Steroids-producing nodules: a two-layered adrenocortical nodular structure as a precursor lesion of cortisol-producing adenoma

Background The human adrenal cortex consists of three functionally and structurally distinct layers; zona glomerulosa, zona fasciculata (zF), and zona reticularis (zR), and produces adrenal steroid hormones in a layer -speci fi c manner; aldosterone, cortisol, and adrenal androgens, respectively. Cortisol-producing adenomas (CPAs) occur mostly as a result of somatic mutations associated with the protein kinase A pathway. However, how CPAs develop after adrenocortical cells acquire genetic mutations, remains poorly understood. Methods We conducted integrated approaches combining the detailed histopathologic studies with genetic, RNAsequencing, and spatially resolved transcriptome (SRT) analyses for the adrenal cortices adjacent to human adrenocortical tumours. Findings Histopathological analysis revealed an adrenocortical nodular structure that exhibits the two -layered zF- and zR-like structure. The nodular structures harbour GNAS somatic mutations, known as a driver mutation of CPAs, and confer cell proliferative and autonomous steroidogenic capacities, which we termed steroids -producing nodules (SPNs). RNA -sequencing coupled with SRT analysis suggests that the expansion of the zF-like structure contributes to the formation of CPAs, whereas the zR-like structure is characterised by a macrophage -mediated immune response. Interpretation We postulate that CPAs arise from a precursor lesion, SPNs, where two distinct cell populations might contribute differently to adrenocortical tumorigenesis. Our data also provide clues to the molecular mechanisms underlying the layered structures of human adrenocortical tissues.