6 research outputs found

    The molecular pathogenesis of GIP-dependent adrenal hyperplasia and invasive pituitary adenomas

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    Partie I – L’HBMS GIP-dĂ©pendante est secondaire Ă  l’expression illĂ©gitime du rĂ©cepteur du GIP (GIPR) dans les lĂ©sions surrĂ©naliennes. La nature bilatĂ©rale de cette pathologie suggĂšre une prĂ©disposition gĂ©nĂ©tique. Notre objectif Ă©tait d’identifier le mĂ©canisme molĂ©culaire Ă  l’origine de l’expression ectopique surrĂ©nalienne du GIPR. Pour cela, nous avons menĂ© une Ă©tude de cohorte et nous avons sĂ©quencĂ© l’ADN germinal et somatique issu de surrĂ©nalectomie de 17 patients ayant une forme familiale ou sporadique d’HBMS GIP-dĂ©pendante. Nous avons rĂ©alisĂ© des analyses transcriptomiques dans les Ă©chantillons surrĂ©naliens et des analyses fonctionnelles in vitro afin de caractĂ©riser les anomalies gĂ©nĂ©tiques identifiĂ©es dans des cellules corticosurrĂ©naliennes humaines. Nous avons identifiĂ© des variants germinaux pathogĂšnes du gĂšne codant la lysine dĂ©mĂ©thylase 1A (KDM1A) chez les 17 patients Ă©tudiĂ©s et nous avons retrouvĂ© une perte de bras court du chromosome 1 porteur du gĂšne KDM1A dans les lĂ©sions surrĂ©naliennes entrainant ainsi la perte complĂšte de l’expression surrĂ©nalienne de KDM1A. L’étude par RNA-seq a permis d’identifier une rĂ©gulation diffĂ©rentielle des rĂ©cepteurs couplĂ©s aux protĂ©ines G chez les patients porteurs d’un variant de KDM1A parmi lesquels GIPR Ă©tait le plus surexprimĂ©. L’inhibition pharmacologique in vitro et le knock-out de KDM1A par l’approche CRISPR-Cas9 dans des cellules corticosurrĂ©naliennes humaines entraĂźnaient une augmentation significative de l’expression du GIPR. Nous avons pu montrer que l’HBMS avec expression illĂ©gitime du GIPR est une maladie gĂ©nĂ©tique liĂ©e Ă  une inactivation en deux Ă©tapes de KDM1A selon un modĂšle de gĂšne suppresseur de tumeur. Partie II : L’envahissement des structures adjacentes, notamment des sinus caverneux par les adĂ©nomes hypophysaires limite le rĂ©sultat du traitement chirurgical. Notre objectif Ă©tait d’identifier les acteurs molĂ©culaires responsables de l’invasion du sinus caverneux par les adĂ©nomes hypophysaires. Pour cela, nous avons comparĂ© par RNA-seq les profils d’expression gĂ©nique des portions non-invasives et des portions envahissant le sinus caverneux de 19 adĂ©nomes hypophysaires, et identifiĂ© une surexpression de la DPP4 (Dipeptidylpeptidase 4) dans les parties invasives. Nous avons Ă©tudiĂ© le rĂŽle de l’inhibition pharmacologique de la DPP4 par la sitagliptine dans la migration et dans l'invasion de cellules hypophysaires murines in vitro. Nous avons mis au point un modĂšle d’adĂ©nomes hypophysaires invasifs murins par injection stĂ©rĂ©otaxique orthotopique de cellules hypophysaires GC dans l’hypophyse de rats Wistar Furth. AprĂšs l’injection, les animaux Ă©taient monitorĂ©s de maniĂšre hebdomadaire et le dĂ©veloppement tumoral Ă©valuĂ© toutes les deux semaines par imagerie par rĂ©sonance magnĂ©tique 7Tesla. La moitiĂ© des animaux ont Ă©tĂ© traitĂ©s par sitagliptine. L’inhibition de la DPP4 endogĂšne par la sitagliptine diminuait l’invasion et la migration cellulaire in vitro dans deux lignĂ©es cellulaires hypophysaires murines. Nous avons Ă©galement montrĂ© que les injections stĂ©rĂ©otaxiques intra-hypophysaires conduisaient au dĂ©veloppement de tumeurs en 7 semaines. L’étude du volume tumoral nous a permis d’identifier une diminution de la croissance tumorale et une amĂ©lioration de la survie dans un sous-groupe d’animaux traitĂ©s par sitagliptine. Nous avons dĂ©crit la signature molĂ©culaire des adĂ©nomes hypophysaires invasifs et montrĂ© une surexpression de la DPP4, dont nous avons prouvĂ© le rĂŽle dans la migration et l’invasion cellulaire in vitro. La sitagliptine in vivo permettait Ă©galement de diminuer la croissance tumorale et d’amĂ©liorer la survie des animaux. Un plus grand nombre d’animaux seront nĂ©cessaires pour confirmer cette observation.Part I: GIP-dependent PBMAH is caused by ectopic expression of GIP receptor (GIPR) in the adrenal tissue. The bilateral nature of this adrenal disease suggests germline genetic predisposition. We aimed to identify the molecular driver event responsible for ectopic GIPR expression in PBMAH. We conducted a cohort study to study germline and somatic DNA from patients who had undergone adrenalectomy for familial or sporadic GIP-dependent PBMAH with Cushing’s syndrome. RNA-sequencing on adrenal samples was performed to study gene expression in GIP-dependent Cushing’s syndrome and in control samples. Functional in vitro studies were performed to study the impact of the genetic event identified in human adrenocortical cells.We identified germline heterozygous pathogenic or likely pathogenic variants in the lysine demethylase 1A (KDM1A) gene in all 17 studied patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. Transcriptome analysis of adrenals from affected patients with loss of KDM1A identified differentially regulated genes, including those encoding for GIPR and some other G protein-coupled receptors. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical cells. We found that familial and sporadic GIP-dependent PBMAH is a genetic disease caused in 100% of cases studied by germline inactivating pathogenic variants of the KDM1A with a loss of heterozygosity of the second KDM1A locus in the adrenal lesions. This stepwise inactivation of KDM1A is suggestive of a tumor suppressor gene model of tumorigenesis. Part II: Surgical treatment of pituitary adenomas is frequently incomplete because of invasion of extrasellar cerebral structures, notably, the cavernous sinus. Our aim was to identify the molecular mechanisms of the invasion of cavernous sinus by pituitary adenomas. We obtained a unique tissue collection of 19 invasive pituitary adenomas with a sample from the intrasellar portion and a sample from the portion invading the cavernous sinus for each adenoma. We used RNA-sequencing to compare the gene expression patterns of the invading and intrasellar portions and identified an overexpression of Dipeptidyl-peptidase 4 (DPP4) in the invasive part of pituitary adenomas. We studied the consequences of DPP4 inhibition by sitagliptine on migration and invasion of murine pituitary cell lines using in vitro Transwell Assay. We elaborated a model of invasive pituitary adenomas by stereotactic injection of murine somatotroph GC cell into the lobes of the pituitary gland of female Wistar Furth rats. After surgery, rats were monitored weekly, tumor development was assessed fortnightly by 3 successive 7Tesla MRI (magnetic resonance imaging) and half of the animals were treated by sitagliptine. In vitro pharmacological inhibition of DPP4 decreased cell migration and invasion in GH3 cells and LbT2 cell. Animals injected with GC cells developed invasive pituitary tumors in 7 weeks. Pharmacological inhibition by sitagliptine tended to slow tumor growth and decreased cumulative mortality. We described the molecular signature associated with the invasion of cavernous sinus by pituitary adenomas and identified over expression of DPP4 in invasive part of pituirary adenomas, which was related to pituitary cells migration and invasion in functional in vitro studies. Sitagliptine in vivo tended to decrease tumor growth and mortality from intracranial hypertension, however larger numbers of animals are necessary to confirm this observation

    Prevalence and clinical correlations of SF3B1 variants in lactotroph tumours.

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    OBJECTIVE: A somatic mutational hotspot in the SF3B1 gene was reported in lactotroph tumours. The aim of our study was to examine the prevalence of driver SF3B1 variants in a multicentre independent cohort of patients with lactotroph tumours and correlate with clinical data. DESIGN AND METHODS: This was a retrospective, multicentre study involving 282 patients with lactotroph tumours (including 6 metastatic lactotroph tumours) from 8 European centres. We screened SF3B1 exon 14 hotspot for somatic variants using Sanger sequencing and correlated with clinicopathological data. RESULTS: We detected SF3B1 variants in seven patients with lactotroph tumours: c.1874G > A (p.Arg625His) (n = 4, 3 of which metastatic) and a previously undescribed in pituitary tumours variant c.1873C > T (p.Arg625Cys) (n = 3 aggressive pituitary tumours). In two metastatic lactotroph tumours with tissue available, the variant was detected in both primary tumour and metastasis. The overall prevalence of likely pathogenic SF3B1 variants in lactotroph tumours was 2.5%, but when we considered only metastatic cases, it reached the 50%. SF3B1 variants correlated with significantly larger tumour size; higher Ki67 proliferation index; multiple treatments, including radiotherapy and chemotherapy; increased disease-specific death; and shorter postoperative survival. CONCLUSIONS: SF3B1 variants are uncommon in lactotroph tumours but may be frequent in metastatic lactotroph tumours. When present, they associate with aggressive tumour behaviour and worse clinical outcome

    Loss of KDM1A in GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome: a multicentre, retrospective, cohort study

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    Background: GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome is caused by aberrant expression of the GIP receptor in adrenal lesions. The bilateral nature of this disease suggests germline genetic predisposition. We aimed to identify the genetic driver event responsible for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Methods: We conducted a multicentre, retrospective, cohort study at endocrine hospitals and university hospitals in France, Canada, Italy, Greece, Belgium, and the Netherlands. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Adrenal samples from patients with primary bilateral macronodular adrenal hyperplasia who had undergone an adrenalectomy for overt or mild Cushing's syndrome without evidence of food-dependent cortisol production and those with GIP-dependent unilateral adrenocortical adenomas were used as control groups. We performed whole genome, whole exome, and targeted next generation sequencing, and copy number analyses of blood and adrenal DNA from patients with familial or sporadic disease. We performed RNA sequencing on adrenal samples and functional analyses of the identified genetic defect in the human adrenocortical cell line H295R. Findings: 17 patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome were studied. The median age of patients was 43·3 (95% CI 38·8–47·8) years and most patients (15 [88%]) were women. We identified germline heterozygous pathogenic or most likely pathogenic variants in the KDM1A gene in all 17 patients. We also identified a recurrent deletion in the short p arm of chromosome 1 harboring the KDM1A locus in adrenal lesions of these patients. None of the 29 patients in the control groups had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in adrenal lesions. Global gene expression analysis showed GIP receptor upregulation with a log2 fold change of 7·99 (95% CI 7·34–8·66; p=4·4 × 10−125), and differential regulation of several other G protein-coupled receptors in GIP-dependent primary bilateral macronodular hyperplasia samples compared with control samples. In vitro pharmacological inhibition and inactivation of KDM1A by CRISPR-Cas9 genome editing resulted in an increase of GIP receptor transcripts and protein in human adrenocortical H295R cells. Interpretation: We propose that GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome results from a two-hit inactivation of KDM1A, consistent with the tumour suppressor gene model of tumorigenesis. Genetic testing and counselling should be offered to these patients and their relatives. Funding: Agence Nationale de la Recherche, Fondation du Grand dĂ©fi Pierre Lavoie, and the French National Cancer Institute

    OR04-4 Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose

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    CONTEXT : Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas. [...
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