Adrenal Tissue Research Articles

Characterizing and Quenching Autofluorescence in Fixed Mouse Adrenal Cortex Tissue.

Tissue autofluorescence of fixed tissue sections is a major concern of fluorescence microscopy. The adrenal cortex emits intense intrinsic fluorescence that interferes with signals from fluorescent labels, resulting in poor-quality images and complicating data analysis. We used confocal scanning laser microscopy imaging and lambda scanning to characterize the mouse adrenal cortex autofluorescence. We evaluated the efficacy of tissue treatment methods in reducing the intensity of the observed autofluorescence, such as trypan blue, copper sulfate, ammonia/ethanol, Sudan Black B, TrueVIEWTM Autofluorescence Quenching Kit, MaxBlockTM Autofluorescence Reducing Reagent Kit, and TrueBlackTM Lipofuscin Autofluorescence Quencher. Quantitative analysis demonstrated autofluorescence reduction by 12-95%, depending on the tissue treatment method and excitation wavelength. TrueBlackTM Lipofuscin Autofluorescence Quencher and MaxBlockTM Autofluorescence Reducing Reagent Kit were the most effective treatments, reducing the autofluorescence intensity by 89-93% and 90-95%, respectively. The treatment with TrueBlackTM Lipofuscin Autofluorescence Quencher preserved the specific fluorescence signals and tissue integrity, allowing reliable detection of fluorescent labels in the adrenal cortex tissue. This study demonstrates a feasible, easy-to-perform, and cost-effective method to quench tissue autofluorescence and improve the signal-to-noise ratio in adrenal tissue sections for fluorescence microscopy.

An adrenal incidentaloma that had appeared to produce dehydroepiandrosterone-sulfate in excess before immunohistochemical study of the tumor.

Adrenal incidentaloma is a clinically unapparent adrenal mass more than one cm in diameter detected during imaging performed not for adrenal disease. A 34-year-old man was evaluated for AI with a diameter of 3.5 cm in the left adrenal. He was obese with body mass index of 33,9. Blood pressure was 110-120/90 mmHg. The general laboratory tests were unremarkable. An adrenal hormone screening set revealed that ACTH was 6.9 pg/mL, cortisol 14.9 μg/dL, renin activity 0.9 ng/mL/h, aldosterone 79.4 pg/mL, dehydroepiandrosterone-sulfate (DHEA-S) measured on two occasions 5,217 ng/mL and 6,477 ng/mL (gender- and age-adjusted reference values, 1,060-4,640 ng/mL). The levels of metanephrine and normetanephrine were normal. The tumor was thought to produce solely DHEA-S. The excised left adrenal tissue contained a tumor with a diameter of 26 mm and neighboring adrenal tissue. The tumor consisted mostly of acidophil cells without necrosis, capsular or vascular invasion, and mitosis. Immunohistochemical study revealed followings: the cells of the tumors were stained positive for 3β-hydroxysteroid dehydrogenase, and 17α-hydroxylase, and 11β-hydroxylase, weakly positive for DHEA sulphotransferase, and negative for aldosterone synthetase. The atrophy of neighboring tissue was presumably caused by excess cortisol production. Four months after surgery, the cortisol level was 11.2 μg/dL and DHEA-S level 1,462 ng/mL. The tumor is considered to be a cortisol-producing adenoma with modestly excessive DHEA-S production rather than isolated DHEA-S-producing adenoma. Immunohistochemical study of steroidogenic enzymes is a valuable addition to blood hormone measurement to clarify steroid production profile.

PS-BPR01-7: CONCURRENT SOMATIC MUTATIONS IN CTNNB1 AND GNA11 IN AN ALDOSTERONE-PRODUCING ADENOMA

Background: Aldosterone-producing adenoma (APA) is a major subtype of primary aldosteronism (PA), a common form of secondary hypertension. In the past decade, there has been significant progress in the determination of genetic causes of APA. The use of next-generation sequencing (NGS) has led to the identification of somatic mutations in APA that cause excess aldosterone production. Most of the affected genes encode ion channels or transporters. Somatic activating mutations in CTNNB1 (β;-catenin) have also been detected in 2–5% of APA. As a possible pathogenesis, an association of CTNNB1-mutated APA with pregnancy and menopause has been proposed. A recent study reported double somatic mutations in CTNNB1 and GNA11/Q in APA. However, due to its rare incidence, characteristics of APA with these mutations are not well determined. Herein, we report a case of APA harboring somatic CTNNB1 and GNA11 mutations. Case presentation: A 46-year-old Japanese woman presented with hypertension and spontaneous hypokalemia. She had no history of pregnancy-induced hypertension. She had regular menstrual cycle at presentation. Endocrine testing showed elevated plasma aldosterone concentration (677.5 pg/mL) under suppressed renin (plasma renin activity, 0.3 ng/mL/h). Computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling indicated excess aldosterone production from the right adrenal gland. NP-59 scintigraphy further demonstrated increased tracer uptake in the right adrenal lesion. She underwent right laparoscopic adrenalectomy. Histologic diagnosis of the resected tumor was adrenocortical adenoma based on the Weiss criteria. Notably, Ki-67 labeling index was high (6% at hotspots). After surgery, her blood pressure and serum potassium both normalized. Based on the PASO (the primary aldosteronism surgical outcome) study criteria, PA remained clinically and biochemically cured at follow-up 18 months after surgery. Methods: Formalin-fixed, paraffin-embedded adrenal tumor tissue was used for immunohistochemical and genetic analysis. Results: Immunohistochemistry (IHC) showed high and diffuse expression of CYP11B2 (aldosterone synthase) within the tumor, whereas 11β;-hydroxylase (CYP11B1, a steroidogenic enzyme for cortisol biosynthesis) was mostly negative. VSNL1, a marker for the zona glomerulosa where physiologic aldosterone biosynthesis occurs, was highly expressed within the tumor. CYP11B2 IHC-guided targeted NGS identified concurrent somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations with similar variant allele frequencies (29.5% and 29.7%, respectively). Conclusions: To best of our knowledge, this is the first Asian case of APA with CTNNB1 and GNA11 mutations. Detailed clinical and histologic examination would provide useful information for better characterization of patients with PA due to these rare mutations.

Dexamethasone affects human fetal adrenal steroidogenesis and subsequent ACTH response in an ex vivo culture model.

Administration of dexamethasone (DEX) has been used experimentally to suppress androgenization of external genitalia in 46,XX fetuses with congenital adrenal hyperplasia. Despite this, the prenatal biological mechanism-of-action of DEX on fetal development is not known. This study aimed to examine direct effects of DEX on human fetal adrenal (HFA) steroidogenic activity including possible effects on the subsequent response to ACTH-stimulation. Human fetal adrenal (HFA) tissue from 30 fetuses (1st trimester) were cultured ex vivo with A) DEX (10 µm) for 14 days, or B) DEX (10 µm) for 10 days followed by ACTH (1 nM) for 4 days. DEX-mediated effects on HFA morphology, viability, and apoptosis (immunohistochemistry), gene expression (quantitative PCR), and steroid hormone secretion (LC-MS/MS) were investigated. DEX-treatment caused decreased androstenedione (p<0.05) and increased cortisol (p<0.01) secretion suggesting that direct effects on the adrenal gland may contribute to the negative feedback on the hypothalamic-pituitary-adrenal axis in vivo. An altered response to ACTH stimulation in HFA pre-treated with DEX included increased androgen (p<0.05) and reduced cortisol production (p<0.05), supporting clinical observations of a temporary decreased ACTH-response following prenatal DEX-treatment. Additionally, the secretion of corticosterone was decreased (p<0.0001) following ACTH-stimulation in the initially DEX-treated HFAs. The observed effects suggest that prenatal DEX-treatment can cause direct effects on HFA steroidogenesis and in the subsequent response to ACTH-stimulation. This may indicate a requirement for careful monitoring of adrenal function in prenatally DEX-treated neonates, with particular focus on their mineralocorticoid levels.

PS-BPR01-2: PHEOCHROMOCYTOMA WITH HYPERALDOSTERONISM-LINKED MUTATIONS IN THE POTASSIUM CHANNEL KCNJ5

Context: Recent advances in genetic analysis techniques have led to the genetic analysis of adrenal tumors with hormone excess. In pheochromocytomas, many genetic abnormalities have been identified. Furthermore, several causative genes have been also reported in primary aldosteronism. We performed a comprehensive genetic analysis of functional adrenal tumors using next-generation sequencing. We report here a case of pheochromocytoma with KCNJ5 gene abnormality, which is considered to be specific for primary aldosteronism. Case: A 53-year-old woman with high blood pressure since age 48, excessive sweating and weight loss since age 52, and left ventricular hypertension noted on electrocardiogram during a medical checkup presented to her previous physician. A computed tomography scan of the abdomen showed a right adrenal tumor 30 mm in diameter, and an urinary test showed high levels of urinary metanephrine and normetanephrine (1.42 mg/day) and I-123 MIBG scintigraphy showed an accumulation at the tumor. Plasma renin activity was 0.2 ng/mL/hr, plasma aldosterone concentration was 73.9 pg/mL, and a positive captopril challenge test was performed. Adrenal scintigraphy using 131 I-adosterol showed bilateral adrenal accumulation, suggesting bilateral lesions. Laparoscopic right adrenalectomy was performed for pheochromocytoma. Histology showed focal proliferation of enlarged medullary-like cells and calcification of the adjacent adrenal tissue. The tumor tissue was positive for KCNJ5 gene mutation. A postoperative captopril challenge test was performed again, which was negative. Conclusion: KCNJ5 gene abnormality is considered specific for primary aldosteronism, but was also found in pheochromocytoma. However, the contribution of KCNJ5 gene abnormality to catecholamine production is not yet elucidated. This is a case of pheochromocytoma in which coexistence of primary aldosteronism is also suspected based on the captopril challenge test and imaging findings, and the finding of a gene mutation associated with primary aldosteronism in such a case is very interesting and will warrant further investigation.

High expression of GMNN predicts malignant progression and poor prognosis in ACC

BackgroundAdrenocortical carcinoma (ACC) is a rare endocrine neoplasm, which is characterized by poor prognosis and high recurrence rate. Novel and reliable prognostic and metastatic biomarkers are lacking for ACC patients. This study aims at screening potential prognostic biomarkers and therapeutic targets of ACC through bioinformatic methods and immunohistochemical (IHC) analysis.MethodsIn the present study, by using the Gene Expression Omnibus (GEO) database we identified differentially expressed genes (DEGs) in ACC and validated these DEGs in The Cancer Genome Atlas (TCGA) ACC cohort. A DEGs-based signature was additionally constructed and we assessed its prognosis and prescient worth for ACC by survival analysis and nomogram. Immunohistochemistry (IHC) was used to verify the relationship between hub gene–GMNN expressions and clinicopathologic outcomes in ACC patients.ResultsA total of 24 DEGs correlated with the prognosis of ACC were screened from the TCGA and GEO databases. Five DEGs were subsequently selected in a signature which was closely related to the survival rates of ACC patients and GMNN was identified as the core gene in this signature. Univariate and multivariate Cox regression showed that the GMNN was an independent prognostic factor for ACC patients (P < 0.05). Meanwhile, GMNN was closely related to the OS and PFI of ACC patients treated with mitotane (P < 0.001). IHC confirmed that GMNN protein was overexpressed in ACC tissues compared with normal adrenal tissues and significantly correlated with stage (P = 0.011), metastasis (P = 0.028) and Ki-67 index (P = 0.014).ConclusionsGMNN is a novel tumor marker for predicting the malignant progression, metastasis and prognosis of ACC, and may be a potential therapeutic target for ACC.

Unusual case of infrarenal pheochromocytoma developed on ectopic adrenal tissue: An autopsy case report

Abstract Introduction: Pheochromocytomas are catecholamine-secreting tumors arising from the chromaffin cells of the adrenal medulla. We describe an unusual case of pheochromocytoma located in the infrarenal area and associated with bilateral adrenal hyperplasia. Case presentation: A 70-year-old patient was admitted for syncope, diaphoresis, and high blood pressure. Computed tomography showed a 73×70×72 mm retroperitoneal mass of the left infrarenal area. High levels of metanephrine and noradrenaline were found in the urine. A tumor resection was performed. A few days after surgery, the blood pressure suddenly decreased and could not be restored, resulting in the death of the patient. Histopathological examination of the surgical specimen revealed a proliferation of monotonous cells, with eosinophilic cytoplasm, round nuclei with prominent nucleoli arranged in clusters, and capsular and vascular invasion. The tumor cells expressed synaptophysin and chromogranin, without positivity for inhibin A or S100 protein. At the autopsy, both adrenal glands showed hyperplasia but unrelated to the tumor mass. The histological aspect, location, and immunophenotype indicated an ectopic infrarenal pheochromocytoma. Conclusions: In patients with pheochromocytoma developed on ectopic adrenal tissue, clinical management might be difficult, and diagnosis can be sometimes established only based on post-mortem histopathological examination. Autopsy can be extremely useful in such cases with unexplained evolution.

COMPARATIVE CHARACTERISTICS OF CORD DENSITY IN FASCICULAR AND RETICULAR ZONES OF ADRENAL GLANDS IN WHITE RATS UNDER CENTRAL TESTOSTERONE BLOCKADE

Introduction. Gonadotropin-releasing hormone has been proven to act as an important regulator of the hypothalamic-pituitary-gonadal axis. Analogues of its agonists and antagonists are effective in the treatment of diseases of the male and female reproductive systems. Clinicians pay special attention to the study of the activity of biochemical regulators in the structures of the hypothalamic-pituitary-adrenocortical system under the administration of different medications, for instance, methanandamide, dapagliflozin, triptorelin acetate, and others. Knowing reactive changes in the fascicular and reticular zones of the adrenal glands under central testosterone deprivation by administering "Diferelin", a synthetic analogue of gonadotropin-releasing hormone enables to demonstrate the relationship between the male gonads, cortical zones of the adrenal glands and hypothalamus at the microscopic level. The purpose of this study is to investigate the density of cords in the fascicular and reticular zones of the adrenal glands of white rats under central testosterone blockade in 1, 3, 6, and 9 month periods of the experiment.&#x0D; Materials and methods. The study included 50 white male rats divided into 5 groups: 1 group included 10 intact rats and 2, 3, 4, 5 groups (10 test animals in each) were injected with the "Diferelin". Adrenal tissues were taken at the end of 1, 3, 6 and 9 months of the experiment.&#x0D; Results and discussion. For the entire duration of the experiment the quantitative composition of corticosterocytes in the studied areas of the fascicular zone (FZ) and reticular zone (RZ) reflected the density index (DI) of the cords of the studied zones in the area with average index of 36422.511±2082.09 μm².&#x0D; Morphometric study of the density of the adrenal medulla and cortical substance enabled to identify the main terms of the development of reactive changes in the adrenal glands under central testosterone deprivation.&#x0D; Conclusion. Increased reactivity of corticosterocytes in the fascicular zone from the 1st month of the experiment with the peak value at the 6th month proves the participation of this zone in the stress response under central testosterone deprivation.&#x0D; A significant increase in the parenchymal component of the reticular zone from 3 to 6 months indicates a compensatory activation of corticosteroids in response to testosterone blockade.

Application of TFE3 Immunophenotypic and TFE3 mRNA Expressions in Diagnosis and Prognostication of Adrenal Cortical Neoplasms and Distinction From Kidney Tumors.

We explored the application of TFE3 immunostaining and TFE3 mRNA expression in the differential diagnosis and prognostication of adrenal cortical tumors and distinction of the latter from clear cell renal cell carcinoma (ccRCC) which show significant morphologic overlap. TFE3 immunostaining was performed on a large cohort of samples including 40 adrenal cortex tissues, 95 adrenocortical adenoma (ACA), 11 adrenocortical carcinoma (ACC), 53 ccRCC, and 18 pheochromocytomas. TFE3 was compared with other immunomarkers melan-A, inhibin-α, synaptophysin, chromogranin A, CAIX and CD10. One hundred percent normal adrenal cortices and 94% ACA were strongly and diffusely stained for TFE3 while no ACC showed diffuse staining. TFE3 is thus useful in distinguishing ACA from ACC. TFE3 is also useful in separating ACC from ccRCC as 64% ACC showed partial, while only 7% of ccRCC showed partial TFE3 staining. Only 1 pheochromocytoma showed focal weak TFE3 staining. Results also demonstrated superiority of TFE3 over other commonly used immunomarkers. TFE3 gene rearrangement testing by fluorescence in situ hybridization showed no rearrangement in 6 TFE3 positive adrenal tumors. TFE3 mRNA were analyzed by the Cancer Genome Atlas database and we found TFE3 mRNA expression correlated with overall patient survival in ACC. Our study showed usefulness of TFE3 in distinguishing ACA from ACC, and ACC from ccRCC. TFE3 is superior over other commonly used immunomarkers for adrenal tumors. In addition, decreased TFE3 immunoexpression and TFE3 mRNA expression may carry poor prognostic implication in adrenal tumors.

Expression of the angiogenesis markers endocan in Cushing's syndrome due to cortisol producing adenoma.

In this study, we aimed to investigate the endocan expression in tissue samples of patients diagnosed with Cushing's syndrome (CS) due to Cortisol-Producing-Adenoma (CPA) and compare it with normal adrenal cortex tissue, and also to evaluate the correlations of endocan expression with clinical and histopathological features. 11 patients who were operated for CS due to CPA between 2009-2021 and 14 control subjects with normal adrenal cortex were included in the study. Demographic, laboratory and clinicopathological data of the patients were recorded. Sections of 4-5 µm thickness were taken from paraffin blocks of patients diagnosed with CS due to CPA and control subjects with normal adrenal cortex tissue. The sections were then stained in a closed system automatic immunohistochemical staining device to perform immunohistochemical analysis. The endocan positive grade were determined based on the proportion of stained cells on a scale of negative to strong. The number of subjects with positive endocan expression and the mean endocan expression level in the CS group were significantly higher than the control group (p = 0.005, p < 0.001, respectively). No correlation was found between endocan expression and clinical and histopathological features. According to the results of our study, endocan overexpression in CPA tissues may be related to the hormonal functionality of CPA.

OR04-4 Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose

Abstract Context Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas. Methods We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France. Results 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss. Discussion We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

LBMON299 Aldosterone Production Is Regulated By Gap Junctions In Human Adrenal Cells

Abstract Aldosterone plays a critical role in blood pressure regulation via the renin-angiotensin-aldosterone system. Autonomous production of aldosterone, primary aldosteronism (PA), is the commonest curable cause of hypertension worldwide. Two cell types in the adrenal cortex produce aldosterone: zona glomerulosa (ZG) cells and aldosterone producing nodules (APN), and in cells of aldosterone producing adenomas (APA). Adrenal cortex cells undergo phenotypic metamorphosis as they migrate from the subcapsular region, centripetally, towards the corticomedullary junction, changing from aldosterone- to cortisol- producing cells as they go. Gap junctions (GJs) are specialised channels of cytoplasmic communication between cells, permitting the regulated transfer of substrates. Loss-of-function mutations in the CADM1 gene, which affects GJ protein CX43, have been found in APAs, implicating CX43 in aldosterone regulation. This study aims to demonstrate the dysregulation of aldosterone production by CX43 inhibition in human adrenal cell line, H295R, and elucidate GJ distribution in human adrenal cortex. Methodology H295R cells were treated with 1×10-8M angiotensin II and connexin mimetic GJ inhibitor, GAP27, at 0µM, 25µM, 83µM and 250µM. Immunofluorescence (IF) was used to identify the abundance of CX43 in different cell types of the adrenal cortex, performed on ex-vivo para-APA adrenal tissue, using antibodies against CX43 (GJA1), colocalised with markers of aldosterone-producing cells (aldosterone synthase, CYP11B2), cells of the zona glomerulosa (ZG) (VSNL1, and DAB2) and zona fasciculata (ZF) (CYP7A1). Results Inhibition of CX43 with Gap27 results in a dose-dependent increase in CYP11B2 mRNA expression and aldosterone production. At the highest concentration of 250µM GAP27, CYP11B2 mRNA expression increased by 133-fold (p=&amp;lt;0. 00001) and aldosterone production by 29-fold (p=0. 0006). IF studies demonstrate a crescendo gradient of CX43 in a centripetal direction from cells of APN to ZF. The abundance was lowest in aldosterone-producing APN cells, moderate in non-aldosterone-producing ZG cells and highest in cortisol-producing ZF cells. GJ distribution was pan-membranous in ZF, but, where present, mostly cytoplasmic in APN. Evidence of recent active communication, in the form of annular gap junctions (AJG), was present in all three cell types. Conclusion The CX43 inhibition-induced upregulation of CYP11B2 expression and aldosterone synthesis indicates that it has an important regulatory role in physiological aldosterone production via CYP11B2 expression. The distribution of CX43 in adrenal tissue suggests its transport substrate not only regulates aldosterone synthesis, but also in adrenal cortex cell phenotypic metamorphosis/migration. Future work will investigate the substrate of CX43 in adrenal cells and its possible effect on adrenal cell phenotype. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ODP055 The methylation pattern of a unique Bilateral para-overian Adrenal Rest Tumor in a girl with Nicotinamide Nucleotide Transhydrogenase mutation

Abstract Background Patients with NNT (Nicotinamide Nucleotide Transhydrogenase) gene mutations, a rare cause of glucocorticoid and mineralocorticoid deficiency require hormone replacement therapy. Adrenal Rest Tumor (ART) in females, reported so far only in noncompliant patients with congenital adrenal hyperplasia and elevated ACTH levels, is very rare (&amp;lt;20 cases world-wide). This study characterizes the pathophysiology, the molecular ontogeny and methylation analysis of a unique ART in a female with adrenal failure due to the G200S mutation in NNT Clinical presentation and Method: A 15-year-old girl, with homozygous G200S NNT-mutation followed for adrenal insufficiency reappeared to follow-up with severe virilization and elevated serum testosterone (28.3 nmol/l) and ACTH (&amp;gt; 1500 pmol/l). Pelvic MRI and Ultrasound demonstrated one sided paraovarian round tumor with pathological vascularization. Laparoscopic exploration revealed bilateral para ovarian mesosalpinx masses involving the serosa of the Fallopian tube, (3 and 1 cm in diameter). The testosterone level normalized within one day after surgical removal of those masses (0.2 nmol/l). Results Histopathology demonstrated a pattern of adrenal rest tissue with strong intracellular positive staining for adrenal markers such as SF-1, calretinin, MART1, inhibin and the pituitary marker ACTH. The staining for ovarian characteristic markers such as PAX 8 was negative. Studying mRNA extracted from the tissue by RT-PCR revealed the positive Gene expression of Cyp17a1, Cyp21a2 and Mc2r cDNA but not Pomc suggesting adrenal but not pituitary origin of the tissue. We further profiled the epigenomic profile of several adrenal rest tumors from both ovarian and testes origins using the Infinium Methylation EPIC array. We characterized the adrenal-specific features by comparing the tumors from the two different originating sites to published methylation array data on healthy adrenal tissue. We further investigated cancer-specific methylation changes to identify activated cancer pathways, and used the methylation arrays to identify somatic copy number alterations. Finally, we analyzed the likely developmental origin of these tumors by comparing to published methylation array data of developmental and adult reproductive tissues. Conclusion This study exemplifies severe virilization that resulted from a unique and rare type of ART in ovarian related tissue that was caused by incompliance to treatment in a patient with NNT gene mutation. The laparoscopic surgical findings indicate that imaging techniques may be insufficient in identification of such rest tumors and call for laparoscopy when clinical findings are suggestive. An early detection of this tumor could preserve fertility. Using histopathology markers cDNA studies and epigenomic profiling by methylation studies, our study shows for the first time that female ART originates from adrenal cells. The growth of a functional androgen producing "tumor" indicates that functional NNT protein is NOT required for androgen synthesis in contrast to glucocorticoids and that a zona reticularis similar tissue in ART is responsive to ACTH stimulation. Presentation: No date and time listed

PSAT012 Endogenous Hypercortisolism Concealing an Autoimmune Disease

Abstract Background Endogenous hypercortisolism may mask symptoms of autoimmune diseases by suppressing immune responses. These conditions can flare upon resolution of hypercortisolism. We report a case of a patient with adrenal Cushing's syndrome (CS) who was diagnosed with seropositive rheumatoid arthritis (RA) after resolution of hypercortisolism post adrenalectomy. Clinical Case A 38-year-old woman presented with a two-year history of a 30-pound weight gain, purple striae, acne, easy bruising, and worsening anxiety. Evaluation was consistent with an ACTH-independent CS: cortisol 33 ug/dL (3-17), ACTH &amp;lt;1 pg/mL (7.2-63.3), DHEA-S 53 ug/dL (45-270), elevated 24-hour urine free cortisol 354.1 ug/day (&amp;lt;45), and an abnormal 1mg dexamethasone suppression with cortisol 23 ug/dL and dexamethasone level 215.1 ng/dL (140-295). CT abdomen revealed bilateral adrenal nodules: right 2.3cm×2.9cm with HU of 19 and left 2.1cm×2.3cm with HU of -16 consistent with bilateral macronodular hyperplasia. A laparoscopic right adrenalectomy and a left cortical-sparing adrenalectomy was performed with the goal of preventing adrenal insufficiency by preserving sufficient adrenal cortex tissue. She was discharged on a hydrocortisone taper following perioperative stress doses. While on hydrocortisone 15mg in the morning and 5mg in the afternoon, no evidence of hypothalamus-pituitary-adrenal (HPA) axis recovery was seen: cortisol &amp;lt;1 ug/DL with ACTH 2.5 pg/mL. Electrolytes and plasma renin activity remained normal. However, once glucocorticoids were tapered to a physiologic dose, she experienced fatigue, diffuse muscle aches, polyarticular joint pain, swelling, and stiffness concerning for an underlying inflammatory disorder. Rheumatology evaluation was consistent with seropositive RA: cyclic citrullinated peptide &amp;gt;200 units (&amp;lt;200) and rheumatoid factor 34 IU/mL (0-14) in the setting of tenosynovitis of her wrists and fingers. Her symptoms rapidly improved on methotrexate 15mg weekly and prednisone 20mg daily. Prednisone was then tapered by 5mg every 2 weeks to physiologic, 5mg daily, without issues while continuing methotrexate. Repeat assessment of her HPA axis is pending. Conclusion Untreated CS with hypercortisolism may mask autoimmune diseases by suppressing inflammation until the hypercortisolism is reversed, and patients no longer have endogenous glucocorticoid excess. The incidence of new onset autoimmune diseases after treatment of CS is not well described in the literature. One single center retrospective analysis of 147 CS patients found 9 cases of patients developing autoimmune diseases such as thyroiditis and vasculitis after treatment of their CS. Here we add to these described cases with the uncovering of RA after normalization of cortisol levels. It is important for clinicians to distinguish activation of autoimmune disorders after CS is treated from manifestations of glucocorticoid withdrawal postoperatively. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

PMON82 SARS-CoV-2 Associated Hypophysitis and Central Diabetes Insipidus

Abstract Background The SARS-CoV-2 infection, known for severe respiratory complications, has also been found to cause multiple endocrinopathies. SARS-CoV-2 enters pneumocytes through the angiotensin-converting enzyme 2 receptor. The adrenal, thyroid, testes, ovaries, hypothalamic, and pituitary tissues express ACE2 receptors, making them putative targets.2,4 Hypothalamic and pituitary involvement was first reported in 2005 and cases continue to be reported during the newest pandemic.1,3 We present a case of central DI following positive RT-PCR testing for SARS-CoV-2. Clinical Case A 44-year-old man presented with headache, multiple neurologic symptoms, polyuria, and polydipsia approximately three weeks following a positive SARS-CoV-2 test. A pituitary MRI showed thickening of the infundibular stalk and inhomogeneity of the pituitary gland, suggestive of hypophysitis. A water deprivation study demonstrated undetectable ADH and inability to concentrate urine spontaneously, but increased urine osmolality in response to DDAVP consistent with central DI. He was started on oral DDAVP with improvement of polyuria and polydipsia. Additional hormone testing showed normal thyroid function and Cosyntropin stimulation, mild hypogonadotropic hypogonadism, and mild elevation in IGF-1. Pituitary hormone abnormalities were transient and DDAVP was discontinued three months later. However, neurologic symptoms persisted. Repeat pituitary MRI three months later was unchanged. Due to spontaneous resolution of pituitary hormone abnormalities, no biopsy of the hypothalamic pituitary area was performed. Conclusion We present a case of central DI following known SARS-CoV-2 infection. The patient presented with sudden onset polyuria and polydipsia prompting need for pituitary evaluation. Testing confirmed central DI which resolved within three months. Involvement of the HPA was first noted in 2005 by Leow et al. who reported SARS causing reversible hypophysitis.1 We found three similar cases of patients developing DI approximately four weeks following SARS-CoV-2 infection; however only one case with evidence of infundibular hypophysitis.2,5,6 Two contributing mechanisms of HPA dysfunction are thought to be the result of direct hypothalamic damage or reversible hypophysitis.4 The worldwide presence of SARS-CoV-2 and myriad of complications following infection prompts need for further research and follow-up to better determine clinical recovery for SARS-CoV-2. Reference: 1. Leow MK, et al. Hypocortisolism in survivors of severe acute respiratory syndrome (SARS). Clin Endocrinol (Oxf). 2005;63(2): 197-202. 2. Misgar RA, et al. Central diabetes insipidus (Infundibuloneuro hypophysitis): A late complication of COVID-19 infection. J Endocrinol Invest. 2021;44(12): 2855-2856. 3. Nonglait PL, et al. Hypophysitis after COVID-19 Infection. Indian J Endocrinol Metab. 2021;25(3): 255-256. 4. Pal R. COVID-19, hypothalamo-pituitary-adrenal axis and clinical implications. Endocrine. 2020;68(2): 251-252. 5. Rajevac H, et al. DIABETES INSIPIDUS AS A SYMPTOM OF COVID-19 INFECTION: CASE REPORT. Chest. 2020;158(4): A2576. 6. Sheikh AB, et al. Diabetes Insipidus and Concomitant Myocarditis: A Late Sequelae of COVID-19 Infection. Journal of Investigative Medicine High Impact Case Reports. January 2021. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ODP048 Large Bilateral Adrenal Lesions Abutting Right Hemidiaphragm in a Man from Texas

Abstract Sporadic cases of disseminated histoplasmosis (DHI) have been reported in several regions of the US, mainly inimmunocompromised patients where involvement of the adrenal gland, either unilaterally or bilaterally, is often being the only demonstrable site of active fungal disease in the setting of nonspecific clinical complaints. While DHI is not immediately life-threatening, adrenal involvement might be the most concerning complications due to potential risk of the gland destruction by the expanding infection or its treatment; it is considered that adrenal insufficiency is one of the most common causes of death in patients with DHI. We present a case of 78-year-old immunocompetent gentleman with history of hypertension, ischemic heart disease, atrial fibrillation and 30 pack-years of cigarette smoking who presented with low-grade fever, anorexia and 30 lbs weight loss. As a part of diagnostic evaluation, CT abdomen was performed and bilateral adrenal lesions were reported: right adrenal mass measured 4.6×3.4 cm, and the left adrenal mass 4.1×3.2 cm in maximal axial dimensions. Of note, the right adrenal mass abutted the crus of the right hemidiaphragm and undersurface of the liver. Hormonal evaluation was performed and did not show any excess secretions. GI consult was called for evaluation of adrenal gland access and biopsy: endoscopic ultrasound guided FNA biopsy access was performed and revealed findings compatible with adrenal histoplasmosis and no malignant cells. ID consult was sought: evaluation and prompt start of antifungal therapy were undertaken: IV Amphotericin B, followed by oral Itraconazole for total of 12 months. Patient has remained stable during initial evaluation: due to his age and comorbid conditions, he was admitted to the hospital during which Cosyntropin stimulation test was performed: results have revealed 12.9 at baseline and 17.6 after stimulation. Interdisciplinary discussion was held and resulted in decision to start steroid replacement therapy due to rather mild response to stimulation and significant risk of collateral damage to the remaining adrenal tissue during the antifungal therapy. Patient has responded to therapy well, though had some worsening of his cardiac function which required optimization of diuretic therapy. Patient's vital signs have remained stable, he has reported improved appetite and started to regain some of the lost weight; his electrolytes remained stable. Six-month interval imaging has shown reduction of the size of the adrenal gland sizes and no evidence of gland hemorrhage; notably, adrenal gland improvement has generally paralleled imaging improvement in other involved organs. Steroid therapy is being continued for total of 12 months. DHI still remains a serious problem in certain regions of the US and the world and has to remain an essential part of the differential diagnosis of the adrenal lesions allowing earlier treatment, gland preservation and avoidance of life-threatening complications. Presentation: No date and time listed

OR20-2 Bilateral Primary Aldosteronism Is Not Excluded by High Adrenal Vein Sampling Lateralization Indices

Abstract Background Primary aldosteronism (PA) is broadly divided into unilateral and bilateral forms, and the most reliable subtyping tool available is adrenal vein sampling (AVS). Unilateral PA is generally diagnosed based on AVS lateralization indices (LI=[aldosterone/cortisol] ratio between the dominant and contralateral adrenal vein) ≥4, or even ≥2 when AVS is performed without cosyntopin stimulation. Long-term follow-up data from patients with PA treated with unilateral adrenalectomy have been minimal. Objective To assess the rates of residual and recurrent PA after AVS-guided unilateral adrenalectomy. Methods We enrolled patients with PA who underwent unilateral adrenalectomy between 09/2017 and 08/2021 in a single tertiary referral center and followed them longitudinally. Demographics, laboratory, imaging, and pathology data were collected. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue blocks. Results During the study period, 80 patients (52 men) with median age 53 (range, 19-76) years underwent unilateral adenectomy, and were followed for a median 80 days (range, 6-653 days). In total 14 (17.5%) patients had evidence of residual PA following surgery, including 3 who had complete biochemical failure, and 1 who had apparent cure of PA immediately post-operatively. All 14 patients had severe PA, with baseline plasma aldosterone concentrations between 23.4 and 496 ng/dL and suppressed renin. Baseline and post-cosyntropin stimulation LIs ranged from 2.7- 187.5 and 1.3-53.7. The LI was &amp;gt;4 only at baseline in 3 cases, and only after cosyntropin stimulation in 2 cases. Histology showed single adenomas, ranging from 0.7-5.3 cm in 7 (50%) patients, and multiple adenomas and/or nodular hyperplasia in 7 (50%) patients. Of the 7 cases analyzed for aldosterone-driver mutations so far, 4 harbored CACNA1D mutations, 2 ATP1A1 mutations, and 1 a KCNJ5 mutation. Conclusions Patients with severe PA and frank AVS aldosterone lateralization might have multifocal, bilateral disease. Long-term follow up and targeted medical therapy should be pursued after unilateral adrenalectomy for presumed unilateral PA. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.

ODP050 Oncocytic Adrenocortical Carcinoma Presenting with Hematuria

Abstract Introduction Adrenocortical carcinoma (ACC) is rare and aggressive endocrine tumor. It is commonly noticed due to symptoms of hormone excess, mass-effect, or incidentalomas. Different histological variants are noted, which include: oncocytic, myxoid, sarcomatoid, and the conventional subtype. The oncocytic variant is usually seen on the left side, and usually presents with symptoms of mass-effect. Only around 17% were noted to be functional. We present an interesting case of ACC (oncocytic type) which presented with hematuria. Case Presentation 70-year-old female with a history of morbid obesity, hypertension, anxiety, GERD, tubular adenoma, hyperlipidemia, and lumbar radiculopathy came in to see her primary care for hematuria and back pain. She has a long-standing history of back pain, for which she has had an EMG, which showed mild axonal polyneuropathy and bilateral L5 radiculopathy. Apart from hematuria, she denies any other urinary complaints. Additionally, she denied any fever, chills, nausea, abdominal pain, sweating, tremor, headache, palpitations, easy bruising, fractures, muscle weakness, hirsutism, or changes in her weight. She has a family history of pancreatic cancer and is currently retired, and denies alcohol, tobacco, or recreational drug use. A KUB X-ray was ordered which showed calcification in the left upper quadrant, concerning for a splenic artery aneurysm; this led to a CTA abdomen, which showed a large 7.2 cm heterogenous enhancing solid left adrenal mass, concerning for primary adrenal carcinoma, which was new compared to imaging 2 years ago. Also seen was a 0.7 cm enhancing right hepatic lobe lesion, along with a 1.5 cm splenic artery aneurysm. Following this, an abdomen MRI with contrast showed a 7×7×7 cm solid heterogeneously enhancing left upper quadrant mass beside the left adrenal gland and pancreas. Subsequently, a CT guided needle biopsy was done, which revealed scant adrenal tissue. PET scan showed a hypermetabolic mass, arising from the left adrenal, consistent with adrenal carcinoma. At this point, an endocrinology referral was placed, and other lab work including 24-hour urine free cortisol, DHEA-S, 24-hour urine metanephrine, and aldosterone/renin ratio was done. DHEA-S level was elevated at 264 mcg/dl (12-133). She was then transferred to a tertiary care facility, where she underwent a left adrenalectomy, para-aortic lymphadenectomy, nephrectomy, splenectomy, and distal pancreatectomy. Pathology confirmed adrenal cortical carcinoma (oncocytic type), with negative margins, Ki-67 labelling index of 25%, and no lymphovascular invasion, pT2pNO. She was then discharged home with visiting nurses. Conclusion Oncocytic ACC is an infrequent tumor, which presents with symptoms of mass effect, and definitive diagnosis requires histopathological assessment. The Lin–Weiss–Bisceglia (LWB) system and Helsinki score help establish malignant potential. Fortunately, it has a decreased tendency to metastasize and a better prognosis than other ACC subtypes. Further studies to understand oncocytic ACC are needed. Presentation: No date and time listed

Hypertension in adrenal pathology: clinical cases

Hyperproduction of hormones by adrenal tissues, or (less often) by their cells ectopic into other organs, naturally leads to the development of secondary hypertension, usually refractory to classical antihypertensive drugs. Adrenal glands produce mineralocorticoids (aldosterone), glucocorticoids (cortisol), androgens and catecholamines (adrenaline, noradrenaline). Excessive secretion of these hormones, mainly each in particular, due to a tumor/hyperplasia of the corresponding structures of the adrenal gland (or as a result of ectopy) leads to a number of diseases one of the leading syndromes of which is symptomatic hypertension, usually refractory to classical antihypertensive drugs. Endocrine disorders can sometimes manifest as hypertension. The pathogenesis of hypertension is determined by the effect of adrenal hormones on vascular tone and directly on the myocardium, mineral metabolism disorders, activation of the renin-angiotensin-aldosterone system, increased production of catecholamines, and the development of atherosclerotic changes in blood vessels. Aldosterone, for example, inhibits renin synthesis, accumulates sodium in the vascular wall and increases its sensitivity to vasoconstrictor stimuli, and hypokalemia leads to the dystrophic changes in nerve fibers and the development of muscle syndrome and nephropathy. Brief excerpts from published own observations are given: primary aldosteronism, ectopic ACTH-dependent Cushing’s syndrome, androsteroma, pheochromocytoma (of the bladder, and a case with stable hypertension). A long-term increase in blood pressure was verified by corresponding hormonal status, electrocardiography data (left ventricular hypertrophy with overload) and ophthalmoscopy (hypertensive retinopathy). The timely diagnosis and adequate treatment of these diseases remains one of the urgent tasks of clinical medicine.

Melatonin ameliorates the adrenal and pancreatic alterations in streptozotocin-induced diabetic rats: Clinical, biochemical, and descriptive histopathological studies.

Previous studies have demonstrated the beneficial effects of melatonin in diabetic rats. However, limited studies have been conducted on the potential effects of melatonin on the descriptive histopathological and morphometric findings in different compartments of the adrenal glands in diabetic animal models. In this study, using a streptozotocin (STZ)-induced diabetic rat model, we sought to examine histological alterations in the pancreas and adrenal glands and observe the effect of the administration of melatonin on the histopathology and morphology of the pancreas and the adrenal gland cortex and medulla that are altered by STZ-induced hyperglycemia. Rats were randomly assigned to four different groups: Group I, normal control; Group II, melatonin group (MT) (10 mg/kg/day); Group III, (diabetic STZ group), and Group IV, diabetic (STZ) + melatonin group (MT). Throughout the experiment, the animals' fasting blood sugar levels were measured. Blood was obtained to determine the animals' cumulative blood sugar levels after sacrification. For histological and morphometrical evaluations, the pancreatic and adrenal gland tissues were dissected and processed. Our results showed that diabetic rats receiving melatonin significantly (P < 0.05) improved their fasting blood sugar and cumulative blood sugar levels compared to the diabetic group not receiving melatonin. Furthermore, histopathological examinations of the pancreatic and adrenal tissues of the diabetic rats indicated the occurrence of severe histopathological and morphometric changes. Morphometric analysis of the adrenals indicated a significant increase (P < 0.05) in the thickness of the cortex zones [zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR)] for the diabetic STZ group compared with other groups, and a significant decrease (P < 0.05) in the diameter of the in adrenal gland medullas in the diabetic STZ rats compared to the other groups. Furthermore, treatment with melatonin restored these changes in both the pancreatic and adrenal gland tissues and produced a significant (P < 0.05) improvement in the cortex and medulla thickness compared to the untreated diabetic rats. Overall, melatonin significantly reduced the hyperglycemic levels of glucose in diabetic rats and reversed the majority of histopathological alterations in the tissues of the pancreas and adrenals, demonstrating its anti-diabetic activity.