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 pheochromocytoma that included the proto- oncogene RET (associated with multiple endocrine neoplasia type 2 [MEN-2]) and the tumor- suppressor gene VHL (associated with von Hippel – Lindau disease) now also encompasses the newly identified genes for succinate dehydrogenase subunit 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 patients with pheochromocytoma with respect to the presence or absence of mutations of one of these four genes and to investigate the relevance of genetic analyses to clinical practice. Methods Peripheral blood from unrelated, consenting registry patients with pheochromocytoma was tested for mutations of RET, VHL, SDHD, and SDHB. Clinical data at first presentation and follow-up were evaluated. Results Among 271 patients who presented with nonsyndromic pheochromocytoma and without a family history of the disease, 66 (24 percent) were found to have mutations (mean age, 25 years; 32 men and 34 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 the presence of a mutation. However, among the 66 patients who were positive 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 molecular testing of VHL, RET, SDHD, and SDHB; these patients had no associated signs and symptoms at presentation. Conclusions Almost one fourth of patients with apparently sporadic pheochromocytoma may be carriers of mutations; routine analysis for mutations of RET, VHL, SDHD, and SDHB is indicated to identify pheochromocytoma-associated syndromes that would otherwise be missed. 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 3000–4000 people and characterized by neurofibromas, lightly pigmented birthmarks (café au lait spots), iris hamartomas (Lish nodules), and skin- fold freckling. NF1 is caused by inactivating mutations of neurofibromin, a tumor suppressor gene that encodes a GTPase-activating protein involved in the inhibition of Ras activity. Pheochromocytomas are rare, with frequency estimates of 0.1–5.7% but elevated to 20–50% in hypertensive patients with NF1. In most reported NF1 catecholamine-producing tumors, single pheochromocytomas are the most common presentation (84%), followed by bilateral pheochromocytomas (10%) and sympathetic paragangliomas (6%). Most are benign tumors (90%), although malignant pheochromocytomas and sympathetic paragangliomas have also been found. These tumors have a presentation and course similar to those of the sporadic ones. Most of them occur in adults (mean age, 42 yr), with rare examples of multigenerational pheochromocytomas. Most pheochromocytomas produce a predominance of norepinephrine and therefore most commonly present with hypertension and adrenergic symptomatology. However, 22% of pheochromocytomas have no symptoms related to excessive catecholamine secretion. NF1 can be diagnosed simultaneously with pheochromocytoma; however, the typical skin lesions lead to the diagnosis of NF1 during childhood, making NF1 unlikely to present as apparently sporadic pheochromocytoma.

6. VHL VHL is an autosomal-dominant syndrome with an incidence of one in 36,000 births. VHL is caused by mutations of the VHL gene, a tumor suppressor gene that encodes a protein (pVHL) that regulates hypoxia- inducible genes, the fibronectin matrix assembly, and angiogenesis. This protein inhibits the accumulation of hypoxia-induced proteins through ubiquitin-mediated degradation of hypoxia-inducible factor-1 subunits under conditions of normoxemia. In carriers of VHL gene germline mutations, the regulation of genes such as the vascular endothelial growth factor and other genes involved in cellular growth seems to be lost, predisposing the VHL carriers to both benign and malignant tumors in multiple organs (i.e. renal, testicular, and pancreatic cysts, renal cell cancer, islet cell tumors, central nervous system hemangioblastomas, endolymphatic sac tumors, and adrenal tumors). Pheochromocytomas occur in 10–34% of patients with VHL with a mean age at presentation of 18.3 yr. The prevalence is 6–9% in people with mutations caused by partial or complete deletions of the VHL gene (VHL type 1), whereas those with missense mutations have a prevalence of 40–59% (VHL type 2), exhibiting genotype-phenotype correlation. These catecholamine- producing tumors could present as the first or only manifestation of VHL (VHL type 2C). Consequently, VHL carriers can present as apparently sporadic pheochromocytoma. VHL catecholamine-producing tumors are most commonly pheochromocytomas (90%), although sympathetic paragangliomas have been described (abdomen 8%, chest 2%, and neck 0.1%). Approximately half of pheochromocytomas are bilateral, and most produce norepinephrine. There are uncommon examples of malignant catecholamine-producing tumors in 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 (Pro 564 ) 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 primary hyperparathyroidism, pancreatic islet cell neoplasms, and pituitary adenomas caused by inactivating mutations of the MEN1 locus coding for the suppressor protein menin. MEN1 may be associated with pheochromocytomas. Fewer than 10 cases of pheochromocytoma have been identified in MEN1, all unilateral, rarely malignant, and most characterized by hypertension and predominant norepinephrine production. None presented as apparently sporadic pheochromocytomas. Given the extremely rare association between MEN1 and pheochromocytomas, and the much higher prevalence of parathyroid, pancreatic, and pituitary diseases in this syndrome, it is not surprising that MEN1 has not yet been reported to present clinically as apparently sporadic pheochromocytoma.

9. MEN2 MEN2 is an autosomal-dominant syndrome caused by activating mutations in the RET protooncogene, which encodes a transmembrane receptor tyrosine kinase involved in the regulation of cell proliferation and apoptosis. MEN2A is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. MEN2B is characterized by MTC, mucosal ganglioneuromas, and pheochromocytoma. Pheochromocytoma occurs in approximately half of gene carriers and is almost always located within the adrenal glands. There have been rare reports of sympathetic paragangliomas, although most of these have been found in the adrenal region and may represent a tumor that has developed in an adrenal rest, recurrence of a previously excised adrenal medullary tumor, or seeding from a malignant pheochromocytoma. Bilateral pheochromocytomas occur in approximately half of patients with MEN2 who have pheochromocytomas; their development is frequently asynchronous, with separation by as much as 15 yr. Pheochromocytomas tend to develop after MTC is identified; however, there are well-documented examples of MEN2-related pheochromocytomas presenting before MTC is found as the initial manifestation of this syndrome. Even so, most such cases do not present clinically as apparently sporadic pheochromocytoma, given that the MEN2 family history or nonsolitary tumor focus is known or suspected. Thus, whereas MEN2 has been reported to present as an apparently sporadic pheochromocytoma, such cases are rare. Pheochromocytomas occur most commonly with codon 634 (MEN2A) or 918 (MEN2B) RET protooncogene mutations and with lesser frequency 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 with mutations of codons 532–534, 630, and 912. Malignant pheochromocytomas are uncommon and are generally found in patients with large tumors. The pattern of catecholamine production in MEN2 pheochromocytoma differs from that seen in other hereditary forms of pheochromocytoma. Epinephrine is produced in disproportionately large amounts, resulting in an early clinical phenotype characterized by attacks of palpitations, nervousness, anxiety, and headaches, rather than the more common pattern of hypertension seen with sporadic or 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 parasympathetic paragangliomas and less frequently by sympathetic paragangliomas and pheochromocytomas. PGL1 is caused by inactivating mutations in the mitochondrial complex II SDHD gene, a tumor suppressor gene involved in the electron transport chain and the tricarboxylic acid cycle. SDHD mutations result in destabilization and loss of structural integrity of the complex II. Consequently, oxygen free radical production increases, stabilizing the hypoxia-inducible factor-1 with subsequent activation of TGF-ß, platelet-derived growth factor receptor-ß, and a ligand for the epidermal growth factor receptor, predisposing to tumor formation. SDHD mutations represent 97% of total germline mutations observed in pheochromocytoma/paraganglioma kindreds found with succinate dehydrogenase mutations. However, this percentage must be certainly an overstatement; more recent literature indicates that mutations of other subunits of the succinate dehydrogenase complex, mainly SDHB, account for at least one half of the mutations. These mutations exhibit a genotype-phenotype correlation. Approximately 75% of pheochromocytomas and sympathetic paragangliomas occur when mutations are localized in the 5' portion of SDHD. Most of these tumors exhibit a benign behavior; however, they can also be malignant. Pheochromocytomas can be unilateral or bilateral. The mean age at diagnosis is 43 yr, with rare cases reported in people younger than 20 yr. SDHD mutations can present as apparently sporadic pheochromocytoma because of the maternal imprinting and the lack of more clearly defined manifestations. PGL2 PGL2 is an autosomal-dominant syndrome defined by familial head and neck parasympathetic paragangliomas. Hereditary transmission occurs exclusively in children of fathers carrying the gene, pointing to the importance of maternal imprinting. The causative gene has been mapped to chromosome 11q13.1 but has not yet been identified. It is unlikely that people with this syndrome will present with an apparently sporadic pheochromocytoma because of its rarity and parasympathetic lineage. So far, no cases 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 neck parasympathetic paragangliomas. PGL3 was initially reported in only one family. The investigators who evaluated this single kindred identified a missense mutation of SDHC, another component of the mitochondrial complex II. None of these family members had a catecholamine-producing tumor. Recently, an international registry of 121 individuals with head and neck paragangliomas and 371 individuals with pheochromocytomas described a prevalence of germline SDHC mutations in 4% of patients with head and neck parasympathetic paragangliomas; no SDHC mutations were identified in sympathetic paragangliomas or pheochromocytomas. Therefore, at present, it is unnecessary to consider this genetic disorder in patients with apparently sporadic pheochromocytomas. PGL4 PGL4 is an autosomal-dominant syndrome, characterized by parasympathetic paragangliomas and frequently by sympathetic paragangliomas and/or pheochromocytomas. Inactivating mutations in the tumor suppressor SDHB gene are responsible for PGL4 syndrome. These mutations cause mitochondrial complex II destabilization and may activate the hypoxic/angiogenic pathway predisposing to tumor formation, with a very strong association with a malignant intra- or extraadrenal phenotype. Apparently sporadic pheochromocytoma has been found in carriers of SDHB mutations, with a mean age of presentation at 34 yr. Although SDHB mutations have been recently described in association with renal 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 mutations may 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. TABLE 1. Hereditary tumor syndromes that include paraganglioma or pheochromocytoma Syndrome, OMIM classification Caus ative gene Gen e locu s Maternal imprintin g Protein productProtein function Mecha nism Catecholamine production by tumorsPhenotype NF1 17q 11.2 – NeurofibrominGTP hydrolysisTumor suppre ssor + Pheochromocytoma/sympatheti c paraganglioma VHL 3p2 5– 26 – VHLSuppressor of transcription elongation Tumor suppre ssor + Pheochromocytoma/sympatheti c paraganglioma MEN1 MENI N 11q 13 – MeninTranscription regulation Tumor suppre ssor + Pheochromocytoma MEN2 RET10q 11.2 – RETTyrosine kinase receptor Proto- oncog ene + Pheochromocytoma PGL1 SDH D 11q 23 ± Succinate dehydrogenase subunit D Regulation of mitochondrial ATP production Tumor suppre ssor + Pheochromocytoma/sympatheti c and parasympathetic 1 paragangliomas PGL2 Unkn own 11q 13.1 ± Unknown – Parasympathetic 1 paragangliomas PGL3 SDHC1q2 1 – Succinate dehydrogenase subunit C Regulation of mitochondrial ATP production Tumor Suppre ssor – Parasympathetic 1 paragangliomas PGL4 SDHB1p3 6.1– 35 – Succinate dehydrogenase subunit B Regulation of mitochondrial ATP production Tumor suppre ssor + Pheochromocytomas/sympathet ic and parasympathetic 1 paragangliomas

18. Investigator (first author/year)LocationMEN2, RETVHL, VHLPGL1, SDHDPGL4, SDHB Eng/1995United States/England/Canada1/460/46 Lindor/1995United States0/29 Beldjord/1995France0/28 Brauch/1997Germany0/622/62 Bar/1997Israel0/26 Rodien/1997France1/120 Van der Harst/1998The Netherlands 5/67 Gimm/2000Germany 1/16 Astuti/2001England 0/241/24 Neumann/2002Poland and Germany8/25018/2507/25012/250 Gimenez/2003France0/842/846/848/84 Benn/2003Australia 0/22/2 Totals 10/645 = 1.55%27/535 = 5.04%14/376 = 3.72%23/360 = 6.38% TABLE 2. Reported frequencies of germline mutations in specific endocrine syndromes associated with apparently sporadic pheochromocytomas

19. 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 TABLE 3. Clinical features suggestive of hereditary pheochromocytoma

20. Rank of order 1 When to do genetic testing Unilateral pheochromocytomas in individuals < 20 yr 2 VHL > RET > SDHB = SDHD Bilateral pheochromocytomas 2 VHL > RET > SDHB = SDHD Sympathetic paragangliomas < 20 yrVHL > SDHB > SDHD Sympathetic paragangliomas >20 yrSDHB > 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 disease 2 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. TABLE 4. When should genetic testing be considered?

21. Conclusion: We recommend genetic testing for patients with an apparently sporadic pheochromocytoma under the age of 20 yr with family history or features suggestive of hereditary pheochromocytoma or for patients with sympathetic paragangliomas. For individuals who do not meet these criteria, genetic testing is optional.