Faculty Bibliography

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.

Patients with adrenocortical carcinoma (ACC) need new treatment options. The aim of this study was to evaluate the effects of the mTOR inhibitors sirolimus and temsirolimus on human ACC cell growth and cortisol production. In H295, HAC15, and SW13 cells, we have evaluated mTOR, IGF2, and IGF1 receptor expressions; the effects of sirolimus and temsirolimus on cell growth; and the effects of sirolimus on apoptosis, cell cycle, and cortisol production. Moreover, the effects of sirolimus on basal and IGF2-stimulated H295 cell colony growth and on basal and IGF1-stimulated phospho-AKT, phospho-S6K1, and phospho-ERK in H295 and SW13 were studied. Finally, we have evaluated the effects of combination treatment of sirolimus with an IGF2-neutralizing antibody. We have found that H295 and HAC15 expressed IGF2 at a >1800-fold higher level than SW13. mTOR inhibitors suppressed cell growth in a dose-/time-dependent manner in all cell lines. SW13 were the most sensitive to these effects. Sirolimus inhibited H295 colony surviving fraction and size. These effects were not antagonized by IGF2, suggesting the involvement of other autocrine regulators of mTOR pathways. In H295, sirolimus activated escape pathways. The blocking of endogenously produced IGF2 increased the antiproliferative effects of sirolimus on H295. Cortisol production by H295 and HAC15 was inhibited by sirolimus. The current study demonstrates that mTOR inhibitors inhibit the proliferation and cortisol production in ACC cells. Different ACC cells have different sensitivity to the mTOR inhibitors. mTOR could be a target for the treatment of human ACCs, but variable responses might be expected. In selected cases of ACC, the combined targeting of mTOR and IGF2 could have greater effects than mTOR inhibitors alone.

CONTEXT/BACKGROUND:ACTH stimulates adrenocortical steroid production through the melanocortin 2 receptor (MC2R). MC2R trafficking and signaling are dependent on the MC2R accessory protein (MRAP). The MRAP homolog MRAP2 also transports the MC2R to the cell surface but might prevent activation. OBJECTIVE:The objective of the investigation was to study the regulatory pathways of MRAP and MRAP2 and their contributions to ACTH responsiveness in human adrenal tissues. DESIGN AND SETTING/METHODS:MRAP, MRAP2, and MC2R expression levels were studied in 32 human adrenocortical samples. Regulation of these mRNAs was investigated in 43 primary adrenal cultures, stimulated with ACTH, forskolin, angiotensin II (AngII), phorbol-12-myristate-13-acetate (PMA), or dexamethasone. The induction of cortisol, cAMP, and ACTH-responsive genes after treatment with ACTH was related to MRAP, MRAP2, and MC2R expression levels. RESULTS:MRAP and MRAP2 levels were lower in adrenocortical carcinomas (ACC) than in other adrenal tissues (P < 0.001). Patient ACTH and cortisol levels were associated with adrenal levels of MRAP and MC2R in adrenal hyperplasia samples (P < 0.05) but not in tumors. ACTH induced the expression of MRAP 11 ± 2.1-fold and MC2R 20 ± 3.8-fold in all adrenal tissue types (mean ± SEM, both P < 0.0001), whereas AngII augmented these mRNAs 4.0 ± 1.2-fold and 12.6 ± 3.2-fold (P < 0.0001) in all but the ACC. MRAP2 expression was suppressed by forskolin (-24 ± 15%, P = 0.013) and PMA (-22 ± 7%, P = 0.0007). MRAP, MRAP2, or MC2R levels were not associated with the induction of cortisol, cAMP, or gene expression by ACTH in vitro. CONCLUSION/CONCLUSIONS:MRAP and MC2R expression is induced by ACTH and AngII, which would facilitate cell surface receptor availability. Physiological expression levels of MRAP, MRAP2, and MC2R were not limiting for ACTH sensitivity.

BACKGROUND:Bilateral adrenalectomy (BLA) is a treatment option to alleviate symptoms in patients with ectopic Cushing's syndrome (ECS) for whom surgical treatment of the responsible nonpituitary tumor is not possible. ECS patients have an increased risk for complications, because of high cortisol levels, poor clinical condition, and metabolic disturbances. This study aims to evaluate the safety and long-term efficacy of endoscopic BLA for ECS. METHODS:From 1990 to present, 38 patients were diagnosed and treated for ECS in the Erasmus University Medical Center, a tertiary referral center. Twenty-four patients were treated with BLA (21 endoscopic, 3 open), 9 patients were treated medically, and 5 patients could be cured by complete resection of the adrenocorticotropic hormone (ACTH)-producing tumor. The medical records were retrospectively reviewed and entered into a database. For evaluation of the efficacy of BLA, preoperative biochemical and physical symptoms were assessed and compared with postoperative data. RESULTS:Endoscopic BLA was successfully completed in 20 of the 21 patients; one required conversion to open BLA. Intraoperative complications occurred in two (10%) patients, and postoperative complications occurred in three (14%) patients. Median hospitalization was 9 (2-95) days, and median operating time was 246 (205-347) min. Hypercortisolism was resolved in all patients. Improvements of hypertension, body weight, Cushingoid appearance, impaired muscle strength, and ankle edema were achieved in 87, 90, 65, 61, and 78% of the patients, respectively. Resolution of diabetes, hypokalemia, and metabolic alkalosis was achieved in 33, 89, and 80%, respectively. CONCLUSION/CONCLUSIONS:Endoscopic BLA is a safe and effective treatment for patients with ectopic Cushing's syndrome.

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.

An increased association between neuroendocrine tumors of the gastrointestinal tract and pancreas (GEP-NET) and other second primary malignancies has been suggested. We determined whether there is indeed an increased risk for second primary malignancies in GEP-NET patients compared with an age- and sex-matched control group of patients with identical malignancies. The series comprised 243 men and 216 women, diagnosed with a GEP-NET between 2000 and 2009 in a tertiary referral center. The timeline, before-at-after diagnosis, and the type of other malignancies were studied using person-year methodology. Poisson distributions were used for testing statistical significance. All data were cross-checked with the Dutch National Cancer Registry. Out of 459 patients with GEP-NET, 67 (13.7%) had a second primary cancer diagnosis: 25 previous cancers (5.4%), 13 synchronous cancers (2.8%), and 29 metachronous cancers (6.3%). The most common types of second primary cancer were breast cancer (n=10), colorectal cancer (n=8), melanoma (n=6), and prostate cancer (n=5). The number of patients with a cancer history was lower than expected, although not significant (n=25 vs n=34.5). The diagnosis of synchronous cancers, mainly colorectal tumors, was higher than expected (n=13 vs n=6.1, P<0.05). Metachronous tumors occurred as frequent as expected (n=29 vs n=25.2, NS). In conclusion, our results are in contrast to previous studies and demonstrate that only the occurrence of synchronous second primary malignancies, mainly colorectal cancers, is increased in GEP-NET patients compared with the general population.

OBJECTIVE:Active Cushing's syndrome (CS) is associated with cardiomyopathy, characterized by myocardial structural, and ultrastructural abnormalities. The extent of myocardial fibrosis in patients with CS has not been previously evaluated. Therefore, the objective of this study was to assess myocardial fibrosis in CS patients, its relationship with left ventricular (LV) hypertrophy and function, and its reversibility after surgical treatment. DESIGN AND METHODS/METHODS:Fifteen consecutive CS patients (41±12 years) were studied together with 30 hypertensive (HT) patients (matched for LV hypertrophy) and 30 healthy subjects. Echocardiography was performed in all patients including i) LV systolic function assessment by conventional measures and by speckle tracking-derived global longitudinal strain, ii) LV diastolic function assessment using E/E', and iii) myocardial fibrosis assessment using calibrated integrated backscatter (IBS). Echocardiography was repeated after normalization of cortisol secretion (14±3 months). RESULTS:CS patients showed the highest value of calibrated IBS (-15.1±2.5  dB) compared with HT patients (-20.0±2.6  dB, P<0.01) and controls (-23.8±2.4  dB, P<0.01), indicating increased myocardial fibrosis independent of LV hypertrophy. Moreover, calibrated IBS in CS patients was significantly related to both diastolic function (E/E', r=0.79, P<0.01) and systolic function (global longitudinal strain, r=0.60, P=0.02). After successful surgical treatment, calibrated IBS normalized (-21.0±3.8 vs -15.1±2.5  dB, P<0.01), suggestive of regression of myocardial fibrosis. CONCLUSIONS:Patients with CS have increased myocardial fibrosis, which is related to LV systolic and diastolic dysfunction. Successful treatment of CS normalizes the extent of myocardial fibrosis. Therefore, myocardial fibrosis appears to be an important factor in the development and potential regression of CS cardiomyopathy.