Frederieke Brouwers, MD, Postdoctoral Fellow
Shoichiro Ohta, MD, PhD, Postdoctoral Fellow
Amanda F. Nave, Postbaccalaureate Fellow

Biochemical diagnosis of pheochromocytoma

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

The diagnosis of pheochromocytoma typically requires confirmation by several tests, perhaps the most important of which is biochemical evidence of excessive catecholamine production by the tumor. Such evidence usually derives from measuring 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 pheochromocytoma. Sometimes pheochromocytomas may be biochemically "silent," 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 pheochromocytoma in the tested population and inadequate specificity of biochemical tests, positive results in patients without pheochromocytoma are a common and troublesome occurrence. We have developed a biochemical test involving measurements of plasma free normetanephrine and metanephrine, respective metabolites of norepinephrine and epinephrine, which, in contrast to catecholamines, 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 pheochromocytoma was confirmed and of 644 patients who were determined not to have the tumor. Sensitivities of plasma free 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 plasma free metanephrines, urinary catecholamines, and plasma catecholamines; and lowest for urinary fractionated metanephrines. Currently, we are also attempting to determine the diagnostic utility of measurements of plasma free metanephrines in patients with metastatic pheochromocytoma before and after treatment.

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, Lenders JWM, Pacak K. Biochemical diagnosis of pheochromocytoma. Front Horm Res;in press.

Eisenhofer G, Lenders JW, Pacak K. Choice of biochemical test for diagnosis of pheochromocytoma:

validation of plasma metanephrines. Curr Hypertens Rep 2002;4:250-255.

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.

Molecular genetic basis of tumorigenesis in familial and sporadic benign and malignant pheochromocytomas

Brouwers, Ohta, Koch,* Pacak; in collaboration with Breza, Eisenhofer, Ksinantova,, Kvetnansky, Linehan, Walther, Zhuang

According to Knudson's well-established "two hit" hypothesis of hereditary tumor development, inactivation of both copies of a tumor suppressor gene is required to promote tumor growth. In contrast, the concept of tumorigenesis for oncogenes concerns mutation in the respective oncogene on one allele to be sufficient. Our collaborators from NINDS have recently proposed a new model of oncogene-driven tumor initiation. The model suggests overrepresentation of the mutated c-met oncogene as an important mechanism of hereditary papillary renal cell tumorigenesis. Homology between MET and RET led us to hypothesize that tumor formation in patients with multiple endocrine neoplasia type 2 (MEN 2)-associated tumors might develop by a similar mechanism. In this syndrome, which mainly consists of pheochromocytoma and medullary thyroid carcinoma, patients with a RET germline mutation develop tumors as early as in the first month of life and as late as in the seventh decade. In a series of experiments headed by Drs. Koch and Zhuang, we found that duplication of the mutated RET allele in trisomy 10 may represent a possible mechanism of tumorigenesis in MEN 2-associated pheochromocytomas. However, some of the pheochromocytomas did not reveal overrepresentation of mutated RET. Instead, the tumors had lost the wild-type RET allele, also leading to a dominant effect of mutant RET. This allelic imbalance between the mutant and wild-type RET allele could also be demonstrated in MEN 2-associated medullary thyroid carcinoma and in the TT cell line. In this cell line, we did not find trisomy 10 or loss of the wild-type RET allele as mechanisms of overrepresentation of mutant RET but, instead, a tandem duplication of RET as a third mechanism for imbalance between mutant and wild-type RET. Some of these MEN 2-associated tumors, however, did not reveal mutant and wild-type RET imbalance. We therefore searched for other mechanisms of a "second hit" in these tumors and found somatic VHL gene deletion and mutation. We are also investigating genetic mechanisms that may be responsible for a malignant potential of pheochromocytoma. For example, we are conducting experiments using gene expression profiling and proteomics to find out which genes and proteins may distinguish malignant pheochromocytomas from benign ones and to search for new markers of malignant pheochromocytoma.

Koch CA, Huang SC, Moley JF, Azumi N, Chrousos GP, Gagel RF, Zhuang Z, Pacak K, Vortmeyer

AO. Allelic imbalance of the mutant and wild-type RET allele in MEN 2-associated medullary thyroid carcinoma. Oncogene 2001;20:7809-7811.

Koch CA, Huang SC, Zhuang Z, Stolle C, Azumi N, Chrousos GP, Vortmeyer AO, Pacak K. Somatic

VHL gene deletion and point mutation in MEN 2A-associated pheochromocytoma. Oncogene 2002;21:479-482.

Pacak K, Linehan WM, Walther M, Goldstein DS, Eisenhofer G. Recent advances in genetics, diagnosis,

 localization, and treatment of pheochromocytoma. Ann Intern Med 2001;134:114-129.

Imaging modalities in the evaluation of patients with pheochromocytoma

Pacak; in collaboration with Carrasquillo, Chen, Reynolds

Pheochromocytoma 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. Positron emission tomographic scanning, which uses short-lived positron-emitting radionuclides, allows administration of large tracer doses and results in high count density and thus better resolution than 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 pheochromocytoma 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 pheochromocytoma (including metastatic disease), 6-[18F]-fluorodopamine PET scanning could detect and localize pheochromocytomas with high sensitivity. In patients in whom the diagnosis of pheochromocytoma 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 pheochromocytoma. Currently, we are comparing the sensitivity and specificity of 6-[18F]-fluorodopamine PET scanning with 123I-metaiodobenzylguanidine scintigraphy and Octreoscan in the diagnostic localization of malignant pheochromocytoma.

Gourgiotis L, Sarlis M, Reynolds JC, van Waes C, Merino MJ, Pacak K. Localization of medullary thyroid carcinoma

metastasis in a multiple endocrine neoplasia type 2A patient by 6-[18F]fluorodopamine positron emission tomography. J Clin Endocrinol Metab 2003;88:637-641.

Illias I, Pacak K. Current approaches and recommended algorithm for the diagnostic localization of

pheochromocytoma. J Clin Endocrinol Metab; in press.

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; in press.

Pacak K, Eisenhofer G, Carrasquillo JA, Chen CC, Li ST, Goldstein DS. 6-[18F]fluorodopamine positron

emission tomographic (PET) scanning for diagnostic localization of pheochromocytoma. Hypertension 2001;38:6-8.

Pacak K, Eisenhofer G, Illias I. Diagnostic imaging of pheochromocytoma. Front Horm Res; in press.

Mechanisms that link different pheochromocytoma tumor cell phenotypes and clinical presentation of disease to specific underlying mutations

Pacak, Brouwers; in collaboration with Eisenhofer, Zhuang

Our recent results indicated that pheochromocytomas from patients with von-Hippel Lindau (VHL) syndrome and MEN 2 display distinct neurochemical and histopathological phenotypic features accompanied by differences in the clinical presentations of the disease. Patients with MEN2 and pheochromocytoma have a high incidence of paroxysmal attacks and exhibit more robust adrenergic and hemodynamic responses to glucagon than do patients with VHL disease and pheochromocytoma. Pheochromocytomas from patients with VHL disease showed a distinctly noradrenergic phenotype, i.e., the tumor produces norepinephrine specifically, while those from patients with MEN2 showed an adrenergic phenotype, i.e., the tumor also produces epinephrine. These phenotypic variations result from differences in expression of certain genes, such as that encoding phenolethanolamine-N-methyltransferase (PNMT), the enzyme that converts norepinephrine to epinephrine. We plan to relate these results and neurochemical analysis of tumor catecholamine content to the underlying germline and somatic mutations. Morphologic studies in conjunction with genetic analysis will attempt to separate the tumors into morphologically and genetically distinct subsets. Follow-up studies combining laser-capture microdissection with differential display or microarray analysis will trace back phenotypic differences in tumors to underlying differences in gene expression and, ultimately, to the basic somatic or germline mutations responsible for the tumor.

Eisenhofer G, Walther MM, Huynh T-T, Li ST, Bornstein S, Mannelli M, Goldstein DS, Vortmeyer A,

Lenders JWM, Linehan WM, Pacak K. Pheochromocytoma in von-Hippel Lindau syndrome and multiple endocrine neoplasia type 2 display distinct noradrenergic and adrenergic phenotype. J Clin Endocrinol Metab 2001;86:1999-2008.

Chromaffin and pheochromocytoma cell cultures and animal model of metastatic pheochromocytoma

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

There is no known effective treatment for malignant pheochromocytoma. 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 pheochromocytoma 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 pheochromocytoma and other tumors of neural crest origin. We are identifying potential radiotherapeutic or chemotherapeutic drugs that might bind to or be taken up by and concentrated in the cells, oncolytic viruses or viral vectors for gene therapy, or proteins expressed specifically in pheochromocytoma tumor cells; determining the cytotoxic efficacy of the drugs or treatments; verifying that the drugs or treatments are not toxic to other cell lines; and developing new clinical research protocols to test the efficacy of the identified potential treatments of pheochromocytoma, especially malignant or unresectable pheochromocytoma. To generate an animal model of metastatic pheochromocytoma, we plan to give mice subcutaneous, intraperitoneal, and intravenous injections of both human and mouse pheochromocytoma cells and screen for the development of metastatic lesions in the lymph nodes, lungs, liver, and spleen. The 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 then image tumors by using Atlas scanner. Tumors that develop in the mice will be removed for subcloning and used for genetic testing.

Pacak K, Fojo T, Goldstein DS, Eisenhofer G, Walther MM, Linehan WM, Bachemeicher L, Abraham J, Wood BJ.

Radiofrequency ablation (RFA): a novel approach for the treatment of metastatic pheochromocytoma. J NCI 2001;93:648-649.

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, NIHCC, Bethesda MD 
Wai-Yee Chan, MD, Laboratory of Clinical Genomics, NICHD, Bethesda MD 
Clara C. Chen, MD, Nuclear Medicine Department, NIHCC, Bethesda MD
Graeme Eisenhofer, PhD, Clinical Neuroscience Branch, NINDS, Bethesda MD 
David S. Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, 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, The Netherlands 
W. Marston Linehan, MD, Urologic Oncology Branch, NCI, Bethesda MD 
Peter J. Munson, PhD, Mathematical Computing Program, Center for Information Technology, NIH, 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, NIHCC, Bethesda MD
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
 Zhengping Zhuang, MD, PhD, Surgical Neurology Branch, NINDS, Bethesda MD

*Christian A Koch, MD, FACE, former Clinical Fellow, now University Hospital, Faculty of Medicine, Leipzig, Germany

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