Disorganized Steroidogenesis in Adrenocortical Carcinoma, a Case Study
- 268 Downloads
Most adrenocortical carcinomas (ACCs) produce excessive amounts of steroid hormones including aldosterone, cortisol, and steroid precursors. However, aldosterone- and cortisol-producing cells in ACCs have not yet been immunohistochemically described. We present a case of ACC causing mild primary aldosteronism and subclinical Cushing’s syndrome. Removal of the tumor cured both conditions. In order to examine the expression patterns of the steroidogenic enzymes responsible for adrenocortical hormone production, 10 tumor portions were immunohistochemically analyzed for aldosterone synthase (CYP11B2), 11β-hydroxylase (CYP11B1, cortisol-synthesizing enzyme), 3β-hydroxysteroid dehydrogenase (3βHSD, upstream enzyme for both CYP11B2 and CYP11B1), and 17α-hydroxylase/C17-20 lyase (CYP17, upstream enzyme for CYP11B1, but not for CYP11B1). CYP11B2, CYP11B1, and 3βHSD were expressed sporadically, and their expression patterns varied significantly among the different tumor portions examined. The expression of these enzymes was random and not associated with each other. CYP17 was expressed throughout the tumor, even in CYP11B2-positive cells. Small tumor cell populations were aldosterone- or cortisol-producing cells, as judged by 3βHSD coinciding with either CYP11B2 or CYP11B1, respectively. These results suggest that the tumor produced limited amounts of aldosterone and cortisol due to the lack of the coordinated expression of steroidogenic enzymes, which led to mild clinical expression in this case. We delineated the expression patterns of steroidogenic enzymes in ACC. The coordinated expression of steroidogenic enzymes in normal and adenoma cells was disturbed in ACC cells, resulting in the inefficient production of steroid hormones in relation to the large tumor volume.
KeywordsAdrenocortical carcinoma Aldosterone synthase Primary aldosteronism 11β-hydroxylase Subclinical Cushing’s syndrome
Adrenocortical carcinomas (ACC) frequently produce adrenocortical hormones including cortisol, early steroid precursors and, to a lesser extent, aldosterone . Nishimoto et al. previously performed immunohistochemical examinations on formalin-fixed paraffin-embedded (FFPE) adrenal sections for human 11β-hydroxylase (CYP11B1, a cortisol-synthesizing enzyme) and aldosterone synthase (CYP11B2) . The expression patterns of these enzymes in a normal adrenal gland, aldosterone-producing adenoma (APA), and cortisol-producing adenoma (CPA) were described. In the zona glomerulosa (ZG) and APA, CYP11B2-positive cells co-express 3β-hydroxysteroid dehydrogenase (3βHSD), an enzyme upstream of CYP11B2 in the aldosterone synthetic pathway . In the zona fasciculata (ZF), APA, and CPA, CYP11B1-positive cells co-express 3βHSD and 17α-hydroxylase/C17-20 lyase (CYP17), both of which are enzymes upstream of CYP11B1 in the cortisol synthetic pathway . Gomez-Sanchez et al. recently developed monoclonal antibodies for human CYP11B2 and CYP11B1 . Despite advances in adrenocortical pathohistology, the distribution of cortisol- or aldosterone-producing cells in ACC has not yet been fully described. We herein performed immunohistochemical analyses for CYP11B1, CYP11B2, 3βHSD, and CYP17 in a case of ACC that presented with subclinical Cushing’s syndrome (SCS) and mild primary aldosteronism (PA).
The molecular studies in the current case report were approved by the Medical Ethics Committee of the School of Medicine, Keio University (approval#: 20090018).
Immunohistochemistry for CYP11B1, CYP11B2, 3βHSD, and CYP17
Sections from archival FFPE surgical specimens of the case were immunostained using a mouse monoclonal anti-human CYP11B2 antibody , rat monoclonal anti-human CYP11B1 antibody , mouse monoclonal anti-human CYP17 antibody which prepared as described below, and polyclonal rabbit anti-human 3βHSD antibody (a gift from Dr. Takeshi Yamazaki at Hiroshima University) . Single staining for CYP11B2, CYP11B1, 3βHSD, and CYP17 was performed as previously reported [2, 5], in which the nucleus was counterstained by hematoxylin. Double staining for CYP11B2 with 3,3′-diaminobenzidine (brown) and 3βHSD with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (blue) was performed as previously reported , and the nucleus was not counterstained.
Mouse Monoclonal Antibody Preparation Against the Human CYP17 Enzyme
Six-week-old Swiss-Webster female mice were initially immunized intraperitoneally with a mixture of 50 μg of the plasmid pcDNA3.1-hCYP17A and 10 μg of a poly(I:C) HMW adjuvant (Cat. Code tlrl-pic, Invivogen.com, San Diego, CA), followed by subcutaneous immunization at multiple sites with 10 μg of recombinant human CYP17 in complete AdjuLite Freund’s adjuvant (catalog#: A5001, Pacific Immunology Corp, Ramona, CA) (total volume 0.1 ml) and 4 weeks later with the recombinant CYP17 enzyme in incomplete AdjuLite Freund’s adjuvant (catalog#: A5002, Pacific Immunology Corp, Ramona, CA). Four weeks after the final immunization, animals were intraperitoneally injected under isoflurane anesthesia with 10 μg of the recombinant enzyme, and blood and the spleen were obtained under isoflurane anesthesia 3 days later.
The spleen cells from the animal with the highest titer to CYP17, as determined by an enzyme-linked immunosorbent assay (ELISA) using plates coated with 20 ng/0.1 ml of recombinant enzyme/well in 1 M sodium chloride and 0.05 M sodium phosphate buffer (pH 7.4), were selected for fusion. Spleen cells were fused to mouse myeloma SP2-mIL6-hIL21 cells [SP2 cells from ATCC (Manassas, VA) were transduced with the retrovirus pMSCV-mIL6-puro (kindly provided by Dr. Scott K. Dessain from Thomas Jefferson University), selected with 5 μg/ml, and then transduced with the lentivirus p6NST50-hIL21-IRES-GFPzeocin (created by cloning a plasmid from DNASU.org, HsCD00288055 into p6NST50-MCS-GFPzeocin, which was kindly provided by Dr. Monika Valink from the Institute of Anatomy, Medical Faculty Carl Gustav Carus in Dresden, Germany, and selected with 0.5 mg/ml of zeocin)] and cultured in Iscove’s media (I7633, Sigmaaldrich.com) with 15 % Fetal Clone I sera (Hyclone, Provo, UT) with HAT (H0262, Sigmaaldrich.com) and 10 % of conditioned media from the same myeloma cell line (1). Clones were screened after 2 weeks using ELISA, and those exhibiting positivity were then subjected to a Western blot analysis using an extract from H293TN cells transfected with the plasmid pcDNA3.1-CYP17A (kindly provided by Dr. Richard Auchus at the University of Texas, Southwestern Medical School). A clone (isotype IgG2b) that gave a single band was then used for immunohistochemistry on normal human adrenal glands and stained the ZF and ZR only.
Positive Cell Area to Total Area (PCA/TA) Measurement
PCA/TA was measured as follows: (1) High resolution images (2400 dpi) of immunostained sections for CYP11B2, CYP11B1, and 3βHSD were captured using a scanner machine. Positive cell area (PCA) was isolated using Colour Deconvolution Software , and PCA was measured using ImageJ software at the same threshold. Each section was traced using Adobe Photoshop CS6 Extended software and the traced areas were measured by ImageJ software (total area, TA). PCA/TA was calculated as PCA divided by the corresponding TA.
Mitotic Cell Count
Five sites of CYP11B2-positive area, CYP11B1-positive area, 3βHSD-positive area, and area negative for these enzymes (black circles in Figs. 3, 4, 5, and 6, respectively) were selected by KN. Mitotic cells were counted in 5 microscopic high power field of each site by three pathologists (YF, MA, and TY). The average values of these counts were used for statistical analysis.
DNA and RNA Isolation from FFPE Tissues, cDNA Generation from RNA, and a Quantitative Real-Time Polymerase Chain Reaction (qPCR) Analysis Using cDNA
Whole FFPE adrenocortical tissues including connective tissue were scraped out from the glass slides. RNAs were isolated from these tissues using the Qiagen Allprep FFPE DNA/RNA kit (catalog#: 80234, Qiagen), according to the manufacturer’s instructions. The isolation protocol was modified by extending the xylene incubation to 5 min, centrifugation during deparaffinization to 5 min, and eluting in a volume of 30 μl. cDNA samples were generated from RNA using the High-Capacity cDNA Reverse Transcription Kit (catalog#: 4368814, Thermo Fisher Scientific). cDNAs were used in the qPCR analysis of CYP11B2 and the 18S ribosomal RNA gene with the primer/TaqMan probe mix for CYP11B2  and TaqMan ribosomal RNA control reagents (catalog#: 4308329, Thermo Fisher Scientific).
Relationships between values having a non-normal distribution were analyzed by Spearman’s rank-order correlation. Non-normal distribution values were compared by a Kruskal-Wallis one-way analysis of variance on ranks. In these analyses, a p value <0.05 was considered to be significant.
Immunohistochemistry and qPCR results
Block (section) #
CYP11B2-fold (S.E. range)
25th percentile value:
75th percentile value:
We delineate for the first time the expression patterns of steroidogenic enzymes including CYP11B2 and CYP11B1 in ACC. The coordinated expression of steroidogenic enzymes found in normal and adenoma cells was disturbed in ACC cells, resulting in the inefficient production of steroid hormones.
We have found only an old report describing ACC immunohistochemistry using steroidogenic enzyme antibodies including cholesterol side chain cleavage, 3βHSD, steroid 21-hydroxylase (CYP21), CYP17, and CYP11B1 . 3βHSD and CYP21 are upstream enzymes of both CYP11B1 and CYP11B2. The antibody for CYP11B1 used in that study was targeted for bovine CYP11B1, which presumably detect both human CYP11B1 and CYP11B2 . The report shows that ACC cells positive for a steroidogenic enzyme do not necessarily express other up/down-stream enzymes. For example, 3βHSD positive ACC area does not express CYP21. And they concluded the expression of steroidogenic enzymes in individual carcinoma cells was disorganized.
Approximately 60–70 % of ACC produce excessive amounts of steroid hormones and they are not clinically apparent in many cases . ACC have been shown to be relatively inefficient in steroid production and the lack of clear hormonal manifestations is due to increased secretion of steroid precursors . Urine steroid metabolomics measuring 32 distinct adrenal-derived steroids has revealed a pattern of predominantly immature, early stage steroidogenesis in most ACC cases, including androgen metabolites and precursors, mineralocorticoid precursor metabolites, and glucocorticoid precursor metabolites . PA in the context of ACC is rare. The immunohistochemical findings in this study where there is a lack of coordination in the expression of steroidogenic enzymes explain that for such a large tumor, steroid hyper-production was disproportionate to the size of the tumor. Although further analyses are needed to contrast with benign adenoma and normal adrenal cortex [2, 14], the steroidogenic enzyme expression may generally be disorganized in ACC causing variable phenotypes including Cushing’s syndrome and androgen excess.
We thank Dr. Takeshi Yamazaki at Hiroshima University for providing us with the anti-3βHSD antibody; Mr. Shinya Sasai at Tachikawa Hospital for his technical assistance with immunohistochemistry; as well as funding support from the Japan Society for the Promotion of Science (KAKENHI-Grants to T.U [#23791043], K.N [#26893261], and KM [#26461387]), the Suzuken Memorial Foundation (to KN), Yamaguchi Endocrine Research Foundation (to KN), Okinaka Memorial Institute for Medical Research (to KN), Federation of National Public Service Personnel Mutual Aid Associations (to KN), NIH HL27255 (to CEG-S), and Initiative for Rare and Undiagnosed Diseases (IRUD) by AMED (to YK).
Compliance with Ethical Standards
Conflict of Interest
- 6.Ruifrok AC, Johnston DA Quantification of histochemical staining by color deconvolution. Anal Quant Cytol Histol 23: 291–299, 2001.Google Scholar