Non-functioning Pituitary Neuroendocrine Tumors Research Articles

MiRNA Signatures Related to Invasiveness and Recurrence in Patients With Non-Functioning Pituitary Neuroendocrine Tumors.

This preliminary study aimed to analyze and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary neuroendocrine tumors (NFPitNET). The relationship between deregulated miRNAs and tumor invasiveness, recurrence, and size was determined. Twenty patients with NFPitNET were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analyzed by quantitative real-time polymerase chain reaction (PCR) using LNA miRNA Cancer-Focus PCR Panel (Qiagen). Three miRNAs (miR-210-3p, miR-149-3p, and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPitNETs. Differential expression of four-miRNA signatures - miRNA-17, miR-19, miR-106a, and miR-20, correlated with patient recurrence. This prospective pilot study selected a unique miRNA expression profile, that correlates with invasiveness and recurrence in non-functioning pituitary neuroendocrine tumors. Moreover, some of the selected miRNAs are reported for the first time in patients with this disease, shedding light on the molecular mechanisms involved in pituitary pathogenesis. The identified miRNAs demonstrate potential as biomarkers, deserving further investigation in a larger cohort to validate their clinical applicability.

Risk Factors for Isolated Sphenoid Sinusitis after Endoscopic Endonasal Transsphenoidal Pituitary Surgery.

(1) Background: Transsphenoidal pituitary surgery can be conducted via microscopic or endoscopic approaches, and there has been a growing preference for the latter in recent years. However, the occurrence of rare complications such as postoperative sinusitis remains inadequately documented in the existing literature. (2) Methods: To address this gap, we conducted a comprehensive retrospective analysis of medical records spanning from 2018 to 2023, focusing on patients who underwent transsphenoidal surgery for pituitary neuroendocrine tumors (formerly called pituitary adenoma). Our study encompassed detailed evaluations of pituitary function and MRI imaging pre- and postsurgery, supplemented by transnasal endoscopic follow-up assessments at the otolaryngology outpatient department. Risk factors for sinusitis were compared using univariate and multivariate logistic regression analyses. (3) Results: Out of the 203 patients included in our analysis, a subset of 17 individuals developed isolated sphenoid sinusitis within three months postoperation. Further scrutiny of the data revealed significant associations between certain factors and the occurrence of postoperative sphenoid sinusitis. Specifically, the classification of the primary tumor emerged as a notable risk factor, with patients exhibiting nonfunctioning pituitary neuroendocrine tumors with 3.71 times the odds of developing sinusitis compared to other tumor types. Additionally, postoperative cortisol levels demonstrated a significant inverse relationship, with lower cortisol levels correlating with an increased risk of sphenoid sinusitis postsurgery. (4) Conclusions: In conclusion, our findings underscore the importance of considering tumor classification and postoperative cortisol levels as potential predictors of postoperative sinusitis in patients undergoing transsphenoidal endoscopic pituitary surgery. These insights offer valuable guidance for clinicians in identifying at-risk individuals and implementing tailored preventive and management strategies to mitigate the occurrence and impact of sinusitis complications in this patient population.

499 Silent Corticotroph Staining Pituitary Neuroendocrine Tumors: Prognostic Significance in Radiosurgery

INTRODUCTION: There is conflicting evidence on the significance of adrenocorticotrophic hormone (ACTH) staining in the prognosis of nonfunctioning pituitary neuroendocrine tumors (NFpitNETs). METHODS: This retrospective, multicenter study included patients managed with SRS for NFpitNET residuals. The patients were divided into two cohorts: 1) silent corticotroph (SC) for NFpitNETs with positive ACTH immunostaining and 2) non-SC NFpitNETs. Rates of local tumor control, as well as the incidence of post-treatment pituitary and neurological dysfunction were documented. Factors associated with radiological and clinical outcomes were also analyzed. RESULTS: The cohort included 535 patients from fourteen centers with 84 (15.7%) patients harboring silent corticotroph NFpitNETs (SCs). At last follow-up, local tumor progression occurred in 11.9% of patients in the SC compared to 8.1% of patients in the non-SC cohort (p = 0.27). No statistically significant difference was noted in new-onset hypopituitarism rates (10.7% vs 15.4%, p = 0.25) or visual deficits (3.6% vs 1.1%, p = 0.088) between the two cohorts at last follow-up. When controlling for residual tumor volume, maximum dose, and patient age and gender, positive ACTH immunostaining did not have a significant correlation with local tumor progression (HR = 1.69, 95% CI = 0.8 - 3.61, p = 0.17). CONCLUSIONS: In contemporary radiosurgical practice with a single fraction dose of 8-25 Gy (median 15 Gy), ACTH immunostaining in NFpitNETs did not appear to confer a significantly reduced rate of local tumor control after SRS.

Erythropoietin-producing hepatocellular receptor B6 is highly expressed in non-functioning pituitary neuroendocrine tumors and its expression correlates with tumor size

BackgroundErythropoietin-producing hepatocellular (EPH) receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The Erythropoietin-producing hepatocellular receptor tyrosine kinase member EphB6 is a pseudokinase which has not attracted an equivalent amount of interest as its enzymatically-active counterparts. The aim of this study was to assess the expression of EphB6 in pituitary tumors.Methods and ResultsHuman normal pituitaries and pituitary tumors were examined for EphB6 mRNA expression using real-time PCR and for EphB6 protein by immunohistochemistry and Western blotting. EphB6 was highly expressed in non-functioning pituitary neuroendocrine tumors (NF-PitNETs) versus the normal pituitary and GH-secreting PitNETs. EphB6 mRNA expression was correlated with tumor size.ConclusionsOur results suggest EphB6 aberrant expression in NF-PitNETs. Future studies are warranted to determine the role and significance of EphB6 in NF-PitNETs tumorigenesis.

Differences in invasiveness and recurrence rate among nonfunctioning pituitary neuroendocrine tumors depending on tumor subtype.

To clarify the invasiveness to surrounding structures and recurrence rate of each subtype of nonfunctioning pituitary neuroendocrine tumor (Pit-NETs) according to the WHO 2022 classification. This retrospective study utilized data from 292 patients with nonfunctioning Pit-NETs treated with initial transsphenoidal surgery. Recurrence was evaluated on 113 patients who were available for a magnetic resonance imaging follow-up ≥ 60months. All tumors were assessed by immunohistochemical staining for Pit-1, T-PIT, and GATA3. Invasiveness to surrounding structures was evaluated based on intraoperative findings. Cavernous sinus invasion was found in 47.5% of null cell tumors, 50.0% of Pit-1 lineage tumors, 31.8% of corticotroph tumors, and 18.3% of gonadotroph tumors. Dura mater defects in the floor of sellar turcica, indicating dural invasion, were found in 44.3% of null cell tumors, 36.4% of corticotroph tumors, 16.7% of Pit-1 lineage tumors, and 17.3% of gonadotroph tumors. In logistic regression analysis, Pit-1 (OR 5.90, 95% CI 1.71-20.4, P = 0.0050) and null tumors (OR 4.14, 95% CI 1.86-9.23, P = 0.0005) were associated with cavernous sinus invasion. Recurrence was found in 8 (4.9%) patients, but without significant differences between tumor subtypes. The presence of cavernous sinus invasion was correlated with recurrence (HR = 1.95, 95% CI 1.10-3.46, P = 0.0227). Among nonfunctioning Pit-NETs, Pit-1 lineage tumors tend to invade the cavernous sinus, corticotroph tumors may produce dura mater defects, and null cell tumors tend to cause both. Pit-NETs with cavernous sinus invasion require a careful attention to recurrence.

THU084 Corticotroph Tumor Type Influences Clinical Presentation In Patients With Nonfunctioning Pituitary Neuroendocrine Tumors

Abstract Disclosure: N. Al-Shamkhi: None. B. Edén Engström: None. O. Gudjonsson: None. J. Wikström: None. O. Casar-Borota: None. E. Rask: None. Introduction: According to the current World Health Organization classification of endocrine tumors the classification of non-functioning pituitary neuroendocrine tumors (NF-PitNET) is based on pituitary cell lineages, defined by immunohistochemical expression of anterior pituitary hormones and pituitary specific transcription factors. The second most common NF-PitNET type, after silent gonadotroph tumors, are silent corticotroph tumors that are regarded as a potentially more aggressive type. If there is indication for treatment, the first-line treatment is transsphenoidal surgery. Some, but not all studies have shown a higher degree of preoperative pituitary failure or more frequent postoperative pituitary failure in patients with silent corticotroph tumors compared to the other NF-PitNET. Objectives: We compared patients with silent corticotroph and silent gonadotroph tumors, and explored whether tumor type and radiological findings were associated with pre- or postoperative pituitary failure. Design and method: One hundred patients who underwent surgery for NF-PitNET at the same tertiary center were included. Data regarding age at surgery, sex, pre- and postoperative pituitary function and tumor type, were collected after chart review. Preoperative MRI examinations were revised regarding tumor volume and Knosp classification. Results: The majority, 72% (72/100) were classified as silent gonadotroph tumors followed by 18% (18/100) silent corticotroph tumors. Patients with silent corticotroph tumors were younger at surgery, P=0.003. There were no differences regarding sex, preoperative tumor volume, invasive growth on MRI, defined as Knosp grade > 3, pituitary failure or prolactin elevation between the two tumor types. Binary logistic regression showed that having a silent corticotroph tumor, P=0.04, odds ratio 5.7 (CI: 1.06-30.71), higher age at surgery, P=0.003, odds ratio 1.07 (CI: 1.02-1.13), and a larger preoperative tumor volume, P=0.01, odds ratio 1.18 (CI: 1.04-1.33), were all factors associated with an increased likelihood of having postoperative pituitary failure. Sex, invasive growth and preoperative prolactin elevation were not significant predictors of postoperative pituitary failure in the binary logistic regression. Conclusion: The results indicate that silent corticotroph tumors influence the clinical presentation of NF-PitNET. Patients with silent corticotroph tumors were significantly younger at surgery, and demonstrated an increased likelihood of postoperative pituitary failure. Presentation: Thursday, June 15, 2023

A Novel Magnetic Resonance Imaging-Based Radiomics and Clinical Predictive Model for the Regrowth of Postoperative Residual Tumor in Non-Functioning Pituitary Neuroendocrine Tumor.

Background and Objectives: To develop a novel magnetic resonance imaging (MRI)-based radiomics-clinical risk stratification model to predict the regrowth of postoperative residual tumors in patients with non-functioning pituitary neuroendocrine tumors (NF-PitNETs). Materials and Methods: We retrospectively enrolled 114 patients diagnosed as NF-PitNET with postoperative residual tumors after the first operation, and the diameter of the tumors was greater than 10 mm. Univariate and multivariate analyses were conducted to identify independent clinical risk factors. We identified the optimal sequence to generate an appropriate radiomic score (Rscore) that combined pre- and postoperative radiomic features. Three models were established by logistic regression analysis that combined clinical risk factors and radiomic features (Model 1), single clinical risk factors (Model 2) and single radiomic features (Model 3). The models' predictive performances were evaluated using receiver operator characteristic (ROC) curve analysis and area under curve (AUC) values. A nomogram was developed and evaluated using decision curve analysis. Results: Knosp classification and preoperative tumor volume doubling time (TVDT) were high-risk factors (p < 0.05) with odds ratios (ORs) of 2.255 and 0.173. T1WI&T1CE had a higher AUC value (0.954) and generated an Rscore. Ultimately, the AUC of Model 1 {0.929 [95% Confidence interval (CI), 0.865-0.993]} was superior to Model 2 [0.811 (95% CI, 0.704-0.918)] and Model 3 [0.844 (95% CI, 0.748-0.941)] in the training set, which were 0.882 (95% CI, 0.735-1.000), 0.834 (95% CI, 0.676-0.992) and 0.763 (95% CI, 0.569-0.958) in the test set, respectively. Conclusions: We trained a novel radiomics-clinical predictive model for identifying patients with NF-PitNETs at increased risk of postoperative residual tumor regrowth. This model may help optimize individualized and stratified clinical treatment decisions.

Identification of cholesterol metabolism-related subtypes in nonfunctioning pituitary neuroendocrine tumors and analysis of immune infiltration

ObjectiveThis study aimed to investigate the role of cholesterol metabolism-related genes in nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs) invading the cavernous sinus and analyze the differences in immune cell infiltration between invasive and noninvasive NF-PitNETs.MethodsFirst, a retrospective analysis of single-center clinical data was performed. Second, the immune cell infiltration between invasive and noninvasive NF-PitNETs in the GSE169498 dataset was further analyzed, and statistically different cholesterol metabolism-related gene expression matrices were obtained from the dataset. The hub cholesterol metabolism-related genes in NF-PitNETs were screened by constructing machine learning models. In accordance with the hub gene, 73 cases of NF-PitNETs were clustered into two subtypes, and the functional differences and immune cell infiltration between the two subtypes were further analyzed.ResultsThe clinical data of 146 NF-PitNETs were evaluated, and the results showed that the cholesterol (P = 0.034) between invasive and noninvasive NF-PitNETs significantly differed. After binary logistic analysis, cholesterol was found to be an independent risk factor for cavernous sinus invasion (CSI) in NF-PitNETs. Bioinformatics analysis found three immune cells between invasive and noninvasive NF-PitNETs were statistically significant in the GSE169498 dataset, and 34 cholesterol metabolism-related genes with differences between the two groups were obtained 12 hub genes were selected by crossing the two machine learning algorithm results. Subsequently, cholesterol metabolism-related subgroups, A and B, were obtained by unsupervised hierarchical clustering analysis. The results showed that 12 immune cells infiltrated differentially between the two subgroups. The chi-square test revealed that the two subgroups had statistically significance in the invasive and noninvasive samples (P = 0.001). KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in the neural ligand–receptor pathway. GSVA analysis showed that the mTORC signaling pathway was upregulated and played an important role in the two-cluster comparison.ConclusionBy clinical data and bioinformatics analysis, cholesterol metabolism-related genes may promote the infiltration abundance of immune cells in NF-PitNETs and the invasion of cavernous sinuses by NF-PitNETs through the mTOR signaling pathway. This study provides a new perspective to explore the pathogenesis of cavernous sinus invasion by NF-PitNETs and determine potential therapeutic targets for this disease.

Identification of biomarkers associated with the invasion of nonfunctional pituitary neuroendocrine tumors based on the immune microenvironment.

The invasive behavior of nonfunctioning pituitary neuroendocrine tumors (NF-PitNEts) affects complete resection and indicates a poor prognosis. Cancer immunotherapy has been experimentally used for the treatment of many tumors, including pituitary tumors. The current study aimed to screen the key immune-related genes in NF-PitNEts with invasion. We used two cohorts to explore novel biomarkers in NF-PitNEts. The immune infiltration-associated differentially expressed genes (DEGs) were obtained based on high/low immune scores, which were calculated through the ESTIMATE algorithm. The abundance of immune cells was predicted using the ImmuCellAI database. WGCNA was used to construct a coexpression network of immune cell-related genes. Random forest analysis was used to select the candidate genes associated with invasion. The expression of key genes was verified in external validation set using quantitative real-time polymerase chain reaction (qRT‒PCR). The immune and invasion related DEGs was obtained based on the first dataset of NF-PitNEts (n=112). The immune cell-associated modules in NF-PitNEts were calculate by WGCNA. Random forest analysis was performed on 81 common genes intersected by immune-related genes, invasion-related genes, and module genes. Then, 20 of these genes with the highest RF score were selected to construct the invasion and immune-associated classification model. We found that this model had high prediction accuracy for tumor invasion, which had the largest area under the receiver operating characteristic curve (AUC) value in the training dataset from the first dataset (n=78), the self-test dataset from the first dataset (n=34), and the independent test dataset (n=73) (AUC=0.732/0.653/0.619). Functional enrichment analysis revealed that 8 out of the 20 genes were enriched in multiple signaling pathways. Subsequently, the 8-gene (BMP6, CIB2, FABP5, HOMER2, MAML3, NIN, PRKG2 and SIDT2) classification model was constructed and showed good efficiency in the first dataset (AUC=0.671). In addition, the expression levels of these 8 genes were verified by qRT‒PCR. We identified eight key genes associated with invasion and immunity in NF-PitNEts that may play a fundamental role in invasive progression and may provide novel potential immunotherapy targets for NF-PitNEts.

The Significance of the Intercarotid Distances for Transsphenoidal Pituitary Surgery: A Magnetic Resonance Imaging Study

We performed MRI to analyze the distance between the left and right internal carotid arteries (ICD) around pituitary tumors for transsphenoidal surgery (TSS). Measurements were performed using thin slice T2-weighted MRI, and/or time-of-flight MR angiography imaging for 64 non-functioning pituitary neuroendocrine tumors (NF-pitNETs), 22 growth hormone-producing pitNETs (GH producing pitNETs), and 46 normal controls. We measured the ICD at 3 anatomic levels: at the distal dural ring (ICD-A); at the most concave point of the C4-C5 bend (ICD-B); and at the most convex point of the C4 bend (ICD-C). Additionally, we measured the sagittal distance between the tuberculum selle and the junction of the sellar floor and clivus on the mid sagittal MRI (TS-C). We found that ICD-B was the longest and that ICD-A was the shortest among the three distance parameters in all groups (P<0.001). The comparison of the groups revealed that the NF-pitNET group had longer distances than the normal control group at all ICDs (P<0.001). The GH producing pitNET group had longer distance than the normal control group at ICD-B (P<0.001). Tumor volume was correlated with ICD-A and ICD-B in the NF-pitNET, and was correlated with ICD-C in the GH producing pitNET group. Among the distance parameters, ICD-B is the longest, and was approximately twice the width of the TS-C. The shape of the sella is an ellipse that is widest in the lateral dimension in TSS. A horizontal based dural incision may be more rational than a conventional X-shaped dural incision.

Molecular characterization of epithelial-mesenchymal transition and medical treatment related-genes in non-functioning pituitary neuroendocrine tumors.

Different medical therapies have been developed for pituitary adenomas. However, Non-Functioning Pituitary Neuroendocrine Tumors (NF-PitNET) have shown little response to them. Furthermore, epithelial-mesenchymal transition (EMT) has been linked to resistance to medical treatment in a significant number of tumors, including pituitary adenomas. We aimed to evaluate the expression of EMT-related markers in 72 NF-PitNET and 16 non-tumoral pituitaries. To further explore the potential usefulness of medical treatment for NF-PitNET we assessed the expression of somatostatin receptors and dopamine-associated genes. We found that SNAI1, SNAI2, Vimentin, KLK10, PEBP1, Ki-67 and SSTR2 were associated with invasive NF-PitNET. Furthermore, we found that the EMT phenomenon was more common in NF-PitNET than in GH-secreting pituitary tumors. Interestingly, PEBP1 was overexpressed in recurrent NF-PitNET, and could predict growth recurrence with 100% sensitivity but only 43% specificity. In parallel with previously reported studies, SSTR3 is highly expressed in our NF-PitNET cohort. However, SSTR3 expression is highly heterogeneous among the different histological variants of NF-PitNET with very low levels in silent corticotroph adenomas. NF-PitNET showed an enhanced EMT phenomenon. SSTR3 targeting could be a good therapeutic candidate in NF-PitNET except for silent corticotroph adenomas, which express very low levels of this receptor. In addition, PEBP1 could be an informative biomarker of tumor regrowth, useful for predictive medicine in NF-PitNET.

Expression and Clinical Significance of MDM2 in Non-Functioning PitNETs.

Background and Objective: Non-functioning pituitary neuroendocrine tumors (NF-PitNETs) represent a heterogeneous tumor type that lacks effective medical treatment. MDM2, the main negative regulator of p53, binds to and forms a stable complex with p53 to regulate its activity. In this study, we measured the expression levels and role of MDM2 in non-functioning PitNET patients' combined clinical features and investigated the effect of etoposide on the cell bioactivity of the GT1-1 cell line in vivo and in vitro. Methods: RT-PCR and immunochemistry measured the expression levels and role of MDM2 in 103 NF-PitNET patients' combined clinical features. Cell proliferation, migration, colony and apoptosis experiments measured the effect of etoposide on the GT1-1 cell line in vivo and in vitro. Results: There was more invasive behavior (p = 0.013) in patients with high MDM2, who were also younger (p = 0.007), were more frequently female (p = 0.049) and had larger tumor sizes (p = 0.018) compared with patients with low MDM2. Patients with high p53 were younger (p = 0.017) and had larger tumor sizes (p = 0.034) compared with patients with low p53. Univariate (p = 0.018) and multivariate (p = 0.023) Cox regression analysis showed that MDM2 was the independent factor for invasive behavior in NF-PitNET patients. Log-rank analysis showed that the average progression-free survival (PFS) time in the low MDM2 patients was longer than that in the high MDM2 patients (p = 0.044). Functional studies indicated that etoposide inhibited cell proliferation and cell migration and induced apoptosis in p53 independence in GT1-1 cells. Furthermore, etoposide significantly inhibited the growth of GT1-1-xenograft in BALB/c nude mice. The tumor growth inhibition rate of etoposide was 67.4 ± 4.6% after 14 d of treatment, which suggested the anti-tumor activity of etoposide. Conclusions: MDM2 played the role of tumorigenesis of NF-PitNET in a p53 independence manner, and an MDM2 inhibitor could be a potential choice for the treatment of NF-PitNET patients.

Epigenomic and transcriptomic landscaping unraveled candidate repositioned therapeutics for non-functioning pituitary neuroendocrine tumors.

Non-functioning pituitary neuroendocrine tumors are challengingly diagnosed tumors in the clinic. Transsphenoidal surgery remains the first-line treatment. Despite the development of state-of-the-art techniques, no drug therapy is currently approved for the treatment. There are also no randomized controlled trials comparing therapeutic strategies or drug therapy for the management after surgery. Therefore, novel therapeutic interventions for the therapeutically challenging NF-PitNETs are urgently needed. We integrated epigenome and transcriptome data (both coding and non-coding) that elucidate disease-specific signatures, in addition to biological and pharmacological data, to utilize rational pathway and drug prioritization in NF-PitNETs. We constructed an epigenome- and transcriptome-based PPI network and proposed hub genes. The signature-based drug repositioning based on the integration of multi-omics data was performed. The construction of a disease-specific network based on three different biological levels revealed DCC, DLG5, ETS2, FOXO1, HBP1, HMGA2, PCGF3, PSME4, RBPMS, RREB1, SMAD1, SOCS1, SOX2, YAP1, ZFHX3 as hub proteins. Signature-based drug repositioning using hub proteins yielded repositioned drug candidates that were confirmed in silico via molecular docking. As a result of molecular docking simulations, palbociclib, linifanib, trametinib, eplerenone, niguldipine, and zuclopenthixol showed higher binding affinities with hub genes compared to their inhibitors and were proposed as potential repositioned therapeutics for the management of NF-PitNETs. The proposed systems' biomedicine-oriented multi-omics data integration for drug repurposing to provide promising results for the construction of effective clinical therapeutics. To the best of our knowledge, this is the first study reporting epigenome- and transcriptome-based drug repositioning for NF-PitNETs using in silico confirmations.

Aggressive nonfunctioning pituitary neuroendocrine tumors.

Nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs) are tumors that are not associated with clinical evidence of hormonal hypersecretion. According to the World Health Organization (WHO), there are some subtypes of PitNETs that exhibit more aggressive behavior than others. Among the types of potentially aggressive PitNETs, three are nonfunctional: silent sparsely granulated somatotropinomas, silent corticotropinomas, and poorly differentiated PIT-1 lineage tumors. Several biological markers have been investigated in NF-PitNETs. However, there is no single biomarker able to independently predict aggressive behavior in NF-PitNETs. Thus, a more complex and multidisciplinary proposal of a comprehensive definition of aggressive NF-PitNETs is necessary. Here, we suggest a combined and more complete criterion for the NF-PitNETs classification. We propose that aggressiveness is due to a multifactorial combination, and we emphasize the need to include new emerging markers that are involved in the aggressiveness of NF-PitNETs and the need to identify.

Angpt2/Tie2 autostimulatory loop controls tumorigenesis.

Invasive nonfunctioning (NF) pituitary neuroendocrine tumors (PitNETs) are non‐resectable neoplasms associated with frequent relapses and significant comorbidities. As the current therapies of NF‐PitNETs often fail, new therapeutic targets are needed. The observation that circulating angiopoietin‐2 (ANGPT2) is elevated in patients with NF‐PitNET and correlates with tumor aggressiveness prompted us to investigate the ANGPT2/TIE2 axis in NF‐PitNETs in the GH3 PitNET cell line, primary human NF‐PitNET cells, xenografts in zebrafish and mice, and in MENX rats, the only autochthonous NF‐PitNET model. We show that PitNET cells express a functional TIE2 receptor and secrete bioactive ANGPT2, which promotes, besides angiogenesis, tumor cell growth in an autocrine and paracrine fashion. ANGPT2 stimulation of TIE2 in tumor cells activates downstream cell proliferation signals, as previously demonstrated in endothelial cells (ECs). Tie2 gene deletion blunts PitNETs growth in xenograft models, and pharmacological inhibition of Angpt2/Tie2 signaling antagonizes PitNETs in primary cell cultures, tumor xenografts in mice, and in MENX rats. Thus, the ANGPT2/TIE2 axis provides an exploitable therapeutic target in NF‐PitNETs and possibly in other tumors expressing ANGPT2/TIE2. The ability of tumor cells to coopt angiogenic signals classically viewed as EC‐specific expands our view on the microenvironmental cues that are essential for tumor progression.

Transcriptomic analysis identifies a tumor subtype mRNA classifier for invasive non-functioning pituitary neuroendocrine tumor diagnostics.

Rationale: The invasive behavior of non-functioning pituitary neuroendocrine tumors (NF-PitNEts) presents obstacles for complete surgical resection and is indicative of poor prognosis. Therefore, developing reliable diagnostic tools for identifying invasive PitNEts would be helpful in guiding surgical decisions and, in particular, the follow-up treatment.Methods: We analyzed differential gene expression profiles between 39 non-invasive and 22 invasive NF-PitNEts by high-throughput sequencing, gene co-expression, and functional annotation. Twenty-one transcripts were further validated by Taqman-qPCR in another 143 NF-PitNEt samples. The histological expression and serum-exosomal mRNA of three candidate genes were examined by tissue microarray and droplet digital PCR.Results: Non-invasive and invasive NF-PitNEts were clustered into distinct groups with a few outliers because of their gonadotroph, corticotroph, or null cell lineages. The gene signature with strong invasive potential was enriched in 'Pathways in cancers' and 'MAPK pathway', with significantly higher in situ INSM1 and HSPA2 protein expression in invasive NF-PitNEts. Further integration of the 20 qPCR-validated differentially expressed genes and pituitary cell lineages provided a gene-subtype panel that performed 80.00-90.24% diagnostic accuracy for the invasiveness of NF-PitNEts.Conclusion: Our approach defined new characteristics in the core molecular network for patients at risk for invasive NF-PitNEt, representing a significant clinical advance in invasive PitNEt diagnostics.

Estrogen Receptors in Nonfunctioning Pituitary Neuroendocrine Tumors: Review on Expression and Gonadotroph Functions.

Estrogen (17β-estradiol or E2) is a crucial regulator of the synthesis and secretion of pituitary reproductive hormones luteinizing hormone, follicle-stimulating hormone, and prolactin. In this review, we summarize the role of estrogen receptors in nonfunctioning pituitary neuroendocrine tumors (NF-Pitnets), focusing on immunoexpression and gonadotroph cell proliferation and apoptosis. Gonadotroph tumors are the most common subtype of NF-Pitnets. Two major estrogen receptor (ER) isoforms expressed in the pituitary are estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Overall, estrogen actions are mostly exerted through the ERα isoform on the pituitary. The G protein–coupled estrogen receptor (GPER) located at the plasma membrane may contribute to nongenomic effects of estrogen. Nuclear immunoreactivity for ERα and ERβ was highest among gonadotroph and null cell tumors. Silent corticotroph tumors are the least immunoreactive for both receptors. A significantly elevated ERα expression was observed in macroadenomas compared with microadenomas. ERα and ERβ may act in opposite directions to regulate the Slug-E-cadherin pathway and to affect invasiveness of NF-Pitnets. In the cellular pathway, ERs regulate estrogen-induced proliferation and differentiation and impact several signaling pathways including the MAPK and PI3K/Akt pathway. Estrogen was the first-discovered inducer of pituitary tumor transforming gene 1 that was abundantly expressed in NF-Pitnets. ERα can be a potential biomarker for predicting tumor size and invasiveness as well as therapeutic target for NF-Pitnets. Selective estrogen receptor modulators or antiestrogen may represent as an alternative choice for the treatment of NF-Pitnets.

Beta-Arrestin 2 Is Required for Dopamine Receptor Type 2 Inhibitory Effects on AKT Phosphorylation and Cell Proliferation in Pituitary Tumors

Dopamine receptor type 2 (DRD2) agonists are the first-choice treatment for prolactin-secreting pituitary tumors but are poorly effective in nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs). DRD2 reduces AKT phosphorylation in lactotrophs, but no data are available in NF-PitNETs. DRD2 effects on AKT are mediated by a β-arrestin 2-dependent mechanism in mouse striatum. The aim of this study was to investigate DRD2 effects on AKT phosphorylation and cell proliferation in human primary cultured NF-PitNET cells and in rat tumoral lactotroph cells MMQ, and to test β-arrestin 2 involvement. We found that the DRD2 agonist BIM53097 induced a reduction of the p-AKT/total-AKT ratio in MMQ (–32.8 ± 17.6%, p < 0.001 vs. basal) and in a subset (n = 15/41, 36.6%) of NF-PitNETs (subgroup 1). In the remaining NF-PitNETs (subgroup 2), BIM53097 induced an increase in p-AKT. The ability of BIM53097 to reduce p-AKT correlated with its antimitotic effect, since the majority of subgroup 1 NF-PitNETs was responsive to BIM53097, and nearly all subgroup 2 NF-PitNETs were resistant. β-Arrestin 2 was expressed in MMQ and in 80% of subgroup 1 NF-PitNETs, whereas it was undetectable in 77% of subgroup 2 NF-PitNETs. In MMQ, β-arrestin 2 silencing prevented DRD2 inhibitory effects on p-AKT and cell proliferation. Accordingly, β-arrestin 2 transfection in subgroup 2 NF-PitNETs conferred to BIM53097 the ability to inhibit both p-AKT and cell growth. In conclusion, we demonstrated that β-arrestin 2 is required for DRD2 inhibitory effects on AKT phosphorylation and cell proliferation in MMQ and NF-PitNETs, paving the way for a potential role of β-arrestin 2 as a biomarker predicting NF-PitNETs’ responsiveness to treatment with dopamine agonists.

SAT-312 Beta-Arrestin 2 Is Required for Dopamine Receptor Type 2 Inhibitory Effects on Akt Phosphorylation and Cell Proliferation in Pituitary Tumors

Dopamine receptor type 2 (DRD2) agonists are the first-choice treatment for PRL-secreting pituitary tumors but are poorly effective in non-functioning pituitary neuroendocrine tumors (NF-PitNETs). DRD2 reduces AKT phosphorylation in lactotrophs, but no data are available in NF-PitNETs. DRD2 effects on AKT are mediated by a β-arrestin 2-dependent mechanism in mouse striatum.The aim of this study was to investigate DRD2 effects on AKT phosphorylation and cell proliferation in human primary cultured NF-PitNET cells and in rat tumoral lactotroph cells MMQ, and to test β-arrestin 2 involvement.We found that DRD2 agonist BIM53097 induced a reduction of p- AKT /total-AKT ratio in MMQ (-32.8±17.6%, p<0.001 vs basal) and in a subset (n=15/41,36.6%) of NF-PitNETs (subgroup 1). In the remaining NF-PitNETs (subgroup 2), BIM53097 induced an increase of p- AKT. The ability of BIM53097 to reduce p-AKT correlated to its antimitotic effect, since the majority of subgroup 1 NF-PitNETs was responsive to BIM53097 and nearly all subgroup 2 NF-PitNETs were resistant. β-arrestin2 was expressed in MMQ and in 80% of subgroup 1 NF-PitNETs, whereas it was undetectable in 77% of subgroup 2 NF-PitNETs.In MMQ, β-arrestin 2 silencing prevented DRD2 inhibitory effects on p-AKT and cell proliferation. Accordingly, β-arrestin 2 transfection in subgroup 2 NF-PitNETs conferred to BIM53097 the ability to inhibit both p-AKT and cell growth.In conclusion, we demonstrated that β-arrestin 2 is required for DRD2 inhibitory effects on AKT phosphorylation and cell proliferation in MMQ and NF-PitNETs, paving the way for a potential role of β-arrestin 2 as a biomarker predicting NF-PitNETs responsiveness to treatment with dopamine agonists.

Gonadotroph Tumors Show Subtype Differences That Might Have Implications for Therapy.

Gonadotroph tumors, although frequent, are poorly studied and understood, being usually included in the larger nonfunctioning pituitary neuroendocrine tumors (PitNETs) group. Moreover, in comparison to the other types of PitNETs, no established medical treatment is currently available for gonadotroph tumors. Here, we performed a retrospective study and analyzed the clinicopathological characteristics of 98 gonadotroph tumors operated in a single large pituitary center. Although being larger in men (p = 0.01), the aggressiveness of gonadotroph tumors did not appear to be sex-related. LH tumors were rare (4/98) and exclusively encountered in men. Somatostatin receptor type 5 (SST5) was absent in all analyzed tumors. The immunoreactive score (IRS) of somatostatin receptor type 2 (SST2) and of estrogen receptor alpha (ERα) was associated with the histological subtype (p = 0.01 and p = 0.02). IRS ERα correlated moderately with IRS SST2 in all (rho = 0.44, adjusted p-value = 0.0001) and in male (rho = 0.51, adjusted p-value = 0.0002) patients, and with follicle-stimulating hormone (FSH) percentage in all (rho = 0.40, adjusted p-value = 0.0005) and in female (rho = 0.58, adjusted p-value = 0.004) patients. In conclusion, gonadotroph tumors exhibit histological characteristics pinpointing the existence of several subtypes. Their heterogeneity warrants further investigations and may have to be taken into account when studying these tumors and investigating treatment options.