Genome-wide evaluation of change in lncRNA expression in non-functioning pituitary adenoma using formalin-fixed and paraffin-embedded tissue specimens
ABSTRACT: Long noncoding RNAs (lncRNAs) have been implicated in the formation of many different types of tumors. However, expression profiles and potential functions of lncRNAs in non-functioning pituitary adenomas (NFPAs) have not been systematically evaluated. We evaluated the expression profiles and potential functions of lncRNAs in non-functioning pituitary adenomas (NFPAs). 10 formalin-fixed and paraffin-embedded (FFPE) tissue specimens (5 non-functioning pituitary adenomas (NFPAs) and 5 normal pituitaries(NPs)) were selected for RNA extraction and hybridization on Affymetrix microarrays. The NFPAs team was designed as the Tumor group (T), while the NPs team was designed as Normal group (N).
Project description:Subsets of pituitary tumors exhibit an aggressive clinical courses and recur despite surgery, radiation, and chemotherapy. Because modulation of the immune response through inhibition of T-cell checkpoints has led to durable clinical responses in multiple malignancies, we explored whether pituitary adenomas express immune-related biomarkers that could suggest suitability for immunotherapy. Specifically, programmed death ligand 1 (PD-L1) has emerged as a potential biomarker whose expression may portend more favorable responses to immune checkpoint blockade therapies. We thus investigated the expression of PD-L1 in pituitary adenomas.PD-L1 RNA and protein expression were evaluated in 48 pituitary tumors, including functioning and non-functioning adenomas as well as atypical and recurrent tumors. Tumor infiltrating lymphocyte populations were also assessed by immunohistochemistry.Pituitary tumors express variable levels of PD-L1 transcript and protein. PD-L1 RNA and protein expression were significantly increased in functioning (growth hormone and prolactin-expressing) pituitary adenomas compared to non-functioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary adenomas harbored higher levels of PD-L1 mRNA compared to recurrent tumors. Tumor infiltrating lymphocytes were observed in all pituitary tumors and were positively correlated with increased PD-L1 expression, particularly in the functional subtypes.Human pituitary adenomas harbor PD-L1 across subtypes, with significantly higher expression in functioning adenomas compared to non-functioning adenomas. This expression is accompanied by the presence of tumor infiltrating lymphocytes. These findings suggest the existence of an immune response to pituitary tumors and raise the possibility of considering checkpoint blockade immunotherapy in cases refractory to conventional management.
Project description:Recent studies suggest that adult pituitary stem cells may play a role in pituitary tumorigenesis. We sought to explore whether the Glial cell-line derived neurotrophic factor receptor alpha 2 (GFR?2), a recently described pituitary stem/progenitor marker, might be differentially expressed in pituitary adenomas versus normal pituitary.The expression of GFR?2 and other members of the GFR receptor family (GFR?1, ?3, ?4) were analyzed using RT-PCR, western blot, and immunohistochemistry in 39 pituitary adenomas, 14 normal pituitary glands obtained at autopsy, and cDNA from 3 normal pituitaries obtained commercially.GFR?2 mRNA was ~2.6 fold under-expressed in functioning adenomas (p < 0.01) and ~3.5 fold over-expressed in non-functioning adenomas (NFAs) (p < 0.05) compared to normal pituitary. Among NFAs, GFR?2 was significantly over-expressed (~5-fold) in the gonadotropinoma subtype only (p < 0.05). GFR?2 protein expression appeared to be higher in most NFAs, although there was heterogeneity in protein expression in this group. GFR?2 protein expression appeared consistently lower in functioning adenomas by IHC and western blot. In normal pituitary, GFR?2 was localized in Rathke's remnant, the putative pituitary stem cell niche, and in corticotropes.Our results suggest that the pituitary stem cell marker GFR?2 is under-expressed in functioning adenomas and over-expressed in NFAs, specifically gonadotropinomas. Further studies are required to elucidate whether over-expression of GFR?2 in gonadotropinomas might play a role in pituitary tumorigenesis.
Project description:Many long non-coding RNAs (lncRNAs) have been identified in several types of human pituitary adenomas and normal anterior pituitary, some of which are involved in the pathogenesis of pituitary adenomas. However, a systematic analysis of lncRNAs expressed at different developmental stages of normal pituitary, particularly in rats, has not been performed. Therefore, we contrasted two cDNA libraries of immature (D15) and mature (D120) anterior pituitary in rat that were sequenced on an Illumina HiSeq Xten platform, and a total of 29,568,806,352 clean reads were identified. Notably, 7039 lncRNA transcripts corresponded to 4442 lncRNA genes, and 1181 lncRNA transcripts were significantly differentially expressed in D15 and D120. In addition, 6839 protein-coding genes (<100?kb upstream and downstream) were the nearest neighbors of 4074 lncRNA genes. An interaction network of lncRNAs and the follicle-stimulating hormone beta-subunit (FSHb) gene was constructed using the lncRNATargets platform, and three novel lncRNAs were obtained. Furthermore, we detected the expression of the novel lncRNAs and ten highly expressed lncRNAs that were randomly selected through quantitative PCR (qPCR). The rat anterior pituitary lncRNA content identified in this study provides a more in-depth understanding of the roles of these lncRNAs in hormone and reproduction development and regulation in mammals.
Project description:Recently, long non-coding RNAs (lncRNAs) have received increased research interest owing to their participation via distinct mechanisms in the biological processes of nonfunctional pituitary adenomas. However, changes in the expression of lncRNAs in gonadotrophin adenoma, which is the most common nonfunctional pituitary adenomas, have not yet been reported. In this study, we performed a genome-wide analysis of lncRNAs and mRNAs obtained from gonadotrophin adenoma patients' samples and normal pituitary tissues using RNA-seq. The differentially expressed lncRNAs and mRNAs were identified using fold-change filtering. We identified 839 lncRNAs and 1015 mRNAs as differentially expressed. Gene Ontology analysis indicated that the biological functions of differentially expressed mRNAs were related to transcription regulator activity and basic metabolic processes. Ingenuity Pathway Analysis was performed to identify 64 canonical pathways that were significantly enriched in the tumor samples. Furthermore, to investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we constructed general co-expression networks for 100 coding and 577 non-coding genes that showed significantly correlated expression patterns in tumor cohort. In particular, we built a special sub-network of co-expression involving 186 lncRNAs interacting with 15 key coding genes of the mTOR pathway, which might promote the pathogenesis of gonadotrophin tumor. This is the first study to explore the patterns of genome-wide lncRNAs expression and co-expression with mRNAs, which might contribute to the molecular pathogenesis of gonadotrophin adenoma.
Project description:Pituitary adenomas are benign tumours comprising approximately 16% of all primary cranial neoplasms. Functioning pituitary adenomas (prolactinomas, somatotroph, corticotroph, thyrotroph and rarely gonadotroph adenomas) cause complex clinical syndromes and require prompt treatment to reduce associated morbidity and mortality. Treatment approaches include transsphenoidal surgery, medical therapy and radiation. Medical therapy is the primary therapy for prolactinomas, and surgery by a skilled neurosurgeon is the first-line approach for other functioning pituitary adenomas. A multimodal treatment is frequently necessary to achieve biochemical and clinical control, especially, when surgery is not curative or when medical therapy fails. Several emerging, novel, medical treatments for acromegaly, Cushing's disease and prolactinomas are in phase II and III clinical trials and may become effective additions to the current drug armamentarium. The availability of various management options will allow an individualised treatment approach based on the unique tumour type, clinical situation and patient preference.
Project description:Pituitary adenomas are classified into functioning and nonfunctioning (silent) tumors on the basis of hormone secretion. However, the mechanism of tumorigenesis and the cell of origin for pituitary adenoma subtypes remain to be elucidated. Employing a tamoxifen-inducible mouse model, we demonstrate that a novel postnatal Pax7(+) progenitor cell population in the pituitary gland gives rise to silent corticotroph macro-adenomas when the retinoblastoma tumor suppressor is conditionally deleted. While Pax transcriptional factors are critical for embryonic patterning as well as postnatal stem cell renewal for many organs, we have discovered that Pax7 marks a restricted cell population in the postnatal pituitary intermediate lobe. This Pax7(+) early progenitor cell population is overlapping but ontologically downstream of the Nestin(+) pituitary stem cell population, yet upstream of another newly discovered Myf6(+) late progenitor cell population. Interestingly, the Pax7(+) progenitor cell population is evolutionarily conserved in primates and humans, and Pax7 expression is maintained not only in murine tumors but also in human functioning and silent corticotropinomas. Taken together, our results strongly suggest that human silent corticotroph adenomas may in fact arise from a Pax7 lineage of the intermediate lobe, a region of the human pituitary bearing closer scientific interest as a reservoir of pituitary progenitor cells.
Project description:Management of pituitary tumors is multidisciplinary, with medical therapy playing an increasingly important role. With the exception of prolactin-secreting tumors, surgery is still considered the first-line treatment for the majority of pituitary adenomas. However, medical/pharmacological therapy plays an important role in controlling hormone-producing pituitary adenomas, especially for patients with acromegaly and Cushing disease (CD). In the case of non-functioning pituitary adenomas (NFAs), pharmacological therapy plays a minor role, the main objective of which is to reduce tumor growth, but this role requires further studies. For pituitary carcinomas and atypical adenomas, medical therapy, including chemotherapy, acts as an adjuvant to surgery and radiation therapy, which is often required to control these aggressive tumors. In the last decade, knowledge about the pathophysiological mechanisms of various pituitary adenomas has increased, thus novel medical therapies that target specific pathways implicated in tumor synthesis and hormonal over secretion are now available. Advancement in patient selection and determination of prognostic factors has also helped to individualize therapy for patients with pituitary tumors. Improvements in biochemical and "tumor mass" disease control can positively affect patient quality of life, comorbidities and overall survival. In this review, the medical armamentarium for treating CD, acromegaly, prolactinomas, NFA, and carcinomas/aggressive atypical adenomas will be presented. Pharmacological therapies, including doses, mode of administration, efficacy, adverse effects, and use in special circumstances are provided. Medical therapies currently under clinical investigation are also briefly discussed.
Project description:Ghrelin, as a brain-gut peptide, has growth hormone (GH)-releasing and appetite-inducing activities and a widespread tissue distribution. Furthermore, ghrelin is an endogenous ligand of the GH secretagogue receptor (GHSR), and both ghrelin and GHSR are expressed in the pituitary; however, the data regarding the expression of ghrelin and GHSR in pituitary adenomas are divergent and conflicting. In the present study, therefore, the expression of ghrelin and GHSR was examined in the full spectrum of human pituitary adenoma subtypes (n=34) and in normal pituitary tissue (n=3). The mRNA and protein expression levels were quantified using a competitive reverse transcription-polymerase chain reaction and western blotting and the correlation of the results with the clinical parameters was assessed. mRNA and protein expression of ghrelin and GHSR was detected in all samples with the highest mean level in GH adenomas, a moderate level in clinically non-functioning adenomas and the lowest level in adrenocorticotropin adenomas. A significant correlation between the ghrelin and GHSR mRNA expression levels was observed in the GH adenomas (n=12) (r=0.8435, P=0.0006). The ghrelin mRNA expression level in the GH adenomas correlated positively with the basic serum GH level (n=12) (r=0.6488, P=0.0225). Furthermore, the mean level of ghrelin mRNA expression was significantly higher in invasive adenomas than in noninvasive adenomas (P<0.01). Collectively, the results of the study provided evidence that ghrelin and GHSR are expressed in the various subtypes of pituitary adenoma, with specific overexpression in GH adenomas. The study suggests that the binding of ghrelin to GHSR promotes the secretion of GH and plays an important role in the development of GH adenomas via autocrine and/or paracrine effects.
Project description:The WHO Classification of Tumours of Endocrine Organs considers the inmunohistochemical characterization of pituitary adenomas (PA) as mandatory for patient diagnosis. Recent advances in the knowledge of the molecular patterns of these tumours could complement this classification with gene expression profiling.Within the context of the Spanish Molecular Registry of Pituitary Adenomas (REMAH), a multicentre clinical-basic research project, we analysed the molecular phenotype of 142 PAs with complete IHC and clinical information. Gene expression levels of all pituitary hormones, type 1 corticotrophin-releasing hormone receptor, dopamine receptors and arginine vasopressin receptor 1b were measured by quantitative real-time polymerase chain reaction. In addition, we used three housekeeping genes for normalization and a pool of nine healthy pituitary glands from autopsies as calibration reference standard.Based on the clinically functioning PA (FPA: somatotroph, corticotroph, thyrotroph and lactotroph adenomas), we established the interquartile range of relative expression for all genes studied in each PA subtype. That allowed molecularly the different PA subtypes, including the clinically non-functioning PA (NFPA). Afterwards, we estimated the concordance of the molecular and immunohistochemical classification with clinical diagnosis in FPA and between them in NFPA. The kappa values were higher in molecular than in immunohistochemical classification in FPA and showed a bad concordance in all NFPA subtypes.According to these results, the molecular characterization of the PA complements the IHC analysis, allowing a better typification of the NFPA.