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

Figure 1.. Adrenal gland collection of a nonhypertensive cohort. One hundred seven adrenal glands from patients without evidence of hypertension (exclusion criteria shown in figure) were collected from a consecutive single institution Japanese autopsy cohort. Sixty-one APCCs were detected in 31 cases, all of which underwent NGS analysis.

Figure 2.. Age-associated increase in APCC score. APCC score (APCC number per adrenal/adrenal cortex area) for each of our 107 nonhypertensive cases were plotted vs age. A significant positive correlation was observed (Spearman rank correlation, r = 0.50, P < 0.0001). Men and women are shown in blue and red, respectively.

Figure 3.. NGS approach to robustly detect somatic mutations in aldosterone-regulating genes from APCCs. FFPE blocks from our cohort were serially sectioned, and careful macrodissection of APCCs under a dissecting microscope was performed using H&E and CYP11B2 IHC to localize APCCs on unstained slides. After DNA extraction, separate libraries were prepared from each APCC using two distinct NGS panels, which target the same genes but differ in amplicon composition. After stringent filtering, variants present in both APCC libraries but absent in normal adrenal cortex were considered true somatic mutations. Variants that were present only in one library were considered panel-specific errors. Variants that were present in any normal adrenal sample were considered nonpathogenetic changes, panel-specific errors, or germline variants. For the genes only on APAv2 (GNAS/CTNNB1/CACNA1H/ARMC5), the variants were filtered by using an APCC library on APAv2 and matched normal tissue.

Figure 4.. Somatic mutations identified in APCCs by NGS. (a) Somatic mutations in CACNA1D, ATP2B3, ATP1A1, and KCNJ5 were observed in 16 of 61 (26%), 5 of 61 (8%), 2 of 61 (3%), and none of 61 (0%) of the 61 total APCCs in our Japanese normotensive cohort, respectively. Two APCCs (26A1 and 39A1) harbored both CACNA1D and ATP2B3 somatic mutations. (b) Of 30 somatic variants, 9 of 20 (45%), none of eight (0%), and one of two (50%) in CACNA1D, ATP2B3, ATP1A1, respectively, were previously reported in aldosterone-producing adenomas.