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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.

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 β-thalassemia major. Methods Cross-sectional study including 72 adults with β-thalassemia major. Patients were tested with 1 µg ACTH for serum and salivary cortisol. Results Subnormal serum cortisol responses to ACTH stimulation (i.e.

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.

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.

Recurrence of Cushing’s disease after successful transsphenoidal surgery occurs in some 30% of the patients and the response to desmopressin shortly after surgery has been proposed as a marker for disease recurrence. The aim of the present study was to evaluate the response to desmopressin over time after surgery. We tested 56 patients with Cushing’s disease in remission after transsphenoidal surgery with desmopressin for up to 20 years after surgery. The ACTH and cortisol response to desmopressin over time was evaluated in patients on long-term remission or undergoing relapse; an increase by at least 27 pg/mL in ACTH levels identified responders. The vast majority of patients who underwent successful adenomectomy failed to respond to desmopressin after surgery and this response pattern was maintained over time in patients on long-term remission. Conversely, a response to desmopressin reappeared in patients who subsequently developed a recurrence of Cushing’s disease, even years prior to frank hypercortisolism. It appears therefore that a change in the response pattern to desmopressin proves predictive of recurrence of Cushing’s disease and may indicate which patients require close monitoring.

Objective Evidence is limited regarding outcome of patients with ectopic Cushing’s syndrome (ECS) due to neuroendocrine tumors (NETs). Design We assessed the prognostic factors affecting the survival of patients with NETs and ECS. Methods Retrospective analysis of clinicopathological features, severity of hormonal syndrome, treatments from a large cohort of patients with NETs and ECS collected from 17 Italian centers. Results Our series included 110 patients, 58.2% female, with mean (±s.d.) age at diagnosis of 49.5 ± 15.9 years. The main sources of ectopic ACTH were bronchial carcinoids (BC) (40.9%), occult tumors (22.7%) and pancreatic (p)NETs (15.5%). Curative surgery was performed in 56.7% (70.2% of BC, 11% of pNETs). Overall survival was significantly higher in BC compared with pNETs and occult tumors (P = 0.033) and in G1-NETs compared with G2 and G3 (P = 0.007). Negative predictive factors for survival were severity of hypercortisolism (P P = 0.001), diabetes mellitus (P = 0.0146) and distant metastases (P P Conclusions Multiple factors affect prognosis of ECS patients: type of NET, grading, distant metastases, severity of hypercortisolism, hypokalemia and diabetes mellitus. BCs have the highest curative surgical rate and better survival compared with occult tumors and pNETs. Hypercortisolism plays a primary role in affecting outcome and quality of life; therefore, prompt and vigorous treatment of hormonal excess by NET surgery and medical therapy should be a key therapeutic goal. In refractory cases, adrenalectomy should be considered as it affects outcome positively at least in the first 2 years.

ObjectiveGlucocorticoids stimulate several steps in red blood cell (RBC) development; however, little is known on changes in erythroid parameters in patients with Cushing's disease. The aim of this study was to assess both RBC and white blood cell (WBC) parameters in a large cohort of patients with Cushing's disease and report on alterations in the active phase and after surgical remission.Design and methodsA total of 80 patients with Cushing's disease (63 women and 17 men) were studied before and for up to 254 months' follow-up (mean follow-up 65.8±6.71 months) after pituitary/adrenal surgery. Details of blood counts were reviewed and compared with data obtained from a database of healthy subjects.ResultsThe RBC counts and haemoglobin levels were low in men with active Cushing's disease (over 80% of values in the lowest quartile) and four patients were overtly anaemic, whereas erythrocyte counts and haemoglobin levels were evenly distributed across the normal range in women with active Cushing's disease. Low erythroid parameters were linked to hypogonadism in men with Cushing's disease. Recovery in erythroid parameters occurred slowly after remission of hypercortisolism in men, in parallel with improvements in testosterone levels. Over 50% of patients with active disease presented increased WBC counts, irrespective of gender, and prompt normalisation within 1 month after surgery.ConclusionsMale patients with Cushing's disease present reduced RBC counts and haemoglobin levels, associated with low testosterone concentrations, which resolve over time after remission of hypercortisolism. Anaemia should therefore be regarded as another unfavourable feature in men with Cushing's disease.

Previous evidence supports a role for growth hormone (GH)-insulin-like growth factor (IGF)-I deficiency in the pathophysiology of osteopenia/osteoporosis in adult thalassemia. Moreover, serum IGF-II has never been studied in this clinical condition. Thus, we elected to study the GH secretory status and the levels of circulating somatomedins, correlating these parameters with bone mineral density (BMD) and biochemical markers of bone turnover. A hundred and thirty-nine normal weight adult thalassemic patients (72 men and 67 women) were studied. Lumbar and femoral neck BMD were measured in 106/139 patients. Sixty-eight patients underwent growth hormone releasing hormone plus arginine testing. Measurement of baseline IGF-I and IGF-II was performed in all patients, while osteocalcin, C-terminal telopeptide of type I collagen (CTx), and urinary cross-linked N-telopeptides of type I collagen (NTx) were assayed in 95 of them. Femoral and lumbar osteoporosis/Z score below the expected range for age were documented in 61.3 and in 56.6 % of patients, respectively. Severe GH deficiency (GHD) was demonstrated in 27.9 % of cases, whereas IGF-I SDS was low in 86.3 %. No thalassemic patients displayed circulating levels of IGF-II below the reference range. GH peaks were positively correlated with femoral, but not lumbar, Z score. No correlations were found between GH peaks and osteocalcin, CTx and NTx. GH peaks were positively correlated with IGF-I values, which in their turn displayed a positive correlation with osteocalcin, CTx, and NTx. No correlations emerged between IGF-I values and either femoral or lumbar Z scores. No correlations were found between IGF-II and any of the following parameters: GH peaks, osteocalcin, CTx, NTx, femoral Z score, and lumbar Z score. Our study, besides providing for the first time evidence of a normal IGF-II production in thalassemia, contributes to a better understanding of the involvement of the somatotropin-somatomedin axis in the pathophysiology of bone demineralization in this disease. In particular, the contribution of GHD to femoral osteoporosis appears to be likely mediated by locally produced rather than circulating IGF-I.

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.

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 admininistration 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.

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.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.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.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.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.

Cushing's disease, i.e. cortisol excess due to an ACTH-secreting pituitary adenoma, is a rare disorder with considerable morbidity and mortality but no satisfactory medical treatment as yet. Experimental data have recently shown that retinoic acid restrains ACTH secretion by tumoral corticotropes.Our objective was to evaluate the efficacy and safety profile of retinoic acid treatment in patients with Cushing's disease.This is a prospective, multicenter study. Seven patients with Cushing's disease (three men, four postmenopausal women) were started on 10 mg retinoic acid daily and dosage increased up to 80 mg daily for 6-12 months. ACTH, urinary free cortisol (UFC), and serum cortisol as well as clinical features of hypercortisolism and possible side effects of retinoic acid were evaluated at baseline, during retinoic acid administration, and after drug withdrawal.A marked decrease in UFC levels was observed in five patients; mean UFC levels on retinoic acid were 22-73% of baseline values and normalization in UFC was achieved in three patients. Plasma ACTH decreased in the first month of treatment and then returned to pretreatment levels in responsive patients whereas no clear-cut pattern could be detected for serum cortisol. Blood pressure, glycemia, and signs of hypercortisolism, e.g. body weight and facial plethora, were ameliorated to a variable extent on treatment. Patients reported only mild adverse effects, e.g. xerophthalmia and arthralgias.Long-term treatment with retinoic acid proved beneficial and well tolerated in five of seven patients with Cushing's disease. This represents a novel, promising approach to medical treatment in Cushing's disease.

ObjectiveWe previously described in young thalassaemic patients an altered cortisol and ACTH responsiveness suggesting an impaired adrenocortical reserve. Owing to iron overload, a worsening of adrenal function should be expected in adult patients.DesignIn 124 adults with β-thalassaemia, urinary free cortisol (UFC) and plasma ACTH levels were determined and compared with those measured in 150 controls. In 45 patients, cortisol was measured in response to: i) tetracosactide 1 μg as an i.v. bolus (low-dose test, LDT) and ii) tetracosactide 250 μg infused i.v. over 8 h (high-dose test, HDT).ResultsUFC and serum cortisol were within the reference range in all patients. Conversely, basal plasma ACTH values were above the upper limit of the normal range in 19 patients. There were no statistically significant differences in the mean values of UFC, basal serum cortisol and plasma ACTH between patients and controls. A subnormal cortisol response to the LDT was registered in 18 out of 56 patients. Three of these patients also displayed a subnormal response to the HDT, together with elevated baseline plasma ACTH levels. In the LDT, a positive correlation was found between basal and peak cortisol values (PPPConclusionsAdult thalassaemic patients often present a subtle impairment of adrenocortical function. This may become clinically relevant in case of major stressful events. Thus, we recommend an assessment of adrenocortical function in all adult thalassaemic patients.

This paper outlines the interferences of the most widely used drugs with hypothalamo-pituitary-adrenal function and the related laboratory parameters, with the purpose of providing practical help to clinicians during testing for hypo- or hypercortisolemic states.

Summary Objective Cushing's syndrome (CS), when fully expressed, is easily diagnosed. Mild cases, however, may require careful distinction from pseudo-Cushing's states as may occur in depression, alcoholism, polycystic ovary disease and visceral obesity. The aim of the present study is a reappraisal of the diagnostic accuracy of the two tests most commonly used to differentiate CS from pseudo-Cushing's: corticotropin-releasing hormone (CRH) stimulation after low dose dexamethasone administration and desmopressin stimulation. Design The study population comprised 32 patients with CS and 23 with pseudo-Cushing's evaluated retrospectively. Methods Urinary free cortisol (UFC), serum cortisol at midnight and after low dose dexamethasone (1 mg overnight and 2 mg over two days) were measured. Further, patients were tested with dexamethasone + CRH and desmopressin and the diagnostic performances of the two tests were compared in the entire series as well as in patients with mild hypercortisolism only (i.e. UFC

Despite recent advances in the management of endogenous Cushing’s syndrome (CS), its treatment remains a challenge. When surgery has been unsuccessful or unfeasible as well in case of recurrence, the “old” pharmacological agents represent an important alternative for both ACTH-dependent and independent hypercortisolism. Especially in the latter, the advent of novel molecules directly targeting ACTH secretion has not outweighed the “old” drugs, which continue to be largely employed and have recently undergone a reappraisal. This review provides a survey of the “old” pharmacological agents in the treatment of CS.