Pheochromocytomas (Pheos) and paragangliomas (PGLs) are neuroendocrine tumors. Approximately 30–40% of Pheos/PGLs are due to germline mutations in one of the susceptibility genes, including those encoding the succinate dehydrogenase subunits A-D (SDHA-D). Up to 2/3 of patients affected by SDHB mutated Pheo/PGL develop metastatic disease with no successful cure at present. Here, for the first time, we evaluated the effects of SDHB silencing in a three dimension (3D) culture using spheroids of a mouse Pheo cell line silenced or not (wild type/wt/control) for the SDHB subunit. We investigated the role of the microenvironment on spheroid growth and migration/invasion by co-culturing SDHB-silenced or wt spheroids with primary cancer-activated fibroblasts (CAFs). When spheroids were co-cultured with fibroblasts, SDHB-silenced cells showed a significant increase in matrigel invasion as demonstrated by the computation of the migratory areas (P < 0.001). Moreover, cells detaching from the SDHB-silenced spheroids moved collectively, unlike the cells of wt spheroids that moved individually. Additionally, SDHB-silenced spheroids developed long filamentous formations along which clusters of cells migrated far away from the spheroid, whereas these structures were not present in wt spheroids. We found that lactate, largely secreted by CAFs, plays a specific role in promoting migration only of SDHB-silenced cells. In this study, we demonstrated that SDHB silencing per se increases tumor cell migration/invasion and that microenvironment, as represented by CAFs, plays a pivotal role in enhancing collective migration/invasion in Pheo SDHB-silenced tumor cells, suggesting their role in increasing the tumor metastasizing potential.
Vanessa D'Antongiovanni, Serena Martinelli, Susan Richter, Letizia Canu, Daniele Guasti, Tommaso Mello, Paolo Romagnoli, Karel Pacak, Graeme Eisenhofer, Massimo Mannelli and Elena Rapizzi
Martin Ullrich, Josephine Liers, Mirko Peitzsch, Anja Feldmann, Ralf Bergmann, Ulrich Sommer, Susan Richter, Stefan R Bornstein, Michael Bachmann, Graeme Eisenhofer, Christian G Ziegler and Jens Pietzsch
Somatostatin receptor-targeting endoradiotherapy offers potential for treating metastatic pheochromocytomas and paragangliomas, an approach likely to benefit from combination radiosensitization therapy. To provide reliable preclinical in vivo models of metastatic disease, this study characterized the metastatic spread of luciferase-expressing mouse pheochromocytoma (MPC) cells in mouse strains with different immunologic conditions. Bioluminescence imaging showed that, in contrast to subcutaneous non-metastatic engraftment of luciferase-expressing MPC cells in NMRI-nude mice, intravenous cell injection provided only suboptimal metastatic spread in both NMRI-nude mice and hairless SCID (SHO) mice. Treatment of NMRI-nude mice with anti-Asialo GM1 serum enhanced metastatic spread due to substantial depletion of natural killer (NK) cells. However, reproducible metastatic spread was only observed in NK cell-defective SCID/beige mice and in hairless immunocompetent SKH1 mice bearing disseminated or liver metastases, respectively. Liquid chromatography tandem mass spectrometry of urine samples showed that subcutaneous and metastasized tumor models exhibit comparable renal monoamine excretion profiles characterized by increasing urinary dopamine, 3-methoxytyramine, norepinephrine and normetanephrine. Metastases-related epinephrine and metanephrine were only detectable in SCID/beige mice. Positron emission tomography and immunohistochemistry revealed that all metastases maintained somatostatin receptor-specific radiotracer uptake and immunoreactivity, respectively. In conclusion, we demonstrate that intravenous injection of luciferase-expressing MPC cells into SCID/beige and SKH1 mice provides reproducible and clinically relevant spread of catecholamine-producing and somatostatin receptor-positive metastases. These standardized preclinical models allow for precise monitoring of disease progression and should facilitate further investigations on theranostic approaches against metastatic pheochromocytomas and paragangliomas.
Frederieke M Brouwers, Sven Gläsker, Amanda F Nave, Alexander O Vortmeyer, Irina Lubensky, Steven Huang, Mones S Abu-Asab, Graeme Eisenhofer, Robert J Weil, Deric M Park, W Marston Linehan, Karel Pacak and Zhengping Zhuang
Pheochromocytomas are catecholamine-producing tumors that can occur in the context of von Hippel–Lindau syndrome (VHL) and multiple endocrine neoplasia type 2 (MEN2). Pheochromocytomas in these two syndromes differ in histopathological features, catecholamine metabolism, and clinical phenotype. To further investigate the nature of these differences, we compared the global protein expressions of 8 MEN2A-associated pheochromocytomas with 11 VHL-associated pheochromocytomas by two-dimensional gel electrophoresis proteomic profiling followed by sequencing and identification of differentially expressed proteins. Although both types of pheochromocytoma shared similarities in their protein expression patterns, the expression of several proteins was distinctly different between VHL- and MEN2A-associated pheochromocytomas. We identified several of these differentially expressed proteins. One of the proteins with higher expression in MEN2-associated tumors was chromogranin B, of which the differential expression was confirmed by western blot analysis. Our results expand the evidence for proteomic differences between these two tumor entities, and suggest that VHL-associated pheochromocytomas may be deficient in fundamental machinery for catecholamine storage. In light of these new findings, as well as existing evidence for differences between both types of pheochromocytomas, we propose that these tumors may have different developmental origins.
Hans K Ghayee, Bas Havekes, Eleonora P M Corssmit, Graeme Eisenhofer, Stephen R Hammes, Zahid Ahmad, Alexander Tessnow, Ivica Lazúrová, Karen T Adams, Antonio T Fojo, Karel Pacak and Richard J Auchus
Extra-adrenal pheochromocytomas, otherwise known as paragangliomas (PGLs), account for about 20% of catecholamine-producing tumors. Catecholamine excess and mutations in the genes encoding succinate dehydrogenase subunits (SDHx) are frequently found in patients with PGLs. Only 2% of PGLs are found in the mediastinum, and little is known about genetic alterations in patients with mediastinal PGLs, catecholamine production by these tumors, or their clinical behavior. We hypothesized that most mediastinal PGLs are associated with germ line SDHx mutations, norepinephrine and/or dopamine excess, and aggressive behavior. The objective of this study was to characterize genetic, biochemical, and clinical data in a series of ten patients with mediastinal PGLs. All ten primary mediastinal PGL patients had germ line SDHx mutations, six in SDHB, and four in SDHD genes. Chest or back pain were the most common presenting symptoms (five patients), and catecholamines and/or their metabolites were elevated in seven patients. Additional tumors included head and neck PGLs in four patients, pheochromocytoma in one patient, and bladder PGL in another. Metastatic disease was documented in six patients (60%), and a concurrent abdominal mass was found in one patient. We conclude that mediastinal PGLs are strongly associated with SDHB and SDHD gene mutations, noradrenergic phenotype, and aggressive behavior. The present data suggest that all patients with mediastinal PGLs should be screened for SDHx gene mutations, regardless of age.
Graeme Eisenhofer, Karel Pacak, Thanh-Truc Huynh, Nan Qin, Gennady Bratslavsky, W Marston Linehan, Massimo Mannelli, Peter Friberg, Stefan K Grebe, Henri J Timmers, Stefan R Bornstein and Jacques W M Lenders
Phaeochromocytomas and paragangliomas (PPGLs) are highly heterogeneous tumours with variable catecholamine biochemical phenotypes and diverse hereditary backgrounds. This analysis of 18 catecholamine-related plasma and urinary biomarkers in 365 patients with PPGLs and 846 subjects without PPGLs examined how catecholamine metabolomic profiles are impacted by hereditary background and relate to variable hormone secretion. Catecholamine secretion was assessed in a subgroup of 156 patients from whom tumour tissue was available for measurements of catecholamine contents. Among all analytes, the free catecholamine O-methylated metabolites measured in plasma showed the largest tumour-related increases relative to the reference group. Patients with tumours due to multiple endocrine neoplasia type 2 and neurofibromatosis type 1 (NF1) showed similar catecholamine metabolite and secretory profiles to patients with adrenaline-producing tumours and no evident hereditary background. Tumours from these three patient groups contained higher contents of catecholamines, but secreted the hormones at lower rates than tumours that did not contain appreciable adrenaline, the latter including PPGLs due to von Hippel–Lindau (VHL) and succinate dehydrogenase (SDH) gene mutations. Large increases of plasma dopamine and its metabolites additionally characterised patients with PPGLs due to the latter mutations, whereas patients with NF1 were characterised by large increases in plasma dihydroxyphenylglycol and dihydroxyphenylacetic acid, the deaminated metabolites of noradrenaline and dopamine. This analysis establishes the utility of comprehensive catecholamine metabolite profiling for characterising the distinct and highly diverse catecholamine metabolomic and secretory phenotypes among different groups of patients with PPGLs. The data further suggest developmental origins of PPGLs from different populations of chromaffin cell progenitors.
Tomáš Zelinka, Henri J L M Timmers, Anna Kozupa, Clara C Chen, Jorge A Carrasquillo, James C Reynolds, Alexander Ling, Graeme Eisenhofer, Ivica Lazúrová, Karen T Adams, Millie A Whatley, Jiří Widimský Jr and Karel Pacak
We performed a retrospective analysis of 71 subjects with metastatic pheochromocytoma and paraganglioma (30 subjects with mutation of succinate dehydrogenase enzyme subunit B (SDHB) gene and 41 subjects without SDHB mutation). Sixty-nine percent presented with bone metastases (SDHB +/−: 77% vs 63%), 39% with liver metastases (SDHB +/−: 27% vs 47%), and 32% with lung metastases (SDHB +/−: 37% vs 29%). The most common sites of bone involvement were thoracic spine (80%; SDHB+/−: 83% vs 77%), lumbar spine (78%; SDHB +/−: 78% vs 75%), and pelvic and sacral bones (78%; SDHB +/−: 91% vs 65%, P=0.04). Subjects with SDHB mutation also showed significantly higher involvement of long bones (SDHB +/−: 78% vs 30%, P=0.007) than those without the mutation. The best overall sensitivity in detecting bone metastases demonstrated positron emission tomography (PET) with 6-[18F]-fluorodopamine ([18F]-FDA; 90%), followed by bone scintigraphy (82%), computed tomography or magnetic resonance imaging (CT/MRI; 78%), 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]-FDG) PET (76%), and scintigraphy with [123/131I]-metaiodobenzylguanidine (71%). In subjects with SDHB mutation, imaging modalities with best sensitivities for detecting bone metastases were CT/MRI (96%), bone scintigraphy (95%), and [18F]-FDG PET (92%). In subjects without SDHB mutations, the modality with the best sensitivity for bone metastases was [18F]-FDA PET (100%). In conclusion, bone scintigraphy should be used in the staging of patients with malignant pheochromocytoma and paraganglioma, particularly in patients with SDHB mutations. As for PET imaging, [18F]-FDG PET is highly recommended in SDHB mutation patients, whereas [18F]-FDA PET is recommended in patients without the mutation.
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.