Objective Develop a consensus for the nomenclature and definition of adrenal histopathologic features in unilateral primary aldosteronism (PA) Context Unilateral PA is the most common surgically-treated form of hypertension. Morphologic examination combined with CYP11B2 (aldosterone synthase) immunostaining reveals diverse histopathologic features of lesions in the resected adrenals. Patients and Methods Surgically removed adrenals (n= 37) from 90 patients operated from 2015 to 2018 in Munich, Germany, were selected to represent the broad histologic spectrum of unilateral PA. Five pathologists (Group 1 from Germany, Italy and Japan) evaluated the histopathology of haematoxylin-eosin and CYP11B2 immunostained sections and a consensus was established to define the identifiable features. The consensus was subsequently used by six additional pathologists (Group 2 from Australia, Brazil, Canada, Japan, UK, USA) for the assessment of all adrenals with disagreement for histopathologic diagnoses among group 1 pathologists. Results Consensus was achieved to define histopathologic features associated with PA. Use of CYP11B2 immunostaining resulted in a change of the original haematoxylin-eosin morphology-driven diagnosis in 5 (14%) of 37 cases. Using the consensus criteria, group 2 pathologists agreed for the evaluation of 11 of the 12 cases of disagreement among group 1 pathologists. Conclusion The HISTALDO (histopathology of primary aldosteronism) consensus is useful to standardize nomenclature and achieve consistency among pathologists for the histopathologic diagnosis of unilateral PA. CYP11B2 immunohistochemistry should be incorporated into the routine clinical diagnostic workup to localize the likely source of aldosterone production.

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.

Primary aldosteronism (PA) affects ~5–10% of hypertensive patients and has unilateral and bilateral forms. Most unilateral PA is caused by computed tomography (CT)-detectable aldosterone-producing adenomas (APA), which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone-regulating genes. The etiology of the most common bilateral form of PA, idiopathic hyperaldosteronism (IHA), is believed to be diffuse hyperplasia of aldosterone-producing cells within the adrenal cortex. Herein, a multi-institution cohort of fifteen IHA adrenals were examined with CYP11B2 immunohistochemistry and next generation sequencing (NGS). 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 one CYP11B2-positive aldosterone-producing cell cluster (APCC) or a micro-APA. The number of APCCs per case (and individual APCC area) in IHA adrenals was significantly larger than in normotensive controls. NGS of DNA from 99 IHA APCCs demonstrated somatic mutations in genes encoding the L-type calcium voltage-gated channel subunit alpha 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 CT-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.

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.

Context Results of previous studies demonstrated clear racial differences in the prevalence of somatic mutations among aldosterone-producing adenoma (APA) patients. 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. Objectives To determine somatic mutation prevalence in Japanese APA patients using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided sequencing approach. Method 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. Results 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 two 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]. Conclusion 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.

APAs with ATPase mutations demonstrated a potentially severe primary aldosteronism phenotype, whereas those with CACNA1D mutations displayed characteristics similar to wild-type APAs. The status of stimulated aldosterone production was also different according to the cell types, suggesting that the regulatory effects of adrenocorticotropic hormone on aldosterone synthesis could possibly vary according to the intracellular signaling involved in hormone production.

Context Aldosterone-producing adrenocortical adenomas (APAs) are mainly composed of clear (lipid rich) and compact (eosinophilic) tumor cells. The detailed association between these histological features and somatic mutations (KCNJ5, ATP1A1, ATP2B3, and CACNA1D) in APAs is unknown. Objective To examine the association between histological features and individual genotypes in APAs. Methods Examination of 39 APAs subjected to targeted next-generation sequencing (11 KCNJ5, 10 ATP1A1, 10 ATP2B3, and 8 CACNA1D) and quantitative morphological and immunohistochemical (CYP11B2 and CYP17A1) analyses using digital imaging software. Results KCNJ5- and ATP2B3-mutated APAs had clear cell dominant features (KCNJ5: clear 59.8% [54.4–64.6%] vs compact 40.2% (35.4–45.6%), P = .0022; ATP2B3: clear 54.3% [48.2–62.4 %] vs compact 45.7% (37.6–51.8 %), P = .0696). ATP1A1- and CACNA1D-mutated APAs presented with marked intratumoral heterogeneity. A significantly positive correlation of immunoreactivity was detected between CYP11B2 and CYP17A1 in tumor cells of KCNJ5-mutated APAs (P = .0112; ρ = 0.7237), in contrast, significantly inverse correlation was detected in ATP1A1-mutated APAs (P = .0025; ρ = −0.8667). Conclusion KCNJ5-mutated APAs, coexpressing CYP11B2 and CYP17A1, were more deviated in terms of zonation-specific differentiation of adrenocortical cells than ATP1A1- and ATP2B3-mutated APAs.

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