Evaluating the efficacy of surgical and conservative approaches in mild autonomous cortisol secretion: a meta-analysis.

Introduction: The management of Mild Autonomous Cortisol Secretion (MACS) remains a topic of debate among clinicians, with differing opinions on the effectiveness of surgical intervention compared to conservative treatment methods. This meta-analysis provides a comprehensive assessment of available literature to determine the most effective approach for treating this condition. Methods: On December 1, 2023, an exhaustive literature search of English databases Embase, PubMed, the Cochrane Library, Scopus, Web of Science, as well as the Chinese databases China HowNet, Wanfang Database, SinoMed Database, and Weipu Database using the keywords "Mild Autonomous Cortisol Secretion", "Subclinical Cushing's Syndrome", "Subclinical Hypercortisolism", "Mild Cortisol Autonomous Secretion", "Adrenal Incidentaloma", "Surgical Treatment" and "Adrenalectomy". The data were statistically analyzed using STATA version 15.0. Results: In this comprehensive analysis involving 629 patients with MACS, the therapeutic efficacy of adrenalectomy was evident. The meta-analysis results indicate that compared to conservative treatment, surgical intervention more effectively improves obesity indicators in patients: waist circumference (SMD=-0.62, 95% CI: -1.06 to -0.18), BMI (SMD=-0.41, 95% CI: -0.62 to -0.20), enhances glycemic control: fasting blood glucose (SMD=-0.47, 95% CI: -0.68 to -0.26), glycated hemoglobin (SMD=-0.66, 95% CI: -0.95 to -0.38), improves lipid metabolism: triglycerides (SMD=-0.45, 95% CI: -0.73 to -0.16), lowers blood pressure: systolic blood pressure (SMD=-1.04, 95% CI: -1.25 to -0.83), diastolic blood pressure (SMD=-0.89, 95% CI: -1.12 to -0.65), and ameliorates hormonal metabolic disorder: 24h urinary free cortisol (SMD=-1.10, 95% CI: -1.33 to -0.87), ACTH (SMD=2.30, 95% CI: 1.63 to 2.97). All these differences are statistically significant. Conclusion: This meta-analysis shows that, compared to conservative treatment, surgical treatment is more effective in improving obesity indicators, glycemic control, lipid metabolism, reducing blood pressure, and ameliorating hormonal metabolic disorders in patients with MACS. These statistically significant results highlight the importance of considering surgical intervention in the management of patients with MACS. Systematic review registration: https://www.crd.york.ac.uk/prospero, identifier CRD42023492527.

Circulating miR-28-5p is overexpressed in patients with sarcopenia despite long-term remission of Cushing's syndrome: a pilot study.

Introduction: Patients with Cushing's syndrome (CS) in remission show sustained fatigue, myopathy, and an increased prevalence of sarcopenia. The mechanisms that determine these persistent muscle problems are not well known. We aimed to identify circulating microRNAs (miRNAs) with differential expression that could be potential biomarkers for the diagnosis and/or prognosis in CS. Patients and methods: Thirty-six women in sustained remission for 13 ± 7 years (mean ± SD) from CS, with a median age (IQ range) of 51 (45.2-60) years and mean ± SD BMI of 27 ± 4 Kg/m2, and 36 matched healthy controls were investigated. In 7 patients sarcopenia was present according to the European Working Group on Sarcopenia in Older People (EWGSOP) criteria. Small RNA libraries were generated and indexed using a modified Illumina TruSeq small RNA-sequencing protocol. MiRNAs were identified in plasma using bioinformatic analysis, and validation was carried out using RT-qPCR. For the validation, Taqman probes were performed on QuantStudio 5 equipment (Applied Biosystems). Results: In a first discovery group using RNA-sequencing, plasma samples of 18 CS patients and 18 healthy subjects were investigated; circulating miR-28-5p, miR-495-3p and miR-654-5p were upregulated in CS patients as compared with controls (p<0.05). In a validation study of the 3 upregulated miRNAs in 36 patients and 26 controls, no differences were observed by RT-qPCR; however, the expression of circulating miR-28-5p was upregulated in CS patients with sarcopenia as compared with those without (AUC for fold-change in the ROC analysis, 0.798; p=0.0156). The optimized cut-off value for miR-28-5p to identify CS patients with sarcopenia was 3.80, which yielded a sensitivity of 86% and a specificity of 69%. Conclusion: MiR-28-5p, a muscle-specific microRNA involved in myotube proliferation and differentiation in vivo, may serve as an independent non-invasive biomarker for identifying CS patients at high-risk of sarcopenia despite biochemical remission.

Whole blood transcriptomic signature of Cushing's syndrome.

OBJECTIVE: Cushing's syndrome is characterized by high morbidity and mortality with high interindividual variability. Easily measurable biomarkers, in addition to the hormone assays currently used for diagnosis, could reflect the individual biological impact of glucocorticoids. The aim of this study is to identify such biomarkers through the analysis of whole blood transcriptome. DESIGN: Whole blood transcriptome was evaluated in 57 samples from patients with overt Cushing's syndrome, mild Cushing's syndrome, eucortisolism, and adrenal insufficiency. Samples were randomly split into a training cohort to set up a Cushing's transcriptomic signature and a validation cohort to assess this signature. METHODS: Total RNA was obtained from whole blood samples and sequenced on a NovaSeq 6000 System (Illumina). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore the transcriptome profile. Ridge regression was used to build a Cushing's transcriptome predictor. RESULTS: The transcriptomic profile discriminated samples with overt Cushing's syndrome. Genes mostly associated with overt Cushing's syndrome were enriched in pathways related to immunity, particularly neutrophil activation. A prediction model of 1500 genes built on the training cohort demonstrated its discriminating value in the validation cohort (accuracy .82) and remained significant in a multivariate model including the neutrophil proportion (P = .002). Expression of FKBP5, a single gene both overexpressed in Cushing's syndrome and implied in the glucocorticoid receptor signaling, could also predict Cushing's syndrome (accuracy .76). CONCLUSIONS: Whole blood transcriptome reflects the circulating levels of glucocorticoids. FKBP5 expression could be a nonhormonal marker of Cushing's syndrome.

Can neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios be used as markers for hypercortisolism in dogs?

Changes in neutrophil-to-lymphocite ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been identified in dogs with hypercortisolism (HC), but, no studies have investigated the changes in these inflammatory biomarkers as cost-effective and available parameters for the diagnosis and management of HC. This study was performed to evaluate whether NLR and PLR could be used as biomarkers for the diagnosis and treatment response in dogs with HC. This retrospective study included 67 dogs with HC, 58 dogs with non-adrenal illness (NAI), and 39 healthy dogs. NLR and PLR were compared among the three groups. Cut-off values of NLR and PLR for HC screening and percent change in biomarkers for assessing treatment response were evaluated. In addition, the NLR and PLR were compared before and after trilostane treatment. NLR and PLR were significantly higher in the HC group than in the NAI and healthy groups. The NLR cut-off value of 4.227 had a sensitivity of 67.16% and specificity of 65.52%, and the PLR cut-off value of 285.0 had a sensitivity of 56.72% and specificity of 70.69% for differentiating between dogs with HC and those with NAI, respectively. Furthermore, a significant decline in NLR was observed after treatment in the well-controlled HC group. The cutoff value of percent change in NLR to identify well-controlled HC was -7.570%; sensitivity and specificity were 100% and 63.64%, respectively. Therefore, NLR and PLR might be used cautiously as supportive biomarkers for HC diagnosis, and NLR could be a potential monitoring tool in assessing the treatment response of HC in dogs.

Prevalence and outcome of secondary hypogonadism in male patients with Cushing's syndrome and mild autonomous cortisol secretion.

BACKGROUND: Secondary hypogonadism (SH) is common in men with Cushing's syndrome (CS), but its impact on comorbidities is largely unknown and longitudinal data are scarce. If SH also affects men with mild autonomous cortisol secretion (MACS) is unknown. METHODS: We included 30 treatment-naïve adult men with CS and 17 men with MACS diagnosed since 2012. Hypogonadism was diagnosed based on total testosterone (TT) concentrations < 10.4 nmol/L and age-specific cut-offs. Outcomes were compared to age- and BMI-matched controls. In 20 men in remission of CS, a longitudinal analysis was conducted at 6, 12, and 24 months. RESULTS: Men with CS had significantly lower concentrations of TT, bioavailable T, and free T compared to controls (P < .0001) with lowest concentrations in ectopic CS. Likewise, TT was lower in men with MACS compared to controls. At baseline, 93% of men with CS and 59% of men with MACS had SH. Testosterone correlated negatively with late night salivary cortisol and serum cortisol pre- and post-1 mg dexamethasone suppression test. Following successful surgery, TT increased significantly (P = .001), normalising within 6 months. Despite normalisation, several RBC parameters remained lower in men with CS even 2 years after successful surgery. CONCLUSIONS: Secondary hypogonadism is common in men with CS and MACS but usually reversible after successful surgery. The persisting changes observed in RBC parameters need to be further investigated in larger cohorts and longer follow-up durations.

Thyroid function spectrum in Cushing's syndrome.

PURPOSE: Thyroid disorders have been reported in hypercortisolism patients. Endogenous Cushing's syndrome (CS) potentially complicates its metabolic sequelae. We investigated thyroid function in CS patients to determine this relationship. METHODS: In this cross-sectional study, we screened CS patients from 2016 to 2019 at our hospital. Patient demographic, medical history, and laboratory data were collected. Additionally, we performed a meta-analysis to demonstrate the prevalence of thyroid dysfunction in patients with CS. RESULTS: Among 129 CS patients, 48.6% had triiodothyronine (TT3), 27.9% had thyroxine (TT4), 24.6% had free T3 (FT3), 27.7% had free T4 (FT4), and 6.2% had thyroid-stimulating hormone (TSH) levels below the reference values. Those with clinical CS showed more pronounced thyroid suppression than did those with subclinical CS. Cortisol levels were markedly greater in patients with pituitary hypothyroidism (P < 0.001). Serum cortisol levels throughout the day and post low-dose dexamethasone-suppression test (LDDST) results correlated with thyroid hormone levels, particularly in ACTH-independent CS. Correlations varied by thyroid status; FT3 and TSH were linked to cortisol in euthyroid individuals but not in those with low T3 or central hypothyroidism. TSH levels notably halved from the lowest to highest cortisol tertile post-LDDST. Finally, meta-analysis showed 22.7% (95% CI 12.6%-32.9%) central hypothyroidism in 528 CS patients of nine studies. CONCLUSION: Thyroid hormone levels are significantly correlated with cortisol levels and are impaired in patients with CS. However, the physiological adaptation and pathological conditions need further study.

68Ga-pentixafor PET/CT in the localization diagnosis of primary aldosteronism concurrent subclinical cushing's syndrsome: two case reports.

PURPOSE: Adrenal venous sampling (AVS) is recommended for subtyping primary aldosteronism (PA). However, in cases of PA, concurrent subclinical Cushing's syndrome (SCS) has the potential to confound AVS results. Pentixafor, a CXC chemokine receptor type 4-specific ligand, has been reported as a promising marker to evaluate functional nature of adrenal adenomas. This study aims to investigate the clinical value of Gallium-68 Pentixafor Positron Emission Tomography-Computed Tomography (68Ga-Pentixafor PET/CT) in the localization diagnosis of patients with PA plus SCS. METHODS: Two patients with a confirmed diagnosis of PA plus SCS underwent AVS and 68Ga-Pentixafor PET/CT. RESULTS: AVS results revealed no lateralization for both patients while 68Ga-Pentixafor PET/CT showed a unilateral adrenal nodule with increased uptake of 68Ga-Pentixafor. Unilateral adrenalectomy was performed based on the results of 68Ga-Pentixafor PET/CT. Subsequently, complete biochemical remission of autonomous aldosterone and cortisol secretion were achieved in both cases. CONCLUSIONS: 68Ga-Pentixafor PET/CT shows promising potential for the localization of aldosterone and cortisol co-secreting adrenal adenoma in patients with PA plus SCS.

Impact of etiology, sex, diabetes mellitus and remission status on erythrocytic profile in patients with cushing's syndrome: a large population database study.

PURPOSE: The study aimed to characterize the erythrocytic profile in patients with cushing's syndrome (CS) versus controls from the normal population according to etiology, sex, presence of diabetes mellitus (DM) and hypercortisolemia remission status. METHODS: This retrospective cohort analysis compared erythrocytic parameters between patients with CS of pituitary (CD) and adrenal (aCS) etiology and age, sex, body mass index (BMI) and socioeconomic status-matched controls in a 1:5 ratio. Laboratory values at baseline were calculated as mean values during the year preceding CS diagnosis, and over one year thereafter. RESULTS: The cohort included 397 CS patients (68.26% female; mean age 51.11 ± 16.85 years) and 1970 controls. Patients with CS had significantly higher baseline median levels of hemoglobin (Hgb) (13.70 g/dL vs. 13.12 g/dL [p < 0.0001]) and hematocrit (Hct) (41.64% vs. 39.80% [p < 0.0001]) compared to controls. These differences were observed for both CD and aCS and for both sexes. Patients who attained remission had Hgb and Hct levels comparable to controls (13.20 g/dL and 40.08% in patients with CD and aCS vs. 13.20 g/dL and 39.98% in controls). Meanwhile, those with persistent/recurrent disease maintained elevated levels. Patients with comorbid DM had similar Hgb but higher Hct (p = 0.0419), while patients without DM showed elevated erythrocytic values compared to controls (p < 0.0001). CONCLUSION: Our data illustrates that erythrocytic parameters are directly influenced by glucocorticoid excess as Hgb and Hct are higher in patients with CS, and normalize after remission. We have identified the influence of DM on erythrocytic parameters in patients with CS for the first time.

Selective venous sampling for secondary hypertension.

Selective venous sampling (SVS), an invasive radiographic procedure that depends on contrast media, holds a unique role in diagnosing and guiding the treatment of certain types of secondary hypertension, particularly in patients who may be candidates for curative surgery. The adrenal venous sampling (AVS), in particular, is established as the gold standard for localizing and subtyping primary aldosteronism (PA). Throughout decades of clinical practice, AVS could be applied not only to PA but also to other endocrine diseases, such as adrenal Cushing syndrome (ACS) and Pheochromocytomas (PCCs). Notably, the application of AVS in ACS and PCCs remains less recognized compared to PA, with the low success rate of catheterization, the controversy of results interpretation, and the absence of a standardized protocol. Additionally, the AVS procedure necessitates enhancements to boost its success rate, with several helpful but imperfect methods emerging, yet continued exploration remains essential. We also observed renal venous sampling (RVS), an operation akin to AVS in principle, serves as an effective means of diagnosing renin-dependent hypertension, aiding in the identification of precise sources of renin excess and helping the selection of surgical candidates with renin angiotensin aldosterone system (RAAS) abnormal activation. Nonetheless, further basic and clinical research is needed. Selective venous sampling (SVS) can be used in identifying cases of secondary hypertension that are curable by surgical intervention. Adrenal venous sampling (AVS) and aldosterone measurement for classificatory diagnosis of primary aldosteronism (PA) are established worldwide. While its primary application is for PA, AVS also holds the potential for diagnosing other endocrine disorders, including adrenal Cushing's syndrome (ACS) and pheochromocytomas (PCCs) through the measurements of cortisol and catecholamine respectively. In addition, renal venous sampling and renin measurement can help to diagnose renovascular hypertension and reninoma.

No requirement of perioperative glucocorticoid replacement in patients with endogenous Cushing's syndrome - a pilot study.

PURPOSE: Surgical therapy represents the first-line treatment for endogenous Cushing's syndrome (CS). While postoperative glucocorticoid replacement is mandatory after surgical remission, the role of perioperative glucocorticoid therapy is unclear. METHODS: We recruited patients with central or adrenal CS in whom curative surgery was planned and patients who underwent pituitary surgery for other reasons than CS as a control group. Patients did not receive any perioperative glucocorticoids until the morning of the first postoperative day. We performed blood samplings in the morning of surgery, immediately after surgery, in the evening of the day of surgery, and in the morning of the first and third postoperative day before any morning glucocorticoid intake. We continued clinical and biochemical monitoring during the following outpatient care. RESULTS: We recruited 12 patients with CS (seven with central CS, five with adrenal CS) and six patients without CS. In patients with CS, serum cortisol concentrations <5.0 µg/dL (<138 nmol/L) were detected in the morning of the first and third postoperative day in four (33%) and six (50%) patients, respectively. Morning serum cortisol concentrations on the third postoperative day were significantly lower when compared to preoperative measurements (8.5 ± 7.6 µg/dL vs. 19.9 ± 8.9 µg/dL [235 ± 210 nmol/L vs. 549 ± 246 nmol/L], p = 0.023). No patient developed clinical or biochemical signs associated with hypocortisolism. During follow-up, we first observed serum cortisol concentrations >5.0 µg/dL (>138 nmol/L) after 129 ± 97 days and glucocorticoids were discontinued after 402 ± 243 days. Patients without CS did not require glucocorticoid replacement at any time. CONCLUSION: Perioperative glucocorticoid replacement may be unnecessary in patients with central or adrenal CS undergoing curative surgery as first-line treatment.

Cushing Syndrome: A Review.

Importance: Cushing syndrome is defined as a prolonged increase in plasma cortisol levels that is not due to a physiological etiology. Although the most frequent cause of Cushing syndrome is exogenous steroid use, the estimated incidence of Cushing syndrome due to endogenous overproduction of cortisol ranges from 2 to 8 per million people annually. Cushing syndrome is associated with hyperglycemia, protein catabolism, immunosuppression, hypertension, weight gain, neurocognitive changes, and mood disorders. Observations: Cushing syndrome characteristically presents with skin changes such as facial plethora, easy bruising, and purple striae and with metabolic manifestations such as hyperglycemia, hypertension, and excess fat deposition in the face, back of the neck, and visceral organs. Cushing disease, in which corticotropin excess is produced by a benign pituitary tumor, occurs in approximately 60% to 70% of patients with Cushing syndrome due to endogenous cortisol production. Evaluation of patients with possible Cushing syndrome begins with ruling out exogenous steroid use. Screening for elevated cortisol is performed with a 24-hour urinary free cortisol test or late-night salivary cortisol test or by evaluating whether cortisol is suppressed the morning after an evening dexamethasone dose. Plasma corticotropin levels can help distinguish between adrenal causes of hypercortisolism (suppressed corticotropin) and corticotropin-dependent forms of hypercortisolism (midnormal to elevated corticotropin levels). Pituitary magnetic resonance imaging, bilateral inferior petrosal sinus sampling, and adrenal or whole-body imaging can help identify tumor sources of hypercortisolism. Management of Cushing syndrome begins with surgery to remove the source of excess endogenous cortisol production followed by medication that includes adrenal steroidogenesis inhibitors, pituitary-targeted drugs, or glucocorticoid receptor blockers. For patients not responsive to surgery and medication, radiation therapy and bilateral adrenalectomy may be appropriate. Conclusions and Relevance: The incidence of Cushing syndrome due to endogenous overproduction of cortisol is 2 to 8 people per million annually. First-line therapy for Cushing syndrome due to endogenous overproduction of cortisol is surgery to remove the causative tumor. Many patients will require additional treatment with medications, radiation, or bilateral adrenalectomy.

Pregnancy complicated with adrenal adenoma causing ACTH-independent Cushing's syndrome, accompanied by obstetric antiphospholipid syndrome and severe pre-eclampsia: case report and literature review.

This case report shares the management experience of a patient with pregnancy combined with adrenal adenoma causing ACTH-independent Cushing's syndrome (CS), accompanied by obstetric antiphospholipid syndrome (OAPS) and severe pre-eclampsia. The case was a 26-year-old that presented with typical clinical symptoms and signs of CS. The patient had a history of 4 spontaneous abortions in the last 4 years. The 24-hour urinary free cortisol was significantly increased, an abnormal cortisol circadian rhythm was demonstrated by a high late-night salivary cortisol, blood ACTH was suppressed (< 1ng/dL), anticardiolipin antibody was positive, and imaging examination showed an adrenal tumor. The patient underwent laparoscopic adrenal tumor resection under general anesthesia at 23 weeks of gestation. The tumor was pathologically confirmed to be an adrenocortical adenoma. The patient underwent a cesarean section at 39 weeks of gestation to give birth to a healthy baby girl with an Apgar score of 10. Pregnancy complicated by CS is clinically rare, easily masked by normal physiological changes of pregnancy, and is difficult to diagnose. The determination of 24-hour urinary free cortisol, the circadian rhythm of serum cortisol, ultrasound, and MRI can be helpful in the diagnosis of CS during pregnancy. Surgery is the first choice for the treatment of CS during pregnancy. As a subtype of antiphospholipid syndrome, patients with OAPS are prone to thrombotic events and recurrent miscarriages if not treated accordingly. To our knowledge no cases of CS with OAPS and severe pre-eclampsia have been reported. We summarize the experience of the treatment of this patient and review the literature to improve clinicians' awareness of this disease.

Eleven-Year Experience with Selective Adrenal Vein Sampling in Management of Primary Adrenal Hormonal Hypersecretion.

Introduction: Nearly half of the adult population in the United States has been diagnosed with hypertension. Adrenal hormonal hypersecretion is a leading cause of secondary hypertension. Adrenal vein sampling (AVS) may assist in differentiating between unilateral and bilateral adrenal hormonal hypersecretion to identify patients who are candidates for adrenalectomy. We reviewed the use of AVS at our institution along with associated outcomes after adrenalectomy. Materials and Methods: A retrospective chart review was conducted of patients with a diagnosis of primary hyperaldosteronism (PA) or adrenocorticotropic hormone-independent Cushing syndrome (AICS) and who underwent adrenalectomy between January 1, 2010, and December 1, 2021. Patient data of baseline characteristics, preoperative workup, including AVS, and postoperative outcomes were collected and analyzed. Results: Seventy-one patients were identified in the study period (48 PA and 23 AICS). Computed tomography scan identified unilateral adrenal nodules in 52 patients (29 left; and 23 right), bilateral nodules in 13 patients, and no nodules in 6 patients. AVS was performed in 45 patients with PA (93%) and 5 patients with AICS (21%). After surgery, the number of PA patients with hypokalemia or requiring potassium supplementation significantly decreased after adrenalectomy (before surgery: 33 [68.7%]; and after surgery: 5 [10.4%], P < .01). The number of medications required for hypertension in AICS patients also significantly decreased. No major adverse events were noted. Conclusions: Our long-term experience demonstrates the ongoing use of AVS during workup of patients with primary hyperaldosteronism and for select patients with adrenocorticotropic hormone-independent Cushing syndrome. However, a low level of discordance between imaging and AVS findings in PA patients suggests that there may be a subset of patients in whom preoperative AVS is not necessary.

Recent Advances in the Clinical Application of Adrenal Vein Sampling.

We reviewed clinical research investigating the applications of adrenal vein sampling (AVS). AVS could be applied not only to primary aldosteronism (PA) but also to other endocrine diseases, such as adrenocorticotropic hormone (ACTH) independent Cushing syndrome (AICS) and hyperandrogenemia (HA). However, the AVS protocol requires improvements to increase its success rate. Using the computed tomography image fusion, coaxial guidewire technique, and fast intraprocedural cortisol testing (CCF) technique could improve the success rate of catheterization in AVS for PA. ACTH loading could be considered in medical centers with a low selectivity of AVS for PA but is not essential in those with mature AVS technology. The continuous infusion method should be recommended for ACTH stimulation in AVS for PA to reduce adverse events. AVS has not been routinely recommended before management decisions in AICS, but several studies verified that AVS was useful in finding out the source of excess cortisol, especially for distinguishing unilateral from bilateral disease. However, it is necessary to reassess the results of AVS in AICS with the use of reference hormones to fully normalize cortisol levels. In addition, it is essential to determine the optimal model that combines AVS results and mass size to guide the selection of surgical plans, including identifying the dominant gland and presenting the option of staged adrenalectomy, to minimize the impact of bilateral resection. For HA, AVS combined with ovarian intravenous sampling to locate excess androgens could be considered when imaging results are equivocal.

Inflammatory biomarkers in the evaluation of pediatric endogenous Cushing syndrome.

OBJECTIVE: Inflammatory biomarkers, such as absolute neutrophil and lymphocyte counts, neutrophil-to-lymphocyte ratio (NLR), platelet (PLT)-to-lymphocyte ratio (PLR) and monocyte-to-lymphocyte ratio (MLR), are associated with the progression and development of several disorders. Although patients with Cushing syndrome (CS) have immunosuppression with altered leucocyte counts, the profile of the inflammatory biomarkers in these patients has not been extensively studied. DESIGN: We compared a panel of inflammatory biomarkers in patients with active endogenous CS (n of complete blood count (CBC) reports = 319) and eucortisolemic subjects of similar age, gender and BMI (n of CBC reports = 93). Patients were divided into two age groups (6-12 years at the time of CBC and >12 years at the time of CBC) based on age differences in normal reference ranges. RESULTS: Patients with CS had higher NLR vs controls (6-12 years: 2.47 (1.86, 3.32) vs 1.35 (1.11, 2.27), P < 0.0001; >12 years: 3.00 (2.23-4.17) vs 1.80 (1.23-2.31), P < 0.0001). Similarly, absolute neutrophil and lymphocyte counts, MLR and PLR differed between patients with CS and controls. The inflammatory biomarkers correlated with indices of cortisol secretion, such as midnight serum cortisol, 24-h urinary free cortisol and morning cortisol. On receiver operating characteristic analysis, NLR showed high area under the curve (AUC) (6-12 years: cutoff of 1.72 had AUC: 0.77, >12 years: cutoff of 2.35 had AUC: 0.81). CONCLUSIONS: We conclude that multiple inflammatory biomarkers differed between patients with CS and controls suggesting substantial effects of hypercortisolemia on the immune system.

Identification of glucocorticoid-related molecular signature by whole blood methylome analysis.

OBJECTIVE: Cushing's syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing's syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing's syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. DESIGN: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing's syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. METHODS: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850 000 CpG sites). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. RESULTS: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing's syndrome correction. In Cushing's syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. CONCLUSIONS: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assessment of glucocorticoid action beyond hormone assays.

Targeted Mutational Analysis of Cortisol-Producing Adenomas.

CONTEXT: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. OBJECTIVE: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. METHODS: We analyzed FFPE adrenal tissue from 77 patients (n = 12 men, 65 women) with either OCS (n = 32) or MACE (n = 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations. RESULTS: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). CONCLUSION: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs.

Interpretation of Abnormal Dexamethasone Suppression Test is Enhanced With Use of Synchronous Free Cortisol Assessment.

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.