Relevance of mutations in protein deubiquitinases genes and TP53 in corticotroph pituitary tumors.

Introduction: Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Methods: Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. Results: USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. Conclusions: We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.

A heterozygous germline deletion within USP8 causes severe neurodevelopmental delay with multiorgan abnormalities.

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in deubiquitinating the enhanced epidermal growth factor receptor for escape from degradation. Somatic variants at a hotspot in USP8 are a cause of Cushing's disease, and a de novo germline USP8 variant at this hotspot has been described only once previously, in a girl with Cushing's disease and developmental delay. In this study, we investigated an exome-negative patient with severe developmental delay, dysmorphic features, and multiorgan dysfunction by long-read sequencing, and identified a 22-kb de novo germline deletion within USP8 (chr15:50469966-50491995 [GRCh38]). The deletion involved the variant hotspot, one rhodanese domain, and two SH3 binding motifs, and was presumed to be generated through nonallelic homologous recombination through Alu elements. Thus, the patient may have perturbation of the endosomal sorting system and mitochondrial autophagy through the USP8 defect. This is the second reported case of a germline variant in USP8.

Clinical Spectrum of USP8 Pathogenic Variants in Cushing's Disease.

Cushing's disease (CD) is a life-threatening condition with a challenging diagnostic process and scarce treatment options. CD is caused by usually benign adrenocorticotrophic hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs), known as corticotropinomas. These tumors are predominantly of sporadic origin, and usually derive from the monoclonal expansion of a mutated cell. Somatic activating variants located within a hotspot of the USP8 gene are present in 11-62% of corticotropinomas, making USP8 the most frequent genetic driver of corticotroph neoplasia. In contrast, other somatic defects such as those affecting the glucocorticoid receptor gene (NR3C1), the BRAF oncogene, the deubiquitinase-encoding gene USP48, and TP53 are infrequent. Moreover, patients with familial tumor syndromes, such as multiple endocrine neoplasia, familial isolated pituitary adenoma, and DICER1 rarely develop corticotropinomas. One of the main molecular alterations in USP8-driven tumors is an overactivation of the epidermal growth factor receptor (EGFR) signaling pathway, which induces ACTH production. Hotspot USP8 variants lead to persistent EGFR overexpression, thereby perpetuating the hyper-synthesis of ACTH. More importantly, they condition a characteristic transcriptomic signature that might be useful for the clinical prognosis of patients with CD. Nevertheless, the clinical phenotype associated with USP8 variants is less well defined. Hereby we discuss the current knowledge on the molecular pathogenesis and clinical picture associated with USP8 hotspot variants. We focus on the potential significance of the USP8 mutational status for the design of tailored clinical strategies in CD.

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

PURPOSE: Clinical 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. METHODS: It was assessed cell viability, POMC expression and ACTH secretion in AtT20/D16v-F2 cells over- or under-expressing PRKCD. RESULTS: We 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. CONCLUSION: Our 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.

The addition of corticotropin-releasing hormone to 2-day low dose dexamethasone suppression test provides additional case detection.

ABSTARCT: PURPOSE: The diagnostic value of adding a Corticotropin-Releasing Hormone (CRH) Stimulation Test to the 2-day Low Dose Dexamethasone Suppression Test (Dex-CRH Test) has been debated in the literature. METHODS: We identified 65 patients with Cushing disease (CD) and 42 patients in whom a diagnosis of Cushing disease could not be confirmed (NCD) after a minimum follow-up of 14 months who underwent the Dex-CRH test. RESULTS: The female sex ratio, median (range) age, and BMI were similar between the two groups. The follow-up for patients with CD and NCD was 74 (4-233) and 52 (14-146) months, respectively. Among 65 patients with CD, 5 (7.7%) had a cortisol level ≤1.4 µg/dl after LDDST but were appropriately classified as CD with a cortisol level >1.4 µg/dL at 15-min post CRH stimulation. In contrast, 3/42 patients (7.1%) in NCD had an abnormal Dex-CRH test. In only one of three patients, the LDDST was marginally normal (cortisol was 1.4 µg/dL and increased to 3.1 µg/dL 15-min post CRH). A cortisol cutoff value of >1.4 µg/dL during the Dex-CRH test provided a sensitivity of 100%, specificity of 93%, and diagnostic accuracy of 97% to diagnose CD. When patients without a Dex level were excluded (n = 74), the sensitivity did not change, but the specificity and accuracy of the Dex-CRH test increased to 97 and 99%, respectively. CONCLUSION: The Dex-CRH Test provided additional case detection in 5/65 (7.7%) patients with CD. It resulted in one false-positive case compared to LDDST. Measurement of dexamethasone improved diagnostic accuracy of the test.

Genetically engineered human pituitary corticotroph tumor organoids exhibit divergent responses to glucocorticoid receptor modulators.

Cushing's disease (CD) is a serious endocrine disorder attributed to an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that that subsequently leads to chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSCUSP48 and iPSCUSP8 cultures, mifepristone induced a predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of Cushing's disease patients.

Characterization of Adrenal miRNA-Based Dysregulations in Cushing's Syndrome.

MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing's syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing's disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes.

Genetics of Cushing's disease: from the lab to clinical practice.

Cushing's disease is a rare, but devastating condition, caused by corticotroph tumors. It rarely manifests as syndrome and very few isolated cases present with germline mutations. Instead, the vast majority of corticotroph tumors are sporadic monoclonal neoplasms. At present, the major recurrent somatic driver mutations are found in the USP8 gene, which encodes for a deubiquitinase that rescues proteins regulating ACTH synthesis. Almost half of functional corticotroph tumors carry somatic USP8 mutations that associate with a distinct transcriptomic and clinical profile. Other genes mutated in a small fraction of corticotroph tumors include the deubiquitinase encoding gene USP48 and the glucocorticoid receptor expressing NR3C1. Recent reports on somatic TP53 and ATRX mutations in corticotroph macroadenomas and carcinomas indicate that within specific patient subpopulations they are not as rare as assumed.

Responsiveness to DDAVP in Cushing's disease is associated with USP8 mutations through enhancing AVPR1B promoter activity.

PURPOSE: To clarify the characteristics of Cushing's disease (CD) patients who respond to the desmopressin (DDAVP) test and its underlying mechanisms. METHODS: Forty-seven patients with CD who underwent DDAVP testing were included. Patients were divided into two groups: DDAVP test (+) (adrenocorticotropic hormone [ACTH] levels increased by ≥ 1.5-fold during the DDAVP test) and DDAVP test (-) (ACTH levels increased by < 1.5-fold). AVP receptor expression levels in these tumors were quantified using quantitative RT-PCR and immunohistochemistry. AVP receptor promoter activity was analyzed using a dual-luciferase reporter assay system. RESULTS: Females (96.9%) and USP8 mutants (85.7%) were more prevalent in the DDAVP test (+) than in the DDAVP test (-). Indeed, the ACTH and cortisol responsiveness to DDAVP was greater in USP8 mutation positive tumors than that in USP8 wild type tumors (3.0-fold vs. 1.3-fold, 1.6-fold vs. 1.1-fold, respectively). Responsiveness to DDAVP was correlated with the expression levels of AVPR1B, but not with those of AVPR2. Comparably, Avpr1b promoter activity was enhanced by the overexpression of mutant USP8 compared to the wild type. CONCLUSIONS: We found that the responsiveness of ACTH to DDAVP in CD was greater in tumors with USP8 mutations. The present data suggest that USP8 mutations upregulate the AVPR1B promoter activity. Additionally, we showed that the DDAVP test can predict the presence of USP8 mutations.

Effects of tubastatin A on adrenocorticotropic hormone synthesis and proliferation of AtT-20 corticotroph tumor cells.

Cushing's disease is an endocrine disorder characterized by hypercortisolism, mainly caused by autonomous production of ACTH from pituitary adenomas. Autonomous ACTH secretion results in excess cortisol production from the adrenal glands, and corticotroph adenoma cells disrupt the normal cortisol feedback mechanism. Pan-histone deacetylase (HDAC) inhibitors inhibit cell proliferation and ACTH production in AtT-20 corticotroph tumor cells. A selective HDAC6 inhibitor has been known to exert antitumor effects and reduce adverse effects related to the inhibition of other HDACs. The current study demonstrated that the potent and selective HDAC6 inhibitor tubastatin A has inhibitory effects on proopiomelanocortin (Pomc) and pituitary tumor-transforming gene 1 (Pttg1) mRNA expression, involved in cell proliferation. The phosphorylated Akt/Akt protein levels were increased after treatment with tubastatin A. Therefore, the proliferation of corticotroph cells may be regulated through the Akt-Pttg1 pathway. Dexamethasone treatment also decreased the Pomc mRNA level. Combined tubastatin A and dexamethasone treatment showed additive effects on the Pomc mRNA level. Thus, tubastatin A may have applications in the treatment of Cushing's disease.

PI3K inhibition by BKM120 results in anti-proliferative effects on corticotroph tumor cells.

PURPOSE: Cushing's disease is associated with significant morbidity; thus, additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway, which plays regulatory role in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, on cell viability, apoptosis, cell cycle phase distribution, and ACTH production in mouse corticotroph tumor cells. METHODS: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using an MTS-based assay. Apoptosis was evaluated by Annexin V staining. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Ccnd1, Ccne1, Cdk2, Cdk4, Myc, and Rb1) was assessed by qPCR. Protein expression of p27, total and phosphorylated Akt was assessed by Western blot. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. RESULTS: Treatment with BKM120 decreased AtT-20/D16v-F2 cell viability, induced a G0/G1 cell cycle arrest, reduced the phosphorylation of Akt at Serine 473, and increased p27 expression. Furthermore, BKM120 treatment diminished ACTH levels in the cell culture supernatants. CONCLUSION: In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in anti-proliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing's disease.

Metformin and Bone Metabolism in Endogenous Glucocorticoid Excess: An Exploratory Study.

Context: Glucocorticoid excess exhibits multiple detrimental effects by its catabolic properties. Metformin was recently suggested to protect from adverse metabolic side-effects of glucocorticoid treatment. Whether metformin is beneficial in patients with endogenous glucocorticoid excess has not been clarified. Objective: To evaluate the phenotype in patients with endogenous Cushing's syndrome (CS) treated with metformin at the time of diagnosis. Patients and Methods: As part of the German Cushing's Registry we selected from our prospective cohort of 96 patients all 10 patients who had been on pre-existing metformin treatment at time of diagnosis (CS-MET). These 10 patients were matched for age, sex and BMI with 16 patients without metformin treatment (CS-NOMET). All patients had florid CS at time of diagnosis. We analyzed body composition, metabolic parameters, bone mineral density and bone remodeling markers, muscle function and quality of life. Results: As expected, diabetes was more prevalent in the CS-MET group, and HbA1c was higher. In terms of comorbidities and the degree of hypercortisolism, the two groups were comparable. We did not observe differences in terms of muscle function or body composition. In contrast, bone mineral density in metformin-treated patients was superior to the CS-NOMET group at time of diagnosis (median T-Score -0.8 versus -1.4, p = 0.030). CS-MET patients showed decreased ß-CTX levels at baseline (p = 0.041), suggesting reduced bone resorption under metformin treatment during glucocorticoid excess. Conclusion: This retrospective cohort study supports potential protective effects of metformin in patients with endogenous glucocorticoid excess, in particular on bone metabolism.

The genomic profiling and MAMLD1 expression in human and canines with Cushing's disease.

BACKGROUND: Cushing's disease (CD) is defined as hypercortisolemia caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (corticotroph PA) that afflicts humans and dogs. In order to map common aberrant genomic features of CD between humans and dogs, we performed genomic sequencing and immunostaining on corticotroph PA. METHODS: For inclusion, humans and dog were diagnosed with CD. Whole exome sequencing (WES) was conducted on 6 human corticotroph PA. Transcriptome RNA-Seq was performed on 6 human and 7 dog corticotroph PA. Immunohistochemistry (IHC) was complete on 31 human corticotroph PA. Corticotroph PA were compared with normal tissue and between species analysis were also performed. RESULTS: Eight genes (MAMLD1, MNX1, RASEF, TBX19, BIRC5, TK1, GLDC, FAM131B) were significantly (P < 0.05) overexpressed across human and canine corticotroph PA. IHC revealed MAMLD1 to be positively (3+) expressed in the nucleus of ACTH-secreting tumor cells of human corticotroph PA (22/31, 70.9%), but absent in healthy human pituitary glands. CONCLUSIONS: In this small exploratory cohort, we provide the first preliminary insights into profiling the genomic characterizations of human and dog corticotroph PA with respect to MAMLD1 overexpression, a finding of potential direct impact to CD microadenoma diagnosis. Our study also offers a rationale for potential use of the canine model in development of precision therapeutics.

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.

Metabolic profiling of serum from dogs with pituitary-dependent hyperadrenocorticism.

Hyperadrenocorticism (HAC) is one of the most common endocrine diseases in dogs characterized by excessive cortisol production caused by an adrenocorticotropic hormone (ACTH)-secreting tumor, namely pituitary-dependent HAC (PDH) or cortisol-secreting adrenal tumor. Metabolomics presents the ability to identify small molecule metabolites. Thus, the use of metabolomics techniques in canine PDH can provide information about the pathophysiology and metabolic changes in this disease. This study aimed to identify and compare differences in serum metabolites between dogs with PDH and healthy dogs. The metabolomic profile of 20 dogs diagnosed with PDH was compared with 20 healthy dogs using liquid chromatography/mass spectrometry (LC/MS), and metabolite discrimination was performed using partial least squares-discriminant analysis (PLS-DA), the variable important in projection (VIP) and fold changes (FC) group-wise comparisons. The hypergeometric test identified the significantly altered pathways. A total of 21 metabolites were found to be significantly different between the two groups. The major alterations were found in arachidonic and decanoic acid, and phospholipids related to phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). These metabolites are related to insulin resistance and other complications (i.e. hypertension). Our results indicate that PDH produces changes in serum metabolites of dogs, and the knowledge of these changes can aid to better understanding of pathophysiological processes involved and contribute to potentially detect new biomarkers for this disease.

New types of localization methods for adrenocorticotropic hormone-dependent Cushing's syndrome.

The management of endogenous Cushing's syndrome (CS) typically involves two key steps: (i) confirmation of autonomous hypercortisolism and (ii) localization of the cause to guide treatment. Adrenocorticotropic hormone (ACTH)-dependent CS is most commonly due to a pituitary corticotrope tumor which may be so small as to evade detection on conventional magnetic resonance imaging (MRI). Although biochemical testing (e.g., corticotropin stimulation; dexamethasone suppression) can provide an indication of the likely origin of ACTH excess, bilateral inferior petrosal sinus catheterization offers greater accuracy to distinguish pituitary-driven CS [Cushing's Disease (CD)] from the ectopic ACTH syndrome [EAS, e.g., due to a bronchial or pancreatic neuroendocrine tumor (NET)]. In patients with CD, 40-50% may not have a pituitary adenoma (PA) readily visualized on standard clinical MRI. In these subjects, alternative MR sequences (e.g., dynamic, volumetric, fluid attenuation inversion recovery) and higher magnetic field strength (7T > 3T > 1.5T) may aid tumor localization but carry a risk of identifying coincidental (non-causative) pituitary lesions. Molecular imaging is therefore increasingly being deployed to detect small ACTH-secreting PA, with hybrid imaging [e.g., positron emission tomography (PET) combined with MRI] allowing precise anatomical localization of sites of radiotracer (e.g., 11C-methionine) uptake. Similarly, small ACTH-secreting NETs, missed on initial cross-sectional imaging, may be detected using PET tracers targeting abnormal glucose metabolism (e.g., 18F-fluorodeoxyglucose), somatostatin receptor (SSTR) expression (e.g., 68Ga-DOTATATE), amine precursor (e.g., 18F-DOPA) or amino acid (e.g., 11C-methionine) uptake. Therefore, modern management of ACTH-dependent CS should ideally be undertaken in specialist centers which have an array of cross-sectional and functional imaging techniques at their disposal.

Asymptomatic pituitary apoplexy induced by corticotropin-releasing hormone in a 14 year-old girl with Cushing's disease.

OBJECTIVES: Pituitary apoplexy is a rare complication of Cushing's disease (CD), especially in the paediatric age and even more rarely it can occur following anterior pituitary stimulation tests. CASE PRESENTATION: We report a case of a 14-year-old girl who was admitted to our Hospital for evaluation of a possible Cushing's syndrome (CS). Her symptoms and initial laboratory tests were suggestive of CD. Magnetic resonance imaging (MRI) revealed a microadenoma of the pituitary gland. As part of her evaluation she was submitted to a corticotropin-releasing hormone (CRH) stimulation test. Two and a half months later the patient was re-evaluated and presented with both clinical improvement of CS, biochemical resolution of hypercortisolism and tumour size reduction in the MRI, also evidencing a haemorrhagic component favouring the diagnosis of pituitary apoplexy after CRH stimulation test. The patient denied any episodes of severe headache, nausea, vomiting or visual changes. CONCLUSIONS: To our knowledge, the authors report the first case of a pituitary apoplexy after a CRH stimulation test in the paediatric age.

Updates in adrenal steroidogenesis inhibitors for Cushing's syndrome - A practical guide.

Medical therapy is essential in the management of patients with Cushing's syndrome (CS) when curative surgery has failed, surgery is not feasible, when awaiting radiation effect, and in recurrent cases of CS. Steroidogenesis inhibitors have a rapid onset of action and are effective in reducing hypercortisolism, however, adverse effects, including adrenal insufficiency require very close patient monitoring. Osilodrostat is the only steroidogenesis inhibitor to have been assessed in prospective randomized controlled trials and approved for Cushing's disease (CD) by the US Food and Drug Administration and for CS by the European Medical Agency (EMA). Osilodrostat has been shown to be highly effective at maintaining normal urinary free cortisol in patients with CD. Drugs such as metyrapone, ketoconazole (both EMA approved), and etomidate lack prospective evaluation(s). There is, however, considerable clinical experience and retrospective data that show a very wide efficacy range in treating patients with CS. In the absence of head-to-head comparative clinical trials, therapy choice is determined by the specific clinical setting, risk of adverse events, cost, availability, and other factors. In this review practical points to help clinicians who are managing patients with CS being treated with steroidogenesis inhibitors are presented.

A human ACTH-secreting corticotroph tumoroid model: Novel Human ACTH-Secreting Tumor Cell in vitro Model.

BACKGROUND: Cushing disease (CD), although rare, is a life-threatening disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, which leads to excess adrenal-derived cortisol. Efficacious and safe medical therapies that control both hormonal hypersecretion and pituitary corticotroph tumor growth remain an unmet need in the management of CD. Translational research in pituitary tumors has been significantly hampered by limited quantities of surgically resected tissue for ex vivo studies, and unavailability of human pituitary tumor cell models. METHODS: To characterize human corticotroph tumors at the cellular level, we employed single cell RNA-sequencing (scRNA-seq) to study 4 surgically resected tumors. We also used microarrays to compare individualized paired consecutive culture passages to understand transcriptional shifts as in vitro cultures lost ACTH secretion. Based on these findings, we then modified our in vitro culture methods to develop sustained ACTH-secreting human corticotroph tumoroid cultures. FINDINGS: scRNA-seq identified 4 major cell populations, namely corticotroph tumor (73.6%), stromal (11.2%), progenitor (8.3%), and immune cells (6.8%). Microarray analysis revealed striking changes in extracellular matrix, cell adhesion and motility-related genes concordant with loss of ACTH secretion during conventional 2D culture. Based on these findings, we subsequently defined a series of crucial culture nutrients and scaffold modifications that provided a more favorable trophic and structural environment that could maintain ACTH secretion in in vitro human corticotroph tumor cultures for up to 4 months. INTERPRETATION: Our human corticotroph tumoroid model is a significant advance in the field of pituitary tumors and will further enable translational research studies to identify critically needed therapies for CD. FUNDING: This work was partly funded by NCI P50-CA211015 and the Warley Trust Foundation.

Somatostatin Receptor as a Molecular Imaging Target in Human and Canine Cushing Disease.

OBJECTIVES: Fluorescence-guided surgery may improve completeness of resection in transsphenoidal surgery for Cushing disease (CD) by enabling visualization of residual tumor tissue at the margins. In this review we discuss somatostatin receptors (SSTRs) as targets for fluorescence-guided surgery and overview existing SSTR-specific imaging agents. We also compare SSTR expression in normal pituitary and corticotrophinoma tissues from human and canine CD patients to assess canines as a translational model for CD. METHODS: A PubMed literature search was conducted for publications containing the terms canine, somatostatin receptor, Cushing's disease, and corticotroph adenoma. SSTR expression data from each study was documented as the presence or absence of expression or, when possible, the number of tumors expressing a given SSTR subtype within a group of tumors being studied. Studies that used reverse transcription polymerase chain reaction to quantify SSTR expression were selected for additional comparative analysis. RESULTS: SSTR5 is strongly expressed in human corticotroph adenomas and weakly expressed in surrounding pituitary parenchyma, a pattern not conclusively observed in canine patients. SSTR2 mRNA expression is similar in human normal pituitary and corticotrophinoma cells but may be significantly higher in canine normal pituitary tissue than in corticotroph tumoral tissue. Limited data were available on SSTR subtypes 1, 3, and 4. CONCLUSIONS: Further studies must fill the knowledge gaps related to species-specific SSTR expression, so using canine CD as a translational model may be premature. We do conclude that the expression profile of SSTR5 (i.e., high local expression in pituitary adenomas relative to normal surrounding tissues) makes SSTR5 a promising molecular target for FGS.