Immunohistochemistry-guided analyses of steroidogenesis in primary bilateral macronodular adrenal hyperplasia

Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of Cushing's syndrome. The study aimed to characterize changes in steroidogenesis in PBMAH through immunohistochemistry (IHC), transcriptomics and exome analysis with detailed clinical data assessment. Our cohort included 22 PBMAH patients who underwent adrenalectomy, and we investigated IHCs for five key steroidogenic enzymes (CYP11B1, CYP11B2, CYP17A1, HSD3B2 and CYB5A), covering the adrenocortical hormone secretion pathways. Unsupervised clustering of IHC staining identified three clusters: Cluster 1 exhibited low CYP11B1 and CYP17A1 expression, elevated HSD3B2 expression, smaller tumors, and included ARMC5 mutants. Cluster 2 showed higher enzyme staining for CYP11B1, CYP17A1 and CYB5A and included KDM1A mutants and female-only cases. Cluster 3 was characterized by slightly increased CYP11B2 staining and comprised of wild-type samples lacking ARMC5 and KDM1A variants. Exome sequencing identified steroidogenic pathway germline variants across the three clusters. Importantly, in Cluster 1, a higher burden of predicted damaging variants across various steroidogenic genes were found potentially leading to reduced enzyme staining through altered functions of the variant. However, this distinctive variant pattern was less evident in Cluster 2 and Cluster 3. Transcriptomics identified no differences between the clusters in terms of gene expression, hinting the influence of possible epigenetic factors on IHC. In summary, our study identified distinct groups in PBMAH based on their IHC staining patterns and highlights the importance for integrated molecular profiling for comprehensive characterization of heterogeneity in PBMAH.