Normal Adrenal Glands Research Articles

ALDOSTERONE INDUCES CYP11B2 GENE EXPRESSION VIA GPER-1 ACTIVATION IN HUMAN ADRENOCORTICAL CELL (HAC15) AND STRIPS FROM ALDOSTERONE PRODUCING ADENOMA

Objective: We previously reported that GPER-1 is mostly expressed in the zona glomerulosa of normal human adrenal gland and in aldosterone-producing adenoma (APA). In the adrenocortical carcinoma cell line HAC15 17b-estradiol induces aldosterone synthesis via GPER-1 activation. Since GPER-1 was found to bind not only 17b-estradiol, but also aldosterone in endothelial and vascular smooth muscle cells, we wondered if aldosterone binds GPER-1 receptor also in the adrenocortical cells, thereby modulating its own synthesis. Design and method: APA strips and adrenocortical carcinoma cell line HAC15 were exposed to [100 nM] aldosterone for 12 hours in the presence or absence of the selective mineralocorticoid receptor (MR) antagonist canrenone and/or of the selective GPER-1 antagonist G36. HAC15 cells were silenced for GPER-1 with specific small interfering RNA (siRNA) and then exposed to aldosterone. The changes of aldosterone synthase (CYP11B2) mRNA and protein levels were measured by real time qRT-PCR and immunoblotting. Results: Aldosterone increased CYP11B2 gene and protein expression (+200% and +130%, respectively) in HAC15 cells (p < 0.001 vs untreated). Pretreatment with canrenone did not prevent such increase, while G36 abolished aldosterone-induced CYP11B2 activation (p < 0.01). In APA strips aldosterone increased CYP11B2 gene expression (+330%, p < 0.01 vs untreated); pre-treatment with G36 blunted this effect of aldosterone. Silencing of GPER-1 with siRNA not only reduced GPER-1 expression (p < 0.01), but also abolished the CYP11B2 increase in response to aldosterone. Conclusions: Aldosterone enhances the expression of CYP11B2 acting via GPER-1, but not MR. Binding of aldosterone to GPER-1 could contribute to perpetuating the autonomous aldosterone excess, which is a hallmark of primary aldosteronism, via an autocrine-paracrine positive feed-back loop.

Role of DOTATATE-PET/CT in preoperative assessment of phaeochromocytoma and paragangliomas.

Diagnosis of paragangliomas (PGL) and phaeochromocytomas (PC) can be challenging particularly if the tumour is small. Detection of metastatic disease is important for comprehensive management of malignant PC/PGL. Somatostatin receptor imaging (SRI) agents have high sensitivity for these tumours, particularly theDOTA family of radiopharmaceuticals labelled with 68 Gallium. To describe the utility of SRI in primary assessment (ie before surgery) for PC/PGL and whether measures of maximum standardized uptake (SUVmax) could be used to distinguish between adrenal adenomas and PCs. Retrospective analysis of patients with PC and PGL between 2012 and 2017. Somatostatin receptor imaging (SRI) was performed for suspected PC (n=46) or PGL (n=27) of which 36 were during primary assessment and 37 during secondary assessment (follow-up after surgery). For comparison of adrenal SUVmax, scans from 30 patients without suspected PC/PGL (20 with normal adrenals; 10 with incidental adenomas) were evaluated. Baseline description, sensitivity, specificity, Youden's index. Sensitivity of DOTATATE-PET was 88% for PC and 100% for PGL. False-negative scans were seen in 2/10 PCs<28mm and in 1/14 PCs>28mm which had features of cystic degeneration. SUVmax of PCs and PGLs was more than double compared to adrenal adenomas (P>.001). Somatostatin receptor imaging (SRI) has high sensitivity in primary assessment for PC and PGL. We recommend that SRI should be performed as part of primary assessment in all suspected PGLs (due to higher risk of multifocal lesions) and in PCs suspected to be associated with hereditary syndromes or metastases.

MiR-193a-3p functions as a tumour suppressor in human aldosterone-producing adrenocortical adenoma by down-regulating CYP11B2.

The mechanism of aldosterone-producing adrenocortical adenoma (APA) pathogenesis and the role of microRNAs (miRNAs) in APA pathogenesis have not been completely clarified. We examined the expression and function of miR-140-3p, miR-193a-3p and miR-22-3p, which have binding sites in CYP11B2. Expression of miRNAs and CYP11B2 mRNA was measured by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation was monitored by colorimetric analysis, and cell apoptosis and cell cycle progression were analysed by flow cytometry. ELISA was carried out to detect aldosterone levels in cell culture supernatants. Luciferase reporter assays, qRT-PCR and Western blotting were performed to identify CYP11B2 as a target of miR-193a-3p. Of the three miRNAs examined, miR-193a-3p exhibited a significant decrease and CYP11B2 mRNA exhibited a significant increase in expression in APA compared with adjacent normal adrenal gland tissue. Transfection of miR-193a-3p mimic into the human adrenocortical cell line H295R showed that elevated miR-193a-3p expression inhibits proliferation and aldosterone secretion, induces G1-phase arrest and promotes apoptosis in H295R cells. Furthermore, in luciferase reporter assays, overexpression of miR-193a-3p in H295R cells significantly reduced the luciferase activity of the wild-type CYP11B2 3'-UTR construct, which could be reversed by mutation of the miR-193a-3p-binding site. Moreover, miR-193a-3p overexpression downregulated CYP11B2 mRNA and protein expression. Finally, overexpression of CYP11B2 diminished the effects of miR-193a-3p on H295R cells. Taken together, our results suggest that CYP11B2 levels may be modulated by miR-193a-3p in APA, which could explain, at least partially, why downregulation of miR-193a-3p during APA formation may promote cell growth and suppress apoptosis.

Secreting ectopic adrenal adenoma: A rare condition to be aware of

Ectopic adrenal adenoma causing chronic Cushing's syndrome (CS) is a rare phenomenon. Diagnosis is usually made years after disease onset because of the insidious nature of the ectopic adrenal gland and because it overlaps with common symptoms, such as overweight and hypertension, in the general population (Kreitschmann-Andermahr et al., 2015). Here, we report the case of a 46-year-old male with a 15-year history of severe hypertension, facial plethora, and centripetal obesity. During treatment for herpes zoster, the patient presented with severe hypokalemia and flaccid paralysis, characteristic changes associated with CS. The serum cortisol level was elevated and baseline adrenocorticotrophic hormone (ACTH) was suppressed. After administration of the low-dose overnight dexamethasone suppression test, plasma cortisol was 38.9μg/dL. A contrast computed tomography (CT) scan revealed normal adrenal glands and significant tumor adjacent to the right renal hilum measuring ∼3.6×2.3cm. The tumor was removed through retroperitoneoscopy, and pathological examination confirmed adrenocortical adenoma and myelolipoma metaplasia. The serum potassium level gradually became normal after surgical removal of the mass. In the current report, we have reviewed the pertinent literature and highlighted the importance of considering ectopic adrenal adenoma in the differential diagnosis of chronic CS with nonspecific symptoms.

Iodine and Fat Quantification for Differentiation of Adrenal Gland Adenomas From Metastases Using Third-Generation Dual-Source Dual-Energy Computed Tomography.

The aim of this study was to investigate the value of third-generation dual-source dual-energy computed tomography (DECT) iodine and fat quantification in differentiating adrenal gland adenomas from metastases. Sixty-two patients (38 men and 24 women; mean age, 69.1 years) underwent clinically indicated DECT of the abdomen on a third-generation dual-source scanner. Examinations were retrospectively included due to detected adrenal masses. For each adrenal lesion, unenhanced and contrast-enhanced attenuation values, as well as dual-energy iodine density and fat fraction, were recorded. Additional magnetic resonance imaging data, positron emission tomography/computed tomography scans, interval imaging follow-up, and histopathological analysis were used as the reference standard for all adrenal lesions. Mean values of unenhanced and contrast-enhanced attenuation, as well as material densities, were compared between adenomas, metastases, and normal adrenal glands. Furthermore, the diagnostic accuracy of unenhanced, contrast-enhanced, and material density analysis was assessed between adrenal adenomas and metastases. Adrenal adenomas showed significant differences regarding iodine density and fat fraction values (1.3 ± 0.4 mg/mL and 34.2% ± 12.6%) in comparison with adrenal metastases (3.2 ± 1.4 mg/mL and 10.7% ± 7.8%) and normal adrenal glands (1.7 ± 0.6 mg/mL and 18.7% ± 12.0%) (all P ≤ 0.004). Analysis of unenhanced attenuation values revealed no significant differences between healthy adrenal parenchyma (19.1 ± 15.6 HU) and adrenal metastases (26.9 ± 16.2 HU) (P = 0.135). Iodine density and fat fraction analysis showed significantly higher diagnostic accuracy for the diagnosis of adenomas (sensitivity, 97% and 89%; specificity, 96% and 89%, respectively) compared with unenhanced and contrast-enhanced evaluation (sensitivity, 65% and 58%; specificity, 73% and 85%, respectively) (P ≤ 0.023). The combined diagnostic value of iodine density and fat fraction analysis revealed a sensitivity of 97% and a specificity of 100%. Third-generation dual-source DECT iodine and fat quantification allow for differentiation between adrenal adenomas and metastases with high diagnostic accuracy.

High-Resolution Tissue Mass Spectrometry Imaging Reveals a Refined Functional Anatomy of the Human Adult Adrenal Gland.

In the adrenal gland, neuroendocrine cells that synthesize catecholamines and epithelial cells that produce steroid hormones are united beneath a common organ capsule to function as a single stress-responsive organ. The functional anatomy of the steroid hormone-producing adrenal cortex and the catecholamine-producing medulla is ill defined at the level of small molecules. Here, we report a comprehensive high-resolution mass spectrometry imaging (MSI) map of the normal human adrenal gland. A large variety of biomolecules was accessible by matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI, including nucleoside phosphates indicative of oxidative phosphorylation, sterol and steroid metabolites, intermediates of glycolysis and the tricarboxylic acid cycle, lipids, and fatty acids. Statistical clustering analyses yielded a molecularly defined adrenal anatomy of 10 distinct molecular zones including a highly structured corticomedullary interface. By incorporating pathway information, activities of carbohydrate, amino acid, and lipid metabolism as well as endocrine bioactivity were revealed to be highly spatially organized, which could be visualized as different molecularly defined zones. Together, these findings provide a molecular definition of human adult adrenal gland structure beyond classical histological anatomy.

Bilateral Adrenal-Renal Fusion: A Radiological Diagnosis

In normal anatomy, the kidneys and adrenal glands are contained within the renal fascia and separated by a connective tissue capsule derived from mesenchymal tissue. Incomplete encapsulation can occur during embryonic development, resulting in adrenal-renal fusion. The true incidence of this developmental anomaly is unknown, as it has primarily been described in the literature following incidental detection on surgical or histological examination. We report the first documented case of bilateral adrenal-renal fusion, diagnosed radiologically.

TRH-induced secretion of adrenocorticotropin and cortisol in dogs with pituitary-dependent hypercortisolism

Background: In dogs, spontaneous Cushing’s syndrome is most often pituitary-dependent and caused by hypersecretion of adrenocorticotropic hormone (ACTH), resulting in increased adrenocortical glucocorticoid secretion similar to horses. In horses with Cushing’s syndrome (or pituitary pars intermedia dysfunction [PPID]) a thyrotropin-releasing hormone (TRH) stimulation test can be used for diagnosis, as TRH administration results in increased circulating ACTH and cortisol concentrations in affected horses.Objective: The aim of this study was to investigate the effect of TRH administration on the circulating ACTH and cortisol concentrations in dogs with pituitary-dependent hypercortisolism (PDH).Methods: Ten clinically normal control dogs and 10 dogs with PDH, all client owned, underwent a TRH stimulation test with measurement of plasma concentrations of ACTH and cortisol, before and after intravenous administration of 10 μg TRH/kg bodyweight.Results: Plasma ACTH concentration did not rise significantly after TRH stimulation, neither in PDH dogs nor in clinically normal dogs. In contrast, the plasma cortisol concentration did increase significantly after TRH stimulation in both groups (p = .003 in PDH and p < .001 in control). Immunohistochemistry of normal adrenal glands demonstrated the presence of TRH receptors in the whole adrenal cortex.Conclusions: The results of this study demonstrate that the TRH stimulation test should be rejected as a tool to diagnose PDH in dogs. The observed TRH-induced increase in plasma cortisol concentration without a significant rise in plasma ACTH concentration may be explained by a direct effect of TRH on adrenocortical cells mediated by adrenocortical TRH receptors.

Targeting CXCR4 (CXC Chemokine Receptor Type 4) for Molecular Imaging of Aldosterone-Producing Adenoma.

Primary aldosteronism is the most frequent cause of secondary hypertension and is associated with increased morbidity and mortality compared with hypertensive controls. The central diagnostic challenge is the differentiation between bilateral and unilateral disease, which determines treatment options. Bilateral adrenal venous sampling, currently recommended for differential diagnosis, is an invasive procedure with several drawbacks, making it desirable to develop novel noninvasive diagnostic tools. When investigating the expression pattern of chemokine receptors by quantitative real-time polymerase chain reaction and immunohistochemistry, we observed high expression of CXCR4 (CXC chemokine receptor type 4) in aldosterone-producing tissue in normal adrenals, adjacent adrenal cortex from adrenocortical adenomas, and in aldosterone-producing adenomas (APA), correlating strongly with the expression of CYP11B2 (aldosterone synthase). In contrast, CXCR4 was not detected in the majority of nonfunctioning adenomas that are frequently found coincidently. The specific CXCR4 ligand 68Ga-pentixafor has recently been established as radiotracer for molecular imaging of CXCR4 expression and showed strong and specific binding to cryosections of APAs in our study. We further investigated 9 patients with primary aldosteronism because of APA by 68Ga-pentixafor-positron emission tomography. The tracer uptake was significantly higher on the side of increased adrenocortical aldosterone secretion in patients with APAs compared with patients investigated by 68Ga-pentixafor-positron emission tomography for other causes. Molecular imaging of aldosterone-producing tissue by a CXCR4-specific ligand may, therefore, be a highly promising tool for noninvasive characterization of patients with APAs.

Protein Expression of PTTG1 as a Diagnostic Biomarker in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) has a poor prognosis and there is an unmet clinical need for biomarkers to improve both diagnostic and prognostic assessment. Pituitary-tumor transforming gene (PTTG1) has been shown to modulate cancer invasiveness and response to therapy. The potential role of PTTG1 protein levels in ACC has not been previously addressed. We assessed whether increased nuclear protein expression of PTTG1 distinguished ACCs from adrenocortical adenomas (ACAs). Patients with ACC or ACA were identified from prospective tissue banks at two independent institutions. Two tissue microarrays (TMAs) consisting of adrenal specimens from 131 patients were constructed and clinically annotated. Immunohistochemical analysis for PTTG1 and Ki-67 was performed on each TMA. TMA-1 (n=80) contained 20 normal adrenals, 20 ACAs, and 40 ACCs, and the validation, TMA-2 (n=51), consisted of 10 normal adrenals, 14 ACAs, and 27 ACCs. On TMA-1, nuclear staining of PTTG1 was detected in 12 (31%) ACC specimens, while all ACAs and normal adrenal glands were negative for PTTG1. On TMA-2, 20 (74%) of the ACC tumors demonstrated PTTG1 nuclear staining of PTTG1, and 13 (93%) ACA and 4 (44%) normal adrenal glands were negative for PTTG1. ACC tumors with increased PTTG1 protein staining had a significantly higher Ki-67 index (p<0.001) than those with lower levels of PTTG1. Increased nuclear protein expression of PTTG1 was observed in malignant adrenal tumors. PTTG1 correlated with Ki-67 in two independent TMAs. PTTG1 is a promising biologic marker in the evaluation of adrenal tumors.

Disordered CYP11B2 Expression in Primary Aldosteronism.

Primary aldosteronism is the most common type of secondary hypertension affecting 6-10% of patients with primary hypertension. PA is mainly caused by unilateral hyperaldosteronism due to an aldosterone-producing adenoma, unilateral hyperplasia with or without micronodules or bilateral zona glomerulosa hyperplasias with or without macro or micronodules. The development of antibodies against the terminal enzyme of aldosterone biosynthesis (CYP11B2) has permitted the further characterization of normal adrenals and resected adrenals from patients with primary aldosteronism. Normal adrenals exhibit two different patterns of cellular expression of CYP11B2: young individuals display a relatively uniform expression of the enzyme throughout the zona glomerulosa while the adrenals of older individuals have dispersed CYP11B2-expressing cells but have more groups of cells called aldosterone-producing cell clusters (APCC). APAs exhibit different patterns of CYP11B2 staining that vary from uniform to homogeneous. There are also a proportion of cells within the APA that co-express different enzymes that are not normally co-expressed in normal individuals. Approximately 30% of patients with unilateral hyperaldosteronism do not have an APA, but either have an increased number of CYP11B2 expressing micronodules or hyperplasia of the zona glomerulosa. In summary, the studies reported in this review are shedding new light on the pathophysiology of primary aldosteronism. The wide variation in histopathological features of the adenomas and concurrent presence of APCCs raises the possibility that most cases of unilateral production of aldosterone actually might represent bilateral asymmetric hyperplasia with nodules frequently due to the development of somatic aldosterone-driving mutations.

Steroidogenic factor-1 inverse agonists as a treatment option for canine hypercortisolism: in vitro study

Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non–ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.

GSTA1 Expression Is Correlated With Aldosterone Level in KCNJ5-Mutated Adrenal Aldosterone-Producing Adenoma.

KCNJ5 mutation is a major cause of aldosterone-producing adenomas (APAs). The development of APA apart from KCNJ5 mutation is less investigated. To investigate other mechanisms affecting aldosterone secretion apart from KCNJ5. Six pairs of KCNJ5-mutated, high and low aldosterone-secreting APAs, five non-KCNJ5-mutated APAs, and four normal adrenal glands were assayed by Affymetrix GeneChip Human Transcriptome Array 2.0. A total of 113 APA samples were investigated to explore the expression of glutathione-S-transferase A1 (GSTA1). H295R cells were used to verify the function of GSTA1. GSTA1 was the top gene downregulated in high-aldosterone KCNJ5-mutated APAs. GSTA1 was also downregulated in KCNJ5-mutated APAs compared with wild-type KCNJ5 APAs. Accordingly, mutant KCNJ5 decreased GSTA1 messenger RNA and protein expression levels. GSTA1 overexpression suppressed aldosterone secretion whether in wild-type or mutant KCNJ5 H295R cells. Adding ethacrynic acid or silencing of GSTA1 increased aldosterone secretion by increasing reactive oxygen species (ROS), superoxide, H2O2 levels, and Ca2+ influx. The expression of the transcription factors NR4A1, NR4A2, and CAMK1 and intracellular Ca2+ were significantly upregulated by GSTA1 inhibition. The reduced form of NAD phosphate oxidase inhibitor or H2O2 scavenger or blocking calmodulin or calcium channels could significantly reduce aldosterone secretion in GSTA1-inhibited cells. (1) GSTA1 expression is reversely correlated with aldosterone level in KCNJ5-mutated APAs, (2) GSTA1 regulates aldosterone secretion by ROS and Ca2+ signaling, and (3) KCNJ5 mutation downregulates GSTA1 expression, and overexpression of GSTA1 reverses increased aldosterone in KCNJ5-mutated adrenal cells.

Favorable surgical outcomes of aldosterone-producing adenoma based on lateralization by CT imaging and hypokalemia: a non-AVS-based strategy

To test the efficacy of a strategy based on CT imaging and clinical characteristics on lateralizing origin of excess aldosterone secretion in primary aldosteronism. Consecutive patients with diagnosed primary hyperaldosteronism from June 2006 to July 2012 in our center underwent adrenal surgeries without pre-operational adrenal venous sampling (AVS) if all the three criteria were met: (1) round- or oval-shaped occupational lesion of low density after contrast enhancement with diameter >1cm on CT scan was located in one adrenal gland; (2) unequivocally normal contralateral adrenal gland; (3) serum potassium level lower than 3.5mmol/L. Subjects who had received operation were taken into analysis and follow-ups. One hundred and twenty-five patients fulfilled the criteria and were recruited into our research. One hundred and twenty-two operated patients (97.6%) experienced complete resolution of hypokalemia as well as resolution or improvement in hypertension with reduction in antihypertensive medication, while 3 patients (2.4%) failed to obtain normal kalemia and continued on spironolactone therapy. At a median of 65-month (range 21-93) follow-up of these 122 subjects, 27 patients dropped out (22.1%). The 95 responding patients reported no episodes of paralysis or confirmed hypokalemia or any supplementation of potassium. Multivariate linear correlation analysis showed that plasma potassium level was correlated inversely with tumor diameter (r=-0.258, 95% CI -0.076, -0.514, p=0.037) and basal plasma aldosterone level (r=-0.251, 95% CI -0.040, -0.464, p=0.042). Most patients with typical unilateral adrenal macroadenomas, normal contralateral glands and hypokalemia could attain favorable surgical therapeutic outcomes without pre-operational AVS lateralization.

Is there a role for epithelial-mesenchymal transition in adrenocortical tumors?

Epithelial-mesenchymal transition (EMT) is a biological dynamic process by which epithelial cells lose their epithelial phenotype and acquire mesenchymal invasive and migratory characteristics. This has been postulated as an essential step during cancer progression and metastasis. Although this is well described in other tumors, the role of EMT in adrenocortical tumors (ACT) has yet to be addressed. The aim of this study was to evaluate the expression of EMT markers e-cadherin, vimentin, and fibronectin, along with EMT-transcription factors (EMT-TFs), TWIST1, SIP1, and SNAIL in 24 adrenocortical carcinoma (ACC), 19 adrenocortical adenomas (ACA), 27 childhood-onset adrenocortical tumors (CAT), and 12 normal adrenal glands. The association of EMT and EMT-TFs with clinical outcomes and pathology features were also evaluated. Cytoplasmic vimentin expression was increased among CAT samples when compared to ACC, ACA, and normal adrenal samples (p < 0.001). There was no difference in e-cadherin and fibronectin expression observed between groups. Nuclear and cytoplasmic expression of TWIST1 and SIP1 was stronger in CAT and ACC vs. ACA and normal tissue samples (all, p < 0.05). ACT, regardless of classification, exhibited increased SNAIL expression when compared to normal tissue (p < 0.05). A significant correlation was observed between vimentin and TWIST1 (r s = 0.44, p < 0.001); SIP1 (r s = 0.51, p < 0.001); and SNAIL (r s = 0.23, p < 0.05). TWIST1 and SIP1 expressions demonstrated a significant correlation (r s = 0.56, p < 0.001). High SIP1 expression was associated with a lower survival rate among ACC cases (p < 0.05). Vimentin, TWIST1, and SIP1 expressions are increased in aggressive ACT. Therefore, EMT may play a relevant role in adrenal tumorigenesis.

Quantitative analysis of normal and pathologic adrenal glands with 18F-FDOPA PET/CT: focus on pheochromocytomas.

Many studies have reported the high performance of 6-fluorine-18-fluorodihydroxyphenilalanine (F-FDOPA) PET/CT in the diagnosis of pheochromocytomas but nobody seems to have investigated physiological and pathological adrenal glands from a quantitative point of view. The purpose of the present study was to assess the quantitative F-FDOPA uptake of normal and pathologic adrenal glands and to establish thresholds to characterize pheochromocytomas. We were especially interested in characterizing the remaining adrenal glands captation after an adrenalectomy. We reviewed 112 F-FDOPA PET/CT scans taken for different indications. A total of 212 adrenal glands, of which 17 were pheochromocytomas, were analyzed on the basis of their functional and morphological features. The final diagnosis was based on histologic proof when available (six pheochromocytomas) or after synthesis of clinical, biological, morphological, and functional results. Maximum standardized uptake value (SUVmax), mediastinum, and liver ratios in case of pheochromocytomas, adenomas, and solitary adrenal glands were determined and compared with those of healthy glands. Receiver operating characteristic curves were determined and areas under the curve were compared for different cutoffs of each index. Pheochromocytomas demonstrated a higher F-FDOPA uptake compared with normal adrenal glands (mean SUVmax: 7.5, SD 4.0, range: 3.5-20.0 vs. mean SUVmax: 2.6, SD: 0.8, range: 1.0-6.9) (P<0.0001). An SUVmax threshold of 4.2 has a sensitivity and specificity of 94 and 98%, respectively. The areas under the curve were 0.988, 0.991, and 0.987 for an SUVmax of 4.2, a mediastinum ratio of 3.0, and a liver ratio of 1.7, respectively. A large number of nonsecreting pheochromocytomas were noticed. On the basis of the SUVmax no statistically significant difference was found between secreting (SUVmax: 8.9, SD: 5.3) and nonsecreting pheochromocytomas (SUVmax: 5.1, SD: 0.9) (P=0.141). After unilateral adrenalectomy, solitary glands presented no increased uptake compared with healthy adrenal glands. An unexpected lower captation was also observed (SUVmax: 2.0, P=0.047). We confirm the high affinity of F-FDOPA for secreting or nonsecreting pheochromocytoma. Indeed within a series of various adrenal glands, only these tumors presented a significant increased uptake compared with normal adrenal glands. Because of a high rate of nonhypersecreting lesions, F-FDOPA can act as a surrogate to biological assays. After an adrenalectomy, the remaining glands did not demonstrate compensatory accumulation of F-FDOPA. To our knowledge this last point has never been addressed.

Development of monoclonal antibodies against the human 3β-hydroxysteroid dehydrogenase/isomerase isozymes

The human 3β-hydroxysteroid dehydrogenase/isomerase (HSD3B) enzymes catalyze the conversion of 3β-hydroxy Δ5-6 steroids into 3-keto Δ4-5 steroids, which is required for the synthesis of the mature steroid hormones secreted by the adrenal and gonads. The human has 2 isozymes, the HSD3B1 that is traditionally located in placenta and extra-adrenal tissues and the HSD3B2 that is expressed in the adrenal and gonads. Mice with both cryptochrome 1 and 2 genes deletion were recently found to have salt-sensitive hypertension and hyperaldosteronism. These deletions were also associated with overexpression of the Hsd3b6 enzyme, the homolog of the human HSD3B1, in the zona glomerulosa which was believed to explain the hyperaldosteronism. A report using antibodies against human HSD3B1 suggested that it was expressed in the zona glomerulosa of normal human adrenals and in patients with idiopathic hyperaldosteronism and the HSD3B2 expressed in both the zona fasciculata and glomerulosa. We have developed specific monoclonal antibodies against the human HSD3B1 and HSD3B2 isozymes and found that the main enzyme expressed in the zona glomerulosa was the HSD3B2. Faint staining of the adrenal was also obtained using the anti-HSD3B1antibody only at high concentrations of antibody. This study fails to confirm that HSD3B1 expression in the human zona glomerulosa and double immunofluorescence clearly shows that the HSD3B2 is expressed in the zona glomerulosa and fasciculata and in the zona glomerulosa HSD3B2 is co-expressed with aldosterone synthase (CYP11B2).

Late onset asymptomatic pancreatic neuroendocrine tumor \u2013 A case report on the phenotypic expansion for MEN1

BackgroundMultiple endocrine neoplasia type 1 (MEN1) is a hereditary cancer syndrome associated with several endocrine as well as non-endocrine tumors and is caused by mutations in the MEN1 gene. Primary hyperparathyroidism affects the majority of MEN1 individuals by age 50 years. Additionally, MEN1 mutations trigger familial isolated hyperparathyroidism. We describe a seemingly unaffected 76-year-old female who presented to our Genetics Clinic with a family history of primary hyperparathyroidism and the identification of a pathogenic MEN1 variant.Case PresentationThe patient was a 76 year-old woman who appeared to be unaffected. She had a family history of a known MEN1 pathogenic variant. Molecular testing for the known MEN1 mutation c.1A > G, as well as, biochemical testing, MRI of the brain and abdomen were all performed using standard methods. Molecular testing revealed our patient possessed the MEN1 pathogenic variant previously identified in her two offspring. Physical exam revealed red facial papules with onset in her seventies, involving her cheeks, nose and upper lip. Formerly, she was diagnosed with rosacea by a dermatologist and noted no improvement with treatment. Clinically, these lesions appeared to be facial angiofibromas. Brain MRI was normal. However, an MRI of her abdomen revealed a 1.5 cm lesion at the tail of the pancreas with normal adrenal glands. Glucagon was mildly elevated and pancreatic polypeptide was nearly seven times the upper limit of the normal range. The patient underwent spleen sparing distal pancreatectomy and subsequent pathology was consistent with a well-differentiated pancreatic neuroendocrine tumor (pNET).ConclusionsAge-related penetrance and variable expressivity are well documented in families with MEN1. It is thought that nearly all individuals with MEN1 manifest disease by age 40. We present a case of late-onset MEN1 in the absence of the most common feature, primary hyperparathyroidism, but with the presence of a pNET and cutaneous findings. This family expands the phenotype associated with the c.1A > G pathogenic variant and highlights the importance of providing comprehensive assessment of MEN1 mutation carriers in families that at first blush may appear to have isolated hyperparathyroidism.

Age-Related Autonomous Aldosteronism.

Both aging and inappropriate secretion of aldosterone increase the risk for developing cardiovascular disease; however, the influence of aging on aldosterone secretion and physiology is not well understood. The relationship between age and adrenal aldosterone synthase (CYP11B2) expression was evaluated in 127 normal adrenal glands from deceased kidney donors (age, 9 months to 68 years). Following immunohistochemistry, CYP11B2-expressing area and areas of abnormal foci of CYP11B2-expressing cells, called aldosterone-producing cell clusters, were analyzed. In a separate ancillary clinical study of 677 participants without primary aldosteronism, who were studied on both high and restricted sodium diets (age, 18-71 years), we used multivariable linear regression to assess the independent associations between age and renin-angiotensin-aldosterone system physiology. In adrenal tissue, the total CYP11B2-expressing area was negatively correlated with age (r=-0.431, P<0.0001), whereas the total aldosterone-producing cell cluster area was positively correlated with age (r=0.390, P<0.0001). The integrated ratio of aldosterone-producing cell cluster to CYP11B2-expressing area was most strongly and positively correlated with age (r=0.587, P<0.0001). When participants in the clinical study were maintained on a high sodium balance, renin activity progressively declined with older age, whereas serum and urinary aldosterone did not significantly decline. Correspondingly, the aldosterone-to-renin ratio was positively and independently associated with older age (adjusted β=+5.54 ng/dL per ng/mL per hour per 10 years, P<0.001). In contrast, when participants were assessed under sodium-restricted conditions, physiological stimulation of aldosterone was blunted with older age (β=-4.6 ng/dL per 10 years, P<0.0001). Aging is associated with a pattern of decreased normal zona glomerulosa CYP11B2 expression and increased aldosterone-producing cell cluster expression. This histopathologic finding parallels an age-related autonomous aldosteronism and abnormal aldosterone physiology that provides 1 potential explanation for age-related cardiovascular risk.

Expression and Histopathological Significance of Disabled-2 in Aldosterone-Producing Adenoma.

The current pathological diagnosis of aldosterone-producing adenoma (APA) is challenging because no histological markers of aldosterone production are available in routine practice. A previous study demonstrated that Disabled-2 (DAB2) is a specific marker of the zona glomerulosa (ZG) in rodents. The aim of the present study was to investigate the significance of immunohistochemical staining to detect DAB2 in the adrenal tissue of patients with APA. We investigated the expression of DAB2 in 36 adrenal glands with APA, 23 adrenal glands with cortisol-producing adenoma (CPA), and 33 adrenal glands with non-functioning adenoma (NFA). Immunohistochemical staining was performed using anti-DAB2 antibodies on paraffin-embedded sections. We analysed the expression of DAB2 semi-quantitatively by scoring staining intensity, and assessed the correlation of this information with the clinical findings. DAB2 mRNA expression in adenoma tissues was evaluated by RT-PCR. DAB2 was highly expressed in the ZG in normal human adrenal glands. DAB2 expression was heterogeneous in APA, with spotted, strong staining noted in most samples (25 of 36 APA). CPA and NFA also exhibited extensive low or moderate DAB2 expression. DAB2 mRNA was significantly increased and positively correlated with CYP11B2 in APA (p<0.05). In APA, the DAB2 score adjusted for tumour volume was positively correlated with plasma aldosterone (p<0.05). Patients with low or moderate DAB2 staining more frequently exhibited high blood pressure and were diagnosed at a younger age compared with patients with high DAB2 staining. The present study clearly demonstrates that DAB2 is a specific marker of the ZG in normal human adrenal glands but that DAB2 immunostaining is not sufficiently powerful for histopathological diagnosis of APA. DAB2 might be involved in excessive aldosterone biosynthesis and correlate with specific clinical characteristics of APA patients.