Pituitary ACTH Secretion Research Articles

7010 The Incidence Of Cushing Syndrome In Wisconsin Is More Common Than Population-Based Studies From Europe

Abstract Disclosure: T. Matoska: None. T.B. Carroll: Consulting Fee; Self; Corcept Therapeutics. Research Investigator; Self; Corcept Therapeutics. T.S. Wang: None. S. Dream: None. N. Zwagerman: None. J.W. Findling: Advisory Board Member; Self; Corcept Therapeutics, Recordati, Diurnal. Introduction: Endogenous neoplastic hypercortisolism [Cushing syndrome (CS)] is considered to be a rare disorder. Population-based studies from Europe estimate the incidence of CS to range from 1.8-3.2 cases/million patient-years, and there is limited data reporting the incidence of CS in the United States. All previous studies have shown that pituitary ACTH secreting tumors [Cushing disease (CD)] are the most common cause of CS. Based on our clinical experience, we speculated that the incidence of CS was more common than reported and that adrenal CS (ACS) was more common than CD. This study reports the incidence and etiology of CS from a single tertiary care center in Wisconsin (WI). Methods: Institutional clinic records between May 1, 2017, and December 31, 2022, were queried to identify WI residents treated for CS. Only patients diagnosed and treated at our institution during the study period were included. The diagnosis of CS was established with standard guideline-supported biochemical testing and appropriate imaging. Patients diagnosed with exogenous CS and those who did not undergo therapy for CS were excluded. Results: The query identified 185 patients who were diagnosed and treated for CS. These patients resided in 27 of the 72 WI counties representing a population of 4.5 million of the total WI population of 5.9 million. This suggests that, at a minimum, the incidence of CS in WI is 7.2 cases/million patient-years. Moreover, data from the WI Hospital Association show our institution treated <50% of all patients with CS discharged from WI hospitals from 2019-2023. Mean age at diagnosis was 52.4 years (SD 14.7) and 135 (73%) were female. Of the total cohort, 111 (60%) had ACS, 68 (36.8%) had CD, and 6 (3.2%) had ectopic ACTH syndrome (EAS). Patients with ACS had significantly lower LNSC, DST cortisol, and UFC when compared to those with CD and EAS. ACS had mean (SD) LNSC, DST cortisol, and UFC of 0.236 µg/dL (0.367), 6.5 µg/dL (8.7), and 64.2 µg/24h (92.8), respectively. CD had mean (SD) LNSC, DST cortisol, and UFC of 0.425 µg/dL (0.403), 11.7 µg/dL (7.5), and 151.1 µg/24h (287.4), respectively. EAS had mean (SD) LNSC, DST cortisol, and UFC of 3.189 µg/dL (0.931), 42.5 µg/dL (40.5), and 1514.2 µg/24h (1278.9), respectively. Overt physical features of CS were documented in the medical record of 117 (63.2%) patients at diagnosis ((ACS: 49 (44%); CD: 62 (91%); EAS: 6 (100%)). Conclusion: This study suggests that ACS is more common than CD as a cause of CS, and patients with ACS more frequently presented without physical features of hypercortisolism, probably due to the milder degree of cortisol excess. The incidence of CS in WI is at least 7.2 cases/million patient-years. As our institution cares for <50% of patients with CS in WI, the true incidence is likely far greater and may approach 14-15 cases/million patient-years. Presentation: 6/1/2024

Cushing syndrome in paediatric population: who and how to screen.

Cushing's syndrome (CS) is characterised by signs and symptoms resulting from excessive and prolonged exposure to exogenous glucocorticoids or endogenous hypercortisolism. In childhood, exogenous CS represents the main cause of CS due to the widespread therapeutic use of glucocorticoids, while endogenous CS is very rare and accounts for about 10% of CS cases. According to the origin of the hypercortisolism, the ACTH-dependent form due to pituitary ACTH-secreting tumours is the most common form of endogenous CS in paediatric age (about 75-80% of cases), following by adrenal causes (about 15-20% of cases) including adenoma, carcinoma (which has a peak of incidence in the first decade), bilateral adrenal hyperplasia or Carney complex, with a different distribution by age. Ectopic ACTH-secreting CS, genetic forms of pituitary adenomas are more uncommon. The insidious onset of hypercortisolism and the absence of salient early signs make the diagnosis of endogenous CS difficult. Facial changes, weight gain with simultaneous growth failure, prepubertal virilisation, or hypogonadism in adolescence represent some of the key features of CS. The diagnostic workup is essentially aimed at confirming hypercortisolism through screening tests whose diagnostic accuracy is not 100% and therefore the combination of more than two tests is mandatory to confirm the diagnosis of CS.

Global Adrenal Insufficiency in Two Independent Patients Carrying the Same Homozygous c.172A>G, p.(Thr58Ala) Mutation in the TBX19 Gene.

TBX19 mutations cause isolated ACTH-deficiency. While this classically results in severe hypocortisolism, potential consequences for mineralocorticoid biosynthesis have not been described to date. Liquid chromatography mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) allow novel insights into the steroid metabolism of pediatric endocrine diseases. Patient 1 (female) presented right after birth with hypoglycemia and hyponatremia (minimum sodium 126 mmol/L). She recovered under therapy with hydrocortisone, fludrocortisone and initial NaCl. Patient 2 (male) presented after birth with prolonged cholestatic jaundice. Only at the age of 3.5 months, repeated episodes of hypoglycemia occurred. Both patients showed severely reduced ACTH. LC-MS/MS analyses on plasma samples demonstrated combined reduced glucocorticoid- and mineralocorticoid biosynthesis confirmed by GC-MS analyses on spot urine. In contrast to patient 1, patient 2 (currently 8 years old) never suffered from hyponatremia. Both patients carry the same homozygous c.172A>G, p.(Thr58Ala) mutation in the TBX19 gene proving isolated ACTH-deficiency. Isolated ACTH-deficiency can be associated with reduced mineralocorticoids and hyponatremia. We hypothesize that sufficient pituitary ACTH secretion is an important predisposition for regular adrenal mineralocorticoid biosynthesis.

Current and Emerging Pharmacological Therapies for Cushing's Disease.

Cushing's Disease (CD), hypercortisolism due to pituitary ACTH secreting neuroendocrine neoplasm, is associated with increased morbidity and, if untreated, mortality in about half of the affected individuals. Consequently, the timely initiation of effective treatment is mandatory. Neurosurgery is the first line and the only potentially curative treatment; however, 30% of patients will have persistent disease post-surgery. Furthermore, a small percentage of those initially controlled will develop hypercortisolism during long-term follow- up. Therefore, patients with persistent or recurrent disease, as well as those considered non-eligible for surgery, will need a second-line therapeutic approach, i.e., pharmacotherapy. Radiation therapy is reserved as a third-line therapeutic option due to its slower onset of action and its unfavorable profile regarding complications. During the past few years, the understanding of molecular mechanisms implicated in the physiology of the hypothalamus-pituitary-adrenal axis has evolved, and new therapeutic targets for CD have emerged. In the present review, currently available treatments, compounds currently tested in ongoing clinical trials, and interesting, potentially new targets emerging from unraveling molecular mechanisms involved in the pathophysiology of Cushing's disease are discussed.

SAT275 Generation And Characterization Of A Humanized CYP21A2 Mouse Model For Congenital Adrenal Hyperplasia

Abstract Disclosure: A. Huebner: None. S. Thirumalasetty: None. T. Schubert: None. R. Naumann: None. I. Reichardt: None. M. Rohm: None. D. Landgraf: None. F. Gembardt: None. M.F. Hartmann: None. S.A. Wudy: None. M. Peitzsch: None. N. Reisch: None. K. Koehler, PhD: None. 21-hydroxylase deficiency (21OHD) is the most common form of congenital adrenal hyperplasia (CAH) and is caused by mutations in the CYP21A2 gene. 21OHD causes a wide array of clinical symptoms that result from gluco- and mineralocorticoid deficiency and adrenal androgen excess. Treatment firstly aims to substitute lacking steroid hormones, and secondly to restore negative feedback towards CRH and pituitary ACTH secretion to diminish adrenal androgen overproduction. In most cases supra-physiological glucocorticoid doses are necessary which may cause short stature, obesity, hypertension, and cardiovascular and metabolic co-morbidity with reduced quality of life. Hence, current steroid substitution regimens have significant limitations, so novel therapeutic strategies are required. In recent years new therapeutic approaches have emerged including new non-glucocorticoid substances interfering with the HPA axis to minimize adrenal androgen production and to lower external glucocorticoid substitution to physiological levels. However, valuable in-vivo models for pre-clinical testing of such drugs are lacking. Here we present the first viable and humanized mouse model in which the mouse gene Cyp21a1 is replaced by the human orthologue CYP21A2 in which the human point mutation R484Q is integrated. Twenty-weeks-old homozygous mice show marked adrenal hyperplasia, enhanced expression of the CYP21A2 gene and a weak increase of Cyp11a1 and Cyp11b2 gene expression. Tandem mass spectrometry measurements of the mice plasma at 20 weeks show decreased corticosterone and 11-deoxycorticosterone levels in the presence of increased ACTH levels in both male and female homozygous animals. Progesterone levels in homozygous mice are significantly higher (p<0.01) than in wildtype mice. We also observed increased aldosterone levels in female mutants whereas blood pressure does not differ between wildtype and mutant mice strains. Histologically, mutants exhibit adrenocortical hyperplasia. While mutant male mice are fertile with normal appearing testes, females are infertile, remain in the diestrus phase and present with a reduced number of ovarian follicles. In parallel, a second mouse strain bearing the I173N mutation was developed. This mutation is frequent in human patients causing simple virilizing or rarely salt wasting CAH. Homozygous mice require dexamethasone treatment during pregnancy and until weaning but are viable without treatment afterwards. Preliminary results show adrenal hyperplasia and alteration in steroidogenic gene expression and steroid profiles. In conclusion, we demonstrate that the humanized mutant CYP21A2 mice may represent an excellent animal CAH model to test novel treatment strategies for CAH patients. We believe that this model(s) will facilitate the transition from basic research into clinical application. Presentation: Saturday, June 17, 2023

Age-related and individual features of the HPA axis stress responsiveness under constant light in nonhuman primates.

The hypothalamic-pituitary-adrenal (HPA) axis is a key adaptive neuroendocrine system, dysfunction of which plays an important role in the increasing incidence of stress-dependent age-related pathology. Among the environmental factors effecting increase age-related diseases, great importance is given to disturbances of the light-dark schedule, particularly with increased illumination at night. While disruption of the light-dark schedule has long been recognized as a powerful behavioral stressor, little is known regarding stress reactivity of the HPA under constant light (CL) conditions, especially with aging and depending on the features of stress behavior. The purpose of this investigation was to study the age-related and individual features of the HPA axis response to acute stress exposure (ASE) under chronic CL in nonhuman primates that are known to differ in behavioral responsiveness to stress. Young and old female rhesus monkeys (with control standard behavior or anxiety and depression-like behavior) were exposed to CL (24h light/day, 330-400 lux for 4 to 8 weeks). Control young and old monkeys were exposed to standard lighting (SL) with natural light during the day and darkness at night. All animals were subjected to ASE (restriction of mobility for 2 hours), functional tests with corticotrophin-releasing hormone and arginine-vasopressin, and study of circadian rhythms of cortisol and pineal melatonin secretion. For the first time an inhibitory effect of CL on the reaction of the adrenal cortex to ASE was revealed in all individuals, regardless of age and preexisting behavior stress reactivity, the mechanisms of which were age-dependent: due to inhibition of the pituitary ACTH secretion in young animals and mainly not affecting the ACTH secretion in old individuals. There were no significant changes in melatonin secretion both in young and old animals. The observed CL inhibition of adrenal cortical reactivity to ASE may be useful to correct increased vulnerability to ASE observed in individuals with preexisting anxiety and depression-like stress behaviors. On the other hand, the CL induced decrease in adrenal stress reactivity of behaviorally normal animals suggests a potential risk of reducing the adaptive capacity of the organism under conditions of continuous light exposure.

RF01 | PMON168 Hedgehog-Induced Somatostatin Receptor Expression is Inhibited by the Activation of the Glucocorticoid Receptor Signaling Pathway in Human Pituitary Adenoma Organoids

Abstract Background Cushing's disease (CD) is an endocrine disorder caused by an ACTH-secreting pituitary adenoma that is associated with increased morbidity and mortality. Because somatostatin inhibits pituitary ACTH secretion, the somatostatin receptor subtypes (SSTR) 2 and 5 have been targets of medical therapies for CD. The SSTR5 is most consistently and strongly expressed in corticotroph pituitary tumors but the SSTR dynamics in CD during treatment and responses to cortisol level changes are far from being understood. Studies in adult stomach demonstrated that the Hedgehog (Hh) signaling pathway is critical for the regulation of somatostatin and SSTR signaling. While Hh signaling is known to be essential during the embryonic development of the pituitary and in the adult gland, the mechanism by which Hh signaling regulates SSTR expression in CD is unknown. Hypothesis Activation of glucocorticoid receptor (NR3C1, GR) results in the inhibition of Hh transcription factor GLI1 leading to reduced SSTR expression. Methods We developed a human pituitary adenoma organoid model (hPITOiPSC) from induced pluripotent stem cells treated with the glucocorticoid receptor (GR) antagonist mifepristone, and co-treated them with or without SSTR agonists pasireotide or octreotide. In a separate series of experiments, the role of Hh transcription factor GLI1 was identified using hPITOiPSC treated with mifepristone with or without GANT61 (GLI inhibitor) or ketoconazole (Smoothened, SMO inhibitor). CRISPR-Cas9 gene editing of hPITOiPSC pituitary organoids were used to model the development of pituitary corticotroph adenomas in the presence of BRAF, USP48 and USP8 mutations. Human pituitary adenoma tissue harvested fresh during pituitary surgery was used to generate pituitary corticotroph subtype adenoma organoids (hPITOs). Dose responses using standard of care drugs were performed using the hPITOs. Results 1) At baseline SSTR2 was abundantly expressed within hPITOiPSC. Mifepristone induced significant increases in ACTH secretion and POMC expression that correlated with induced SSTR2 and 5. Mifepristone-induced SSTR2 and 5 expression was significantly inhibited in the presence of GANT61, whereas SMO inhibitor ketoconazole had a minimal effect on mifepristone-induced SSTR2/5, POMC expression and ACTH secretion. Dexamethasone alone significantly inhibited both GLI1 and SSTR2 and 5. 2) While pituitary organoids that were differentiated from control iPSCs (iPSCCtrl) expressed all major hormone-producing cell lineages, there was a significant increase in ACTH expression with loss of PIT1, GH, FSH, LH and PRL in iPCSs expressing mutated BRAF, USP48 and USP8. Organoids expressing a mutation in BRAF had significantly higher SSTR2 expression levels compared to controls. 3) HPITOs generated from pituitary adenomas of CD patients showed differential organoid responses to pasireotide, mifepristone, and ketoconazole. Conclusion Within pituitary adenomas, the SSTR is a transcriptional target of Gli1, and this response is blocked by the activation of the GR. These data form the basis of combination therapy for CD with mifepristone and pasireotide. Presentation: Saturday, June 11, 2022 1:37 p.m. - 1:42 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

PMON102 Utility of the Desmopressin Stimulation Test to Detect Cyclic Cushing's Disease

Abstract Background Arginine-vasopressin stimulates pituitary ACTH secretion after binding to arginine-vasopressin receptor 1B on corticotropes. Desmopressin (DDAVP), which preferentially binds to arginine-vasopressin receptor 2, stimulates the release of ACTH in most patients with pituitary corticotrope adenomas (Cushing's disease, CD), but not in most patients with ectopic ACTH-producing tumors or healthy people. Cyclic CD is characterized by periods of normal or low cortisol and ACTH, interspersed with hypercortisolism. We postulated that the DDAVP stimulation test, with measurement of ACTH and cortisol after a 10 mcg injection of DDAVP, might confirm the presence of an ACTH-producing pituitary adenoma even during periods without hypercortisolemia. Clinical Case A 27-year-old woman was diagnosed with Cushing's disease one year after developing weight gain, striae, intermittent acne, balding, irregular menses, a dorsal fat pad and facial rounding. Pituitary MRI revealed a right-sided 7 mm microadenoma; inferior petrosal sinus sampling confirmed a pituitary source of ACTH. Transsphenoidal resection was performed 6 months later. Histology showed an ACTH-positive adenoma and cortisol levels fell to 33nmol/L (1.2 ug/dL). Ten months later, due to intermittent symptoms, testing confirmed elevated UFC, with midnight cortisol of 565 nmol/L (20.3 ug/dL). Pituitary MRI showed a new right-sided lesion, interpreted as a residual adenoma or postoperative changes. Repeat surgical resection was recommended. She presented to our institution for a second opinion. On evaluation three weeks after recurrent hypercortisolism was confirmed, ACTH was low-normal (11.9 pg/mL, RR 5.0-46.0) and serum cortisol was undetectable. This rapid decrease was thought to signify apoplexy of the remaining adenoma or cyclic Cushing's disease. An MRI excluded apoplexy. However, surgery seemed inappropriate given hypocortisolism. To evaluate for the possibility of cyclic Cushing's disease due to residual adenoma, we performed a DDAVP stimulation test. This showed significant increases in ACTH and cortisol levels from 0 min to a 30-minute peak (ACTH 31.6 to 73.1 pg/mL, 131% increase, nl <50%; cortisol 2.6 to 8.1 mcg/dL [72 - 225 nmol/L], 212% increase, nl <20%). This led to transsphenoidal resection of a right-sided lesion. Cortisol levels were undetectable on post-operative day 3 and pathology showed an ACTH-producing adenoma. One week postoperatively, a repeat DDAVP stimulation test showed an insignificant rise in ACTH, and undetectable cortisol throughout (ACTH 11.1 to 12.4 pg/mL, 12% increase; cortisol <1.0 mcg/dL, 0% increase), which was deemed to indicate successful resection of the residual adenoma. She continues to lose weight and her clinical features of cortisol excess are involuting. Conclusion This case demonstrates how the DDAVP stimulation test can be used to both confirm the presence of ACTH-producing pituitary adenomas and assess the response to surgical resection. It may be particularly useful in the setting of cyclic Cushing's disease when cortisol and ACTH levels are not persistently elevated. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Celastrol Induces Apoptosis and Autophagy via the AKT/mTOR Signaling Pathway in the Pituitary ACTH-secreting Adenoma Cells.

Pituitary adrenocorticotropic hormone (ACTH)-secreting adenoma is a relatively intractable endocrine adenoma that can cause a range of severe metabolic disorders and pathological changes involving multiple systems. Previous studies have shown that celastrol has antitumor effects on a variety of tumor cells via the AKT/mTOR signaling. However, whether celastrol has pronounced antitumor effects on pituitary ACTH-secreting adenoma is unclear. This study aimed to identify a new effective therapeutic drug for pituitary ACTH-secreting adenoma. Mouse pituitary ACTH-secreting adenoma cells (AtT20 cells) were used as an experimental model in vitro and to establish a xenograft tumor model in mice. Cells and animals were administered doses of celastrol at various levels. The effects of celastrol on cell viability, migration, apoptosis and autophagy were then examined. Finally, the potential involvement of AKT/mTOR signaling in celastrol's mechanism was assessed. Celastrol inhibited the proliferation and migration of pituitary adenoma cells in a time- and concentration-dependent manner. It blocked AtT20 cells in the G0/G1 phase, and induced apoptosis and autophagy by downregulating the AKT/mTOR signaling pathway. Similar results were obtained in mice. Celastrol exerts potent antitumor effects on ACTH-secreting adenoma by downregulating the AKT/mTOR signaling in vitro and in vivo.

Dual effects of 9-cis retinoic acid on ACTH-dependent hyperplastic adrenal tissues

Retinoids play a pivotal role in adrenal development and differentiation. Recent clinical trials revealed therapeutic potential of both all-trans and 9-cis retinoic acid in patients with cortisol excess due to a pituitary ACTH-secreting adenoma and indicated that retinoids might act also on the adrenal. Aim of the present study was to evaluate the effect of 9-cis retinoic acid on adrenals from patients with ACTH-dependent Cushing’s syndrome. Adrenal specimens from six patients with Cushing’s disease were incubated with 10 nM–1 µM 9-cis retinoic acid with and without 10 nM ACTH. Cortisol secretion was measured by immunoassay and expression of genes involved in steroidogenesis as well as retinoic acid action were evaluated by real-time RT-PCR. Incubation with 10–100 nM 9-cis retinoic acid increased spontaneous cortisol secretion and expression of STAR and CYP17A. On the other hand, in wells treated with ACTH, 9-cis retinoic acid markedly diminished ACTH receptor upregulation and no stimulatory effect on cortisol secretion or steroidogenic enzyme synthesis was observed. ACTH itself increased ligand-induced retinoic acid receptor expression, possibly enhancing sensitivity to retinoic acid. Our findings indicate that the effect of 9-cis retinoic acid in presence of ACTH is distinct from unchallenged wells and support the hypothesis of a direct adrenal action in patients with Cushing’s disease.

Two Distinctive POMC Promoters Modify Gene Expression in Cushing Disease.

ContextMechanisms underlying pituitary corticotroph adenoma adrenocorticotropin (ACTH) production are poorly understood, yet circulating ACTH levels closely correlate with adenoma phenotype and clinical outcomes.ObjectiveWe characterized the 5′ ends of proopiomelanocortin (POMC) gene transcripts, which encode the precursor polypeptide for ACTH, in order to investigate additional regulatory mechanisms of POMC gene transcription and ACTH production.MethodsWe examined 11 normal human pituitary tissues, 32 ACTH-secreting tumors, as well as 6 silent corticotroph adenomas (SCAs) that immunostain for but do not secrete ACTH.ResultsWe identified a novel regulatory region located near the intron 2/exon 3 junction in the human POMC gene, which functions as a second promoter and an enhancer. In vitro experiments demonstrated that CREB binds the second promoter and regulates its transcriptional activity. The second promoter is highly methylated in SCAs, partially demethylated in normal pituitary tissue, and highly demethylated in pituitary and ectopic ACTH-secreting tumors. In contrast, the first promoter is demethylated in all POMC-expressing cells and is highly demethylated only in pituitary ACTH-secreting tumors harboring the ubiquitin-specific protease 8 (USP8) mutation. Demethylation patterns of the second promoter correlate with clinical phenotypes of Cushing disease.ConclusionWe identified a second POMC promoter regulated by methylation status in ACTH-secreting pituitary tumors. Our findings open new avenues for elucidating subcellular regulation of the hypothalamic-pituitary-adrenal axis and suggest the second POMC promoter may be a target for therapeutic intervention to suppress excess ACTH production.

Acute elevation of interleukin 6 and matrix metalloproteinase 9 during the onset of pituitary apoplexy in Cushing's disease.

Pituitary apoplexy is a rare endocrine emergency. The purpose of this study is to characterize physiological changes involved in pituitary apoplexy, especially during the acute phase. A Cushing's disease patient experienced corticotroph releasing hormone (CRH)-induced pituitary apoplexy during inferior petrosal sinus sampling (IPSS). The IPSS blood samples from the Cushing's disease patient were retrospectively analyzed for cytokine markers. For comparison, we also analyzed cytokine markers in blood samples from two pituitary ACTH-secreting microadenoma patients and one patient with an ectopic ACTH-secreting tumor. Acute elevation of interleukin 6 (IL-6) and matrix metalloproteinase 9 (MMP9) was observed in the IPSS blood sample on the apoplectic hemorrhagic site of the tumor. In contrast, such a change was not observed in the blood samples from the contralateral side of the apoplexy patient and in other IPSS samples from two non-apoplexy Cushing's disease patient and a patient with ectopic Cushing's syndrome. IL-6 and MMP9 may be involved in the acute process of pituitary apoplexy in Cushing's disease.

D-Amino acids in mammalian endocrine tissues.

D-Aspartate, D-serine and D-alanine are a regular occurrence in mammalian endocrine tissues, though in amounts varying with the type of gland. The pituitary gland, pineal gland, thyroid, adrenal glands and testis contain relatively large amounts of D-aspartate in all species examined. D-alanine is relatively abundant in the pituitary gland and pancreas. High levels of D-serine characterize the hypothalamus. D-leucine, D-proline and D-glutamate are generally low. The current knowledge of physiological roles of D-amino acids in endocrine tissues is far from exhaustive, yet the topic is attracting increasing interest because of its potential in pharmacological application. D-aspartate is known to act at all levels of the hypothalamus-pituitary-testis axis, playing a key role in reproductive biology in several vertebrate classes. An involvement of D-amino acids in the endocrine function of the pancreas is emerging. D-Aspartate has been immunolocalized in insulin-containing secretory granules in INS-1 E clonal β cells and is co-secreted with insulin by exocytosis. Specific immunolocalization of D-alanine in pituitary ACTH-secreting cells and pancreatic β-cells suggests that this amino acid participates in blood glucose regulation in mammals. By modulating insulin secretion, D-serine probably participates in the control of systemic glucose metabolism by modulating insulin secretion. We anticipate that future investigation will significantly increase the functional repertoire of D-amino acids in homeostatic control.

Surgical Indications and Techniques for Adrenalectomy.

Indications for adrenalectomy are malignancy suspicion or malignant tumors, non-functional tumors with the risk of malignancy and functional adrenal tumors. Regardless of the size of functional tumors, they have surgical indications. The hormone-secreting adrenal tumors in which adrenalectomy is indicated are as follows: Cushing’s syndrome, arises from hypersecretion of glucocorticoids produced in fasciculata adrenal cortex, Conn’s syndrome, arises from an hypersecretion of aldosterone produced by glomerulosa adrenal cortex, and Pheochromocytomas that arise from adrenal medulla and produce catecholamines. Sometimes, bilateral adrenalectomy may be required in Cushing’s disease due to pituitary or ectopic ACTH secretion. Adenomas arise from the reticularis layer of the adrenal cortex, which rarely releases too much adrenal androgen and estrogen, may also develop and have an indication for adrenalectomy. Adrenal surgery can be performed by laparoscopic or open technique. Today, laparoscopic adrenalectomy is the gold standard treatment in selected patients. Laparoscopic adrenalectomy can be performed transperitoneally or retroperitoneoscopically. Both approaches have their advantages and disadvantages. In the selection of the surgery type, the experience and habits of the surgeon are also important, along with the patient’s characteristics. The most common type of surgery performed in the world is laparoscopic transabdominal lateral adrenalectomy, which most surgeons are more familiar with. The laparoscopic anterior transperitoneal approach is the least preferred laparoscopic method in adrenalectomy. Retroperitoneal laparoscopic adrenalectomy can be performed with a posterior or lateral approach. In addition to conventional laparoscopy, laparoscopic surgery is robot-assisted, which can be administered by transperitoneal or retroperitoneal approach. In addition, conventional or robot-assisted laparoscopic adrenalectomy can be performed transabdominally or retroperitoneally using the single-port method. Today, partial adrenalectomy can be performed using laparoscopic techniques in bilateral adrenal masses, hereditary diseases with the risk of developing multiple adrenal tumors, and solitary masses of the adrenal gland. Open surgery is indicated in the case of malignancy or suspected malignancy and large tumors when laparoscopic surgery is contraindicated. The risk of conversion to open surgery is low (approximately 5%). The open transperitoneal anterior approach is the most common open intervention, especially in large tumors with malignancy or suspected malignancy. This procedure can be performed using a midline incision, bilateral or unilateral subcostal incision, Makuuchi or modified Makuuchi incision. Thoracoabdominal incision may be required, especially in the removal of large malignant lesions as a block. The open retroperitoneal approach can be applied posteriorly or laterally.

FDG-PET/CT in the detection of pituitary stalk ACTH-secreting adenoma.

FDG-PET/CT in the detection of pituitary stalk ACTH-secreting adenoma.

Identification of a Novel Functional Corticotropin-Releasing Hormone (CRH2) in Chickens and Its Roles in Stimulating Pituitary TSHβ Expression and ACTH Secretion.

Corticotropin-releasing hormone (CRH), together with its structurally and functionally related neuropeptides, constitute the CRH family and play critical roles in multiple physiological processes. Recently, a novel member of this family, namely CRH2, was identified in vertebrates, however, its functionality and physiological roles remain an open question. In this study, using chicken (c-) as the animal model, we characterized the expression and functionality of CRH2 and investigated its roles in anterior pituitary. Our results showed that (1) cCRH2 cDNA is predicted to encode a 40-aa mature peptide, which shares a higher amino acid sequence identity to cCRH (63%) than to other CRH family peptides (23–38%); (2) Using pGL3-CRE-luciferase reporter system, we demonstrated that cCRH2 is ~15 fold more potent in activating cCRH receptor 2 (CRHR2) than cCRHR1 when expressed in CHO cells, indicating that cCRH2 is bioactive and its action is mainly mediated by CRHR2; (3) Quantitative real-time PCR revealed that cCRH2 is widely expressed in chicken tissues including the hypothalamus and anterior pituitary, and its transcription is likely controlled by promoters near exon 1, which display strong promoter activity in cultured DF-1 and HEK293 cells; (4) In cultured chick pituitary cells, cCRH2 potently stimulates TSHβ expression and shows a lower potency in inducing ACTH secretion, indicating that pituitary/hypothalamic CRH2 can regulate pituitary functions. Collectively, our data provides the first piece of evidence to suggest that CRH2 play roles similar, but non-identical, to those of CRH, such as its differential actions on pituitary, and this helps to elucidate the roles of CRH2 in vertebrates.

Surgical treatment for Cushing's disease with negative results in high dose dexamethasone suppression tests

Objective: To analyze the indication and the outcome of trans-sphenoidal surgery (TSS) in Cushing's disease (CD) with negative high dose dexamethasone suppression tests (HDDST) results. Methods: Eighteen cases of ACTH-dependent Cushing's syndrome (CS) with negative HDDST results in the Department of Neurosurgery in Shanghai Ruijin Hospital from January 2015 to December 2017 were retrospectively reviewed. All patients underwent TSS. There were 5 males and 13 females, with an average age of (41±14) years. Results: All patients underwent bilateral inferior petrosal sinus sampling (BIPSS) before the surgery and got evidence of pituitary origin of ACTH secretion. They were thus indicated for TSS. Immediate post-operative remission was achieved in ratio 17/18. There were no recurrences within a flow-up of 1 to 3 years. Pituitary ACTH secreting adenomas were pathologically confirmed in 15 cases, including the one who did not achieve post-operative remission. Thus, all 18 patients with negative HDDST results can finally be confirmed as CD. Conclusions: HDDST alone is not sufficient to eliminate CD. For patients with ACTH-dependent CS with negative HDDST results, BIPSS should be further performed. The fact of post-operative remission and the pathological confirm of ACTH secreting pituitary adenoma may add final evidence to the diagnosis of CD.

OR17-1 Changes in the Hypothalamic-Pituitary-Adrenal Axis during Pediatric Critical Illness

Critical illness in adults is hallmarked by prolonged hypercortisolism and a rapid decrease in ACTH. The high cortisol is explained by reduced cortisol breakdown and a decrease in cortisol binding proteins, rather than by adrenal production. In critically ill children, the evolution of the changes in the HPA axis and underlying mechanisms remain to be studied. Also, the impact hereon of nutritional management is unknown. In the PEPaNIC RCT, accepting low macronutrient intake up to day 8 in the pediatric intensive care unit (PICU) by withholding early supplemental parenteral nutrition (PN) accelerated recovery as compared with initiating supplemental PN early. In this preplanned secondary analysis of the PEPaNIC RCT, we assessed the changes over time in the HPA axis and their prognostic value during critical illness in children and investigated the impact on these changes of withholding early PN. We quantified plasma ACTH, total cortisol, CBG and albumin and calculated free cortisol upon PICU admission, day 3 and last PICU day for 223 short-stay (<3 days) and 309 long-stay patients (≥3 days) who did not receive corticosteroids before sampling, in comparison with 64 matched healthy children. Upon admission, ACTH was elevated in short-stay patients (P=0.02) and comparable with healthy children in long-stay patients (P=0.75), whereas total and free cortisol were elevated in both groups (P<0.0001). In short-stay patients, ACTH became subnormal on the last day (P<0.0001). Also total and free cortisol decreased, but remained higher than in healthy children (P=0.003-0.005). In long-stay patients, ACTH decreased towards day 3 (P<0.0001) and remained low on the last day (P<0.0001), whereas total and free cortisol were normal on day 3 (P=0.37-0.62) and on the last day (P=0.30-0.74). CBG and albumin were low throughout PICU stay in both groups. The rapid decrease in cortisol over time in PICU likely excludes negative feedback by cortisol on pituitary ACTH secretion as cause of the decrease in ACTH. In multivariable analysis, the decrease in ACTH was also not associated with drugs commonly used in PICU, that have been suggested to affect the HPA axis in adults. High total and free cortisol upon admission were associated with 90-day mortality and prolonged length of stay in univariable analysis. This association disappeared when adjusting for baseline risk factors. Withholding early PN did not affect the changes in the HPA axis from admission to day 3 or to last day for short-stay patients. In conclusion, the time course of the changes in the HPA axis during critical illness in children differs from that in adults, with a rapid normalization of cortisol together with a decrease in ACTH over time. Further investigation of the mechanism underlying this ACTH decrease is warranted, since it might be iatrogenic or related to a possibly more immature central secretion during critical illness in children as compared with adults.

MON-409 Ectopic ACTH Syndrome with a Pituitary Mass

Background: The ectopic ACTH syndrome comprises 10% of Cushing’s syndrome cases. It is associated with an array of both highly malignant and more indolent tumors. Clinical features develop rapidly in ectopic ACTH syndrome. Distinguishing ectopic ACTH production from pituitary ACTH secretion is essential in determining appropriate management and may be challenging in the case of an occult neuroendocrine tumor. Clinical Case: A 46 year-old male presented to the ER after his PCP identified hypokalemia to 1.6 mmol/L (3.5- 5.0 mmol/L). He endorsed two weeks of proximal muscle weakness, lower extremity edema, a retro-orbital headache, and 10lb weight loss. Exam revealed mild facial fullness and pitting edema to the knees. No supraclavicular or dorsal cervical adiposity, violaceous abdominal striae or hyperpigmentation was observed. His blood pressure was elevated at 177/92 and an EKG revealed sinus bradycardia at a rate of 44 bpm. An 8AM cortisol was elevated at 67.4 mcg/dL (2.5-19.5 mcg/dL). ICU admission for blood pressure control and IV potassium supplementation was required. There was concern for Cushing’s disease given an inappropriately elevated ACTH level of 159.3 pg/mL (6-50 pg/mL). However, ectopic ACTH was highly suspected given the rapid development of symptoms and a smoking history of roughly 60 pack yrs. A CT of the chest, abdomen, and pelvis revealed no findings suspicious for malignancy. An MRI brain revealed a 19mm sellar and suprasellar mass atop the pituitary extending superiorly in the supra-diaphragmatic region adjacent to the pituitary stalk with a thickened, enhancing infundibulum. The patient required 60meq TID of oral KCl at discharge. Insulin dependent DMII rapidly developed in the weeks following discharge. An 8mg dexamethasone suppression test was performed with a baseline 8AM cortisol of 88 mcg/dL. Post dexamethasone, 8AM cortisol was 62 mcg/dL. Although these results were not consistent with the 50% reduction in cortisol levels expected by a pituitary adenoma in response to high dose dexamethasone administration, a trans-sphenoidal hypophysectomy was undertaken per patient preference. A pale discohesive tissue was identified intra-operatively which stained for ACTH, consistent with a corticotroph macroadenoma. An Indium-111 pentetreotide scan was negative for evidence of a somatostatin receptor expressing neoplasm. Two months after surgery, early-afternoon cortisol decreased to 7.5 mcg/dL. The patient’s insulin dependence gradually resolved as did his hypokalemia, edema, weakness, and weight loss. Conclusion: We describe an unusual case wherein clinical features of the ectopic ACTH syndrome developed over the course of two weeks in a lifelong smoker. Despite failing an 8mg dexamethasone test, a corticotroph macroadenoma was discovered. There was no identifiable source of ectopic ACTH on an octreotide scan despite a highly suggestible presentation.

MON-407 Tumor Control after Radiotherapy and Temozolomide in a Cushing's Disease Patient with Giant Aggressive Pituitary Tumor

Introduction: Typical ACTH-producing pituitary adenomas are benign tumors, microadenomas, successfully treated by neurosurgery. However, 10-20% of Cushing’s disease (CD) patients have macroadenomas, sometimes invasive and aggressive cases with poorer surgical prognosis needing additional approaches. Temozolomide (TMZ), second generation alkylating agent, has been used alone or in combination to other medications/radiotherapy (RTX) for tumor control of aggressive pituitary adenomas and carcinomas. We report one patient with giant aggressive corticotropinoma that presented control tumor with RTX and TMZ after four pituitary surgeries unable to uncontroll tumor growth. Clinical Case: A 18-year-old female patient presented with weight gain, red moon face, supraclavicular fullness, dorsal hump, large purple striae, acanthosis nigricans, secondary amenorrhea, frontotemporal headache, visual impairment and right palpebral ptosis. Hormonal analysis confirmed ACTH-dependent Cushing's syndrome. Pituitary MRI showed the presence of a large selar and suprasellar expansive lesion of 5.2X5.1X4.0 cm with displacement of third ventricle, hypothalamus and lateral ventricles. The patient was submitted to the first neurosurgery (transsphenoidal route) with partial resection. Pathological analyses confirmed pituitary adenoma positive for ACTH, Ki-67 8%, negative p53 and 7 mitoses/10 field of great increase. Subsequent surgery was done by craniotomy with partial resection. Cabergoline 2 mg/week was started but the tumor keeps growing with worsening of visual complaints and increase of cortisol levels. At admission in our center, we planned new surgeries do remove the maximal tumor possible (debulking) and subsequent RTX and TMZ. She was submitted to third surgery by transsphenoidal route and the last surgical procedure by craniotomy. MRI pre-RTX done three months after the 4th surgery showed remnant tumor of 3.5X3.0X3.0 cm. Fractioned stereotaxic radiotherapy was done with linear accelerator in a total of 54 Gy divided in 30 sessions. TMZ was initiated in a dose of 150 m2/day, cycles of five days/month, using for 14 months. Pituitary MRI 3, 6 and 10 months after TMZ showed a small reduction of tumor with increase of intratumor cystic areas. Conclusion: The combined use of TMZ and radiotherapy is an important approach for the treatment of rapidly growing, aggressive pituitary ACTH-secreting tumors resistant to conventional treatment.