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.

Multifocal, Asymmetric Bilateral Primary Aldosteronism Cannot be Excluded by Strong Adrenal Vein Sampling Lateralization: An International Retrospective Cohort Study.

BACKGROUND: Primary aldosteronism (PA) has been broadly dichotomized into unilateral and bilateral forms. Adrenal vein sampling (AVS) lateralization indices (LI) ≥2 to 4 are the standard-of-care to recommend unilateral adrenalectomy for presumed unilateral PA. We aimed to assess the rates and characteristics of residual PA after AVS-guided adrenalectomy. METHODS: We conducted an international, retrospective, cohort study of patients with PA from 7 referral centers who underwent unilateral adrenalectomy based on LI≥4 on baseline and/or cosyntropin-stimulated AVS. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue. RESULTS: The cohort included 283 patients who underwent AVS-guided adrenalectomy, followed for a median of 326 days postoperatively. Lack of PA cure was observed in 16% of consecutive patients, and in 22 patients with lateralized PA on both baseline and cosyntropin-stimulated AVS. Among patients with residual PA postoperatively, 73% had multiple CYP11B2 positive areas within the resected adrenal tissue (versus 23% in those cured), wherein CACNA1D mutations were most prevalent (63% versus 33% in those cured). In adjusted regression models, independent predictors of postoperative residual PA included Black versus White race (odds ratio, 5.10 [95% CI, 1.45-17.86]), AVS lateralization only at baseline (odds ratio, 8.93 [95% CI 3.00-26.32] versus both at baseline and after cosyntropin stimulation), and CT-AVS disagreement (odds ratio, 2.75 [95% CI, 1.20-6.31]). CONCLUSIONS: Multifocal, asymmetrical bilateral PA is relatively common, and it cannot be excluded by robust AVS lateralization. Long-term postoperative monitoring should be routinely pursued, to identify residual PA and afford timely initiation of targeted medical therapy.

CBT for Generalized Anxiety Disorder in Parkinson's Disease: A Case Study.

OBJECTIVES: To examine the use of cognitive behavioral therapy (CBT) in a case of co-occurring generalized anxiety disorder (GAD) and Parkinson's disease (PD). METHODS: This case study refers to a male aged 75 years with a diagnosis of Idiopathic Parkinson's disease. It focuses on applying a CBT model to address the psychological difficulties with PD and GAD. RESULTS: This case study reveals key aspects in presentation, diagnosis, and psychological treatment between PD and GAD, and is one of few studies published in this area. CONCLUSIONS: Symptoms of anxiety in an older adult with PD decreased during a course of CBT. The implications of the treatment outcome of this study and further considerations of treatment plans for comorbid PD and anxiety have been discussed. CLINICAL IMPLICATIONS: Using CBT could positively impact non-motor symptoms of Parkinson's, such as sleep difficulties and speech impediments. Using CBT for the catastrophic thinking and worry content in GAD seems to act as a complementary therapy for psychological/non-motor symptoms of PD.

Postoperative ACTH-stimulated aldosterone predicts biochemical outcome in primary aldosteronism.

OBJECTIVE: Primary aldosteronism (PA) is the most common surgically curable cause of hypertension. Unilateral aldosterone-producing adenoma can be treated with adrenalectomy. Clinical and biochemical outcomes are assessed 6-12 months after adrenalectomy according to primary aldosteronism surgical outcome (PASO) consensus criteria. Earlier prediction of biochemical remission would be desirable as it could reduce cumbersome follow-up visits. We hypothesized that postoperative adrenocorticotropic hormone (ACTH) stimulated plasma aldosterone concentrations (PAC) measured shortly after adrenalectomy can predict PASO outcomes. DESIGN: Retrospective cohort study. METHODS: We analyzed 100 patients of the German Conn's registry who underwent adrenalectomy and postoperative ACTH stimulation tests within the first week after adrenalectomy. Six to twelve months after adrenalectomy we assessed clinical and biochemical outcomes according to PASO criteria. Serum cortisol and PAC were measured by immunoassay at baseline and 30 min after the intravenous ACTH infusion. We used receiver operating characteristics (ROC) curve analysis and matched the parameters to PASO outcomes. RESULTS: Eighty-one percent of patients had complete, 13% partial, and 6% absent biochemical remission. Complete clinical remission was observed in 28%. For a cut-off of 58.5 pg/mL, stimulated PAC could predict partial/absent biochemical remission with a high sensitivity (95%) and reasonable specificity (74%). Stimulated PAC's area under the curve (AUC) (0.89; confidence interval (CI) 0.82-0.96) was significantly higher than other investigated parameters. CONCLUSIONS: Low postoperative ACTH stimulated PAC was predictive of biochemical remission. If confirmed, this approach could reduce follow-up visits to assess biochemical outcome.

PPARG dysregulation as a potential molecular target in adrenal Cushing's syndrome.

Background: We performed a transcriptomic analysis of adrenal signaling pathways in various forms of endogenous Cushing's syndrome (CS) to define areas of dysregulated and druggable targets. Methodology: Next-generation sequencing was performed on adrenal samples of patients with primary bilateral macronodular adrenal hyperplasia (PBMAH, n=10) and control adrenal samples (n=8). The validation groups included cortisol-producing adenoma (CPA, n=9) and samples from patients undergoing bilateral adrenalectomy for Cushing's disease (BADX-CD, n=8). In vivo findings were further characterized using three adrenocortical cell-lines (NCI-H295R, CU-ACC2, MUC1). Results: Pathway mapping based on significant expression patterns identified PPARG (peroxisome proliferator-activated receptor gamma) pathway as the top hit. Quantitative PCR (QPCR) confirmed that PPARG (l2fc<-1.5) and related genes - FABP4 (l2fc<-5.5), PLIN1 (l2fc<-4.1) and ADIPOQ (l2fc<-3.3) - were significantly downregulated (p<0.005) in PBMAH. Significant downregulation of PPARG was also found in BADX-CD (l2fc<-1.9, p<0.0001) and CPA (l2fc<-1.4, p<0.0001). In vitro studies demonstrated that the PPARG activator rosiglitazone resulted in decreased cell viability in MUC1 and NCI-H295R (p<0.0001). There was also a significant reduction in the production of aldosterone, cortisol, and cortisone in NCI-H295R and in Dihydrotestosterone (DHT) in MUC1 (p<0.05), respectively. Outcome: This therapeutic effect was independent of the actions of ACTH, postulating a promising application of PPARG activation in endogenous hypercortisolism.

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.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

BACKGROUND: Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. METHODS: Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. RESULTS: We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. CONCLUSIONS: Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

Polycystic Ovary Syndrome: Common Questions and Answers.

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy affecting women of childbearing age. Its complex pathophysiology includes genetic and environmental factors that contribute to insulin resistance in patients with this disease. The diagnosis of PCOS is primarily clinical, based on the presence of at least two of the three Rotterdam criteria: oligoanovulation, hyperandrogenism, and polycystic ovaries on ultrasonography. PCOS is often associated with hirsutism, acne, anovulatory menstruation, dysglycemia, dyslipidemia, obesity, and increased risk of cardiovascular disease and hormone-sensitive malignancies (e.g., at least a twofold increased risk of endometrial cancer). Lifestyle modification, including caloric restriction and increased physical activity, is the foundation of therapy. Subsequent management decisions depend on the patient's desire for pregnancy. In patients who do not want to become pregnant, oral contraceptives are first-line therapy for menstrual irregularities and dermatologic complications such as hirsutism and acne. Antiandrogens such as spironolactone are often added to oral contraceptives as second-line agents. In patients who want to become pregnant, first-line therapy is letrozole for ovulation induction. Metformin added to lifestyle management is first-line therapy for patients with metabolic complications such as insulin resistance. Patients with PCOS are at increased risk of depression and obstructive sleep apnea, and screening is recommended.

TSPAN12 (Tetraspanin 12) Is a Novel Negative Regulator of Aldosterone Production in Adrenal Physiology and Aldosterone-Producing Adenomas.

BACKGROUND: Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, a condition of low-renin hypertension, in which aldosterone overproduction is usually driven by a somatic activating mutation in an ion pump or channel. TSPAN12 is differentially expressed in different subgroups of APAs suggesting a role in APA pathophysiology. Our objective was to determine the function of TSPAN12 (tetraspanin 12) in adrenal physiology and pathophysiology. METHODS: APA specimens, pig adrenals under dietary sodium modulation, and a human adrenocortical cell line HAC15 were used for functional characterization of TSPAN12 in vivo and in vitro. RESULTS: Gene ontology analysis of 21 APA transcriptomes dichotomized according to high versus low TSPAN12 transcript levels highlighted a function for TSPAN12 related to the renin-angiotensin system. TSPAN12 expression levels in a cohort of 30 APAs were inversely correlated with baseline plasma aldosterone concentrations (R=-0.47; P=0.009). In a pig model of renin-angiotensin system activation by dietary salt restriction, TSPAN12 mRNA levels and TSPAN12 immunostaining were markedly increased in the zona glomerulosa layer of the adrenal cortex. In vitro stimulation of human adrenocortical human adrenocortical cells with 10 nM angiotensin II for 6 hours caused a 1.6-fold±0.13 increase in TSPAN12 expression, which was ablated by 10 µM nifedipine (P=0.0097) or 30 µM W-7 (P=0.0022). Gene silencing of TSPAN12 in human adrenocortical cells demonstrated its inverse effect on aldosterone secretion under basal and angiotensin II stimulated conditions. CONCLUSIONS: Our findings show that TSPAN12 is a negative regulator of aldosterone production and could contribute to aldosterone overproduction in primary aldosteronism.

Whole blood methylome-derived features to discriminate endocrine hypertension.

BACKGROUND: Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT (n = 42) or EHT (n = 213), and at identifying specific discriminating signatures using machine learning approaches. RESULTS: Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods-Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine-predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL. CONCLUSIONS: The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder.

Differential central regulatory mineralocorticoidreceptor systems for anxiety and depression - Could KCNJ5 be an interesting target for further investigations in major depression?

The mineralocorticoid receptor (MR) is suggested to play a role in the pathophysiology of depression and anxiety. Main support comes from studies in patients with primary aldosteronism (PA) which suggested different central pathways for depression and anxiety mediated via the MR and gender differences. We investigated 118 patients with PA over 3 years using self-rating questionnaires for anxiety (GAD-7) and depression (PHQD) at baseline and once a year under specific treatment with adrenalectomy (ADX; n = 48) or a MR antagonist (MRA; n = 70). Genotyping for KCNJ5 mutation was performed in resected tumors. At baseline, patients treated by ADX or MRA had comparable scores for anxiety and depression. Females showed a better metabolic profile but higher scores of depression and anxiety, compared to males. Initiation of specific treatment for PA resulted in a better response in depressive symptoms after ADX and of anxiety under MRA treatment. However, GAD-7 and PHQD remained high in women over the three-year follow-up. KCNJ5 mutation, linked to co-secretion of hybrid steroids as 18-oxocortisol and 18-hydroxycortisol, was detected in 10 female and 2 male patients. They tended to have higher GAD and PHQD scores at baseline compared to patients without KNCJ5 mutation, but showed a significant better reduction in symptoms of anxiety during the 3-year follow up compared to patients without this mutation (all p < 0.05). These data support a differentiated regulation of depression and anxiety by the MR. Moreover, genetic mutations such as KCNJ5 could affect the pathophysiology of these disorders by impacting in adrenal steroidogenesis.

Leveraging patient-centric sampling for clinical drug development and decentralized clinical trials: Promise to reality.

Advances in the technologies to enable patient-centric sampling (PCS) have the potential to improve blood sample collection by enabling clinical trial participants to collect samples via self-collection or with the help of a caregiver in their home. Typically, blood samples to assess pharmacokinetics and pharmacodynamics of a drug during clinical development are collected at a clinical site via venous blood draw. In this position paper by the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), the potential value PCS can bring to patients, to the clinical datasets generated, and to clinical trial sponsors is discussed, along with considerations for program decision making, bioanalytical feasibility, operations, and regulatory implications. With an understanding of the value of PCS and considerations when implementing during clinical drug development, we can bring the promise of PCS closer to reality and enable decentralized clinical trials.

Primary aldosteronism: Pathophysiological mechanisms of cell death and proliferation.

Primary aldosteronism is the most common surgically curable form of hypertension. The sporadic forms of the disorder are usually caused by aldosterone overproduction from a unilateral adrenocortical aldosterone-producing adenoma or from bilateral adrenocortical hyperplasia. The main knowledge-advances in disease pathophysiology focus on pathogenic germline and somatic variants that drive the excess aldosterone production. Less clear are the molecular and cellular mechanisms that lead to an increased mass of the adrenal cortex. However, the combined application of transcriptomics, metabolomics, and epigenetics has achieved substantial insight into these processes and uncovered the evolving complexity of disrupted cell growth mechanisms in primary aldosteronism. In this review, we summarize and discuss recent progress in our understanding of mechanisms of cell death, and proliferation in the pathophysiology of primary aldosteronism.

Preanalytical Pitfalls in Untargeted Plasma Nuclear Magnetic Resonance Metabolomics of Endocrine Hypertension.

Despite considerable morbidity and mortality, numerous cases of endocrine hypertension (EHT) forms, including primary aldosteronism (PA), pheochromocytoma and functional paraganglioma (PPGL), and Cushing's syndrome (CS), remain undetected. We aimed to establish signatures for the different forms of EHT, investigate potentially confounding effects and establish unbiased disease biomarkers. Plasma samples were obtained from 13 biobanks across seven countries and analyzed using untargeted NMR metabolomics. We compared unstratified samples of 106 PHT patients to 231 EHT patients, including 104 PA, 94 PPGL and 33 CS patients. Spectra were subjected to a multivariate statistical comparison of PHT to EHT forms and the associated signatures were obtained. Three approaches were applied to investigate and correct confounding effects. Though we found signatures that could separate PHT from EHT forms, there were also key similarities with the signatures of sample center of origin and sample age. The study design restricted the applicability of the corrections employed. With the samples that were available, no biomarkers for PHT vs. EHT could be identified. The complexity of the confounding effects, evidenced by their robustness to correction approaches, highlighted the need for a consensus on how to deal with variabilities probably attributed to preanalytical factors in retrospective, multicenter metabolomics studies.

Histopathology and Genetic Causes of Primary Aldosteronism in Young Adults.

CONTEXT: Due to its rare incidence, molecular features of primary aldosteronism (PA) in young adults are largely unknown. Recently developed targeted mutational analysis identified aldosterone-driver somatic mutations in aldosterone-producing lesions, including aldosterone-producing adenomas (APAs), aldosterone-producing nodules (APNs), and aldosterone-producing micronodules, formerly known as aldosterone-producing cell clusters. OBJECTIVE: To investigate histologic and genetic characteristics of lateralized PA in young adults. METHODS: Formalin-fixed, paraffin-embedded adrenal tissue sections from 74 young patients with lateralized PA (<35 years old) were used for this study. Immunohistochemistry (IHC) for aldosterone synthase (CYP11B2) was performed to define the histopathologic diagnosis. Somatic mutations in aldosterone-producing lesions were further determined by CYP11B2 IHC-guided DNA sequencing. RESULTS: Based on the CYP11B2 IHC results, histopathologic classification was made as follows: 48 APAs, 20 APNs, 2 multiple aldosterone-producing nodules (MAPN), 1 double APN, 1 APA with MAPN, and 2 nonfunctioning adenomas (NFAs). Of 45 APAs with successful sequencing, 43 (96%) had somatic mutations, with KCNJ5 mutations being the most common genetic cause of young-onset APA (35/45, 78%). Of 18 APNs with successful sequencing, all of them harbored somatic mutations, with CACNA1D mutations being the most frequent genetic alteration in young-onset APN (8/18, 44%). Multiple CYP11B2-expressing lesions in patients with MAPN showed several aldosterone-driver mutations. No somatic mutations were identified in NFAs. CONCLUSION: APA is the most common histologic feature of lateralized PA in young adults. Somatic KCNJ5 mutations are common in APAs, whereas CACNA1D mutations are often seen in APNs in this young PA population.

Integration of artificial intelligence and plasma steroidomics with laboratory information management systems: application to primary aldosteronism.

OBJECTIVES: Mass spectrometry-based steroidomics combined with machine learning (ML) provides a potentially powerful approach in endocrine diagnostics, but is hampered by limitations in the conveyance of results and interpretations to clinicians. We address this shortcoming by integration of the two technologies with a laboratory information management systems (LIMS) model. METHODS: The approach involves integration of ML algorithm-derived models with commercially available mathematical programming software and a web-based LIMS prototype. To illustrate clinical utility, the process was applied to plasma steroidomics data from 22 patients tested for primary aldosteronism (PA). RESULTS: Once mass spectrometry data are uploaded into the system, automated processes enable generation of interpretations of steroid profiles from ML models. Generated reports include plasma concentrations of steroids in relation to age- and sex-specific reference intervals along with results of ML models and narrative interpretations that cover probabilities of PA. If PA is predicted, reports include probabilities of unilateral disease and mutations of KCNJ5 known to be associated with successful outcomes of adrenalectomy. Preliminary results, with no overlap in probabilities of disease among four patients with and 18 without PA and correct classification of all four patients with unilateral PA including three of four with KCNJ5 mutations, illustrate potential utility of the approach to guide diagnosis and subtyping of patients with PA. CONCLUSIONS: The outlined process for integrating plasma steroidomics data and ML with LIMS may facilitate improved diagnostic-decision-making when based on higher-dimensional data otherwise difficult to interpret. The approach is relevant to other diagnostic applications involving ML.