested encephalitis involving the left frontal lobe. During anesthesia to obtain cerebrospinal fluid, the cat died.
At necropsy, gross lesions were seen only in the brain in all three animals. Meninges of the dogs were diffusely opaque, congested and multifocally adherent to the cerebrum. Case 1 had a discrete, focal, green-brown lesion, 1.0-1.5 cm in diameter, in the right thalamus. Case 2 had a well circum- scribed, red-green malacic lesion, 0.5-1 cm in diameter, on the dorsal surface of the right parietal lobe. In Cases 2 and 3, there were multifocal, discrete to coalescing, green-brown foci varying in diameter from 0.1 to 0.5 cm in both white and gray matter of the dorsolateral area of the frontal lobe (Fig. 1).
Microscopically, lesions varied from abscesses, some of which were encapsulated by thick fibrous tissue, to less dis- crete and often coalescing foci of pyogranulomatous inflam- mation. Within epithelioid and multinucleated giant cells, and extracellularly, were moderate numbers of septate, pig- mented hyphae 3-6 um in width. Hyphae were light brown to basophilic in hematoxylin and eosin (HE)-stained sections and had parallel contoured walls with occasional branching (Fig. 2). Hyphae were also within blood vessels, some of which were thrombosed. All three animals had multifocal suppurative meningitis. Case 3 had diffuse pyogranuloma- tous ependymitis with hyphae in the lateral ventricles. Phaeo- hyphomycotic lesions were not present in extraneural sites. In Case 1, other lesions included a diffuse lymphocytic