Pituitary Corticotroph Research Articles

Long-term, dynamic remodelling of the corticotroph transcriptome and excitability after a period of chronic stress.

Chronic stress results in long-term dynamic changes at multiple levels of the hypothalamic-pituitary-adrenal (HPA) axis resulting in stress axis dysregulation with long term impacts on human and animal health. However, the underlying mechanisms and dynamics of altered of HPA axis function, in particular at the level of pituitary corticotrophs, during a period of chronic stress and in the weeks after its cessation (defined as "recovery") are very poorly understood. Here we address the fundamental question of how a period of chronic stress results in altered anterior pituitary corticotroph function and whether this persists in recovery, as well as the transcriptomic changes underlying this. We demonstrate that in mice spontaneous and corticotrophin releasing hormone (CRH)-stimulated electrical excitability of corticotrophs, essential for ACTH secretion, is suppressed for weeks-months of recovery following a period of chronic stress. Surprisingly, there are only modest changes in the corticotroph transcriptome during the period of stress but major alterations occur in recovery. Importantly, while transcriptional changes for a large proportion of mRNAs follow the time course suppression of corticotroph excitability, many other genes display highly dynamic transcriptional changes with distinct time courses throughout recovery. Taken together, this suggests that chronic stress results in complex dynamic transcriptional and functional changes in corticotroph physiology, which are highly dynamic for weeks following cessation of chronic stress. These insights provide a fundamental new framework to further understand underlying molecular mechanisms as well approaches to both diagnosis and treatment of stress-related dysfunction of the HPA axis.

8133 Spatial Transcriptomic Profiling of Pituitary Corticotroph Carcinoma: Uncovering Architectural Complexity

Abstract Disclosure: D. Zhang: None. M. Bergsneider: None. M.B. Wang: None. W. Kim: None. H. Vinters: None. A.P. Heaney: None. Most pituitary corticotroph adenomas are benign but ∼15% are refractory to conventional surgical, medical and even radiotherapeutic management. This tumor subset invade sellar adjacent structures, and in rare cases metastasize locally within the central nervous system or to neck lymph nodes (LN) or more distant organs such as the liver. To better understand the potential interactions between tumor cells and neighboring tissues during these progressive stages, we used spatial transcriptomics (ST, Visium, 10x Genomics) to profile the genetic composition and native architecture of 4 surgically resected corticotroph tumor paraffin-embedded samples collected from the same patient over several years. Samples 1& 2 invaded the sino-nasal mucosa, sample 3 the parietal dura, sample 4 the clival bone, and sample 5 had metastasized locally to a neck LN. Quality of the 4 samples was confirmed by DV200 score (51 ± 7%). An average of 2,473 tissue spots per tissue sample were obtained after sequencing with a median of 4,893 genes detected per spot. We then used the Seurat analytic pipeline to reconstitute spatial transcriptional distribution that authentically mirrored histologically verifiable structures. These included the main tumor area itself, it’s invading tumor edge, adjacent normal epithelium, dura, and LN tissue. Further deconvolution analysis revealed a distinct cellular composition at the invading tumor edge which was tissue context specific. For instance, a proliferative cell population was noted between the central tumor area and it’s invasive margin, B cell and fibroblast cell enrichment was prominent at sino-nasal mucosal invasive margins, whereas endothelial and pericyte cells were prominent along the tumor/ normal pituitary border. . As expected, the central tumor area expressed genes associated with corticotroph hormone production including POMC, NNAT, GAL and GNAS, genes associated with mitochondrial energy metabolism such as ATP5F1E, NDUFA8 and COX7B as well as transcripts related to lipid kinase activity (GKG, CERKL, PIK3C2G, DGKK). Most intriguingly, tumor cells at the fore-front of the invading edge expressed high level of cyclin-dependent kinases, potentially reflecting higher proliferative rates of these invasive margin cells. Additional studies to validate and define the role of theses invasive margin transcripts are now warranted to translate our profiling findings into potential drug targets. In summary, this is the first use of spatial transcriptomics to delineate the evolving transcriptomic features in a series of surgically resected pituitary corticotroph tumors from the same patient that progressed over time to a locally metastatic corticotroph carcinoma. It provides unparalleled insights into the differential transcriptional topography of corticotroph tumor tissue and most importantly, its invading edge and may identify novel therapeutic targets. Presentation: 6/3/2024

6589 Antisense Oligonucleotides for Reducing ACTH Secretion in a Model of Cushing's Disease

Abstract Disclosure: M.S. Varughese: None. H. Eltumi: None. E.H. Kemp: None. J.D. Newell-Price: None. Antisense Oligonucleotides for Reducing ACTH Secretion in a Model of Cushing’s Disease Background: Cushing’s disease (CD) is a multi-system disorder with high morbidity and mortality characterised by pathologically raised serum cortisol due excess expression of proopiomelanocortin (Pomc) and overproduction of ACTH by a pituitary corticotroph adenoma. Trans-sphenoidal adenomectomy is the treatment of choice, but there is a 30% recurrence rate over time. Advances in medical therapy are needed. Antisense oligonucleotides (ASOs) can silence mRNA translation by specific base-pairing. Locked nucleic acid (LNA) and 2’-O-methyl (OMe) ribose terminals make ASOs more effective by improving affinity and resistance to nuclease degradation. Aims: To investigate the effectiveness of LNA- and OMe-modified anti-Pomc ASOs at reducing ACTH secretion from murine adrenotropic tumour (AtT20) cells. To analyse the nuclease resistance of Pomc ASOs and their ability to drive an immune response. Methods: Two ASOs (ASO2 and ASO3) were designed against Pomc with phosphorothioate backbones modified with either LNA or OMe ribose terminals. These were used in lipofectamine-mediated cell transfections of AtT20 cells and ACTH in the culture medium measured by immunoassay. ASO stability was assessed by nuclease degradation assays and immunogenicity by cytokine ELISAs. Results: When compared with untreated AtT20 cells, LNA- and OMe-modified Pomc ASOs at 1 nM significantly suppressed ACTH secretion for up to 120 h and 96 h, respectively (n = 4; Unpaired t tests, all P values < 0.05). In contrast, control treatments, including scrambled and mismatched ASOs, did not cause a significant reduction. For comparison, 1 nM of ASO2-OMe, ASO2-LNA, ASO3-OMe and ASO3-LNA lowered ACTH at 24 h by 62%, 71%, 63% and 79%, respectively. Modified Pomc ASOs showed 14-31% degradation over a 120-min incubation period with 3’- and 5’-exonucleases, whilst equivalent unmodified Pomc ASOs were completely degraded. No secretion of IFN-α, IFN-β, TNF-α, IL-6 or IL-1β was detected following transfection of AtT20 cells with Pomc ASOs. Conclusions:Pomc ASOs caused significant reduction of ACTH secretion from AtT-20 cells. LNA-modified Pomc ASOs were more effective than those with OMe modifications, with neither stimulating a detectable immune response. These ASOs hold promise as a systemic therapy for Cushing’s disease. Presentation: 6/3/2024

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.

Spatial Transcriptomic Analysis of Pituitary Corticotroph Tumors.

Spatial transcriptomic (ST) analysis of tumors provides a novel approach to studying gene expression along with the localization of tumor cells in their environment to uncover spatial interactions. We present ST analysis of corticotroph pituitary neuroendocrine tumors (PitNETs) from formalin-fixed, paraffin-embedded tissues. ST data were compared to immunohistochemistry results. Gene expression profiles were reviewed for cluster annotations, and differentially expressed genes were used for pathway analysis. Seven tumors were used for ST analysis. In situ annotation of tumor tissue was inferred from the gene expression profiles and was in concordance with the annotation made by a pathologist. Furthermore, relative gene expression in the tumor corresponded to common protein staining used in the evaluation of PitNETs, such as reticulin and Ki-67 index. Finally, we identified intratumor heterogeneity; clusters within the same tumor may present with different transcriptomic profiles, unveiling potential intratumor cell variability. Together, our results provide the first attempt to clarify the spatial cell profile in PitNETs.

Conversion of spikers to bursters in pituitary cell networks: Is it better to disperse for maximum exposure or circle the wagons?

The endocrine cells of the pituitary gland are electrically active, and in vivo they form small networks where the bidirectional cell-cell coupling is through gap junctions. Numerous studies of dispersed pituitary cells have shown that typical behaviors are tonic spiking and bursting, the latter being more effective at evoking secretion. In this article, we use mathematical modeling to examine the dynamics of small networks of spiking and bursting pituitary cells. We demonstrate that intrinsic bursting cells are capable of converting intrinsic spikers into bursters, and perform a fast/slow analysis to show why this occurs. We then demonstrate the sensitivity of network dynamics to the placement of bursting cells within the network, and demonstrate strategies that are most effective at maximizing secretion from the population of cells. This study provides insights into the in vivo behavior of cells such as the stress-hormone-secreting pituitary corticotrophs that are switched from spiking to bursting by hypothalamic neurohormones. While much is known about the electrical properties of these cells when isolated from the pituitary, how they behave when part of an electrically coupled network has been largely unstudied.

Ion channels and the diversity of spontaneous firing in anterior pituitary corticotrophs: A dynamical analysis

Pituitary cells release hormones based on firing patterns, mediated by multiple ionic currents. Analyzing a single channel’s effect on firing is insufficient. We used a four-dimensional Hodgkin–Huxley-type corticotroph model to analyze its dynamics from a geometric perspective. We conducted a two-parameter bifurcation analysis of the system and found that activation levels of BK, NS, Kir, Ca, and LCa channels are crucial in generating and transitioning between different firing patterns. Chaotic firing can arise during the transition between firing patterns. We have employed the Poincaré map method and sequence reconstructions to construct return maps to characterize chaos-firing. The membrane potential time series is affected by fluctuations in intracellular calcium ([Ca2+]i). We studied the variable c using fast–slow analysis techniques and phase portraits to explore its relationship with phase portraits. The study revealed that the system exhibits bistability, which is dependent on the initial values of c or V. The findings indicate that firing activities can arise from either a supercritical or subcritical Hopf bifurcation. Analyses show the interaction between the membrane potential and [Ca2+]i at various BK channel activation levels.

Correction: FAF1 Gene Involvement in Pituitary Corticotroph Tumors.

Correction: FAF1 Gene Involvement in Pituitary Corticotroph Tumors.

Metformin inhibits cell proliferation and ACTH secretion in AtT20 cells via regulating the MAPK pathway

We investigated the impact of metformin on ACTH secretion and tumorigenesis in pituitary corticotroph tumors. The mouse pituitary tumor AtT20 cell line was treated with varying concentrations of metformin. Cell viability was assessed using the CCK-8 assay, ACTH secretion was measured using an ELISA kit, changes in the cell cycle were analyzed using flow cytometry, and the expression of related proteins was evaluated using western blotting. RNA sequencing was performed on metformin-treated cells. Additionally, an in vivo BALB/c nude xenograft tumor model was established in nude mice, and immunohistochemical staining was conducted for further verification. Following metformin treatment, cell proliferation was inhibited, ACTH secretion decreased, and G1/S phase arrest occurred. Analysis of differentially expressed genes revealed cancer-related pathways, including the MAPK pathway. Western blotting confirmed a decrease in phosphorylated ERK1/2 and phosphorylated JNK. Combining metformin with the ERK1/2 inhibitor Ulixertinib resulted in a stronger inhibitory effect on cell proliferation and POMC (Precursors of ACTH) expression. In vivo studies confirmed that metformin inhibited tumor growth and reduced ACTH secretion. In conclusion, metformin inhibits tumor progression and ACTH secretion, potentially through suppression of the MAPK pathway in AtT20 cell lines. These findings suggest metformin as a potential drug for the treatment of Cushing's disease.

FAF1 Gene Involvement in Pituitary Corticotroph Tumors.

Cushing's disease (CD) is caused by rare pituitary corticotroph tumors that lead to corticotropin (ACTH) excess. Variants in FAF1, a pro-apoptotic protein involved in FAS-induced cell death, have been implicated in malignant disorders but the involvement of FAF1 in pituitary tumors has not been studied. Genetic data from patients with CD were reviewed for variants in FAF1 gene. Knockout mice (KO) were followed to assess the development of any pituitary disorder or cortisol excess. AtT-20 cells were used to study the effects of the variants of interest on ACTH secretion and cell proliferation. Three variants of interest were identified in 5 unique patients, two of which had rare allele frequency in genomic databases and were predicted to be likely pathogenic. KO mice were followed over time and no difference in their length/weight was noted. Additionally, KO mice did not develop any pituitary lesions and retained similar corticosterone secretion with wild type. AtT-20 cells transfected with FAF1 variants of interest or WT expression plasmids showed no significant difference in cell death or Pomc gene expression. However, in silico prediction models suggested significant differences in secondary structures of the produced proteins. In conclusion, we identified two FAF1 variants in patients diagnosed with CD with a potential pathogenic effect on the protein function and structure. Our in vitro and in vivo studies did not reveal an association of FAF1 defects with pituitary tumorigenesis and further studies may be needed to understand any association.

Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

OR2803 Corticotroph Fate Weighted Cell Differentiation Paradigm For Human Induced Pluripotent Stem Cells

Abstract Disclosure: M.E. Moore: None. K.G. Chen: None. K. Park: None. D. Asuzu: None. N. Ramavenkat: None. S. Walbridge: None. D. Mullaney: None. M. Arhin: None. D. Maric: None. D. Mandal: None. P. Chittiboina: None. Introduction: The vertebrate anterior pituitary gland is a highly conserved ‘master’ organ that controls hormonal milieu in real-time. Pituitary adenomas and their treatments (surgery, chemotherapy or radiation) can result in pituitary organ failure, specifically hypocortisolism, a challenging condition to manage. Here, we created an optimized paradigm (anterior pituitary corticotroph or APcor) to preferentially drive human induced pluripotent stem cells (hiPSCs) towards a pituitary corticotroph fate. Methods: We directed U112I hiPSCs towards terminally differentiated cell types using a combination of rho-associated kinase inhibitor (Y-27632), bone morphogenic protein 4 (BMP4), transforming growth factor β inhibitor (SB431542), sonic hedgehog (SHH), fibroblast growth factor 8b (FGF8b), fibroblast growth factor 10 (FGF10) and leukemia inhibitory factor (LIF). Markers for pluripotent stem cells, cranial placode (CP), Rathke’s Pouch (RP), anterior pituitary progenitors and mature cell types were assessed at 10-day intervals using quantitative qRT-PCR and multiplex immunocytochemistry (mICC) or immunohistochemistry (IHC). ACTH activity was measured using ELISA (MD Bio). Results: We tested initial differentiation conditions and found optimal CP initiation in presence of SB431542. We observed decrease in embryonic stem cell markers OCT4 and NANOG by day 10, with concomitant increase in CP/RP markers SIX1, PAX6 and SIX6 using mICC/IHC and qRT-PCR. Continuation of differentiation paradigm with LIF showed strong expression of anterior pituitary progenitor markers PITX1, TBX19 and POU1F1 at day 20. pRT-PCR analysis at this time point confirms differentiation towards pituitary cell types without needing enrichment for SIX1. At day 30, POMC expression was observed by IHC in 10-15% of cells as measured by quantitative analysis (Qupath 0.2.0), indicating mature corticotrophs. Corticotroph cell fate was confirmed with corticotropin releasing hormone (CRH) stimulation which led to up to 40x-fold increase in ACTH expression detected by ELISA (D30 w/CRH vs Pluripotent w/CRH). Conclusions: We found with the APcor paradigm, we can reliably drive towards corticotroph cell fate. We successfully differentiated hiPSCs into mature functioning corticotrophs with ability to recapitulate pituitary organ function. Our method is amenable to 3D organoid formation and pre-clinical implantation in animal models of hypophysectomy as a novel hormone replacement therapy. Presentation: Sunday, June 18, 2023

FRI324 Pituitary Macroadenoma With Carcinomatous Transformation And Multiple Cranial Masses And Spinal Leptomeningeal Carcinomatosis: A Case Report

Abstract Disclosure: C. Cristancho: None. G. Szabo: None. Background: Aggressive pituitary tumors are invasive tumors not responding to standard therapies and presenting with multiple local recurrences; if metastases occur, the tumors are defined as pituitary carcinoma (PC) Clinical Case: 51- year-old Hispanic woman with history of pituitary macroadenoma diagnosed at the age of 41, when presented with weight gain and decrease in visual acuity. Per records initial Brain MRI reported hyperintense sellar lesion of 2.1x2.3x2.6 cm with supra-sella extension and shift of optic chiasm. Patient had 4 pituitary surgeries between 2014-2018 due to growth and had radiation and Temozolomide between 2018-2020. Pathology was diffusely positive for ACTH, PRL, and Ki-67 was 10-20%, P 55 positive staining was 25-30%. As a result from pituitary surgeries patient developed central hypothyroidism, and central AI. Subsequent mornings cortisol were nl between 16-18. No UFC or LNSC were available. MEN1 negative. Brain MRIs done between 2019 and 2020 showed stable 13x8 mm residual pituitary mass with postoperative changes. Patient denied history of diabetes, fragility fractures, HTN, DVTs. She had regular menstrual periods until age of 42. Had left adrenal mass resection in 2019 with benign pathology results. During her initial endocrinology visit her ROS was positive for lower back pain and mild proximal muscle weakness. Patient denied visual changes, headaches, skin changes, polydipsia or polyuria. Physical exam was remarkable for small buffalo hump, and 5-/5 proximal muscle weakness. Negative for moon face, purple striae, acanthosis nigricans, hirsutism, acne. Initial tests were consistent with ACTH-dependent CS: elevated cortisol PM: 18.2 (n <10ug/dL) and LNSC 0.58 and 0.64 mcg/dl (n <0.09), 24-hr UFC 264 (n <50 mcg/dl), abnormal ACTH 150 (n <50 pg/ml).Brain MRI revealing extra-axial mass of 1.6 x1.1 cm overlying posterior aspect of right cerebellar hemisphere and focal lesion in left prepontine cistern of 0.8 cm. Leptomeningeal Enhancement. No sellar mass. Spine MRI showed scattered nodules lining the cord throughout the cervical and upper thoracic spine concerning for metastatic disease. Right cerebellar mass biopsy positive for metastatic pituitary neuroendocrine tumor corticotroph cell subtype, ACTH, chromogranin A, synaptophysin positive; PRL, TSH, GnRH, alpha subunit staining was negative; Ki-67 of 2%. Temozolomide, Capecitabin, and Ketoconazole 200 mg TID were started. Subsequent LNSC <0.03 and <0.03 mcg/dl, UFC at 3 different times were 12.8, 11.2 and 8.1 mcg/dl showing rapid decrease of cortisol levels in a 4-week period. A block-replace approach was decided with Dexamethasone 1 mg daily and a lower dose of Ketoconazole 200 mg BID. Patient reports no side effects. Presentation: Friday, June 16, 2023

Crooke Cell Adenoma Confers Poorer Endocrinological Outcomes Compared with Corticotroph Adenoma: Results of a Multicenter, International Analysis

Crooke cell adenomas (CCAs) are a rare, aggressive subset of secretory pituitary corticotroph adenomas (sCTAs) found in 5%-10% of patients with Cushing disease. Multiple studies support worse outcomes in CCAs but are limited by small sample size and single-institution databases. We compared outcomes in CCA and sCTA using a multicenter, international retrospective database of high-volume skull base centers. Patients surgically treated for pituitary adenoma from January 2017 through December 2020 were included. Among 2826 patients from 12 international centers, 20 patients with CCA and 480 patients with sCTA were identified. No difference in baseline demographics, tumor characteristics, or postoperative complications was seen. Microsurgical approaches (60% CCA vs. 62.3% sCTA) were most common. Gross total resection was higher in CCA patients (100% vs. 83%, P= 0.05). Among patients with gross total resection according to intraoperative findings, fewer CCA patients had postoperative hormone normalization of pituitary function (50% vs. 77.8%, P < 0.01) and remission of hypersecretion by 3-6 months (75% vs. 84.3%, P < 0.01). This was the case despite CCA having better local control rates (100% vs. 96%, P < 0.01) and fewer patients with remnant on magnetic resonance imaging (0% vs. 7.2%, P < 0.01). A systematic literature review of 35 studies reporting on various treatment strategies reiterated the high rate of residual tumor, persistent hypercortisolism, and tumor-related mortality in CCA patients. This modern, multicenter series of patients with CCA reflects their poor prognosis and reduced postsurgical hormonal normalization. Further work is necessary to better understand the pathophysiology of CCA to devise more targeted treatment approaches.

An individualized approach to the management of Cushing disease.

Cushing disease caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary corticotroph adenoma leads to hypercortisolaemia with high mortality due to metabolic, cardiovascular, immunological, neurocognitive, haematological and infectious conditions. The disorder is challenging to diagnose because of its common and heterogenous presenting features and the biochemical pitfalls of testing levels of hormones in the hypothalamic-pituitary-adrenal axis. Several late-night salivary cortisol and 24-h urinary free cortisol tests are usually required as well as serum levels of cortisol after a dexamethasone suppression test. MRI might only identify an adenoma in 60-75% of patients and many adenomas are small. Therefore, inferior petrosal sinus sampling remains the gold standard for confirmation of ACTH secretion from a pituitary source. Initial treatment is usually transsphenoidal adenoma resection, but preoperative medical therapy is increasingly being used in some countries and regions. Other management approaches are required if Cushing disease persists or recurs following surgery, including medications to modulate ACTH orblock cortisol secretion or actions, pituitary radiation, and/or bilateral adrenalectomy. All patients require lifelong surveillance for persistent comorbidities, clinical and biochemical recurrence, and treatment-related adverse effects (including development of treatment-associated hypopituitarism). In this Review, we discuss challenges in the management of Cushing disease in adults and provide information to guide clinicians when planning an integrated and individualized approach for each patient.

Sex differences in pituitary corticotroph excitability.

Stress-related illness represents a major burden on health and society. Sex differences in stress-related disorders are well documented, with women having twice the lifetime rate of depression compared to men and most anxiety disorders. Anterior pituitary corticotrophs are central components of the hypothalamic-pituitary-adrenal (HPA) axis, receiving input from hypothalamic neuropeptides corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), while regulating glucocorticoid output from the adrenal cortex. The dynamic control of electrical excitability by CRH/AVP and glucocorticoids is critical for corticotroph function; however, whether corticotrophs contribute to sexually differential responses of the HPA axis, which might underlie differences in stress-related disorders, is very poorly understood. Using perforated patch clamp electrophysiology in corticotrophs from mice expressing green fluorescent protein under the control of the Pomc promoter, we characterized basal and secretagogue-evoked excitability. Both male and female corticotrophs show predominantly single-spike action potentials under basal conditions; however, males predominantly display spikes with small-amplitude (<20mV) afterhyperpolarizations (B-type), whereas females displayed a mixture of B-type spikes and spikes with a large-amplitude (>25mV) afterhyperpolarization (A-type). In response to CRH, or CRH/AVP, male cells almost exclusively transition to a predominantly pseudo-plateau bursting, whereas only female B-type cells display bursting in response to CRH±AVP. Treatment of male or female corticotrophs with 1nM estradiol (E2) for 24-72h has no effect on the proportion of cells with A- or B-type spikes in either sex. However, E2 results in the cessation of CRH-induced bursting in both male and female corticotrophs, which can be partially reversed by adding a BK current using a dynamic clamp. RNA-seq analysis of purified corticotrophs reveals extensive differential gene expression at the transcriptional level, including more than 71 mRNAs encoding ion channel subunits. Interestingly, there is a two-fold lower level (p < 0.01) of BK channel pore-forming subunit (Kcnma1) expression in females compared to males, which may partially explain the decrease in CRH-induced bursting. This study identified sex differences at the level of the anterior pituitary corticotroph ion channel landscape and control of both spontaneous and CRH-evoked excitability. Determining the mechanisms of sex differences of corticotroph and HPA activity at the cellular level could be an important step for better understanding, diagnosing, and treating stress-related disorders.

Severe Obesity Associated With Pituitary Corticotroph Hyperplasia and Neoplasia

ObjectiveTo investigate the incidence of corticotroph hyperplasia (CH) or lymphocyte infiltration in the pituitary of patients with obesity. MethodsThe pituitary and adrenal glands from 161 adult autopsies performed between 2010 and 2019 at our institution were reviewed. The clinical history, body mass index (BMI), and cause of death were recorded. Routine hematoxylin and eosin staining, reticulin staining, and immunohistochemical staining for adrenocorticotropic hormone, CD3, and CD20 were performed. The results were analyzed using the Fisher and chi-square statistics. Decedents were separated into 4 groups based on BMI (kg/m2): (1) lean (BMI, <25.0), (2) overweight (BMI, 25.0-29.9), (3) obesity class I (BMI, 30.0-34.9), and (4) obesity classes II to III (BMI, >34.9). ResultsCH/neoplasia was identified in 44 of 161 pituitary glands. Four (9.1%) of 53 lean patients had pituitary lesions, whereas 27.3% (12) of overweight, 22.7% (10) of obesity class I, and 40.9% (18) of obesity class II patients had hyperplasia (P < .0001). Small corticotroph tumors were identified in 15 patients; only 1 was a lean patient, and the tumor was associated with the Crooke hyaline change of nontumorous corticotrophs. The presence of CH and neoplasia was associated with adrenal cortical hyperplasia and lipid depletion. Microscopic foci of T and B lymphocytes were identified in the pituitaries of patients in each weight category; no independent association between BMI and lymphocyte inflammation was found. ConclusionOur data indicate an association between CH/neoplasia and obesity. It remains unclear whether obesity is the cause or effect of adrenocorticotropic hormone and cortisol excess.

Insights Obtained from the Nontumorous Glandular Tissue in Patients with Endocrine Tumors.

The pathology of neoplasia tends to focus on the tumor that requires characterization, grading, and staging. However, nontumorous tissue surrounding the lesion can also provide information, particularly about pathogenetic mechanisms. In endocrine tissues, this takes the form of precursor lesions that characterize several genetic predisposition syndromes. In addition, because of the unique functional aspects of endocrine neoplasia, the nontumorous tissue provides evidence of hormone excess, with hyperplasia and/or atrophy and other involutional changes allowing the pathologist to confirm both hormone function by the tumor and the effects of medical therapies. In this article, we review the various clinically relevant features that should be assessed and reported to enhance clinical management of patients with endocrine neoplasms. For example, in thyroid there may be inflammatory thyroiditis or goiter of various etiologies; there may be C-cell hyperplasia either as a preneoplastic lesion in patients with genetic predisposition to medullary thyroid carcinoma or as a reactive phenomenon. Drug-induced changes can be seen in thyroid and adrenal cortex. In neuroendocrine tissues, the nontumorous tissues may show precursor lesions such as endocrine cell hyperplasia/dysplasia; there may be related or unrelated hyperplastic or neoplastic lesions. Some tissues, such as pituitary corticotrophs and adrenal cortex, develop changes that reflect feedback suppression by hormone excess that can serve as biomarkers of tumor functionality and provide enhanced clinicopathologic correlates.

Comparison of the preoperative diagnostic accuracy of BIPSS versus MRI for Cushing disease: a single-centre experience

BackgroundCushing disease (CD) arises due to a pituitary corticotroph adenoma, which is the most common cause of Cushing syndrome (CS). Bilateral inferior petrosal sinus sampling (BIPSS) is a safe method for differentiating CD from ectopic adrenocorticotropic hormone (ACTH)-dependent CS. Enhanced high-resolution magnetic resonance imaging (MRI) can localize tiny pituitary lesions. The aim of this study was to compare the preoperative diagnostic accuracy of BIPSS versus MRI for CD in CS patients. We performed a retrospective study of patients who underwent BIPSS and MRI between 2017 and 2021. Low- and high-dose dexamethasone suppression tests were performed. Blood samples were collected simultaneously from the right and left catheter and femoral vein before and after desmopressin stimulation. MRI images were obtained, and endoscopic endonasal transsphenoidal surgery (EETS) was performed in confirmed CD patients. Dominant sides of ACTH secretion during BIPSS and MRI were compared with surgical findings.ResultsTwenty-nine patients underwent BIPSS and MRI. CD was diagnosed in 28 patients, 27 of whom received EETS. Localizations of microadenomas by MRI and BIPSS agreed with the EETS findings in 96% and 93% of the cases, respectively. BIPSS and EETS were successfully performed on all patients.ConclusionBIPSS was the most accurate method (gold standard) for establishing a preoperative diagnosis of pituitary-dependent CD and was more sensitive than MRI in diagnosing microadenoma. High-resolution MRI with enhancement had an advantage over BIPSS in microadenoma lateralization diagnostics. The combined use of MRI and BIPSS could improve the preoperative diagnosis accuracy in ACTH-dependent CS patients.

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.