Lionel Groussin

Mutations of β-Catenin in Adrenocortical Tumors: Activation of the Wnt Signaling Pathway Is a Frequent Event in both Benign and Malignant Adrenocortical Tumors

Adrenocortical cancer is a rare cancer with a very poor prognosis. The genetic alterations identified to date in adrenocortical tumors are limited. Activating mutations of the Wnt signaling pathway have been observed in more frequent cancers, particularly digestive tract tumors. We investigated whether Wnt pathway activation is involved in adrenocortical tumorigenesis. In a series of 39 adrenocortical tumors, immunohistochemistry revealed abnormal cytoplasmic and/or nuclear accumulation of beta-catenin in 10 of 26 adrenocortical adenomas and in 11 of 13 adrenocortical carcinomas. An activating somatic mutation of the beta-catenin gene was shown in 7 of 26 adrenocortical adenomas and in 4 of 13 adrenocortical carcinomas; these mutations were observed only in adrenocortical tumors with abnormal beta-catenin accumulation and most were point mutations altering the Ser45 of exon 3 (in the consensus GSK3-beta/CK1 phosphorylation site). Functional studies showed that the activating Ser45 beta-catenin mutation found in the adrenocortical cancer H295R cell line leads to constitutive activation of T-cell factor-dependent transcription. This is the first molecular defect to be reported with the same prevalence in both benign (27%) and malignant (31%) adrenocortical tumors. beta-Catenin mutations are also the most frequent genetic defect currently known in adrenocortical adenomas. In adrenocortical adenomas, beta-catenin alterations are more frequent in nonfunctioning tumors, suggesting that beta-catenin pathway activation might be mostly involved in the development of nonsecreting adrenocortical adenomas and adrenocortical carcinomas. The very frequent and substantial accumulation of beta-catenin in adrenocortical carcinomas suggests that other alterations might also be involved. This finding may contribute to new therapeutic approaches targeting the Wnt pathway in malignant adrenocortical tumors, for which limited medical therapy is available.

A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 ( PDE11A ) in individuals with adrenocortical hyperplasia

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31–2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31–2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.

Gene Expression Profiling Reveals a New Classification of Adrenocortical Tumors and Identifies Molecular Predictors of Malignancy and Survival

PURPOSE Adrenocortical tumors, especially cancers, remain challenging both for their diagnosis and prognosis assessment. The aim of this article is to identify molecular predictors of malignancy and of survival. PATIENTS AND METHODS One hundred fifty-three unilateral adrenocortical tumors were studied by microarray (n = 92) or reverse transcription quantitative polymerase chain reaction (n = 148). A two-gene predictor of malignancy was built using the disease-free survival as the end point in a training cohort (n = 47), then validated in an independent validation cohort (n = 104). The best candidate genes were selected using Cox models, and the best gene combination was validated using the log-rank test. Similarly, for malignant tumors, a two-gene predictor of survival was built using the overall survival as the end point in a training cohort (n = 23), then tested in an independent validation cohort (n = 35). RESULTS Unsupervised clustering analysis discriminated robustly the malignant and benign tumors, and identified two groups of malignant tumors with very different outcome. The combined expression of DLG7 and PINK1 was the best predictor of disease-free survival (log-rank P approximately 10(-12)), could overcome the uncertainties of intermediate pathological Weiss scores, and remained significant after adjustment to the Weiss score (P < 1.3 x 10(-2)). Among the malignant tumors, the combined expression of BUB1B and PINK1 was the best predictor of overall survival (P < 2 x 10(-6)), and remained significant after adjusting for MacFarlane staging (P < .005). CONCLUSION Gene expression analysis unravels two distinct groups of adrenocortical carcinomas. The molecular predictors of malignancy and of survival are reliable and provide valuable independent information in addition to pathology and tumor staging. These original tools should provide important improvements for adrenal tumors management.

Integrated genomic characterization of adrenocortical carcinoma

Adrenocortical carcinomas (ACCs) are aggressive cancers originating in the cortex of the adrenal gland. Despite overall poor prognosis, ACC outcome is heterogeneous. We performed exome sequencing and SNP array analysis of 45 ACCs and identified recurrent alterations in known driver genes (CTNNB1, TP53, CDKN2A, RB1 and MEN1) and in genes not previously reported in ACC (ZNRF3, DAXX, TERT and MED12), which we validated in an independent cohort of 77 ACCs. ZNRF3, encoding a cell surface E3 ubiquitin ligase, was the most frequently altered gene (21%) and is a potential new tumor suppressor gene related to the β-catenin pathway. Our integrated genomic analyses further identified two distinct molecular subgroups with opposite outcome. The C1A group of ACCs with poor outcome displayed numerous mutations and DNA methylation alterations, whereas the C1B group of ACCs with good prognosis displayed specific deregulation of two microRNA clusters. Thus, aggressive and indolent ACCs correspond to two distinct molecular entities driven by different oncogenic alterations.

ARMC5 Mutations in Macronodular Adrenal Hyperplasia with Cushing's Syndrome

BACKGROUND Corticotropin-independent macronodular adrenal hyperplasia may be an incidental finding or it may be identified during evaluation for Cushings syndrome. Reports of familial cases and the involvement of both adrenal glands suggest a genetic origin of this condition. METHODS We genotyped blood and tumor DNA obtained from 33 patients with corticotropin-independent macronodular adrenal hyperplasia (12 men and 21 women who were 30 to 73 years of age), using single-nucleotide polymorphism arrays, microsatellite markers, and whole-genome and Sanger sequencing. The effects of armadillo repeat containing 5 (ARMC5) inactivation and overexpression were tested in cell-culture models. RESULTS The most frequent somatic chromosome alteration was loss of heterozygosity at 16p (in 8 of 33 patients for whom data were available [24%]). The most frequent mutation identified by means of whole-genome sequencing was in ARMC5, located at 16p11.2. ARMC5 mutations were detected in tumors obtained from 18 of 33 patients (55%). In all cases, both alleles of ARMC5 carried mutations: one germline and the other somatic. In 4 patients with a germline ARMC5 mutation, different nodules from the affected adrenals harbored different secondary ARMC5 alterations. Transcriptome-based classification of corticotropin-independent macronodular adrenal hyperplasia indicated that ARMC5 mutations influenced gene expression, since all cases with mutations clustered together. ARMC5 inactivation decreased steroidogenesis in vitro, and its overexpression altered cell survival. CONCLUSIONS Some cases of corticotropin-independent macronodular adrenal hyperplasia appear to be genetic, most often with inactivating mutations of ARMC5, a putative tumor-suppressor gene. Genetic testing for this condition, which often has a long and insidious prediagnostic course, might result in earlier identification and better management. (Funded by Agence Nationale de la Recherche and others.).

18F-Fluorodeoxyglucose Positron Emission Tomography for the Diagnosis of Adrenocortical Tumors: A Prospective Study in 77 Operated Patients

CONTEXT Most adrenal incidentalomas are nonfunctioning adrenocortical adenomas (ACAs). Adrenocortical carcinomas (ACCs) are rare but should be recognized at an early stage. OBJECTIVE The objective of the study was to evaluate the usefulness of (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG PET) to predict malignancy in patients without a previous history of cancer. DESIGN This was a prospective, multicenter study from 2001 to 2006. SETTING The study was conducted at a network of seven university hospitals in Paris. PATIENTS Seventy-seven patients were included. All underwent surgery because of hypersecretory and/or growing benign lesions (n = 18), obvious ACCs (n = 21), or radiologically indeterminate lesions (n = 38). MAIN OUTCOME MEASURE The degree of (18)F-FDG PET uptake [maximum standardized uptake value (maxSUV)] was related to the pathological findings serving as a reference, and its diagnostic value was compared with that of computerized tomography (CT) scan. RESULTS Pathology eventually diagnosed 43 ACAs, 22 ACCs, and 12 nonadrenocortical lesions. Using a cutoff value above 1.45 for adrenal to liver maxSUV ratio, the sensitivity and specificity to distinguish ACAs from ACCs were, respectively, 1.00 (95% confidence interval 0.85-1.00) and 0.88 (95% confidence interval 0.75-0.96). Among the 38 indeterminate lesions at CT scan, we could analyze a subgroup of 16 adrenocortical tumors with high unenhanced density (>10 HU) and an inappropriate washout: (18)F-FDG PET correctly predicted the benignity in 13 of 15 ACAs. CONCLUSIONS In a multidisciplinary team approach, (18)F-FDG PET helps to manage suspicious CT scan lesions. An adrenal to liver maxSUV ratio less than 1.45 is highly predictive of a benign lesion.

Role of the PKA‐Regulated Transcription Factor CREB in Development and Tumorigenesis of Endocrine Tissues

Abstract: The cAMP pathway plays a major role in the development of endocrine tissues and various molecular defects of key components of this pathway (G protein, receptors, PKA, etc.) have been observed in endocrine tumors. The ubiquitous transcription factor CREB (cAMP‐response element binding protein) binds to the cAMP response element (CRE) and stimulates transcription after phosphorylation on Ser133 by PKA. The CREB family of transcription factors contains three members: CREB, CREM, and ATF‐1. Targeted expression of dominant‐negative mutants of CREB in transgenic mice leads to somatotrophs or thyroid hypoplasia. GH‐secreting adenomas are benign secreting tumors expressing an activated mutant Gαs protein (Gsp) in about 40% of cases. In GH‐secreting adenomas CREB is always expressed and often highly phosphorylated. The CREM isoform ICER is stimulated by cAMP, and its expression is increased in Gsp‐harboring tumors. After transfection in pituitary somatotroph cells, activating mutations of Gs protein (Gsp) and overexpression of wild‐type GαS stimulate transcription of various CRE‐containing promoters via CREB in a Ser133‐specific‐dependent manner. Activation of the cAMP pathway by ACTH is required for adrenal cortex (AdCx) maintenance and steroidogenesis. CREB is expressed in normal AdCx. Alterations of CRE binding proteins with loss of CREB expression and compensatory overexpression of CREMτ is observed in the human adrenocortical cancer cell line H295R. Similar alterations are found at the protein level in human malignant adrenocortical tumors. In conclusion, the CREB family of transcription factors plays an important role in the development, differentiation, and proliferation of endocrine tissues. Various alterations of the CREB family of transciption factors can be observed in endocrine tumors.

Mutations and Polymorphisms in the Gene Encoding Regulatory Subunit Type 1-alpha of Protein Kinase A (PRKAR1A): An Update

PRKAR1A encodes the regulatory subunit type 1‐alpha (RIα) of the cyclic adenosine monophosphate (cAMP)‐dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIα haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIα haploinsufficiency, but also by the expression of altered RIα protein, as proven by analysis of expressed mutations in the gene, consisting of aminoacid substitutions and in‐frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed—these are frame‐shifts in the 3′ end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation‐negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype. Hum Mutat 31:369–379, 2010. Published 2010 Wiley‐Liss, Inc.

Wnt/β-Catenin and 3′,5′-Cyclic Adenosine 5′-Monophosphate/Protein Kinase A Signaling Pathways Alterations and Somatic β-Catenin Gene Mutations in the Progression of Adrenocortical Tumors

BACKGROUND The Wnt/beta-catenin and cAMP signaling pathways play an important role in adrenal cortex tumorigenesis. Somatic activating mutations of the beta-catenin gene (CTNNB1) are the most frequent genetic defects identified both in adrenocortical adenomas (ACAs) and adrenocortical cancers (ACCs). PRKAR1A mutations leading to cAMP pathway dysregulation are observed in primary pigmented nodular adrenocortical diseases (PPNADs) and some sporadic ACAs. OBJECTIVE The objective of the investigation was to study Wnt/beta-catenin dysregulation in adrenocortical tumors (ACTs) with cAMP pathway genetic alteration and search for secondary CTNNB1 somatic mutations in heterogeneous tumors. PATIENTS AND METHODS Nine PPNADs, including five with macronodules, three ACAs with PRKAR1A somatic mutations, and one heterogeneous tumor with ACC developed within an ACA, were studied by immunohistochemistry and DNA sequencing. RESULTS beta-Catenin accumulation was observed in all PPNADs, ACAs with PRKAR1A mutations, and the ACC component of the heterogeneous tumor. CTNNB1 somatic activating mutations were found in the macronodule of two of the five macronodular PPNADs, in one ACA with a PRKAR1A somatic mutation, and in the malignant part of the heterogeneous ACT. CONCLUSIONS The Wnt/beta-catenin pathway is activated in PPNADs and ACAs with PRKAR1A mutations, suggesting a cross talk between the cAMP and Wnt/beta-catenin pathways in ACT development. In addition, the occurrence as an additional hit of a CTNNB1 somatic mutation is associated with larger or more aggressive ACTs. This underlines the importance of the Wnt/beta-catenin pathway in adrenal cortex tumorigenesis and the importance of genetic accumulation in the progression of ACTs.

Intraadrenal Corticotropin in Bilateral Macronodular Adrenal Hyperplasia

BACKGROUND Bilateral macronodular adrenal hyperplasia is a rare cause of primary adrenal Cushings syndrome. In this form of hyperplasia, hypersecretion of cortisol suppresses the release of corticotropin by pituitary corticotrophs, which results in low plasma corticotropin levels. Thus, the disease has been termed corticotropin-independent macronodular adrenal hyperplasia. We examined the abnormal production of corticotropin in these hyperplastic adrenal glands. METHODS We obtained specimens of hyperplastic macronodular adrenal tissue from 30 patients with primary adrenal disease. The corticotropin precursor proopiomelanocortin and corticotropin expression were assessed by means of a polymerase-chain-reaction assay and immunohistochemical analysis. The production of corticotropin and cortisol was assessed in 11 specimens with the use of incubated explants and cell cultures coupled with hormone assays. Corticotropin levels were measured in adrenal and peripheral venous blood samples from 2 patients. RESULTS The expression of proopiomelanocortin messenger RNA (mRNA) was detected in all samples of hyperplastic adrenal tissue. Corticotropin was detected in steroidogenic cells arranged in clusters that were disseminated throughout the adrenal specimens. Adrenal corticotropin levels were higher in adrenal venous blood samples than in peripheral venous samples, a finding that was consistent with local production of the peptide within the hyperplastic adrenals. The release of adrenal corticotropin was stimulated by ligands of aberrant membrane receptors but not by corticotropin-releasing hormone or dexamethasone. A semiquantitative score for corticotropin immunostaining in the samples correlated with basal plasma cortisol levels. Corticotropin-receptor antagonists significantly inhibited in vitro cortisol secretion. CONCLUSIONS Cortisol secretion by the adrenals in patients with macronodular hyperplasia and Cushings syndrome appears to be regulated by corticotropin, which is produced by a subpopulation of steroidogenic cells in the hyperplastic adrenals. Thus, the hypercortisolism associated with bilateral macronodular adrenal hyperplasia appears to be corticotropin-dependent. (Funded by the Agence Nationale de la Recherche and others.).