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Clinically non-functioning pituitary adenomas (NFPAs) represent the second most common subtype of pituitary tumours, representing 15-43% of all pituitary adenomas. NFPAs are usually benign tumours with no clinical evidence of hormonal hypersecretion. In most of the cases the tumours are arising from the gonadotroph linage, but silent corticotroph, somatotroph, thyrotroph and rarely lactotroph cell characteristics can be discovered with immunostaining. As a result of lack of hormonal hyper-secretion, the diagnosis of NFPAs is made most often when the patient presents with mass effects due to a macroadenoma. NFPAs are most common tumour types in surgical series, and their primary treatment is indeed surgery. However, complete resection is achieved only in about 66% of the cases, and 20% of gross total resected tumours recur after 10 years. Over the past few decades, remarkable progress has been achieved regarding medical treatment options for pituitary tumours. However, NFPAs remain the only subtype with no widely accepted pharmacological treatment. Dopamine receptor type 2 (DRD2) and somatostatin receptor (SST) expression have been demonstrated in NFPAs, prompting the investigation of dopamine agonists and somatostatin analogues as potential treatment strategies. The DRD2 agonist cabergoline is already used as first-line treatment of prolactinomas to induce tumour shrinkage and reduce prolactin secretion. Here we aim to investigate the efficacy of cabergoline on human NFPA tissue. We assessed DRD2 expression levels via immunohistochemistry and qPCR in a large cohort of NFPAs (n=40) and in few prolactinomas (n=10). D2DR has two different isoforms, long (D2RL) and short (D2RS), which are differently expressed and can induce different effects on cell viability. In cabergoline-sensitive prolactinomas D2RS is the predominant isoform. NFPAs shown 10-fold and 5-fold decrease expression of D2RS and D2RL compared to prolactinomas, respectively. Additionally, in NFPAs we observed a significant decrease of cAMP production after cabergoline treatment (45% ±7; p We have also seen in NFPAs downregulation of Ga i2 protein expression levels, highlighting the general decrease of expression levels in the dopamine pathway (50% less, p Our data suggest that the difference in cabergoline responses between NFPAs and prolactinomas may be due to their distinct expression of D2DR isoforms, which differ by 29 amino acids in the third cytoplasmic loop, essential for G-protein binding. It has been shown that D2RL, but not D2RS, requires Gai2. The difference in D2R isoform expression could translate to distinct G-protein activation, ultimately leading to the contrasting viability results after cabergoline treatment. Taken together, these data will help inform future treatment strategies for patients with NFPAs. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Introduction: Tumour-associated fibroblasts (TAFs) are important elements of the tumour microenvironment (TME) well-studied in several cancers, but their role in pituitary adenomas (PAs) has never been shown. We aimed to study the role of TAFs in PAs and its response to somatostatin analogues (SSA). Methods: TAFs were isolated from 11 freshly-cultured NFPAs and 5 GHomas as confirmed by vimentin and αSMA immunostaining. TAF presence in PAs was confirmed by immunohistochemistry, and further assessed with the gene-signature based method xCELL on a different set of samples. Macrophage infiltration, angiogenesis and epithelial-to-mesenchymal pathways were evaluated by immunohistochemistry for CD68, CD163, HLA-DR, E-cadherin, ZEB1 and CD31. TAFs secretome was assessed in vitro on culture supernatants, at baseline and after treatment with pasireotide (10-7M) using Millipore MILLIPLEX human cytokine 42-plex. GH3 cells were treated with TAF-conditioned media (CM) and normal skin fibroblasts-CM (F-CM). Cell morphology (analysed by ImageJ), invasion and epithelial-to-mesenchymal pathways were assessed. Results: CCL2 and eotaxin-1 were identified as the top secreted cytokines in TAF supernatants, followed by VEGF-A, CCL22, IL-6, FGF-2 and IL-8. TAF secretomes from NFPAs and GHomas did not differ significantly. PAs with cavernous sinus invasion had higher TAF-derived IL-6 levels compared to non-invasive PAs (72.7±10.7 vs 43.9±6.3pg/mL; p=0.027), while there was a trend for TAFs from PAs with higher Ki67 to secrete more CCL2 (p=0.058). Correlation between macrophages and TAF-derived FGF-2 was found (r=0.499, p=0.049), and increased FGF-2 and CXCL1 levels were seen in PAs with a macrophage M2:M1 ratio≥2. CCL2 levels were correlated with microvessel area (r=0.672, p=0.004), whereas PDGF-AA was negatively correlated with E-cadherin immunoreactivity (r=-0.564, p=0.023). RT-qPCR analyses indicated that the sst1 receptor is the predominant somatostatin receptor expressed in TAFs, while sst2 and sst5 receptors are poorly expressed. Pasireotide treatment decreased TAF-derived IL-6 by 80% (p

Background: Cushing’s disease is a challenging endocrine disorder caused by non-physiological cortisol excess from adrenocorticotropic hormone (ACTH) secreting pituitary adenoma. Inferior petrosal venous sampling (IPSS) is considered the gold standard investigation to differentiate Cushing’s disease from ectopic ACTH syndrome. Methods: This retrospective study included all patients who underwent IPSS between January 2011 and October 2020 at The National Hospital for Neurology and Neurosurgery in London. Patients demographics, radiological, surgical, endocrinological and, histological data were retrieved. We assessed the accuracy of IPSS in localizing ACTH secreting pituitary adenoma and its concordance with neuroimaging and surgical findings at the time of tumor resection. Results: In total 68 patients underwent IPSS, 22 males and 46 females. The median age was 42 years. IPSS was performed prior to primary surgery in 61 patients (90%) and before secondary surgery in 4 patients (6%). Three patients (4%) are awaiting surgery at the time of our study. Fifty-two patients (80%) had positive histology of ACTH expressing adenoma. Four patients (8%) had ectopic ACTH syndrome. The sensitivity of IPSS in predicting Cushing’s disease prior to corticotropin-releasing hormone (CRH) stimulation was 91% (95% CI [83% to 97%]) and accuracy of 88% (95% CI [77% to 95%]). The sensitivity of IPSS post CRH administration was 96% (95% CI [87% to 100%]) with accuracy of 86% (95% CI [75% to 93%]). Data on lateralization of pituitary adenoma were available for 63 patients. Prior to CRH stimulation, lateralization was right sided in 35 patients (56%), left sided in 15 (24%), and 13 patients (20%) did not have adequate interpetrosal sinus ACTH ratio. Lateralization post CRH stimulation was right sided in 40 patients (64%), left sided in 20 (32%), and 3 patients (4%) did not achieve adequate ACTH gradient ratio between two sides. Eighteen patients (30%) switched adenoma lateralization between pre and post CRH stimulation. Post CRH IPSS was consistent with neuroradiology in localizing pituitary adenomas in 59% (29 out of 49 patients) and concordant with surgical findings in 41% (25 out of 61 patients). Patients with ectopic ACTH syndrome had negative IPSS at all stages. IPSS procedure failed in 2 patients (3%). No post procedure complications were reported. Conclusion: IPSS has high sensitivity in diagnosing pituitary driven Cushing’s disease with good safety profile. However, the reliability in lateralizing pituitary adenoma is debated. One third of patients in this cohort switched lateralization before and after CRH administration. Studies on monkeys and rats showed that CRH induces coronary vasodilation and reduction in systemic vascular resistance. This suggest that CRH might have vasoactive effect on pituitary blood vessels with subsequent influence on IPS:P ACTH gradient ratio between the two sides.