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This is the first study examining the impact of both clinical, biochemical, and genetic determinants in the occurrence of bone complications in patients with overt Cushing’s syndrome (CS). It demonstrates that the degree and duration of hypercortisolism seem to play a major role in bone loss and fractures development in these patients. Bone loss and fractures are a common complication of CS. We investigate the role of gender, disease etiology, duration, and degree of hypercortisolism as well as the impact of glucocorticoid receptor (GR) polymorphisms on the development of bone complications in CS. Fifty-two patients with active CS (38 Cushing’s disease and 14 with cortisol-secreting adrenal adenoma) were genotyped for GR polymorphisms (BclI, N363S, ER22/23EK, and A3669G). In all patients, clinical, hormonal, and biochemical markers of bone turnover, densitometric parameters [lumbar spine and left femur bone mineral density (BMD), T-score, Z-score] as well as the prevalence of bone demineralization and both vertebral and peripheral fractures were assessed. No differences were found in bone complications according to gender, disease etiology, and genetic variants distribution. Fractured patients compared to non-fractured ones showed increased levels of urinary free cortisol (UFC) and a more compromised densitometric profile. UFC levels correlated with the occurrence of vertebral fractures (r = 0.43, p = 0.009) while midnight serum cortisol correlated with L1–L4 BMD values (r = −0.35, p = 0.04). Disease duration correlated with the presence of peripheral fractures (r = 0.36, p = 0.04). While GR gene variants as well as gender and disease etiology seem not to play a role, the degree and duration of hypercortisolism seem to be the major determinants of bone loss and fractures in this group of patients. More investigations are needed to understand the real impact of these determinants on the development of bone complications in patients with hypercortisolism.

A hypercoagulable state and its consequent increased incidence of thromboembolic complications are reported in patients with Cushing’s syndrome (CS). These alterations are related to cortisol excess that induces prothrombotic changes in blood by several and complex mechanisms including increased levels of clotting factors, mainly factor VIII and von Willebrand factor (VWF) and impaired fibrinolytic capacity. However, it has recently been observed that the increase in VWF levels is not a constant feature of CS and that VWF response to glucocorticoids is genetically determined and depends on the presence of particular polymorphisms in the VWF gene promoter. The risk of venous thromboembolism is moreover enhanced in patients with CS by additional endogenous and exogenous risk factors such as obesity, bed rest, surgery and invasive diagnostic procedures like inferior petrosal sinus (IPS) sampling. In line with all these data, patients with active CS should be treated as having a prothrombotic disorder and undergo antithrombotic prophylaxis during IPS sampling. Special care should be taken in the immediate perioperative period in order to avoid thromboembolic events. In the absence of prospective randomized trials, preventive antithrombotic treatment (best with heparin) during IPS sampling and low-dose heparin treatment early after surgery should be suggested.

Summary Objective Several splice variants (SVs) of GHRH receptor (GHRH-R) have been identified in various human cancers through which GHRH antagonists may exert their IGF-II-mediated antiproliferative action. Because the overexpression of the IGF-II gene is a frequent feature of adrenal carcinoma, we searched for the presence of GHRH-R SVs in these tumours. Methods and Results The expression of GHRH-R SVs was assessed by nested PCR in 45 human adrenocortical tumours. We have amplified 720-, 566- and 335-bp PCR products only in carcinomas. Their sequence revealed three open reading frames, corresponding to SV1, SV2 and SV4 of GHRH-R. SV2 was detected in five of 24 cancers examined, whereas the incidence of SV1 and SV4 was lower. Their simultaneous expression was observed in one carcinoma. No PCR products for SV3 or wild-type GHRH-R were found in carcinomas; mRNA for wild-type GHRH-R or SVs of GHRH-R were not observed either in adenomas or in normal adrenal or in NCI-H295R cells. Interestingly, all carcinomas which expressed SVs were also positive for the presence of GHRH mRNA. Conclusion This is the first time that the expression of splice variants of GHRH-R has been demonstrated in human adrenal carcinoma. This study raises the possibility that splice variants might play a role in adrenal carcinogenesis and might offer the possibility for new therapeutic strategies at least in a subgroup of adrenal carcinomas.

The Cancer Stem Cells (CSCs) theory suggests that genetic alterations in stem cells are the direct cause for cancer. The evidence for a CSC population that results in pituitary tumors is poor. Some studies report the isolation of CSCs, but a deep characterization of the stemness of these cells is lacking. Here, we report the isolation and detailed characterization of progenitor mesenchymal cells (PMCs) from both growth hormone-secreting (GH(+)) and non-secreting (NS) pituitary adenomas, determining the immunophenotype, the expression of genes related to stemness or to pituitary hormone cell types, and the differentiative potential towards osteo-, chondro- and adipogenic lineages. Finally, the expression of CD133, known as a marker for CSCs in other tumors, was analyzed. Isolated cells, both from GH(+) and NS tumors, satisfy all the criteria for the identification of PMCs and express known stem cell markers (OCT4, SOX2, KLF4, NANOG), but do not express markers of pituitary hormone cell types (PITX2, PROP1, PIT1). Finally, PMCs express CD133. We demonstrated that pituitary tumors contain a stem cell population that can generate cell types characteristic of mesenchymal stem cells, and express CD133, which is associated with CSCs in other tumors.

ObjectiveGlucocorticoid receptor (GR) polymorphisms alter glucocorticoid (GC) sensitivity and have been associated with altered metabolic profiles. We evaluate the prevalence of the fourGR(NR3C1) polymorphisms BclI, N363S, ER22/23EK, and A3669G in patients with Cushing's syndrome (CS) compared with healthy controls (HC) and we investigate their role in the development of metabolic abnormalities in patients with CS according to their hormonal profile.Patients and methodsSixty-one patients with CS and 71 sex- and age-matched HC were genotyped.ResultsBclI variant was markedly higher in patients with CS compared with HC (62 vs 41%,PIn CS patients, despite the significantly increased levels of morning serum cortisol in BclI carriers compared with wild type no clinical or metabolic differences were found.In contrast, A3669GGRcarriers showed a significantly reduced prevalence of type 2 diabetes mellitus compared with wild type (19 vs 68%,P=0.001) despite the higher levels of both serum morning (21.7±6 vs 27.3±8.6 μg/dl,P=0.009) and midnight cortisol (18.8±5.8 vs 24.0±8.0 μg/dl,P=0.01). The negative association between diabetes and A3669GGRpolymorphism remained significant when data were adjusted for potential confounding factors.ConclusionsThe A3669G polymorphism of theGRgene plays a protective role in patients with CS, attenuating the effects of GC excess on glucose metabolism as shown by their reduced risk of diabetes.

Several lines of evidence suggest a detrimental effect of aldosterone excess on the development of metabolic alterations. Glucose metabolism derangements due to aldosterone action are frequently observed not only in patients with primary aldosteronism but also in patients with obesity. A contribution to the hyperaldosteronism observed in obese subjects can be attributed, at least in part, to the action of still unidentified adipocyte-derived factor. Aldosterone, through genomic and non-genomic actions contributes to induce several abnormalities: pancreatic fibrosis, impaired beta cell function, as well as reduced skeletal muscle and adipose tissue insulin sensitivity. Oxidative stress, systemic inflammation, together with these metabolic alterations may explain the appearance of the cardiometabolic syndrome and the progression of cardiovascular and renal diseases, in the presence of inappropriate aldosterone levels. The biological actions of aldosterone are mediated by mineralocorticoid receptor (MR), although MR can be activated through an aldosterone independent fashion. Besides salt-water homeostasis, MR activation promotes inflammation, endothelial dysfunction, cardiovascular remodelling and affects adipose tissue differentiation and function. Clinical and experimental studies have shown that MR blockade is able to suppress inflammation, to improve endothelium- dependent vasorelaxation, but most interestingly, to improve pancreatic insulin release as well as insulin-mediated glucose utilization. These actions indicate MR antagonists as a useful therapeutic tool able not only to reduce cardiovascular risk and renal damage, but also to improve metabolic sequaelae.

BACKGROUND: A novel Cys-Ser Ret germline point mutation in a 58-year-old woman with bilateral medullary thyroid carcinoma (MTC) prompted us to perform genetic analysis of the family and evaluate the biological consequences of such a mutation. METHODS: Ret analysis by direct sequencing was performed in five family members. The biological activity and biochemical properties of the Ret- Cys515Ser mutant were analyzed in NIH-3T3 cells. RESULTS: The proband's son, age 35, had the Ret- Cys515Ser mutation and the L769 CTT/CTG exon 13 polymorphic variant, which was also found in his father. Clinical evaluation of the son also revealed bilateral multifocal microscopic MTC and papillary thyroid carcinoma (PTC). In vitro and in vivo analysis indicated ligand-independent activation of the Ret-Cys515Ser mutant due to aberrant disulfide homodimerization, increased mitogenic activity, and ability to induce anchorage-independent growth in NIH-3T3 cells in comparison to wild-type Ret, suggesting a possible role of Cys515Ser in tumor development. CONCLUSIONS: The Cys515Ser mutation adds to cysteine substitution groups that have been described in association with MTC. Our data also highlight the importance of performing a complete genetic analysis in patients who present with MTC.

A 56-yr-old woman was referred with a diagnosis of Cushing’s disease. Hypertension and severe hypokalemia were present and high urinary free cortisol/cortisone ratio was detected, raising a suspicion of an ectopic ACTH syndrome. Inferior petrosal sinus sampling, thoracic computed tomography, and octreotide scans were negative. Remission and relapse periods lasting 3–4 months were observed during the 3.5 yr of follow-up. Finally a thoracic computed tomography scan showed a basal paracardic nodule in the left lung. After surgery, a well-differentiated neuroendocrine tumor (typical bronchial carcinoid) was diagnosed, staining positively for ACTH. RT-PCR revealed expression of proopiomelanocortin, CRH receptor, and V3 vasopressin receptor. Somatostatin receptor type 1, 2, 3, and 5 mRNA was detected only in tumoral tissue. Interestingly, we observed the simultaneous presence of ghrelin and both GH secretagogue (GHS) receptors (1a and 1b) mRNA in tumoral tissue but not in the normal lung. This finding correlates with the in vivo ACTH hyperresponsiveness to hexarelin (a GHS). This is the first report of a cyclical ectopic ACTH-secreting tumor with an in vivo ACTH response to hexarelin coupled with the tumoral expression of ghrelin and GHS receptors. This finding might imply an autocrine/paracrine modulatory effect of ghrelin in bronchial ACTH-secreting tumors.