Aldosterone-producing Cell Clusters Research Articles

A Case of Idiopathic Hyperaldosteronism with Cortisol Producing Adenoma and Aldosterone Producing Cell Clusters in the Removed Adrenal Gland

A Case of Idiopathic Hyperaldosteronism with Cortisol Producing Adenoma and Aldosterone Producing Cell Clusters in the Removed Adrenal Gland

Expression of aldosterone synthase CYP11B2 was inversely correlated with longevity

Immunohistochemistry of human aldosterone synthase (CYP11B2) has revealed that most of aldosterone is autonomously produced in aldosterone-producing cell clusters (APCCs) beneath the capsule of adult adrenals rather than physiologically in the zona glomerulosa (ZG). APCCs have been occasionally found to harbor a somatic mutation of ion channel/pump genes, and number and size of APCCs increase with age until 50 years old. Herein, the objective of the study was to examine APCC development in 106 autopsied adrenals from 85 elderly individuals who died at ages from 50 to 103 years. We obtained the following results: (1) physiological CYP11B2 expression in ZG were attenuated in more elderly persons; (2) number and size of APCCs decreased with age; (3) detachment of APCC from the capsule appeared to occur occasionally over the wide range of the ages; and (4) incidental micro aldosterone-producing adenomas (APAs) and possible APCC-to-APA transitional lesions (pAATLs) were found primarily in samples from persons aged 50–60 years but not in samples from more elderly persons; pAATL was a putative designation based on our previous results indicating that it consisted of subcapsular APCC-like portion and inner APA-like portions. Thus, the formation of the CYP11B2-expressing lesions as well as thickening of the ZG in the adrenals were inversely correlated with age of death in the individuals aged over 50 years. Considering that autopsy samples were used in this study, inactive production of aldosterone regardless of autonomous or physiological manners may have survival advantages in individuals aged over 50 years.

Immunohistochemistry for aldosterone synthase CYP11B2 and matrix-assisted laser desorption ionization imaging mass spectrometry for in-situ aldosterone detection

Immunohistochemistry for aldosterone synthase (CYP11B2) has markedly provided a comprehensive picture of the adrenocortical diseases, particularly primary aldosteronism. The findings from CYP11B2-immunohistochemistry are consistent with the clinical courses of most patients with primary aldosteronism. We herein review the updated pathophysiology and usefulness of the method for understanding individual patients with different subtypes of primary aldosteronism. After our discovery of aldosterone-producing cell clusters (APCCs) using the immunohistochemistry for CYP11B2, we found possible APCC-to-APA transitional lesions (pAATLs) in a few cases that had been hitherto classified as unilateral hyperplasia or multiple nodules. On the basis of morphological and functional features of pAATLs as well as distributions of somatic mutations within the lesions, we have made a hypothesis that APCC grows to APA via pAATL for one of developmental courses of APA. Recently, we successfully performed in-situ detection of aldosterone on adrenal tissue sections using a state-of-the-art technique, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-imaging). This method revealed in-situ synthesis of aldosterone in APCCs and APAs in addition to several other steroids. CYP11B2 immunohistochemistry revealed the pathophysiology of aldosterone production in the past decade, especially formation of APCC in normal adrenals and pAATL that is a possible lesion developing from APCC to APA. The term 'idiopathic hyperaldosteronism' may soon become obsolete.

Aldosterone-Producing Adenomas.

Aldosterone-producing adenomas (APA) are more common than initially anticipated. APA cause primary aldosteronism (PA), which affect 3-10% of the hypertensive population. Research during recent years has led to an increased knowledge of the background dysregulation of the increased aldosterone release, where mutation in the gene encoding the potassium channel GIRK4-KCNJ5-is the most common. Moreover, the discovery of aldosterone-producing cell clusters in apparently normal adenomas has also led to increased understanding of the development of PA, and presumably also APA. A continuum ranging from low-renin hypertension to APA and overt PA is reasoned, and the secondary effects of aldosterone on especially the cardiovascular system have also become more evident. Diagnostics of PA and APA is important in order to reduce cardiovascular morbidity and mortality, but the diagnostic methods are somewhat unspecific and insensitive, indicating the need for novel methods.

Aldosterone and 18-Oxocortisol Coaccumulation in Aldosterone-Producing Lesions.

Primary aldosteronism is a secondary hypertensive disease caused by autonomous aldosterone production that often caused by an aldosterone-producing adenoma (APA). Immunohistochemistry of aldosterone synthase (CYP11B2) shows the presence of aldosterone-producing cell clusters (APCCs) even in non-primary aldosteronism adult adrenal cortex. An APCC-like structure also exists as possible APCC-to-APA transitional lesions (a speculative designation) in primary aldosteronism adrenals. However, whether APCCs produce aldosterone or 18-oxocortisol, a potential serum marker of APA, remains unknown because of lack of technology to visualize adrenocorticosteroids on tissue sections. To address this obstacle, in this study, we used highly sensitive Fourier transform ion cyclotron resonance mass spectrometry to image various adrenocorticosteroids, including 18-oxocortisol, in adrenal tissue sections from 8 primary aldosteronism patients with APCC (cases 1-4), possible APCC-to-APA transitional lesions (case 5), and APA (cases 6-8). Further analyses by tandem mass spectrometry imaging allowed us to differentially visualize aldosterone from cortisone, which share identical mass-to-charge ratio value ( m/z). In conclusion, these advanced imaging techniques revealed that aldosterone and 18-oxocortisol coaccumulated within CYP11B2-expressing lesions. These imaging outcomes along with a growing body of aldosterone research led us to build a progressive development hypothesis of an aldosterone-producing pathology in the adrenal glands.

Cellular and Genetic Causes of Idiopathic Hyperaldosteronism.

Primary aldosteronism affects ≈5% to 10% of hypertensive patients and has unilateral and bilateral forms. Most unilateral primary aldosteronism is caused by computed tomography-detectable aldosterone-producing adenomas, which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone-regulating genes. The cause of the most common bilateral form of primary aldosteronism, idiopathic hyperaldosteronism (IHA), is believed to be diffuse hyperplasia of aldosterone-producing cells within the adrenal cortex. Herein, a multi-institution cohort of 15 IHA adrenals was examined with CYP11B2 immunohistochemistry and next-generation sequencing. CYP11B2 immunoreactivity in adrenal glomerulosa harboring non-nodular hyperplasia was only observed in 4/15 IHA adrenals suggesting that hyperplasia of CYP11B2-expressing cells may not be the major cause of IHA. However, the adrenal cortex of all IHA adrenals harbored at least 1 CYP11B2-positive aldosterone-producing cell cluster (APCC) or micro-aldosterone-producing adenomas. The number of APCCs per case (and individual APCC area) in IHA adrenals was significantly larger than in normotensive controls. Next-generation sequencing of DNA from 99 IHA APCCs demonstrated somatic mutations in genes encoding the L-type calcium voltage-gated channel subunit α 1-D ( CACNA1D, n=57; 58%) and potassium voltage-gated channel subfamily J-5 ( KCNJ5, n=1; 1%). These data suggest that IHA may result from not only hyperplasia but also the accumulation or enlargement of computed tomography-undetectable APCC harboring somatic aldosterone-driver gene mutations. The high prevalence of mutations in the CACNA1D L-type calcium channel provides a potential actionable therapeutic target that could complement mineralocorticoid blockade and inhibit aldosterone overproduction in some IHA patients.

Immunohistopathology and Steroid Profiles Associated With Biochemical Outcomes After Adrenalectomy for Unilateral Primary Aldosteronism.

Unilateral primary aldosteronism (PA) is the most common surgically curable form of hypertension that must be accurately differentiated from bilateral PA for therapeutic management (surgical versus medical). Adrenalectomy results in biochemical cure (complete biochemical success) in almost all patients diagnosed with unilateral PA; the remaining patients with partial or absent biochemical success comprise those with persisting aldosteronism who were misdiagnosed as unilateral PA preoperatively. To identify determinants of postsurgical biochemical outcomes, we compared the adrenal histopathology and the peripheral venous steroid profiles of patients with partial and absent or complete biochemical success after adrenalectomy for unilateral PA. A large multicenter cohort of adrenals from patients with absent and partial biochemical success (n=43) displayed a higher prevalence of hyperplasia (49% versus 21%; P=0.004) and a lower prevalence of solitary functional adenoma (44% versus 79%; P<0.001) compared with adrenals from age- and sex-matched patients with PA with complete biochemical success (n=52). We measured the peripheral plasma steroid concentrations in a subgroup of these patients (n=43) and in a group of patients with bilateral PA (n=27). Steroid profiling was associated with histopathologic phenotypes (solitary functional adenoma, hyperplasia, and aldosterone-producing cell clusters) and classified patients according to biochemical outcome or diagnosis of bilateral PA. If validated, peripheral venous steroid profiling may be a useful tool to guide the decision to perform surgery based on expectations of biochemical outcome after the procedure.

Abstract 098: Aldosterone-Producing Cell Clusters in Essential Hypertension

Introduction: Primary aldosteronism (PA) is one of the major causes of secondary hypertension and has two major subtypes. One is unilateral aldosterone-producing adenoma (APA), which is positive for aldosterone synthase (CYP11B2) by immunohistochemistry (IHC), and the other is bilateral idiopathic hyperaldosteronism (IHA), which has image-undetectable CYP11B2-positive cell clusters (termed aldosterone-producing cell clusters, APCC) beneath the adrenal capsule. We and others have previously shown that APCC present in normotensive adrenals and the number is significantly less than IHA adrenals, supporting the disease-causing role of APCC in hyperaldosteronism of IHA. Furthermore, we have also shown that most of APCCs in the IHA and normotensive adrenals harbor somatic mutations in a L-type calcium channel, CACNA1D , whereas most of APA has been previously shown to harbor those in a potassium channel, KCNJ5 . These mutations are reported to increase intracellular calcium levels, resulting in aldosterone overproduction. Here, we seek for the first time to identify the potential role of APCC in non-PA hypertensive patients and to clarify whether these adrenals could be precursor of APA or IHA by performing next-generation sequencing (NGS) on observed APCC. Method and Results: We selected fifteen adrenal glands with the evidence of hypertension and/or antihypertensive agents from a cohort of serial Japanese autopsy cases. None of the cases harbored adrenal tumors, but instead nine cases harbored 23 APCCs. We then isolated DNA from each APCC and performed NGS targeting genes mutated in APA. Of observed APCCs, 9 (39%) APCCs harbored somatic mutations in CACNA1D . Interpretations: These results show that autonomous aldosterone production in a part of patients with essential hypertension is caused by APCCs with aldosterone-driving somatic mutations. In addition, the mutation spectrum observed in this cohort suggests that essential hypertension could potentially develop into IHA through aldosterone overproduction by APCC.

Molecular genetics of Conn adenomas in the era of exome analysis

Aldosterone-producing adenomas (APA) are a major cause of primary aldosteronism (PA), the most common form of secondary hypertension. Exome analysis of APA has allowed the identification of recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3in more than 50% of sporadic cases. These gain of function mutations in ion channels and pumps lead to increased and autonomous aldosterone production. In addition, somatic CTNNB1mutations have also been identified in APA. The CTNNB1mutations were also identified in cortisol-producing adenomas and adrenal cancer, but their role in APA development and the mechanisms specifying the hormonal production or the malignant phenotype remain unknown. The role of the somatic mutations in the regulation of aldosterone production is well understood, while the impact of these mutations on cell proliferation remains to be established. Furthermore, the sequence of events leading to APA formation is currently the focus of many studies. There is evidence for a two-hit model where the somatic mutations are second hits occurring in a previously remodeled adrenal cortex. On the other hand, the APA-driver mutations were also identified in aldosterone-producing cell clusters (APCC) in normal adrenals, suggesting that these structures may represent precursors for APA development. As PA due to APA can be cured by surgical removal of the affected adrenal gland, the identification of the underlying genetic abnormalities by novel biomarkers could improve diagnostic and therapeutic approaches of the disease. In this context, recent data on steroid profiling in peripheral venous samples of APA patients and on new drugs capable of inhibiting mutated potassium channels provide promising preliminary data with potential for translation into clinical care.

Overview of aldosterone-related genetic syndromes and recent advances.

Primary aldosteronism is the most common form of secondary hypertension. Early diagnosis and treatment are key to cure of hypertension and prevention of cardiovascular complications. Recent genetic discoveries have improved our understanding on the pathophysiology of aldosterone production and triggered the development of new diagnostic procedures and targeted treatments for primary aldosteronism. Different inherited genetic abnormalities distinguish specific forms of familial hyperaldosteronism. Somatic mutations are found not only in aldosterone-producing adenoma (APA), leading to primary aldosteronism, but also in aldosterone producing cell clusters of normal and micronodules from image-negative adrenal glands. Genetic knowledge has allowed the discovery of surrogate biomarkers and specific pharmacological inhibitors. Ageing appears to be associated with dysregulated and relatively autonomous aldosterone production. New biochemical markers and pharmacological approaches may allow preoperative identification of somatic mutation carriers and use of targeted treatments.

Impact of aldosterone-producing cell clusters on diagnostic discrepancies in primary aldosteronism.

Adrenocorticotropic hormone (ACTH) stimulation is recommended in adrenal vein sampling (AVS) for primary aldosteronism (PA) to improve the AVS success rate. However, this method can confound the subtype diagnosis. Gene mutations or pathological characteristics may be related to lateralization by AVS. This study aimed to compare the rate of diagnostic discrepancy by AVS pre- versus post-ACTH stimulation and to investigate the relationship between this discrepancy and findings from immunohistochemical and genetic analyses of PA. We evaluated 195 cases of AVS performed in 2011–2017. All surgical specimens were analyzed genetically and immunohistochemically. Based on the criteria, AVS was successful in 158 patients both pre- and post-ACTH; of these patients, 75 showed diagnostic discrepancies between pre- and post-ACTH. Thus, 19 patients underwent unilateral adrenalectomy, of whom 16 had an aldosterone-producing adenoma (APA) that was positive for CYP11B2 immunostaining. Of them, 10 patients had discordant lateralization between pre- and post-ACTH. In the genetic analysis, the rate of somatic mutations was not significantly different between APA patients with versus without a diagnostic discrepancy. In the immunohistochemical analysis, CYP11B2 levels and the frequency of aldosterone-producing cell clusters (APCCs) in APAs were almost identical between patients with versus without a diagnostic discrepancy. However, both the number and summed area of APCCs in APAs were significantly smaller in patients with concordant results than in those whose diagnosis changed to bilateral PA post-ACTH stimulation. In conclusion, lateralization by AVS was affected by APCCs in the adjacent gland, but not by APA-related factors such as somatic gene mutations.

Aging and Adrenal Aldosterone Production.

Aldosterone, the primary mineralocorticoid, is synthesized in the outer zone of the adrenal cortex called the zona glomerulosa (ZG). The production of aldosterone is tightly regulated by angiotensin II (Ang II) and circulating potassium levels.1 Physiologically, aldosterone plays a key role in the maintenance of intravascular volume and blood pressure through sodium retention in the kidney. Excess aldosterone causes hypertension and induces cardiovascular complications.2–5 The autonomous secretion of aldosterone, independent of Ang II and sodium status, is known as primary aldosteronism (PA). PA is the most common cause of endocrine-related hypertension with a prevalence of 5% to 10% in hypertensive population6–10 and ≈20% in resistant hypertension.11–13 Because of the increased risk for cardiovascular complications in patients with PA, early detection of the disease and targeted treatment is recommended.14 The molecular pathogenesis of PA was largely unknown until recently. The development of specific antibodies against aldosterone synthase (CYP11B2), which is required for the final steps of aldosterone production, has allowed the detection of aldosterone-producing cells in resected adrenals.15,16 Using these antibodies, non-neoplastic foci of CYP11B2-expressing cells called aldosterone-producing cell clusters (APCC)15 have been identified in adrenal tissues adjacent to aldosterone-producing adenomas (APA) and in normal human adrenal glands without tumor or hyperplasia.15–20 Studies have shown that APCC are a common occurrence in normal human adrenals.21–23 Because circulating renin and Ang II levels are suppressed in patients with PA, the observation of APCC adjacent to APA suggested that APCC may represent a source of autonomous and renin-independent aldosterone secretion, perhaps even a precursor to APA.24 Over the past 6 years, somatic and germline mutations that cause inappropriate aldosterone production have been identified in patients with PA. Most of the mutations …

Aldosterone-Producing Cell Clusters in Normal and Pathological States.

Primary aldosteronism (PA) significantly increases the risk of cardiovascular complications, and early diagnosis and targeted treatment based on its pathophysiology is warranted. Next-generation sequencing (NGS) has revealed recurrent somatic mutations in aldosterone-driving genes in aldosterone-producing adenoma (APA). By applying CYP11B2 (aldosterone synthase) immunohistochemistry and NGS to adrenal glands from normal subjects and PA patients, we and others have shown that CYP11B2-positive cells make small clusters, termed aldosterone-producing cell clusters (APCC), beneath the adrenal capsule, and that APCC harbor somatic mutations in genes mutated in APA. We have shown that APCC are increased in CT-negative PA adrenals, while others showed potential progression from APCC to micro APA through mutations. These results suggest that APCC are a key factor for understanding the origin of PA, and further investigation on the relation between APCC and PA is highly needed.

Disordered CYP11B2 Expression in Primary Aldosteronism.

Primary aldosteronism is the most common type of secondary hypertension affecting 6-10% of patients with primary hypertension. PA is mainly caused by unilateral hyperaldosteronism due to an aldosterone-producing adenoma, unilateral hyperplasia with or without micronodules or bilateral zona glomerulosa hyperplasias with or without macro or micronodules. The development of antibodies against the terminal enzyme of aldosterone biosynthesis (CYP11B2) has permitted the further characterization of normal adrenals and resected adrenals from patients with primary aldosteronism. Normal adrenals exhibit two different patterns of cellular expression of CYP11B2: young individuals display a relatively uniform expression of the enzyme throughout the zona glomerulosa while the adrenals of older individuals have dispersed CYP11B2-expressing cells but have more groups of cells called aldosterone-producing cell clusters (APCC). APAs exhibit different patterns of CYP11B2 staining that vary from uniform to homogeneous. There are also a proportion of cells within the APA that co-express different enzymes that are not normally co-expressed in normal individuals. Approximately 30% of patients with unilateral hyperaldosteronism do not have an APA, but either have an increased number of CYP11B2 expressing micronodules or hyperplasia of the zona glomerulosa. In summary, the studies reported in this review are shedding new light on the pathophysiology of primary aldosteronism. The wide variation in histopathological features of the adenomas and concurrent presence of APCCs raises the possibility that most cases of unilateral production of aldosterone actually might represent bilateral asymmetric hyperplasia with nodules frequently due to the development of somatic aldosterone-driving mutations.

Unanswered Questions in the Genetic Basis of Primary Aldosteronism

Over the past six years, the genetic basis of a significant fraction of primary aldosteronism (PA) cases has been solved. Breakthrough discoveries include the role of somatic variants in the KCNJ5, CACNA1D, ATP1A1, and ATP2B3 genes as causes of aldosterone-producing adenomas (APAs), and the recognition of three novel hyperaldosteronism syndromes with germline variants in the KCNJ5, CACNA1D, and CACNA1H genes. The description of somatic variants in CACNA1D and ATP1A1 in aldosterone-producing cell clusters (APCCs) suggests that these clusters are precursors of some aldosterone-producing adenomas. Yet, a number of questions remain unanswered. These include the genetic basis of about 40% of APAs without somatic variants in known genes. Do technical issues explain this finding, or are the unexplained APAs due to somatic copy number variation or rare variants in thus-far undiscovered genes? Similarly, the role of CTNNB1 (beta catenin) variants in APA pathogenesis is still unclear. The major question to be solved is the genetic basis of bilateral adrenal hyperplasia (BAH). Is BAH due to the bilateral occurrence of APCCs, to germline variants, or perhaps due to unknown serum factors? Lastly, the etiology of unsolved cases of apparently familial hyperaldosteronism remains to be discovered. It is expected that genetic studies over the next few years will lead to answers to at least some of the questions raised.

Aldosterone in Advancing Age

Article, see p 347 The study by Nanba et al, “Age-related autonomous aldosteronism,”1 in this issue of Circulation , addresses 2 areas of current major interest in cardiovascular endocrinology. The first is that of aldosterone-producing cell clusters (APCCs), distinct from a more or less continuous outer layer of aldosterone-producing cells familiar as the zona glomerulosa of the adrenal gland. The second is that of the changes in plasma renin activity (PRA) and serum aldosterone concentration with age in a group of normotensive and stage 1 hypertensive patients from the HyperPATH study (Hypertensive Pathotype), with primary aldosteronism based on current criteria excluded. The study is novel in terms not only of the numbers involved (adrenal glands from kidney donors n=127, HyperPATH cohort n=677), but also in the possibilities of linking the 2 lines of study into a coherent explanation of the changes seen in human aging. The presentation is also straightforward, crisp, and clear, with the discussion, in particular, mercifully free of padding and nit-picking: for this, and the novelty and possible implications of their findings, as well, we owe the authors our thanks. What the study shows is that there are 2 apparently related changes in aldosterone secretion with age. In adrenal glands from young organ donors, the zona glomerulosa appears as a thin continuous outer layer, as identified by immunohistochemistry with antibody specific for the enzyme aldosterone synthase (CYP11B2). When adrenal glands from older donors are examined, the previously continuous zona glomerulosa appears disrupted (or discontinuous), progressively with increasing age. At the same time, isolated aldosterone-producing cell clusters appear more and more commonly. Whereas the factors controlling aldosterone secretion from zona glomerulosa cells are well established, those from APCCs are not; on the basis that secretion from APCCs may be constitutive, the authors suggest that this may represent …

Age-Related Autonomous Aldosteronism.

Both aging and inappropriate secretion of aldosterone increase the risk for developing cardiovascular disease; however, the influence of aging on aldosterone secretion and physiology is not well understood. The relationship between age and adrenal aldosterone synthase (CYP11B2) expression was evaluated in 127 normal adrenal glands from deceased kidney donors (age, 9 months to 68 years). Following immunohistochemistry, CYP11B2-expressing area and areas of abnormal foci of CYP11B2-expressing cells, called aldosterone-producing cell clusters, were analyzed. In a separate ancillary clinical study of 677 participants without primary aldosteronism, who were studied on both high and restricted sodium diets (age, 18-71 years), we used multivariable linear regression to assess the independent associations between age and renin-angiotensin-aldosterone system physiology. In adrenal tissue, the total CYP11B2-expressing area was negatively correlated with age (r=-0.431, P<0.0001), whereas the total aldosterone-producing cell cluster area was positively correlated with age (r=0.390, P<0.0001). The integrated ratio of aldosterone-producing cell cluster to CYP11B2-expressing area was most strongly and positively correlated with age (r=0.587, P<0.0001). When participants in the clinical study were maintained on a high sodium balance, renin activity progressively declined with older age, whereas serum and urinary aldosterone did not significantly decline. Correspondingly, the aldosterone-to-renin ratio was positively and independently associated with older age (adjusted β=+5.54 ng/dL per ng/mL per hour per 10 years, P<0.001). In contrast, when participants were assessed under sodium-restricted conditions, physiological stimulation of aldosterone was blunted with older age (β=-4.6 ng/dL per 10 years, P<0.0001). Aging is associated with a pattern of decreased normal zona glomerulosa CYP11B2 expression and increased aldosterone-producing cell cluster expression. This histopathologic finding parallels an age-related autonomous aldosteronism and abnormal aldosterone physiology that provides 1 potential explanation for age-related cardiovascular risk.

Aldosterone-Producing Cell Clusters Frequently Harbor Somatic Mutations and Accumulate With Age in Normal Adrenals.

Context:Aldosterone synthase (CYP11B2) immunohistochemistry and next-generation sequencing (NGS) have revealed the frequent presence of aldosterone-producing cell clusters (APCCs) harboring somatic mutations in aldosterone-regulating genes in adrenals from Americans without defined hypertension status.Objective:Determine the frequency and somatic mutation status of APCCs in a Japanese nonhypertensive cohort.Design, Setting, Patients, and Interventions:Adrenals from 837 consecutive autopsies at a Japanese institution, Tohoku University Hospital, were screened to select 107 unilateral adrenal glands from nonhypertensive patients. APCC score (APCC number/adrenal cortex area per case) was assessed by CYP11B2 immunohistochemistry. DNA from all APCCs and adjacent adrenal cortex was subjected to NGS using two panels targeting aldosterone-regulating genes.Primary Outcome Measure:APCC frequency and somatic mutation spectrum.Results:In 107 adrenals, 61 APCCs were detected (average of 0.6 APCCs per gland). APCC score was positively correlated with age (r = 0.50, P < 0.0001). NGS demonstrated high confidence somatic mutations in 21 of 61 APCCs (34%). Notably, 16 of 21 APCCs (76%) harbored somatic mutations in CACNA1D, the most frequently mutated gene in our previous studies of APCCs in Americans and CYP11B2-positive micronodules in cross-sectional imaging (computed tomography) negative primary aldosteronism (PA), whereas no APCCs harbored mutations in KCNJ5, the most frequently mutated gene in aldosterone-producing adenoma. APCC score was significantly lower than our previous cohort of unilateral computed tomography–negative PA.Conclusions:APCCs are frequent in nonhypertensive Japanese adrenals, accumulate with age, and frequently harbor somatic mutations (most commonly in CACNA1D). The role of APCCs in PA pathobiology and non-PA hypertension warrants further investigation.

Abstract #1136: Postmenopausal Adrenal Expression of the Luteinizing Hormone/Human Chorionic Gonadotropin Receptor in Aldosterone-Producing Cell Clusters

Abstract #1136: Postmenopausal Adrenal Expression of the Luteinizing Hormone/Human Chorionic Gonadotropin Receptor in Aldosterone-Producing Cell Clusters

Histopathological Classification of Cross-Sectional Image-Negative Hyperaldosteronism.

Approximately half of patients with primary aldosteronism (PA) have clinically evident disease according to clinical (hypertension) and/or laboratory (aldosterone and renin levels) findings but do not have nodules detectable in routine cross-sectional imaging. However, the detailed histopathologic, steroidogenic, and pathobiological features of cross-sectional image-negative PA are controversial. To examine histopathology, steroidogenic enzyme expression, and aldosterone-driver gene somatic mutation status in cross-sectional image-negative hyperaldosteronism. Twenty-five cross-sectional image-negative cases were retrospectively reviewed. In situ adrenal aldosterone production capacity was determined using immunohistochemistry (IHC) of steroidogenic enzymes. Aldosterone-driver gene somatic mutation status (ATP1A1, ATP2B3, CACNA1D, and KCNJ5) was determined in the CYP11B2 immunopositive areas [n = 35; micronodule, n = 32; zona glomerulosa (ZG), n = 3] using next-generation sequencing after macrodissection. Cases were classified as multiple adrenocortical micronodules (MN; n = 13) or diffuse hyperplasia (DH) of ZG (n = 12) based upon histopathological evaluation and CYP11B2 IHC. Aldosterone-driver gene somatic mutations were detected in 21 of 26 (81%) of CYP11B2-positive cortical micronodules in MN; 17 (65%) mutations were in CACNA1D, 2 (8%) in KCNJ5, and 1 each (4% each) in ATP1A1 and ATP2B. One of 6 (17%) of nodules in DH harbored somatic aldosterone-driver gene mutations (CACNA1D); however, no mutations were detected in CYP11B2-positive nonnodular DH areas. Morphologic evaluation and CYP11B2 IHC enabled the classification of cross-sectional image-negative hyperaldosteronism into MN and DH. Somatic mutations driving aldosterone overproduction are common in micronodules of MN, suggesting a histological entity possibly related to aldosterone-producing cell cluster development.