DIAGNOSIS, LOCALIZATION, PATHOPHYSIOLOGY, AND MOLECULAR BIOLOGY OF PHEOCHROMOCYTOMA
, Head, Section on Medical Neuroendocrinology
Jie He, MD, Clinical Associate
Anna Kozupa, MD, Clinical Associate
Henri Timmers, MD, PhD, Visiting Fellow
Karen T. Adams, MSc, CRNP, Research Nurse
Shiromi M. Perera, BS, Technician
Daniel C. Solis, BA, NIH Academy Student
Stephanie Fliedner, MS, Predoctoral Fellow
Edwin W. Lai, BS, Predoctoral Fellow
Lucia Martiniova, MS, Predoctoral Fellow
Figure 11.6
Genotype-phenotype correlations in various pheochromocytomas
Advances in genetics and the recognition of the high prevalence of pheochromocytoma in certain familial syndromes dictate mandatory tumor screening in patients with those mutations associated with the disease, irrespective of the presence of classical clinical signs and symptoms. Accumulating data also indicate that many more pheochromocytomas are attributable to germline mutations than previously recognized, raising the importance of considering an underlying hereditary condition even in those patients without an obvious family history.
To date mutations in five main genes have been identified as responsible for familial pheochromocytomas (Table 11.1): (1) the von Hippel-Lindau (VHL) gene in VHL syndrome; (2) the RET gene in multiple endocrine neoplasia type 2 (MEN 2); (3) the neurofibromatosis type 1 (NF-1) gene associated with von Recklinghausen’s disease; and (4) mutations of genes encoding mainly the B and D subunits of mitochondrial succinate dehydrogenase (SDHB and SDHD) associated with familial pheochromocytomas.
Table 11.1
SDHB-related pheochromocytoma and paraganglioma
Patients with mutations in the SDHB and SDHD genes are more likely to develop extra-adrenal than adrenal tumors. Furthermore, SDHB mutations appear to be associated with more aggressive tumor behavior and a higher rate of malignancy. In an initial study, we examined the frequency of SDHB mutations in patients with malignant pheochromocytomas/paragangliomas. We found pathogenic SDHB mutations in 30 percent of patients. In those patients presenting initially with primary abdominal paragangliomas, mutations of the SDHB gene were associated with about one-half of all malignancies. The high frequency of SDHB germline mutations among patients with malignant disease, particularly when originating from paraganglioma, justifies a high priority for SDHB germline mutation testing in such patients.
In a further study, we aimed to gain deeper insight into the clinical and biochemical characteristics of SDHB-associated paragangliomas. We studied thirty patients with abdominal or thoracic paragangliomas and SDHB mutations. At presentation, 21 percent of the patients lacked any symptoms of catecholamine excess. Family history was positive for paraganglioma in only 10 percent of the patients. We found primary tumors in extra-adrenal locations in 97 percent of the patients, with a tumor mean diameter of about 8 cm. In 30 percent of the patients, metastatic disease was already apparent at initial diagnosis, and 97 percent of the patients eventually developed metastases after 2.6 years on average. The biochemical phenotype indicated hypersecretion of both norepinephrine (NE) and dopamine in about half of the patients. We observed no correlation between the SDHB gene mutation type and clinical presentations, including malignant potential.
We also found that, despite the presence of extensive metastatic disease, 10 percent of the patients with SDHB mutations had so-called biochemically silent pheochromocytomas (plasma and urine concentrations of catecholamines and their metabolites were consistently normal). Therefore, we investigated the mechanism underlying the lack of catecholamine production and/or secretion. In four patients with silent tumors, electron microscopy of tumor cells showed the presence of normal secretory granules. We found that the lack of catecholamine production by these tumors was attributable to the absence of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis.
In two additional studies, we demonstrated the importance of functional imaging. SDHB gene mutations carry a high malignant potential; thus, timely and accurate localization of SDHB-related pheochromocytomas and paragangliomas is critical for implementing optimal treatment. In 30 patients with SDHB-associated paragangliomas, we compared the sensitivities of the following methods for detection of metastases: 18F-fluorodopamine ([18F]-FDA) and 18F-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET), [123I]- and [131I]-metaiodobenzylguanidine (MIBG), 111In-pentetreotide (Octreoscan), and Tc-99m-methylene diphosphonate (MDP) bone scintigraphy. We used lesions detected by CT and MRI as a standard of reference. Sensitivity according to patient/body region were as follows: 123/131I-MIBG: 57/68; Octreoscan: 59/81; 18F-FDA: 70/99; and FDG: 97/100. FDG PET detected at least 90 percent of regions that were false negative on 123I-MIBG scintigraphy or 18F-FDA PET (Figure 11.7). We concluded that FDG PET is the preferred functional imaging modality for staging and treatment monitoring of SDHB-related metastatic paraganglioma.
Figure 11.7
Metastatic SDHB-related pheochromocytoma positive on FDG (arrows) but not on 18F-FDA PET or 123I-MIBG scintigraphy.
In a retrospective analysis of 71 subjects with metastatic pheochromocytoma (30 sub-jects with SDHB mutations), we compared the utility of bone scintigraphy with functional imaging in the detection of bone metastases. Seventy-seven percent of patients with SDHB mutations and 63 percent of patients without SDHB mutations presented with bone metastatic disease. The most common sites of bone involvement were thoracic spine (80 percent), lumbar spine (78 percent), and pelvic and sacral bones (78 percent). Subjects with SDHB mutations showed significantly higher involvement of long bones and pelvic and sacral bones than those without the mutation. The best overall sensitivity in detecting bone metastases was [18F]-FDA PET (90 percent), followed by bone scintigraphy (82 percent), CT or MRI (CT/MRI) (78 percent), and [123I]-MIBG (71 percent). In subjects with SDHB mutations, imaging modalities with the highest sensitivities for detecting bone metastases were CT/MRI (96 percent), bone scintigraphy (95 percent), and FDG PET (92 percent). In contrast, for subjects without SDHB mutations, the modality with the best sensitivity for bone metastases was [18F]-FDA PET (100 percent). We concluded that bone scintigraphy should be used for the detection of bone metastatic disease, particularly in patients with SDHB mutations. For the overall staging of patients with metastatic pheochromocytoma, we highly recommend FDG PET in patients with SDHB gene mutations, whereas we recommend [18F]-FDA PET in patients without SDHB gene mutations.
Brouwers FM, Kant J, Tao J, Eisenhofer G, Pacak K. High frequency of SDHB germline mutations in patients with malignant catecholamine-producing paragangliomas: implications for genetic testing. J Clin Endocrinol Metab 2006;91:4505-9.
Hadi M, Chen CC, Whatley M, Pacak K, Carrasquillo JA. Brown fat imaging with 18F-6-fluorodopamine PET/CT, 18F-FDG PET/CT, and 123I-MIBG SPECT: a study of patients being evaluated for pheochromocytoma. J Nucl Med 2007;48:1077-83.
Timmers HJLM, Hadi M, Carrasquillo JA, Chen CC, Martiniova L, Whatley M, Ling A, Eisenhofer G, Adams KT, Pacak K. The effects of carbidopa on uptake of 6-18F-Fluoro-L-DOPA in PET of pheochromocytoma and extraadrenal abdominal paraganglioma. J Nucl Med 2007;48:1599-606.
Timmers HJLM, Kozupa A, Chen CC, Carrasquillo JA, Ling A, Eisenhofer G, Adams KT, Solis D, Lenders JWM, Pacak K. Superiority of fluorodeoxyglucose positron emission tomography to other functional imaging techniques in the evaluation of metastatic Succinate Dehydrogenase Subunit B-associated pheochromocytoma and paraganglioma. J Clin Oncol 2007;5:2262-9.
Timmers HJLM, Kozupa A, Eisenhofer G, Raygada M, Adams KT, Solis D, Lenders JWM, Pacak K. Clinical presentations, biochemical phenotypes, and genotype-phenotype correlations in patients with SDHB-associated pheochromocytomas and paragangliomas: report from the NIH. J Clin Endocrinol Metab 2007;92:779-86.
VHL- and MEN-related pheochromocytoma
Clinical manifestations of pheochromocytomas in patients with VHL syndrome differ from manifestations in patients with MEN 2. Pheochromocytomas in VHL syndrome produce NE; those in MEN 2 produce epinephrine. Patients with MEN 2 pheochromocytomas have a high incidence of paroxysmal attacks and a higher prevalence of hypertension and other cardiovascular problems than do patients with VHL pheochromocytomas. Therefore, we hypothesized that these distinctions relate to differences in expression of the transporters responsible for uptake and storage of (1) catecholamines, namely, neuropeptide Y (NPY), a vasoactive peptide that influences blood pressure, and of (2) chromogranin A and B (CGA and CGB), major secretory proteins of chromaffin granules. We demonstrated that MEN 2–related pheochromocytomas expressed more cell membrane noradrenergic transporter mRNA and protein than tumors from VHL patients. The difference was associated with larger numbers of storage vesicles and higher tissue content of catecholamines in MEN 2 than in VHL tumors. In another study, we found that tumor NPY levels in VHL patients were significantly lower than in those from MEN 2 patients for both mRNA and the peptide. We also showed that pheochromocytomas from MEN 2 patients expressed substantially more CGA and CGB at both the mRNA and protein levels than tumors from VHL patients. We concluded that the differences in tumor CGA expression could contribute to differences in secretory vesicle formation and secretion in the two tumor types. Thus, VHL tumors possess deficiencies in the mechanisms governing the regulated pathway of secretory vesicle formation and the storage and release of granular contents. These deficiencies may contribute, for example, to the lower prevalence of hypertension in VHL patients versus patients with MEN 2–related pheochromocytoma.
[123I]-MIBG is used in the localization of pheochromocytoma/paraganglioma because of its excellent specificity. Suboptimal sensitivity may be caused by MIBG’s low affinity for the cell-membrane NE-transporter system, its loss by tumor cell dedifferentiation, the lack of storage granules, altered vesicle transporter, or drug interferences. Using CT, MRI, [123/131I]-MIBG scintigraphy, and [18F]-FDA PET, we evaluated seven VHL patients with adrenal pheochromocytoma. [18F]-FDA PET localized pheochromocytoma in all seven patients, as did CT. In contrast, three out of the seven had negative results with the use of [123/131I]-MIBG scintigraphy, and one out of six had negative MRI results. We concluded that [18F]-FDA PET in conjunction with CT/MRI should be considered an effective method for the proper localization of VHL-related adrenal pheochromocytoma.
Brouwers MF, Glasker S, Nave AF, Vogel T, Vortmeyer AO, Lubensky I, Huang S, Abu-Asab MS, Eisenhofer G, Weil RJ, Park DM, Linehan WM, Pacak K, Zhuang Z. Proteomic profiling of VHL and MEN2 pheochromocytomas reveals different expression of chromogranin B. Endocr Relat Cancer 2006;14:463-71.
Cleary S, Phillips JK, Huynh TT, Pacak K, Elkahloun AG, Barb J, Worrell RA, Goldstein DS, Eisenhofer G. Neuropeptide Y expression in phaeochromocytomas: relative absence in tumours from patients with von Hippel-Lindau syndrome. J Endocrinol 2006;193:225-33.
Cleary S, Phillips JK, Huynh TT, Pacak K, Fliedner S, Elkahloun AG, Munson P, Worrell RA, Eisenhofer G. Chromogranin A expression in phaeochromocytomas associated with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2. Horm Metab Res 2007;39:876-83.
Kaji P, Carrasquillo JA, Linehan WM, Chen CC, Eisenhofer G, Pinto PA, Lai EW, Pacak K. The role of 6-[18F]fluorodopamine positron emission tomography in the localization of adrenal pheochromocytoma associated with von Hippel-Lindau syndrome. Eur J Endocrinol 2006;156:483-7.
An animal model of pheochro-mocytoma: imaging and gene-tic results
The use of suitable laboratory animal models should speed evaluations of new treatments for pheochromocytoma, particularly in the case of uncommon malignancies, which often are not the subject of comprehensive clinical trials. Previously, we reported that we generated a new mouse model of metastatic pheochromocytoma by using tail vein injection of a mouse pheochromocytoma cell (MPC) line, reproducibly producing several liver tumors in the animals. Furthermore, we showed that in vivo microCT imaging, enhanced with the hepatobillary-specific contrast agent glyceryl-2-oleyl-1,3-di-7-(3-amino-2,4,6-triiodophenyl)-heptanoate (FenestraTMLC), detected liver lesions as small as 0.35 mm as early as four weeks after injection of the tumor cells.
As a continuation of our first anatomical studies, we have further optimized the use of microCT and MRI to localize organ and bone metastatic lesions in the mouse model of pheochromocytoma. To improve localization of liver lesions with microCT, we combined two time points of scanning. We performed the first microCT scan immediately after FenestraTMLC injection, when both liver lesions and vessels were detected. We performed the second scan three hours after FenestraTMLC injection when the contrast disappeared from the vessels and allowed detection of the liver metastatic lesions. By combining the two scans, we were able to localize liver lesions precisely at sizes of 300 to 500 µm in diameter (Figure 11.8A). We used MRI to detect other organ lesions. To optimize the signal-to-noise ratio, we used a small animal–dedicated radiofrequency coil on 3T MRI; to reduce the motion artifacts, we applied respiratory triggering on anesthetized mice while scanning (Figure 11.8B).
Figure 11.8
(A) Represents microCT with FenestraTMLC contrast agent. FenestraTMLC is still circulating in vessels and it is difficult to distinguish between liver lesions and vessels; right: represents scan 3 hours after FenestraTMLC was administered; (B) (a) Represents T2 weighted MRI image without respiratory triggering, (b) with respiratory triggering, achieving much better image quality and detection of liver lesions; (c) Represents necropsy of the animal at the end of the experiment.
We also introduced the use of [18F]-FDA, 18F-fluorodihydroxyphenylalanine ([18F]-FDOPA), and FDG PET scanning in our animal model. An Advanced Technology Laboratory Animal Scanner (ATLAS) collected the PET image data. The spatial resolution of the system is 1.8 mm FWHM (full width at half maximum) in the axial FOV (field of view). [18F]-6F-DA and [18F]-DOPA PET, but only rarely [18F]-FDG PET, detected hepatic metastasis in all animals, and those findings were comparable to microCT and MRI results. We concluded that the combination of MRI and PET scanning using pheochromocytoma-specific radiopharmaceuticals is the best approach to assessing the extent of metastatic disease in mice.
In addition to developing and optimizing methods for tumor imaging in mice, we used the mouse model of metastatic pheochromocytoma to study the gene expression profile of hepatic and subcutaneous lesions derived from mouse pheochromocytoma cells. We cannot overstate the importance of developing biomarkers or genomic screens that may predict the aggressiveness of pheochromocytoma/paraganglioma and, ultimately, a patient’s prognosis. Comparison of subcutaneous and liver tumors revealed eight genes (Pten, Mdm2, Metap2, Rb1, Reck, S100a4, Timp2, and Timp3) with two-fold increases in expression in the liver compared with subcutaneous tumors. QT-PCR analysis confirmed five of these genes (Metap2, Reck, S100a4, Timp2, and Timp3). The study provided initial information about which genes could play an important role in the aggressive behavior of metastatic pheochromocytoma cells.
Martiniova L, Ohta S, Guion P, Schimel D, Lai EW, Klaunberg B, Jagoda E, Pacak K. Anatomical and functional imaging of tumors in animal models: focus on pheochromocytoma. Ann NY Acad Sci 2006;1073:392-404.
Ohta S, Lai EW, Morris JC, Klaunberg B, Green J, Bakan DA, Kukura C, Shun’ichiro T, Tischler AS, Powers JF, Alesci S, Schimel D, Pacak K. MicroCT for high-resolution imaging of ectopic pheochromocytoma tumors in the liver of nude mice. Int J Cancer 2006;119:2236-41.
Ohta S, Lai EW, Morris JC, Pang ALY, Watanabe M, Yazawa H, Zhang R, Green JE, Chan W-Y, Sirajuddin P, Taniguchi S, Powers JF, Tischler AS, Pacak K. Metastasis-associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells. Mol Carcinogenesis [E-pub ahead of print].
Ohta S, Lai EW, Taniguchi S, Tischler AS, Alesci S, Pacak K. Animal models of pheochromocytoma including NIH initial experience. Ann NY Acad Sci 2006;1073:300-5.
Pacak K, Eisenhofer G, eds. Pheochromocytoma. Ann NY Acad Sci 2006;1073 pages.
Effective pheochromocytoma teamwork: action through multidisciplinary and multi-institutional approaches and the involvement of patients
The rarity of pheochromocytoma and the resulting fragmented nature of studies, which typically involve small numbers of patients, place limits on the development of effective treatment options, diagnostic approaches, and prognostic markers for pheochromocytoma. Yet, the development of needed treatments and diagnostic approaches may be facilitated by comprehensive clinical studies involving large numbers of patients, stringently collected clinical data and tumor and blood samples, and interdisciplinary collaborations among several specialist centers. Therefore, by initiating the Pheochromocytoma RESearch Support ORganization (PRESSOR; http:www.pressor.org), we expedited collaborations and the sharing of materials, information, and expertise among investigators and clinicians interested in pheochromocytoma-related research. Through its support network and structure, PRESSOR permits a concerted and coordinated approach to basic and clinical research into pheochromocytoma that should lead to an improved understanding of the biology of the tumor and more rapid advances in treatments for patients with malignancies. We organized several symposia for health professionals (see Workshops and Conferences at the beginning of this volume) to establish consensus on evidence-based guidelines for effective biochemical diagnosis, localization, and treatment of various types of pheochromocytoma and developed strategies for future effective treatments for metastatic pheochromocytoma.
Pacak K, Eisenhofer G, Ahlman H, Bornstein SR, Gimenez-Raqueplo A-P, Grossman AB, Kimura N, Mannelli M, McNicol AM, Tischler AT. Pheochromocytoma: recommendations for clinical practice from the First International Symposium. Nat Clin Pract Endocrinol Metab 2007;3:92-102.
COLLABORATORS
Mones Abu-Asab, MD, PhD, Laboratory of Pathology, NCI, Bethesda, MD
Chris Albanese, PhD, Georgetown University, Washington, DC
Salvatore Alesci, MD, PhD, Clinical Neuroendocrinology Branch, NIMH, Bethesda, MD
Jan Breza, MD, PhD, DSc, Komensky University, Bratislava, Slovak Republic
Jorge Carrasquillo, MD, Nuclear Medicine Department, NIH Clinical Center, Bethesda, MD
Wai-Yee Chan, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Clara Chen, MD, Nuclear Medicine Department, NIH Clinical Center, Bethesda, MD
Susannah Cleary, BS, Clinical Neuroscience Branch, NINDS, Bethesda, MD
Graeme Eisenhofer, PhD, Clinical Neuroscience Branch, NINDS, Bethesda, MD
Abdel Elkahloun, PhD, Genome Technology Branch, NHGRI, Bethesda, MD
Tito Fojo, MD, PhD, Cancer Therapeutics Branch, NCI, Bethesda, MD
David Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, Bethesda, MD
Jeff Green, MD, PhD, Laboratory of Cell Regulation and Carcinogenesis, NCI, Bethesda, MD
Thanh-Truc Huynh, BS, Clinical Neurocardiology Section, NINDS, Bethesda, MD
Ioannis Ilias, MD, University of Patras, Patras, Greece
Jacques Lenders, MD, PhD, Universiteit Nijmegen, Nijmegen, Netherlands
Stephen Libutti, MD, Center for Cancer Research, NCI, Bethesda, MD
W. Marston Linehan, MD, Urologic Oncology Branch, NCI, Bethesda, MD
Alexander Ling, MD, Radiology Department, NIH Clinical Center, Bethesda, MD
Irina A. Lubensky, MD, Surgical Neurology Branch, NINDS, Bethesda, MD
Maria Merino, MD, Laboratory of Pathology, NCI, Bethesda, MD
John Morris, MD, PhD, Metabolism Branch, NCI, Bethesda, MD
Peter J. Munson, PhD, Mathematical Computing Program, CIT, NIH, Bethesda, MD
Alan L.Y. Pang, MD, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Jacqueline Phillips, PhD, Murdoch University, Perth, Australia
Raj K. Puri, MD, PhD, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Margarita Raygada, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
James Reynolds, MD, PhD, Nuclear Medicine Department, NIH Clinical Center, Bethesda, MD
Arthur S. Tischler, MD, New England Medical Center, Boston, MA
Alexander O. Vortmeyer, MD, PhD, Surgical Neurology Branch, NINDS, Bethesda, MD
Robert A. Wesley, PhD, Biostatistics and Epidemiology Service, NIH Clinical Center, Bethesda, MD
Jiri Widimsky, MD, PhD, Charles University, Prague, Czech Republic
Zhengping Zhuang, MD, PhD, Surgical Neurology Branch, NINDS, Bethesda, MD
For further information, contact karel@mail.nih.gov.
