Diagnostic Paediatric Pathology

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1 Annals of Diagnostic Paediatric Pathology 2007, 11(1 2):15 19 Copyright by Polish Paediatric Pathology Society Annals of Pheochromocytoma in children and adolescents based on Polish Pheochromocytoma Registry Mariola Peczkowska 1, Andrzej Januszewicz 1, Barbara Jarz¹b 3, Hartmut Neumann 4, Agata Kubaszek 1, Hanna Janaszek-Sitkowska 1, Mieczys³aw Litwin 5, Jolanta Antoniewicz 5, Ewa Aksamit-Bialoszewska 6, El bieta Ros³onowska 7, Aleksander Prejbisz 1, Magdalena Januszewicz 8, Ilona Micha³owska 2, Jaros³aw Æwik³a 9, Mariusz Furmanek 9, Jerzy Walecki 9 Diagnostic Paediatric Pathology 1 Department of Hypertension 2 Department of Radiology Institute of Cardiology 3 Nuclear Medicine and Endocrine Oncology Department Maria Sklodowska Curie Memorial Institute Gliwice, Poland 4 Medizinische Universitatsklinik Abteilung IV Freiburg, Germany 5 Department of Nephrology, Kidney Transplantation and Arterial Hypertension The Children's Memorial Health Institute 6 Endocrinology and Diabetology Ward General District Hospital Olsztyn, Poland 7 Department of Endocrinology Centre for Postgraduate Medical Education 8 II Department of Clinical Radiology Medical Academy in Warsaw Poland 9 Department of Radiology Central Clinical Hospital of the Ministry of Internal Affairs and Administration Abstract Pheochromocytomas and paragangliomas occur as sporadic tumors or in a familial context. It has been shown that approximately 25% of patients with pheochromocytoma and paraganglioma carry germline mutations in one of 4 susceptibility genes. Peripheral blood from unrelated, registry patients with pheochromocytoma was tested for mutations of RET, VHL, SDHD, and SDHB. Clinical data at first presentation and follow-up were evaluated. Among 136 patients (93 female and 43 male, 68,4% and 31,6% respectively; mean age, 45±14 years) who presented with pheochromocytoma, 34 (24,8%) were found to have mutations. Younger age, multifocal tumors, and extra-adrenal tumors were significantly associated with the presence of a mutation. There were 16 patients with pheochromocytoma under 20 years of age (9 female and 7 male, mean age 15,9±4,6 years, age range 5-20 years). Among them, we identified 15 patients with germ-line mutations (94 percent); 1 had M918T mutation of RET proto-oncogene resulting in MEN 2B syndrome, 6 had mutations of VHL gene, 5 mutations of SDHD gene, and 3 mutations of SDHB gene. Only two patients had positive family history at presentation. One fourth of patients with pheochromocytoma are carriers of mutations. Incidence of hereditary syndromes in patients under 20 years is 94%. Paraganglioma pheochromocytoma syndrome resulting from SDHD or SDHB gene mutations (PGL1 and PGL4 syndrome, respectively) seems to be most common hereditary syndrome in Polish population of patients with pheochromocytoma under 20 years of age. Second major hereditary syndrome in this group was von Hippel-Lindau disease. Key words: Hereditary syndromes, paraganglioma, pheochromocytoma Address for correspondence Mariola Peczkowska, MD, PhD phone: Department of Hypertension fax: Institute of Cardiology mpeczkowska@ikard.pl Warsaw Alpejska 42

2 16 Introduction Familial pheochromocytomas Pheochromocytomas are rare tumors of the adrenal gland that arise from chromaffin cells in the adrenal medulla. Paragangliomas arise from extra adrenal chromaffin cells and can originate in either the sympathetic nervous system or parasympathetic ganglia. Paragangliomas that arise from sympathetic nervous system occur most frequently in the retroperitoneum and are traditionally termed extra adrenal pheochro- Table 1 mocytoma. Paragangliomas have been broadly categorized into two groups: those in the head and neck region with carotid body as the major site and those located elsewhere, with adrenal medulla as the major site. Tumors in the head and neck are anatomically associated with parasympathetic nervous system and are located close to the major arteries and nerves, whereas the adrenal medulla and other paraganglia below neck and head are closer associated with the sympathetic nervous system. Parasympathetic tumors are usually present as an asymptomatic slow growing mass, lacking endocrine activity, whereas sympathetic tumors are hormonally active and secret excess amount of catecholamines. Pheochromocytomas and paragangliomas occur as sporadic tumors or may be associated with hereditary syndromes [2]. Several genetic syndromes are known to be associated with increased risk for pheochromocytoma, including von Hippel Lindau (VHL) syndrome, multiple endocrine neoplasia typ 2 (MEN 2) and neurofibromatosis type 1 (NF1), occurring as a result of germline mutations in VHL, RET, or NF1 genes, respectively. More recently, nuclear genes encoding mitochondrial complex II subunit proteins have been associated with the development of pheochromocytomas and paragangliomas and newly discovered paraganglioma/pheochromocytoma syndrome (PPS) was added to the list of hereditary syndrome in pheochromocytoma and paraganglioma. Neumann et al. showed that close to 24% of patients with pheochromocytomas and paragangliomas carry a germline mutation [8]. This was confirmed by Amar et al. who even found 27% carrying germline mutations [1]. Therefore, the historically established rule of tens, stating that approximately 10% of pheochromocytomas are hereditary, 10% are malignant, 10% are bilateral, 10% are extra-adrenal, 10% are not associated with hypertension and 10% occur in children, is no longer valid concerning genetics. Molecular medicine makes it possible to differentiate sporadic from hereditary disease, which will affect medical management not only for the patient but also for the family. Familial syndromes of pheochromocytoma are presented in Table 1. Syndrome and prevalence Gene Gene locus of pheochromocytomas Multiple endocrine neoplasia Type 2A Ret-protooncogene 10q11.2 (30 60%) Medullary thyroid carcinoma Pheochromocytoma Hyperparathyroidism Multiple endocrine neoplasia Type 2B Ret-protooncogene 10q11.2 (MEN2B) (30-60%) Medullary thyroid carcinoma Pheochromocytoma Marfanoid habitus Skeletal deformation Mucosal neuromas Ganglioneuromatosis of the intestinal tract Von Hippel-Lindau disesase (VHL) VHL-tumor 3p25-26 (15 20%) type 2 suppressor gene A: Retinal and CNS haemangioblastomas Pheochromocytomas Endolymphatic sac tumors Epididymal cystadenomas B: As in VHL type 2A + renal cell cysts and carcinomas As in VHL type 2A + pancreatic neoplasmas and cysts C: Pheochromocytomas only Neurofibromatosis type 1 (NF1) NF-1-gene 1 7q11.2 (3 5%) Multiple fibromas on skin Cafe au lait spots Lisch nodules of the iris Pheochromocytoma-paraganglioma SDHB-gene 1p35-36 syndrome (PGL, 70-80%) SDHD-gene 11q21-23 Head and neck tumors Extra adrenal and adrenal pheochromocytomas

3 17 In the present study, we analyzed the known susceptibility genes for pheochromocytoma VHL, RET, SDHD, and SDHB in a large, unselected series of patients collected in Polish Registry of Pheochromocytoma who presented with this tumor in order to classify them as having either sporadic or hereditary disease. In addition, we analyzed hereditary syndrome prevalence and clinical characteristic in young pheochromocytoma patients under 20 years of age. Material and methods The population based registry for pheochromocytoma was conducted in Poland. Patients were recruited from thirty five centres and clinical, radiological and demographical data was collected. One hundred thirty-six consecutive, unrelated patients with histologically confirmed pheochromocytoma from whom blood-leukocyte DNA was available were enrolled. All patients provided written or oral informed consent. For classification we used term paraganglioma for tumors of location in the head and neck area whereas those of adrenal, extraadrenal abdominal and thoracic location were named pheochromocytomas. All eight exons of SDHB, all four exons of SDHD, all three exons of VHL, and exons 10, 11, and 13 through 16 of RET were examined by analysis of single-strand conformation polymorphisms and direct sequencing, as previously described [5]. Missense mutations were diagnosed if the DNA variants were absent in 100 healthy controls. All molecular examinations were performed in Department of Nephrology and Hypertension, Albert Ludwigs University, Freiburg, Germany. Results A total of 136 patients (93 female and 43 male, 68,4% and 31,6% respectively; mean age, 45±14 years) with pheochromocytoma were enrolled in the study. We identified 34 patients with deleterious germ-line mutations (24,8%). Only 3 patients had family history at the presentation. Among the 34 patients with mutations, 32% had mutations of RET, 18% had germ-line mutations of VHL, 12% had clinical signs of NF1 syndrome, and 18 and 21% presented mutations of two newly identified genes, SDHD and SDHB. The age at the onset of symptoms was statistically lower in all carriers of mutations than in patients with sporadic disease. Multiple, extra adrenal, and bilateral adrenal pheochromocytomas as well as recurrences of the disease were statistically more frequent among patients with mutations than among patients without mutations. Incidence of malignant pheochromocytoma did not differ between study groups (Table 2). The most frequent symptom of the disease was hypertension (86%), then palpitations (67%), sweating (56%), headache (40%), blanching of the skin (40%), severe cardiovascular complications (myocardial infarction, stroke, severe arrhythmia, etc.) (7,4%). Asymptomatic clinical course was observed in 8,8% of patients (Table 3). Table 2 Comparison between hereditary pheochromocytoma and sporadic pheochromocytoma groups Hereditary Sporadic p Age (years) 34,3±13,2 48,7±13,4 p<0,0001 Multiple 17 of 34 [50%] 4 of 102 [3,9%] p<0,0001 Extra-adrenal 8 of 43 6 of 102 p<0,0001 [23,6%] [5.9%] Bilateral adrenal 10 of 34 2 of 102 p<0,0001 [29,4%] [2%] Recurrence 10 of 34 0 of 102 p<0,0001 [29,4%] Malignant 4 of 34 4 of 102 NS [11,8%] [3,9%] Table 3 Major symptoms in pheochromocytoma Symptoms Frequency Hypertension 86% Palpitations 67% Sweating 56% Headache 40% Blanching of the skin 40% Severe cardio-vascular complications 7,4% Asymptomatic 8,8% Then we analyzed the subgroup of 16 patients with pheochromocytoma under 20 years of age (9 female and 7 male, mean age 15,9±4,6 years, age range 5 20 years). Among them, we identified 15 patients with germ-line mutations (94 percent); 1 had M918T mutation of RET proto-oncogene resulting in MEN 2B syndrome, 6 had mutations of VHL gene, 5 mutations of SDHD gene, and 3 mutations of SDHB gene. Only two patients had positive family history at presentation. Paraganglioma pheochromocytoma syndrome resulting from SDHD or SDHB gene mutations (PGL1 and PGL4 syndrome, respectively) seems to be most common hereditary syndrome in Polish population. Second major hereditary syndrome in this group was von Hippel-Lindau disease. Type of mutations is presented in Table 5. In group with hereditary syndromes, multiple pheochromocytomas were observed in 12 patients (80%), extra adrenal in 7 patients (47%) and bilateral adrenal tumors occurred in 6 patients (40%). Thoracic pheochromocytomas were found in 2 patients with SDHD gene mutations. Multiple or extra adrenal or bilateral adrenal pheochromcytomas were found in 13 patients (87%). Recurrence of the disease occurred in 5 patients (33%). We did not observe malignant pheochromocytoma in this group. Several additional tumor

4 18 types were diagnosed in participating patients. A medullary thyroid carcinoma was present in one patient with MEN 2B syndrome. Renal cell carcinoma (RCC) occurred in two patients in SDHB mutation carrier and in patient with von Hippel Lindau disease. Pancreatic islet tumors and haemangioblastoma of IV brain ventricle were found in patient with VHL. In the cohort presented here, there were also two reports of multiple head and neck paraganglioma in SDHD gene mutation carriers [Table 4]. Table 4 Clinical characteristic of patients with hereditary syndromes of pheochromocytoma Characteristics of pheochromocytoma patients with hereditary syndromes under 20 years of age Multiple tumors 12 patients (80%) Extra adrenal tumors 7 patients (47%) Bilateral adrenal tumors 6 patients (40%) Thoracic localization of tumors 2 patients (13%) only in SDHD gene mutations Recurrence of the disease 5 patients (33%) Malignant 0 Associated tumors:! Renal cell carcinoma 2 patients: VHL and SDHB! Pancreatic islet tumors 1 patient VHL! haemangioblastoma of IV brain ventricle! Multiple head and neck paraganglioma 2 patients SDHD! Medullary thyroid carcinoma 1 patient MEN 2B Multiple or extraadrenal or bilateral adrenal pheochromocytoma 13 patients (87%) Discussion Our clinical and molecular evaluation of 136 unrelated patients who presented with pheochromocytoma revealed that 34 patients (24,8%) had a hereditary predisposition to von Hippel Lindau disease, MEN-2, neurofibromatosis type 1, or the syndromes associated with pheochromocytoma and paraganglioma. Among the 34 patients with mutations, 32% had mutations of RET, 18% had germ-line mutations of VHL, 12 percent had clinical signs of NF1 syndrome, and 18 and 21% had mutations of two newly identified genes, SDHD and SDHB. Patients with hereditary syndromes were younger that those with sporadic disease and multiple and extra- adrenal tumors were more frequent in this group. Our results are consistent with other population-based studies [2, 4, 7, 8, 9]. In our registry almost one half of patients with hereditary pheochromocytoma was under 20 years of age. We can state that 87% of all multifocal (including bilateral tumors) and extra adrenal tumors in patients with onset of the disease at the age of 20 years or younger were found to be hereditary. All these findings po- inted out, that extra adrenal and/or multiple pheochromocytomas as well as young age at presentation may be striking features of hereditary disease [9]. Interestingly, 91% of probands found to have hereditary disease with the use of molecular testing had no associated signs and symptoms at presentation. Only three (9%) of them had positive family history at presentation. A partial explanation for the high frequency of hereditary pheochromocytoma without family history of disease might include spontaneous mutation in one of the susceptibility genes, decreased penetrance, and maternal imprinting [9]. In our registry, spontaneous mutations in one of four susceptibility genes occurred in two patients with hereditary von Hippel Lindau disease, one with MEN 2B syndrome, one with SDHD gene mutation and one with SDHB mutation (Table 5). Today, more than 200 different VHL mutations have been identified which appear to be equally distributed throughout the gene. De novo mutations at hypermutable sequences result in most of the recurrent mutations. In large German registry of von Hippel Lindau disease, frequency of spontaneous mutation in VHL is thirteen percent of all cases [9, 10]. In MEN 2B syndrome, which is the most distinct and aggressive of the MEN 2 variants, de novo mutations are very often and reach fifty percent [12]. A little is known about spontaneous mutations in SDHD and SDHB genes because they are newly identified genes. Penetrance is known to be relatively high (approximately 70 percent by the age of 70) among patients with MEN- 2 and von Hippel Lindau disease, overall [9, 10]. Benn et al. reported a statistically significant age-related penetrance difference for SDHB and SDHD mutation carriers. By age 30 yr, 29% of SDHB mutation carriers and 48% of SDHD mutation carriers were diagnosed with paraganglioma; by age 40 yr, 45% of SDHB mutation carriers and 73% of SDHD mutation carriers were diagnosed with paraganglioma [3, 13]. As discussed by authors, SDHD and SDHB mutations have an age-related penetrance, and the lifetime risk of developing paraganglioma (s)/pheochromocytoma (s) approaches 100% by age 70 yr [3, 13]. In patients with SDHD mutations, penetrance depends on whether the individual inherited the mutation from the mother or the father [11, 13]. The disease is not manifested when the mutation is inherited from the mother, but is highly penetrant when inherited from the father. This phenomenon is known as maternal imprinting. Overall, therefore, pheochromocytomas in patients without family histories are due to spontaneous mutations, decreased penetrance, or maternal imprinting, although other causes such as gene gene interactions and gene environment interactions may be possible. Genetic testing can be a powerful aid to the identification of a syndrome in such cases. Since disease is likely to develop in virtually all patients with a family specific mutation, it seems reasonable to subject such patients to lifelong surveillance. This report highlights the value of genetic testing for affected patients and at-risk asymptomatic family members. Genetic testing should be considered in all patients with pa-

5 19 Table 5 Germ-line mutations in the four genes detected in the series of patients with pheochromocytoma under 20 years of age Independent cases Gene Exon cdna nucleotide Amino-acid change Mutation type Family history 1. SDHB C>T R90X Stopcodon hereditary G>A C196Y Missense unknown delTCTC F238frameshift Frameshift spontaneous 4. SDHD 1 33 C>A C11X Stopcodon spontaneous C>A C11X Stopcodon hereditary C>A C11X Stopcodon unknown C>A C11X Stopcodon hereditary C>A C11X Stopcodon hereditary 9. VHL C>T A167Y Missense spontaneous T>A L184H Missense unknown A>G S80G Missense hereditary A>G S80G Missense hereditary A>G S80G Missense hereditary G>C R64P Missense spontaneous 15. RET ATG>ACG M918T Missense spontaneous raganglioma and/or pheochromocytoma [6, 9, 13]. Familial pheochromocytoma/paraganglioma is inherited in an autosomal dominant manner; thus, an affected person has a 50% chance of passing the mutation on to each child. The surveillance strategy for a germline mutation-positive asymptomatic patient should include: annual history and physical examination by a clinician experienced with pheochromocytoma and paraganglioma; annual biochemical testing, e. g. 24-h urine for metanephrines and catecholamines and computed tomography or magnetic resonance imaging every 2 yr taking into consideration the tumour locations typically associated with the mutated gene [6, 13]. Given the observation that children of female SDHD mutation carriers do not manifest the disease when the mutation has been inherited, rigorous clinical surveillance is probably not warranted for these offspring. References 1. Amar L, Bertherat J, Baudin E, et al (2005) Genetic testing in pheochromocytoma or functional paraganglioma. J Clin Oncol 23: Baysal BE, Ferrell RE, Willett-Brozick JE, et al (2000) Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287: al (2006) Clinical presentation and penetrance of pheochromocytoma/paraganglioma syndromes. J Clin Endocrinol Metab 91 (3): Elder EE, Elder G, Larsson C (2005) Pheochromocytoma and functional paraganglioma syndrome: No longer the 10% tumor. J Surg Oncol 89: Gimm O, Armanios M, Dziema H, Neumann HPH, Eng C (2000) Somatic and occult germ-line mutations in SDHD, a mitochondrial complex II gene, in nonfamilial pheochromocytoma. Cancer Res 60: Grupa robocza PTNT (2006) Zalecenia Polskiego Towarzystwa Nadcisnienia Tetniczego dotyczace diagnostyki i leczenia pheochromocytoma. Nadcisnienie Tetnicze (in Polish) 7. Januszewicz W, Wocial B, Sznajderman M, Januszewicz A (2000) Guz chromochlonny. Wydawnictwo Lekarskie PZWL, Warszawa (in Polish) 8. Jarzab B (2004) Dziedziczne uwarunkowania guzow chromochlonnych. In: Nadcisnienie tetnicze. Red. Januszewicz A, Januszewicz W, Szczepanska- -Sadowska E, Sznajderman M. Medycyna Praktyczna, Krakow (in Polish) 9. Neumann HP, Bausch B, McWhinney SR, et al (2002) Germ-line mutations in nonsyndromic pheochromocytoma. N Engl J Med 346: Neumann HP, Berger DP, Sigmund G, et al. (1993) Pheochromocytomas, multiple endocrine neoplasia type 2, and von Hippel-Lindau disease. N Engl J Med 329: Neumann HPH, Pawlu C, Peczkowska M, et al. (2004) Distinct clinical features of paraganglioma syndromes associated with SDHB and SDHD gene mutations. JAMA 292: Peczkowska M, Januszewicz A (2005) Multiple endocrine neoplasia type 2. Familial Cancer 4: Young WF, Abboud AL (2006) Paraganglioma All in the family J Clin Endocrinol Metab 91: