OR19-01 IHC-Guided Capture of Adrenocortical Zones from Archival Formalin-Fixed, Paraffin-Embedded Adrenal Tissue Allows the Study Of RNA Expression Profiles

Abstract

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.