Double somatic mutations in CTNNB1 and GNA11 in an aldosterone-producing adenoma.

Double somatic mutations in CTNNB1 and GNA11/Q have recently been identified in a small subset of aldosterone-producing adenomas (APAs). As a possible pathogenesis of APA due to these mutations, an association with pregnancy, menopause, or puberty has been proposed. However, because of its rarity, characteristics of APA with these mutations have not been well characterized. A 46-year-old Japanese woman presented with hypertension and hypokalemia. She had two pregnancies in the past but had no history of pregnancy-induced hypertension. She had regular menstrual cycle at presentation and was diagnosed as having primary aldosteronism after endocrinologic examinations. Computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling demonstrated excess aldosterone production from the right adrenal gland. She underwent right laparoscopic adrenalectomy. The resected right adrenal tumor was histologically diagnosed as adrenocortical adenoma and subsequent immunohistochemistry (IHC) revealed diffuse immunoreactivity of aldosterone synthase (CYP11B2) and visinin like 1, a marker of the zona glomerulosa (ZG), whereas 11ß-hydroxylase, a steroidogenic enzyme for cortisol biosynthesis, was mostly negative. CYP11B2 IHC-guided targeted next-generation sequencing identified somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations. Immunofluorescence staining of the tumor also revealed the presence of activated ß-catenin, consistent with features of the normal ZG. The expression patterns of steroidogenic enzymes and related proteins indicated ZG features of the tumor cells. PA was clinically and biochemically cured after surgery. In conclusion, our study indicated that CTNNB1 and GNA11-mutated APA has characteristics of the ZG. The disease could occur in adults with no clear association with pregnancy or menopause.

Unilateral Primary Aldosteronism: Long-Term Disease Recurrence After Adrenalectomy.

BACKGROUND: Primary aldosteronism (PA) is frequently caused by a unilateral aldosterone-producing adenoma with a PA-driver mutation. Unilateral adrenalectomy has a high probability of short-term biochemical remission, but long-term postsurgical outcomes are relatively undefined. Our objective was to investigate the incidence of long-term recurrence of PA in individuals with postsurgical short-term biochemical remission. METHODS: Adrenalectomized patients for unilateral PA were included from a single referral center. Histopathology and outcomes were assessed according to international histopathology of unilateral primary aldosteronism and PASO (Primary Aldosteronism Surgical Outcome) consensuses. Genotyping was performed using CYP11B2 (aldosterone synthase)-guided sequencing. RESULTS: Classical adrenal histopathology, exemplified by a solitary aldosterone-producing adenoma, was observed in 78% of 90 adrenals, compared with 22% with nonclassical histopathology. The classical group displayed higher aldosterone-to-renin ratios (P=0.013) and lower contralateral ratios (P=0.008). Outcome assessments at both short (12 months [7; 12]) and long (89 months [48; 124]) terms were available for 57 patients. At short-term assessment, 53 (93%) displayed complete biochemical success (43 classical and 10 nonclassical), but long-term assessment demonstrated biochemical PA recurrence in 12 (23%) with an overrepresentation of the nonclassical histopathology (6 [60%] of 10 nonclassical histopathology versus 6 [14%] of 43 classical histopathology; P=0.005). PA-driver mutations were identified in 97% of 64 aldosterone-producing adenomas; there was no association of the aldosterone-producing adenoma genotype with PA recurrence. CONCLUSIONS: A substantial proportion of individuals display postsurgical biochemical recurrence of PA, which is related to the histopathology of the resected adrenal gland. These findings emphasize the role of histopathology and the requirement for continued outcome assessment in the management of surgically treated patients for PA.

Single-Nucleus Analysis Reveals Tumor Heterogeneity of Aldosterone-Producing Adenoma.

BACKGROUND: Recent advances in omics techniques have allowed detailed genetic characterization of aldosterone-producing adenoma (APA). The pathogenesis of APA is characterized by tumorigenesis-associated aldosterone synthesis. The pathophysiological intricacies of APAs have not yet been elucidated at the level of individual cells. Therefore, a single-cell level analysis is speculated to be valuable in studying the differentiation process of APA. METHODS: We conducted single-nucleus RNA sequencing of APAs with KCNJ5 mutation and nonfunctional adenomas obtained from 3 and 2 patients, respectively. RESULTS: The single-nucleus RNA sequencing revealed the intratumoral heterogeneity of APA and identified cell populations consisting of a shared cluster of nonfunctional adenoma and APA. In addition, we extracted 2 cell fates in APA and obtained a cell population specialized in aldosterone synthesis. Genes related to ribosomes and neurodegenerative diseases were upregulated in 1 of these fates, whereas those related to the regulation of glycolysis were upregulated in the other fate. Furthermore, the total RNA reads in the nucleus were higher in hormonally activated clusters, indicating a marked activation of transcription per cell. CONCLUSIONS: The single-nucleus RNA sequencing revealed intratumoral heterogeneity of APA with KCNJ5 mutation. The observation of 2 cell fates in KCNJ5-mutated APAs provides the postulation that a heterogeneous process of cellular differentiation was implicated in the pathophysiological mechanisms underlying APA tumors.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism.

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

Somatic SLC30A1 mutations altering zinc transporter ZnT1 cause aldosterone-producing adenomas and primary aldosteronism.

Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.

Molecular and Epigenetic Control of Aldosterone Synthase, CYP11B2 and 11-Hydroxylase, CYP11B1.

Aldosterone and cortisol serve important roles in the pathogenesis of cardiovascular diseases and metabolic disorders. Epigenetics is a mechanism to control enzyme expression by genes without changing the gene sequence. Steroid hormone synthase gene expression is regulated by transcription factors specific to each gene, and methylation has been reported to be involved in steroid hormone production and disease. Angiotensin II or potassium regulates the aldosterone synthase gene, CYP11B2. The adrenocorticotropic hormone controls the 11b-hydroxylase, CYP11B1. DNA methylation negatively controls the CYP11B2 and CYP11B1 expression and dynamically changes the expression responsive to continuous stimulation of the promoter gene. Hypomethylation status of the CYP11B2 promoter region is seen in aldosterone-producing adenomas. Methylation of recognition sites of transcription factors, including cyclic AMP responsive element binding protein 1 or nerve growth factor-induced clone B, diminish their DNA-binding activity. A methyl-CpG-binding protein 2 cooperates directly with the methylated CpG dinucleotides of CYP11B2. A low-salt diet, treatment with angiotensin II, and potassium increase the CYP11B2 mRNA levels and induce DNA hypomethylation in the adrenal gland. A close association between a low DNA methylation ratio and an increased CYP11B1 expression is seen in Cushing's adenoma and aldosterone-producing adenoma with autonomous cortisol secretion. Epigenetic control of CYP11B2 or CYP11B1 plays an important role in autonomic aldosterone or cortisol synthesis.

Cardiovascular and metabolic characters of KCNJ5 somatic mutations in primary aldosteronism.

Background: Primary aldosteronism (PA) is the leading cause of curable endocrine hypertension, which is associated with a higher risk of cardiovascular and metabolic insults compared to essential hypertension. Aldosterone-producing adenoma (APA) is a major cause of PA, which can be treated with adrenalectomy. Somatic mutations are the main pathogenesis of aldosterone overproduction in APA, of which KCNJ5 somatic mutations are most common, especially in Asian countries. This article aimed to review the literature on the impacts of KCNJ5 somatic mutations on systemic organ damage. Evidence acquisition: PubMed literature research using keywords combination, including "aldosterone-producing adenoma," "somatic mutations," "KCNJ5," "organ damage," "cardiovascular," "diastolic function," "metabolic syndrome," "autonomous cortisol secretion," etc. Results: APA patients with KCNJ5 somatic mutations are generally younger, female, have higher aldosterone levels, lower potassium levels, larger tumor size, and higher hypertension cure rate after adrenalectomy. This review focuses on the cardiovascular and metabolic aspects of KCNJ5 somatic mutations in APA patients, including left ventricular remodeling and diastolic function, abdominal aortic thickness and calcification, arterial stiffness, metabolic syndrome, abdominal adipose tissue, and correlation with autonomous cortisol secretion. Furthermore, we discuss modalities to differentiate the types of mutations before surgery. Conclusion: KCNJ5 somatic mutations in patients with APA had higher left ventricular mass (LVM), more impaired diastolic function, thicker aortic wall, lower incidence of metabolic syndrome, and possibly a lower incidence of concurrent autonomous cortisol secretion, but better improvement in LVM, diastolic function, arterial stiffness, and aortic wall thickness after adrenalectomy compared to patients without KCNJ5 mutations.

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.

In Situ Metabolomics of Cortisol-Producing Adenomas.

BACKGROUND: Recent advances in omics techniques have allowed detailed genetic characterization of cortisol-producing adrenal adenoma (CPA). In contrast, the pathophysiology of CPAs has not been elucidated in detail on the level of tumor metabolic alterations. METHODS: The current study conducted a comprehensive mass spectrometry imaging (MSI) map of CPAs in relation to clinical phenotypes and immunohistochemical profiles of steroidogenic enzymes. The study cohort comprised 46 patients with adrenal tumors including CPAs (n 35) and nonfunctional adenomas (n 11). RESULTS: Severity of cortisol hypersecretion was significantly correlated with 29 metabolites (adjusted P 0.05). Adrenal androgens derived from the classic androgen pathway were inversely correlated with both cortisol secretion (rs 0.41, adjusted P 0.035) and CYP11B1 expression (rs 0.77, adjusted P 2.00E-08). The extent of cortisol excess and tumor CYP11B1 expression further correlated with serotonin (rs 0.48 and 0.62, adjusted P 0.008 and 2.41E-05). Tumor size was found to be correlated with abundance of 13 fatty acids (adjusted P 0.05) and negatively associated with 9 polyunsaturated fatty acids including phosphatidic acid 38:8 (rs 0.56, adjusted P 0.009). CONCLUSIONS: MSI reveals novel metabolic links between endocrine function and tumorigenesis, which will further support the understanding of CPA pathophysiology.

Heterogeneous circulating miRNA profiles of PBMAH.

Objective: Primary bilateral macronodular adrenal hyperplasia (PBMAH), a rare cause of Cushing syndrome, is often diagnosed as a bilateral adrenal incidentaloma with subclinical cortisol production. Circulating microRNAs (miRNAs) are a characteristic of adrenocortical adenomas, but miRNA expression in PBMAH has not been investigated. We aimed to evaluate the circulating miRNA expression in patients with PBMAH and compare them with those in patients with non-functioning adrenocortical adenoma (NFA) and cortisol-producing adrenocortical adenoma (CPA). Methods: miRNA profiling of plasma samples from four, five, and five patients with NFA, CPA, and PBMAH, respectively, was performed. Selected miRNA expressions were validated using quantitative RT-PCR. Results: PBMAH samples showed distinct miRNA expression signatures on hierarchical clustering while NFA and CPA samples were separately clustered. PBMAH was distinguished from the adenoma group of NFA and CPA by 135 differentially expressed miRNAs. Hsa-miR-1180-3p, hsa-miR-4732-5p, and hsa-let-7b-5p were differentially expressed between PBMAH and adenoma (P = 0.019, 0.006, and 0.003, respectively). Furthermore, PBMAH could be classified into two subtypes based on miRNA profiling: subtype 1 with a similar profile to those of adenoma and subtype 2 with a distinct profile. Hsa-miR-631, hsa-miR-513b-5p, hsa-miR-6805-5p, and hsa-miR-548av-5p/548k were differentially expressed between PBMAH subtype 2 and adenoma (P = 0.027, 0.027, 0.027, and 1.53E-04, respectively), but not between PBMAH, as a whole, and adenoma. Conclusion: Circulating miRNA signature was identified specific for PBMAH. The existence of subtype-based miRNA profiles may be associated with the pathophysiological heterogeneity of PBMAH.

Primary aldosteronism caused by a pI157S somatic KCNJ5 mutation in a black adolescent female with aldosterone-producing adenoma.

Aldosterone-producing adenoma is a rare cause of hypertension in children. Only a limited number of cases of aldosterone-producing adenomas with somatic KCNJ5 gene mutations have been described in children. Blacks are particularly more susceptible to developing long-standing cardiovascular effects of aldosterone-induced severe hypertension. Somatic CACNA1D gene mutations are particularly more prevalent in black males whereas KCNJ5 gene mutations are most frequently present in black females. We present here a novel somatic KCNJ5 p.I157S mutation in an aldosterone-producing adenoma from a 16-year-old black female whose severe drug-resistant hypertension significantly improved following unilateral adrenalectomy. Prompt diagnosis of aldosterone-producing adenoma and early identification of gene mutation would enable appropriate therapy and significantly reduce cardiovascular sequelae.

Genetic Dissection of Primary Aldosteronism in a Patient With MEN1 and Ipsilateral Adrenocortical Carcinoma and Adenoma.

BACKGROUND: Adrenal tumors are found in up to 40% of patients with multiple endocrine neoplasia type 1 (MEN1). However, adrenocortical carcinomas (ACC) and primary aldosteronism (PA) are rare in MEN1. CASE: A 48-year-old woman known to have primary hyperparathyroidism and hypertension with hypokalemia was referred for a right complex 8-cm adrenal mass with a 38.1 SUVmax uptake on 18F-FDG PET/CT. PA was confirmed by saline suppression test (aldosterone 1948 pmol/L-1675 pmol/L; normal range [N]: <165 post saline infusion) and suppressed renin levels (<5 ng/L; N: 5-20). Catecholamines, androgens, 24-hour urinary cortisol, and pituitary panel were normal. A right open adrenalectomy revealed a concomitant 4-cm oncocytic ACC and a 2.3-cm adrenocortical adenoma. Immunohistochemistry showed high expression of aldosterone synthase protein in the adenoma but not in the ACC, supporting excess aldosterone production by the adenoma. GENETIC ANALYSIS: After genetic counseling, the patient underwent genetic analysis of leucocyte and tumoral DNA. Sequencing of MEN1 revealed a heterozygous germline pathogenic variant in MEN1 (c.1556delC, p.Pro519Leufs*40). The wild-type MEN1 allele was lost in the tumoral DNA of both the resected adenoma and carcinoma. Sequencing analysis of driver genes in PA revealed a somatic pathogenic variant in exon 2 of the KCNJ5 gene (c.451G>A, p.Gly151Arg) only in the aldosteronoma. CONCLUSION: To our knowledge, we describe the first case of adrenal collision tumors in a patient carrying a germline pathogenic variant of the MEN1 gene associated with MEN1 loss of heterozygosity in both oncocytic ACC and adenoma and a somatic KCNJ5 pathogenic variant leading to aldosterone-producing adenoma. This case gives new insights on adrenal tumorigenesis in MEN1 patients.

Identification of risk loci for primary aldosteronism in genome-wide association studies.

Primary aldosteronism affects up to 10% of hypertensive patients and is responsible for treatment resistance and increased cardiovascular risk. Here we perform a genome-wide association study in a discovery cohort of 562 cases and 950 controls and identify three main loci on chromosomes 1, 13 and X; associations on chromosome 1 and 13 are replicated in a second cohort and confirmed by a meta-analysis involving 1162 cases and 3296 controls. The association on chromosome 13 is specific to men and stronger in bilateral adrenal hyperplasia than aldosterone producing adenoma. Candidate genes located within the two loci, CASZ1 and RXFP2, are expressed in human and mouse adrenals in different cell clusters. Their overexpression in adrenocortical cells suppresses mineralocorticoid output under basal and stimulated conditions, without affecting cortisol biosynthesis. Our study identifies the first risk loci for primary aldosteronism and highlights new mechanisms for the development of aldosterone excess.

Clinical Translationality of KCNJ5 Mutation in Aldosterone Producing Adenoma.

Hypertension due to primary aldosteronism poses a risk of severe cardiovascular complications compared to essential hypertension. The discovery of the KCNJ5 somatic mutation in aldosteroene producing adenoma (APA) in 2011 and the development of specific CYP11B2 antibodies in 2012 have greatly advanced our understanding of the pathophysiology of primary aldosteronism. In particular, the presence of CYP11B2-positive aldosterone-producing micronodules (APMs) in the adrenal glands of normotensive individuals and the presence of renin-independent aldosterone excess in normotensive subjects demonstrated the continuum of the pathogenesis of PA. Furthermore, among the aldosterone driver mutations which incur excessive aldosterone secretion, KCNJ5 was a major somatic mutation in APA, while CACNA1D is a leading somatic mutation in APMs and idiopathic hyperaldosteronism (IHA), suggesting a distinctive pathogenesis between APA and IHA. Although the functional detail of APMs has not been still uncovered, its impact on the pathogenesis of PA is gradually being revealed. In this review, we summarize the integrated findings regarding APA, APM or diffuse hyperplasia defined by novel CYP11B2, and aldosterone driver mutations. Following this, we discuss the clinical implications of KCNJ5 mutations to support better cardiovascular outcomes of primary aldosteronism.

Update on the Genetics of Primary Aldosteronism and Aldosterone-Producing Adenomas.

PURPOSE OF THE REVIEW: Primary aldosteronism (PA) is the leading cause of secondary hypertension, accounting for over 10% of patients with high blood pressure. It is characterized by autonomous production of aldosterone from the adrenal glands leading to low-renin levels. The two most common forms arise from bilateral adrenocortical hyperplasia (BAH) and aldosterone-producing adenoma (APA). We discuss recent discoveries in the genetics of PA. RECENT FINDINGS: Most APAs harbor variants in the KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1 genes. With the exception of ß-catenin (CTNNB1), all other causative genes encode ion channels; pathogenic variants found in PA lead to altered ion transportation, cell membrane depolarization, and consequently aldosterone overproduction. Some of these genes are found mutated in the germline state (CYP11B2, CLCN2, KCNJ5, CACNA1H, and CACNA1D), leading then to familial hyperaldosteronism, and often BAH rather than single APAs. Several genetic defects in the germline or somatic state have been identified in PA. Understanding how these molecular abnormalities lead to excess aldosterone contributes significantly to the elucidation of the pathophysiology of low-renin hypertension. It may also lead to new and more effective therapies for this disease acting at the molecular level.

Pathogenesis of Primary Aldosteronism: Impact on Clinical Outcome.

Primary aldosteronism (PA) is the most common form of secondary arterial hypertension, with a prevalence of approximately 20% in patients with resistant hypertension. In the last decade, somatic pathogenic variants in KCNJ5, CACNA1D, ATP1A1 and ATP2B3 genes, which are involved in maintaining intracellular ionic homeostasis and cell membrane potential, were described in aldosterone-producing adenomas (aldosteronomas). All variants in these genes lead to the activation of calcium signaling, the major trigger for aldosterone production. Genetic causes of familial hyperaldosteronism have been expanded through the report of germline pathogenic variants in KCNJ5, CACNA1H and CLCN2 genes. Moreover, PDE2A and PDE3B variants were associated with bilateral PA and increased the spectrum of genetic etiologies of PA. Of great importance, the genetic investigation of adrenal lesions guided by the CYP11B2 staining strongly changed the landscape of somatic genetic findings of PA. Furthermore, CYP11B2 staining allowed the better characterization of the aldosterone-producing adrenal lesions in unilateral PA. Aldosterone production may occur from multiple sources, such as solitary aldosteronoma or aldosterone-producing nodule (classical histopathology) or clusters of autonomous aldosterone-producing cells without apparent neoplasia denominated aldosterone-producing micronodules (non-classical histopathology). Interestingly, KCNJ5 mutational status and classical histopathology of unilateral PA (aldosteronoma) have emerged as relevant predictors of clinical and biochemical outcome, respectively. In this review, we summarize the most recent advances in the pathogenesis of PA and discuss their impact on clinical outcome.

Association of DNA methylation with steroidogenic enzymes in Cushing's adenoma.

DNA methylation and demethylation regulate the transcription of genes. DNA methylation-associated gene expression of adrenal steroidogenic enzymes may regulate cortisol production in cortisol-producing adenoma (CPA). We aimed to determine the DNA methylation levels of all genes encoding steroidogenic enzymes involved in CPA. Additionally, the aims were to clarify the DNA methylation-associated gene expression and evaluate the difference of CPA genotype from others using DNA methylation data. Twenty-five adrenal CPA and six nonfunctioning adrenocortical adenoma (NFA) samples were analyzed. RNA sequencing and DNA methylation array were performed. The methylation levels at 118 methylation sites of the genes were investigated, and their methylation and mRNA levels were subsequently integrated. Among all the steroidogenic enzyme genes studied, CYP17A1 gene was mainly found to be hypomethylated in CPA compared to that in NFA, and the Benjamini-Hochberg procedure demonstrated that methylation levels at two sites in the CYP17A1 gene body were statistically significant. PRKACA mutant CPAs predominantly exhibited hypomethylation of CYP17A1 gene compared with the GNAS mutant CPAs. Inverse associations between CYP17A1 methylation in three regions of the gene body and its mRNA levels were observed in the NFAs and CPAs. In applying clustering analysis using CYP17A1 methylation and mRNA levels, CPAs with PRKACA mutation were differentiated from NFAs and CPAs with a GNAS mutation. We demonstrated that CPAs exhibited hypomethylation of the CYP17A1 gene body in CPA, especially in the PRKACA mutant CPAs. Methylation of CYP17A1 gene may influence its transcription levels.

Mutational landscape of non-functional adrenocortical adenomas.

Adrenal incidentalomas are the most frequent human neoplasms. Recent genomic investigations on functional adrenocortical tumors have demonstrated that somatic mutations in PRKACA and KCNJ5 responsible for the development of adrenocortical adenomas (ACAs) are associated with hypercortisolism and aldosteronism, respectively. Several studies have identified CTNNB1 mutations in ACAs and have been mostly involved in the tumorigenesis of non-functional ACA (NFACA). However, integrated genomic characterization of NFACAs is lacking. In the current study, we utilized pan-genomic methods to comprehensively analyze 60 NFACA samples. A total of 1264 somatic mutations in coding regions among the 60 samples were identified, with a median of 15 non-silent mutations per tumor. Twenty-two NFACAs (36.67%) had genetic alterations in CTNNB1. We also identified several somatic mutations in genes of the cAMP/PKA pathway and KCNJ5. Histone modification genes (KMT2A, KMT2C, and KMT2D) were altered in 10% of cases. Germline mutations of MEN1 and RET were also found. Finally, by comparison of our transcriptome data with those available in the TCGA, we illustrated the molecular characterization of NFACA. We revealed the genetic profiling and molecular landscape of NFACA. Wnt/ß-catenin pathway activation as shown ssby nuclear and/or cytoplasmic ß-catenin accumulation is frequent, occurring in about one-third of ACA cases. cytochrome P450 enzymes could be markers to reveal the functional status of adrenocortical tumors. These observations strongly suggest the involvement of the Wnt/ß-catenin pathway in benign adrenal tumorigenesis and possibly in the regulation of steroid secretion.

Association of Nrf2 expression and mutation with Weiss and Helsinki scores in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignant tumor. Genetic abnormalities that may represent therapeutic targets and prognostic factors in ACC remain unclear. Besides being one of the main cellular defense mechanisms that regulates antioxidant pathways for detoxifying reactive oxygen species (ROS), the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) promotes tumor proliferation by increasing metabolic activity. In surgical specimens from 12 cases of nonmetastatic ACCs and nine cases of benign adrenocortical adenoma (ACA), we investigated gene mutation and protein expressions for Nrf2 and the preoperative maximum standard glucose uptake (SUVmax) on [18 F]fluorodeoxy-glucose positron emission tomography. Three of five ACCs with a Weiss score of 7 to 9 were Nrf2 mutants; these ACCs had higher expression of Nrf2 and higher preoperative SUVmax. The other seven ACCs had a Weiss score of 3 to 6; these seven ACCs and all the ACAs were non-Nrf2 gene mutants. Patients with a Weiss score of 7 to 9 and Nrf2 mutant ACC had shorter overall survival. Based on Helsinki scoring, three ACCs with a Helsinki score greater than 17 had Nrf2 mutants, higher expression of Nrf2, higher preoperative SUVmax, and shorter overall survival. Our findings indicate that Nrf2 activation and the associated increase in metabolism play roles in ACC, in particular in ACC with a Weiss score of 7 to 9 and a Helsinki score of greater than 17.