Steroids-producing nodules: a two-layered adrenocortical nodular structure as a precursor lesion of cortisol-producing adenoma.

BACKGROUND: The human adrenal cortex consists of three functionally and structurally distinct layers; zona glomerulosa, zona fasciculata (zF), and zona reticularis (zR), and produces adrenal steroid hormones in a layer-specific manner; aldosterone, cortisol, and adrenal androgens, respectively. Cortisol-producing adenomas (CPAs) occur mostly as a result of somatic mutations associated with the protein kinase A pathway. However, how CPAs develop after adrenocortical cells acquire genetic mutations, remains poorly understood. METHODS: We conducted integrated approaches combining the detailed histopathologic studies with genetic, RNA-sequencing, and spatially resolved transcriptome (SRT) analyses for the adrenal cortices adjacent to human adrenocortical tumours. FINDINGS: Histopathological analysis revealed an adrenocortical nodular structure that exhibits the two-layered zF- and zR-like structure. The nodular structures harbour GNAS somatic mutations, known as a driver mutation of CPAs, and confer cell proliferative and autonomous steroidogenic capacities, which we termed steroids-producing nodules (SPNs). RNA-sequencing coupled with SRT analysis suggests that the expansion of the zF-like structure contributes to the formation of CPAs, whereas the zR-like structure is characterised by a macrophage-mediated immune response. INTERPRETATION: We postulate that CPAs arise from a precursor lesion, SPNs, where two distinct cell populations might contribute differently to adrenocortical tumorigenesis. Our data also provide clues to the molecular mechanisms underlying the layered structures of human adrenocortical tissues. FUNDING: KAKENHI, The Uehara Memorial Foundation, Daiwa Securities Health Foundation, Kaibara Morikazu Medical Science Promotion Foundation, Secom Science and Technology Foundation, ONO Medical Research Foundation, and Japan Foundation for Applied Enzymology.

Cardiovascular risk assessments in patients with cortisol-producing adenoma: impact of clinical features and genetic characteristics.

The causes of adrenal Cushing's syndrome (CS) encompass a wide spectrum of adrenal cortisol proliferations that exhibit clinical and molecular heterogeneity. The aims of our study were to investigate whether clinical and molecular heterogeneity influences endothelial function and metabolic abnormalities in patients with cortisol-producing adenoma (CPA). We retrospectively enrolled 25 patients with CPA and 45 patients with essential hypertension (EH). All CPAs were studied by direct sequencing of PRKACA. Flow-mediated vasodilation (FMD), an index of vascular endothelial function, was significantly lower in CS and subclinical CS (SCS) groups than in the EH group. FMD impairment did not differ significantly between CS and SCS groups. No differences in FMD were seen between PRKACA mutant and wild-type groups. FMD correlated negatively with hemoglobin A1c (HbA1c) in both PRKACA mutant and wild-type groups, as well as in CS and SCS groups. After adrenalectomy, systolic blood pressure (SBP) and HbA1c decreased significantly from baseline in the CS group, and SBP and low-density lipoprotein cholesterol (LDL-C) decreased significantly from baseline in the SCS group. While SBP and LDL-C decreased significantly from baseline in patients with wild-type PRKACA, only HbA1c decreased from baseline in patients harboring PRKACA mutations. Our data showed that patients with CPA have impaired endothelial function compared with EH patients and suggest the need for strict monitoring of atherosclerosis, even in patients with SCS or without PRKACA mutation.

Different cell compositions and a novel somatic KCNJ5 variant found in a patient with bilateral adrenocortical adenomas secreting aldosterone and cortisol.

Introduction: This study aimed to explore the possible pathogenesis of a rare case of co-existing Cushing's syndrome (CS) and primary aldosteronism (PA) caused by bilateral adrenocortical adenomas secreting aldosterone and cortisol, respectively. Methods: A 41-year-old Chinese woman with severe hypertension and hypokalemia for 5 and 2 years, respectively, was referred to our hospital. She had a Cushingoid appearance. Preoperative endocrinological examinations revealed autonomous cortisol and aldosterone secretion. Computed tomography revealed bilateral adrenal adenomas. Subsequently, adrenal vein sampling and sequential left and right partial adrenalectomy indicated the presence of a left aldosterone-producing tumor and a right cortisol-producing tumor. Pathological examination included immunohistochemical analysis of the resected specimens. Secretions of aldosterone and cortisol were observed both in vivo and in vitro. Further, whole-exome sequencing was performed for DNA that was extracted from peripheral blood leukocytes and bilateral adrenal adenomas in order to determine whether the patient had relevant variants associated with PA and CS. Results: Immunohistochemical staining revealed that the left adenoma primarily comprised clear cells expressing CYP11B2, whereas the right adenoma comprised both eosinophilic compact and clear cells expressing CYP11B1. The mRNA levels of steroidogenic enzymes (including CYP11B1 and CYP17A1) were high in the right adenoma, whereas CYP11B2 was highly expressed in the left adenoma. A novel somatic heterozygous missense variant-KCNJ5 c.503T > G (p.L168R)-was detected in the left adrenal adenoma, but no other causative variants associated with PA and CS were detected in the peripheral blood or right adrenocortical adenoma. In the primary cell culture of the resected hyperplastic adrenal adenomas, verapamil and nifedipine, which are two calcium channel blockers, markedly inhibited the secretion of both aldosterone and cortisol. Conclusion: We present an extremely rare case of bilateral adrenocortical adenomas with distinct secretion of aldosterone and cortisol. The heterogeneity of the tumor cell compositions of aldosterone- and cortisol-producing adenoma (A/CPA) and somatic mutation of KCNJ5 may have led to different hormone secretions in the bilateral adrenal adenomas.

Expression of the angiogenesis markers endocan in Cushing's syndrome due to cortisol producing adenoma.

PURPOSE: In this study, we aimed to investigate the endocan expression in tissue samples of patients diagnosed with Cushing's syndrome (CS) due to Cortisol-Producing-Adenoma (CPA) and compare it with normal adrenal cortex tissue, and also to evaluate the correlations of endocan expression with clinical and histopathological features. METHODS: 11 patients who were operated for CS due to CPA between 2009-2021 and 14 control subjects with normal adrenal cortex were included in the study. Demographic, laboratory and clinicopathological data of the patients were recorded. Sections of 4-5 µm thickness were taken from paraffin blocks of patients diagnosed with CS due to CPA and control subjects with normal adrenal cortex tissue. The sections were then stained in a closed system automatic immunohistochemical staining device to perform immunohistochemical analysis. The endocan positive grade were determined based on the proportion of stained cells on a scale of negative to strong. RESULTS: The number of subjects with positive endocan expression and the mean endocan expression level in the CS group were significantly higher than the control group (p = 0.005, p < 0.001, respectively). No correlation was found between endocan expression and clinical and histopathological features. CONCLUSION: According to the results of our study, endocan overexpression in CPA tissues may be related to the hormonal functionality of CPA.

The Association of Cholesterol Uptake and Synthesis with Histology and Genotype in Cortisol-Producing Adenoma (CPA).

Cortisol-producing adenoma (CPA) is composed of clear and compact cells. Clear cells are lipid abundant, and compact ones lipid poor but associated with higher production of steroid hormones. PRKACA mutation (PRKACA mt) in CPA patients was reported to be associated with more pronounced clinical manifestation of Cushing's syndrome. In this study, we examined the association of histological features and genotypes with cholesterol uptake receptors and synthetic enzymes in 40 CPA cases, and with the quantitative results obtained by gas chromatography-mass spectrometry (GC-MS) analysis in 33 cases to explore their biological and clinical significance. Both cholesterol uptake receptors and synthetic enzymes were more abundant in compact cells. GC-MS analysis demonstrated that the percentage of compact cells was inversely correlated with the concentrations of cholesterol and cholesterol esters, and positively with the activity of cholesterol biosynthesis from cholesterol esters. In addition, hormone-sensitive lipase (HSL), which catalyzes cholesterol biosynthesis from cholesterol esters, tended to be more abundant in compact cells of PRKACA mt CPAs. These results demonstrated that both cholesterol uptake and biosynthesis were more pronounced in compact cells in CPA. In addition, more pronounced HSL expression in compact cells of PRKACA mt CPA could contribute to their more pronounced clinical manifestation.

Targeted Mutational Analysis of Cortisol-Producing Adenomas.

CONTEXT: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. OBJECTIVE: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. METHODS: We analyzed FFPE adrenal tissue from 77 patients (n = 12 men, 65 women) with either OCS (n = 32) or MACE (n = 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations. RESULTS: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). CONCLUSION: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs.

Molecular Mechanisms of Functional Adrenocortical Adenoma and Carcinoma: Genetic Characterization and Intracellular Signaling Pathway.

The adrenal cortex produces steroid hormones as adrenocortical hormones in the body, secreting mineralocorticoids, glucocorticoids, and adrenal androgens, which are all considered essential for life. Adrenocortical tumors harbor divergent hormonal activity, frequently with steroid excess, and disrupt homeostasis of the body. Aldosterone-producing adenomas (APAs) cause primary aldosteronism (PA), and cortisol-producing adenomas (CPAs) are the primary cause of Cushing's syndrome. In addition, adrenocortical carcinoma (ACC) is a highly malignant cancer harboring poor prognosis. Various genetic abnormalities have been reported, which are associated with possible pathogenesis by the alteration of intracellular signaling and activation of transcription factors. In particular, somatic mutations in APAs have been detected in genes encoding membrane proteins, especially ion channels, resulting in hypersecretion of aldosterone due to activation of intracellular calcium signaling. In addition, somatic mutations have been detected in those encoding cAMP-PKA signaling-related factors, resulting in hypersecretion of cortisol due to its driven status in CPAs. In ACC, mutations in tumor suppressor genes and Wnt-ß-catenin signaling-related factors have been implicated in its pathogenesis. In this article, we review recent findings on the genetic characteristics and regulation of intracellular signaling and transcription factors in individual tumors.

Intratumoral steroid profiling of adrenal cortisol-producing adenomas by liquid chromatography- mass spectrometry.

Endogenous Cushing syndrome (CS) is an endocrine disorder marked by excess cortisol production rendering patients susceptible to visceral obesity, dyslipidemia, hypertension, osteoporosis and diabetes mellitus. Adrenal CS is characterized by autonomous production of cortisol from cortisol-producing adenomas (CPA) via adrenocorticotropic hormone-independent mechanisms. A limited number of studies have quantified the steroid profiles in sera from patients with CS. To understand the intratumoral steroid biosynthesis, we quantified 19 steroids by mass spectrometry in optimal cutting temperature compound (OCT)-embedded 24 CPA tissue from patients with overt CS (OCS, n = 10) and mild autonomous cortisol excess (MACE, n = 14). Where available, normal CPA-adjacent adrenal tissue (AdjN) was also collected and used for comparison (n = 8). Immunohistochemistry (IHC) for CYP17A1 and HSD3B2, two steroidogenic enzymes required for cortisol synthesis, was performed on OCT sections to confirm the presence of tumor tissue and guided subsequent steroid extraction from the tumor. LC-MS/MS was used to quantify steroids extracted from CPA and AdjN. Our data indicated that CPA demonstrated increased concentrations of cortisol, cortisone, 11-deoxycortisol, corticosterone, progesterone, 17OH-progesterone and 16OH-progesterone as compared to AdjN (p < 0.05). Compared to OCS, MACE patient CPA tissue displayed higher concentrations of corticosterone, 18OH-corticosterone, 21-deoxycortisol, progesterone, and 17OH-progesterone (p < 0.05). These findings also demonstrate that OCT-embedded tissue can be used to define intra-tissue steroid profiles, which will have application for steroid-producing and steroid-responsive tumors.

Circulating microRNA Expression in Cushing's Syndrome.

Context: Cushing's syndrome (CS) is a rare disease of endogenous hypercortisolism associated with high morbidity and mortality. Diagnosis and classification of CS is still challenging. Objective: Circulating microRNAs (miRNAs) are minimally invasive diagnostic markers. Our aim was to characterize the circulating miRNA profiles of CS patients and to identify distinct profiles between the two major CS subtypes. Methods: We included three groups of patients from the German Cushing's registry: ACTH-independent CS (Cortisol-Producing-Adenoma; CPA), ACTH-dependent pituitary CS (Cushing's Disease; CD), and patients in whom CS had been ruled out (controls). Profiling of miRNAs was performed by next-generation-sequencing (NGS) in serum samples of 15 CS patients (each before and after curative surgery) and 10 controls. Significant miRNAs were first validated by qPCR in the discovery cohort and then in an independent validation cohort of 20 CS patients and 11 controls. Results: NGS identified 411 circulating miRNAs. Differential expression of 14 miRNAs were found in the pre- and postoperative groups. qPCR in the discovery cohort validated 5 of the significant miRNAs from the preoperative group analyses. Only, miR-182-5p was found to be significantly upregulated in the CD group of the validation cohort. Comparing all CS samples as a group with the controls did not reveal any significant differences in expression. Outcome: In conclusion, our study identified miR-182-5p as a possible biomarker for CD, which has to be validated in a prospective cohort. Furthermore, our results suggest that presence or absence of ACTH might be at least as relevant for miRNA expression as hypercortisolism itself.

Intratumoral heterogeneity of the tumor cells based on in situ cortisol excess in cortisol-producing adenomas; ∼An association among morphometry, genotype and cellular senescence∼.

Cortisol-producing adrenocortical adenomas (CPAs) are associated with ACTH-independent Cushing's syndrome and histologically composed of two cellular subtypes: compact (lipid-poor) and clear (lipid-rich) tumor cells. However, the details of hormonal and biological activities of these tumor cells have remained unknown, especially in CPAs. CPAs frequently harbored unique histological features different from those of aldosterone-producing adenomas (APAs) including a senescent phenotype. Therefore, we explored the association between morphological features and the immunoreactivity of steroidogenic enzymes in CPAs with different genotypes and compared them with cellular senescence markers as well as clinicopathological factors of the cases. Hormonal activities (3ßHSD, CYP21A, CYP17A1, CYP11B1 and DHEA-ST) and cellular senescence markers (p16, p21 and Ki-67) within different morphological features (clear and compact) were evaluated in 40 CPAs. CPA genotypes (PRKACA, GNAS and CTNNB1) were examined by Sanger sequencing and then compared them with the factors above. p21 immunoreactivity was significantly positively correlated with that of CYP21A (p = 0.0110), CYP17A1 (p = 0.0356) and DHEA-ST (p = 0.0420) but inversely with tumor size (p = 0.0015). CYP21A (p = 0.0016), CYP11B1 (p = 0.0001), CYP17A1 (p < 0.0001) and p16 (p = 0.0137) immunoreactivity were all significantly higher in compact cells than those in clear cells. CYP17A1 (p = 0.0056) and 3ßHSD (p = 0.0437) immunoreactivity was significantly higher in PRKACA-mutated than wild type CPAs. p16 immunoreactivity and serum DHEA-S level were both significantly higher in GNAS-mutated than PRKACA-mutated (p = 0.0250) and wild type (p = 0.0180) CPAs. Results of our present study did demonstrate that compact tumor cells were hormonally active and more senescent than clear tumor cells in CPAs. PRKACA- and GNAS-mutated tumor cells were more hormonally active and senescent than those without mutations despite the similar morphological features. We herein proposed a novel histological classification of the tumor cell subtypes based on in situ cortisol excess, genotypes and the status of cell senescence in CPAs.

Association between thyroid function and serum cortisol in cortisol-producing adenoma patients.

PURPOSE: Thyroid dysfunction has been reported in hypercortisolism. Previous findings regarding changes in thyroid function due to cortisol-producing adenoma (CPA) have been inconsistent. The study aimed to investigate the association between thyroid function and excessive cortisol secretion in patients with CPA and to explore the changes in pituitary function after adrenalectomy. METHODS: We conducted a retrospective study; thyroid function was evaluated in 94 patients with CPA and 94 healthy controls (HC) matched for age and sex. A total of 94 patients with nonfunctioning adrenal incidentalomas (NFAIs) were recruited as a second control group. RESULTS: Serum thyroid stimulating hormone (TSH) and free thyroxine (T4) levels were significantly lower in the CPA group than in the HC and NFAIs groups (P < 0.001). The prevalence of central hypothyroidism was 12.8% in the CPA group and increased according to serum cortisol quartiles (P for trend = 0.025). According to the stepwise multiple linear regression analysis, serum cortisol was negatively associated with TSH and free T4 levels in the CPA group after adjustment for body mass index and age. Furthermore, decreased TSH levels were corrected by adrenalectomy [0.75 (0.50, 1.14) vs. 1.91 (1.36, 2.71) µIU/ml, P < 0.001], in parallel with a recovery in free T4 levels [11.20 (10.00, 12.43) vs. 12.04 (11.24, 13.01), P < 0.001]. Postoperative growth hormone and prolactin levels did not change compared with baseline. CONCLUSION: Serum TSH and free T4 levels were decreased in patients with CPA, and dysfunction of the hypothalamic-pituitary-thyroid axis might be reversible after surgery.

A nationwide survey of adrenal incidentalomas in Japan: the first report of clinical and epidemiological features.

The aim of this study was to reveal clear epidemiologic and clinical characteristics of incidentally discovered adrenal masses, termed adrenal incidentalomas (AIs), and to establish appropriate managemental and therapeutic regimens in Japan. This study had been originally carried out as a project of a research proposed on behalf of the Japanese Ministry of Health, Labour and Welfare, from 1999 to 2004. This nationwide multicenter study on AIs included 3,672 cases with clinically diagnosed AIs, involving 1,874 males and 1,738 females, with mean age 58.1 ± 13.0 years (mean ± SD). In the present study, we focused on the investigation of the real prevalence of various adrenal disorders with AI. The mean nodule size of AI based on computed tomography was 3.0 ± 2.0 cm. Compared to non-functioning adenomas (NFAs), tumor diameters were significantly larger in adrenocortical carcinomas (ACCs), pheochromocytomas, cortisol-producing adenomas (CPAs), myelolipomas, metastatic tumors, cysts, and ganglioneuromas (p < 0.01). Endocrinological evaluations demonstrated that 50.8% of total AIs were non-functioning adenomas, while 10.5%, including 3.6% with subclinical Cushing's syndrome, were reported as CPAs, 8.5% as pheochromocytomas, and 5.1% as aldosterone-producing adenomas. ACCs were accounted for 1.4% (50 cases) among our series of AIs. In conclusion, while almost 50 % of AIs are non-functional adenomas, we must be particularly careful as AIs include pheochromocytomas or adrenal carcinomas, because they may be asymptomatic. To our knowledge, this is the first and the largest investigation of AI, thus providing basic information for the establishment of clinical guidelines for the management of AI.

Aberrant DNA methylation of synaptophysin is involved in adrenal cortisol-producing adenoma.

Cortisol-producing adenoma (CPA) is the main cause of Adrenal Cushing syndrome. However, its molecular mechanism is not fully understood. Previous study revealed Synaptophysin (SYP) is ubiquitously expressed in adrenocortical tumors, but its function in CPA still need to be discovered. In the present study we determine the molecular mechanism involved in SYP dysregulation in CPA and how SYP affects the secretion of cortisol in CPA. Our results showed that aberrant DNA methylation of SYP is involved in CPA progress. Using a miRNA microarray and qRT-PCR, we found decreased expression of miR-27a-5p in CPA compared with normal adrenal tissue. Moreover, the expression of TET3, the target gene of miR-27a-5p, increased in CPA compared with normal adrenal tissue. Knock-down of TET3 resulted in hypermethylation of SYP which reducing the expression level of SYP in H295R cells. The miR-27a-5p-TET3-SYP signalling pathway may regulate proliferation and cortisol secretion in H295R cells and, thus, play a key role in CPA development.

Glutamate receptors and the regulation of steroidogenesis in the human adrenal gland: the metabotropic pathway.

BACKGROUND: l-glutamate is a major excitatory neurotransmitter in the mammalian brain. Glutamate receptors have been reported in the rat adrenal cortex and in human aldosterone-producing adenomas (APA). However, details regarding the expression levels and functions of these receptors in human adrenocortical tissues remain unknown. METHODS: The mRNA levels of glutamate receptors were evaluated by qPCR in: 12 normal adrenal cortex (NAC), 11 APA, and 12 cortisol-producing adenoma (CPA) tissues. Protein localization was evaluated by immunohistochemistry for 15 NAC, 5 idiopathic hyperaldosteronism cases, 15 APA and 15 CPA. H295R cells were treated with angiotensin-II or forskolin alone or combined with the GRM2/3 agonist LY354740. RESULTS: The level of GRM3 mRNA was higher in APA than in CPA (P=0.0086) or NAC (P=0.0022). GRM1, IGLUR2, and IGLUR3 were also detected in adrenocortical tissues. When added to angiotensin-II/forskolin treatments, LY354740 decreased aldosterone and cortisol production in H295R cells. CONCLUSIONS: GRM3 is considered to regulate steroidogenesis in adrenocortical tissues.