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DIAGNOSIS, LOCALIZATION, PATHOPHYSIOLOGY, AND MOLECULAR BIOLOGY OF PHEOCHROMOCYTOMA

Karel Pacak, MD, PhD, DSc, Head, Unit on Clinical Neuroendocrinology

Frederieke M. Brouwers, MD, Postdoctoral Fellow

Shoichiro Ohta, MD, PhD, Postdoctoral Fellow

Dnyanesh Tipre, PhD, Postdoctoral Fellow

Edwin W. Lai, BS, Predoctoral Fellow

Shiromi Perera, BS, Technician

Karen T. Adams, RN, Research Nurse

We are conducting patient-oriented, conceptually innovative research into the etiology, pathophysiology, diagnosis, prognosis, and treatment of pheochromocytoma (PHEO). Projects include not only translational research. i.e., applying basic science knowledge to clinical diagnosis, pathophysiology, and treatment, but also “reverse translation research,” by which appreciation of clinical findings leads to new concepts that basic researchers can pursue in the laboratory. The long-range goal is to develop new and improved approaches in the diagnosis and treatment of PHEO. As an initial step toward this goal, we focus on molecular and genetic mechanisms and proteomics that may elucidate the bases for the malignant potential of PHEOs, for the predisposition to develop these tumors, and for expression of different neurochemical phenotypes. We also focus on new imaging approaches based on 6-[¹⁸F]fluorodopamine positron emission tomographic (PET) scanning and on new biochemical diagnostic criteria based on measurement of plasma metanephrines. Our research team will conduct clinical studies related to treatment of malignant PHEO using ¹³¹I-metaiodobenzylguanidine.

Biochemical diagnosis of pheochromocytoma

Pacak; in collaboration with Eisenhofer, Lenders, Linehan, Walther

The diagnosis of PHEO typically requires confirmation by several tests, perhaps the most important of which is biochemical evidence of excessive catecholamine production by the tumor. Such evidence is usually based on measurements of catecholamines and certain catecholamine metabolites in urine or plasma. However, the catecholamines norepinephrine and epinephrine are also produced by sympathetic nerves and the adrenal medulla and are thus not specific to PHEO. Sometimes PHEO may be biochemically “silent,” either not producing catecholamines in amounts sufficient to obtain a positive biochemical test result or secreting catecholamines episodically; between episodes, catecholamine levels may be normal. Moreover, due to the low prevalence of PHEO in the tested population and inadequate specificity of biochemical tests, positive results in patients without PHEO are a common and troublesome occurrence. In collaboration with G. Eisenhofer, we have developed a biochemical test involving measurements of free plasma normetanephrine and metanephrine, the metabolites, respectively, of norepinephrine and epinephrine, which offers advantages over other tests for diagnosis of PHEO. In contrast to catecholamines, metanephrines are produced continuously and independently of exocytotic catecholamine release.

We carried out a large prospective multicenter cohort study of 214 patients in whom the diagnosis of PHEO was confirmed and of 644 patients in whom the diagnosis of PHEO was excluded. Sensitivities of free plasma and urinary fractionated metanephrines were higher than those for plasma catecholamines, urinary catecholamines, urinary total metanephrines, and urinary vanillylmandelic acid. Specificity was highest for urinary vanillylmandelic acid and urinary total metanephrines; intermediate for free plasma metanephrines, urinary catecholamines, and plasma catecholamines; and lowest for urinary fractionated metanephrines. Recently, we also reported that free plasma metanephrines are relatively independent of renal function and are therefore more suitable for diagnosis of PHEO among patients with renal failure than measurements of deconjugated metanephrines. Currently, we are also attempting to determine the diagnostic utility of measuring free plasma metanephrines in patients with metastatic PHEO before and after treatment.

Eisenhofer G, Pacak K. Diagnosis of pheochromocytoma. In: Harrison’s Textbook online, McGraw-Hill:2004.

Lenders JW, Pacak K, Walther MM, Linehan WM, Friberg P, Keiser HK, Goldstein DS, Eisenhofer G. Biochemical diagnosis of pheochromocytoma: which test is best? JAMA 2002;287:1427-1434.

Eisenhofer G, Goldstein DS, Walther MM, Lenders JWM, Friberg P, Keiser HR, Pacak K. Biochemical diagnosis of pheochromocytoma: how to distinguish true- from false-positive test results. J Clin Endocrinol Metab 2003;88:2656-2666.

Eisenhofer G, Huysmans F, Pacak K, Walther MM, Sweep CGJ, Lenders JWM. Plasma metanephrines in renal failure. Kidney Int, 2005, in press.

The molecular genetic basis of tumorigenesis in malignant pheochromocytomas

Ohta, Lai, Pacak; in collaboration with Breza, Chan, de Krijger, Eisenhofer, Ksinantova, Kvetnansky, Munson, Pang

Currently, we lack a reliable method for predicting the malignant potential of PHEO based on conventional histology or genetic, molecular, or immunohistochemical markers. Metastasis suppressor genes affect the spread of several cancers and therefore may hold promise as prognostic markers or therapeutic targets for malignant PHEO. Thus, we hypothesized that the downregulation of metastasis suppressor genes in malignant PHEO may play a role in malignant behavior.

We applied a quantitative real-time polymerase chain reaction to 11 metastasis suppressor genes known to be involved in the regulation of important cancer-related cell events, such as cell growth regulation and apoptosis (nm23-HI, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TXNIP, and CRSP-3), cell-cell communication (BRSM-1), invasion (CRMP-1), and cell adhesion (E-Cad and KiSS1). Following cross-validation, the nonlinear rule produced zero errors in 10 malignant samples and three errors in 15 benign samples, with an overall error rate of 12 percent. The results suggest that downregulation of metastasis suppressor genes reflects malignant PHEO with a high degree of sensitivity.

Eisenhofer G, Huynh TT, Pacak K, Brouwers FM, Walther MM, Linehan WM, Munson PJ, Mannelli M, Goldstein DS, Elkahloun AG. Distinct gene expression profiles in norepinephrine and epinephrine producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel-Lindau syndrome. Endocr Relat Cancer 2004;11:897-911.

Ohta S, Lai EW, Pang ALY, Brouwers FM, De Krijger R, Ksinantova L, Blazicek P, Breza J, Kvetnansky R, Wesley RA, Pacak K. Down-regulation of metastasis suppressor genes in malignant pheochromocytoma. Int J Cancer 2004, Nov 2;[Epub ahead of print].

Molecular mechanisms that link pheochromocytoma tumor cell phenotypes and clinical presentation of disease to specific underlying mutations

Brouwers, Pacak; in collaboration with Eisenhofer, Elkahloun, Munson

Our recent results indicated that PHEOs from patients with von Hippel-Lindau (VHL) syndrome and MEN2 display distinct neurochemical and histopathological phenotypic features accompanied by differences in the clinical presentations of the disease. Patients with PHEOs and MEN2 have a high incidence of paroxysmal attacks and exhibit more robust adrenergic and hemodynamic responses to glucagon than do patients with VHL disease and PHEO. PHEOs from patients with VHL disease showed a distinctly noradrenergic phenotype in which the tumor specifically produced norepinephrine while PHEOs from patients with MEN2 showed an adrenergic phenotype in which the tumor also produced epinephrine.

Given that these hereditary tumors are characterized biochemically by differences in synthesis of epinephrine, we also compared gene expression in the two types of PHEO. The analysis focused mainly on the genes and pathways activated in VHL-associated PHEOs in relationship to those activated in MEN2 and sporadic norepinephrine-producing tumors. Many of the genes overexpressed in VHL compared with MEN2 tumors were clearly linked to the hypoxia-driven angiogenic pathways that are activated in VHL-associated tumorigenesis. Such genes include those encoding the glucose transporter (GLUT1), vascular endothelial growth factor (VEGF), placental growth factor, angiopoietin 2, tie-1, and VEGF receptors (VEGFR-2 and neuropilin-1). Other upregulated genes, such as connective tissue growth factor, cysteine-rich 61, matrix metalloproteinase 1, VE-cadherin, tenascin C, stanniocalcin 1, and cyclooxygenases 1 and 2, are known to be involved in VEGF-regulated angiogenesis. Shared differences in the expression of subsets of genes in norepinephrine versus epinephrine producing hereditary and sporadic PHEOs indicated other differences in gene expression that may underlie the biochemical phenotype. Overexpression of the hypoxia-inducible transcription factor HIF-2alpha (EPAS-1) in norepinephrine-predominant sporadic and VHL tumors compared with epinephrine-producing tumors indicates that expression of EPAS-1 depends on the noradrenergic biochemical phenotype. The findings fit with the known expression of HIF-2 alpha in norepinephrine-producing cells of the sympathetic nervous system and might explain both the development and noradrenergic biochemical phenotype of PHEOs in VHL syndrome.

The role of serum protein patterns in the diagnosis of malignant and benign pheochromocytomas

Brouwers, Pacak; in collaboration with Eisenhofer, Liotta, Mannelli, Petricoin, Shulkin, Walther

In recent years, new developments have made proteomic technologies such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) highly applicable to tumor biomarker discovery. Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS and surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) MS, alone or in tandem with other proteomics techniques, have successfully identified biomarkers in diseases such as breast cancer, rheumatoid arthritis, and lung cancer. Recently, investigators have successfully employed pattern recognition software programs for the analysis of the MS spectrum itself to discriminate biological states. Based on distinct serum protein patterns, artificial intelligence–type computer algorithms detected ovarian cancer and prostate cancer with high sensitivity (100 and 95 percent, respectively) and specificity (95 and 95 percent, respectively). Therefore, in the present study, we combined protein chips with a high-resolution mass spectrometer to generate protein profiles from serum of 64 patients with PHEO, including 31 patients with metastatic disease and 64 age- and gender-matched controls. We analyzed the profiles with the use of pattern recognition algorithms to detect protein fingerprints that distinguish malignant from benign PHEOs and malignant PHEOs from healthy controls. Four separate models could each distinguish malignant from benign PHEOs, and two models could separate patients with malignant PHEOs from healthy controls—all with 100 percent sensitivity and specificity in blinded test sets. The results indicate that malignant and benign PHEOs can be distinguished with high specificity and sensitivity based on a number of distinct serum protein patterns.

Imaging modalities in the evaluation of patients with pheochromocytoma

Pacak; in collaboration with Carrasquillo, Chen, Reynolds

PHEO constitutes a form of surgically curable hypertension, and failure to diagnose and localize the tumor can result in sudden, unexpected, and potentially lethal complications. Computed tomography and magnetic resonance imaging have good sensitivity but poor specificity, and commonly available nuclear imaging modalities, such as ¹³¹I-metaiodobenzylguanidine scintigraphy, have high specificity but limited sensitivity.PET scanning uses short-lived positron-emitting radionuclides and allows administration of large tracer doses, resulting in high-count density and thus superior resolution compared with conventional single-photon emitters used in nuclear medicine. 6-[¹⁸F]-fluorodopamine, a sympathoneural imaging agent developed in the NIH intramural research program, is a positron-emitting analog of dopamine. In catecholamine-synthesizing cells including PHEO cells, 6-[¹⁸F]-fluorodopamine is transported actively and avidly by both the plasma membrane norepinephrine transporter and intracellular vesicular monoamine transporter.

Our results showed that in patients with PHEO (including metastatic disease), 6-[¹⁸F]-fluorodopamine PET scanning could detect and localize PHEOs with high sensitivity. In patients in whom the diagnosis of PHEO is considered but excluded because of negative biochemical results, 6-[¹⁸F]-fluorodopamine PET scans are consistently negative. Furthermore, our results showed that 6-[¹⁸F]-fluorodopamine was superior to ¹³¹I-metaiodobenzylguanidine in diagnostic localization of benign and metastatic PHEO. We also conducted the study to compare the sensitivity of 6-[¹⁸F]-fluorodopamine PET scanning with ¹²³I-metaiodobenzylguanidine scintigraphy and Octreoscan in the diagnostic localization of malignant PHEO. The comparison of scintigraphic modalities showed most foci of uptake with [¹⁸F]-fluorodopamine PET scanning for both nonmetastatic and metastatic PHEO. In nonmetastatic PHEOs, ¹²³I-metaiodobenzylguanidine scintigraphy showed more foci than with Octreoscan, but in metastatic PHEOs, it was Octreoscan that showed more foci than ¹²³I-metaiodobenzylguanidine scintigraphy. The calculated sensitivities were 87 percent with 6-[¹⁸F]-fluorodopamine PET, 64 percent with ¹²³I-metaiodobenzylguanidine scintigraphy, and 67 percent with Octreoscan.

Eisenhofer G, Pacak K. Diagnosis of pheochromocytoma. In: Harrison’s Textbook online, McGraw-Hill, 2004.

Illias I, Pacak K. Current approaches and recommended algorithm for the diagnostic localization of pheochromocytoma. J Clin Endocrinol Metab 2004;89:479-491.

Illias I, Yu J, Carrasquillo JA, Chen CC, Eisenhofer G, Whatley M, McElroy B, Pacak K. Superiority of 6-[18-F]-fluorodopamine positron emission tomography versus [131]-metaiodobenzylguanidine scintigraphy in the localization of metastatic pheochromocytoma. J Clin Endocrinol Metab 2003;88:4083-4087.

Pacak K, Eisenhofer G, Goldstein DS. Imaging of endocrine tumors: the role of positron emission tomography. Endocr Rev 2004;25:568-580.

Chromaffin and pheochromocytoma cell cultures

Brouwers, Ohta, Pacak; in collaboration with Alesci, Morris, Tischler

There is no known effective treatment for malignant PHEO. Developing effective treatments requires a means to test new drugs for their ability to kill tumor cells selectively. New therapeutic options, such as gene therapy and immunotherapy, should also be considered. This project is designed to establish human PHEO cell lines to be used as in vitro models for testing radiotherapeutic or chemotherapeutic compounds, genetic manipulations, or vaccines that could be applied to the treatment or prevention of metastatic PHEO and other tumors of neural crest origin. The general goal is to validate the feasibility and effectiveness of any of these therapeutic approaches in new clinical trials at the NIH. Several studies are under way to establish human PHEO cell lines; identify potential radiotherapeutic or chemotherapeutic drugs that might bind to or be taken up by and concentrated in the cells, oncolytic viruses, viral vectors for gene therapy, or proteins expressed specifically in PHEO tumor cells; determine the cytotoxic efficacy of such drugs or treatments; verify that the drugs or treatments are not toxic to other cell lines; and develop new clinical research protocols to test the efficacy of the identified potential treatments of PHEO, especially malignant or unresectable PHEO.

Animal model of metastatic pheochromocytoma

Brouwers, Ohta, Lai, Pacak; in collaboration with Green, Tischler

The goal of our animal study is to generate an animal model of metastatic PHEO by testing subcutaneous, intraperitoneal, and intravenous injection of both human and mouse PHEO cells and then screening the mice for the development of metastatic lesions in the lymph nodes, lungs, liver, and spleen. These metastatic lesions will be serially passaged and screened to select for subclones of high metastatic potential. We will follow the animals for tumor growth, measure blood catecholamine and metanephrine levels as surrogate markers for tumor burden, and image tumors by using an Atlas scanner. We will remove any tumors that develop and subject them to subcloning and genetic testing.

COLLABORATORS

Salvatore Alesci, MD, Clinical Neuroendocrinology Branch, NIMH, Bethesda, MD

Jan Breza, MD, PhD, DSc, Faculty of Medicine, Komensky University, Bratislava, Slovakia

Jorge A. Carrasquillo, MD, Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Wai-Yee Chan, MD, Laboratory of Clinical Genomics, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Clara C. Chen, MD, Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Ronald de Krijger, MD, PhD, Josephine Nefkens Institute, Rotterdam, Netherlands

Graeme Eisenhofer, PhD, Clinical Neuroscience Branch, NINDS, Bethesda, MD

Abdel G. Elkahloun, PhD, Genome Technology Branch, NHGRI, Bethesda, MD

David S. Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, Bethesda, MD

Jeff Green, MD, PhD, Laboratory of Cell Regulation and Carcinogenesis, NCI, Bethesda, MD

Lucia Ksinantova, MD, PhD, Institute of Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia

Richard Kvetnansky, PhD, DSc, Institute of Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia

Jacques Lenders, MD, PhD, Nijmegen University, Netherlands

W. Marston Linehan, MD, Urologic Oncology Branch, NCI, Bethesda, MD

Lance Liotta, MD PhD, Laboratory of Pathology, NCI, Bethesda, MD

Massimo Mannelli, MD, University of Florence, Italy

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, Laboratory of Clinical Genomics, NICHD, Bethesda, MD

Emanuel Petricoin, MD, FDA-NCI Clinical Proteomics Program, Center for Biologics Evaluation and Research, Bethesda, MD

James Reynolds, MD, PhD, Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Barry L. Shulkin, MD, MBA, St. Jude Children’s Research Hospital, Memphis, TN

Arthur S. Tischler, MD, New England Medical Center, Boston, MA

Alexander Vortmeyer, MD, Surgical Neurology Branch, NINDS, Bethesda, MD

McClellan M. Walther, MD, Urologic Oncology Branch, NCI, Bethesda, MD

Robert A. Wesley, PhD, Biostatistics and Clinical Epidemiology Service, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD

Zhengping Zhuang, MD, PhD, Surgical Neurology Branch, NINDS, Bethesda, MD

For further information, contact karel@mail.nih.gov