Somatic Mutations In Aldosterone-producing Adenomas Research Articles

PS-BPR01-7: CONCURRENT SOMATIC MUTATIONS IN CTNNB1 AND GNA11 IN AN ALDOSTERONE-PRODUCING ADENOMA

Background: Aldosterone-producing adenoma (APA) is a major subtype of primary aldosteronism (PA), a common form of secondary hypertension. In the past decade, there has been significant progress in the determination of genetic causes of APA. The use of next-generation sequencing (NGS) has led to the identification of somatic mutations in APA that cause excess aldosterone production. Most of the affected genes encode ion channels or transporters. Somatic activating mutations in CTNNB1 (β;-catenin) have also been detected in 2–5% of APA. As a possible pathogenesis, an association of CTNNB1-mutated APA with pregnancy and menopause has been proposed. A recent study reported double somatic mutations in CTNNB1 and GNA11/Q in APA. However, due to its rare incidence, characteristics of APA with these mutations are not well determined. Herein, we report a case of APA harboring somatic CTNNB1 and GNA11 mutations. Case presentation: A 46-year-old Japanese woman presented with hypertension and spontaneous hypokalemia. She had no history of pregnancy-induced hypertension. She had regular menstrual cycle at presentation. Endocrine testing showed elevated plasma aldosterone concentration (677.5 pg/mL) under suppressed renin (plasma renin activity, 0.3 ng/mL/h). Computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling indicated excess aldosterone production from the right adrenal gland. NP-59 scintigraphy further demonstrated increased tracer uptake in the right adrenal lesion. She underwent right laparoscopic adrenalectomy. Histologic diagnosis of the resected tumor was adrenocortical adenoma based on the Weiss criteria. Notably, Ki-67 labeling index was high (6% at hotspots). After surgery, her blood pressure and serum potassium both normalized. Based on the PASO (the primary aldosteronism surgical outcome) study criteria, PA remained clinically and biochemically cured at follow-up 18 months after surgery. Methods: Formalin-fixed, paraffin-embedded adrenal tumor tissue was used for immunohistochemical and genetic analysis. Results: Immunohistochemistry (IHC) showed high and diffuse expression of CYP11B2 (aldosterone synthase) within the tumor, whereas 11β;-hydroxylase (CYP11B1, a steroidogenic enzyme for cortisol biosynthesis) was mostly negative. VSNL1, a marker for the zona glomerulosa where physiologic aldosterone biosynthesis occurs, was highly expressed within the tumor. CYP11B2 IHC-guided targeted NGS identified concurrent somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations with similar variant allele frequencies (29.5% and 29.7%, respectively). Conclusions: To best of our knowledge, this is the first Asian case of APA with CTNNB1 and GNA11 mutations. Detailed clinical and histologic examination would provide useful information for better characterization of patients with PA due to these rare mutations.

Colocalization of Wnt/β-Catenin and ACTH Signaling Pathways and Paracrine Regulation in Aldosterone-producing Adenoma.

Aldosterone-producing adenomas (APAs) are a common cause of primary aldosteronism (PA). Despite the discovery of somatic mutations in APA and the characterization of multiple factors regulating adrenal differentiation and function, the sequence of events leading to APA formation remains to be determined. We investigated the role of Wnt/β-catenin and adrenocorticotropin signaling, as well as elements of paracrine regulation of aldosterone biosynthesis in adrenals with APA and their relationship to intratumoral heterogeneity and mutational status. We analyzed the expression of aldosterone-synthase (CYP11B2), CYP17A1, β-catenin, melanocortin type 2 receptor (MC2R), phosphorlyated cAMP response element-binding protein (pCREB), tryptase, S100, CD34 by multiplex immunofluorescence, and immunohistochemistry-guided reverse transcription-quantitative polymerase chain reaction. Eleven adrenals with APA and 1 with micronodular hyperplasia from patients with PA were analyzed. Main outcome measures included localization of CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, tryptase, S100, CD34 in APA and aldosterone-producing cell clusters (APCCs). Immunofluorescence revealed abundant mast cells and a dense vascular network in APA, independent of mutational status. Within APA, mast cells were localized in areas expressing CYP11B2 and were rarely colocalized with nerve fibers, suggesting that their degranulation is not controlled by innervation. In these same areas, ß-catenin was activated, suggesting a zona glomerulosa cell identity. In heterogeneous APA with KCNJ5 mutations, MC2R and vascular endothelial growth factor A expression was higher in areas expressing CYP11B2. A similar pattern was observed in APCC, with high expression of CYP11B2, activated β-catenin, and numerous mast cells. Our results suggest that aldosterone-producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status, suggesting common developmental mechanisms.

KCNJ5 Somatic Mutations in Aldosterone-Producing Adenoma Are Associated with a Greater Recovery of Arterial Stiffness.

Simple SummaryPrimary aldosteronism (PA) is the most common form of secondary hypertension and induces various cardiovascular injuries. Aldosterone-producing adenoma (APA) is one of the major forms of PA. The occurrence of APA is closely correlated with somatic mutations, including KCNJ5. We described here the impact of KCNJ5 somatic mutations on arterial stiffness excluding the influence of age, sex, and blood pressure status. We found KCNJ5 mutation carriers had similar arterial stiffness before surgery, but greater improvement of arterial stiffness after adrenalectomy compared with non-carriers. Hence, APA patients with KCNJ5 mutations had a greater improvement in arterial stiffness after adrenalectomy than those without mutations.Primary aldosteronism is the most common form of secondary hypertension and induces various cardiovascular injuries. In aldosterone-producing adenoma (APA), the impact of KCNJ5 somatic mutations on arterial stiffness excluding the influence of confounding factors is uncertain. We enrolled 213 APA patients who were scheduled to undergo adrenalectomy. KCNJ5 gene sequencing of APA was performed. After propensity score matching (PSM) for age, sex, body mass index, blood pressure, number of hypertensive medications, and hypertension duration, there were 66 patients in each group with and without KCNJ5 mutations. The mutation carriers had a higher aldosterone level and lower log transformed brachial–ankle pulse wave velocity (baPWV) than the non-carriers before PSM, but no difference in log baPWV after PSM. One year after adrenalectomy, the mutation carriers had greater decreases in log plasma aldosterone concentration, log aldosterone–renin activity ratio, and log baPWV than the non-carriers after PSM. Only the mutation carriers had a significant decrease in log baPWV after surgery both before and after PSM. KCNJ5 mutations were not correlated with baseline baPWV after PSM but were significantly correlated with ∆baPWV after surgery both before and after PSM. Conclusively, APA patients with KCNJ5 mutations had a greater regression in arterial stiffness after adrenalectomy than those without mutations.

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-β-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.

The Impact of ACTH on Peripheral Steroids Differs between Unilateral and Bilateral Primary Aldosteronism

Background: ACTH is thought to contribute to aldosterone excess in primary aldosteronism (PA), possibly via aberrant melanocortin type 2 receptor (MC2R) expression in aldosterone producing adenomas (APAs). Dynamic manipulation of the hypothalamic-pituitary-adrenal (HPA) axis has been proposed as a non-invasive tool for distinguishing unilateral PA (UPA) from bilateral PA (BPA), but existing data are minimal. Objective: To characterize the steroid responses to intrinsic ACTH variations and extrinsic HPA manipulation in UPA and BPA. Methods: We conducted comprehensive dynamic testing in PA patients, who were subtyped based on adrenal vein sampling. Peripheral plasma samples were collected from each patient at 6 time-points: morning; midnight; after 1 mg dexamethasone suppression (DST); and after cosyntropin stimulation (at 15’, 30’, and 60’). We quantified 15 steroids by mass spectrometry in each sample. Next generation sequencing was used to detect aldosterone-driver somatic mutations in APAs from 39 cases with available tissue. The Mann-Whitney test, Wilcoxon signed rank test, and repeated measures two-way ANOVA were employed, as appropriate. Penalized logistic regression was used to select steroids that best distinguished UPA from BPA. Receiver operating characteristic (ROC) curves were then plotted using the predicted score from the logistic regression model with the selected steroids, and area under the curves (AUC) were computed. Results: We included 80 PA patients, median age 51 (range, 26–76), 50% men, 40 with each subtype, both groups with similar age and sex distribution. Morning and midnight concentrations of 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), aldosterone, and 18-hydroxycorticosterone (18OHB) were higher in patients with UPA vs. BPA (p<0.001 for all). In response to cosyntropin stimulation, the UPA group had larger increments of aldosterone, 18oxoF, 11-deoxycorticosterone, corticosterone, and 11-deoxycortisol than the BPA group (p<0.05 for all). Following DST, aldosterone,18OHF, and 18oxoF were higher in UPA than in BPA patients (p<0.01 for all). Overall, cortisol and cortisone serum concentrations were similar between the two subtypes. Of the UPA cases, 27 (69%) had KCNJ5 mutations. Relative to UPA patients with other mutations, the KCNJ5 group had higher 18oxoF and 18OHF at baseline; higher 18oxoF and corticosterone after both dynamic tests; and lower aldosterone after DST. The highest AUC for PA subtyping was achieved using cosyntropin stimulated steroids (0.957), while baseline data reached an AUC of 0.909. Conclusions: Steroid responses to dynamic HPA testing differs between UPA and BPA: 18oxoF and 18OHF are less suppressible, while several steroids are disproportionally amplified by ACTH in patients with UPA vs. BPA. Such non-invasive tests could circumvent the need for adrenal vein sampling in a subset of PA patients.

GENETICS IN ENDOCRINOLOGY: Impact of race and sex on genetic causes of aldosterone-producing adenomas.

Primary aldosteronism (PA) is a common cause of secondary hypertension. Recent technological advances in genetic analysis have provided a better understanding of the molecular pathogenesis of this disease. The application of next-generation sequencing has resulted in the identification of somatic mutations in aldosterone-producing adenoma (APA), a major subtype of PA. Based on the recent findings using a sequencing method that selectively targets the tumor region where aldosterone synthase (CYP11B2) is expressed, the vast majority of APAs appear to harbor a somatic mutation in one of the aldosterone-driver genes, including KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CLCN2. Mutations in these genes alter intracellular ion homeostasis and enhance aldosterone production. In a small subset of APAs, somatic activating mutations in the CTNNB1 gene, which encodes β-catenin, have also been detected. Accumulating evidence suggests that race and sex impact the somatic mutation spectrum of APA. Specifically, somatic mutations in the KCNJ5 gene, encoding an inwardly rectifying K+ channel, are common in APAs from Asian populations as well as women regardless of race. Associations between APA histology, genotype, and patient clinical characteristics have also been proposed, suggesting a potential need to consider race and sex for the management of PA patients. Herein, we review recent findings regarding somatic mutations in APA and discuss potential roles of race and sex on the pathophysiology of APA as well as possible clinical implications.

Hypertension Editors' Picks: Hyperaldosteronism.

Hypertension Editors' Picks: Hyperaldosteronism.

Prevalence of Somatic Mutations in Aldosterone-Producing Adenomas in Japanese Patients.

Results of previous studies demonstrated clear racial differences in the prevalence of somatic mutations among patients with aldosterone-producing adenoma (APA). For instance, those in East Asian countries have a high prevalence of somatic mutations in KCNJ5, whereas somatic mutations in other aldosterone-driving genes are rare. To determine somatic mutation prevalence in Japanese APA patients using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided sequencing approach. Patients with a unilateral form of primary aldosteronism who underwent adrenalectomy at the Tohoku University Hospital were studied. Based on CYP11B2 immunolocalization of resected adrenals, genomic DNA was isolated from the relevant positive area of 10% formalin-fixed, paraffin-embedded tissue of the APAs. Somatic mutations in aldosterone-driving genes were studied in APAs by direct Sanger sequencing and targeted next-generation sequencing. CYP11B2 IHC-guided sequencing determined APA-related somatic mutations in 102 out of 106 APAs (96%). Somatic KCNJ5 mutation was the most frequent genetic alteration (73%) in this cohort of Japanese patients. Somatic mutations in other aldosterone-driving genes were also identified: CACNA1D (14%), ATP1A1 (5%), ATP2B3 (4%), and CACNA1H (1%), including 2 previously unreported mutations. KCNJ5 mutations were more often detected in APAs from female patients compared with those from male patients [95% (36/38) vs 60% (41/68); P < 0.0001]. IHC-guided sequencing defined somatic mutations in over 95% of Japanese APAs. While the dominance of KCNJ5 mutations in this particular cohort was confirmed, a significantly higher KCNJ5 prevalence was detected in female patients. This study provides a better understanding of genetic spectrum of Japanese APA patients.

The Potential Role of Aldosterone-Producing Cell Clusters in Adrenal Disease.

Primary aldosteronism (PA) is the most common cause of secondary hypertension. The hallmark of PA is adrenal production of aldosterone under suppressed renin conditions. PA subtypes include adrenal unilateral and bilateral hyperaldosteronism. Considerable progress has been made in defining the role for somatic gene mutations in aldosterone-producing adenomas (APA) as the primary cause of unilateral PA. This includes the use of next-generation sequencing (NGS) to define recurrent somatic mutations in APA that disrupt calcium signaling, increase aldosterone synthase (CYP11B2) expression, and aldosterone production. The use of CYP11B2 immunohistochemistry on adrenal glands from normal subjects, patients with unilateral and bilateral PA has allowed the identification of CYP11B2-positive cell foci, termed aldosterone-producing cell clusters (APCC). APCC lie beneath the adrenal capsule and like APA, many APCC harbor somatic gene mutations known to increase aldosterone production. These findings suggest that APCC may play a role in pathologic progression of PA. Herein, we provide an update on recent research directed at characterizing APCC and also discuss the unanswered questions related to the role of APCC in PA.

Genetic Characteristics of Aldosterone-Producing Adenomas in Blacks.

Somatic mutations have been identified in aldosterone-producing adenomas (APAs) in genes that include KCNJ5, ATP1A1, ATP2B3, and CACNA1D. Based on independent studies, there appears to be racial differences in the prevalence of somatic KCNJ5 mutations, particularly between East Asians and Europeans. Despite the high cardiovascular disease mortality of blacks, there have been no studies focusing on somatic mutations in APAs in this population. In the present study, we investigated genetic characteristics of APAs in blacks using a CYP11B2 (aldosterone synthase) immunohistochemistry-guided next-generation sequencing approach. The adrenal glands with adrenocortical adenomas from 79 black patients with primary aldosteronism were studied. Seventy-three tumors from 69 adrenal glands were confirmed to be APAs by CYP11B2 immunohistochemistry. Sixty-five of 73 APAs (89%) had somatic mutations in aldosterone-driver genes. Somatic CACNA1D mutations were the most prevalent genetic alteration (42%), followed by KCNJ5 (34%), ATP1A1 (8%), and ATP2B3 mutations (4%). CACNA1D mutations were more often observed in APAs from males than those from females (55% versus 29%, P=0.033), whereas KCNJ5 mutations were more prevalent in APAs from females compared with those from males (57% versus 13%, P<0.001). No somatic mutations in aldosterone-driver genes were identified in tumors without CYP11B2 expression. In conclusion, 89% of APAs in blacks harbor aldosterone-driving mutations, and unlike Europeans and East Asians, the most frequently mutated aldosterone-driver gene was CACNA1D. Determination of racial differences in the prevalence of aldosterone-driver gene mutations may facilitate the development of personalized medicines for patients with primary aldosteronism.

Targeted Molecular Characterization of Aldosterone-Producing Adenomas in White Americans.

Somatic mutations have been identified in more than half of aldosterone-producing adenomas (APAs) through mutation hotspot sequencing. The underlying pathogenesis of inappropriate aldosterone synthesis in the remaining population is still unknown. To investigate the prevalence and spectrum of somatic mutations in APAs using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)‒guided next-generation sequencing (NGS) approach. Formalin-fixed paraffin-embedded adrenal tissue from white American patients with primary aldosteronism who underwent adrenalectomy at the University of Michigan was used. Genomic DNA was isolated from 75 APAs (identified by CYP11B2 IHC). NGS was performed to identify somatic mutations by sequencing the entire coding region of a panel of genes mutated in APAs. Somatic mutations were identified in 66 of 75 APAs (88%). Of the APAs with somatic mutations, six were smaller than coexisting CYP11B2-negative adrenocortical adenomas. The most frequently mutated gene was KCNJ5 (43%), followed by CACNA1D (21%), ATP1A1 (17%), ATP2B3 (4%), and CTNNB1 (3%). In addition to identification of previously reported mutations, we identified five previously unreported mutations (two in KCNJ5, one in ATP1A1, one in ATP2B3, and one in CACNA1D genes). KCNJ5 mutations were more frequent in women (70% vs 24% in men). Comprehensive NGS of CYP11B2-expressing adrenal tumors identified somatic mutations in aldosterone-driving genes in 88% of APAs, a higher rate than in previous studies using conventional approaches.

An Update on Familial Hyperaldosteronism.

Familial forms of primary aldosteronism have been suggested to account for up to 6% of cases in referral centers. For many years, the genetics of familial hyperaldosteronism remained unknown, with the notable exception of glucocorticoid-remediable aldosteronism, due to unequal crossing over and formation of a chimeric 11β-hydroxylase/aldosterone synthase gene. Over the past 5 years, mutations in 3 additional genes have been shown to cause familial forms of primary aldosteronism. Gain-of-function heterozygous germline mutations in KCNJ5, which encodes an inward rectifier potassium channel, cause autosomal dominant syndromes of PA and hypertension with or without adrenal hyperplasia. Germline mutations in CACNA1D, which codes for an L-type calcium channel, have so far only been found in 2 cases with a syndrome of primary aldosteronism, seizures, and neurologic abnormalities. Both KCNJ5 and CACNA1D mutations in familial hyperaldosteronism were only discovered following identification of similar or identical somatic mutations in aldosterone-producing adenomas. In contrast, a recent exome sequencing study identified germline mutations in CACNA1H (a T-type calcium channel), previously undescribed in adenomas, in 5 unrelated families with early-onset primary aldosteronism and hypertension, without any additional shared symptoms. Future exome or genome sequencing studies are expected to shed light on the genetic basis of many cases of familial hyperaldosteronism that remain unexplained.

Prevalence and Clinical Correlates of Somatic Mutation in Aldosterone Producing Adenoma-Taiwanese population

Primary aldosteronism (PA) is a common form of secondary hypertension and has significant cardiovascular consequences. Mutated channelopathy due to activation of calcium channels has recently been described in aldosterone-producing adenoma (APA). The aim of this study was to investigate the prevalence of somatic mutations in these key genes in unselected patients and their clinical and cardio-metabolic parameters and molecular correlates. The study involved 148 consecutive PA patients, (66 males; aged 56.3 ± 12.3years) identified by modified four corners rules who received adrenalectomy, collected from the Taiwan PA investigator (TAIPAI) group, A high rate of somatic mutation in APA was found (n = 91, 61.5%); including mutations in KCNJ5 (n = 88, 59.5%), ATP1A1 (n = 2, 1.4%), and ATP2B3(n = 1, 0.7%); however, no mutations in CACNA1D were identified. Mutation-carriers were younger (<0.001), had lower Cyst C (p = 0.042), pulse wave velocity (p = 0.027), C-reactive protein (p = 0.042) and a lower rate of proteinuria (p = 0.031) than non-carriers. After multivariate adjustment, mutation carriers had lower serum CRP levels than non-carriers. Patients with mutation also had a greater chance of recovery from hypertension after operation (p = 0.005) A high incidence of somatic mutations in APA was identified in Taiwanese population. Mutation- carriers had lower CRP levels and a higher rate of cure of hypertension after adrenalectomy. This raises the possibility of using mutation screening as a tool in predicting long term outcome after adrenalectomy.

Genetic spectrum and clinical correlates of somatic mutations in aldosterone-producing adenoma.

Primary aldosteronism is the most common form of secondary hypertension. Somatic mutations in KCNJ5, ATP1A1, ATP2B3, and CACNA1D have been described in aldosterone-producing adenomas (APAs). Our aim was to investigate the prevalence of somatic mutations in these genes in unselected patients with APA (n=474), collected through the European Network for the Study of Adrenal Tumors. Correlations with clinical and biochemical parameters were first analyzed in a subset of 199 patients from a single center and then replicated in 2 additional centers. Somatic heterozygous KCNJ5 mutations were present in 38% (180/474) of APAs, whereas ATP1A1 mutations were found in 5.3% (25/474) and ATP2B3 mutations in 1.7% (8/474) of APAs. Previously reported somatic CACNA1D mutations as well as 10 novel CACNA1D mutations were identified in 44 of 474 (9.3%) APAs. There was no difference in the cellular composition of APAs or in CYP11B2, CYP11B1, KCNJ5, CACNA1D, or ATP1A1 gene expression in APAs across genotypes. Patients with KCNJ5 mutations were more frequently female, diagnosed younger, and with higher minimal plasma potassium concentrations compared with CACNA1D mutation carriers or noncarriers. CACNA1D mutations were associated with smaller adenomas. These associations were largely dependent on the population structure of the different centers. In conclusion, recurrent somatic mutations were identified in 54% of APAs. Young women with APAs are more likely to be KCNJ5 mutation carriers; identification of specific characteristics or surrogate biomarkers of mutation status may lead to targeted treatment options.

KCNJ5 Mutations in European Families With Nonglucocorticoid Remediable Familial Hyperaldosteronism

Primary aldosteronism is the most frequent cause of endocrine hypertension. Three forms of familial hyperaldosteronism (FH) have been described, named FH-I to -III. Recently, a mutation of KCNJ5 has been shown to be associated with FH-III, whereas the cause of FH-II is still unknown. In this study we searched for mutations in KCNJ5 in 46 patients from 21 families with FH, in which FH-I was excluded. We identified a new germline G151E mutation in 2 primary aldosteronism-affected subjects from an Italian family and 3 somatic mutations in aldosterone-producing adenomas, T158A described previously as a germline mutation associated with FH-III, and G151R and L168R both described as somatic mutations in aldosterone-producing adenoma. The phenotype of the family with the G151E mutation was remarkably milder compared with the previously described American family, in terms of both clinical and biochemical parameters. Furthermore, patients with somatic KCNJ5 mutations displayed a phenotype indistinguishable from that of sporadic primary aldosteronism. The functional characterization of the effects of the G151E mutation in vitro showed a profound alteration of the channel function, with loss of K(+) selectivity, Na(+) influx, and membrane depolarization. These alterations have been postulated to be responsible for voltage gate Ca(2+) channel activation, increase in cytosolic calcium, and stimulation of aldosterone production and adrenal cell proliferation. In conclusion, we describe herein a new mutation in the KCNJ5 potassium channel associated with FH-III, responsible for marked alterations of channel function but associated with a mild clinical and hormonal phenotype.