GNAS mutations suppress cell invasion by activating MEG3 in growth hormone-secreting pituitary adenoma.

Approximately 30%-40% of growth hormone-secreting pituitary adenomas (GHPAs) harbor somatic activating mutations in GNAS (α subunit of stimulatory G protein). Mutations in GNAS are associated with clinical features of smaller and less invasive tumors. However, the role of GNAS mutations in the invasiveness of GHPAs is unclear. GNAS mutations were detected in GHPAs using a standard polymerase chain reaction (PCR) sequencing procedure. The expression of mutation-associated maternally expressed gene 3 (MEG3) was evaluated with RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Cell invasion ability was measured using a Transwell assay, and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by immunofluorescence and western blotting. Finally, a tumor cell xenograft mouse model was used to verify the effect of MEG3 on tumor growth and invasiveness. The invasiveness of GHPAs was significantly decreased in mice with mutated GNAS compared with that in mice with wild-type GNAS. Consistently, the invasiveness of mutant GNAS-expressing GH3 cells decreased. MEG3 is uniquely expressed at high levels in GHPAs harboring mutated GNAS. Accordingly, MEG3 upregulation inhibited tumor cell invasion, and conversely, MEG3 downregulation increased tumor cell invasion. Mechanistically, GNAS mutations inhibit EMT in GHPAs. MEG3 in mutated GNAS cells prevented cell invasion through the inactivation of the Wnt/ß-catenin signaling pathway, which was further validated in vivo. Our data suggest that GNAS mutations may suppress cell invasion in GHPAs by regulating EMT through the activation of the MEG3/Wnt/ß-catenin signaling pathway.

Integrative clinical, hormonal, and molecular data associate with invasiveness in acromegaly: REMAH study.

INTRODUCTION: Growth hormone (GH)-secreting pituitary tumors (GHomas) are the most common acromegaly cause. At diagnosis, most of them are macroadenomas, and up to 56% display cavernous sinus invasion. Biomarker assessment associated with tumor growth and invasion is important to optimize their management. OBJECTIVES: The study aims to identify clinical/hormonal/molecular biomarkers associated with tumor size and invasiveness in GHomas and to analyze the influence of pre-treatment with somatostatin analogs (SSAs) or dopamine agonists (DAs) in key molecular biomarker expression. METHODS: Clinical/analytical/radiological variables were evaluated in 192 patients from the REMAH study (ambispective multicenter post-surgery study of the Spanish Society of Endocrinology and Nutrition). The expression of somatostatin/ghrelin/dopamine system components and key pituitary/proliferation markers was evaluated in GHomas after the first surgery. Univariate/multivariate regression studies were performed to identify association between variables. RESULTS: Eighty percent of patients harbor macroadenomas (63.8% with extrasellar growth). Associations between larger and more invasive GHomas with younger age, visual abnormalities, higher IGF1 levels, extrasellar/suprasellar growth, and/or cavernous sinus invasion were found. Higher GH1 and lower PRL/POMC/CGA/AVPR1B/DRD2T/DRD2L expression levels (P < .05) were associated with tumor invasiveness. Least Absolute Shrinkage and Selection Operator's penalized regression identified combinations of clinical and molecular features with areas under the curve between 0.67 and 0.82. Pre-operative therapy with DA or SSAs did not alter the expression of any of the markers analyzed except for DRD1/AVPR1B (up-regulated with DA) and FSHB/CRHR1 (down-regulated with SSAs). CONCLUSIONS: A specific combination of clinical/analytical/molecular variables was found to be associated with tumor invasiveness and growth capacity in GHomas. Pre-treatment with first-line drugs for acromegaly did not significantly modify the expression of the most relevant biomarkers in our association model. These findings provide valuable insights for risk stratification and personalized management of GHomas.

Effectiveness of combined first-line medical treatment in acromegaly with prolactin cosecretion.

OBJECTIVE: The aim of this study is to compare the response to first-line medical treatment in treatment-naive acromegaly patients with pure growth hormone (GH)-secreting pituitary adenoma (GH-PA) and those with GH and prolactin cosecreting PA (GH&PRL-PA). DESIGN: This is a retrospective multicentric study of acromegaly patients followed from 2003 to 2023 in 33 tertiary Spanish hospitals with at least 6 months of first-line medical treatment. METHODS: Baseline characteristics, first-line medical treatment strategies, and outcomes were analyzed. We employed a multiple logistic regression full model to estimate the impact of some baseline characteristics on disease control after each treatment modality. RESULTS: Of the 144 patients included, 72.9% had a GH-PA, and 27.1% had a GH&PRL-PA. Patients with GH&PRL-PA were younger (43.9 ± 15.0 vs 51.9 ± 12.7 years, P < .01) and harboring more frequently macroadenomas (89.7% vs 72.1%, P = .03). First-generation somatostatin receptor ligand (fgSRL) as monotherapy was given to 106 (73.6%) and a combination treatment with fgSRL and cabergoline in the remaining 38 (26.4%). Patients with GH&PRL-PA received more frequently a combination therapy (56.4% vs 15.2%, P < .01). After 6 months of treatment, in the group of patients under fgSRL as monotherapy, those patients with GH&PRL-PA had worse control compared to GH-PAs (29.4% vs 55.1%, P = .04). However, these differences in the rate of disease control between both groups disappeared when both received combination treatment with fgSRL and cabergoline. CONCLUSION: In GH&PRL-PA, the biochemical control achieved with fgSRL as monotherapy is substantially worse than in patients harboring GH-PA, supporting the inclusion of cabergoline as first-line medical treatment in combination with fgSRLs in these subgroups of patients.

Histopathology of growth hormone-secreting pituitary tumors: State of the art and new perspectives.

Somatotroph (GH) adenomas/PitNETs typically arise from adenohypophysis and are biochemically active, leading to acromegaly and gigantism. More rarely, they present with ectopic origin and do not present overt biochemical or clinical features (silent variants). Histopathological examination should consider the clinical and radiological background, and include multiple steps assessing tumor morphology, pituitary transcription factors (PTFs), hormone secretion, proliferation markers, granulation, and somatostatin receptors (STRs), aimed at depicting as better as possible tumor origin (in case of non-functioning and/or metastatic tumor), and clinical behavior, including response to treatment. GH-secreting tumors are part of the Pit-1 family tumors and can secrete GH only (pure somatotrophs) or co-secrete prolactin (mixed tumors; in this case, various histological subtypes have been identified). Each subtype presents unique radiological, biochemical, and clinical characteristic. Therefore, the integration of biochemical, clinical, radiological, and histopathological elements is fundamental for proper diagnosis and management of pituitary adenomas/PitNETs, to be performed in referral Centers. In more recent times, the importance of genetic and epigenetic evaluation in the characterization of pituitary tumors (i.e., early identification of aggressive variants) has been outlined by some large studies, with the intention of improving targeted treatments.

Lysine Methyltransferase 5A Promotes the Progression of Growth Hormone Pituitary Neuroendocrine Tumors through the Wnt/ß-Catenin Signaling Pathway.

INTRODUCTION: Growth hormone (GH) secreting pituitary adenoma is considered one of the most harmful types of Pituitary Neuroendocrine Tumors (PitNETs). Our previous research has found that high expression of Lysine methyltransferase 5A (KMT5A) is closely related to the proliferation of PitNETs. The aim of this study was to investigate the role and molecular mechanism of KMT5A in the progression of GH PitNETs. METHODS: Immunohistochemistry, qRT-PCR, and Western blot (WB) were used to assess the expression levels of KMT5A in human normal pituitary and GH PitNETs, as well as in rat normal pituitary and GH3 cells. Additionally, we utilized RNA interference technology and treatment with a selective KMT5A inhibitor to decrease the expression of KMT5A in GH3 cells. CCK-8, EdU, flow cytometry (FCM), clone formation, and WB assay were further employed to evaluate the impact of KMT5A on the proliferation of GH3 cells in vitro. A xenograft model was established to evaluate the role of KMT5A in GH PitNETs progression in vivo. RESULTS: KMT5A was highly expressed in GH PitNETs and GH3 cells. Moreover, the reduction of KMT5A expression led to inhibited growth of GH PitNETs and increased apoptosis of tumor cells, as indicated by the findings from CCK-8, EdU, clone formation, and FCM assays. Additionally, WB analysis identified the Wnt/ß-catenin signaling pathway as a potential mechanism through which KMT5A promotes GH PitNETs progression. CONCLUSION: Our research suggests that KMT5A may facilitate the progression of GH PitNETs via the Wnt/ß-catenin signaling pathway. Therefore, KMT5A may serve as a potential therapeutic target and molecular biomarker for GH PitNETs.

Immunological signatures and predictive biomarkers for first-generation somatostatin receptor ligand resistance in Acromegaly.

PURPOSE: Predicting resistance to first-generation Somatostatin Receptor Ligands (fg-SRL) in Acromegaly patients remains an ongong challenge. Tumor-associated immune components participate in various pathological processes, including drug-resistance. We aimed to identify the immune components involved in resistance of fg-SRL, and to investigate biomarkers that can be targeted to treat those drug-resistant Acromegaly. METHODS: We conducted a retrospective study involving 35 Acromegaly patients with somatotropinomas treated postoperatively with fg-SRL. Gathering clinicopathological data, SSTR2 expression, and immunological profiles, we utilized univariate, binary logistic regression, and ROC analyses to assess their predictive roles in fg-SRL resistance. Spearman correlation analysis further examined interactions among interested characteristics. RESULTS: 19 patients (54.29%) exhibited resistance to postoperative fg-SRL. GH level at diagnosis, preoperative tumor volume, T2WI-MRI intensity, granularity, PD-L1, SSTR2, and CD8 + T cell infiltration showed association with clinical outcomes of fg-SRL. Notably, T2WI-MRI hyperintensity, PD-L1-IRS > 7, CD8 + T cell infiltration < 14.8/HPF, and SSTR2-IRS < 5.4 emerged as reliable predictors for fg-SRL resistance. Correlation analysis highlighted a negative relationship between PD-L1 expression and CD8 + T cell infiltration, while showcasing a positive correlation with preoperative tumor volume of somatotropinomas. Additionally, 5 patients with fg-SRL resistance underwent re-operation were involved. Following fg-SRL treatment, significant increases in PD-L1 and SSTR5 expression were observed, while SSTR2 expression decreased in somatotropinoma. CONCLUSION: PD-L1 expression and CD8 + T cell infiltration, either independently or combined with SSTR2 expression and T2WI-MRI intensity, could form a predictive model guiding clinical decisions on fg-SRL employment. Furthermore, targeting PD-L1 through immunotherapy and embracing second-generations of SRL with higher affinity to SSTR5 represent promising strategies to tackle fg-SRL resistance in somatotropinomas.

Clinicopathological Analysis of Densely and Sparsely Granulated Somatotroph Tumors of Pituitary.

OBJECTIVE: Somatotroph tumors are the second most common type of pituitary neuroendocrine tumors, which can be further classified into 2 subtypes-densely granulated somatotroph tumors (DGSTs) and sparsely granulated somatotroph tumors (SGSTs). The aim of this study was to investigate the clinical significance of the 2 subtypes in a retrospective analysis. METHODS: From the database of the Ningbo Clinical Pathology Diagnosis Center, we collected patients diagnosed with pituitary somatotroph tumors. We then compiled pertinent clinical and radiological data and proceeded with histopathological examination involving hematoxylin-eosin staining and immunohistochemical staining. Subsequent analysis compared the 2 subtypes using either χ2 test or Fisher exact test. RESULTS: We analyzed 40 cases of somatotroph tumors, 18 cases DGSTs and 22 SGSTs. Male-to-female ratio was 5:4 for DGSTs and 4:7 for SGSTs. Mean age was 52.83 years for DGSTs and 47.18 years for SGSTs. Statistically significant differences were observed between the DGST and SGST groups in invasiveness (P = 0.0267) and postoperative remission (P = 0.007). Cells of both DGSTs and SGSTs exhibited coexpression of PIT1, growth hormone, and CAM5.2, although the patterns of CAM5.2 expression differed between the 2 subtypes. CONCLUSIONS: The efficacy of CAM5.2 staining in distinguishing between DGSTs and SGSTs was demonstrated. SGSTs, with their increased invasiveness and lower remission rate, are a high-risk subtype. The histological subtype of somatotroph tumors plays a crucial role in guiding treatment decisions and prognostic evaluation in affected patients.

Clinical Characteristics and Management of Cosecreting Thyroid Stimulating Hormone or Prolactin Pituitary Growth Hormone Adenomas: A Case-Control Study.

OBJECTIVE: Cosecreting thyroid stimulating hormone (TSH) or prolactin (PRL) in patients with pituitary growth hormone (GH) adenomas has been rarely reported. Our study aimed to elucidate their clinical characteristics. METHODS: We retrospectively collected data of 22 cases of cosecreting GH and TSH pituitary adenomas [(GH+TSH)oma] and 10 cases of cosecreting GH and PRL pituitary adenomas [(GH+PRL)oma] from Beijing Tiantan Hospital, Capital Medical University between January 2009 and January 2023. The clinical manifestation, preoperative hormone levels, imaging features, pathologic characteristics, and biochemical remission rates were compared among 335 patients with solo-secreting GH adenomas (GHoma) and 49 patients with solo-secreting TSH adenoma (TSHoma). Patients with (GH+TSH)oma and (GH+PRL)oma were grouped according to biochemical remission to explore the risk factors leading to biochemical nonremission. RESULTS: Cosecreting pituitary GH adenomas had various clinical manifestations and a larger tumor volume and were more likely to invade the cavernous sinus bilaterally and compress the optic chiasm. GH and TSH levels were lower in (GH+TSH)oma than in GHoma or TSHoma. Solo part remission was observed both in (GH+TSH)oma and (GH+PRL)oma. Cavernous sinus invasion was an independent risk factor for biochemical nonremission in patients with (GH+TSH)oma and (GH+PRL)oma. CONCLUSIONS: The clinical manifestation of (GH+TSH)oma and (GH+PRL)oma may be atypical. When screening for pituitary adenomas, a comprehensive evaluation of all pituitary target gland hormones is needed. Cosecreting pituitary GH adenomas are more aggressive and surgery is often unable to completely remove the tumor, requiring pharmacologic or radiological treatment if necessary. Clinicians should give high priority to biochemical remission, although solo part remission may occur.

KDM1A genotyping and expression in 146 sporadic somatotroph pituitary adenomas.

IMPORTANCE: A paradoxical increase of growth hormone (GH) following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants and somatic loss of heterozygosity of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. OBJECTIVE: We aimed to analyze KDM1A gene sequence and KDM1A and GIPR expressions in somatotroph pituitary adenomas. SETTINGS: We conducted a cohort study at university hospitals in France and in Italy. We collected pituitary adenoma specimens from acromegalic patients who had undergone pituitary surgery. We performed targeted exome sequencing (gene panel analysis) and array-comparative genomic hybridization on somatic DNA derived from adenomas and performed droplet digital PCR on adenoma samples to quantify KDM1A and GIPR expressions. RESULTS: One hundred and forty-six patients with sporadic acromegaly were studied; 72.6% presented unsuppressed classical GH response, whereas 27.4% displayed a paradoxical rise in GH after oral glucose load. We did not identify any pathogenic variant in the KDM1A gene in the adenomas of these patients. However, we identified a recurrent 1p deletion encompassing the KDM1A locus in 29 adenomas and observed a higher prevalence of paradoxical GH rise (P = .0166), lower KDM1A expression (4.47 ± 2.49 vs 8.56 ± 5.62, P < .0001), and higher GIPR expression (1.09 ± 0.92 vs 0.43 ± 0.51, P = .0012) in adenomas from patients with KDM1A haploinsufficiency compared with those with 2 KDM1A copies. CONCLUSIONS AND RELEVANCE: Unlike in GIP-dependent primary bilateral macronodular adrenal hyperplasia, KDM1A genetic variations are not the cause of GIPR expression in somatotroph pituitary adenomas. Recurrent KDM1A haploinsufficiency, more frequently observed in GIPR-expressing adenomas, could be responsible for decreased KDM1A function resulting in transcriptional derepression on the GIPR locus.

Metabolomics in acromegaly: a systematic review.

The therapeutic response heterogeneity in acromegaly persists, despite the medical-surgical advances of recent years. Thus, personalized medicine implementation, which focuses on each patient, is justified. Metabolomics would decipher the molecular mechanisms underlying the therapeutic response heterogeneity. Identification of altered metabolic pathways would open new horizons in the therapeutic management of acromegaly. This research aimed to evaluate the metabolomic profile in acromegaly and metabolomics' contributions to understanding disease pathogenesis. A systematic review was carried out by querying four electronic databases and evaluating patients with acromegaly through metabolomic techniques. In all, 21 studies containing 362 patients were eligible. Choline, the ubiquitous metabolite identified in growth hormone (GH)-secreting pituitary adenomas (Pas) by in vivo magnetic resonance spectroscopy (MRS), negatively correlated with somatostatin receptors type 2 expression and positively correlated with magnetic resonance imaging T2 signal and Ki-67 index. Moreover, elevated choline and choline/creatine ratio differentiated between sparsely and densely granulated GH-secreting PAs. MRS detected low hepatic lipid content in active acromegaly, which increased after disease control. The panel of metabolites of acromegaly deciphered by mass spectrometry (MS)-based techniques mainly included amino acids (especially branched-chain amino acids and taurine), glyceric acid, and lipids. The most altered pathways in acromegaly were the metabolism of glucose (particularly the downregulation of the pentose phosphate pathway), linoleic acid, sphingolipids, glycerophospholipids, arginine/proline, and taurine/hypotaurine. Matrix-assisted laser desorption/ionization coupled with MS imaging confirmed the functional nature of GH-secreting PAs and accurately discriminated PAs from healthy pituitary tissue.

Molecular targets in acromegaly.

Molecular therapeutic targets in growth hormone (GH)-secreting adenomas range from well-characterized surface receptors that recognize approved drugs, to surface and intracellular markers that are potential candidates for new drug development. Currently available medical therapies for patients with acromegaly bind to somatostatin receptors, GH receptor, or dopamine receptors, and lead to attainment of disease control in most patients. The degree of control is variable: however, correlates with both disease aggressiveness and tumor factors that predict treatment response including somatostatin receptor subtype expression, granulation pattern, kinases and their receptors, and other markers of proliferation. A better understanding of the mechanisms underlying these molecular markers and their relationship to outcomes holds promise for expanding treatment options as well as a more personalized approach to treating patients with acromegaly.

Digital quantification of somatostatin receptor subtype 2a immunostaining: a validation study.

Objective: The aim of this study was to develop an open-source and reproducible digital quantitative analysis (DIA) of somatostatin receptor subtype 2a (SST2) staining in formalin-fixed paraffin-embedded tissues of pancreatic neuroendocrine tumors (panNETs) and growth hormone (GH)-secreting pituitary adenomas (GHomas). Design: SST2 immunostaining of 18 panNETs and 39 GHomas was assessed using a novel DIA protocol and compared with a widely used semi-quantitative immunoreactivity score (IRS). Methods: The DIA software calculates the staining intensity/area and the percentage of positive cells (%PC). Four representative images were selected for each sample by two independent selectors (S1 and S2), with the analysis performed by two independent analyzers (A1 and A2). Agreement between observers was calculated using the concordance correlation coefficient (CCC). Results: In panNETs, the CCC ranged 0.935-0.977 for intensity/area and 0.942-0.983 for %PC. In GHomas, the CCC ranged 0.963-0.997 for intensity/area and 0.979-0.990 for %PC. In both panNETs and GHomas, the DIA staining intensity was strongly correlated with the IRS (Spearman rho: 0.916-0.969, P < 0.001), as well as the DIA %PC with the IRS %PC (Spearman rh: 0.826-0.881, P < 0.001). In GHomas, the biochemical response to somatostatin receptor ligands correlated with SST2 expression, evaluated both as DIA intensity/area (Spearman rho: -0.448 to -0.527, P = 0.007-0.004) and DIA %PC (Spearman rho: -0.558 to -0.644, P ≤ 0.001). Conclusions: The DIA has an excellent inter-observer agreement and showed a strong correlation with the widely used semi-quantitative IRS. The DIA protocol is an open-source, highly reproducible tool and provides a reliable quantitative evaluation of SST2 immunohistochemistry.

Machine learning as a clinical decision support tool for patients with acromegaly.

OBJECTIVE: To develop machine learning (ML) models that predict postoperative remission, remission at last visit, and resistance to somatostatin receptor ligands (SRL) in patients with acromegaly and to determine the clinical features associated with the prognosis. METHODS: We studied outcomes using the area under the receiver operating characteristics (AUROC) values, which were reported as the performance metric. To determine the importance of each feature and easy interpretation, Shapley Additive explanations (SHAP) values, which help explain the outputs of ML models, are used. RESULTS: One-hundred fifty-two patients with acromegaly were included in the final analysis. The mean AUROC values resulting from 100 independent replications were 0.728 for postoperative 3 months remission status classification, 0.879 for remission at last visit classification, and 0.753 for SRL resistance status classification. Extreme gradient boosting model demonstrated that preoperative growth hormone (GH) level, age at operation, and preoperative tumor size were the most important predictors for early remission; resistance to SRL and preoperative tumor size represented the most important predictors of remission at last visit, and postoperative 3-month insulin-like growth factor 1 (IGF1) and GH levels (random and nadir) together with the sparsely granulated somatotroph adenoma subtype served as the most important predictors of SRL resistance. CONCLUSIONS: ML models may serve as valuable tools in the prediction of remission and SRL resistance.

Diagnosis and Treatment of Acromegaly: An Update.

Acromegaly is typically caused by a growth hormone-secreting pituitary adenoma, driving excess secretion of insulin-like growth factor 1. Acromegaly may result in a variety of cardiovascular, respiratory, endocrine, metabolic, musculoskeletal, and neoplastic comorbidities. Early diagnosis and adequate treatment are essential to mitigate excess mortality associated with acromegaly. PubMed searches were conducted using the keywords growth hormone, acromegaly, pituitary adenoma, diagnosis, treatment, pituitary surgery, medical therapy, and radiation therapy (between 1981 and 2021). The diagnosis of acromegaly is confirmed on biochemical grounds, including elevated serum insulin-like growth factor 1 and lack of growth hormone suppression after glucose administration. Pituitary magnetic resonance imaging is advised in patients with acromegaly to identify an underlying pituitary adenoma. Transsphenoidal pituitary surgery is generally first-line therapy for patients with acromegaly. However, patients with larger and invasive tumors (macroadenomas) are often not in remission postoperatively. Medical therapies, including somatostatin receptor ligands, cabergoline, and pegvisomant, can be recommended to patients with persistent disease after surgery. Select patients may also be candidates for preoperative medical therapy. In addition, primary medical therapy has a role for patients without mass effect on the optic chiasm who are unlikely to be cured by surgery. Clinical, endocrine, imaging, histologic, and molecular markers may help predict the response to medical therapy; however, confirmation in prospective studies is needed. Radiation therapy is usually a third-line option and is increasingly administered by a variety of stereotactic techniques. An improved understanding of the pathogenesis of acromegaly may ultimately lead to the design of novel, efficacious therapies for this serious condition.

Long-term outcomes of patients with acromegaly: a report from the Swedish Pituitary Register.

OBJECTIVE: To describe the treatment and long-term outcomes of patients with acromegaly from all healthcare regions in Sweden. DESIGN AND METHODS: Analysis of prospectively reported data from the Swedish Pituitary Register of 698 patients (51% females) with acromegaly diagnosed from 1991 to 2011. The latest clinical follow-up date was December 2012, while mortality data were collected for 28.5 years until June 2019. RESULTS: The annual incidence was 3.7/million; 71% of patients had a macroadenoma, 18% had visual field defects, and 25% had at least one pituitary hormone deficiency. Eighty-two percent had pituitary surgery, 10% radiotherapy, and 39% medical treatment. At the 5- and 10-year follow-ups, insulin-like growth factor 1 levels were within the reference range in 69 and 78% of patients, respectively. In linear regression, the proportion of patients with biochemical control including adjuvant therapy at 10 years follow-up increased over time by 1.23% per year. The standardized mortality ratio (SMR) (95% CI) for all patients was 1.29 (1.11-1.49). For patients with biochemical control at the latest follow-up, SMR was not increased, neither among patients diagnosed between 1991 and 2000, SMR: 1.06 (0.85-1.33) nor between 2001 and2011, SMR: 0.87 (0.61-1.24). In contrast, non-controlled patients at the latest follow-up from both decades had elevated SMR, 1.90 (1.33-2.72) and 1.98 (1.24-3.14), respectively. CONCLUSIONS: The proportion of patients with biochemical control increased over time. Patients with biochemically controlled acromegaly have normal life expectancy, while non-controlled patients still have increased mortality. The high rate of macroadenomas and unchanged age at diagnosis illustrates the need for improvements in the management of patients with acromegaly.

Characteristics of Gut Microbiota in Patients with GH-Secreting Pituitary Adenoma.

Prior study has demonstrated that gut microbiota at the genus level is significantly altered in patients with growth hormone (GH)-secreting pituitary adenoma (GHPA). Yet, no studies exist describing the state of gut microbiota at species level in GHPA. We performed a study using 16S rRNA amplicon sequencing in a cohort of patients with GH-secreting pituitary adenoma (GHPA, n = 28) and healthy controls (n = 67). Among them, 9 patients and 10 healthy controls were randomly chosen and enrolled in metagenomics shotgun sequencing, generating 280,426,512 reads after aligning to NCBI GenBank DataBase to acquire taxa information at the species level. Weighted UniFrac analysis revealed that microbial diversity was notably decreased in patients with GHPA, consistent with a previous study. With 16S rRNA sequencing, after correction for false-discovery rate (FDR), rank-sum test at the genus level revealed that the relative abundance of Oscillibacter and Enterobacter was remarkably increased in patients and Blautia and Romboutsia genera predominated in the controls, augmented by additional LEfSe (linear discriminant analysis effect size) analysis. As for further comparison at the species level with metagenomics sequencing, rank-sum test together with LEfSe analysis confirmed the enrichment of Alistipes shahii and Odoribacter splanchnicus in the patient group. Notably, LEfSe analysis with metagenomics also demonstrated that Enterobacter sp. DC1 and Enterobacter sp. 940 PEND, derived from Enterobacter, were both significantly enriched in patients. Functional analysis showed that amino acid metabolism pathway was remarkably enriched in GHPA, while carbohydrate metabolism pathway was notably enriched in controls. Further, significant positive correlations were observed between Enterobacter and baseline insulin-like growth factor 1 (IGF-1), indicating that Enterobacter may be strongly associated with GH/IGF-1 axis in GHPA. Our data extend our insight into the GHPA microbiome, which may shed further light on GHPA pathogenesis and facilitate the exploration of novel therapeutic targets based on microbiota manipulation. IMPORTANCE Dysbiosis of gut microbiota is associated not only with intestinal disorders but also with numerous extraintestinal diseases. Growth hormone-secreting pituitary adenoma (GHPA) is an insidious disease with persistent hypersecretion of GH and IGF-1, causing increased morbidity and mortality. Researches have reported that the GH/IGF-1 axis exerts its own influence on the intestinal microflora. Here, the results showed that compared with healthy controls, GHPA patients not only decreased the alpha diversity of the intestinal flora but also significantly changed their beta diversity. Further, metagenomics shotgun sequencing in the present study exhibited that Enterobacter sp. DC1 and Enterobacter sp. 940 PEND were enriched in patients. Also, we were pleasantly surprised to find that the Enterobacter genus was strongly positively correlated with baseline IGF-1 levels. Collectively, our work provides the first glimpse of the dysbiosis of the gut microbiota at species level, providing a better understanding of the pathophysiological process of GHPA.

Pituitary Somatotroph Adenoma-derived Exosomes: Characterization of Nonhormonal Actions.

CONTEXT: The identification and biological actions of pituitary-derived exosomes remain elusive. OBJECTIVE: This work aimed to validate production of exosomes derived from human and rat pituitary and elucidate their actions. METHODS: Isolated extracellular vesicles (EVs) were analyzed by Nanoparticle Tracking Analysis (NTA) and expressed exosomal markers detected by Western blot, using nonpituitary fibroblast FR and myoblast H9C2 cells as controls. Exosome inhibitor GW4869 was employed to detect attenuated EV release. Exosomal RNA contents were characterized by RNA sequencing. In vitro and in vivo hepatocyte signaling alterations responding to GH1-derived exosomes (GH1-exo) were delineated by mRNA sequencing. GH1-exo actions on protein synthesis, cAMP (3',5'-cyclic adenosine 5'-monophosphate) response, cell motility, and metastases were assessed. RESULTS: NTA, exosomal marker detection, and GW4869 attenuated EV release, confirming the exosomal identity of pituitary EVs. Hydrocortisone increased exosome secretion in GH1 and GH3 cells, suggesting a stress-associated response. Exosomal RNA contents showed profiles distinct for pituitary cells, and rat primary hepatocytes exposed to GH1-exo exhibited transcriptomic alterations distinct from those elicited by growth hormone or prolactin. Intravenous GH1-exo injection into rats attenuated hepatic Eif2ak2 and Atf4 mRNA expression, both involved in cAMP responses and amino acid biosynthesis. GH1-exo suppressed protein synthesis and forskolin-induced cAMP levels in hepatocytes. GH1-exo-treated HCT116 cells showed dysregulated p53 and mitogen-activated protein kinase (MAPK) pathways and attenuated motility of malignant HCT116 cells, and decreased tumor metastases in nude mice harboring splenic HCT116 implants. CONCLUSION: Our findings elucidate biological actions of somatotroph-derived exosomes and implicate exosomes as nonhormonal pituitary-derived messengers.

The intestinal flora of patients with GHPA affects the growth and the expression of PD-L1 of tumor.

CONTEXT: Pituitary adenoma (PA) is a common intracranial tumor. The evidence indicates that the tumor immune microenvironment (TIME) is associated with PA and that the intestinal flora influences other tumors' growth through interacting with the TIME. However, how the intestinal microbial flora contributes to the development of PA through the immune response is unknown. OBJECTIVE AND METHODS: Here we used high-throughput Illumina MiSeq sequencing targeting the V3-V4 region of the 16S ribosomal RNA gene to investigate the intestinal flora of patients with growth hormone-secreting pituitary adenoma (GHPA), nonfunctional pituitary adenoma (NFPA), and healthy controls. We determined their effects on tumor growth and the TIME. Fecal microbiota transplantation (FMT) was performed after adoptive transfer via peripheral blood mononuclear cells to tumor-bearing nude mice, which allowed the study of the immune response. RESULT: We discovered differences in the structures and quantities of intestinal flora between patients with GHPA, patients with NFPA, and healthy controls. After FMT, the intestinal flora of GHPA patients promoted the growth of tumors in mouse models. The number of programmed cell death ligand 1 (PD-L1)-positive cells increased in tumor tissues as well as the extent of infiltration of CD8+ cells. Increased numbers of CD3+CD8+ cells and increased levels of sPD-L1 were detected in peripheral blood. CONCLUSION: These findings indicated that the intestinal flora of patients with GHPA promoted tumor growth and that the immune system may mediate this change.

METTL3-mediated RNA m6A Hypermethylation Promotes Tumorigenesis and GH Secretion of Pituitary Somatotroph Adenomas.

INTRODUCTION: Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs. While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As N6-methyladenosine (m6A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. METHODS: Protein expression of m6A regulators was measured by immunohistochemistry. Global levels and distribution of m6A methylation were separately analyzed by m6A enzyme-linked immunosorbent assay and m6A sequencing (m6A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its m6A- dependent regulatory mechanism in tumor progression and GH secretion. RESULTS: We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of m6A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45γ act as the downstream targets in this process. CONCLUSION: Our findings strongly suggest that m6A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45γ in a m6A-dependent manner. Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs.

Transcription factor GTF2B regulates AIP protein expression in growth hormone-secreting pituitary adenomas and influences tumor phenotypes.

BACKGROUND: Clinically, the low expression of wild-type aryl hydrocarbon receptor-interacting protein (AIP) in patients with sporadic growth hormone (GH)-secreting pituitary adenoma (GHPA) is associated with a more aggressive phenotype. However, the mechanism by which AIP expression is regulated in GHPA remains unclear. Herein, we investigated a transcription factor that regulates AIP expression and explored its role in tumor phenotypes. METHODS: General transcription factor IIB (GTF2B) was predicted by several bioinformatic tools to regulate AIP expression transcriptionally. Regulation by GTF2B was evaluated using chromatin immunoprecipitation (ChIP), reverse transcription PCR, luciferase reporter, and western blot experiments in SH-SY5Y cells. Furthermore, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, transwell invasive assay, ELISA, western blot, immunohistochemical staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling were performed to investigate the effects of GTF2B and AIP on tumor cell proliferation, apoptosis, growth hormone secretion, and invasiveness in GH3 cells and mouse xenograft models. Moreover, correlations between GTF2B and AIP expression were explored in GHPA cases. RESULTS: ChIP and luciferase reporter studies demonstrated that the regulation of AIP expression by GTF2B was dependent on the intergenic-5' untranslated region element of AIP and the initial residual S65 of GTF2B. In vitro and in vivo experiments indicated that GTF2B regulated AIP expression to impact the GHPA phenotype; this was confirmed by data from 33 GHPA cases. CONCLUSIONS: We determined the regulation by GTF2B of AIP transcription in GHPA and its impact on tumor phenotype. Our findings suggest that GTF2B may be a potential therapeutic target for GHPA with low AIP expression.