Morphologic and Molecular Characterization of Adrenals and Adrenal Rest Affected by Congenital Adrenal Hyperplasia

Abstract

IntroductionAdrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues.Patients/MethodsCAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses.ResultsBased on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue.ConclusionsCAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

Evidence of the Role of Inflammation and the Hormonal Environment in the Pathogenesis of Adrenal Myelolipomas in Congenital Adrenal Hyperplasia

“…Our data support the theory introduced in 1950 that adrenal tissue has common precursor progenitor cells for both hematopoietic and mesenchymal lineages, especially adipose and immune cells [ 14 ]. We previously reported a lack of zonation [ 15 ] and lymphocytic infiltration [ 16 ] in the adrenals of patients with classic CAH.…”

“…Multiple tissue sections with a thickness 5 µm from formalin-fixed paraffin-embedded (FFPE) tissue blocks of CAH adrenal, normal adrenal, normal adipose tissue, and tumor myelolipoma tissue sections were mounted on Superfrost plus slides (Erie Scientific, Ramsey, MN, USA) and hematoxylin and eosin (H&E) staining was performed on tissue sections from CAH adrenal, normal adrenal, and adrenal myelolipoma, as previously described [ 16 ]. Histopathological assessment of inflammation and expression of inflammatory markers was performed using prediluted antibodies as markers (Roche Tissue Diagnostics, Marlborough, MA, USA), CD3 (T lymphocyte-specific) (790–4341: clone 2GV6), CD20 (B-lymphocyte-specific) (760–2531: clone L26), and CD68 (macrophage/monocyte histiocyte-specific (790–2931: clone KP-1).…”

“…Cells were divided and seeded in six-well cell culture plates in triplicates and then treated with hormones (17OHP: 10 μM, ACTH: 0.68 nM, testosterone: 100 nM). Cells were treated for 72 h and total RNA was isolated, quality and quantity were checked using a Bioanalyzer, sequencing libraries were constructed via the TruSeq stranded RNA kit, and RNA-sequencing was performed as described previously [ 16 ]. The output data files were used to identify and compare any differentially expressed genes using DESeq2 (v3.16).…”

11-Oxygenated androgens are not secreted by the human ovary: in-vivo data from four different cases of hyperandrogenism

“…Although it has been shown that 11β-hydroxysteroid dehydrogenase types 1 and 2 are expressed in testicular Leydig and ovarian theca cells ( 15 ), our findings contradict the idea that gonadal 11-KT production significantly contributes to the systemic pool in females because CYP11B1 is usually only expressed in negligible amounts in the gonads ( 23 ). An exception to this are patients with CAH in which aberrant adrenal tissue can be found within the testicles or ovaries ( 24 , 25 ). The idea that 11-KT is produced in peripheral tissue from 11-OHA4 produced in the adrenal is supported by the observation that 11-KT levels did not differ between the sampling sites, considering the significantly higher 11-OHA4 levels in the adrenal vein samples, especially.…”

Management challenges and therapeutic advances in congenital adrenal hyperplasia

“…Histological and biochemical profiles highlight zone-specific alterations in the adrenal cortex in CAH. For example, immunostaining of CAH-affected adrenal glands shows a strong presence of zona reticularis enzyme CYB5A compared with other zones 20 , 21 (Fig. 2 ).…”