Cushing Syndrome Workup

Laboratory abnormalities that may be associated with Cushing syndrome include the following:

• Elevated white blood cell count greater than 11,000/mm³
• Hyperglycemia
• Hypokalemic metabolic alkalosis may occur in patients with markedly elevated cortisol due to cortisol activation of the renal mineralocorticoid receptor.

Biochemical evaluation of Cushing syndrome

• The diagnosis of Cushing syndrome due to endogenous overproduction of cortisol requires the demonstration of inappropriately high serum or urine cortisol levels, as shown in the algorithm below. Currently, 4 methods are accepted for the diagnosis of Cushing syndrome: urinary free cortisol level, low-dose dexamethasone suppression test, evening serum and salivary cortisol level, and dexamethasone–corticotropin-releasing hormone test. Diagnosis of Cushing syndrome.

  • Urinary free cortisol (UFC) determination has been widely used as an initial screening tool for Cushing syndrome because it provides measurement of cortisol over a 24-hour period. A valid result depends on adequate collection of the specimen. Urinary creatinine excretion can be used to assess the reliability of the collection. Urine free cortisol values higher than 3-4 times the upper limit of normal are highly suggestive of Cushing syndrome.^{[4, 5]} Values higher than the normal reference range but less than 3-4 times the upper limit of normal are inconclusive. Values that fall within this range may indicate pseudo–Cushing syndrome or Cushing syndrome and require further testing. Multiple collections are necessary because patients with disease may have values that fall within the normal range. Three urine free cortisol levels in the normal range exclude the diagnosis of endogenous Cushing syndrome.
  • The rationale for the dexamethasone suppression tests is based on the normal physiology of the hypothalamic-pituitary-adrenal axis; glucocorticoids inhibit secretion of hypothalamic CRH and pituitary ACTH but do not directly affect adrenal cortisol production. Since cortisol production is controlled by ACTH, decreases in ACTH lead to decreases in plasma and urine cortisol. The overnight 1-mg dexamethasone suppression test requires administration of 1 mg of dexamethasone at 11 PM with subsequent measurement of cortisol level at 8 am.^[6] In healthy individuals, the serum cortisol level should be less than 2-3 mcg/dL. To enhance the sensitivity of the test, a cutoff value of less than 1.8 mcg/dL (50 nmol/L) excludes Cushing syndrome. Its ease of administration makes the 1-mg dexamethasone suppression test a widely used screening tool.
  • Late-night serum and salivary cortisol levels take advantage of the alterations in circadian rhythm of cortisol secretion in patients with Cushing syndrome. Normally, cortisol values are at their lowest level late at night.
    • In patients with Cushing syndrome, an elevated serum cortisol at 11 PM can be an early, but not definitive, finding. Measuring serum cortisol levels requires hospitalization, with blood samples obtained within 5-10 minutes of waking a patient, and is not a practical test.
    • Measuring salivary cortisol level has gained interest, as it is a simple and convenient way of obtaining a nighttime sample.^[7] This measurement allows patients to collect their own samples at home. With repeated measurements, levels less than 1.3 ng/mL (radioimmunoassay) or 1.5 ng/mL (competitive protein-binding assay) exclude Cushing syndrome. Less experience has been gathered for this assay, and it is expensive. Most physicians who do use this test obtain readings over several evenings to increase accuracy.
    • Results from a meta-analysis of 7 studies relating to late-night salivary cortisol testing in the diagnosis of Cushing syndrome (947 patients aged >18 years, including 339 persons with Cushing syndrome) indicated that such testing has a sensitivity of 92%, a specificity of 96%, and a diagnostic odds ratio of 311.^[8] The report's authors concluded that late-night salivary cortisol measurement "is a robust, convenient test for screening and diagnosis of Cushing syndrome."
  • The dexamethasone-CRH test is intended to distinguish patients with Cushing syndrome from those with pseudo-Cushing states. It combines a 48-hour low-dose dexamethasone suppression test with CRH stimulation. Dexamethasone (0.5 mg every 6 hours) is given 8 times starting at about 8 AM, CRH is administered intravenously 6 hours after the last dose of dexamethasone and plasma cortisol and ACTH levels are obtained at 15-minute intervals for 1 hour. A cortisol value greater than 50 nmol/L (1.4 mcg/dL) identifies Cushing syndrome. This test is reserved for patients with high clinical suspicion for Cushing syndrome but equivocal results on other diagnostic tests.

Unfortunately, mild Cushing syndrome is often difficult to distinguish from normal cortisol secretion or pseudo-Cushing states. The aforementioned tests can produce both false-positive and false-negative results. False-positive results are associated with obesity, alcoholism, chronic renal failure, affective disorders, strenuous exercise, or eating disorders. Other potential confounders in the interpretation of tests include the following:

• Medications that increase corticosteroid-binding globulin, such as estrogen and tamoxifen, may cause appropriate increases in serum cortisol levels.
• Medications that facilitate the metabolism of dexamethasone, such as phenobarbital, phenytoin, and rifampin, may cause false-positive results with the dexamethasone suppression test.

Acute illness activates the HPA axis, resulting in increases in ACTH and cortisol. The laboratory workup for Cushing syndrome should not be performed when subjects are acutely ill.

Table. States of Increased and Decreased HPA Activity (Open Table in a new window)

Once the diagnosis is established, the next step requires determining the etiology of Cushing syndrome. See the flow chart below. The logical first step involves identifying if the hypercortisolism is an ACTH-dependent or ACTH-independent disorder.

• In a patient in whom the diagnosis of Cushing syndrome has been established, an undetectable plasma ACTH with a simultaneously elevated serum cortisol level is diagnostic of ACTH-independent Cushing syndrome. ACTH-independent Cushing syndrome is due to a primary cortisol-producing adrenal adenoma or carcinoma, assuming exogenous glucocorticoid use has been excluded. A plasma ACTH (measured by an immunoradiometric assay) of less than 5 pg/mL is suggestive of a primary adrenal tumor. An ACTH level greater than 10-20 pg/mL is consistent with ACTH-dependent Cushing syndrome.
• The 8-mg overnight dexamethasone suppression test and the 48-hour high-dose dexamethasone test may be useful when baseline ACTH levels are indeterminate. These studies also help in determining whether a patient who has ACTH-dependent disease has pituitary-dependent or ectopic ACTH disease.

  • In the 8-mg overnight dexamethasone suppression test, individuals ingest 8 mg dexamethasone orally at 11 PM, with measurement of an 8 am cortisol level the next day. A baseline 8 am cortisol measurement is also obtained the morning prior to ingesting dexamethasone. Suppression of serum cortisol level to less than 50% of baseline is suggestive of a pituitary source of ACTH rather than ectopic ACTH or primary adrenal disease. However, the diagnostic accuracy is only 70-80%.
  • With the 48-hour high-dose dexamethasone suppression test, patients ingest 2 mg dexamethasone every 6 hours for 8 doses. A decrease in urinary free cortisol of greater than 50% is suggestive of an anterior pituitary adenoma rather than ectopic ACTH or a primary adrenal tumor. Unfortunately, the sensitivity of this test is only 80%, with a specificity of 70-80%. The more stringent criterion of a 90% decrease in urinary free cortisol levels excludes the diagnosis of ectopic ACTH and has almost 100% specificity for anterior pituitary disease.
• Testing with CRH is used in the differential diagnosis of ACTH-dependent Cushing syndrome. In most patients with pituitary ACTH secretion, the intravenous administration of CRH causes a rise in plasma ACTH and cortisol levels. In patients with ectopic secretion of ACTH, CRH does not affect ACTH or cortisol levels. ACTH and cortisol samples are obtained before administration of ovine CRH (oCRH), and subsequently at 15, 30, 45, 60, 90, and 120 minutes after administration of 1 mcg/kg of CRH. A rise of more than 20% in peak plasma cortisol level or a rise of more than 50% in peak ACTH level after oCRH is consistent with pituitary ACTH-dependent Cushing syndrome. Sensitivity and specificity are 91% and 95%, respectively, for cortisol measurements and 86% and 95% for ACTH measurements, respectively.
• If concern for adrenal carcinoma exists, measurements of adrenal androgen production, such as serum dehydroepiandrosterone sulfate [DHEAS], and 24-hour urinary 17-ketosteroid measurements may be helpful.

• Imaging studies for Cushing syndrome should be performed after the biochemical evaluation has been performed. The rationale for this is that unguided imaging of the pituitary or adrenal glands may yield a 10% incidence of incidental nonfunctioning pituitary or adrenal adenomas, which may mislead one from proper therapy and surgery. Ideally, the biochemical abnormalities should reconcile with the anatomic abnormalities before definitive therapy is offered.
• An abdominal CT scan is recommended if a primary adrenal problem is suspected. The presence of an adrenal mass larger than 4-6 cm raises the possibility that the mass is an adrenal carcinoma.
• If a pituitary source of excess ACTH is suspected, patients should undergo a contrast-enhanced magnetic resonance imaging (MRI) study of the pituitary. Unfortunately, normal-appearing pituitaries may occur in some patients with Cushing disease due to both diffuse hyperplasia of ACTH-producing cells and small microadenomas that do not appear on imaging studies. In the latter case, ACTH lateralization during an inferior petrosal sinus sampling (IPSS) study may be useful in lateralizing the occult lesion and in guiding surgical therapy.
• Chest and abdominal CT scans should be performed in patients with suspected ectopic ACTH production.
• Octreotide scintigraphy may be helpful in detecting ectopic ACTH tumors because some neuroendocrine tumors typically have cell surface receptors for somatostatin.

• Inferior petrosal sinus sampling (IPSS) is useful in distinguishing a pituitary source from an ectopic source of ACTH. An experienced interventional radiologist should perform this procedure to decrease the incidence of neurological complications. This study should not be used to establish the diagnosis of Cushing syndrome.

  • Bilateral IPSS and simultaneous peripheral ACTH measurements are made at baseline and 2-3 minutes, 5 minutes, and 10 minutes after intravenous administration of oCRH at 1 mcg/kg.
  • A baseline and/or stimulated IPS-to-peripheral ACTH ratio of less than 1.8 is suggestive of ectopic ACTH, while an IPS-to-peripheral ACTH ratio of greater than 2 is consistent with Cushing disease.
  • In approximately 70% of patients, a ratio of greater than 1.4 between the right and left inferior petrosal sinuses is predictive of the location of the microadenoma.
  • This study is not interpretable if pituitary venous drainage anatomy is anomalous.
  • A study by Mulligan et al concluded that prolactin measurement during IPSS may reduce false-negative results in patients with Cushing disease who do not have an appropriate central-to-peripheral ACTH gradient.^[10]