Rossella Libé

SDHA is a tumor suppressor gene causing paraganglioma

Mitochondrial succinate-coenzyme Q reductase (complex II) consists of four subunits, SDHA, SDHB, SDHC and SDHD. Heterozygous germline mutations in SDHB, SDHC, SDHD and SDHAF2 [encoding for succinate dehydrogenase (SDH) complex assembly factor 2] cause hereditary paragangliomas and pheochromocytomas. Surprisingly, no genetic link between SDHA and paraganglioma/pheochromocytoma syndrome has ever been established. We identified a heterozygous germline SDHA mutation, p.Arg589Trp, in a woman suffering from catecholamine-secreting abdominal paraganglioma. The functionality of the SDHA mutant was assessed by studying SDHA, SDHB, HIF-1alpha and CD34 protein expression using immunohistochemistry and by examining the effect of the mutation in a yeast model. Microarray analyses were performed to study gene expression involved in energy metabolism and hypoxic pathways. We also investigated 202 paragangliomas or pheochromocytomas for loss of heterozygosity (LOH) at the SDHA, SDHB, SDHC and SDHD loci by BAC array comparative genomic hybridization. In vivo and in vitro functional studies demonstrated that the SDHA mutation causes a loss of SDH enzymatic activity in tumor tissue and in the yeast model. Immunohistochemistry and transcriptome analyses established that the SDHA mutation causes pseudo-hypoxia, which leads to a subsequent increase in angiogenesis, as other SDHx gene mutations. LOH was detected at the SDHA locus in the patients tumor but was present in only 4.5% of a large series of paragangliomas and pheochromocytomas. The SDHA gene should be added to the list of genes encoding tricarboxylic acid cycle proteins that act as tumor suppressor genes and can now be considered as a new paraganglioma/pheochromocytoma susceptibility gene.

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.

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.

Adrenocortical carcinoma: a clinician's update

Adrenocortical carcinoma is a rare heterogeneous neoplasm with an incompletely understood pathogenesis and a poor prognosis. Previous studies have identified overexpression of insulin-like growth factor 2 (IGF-2) and constitutive activation of β-catenin as key factors involved in the development of adrenocortical carcinoma. Most patients present with steroid hormone excess, for example Cushing syndrome or virilization, or abdominal mass effects, but a growing proportion of patients with adrenocortical carcinoma (currently >15%) is initially diagnosed incidentally. No general consensus on the diagnostic and therapeutic measures for adrenocortical carcinoma exists, but collaborative efforts, such as international conferences and networks, including the European Network for the Study of Adrenal Tumors (ENSAT), have substantially advanced the field. In patients with suspected adrenocortical carcinoma, a thorough endocrine and imaging work-up is recommended to guide the surgical approach aimed at complete resection of the tumor. To establish an adequate basis for treatment decisions, pathology reports include the Weiss score to assess malignancy, the resection status and the Ki67 index. As recurrence is frequent, close follow-up initially every 3 months is mandatory. Most patients benefit from adjuvant mitotane treatment. In metastatic disease, mitotane is the cornerstone of initial treatment, and cytotoxic drugs should be added in case of progression. Results of a large phase III trial in advanced adrenocortical carcinoma are anticipated for 2011 and will hopefully establish a benchmark therapy. New targeted therapies, for example, IGF-1 receptor inhibitors, are under investigation and may soon improve current treatment options.

Urine Steroid Metabolomics as a Biomarker Tool for Detecting Malignancy in Adrenal Tumors

Context: Adrenal tumors have a prevalence of around 2% in the general population. Adrenocortical carcinoma (ACC) is rare but accounts for 2–11% of incidentally discovered adrenal masses. Differentiating ACC from adrenocortical adenoma (ACA) represents a diagnostic challenge in patients with adrenal incidentalomas, with tumor size, imaging, and even histology all providing unsatisfactory predictive values. Objective: Here we developed a novel steroid metabolomic approach, mass spectrometry-based steroid profiling followed by machine learning analysis, and examined its diagnostic value for the detection of adrenal malignancy. Design: Quantification of 32 distinct adrenal derived steroids was carried out by gas chromatography/mass spectrometry in 24-h urine samples from 102 ACA patients (age range 19–84 yr) and 45 ACC patients (20–80 yr). Underlying diagnosis was ascertained by histology and metastasis in ACC and by clinical follow-up [median duration 52 (range 26–201) months] without evidence of metastasis in ACA. Steroid excretion data were subjected to generalized matrix learning vector quantization (GMLVQ) to identify the most discriminative steroids. Results: Steroid profiling revealed a pattern of predominantly immature, early-stage steroidogenesis in ACC. GMLVQ analysis identified a subset of nine steroids that performed best in differentiating ACA from ACC. Receiver-operating characteristics analysis of GMLVQ results demonstrated sensitivity = specificity = 90% (area under the curve = 0.97) employing all 32 steroids and sensitivity = specificity = 88% (area under the curve = 0.96) when using only the nine most differentiating markers. Conclusions: Urine steroid metabolomics is a novel, highly sensitive, and specific biomarker tool for discriminating benign from malignant adrenal tumors, with obvious promise for the diagnostic work-up of patients with adrenal incidentalomas.

Integrative Genomic Analysis Reveals Somatic Mutations in Pheochromocytoma and Paraganglioma

Pheochromocytomas and paragangliomas are neuroendocrine tumors that occur in the context of inherited cancer syndromes in ∼30% of cases and are linked to germline mutations in the VHL, RET, NF1, SDHA, SDHB, SDHC, SDHD, SDHAF2 and TMEM127 genes. Although genome-wide expression studies have revealed some of the mechanisms likely to be involved in pheochromocytoma/paraganglioma tumorigenesis, the complete molecular distinction of all subtypes of hereditary tumors has not been solved and the genetic events involved in the generation of sporadic tumors are unknown. With these purposes in mind, we investigated 202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. Gene expression signatures defined the hereditary tumors according to their genotype and notably, led to a complete subseparation between SDHx- and VHL-related tumors. In tumor tissues, the systematic characterization of somatic genetic events associated with germline mutations in tumor suppressor genes revealed loss of heterozygosity (LOH) in a majority of cases, but also detected point mutations and copy-neutral LOH. Finally, guided by transcriptome classifications and LOH profiles, somatic mutations in VHL or RET genes were identified in 14% of sporadic pheochromocytomas/paragangliomas. Overall, we found a germline or somatic genetic alteration in 45.5% (92/202) of the tumors in this large series of pheochromocytomas/paragangliomas. Regarding mutated genes, specific molecular pathways involved in tumorigenesis mechanisms are identified. Altogether, these new findings suggest that somatic mutation analysis is likely to yield important clues for personalizing molecular targeted therapies.

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.

Germline inactivating mutations of the aryl hydrocarbon receptor-interacting protein gene in a large cohort of sporadic acromegaly: mutations are found in a subset of young patients with macroadenomas

OBJECTIVE Germline mutations of the aryl hydrocarbon receptor-interacting protein gene (AIP) have recently been described in three families with GH or prolactin-secreting tumors, as well as in a few patients with apparently sporadic somatotropinomas. The aim of the study was to determine the prevalence of AIP mutations in a large cohort of patients with apparently sporadic GH-secreting tumors. DESIGN One hundred and fifty-four patients were included in a prospective cohort designed to study the genetic predisposition to GH-secreting tumors together with 270 controls. METHODS In all these subjects, the entire coding sequence of the AIP gene was screened for germline mutations. RESULTS AIP mutations were detected in 5 out of 154 patients (3%): nonsense mutations in exon 4 (p.Lys201X; n = 2) and in exon 6 (p.Arg304X), one deletion in exon 3 (c.404delA; pHis135LeufsX21), and one mutation affecting the splice acceptor site of exon 4 (c.469-2 A > G). The five patients with an AIP mutation were significantly younger (mean age +/- S.D.: 25 +/- 10 vs 43 +/- 14 years, P = 0.005) and three of them presented with gigantism. One missense mutation (p.Arg304Gln) was found in a single patient that was absent in all controls. CONCLUSIONS Germline mutations of the AIP gene were found in a small proportion of patients with sporadic pituitary somatotropinomas. This study shows that age and gigantism are simple clinical features which can help to select patients for mutation screening. It also supports the role of AIP in pituitary tumorigenesis.

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.