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Adequate access to fresh or frozen normal adrenal tissue has been a primary limitation to the enhanced characterization of the adrenal zones via RNA sequencing (RNAseq). Herein, we describe the application of targeted RNAseq to formalin-fixed paraffin-embedded (FFPE) normal adrenal gland specimens. Immunohistochemistry (IHC) was used to visualize and guide the capture of the adrenocortical zones and medulla. Following IHC-based tissue capture and isolation of RNA, high-throughput targeted RNAseq highlighted clear transcriptomic differences and identified differentially expressed genes among the adrenal zones. Our data demonstrate the ability to capture FFPE adrenal zone tissue for targeted transcriptomic analyses. Future comparison of normal adrenal zones will improve our understanding of transcriptomic patterns and help identify potential novel pathways controlling zone-specific steroid production.

Somatic mutations driving aldosterone production have been identified in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected 2 somatic variants in CLCN2 in 2 APAs that were negative for currently known aldosterone-driver mutations. The CLCN2 gene encodes the voltage-gated chloride channel ClC-2. CLCN2 germline variants have previously been shown to cause familial hyperaldosteronism type II. Somatic mutations in CLCN2 were identified in 2 of 115 APAs, resulting in a prevalence of 1.74%. One of the CLCN2 somatic mutations (c.G71A,p.G24D) was identical to a previously described germline variant causing early-onset PA, but was present only as a somatic mutation. The second CLCN2 mutation, which affects the same region of the gene, has not been reported previously (c.64-2_74del). These findings prove that WES of CYP11B2-guided mutation-negative APAs can help determine rarer genetic causes of sporadic PA.

Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2, IGF2, etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.

Background: The mechanisms leading to elevated aldosterone synthesis in aldosterone-producing adenomas (APAs) remain an area of active research. Aldosterone-driver somatic gene mutations that allow inappropriate intracellular calcium entrance have been identified in most APAs. Cell-based studies of such mutations indicate that responses to physiologic stimuli, such as angiotensin II or ACTH, are increased. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations. Herein, we analyzed the transcript expression of the ACTH receptor (MC2R), the melanocortin 2 receptor accessory protein (MRAP) and the type 1 angiotensin II receptor (AGTR1) in APAs with known aldosterone-driver somatic mutations. Methods: RNA was isolated from normal adrenal glands (n=8), and from APAs with mutations in: KCNJ5 (n=14), ATP1A1 (n=14), CACNA1D (n=14), and ATP2B3 (n=5). The gene expressions of MC2R, MRAP, AGTR1 and aldosterone synthase (CYP11B2) were quantified using qPCR and normalized to β-actin. Results: All APA mutation groups had significantly higher transcript levels of CYP11B2, MC2R and AGTR1 as compared to whole normal adrenals. While MRAP and AGTR1 transcripts were comparable between tumor mutation groups, MC2R expression was significantly lower in KCNJ5-mutated APAs compared to other APAs. Overall, CYP11B2 expression demonstrated positive correlations with MC2R (R=0.728, p

Introduction: Characterization of human adrenal RNA has relied on fresh tissue, or laser capture from OCT-embedded frozen tissue. These approaches have allowed identification of differences between zones of the adrenal cortex, but the need for fresh or frozen material has limited access for most researchers and has limited the number of samples that can be studied. In order to expand tissue availability and researcher access, we examined the use of archival formalin-fixed, paraffin-embedded (FFPE) adrenals for the capture and analysis of RNA from each adrenal zone. Methods: 13 normal adrenals were obtained from deceased renal donors. Following FFPE, tissue serial sections were examined for zonal histology. Immunohistochemistry (IHC) was used to guide the capture of specific adrenal zones from FFPE tissue. CYP11B2, CYP17A1, and CYB5A IHC was used to visualize the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) respectively. Serial unstained sections were then dissected under a dissection microscope based on IHC-guided visualization of the ZG, ZF, and ZR. Following capture of zones and isolation of RNA, quantitative PCR (qPCR) was used to examine zonal expression of transcripts for the IHC markers. Using housekeeping genes for normalization, specific zonal markers identified successfully isolated zones. Results: Adrenals with clear zonal expression of CYP11B2 (ZG), HSD3B2 (ZG/ZF), and CYB5A (ZR) were included for study. Based on marker inclusion criteria, 3 ZG, 3 ZF, 3 ZR, and 3 medulla samples were studied. Using these samples, confirmatory markers of ZG (VSNL1), ZF (CYP17A1), ZR (SLC27A2), and medulla (TH) were analyzed by qPCR. Levels of these transcripts segregated as expected in the adrenal zones. Discussion: The use of archival normal and pathologic adrenal tissue for RNA analysis would increase the number of samples available to many research groups. Our data demonstrates the ability to capture and use FFPE tissue for the capture and analysis of RNA. Future comparison of normal adrenal zones to adrenal tumor phenotypes will improve our understanding of tumor biology and provide potential genetic markers for targeted therapeutics.