ACTH-secreting Pituitary Tumors Research Articles

A long-term prognosis study of human USP8-mutated ACTH-secreting pituitary neuroendocrine tumours.

Somatic variants in the ubiquitin-specific protease 8 (USP8) gene are the most common genetic cause of Cushing disease. We aimed to explore the relationship between clinical outcomes and USP8 status in a single centre. We investigated the USP8 status in 48 patients with pituitary corticotroph tumours. A median of 62 months of follow-up was conducted after surgery from November 2013 to January 2015. The clinical, biochemical and imaging features were collected and analysed. Seven USP8 variants (p.Ser718Pro, p.Ser719del, p.Pro720Arg, p.Pro720Gln, p.Ser718del, p.Ser718Phe, p.Lys713Arg) were identified in 24 patients (50%). USP8 variants showed a female predominance (100% vs. 75% in wild type [WT], p = .022). Patients with p.Ser719del showed an older age at surgery compared to patients with the p.Pro720Arg variant (47- vs. 24-year-olds, p = .033). Patients with p.Pro720Arg showed a higher rate of macroadenoma compared to patients harbouring the p.Ser718Pro variant (60% vs. 0%, p = .037). No significant differences were observed in serum and urinary cortisol andadrenocorticotropin hormone (ACTH) levels. Immediate surgical remission (79% vs. 75%) and long-term hormone remission (79% vs. 67%) were not significantly different between the two groups. The recurrence rate was 21% (4/19) in patients harbouring USP8 variants and 13% (2/16) in WT patients. Recurrence-free survival presented a tendency to be shorter in USP8-mutated individuals (76.7 vs. 109.2 months, p = .068). Somatic USP8 variants accounted for 50% of the genetic causes in this cohort with a significant female frequency. A long-term follow-up revealed a tendency toward shorter recurrence-free survival in USP8-mutant patients.

Mesenchymal stem cells induce an immunosuppressive microenvironment in pituitary tumors.

The tumor microenvironment (TME) includes diverse cellular components such as mesenchymal stem cells (MSC) and immune cells among others. MSC have been isolated from different tumors and they favor tumor cell growth, however, their role in pituitary tumors (PT) remains unknown. Herein we report the presence of MSCs in 2 ACTH-secreting PT causing Cushing disease (MCU), 2 nonfunctioning adenomas of gonadotrope differentiation (MNF) and 2 non tumoral pituitary glands (MS). We have analyzed their transcriptomic profiles by RNAseq and compared MSC in terms of their immunosuppressive effects against lymphoid T cell and macrophage populations by means of co-cultures and flow cytometry. Our transcriptomic analysis revealed molecular differences between MSC derived from non-tumoral pituitaries and MSC derived from PT. Two distinct subpopulations of MSC, one displaying immunosuppressive properties and the other with increased pro-proliferative capabilities, regardless of their origin. MSC derived from ACTH- and nonfunctioning PT, but not those derived from non-tumoral glands significantly inhibited the proliferation of activated T cells, favored the generation of Tregs and promote M2 macrophage polarization. Such immunosuppressive effects were correlated with an upregulation of programmed death ligand 1 and intracellular expression of macrophage colony stimulating factor (M-CSF) and IL-10. Importantly, MSC derived from ACTH-PT showed a higher immunosuppressive potential than MSC isolated from nonfunctioning tumors. This study demonstrates the presence of at least two MSC subpopulations in the pituitary gland and suggests that immunosuppressive effects of MSC may have important implications in PT growth.

THU520 Successful Treatment Of Ectopic ACTH From Metastatic Gastrinoma With Temozolomide Monotherapy

Abstract Disclosure: A. Syeda: None. A. Khan: None. D. Larosa: None. P. Kunz: None. B. Esayag-Tendler: None. Background: Temozolomide is a promising therapeutic agent for the treatment of refractory ACTH-secreting pituitary tumors. However, its role in the management of ectopic ACTH secretion due to neuro endocrine tumors (NETs) has not been well elucidated. Case: A 46-year-old man with a refractory functional pancreatic NET/ high grade metastatic gastrinoma, post distal pancreatectomy and splenectomy, briefly on everolimus therapy, presented with altered mentation and worsening edema. Clinically he had proximal muscle weakness, anasarca and hypokalemia with a potassium of 2.8mmol/L (n:3.6-5.1 mmol/L). Further work up revealed AM cortisol 79.6 ug/dL (n:7-23 ug/dL), 24-hour urine cortisol was 1820 ug/d (n: < 60 ug/d), ACTH 807 pg/mL (n:7.2-63.3 pg/mL) and gastrin level >10,000 pg/mL (n:0-100 pg/mL). Imaging studies of the brain, chest, abdomen, and pelvis were unremarkable. Octreotide infusion at 100 mg/hour was started with a goal to decrease gastrin and ectopic ACTH production. There was no response to Octreotide even after 5 days of continuous infusion, hence it was switched to subcutaneous injection form to control diarrhea. Lutathera/Peptide receptor radionuclide therapy was considered but held off due to the patient's delirium. As hypercortisolism is associated with multiple risks including thromboembolic events, rapid reduction of cortisol biosynthesis was achieved with Ketoconazole. Other options to inhibit steroidogenesis were available; the goal was to decrease both ACTH and Gastrin levels and for this reason Temozolomide was started. It crosses the blood-brain-barrier and hence was a preferred agent in this patient as he was noted to have atypical cells in the CSF. He received Temozolomide 150 mg/m² for 5 days of a 28-day cycle. Subsequently, AM cortisol decreased to 3.4 ug/dL, ACTH and gastrin started to trend down to 144 pg/mL and 3505 pg/mL respectively. Thereafter, the patient was given steroids and ketoconazole as part of a block and replace strategy to avoid adrenal insufficiency and hypercortisolism respectively. Temozolomide was very well tolerated; there were different reasons to hold Capecitabine including severe thrombocytopenia. Bilateral adrenalectomy was discussed but deferred as the ACTH levels decreased. Clinically, the patient reported improved muscle strength, edema and weight decreased significantly. The hospital stay was complicated by several other comorbidities which were managed appropriately by a multidisciplinary approach. Conclusion: Temozolomide should be considered in ACTH producing NETs refractory to conventional treatment strategies. Presentation: Thursday, June 15, 2023

Protein kinase C delta mediates Pasireotide effects in an ACTH-secreting pituitary tumor cell line

PurposeClinical control of corticotroph tumors is difficult to achieve since they usually persist or relapse after surgery. Pasireotide is approved to treat patients with Cushing’s disease for whom surgical therapy is not an option. However, Pasireotide seems to be effective only in a sub-set of patients, highlighting the importance to find a response marker to this approach. Recent studies demonstrated that the delta isoform of protein kinase C (PRKCD) controls viability and cell cycle progression of an in vitro model of ACTH-secreting pituitary tumor, the AtT-20/D16v-F2 cells. This study aims at exploring the possible PRKCD role in mediating Pasireotide effects.MethodsIt was assessed cell viability, POMC expression and ACTH secretion in AtT20/D16v-F2 cells over- or under-expressing PRKCD.ResultsWe found that Pasireotide significantly reduces AtT20/D16v-F2 cell viability, POMC expression and ACTH secretion. In addition, Pasireotide reduces miR-26a expression. PRKCD silencing decreases AtT20/D16v-F2 cell sensitivity to Pasireotide treatment; on the contrary, PRKCD overexpression increases the inhibitory effects of Pasireotide on cell viability and ACTH secretion.ConclusionOur results provide new insights into potential PRKCD contribution in Pasireotide mechanism of action and suggest that PRKCD might be a possible marker of therapeutic response in ACTH-secreting pituitary tumors.

A Severe Case of Hypokalemic Metabolic Alkalosis: A Quiz

A Severe Case of Hypokalemic Metabolic Alkalosis: A Quiz

Cyclic Cushing's syndrome: difficulties of diagnostic search and choice of treatment tactics. A clinical case

Cyclic Cushing's syndrome is a pathological condition characterized by alternating periods of excessive cortisol secretion with corresponding clinical manifestations and periods of spontaneous remission of the disease.To diagnose Cyclic Cushing's syndrome it is necessary to record at least three episodes of excessive cortisol secretion alternating with periods of normalization of its production.In most cases, this pathology is diagnosed in patients with ACTH-secreting pituitary tumor, however, there are rare cases of cyclic hypercorticism with ectopic ACTH secretion by tumors of different localization and without verification of pathological hormonal secretion focus. In addition, cyclic hyperproduction of cortisol can be also observed in ACTH-independent Cushing's syndrome associated with the presence of corticosteroma or adrenal hyperplasia. The exact causes and mechanisms of the cyclic hypercorticism are currently insufficiently studied.Due to the atypical course of the disease, the unpredictability of the occurrence of a new «cycle», the variability of its duration and manifestations (not only in different patients, but also in the same patient), verification of the diagnosis and determination of treatment tactics may be difficult in the daily practice of specialists, and the prevalence of this condition can be undervalued.

ODP293 Aberant Expression of SF-1 Transcription Factor in ACTH-secreting Pituitary Tumors: Possible Association With Aggressive Disease Evolution

Abstract Cushing's disease represents a rare endocrine disorder caused by hypercortisolism due to an ACTH-secreting pituitary tumor. Pituitary adenomas (PAs) are mostly benign tumors but may have features of invasiveness and aggressiveness. They are clinically classified by their hormone-secreting characteristics. Expression of pituitary hormones in adenohypophyseal cells is controlled by several transcription factors (PIT1 for GH, PRL and TSH lineage, SF1 for gonadotroph lineage and TPIT for ACTH lineage). Eight cases of Cushing's disease (CD) were included in the current study. The diagnostic was sustained by clinical features, hormonal assessment, and radiological findings (pituitary CT or MRI). All patients underwent surgical removal of pituitary tumor using transsphenoidal resection. All tissue specimens were evaluated using histological routine staining (hematoxylin and eosin) and immunohistochemical staining for vimentin, pituitary hormones (GH, PRL, TSH, FSH, LH, ACTH), and transcription factors (PIT1, TPIT, SF1). The intensity IHC scores ranged from 0 to 3+ and a staining higher than 30% (2+) was considered positive for interpreting the results. Based on the histopathological and immunohistochemical data correlated with the evolution of the disease (remission versus persistent disease), five molecular subgroups were identified. Subgroups 1 to 3 had a positive expression of ACTH and TPIT, with variations regarding SF1 expression: there was no expression in subgroup 1, nuclear positivity in 2, and cytoplasmic reaction in 3. Subgroups 4 and 5 consisted of plurihormonal adenomas with unusual immunohistochemical combination. They had IHC positive score for ACTH, GH, TPIT and were differentiated by the expression of SF1: lack of expression in subgroup 4 and intense cytoplasmic reaction in 5. The expression of SF1 was linked to the evolution of the disease, patients from subgroups which did not have SF1 cytoplasmic reaction (1, 2, 4) had remission of CD, while patients in subgroups 3 and 5 had persistent disease (including one case who evolved to exitus due to cardiovascular complications of the severe hypercortisolism). Transcription factors evaluation is important for classifying pituitary adenomas and finding clinicopathological correlations with the aim of providing a better therapeutical approach. In the current study, although SF1 is a marker of gonadotroph lineage (1), its aberrant cytoplasmic expression in CD may contribute to an aggressive evolution and to other cell lineage differentiation (PIT1, TPIT). Neou M et al, Pangenomic Classification of Pituitary Neuroendocrine Tumors. Cancer Cell. 2020 Jan 13;37(1): 123-134. e5. doi: 10.1016/j. ccell.2019.11. 002. Epub 2019 Dec 26. PMID: 31883967. Presentation: No date and time listed

The Role of Glucocorticoid Receptor in the Pathophysiology of Pituitary Corticotroph Adenomas.

Adrenocorticotropic Hormone (ACTH)-secreting pituitary adenomas are rare tumors characterized by autonomous ACTH secretion with a consequent increase in circulating cortisol levels. The resulting clinical picture is called Cushing’s disease (CD), a severe condition burdened with high morbidity and mortality. Apart from increased cortisol levels, CD patients exhibit a partial resistance to the negative glucocorticoid (GC) feedback, which is of paramount clinical utility, as the lack of suppression after dexamethasone administration is one of the mainstays for the differential diagnosis of CD. Since the glucocorticoid receptor (GR) is the main regulator of negative feedback of the hypothalamic–pituitary–adrenal axis in normal conditions, its implication in the pathophysiology of ACTH-secreting pituitary tumors is highly plausible. In this paper, we review GR function and structure and the mechanisms of GC resistance in ACTH-secreting pituitary tumors and assess the effects of the available medical therapies targeting GR on tumor growth.

P720R USP8 Mutation Is Associated with a Better Responsiveness to Pasireotide in ACTH-Secreting PitNETs.

Simple SummarySomatic mutations in USP8 gene have been identified in 11–60% of patients with ACTH-secreting pituitary tumors. This study investigated the impact of USP8 mutations on corticotroph tumor cells responsiveness to the treatment with the somatostatin analog pasireotide. Although we found that the somatostatin receptor targeted by pasireotide, SSTR5, was upregulated in cells transfected with USP8 mutants, pasireotide failed to reduce ACTH secretion in cells expressing mutated USP8, except for the mutation P720R. Overall these data demonstrate that different USP8 mutations exert opposite effects on the responsiveness of ACTH-secreting pituitary tumors to pasireotide.Somatic mutations in the ubiquitin specific peptidase 8 (USP8) gene have been associated with higher levels of somatostatin (SS) receptor subtype 5 (SSTR5) in adrenocorticotroph hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs). However, a correlation between the USP8 mutational status and favourable responses to pasireotide, the somatostatin multi-receptor ligand acting especially on SSTR5, has not been investigated yet. Here, we studied the impact of USP8 mutations on pasireotide responsiveness in human and murine corticotroph tumor cells. SSTR5 upregulation was observed in USP8 wild-type primary tumor cells transfected with S718del USP8 mutant. However, cell transfection with S718del USP8 and C40-USP8 mutants in in vitro sensitive cultures from USP8 wild-type tumors abolished their ability to respond to pasireotide and did not confer pasireotide responsiveness to the in vitro resistant culture. Pasireotide failed to reduce ACTH secretion in primary cells from one S718P USP8-mutated tumor but exerted a strong antisecretory effect in primary cells from one P720R USP8-mutated tumor. In agreement, AtT-20 cells transfection with USP8 mutants led to SSTR5 expression increase but pasireotide could reduce ACTH production and cyclin E expression in P720R USP8 overexpressing cells, only. In situ Proximity Ligation Assay and immunoflurescence experiments revealed that P720R USP8 mutant is still able to bind 14-3-3 proteins in AtT-20 cells, without affecting SSTR5 localization. In conclusion, P720R USP8 mutation might be considered as a molecular predictor of favourable response to pasireotide in corticotroph tumor cells.

Course of Cushing`s disease and treatment outcomes in correlation with pituitary MRI in children.

Cushing's disease (CD) is a rare disorder of a persistent cortisol excess caused by ACTH-secreting pituitary tumor (corticotropinoma). Transsphenoidal surgery (TSS) is a treatment of choice for СD, which effectiveness range is from 70 to 90%. Recurrence rate after successful treatment is about 25%. If surgical treatment is unsuccessful or recurrence appear, radiation treatment is the next therapeutic option, which effectiveness range is also 90%, but the hypopituitarism rate as side effect of treatment is higher. Preoperative predictors of remission and recurrence are still unexplored what leads to further investigations. Analysis of remission and recurrence rates of pediatric CD after successful treatment according to preoperative MRI and therapeutic option. We conducted a retrospective analysis of 90 pediatric patients with CD who were observed between 1992 and 2020 at the Endocrinology Research Centre. The most common clinical symptoms of CD were weight gain [94%] and growth retardation [72%]. Pituitary tumor was detected on radiological imaging in 53/90 patients [59%], there were no signs of visible adenoma in 37/90 of patients[41%]. 63 of 90 patients underwent TSS (70%), 27 patients underwent radiosurgery (30%). Remission rate after TSS was 71%[45/63], after radiosurgery - 85% [23/27]. There were no significant differences in remission rates after radical treatment according to preoperative MRI results (P=0.21 after TSS and P=0.87 after radiosurgery, х2 analysis). Recurrence after successful treatment was diagnosed in 10 patients. There were no significant differences in time to recurrence according to preoperative MRI results (P=0.055, х2 analysis). Time to recurrence was statistically different after TSS compared to radiosurgery (P=0.007, Kaplan-Meier analysis) and in the group with developed adrenal insufficiency in the early postoperative period (P=0.04, Kaplan-Meier analysis). Analysis of side effect of treatment showed that the frequency of growth hormone and gonadotrophin deficiency was statistically higher after radiosurgery (р<0.01, Kruskel-Wallis ANOVA test). Diabetes insipidus was diagnosed only after TSS. Results of our study didn`t allow to use MRI-results as predictor of effectiveness treatment in pediatric CD. Therapeutic option has an impact on time to recurrence, not on recurrence rates. The frequency of growth hormone and gonadotrophin deficiency was statistically higher after radiosurgery compared to TSS. Further studies are needed to identify predictors of remission and recurrence in CD.>< 0.01, Kruskel-Wallis ANOVA test). Diabetes insipidus was diagnosed only after TSS. Results of our study didn`t allow to use MRI-results as predictor of effectiveness treatment in pediatric CD. Therapeutic option has an impact on time to recurrence, not on recurrence rates. The frequency of growth hormone and gonadotrophin deficiency was statistically higher after radiosurgery compared to TSS. Further studies are needed to identify predictors of remission and recurrence in CD.

Glucocorticoid Receptor Antagonism Upregulates Somatostatin Receptor Subtype 2 Expression in ACTH-Producing Neuroendocrine Tumors: New Insight Based on the Selective Glucocorticoid Receptor Modulator Relacorilant.

Somatostatin exhibits an inhibitory effect on pituitary hormone secretion, including inhibition of growth hormone and adrenocorticotropic hormone (ACTH), and it can have antisecretory and antitumor effects on neuroendocrine tumors (NETs) that express somatostatin receptors. Although the precise mechanism remains unclear, the finding that glucocorticoids downregulate somatostatin receptor subtype 2 (SSTR2) expression has been used to explain the lack of efficacy of traditional SSTR2-targeting analogs in patients with ACTH-secreting NETs. Glucocorticoid receptor (GR) antagonism with mifepristone has been shown to reverse the glucocorticoid-induced downregulation of SSTR2; however, the effects of GR modulation on SSTR2 expression in ACTH-secreting NETs, particularly corticotroph pituitary tumors, are not well known. The current study presents new insight from in vitro data using the highly selective GR modulator relacorilant, showing that GR modulation can overcome dexamethasone-induced suppression of SSTR2 in the murine At-T20 cell line. Additional data presented from clinical case observations in patients with ACTH-secreting NETs suggest that upregulation of SSTR2 via GR modulation may re-sensitize tumors to endogenous somatostatin and/or somatostatin analogs. Clinical, laboratory, and imaging findings from 4 patients [2 ACTH-secreting bronchial tumors and 2 ACTH-secreting pituitary tumors (Cushing disease)] who were treated with relacorilant as part of two clinical studies (NCT02804750 and NCT02762981) are described. In the patients with ectopic ACTH secretion, SSTR2-based imaging (Octreoscan and 68Ga-DOTATATE positron emission tomography) performed before and after treatment with relacorilant showed increased radiotracer uptake by the tumor following treatment with relacorilant without change in tumor size at computed tomography. In the patients with Cushing disease who received relacorilant prior to scheduled pituitary surgery, magnetic resonance imaging after a 3-month course of relacorilant showed a reduction in tumor size. Based on these findings, we propose that GR modulation in patients with ACTH-secreting NETs upregulates previously suppressed SSTR2s, resulting in tumor-specific antisecretory and anti-proliferative effects. The effect of relacorilant on pituitary corticotroph tumors is being investigated in an ongoing phase 3 study (NCT03697109; EudraCT 2018-003096-35).

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant.

Simple SummaryCushing’s Disease (CD) is a rare but severe endocrine disorder due to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, and pathogenetics remained a puzzling issue for a long time. The recent identification of somatic mutations in the 14-3-3 protein binding motif of ubiquitin specific peptidase 8 gene (USP8), present in a consistent subgroup of ACTH-secreting pituitary tumors, have represented a major advance in the understanding of CD pathogenesis. In our cohort of 60 patients we found an incidence of 11.7% of USP8 recurrent somatic mutations whereas a novel USP8 variant (G664R) located upstream the canonical USP8 mutational hotspot was identified in one case. This alteration has never been reported by previous records. The present study provides USP8 G664R variant in vitro functional characterization in AtT-20 cells and demonstrates its possible implication in ACTH-secreting tumor pathogenesis, contributing to enlarge the genetic landscape of CD.Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases USP8 and USP48, and in BRAF genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in USP8, USP48, and BRAF hotspot regions. Seven patients were found to carry USP8 somatic mutations in the well-characterized 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of USP8 G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the USP8 G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, p < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, p < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, p < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in USP8 (13.3% vs. 36.5% incidence of all published mutations) and USP48 (3.3% vs. 13.3% incidence) hotspot regions. A novel USP8 variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains.

Giant silent corticotrope pituitary adenoma in a patient with complicated clinical course

Introduction Somatostatin analogues are a cornerstone in the treatment of acromegaly. Somatostatin analogues of the first generation, mainly targeting the somatostatin receptor (SSTR) subtype 2, proofed to be efficient in most patients with acromegaly. With the development of somatostatin analogues targeting the subtype 5 additionally to the subtype 2, i.e. pasireotide, an efficient drug for ACTH secreting pituitary tumors and suboptimal responding acromegaly patients became available. Methods We investigated immunohistochemically SSTR subtypes expression in three pituitary adenomas: two from operated acromegaly patients with clinical relapse and one from a patient with clinically silent ACTH-positive macroadenoma with unfavourable clinical course. Results The predictive value of SSTR subtypes immunhistochemical analysis for the therapeutic response was discussed. The one silent corticotroph adenoma case presented here was negative for subtype 2 as well as for subtype 5 and therefore we did not see an indication for somatostatin analogue therapy. In acromegaly high Ki-67 appeared to have a negative impact upon therapeutic response in the two cases. Conclusion The immunohistochemical analysis of SSTR subtypes expression in pituitary adenoma patients especially with acromegaly and complicated clinical course might be helpful for decision making or prognostic significance. Our case reports point to the need of clinical trials for further investigation.

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.

Genetic profiling of a cohort of Italian patients with ACTH-secreting pituitary tumors and characterization of a novel USP8 gene variant

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Novel insight into ACTH-secreting pituitary tumors biological behavior: hormone secretion and cell proliferation modulation by Ubiquitin Specific Peptidase 8 inhibitor RA-9

Novel insight into ACTH-secreting pituitary tumors biological behavior: hormone secretion and cell proliferation modulation by Ubiquitin Specific Peptidase 8 inhibitor RA-9

Effects of CRN04894, a Nonpeptide Orally Bioavailable ACTH Antagonist, on Corticosterone in Rodent Models of ACTH Excess

CRN04894 is an orally administered nonpeptide that is a potent and selective antagonist for adrenocorticotropic hormone (ACTH) acting at the melanocortin 2 receptor (MC2R) and is currently under development for the treatment of diseases of ACTH excess such as Cushing’s disease, congenital adrenal hyperplasia, and ectopic ACTH-secreting tumors. Cushing’s disease results from an adenoma derived from pituitary corticotropic cells that secrete excess ACTH, whereas ectopic ACTH syndrome arises from nonpituitary ACTH secreting tumors. Congenital adrenal hyperplasia is a genetic disease that results in cortisol deficiency leading to high levels of ACTH and adrenal androgens. Each of these indications is characterized by high ACTH levels that act on MC2R expressed in the adrenal cortex to drive pathological elevations of adrenally derived steroid hormones. CRN04894 blocks the action of ACTH at MC2R, providing a potential novel treatment for these diseases. Preclinical models of chronic hypercortisolemia include implantation of ACTH-secreting pituitary tumor cells in mice and continuous administration of ACTH via subcutaneously implanted osmotic pumps in rats. These models induce features consistent with human diseases of ACTH excess including hypercortisolemia and hypertrophy of the adrenal glands. We employed both rodent models to examine the pharmacodynamic effects of CRN04894 on corticosterone levels and adrenal gland morphology. In the mouse pituitary tumor model, subcutaneous inoculation of the ACTH-secreting mouse pituitary tumor cell line, AtT-20, into immunodeficient mice resulted in formation of tumors and increased plasma ACTH and corticosterone levels. Repeated daily oral administration of CRN04894 for 14 days dose-dependently and robustly suppressed plasma corticosterone levels in mice with AtT-20 tumors. In the rat model, subcutaneous implantation of osmotic pumps delivering ACTH resulted in increased corticosterone levels, reduction in body weight, and hypertrophy of the adrenal glands after 7 days. Daily oral administration of CRN04894 over 7 days dose-dependently suppressed corticosterone levels, mitigated the effect of ACTH excess on body weight, and rescued the adrenal gland hypertrophy. These findings provide evidence that CRN04894 functions as an effective ACTH antagonist at MC2R to suppress adrenal corticosterone secretion in both mouse and rat models of ACTH excess and hypercortisolemia, thus providing a strong rationale for its potential therapeutic utility in diseases of ACTH excess. This work was supported in part by an SBIR grant from the NIH awarded to Dr. Struthers (R43- DK115245)

ACTH-secreting pituitary tumor with delayed aggressive clinical recurrence – management challenges

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Somatostatin receptor expression and patients' response to targeted medical treatment in pituitary tumors: evidences and controversies.

Somatostatin receptors (SSTs) are widely co-expressed in pituitary tumors. SST2 and SST5 are the most represented SST subtypes. First-generation somatostatin receptor ligands (SRLs) mainly target SST2, while pasireotide, a multi-receptor ligand, shows high binding affinity for both SST5 and SST2. Therefore, SRLs are routinely used as medical treatment for GH-, TSH-, and ACTH-secreting pituitary tumors. Critical revision of literature data correlating SST expression with patients' response to SRLs. SST2 expression in somatroph tumors directly correlates with GH and IGF-1 decrease after first-generation SRL treatment. SST2 immunohistochemistry represents a valuable tool to predict biochemical response to first-generation SRLs in acromegalic patients. Pasireotide seems to exert its biological effects via SST2 in unselected patients. However, in those subjects resistant to first-generation SRLs, harbouring tumors with negligible SST2 expression, pasireotide can act throughout SST5. More than somatotroph tumors, TSH-omas represent the paradigm of tumors showing a satisfactory response to SRLs. This is probably due to the high SST2 expression observed in nearly 100% of cases, as well as to the balanced amount of SST5. In corticotroph tumors, pasireotide mainly act via SST5, although there is a need for translational studies correlating its efficacy with SST expression in this peculiar tumor histotype. The assumption "more target receptor, more drug efficacy" is not straightforward for SRLs. The complex pathophysiology of SSTs, and the technical challenges faced to translate research findings into clinical practice, still need our full commitment to make receptor evaluation a worthwhile procedure for individualizing treatment decisions.

SAT-203 Misleading Diagnosis of Cushing’s Syndrome on Pramipexole

We present an intriguing case of a patient incorrectly diagnosed with Cushing’s disease secondary to pramipexole use for restless leg syndrome (RLS). 61-year-old male with symptoms of 55 lb weight gain, progressive fatigue, and subjective muscle weakness presented to endocrinology office for work-up of possible Cushing’s syndrome. Physical exam was significant for mild facial plethora, purple striae, and central obesity. Patient’s history includes diagnosis of prolactin-producing pituitary microadenoma 30 years ago treated with bromocriptine with normalization of prolactin and resolution of adenoma. Patient notes ten years ago, he was started on pramipexole 1 mg TID for severe RLS. Given patient’s clinical symptoms he underwent hormonal testing which was notable for an abnormal 1 mg ONDST with morning cortisol 2.44 mcg/dL (normal < 1.8 mg/dL) and two abnormal late night salivary cortisol 0.177 mcg/dL and 0.199 mcg/dL (normal < 0.122). Repeat MRI showed a 0.4 cm hypoenhancing lesion in the left side of the anterior pituitary gland. Patient was referred for IPSS. His central-to-peripheral plasma ACTH ratio was as follows: before CRH administration: left 5.8, right 1.2; after CRH administration: left 16.4, right 2.7. These results demonstrated positive IPSS indicating centralization or a pituitary source of ACTH excess. Patient was going to be referred to neurosurgery for left pituitary adenoma resection, but literature search revealed a study showing pramipexole causing an increase in cortisol levels in physiologic studies involving healthy subjects. Due to these findings, labs were repeated while holding pramipexole. His morning cortisol and ACTH levels were normal. Late night salivary cortisol x3 were normal: 0.038, 0.071, and 0.024 mcg/dL (normal < 0.090 mcg/dL). The 24 hour urine free cortisol x2 was also normal. These negative biochemical findings showed no evidence of hypercortisolism off of pramipexole. This further complicated the case as the patient had positive IPSS. In order to help exclude the diagnosis of an ACTH secreting pituitary tumor, a DDAVP stimulation test was performed showing a normal response. Based on these tests it was concluded he does not have Cushing’s and that his hypercortisolism was related to pramipexole use. Repeat brain MRI showed no structural evidence of any pituitary lesion. We report a very rare case of pramipexole use leading to a false diagnosis of Cushing’s syndrome. This was fortunately identified prior to patient undergoing unnecessary surgery. We recommend awareness of pramipexole use associated with hypercortisolism to prevent incorrect diagnosis of Cushing’s syndrome.Schilling, J. C., Adamus, W. S., & Palluk, R. (1992). Neuroendocrine and side effect profile of pramipexole, a new dopamine receptor agonist, in humans. Clinical Pharmacology & Therapeutics, 51(5), 541-548.