JPC SYSTEMIC PATHOLOGY
ENDOCRINE SYSTEM
February 2022
E-N06

SLIDE A: Signalment (JPC #1441190): 14-year-old Holstein bull

HISTORY: None

HISTOPATHOLOGIC DESCRIPTION: Adrenal gland: Expanding and replacing the medulla, multifocally compressing and infiltrating the adjacent cortex, and extending to one section margin is an unencapsulated, well demarcated, densely cellular, multilobulated neoplasm composed of polygonal cells arranged in nests and packets, often palisading along a fine fibrovascular stroma. Neoplastic cells have distinct cell borders, abundant finely granular, amphophilic cytoplasm, and an irregularly round, anti-basilar nucleus with finely stippled chromatin and one distinct nucleolus. Occasionally, neoplastic cells palisade around blood vessels forming pseudo-rosettes. There is moderate anisocytosis and anisokaryosis. Mitoses average less than 1 per 2.37 mm². Within the center of the neoplasm, there are occasional areas of hemorrhage, fibrin, and edema up to 0.5 mm in diameter and few hemosiderin-laden macrophages.

MORPHOLOGIC DIAGNOSIS: Adrenal gland: Pheochromocytoma.

SLIDE B: Signalment (JPC #1894867): A dog

HISTORY: None

HISTOPATHOLOGIC DESCRIPTION: Adrenal gland: Replacing the medulla and multifocally compressing and invading the adjacent severely atrophied cortex and extending to cut borders is an unencapsulated, well demarcated, densely cellular, multilobulated neoplasm composed of polygonal cells arranged in nests and packets, often palisading along a fine fibrovascular stroma. Neoplastic cells have distinct cell borders, amphophilic to brownish, granular cytoplasm, and irregularly round, often anti-basilar nuclei with finely stippled chromatin and one nucleolus. Occasionally, neoplastic cells palisade around blood vessels, forming pseudo-rosettes. There is moderate anisokaryosis. There are up 4 mitotic figures per individual high power field (0.237 mm²). There is mild multifocal hemorrhage, fibrin, and edema with few hemosiderin-laden macrophages.

MORPHOLOGIC DIAGNOSIS: Adrenal gland: Pheochromocytoma.

GENERAL DISCUSSION:

The adrenal medulla:

• Arises from the neuroectoderm of the neural crest, unlike the adrenal cortex
• Secretes catecholamine hormones (epinephrine and norepinephrine)
• Composed of chromaffin cells (have a positive chromaffin reaction, which is the oxidation of catecholamines by potassium dichromate à brown-yellow pigment; low sensitivity test)
• Blood flows from the cortex to the medulla, therefore the adrenal medulla sees blood with the highest concentration of corticosteroids of any location in the body; the enzyme that converts norepinephrine into epinephrine (phenylethanolamine-N-methyl transferase, or PMNT) is corticosteroid dependent
• Catecholamine synthesis: Tyrosine (dietary or from hydroxylation of phenylalanine) à 1-dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase à decarboxylated to 1-dihydroxyphenylethylamine (dopamine) by 1-amino acid decarboxylase à beta hydroxylation to norepinephrine by dopamine beta-hydroxylase à methylated to epinephrine by PMNT in epinephrine-producing chromaffin cells
• Sympathetic nerve fibers synapse directly on adrenal medullary secretory cells à increased activity of tyrosine hydroxylase and dopamine beta-hydroxylase activity à catecholamine synthesis is stimulated
• Catecholamine breakdown products are excreted in the urine and can be measured

Pheochromocytomas:

• The most common neoplasms arising in the adrenal medulla of animals
• Most frequently reported in cattle and dogs; rare in other species except clouded leopards; usually middle-aged dogs with no breed or sex predilection
• Arise from the catecholamine-secreting chromaffin cells of the adrenal medulla, either epinephrine-secreting, norepinephrine-secreting, or both (differentiated ultrastructurally)
  • Although the normal adult medulla secretes predominantly epinephrine, pheochromocytomas tend to produce mainly norepinephrine
• Always located in the adrenal gland; non-adrenal (extramedullary) neoplasms arising from chromaffin cells are termed paragangliomas
• May be functional or nonfunctional; functional tumors are infrequent, they secrete epinephrine and/or norepinephrine
• May be benign or malignant; malignant pheochromocytomas invade through the adrenal capsule and into adjacent structures or metastasize to distant sites
• In dogs, pheochromocytomas carry a guarded prognosis, more related to surgical factors associated with adrenalectomy; the relationship between survival and invasiveness is unclear
• In dogs, pheochromocytomas are locally invasive in 39-50% of cases and have distant metastasis in 13-24% of cases, with metastasis to liver, regional lymph nodes, spleen, lungs, kidney, bone
• In humans, benign versus malignant designation is determined using a pheochromocytoma of the adrenal gland scaled score (PASS) based on histologic parameters and immunohistochemistry (worse prognosis with higher expression of Ki-67, COX-2, p53, BCL-2, c-erbB-2, VEGF, and decreased expression of S100); a recent study in dogs (Zini, Vet Pathol, 2018) indicated these as well as chromogranin A expression were not prognostically significant
• Multiple endocrine neoplasia-like (MEN) syndrome: Pheochromocytomas may develop concurrent with calcitonin-producing C cell thyroid gland tumors (E-N05), +/- other endocrine neoplasms (e.g. pituitary adenoma, parathyroid adenoma) and may represent neoplasia of multiple neuroectodermal origin endocrine cells; this is most commonly reported in bulls (and humans), but has also been reported in dogs, cats, ferrets, horses, and baboons

PATHOGENESIS:

• The cause in domestic animals is unknown; there may be a familial predisposition in cattle with an autosomal dominant inheritance pattern in Guernsey cattle
• Primary endocrine hyperfunction: Functional pheochromocytoma à production of catecholamines (epinephrine and/or norepinephrine) à tachycardia, edema, cardiac hypertrophy, paroxysmal hypertension-induced arteriolar sclerosis and arteriolar medial hypertrophy
• In primary endocrine hyperfunction (in general), cells autonomously produce hormone without trophic stimulation, as opposed to secondary hyperfunction in which there is increased hormone release in response to a trophic hormone
• Pheochromocytomas often invade adjacent tissues and can reach the caudal vena cava à formation of large thrombi à partial to total occlusion à abdominal effusion or sudden death due to acute hemorrhage

TYPICAL CLINICAL FINDINGS:

• Nonfunctional tumors: Space occupying mass or local vascular invasion; may cause vomiting, abdominal pain, and abdominal distension; can rupture and cause hemoperitoneum
• Functional tumors: Catecholamine release causes tachycardia, hypertension, hyperthermia, and sweating
• Caudal vena cava invasion: Edema and/or abdominal hemorrhage and/or sudden death due to hemorrhage

TYPICAL GROSS FINDINGS:

• Usually dark reddish-brown, often large (>10 cm), soft, well demarcated nodules, which often incorporate the majority of the gland leaving a small remnant of the adrenal gland at one pole
• Areas of necrosis and hemorrhage
• Most often unilateral but may be bilateral
• Large, malignant pheochromocytomas may compress or invade adjacent structures (e.g. caudal vena cava in which it can create an intravascular tumor embolus, aorta)
• With functional tumors, dogs and horses can have cardiac hypertrophy

TYPICAL CYTOLOGIC FINDINGS:

• Similar cytologic appearance as other neuroendocrine tumors
• Naked nuclei against a pale basophilic background
• Intact cells are usually identified in carefully prepared specimens
• Lack of nuclear features of malignancy are unreliable in predicting benign biologic behavior; tumors of well differentiated cells are known to metastasize or invade surrounding tissue
• However, when nuclear features of malignancy are present, they strongly suggest potential for local invasion or metastasis

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Characteristic neuroendocrine pattern with irregular cords and arcuate arrays of small cuboidal to large polygonal cells separated into small nests and packets or lobules by fine connective tissue septa with capillaries
• Cells have amphophilic (or “muddy” brownish purple) pale, finely granular cytoplasm with hyperchromatic nuclei, often with indistinct cell membranes due to rapid autolysis of the adrenal medulla
• There is histologic overlap between benign and malignant pheochromocytomas, nuclear pleomorphism is not a reliable indicator of malignancy; proof of malignancy requires invasion through the adrenal capsule or distant metastasis
• In other organs, there may be histologic evidence of hypertension:
• Cardiovascular lesions: cardiomyocyte hypertrophy, coronary artery degeneration and fibrosis with medial thickening, arteriolar sclerosis
• Retinal lesions: detachment and/or degeneration

ULTRASTRUCTURAL FINDINGS:

• The principle distinguishing feature between norepinephrine- and epinephrine-secreting pheochromocytomas is the ultrastructural appearance of the cytoplasmic secretory granules:
• Norepinephrine secretory granules have an eccentric electron dense core surrounded by a wide submembranous space
• Epinephrine secretory granules have a coarsely granular internal core of lower density than norepinephrine granules and a narrow submembranous space

ADDITIONAL DIAGNOSTIC TESTS:

• Henle chromaffin reaction: Application of Zenker’s solution (which includes potassium dichromate) to a freshly cut tumor causes oxidation of catecholamines forming dark brown pigment within 20 minutes
• Special stains: Churukian Schenk method: neuroendocrine cell granules (argyrophil and argentaffin) stain dark brown to black
• Immunohistochemistry: Immunoreactive for neuroendocrine markers (Chromogranin A, synaptophysin, protein gene product 9.5 (PGP 9.5), neuron-specific enolase), catecholamines, catecholamine-synthesizing enzymes, and opioid peptides (met-enkephalin, leu-enkephalin, beta-endorphin, dynorphin B)
• Urinalysis: increased detection of free catecholamines and breakdown products of catecholamines (vanillylmandelic acid, +/- metanephrine, normetanephrine) indicates functional pheochromocytoma (especially in bulls); ELISA for normetanephrine in urine samples has shown consistent results in canine urine (Höglund, J Vet Diagn Invest, 2022)

DIFFERENTIAL DIAGNOSIS:

• Adrenal medullary hyperplastic nodules: Multiple nodules (pheochromocytoma is usually solitary although may be bilateral)
• Poorly differentiated adrenocortical carcinoma: May require immunohistochemistry to differentiate (adrenal cortical cells do not express neuroendocrine markers or opioid peptides, and may express α-inhibin and melan-A, calreticulin, steroidogenic factor 1, S-100)
• Neuroblastoma: Primitive neuroectodermal tumor typically of young animals that can develop in the central or peripheral nervous system (PNS); in the PNS it often arises in the adrenal medulla and can form a large intra-abdominal mass
• Ganglioneuroma: Benign tumor that can develop in the adrenal medullary tumor in young animals composed of multipolar ganglion cells and neurofibrils separated by a prominent connective tissue stroma
• Mixed adrenal medullary tumor (complex pheochromocytoma): Features of pheochromocytoma and ganglioneuroma and/or neuroblastoma with nerve fibers and Schwann cells, intermingled or in separate areas; represents divergent differentiation from a common stem cell

COMPARATIVE PATHOLOGY:

• Pheochromocytomas occur in all animals but are most common in cattle, dogs, non-human primates, and clouded leopards, with rare other individual reports in the literature
• Non-domestic felids:
• Pheochromocytomas are generally uncommon
• Clouded leopards (endangered) seem to have a species predilection; a recent report (Corner, Vet Pathol, 2017) indicates that historically 45% of necropsied clouded leopards had pheochromocytomas; in this report, pheochromocytomas were bilateral in 5/20, the cause of death due to vena cava invasion in 4/20 one of which also had pulmonary metastasis, all contained both norepinephrine and epinephrine granules, all were immunoreactive for chromogranin A, PGP 9.5, and synaptophysin, and 7/20 animals had histologic evidence suggestive of hypertension in other organs (e.g. retinal detachment, retinal degeneration, myocardial muscular arteriosclerosis)
• Non-human primates:
• New World Primates: Reported more commonly than in Old World primates, and a high frequency has been reported in a colony of cotton-top tamarins, several of which had concurrent myocardial degeneration and vascular proliferation and thickening that may indicate tumor functionality
• Old World Primates: Rarely reported; the first report in African green monkeys and the first of malignant pheochromocytoma in Old World primates described pulmonary metastasis in 2 rhesus macaques and portal vein invasion in 1 African green monkey (Colgin, Vet Pathol. 2016)
• Aye-ayes (endangered prosimian): A recent report describes the first reports of neoplasia in aye-ayes, one of these neoplasms was an incidental pheochromocytoma (Rodriguez Barbon, J Comp Pathol, 2018)
• Rats: Some strains of rats have a high incidence of pheochromocytoma, suggesting a genetic of familial predisposition; chronic adrenal medullary stimulation from stress and hypercalcemia increases the incidence of pheochromocytoma
• Gerbils: Breeding females and to a lesser extent males develop a hyperadrenocorticism/cardiovascular disease syndrome; some of these animals have pheochromocytomas; cause and effect relationships have not been determined

REFERENCES:

1. Bakthavatchalu V, Muthupalani S, Marini RP, Fox JG. Endocrinopathy and aging in ferrets. Vet Pathol. 2016;53(2):349-365.
2. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits, 4th ed. Ames, IA:Wiley Blackwell; 2016:207-208.
3. Choi US, Arndt T. Endocrine/Neuroendocrine System. In: Raskin RE, Meyer DJ, eds. Canine and Feline Cytology: A Color Atlas and Interpretation Guide, 3^rd St. Louis, MO:Elsevier. 2016:444.
4. Colgin LM, Schwahn DJ, Castillo-Alcala F, Kiupel M, Lewis AD. Pheochromocytoma in old world primates (Macaca mulatta and Chlorocebus aethiops). Vet Pathol. 2016;53(6):1259-1263.
5. Corner S, Walsh T, Padilla L, MacNeill A, Wallig M, Kiupel M, Terio K. Histologic and immunohistochemical characterization of pheochromocytomas in 20 clouded leopards (Neofelis nebulosa). Vet Pathol. 2017;54(2):269-276.
6. Höglund K, Palmqvist H, Ringmark S, Svensson A. Quantification of normetanephrine in canine urine using ELISA: evaluation of factors affecting results. J Vet Diagn Invest. 2022;34(1):28-35.
7. Miller AD. Neoplasia and proliferative disorders of nonhuman primates. In: Abee CR, Mansfield K, Tardiff S, Morris T, eds. Nonhuman Primates in Biomedical Research: Diseases, Vol. 2, 2nd ed. Waltham, MA: Academic Press; 2012:338-339.
8. Miller MA. Endocrine system. In: McGavin MD, Zachary JF, eds. Pathologic Basis of Veterinary Disease. 6th ed. St. Louis, MO: Elsevier; 2017:693-694, 709-710.
9. Rodriguez Barbon A, Cowen R, Knott C, et. al. Neoplasia in three Aye-Ayes (Daubentonia madagascariensis). J Comp Pathol. 2018;159:16-20.
10. Rosol TJ, Gröne A. Endocrine glands. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 3, 6th ed. Philadelphia, PA: Elsevier; 2016:348-352.
11. Terio KA, McAloose D, Mitchell E. Felindae. In: Terio KA, McAllose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:269.
12. Zini E, Nolli S, Ferri F, et. al. Pheochromocytoma in dogs undergoing adrenalectomy. Vet Pathol. 2018;56(3):358-368.

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