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Background: Obesity, diabetes and hypertension are common in the general population, and do not discriminate between patients (pts) with or without Cushing’s syndrome (CS). Thus, Endocrine Society guidelines recommend biochemical testing in pts with multiple features compatible with the diagnosis (1). A 24-hour (d) urine cortisol (UF) is one of the recommended screening tests. However, tandem mass spectrometry (MS/MS) measurement of UF may be less sensitive than immunoassay because of antibody cross-reactivity with cortisol-like compounds. Cortisone (E), the product of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivation of cortisol, can be measured in the urine (UE) by MS/MS and might improve the sensitivity of UF alone. The goal of this study was to compare the diagnostic utility of UE, UF, their sum (UF+UE) and their ratio (rUF/UE) for the detection of CS. Methods: We retrospectively reviewed 35 pts evaluated for CS from 1/2017 to 9/2019. UF (reference range (RR), 3.5-45 ug/d) and UE (RR 17-129 ug/d) were measured by MS/MS. CS etiology was later confirmed in 24 pts by pathology and/or tumor localization [14 female; 14 Cushing’s disease, 7 ectopic ACTH secretion (EAS), 3 presumed EAS awaiting surgery; 46 UF and UEs], and was excluded (noCS) in 11 pts followed over time [8 female; 65 UF and UEs with salivary cortisol and/or dexamethasone suppression]. For CS patients with multiple results, the sample with the lowest UF was compared with all available samples from the NoCS group. Results: Using a single result for each CS pt, the median (range) for UF, UE, UF+UE and rUF/UE were 281 ug/d (34 - 32,444), 358 ug/d (171 - 802), 642 ug/d (232 - 33246) and 0.78 (0.13 - 40.5). By contrast, using all observations in noCS pts, median (range) for UF, UE, UF+UE and rUF/UE were 13.6 ug/d (2.2 - 88.8), 79.5 ug/d (4.9 - 253), 98.2 ug/d (7.1 - 342) and 0.2 (0.05-0.67). Using the upper RR (or their sum) as a criterion for abnormality, all CS pts had abnormal UE and UF+UE; 45/46 had abnormal UF. In noCS samples, abnormal values were seen in 12/65 UF (3 pts), 14/65 UE (4 pts), and 13/65 UF+UE (3 pts). The rUF/UE was &amp;lt; 0.70 in all noCS pts and in 9/24 CS patients. Within this group, the UF+UE was &amp;gt; 230 ug/d in two noCS patients (2/3 and 2/7 samples/pt), and in all 9 CS patients, including two with normal UF and two with UF &amp;lt;50% above the RR. Alternatively, a UE &amp;gt; 170 ug/d identified the same noCS and CS patients. Conclusion: UF remains an important tool for screening of CS. The addition of UE and UF+UE may provide additional information to discriminate CS from noCS in patients with rUF/UE &amp;lt; 0.70. In contrast to CS pts, noCS pt consistently had UF+UE &amp;gt; 230 ug/d or UE &amp;gt; 170/d, underscoring the importance of multiple samples. Additional study, especially of pts with mild hypercortisolism, is needed to determine optimal diagnostic criteria. 1.Nieman L et al. J Clin Endocrinol Metab 93:1526, 2008

MON-162 Utility of 24-Hour Urine Cortisone for the Diagnosis of Cushing’s Syndrome