Genetic, Cellular, and Molecular Heterogeneity in Adrenals With Aldosterone-Producing Adenoma

Abstract

Aldosterone-producing adenoma (APA) cause primary aldosteronism-the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying KCNJ5 mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated β-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying KCNJ5 mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in KCNJ5-mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known KCNJ5 mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development.

Keywords: adrenal cortex; aldosterone; arterial hypertension; mineralocorticoids; mutation; primary aldosteronism.

Figure 1.

Molecular heterogeneity of aldosterone-producing adenoma (APA). A, Example of aldosterone synthase (CYP11B2), 11β-hydroxylase (CYP11B1), GIRK4, and β-catenin staining in APA. B, Colocalization of CYP11B2, CYP17A1 (17α-hydroxylase), GIRK4 (G protein-activated inward rectifier potassium channel 4), and β-catenin by multiplex immunofluorescence in APA. C–F, Automatic quantification of colocalization of different proteins in 13 APAs carrying mutations in KCNJ5 (C; n=2), CACNA1D (D; n=5), ATP2B3 (E; n=3), and ATP1A1 (F; n=3). For each couple of colocalized proteins, marker 1 refers to the first marker indicated in the legend and marker 2 to the second one.

Figure 2.

Genetic and molecular heterogeneity in aldosterone-producing adenoma (APA). A, Heterogeneous aldosterone synthase (CYP11B2) staining in APA from 3 patients. DNA was extracted from 4 CYP11B2-positive regions and 4 regions with mostly negative expression of CYP11B2 from patient 40, 1 CYP11B2-positive region and 1 region mostly negative for CYP11B2 from patient 50 (top), and 2 CYP11B2-positive regions with different degree of its expression and 1 tumor region mostly negative for CYP11B2 from case 2 (bottom, right). Determination of CYP11B2 mRNA expression from different tumor regions from case 2 (bottom, left). B, Results of next-generation sequencing (NGS) performed in multiple tumor regions with different CYP11B2 expression levels in APA. B2T indicates CYP11B2-expressing tumor region; FFPE, formalin fixed paraffin embedded; N, adjacent normal adrenal; and VAF, variant allele frequency. *Flow-corrected read depth.

Figure 3.

Different structures expressing CYP11B2 in adrenals with aldosterone-producing adenoma (APA). Immunohistochemistry showing CYP11B2 expression in APA and aldosterone-producing cell cluster (APCC) in patient 5 (A, low magnification; C, high magnification). One structure expressing CYP11B2 extends deep into the adrenal cortex (A, low magnification; B, high magnification). A secondary nodule expressing CYP11B2 in patient 4 (D).