Pituitary (2013) 16:139–145 DOI 10.1007/s11102-012-0387-7
Reliability of the diagnostic tests for Cushing’s syndrome performed in a tertiary referral center Mutlu Gu¨nes • Ozlem Celik • Pinar Kadioglu
Published online: 1 April 2012 Ó Springer Science+Business Media, LLC 2012
Abstract The study aimed to retrospectively evaluate the reliability of the diagnostic and location tests in Cushing’s Syndrome (CS). Eighty-seven patients diagnosed with CS between 1995 and 2007 by Endocrinology Metabolism Department of Cerrahpasa Medical School were included in the study. The control group consisted of 91 patients who presented to the outpatient clinic because of obesity. The diagnostic tests were as follows: 1 mg dexamethasone suppression test (DST), 24-h urinary free cortisol (UFC), midnight cortisol level (MCL), ACTH level and overnight 8 mg DST. The sensitivity and specificity of UFC were 81 and 66 % respectively for the cut-off point of 50 lg/day, whereas they were 64 and 76 % respectively for the cut-off point of 100 lg/day. For the cut-off value of 1.8/lg/dL for MCL and 1 mDST, the sensitivity rates were 100 and 98 %, while the specificity rates were 88 and 33 %, respectively. Among the location tests, the sensitivity and specificity of ACTH under 10 pg/mL for adrenal CS were 92 and 94 % respectively. The sensitivity and specificity of ACTH higher than 30 pg/mL for ACTH-dependent CS were 69 and 100 % respectively. The sensitivity rates of 8 mg DST for 50 and 60 % suppressions were 83 and 79 % respectively, whereas the specificity rates were 75 and 88 % respectively. 1 mg DST (cut-off \1.8 lg/dL) and UFC (50 lg/24 h) are appropriate tests for screening CS.
M. Gu¨nes O. Celik P. Kadioglu Division of Endocrinology and Metabolism, Department of Internal Medicine, Cerrahpasa Medical School, University of Istanbul, Istanbul, Turkey P. Kadioglu (&) Cerrahpasa Tip Faku¨ltesi, Ic Hastalıkları Anabilim Dali, Endokrinoloji-Metabolizma ve Diyabet Bilim Dali, 34303 Cerrahpasa, Istanbul, Turkey e-mail:
[email protected]
Overnight 8 mg DST with 60 % suppression for Cushing’s Disease (CD) and ACTH levels\10 pg/mL for adrenal CS, ACTH levels [30 pg/mL for ACTH dependency were identified as the best tests for the differential diagnosis of the subtypes. Keywords Cushing’s disease Diagnostic tests Midnight cortisol Urinary free cortisol ACTH
Introduction Cushing’s syndrome (CS) develops due to endogenous excess cortisol production or more commonly, as a result of long-term glucocorticoid therapy [1]. The annual incidence of endogenous CS among general population is 0.7–2.4 per million [1, 2]. Insulin resistance, central obesity, dyslipidemia, hypertension, impaired glucose tolerance, diabetes mellitus, atherosclerosis and early cardiovascular disease are associated with CS. It has been shown that standard mortality rate was increased by 3.8–5 folds in the patients whose moderate hypercortisolemia persisted despite therapy, as compared to the general population [3–5]. Symptoms and signs which were characteristic for CS such as easy bruising, plethora, strias, weight gain, edema, dorsocervical fat pad, proximal myopathy and depression are also seen in the pseudo-Cushing states [3]. The incidence of CS is increased since the screening tests are performed not only in those with the symptoms of CS but also in those with obesity, metabolic syndrome, polycystic ovary syndrome and/or osteoporosis [6–8]. The incidence of CS in uncontrolled diabetic patients has increased up to 2–3 % [9]. Considering that they overlap in terms of clinical characteristics and they concern a large patient population, the test that would be used for the differentiation of pseudo-
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Cushing and CS should be cheap, reliable, and easy to apply. Which is the best screening test and what the cut-off point should be have remained controversial. Thus, a guideline aiming the diagnosis of CS was published in 2008. However, it has been highlighted that studies are needed to standardize the measurements for the diagnosis of CS and to form an information pool about diagnostic tests by means of screening the consecutive patients [3]. In our study, we aimed to retrospectively evaluate the specificity, sensitivity, and reliability of the diagnostic tests used for the patients diagnosed with CS in a single tertiary referral center.
Subjects and methods Eighty-seven patients, who presented to Endocrinology, Metabolism Out-patient Clinic of Cerrahpasa Medical School between 1995 and 2007 were included in the study. All the patients were operated, and diagnoses were verified as CS based on the pathology studies. The control group comprised 91 obese patients that presented to the outpatient clinic between the same years. The study was approved by the Ethical Committee of Cerrahpasa Medical School. The reliability of the diagnostic tests was retrospectively evaluated. The diagnostic and location tests were evaluated as follows: 1 mg dexamethasone suppression test (DST), 24-h urinary free cortisol (UFC), midnight cortisol level (MCL), ACTH level, and overnight 8 mg DST. The cut-off value was considered 1.8 lg/dL for MCL and for 1 mg DST. The blood samples were obtained 48 h after the hospitalization of the patients, at midnight within 5–10 min, and from the catheter inserted in the evening for the measurement of MCL. In the 1 mg overnight DST, 1 mg dexamethasone was given at 2300 hours and the concentration of cortisol in serum was measured the next day at about 0800 hours. The cut-off value was considered C100 lg/24 h for UFC. High dose (8 mg) dexamethasone was given at 2300 hours and serum cortisol levels were measured before and after dexamethasone administration. Suppression of more than 50 % as compared to the baseline in the 8 mg DST was considered significant in favor of pituitary origin, whereas suppression of less than 50 % was considered significant in favor of ectopic and adrenal origin. Plasma cortisol levels were analyzed via solid phase competitive chemiluminescent enzyme immunoassay (normal range = 5–25 lg/dL). ACTH levels were analyzed via Radioimmunoassay (RIA) method (normal range = 10–46 pg/mL) until 2002 and have been analyzed via solid phase two-site sequential chemiluminescent immunometric assay (normal range = 5–46 pg/mL) since 2002. Urinary
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free cortisol levels were measured by RIA method (normal range = 9–156 lg/day). SSPS 13 program was used for the statistical analyses of the data. When the distribution was not normal, nonparametric tests were used. Kruskal–Wallis and Mann– Whitney U tests were used to compare the data. p \ 0.05 was considered statistically significant. Sensitivity, specificity, positive and negative predictive values, diagnostic accuracy were calculated according to standard statistical methods. Sensitivity and specificity were calculated using [true positive/(true positive ? false negative)] and [true negative/(true negative ? false positive)] formulas respectively. In addition, positive predictive and negative predictive values were calculated using [true positive/(true positive ? false positive)] and [true negative/(true negative ? false negative)] formulas respectively. Diagnostic accuracy was calculated using the formula as [(true positive ? true negative)/(true positive ? false positive ? true negative ? false negative)]. The area under each receiver operating characteristics (ROC) was calculated to define the best cutoff values.
Results Eighty patients diagnosed with CS (68 female, 12 male, median age = 38.0 years [IQR (Interquartile range): 29.0–48.0]) were included in the study. The control group consisted of 98 obese patients (83 female, 15 male, median age = 45.0 years [IQR: 35.00–53.0]) that presented to the outpatient clinic between the same years. When the obese patients were evaluated, seven patients were diagnosed with CS. Thus, totally 87 patients (74 female, 13 male, median age = 40.0 years [IQR: 29.8–48.0]) were included in the CS group. CS in 2 of these patients (n = 7) with subclinical CS was of adrenal origin and in 5, of pituitary origin. The comparisons of the patients in CS group (Cushing’s disease, adrenal CS and ectopic CS) and the obese patients showed that in the CS group, UFC and the cortisol levels after 1 mg DST were higher. However, for ACTH levels, no differences were determined between the groups (p \ 0.001, p \ 0.001, and p = 0.14 respectively). Distribution of the patients whose diagnosis of CS was pathologically verified was shown in Table 1. Data on UFC were obtained in 54 of 66 patients with CD and in 10 of 15 patients with adrenal CS. Statistically significant difference was observed between the three groups in terms of UFC levels (p = 0.004). Inter-group comparisons revealed that the UFC levels of the patients with ectopic CS (median = 1692 lg/day [IQR = 1000– 1830]) were higher than those in both the patients with CD (median = 127.5 lg/day [IQR = 54.5–333.5]) and the
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141 DST
Table 1 Etiologic distrubition of the patients with Cushing’s syndrome (CS) Subtype
n
%
ACTH-dependent CS
72
82.7
Cushing’s disease
65
74.71
6
6.89
1,0
Ectopic CS Small cell lung cancer
2
Bronchial carcinoid tumor
2
Pheochromocytoma
1
Unknown localisation Hypotalamic CS (ectopic hypotalamic ACTH-secreting adenoma) ACTH-independent CS
1 1
17.2
11
12.66
Adrenal hyperplasia
2
2.29
Adrenal carcinoma
2
2.29
0,6
0,4
0,2
1.16
15
Adrenal adenoma
Sensitivity
0,8
0,0 0,0
0,2
0,4
0,8
1,0
UFC
1,0
Sensitivity
0,8
0,6
0,4
0,2
0,0 0,0
0,2
0,4
0,6
0,8
1,0
1 -Specificity MCL 1,0
0,8
Sensitivity
patients with adrenal CS (median = 154.7 lg/day [IQR = 94.0–527.6]) (p = 0.001 and p = 0.011 respectively). Contrarily, no significant differences were found between the UFC levels of the patients with CD and adrenal CS (p = 0.38). UFC levels were below 100 lg/day in 39 % of the patients with CD and in 20 % of the patients with adrenal CS. Although the midnight cortisol level (MCL) was significantly high in the patients with ectopic CS (median = 41.0 lg/dL [IQR = 31.9–50.0]) as compared to the other groups, no statistically significant differences were observed among the three groups (p = 0.06). The ROC curves of 1 mg DST, UFC, and MCL in the diagnosis of CS were shown in Fig. 1. For 1 mg DST, the cut-off value was 2.2 lg/dL [area under curve (AUC): 0.97; (95 % CI: 0.94–1)]; the cut-off value of UFC was 158 lg/day [AUC: 0.82; (95 % CI: 0.75–0.89)], the cut-off value of MCL was 1.66 lg/dL [(AUC: 0.86; (95 % CI: 0.62–1)]. The comparison between the areas under the ROC curves did not demonstrate significant differences between either 1 mg DST, UFC, or MCL, respectively. Statistically significant difference was observed among the three groups in terms of suppression levels for 8 mg DST (p \ 0.001). Inter-group comparisons revealed statistically significant difference between the suppression level of the CD group (median = 83.6 % [IQR = 66.8– 90.3]) and the suppression level of the adrenal CS group (median = 17.0 % [IQR = 6.5–28.4]), (p \ 0.001). However, no statistically significant difference was found between the groups with CD and ectopic CS in terms of suppression levels (median = 52.5 % [IQR = -4.1–68.7]) (p = 0.17). There was no significant difference between the suppression levels of the patients with ectopic CS and with adrenal CS (p = 0.60).
0,6
1 -Specificity
0,6
0,4
0,2
0,0 0,0
0,2
0,4
0,6
0,8
1,0
1 -Specificity
Fig. 1 ROC curves of 1 mg dexamethasone suppression test (DST), 24-h urinary free cortisol (UFC), midnight cortisol level (MCL)
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Table 2 Sensitivity, specificity, positive predictive, negative predictive, diagnostic accuracy values of the screening tests according to the cutoff values in patients with Cushing’s syndrome Tests and cut-off value
Sensitivity %
Specificity %
Positive predictive %
Negative predictive %
Diagnostic accuracy %
UFC [50 lg/24 h
81
66
66
81
73
[100 lg/24 h [195 lg/24 h
64 40
76 100
68 100
72 67
71 73
100
88
96
100
97
98
33
96
50
95
1 mg DST ([1.8 lg/dL) MCL ([1.8 lg/dL)
UFC urinary free cortisol, DST dexamethasone suppression test, MCL midnight cortisol level
Table 3 Sensitivity, specificity, positive predictive, negative predictive, diagnostic accuracy values of the localisation tests according to the cut-off values in patients with Cushing’s syndrome Tests and cut-off value
Sensitivity %
Specificity %
Positive predictive %
Negative predictive %
Diagnostic accuracy %
23
100
100
87
88
ACTH \7 pg/mLa a
92
94
75
95
94
[30 pg/mLb
69
100
100
38
74
[%50
83
75
92
57
81
[%60
79
88
95
58
81
[%79
60
100
100
45
70
\10 pg/mL 8 mg DST
a
ACTH independent CS
b
ACTH dependent CS
Significant difference was observed among the three groups in terms of ACTH levels (p \ 0.001). Inter-group comparisons showed that ACTH levels were significantly higher in the patient group with ectopic CS (median = 157.5 pg/mL [IQR = 63.8–185.5]) than in the patients with CD (median = 42.0 pg/mL [IQR = 24.5– 73.5]) (p = 0.005). The ACTH levels were significantly lower in the patients with adrenal CS (median = 10.0 pg/ mL [IQR = 7.0–10.0]) than the levels of the other groups (p \ 0.001 and p \ 0.001 respectively). The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the screening tests are shown in Table 2. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the tests performed for location are shown in Table 3.
Discussion The study showed that when the cut-off point was considered 50 lg/24 h for the measurement of the UFC and 1.8 lg/dL for the 1 mg DST, both tests were appropriate
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for screening CS. In terms of location tests, ACTH level lower than 10 pg/mL is a good indicator for adrenal CS, and ACTH level over 30 pg/mL is a good indicator in differentiating ACTH-dependent CS. Whereas differentiation could be made between pituitary and adrenal locations with 8 mg DST, differentiation of pituitary and ectopic or ectopic and adrenal locations was not possible. UFC measures the cortisol that is not bound to cortisol binding globulin, which is filtered by the kidney. When the assay upper limit of normal is used as a criterion, the overall evidence supports the diagnostic accuracy of UFC in adults suspected of having CS [3]. In the study conducted by Putignano et al. [10] on 41 patients with CS and 33 patients with pseudo-Cushing syndrome, as well as 199 simple obese patients and 27 healthy subjects, midnight salivary cortisol, UFC and MCL were used as the screening tests. Whereas they identified significant difference between the obese patients and the patients with CS in terms of UFC levels, no significant difference was determined between the UFC levels of obese patients and of healthy subjects. When the cut-off point for UFC was considered 80 lg/24 h in their study, the sensitivity and the specificity were 97.6 and 85.3 % respectively. Kidambi
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et al. [11] found that UFC levels were normal in 7 of 11 patients with CS without remarkable clinical symptoms but with mild or moderate increase in cortisol levels retrospectively, who had been referred because of weight gain, uncontrolled diabetes and incidentaloma. They suggested that the patients with CS who present only with mild or moderate increase in UFC levels might, therefore, be missed out. In patients with mild CS, who present the vast majority of cases in modern series, it is not uncommon to detect normal UFC measurements. Reimondo et al. [12] reported that measurement of UFC is significantly inferior to other screening tests. Moreover, they have agreed that recent studies have demonstrated a less than ideal sensitivity ranging from 45 to 71 % when 100 % specificity is considered. In Turkey, patients usually find it difficult to collect urine during 24 h for 3 times so it is not practical to assess hypercortisolemia with multiple urine specimens. Therefore we aimed to find a cut-off value for cortisol level in a single 24 h urine specimen in order to detect hypercortisolemia (such as [50 lg/24 h, [100 lg/24 h, [195 lg/24 h). When the cut-off point was considered 50 lg/24 h, it was determined that the UFC levels of 22 % of the patients in the CD were under this value, whereas the UFC levels of 100 % of the patients with adrenal CS were over 50 lg/24 h in our study. We also determined that when the cut-off point was considered 100 lg/24 h, UFC levels of 39 % of the patients in the CD and 20 % of the patients with adrenal CS were under this value. Both the sensitivity and the specificity of UFC found in our study were quite low as compared to the studies performed with other RIA methods and to the high performance liquid chromatography (HPLC). The sensitivity and the specificity of HPLC method are higher than those of RIA method [3, 13]. Low sensitivity and specificity of UFC levels in this study may be attributed to the use of RIA method, measurement of UFC with unextracted urine and a single urine collection. It may be expected that when the percentage of the false negativity of UFC levels was taken into consideration, using a cut-off point of 50 lg/24 h instead of 100 lg/24 h would enhance the diagnostic value when evaluating the patients. In Turkey, patients usually find it difficult to collect urine during 24 h for 3 times so it is not practical to assess hypercortisolemia with multiple urine specimens. One mg DST is a test indicating that the normal physiology has been impaired and the glucocorticoid feedback has disappeared. It is a cheap test that can be easily applied in the outpatient clinics [3]. Cronin et al. [14] reported the sensitivity of 1 mg DST as 87.5 %. It has been shown that the sensitivity and the specificity were 95 and 80 % respectively by reducing the cut-off point below 1.8 lg/dL [3, 15]. In our study, it was observed that the plasma cortisol levels after 1 mg DST were over 1.8 lg/dL in all of
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the patients. One of the reasons for high sensitivity in this study might be the patients with suspected CS that had been referred to our center. If population-based patients had been used in this present study, more accurate information could have been obtained about the exact sensitivity and specificity of this test. Measuring midnight salivary or blood cortisol levels is based upon disappearance of diurnal rhythm in the CS. Although it is easier to collect saliva and does not require hospital stay, it is difficult to comment because of undefined standards [3–16]. In Turkey, salivary cortisol is not routinely used except for clinical studies. Therefore, MCL was measured in this study. In a study, it was shown that the sensitivity of MCL was 100 % when the cut-off point was considered 1.8 lg/dL [17]. Current studies have reported that the specificity is decreased down to 20.2 % with the same criteria but is increased up to 87 % when the cut-off point is considered 7.5 lg/dL [18]. Measurement of MCL requires hospital stay and the measurements need to be performed after 48 h in order to prevent false positivity, which are the disadvantages of the test. Although this study showed that diurnal test is appropriate as a screening test for CS, but considering its cost, it would be more appropriate to use it as a supporting test for Turkey rather than as the first choice. The most common tests used for location include ACTH, single dose 8 mg DST, CRH stimulation, and inferior petrosal sinus sampling (IPSS) [19]. CRH ampoule is used for only IPSS examination because of its high cost in Turkey. IPSS was not included in this study. Serum ACTH level lower than 5 pg/mL most likely indicates adrenal CS [20]. However, ACTH levels are usually higher in the ectopic CS, but only in a minority of patients, serum ACTH is able to differentiate between pituitary and ectopic origin. In some cases with a definitive diagnosis of pituitary dependent CS it is possible to detect ACTH levels in the low—normal range and, conversely in some patients with adrenal CS, ACTH levels cannot be fully suppressed. However the variability of ACTH assay must be kept in mind. Pecori Giraldi et al. [21] collected 25 fresh-frozen plasma samples from patients with either high, low, or normal ACTH concentrations which were measured using 7 different assays by 35 different laboratories. High and normal ACTH values were interpreted correctly in 98.4 and 89.6 % of measurements respectively, whereas only 59.5 % of measurements in patients in whom ACTH was expected to be suppressed were classified correctly. An occult ectopic source of ACTH may mimic CD because such tumors may express glucocorticoid, CRH and/or vasopressin (AVP) receptors [20, 22]. In a recent study, plasma ACTH levels were normal in 32 % of 79 patients with ectopic CS [23]. Invitti et al. [24] reported that 55 % of patients with CD and 17 % of the patients with ectopic
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CS exhibited normal morning plasma ACTH levels, with values as low as 9 pg/mL. It was found that ACTH levels were detectable in 58 % and within normal range in 28 % of patients with ACTH independent CS in their study. Vilar et al. [25] evaluated laboratory features of 74 patients with CS. ACTH levels were reduced (\10 pg/mL) in 100 % of the patients with adrenal CS, normal in 37 %, or elevated in 63 % of the patients with CD and invariably high in ectopic CS. In addition, they found a great overlap in the hormone values in patients with CD or ectopic CS. In our study, the cut-off point for ACTH was considered below 10 pg/mL; accordingly, the sensitivity and the specificity for adrenal CS were 92 and 94 % respectively. When the ACTH level is over 30 pg/mL, it indicates ACTHdependent origin with 100 % specificity. However, no cutoff point could be obtained to be used in practice in the differentiation of pituitary and ectopic locations. The sensitivity of the 8 mg overnight DST in the differential diagnosis between pituitary and ectopic CS has been reported to be 59–92 %, but with poor specificity 29–60 % [26]. In the study conducted by Dicheck et al. [27] the specificity and the sensitivity of the test were 57 and 88 % respectively when the cut-off point for suppression was considered 50 % after 8 mg DST. The specificity was 100 % with the same method when the cut-off point for suppression was considered 80 %. Erem et al. [28] retrospectively evaluated 55 patients with CS. Sensitivity and specificity of 8 mg DST (when serum cortisol suppression [50 %) in distinguishing CD were found to be 82 and 100 %, respectively. Vilar et al. [25] showed that serum cortisol suppression [50 % after 8 mg DST was observed in 79.5 % of the patients with CD and in 28. 6% of the patients with ectopic CS. Recently, Suda et al. [29] have reported that a high dose (8 mg) dexamethasone suppressed the morning cortisol secretion in 82 % of all the patients with CD, while this occurred in only 20 % of the patients with ectopic CS. Isidori et al. [30] suggested that the differentiation of ectopic CS could be made more accurately with 60 % suppression, and they determined 80 % sensitivity and 90 % specificity for the test. In our study, the sensitivity and the specificity of the test in differentiating CD were 83 and 75 % respectively when the cut-off point for suppression was considered 50 % and over. When the suppression was considered 60 %, the sensitivity and the specificity of the test were 79 and 88 % respectively. Although the results of this study were similar to the results of the above mentioned studies, estimation CD with 50 % suppression level was found to be more sensitive than that found in the study conducted by Dicheck et al. In conclusion, 1 mg DST (cut-off \1.8 lg/dL) and UFC (50 lg/24 h) are appropriate tests for screening CS in our country. Overnight 8 mg DST with 60 %
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suppression for CD and ACTH levels \10 pg/mL for adrenal CS, and ACTH levels [30 pg/mL for ACTH dependency were identified as the most reliable tests for the differential diagnosis of the subtypes. However, there is no consensus on the specificity, sensitivity, or cut-off points of the diagnostic and location tests used in the CS. Therefore, the optimal test and cut-off value is still a matter of debate. Although this study was conducted at a university hospital, the data from the centers focusing on this issue may provide information about screening and location tests for CS. Acknowledgments This research did not receive any specific grant from any funding agencies in the public, commercial, or non-profit sector. Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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