The clinical manifestations of Cushing s syndrome (CS)

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1 ORIGINAL ARTICLE Endocrine Care Late-night Salivary Cortisol Has a Better Performance Than Urinary Free Cortisol in the Diagnosis of Cushing s Syndrome Paula C. L. Elias, Edson Z. Martinez, Bruno F. C. Barone, Livia M. Mermejo, Margaret Castro, and Ayrton C. Moreira Division of Endocrinology Department of Medicine (P.C.L.E., B.F.C.B., L.M.M., M.C., A.C.M.) and Division of Statistics Department of Social Medicine (E.Z.M.), Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Sao Paulo , Brazil Context: The comparison of variability, reproducibility, and diagnostic performance of late-night salivary cortisol (LNSF) and urinary free cortisol (UFC) using concurrent and consecutive samples in Cushing s syndrome (CS) is lacking. Objectives, Patients, and Methods: In a prospective study, we evaluated 3 simultaneous and consecutive samples of LNSF by RIA and UFC by liquid chromatography associated with tandem mass spectrometry in Cushing s disease (CD) patients (n 43), adrenal CS patients (n 9), and obese subjects (n 18) to compare their diagnostic performances. In CS patients, we also performed a modified CS severity index. Results: There was no difference in the coefficient of variation (percentage) between LNSF and UFC among the 3 samples obtained for each patient with Cushing s disease (35 26 vs 31 24), adrenal CS (28 14 vs 22 14), and obesity (39 37 vs 48 20). LNSF confirmed the diagnosis of hypercortisolism even in the presence of normal UFC in 17.3% of CS, whereas the inverse situation was not observed for UFC. The area under the receiver-operating characteristic curves for LNSF was (95% credible interval [CI] ) and for UFC was (95% CI ). The ratio between areas under the curve was (95% CI ), indicating better performance of LNSF than UFC in diagnosing CS. There was no association between the CS severity index and the degree of biochemical hypercortisolism. Conclusion: Our data show that despite similar variability between both methods, LNSF has a superior diagnostic performance than UFC and should be used as the primary biochemical diagnostic test for CS diagnosis. (J Clin Endocrinol Metab 99: , 2014) The clinical manifestations of Cushing s syndrome (CS) can be variable and cyclical and has been suspected before the classical clinical picture. Moreover, diagnostic difficulties can arise due to the relative rarity of the disease (1). The diagnostic strategies have changed throughout the last decades. In the 1970s, the diagnosis was established by daily urinary free cortisol (UFC) and late evening plasma cortisol levels with low-dose (2 mg/d for 48 hours) dexamethasone suppression test (DST) (2). Two decades later, ISSN Print X ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received November 27, Accepted February 25, First Published Online March 14, 2014 biochemical confirmation of CS was best achieved by still using low-dose DST, UFC, and sleeping midnight plasma cortisol measurements (3). At that time, evaluation of latenight salivary cortisol (LNSF) levels was demonstrated to be as sensitive as the conventional gold standard tests (4, 5). The last Endocrine Society clinical practice guideline for the diagnosis of CS recommends one of the following tests: at least 2 measurements of UFC or LNSF, 1-mg DST, or 2-mg low-dose DST. However, patients with inciden- Abbreviations: AUC, area under the curve; BMI, body mass index; BP, blood pressure; CD, Cushing s disease; CI, credible interval; CS, Cushing s syndrome; CSI, CS severity index; CV, coefficient of variation; DST, dexamethasone suppression test; LC-MS/MS, liquid chromatography associated with tandem mass spectrometry; LNSF, late-night salivary cortisol; LR, likelihood ratio; PPNAD, primary pigmented nodular adrenocortical disease; ROC, receiveroperating characteristic; UFC, urinary free cortisol. doi: /jc J Clin Endocrinol Metab, June 2014, 99(6): jcem.endojournals.org 2045

2 2046 Elias et al Salivary Cortisol vs UFC in Hypercortisolism J Clin Endocrinol Metab, June 2014, 99(6): talomas usually do not have consistently elevated UFC and LNSF. In such conditions, the 1-mg DST has been preferable to UFC and LNSF (6). UFC provides an integrated assessment of cortisol secretion over a 24-hour period; however, urine measurements may be inaccurate because of improper collection or renal insufficiency. The introduction of salivary cortisol as a first line test for CS (4, 5, 7, 8), irrespective of whether patients are investigated on an inpatient or an outpatient basis (8, 9) has allowed the study of hypothalamic-pituitary-adrenal axis response patterns without the need of repeated blood or urinary samples. Two recent meta-analyses support the use of LNSF for screening and diagnosis of CS (10, 11). Studies using receiver-operating characteristic (ROC) curves have addressed the comparison of LNSF with traditional tests in a large population of CS and obese subjects. In terms of sensitivity, specificity, and diagnostic accuracy or predictive values, no statistically significant difference among midnight plasma cortisol, LNSF, and UFC has been observed (12). LNSF seems to help in diagnosing patients with a high index of clinical suspicion presenting mild endogenous CS with either normal or only slightly elevated UFC (13). Patients with CS with at least one normal UFC or LNSF have been observed in other series (14). The limitation of these studies was the long time interval of serial measurement of either UFC or LNSF and/or nonsimultaneous and nonconsecutive collection of urine and saliva. Thus, intermittent hypercortisolism could lead to normal values of these tests. One study evaluated the dayto-day variability of LNSF by 3 repeated measurements in healthy controls, but not in CS patients (15). More recently, the variability of baseline UFC calculated from four 24-hour urine samples collected over 2 weeks was evaluated in patients with persistent/recurrent or de novo Cushing s disease. Of note, patients with mild degrees of cortisol excess were not included in the study; in addition, UFC was analyzed at 3 different laboratories (16). The cyclical characteristics of CS observed in some patients and the pulsatile and circadian nature of cortisol secretion may contribute to the variability of UFC and LNSF. The comparison of variability, reproducibility, and diagnostic performance of LNSF and UFC using simultaneous and consecutive samples in CS is lacking. In the present prospective study, we evaluated 3 concurrent and consecutive samples of LNSF and UFC in a large group of patients with CS to compare their diagnostic performances. Subjects and Methods Subjects This study was approved by the Ethical Committee of the Ribeirao Preto Medical School, University of Sao Paulo, and according to the requirements of the Declaration of Helsinki, written informed consent was obtained from all participants. Seventy-five patients with clinical features suspicious for hypercortisolism were consecutively referred between 2005 and 2012 to our endocrine center in the Division of Endocrinology, University Hospital of the Ribeirao Preto Medical School, University of Sao Paulo, one of the referral services for CS in Brazil. We adopted as cutoff values an 11:00 PM salivary cortisol value lower than 350 ng/dl (or 9.8 nmol/l) and salivary cortisol levels after the overnight 1-mg DST lower than 150 ng/dl (or 4.2 nmol/l) to exclude the biochemical diagnosis of endogenous hypercortisolism, as previously described (4, 17). Based on the adopted parameters for biochemical diagnosis of hypercortisolism, CS was diagnosed in 57 patients, whereas 18 patients (17 females and 1 male; age [range 18 71] years) were classified as Cushingoid obesity. All obese patients, used as controls, also presented essential hypertension, diabetes, and/or hirsutism and suppressed salivary cortisol after the 1-mg DST ( ng/dl) (4). Furthermore, this group had no progression of hypercortisolism clinical signs during a mean follow-up of 22.5 months. To evaluate the etiology of CS, standard tests of pituitary-adrenal function, including plasma ACTH levels, low- and high-dose DST, CRH test, bilateral inferior petrosal sinus sampling (BIPSS), and image studies were performed. Diagnosis of ACTH/pituitary-dependent disease was confirmed by the presence of pituitary tumor larger than 5 mm on magnetic resonance imaging or by bilateral inferior petrosal sinus sampling when there is no clear evidence of pituitary tumor on magnetic resonance imaging. The diagnosis of Cushing s disease (CD) was established in 43 patients (38 females and 5 males; age [range 9 64] years). Nine patients had ACTH-independent CS (9 females; age [range 13 50] years). Five patients who presented CS due to ectopic ACTH secretion were excluded from the study because of disease severity and the small number of samples to be analyzed. All CD patients underwent transsphenoidal surgery by the same surgeon, and adenoma was identified in all 43 patients. Among 43 patients, 21 CD patients presented postoperative morning serum cortisol 2 g/dl and 6 had 5 g/dl. All these patients (62.8%) had adrenal insufficiency and were considered on surgical remission. His-

3 doi: /jc jcem.endojournals.org 2047 topathology confirmed the presence of tumor in 40 patients. An immunohistochemistry study was performed in 38 patients, and ACTH-positive staining was found in 37 of them. In the remaining 3 hypercortisolemic patients without histopathology confirmation, 1 presented clinical and biochemical remission after transsphenoidal surgery, 1 developed Nelson s syndrome after adrenalectomy, and 1 died by postoperative complications. Adrenal CS was confirmed by a surgeon during a laparotomy procedure and by histopathology. In all 3 groups, we evaluated the body mass index (BMI), systolic and diastolic blood pressure (BP), and the presence of impaired glucose tolerance or diabetes. In CS patients we performed a modified CS severity index (CSI), as previously reported (18). CSI was summarized in an overall assessment of 8 clinical features: fat distribution, skin lesions, muscle weakness, mood disorder, hypertension, diabetes, hypokalemia, and sex-related disturbances, scored on a scale of 0, 1, or 2 points (absent, mild, or severe forms, respectively) with a maximum of 16 points. Methods Inpatient salivary cortisol samples were collected by Salivette sampling devices (Sarstedt Nuembrecht, Germany) at 9:00 AM and 11:00 PM (LNSF) on 3 consecutive days. On the same days, 24-hour urine collections for UFC measurements were performed for each subject. Renal function was normal in all patients. The salivary and urinary samples were stored at 20 C until analysis. Salivary cortisol measurements were performed by a previously described RIA method on 25- L samples of saliva without previous extraction or chromatography. The antiserum specificity was 100% for cortisol and 15% for cortisone. The mean intra-assay coefficient of variation (CV) was 5.5% (19). All samples obtained from each subject were analyzed in duplicate in the same assay. UFC was measured by liquid chromatography associated with tandem mass spectrometry (LC-MS/MS) (normal range, 3 43 g/24 hours). The mean intra-assay CV was 4.4% (20). Statistical analysis Data are expressed as mean and SD. A Kruskal-Wallis test was performed for multiple comparisons among different groups and the Dunn s multiple-comparison test was used as a post hoc test. The Wilcoxon-Mann-Whitney test was used when appropriate. The performance of CSI in reflecting severity of the disease was correlated with the values of LNSF and UFC by Spearman s method. Fisher s exact test was used for categorical data such as the CSI and the degree of biochemical hypercortisolism evaluated by LNSF and UFC. Statistical analyses were carried out using SAS for Windows software (SAS Institute). The level of significance was set at.05. ROC curves for LNSF and UFC were obtained to discriminate between states of hypercortisolism and obesity. In addition, the comparison of the area under ROC curves was performed by a parametric approach using Bayesian estimation with noninformative previous distributions (21), considering repeated measures. This statistical model is very similar to the one presented in Box and Tiao (Section 7.2) (22) in assuming that the logarithm of LNSF and UFC follows a normal distribution and treating the multiple observations per individual as blocks. This analysis was performed using the software OpenBugs (version 3.2.1; Medical Research Council UK, 2011) based on Markov chain Monte Carlo (MCMC) estimation. The area under the curve (AUC) was estimated by integration, and inferences were obtained using the Markov chain Monte Carlo method. Summaries for each parameter of interest were reported as the mean of the respective simulated chains and its 95% credible intervals (CI). CIs are Bayesian analogs of classical confidence intervals. Results The clinical characteristics of the patients are presented in Table 1. There were no differences in age and gender among CS and obese patients. The BMIs were not different between CD and adrenal CS or obese patients. However, adrenal CS patients had lower BMI compared with obese patients (P.03). Regarding systolic/diastolic BP, no differences were observed among the groups. Most CD (67.4%), adrenal CS (55.6%), and obese (72.2%) patients had high BP. Diabetes mellitus was observed in 67.4% of CD, 33.3% of adrenal CS, and 27.8% of obese patients. The mean global CSI score was not different between CD ( , range 3 13) and adrenal CS ( , range 2 10). In addition, there was no evidence of correlation between individual CSIs with the respective mean of LNSF and UFC, either for CD (P.32 and P.48, respectively) or adrenal CS (P.36 and P.33, respectively). In addition, we did not observe an association between the CSI ( 8 or 8) and the degree of biochemical hypercortisolism considering LNSF and UFC below or above the respective mean values (LNSF, P.55; UFC, P.73). LNSF levels (ng/dl) were similar between CD ( ) and adrenal CS ( ) patients (P.6); Table 1. Main Clinical Characteristics of the Patients CD Adrenal CS Obese n (F/M) 43 (38/5) 9 (9/0) 18 (17/1) Age, y BMI, kg/m 2 (mean a SD) Systolic BP, mm Hg Diastolic BP, mm Hg Hypertension, % Glucose intolerance, % Diabetes mellitus, % Abbreviations: F, female; M, male. a P.05 vs adrenal CS.

4 2048 Elias et al Salivary Cortisol vs UFC in Hypercortisolism J Clin Endocrinol Metab, June 2014, 99(6): Figure 3. Correlation between mean individual values of LNSF and UFC in CS patients (n 52; r 0.59, P.0001): CD, n 43 (E); adrenal CS, n 9(Œ). The dotted lines represent the cutoff values of LNSF (350 ng/dl) and UFC (43 g/24h). The quadrants are numbered from I to IV. Figure 1. Individual values of 3 consecutive samples of LNSF and UFC obtained from each patient with CD. The dotted line represents the cutoff values of LNSF (350 ng/dl) and UFC (43 g/24 h). both groups had higher LNSF than obese patients ( ; P.001). UFC levels ( g/24 h) were also similar between CD ( ) and adrenal CS ( ) patients; both groups had higher UFC than obese patients ( ; P.001). Figures 1 and 2 show samples of LNSF and UFC obtained from each patient with CD and adrenal CS, respectively. There was no difference among the 3 LNSF (P.16) and UFC (P.14) samples for each subject with CD. No difference was also observed among the 3 LNSF (P.57) and the UFC (P.11) samples for each subject with adrenal CS as well as for obese patients (P.12). Using the cutoff value of 350 ng/dl for LNSF (17) and 43 g/24 h for UFC (6), LNSF samples diagnosed all but 1 CD patient (2.3%), whereas UFC failed in 8 of 43 (18.6%). Regarding adrenal CS patients, LNSF failed in 1 of 9 (11%) and UFC in 2 of 9 (22%). Figure 2. Individual values of 3 consecutive samples of LNSF and UFC obtained from each patient with different etiologies of adrenal CS: carcinoma, ACTH-independent macronodular adrenal hyperplasia (AIMAH), PPNAD, and adenoma. The dotted lines represent the cutoff values of LNSF (350 ng/dl) and UFC (43 g/24 h). The mean of LNSF intrapatient CV in CD group (calculated using 3 samples) was 35% (95% CI, 27 44) compared with 28% (95% CI, 18 39) in the adrenal CS patients and with 39% (95% CI, 26 57) in obese patients. Regarding the mean of UFC intrapatient CV (calculated using 3 samples), CV in the CD patients was 31% (95% CI, 24 39) compared with 22% (95% CI, 11 33) in the adrenal CS patients and with 48% (95% CI, 36 61) in obese patients. There was no difference in the CV between LNSF and UFC among the 3 samples obtained for each patient with CD (35% 26% vs 31% 24%), adrenal CS (28% 14% vs 22% 14%) and obesity (39% 37% vs 48% 20%). A positive correlation was found between the individual mean of LNSF and the respective UFC mean in CS patients (r 0.59, P.0001; Figure 3) but not in obese patients (data not shown). The axes of a 2-dimensional Cartesian system divide Figure 3 into 4 counterclockwise quadrants based on the cutoff values of LNSF (350 ng/dl) and UFC (43 g/24 h). Quadrant I shows that above the cutoff values, either LNSF or UFC was able to diagnose CS in most patients. Quadrant II shows that LNSF confirmed the diagnosis even in the presence of UFC 43 g/24 h in 9 patients (17.3%): 8 with CD and 1 with primary pigmented nodular adrenocortical disease (PPNAD). Of note, CSI was not different between patients with high LNSF and UFC values (quadrant I, CSI ) and patients with high LNSF and normal UFC values (quadrant II, CSI ). Quadrant III shows that neither LNSF nor UFC was able to diagnose CS in 1 adrenal CS patient. Finally, quadrant IV shows that UFC alone did not help to diagnose CS in any patient. ROC curves were created to establish the optimal threshold values for each test and its diagnostic efficiency (Figure 4). For CS, a cutoff value of 485 ng/dl for LNSF gave the best sensitivity and specificity (94.4% and 94.6%, respectively) with a positive likelihood ratio (LR ) and negative LR (LR ) of 17.5 and 0.06, respectively. For a cutoff value of 45 g/24 h for UFC, sensitivity and specificity were 93.2% and 79.2% with LR and LR

5 doi: /jc jcem.endojournals.org 2049 Figure 4. ROC curves showing the diagnostic performance of LNSF (solid line) and UFC (dotted line) in diagnosis of CS. Cutoff values of 485 ng/dl for LNSF and 45 g/24 h for UFC show sensitivity/specificity of 94.4%/94.6% and 93.2%/79.2%, respectively, to predict CS. of 4.5 and 0.06, respectively. The AUC by the Bayesian approach for LNSF was (95% CI, ) and for UFC was (95% CI, ). The ratio between AUCs was (95% CI, ) indicating a better performance of LNSF than UFC in diagnosing CS. Discussion In the present study, we evaluated 3 simultaneous and consecutive samples of LNSF and UFC in a large group of patients with CS. Our data demonstrated no difference among the 3 absolute values of samples obtained in each patient, either for LNSF or UFC. In addition, there was no difference between LNSF and UFC CV among the 3 samples. Finally, the comparison of ROC curves indicated a better performance of LNSF than UFC in diagnosing CS. Altogether, these data suggest that, in clinical practice, LNSF should replace the well-established, but cumbersome, UFC for diagnosing CS. In the present study, the occurrence of obesity and hypertension were similar among CD, adrenal CS, and obese patients, whereas diabetes was observed more frequently in CD patients. These findings can be ascribed by the low LR of these symptoms/signs in diagnosing CS due to overlap in clinical findings between CS and Cushingoid obesity (1). The pretest probability in diseases with a high prevalence is equal to the prevalence of the disease (23, 24). However, it is not applicable for rare endogenous CS. Recently, applying a Bayesian approach to evaluate the clinical diagnostic probability of CS, the pretest probability showed a linear increase throughout the physician endocrine experience (25). Clinimetrics employ indexes and rating scales to measure clinical phenomena such as severity of the disease, rate of progression, and the magnitude of changes upon treatment. CSI was used to evaluate disease severity in 14 CS patients (18). The mean of the CSI scores observed by us is similar to those observed in that previous study ( ) with no difference in the CSI score between CD ( ) and adrenal CS ( ) patients. In addition, there was no evidence of correlation between severity of the disease evaluated by individual CSI with the respective mean of LNSF and UFC, either for CD or adrenal CS. Our data obtained in a larger series of CS patients confirm previous findings evaluating only UFC (18) and also extend this information to LNSF. Overall, these data indicate that clinical score and biochemical measures do not share the same properties in diagnosing hypercortisolism. It can be ascribed to the interindividual variability and to a spectrum of glucocorticoid sensitivity and glucocorticoid tissue specificity observed in several pathological conditions and even in healthy individuals (26, 27). Moreover, the quality of CSI as a clinical discriminatory tool might be questioned because it gives the same score for each sign or symptom, independently of its prevalence (eg, obesity, diabetes, and signs of catabolism) in the general population, bringing inherent problems to this method. Elamin et al (28) conducted a systematic review and a meta-analysis and showed that UFC, LNSF, and 1 mg- DST had a similar high accuracy when analyzed by classical statistical methods. Although the meta-analysis evaluated a very large number of CS patients, its heterogeneous nature due to the fact of isolated collection of UFC, plasma, or saliva obtained in different centers with different patients can make a more precise comparison between each test difficult. In the present study, evaluating LNSF and UFC simultaneously on 3 consecutive days, no difference was observed among values of the samples for LNSF and for UFC obtained from each CD, adrenal CS, and obese patient. In addition, there was no difference in the CV (percentage) of LNSF or UFC within the 3 samples obtained for each patient. In CD, adrenal CS, and obese patients, the mean CV of LNSF varied from 28% to 39% and for UFC from 22% to 48%. Previous studies evaluated the variability of LNSF in a subset of CS patients collecting LNSF on 2 consecutive nights. One observed variability of 35% in CS (8), whereas the other observed 22% in normal subjects, 32% in a suspected CS group, and 51% in CS patients, being higher in the latter group (29). In addition, the variability of LNSF in a subset of CS patients, in which the sample collection occurred on 2 nonconsecutive days, was 17% (30). Concerning UFC, a recent study evaluated its variability on 4 nonconsecutive samples collected over a 2-week period and found intrapatient CV of 52% (16). Our results on LNSF and UFC CV are similar to those that used consecutive sampling collection.

6 2050 Elias et al Salivary Cortisol vs UFC in Hypercortisolism J Clin Endocrinol Metab, June 2014, 99(6): One aspect that has been claimed by some authors is the putative overlap in LNSF in later life, especially in diabetic patients, rendering less specificity for this method. A previous study suggested age-dependent LNSF cutoff levels in diagnosing CS (31). In addition, a recent study showed that older age was independently associated with higher cortisol peak, nadir, and AUC (32). However, in both studies, independently of the age, the cutoff values were below 350 ng/dl, the value used in our study to differentiate CS from pseudo-cs. Another interesting point is that the cortisol awakening response was significantly lower in diabetic patients. However, there was no difference in late cortisol decline (2 hours after awakening to bedtime) by diabetes status (33), suggesting that neither age nor diabetes may influence the diagnostic performance of LNSF in screening CS. A significant positive correlation was found between the individual mean of LNSF and the respective UFC mean in CS patients as a group. Either LNSF or UFC was able to diagnose CS in most patients. However, LNSF confirmed the diagnosis even in the presence of normal UFC in a subset of patients (17.3%), whereas UFC alone did not help any patient. In one patient with PPNAD neither LNSF nor UFC was able to diagnose CS, probably due to cyclical hypercortisolemia (34). ROC curves were obtained to establish the optimal threshold values for each test for CS diagnosis. The LNSF cutoff obtained from the ROC curve using our samples was 485 ng/dl. Recently, Ceccato et al (35) using 1 LNSF sample described that values above 524 ng/dl, assayed by RIA, differentiated CS patients from controls with high sensitivity and specificity. These values should be further cross-validated by other studies. Of note, our data demonstrate that the sensitivity and specificity of LNSF were not improved using 3 consecutive samples compared with previous studies using fewer samples (4, 5, 35). In our study, although LNSF and UFC sensitivities were similar, the specificity was higher for LNSF. Unlike sensitivity and specificity, LRs are independent of disease prevalence and can be used at an individual level; the larger the positive LR, the greater the likelihood of disease (36). Indeed, our data show that LNSF had an LR of 17.5 compared with an LR of 4.5 for UFC, indicating a superior power of LNSF compared with UFC as a diagnostic test for CS. In addition, the Bayesian analysis of the AUCs clearly indicated better performance of LNSF than UFC in diagnosing CS. It is important to point out that the better LNSF performance cannot be ascribed to the variability of these methods, because the CV were similar in LNSF and UFC tests. One hypothesis would be that small increases in cortisol production at the circadian nadir may not be detected as an increase in UFC (37). Another possibility is that there are intrinsic differences between methodologies. In the present study, LNSF was measured by RIA, and thus, the antibody against cortisol also cross-reacts against cortisone. As known, there is significant conversion of cortisol to cortisone in salivary glands by 11 -hydroxysteroid dehydrogenase II leading to a high concentration of cortisone in saliva (38). Indeed, a recent study demonstrated lower sensitivity (74.5%) and specificity (90.1%) of LNSF in diagnosing CS using LC-MS/MS (39). Conversely, in our study, UFC was measured by LC-MS/MS, which detects cortisol and eliminates the interference of cortisol metabolites, increasing its biochemical specificity. Therefore, what could be a limitation of salivary cortisol assayed by immunoassay-based methods turns into an advantage for diagnosing CS. However, it is important to be aware of different results generated by the commercially available methods and to interpret the published reference intervals appropriately (17, 40). In conclusion, the present study, using simultaneous and consecutive samples of LNSF and UFC compared the variability, reproducibility, and the diagnostic performance of both methods in CS diagnosis. Our data show that despite similar variability between both methods, LNSF has a superior diagnostic performance than UFC and should be used as the primary biochemical diagnostic test for CS diagnosis. Acknowledgments We thank Mr José Roberto Silva, Ms Adriana Rossi, and Ms Lucimara Bueno for technical support. Address all correspondence and requests for reprints to: Ayrton C. Moreira, Department of Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Sao Paulo , Brazil. acmoreir@fmrp.usp.br. This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de Sao Paulo (07/ and 10/ ) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) (314279/2009-1). Disclosure Summary: The authors have nothing to disclose. References 1. Aron DC. Cushing s syndrome: why is diagnosis so difficult? Rev Endocr Metab Disord. 2010;11: Crapo L. Cushing s syndrome: a review of diagnostic tests. Metabolism. 1979;28: Newell-Price J, Trainer P, Besser M, Grossman A. The diagnosis and differential diagnosis of Cushing s syndrome and pseudo-cushing s states. Endocr Rev. 1998;19: Castro M, Elias PC, Quidute AR, Halah FP, Moreira AC. Outpatient screening for Cushing s syndrome: the sensitivity of the combination of circadian rhythm and overnight dexamethasone sup-

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