Once the diagnosis of CS has been confirmed, a combination of blood tests and MRI/CT scans can be employed to determine the cause of the CS (Newell-Price et al., 2006; Sharma et al., 2015).
Plasma ACTH levels are measured to determine whether CS is ACTH-dependent or independent. Values of <10 pg/ml (2 pmol/l) suggest CS is ACTH-independent (Arnaldi et al., 2003). An inappropriately normal or elevated ACTH level (>20 pg/ml, 4.4 pmol/l) is consistent with an ACTH-dependent form of Cushing’s Syndrome (Sharma et al., 2015). Levels between 10 and 20 are indeterminate, and should be repeated several times, and when still uncertain a CRH test should be performed.
The CRH stimulation test is performed by intravenous injection of 1 µg/kg or 100 µg of synthetic, ovine or human CRH. Most pituitary tumours (and some ectopic ACTH-secreting tumours) will respond with an increase in plasma ACTH and cortisol levels. In ACTH-independent, adrenal CS, there is usually very little or no cortisol or ACTH response to CRH. However as ectopic ACTH-dependent tumours can also respond to CRH, this test is not reliable enough to be used in isolation (Arnaldi et al., 2003). It is also useful to determine ACTH-dependence or independence.
HDDST can be used to distinguish between pituitary and ectopic tumours. Several versions of this test exist; standard 2-day oral high dose (2 mg every 6 hours for eight doses), 8 mg overnight oral, and 4–7 mg intravenous tests. Plasma and/or urinary cortisol levels are evaluated before, during, and/or after high dose DST (Arnaldi et al., 2003).
These tests distinguish pituitary from ectopic sources of ACTH with a sensitivity of 60–80% and with high specificity when a cut-off of plasma cortisol suppression above 50% is used (Arnaldi et al., 2003). Nevertheless, it involves the administration of high doses of corticosteroid to a patient with on-going steroid excess, and its use is decreasing with the availability of other test procedures. It has also been suggested that the relative fall in cortisol seen in the low-dose 48-hour test can be just as useful in discriminating between Cushing’s Disease and the ectopic ACTH syndrome.
The metyrapone stimulation test is another non-invasive test to differentially diagnose CS (Avgerinos et al., 1994). Metyrapone is a drug that lowers cortisol levels and the test is used to help evaluate the pituitary gland's ability to produce ACTH in response to a decreased cortisol level. It involves patients receiving six doses of metyrapone (750 mg per dose) at 4-hour intervals. Plasma and urinary levels of the steroid metabolites 17-hydroxysteroid or 11-deoxycortisol are evaluated. The metyrapone test can also be used in combination with the high dose dexamethasone test to improve accuracy (Avgerinos et al., 1994).
If ACTH levels are suppressed, CT/MRI scanning of the adrenals can be used to identify the type of lesion responsible for CS (Arnaldi et al., 2003; Sharma et al., 2015).
Pituitary MRIs with gadolinium enhancement should be performed in all patients with ACTH-dependent CS; this can be used to identify a pituitary tumour in up to 60% of patients (Arnaldi et al., 2003). The majority of ACTH-secreting pituitary tumours, however, are small and may not be detected even using more advanced MRI techniques such as spoiled gradient-recalled acquisition or dynamic MRI sequences (Sharma et al., 2015). In addition, pituitary ‘incidentalomas' are increasingly recognised in otherwise normal patients, so the presence of a lesion in a patient with ACTH-dependent CS may not necessarily indicate Cushing’s Disease (Ioachimescu et al., 2015).
BIPPS testing is recommended in patients with ACTH-dependent CS, if clinical, biochemical and radiological studies are inconclusive. Blood samples are taken from both inferior petrosal sinuses (IPS) and a peripheral vein at baseline and after stimulation with ovine or human CRH. ACTH levels across the two sites are then compared. An IPS: peripheral site ratio of >2 at baseline, or >3 after CRH is consistent with a diagnosis of Cushing’s Disease (Arnaldi et al., 2003). Unless a pituitary lesion is obvious and the other data are entirely compatible with Cushing’s Disease, it may be of use in most patients.
When performing diagnostics tests, the results and the likelihood of adverse events are related to the experience of the radiology team, and should therefore only be performed in specialist centres.
Table 1. Overview of diagnostic test results (adapted from Arnaldi et al., 2003).
For patients with suspected adrenal cell carcinoma, patients may also undergo additional tests, including serum steroid levels, mineralocorticoid levels and catecholamine excess. The European Network for the Study of Adrenal Tumors recommend the following tests in patients with suspected adrenal cell cancer (Berruti et al., 2012).