Endocrine Care

LIMITS OF DEXAMETHASONE SUPPRESSION TESTS IN THE

DIAGNOSIS OF CUSHING’S SYNDROME

Monica Gheorghiu*, Simona Galoiu, Andra Caragheorgheopol, M. Coculescu

Department of Endocrinology, “Carol Davila” University of Medicine and Pharmacy, “C.

I. Parhon” Institute of Endocrinology Bucharest, Romania

Introduction: The dexamethasone suppression tests (DST) in the diagnosis of Cushing's

syndrome (CS) give frequently equivocal results. Our study evaluated the precision of DST in
the diagnosis of CS. Patients and methods: 223 patients (15 - 77 years, 130 F / 93 M) were
studied for putative CS by morning and midnight serum cortisol, urinary free cortisol (UFC) and
17 hydroxycorticosteroids (17OHCS) levels at baseline and after DST as follows: 1 mg overnight
(oDST), 0.5mg q.d., 2 days (LDDST) and 2 mg q.d. 2 days (HDDST). Since not all cases were
evaluated by all tests, statistical analysis used available results.
Results: 79 patients had CS (47 pituitary, 3 ectopic, 21 adrenal adenoma and 8 adrenal
carcinoma), 45 had adrenal tumor without all the criteria for CS (NCT) and 99 were controls. All
patients with CS had abnormal cortisol biorhythm, but also 31.8% patients with NCT and 50%
evaluated controls. The best basal screening test for CS is UFC, with a cut-off value of 100 g/24
h. For DST with a serum cortisol cutoff level (CCL) of 5 µg/dl, oDST correctly diagnosed all CS,
while lowering the CCL at 1.8 µg/dl increased the false positive rate to 6.8%. At LDDST, the
serum CCL of 5 µg/dl correctly identified all patients with CS, but was 2.5 % false positive in
controls. A significant correlation of serum cortisol values after oDST or LDDST with basal
17OHCS and UFC was found (r=0.6, p<0.001), suggesting that patients with mild CS are more
prone to test as false negatives. The classical criterion of 50% suppression for UFC after LDDST
correctly identified 12/14 CS patients and all 8 controls. Better sensitivity (Sn) had an UFC cut-
off level of 10µg/24h, p<0.001. A 50% suppression of 17OHCS identified 45/61 CS patients and
excluded the disease in 30/37 patients. In HDDST, serum cortisol suppression by > 50%
diagnosed Cushing's disease (CD) with 85 % Sn and 57% specificity (Sp). The best HDDST
accuracy had UFC (83%), with 27% false negative results (3/11 patients) for a cut-off value of
50% from baseline. 17OHCS were suppressed by 50% in 19/35 patients with CD (54%) and in
3/33 (9%) patients with other causes of CS. NCT patients had higher basal values of UFC and
17OHCS than controls (p<0.01) and up to 50% had an abnormal biorhythm, suggesting a degree
of hypercortisolism, with an inadequate oDST or LDDST . Therefore, only 6/45 NCT fulfilled
the criteria for subclinical CS.
Conclusion: The best screening test was oDST (100% Sn and Sp), followed by UFC
(100% Sn and 92% Sp). LDDST with serum CCL at 5 µg/dl had 100% Sn and 97% Sp.
*Correspondence to: Monica Gheorghiu “C. I. Parhon” Institute of Endocrinology, Bd. Aviatorilor
34-36, Bucharest, Romania, fax: +4021.3198718, e-mail:monicagheorghiu@yahoo.com

Acta Endocrinologica (Buc), vol. II, no. 1, p. 45-61, 2006

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 Monica Gheorghiu et al.

HDDST identified CD by UFC with 73% Sn and 92% Sp and by serum cortisol with 85%
Sn and 57% Sp. For NCT, the standard tests identified SCS in 13%. However, a thorough
evaluation including multiple tests should be undertaken for the positive and differential
diagnosis of Cushing's syndrome.
Key words: Cushing’s syndrome, dexamethasone, cortisol, adrenal tumor.

INTRODUCTION

Cushing’s syndrome (CS) is difficult to diagnose because there is no “gold

standard“ test either for the positive, or for the etiological diagnosis. Since the
original description by Liddle in 1960 (1), the dexamethasone suppression tests
have been widely used as a main diagnostic tool in CS. Unfortunately, these tests
give frequently equivocal results. In the original suppression test, the standard
criterion of 50% suppression has been applied to the 24h urinary 17 hydroxy-
corticosteroids (17OHCS). In an attempt to improve the test’s sensitivity and
specificity over the pre-test probability, clinicians have replaced the measurement
of 17OHCS with 24h-urinary free cortisol (UFC) and with the absolute value of
post-test morning serum cortisol. Controversy exists also regarding the serum
cortisol cut-off level. Lowering the cut-off from 5 to 1.8 µg/dl improves the test’s
sensitivity, but enhances the rate of false positive results (2).
The high dose dexamethasone suppression test (HDDST) has been
traditionally used to distinguish Cushing’s disease from ectopic ACTH secretion.
However, its reported sensitivity of 50-60% brought several authors to doubt its
efficacy in clinical practice (3, 4).
In the last decade, it became more evident that the spectrum of autonomous
hypercortisolism ranges from mild to overt clinical Cushing’s syndrome (CS). About
10-20% of the incidentally discovered adrenal tumors show some degree of abnormality
of hypothalamo-pituitary -adrenal (HPA) axis (named subclinical Cushing’s syndrome
– SCS) (5, 6). A higher accuracy of the biochemical tests is required, especially for the
mild hypercortisolism. This would avoid the high morbidity rate induced by a long-
standing CS and would allow a less invasive laparoscopic surgery to be performed in
our patients.
In the current study, we evaluated the efficacy of the classical dexamethasone
suppression tests for the positive and differential diagnosis of Cushing’s syndrome
(including the subclinical type) in patients who were referred for putative
hypercortisolism to our academic center.

PATIENTS AND METHODS

We retrospectively studied 223 patients investigated for suspected

autonomous hypercortisolism, due to clinical features or an incidentally discovered

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 Dexamethasone tests in Cushing’s syndrome

adrenal tumor. They have been evaluated in the Department of

Neuroendocrinology, “C. I. Parhon” Institute of Endocrinology, Bucharest. Patients
had a mean age of 34 (range 15 – 77 years), 130 women and 93 men. Endocrine
evaluation consisted of baseline measurements of serum cortisol at 08.00 h and/or
23.00 h, 24 h urinary free cortisol (UFC) and 24 h urinary 17 hydroxycorticosteroids
(17OHCS), overnight 1 mg dexamethasone suppression test (oDST) or 2 × 2 mg
low dose dexamethasone suppression test (LDDST). Patients who failed to suppress
to oDST or LDDST underwent further endocrine evaluation, including a 2 × 8 mg
high dose dexamethasone suppression test (HDDST).
Based on this work-up, patients have been retrospectively included in 3 groups:
- Cushing’s syndrome (CS): 79 patients with documented Cushing’s
syndrome, including 47 patients with Cushing’s disease (CD), 3 with ectopic ACTH
secretion (EAS), 21 with adrenal adenomas and 8 adrenal carcinomas. These
patients have been investigated between 1978-2006.
- Non-Cushing adrenal tumors (NCT): 45 patients with adrenal tumors
including 22 patients with incidentally discovered adrenal tumors and 23 patients
with clinical suspicion of CS, who did not fulfill all the biochemical criteria for
Cushing’s syndrome. Patients with pheochromocytoma, adrenal cysts, metastatic
lesions, or primary aldosteronism, based on endocrine testing, imaging studies or
histopathologic examination of resected tumors were excluded. These patients have
been investigated between 1994-2006. They were analyzed separately and were
excluded from the statistical analysis with regard to test characteristics.
- Controls (C): 99 patients with few clinical signs suggestive for
hypercortisolism, adequate suppression to DST, without known adrenal tumor,
consisting of 85% obese patients (BMI ≥30 Kg/m2) and 7% overweight patients
(25≤BMI<30 Kg/m2). These patients have been investigated between 2004-2006.
Diagnostic tests
Cortisol biorhythm has been evaluated by sampling at 23.00h, most patients
being awake or awakened for this purpose.
oDST: serum cortisol was obtained at 8.00 h after 1mg dexamethasone was
given at 23.00h.
LDDST and HDDST: 24h urine was collected for UFC and/or 17OHCS during
the day preceding the test and serum cortisol was obtained at 08.00h on day 0 (basal
values). Dexamethasone orally was administered at a dose of 0.5 mg for the LDDST
and at a dose of 2 mg for the HDDST every 6 h for 48 h, after the first blood sample
was taken. 24 h urine was collected for urinary free cortisol (UFC) and 17OHCS in
day 2, and blood samples at 8.00h (48h after the first dose of dexamethasone) were
collected for serum cortisol measurement. 17OHCS values have been divided by 24
h creatininuria, in order to avoid variation due to obesity. In patients in whom the
HDDST followed the LDDST immediately, the basal serum cortisol value of the
LDDST was considered the basal value for the HDDST (7).
In patients with NCT, subclinical Cushing’s syndrome (SCS) has been defined
as an abnormal response to oDST or LDDST and at least another abnormal test of

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the HPA axis function (serum cortisol rhythm, urinary cortisol/17OHCS, serum
ACTH, but not HDDST) in the absence of overt clinical signs or symptoms of
hypercortisolism.
Pituitary and/or adrenal imaging were performed using computed tomography
or magnetic resonance imaging for the pituitary (n=124) and/or the adrenals (n=43).
Assays
Serum and urinary cortisol were measured using chemiluminescence cortisol
assay ACS: 180. The intra-assay coefficient of variability was 5 – 7.6 %. Normal
reference range for 8.00h serum cortisol = 4.3 – 22.4 µg/dl and for 24h UFC = 28.5
– 213.7 µg/24h. There was a 7.4% cross reactivity with cortisone, 11 deoxycortisol,
0.2% with dexamethasone. Urinary metabolites of cortisol, 17OHCS were
measured by spectrocolorimetric, Porter Silber method.
Statistical analyis
We used descriptive tests, ANOVA, Mann Whitney test for inter-group
differences, ROC (receiver operating characteristic) curves. A p <0.05 was
considered statistically significant. Since not all cases were evaluated by all tests,
statistical analysis used the available results.

RESULTS

Patient characteristics
The investigated patients had one or more features suggestive for Cushing’s
syndrome, most of them being non-specific: obesity, hypertension and menstrual
disorders. Only 15 out of the 22 patients with incidentally discovered adrenal
tumors had no suggestive signs or symptoms.
The patient characteristics for the entire series, stratified by diagnosis (n=223),
are shown in Table 1. Controls were significantly younger than patients with CS and
NCT (31+12.8 vs. 37.2+11.9 and 53.5+12.6, respectively, p<0.001 and 0.03,
respectively). Of note, 91 % of controls were overweight or obese. In patients with
CD, 23 patients had a pituitary microadenoma, 12 patients had a macroadenoma,
and 12 patients had no identified pituitary tumor.
Basal hormonal values
Basal values of 23.00h serum cortisol, urinary 17OHCS and UFC are shown
in Table 2. Basal values of all tests were similar in controls and NCT patients, and
significantly higher in patients with CS.
The 23.00h serum cortisol was > 5 µg/dl in all evaluated patients with CS
(n=51), but also in 31.8% (7/22) patients with NCT and in 50% (6/12) controls,
resulting in 100% sensitivity with only 50% specificity (Table 3). Considering a
cut-off level of 1.8 µg/dl, a normal biorhythm showed none of CS patients, and only
6/22 patients with NCT and 3/12 controls, lowering the specificity to 25%.
24h UFC is currently considered a valuable screening tool for CS. UFC in CS
(range 101.3 – 1333 µg/24h) is statistically higher than in controls (46.6 –

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 Dexamethasone tests in Cushing’s syndrome

180.6 µg/24h). The upper limit of our commercial assay for UFC evaluation is
213.7 µg/24h. This is at least twice higher than the reported normal range for most
UFC assays (80-120 µg/24h) (8). Using our assay’s upper limit, only 7/19 (36.8%)
of CS patients had abnormal values and none of controls, giving only 37%
sensitivity and 100% specificity. The optimal UFC cut-off level revealed by ROC
analysis on our patients is 100 µg/dl, with 100% sensitivity and 82% specificity
(Fig. 1). This leads to18% false positive controls.
Urinary 17OHCS are less discriminative than UFC. Even divided by the 24h
urinary creatinine, in order to avoid false positive results in obese patients, there is
a remarkable overlap between controls (range 0.5-10.9 mg/24h) and CS patients
(range 2.6-58 mg/24h). The upper limit of normal range (5.5 mg/24h) had 94%
sensitivity, but only 70% specificity for the diagnosis of CS. A better specificity
(98%) is obtained with a cut-off of 10 mg/24h, with a fall in sensitivity at 73%.
The accuracy of basal tests is illustrated in Table 3.
oDST
Serum and urinary cortisol values and 17OHCS values after oDST and
LDDST are shown in Table 4.
Considering a post-test serum cortisol cut-off level of 5 µg/dl, oDST correctly
discriminated Cushing’s syndrome (n=5) from controls (n=44) in all evaluated

Table 1. Characteristics of patients of the studied groups

Controls Non-Cushing Cushing's Adrenal Adrenal Ectopic

adrenal disease adenoma carcinoma ACTH
tumors Syndrome
n 99 45 47 21 8 3
Sex (F/M) 35/64 30/15 38/9 21/0 5/3 1/2
Age (years) 31 + 12.8 a 53 + 12.096 a, b 34 + 1.7 b 42 + 11.3 b 43.5 + 7.8 b 34 + 21 b
(median + SD)
BMI > 25 91.9 73.3 81 76.2 75 66.7
kg/m2 (% of
patients)
a p<0.001 controls vs. Non-Cushing adrenal tumors;
b p<0.03 controls vs. patients with Cushing's syndrome (including Cushing's disease)

patients. ROC analysis showed 100% sensitivity and 100 % specificity for this cut-
off in the CS diagnosis (area under curve, AUC=1, p<0.001).
Lowering the cut-off at 1.8 µg/dl, the false positive rate increases to 6.8% (3/44
controls) and the specificity of the test falls to 93 %.
LDDST
The serum cortisol cut-off level of 5 µg/dl correctly identified all patients with CS
(57/57), but yielded 2.5 % (1/40) false positive results in controls (1 patient who had
an adequate suppression at repeated testing during follow-up). ROC analysis showed

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Table 2. Basal values of 23.00h serum cortisol, 24h urinary free cortisol (UFC) and 17
hydroxycorticosteroids (17 OHCS) in patients evaluated for suspected Cushing's syndrome

Basal Values Controls Cushing's syndrome Non-Cushing

(Median + SD) (n = 99) adrenal tumors
Cushing's disease ACTH independent Cushing's (n = 45)
(n = 47) syndrome and EAS (n = 32)
23.00h serum 4.9 + 7.5 25.8 + 12.1 a 21.29 + 10.1 b 4.3 + 3.5 a, b
cortisol (µg/dl) n = 12 n = 42 n=9 n = 22

UFC (µg/24h) 85.1 + 35.1 198.6 + 348.3 a 145.3 + 282.6 b 103.1 + 41.3 a, b
UFC >100 2/11 11/11 8/8 10/17
µg/24h
(no. of patients)
17 OHCS 4.7 + 2.3 15.7 + 8.03 a 15.8 + 12.8 b 5.7 + 2.3 a, b
(mg/24h/g
creatininuria)
17OHCS > 5.5 15/50 38/40 26/28 20/36
mg/ 24h/g
creatininuria
(no. of patients)
a p<0.001 Cushing's disease vs. non-Cushing adrenal tumors
b p<0.01 Cushing's syndrome vs. non-Cushing adrenal tumors

100% sensitivity and 97% specificity for this cut-off in the CS diagnosis, (AUC) =
0.99, p<0.001.
Serum cortisol after LDDST in patients with CS ranged 7.3 –121.5 µg/dl, with no
significant difference between patients with CD and patients with other etiologies of CS,
but significantly higher than in controls (range 0.03-18.5 µg/dl) p<0.001 (Fig. 2).
Considering a cut-off level of 1.8 µg/dl, 4/40 (10%) of the controls were false
positive, with 90% specificity (Table 5).
The historical criterion of 50% suppression for serum cortisol was less
sensitive, leading to 92% sensitivity and 91% specificity.
In 9 patients, both oDST and LDDST were performed and 8.00h serum
cortisol had similar values (p = ns).
In the LDDST, urinary tests had a lower sensitivity or specificity compared to
serum cortisol.
The classical criterion of 50% suppression for UFC correctly identified 12/14
CS patients, and all 8 controls. The test showed an 85% sensitivity and 100%
specificity with only 15% false negatives, but the number of patients was small. A
UFC value above 10 µg/24h in LDDST has 100% sensitivity and 88% specificity
for Cushing’s syndrome diagnosis, area=1, p<0.001.
A 50% suppression of 17OHCS identified 45/61 CS patients and excluded the
disease in 30/37 patients, showing a 74% sensitivity and 81% specificity (Fig. 3).
The false positive controls (n=7) had low (n=5) or normal (n=2) basal 17OHCS

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 Dexamethasone tests in Cushing’s syndrome

values. An optimal cut-off in our patients was an absolute value of 17OHCS after
LDDST of 3.3 mg/24h/g urinary creatinine, with 100% sensitivity and 98%
specificity in diagnosing autonomous hypercortisolism, area under curve=0.99,
p<0.001. Although the post-test serum cortisol value did not differ in CD patients
Table 3. Accuracy of 23.00h serum cortisol, basal 24h urinary free cortisol (UFC) and
17 hydroxycorticosteroids (17 OHCS) for the diagnosis of Cushing's syndrome
Cut-off Sensitivity Specificity False positive False negative Accuracy
(%) (%) (%) (%) (%)
23.00 h 5 (µg/dl) 100 50 50 0 90
cortisol 1.8 (µg/dl) 100 25 75 0 86
Basal > 5.5 94 70 30 6 84
17OHCS mg/24h/g
creatininuria
Basal UFC > 100 g/24h 100 82 18 0 93

Table 4. Characteristics of oDST and LDDST in patients evaluated for suspected Cushing's
syndrome
Cushing's syndrome Non-Cushing adrenal tumors

Cushing's ACTH Nonfunctioning Adrenal tumors

(Median + SD) Controls disease independent adrenal tumors with subclinical
(n = 99) (n = 47) Cushing's (n = 39) hypercortisolism
syndrome and (n = 6)
EAS (n = 32)
oDST
Cortisol (µg/dl) 0.5 ± 0.6 a 20.3 ± 6.4 26.09 1.2 ± 1.2 a 5.8
n = 44 n=4 n=1 n = 15 n=1
LDDST
Cortisol (µg/dl) 0.3 ± 2.9 a 19.8 ± 8.8 b 23.2 ± 23.4 c 1.7 ± 1.9 a 6 ± 2.6 b, c
n = 40 n = 32 n = 25 n = 17 n=3
Cortisol 97 ± 151 a 15 ± 25.3 b -1.2 ± 33.7 c 88 ± 13 a 65 ± 11 b, c
suppression %
UFC (µg/24h) 6.4 ± 3.5 243 ± 406 188 ± 84 11.2 ± 11 61.6
n=9 n = 10 n=5 n=6 n=1
UFC 94 ± 4 -5.3 ± 34 -9 ± 63.7 91 ± 11 34
suppression %
17 OHCS 1.02 ± 0.9 a 8.8 ± 6.4 b 14.4 ± 13.3 c 2.2 ± 1.7 a 4.9 ± 2.2 b, c
(mg/24h/g n=37 n = 34 n = 27 n = 22 n=5
creatininuria)
17 OHCS 68 ± 28 a 36 ± 78 -4.3 ± 43 57 ± 87 a 32 ± 93
suppression%

a p<0.05 controls vs. nonfunctioning adrenal tumors

b p<0.05 Cushing's disease vs. adrenal tumors with subclinical hypercortisolism
c p<0.05 Cushing's syndrome vs. adrenal tumors with subclinical hypercortisolism.

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compared with other etiologies of CS, the suppression of serum cortisol and
17OHCS is higher in CD patients, p<0.05, (Table 3).
There is a similar and significant correlation of serum cortisol values after
oDST or LDDST with basal 17OHCS and UFC (r=0.6, p<0.001, Fig. 4). In our
series, none of the patients with EAS showed >50% suppression.
High dose dexamethasone suppression test
HDDST has been traditionally used for the differential diagnosis between
Cushing’s disease and ACTH ectopic secretion or a cortisol secreting adrenal
tumor. The classical 50% suppression criterion for serum cortisol correctly
identified only 11/34 (32%) patients with CD (68% false negative), and 30/32
(93%) patients with other causes of CS (6% false positive, Fig. 5). Thus, in our

Figure 1. ROC curve for 24 h

urinary free cortisol in patients
investigated for putative
hypercortisolism. The optimal cut-
off level is 100 µg/dl, with 100%
sensitivity and 92% specificity.

Figure 2. Serum cortisol after low

dose dexamethasone suppression
test (LDDST) in studied patients.
CD = Cushing's disease, CS =
Cushing's syndrome, NCT = Non-
Cushing adrenal tumors, SCS =
Subclinical Cushing's syndrome.
Horizontal red line indicates the
cut-off value of 5 µg/dl for serum
cortisol. Green squares represent
patients with ectopic ACTH
secretion.

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 Dexamethasone tests in Cushing’s syndrome

Table 5. Accuracy of oDST and LDDST in patients evaluated for suspected Cushing's syndrome

Cut-off Sensitivity Specificity False False Accuracy

(%) (%) positive (%) negative (%) (%)
oDST 5 (µg/dl) 100 100 0 0 100
Cortisol)
1.8 (µg/dl) 100 93 7 0 92
LDDST 5 (µg/dl) 100 97 3 0 99
Serum
1.8 (µg/dl) 100 90 10 0 96
cortisol
LDDST 50% 85 100 0 15 91
UFC suppression
10 (µg/24h) 100 88 12 0 96
LDDST 50% 74 81 9 26 76
17OHCS suppression
3.3 mg/24h/ 100 97 3 0 99
creatininuria

patients, in HDDST, serum cortisol suppression by > 50% from baseline can
diagnose Cushing’s disease with only 85 % sensitivity and 57% specificity,
AUC=0.5, p=ns (Table 7).
In CD, 17OHCS were suppressed by 50% in 19/35 patients with CD (54%)
and in 3/33 (9%) patients with other causes of CS (54% sensitivity and 91 %
specificity, Fig. 6). UFC was suppressed by > 50% in 8/11 (73%) of CD patients
(27% false negative) and in 1/12 (8%) patients with other CS etiologies. The test
sensitivity was 73% and specificity 92% (Fig. 7).
We found no statistical differences in basal and LDDST hormonal
characteristics or tumor size in patients with CD who did not suppress at HDDST.
There was a significant correlation between the percent of fall in serum
cortisol after LDDST and HDDST, r=0,69, p<0.001. In patients with CD, serum
cortisol after HDDST was significantly lower as compared to other CS etiologies
(p<0.01), with a better suppression percent after HDDST (34+33% versus 9.4 +
106%, p<0.01), but also after LDDST (15 + 25% versus -1.2 + 33%, p=0.04). The
same characteristics are true for UFC and 17OHCS (Table 6). However, the range
of suppression was very large in each group of CS (0 to > 90%). None of the
patients with EAS showed a cortisol or 17OHCS suppression > 50% at HDDST.
Subclinical Cushing’s syndrome
In the group of patients with adrenal tumors who did not fulfill all the
biochemical criteria of CS, 59% (10/17) patients had high basal UFC values (>100
µg/dl), 55% (20/36) had high 17OHCS basal levels and 31.8 - 50% (7 - 11/22) had
an abnormal biorhythm, when a serum cortisol cut-off of 5 and 1.8 µg/dl,
respectively, is considered. These numbers are higher than those recorded in the
control population (p<0.05 for the urinary figures), suggesting that at least some of
the NCT patients may have a degree of hypercortisolism (Table 2).

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LDDST indicated a lack of suppression by the standard 50% criterion in 14%

(1/7) for UFC, 40% (11/27) for 17OHCS and 15-30% (3-6/20) for cortisol (cut-off 5 -
1.8 µg/dl). Significantly more patients from this group showed inadequate suppression
compared to controls (p<0.01 for serum cortisol and 17OHCS suppression).
However, only 6/45 (13% of the patients with adrenal tumors) fulfilled the
criteria for SCS (i.e., aside lack of suppression in LDDST, they had at least another

Figure 3. 17OHCS
suppression after low dose
dexamethasone suppression
test (LDDST) in studied
patients. CD = Cushing's
disease, CS = Cushing's
syndrome, NCT = Non-
Cushing adrenal tumors, SCS
= Subclinical Cushing's
syndrome. Horizontal red line
indicates the cut-off value of
50% 17OHCS suppression.
Green squares represent
patients with ectopic ACTH
secretion.

Figure 4. Correlation between

basal 17 OHCS and serum
cortisol after overnight or low
dose dexamethasone
suppression tests (oDST or
LDDST) in patients with
Cushing's syndrome (n=51)
and controls (n=38), r=0.6,
p<0.01. Horizontal red line
indicates the cut-off value of 5
µg/dl for serum cortisol.

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 Dexamethasone tests in Cushing’s syndrome

abnormal test of the HPA axis function, as serum cortisol rhythm, basal UFC,
17OHCS or serum ACTH, but not HDDST). Four of them had non-symptomatic
incidentally discovered tumors. Even excluding the SCS patients from the statistical
analysis, the median values after oDST / LDDST for serum cortisol and 17OHCS
were significantly higher and the degree of suppression was lower in patients with

Figure 5. Serum cortisol after high dose
dexamethasone suppression test (HDDST) in
patients with Cushing's syndrome. Horizontal red
line indicates the cut-off value of 50% serum
cortisol suppression.
Figure 6. 17OHCS after HDDST in patients with
Cushing's syndrome. Horizontal red line
indicates the cut-off value of 50% for 17OHCS
suppression.

adrenal non-Cushing tumors, compared to controls. Patients with incidentally

discovered adrenal tumors have nonCushing similar hormonal values with patients
with adrenal tumors with clinical suspicion of hypercortisolism, except lower basal
levels of 17OHCS (4.03 ± 2.2 vs. 7.4±1.58, p= 0.002).
Nevertheless, the median values for all the tests are significantly lower in
patients with SCS as compared to patients with CS (Table 3).
Eight patients with adrenal tumor who did not suppress their UFC or 17OHCS
> 50% at the initial evaluation, suppressed their plasma cortisol and/or UFC at
subsequent testing.

DISCUSSION

Since the 60’s, when Liddle described the dexamethasone suppression testing
for Cushing’s syndrome, controversies have persisted about the best diagnostic tests
and the optimal test cut-off levels to be used. This study evaluated the limits of the

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classical diagnostic procedures, when both standard and modern cut-off levels were
applied to patients referred to our center for evaluation of putative hypercortisolism.
We should keep in mind that screening procedures must have a high
sensitivity, in order to identify even the patients with mild disease, while tests for
the diagnosis of the etiological form of CS should have a higher specificity.
Nevertheless, the test performance should be better than the pretest probability of
having the disease, in order to be useful.
Ideally, the evaluation of test accuracy should be done using the general
population as control. In this study, the controls have been referred to our center
because they had some clinical signs suggestive for hypercortisolism, like
overweight or obesity (in 91% of controls), hypertension or menstrual irregularities.
As obesity is a well-known condition that may interfere with the biochemical tests
for CS, this selection bias may result in test sensitivity and specificity values that
are lower than in the reference studies, but more representative for the clinical
practice.
The biochemical screening tests for CS include the evaluation of cortisol
biorhythm, basal UFC or 17OHCS and the 1 mg overnight dexamethasone testing
(oDST).

Figure 7. ROC curve for

suppression (%) of 24 h urinary
free cortisol after high dose
dexamethasone suppression
test (HDDST) in patients with
Cushing's syndrome. For the
classical cut-off of 50%
suppression, the test sensitivity
was 73% and specificity 92%.

It has been reported that only 3.4% of patients with CS may have normal
serum cortisol values at 23.00 h (9). Indeed, none of our patients with CS had a
23.00h cortisol below 5 µg/dl. However, the false positive rate in controls was about
50% and increased if a lower cut-off of 1.8 µg/dl was used. Hospitalization stress,
venipuncture stress or depression may have contributed to these results.
Even considering the well-known difficulties in urine collection, the
evaluation of UFC and 17OHCS/g creatininuria showed also a good sensitivity. In

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his review of 14 separate studies assessing the utility of 17OHCS measurement for
the diagnosis of Cushing’s syndrome, Crapo (9) reported a false negative rate of
11% of 315 patients with CS, while in obese controls the false positive rate was
27%. This compares favorably with our results (5.8% false negative and 30% false
positive rate).
UFC has a better reported sensitivity (95 to 100%) and specificity (94 – 98%)
(8, 9). It may be elevated in 40% of patients with depression and in 50% of women
with polycystic ovarian syndrome (10), as well as in patients treated with
fenofibrate, carbamazepine, digoxine, corticosteroids. On the other hand, up to 11%
of CS patients may have normal values at one of four 24 h urine collections (8)
Although few of our patients underwent UFC testing, a cut-off level of 100 µg/24h
resulted in 100% sensitivity and 92% specificity, which made it the best of the non-
dynamic screening tools.
The oDST is widely used as a reliable and easily to perform screening test for
CS. It has a reported sensitivity between 83 – 100% at a serum cortisol cut-off range
of 3.6 – 7.2 µg/dl (2). Lowering the cut-off to 1.8 µg/dl, as recommended by an
international consensus on CS diagnosis and treatment (11) will identify some of
the few CS patients with an unusual high suppression to dexamethasone, but
increases the false positive rate, sometimes up to a point where the overall utility of
the test is diminished. This was also the case in our series, where all the CS patients
had cortisol values > 5 µg/dl and the false positive rate increased from 0 to 6.8% in
controls at the lower cut-off.
Although both oDST and LDDST were performed only in a small number of
patients (n=9), the similar values of morning serum cortisol confirmed that oDST is
a valuable tool in the diagnosis of autonomous hypercortisolism (8).
The LDDST is used for the confirmation of CS in patients with positive
screening tests. The classical 2 day LDDST evaluated in patients with mild CS
compared to patients with pseudoCushing’s conditions yielded a sensitivity of only
79%, a specificity of 74% and a diagnostic accuracy of 71% using urinary steroids
(12). Findling et al (2) reported false-negative results in 38% of the patients when
UFC was used and 28% when 17OHCS were used.
These were similar to our results (table 5). A 17OHCS value above 3.3
mg/24h/g creatininuria and a UFC above 10 mg/24h after LDDST showed 100%
sensitivity in our patients and deserve to be taken into consideration when
interpreting the results of a LDDST.
Using the post-test 8.00h serum cortisol, at a cut-off of 5 µg/dl, it has been
reported an increase of the test’s sensitivity to 93 –94%, which is more appropriate
for a confirmation test such as LDDST (7, 13). In our series, the overall accuracy of
LDDST using this criterion was 99%.
As previously reported by others (2), we found a significant correlation
between serum cortisol values after oDST and LDDST with basal 17OHCS and
UFC (r = 0.6, p< 0.01), suggesting that patients with mild CS are more likely to
suppress their serum cortisol to low levels after a low dose DST.

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 Monica Gheorghiu et al.

On the other hand, controls with low levels of basal urine steroids (10 OHCS
< 3.5 mg/24h/g creatininuria) may not suppress adequately after LDDST, possibly
due to the method’s limit of detection at low urinary values.
The HDDST is a non-invasive means of differentiating CD from other CS
etiologies, particularly from EAS. The sensitivity of HDDST has been reported to
range from 65-100%, and the specificity from 60-100% (3). These values have
often been lower than the pretest probability of having CD (which has been 80-90%
in most series). As a consequence, several authors doubt its efficacy in the clinical
practice (3, 4).
In our series, EAS has been identified in only 3 of the patients and could not
be used as a distinct variable for the statistical analysis. Comparing patients with

Table 6. Characteristics of high dose dexamethasone suppression test in patients with

Cushing's syndrome

Median + SD Cushing's All other Adrenal Adrenal Ectopic ACTH

Disease etiologies of adenoma carcinoma Secretion
(n = 47) CS (n = 42) (n = 21) (n = 8) (n = 3)
Cortisol (µg/dl) 16.9 ± 9.1 a 23.5 ± 27.06 a 23.2 ± 32.8 23.2 ± 9.2 28.4 ± 19.3
n = 37 n = 22 n = 14 n=5 n=3
Cortisol 34 ± 33 a 9.4 ± 106.8 a 4.7 ± 132 17 ± 18 0.6 ± 46
suppression %
UFC (µg/24h) 95 ± 159 a 237.4 ± 130.3 a 334 ± 142 237 _
n = 10 n=5 n=4 n=1
UFC 73 ± 47 a -40.2 ± 109.5 a -11 ± 117 -135 _
suppression %
17 OHCS 5.7 ± 6 a 15.5 ± 18.7 a 14 ± 8 24.7 ± 15.6 58.7 ± 52.9
(mg/24h/g n = 36 n = 26 n = 17 n=7 n=2
creatininuria)
17 OHCS 54 ± 92a -6 ± 93.2 a -23 ± 90 5.7 ±104 -49 ± 130
suppression %
a p<0.05 Cushing's disease vs. all other etiologies of Cushing's syndrome

Table 7. Diagnostic accuracy of high dose dexamethasone suppression test (HDDST) for the
diagnosis of Cushing's disease

HDDST Cut-off Sensitivity Specificity False False Accuracy

(%) (%) positive (%) negative (%) (%)
Serum 50% 85 57 43 15 65
cortisol suppression
(µg/dl)
UFC 50% 73 92 27 8 83
(µg/24h) suppression
17OHCS 50% 54 90 46 10 72
(mg/24h/g suppression
creatininuria)

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 Dexamethasone tests in Cushing’s syndrome

CD to patients with all other etiologies of CS, the classical criterion of 50%
suppression in HDDST yielded a sensitivity of 85, 73, 54% and a specificity of 57,
90, 92%, for serum cortisol, UFC and 17OHCS respectively. The accuracy was
better for UFC measurements, but is still quite low. Apart from the difficulties in
collecting 24 h urine (which should be similar in both LDDST and HDDST), other
conditions may lead to an inadequate suppression in DST: psychiatric illness,
obesity, alcoholism, stress, elevated corticosteroid-binding globulin (i.e.,
pregnancy, estrogen treatment), glucocorticoid resistance, or decreased absorption
of dexamethasone, drugs that speed enzymatic liver activity (i.e., phenobarbital,
phenytoin), abnormal cortisol metabolism, or the inability to follow directions (11).
On the other hand, false-negative test results may occur in chronic renal failure and
hypothyroidism.
However, we doubt that these conditions could explain the great discrepancy
between sensitivities of LDDST and HDDST in our series. Patients with pituitary
corticotroph macroadenomas have been reported to have a lower suppressibility of
urinary steroids in HDDST (14, 15). In our patients we found no statistical difference
regarding the tumor size or other basal hormonal evaluation in patients with CD who
did not suppress at HDDST. Although patients with CD have significantly lower serum
cortisol levels either after LDDST or after HDDST and a greater percent of cortisol
suppression as compared to patients with other CS etiologies, there is a remarkable
overlap between the 2 categories (Figs. 3, 4). These results confirm that HDDST alone
is not able to differentiate all the patients with CD.
Multiple tests should be undertaken in order to establish the etiological form of
CS, as recomended also by a recent international consensus (11) Taking into account
all these test limitations, the question is whether the standard tests are sufficiently
accurate to detect the mild degree of hypercortisolism that have been reported in 5 –
20% of the incidentally discovered adrenal tumors (6, 16). Since these patients may
exhibit an adverse cardiovascular and metabolic risk compared to controls (17) or may
progress to overt CS (estimated cumulative risk of 12.5% after one year) (18), they
should be identified and enrolled in a careful long-term follow-up.
We evaluated 22 patients with incidentally discovered adrenal tumors and 23
patients with adrenal tumors and some with clinical suspicion of CS who did not fulfill
all the biochemical criteria for Cushing’s syndrome. Using the standard tests, we
showed that up to 60% of these patients had at least one abnormal hormonal value at
baseline, and 15 to 40% do not suppress adequately at oDST or LDDST. These patients
showed a significant degree of hypercortisolism compared to controls, although basal
and suppressed hormonal levels are much lower as compared to patients with CS.
The definition of this subclinical Cushing’s syndrome is currently controversial.
Several authors recommend that at least 2 tests that show an abnormality of the HPA
axis, in the absence of overt signs and symptoms of CS, are needed for the diagnosis.
We considered that an abnormal oDST or LDDST is compulsory for defining SCS
taking into account the high false positive rate of basal evaluations in our controls. By
this definition, only 6 patients (13%) of this group were diagnosed with SCS (4 of them

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 Monica Gheorghiu et al.

having incidentally discovered adrenal tumors and no suggestive clinical signs). It is

not yet established whether patients with SCS do actually benefit from adrenalectomy
(19). Further studies are needed in order to establish the optimal management in these
patients.

CONCLUSION

Our results confirm that none of the classical tests used to diagnose Cushing’s
syndrome is perfect. A thorough evaluation including multiple tests should be
undertaken for the positive or the differential diagnosis of Cushing’s syndrome,
recommended also by a recent international consensus. Despite their imperfection,
the standard tests succeed to detect even mild degrees of hypercortisolism in
incidentally discovered adrenal tumors. Long-term prospective longitudinal studies
are needed in order to establish the optimal test cut-offs that would identify those
patients with adrenal “incidentalomas” in whom surgery is warranted.

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