Bilateral Adrenocortical Nodular Disease and Cushing's Syndrome.

Primary Pigmented Nodular Adrenocortical Disease (PPNAD) and Bilateral Macronodular Adrenocortical Disease (BMAD) are two forms of Adrenocortical Nodular Diseases causing Cushing's syndrome, but are two very distinct conditions. PPNAD, affecting mostly young patients with an almost constant severe Cushing's syndrome, is characterized by pigmented micronodules, usually less than 1 cm, not always visible on imaging. On the contrary, BMAD is predominantly diagnosed in the fifth and sixth decades, with highly variable degrees of cortisol excess, from mild autonomous cortisol secretion to overt Cushing's syndrome. BMAD presents as large bilateral adrenal macronodules, easily observed on imaging. Both diseases are often genetically determined: frequently PPNAD is observed in a multiple neoplasia syndrome, Carney complex (CNC), and a germline genetic defect is identified in around 80% of index cases, always affecting key actors of the cAMP/PKA pathway: mostly PRKAR1A, encoding the PKA 1-alpha regulatory subunit. On the other hand, BMAD appears mostly isolated, and two predisposing genes are known at present: ARMC5, accounting for around 20% of index cases, and the recently identified KDM1A, causing the rare presentation with food-dependent Cushing's syndrome, mediated by the ectopic expression of the Glucose-dependent Insulinotropic Polypeptide receptor (GIPR) in adrenal nodules. GIPR was the first demonstrated receptor to illegitimately regulate cortisol secretion in nodular adrenocortical diseases, and a myriad of other receptors and paracrine signals were discovered afterward. The last 30 years were pivotal in the understanding of the genetics and pathophysiology of Bilateral Adrenocortical Nodular Diseases, leading to a personalized approach of these fascinating conditions.

Steroidogenesis and androgen/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice.

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.

Immunohistochemical characterization of a steroid-secreting oncocytic adrenal carcinoma responsible for paraneoplastic hyperparathyroidism.

We report a unique case of a 44-year-old man with paraneoplastic hyperparathyroidism due to an oncocytic adrenocortical carcinoma (stage pT3N0R0M0, ENSAT 2 with a 4% Ki-67). Paraneoplastic hyperparathyroidism was associated with mild adrenocorticotropic hormone (ACTH)-independent hypercortisolism and increased estradiol secretion responsible for gynecomastia and hypogonadism. Biological investigations performed in blood samples from peripheral and adrenal veins revealed that the tumor secreted parathyroid hormone (PTH) and estradiol. Ectopic PTH secretion was confirmed by abnormally high expression of PTH mRNA and clusters of PTH immunoreactive cells in the tumor tissue. Double-immunochemistry studies and analysis of contiguous slides for the expression of PTH and steroidogenic markers (scavenger receptor class B type 1 [SRB1], 3ß-hydroxysteroid dehydrogenase [3ß-HSD], and aromatase) were performed. The results suggested the presence of two tumor cells subtypes with large cells with voluminous nuclei producing only PTH and that were distinct from steroid-producing cells.

HCG-responsive aldosteronoma with transient secretion during pregnancy confirmed through HCG-stimulated adrenal venous sampling.

Primary aldosteronism can be regulated by the ectopic expression of G-protein coupled receptors in aldosteronomas or bilateral hyperplasias. We report a rare case of a young woman in whom 2 pregnancies were complicated by pre-eclampsia and 1 miscarriage. The transient primary aldosteronism during pregnancies suggested the possibility of HCG stimulated aberrant adrenal expression of LHCG receptor in her adrenal tissues. This was supported by increased aldosterone and renin suppression during 5-day HCG stimulation test outside of pregnancy. Following a second 5-day HCG stimulation test, bilateral simultaneous adrenal vein sampling identified a lateralized source of aldosterone from an 8 mm right adrenal nodule. A right laparoscopic adrenalectomy resulted in clinical and biochemical cure and allowed a further uneventful pregnancy a few years later. This case illustrates the indication to investigate for potential primary aldosteronism in woman with transient hypertension during pregnancy.

Expression of LHCGR in Pheochromocytomas Unveils an Endocrine Mechanism Connecting Pregnancy and Epinephrine Overproduction.

BACKGROUND: The mechanisms by which pregnancy may unmask pheochromocytomas and paragangliomas are not totally understood. We hypothesized that gestational hormones may participate in the pathophysiology of catecholamine excess during pregnancy. We report a case of silent pheochromocytoma revealed in a pregnant woman by life-threatening adrenergic myocarditis. METHODS: In vitro studies were conducted to investigate the effect of estradiol and the pregnancy hormone hCG (human chorionic gonadotropin) on epinephrine secretion by cultured cells derived from the patient's tumor. Expression of LHCG (luteinizing hormone/chorionic gonadotropin) receptor was searched for in the patient's tumor, and a series of 12 additional pheochromocytomas by real-time reverse transcription polymerase chain reaction and immunohistochemistry. LHCGR expression was also analyzed in silico in the pheochromocytomas and paragangliomas cohorts of the Cortico et Médullosurrénale: les Tumeurs Endocrines and The Cancer Genome Atlas databases. RESULTS: hCG stimulated epinephrine secretion by cultured cells derived from the patient's pheochromocytoma. The patient's tumor expressed the LHCG receptor, which was colocalized with catecholamine-producing enzymes. A similar expression pattern of the LHCG receptor was also observed in 5 out of our series of pheochromocytomas. Moreover, in silico studies revealed that pheochromocytomas and paragangliomas display the highest expression levels of LHCG receptor mRNA among the 32 solid tumor types of The Cancer Genome Atlas cohort. CONCLUSIONS: Pregnancy may thus favor surges in plasma catecholamine and hypertensive crises through hCG-induced stimulation of epinephrine production by pheochromocytomas.

Cushing's syndrome caused by intra-adrenocortical adrenocorticotropic hormone in a dog.

A 13-year-old Labrador retriever was diagnosed with Cushing's syndrome (CS) caused by primary bilateral nodular adrenocortical hyperplasia with adrenocorticotropic hormone (ACTH) expression. The pituitary origin of CS was ruled out by suppression of plasma ACTH concentration and absence of a proliferative lesion on histological evaluation of the pituitary gland using periodic acid-Schiff (PAS) staining, reticulin staining, and immunostaining for ACTH. A pheochromocytoma also was found at necropsy examination. On histological evaluation of both adrenal glands, at the junction of the fascicular and glomerular zones, multiple cell clusters distributed in both hyperplastic adrenal cortices expressed ACTH, whereas the pheochromocytoma cells did not. These results indicate that a disease similar to primary bilateral macronodular adrenocortical hyperplasia in humans also occurs in dogs, with intra-adrenocortical expression of ACTH, glucocorticoids excess, and clinical signs of CS. Therefore, the term ACTH-independent could be inappropriate in some cases of bilateral adrenocortical hyperplasia and suppressed plasma ACTH concentration in dogs.

ß-Catenin activation and illicit receptor expression in adrenocortical cells.

Adrenal cortisol-producing tumors can express illicit membrane receptors such as luteinizing hormone (LH), glucose-dependent insulinotropic peptide (GIP) or type 4 and 7 serotonin (5-HT4/7) receptors. Abnormal expression of the LH receptor (LH-R) has been ascribed to the activation of the Wnt/ß-catenin signaling pathway in adrenocortical cells. In the present study, we have investigated whether ß-catenin activation may also trigger the illegitimate expression of GIP and 5-HT receptors. Three models of ß-catenin activation in adrenocortical cells were used: an APC-mutated adrenocortical tumor, human-transfected adrenocortical cells and genetically modified mouse adrenal glands. The methods employed include quantitative reverse transcription PCR, immunohistochemistry and measurement of cortisol secretion by cultured tumor cells. Abnormal expression of the GIP, 5-HT7and LH receptors was observed in the APC-mutated adrenocortical tumor tissue. In addition, GIP, 5-HT and human chorionic gonadotropin stimulated cortisol production from tumor cells in primary culture. Conversely, only the LHCGR was upregulated in human and mouse adrenocortical cells harboring the activation of ß-catenin. Moreover, LH-R immunoreactivity was detected in clusters of zona fasciculata cells in the ß-catenin-activated mouse model. Our data indicate that activation of the ß-catenin signaling pathway can promote the illicit expression of functional LH-Rs in adrenal zona fasciculata cells but does not favor the abnormal expression of GIP and 5-HT receptors.

Steroidogenic cell microenvironment and adrenal function in physiological and pathophysiological conditions.

The human adrenal cortex is a complex organ which is composed of various cell types including not only steroidogenic cells but also mesenchymal cells, immunocompetent cells and neurons. Intermingling of these diverse cell populations favors cell-to-cell communication processes involving local release of numerous bioactive signals such as biogenic amines, cytokines and neuropeptides. The resulting paracrine interactions play an important role in the regulation of adrenocortical cell functions both in physiological and pathophysiological conditions. Especially, recent evidence indicates that adrenocortical cell microenvironment is involved in the pathogenesis of adrenal disorders associated with corticosteroid excess. The paracrine factors involved in these intraadrenal regulatory mechanisms may thus represent valuable targets for future pharmacological treatments of adrenal diseases.

The neuropeptide substance P regulates aldosterone secretion in human adrenals.

Aldosterone, produced by the adrenals and under the control of plasma angiotensin and potassium levels, regulates hydromineral homeostasis and blood pressure. Here we report that the neuropeptide substance P (SP) released by intraadrenal nerve fibres, stimulates aldosterone secretion via binding to neurokinin type 1 receptors (NK1R) expressed by aldosterone-producing adrenocortical cells. The action of SP is mediated by the extracellular signal-regulated kinase pathway and involves upregulation of steroidogenic enzymes. We also conducted a prospective proof-of-concept, double blind, placebo-controlled clinical trial aimed to investigate the impact of the NK1R antagonist aprepitant on aldosterone secretion in healthy male volunteers (EudraCT: 2008-003367-40, ClinicalTrial.gov: NCT00977223). Participants received during two 7-day treatment periods aprepitant (125 mg on the 1st day and 80 mg during the following days) or placebo in a random order at a 2-week interval. The primary endpoint was plasma aldosterone levels during posture test. Secondary endpoints included basal aldosterone alterations, plasma aldosterone variation during metoclopramide and hypoglycaemia tests, and basal and stimulated alterations of renin, cortisol and ACTH during the three different stimulatory tests. The safety of the treatment was assessed on the basis of serum transaminase measurements on days 4 and 7. All pre-specified endpoints were achieved. Aprepitant decreases aldosterone production by around 30% but does not influence the aldosterone response to upright posture. These results indicate that the autonomic nervous system exerts a direct stimulatory tone on mineralocorticoid synthesis through SP, and thus plays a role in the maintenance of hydromineral homeostasis. This regulatory mechanism may be involved in aldosterone excess syndromes.

Role of Mast Cells in the Control of Aldosterone Secretion.

Mast cells are immune cells present in adrenals from various species. Proliferation and activation of adrenal mast cells seem to be influenced by environment, since they increase during summer and in response to sodium restriction in frogs and mouse, respectively. Although the physiological factors regulating adrenal mast cell activity have not been identified, they might involve neurotransmitters and the renin-angiotensin system. Some data indicate that adrenal mast cells stimulate proliferation of steroidogenic cells in the zona glomerulosa and activate the mineralocorticoid production. In human, mast cell degranulation stimulates aldosterone synthesis through the release of serotonin (5-HT) and activation of 5-HT4 receptors. Increase in mast cell population and upregulation of the 5-HT signaling pathway occur in aldosterone-producing adenomas. In particular, aldosterone-producing adenoma cells overexpress 5-HT4 receptors and are hyper-responsive to 5-HT4 receptor agonists. These data suggest that the intra-adrenal serotonergic regulatory system represents a potential target for development of both adrenal imaging methods to evaluate the lateralization of aldosterone production, and pharmacological treatments of primary aldosteronism.

Illicit Upregulation of Serotonin Signaling Pathway in Adrenals of Patients With High Plasma or Intra-Adrenal ACTH Levels.

CONTEXT: In the human adrenal, serotonin (5-HT), released by mast cells stimulates corticosteroid secretion through activation of type 4 serotonin receptors (5-HT4R). In primary pigmented nodular adrenocortical disease cells, activation of the cAMP/protein kinase A (PKA) pathway by PRKAR1A mutations triggers upregulation of the 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and the 5-HT4, 5-HT6, and 5-HT7 receptors. Because ACTH stimulates cortisol secretion through activation of PKA, adrenocortical tissues exposed to sustained stimulation by ACTH may harbor increased expression of TPH and 5-HT4/6/7 receptors. OBJECTIVE: To investigate the effects of long-term ACTH stimulation on the serotonergic pathway in adrenals of patients with high plasma or intra-adrenal ACTH levels. METHODS: Adrenal tissues were obtained from patients with Cushing disease, ectopic secretion of ACTH [paraneoplastic Cushing syndrome; (paraCS)], 21-hydroxylase deficiency (21-OHD), primary bilateral macronodular adrenal hyperplasia with intra-adrenal ACTH presence, or cortisol-producing adenomas. TPH and 5-HT4/6/7 receptor expression was investigated using RT-PCR and immunochemistry in comparison with normal adrenals. Primary cultured adrenocortical cells originating from a patient with paraCS were incubated with 5-HT and 5-HTR agonists/antagonists. RESULTS: TPH and/or 5-HT4/6/7 receptors were overexpressed in the different types of tissues. In paraCS cultured cells, the cortisol response to 5-HT was exaggerated compared with normal adrenal cells and the stimulatory action of 5-HT was reduced by 5-HT4R antagonist. CONCLUSION: Our results indicate that prolonged activation of the cAMP/PKA pathway by ACTH induces an aberrant serotonergic stimulatory loop in the adrenal cortex that likely participates in the pathogenesis of corticosteroid hypersecretion.

Paracrine Regulation of Aldosterone Secretion in Physiological and Pathophysiological Conditions.

Aldosterone secretion by the zona glomerulosa of the adrenal cortex is controlled by circulating factors including the renin angiotensin system (RAS) and potassium. Mineralocorticoid production is also regulated through an autocrine/paracrine mechanism by a wide variety of bioactive signals released in the vicinity of adrenocortical cells by chromaffin cells, nerve endings, cells of the immune system, endothelial cells and adipocytes. These regulatory factors include conventional neurotransmitters and neuropeptides. Their physiological role in the control of aldosterone secretion is not fully understood, but it is likely that they participate in the RAS-independent regulation of zona glomerulosa cells. Interestingly, recent observations indicate that autocrine/paracrine processes are involved in the pathophysiology of primary aldosteronism. The intraadrenal regulatory systems observed in aldosterone-producing adenomas (APA), although globally similar to those occurring in the normal adrenal gland, harbor alterations at different levels, which tend to strengthen the potency of paracrine signals to activate aldosterone secretion. Enhancement of paracrine stimulatory tone may participate to APA expansion and aldosterone hypersecretion together with somatic mutations of driver genes which activate the calcium signaling pathway and subsequently aldosterone synthase expression. Intraadrenal regulatory mechanisms represent thus promising pharmacological targets for the treatment of primary aldosteronism.

Autoantibodies reactive to adrenocorticotropic hormone can alter cortisol secretion in both aggressive and nonaggressive humans.

Violent aggression in humans may involve a modified response to stress, but the underlying mechanisms are not well understood. Here we show that naturally present autoantibodies reactive to adrenocorticotropic hormone (ACTH) exhibit distinct epitope-binding profiles to ACTH peptide in subjects with a history of violent aggression compared with controls. Namely, while nonaggressive male controls displayed a preferential IgG binding to the ACTH central part (amino acids 11-24), subjects who had committed violent acts of aggression had IgG with increased affinity to ACTH, preferentially binding to its N terminus (amino acids 1-13). Purified IgGs from approximately half of the examined sera were able to block ACTH-induced cortisol secretion of human adrenal cells in vitro, irrespective of the source of sample (from a control subject or a violent aggressor). Nevertheless, in the resident-intruder test in mice, i.p. injection of residents with ACTH and IgG from aggressive subjects, but not from control subjects, shortened latency for the first attack against intruders. Immunohistochemical screening of violent aggressors' sera on rat brain and pituitary sections did not show IgG binding to ACTH-producing cells, but 4 of 16 sera revealed selective binding to a nonidentified antigen in vasopressinergic neurons of the hypothalamic paraventricular and supraoptic nuclei. Thus, the data show that ACTH-reactive plasmatic IgGs exhibit differential epitope preference in control and violently aggressive subjects. These IgGs can modulate ACTH-induced cortisol secretion and, hence, are involved in the regulation of the stress response. However, the possible role of ACTH-reactive autoantibodies in aggressive behavior needs further investigation.

Reversal of a Blunted Follicle-Stimulating Hormone by Chemotherapy in an Inhibin B-Secreting Adrenocortical Carcinoma.

CONTEXT: Adrenocortical carcinomas (ACCs) are revealed in 60% of cases by steroid hypersecretion. Alternatively, it is uncommon to observe a paraneoplastic syndrome due to a peptide oversecretion. CASE DESCRIPTION: We describe a 60-year-old man with a right adrenal mass. Hormonal evaluation showed an ACTH-independent Cushing syndrome. Surprisingly, follicle-stimulating hormone (FSH) levels were suppressed and blunted during gonadotropin-releasing hormone stimulation, despite normal luteinizing hormone levels. Levels of inhibin B, which negatively regulates the pituitary FSH, were very high. Given the atypical hormonal findings, an adrenal mass biopsy was performed, which allowed the diagnosis of an adrenocortical tumor (positive for steroidogenic factor-1 immunostaining). Moreover, an intense α-inhibin subunit immunostaining was observed. Because of the presence of metastases, the patient received mitotane and chemotherapy (etoposide and cisplatin). After 2 cycles, the inhibin B dropped. After 5 cycles, tumor size was reduced by 15%. Inhibin B levels remained low, and basal and gonadotropin-releasing hormone-stimulated FSH levels normalized. The patient underwent tumor resection, and pathology confirmed the ACC diagnosis (Weiss score of 9). The intensity of the α-inhibin subunit immunostaining was significantly decreased. CONCLUSIONS: We report the case of an inhibin B-secreting ACC in which the response to chemotherapy and mitotane was associated with a normalization of inhibin B secretion, allowing the reversal of the blunted FSH secretion. Inhibin B should be measured in case of suppressed FSH levels despite normal luteinizing hormone levels and may be considered a tumoral marker in some ACCs, even during treatment follow-up.

Dysregulation of Aldosterone Secretion in Mast Cell-Deficient Mice.

Resident adrenal mast cells have been shown to activate aldosterone secretion in rat and man. Especially, mast cell proliferation has been observed in adrenal tissues from patients with aldosterone-producing adrenocortical adenoma. In the present study, we show that the activity of adrenal mast cells is stimulated by low-sodium diet and correlates with aldosterone synthesis in C57BL/6 and BALB/c mice. We have also investigated the regulation of aldosterone secretion in mast cell-deficient C57BL/6 KitW-sh/W-sh mice in comparison with wild-type C57BL/6 mice. KitW-sh/W-sh mice submitted to normal sodium diet had basal plasma aldosterone levels similar to those observed in wild-type animals. Conversely, low-sodium diet unexpectedly induced an exaggerated aldosterone response, which seemed to result from an increase in adrenal renin and angiotensin type 1 receptor expression. Severe hyperaldosteronism was associated with an increase in systolic blood pressure and marked hypokalemia, which favored polyuria. Adrenal renin and angiotensin type 1 receptor overexpression may represent a compensatory mechanism aimed at activating aldosterone production in the absence of mast cells. Finally, C57BL/6 KitW-sh/W-sh mice represent an unexpected animal model of primary aldosteronism, which has the particularity to be triggered by sodium restriction.

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing's syndrome.

GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing's syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

Long-Term Control of Hypercortisolism by Vandetanib in a Case of Medullary Thyroid Carcinoma with a Somatic RET Mutation.

BACKGROUND: Medullary thyroid carcinomas (MTCs) complicated by ectopic Cushing's syndrome (CS) have a poor prognosis, partially due to the difficulty in controlling hypercortisolism by adrenal blocking drugs. Recent reports (including the initial follow-up of this patient) have suggested that tyrosine kinase inhibitors (TKIs) may be a therapeutic option due to an anti-secretory action on ACTH. However, there is a lack of long-term follow-up studies. PATIENT FINDINGS: The case is reported of a 58-year-old man with MTC-related CS resistant to a combination of several anti-cortisolic drugs. Vandetanib, an oral multi-TKI that targets RET in particular, was initiated, and a rapid reversal of the hypercortisolism was observed without any change in tumor size. Vandetanib was briefly interrupted twice, once for 45 days because of side effects and a second time for 10 days to schedule surgical debulking. Each time, plasma cortisol and calcitonin levels increased after TKI withdrawal and were rapidly lowered by vandetanib reintroduction. As described in other cases of CS caused by MTC, a marked ACTH increase after desmopressin stimulation was observed before vandetanib therapy. In contrast, a blunted ACTH response to desmopressin was documented throughout the course of vandetanib treatment. This modulation of the tumoral ACTH production is a strong argument in favor of a TKI anti-secretory action. A left thyroid lobectomy and a modified neck dissection were performed one year after the initiation of vandetanib in order to reduce the tumor mass. An activating M918T RET (c.2753T>C) somatic mutation was identified in a lymph node metastasis. CONCLUSION: Three years and eight months after vandetanib initiation, there was no sign of recurrence of hypercortisolism. This case illustrates the long-term effectiveness of vandetanib in maintaining the control of hypercortisolism in MTC-related CS.