Faculty Bibliography

Glucocorticoids regulate many physiological processes and have an essential role in the systemic response to stress. For example, gene transcription is modulated by the glucocorticoid-glucocorticoid receptor complex via several mechanisms. The ultimate biologic responses to glucocorticoids are determined by not only the concentration of glucocorticoids but also the differences between individuals in glucocorticoid sensitivity, which is influenced by multiple factors. Differences in sensitivity to glucocorticoids in healthy individuals are partly genetically determined by functional polymorphisms of the gene that encodes the glucocorticoid receptor. Hereditary syndromes have also been identified that are associated with increased and decreased sensitivity to glucocorticoids. As a result of their anti-inflammatory properties, glucocorticoids are widely used in the treatment of allergic, inflammatory and haematological disorders. The variety in clinical responses to treatment with glucocorticoids reflects the considerable variation in glucocorticoid sensitivity between individuals. In immune-mediated disorders, proinflammatory cytokines can induce localized resistance to glucocorticoids via several mechanisms. Individual differences in how tissues respond to glucocorticoids might also be involved in the predisposition for and pathogenesis of the metabolic syndrome and mood disorders. In this Review, we summarize the mechanisms that influence glucocorticoid sensitivity in health and disease and discuss possible strategies to modulate glucocorticoid responsiveness.

CONTEXT/BACKGROUND:Primary pigmented nodular adrenocortical disease (PPNAD) can lead to steroid hormone overproduction. Mutations in the cAMP protein kinase A regulatory subunit type 1A (PRKAR1A) are causative of PPNAD. Steroidogenesis in PPNAD can be modified through a local glucocorticoid feed-forward loop. OBJECTIVE:Investigation of regulation of steroidogenesis in a case of PPNAD with virilization. MATERIALS AND METHODS/METHODS:A 33-year-old woman presented with primary infertility due to hyperandrogenism. Elevated levels of testosterone and subclinical ACTH-independent Cushing's syndrome led to the discovery of an adrenal tumor, which was diagnosed as PPNAD. In vivo evaluation of aberrantly expressed hormone receptors showed no steroid response to known stimuli. Genetic analysis revealed a PRKAR1A protein-truncating Q28X mutation. After adrenalectomy, steroid levels normalized. Tumor cells were cultured and steroidogenic responses to ACTH and dexamethasone were measured and compared with those in normal adrenal and adrenocortical carcinoma cells. Expression levels of 17β-hydroxysteroid dehydrogenase (17β-HSD) types 3 and 5 and steroid receptors were quantified in PPNAD, normal adrenal, and adrenal adenoma tissues. RESULTS:Isolated PPNAD cells, analogous to normal adrenal cells, showed both increased steroidogenic enzyme expression and steroid secretion in response to ACTH. Dexamethasone did not affect steroid production in the investigated types of adrenal cells. 17β-HSD type 5 was expressed at a higher level in the PPNAD-associated adenoma compared with control adrenal tissue. CONCLUSION/CONCLUSIONS:PPNAD-associated adenomas can cause virilization and infertility by adrenal androgen overproduction. This may be due to steroidogenic control mechanisms that differ from those described for PPNAD without large adenomas.

Cushing's disease (CD) is a rare endocrine disorder that is caused by an adrenocorticotropin (ACTH)-producing pituitary adenoma that chronically stimulates adrenocortical cortisol production. CD is primarily treated by transsphenoidal surgery, resulting in long-term biochemical remission in approximately 60-90% of the patients. Patients who are ineligible for surgery or have undergone unsuccessful surgery are candidates for drug therapy. Medical treatment can aim to inhibit the excess ACTH production at the level of the pituitary adenoma, to decrease adrenocortical steroidogenesis or to antagonize the effects of cortisol at the level of its receptor. In recent years, a number of studies have been published that evaluated the efficacy of medical therapy, either as monotherapy or combination therapy, in patients with CD. In particular, the effects of the somatostatin analog pasireotide and the glucocorticoid receptor antagonist mifepristone have been investigated in two large clinical trials. Moreover, the first clinical experiences with the novel steroidogenesis inhibitor LCI699 and the pituitary-directing retinoic acid have become available. Finally, an in vitro study with gefitinib, an epidermal growth factor receptor-antagonist, showed promising results that warrant further exploration. In this review, the efficacy and limitations of drugs that have been used in the treatment of CD will be discussed as well as recent developments with respect to new pituitary- and adrenal-targeting compounds.

CONTEXT/BACKGROUND:Cushing's disease (CD) is associated with serious morbidity and, when suboptimally treated, an increased mortality. Although surgery is the first-line treatment modality for CD, hypercortisolism persists or recurs in an important subset of patients. Considering the deleterious effects of uncontrolled CD, there is a clear need for effective medical therapy. OBJECTIVE:In this review, we discuss molecular targets for medical therapy, efficacy, and side effects of the currently used drugs to treat hypercortisolism and focus on recent developments resulting from translational and clinical studies. EVIDENCE ACQUISITION/METHODS:Selection of publications related to the study objective was performed via a PubMed search using relevant keywords and search terms. MAIN FINDINGS/RESULTS:Medical therapy for CD can be classified into pituitary-directed, adrenal-blocking, and glucocorticoid receptor-antagonizing drugs. Recent studies demonstrate that somatostatin receptor subtype 5 (sst(5)) and dopamine receptor subtype 2 (D(2)) are frequently (co-)expressed by corticotroph adenomas. Pituitary-directed therapy with pasireotide and cabergoline, targeting sst(5) and D(2), respectively, is successful in approximately 25-30% of patients. Adrenal-blocking drugs can be effective by inhibiting steroidogenic enzyme activity. Finally, the glucocorticoid receptor antagonist mifepristone induces clinical and metabolic improvement in the majority of patients. Each drug can have important side effects that may impair long-term treatment. Generally, patients with moderate to severe hypercortisolism need combination therapy to normalize cortisol production. CONCLUSION/CONCLUSIONS:Medical therapy for CD can be targeted at different levels and should be tailored in each individual patient. Future studies should examine the optimal dose and combination of medical treatment modalities for CD.

Adrenocortical carcinoma (ACC) is an aggressive tumor with very poor prognosis. Novel medical treatment opportunities are required. We investigated the effects of interferon-β (IFN-β), alone or in combination with mitotane, on cell growth and cortisol secretion in primary cultures of 13 human ACCs, three adrenal hyperplasias, three adrenal adenomas, and in two ACC cell lines. Moreover, the interrelationship between the effects of IGF2 and IFN-β was evaluated. Mitotane inhibited cell total DNA content/well (representing cell number) in 7/11 (IC50: 38±9.2 μM) and cortisol secretion in 5/5 ACC cultures (IC50: 4.5±0.1 μM). IFN-β reduced cell number in 10/11 (IC50: 83±18 IU/ml) and cortisol secretion in 5/5 ACC cultures (IC50: 7.3±1.5 IU/ml). The effect of IFN-β on cell number included the induction of apoptosis. IFN-β strongly inhibited mRNA expression of STAR, CYP11A1, CYP17A1, and CYP11B1. Mitotane and IFN-β induced an additive inhibitory effect on cell number and cortisol secretion. IGF2 (10 nM) inhibited apoptosis and increased cell number and cortisol secretion. These effects were counteracted by IFN-β treatment. Finally, IFN-β inhibited IGF2 secretion and mRNA expression. In conclusion, IFN-β is a potent inhibitor of ACC cell growth in human primary ACC cultures, partially mediated by an inhibition of the effects of IGF2, as well as its production. The increased sensitivity of ACC cells to mitotane induced by treatment with IFN-β may open the opportunity for combined treatment regimens with lower mitotane doses. The inhibition of the expression of steroidogenic enzymes by IFN-β is a novel mechanism that may explain its inhibitory effect on cortisol production.

CONTEXT/BACKGROUND:Somatostatin receptor subtype 2 (sst2A) protein expression has been demonstrated to positively correlate with somatostatin analog treatment outcome in GH-secreting adenomas. Recently, a new rabbit monoclonal anti-sst2A antibody (clone UMB-1) has been validated as a reliable method to selectively detect sst2A protein levels in formalin-fixed tissues. OBJECTIVE:The aim of the study was to establish whether the evaluation of sst2A protein levels, assessed with a routine reproducible immunohistochemistry protocol using UMB-1 antibody, may predict the successful adjuvant therapy with somatostatin analogs in acromegalic patients. DESIGN, SETTING, AND PATIENTS/METHODS:Thirty-six acromegalic patients from our referral hospital were evaluated retrospectively. Sst2A expression analysis was performed by immunohistochemistry in 25 patients and by quantitative RT-PCR in 26 patients. Sst2A immunoreactivity was evaluated using an immunoreactivity score (IRS), which takes into account both the percentage of positive cells and staining intensity. INTERVENTIONS/METHODS:Patients with persistent disease after surgery (n = 26) were treated with somatostatin analogs for a median duration of 6 months. MAIN OUTCOME MEASURE/METHODS:GH and IGF-I levels were measured before and after postoperative treatment. RESULTS:Sst2A IRS showed a significant positive correlation with both GH (P = 0.039) and IGF-I (P = 0.001) suppression by octreotide. Sst2A IRS was negatively associated with IGF-I levels reached after treatment (P = 0.001), and patients that achieved IGF-I normalization showed significantly higher sst2A IRS compared to the group that was not normalized (P = 0.002). A sst2A IRS of at least 5 showed a sensitivity of 86% and a specificity of 91% in predicting IGF-I normalization during adjuvant octreotide treatment. CONCLUSION/CONCLUSIONS:Sst2A IRS with the anti-sst2A antibody UMB-1 represents a valid tool in the clinical practice to identify acromegalic patients likely to be responders to adjuvant therapy with the currently available somatostatin analogs.

INTRODUCTION/BACKGROUND:The mechanism underlying the spontaneous improvement of rheumatoid arthritis (RA) during pregnancy and the subsequent postpartum flare is incompletely understood, and the disease course varies widely between pregnant RA patients. In pregnancy, total and free levels of cortisol increase gradually, followed by a postpartum decrease to prepregnancy values. The glucocorticoid receptor (GR) polymorphisms BclI and N363S are associated with relatively increased glucocorticoid (GC) sensitivity, whereas the 9β and ER22/23EK polymorphisms of the GR gene are associated with a relatively decreased GC sensitivity. We examined the relation between the presence of these GR polymorphisms and level of disease activity and disease course of RA during pregnancy and postpartum. METHODS:We studied 147 participants of the PARA study (Pregnancy-Induced Amelioration of Rheumatoid Arthritis study), a prospective study investigating the natural improvement during pregnancy and the postpartum flare in women with RA. Patients were visited, preferably before pregnancy, at each trimester and at three postpartum time points. On all occasions, disease activity was scored by using DAS28. All patients were genotyped for the GR polymorphisms BclI, N363S, 9β, and ER22/23EK and divided in groups harboring either polymorphisms conferring increased GC sensitivity (BclI and N363S; GC-S patients) or polymorphisms conferring decreased GC sensitivity (9β or 9β + ER22/23EK; GC-I patients). Data were analyzed by using a mixed linear model, comparing GC-S patients with GC-I patients with respect to improvement during pregnancy and the postpartum flare. The cumulative disease activity was calculated by using time-integrated values (area under the curve, AUC) of DAS28 in GC-I patients versus GC-S patients. Separate analyses were performed according to the state of GC use. RESULTS:GC-S patients treated with GC had a significantly lower AUC of DAS28 in the postpartum period than did GC-I patients. This difference was not observed in patients who were not treated with GCs. During pregnancy, GC-S and GC-I patients had comparable levels of disease activity and course of disease. CONCLUSIONS:Differences in relative GC sensitivity, as determined by GR polymorphisms, are associated with the level of disease activity in the postpartum period in GC-treated patients, but they do not seem to influence the course of the disease per se.

OBJECTIVE:The use of ergot-derived dopamine agonists (DA) to treat patients with prolactinomas has not been associated with an increased risk of significant heart valve dysfunction. Accordingly, the present study evaluated whether the long-term use of DA for hyperprolactinaemia may be associated with increased risk of significant valvular heart disease. METHODS:A total of 74 patients (mean age 48 ± 1·4 years, 23% male) with prolactinoma treated with DA for at least 1 year were evaluated with 2-dimensional echocardiography at baseline. After 2 years of follow-up, a repeat echocardiography was performed to evaluate significant changes in valvular heart structure (thickening, calcifications and leaflet motion abnormalities) and function (regurgitation or stenosis). Patients were classified according to treatment: patients treated with cabergoline (group 1: n = 45), and patients not treated with cabergoline (group 2: n = 29). RESULTS:At 2-year follow-up, no significant valvular stenosis was observed in any patient. In addition, the prevalence of any significant valve regurgitation did not change significantly (from 12% to 15%, P = NS). However, there was a significant increase in the prevalence of valvular calcifications (from 48% to 58%, P = 0·004) and, particularly, in the prevalence of aortic valve calcifications (from 39% to 53%, P = 0·002). In a per-treatment-based analysis, the group of patients treated with cabergoline had significantly higher prevalence of aortic valve calcification at 2 years follow-up as compared to the group of patients not treated with cabergoline (63%vs 38%, P = 0·016). CONCLUSIONS:The long-term therapy with DA (cabergoline) of patients with prolactinoma is associated with an increased prevalence of valvular calcification. However, these structural changes were not accompanied by an increased prevalence of valvular dysfunction.

OBJECTIVE:The insufficient diagnostic accuracy for differentiation between benign and malignant adrenocortical disease and lack of sensitive markers reflecting tumor load emphasize the need for novel biomarkers for diagnosis and follow-up of adrenocortical carcinoma (ACC). DESIGN/METHODS:Since the inhibin α-subunit is expressed within the adrenal cortex, the role of serum inhibin pro-αC as a tumor marker for ACC was studied in patients. METHODS:Regulation of adrenal pro-αC secretion was investigated by adrenocortical function tests. Serum inhibin pro-αC levels were measured in controls (n=181) and patients with adrenocortical hyperplasia (n=45), adrenocortical adenoma (ADA, n=32), ACC (n=32), or non-cortical tumors (n=12). Steroid hormone, ACTH, and inhibin A and B levels were also estimated in patient subsets. RESULTS:Serum inhibin pro-αC levels increased by 16% after stimulation with ACTH (P=0.043). ACC patients had higher serum inhibin pro-αC levels than controls (medians 733 vs 307 ng/l, P<0.0001) and patients with adrenocortical hyperplasia, ADA, or non-adrenocortical adrenal tumors (148, 208, and 131 ng/l, respectively, P=0.0003). Inhibin pro-αC measurement in ACC patients had a sensitivity of 59% and specificity of 84% for differentiation from ADA patients. Receiver operating characteristic analysis displayed areas under the curve of 0.87 for ACC vs controls and 0.81 for ACC vs ADA (P<0.0001). Surgery or mitotane therapy was followed by a decrease of inhibin pro-αC levels in 10/10 ACC patients tested during follow-up (P=0.0065). CONCLUSIONS:Inhibin pro-αC is produced by the adrenal gland. Differentiation between ADA and ACC by serum inhibin pro-αC is limited, but its levels may constitute a novel tumor marker for ACC.