Grebe, Stefan K.

Abstract Disclosure: S. Ahmadian: None. C.A. Villavicencio Torres: None. V.M. Fell: None. K.L. Moehnke: None. Y. Chai: None. R.L. Taylor: None. S.K. Grebe: None. A.D. Maus: None. I. Bancos: None. Background: Salivary cortisol (SalF), and cortisone (SalE) interpretation can be influenced by intra-individual variability arising from cortisol secretion cyclicity, assay performance, and sampling techniques. No studies to date have examined the impact of sampling on intra-individual variability. Methods: We conducted a single-center cross-sectional study of adults evaluated with total serum cortisol measurements as a part of the overnight dexamethasone suppression test (DST) or cosyntropin stimulation test (CST), November 2024- January 2025. Participants were provided with two cotton swabs (salivettes) and asked to collect two consecutive saliva samples immediately after the venipuncture for total cortisol. Each cotton swab was kept in the mouth for 2-2.5 minutes. Saliva was tested for SalF and SalE by mass spectrometry. Variability was examined based on cortisol tertiles. Results: A total of 78 paired saliva samples were obtained from 60 participants (45 [75%] women; median age 55 years [IQR 39-66]), who were undergoing either DST (42 [70%]) or CST (18 [30%]). A total of 10 (17%) saliva samples did not contain a sufficient sample for analysis during the first or second saliva collection (7 [12%] from DST group, and 3 [5%] from CST group).Among 53 patients undergoing DST, the median serum total cortisol was 1.3 mcg/dL [IQR 0.9-3], the median first SalF was 20 ng/dL [IQR 7-28], the median second SalF was 12 ng/dL [IQR 6-23], and the median variability was 11% [IQR 3-22%]. The median first SalE was 69 ng/dL [IQR 54-185], while the median second SalE was 63 ng/dL [IQR 56-178], with a median variability of 6% [IQR 4-10].In patients undergoing CST, peak median serum total cortisol was 20 mcg/dL [IQR 18-24], median first SalF was 809 ng/dL [IQR 536-1227], and median second SalF was 1063 ng/dL [IQR 667-1503], with the median variability of 14% [IQR 8-22]. The median first SalE was 2507 ng/dL [IQR1697-2966], while the median second SalE was 2485 ng/dL [IQR 1530-2976], with a median variability of 9% [IQR 3-12].When examining intra-individual variability of SalF, the variability was lowest in the middle tertile, corresponding to serum cortisol of 1.8-8.9 mcg/dL, at 9% [IQR 5-16]. Variability was higher in the lowest tertile (14% [IQR 0-52]) and the highest tertile (12% [IQR 6-16]). Overall, the variability of SalE was lower than that of SalF: 7% [IQR 4-13] in the lowest tertile, 5% [IQR 2-9] in the middle tertile, and 10% [IQR 4-12] in the highest tertile. Conclusion: We show that saliva sampling may impact the results, with 17% patients providing insufficient sample for analysis, and variability of 9-14% for SalF and 5-10% for SalE. Same time-point sampling-related variability should be considered in interpretation of SalF and SalE measurements. Presentation: Sunday, July 13, 2025

Abstract Disclosure: C.A. Villavicencio Torres: None. Y. Chai: None. S.K. Grebe: None. T.D. Maus: None. I. Bancos: None. Background: Mild autonomous cortisol secretion (MACS) is diagnosed based on abnormal post-dexamethasone cortisol >1.8 mcg/dL. Late-night salivary cortisol (SalF) measurements in MACS are usually within normal ranges, and as such are not recommended by the adrenal incidentaloma guidelines. Late-night salivary cortisone (SalE) measurement may have a higher accuracy than SalF in diagnosing MACS. Objectives: To determine associations between SalF and SalE and post-dexamethasone cortisol concentrations in patients with MACS. Methods: We conducted a single-center retrospective cohort study of data between 2020-2023 that included patients with MACS who had concomitant SalF and SalE measurements at the time of diagnosis. Electronic medical records were reviewed for clinical, imaging, biochemical characteristics, and outcome data. Results: In 45 patients with MACS (31, 69% women) evaluated at a median age of 62 years (IQR 54-70), median post-dexamethasone cortisol was 3 mcg/dL (IQR 2.5-5.7), median corticotropin (ACTH) was 11 pg/mL (IQR 5.2-30), and median dehydroepiandrosterone sulfate (DHEAS) was 47.5 mcg/dL (IQR 27.8-98.8). In 36 patients, 24h-urine cortisol was also available, with a median cortisol of 14 mcg/dL (IQR 11.3-18), normal ranges <45 mcg/24h. Median adrenal adenoma size was 2.2 cm (IQR 1.6-3.0), and median unenhanced HU was 10 (IQR -5 to 22).Median SalF was 52 ng/dL (IQR 23.9-97.5), and median SalE was 332 ng/dL (IQR 222.5-607.0), with a median SalE/SalF ratio of 6.6 (IQR 5.7-9.0). SalF was normal (<50 ng/dL) in 22 (49%) patients with MACS, without differences in the post-dexamethasone cortisol concentrations between those with normal and abnormal SalF (median of 3 mcg/dL vs 3 mcg/dL, P=0.94). SalF correlated with SalE (R2=0.83, P<0.0001). However, only SalE was associated with post-dexamethasone cortisol (R2=0.09, P=0.04). Conclusion: SalE is associated with post-dexamethasone cortisol in patients with MACS and, after an appropriate validation, may provide additional value in support of diagnosis of MACS. Presentation: 6/3/2024

Abstract Context Guidelines suggest performing urine steroid profiling in patients with indeterminate adrenal tumors to make a noninvasive diagnosis of adrenocortical carcinoma (ACC). However, urine steroid profiling is not widely available. Objective To determine the accuracy of clinically available serum 11-deoxycortisol, 17OH-progesterone, and 17OH-pregnenolone in diagnosing ACC. Methods We conducted a prospective single-center cohort study of patients with adrenal masses evaluated between 2015 and 2023. Serum was analyzed by liquid chromatography–mass spectrometry for 17OH-pregnenolone, 17OH-progesterone, and 11-deoxycortisol. Reference standard for adrenal mass included histopathology, imaging characteristics, imaging follow up of 2 years, or clinical follow up of 5 years. Localized Generalized Matrix Learning Vector Quantization analysis was used to develop serum steroid score and assessed with area under receiver operating curve. Results Of 263 patients with adrenal masses, 44 (16.7%) were diagnosed with ACC, 161 (61%) with adrenocortical adenomas (ACAs), 27 (10%) with other adrenal malignancies, and 31 (12%) with other. Hounsfield unit ≥ 20 was demonstrated in all ACCs, in all but 1 other adrenal malignancy, and in 58 (31%) ACAs. All 3 steroids were higher in patients with ACCs vs non-ACCs, including when comparing ACCs with functioning ACAs, and with ACAs with Hounsfield unit ≥ 20 (P < .0001 for all). Localized Generalized Matrix Learning Vector Quantization analysis yielded a serum steroid score that discriminated between ACC and non-ACC groups with a mean threshold fixed area under receiver operating curve of 0.823. Conclusion We showed that measurements of 11-deoxycortisol, 17OH-progesterone, and 17OH-pregnenolone could be valuable in diagnosing ACC. After appropriate validation, serum steroid score could be integrated in clinical practice.

Abstract Context Interpretation of dexamethasone suppression test (DST) may be influenced by dexamethasone absorption and metabolism and by the altered cortisol binding. Objective We aimed to determine the normal ranges of free cortisol during DST in participants without adrenal disorders and to identify the population of patients where post-DST free cortisol measurements add value to the diagnostic workup. Design and Setting Cross-sectional study conducted in a tertiary medical center. Participants Adult volunteers without adrenal disorders (n = 168; 47 women on oral contraceptive therapy [OCP], 66 women not on OCP, 55 men) and patients undergoing evaluation for hypercortisolism (n = 196; 16 women on OCP). Measurements Post-DST dexamethasone and free cortisol (mass spectrometry) and total cortisol (immunoassay). Main Outcome Measures Reference range for post-DST free cortisol, diagnostic accuracy of post-DST total cortisol. Results Adequate dexamethasone concentrations (≥0.1 mcg/dL) were seen in 97.6% volunteers and 96.3% patients. Only 25.5% of women volunteers on OCP had abnormal post-DST total cortisol (>1.8 mcg/dL). In volunteers, the upper post-DST free cortisol range was 48 ng/dL in men and women not on OCP, and 79 ng/dL in women on OCP. When compared with post-DST free cortisol, diagnostic accuracy of post-DST total cortisol was 87.3% (95% CI, 81.7-91.7); all false-positive results occurred in patients with post-DST cortisol between 1.8 and 5 mcg/dL. OCP use was the only factor associated with false-positive results (21.1% vs 4.9%, P = 0.02). Conclusions Post-DST free cortisol measurements are valuable in patients with optimal dexamethasone concentrations and post-DST total cortisol between 1.8 and 5 mcg/dL.

Introduction Genotype-phenotype discordance occurs occasionally in congenital adrenal hyperplasia (CAH). Its causes are largely unknown. We describe a case of monochorionic, diamniotic twins with discordant clinical presentations of CAH, and show evidence for this being due to mosaicism resulting from a postzygotic full gene deletion of CYP21A2 prior to twinning. Case description A 7-day-old 36-week gestation female infant (Twin A) presented to the emergency department with elevated 17-hydroxyprogesterone (17-OHP). Her identical twin (Twin B) had normal 17-OHP on newborn screening. Both twins showed signs of virilization, more pronounced in Twin B. Molecular genetic testing of both twins and their parents showed a WT paternally-inherited CYP21A2 and a maternally-inherited copy containing the c.293-13C>G mutation. Both twins were also found to have a 5′-CYP21A1P/CYP21A2-3′ hybrid (product of the common 30-kb deletion), derived from the deletion of the paternally-inherited CYP21A2. Neither mother nor father carried the deletion. Conclusions The genetic findings are consistent with mosaicism for two CYP21A2 cell lines in both twins. The first cell line is expected, based on parental results, while the second line is due to a postzygotic full gene deletion of the paternally-inherited WT CYP21A2. The resultant genotype, compound heterozygosity for c.293-13C>G and a CYP21A2 full gene deletion, is consistent with a salt-wasting CAH phenotype. Differential distribution of the second cell line between the twins is most likely the cause for their discrepant phenotypes. We believe this is the first report of somatic CYP21A2 mosaicism, and represents a novel cause for discrepant CAH phenotypes in monozygotic twins.

Abstract Background: Mechanisms of hereditary erythrocytosis have been elucidated recently. These include high oxygen affinity (HOA) hemoglobin (Hb) variants, bisphosphoglycerate mutase (BPGM) deficiency, abnormalities in the erythropoietin receptor (EPOR) and oxygen-sensing pathway (OSP) proteins prolyl hydroxylase domain-2 (PHD2), hypoxia-inducible factor-2 alpha subunit (HIF2A), and von Hippel-Lindau (VHL). We present our experience with these disorders. Methods: Evaluation of erythrocytosis patients' blood samples for HOA Hb variants by protein or DNA sequencing methods has been performed in our laboratory for over 30 years. Testing has included alkaline/acid electrophoresis, isoelectric focusing, capillary electrophoresis, high performance liquid chromatography, mass spectrometry and Sanger sequencing. BPGM deficiency was determined by enzyme activity assay and/or genetic testing. Since 2012 we have evaluated the EPOR, EGLN1 (PHD2), EPAS1(HIF2A), and VHL genes. Reflexive evaluations, which include testing for p50 and pertinent Sanger sequencing of HBB, HBA1, HBA2, EPOR (exon 8), EGLN1 (exons 1-5), EPAS1 (exon 12) and VHL, have been performed on a subset of cases. Genetic results were correlated with clinical and phenotypic data. Results: Retrospective database review identified 62 confirmed HOA Hb variants (48β, 14α) with clinical erythrocytosis (Table 1). Multiple Hb variants were silent on at least one protein method but showed decreased p50 values. One homozygous BPGM mutation case was identified. Of the 394 cases evaluated for EPOR and OSP abnormalities, 39 cases (10%) contained genetic alterations. Of these, 11 were known pathogenic mutations and 28 were novel alterations including 4 pathogenic, 12 likely pathogenic and 12 variants of unknown significance (VUS). Eighteen EGLN1, 10 EPAS1, and 7 EPOR alterations were detected, all heterozygous. Four VHL mutation cases were identified: 3 homozygous/compound heterozygous known mutations and one novel heterozygous VUS (p.R200Q). All EPOR mutations resulted in a premature stop codon. Most of the EPAS1 alterations were located near proline 531, although two predicted splice site disturbances were found. The EGLN1 alterations were novel, variable, and affect the enzyme's catalytic domain. All 4 VHL cases involved the R200 amino acid position in at least one allele. Serum Epo levels varied: decreased in EPOR (5/5); normal in PHD2 (11/11) and most HIF2A (6/7) and VHL (3/4) cases; increased in 1 HIF2A and 1 VHL case. p50 was normal in EPOR and OSP cases (31/31 tested). Conclusion: A spectrum of abnormalities was identified. Salient points include: 1) Diagnostically, a high index of suspicion for HOA Hb variants is required as they can give false negative results by routine protein detection methods. p50 and Epo levels were useful for triage. 2) Phenotypically, serum Epo levels were low in EPOR and normal in PHD2 and HIF2A cases. Unexpectedly, the two classic Chuvash polycythemia VHL cases were associated with normal Epo levels. 3) Mechanistically, the alterations were truncating in EPOR, variable and scattered in EGLN1 and clustered near proline 531 in EPAS1. VHL mutations were rare and consistently affected the R200 amino acid position in at least one allele. Novel alterations were frequent, especially in EGLN1. 4) Clinically, many patients were asymptomatic but a subset demonstrated recurrent headaches and one had chest pain. Clotting complications included cerebrovascular accident in one EPAS1 and EGLN1 case each and portal vein thrombosis in one EGLN1 case. Table 1. Clinically significant high oxygen affinity Hb variants at Mayo Clinic n Beta Alpha >30 Malmo Tarrant 10 - 20 Bethesda, San Diego, Andrew-Minneapolis, M-Saskatoon, Olympia, Syracuse, Abruzzo Dallas 5 - 10 Ty Gard, Ypsilanti, Alberta, Coimbra, North Chicago J-Cape Town 2 - 4 Brigham, Kempsey, Little Rock, Puttelange, Wood, Brisbane, Cowtown, Creteil, Linkoping, Osler, Potomac, Providence, Ranier, Sparta, Vanderbilt, Heathrow, Helsinki, Hiroshima, Homozygous HPFH, McKees-Rocks, Pierre Benite, Regina Chesapeake, Ethiopia, Chiapas, Columbia Missouri, Burlington, Legnano 1 Alcorn County*, Bologna-St. Orsola, Bunbury, Cambridge-MA, Cardarelli, Chandigarh, Johnstown, Nantes, Nebraska, Olomouc, Palmerston North, Pitie-Salpetriere, Saint-Jacques, South Milwaukee Linwood, Longview*, Milledgeville, Sarasota Springs, Voorhees * novel Disclosures Pardanani: Stemline: Research Funding.

Over the past two decades, vitamin D level measurements have become some of the most frequently ordered tests in the laboratory. This increase is due to a growing awareness of widespread vitamin D deficiency and scientific data suggesting the beneficial effects of vitamin D in various diseases. A literature search was carried out in PubMed for cases reporting vitamin D intoxication and overdose. Thirteen articles were included in this review. Intoxication was severe in the reported cases. Patients presented with serum vitamin D concentrations ranging between 150 and 1220 ng/mL and serum calcium concentrations between 11.1 and 23.1 mg/dL. Most of the reported patients showed symptoms of vitamin D toxicity such as vomiting, dehydration, pain, and loss of appetite. The underlying causes included manufacturing errors, overdosing by patients or prescribers, and combinations of these factors. Our literature search highlights the fact that even though vitamin D intoxication is rare, it does occur and therefore patients and prescribers should be more cognizant of the potential dangers of vitamin D overdose.