Integrative Clinical, Radiological, and Molecular Analysis for Predicting Remission and Recurrence of Cushing Disease.

CONTEXT: Adrenocorticotropin (ACTH)-secreting pituitary tumors (ACTHomas) are associated with severe comorbidities and increased mortality. Current treatments mainly focus on remission and prevention of persistent disease and recurrence. However, there are still no useful biomarkers to accurately predict the clinical outcome after surgery, long-term remission, or disease relapse. OBJECTIVES: This work aimed to identify clinical, biochemical, and molecular markers for predicting long-term clinical outcome and remission in ACTHomas. METHODS: A retrospective multicenter study was performed with 60 ACTHomas patients diagnosed between 2004 and 2018 with at least 2 years' follow-up. Clinical/biochemical variables were evaluated yearly. Molecular expression profile of the somatostatin/ghrelin/dopamine regulatory systems components and of key pituitary factors and proliferation markers were evaluated in tumor samples after the first surgery. RESULTS: Clinical variables including tumor size, time until diagnosis/first surgery, serum prolactin, and postsurgery cortisol levels were associated with tumor remission and relapsed disease. The molecular markers analyzed were distinctly expressed in ACTHomas, with some components (ie, SSTR1, CRHR1, and MKI67) showing instructive associations with recurrence and/or remission. Notably, an integrative model including selected clinical variables (tumor size/postsurgery serum cortisol), and molecular markers (SSTR1/CRHR1) can accurately predict the clinical evolution and remission of patients with ACTHomas, generating a receiver operating characteristic curve with an area under the curve of 1 (P < .001). CONCLUSION: This study demonstrates that the combination of a set of clinical and molecular biomarkers in ACTHomas is able to accurately predict the clinical evolution and remission of patients. Consequently, the postsurgery molecular profile represents a valuable tool for clinical evaluation and follow-up of patients with ACTHomas.

Sex Hormone Receptor Expression in Craniopharyngiomas and Association with Tumor Aggressiveness Characteristics.

Craniopharyngiomas (CPs) are rare tumors of the sellar and suprasellar regions of embryonic origin. The primary treatment for CPs is surgery but it is often unsuccessful. Although CPs are considered benign tumors, they display a relatively high recurrence rate that might compromise quality of life. Previous studies have reported that CPs express sex hormone receptors, including estrogen and progesterone receptors. Here, we systematically analyzed estrogen receptor α (ERα) and progesterone receptor (PR) expression by immunohistochemistry in a well-characterized series of patients with CP (n = 41) and analyzed their potential association with tumor aggressiveness features. A substantial proportion of CPs displayed a marked expression of PR. However, most CPs expressed low levels of ERα. No major association between PR and ERα expression and clinical aggressiveness features was observed in CPs. Additionally, in our series, ß-catenin accumulation was not related to tumor recurrence.

GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells.

In response to liver injury, hepatic stellate cells activate and acquire proliferative and contractile features. The regression of liver fibrosis appears to involve the clearance of activated hepatic stellate cells, either by apoptosis or by reversion toward a quiescent-like state, a process called deactivation. Thus, deactivation of active hepatic stellate cells has emerged as a novel and promising therapeutic approach for liver fibrosis. However, our knowledge of the master regulators involved in the deactivation and/or activation of fibrotic hepatic stellate cells is still limited. The transcription factor GATA4 has been previously shown to play an important role in embryonic hepatic stellate cell quiescence. In this work, we show that lack of GATA4 in adult mice caused hepatic stellate cell activation and, consequently, liver fibrosis. During regression of liver fibrosis, Gata4 was reexpressed in deactivated hepatic stellate cells. Overexpression of Gata4 in hepatic stellate cells promoted liver fibrosis regression in CCl4-treated mice. GATA4 induced changes in the expression of fibrogenic and antifibrogenic genes, promoting hepatic stellate cell deactivation. Finally, we show that GATA4 directly repressed EPAS1 transcription in hepatic stellate cells and that stabilization of the HIF2α protein in hepatic stellate cells leads to liver fibrosis.

Quantitative Analysis of Somatostatin and Dopamine Receptors Gene Expression Levels in Non-functioning Pituitary Tumors and Association with Clinical and Molecular Aggressiveness Features.

The primary treatment for non-functioning pituitary tumors (NFPTs) is surgery, but it is often unsuccessful. Previous studies have reported that NFPTs express receptors for somatostatin (SST1-5) and dopamine (DRDs) providing a rationale for the use of dopamine agonists and somatostatin analogues. Here, we systematically assessed SST1-5 and DRDs expression by real-time quantitative PCR (RT-qPCR) in a large group of patients with NFPTs (n = 113) and analyzed their potential association with clinical and molecular aggressiveness features. SST1-5 expression was also evaluated by immunohistochemistry. SST3 was the predominant SST subtype detected, followed by SST2, SST5, and SST1. DRD2 was the dominant DRD subtype, followed by DRD4, DRD5, and DRD1. A substantial proportion of NFPTs displayed marked expression of SST2 and SST5. No major association between SSTs and DRDs expression and clinical and molecular aggressiveness features was observed in NFPTs.

GATA factors in pancreas development and disease.

There is an urgent need for the development of novel therapeutics options for diabetic patients given the high prevalence of diabetes worldwide and that, currently, there is no cure for this disease. The transplantation of pancreatic islets that contain insulin-producing cells is a promising therapeutic alternative, particularly for type 1 diabetes. However, the shortage of organ donors constitutes a major limitation for this approach; thus, developing alternative sources of insulin-producing cells is of critical importance. In the last decade, our knowledge of the molecular mechanisms controlling embryonic pancreas development has significantly advanced. More importantly, this knowledge has provided the basis for the in vitro generation of insulin-producing cells from stem cells. Recent studies have revealed that GATA transcription factors are involved in various stages of pancreas formation and in the adult ß cell function. Here, we review the fundamental role of GATA transcription factors in pancreas morphogenesis and their association with congenital diseases associated with pancreas.

Chronic adult-onset of growth hormone/IGF-I hypersecretion improves cognitive functions and LTP and promotes neuronal differentiation in adult rats.

AIM: Besides their metabolic and endocrine functions, the growth hormone (GH) and its mediated factor, the insulin-like growth factor I (IGF-I), have been implicated in different brain functions, including neurogenesis. Long-lasting elevated GH and IGF-I levels result in non-reversible somatic, endocrine and metabolic morbidities. However, the subcutaneous implantation of the GH-secreting (GH-S) GC cell line in rats leads to the controllable over-secretion of GH and elevated IGF-I levels, allowing the experimental study of their short-term effects on brain functions. METHODS: Adult rats were implanted with GC cells and checked 10 weeks later, when a GH/IGF-I-secreting tumour was already formed. RESULTS: Tumour-bearing rats acquired different operant conditioning tasks faster and better than controls and tumour-resected groups. They also presented better retentions of long-term memories in the passive avoidance test. Experimentally evoked long-term potentiation (LTP) in the hippocampus was also larger and longer lasting in the tumour bearing than in the other groups. Chronic adult-onset of GH/IGF-I hypersecretion caused an acceleration of early progenitors, facilitating a faster neural differentiation, maturation and integration in the dentate gyrus, and increased the complexity of dendritic arbours and spine density of granule neurons. CONCLUSION: Thus, adult-onset hypersecretion of GH/IGF-I improves neurocognitive functions, long-term memories, experimental LTP and neural differentiation, migration and maturation.

Loss of GATA4 causes ectopic pancreas in the stomach.

Pancreatic heterotopia is defined as pancreatic tissue outside its normal location in the body and anatomically separated from the pancreas. In this work we have analyzed the stomach glandular epithelium of Gata4 flox/flox ; Pdx1-Cre mice (Gata4KO mice). We found that Gata4KO glandular epithelium displays an atypical morphology similar to the cornified squamous epithelium and exhibits upregulation of forestomach markers. The developing gastric units fail to form properly, and the glandular epithelial cells do not express markers of gastric gland in the absence of GATA4. Of interest, the developing glands of the Gata4KO stomach express pancreatic cell markers. Furthermore, a mass of pancreatic tissue located in the subserosa of the Gata4KO stomach is observed at adult stages. Heterotopic pancreas found in Gata4-deficient mice contains all three pancreatic cell lineages: ductal, acinar, and endocrine. Moreover, Gata4 expression is downregulated in ectopic pancreatic tissue of some human biopsy samples. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A Somatostatin Receptor Subtype-3 (SST3) Peptide Agonist Shows Antitumor Effects in Experimental Models of Nonfunctioning Pituitary Tumors.

PURPOSE: Somatostatin analogues (SSA) are efficacious and safe treatments for a variety of neuroendocrine tumors, especially pituitary neuroendocrine tumors (PitNET). Their therapeutic effects are mainly mediated by somatostatin receptors SST2 and SST5. Most SSAs, such as octreotide/lanreotide/pasireotide, are either nonselective or activate mainly SST2. However, nonfunctioning pituitary tumors (NFPTs), the most common PitNET type, mainly express SST3 and finding peptides that activate this particular somatostatin receptor has been very challenging. Therefore, the main objective of this study was to identify SST3-agonists and characterize their effects on experimental NFPT models. EXPERIMENTAL DESIGN: Binding to SSTs and cAMP level determinations were used to screen a peptide library and identify SST3-agonists. Key functional parameters (cell viability/caspase activity/chromogranin-A secretion/mRNA expression/intracellular signaling pathways) were assessed on NFPT primary cell cultures in response to SST3-agonists. Tumor growth was assessed in a preclinical PitNET mouse model treated with a SST3-agonist. RESULTS: We successfully identified the first SST3-agonist peptides. SST3-agonists lowered cell viability and chromogranin-A secretion, increased apoptosis in vitro, and reduced tumor growth in a preclinical PitNET model. As expected, inhibition of cell viability in response to SST3-agonists defined two NFPT populations: responsive and unresponsive, wherein responsive NFPTs expressed more SST3 than unresponsive NFPTs and exhibited a profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT signaling pathways upon SST3-agonist treatments. Concurrently, SSTR3 silencing increased cell viability in a subset of NFPTs. CONCLUSIONS: This study demonstrates that SST3-agonists activate signaling mechanisms that reduce NFPT cell viability and inhibit pituitary tumor growth in experimental models that expresses SST3, suggesting that targeting this receptor could be an efficacious treatment for NFPTs.

Surgical Outcome and Treatment of Thyrotropin-Secreting Pituitary Tumors in a Tertiary Referral Center.

OBJECTIVE: Thyrotropin (TSH)-secreting pituitary tumors are rare and typically present with hyperthyroidism. Here we report the diagnosis, treatment, and surgical outcomes in a series of patients with TSH-secreting pituitary tumors in a tertiary referral center. METHODS: Descriptive retrospective study that included all patients with TSH-secreting pituitary tumors who underwent transsphenoidal surgery in the endocrinology and nutrition unit of the Virgen del Rocío University Hospital (Seville, Spain) between 2004 and 2016. RESULTS: The mean age at diagnosis was 42.8 ± 17 years. The mean time from onset of symptoms to diagnosis was 13 ± 10 months. Four patients displayed symptoms indicating hyperthyroidism (1 suffered from tachycardia); 3 patients showed symptoms because of mass effect (visual impairment and headache) and 3 patients were diagnosed based on incidental findings after routine blood tests (high free thyroxine levels). Eight patients had macroadenomas, and 2 patients had microadenomas. Five patients underwent conventional pituitary surgery, and 5 patients underwent expanded endoscopic transsphenoidal surgery. Six patients achieved cure after surgery. The other patients received radiotherapy and/or treatment with somatostatin analogs. Analysis of somatostatin receptor (SSTR) expression by immunohistochemistry could be performed in 6 tumors. CONCLUSIONS: Our results confirm the clinical and hormonal heterogeneity caused by TSH-secreting pituitary adenomas. Surgery is considered the first choice of treatment for these tumors. We observed surgical cure rates similar to those reported in recent published series. SSTR2 and SSTR3 are highly expressed in TSH-secreting pituitary adenomas. Our results suggest that somatostatin analog treatment may be also helpful in the treatment of TSH-secreting pituitary adenomas.

Surgical Outcomes and Comorbidities in Cushing Disease: 30 Years of Experience in a Referral Center.

OBJECTIVE: Cushing disease (CD) is a rare, poorly understood entity. Our aim was to add our clinical experience of >30 years in a multidisciplinary specialized unit to the global knowledge of CD. METHODS: This descriptive retrospective study included all patients admitted to the Endocrinology and Nutrition Department of the Virgen del Rocío University Hospital, Seville, Spain, from January 1980 to May 2016. All patients had a definitive diagnosis of CD. RESULTS: Total sample included 119 patients; 100 (84%) were female. Median age at diagnosis was 37.97 years (interquartile range [IQR]: 25.89-45.07 years). Median follow-up was 88 months (IQR: 45.50-157.00 months). Most tumors were microadenomas (62/95) (5.1 mm [IQR: 4.0-7.0 mm]) without sinus invasion. Surgical procedures were conventional transsphenoidal surgery (CTSS) (101/108; cured 70 after first attempt) and expanded endoscopic transsphenoidal surgery (EETSS) (7/108; cured 5 after first attempt); 11 patients did not receive surgical treatment. Fourteen patients received radiotherapy after a first surgery and 5 patients after a second surgical removal attempt. In 13 patients (12.04%), CD relapse was demonstrated after initial CTSS (median disease-free period 65 months [IQR: 45-120 months]). Ten patients developed panhypopituitarism owing to the surgical procedure (CTSS); 8 patients developed panhypopituitarism after adjuvant radiotherapy. CONCLUSIONS: We observed slightly inferior cure rate after first surgery compared with moderately better relapse rates and time to relapse. Radiotherapy after surgery failure seemed to be more effective than CTSS; however, EETSS may be a valid alternative. Postoperative panhypopituitarism rate after first surgery was lower than expected; after radiotherapy, our results were comparable to other series.

AIP and MEN1 mutations and AIP immunohistochemistry in pituitary adenomas in a tertiary referral center.

BACKGROUND: Pituitary adenomas have a high disease burden due to tumor growth/invasion and disordered hormonal secretion. Germline mutations in genes such as MEN1 and AIP are associated with early onset of aggressive pituitary adenomas that can be resistant to medical therapy. AIMS: We performed a retrospective screening study using published risk criteria to assess the frequency of AIP and MEN1 mutations in pituitary adenoma patients in a tertiary referral center. METHODS: Pituitary adenoma patients with pediatric/adolescent onset, macroadenomas occurring ≤30 years of age, familial isolated pituitary adenoma (FIPA) kindreds and acromegaly or prolactinoma cases that were uncontrolled by medical therapy were studied genetically. We also assessed whether immunohistochemical staining for AIP (AIP-IHC) in somatotropinomas was associated with somatostatin analogs (SSA) response. RESULTS: Fifty-five patients met the study criteria and underwent genetic screening for AIP/MEN1 mutations. No mutations were identified and large deletions/duplications were ruled out using MLPA. In a cohort of sporadic somatotropinomas, low AIP-IHC tumors were significantly larger (P = 0.002) and were more frequently sparsely granulated (P = 0.046) than high AIP-IHC tumors. No significant relationship between AIP-IHC and SSA responses was seen. CONCLUSIONS: Germline mutations in AIP/MEN1 in pituitary adenoma patients are rare and the use of general risk criteria did not identify cases in a large tertiary-referral setting. In acromegaly, low AIP-IHC was related to larger tumor size and more frequent sparsely granulated subtype but no relationship with SSA responsiveness was seen. The genetics of pituitary adenomas remains largely unexplained and AIP screening criteria could be significantly refined to focus on large, aggressive tumors in young patients.

E-cadherin expression is associated with somatostatin analogue response in acromegaly.

Acromegaly is a rare disease resulting from hypersecretion of growth hormone (GH) and insulin-like growth factor 1 (IGF1) typically caused by pituitary adenomas, which is associated with increased mortality and morbidity. Somatostatin analogues (SSAs) represent the primary medical therapy for acromegaly and are currently used as first-line treatment or as second-line therapy after unsuccessful pituitary surgery. However, a considerable proportion of patients do not adequately respond to SSAs treatment, and therefore, there is an urgent need to identify biomarkers predictors of response to SSAs. The aim of this study was to examine E-cadherin expression by immunohistochemistry in fifty-five GH-producing pituitary tumours and determine the potential association with response to SSAs as well as other clinical and histopathological features. Acromegaly patients with tumours expressing low E-cadherin levels exhibit a worse response to SSAs. E-cadherin levels are associated with GH-producing tumour histological subtypes. Our results indicate that the immunohistochemical detection of E-cadherin might be useful in categorizing acromegaly patients based on the response to SSAs.

Stabilization of HIF-2α impacts pancreas growth.

Hypoxia inducible factors (HIFs) are critical regulators of the response to oxygen deficiency by activating target genes involved in a variety of biological functions. HIFs have been implicated in the pathophysiology of numerous pathologies including cancer. Patients with mutations in the von Hippel-Lindau (VHL) gene, an essential regulator of HIF activity, develop tumors in several organs including the pancreas. Previous functional studies of HIF activation in the pancreas have used Vhlh (the murine homolog of VHL) deficient mice. However, the role of each specific HIF transcription factors in the pancreas has not been thoroughly examined. We derived mice that constitutively express a normoxia-stable form of HIF2α in the pancreas. Activation of HIF2α in the pancreas severely impairs postnatal exocrine pancreas. Mice with pancreas-specific activation of HIF2α develop histological features reminiscent of pancreatitis including loss of acinar cells, ductal dilation and fibrosis. Moreover, we provide evidence that signaling pathways important for acinar cell homeostasis are altered in HIF2α-overexpressing pancreata.

Association between dopamine and somatostatin receptor expression and pharmacological response to somatostatin analogues in acromegaly.

Acromegaly is a hormonal disorder resulting from excessive growth hormone (GH) secretion frequently produced by pituitary adenomas and consequent increase in insulin-like growth factor 1 (IGF-I). Elevated GH and IGF-I levels result in a wide range of somatic, cardiovascular, endocrine, metabolic and gastrointestinal morbidities. Somatostatin analogues (SSAs) form the basis of medical therapy for acromegaly and are currently used as first-line treatment or as second-line therapy in patients undergoing unsuccessful surgery. However, a considerable percentage of patients do not respond to SSAs treatment. Somatostatin receptors (SSTR1-5) and dopamine receptors (DRD1-5) subtypes play critical roles in the regulation of hormone secretion. These receptors are considered important pharmacological targets to inhibit hormone oversecretion. It has been proposed that decreased expression of SSTRs may be associated with poor response to SSAs. Here, we systematically examine SSTRs and DRDs expression in human somatotroph adenomas by quantitative PCR. We observed an association between the response to SSAs treatment and DRD4, DRD5, SSTR1 and SSTR2 expression. We also examined SSTR expression by immunohistochemistry and found that the immunohistochemical detection of SSTR2 in particular might be a good predictor of response to SSAs.

GATA6 Controls Insulin Biosynthesis and Secretion in Adult ß-Cells.

GATA4 and GATA6 play essential, but redundant, roles in pancreas formation in mice, and GATA6 mutations cause pancreatic agenesis in humans. GATA6 mutations have also recently been linked to adult-onset diabetes, with subclinical or no exocrine insufficiency, suggesting an important role for GATA6 in human ß-cell physiology. To investigate the role of GATA6 in the adult endocrine pancreas, we generated mice in which Gata6 is specifically inactivated in the pancreas. These mice develop glucose intolerance. Islets deficient in GATA6 activity display decreased insulin content and impaired insulin secretion. Gata6-deficient ß-cells exhibit ultrastructural abnormalities, including increased immature insulin granules, swollen mitochondria, and disorganized endoplasmic reticulum. We also demonstrate that Pdx1 expression in adult ß-cells depends on GATA sites in transgenic reporter mice and that loss of GATA6 greatly affects ß-cell-specific gene expression. These findings demonstrate the essential role of GATA6 in ß-cell function.

An approach to screening for Cushing's syndrome in non-specialized health care settings / Una aproximación al cribado del síndrome de Cushing en un entorno asistencial no especializado

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Mitochondrial Dynamics Mediated by Mitofusin 1 Is Required for POMC Neuron Glucose-Sensing and Insulin Release Control.

Proopiomelanocortin (POMC) neurons are critical sensors of nutrient availability implicated in energy balance and glucose metabolism control. However, the precise mechanisms underlying nutrient sensing in POMC neurons remain incompletely understood. We show that mitochondrial dynamics mediated by Mitofusin 1 (MFN1) in POMC neurons couple nutrient sensing with systemic glucose metabolism. Mice lacking MFN1 in POMC neurons exhibited defective mitochondrial architecture remodeling and attenuated hypothalamic gene expression programs during the fast-to-fed transition. This loss of mitochondrial flexibility in POMC neurons bidirectionally altered glucose sensing, causing abnormal glucose homeostasis due to defective insulin secretion by pancreatic ß cells. Fed mice lacking MFN1 in POMC neurons displayed enhanced hypothalamic mitochondrial oxygen flux and reactive oxygen species generation. Central delivery of antioxidants was able to normalize the phenotype. Collectively, our data posit MFN1-mediated mitochondrial dynamics in POMC neurons as an intrinsic nutrient-sensing mechanism and unveil an unrecognized link between this subset of neurons and insulin release.

Loss of Pancreas upon Activated Wnt Signaling Is Concomitant with Emergence of Gastrointestinal Identity.

Organ formation is achieved through the complex interplay between signaling pathways and transcriptional cascades. The canonical Wnt signaling pathway plays multiple roles during embryonic development including patterning, proliferation and differentiation in distinct tissues. Previous studies have established the importance of this pathway at multiple stages of pancreas formation as well as in postnatal organ function and homeostasis. In mice, gain-of-function experiments have demonstrated that activation of the canonical Wnt pathway results in pancreatic hypoplasia, a phenomenon whose underlying mechanisms remains to be elucidated. Here, we show that ectopic activation of epithelial canonical Wnt signaling causes aberrant induction of gastric and intestinal markers both in the pancreatic epithelium and mesenchyme, leading to the development of gut-like features. Furthermore, we provide evidence that ß -catenin-induced impairment of pancreas formation depends on Hedgehog signaling. Together, our data emphasize the developmental plasticity of pancreatic progenitors and further underscore the key role of precise regulation of signaling pathways to maintain appropriate organ boundaries.