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International audience; Cushing's syndrome is defined by prolonged exposure to glucocorticoids, leading to excess morbidity and mortality. Diagnosis of this rare pathology is difficult due to the low specificity of the clinical signs, the variable severity of the clinical presentation, and the difficulties of interpretation associated with the diagnostic methods. The present consensus paper by 38 experts of the French Society of Endocrinology and the French Society of Pediatric Endocrinology and Diabetology aimed firstly to detail the circumstances suggesting diagnosis and the biologic diagnosis tools and their interpretation for positive diagnosis and for etiologic diagnosis according to ACTH-independent and -dependent mechanisms. Secondly, situations making diagnosis complex (pregnancy, intense hypercortisolism, fluctuating Cushing's syndrome, pediatric forms and genetically determined forms) were detailed. Lastly, methods of surveillance and diagnosis of recurrence were dealt with in the final section.

International audience; PURPOSE: This study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS. METHODS: A multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing). RESULTS: The integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10(-12) and P < .01, respectively). Subsequently, KDM1A pathogenic variants were identified in 4 of 4 additional index cases with FDCS. CONCLUSION: KDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management.

Context Despite the growing evidence of the clinical value of somatostatin receptor (SSTR) positron emission tomography (PET) in the evaluation of neuroendocrine tumors (NETs), its role remains to be clarified at different time points in the journey of patients with multiple endocrine neoplasia type 1 (MEN1). The rarity of the disease is however a significant impediment to prospective clinical trials. Objective The goals of the study were to assess the indications and value of SSTR PET/computed tomography (CT) in patients with MEN1. Methods We retrospectively included patients from 7 French expert centers for whom data on SSTR PET/CT and morphological imaging performed at the same period were available. Detection rates of PET study were analyzed. Results One hundred and 8 patients were included. SSTR PET/CT was performed at screening (n = 33), staging (n = 34), restaging (n = 37), and for peptide receptor targeted radiotherapy selection (n = 4). PET detected positive pancreatic lesions in 91% of cases at screening, with results comparable with magnetic resonance imaging but superior to CT (P = .049). Metastases (mostly lymph node [LN]) were present at the screening phase in 28% of cases, possibly due to the suboptimal value of screening morphological imaging in the assessment of nodal metastases and/or a long delay between imaging studies. SSTR PET/CT was considered superior or complementary to the reference standard in the assessment of LN or distant metastases in the vast majority of cases and regardless of the clinical scenario. Conclusion This study shows the potential added value of SSTR PET in the assessment of MEN1-associated NETs and provides great impetus toward its implementation in the evaluation of patients with MEN1.

Context Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1. The uncertainty of pathogenicity of MEN1 variants complexifies the selection of the patients likely to benefit from specific care. Objective MEN1-mutated patients should be offered tailored tumor screening and genetic counseling. We present a patient with hyperparathyroidism for whom genetic analysis identified a variant of uncertain significance in the MEN1 gene (NM_130799.2): c.654G > T p.(Arg218=). Additional functional genetic tests were performed to classify the variant as pathogenic and allowed prenatal testing. Design Targeted next generation sequencing identified a synonymous variant in the MEN1 gene in a 26-year-old male with symptomatic primary hyperparathyroidism. In silico and in vitro genetic tests were performed to assess variant pathogenicity. Results Genetic testing of the proband’s unaffected parents showed the variant occurred de novo. Transcript study showed a splicing defect leading to an in-frame deletion. The classification of the MEN1 variant as pathogenic confirmed the diagnosis of MEN1 and recommended an adapted medical care and follow-up. Pathogenic classification also allowed to propose a genetic counseling to the proband and his wife. Noninvasive prenatal diagnosis was performed with a personalized medicine-based protocol by detection of the paternally inherited variant in maternal plasmatic cell free DNA, using digital PCR. Conclusion We showed that functional genetic analysis can help to assess the pathogenicity of a MEN1 variant with crucial consequences for medical care and genetic counseling decisions.

Objective Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a heterogeneous disease characterized by adrenal macronodules and variable levels of cortisol excess, with not clearly established clinical diagnostic criteria. It can be caused by ARMC5 germline pathogenic variants. In this study, we aimed to identify predictive criteria for ARMC5 variants. Methods We included 352 consecutive index patients from 12 European centers, sequenced for germline ARMC5 alteration. Clinical, biological and imaging data were collected retrospectively. Results 52 patients (14.8%) carried ARMC5 germline pathogenic variants and showed a more distinct phenotype than non-mutated patients for cortisol excess (24-h urinary free cortisol 2.32 vs 1.11-fold ULN, respectively, P < 0.001) and adrenal morphology (maximal adrenal diameter 104 vs 83 mm, respectively, P < 0.001) and were more often surgically or medically treated (67.9 vs 36.8%, respectively, P < 0.001). ARMC5-mutated patients showed a constant, bilateral adrenal involvement and at least a possible autonomous cortisol secretion (defined by a plasma cortisol after 1 mg dexamethasone suppression above 50 nmol/L), while these criteria were not systematic in WT patients (78.3%). The association of these two criteria holds a 100% sensitivity and a 100% negative predictive value for ARMC5 pathogenic variant. Conclusion We report the largest series of index patients investigated for ARMC5 and confirm that ARMC5 pathogenic variants are associated with a more severe phenotype in most cases. To minimize negative ARMC5 screening, genotyping should be limited to clear bilateral adrenal involvement and autonomous cortisol secretion, with an optimum sensitivity for routine clinical practice. These findings will also help to better define PBMAH diagnostic criteria.

Primary hyperparathyroidism (HPT1) is the most frequent endocrinopathy in multiple endocrine neoplasia type 1 (MEN1). Its surgical management is challenging. We aimed to describe and compare the imaging findings of parathyroid ultrasound (US), sestaMIBI scintigraphy (sestaMIBI), and 18F-fluorocholine (FCH) PET/CT in a series of MEN1 patients with HPT1. Retrospective analysis of a cohort of MEN1 patients with HPT1 assessed by parathyroid US, sestaMIBI scintigraphy and SPECT/CT, and FCH-PET/CT for potential surgery between 2015 and 2019. Twenty-two patients with a confirmed diagnosis of MEN1 who presented with HPT1 and were assessed by the 3 imaging modalities were included. After imaging workups, 11 patients were operated on for the first time, 4 underwent a redo surgery, and 7 did not undergo an operation. The overall patient-based positivity rate of imaging was 91% (20 of 22) for parathyroid US and 96% (21 of 22) for both sestaMIBI and FCH-PET/CT. The 3 imaging modalities demonstrated negative findings in 1/22 patient who did not undergo surgery. Overall, 3 pathologic glands were not detected by any imaging technique. SestaMIBI and FCH-PET/CT both resulted in the same 3 false-positive results in ectopic areas with a significant uptake on two thymic carcinoid tumors and one inflammatory lymph node. FCH-PET/CT provided more surgically relevant data than sestaMIBI in 4/11 patients with initial surgery and in 1/4 patient who underwent redo surgery. Compared to sestaMIBI scintigraphy, FCH-PET/CT provides additional information regarding the number of pathologic parathyroid glands and their localization in MEN1 patients with HPT1.

Introduction Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a heterogeneous disease characterized by bilateral adrenal macronodules responsible for adrenal Cushing. To date, two genetic causes of PBMAH are known: germline inactivating variants of the tumor suppressor genes ARMC5 identified in 2013 (Assié, N Eng J Med 2013), responsible for 20 to 25% of index cases, and KDM1A, identified recently (Vaczlavik, GIM 2021; Chasseloup, Lancet D&E 2021), responsible for the rare presentation associated with food-dependent Cushing's syndrome (FDCS) due to aberrant expression of the GIP receptor (GIPR) in adrenocortical cells. Multiple other illegitimate receptors are known to be responsible for abnormal cortisol response to various physiological stimuli in PBMAH. A recent multiomic analysis, identified three distinct molecular PBMAH groups: G1 with ARMC5-mutated tumors, G2 with KDM1A-mutated tumors from FDCS patients, and G3 with no identified genetic cause at present. We aimed to identify specific expression profiles of illegitimate receptors in the three groups. Methods Based on the transcriptome data obtained by RNA sequencing (Illumina) of the tumors from 31 patients (G1/ARMC5, 16 patients; G2/KDM1A, 6 patients; G3, 9 patients), expression of the following genes, encoding potential illegitimate receptors, were compared: ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, AVPR1A, AVPR1B, AVPR2, GCGR, GIPR, HTR4, HTR7, LHCGR. Calculations were performed using R statistical software. The Bioconductor limma package was used to analyze mRNA differential expression. Results G1/ARMC5 tumors showed a relative overexpression of the vasopressin receptors AVPR1A and AVPR1B compared to the two other groups (fold-change [FC] =7.39, p Conclusion This study reveals specific expression profiles of illegitimate receptors related to the three molecular groups. ARMC5 tumors are associated with the overexpression of two vasopressin receptors, while, besides GIPR, KDM1A inactivation seems to drive the overexpression of the LH/hCG receptor, as previously suggested in patients with FDCS (Bertherat, JCE&M 2005), potentially responsible for Cushing's syndrome associated with pregnancy and menopause. These molecular patterns need to be corroborated by clinical data with a systematic testing of the aberrant cortisol responses. Additionally, further studies would be needed to investigate the clinical relevance and significance of moderate fold-changes in gene expression (e.g. Presentation: Saturday, June 11, 2022 12:00 p.m. - 12:15 p.m.

MEN1 is an autosomal dominant hereditary syndrome characterized by several endocrine tumors, in most cases affecting the parathyroid glands, pancreas, and anterior pituitary. It is the result of inactivating mutations in the tumor suppressor gene MEN1. More than 1300 different mutations have been identified in this gene. Mosaic MEN1 mutations have been previously described in only a few patients in the literature. In this paper, we provide a review of six cases of MEN1 mosaicism reported in the literature supplemented with six additional cases described by the French TENgen network of laboratories. This review highlights that (i) MEN1 mosaicism is not associated with a mild phenotype and results in the same natural history as heterozygous MEN1 mutation and (ii) that more systematic detection of MEN1 mosaic mutation enables improvements in both patient monitoring and genetic counseling.

Design Molecular classification is important for the diagnosis and prognosis of adrenocortical tumors (ACT). Transcriptome profiles separate adrenocortical adenomas ‘C2’ from carcinomas, and identify two groups of carcinomas ‘C1A’ and ‘C1B’, of poor and better prognosis respectively. However, many ACT cannot be profiled because of improper or absent freezing procedures, a mandatory requirement so far. The main aim was to determine transcriptome profiles on formalin-fixed paraffin-embedded (FFPE) samples, using the new 3’-end RNA-sequencing technology. A secondary aim was to demonstrate the ability of this technique to explore large FFPE archives, by focusing on the rare oncocytic ACT variants. Methods We included 131 ACT: a training cohort from Cochin hospital and an independent validation cohort from Wuerzburg hospital. The 3’ transcriptome was generated from FFPE samples using QuantSeq (Lexogen, Vienna, Austria) and NextSeq500 (Illumina, San Diego, CA, USA). Results In the training cohort, unsupervised clustering identified three groups: ‘C1A’ aggressive carcinomas (n = 28, 29%), ‘C1B’ more indolent carcinomas (n = 28, 29%), and ‘C2’ adenomas (n = 39, 41%). The prognostic value of FFPE transcriptome was confirmed in the validation cohort (5-year OS: 26% in ‘C1A’ (n = 26) and 100% in ‘C1B’ (n = 10), P = 0.003). FFPE transcriptome was an independent prognostic factor in a multivariable model including tumor stage and Ki-67 (OS HR: 7.5, P = 0.01). Oncocytic ACT (n = 19) did not form any specific cluster. Oncocytic carcinomas (n = 6) and oncocytic ACT of uncertain malignant potential (n = 4) were all in ‘C1B’. Conclusions The 3’ RNA-sequencing represents a convenient solution for determining ACT molecular class from FFPE samples. This technique should facilitate routine use and large retrospective studies.

Purpose: To investigate the genetic cause of Food-dependent Cushing’s syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the GIP receptor (GIPR). Germline ARMC5 alterations have been reported in about 25 % of PBMAH index cases but are absent in patients with FDCS. Methods: A multi-omics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNAseq, SNP array, methylome, miRNome, exome sequencing). Results: The integrative analysis revealed three molecular groups with different clinical features: G1, PBMAH due to ARMC5 inactivating variants; G2, with GIPR ectopic expression and FDCS; and G3, with a less severe phenotype. Exome sequencing revealed germline truncating variants of LSD1/KDM1A in the G2 group, constantly associated with a somatic loss of the LSD1/KDM1A wildtype allele on 1p, leading to a loss of LSD1/KDM1A expression both at mRNA and protein levels (p=1.2x10-12 and p

Objective Primary hyperparathyroism (PHPT) is a disease with either sporadic or inherited presentation. Germline mutations responsible for this disease can be found in different genes, the most frequently involved being MEN1, CDC73 = HRPT2 and CASR. During the last few years, new genes have been described as responsible for the development of PHPT such as GCM2. These genes are not systematically included in PHPT genetic screening yet. The aim of this work was to assess the importance of GCM2 genetic analysis in PHPT to determine if this gene should be included in gene panel investigated for this disease. Design and methods The TENGEN network (French Oncogenetic Network of Neuroendocrine Tumors) collected and interpreted allelic variants according to the clinical characteristics of the GCM2-positive patients identified through genetic testing performed in French laboratories (713 patients with PHPT). Results From 713 patients with PHPT included in this study, 85 (6.6%) carried at least one GCM2 variant. A total of 12 variants classified as uncertain significance or likely pathogenic were reported in 47 patients. Their mean age at PHPT diagnosis was 49 years. Additionally, the investigation of a large family showed that GCM2 variants could be associated with low penetrance. Conclusion We provide a description and interpretation for GCM2 variants identified in a French population. We suggest that this gene should be included in genetic screening of patients with PHPT and propose the follow-up of asymptomatic patients carrying such variants for calcemia.

Context Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the MEN1 gene characterized by a broad spectrum of clinical manifestations, of which the most frequent are primary hyperparathyroidism, pituitary adenomas, and neuroendocrine tumors. Objective The aim of this work was to facilitate interpretation of variants and improve the genetic counseling and medical care of families of patients with MEN1. Design, Setting, and Patients The TENGEN network (Oncogenetics Network of Neuroendocrine Tumors) has interpreted and collected all allelic variants and clinical characteristics of the MEN1-positive patients identified through genetic testing performed in the French population from 1997 to 2015. Patients and their variants were registered in the locus-specific UMD-MEN1 database (www.umd.be/MEN1/). Main Outcomes Variant classification, age-related penetrance, and odds ratios. Results A total of 370 distinct variants reported in 1676 patients, including 181 unpublished variants, have been registered. This database analysis revealed a low frequency (6.6%) of benign or likely benign missense variants in MEN1. Eight families (1.9%) had members with familial isolated hyperparathyroidism and harbored the same mutations as that found in families with authentic MEN1. An association existed between large rearrangements and an earlier onset of the disease, whereas no difference was observed between truncating and nontruncating variants. Conclusion The UMD-MEN1 database provides an exhaustive overview of the MEN1 variants present in the French population. For each variant, a classification is publicly available. Clinical data collections allow the determination of genotype-phenotype correlation and age-related penetrance of lesions in the cohort.

Contexte L’hyperparathyroidie primaire (HPT1) est l’atteinte endocrinienne la plus frequente de la Neoplasie Endocrinienne Multiple de type 1 (NEM1). Sa prise en charge chirurgicale est delicate. Methode Nous avons evalue entre 2015 et 2019, l’interet de l’echographie parathyroidienne, la scintigraphie au sesta-MIBI avec acquisition TEMP/TDM (sesta-MIBI) et la TEP/TDM a la 18F-fluorocholine (FCH-TEP/TDM) avant chirurgie parathyroidienne de patients presentant une HPT1 dans un contexte de NEM1 et correle leurs donnees aux resultats histologiques des patients operes. Resultats Vingt-deux patients ont ete inclus. Onze patients ont ete operes pour la premiere fois, 4 ont beneficie d’une reprise et 7 n’ont pas ete operes. Le taux global de positivite de l’imagerie etait de 91 % (21/22 patients) pour l’echographie et 96 % (20/22 patients) pour la sesta-MIBI et la FCH-TEP/TDM. Les 3 modalites d’imagerie etaient negatives chez un patient, non opere. Chez les patients operes, 3 glandes pathologiques n’avaient ete detectees par aucune des techniques d’imagerie. La sesta-MIBI et la FCH-TEP/TDM ont realise de facon superposable 3 faux-positifs dans des regions evocatrices de localisation parathyroidienne ectopique : deux etaient des tumeurs thymiques et un, un ganglion inflammatoire. La FCH-TEP/TDM comparee a la sesta-MIBI a permis de mieux localiser en preoperatoire les glandes pathologiques chez 4/11 patients operes pour la premiere fois et 1/4 patient repris chirurgicalement. Conclusion Comparee a la sestaMIBI, la FCH-TEP/TDM fournit des informations supplementaires concernant le nombre de glandes parathyroidiennes pathologiques et leur localisation chez les patients atteints d’HPT1 dans le cadre d’une NEM1. Elle permet de mieux planifier leur prise en charge chirurgicale.

Introduction L’HMBS est une pathologie rare et heterogene, caracterisee par de multiples macronodules surrenaliens, responsables d’un hypercortisolisme d’intensite variable. La moitie des patients avec forme severe, traites par surrenalectomie bilaterale, presente une mutation germinale inactivatrice heterozygote du gene suppresseur de tumeurs ARMC5, et 80 % pour les formes clairement familiales, contre seulement 20 % des formes sporadiques. L’objectif de cette etude etait d’etablir des criteres predictifs de mutation d’ARMC5. Methodes 454 cas index francais et europeens porteurs d’HMBS ont ete genotypes pour ARMC5 a Cochin. Parmi ces 454 patients, nous avons analyse retrospectivement les donnees cliniques, radiologiques et biologiques de 264 patients suivis dans cinq services d’endocrinologie universitaires francais (reseau COMETE). Resultats 40 cas index sur les 264 analyses (15,2 %) presentent une mutation germinale inactivatrice d’ARMC5, coherent avec le taux de mutation observe dans la cohorte de 454 patients (14,3 %). Les patients mutes ont une pathologie plus severe que les patients non-mutes, en termes d’intensite de l’hypercortisolisme biologique, de morphologie surrenalienne et de complications. 100 % des patients mutes presentent des nodules surrenaliens bilateraux ET un cortisol plasmatique apres freinage minute a la dexamethasone superieur a 50 nmol/L, alors que seulement 61,6 % des patients non-mutes ont l’association de ces deux criteres. Du fait de sa valeur predictive negative de 100 %, l’absence de cette association permet d’exclure une mutation d’ARMC5. Conclusion Restreindre l’indication du sequencage d’ARMC5 aux patients presentant des nodules surrenaliens bilateraux et une secretion autonome de cortisol permettrait d’eviter un genotypage negatif chez un tiers des patients sans perte de sensibilite.

Introduction Le complexe de Carney (CNC) est un syndrome regroupant des neoplasies endocriniennes et non endocriniennes. Cette etude decrivant les caracteristiques des lesions mammaires des patientes avec CNC, les autres manifestations du CNC et le genotype PRKAR1A, s’est particulierement interessee aux carcinomes mammaires, cancer le plus frequent chez la femme et principale cause de deces par cancer dans le monde. Methodes Cette cohorte de 70 patients avec CNC provient d’un suivi prospectif multicentrique francais de 3 ans (Espiard et al. JCEM 2020). L’analyse des 50 femmes incluses a porte sur les manifestations de CNC (lesions mammaires particulierement), le genotype et les caracteristiques hormonales. Resultats Parmi les 38 femmes avec imagerie mammaire, dix presentaient des lesions benignes et six des carcinomes : un carcinome in-situ a 54 ans et cinq cancers invasif avant 50 ans et une recurrence controlaterale. La survenue du cancer mammaire chez les patientes avec CNC etait plus frequente et l’âge moyen 17 ans plus precoce (44,7 ans) que la population generale. Tous les cancers mammaires sont survenus chez des patientes mutees PRKAR1A, avec de bons facteurs pronostic : recepteurs hormonaux positifs, HER2 negatif, un seul cancer metastatique. Il n’y avait pas d’association entre les carcinomes mammaires et les manifestations du CNC ou facteurs de risque conventionnels. Discussion Les lesions mammaires sont frequentes chez les femmes atteintes de CNC. Le carcinome mammaire pourrait etre une « nouvelle » manifestation de cette maladie, une strategie de depistage et un suivi adequat pourraient etre proposes.

International audience; In 2015, the ACMG-AMP guidelines provided a general procedure for the objective and reproducible classification of genomic variants. While the benefits of this framework are of major importance, its adaptation for locus-specific use is needed. Multiple Endocrine Neoplasia type 1 (MEN1) occurs due to inactivating mutations in the tumour suppressor gene MEN1, including 20% of missense variants. The classification of these variants may be extremely challenging. Here, we compared the interpretation of the 122 MEN1 missense variants, identified in the French population over the past 15 years by the TENGEN network (French oncogenetics network of neuroendocrine tumors) versus by using the ACMG-AMP guidelines, and analyzed the causes of discordance. A total of 59.8% of missense variants were termed as (likely)-pathogenic variants by TENGEN versus only 28.7% using ACMG-AMP guidelines. Actually, 53.4% (39/73) of TENGEN (likely)-pathogenic variants were declassified in variant of uncertain significance (VUS) by using ACMG-AMP guidelines, thereby affecting the clinical management of patients and their families. Twenty of these ACMG-AMP VUS were found in patients with a clinically authentic MEN1 disease. Here, TENGEN proposes adjustments to the ACMG-AMP framework for the interpretation of MEN1 missense variants. These propositions merge both the classification systems, and are particularly interesting, as MEN1 is included in the ACMG secondary findings list for reporting in clinical genomic sequencing.

Introduction L’insulinome est le plus souvent sporadique mais peut egalement s’integrer dans le cadre d’une neoplasie endocrinienne multiple de type 1 (NEM1) avec une prevalence importante des formes multiples dans ce contexte. Un seul cas d’insulinome multiple en dehors du contexte de NEM1 a deja ete rapporte (Babic et al., JCEM, 2016). Description Nous rapportons ici deux cas d’insulinome multiple, chez respectivement, un homme âge de 51 ans et une femme âgee de 69 ans. Les deux patients rapportaient la survenue de malaises atypiques, post-prandiaux et presenterent une hypoglycemie severe apres charge en glucose, respectivement 0,8 mmol/L apres charge IV chez le premier cas et 2 mmol/L apres charge orale chez le deuxieme cas, associee a des taux d’insulinemie inadaptes, respectivement 157,6 mUI/L et 28,2 mUI/L. L’imagerie pancreatique, conventionnelle et basee sur l’expression des recepteurs de la somatostatine, identifia 4 lesions caudales chez les deux patients. L’histologie confirma la presence de 4 tumeurs endocrines benignes dans les deux cas, toutes positives pour l’insuline a l’exception d’une lesion chez le 2e cas. Aucune mutation du gene MEN1 ne fut identifiee chez aucun des 2 patients. L’analyse des genes ABCC8, KCNJ11, GCK, HNF4A et HNF1A fut egalement negative chez le premier cas. Conclusion Nous decrivons ici une nouvelle entite d’insulinome multiple non syndromique, caracterisee par un debut des symptomes apres 50 ans et la presence de malaises hypoglycemiques atypiques, post-prandiaux. La recherche d’une origine moleculaire permettrait de progresser dans la comprehension de cette nouvelle entite diagnostique.

Sex chromosome distribution and aneuploidy as well as germ cell degeneration were evaluated in meiotic and post-meiotic cells from an infertile XYY male. Sex chromosome distribution was assessed by multicolour fluorescence in situ hybridization on meiotic preparations. Post-meiotic cell aneuploidy was characterized by a method combining multicolour fluorescence in situ hybridization and immunocytochemistry using the proacrosin-specific monoclonal antibody (mAb 4D4). TUNEL assay was carried out on seminiferous tubules to evaluate germ cell degeneration. At the prophase stage of the first meiotic division, 63.64% of cells at the pachytene stage carried three sex chromosomes. The ratio of X-bearing to Y-bearing spermatids and spermatozoa differed significantly from 1 : 1 with an excess of Y-bearing spermatids and spermatozoa. The frequency of hyperhaploid XY spermatids was increased in the XYY male, as well as the incidence of YY, XY and disomic 18 ejaculated spermatozoa. A preferential elimination of germ cells by apoptosis occurred in spermatocytes I. The persistence of the extra Y chromosome during meiosis of an XYY male is associated with a high rate of spermatocyte I degeneration and a low rate of aneuploid spermatozoa.

To identify meiotic criteria for infertility management in non-obstructive azoospermic men, a prospective and multicentric study was organized in Andrological Departments of Paris (France), Roma (Italy) and Budapest (Hungary). In 117 non-obstructive azoospermic men with normal karyotype and no Y-chromosome microdeletion, histology and meiotic studies on bilateral bipolar testicular biopsies were done. Histologically, 40 patients (34%) presented spermatocyte or spermatid arrest, 39 (33%) hypospermatogenesis whereas no meiotic cell could be observed in the remaining patients (33%). Cytogenetically, meiotic figures could only be obtained from the two first histological groups. Meiotic abnormalities were observed in a total of 44 patients (37.6%) including nine patients (7.7%) with severe class I and class IIB anomalies and 19 patients (16.2%) with class IIC environmentally linked meiotic abnormalities. These results provided essential clues for an accurate clinical management. For patients with no meiotic figures and patients with class I and class IIB anomalies, an hormonal stimulation is illusory and a sperm gift should be directly proposed. An hormonal stimulation should be proposed to all the other patients, either directly or following the treatment of the testicular microenvironment for the patients presenting class IIC anomalies. The genetic risk and possibility of prenatal chromosomal analysis in case of pregnancy should be clearly exposed to all the couples in all the cases where type IIA, III or IV anomalies are present. This therapeutical strategy has been applied to all the patients in our series.

BackgroundPoint mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected.ObjectiveSet up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations.MethodsMultiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit.ResultsMLPA allowed identification of exons 3–6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit.ConclusionsMLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.

The expanding field of in vitro fertilization (IVF) and assisted reproduction technologies (ART) takes advantage of advanced microtechnologies and microoptics for laboratory microscopy as micromanipulators and microlasers for cell photoactivation and drilling (1).

Some controversy has existed regarding the nosology of meningeal hemangiopericytoma. In the WHO's classification of 1979 these tumours were included as a subgroup of meningiomas, but for some authors, they should not be classified as meningiomas. Cytogenetic studies on meningioma demonstrate monosomy or partial deletion of chromosome 22 in 60% of these tumors. There have been few cytogenetic studies about meningeal hemangiopericytoma. We present here the results of cytogenetic studies and fluorescence in situ hybridization in six cases of meningeal hemangiopericytoma. In these tumours we have never found monosomy 22, but all six cases were hyperdiploid. These cytogenetic data might provide additional evidence to differenciate the meningeal hemangiopericytoma from the meningioma.

e21014 Background: The efficacy of anti-BRAF therapies in patients with melanoma has emphasized the need to identify BRAF mutations in the tumour. As circulating tumour cells (CTC) derive from the primary tumour (PT), their analysis may represent a non-invasive tool to follow the molecular-target in blood after tumour removal. Methods: This prospective study included 26 patients: 24 patients had confirmed melanoma, 2 patients had benign lesions (1 naevus and 1 pyogenic granuloma). Blood samples were taken at time of the PT surgical excision; 12 healthy donors were included as controls. The 24 patients, 13 females and 11 males, had invasive melanoma (13 superficial spreading melanoma, 7 nodular melanoma, 1 lentigo malignant melanoma, 1 desmoplastic melanoma, 2 unclassified melanoma.). The mean thickness was 7.8 mm. Cytology, immunophenotyping and analysis of BRAF mutations on exon 15 were performed comparatively in primary tumour and CTC from the same patient. CTC were selected by the ScreenCell method, a single-use innovative system to isolate rare tumour cells through a filter-membrane on the basis of their size. Nucleic acids DNA/RNA were extracted from live CTC after filtration. Results: Isolated CTC and microemboli were found in 21/24 patients and were found comparable to the primary melanoma cells. No CTC was found in 10 healthy donors and 2 patients with benign lesions. The mean number of CTC was 2 CTC per ml, the maximum was 4 CTC and 7 microemboli per ml. Malignancy of CTC was assessed on classic criteria: cell size larger than 15 microns, nucleo-cytoplasmic ratio > 0.5, presence of clusters composed of pleomorphic cells. CTC were positively immunostained either by S-100 or MART1/melanA or HMB45 antibodies; 4 patients were studied for BRAF mutations in both melanoma and CTC. The results (2 negative and 2 positive) were concordant in primary melanoma cells and CTC. The V600E-BRAF mutation was found in the 2 positive cases. Conclusions: The ScreenCell method of CTC selection by size allows to detect BRAF mutations in CTC of patients with melanoma. This simple method could be proposed to follow the molecular-target during the course of evolution and treatment after tumour removal.