Normal Adrenal Glands Research Articles

Therapeutic potential of targeting the FLNA-regulated Wee1 kinase in adrenocortical carcinomas.

Filamin A (FLNA) is poorly expressed in adrenocortical carcinomas (ACC) compared to adenomas (ACA). Its presence is associated to a less aggressive tumour behaviour, potentially due to its role in negatively regulating IGF1R signalling. Upregulation of G2/M Wee1 kinase was shown in FLNA-deficient mouse neural progenitor cells, and it has been reported in several tumours. This study explored Wee1 expression in ACC and its regulation by FLNA, the effects of Wee1 inhibitor AZD1775, and the impact of FLNA on its efficacy in ACC cell lines and primary cells. Analysis of FLNA and Wee1 proteins revealed elevated Wee1 and reduced FLNA in ACC compared to normal adrenal gland. FLNA knockdown increased Wee1 protein in NCI-H295R, MUC-1, and in primary ACC cells. Higher p-CDK1 and cyclin B1 were shown in FLNA-silenced MUC-1, while decreased Wee1, p-CDK1 and cyclin B1 resulted after FLNA overexpression. Wee1 reduction was reverted by lactacystin treatment and FLNA transfection increased p-Wee1 (Ser123), suggesting FLNA's role in targeting Wee1 for degradation. AZD1775 dose-dependently reduced proliferation and viability in ACC cell lines and primary cultures, and it triggered MUC-1 cell death. Similar effects were induced by Wee1 silencing. FLNA depletion augmented AZD1775's efficacy in reducing proliferation and potentiating apoptosis in MUC-1 and primary cells. In conclusion, we demonstrated that FLNA regulates Wee1 expression by promoting its degradation, suggesting that low FLNA typical of ACC leads to increased Wee1 with consequent cancer cells growth. It proposes Wee1 inhibition as a new potential therapeutic approach for ACC, particularly for those lacking FLNA.

Somatic GNAQ, CTNNB1, and CACNA1C Mutations in Cat Aldosterone-Secreting Tumors.

Primary aldosteronism (PA) is a common cause of human hypertension. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3 are found in at least 80% of aldosterone-producing adenomas, which cause unilateral PA in humans. Somatic mutations have been identified infrequently in 7 other genes; few of these were known to play a role in aldosterone secretion before the discovery of their mutations. Interrogating somatic mutations in the domestic cat, in which spontaneous PA is also known to occur, might improve the understanding of normal adrenal gland physiology and the pathophysiology of PA. DNA and RNA extracted from tissue from 13 cats with unilateral aldosterone-secreting tumors, including 8 carcinomas and 5 adenomas, underwent whole genome sequencing, targeted Sanger sequencing, and RNA sequencing. Single-nucleotide substitution variants were filtered to select those with a predicted deleterious effect on protein function and a suspected role in aldosterone secretion. Probable functional somatic single-nucleotide polymorphisms (n=8) were found in 3 adenomas and 2 carcinomas. Mutations with predicted significant effects were identified in 2 genes also mutated in human PA; GNAQ and CTNNB1, and in a residue of CACNA1C analogous to a common CACNA1D mutation. In contrast to humans, CACNA1C expression was much greater than CACNA1D in both feline tumor and nontumor adrenal tissue. No mutations were identified in KCNJ5, CACNA1D, ATP1A1, or ATP2B3. Similar mutations were identified in cats to those found in humans. It is, therefore, likely that both species have shared underlying selection pressures for mutations that increase aldosterone secretion.

Immunohistochemical expression of CYP11A1, CYP11B, CYP17, and HSD3B2 in functional and nonfunctional canine adrenocortical tumors.

Functionality of human adrenal tumors is inferred by CYP11B1 (cortisol synthase) expression, CYP11B2 (aldosterone synthase) expression, or both. Nonfunctional canine adrenal tumors have low expression of steroidogenic enzymes, whereas aldosterone-producing tumors express CYP11B, and cortisol-producing tumors express both CYP11B and CYP17. Twenty-two client-owned dogs with adrenocortical tumors (ACT) (8 nonfunctional, 7-cortisol producing, 2 aldosterone-producing and 5 functional noncortisol producing) and 2 dogs with normal adrenal glands. Retrospective case series. Adrenal functionality was determined from clinical signs and endocrine testing. CYP11A1, CYP11B, CYP17, and HSD3B2 expression was detected by immunohistochemistry on formalin-fixed paraffin-embedded adrenal tissue. Protein expression was semiquantified by 2 blinded observers using H-scoring (results reported as median [range]) and compared in nonfunctional and cortisol-producing adrenal tumors by Mann-Whitney U tests. CYP11A1, CYP11B, and HSD3B2 were present within all cortical layers of normal adrenal glands, and CYP17 was expressed within the zona fasciculata and zona reticularis. Expression of CYP11A1 (191.25 [97.5-270] vs. 175 [102.5-295] P = .69), CYP11B (190 [130-265] vs. 147.5 [95-202.5]; P = .07), CYP17 (177.5 [87.5-240] vs. 247.5 [55-292.5]; P = .40), and HSD3B2 (230 [47.5-295] vs. 277.5 [67.5-295]; P = .34) were not significantly different between cortisol-producing and nonfunctional ACT. Our findings suggest it is not possible to determine functionality of canine ACT by immunohistochemistry for steroidogenic enzymes. Tumor size cannot be used to infer functionality of adrenal tumors.

7648 Bilateral Adrenal Hemorrhage After Laminectomy: A Rare Complication

Abstract Disclosure: M. Rizk: None. S. Sharma: None. H. Qadeer: None. R. Paudel: None. C. Sherpa: None. Introduction: Bilateral Adrenal Hemorrhage (BAH) is an exceedingly uncommon complication of laminectomy, with few cases reported in the literature. In patients with history of any surgical procedure who present with unexplained symptoms of abdominal pain, back pain, hypotension, fever, confusion, or electrolyte abnormalities especially hyponatremia, acute adrenal insufficiency (AI) should be highly suspected. We herein describe a case of BAH following laminectomy. Clinical Case: 63-year-old male with past medical history of hypertriglyceridemia was admitted for post traumatic L1 burst fracture and was treated with T11-L3 fusion and T12-L3 laminectomy. The patient had normal bilateral adrenal glands after the fall on CT thoracic/lumbar spine imaging. The course was complicated by thrombocytopenia initially thought to be induced by therapeutic heparin since heparin antibody was positive. Serotonin-release assay returned negative weeks later ruling out heparin induced thrombocytopenia. Two weeks later, he developed unexplained tachycardia and CT angiogram was done which showed segmental pulmonary embolism and bilateral adrenal nodules measuring at least 3.9 cm on right side and 3.3 cm on left side. He was treated with Eliquis and was discharged home. Two weeks later, he was readmitted with weight loss of 30 lbs, weakness and altered mental status. His vital signs were significant for hypotension and tachycardia. Initial work up showed profound hyponatremia (sodium 121 mEq/L), hyperkalemia (potassium 6.1 mEq/L), hypoglycemia (Glucose 58 mg/dL), hypercalcemia (calcium 11.7 mg/dL) and acute kidney injury (creatinine 2.58 mg/dL). Repeat CT scan of chest/abdomen/pelvis without contrast showed bilateral hyperdense 4 cm adrenal mass/hematoma. Since initial CT of thorax/spine done after the fall showed normal adrenal glands, the new bilateral hyperdense adrenal masses noted post procedure is suggestive of adrenal hemorrhage/hematoma. Undetectable cortisol and high ACTH confirmed AI. Patient was treated with stress dose hydrocortisone followed by slow taper. MRI of pituitary gland showed partial empty Sella however since ACTH was high, empty sella was only an incidental finding and not the cause of AI. He is maintained on hydrocortisone 15 mg in the morning and 5 mg in the evening plus fludrocortisone 0.1 mg daily. His fatigue has improved, he is gaining weight and electrolytes are normal. Conclusions: This case report emphasizes the significance of timely diagnosis and management of BAH, which leads to primary AI. BAH is a rare complication following laminectomy. Clinicians should be aware of this possibility and consider it in the differential diagnosis of postoperative abdominal pain and electrolyte abnormalities, especially hyponatremia. Timely recognition and high index of suspicion should prompt empiric glucocorticoid replacement to decrease mortality. Presentation: 6/1/2024

7745 Hirsutism in young girls, Beyond PCOS

Abstract Disclosure: I. Mishra: None. A. Baliarsinha: None. Hirsutism is excessive terminal hair that appears in a male pattern in women.Though obesity,insulin resistance and PCOS are common cause of hirsutism in young girls,suspicion of other etiologies must be made on a background of overlapping features.We describe 3 cases of young females presenting with hirsutism with varied etiologies. Case 1.16 year 11months old female presented with oligomenorrhea -3years,excessive hair growth over face -2 years.On examination,BMI was 25.2 kg/m2,Grade 2 acanthosis,A3P5B5,modified FG- 16/36,clitoral length-1.3cm and clitoral index - 70 mm2.Biochemical evaluation revealed normal thyroid function tests,s.prolactin- 23.65 ng/ml,ONDST- 1.2 mcg/dl,LH - 1.49 IU/ml,FSH- 7.44IU/ml,serum testosterone- 70ng/dl,17OHP- 7.03 ng/ml,DHEAS- 553mcg/dl and post synacthen 17OHP- 36.89 ng/ml respectively. USG abdomen & pelvis was normal.HOMA-IR was 4.1. Diagnosis was LOCAH Case 2.19 year old female presented with hair growth in androgen dependent areas-5years, regular predictable menstrual cycles and was treated as a case of PCOS for 5 years with no changes in hair growth pattern.She was off therapy since 1 year prior presenting to us.On examination,BMI was 24 kg/m2,acanthosis absent,A3P5B5,modified FG- 18/36, clitoral length- 6 mm,clitoral index - 25mm2.Biochemical evaluation revealed normal thyroid function tests,s.prolactin- 22.9 ng/ml,ONDST- 0.508 mcg/dl,LH - 5.98 IU/ml,FSH- 5.29 IU/ml, serum testosterone- 23.4 ng/dl,17OHP- 1.01 ng/ml and DHEAS- 298 mcg/dl respectivelyUSG Abdomen & pelvis;RO - 5.7cc,LO - 5.6 cc with B/L PCOM.Review TVS was normal.In view of discordant results DHT and luteal phase serum progesterone were done which were 80.5 pg/ml and 2.1ng/ml respectively.HOMA IR was 1.9.Diagnosis was IDIOPATHIC HIRSUTISM. Case 3.12 year old female child presented with excessive hair growth over face,chest,back and abdomen- 1yr,hoarseness of voice - 1 yr,onset of breast enlargement apprx 8 to 9yrs with non-attainment of menarche.On examination BMI was 30 kg/m2,Grade 3 acanthosis, A3P5B3,modified FG- 15/36,clitoral length- 2.8 cm & clitoral index - 120 mm2. Biochemical evaluation revealed normal thyroid function tests,LH - 15.3 IU/ml,FSH- 6 IU/ml,s.prolactin- 11.2 ng/ml,ONDST- 0.38 mcg/dl,DHEAS- 161 mcg/dl,serum testosterone- 140 ng/dl,17OHP- 3.9 ng/ml and post synacthen 17OHP- 4.10 ng/ml respectively.USG abdomen-s/o PCOS with normal adrenals. In view of discordant results short DAST was done which was Testosterone(ng/dl)baseline-125;4hr-91.1, DHEAS (mcg/dl) baseline-180;4hr-188 cortisol (mcg/dl) baseline-7.04;4hr-0.95.HOMA-IR was 5.4. Diagnosis was PCOS(OVARIAN THECOSIS).Varied presentations of hyperandrogenic disorders occurs in young females.A need to revise the diagnosis must be made if no response is obtained with initial drug regimes. Presentation: 6/3/2024

6426 Pregnancy-induced ACTH-independent Cushing Syndrome: A Rare Phenomenon Secondary To Aberrant Receptors On Adrenal Glands

Abstract Disclosure: K. Manglani: None. S.T. Sharma: None. Pregnancy-induced hypercortisolism is a rare phenomenon with very few cases reported in the literature. The pathogenesis is thought to be secondary to the expression of aberrant LH/hCG receptors on the adrenal gland that stimulate cortisol production in pregnancy. We present a case of a 31-year-old female with no past comorbidities until her first pregnancy in 2017 when she gained around 50lbs, developed violaceous striae, acne, hirsutism, depression with suicidal ideation for which she was hospitalized twice, and underwent emergent C-section at 34 weeks’ gestation due to pre-eclampsia. She was referred to the Endocrinology clinic post-delivery, and screening for Cushing syndrome was negative at that time: 24-hour urinary free cortisol (UFC), late-night salivary cortisol (LNSC) and 1-mg dexamethasone (dex) suppression test (DST) were normal. She presented again in 2020 during her second pregnancy at 14 weeks gestation, with 20lb weight gain in the first trimester, acne, hirsutism, and easy bruising. Labs showed cortisol of 22.4 mcg/dL (6.2-19.4), ACTH <1.5 pg/mL, total testosterone 168 ng/dL (8-48), androstenedione 751 ng/dL (41-262), DHEAS 91.2 mcg/dL (84.8-378), 24-hour UFC 277 mcg/day (6-42), LNSC 0.671 mcg/dL, 1mg DST with post-dex cortisol 24 mcg/dL (dex level 58 ng/dL), and 8-mg DST with post-dex cortisol level of 28.2 mcg/dL (dex level 457 ng/dL). Testing was consistent with ACTH-independent Cushing syndrome. Ultrasound and MRI of the abdomen/pelvis showed normal adrenal glands with no hyperplasia or mass. Given worsening hypercortisolism and her course during a prior pregnancy, medical treatment with metyrapone was initiated. We started at a dose of 500 mg daily and gradually increased to 1500 mg with close monitoring of her blood pressure and cortisol levels. She underwent vaginal delivery at 38 weeks’ gestation, and the baby required a brief stay in the NICU for respiratory distress but was otherwise doing well. The patient developed secondary adrenal insufficiency post-delivery requiring hydrocortisone, with recovery of her HPA axis in around 10 weeks. Since then, she has been off hydrocortisone with normal UFC, ACTH, and cortisol levels. This case enables discussion regarding the underlying etiology, diagnostic difficulties, and management of this rare condition. Although the exact etiology is unknown, the postulated mechanism is aberrant LH/hCG receptor expression given the patient’s presentation during each pregnancy, normal cortisol levels between both pregnancies, and a brief period of adrenal insufficiency post-delivery. Management of hypercortisolism during pregnancy remains challenging. Metyrapone, a steroidogenesis inhibitor, can be considered for control of hypercortisolemia during pregnancy (off-label) in select cases to prevent maternal and fetal complications. Presentation: 6/1/2024

7958 Organ Of Zuckerkandl Paragangliomas

Abstract Disclosure: L. McVicker: None. S. Egan: None. K.C. Conlon: None. J. Gibney: None. J. Feeney: None. M. Whelan: None. A. Garrahy: None. Background: The Organ of Zuckerkandl (OoZ) was first described in 1901 by an Austrian anatomist Emile Zuckerkandl as a para-aortic paraganglia located laterally to the abdominal aorta at the level of the inferior mesenteric artery. It represents the largest accumulation of extra-adrenal chromaffin cells in mammals. Paragangliomas (PGL) of the OoZ are rare entities with prior retrospective study of 371 patients with phaeochromocytoma or sympathetic PGL revealing only 21 cases of OoZ PGLs of which 14 of which (66%) had metastases at diagnosis (1). 70% of cases are in association with SDHB or less commonly, SDHD mutations (2). Clinical Case: A 48-year-old female presented with a year long history of palpitations and headache. Plasma normetanephrine level was 6340 pmol/L (lab reference 0-1180), plasma metanephrine 412 pmol/L (lab reference 0-510) and plasma 3-methoxytyramine <100 pmol/L (lab reference 0-180). CT adrenals showed normal adrenal glands. A 4.5 cm x 2.1 cm hyper-vascular retroperitoneal mass was visualised. A 68Ga-DOTATOC PET/CT showed no abnormal uptake in the area of the mass. [1]23I-MIBG scan showed increased uptake within the mass compatible with a PGL of the OoZ. Following surgery, histology confirmed PGL of the OoZ and plasma metanephrines normalised following. Genetic testing for predisposing mutation was negative. A 38-year-old male presented with sweating episodes, palpitations, and hypertension. Plasma normetanephrine level was 2,230 pmol/L (lab reference 0-1180) and plasma metanephrines 223 pmol/L (lab reference 0-520). CT adrenals showed normal adrenal glands with no other abnormalities noted. [1]23I-MIBG showed increased uptake in the region of the origin of the inferior mesenteric artery from the aorta consistent with PGL of the OoZ. The patient underwent laparoscopic resection with resultant normalisation of metanephrine levels. Histology showed a 21 mm encapsulated tumour in keeping with PGL with a PASS Score of 7. As with the first case, genetic testing for predisposing mutations returned negative. Conclusion: OoZ PGLs represent rare presentations of phaeochromocytoma-paraganglioma disease. This small case series is notable as neither case was associated with a predisposing genetic mutation, which has previously been shown to be more common in OoZ PGLs. There was also no evidence of metastasis in either case which is more commonly associated with this OoZ disease. The first case is also remarkable in terms of the tumour’s imaging characteristics. Previous studies have shown greater sensitivity using 68Ga-DOTA-Somatostatin Analogue PET/CT in comparison to [1]23I-MIBG in the detection of PGLs (3). In the first case, the tumour did not show any significant avidity on 68Ga-DOTATOC PET/CT but was avid on [1]23I-MIBG. Reference: 1. Ayala-Ramirez et al. J Clin Endocrinol Metab. 2011 2. Lodish et al. Endocr Relat Cancer. 2010 3. Carrasquillo et al. J Nuclear Medicine. 2021 Presentation: 6/2/2024

7452 A Diagnostically Challenging Case of Hyperandrogenism in Postmenopausal Woman

Abstract Disclosure: M.S. Hossain: None. S. Hossain: None. B. Gautam: None. H. liao: None. S.C. Kumar: None. D.S. Rosenthal: None. Background: The diagnosis of new-onset severe hyperandrogenism in postmenopausal women is rarely encountered, posing a unique challenge for clinicians. Ovarian hyperthecosis and androgen-secreting tumors are the usual suspects in post menopause. In our case ovarian hyperthecosis is the primary consideration but elevated 17-OH Progesterone (17-OHP) introduces the potential co-existence of Non classical congenial adrenal hyperplasia (NCCAH) or rare instances of Sertoli-Leydig Cell Tumor (SLCT). Case Presentation: A 70 years old female, with no past medical history, referred to the Endocrinology clinic for significantly elevated testosterone level. She reported excessive hair growth in various areas of her body since 2022, which required shaving almost every day. She also reported male pattern hair loss and weight loss of 15 lbs over the last three months. She denied headache, vision changes, deepening of voice, history of fertility problems and use of exogenous testosterone. Her vital sign were normal. On examination, she had clitoromegaly, terminal hair growth on her upper lip and chin, breast atrophy, male pattern baldness, but no cushingoid or acromegaly features. Blood tests showed significantly elevated total testosterone: 1422 ng/dl (2-45); free testosterone: 147.9 pg/ml (0.2-3.7) and Estradiol: 64 pg/ml (Postmenopausal <31) with low LH: 7.6 mIU/ml (Postmenopausal 10.0-54.7) and FSH: 22.2 mIU/mL (Postmenopausal 23-116). Her serum ACTH, DHEA Sulfate, 11-deoxycortisol, 17 OH pregnenolone, TSH, Free T4, prolactin, IGF-1 were normal while her 17-OHP: 396 ng/dl (<45 ng/dl) was significantly elevated. An ACTH stimulation test showed 17-OHP level of 734 ng/dl at 30 minutes and 901 ng/dl at 1 hour. Pelvic ultrasonography: right ovary of 2.3 x 1.6 x 1.9 cm and the left ovary of 3.1 x 1.8 x 3.0 cm. MRI showed normal adrenal glands but a prominent left ovary. Patient was referred to the Obstetrics department to undergo bilateral oophorectomy for the definitive diagnosis and treatment. Discussion: The current ACTH stimulation test for non-classical congenital adrenal hyperplasia is validated for premenopausal women but lacks standardization in post-menopausal state. In our case, distinguishing the source of elevated 17-OHP between adrenal (NCCAH) and ovarian (SLCT) was difficult. Unexpected asymmetry in ovarian size observed in our case further complicates the scenario, contrasting with the typical symmetrical enlargement seen in ovarian hyperthecosis. Pending surgical pathology will further guide the management. This case highlights the complexity of diagnosing hyperandrogenism in postmenopausal women, emphasizing the significance of thoroughly exploring differential diagnoses and meticulously analyzing laboratory results. Presentation: 6/2/2024

Novel repurposing of sulfasalazine for the treatment of adrenocortical carcinomas, probably through the SLC7A11/xCT-hsa-miR-92a-3p-OIP5-AS1 network pathway

BackgroundRecent multigenomic analysis of adrenocortical carcinomas (ACCs) identified SLC7A11/xCT as a novel biomarker. The Food and Drug Administration–approved anti-inflammatory drug, sulfasalazine (SAS), induces ferroptosis by blocking SLC7A11 expression. We hypothesize that SAS could be repurposed to target ACC cells. MethodsExpression of SLC7A11 and its association with ACC survival was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA). The validated ACC cell lines NCI-H295R, ACC1, and ACC2 were grown in 2D culture. In vitro studies included the CellTiter-Glo assay to calculate viability, Western blot (WB) analysis for apoptosis and other target protein changes, reverse transcriptase polymerase chain reaction for steroidogenic enzyme changes, C11BODIPY for lipid peroxidation, and mass spectrometry for changes in lipids. ResultsThe Cancer Genome Atlas Program database analysis in GEPIA showed that SLC7A11 and linked long noncoding RNA OAP5-AS1 are highly expressed in ACC tumors versus normal adrenals (n = 77 vs 128; P < .05). This was associated with poor overall and disease-free survival with hazard ratios of 4.3 and 5.2 for SLC7A11 and 4.8 and 2.7 for OAP5-AS1, respectively. ACC cell line half-inhibitory maximum concentration values after 72-hour SAS treatment ranged from 412 nM (ACC1) to 799 nM (ACC2), and all showed cleavage of poly (ADP-ribose) polymerase, upregulation of p-Akt and p-ERK, and downregulation of GPX4 and SLC7A11 (P < .05) by WB analysis. Sphere formation, migration, and invasion assay showed inhibition, and lipid peroxidation using C11BODIPY, increase in intracellular iron, induction of oxidative stress, and significant upregulation of oxidized polyunsaturated fatty acid phospholipids (P < .05 each) by mass spectrometry suggests induction of ferroptosis. ConclusionSAS downregulates tSLC7A11 in ACCs, targets the Akt/ERK pathway and lipid metabolism, and induces cell death in vitro, warranting additional translational studies to define its therapeutic potential in ACC.

Primary unilateral macronodular adrenal hyperplasia with concomitant glucocorticoid and androgen excess and KDM1A inactivation.

Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of Cushing's syndrome. Individuals with PBMAH and glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome due to ectopic expression of the GIP receptor (GIPR) typically harbor inactivating KDM1A sequence variants. Primary unilateral macronodular adrenal hyperplasia (PUMAH) with concomitant glucocorticoid and androgen excess has never been encountered or studied. We investigated a woman with a large, heterogeneous adrenal mass and severe adrenocorticotropic hormone-independent glucocorticoid and androgen excess, a biochemical presentation typically suggestive of adrenocortical carcinoma. The patient presented during pregnancy (22nd week of gestation) and reported an 18-month history of oligomenorrhea, hirsutism, and weight gain. We undertook an exploratory study with detailed histopathological and genetic analysis of the resected adrenal mass and leukocyte DNA collected from the patient and her parents. Histopathology revealed benign macronodular adrenal hyperplasia. Imaging showed a persistently normal contralateral adrenal gland. Whole-exome sequencing of 4 representative nodules detected KDM1A germline variants, benign NM_001009999.3:c.136G > A:p.G46S, and likely pathogenic NM_001009999.3:exon6:c.865_866del:p.R289Dfs*7. Copy number variation analysis demonstrated an additional somatic loss of the KDM1A wild-type allele on chromosome 1p36.12 in all nodules. RNA sequencing of a representative nodule showed low/absent KDM1A expression and increased GIPR expression compared with 52 unilateral sporadic adenomas and 4 normal adrenal glands. Luteinizing hormone/chorionic gonadotropin receptor expression was normal. Sanger sequencing confirmed heterozygous KDM1A variants in both parents (father: p.R289Dfs*7 and mother: p.G46S) who showed no clinical features suggestive of glucocorticoid or androgen excess. We investigated the first PUMAH associated with severe Cushing's syndrome and concomitant androgen excess, suggesting pathogenic mechanisms involving KDM1A.

Metabolomic profiling of pheochromocytomas in dogs: Catecholamine phenotype and tricarboxylic acid cycle metabolites.

In humans with pheochromocytomas (PCCs), targeted metabolomics is used to determine the catecholamine phenotype or to uncover underlying pathogenic variants in tricarboxylic acid (TCA) cycle genes such as succinate dehydrogenase subunits (SDHx). To analyze catecholamine contents and TCA cycle metabolites of PCCs and normal adrenals (NAs). Ten healthy dogs, 21 dogs with PCC. Prospective observational study. Dogs diagnosed with PCC based on histopathological and immunohistochemical confirmation were included. Tissue catecholamine contents and TCA metabolites in PCCs and NAs were measured by liquid chromatography with mass spectrometry or electrochemical detection. Compared to NAs, PCCs had significantly higher tissue proportion of norepinephrine (88% [median: range, 38%-98%] vs 14% [11%-26%]; P < .001), and significantly lower tissue proportion of epinephrine (12% [1%-62%] vs 86% [74%-89%]; P < .001). Pheochromocytomas exhibited significantly lower fumarate (0.4-fold; P < .001), and malate (0.5-fold; P = .008) contents than NAs. Citrate was significantly higher in PCCs than in NAs (1.6-fold; P = .015). One dog in the PCC group had an aberrant succinate : fumarate ratio that was 25-fold higher than in the other PCCs, suggesting an SDHx mutation. This study reveals a distinct catecholamine content and TCA cycle metabolite profile in PCCs. Metabolite profiling might be used to uncover underlying pathogenic variants in TCA cycle genes in dogs.

Single-nucleus and spatial transcriptome reveal adrenal homeostasis in normal and tumoural adrenal glands.

The human adrenal gland is a complex endocrine tissue. Studies on adrenal renewal have been limited to animal models or human foetuses. Enhancing our understanding of adult human adrenal homeostasis is crucial for gaining insights into the pathogenesis of adrenal diseases, such as adrenocortical tumours. Here, we present a comprehensive cellular genomics analysis of the adult human normal adrenal gland, combining single-nuclei RNA sequencing and spatial transcriptome data to reconstruct adrenal gland homeostasis. As expected, we identified primary cells of the various zones of the adrenal cortex and medulla, but we also uncovered additional cell types. They constitute the adrenal microenvironment, including immune cells, mostly composed of a large population of M2 macrophages, and new cell populations, including different subpopulations of vascular-endothelial cells and cortical-neuroendocrine cells. Utilizing spatial transcriptome and pseudotime trajectory analysis, we support evidence of the centripetal dynamics of adrenocortical cell maintenance and the essential role played by Wnt/β-catenin, sonic hedgehog, and fibroblast growth factor pathways in the adult adrenocortical homeostasis. Furthermore, we compared single-nuclei transcriptional profiles obtained from six healthy adrenal glands and twelve adrenocortical adenomas. This analysis unveiled a notable heterogeneity in cell populations within the adenoma samples. In addition, we identified six distinct adenoma-specific clusters, each with varying distributions based on steroid profiles and tumour mutational status. Overall, our results provide novel insights into adrenal homeostasis and molecular mechanisms potentially underlying early adrenocortical tumorigenesis and/or autonomous steroid secretion. Our cell atlas represents a powerful resource to investigate other adrenal-related pathologies.

Cellular landscape of adrenocortical carcinoma at single-nuclei resolution

Adrenocortical carcinoma (ACC) is a rare yet devastating tumour of the adrenal gland with a molecular pathology that remains incompletely understood. To gain novel insights into the cellular landscape of ACC, we generated single-nuclei RNA sequencing (snRNA-seq) data sets from twelve ACC tumour samples and analysed these alongside snRNA-seq data sets from normal adrenal glands (NAGs). We find the ACC tumour microenvironment to be relatively devoid of immune cells compared to NAG tissues, consistent with known high tumour purity values for ACC as an immunologically “cold” tumour. Our analysis identifies three separate groups of ACC samples that are characterised by different relative compositions of adrenocortical cell types. These include cell populations that are specifically enriched in the most clinically aggressive and hormonally active tumours, displaying hallmarks of reorganised cell mechanobiology and dysregulated steroidogenesis, respectively. We also identified and validated a population of mitotically active adrenocortical cells that strongly overexpress genes POLQ, DIAPH3 and EZH2 to support tumour expansion alongside an LGR4+ progenitor-like or cell-of-origin candidate for adrenocortical carcinogenesis. Trajectory inference suggests the fate adopted by malignant adrenocortical cells upon differentiation is associated with the copy number or allelic balance state of the imprinted DLK1/MEG3 genomic locus, which we verified by assessing bulk tumour DNA methylation status. In conclusion, our results therefore provide new insights into the clinical and cellular heterogeneity of ACC, revealing how genetic perturbations to healthy adrenocortical renewal and zonation provide a molecular basis for disease pathogenesis.

POLYCYSTIC OVARY SYNDROME (PCOS) AND ITS TREATMENT AS CAUSE OF SECONDARY ALDOSTERONISM: A DOG CHASING ITS TAIL

Objective: Polycystic Ovary Syndrome (PCOS) is a complex endocrine-metabolic disorder affecting about 7% of childbearing women. PCOS is characterized by anovulation, hyperandrogenism, insulin resistance and it is associated with an increased cardiovascular risk. PCOS patients may exhibit high blood pressure and signs of hydro-saline retention. Therefore, clinicians would be interested in performing hormonal screening for secondary hypertension. Design and method: We perform the aldosterone/direct renin concentration ratio as a screening test for primary aldosteronism (PA). The test was proceed after serum potassium correction and previous a wash-out period of interfering medications. The recumbent saline suppression test was performed with infusion of 2L NaCl 0.9% in 4 hours under standardized salt intake (9gr/24h). Aldosterone and direct renin were determined by chemiluminescent immunoassay technology (DiaSorin®). Results: A 33yrs old woman attended our renal outpatient clinic because of recurrent episodes of generalized swelling, diuresis contraction and fluid retention. The patient, known for PCOS, was treated with combined oral contraceptives and loop diuretics. She had no history of hypertension, but she repeatedly displayed very high aldosterone values. Clinical examination was unremarkable, in-office and home blood pressure values were normal. Laboratory data showed normal renal function and the absence of acid-base disorders; serum potassium was at lower limit of normal. A CT-scan showed normal adrenal glands. We performed a recumbent saline suppression test, previous a wash-out period of oral contraceptives (&gt; 6 weeks) and loop diuretics (&gt; 2 weeks). The results listed below are consistent with a baseline hyperaldosteronism with normal aldosterone adrenal response to volume expansion, excluding PA. Several years before, the same results were presented, without suspension of the oral contraceptives. Conclusions: In patients with PCOS the increased adrenal production of aldosterone is usually secondary and independent from the renin-aldosterone axis (direct stimulation of adrenal zona glomerulosa via testosterone). Other iatrogenic factors may affect the renin-aldosterone values, such us the use of oral contraceptives and diuretics. Therefore, a confirmatory test performed under standardized conditions is often required, in order to exclude primary versus secondary forms of hyperaldosteronism.

Retrospective application of a validated algorithm for estimation of adrenal gland volume after computed tomography on 46 dogs undergoing adrenalectomy.

Canine adrenal gland volume can be predicted based on body weight and computed tomography (CT) measurements using a validated algorithm. Use of this algorithm to detect adrenal pathology, including hyperplasia, hypoplasia and neoplasia, in clinical cases has not been described. The objective of this study was to illustrate application of the algorithm by estimating subject-specific adrenal gland volume in a historical cohort of dogs with known adrenal disease. Forty-six dogs that underwent CT and subsequent adrenalectomy were included. Clinical records and CT images from dogs that underwent adrenalectomy and histologic examination of the excised adrenal gland(s) were reviewed. Normal adrenal gland volumes for each dog were estimated using the algorithm, and compared with measured volumes of the affected glands. Linear measurement of the largest lesion diameter was also recorded. Fifty-eight adrenal glands were removed from 46 dogs, with pathology confirmed in all glands. Pathology included 28 adenomas, 13 carcinomas, 11 pheochromocytomas and 6 other benign pathologies. The volume of all removed adrenal glands was measured to be larger than the expected normal volume estimated by the algorithm, ranging from 1.1 to 212.9 times larger than estimated. Adrenal glands with malignant and benign pathology showed variable volumes with overlapping ranges recorded. Assessment of the dimensions of any focal lesion against a cut-off of 20 mm failed to discriminate malignancy. This study illustrates and supports the application of a validated volumetric algorithm for estimation of subject-specific adrenal gland volume to identify the presence of pathology and as a tool to assist clinical decision-making.

Abstract 1896: Therapeutic potential of TORL-4-500, an antibody-drug conjugate directed against delta like non-canonical notch ligand 1 (DLK1)

Abstract Delta like non-canonical Notch ligand 1 (DLK1) is a transmembrane protein that belongs to the NOTCH non-canonical ligand family. It has been implicated in adipogenesis, the regulation of stem cell pools, tissue differentiation during development, cancer differentiation, and cancer stem-like cell maintenance. DLK1 is highly expressed in adrenocortical, uterine, and testicular cancers as well as in a large portion of pancreatic, sarcoma, liver and squamous lung cancer patient samples. In contrast there is limited normal tissue expression beyond the normal adrenal gland, pituitary, and ovarian tissue samples. This expression profile makes DLK1 an attractive target for development of a therapeutic antibody-drug conjugate (ADC). This study describes the generation and preclinical characterization of TORL-4-500, an ADC consisting of a humanized anti-DLK1 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker. TORL-4-500 showed strong binding to DLK1 by flow cytometry in DLK1 native and artificial overexpressing cell lines. In contrast no binding was observed in DLK1 non-expressing cell lines. TORL-4-500 exhibits nanomolar binding affinity for both human and cynomolgus monkey DLK1 and is rapidly internalized in DLK1 expressing cells. TORL-4-500 exhibited selective efficacy in cell line xenograft models of DLK1 positive human cancers. Treatment with TORL-4-500 induced significant regressions in four DLK1 expressing human cancer cell line xenograft studies encompassing liver, small cell lung cancer (SCLC) and sarcoma cancer. Furthermore, anti-tumor responses were sustained in each of the DLK1 expressing models for several weeks post-final dose. In the case of the two SCLC cell lines, complete regressions of xenograft tumors were measured out past 100 days in the TORL-4-500 treated animals. No significant impact on xenograft tumor growth was observed in either a DLK1 non-expressing colon cell line xenograft or in a DLK1 non-expressing melanoma xenograft study. Each of the doses tested in this study was well tolerated in mice with no dose-limiting toxicity observed. The nonclinical pharmacokinetics and toxicokinetics of TORL-4-500 were characterized in mice and monkeys and results support dosing in humans. In summary, TORL-4-500 is a novel therapeutic for DLK1 positive cancers and on the basis of these promising preclinical efficacy results, a first in human trial to evaluate safety, tolerability, pharmacokinetics, and antitumor activity of TORL-4-500 has been launched in patients with advanced cancer and is currently ongoing (NCT06005740). Citation Format: Martina S. McDermott, Neil A. O'Brien, Ming Lu, Jun Zhang, KeWei Gong, Benjamin Hoffstrom, Tong Luo, Min Liang, Weiping Jia, Kevin Chau, Leonard Presta, John Glaspy, Dennis J. Slamon. Therapeutic potential of TORL-4-500, an antibody-drug conjugate directed against delta like non-canonical notch ligand 1 (DLK1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1896.

Clinical Value of 68Ga-Pentixafor PET/CT in Subtype Diagnosis of Primary Aldosteronism Patients with Adrenal Micronodules.

Our objective was to investigate the clinical value of 68Ga-pentixafor PET/CT in subtype diagnosis of primary aldosteronism (PA) patients with adrenal micronodules less than 1 cm in diameter and compare it with the routine clinical methods. Methods: We used prospective enrollment of PA patients with adrenal micronodules identified by adrenal CT scans to undergo 68Ga-pentixafor PET/CT. Patients were divided into surgically eligible and ineligible groups based on surgical pathology and postoperative follow-up or adrenal venous sampling (AVS) results. Patient management was discussed by a multidisciplinary team. The semiquantitative parameters of PET/CT included SUVmax for adrenal lesion and SUV ratios for lesion to liver and lesion to normal adrenal gland. Results: In total, 123 PA patients with adrenal micronodules were examined using 68Ga-pentixafor PET/CT, and 104 patients who underwent surgery or successful AVS were included in the analysis (48 ± 10 y old). The sensitivity, specificity, and accuracy of visual analysis using 68Ga-pentixafor PET/CT to identify surgically eligible patients were 90.2%, 72.7%, and 86.5%, respectively, which were significantly higher than those of adrenal CT (73.1%, 53.8%, and 68.3%, respectively) and yielded consistent results in different CT morphologic or age subgroups. In 36 patients who had both AVS and 68Ga-pentixafor PET/CT, the tests showed a 66.7% concordance rate. However, PET/CT was significantly more concordant with surgical outcomes than was AVS in 17 patients who underwent adrenalectomy (82.4% vs. 68.86%). Among the 183 adrenal micronodules included in the study, the semiquantitative diagnostic thresholds for 92 lesions eligible for surgical treatment were an SUVmax of at least 4.55, an SUV ratio of at least 2.17 for lesion to liver, and an SUV ratio of at least 1.90 for lesion to normal adrenal gland. All patients benefited from surgical removal of 68Ga-pentixafor-avid microlesions. Conclusion: In PA patients with adrenal micronodules, 68Ga-pentixafor PET/CT demonstrated promising diagnostic accuracy in classification and appeared to perform better than adrenal CT. Furthermore, there was also a suggestion of some potential in predicting postoperative efficacy compared with AVS, although these observations require further investigation and verification in larger cohorts.

The Carney Triad

Abstract Introduction/Objective Carney triad is a multi-tumor syndrome comprising of gastrointestinal stromal tumor (GIST), extra-adrenal paraganglioma and pulmonary chondroma that was first described in 1977. The purpose of this report of this to remind the pathologists about this triad and about the fact that pathology is far beyond just giving a diagnosis on a surgical specimen. Methods/Case Report A 41-year-old female presented with dizziness, nausea, vomiting, weakness, early satiety, and palpitations. She was found to be pregnant, and an ultrasound showed an intrauterine pregnancy along with a large abdominal mass. A subsequent computed tomography (CT) scan demonstrated a left adrenal heterogeneous mass. CT chest was also performed which demonstrated a 0.3 cm right middle lobe pulmonary nodule, compatible with a benign tumor. Lab work showed cortisol, dehydroepiandrosterone sulfate (DHEAS), and adrenocorticotropic hormone (ACTH) to be within normal limits, but both metanephrine and normetanephrine were elevated. The patient was treated with an alpha and beta blockade prior to undergoing an adrenalectomy. During adrenalectomy an incidental mass was found at the greater curvature of the stomach. On gross examination, the adrenal mass was a large, encapsulated, brown-black, multi-lobulated, weighing 1,427 grams and measuring 19.8 x 13.7 x 11.6 cm. Microscopically, the mass was consistent with an extra-adrenal paraganglioma, compressing the adjacent normal adrenal gland and not invading or arising from it. The stomach mass was grossly tan-yellow, multi-lobulated, and firm. The cut sections were variegated tan, white. Microscopically, the mass was consistent with a GIST. Results (if a Case Study enter NA) NA Conclusion Individual diagnoses were rendered for the adrenalectomy and gastric mass specimens. Additionally, possibility of carney’s triad was suggested based on patient’s chest imaging findings.

OR02-05 Differences In Genomic Characteristics Of Small Aldosterone-producing Lesions Between Normal And Pathologic Adrenals

Abstract Disclosure: J. Lim: None. S. Plaska: None. &amp;. Liu: None. J. Baker: None. A. Blinder: None. W.E. Rainey: None. A.M. Udager: None. Introduction: Primary aldosteronism (PA) – the most common form of secondary hypertension – is characterized by inappropriate adrenal aldosterone production under suppressed renin conditions. The majority of PA is caused by unilateral aldosterone-producing adenomas (APA) and bilateral hyperaldosteronism (BHA). Emerging evidence suggests that BHA is associated with the accumulation of small aldosterone-producing lesions (less than 1 cm in diameter), including aldosterone-producing micronodules (APM) and/or aldosterone-producing nodules (APN). However, previous studies have shown that APM may also be observed in normal adrenals, and thus, the impact of APM and APN on the development and progression of aldosterone overproduction is not well established. In this study, we compare the frequency and distribution of somatic aldosterone-driver mutations in small aldosterone-producing lesions from normal and pathologic adrenals. Methods: Twenty-eight normal adrenals from deceased renal donors (n=24) and patients undergoing radical nephrectomy for renal cell carcinoma (n=4) were compared with 24 pathologic adrenals from patients with PA who underwent adrenalectomy at the University of Michigan. CYP11B2 immunohistochemistry-guided capture was performed to selectively obtain APM and APN DNA from formalin-fixed paraffin embedded tissue, and mutation status for all known aldosterone-driver genes was determined by Ion Torrent-based targeted next-generation sequencing. Results: From normal adrenals, 57 APM from 12 female and 16 male subjects (median age = 49 years) were analyzed, and approximately 46% of these lesions harbored an aldosterone-driver mutation. The most commonly mutated gene in normal samples was CACNA1D (n = 23; 40.4%), but no KCNJ5 mutations were identified, and no significant sex differences were observed. From pathologic adrenals, 56 APM and 7 APN from 17 female and 7 male subjects (median age = 46.5 years) were analyzed, and the frequency of aldosterone-driver mutations (approximately 70%) was significantly higher than normal adrenals (p-value &amp;lt; 0.05). Although CACNA1D mutations were still predominant in pathologic adrenals (n = 33; 52.4%), a number of KCNJ5 mutations were also observed (n = 5; 7.9%) – particularly in APN. Finally, there was a higher rate of aldosterone-driver mutations in small aldosterone-producing lesions in pathologic adrenals from men compared to women (93.3% vs. 62.5%; p-value = 0.051). Conclusions: The frequency and distribution of aldosterone-driver mutations in small aldosterone-producing lesions (APM and APN) in pathologic adrenals are different from those in normal adrenals and may also vary by sex. Further research is needed on how these different APM patterns affect autonomous aldosterone production in patients with PA. Presentation: Thursday, June 15, 2023

FRI167 Implication Of Serotonin In The Physiopathology Of Aldosterone-Producing Adenoma

Abstract Disclosure: C. Duparc: None. M. D'agostino: None. A. Lopez: None. S. Renouf: None. E. Louiset: None. H. Lefebvre: None. In the human adrenal gland, serotonin (5-HT), released by subcapsular mast cells strongly stimulates expression of CYP11B2, encoding aldosterone-synthase and aldosterone production. The effect of 5-HT on adrenocortical cells is mediated by activation of type 4 serotonin receptor (5-HT4R) positively coupled to cAMP/protein kinase A (PKA) signaling pathway and calcium influx. Consistently, administration of cisapride, a 5-HT4R agonist, to healthy volunteers induces a significant increase in plasma aldosterone levels. In aldosterone producing adenomas (APA), the density of mast cells is enhanced in comparison with normal adrenals. Moreover, molecular studies have shown overexpression of HTR4 gene, encoding 5-HT4R, in APA tissues. Administration of cisapride to patients with APA induced more robust plasma aldosterone responses than in healthy voluntaries. These results suggest that 5-HT could play a role in physiopathology of APA. The aim of our study was to examine expression of genes and proteins involved in aldosterone production and actors of the serotonergic pathway in a series of 31 adenomas removed from patients with primary aldosteronism. We have also investigated the in vitro effect of various 5-HT4R ligands on aldosterone production by explants or cultured cells derived from adenoma tissues collected at surgery. Firstly, aldosterone synthase (AS) immunostaining allowed identification of adenoma phenotypes accordingly to the new international histopathology consensus for unilateral Primary aldosteronism. Among the 31 tissues studied, 20 were classified as classical APAs, i.e. presenting with homogenous or heterogenous AS distribution, remaining being considered as 11 non-classical APA. Expression of the gene encoding the 5-HT-synthesizing enzyme tryptophan hydroxylase was similar in APA and normal adrenals. HTR4 overexpression was more robust in classical than non-classical APAs. HTR4 mRNA levels were positively correlated to those encoding CYP11B2. In addition, 5-HT4R immunostaining was widely distributed in most APA tissues, a pattern which was not exclusively noticed in AS-positive adenomas. BIMU-8, a potent 5-HT4R agonist, strongly increased aldosterone production by perifused APA explants. This effect was blocked by administration of GR-113808, a 5-HT4R antagonist. In cultured APA cells, BIMU-8 stimulated aldosterone synthesis in a dose-dependent manner with a higher efficiency than that observed in normal adrenocortical cells. Aldosterone secretion was less strongly stimulated by prucalopride, a partial 5-HT4R agonist used in the treatment of chronic constipation. These data reveal an enhancement of the 5-HT signaling pathway in APAs in comparison with normal adrenals, suggesting that 5-HT and the 5-HT4R may participate in the pathophysiology of primary aldosteronism. Presentation: Friday, June 16, 2023