Delineating endogenous Cushing's syndrome by GC-MS urinary steroid metabotyping.

BACKGROUND: Diagnosing Cushing's syndrome (CS) is highly complex. As the diagnostic potential of urinary steroid metabolome analysis by gas chromatography-mass spectrometry (GC-MS) in combination with systems biology has not yet been fully exploited, we studied a large cohort of patients with CS. METHODS: We quantified daily urinary excretion rates of 36 steroid hormone metabolites. Applying cluster analysis, we investigated a control group and 168 patients: 44 with Cushing's disease (CD) (70% female), 18 with unilateral cortisol-producing adrenal adenoma (83% female), 13 with primary bilateral macronodular adrenal hyperplasia (PBMAH) (77% female), and 93 ruled-out CS (73% female). FINDINGS: Cluster-Analysis delineated five urinary steroid metabotypes in CS. Metabotypes 1, 2 and 3 revealing average levels of cortisol and adrenal androgen metabolites included patients with exclusion of CS or and healthy controls. Metabotype 4 reflecting moderately elevated cortisol metabolites but decreased DHEA metabolites characterized the patients with unilateral adrenal CS and PBMAH. Metabotype 5 showing strong increases both in cortisol and DHEA metabolites, as well as overloaded enzymes of cortisol inactivation, was characteristic of CD patients. 11-oxygenated androgens were elevated in all patients with CS. The biomarkers THS, F, THF/THE, and (An + Et)/(11ß-OH-An + 11ß-OH-Et) correctly classified 97% of patients with CS and 95% of those without CS. An inverse relationship between 11-deoxygenated and 11-oxygenated androgens was typical for the ACTH independent (adrenal) forms of CS with an accuracy of 95%. INTERPRETATION: GC-MS based urinary steroid metabotyping allows excellent identification of patients with endogenous CS and differentiation of its subtypes. FUNDING: The study was funded by the Else Kröner-Fresenius-Stiftung and the Eva-Luise-und-Horst-Köhler-Stiftung.

PPARG dysregulation as a potential molecular target in adrenal Cushing's syndrome.

Background: We performed a transcriptomic analysis of adrenal signaling pathways in various forms of endogenous Cushing's syndrome (CS) to define areas of dysregulated and druggable targets. Methodology: Next-generation sequencing was performed on adrenal samples of patients with primary bilateral macronodular adrenal hyperplasia (PBMAH, n=10) and control adrenal samples (n=8). The validation groups included cortisol-producing adenoma (CPA, n=9) and samples from patients undergoing bilateral adrenalectomy for Cushing's disease (BADX-CD, n=8). In vivo findings were further characterized using three adrenocortical cell-lines (NCI-H295R, CU-ACC2, MUC1). Results: Pathway mapping based on significant expression patterns identified PPARG (peroxisome proliferator-activated receptor gamma) pathway as the top hit. Quantitative PCR (QPCR) confirmed that PPARG (l2fc<-1.5) and related genes - FABP4 (l2fc<-5.5), PLIN1 (l2fc<-4.1) and ADIPOQ (l2fc<-3.3) - were significantly downregulated (p<0.005) in PBMAH. Significant downregulation of PPARG was also found in BADX-CD (l2fc<-1.9, p<0.0001) and CPA (l2fc<-1.4, p<0.0001). In vitro studies demonstrated that the PPARG activator rosiglitazone resulted in decreased cell viability in MUC1 and NCI-H295R (p<0.0001). There was also a significant reduction in the production of aldosterone, cortisol, and cortisone in NCI-H295R and in Dihydrotestosterone (DHT) in MUC1 (p<0.05), respectively. Outcome: This therapeutic effect was independent of the actions of ACTH, postulating a promising application of PPARG activation in endogenous hypercortisolism.

Steroid profiling using liquid chromatography mass spectrometry during adrenal vein sampling in patients with primary bilateral macronodular adrenocortical hyperplasia.

Introduction: Adrenal vein sampling (AVS) is not a routine procedure in patients with primary bilateral macronodular adrenocortical hyperplasia (PBMAH), but has been used to determine lateralization of cortisol secretion in order to guide decision of unilateral adrenalectomy. Our aim was to characterize the steroid fingerprints in AVS samples of patients with PBMAH and hypercortisolism and to identify a reference hormone for AVS interpretation. Method: Retrospectively, we included 17 patients with PBMAH from the German Cushing's registry who underwent AVS. 15 steroids were quantified in AVS and peripheral blood samples using LC-MS/MS. We calculated lateralization indices and conversion ratios indicative of steroidogenic enzyme activity to elucidate differences between individual adrenal steroidomes and in steroidogenic pathways. Results: Adrenal volume was negatively correlated with peripheral cortisone (r=0.62, p<0.05). 24-hour urinary free cortisol correlated positively with peripheral androgens (rDHEA=0.57, rDHEAS=0.82, rA=0.73, rT=0.54, p<0.05). DHEA was found to be a powerful reference hormone with high selectivity index, which did not correlate with serume cortisol and has a short half-life. All investigated steroids showed lateralization in single patients indicating the heterogenous steroid secretion pattern in patients with PBMAH. The ratios of corticosterone/aldosterone (catalyzed by CYP11B2), androstenedione/dehydroepiandrosterone (catalyzed by HSD3B2) and cortisone/cortisol (catalyzed by HSD11B2) in adrenal vein samples were higher in smaller adrenals (p<0.05). ARMC5 mutation carriers (n=6) showed lower androstenedione/17-hydroxyprogesterone and higher testosterone/androstenedione (p<0.05) ratios in peripheral blood, in line with lower peripheral androstenedione concentrations (p<0.05). Conclusion: Steroid profiling by LC-MS/MS led us to select DHEA as a candidate reference hormone for cortisol secretion. Lateralization and different steroid ratios showed that each steroid and all three steroidogenic pathways may be affected in PBMAH patients. In patients with germline ARMC5 mutations, the androgen pathway was particularly dysregulated.

Characterization of Adrenal miRNA-Based Dysregulations in Cushing's Syndrome.

MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing's syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing's disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes.

Chemosensory dysfunction in Cushing's syndrome.

PURPOSE: Cushing's syndrome (CS) can lead to structural changes in the brain and cognitive impairment, but chemosensory function has not been investigated yet. The aim was to analyze sense of smell and taste in patients with CS and explore the effect of therapy. METHODS: The study cohort comprised 20 patients with florid CS treated between 2018 and 2020 in the outpatient clinic of the LMU Munich. We compared these 20 patients with CS to 40 healthy subjects matched for age, sex, and smoking status. Patients' sense of smell and taste was examined at diagnosis and 3 months after successful therapeutic surgery leading to clinical and biochemical remission. Odor threshold, discrimination, and identification were measured with "Sniffin' Sticks", taste was measured with "Taste Strips". Perceived sense of smell and taste was retrieved via a questionnaire. RESULTS: Patients with florid CS had significantly reduced smell (total smell score 30.3 vs. 34.4, p < 0.0005) and taste scores (9.5 vs. 12.0, p < 0.0005) compared to controls and significantly more frequently hyposmia (55 vs. 2.5%, p < 0.0005), hypogeusia (40 vs. 0%, p < 0.0005), and self-reported chemosensory impairment (60 vs. 0%, p < 0.0005). Three months after successful surgery, CS patients showed significant improvement of odor threshold (8.1 vs. 7.0, p < 0.0005), odor discrimination (12.0 vs. 11.0, p = 0.003), total smell score (33.4 vs. 30.3, p < 0.0005), and taste (11.5 vs. 9.5, p = 0.003). CONCLUSIONS: Chemosensory dysfunction is a novel and clinically relevant feature of CS.

Circulating microRNA Expression in Cushing's Syndrome.

Context: Cushing's syndrome (CS) is a rare disease of endogenous hypercortisolism associated with high morbidity and mortality. Diagnosis and classification of CS is still challenging. Objective: Circulating microRNAs (miRNAs) are minimally invasive diagnostic markers. Our aim was to characterize the circulating miRNA profiles of CS patients and to identify distinct profiles between the two major CS subtypes. Methods: We included three groups of patients from the German Cushing's registry: ACTH-independent CS (Cortisol-Producing-Adenoma; CPA), ACTH-dependent pituitary CS (Cushing's Disease; CD), and patients in whom CS had been ruled out (controls). Profiling of miRNAs was performed by next-generation-sequencing (NGS) in serum samples of 15 CS patients (each before and after curative surgery) and 10 controls. Significant miRNAs were first validated by qPCR in the discovery cohort and then in an independent validation cohort of 20 CS patients and 11 controls. Results: NGS identified 411 circulating miRNAs. Differential expression of 14 miRNAs were found in the pre- and postoperative groups. qPCR in the discovery cohort validated 5 of the significant miRNAs from the preoperative group analyses. Only, miR-182-5p was found to be significantly upregulated in the CD group of the validation cohort. Comparing all CS samples as a group with the controls did not reveal any significant differences in expression. Outcome: In conclusion, our study identified miR-182-5p as a possible biomarker for CD, which has to be validated in a prospective cohort. Furthermore, our results suggest that presence or absence of ACTH might be at least as relevant for miRNA expression as hypercortisolism itself.

RNA Sequencing and Somatic Mutation Status of Adrenocortical Tumors: Novel Pathogenetic Insights.

CONTEXT: Pathogenesis of autonomous steroid secretion and adrenocortical tumorigenesis remains partially obscure. OBJECTIVE: To investigate the relationship between transcriptome profile and genetic background in a large series of adrenocortical tumors and identify new potential pathogenetic mechanisms. DESIGN: Cross-sectional study. SETTING: University Hospitals of the European Network for the Study of Adrenal Tumors (ENSAT). PATIENTS: We collected snap-frozen tissue from patients with adrenocortical tumors (n = 59) with known genetic background: 26 adenomas with Cushing syndrome (CS- cortisol-producing adenoma [CPA]), 17 adenomas with mild autonomous cortisol secretion (MACS-CPAs), 9 endocrine-inactive adenomas (EIAs), and 7 adrenocortical carcinomas (ACCs). INTERVENTION: Ribonucleic acid (RNA) sequencing. MAIN OUTCOME MEASURES: Gene expression, long noncoding RNA (lncRNA) expression, and gene fusions. Correlation with genetic background defined by targeted Sanger sequencing, targeted panel- or whole-exome sequencing. RESULTS: Transcriptome analysis identified 2 major clusters for adenomas: Cluster 1 (n = 32) mainly consisting of MACS-CPAs with CTNNB1 or without identified driver mutations (46.9% of cases) and 8/9 EIAs; Cluster 2 (n = 18) that comprised CP-CPAs with or without identified driver mutation in 83.3% of cases (including all CS-CPAs with PRKACA mutation). Two CS-CPAs, 1 with CTNNB1 and 1 with GNAS mutation, clustered separately and relatively close to ACC. lncRNA analysis well differentiate adenomas from ACCs. Novel gene fusions were found, including AKAP13-PDE8A in one CS-CPA sample with no driver mutation. CONCLUSIONS: MACS-CPAs and EIAs showed a similar transcriptome profile, independently of the genetic background, whereas most CS-CPAs clustered together. Still unrevealed molecular alterations in the cAMP/PKA or Wnt/beta catenin pathways might be involved in the pathogenesis of adrenocortical tumors.

Therapeutic options after surgical failure in Cushing's disease: A critical review.

Cushing's disease (CD) is the most common etiology of Cushing's syndrome (CD) due to corticotroph pituitary adenoma, which are in most cases small (80-90% microadenomas) and in about 40% cannot be visualized on imaging of the sella. First-line treatment for CD is transsphenoidal surgery (TSS) with the aim of complete adenoma removal and preservation of pituitary gland function. As complete adenoma resection is not always possible, surgical failure is a common problem. This can be the case either due to persistent hypercortisolism after first TSS or recurrence of hypercortisolism after initially achieving remission. For these scenarios exist several therapeutic options with their inherent characteristics, which will be covered by this review.

Long-Term Outcome of Primary Bilateral Macronodular Adrenocortical Hyperplasia After Unilateral Adrenalectomy.

CONTEXT: Unilateral adrenalectomy has been proposed in selected patients with primary bilateral macronodular adrenocortical hyperplasia (PBMAH), but its long-term outcome is unclear. OBJECTIVE: The aim of this study was to analyze long-term clinical and biochemical outcomes of unilateral adrenalectomy vs bilateral adrenalectomy in patients with PBMAH in comparison with the outcome of cortisol-producing adenoma (CPA) treated with unilateral adrenalectomy. DESIGN: Retrospective observational study in three German and one Italian academic tertiary care center. PATIENTS AND METHODS: Twenty-five patients with PBMAH after unilateral adrenalectomy (unilat-ADX-PBMAH), nine patients with PBMAH and bilateral adrenalectomy (bilat-ADX-PBMAH), and 39 patients with CPA and unilateral adrenalectomy (unilat-ADX-CPA) were included. RESULTS: Baseline clinical and biochemical parameters were comparable in patients with unilat-ADX-PBMAH, bilat-ADX-PBMAH, and unilat-ADX-CPA. Directly after surgery, 84% of the patients with unilat-ADX-PBMAH experienced initial remission of Cushing syndrome (CS). In contrast, at last follow-up (median, 50 months), 32% of the patients with unilat-ADX-PBMAH were biochemically controlled compared with nearly all patients in the other two groups (P = 0.000). Adrenalectomy of the contralateral side had to be performed in 12% of the initial patients with unilat-ADX-PBMAH. Three of 20 patients with unilat-ADX-PBMAH (15%) died during follow-up, presumably of CS-related causes; no deaths occurred in the other two groups (P = 0.008). Deaths occurred exclusively in patients who were not biochemically controlled after unilateral ADX. CONCLUSIONS: Our data suggest that unilateral adrenalectomy of patients with PBMAH leads to clinical remission and a lower incidence of adrenal crisis but in less sufficient biochemical control of hypercortisolism, potentially leading to higher mortality.

Surviving ectopic Cushing's syndrome: quality of life, cardiovascular and metabolic outcomes in comparison to Cushing's disease during long-term follow-up.

OBJECTIVE: Aim of our study was to analyze long-term outcome of patients with the ectopic Cushing's syndrome (ECS) compared to patients with Cushing's disease (CD) regarding cardiovascular, metabolic, musculoskeletal and psychiatric comorbidities. DESIGN: Cross-sectional study in patients with ECS and CD in two German academic tertiary care centers. METHODS: Standardized clinical follow-up examination was performed including health-related quality of life (QoL) in 21 ECS patients in long-term remission (≥18 months since successful surgery). Fifty-nine patients with CD in remission served as controls. RESULTS: Time from first symptoms to diagnosis of Cushing's syndrome (CS) was shorter in ECS than in CD (8.5 (IQR: 30.3) vs 25 (IQR: 39.0) months, P = 0.050). ECS patients had lower self-reported psychiatric morbidity compared to CD (19% vs 43%, P = 0.050) at follow-up. Moreover, female ECS patients reported favorable scores for QoL in the SF-36 questionnaire (mental health: 92 (IQR: 30) vs 64 (IQR: 32) in CD, P = 0.010) and a Cushing-specific QoL questionnaire (73 (IQR: 18) vs 59 (IQR: 36) in CD, P = 0.030). In a pooled analysis of ECS and CD patients, QoL correlated with time from first symptoms until diagnosis of CS, but not with urinary free cortisol levels or serum cortisol after dexamethasone at the time of diagnosis. Long-term outcomes regarding hypertension, metabolic parameters, bone mineral density and grip strength were comparable in ECS and CD. CONCLUSIONS: Our data support the concept that time of exposure to glucocorticoid excess appears to be a better predictor than peak serum cortisol levels at the time of diagnosis regarding long-term psychiatric morbidity and QoL.

Cushing's syndrome: a model for sarcopenic obesity.

PURPOSE: Obesity and its metabolic impairments are discussed as major risk factors for sarcopenia leading to sarcopenic obesity. Cushing's syndrome is known to be associated with obesity and muscle atrophy. We compared Cushing's syndrome with matched obese controls regarding body composition, physical performance, and biochemical markers to test the hypothesis that Cushing's syndrome could be a model for sarcopenic obesity. METHODS: By propensity score matching, 47 controls were selected by body mass index and gender as obese controls. Fat mass and muscle mass were measured by bioelectrical impedance analysis. Muscle function was assessed by chair rising test and hand grip strength. Biochemical markers of glucose and lipid metabolism and inflammation (hsCRP) were measured in peripheral blood. RESULTS: Muscle mass did not differ between Cushing's syndrome and obese controls. However, Cushing's syndrome patients showed significantly greater chair rising time (9.5 s vs. 7.3 s, p = 0.008) and significantly lower hand grip strength (32.1 kg vs. 36.8 kg, p = 0.003). Cushing's syndrome patients with impaired fasting glucose have shown the highest limitations in hand grip strength and chair rising time. CONCLUSIONS: Similar to published data in ageing medicine, Cushing's syndrome patients show loss of muscle function that cannot be explained by loss of muscle mass. Impaired muscle quality due to fat infiltration may be the reason. This is supported by the observation that Cushing's syndrome patients with impaired glucose metabolism show strongest deterioration of muscle function. Research in sarcopenic obesity in elderly is hampered by confounding comorbidities and polypharmacy. As Cushing's syndrome patients are frequently free of comorbidities and as Cushing's syndrome is potentially curable we suggest Cushing's syndrome as a clinical model for further research in sarcopenic obesity.

Persistence of myopathy in Cushing's syndrome: evaluation of the German Cushing's Registry.

BACKGROUND: Cushing's syndrome (CS) is characterized by an excessive secretion of glucocorticoids that results in a characteristic clinical phenotype. One feature of clinical hypercortisolism is breakdown of protein metabolism translating into clinical consequences including glucocorticoid-induced myopathy. While surgery is effective in control of cortisol excess, the effect of biochemical remission on muscular function is yet unclear. METHODS: In a cross-sectional study we analyzed 47 patients with CS during the florid phase (ActiveCS). 149 additional patients were studied 2-53 years (mean: 13 years) after surgery in biochemical long-term remission (RemissionCS). Also, 93 rule-out CS patients were used as controls (CON). All subjects were assessed for grip strength using a hand grip dynamometer and underwent the chair rising test (CRT). RESULTS: Hand grip strength (85% vs 97% of norm, P = 0.002) and the CRT performance (9.5 s vs 7.1 s, P = 0.001) were significantly lower in ActiveCS compared to the CON group. Six months after treatment grip strength further decreased in CS (P = 0.002) and CRT performance remained impaired. The RemissionCS group (mean follow-up 13 years) had reduced hand grip strength (92% compared to normal reference values for dominant hand, P < 0.001). The chair rising test performance was at 9.0 s and not significantly different from the ActiveCS group (P = 0.45). CONCLUSION: CS affects muscle strength in the acute phase, but functional impairment remains detectable also during long-term follow-up despite biochemical remission.

Volumetric and densitometric evaluation of the adrenal glands in patients with primary aldosteronism.

OBJECTIVE: To evaluate volumetric and densitometric properties of the adrenal glands in patients with unilateral and bilateral disease in comparison with normal controls. DESIGN: A total of 77 patients (56 males and 21 females) diagnosed with primary aldosteronism (PA) with a mean age of 53 ± 10 years were prospectively enrolled. Unenhanced and contrast-enhanced computed tomography scans were analysed for adrenal volumes and mean densities. These values were compared with normal controls and between PA subtypes. RESULTS: Adrenals containing an aldosterone-producing adenoma (APA, n = 56) had on average higher attenuation values as compared to adrenals with bilateral adrenal hyperplasia (n = 21). Mean adrenal gland volume in PA patients was not significantly different between PA subtypes. In comparison with normal adrenal glands, volumes were significantly higher in PA patients (P < 0·0001) including adrenals contralateral to APAs, which were significantly larger in comparison with controls. CONCLUSION: Independent of subtype differentiation, adrenal volumetry reveals higher adrenal volumes in PA patients in comparison with normal controls. These findings provide indirect evidence for a general adrenal growth dysregulation in the context of PA.

A critical reappraisal of bilateral adrenalectomy for ACTH-dependent Cushing's syndrome.

OBJECTIVE: Our aim was to review short- and long-term outcomes of patients treated with bilateral adrenalectomy (BADx) in ACTH-dependent Cushing's syndrome. METHODS: We reviewed the literature and analysed our experience with 53 patients treated with BADx since 1990 in our institution. RESULTS: BADx is considered if ACTH-dependent Cushing's syndrome is refractory to other treatment modalities. In Cushing's disease (CD), BADx is mainly used as an ultima ratio after transsphenoidal surgery and medical therapies have failed. In these cases, the time span between the first diagnosis of CD and treatment with BADx is relatively long (median 44 months). In ectopic Cushing's syndrome, the time from diagnosis to BADx is shorter (median 2 months), and BADx is often performed as an emergency procedure because of life-threatening complications of severe hypercortisolism. In both situations, BADx is relatively safe (median surgical morbidity 15%; median surgical mortality 3%) and provides excellent control of hypercortisolism; Cushing's-associated signs and symptoms are rapidly corrected, and co-morbidities are stabilised. In CD, the quality of life following BADx is rapidly improving, and long-term mortality is low. Specific long-term complications include the development of adrenal crisis and Nelson's syndrome. In ectopic Cushing's syndrome, long-term mortality is high but is mostly dependent on the prognosis of the underlying malignant neuroendocrine tumour. CONCLUSION: BADx is a relatively safe and highly effective treatment, and it provides adequate control of long-term co-morbidities associated with hypercortisolism.

Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resection.

BACKGROUND: Recurrence of adrenocortical carcinoma (ACC) even after complete (R0) resection occurs frequently. OBJECTIVE: The aim of this study was to identify markers with prognostic value for patients in this clinical setting. DESIGN, SETTING, AND PARTICIPANTS: From the German ACC registry, 319 patients with the European Network for the Study of Adrenal Tumors stage I-III were identified. As an independent validation cohort, 250 patients from three European countries were included. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Clinical, histological, and immunohistochemical markers were correlated with recurrence-free (RFS) and overall survival (OS). RESULTS: Although univariable analysis within the German cohort suggested several factors with potential prognostic power, upon multivariable adjustment only a few including age, tumor size, venous tumor thrombus (VTT), and the proliferation marker Ki67 retained significance. Among these, Ki67 provided the single best prognostic value for RFS (hazard ratio [HR] for recurrence, 1.042 per 1% increase; P < .0001) and OS (HR for death, 1.051; P < .0001) which was confirmed in the validation cohort. Accordingly, clinical outcome differed significantly between patients with Ki67 <10%, 10-19%, and ≥20% (for the German cohort: median RFS, 53.2 vs 31.6 vs 9.4 mo; median OS, 180.5 vs 113.5 vs 42.0 mo). Using the combined cohort prognostic scores including tumor size, VTT, and Ki67 were established. Although these scores discriminated slightly better between subgroups, there was no clinically meaningful advantage in comparison with Ki67 alone. CONCLUSION: This largest study on prognostic markers in localized ACC identified Ki67 as the single most important factor predicting recurrence in patients following R0 resection. Thus, evaluation of Ki67 indices should be introduced as standard grading in all pathology reports of patients with ACC.

Mutations in the deubiquitinase gene USP8 cause Cushing's disease.

Cushing's disease is caused by corticotroph adenomas of the pituitary. To explore the molecular mechanisms of endocrine autonomy in these tumors, we performed exome sequencing of 10 corticotroph adenomas. We found somatic mutations in the USP8 deubiquitinase gene in 4 of 10 adenomas. The mutations clustered in the 14-3-3 protein binding motif and enhanced the proteolytic cleavage and catalytic activity of USP8. Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling. USP8 mutants enhanced promoter activity of the gene encoding proopiomelanocortin. In summary, our data show that dominant mutations in USP8 cause Cushing's disease via activation of EGF receptor signaling.

Time to recovery of adrenal function after curative surgery for Cushing's syndrome depends on etiology.

CONTEXT: Successful tumor resection in endogenous Cushing's syndrome (CS) results in tertiary adrenal insufficiency requiring hydrocortisone replacement therapy. OBJECTIVE: The aim was to analyze the postsurgical duration of adrenal insufficiency of patients with Cushing's disease (CD), adrenal CS, and ectopic CS. DESIGN: We performed a retrospective analysis based on the case records of 230 patients with CS in our tertiary referral center treated from 1983-2014. The mean follow-up time was 8 years. PATIENTS: We included 91 patients of the three subtypes of CS undergoing curative intended surgery and documented followup after excluding cases with persistent disease, pituitary radiation, concurrent adrenostatic or somatostatin analog treatment, and malignant adrenal disease. RESULTS: The probability of recovering adrenal function within a 5 years followup differed significantly between subtypes (P = .001). It was 82% in ectopic CS, 58% in CD and 38% in adrenal CS. In the total cohort with restored adrenal function (n = 52) the median time to recovery differed between subtypes: 0.6 years (interquartile range [IQR], 0.03-1.1 y) in ectopic CS, 1.4 years (IQR, 0.9-3.4 y) in CD, and 2.5 years (IQR, 1.6-5.4 y) in adrenal CS (P = .002). In CD the Cox proportional-hazards model showed that the probability of recovery was associated with younger age (hazard ratio, 0.896; 95% confidence interval, 0.822-0.976; P = .012), independently of sex, body mass index, duration of symptoms, and basal ACTH and cortisol levels. There was no correlation with length and extend of hypercortisolism or postoperative glucocorticoid replacement doses. CONCLUSIONS: Time to recovery of adrenal function is dependent on the underlying etiology of CS.

Favorable long-term outcomes of bilateral adrenalectomy in Cushing's disease.

OBJECTIVE: Bilateral adrenalectomy (BADX) is an important treatment option for patients with Cushing's syndrome (CS). Our aim is to analyze the long-term outcomes, surgical, biochemical, and clinical as well as morbidity and mortality, of patients who underwent BADX. DESIGN: A total of 50 patients who underwent BADX since 1990 in two German centers were identified. Of them, 34 patients had Cushing's disease (CD), nine ectopic CS (ECS), and seven ACTH-independent bilateral adrenal hyperplasia (BAH). METHODS: Standardized follow-up examination was performed in 36 patients with a minimum follow-up time of 6 months after BADX and a median follow-up time of 11 years. RESULTS: Surgical morbidity and mortality were 6 and 4% respectively. All patients were found to be in remission after BADX. Almost all Cushing's-specific comorbidities except for psychiatric diseases improved significantly. Health-related quality of life remained impaired in 45.0% of female and 16.7% of male patients compared with a healthy population. The median number of adrenal crises per 100 patient-years was four. Nelson tumor occurred in 24% of CD patients after a median time span of 51 months. Long-term mortality after 10 years was high in ECS (44%) compared with CD (3%) and BAH (14%). CONCLUSIONS: BADX is an effective and relatively safe treatment option especially in patients with CD. The majority of patients experience considerable improvement of Cushing's symptoms.

Constitutive activation of PKA catalytic subunit in adrenal Cushing's syndrome.

BACKGROUND: Corticotropin-independent Cushing's syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood. METHODS: We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro. RESULTS: Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing's syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased. CONCLUSIONS: Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).