Late-night Salivary Cortisol Research Articles

Prospective Evaluation of Late-Night Salivary Cortisol and Cortisone by EIA and LC-MS/MS in Suspected Cushing Syndrome.

ContextLate-night salivary cortisol (LNSC) measured by enzyme immunoassay (EIA-F) is a first-line screening test for Cushing syndrome (CS) with a reported sensitivity and specificity of >90%. However, liquid chromatography-tandem mass spectrometry, validated to measure salivary cortisol (LCMS-F) and cortisone (LCMS-E), has been proposed to be superior diagnostically.Objective, Setting, and Main Outcome MeasuresProspectively evaluate the diagnostic performance of EIA-F, LCMS-F, and LCMS-E in 1453 consecutive late-night saliva samples from 705 patients with suspected CS.DesignPatients grouped by the presence or absence of at least one elevated salivary steroid result and then subdivided by diagnosis.ResultsWe identified 283 patients with at least one elevated salivary result; 45 had an established diagnosis of neoplastic hypercortisolism (CS) for which EIA-F had a very high sensitivity (97.5%). LCMS-F and LCMS-E had lower sensitivity but higher specificity than EIA-F. EIA-F had poor sensitivity (31.3%) for adrenocorticotropic hormone (ACTH)-independent CS (5 patients with at least 1 and 11 without any elevated salivary result). In patients with Cushing disease (CD), most nonelevated LCMS-F results were in patients with persistent/recurrent CD; their EIA-F levels were lower than in patients with newly diagnosed CD.ConclusionsSince the majority of patients with ≥1 elevated late-night salivary cortisol or cortisone result did not have CS, a single elevated level has poor specificity and positive predictive value. LNSC measured by EIA is a sensitive test for ACTH-dependent Cushing syndrome but not for ACTH-independent CS. We suggest that neither LCMS-F nor LCMS-E improves the sensitivity of late-night EIA-F for CS.

Pituitary-adrenal axis and peripheral cortisol metabolism in obese patients.

A close relationship between adiposity and increased cortisol levels is well established in patients with endogenous hypercortisolism. Nevertheless, hypothalamic-pituitary-adrenal (HPA) axis regulation in overweight subjects is still a matter of concern. We studied free cortisol (urinary free cortisol, UFC and late night salivary cortisol, LNSC), pituitary feedback (serum cortisol after 1 mg dexamethasone suppression test, 1 mg DST) and peripheral cortisol metabolism (urinary cortisol to cortisone ratio, F/Eratio) in a large series of overweight subjects without Cushing's Syndrome. We considered 234 patients divided in 5 BMI classes, matched for age and gender (BMI ≤ 25 kg/m2n = 38; 25-30 n = 58; 30-35 n = 52; 35-40 n = 52; >40 n = 34). UFC, LNSC and urinary F/Eratio were assessed with LC-MS. We collected 183 LNSC, 176 UFC, 152 1 mg DST and 64 F/Eratio tests. UFC levels were higher in lean subjects, and they decreased according to the BMI classes (p = 0.022). Non-suppressed cortisol levels (>50 nmol/L) after 1 mg DST were observed especially in patients with normal weight or mild obesity. Patients with BMI ≥ 35 kg/m2 revealed a reduced F/Eratio (0.39 vs. 0.61, p = 0.006). The specificity of tests (false positive results) was higher considering 1 mg DST or UFC in obese patients, on the contrary impaired cortisol rhythm (LNSC above normality) was observed in 47 subjects, irrespective of weight. Overweight and obese subjects are characterised by an original regulation of HPA axis (reduced UFC levels, increased suppression after 1 mg DST) and peripheral cortisol metabolism (reduced F/Eratio), suggesting an effort to counteract hypercortisolism.

Diagnostic value of salivary cortisol in 1-mg dexamethasone suppression test

BACKGROUND: Late-night salivary cortisol and serum cortisol measurements after 1-mg Dexamethasone Suppression Test (1-mg DST) are routinely used to diagnose Cushings syndrome (CS). Measuring morning salivary instead of serum cortisol after 1-mg DST would make the diagnostics of CS fully non-invasive.
 AIM: To evaluate the diagnostic accuracy of salivary cortisol in 1-mg DST as measured by electrochemiluminescence assay (ECLIA).
 MATERIALS AND METHODS: We combined a cohort diagnostic study, including 164 participants (132 females, 32 males) aged from 18 to 77 years: 110 were overweight or obese as increased BMI is the most common sign of Cushings Syndrome (CS), and 54 healthy volunteers. In each cohort late-night salivary cortisol was measured (at 23:00) followed by 1-mg DST and blood and salivary sampling for cortisol measurement the next morning at 08:00-09:00. Cortisol in saliva and serum were measured on automatic analyzer Cobas е 601 by F. Hoffmann-La Roche Ltd, using ECLIA. The final diagnosis was confirmed by the histological evaluation after surgery or using a follow-up observation in patients with obesity to exclude Cushings syndrome manifestation.
 RESULTS: Among 110 patients, 54 subjects were finally confirmed as having Cushing's syndrome. Reference interval for salivary cortisol after 1-mg DST was estimated to be 0,512,7 nmol/l (595 procentile). Maximal salivary cortisol level in 1-mg DST registered in healthy person was 29,6 mmol/l. Areas under the curve (AUC) were as following: for salivary cortisol in 1-mg DST 0,838 (95% СI 0,7720,905), for blood cortisol in 1-mg DST 0,965 (95% CI 0,9390,992) and for late-night salivary cortisol 0,925 (95% CI 0,8820,969). The optimal cut-off point for salivary cortisol after 1-mg DST was estimated as 12.1 nmol/l (sensitivity 60%, specificity 92,9%) among CS versus healthy subjects; 12,6 (sensitivity 58,2%, specificity 96,2%) among patients with obesity and CS; and 12,2 nmol/l (sensitivity 60,7%, specificity 93,4%) among CS and both obese and healthy control subjects. Considering small difference between cut-off points, the recommended cut-off value for salivary cortisol after 1-mg DST is recommended to be 12,0 nmol/l if measured by ECLIA.
 CONCLUSION: Although salivary cortisol after 1-mg DST is inferior to serum cortisol after 1-mg DST in the diagnostic performance and diagnostic accuracy, it can be used as a low-invasive screening test with superior specificity.

Incidence of Adrenal Insufficiency and Cushing's Syndrome After Long-Term Epidural Steroid Injections Over Six Months or Longer: A Preliminary Study.

PurposeEndocrinological complications of an epidural steroid injection (ESI) are rare but dangerous. Nevertheless, despite the associated risks, repeated long-term ESIs are indispensable in some clinical situations. However, only a few reports to date have assessed the safety of this procedure. In this study, we evaluate the incidence of adrenal insufficiency (AI) and Cushing’s syndrome after long-term ESIs.MethodsThis clinical observational study enrolled herniated nucleus pulposus or spinal stenosis patients who had received ESIs over a period of six months or longer. The adrenocorticotropic hormone (ACTH) stimulation test was performed to confirm AI and the late-night salivary cortisol (LNSC) test was performed to diagnose Cushing’s syndrome. To evaluate the hypothalamus pituitary adrenal axis suppression, salivary cortisol (SC) levels were measured on days 0, 1, 7, 21, 28, 35, and 42.ResultsThis study included 17 patients. Among these, two patients (11.8%) developed AI, but no cases of Cushing’s syndrome were reported. There was no predictor for the development of AI. The SC levels tended to increase with time after an ESI (β = 0.003). The slope of the mixed effect model between the AI and non-AI groups showed a significant difference (p value = 0.015). However, the differences in mean SC levels at each time point between the two groups were not significant (adjusted p value = 0.053).ConclusionLong-term ESI use may be associated with AI development. An unexpected adrenal crisis due to AI could be life threatening. Therefore, regular monitoring of adrenal function in patients who have received long-term ESIs may be prudent.

Late-Night Salivary Cortisol in Healthy, Community-Dwelling Asian Indians Assessed by Second-Generation ECLIA.

Late-night salivary cortisol (LNSC) is used as a screening test for Cushing syndrome (CS), but there is no community-derived normative data for the normal upper limit in the South Asian population. This study aimed to determine the upper limit of normal (97.5th percentile) for LNSC in an Asian Indian population using a commercially available second-generation electrochemiluminiscence immunoassay (ECLIA). LNSC in apparently healthy community-dwelling individuals was assessed by multistage cluster sampling. Healthy individuals age 18 to 60 years from 8 urban and 8 rural clusters of Thiruvananthapuram district were studied. Thirty people from an approximate population of 1000 individuals from each cluster participated in the study. A saliva sample was collected between 11 PM and 12 midnight and analyzed using Roche COBAS-e-411 and ultrasensitive Cortisol II kits the next day. Cortisol values from 474 salivary samples were available for final analysis after exclusion of improperly collected samples. The 97.5th percentile of the LNSC concentrations was 0.25 μg/dL (6.89 nmol/L) (90% CI, 0.23-0.27 μg/dL; ie, 6.34-7.45 nmol/L). In postmenopausal women, median LNSC was significantly higher but the 90% CI for the upper limit of their LNSC (0.28μg/dL or 7.72 nmol/L) overlapped with that of premenopausal women. This study establishes the normal value of LNSC estimated by second-generation ECLIA in healthy community-dwelling Asian Indian individuals for the first time. Salivary cortisol at 11 pm to 12 am is less than 0.25μg/dL (6.89 nmol/L) in the general Asian Indian population. Menopause causes a significant increase in LNSC and may lead to overdiagnosis of CS if not interpreted carefully.

The Impact of Glucocorticoid Co-Secretion in Primary Aldosteronism on Thyroid Autoantibody Titers During the Course of Disease.

Excess aldosterone is associated with the increased risk of cardio-/cerebrovascular events as well as metabolic comorbidities not only due to its hypertensive effect but also due to its proinflammatory action. Autonomous cortisol secretion (ACS) in the setting of primary aldosteronism (PA) is known to worsen cardiovascular outcome and potentially exhibit immunosuppressive effects. The aim of this study was to determine the impact of ACS status in patients with PA on kinetics of thyroid autoantibodies (anti-TPO, anti-TG) pre and post therapy initiation. Ninety-seven PA patients (43 unilateral, 54 with bilateral PA) from the database of the German Conn’s Registry were included. Anti-TPO and anti-TG levels were measured pre and 6–12 months post therapeutic intervention. Patients were assessed for ACS according to their 24- hour urinary cortisol excretion, late night salivary cortisol and low-dose dexamethasone suppression test. Abnormal test results in line with ACS were identified in 74.2% of patients with PA. Following adrenalectomy, significant increases in anti-TPO levels were observed in patients with at least one abnormal test (p = 0.049), adrenalectomized patients with at least two pathological ACS tests (p = 0.015) and adrenalectomized patients with pathologic dexamethasone suppression tests (p = 0.018). No antibody increases were observed in unilateral PA patients without ACS and in patients with bilateral PA receiving mineralocorticoid antagonist therapy (MRA). Our data are in line with an immunosuppressive effect of mild glucocorticoid excess in PA on thyroid autoantibody titers. This effect is uncovered by adrenalectomy, but not by MRA treatment.

SAT-166 Advantage and Trustworthy of Cortisol and Dexamethasone Evaluation in Different Biological Matrices in Patients with Adrenal Masses

Biochemical function of adrenal masses is currently based on 1mg post-overnight dexamethasone suppression test (pDST). Several approaches are recently developed, in order to reduce false positive/negative samples, only in retrospective series. They are based on the correlation of some different parameters, i.e. late-night salivary cortisol (LNSC) vs serum and salivary cortisol pDST; LNSC vs serum and salivary cortisol and serum dexamethasone pDST; LNSC and cortisone vs serum cortisol and salivary cortisol and cortisone pDST. Although these findings offer a better diagnostic performance, several conditions are still disappointed. No information is traceable about the harvest time of diurnal salivary and serum samples and no study include neither the levels of salivary nor urinary dexamethasone pDST. Aim of our study is to combine all these strategies in order to avoid the underestimated biases and obtain more precise information about the true “cortisol condition” of the patients.To reach this purpose we assess both cortisol and dexamethasone concentrations in several samples: saliva at 11PM before the drug administration, diurnal saliva and serum at 8AM and also the urine collection from 11PM to 8AM. Analytes levels are measured using a validated liquid chromatography-tandem mass spectrometry method. In this study we included 20 subjects without morphological adrenal alteration (MRI assessment), dyslipidemia, hypertension and impaired glucose tolerance (healthy controls) and 20 patients with adrenal incidentaloma showing different cortisol levels ranging from normal to ACTH-independent hypercortisolism. In both series, LNSC were similar to salivary cortisol pDST, even if they were greater in the patients with adrenal incidentalomas and subclinical cortisol secretion. Serum dexamethasone levels were in reference ranges, while salivary and urinary dexamethasone found in these matrices require additional sample numbers in order to establish appropriate cut-offs. Our preliminary results suggest that the combination of these findings could represent an improvement to assess the individual cortisol status.

SAT-203 Misleading Diagnosis of Cushing’s Syndrome on Pramipexole

We present an intriguing case of a patient incorrectly diagnosed with Cushing’s disease secondary to pramipexole use for restless leg syndrome (RLS). 61-year-old male with symptoms of 55 lb weight gain, progressive fatigue, and subjective muscle weakness presented to endocrinology office for work-up of possible Cushing’s syndrome. Physical exam was significant for mild facial plethora, purple striae, and central obesity. Patient’s history includes diagnosis of prolactin-producing pituitary microadenoma 30 years ago treated with bromocriptine with normalization of prolactin and resolution of adenoma. Patient notes ten years ago, he was started on pramipexole 1 mg TID for severe RLS. Given patient’s clinical symptoms he underwent hormonal testing which was notable for an abnormal 1 mg ONDST with morning cortisol 2.44 mcg/dL (normal < 1.8 mg/dL) and two abnormal late night salivary cortisol 0.177 mcg/dL and 0.199 mcg/dL (normal < 0.122). Repeat MRI showed a 0.4 cm hypoenhancing lesion in the left side of the anterior pituitary gland. Patient was referred for IPSS. His central-to-peripheral plasma ACTH ratio was as follows: before CRH administration: left 5.8, right 1.2; after CRH administration: left 16.4, right 2.7. These results demonstrated positive IPSS indicating centralization or a pituitary source of ACTH excess. Patient was going to be referred to neurosurgery for left pituitary adenoma resection, but literature search revealed a study showing pramipexole causing an increase in cortisol levels in physiologic studies involving healthy subjects. Due to these findings, labs were repeated while holding pramipexole. His morning cortisol and ACTH levels were normal. Late night salivary cortisol x3 were normal: 0.038, 0.071, and 0.024 mcg/dL (normal < 0.090 mcg/dL). The 24 hour urine free cortisol x2 was also normal. These negative biochemical findings showed no evidence of hypercortisolism off of pramipexole. This further complicated the case as the patient had positive IPSS. In order to help exclude the diagnosis of an ACTH secreting pituitary tumor, a DDAVP stimulation test was performed showing a normal response. Based on these tests it was concluded he does not have Cushing’s and that his hypercortisolism was related to pramipexole use. Repeat brain MRI showed no structural evidence of any pituitary lesion. We report a very rare case of pramipexole use leading to a false diagnosis of Cushing’s syndrome. This was fortunately identified prior to patient undergoing unnecessary surgery. We recommend awareness of pramipexole use associated with hypercortisolism to prevent incorrect diagnosis of Cushing’s syndrome.Schilling, J. C., Adamus, W. S., & Palluk, R. (1992). Neuroendocrine and side effect profile of pramipexole, a new dopamine receptor agonist, in humans. Clinical Pharmacology & Therapeutics, 51(5), 541-548.

SAT-146 A Rare Combination of Severe Ectopic Cushing’s Syndrome and Graves Hyperthyroidism: A Case Report

BACKGROUND: Ectopic ACTH secretion (EAS) is a rare cause of Cushing’s syndrome. Olfactory neuroblastoma (ONB) is a malignant tumor derived from the olfactory epithelium and can rarely be a source of ectopic hormone production. There are only 19 reported cases of EAS from ONB. We report a case of severe ectopic Cushing’s due to ONB. Interestingly, the patient also presented with Graves disease, which is an unusual pathophysiologic combination since supraphysiologic levels of glucocorticoids suppress the immune system, thereby ameliorating autoimmune processes. Remarkably, Graves disease improved following the removal of the source of ectopic ACTH. CASE PRESENTATION: A 41year old male presented with epistaxis, anosmia, and headaches. He also reported recent weight gain, muscle weakness, and new onset hypertension. Cross-sectional imaging revealed a right nasal cavity mass with intracranial extension. Endonasal biopsy was consistent with a diagnosis of ONB. Biochemical evaluation demonstrated hypokalemic alkalosis, hyperglycemia, and severe hypercortisolism [ACTH 734 (9-46 pg/ml), am cortisol 110 (2-15 ug/ml), late night salivary cortisol 9.8 and 22.53 (<0.09 ug/dl), 24 hour UFC 41,337.3 (4-50 mcg) and non-suppressed cortisol 110 (1.8 mcg/dl) by 1 mg dexamethasone]. Pituitary MRI showed no sellar pathology. Following a period of cortisol suppression with Ketoconazole then with Etomidate infusion, the patient underwent resection of the ONB, followed by chemo- and radiotherapy, which resulted in improvement of severe hypercortisolism. Histopathology showed positive ACTH immunostaining. Pre-operative evaluation also demonstrated hyperthyroidism: TSH <0.010 (0.3-5uIu/ml), FT4 3.11 (0.89-1.76 ng/dl), normal TT3 0.81 (0.6-1.81 ng/ml). Thyroid auto-antibodies were negative; however, radioiodine scan and uptake demonstrated diffusely increased uptake in the enlarged thyroid gland. Pre-operative euthyroidism was achieved with a combination of methimazole, SSKI and cholestyramine. After 6 months of methimazole therapy the patient presented with hypothyroidism (TSH 72.37, FT4 0.1). Methimazole was discontinued and he achieved euthyroidism on subsequent evaluations. CONCLUSION: EAS due to ONB is a very rare cause of Cushing’s syndrome. To our knowledge, this is the first reported case of a male presenting with EAS and concurrent Graves hyperthyroidism. An extensive review of the literature and seeking expert opinion did not provide a convincing pathophysiologic explanation to this unusual concurrence. We therefore hypothesize that, while the two endocrine conditions were simultaneous, they were likely unrelated.

OR23-03 Post-Operative Day One Morning Cortisol Value as a Biomarker to Predict the Recurrence of Cushing Disease

Tumor removal by transsphenoidal surgery (TSS) is the first line treatment for Cushing disease (CD). However, recurrence is relatively common. A one week post-operative (post-op) nadir cortisol has been used as a biomarker to predict recurrence1. We identified 299 CD patients from our longitudinal multidisciplinary clinic or our institutional RPDR search tool who met biochemical diagnostic criteria1 and had undergone TSS between May 2008 and May 2018, to evaluate post-op cortisol levels as biomarkers to predict long-term remission and to characterize clinical features of Cushing syndrome. Predictors of recurrence were identified with logistic regression, using recurrence as the dependent variable, and a Kaplan-Meier survival curve analysis was performed to compare long-term remission after TSS among the 202 patients who reached initial remission and had at least 1 year of follow-up. The post-op day 1 morning (AM) cortisol had significant association with CD recurrence (OR=1.025, 95%CI:1.002-1.048, p=0.032). The time to recurrence was significantly longer in patients with post-op day 1 AM cortisol <5 μg/dL. In contrast, one week post-op nadir cortisol (OR=1.081, 95%CI: 0.989-1.181, p=0.086), urinary free cortisol (OR=1.032,95%CI: 0.994-1.07, p=0.098), or late night salivary cortisol (OR=1.383, 95%CI:0.841-2.274, p=0.201) had no significant correlation with recurrence. There were no significant differences in time to recurrence for post-op day 2 AM cortisol <5 μg/dL. Among patients who developed post-op adrenal insufficiency, recurrence was significantly lower if glucocorticoid replacement continued for more than one year. In addition, tumor proliferative index (MIB-1) had a significant correlation with recurrence (OR=1.287, 95%CI:1.106-1.498, p=0.001). The most common symptoms and signs of initial presentation of CD were weight gain (91.6%), central obesity (79.6%), menstrual disorders (77.9%), round face (65.9%), hypertension (63.2%), mood disorders (60.2%), dorsocervical fat deposition (59.9%), supraclavicular fat deposition (59.9%), osteoporosis (58.9%), fatigue (58.2%), bruising (55.9%) and facial hirsutism (54.2%). Most of the best discriminating CD features did not have high sensitivity, such as purple striae (31.4%), facial plethora (33.4%) and proximal muscle weakness (30.8%). Our data show that post-op day one morning cortisol level above 5 μg/dL had significant association with recurrence. In contrast, the one week post-op nadir cortisol level had no significant value to predict recurrence. Our data also suggest that nonspecific symptoms and signs of CD are more common than stereotypical signs. Reference: Nieman LK, et al. Treatment of Cushing’s Syndrome: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2015; 100:2807-2831

SAT-LB76 Impact of Glucocorticoid Cosecretion in Primary Aldosteronism on Thyroid Autoantibody Titers During the Course of Disease

Context: Excess aldosterone is associated with the increased risk of cardio- and cerebrovascular events as well as metabolic comorbidities not only due to its hypertensive effect but also due to its proinflammatory action. Autonomous cortisol secretion (ACS) in the setting of primary aldosteronism (PA) is known to worsen cardiovascular outcome and potentially exhibit immunosuppressive effects.The aim of this study was to determine the impact of ACS status in patients with PA on kinetics of thyroid autoantibodies (anti-TPO, anti-TG) pre and post therapy initiation.Patients and Methods: 97 PA patients (43 with unilateral, 54 with bilateral PA) from the database of the German Conn’s Registry were included. Anti-TPO and anti-TG levels were measured pre and 6 to 12 months post therapeutic intervention. Patients were assessed for ACS according to their 24h urinary cortisol excretion, late night salivary cortisol and low-dose dexamethason suppression test.Results: Abnormal test results in line with ACS were identified in 74.2% of patients. Significant increases in anti-TPO levels were observed in adrenalectomized patients with at least one abnormal test (p = 0.049), adrenalectomized patients with at least two pathological ACS tests (p = 0.015) and adrenalectomized patients with pathologic dexamethasone suppression tests (p = 0.018). No antibody increases were observed in unilateral PA patients without ACS and in patients with bilateral PA receiving mineralocorticoid antagonist therapy.Conclusion: ACS appears to be a relevant factor in PA affecting thyroid autoimmune disease. The biochemical and clinical course maybe be exacerbated after resolution of hypercortisolism by adrenalectomy in PA.

MON-332 Safety and Efficacy of Levoketoconazole in the Treatment of Endogenous Cushing’s Syndrome (LOGICS): A Double-Blind, Placebo-Controlled, Withdrawal Study

Endogenous Cushing’s syndrome (CS) is a rare, serious disorder caused by chronically elevated cortisol. A phase 3, open-label study (SONICS) of levoketoconazole in adults with CS and mean urinary free cortisol (mUFC) ≥1.5 × upper limit of normal (ULN) at baseline demonstrated normalization of mUFC in 62% of those completing 6 months of maintenance treatment (Fleseriu M, et al. Lancet Diabetes Endocrinol. 2019;7[11]:855-865). LOGICS is an ongoing, phase 3, double-blind, placebo-controlled, randomized-withdrawal study further investigating the safety and efficacy of levoketoconazole in patients who completed the SONICS study, or patients with CS who are levoketoconazole treatment-naive (ClinicalTrials.gov identifier: NCT03277690). The primary objective of LOGICS is to evaluate the effect of withdrawing levoketoconazole treatment to placebo, versus continuing treatment with levoketoconazole, on the cortisol therapeutic response established during open-label levoketoconazole therapy. The study includes (1) a screening phase (up to 13 weeks to allow for washout of CS medications); (2) a dose titration-maintenance phase (150-600 mg BID [dosed as needed to target mUFC normalization]) of ≥14 weeks, with at least the final 4 weeks demonstrating mUFC normalization prior to advancing to the randomized-withdrawal (R-W) phase; (3) a double-blind, placebo-controlled R-W phase (levoketoconazole or placebo; up to 8 weeks); and (4) a double-blind restoration phase (levoketoconazole and placebo; 8 weeks). Patients are randomized 1:1 in the R-W phase to continue their therapeutic dose of levoketoconazole, established during the dose titration-maintenance phase, or to receive an equivalent number of placebo tablets. Up to ~54 subjects are targeted for randomization. The primary efficacy endpoint, assessed at the completion of R-W phase, is the proportion of patients with loss of therapeutic response to levoketoconazole (mUFC ≥1.5x ULN, >40% above baseline if baseline value is >ULN [SONIC-completer cohort only], or early rescue criterion met) upon withdrawing to placebo, compared with continuing treatment with levoketoconazole, during the R-W phase. Secondary endpoints include changes from baseline to all postbaseline visits in R-W phase for mUFC, late-night salivary cortisol, CS cardiovascular biomarkers (fasting glucose, fasting insulin, hemoglobin A1c, homeostatic model assessment-insulin resistance, total and LDL cholesterol, high-sensitivity C-reactive protein), CS clinical signs and symptoms (acne, hirsutism [women only], and peripheral edema scores), and patient-reported outcomes of QoL (Cushing QoL questionnaire score) and depression (Beck Depression Inventory II). Potential liver toxicity, QT prolongation, and adrenal insufficiency are prespecified adverse events of special interest.

MON-172 Comparison of a Late-night Salivary Cortisol Immunoassay and a Cortisol and Cortisone LCMS Assay in Patients with Cushing’s Disease

Late-night salivary cortisol (LNSC) measured by enzyme immunoassay (EIA) is established as a reliable screening test and recommended as a first-line test for Cushing’s syndrome.1,2 However, liquid chromatography-tandem mass spectrometry (LCMS), validated to measure salivary cortisol (F) and cortisone (E), has been proposed to be superior diagnostically as well as helpful in detecting saliva contaminated with topical hydrocortisone (i.e. cortisol).3,4 We measured EIA-F, LCMS-F, and LCMS-E in 913 consecutive late-night saliva samples from patients suspected of Cushing’s syndrome. For the current report, we focus only on the patients with Cushing’s disease (CD) who had at least one elevated salivary result using cutoffs established previously [EIA-F ≥3.3 nmol/L, LCMS-F ≥2.8 nmol/L, and LCMS-E ≥8.7 nmol/L].5 We identified 27 patients who had newly diagnosed CD (n-CD) or had persistent/recurrent CD after pituitary surgery (r-CD). Data are shown as mean (SD). There were 19F/8M patients aged 50 (17) y; their BMI was 35 (11) kg/m2. 8 patients had n-CD and 19 had r-CD. In all 27 patients, EIA-F was 9.1 (6.0), LCMS-F was 5.1 (4.0), and LCMS-E was 19.6 (12.3) nmol/L. In n-CD, EIA-F was 14.3 (7.7), LCMS-F was 7.7 (4.6), and LCMS-E was 29.4 (12.8) nmol/L. In r-CD, EIA-F was 6.9 (3.6), LCMS-F was 4.0 (3.2), and LCMS-E was 15.5 (10.0) nmol/L. Mean EIA-F was greater than mean LCMS-F in all patients, and mean LNSC in n-CD patients was greater than mean LNSC in r-CD patients. The LCMS-F/E ratio was <0.8 in all subjects indicating that no saliva samples were contaminated with topical hydrocortisone.3 Furthermore, there was no difference in the LCMS-F/E ratio between n-CD [0.3 (0.1)] and r-CD [0.3 (0.2)] patients. Using previously identified cutoffs, 9 of the 27 CD patients had normal LCMS-F whereas only 1 patient had a normal EIA-F (P=0.011). The EIA-F results of patients with normal LCMS-F were significantly lower than patients with increased LCMS-F demonstrating that LCMS-F was more likely to provide false negative results with milder hypercortisolism. Most of these false-negative LCMS-F results occurred in the r-CD patients (8 out of 19) who had milder hypercortisolism compared to the n-CD patients (P=0.006). No differences in diagnostic performance were found between EIA-F and LCMS-E. In summary, EIA-F appears to be superior to LCMS-F in identifying CD patients with milder hypercortisolism using established diagnostic cutoffs.5 Neither LCMS-E nor F/E ratio improved the diagnostic sensitivity in newly diagnosed CD or persistent/recurrent CD after pituitary surgery. We suggest that late-night salivary cortisol measured by EIA provides the best sensitivity for CD diagnosis. 1J Clin Endocrinol Metab 2008;93:1526–1540. 2Clin Chem 2003;49:203–204. 3Clin Chem 2012;58:947–948. 4J Clin Endocrinol Metab 2010;95:4951–4958. 5J Endocr Soc 2019;3:1631–1640.

SUN-162 Isolated Primary Pigmented Micronodular Adrenal Disease. Unilateral or Bilateral Adrenalectomy? Two Cases Report

Background: Isolated primary pigmented nodular adrenal disease (i-PPNAD) is a particular case of micronodular bilateral hyperplasia (MiBAH) leading to Cushing syndrome (CS). We present two patients with i-PPNAD who undergone unilateral adrenalectomy with different disease outcomes. Case 1: A 56 yo man presented with typical signs and symptoms of CS, including central weight gain, proximal muscle weakness, hypertension and marked osteoporosis. A biochemical investigation showed an assessment of late-night salivary cortisol - 23 nmol/l (n <9), 24h urinary free cortisol (UFC)- 1206 nmol/24h (n-60–413). The 1 mg DST showed post-DST cortisol 617 nmol/l (n<50). ACTH concentration was suppressed at 1.1 pg/ml (n-7–66). Adrenal CT-scan was normal. PPNAD was assumed and the screening for Carney complex components was negative. After left adrenalectomy, histological examination confirmed the diagnosis of PPNAD. However, biochemical remission wasn’t achieved: postoperative UFC-860 nmol/24h. Thus, the right adrenalectomy was conducted. Case 2: A 40 yo female presented with long term drug-corrected (beta-blockers, ACE inhibitor) arterial hypertension, menstrual dysfunction and clinical features of CS (central obesity, proximal muscle weakness, hirsutism), without osteoporosis. Baseline hormonal evaluation documented slightly increased level of salivary cortisol- 9,8 nmol/l (n <9), post 1 mg DST cortisol - 470 nmol/l (n<50) and ACTH-concentration below detection <1 pg/ml (n-7–66). However, UFC was normal – 215 nmol/l (n-60–413). Abdominal CT-scan revealed micronodular hyperplasia of the left adrenal and was suggestive of possible right adrenal gland hyperplasia. We performed left adrenalectomy. In the early postoperative period, adrenal insufficiency was diagnosed and thus it required hydrocortisone replacement. Discussion: We obtained two opposite results: in case 1, an expected remission after unilateral adrenalectomy was not achieved, while patient No.2 developed adrenal insufficiency, which was a more favourable prognostic sign and attested to the successful outcome of surgical treatment. A possible explanation of the ineffectiveness of surgical intervention in patient No.1 was primary more severe hypercortisolism. It is also necessary to take into account that according to the CT data of the patient No.1, both adrenal glands were within normal ranges and did not differ from one another, while in the patient No.2, the lesion was more prominent on the right side. Conclusion: Diagnostic of i-PPNAD and choice of intervention can be precarious, consequently more studies are needed to define to which patients unilateral adrenalectomy could be an efficient treatment modality. It is possible to perform unilateral adrenalectomy with subsequent assessment of cortisol levels, in case of lack of remission the contralateral adrenalectomy is required.

SUN-205 Pseudo-Cushing Syndrome Secondary to Malnutrition and Gluco-Toxicity Mimicking Type 1 Diabetes Mellitus

Background: Pseudo-Cushing Syndrome (PCS) is an under-recognized clinical entity that is a reversible consequence of alterations in cortisol production. We present a case of a patient with presumed type 1 Diabetes Mellitus (DM) who was found to have PCS secondary to malnutrition. Once the nutritional status normalized, the patient’s glycemic control remarkably improved and became well-controlled on metformin alone. Clinical case: A 54-year-old female with poorly controlled insulin-dependent DM for 10 years was referred for concern for adrenal insufficiency after an ACTH came back elevated in the setting of intractable nausea, vomiting and considerable weight loss over 1 year. Prior HbA1c was 16.2% (RR 4.4–6.7). On exam her vitals were normal, body mass index (BMI) was 15 kg/m2. Workup confirmed an elevated ACTH of 100 pg/ml (RR 6–50 pg/ml), however, random PM cortisol was unexpectedly elevated at 26.58 ug/dL (RR 4.46 – 22.7). 8 AM labs for ACTH and cortisol were similarly elevated at 91 pg/ml and 28.33 ug/dl, respectively. She had no evidence of classic Cushingoid features. Subsequent low dose dexamethasone suppression test and 24-hour urine free cortisol were negative. Over 18 months, with optimization of her insulin therapy, BMI improved to 19 kg/m2, ACTH and cortisol started to downtrend spontaneously. After 30 months, her BMI improved to 20 kg/m2. Repeat blood work showed A1C 6.5%, ACTH and cortisol completely normalized to 42 pg/dl and 8 ug/dL, respectively. After being adherent to insulin for a few years, her gluco-toxicity state resolved. A month prior to following up, she self-discontinued insulin due to hypoglycemia but continued on metformin. Currently she continues to remain off insulin. Discussion: PCS is a challenging diagnosis to recognize and differentiate from Cushing Disease (CD) especially due to overlap in biochemical profile. It is important to be aware of this clinical condition to avoid misdiagnosis, delay in treatment or over-treatment. Common etiologies causing PCS include depression, chronic alcoholism, obesity, physical stress, malnutrition, eating disorders, uncontrolled DM, obstructive sleep apnea. PCS occurs due to chronic activation of the hypothalamic-pituitary-adrenal axis, it is usually mild and resolves with treatment of underlying etiology. In our case, first-line screening tests could differentiate between PCS and CD hence she did not require late-night salivary cortisol testing or corticotropic-releasing hormone testing. Conclusion: In our patient, PCS occurred secondary to malnutrition and severe gluco-toxicity which mimicked insulin-dependent type 1 DM. Interestingly, once her nutritional status and insulin compliance improved, cortisol levels normalized, gluco-toxicity state resolved and she no longer required exogenous insulin therapy.

Accuracy of Laboratory Tests for the Diagnosis of Cushing Syndrome.

The diagnosis of Cushing syndrome (CS) can be challenging. It remains to be determined which diagnostic tests are the most accurate. To summarize the accuracy of diagnostic tests for CS using contemporary meta-analytic techniques (hierarchical models). PubMed, Embase, Scopus, Web of Science, and the Cochrane Database of Systemic Reviews (inception until August 3, 2018). Studies performed in adults that determined the accuracy of one or more diagnostic tests: overnight 1-mg dexamethasone suppression test (DST), 2-day low-dose DST (2d DST), 24-hour urinary free cortisol (UFC), late-night salivary cortisol (LNSC), midnight serum cortisol (MSC), and the dexamethasone-suppressed CRH (dex-CRH) and desmopressin (dex-DDAVP) tests. Two authors independently extracted data and performed methodological assessments. One hundred thirty-nine studies (14 140 participants) were included in the analysis. The respective sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio (95% confidence interval [CI]) estimates include the following: DST 98.6% (96.9%-99.4%), 90.6% (86.4%-93.6%), 10.5 (7.2-15.3), and 0.016 (0.007-0.035); 2d DST 95.3% (91.3%-97.5%), 92.8% (85.7%-96.5%), 13.2 (6.47-27.1), and 0.051 (0.027-0.095); UFC 94.0% (91.6%-95.7%), 93.0% (89.0%-95.5%), 13.3 (8.47-21.0), and 0.065 (0.046-0.092); LNSC 95.8% (93.%-97.2%), 93.4% (90.7%-95.4%), 14.6 (10.3-20.7), and 0.045 (0.030-0.066); MSC 96.1% (93.5%-97.6%), 93.2% (88.1%-96.3%), 14.2 (7.96-25.2), and 0.042 (0.026-0.069); and dex-CRH 98.6% (90.4%-99.8%), 85.9% (67.6%-94.7%), 7.0 (2.80-17.6), and 0.016 (0.002-0.118). A single study evaluated dex-DDAVP. Meta-regression and a novel network meta-analytic approach suggest that DST is the most sensitive while UFC is the least sensitive. All of the included diagnostic tests for CS are highly sensitive and specific. It appears that the DST is the most sensitive while the UFC is less sensitive. The specificity of all first-line tests appears comparable.

Dexamethasone measurement during low-dose suppression test for suspected hypercortisolism: threshold development with and validation.

Dexamethasone Suppression Test (DST), recommended for Cushing's Syndrome (CS) diagnosis, explores the pituitary feedback to glucocorticoids. Its diagnostic accuracy could be affected by dexamethasone bioavailability, and therefore, we have developed and validated a dexamethasone threshold after 1-mg DST. We studied 200 subjects: 125 patients were considered retrospectively and 75 were enrolled prospectively as the validation cohort. Serum dexamethasone, Late Night Salivary Cortisol (LNSC), and Urinary Free Cortisol (UFC) were measured with LC-MS/MS. Normal LNSC and UFC levels were used to exclude CS. The lower 2.5th percentile of dexamethasone distribution in non-CS patients with cortisol ≤ 50nmol/L after 1-mg DST was used as threshold. 16 patients were CS and 184 non-CS (108 adrenal incidentaloma and 76 excluded CS); 4.5nmol/L resulted the calculated threshold. Cortisol after 1-mg DST confirmed high sensitivity (100% at 50nmol/L cut-off) and moderate-low specificity (63%, increased to 91% at 138nmol/L) to diagnose CS in the whole cohort of patients. We could reduce the number of false-positive results (from 10 to 6 and from 7 to 4 in AI and excluded CS) considering adequate dexamethasone levels. Dexamethasone levels were not affected by hypercortisolism, age, gender, smoke, weight, and creatinine. 6% of non-CS patients did not achieve adequate dexamethasone levels (40% of tests with serum cortisol > 138nmol/L after 1-mg DST). We developed and validated the routine dexamethasone measurement during 1-mg DST: it is independent from patient's clinical presentation, and it should be used to increase the specificity of serum cortisol levels.

Hypercortisolemia Recurrence in Cushing's Disease; a Diagnostic Challenge.

Cushing's disease recurrence following successful pituitary surgery is common and merits prompt and careful diagnosis, as untreated hypercortisolism leads to increased morbidity and mortality. However, an established recurrence definition has not been forthcoming. This poses a diagnostic challenge especially early in the course of returning hypercortisolemia and/or in the presence of non-neoplastic hypercortisolemia. A late-night salivary cortisol (LNSC) test is the first test to reveal abnormal results, however, has limitations related to assay performance as well as individual patient variability. Dexamethasone suppression tests and 24-h urinary free cortisol (UFC) results are next to reveal abnormal results. Other tests including, corticotropin-releasing hormone (CRH) stimulation test and combined CRH-dexamethasone test, as well as desmopressin stimulation test with/without dexamethasone are also used, although, none have proven to be the preeminent diagnostic test for recurrence determination. There is a possible role for these tests in predicting recurrence in patients who have experienced remission, though, this also remains challenging due to lack of established cutoff values. This article details and summarizes evidence about different diagnostic tests currently used to diagnose and predict Cushing's disease recurrence.

Toward a Diagnostic Score in Cushing's Syndrome.

Cushing's syndrome (CS) is a classical rare disease: it is often suspected in patients who do not have the disease; at the same time, it takes a mean of 3 years to diagnose CS in affected individuals. The main reason is the extreme rarity (1–3/million/year) in combination with the lack of a single lead symptom. CS has to be suspected when a combination of signs and symptoms is present, which together make up the characteristic phenotype of cortisol excess. Unusual fat distribution affecting the face, neck, and trunk; skin changes including plethora, acne, hirsutism, livid striae, and easy bruising; and signs of protein catabolism such as thinned and vulnerable skin, osteoporotic fractures, and proximal myopathy indicate the need for biochemical screening for CS. In contrast, common symptoms like hypertension, weight gain, or diabetes also occur quite frequently in the general population and per se do not justify biochemical testing. First-line screening tests include urinary free cortisol excretion, dexamethasone suppression testing, and late-night salivary cortisol measurements. All three tests have overall reasonable sensitivity and specificity, and first-line testing should be selected on the basis of the physiologic conditions of the patient, drug intake, and available laboratory quality control measures. Two normal test results usually exclude the presence of CS. Other tests and laboratory parameters like the high-dose dexamethasone suppression test, plasma ACTH, the CRH test, and the bilateral inferior petrosal sinus sampling are not part of the initial biochemical screening. As a general rule, biochemical screening should only be performed if the pre-test probability for CS is reasonably high. This article provides an overview about the current standard in the diagnosis of CS starting with clinical scores and screenings, the clinical signs, relevant differential diagnoses, the first-line biochemical screening, and ending with a few exceptional cases.

Cushing Disease Masquerading as Glaucoma

Glaucoma is a well-recognized side effect of corticosteroids. However, steroid-induced glaucoma typically refers to that caused by exogenous corticosteroid administration. Glaucoma secondary to endogenous overproduction of corticosteroids has only been reported in a few case reports. We aim to bring attention to glaucoma as a rare but important manifestation of endogenous hypercortisolism. Patient history, physical exam, laboratory results, and imaging studies were reviewed. We report a case of glaucoma as the initial presentation of Cushing disease (CD). The patient was diagnosed with glaucoma 16 months prior to his endocrinology evaluation. At our initial encounter, the patient had a cushingoid appearance. Levels of 24-hour urinary cortisol and late-night salivary cortisol were elevated. Serum cortisol was not suppressed by 1 mg of dexamethasone overnight, but it was suppressed by 8 mg of dexamethasone. Adrenocorticotropic hormone was also elevated. All other pituitary hormone axes were unremarkable (thyroid-stimulating hormone, free thyroxine, follicle-stimulating hormone, luteinizing hormone, growth hormone, prolactin, and insulin-like growth factor). Pituitary magnetic resonance imaging suggested a small adenoma (2 to 3 mm); therefore, the patient underwent inferior petrosal sinus sampling. The results were consistent with CD. Transsphenoidal resection was performed and final pathology confirmed an adrenocorticotropic hormone-positive adenoma. Hypercortisolism and intraocular pressures improved after the surgery. Glaucoma can lead to irreversible blindness if left untreated or uncontrolled. However, endogenous hypercortisolism-induced glaucoma can be reversed with treatment of the underlying CD. Thus, heightened awareness of extraocular manifestations of secondary causes of glaucoma such as endogenous hypercortisolism is necessary in order to promote prompt evaluation and treatment.