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Summary Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of ACTH-independent Cushing syndrome (CS). This condition is characterized by glucocorticoid and/or mineralocorticoid excess, and is commonly regulated by aberrant G-protein coupled receptor expression may be subclinical, allowing the disease to progress for years undetected. Inhibin A is a glycoprotein hormone and tumor marker produced by certain endocrine glands including the adrenal cortex, which has not been previously investigated as a potential tumor marker for PBMAH. In the present report, serum inhibin A levels were evaluated in three patients with PBMAH before and after adrenalectomy. In all cases, serum inhibin A was elevated preoperatively and subsequently fell within the normal range after adrenalectomy. Additionally, adrenal tissues stained positive for inhibin A. We conclude that serum inhibin A levels may be a potential tumor marker for PBMAH. Learning points: PBMAH is a rare cause of CS. PBMAH may have an insidious presentation, allowing the disease to progress for years prior to diagnosis. Inhibin A is a heterodimeric glycoprotein hormone expressed in the gonads and adrenal cortex. Inhibin A serum concentrations are elevated in some patients with PBMAH, suggesting the potential use of this hormone as a tumor marker. Further exploration of serum inhibin A concentration, as it relates to PBMAH disease progression, is warranted to determine if this hormone could serve as an early detection marker and/or predictor of successful surgical treatment.

Summary Mifepristone is a promising option for the management of hypercortisolism associated with hyperglycemia. However, its use may result in serious electrolyte imbalances, especially during dose escalation. In our patient with adrenocorticotropic hormone-independent macro-nodular adrenal hyperplasia, unilateral adrenalectomy resulted in biochemical and clinical improvement, but subclinical hypercortisolism persisted following adrenalectomy. She was started on mifepristone. Unfortunately, she missed her follow-up appointments following dosage escalation and required hospitalization at an intensive care level for severe refractory hypokalemia. Learning points: Mifepristone, a potent antagonist of glucocorticoid receptors, has a high risk of adrenal insufficiency, despite high cortisol levels. Mifepristone is associated with hypokalemia due to spill-over effect of cortisol on unopposed mineralocorticoid receptors. Given the lack of a biochemical parameter to assess improvement, the dosing of mifepristone is based on clinical progress. Patients on mifepristone require anticipation of toxicity, especially when the dose is escalated. The half-life of mifepristone is 85 h, requiring prolonged monitoring for toxicity, even after the medication is held.

Context: The literature on outcomes of bilateral adrenalectomy (BADx) in Cushing's syndrome (CS) is scant. Aims: The aim of this study is to analyze the short- and long-term outcomes of patients who underwent BADx and to compare the outcomes among different etiologies of CS. Settings and Design: This is a retrospective analysis of patients who underwent BADx for CS at our center between 2005 and 2018. Materials and Methods: In all, 33 patients were studied for clinical outcomes, survival rates, and long-term complications. Statistical Analysis: All analyses were performed with SPSS software (version 21.0). Results: The mean age at surgery was 39.33 ± 15.67 years. The primary etiology for CS was Cushing's disease (CD) in 42.42%, ectopic source in 36.36%, primary pigmented nodular adrenocortical disease (PPNAD) in 12.12%, and adrenocorticotrophin hormone–independent macronodular adrenal hyperplasia (AIMAH) in 9.09% of patients. The median follow-up time was 72.77 months. Improvement in hypertension and diabetes status after surgery was seen in 78% and 76.19% of patients, respectively. Proximal myopathy improved in 68% of patients. Nelson's syndrome and adrenal crisis were seen in 21.4% of patients each on long-term follow-up. Total mortality after BADx was 33.3%. Mortality in the first 30 days after surgery was seen in five patients (15.15%). Higher cortisol levels at presentation and age more than 40 years were predictors of mortality. Among the Cushing's subtypes, PPNAD had the best prognosis followed by CD. Perioperative Infections were a major cause of mortality. Conclusion: BADx is an effective treatment for CS especially in patients with PPNAD and CD but carries a significant mortality rate too.

L´hyperplasie macronodulaire bilatérale des surrénales (HMBS) est une cause rare de syndrome de Cushing d´origine surrénalienne, représentant moins de 1% des cas. Nous rapportons le cas d´un patient âgé de 48 ans, diabétique et hypertendu. Présentant un syndrome de Cushing clinique. Le bilan étiologique a permis de retenir le diagnostic d´hypercorticisme «Adrenocorticotropic hormone» (ACTH) indépendant en rapport avec une HMBS. Le choix thérapeutique était en faveur d´une surrénalectomie unilatérale gauche orientée par la scintigraphie au noriodocholestérol, avec une bonne évolution. Cependant, vu le risque de récidive et de complications cardiovasculaires, une surveillance au long cours a été programmée.

Primary bilateral macronodular adrenal hyperplasia (PBMAH) is characterized by bilateral multiple adrenal macro-nodules that often cause mild over-secretion of cortisol in the form of subclinical Cushing’s syndrome. We herein describe a case, wherein unilateral adrenalectomy partially improved hyperglycemia in a patient with PBMAH and suggest the usefulness and limitations of this surgical strategy. A 64-year-old woman with type 2 diabetes had an incidental diagnosis of bilateral adrenal lesions. She had a family history of type 2 diabetes, and her HbA1c level was 8.9% under insulin therapy. She did not present with any symptoms associated with Cushing’s syndrome. The basal cortisol level was in the normal range (12.0 μg/dL); however, the adrenocorticotropic hormone (ACTH) level was suppressed (2.1 pg/mL) and the serum cortisol level was not suppressed in the dexamethasone test. Computed tomography and magnetic resonance imaging showed bilateral adrenal macro-nodules and (131)I-adosterol accumulated in the bilateral adrenal lesions. Collectively, she was diagnosed with subclinical Cushing’s syndrome due to PBMAH complicated with diabetes mellitus, hypertension, and dyslipidemia. Laparoscopic left adrenalectomy was performed, and the pathologic findings were consistent with PBMAH. After unilateral adrenalectomy, serum cortisol levels decreased, and hypertension improved. Both HbA1c levels and insulin requirement also decreased, but insulin therapy was continuously needed. It should be noted that hyperglycemia may not be cured after successful surgery in a patient with PBMAH. Additional operation or medical therapy should be considered if unilateral adrenalectomy is unable to correct hypercortisolism in PBMAH patients.

The genetic alterations that cause the development of glucocorticoid and/or mineralocorticoid producing benign adrenocortical tumors and hyperplasias have largely been elucidated over the past two decades through advances in genomics. In benign aldosterone-producing adrenocortical tumors and hyperplasias, alteration of intracellular calcium signaling has been found to be significant in aldosterone hypersecretion, with causative defects including those in KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CLCN2. In benign cortisol-producing adrenocortical tumors and hyperplasias abnormal cyclic adenosine monophosphate-protein kinase A signaling has been found to play a central role in tumorigenesis, with pathogenic variants in GNAS, PRKAR1A, PRKACA, PRKACB, PDE11A, and PDE8B being implicated. The role of this signaling pathway in the development of Cushing’s syndrome and adrenocortical tumors was initially discovered through the study of the underlying genetic defects causing the rare multiple endocrine neoplasia syndromes McCune-Albright syndrome and Carney complex with subsequent identification of defects in genes affecting the cyclic adenosine monophosphate-protein kinase A pathway in sporadic tumors. Additionally, germline pathogenic variants in ARMC5, a putative tumor suppressor, were found to be a cause of cortisol-producing primary bilateral macronodular adrenal hyperplasia. This review describes the genetic causes of benign cortisol- and aldosterone-producing adrenocortical tumors.

Summary Background GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome is caused by aberrant expression of the GIP receptor in adrenal lesions. The bilateral nature of this disease suggests germline genetic predisposition. We aimed to identify the genetic driver event responsible for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Methods We conducted a multicentre, retrospective, cohort study at endocrine hospitals and university hospitals in France, Canada, Italy, Greece, Belgium, and the Netherlands. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Adrenal samples from patients with primary bilateral macronodular adrenal hyperplasia who had undergone an adrenalectomy for overt or mild Cushing's syndrome without evidence of food-dependent cortisol production and those with GIP-dependent unilateral adrenocortical adenomas were used as control groups. We performed whole genome, whole exome, and targeted next generation sequencing, and copy number analyses of blood and adrenal DNA from patients with familial or sporadic disease. We performed RNA sequencing on adrenal samples and functional analyses of the identified genetic defect in the human adrenocortical cell line H295R. Findings 17 patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome were studied. The median age of patients was 43·3 (95% CI 38·8–47·8) years and most patients (15 [88%]) were women. We identified germline heterozygous pathogenic or most likely pathogenic variants in the KDM1A gene in all 17 patients. We also identified a recurrent deletion in the short p arm of chromosome 1 harboring the KDM1A locus in adrenal lesions of these patients. None of the 29 patients in the control groups had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in adrenal lesions. Global gene expression analysis showed GIP receptor upregulation with a log2 fold change of 7·99 (95% CI 7·34–8·66; p=4·4 × 10−125), and differential regulation of several other G protein-coupled receptors in GIP-dependent primary bilateral macronodular hyperplasia samples compared with control samples. In vitro pharmacological inhibition and inactivation of KDM1A by CRISPR-Cas9 genome editing resulted in an increase of GIP receptor transcripts and protein in human adrenocortical H295R cells. Interpretation We propose that GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome results from a two-hit inactivation of KDM1A, consistent with the tumour suppressor gene model of tumorigenesis. Genetic testing and counselling should be offered to these patients and their relatives. Funding Agence Nationale de la Recherche, Fondation du Grand defi Pierre Lavoie, and the French National Cancer Institute.

Primary bilateral adrenal macronodular hyperplasia is characterized by functioning adrenal macronodules and variable cortisol secretion. Familial clustering suggests a genetic cause that has been confirmed with the identification of some genetic mutations, including inactivating germline mutations, in armadillo repeat containing 5 (ARMC5) gene. The identification of the pathogenic variant enables the physician to identify and treat these patients earlier and more effectively. It has also been noticed that patients with germline causative variants show a different clinical spectrum, presenting specific clinical characteristics, as the association with the presence of meningiomas.

This review reports the main molecular alterations leading to development of benign cortisol- and/or aldosterone-secreting adrenal tumors. Causes of adrenal Cushing syndrome can be divided in 2 groups: multiple bilateral tumors or adenomas secreting cortisol. Bilateral causes are mainly primary pigmented nodular adrenocortical disease, most of the time due to PRKAR1A germline-inactivating mutations, and primary bilateral macronodular adrenal hyperplasia that can be caused in some rare syndromic cases by germline-inactivating mutations of MEN1, APC, and FH and of ARMC5 in isolated forms. PRKACA somatic-activating mutations are the main alterations in unilateral cortisol-producing adenomas. In primary hyperaldosteronism (PA), familial forms were identified in 1% to 5% of cases: familial hyperaldosteronism type I (FH-I) due to a chimeric CYP11B1/CYP11B2 hybrid gene, FH-II due to CLCN-2 germline mutations, FH-III due to KCNJ5 germline mutations, FH-IV due to CACNA1H germline mutations and PA, and seizures and neurological abnormalities syndrome due to CACNA1D germline mutations. Several somatic mutations have been found in aldosterone-producing adenomas in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 genes. In addition to these genetic alterations, genome-wide approaches identified several new alterations in transcriptome, methylome, and miRnome studies, highlighting new pathways involved in steroid dysregulation.

Context. Approximately 50% of familial cases of primary bilateral macronodular adrenal hyperplasia (PBMAH) are caused by mutations in the ARMC5 gene. Case report. We report the case of a 37 year-old patient of Haitian origin, who presented with resistant hypertension. His workup showed high aldosterone (410 pmol/L) with suppressed renin levels (0.2 ng/mL/h) with an aldosterone to renin ratio (ARR) of 2050. Patient also had suppressed ACTH levels ( Genetic analyses. Following genetic counselling, MEN1 gene analysis was performed using sequencing/MLPA techniques but did not reveal a mutation. Initial genetic testing included ARMC5 gene analysis using direct Sanger sequencing which was negative. However, using Next-Generation Sequencing (NGS) and MLPA analysis, a heterozygous germline ARMC5 deletion of exons 5-8 was identified. The deletion is predicted to prematurely truncate the protein product and cause loss of function. The ARMC5 deletion segregated with the disease in his 24 yo son who had bilateral adrenal adenomas that appeared to be non-functional. The patient’s father was also known for having bilateral adrenal masses and hypertension. To our knowledge we report the second case of ARMC5 deletion in familial PBMAH. Suzuki et al. reported two patients, a mother and her son, carrying ARMC5 deletion of exons1-5 and interestingly they were also affected by PBMAH co-secreting cortisol and aldosterone (1). As in this case report, the ARMC5 deletion was missed using Sanger sequencing initially. Conclusion. These cases demonstrate that large deletions may be missed by Sanger sequencing and that the real prevalence of ARMC5 mutations may have been underestimated. The link between deletion of ARMC5 and correlation with PBMAH co-secreting aldosterone and cortisol remains to be determined but may be a step forward for genotype-phenotype correlation. 1.Suzuki S, et al. Endocrine practice: official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists. 2015;21 (10):1152-60.

BACKGROUND: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a known rare cause of Cushing’s syndrome (CS). A mutation in the armadillo repeat containing 5 (ARMC5) sequence is associated with up to 55% of PBMAH cases. Recent studies have linked ARMC5 mutations to presence of other benign neoplasias suggesting that ARMC5 could be a tumor suppressor gene. Case: 72-year-old female with a history of obesity, HTN, DM2, osteoporosis, multiple meningiomas, and hypercalcemia with recurrent kidney stones was incidentally found to have bilateral adrenal nodules on CT imaging. She had mild cushingoid features with truncal obesity and moon facies. She had multiple low dose dexamethasone suppression tests with AM cortisol levels in 17-21 ug/dL range (

Summary PBMAH is a rare etiology of Cushing syndrome (CS). Familial clustering suggested a genetic cause that was recently confirmed, after identification of inactivating germline mutations in armadillo repeat-containing 5 (ARMC5) gene. A 70-year-old female patient was admitted due to left femoral neck fracture in May 2014, in Orthopedics Department. During hospitalization, hypertension (HTA) and hypokalemia were diagnosed. She presented with clinical signs of hypercortisolism and was transferred to the Endocrinology ward for suspected CS. Laboratory workup revealed: ACTH Learning points: PBMAH is a rare etiology of CS, characterized by functioning adrenal macronodules and variable cortisol secretion. The asymmetric/asynchronous involvement of only one adrenal gland can also occur, making disease diagnosis a challenge. Familial clustering suggests a genetic cause that was recently confirmed, after identification of inactivating germline mutations in armadillo repeat-containing 5 (ARMC5) gene. The insidious nature of this disease and the well-known deleterious effect of hypercortisolism favor genetic study of other family members, to diagnose and treat these patients timely. As ARMC5 is expressed in many organs and recent findings suggest an association of PBMAH and meningioma, a watchful follow-up is required.

Introduction: Adrenal myelolipomas are usually isolated benign adrenal lesions, but can be found adjacent to steroid-secreting adrenocortical tumors. We studied the aberrant regulation of cortisol secretion in a 61 year-old woman with combined bilateral myelolipomas and primary bilateral macronodular adrenal hyperplasia (BMAH) causing Cushing’s syndrome. The goal was to identify potential aberrant ligands and hormone receptors regulating cortisol excess, using both an in vivo investigation protocol and in vitro studies on resected adrenals. Materials and methods: Cortisol response was measured during in vivo tests that transiently modulated the levels of ligands for potential aberrant receptors, including GIP. Response to medical therapies decreasing GIP was monitored. Expression of ACTH and of GIP receptors were examined in resected adrenal tissues by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Results: In vivo, cortisol increased in response to mixed meals (+353%), oral 75 g glucose (+71%), GIP infusion (+416%) and hLH IV (+243%). Suppression of GIP by pasireotide improved cortisol secretion but produced hyperglycemia. The left adrenal was predominantly composed of myelolipoma and strands of BMAH, while the right was mainly composed of BMAH with some foci of myelolipoma on pathology. No ACTH was detectable by immunohistochemistry in BMAH or myelolipomas tissue. Ectopic GIP receptor was confirmed by RT-PCR and immunohistochemistry in BMAH tissues but not in the myelolipomas. Conclusion: This is the first report of interspersed myelolipoma and BMAH with GIP-dependent Cushing’s syndrome. Their simultaneous occurrence may suggest yet to be identified adrenocortical tissue paracrine factors regulated by aberrant receptors which could stimulate adipocyte and hematopoietic cell proliferation.

Introduction: Asymptomatic adrenal masses are often discovered incidentally while testing for unrelated conditions. A very uncommon finding is thickening and increased nodularity of both adrenal glands, which can lead to the patient being diagnosed with subclinical Cushing's syndrome, Conn's syndrome, or both. Case presentation: A 45-year-old African-American man presented to the hospital with a one-month history of increased abdominal girth, bilateral lower extremity edema and shortness of breath, with progressive epigastric abdominal pain which prompted admission. His medical history was relevant for obesity, coronary artery disease and heart failure with reduced ejection fraction. On examination he had a blood pressure of 152/99 mmHg, was diaphoretic, and complained of constant epigastric pain. Multiple purple and pale striae were visible along his abdomen, and he had pitting edema in both lower extremities. His initial laboratory studies showed a potassium of 2.6 mEq/L. A CT scan of the abdomen was relevant for bilaterally enlarged and tortuous adrenal glands, which the patient had no previous knowledge of. Diuresis greatly improved his edema and shortness of breath. Additional laboratory tests showed persistent hypokalemia, normal plasma renin activity and aldosterone, elevated fasting cortisol, normal metanephrines in 24-hour urine collection, and low ACTH level. A suppression test done by administering dexamethasone 0.5 mg every 6 hours for 2 days, was able to suppress the elevated cortisol levels. He was started on spironolactone, and underwent MRI of the abdomen, which confirmed bilateral multinodular adrenal hyperplasia with no imaging characteristics of pheochromocytoma. The patient was discharged home once his clinical status improved. Laboratory studies done at follow-up appointments showed persistently elevated cortisol, normal aldosterone to renin ratio, normal levels of plasma metanephrines and suppressed levels of ACTH. He was also referred to surgery for eventual bilateral adrenalectomy. Discussion: Primary bilateral macronodular adrenal hyperplasia (BMAH) is a very rare cause of endogenous Cushing's syndrome, representing less than 2% of cases. It is characterized by enlarged adrenal glands containing pigmented nodules that secrete excess cortisol and/or aldosterone (1). BMAH is often an incidental finding in patients with subclinical hyper-cortisolism. A low level of plasma ACTH along with bilaterally enlarged adrenal glands on imaging suggest the diagnosis, and this is confirmed with a dexamethasone suppression test. Accurate detection is important for guiding management, which includes pharmacological therapy and bilateral adrenalectomy. Reference: 1. De Venanzi A, Alencar GA, Bourdeau I, Fragoso MCBV, Lacroix A. Primary bilateral macronodular adrenal hyperplasia. Curr Opin Endocrinol Diabetes Obes. 2014 Jun;21(3):177-84.

To investigate Armadillo repeat-containing 5 (ARMC5) mutations in Chinese patients with familial and sporadic primary bilateral macronodular adrenal hyperplasia (PBMAH), we performed clinical data collection and ARMC5 sequencing for three PBMAH families and 23 sporadic PBMAH patients. ARMC5 pathogenic germline mutations were identified in all 3 PBMAH families. Secondary ARMC5 somatic mutations were found in two adrenal nodules from two PBMAH family members with ARMC5 germline mutations. PBMAH family members with ARMC5 pathogenic germline mutations displayed various clinical manifestations. ARMC5 pathogenic germline mutations were identified in 5 sporadic PBMAH patients among whom one patient displayed both hypercortisolism and primary aldosteronism. We detected a total of 10 ARMC5 pathogenic mutations, of which 8 had not been previously reported. Our results suggest that ARMC5 pathogenic germline mutations are common in familial and sporadic Chinese PBMAH patients, and demonstrate the importance of ARMC5 screening in PBMAH family members to detect patients with insidious PBMAH.

Bilateral macronodular adrenal hyperplasia (BMAH) is a relatively rare cause of Cushing's Syndrome (CS). In recent years, growing evidence has shown that steroidogenesis is regulated by aberrant G-protein-coupled receptors (GPCRs) expression and their ligands, in a significant proportion of patients with BMAH. The screening of patients with overt or subclinical CS demonstrates the frequent expression of several GPCRs that opened the option to potential therapeutic applications. Thus, several studies have demonstrated that targeting the involved receptor with specific antagonists may result in a more or less effective control of cortisol excess. Bilateral adrenalectomy has traditionally been considered the treatment of choice for BMAH. However, unilateral adrenalectomy has been recently proposed as an alternative in selective patients to avoid the long-term necessity of gluco/mineralocorticoid replacement. Adrenal steroidogenesis inhibitors remain a valid option when medical treatment is needed due to high surgical risk.

Context: Elevated human choriogonadotropin (hCG) may stimulate aberrantly expressed luteinizing hormone (LH)/hCG receptor (LHCGR) in adrenal glands, resulting in pregnancy-induced bilateral macronodular adrenal hyperplasia and transient Cushing syndrome (CS). Objective: To determine the role of LHCGR in transient, pregnancy-induced CS. Design, Setting, Patient, and Intervention: We investigated the functional implications of LHCGRs in a patient presenting, at a tertiary referral center, with repeated pregnancy-induced CS with bilateral adrenal hyperplasia, resolving after parturition. Main Outcome Measures and Results: Acute testing for aberrant hormone receptors was negative except for arginine vasopressin (AVP)–increased cortisol secretion. Long-term hCG stimulation induced hypercortisolism, which was unsuppressed by dexamethasone. Postadrenalectomy histopathology demonstrated steroidogenically active adrenocortical hyperplasia and ectopic cortical cell clusters in the medulla. Quantitative polymerase chain reaction showed upregulated expression of LHCGR, transcription factors GATA4, ZFPM2, and proopiomelanocortin (POMC), AVP receptors (AVPRs) AVPR1A and AVPR2, and downregulated melanocortin 2 receptor (MC2R) vs control adrenals. LHCGR was localized in subcapsular, zona glomerulosa, and hyperplastic cells. Single adrenocorticotropic hormone–positive medullary cells were demonstrated in the zona reticularis. The role of adrenal adrenocorticotropic hormone was considered negligible due to downregulated MC2R. Coexpression of CYP11B1/CYP11B2 and AVPR1A/AVPR2 was observed in ectopic cortical cells in the medulla. hCG stimulation of the patient’s adrenal cell cultures significantly increased cyclic adenosine monophosphate, corticosterone, 11-deoxycortisol, cortisol, and androstenedione production. CTNNB1, PRKAR1A, ARMC5, and PRKACA gene mutational analyses were negative. Conclusion: Nongenetic, transient, somatic mutation-independent, pregnancy-induced CS was due to hCG-stimulated transformation of LHCGR-positive undifferentiated subcapsular cells (presumably adrenocortical progenitors) into LHCGR-positive hyperplastic cortical cells. These cells respond to hCG stimulation with cortisol secretion. Without the ligand, they persist with aberrant LHCGR expression and the ability to respond to the same stimulus., Précis: Pregnancy-induced CS due to hCG-stimulated transformation of LHCGR-positive undifferentiated subcapsular cells into LHCGR-positive hyperplastic cortical cells with hCG-stimulated cortisol secretion.

Germline loss-of-function mutations in the armadillo-repeat-containing 5 (ARMC5) gene are an established cause of Cushing's syndrome due to bilateral macronodular adrenal hyperplasia (BMAH), 1,2 and may play a role in primary aldosteronism. 3 As familial hyperaldosteronism type II (FH-II) has a presumed genetic basis, 4 we hypothesised that germline ARMC5 mutations underlie FH-II. We interrogated whole-exome sequencing data from four FH-II families. We did not identify any pathogenic ARMC5 variants which segregated with the phenotype of primary aldosteronism. Refereed/Peer-reviewed

Context: Maffucci syndrome is a rare, nonhereditary, mesodermal dysplastic disease characterized by the presence of multiple hemangiomas and enchondromas. This pathological condition, which is often unrecognized, is associated with a high prevalence of benign and malignant endocrine tumors involving pituitary, adrenal, thyroid, and parathyroid glands. Case Description: We describe the case of a young patient presenting a history suggestive of secondary arterial hypertension and typical features of Maffucci syndrome (multiple hemangiomas and enchondromas), which were unrecognized over the previous 3 decades. Given that endocrine diseases are common causes of secondary arterial hypertension and are often associated with Maffucci syndrome, a comprehensive diagnostic workup was performed, revealing the presence of large bilateral adrenal masses (70 mm right, 35 mm left) and autonomous cortisol secretion (adrenocorticotropic hormone–independent Cushing syndrome). The patient underwent a bilateral adrenalectomy, and steroid replacement therapy was initiated. Surgery resulted in a normalization of arterial blood pressure, and antihypertensive treatment was discontinued. Histological examinations revealed morphological features of primary bilateral macronodular adrenal hyperplasia. Conclusions: Early recognition and lifelong monitoring of Maffucci syndrome is required to identify and treat possible associated endocrine diseases and malignancies. Among them, unilateral cortical adrenal masses have been previously described, but to our knowledge, this is the first reported case of Maffucci syndrome associated with primary bilateral macronodular adrenal hyperplasia. Additional studies are needed to establish the etiopathological link between these 2 entities and, more in general, between Maffucci syndrome and endocrine diseases, but possible common genetic alterations may be suggested., Précis: Establishing the diagnosis of Maffucci syndrome in a patient with suspected secondary arterial hypertension prompted an extensive workup revealing primary bilateral macronodular adrenal hyperplasia.

Context: Recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3 have been identified in aldosterone-producing adenomas (APAs). The question as to whether they are responsible for both nodulation and aldosterone production is not solved. Case Description: We describe the case of a young patient who was diagnosed with severe arterial hypertension due to primary aldosteronism at age 26 years, followed by hemorrhagic stroke 4 years later. Abdominal computed tomography showed bilateral macronodular adrenal hyperplasia. Identification of lateralized aldosterone secretion led to right adrenalectomy, followed by normalization of biochemical and hormonal parameters and amelioration of blood pressure. The resected adrenal showed three nodules, one of them expressing aldosterone synthase and harboring a somatic KNCJ5 mutation. A Weiss revisited index of 3 of the APA prompted us to perform a second 18F-2-fluoro-2-deoxy-D-glucose-positron emission tomography after surgery, which revealed abnormal rectal activity despite the absence of clinical symptoms. Gastrointestinal exploration showed multiple polyps with severe dysplasia, and the diagnosis of familial adenomatous polyposis was established in the presence of a germline heterozygous APC gene mutation. Sequencing of somatic DNA from the APA and a second adrenal nodule revealed biallelic APC inactivation due to loss of heterozygosity in both nodules. Conclusions: This case report underlines the need for establishing the frequency of germline APC variants in patients with primary aldosteronism and bilateral macronodular adrenal hyperplasia because their presence may predispose to APA development and severe hypertension well before the first familial adenomatous polyposis symptoms appear. From a mechanistic point of view, it supports a two-hit model for APA development, whereby the first hit drives increased cell proliferation whereas the second hit specifies the pattern of hormonal secretion.

International audience; In the normal human adrenal gland, steroid secretion is regulated by a complex network of autocrine/paracrine interactions involving bioactive signals released by endothelial cells, nerve terminals, chromaffin cells, immunocompetent cells, and adrenocortical cells themselves. ACTH can be locally produced by medullary chromaffin cells and is, therefore, a major mediator of the corticomedullary functional interplay. Plasma ACTH also triggers the release of angiogenic and vasoactive agents from adrenocortical cells and adrenal mast cells and, thus, indirectly regulates steroid production through modulation of the adrenal blood flow. Adrenocortical neoplasms associated with steroid hypersecretion exhibit molecular and cellular defects that tend to reinforce the influence of paracrine regulatory loops on corticosteroidogenesis. Especially, ACTH has been found to be abnormally synthesized in bilateral macronodular adrenal hyperplasia responsible for hypercortisolism. In these tissues, ACTH is detected in a subpopulation of adrenocortical cells that express gonadal markers. This observation suggests that ectopic production of ACTH may result from impaired embryogenesis leading to abnormal maturation of the adrenogonadal primordium. Globally, the current literature indicates that ACTH is a major player in the autocrine/paracrine processes occurring in the adrenal gland in both physiological and pathological conditions.

The Editorial on the Research Topic Adrenal cortex: from physiology to disease The adrenal gland plays essential roles in the control of body homeostasis, stress, and immune responses. The adrenal cortex represents up to 90% of the gland and is specialized in the production of adrenal steroids. The coordinate production of these steroids relies on adrenal cortex zonation, which corresponds to the establishment of distinct concentric functional zones in the perinatal period: outermost zona glomerulosa synthesizes mineralocorticoids, zona fasciculata produces glucocorticoids, and innermost zona reticularis synthesizes both glucocorticoids and adrenal androgens. This zonal organization has to be maintained throughout the life of the individual, despite constant centripetal tissue renewal. The review by Pihlajoki et al. summarizes the latest findings on the mechanisms of adrenal cortex renewal, which relies on outer cortex progenitors recruitment and lineage conversion along cell migration within the cortex. This paper also provides a comprehensive overview of the hormones, signaling pathways, and transcription factors that control these processes to allow for on-demand adaptation of cortical function and maintenance of adrenal homeostasis. Defects in adrenal development and maintenance are associated with adrenal insufficiency, a life threatening condition for which lifelong hormonal replacement therapies can be challenging. The review by Ruiz-Babot and colleagues sheds light on novel developments in the field of adrenal replacement, including pluripotent cell reprograming and the use of encapsulating devices with semi-permeable membranes to avoid immune rejection of grafts. These promising approaches could pave the way for future clinical management of adrenal-insufficiency patients. While adrenal insufficiency is clinically problematic, the opposite situation in which adrenal steroid production is increased also raises significant clinical concerns. Hypercortisolism results in Cushing’s syndrome associated with central obesity, arterial hypertension, immunosuppression, and depression. Hyperaldosteronism is associated with high blood pressure and profound cardiovascular and renal alterations, which result in increased risk of cardiovascular failure. These highly morbid syndromes are the consequence of either benign hyperplasia and tumors or adrenocortical cancer (ACC). The review by Boulkroun and colleagues establishes the molecular bases of normal control of aldosterone production and elaborates on recent next-generation sequencing (NGS) analyses that allowed identification of mutations in potassium and calcium channels as key players in the development of hyperaldosteronism. Even if these mutations can explain increased aldosterone secretion, they are unlikely to account for tumor growth. Boulkroun et al. summarize data showing that deregulated cell growth in aldosterone-producing adenomas is likely to result from WNT and SHH signaling pathway activation in these tumors. Deregulated protein kinase A (PKA) signaling is a common theme in adrenal tumors associated with ACTH-independent hypercortisolism. These include primary pigmented adrenocortical disease (PPNAD), adrenal adenomas, bilateral macronodular adrenal hyperplasia (BMAH), and adrenal cancer. The review by Berthon et al. provides in-depth insight into the genetic causes of deregulated PKA signaling, including inactivating PRKAR1A mutations in PPNAD and activating PRKACA mutations in cortisol-producing adenomas. Interestingly, mutations in either PRKAR1A or PRKACA were not found in BMAH. The review by Drougat et al. emphasizes the discovery of mutations in ARMC5 as a likely cause of these particular benign adrenal tumors and elaborates on potential pathogenic mechanisms. Lefebvre et al. shed another light on BMAH by focusing on the paracrine regulation of cortisol secretion. They gather data showing that cortisol secretion is stimulated by the release of a number of factors either produced by non-steroidogenic cells within the cortex (mast, chromaffin, and endothelial cells) or by a subset of aberrantly differentiated steroidogenic cells that can release serotonin or even ACTH within the hyperplastic tissue. They further suggest that aberrant ACTH production and expression of ectopic receptors, such as the receptors of LH, GIP, and 5-HT7, may be the result of aberrant differentiation of gonadal-like cells, triggered by driver mutations, such as ARMC5 inactivation. Beyond steroid hormone excess, which is also observed in about 40–60% of patients, ACC still represents a major therapeutic challenge. The review by Libe and colleagues provides an overview of current diagnosis and treatment of ACC, which emphasizes the need for novel therapeutic targets in a cancer with dismal prognosis. The review by Drougat and colleagues provides insight into the role of mutations targeting the WNT signaling pathway (essentially activating mutations of CTNNB1 and deletions of ZNRF3) and their pathogenic role in ACC. This paper on adult ACC is nicely complemented by Lalli and Figueiredo’s review that focuses on pediatric ACC. These are rare tumors that generally occur in the context of TP53 alterations, in particular the specific R337H mutation found with high frequency in Southern Brazil. The authors present evidence that these tumors are likely to derive from the fetal adrenal and discuss the common and divergent alterations found in pediatric and adult ACC, which highlights the lack of effective prognosis markers in the former. Deregulation of miRNA production is a common theme in most cancers. Nadia Cherradi provides a comprehensive overview of miRNA deregulation in ACC and shows that they can provide novel insight into the pathogenesis of ACC and may constitute interesting therapeutic targets. This review also highlights the usefulness of circulating miRNAs as novel non-invasive diagnostic and prognostic biomarkers in ACC. It further elaborates on an exciting aspect of miRNAs biology that involves their circulation within ACC cell-derived exosomes, which would allow communication with tumor microenvironment. We hope that you will find this topic inspiring and that it will shed light on exciting aspects of adrenal physiology and disease.

Objective Bilateral adrenalectomy or unilateral adrenalectomy and contralateral partial adrenalectomy are indicated for the treatment of ACTH-independent macronodular adrenal hyperplasia. Independent of the surgical procedure, the prognosis is poor. This paper discusses a new treatment method and its efficacy for treating nodular adrenal hyperplasia. Methods We performed a retrospective review of the medical records of 12 patients operated on between January 2008 and October 2014 at the First Affiliated Hospital of Zhengzhou University. All patients were treated by laparoscopic subcutaneous transposition of a pedicled adrenal. We performed postoperative monitoring of patients including clinical symptoms and 24-hour levels of serum free and urinary free cortisol. Results All twelve patients were pathologically confirmed to have nodular adrenal hyperplasia, and were followed for an average of 45.5 months (range, 24–60 months). The clinical symptoms of all patients disappeared, and the 24-hour plasma free cortisol and urinary free cortisol levels were within the normal range. Conclusions Laparoscopic subcutaneous transposition of a pedicled adrenal is a new and effective method for treating bilateral macronodular adrenal hyperplasia, and can achieve long-term remission of Cushing’s syndrome.

International audience; CONTEXT:Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of primary adrenal Cushing's syndrome (CS). ARMC5 germline mutations have been identified recently in PBMAH.OBJECTIVE:To determine the prevalence of ARMC5 mutations and analyze genotype-phenotype correlation in a large cohort of unrelated PBMAH patients with subclinical or clinical CS.PATIENTS AND METHODS:ARMC5 was sequenced in 98 unrelated PBMAH index cases. PBMAH was identified by bilateral adrenal nodular enlargement on computed tomography scan. The effect on apoptosis of ARMC5 missense mutants was tested in H295R and HeLa cells. Clinical and hormonal data were collected including midnight and urinary free cortisol levels, ACTH, androgens, renin/aldosterone ratio, cortisol after overnight dexamethasone suppression test, cortisol and 17-hydroxyprogesterone after ACTH 1-24 stimulation and illegitimate receptor responses. Computed tomography and histological reports were analyzed.RESULTS:ARMC5-damaging mutations were identified in 24 patients (26%). The missense mutants and the p.F700del deletion were unable to induce apoptosis in both H295R and HeLa cell lines, unlike the wild-type gene. ARMC5-mutated patients showed an overt CS more frequently, compared to wild-type patients: lower ACTH, higher midnight plasma cortisol, urinary free cortisol, and cortisol after dexamethasone suppression test (P = .003, .019, .006, and

To evaluate the pathophysiology of altered cortisol secretion in patients with primary adrenal hypercortisolism, cortisol secretion was investigated in 12 patients, seven with a unilateral adenoma and five with ACTH-independent macronodular adrenal hyperplasia compared with age- and gender-matched controls and with patients with pituitary-dependent hypercortisolism. Pulsatile secretion was increased 2-fold (P = 0.04), attributable to increased event frequency (P = 0.002). All patients showed a significant diurnal rhythm with a delay in phase shift of 3 h (P = 0.01). Approximate entropy ratio, a feedback-sensitive measure, was increased compared with controls (P = 0.00003) but similar to that of pituitary-dependent hypercortisolism (P = 0.77), denoting loss of autoregulation. Cortisol burst-mass tended to be smaller in patients with ACTH-independent macronodular adrenal hyperplasia than in unilateral adenoma (P = 0.06). In conclusion, increased cortisol secretion in patients with primary adrenal Cushing’s syndrome is caused by amplified pulsatile secretion via event frequency modulation. We speculate that partial preservation of secretory regularity and diurnal rhythmicity point to incomplete autonomy of these tumors.

Summary Medical therapy for Cushing's syndrome due to bilateral macronodular adrenal hyperplasia (BMAH) is generally administered for a limited time before surgery. Aberrant receptors antagonists show inconsistent efficacy in the long run to prevent adrenalectomy. We present a patient with BMAH, treated for 10 years with low doses of ketoconazole to control cortisol secretion. A 48-year-old woman presented with headaches and hypertension. Investigations showed the following: no clinical signs of Cushing's syndrome; enlarged lobulated adrenals; normal creatinine, potassium, and aldosterone; normal urinary aldosterone and metanephrines; elevated urinary free cortisol and steroid metabolites; and suppressed plasma renin activity and ACTH. A screening protocol for aberrant adrenal receptors failed to show any illegitimate hormone dependence. Ketoconazole caused rapid normalisation of cortisol and ACTH that persists over 10 years on treatment, while adrenals show no change in shape or size. Ketoconazole decreases cortisol in patients with Cushing's syndrome, and may prevent adrenal overgrowth. Steroid secretion in BMAH is inefficient as compared with normal adrenals or secreting tumours and can be controlled with low, well-tolerated doses of ketoconazole, as an alternative to surgery. Learning points Enlarged, macronodular adrenals are often incidentally found during the investigation of hypertension in patients harboring BMAH. Although laboratory findings include low ACTH and elevated cortisol, the majority of patients do not display cushingoid features. Bilateral adrenalectomy, followed by life-long steroid replacement, is the usual treatment of this benign condition, and alternative medical therapy is sought. Therapy based on aberrant adrenal receptors gives disappointing results, and inhibitors of steroidogenesis are not always well tolerated. However, ketoconazole at low, well-tolerated doses appeared appropriate to control adrenal steroid secretion indefinitely, while preventing adrenal overgrowth. This treatment probably constitutes the most convenient long-term alternative to surgery.

Context: Bilateral macronodular adrenal hyperplasia (BMAH) is a rare form of adrenal Cushing's syndrome. Familial cases have been reported, but at the time we conducted this study, the genetic basis of BMAH was unknown. Recently, germline variants of armadillo repeat containing 5 (ARMC5) in patients with isolated BMAH and somatic, second-hit mutations in tumor nodules, were identified. Conclusions: Our studies have detected ARMC5 mutations in 4 of 5 BMAH families tested, confirming that these mutations are a frequent cause of BMAH. Two of the 4 families had novel mutations, indicating allelic heterogeneity. Preclinical evaluation did not predict mutation status. The ARMC5-negative family had unusual prominent hyperaldosteronism. Further studies are needed to determine the penetrance of BMAH in ARMC5 mutation-positive relatives of affected patients, the practical utility of genetic screening and genotype-phenotype correlations. Design: We performed whole exome capture and sequencing of 2 affected individuals from each of 4 BMAH families (BMAH-01, BMAH-02, BMAH-03, and BMAH-05). Based on clinical evaluation, there were 7, 3, 3, and 4 affected individuals in these families, respectively. Sanger sequencing of ARMC5 was performed in 1 other BMAH kindred, BMAH-06. Objective: Our objective was to identify the genetic basis of familial BMAH. Results: Exome sequencing identified novel variants Chr16:g.31477540, c.2139delT, p.(Thr715Leufs*1) (BMAH-02) and Chr16:g.31473811, c.943C-->T, p.(Arg315Trp) (BMAH-03) in ARMC5 (GRch37/hg19), validated by Sanger sequencing. BMAH-01 had a recently reported mutation Chr16:g.31476121, c.1777C-->T, p.(Arg593Trp). Sanger sequencing of ARMC5 in BMAH-06 identified a previously reported mutation, Chr16:g.31473688; c.799C-->T, p.(Arg267*). The genetic basis of BMAH in BMAH-05 was not identified. Refereed/Peer-reviewed

Background Corticotropin-independent macronodular adrenal hyperplasia may be an incidental finding or it may be identified during evaluation for Cushing’s syndrome. Reports of familial cases and the involvement of both adrenal glands suggest a genetic origin of this condition. Methods We genotyped blood and tumor DNA obtained from 33 patients with corticotropinindependent macronodular adrenal hyperplasia (12 men and 21 women who were 30 to 73 years of age), using single-nucleotide polymorphism arrays, microsatellite markers, and whole-genome and Sanger sequencing. The effects of armadillo repeat containing 5 (ARMC5) inactivation and overexpression were tested in cell-culture models. Results The most frequent somatic chromosome alteration was loss of heterozygosity at 16p (in 8 of 33 patients for whom data were available [24%]). The most frequent mutation identified by means of whole-genome sequencing was in ARMC5, located at 16p11.2. ARMC5 mutations were detected in tumors obtained from 18 of 33 patients (55%). In all cases, both alleles of ARMC5 carried mutations: one germline and the other somatic. In 4 patients with a germline ARMC5 mutation, different nodules from the affected adrenals harbored different secondary ARMC5 alterations. Transcriptome-based classification of corticotropin-independent macronodular adrenal hyperplasia indicated that ARMC5 mutations influenced gene expression, since all cases with mutations clustered together. ARMC5 inactivation decreased steroidogenesis in vitro, and its overexpression altered cell survival. Conclusions Some cases of corticotropin-independent macronodular adrenal hyperplasia appear to be genetic, most often with inactivating mutations of ARMC5, a putative tumorsuppressor gene. Genetic testing for this condition, which often has a long and insidious prediagnostic course, might result in earlier identification and better management. (Funded by Agence Nationale de la Recherche and others.)

ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a distinctive subtype of Cushing’s syndrome (CS), with different clinical manifestations according to the level of serum cortisol. Based on clinical manifestations and serum cortisol, we divide AIMAH into three types, subclinical AIMAH, clinical AIMAH and high-risk AIMAH, with varied treatment methods being adapted to different subtypes. At the same time, we describe 3 patients who represent these subtypes of this disease, and review some cases of AIMAH which have been previously reported in the English literature. To our knowledge, this is the first article discussing classification, diagnosis and treatment of this disease and should be useful for future therapy of AIMAH.

BACKGROUND Bilateral macronodular adrenal hyperplasia is a rare cause of primary adrenal Cushing’s syndrome. In this form of hyperplasia, hypersecretion of cortisol suppresses the release of corticotropin by pituitary corticotrophs, which results in low plasma corticotropin levels. Thus, the disease has been termed corticotropin-independent macronodular adrenal hyperplasia. We examined the abnormal production of corticotropin in these hyperplastic adrenal glands. METHODS We obtained specimens of hyperplastic macronodular adrenal tissue from 30 patients with primary adrenal disease. The corticotropin precursor proopiomelanocortin and corticotropin expression were assessed by means of a polymerase-chainreaction assay and immunohistochemical analysis. The production of corticotropin and cortisol was assessed in 11 specimens with the use of incubated explants and cell cultures coupled with hormone assays. Corticotropin levels were measured in adrenal and peripheral venous blood samples from 2 patients. RESULTS The expression of proopiomelanocortin messenger RNA (mRNA) was detected in all samples of hyperplastic adrenal tissue. Corticotropin was detected in steroidogenic cells arranged in clusters that were disseminated throughout the adrenal specimens. Adrenal corticotropin levels were higher in adrenal venous blood samples than in peripheral venous samples, a finding that was consistent with local production of the peptide within the hyperplastic adrenals. The release of adrenal corticotropin was stimulated by ligands of aberrant membrane receptors but not by corticotropinreleasing hormone or dexamethasone. A semiquantitative score for corticotropin immunostaining in the samples correlated with basal plasma cortisol levels. Corticotropin-receptor antagonists significantly inhibited in vitro cortisol secretion. CONCLUSIONS Cortisol secretion by the adrenals in patients with macronodular hyperplasia and Cushing’s syndrome appears to be regulated by corticotropin, which is produced by a subpopulation of steroidogenic cells in the hyperplastic adrenals. Thus, the hypercortisolism associated with bilateral macronodular adrenal hyperplasia appears to be corticotropin-dependent. (Funded by the Agence Nationale de la Recherche and others.)

Phosphodiesterases (PDEs) are key regulatory enzymes of intracellular cAMP levels. PDE11A function has been linked to predisposition to adrenocortical tumors.The aim of the study was to study the PDE11A gene in a large cohort of patients with ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in control subjects.The PDE11A entire coding region was sequenced in 46 patients with AIMAH and 192 controls. Two variants found in AIMAH patients were transiently expressed in HEK 293 and adrenocortical H295R cells for further functional studies.The frequency of all PDE11A variants was significantly higher among patients with AIMAH (28%) compared to controls (7.2%) (P = 5 × 10(-5)). Transfection of the two PDE11A variants found in AIMAH patients only (D609N or M878V) showed that cAMP levels were higher, after forskolin stimulation, in cells transfected with the PDE11A mutants, compared to cells transfected with the wild-type PDE11A in HEK 293 cells (P0.05). Moreover, transfection with mutants PDE11A increased transcriptional activity of a cAMP-response element reporter construct compared to wild-type PDE11A in HEK 293 cells (P0.0004 for D609N and P0.003 for M878V) and in the adrenocortical H295R cells (P0.05 for D609N and M878V). In addition, analysis of cAMP levels in intact living culture cells by fluorescence resonance energy transfer probes showed increased cAMP in forskolin-treated cells transfected with PDE11A variants compared with wild-type PDE11A (P0.05).We conclude that PDE11A genetic variants may increase predisposition to AIMAH.

Among the first tissues to be studied for their micro-RNA expression is the adrenal cortex; in this brief overview, we provide the first update on micro-RNAs for this tissue, which a useful trailblazer for other endocrine and steroidogenic glands. Our laboratory studied both normal adrenal cortex and benign adrenal tumors, such as bilateral adrenal hyperplasias (BAH). Massive macronodular adrenocortical disease (MMAD) [also known as ACTH-independent macronodular adrenal hyperplasia (AIMAH)] causes Cushing syndrome and is characterized by multiple bilateral cortical nodules or adenomas that lead to significant enlargement of the adrenal glands.1 Primary pigmented nodular adrenocortical disease (PPNAD) is a relatively unique form of BAH that is often associated with Carney complex, a multiple neoplasia syndrome; unlike MMAD, a disease of mostly adults, PPNAD presents with Cushing syndrome in early life.2 MicroRNAs (miRNAs) are RNAs of about 21–26 nucleotides in length and comprise a class of non-coding RNAs that are derived by cleavage from much larger precursor RNAs. Until now we can find up to 600 miRNAs in the miRBase Sequence Database but between 1,000 and 10,000 could be predicted.3 miRNAs have an important role in several biological processes, from regulation of immunity to organ biogenesis and cancer. miRNA genes can function as promoters of tumorigenesis (oncogenes) or suppressors of tumor formation (tumor suppressors), and hitherto have been reported to be altered in many tumors. An aberrant miRNA expression signature that is often shared has now been found in tumors from several tissues.4,5 We recently obtained miRNA expression analysis, in MMAD/AIMAH and PPNAD.4,6 Some downregulated or upregulated miRNAs were found to be implicated in adrenal tissue tumorigenesis and, like in other tissues, we found alterations in some miRNAs that are known to be altered in other tumors. Such an example was the family of miRNA let-7, members of which (let-7a, let-7b, let-7c and let-7g) were downregulated in PPNAD (but not in the MMAD) and have been significantly downregulated in lung and other cancers.7 A recent study shows that this miRNA is important as a regulator of the self-renewal of breast cancer stem cells.8 In addition, we detected other miRNAs to be expressed at high levels in MMAD and in PPNAD (despite their histologic differences) such as miR-210; this miRNA has been found to be upregulated in breast cancer and is regulated by the hypoxia-inducible factor.9 MiR-203, whose putative target gene is ABL1, a well known oncogene that is activated in chronic myelogenous leukemia,10 was downregulated in both MMAD and PPNAD4 and, thus, may act as a tumor suppressor in adrenocortical tumors. MiR-203 also targets the transcription factor p63 involved in proliferation and differentiation of epithelial cells.11 Notably, there are some miRNAs whose pattern of expression appears to be unique for adrenal tumors, such as the miR-449; one of the highest downregulated miRNA in PPNAD. Interestingly, when we used a bioinformatics algorithm (that was constructed to predict miRNA gene targets) that integrated expression data on PPNAD that we have previously published,12 a correlation was found between miR-449 and WISP2 expression levels, supporting the potential involvement of Wnt signaling in PPNAD that has been suggested by other studies.12,13 The Wnt signaling pathway, now under study for its wide regulation by miRNAs, is a complex network of proteins and has an important role in embryogenesis and cancer.14 We have shown that inhibition of PKA activity using PKA inhibitor H89 increased miR-449 expression and decreased WISP2 mRNA expression.4 Although the phenotype of the adrenal tumors may be immunologically or histologically widely different, these tumors can vary greatly in their clinical behavior and prognosis. When a more carefully and broader view is applied to the analysis of miRNA patterns of expression in the adrenal tumors, a very interesting correlation with clinicopathologic variables, which are important diagnostic/prognostic factors, can be observed. It seems that two miRNAs (miR-130a and miR-382) are showing an expression pattern that correlates with the diurnal cortisol levels in patients with MMAD, an index of the severity of their disease. We can conclude that, if carefully analyzed, miRNAs may show a specific signature in basically all tissues and their diseases; this property makes the analysis of the expression of miRNAs useful in differential diagnosis, prognosis and maybe the treatment of a variety of disorders.

Stimulation of cortisol secretion through abnormally expressed G protein-coupled receptors (GPCRs) is a frequent feature of ACTH-independent macronodular adrenal hyperplasia (AIMAH). This has opened a pharmacological strategy that targets GPCRs for the treatment of Cushing's syndrome in AIMAH. However, only few drugs are available for the presently described GPCRs.The objective of the study was to identify new GPCR targets for the pharmacological treatment of adrenal Cushing's syndrome.We designed a cDNA chip containing 865 nucleotidic sequences of GPCRs. mRNAs were extracted from three normal adrenals, 18 AIMAHs, four adrenals from Cushing's disease patients, and 13 cortisol-secreting adenomas. A set of GPCR mRNAs that showed significantly higher or lower expression in AIMAH than in normal adrenal were studied by quantitative RT-PCR analysis. Analysis of protein expression and function were performed on selected GPCRs.The study was conducted at a tertiary care center and basic research laboratories.The ACTH MC2 receptor showed a low expression in 15 of 18 AIMAHs samples, whereas several previously undescribed GPCR genes were found highly expressed in a subset of AIMAH, such as the receptors for motilin (MLNR; three of 18 AIMAHs) and γ-aminobutyric acid (GABBR1; five of 18 AIMAHs), and the α2A adrenergic receptor (ADRA2A; 13 of 18 AIMAHs), on which we focused our attention. Western blot and immunochemistry analyses showed expression of ADRA2A protein in AIMAH but not in normal adrenal cortex. The ADRA2A agonist clonidine enhanced both basal and stimulated cortisol production. Clonidine-induced increase in basal cortisol levels was blocked by the ADRA2A antagonist yohimbine.ADRA2A is a potential target for pharmacological treatment of Cushing's syndrome linked to AIMAH.

Cushing's syndrome due to ACTH-independent macronodular adrenal hyperplasia (AIMAH) can be associated with abnormal responses of aberrantly expressed adrenocortical receptors. This study aimed to characterize in vitro the pathophysiology of hypercortisolism in a b-blocker-sensitive Cushing's syndrome due to AIMAH. Cortisol secretion profile under aberrant receptors stimulation revealed hyperresponsiveness to salbutamol (beta2-adrenoceptor agonist), cisapride (5-HT4 receptor agonist), and vasopressin in AIMAH cultured cells, but not in normal adrenocortical cells. By RT-PCR, AIMAH tissues revealed beta2-adrenoceptor overexpression rather than ectopical expression. MC2R expression was similar in both AIMAH and normal adrenocortical tissues. Curiously, cortisol levels of AIMAH cells under basal condition were 15-fold higher than those of control cells and were not responsive to ACTH. Analysis of culture medium from AIMAH cells could detect the presence of ACTH, which was immunohistochemically confirmed. Finally, the present study of AIMAH cells has identified: a) cortisol hyperresponsiveness to catecholamines, 5-HT4 and vasopressin in vitro, in agreement with clinical screening tests; b) abnormal expression of beta2-adrenoceptors in some areas of the hyperplastic adrenal tissue; c) autocrine loop of ACTH production. Altogether, the demonstration of aberrant responses to hormonal receptors and autocrine hormone production in the same tissue supports the assumption of multiple molecular alterations in adrenal macronodular hyperplasia. A síndrome de Cushing secundária à hiperplasia adrenal macronodular independente de ACTH (AIMAH) pode estar associada com respostas anômalas a estímulos sobre receptores hormonais expressos de maneira aberrante no córtex adrenal. O objetivo deste trabalho foi caracterizar a fisiopatologia do hipercortisolismo in vitro na síndrome de Cushing responsiva a beta-bloqueadores decorrente de AIMAH. Em cultura de células, a secreção de cortisol apresentou resposta aumentada ao salbutamol (agonista beta2-adrenérgico), à cisaprida (agonista de receptor 5-HT4) e à vasopressina, na AIMAH mas não no córtex adrenal normal. O estudo de receptores aberrantes por RT-PCR demonstrou que o gene do receptor beta2-adrenérgico estava superexpresso (e não expresso ectopicamente) nos fragmentos da AIMAH quando comparado ao tecido normal. A expressão de MC2R foi semelhante em ambos. Curiosamente, o nível basal de secreção de cortisol pelas células da AIMAH foi 15 vezes superior às células normais, não havendo resposta das células AIMAH ao estímulo com ACTH. A análise do meio de cultura das células AIMAH revelou a presença de ACTH, que foi confirmada por estudo imuno-histoquímico. Em suma, este estudo demonstrou: a) aumento dos níveis de cortisol in vitro em resposta a catecolaminas, 5-HT4 e vasopressina, correspondendo aos resultados dos testes clínicos para pesquisa de receptores aberrantes; b) expressão anormal de receptores beta2-adrenérgicos em algumas áreas de hiperplasia; c) produção autócrina de ACTH. Estes resultados envolvendo ativação de receptores aberrantes e estímulo hormonal autócrino no mesmo tecido favorecem a hipótese da existência de alterações moleculares múltiplas na hiperplasia adrenal macronodular.

International audience; CONTEXT: Aberrant expression of LH/human chorionic gonadotropin (hCG) receptor has been suggested in several cases of bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The cortisol production is then directly controlled by endogenous secretion of LH/hCG. However, the direct involvement of this aberrant LH/hCG receptor expression in the development of the hyperplasia has not been demonstrated. Moreover in most cases, whenever investigated, the aberrant expression of LH/hCG receptor has been associated with the ectopic expression of other G protein-coupled receptors such as gastric inhibitory polypeptide, serotonin, or vasopressin receptors. OBJECTIVE: The aim of this study was to explore the action of LH/hCG receptor on the development of adrenal hyperplasia. RESULTS: The ectopic expression of this single nonmutated gene transduced into bovine adrenocortical cells was sufficient to induce not only the aberrant cortisol secretion but also hyperproliferation and benign transformation. The cells were transplanted beneath the kidney capsule of adrenalectomized immunodeficient mice. Only the cells expressing the LH/hCG receptor gene formed an enlarged tissue with a high proliferation rate. The tissue expressing LH/hCG receptor was responsible for elevated plasma cortisol and decreased plasma ACTH levels in transplanted mice. These animals displayed physiological changes similar to those of patients with Cushing's syndrome, including muscle atrophy, thin skin, spleen atrophy, and hyperglycemia. CONCLUSIONS: These results demonstrate that a single genetic event such as the inappropriate expression of the nonmutated LH/hCG receptor gene is sufficient to initiate the phenotypic changes that cause the development of a benign adrenocortical tumor.

International audience; In ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushing's syndrome, cortisol production can be controlled by illegitimate membrane receptors. The aim of the present study was to evaluate in vivo and in vitro the sensitivity of AIMAH to various regulatory factors to detect the expression of illegitimate receptors by the tissues. Four consecutive patients with AIMAH and hypercortisolism (H1-H4) preoperatively underwent a series of pharmacological and/or physiological tests. After adrenalectomy, in vitro studies were conducted to investigate the cortisol responses of cultured cells, derived from hyperplastic tissues, to various membrane receptor ligands. The adrenal tissues of the two patients who responded in vivo to food intake (H2 and H4) were stimulated in vitro by gastric inhibitory polypeptide. GnRH and human chorionic gonadotropin, but not FSH, stimulated cortisol secretion in patients H2 and H4. In these two cases, human chorionic gonadotropin but not GnRH stimulated cortisol production from cultured adrenocortical cells. Cisapride induced a significant increase in cortisol levels in patient H1. In addition, serotonin (5-HT) was more efficient to stimulate cortisol production in H1 cells than in normal adrenocortical cells. Upright stimulation test provoked an increase in cortisol levels in patients H1, H2, and H3. H1 and H2 cells were more sensitive to the stimulatory action of angiotensin II than normal cells. Similarly, arginine vasopressin (AVP) more efficiently activated steroidogenesis in H1 cells than in normal cells. In H1 tissue, immunohistochemical studies revealed the presence of 5-HT- and AVP-like immunoreactivities within clusters of steroidogenic cells, suggesting that these two factors acted through an autocrine/paracrine mechanism to stimulate cortisol secretion. The present study provides the first demonstration of primary adrenal Cushing's syndrome dependent on both gonadotropin and gastric inhibitory polypeptide. Our data also show a hyperresponsiveness of hyperplastic adrenal tissues to 5-HT, angiotensin II, and AVP. Finally, they reveal for the first time the presence of paracrine regulatory signals in adrenal hyperplasia tissues.

International audience; The serotonin4 (5-HT4) receptor agonists cisapride and/or metoclopramide have been shown to stimulate cortisol secretion in some patients with ACTH-independent bilateral macronodular adrenal hyperplasias (AIMAH) causing Cushing's syndrome. In the present study, we have investigated quantitatively and qualitatively the expression of the 5-HT4 receptor in both normal adrenal cortex and tissues removed from six patients (P1-P6) with cisapride-responsive AIMAH and Cushing's syndrome. Real-time quantitative PCR assay revealed that the 5-HT4 receptor was overexpressed in four of the six hyperplasias studied when compared with normal adrenal cortex. In these tissues, 5-HT4 receptor mRNA expression was 3 to 16 times higher than in normal glands, likely explaining the abnormal in vivo cortisol response to cisapride. Characterization of 5-HT4 receptor splice variants by RT-PCR in both hyperplastic and normal adrenals showed that the variants present in the two hyperplasias that did not overexpress the 5-HT4 receptor, i.e. P2 and P5, could also be detected in the normal adrenal tissue. In addition, sequencing of the full-length cDNAs encoding 5-HT4 receptors in hyperplasias P2 and P5 did not reveal any mutation. Taken together, our results show an overexpression of the 5-HT4 receptor in cisapride-responsive AIMAH. However, in two cases, the level of expression of the receptor in the hyperplastic adrenal cortex was similar to that of normal adrenal gland. The enhanced sensitivity of these two tissues to 5-HT4 receptor agonists was not due to ectopic expression of 5-HT4 receptor isoforms or to the occurrence of somatic gain-of-function mutation of the receptor.

Recent studies from several groups have indicated that abnormal or ectopic expression and function of adrenal receptors for various hormones may regulate cortisol production in ACTH-independent hypercortisolism. Gastric inhibitory polypeptide (GIP)-dependent Cushing's syndrome has been described in patients with either unilateral adenoma or bilateral macronodular adrenal hyperplasia; this syndrome results from the large adrenal overexpression of the GIP receptor without any activating mutation. We have conducted a systematic in vivo evaluation of patients with adrenal Cushing's syndrome in order to identify the presence of abnormal hormone receptors. In macronodular adrenal hyperplasia, we have identified, in addition to GIP-dependent Cushing's syndrome, other patients in whom cortisol production was regulated abnormally by vasopressin, ss-adrenergic receptor agonists, hCG/LH, or serotonin 5HT-4 receptor agonists. In patients with unilateral adrenal adenoma, the abnormal expression or function of GIP or vasopressin receptor has been found, but the presence of ectopic or abnormal hormone receptors appears to be less prevalent than in macronodular adrenal hyperplasia. The identification of the presence of an abnormal adrenal receptor offers the possibility of a new pharmacological approach to control hypercortisolism by suppressing the endogenous ligands or by using specific antagonists for the abnormal receptors.

ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a rare cause of endogenous Cushing's syndrome (CS). The diagnosis is suggested by bilateral adrenal nodular enlargement on conventional imaging and the demonstration of ACTH-independent hypercortisolism. Cortisol secretion in AIMAH can be regulated by the aberrant adrenal expression of receptors for gastric inhibitory polypeptide, vasopressin, catecholamines, luteinizing hormone/human chorionic gonadotropin and serotonin. We report on a 47-year-old man with CS due to AIMAH. The cortisol levels were increased after vasopressin stimulation, suggesting aberrant expression of vaso-pressin receptor in the adrenal glands. Total adrenalectomy is considered a standard therapy for AIMAH.