E-N09

SIGNALMENT (JPC #2064150): A dog; age, sex and breed unknown

HISTORY: This dog had clinical signs of hyperadrenocorticism.

HISTOPATHOLOGIC DESCRIPTION: Adrenal gland: Infiltrating and effacing normal adrenal gland architecture and compressing the remaining adrenal cortex is an unencapsulated, poorly demarcated, multilobular, densely cellular neoplasm composed of polygonal cells arranged in cords, nests, and packets on a fine fibrovascular stroma which forms lobules separated by dense reticulations of collagen. Neoplastic cells have indistinct borders, a moderate amount of microvacuolated eosinophilic cytoplasm, and a round to oval nucleus with finely stippled chromatin and one magenta nucleolus. There is mild to moderate anisocytosis and anisokaryosis and the mitotic count is 3 per 0.237sq mm (individual 40x HPF). Multifocally, neoplastic cells and adrenocortical cells contain large, clear cytoplasmic vacuoles and have hyperchromatic to pyknotic nuclei (lipid degeneration). Within the neoplasm there is multifocal single cell necrosis and larger areas of lytic necrosis, hemorrhage, fibrin, edema, and mineral deposition.

MORPHOLOGIC DIAGNOSIS: Adrenal gland: Adrenocortical carcinoma, breed unspecified, canine.

GENERAL DISCUSSION:

Adrenocortical neoplasms cells can be benign adenomas or malignant carcinomas and may be and functional or nonfunctional
1. Adenomas are more common than carcinomas
  - Most frequent in old dogs (>8 yrs old), usually incidental findings
2. Carcinomas reported most often in cattle and older dogs
  - Metastasis common in the lungs (especially in bovines) or invasion into the caudal vena cava with spread to kidney, liver and lymph node (especially in canines)
Functional adrenocortical neoplasms (adenoma or carcinoma)
1. Most commonly from the zona fasciculata and secrete cortisol, resulting in hypercortisolism and hyperadrenocorticism
2. Less commonly, can arise from zona glomerulosa (secrete aldosterone), seen in cats, or from the zona reticularis (secrete estrogen), most common in ferrets
Hypercortisolism / hyperadrenocorticism (HAC) (Cushing’s disease)
1. One of the most common endocrinopathies in adult dogs (small breed dogs, such as poodles), occasionally in cats, due to long-term cortisol excess leading to a spectrum of clinical signs
2. 15% of dogs with hyperadrenocorticism have function adrenal tumors; 85% have a functional pituitary corticotroph adenoma (E-N01) which results in bilateral adrenocortical hypertrophy (known as pituitary dependent hyperadrenocorticism)

PATHOGENESIS:

Carcinoma: PI3 kinase activation through insulin growth factor-1 (IGF-1) is thought to be associated with malignancy in dogs and humans
Hypercortisolism / hyperadrenocorticism due to functional adrenal neoplasm:
1. Atrophy of contralateral adrenal gland:
  - Increased circulating glucocorticoids -> Increased negative feedback (hypothalamic-pituitary-adrenocortical axis) -> Decreased ACTH -> Adrenocortical trophic atrophy of the zonae fasciculata and reticularis (zona glomerulosa not affected by ACTH; mineralocorticoid levels normal)
  - Similar atrophy may be present in the compressed cortex surrounding the neoplasm
2. One-third of dogs with HAC due to functional adrenal tumors have activating mutations in the stimulatory ACTH receptor (melanocortin 2 receptor [MC2R]), specifically the G protein alpha-subunit), which may allow functional tumors to be independent of ACTH stimulation
3. Systemic effects of cortisol: Disturbances and lesions from gluconeogenic, lipolytic, protein catabolic, and anti-inflammatory effects on many organs

TYPICAL CLINICAL FINDINGS:

Clinical signs of neoplasm may be due to invasion, metastasis, and compression of adjacent tissue (e.g., abdominal hemorrhage)
Hypercortisolism / hyperadrenocorticism:
1. Polyuria/polydipsia, polyphagia, pendulous abdomen, muscle weakness, lethargy
2. Mature neutrophilia, lymphopenia, monocytosis, eosinopenia (steroid stress leukogram)
3. Increased serum alkaline phosphatase (levamisole-resistant steroid isoenzyme), elevated cortisol, decreased urine specific gravity, hyperglycemia, lipemia, hypercholesterolemia
4. Skin lesions: Bilateral, symmetric alopecia; skin fragility syndrome in cats
5. Increased susceptibility to bacterial infections

TYPICAL GROSS FINDINGS:

Adenoma:
1. Usually unilateral, <2 cm, well circumscribed, solitary yellow (due to lipid content) to red nodule which expands the cortex and compresses adjacent normal cortex and medulla
Carcinoma:
1. May be bilateral, >2 cm, variegated, yellow to red, friable with poorly circumscribed areas of necrosis and cystic degeneration
2. +/- vascular invasion of caudal vena cava or aorta, tumor cell thrombi, mineralization or ossification; metastasis to the liver and lungs is most common
Hypercortisolism / hyperadrenocorticism:
1. Contralateral adrenal cortex atrophy
2. Muscle atrophy (abdominal, extremity, temporal muscles), hepatomegaly
3. Epidermal and dermal atrophy (described as "paper thin"), especially bilaterally symmetrical and over points of wear (see I-M23)
4. Cutaneous mineralization (calcinosis cutis), especially dorsal midline, ventral abdomen, and inguinal region)
  - Calcium can also be deposited in other tissues, such as lungs, skeletal muscle, and stomach

TYPICAL LIGHT MICROSCOPIC FINDINGS:

Adenoma:
1. Single discrete nodule, partially or completely encapsulated, composed of polygonal cells arranged in broad trabeculae or nests resembling zonae fasciculata or reticularis with vacuolated cytoplasm; surrounded by compressed cortex
2. Nodular hyperplasia may also be present
3. +/- telangiectasis, hematopoiesis, mineralization, hemosiderosis, adipose, and fibrosis may be present
Carcinoma:
1. Adrenocortical architecture obliterated; multinodular, divided by fibrovascular stroma; cells more pleomorphic; hemorrhage and necrosis common (due to thin-walled sinusoids)
2. Capsular and peripheral invasion, peripheral fibrosis, trabecular growth pattern
3. Differentiation from adenoma on histology alone is challenging
Contralateral adrenal cortex: Atrophy of zonae fasciculata and reticularis (see E-M04) with functional adrenal tumors
Hypercortisolism / hyperadrenocorticism:
1. Lesions may be seen throughout the body, commonly involving the skin (epidermal atrophy, mineralization)
2. HAC is often treated with mitotane (o,p'-DDD) or Trilostane
  - Mitotane is cytotoxic to the zonae fasciculata and reticularis and results in necrosis, followed by collapse of the 2 inner zones
  - Trilostane is an inhibitor of 3"-hydroxysteroid dehydrogenase (necessary for the synthesis of steroid hormones like cortisol); treated adrenal cortices may have accumulation of cytoplasmic lipid vacuoles, apoptosis, necrosis, and hemorrhage

ADDITIONAL DIAGNOSTIC TESTS:

Cytology: Cells resembling normal secretory cells of adrenal cortex, with most cells appearing as naked nuclei in a background of abundant free cytoplasm; tumor cells from adrenocortical adenocarcinomas may be more pleomorphic than those from adenomas
Immunohistochemistry: Adenomas and carcinomas are variably positive for the normal adrenal cortical immunohistochemical antigens: steroidogenic factor-1, calreticulin, α-inhibin, melan-A, and S-100
1. Typically negative for cytokeratin and chromogranin
2. No biochemical or clinical features can distinguish between adenomas and carcinomas
Hyperadrenocorticism: Laboratory confirmation accomplished by a combination of suppression and stimulation tests of the hypothalamic-pituitary-adrenocortical axis:

Parameter	Adrenal-dependent	Pituitary-dependent
Tests to confirm hyperadrenocorticism regardless of cause include:
Resting cortisol	Normal to Elevated	Normal to Elevated
ACTH stimulation	Normal to exaggerated (up to 50% normal)	Normal to exaggerated
Low dose dex suppression	Will not suppress	Will not suppress
Urine cortisol:creatinine (best to rule OUT cushings)	Increased (may also increase with stress)	Increased (may also increase with stress)
Tests to distinguish the cause:
Low dose dex suppression at 4 and 8 hours	No suppression (escape) at 4 hours	Most (75-98%) escape by 8 hours (so if suppressed at 4hrs and escape by 8 hrs= PDH)
High dose dex suppression	No suppression	75% suppress
Endogenous ACTH	Low	Normal to high
Corticotrophin releasing hormone test	No response in either cortisol or ACTH	Increase in cortisol and ACTH

DIFFERENTIAL DIAGNOSIS:

For gross finding of adrenal cortical masses:

Adrenal cortical hyperplasia
- Nodular cortical hyperplasia (E-N08): Multiple expansile nodules of unencapsulated hyperplastic cells often in bilateral adrenal glands and often concurrent extracapsular nodules
- Nodular hyperplasia of the zona reticularis: May present as a discreet focus of zona reticularis extending into the medulla and/or irregular corticomedullary boundaries; associated with androgen excess
- Diffuse cortical hyperplasia: Often due to excess ACTH secondary to a functional pituitary corticotroph adenoma (E-N01)
- Hyperplasia of the zona glomerulosa: Due to chronic increased renin (secondary to chronic sodium depletion or potassium excess) (Renin -> increased angiotensinogen II, which is trophic for the zona glomerulosa)
Myelolipoma: A benign lesion of adipose cells and hematopoietic tissue; most common in cattle and NHPs (also common in the spleen, see H-N04) likely formed due to metaplastic transformation of adrenal cortical cells
Pheochromocytoma: Cells with darker, granular cytoplasm with hyperchromatic nuclei; Henle chromaffin reaction on gross sections using Zenker’s solution; immunohistochemical profile may include 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)
Metastatic neoplasia: The adrenal corticomedullary junction is a common site of tumor metastasis

COMPARATIVE PATHOLOGY:

Ferrets: Adrenal-associated endocrinopathy (AAE) is a common metabolic disease of middle-aged, early gonadectomized ferrets and may be caused by either hyperplastic or neoplastic adrenocortical lesions
1. Functional proliferative lesions of the adrenal cortex (in order of frequency): Hyperplasia (unilateral or bilateral) > Carcinoma > Adenoma; occurs in females > males
  - Note: Ectopic adrenocortical tissue is common in ferrets (do not interpret as metastasis)
2. Pathogenesis: Linked to early neutering: Early gonadectomy disrupts negative feedback of hypothalamic–pituitary–gonadal axis -> Persistently elevated levels of luteinizing hormone (LH) -> Proliferation of primitive adrenocortical cells, upregulation of LH receptors and increased GATA4 expression (transcription factor for sex steroids) in ZF and ZR -> Secretion of estradiol-17β or its intermediates -> Hyperestrogenism
3. Clinical signs/gross lesions (due to hyperestrogenism): Vulvar enlargement, bilaterally symmetric alopecia, PU/PD, anemia, thrombocytopenia, pyometra, endometrial hyperplasia, squamous metaplasia of prostatic ductular epithelium, cystic prostatic disease (+/-urinary obstruction), return of sexual behavior
  - May also be insulin-producing tumors of pancreatic islets β-cells the (1/3 of cases) signs of hypoglycemia
4. Histo:
  - Nodular hyperplasia: Usually multiple variably-sized foci
  - Cortical adenoma: Usually well-demarcated (usually unilateral) nodules
  - Adenomas and carcinoma: Typically pleomorphic with variably differentiated cells + spindle cell differentiation (α-smooth muscle actin and desmin positive); occasional myxoid variants exist; tumor cells are vimentin, α-inhibin, synaptophysin, estrogen receptor, and GATA-4 positive)
5. No hypercortisolism (release sex steroids NOT cortisol), therefore no atrophy of the contralateral cortex
Sheep: A case of Multiple Endocrine Neoplasia (MEN), composed of insulinoma, adrenocortical carcinoma with myxoid differentiation, and thyroid C-cell carcinoma reported (Tanaka, J Vet Diagn Invest. 2023)
1. MEN is associated with one (of a number of) genetic mutations resulting in concurrent endocrine tumors and is most commonly reported in dogs, cats, horses, and cattle; often involving thyroid (C-cell), parathyroid, and/or pituitary tumors
Lab animal: Reported in mice
Hamster: Adrenocortical tumors are one of the most common tumors in hamsters and a common cause of hyperadrenocorticism
Cats: Functional neoplasms of the zona glomerulosa rarely reported, leading to hyperaldosteronism (Conn syndrome); may present with hyperkalemia and hypertension
Avian: Adenomas and carcinomas of interrenal cell origin (avian adrenal gland) occur sporadically, they may be functional (secreting corticosterone [the dominant avian glucocorticoid]), however levels must be compared with healthy same species
Reptiles: Interrenal (adrenocortical) adenocarcinomas reported
Gonadectomy increases incidence of adrenal tumors in mice and goats

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