Ubiquitin-Specific Protease 8 Mutant Corticotrope Adenomas Present Unique Secretory and Molecular Features and Shed Light on the Role of Ubiquitylation on ACTH Processing.

BACKGROUND: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been shown to occur in ACTH-secreting pituitary adenomas, thus calling attention to the ubiquitin system in corticotrope adenomas. OBJECTIVES: Assess the consequences of USP8 mutations and establish the role of ubiquitin on ACTH turnover in human ACTH-secreting pituitary adenomas. METHODS: USP8 mutation status was established in 126 ACTH-secreting adenomas. Differences in ACTH secretion and POMC expression from adenoma primary cultures and in microarray gene expression profiles from archival specimens were sought according to USP8 sequence. Ubiquitin/ACTH coimmunoprecipitation and incubation with MG132, a proteasome inhibitor, were performed in order to establish whether ubiquitin plays a role in POMC/ACTH degradation in corticotrope adenomas. RESULTS: USP8 mutations were identified in 29 adenomas (23%). Adenomas presenting USP8 mutations secreted greater amounts of ACTH and expressed POMC at higher levels compared to USP wild-type specimens. USP8 mutant adenomas were also more sensitive to modulation by CRH and dexamethasone in vitro. At microarray analysis, genes associated with endosomal protein degradation and membrane components were downregulated in USP8 mutant adenomas as were AVPR1B, IL11RA, and PITX2. Inhibition of the ubiquitin-proteasome pathway increased ACTH secretion and POMC itself proved a target of ubiquitylation, independently of USP8 sequence status. CONCLUSIONS: Our study has shown that USP8 mutant ACTH-secreting adenomas present a more "typical" corticotrope phenotype and reduced expression of several genes associated with protein degradation. Further, ubiquitylation is directly involved in intracellular ACTH turnover, suggesting that the ubiquitin-proteasome system may represent a target for treatment of human ACTH-secreting adenomas.

Gene expression profiling in human corticotroph tumours reveals distinct, neuroendocrine profiles.

Adrenocorticotrophic hormone (ACTH)-secreting pituitary adenomas give rise to a severe endocrinological disorder, comprising Cushing's disease, with multifaceted clinical presentation and treatment outcomes. Experimental studies suggest that the disease variability is inherent to the pituitary tumour, thus indicating the need for further studies into tumour biology. The present study evaluated transcriptome expression pattern in a large series of ACTH-secreting pituitary adenoma specimens in order to identify molecular signatures of these tumours. Gene expression profiling of formalin-fixed, paraffin-embedded specimens from 40 human ACTH-secreting pituitary adenomas revealed the significant expression of genes involved in protein biosynthesis and ribosomal function, in keeping with the neuroendocrine cell profile. Unsupervised cluster analysis identified 3 distinct gene profile clusters and several genes were uniquely overexpressed in a given cluster, accounting for different molecular signatures. Of note, gene expression profiles were associated with clinical features, such as the age and size of the tumour. Altogether, the findings of the present study show that corticotroph tumours are characterised by a neuroendocrine gene expression profile and present subgroup-specific molecular features.

Low-dose Synachten test with measurement of salivary cortisol in adult patients with ß-thalassemia major.

PURPOSE: Beta-thalassemia major is a severe, congenital hematological disorder and, if untreated, leads to early mortality. Progress in therapeutical strategies improved clinical outcomes and life expectancy; however, increased survival led to the development of new disorders, including endocrinopathies. Little is known on the possible impairment of adrenocortical function, a potentially life-threatening condition, in long-term thalassaemic survivors. We therefore decided to assess adrenal reserve and the value of salivary cortisol during ACTH stimulation in the diagnosis of adrenocortical insufficiency in adult patients with ß-thalassemia major. METHODS: Cross-sectional study including 72 adults with ß-thalassemia major. Patients were tested with 1 µg ACTH for serum and salivary cortisol. RESULTS: Subnormal serum cortisol responses to ACTH stimulation (i.e., <500 nmol/l) were registered in 15 out of 72 patients. Salivary cortisol increased in parallel with serum cortisol and a clear-cut positive correlation was detected at each timepoint. Moreover, peak salivary cortisol values after ACTH stimulation were significantly lower in patients with impaired adrenal reserve (513.6 ± 52.33 vs. 914.1 ± 44.04 nmol/l p < 0.0001). CONCLUSIONS: Our results attest to the need for testing for adrenal insufficiency among adult thalassaemic patients, as up to 20% presented impaired adrenal reserve. Salivary and serum cortisol levels during stimulation with ACTH were closely correlated and the use of salivary cortisol sampling during ACTH testing may represent a surrogate to serum cortisol in these patients.

Pseudo-Cushing - A Clinical Challenge?

The distinction between Cushing's syndrome and pseudo-Cushing is a major clinical challenge. Indeed, any endocrinologist used to dealing with Cushing's syndrome has certainly faced this dilemma more than once and is aware that there are no clear-cut solutions. Several factors contribute to this ongoing quandary, such as unbalanced epidemiology, overlap in clinical features and inherent variability in test responses. Thus, extreme care has to be taken in both excluding and confirming Cushing's syndrome in patients with mild clinical features and borderline laboratory alterations.

Variability in laboratory parameters used for management of Cushing's syndrome.

The progress in assay methodology, from the use of radioactive tracers to chemiluminescent signals, from competitive to chromatographic techniques and from serum or urine to saliva has considerably impacted on hormonal measurements. The clinician now may choose among multiple tests but the inherent variability in cortisol and ACTH secretion, coupled to lack of harmonization among assay procedures and normal ranges mandates careful interpretation of any result. The present review will examine factors which affect interpretation of cortisol and ACTH measurements and their impact on tests used for management of Cushing's syndrome.

The hypertension of Cushing's syndrome: controversies in the pathophysiology and focus on cardiovascular complications.

Cushing's syndrome is associated with increased mortality, mainly due to cardiovascular complications, which are sustained by the common development of systemic arterial hypertension and metabolic syndrome, which partially persist after the disease remission. Cardiovascular diseases and hypertension associated with endogenous hypercortisolism reveal underexplored peculiarities. The use of exogenous corticosteroids also impacts on hypertension and cardiovascular system, especially after prolonged treatment. The mechanisms involved in the development of hypertension differ, whether glucocorticoid excess is acute or chronic, and the source endogenous or exogenous, introducing inconsistencies among published studies. The pleiotropic effects of glucocorticoids and the overlap of the several regulatory mechanisms controlling blood pressure suggest that a rigorous comparison of in-vivo and in-vitro studies is necessary to draw reliable conclusions. This review, developed during the first 'Altogether to Beat Cushing's syndrome' workshop held in Capri in 2012, evaluates the most important peculiarities of hypertension associated with CS, with a particular focus on its pathophysiology. A critical appraisal of most significant animal and human studies is compared with a systematic review of the few available clinical trials. A special attention is dedicated to the description of the clinical features and cardiovascular damage secondary to glucocorticoid excess. On the basis of the consensus reached during the workshop, a pathophysiology-oriented therapeutic algorithm has been developed and it could serve as a first attempt to rationalize the treatment of hypertension in Cushing's syndrome.

Von Willebrand factor and fibrinolytic parameters during the desmopressin test in patients with Cushing's disease.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Desmopressin is a known haemostatic agent and is also being used, albeit at lower doses, during the diagnostic work-up of Cushing's syndrome, a condition characterized by excess cortisol concentrations and frequent thromboembolic events. No study has yet evaluated whether administration of desmopressin for diagnostic purposes induces significant, adverse changes in endothelial cell markers in these patients. WHAT THIS STUDY ADDS: Administration of desmopressin to patients with Cushing's disease induces changes in endothelial cell markers comparable with those observed in obese and normal weight subjects. It follows, that desmopressin testing does not induce disease-specific untoward changes in coagulatory markers in patients with endogenous hypercortisolism and its use in this context appears safe. AIMS: Desmopressin, a vasopressin analogue, is used for various clinical purposes, including haemostasis and, in recent times, the diagnostic work-up of patients with Cushing's syndrome, a condition associated with a known prothrombotic profile. We decided to evaluate whether and to what extent a diagnostic dose of desmopressin induces significant changes in endothelial parameters in patients with Cushing's disease (CD) and obese and normal weight controls. METHODS: Twelve patients with CD, 10 obese and five normal weight controls were studied. Von Willebrand antigen (VWF:Ag), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) were measured at baseline and up to 4 h after 10 µg desmopressin i.v. RESULTS: Desmopressin 10 µg transiently increased VWF:Ag and t-PA and decreased PAI-1 in all subjects. The magnitude of the VWF:Ag and t-PA increases after desmopressin was comparable in the three groups (VWF:Ag peak-to-basal ratio 1.9 ± 0.17, 1.5 ± 0.11 and 1.8 ± 0.13 and t-PA peak-to-basal ratio 1.6 ± 0.18, 1.6 ± 0.20 and 1.8 ± 0.24 for CD, obese and controls, respectively, all NS). The PAI-1 decrease observed in patients with CD was comparable with obese (0.7 ± 0.07 and 0.6 ± 0.09, NS) and controls (0.7 ± 0.07 vs. 0.4 ± 0.09, P= 0.08). CONCLUSIONS: Administration of desmopressin to patients with CD for diagnostic purposes induces a transitory increase in VWF:Ag counterbalanced by a decrease in PAI-1 and increase in t-PA. The magnitude of these changes is largely comparable with that observed in obese and normal weight controls. Our data show that testing with desmopressin does not induce disease-specific changes in endothelial markers in patients with CD.

Specificity of first-line tests for the diagnosis of Cushing's syndrome: assessment in a large series.

CONTEXT: The diagnosis of Cushing's syndrome requires highly sensitive screening tests. Therefore, diagnostic cutoffs have been lowered to maximize sensitivity and identify all patients. However, few studies have investigated the impact of these refinements on the specificity of first-line tests. OBJECTIVE: The aim of the study was the assessment of the specificity of three widely used screening tests in a large series of Cushing's syndrome suspects referred to our endocrine service. PATIENTS: We retrospectively reviewed the results of urinary free cortisol (UFC), 1-mg dexamethasone suppression test [overnight suppression test (OST)], and serum cortisol at midnight in 3,461, 357, and 864 patients, respectively, with clinical features suggestive of Cushing's syndrome but in whom this diagnosis was subsequently excluded. RESULTS: UFC and OST at the 5-microg/dl cutoff exhibited the highest specificities [91% (95% confidence intervals [CI] 90.2-92.1%) and 97% (95% CI 96.3-98.5%), respectively]. Conversely, midnight serum cortisol yielded 87% (95% CI 84.3-91.1%) specificity only with the 7.5-microg/dl cutoff, whereas the 1.8-microg/dl threshold resulted in an unacceptably high proportion of false positives at only 20% specificity (95% CI 16.0-24.4%). Gender and age may lead to misleading results in all three screening tests. CONCLUSIONS: Specificity of tests for Cushing's syndrome varies considerably, with OST and UFC presenting the best performances, and circadian rhythm appearing heavily impaired by lowering of diagnostic cutoffs. Indeed, the vast majority of individuals in our series presented midnight serum cortisol values greater than 1.8 microg/dl; thus, caution has to be exercised when this criterion is used to exclude Cushing's syndrome.