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Free access

Eva Szarek, Evan R Ball, Alessio Imperiale, Maria Tsokos, Fabio R Faucz, Alessio Giubellino, François-Marie Moussallieh, Izzie-Jacques Namer, Mones S Abu-Asab, Karel Pacak, David Taïeb, J Aidan Carney, and Constantine A Stratakis

Carney triad (CTr) describes the association of paragangliomas (PGL), pulmonary chondromas, and gastrointestinal (GI) stromal tumors (GISTs) with a variety of other lesions, including pheochromocytomas and adrenocortical tumors. The gene(s) that cause CTr remain(s) unknown. PGL and GISTs may be caused by loss-of-function mutations in succinate dehydrogenase (SDH) (a condition known as Carney–Stratakis syndrome (CSS)). Mitochondrial structure and function are abnormal in tissues that carry SDH defects, but they have not been studied in CTr. For the present study, we examined mitochondrial structure in human tumors and GI tissue (GIT) of mice with SDH deficiency. Tissues from 16 CTr tumors (n=12), those with isolated GIST (n=1), and those with CSS caused by SDHC (n=1) and SDHD (n=2) mutations were studied by electron microscopy (EM). Samples of GIT from mice with a heterozygous deletion in Sdhb (Sdhb + /−, n=4) were also studied by EM. CTr patients presented with mostly epithelioid GISTs that were characterized by plump cells containing a centrally located, round nucleus and prominent nucleoli; these changes were almost identical to those seen in the GISTs of patients with SDH. In tumor cells from patients, regardless of diagnosis or tumor type, cytoplasm contained an increased number of mitochondria with a ‘hypoxic’ phenotype: mitochondria were devoid of cristae, exhibited structural abnormalities, and were of variable size. Occasionally, mitochondria were small and round; rarely, they were thin and elongated with tubular cristae. Many mitochondria exhibited amorphous fluffy material with membranous whorls or cystic structures. A similar mitochondrial hypoxic phenotype was seen in Sdhb + /− mice. We concluded that tissues from SDH-deficient tumors, those from mouse GIT, and those from CTr tumors shared identical abnormalities in mitochondrial structure and other features. Thus, the still-elusive CTr defect(s) is(are) likely to affect mitochondrial function, just like germline SDH-deficiency does.

Open access

Trisha Dwight, Edward Kim, Karine Bastard, Diana E Benn, Graeme Eisenhofer, Susan Richter, Massimo Mannelli, Elena Rapizzi, Aleksander Prejbisz, Mariola Pęczkowska, Karel Pacak, and Roderick Clifton-Bligh

Mosaic or somatic EPAS1 mutations are associated with a range of phenotypes including pheochromocytoma and/or paraganglioma (PPGL), polycythemia and somatostatinoma. The pathogenic potential of germline EPAS1 variants however is not well understood. We report a number of germline EPAS1 variants occurring in patients with PPGL, including a novel variant c.739C>A (p.Arg247Ser); a previously described variant c.1121T>A (p.Phe374Tyr); several rare variants, c.581A>G (p.His194Arg), c.2353C>A (p.Pro785Thr) and c.2365A>G (p.Ile789Val); a common variant c.2296A>C (p.Thr766Pro). We performed detailed functional studies to understand their pathogenic role in PPGL. In transient transfection studies, EPAS1/HIF-2α p.Arg247Ser, p.Phe374Tyr and p.Pro785Thr were all stable in normoxia. In co-immunoprecipitation assays, only the novel variant p.Arg247Ser showed diminished interaction with pVHL. A direct interaction between HIF-2α Arg247 and pVHL was confirmed in structural models. Transactivation was assessed by means of a HRE-containing reporter gene in transiently transfected cells, and significantly higher reporter activity was only observed with EPAS1/HIF-2α p.Phe374Tyr and p.Pro785Thr. In conclusion, three germline EPAS1 variants (c.739C>A (p.Arg247Ser), c.1121T>A (p.Phe374Tyr) and c.2353C>A (p.Pro785Thr)) all have some functional features in common with somatic activating mutations. Our findings suggest that these three germline variants are hypermorphic alleles that may act as modifiers to the expression of PPGLs.

Free access

James F Powers, Brent Cochran, James D Baleja, Hadley D Sikes, Andrew D Pattison, Xue Zhang, Inna Lomakin, Annette Shepard-Barry, Karel Pacak, Sun Jin Moon, Troy F Langford, Kassi Taylor Stein, Richard W Tothill, Yingbin Ouyang, and Arthur S Tischler

Free access

Graeme Eisenhofer, Stefan R Bornstein, Frederieke M Brouwers, Nai-Kong V Cheung, Patricia L Dahia, Ronald R de Krijger, Thomas J Giordano, Lloyd A Greene, David S Goldstein, Hendrik Lehnert, William M Manger, John M Maris, Hartmut P H Neumann, Karel Pacak, Barry L Shulkin, David I Smith, Arthur S Tischler, and William F Young Jr

Pheochromocytomas are rare catecholamine-producing neuroendocrine tumors that are usually benign, but which may also present as or develop into a malignancy. Predicting such behavior is notoriously difficult and there are currently no curative treatments for malignant tumors. This report follows from a workshop at the Banbury Conference Center, Cold Spring Harbor, New York, on the 16th–18th November 2003, held to review the state of science and to facilitate future progress in the diagnosis and treatment of malignant pheochromocytoma. The rarity of the tumor and the resulting fragmented nature of studies, typically involving small numbers of patients, represent limiting factors to the development of effective treatments and diagnostic or prognostic markers for malignant disease. Such development is being facilitated by the availability of new genomics-based tools, but for such approaches to succeed ultimately requires comprehensive clinical studies involving large numbers of patients, stringently collected clinical data and tumor samples, and interdisciplinary collaborations among multiple specialist centers. Nevertheless, the well-characterized hereditary basis and the unique functional nature of these neuroendocrine tumors provide a useful framework that offers advantages for establishing the pathways of tumorigenesis and malignancy. Such findings may have relevance for understanding the basis of other more common malignancies where similar frameworks are not available. As the relevant pathways leading to pheochromocytoma are established it should be possible to take advantage of the new generation of drugs being developed to target specific pathways in other malignancies. Again the success of this will require well-designed and coordinated multicenter studies.

Open access

James F Powers, Brent Cochran, James D Baleja, Hadley D Sikes, Andrew D Pattison, Xue Zhang, Inna Lomakin, Annette Shepard-Barry, Karel Pacak, Sun Jin Moon, Troy F Langford, Kassi Taylor Stein, Richard W Tothill, Yingbin Ouyang, and Arthur S Tischler

Tumors caused by loss-of-function mutations in genes encoding TCA cycle enzymes have been recently discovered and are now of great interest. Mutations in succinate dehydrogenase (SDH) subunits cause pheochromocytoma/paraganglioma (PCPG) and syndromically associated tumors, which differ phenotypically and clinically from more common SDH-intact tumors of the same types. Consequences of SDH deficiency include rewired metabolism, pseudohypoxic signaling and altered redox balance. PCPG with SDHB mutations are particularly aggressive, and development of treatments has been hampered by lack of valid experimental models. Attempts to develop mouse models have been unsuccessful. Using a new strategy, we developed a xenograft and cell line model of SDH-deficient pheochromocytoma from rats with a heterozygous germline Sdhb mutation. The genome, transcriptome and metabolome of this model, called RS0, closely resemble those of SDHB-mutated human PCPGs, making it the most valid model now available. Strategies employed to develop RS0 may be broadly applicable to other SDH-deficient tumors.

Free access

Roland Därr, Joan Nambuba, Jaydira Del Rivero, Ingo Janssen, Maria Merino, Milena Todorovic, Bela Balint, Ivana Jochmanova, Josef T Prchal, Ronald M Lechan, Arthur S Tischler, Vera Popovic, Dragana Miljic, Karen T Adams, F Ryan Prall, Alexander Ling, Meredith R Golomb, Michael Ferguson, Naris Nilubol, Clara C Chen, Emily Chew, David Taïeb, Constantine A Stratakis, Tito Fojo, Chunzhang Yang, Electron Kebebew, Zhengping Zhuang, and Karel Pacak

Worldwide, the syndromes of paraganglioma (PGL), somatostatinoma (SOM) and early childhood polycythemia are described in only a few patients with somatic mutations in the hypoxia-inducible factor 2 alpha (HIF2A). This study provides detailed information about the clinical aspects and course of 7 patients with this syndrome and brings into perspective these experiences with the pertinent literature. Six females and one male presented at a median age of 28 years (range 11–46). Two were found to have HIF2A somatic mosaicism. No relatives were affected. All patients were diagnosed with polycythemia before age 8 and before PGL/SOM developed. PGLs were found at a median age of 17 years (range 8–38) and SOMs at 29 years (range 22–38). PGLs were multiple, recurrent and metastatic in 100, 100 and 29% of all cases, and SOMs in 40, 40 and 60%, respectively. All PGLs were primarily norepinephrine-producing. All patients had abnormal ophthalmologic findings and those with SOMs had gallbladder disease. Computed tomography (CT) and magnetic resonance imaging revealed cystic lesions at multiple sites and hemangiomas in 4 patients (57%), previously thought to be pathognomonic for von Hippel–Lindau disease. The most accurate radiopharmaceutical to detect PGL appeared to be [18F]-fluorodihydroxyphenylalanine ([18F]-FDOPA). Therefore, [18F]-FDOPA PET/CT, not [68Ga]-(DOTA)-[Tyr3]-octreotate ([68Ga]-DOTATATE) PET/CT is recommended for tumor localization and aftercare in this syndrome. The long-term prognosis of the syndrome is unknown. However, to date no deaths occurred after 6 years follow-up. Physicians should be aware of this unique syndrome and its diagnostic and therapeutic challenges.

Free access

Nicole Bechmann, Mats Leif Moskopp, Martin Ullrich, Bruna Calsina, Pål William Wallace, Susan Richter, Markus Friedemann, Katharina Langton, Stephanie M J Fliedner, Henri J L M Timmers, Svenja Nölting, Felix Beuschlein, Martin Fassnacht, Aleksander Prejbisz, Karel Pacak, Hans K Ghayee, Stefan R Bornstein, Peter Dieterich, Jens Pietzsch, Ben Wielockx, Mercedes Robledo, Nan Qin, and Graeme Eisenhofer

Mutations that drive the stabilization of hypoxia inducible factor 2α (HIF2α) and downstream pseudohypoxic signaling are known to predispose to the development of pheochromocytomas and paragangliomas (PPGLs). However, any role of HIF2α in predisposition to metastatic disease remains unclear. To assess such a role we combined gene-manipulations in pheochromocytoma cell lines with retrospective analyses of patient data and gene expression profiling in tumor specimens. Among 425 patients with PPGLs identified with mutations in tumor-susceptibility genes, those with tumors due to activation of pseudohypoxic pathways had a higher frequency of metastatic disease than those with tumors due to activation of kinase-signaling pathways, even without inclusion of patients with mutations in SDHB (18.6% vs 4.3% in, P < 0.0001). Three out of nine (33%) patients with gain-of-function mutations in HIF2α had metastatic disease. In cell line studies, elevated expression of HIF2α enhanced cell proliferation and led to increased migration and invasion capacity. Moreover, HIF2α expression in HIF2α-deficient cells resulted in increased cell motility, diffuse cluster formation and emergence of pseudopodia indicating changes in cell adhesion and cytoskeletal remodeling. In a mouse liver metastasis model, Hif2a enhanced the metastatic load. Transcriptomics data revealed alterations in focal adhesion and extracellular matrix–receptor interactions in HIF2α-mutated PPGLs. Our translational findings demonstrate that HIF2α supports pro-metastatic behavior in PPGLs, though other factors remain critical for subsequent transition to metastasis. We identified LAMB1 and COL4A2 as new potential therapeutic targets for HIF2α-driven PPGLs. Identified HIF2α downstream targets might open a new therapeutic window for aggressive HIF2α-expressing tumors.