Journal Club 2006.9.21

1. Journal Club 2006.9.21 Diabetes and Endocrine Department, Kameda medical center Masahiro Masuzawa

2. REVIEW ARTICLE Should Patients with Apparently Sporadic Pheochromocytomas or Paragangliomas be Screened for Hereditary Syndromes? Camilo Jiménez, Gilbert Cote, Andrew Arnold and Robert F. Gagel Instituto Nacional de Cancerología/Fundación Santafé de Bogotá (C.J.), Colombia, South America, Joint Baylor College of Medicine/The University of Texas M. D. Anderson Cancer Center Training Program in Endocrinology, Houston, Texas 77030; Department of Endocrine Neoplasia and Hormonal Disorders (G.C., R.F.G.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; and Center for Molecular Medicine (A.A.), University of Connecticut School of Medicine, Farmington, Connecticut 06030 Address all correspondence and requests for reprints to: Robert F. Gagel, M.D., Unit 433, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: rgagel{at}mdanderson.org. The Journal of Clinical Endocrinology & Metabolism, 2006, 91(8):2851-2878

3. ABSTRACT Background The group of susceptibility genes for pheochromocytomathat included the proto-oncogene RET (associated with multipleendocrine neoplasia type 2 [MEN-2]) and the tumor-suppressorgene VHL (associated with von Hippel–Lindau disease) nowalso encompasses the newly identified genes for succinate dehydrogenasesubunit D (SDHD) and succinate dehydrogenase subunit B (SDHB),which predispose carriers to pheochromocytomas and glomus tumors.We used molecular tools to classify a large cohort of patientswith pheochromocytoma with respect to the presence or absenceof mutations of one of these four genes and to investigate therelevance of genetic analyses to clinical practice. Methods Peripheral blood from unrelated, consenting registrypatients with pheochromocytoma was tested for mutations of RET,VHL, SDHD, and SDHB. Clinical data at first presentation andfollow-up were evaluated. Results Among 271 patients who presented with nonsyndromic pheochromocytomaand without a family history of the disease, 66 (24 percent)were found to have mutations (mean age, 25 years; 32 men and34 women). Of these 66, 30 had mutations of VHL,13 of RET,11 of SDHD, and 12 of SDHB. Younger age, multifocal tumors,and extraadrenal tumors were significantly associated with thepresence of a mutation. However, among the 66 patients who werepositive for mutations, only 21 had multifocal pheochromocytoma.Twenty-three (35 percent) presented after the age of 30 years,and 17 (8 percent) after the age of 40. Sixty-one (92 percent)of the patients with mutations were identified solely by moleculartesting of VHL, RET, SDHD, and SDHB; these patients had no associatedsigns and symptoms at presentation. Conclusions Almost one fourth of patients with apparently sporadicpheochromocytoma may be carriers of mutations; routine analysisfor mutations of RET, VHL, SDHD, and SDHB is indicated to identifypheochromocytoma-associated syndromes that would otherwise bemissed. N Engl J Med, 2002, 346:1459-1466

4. The Hereditary Pheochromocytoma or Paraganglioma Syndrome

5. NF1 NF1 is an autosomal-dominant disorder, occurring in one of 3000–4000people and characterized by neurofibromas, lightly pigmentedbirthmarks (café au lait spots), iris hamartomas (Lishnodules), and skin-fold freckling. NF1 is caused by inactivatingmutations of neurofibromin, a tumor suppressor gene that encodesa GTPase-activating protein involved in the inhibition of Rasactivity. Pheochromocytomas are rare, with frequency estimatesof 0.1–5.7% but elevated to 20–50% in hypertensivepatients with NF1 . In most reported NF1 catecholamine-producingtumors, single pheochromocytomas are the most common presentation(84%), followed by bilateral pheochromocytomas (10%) and sympatheticparagangliomas (6%) . Most are benign tumors (90%),although malignant pheochromocytomas and sympathetic paragangliomashave also been found . These tumors have a presentationand course similar to those of the sporadic ones. Most of themoccur in adults (mean age, 42 yr), with rare examples of multigenerationalpheochromocytomas . Most pheochromocytomas produce a predominanceof norepinephrine and therefore most commonly present with hypertensionand adrenergic symptomatology. However, 22% of pheochromocytomashave no symptoms related to excessive catecholamine secretion. NF1 can be diagnosed simultaneously with pheochromocytoma;however, the typical skin lesions lead to the diagnosis of NF1during childhood , making NF1 unlikely to present as apparentlysporadic pheochromocytoma.

6. VHL VHL is an autosomal-dominant syndrome with an incidence of onein 36,000 births . VHL is caused by mutations of the VHLgene, a tumor suppressor gene that encodes a protein (pVHL)that regulates hypoxia-inducible genes, the fibronectin matrixassembly, and angiogenesis . This protein inhibitsthe accumulation of hypoxia-induced proteins through ubiquitin-mediateddegradation of hypoxia-inducible factor-1 subunits under conditionsof normoxemia . In carriers of VHL gene germline mutations,the regulation of genes such as the vascular endothelial growthfactor and other genes involved in cellular growth seems tobe lost, predisposing the VHL carriers to both benign and malignanttumors in multiple organs (i.e. renal, testicular, and pancreaticcysts, renal cell cancer, islet cell tumors, central nervoussystem hemangioblastomas, endolymphatic sac tumors, and adrenaltumors) . Pheochromocytomas occur in 10–34%of patients with VHL with a mean age at presentationof 18.3 yr. The prevalence is 6–9% in people with mutationscaused by partial or complete deletions of the VHL gene (VHLtype 1), whereas those with missense mutations have a prevalenceof 40–59% (VHL type 2), exhibiting genotype-phenotypecorrelation . These catecholamine-producing tumors couldpresent as the first or only manifestation of VHL (VHLtype 2C) . Consequently, VHL carriers can present as apparentlysporadic pheochromocytoma. VHL catecholamine-producing tumorsare most commonly pheochromocytomas (90%), although sympatheticparagangliomas have been described (abdomen 8%, chest 2%, andneck 0.1%) . Approximately half of pheochromocytomas arebilateral , and most produce norepinephrine . Thereare uncommon examples of malignant catecholamine-producing tumorsin VHL (<10%), frequently sympathetic paragangliomas .

7. Figure 1. The ubiquitination of the HIF-1   by the pVHL. (Modified with permission from S. Richard: Atlas of Genetics and Cytogenetics in Oncology and Haematology, VHL, January 2002; http://www.infobiogen.fr/services/chromcancer/Genes/VHLID132.html.) HIF-1   heterodimerizes with HIF-1ß to form HIF that functions as a transcription factor. HIF activates the expression of genes involved in angiogenesis, erythropoiesis, energy metabolism, apoptosis, and/or proliferation in response to low-oxygen tension (hypoxia) conditions. pVHL inhibits HIF activity under normal oxygen conditions (normoxia) by targeting the HIF-1   subunits for ubiquitination and proteasomal degradation (left). pVHL binds to HIF only when a conserved proline (Pro564) in HIF is hydroxylated, a modification that is oxygen-dependent (27 ). Under hypoxic conditions (right), the nonhydroxylated HIF-1   subunits are not recognized by pVHL; HIF consequently accumulates and thereby restores normoxia. Among the possible target genes activated by HIF are the genes encoding the following: VEGF, erythropoietin (Epo), glucose transporter-1 (Glut-1), platelet-derived growth factor-ß (PDGF), plasminogen activator inhibitor 1 (PAI-1), and TGF-  . EloB, Elongin B; EloC, Elongin C; Cul2, Cullin2; Rbx1, RBx1-protein; E2, ubiquitin conjugating enzyme.

8. MEN1 MEN1 is an autosomal-dominant syndrome characterized by primaryhyperparathyroidism, pancreatic islet cell neoplasms, and pituitaryadenomas caused by inactivating mutations of the MEN1 locuscoding for the suppressor protein menin. MEN1 may be associatedwith pheochromocytomas. Fewer than 10 cases of pheochromocytomahave been identified in MEN1, all unilateral, rarely malignant,and most characterized by hypertension and predominant norepinephrineproduction . None presented as apparentlysporadic pheochromocytomas. Given the extremely rare associationbetween MEN1 and pheochromocytomas, and the much higher prevalenceof parathyroid, pancreatic, and pituitary diseases in this syndrome,it is not surprising that MEN1 has not yet been reported topresent clinically as apparently sporadic pheochromocytoma.

9. MEN2 MEN2 is an autosomal-dominant syndrome caused by activatingmutations in the RET protooncogene, which encodes a transmembranereceptor tyrosine kinase involved in the regulation of cellproliferation and apoptosis. MEN2A is characterized by medullarythyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism.MEN2B is characterized by MTC, mucosal ganglioneuromas, andpheochromocytoma. Pheochromocytoma occurs in approximately halfof gene carriers and is almost always located within the adrenalglands. There have been rare reports of sympathetic paragangliomas, although most of these have been found in the adrenalregion and may represent a tumor that has developed in an adrenalrest , recurrence of a previously excised adrenal medullarytumor , or seeding from a malignant pheochromocytoma .Bilateral pheochromocytomas occur in approximately half of patientswith MEN2 who have pheochromocytomas; their development is frequentlyasynchronous, with separation by as much as 15 yr . Pheochromocytomastend to develop after MTC is identified; however, there arewell-documented examples of MEN2-related pheochromocytomas presentingbefore MTC is found as the initial manifestation of this syndrome. Even so, most such cases do not present clinically asapparently sporadic pheochromocytoma, given that the MEN2 familyhistory or nonsolitary tumor focus is known or suspected. Thus,whereas MEN2 has been reported to present as an apparently sporadicpheochromocytoma, such cases are rare. Pheochromocytomas occur most commonly with codon 634 (MEN2A)or 918 (MEN2B)RET protooncogene mutations and with lesserfrequency in kindreds with mutations of codons 609, 611, 618,620, 768, 790, 791, V804L, V804M, 883, and 891. Pheochromocytomas have not been found in kindreds withmutations of codons 532–534, 630, and 912.Malignant pheochromocytomas are uncommon and are generally foundin patients with large tumors. The patternof catecholamine production in MEN2 pheochromocytoma differsfrom that seen in other hereditary forms of pheochromocytoma.Epinephrine is produced in disproportionately large amounts,resulting in an early clinical phenotype characterized by attacksof palpitations, nervousness, anxiety, and headaches, ratherthan the more common pattern of hypertension seen with sporadicor other hereditary tumors.

10. Fig 2. The RET receptor tyrosine kinase is positioned in the cell membrane. It is activated when its ligand binds a co-receptor and the complex in turn interacts with RET.

11. Fig 3. Schematic representation of the RET gene showing the codons involved in germline mutation in MEN 2.

12. PGL1 PGL1 is an autosomal-dominant syndrome with maternal imprinting,characterized by familial and isolated head and neck parasympatheticparagangliomas and less frequently by sympathetic paragangliomasand pheochromocytomas. PGL1 is caused by inactivatingmutations in the mitochondrial complex II SDHD gene,a tumor suppressor gene involved in the electron transport chainand the tricarboxylic acid cycle. SDHD mutations resultin destabilization and loss of structural integrity of the complexII. Consequently, oxygen free radical production increases,stabilizing the hypoxia-inducible factor-1 with subsequent activationof TGF-ß, platelet-derived growth factor receptor-ß,and a ligand for the epidermal growth factor receptor, predisposingto tumor formation. SDHD mutations represent 97% of totalgermline mutations observed in pheochromocytoma/paragangliomakindreds found with succinate dehydrogenase mutations. However, this percentage must be certainly an overstatement;more recent literature indicates that mutations of other subunitsof the succinate dehydrogenase complex, mainly SDHB, accountfor at least one half of the mutations. These mutations exhibita genotype-phenotype correlation. Approximately 75% of pheochromocytomasand sympathetic paragangliomas occur when mutations are localizedin the 5' portion of SDHD. Most of these tumors exhibita benign behavior; however, they can also be malignant.Pheochromocytomas can be unilateral or bilateral. The meanage at diagnosis is 43 yr, with rare cases reported inpeople younger than 20 yr. SDHD mutations can present asapparently sporadic pheochromocytoma because of the maternalimprinting and the lack of more clearly defined manifestations. PGL2 PGL2 is an autosomal-dominant syndrome defined by familial headand neck parasympathetic paragangliomas. Hereditary transmissionoccurs exclusively in children of fathers carrying the gene,pointing to the importance of maternal imprinting. Thecausative gene has been mapped to chromosome 11q13.1 but hasnot yet been identified. It is unlikely that people withthis syndrome will present with an apparently sporadic pheochromocytomabecause of its rarity and parasympathetic lineage. So far, nocases of PGL2 presenting as pheochromocytoma have been described.

13. PGL3 PGL3 is an autosomal-dominant syndrome without maternal imprinting,characterized by benign and seldom multifocal head and neckparasympathetic paragangliomas. PGL3 was initially reportedin only one family. The investigators who evaluated this singlekindred identified a missense mutation of SDHC, another componentof the mitochondrial complex II. None of these family membershad a catecholamine-producing tumor. Recently, an internationalregistry of 121 individuals with head and neck paragangliomasand 371 individuals with pheochromocytomas described a prevalenceof germline SDHC mutations in 4% of patients with head and neckparasympathetic paragangliomas; no SDHC mutations were identifiedin sympathetic paragangliomas or pheochromocytomas. Therefore,at present, it is unnecessary to consider this genetic disorderin patients with apparently sporadic pheochromocytomas. PGL4 PGL4 is an autosomal-dominant syndrome, characterized by parasympatheticparagangliomas and frequently by sympathetic paragangliomasand/or pheochromocytomas. Inactivating mutations in the tumorsuppressor SDHB gene are responsible for PGL4 syndrome. These mutations cause mitochondrial complex II destabilizationand may activate the hypoxic/angiogenic pathway predisposingto tumor formation, with a very strong association with a malignantintra- or extraadrenal phenotype. Apparently sporadicpheochromocytoma has been found in carriers of SDHB mutations, with a mean age of presentation at 34 yr. AlthoughSDHB mutations have been recently described in association withrenal cell carcinomas and papillary thyroid carcinomas, the lack of a frequent association with these disorders,a possible low penetrance, and a risk for new-onset SDHB mutationsmay explain the subset presenting as apparently sporadic pheochromocytoma.

16. Figure ４. The proposed model of WT and mutant SDHC containing complex II with the proposed site of superoxide production. Proposed normal complex II existing in B1 cells (A) and the proposed SDHC mutant complex II present in B9 cells (B). Arrows, flow of electrons as they are passed from succinate through the flavin to the FeS groups and then to the CoQ-binding site or the heme b site.

17. Syndrome, OMIM classification Causative gene Gene locus Maternal imprinting Protein product Protein function Mechanism Catecholamine production by tumors Phenotype NF1 NF1 17q11.2 – Neurofibromin GTP hydrolysis Tumor suppressor + Pheochromocytoma/sympathetic paraganglioma VHL VHL 3p25–26 – VHL Suppressor of transcription elongation Tumor suppressor + Pheochromocytoma/sympathetic paraganglioma MEN1 MENIN 11q13 – Menin Transcription regulation Tumor suppressor + Pheochromocytoma MEN2 RET 10q11.2 – RET Tyrosine kinase receptor Proto-oncogene + Pheochromocytoma PGL1 SDHD 11q23 ± Succinate dehydrogenase subunit D Regulation of mitochondrial ATP production Tumor suppressor + Pheochromocytoma/sympathetic and parasympathetic1 paragangliomas PGL2 Unknown 11q13.1 ± Unknown Unknown Unknown – Parasympathetic1 paragangliomas PGL3 SDHC 1q21 – Succinate dehydrogenase subunit C Regulation of mitochondrial ATP production Tumor Suppressor – Parasympathetic1 paragangliomas PGL4 SDHB 1p36.1–35 – Succinate dehydrogenase subunit B Regulation of mitochondrial ATP production Tumor suppressor + Pheochromocytomas/sympathetic and parasympathetic1 paragangliomas TABLE 1. Hereditary tumor syndromes that include paraganglioma or pheochromocytoma

18. TABLE 2.Reported frequencies of germline mutations in specific endocrine syndromes associated with apparently sporadic pheochromocytomas Investigator (first author/year) Location MEN2, RET VHL, VHL PGL1, SDHD PGL4, SDHB Eng/1995 United States/England/Canada 1/46 0/46 Lindor/1995 United States 0/29 Beldjord/1995 France 0/28 Brauch/1997 Germany 0/62 2/62 Bar/1997 Israel 0/26 0/26 Rodien/1997 France 1/120 Van der Harst/1998 The Netherlands 5/67 Gimm/2000 Germany 1/16 Astuti/2001 England 0/24 1/24 Neumann/2002 Poland and Germany 8/250 18/250 7/250 12/250 Gimenez/2003 France 0/84 2/84 6/84 8/84 Benn/2003 Australia 0/2 2/2 Totals 10/645 = 1.55% 27/535 = 5.04% 14/376 = 3.72% 23/360 = 6.38%

19. TABLE 3.Clinical features suggestive of hereditary pheochromocytoma Clinical features Pheochromocytoma     Sudden death, particularly at a young age     Hypertension or stroke, particularly at a young age or during pregnancy     Hypertensive response to anesthesia VHL  Kidney or pancreatic cysts or cancer     Testicular mass or cyst in children  Early onset of deafness     Early onset of blindness     Central nervous system tumors MEN2     Thyroid cancer, goiter     High blood calcium or kidney stones PGL1 and PGL4     Head and neck tumors with signs and symptoms mainly related to their location (dysphonia, dysphagia, etc.) more than excessive production of catecholamines   Abdominal tumors

20. TABLE 4. When should genetic testing be considered? Rank of order1 When to do genetic testing     Unilateral pheochromocytomas in individuals < 20 yr2 VHL > RET > SDHB = SDHD     Bilateral pheochromocytomas2 VHL > RET > SDHB = SDHD     Sympathetic paragangliomas < 20 yr VHL > SDHB > SDHD     Sympathetic paragangliomas >20 yr SDHB > VHL > SDHD When not to do genetic testing     Age > 50 yr Genetic testing is optional (not routinely recommended)  Patients with unilateral pheochromocytomas, ages 20–50 yr, and no suspicious clinical findings or family history for hereditary disease2 SDHB > VHL > SDHD >>> RET 1 The rank of order may vary if there is a clinical individual or familiar suspicion for a specific hereditary disease. 2 If the biochemical profile indicates an increased production of epinephrine/metanephrines, RET should be tested first.

21. Conclusion: We recommend genetic testing for patients with anapparently sporadic pheochromocytoma under the age of 20 yrwith family history or features suggestive of hereditary pheochromocytomaor for patients with sympathetic paragangliomas. For individualswho do not meet these criteria, genetic testing is optional.