The role of the laboratory in the diagnosis of Cushing's syndrome

Transcription

1 Review Article Ann Clin Biochem 1997; 34: The role of the laboratory in the diagnosis of Cushing's syndrome L A Perry and A B GrossmanI From the Immunoassay Laboratory, Departments of Clinical Biochemistry and Endocrinology", St Bartholomew's Hospital, London ECIA 7BE, UK Additional key phrases: cortisol; urine free cortisol; dexamethasone suppression test; corticotrophin releasing hormone stimulation test Cushing's syndrome remains one of the most difficult diagnostic problems in endocrinology. Patients with suspected ACTH-dependent Cushing's syndrome continue to challenge the diagnostic skills of the clinical scientist and clinician, even with the array of biochemical tests and imaging techniques now available. The diagnosis of Cushing's syndrome rests on demonstrating over-production of cortisol and relative resistance of this over-production to biochemical manipulation. However, there are causes of hypercortisolism other than Cushing's syndrome: these and the types of Cushing's syndrome have been summarized in Table I. Clinical suspicion of endogenous hypercortiso Iism can only be confirmed biochemically. To initiate the most appropriate and effective treatment it is essential, even in the most overt cases of Cushing's syndrome, to identify the primary abnormality causing the hypercortisolism. In many cases, the initial tests can be conducted on an out-patient basis, but confirmation will usually require in-patient assessment. Differential diagnosis is often possible only under strictly controlled in-patient provocative testing. Biochemical tests for the existence and differential diagnosis of Cushing's syndrome have been extensively reviewed 1-8 and consist of a variety of first-line and definitive tests. Commonly used first-line tests include the overnight low-dose dexamethasone suppression test (LDDST) and measurement of the 24 h urinary excretion of free cortisol (UFC). Although these tests are far from infallible, negative results markedly decrease the likelihood Correspondence: Dr L A Perry, Immunoassay Laboratory, Bartholomew Close, London EClA 7BE, UK This article was commissioned by the Clinical Laboratory Investigation Standing Committee of the Scientific Committee of the Association of Clinical Biochemists. of disease, particularly when clinical evidence for its presence is weak. Once hypercortisolism is confirmed and causes other than Cushing's syndrome have been excluded, one must ascertain the precise aetiology of the Cushing's syndrome in order to expedite appropriate treatment. Pituitary-dependent ACTH hypersecretion (Cushing's disease) is the commonest (70%) cause of endogenous Cushing's syndrome in adults, followed by the 'ectopic ACTH syndrome' (12%). ACTH-independent causes of Cushing's syndrome include adrenal adenomas (10%) and carcinomas (8%).8 In children over the age of 10, 75% of cases are due to Cushing's disease," while in children under the age of 10, Cushing's disease accounts for only 50%; although based on 12 cases, these proportions reflect the series recently reported from the National Institutes of Health." The ectopic ACTH syndrome is very rare in children, although individual cases have been published.? Definitive diagnostic tests to elucidate the aetiology of the syndrome include the high-dose dexamethasone suppression test (HDDST), basal plasma ACTH determination and the corticotrophin releasing hormone (CRH) stimulation test. 2,4,8 When biochemical testing suggests the presence of specific lesions, further investigative protocols have been defined.!':" The tests include catheterization and selective blood sampling from veins draining the area of the suspected lesion, such as petrosal sinus sampling for pituitary lesions (with or without injection of CRH), and radiological imaging techniques such as computerized tomography (CT) and magnetic resonance imaging (MRI) scanning. INITIAL OUT-PATIENT INVESTIGATIONS Urinary free cortisol (UFC) All urine collections have the disadvantage of relying on the patient managing a complete and uncontaminated collection, and this remains a major drawback in the use of urinary assays in the diagnosis of Cushing's syndrome. 345

2 346 Perry and Grossman TABLE I. Causes ofhypercortisolism Cushing's syndrome ACTH-dependent Pituitary-dependent bilateral adrenocortical hyperplasia: due to increased pituitary ACTH secretion (Cushing's disease) 'Ectopic ACTH syndrome': secretion of ACTH by benign or malignant tumours of non-endocrine origin Rarely, corticotrophin releasing hormone or bombesin-secreting tumours Iatrogenic: following treatment with ACTH or synthetic analogues Non-ACTH-dependent Adenomas of the adrenal cortex Carcinomas of the adrenal cortex Iatrogenic: following treatment with supraphysiological doses of natural or synthetic glucocorticosteroids Others Alcoholic pseudo-cushing's syndrome Severe depression Severe stress/critical illness (e.g. following myocardial infarction) Macronodular adrenocortical hyperplasia (very rare, <0'5%) The unacceptably high false-negative rate (11%) and false-positive rate (27%) excluded urinary l7-hydroxycorticosteroid and 17-oxogenic steroids (false negative rate 24%) as initial screening tests for Cushing's syndrome.? The most widely used test to confirm hypercortisolism is assessment of cortisol excretion, utilizing the 24 h UFC determination.j-v'<!" Plasma cortisol concentrations rise and fall episodically in both normal subjects and those with Cushing's syndrome. 15 Measurement of 24 h excretion of cortisol in urine provides an 'integrated' measure of cortisol although it is of limited value in patients with abnormal renal function. The cortisol secretion rate (determined only by isotope dilution studies) in normal adults is Jlmol/24 h, 16 of which nmol is filtered by the kidney and excreted as un metabolized cortisol (usually called urinary free cortisol}!" In a meta-analysis of 15 separate studies, UFC measurement had a sensitivity of 96 6% and a specificity of 94 3% in the diagnosis of Cushing's syndrome.? However, 3,3% of 479 lean, obese and chronically ill controls had an elevated 24 h UFC excretion, while 5,6% of 248 patients with Cushing's syndrome had normal 24 h excretion. A recent survey of 146 patients with Cushing's syndrome confirmed a sensitivity of 95% but also noted that II % of patients had at least one out of four UFC measurements within the reference range. 18 Since most assays define the upper limit of normal as nmol/24 h, these assays cannot be specific. The reference range depends on whether or not the sample is collected into acid'? and the type of assay employed. Most methods use an initial organic solvent extraction of cortisol from urine followed by immunoassay,20 but techniques such as HPLC are also used External quality assessment of UFC methods in the UK compared with a gas chromatography-mass spectrometry (OCMS) reference method 23 identified specificity as a major problem with UFC assays. The majority ofufc methods have not been designed/validated for urine samples but are an 'add-on' to a method protocol designed for serum cortisol. Importantly, the assessment showed 27% of laboratories performing outside the acceptable limits of UK National External Quality Assurance Scheme (NEQAS) performance criteria, which suggests that the routine UFC measurement should be less frequently employed than previously recommended. This poor performance is in contrast to that obtained in the plasma cortisol assessment scheme where in the 6 months up to March 1995, only 10% of laboratories fell outside the stricter acceptable performance limits. However, if the UFC test is used, at least two and preferably four consecutive 24 h specimens should be collected. Several measurements are needed because even carefully instructed patients make mistakes (e.g. not discarding the urine voided before beginning the first day's collection),24 and there is also the possibility of episodic activity of the disease.p If a patient has renal failure with a glomerular filtration rate below 30 ml.jmin, UFC measurements are no longer valid." Ann cu«biochem 1997: 34

3 Diagnosis of Cushing's syndrome 347 One North American group with very extensive experience has recently even suggested that the UFC is only of value in the confirmation of hypercortisolism if the result is higher than four times the upper limit ofnormal." Creatinine measurement to determine the completeness of a 24 h collection has been questioned because of the wide range of values that occur. Creatinine excretion is fairly constant while cortisol is excreted episodically during the 24 h. The dependence of creatinine excretion on skeletal muscle mass and the added imprecision intrinsic to ratio calculations are important reasons why UFC results should not be expressed as a cortisol/creatinine ratio. Using the first morning urine collection and measuring the cortisol/creatinine ratio-? is subject to the same criticism, which is why it is not recommended as a diagnostic test. 2M To compound the problems, the biochemical and/or clinical changes of Cushing's syndrome can be closely mimicked in other pathological states (depression, obesity, alcoholism)," and special attention is required to ensure that the correct diagnosis is reached. UFC determinations will often provide a decisive distinction between patients with hypercortisolism and obese non-cushing's patients. Pseudo-Cushingoid states such as alcoholism and depressive psychosis may yield UFC results which are indistinguishable from patients with true Cushing's syndrome.l? Patients with Cushing's syndrome often (over 60%)30 have depression as a feature of their illness and may require further tests to differentiate whether the emotional disorder is due to hypercortisolism per se or a primary disturbance. Low-dose dexamethasone suppression test (LDDST) Dexamethasone is a synthetic glucocorticoid 64 times more potent than cortisol. It is used to test the integrity of the hypothalamo-pituitaryadrenal axis. When dexamethasone is given to a healthy subject, ACTH release is suppressed by negative feedback to the pituitary and the hypothalamus, and consequently adrenal cortisol production will fall to very low levels. Patients with Cushing's syndrome, from whatever cause, lose this normal negative feedback control by circulating glucocorticoids and thus exhibit detectable plasma cortisol concentrations after dexamethasone administration. Several versions of the low-dose dexamethasone suppression test for the diagnosis of Cushing's syndrome exist. The differences are in the quantity of dexamethasone ingested and the timing of sample collection and duration of the test. The overnight dexamethasone suppression test!' and the classical Liddle dexamethasone suppression test (in which a basal plasma cortisol sample is collected at 0900 h, dexamethasone (0 5 mg) is then taken every 6 h for 48 h and the final cortisol sample (48h) is collected at 0900 h, 6 h after the last dexamethasone dose)32 are the most widely used, simplest and most reliable tests for the diagnosis of Cushing's syndrome. Our experience is that both protocols can be applied reliably in the outpatient clinic. The overnight dexamethasone suppression test has been described using different doses of dexamethasone (1, I 5 or 2 mg), in each case administered orally at 2300 h or midnight with measurement of serum or plasma cortisol 8 or 9 h later; this is based on the principle that the sensitivity of the pituitary is greatest at night.p Definition of normality following either LDDST protocol is contentious. Early studies showed that in healthy subjects plasma cortisol fell below 140nmol/U on overnight dexamethasone suppression, and below 150 nmol/l after the Liddle dexamethasone suppression protocol.32,34 The ranges for normality described by Liddle 32 relate to UFC and urine 17-hydroxycorticoid measurements. Ranges subsequently derived for plasma cortisol were based on the Mattingly fluorometric assay.35 Isotopic and non-isotopic immunoassays for plasma cortisol have since become more sensitive and specific, but most published normal ranges still relate to the Mattingly fluorometric method with which it was difficult to measure plasma cortisol concentrations below 150 nmol/l reliably. In a survey of UK laboratories of the first-line investigation of Cushing's syndrome, 131/191 used the overnight LDDST whereas only 11 used the Liddle 48 h LDDST protocol." The dose of dexamethasone recommended for the overnight test in 127/131 laboratories was: 1mg (91 laboratories), I 5 mg (3 laboratories) and 2 mg (33 laboratories). The cut-oft concentration reported by laboratories as indicative of adequate suppression ranged from less than 50 to less than 200 nmol/l, with 83 laboratories quoting cortisol results between nmol/ L as adequately suppressed. Whether 1, 1 5 or 2 mg of dexamethasone gives the best discrimination is hard to define. There is a need for more comparative data for the 1 and 2 mg test, with

4 348 Perry and Grossman particular emphasis in sensitivity (false negatives in patients with Cushing's syndrome) between the two protocols. However, the Liddle 48 h test has been reported as having a % true positive rate,37,38 while in our experience false negatives are extremely rare and certainly not greater than 2%.39 Increasingly, automated cortisol assays are offering increased assay sensitivity, therefore can a cut-off concentration lower than 50 nmol/l be used for defining normality? Our experience (down to 25 nmol/l-unpublished data) is that lowering the cut-off does not increase diagnostic sensitivity but does decrease specificity (e.g. some patients with simple obesity can have cortisol concentrations that only fall to 35 nmol/l), If the LDDST protocol is followed, normal suppression should now be defined as a plasma cortisol < 50 nmol/l at 0900 h after 48 h of dexamethasone at 2 mg/day, By this criterion we have found a sensitivity of LDDST for the diagnosis of Cushing's syndrome of 98%.40 In a review of 150 patients investigated with histologically-confirmed Cushing's syndrome, only three (i.e. 2%) suppressed their cortisol levels below 50 nmol/l on the Liddle protocol.t? Although the overnight or the 48 h dexamethasone suppression tests can be used as a first line out-patient procedure for Cushing's syndrome, we prefer the Liddle 48 h protocol because of its higher sensitivity and lower false positive rates. Either LDDST protocol may be readily performed by general practitioners (GP) provided that adequate explanation is given to doctor and patient emphasizing the strict adherence to timing, namely starting the test at 0900 h, administration of dexamethasone every 6 h rather than four times a day, and returning for the final blood sample at 0900 h (6 h after the last dexamethasone dose). If a suspicion of Cushing's syndrome remains in spite ofapparent suppression, we recommend that the patient be admitted to hospital for further confirmatory tests. False positive results using either dexamethasone suppression regimen may occur in patients receiving drugs that accelerate dexamethasone metabolism by the liver (e.g. phenytoin, phenobarbitone, rifampicin, etc.), oestrogen therapy or tamoxifen [because circulating corticosteroidbinding globulin (CBG) levels are increased], patients with endogenous depression, critical illness (e.g. following recent myocardial infarction) or in patients with alcoholic pseudo Cushing's syndrome."! However, it must be remembered that failure to suppress plasma cortisol is often due to failure to comply fully with dexamethasone intake; this may be more common with the 48 h protocol. Conclusion The overnight or the 48 h dexamethasone suppression test can be used as a first line outpatient screen for suspected Cushing's syndrome, but we would recommend the classical low dose dexamethasone suppression test (0,5 mg dexamethasone every 6 h for 48 h starting at 0900 h) for confirming hypercortisolism. Plasma cortisol concentrations at the end of the test (less than 50 nmol]l) essentially exclude Cushing's syndrome. This test is preferred over UFC because immunoassays for plasma cortisol have greater specificity, and the test is easier for the GP, the patient and the laboratory to perform on an outpatient basis. However, there is a small number of false negatives, and if a high index of suspicion persists the patient should be subjected to further evaluation. IN-PATIENT TESTS FOR CUSHING'S SYNDROME Circadian rhythm The absence of a circadian rhythm has been considered as the hallmark of the diagnosis of Cushing's syndrome. Normally, cortisol secretion parallels the circadian rhythm of ACTH secretion, with concentration highest in the morning (0800 h) and lowest at night (between OOOQ--{)400 h). Random plasma cortisol concentrations have proved to be of little diagnostic value. This does not mean that timed samples at other times should not be taken. A sample at 0900 h is still essential to act as the basal sample for the subsequent dynamic tests such as the LDDST and HDDST, and provides an estimate of the degree of activity and/or cyclicity of the disease. There is considerable overlap between the 0900 h plasma cortisol in patients with Cushing's syndrome and the quoted reference range." The discrimination improves with advancing clock time, although even at 1800 h 17% of patients with Cushing's syndrome may still have values that fall within the normal range.? while the value at midnight has, in contrast, been shown to overlap with normal subjects in only 3 4% of cases? A single sleeping midnight cortisol, rather than extensive documentation of the circadian

5 Diagnosis ofcushing's syndrome 349 rhythm, produces the highest sensitivity for the diagnosis of Cushing's syndrome and no overlap with controls. A midnight plasma cortisol from a hospitalized patient not suffering from disease of the hypothalamic-pituitary-adrenal axis, 48 h after admission to hospital and asleep prior to venepuncture, is in our experience < 50 nrnol/ L.39 Patients with intercurrent illness (e.g. acute heart failurej." depressionf or on oestrogen therapy will have a midnight plasma cortisol concentration above 50 nmol/l, A sleeping midnight cortisol of < 50 nmol/l excludes the diagnosis of Cushing's syndrome.'? To avoid elevation of plasma cortisol at midnight because of anticipation of venesection, it is routine clinical practice not to insert a venous cannula, and patients are not informed that the midnight sample is due to be drawn. Patients are requested to retire to bed by 2230 h on every night of their admission. The midnight plasma cortisol is sampled within 5 min of waking the patient. However, in patients who wake when the blood is about to be collected, provided venepuncture is completed within 5 min, the sample may still be deemed as 'asleep' since it takes longer for the adrenal cortex to respond to the ACTH release induced by waking." Where possible, two midnight samples are attempted during the hospital admission. It is recommended that both the LDDST and a sleeping midnight cortisol should be performed in every patient for the confirmation of Cushing's syndrome, especially since the LDDST may miss 2% ofcases if used alone. Insulin-induced hypoglycaemia test Patients with Cushing's syndrome of any aetiology characteristically fail to show a plasma cortisol response during a standard insulinhypoglycaemia test." Therefore this is a valuable test in differentiating patients with depression who may show no suppression of plasma cortisol during LDDST and no circadian rhythmicity, but exhibit a rise in plasma cortisol of ~ 170 nrnol/l in response to hypoglycaemia (blood glucose < 2 2 mmol/l).43 However, the usefulness of the test is limited as approximately 18% of patients with true Cushing's syndrome may also show a response." It has long been recognized that the insulinhypoglycaemia test may have serious adverse effects and it is recommended that it be performed by experienced staff and not on the very young. The test should not be performed on patients with epilepsy or ischaemic heart disease, and a normal electrocardiogram is mandatory. We recommend that the test not be performed in any young children, even if all the pre-test checks are satisfactory, because of two deaths and one severe neurological consequence following testing with hypoglycaemia which were due in part to over-enthusiastic correction of hypoglycacmia.t" Liver function tests It is recommended that liver function tests (LFTs) including y-glutamyl transferase are measured, particularly in patients who present with a history of regular alcohol intake. Patients with alcoholic pseudo-cushing's syndrome will have deranged LFTs. Alcohol withdrawal by hospitalization of the patient for 5 days will show loss of the hypercortisolism. In patients where Cushing's syndrome is still suspected, tests to confirm the diagnosis can be done after the fifth day. DIFFERENTIAL DIAGNOSIS OF CUSHING'S SYNDROME When hypercortisolism and Cushing's syndrome have been confirmed with reasonable certainty, defining the precise aetiology of the Cushing's syndrome is essential in order to institute appropriate treatment. Differentiation of ACTH-dependent from ACfHindependent turnnours Before the introduction of reliable ACTH assays, the HDDST was used to differentiate adrenal tumours from ACTH-dependent lesions. Thus, in the presence of documented Cushing's syndrome, consistently undetectable plasma ACTH strongly suggests the presence of a functioning adrenal tumour. Sample collection for ACTH is important. We strongly recommend that blood collection in glass tubes should always be avoided, irrespective of the type ofassay, because ACTH sticks to glass."? Blood should be collected into tubes with appropriate anti-coagulant, centrifuged at 4 C and the plasma snap-frozen to prevent endogenous peptidase degradation of the ACTH present. A variety of ACTH assays exist, predominantly of the non-extraction immunometric type. The NEQAS specificity and analytical performance of these methods is highly variable. Caution must still be exercised with interpretation of ACTH results because of problems with the sample collection procedure (ACTH is susceptible to endogenous peptidases) and assay

6 350 Perry and Grossman specificity. As a further complication, some tumours, notably the ectopic ACTH secretors, produce not only (1-39)-ACTH but other precursor molecules, sometimes in greater quantities. The specificity of the ACTH assay with respect to these other species is critical to the interpretation of the assay result. Assay matrix, standard, antiserum specificity and sample collection all affect the ability to differentiate normal individuals from patients with border-line detectable ACTH concentration (as seen in patients with adrenal tumours or in patients with nodular adrenocortical hyperplasia). In patients with Cushing's syndrome found to have an undetectable plasma ACTH concentration, CT or MRI scanning of the abdomen should be used. This will simply confirm and localize the lesion. CT scanning after intravenous administration of contrast medium is gradually being displaced by MRI for this purpose. Differentiation of pituitary from ectopic ACTH secretion While the diagnosis of adrenal tumours has become simpler, differentiation ofpituitary from ectopic ACTH production still remains a major challenge. If the basal ACTH is generally under 200 ngjl this usually indicates pituitary ACTH production, while an ACTH above 300 ngjl is typical of malignant ectopic ACTH secretion, as in small-cell carcinoma of the lung. Nonmalignant ectopic ACTH secretion can generate any level of ACTH. Many of the ectopic ACTH-secreting tumours are radiologically 'occult' in that they are not immediately obvious with simple imaging techniques: most are bronchial carcinoids, and may not become apparent for many years after the initial diagnosis of Cushing's syndrome.f Since pituitary microsurgery is the treatment of choice in Cushing's disease, an accurate diagnosis is vita1. 4s,49 The principal diagnostic tests are the HDDST and a test based on hypothalamic hormone stimulation. Other biochemical tests such as the short Synacthen test, adrenal androgen secretion and urinary steroid profiles are generally unhelpful and are not recommended. High-dose dexamethasone suppression test (HDDST) This test was originally described to differentiate Cushing's disease from adrenal tumours.f However, it is now generally used to distinguish Cushing's disease from ectopic ACTH secretion, as the ectopic ACTH tumour generally resists glucocorticoid negative feedback. The efficacy of this test has been repeatedly reviewed Since pituitary tumours secreting ACTH are only moderately resistant to dexamethasone, 78% will show at least a 50% fall in 0900 h plasma cortisol concentration from baseline after dexamethasonc.f The high dose dexamethasone regimen is 2 mg dexamethasone (orally) given every 6 h for 48 h starting at 0900 h, with blood taken for cortisol at the beginning of the test, i.e. immediately prior to the first dose of dexamethasone and at the end, 48 h later. Some groups measure cortisol and ACTH 24 h after the first dose which is unnecessary for diagnosis but may be useful for research. Suppression (i.e. the percentage suppression of the 48 h sample over the basal level immediately prior to the test) of more than 50% in the serum cortisol result was seen in 78% of patients with Cushing's disease and 11% of patients with ectopic ACTH syndrome. Relaxation of the criteria to require only 40% suppression would merely reduce differentiation, while increasing the criterion of suppression to 60% would increase the sensitivity but at the expense of specificity.v'" Plasma potassium Patients with ectopic ACTH production are almost always hypokalaemic, with associated metabolic alkalosis.40,54,55 Suspected Cushing's syndrome patients are often receiving treatment for hypertension when referred to hospital; if possible, review plasma potassium concentrations prior to the taking of anti-hypertensive drugs, particularly thiazide diuretics. Unprovoked hypokalaemia should increase the suspicion of ectopic ACTH production. However, about 10% of patients with Cushing's disease may on occasion show hypokalaemia. Glucose intolerance Increased gluconeogenesis may lead to impaired glucose tolerance. This is much more common in the ectopic ACTH syndrome secondary to a small cell carcinoma of the bronchus, in which plasma cortisol concentrations tend to be very high. However, the test has poor differential diagnostic value. Tumour markers If a patient diagnosed as having Cushing's syndrome fails to suppress plasma cortisol on

7 Diagnosis ofcushing's syndrome 351 ~ Cl.s 100 lō J: <: III E 50 Ul III it 150 (a) ~N-1 0 I I I I ( Time (min) 1000 (b) ~ 0E.s 0 Ul 500 'E 8 E 2 CD en 0 I I I I ( Time (min) FIGURE I. (a) Plasma ACTH and (b) Serum cortisol responses to human corticotrophin releasing hormone in normals (.). obese subjects (Ji..) and patients with Cushing's disease (.). Note that patients with Cushing's disease typically show an exaggerated cortisol and ACTH response to corticotrophin releasing hormone (CRH), regardless of whether they show suppression with dexamethasone. However, some 20% ofpatients with proven Cushing's disease show no clear response to CRH. LDDST and HDDST, has detectable ACTH levels, and on initial presentation had a low potassium, a diagnosis of ectopic ACTH secretion should be seriously considered. In an attempt to locate the source of the ACTH secretion, additional biochemical investigations should include measurement of plasma calcitonin (for medullary carcinoma of the thyroid), urinary catecholamines (for ACTH-secreting phaeochromocytomas), urinary 5-HIAA (for carcinoid tumours) and a gastrointestinal hormone screen for gastrointestinal neuroendocrine tumours. This is because 70% of patients with the ectopic-acth secreting tumour are likely to secrete one or more other hormones as weipo Other hormone profiles may also be valuable if the patient is suspected of having multiple endocrine neoplasia (MEN 1). Such patients may have an ACTH-secreting pituitary tumour in association with primary hyperparathyroidism and gastrointestinal neuroendocrine tumours. Others may have cortisol-secreting adrenal tumours or ACTH-secreting carcinoids. Any positive tumour marker can be serially followed as a measure of tumour activity. Provided the recommended sample collection procedure is followed, these samples can be collected at the local hospital even if the analyses are not available on that site. Metyrapone stimulation test Metyrapone blocks the conversion of 11-deoxycortisol to cortisol. As a consequence, the plasma cortisol concentration falls, the pituitary secretes more ACTH and plasma 11-deoxycortisol level increases. 56 Patients with Cushing's disease have a supranormal increase of plasma 11-deoxycortisol whereas patients with ectopic ACTH-secreting tumours show little or no response because the ectopic tumour does not respond to the induced low plasma cortisol and pituitary ACTH production is suppressed.s' Using a positive result in either the metyrapone test or the HDDST, one study has reported an increased sensitivity of diagnosing the aetiology of the Cushing's syndrome to 88% while maintaining the specificity at 100%.51 These observations are in contrast to our earlier report. In our experience 60% of ectopic ACTH patients responded to the metyrapone test,40 and we have not found this to be a useful discriminator and have abandoned it. Most centres confirm that this test has little use in the differential diagnosis of Cushing's syndrome. CRH test The CRH test provides help in the differential diagnosis of Cushing's syndrome.50 CRH was isolated from sheep hypothalami in 1981 and shown to be a 41-amino acid peptide,58 which differs from human CRH by seven residues. This peptide is a potent stimulus to pituitary ACTH secretion in man.t? It was first demonstrated in 1982 that synthetic ovine CRH (ocrh) was able to stimulate ACTH secretion in patients with Cushing's disease but not ectopic ACTH secretion,60 and as a consequence CRH has become established as a powerful tool in the investigation of patients with Cushing's syndrome. Its use as a stimulation test in Cushing's syndrome has since Ann cu«biochem 1997: 34

8 352 Perry and Grossman been reported by many clinical groupsll,60,61 using either ovine or human CRH. The test protocol is described in Appendix I. ACTH-secreting pituitary tumours usually have CRH receptors and exhibit exaggerated ACTH and cortisol responses to CRH administration (Fig. I): non-pituitary tumours mayor may not have these receptors and a blunted ACTH and cortisol response is typically seen. There are data for both ovine and human CRH tests in clinical studies ,63 Synthetic ovine CRH (ocrh) given intravenously has a longer half-life and produces a greater and more prolonged ACTH and cortisol response than synthetic human CRH (hcrh) in normal subjects.s" However, a recent report'" showed no difference between peptides in Cushing's disease, and in addition points out that only synthetic hcrh is commercially available to perform the test. Of more concern is that ocrh may be antigenic. Nevertheless, while there are extensive data on CRH responsiveness in small numbers of patients, there is limited clinical experience in the comparison of the CRH test with more conventional tests such as the dexamethasone suppression test in the differential diagnosis of Cushing's syndrorne.l'v" most studies have used ovine CRH. Findling and Doppman? have critically reviewed the data in one study,65 questioning the interpretation of the ACTH response in four of the 16 patients with ectopic ACTH-secreting tumours. The argument centres on what basal ACTH value was used. Occasionally, however, patients with the ectopic ACTH syndrome do show a clear ACTH response to ocrh administration.s'' However, 10-15% of patients with Cushing's disease may not respond to hcrh, The very variable baseline serum cortisol in Cushing's syndrome means that the peak serum cortisol after hcrh alone cannot discriminate between ectopic and pituitary ACTH secretion unless at least a 20% increase in level above baseline is seen. Patients with depression show a normal cortisol but reduced ACTH response, According to one detailed analysis of the response to ocrh, the most sensitive indicator of responsiveness is an increase in mean cortisol concentration at 30 and 45 min of more than 20%.65 In some patients the high-dose dexamethasone regimen can suppress pituitary ACTH secretion for a long time, which would result in a poor ACTHjcortisol response and an erro- neous differential diagnosis if the CRH test is performed the following day. We recommend that in the ward protocol for the assessment of Cushing's syndrome, the CRH test is performed before commencing the HDDST. Venous sampling catheterization The successful use of selective venous sampling from major systemic veins to locate the source of ACTH secretion has been described. 40,67,68 A catheter is introduced into the femoral vein and manipulated via the great veins to allow samples to be obtained for ACTH, or any other 'tumour marker', from various sites throughout the body as well as from the high internal jugular and the inferior petrosal sinuses to detect pituitary ACTH secretion. Simultaneous peripheral samples are required to ensure that variations seen in plasma ACTH do not simply reflect the pulsatile nature of its secretion. Facilities must exist within the radiology theatre for the plasma ACTH samples to be centrifuged at low temperature and rapidly frozen. This highly invasive technique with its associated morbidity should be performed only in experienced centres. It is becoming less popular not only because of its invasiveness and poor success rate but because imaging techniques are becoming more accurate. Inferior petrosal sinus catheterization combined with CRR A patient with ACTH-dependent Cushing's syndrome who fails to suppress cortisol and ACTH on LDDST but suppresses with the HDDST, and shows an exaggerated cortisol and ACTH response to the CRH test, probably has Cushing's disease. The diagnosis can be confirmed by means of inferior petrosal sinus catheterization and sampling for ACTH after the administration of CRH. If CRH is not used during the test, the power to discriminate pituitary from non-pituitary ACTH secretion is significantly lower." This test provides the most accurate and reliable means of distinguishing pituitary from non-pituitary ACTH-dependent Cushing's syndrome The technique, developed over the last decade, will confirm the pituitary source of ACTH secretion and may help to localize the tumour within the pituitary fossa. As each petrosal sinus receives venous drainage from the ipsilateral side of the pituitary with little intercavernous mixing, ACTH-secreting tumours with a lateral location

9 Diagnosis ofcushing's syndrome 353 should secrete ACTH mostly into the corresponding petrosal sinus. The treatment ofchoice in Cushing's disease is selective transsphenoidal adenornectomy;" as most pituitary ACTHsecreting tumours are microadenomas and situated laterally in the fossa," pre-operative localization of the tumour should improve the surgeon's ability to remove the whole tumour without compromising residual pituitary function. Unfortunately, the results to date do not entirely support this concept (see below). Confirmation of Cushing's disease has been based on the ACTH gradient between the petrosal sinuses and peripheral vein samples; variously reported as being greater than 1-4 or ,75 However, we have seen spontaneous variation between the highest and lowest value of three peripheral vein samples of up to 1 8; in another report," a peripheral to petrosal ratio of 1 7 was noted in a patient with ectopic ACTH secretion. We believe a ratio of > 2 basally (central to peripheral) and > 3 after CRH is necessary to diagnose Cushing's disease with confidence. Using this ratio we have had no false positives. However, it is vitally important to achieve accurate positioning of the catheters and to use CRH. With the criterion of a peripheral to petrosal ratio of > 2, in 23 patients who were subsequently proved histologically to have Cushing's disease a significant gradient was found in only 26% of cases where the internal jugulars were sampled. This rose to 46% when samples were obtained from the inferior petrosal sinus and to 85% following CRH.4 In the largest series to date (203 patients with Cushing's disease and 17 with ectopic ACTH secretion), a CRH-induced rise in the petrosal sinus plasma ACTH more than three times the peripheral level showed 100% diagnostic accuracy, sensitivity and specificity." Our experience" is that while localization is more than 90% accurate in determining whether the patient has a pituitary source, lateralization is probably no more than 70% accurate, a finding supported by others.?5.76 Assessment of the value of inferior petrosal sinus sampling in the lateralization of ACTH-secreting tumours is difficult, as tumours may be midline or have anomalous drainage. Patients with ectopic ACTH-secreting neoplasms fail to respond to the CRH test and to inferior petrosal sinus catheterization with CRH injection. This test should be used only for patients with clear clinical and biochemical evidence of ACTH-dependent hypercortisolism, not to distinguish patients with mild or intermittent Cushing's disease from normal subjects or from individuals with pseudo-cushingoid states." As with venous sampling catheterization, facilities must exist within the radiology theatre for the ACTH samples to be centrifuged cold and rapidly frozen. The success ofthis procedure is dependent on the radiologist's skills and the morbidity means that it is still limited to specialist centres." Inferior petrosal sinus sampling after CRH administration is a powerful aid in differentiating the aetiology of ACTH-dependent Cushing's syndrome and should be performed where doubt as to the correct diagnosis persists after CRH stimulation test and HDDST, but it should be confined to specialist centres. RADIOLOGICAL IMAGING TECHNIQUES Imaging techniques are useful for locating a tumour, but provide no information as to function. Cushing's syndrome patients with undetectable plasma ACTH (therefore adrenal adenoma or carcinoma) should proceed to a CT scan of the adrenals. The CT scan will rapidly confirm the diagnosis, locate the lesion and generally distinguish adenoma (unilateral mass, small, rounded, well circumscribed) and carcinoma (usually larger than 5 em in diameter [and therefore likely to weigh > 100 g], irregular/ lobulated, infiltrating with metastases)." The uninvolved contralateral adrenal gland may be normal or atrophic. CT of the adrenals in patients with ACTHdependent Cushing's syndrome shows bilaterally hyperplastic glands. About 10-15% of ACTHdependent Cushing's syndrome patients may also have nodular hyperplasia of the adrenal gland." while up to 30% of proven Cushing's disease patients have normal adrenals.p A CT or MR! scan of the pituitary demonstrating an adenoma is useful to the neurosurgeon in a patient with clinical and biochemical Cushing's disease. Neither the CT nor the MR! has any diagnostic value; CT of the pituitary detects only 30% of corticotroph adenomas.p MRI is more sensitive in detecting corticotroph adenomas but still detects only 50-60% of microadenomas which cause Cushing's disease.p' Furthermore, up to 10% of the population aged have incidental tumours of the pituitary revealed by MRI.84

10 354 Perry and Grossman Patients with clinical and biochemical signs of ectopic ACTH production should receive a CT of the chest and abdomen to reveal the other potential sources such as bronchial or thymic carcinoids,85.86 pancreatic islet cell tumours and intestinal carcinoids It is increasingly our experience that all patients with ACTH-dependent Cushing's syndrome should have high resolution CT scanning of the thorax. CUSHING'S SYNDROME DURING PREGNANCY Cushing's syndrome during pregnancy is very rare (less than 100 cases reported in the literature). In the largest series (22 patients), the majority had adrenal tumours; only four had Cushing's disease (none of whom were diagnosed during pregnancyj.f" The diagnosis of Cushing's syndrome during pregnancy is complex because the biochemical features are obscured by changes in the normal hypothalamo-pituitary-adrenal (HPA) axis that occur during gestation. These include the increased plasma levels of cortisol and cortisol-binding globulin and urinary free cortisol, the failure of suppression of the HPA axis by dexamethasone," enlargement of the pituitary during normal pregnancy'? and the presence of placental CRR.93 The diagnosis is important because Cushing's syndrome during pregnancy is associated with a high rate of complications for both mother and fetus. Differentiating Cushing's syndrome during pregnancy from a normal pregnancy is difficult. A recent case report demonstrated the diagnosis and cure of Cushing's syndrome during pregnancy.p' Diagnosis was aided by the CRH test and MRI. Magnetic resonance scanning was particularly valuable and avoided the risk of unnecessary exposure to irradiation during pregnancy. Therefore, if a pregnant patient is suspected to have Cushing's syndrome, the recommended two procedures most likely to confirm the diagnosis and possible aetiology are the CRR test and an MRI scan. CONCLUSION When the patient's history and clinical presentation suggest Cushing's syndrome, an outpatient low-dose dexamethasone suppression test (0'5 mg every 6 h for 48 h) should be the initial investigation. If the plasma cortisol concentration is lower than 50 nmol/l at the end of the test, then Cushing's syndrome is effectively excluded. Detectable plasma cortisol levels should be correlated with other factors to exclude a false-positive result (e.g. poor compliance with prescribed medication, concurrent medication with oestrogen or drugs which will increase the metabolism of the dexamethasone). The patient is next admitted to hospital for further tests (see the algorithm Appendix 3), such as the 'midnight sleeping plasma cortisol' (to confirm Cushing's syndrome), measurement of electrolytes (hypokalaemia strongly suggests ectopic ACTH-dependent Cushing's syndrome) and plasma ACTH (a persistently undetectable ACTH level in the presence of elevated plasma cortisol strongly suggests an adrenal tumour, which can be rapidly confirmed by CT scanning of the abdomen). CRH/HDDST should not be done unless plasma ACTH levels are clearly undetectable. The LDDST is performed a second time in our unit either if the clinical suspicion is that the patient is not Cushingoid or where the patient is a tertiary referral and the LDDST had not been performed prior to admission. If the hcrh test shows a plasma cortisol rise of > 20% above basal, and the patient suppresses plasma cortisol by more than 50% of basal level on high-dose dexamethasone, the combined results strongly suggest pituitarydependent Cushing's syndrome (i.e. Cushing's disease). CT or MR imaging may also demonstrate a pituitary lesion. Borderline results of the CRH or HDDST require further testing using inferior petrosal sinus catheterization with CRH to confirm Cushing's disease, although it may also be argued that all patients with ACTHdependent Cushing's syndrome should have this procedure. The test results together with the CT scanning will confirm the diagnosis and are used by the neurosurgeon when proceeding with transsphenoidal surgery, the therapy of choice in these cases. Finally, the most difficult category to diagnose are patients with occult ectopic ACTH-dependent Cushing's syndrome. These patients typically fail to respond to the CRH and HDDST, have detectable plasma ACTH, and a low K + with alkalosis and impaired glucose tolerance. These patients must be investigated with a detailed CT of the chest and abdomen and an analysis of blood tumour markers. If a suspicious lesion is detected by Ann cu«biochem 1997: 34

11 Diagnosis of Cushing's syndrome 355 TABLE 2. ACTH results from a patient who underwent bilateral simultaneous petrosal sinus sampling with corticotrophin releasing hormone (CRH). Baseline plasma ACTH results in the right and left low and high jugular veins as well as in the peripheral circulation show that the right and left low jugular sites have a gradient of approximately 1 with the simultaneous peripheral. The right high jugular has a gradient of1 with the peripheral, but the left high jugular has a gradient of2 8 with the simultaneous peripheral. This is consistent with a diagnosis ofcushing's disease. The gradient is amplified when the catheter samples the petrosal sinus and following CRH injection. The right petrosal sampling sites do not show a significant gradient, whereas the left petrosal sites show a basal gradient of 3 3 with the peripheral sample which 5 min after CRH, increases to 166. The technique not only confirms Cushing's disease but also lateralizes the tumour to the left in the pituitary fossa. Tumour histology confirmed Cushing's disease ACTH (ng/l) Peripheral High jugular Low jugular Petrosal Left Right Left Right Left Right Baseline results CRH test Time (min) imaging, this could be assessed by a venous sampling catheter to locate the source of the ectopic ACTH secretion or by biopsy of the lesion and establishing a histological diagnosis and immunocytochemistry. The diagnosis and differential diagnosis of Cushing's syndrome continues to challenge even the most experienced clinical centres, particularly because of the need for serial stimulation and suppression tests (and their interpretation) and the cyclicity of some tumours. The expertise required to perform the inferior petrosal sinus and whole-body venous sampling catheterizations continues to restrict evaluation of the more difficult cases of Cushing's syndrome to specialist centres. APPENDIX 1 Protocol for performing the CRH test 1 In the standard CRH test, the patient is fasted overnight, and an intravenous cannula is inserted into a forearm vein at 0815 h. Blood is sampled basally at 0845 hand 0900 h for ACTH and cortisol. Synthetic human CRH, either 100j.Lg or I j.lgjkg, in I ml of acid saline 59 is injected rapidly. Blood is sampled at 15min intervals for 1h, then at 30 min intervals for the second hour and assayed for plasma ACTH and cortisol. ACTH peaks at about 30 min and cortisol at 45 to 60 min after CRH administration. APPENDIX 2 Protocol for simultaneous bilateral inferior petrosal sinus sampling with CRH stimulationi Patients being prepared for petrosal catheterization should not be given sedatives, as these may inhibit the ACTH response to CRR. A suitable period (at least 72 h) should have elapsed between the high-dose dexamethasone test and the sampling to ensure that the pituitary is not suppressed. When a catheter has been placed in each inferior petrosal sinus, blood samples are simultaneously withdrawn from each sinus and from a peripheral vein, immediately before peripheral venous injection of hcrh (100 j.lg). For 2min intervals, that is 3-5, 8-10 and min after the CRH injection, blood is again simultaneously sampled from each site and assayed for ACTH (Table 2). In patients who have rapidly fluctuating states of hypercortisolism, a basal peripheral cortisol level should be used to assess whether their disease has 'switched off; otherwise, it is unnecessary to measure plasma cortisol during this test. APPENDIX 3 A simplified algorithm for the recommended daily protocol for evaluating a patient with Cushing's syndrome Pre-admission: Diagnosis of Cushing's syndrome established by low-dose dexamethasone

12 356 Perry and Grossman suppression test (LDDST) (0,5 mg every 6 h for 48 h); if cortisol is < 50 nmol/l then Cushing's syndrome is unlikely; if > 50 nmol/l obtain outpatient plasma ACTH to determine whether detectable. Ifplasma cortisol after LDDST is > 50 nmol]l: admit to hospital Monday Routine chemistry (plasma K +, glucose and LFTs) Tuesday If ACTH detectable do CRH test: give 100 J1.g hcrh intravenously and measure cortisol and ACTH for 2h Wednesday Circadian rhythm study: sample for cortisol and ACTH at 0900 h, 1800 h and midnight (asleep) Thursday- Monday Low-dose followed by high-dose dexamethasone suppression test (0,5 mg dexamethasone orally every 6 h for 48 h followed by 2 mg every 6h for 48 h). By the following Monday/Tuesday all results of tests are available and provide provisional diagnoses: A No suppression on LDDST = Cushing'S syndrome. If pseudo-cushing's syndrome due to depression is suspected, perform an insulin-induced hypoglycaemia test B Plasma ACTH is undetectable: diagnostic of adrenal tumour. Proceed to imaging of abdomen and adrenalectomy; omit the CRH test if the outpatient plasma ACTH level is undetectable C Plasma ACTH detectable but not grossly elevated: diagnosis of pituitary or ectopic ACTH secretion: (i) (ii) Plasma cortisol rises during the CRH test and is suppressed during HDDST = pituitary tumour; Cushing's disease. Proceed to pituitary imaging, petrosal sinus catheter study and transsphenoidal adenomectomy. Serum cortisol does not rise during CRH test and does not suppress on HDDST. = either Cushing'S disease or ectopic ACTHproducing tumour. Plasma ACTH may be grossly elevated (> 300 ng/l) plus hypokalaemia, alkalosis and hyperglycaemia. Tumour markers may be positive. Proceed with imaging studies of the chest, abdomen and pituitary and a petrosal sinus catheter study. (iii) If the results of the CRH test and HDDST are discrepant i.e. do not point to a uniform diagnosis and imaging studies are not helpful, it becomes mandatory to proceed to petrosal sinus catheter (with CRH) since pituitary disease is much more common than the ectopic ACTH syndrome. If petrosal sinus sampling is not possible locally, refer the patient to a specialist centre capable ofperforming the procedure. (iv) Biochemical and radiological investigations make it impossible to decide on the aetiology. Proceed with controlling the hypercortisolism medically (using ketoconazole or metyrapone) and after an interval the relevant investigations can be repeated. REFERENCES I Trainer PJ, Besser GM. The Bart's Endocrine protocols. London: Churchill Livingstone, Crapo L. Cushing's syndrome a review of diagnostic tests. Metabolism 1979; 28: Carpenter PC. Cushing's syndrome: update of diagnosis and management. Mayo Clin Proc 1986; 61: Trainer PJ, Grossman AB. The diagnosis and differential diagnosis of Cushing's syndrome. Clin Endocrinol 1991; 34: Muller OA, von Werder K. Ectopic production of ACTH and corticotrophin releasing hormone (CRH). J Steroid Biochem Molec Bioi 1992; 43: Danese RD, Aron DC. Cushing's syndrome and hypertension. Endocrinol Metah CUn N Am 1994; 23: Findling JW, Doppman JL. Biochemical and radiologic diagnosis of Cushing's syndrome. Endocrinol Metab Clin N Am 1994; 23: Orth DN. Cushing's syndrome. N Eng! J Med 1995; 332: Weber A, Trainer PJ, Grossman AB, Afshar F, Medbak S, Perry LA, et al. Investigation, management and therapeutic outcome in 12 cases of childhood and adolescent Cushing's syndrome. Clin Endocrinol 1995; 43: Magiakou MA, Mastorakos G, Oldfield EH, Gomez MT, Doppman JL, Cutler GB, et al. Cushing's syndrome in children and adolescents. Presentation, diagnosis and therapy. N Engl J Med 1994; 331: II Chrousos GP, Schulte HM, Oldfield EH, Gold PW, Cutler GB, Loriaux DL. The corticotrophinreleasing factor stimulation test: An aid in the evaluation of patients with Cushing's syndrome. N Engl J Med 1984; 310: Oldfield EH, Chrousos GP, Schulte HM, Schaaf M, McKeever PE, Krudy AG, et al. Pre-operative

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