CLINICS SPECIAL SUPPLEMENT

I S S N - 1807-5932 printed version I S S N - 1980-5322 online version

SUPPLEMENT 1 Multiple Endocrine Neoplasias

Official Scientific Journal of Faculdade de Medicina and Hospital das ClĂ­nicas Universidade de SĂŁo Paulo

CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonc¸a Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Carlos Serrano Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Gustavo Franco Carvalhal Faculdade de Medicina da Pontifı´cia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ivete Bedin Prado Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ludhmila Abrahao Hajjar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Emilia Inoue Sato Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Olavo Pires de Camargo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Fulvio Alexandre Scorza Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Silvia Vanessa Lourenc¸o Faculdade de Odontologia da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Thelma Suely Okay Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Board Fa´bio Biscegli Jatene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Marcelo Zugaib Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Francisco Laurindo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan

Daniel Romero Mun˜oz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil

Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Abhijit Chandra King George’s Medical College Lucknow, India

Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´

Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil

Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Adauto Castelo Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ademar Lopes Fundac¸a˜o Antoˆnio Prudente, Hospital do Caˆncer Sa˜o Paulo, SP, Brazil Alberto Azoubel Antunes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Andrea Schmitt University of Goettingen Goettingen, Germany

Ernest Eugene Moore University of Colorado Denver Denver, CO, USA

Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Euclides Ayres Castilho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Mauro Perretti William Harvey Research Institute London, UK

Irshad Chaudry University of Alabama Birmingham, AL, USA

Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

Ivan Cecconello Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Milton de Arruda Martins Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ke-Seng Zhao Southern Medical University Guangzhou, China

Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA

Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK

Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA

Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Pedro Puech-Lea˜o Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center

Houston, Texas, USA

Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´ Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica

Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Board of Governors Alberto Jose´ da Silva Duarte Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Neto Dalton de Alencar Fischer Chamone Daniel Romero Mun˜oz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euripedes Constantino Miguel Fa´bio Biscegli Jatene Flair Jose´ Carrilho Francisco Vargas Suso Gerson Chadi Gilberto Luis Camanho Irene de Lourdes Noronha Irineu Tadeu Velasco Ivan Cecconello Jorge Elias Kalil

Jose´ Antonio Franchini Ramires Jose´ Antonio Sanches Jose´ Eduardo Krieger Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Lenine Garcia Branda˜o Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Srougi Milton de Arruda Martins Nelson de Luccia Noedir Antonio Groppo Stolf Olavo Pires de Camargo Paulo Andrade Lotufo

Editorial Director Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Roberto Kalil Roberto Zatz Roger Chammas Samir Rasslan Sandra Josefina Ferraz Ellero Grisi Selma Lancman Tarcı´sio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Venaˆncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistant Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ariane Maris Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovı´dio Pires de Campos, 225 - 6 ˚ Andar Email: clinics@hcnet.usp.br Website: www.scielo.br/clinics

CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-2661-6235 Submission: http://www.editorialmanager.com/clinics

Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of Knowledge); Scopus; Ulrich’s Periodical Directory; Qualis/Capes - Classified as an International Circulation Journal in Medicine.

Clinics. Sa˜o Paulo: Scientific Journal of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clı´nicas da FMUSP, 1946–2004.

1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo.

CDD 610

PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central and SciELO. This complies with the policies of funding agencies, such as, the Wellcome Trust, the Research Councils UK - RCUK, the National Institutes of Health(NIH), and the DFG, German Research Foundation, which request or require deposition of the published articles that they fund into such publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (http://www.icmje.org/) on trial registration.

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ISSN-1807-5932

CLINICS CONTENTS Clinics 2012 67(S1):1–178

EDITORIAL

Multiple endocrine neoplasias: an overview of recent progress Mauricio Rocha-e-Silva, Sergio P. A. Toledo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

REVIEWS

Narrowing the gap of personalized medicine in emerging countries: the case of multiple endocrine neoplasias in Brazil Rodrigo A. Toledo, Tomoko Sekiya, Viviane C. Longuini, Flavia L. Coutinho, Delmar M. Lourenc¸o Jr, Sergio P. A. Toledo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Multiple endocrine neoplasia: the Chilean experience Rene´ E. Diaz, Nelson Wohllk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

MENX and MEN4 Natalia S. Pellegata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Head and neck paragangliomas: clinical and molecular genetic classification Christian Offergeld, Christoph Brase, Svetlana Yaremchuk, Irina Mader, Hans Christian Rischke, Sven Gla¨sker, Kurt W Schmid, Thorsten Wiech, Simon F Preuss, Carlos Sua´rez, Tomasz Kopec´, Attila Patocs, Nelson Wohllk, Mahdi Malekpour, Carsten C Boedeker, Hartmut PH Neumann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Familial gigantism Wouter W. de Herder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

RET codon 609 mutations: a contribution for better clinical managing Caterina Mian, Paola Sartorato, Susi Barollo, Mariangela Zane, Giuseppe Opocher . . . . . . . . . . . . . . . . . . . . . . . . . 33

Clinical and genetic aspects of familial isolated pituitary adenomas Vladimir Vasilev, Adrian Daly, Luciana Naves, Sabina Zacharieva, Albert Beckers . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Pituitary tumors in patients with MEN1 syndrome Luis V. Syro, Bernd W. Scheithauer (in memoriam), Kalman Kovacs, Rodrigo A. Toledo, Francisco J. London˜o, Leon D. Ortiz, Fabio Rotondo, Eva Horvath, Humberto Uribe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Variable clinical expression in patients with a germline MEN1 disease gene mutation: clues to a genotype–phenotype correlation Cornelis J. Lips, Koen M. Dreijerink, Jo W. Ho¨ppener . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

RET haplotype, not linked to the C620R activating mutation, associated with Hirschsprung disease in a novel MEN2 family Elisangela P. S. Quedas, Viviane C. Longuini, Tomoko Sekiya, Flavia L. Coutinho, Sergio P. A. Toledo, Uenis Tannuri, Rodrigo A. Toledo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

The Hirschsprung’s–multiple endocrine neoplasia connection Sam W. Moore, Monique Zaahl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Genotype-phenotype correlation in multiple endocrine neoplasia type 2 Friedhelm Raue, Karin Frank-Raue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Molecular mechanisms of RET receptor-mediated oncogenesis in multiple endocrine neoplasia 2 Simona M. Wagner, ShuJun Zhu, Adrian C. Nicolescu, Lois M. Mulligan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

CLINICAL SCIENCE

Over-representation of the G12S polymorphism of the SDHD gene in patients with MEN2A syndrome ´ va Csajbo´k, Pe´ter Igaz, Nikoletta Lendvai, Miklos To´th, Zsuzsanna Valkusz, Gabriella Beko˝, Nikolette Szu¨cs, E Bala´zs Kriszt, Bala´zs Kova´cs, Ka´roly Ra´cz, Attila Pato´cs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

REVIEWS

Personalized medicine: caught between hope, hype and the real world Marc Dammann, Frank Weber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Biochemical, bone and renal patterns in hyperparathyroidism associated with multiple endocrine neoplasia type 1 Delmar M. Lourenc¸o Jr, Flavia L. Coutinho, Rodrigo A. Toledo, Tatiana Denck Gonc¸alves, Fabio L. M. Montenegro, Sergio P. A. Toledo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Diagnostic work-up of gastroenteropancreatic neuroendocrine tumors ¨ berg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Kjell O

Multiple endocrine neoplasia type 2: achievements and current challenges Andreas Machens, Henning Dralle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Temozolomide in aggressive pituitary adenomas and carcinomas Leon D. Ortiz, Luis V. Syro, Bernd W. Scheithauer (in memoriam), Fabio Rotondo, Humberto Uribe, Camilo E. Fadul, Eva Horvath, Kalman Kovacs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Kinase inhibitors for advanced medullary thyroid carcinoma Martin Schlumberger, Marie-He´le`ne Massicotte, Camila L. Nascimento, Ce´cile Chougnet, Eric Baudin, Sophie Leboulleux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Total parathyroidectomy in a large cohort of cases with hyperparathyroidism associated with multiple endocrine neoplasia type 1: experience from a single academic center Fabio Luiz de Menezes Montenegro, Delmar Muniz Lourenc¸o Junior, Marcos Roberto Tavares, Sergio Samir Arap, Climerio Pereira Nascimento Junior, Ledo Mazzei Massoni Neto, Andre´ D’Alessandro, Rodrigo Almeida Toledo, Fla´via Lima Coutinho, Lenine Garcia Branda˜o, Gilberto de Britto e Silva Filho, Ano´i Castro Cordeiro, Sergio Pereira Almeida Toledo . . . . . . 131

Primary hyperparathyroidism in multiple endocrine neoplasia type 1: when to perform surgery?

Francesca Giusti, Francesco Tonelli, Maria Luisa Brandi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Surgical treatment of pancreatic endocrine tumors in multiple endocrine neoplasia type 1 Marcel Cerqueira Cesar Machado . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Surgical approach to medullary thyroid carcinoma associated with multiple endocrine neoplasia type 2 Marcos R. Tavares, Se´rgio P. A. Toledo, Fa´bio L. M. Montenegro, Raquel A. Moyses, Rodrigo A. Toledo, Tomoko Sekyia, Claudio R. Cernea, Lenine G. Branda˜o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Surgical approach in patients with hyperparathyroidism in multiple endocrine neoplasia type 1: total versus partial parathyroidectomy Francesco Tonelli, Francesco Giudici, Tiziana Cavalli, Maria Luisa Brandi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Retroperitoneoscopic adrenalectomy in pheochromocytoma Marcelo Hisano, Fabio Carvalho Vicentini, Miguel Srougi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Post-surgical follow-up of primary hyperparathyroidism associated with multiple endocrine neoplasia type 1 Flavia L. Coutinho, Delmar M. Lourenco Jr, Rodrigo A. Toledo, Fabio L. M. Montenegro, Sergio P. A. Toledo . . . 169

Surgical management of pancreatico-duodenal tumors in multiple endocrine neoplasia syndrome type 1 ˚ kerstro¨m, Peter Sta˚lberg, Per Hellman. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Go¨ran A

CLINICS 2012;67(S1):1

DOI:10.6061/clinics/2012(Sup01)01

EDITORIAL

Multiple endocrine neoplasias: an overview of recent progress Mauricio Rocha-e-Silva,I Sergio P. A. ToledoII I

Editor, Clinics. II Guest Editor.

This is the second special supplement published by CLINICS, following hard upon the heels of our issue on Neural and Behavioral aspects of the human mind (1-8). It is dedicated to the theme of multiple endocrine neoplasias (MENs) types 1 and 2, and we believe, it is especially valuable because we have collected the views of several well-known specialists in this highly specific field from 12 different countries. Thus, a global overview of the subject could be put together. The idea was to offer the reader an update on important aspects of MENs such as those concerning early diagnosis, family screening, early surgical intervention, follow up, medical treatment, and genetic counseling.

Two articles cover specific national aspects of the manner in which MENs are dealt with in Brazil and Chile, which are of course, of special interest to readers in Latin America, because of the many common features of medicine in this part of the world.

Twelve articles cover aspects of the genetic fundamentals behind a number of these diseases. One is an original piece of research on the genetics of MEN 2A. Diagnosis and general aspects leading thereto are the subject of three articles. Three others discuss clinical approaches and eight cover surgical treatment.

We sincerely hope that we are putting forward a useful collection, which may serve as a guide to all those working in this rapidly evolving field. We believe this volume will

be of interest to scientists, clinicians, and to all other professionals directly or indirectly involved with the problems arising from MENs. Our long term goal is to update this collection when a sufficiently new situation has evolved in the general knowledge in this fascinating field.

REFERENCES 1. Bliss TV, Cooke SF. Long-term potentiation and long-term depression: a clinical perspective. Clinics. 2011;66(S1):3–17, http://dx.doi.org/ 10.1590/S1807-59322011001300002.

2. Ferreira LK, Busatto GF. Neuroimaging in Alzheimer’s disease: current role in clinical practice and potential future applications. Clinics.

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3. Lebedev MA, Tate AJ, Hanson TL, Li Z, O’Doherty JE, Winas JA, et al.

Future developments in brain-machine interface research. Clinics.

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4. Marie SK, Oba-Shinjo SM. Metabolism and Brain Cancer. Clinics.

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7. Scorza FA, Cysneiros RM, de Albuquerque M, Scattolini M, Arida RM. Sudden unexpected death in epilepsy: an important concern. Clinics.

2011;66(S1):65–9, http://dx.doi.org/10.1590/S1807-59322011001300008.

8. Yeganeh-Doost P, Gruber O, Falkai P, Schmitt A. The role of the cerebellum in schizophrenia: from cognition to molecular pathways. Clinics.

2011;66(S1):71–7, http://dx.doi.org/10.1590/S1807-59322011001300009.

Email: toldo@usp.br Tel.: 55 11 3133.9694 Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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CLINICS 2012;67(S1):3-6

DOI:10.6061/clinics/2012(Sup01)02

REVIEW

Narrowing the gap of personalized medicine in emerging countries: the case of multiple endocrine neoplasias in Brazil Rodrigo A. Toledo, Tomoko Sekiya, Viviane C. Longuini, Flavia L. Coutinho, Delmar M. Lourenc¸o Jr., Sergio P. A. Toledo Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Endocrine Genetics Unit (LIM-25), Endocrinology Division, Sa˜o Paulo, SP/Brazil.

The finished version of the human genome sequence was completed in 2003, and this event initiated a revolution in medical practice, which is usually referred to as the age of genomic or personalized medicine. Genomic medicine aims to be predictive, personalized, preventive, and also participative (4Ps). It offers a new approach to several pathological conditions, although its impact so far has been more evident in mendelian diseases. This article briefly reviews the potential advantages of this approach, and also some issues that may arise in the attempt to apply the accumulated knowledge from genomic medicine to clinical practice in emerging countries. The advantages of applying genomic medicine into clinical practice are obvious, enabling prediction, prevention, and early diagnosis and treatment of several genetic disorders. However, there are also some issues, such as those related to: (a) the need for approval of a law equivalent to the Genetic Information Nondiscrimination Act, which was approved in 2008 in the USA; (b) the need for private and public funding for genetics and genomics; (c) the need for development of innovative healthcare systems that may substantially cut costs (e.g. costs of periodic medical followup); (d) the need for new graduate and postgraduate curricula in which genomic medicine is emphasized; and (e) the need to adequately inform the population and possible consumers of genetic testing, with reference to the basic aspects of genomic medicine.

KEYWORDS: Genomics; Multiple Endocrine Neoplasia; RET; MEN1; AIP; Medical Genetics.

Toledo RA, Sekiya T, Longuini VC, Coutinho FL, Lourenc¸o DM Jr, Toledo SPA. Narrowing the gap of personalized medicine in emerging countries: the case of multiple endocrine neoplasias in Brazil. Clinics. 2012;67(S1):3-6.

E-mail: toledorodrigo@usp.br Tel.: 55 11 3061-7252

and may achieve the expected US$1,000 in the near future. It is anticipated that genomic medicine may help to improve the quality of life and prolong human survival. However, the genetic analyses that have been developed and which have been ready to use for some time now, have been underutilized in the past decade (3). Although the fields of genetics and genomics are well established in developed countries, important progress has been made in other countries in recent years, especially in the emerging countries (5). In order to the increase this development and growth, these countries should immediately begin to tackle essential issues, some of which are discussed below (1–4).

INTRODUCTION The finished version of the human genome sequence was completed in 2003, and this event initiated a revolution in medical practice, which is usually referred to as the age of genomic medicine or personalized medicine (1,2). Genomic medicine aims to be predictive, personalized, and preventive, mostly with regard to mendelian inherited disease forms, but also to complex disorders (3). Further, it must be participative, because patients, their at-risk relatives, and the potential consumers of genetic testing should be aware of the pros and cons of this new technology. Thus, genomic medicine is the art and science of the four Ps (4Ps) (4). The effect of genomic medicine on clinical diagnosis and treatment is currently limited; however, it is almost certain that its impact will increase exponentially in the coming years, as costs of genome sequencing are decreasing rapidly

Reimbursement A crucial issue is the reimbursement of genetic testing by the health insurance companies. Although the costs of genetic testing are reducing, it remains expensive for most of the population. In addition, an adult positive-mutation carrier may have several at-risk children to be tested.

However, extensive data evaluating the economics of genetic testing are lacking (http://www.genome.gov/ 19016729). Hopefully, in the near future, health insurance companies that are established in emerging countries may

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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agree to cover the costs of genetic diagnostic testing of their clients, and thus offer this new technology to patients and their at-risk relatives.

Information to the community Finally, the public should be informed about the basic findings and advantages to be gained from the enormous amount of research data related to genomic medicine. This information may bring several benefits. First, it would make it easier for a person to search their family medical history to find out whether there is an inherited condition in the family, facilitate the personal clinical and molecular diagnosis for any affected members, and allow members to take advantage of genetic screening, early diagnosis, and preventive treatment.

This also raises new challenges, however. The US Food and Drug Administration has been investigating how best to oversee and validate direct-to-consumer test kits. There is concern that doubts over the validity of these tests, and inconsistency in results from different companies, may result in a strict regulatory framework that will hamper scientific innovation. Nature has examined and reported these issues (http://dx.doi.org/10.1038/news.2010.382).

Confidentiality An important issue is the confidentially of genetic test results. In 2008, the Genetic Information Nondiscrimination Act (GINA) was approved in the USA. Approval of this law was strongly supported by Senator Edward Kennedy, among others (http://help.senate.gov/old_site/Min_press/2008_ 04_24_a.pdf). GINA is considered to be one of the most important antidiscrimination laws approved so far in the USA. The law has two parts: one that prohibits genetic discrimination in health insurance, and one that prohibits genetic discrimination in employment.

In large areas of the world, genetic testing is not properly regulated. The lack of a GINA-like law in many countries may limit the interest of many healthy individuals and patients in participating in genetic testing. Presently, the approval of such a law constitutes a major challenge to legislators all over the world. The global approval of antidiscrimination laws would constitute one of the bases for a modern and humanized genomic medicine for the 21st century.

However, genetic testing is a fast-growing field and present laws do not cover every situation (http://health. nih.gov/topic/GeneticTestingCounseling). A critical issue is the routine inclusion of genetic testing results in a person’s medical records, with the possibility that these data may be accessed by employers and health insurance companies. As far as we know, this specific and critical aspect requires further regulation in many countries. A valid approach would be to include genetic testing results in personal records only after obtaining written, specific informed consent from the patient or his/her legal guardian.

Genomic medicine in emerging countries: the Brazilian experience Recently, a series of six papers discussing the Brazilian public health system were published in The Lancet (6–10).

These papers identified important advances in the Brazilian healthcare system over the past decade, as well as several future challenges in this field. Brazil is presently the second largest emerging country within the BRIC (Brazil, Russia, India, China) countries, with more than 190 million people.

However, a large proportion of the population does not have private healthcare insurance, and thus depends on the Brazilian public health system, the Unified Health System (SUS). As a result of the economic stability and growth of the last decade, the Brazilian government is presently funding projects relating to human genetics and genomics.

The first whole-sequence determination of a plant pathogen, Xyllela fastidiosa, carried out by the Brazilian ONSA (Organization for Nucleotide Sequencing and Analysis) Network, funded by the Sa˜o Paulo State Research Foundation (FAPESP), was a landmark in genomics science in this country (11). Further investments have been made by FAPESP and the National Research Agency (CNPq) in programs such as the Human Cancer Genome, as well as medical genetics initiatives that are being developed into potential applications in the SUS. In Brazil, the possibility of applying genomic medicine to clinical practice has not yet had a significant impact. Brazil has achieved substantial economic growth in the last decade. Nevertheless, the vast majority of the population has limited access to modern medicine and especially to genomic medicine, although some initiatives have been performed.

An example of a pioneering initiative is the screening of some of the inherited endocrine neoplasia susceptibility genes (RET, MEN1, p27Kip1, AIP, and TMEM127). This program was supported by the SUS, through the FFM (School of Medicine Foundation), and partially by FAPESP. We conducted this project from 2000 to 2011 in the largest non-profit academic hospital in Brazil: the Hospital das Clı´nicas of the University of Sa˜o Paulo School of Medicine.

DNA samples from 871 patients and their at-risk relatives were studied. Informed consent was obtained from all individuals or their parents. DNA from patients diagnosed with multiple endocrine neoplasia type 1 or 2 (MEN1 or

Public and private funding Public healthcare systems from different countries should consider the potential benefits of funding programs, probably based initially on searching for mendelian germline genetic mutations, such as those occurring in cancer genetics. Extensive data evaluating the economics of genetic testing are needed to give further support to these initiatives. Family screening for mendelian diseases could be the first choice (3). However, large portions of the world’s population do not have access to a private healthcare system and cannot afford the expense involved in genetic testing.

It is possible that private groups supplying investment funds may play an important role by supporting small innovative companies who are researching into new and informative genetic tests.

New medical curricula In general, medical schools in many countries have not yet developed innovative curricula that introduce medical doctors to the world of genomic medicine. Therefore, there should be a strong effort, particularly in emerging countries, to educate graduate and postgraduate medical students in the rapidly advancing field of medical genetics and genomic medicine. In this century, clinical practitioners will be required to have substantial knowledge of personalized medicine to better manage patients with inherited conditions.

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MEN2), familial isolated pituitary adenoma (FIPA), and non-syndromic familial pheochromocytoma (F-PHEO) was genetically sequenced. Out of the 871 individuals, 346 carriers of disease-causing germline mutations and 525 noncarriers were identified (12–26). The positive mutation carriers have greatly benefited from the genetic testing for the five tumor susceptibility genes. Early diagnosis, genetic counseling, and recommendation of appropriate and potentially preventive/prophylactic surgeries have been offered to patients. Consequently, high cure rates have been observed in this group of patients, particularly in RETpositive mutation carriers who were genetically detected at an early age (,10 years) (12–15,24). This strategy demonstrated that public familial screening programs, attempting to apply current knowledge from genomic medicine to medical practice, may have a positive impact on the quality of healthcare systems in emerging countries.

The feedback of the referral physicians was positive and demonstrated an evident improvement in their capacity to manage the genetically diagnosed individuals and their at-risk relatives. These results confirm that Brazil needs new medical curricula emphasizing recent advances in the study of the human genomics, proteomics, and many other ‘‘-omics’’.

In 2009, the integration of genetic medicine into Brazil’s universal healthcare system was approved by the government as the National Policy to Clinical Genetics Care (http://www. in.gov.br/imprensa/visualiza/index.jsp?jornal = 1&pagina = 50&data = 21/01/2009). Under this law, public healthcare systems and private insurance companies are requested to reimburse a limited amount of genetic testing. However, the implementation of this law requires regulation. Its final approval/regulation will be welcomed, as there will be better medical care for patients and their at-risk relatives.

In conclusion, genomic medicine should be predictive, personalized, preventive, and participatory. It has been implemented mostly in developed countries, and several issues remain in applying genomic medicine to clinical practice in emerging countries, such as Brazil. However, this needs to be achieved quickly in order to patients take advantage of this era of evidence-based molecular diagnosis. The effort is worthwhile; after all Whoever saves a life, it is considered as if he saved an entire world (Mishnah Sanhedrin 455).I If the purpose of applying the accumulating data from genomic medicine to clinical practice in emerging countries is achieved, it will be another step in reaching the main goal of medicine – to save lives.

ACKNOWLEDGMENTS We thank all patients and their families who participated in th program.

We also thank Maria das Grac¸as Cavalcanti for providing excellent social support to the patients during the last 20 years. We thank the technicians, administrative staff, students, and research fellows who participated in the activities of the Endocrine Genetics Unit during the period of this program.

We are also grateful to all the colleagues who referred patients to the Neoplasia Endocrine Syndromes Genetic Screening Program in recent years. We are very grateful for the constant support given to this project by Dr. Berenice B. Mendonc¸a, Professor of Endocrinology at the Faculdade de Medicina da Universidade de Sa˜o Paulo (FMUSP).

AUTHOR CONTRIBUTIONS Toledo RA, Sekiya T, and Longuini VC designed and performed the experiments, and analyzed the data. Lourenc¸o DM Jr, Coutinho FL, and Toledo SPA contributed to the clinical information and discussion. Toledo RA wrote the paper. Toledo SPA was responsible for critical revision of the manuscript regarding intellectual content.

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2. Guttmacher AE, McGuire AL, Ponder B, Stefa´nsson K. Personalized genomic information: preparing for the future of genetic medicine. Nat Rev Genet. 2010;11(2):161-5, http://dx.doi.org/10.1038/nrg2735.

3. Eng C. Mendelian genetics of rare—and not so rare—cancers. Ann N Y Acad Sci. 2010;1214:70-82, http://dx.doi.org/10.1111/j.1749-6632.2010.05789.x.

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7. Victora CG, Aquino EM, do Carmo Leal M, Monteiro CA, Barros FC, Szwarcwald CL. Maternal and child health in Brazil: progress and challenges. Lancet. 2011;377:2042-53, http://dx.doi.org/10.1016/S01406736(11)60055-X. 8. Barreto ML, Teixeira MG, Bastos FI, Ximenes RA, Barata RB, Rodrigues LC. Successes and failures in the control of infectious diseases in Brazil: social and environmental context, policies, interventions, and research needs. Lancet. 2011;377:1877-89, http://dx.doi.org/10.1016/S01406736(11)60202-X. 9. Schmidt MI, Duncan BB, E Silva GA, Menezes AM, Monteiro CA, Barreto SM, et al. Chronic non-communicable diseases in Brazil: burden and current challenges. Lancet. 2011;377:1949-61, http://dx.doi.org/ 10.1016/S0140-6736(11)60135-9.

10. Victora CG, Barreto ML, do Carmo Leal M, Monteiro CA, Schmidt MI, Paim J, et al. Health conditions and health-policy innovations in Brazil: the way forward. Lancet. 2011;377:2042-53, http://dx.doi.org/10.1016/S01406736(11)60055-X. 11. Simpson AJ, Reinach FC, Arruda P, Abreu FA, Acencio M, Alvarenga R, et al. The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis. Nature. 2000;406:151-9, http://dx.doi.org/ 10.1038/35018003.

12. Lourenc¸o DM Jr, Toledo RA, Coutinho FL Margarido LC, Siqueira SA, dos Santos MA, et al. The impact of clinical and genetic screenings on the management of the multiple endocrine neoplasia type 1. Clinics.

2007;62:465-76, http://dx.doi.org/10.1590/S1807-59322007000400014.

13. Toledo RA, Lourenc¸o DM, Coutinho FL, Quedas E, Mackowiack I, Machado MC, et al. Novel MEN1 germline mutations in Brazilian families with multiple endocrine neoplasia type 1. Clin Endocrinol.

2007;67:377-84, http://dx.doi.org/10.1111/j.1365-2265.2007.02895.x.

14. Toledo RA, Lourenc¸o DM Jr, Liberman B, Cunha-Neto MB, Cavalcanti MG, Moyses CB, et al. Germline mutation in the aryl hydrocarbon receptor interacting protein gene in familial somatotropinoma. J Clin Endocrinol Metab. 2007;92:1934-7, http://dx.doi.org/10.1210/jc.20062394.

15. Toledo RA, Wagner SM, Coutinho FL, Lourenc¸o DM Jr, Azevedo JA, Longuini VC, et al. High penetrance of pheochromocytoma associated with the novel C634Y/Y791F double germline mutation in the RET

ETHICS APPROVAL The genetic studies described here were approved by the institutional ethics committee (Cappesq – HC-FMUSP).

RESEARCH FUNDING Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Coordenac¸a˜o de aperfeic¸oamento de pessoal de nı´vel superior (Capes) and Fundac¸a˜o Faculdade de Medicina (FFM). Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, numbers 2009/11942 and 2009/15386-6), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq, number 401990/2010-9), Coordenac¸a˜o de aperfeic¸oamento de pessoal de nı´vel superior (Capes) and Fundac¸a˜o Faculdade de Medicina (FFM).

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16.

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21. Qin Y, Yao L, King EE, Buddavarapu K, Lenci RE, Chocron ES. Germline mutations in TMEM127 confer susceptibility to pheochromocytoma. Nat Genet. 2010;42:229-33, http://dx.doi.org/10.1038/ng.533.

22. Lourenc¸o DM Jr, Toledo RA, Mackowiak II, Coutinho FL, Cavalcanti MG, Correia-Deur JE, et al. Multiple endocrine neoplasia type 1 in Brazil: MEN1 founding mutation, clinical features, and bone mineral density profile. Eur J Endocrinol. 2008;159:259-74.

23. Santos MA, Quedas EP, Toledo Rde A, Lourenc¸o DM Jr, Toledo SP. Screening of RET gene mutations in multiple endocrine neoplasia type-2 using conformation sensitive gel electrophoresis (CSGE). Arq Bras Endocrinol Metabol. 2007;51:1468-76, http://dx.doi.org/10.1590/S000427302007000900009.

24. Santos MA, Nunes AB, Abelin N, Ezabella MC, Toledo Rde A, Lourenc¸o D M Jr, et al. Genetic screening of multiple endocrine neoplasia type 2: experience of the USP Endocrine Genetics Unit. Arq Bras Endocrinol Metabol. 2006;50:7-16, http://dx.doi.org/10.1590/S0004-27302006000 100003.

25. Costa MH, Domenice S, Toledo RA, Lourenc¸o DM Jr, Latronico AC, Pinto EM, et al. Glucose-dependent insulinotropic peptide receptor overexpression in adrenocortical hyperplasia in MEN1 syndrome without loss of heterozygosity at the 11q13 locus. Clinics. 2011;66:529-33.

26. Toledo SP, Lourenc¸o DM Jr, Santos MA, Tavares MR, Toledo RA, Correia-Deur JE. Hypercalcitoninemia is not pathognomonic of medullary thyroid carcinoma. Clinics. 2009;64:699-706, http://dx.doi.org/ 10.1590/S1807-59322009000700015.

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Toledo RA, Mendonca BB, Fragoso MC, Soares IC, Almeida MQ, Moraes MB. Isolated familial somatotropinoma: 11q13-loh and gene/protein expression analysis suggests a possible involvement of aip also in nonpituitary tumorigenesis. Clinics. 2010;65:407-15, http://dx.doi.org/ 10.1590/S1807-59322010000400010.

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CLINICS 2012;67(S1):7-11

DOI:10.6061/clinics/2012(Sup01)03

REVIEW

Multiple endocrine neoplasia: the Chilean experience Rene´ E. Diaz,I,II Nelson WohllkII I

Fellow in Endocrinology, Universidad de Chile, Santiago de Chile. II Department of Endocrinology, Hospital del Salvador, Universidad de Chile, Santiago de Chile.

Multiple endocrine neoplasia (MEN) types 1 and 2 are genetic diseases that are inherited as autosomal traits. The major clinical manifestations of multiple endocrine neoplasia type 1 include the so-called ‘‘3 P’s’’: parathyroid, pituitary, and pancreatic tumors, including gastroenteroneuroendocrine tumors. Genetic testing can be performed on patients and the potential carriers of the menin gene mutation, but the genotype-phenotype correlation in multiple endocrine neoplasia type 1 is less straightforward than multiple endocrine neoplasia type 2.

Most likely, the main advantage of genetic testing in MEN1 is to exclude from further studies those who are negative for the genetic mutation if they belong to a family with a known history of MEN1. In Chile, we started with rearranged during transfection proto-oncogene genetic testing (MEN2) 15 years ago. We carried out a prophylactic total thyroidectomy to prevent medullary thyroid carcinoma in a three-year-old girl who presented with microscopic medullary thyroid carcinoma. More than 90% of the individuals who tested positive using a genetic test achieved a biochemical cure compared with only 27% of patients who receive a clinical diagnosis.

Mutations are mainly located in exon 11; the most common is C634W, rather than C634R. Hypertensive crisis was the cause of death in three patients, and extensive distant metastases occurred in nine (including two patients with multiple endocrine neoplasia type 2B) of 14 patients. Earlier recognition of medullary thyroid carcinoma and the other features of the disease, especially pheochromocytoma, will improve the survival rate of patients with multiple endocrine neoplasia.

KEYWORDS: RET; Menin; Mutations; MEN1; MEN2.

Diaz RE, Wohllk N. Multiple endocrine neoplasia: the Chilean experience. Clinics. 2012;67(S1):7-11.

E-mail: nwohllk@gmail.com Tel.: 562 378 0517

fifth decade), high mortality, and no clear genotypephenotype correlation compared with MEN2. De novo mutations have been described in up to 10% of cases (5–9).

The MEN1 gene consists of 10 exons that encode a 610amino acid protein, Menin, a nuclear protein that is ubiquitously expressed. Menin is a tumor suppressor protein and also plays a role in maintaining DNA stability and gene regulation. Approximately 1133 germline mutations in the MEN1 gene have been reported (9). The model for tumorigenesis in familial MEN1, according to Knudson’s ‘‘two hits’’ theory, is that a mutated copy of MEN1 is inherited at the germline level from an affected parent (first hit), whereas the wild-type copy, which is inherited from the healthy parent, is lost at the somatic level (second hit), thereby resulting in a tumor. This mechanism is supported by the observation that heterozygosity is lost, or allelic loss occurs, when tumor DNA is compared with constitutive DNA (2).

Genetic studies should be offered to patients who meet the clinical criteria for MEN1 (sporadic or familial), their asymptomatic relatives, and patients who do not meet MEN1 criteria but have suspicious/atypical features that are suggestive of MEN1. Genetic testing has enabled the confirmation of clinical diagnosis, identification of asymptomatic carriers who require screening for tumor detection and early treatment and identification of family members who do not have the MEN1 mutation and, therefore, do not require further clinical examinations (10).

MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 Multiple endocrine neoplasia type 1 (MEN1; MIM 131100) is an autosomal dominant disease that predisposes individuals to develop parathyroid tumors (100% penetrance by age 50), pancreatic islet cells tumors (30–75%), and pituitary adenoma (10–60%) (1). Some patients may also develop adrenal cortical tumors, carcinoid tumors, facial angiofibromas, collagenomas and lipomas. They can also develop a variable combination of more than 20 endocrine and nonendocrine tumors (Table 1) (2,3).

A diagnosis of MEN1 should be considered when two of the three principal MEN1 tumors affecting the parathyroids, pancreatic islets, or anterior pituitary are present.

Familial MEN1 is diagnosed when at least one other firstdegree relative has one of the three main MEN1 tumors (4).

MEN1 is a rare disease (the prevalence has been estimated at 2–3/100,000) caused by a mutation in the MEN1 gene (menin, MIM 613733) located on chromosome 11q13 and is characterized by a high penetrance (94% by the

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported

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CLINICS 2012;67(S1):7-11

Table 1 - MEN1-related tumors (penetrance at age 40).

Organ/tumor Parathyroid hyperplasia/adenoma Gastroenteropancreatic tract

Pituitary

Foregut carcinoids

Adrenal gland

Skin tumors

Central nervous system Others

Features

%

Multiglandular disease Gastrinoma Insulinoma Others (VIPoma, PPoma, SSoma, glucagonoma) Nonfunctioning Prolactinoma Acromegaly GH/PRL Nonfunctioning adenomas Thymic Bronchial Gastric enterochromaffin tumor Nonfunctioning Pheochromocytoma Cortisol, aldosterone Facial angiofibromas Collagenomas Lipomas Meningiomas Ependymomas Leyomiomas

.90 40 10 2 20 20 5 5 17 2 2 10 20–40 ,1 Rare 85 70 30 5–8 1 10

VIP: vasoactive intestinal peptide. PP: pancreatic polypeptide. SS: somatostatin. GH: growth hormone. PRL: prolactin.

onset was 39 years (19–86 years) and two patients were also diagnosed with concomitant parathyroid carcinoma (at ages of 31 and 75 years).

With regard to the other manifestations of MEN, their prevalence does not differ significantly from other published studies, with the exception of carcinoid tumors, which our data indicate are more frequent even though all of these cases occurred in the same family (7). One patient had papillary thyroid microcarcinoma, a finding that could be incidental. Regarding cutaneous manifestations, we observed collagenoma, lipomas, and angiofibromas, but not all patients were meticulously examined; therefore, definitive conclusions cannot be reached.

Parathyroid tumors are the first manifestation of MEN1 in more than 87% of patients; however, other tumors, like pancreatic or pituitary adenoma, have also been reported as the first manifestation (7).

Physical examination for dermal tumors should start in childhood and be carried out periodically. Annual biochemical screening is recommended after five years of age, and tumor imaging should be performed less frequently (Table 2) (4).

We started MEN1 genetic testing in 2001, initially with one family, and then helped other researchers in Chile. We gathered 22 patients from four families and three sporadic cases, who were the youngest and the oldest patients included (two patients). Genetic testing was performed on two families (30 individuals), which identified 13 members with the IVS9 + 1G.A mutation and three members with the IVS6 + 1G.A mutation. Partial data from one family was published in 2004 (11).

Table 3 shows the prevalence of each manifestation and the age at diagnosis. Regarding hyperparathyroidism (HPT), all three of the unaffected individuals were younger than 24 years and the diagnosis was confirmed by genetic testing. They were followed according to the criteria shown in Table 2. Of the 22 other HPT individuals, the mean age at

MULTIPLE ENDOCRINE NEOPLASIA TYPE 2 MEN type 2 (MEN2) is an autosomal dominant syndrome that affects approximately one in 30,000 individuals (4,12–14).

The major components of the syndrome are medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO) and HPT. Germline mutations in the proto-oncogene RET (MIM 164761), located on chromosome 10q11.2, are responsible for the three subtypes: MEN2A (80%; MIM 171400), MEN2B (5%; MIM 162300) and familial MTC (FMTC, 15%; MIM 155240).

MTC commonly presents alongside the three subtypes, and

Table 2 - Clinical surveillance for subjects known to have MEN1 Adapted from (2,4).

Tumor Hyperparathyroidism Insulinoma Anterior pituitary Gastrinoma Tumor Enteropancreatic and carcinoid tumor Anterior pituitary Other enteropancreatic

Biochemical tests

Age to begin (yr)

Frequency

Serum calcium PTH Glycemia, fasting insulin, proinsulin PRL, IGF-1 Fasting and/or stimulated gastrin

5 8 5 5 20

Yearly Yearly Yearly Yearly Yearly

Imaging tests

Age to begin (yr)

Frequency

Chest and Abdominal CT or MRI Head MRI Somatostatin Receptor Scintigraphy (SRS) Octreotide scan

20 5 Yet to be defined

Yearly to every 3–5 yr Yearly to every 3–5 yr Yet to be defined

CT: computed tomography. MRI: magnetic resonance imaging.

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CLINICS 2012;67(S1):7-11

Multiple endocrine neoplasia: the Chilean experience Diaz RE and Wohllk N

Table 3 - Prevalence of manifestations and age of diagnosis of MEN1 Chilean families. % (affected/total)

Median age at diagnosis, years (range)

Hyperparathyroidism Pituitary tumor

88 (22/25) 28 (7/25)

39 (19–86) 40 (18–86)

Gastroenteropancreatic tumor

32 (8/25)*

35 (19–60)

Foregut carcinoids

16 (4/25){

52 (23–67)

Adrenal gland

24 (6/25)

47 (23–86)

Manifestation

N 22 3 1 1 2 5 2 3 3 2 4 2

Acromegaly Prolactinoma GH/PRL Nonfunctioning adenomas Gastrinoma Nonfunctioning enterochromaffin Bronchial Duodenal Nonfunctioning Bilateral Hyperplasia

*

Two patients presented with gastrinoma and pancreatic neuroendocrine tumor concomitantly.

One patient had lung and duodenal carcinoids.

{

respectively (12,18–20). Therefore, identification of the specific RET mutation that is present in at-risk patients allows for effective clinical screening and optimal clinical and surgical management. In 2009, after extensive review of the literature, the American Thyroid Association (ATA) replaced the consensus guidelines for diagnosis that were released in 2001 and developed a categorization system based on new evidence in order to offer age-based recommendations for performing prophylactic thyroidectomy, predict phenotypes and to establish who should be screened for PHEO and HPT. Table 4 shows the adapted ATA guidelines and other guidelines (12,13).

We started RET genetic testing in 1997 and the most important findings have been published (16,21,22). Sequencing of the RET proto-oncogene was carried out in 60 sporadic MTC patients and 15 families with the MEN2 phenotype, totaling almost 200 individuals. In one family, genetic testing is pending but their phenotype is MEN2A. From the apparently sporadic patients, we found three new MEN2 (probably FMTC) individuals (5%), one with codon 618, one with codon V804M, and another with codon S891A. This percentage is within the range of occurrence of apparently sporadic hereditary cases (4–7%) (23,24). Table 5 shows the main clinical features of 19 families. MEN2A accounted for 73% of all cases, while FMTC and MEN2B account for 21% and 6% of cases, respectively. The femalemale ratio was 1.2751. For patients who received genetic testing (61 out of 63), the most common mutation was located in exon 11 (62%), followed by exon 10 (25%), exon 16 (6%), exon 14 (3.5%), and exon 15 (3.5%). In our population, the C634W mutation was the most common (28%), followed by mutations on exon 10 at codon C620R (21%) and the C634R

the risk of developing MTC is nearly 100%. Patients with MEN2A/2B have a 30–50% risk of developing PHEO, especially those who harbor mutations in codon 634, codon A883F, and M918T (12–15). Diverse features differentiate both subtypes: on one hand, only patients with MEN2A are at risk of developing HPT and cutaneous lichen amyloidosis (CLA) (10–30% and 12%, respectively) and only patients with MEN2B have marfanoid habitus, mucosal neuromas on the tongue, lips and subconjunctival areas and diffuse ganglioneuromas of the gastrointestinal tract (14). In FMTC, affected patients suffer exclusively from MTC; to prove that a particular member of a family has FMTC, it is necessary to demonstrate the absence of PHEO and HPT in two or more generations within a family or provide evidence of an identified RET mutation in families with FMTC (American Thyroid Association) (13). Rarely, families with both MEN2 and Hirschsprung’s disease have been found to have MEN2specific codons 609, 611, 618, and 620 while also presenting with the MEN2A or FMTC subtypes (15,16). MTC, when presenting along with MEN2B, develops earlier and has a more aggressive course than MTC in the other two subtypes.

During the first year of life, the classic features of MEN2B may be lacking, so physicians must recognize the more subtle features of this condition, such as the inability to cry and constipation (17). Straightforward genotype-phenotype correlations in MEN2 have also been described, and, as expected, the aggressiveness of MTC is influenced by the specific RET mutation. For instance, MTC has been confirmed using thyroidectomy specimens collected from MEN2A patients as young as 15 months and in a 9-monthold child with MEN2B (M918T); their conditions progressed to N1 (TNM staging AJCC 2002) at 5 and 2.7 years old,

Table 4 - Recommendations for screening procedures and time of prophylactic thyroidectomy. Adapted from (12,13).

ATA Level RET Mutation codon Age of prophylactic thyroidectomy

Age of screening for PHEO Age of screening for PHP

D

C

B

A

918, 883

634

609, 611, 618, 620, 630

768, 790, 804, 891

ASAP or within the first year of life

,5 years

Start at 8 years, then annually Does not apply

Start at 8 years, then annually Start at 8 years, then annually

Consider surgery before age 5.

May delay surgery beyond age of 5 years if stringent criteria are met* Start at 20 years, then annually Start at 20 years, then annually

May delay surgery beyond age 5 years if stringent criteria are met* Start at 20 years, then periodically Start at 20 years, then periodically

ASAP: as soon as possible.

Normal annual basal/stimulated serum calcitonin level, normal annual neck ultrasound, less aggressive MTC family history and family preference.

*

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Multiple endocrine neoplasia: the Chilean experience Diaz RE and Wohllk N

CLINICS 2012;67(S1):7-11

Table 5 - Clinical characteristics and RET mutations in families with MEN2.

Family number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 total

Individuals analyzed/ mutated

MTC/CCH

26/11 7/5 3/1 4/1 6/5 7/4 3/1 6/4 9/5 9/2 2/2 17/6 11/7 6/2 3/2 2/2 3/1 3/1 2/2 129/63

8/3 5/0 1/0 1/0 5/0 4/0 1/0 4/0 5/0 2/0 2/0 5/1 5/2 1/1 2/0 2/0 1/0 1/0 2/0 56/7

ATA risk

Age of proband at diagnosis (yr) of any tumor

Phenotype

Codon/protein substitution

C C C C C C C C C C B B B A A D D D pending

42 28 43 23 38 33 26 35 29 37 40 50 18 40 47 32 14 4 16

MEN2A/CLA MEN2A MEN2A/CLA MEN2A MEN2A MEN2A MEN2A MEN2A MEN2A FMTC FMTC FMTC MEN2A FMTC FMTC MEN2B MEN2B MEN2B MEN2A

C634W C634W C634W C634R C634R C634R C634R C634G C634F C634Y C618R C620R C620R V804M S891A M918T M918T M918T pending

Table 6 - Outcome according to method of diagnosis and serum calcitonin level.

Method of Diagnosis Genetic Testing

Clinical

Total

Cured % Persistent % Dead % Total %

28 90.3 2 6.5 1 3.2 31 100

8 27.6 8 28 13 44.8 29 100

36 60 10 16.7 14 23 60 100

3 2 1 1 1 1 1 2 2 0 0 0 2 0 0 1 0 0 1 17

0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2

patients who were prophylactically operated on were more likely to be biochemical cured and demonstrated less persistent disease states than those who were operated on after clinical diagnosis. Regarding the other main features, PHEO was present in 27% of patients, which presented bilaterally in most patients with PHEO (70%), and the patients who developed PHEO had a median age of 34 years (range: 26–47 years). HPT was very uncommon (3%) and only seen in two patients with the C634R mutation, as has been reported in other studies (13,14,25). CLA was seen in three patients with the C634W mutation, which is in agreement with other reports in the literature (12,13,26). We did not find any cases of Hirschsprung’s disease, a rarely associated condition that has been described in patients with mutations in exon 10 (16,27,28).

We found four patients with the MEN2B phenotype; de novo mutation was present in two of these patients and, retrospectively, both displayed the precocious symptoms of the inability to cry and constipation during the first year of life.

In summary, the various types of MEN are inherited autosomal dominant syndromes. Molecular biology techniques have made it possible to identify the genes responsible for causing MEN1 and 2. The identification of the carriers of mutant MEN1 and 2 genes who are at risk of developing these syndromes requires frequent biochemical screening for the development of endocrine tumors. The identification of those who are negative will save resources and reassure both the patient and the family. Carriers of the genes responsible for MEN2 should be advised to have thyroidectomy in childhood, according with the ATA risk level of the patient. Some cases of sporadic MTC are determined to actually be MEN2 following RET proto-oncogene testing; therefore, the routine application of this test is recommended in all cases of apparent sporadic MTC.

mutation (18%). These frequencies differ from the largest published series, where C634R is the reported as the most common mutation (13,15). We identified 56 patients with MTC, with a median age of 28.5 years (range: 1–73 years), and seven patients with CCH, with a median age of 7 years (range: 4–28 years). Interestingly, a V804M carrier who was operated on at 28 years of age showed only CCH, demonstrating that this mutation may have low penetrance in some cases (12,14).

The median preoperatory serum calcitonin level was 11 pg/ ml (range: 2.9–27 pg/ml) in patients with CCH and 149 pg/ml (range: 13–5549 pg/ml) in patients with MTC. There was a high correlation between the preoperatory serum calcitonin level and tumor size and stage (0.79 and 0.69 respectively, p,0.005). Table 6 shows clinical outcomes and compares biochemical cure, persistent disease, and death with the method of diagnosis (e.g., genetic testing or clinical diagnosis): 14 patients died in total, and of these three died from hypertensive crisis, two from surgical complications (one was diagnosed by genetic testing) and nine from distant metastases (including two out of the four patients with MEN2B). Forty-nine patients are still alive: 36 are biochemically cured, 10 have persistent disease, two have not been operated on yet and one has unknown condition that is affecting his postoperatory calcitonin level. As expected,

Outcome

PHEO (number) HPT (number)

AUTHOR CONTRIBUTIONS Wohllk N and Dı´az R were responsible for the data collection, statistical analysis, supervision of the study, and writing of the manuscript. Wohllk N was also responsible for the genetic testing results and communications with patients and their parents.

Fisher’s exact test = 0.000.

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Multiple endocrine neoplasia: the Chilean experience Diaz RE and Wohllk N

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CLINICS 2012;67(S1):13-18

DOI:10.6061/clinics/2012(Sup01)04

REVIEW

MENX and MEN4 Natalia S. Pellegata Institute of Pathology, Helmholtz Zentrum Mu¨nchen-German Research Center for Environmental Health, Ingolstaedter Landstrasse, Neuherberg/Germany.

Multiple endocrine neoplasias are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Two MEN syndromes have long been known and are well characterized: the MEN type 1 (MEN1) and type 2 (MEN2). These syndromes are caused by germline mutations in the MEN1 and RET genes, respectively, and have a different tumor spectrum. Recently, a variant of the MEN syndromes arose spontaneously in a rat colony and was named MENX. Affected animals consistently develop multiple endocrine tumors, with a spectrum that shares features with both MEN1 and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for MENX. Capitalizing on these findings, heterozygous germline mutations in the human homologue, CDKN1B, were searched for and identified in patients with multiple endocrine tumors. As a consequence of this discovery, a novel human MEN syndrome, named MEN4, was recognized, which is caused by mutations in p27. Altogether, these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. Here we review the characteristics of the MENX and MEN4 syndromes and we briefly address the main function of p27 and how they are affected by MENX/4-associated mutations.

KEYWORDS: Multiple Endocrine Neoplasias; MENX; MEN4; p27; Cdkn1b Gene.

Pellegata NS. MENX and MEN4. Clinics. 2012;67(S1):13-18.

E-mail: natalia.pellegata@helmholtz-muenchen.de Tel.: 49 893187-2633

10¡2 months, whereas their wild-type littermates live approximately 24–30 months (2). Based on the symptomatology of affected rats, high blood pressure seems to be the leading cause of morbidity, although formal proof is still lacking.

Additional phenotypic characteristics of the MENXaffected rats include: macroscopically visible bilateral juvenile cataracts (1), increased body size, and organomegaly, particularly of spleen and thymus, in mutants versus wild-type littermates (2).

The predisposition of MENX-affected rats to develop multiple endocrine tumors is inherited as a recessive trait, indicating that they are homozygous for the underlying genetic mutation (1). Linkage analysis followed by a positional cloning approach allowed us to identify the gene responsible for the MENX syndrome: the Cdkn1b gene encoding the cell cycle inhibitor p27. Affected rats carry a tandem duplication of eight nucleotides (c.520-528dupTTCAGAC, RefSeq: NM_031762 from base 77 to 670) in exon 2 of Cdkn1b, which causes a frameshift (2). At the protein level, the mutated allele encodes a protein predicted to have a novel C-terminal sequence starting at codon 177, referred to as p27fs177. The wild-type p27 (p27wt) protein is 198 amino acids long, whereas the mutated protein, as a result of the frameshift, is predicted to be 221 amino acids long. This Cdkn1b mutation was identified in homozygosity in all MENX-affected rats tested (n.200), whereas it was never observed in unaffected littermates or in control wild-type rats of commercially available inbred strains (2).

Following the identification of the causative genetic mutation, the expression of the Cdkn1b gene in MENX rats was explored in more detail. The analysis of various tissues of 2-month-old affected rats (before they develop malignancies)

The MENX syndrome in the rat The multiple endocrine neoplasia X (MENX) multi-tumor syndrome was discovered by accident when we observed that some rats belonging to a Sprague–Dawley colony started to spontaneously develop multiple endocrine tumors. Specifically, these rats presented with multifocal anterior pituitary adenoma and bilateral adrenal pheochromocytoma, as well as extra-adrenal pheochromocytoma (paraganglioma), thyroid C-cell hyperplasia, parathyroid hyperplasia, and pancreatic islet cells hyperplasia (Figure 1) (1). This multi-tumor syndrome was named MENX, as it shares phenotypic features with both MEN1 and MEN2 human tumor syndromes (1).

Malignancies in MENX-affected rats show a clear progression with time: these animals develop with complete penetrance adrenal medullary hyperplasia at 3–4 months of age, which progresses to pheochromocytoma by 6–8 months of age. They also present with multifocal anterior pituitary neoplasms at about 4 months of age, which progress to larger adenomas by 8–12 months of age (frequency 100%).

MENX mutant rats also develop pancreatic islet cell hyperplasia (frequency 80%) and from these hyperplastic islets, if the animal lives long enough, insulinomas may arise (Figure 1). Due to the development of multiple malignancies, affected rats have an average life span of

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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Cdkn1b mRNA, but affects the amount of the encoded protein through post-transcriptional or post-translational mechanisms.

The discovery of CDKN1B mutations in human patients: the MEN4 syndrome Approximately 30% of patients who have a MEN1–like phenotype do not carry detectable MEN1 gene mutations (3). Although this could be because of the presence of mutations in noncoding or regulatory sequences of the gene that are not commonly analyzed, it also suggests that these patients could potentially bear mutations in additional, not yet identified, susceptibility genes. Following the identification of the genetic mutation causing MENX syndrome in rats, we decided to investigate whether mutations in the human homologue CDKN1B could explain some of the MEN1-like cases without mutations in MEN1. We screened for the presence of CDKN1B germline mutations several patients fulfilling these criteria and we identified a germline heterozygous TGG.TAG (c.227G.A; RefSeq: AY890407) nonsense mutation at codon 76 (p.Trp76stop, W76X) in a female proband with growth hormone (GH)-secreting pituitary adenoma (causing acromegaly; age 30 years) and primary hyperparathyroidism (age 46 years) (2). Analysis of the proband’s relatives confirmed that in her family, the p27W76X mutation segregates with the predisposition to tumors belonging to the MEN1 tumor spectrum. Indeed, one of the proband’s sisters, also a carrier of the germline c.227G.A mutation, developed renal angiomyolipoma (age 55 years), a tumor type that has been previously associated with the MEN1 syndrome.

As a consequence of this discovery, other groups identified germline mutations in patients with features suggestive of MEN1. First, Georgitsi et al. (4) identified a heterozygous 19-bp duplication (c.59_77dup19) in exon 1 of the CDKN1B gene in one out of 36 European patients with a MEN1-like phenotype and no MEN1 gene mutations. The mutation carrier was a woman who had developed a smallcell neuroendocrine cervical carcinoma (aged 45 years), adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma (Cushing’s disease) (aged 46 years), and hyperparathyroidism (aged 47 years). The c.59_77dup19 duplication causes a frameshift, and the variant mRNA is predicted to encode a p27 protein 69 amino acids shorter than p27wt and with a different amino acid sequence after codon 25 (p.Lys25fs, K25fs).

Subsequently, in a study of American patients with a family history of endocrine tumors and showing primary hyperparathyroidism (1 ˚ HPT) as a common feature, three new germline CDKN1B changes were identified: ATG-7 (g.c); c.283C.T (Pro95Ser, P95S); and c.592G.G (stop.Gln, stop.Q) (5). The patient who had the ATG7(g.c) nucleotide change presented with primary hyperparathyroidism (aged 61 years) and a non-functioning bilateral adrenal mass (aged 63 years). The patient with the missense variant at codon 95 presented with 1 ˚ HPT, Zollinger-Ellison syndrome (ZES) and masses in the duodenum and pancreas (aged 50 years). The individual with the stop.Q mutation showed primary hyperparathyroidism (aged 50 years).

By screening 27 Italian patients with a tumor phenotype suggestive of MEN1, we identified a heterozygous germline change (c.206C.T) which, at the protein level, translates to a Pro.Leu amino acid substitution at codon 69 (p.Pro69Leu,

Figure 1 - Histology of rat tissues affected by the MENX syndrome. Adrenal (A, B), pituitary (C, D) and thyroid (E, F) glands, and pancreas (G, H) of wild-type and age-matched MENX-affected (mutant) rats. Multifocal hyperplastic lesions in the anterior pituitary are indicated by arrows (D). A nodule of thyroid C cell hyperplasia is also indicated (F). Pancreatic islet cells are indicated by arrows in panels G and H: as the pictures were taken with the same magnifications the increase in size of the islets in mutant rats can be readily appreciated. Hematoxylin and eosin stain (H&E) original magnification: 20X (A–D); 25X (E, F); 40X (G, H). (Reproduced with permission from: Molatore & Pellegata, 2010 Copyright ß 2010 by Elsevier).

and wild-type age-matched littermates (controls) showed that the level of Cdkn1b mRNA is highly similar in both animal groups. Furthermore, correct splicing of the mutant mRNA was demonstrated in mutant rat tissues (2). When the expression of the encoded p27 protein was assessed by western blotting, a faint band corresponding in size to the predicted p27 mutant protein (p27fs177) could be observed in only some tissues (i.e. thymus and thyroid).

Immunohistochemical staining, performed with an antip27-specific antibody on a broader variety of tissues, showed extremely low (thyroid, pituitary, thymus, parathyroid, and brain) or absent (adrenals, lung, kidney, liver, and testis) p27 immunoreactivity in tissues of affected rats before they eventually developed neoplasias, whereas the same tissues obtained from wild-type rats showed strong nuclear positivity for p27 (2). This suggests that the MENX mutation does not affect the transcription or processing of

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accumulates in the nucleus, where it binds the cyclin/CDK complexes to block cell cycle progression (13). Upon mitogenic stimulation, the cells need to re-enter the cell cycle, so they rapidly eliminate p27 through proteasome-mediated degradation following two molecular pathways. The first involves the ubiquitylation-promoting complex KPC1/KPC2 and occurs in the cytoplasm (14), while the second pathway takes place in the nucleus and is mediated by the ubiquitin ligase SKP2 (15).

Degradation through the KPC1 pathway occurs following phosphorylation of p27 at the Ser10 residue and its export to the cytoplasm (16). SKP2-mediated degradation requires the phosphorylation of p27 at the Thr187 residue by cyclinE,A/ CDK2 complexes, an event that takes place in late G1. The phosphorylation at Thr187 creates a recognition site for the SKP2 ubiquitin ligase which induces p27 poly-ubiquitylation and subsequent degradation by the proteasome (Figure 3) (15).

The complex regulation of the intracellular amount of p27 suggests that its dose is critical for tissue homeostasis. This hypothesis is supported by the observation that mice heterozygous for a null Cdkn1b allele are predisposed to tumor formation, thereby indicating that p27 is a dose-dependent (haplo-insufficient) tumor suppressor in mice (17).

It is important to mention that the amount of p27 is reduced/lost in many cancers, including those of the colon, breast, prostate, and stomach, and this down-regulation is associated with poor prognosis (18). It was demonstrated that in aggressive colorectal carcinoma samples, the loss/ reduction of p27 expression is caused by accelerated proteasome-dependent degradation of the protein (19), probably caused by increased expression of the ubiquitin ligase SKP2 (20). This molecular mechanism to reduce the amount of p27 has been subsequently identified in other human tumors (18). The observation that low p27 protein levels are often associated with no change in CDKN1B mRNA expression in many human tumors has been considered indirect evidence that enhanced proteolysis is responsible for p27 down-regulation in such tumors, even when formal evidence has not been presented.

In addition to the amount of protein, the intracellular localization of p27 can also profoundly affect the protein’s function. Indeed, p27wt is mainly localized in the nucleus and in this compartment it can bind to, and inhibit, cyclin/ CDK complexes, thereby acting as a cell cycle inhibitor.

However, p27 can be mislocalized in the cytoplasm through phosphorylation or protein binding (21), and, once confined in this compartment, p27 may acquire additional functions that are not completely understood. Studies on engineered mouse strains with mutations in p27 that abolish cyclin/ CDK binding have suggested that cytoplasmic p27 may play a pro-oncogenic role (22), possibly by interacting with Rac and RhoA, molecules, which are involved in cell migration (23,24). Mislocalization of p27 in the cytoplasm through phosphorylation of Thr157 by protein kinase b (AKT) has frequently been observed in carcinoma of the breast, and is associated with poorer prognosis (25).

P69L) (6). The mutation-positive patient developed bilateral multiple metastases of bronchial carcinoid and pituitary non-functioning microadenoma (aged 79 years), 1 ˚ HPT caused by a parathyroid adenoma (aged 67 years), and also a papillary thyroid carcinoma (aged 64 years). The patient’s relatives refused to undergo genetic screening, therefore the inheritance of this mutation could not be assessed.

Recently, two novel germline CDKN1B mutations were found by analyzing 90 common sporadic parathyroid adenomas with non-familial presentation (7). Specifically, one patient carried a heterozygous germline single nucleotide change c.25G.A at base 25 in CDKN1B exon 1, which would result in a Gly9Arg (G9R) substitution in the translated p27 protein product. This patient, a 68-year old man, had a typical, single-gland, mildly symptomatic presentation. Another tumor contained a heterozygous single nucleotide substitution c.397C.A, directing a Pro133Thr (P133T) substitution in the translated p27 protein. This alteration was also present in the germline DNA of another patient, a 53-year-old woman presenting with fatigue, slightly elevated serum calcium and parathyroid hormone levels, and a single parathyroid adenoma (7).

The CDKN1B mutations above described and the phenotype of the mutation carriers are summarized in Table 1, while the location of these changes within the p27 protein is schematically outlined in Figure 2. It is important to point out that the CDKN1B changes described here were not found in 192–650 alleles (depending on the study) from healthy control individuals with no history of endocrine tumors, nor were they identified in .2000 CDKN1B alleles sequenced in the world literature.

As a result of the identification of patients bearing germline CDKN1B mutations and showing multiple endocrine tumors, a novel MEN syndrome was recognized and submitted to the Online Mendelian Inheritance in Man (OMIM) database in 2007 under the name of MEN4. Owing to the limited number of patients with MEN4 so far reported, the precise tumor spectrum associated with this syndrome is still not well defined (Table 1).

Function of p27 protein p27 belongs to the kinase inhibitory protein/cyclin-dependent kinase (CDK) inhibitor interacting protein (KIP/CIP) family of cell cycle inhibitors, and its main function is to control the progression from G1 to S phase (8). p27 binds to, and inhibits, cyclinE/CDK2 and cyclinA/CDK2 complexes in response to either mitogenic or anti-mitogenic stimuli. The targets of the CDK2 kinase are members of the retinoblastoma (pRb) family, which upon phosphorylation, release E2F transcription factors, thereby allowing the transcription of genes responsible for the progression into the S phase (8).

Thus, binding of p27 to cyclinE,A/CDK2 prevents pRb phosphorylation and stops the cells in the G1 phase (Figure 3). p27 is also required for the cytoplasmic assembly and nuclear import of cyclinD/CDK complexes, molecules that promote cell cycle progression (8). Because p27 is a critical regulator of cell cycle progression, its activity is tightly regulated at different levels: transcriptional, translational, and post-translational. Transcriptional regulation of the Cdkn1b promoter (9,10) and control of mRNA translation (11) have been described, but the best known mechanism that regulates p27 function is post-translational, and it modulates the abundance of the protein through ubiquitin-mediated proteasomal degradation (12). In growth-arrested cells, p27

Germline mutations in p27 MENX-associated mutation. Functional in vitro studies have been performed to understand the effect of the Cdkn1b mutation causing MENX in rats. These analyses have demonstrated that the p27fs177 mutant protein retains some properties of the p27wt protein: it can localize to the nucleus and interact with cyclin-dependent kinases (Cdk2 and Cdk4) and, to a lesser extent, with cyclins (26). However, differently

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Table 1 - Germline mutations in the CDKN1B gene, phenotype of the mutation carriers and molecular phenotype of the p27 variant proteins. Mutations are numbered referenced to the cDNA sequence AY890407 (GenBank).

Location number Codon

Mutation

Clinical Predicted Phenotype effect of proband

Molecular Phenotype

Protein Interaction

Protein expression in patient’s tumor

N.D.

N.D.

N.D.

N.D.

Agarwal et al., 2009

Nuclear

N.D.

Reduced

Nuclear

N.D.

N.D.

Absent

-

Costa-Guda et al., 2011 Georgitsi et al., 2007

Nuclear cytoplasm

GRB2/Reduced

Absent/reduced*

Nuclear

Molatore et al., 2010

Cytoplasm

N.D.

Absent

-

Pellegata et al., 2006

N.D.

GRB2/Reduced

N.D.

N.D.

Agarwal et al., 2009

Nuclear

N.D.

N.D.

N.D.

Costa-Guda et al., 2011

N.D.

CDK2/wt

N.D.

N.D.

Agarwal et al., 2009

Protein Protein expression localization in vitro in vitro 59 UTR -7

-

Exon 1 25

9

59-77

25

206

69

227

76

283

95

397

133

Exon 2 592

198

1 ˚HPT (1 PT Reduced tumor), bilateral adrenal mass NF, uterine fibroids

ATG-7(g.c)

1 ˚HPT (1 PT Reduced tumor) N.D. nt59_77dup19 fs, K25fs 1 ˚HPT, ACTHpituitary adenoma, carcinoid tumor of the uterine cervix CCC.CTC Reduced ms, P69L 1 ˚HPT, NF pituitary adenoma, bronchial carcinoids, PTC TGG.TAG ns, W76X 1 ˚HPT, GHWt pituitary adenoma CCC.TCC ms, P95S 1 ˚HPT (2 PT Wt tumors), ZES, mass in duodenum and tail of pancreas CCC.ACA ms, P133T 1 ˚HPT Wt (1 PT tumor) GGG.AGG

TAG.CAG

ms, G9R

if, stop.Q 1 ˚HPT (3 PT tumors)

Reference Protein localization in patient’s tumor

Reduced

*

From this patient 2 tumor tissues were available (PT and pituitary adenoma).

UTR, untranslated region; 1 ˚HPT, primary hyperparathyroidism; PT, parathyoir tumor; NF, non-functioning; ms, missense; fs, frameshift; PTC, papillary thyroid carcinoma; ns, nonsense, if, in frame; ZES, Zollinger-Ellison syndrome.

ATG-7(g.c) change affects the Kozac consensus sequence, which plays a major role in the initiation of mRNA translation. The ATG-7(g.c) nucleotide substitution reduces the translation efficiency of the variant allele, which, in turn, causes a reduced amount of the encoded p27 protein compared with the wild-type CDKN1B allele (5). In vitro studies showed that the p27G9R variant is less stable than p27wt, and that this associates with strongly reduced expression of p27 in the tumor tissue of the mutationcarrier individual. Moreover, p27G9R mimics a p27 protein phosphorylated at the Ser10 residue, the major phosphorylation site of the protein, which regulates its localization and stability (7). The P69L change substitutes one of the proline residues mediating the interaction between p27wt and the CDK2 protein. Functional studies on the p27P69L mutant protein have demonstrated that, indeed, this protein does not efficiently bind CDK2 (6). Moreover, p27P69L is quite unstable in vitro, and is expressed at a reduced level both in transfected cells and in the tissues of mutation-positive

from p27wt, p27fs177 is highly unstable and rapidly degraded, so that its steady-state level in vitro is always very low. The p27unrelated C-terminal domain of p27fs177 is responsible for the rapid degradation of the protein, probably through protein misfolding. Studies of primary rat newborn fibroblasts established from wild-type unaffected and mutant rats confirmed the rapid degradation of p27fs177, which can be rescued by proteasome inhibition (i.e. MG132, epoxomycin, or bortezomib treatment) and thus is mediated by the proteasome.

Specifically, degradation of p27fs177 is, at least in part, mediated by Skp2-dependent proteasomal proteolysis (26).

Based on these findings, we postulate that reduced p27 levels, not newly acquired molecular phenotypes, trigger tumor formation in MENX-affected rats. This establishes a parallel with p27-deficient mice, which are also predisposed to tumor formation (27).

MEN4-associated mutations. Most of the germline mutations identified in patients have been studied in vitro to understand their link to tumor predisposition. The

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Figure 2 - Schematic structure of the p27 protein and position of the germline mutations identified. The regions mediating the binding of p27 to its major interacting partners, which are reported below the protein, are indicated. The germline mutations so far identified in MEN4 patients are indicated above the protein sequence. NLS = nuclear localization signal.

adenoma cells (6). The p27P95S variant changes one of the residues mediating the binding of p27 to the Grb2 adaptor protein. As a consequence of this change, the p27P95S mutant protein does not efficiently bind to Grb2, and this may ultimately impair the activation of the Ras signal transduction pathway (5). By contrast, no molecular phenotype could be assigned to the p27P133T variant using various in vitro assays (7). The p27 stop.Q variant encodes a protein

patients. p27P69L is also less efficient than p27wt in blocking cell growth (6). The p27W76X peptide has lost the nuclear localization signal (Figure 2), and therefore it is mislocalized to the cytoplasm in vitro and in vivo (in the tissues of a mutation-positive patient) (2,6). Therefore, p27W76X can no longer bind and inhibit cyclin–CDK complexes and, as a consequence, this peptide has lost the ability to inhibit cell growth when over-expressed in p27-negative GH3 pituitary

Figure 3 - Graphic representation of the nuclear and cytoplasmic interactions of p27. Upon mitogenic stimulation, p27 is released from cyclinE/CDK2 complexes and this dissociation from p27 activates CDK2, which, in turn, phosphorylates pRb. Phosphorylated pRb releases the transcription factor E2F, which induces the expression of genes required for the G1 to S progression. After the dissociation of p27 from the cyclinE/CDK2 complex in early G1 a portion of p27 is phosphorylated on Ser10 and exported into the cytoplasm through the interaction with CRM1 (exportin). Once in the cytoplasm, p27 is ubiquitylated by the KPC1/KPC2 complex and degraded by the proteasome. Upon mitogenic stimulation p27 becomes the substrate of the cyclinE/CDK2 complex, which phosphorylates the protein at the Thr187 residue, thereby creating a recognition site for the SKP2 ligase, which promotes ubiquitylation-mediated degradation of p27 by the proteasome in S phase.

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containing 60 amino acids more than p27wt. The amount of the p27stop.Q mutant protein was lower than that of p27wt in vitro, and it could be rescued by proteasome inhibitor treatment. Therefore, the stop.Q variation encodes a quite unstable p27 protein (5).

Based on how severely they affect the molecular characteristics of p27, the various genetic changes illustrated here have been classified as pathogenic mutations (K25fs, P69L, W76X), potentially pathogenic mutations [ATG7(g.c), G9R, P95S, stop.Q] or variants of unknown pathogenic significance (P133T). The molecular phenotype of the various p27 mutations is summarized in Table 1.

The recognition of both the MENX (rat) and the MEN4 (human) syndromes has demonstrated that Cdkn1b/ CDKN1B is a new tumor susceptibility gene for multiple neuroendocrine tumors in both species. The observations summarized here, together with studies of engineered mouse models with defective or mutant p27, confirm a critical role for p27 in regulating cell proliferation in neuroendocrine cells. Our hypothesis is that in these cells, aberrant/absent p27 activity cannot be compensated for by other cyclin–CDK inhibitors, as it occurs in tissues less affected by lack of functional p27. This would explain the tissue-specific susceptibility to tumor formation associated with p27 germline mutations. As novel CDKN1B mutations are discovered, our understanding of the relationship between p27 and neuroendocrine tumor predisposition will increase. The characterization of the molecular properties of mutant p27 proteins associated with MEN4 may facilitate the development of more effective targeted therapeutic strategies for the patients carrying those mutations.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

ACKNOWLEDGMENTS

19.

I thank all members of the laboratory for stimulating discussions. This work was supported by SFB 824 (DFG Sonderforschungsbereich 824) from the Deutsche Forschungsgemeinschaft, Bonn, Germany, and by grant #109223 from the Deutsche Krebshilfe, Bonn, Germany.

20.

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CLINICS 2012;67(S1):19-28

DOI:10.6061/clinics/2012(Sup01)05

REVIEW

Head and neck paragangliomas: clinical and molecular genetic classification Christian Offergeld,I Christoph Brase,II Svetlana Yaremchuk,III Irina Mader,IV Hans Christian Rischke,V Sven Gla¨sker,VI Kurt W Schmid,VII Thorsten Wiech,VIII Simon F Preuss,IX Carlos Sua´rez,X Tomasz Kopec´,XI Attila Patocs,XII,XIII Nelson Wohllk,XIV Mahdi Malekpour,XV,XVI Carsten C Boedeker,I Hartmut PH NeumannXVII I Department of Otorhinolaryngology, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany. II Department of Otorhinolarnygology, Friedrich-Alexander-University of Erlangen-Nu¨rnberg, Erlangen, Germany. III Institute of Otolaryngology of the Academy of Medical Science of Ukraine, Kiev, Ukraine. IV Department of Neuroradiology, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany. V Department of Nuclear Medicine, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany. VI Department of Neurosurgery, University Medical Center, AlbertLudwigs-University, Freiburg, Germany. VII Institute of Pathology and Neuropathology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany. VIII Institute of Pathology, University Hospital Freiburg, Albert-Ludwigs-University, Freiburg, Germany. IX Department of Otorhinolarnygology, University of Cologne, Cologne, Germany. X Department of Otolaryngology, Hospital Universitario Central de Asturias, Oviedo, Spain. XI Poznan University of Mediacal Sciences, Department of Otolaryngology, Poznan, Poland. XII 2nd Department of Medicine, Semmelweis-University, Budapest, Hungary.

XIII Molecular Medicine Research Group, Hungarian Academy of Sciences, Semmelweis-University, Budapest,Hungary. XIV Department of Endocrinology, Hospital del Salvador. University of Chile, Santiago de Chile, Chile. XV Department of Otorhinolaryngology, Tehran University of Medical Sciences, Tehran, Iran. XVI Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA. XVII Department of Nephrology and General Medicine, University Medical Center, Albert-Ludwigs- Albert-Ludwigs-University, Freiburg, Germany.

Head and neck paragangliomas are tumors arising from specialized neural crest cells. Prominent locations are the carotid body along with the vagal, jugular, and tympanic glomus. Head and neck paragangliomas are slowly growing tumors, with some carotid body tumors being reported to exist for many years as a painless lateral mass on the neck. Symptoms depend on the specific locations. In contrast to paraganglial tumors of the adrenals, abdomen and thorax, head and neck paragangliomas seldom release catecholamines and are hence rarely vasoactive. Petrous bone, jugular, and tympanic head and neck paragangliomas may cause hearing loss. The internationally accepted clinical classifications for carotid body tumors are based on the Shamblin Class I–III stages, which correspond to postoperative permanent side effects. For petrous-bone paragangliomas in the head and neck, the Fisch classification is used. Regarding the molecular genetics, head and neck paragangliomas have been associated with nine susceptibility genes: NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2 (SDH5), and TMEM127. Hereditary HNPs are mostly caused by mutations of the SDHD gene, but SDHB and SDHC mutations are not uncommon in such patients. Head and neck paragangliomas are rarely associated with mutations of VHL, RET, or NF1. The research on SDHA, SDHAF2 and TMEM127 is ongoing. Multiple head and neck paragangliomas are common in patients with SDHD mutations, while malignant head and neck paraganglioma is mostly seen in patients with SDHB mutations.

The treatment of choice is surgical resection. Good postoperative results can be expected in carotid body tumors of Shamblin Class I and II, whereas operations on other carotid body tumors and other head and neck paragangliomas frequently result in deficits of the cranial nerves adjacent to the tumors. Slow growth and the tendency of hereditary head and neck paragangliomas to be multifocal may justify less aggressive treatment strategies.

KEYWORDS: Paraganglioma; Susceptibility Genes; Shamblin Classification; Fisch Classification.

Offergeld C, Brase C, Yaremchuk S, Mader I, Rischke HC, Gla¨sker S, et al. Head and neck paragangliomas: clinical and molecular genetic classification. Clinics. 2012;67(S1):19-28.

E-mail: hartmut.neumann@uniklinik-freiburg.de Tel.: +49 761 270 35780

parasympathetic paraganglia of the skull base and neck, and are also called glomus tumors (1–5). Most well known is the tumor of the glomus caroticum, the carotid body tumor (CBT). HNP surgery is a challenge to the surgeon because of the tumor’s location in the vicinity of important blood vessels and cranial nerves. As well as pre-operative impairment of nerve function, surgery may result in deficits of cranial nerves VII, VIII, IX, X, XI, and XII. In this review, we present an overview of the clinical presentation and radiologic imaging of HNPs, topographical classification of HNPs, and recent advances in the

INTRODUCTION Head and neck paragangliomas (HNPs) are tumors of the autonomic system. They arise from chromaffin cells of the

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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genetics of HNPs in the context of nine susceptibility genes predisposing to tumors of the paraganglial system. These genetic data not only enrich our understanding of HNPs, but also provide us with a tool for risk assessment of tumors outside the head and neck region.

(XII) (10,11). Intracranial extension, which is the main cause of death, occurs in 22% of the cases (4). Oropharyngeal involvement caused by bulging of the pharyngeal wall into the pharyngeal lumen and medial displacement of the tonsil, may occasionally be observed.

PREVALENCE

Temporal paragangliomas Temporal paragangliomas entail tympanic and jugular paragangliomas. They arise from structures in close vicinity and are sometimes summarized in one group.

HNPs are rare tumors, representing less than 0.5% of all head and neck tumors. Approximately 3% of paragangliomas occur in the head and neck area (4); other estimates suggest that 1 in 30,000 head and neck tumors are HNPs (5).

Tympanic paragangliomas Tympanic paragangliomas (TPs) are mostly small-sized tumors originating in the middle ear. These tumors become symptomatic as pulsatile tinnitus in the vast majority of patients. Hearing loss is initially present in about half of patients (1,3–5,12). Most TPs are visible as a vascular middle ear mass. They are diagnosed by careful examination of the tympanic membrane and identification of the tumor through the translucent eardrum. Introducing positive pressure in the ear canal stops the pulsations of the tumor.

Frequently, it is impossible to visualize the entire tumor clinically, thus computed tomography (CT) or magnetic resonance imaging (MRI) scans are diagnostic.

TOPOGRAPHY Most HNPs arise in the bifurcation of the carotid artery (i.e. CBTs). Vagal paragangliomas arise from the autonomic nervous part of the vagal nerve (cranial nerve X). HNPs arising in the petrous bone originate from the tympanic glomus or the jugular glomus. When these tumors cannot be differentiated, they are referred to as jugulotympanic paragangliomas. Given the extensive spreading of the autonomic nervous structure, HNPs can arise at other locations, such as the ciliary glomus or laryngic glomus.

SIGNS AND SYMPTOMS Jugular paragangliomas

HNPs are space-occupying tumors. Signs and symptoms are induced by the topography of the anatomical structures from which the tumors originate. In contrast to extra-adrenal, abdominal, and thoracic pheochromocytomas, HNPs rarely secrete active substances. Only 1–3% of HNPs are vasoactive and associated with elevated catecholamines. Such tumors can induce tachycardia, hypertension and sweating attacks.

Jugular paragangliomas (JPs) arise from paraganglia in or around the jugular bulb (12,13). Occlusion of venous flow happens when these tumors increase in size. Consequently, venous blood from the brain shifts toward the unaffected sigmoid sinus and jugular bulb. Symptoms are similar to those of TP. Pulsatile tinnitus is a frequent symptom. A bruit can be heard by placing a stethoscope over the ear. Conductive hearing loss is seen with progression of the tumor, which either causes impairment of vibration of the ossicles or invades the bones behind the eardrum. Sensorineural hearing loss and/or dizziness is reported by patients when the tumor has invaded the inner ear. Occasionally, JPs can cause deficits of other cranial nerves and create dysfunctional swallowing and huskiness of the voice. However, due to the relatively slow growth, the swallowing mechanism and vocal cord function of the opposite side may initially compensate and mask the disease symptoms. When these tumors grow, they can also invade the facial nerve leading to facial paralysis, or they can encompass the hypoglossal nerve, leading to paralysis of half of the tongue. Further growth can lead to compression of the brain and/or brainstem.

Carotid body tumors (CBTs) CBTs arise from paraganglia located within the covering of the carotid artery in the neck. CBTs often remain clinically silent before presenting as a painless, slowly enlarging mass in the lateral neck (1,6,7). Approximately 60–70% of all CBTs take this route of clinical presentation (6,7). The initial symptoms can also be a pulsating mass in the neck. CBTs can be moved horizontally rather than vertically, a finding known as a positive Fontaine’s sign (1). Large CBTs may induce dysfunction of the vagal nerve and, less frequently, of cranial nerves IX, XI, and XII. Occasionally, Horner’s syndrome or deficits of the facial nerve may result from extremely large tumors (1,6). Sometimes a carotid bruit or a pulsating mass can be detected.

RADIOLOGY

Vagal paragangliomas

B-mode sonography and color duplex sonography are inexpensive, noninvasive diagnostic tools that are often used as the first imaging step for cervical HNPs (1,14). A CBT is usually sonographically seen as a solid, well-defined, hypoechoic mass with a splaying of the carotid bifurcation (1). The external carotid artery (ECA) is usually displaced anteromedially, and the internal carotid artery (ICA) is typically displaced posterolaterally (15).

VPs tend to displace both the ECA and the ICA anteriorly.

The internal jugular vein is typically compressed and displaced posteriorly (15). In large VPs there may also be a splaying of the carotid bifurcation, thereby mimicking a CBT. In color duplex sonography scans, cervical HNPs present as hypervascular tumors in most cases. It is important to

Vagal paragangliomas (VPs) are located more cephalad in the neck, between the jugular vein and the internal carotid artery, sometimes extending to the base of the skull through the jugular foramen or posterior to the mastoid tip (8). The symptoms of a VP depend on the location of the tumor along the vagal nerve from the skull base to the lower neck (8). Most VPs arise from the glomus nodosum, that is, the inferior ganglion. Typically, VPs present with an asymptomatic neck mass behind the angle of the mandible. Other symptoms of VPs are pulsatile tinnitus or ringing in the ear heard with each heartbeat (9). At times, this can be heard by placing the stethoscope over the ear. Less than 50% of VPs present with deficits of cranial nerves, which manifest as hoarseness (X), dysphagia (IX), shoulder drop (XI), nasal reflux of fluids, aspiration and hemiatrophy of the tongue

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mention, however, that absence of hypervascularity does not exclude a cervical HNP (16). In a study on 18 CBTs, 15 (83.3%) presented as a hypervascular mass in color duplex sonography scans (16).

Carotid, tympanic, and jugular paragangliomas are easily detected using CT and MR angiography. They contain many arterio-venous shunts and show strong enhancement with CT-angiography. Using time-resolved contrast-enhanced MR-angiography, they are detected in the early arterial phase (Figure 2D). Tympanic paragangliomas are seen on high resolution CT images of the petrous bone as a focal soft-tissue mass in the tympanon (17). CT is superior for the detection of bone destruction in the temporal bone (18). For jugular paragangliomas both MR (17) and CT (19) are recommended. CT-angiography depicts the anatomy more accurately than MR-angiography because of the better spatial resolution (19). However, contrast-enhanced MRangiography is better suited for screening and detection of multiple lesions. Both cross-sectional methods can be used for operative navigation. Pre-operative embolization of the paragangliomas aims for a reduction of blood loss during operation, but the literature is controversial (7,20).

advantage in identifying patients likely to benefit from therapy with 90Y-labelled DOTA-TATE. 18F-FDOPA PET, in particular, has been proved to be highly sensitive in different series and may be considered a good imaging technique for HNPs (23) but may perform poorly in SDHBassociated metastatic disease; under these conditions, 18FFDG-PET/CT provides higher sensitivities (24).

CLINICAL CLASSIFICATION OF CAROTID BODY TUMORS CBTs are usually classified using the criteria described by Shamblin and co-workers (25).

Shamblin classification Shamblin Class I CBTs are localized tumors with splaying of the carotid bifurcation, but little attachment to the carotid vessels. Complete surgical resection is generally possible with only minimal risk of vascular or cranial nerve complications (1).

Shamblin Class II CBTs partially surround the carotid vessels and complete resection is more challenging.

Shamblin class III CBTs intimately surround the carotid.

Complete resection is very challenging, and often requires temporary interruption of the cerebral circulation for vascular reconstruction. The risk of permanent vascular and neural defects is significantly higher than for Class I and Class II tumors (26). Figure 1A–C demonstrates three Shamblin classes of CBTs.

NUCLEAR MEDICINE IMAGING HNPs typically show avid uptake with different functional imaging techniques, namely 18F-fluorodihydroxyphenylalanine (18F-FDOPA) positron emission tomography/computed tomography (PET/CT); 18F-fluorodopamine (18FFDA) PET/CT and 123I-metaiodobenzylguanidine (123IMIBG) scintigraphy; the latter is paraganglioma specific.

Furthermore, somatostatin receptor imaging has a sensitivity of greater than 90% for HNP (21) using 111In-octreotide scintigraphy. Positron emitting somatostatin receptor imaging agents, such as 68Ga-DOTA-peptides [e.g. 68Ga-DOTATATE (DOTA-Tyr3-Thr8-octreotide)] have shown promising sensitivities in small series (22). It offers an additional

CLINICAL CLASSIFICATION OF TEMPORAL PARAGANGLIOMAS CT is almost crucial to evaluate temporal paragangliomas.

The extent of temporal bone destruction is used to classify these tumors according to the Fisch classification (Table 1) (27).

Figure 1 - Shamblin classification. The Shamblin classification is based on involvement of the carotid arteries, which are shown by white arrows in A–C. The tumors are indicated by yellow arrows. (A) CBT (class I): the axial T2-weighted MRI shows a CBT in a typical location.

Bulging of the left carotid bifurcation without any encasement of the carotid vessels. (B) CBT (class II): axial T2-weighted MRI showing a right-sided CBT with a partial encasement of the internal and external carotid artery. (C) CBT (class III): an axial slice of CT-angiography reveals a huge left-sided CBT with a complete encasement of the carotid vessels. In this case, bone destruction of the skull base, attributed to the enormous CBT, was also detected by CT (not shown).

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Figure 2 - Fisch classification. The tumors are indicated by yellow arrows. (A) Tympanic paraganglioma (Fisch class A): axial CT scan of a tympanic paraganglioma (bone window) on the right promontory. Note the absence of any bony erosion. (B) Tympanic paraganglioma (Fisch class B): the axial CT scan reveals a left-sided tympanic paraganglioma surrounding and partially destroying the ossicles. The malleus and stapes could not be discriminated. The tumor had also invaded the hypotympanon. There was no destruction of the bone wall to the jugular bulb. (C) Jugular paraganglioma (Fisch class C): axial CT scan showing a left-sided jugular paraganglioma. Note the bone destruction between the jugular bulb and the soft tissue tumor in the hypotympanon. (D) Tympanic paraganglioma (Fisch class D): time-resolved contrast-enhanced MR-angiography (upper row) depicts an early venous drainage attributed to arterio-venous fistulas within this tympanic paraganglioma (solid arrows). Coronal CT (lower row left) of the petrous bone shows a soft tissue tumor with encasement of the ossicles within the whole tympanon, and destruction of the tegmen tympani. Coronal T2-weighted and contrast enhanced T1-weighted images show a small, but distinct, intracranial but extradural tumor growth on the lateral skull base (dotted arrows, lower row).

Fisch classification

MOLECULAR GENETICS CLASSIFICATION

TPs arise from paraganglia along the tympanic plexus on the promontory (table 2, figure 2). Fisch class A tumors are limited to the promontory and the mesotympanon. In Fisch class B tumors, there is an invasion of the hypotympanon.

Class B paragangliomas often surround, and frequently destroy, the ossicles. In TPs, the bone over the jugular bulb is intact. JPs arise from paraganglia in, or around, the jugular bulb. There is bone destruction around the jugular bulb in all patients with JPs. Fisch class C tumors are further classified into subgroups C1–C4 according to the extent of bone erosions around the carotid foramen, the carotid canal and the foramen lacerum. Patients with Fisch class D tumors have intracranial extension of the paraganglioma. Further subclassification is based on the extradural versus intradural extension and the depth of intracranial invasion. It is important to note that for JPs, Fisch class D cannot be used separately without also using the subclassification for class C. In paragangliomas with intracranial extension, the D classification should always be accompanied by a C classification (e.g. C3Di2). Fisch classification before surgery is essential as the operative approach is determined by the tumor stage.

Current genetic classification is still incomplete and merits revision. To date, nine different susceptibility genes have been identified for tumors of the paraganglial system. The six relevant genes and associated syndromes are summarized in Table 3. It is important to note that some genes are frequently, and others rarely, mutated. The succinate dehydrogenase subunit D (SDHD) gene was first recognized as the susceptibility gene of paraganglioma syndrome type 1 (PGL 1) in 2000 (28). Subsequently, mutations of the B and C subunit genes (SDHB and SDHC) were described in patients with paraganglioma syndromes that were later designated as PGL type 3 and type 4 (29,30). The susceptibility gene for PGL 2 was identified nine years later (22). The SDHAF2 gene, also called SDH5, affects flavination of SDHA. In 2010, the SDHA gene was identified as a tumor suppressor gene associated with paraganglial tumors (31). Also in 2010, a gene called TMEM127 was found to be mutated in pheochromocytomas, i.e. exclusively adrenal paraganglial tumors (32). Meanwhile, it became evident that the spectrum of TMEM127 mutations extends to HNPs (33). Rarely, mutations of the well-known genes causing multiple

Table 1 - The Shamblin classification of carotid body tumors.

Class I II III

Tumor characteristics Splaying of the carotid bifurcation with little attachment to the carotid vessels; complete resection with very little morbidity Partial surrounding of internal and external carotid artery; complete resection more challenging Complete surrounding of the carotid vessels; complete resection often requires major vessel reconstruction

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SDHD, and showed that the penetrance for SDHD was about twice as high as that of SDHB carriers. SDHD mutations induced HNPs about 20 years earlier compared with SDHB mutations.

A thorough genotype–phenotype study for different HNP loci is currently lacking in the literature. What we currently know is that patients with mutations of SDHB, SDHC, and SDHD genes may develop tumors in the carotid body and the vagal, jugular, and tympanic paraganglia (23,28).

Patients with germline mutations of the SDHAF2 gene have been studied in detail by a Dutch and Spanish group in 2010 (40). So far, only a single mutation has been identified, and potentially all patients are relatives. CBTs have been shown in a patient with a TMEM127 germline mutation (33). More patients and details may be expected in forthcoming similar reports.

endocrine neoplasia type 2 (MEN2), von Hippel-Lindau disease, and neurofibromatosis type 1 (VHL, RET, and NF1) may also predispose to HNPs. The molecular pathomechanisms leading to the development of paraganglial tumors is still largely unknown.

Various processes, including activation of hypoxia-inducible factor 1a (HIF1a)-related neoangiogenesis pathways (in von Hippel-Lindau and in SDHx-associated paraganglioma syndromes), as well as activation of the Ras oncogene pathways (in MEN2 and neurofibromatosis type 1) have been suggested. However, in HNP, the major pathomechanism may stem from the impaired function of mitochondria, manifesting either as activation of the pseudo-hypoxia pathway as a result of accumulation of succinate (which inhibits HIF-alpha prolyl hydroxylases; PHD), leading to stabilization and activation of HIF-1a (34) or as increased oxidative stress and genomic instability (35,36). Systematic evaluation of genes involved in regulation of these pathways may reveal additional susceptibility genes. However, mutation analysis of PHD genes in patients with pheochromocytoma/paraganglioma and renal cell carcinoma showed no alterations (30).

The likelihood of identifying germline mutations in patients with HNP has been addressed by several studies.

Our study, published in 2009 (37), with 598 patients with HNP, found SDHx germline mutations in 31% of patients (52% SDHD, 34% SDHB, and 14% SDHC). Predictors for a positive mutation test included family history, previous adrenal or extra-adrenal pheochromocytoma, multiple HNPs, age #40 years and male gender. The prevalence of germline mutations of the VHL, RET, and NF1 genes among patients with HNP has also been evaluated by our group through extensive collaboration (38). Twelve patients were found to have hereditary non-SDHx HNPs out of a total of 809 patients with HNP and 2084 with VHL: 11 with germline VHL mutations and one with a RET mutation.

The prevalence of hereditary HNP was 5 out of 1,000 in patients with VHL and nine out of 1,000 in patients with non-SDHx HNP. A comprehensive literature review revealed HNPs in five von Hippel-Lindau, two MEN2 and one neurofibromatosis type 1 patients, who previously had symptoms characteristic for von Hippel-Lindau disease, MEN2 or neurofibromatosis type 1. Thus far, the number of cases associated with mutations in SDHA gene is unknown.

This issue could be addressed from the other end, i.e. the prevalence of HNPs in carriers of SDHB and SDHD mutations. This has been recently investigated by a group from Birmingham in the UK (39), who compared the penetrance for HNP for mutation carriers of SDHB and

SURGICAL TREATMENT OF HNP Surgery of carotid body tumors In general, treatment options for HNPs comprise surgical resection, as well as irradiation therapy, stereotactic radiosurgery and permanent embolization (1,7,41). If necessary and promising, a combined treatment strategy could be used. The majority of cases of HNP should undergo complete surgical resection as this is the only therapeutic option potentially offering a cure for the patient and is therefore the treatment of choice (1,6–8,42).

CBTs are the most common type of HNPs, which are categorized according to the Shamblin classification discussed above (25). The principal goal of any surgical attempt concerning CBT is complete tumor resection via a transcervical approach (1,7,41). With complete surgical resection, the tumor is controlled locally in 89–100% of cases (1,43,44). However, the probability of postoperative cranial nerve dysfunction has to be taken into consideration, even in cases of successful surgical removal of CBT (1,7). In a large study on postoperative outcome in 1,181 patients with CBT, Anand et al. found permanent cranial nerve deficit in 21.8% of cases (26). Obviously, complication rates are directly related to tumor size. Therefore, the Shamblin classification is not only essential for description of tumor characteristics, but also for the estimation of risks concerning any kind of surgical removal. As the tumor class in the Shamblin classification increases (i.e. from I to III), surgical removal of CBT is expected to become more difficult and challenging. In cases with Shamblin Class III-rated CBTs, not only does the possibility of cranial nerve deficits increase but also permanent vascular and neural defects are frequently seen, as a result of the need for

Table 2 - Classification of jugulotympanic paragangliomas according to Fisch and Mattox.

Class A B C1 C2 C3 C4 De

1/2

Di

1/2/3

Location and extension of paraganglioma Paragangliomas that arise along the tympanic plexus on promontory Paragangliomas with invasion of the hypotympanon; cortical bone over jugular bulb intact Paragangliomas with erosion of the carotid foramen Paragangliomas with destruction of the vertical carotid canal Paragangliomas with involvement of the horizontal portion of the carotid canal; foramen lacerum intact Paragangliomas with invasion of the foramen lacerum and cavernous sinus Paragangliomas with intracranial but extradural extension; De1/2 according to displacement of the dura (De1 = less than 2 cm, De2 = more than 2 cm) Paragangliomas with intracranial and intradural extension; Di1/2/3 according to depth of invasion into the posterior cranial fossa (Di1 = less than 2 cm, Di 2 = between 2 and 4 cm, Di3 = more than 4 cm)

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intra-operative interruption of the cerebral circulation for complete tumor resection (1,6).

comparison of results emerging from different studies is difficult because parameters such as Fisch classification and follow-up intervals are not consistent between studies (14).

Surgery of vagal paragangliomas Similar to CBTs, most authorities prefer surgical resection of VPs (6–8,42,45). If surgical removal of VP is successfully performed, local tumor control can be achieved in up to 100% of cases (43,44,46). Postoperative cranial nerve dysfunction is considered a major complication of transcervical surgical resection of these tumors (7,8). Unfortunately, in most cases of VP, it is necessary to sacrifice the vagal nerve intra-operatively (47). A promising study by Miller et al. demonstrated higher preservation rates of the vagal nerve by microsurgical techniques during surgical removal (8). As for CBTs, the rate of surgical complications in VP also increases with tumor size, but, unlike CBT, there exists no generally accepted classification system for VP (1).

Therefore, estimation of possible postoperative complications is solely dependent on the surgeon’s experience and pre-operative imaging.

NEUROSURGICAL ASPECTS OF SKULL BASE PARAGANGLIOMAS Skull base paragangliomas are rare tumors, even in major referral neurosurgical centers, and they remain challenging, due to their vascularized nature and deep intra-/extracranial location. These tumors may involve the jugular bulb, middle ear, petrous apex, clivus, carotid artery, infratemporal and posterior fossa. Despite their benign nature in the majority of cases, these tumors tend to invade adjacent structures, which may sometimes preclude complete resection of these lesions. In order to achieve a good clinical outcome, close collaboration between ear, nose, and throat (ENT) surgeons, neurosurgeons, and interventional neuroradiologists is of paramount importance.

The surgical approach is determined by the size and anatomical location of the tumor. After careful analysis of MRI and thin-section CT, the surgical approach is planned with collaborative teamwork to address all aspects of the tumor. The approach should be wide, as these tumors bleed profusely on manipulation. The possibility of severe blood loss needs to be considered in pre-operative planning. Preoperative embolization is generally recommended, especially for larger tumors. This will allow for safer surgery as a result of less intraoperative blood loss and better visualization of the structures.

Small tumors in the jugular bulb may be removed by a transjugular approach. Other approaches include the transmastoid and combined approaches (51). The intracranial part of larger tumors is frequently best removed by an infratemporal approach.

Intraoperative neuromonitoring is mandatory and should include at least electromyography of the involved cranial nerves. For tumors with major intracranial extension, motor, sensory and auditory evoked potentials may become necessary. The anesthesiologist needs to be informed about this, and plan accordingly (no gas, no long-acting muscle relaxants). The introduction of intra-operative neuromonitoring has significantly improved postoperative outcome, particularly in sparing the facial nerve during skull-base surgery (52).

Surgery of skull base paragangliomas Paragangliomas of the temporal bone are usually differentiated into TPs and JPs, based on the Fisch classification (27). Using this classification, the feasibility of (complete) surgical resection can be estimated by the surgeon.

Whenever possible, surgical resection is the treatment of choice for patients with TP or JP, depending on tumor size, patient age, and other possibly intervening individualized factors. TPs are usually categorized as Fisch A and B. Surgical removal is the treatment of choice. Major postoperative complications such as cranial nerve deficits are usually not expected, but minor complications such as postoperative conductive hearing loss may occur (45). In cases categorized as Fisch A and B, a transmeatal approach or a surgical approach via mastoidectomy, including posterior tympanotomy and exposure of the facial nerve, may be adequate (14). In some cases of TP categorized as class B, a juxtacondylar approach might be necessary (14,48).

There is an ongoing debate on treatment modalities when it comes to JP (1,48). These masses are categorized as Fisch C and D tumors (1,7,41). Thanks to development and implementation of microsurgical techniques, it is possible to resect these tumors completely, and local tumor control can be achieved in 80–90% of cases (41,49,50). JP (Fisch classification C and D) are usually resected via an infratemporal approach. Tumors classified as Fisch C1, C2 and De, Di1/2 may demand a variant of the juxtacondylar approach (14). It is important to note that because these tumors are adherent and occasionally invade cranial nerves IX–XII, a higher complication risk concerning cranial nerve deficits is anticipated for the postoperative outcome.

Patients may therefore experience problems, including swallowing, voice, articulation, and shoulder weakness (7).

Consequently, further surgical procedures in an attempt to correct these complications may be necessary in these patients (1,7). In general, tumors classified as Fisch Di1/2 should be resected in a two-stage, team-approach procedure involving neurosurgeons. This therapeutic option is not available for patients categorized as Fisch Di3. In these cases, palliative radiotherapy is advocated (14). Current literature shows that the long-term success rate following surgical therapy of JP is 72–95%; however, it should be noted that

HISTOPATHOLOGY The HNPs described above have a similar histopathologic appearance. Under the microscope, two types of cells displaying typical nest-like or alveolar architectural pattern are seen. Type I cells, or chief cells, are epitheloid cells often with enlarged hyperchromatic nuclei and arranged in solid groups called ‘‘Zellballen’’. These groups of chief cells are surrounded by a flattened layer of type II cells or sustentacular cells, which can be visualized by immunohistochemical staining for S-100 protein (Figure 3). Type I and type II cells characteristically lie within a dense network of capillaries.

Chief cells may be quite uniform or may display pronounced nuclear pleomorphism, such as bizarre and huge multinucleated cells. They are immunohistochemically positive for chromogranins (HNP shows strong expression of chromogranin B, whereas chromogranin A is expressed

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Head and Neck Paraganglioma Offergeld C et al.

Figure 3 - Histology and immunostaining of head and neck paragangliomas. (A) Demonstration of sustentacular cells by S-100 protein in an HNP. The delicate net of sustentacular cells is surrounding the so-called Zellballen of chief cells (x200). (B) Complete immunohistochemical negativity for SDHB strongly suggesting a SDHB germline mutation (x200). (C) Immunohistochemical demonstration of SDHB protein in tumor cells, virtually excluding a germline mutation in SDHB (x200).

to a much lesser degree (53) and synaptophysin, yet are negative to cytokeratin, in contrast to some pheochromocytomas and paragangliomas of the filum terminale.

Unfortunately, nuclear pleomorphism, mitotic activity, necrosis, or vascular or perineural invasion cannot predict the biological behavior, as all these features may also be found in benign HNPs.

Several attempts have been made to histologically distinguish benign tumors from the rare malignant paraganglial tumors. These are based on scoring systems that have been introduced for pheochromocytomas and paragangliomas of the retroperitoneum, although no scoring system has been generally accepted as yet. The most popular scoring system has been suggested by Thompson

Table 3 - Molecular classification of head and neck paragangliomas (HNPs).

Syndrome

VHL

MIM ID Inheritance

193300 Autosomal Dominant

Gene name Protein function

Locus Age at diagnosis of Pheochromocytoma mean and range, in years Exons (n)//amino acids (n) HNP (%) Age at diagnosis HNP Median (range, in years) Pheo risk (%) PG Abdominal extraadrenal (%) Multifocality (%) Malignant (%) Clinical Phenotype

Associated tumors

PGL1

PGL2

PGL3

PGL4

168000 60650 605373 115310 Autosomal Autosomal Autosomal Autosomal Dominant with Dominant with Dominant Dominant parent-of-origin parent-of-origin effect effect VHL SDHD SDHAF2 SDHC SDHB An ubiquitin ligase One of two Mitochondrial One of two The iron-sulfur protein; it plays a membraneassembly factor membraneprotein that form role in the oxygen- anchoring subunits for complex II— anchoring together with sensing pathway of complex II (SDH) interacts subunits of SDHA, the main directly with complex II (SDH) catalytic domain SDHA of complex II 3p25-26 11q23 11q13.1 1q21 1p36 22 (5–67) 27 (5–65) unknown Extremely rare 34 (12–66)

TMEM127 613903 Autosomal dominant

TMEM127 Endosomal trafficking?; mTOR regulation?

2q11 43 (34–54)

3/213 0,5 23 (7-39)

4/159 41 40 (12–74)

4/166 73–86 45 (15–65)

6/169 100 46 (13–73)

8/280 8 42 (9–75)

3/238 1–2 34

10-34 17

53 59

0 0

,3 0

28 62

25 1–2

56 4% Sympathetic Noradrenergic

55 0 Parasympathetic, occasionally noradrenergic Rarely Papillary thyroid cancer, GIST

11 32 Noradrenergic

33 5 Sympathetic Noradrenergic

Eye and CNS hemangioblastomas, clear cell renal cancer, islet cell tumors, endolymphatic sac tumors of the inner ear

0 9 0 0 Parasympa thetic Parasympa thetic

GIST

GIST

Rarely renal cell cancer GIST

Adapted from Bausch et al. (73), Hensen et al. (74) and Neumann and Eng (75).

CNS = central nervous system; GIST = gastrointestinal stromal tumor, mTOR = mammalian target of rapamycin; PGL = Paraganglioma syndrome; SDHAF2 = succinate dehydrogenase complex assembly factor 2; TMEN127 = transmembrane protein 127.

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wait-and-scan policy was chosen (39). Only three of these patients developed cranial nerve palsy during an average follow-up of 8.5 years. It should be kept in mind that the morbidity of a cranial nerve palsy caused by surgery is higher than the morbidity caused by slow fading of the nerve function through the tumor (72). Hence, surgical removal of VPs should be weighed against the patient’s age, size of the tumor, predicted tumor growth and cranial nerve function (72).

(54), and includes a variety of criteria, such as cellular and nuclear features, growth patterns, or occurrence of necrosis.

It has to be emphasized that only the occurrence of lymph node and/or distant metastases are the ultimate criteria for malignancy. Morphologic demonstration of tumor invasion to adjacent structures including bone does not indicate biologic malignancy. Isolated tumor nodules in the vicinity of the main tumor may represent multicentricity of the HNP. Such tumor nodules can only be interpreted as lymph node metastases if they are embedded into (remnants of) lymphatic tissue.

Immunochemical staining with antibodies against SDHB protein has been found to be associated with germline mutations of the SDHB, SDHC, and SDHD genes (Figures 3A and B) (55). As a result, immunostaining has become a screening method to select patients for subsequent molecular genetic testing. The value of immunostaining against SDHB and other proteins is currently under investigation, and the importance for routine histopathology and genetics will be corroborated in expected additional analyses (56).

ACKNOWLEDGMENTS This work is funded, in part, by the German Cancer Foundation (Deutsche Krebshilfe Grant 107995 to HPHN).

AUTHOR CONTRIBUTIONS Offergeld C, Brase C, Yaremchuk S, Preuss SF, Suarez C, Kopec´ T, Malekpour M, Boedeker CC, Schmid KW, Wiech T wrote the sections ‘‘Signs and symptoms’’, ‘‘Clinical classification’’ and ‘‘Surgery’’. Mader I wrote the section ‘‘Radiology’’. Rischke HC wrote the section ‘‘Nuclear imaging’’. Gla¨sker S wrote the section ‘‘Neurosurgery’’. Patocs A, Wohllk N, Neumann HPH wrote the section ‘‘Molecular genetics’’. Neumann HPH coordinated the work. All authors edited the text and approved the final version.

OUTCOME AND CHALLENGES Management of head and neck paragangliomas is challenging for every physician, especially in cases of multiple paragangliomas or tumors in an advanced stage.

The outcome mainly depends on the stage of tumor at initial presentation.

For Shamblin type I and type II CBTs, the peri- and postoperative risk of neurovascular complications is low (57). Complication rate increases strongly for Shamblin type III CBTs (57,58). The current literature shows serious postoperative complications only in 0–2.3% of patients with Shamblin type I and II tumors, while this figure increases to 7–35.7% for patients with a Shamblin type III tumor (57–59).

For JP, surgical control is achieved in about 70–86% of patients, even in Fisch class C and D tumors (45,60–62).

Although postoperative functional deficit of the cranial nerves occurs, patients seem to be able to cope with the new limitations within two years and get back to a normal social life (60). While total resection of the tumors should be performed as a curative treatment for paragangliomas (62,63), a subtotal removal may be required to preserve cranial nerve function. In such cases, subtotal tumor resection, combined with gamma-knife surgery or radiation therapy, has proved to be a safe and effective treatment modality (61,64–66). In the elderly, it is important to weigh up the possible disabilities after surgical therapy, so a more conservative treatment concept might be preferred (61,67).

The outcome after surgical removal of TP (Fisch type A) is normally very good, with preservation of hearing in more than 90% of cases (63,68–70); therefore, surgical resection of these tumors is the therapy of choice (63,68,70).

VPs are special. They originate from the vagus nerve, so surgical removal of these tumors will be associated with a loss of vagal nerve function in the vast majority of cases (39,71,72) despite the fact that the nerve is kept anatomically intact (72). The immediate consequences would be hoarseness, dysphagia, and aspiration. The situation is even worse for extensive VPs, where additional cranial nerve deficits (in about 50–60% of patients) are seen because of the close proximity of the tumor in the jugular foramen to cranial nerves IX, XI, and XII (39,71,72). Bradshaw and Jansen described a group of 40 patients with VPs, in whom a

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DOI:10.6061/clinics/2012(Sup01)06

REVIEW

Familial gigantism Wouter W. de Herder Department of Internal Medicine, Sector of Endocrinology, Erasmus MC, Rotterdam, the Netherlands.

Familial GH-secreting tumors are seen in association with three separate hereditary clinical syndromes: multiple endocrine neoplasia type 1, Carney complex, and familial isolated pituitary adenomas.

KEYWORDS: Acromegaly; Gigantism; Pituitary; History; Hereditary. de Herder WW. Familial gigantism. Clinics. 2012;67(S1):29-32.

E-mail: w.w.deherder@erasmusmc.nl Tel.: 31 10 7035950

(23) were convinced that acromegaly and gigantism were two entirely different disorders. Gigantism was considered an exaggerated variant of normal development, whereas acromegaly was considered as a pathological condition.

However, in 1884, Drs. Christian F. Fritsche and Theodor Albrecht Edwin Klebs (24), supported by the work of Dr. Karl Langer (1872) (25), concluded that in contrast to gigantism, which they considered a congenital disorder, acromegaly is an acquired variety of gigantism that occurs at a later age after growth is completed. In 1894, Dr. M. Sternberg concluded that there are many similarities between acromegaly and gigantism (26). However, in 1897 he changed his view and agreed with Dr. Pierre Marie and others that both disorders have different origins (27). In 1891, Dr. D.J. Cunningham, after studying the skeleton of the Irish giant Cornelius Magrath (1736–1760; height: 2.26 m [7 ft, 5 in]), pointed out the connection between acromegaly and gigantism (28–30). In 1893, Dr. Charles Dana (31) and Dr. Woods Hutchinson (32) described the case report and postmortem studies of the French giantess Emma Aline Bataillard (also known as Lady Aama, 1877–1895; height: 2.03 m [6 ft, 8 in]) and reached the same conclusion. After studying the acromegalic giant Jean-Pierre Mazas (1847– 1901; height: 2.30 m [7 ft, 6K in]), the so-called ‘‘giant of Montastruc’’ (Monastruc, Haute-Garonne, France) in 1895, Drs. E´douard Brissaud and Henry Meige also concluded that acromegaly and gigantism can coexist in the same person (33,34).

It gradually became evident that acromegaly and gigantism have the same pathogenetic mechanism, but differ regarding the age of onset. Gigantism occurs much earlier in life when the skeleton still has the potential to grow, a developmental phase now known as ‘‘prepubertal’’ (33–35).

Finally, the cause of acromegaly and gigantism — the overproduction of pituitary growth hormone — became known in the early years of the 20th century (36).

Familial acromegaly and familial gigantism are extremely rare. The earliest report of familial gigantism may be that of Goliath, described in in the Bible, where it is stated that his father and three brothers were also giants. The first description of familial acromegaly in the medical literature is most likely a report by Fraenkel that was published in 1901 (37,38). The Ugo brothers, or Hugo brothers, also

HISTORICAL OVERVIEW In 1886, Dr Pierre Marie used the term ‘‘acromegaly’’ for the first time and described the full clinical characteristics of this condition (1). However, twenty-two years prior in 1864, Dr. Andrea Verga had already named the same disorder ‘‘prosopo-ectasia’’ [widening of the face] (2). At the postmortem examination of an acromegalic woman (with ‘‘prosopo-ectasia’’), he found a walnut-sized sellar tumor and displacement of the optic nerves (2). A normal pituitary gland was not found. He wondered whether the normal pituitary gland had disappeared because of pressure from the tumor or whether the tumor itself was a degeneration of the gland (2). Also, Dr. V. Brigidi described in 1877 a case report that included findings obtained at the autopsy of the acromegalic Italian actor Ghirlenzoni (3). Dr. V. Brigidi had diagnosed this condition as ‘‘rheumatitis deformans’’ and made the significant observation of a hypertrophied pituitary gland on the first microscopic examination of a pituitary tumor (3). In 1887, Dr. Oskar Minkowski reported that pituitary enlargement is found in all postmortem studies on patients with acromegaly (4). In 1892, Dr. Roberto Massalongo was able to correlate acromegaly with increased pituitary function by demonstrating that a pituitary tumor taken from a patient with acromegaly contained specific granulated cells (5). At the end of the 19th century, the relationship between pituitary hyperfunctionhypertrophy (or a hyperfunctioning pituitary tumor) and acromegaly was clearly established and confirmed by many investigators (6–12). Dr. Henri Henrot had already published in 1877 an autopsy report of a patient with gigantism, in whom a 4.5 6 3.0 cm tumor was found in the position of the pituitary body (13,14).

Initially, it was believed that acromegaly and gigantism were two totally different diseases. Dr. Pierre Marie (15–21), his intern Dr. J.D. Souza-Leite (22) and Dr. Georges Guinon

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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known in France as ‘‘les Ge´ants des Alpes’’ (‘‘the Giants of the Alps’’) (Figure 1), were two famous giants who traveled the world and appeared at fairs and circuses at the end of the 19th century and the beginning of the 20th century.

Battista Ugo (in French: Baptiste Hugo, 1876–1916) attained a height of 2.30 m (7 ft, 7 in) and Paolo Antonio Ugo (in French: Antoine Hugo, 1887–1914) attained a height of 2.25 m (7 ft, 5 in) (39–41). Their parents, three brothers and two sisters were of average size. The postmortem examination of Baptiste Hugo was performed and published by Dr. D. Symmers at the William Parker Hospital in New York.

The report describes a pituitary adenoma measuring 5 6 2.5 6 2.3 cm (2 6 1 6 0.9 in) and weighing 5.94 g with suprasellar expansion that compressed both optic nerves.

Left parasellar and retrosellar expansion were also found.

In his famous monograph, ‘‘The Pituitary Body and its Disorders’’, published in 1912, Dr. Harvey Cushing (42) hinted at ‘‘Mendelian tendencies’’ in two patients with

familial gigantism (case XIII, surgical No. 27784, pp. 8–92; case XXXI, surgical No. 27011K, pp. 158–162) (42).

Since these initial reports, several authors have reported the coincidence of acromegaly and gigantism in first-degree relatives (43–55).

Clinical relevance Growth hormone (GH)-secreting pituitary adenomas have an annual incidence of about 3 per 1,000,000 and a prevalence of about 60 per 1,000,000. The likelihood of multiple GHsecreting tumors presenting among first-degree relatives within a single family is, therefore, statistically very uncommon, and their occurrence has prompted many researchers to consider this an inheritable disorder (56).

Familial GH-secreting tumors are seen in association with three separate hereditary clinical syndromes: (1) multiple endocrine neoplasia type 1 (MEN1), (2) Carney complex (CNC), and (3) familial isolated pituitary adenomas (FIPA).

Multiple endocrine neoplasia type 1 (MEN1) Autosomal-dominant MEN1 syndrome is associated with a loss of heterozygosity (LOH) on chromosome locus 11q13 (57). The MEN1 gene tumor suppressor gene encodes a 610-amino acid protein known as menin. More than 550 mutations of the MEN1 gene have been identified. Pituitary tumors occur in approximately 30–50% of patients with MEN1 (58), but the frequency of GH-producing pituitary tumors in MEN1 patients is only about 10% (59).

Carney complex (CNC) Pituitary adenomas occur in 10–20% of patients with the autosomal-dominant CNC, and these are invariably GHsecreting tumors (60).

CNC is associated with a LOH on chromosomal locus 17q22–24. Germline mutations in the protein kinase A (PKA) regulatory subunit 1 (PRKAR1A) gene have been identified (61). Loss of function of this regulatory subunit results in constitutive activation of PKA that increases signaling through a pathway that enhances proliferation of the GH-producing cells in the pituitary.

Familial isolated pituitary adenomas (FIPA) Inactivating germline mutations in the aryl hydrocarbon receptor that interacts with tumor suppressor protein (AIP) can be found in 40–50% of families that have a case of acromegaly occurring along with FIPA (62–66). To date, about 50 different AIP mutations have been identified.

AIP mutation-positive acromegaly or gigantism is generally diagnosed 10 years earlier than sporadic acromegaly.

GH-producing tumors in AIP-positive patients (mostly males) more frequently display extrasellar growth, exhibit more aggressive characteristics, and have a tendency for higher accelerated growth than sporadic tumors. These tumors are also more frequently associated with onset in childhood and adolescence, thus presenting more frequently with gigantism (67,68). Furthermore, AIP mutation-positive GH-producing tumors appear to be less responsive to medical therapy with somatostatin analogs and dopamine agonists.

In 2010, Chahal and co-workers extracted DNA from a tooth of the Irish giant, Charles Byrne (1761–1783; 2.31 m, [7 ft 7 in]), and identified a germline mutation in the AIP gene. Four contemporary Northern Irish families who

Figure 1 - Familial gigantism in the two Hugo brothers. Top row: Battista Ugo (Baptiste Hugo), 1876–1916, reached a height of 2.30 m (7 ft, 7 in) and Paolo Antonio Ugo (Antoine Hugo), 1887– 1914, reached a height of 2.25 m (7 ft, 5 in). Bottom row: the parents of the Ugo brothers, Teresa Chiardola (1849–1905) and Antonio Ugo (1840–1917), and their sister, Maddalena Ugo (1885–1960). Picture from the collection of Dr. W. W. de Herder.

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53. Stock JL, Warth MR, Teh BT, Coderre JA, Overdorf JH, Baumann G, et al.

A kindred with a variant of multiple endocrine neoplasia type 1 demonstrating frequent expression of pituitary tumors but not linked to the multiple endocrine neoplasia type 1 locus at chromosome region 11q13. J Clin Endocrinol Metab. 1997;82(2):486–92, http://dx.doi.org/ 10.1210/jc.82.2.486.

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presented with gigantism, acromegaly, or prolactinoma have the same mutation and haplotype associated with the mutated gene. Using coalescent theory, they came to the conclusion that these persons share a common ancestor who lived about 57–66 generations earlier (69).

Familial acromegaly and gigantism only account for a very tiny proportion of all pituitary adenomas and can be related to MEN 1 and CNC syndromes as well as to mutations in the AIP gene. AIP mutations are very common in gigantism.

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DOI:10.6061/clinics/2012(Sup01)07

REVIEW

RET codon 609 mutations: a contribution for better clinical managing Caterina Mian,I Paola Sartorato,II Susi Barollo,I Mariangela Zane,II Giuseppe OpocherI,II I University of Padova, Department of Medicine, Via Gattamelata, 64 35128 Padova, Italy. II Veneto Institute of Oncology, Via Gattamelata, 64 35128 Padova, Italy.

Medullary thyroid carcinoma currently accounts for 5–8% of all thyroid cancers. The clinical course of this disease varies from extremely indolent tumors that can go unchanged for years to an extremely aggressive variant that is associated with a high mortality rate. As many as 75% of all medullary thyroid carcinomas are sporadic, with an average age at presentation reported as 60 years, and the remaining 25% are hereditary with an earlier age of presentation, ranging from 20 to 40 years. Germline RET proto-oncogene mutations are the genetic causes of multiple endocrine neoplasia type 2 and a strong genotype-phenotype correlation exists, particularly between a specific RET codon mutation and the (a) age-related onset and (b) thyroid tumor progression, from C-cell hyperplasia to medullary thyroid carcinoma and, ultimately, to nodal metastases. RET mutations predispose an individual to the development of medullary thyroid carcinomas and can also influence the individual response to RET protein receptor-targeted therapies. RET codon 609point mutations are rare genetic events belonging to the intermediate risk category for the onset of medullary thyroid carcinoma. A large genealogy resulting in a less aggressive form of medullary thyroid carcinoma is associated with the high penetrance of pheochromocytoma and has been reported in the literature. In this short review article, we comment on our previous report of a large multiple endocrine neoplasia type 2A kindred with the same Cys609Ser germline RET mutation in which, conversely, the syndrome was characterized by a slightly aggressive, highly penetrant form of medullary thyroid carcinoma that was associated with low penetrance of pheochromocytoma and primary hyperparathyroidism.

KEYWORDS: Pheochromocytoma; MEN2A; Medullary Thyroid Carcinoma; Hereditary Thyroid Cancer.

Mian C, Sartorato P, Barollo S, Zane M, Opocher G. RET codon 609 mutations: a contribution for better clinical managing. Clinics. 2012;67(S1):33-36.

E-mail: giuseppe.opocher@unipd.it Tel.: +39 0498215569

endocrine neoplasia type 2B (MEN2B), or familial MCT (FMTC). When the RET mutation presents at the germline level, the disease, either FMTC or MEN2, is transmitted as an autosomal-dominant trait with complete penetrance. In MEN2A, in addition to MTC (100% of cases), patients develop pheochromocytoma (PHEO) and primary hyperparathyroidism (HPT) in approximately 50% and 20% of cases, respectively. The MEN2B phenotype is characterized by the presence of mucosal neuromas and marfanoid habitus as well as MTC (100% of cases) and PHEO (50% of cases) (1).

In this syndrome, the phenotype-genotype correlation is strong. Thus, different codon mutations along the RET oncogene predispose individuals to different ages of onset and disease aggressiveness. Also, the specific RET codon mutation can influence decisions about surgical prophylactic thyroid intervention.

Complete penetrance, autosomal-dominant transmission, strong phenotype-genotype correlation, and a relatively simple genomic structure are the key points of the success of RET, as an example, in translational medicine. For these reasons, the detection of germline RET mutations is now broadly accepted for the clinical evaluation of subjects with MTC. The RET gene is localized on chromosome 10, band 10q11.2; it is comprised of 21 exons and encodes for the RET transmembrane receptor with intrinsic tyrosine kinase (TK) activity and is stimulated by interaction with different

INTRODUCTION Medullary thyroid carcinoma (MTC) currently accounts for 5–8% of all thyroid cancers. It arises from the parafollicular Ccells originating from the neural crest that are incorporated into the thyroid during fetal development. The clinical course of MTC varies from extremely indolent tumors that can go unchanged for years to extremely aggressive variants that are associated with a high mortality rate.

The relationship between proto-oncogene RET mutations and MTC is one of the best examples of translational medicine. RET mutations predispose an individual to the development of parafollicular C-cell hyperplasia and, subsequently, to the onset of MTC. Most of the sporadic MTC cases harbor a somatic RET mutation that can be correlated with disease aggressiveness. Up to 20–30% of the subjects with an apparently sporadic MTC have a germline mutation (1).

Hereditary MTC can occur as three major subtypes: multiple endocrine neoplasia type 2A (MEN2A), multiple

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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patients with the RET codon 609 mutation is to take into account descriptions of large, affected families and registry studies with sufficiently large numbers of individuals with the RET 609 codon mutation.

Several large families with RET codon 609 mutations have been reported in the literature. In the family reported by Kinlaw et al. (7), carriers of the RET codon 609 cysteine-toserine substitution were characterized by the low penetrance of MTC and the high penetrance of PHEO. Calva et al. (8) described 16 affected patients belonging to a 38member genealogy with a RET Cys609Tyr mutation. The phenotype of these subjects was characterized by MTC in nine out of 16 affected cases, lymph node metastasis in six out of nine cases, parathyroid adenoma in one out of 16 cases; however, PHEO was not found in this family.

We have also described a 5-generation, 48-member family with MEN2A syndrome that also harbored the RET Cys609Tyr mutation (9). Furthermore, a large registry study of individuals carrying RET exon 10 mutations, which also includes this family, was published by Frank-Raue et al. (10).

growth factors belonging to the glial-derived neurotrophic factor family. In animal models, RET is essential for the development of the sympathetic and parasympathetic enteric nervous systems and the kidneys.

In 98% of MEN2A families, germline mutations affect the cysteine-rich extracellular domain by converting a cysteine into another amino acid, and by determining RET spontaneous dimerization and activation. These mutations are located in codon 634 (exon 11) or codons 609, 611, 618, and 620 (exon 10). The most common mutation, accounting for 80% of MEN2A families, affects codon 634, and in this codon a particular point mutation, when a cysteine is substituted for an arginine, accounts for 50% of all MEN2A cases. Approximately half of FMTC kindred are due to germline mutations in exon 10 (codon 618 and 620), but some FMTC families are caused by mutations in exon 11 (codons 630, 631 or 634). Interestingly, the cysteine-arginine substitution in the RET 634 codon has never been found in FMTC families. In an increasing number of FMTC cases, germline mutations have been reported in exons 13 (codons 768, 790 and 791), 14 (codon 804 and 844), and 15 (codon 891), which are located in the TK domain, thereby interfering with intracellular ATP binding. In approximately 95% of patients with MEN2B, a single mutation that converts methionine to threonine at codon 918 (exon 16) has been identified (1). This mutation causes alterations in the substrate recognition pocket of the TK catalytic core.

Other rare intracellular mutations that are associated with MEN2B involve codon 882 (exon 15).

There is a close relationship between genotype and phenotype. Thus, more aggressive phenotypes have been noticed in cases carrying mutations in the extracellular portion of RET rather than the intracellular portion.

Furthermore, the average age at diagnosis for MEN2 patients with C-cell hyperplasia and RET extracellular domain mutations is 8.3 years, whereas the average age at diagnosis is of 11.2 years in patients with RET intracellular domain mutations. In patients with node-negative MTC, the average age at diagnosis is 10.2 years for patients with the associated extracellular domain mutations and 16.6 years for patients harboring intracellular domain mutations (2).

The strong genotype-phenotype correlation and the agerelated progression of MTC based on the type of RET mutation has enabled researchers to identify different classes of risk regarding MTC penetrance and aggressiveness. Thus, a ‘‘codon-directed’’ appropriate timing for surgery in RET mutation carriers has been defined. Patients with level 3 mutations (codons 883, 918 and 922) are at the highest risk of developing aggressive MTC, while patients with level 2 mutations (codons 611, 618, 620 and 634) are at intermediate risk and patients with level 1 mutations (codons 768, 790, 791, 804 and 891) are at the lowest risk (3).

Description of the Italian RET C609S pedigree The proband was a 36-year-old man. He had a 12-mm hypoechogenic, highly vascularized thyroid node of indeterminate cytology and a serum calcitonin (CT) level of 76 pg/ml. His family history was remarkable in terms of MTC and PHEO, but a previous genetic test on one of his affected relatives had failed to identify known any RET mutations.

Urinary metanephrines and adrenal computed tomography revealed no biochemical or radiological signs of PHEO; serum PTH, calcium and phosphorus levels were also within the normal ranges. The patient underwent a total thyroidectomy and central neck dissection, and the histological diagnosis was MTC without lymph node involvement (T1mN0Mx).

His genealogy included five already diagnosed cases of MTC. Two subjects had isolated MTC, three had lymphnode-positive MTC, and one had liver metastasis. Two out of five patients had PHEO (which was the first clinical sign of MEN2A syndrome in one case).

RET analysis, which was reconsidered in the proband, revealed a codon 609 mutation (TGC609TCC) that lead to a cysteine-to-serine substitution, Cys609Ser. This mutation was also confirmed in the members of his family already known to be affected. Another 24 family members underwent genetic testing for this RET mutation, revealing nine carriers (Figure 1). Two at-risk individuals refused genetic investigations.

Phenotypic characterization of the gene mutation carriers In short, clinical investigation revealed that none of the patients had a palpable thyroid node. None of the nodes were larger than 10 mm in diameter, presented with a suspected echographic pattern on ultrasound or had hypertension; one patient had two episodes of nephrolithiasis as the first clinical sign of the syndrome. Serum CT measurements revealed that the older subjects, one of whom was an 86-year-old woman, had the highest basal CT levels, while those of the younger subjects were low or unresponsive to pentagastrin. None of the patients had biochemical or radiological evidence of PHEO. All patients who underwent thyroid surgery demonstrated isolated MTC and/or C-cell hyperplasia.

The RET codon 609 mutation RET mutations in codon 609 are extremely rare genetic events in patients with MEN2A (,1% of all reported cases) and were initially considered as level 1 mutations (4).

However, after the publication of a family pedigree carrying the RET 609 cysteine-to-glycine substitution, in which a RET mutation carrier was diagnosed with MTC at 5 years of age (5), some authors shifted the RET codon 609 mutations from risk level 1 to risk level 2 (6).

One way to overcome the relatively poor information available regarding the genotype-phenotype correlation in

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RET C609S mutation Mian C et al.

Figure 1 - Pedigree analysis. The proband is indicated by the black arrow. Deceased patients are identified by diagonal lines. Subjects with signs of MEN2A syndrome are indicated by black shading, which covers half of the pedigree symbol for patients with MTC alone and three-quarters of the symbol for patients with MTC plus PHEO. All patients who underwent genetic testing are marked with an asterisk. After genotyping, gene mutation carriers are identified by the square shape of their respective pedigree symbols.

clinical consequences of primary HPT); the penetrance of PHEO and primary HPT was low. Furthermore, the paper

by Frank-Raue et al. (10) was a very large registry study on RET exon 10 mutations. In this study, the 45 carriers of RET codon 609 mutations, each with different cysteine substitutions (including 15 individuals belonging to the Italian C609S pedigree), demonstrated a less aggressive MTC but a 50% penetrance of PHEO (10).

In conclusion, the results obtained in this study of large genealogies may be very helpful for establishing a better genotype-phenotype correlation. In particular, the results we obtained by analyzing this very large MEN2 kindred suggest that carriers of the Cys609Ser RET mutation can be considered as risk level 1, for which non-aggressive clinical management may be indicated. Thus, as also reported by Frank-Raue et al. (10), in these cases, prophylactic total thyroidectomy may be postponed until after 5 years of age if careful yearly monitoring of stimulated calcitonin levels is implemented.

Phenotype-genotype correlations in RET 609 carriers and recommendations The 1999 consensus statement on MEN recommended that codon 609 RET mutations should be considered as risk level 1, for which there are no unequivocal clinical management guidelines. Some clinicians recommend a prophylactic total thyroidectomy by the age of 5 years, others by the age of 10 years, whereas others advocate surgery as soon as routine pentagastrin-stimulated test findings become abnormal (3).

Indeed, 609 RET mutations are quite rare genetic events at the onset of MEN2A and few affected families have been described in the literature (7,9,10,11,12). One family was comprised a 5-year-old boy who harbored a Cys609Gly substitution that is associated with an invasive MTC at final histology and warranting the inclusion of such mutations in the intermediate risk category (5,6). On the other hand, in vitro data obtained from transfected NIH3T3 cells seem to suggest that 609 mutations have a smaller capacity for neoplastic transformation than other level 2 RET mutations (13). Only one large family carrying a Cys609Ser mutation was reported by Kinlaw et al. and, regarding its clinical aspects, this genealogy revealed an unusual phenotype characterized by a scarcely penetrant, non-aggressive MTC (20%) and a tendency for cases to present with PHEO rather than MTC (7). We described a kindred with the same RET mutation. Based on the large number of affected subjects with this specific mutation in this family who had undergone total thyroidectomy, we agree that the Cys609Ser mutation carries a level 1 risk: we saw no MTC develop before the age of 17 years, no lymph node metastases up to 30 years of age and no distant metastases up to 60 years of age. There was also an 86-year-old mutation carrier who did not undergo surgery. Moreover, we saw no anticipation effect, which is by no means rare in MEN2A kindreds carrying level 1 mutations, leading to MTC onset at an earlier age in each successive generation (14).

MTC was highly penetrant in our family (75% of cases) and was the first sign of disease in all but two cases (one presented with PHEO and another presented with the

AUTHOR CONTRIBUTIONS Mian C has actively contributed to the clinical and molecular studies on patients and to the drafting of the article. Sartorato P has contributed to the review of the literature on the topic and to the drafting of the article.

Barollo S and Zane M have performed the genetic studies. Opocher G has coordinated the clinical and molecular studies conducted on patients and planned the drafting of the review.

REFERENCES 1. Pacini F, Castagna MG, Cipri C, Schlumberger M. Medullary thyroid carcinoma. Clin Oncol. 2010;22(6):475–85, http://dx.doi.org/10.1016/ j.clon.2010.05.002.

2. Machens A, Dralle H. Genotype-phenotype based surgical concept of hereditary medullary thyroid carcinoma. World J Surg. 2007;31(5):957– 68, http://dx.doi.org/10.1007/s00268-006-0769-y.

3. Brandi ML, Gagel RF, Angeli A, Bilezikian JP, Beck-Peccoz P, Bordi C, et al. Guidelines for diagnosis and therapy of MEN type 1 and type 2.

J Clin Endocrinol Metab. 2001;86(12):5658–71, http://dx.doi.org/ 10.1210/jc.86.12.5658.

4. Machens A, Dralle H. Familial prevalence and age of RET germline mutations: implications for screening. Clin Endocrinol. 2008;69(1):81–7, http://dx.doi.org/10.1111/j.1365-2265.2007.03153.x.

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5. Simon S, Pavel M, Hensen J, Berg J, Hu¨mmer HP, Carbon R. Multiple endocrine neoplasia 2A syndrome: surgical management. J Pediatr Surg.

2002;37(6):897–900, http://dx.doi.org/10.1053/jpsu.2002.32906.

6. Machens A, Ukkat J, Brauckhoff M, Gimm O, Dralle H. Advances in the management of hereditary medullary thyroid cancer. J Intern Med. 2005;257(1):50–9, http://dx.doi.org/10.1111/j.1365-2796.2004.01423.x.

7. Kinlaw WB, Scott SM, Maue RA, Memoli VA, Harris RD, Daniels GH, et al. Multiple endocrine neoplasia 2A due to a unique C609S RET mutation presents with pheochromocytoma and reduced penetrance of medullary thyroid carcinoma. Clin Endocrinol. 2005;63(6):676–82, http://dx.doi.org/10.1111/j.1365-2265.2005.02400.x.

8. Calva D, O’Dorisio TM, Sue O’Dorisio M, Lal G, Sugg S, Weigel RJ, et al.

When is prophylactic thyroidectomy indicated for patients with the RET codon 609 mutation? Ann Surg Oncol. 2009;16(8):2237–44, http:// dx.doi.org/10.1245/s10434-009-0524-3.

9. Mian C, Barollo S, Zambonin L, Pennelli G, Bernante P, Pelizzo MR, et al.

Characterization of the largest kindred with MEN2A due to a Cys609Ser RET mutation. Fam Cancer. 2009;8(4):379–82, http://dx.doi.org/10.1007/ s10689-009-9250-z.

10. Frank-Raue K, Rybicki LA, Erlic Z, Schweizer H, Winter A, Milos I, et al.

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estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10. Hum Mutat. 2011;32(1):51–8, http:// dx.doi.org/10.1002/humu.21385.

Marsh DJ, Andrew SD, Eng C, Learoyd DL, Capes AG, Pojer R, et al.

Germline and somatic mutations in an oncogene: RET mutations in inherited medullary thyroid carcinoma. Cancer Res. 1996;56(6):1241– 3.

Igaz P, Pato´cs A, Ra´cz K, Klein I, Va´radi A, Esik O. Occurrence of pheochromocytoma in a MEN2A family with codon 609 mutation of the RET proto-oncogene. J Clin Endocrinol Metab. 2002;87(6):2994, http:// dx.doi.org/10.1210/jc.87.6.2994-a.

Iwashita T, Kato M, Murakami H, Asai N, Ishiguro Y, Ito S, et al.

Biological and biochemical properties of Ret with kinase domain mutations identified in multiple endocrine neoplasia type 2B and familial medullary thyroid carcinoma. Oncogene. 1999;18(26):3919–22, http://dx.doi.org/10.1038/sj.onc.1202742.

Fattoruso O, Quadro L, Libroia A, Verga U, Lupoli G, Cascone E, et al. A GTG to ATG novel point mutation at codon 804 in exon 14 of the RET proto-oncogene in two families affected by familial medullary thyroid carcinoma. Hum Mutat. 1998;11(Suppl 1):S167–71, http://dx.doi.org/ 10.1002/humu.1380110156.

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DOI:10.6061/clinics/2012(Sup01)08

REVIEW

Clinical and genetic aspects of familial isolated pituitary adenomas Vladimir Vasilev,I,II Adrian Daly,I Luciana Naves,III Sabina Zacharieva,II Albert BeckersI I University of Lie`ge, Centre Hospitalier Universitaire de Lie`ge, Department of Endocrinology, Belgium. II Medical University, Sofia, Clinical Centre of Endocrinology and Gerontology, Bulgaria. III University of Brasilia, Internal Medicine, Department of Endocrinology, Brasilia/DF, Brazil.

Pituitary adenomas represent a group of functionally diverse neoplasms with relatively high prevalence in the general population. Most occur sporadically, but inherited genetic predisposing factors are increasingly recognized.

Familial isolated pituitary adenoma is a recently defined clinical entity, and is characterized by hereditary presentation of pituitary adenomas in the absence of clinical and genetic features of syndromic disease such as multiple endocrine neoplasia type 1 and Carney complex. Familial isolated pituitary adenoma is inherited in an autosomal dominant manner and accounted for approximately 2–3% of pituitary tumors in some series. Germline mutations in the aryl-hydrocarbon interacting protein gene are identified in around 25% of familial isolated pituitary adenoma kindreds. Pituitary adenomas with mutations of the aryl-hydrocarbon interacting protein gene are predominantly somatotropinomas and prolactinomas, but non-functioning adenomas, Cushing disease, and thyrotropinoma may also occur. These tumors may present as macroadenomas in young patients and are often relatively difficult to control. Furthermore, recent evidence indicates that aryl-hydrocarbon interacting protein gene mutations occur in .10% of patients with sporadic macroadenomas that occur before 30 years of age, and in .20% of children with macroadenomas. Genetic screening for aryl-hydrocarbon interacting protein gene mutations is warranted in selected high-risk patients who may benefit from early recognition and follow-up.

KEYWORDS: Familial Isolated Pituitary Adenomas; FIPA; AIP Gene; AIP Mutations.

Vasilev V, Daly A, Naves L, Zacharieva S, Beckers A. Clinical and genetic aspects of familial isolated pituitary adenomas. Clinics. 2012;67(S1):37-41.

E-mail: Albert.Beckers@chu.ulg.ac.be Tel.: 0032 4 3667084

and, rarely, mutations of classic oncogenes (4,5). The vast majority of pituitary adenomas, however, arise sporadically, and inherited germline mutations in different genes are few in number, accounting for approximately 5% of all pituitary tumors (6). Traditionally, familial pituitary adenomas have been associated with some multiple neoplasia syndromes, including multiple endocrine neoplasia type 1 (MEN 1), Carney complex, and the newly defined multiple endocrine neoplasia type 4. By the end of the 20th century, however, only occasional cases of non-syndromic familial pituitary tumors were reported, mostly acromegaly (7). The first single-center study to specifically scout for cases of familial pituitary adenomas unrelated to MEN 1 and Carney complex was performed in Lie`ge, Belgium in the 1990s, and led to the identification of an initial cohort of 27 patients (8). Reports from the same center confirmed the condition as a new clinical entity, and the term familial isolated pituitary adenomas (FIPA) was adopted (9–11). Its definition expanded the search internationally, and by 2011 more than 200 affected families had been reported (12,13). FIPA is currently considered to account for around 2–3% of pituitary adenomas (14).

INTRODUCTION Pituitary adenomas are one of the most common intracranial neoplasms. Questions regarding their prevalence in the general population were recently addressed by a large cross-sectional study in the province of Lie`ge, Belgium, which revealed 94 cases of pituitary adenomas per 100,000 inhabitants (1). Similar findings were later confirmed in Banbury, UK (2). Despite being generally benign, pituitary adenomas still exert significant influence as they combine symptoms of hormonal dysfunction with signs of local compression, and may require complex and costly management and long-term follow-up. Pituitary adenoma formation is generally considered to be the result of the clonal expansion of a single mutated cell (3) and molecular studies have identified a number of genetic and epigenetic abnormalities that may have a possible causative or facilitatory role in pituitary tumorigenesis. These include somatic mutations in the gsp oncogene, overexpression of the pituitary tumor transforming gene (PTTG), disruptions in cell cycle regulation and intracellular signaling pathways

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

CLINICAL FEATURES OF PATIENTS WITH FIPA The syndrome of FIPA is defined as familial presentation of any type of pituitary adenoma in the absence of clinical and genetic evidence for MEN 1 and Carney complex

No potential conflict of interest was reported

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(9,11,14,15). Following the initial description of the condition, the clinical characteristics of a large international cohort of 64 families comprising more than 140 patients from 22 tertiary referral centers were reported in 2006 (14).

Genealogical information suggested that FIPA is inherited in an autosomal dominant pattern with variable penetrance.

Based on the tumor phenotype in the individual families, FIPA can be divided into two almost equal subgroups: homogeneous, when all affected family members experience the same adenoma type, and heterogeneous, with different pituitary tumors within the family. Prolactinomas and somatotropinomas comprise more than 70% of all tumors, and although in heterogeneous FIPA all types of tumors can be seen, there is at least one prolactin- or growth hormonesecreting adenoma in almost all affected families. Females tend to be more frequently affected (62%), which is not unexpected given the fact that prolactinomas are the most common phenotype overall. Prolactin-secreting adenomas comprise 40% of all FIPA tumors, and their characteristics principally match their sporadic counterparts in terms of sexual predisposition, age at presentation, and proportion of microadenomas. In heterogeneous FIPA families, however, these tumors exhibit more aggressive behavior, with significantly higher rates of suprasellar expansion and cavernous sinus invasion compared with sporadic prolactinomas. Growth hormone-secreting adenomas account for 30% of FIPA tumors, and somatoprolactinomas are responsible for another 7%. They are equally distributed between homogeneous and heterogeneous families but, unlike FIPA prolactinomas, somatotropinomas are more aggressive when occurring in a homogeneous setting. In homogeneous FIPA, acromegaly is usually diagnosed 10 years earlier, with tumors more frequently displaying extrasellar growth, compared with heterogeneous relatives and sporadic populations (14). Acromegaly in patients with FIPA also appears to respond poorly to somatostatin analog therapy (16). Non-secreting adenomas, predominantly associated with heterogeneous families, arise in 13% of patients with FIPA and are also characterized by more aggressive evolution, being diagnosed earlier and exhibiting more invasive properties than sporadic adenomas. Gonadotropinomas, corticotropinomas, and thyrotropinomas are rare, and account for 4%, 4%, and 1% of FIPA tumors, respectively. They are usually associated with other adenoma types in heterogeneous families, although individual families with homogeneous presentation have been reported (14). The descendants in FIPA families with multiple affected generations are diagnosed considerably earlier than their parents/grandparents.

mechanisms leading to pituitary tumorigenesis are not known. Homozygous AIP–/– knockout mice die in the early embryonic period as a result of severe cardiovascular abnormalities, suggesting that AIP may play a role in cardiovascular development (21). Heterozygous AIP+/– animals, however, develop a phenotype that is very similar to human pituitary disease with the majority of the mice presenting with aggressive somatotropinomas (22). The AIP gene consists of six exons and codes for a 330-amino-acid protein, the sequence of which is highly conserved between different species. It shares a structural homology with immunophilin proteins because of the presence of a peptidyl-prolyl cis-trans isomerase-like domain, but does not function as such (23,24). Instead, AIP takes part in numerous protein–protein interactions, mediated through its C-terminal, which houses three tetratricopeptide repeats and a final a-helix. Among the first identified partners of AIP is the aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor that modulates cellular responses to various xenobiotic toxins, such as dioxins, as well as some endogenous compounds such as cAMP (25). In the absence of ligands, the AhR binds to two molecules of the 90-kDa heat-shock protein, acting as chaperone, and to AIP and p23 proteins, acting as co-chaperones, to form a multiprotein complex in the cytoplasm (26). The activation of this complex by its xenobiotic ligand results in nuclear translocation, where AhR binds to the aryl hydrocarbon receptor nuclear translocator and promotes the transcription of specific genes coding various drug metabolizing enzymes as well as other proteins (24). The effect of AIP on the functional status of AhR is still a matter of debate because conflicting results have been reported, but it seems that it maintains the stability of the complex by protecting AhR from ubiquitin-dependent degradation (27). Reduced AIP levels in AIP-mutated pituitary adenomas are associated with a lack of nuclear AhR immunostaining, suggesting that down-regulation of AhR may be involved in pituitary tumorigenesis (28). Consistent with this finding, overexpression of wild-type AIP in pituitary and hepatic cell cultures slows down cell proliferation (16). AIP is also thought to interact with two subtypes of phosophodiesterases: PDE4A5 and PDE2A (29,30). These enzymes participate in the regulation of numerous signaling cascades that use cAMP as a second messenger, including the growth hormone-releasing hormone receptor pathway in pituitary cells. Disruptions in signal transduction, which lead to abnormally high cAMP concentrations, are associated with pituitary hyperplasia and adenoma formation in some conditions, such as Carney complex and McCune–Albright syndrome (31). AIP binding to PDE4A5 reduces its catalytic activity, and it is not clear if this interaction plays a role in pituitary tumorigenesis, as loss of AIP would presumably result in low cAMP levels. The interaction with PDE2A does not alter the enzyme activity, and the local reduction of cAMP may impede the nuclear translocation of the AhR complex (30). Recently, AIP was shown to interact with the tyrosine kinase receptor, encoded by the RET proto-oncogene, and the inhibitor of apoptosis, survivin, and therefore to have a potential role in cell cycle regulation. Binding to survivin maintains its stability and promotes cell survival by elevating the antiapoptotic threshold. On the other hand, the interaction with RET prevents the formation of the AIP–survivin

MOLECULAR GENETICS OF FIPA The elucidation of the responsible genetic causes of FIPA started with the identification of loss of heterozygosity in locus 11q13 in relatives with familial acromegaly who lacked mutations in the MEN1 gene (17,18).

Separate research for potential genes in a Finnish cohort of patients with familial pituitary tumors revealed inactivating mutations in the gene for aryl-hydrocarbon receptor interacting protein (AIP) (19). The causative role of AIP in FIPA was confirmed with the discovery of several new germline mutations in a large series of 73 families (20).

Loss of heterozygosity in tumor tissues suggests a tumor suppressor function for AIP, but the exact molecular

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complex, resulting in subsequent survivin degradation and increase in apoptosis (32). These effects, however, are contrary to the proposed tumor-suppressor role of AIP. Apart from stabilizing the AhR complex, AIP may also bind to a set of nuclear receptors including peroxisome proliferator-activated receptor a, the glucocorticoid receptor, and b-thyroid hormone receptor 1. Furthermore, a role has been proposed for AIP in virus-induced tumorigenesis as a potential partner of hepatitis B virus X antigen and Epstein–Barr virus-encoded nuclear antigen 3 (24).

Over 50 different mutations in the AIP sequence have been identified in FIPA families from all over the world, and these mutations are spread through the entire length of the gene (23,33). Most of them affect the C-terminal end and the tetratricopeptide repeat motifs supporting their essential role in AIP function. Nonsense and frameshift mutations lead to premature stop codons with a resultant truncated protein, whereas missense mutations tend to affect the tetratricopeptide repeat domains and the terminal a-helix. Whole gene deletions have also been identified, suggesting the use of a multiple ligation-dependent probe amplification method for patients with FIPA in whom sequencing fails to identify abnormalities (34,35,36).

Mutations in codons R304, R271, and R81 have been reported in independent families with FIPA, indicating possible hotspots. No genotype–phenotype correlations have been observed to date in patients with AIP-mutated FIPA (37).

Mutations in the AIP gene, however, are found in only approximately 25% of all patients with FIPA, and in 40–50% of patients in the subgroup with acromegaly from homogeneous FIPA families (12). The genetic cause for the rest of the cases is still unknown, but several other loci, such as 2p16, 3q28, 4q32, 8q12, 19q13, and 21q22, may be involved in the development of the syndrome although no particular genes have been identified (38). On the other hand, the penetrance of AIP mutations is estimated to be approximately 30% in the largest reported families (15,19,35,39), suggesting the possible existence of genetic or environmental modifying factors (Figure 1).

CLINICAL CHARACTERISTICS OF PATIENTS WITH AIP-MUTATED FIPA Patients with AIP-related pituitary adenomas have also been shown to exhibit some specific clinical features that differentiate them from patients with wild-type AIP alleles (37). In contrast to the overall female predominance in FIPA, male sex is significantly more common in the subgroup of patients harboring AIP mutations. All types of pituitary tumors may occur in association with mutated AIP, but growth hormone-secreting adenomas largely predominate, arising in about 80% of patients, and co-secretion of prolactin is observed in more than 50% of these. A direct comparison of 75 patients with AIP-mutated somatotropinomas with 232 genetically negative control subjects with acromegaly revealed that AIP anomalies are associated with much earlier onset and more aggressive evolution of the disease (Table 1). Invasive macroadenomas are manifested in childhood or adolescence in more than half of patients with AIP mutations, and almost a third of patients with somatotropinomas present with gigantism. Disease control is also harder to achieve and maintain, because somatostatin analogs are less effective for lowering growth hormone and insulin-like growth factor levels and inducing tumor shrinkage in acromegaly caused by AIP mutations.

Moreover, these patients have significantly worse long-term therapeutic control although they frequently undergo multiple surgeries and radiotherapy. Patients with AIPmutated prolactinomas also present with large tumor size and invasive features. Resistance to dopamine agonists may be observed in 50% of these, raising the need for surgery and/or radiotherapy.

CLINICAL IMPLICATIONS AND MANAGEMENT Similar to the other familial presentations of pituitary tumors in MEN 1 and Carney complex, the treatment of FIPA does not differ substantially from the management of sporadic adenomas in terms of indications and therapeutic modalities. However, the aggressive nature of FIPA tumors, especially in patients with AIP mutations requires increased attention from medical specialists. Detailed physical

Figure 1 - Genealogical tree of a familial isolated pituitary adenoma (FIPA) family with E174 frameshift aryl-hydrocarbon receptor interacting protein gene (AIP) mutation. Filled black symbols indicate mutation-positive patients with pituitary tumors. (+) symbols show patients with AIP mutation without clinical, hormonal or radiological evidence for pituitary pathology. (-) is used for patients with wild-type AIP. Subjects marked with (?) did not undergo genetic analysis but had no clinical signs of pituitary disease. Subjects with AIP mutation and elevated insulin-like growth factor-1 levels are shown by a symbol with a filled upper right corner, and an AIPmutation positive girl with premature telarche, ovarian enlargement and advanced bone age is indicated by a symbol with a filled lower left corner. Adapted by the author Naves LA (39).

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AUTHOR CONTRIBUTIONS

Table 1 - Comparison of some demographic and clinical features of acromegaly patients with and without AIP mutations.

AIP mutated group

Control group

75 61.3% 22

232 46.5% 43

90.1% 65.1% 51.7% 32%

80.8% 49.8% 38.8% 6.5%

Number of patients Males* Age at diagnosis, years* Macroadenomas* Extrasellar expansion Invasion* Gigantism*

All the authors participated in data analysis. Vasilev V, Daly A and Beckers A were responsible for the manuscript writing. All the authors approved the final version of the manuscript.

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10. Valdes Socin H, Jaffrain Re´a M, Tamburrano G, Cavagnini F, Cicarelli E, Colao A, et al. Familial isolated pituitary adenomas: clinical and molecular studies in 80 patients. The Endocrine Society’s 84th Annual Meeting; 2002 19-22 june; San Francisco.

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14. Daly AF, Jaffrain-Rea ML, Ciccarelli A, Valdes-Socin H, Rohmer V, Tamburrano G, et al. Clinical characterization of familial isolated pituitary adenomas. J Clin Endocrinol Metab. 2006;91(9):3316-23, http://dx.doi.org/10.1210/jc.2005-2671.

15. Beckers A, Daly AF. The clinical, pathological, and genetic features of familial isolated pituitary adenomas. Eur J Endocrinol. 2007;157(4):37182, http://dx.doi.org/10.1530/EJE-07-0348.

16. Leontiou CA, Gueorguiev M, van der Spuy J, Quinton R, Lolli F, Hassan S, et al. The role of the aryl hydrocarbon receptor-interacting protein gene in familial and sporadic pituitary adenomas. J Clin Endocrinol Metab.

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17. Yamada S, Yoshimoto K, Sano T, Takada K, Itakura M, Usui M, et al.

Inactivation of the tumor suppressor gene on 11q13 in brothers with familial acrogigantism without multiple endocrine neoplasia type 1.

J Clin Endocrinol Metab. 1997;82(1):239-42, http://dx.doi.org/10.1210/ jc.82.1.239.

18. Gadelha MR, Prezant TR, Une KN, Glick RP, Moskal SF, 2nd, Vaisman M, et al. Loss of heterozygosity on chromosome 11q13 in two families with acromegaly/gigantism is independent of mutations of the multiple endocrine neoplasia type I gene. J Clin Endocrinol Metab. 1999;84(1):24956, http://dx.doi.org/10.1210/jc.84.1.249.

19. Vierimaa O, Georgitsi M, Lehtonen R, Vahteristo P, Kokko A, Raitila A, et al. Pituitary adenoma predisposition caused by germline mutations in the AIP gene. Science. 2006;312(5777):1228-30, http://dx.doi.org/ 10.1126/science.1126100.

20. Daly AF, Vanbellinghen JF, Khoo SK, Jaffrain-Rea ML, Naves LA, Guitelman MA, et al. Aryl hydrocarbon receptor-interacting protein gene mutations in familial isolated pituitary adenomas: analysis in 73 families.

J Clin Endocrinol Metab. 2007;92(5):1891-6, http://dx.doi.org/10.1210/ jc.2006-2513.

21. Lin BC, Sullivan R, Lee Y, Moran S, Glover E, Bradfield CA. Deletion of the aryl hydrocarbon receptor-associated protein 9 leads to cardiac

Significant difference is marked with an *. Adapted from Daly et al. (37).

AIP: aryl-hydrocarbon receptor interacting protein gene.

examination should be performed for exclusion of extrapituitary pathology that may imply syndromic disease, and a comprehensive family history taken before referring patients to genetic screening. Genetic testing in relatives of patients with AIP mutations can be especially beneficial in terms of early diagnosis, which may yield better outcomes from treatment. Although no consensus protocols for management of patients with AIP-mutated FIPA currently exist, it may be appropriate to start regular magnetic resonance imaging monitoring and hormonal evaluation from early childhood because macroadenomas and gigantism have been diagnosed in patients as young as 6–8 years (34,36). Widespread screening for AIP mutations among apparently sporadic pituitary adenomas may not be warranted, as the prevalence of AIP alterations is low in such populations. However, AIP mutations are discovered in approximately 12% of young patients (aged ,30 years) and in 20% of pediatric patients, most often presenting with growth hormone-secreting or prolactin-secreting macroadenomas, suggesting that focused screening in this patient group may provide valuable clinical information (33,40).

The definition of FIPA has further widened the spectrum of familial pituitary pathology in addition to the wellknown MEN 1 and Carney complex. The identification of the AIP gene as a causative factor in a subset of patients with FIPA has also provided some new insights into pituitary tumorigenesis. Genetic testing can now be offered to at-risk subjects in affected families, allowing for earlier diagnosis and more successful treatment. Consensus guidelines concerning the management and follow-up of patients with FIPA will hopefully be developed with the accumulation of data from large international cohorts and long-term monitoring studies. However, much remains to be done, as the low prevalence and the uncertain penetrance of AIP mutations suggests that other predisposing or modifying genes are to be expected. It is also unclear whether patients with FIPA additionally have a predisposition for other endocrine or non-endocrine tumors, which could expand its definition beyond the pituitary. Further studies will help to clarify these issues and provide more information on the genetic and molecular basis for the development of pituitary adenomas.

ACKNOWLEDGMENTS The authors would like to thank the assistance of Prof. Albert Beckers from the FIRS of the Centre Hospitalier Universitaire de Lie`ge and from Pfizer.

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Raitila A, Lehtonen HJ, Arola J, Heliovaara E, Ahlsten M, Georgitsi M, et al. Mice with inactivation of aryl hydrocarbon receptor-interacting protein (Aip) display complete penetrance of pituitary adenomas with aberrant ARNT expression. Am J Pathol. 2010;177(4):1969-76, http:// dx.doi.org/10.2353/ajpath.2010.100138.

Ozfirat Z, Korbonits M. AIP gene and familial isolated pituitary adenomas. Mol Cell Endocrinol. 2010;326(1-2):71-9, http://dx.doi.org/ 10.1016/j.mce.2010.05.001.

Trivellin G, Korbonits M. AIP and its interacting partners. J Endocrinol.

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Oesch-Bartlomowicz B, Huelster A, Wiss O, Antoniou-Lipfert P, Dietrich C, Arand M, et al. Aryl hydrocarbon receptor activation by cAMP vs. dioxin: divergent signaling pathways. Proc Natl Acad Sci U S A. 2005;102(26):921823, http://dx.doi.org/10.1073/pnas.0503488102.

Kazlauskas A, Poellinger L, Pongratz I. Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (Aryl hydrocarbon) receptor.

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274.19.13519.

Kazlauskas A, Poellinger L, Pongratz I. The immunophilin-like protein XAP2 regulates ubiquitination and subcellular localization of the dioxin receptor. J Biol Chem. 2000;275(52):41317-24, http://dx.doi.org/10.1074/ jbc.M007765200.

Jaffrain-Rea ML, Angelini M, Gargano D, Tichomirowa MA, Daly AF, Vanbellinghen JF, et al. Expression of aryl hydrocarbon receptor (AHR) and AHR-interacting protein in pituitary adenomas: pathological and clinical implications. Endocr Relat Cancer. 2009;16(3):1029-43, http:// dx.doi.org/10.1677/ERC-09-0094.

Bolger GB, Peden AH, Steele MR, MacKenzie C, McEwan DG, Wallace DA, et al. Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2. J Biol Chem.

2003;278(35):33351-63, http://dx.doi.org/10.1074/jbc.M303269200. de Oliveira SK, Hoffmeister M, Gambaryan S, Muller-Esterl W, Guimaraes JA, Smolenski AP. Phosphodiesterase 2A forms a complex with the co-chaperone XAP2 and regulates nuclear translocation of the aryl hydrocarbon receptor. J Biol Chem. 2007;282(18):13656-63, http:// dx.doi.org/10.1074/jbc.M610942200.

Horvath A, Stratakis CA. Clinical and molecular genetics of acromegaly: MEN1, Carney complex, McCune-Albright syndrome, familial acromegaly and genetic defects in sporadic tumors. Rev Endocr Metab Disord.

2008;9(1):1-11, http://dx.doi.org/10.1007/s11154-007-9066-9.

32. Vargiolu M, Fusco D, Kurelac I, Dirnberger D, Baumeister R, Morra I, et al. The tyrosine kinase receptor RET interacts in vivo with aryl hydrocarbon receptor-interacting protein to alter survivin availability.

J Clin Endocrinol Metab. 2009;94(7):2571-8, http://dx.doi.org/10.1210/ jc.2008-1980.

33. Jaffrain-Rea M-L, Daly AF, Angelini M, Petrossians P, Bours V, Beckers A. Genetic susceptibility in pituitary adenomas: from pathogenesis to clinical implications. Exp Rev Endocrinol Metab. 2011;6(2):195-214, http:// dx.doi.org/10.1586/eem.10.87.

34. Barlier A, Vanbellinghen JF, Daly AF, Silvy M, Jaffrain-Rea ML, Trouillas J, et al. Mutations in the aryl hydrocarbon receptor interacting protein gene are not highly prevalent among subjects with sporadic pituitary adenomas. J Clin Endocrinol Metab. 2007;92(5):1952-5, http:// dx.doi.org/10.1210/jc.2006-2702.

35. Igreja S, Chahal HS, King P, Bolger GB, Srirangalingam U, Guasti L, et al.

Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families. Hum Mutat.

2010;31(8):950-60, http://dx.doi.org/10.1002/humu.21292.

36. Georgitsi M, Heliovaara E, Paschke R, Kumar AV, Tischkowitz M, Vierimaa O, et al. Large genomic deletions in AIP in pituitary adenoma predisposition. J Clin Endocrinol Metab. 2008;93(10):4146-51, http:// dx.doi.org/10.1210/jc.2008-1003.

37. Daly AF, Tichomirowa MA, Petrossians P, Heliovaara E, Jaffrain-Rea ML, Barlier A, et al. Clinical characteristics and therapeutic responses in patients with germ-line AIP mutations and pituitary adenomas: an international collaborative study. J Clin Endocrinol Metab. 2010;95(11): E373-83, http://dx.doi.org/10.1210/jc.2009-2556.

38. Toledo RA, Lourenco DM Jr., Toledo SP. Familial isolated pituitary adenoma: evidence for genetic heterogeneity. Front Horm Res. 2010;38:77-86, http://dx.doi.org/10.1159/000318497.

39. Naves LA, Daly AF, Vanbellinghen JF, Casulari LA, Spilioti C, Magalhaes AV, et al. Variable pathological and clinical features of a large Brazilian family harboring a mutation in the aryl hydrocarbon receptor-interacting protein gene. Eur J Endocrinol. 2007;157(4):383-91, http://dx.doi.org/10.1530/EJE-07-0533.

40. Tichomirowa MA, Bariler A, Daly AF, Jaffrain-Rea M-L, Ronchi CL, Yaneva M, et al. High prevalence of AIP gene mutations following focused screening in young patients with sporadic pituitary macroadenomas. Eur J Endocrinol. 2011;165(4):509-15, http://dx.doi.org/ 10.1530/EJE-11-0304.

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DOI:10.6061/clinics/2012(Sup01)09

REVIEW

Pituitary tumors in patients with MEN1 syndrome Luis V. Syro,I Bernd W. Scheithauer (in memoriam),II Kalman Kovacs,III Rodrigo A. Toledo,IV Francisco J. London˜o,V Leon D. Ortiz,VI Fabio Rotondo,III Eva Horvath,III Humberto UribeVII I Department of Neurosurgery, Hospital Pablo Tobon Uribe and Clinica Medellin, Medellin, Colombia. II Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA. III Department of Laboratory Medicine, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada. IV Endocrine Genetics Unit (LIM25), Endocrinology, Hospital das Clı´nicas, University of Sao Paulo School of Medicine, Sao Paulo/SP, Brazil.

V Department of Neurosurgery, Universidad de Antioquia, Hospital San Vicente de Paul, Medellin, Colombia. VI Division of Neuro-oncology, Instituto de Cancerologia, Clinica Las Americas, Medellin, Colombia. VII Department of Neurosurgery, Clinica SOMA, Medellin, Colombia.

We briefly review the characteristics of pituitary tumors associated with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 is an autosomal-dominant disorder most commonly characterized by tumors of the pituitary, parathyroid, endocrine-gastrointestinal tract, and pancreas. A MEDLINE search for all available publications regarding multiple endocrine neoplasia type 1 and pituitary adenomas was undertaken. The prevalence of pituitary tumors in multiple endocrine neoplasia type 1 may vary from 10% to 60% depending on the studied series, and such tumors may occur as the first clinical manifestation of multiple endocrine neoplasia type 1 in 25% of sporadic and 10% of familial cases. Patients were younger and the time between initial and subsequent multiple endocrine neoplasia type 1 endocrine lesions was significantly longer when pituitary disease was the initial manifestation of multiple endocrine neoplasia type 1. Tumors were larger and more invasive and clinical manifestations related to the size of the pituitary adenoma were significantly more frequent in patients with multiple endocrine neoplasia type 1 than in subjects with non-multiple endocrine neoplasia type 1. Normalization of pituitary hypersecretion was much less frequent in patients with multiple endocrine neoplasia type 1 than in subjects with non-multiple endocrine neoplasia type 1. Pituitary tumors in patients with multiple endocrine neoplasia type 1 syndrome tend to be larger, invasive and more symptomatic, and they tend to occur in younger patients when they are the initial presentation of multiple endocrine neoplasia type 1.

KEYWORDS: Pituitary Neoplasms; Multiple Endocrine Neoplasia Type 1; Review; Genetics.

Syro LV, Scheithauer BW, Kovacs K, Toledo RA, London˜o FJ, Ortiz LD, et al. Pituitary tumors in patients with MEN1 syndrome. Clinics. 2012;67(S1):43-48.

E-mail: lvsyro@une.net.co Tel.: 57 4 231 9099

published the first review of MEN1 syndrome in which they described 14 cases in the literature and eight cases from the Mayo Clinic (8). In 1954, Wermer et al. suggested that this syndrome followed an autosomal-dominant inheritance pattern with high penetrance (9). The name MEN1 syndrome has replaced the original name, Wermer’s syndrome. The MEN1 clinical phenotype was fully characterized in the 1960s. In 1988, the MEN1 locus was mapped to chromosome 11q13 using recombinant DNA probes in two brothers with MEN1 syndrome (10). Linkage analysis in MEN1 families confirmed the 11q13 locus. In 1997, the MEN1 gene was identified by positional cloning, and mutations causing MEN1 syndrome were confirmed (11,12).

INTRODUCTION Multiple endocrine neoplasia type 1 (MEN1, OMIM #131100) is an autosomal-dominant disorder mostly characterized by tumors of the pituitary, parathyroid, endocrinegastrointestinal tract, and pancreas (1). Adrenocortical and thyroid tumors, cutaneous lipomas, meningiomas, facial angiofibromas and gastric, thymic or bronchial carcinoid can also occur (2–5). The phenotype of MEN1 may vary widely, and this neoplasia should be suspected in patients with an endocrinopathy of two of the three main affected glands or with one endocrinopathy of one of these organs plus a first-degree relative who is affected by MEN1 syndrome (6).

At the beginning of the past century, autopsy findings of a patient with acromegaly and four enlarged parathyroid glands were reported (7). In 1953, Underdahl et al.

CLINICAL FEATURES MEN1 is characterized by a combination of tumors of the anterior pituitary, parathyroid, gastrointestinal tract, and pancreas. Some patients may also develop adrenocortical tumors, carcinoid tumors, facial angiofibromas, collagenomas, and lipomas. Tumors of the parathyroid are the first manifestation of MEN1 in more than 85% of patients. In the remaining patients, the first manifestation may be an insulinoma or prolactinoma. Gastrointestinal and pancreatic

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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tumors are gastrinomas, insulinomas, pancreatic polypeptidomas (PPomas), glucagonomas, and vaso-active intestinal polypeptidomas (VIPomas).

The most frequent MEN1-associated endocrinopathy, which occurs in 90% of individuals with MEN1 between the ages of 20 and 25 years, is primary hyperparathyroidism, and it manifests as hypercalcemia in 100% of affected individuals by 50 years of age. It is a multi-glandular disorder with enlargement of all parathyroid glands asynchronously and asymmetrically (7). Thus, increased serum calcium and parathyroid hormone levels, renal calculi, and bone demineralization should be actively searched for in suspected cases (13).

MEN1 patients usually have a family history of MEN1, and MEN1 gene mutations can be identified in 70% to 95% of cases (1). Several endocrine tumors in MEN1 are benign and cause symptoms by hypersecretion of hormones or local mass effects, while other MEN1-related tumors are associated with a very high risk of malignancy as gastrinomas and carcinoid tumors (1,4). About one-third of patients affected with MEN1 will die early from an MEN1-related cancer or metabolic disturbance (14–16).

Consequently, the average age of death in individuals with MEN1 is significantly lower than that in the general population. MEN1 syndrome occurs in approximately 1 in 30,000 individuals, with an equal sex distribution, and there is no ethnic or racial predilection (14).

heterozygosity (LOH), and this has been taken as evidence that the disease follows Knudson’s two-hit model of tumorigenesis (27). The first hit is a heterozygous MEN1 germline mutation inherited from one parent (familial cases) or developed in an early embryonic stage (sporadic cases) and is present in all cells at birth. The second hit is an MEN1 somatic mutation that occurs in the predisposed endocrine cell, giving the survival advantage needed for tumor development (28).

MEN1 syndrome has high penetrance. Half of patients develop signs and symptoms by 20 years of age, and more than 95% will have symptoms by 40 years of age (29). There is significant variability in the age of onset, severity of disease, and tumor types. Despite numerous studies, no genotype-phenotype relation has been established, and no correlations have been detected between MEN1 mutations and clinical manifestations (1).

Ten percent of patients with the MEN1 syndrome may not harbor mutations in the coding region of the MEN1 gene, and whether these individuals have whole gene deletions or mutations in the promoter or untranslated region remains to be investigated. One study showed that 33% of patients who do not have mutations within the coding region have large deletions involving complete exons not easily detected by DNA sequence analysis (30).

MEN1 families without inactivation of the MEN1 gene may also present mutations in other genes. Pellegata et al. first reported a CDKN1B-inactivating germline mutation in a family with an MEN1-like phenotype (MEN4 syndrome) involving cases with acromegaly, primary hyperparathyroidism, renal angiomyolipoma, and testicular cancer (31). A second CDKN1B germline mutation was described in an apparently ‘‘sporadic’’ MEN1 patient with adrenocorticotropic hormone secreting pituitary adenoma (Cushing’s disease) (32). Recently, other CDKN1B mutations were identified in a very large cohort of patients with MEN1related states in whom no detectable MEN1 mutation was found (33). Of note, none of these CDKN1B-mutationpositive patients had developed pituitary adenomas. These data suggested that the tumoral susceptibility caused by CDKN1B inactivation needs to be further evaluated and that the association of CDKN1B mutations with pituitary disease may be less common than initially thought. Agarwal et al. demonstrated that patients with MEN1 states may rarely harbor germline mutations in other genes encoding cyclindependent kinase inhibitors (CDKIs), such as CDKN2B/ p15INK4b, CDKN2C/p18-INK4C, and CDKN1A/p21 CIP1 (33). A mutation in the CDKN1A/p21CIP1 gene was associated with pituitary tumors (macroprolactinomas) in two relatives. In conclusion, our knowledge about the role of the CDKI genes in the susceptibility to familial pituitary adenomas is still limited owing to the small number of patients with mutations reported so far (31–33).

MOLECULAR AND GENETIC CHARACTERISTICS MEN1 gene is a tumor suppressor gene localized on chromosome 11q13. It contains 10 exons spanning about 10 kb and encodes menin, a 610-amino-acid protein. The first exon and part of exon 10 are not translated. A major transcript of 2.8 kb has been described in a wide variety of human tissues such as the pancreas, thymus, adrenals, thyroid, testis, leukocytes, heart, brain, lung, muscle, small intestine, liver, and kidney (7).

Menin is a nuclear protein with a wide variety of molecular interactions with proteins that are involved in transcriptional regulation, genome stability, proliferation, and cell division (17,18). It acts as a scaffold to regulate gene transcription by coordinating chromatin remodeling and is an essential component of the histone methyltransferase complex. The proposed mechanism by which MEN1 mutations lead to tumor formation is by disruption of the interactions between menin and other proteins, thus altering critical events in cell cycle regulation and proliferation (19,20).

Over 1,300 mutations have been reported, scattered in and around the open reading frame without significant clustering, predominantly in coding exons but also within intronic sequences (21,22). The majority of the mutations result in premature stop codon generation (.70%). Most of the mutations are unique, and only a few are reported in more than 2% of cases. About 45% of the mutations are deletions, 25% are nonsense mutations, 15% are insertions, 10% are missense mutations, and less than 5% are splice-site mutations (22). MEN1-founding mutations have been described in Europe (France, Finland, and Sweden), North America (United States, Canada), South America (Brazil), and Oceania (Australia) (7,23–26).

The MEN1 mutations inactivate the gene and are consistent with those expected for a tumor suppressor (22). More than 90% of tumors from MEN1 patients have loss of

PITUITARY TUMORS IN MEN1 Prevalence The prevalence of pituitary tumors may vary widely, from 10% to 76% of MEN1 cases, depending on the studied series (26,29,34). Of note, the prevalence of pituitary tumors in MEN1 in several large series consisting of small families affected by MEN1 (less than five affected cases per family) differs markedly from the few very large MEN1 genealogies (.20 affected cases per family) so far reported (26). MEN1

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Pituitary tumors and MEN1 syndrome Syro LV et al.

cases in the latter families are usually caused by a founding MEN1 gene, and some of them may present very high frequencies of pituitary tumors, such as those reported in Newfoundland and Brazil (26,35).

Morphologic characteristics In a study by Trouillas et al. of 211 MEN1 patients, 77 surgically removed pituitary adenomas were described and compared with 2,509 unselected non-MEN1 sporadic pituitary tumors (40). These authors also compared a control subgroup of 296 cases from the latter group in which nonMEN1 cases were matched with MEN1 cases at the time of surgery and the immunoprofile of the tumor. The results showed that patients with MEN1 were younger and had larger tumors. The tumors of patients with MEN1 syndrome were more often invasive. The types of lesions were significantly different in MEN1 and non-MEN1 patients owing to the higher frequency of multiple adenomas (4% vs.

0.1%) and hyperplasia (4% vs. 0%) in the MEN1 patients. The frequency of functional pituitary adenomas was identical in the two groups (72% MEN1 vs. 64% non-MEN1), but the proportion of plurihormonal adenomas was significantly higher in the MEN1 group. Regarding immunoexpression, they did not find predominance of any functional subtype. It is important to note that in patients with pituitary hyperplasia, tumors producing a releasing hormone such as growthhormone-releasing hormone must be investigated. Adenoma multiplicity may underlie surgical failure in cases in which one adenoma is removed and the other is left behind (41).

General characteristics Amongst 1,500 pituitary adenomas surgically resected at the Mayo Clinic, 41 (2.7%) occurred in the setting of MEN1 (36). In patients with MEN1, the frequency of functional adenomas, mainly prolactin and growth hormoneproducing tumors, was higher than that of sporadic adenomas. There was, however, no difference in age, gender, tumor size, or invasiveness.

In 1980, Farid et al. described a syndrome of prolactinoma, carcinoid, and hyperparathyroidism in four large families from Newfoundland (37). This variant is now referred to as MEN1-Burin, for the region the affected patients were from (the Burin Peninsula). These patients presented frequently with prolactinoma and carcinoid. A nonsense mutation of the MEN1 gene, which was responsible for the disease in these patients, was found later (35).

Burgess et al. described another prolactinoma variant, MEN1-Tasman (34). In this case, prolactinomas occurred in 76% and nonfunctional adenomas in 24% of patients.

Recently, a founding MEN1 gene mutation was detected in a large MEN1 Brazilian family, and a high frequency of pituitary tumors was verified (26).

In a France–Belgium multicenter study, 324 MEN1 patients were analyzed; 136 MEN1 patients with a pituitary adenoma were compared to 110 control patients with sporadic pituitary adenomas. No significant differences were detected in the types of adenomas, but the mean age of patients with pituitary disease as the initial manifestation of MEN1 was significantly lower (33.9¡14 years) than that of patients showing a primary endocrine, intestinal, or pancreatic tumor (41.6¡14 years). The study also showed that the delay between initial and subsequent MEN1 endocrine lesions was significantly longer when pituitary disease was the initial MEN1 manifestation (9.0¡8.1 years) than when the initial manifestation was intestinal and pancreatic tumors (4.1¡4.0 years) or hyperparathyroidism (5.2¡5.1 years) (38).

Treatment Treatment of pituitary tumors depends on clinical presentation, and its aims are tumor volume diminution, normalization of hormone hypersecretion, and preservation of normal pituitary function. Surgery, medication (dopamine agonists, somatostatin analogs, growth hormone receptor antagonists) and radiotherapy are used. The choice of treatment depends on hormone hyperproduction (prolactin or growth hormone), the size and invasion of the tumor, presence of visual impairment, the presence of associated comorbidities, the response or lack of response to medical treatment, contraindications, and the patient’s preference. The treatment is the same for sporadic and MEN1-associated pituitary tumors. There are insufficient data in the literature to propose a different approach at present. A closed surveillance is recommended.

Risk of malignant progression Although it was reported that pituitary tumors were larger and more often invasive in patients with MEN1 syndrome than in patients with sporadic tumors, malignant tumors were not more frequent. However, three cases of pituitary carcinomas were recently reported in patients with MEN1. The first one was a female with sporadic MEN1 and gonadotroph pituitary carcinoma (42). The second was a male with prolactin-producing carcinoma with familial MEN1 (43), and the third was a male with a thyrotropinproducing carcinoma in sporadic MEN1 (44). Owing to the low frequency of pituitary carcinomas, which represent about 0.1–0.2% of all cases of pituitary tumors, it is intriguing that three MEN1-associated cases have been reported.

Clinical characteristics In the France-Belgium multicenter study, the frequency of pituitary macroadenomas was significantly higher in MEN1 patients than in non-MEN1 subjects (85% vs. 42%) (38). Clinical manifestations related to the size of pituitary adenoma were significantly more frequent in MEN1 patients than in non-MEN1 subjects (29% vs. 14%).

Pituitary adenomas with initial manifestation occurred in 17% of patients and were more frequent in women than in men. Normalization of pituitary hypersecretion was much less frequent in MEN1 patients than in non-MEN1 subjects (42% vs. 90%). In a recent publication, the authors found a gender-related difference showing that the prevalence and probability of developing pituitary tumors were significantly greater in females than in males (39). A family history of MEN1 was more frequently found in men than in women at the time of diagnosis. Pituitary tumors occurred as the first clinical manifestation of MEN1 syndrome in 25% of sporadic and in 10% of familial cases (7).

FUTURE PERSPECTIVES Despite significant efforts in the past decade, little is known about genetic alterations in patients with sporadic pituitary tumors. Recent interest in genetics and molecular alterations in patients with pituitary tumors has led to a

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MEN1. Most cases are prolactinomas, somatotropinomas, and non-secreting tumors, although three cases of pituitary carcinoma were reported. In some families, including four families from Newfoundland reported as the so-called MEN1-Burin variant, prolactinoma is unusually common.

Specific cases presenting isolated familial prolactinomas or somatotropinomas need to be differentiated from cases with familial acromegaly/gigantism owing to germline mutations in the AIP gene (54).

When pituitary adenomas present as the initial manifestation of MEN1, they occur at an earlier age than when the initial manifestation is an endocrine, intestinal, or pancreatic tumor. Pituitary tumors in patients with MEN1 syndrome tend to be larger, more frequently invasive and more symptomatic, and they occur in younger patients. The normalization of pituitary hypersecretion occurs much less frequently in MEN1 functional adenomas than in sporadic adenomas. They are more frequently multi-hormonal and mixed tumors. Pituitary hyperplasia and multiple tumors are more frequently found in MEN1 pituitary tumors than in sporadic cases, although no significant differences were noticed.

In summary, pituitary tumors associated with MEN1 may differ markedly from sporadic tumors, and these differences are usually very helpful in performing the differential diagnosis between these entities. Briefly, pituitary tumors in MEN1 (a) are usually diagnosed at earlier ages than sporadic pituitary adenomas and occur more frequently in females; (b) have a higher degree of aggressiveness and invasiveness; (c) have a higher prevalence of macroadenomas; (d) more frequently present as multi-hormonal and mixed tumors; (e) are more often resistant to medical therapy; and (f) have higher tumor recurrence rates.

Furthermore, pituitary hyperplasia and multiple pituitary tumors are more frequently found in MEN1 than in sporadic tumors, although no significant differences were noticed.

Finally, it is important to obtain genetic MEN1 mutation screening in MEN1 family members at risk to present with disease. This has a strong clinical impact that can lead to decreased morbidity rates and possibly decreased mortality rates, and this screening should be routinely performed (55,56).

better understanding of the different syndromes involved in the development of MEN1, Carney complex, McCune– Albright syndrome and familial acromegaly (20,28,45,46). A number of genetic factors, hormones, growth factors, adhesion molecules and cell cycle regulators may be involved in these syndromes. Their study could help to elucidate the process of pituitary tumor initiation and progression.

Information related to human genes is accumulating at an ever-increasing pace, and the growing inventory of genetic variations is facilitating our understanding of the same disease among individuals and populations. MEN1-founding mutations have been described in different places around the world. The prevalence, severity, and resistance to treatment could differ among ethnic groups as a consequence of inherited and noninherited causes. It will be important in the future to analyze whether or not these groups possess ancestral similarities that could explain differences in clinical characteristics (47). Comparison of the prevalence of the two most important components of human genetic variation, single-nucleotide polymorphisms and copy-number variants, among these populations will provide us with better insight into MEN1. The use of ancestry in mapping genes that contribute to the development and progression of pituitary tumors in MEN1 may help us to understand the causes of disease and health disparities between these patients all around the world.

Genomic data present a new array of opportunities and challenges, and data collection should be extended to as many diverse populations as possible.

Future study of MEN1 and pituitary tumors includes the application of ‘‘omic’’ technologies (genomics, transcriptomics, proteomics, and metabolomics). Contrary to traditional molecular biology methods that were used to study the role of a single gene and single protein, ‘‘omic’’ data along with a systems biology approach will help us to apply a multiple-factor model of disease and to clarify the network of interactions and regulatory events that contribute to disease (48,49).

Finally, the use of exome sequencing will offer new opportunities for research on Mendelian disorders. Exome sequencing has now been applied in multiple situations individually where the causal variants for a number of Mendelian disorders have been successfully identified. In addition, exome sequencing has been shown to be more rewarding in the study of disorders with genetic and phenotypic heterogeneity such as MEN1 (50–52). Highthroughput sequencing may also be helpful in uncovering new genetic variants among MEN1 patients with no mutations apparent from conventional PCR and exon sequencing.

ACKNOWLEDGMENTS The authors are grateful to the Jarislowsky and Lloyd Carr Harris foundations for their continuous support. R.A.T. is the recipient of a FAPESP (Sao Paulo State Research Foundation) postdoctoral fellowship.

The authors thank Mrs. Denise Chase of the Mayo Clinic for her assistance and secretarial support.

AUTHOR CONTRIBUTIONS CONCLUSION

Syro LV, Scheithauer BW, and Kovacs K conceived the study. London˜o FJ, Ortiz LD, and Rotondo F searched the literature and extracted the data. Syro LV, London˜o FJ, Ortiz LD, and Uribe H wrote the manuscript.

Scheithauer BW, Rotondo F, Horvath E, and Toledo RA contributed to the initial revision of the manuscript. All authors contributed to the critical revision of the manuscript before publication and approved the final version.

MEN1 is a complex syndrome in which a large number of endocrine and non-endocrine neoplasias may occur. The occurrence of anterior pituitary tumors in MEN1 syndrome may range between 10% and 60%. Pituitary involvement includes the initial manifestation of MEN1 syndrome in 10% to 25% of individuals (38,53). Pituitary adenomas are significantly more frequent in women than in men.

Approximately 65% to 85% of pituitary tumors in MEN1 syndrome are macroadenomas. Plurihormonal adenomas occur more frequently than monohormonal adenomas in

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DOI:10.6061/clinics/2012(Sup01)10

REVIEW

Variable clinical expression in patients with a germline MEN1 disease gene mutation: clues to a genotype–phenotype correlation Cornelis J. Lips,I Koen M. Dreijerink,I Jo W. Ho¨ppenerII I

University Medical Center Utrecht, Department of Internal Medicine & Endocrinology, Utrecht, The Netherlands. II University Medical Center Utrecht, Department of Metabolic & Endocrine Diseases, Utrecht, The Netherlands.

Multiple endocrine neoplasia type 1 is an inherited endocrine tumor syndrome, predominantly characterized by tumors of the parathyroid glands, gastroenteropancreatic tumors, pituitary adenomas, adrenal adenomas, and neuroendocrine tumors of the thymus, lungs or stomach. Multiple endocrine neoplasia type 1 is caused by germline mutations of the multiple endocrine neoplasia type 1 tumor suppressor gene. The initial germline mutation, loss of the wild-type allele, and modifying genetic and possibly epigenetic and environmental events eventually result in multiple endocrine neoplasia type 1 tumors. Our understanding of the function of the multiple endocrine neoplasia type 1 gene product, menin, has increased significantly over the years. However, to date, no clear genotype– phenotype correlation has been established. In this review we discuss reports on exceptional clinical presentations of multiple endocrine neoplasia type 1, which may provide more insight into the pathogenesis of this disorder and offer clues for a possible genotype–phenotype correlation.

KEYWORDS: Multiple Endocrine Neoplasia type 1; MEN1; Menin; Genotype–Phenotype Correlation; Clinical Expression.

Lips CJ, Dreijerink KM, Ho¨ppener JW. Variable clinical expression in patients with a germline MEN1 disease gene mutation: clues to a genotype– phenotype correlation. Clinics. 2012;67(S1):49-56.

E-mail: lips05@zonnet.nl Tel.: +31 703240428

expression can also vary between individual members of the same family, possibly because of additional genetic or epigenetic factors. To date, a clear correlation between genetic events and the variable clinical expression of MEN1 has not been established (1–5). Further understanding of the genetic aspects of MEN1 and the pathogenesis of MEN1related tumors could enable more tailored clinical screening and treatment strategies.

In this review, we discuss recent reports on aberrant clinical expression of MEN1, which may allow us a glimpse into the pathogenesis of this intriguing disorder.

In 1903, Erdheim described the case of an acromegalic patient with a pituitary adenoma and three enlarged parathyroid glands. Fifty years later, Underdahl et al. reported eight patients with a syndrome of pituitary-, parathyroid-, and pancreatic islet adenomas. In 1954, Wermer found that the syndrome was transmitted as a dominant trait (6). In 1968, Steiner et al. introduced the term ‘‘multiple endocrine neoplasia’’ (MEN) to describe disorders featuring combinations of endocrine tumors; they designated the Wermer syndrome as MEN1 and the Sipple syndrome as MEN2. Gorlin subdivided type 2 into A and B. Then, in 1975, Khairi (7) suggested that type 2B be called type 3; however, this was not generally accepted.

More recently, in 2006, families with multiple endocrine tumors but without MEN1 or RET (MEN2) gene mutations were identified (8). This related syndrome is referred to as MEN4.

INTRODUCTION ’’It is an old experience that through her errors, Nature often grants us unexpected insights into her secrets which are otherwise a closed domain‘‘, William Harvey, 1657.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited endocrine tumor syndrome, characterized predominantly by tumors of the parathyroid glands, gastroenteropancreatic tumors, pituitary adenomas, adrenal adenomas, and neuroendocrine tumors of the thymus, lungs or stomach. MEN1 is caused by germline mutations of the MEN1 tumor suppressor gene. It appears that in the MEN1 syndrome, clinical expression differs between families. This may be the result of the specific MEN1 gene mutation in a family (genotype–phenotype correlation). As a rule, the development of a tumor depends on a series of genetic events (multistep tumorigenesis). Thus, additional co-segregating modifying factors such as germline mutations in other genes are likely to play a role in the interfamilial variability of MEN 1. Moreover, clinical

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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inhibiting genes, the human telomerase (hTERT) gene, and nuclear receptor target genes (12–15).

As a transcriptional co-regulator, menin may function as a co-activator or co-repressor by recruiting histone-modifying enzymatic activity (12,15,16). As a co-activator, menin is involved in the regulation of histone methylation by recruiting the mixed-lineage leukaemia (MLL) proteins MLL1 and MLL2 (12,17). In this way, menin can bind to nuclear receptors and activate nuclear receptor-mediated gene transcription (12,15).

By tethering histone deacetylase activity to genes, menin can serve as a repressor of transcription (18).

In order to understand the role of menin as a tumor suppressor protein and as a co-factor of MLL fusion proteins, the structural basis had to be revealed. Recently, the crystal structure of menin in Nematostella vectensis was reported (19).

Knowledge about the three-dimensional structure may elucidate the interactions essential to the function of menin.

It appears that the Leucine, Leucine, Tryptophan, Leucine, Leucine (LLWLL) amino acids nuclear receptor interaction motif of menin is well-conserved and is located in an alphahelix. In general, modeling gene mutations into this structure will be helpful in determining the effects on protein function.

Inactivation of the MEN1 gene results in predisposition to tumor formation (see Figure 1, Table 1).

DISCOVERY OF THE MEN1 GENE In positional cloning, gene mapping precedes, and eventually leads to, gene identification. The first step is mapping the gene to a specific chromosomal region by linkage analysis.

The second step is identifying the correct gene among all possible candidate genes within that particular chromosomal region. After the gene has been identified, it is possible to study its (patho)physiologic function.

In 1996, two groups independently identified the MEN1 gene on chromosome 11q13 (9,10). To date, more than 1336 different MEN1 gene mutations (both germline and sporadic) have been reported in the literature (4). Most of these mutations are clearly inactivating, in accordance with the notion that the MEN1 gene is a tumor suppressor gene.

There are no mutational hot spots in the MEN1 gene.

FUNCTION OF THE MEN1-GENE PRODUCT The MEN1 gene product, menin, functions as an adaptor protein that is involved in interactions with multiple protein partners. Men1 null mutant mice have indicated that menin is essential for viability (11). Menin is involved in neuroendocrine cell development and function. Later on, it is active in many cellular processes, including gene transcription regulation, DNA replication, DNA repair, and signal transduction.

Menin target genes that are important for development and proliferation, including homeobox domain (HOX) genes, the CDKN2C and CDKN1B cyclin-dependent kinase

ABERRANT CLINICAL EXPRESSION OF MEN1 A MEN1 gene mutation may be completely detrimental to gene function. It may also result in a protein product with

Figure 1 - In MEN1 tumors, inactivating mutations in the MEN1 gene result in alterations of histone protein modifications: both deacetylation (left) and trimethylation (right) are repressed. In this way, the normal function of menin acting as co-repressor and coactivator of gene transcription is disabled. Consequently, the normal function of menin (preservation of differentiation of the cell by modification of histone proteins and transcription of genes responsible for inhibition of cell division) is defective. Red arrows indicate inhibition of apoptosis, cell differentiation, DNA repair, and endocrine metabolic functions, whereas stimulators of cell division are indicated in green. Opportunities for tumor treatment are indicated in blue. E2 = estradiol; TZDs = thiazolidinediones; VDR = Vitamin D receptor.

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Table 1 - Menin-interacting proteins.

Protein A Chromatin modification proteins HDAC1* MLL MLL2 mSin3A* LEDGF B Transcription factors JunD NF-kB Pem Smad TGF-b WNT/b-catenin* ERa VDR PPARc FOXO1* C DNA repair proteins RPA2 ASK CHES1(FOXN3) FANCD2 D Cytoplasmic proteins Vimentin AKT1 GFAP NM23 IQGAP1 NMHC *

Function

Reference

Chromatin modification Chromatin modification Chromatin modification Chromatin modification Chromatin-associated

Kim et al. (18) Yokoyama et al. (17) Hughes et al. (12) Kim et al. (18) Yokoyama et al. (20)

Gene Gene Gene Gene Gene Gene Gene Gene Gene Gene

transcription transcription transcription transcription transcription transcription transcription transcription transcription transcription

Agarwal et al. (21) Heppner et al. (22) Lemmens et al. (23) Kaji et al. (24) Shattuck et al. (25) Chen et al. (26) Dreijerink et al. (27) Dreijerink et al. (27) Dreijerink et al. (27) Wuescher et al. (28)

DNA replication Cell cycle regulation DNA repair/ Transcription DNA repair

Sukhodolets et al.(29) Schnepp et al. (30) Busygina et al. (31) Jin et al. (32)

Cytoplasmic Signal transduction Cytoplasmic GTP-ase Cell adhesion Myosin

Lopez-Egido et al. (33) Wang et al. (34) Lopez-Egido et al. (33) Ohkura et al. (35) Yan et al. (36) Obungu et al. (37)

Proteins in a complex with menin, possibly not through a direct interaction.

that either a common ancestral mutation in the MEN1-Burin gene or a modifying gene on 11q13 is responsible for this prolactinoma variant of MEN1 (42–45).

some residual function. An aberrant menin protein may be impaired in its function by several mechanisms: menin can interact with many different proteins. Possibly, germline mutations in the MEN1 gene selectively affect menin binding to its partners, leading to distinct phenotypes.

The type of missense mutation (e.g. replacement of arginine with glycine) may have a differential effect on the function of menin (38): in-frame or missense mutations differ from frameshift/nonsense mutations (39), whereas missense and in-frame mutations may affect the interactions of a menin domain with transcription factors such as JunD, Smad3 and NFkappaB and nuclear receptors (1), or impair sensitization to apoptosis from caspase-3, p53 or p21 (40).

A MEN1 gene missense mutation may result in protein instability, and enhanced and early proteolytic degradation via the ubiquitin–proteasome pathway (41).

It was generally assumed that, in contrast to MEN2, in MEN1 there is no clear genotype–phenotype correlation (1,3–5). However, several reports challenge this assumption.

2. Familiar isolated hyperparathyroidism and MEN1 gene missense mutations. Familial isolated primary hyperpa-

rathyroidism (FIHP) is an autosomal dominant disorder that can represent an early stage of either MEN1 caused by an allelic variant of the MEN1 gene, or of hyperparathyroidism– jaw tumor (HPT-JT) syndromes; alternatively, the condition can be caused by an allelic variant of the hyperparathyroidism 2 (HRPT2) gene, or caused by a mutation at another locus.

Interestingly, the major proportion of MEN1 gene germline mutations that have been found in FIHP are seemingly mild missense mutations or in-frame deletions (46–55). In MEN1, roughly 80% of patients harbor nonsense mutations.

3. Predominant mutations in MEN1 pancreatic neuroendocrine tumors. Schaaf performed mutation analy-

sis of the MEN1 gene in tumors from 306 patients with MEN1, and found that patients with gastroenteropancreatic tumors more often had truncating mutations, very probably leading to completely inactivated menin (56).

4. Mild/late onset versus malignant phenotypes. To date, several disease-related MEN1 gene intron mutations have been reported. These intron mutations may affect mRNA splicing and cause mild phenotypes, with late, and relatively low, penetrance of the disease (57–59). However, clinical expression at a young age may occur. This may be explained by interpatient variations in gene transcription and translation of the MEN1 gene.

Two recent case reports described families with a high penetrance of malignant neuroendocrine tumors of the pancreas (60,61). Both these families carried germline mutations that completely abolish menin function.

Familial aberrant expression 1. MEN1 Burin. Four large kindreds from the Burin peninsula/Fortune Bay area of Newfoundland with prominent features of prolactinomas, in addition to carcinoids, and parathyroid tumors (referred to as MEN1Burin) have been described, and they show linkage to 11q13, the same locus as that of MEN1. Haplotype analysis with 16 polymorphic markers now reveals that representative affected individuals from all four families share a common haplotype over a 2.5 Mb region. A nonsense mutation in the MEN1 gene has been found to be responsible for the disease in the affected members in all four of the MEN1-Burin families. This suggests

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The earliest manifestation of MEN1 was a pituitary adenoma in a 5-year-old boy who had a missense mutation leading to a H139D substitution in the MEN1 protein (62).

Functional analysis of the mutant protein revealed severely reduced protein stability (41), reduced binding to JunD (16), reduced binding to the estrogen receptor alpha and absent histone-methylation recruiting capacity. Thus, functional analysis of this potentially mild missense MEN1 gene mutation shows that the protein product is, in fact, completely inactivated.

deletion of a larger chromosomal region containing multiple genes [loss of heterozygosity (LOH)].

The gene encoding the aryl hydrocarbon receptor interacting protein (AIP) is located on 11q13, in the vicinity of the MEN1 gene. Recently, it was found that inactivating mutations in the AIP gene are the underlying cause of low-penetrance pituitary adenoma predisposition. The finding of a truncated gene and LOH indicates that AIP acts as a tumor suppressor gene (68,69). In northern Finland, AIP-germline mutations accounted for 16% of cases of acromegaly in young patients. The tumor suppressor genes AIP and MEN1 are located 3 Mb apart. Concomitant deletions of these genes may underlie predisposition to MEN1 and pituitary adenoma. To what extent deletion of the AIP gene is present in MEN1 tumors has yet to be established. Inactivation of this gene in animal models may reveal a potential causative role in MEN1-associated tumors.

2. Genetic predisposition for other diseases. Genetic predisposition for other diseases may contribute to enhancement of tumor formation in patients with MEN1 (70). For instance, normally the vitamin D receptor on parathyroid cells inhibits production and release of parathyroid hormone (PTH). In families with inactivating mutations in the gene encoding VDR, this is associated with end-organ resistance to calcitriol.

In the parathyroid glands of patients with MEN1, there exists a decreased activation of the VDR. An additional defect in the VDR or calcium receptor may contribute to hyperactivity, hyperplasia, and adenoma (71).

5. Metabolic effects of aberrant expression of the MEN1 gene. In MEN1 disease-gene carriers, all vitamin D

receptors (VDRs) and peroxisome proliferator-activated receptors (PPARs)-c are expressed but are probably less activated because of impaired menin function.

A. PPARc2 is a transcription factor that plays a key role in adipocyte differentiation. Polymorphisms in this gene may contribute to the variability in body mass index and insulin sensitivity in the general population. PPARc is the receptor for the thiazolidinediones, which act as PPARc agonists and lower the blood glucose levels in patients with type 2 diabetes mellitus by increasing insulin sensitivity. Individuals with dominant-negative PPARc gene mutations manifest a syndrome that combines lipodystrophy with features of the metabolic syndrome, including insulin resistance, type 2 diabetes, hepatic steatosis, dyslipidemia, hypertension and (in women) polycystic ovary syndrome. In patients with MEN1, decreased activation of PPAR may result in insulin resistance and weight gain (63).

B. Vitamin D receptors (VDRs) are found in a large number of tissues beyond the classic target tissues gut, bone and kidney. These tissues include endocrine glands such as pituitary, parathyroid glands, pancreatic islets, etc.

Lourenc¸o et al. discussed the increased bone loss pattern found in patients with MEN1 compared with that of patients with sporadic primary HPT (64). Besides increased bone loss, resistance to vitamin D may be associated with insulin resistance and beta cell dysfunction, leading to increased risk for type 2 diabetes in patients with MEN1 (65,66).

3. Additional, somatic mutations involved in acceleration of tumor growth.

3a). Data from other familial neuroendocrine tumor syndromes. How can we identify acquired mutations that

are responsible for acceleration of tumor growth in MEN1?

Clues for modifier genes may be found in other familial neuroendocrine tumor syndromes such as MEN2 and MEN4 (the latter is also referred to as MENX). Which are their traditional pathogenetic pathways and are these involved in aberrant clinical MEN1 expression? Overlap between MEN1 and MEN2 and additional genetic events have to be explored (e.g. p18/p27 knock-out mice develop both MEN1- and MEN2-associated tumors) (72,73).

Phenotypic overlap between MEN1- and MEN2-like syndromes was identified in the rat and named MENX. The syndrome is caused by a germline inactivating mutation in the CDKN1B gene encoding p27Kip1 (8). p27Kip1 has a key role in cell cycle regulation and is involved in differentiation, apoptosis, and angiogenesis.

Subsequently, germline mutations in the CDKN1B gene were identified in the germline of a MEN1-like family. In these patients, germline mutations of the MEN1 gene could not be detected (8). However, only the menin-coding region and splice junctions were analyzed. The patients were also negative for RET gene mutations (MEN2). Mutations in CDKN1B and related genes, but without MEN1 gene mutations, are a rare cause of MEN1-like phenotype (74– 76). As a consequence of mutations in the p27 gene, a novel human MEN syndrome was recognized and named MEN4.

In mice, the Cdkn2c gene encoding p18Ink4c was shown to collaborate with menin in suppression of neuroendocrine tumor development (77). Whether occurrence of somatic mutations in p18Ink4c and/or p27Kip1 accelerates tumor growth in human MEN1 tumors has yet to be established. In

Effect of gender The prevalence and probability of pancreatic tumors among patients with MEN1 were higher in males than in females. This difference was due to the differential occurrence of gastrinomas. The prevalence and probability of developing pituitary adenomas were significantly greater in females. Thymic tumors are found nearly exclusively in male MEN1 patients (67).

The difference in clinical expression between the genders may be explained by the difference in transcription regulation of estrogen and androgen receptors. Menin can act as a co-activator of nuclear hormone receptors including estrogen (ERa) and possibly androgen (AR) receptors. A defect in the MEN1 gene, together with gender-specific differences in concentrations of the hormones involved and tissue-specific distribution of their receptors, may contribute to the observed gender-specific differences in prevalence of prolactinoma and gastrinoma.

Additional genetic effects 1. Loss of heterozygosity; the AIP gene. In accordance with the tumor suppressor function of menin, MEN1associated tumors exhibit loss of the wild-type allele. This second hit occurs as a somatic mutation and often involves

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epigenetic silencing, upstream activation through receptor tyrosine kinase (RTK) signaling, and/or downstream loss of the tumor suppressors p18 and p27.

A combination of a mutation in the MEN1 disease gene with other specific mutations of genes in the PI3K/AKT pathway may be associated with acceleration of tumor growth.

In addition, inactivated or absent menin promotes RAS expression; activating mutations of RAF, MEK, or ERK may accelerate cell proliferation (89).

transgenic MEN2 mice and human patients with MEN2, inactivating mutations in p18 will promote tumor growth (72,73,78,79). Reduced expression of CDKN1B, but not CDKN2C, has been observed in parathyroid adenomas from patients with MEN1.

3b). Clues from sporadic parathyroid adenomas, pituitary tumors, and pancreatic NETs i) Sporadic Parathyroid adenoma. A high rate of so-

matic MEN1 gene mutations is seen in sporadic parathyroid adenomas. There exists an interaction between menin and the transforming growth factor (TGF)-beta/Smad signaling pathway. In vitro experimentation has demonstrated that the presence of menin is required for TGF-beta to effectively inhibit parathyroid cell proliferation and PTH production (80). ii) Pituitary tumors. The pituitary tumor transforming gene (PTTG; securin) was the first transforming gene found to be highly expressed in pituitary tumor cells, and seems to play an important role in the process of oncogenesis. Cell signaling abnormalities have been identified in pituitary tumors, but their genetic basis is unknown. Both Raf/mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase(PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways are over-expressed and/or over-activated in pituitary tumors (81). These pathways share a common root, including initial activation by a tyrosine kinase receptor.

Pit-1 is a direct transcriptional target of VDR. Recruitment of histone deacetylase 1 is involved in the repressive effect of VDR on Pit-1 expression (82).

There is a critical role for the growth factor activin in regulating inhibition of pituitary cell growth and Pit-1/PRL expression through the Smads and menin (83). Alterations in the activin/TGF-beta downstream signaling pathways may be critical steps towards tumor formation and progression (84). To date, the occurrence of additional TGF-beta, Pit-1, PTTG, or VDR mutations in MEN1-associated tumors has not been published. iii) Pancreatic neuroendocrine tumors. In nonfamilial pancreatic neuroendocrine tumors (PanNETs) the most commonly mutated genes specify proteins implicated in chromatin remodeling: 44% of the tumors had somatic inactivating mutations in MEN1. Clinically, mutations in the MEN1 gene were associated with better prognosis. Also, mutations in genes in the mTOR pathway were found in 14% of the tumors, a finding that could potentially be used to stratify patients for treatment with mTOR inhibitors (85).

Loss of menin expression is associated with overexpression of the Raf/MEK/ERK and P13K/AKT/mTOR pathways in pancreatic tumors (34). Intact menin has an essential role in WNT/b-catenin signaling, and inhibits mouse pancreatic islet tumor proliferation (26). Menin regulates subcellular localization of b-catenin via nuclearcytoplasmic shuttling. Loss of menin leads to Wnt/b-catenin signaling activation. Expression of p27 was found to be repressed in pancreatic islet cell tumors (86).

3c) Pathways in multistep carcinogenesis. It appears that interaction of components of the PI3K/AKT pathway is involved in neuroendocrine tumor formation (87,88).

Deregulation of the PI3K/AKT pathway in neuroendocrine tumors can occur through a range of processes (see Figure 2), including gain of function by oncogenic mutations of PI3K signalling, loss of function of the tumor suppressor PTEN through gene deletion, mutation, micro-RNA expression or

Ecogenetic factors Common environmental factors may interact with genetic predisposition to MEN1 and contribute to enhancement of tumor formation (70). In the parathyroid glands of patients with MEN1, a diet low in vitamin D or calcium may result in tumor growth. In the lactotropic cells of the pituitary gland, estrogenic or neuroleptic drugs may stimulate cell division. In the gastrin-producing cells of the stomach, presence of achlorhydria or use of histamine-H2 receptor or proton pump blockers may promote tumor growth (90).

Lifestyle factors such as smoking and exposure to radiation may have deleterious effects on menin function and tumor growth, as with many types of cancer.

CONCLUDING REMARKS Careful observation of patients and collaboration between disciplines, including molecular endocrinology, has opened new directions in the management of MEN1 syndrome, and has promoted development of novel target-directed therapy. Since 1980, life expectancy and quality of life have improved considerably.

By contrast, thymic tumors and duodenopancreatic tumors, including nonsecreting pancreatic tumors increase the risk of death (91). Rare, but aggressive, adrenal tumors may also cause early death. In MEN1 disease gene carriers in MEN1 families, most deaths were related to the disease, and probably resulted from additional mutations.

It is possible that consecutive and specific pathogenetic pathways are involved in MEN1 tumor formation. We presume that, through the inherited germline mutation resulting in organ-specific cell division, the patient is rendered vulnerable to additional, somatic mutations in these organs. These mutations may occur spontaneously or may be triggered by life style and/or environment. Predisposition and expression of other genetic diseases may also be involved.

A complex genotype–phenotype relationship may be present.

Unfortunately, for the majority of the patients it is not currently possible to predict the course of the disease.

Germline mutations that result in complete inactivation of the gene product apparently cause more severe disease.

Consequently, extensive periodical clinical examination has to be performed in all carriers of the MEN1 disease gene.

In the near future, tumor gene-expression profiles and high throughput sequencing may permit more insight into additional genetic and epigenetic events that cause progression of tumor development. Functional analysis of MEN1 gene mutations is sometimes required to study the true effect of the mutation. In addition, as already suggested by Sir William Harvey, it is very helpful to investigate rare presentations of diseases (92). Eventually, this insight may allow target-directed and mutation-specific therapy.

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Figure 2 - Beyond inactivation of the MEN1 gene, additional mutations in other genes may be responsible for acceleration of tumor growth, thus involving a process of multistep tumorigenesis. Deregulation of normal neuroendocrine development/differentiation can occur through a range of processes, e.g. activation of the PI3K pathway leads to AKT phosphorylation, triggering a downstream cascade of events. Deregulation of this pathway can occur through several mechanisms: 1) gain of function by oncogenic mutations of PIK3; 2) loss of function of the tumor suppressor PTEN through gene deletion, mutation, micro-RNA expression, or epigenetic silencing; 3) amplification or mutation of AKT isoforms; 4) upstream activation through RTK signalling; 5) downstream loss of the tumor suppressors p18 and p27; 6) increased RAS expression, or activating mutations of RAF, MEK, or ERK, which accelerate cell proliferation.

Opportunities for treatment are in blue. NSAIDs = non-steroidal anti-inflammatory drugs; RTK = receptor tyrosine kinase; TZDs = thiazolidinediones.

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AUTHOR CONTRIBUTIONS Lips CJ conceived and designed the study and was also responsible for the manuscript writing and preparation of figures and table. Dreijerink KM searched the literature for important contents, provided assistance to the manuscript writing and to the molecular aspects of the menin protein.

Ho¨ppener JW provided assistance to the study design, manuscript writing and final version of the manuscript.

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DOI:10.6061/clinics/2012(Sup01)11

REVIEW

RET haplotype, not linked to the C620R activating mutation, associated with Hirschsprung disease in a novel MEN2 family Elisangela P. S. Quedas,I Viviane C. Longuini,I Tomoko Sekiya,I Flavia L. Coutinho,I Sergio P. A. Toledo,I Uenis Tannuri,II Rodrigo A. ToledoI I Faculdade de Medicina da Universidade de Sa˜o Paulo, Endocrine Genetics Unit (LIM-25), Division of Endocrinology, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Division of Pediatric Surgery and Laboratory of Pediatric Surgery (LIM-30), Sa˜o Paulo/SP, Brasil.

Hirschsprung disease is a congenital form of aganglionic megacolon that results from cristopathy. Hirschsprung disease usually occurs as a sporadic disease, although it may be associated with several inherited conditions, such as multiple endocrine neoplasia type 2. The rearranged during transfection (RET) proto-oncogene is the major susceptibility gene for Hirschsprung disease, and germline mutations in RET have been reported in up to 50% of the inherited forms of Hirschsprung disease and in 15–20% of sporadic cases of Hirschsprung disease. The prevalence of Hirschsprung disease in multiple endocrine neoplasia type 2 cases was recently determined to be 7.5% and the cooccurrence of Hirschsprung disease and multiple endocrine neoplasia type 2 has been reported in at least 22 families so far. It was initially thought that Hirschsprung disease could be due to disturbances in apoptosis or due to a tendency of the mutated RET receptor to be retained in the Golgi apparatus. Presently, there is strong evidence favoring the hypothesis that specific inactivating haplotypes play a key role in the fetal development of congenital megacolon/Hirschsprung disease. In the present study, we report the genetic findings in a novel family with multiple endocrine neoplasia type 2: a specific RET haplotype was documented in patients with Hirschsprung disease associated with medullary thyroid carcinoma, but it was absent in patients with only medullary thyroid carcinoma.

Despite the limited number of cases, the present data favor the hypothesis that specific haplotypes not linked to RET germline mutations are the genetic causes of Hirschsprung disease.

KEYWORDS: RET; MEN2; HSCR; Hirschsprung; Haplotypes.

Quedas EPS, Longuini VC, Sekiya T, Coutinho FL, Toledo SPA, Tannuri U. RET haplotype, not linked to the C620R activating mutation, associated with Hirschsprung disease in a novel MEN2 family. Clinics. 2012;67(S1):57-61.

E-mail: toledorodrigo@usp.br / toledorodrigo79@hotmail.com Tel.: 55 11 3061-7252

newborns. HSCR is a neurocristopathy most frequently found in its sporadic form (S-HSCR, ,70% of cases) which is a non-Mendelian disease with low, sex-dependent penetrance and variable clinical expression (1–3). Familial forms of HSCR (F-HSCR) usually occur in association with several inherited syndromes, such as MEN2 (4–13).

Recently, a Hirschsprung disease consortium verified that germline mutations in the RET proto-oncogene have been reported in up to 50% of patients with F-HSCR and in 1520% of patients with S-HSCR (2). RET is the major susceptibility gene for HSCR, although up to 10 genes and five different loci have been associated with HSCR development (1,2). The short-segment form of the condition is more frequent (80%) than the long-segment form [20%], and recently developed surgical approaches for both forms have a high chance of success, markedly decreasing the mortality and morbidity rates of this condition (2,14-17).

Diagnosis of HSCR is usually confirmed by rectal biopsy in newborns with intestinal obstructions with the following findings: failure of meconium delivery in the first 2 days of life, abdominal distention that is alleviated with rectal stimulus and enema, vomiting, and neonatal enterocolitis.

The shorter the aganglionic segment, the later the signals

INTRODUCTION We will briefly review Hirschsprung disease (HSCR), multiple endocrine neoplasia type 2 (MEN2), and the co-segregation of these conditions in the few families that have been studied to date. In addition, we will discuss the existing hypotheses to explain the finding of HSCR associated with MEN2.

HIRSCHSPRUNG DISEASE HSCR (MIM #142623) is a congenital intestinal malformation characterized by aganglionic megacolon in a variable length of the intestine; both short- and longsegment forms of the disease exist (1). The incidence of this congenital megacolon is 1 per 5,000 newborns, although it varies from 1.5 in Caucasians to 2.8 in Asians for each 10,000

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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hypothesis that specific haplotypes play a key role in the fetal development of congenital megacolon (2,32). Recent data suggest that mutations in RET ligand genes may be responsible for a small proportion of HSCR cases (33).

As previously mentioned, the prevalence of HSCR in MEN2 cases has been recently determined to be 7.5% (22).

Borrego et al. (10) reported that 13 HSCR/MEN2 families had been described up to 1998. At present, up to 22 HSCR/ MEN2 families have been reported, although the number of families that have been genetically investigated is relatively limited (2,4,12). S-HSCR or F-HSCR cases have been extensively studied clinically; however, further genetic haplotype analyses may contribute to a better understanding of the molecular causes of familial HSCR associated with MEN2.

and symptoms develop and consequently the more delayed the diagnosis of HSCR (2,3,16).

MEN2 MEN2 (MIM #171400) is an inherited syndrome caused by germline mutations in the RET proto-oncogene, which encodes the RET receptor tyrosine kinase. Three neuroendocrine neoplasias are usually present: medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO), and primary hyperparathyroidism. Two other subtypes of MEN2 are familial MTC (FMTC; MTC only) and MEN2B (MTC; PHEO, and ganglioneuromas) (7,13,18,19).

HIRSCHSPRUNG DISEASE CO-OCCURRING WITH MEN2

LOCAL EXPERIENCE

In MEN2A, MTC and PHEO are the most prevalent causes of morbidity and mortality, although HSCR is also an infrequent but potentially life-threatening MEN2 phenotype (2,18,19). In contrast, in sporadic MTC there are no other associated neuroendocrine tumors and HSCR has not been reported to date (20,21).

Recently, an international consortium on RET exon 10 studied 340 affected subjects from 103 MEN2 families and assessed the risk profiles and penetrance estimations of MEN2 patients who were diagnosed with germline RET mutations in exon 10. In this study, 267 MEN2 patients were investigated for HSCR disease and an overall penetrance of 7.5% was observed (22). Of note, carriers of germline RET mutations in codon 620 were more prone to HSCR (13%), especially those with C620R or C620G substitutions (14– 33%), although RET 618 and 609 codon mutation carriers may also present with HSCR (22).

Although a limited number of affected familial cases from this new FMTC/MEN2 genealogy could be studied, data from patients with either MTC alone or HSCR associated with MTC/MEN2 were available to be analyzed.

A novel HSCR/FMTC family Herein we report the results of the genotyping analysis of a novel HSCR/FMTC family that carries the germline c.1858T.C (p.C620R) RET activating mutation in exon 10.

The patients come from a rural area of northeast Brazil and have been diagnosed, treated and followed for several years at the University of Sa˜o Paulo School of Medicine, Academic Hospital (Hospital das Clı´nicas, southeast Brazil) (Figure 1).

Early recognition of HSCR in the studied cases was achieved with routine investigations for HSCR (15,16). The recommended surgical approach for HSCR was used, allowing the affected patients in the study to survive longer (14,17). The two patients with HSCR/MEN2 who were operated on in our hospital had the long-segment form of HSCR. No renal/urinary disturbances were observed in our cases, consistent with the findings from the HSCR mouse model (34). Several RET gene mutation carriers from this family underwent preventive or curative total thyroidectomy. We have recently reported partial phenotypic data obtained from this large FMTC/MEN2 family (22).

Present hypotheses The co-occurrence of the HSCR and MEN2 (HSCR/ MEN2) phenotypes raises an apparent paradox because MTC is caused by activation of cellular growth signaling of the RET tyrosine kinase receptor, while HSCR is largely associated with disruption of this pathway because of inactivating RET mutations (1). Initially it was thought that disturbed mechanisms of apoptosis might lead to HSCR (23). Several investigations have examined this topic (24– 33).

At present, there are two main hypotheses to explain the occurrence of HSCR/MTC within MEN2 families. The first states that MEN2-associated RET mutations, mostly in exon 10, may play a dual activating and inactivating role in the RET tyrosine kinase molecular pathway, leading to constitutive activation of the RET receptor. The mutated RET receptor would present a tissue-specific tendency to be retained in the Golgi apparatus, leading to a reduced number of RET membrane receptors in the intestine and, therefore, decreased intracellular tyrosine signaling (24).

The second hypothesis is that the RET-activating mutations play either no role, or only a minor role, in HSCR development (2). In this scenario, low-penetrant and dosedependent, specific predisposition to RET haplotypes may lead to a reduction in RET tyrosine receptor activation during embryonic life, leading to neuronal and intestinal cell disturbances and, finally, HSCR. Of note, both protective and predisposing haplotypes to HSCR have been described (2). There is strong evidence favoring the

Visit to the area During a recent visit to the area where the family originally lived and where most patients still live (2,500 km from Sa˜o Paulo), written informed consent was obtained and blood samples for DNA analysis were collected from several at-risk family members. The researchers were informed that the family had experienced three neonatal deaths of babies who were operated on for congenital megacolon at a local hospital, but not in time to save their lives. The institutional ethics committee has approved this research project.

Haplotype investigation To verify if an additional and inactivating RET mutation was present in the C620R-mutated HSCR patients in this family, the 21 exons of the RET proto-oncogene were analyzed by polymerase chain reaction amplification and bidirectional sequencing, as previously reported (35). No germline RET mutation other than C620R was observed. We also investigated whether RET single nucleotide poly-

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Hirschprung disease and MEN2 Quedas EPS et al.

Figure 1 - Identification of different haplotypes, including the rs2505533 and rs34827976 SNPs, in relatives with MTC/HSCR or MTC alone (non-HSCR) who carry the C620R RET mutation. The observed haplotype in the HSCR/MTC patients (red) contains the region previously recognized as related to the sporadic and familial forms of HSCR (1,2). Four SNPs within this region are only homozygous (aa) in patients with HSCR.

strengthened the proposal that this RET gene region is involved in the development of HSCR (n = 9; p = 0.012; Fisher exact test). It is worth noting that only HSCR patients were homozygous for the four SNPs located in the implicated region: rs2565206, rs2505532, rs72781232, and rs2505531 (Figure 1). Concordantly, RET exon 2 A45A SNP has been found in the homozygous state in cases with HSCR

morphisms (SNPs) or specific haplotypes were involved.

Haplotypes were estimated using the EHPlus and Hapi software programs (http://linkage.rockefeller.edu). Genotyping data on 16 RET intragenic SNPs were as follows: rs2505533, novel SNP-1, rs2505532, rs72781232, rs2505531, rs2565206, rs1800858, rs1864404, novel SNP-2, rs1800860, rs3026750, rs34827976, rs1799939, rs1800861, rs1800862, and rs1800863 (Table 1).

Initially, the haplotype CTCGTGGTGAdel (rs2505533, IVS1-126, rs2505532, rs72781232, rs2505531, rs2565206, rs1800858, rs1864404, rs1800860, rs3026750 and rs34827976) was documented in all of the studied C620R-mutated relatives in the present family, indicating that this haplotype is linked to the RET mutation.

Because not all of family members with the RET mutation had developed HSCR, the emerging hypothesis was that HSCR disease maybe associated with genetic variants in the remaining chromosome region. In accordance with this assumption, the MEN2-affected relatives who did not have HSCR were believed to have inherited the CATACTGTAAdel haplotype, while the HSCR/MEN2 patients had inherited a different haplotype, TTCGTTAAAGC (Figure 1).

The RET gene region that includes the observed haplotypes in this new MEN2 family, especially the combination of the SNPs from rs2505533 to rs2565206 in linkage disequilibrium with SNPs rs1800858 and rs1800861, has been previously implicated in the development of both sporadic and familial forms of HSCR (1,2). Furthermore, analysis of four additional members of this family with the C620R mutation (three cases without and one with HSCR)

Table 1 - Sixteen RET intragenic SNPs that were genotyped in patients with MTC alone or HSCR/MTC from a newly reported FMTC/MEN2 family.

*

59

SNP*

Location

rs2505533 Novel SNP rs2505532 rs72781232 rs2505531 rs2565206 rs1800858 rs1864404 Novel SNP rs1800860 rs3026750 rs34827976 rs1799939 rs1800861 rs1800862 rs1800863

Intron 1

Exon 2 Intron 5 Intron 7 Exon 7 Intron 8 Exon Exon Exon Exon

11 13 14 15

DNA c.74-1454T.C c.74-1379T.A c.74-1362C.T c.74-1329G.A c.74-1290T.C c.74-126G.T c.135G.A c.1064-89A.T c.1522+58C.A c.1296A.G c.1648+84G.A c.1648+88delC c.2071G.A c.2307T.A c.2508C.T c.2712C.G

Please see http://www.ncbi.nlm.nih.gov/snp.

Protein / / / / / / p.A45A / / p.A432A / / p.G691S p.L769L p.S836S p.S904S

Hirschprung disease and MEN2 Quedas EPS et al.

CLINICS 2012;67(S1):57-61

only, whereas MEN2 patients and healthy controls are frequently heterozygous for this SNP (2,31).

In summary, we have presented new evidence supporting the role of a RET haplotype, which is not linked to the C620R mutation, in the development of HSCR in a newly reported FMTC/MEN2 family. Our findings support the hypothesis that the occurrence of HSCR in MEN2 families is not caused by any additional RET-inactivating mutation. Rather, it is most likely due to the combination of low-penetrance RET SNPs in the previously recognized HSCR-risk region between SNPs 2505533 and 2565206. The TTCGTTAAAGC RET haplotype may be helpful as a genetic marker for HSCR, which would ultimately translate into improved medical care for these patients.

Funding: RAT is the recipient of a postdoctoral fellowship from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (2009/15386-6), TS is the recipient of a doctoral FAPESP fellowship (11942/2009), FLC is the recipient of a Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) fellowship and SPAT is supported by a CNPq senior research productivity fellowship. This work was supported by a CNPq grant (401990/2010-9).

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AUTHOR CONTRIBUTIONS Toledo RA designed and conceived the study, performed the experiments, conducted the analysis, and contributed to discussions. Toledo SPA designed and conceived the study, provided important input, supplied the clinical data, and contributed to discussions. Quedas EPS, VC Longuini, and Sekiya T performed the experiments, conducted the analysis and contributed to discussions. Coutinho FL and Tannuri U provided clinical data and contributed to discussions.

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Cys618Arg mutation in the RET proto-oncogene associated with familial medullary thyroid carcinoma and maternally transmitted Hirschsprung’s disease suggesting a role for imprinting. Hum Mutat. 1997;10(2):155-9, http://dx.doi.org/10.1002/(SICI)1098-1004(1997)10:2,155::AIDHUMU7.3.0.CO;2-J. 10. Borrego S, Eng C, Sanchez B, Saez ME, Navarro E, Antinolo G. Molecular analysis of the ret and GDNF genes in a family with multiple endocrine

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Cohen MS, Phay JE, Albinson C, DeBenedetti MK, Skinner MK, Lairmore TC, et al. Gastrointestinal manifestations of multiple endocrine neoplasia type 2. Ann Surg. 2002;235(5):648-55, http://dx.doi.org/10.1097/ 00000658-200205000-00006.

Toledo SP, dos Santos MA, Toledo RA, Lourenc¸o DM Jr. Impact of RET proto-oncogene analysis on the clinical management of multiple endocrine neoplasia type 2. Clinics. 2006;61(1):59-70, http://dx. doi.org/10.1590/S1807-59322006000100011.

Tannuri U, Tannuri AC. Congenital megacolon in the newborn: which is the best treatment? Rev Assoc Med Bras. 2007;53(2):100-1, http:// dx.doi.org/10.1590/S0104-42302007000200008.

Coelho MC, Tannuri U, Benditt I, Santos MM. Studies of RET gene expression and acetylcholinesterase activity in a series of sporadic Hirschsprung’’s disease. Pediatr Surg Int. 2008;24(9):1017-21, http:// dx.doi.org/10.1007/s00383-008-2207-8.

Santos MM, Tannuri U, Coelho MC. Study of acetylcholinesterase activity in rectal suction biopsy for diagnosis of intestinal dysganglionoses: 17-year experience of a single center. Pediatr Surg Int. 2008;24(6):715-9, http://dx.doi.org/10.1007/s00383-008-2141-9.

Tannuri AC, Tannuri U, Roma˜o RL. Transanal endorectal pull-through in children with Hirschsprung’s disease: technical refinements and comparison of results with the Duhamel procedure. J Pediatr Surg.

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Correia-Deur JE, Toledo RA, Imazawa AT, Lourenc¸o DM Jr, Ezabella MC, Tavares MR, et al. Sporadic medullary thyroid carcinoma: clinical data from a university hospital. Clinics (Sao Paulo). 2009;64(5):379-86, http://dx.doi.org/10.1590/S1807-59322009000500002.

Toledo SP, Lourenc¸o DM Jr, Santos MA, Tavares MR, Toledo RA, Correia-Deur JE. Hypercalcitoninemia is not pathognomonic of medullary thyroid carcinoma. Clinics (Sao Paulo). 2009;64(7):699-6, http:// dx.doi.org/10.1590/S1807-59322009000700015.

Frank-Raue K, Rybicki LA, Erlic Z, Schweizer H, Winter A, Milos I, et al.

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Bordeaux MC, Forcet C, Granger L, Corset V, Bidaud C, Billaud M, et al.

The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease. EMBO J. 2000;19(15):4056-63, http://dx.doi.org/ 10.1093/emboj/19.15.4056.

Nishikawa M, Murakumo Y, Imai T, Kawai K, Nagaya M, Funahashi H, et al. Cys611Ser mutation in RET proto-oncogene in a kindred with medullary thyroid carcinoma and Hirschsprung’s disease. Eur J Hum Genet. 2003;11(5):364-8, http://dx.doi.org/10.1038/sj.ejhg.5200971.

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Carlomagno F, De Vita G, Berlingieri MT, De Franciscis V, Mellilo RM, Colantuoni V, et al. Molecular heterogeneity of RET loss of function in Hirschsprung’s disease. EMBO J. 1996;15(11):2717-25.

Iwashita T, Murakami H, Asai N, Takahashi M. Mechanism of Ret dysfunction by Hirschsprung mutations affecting its extra cellular domain. Hum Mol Genet. 1996;5(10):1578-80, http://dx.doi.org/ 10.1093/hmg/5.10.1577.

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Association of multiple endocrine neoplasia type 2 and Hirschsprung disease. J Intern Med. 1998;243(6):515-20, http://dx.doi.org/10.1046/j.13652796.1998.00332.x.

Carrasquillo MM, McCallion AS, Puffenberger EG, Kashuk CS, Nouri N, Chakravarti A. Genome-wide association study in Mennonites identifies multiple genes for oligogenic Hirschsprung disease. Am J Hum Genet.

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32. Ferna´ndez RM, Antin˜olo G, Eng C, Borrego S. The RET C620S mutation causes multiple endocrine neoplasia type 2A (MEN2A) but not Hirschsprung disease (HSCR) in a family cosegregating both phenotypes.

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33. Ruiz-Ferrer M, Torroglosa A, Luzo´n-Toro B, Fernandez RM, Antinolo G, Mulligan LM, et al. Novel mutations at RET ligand genes preventing receptor activation are associated to Hirschsprung’s disease. J Mol Med (Berl). 2011;89(5):471-80, http://dx.doi.org/ 10.1007/s00109-010-0714-2.

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35. Toledo RA, Wagner SM, Coutinho FL, Lourenc¸o DM Jr, Azevedo JA, Longuini VC, et al. High penetrance of pheochromocytoma associated with the novel C634Y/Y791F double germline mutation in the RET protooncogene. J Clin Endocrinol Metab. 2010;95(3):1318-27, http://dx.doi.org/ 10.1210/jc.2009-1355.

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DOI:10.6061/clinics/2012(Sup01)12

REVIEW

The Hirschsprung’s–multiple endocrine neoplasia connection Sam W. Moore,I Monique ZaahlII I Division of Pediatric Surgery, Faculty of Medicine, University of Stellenbosch, Tygerberg, South Africa. II Division of Human Genetics, Faculty of Medicine, University of Stellenbosch, Tygerberg, South Africa.

The risk of patients with Hirschsprung’s disease later developing multiple endocrine neoplasia remains a matter of concern. The multiple endocrine neoplasia 2–Hirschsprung’s disease association has been shown to cosegregate in Hirschsprung’s disease patients with both short- and long-segment aganglionosis, although patients with longsegment aganglionosis a to carry the greatest risk. The Hirschsprung’s disease–medullary thyroid carcinoma relationship also appears to be bi-directional, and activation or suppression of the rearranged during transfection gene appeared to vary over succeeding generations within the same family. Rearranged during transfection gene variations are associated with both conditions. The cosegregation of Hirschsprung’s disease and multiple endocrine neoplasia 2 is particularly interesting as it involves both ‘‘switch off’’ and ‘‘switch on’’ of the rearranged during transfection proto-oncogene in the same patient. This cosegregation mostly relates to the cysteine-rich area on RET620 (the ‘‘Janus gene’’). The mechanism whereby rearranged during transfection influences gene activation in multiple endocrine neoplasia 2 is complex, but genetic variations impair the rearranged during transfection tyrosine kinase response to tyrosine kinase activation, thus appearing to dictate downstream signaling cascade responses.

Better understanding of the RET-620 relationship allows for a more cost-effective method of identifying those at risk by focusing rearranged during transfection gene testing to this specific area as a ‘‘hot spot’’. The clinical awareness of possible medullary thyroid carcinoma has led to timely intervention and early treatment of this chemo- and radioresistant tumor with poor prognosis. Establishment of ‘‘risk’’ by genetic testing has become a classic model of molecular medicine being integrated into patient care and offering rearranged during transfectiondirected prophylactic surgical management. In addition, novel approaches to treatment based on this genetic knowledge have already shown early promise in randomized clinical trials.

KEYWORDS: Hirschsprung’s disease; Genetics; Novel mutations; RET; Endothelin receptor B gene.

Moore SW, Zaahl M. The Hirschsprung’s–multiple endocrine neoplasia connection. Clinics. 2012;67(S1):63-67.

E-mail: swm@sun.ac.za Tel.: 27 21 9389280

transfection) proto-oncogene tyrosine kinase which is situated at chromosome 10q11.2.

There are early reports of associations between HSCR and a number of associated anomalies related to neural cell development. These include neuroblastoma (1), phaeochromocytoma (1,4,5) and multiple endocrine neoplasia (MEN) type 2 A and B syndromes (5–7), among others (1). Reports on this relatively uncommon cosegregation of HSCR and MEN2 in the same patient (6,8–20) exist because of the common factor of the RET gene being associated with both conditions (HSCR, MEN type 2 and medullary thyroid carcinoma (MTC)). This is an extremely interesting observation, as it involves both gain and loss of function of the same gene in the same patient.

INTRODUCTION Hirschsprung’s disease (HSCR), or aganglionic megacolon, occurs due to the congenital absence of parasympathetic neuronal ganglia in a segment of the hindgut, resulting in malfunction of the affected segment. The condition presents as a common cause of intestinal obstruction in neonates, with an incidence of 1 in 5,000 (0.02%) live births and an overall risk to siblings of 4% (1– 3). A study of HSCR indicates several clear-cut associations that are known or are suspected to be related to an increased risk of HSCR. These include its recurrence in families and the association with the major susceptibility genes points convincingly to genetic factors in its multifactorial pathogenesis. The main susceptibility gene for Hirschsprung’s disease is the RET (rearranged during

Which patients are at risk?

The MEN–HSCR association has been shown to cosegregate in HSCR patients with both short- and long-segment aganglionosis (21). It seems to be particularly associated with long-segment aganglionic segments, and Decker et al.

(21) reported a long aganglionic segment (L-HSCR) in seven out of 13 patients (54%). Our experience is of total colonic aganglionosis (TCA) in the index patient in all three families

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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CLINICS 2012;67(S1):63-67

appear later in life without the connection being made. As a result, the risk of HSCR patients later developing features of an MEN2 syndrome (with MTC) is thought to be underevaluated. The risk of a future thyroid carcinoma remains a matter of general concern in long-term HSCR management.

It is becoming clear that there is a need for screening in order to determine which patients require RET gene analysis as a means of determining risk.

The most frequently identified mutation responsible for MEN2A involves the substitution of the cysteine amino acid at position 634 (Cys634Arg or C634R) by the amino acid arginine. However, point mutations in the cysteine-rich RET area at the 620 position, along with some other RET gene areas (e.g. C609, C611 and C618) have been reported as being mostly associated with the HSCR–MEN2 phenotype.

By way of contrast, in .90% of MEN2B patients, the amino acid methionine is replaced by threonine at position 918 (M918T) in the intracellular portion of the gene. There are also a number of other RET variations that are associated with thyroid cancers and familial medullary thyroid carcinoma which may give rise to an overactive abnormal RET protein.

The cosegregation of HSCR and MEN2 is particularly interesting as it involves both a ‘‘switch off’’ and a ‘‘switch on’’ of the gene in the same patient. Some remarkable family lineages have been reported which demonstrate the occurrence of the two conditions in family lineages (17,22).

identified in our series (22). It therefore appears that patients with long-segment HSCR carry the highest risk of developing MTC and should have a detailed family history taken: the presence of a long-segment HSCR should be an important selection criterion for gene testing in HSCR. It is important to explore this concept further in families where HSCR and MTC coexist, as it will yield possible RET gene associations and insights into possible molecular reasons for the phenotypic expression. It is generally accepted that aberrant RET protein synthesis, due to inactivating genetic variations, lead to the congenital malformation of the enteric nervous system (ENS) which we call Hirschsprung’s disease.

RET gene activation occurs in MEN2A. The HSCR–MTC relationship also appears to be bidirectional, and RET gene activation or suppression appeared to vary over succeeding generations within the same family (22). Butter et al. (23) reported a 50% incidence of HSCR in 20 patients undergoing a prophylactic thyroidectomy for RET-associated MTC risk (a RET C620 mutation). In one further reported case of familial MTC (24) with a C620S point mutation, the MTC developed 12 years after surgical correction of HSCR in the child plus a maternal MTC 7 years after the child’s birth. In our reported series (22), MTC was detected in the parent 5 years after the birth of the affected child.

Hirschsprung’s, MEN and the RET proto-oncogene The RET proto-oncogene [10q11.22] is the major gene involved in the pathogenesis of HSCR with causative lossof-function mutations being identified in more than 70% of cases (25). In essence, the extracellular domain RET mutations alter the protein and possibly its processing in the endoplasmic reticulum (26). As a result, RET transport to and its expression at the cell surface is decreased.

Multiple endocrine neoplasia also results from autosomally dominant, inherited, highly penetrant germline RET mutations that predispose patients to the development of tumors in cells derived from neural crest origin. Of the more than 25 RET proto-oncogene gene mutations which have been described in association with MEN type 2 syndromes, the most important are associated with the six cysteine alleles of the extracellular portion of the RET protooncogene in MEN2A. RET is a vital gene which directs the migration, proliferation and survival of the enteric neural crest-derived cells of the enteric nervous system (ENS) during embryogenesis. It is also responsible for the development of the autonomic nervous system as well as for controlling kidney development and spermatogenesis, among other functions.

Genetic mutation and/or variation may result in RET malfunction, which has been associated with at least four clinical conditions (HSCR, MEN type 2 syndromes (A and B) and familial medullary thyroid carcinoma (FMTC)). In HSCR, hypomorphic RET alleles may cause delayed neuroblast migration and non-apoptotic cell death. This causes ENS maldevelopment, resulting in aganglionosis (HSCR) and other related conditions of the ENS. The presence of a HSCR–MEN2 association raises the question of which HSCR patients are at greatest risk and should therefore be further investigated by RET gene analysis. Hereditary MTC can, with rare exceptions, be traced back in family trees (27) and the HSCR–MEN2 familial association may be missed because the MTC may

Which RET gene variations are important?

Like other receptor tyrosine kinases (RTKs), RET is made up of at least three functional areas (extracellular, transmembrane and intracellular regions) which appear to have specific roles in cellular function (28). It is interesting that, in contrast to other susceptibility areas, the RET mutations which have been identified as carrying both a risk for HSCR as well as MEN2 approximate the transmembrane domain of the gene (mostly C620R and occasionally C620S and rarely C620W) (21). Although the majority of reports connect these two conditions with point mutations in the cysteinerich RET-area at the 620 position, other RET gene areas (e.g.

C609, C611 and C618) have been reported as alternative sites. The high frequency with which the C620 RET mutation occurs in HSCR–MEN patients suggests the concept of the so-called ‘‘Janus gene’’ mutation in this position, which, like the Roman god of doorways, can face in both directions (i.e. activation (MEN/MTC) and inactivation (HSCR)).

Our own studies (22) have shown the RET-620 cysteine variations to be present in 2.5% of HSCR families (three out of 118 families investigated), which is in keeping with other reports (21). There was, however, an uncertain prevalence of HSCR–MEN in related family members as not all could be tested.

Studies have also shown that the closer mutations are to the transmembrane domain, the higher their importance in terms of the development of cellular proliferation and tumor activation (29). A strong self-association has been shown in the transmembrane portion of the gene (RET-TM), which has been suggested as a possible explanation of the oncogenic activation due to RET–cysteine mutations in oncogenesis (29). This suggests an area that plays an important role in MTC risk evaluation and may identify a target area for novel therapeutic strategies.

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Hirschsprung’s–MEN connection Moore SW and Zaahl M

RET expression in HSCR (26). Changes in polarity in MEN2A–HSCR may result in the unfolding of the RET protein initially failing to achieve homeostasis via the unfolded protein response (UPR) of the endoplasmic reticulum, leading to apoptosis and HSCR. Subsequent upregulation of the UPR may then induce changes which allow gene activation, thus providing a growth advantage to tumor cells.

Oncogenic RET mutations affect protein synthesis and function, which then influence a number of downstream molecular signaling pathways (43). Studies have demonstrated that Ret influences the downstream phosphorylation of the insulin receptor substrate-2 (IRS 2) with subsequent activation of phosphatidylinositol 3 kinase (PI 3-kinase) and protein kinase B (PKB/AKT) signaling pathways (44). In addition, the induction of phosphorylation of SHC results in growth factor receptor binding protein 2 (Grb-2) binding and activation of the mitogen-activating protein (MAP) kinase pathway (45). The resulting MAPK IRS-2, PI 3-kinase and PKB/AKT activation could transduce the oncogenic Ret signal to promote gene transcription, possibly through activation of the jun/AP-1 transcription cascades (44).

How does RET gene variation result in cellular activation?

Because MEN2A relates mostly to the RET alleles at the six cysteine positions in the extracellular domain of the gene, it appears to induce a different set of downstream signaling genes from that carrying the MEN 2B mutation.

The multidocking intracellular portion of the RET gene where MEN2B RET variations are situated appears to be vital to both tyrosine kinase function and downstream signaling due to the number of signaling molecules that interact there (e.g. Shc, Src, FRS2, IRS1, Gab1/2, and Enigma) (30–32). It is therefore easier to understand how mutations within this tyrosine-kinase rich region (e.g. in 95% of MEN2B patients) alters the substrate specificity of RET tyrosine kinase and results in gene activation.

The mechanism whereby RET influences gene activation in MEN2A is more complex. Genetic variations impair the RET tyrosine kinase response to tyrosine kinase activation, thus appearing to dictate downstream signaling cascade response (33). The induction of a disulfide-linked homodimerization resulting from the cysteine-related mutations within the extracellular portion of the RET proto-oncogene promote dimer formation within the gene as covalent receptor dimers are linked by disulfide bonds between unpaired cysteines (34,35). It is known that the RET protein targets a number of intracellular signaling cascades, including the RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription pathways (36). The activation of the MAPK and Ras/ ERK molecular signaling cascades involves grb2/mSOS recruitment (37). In addition, recent studies have suggested that the mammalian target of the rapamycin (mTOR) intracellular signaling pathway is functionally activated in MTC (especially in those with germline RET mutations) (38), and that the role of the other downstream RET tyrosine kinase signaling cascades (e.g. BRAF, RAS isoforms and PI3 kinase) is less certain.

Impact on treatment This paper draws attention to the apparent genotype– phenotype correlation which exists between RET and the MEN2 syndromes (46,47). Clinical awareness of possible MTC has led to timely intervention and early treatment of this poor prognosis chemo- and radioresistant tumor.

Establishment of ‘‘risk’’ by genetic testing has become a classic model of molecular medicine being integrated into patient care. It is therefore fairly clear that RET gene screening is of considerable value in familial screening, and offering RET testing is considered best practice for the clinical management of patients at risk of MEN2A and MEN2B. Gene mutation in the RET 620 position carries significant risk of developing HSCR and/or MTC. In the interests of cost-effectiveness, it may be part of a targeted investigation of high-risk areas of the gene in HSCR patients at risk, thus facilitating follow-up and future management. Genetic screening is an extremely sensitive method of identifying risk in MEN2 syndromes (¡98%) and prophylactic surgical intervention may prevent the onset of the radio- and chemoresistant MTC (48).

Better understanding of the RET-620 relationship would also allow for a more cost-effective method of identifying those at risk by focusing RET gene testing to this specific area as a ‘‘hot spot’’. In addition, a number of attractive targets are being identified which may be suitable for novel therapeutic signaling pathway-specific drug design (49).

Novel approaches to treatment based on this genetic knowledge have already resulted in randomized clinical trials into the effectiveness of multi-kinase inhibitors such as axitinib, sorafenib, motesanib, and XL-184. These have shown early promise in selected cases which may soon change the management of thyroid cancer (50).

How does the RET-620 relationship work?

Oncogenic RET mutations mostly occur de novo in sporadic MTC, and it is now well accepted that RET gene variations produce aberrant proteins which may bypass the normal linking to growth factors outside the cell, most probably triggering cells to grow and divide abnormally and, in turn, promoting tumor formation. Ret activation then promotes Ret-dimer formation and activation of the tyrosine kinase outside of the normal ligand binding system.

However, little is known of the actual mechanisms whereby extracellular domain mutations (e.g. C620S) may result in C-cell proliferation (39). Current understanding of RET gene activation in MTC is that the gene is activated by alterations in autophosphorylation, or alternatively by the modification of the subcellular distribution of the active kinase or the induction of unusual interactions with other proteins (e.g. RET/PTC) (40).

It is possible that the MEN2A–HSCR connection represents a second alternative intracellular mechanism in the pathogenesis of MTC (41). The less common codons C620, C618 and C611 within exon 10 confer a weaker transforming activity in vitro than exon 11 (C634) RET mutations (42). It has been suggested that the MEN2A–HSCR connection represents a second alternative intracellular mechanism in the pathogenesis of MTC involving the endoplasmic reticulum which has been shown to decrease cell surface

AUTHOR CONTRIBUTIONS Moore SW is an expert on the genetic connections and other aspects of Hirschsprung’s disease and MEN2 syndromes. He wrote the review and was responsible for the clinical component and interpretation of results.

Zaahl M is a scientist who collaborated for a long period on this project.

She is the head of the Department of Genetics at Stellenbosch University

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22. Moore SW, Zaahl M. Familial associations in medullary thyroid carcinoma with Hirschsprung disease: the role of the RET-C620 ‘‘Janus’’ genetic variation. J Pediatr Surg. 2010;45:393–6.

23. Butter A, Gagne J, Al Jazaeri A, Emran MA, Deal C, St Vil D. Prophylactic thyroidectomy in pediatric carriers of multiple endocrine neoplasia type 2A or familial medullary thyroid carcinoma: mutation in C620 is associated with Hirschsprung’s disease. J Pediatr Surg.

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and runs the laboratory where the genetic work was carried out. She personally oversaw most of the laboratory work presented in this study.

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11. Peretz H, Luboshitsky R, Baron E, Biton A, Gershoni R, Usher S, et al. Cys 618 Arg mutation in the RET proto-oncogene associated with familial medullary thyroid carcinoma and maternally transmitted Hirschsprung’s disease suggesting a role for imprinting. Hum Mutat. 1997;10:155–9, http://dx.doi.org/10.1002/(SICI)1098-1004(1997)10:2,155::AIDHUMU7.3.0.CO;2-J. 12. Romeo G, Ceccherini I, Celli J, Priolo M, Betsos N, Bonardi G, et al.

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DOI:10.6061/clinics/2012(Sup01)13

REVIEW

Genotype-phenotype correlation in multiple endocrine neoplasia type 2 Friedhelm Raue, Karin Frank-Raue Endocrine Practice, Heidelberg, Germany.

Multiple endocrine neoplasia type 2 is an autosomal-dominant hereditary cancer syndrome caused by missense gain-of-function mutations of the rearranged during transfection proto-oncogene, which encodes the receptor tyrosine kinase, on chromosome 10. It has a strong penetrance of medullary thyroid carcinomas and can be associated with bilateral pheochromocytoma and primary hyperparathyroidism. Multiple endocrine neoplasia type 2 is divided into three varieties depending on its clinical features: multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, and familial medullary thyroid carcinoma. The specific rearranged during transfection mutation may suggest a predilection toward a particular phenotype and clinical course of medullary thyroid carcinoma, with strong genotype-phenotype correlations. Offering rearranged during transfection testing is the best practice for the clinical management of patients at risk of developing multiple endocrine neoplasia type 2, and multiple endocrine neoplasia type 2 has become a classic model for the integration of molecular medicine into patient care. Recommendations on the timing of prophylactic thyroidectomy and extent of surgery are based on the classification of rearranged during transfection mutations into risk levels according to genotype-phenotype correlations. Earlier identification of patients with hereditary medullary thyroid carcinoma can change the presentation from clinical tumor to preclinical disease, resulting in a high cure rate of affected patients and a much better prognoses.

KEYWORDS: Medullary Thyroid Carcinoma; Pheochromocytoma; Primary Hyperparathyroidism; RET-protooncogene; Prophylactic Thyroidectomy.

Raue F, Frank-Raue K. Genotype-phenotype correlation in multiple endocrine neoplasia type 2. Clinics. 2012;67(S1):69-75.

E-mail: friedhelm.raue@raue-endokrinologie.de Tel.: 00446221-439090

much better prognoses (2). Each variant of MEN2 results from a different mutation of the RET gene with good genotype-phenotype correlations.

INTRODUCTION Multiple endocrine neoplasia type 2 (MEN2) is an autosomal-dominant hereditary cancer syndrome caused by missense mutations in the RET (REarranged during Transfection) proto-oncogene, and these result in the gainof-function of the encoding receptor tyrosine kinase (1). The estimated prevalence is 2.5 per 100,000 in the general population. MEN2 shows a high penetrance for medullary thyroid carcinoma (MTC), which is a rare calcitonin (Ct)secreting tumor derived from thyroid parafollicular or Ccells of the thyroid; these cells are themselves derived from the neural crest. Most patients with MTC have the sporadic (i.e., non-familial) form of the tumor, while 25–30% present with the hereditary form. MEN2 syndrome occurs in three clinically distinct varieties with variable penetrance of MTC, pheochromocytoma, and hyperparathyroidism. Early identification of patients with MTC changes the presentation from a clinically evident tumor to a preclinical disease, resulting in a higher cure rate of the affected patients with

Phenotype: clinical description of MEN2 Three distinct clinical subtypes of MEN2 have been characterized (Table 1). They differ with respect to incidence, genetics, age-related penetrance, association with other diseases, aggressiveness of MTC, and prognosis (3,4).

MEN2A syndrome (OMIM#171400) is characterized by the presence of MTC, bilateral pheochromocytoma and/or multiple tumors of the parathyroid glands (primary hyperparathyroidism) within a single patient or family. It is the most common form of MEN2 syndrome, representing 55% of cases (5). The frequency of MTC is .90% among patients with MEN2A, while the frequencies of pheochromocytoma and multiple parathyroid gland hyperplasia are 40–50% and 10–20%, respectively. MTC is generally the first manifestation of MEN2A and presents when patients are between 5 and 25 years of age. Rare variants of MEN2A exist, including MEN2A with cutaneous lichen amyloidosis (this skin lesion is located over the upper portion of the back) and MEN2A/ familial MTC (FMTC) with Hirschsprung’s disease (see below).

MEN2B (OMIM#162300) is characterized by the association between MTC, pheochromocytoma, the absence of hyperparathyroidism, and visible physical stigmata such as

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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Table 1 - Clinical classification of MEN2 and FMTC, and occurrence of MTC, associated tumors and other diseases.

Subtype

Percentage of total cases

Typical age of onset (years)

MTC (%)

Pheo (%)

HPT (%)

Associated diseases

MEN2A MEN2B FMTC

56 9 35

10 2 30

100 100 95

50 50

25

Cutaneous lichen amyloidosis, Hirschsprung disease Ganglioneuromatosis, marfanoid habitus Rare

FMTC: familial medullary thyroid carcinoma. HPT: primary hyperparathyroidism. MEN2: multiple endocrine neoplasia type 2. MTC: medullary thyroid carcinoma. Pheo: pheochromocytoma.

raised bumps on the lips and tongue (caused by cutaneous neuromas), ganglioneuromas of the intestine, and asthenic marfanoid body habitus with skeletal deformations and joint laxity. Pheochromcytomas occur in 50% of individuals with MEN2B, approximately half of these are multiple and often bilateral; the development of mucosal neuromas on the tongue and lips (bumpy lips) (resulting in a distinctive facial appearance), together with a marfanoid body habitus, allows for diagnosis at a first glance. Early diagnosis by clinical means is difficult because of the gradual development of the typical clinical appearance during childhood.

Diffuse ganglioneuromatosis of the gastrointestinal tract is responsible for the megacolon, constipation and diarrhea that often present. Because of their similar clinical presentations, the clinician should carefully differentiate the diagnosis of Hirschsprung disease as a result of aganglioneuromatosis from MEN2B. Brauckhoff et al. reported that 86% of patients with MEN2B demonstrate an inability to cry tears (6). It is the rarest form of MEN2 and accounts for 5– 10% of MEN2 cases. Patients with MEN2B typically present with MTC during the first year of life and have a more aggressive form of MTC with higher morbidity and mortality rates than patients with MEN2A (4). Patients with MEN2B often do not have a family history of the disease; in more than 50% of cases the syndrome is due to a de novo germline RET mutation.

FMTC (OMIM#155240) is characterized by a strong predisposition to MTC in families with a very low incidence of other endocrinopathies related to MEN2. It is clinically diagnosed in families with four or more cases of MTC. It is viewed as a phenotypic variant of MEN2A with decreased penetrance of pheochromocytoma and primary hyperparathyroidism. It has been diagnosed more frequently in recent years and is reported to account for 35–60% of all MEN2 cases (5,7-9). Regarding MTC in FMTC, penetrance is lower and the clinical course is more benign than in MEN2A and MEN2B, with a late onset or no clinically manifesting disease and the prognosis is relatively good. Therefore, a family history is often inadequate for establishing familial disease.

to MTC varies with different germline RET mutations. MTC is diagnosed when nests of C-cells extend beyond the basement membrane and infiltrate and destroy thyroid follicles. MTC in MEN2 patients is multicentric and concentrated in the upper third of the thyroid gland. The peak incidence of sporadic MTC occurs in the fifth decade of life, while hereditary MTC can be diagnosed earlier, depending on the availability of genetic and biochemical screenings. The mode of discovery of MTC has changed, based on the use of specific strategies: serum Ct screening is used for patients with thyroid nodules (11) and genetic screening for RET proto-oncogene mutations is used for all patients with MTC (9). The earlier identification of patients with MTC has altered the presentation from symptomatic tumors to a preclinical disease, resulting in a much better prognosis with a higher cure rate. The average 5-year survival for MTC is 83%, which is lower than that for papillary and follicular thyroid cancers. The most important predictor of survival is the tumor stage at diagnosis.

Metastasis may be found first in the central, lateral cervical and mediastinal lymph nodes of the neck in 30% of patients with MTC ,1 cm in diameter. Metastases outside the neck and mediastinum may develop during the course of the disease, predominantly in the lung, liver, and bone.

The primary secretory product of MTC is Ct, which serves as a tumor marker for MTC. The normal concentration of mature Ct is ,10 pg/ml and after stimulation it is ,100 pg/ml. The Ct secretagogues are pentagastrin (0.5 mg/kg body weight), which is no longer available in the USA, and calcium (0.25 mg/kg body weight) (12). Basal Ct levels are elevated in some patients with renal insufficiency, autoimmune thyroid disorders or hypergastrinemia. This has to be taken into account when discussing elevated Ct levels. Either basal or stimulated plasma Ct levels using pentagastrin or calcium are elevated in virtually all patients with MTC. Basal Ct concentrations usually correlate with tumor mass and are almost always high in patients with palpable tumors (13). Similarly, following surgery to remove a tumor, elevated plasma Ct levels are indicative of persistent or recurrent disease.

Medullary thyroid carcinoma (MTC)

Pheochromocytoma

MTC originates from neural crest-derived Ct-producing cells (C-cells). Hereditary MTC characteristically presents as a multifocal process with C-cell hyperplasia (CCH) as the precursor lesion to MTC. Only familial primary CCH is a preneoplastic lesion. Secondary CCH, which has been associated with chronic lymphocytic thyroiditis, hypergastrinemia, other follicular-cell-derived thyroid tumors and even aging, has a much lower, if any, potential for malignancy (10). The time frame for the progression from CCH to microscopic carcinoma in MEN2 remains unclear, but it may take years; the age of transformation from CCH

The clinical manifestation of pheochromocytoma associated with MEN2 is similar to that seen in sporadic cases, with signs and symptoms such as headache, palpitations, nervousness, tachycardia, and hypertension. Measurement of plasma and/or 24-h urinary excretion of catecholamine metabolites (e.g., epinephrine, norepinephrine, metanephrine and normetanephrine) should be performed. Once the biochemical diagnosis is made, imaging studies, such as abdominal magnetic resonance imaging and/or computed tomography, in addition to metaiodobenzylguanidine scanning and/or fluorodopamine positron emission tomography,

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are appropriate. Pheochromocytoma in MEN2 is almost always located in the adrenal and is almost always benign.

The possibility of bilateral disease must be carefully evaluated. The presence of pheochromocytoma must be ruled out prior to any surgical procedure because if it is untreated it can be lethal. Endoscopic adrenal-sparing surgery has become the method of choice. The penetrance of pheochromocytoma among different MEN2 kindreds depends on specific RET germline mutations (see below) (14). Pheochromocytoma usually becomes evident approximately 10 years later than the manifestation of MTC. Therefore, it is usually identified when screening patients with MEN2, often before clinical symptoms present.

Biochemical screening for pheochromocytoma should be performed annually in MEN2 patients depending on their genotype (see below), beginning at 8 years of age for carriers of RET mutations associated with MEN2B and codon 634 mutations and by the age of 20 years for carriers of other codon mutations. Because of the high risk to the fetus and the mother, women with a RET mutation associated with MEN2 should be screened for pheochromocytoma before pregnancy.

and is considered to be standard care for all first-degree relatives of patients with newly diagnosed MTC. It is also recommended for all patients with MTC, regardless of other features or family history, especially those with clinical features that are indicative of MEN2 and those with primary CCH. About 4–10% of patients with apparently sporadic MTC have germline mutations in the RET proto-oncogene and, consequently, present with the hereditary form (8).

The RET gene consists of 21 exons and encodes a receptor tyrosine kinase that appears to transduce growth and differentiation signals in several developing tissues, including those derived from the neural crest. The transcript length is 5659 base pairs and is translates to a 1114 amino acid-residue protein. The RET protein consists of an extracellular segment with a ligand-binding domain, a cadherin (Ca2+-dependent cell adhesion)-like domain, and a cysteine-rich domain that is positioned near the cell membrane. The RET protein has a single transmembrane domain and an intracellular segment with two tyrosine kinase subdomains, TK1 and TK2. It is activated by ligandinduced dimerization (19). RET binds ligands of the glialderived neurotrophic factor (GDNF) family in conjunction with a co-receptor, designated GDNF-family receptor a, to a complex that triggers autophosphorylation and intracellular signaling. RET is produced in neuroendocrine cells, including C-cells of the thyroid (the precursors of MTC) and pheochromocytomas.

Hereditary MTC is caused by autosomal-dominant gainof-function mutations in the RET proto-oncogene. Mutation of the extracellular cysteine in codon 634 in exon 11 causes ligand-independent dimerization of receptor molecules, enhanced phosphorylation of intracellular substrates and cell transformation. Mutation of the intracellular tyrosine kinase (codon 918) has no effect on receptor dimerization but does cause constitutive activation of intracellular signaling pathways and also results in cellular transformation. There is a significant age-related progression from CCH to MTC that correlates with the transformative capacity of the particular RET mutation (2). Mutations that strongly activate the RET proto-oncogene are associated with a more aggressive form of MTC that presents earlier, and mutations that provide weaker RET activation result in a less aggressive form of the disease that presents later. On the basis of this finding, a risk-stratification strategy has been developed (see below). MTC is generally the first neoplastic manifestation seen in patients with MEN2A because of its earlier age of onset and higher rate of penetrance compared with pheochromocytoma or parathyroid hyperplasia. This indicates that C-cells are more susceptible to oncogenic RET activation than adrenal medullary or parathyroid cells.

RET has also been implicated in congenital aganglionosis (absence of enteric nerve cells) in the gastrointestinal tract (Hirschsprung disease). The lack of neuroenteric plexi impairs smooth muscle activity of the intestines (particularly the colon), resulting in refractory constipation. Up to 50% of familial cases and up to 35% of simplex cases (i.e., a single occurrence in a family) of Hirschsprung disease are caused by germline loss-of-function mutations in the RET proto-oncogene (20). There are some families and individuals harboring germline RET mutations in exon 10 that cosegregate with MEN2A/FMTC and Hirschsprung disease.

Because of a seemingly similar clinical presentation, clinicians should be careful to differentiate the diagnosis

Primary Hyperparathyroidism Primary hyperparathyroidism is often clinically occult and does not differ from that seen in patients with mild sporadic primary hyperparathyroidism. It has been reported in 10–25% of patients with MEN2A, but is rarely the first manifestation of this syndrome (15,16). Diagnosis is established by detecting high concentrations of intact parathyroid hormone in the presence of hypercalcemia, usually after the third decade of life. Pathological findings show chief cell hyperplasia involving multiple glands and sometimes multiple adenomas. Annual measurement of the serum calcium concentration and parathyroid hormone in gene carriers is probably adequate for screening purposes, and should begin by age 8 years for carriers of RET mutations in exon 10 and 11 and by age 20 years for carriers of other MEN2A RET mutations. The goal for MEN2 patients with primary hyperparathyroidism is to excise the enlarged glands and to leave at least one normal parathyroid gland intact. If the glands are all enlarged, a subtotal parathyroidectomy or total parathyroidectomy with autotransplantation should be performed.

Genotype of MEN2: RET proto-oncogene structure, function and abnormalities The genes responsible for MEN2 were found to be localized in centromeric chromosome 10 (10q11.2) by genetic linkage analysis in 1987. Subsequently, point mutations of the RET (REarranged during Transfection) proto-oncogene were identified in MEN2A, MEN2B, and FMTC in eight exons located near this region (Figure 1) (17,18). Analysis of RET in families with MEN2A and FMTC revealed that nearly all of these families have germline mutations, and that only those family members with the germline missense mutations have the disease. This discovery prompted major advances in the understanding of the molecular and genetic basis of MTC, and has significantly changed the clinical management of families with hereditary tumors. Missense mutations have been reported in exons 5, 8, 10, 11, and 13–16 of RET (http://arup.utah.edu/database/MEN2/MEN2_welcome.php) (Figure 1) (5). Genetic testing detects about 98% of mutation carriers

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Figure 1 - Germline mutations in the RET proto-oncogene associated with MEN2 and FMTC. Numbers indicate the mutated codons of the RET gene.

of Hirschsprung disease from the constipation/obstipation that result from ganglioneuromatosis associated with MEN2B.

other half are benign or uncertain variants (http://arup. utah.edu/database/MEN2/MEN2_welcome.php).

In newly detected germline RET sequence changes it is mandatory to clarify if the ‘‘mutation’’ is causative of MEN2 and if it segregates with the MEN2 disease symptoms within a family. The web-based Associated Regional and University Pathologists (ARUP) online Scientific Resource RET database (http://arup.utah.edu/database/MEN2/ MEN2_welcome.php) uses the following classification definition: mutation, polymorphism, and variant of unknown significance (VUS). Mutation is defined as causative for the disease and segregates with the disease if the following conditions are met: (1) if there are at least two affected family members and at least one has MTC and one other family member has one clinical feature of MEN2; (2) if one patient has MTC and another has MEN2 clinical features; or (3) if at least three unrelated individuals with MTC have the same germline RET sequence variant. Polymorphisms are harmless germline RET sequence changes that are not causative of MEN2, and they include G691S, L769L, S836S, S904S and intron 14 c.2608-24G.A. It is speculated that some of the normal allelic variants may be modifying factors in patients with pathologic RET mutations (23-25). VUS are RET sequence changes in which there is not enough clinical evidence to indicate a causative role, so further clinical studies, in vitro experiments (e.g., kinase activity assay), and prediction algorithms are necessary to clarify whether the sequence change is a mutation or a polymorphism. One

Genotype-phenotype correlations in MEN2 Clear associations have been documented between specific RET mutations (genotype) and the age of onset and aggressiveness of MTC and the presence or absence of other endocrine neoplasms (phenotype), such as pheochromocytoma or hyperparathyroidism (Table 2) (2,21,22). Some overlap exists between RET mutations and the resulting clinical subtype of MEN2. Approximately 98% of families with MEN2A have a RET mutation in exon 10 or 11.

Mutations in the cysteine of codon 634 occur in about 87% of families; 95% of families with FMTC have an identifiable RET mutation. These mutations typically occur in one of the five cysteine residues (codons 609, 611, 618, 620, and 634 in exons 10 and 11) or in exons 5, 8, and 13–16, with an important minority affecting codons 790, 791, 768, and 804; 95% of individuals with the MEN2B phenotype have a single-point mutation in the tyrosine kinase domain of the RET gene at codon 918 in exon 16, and this substitutes a threonine for methionine (M918T) (Figure 2). A second mutation at codon 883 in exon 15, A883F, has been identified in several affected individuals without a M918T mutation (4). In addition to these well-known and clinically well-characterized mutations, more than 140 RET variants are known. Half of these variants are pathogenic, and the

Table 2 - Management of patients with different RET mutations (4).

Characteristic/management a

ATA risk level (2009) MEN2 subtype MTC aggressiveness MTC age of onset Timing of prophylactic thyroidectomy Screening for Pheo Screening for HPT

Codons 321, 515, 533, 600, 603, 606, 635, 649, 666, 768, 776, 790, 791, 804, 819, 833, 844, 861, 891, 912

Codons 609, 611, 618, 620, 630, 631

A B FMTC FMTC/MEN2A Moderate High Adults 5 years When calcitonin rises/age 5 or 10 5 years years Start at 20 years, periodically Start at 20 years, annually Start at 20 years, periodically Start at 20 years, periodically

Codon 634

Codons 918, 883

C MEN2A Higher Before the age of 5 years Before the age of 5 years

D MEN2B Highest First year of life First months of life

Start at 8 years, annually Start at 8 years, annually

Start at 8 years, annually -

ATA: American Thyroid Association. FMTC: familial medullary thyroid carcinoma. HPT: primary hyperparathyroidism. MTC: medullary thyroid carcinoma.

Pheo: pheochromocytoma. a Risk for aggressive MTC; level D is the highest risk (4).

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Figure 2 - Distribution of RET mutations, as determined by the Molecular Genetics Laboratory, Heidelberg, Germany, 2000–2011.

mutations (exon 5, codon 321; exon 8, codons 531, 515 and 533; exon 10, codons 600, 603 and 606; exon 11, codons 649 and 666; exon 13, codons 768, 777, 790 and 791; exon 14, codons 804, 819, 833 and 844; and exon 15, codons 866, 891 and 912), which mainly include phenotypic FMTC, are at the lowest risk for the development and aggressiveness of MTC; patients with ATA B mutations (exon 10, codons 609, 611, 618 and 620; and exon 11, codons 630, 631 and 633) are at intermediate risk; patients with ATA C mutations, the classical MEN2A mutation (exon 11, codon 634) are at a higher risk than patients with ATA B mutations; and patients with ATA D mutations have the typical MEN2B mutation (exon 15, codon 883; and exon 16, codon 918) and are at the highest risk of developing MTC with a high rate of growth.

In the cases of these higher risk mutations, thyroidectomy is recommended before 5 years of age for patients with ATA C mutations and as early as possible, preferably within the first year after birth, for patients with ATA D mutations (4).

For patients with ATA A mutations, there are various recommendations regarding the appropriate age at which to perform prophylactic surgery: some authors suggest thyroidectomy at 5 years of age, some at 10 years, while others, including the present authors (27), suggest that surgery may be postponed until an abnormal C-cell stimulation test result is obtained (i.e., an abnormal Ct response to pentagastrin or calcium stimulation). However, further studies, particularly regarding rare and/or newly diagnosed mutations (26), are necessary before general recommendations can be made. In order to avoid complications such as recurrent laryngeal nerve injuries or hypopathyroidism, the operation should be performed by surgeons who perform a high volume of this type of procedure.

The association between disease phenotype and RET genotype may also have important implications for the management of clinical presentations other than MTC in MEN2 patients and their families. If the genotype could be fully correlated with certain phenotypic features, then a clinician could use a patient’s genotype to decide whether intense screening for pheochromocytoma or HPT is necessary in those patients with mutations associated with a higher risk of disease (Table 2). Pheochromocytoma is associated

example of this is the S649L and Y791F RET germline sequence variants that have been described and verified as mild mutations by some (26-28) and as non-pathogenic by others (29). Using the Bayes classification to predict the disease associations of uncertain gene variants into categories of benign and pathogenic, amino acid-substitution penalties, structural disruption, sequence homology (e.g., ortholog conservation) or neural nets, the RET Y791N sequence changes are classified as pathogenic (30). Further clinical studies are necessary to classify VUS as deleterious mutations before guiding therapy by the codon positions of these RET mutations.

Clinical implications of genotype-phenotype correlations Patients with a codon 918 mutation and MEN2B are at high risk of aggressive MTC occurring at a young age (31).

In contrast, patients with codon 791 mutations are at a relatively low risk of aggressive disease and develop slowgrowing tumors as a late manifestation (27). According to recent data, membrane proximity of the mutation seems to be an important determinant of tumor development in carriers of RET mutations in exon 10 (32,33). These genotype-phenotype correlations between mutation, age of onset, tumor aggressiveness, and onset of pheochromocytoma and parathyroid disease may be used to predict the phenotype and make recommendations regarding the ages at which to perform prophylactic thyroidectomy and begin biochemical screening for pheochromocytoma and hyperparathyroidism (Table 2). This is particularly true in presymptomatic RET mutation carriers because a prophylactic thyroidectomy should be performed before the development of MTC, or at least when the MTC is confined within the thyroid and before the spread of disease beyond the gland. The multicentric and bilateral nature of hereditary MTC implicates a total thyroidectomy.

Recommendations for the timing of prophylactic thyroidectomy and the extent of surgical resection are based on a model that utilizes these genotype-phenotype correlations to stratify mutations into risk levels (A–D) (Table 2) (4).

Patients with American Thyroid Association (ATA) A

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with exon 634 and 918 mutations in approximately 50% of patients, with exon 10 mutations (codons 609, 611, 618 and 620) in up to 17% of patients (33), and rarely with mutations in exons 13–15 (codons 791 and 804; Tables 1 and 2) (14,34).

Hyperparathyroidism in MEN2A is most commonly associated with codon 634 mutations and C634R in particular (35).

All cases of MEN2A with Hirschsprung disease are associated with mutations in exon 10 (codons 609, 611, 618, and 620); in an unselected, multinational study on carriers of exon 10 mutations, 7.5% of informative carriers were found to also have a Hirschsprung disease phenotype (33). MEN2A with cutaneous lichen amyloidosis is associated with mutations in codon 634 (36).

3.

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Perspectives: screening and genetic counseling MEN2 is a monogenic disorder and, according to its autosomal-dominant pattern of inheritance, each affected individual has a 50% probability of transmitting the defective gene to progeny, independent of sex. Predictive DNA tests are available; therefore, it is worthwhile to screen both first- and second-grade family members. In clinically demonstrable MTC, the specific RET mutation gives information on the risk of developing pheochromocytoma and HPT, the aggressiveness of MTC and clinical prognosis.

By screening family members, genetic testing allows for early treatment and, thereby, offers the chance of prophylactic thyroidectomy and curing the patient of MTC. From a medical point of view, MEN2 offers a unique model of how to use genetic information to cure a patient with a hereditary cancer syndrome. From the point of view of the patient, there is a loss of privacy and autonomy and a feeling of stigmatization and discrimination. There appears to be a threshold for patients to inform their relatives about this hereditary disease. The confidentiality of genetic testing must be absolute, with no exception; therefore, the duty to inform relatives who may be at risk depends on the moral obligation of the patient. However, many guidelines do allow for the disclosure of results to at-risk individuals without the patient’s consent when the information that will be disclosed will prevent serious harm (4). Our social attitude to rare hereditary diseases has to be reconsidered.

We need precise laws to prevent discrimination concerning full insurance coverage and employment. Patient interest groups may be able to providing information, support during times of emotional distress and stand up for common political and social aspects.

Early identification of patients with hereditary MTC using DNA screening changes the presentation from a clinical tumor (index case) to a preclinical disease (screening case), resulting in a high cure rate of the affected patients by using presymptomatic treatments, improved life expectancy and quality of life.

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AUTHOR CONTRIBUTIONS

20.

Raue F outlined the paper, wrote all of the sections except the genotypephenotype section, prepared the tables and figures, and checked the final version. Frank-Raue K wrote the genotype-phenotype section, updated the references and read the proof of the manuscript.

21.

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24. Cardot-Bauters C, Leteurtre E, Leclerc L, Vantyghem MC, Do Cao C, Wemeau JL, et al. Does the RET variant G691S influence the features of sporadic medullary thyroid carcinoma? Clin Endocrinol (Oxf).

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DOI:10.6061/clinics/2012(Sup01)14

REVIEW

Molecular mechanisms of RET receptor-mediated oncogenesis in multiple endocrine neoplasia 2 Simona M. Wagner*, ShuJun Zhu*, Adrian C. Nicolescu, Lois M. Mulligan Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen9s University, Kingston, ON, Canada. *contributed equally to the study.

Multiple endocrine neoplasia type 2 is an inherited cancer syndrome characterized by tumors of thyroid and adrenal tissues. Germline mutations of the REarranged during Transfection (RET) proto-oncogene, leading to its unregulated activation, are the underlying cause of this disease. Multiple endocrine neoplasia type 2 has been a model in clinical cancer genetics, demonstrating how knowledge of the genetic basis can shape the diagnosis and treatment of the disease. Here, we discuss the nature and effects of the most common recurrent mutations of RET found in multiple endocrine neoplasia type 2. Current understanding of the molecular mechanisms of RET mutations and how they alter the structure and function of the RET protein leading to its aberrant activation, and the effects on RET localization and signaling are described.

KEYWORDS: RET; Multiple Endocrine Neoplasia Type 2; Genotype–Phenotype.

Wagner SM, Zhu SJ, Nicolescu AC, Mulligan LM. Molecular mechanisms of RET receptor-mediated oncogenesis in multiple endocrine neoplasia 2. Clinics. 2012;67(S1):77-84.

E-mail: mulligal@queensu.ca Tel.: 1 613 533 6000 ext.77475

for predicting clinical course, and the underlying functional effects have been explored. Detection of RET mutations in MEN 2 represents a paradigm for genetically guided patient management, and genotype–phenotype correlations in this disease now inform recommended interventions, patient and family screening, and long-term follow-up (7,8).

Functional characterization of these mutations also has the potential to define optimal therapeutic regimens, and may identify additional phenotypic implications that have not been broadly recognized. Here, we discuss our current understanding of the molecular mechanisms for the more common RET mutations and their potential significance.

INTRODUCTION Multiple endocrine neoplasia type 2 (MEN 2) is an inherited cancer syndrome, characterized by medullary thyroid carcinoma (MTC). The disease has three clinically defined subtypes, as described elsewhere in this volume.

Briefly, familial MTC (FMTC), considered the least aggressive form of MEN 2, exhibits MTC without additional tumors or phenotypes, and frequently shows later onset than other disease subtypes. MEN 2A is characterized by MTC with pheochromocytoma, which occurs in approximately 50% of cases, and parathyroid hyperplasia or adenoma in 10–35%. Finally, MEN 2B is also characterized

by MTC and pheochromocytoma, but parathyroid hyperplasia is rare. This is the most aggressive subtype, with earliest onset of disease and metastasis, and poorest prognosis. In MEN 2B, MTC has been documented in patients as young as 2 months (1). In addition, patients with MEN 2B frequently present with other non-tumor features including ganglioneuromatosis of the mouth and gut, corneal nerve thickening, delayed puberty, and a marfanoid habitus (2,3).

MEN 2 is dominantly inherited, and its genetic cause, mutations of the REarranged during Transfection (RET) protooncogene, was first recognized nearly 20 years ago (4–6).

Since then, the range of mutations identified, their potential

RET RECEPTOR The RET proto-oncogene encodes a receptor tyrosine kinase that is required for the development of neural-crestderived cells, the urogenital system, and the central and peripheral nervous systems, notably the enteric nervous system (9,10). The RET protein has a large extracellular domain containing a cysteine-rich region and a series of cadherin homology domains, a transmembrane domain, and an intracellular tyrosine kinase domain, required for RET phosphorylation and downstream signaling (Figure 1A) (11,12). The RET kinase is structurally similar to other tyrosine kinases, sharing many conserved functional motifs and regulatory residues that have been shown to have importance for kinase enzyme function (13). RET is activated by binding of a multi-protein ligand complex. RET binds a soluble ligand of the glial cell-line-derived neurotrophic factor (GDNF) family but also requires a co-receptor of the GDNF family receptors a (GFRa), which is tethered to the cell membrane via glycosylphosphatidylinositol linkage

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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RET receptor-mediated oncogenesis in MEN 2 Wagner SM et al.

CLINICS 2012;67(S1):77-84

Figure 1 - The RET receptor: structure, activation, and oncogenic mutations. (A) Schematic diagram depicting RET tyrosine kinase receptor domains and the location of recurrent oncogenic mutations. The RET protein has a large extracellular domain containing a cysteine-rich region and a series of cadherin homology domains, a transmembrane domain, and an intracellular tyrosine kinase domain. The positions of the most common mutations found in patients with multiple endocrine neoplasia type 2 (MEN 2) are shown.

(B) Mechanism of RET activation. Wild-type RET activation requires the dimerization of RET, mediated through formation of a multicomponent complex. RET is activated by binding both a soluble ligand (glial cell-line-derived neurotrophic factor; GDNF) and a non-signaling extracellular co-receptor (GDNF family receptor; GFRa). Upon activation of RET, phosphorylation of multiple intracellular tyrosines leads to stimulation of downstream signaling pathways.

ligands and co-receptors. Mutations are primarily amino acid substitutions affecting a very small number of RET codons in either the extracellular domain or within the kinase domain (Table 1; Figure 1A). Mutations are dominant, requiring only a single mutant allele to confer the disease phenotype. Summaries of MEN 2 RET mutation occurrence are well reviewed elsewhere (20–23) or are available online (http://www.arup.utah.edu/database/ MEN2/MEN2_welcome.php). Together, these data suggest strong overall themes as to functional effects of these mutations, but also as to their clinical significance.

Strong associations of disease subtype, and also specific disease phenotypes, with individual RET mutations have made it possible to stratify risk of MEN 2 by genotype (7,8).

The management guidelines of the American Thyroid Association (8) base the recommendations for initial diagnosis, therapeutic intervention, and long-term followup on patient genotype and the current understanding of the natural history of the disease associated with each RET mutation. Mutations of cysteine residues (primarily

(Figure 1B) (14,15). Initially, GDNF binds to GFRa, and these complexes are then able to recruit RET to form heterohexamers that are concentrated in regions of the cell membrane called lipid rafts (14,16). These are membrane domains enriched in glycosylphosphatidylinositol-linked proteins and signaling molecules that provide a platform not only for enhanced cell signaling, but also for regulation of receptor kinase activity and downregulation (17). Activation of RET leads to stimulation of multiple downstream pathways, including mitogen-activated protein kinase and extracellular signal-regulated kinase, phosphoinositide 3-kinase and protein kinase B, signal transducer and activator of transcription 3, proto-oncogene tyrosine-protein kinase Src1, and focal adhesion kinase (18,19), that promote cell growth, proliferation, survival, and/or differentiation.

THE RET PROTO-ONCOGENE IN MEN 2 MEN 2 is associated with point mutations of RET, predictably leading to its activation in the absence of

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Table 1 - Molecular effects of RET mutations in multiple endocrine neoplasia 2.

Mutation location Extracellularcysteine rich domain

Intracellular tyrosine kinase domain

Affected RET Codons

Putative function of the wild-type residue

C609 C611 C618 C620 C630 C634

Contributes to tertiary structure of RET through the formation of intramolecular disulfide bonds

L790, Y791

In the N-terminal lobe of the RET kinase

E768

In close proximity with the ATP binding site

V804

A gatekeeper residue which regulates access to the ATP binding site C-terminal lobe of the kinase, adjacent to the activation loop of the kinase Situated next to activation loop

S891

A883

M918

Phenotype

Weakly activating. Alteration in protein folding and maturation.

Formation of mutant RET dimers that are constitutively active in the absence of ligands Strongly activating. Ligandindependent dimerization of receptor molecules, enhanced phosphorylation of intracellular substrates.

Moderately activating. Affects ATP binding and inter-lobe flexibility.

MEN 2A and FMTC

Prophylactic thyroid surgery before the age of 5. Under some conditions may delay beyond 5 years

MEN 2A

Surgery before age 5

MEN 2A and FMTC

May delay surgery beyond 5 years

Alters interactions within the region and facilitates the transition to an active conformation Alters hinge flexibility and positioning of RET helices for catalysis

FMTC

Alters activation loop conformation and promotes monomeric RET activation Strongly activating. Local conformational change which destabilizes the inactive form of the protein and promotes its activation Strongly activating. Alters protein conformation and substrate specificity.

The mutant can dimerize and become phosphorylated in the absence of ligand stimulation

MEN 2A and FMTC

Role in formation of intramolecular disulfide bonds

Lies in the substrate-binding pocket of the kinase and plays a role in stabilizing the receptor–ATP complex

Recommended intervention (8)

Predicted mutation effects

FMTC

MEN 2B

As early as possible (within first year of life)

MEN 2B

FMTC, familial medullary thyroid carcinoma; MEN 2, multiple endocrine neoplasia 2; RET, REarranged during Transfection.

activation of the RET receptor, autophosphorylation of RET, and aberrant stimulation of downstream signaling pathways. It follows that the molecular mechanisms of mutations associated with these different phenotypes may also be distinct and that these mechanisms may provide clues to disease origin and, potentially, treatment for patients with these mutations. Here, we discuss some of the current understanding of the mechanisms of RET dysfunction seen in MEN 2, and explore the potential implications of these mechanisms.

cysteines 609, 611, 618, 620, 630, and 634) in the RET extracellular domain account for the majority of MEN 2A cases, and are also common in patients with FMTC. Intracellular kinase domain mutations are mainly associated with FMTC and MEN 2B. Mutations in the intracellular codons 768, 790, 791, 804, and 891 underlie FMTC, and occur less commonly in patients with MEN 2A (20,24), while specific mutations of codon 918 (M918T) or 883 (A883F) account for the vast majority of MEN 2B cases, and are exclusive to the subtype (3,25). In addition to association with disease subtype, significant correlations of specific mutations with disease features are reported. For example, RET codon 634 mutations carry a greater patient risk for pheochromocytoma and parathyroid hyperplasia (4,26–28), and are associated with a higher frequency of detection of MTC at the time of early thyroidectomy (29). Variation in clinical presentation has even been observed with different codon 634 substitutions. The specific substitution of an arginine at codon 634 (C634R) is strongly associated with increased risk of parathyroid hyperplasia (4,26–28), increased frequency of distant metastases, earlier onset of both lymph node and distant metastases, and bilaterality of pheochromocytoma (30,31).

RET Extracellular Domain Cysteine Residues The most frequently identified RET mutations in MEN 2 affect cysteines in the extracellular cysteine-rich region (primarily residues between Cys515 and Cys634) (Figure 1A; Table 1). In the normal protein, intramolecular cysteine– cysteine disulfide bonds contribute to the tertiary structure of the RET extracellular domain. Correct positioning of residues in this region is critical to interactions with GDNF– GFRa ligand complexes (14,15,32). Amino acid substitutions, resulting in replacement of a normal cysteine with any amino acid, lead to loss of intramolecular bonds and to an unpaired cysteine that is available for intermolecular interactions with other mutant RET proteins (Figure 2A) (33–35). These mutant RET dimers are constitutively active in the absence of ligands. Furthermore, mutant dimers are not recruited to lipid rafts through GFRa interactions, and may be activated in other membrane compartments, which can affect the nature and intensity of the resultant downstream signals (36,37). Downstream signaling regulation, via

MOLECULAR MECHANISMS OF RET MUTATIONS Evidence-based assessment of MEN 2 genotypic data demonstrate that not all RET mutations have equivalent clinical significance, although all reported mutations are thought to lead to ligand-independent constitutive

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Figure 2 - Molecular mechanisms of pathogenic RET activation. Schematic diagrams showing mechanisms of RET activation in the presence of various multiple endocrine neoplasia type 2 (MEN 2) mutations. (A) Substitutions of extracellular cysteines lead to formation of intermolecular disulfide bonds and to constitutive RET dimerization and activation. (B) The MEN 2B mutation, M918T (star) in the kinase domain, leads to a conformational change with multiple effects including an increase in RET kinase activity and activation of receptors in either dimeric or monomeric form. (C) Intracellular kinase domain mutations asterisk implicated in familial medullary thyroid carcinoma (FMTC) (e.g. residues 791 and 891), permit activation of monomeric RET, allowing for a partially active conformation.

interactions with ubiquitin ligases such as CasitasB-lineage lymphoma proto-oncogene (CBL) (38), or with cellular phosphatases such as SHP1 and SHP2 (39,40) that are involved in limiting or terminating signals, differ from that of the raft-associated wild-type receptor, enhancing the effect of the oncogenic mutation.

Although the molecular mechanisms of activation are similar, cysteine RET mutations also vary in impact. In general, mutations located closer to the RET transmembrane domain have greater transforming ability and are linked to increased risks of more aggressive MEN 2 disease (41) (Table 1). Codon 634 mutations confer the greatest degree of RET activation, with higher levels of autophosphorylation and transforming ability than the other cysteine mutations, and are linked to broader phenotypes and more severe disease, as described above. Interestingly, mutations of other cysteine residues are believed to affect the efficiency of RET protein folding and maturation, and to impair transport to the cell membrane, resulting in decreased levels of cell surface protein and weaker signaling capability (33–35,42,43). In fact, a subset of RET cysteine mutations, sometimes referred to as Janus mutations, can lead to a partial loss-of-function phenotype, as well as to oncogenic effects. These mutations, generally affecting codons 609, 611, 618 or 620, are thought to confer cell-type-specific decreases in functional protein on the cell surface. Inactivating mutations of RET can lead to the congenital abnormality Hirschsprung disease, which is characterized by the absence of the enteric neurons from the distal colon (44). Janus mutations have been linked to an insufficiency of mature

RET protein in the gut, resulting in the Hirschsprung phenotype, yet at the same time, risks remain high for MEN 2 phenotypes, as sufficient mature protein is expressed in the thyroid for development of MTC (45–47).

RET Intracellular Domain Mutations Intracellular RET kinase mutations fall into two groups: high-penetrance mutations causing MEN 2B, and less aggressive mutations that lead to FMTC or, more rarely, MEN 2A (Table 1). These RET mutations fall within the Nterminal and C-terminal lobes of the kinase (Figure 3).

Although the mutations are spread out along the linear protein sequence (Figure 1A), they appear to cluster on either the ATP-binding face or substrate-binding/autoinhibitory face of the protein tertiary structure, suggesting some common themes in their functional effects (Figure 3; Table 1). The precise mechanisms by which these intracellular mutations activate RET are various, but it is suggested that they all do so through destabilizing the inactive form of RET, and shifting the equilibrium of RET receptors towards the active state (48).

MEN 2B Mutations Over 95% of MEN 2B cases are associated with the same methionine to threonine change at codon 918 (M918T) in the RET kinase domain (Figure 3). Structurally, this residue lies in the substrate-binding pocket of the kinase, and the M918T mutation appears to increase RET–ATP binding affinity and the stability of the active ATP-bound form, effectively

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Figure 3 - Structure of the RET tyrosine kinase domain. Ribbon diagrams of the intracellular regions of activated RET, in two orientations, showing the positions of key functional features of the kinase: the ATP binding pocket; the activation or autoinhibitory loop; and the substrate binding pocket. Two orientations of the model, displaying the autoinhibitory/substrate binding face (left) and the ATP-binding face (right), are shown. Amino acid residues that are mutated in patients with multiple endocrine neoplasia type 2 (MEN 2) are represented in the stick form. The three-dimensional representation was based on the crystal structure of the phosphorylated (activated) RET tyrosine kinase domain (residues 709–990).

following ligand stimulation (57) and for cytosolic RET mutants found in papillary thyroid carcinoma (37).

An alanine to phenylalanine substitution at codon 883 (A883F) is the only other recurring MEN 2B mutation (58,59). Structurally, this residue lies between the activation and catalytic loops of the kinase (Figure 3), and would be predicted to increase the flexibility of these domains, so destabilizing the inactive form of the protein and promoting its activation. Although generally considered a high-risk mutation, some studies suggest that it may have a lesser effect than the M918T mutation (60).

A handful of instances of double mutations in MEN 2B have also been reported: V804M/E805K (51), V804M/Y806C (61), and V804M/S904C (62). It appears that the combination of two mild intracellular mutations can cooperate to produce a more severe mutant. Each mutation alone (V804M, E805K, Y806C, S904C) has low or no transforming ability, consistent with the observation that V804M generally leads to FMTC (discussed below), but when coupled together, they exert a synergistic effect on the transforming ability of mutated RET (51,63).

making RET more active, more of the time (48–51). The M918T mutation appears to increase the stability of monomeric active forms of RET, but activation of these mutants can also be further enhanced by binding of GDNF–GFRa complexes, suggesting that these mutant RET forms may induce signal transduction from both within and outside the lipid rafts, perhaps via distinct signaling complexes (Figure 2B). As a result, RET downstream signals are enhanced, and activation of targets is increased, notably including upregulation of gene transcripts that contribute to cell proliferation or to metastasispromoting cell behaviors (52,53). Although it has been postulated that the M918T mutation alters the preferred substrates of the mutant RET protein with respect to both autophosphorylation of RET tyrosine residues, and phosphorylation of downstream signaling molecules (54,55), novel downstream targets that cannot also be stimulated at lower levels by other less active RET mutants or by ligand activation of wild-type RET have not been broadly identified (48–51,53).

An intriguing finding has been that activation of M918T RET begins before the receptor arrives at the cell surface, stimulating signaling pathways from the endoplasmic reticulum before the receptor reaches its fully glycosylated mature form (56), which has not been observed for other mutants. RET signaling from intracellular compartments may differ (in intensity or otherwise) from that at the plasma membrane, which has been shown to be the case when wild-type RET is internalized into endosomes

Lower Risk Intracellular Domain Mutations Recurrent mutations in the intracellular codons 768, 790, 791, 804, and 891 are found in patients with FMTC and, less commonly, MEN 2A (20,64). This group of mutations is the most diverse in functional effects, phenotypic variability, and long-term clinical implications.

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tumors (24,75). Co-occurrence of Y791F and codon 634 mutations has been shown to increase the risk of pheochromocytoma in some families (76), whereas other studies have concluded that this mutation is not pathogenic (77).

Interestingly, a clue is perhaps provided by studies identifying Y791F and Y791N mutations in patients with Hirschsprung disease (75,78,79), possibly suggesting that mutations of tyrosine 791 may act as modifiers of multiple phenotypes.

Mutations of glutamic acid 768 occur almost exclusively in patients with FMTC, whereas leucine 790 mutations have been recognized in both FMTC and MEN 2A families (20–23). These are considered lower penetrance mutations, associated with later-onset disease, as reflected by evidencebased clinical management recommendations suggesting that delayed prophylactic surgery may be acceptable (Table 1) (8). The E768 and L790 residues lie close to the ATP binding site (Figure 3) and may alter interactions in this region, and/or increase flexibility of domains, making the transition to an active conformation relatively easy.

Mutation of serine 891 to an alanine was initially recognized as an FMTC mutation, but more recently has been linked to MEN 2A features (65). Codon 891 lies in a conserved region of the RET protein, and its mutation appears to alter protein autoinhibition and ATP binding, favoring an active conformation. Interestingly, S891A and Y791F mutations are functionally unique in that they do not require RET dimerization for full activation, and so RET autophosphorylation and downstream signaling are not further enhanced by ligand binding (Figure 2C) (66). As for other RET mutants, this means that RET is not recruited into lipid rafts by GFRa, and hence it is likely that the nature, intensity, and duration of signaling is altered for these mutants (36–40).

The most common of these lower-risk mutations is substitution of valine 804 (67). This residue lies in the sequence linking the N-terminal and C-terminal lobes of the kinase domain, in a conserved region critical for RET–ATP binding, which is required for activation of the kinase.

Substitution of valine 804 for a leucine (V804L) or methionine (V804M) changes the conformation of the ATP binding pocket, making it more permissive for binding ATP, and thus enhancing RET activation (51,68,69). Residue 804 represents a classical gatekeeper residue (51), positioned so as to regulate access to the ATP-binding site. Competitive binding to this region is the mechanism of action of multikinase inhibitors such as vandetanib, which has recently been approved for treatment of advanced MTC (70). Although vandetanib effectively inhibits wild-type and other mutant RET forms, the V804L or V804M mutations confer resistance to the drug (48,68). As other kinase inhibitors (such as sorafenib, which is currently under review for managing advanced thyroid carcinoma (71,72)) are not affected by codon 804 mutations (68), RET mutation status can have profound clinical importance for optimizing treatment regimens.

The molecular effects of substitution of phenylalanine for tyrosine at codon 791 (Y791F) of RET are not clearly defined.

This residue is not a known site of tyrosine phosphorylation (73), so direct protein interactions of RET with other molecules are unlikely to be altered by this mutation. The position of the residue, close to the ATP binding pocket, may enhance ATP access, or may again alter protein flexibility, favoring the active conformation. In vitro, Y791F mutations have been shown to enhance signal transducer and activator of transcription 3 (STAT3) signaling (74). Like S891A mutants, the Y791F form of RET appears to exist as an active monomer as it does not require dimerization to be activated, and ligand binding does not further enhance autophosphorylation or downstream signaling (66). The significance of the Y791F mutation remains somewhat controversial. Reports have identified this mutation, alone or in combination with other mutations, in MEN 2A and FMTC, and in sporadic MTC and pheochromocytoma

CONCLUSIONS AND PERSPECTIVES The landscape of MEN 2 disease management has been transformed by the identification and cataloguing of its underlying genetic causes. Mutation genotype has guided the evidence-based diagnosis, prediction, and management of MEN 2 as for few other diseases. However, we are only beginning to reap the benefits of functional characterization of RET mutations. The new crop of anti-RET therapeutics being developed has implications not just for MEN 2, but for thyroid cancer in general, and for other diseases that have recently been linked to RET activity including pancreatic and breast cancers (80–82). Conversely, mutations or altered expression of RET that result in decreased receptor function have been linked to developmental defects, such as Hirschsprung disease (44) and kidney anomalies (83,84), and current research also links GDNF and survival of dopaminergic neurons in Parkinson disease (85). Together, these studies clearly indicate that understanding of the normal functions and physiological role of RET are essential in assessing the short-term and long-term benefits and potential harms of novel RET-targeted therapeutics.

AUTHOR CONTRIBUTIONS Wagner SM prepared the figures and was also responsible for the manuscript writing. Zhu S contributed to the manuscript writing.

Nicolescu A prepared the models and figures. Mulligan LM is the senior author who prepared the final manuscript version. Wagner SM and Zhu S contributed equally to the study.

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CLINICS 2012;67(S1):85-89

DOI:10.6061/clinics/2012(Sup01)15

CLINICAL SCIENCE

Over-representation of the G12S polymorphism of the SDHD gene in patients with MEN2A syndrome Nikoletta Lendvai,I Miklos To´th,I Zsuzsanna Valkusz,II Gabriella Beko˝,III Nikolette Szu¨cs,I E´va Csajbo´k,II Pe´ter Igaz,I Bala´zs Kriszt,IV Bala´zs Kova´cs,IV Ka´roly Ra´cz,I Attila Pato´csIII,V I

2nd Department of Medicine, Semmelweis University, Budapest, Hungary. II 1st Department of Medicine, University of Szeged, Szeged, Hungary.

Department of Laboratory Medicine, Semmelweis University. IV Regional University Center of Excellence in Environmental Industry Based on Natural Resource, Szent Istvan University, Go¨do¨llo˝, Hungary. V Molecular Medicine Research Group, Hungarian Academy of Sciences and 2nd Department of Medicine, Semmelweis University, Budapest, Hungary.

III

OBJECTIVE: To evaluate whether germline variants of the succinate dehydrogenase genes might be phenotypic modifiers in patients with multiple endocrine neoplasia type 2. Mutations of genes encoding subunits of the succinate dehydrogenase are associated with hereditary paraganglioma/pheochromocytoma syndrome.

Pheochromocytoma is one of the main manifestations of multiple endocrine neoplasia type 2 caused by germline mutation of the rearranged during transfection proto-oncogene.

METHODS: Polymorphisms of the succinate dehydrogenase genes were analyzed in 77 rearranged during transfection mutation carriers, 47 patients with sporadic medullary thyroid cancer, 48 patients with sporadic Pheo, and 100 healthy individuals. Exons 10–16 of the rearranged during transfection proto-oncogene were analyzed by direct DNA sequencing, and all exons of the von Hippel-Lindau, succinate dehydrogenase B, and succinate dehydrogenase subunit D genes were tested by direct DNA sequencing and multiple ligation probe analysis. The G12S polymorphism of the succinate dehydrogenase subunit D gene was determined by restriction fragment length polymorphism.

RESULTS: Of the 77 rearranged during transfection mutation carriers, 55 from 16 families had multiple endocrine neoplasia type 2A, three from three families had multiple endocrine neoplasia type 2B, and 19 from two families had familial medullary thyroid carcinoma. Eight of 55 (14.5%) patients with multiple endocrine neoplasia type 2A had this variant whereas it was absent in multiple endocrine neoplasia type 2B, familial medullary thyroid carcinoma, sporadic medullary thyroid carcinoma, and sporadic pheochromocytoma groups, and its prevalence in controls was 1% (p,0.002 multiple endocrine neoplasia type 2A versus controls). No associations between G12S and age of manifestation, incidence of pheochromocytoma or hyperparathyroidism, or level of serum calcitonin were observed.

CONCLUSION: The high prevalence of the G12S variant in patients with multiple endocrine neoplasia type 2A raises questions about its role as a genetic modifier, but this proposal remains to be established.

KEYWORDS: RET proto-oncogene; Succinate dehydrogenase subunit D; Medullary thyroid cancer; Pheochromocytoma.

Lendvai N, To´th M, Valkusz Z, Beko˝ G, Szu¨cs N, Csajbo´k E, et al. Over-representation of the G12S polymorphism of the SDHD gene in patients with MEN2A syndrome. Clinics. 2012;67(S1):85-89.

E-mail: patatt@bel2.sote.hu Tel.: 36 1 266 0926 ext. 55577

dominantly inherited disease with an approximate prevalence of 2.5 per 100.000 in the general population (1). MEN2 has three subtypes: i) MEN2A, characterized by medullary thyroid carcinoma (MTC), pheochromocytoma (Pheo) and primary hyperparathyroidism; ii) MEN2B, which presents with the most aggressive MTC, pheochromocytoma, neuromas and marfanoid phenotype; and iii) familial MTC (FMTC), the mildest form of MTC. Several important genotype–phenotype associations have been determined; the most commonly affected codon, 634 (nearly 85% of MEN2A cases), frequently associates with Pheo and hyperparathyroidism, whereas mutations of codons 609, 611, 618, and 620 (accounting for 10–15% of MEN2A) usually associate with the milder form of MEN2 (1–3).

INTRODUCTION The RET (rearranged during transfection) proto-oncogene located on chromosomal region 10q11.2 consists of 21 exons, and it encodes a receptor tyrosine kinase (1). Germline gainof-function mutations of the RET proto-oncogene cause multiple endocrine neoplasia type 2 (MEN2), an autosomal

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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However, the phenotypic heterogeneity observed even in members of the same family suggests that other factors, for example genetic modifiers, may influence the clinical manifestation of the disease (4–7).

Hereditary paraganglioma/pheochromocytoma (PGL) syndrome is caused by the germline heterozygous mutations of the SDHx genes (SDHB, SDHC, SDHD, encoding subunits B, C and D, respectively) (8–10) and the newly identified SDH5 gene (11) and TMEM127. SDHx genes encode subunits of the mitochondrial complex II (succinate dehydrogenase, SDH), an enzyme involved in oxidative phosphorylation and intracellular oxygen sensing and signaling (12).

Both MTC and paraganglioma/pheochromocytoma arise from neural crest-derived precursor cells. Accumulation of amino-acid coding polymorphisms (S163P in SDHB, G12S, and H50R in SDHD) has been found among patients with MTC, especially in those with familial tumors (13). In addition, these rare genetic variants have been identified in patients with Cowden-like syndrome (14) and the H50R polymorphism has been described in six members of a family with non-RET-associated C-cell hyperplasia and hypercalcitoninemia (15). These previous data may suggest a possible connection between SDHx polymorphisms and familial MTC and/or C-cell hyperplasia/hypercalcitoninemia; however, the occurrence of the SDHx variants among patients harboring germline RET mutations has not been previously examined. Therefore, the aim of the present study was to investigate whether polymorphisms of the SDHx genes could influence clinical manifestations of the disease in a cohort of subjects harboring RET mutations. In addition, we wanted to determine whether the prevalence of SDHx polymorphisms in patients with sporadic MTC, sporadic Pheo and healthy subjects might be different from that found in RET mutation carriers.

years; range: 2–57 years). The presence of Pheo and MTC were confirmed by histologic examination of surgically removed tumors. Total thyroidectomy was performed in all patients with germline RET mutation in the symptomatic group and was also offered to all individuals from the asymptomatic group.

Patients with sporadic MTC The study included 47 unrelated patients with histologically confirmed MTC evaluated consecutively at the 2nd Department of Medicine, Faculty of Medicine, Semmelweis University between 1998 and 2010. There were 15 men (age, mean ¡ SD, 44.7¡13.3; range: 28–82 years) and 32 women (age, mean ¡ SD, 47.7¡12,3; range: 23–76 years). Preoperative evaluation included medical history, physical examination, thyroid and abdominal ultrasonography, CT or magnetic resonance imaging (MRI), MIBG-scintigraphy, routine biochemical testing, serum calcitonin measurements, and mutation analysis of exons 10–14 of the RET gene.

Patients with apparently sporadic Pheo The study included 48 unrelated patients with histologically confirmed sporadic adrenal pheochromocytomas evaluated consecutively at the 2nd Department of Medicine, Faculty of Medicine, Semmelweis University between 1998 and 2010. There were 16 men (age, mean ¡ SD, 36¡14; range: 13–66 years) and 32 women (age, mean ¡ S.D, 42¡14; range: 19–64 years). Pre-operative evaluation included medical history, physical examination, abdominal ultrasonography, CT or MRI, MIBG-scintigraphy, routine biochemical testing, and 24 h urinary catecholamine metabolite determination. The mutation analysis of RET exons 10–14 and the entire VHL, SDHB, and SDHD genes revealed no disease-causing mutations. Patients with confirmed VHL (five patients), SDHB (one patient), or SDHD (one patient) mutations were excluded from the study. Five patients were initially thought to have sporadic pheochromocytoma, but were later identified as having a disease-causing RET mutation and were included in the study as RET mutation carriers. MTC, either by elevated serum calcitonin or by postoperative histology, had been diagnosed in all of these patients. Genetic counseling and genetic screening for all first-degree relatives have been offered.

PATIENTS AND METHODS Written informed consent was obtained from all patients and family members who participated in the study. Patients underwent a complete clinical examination, laboratory testing, including serum basal calcitonin measurement [hCalcitonin IRMA kit (Diagnostic Systems Laboratories, Inc., Budapest, Hungary), reference range: male ,15 pg/ml, female ,10 pg/ml until December 2007; and Liaison (Diasorin SPA, Stillwater, MN, USA), reference value: male ,18.9 pg/ml and female ,6 pg/ml after January 2008], plasma parathyroid hormone (Elecsys; Roche Diagnostics, Basel, Switzerland), urinary catecholamine metabolites (high pressure liquid chromatography with electrochemical detection), and imaging studies, including cervical ultrasonography, thoracal and abdominal computed tomography (CT), and whole-body metaiodobenzylguanidine scintigraphy (MIBG).

Germline mutation screening of the RET, VHL, SDHB, and SDHD genes Genomic DNA was isolated from peripheral blood using the Roche DNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany) and QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA, USA) in accordance with the manufacturers’ instructions. RET proto-oncogene mutations were detected by direct sequencing as previously reported (16,17). Mutation analysis of the VHL, SDHB, and SDHD genes in cases of apparently sporadic Pheo were performed by direct sequencing of the entire coding region of the VHL, SDHB, and SDHD genes, as previously reported (17,18), and large deletion analysis of the VHL, SDHB, SDHC, and SDHD genes performed using multiplex ligation probe amplification (18).

Patients with MEN2 syndrome In total, 77 patients with germline RET proto-oncogene mutations who were members of 21 unrelated families with MEN2 syndrome were identified by genetic screening at our center. Of the 77 patients, 55 had MEN2A (mean age at diagnosis: 33.4¡17 years; range: 7–76 years), three had MEN2B (mean age at diagnosis: 15.6¡5 years; range: 10–20 years), and 19 had FMTC (mean age at diagnosis: 23.7¡16.8

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G12S polymorphism of the SDHD gene Lendvai N et al.

Figure 1 - (A) Gel electrophoresis of PCR fragments after digestion with BanI for identification of the G12S polymorphism of the SDHD gene by RFLP (L = DNA ladder; C+ = positive control; heterozygote for G12S; C- = negative control; G12 normal, P1–P5 = patients. (B) Chromatograms of exon 1 of the SDHD gene showing the wild type and the heterozygote form of the G12S (GGT12AGT) polymorphism.

40¡3 years in probands and 29.6¡19.3 versus 32.5¡20.5 years in non-carriers). Among probands with RET mutations, carriers of the G12S had higher serum calcitonin levels compared with those who did not carry the SDHD G12S variant (6,864¡11,111 versus 1,250¡932 pg/ml), but the difference was not significant. Among family members with RET mutations, serum calcitonin levels were similar in G12S carriers and non-carriers 436¡876 versus 393¡556 pg/ml) (Table 2).

The phenotypic heterogeneity seen in families with different RET mutations, the variation of clinical course within families with the same RET mutation, and the results from RET transgenic mouse models suggest a potential role of genetic components in phenotype modulation (2,19).

Polymorphisms of the RET gene have been analyzed as such genetic modifiers, but the results from these studies are conflicting. Robledo et al. showed that two RET variants (G691S and S904S) may modify the age of onset of MTC in family members (5); and Tamanaha et al. reported that two intronic polymorphisms of RET may modify the phenotype in a large family with G533C RET mutation (7), while Baumgartner-Parzer found that the L769L and the IVS14-24 may act as modifiers in some forms of hereditary and sporadic MTC (20). However, Lesueur et al. were unable to replicate this association in a large cohort of 384 members of MEN2 families from four different European populations.

This latter study showed that of the several polymorphisms of genes encoding the RET protein, its co-receptors and ligands, only the synonymous polymorphism A432A of the RET gene associated weakly with tumor spectra in patients with MEN2A (6). MEN2-related MTC RET variants have been analyzed as genetic susceptibility factors for the development of sporadic MTC: polymorphisms located in coding regions of RET, G691S, L769L, S836S, and S904S have been shown to be over-represented in patients with sporadic

Restriction fragment length polymorphism (RFLP) analysis for identification the G12S polymorphism of the SDHD gene The nucleotide change of G to A, which corresponds to the G12S polymorphism, results in the preservation of the BanI restriction cleavage site. Therefore, digestion with the BanI restriction enzyme (New England BioLabs Inc., Ipswich, MA, USA) for 90 min at 37 ˚C was performed after polymerase chain reaction (PCR) amplification of exon 1 of the SDHD gene for genotyping of RET mutation carriers, sporadic MTC patients, and 100 controls (Figure 1). Samples from patients with positive results were examined by direct DNA sequencing. The results obtained with both methods were the same in all cases.

Statistical analysis Baseline characteristics were compared using the chisquared test or Fisher’s exact test for qualitative variables, and Student’s t test or Mann-Whitney’s U test for quantitative variables. The statistical package SPSS 15.0 (SPSS Inc., Chicago, IL, USA) was used and p,0.05 was considered statistically significant.

RESULTS AND DISCUSSION Eight of the 55 patients with MEN2A (15.5%) had the G12S variant, whereas it was absent in the MEN2B and FMTC groups. No patient with sporadic MTC and/or sporadic Pheo carried this variant. Among the 100 population-based, healthy control individuals, only one individual carried this variant (prevalence, 1%) (Table 1). No association between the G12S polymorphism of the SDHD gene and the incidence of Pheo or hyperparathyroidism in RET mutation carriers was observed, and the age of disease manifestation was similar in G12S carriers and in non-carriers (43¡9 versus

Table 1 - Prevalence of the G12S polymorphism of the SDHD gene among germline RET mutation carriers, patients with sporadic medullary thyroid cancer, patients with sporadic pheochromocytomas, and healthy controls.

MEN2A 8/55 (15.5%)*

FMTC

MEN2B

Spoaradic MTC

Spoaradic Pheo

Control

0/19 (0%)

0/3 (0%)

0/47

0/48

1/100 (1%)

*

p,0.002 versus control group.

FMTC = familiar medullary thyroid cancer; MEN2A = multiple endocrine neoplasia type 2A; MEN2B = multiple endocrine neoplasia type 2B; MTC = medullary thyroid cancer; Pheo = pheochromocytoma.

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Table 2 - Clinical presentation, serum calcitonin concentration and the G12S status of patients with MEN2A. MEN2A (n = 55) Probands (n = 16) Age of presentation (years) Prevalence of: MTC Pheo PHPT Serum calcitonin; mean ¡ SD (range) Affected family members (n = 39) Age of presentation Prevalence of: MTC Pheo PHPT Serum calcitonin mean ¡ SD (range)

G12S negative

G12S positive

n = 13 40.1¡9

n=3 43¡3

12/13 6/13 4/13 1,206¡932 (13–2400) n = 34 32.5¡19.3

3/3 2/3 1/3 6,864¡11,111 (124–19,690) n=5 29.6¡20.5

22/34 8/34 4/34 393.8¡556 (0–1978)

3/5 2/5 1/5 436.2¡876 (0–2000)

SD = standard deviation; MEN2A = multiple endocrine neoplasia type 2A; MTC = medullary thyroid cancer; Pheo = pheochromocytoma; PHPT = primary hyperparathyroidism.

the ethnic background of the different populations tested.

Our patients and controls were of Hungarian origin, representing an independent entity among Caucasian populations. More importantly, in our study, the high incidence of the G12S variant among RET carriers, especially in those with the MEN2A phenotype, raised the possibility that this variant may have a role in the phenotypic modulation of the disease. However, we were unable to detect significant differences in the clinical presentation between G12S carriers and non-carriers.

Whether this failure was a result of the relatively small size of our patient cohorts remains to be further investigated. Interestingly, Waldmann et al. reported an increased prevalence of intronic SDHB polymorphisms among patients with malignant Pheo compared with patients with benign tumors (29).

In conclusion, we found a significantly higher prevalence of the G12S variant of the SDHD gene among germline RET mutation carriers presenting with MEN2A compared with the control group. The high prevalence of the G12S variant in these patients supports its genetic modifier role, but this proposal remains to be established.

MTC (21–23) compared with the general population, but others were unable to confirm these associations (24,25).

Germline mutations of SDHx genes encoding subunits of the mitochondrial complex II represent a genetic susceptibility for Pheo/PGL. These tumors are derived from cells of the neural crest, similar to MTC. RET mutations also cause Pheo, again suggesting a link between the genetic background of Pheo and MTC. Therefore, it has been assumed that mutations of these genes may be involved in the pathogenesis of MTC. Lima et al. reported a family with Ccell hyperplasia, a pre-cancerous state of MTC, who were proved to have the H50R variant of the SDHD gene (15).

Montani et al. demonstrated an increased frequency of amino acid-coding SDHx polymorphisms in patients with sporadic and familial MTC (13). In addition, a systemic evaluation of genetic variants of the SDHx genes among patients with sporadic MTC showed a significant association between the H50R variant and sporadic MTC in Spanish patients, although this observation was absent in an English cohort (26). Variants of the SDHx genes have been implicated in the pathogenesis of various endocrine and non-endocrine tumors, such as Merkel cell carcinoma, carcinoid, papillary thyroid cancer, and renal cell cancer found in patients with Cowden-like syndrome (14).

In the present study, we found that the G12S variant was significantly over-represented among RET mutation carriers compared with sporadic MTC, sporadic Pheo, or control individuals. In our study, this variant occurred mainly in patients with MEN2A, while Montani et al. detected G12S in a patient with MEN2B harboring the M918T mutation of the RET gene (13). Interestingly, the prevalence of alterations of the SDHx genes in patients with RET mutations was similar in our study and the study of Montani et al. (13).

The prevalence of the G12S in the general population is between 2.5% and 5% (27) according to the Leiden Open Variation Database (http://chromium.liacs.nl) (28), which is somewhat higher than in our control population (1%).

This difference may be due to differences in the selection criteria applied for controls. Our control group were evaluated for endocrine dysfunction; none of them had signs or symptoms characteristic of thyroid cancer or Pheo.

By contrast, population-based controls, frequently anonymous blood donors, have never been tested for these rare conditions. Alternatively, the difference between the studies in prevalence of G12S can also be attributed to

AUTHOR CONTRIBUTIONS Pato´cs A was responsible for the statistical analysis and study design, molecular biological analysis, evaluation and collection of clinical data, genetic counseling, and manuscript writing and critical review. Lendvai N has contributed to the statistical analysis and study design, molecular biological analysis, and manuscript writing and critical review. Kova´cs B and Kriszt B have contributed to the molecular biological analysis and to the manuscript writing and critical review. To´th M, Szu¨cs N, Csajbo´k E´ and Ra´cz K have contributed to the evaluation and collection of clinical data and to the manuscript writing and critical review. Igaz P has contributed to the evaluation and collection of clinical data, genetic counseling, and manuscript writing and critical review. Beko˝ G was responsible for the measurement of serum calcitonin levels and to the manuscript writing and critical review.

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CLINICS 2012;67(S1):91-97

DOI:10.6061/clinics/2012(Sup01)16

REVIEW

Personalized medicine: caught between hope, hype and the real world Marc Dammann, Frank Weber University Hospital Essen, Endocrine Surgery, Department of General, Visceral and Transplantation Surgery, Hufelandstrasse/Essen, Germany.

Genomic and personalized medicine have become buzz phrases that pervade all fields of medicine. Rapid advances in ‘‘-omics’’ fields of research (chief of which are genomics, proteinomics, and epigenomics) over the last few years have allowed us to dissect the molecular signatures and functional pathways that underlie disease initiation and progression and to identify molecular profiles that help the classification of tumor subtypes and determine their natural course, prognosis, and responsiveness to therapies. Genomic medicine implements the use of traditional genetic information, as well as modern pangenomic information, with the aim of individualizing risk assessment, prevention, diagnosis, and treatment of cancers and other diseases. It is of note that personalizing medical treatment based on genetic information is not the revolution of the 21st century. Indeed, the use of genetic information, such as human leukocyte antigen-matching for solid organ transplantation or blood transfusion based on ABO blood group antigens, has been standard of care for several decades. However, in recent years rapid technical advances have allowed us to perform high-throughput, high-density molecular analyses to depict the genomic, proteinomic, and epigenomic make-up of an individual at a reasonable cost. Hence, the so-called genomic revolution is more or less the logical evolution from years of bench-based research and bench-to-bedside translational medicine.

KEYWORDS: Inherited Endocrine Neoplasia; Genomic Medicine; Tumor Syndromes.

Dammann M, Weber F. Personalized medicine: caught between hope, hype and the real world. Clinics. 2012;67(S1):91-97.

E-mail: frank.weber@uk-essen.de Tel.: 49 201 7231469

perform high-throughput, high-density molecular analyses to describe the genomic, proteinomic, and epigenomic make-up of an individual at a reasonable cost. Hence, the so-called genomic revolution is more or less the logical evolution from years of bench-based research and bench-tobedside translational medicine. The challenge is to determine how the structural changes, both within the germline and soma, affect how genes globally interact within the germline, how they interact locally between the germline and tumor and, finally, how the environment and lifestyle choices can alter genes at a functional level in order to modulate thyroid cancer susceptibility, aggressiveness, and the likelihood of cure.

Molecular testing for non-inherited genetic profiles (i.e., the somatic alteration of genes such as KIT, BRAF, and EGFR) to tailor and optimize treatment approaches has become implemented in the routine armament for the diagnosis and treatment of various tumors (i.e., lung, colon, breast, gastrointestinal stromal, thyroid, etc.) and will not be discussed in this review (1–4). The impact on practicing personalized medicine will be tremendous. For instance, it will not be long before we can tailor our surgical approaches and surveillance of patients with papillary microcarcinomas based on the BRAF (V600E) mutation status (5).

On the other hand, human disease susceptibility can be seen as the result of either rare germline genetic variations of high penetrance or as common genomic variants of low penetrance. The latter path on the road to practicing genomic medicine is using the technical advances of high-throughput,

INTRODUCTION Genomic medicine and personalized medicine have become buzz phrases that pervade all fields of medicine.

The rapid advances in ‘‘–omics’’ research (chief of which are genomics, proteinomics, and epigenomics) over the last few years have allowed us to dissect the molecular signatures and functional pathways that underlie disease initiation and progression and to identify molecular profiles that help the classification of tumor subtypes and determine their natural course, prognosis, and responsiveness to therapies.

Genomic medicine implements the use of traditional genetic information, as well as modern pangenomic information, with the aim of individualizing risk assessment, prevention, diagnosis, and treatment of cancers and other diseases.

It is of note that personalizing medical treatment based on genetic information is not a revolution of the 21st century.

Indeed, the use of genetic information, such as human leukocyte antigen-matching for solid organ transplantation or blood transfusion based on ABO blood group antigens, has been standard of care for several decades. However, in recent years rapid technical advances have allowed us to

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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high-density analyses (e.g., whole-genome linked analysis) to correlate common genetic variations (e.g., single nucleotide polymorphisms) to common diseases (6). In this way, a vast variety of allelic alterations have been identified that correlate with the risk of cancer, other common diseases (e.g., diabetes, hypertension, autism, obesity, etc.) or even a patient’s ability to respond to severe sepsis (7–10). There is tremendous hype to push forward on this path in order to implement ‘‘–omics’’-based analyses into clinical practice (11,12). However, there are some obstacles to clinical implementation that must not be underestimated and the impact on public health must be critically discussed (12–14). Beyond ethical and legal issues, numerous studies have shown that susceptibility variants for common diseases only marginally contribute to the disease risk with odd ratios of around 1.5 (6). For instance, in coronary heart syndrome, other medical conditions and lifestyle factors, such as blood pressure, diabetes, or smoking, are better predictors of cardiovascular events than are genomic variants (15). Furthermore, easy access to affordable, high-volume sequencing/genotyping techniques (i.e., the ability to sequence thousands of genes or millions of single nucleotide polymorphisms) resulted in a flood of studies reporting the association of one or another genetic variant to disease or disease outcome (6). This harbors a profound statistical dilemma.

Among the major limitations of many association studies is that they are profoundly underpowered. For instance, to detect an association with an odds ratio of 1.5 (i.e., a patient with the variant is 1.5 times more likely to have the disease or outcome) more than 1,000 people need to be tested when the suspected susceptibility variant occurs with a frequency of 0.2 (20% of the population).

Furthermore, none of the technologies used today achieves 100% accuracy. Even if we propose a test accuracy of 99% — to be more realistic, we should estimate it between 80% and 95% — at least one of 100 patients would receive an erroneous result. Hence, there is the widely accepted opinion that clinical implementation should be scrutinized under the framework of analytic validity, clinical validity, clinical utility, and ethical, legal, and social implications.

The last of these implies that for each test there has to be a medical treatment available to prevent the outbreak or influence the course of the disorder in those patients who are identified as being at risk.

However, one can envision that using population-wide ‘‘–omics’’ screening methods should help to establish which genetic changes, whether static (structural genomic) or dynamic (expression profiling), predispose under which environmental conditions to disrupt the usual cellular balances of apoptosis, survival, and maintenance of differentiation. In the future, this will provide us with unique genetic signatures for the prediction of outcomes and choices of therapy or prevention, or, in short, personalized

genomic healthcare. Furthermore, the field of pharmacogenomics allows us to anticipate what the future can hold. For instance the HCP5 single nucleotide polymorphism serves as a predictor for a hypersensitivity to antiviral drug medications, and the CYP2C19 variant is associated with a diminished clopidogrel response (16,17). Hence, patients at risk of adverse events or with limited response to medication can be identified using virtually ‘‘bed-side’’ genetic testing methods and the treatment regimen can be tailored accordingly.

PERSONALIZED MEDICINE FOR HERITABLE ENDOCRINE NEOPLASIAS This review will focus on the implementation of validated personalized healthcare practices and the utilization of evidence-based genetic tests for the individualized treatment of inherited endocrine, especially thyroid, cancers.

Looking back in history, one finds that endocrine neoplasias and those involved in their treatment have pioneered the field of clinical cancer genetics and personalized medicine.

There is an array of heritable endocrine neoplasias for which genes that connote a high degree of penetrance have been identified and validated. In these situations, molecular diagnostic and predictive testing leads to tailored surveillance and clear therapeutic implications that, at best, allow for preventive surgery.

MEN 2 and RET: the paradigm for the practice of genomic medicine The inherited tumor syndrome multiple endocrine neoplasia type 2 (MEN 2) can be considered the role model for the practice of genomic medicine (4,18–20). MEN 2 is an autosomal-dominant, transmitted tumor syndrome caused by germline mutation in the RET (REarrangend during Transfection) proto-oncogene and is comprised of the key endocrine neoplasia components of pheochromocytoma, hyperparathyroidism, and, importantly, medullary thyroid carcinoma (MTC) as the life-limiting alteration (21,22).

About 25% of all MTCs occur as part of the autosomaldominant MEN 2 syndrome, hence all patients treated for MTC should be tested for this mutation regardless of age or known family history. The presence of the RET MTC susceptibility gene enabled us to implement a powerful molecular diagnostic test to identify mutation carriers at premorbid stages (i.e., predictive testing) (Table 1). In this situation, family members who are found to carry the family-specific mutation but who are as yet unaffected can be subjected to life-saving prophylactic surgery and surveillance (18). Importantly, the delineation of a genotype-phenotype correlation allows us to tailor surgical techniques based on the exact amino acids that are altered (23). Furthermore, family members not harboring familyspecific disease-associated mutations can be definitively

Table 1 - Risk stratification in MEN 2.

1

Risk group

Risk type

RET codon

Youngest patient

Age for prophylactic surgery1

3 2a 2b 1

Highest Very high High Low

922, 918, 883 634, 630 620, 618, 611, 609 891, 804, 791, 790, 768

9 months 12 months 5 years 6 to 22 years

,2 years ,2 years ,5 years ,10 years

Based on the youngest observed patient.

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Personalized medicine for endocrine neoplasia Dammann M and Weber F

advised that they only have the same risk as the general population of developing the component neoplasias of MEN 2, so could be excluded from lifelong clinical surveillance and prophylactic surgeries.

There are three forms of MEN 2. First, MEN 2A, the most common form, is characterized by the components MTC: primary hyperparathyroidism and pheochromocytoma.

Because MTC can show a high penetrance at a young age, prophylactic surgery should be offered depending on the risk genotype before the age of 5–10 years. MEN 2B is the most aggressive form caused by germline mutations in codon 918 (98%) or codon 883 (2%) and is associated with an age of onset for MTC in the first year of life. Therefore, genetic testing should be offered shortly after birth in order to identify those infants who require early preventive surgery. Of note, in MEN 2B MTC occurs together with pheochromocytoma (50%) and marfanoid habitus (100%), intestinal ganglioneuromatosis, mucosal neuromas, and thickening of the corneal nerve (all up to 90%), but not primary hyperparathyroidism. Familial MTC is often considered a separate entity giving rise to MTC as the sole clinical manifestation. Whether this holds true today has to be critically discussed. Most likely, familial MTC is just the result of variability in penetrance, thus surveillance for hyperparathyroidism and pheochromocytoma should be offered to these patients as well. Importantly, for the most common exon 11 mutation (RET C634G), a penetrance of 21% by age 50 for hyperparathyroidism and 52% by age 30 and 83% by age 50 for pheochromocytoma have been reported. However, it is not possible to predict precisely if and when an individual patient might suffer from parathyroid hyperplasia or pheochromocytoma (24). Hence, all patients should undergo lifelong surveillance. Furthermore, there are other clinical aspects that cannot be entirely explained by the traditional mutations in RET. For instance, within some MEN 2 families, the age of onset and severity of disease vary considerably among the affected family members despite carrying identical germline RET mutations. For example, for the most common exon 11 mutation (Cys634Trp) the age of onset for MTC varies between

families from 3 years to .60 years. To what extent allelic variants play a role is a matter of continued investigation (25). For instance, the variant IVS1-126G . T in a large family with the G533C mutation was associated with an early onset of disease (26). One might envision that the integration of classic Mendelian cancer predisposition genes with genomic alterations of low penetrance might bring the practice of personalized genomic medicine to the next level (26,27).

Heritable adrenal neoplasias Heritable adrenal neoplasias can affect the medulla (pheochromocytoma) as well as the adrenal cortex. The genetic differential diagnoses for adrenocortical neoplasia are summarized in Table 2. Diagnostic work-up, management, and surveillance of patients with hereditary diseases can differ significantly from their sporadic counterparts, so it is important to identify those patients. For instance, in MEN 1, adrenocortical lesions occur in as many as 40% of patients. Most manifest as non-functional, hyperplastic lesions and present an indolent course. Although the incidence of adrenocortical carcinoma has been reported to be between 2% and 6%, there is no a consensus regarding the management of MEN 1-associated adrenocortical lesions. Even though there is no genotype-phenotype correlation, it is a widely accepted protocol to surgically remove lesions exceeding a cutoff size of 3 cm in diameter.

A considerable proportion of pheochromocytomas are heritable and form part of tumor syndromes other than MEN 2. In addition, studies have shown that pheochromocytomas can precede the development of MTC, so knowledge about differential diagnosis is important. Pheochromocytomas can occur in the adrenal medulla but also extraadrenally in the head-and-neck region as well as being abdominal and thoracic, where they are termed paraganglioma (PGL). It used to be thought that about 10% of all pheochromocytomas occur in a familial setting and are caused by germline genetic alterations. Today, there is growing evidence that about 25–30% of all patients with pheochromocytoma and paraganglioma harbor a germline

Table 2 - Genetic differential diagnosis for adrenocortical neoplasias.

Location Adrenocortical neoplasia

Adrenal medulla/ Pheochromozytoma

Heritable tumor syndrome and phenotype

Gene MEN1

+

Multiple endocrine neoplasia type 1 (MEN 1) Adrenocortical adenoma, HPT, pituitary and neuroendocrine tumors

+

Beckwith–Wiedemann syndrome (BWS) ACC, Wilms’ tumor, hepatoblastoma, rhabdomyosarcoma

+

McCune–Albright syndrome Cushing syndrome, pituitary tumors, polyostotic fibrous dysplasia, patchy skin pigmentation, thyrotoxicosis, gigantism

+

Li–Fraumeni Syndrome (LFS) ACC, breast cancer, sarcomas, leukemia,

+

Adrenocortical hyperplasia Nodular hyperplasia

+

Gardner syndrome ACC, polyps of the colon, osteomas, fibromas, thyroid cancer

+ + + +

Multiple endocrine neoplasia type 2 (MEN 2)

RET

Von Hippel–Lindau disease (VHL)

VHL

CDKN1C (KIP2) GNAS1

TP53 CYP21

Neurofibromatosis type 1 (NF1)

APC

NF1

Pheochromocytoma–paraganglioma syndrome (PC-PGL syndrome)

ACC: adrenocortical carcinoma. HPT: hyperparathyroidism.

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Whereas the clinical and pathologic call of malignancy remains a diagnostic challenge and preventive therapy cannot be advised, it is key to conduct predictive testing of as-yet-unaffected relatives, so that mutation carriers can received a tailored screening and surveillance program (29).

Table 3 - Comparison of pheochromocytoma-associated and paraganglioma-associated syndromes.

Syndrome Gene Mean age of diagnosis (years) Multifocal tumors Location Adrenal Extra-adrenal HNP Malignant

MEN2

VHL

NF1

PGL1

PGL3

PGL4

RET 36

VHL 22

NF1 41

SDHD 27

SDHC 46

SDHB 34

+++

+++

++

+++

+

++

+++ – – +

+++ + – +

+++ – – ++

++ ++ ++ –

– – +++ –

++ +++ + +++

Heritable non-medullary thyroid cancers In contrast to heritable MTC, for which the genetic differential diagnosis is straightforward and results in only MEN 2, heritable non-MTC (NMTC) is more complex (4,20,24,30). In this setting we have to differentiate between NMTCs that are part of a heritable syndrome and those that occur non-syndromically as familial NMTC. There are three well-characterized syndromic forms of heritable NMTC—Cowden syndrome, Gardner syndrome (familial adenomatous polyposis syndrome), and Werner syndrome — all of which are transmitted in an autosomal-dominant fashion (31,32). Cowden syndrome is caused by germline mutations of the PTEN tumor suppressor gene. Patients with Cowden syndrome present with characteristic multiple hamartomas and mucocutaneous lesions (e.g., oral papillomatous papules, trichilemmoma, and acral keratosis). In addition, affected patients are at high risk of developing various benign and malignant neoplasias, especially of the breast (up to 50% lifetime risk in affected females) and thyroid gland. While benign thyroid nodules are observed in up to 75% of patients, NMTC commonly presents as a follicular thyroid carcinoma and is associated with a 10-fold increased lifetime risk compared with the general population.

Gardner syndrome is caused by germline mutations in the APC gene and is characterized by gastrointestinal adenomatous polyps and a high risk of developing gastrointestinal and other cancers. It is estimated that the lifetime risk of developing NMTC ranges between 2% and 10% in those affected with familial adenomatous polyposis syndrome. In contrast, the gastrointestinal manifestation will progress to colorectal cancer in 100% of affected patients and preventive surgery is advised for these patients. Interestingly, NMTC will present in this setting as a unique pathologic variant known as cribriform morular variant of papillary thyroid carcinoma (33). Therefore, any patients with this diagnosis who are operated on for thyroid disease should be screened for familial adenomatous polyposis syndrome as well.

Werner syndrome is a rare autosomal recessive disease that results in premature aging (34). Besides their elderly appearance, affected patients develop various age-related disorders such as diabetes, osteoporosis, heart disease, and other malignancies. The latter two are responsible for a diminished life expectancy of around 50 years. The incidence of thyroid cancer (e.g., papillary, follicular, and anaplastic thyroid cancer) is about 20%. Because of the characteristic physical appearance, genetic testing is commonly used to confirm the clinical diagnosis.

Molecular diagnosis and predictive testing for these syndromes are available and highly accurate, and the results change medical management with regard to prophylaxis and surveillance. What cannot be done currently is accurately predict who will develop thyroid cancer and what the age of maximal risk is. However, the high incidence of malignant thyroid disease warrants regular thyroid screening and a low threshold for recommending thyroidectomy.

+++: very high. ++: high. +: low. –: absent. HNP: head-and-neck paraganglioma.

Modified from reference 7.

mutation. Six tumor syndromes, including MEN 2, Von Hippel–Lindau disease, neurofibromatosis (NF1) and pheochromocytoma-paraganglioma syndrome (PGL1, PGL2 and PGL3), and their predisposing genes have been identified (Table 3). The predominant signs of neurofibromatosis type 1 include characteristic cafe´-au-lait spots, neurofibromas, and other lesions such as freckling of the skin. Although penetrance of pheochromocytoma in NF1 is low (,5%), malignant pheochromocytomas are not infrequent. In patients with NF1 and pheochromocytoma there are no mutation hot spots, instead the alterations are spread all over the gene and include large deletions or rearrangements in about 10% of patients. As outlined above, pheochromocytoma in MEN 2 shows a variable penetrance that can be as high as 50%. Most notably, mutations in codons 634 and 918 harbor a very high risk of developing this disease.

Pheochromocytomas in MEN 2 are commonly benign and located bilaterally in the adrenal medulla. Von HippelLindau syndrome is an autosomal-dominant disorder that predisposes individuals to pheochromocytoma and other important neoplasias such as clear cell renal carcinomas, pancreatic islet cell tumors, and cystic disease of the kidney and pancreas. Interestingly, although the overall penetrance of pheochromocytoma ranges between 20% and 30% of patients with Von Hippel-Lindau syndrome, in certain families the prevalence of pheochromocytoma can be more than 90%. As a rule, all tumorous components of Von Hippel-Lindau syndrome can be effectively treated if diagnosed in time. Three paraganglioma syndrome predisposition genes have been identified so far, namely, SDHB the predisposition gene for PGL4, SDHC (PGL3), and SDHD (PGL1). Patients with PGL1 nearly always display benign but multiple adrenal pheochromocytomas and paragangliomas of the head and neck. Patients with PGL4 often display extra-adrenal, thoracic, or head-and-neck paragangliomas.

In contrast to PGL1, they are frequently malignant and about one-third of patients develop metastases. PGL3 is not associated with malignant pheochromocytoma/paraganglioma and patients display the characteristics of age, manifestation, and tumor number similar to those with sporadic HNPs. One has to be aware of the clinical symptoms and signs that allow for an operational division, which in turn is the cornerstone of prioritized genetic testing and personalized management of patients and their relatives (28). Hereditary pheochromocytoma and paraganglioma syndromes should be recognized as early as possible in order to tailor medical management and follow-up.

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19p13.2 (TCO), only TCO and NMTC1 have been replicated in independent family sets, but the susceptibly genes mapped to these loci have not been identified (42–46).

The NMTC1 locus has been replicated in 10 kindreds and linkage to FNMTC is proposed with a logarithm of the odds (LOD) score of 2.85 (where a LOD score .3 is accepted to confirm linkage) is suggested (42). However, it has been suggested that NMTC1 has a more significant association with the follicular variant of papillary thyroid carcinoma. A linkage between the TCO (thyroid tumors with cell oxyphilia) locus and NMTC has been identified and validated in four studies comprised of 21 families (LOD score of 3.01) (42,43). In contrast, studies with a total of 65 relatives failed to show a linkage, so TCO might contribute only slightly to FNMTC and be associated with cell oxyphilia.

The MNG1 locus has only shown linkage (LOD score of 4.88) in one kindred with two family members who were affected by papillary thyroid carcinoma. Interestingly, 18 family members showed multinodular goiters (44). Given that six other studies that included .100 kindreds failed to reproduce this association, MNG1 might rather account for a familial form of benign nodular disease rather than FNMTC. Linkage of the PRN locus (LOD score of 3.58) was identified in one large kindred with five cases of papillary thyroid carcinoma and two cases of papillary renal neoplasm (45). However, no additional families have been identified so far. Thus, the 1q21 locus might be associated with rare phenotypes of papillary thyroid carcinoma and papillary renal neoplasm. Recently, another association was identified in one kindred with five cases of thyroid cancer and 11 cases of benign nodular disease (47). The putative susceptibility locus was mapped to 8q23.1-p22 with a LOD score of 4.41.

However, a follow-up study by the same group included six families but failed to validate the linkage (48).

Thus, clinical genetic counseling or advice for families segregating NMTC is imprecise. Notably, there is ongoing debate as to whether or not FNMTC displays a more aggressive phenotype compared with its sporadic counterpart (49). Indeed, several studies have found that FNMTC presents at a younger age and displays more aggressive features, such as multifocality, local and lymph node invasion, and diminished tumor-free and overall survival rates (50–53). In contrast, other studies have failed to show a difference between FNMTC and sporadic cancer (49,54,55). A recent study that included 67 patients from 46 families with FNMTC and 375 controls was not able to identify a more aggressive phenotype (49,54). Still, there is a tendency for a lower age of onset (42 vs. 47 years) in the FNMTC group compared with the control group. Others have proposed prophylactic thyroidectomy for FNMTC (42). However, from a surgeon’s standpoint, the current data are too weak and inconsistent for such a general demand. First and foremost ‘‘primum non nocere’’ has to prevail. Thus, in the setting of two or more family members with NMTC, a thyroid work up should be offered to all first-degree relatives and the presence of thyroid nodular disease should have a lower threshold for recommending thyroidectomy to these patients.

It has been suggested that there is an association between non-MTC and two other familial cancer syndromes, Carney complex (PRKAR1a) and Pendred syndrome. Based on a small series of 15 kindreds with Carney complex the prevalence of thyroid cancer is estimated 4% in affected family members (35). For Pendred Syndrome the estimated 1% prevalence for thyroid cancer is deduced from a single family report in which two of the three affected family members developed follicular thyroid cancer (36). Whether these are true associations due to the germline alterations that are involved, or if additional modifying events participate, is a matter of debate. However, patients with Carney complex typically develop nodular thyroid disease in childhood (about 60%) and in Pendred syndrome there is a high frequency of multinodular goiters and hypothyroidism. Hence, while a preventive thyroidectomy cannot be advised for any of these syndromes, a regular thyroid work-up should be part of the routine surveillance for patients identified as being at risk.

Non-syndromic familial non-MTC For non-syndromic familial NMTC (FNMTC), epidemiologic data indicate a very high likelihood of familial aggregation and, hence, a strong genetic component (4,37).

Indeed, it is estimated that about 10% of all cases of NMTC are hereditary, putting affected first-degree relatives at up to a 10-fold increased risk compared with the general population (38,39). In fact, differentiated epithelial thyroid carcinomas have one of the highest familial risks among all cancer sites. FNMTC is characterized by two or more first-degree relatives who are affected by thyroid carcinomas (NMTC) but lack the signs of other hereditary syndromes or exposure to other risk factors (e.g., radiation). This operational definition is based on the assumption that the chance of clustering of three or more relatives with NMTC in one family by chance is less than 6% (40). In contrast, there is a .60% likelihood that two affected first-degree relatives will harbor sporadic disease (40). The standardized incidence ratio is an index that estimates the familial risk of developing a malignancy based on epidemiologic data, and as a result may be viewed as a measure of cancer risk for the children of parents with a specific malignancy. For NMTC, the standardized incidence ratio exceeds an impressive value of 3.8 (38,41). One might argue that the familial aggregation might be the result of environmental factors shared by the family members because environmental factors have been implicated in thyroid carcinogenesis. However, studies comparing cancer risk between spouses showed that the standardized incidence ratio does not exceed 1.4. Therefore, for NMTC, it has been argued that heritability is most likely the main contributor to the high standardized incidence ratios and that familial aggregations of NMTC are most likely the result of germline mutations in susceptibility genes.

Despite the impressive standardized incidence ratio for FNMTC and tremendous efforts over the last 10 years to identify the cancer-associated gene, no susceptibility genes have been identified so far (4,42). Nevertheless, at least five putative susceptibility loci have been identified. The caveat remains that for each locus evidence both in favor and opposing association with FNMTC has been published (42).

This can be explained by the fact that the operational definition for FNMTC varies between studies (some already include families with two affected first-degree relatives) and some include benign thyroid diseases as well. Of the five loci on 1q21 (PRN), 2q21 (NMTC1), 8p23.1-p22 (FTEN), 14q31 (MNG1), and

KNOWING HERITABLE CANCER PHENOMICS: THE FIRST STEP TO PRACTICE PERSONALIZED MEDICINE Over the recent years, interest in the field of personalized medicine has increased. In fact, one has to wonder if

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8. Anney R, Klei L, Pinto D, Regan R, Conroy J, Magalhaes TR, et al. A genome-wide scan for common alleles affecting risk for autism. Hum Mol Genet. 2010;19(20):4072-82.

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12. Guttmacher AE, McGuire AL, Ponder B, Stefansson K. Personalized genomic information: preparing for the future of genetic medicine. Nat Rev Genet. 2010;11(2):161-5.

13. Hall WD, Mathews R, Morley KI. Being more realistic about the public health impact of genomic medicine. PLoS Med. 2010;7(10):e1000347, http://dx.doi.org/10.1371/journal.pmed.1000347 14. Feero WG, Guttmacher AE, Collins FS. Genomic medicine: an updated primer. N Engl J Med. 2010;362(21):2001-11, http://dx.doi.org/10.1056/ NEJMra0907175.

15. Paynter NP, Chasman DI, Buring JE, Shiffman D, Cook NR, Ridker PM. Cardiovascular disease risk prediction with and without knowledge of genetic variation at chromosome 9p21.3. Ann Intern Med. 2009;150(2):6572.

16. Colombo S, Rauch A, Rotger M, Fellay J, Martinez R, Fux C, et al. The HCP5 single-nucleotide polymorphism: a simple screening tool for prediction of hypersensitivity reaction to abacavir. J Infect Dis. 2008;198(6):864-7, http://dx.doi.org/10.1086/591184.

17. Buchan BW, Peterson JF, Cogbill CH, Anderson DK, Ledford JS, White MN, et al. Evaluation of a Microarray-Based Genotyping Assay for the Rapid Detection of Cytochrome P450 2C19 *2 and *3 Polymorphisms From Whole Blood Using Nanoparticle Probes. Am J Clin Pathol.

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19. Frilling A, Weber F. Prophylactic thyroid surgery. Chirurg. 2006;77(1):614, http://dx.doi.org/10.1007/s00104-005-1118-7.

20. Weber F, Eng C. Update on the molecular diagnosis of endocrine tumors: toward -omics-based personalized healthcare? J Clin Endocrinol Metab.

2008;93(4):1097-104, http://dx.doi.org/10.1210/jc.2008-0212.

21. Eng C. RET proto-oncogene in the development of human cancer. J Clin Oncol. 1999;17(1):380-93.

22. Frilling A, Dralle H, Eng C, Raue F, Broelsch CE. Presymptomatic DNA screening in families with multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma. Surgery. 1995;118(6):1099-103; discussion 1103-4, http://dx.doi.org/10.1016/S0039-6060(05)80120-5 23. Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel RF, et al. The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. JAMA. 1996;276(19):1575-9.

24. Milos IN, Frank-Raue K, Wohllk N, Maia AL, Pusiol E, Patocs A, et al.

Age-related neoplastic risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germ line RET Cys634Trp (TGC.TGG) mutation. Endocr Relat Cancer. 2008;15(4):1035-41, http:// dx.doi.org/10.1677/ERC-08-0105.

25. Weber F, Eng C. Editorial: germline variants within RET: clinical utility or scientific playtoy? J Clin Endocrinol Metab. 2005;90(11):6334-6, http:// dx.doi.org/10.1210/jc.2005-2030.

26. Tamanaha R, Camacho CP, Pereira AC, da Silva AM, Maciel RM, Cerutti JM. Evaluation of RET polymorphisms in a six-generation family with G533C RET mutation: specific RET variants may modulate age at onset and clinical presentation. Clin Endocrinol (Oxf). 2009;71(1):56-64, http:// dx.doi.org/10.1111/j.1365-2265.2008.03491.x.

27. Jiang Q, Ho YY, Hao L, Nichols Berrios C, Chakravarti A. Copy number variants in candidate genes are genetic modifiers of Hirschsprung disease. PLoS One. 2011;6(6):e21219.

28. Erlic Z, Rybicki L, Peczkowska M, Golcher H, Kann PH, Brauckhoff M, et al. Clinical predictors and algorithm for the genetic diagnosis of pheochromocytoma patients. Clin Cancer Res. 2009;15(20):6378-85, http://dx.doi.org/10.1158/1078-0432.CCR-09-1237.

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30. Eng C. Familial papillary thyroid cancer: many syndromes, too many genes? J Clin Endocrinol Metab. 2000;85(5):1755-7, http://dx.doi.org/ 10.1210/jc.85.5.1755.

31. Eng C. Mendelian genetics of rare—and not so rare—cancers. Ann N Y Acad Sci. 2010;1214:70-82.

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personalized medicine is snowballing out of control when direct-to-consumer genomic testing is becoming increasingly available (56). Indeed, since 2007 the interested consumer is able to receive a whole-genome scan for less than US$500, without ever having to see a doctor or receive medical advice.

This kind of self-administered genetic testing is problematic, as shown by Bloss et al. (56) who found a correlation between test-related distress and increased lifetime risk. Various professional organizations, including the American Medical Association and the American College of Medical Genetics, state that such genetic testing should not be performed without the supervision of healthcare providers. However, centers for genomic medicine, and even clinical cancer genetics, are far from being ubiquitously established even in high-level medical centers. It is estimated that only 1% of patients that would benefit from this 21st century approach to medical treatment will be referred to professional genetic counseling (31). The implementation of state-of-the-art clinical genetic counseling within the constraints of budget and limited human resources is one of the important tasks that have to be tackled.

Eventually, one has to keep in mind that, notwithstanding all of these technological advances, several tumor syndromes can be identified by rigorous clinical analysis (57).

Especially regarding endocrine-related neoplasia, the medical practitioner has to be aware of the phenomics, or phenotypic profiles, that comes along with the array of inheritable endocrine tumor syndromes. It is of no surprise that several of the inherited tumor syndromes were first described and named long before genetic causes had been identified. In fact, it should be paramount to the medical professional to use meticulous observation to ‘‘personalize’’ the decision for treatment options and, much more importantly, to prioritize medical genetic testing.

ACKNOWLEDGMENTS Frank Weber is deeply grateful to his mentor, Charis Eng, who always encouraged and inspired him. Thank you for your friendship and advice.

AUTHOR CONTRIBUTIONS Weber F and Dammann M were responsible for the selection of relevant literature as well as for writing and revising the manuscript.

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DOI:10.6061/clinics/2012(Sup01)17

REVIEW

Biochemical, bone and renal patterns in hyperparathyroidism associated with multiple endocrine neoplasia type 1 Delmar M. Lourenc¸o Jr.,I Flavia L. Coutinho,I Rodrigo A. Toledo,I Tatiana Denck Gonc¸alves,I Fabio L. M. Montenegro,II Sergio P. A. ToledoI I Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Endocrine Genetics Unit (LIM-25), Division of Endocrinology, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Division of Head and Neck Surgery, Sa˜o Paulo/SP, Brazil.

Primary hyperparathyroidism associated with multiple endocrine neoplasia type I (hyperparathyroidism/multiple endocrine neoplasia type 1) differs in many aspects from sporadic hyperparathyroidism, which is the most frequently occurring form of hyperparathyroidism. Bone mineral density has frequently been studied in sporadic hyperparathyroidism but it has very rarely been examined in cases of hyperparathyroidism/multiple endocrine neoplasia type 1. Cortical bone mineral density in hyperparathyroidism/multiple endocrine neoplasia type 1 cases has only recently been examined, and early, severe and frequent bone mineral losses have been documented at this site. Early bone mineral losses are highly prevalent in the trabecular bone of patients with hyperparathyroidism/ multiple endocrine neoplasia type 1. In summary, bone mineral disease in multiple endocrine neoplasia type 1related hyperparathyroidism is an early, frequent and severe disturbance, occurring in both the cortical and trabecular bones. In addition, renal complications secondary to sporadic hyperparathyroidism are often studied, but very little work has been done on this issue in hyperparathyroidism/multiple endocrine neoplasia type 1. It has been recently verified that early, frequent, and severe renal lesions occur in patients with hyperparathyroidism/multiple endocrine neoplasia type 1, which may lead to increased morbidity and mortality. In this article we review the few available studies on bone mineral and renal disturbances in the setting of hyperparathyroidism/multiple endocrine neoplasia type 1. We performed a meta-analysis of the available data on bone mineral and renal disease in cases of multiple endocrine neoplasia type 1-related hyperparathyroidism.

KEYWORDS: Osteoporosis; Bone Demineralization; Nephrolithiasis; Outcome; Natural History.

Lourenc¸o DM Jr, Coutinho FL, Toledo RA, Gonc¸alves TD, Montenegro FLM, Toledo SPA. Biochemical, bone and renal patterns in hyperparathyroidism associated with multiple endocrine neoplasia type 1. Clinics. 2012;67(S1):99-108.

E-mail: delmarmuniz@usp.br Tel.: 55 11 3061-7252

the disease (2–6). It is worth noting that subtotal or total parathyroidectomy (PTx) is usually recommended for the treatment of inherited HPT cases, whereas adenomectomy is indicated for the vast majority of sporadic HPT cases (6– 8). As part of the familial HPT subset, HPT associated with multiple endocrine neoplasia type 1 (HPT/MEN1) comprises up to 4.5% of all HPT cases (9). MEN1 is a complex autosomal-dominant hereditary disorder characterized by a high predisposition to develop a wide spectrum of endocrine and non-endocrine tumors. MEN1 is defined by the presence of tumors in at least two of the three main endocrine glands involved in this syndrome (i.e., the parathyroids, the pituitary gland, and the endocrine cells of the pancreas/duodenum). Familial MEN1 is determined by the identification of at least one of the major tumors in a family member of the index case with MEN1 (5). HPT is the most frequent tumor in MEN1, reaching nearly complete penetrance by the time the patient is 50 years of age. Sporadic HPT (S-HPT) differs from MEN1-related HPT in many ways and the recognition of these differences may help to reveal the diagnosis of MEN1 (Table 1) (10).

INTRODUCTION Hyperparathyroidism in multiple endocrine neoplasia type 1 Primary hyperparathyroidism (HPT) is one of the most common endocrine diseases, with an estimated prevalence of 1–21 cases per 1,000 and an incidence of 27–30 cases per 100,000 person-years (1). HPT is biochemically characterized by hypercalcemia associated with abnormally high serum levels of parathyroid hormone (PTH). It is caused by parathyroid adenoma in 80–90% of cases, parathyroid hyperplasia in 10–15% of cases, and carcinoma in less than 1% of cases (2–4).

HPT occurs predominantly as a sporadic disease in 95% of cases; the remaining 5% of cases are hereditary forms of

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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Table 1 - Classic comparisons of sporadic HPT and MEN1-related HPT. Clinical characteristic Onset Sex (M:F) Germline MEN1 mutation Pathology Surgery Timectomy Other associated tumors Adverse effects of hypergastrinemia Cure rate HPT recurrence rate after PTx

MEN1-related HPT

Sporadic HPT

Earlier (20–30 years) 1:1 Yes Multiglandular (hyperplasia) Total or subtotal PTx Yes Yes Yes Low High (,50%)

Later (50–70 years) 1:3 No Uniglandular (adenoma) Adenomectomy No No No High (. 90%) Rare

HPT: primary hyperparathyroidism. MEN1: multiple endocrine neoplasia type 1. PTx: parathyroidectomy.

date (22). Also, in case series, new germline MEN1 mutations were documented by our group and all types of mutations have been observed, mostly those leading to the truncation of the Menin protein (23,24). Point mutations in the MEN1 gene represent up to 20% of cases, as recently confirmed by us (www.arup.utah.edu/database/ men1/classification.php). Furthermore, Menin interacts with several partners (25–28).

A marked change in the clinical picture of HPT occurred after the widespread introduction of annual calcium measurements using multichannel analyzers in the mid 1970s in the USA and Europe. Beyond increasing prevalence of S-HPT, this practice resulted in crescent diagnosis of the asymptomatic form of HPT and progressive decreasing of symptomatic cases. Thus, renal, bone and neurological complications secondary to S-HPT are increasingly uncommon nowadays in countries where routine measurements of serum calcium are performed (1–4).

At present, most S-HPT cases are asymptomatic and characterized by vague neurocognitive symptoms such as pain, fatigue, depression, and milder degrees of bone and renal disease mainly characterized for reduced creatinine clearance rate and bone demineralization (1–4).

OBJECTIVE Very few papers have addressed the bone mineral and renal complications that occur secondary to MEN1-related HPT, the shift to the asymptomatic form, and the natural history of this disorder. In this review, we present a metaanalysis of the current literature on this subject.

Other tumors associated with MEN1 Biochemical, bone mineral and renal disease in HPT related to MEN1

Multiple duodenal gastrinomas and non-functioning pancreatic neuroendocrine tumors occur in 40% and 34% of cases, respectively, at around 50 years of age. Because of their high potential for malignancy, these types of tumors are the major causes of MEN1-related morbidity and mortality (5,11,12). Several tumors that may be associated with MEN1 have a high potency for malignancy but occur at low frequencies (2%), such as glucagonomas, somatostatinomas and pancreatic polypeptide or vasoactive intestinal polypeptide secreting tumors. (5).

Pituitary tumors may occur in up to 30–40% of the patients with MEN1, mostly in the form of prolactinomas and non-functioning and co-secreting pituitary adenomas.

In addition, corticotrophin-secreting tumors may potentially lead to Cushing’s disease. It should also be noted that acromegaly/gigantism is less prevalent in MEN1, and other forms of familial acromegaly should be ruled out (13–19).

A wide spectrum of other endocrine tumors may be present, such as thymic, bronchial and gastric carcinoids and nonfunctioning adrenocortical tumors, as well as non-endocrine tumors, such as facial angiofibromas, collagenomas, lipomas, meningeomas, ependimomas, and pinealomas (5,20,21).

Burgess series. The first study on bone mineral status in MEN1 was published by Burgess et al. in 1998 (29). In this paper, 29 women with HPT from a large Tasmanian family with MEN1 were studied. Bone mineral density (BMD) was measured at only two main bone sites: the femoral neck (FN) and the lumbar spine (LS). A high frequency of osteoporosis (T-score ,–2.5) was verified in the FN (44.8%) and LS (25.6%), whereas osteopenia (T-score ,–1.0 and .

–2.5) was also prevalent in the FN (41.4%) and LS (34.5%).

Considering all cases with reduced BMD (T-score ,–1.0), the FN was more compromised (86.2%) than the LS (60.1%). Of note, normal BMD in the LS was found in 37.9% of cases whereas normal BMD in the FN was found in 13.8% of cases, suggesting a preferential demineralization in mixed bone rather than in trabecular bone. The high prevalence of osteopenia and osteoporosis in the LS of these cases might suggest that in HPT/MEN1 patients the trabecular bone is less metabolically ‘‘protected’’ than in asymptomatic S-HPT patients (29– 31). The finding of severe osteopenia at the FN by 35 years of age (T-score ,–2.0) in four of the nine cases (44%) of the MEN1 subset with uncontrolled HPT suggest an early bone disease in this MEN1-related HPT subset (29). One of the illustrations in this study showed that there were 20 women who were younger than 50 years. Half of them (10/20; 50%) had T-scores ,–2.0 for the FN and seven of them had such scores for the LS (35%). The individual Zscores of the studied cases were not published, preventing an analysis of the Z-scores for cases younger than 50 years, as is presently recommended. Also, reduced BMD is

Genetic aspects The gene responsible for MEN1 syndrome, MEN1 gene, is localized to chromosome 11q13 and belongs to a class of tumor suppressor genes. The MEN1 gene codes Menin, a ubiquitously expressed nuclear protein that interacts directly with proteins involved in transcriptional regulation, genome stability, cell division, and cell cycle control (20). More than 1,000 mutations of the MEN1 gene associated with MEN1 syndrome have been described to

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in 56 of the 84 patients and bone demineralization was observed in 47% of them (Z-scores were ,–2.0 in at least one of the analyzed bone sites). These data led the authors to suggest that a more virulent form of HPT presents in MEN1 cases with HPT/gastrinoma. However, the authors did not indicate which bone sites had been examined (38). Of note, Burgess et al. found no association between reduced bone mass and a restricted number of cases with gastrinoma (29), whereas no data on the BMD status of the subset of cases of HPT/gastrinoma were available in our first case series (36).

Eller-Vainicher case series. This study was the first to compare BMD data from a MEN1-related HPT series with BMD data from S-HPT cases (39). Findings from 469 S-HPT cases were compared with those of 64 HPT/MEN1 patients (36 index cases and 28 affected relatives).

There was no significant difference between HPT/MEN1 index cases and their affected family members in terms of clinical, biochemical, and BMD findings. However, HPT/ MEN1 patients had a tendency to be younger and have lower serum levels of phosphate and PTH than patients with S-HPT. Both HPT occurring before 50 years of age and inappropriately normal PTH values were considered independent, predisposing factors to MEN1-related HPT. Also, the likelihood of association with the MEN1 syndrome was 13.5-fold greater when both factors were present (39).

A BMD analysis of LS and FN bone sites was conducted in 432 patients (388 S-HPT and 44 HPT/MEN1 cases). In both sites, bone mineral losses were more compromised in the HPT/MEN1 subset than in the S-HPT subset (Z-scores: LS, –1.33¡1.23 vs. –0.74¡1.40; FN, –1.13¡0.96 vs. –0.60 ¡1.07). The prevalence of nephrolithiasis was similarly high in both groups (57.8% vs. 55.2%) (39).

It was concluded that bone mineral disease is more severe in cases with MEN1-related HPT than in cases with S-HPT. These findings are in agreement with previous evidence indicating the lack of relative protection of the vertebral (trabecular) bone in HPT/MEN1 (10,29,36).

Kann case series. Recently, Kann et al. (40) studied BMD in 23 patients with MEN1-related HPT using peripheral quantitative computed tomography, and they confirmed the previous findings of marked bone demineralization in HPT/MEN1. Despite the limited number of patients and the absence of statistical significance, it was suggested that several other pathological conditions that potentially occur in MEN1 could be responsible, in part, for the bone demineralization observed in these cases. The conditions cited as potentially influencers of the BMD values were gonadotrophic and somatotrophic insufficiency, hypercortisolism and a history of upper gastrointestinal surgery (40).

considered when the Z-score is ,–2.0 in individuals younger than 50 years (32,33).

Although there were unknown numbers of untreated and treated patients, BMD in the subset of patients with controlled HPT was better than that in patients with uncontrolled HPT (as measured by the Z-score), indicating that previous BMD reports could be even more compromised initially suspected. In addition, five patients could be analyzed before and after successful subtotal parathyroidectomy in this study. Although the number of patients was limited, a marked increase in bone mass was noted at the LS and FN over a mean follow-up period of 18 months, suggesting a possible beneficial effect of early surgical treatment for treating HPT/MEN1 cases, similar to that observed in S-HPT cases (29,34,35).

Our first case series. The second paper addressing this topic was published in 2008 and describes a large Brazilian family with MEN1 and 50 clinically and/or genetically affected members over six generations (36). BMD analysis was performed in 20 HPT/MEN1 patients: 17 untreated cases and three cases with uncontrolled HPT due to previously unsuccessful parathyroid surgeries. In the 20 affected family members, we examined cortical bone BMD in HPT/MEN1 for the firstly approach (36).

Although the number of patients studied was limited, it was possible to analyze data from both men and women and a preliminary natural history of bone disease in MEN1 could be developed. Of the 20 cases, 75% and 60% of them had osteoporosis (T ,–2.5) and osteopenia (T ,–1.0 and .

–2.5), respectively, in at least one of the three main bone sites. Considering all cases with reduced BMD (T ,–1.0), the most compromised bone site was the proximal one-third of the distal radius (90%; 1/3DR), followed by the FN (65%), and LS (60%) (36).

Osteoporosis was predominant in 1/3DR (55%), followed by the LS (40%), and FN (20%). The degree of bone demineralization was more severe in the cortical bone (T – 2.87¡0.32 for the 1/3DR), although L1–L4 (T –1.92¡0.39) and the FN (T –1.48¡0.27) were also affected. Moreover, cortical bone was the most compromised bone site in these MEN1-related HPT cases, as it was the first to be affected and presented with the highest prevalence of bone demineralization and the worst BMD values (36). These findings are quite similar to those that have been reported for S-HPT (30,31,35).

Data from our study (36) reinforce the findings of Burgess et al. (29) by reporting a high frequency of reduced BMD of the LS in the subset of MEN1-related HPT cases. In both studies, patients presented with serum PTH levels that were 2- to 3-fold higher than the upper limit of the normal range, as is frequently observed in asymptomatic S-HPT (34,35).

However, the relative protection of vertebral bone sites, secondary to the anabolic effect of these PTH values on trabecular bone, was not observed in the HPT/MEN1 subset, in contrast with data on S-HPT (29,31,36,37).

Norton case series. In a study on the long-term outcome of PTx in 84 consecutive MEN1 cases with HPT associated with gastrinoma (HPT/gastrinoma), Norton et al. (38) reported a more severe form of HPT. In these cases, the disease was characterized by a higher frequency of nephrolithiasis at the time of diagnosis (62% vs. 40¡6% from seven literature series), a higher frequency of recurrent HPT (44% vs. 17% from 15 literature series, subtotal PTx), and higher levels of PTH (2.4-fold vs. 1.67¡0.38 from eight literature series). In addition, BMD studies were performed

BMD after parathyroidectomy in HPT associated with MEN1 The fifth study that examined BMD status in MEN1related HPT described short-term BMD changes that occurred after PTx in 16 HPT/MEN1 cases. A significant recovery in terms of BMD at the LS and FN, but not in the 1/3DR, was documented in 16 HPT/MEN1 patients at 15 months after PTx (41). These data contrast with the findings of BMD recovery that were verified in cases with S-HPT, in which a BMD gain was noticed in all three bone sites (35).

Several factors potentially influence or even determine the differences in the patterns of BMD recovery in HPT/MEN1 compared with S-HPT: different surgical protocols (total PTx in HPT/MEN1 vs. adenomectomy in S-HPT), the

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amount of remnant parathyroid tissue, the degree of success of parathyroid auto-implantations, and the lack of proper PTH action for long periods in patients who undergo total PTx. Most cases reached appropriate PTH levels, as produced by the parathyroid implant, only 9 months after surgery (41).

Further long-term studies are needed to elucidate whether a cortical bone BMD recovery after PTx would occur in HPT/ MEN1 cases. As the metabolic activity of cortical bone is slower than that of trabecular bone (41), it would be expected that a long-term BMD recovery at the 1/3DR may occur in HPT/MEN1 patients under more prolonged PTH action.

Furthermore, we had the opportunity to first report the basal and post-PTx values of bone formation and reabsorption markers osteocalcin and CTX, respectively, in HPT/MEN1 (41). The levels of basal bone markers varied from mildly high to normal, and a significant decrease was observed in these cases after total PTx followed by parathyroid autoimplantation in the non-dominant forearm (41).

Bone disease Seven investigations have addressed this subject to date.

BMD in HPT/MEN1 cases was examined by most of these studies, although different bone sites were analyzed and different parameters were applied limiting an integrated analysis (Table 3).

The LS, a site rich in trabecular bone, and the FN, a site composed of mixed amounts of trabecular and cortical bone, were analyzed in five of the seven studies by bone densitometry (10,29,36,39,41). The 1/3DR, consisting mainly of cortical bone, is the preferentially compromised bone site in S-HPT (30,34,35,42); however, this site was examined in only three of the latter five studies (10,36,41). One study did not mention which bone sites were analyzed (38). A recent study used peripheral quantitative computed tomography instead of dual-energy X-ray absorptiometry to analyze the bone mineral status (40) (Table 3).

Of note, different parameters were used to analyze BMD in these studies. T-score values were preferentially used in some studies, while Z-score values were utilized in others, depending on the objectives and criteria recommended at each time (Table 3) (29,41). At present, the criteria applied for analyzing BMD were defined and officially adopted by the International Society for Clinical Densitometry (ISCD) in 2008 and accepted by consensus on HPT in 2009 (32,33).

According to these criteria, BMD values should be measured by dual-energy X-ray absorptiometry at four major bone sites: the lumbar spine (L1-L4), the FN, total hip (TH), and the 1/3DR (32,33). The Z-score should be used for patients younger than 50 years, whereas the T-score should be used for patients older than 50 years. Z-score values ,– 2.0 or T-score values ,–1.0 indicate a reduced BMD, T-score values between –1.0 and –2.5 indicate osteopenia and Tscores ,–2.5 indicate osteoporosis (32,33). These new criteria have been used in one BMD study of HPT/MEN1 cases so far (10).

The lack of available individual BMD data and the superposition of patients who had been studied in an earlier case sets were noticed in some of these studies, limiting the possibility of a combined BMD analysis using the ISCD criteria (32,33). A combination analysis using the present ISCD criteria, excluding interposition of patients from different studies, could be applied, in part, by using BMD data from two studies (Table 4) (10,29).

ISCD criteria were applied to the findings obtained from patients older than 50 years from the two latter studies; BMD data from the LS and FN were reanalyzed for 24 patients and data from the TH and 1/3DR were reanalyzed for 14 cases. BMD (T-score ,–1.0) was found to be reduced in 79.1%, 83.3%, 100%, and 92.9% of cases at the LS, FN, TH, and 1/3DR sites, respectively. Osteoporosis was more prevalent than osteopenia in the LS (58.3% vs. 20.8%) and in the 1/3DR (57.1% vs. 35.7%), but this condition was equally prevalent in the FN (41.6%) and osteopenia was dominant in TH (78.6% vs. 21.4%) (10,29). In this evaluation, we included the older cases reported by Burgess et al., despite the presence of patients with uncontrolled (4/9 cases) and controlled HPT (5/9 cases). These findings confirm recent data on outcomes in MEN1-related HPT that indicate an increased frequency of age-related bone demineralization in all bone sites in 15 untreated HPT/ MEN1 cases (Table 4) (10).

Presently, it is preferable that BMD data obtained from younger patients be analyzed using the ISCD criteria. To

Our second case series Recently, we reported the first study to examine the early and late outcomes of bone mineral and renal complications in 36 patients with uncontrolled HPT from eight MEN1 families (10). These topics have been extensively studied in sets of cases with S-HPT (30,34,35,42).

The peculiar clinical characteristics of these patients provided the conditions required to study the outcome of HPT cases associated with MEN1 (Table 2). Bone demineralization was early, extensive, severe, and progressive in these cases. The 1/3DR (cortical bone) was the preferentially compromised bone site in younger patients and asymptomatic cases (10).

In addition, the high prevalence of nephrolithiasis in cases younger than 30 years and in most cases older than this age suggested that a relatively short period of asymptomatic HPT may be the rule for MEN1, in contrast to S-HPT cases (Table 2) (10,35). However, as only 15 HPT/MEN1 cases younger than 30 years and nine asymptomatic HPT/MEN1 cases were available to be studied (10,35), further longitudinal BMD data are needed in this set of cases.

In this study, progressive bone mineral loss could be observed with aging and long-standing HPT disease. Thus, in the older group (.50 years) bone demineralization was significantly more frequent, extensive, and severe.

Furthermore, the older group had higher frequencies of previous fractures and a progressive decrease in renal function associated with a high frequency of nephrolithiasis-related co-morbidities (10) (Table 2).

Progressive demineralization of the cortical and trabecular bones was associated with the duration of HPT disease; specifically, in cortical bone, this progression was also observed in patients who had had HPT disease for more than 10–20 years (Table 2). This bone demineralization pattern differs from that reported for S-HPT, in which stable bone disease occurred at these sites for the first 10 years of disease followed by progressive demineralization of cortical bone for up to 15 years (10,35).

DISCUSSION We performed a meta-analysis of data on bone mineral status and renal complications secondary to HPT associated with MEN1.

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Table 2 - Early and late outcomes of renal and bone disease in MEN1-related HPT. Characteristics of 36 MEN1-related HPT cases

N N N N N

Symptomatic cases, predominantly (75%) Untreated cases, mainly (86.1%) All uncontrolled HPT Long-term disease (15.4¡12.8 years) (61%) Presence of young and old cases, including nine asymptomatic cases (25%) Specific bone mineral profile (distinguishing characteristic from the profile of sporadic HPT)

Severe demineralization of cortical (1/3DR) and trabecular bone (LS) Bone mineral outcome Early stages

N

Late stages

N

Preferential demineralization in 1/3DR, which may be the only compromised bone site

N N

compromised

N

Absence of vertebral bone protection

Vertebral and cortical bone are equally compromised, with a predominance of osteoporosis

N

Lower number of compromised bone sites in asymptomatic cases and symptomatic cases with a shorter estimated period of disease (less than 10 years)

N

All bone sites are frequently demineralized and more severely

Higher number of compromised bone sites in symptomatic cases with longer estimated period of disease (after 10 or 20 years)

N

Bone disease may preceed renal disease

Higher frequency of fracture in cases older than 50 years old (40% vs. 4.8%)

Proximal one-third of the distal radius Lower BMD in:

N N N

cases with a history of HPT for more than 10 years and more than 20 years patients with HPT/gastrinoma patients with higher levels of calcium Lumbar spine

Lower BMD in:

N

patients who have had HPT for a longer time Renal disease outcome

N N N

Early onset of urolithiasis (86.2%, ,30 years) High frequency of therapeutic procedures to urolithiasis as surgery and lithotripsy (47.2%) Higher prevalence of different degrees of renal insufficiency in cases older than 50 years old (40%)

Data were obtained from Lourenc¸o et al. (10). MEN1: multiple endocrine neoplasia type 1. HPT: primary hyperparathyroidism. LS: lumbar spine. 1/3DR: proximal one-third of the distal radius. BMD: bone mineral density.

date, these criteria have been applied only to one HPT/ MEN1 case series (10). An early onset of reduced BMD was found in half (6/12) of patients below the age of 30 years.

Furthermore, in this study, 40% of patients younger than 50 years had reduced BMD at the 1/3DR compared with 23.8%, 25%, and 22.2% at the LS, FN, and TH, respectively.

Reduced BMD (Z-score ,–2.0) in at least one bone site was noticed in 62% of all patients younger than 50 years (12/21), confirming the presence of early bone demineralization

in MEN1-related HPT. In addition, this study confirms previous BMD data obtained using T-score values and indicates that bone demineralization in MEN1-related HPT is most likely an early event (Table 4) (10,29). However, further BMD data obtained from younger HPT/MEN1 cases are necessary to clarify this issue.

In the subset of patients with S-HPT, the asymptomatic form is more prevalent in developed countries. The classical bone demineralization pattern of this condition

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Table 3 - The methods used in the seven studies that examined bone mineral status in MEN1-related HPT. Literature/Method

Number of patients

Bone sites

Criteria used in BMD analysis T-scores*: Osteopenia, ,21.0 Severe osteopenia, ,22.0 Osteoporosis, ,22.5 Z-score*: .–1.0; ,–1.0; ,–2.0 T-scores*: Osteopenia, ,21.0 Osteoporosis, ,22.5 Any Z score #–2.0 Z score #–2.0

Burgess et al. (29)/DXA

29

LS FN

Lourenc¸o et al. (36)/DXA

20

Norton et al. (38) Eller-Vainicher et al. (39)/DXA

56 44

Coutinho et al. (41)/DXA

16

Lourenc¸o et al. (10)/DXA

36

LS FN 1/3 DR Not available LS FN LS FN TH 1/3 DR UDR LS FN TH 1/3 DR

Khan et al. (40)/pqCT (g/cm3)

23

Forearm Trabecular BMD/ total BMD

Mean values of: g/cm2, T- and Z-scores before/after PTx

T-scores**: Reduced BMD, ,–1.0 Osteopenia, –1.0 to –2.5 Osteoporosis, ,–2.5 Z score***: Reduced BMD, #–2.0 Z-score: .–1: normal –1 to –2: low ,–2: reduced

BMD: bone mineral density. MEN1: multiple endocrine neoplasia type 1. HPT: primary hyperparathyroidism. DXA: dual-energy X-ray absorptiometry. pqCT: peripheral quantitative computed tomography. LS: lumbar spine. FN: femoral neck. 1/3DR: proximal one-third of the distal radius. TH: total hip.

UDR: ultra distal radius. T-scores: difference from the mean BMD value in a healthy young reference population, in SD units. Z-scores: age-matched comparison, in SD units. *: independent of age. PTx: parathyroidectomy. **: performed in patients older than 50 years of age. ***: performed in patients younger than 50 years of age.

is characterized by the early and preferential loss of cortical bone (1-4,30,31,35). It was speculated that long-term asymptomatic hypercalcemia could occur in HPT/MEN1 cases, similar to that reported in S-HPT (43). However, as far as we know, no prospective study on this specific topic has been performed to date. In this context, the BMD status of asymptomatic HPT/MEN1 cases has been rarely studied (10). So far, only nine asymptomatic HPT/MEN1 cases have been analyzed in a cross-sectional study, although early bone losses were documented in 44.4% of the cases with a predominance of cortical bone demineralization.

Interestingly, eight of these patients (88.9%) were younger than 30 years.

of nephrolithiasis in MEN1-related HPT (10). These two studies revealed a high frequency of kidney co-morbidities secondary to nephrolithiasis (10,45). Taken together, these two studies analyzed a total of 62 patients and 46 of them (74.2%) had renal calculi. In 26 of these 46 cases (56.5%), sessions of extra-corporeal shock-wave lithotripsy and/or surgical procedures were needed to treat nephrolithiasis.

Three cases (6.5%) required unilateral nephrectomy because of pyonephrosis and staghorn calculus presented as the first renal clinical manifestation, acute obstructive urolithiasis resulting in renal abscess and sepsis after only a few crisis of renal calculi and chronic urolithiasis associated with recurrent renal infections leading to complete loss of kidney function, respectively (10,45). Different degrees of renal insufficiency were reported in seven of 29 HPT/MEN1 cases (19.4%). In two patients older than 47 years of age, creatinine clearance rates less than 44 ml/min/1.73 m2 were reported (10).

Renal disease in HPT associated with MEN1 A high frequency of early-onset nephrolithiasis was documented in our patients before 30 years of age (86.2%), suggesting the existence of a relatively short period of asymptomatic hypercalcemia in this condition. Occasionally, two asymptomatic cases became symptomatic soon after the diagnosis of HPT (10). These findings supported the hypothesis that patients with MEN1-related HPT have a higher susceptibility to nephrolithiasis than S-HPT patients (10,39,44,45).

Consistent with this view, Christopoulos et al. (45) reported a high frequency of renal calculi in HPT/MEN1 patients before 30 years of age: 15 of the 17 younger patients (82%) in the report presented with nephrolithiasis. Of note, only three of our nine HPT/MEN1 patients with no renal calculi were older than 30 years, reinforcing the early onset

HPT associated with MEN1 and gastrinoma Norton et al. (38) reviewed seven series of MEN1-related HPT and the frequency of nephrolithiasis in this group was 40¡6%. It was suggested that MEN1 patients with HPT/ gastrinoma had a higher susceptibility to the development of nephrolithiasis (62%) and a higher frequency of reduced BMD (46%). However, other studies revealed similar frequencies for nephrolithiasis (57.8–75%), independent of the presence of gastrinoma (10,39,45). In one of these series, a higher frequency of nephrolithiasis, although not statistically significant, was found in MEN1-related HPT cases with gastrinoma than in MEN1-related HPT cases without

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Table 4 - Comparative analysis of BMD findings using the ISCD (2009) criteria (32,33).

BMD pattern in MEN1-related HPT

LS (n)

FN (n)

TH (n)

1/3DR (n)

Group younger than 50 years of age with reduced BMD (Z , -2.0) Burgess et al. (29) Lourenc¸o et al. (10) Total

23.8 (5/21) 23.8 (5/21)

Burgess et al. (29) Lourenc¸o et al. (10) Total

77.7 (7/9) 80 (12/15) 79.1 (19/24)

25 (5/20) 25 (5/20)

22.2 (4/18) 22.2 (4/18)

40 (8/20) 40 (8/20)

Group older than 50 years of age with reduced BMD (T , -1.0) 100 (9/9) 73.3 (11/15) 83.3 (20/24)

100 (14/14) 100 (14/14)

92.9 (13/14) 92.9 (13/14)

Group older than 50 years of age with osteoporosis (T , -2.5) Burgess et al. (29) Lourenc¸o et al. (10) Total

44.4 (4/9) 67.7 (10/15) 58.3 (14/24)

66.6 (6/9) 26.7 (4/15) 41.6 (10/24)

21.4 (3/14) 21.4 (3/14)

57.1 (8/14) 57.1 (8/14)

Group older than 50 years of age with osteopenia (-2.5 , T , -1.0) Burgess et al. (29) Lourenc¸o et al. (10) Total

33.3 (3/9) 13.3 (2/15) 20.8 (5/24)

33.3 (3/9) 46.7 (7/15) 41.6 (10/24)

78.6 (11/14) 78.6 (11/14)

35.7 (5/14) 35.7 (5/14)

Reduced BMD: Z , -2.0 in younger patients (,50 years) and T , -1 in older patients (.50 years) Burgess et al. (29) Lourenc¸o et al. (10) Total

77.7 (7/9)* 47.2 (17/36) 53.3 (24/45)

100 (9/9)* 45.7 (16/35) 56.8 (25/44)

56.2 (18/32) 56.3 (18/32)

61.8 (21/34) 61.8 (21/34)

Reduced BMD (Z , -2.0), independent of age Burgess et al. (29) Lourenc¸o et al. (10) Total

20.7 (6/29) 36.1 (13/36) 29.2 (19/65)

27.6 (8/29) 20 (7/35) 23.4 (15/64)

18.8 (6/32) 18.8 (6/32)

47.1 (16/34) 47.1 (16/34)

BMD: bone mineral density. ISCD: International Society for Clinical Densitometry. MEN1: multiple endocrine neoplasia type 1. HPT: primary hyperparathyroidism. LS: lumbar spine. FN: femoral neck. TH: total hip. 1/3DR: proximal one-third of the distal radius. T-scores: difference from the mean BMD value in a healthy young reference population, in SD units. Z-scores: age-matched comparison, in SD units. *: only patients older than 50 years were included.

levels of calcium and a predominance of PTH values that are 2- to 3-fold above the upper limit of the normal range.

Two earlier studies demonstrated a direct correlation between age and calcium/PTH values in a subset of patients with MEN1-related HPT, which is probably associated with the progressive development of parathyroid hyperplasia and the asynchronous onset of HPT disease in the different parathyroid glands (46,47). A tendency toward increasing PTH levels was also observed in older patients after long periods of HPT disease (10).

Furthermore, Eller-Vainicher et al. (39) reported a direct association between increasing PTH/calcium levels and aging. These authors also reported an association between inappropriately normal serum PTH values and age below 50 years with presence of HPT/MEN1. Thus, fifteen of the 40 HPT/MEN1 patients younger than 50 years (37.5%) had inappropriately normal PTH levels. Additionally, cases younger than 50 years and presenting inappropriately normal PTH levels had 13.5 fold higher likelihood of association with MEN1 syndrome than patients with 50 years old or more and presenting high serum levels of PTH. (39).

Combining data from Burgess et al. ’s study and our own, the prevalence of inappropriately normal PTH levels was 9.2% (6/65), independent of age (10,29). However, the real prevalence could be even higher once controlled and uncontrolled HPT cases from the study by Burgess et al.

(29) are included in the present evaluation. One of the six

gastrinoma (93.3% vs. 71.4%) (10). Bias might have occurred with these data because of the limited number of patients available for comparisons. Of note, the onset of gastrinomas in patients with MEN1 usually occurs between 30–50 years of age, when most patients have already developed renal calculi (10,45).

In the same study, using ISCD criteria, MEN1 cases with HPT/ gastrinoma had higher frequencies of reduced BMD independent of the bone site than HPT/MEN1 cases without gastrinoma (86.7% vs. 52.4%, p,0.03) (10). Demineralization at the 1/3DR in HPT/gastrinoma cases was significantly more severe than in HPT-only MEN1 cases (p,0.013) (10). In addition, there was a tendency for higher PTH and calcium levels in our HPT/gastrinoma cases, reinforcing data reported by Norton et al. (10,38). When individual data from our second case series were reviewed and the criteria used by Norton et al. were applied (Z ,–2.0, independent of the bone site), 66.7% of the cases presented reduced BMD independent of the presence of gastrinoma. However, no appropriate comparisons could be performed between these two studies, as BMD data for the specific bone sites were not available for the cases reported by Norton et al. (38) and because we had a limited number of gastrinoma/MEN1 cases (10).

Biochemical data on HPT associated with MEN1 Several studies have reported mild biochemical disease associated with MEN1-related HPT, with modestly elevated

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(14,15) and should be differentiated from familial and isolated acromegaly/gigantism caused by other gene mutations, such as AIP (13,16-19). Hypercortisolism occurs infrequently in MEN1 (up to 7% of cases) (14), although several studies involving large series reported no case with hypercortisolism in MEN1 (10,36,39,41).

Of note, Kaan et al. analyzed the potential impact of previous gastrointestinal surgeries on BMD and found no significant correlations (40). Although several factors may potentially worsen bone mineral status in MEN1, a satisfactory BMD recovery could be provided to patients if MEN1related conditions are promptly diagnosed and treated.

cases in our series had asymptomatic HPT and another had recurrent HPT (10). It is possible that HPT/MEN1 cases in these two studies (10,29) were diagnosed later than in the study by Eller-Vainicher et al. (39). In addition, the laboratory diagnosis of HPT in the Eller-Vainicher study may have been more rigorously performed, as serum PTH levels were considered after adjusting for age and sex (39).

Biochemical data and BMD Correlations between biochemical and BMD data in HPT/ MEN1 are contrasting. Burgess et al. reported an inverse correlation between PTH levels and LS and FN BMD findings, but not with regard to calcium levels. Conversely, no correlation between PTH levels with BMD values were observed in other studies (10,39). Of note, an inverse correlation between calcium levels and 1/3 DR BMD values was reported (10).

Genetics and MEN1 BMD studies on HPT/MEN1 have also examined connections with the genetic status of these cases. Burgess et al. studied 29 affected women from a large MEN1 family harboring a known germline MEN1 mutation (29).

However, genotyping could not be performed on all of the studied cases. Thus, the possibility of phenocopies, which may occur in up to 5% of MEN1 families (20,48), could not be fully excluded from these cases.

Eller-Vainicher studied a series of 64 affected patients from 36 unrelated MEN1 families (39). A germline MEN1 mutation was found in 31 index cases but not in the remaining three index cases that were included in this sample of familial MEN1 genealogies because they had typical MEN1-affected relatives.

In three other studies, all affected cases belonging to MEN1 families harboring novel or previously reported germline MEN1 mutations and all index cases, as well as affected family members, were genetically documented (10,23,24,36,41).

Norton et al. reported a familial history of MEN1 in 68 of the 81 (81%) studied cases (38). A germline MEN1 mutation was found in 61 cases (75.3%), nine cases had no MEN1 mutation and the genetic status of 14 other cases was unknown. Kaan found a germline MEN1 mutation in all of the cases studied, except one (22/23; 95.7%) (40).

It is worth noting that an appropriate selection of genetically characterized MEN1 cases is needed, as the pathogeneses of sporadic MEN1 and of familial MEN1 cases that do not harbor a germline MEN1 mutation are different.

Other conditions associated with MEN1 and BMD Recently, Kaan et al. (40) used peripheral quantitative computed tomography to analyze BMD and reported a higher prevalence of bone demineralization in HPT/MEN1 cases (48% in trabecular BMD and 60% in total BMD) than in the normal population (15%). However, most of these cases had been previously submitted to PTx and, thus, a postsurgical BMD gain had probably occurred. Thus, the real prevalence of bone demineralization in this group of patients could be even higher. Despite the limited number of cases and the lack of statistical significance of the findings, it was suggested that other associated factors might influence the BMD status of these cases (40). This conclusion was based on the presence of five cases (22.7%) with hypopituitarism secondary to gonadotrophic and/or somatotrophic insufficiency, three cases with hypercortisolism (13.6%), and 12 cases (52%) with previous surgeries to the upper gastrointestinal tract (40). Other studies that examined this subject produced different results. Burgess et al. (29) found no association between hypogonadism secondary to prolactinoma or menopausal status and reduced BMD. Eller-Vainicher reported 10 cases with hyperprolactinemia but without hypogonadism and no cases with hypopituitarism (39). Also, we recently reported no difference in BMD status between six postmenopausal and 12 premenopausal women, as well as between 10 cases with and 26 cases without prolactinoma (10). In this latter study, the HPT/MEN1 cases with prolactinoma had been successfully treated with bromoergocriptine or a similar agent and a potential BMD recovery might have interfered with the statistical results. It is most likely that prolactinoma and menopause occur much more frequently in MEN1 than hypopituitarism secondary to pituitary macroadenoma or hypercortisolism, in contrast with data from Kaan et al. (40). However, large studies are needed to clarify this issue.

The prevalence of pituitary adenoma in familial MEN1 was evaluated by Verges et al. and found to be 34% (14).

Prolactinoma was the most frequent pituitary tumor (68%), and macroadenoma was highly prevalent in this sample (85%) (14). Based on these data, we could estimate that one-fifth and one-third of MEN1 patients, respectively, might potentially develop prolactinomas and macroadenomas. However, the actual prevalence of hypopituitarism in MEN1 is currently unknown. Somatotropinomas are relatively rare in MEN1

Prospective studies Despite the absence of prospective studies focusing on the early and late outcomes of renal and bone mineral complications in the MEN1-related HPT subset, an integrated analysis of the published data may contribute to a better understanding of several critical topics and allow us to add to the list of differences between HPT/MEN1 and SHPT (Table 5).

Treatment of MEN1 The main challenge associated with the MEN1-related HPT subset is improving the treatment options for this condition, as the recurrence rate of HPT may be as high as 50% in the period of 5–10 years after PTx (7,8,29,49). Also, the increasing survival rate of MEN1 patients may result in an increasing number of MEN1 patients with post-PTx hypoparathyroidism. Calcimimetic drugs are a potential tool that could be used to modify this outcome. HPT was controlled with 30 mg/day cinacalcet over a 12-month period in a MEN1 patient who refused a second PTx because of local recurrence, and a

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Bone mineral and renal diseases in HPT/MEN1 Lourenc¸o DM Jr et al.

Table 5 - Differences and similarities between MEN1-related HPT and sporadic HPT in terms of biochemical patterns, renal comorbidities and bone complications.

Clinical characteristic Hypercalcemia PTH levels PTH levels Nephrolithiasis Bone mineral disease Onset of bone disease Preferentially affected bone site Relative ‘‘protection’’ of the trabecular bone Bone outcome Bone outcome after 10 years

MEN1-related HPT Mildly high Inappropriately normal or mildly high Increasing with age Higher frequency and early onset More severe (++++) Early Cortical bone (1/3 distal radius) Absent Progressive (cortical and trabecular bones) Progressive (cortical)

Sporadic HPT Mildly/mModerately high Mildly/moderately high Unrelated to age Lower frequency Less severe (++) Early Cortical bone (1/3 distal radius) Present Stable (first 10 years) Progressive (cortical)

MEN1: multiple endocrine neoplasia type 1. HPT: primary hyperparathyroidism.

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BMD recovery at the LS (4.7%) and TH (17.8%) was seen 1 year after treatment (50). Recently, Moyes et al. documented metabolic PTH/calcium control 10–35 months after cinacalcet therapy in seven MEN1-related HPT cases (51). Despite the absence of new episodes of nephrolithiasis during the follow-up period, there was no significant BMD gain in five of the seven cases studied (51).

The current evidence shows that HPT/MEN1 is associated with a high prevalence of HPT-related comorbidities.

Severe and frequent bone and renal diseases have been documented in these cases, mostly occurring in earlier but also at later stages of HPT. A better understanding of the natural history of HPT/MEN1 disease; further improvements in surgical techniques; and the development of secure, efficient, and well-tolerated calcimimetic drugs may enable more appropriate therapies to be developed for this subset of patients. It is hoped that in the near future therapeutic improvements may potentially prevent the development of bone and renal complications in HPT/ MEN1 cases, correct metabolic disturbances, and avoid postsurgical hypoparathyroidism, as well as provide better management of recurrences. At present, adequate surveillance of bone, renal, and other co-morbidities in MEN1related HPT should be performed, thereby contributing to an improved determination of the optimal timing for subtotal or total parathyroid surgery.

ACKNOWLEDGMENTS This study was supported by Fundac¸a˜o de Amparo a` Pesquisa (FAPESP) Grants #2008/58552. FLC is the recipient of a National Council for Scientific and Technological Development (CNPq) fellowship (142603/ 2010-4), RAT is the recipient of a Fundac¸a˜o de Amparo a` Pesquisa postdoctoral fellowship (11942/2009) and SPAT is the recipient of a National Council for Scientific and Technological Development senior fellowship and a National Council for Scientific and Technological Development grant (401990/2010-9).

AUTHOR CONTRIBUTIONS All authors included, as this is a review article, have participated actively reviewing in detail the current literature and in the writing process of this manuscript.

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Katai M, Sakurai A, Ikeo Y, Hashizume K. Primary hyperparathyroidism in patients with multiple endocrine neoplasia type 1: comparison with sporadic parathyroid adenomas. Horm Metab Res. 2001;33(8):499-503, http://dx.doi.org/10.1055/s-2001-16944 Burgess JR, Nord B, David R, Greenaway TM, Parameswaran V, Larsson C, et al. Phenotype and phenocopy: the relationship between genotype and clinical phenotype in a single large family with multiple endocrine neoplasia type 1 (MEN 1). Clin Endocrinol. 2000;53(2):205-11, http:// dx.doi.org/10.1046/j.1365-2265.2000.01032.x.

Goudet P, Cougard P, Verge`s B, Murat A, Carnaille B, Calender A, et al.

Hyperparathyroidism in multiple endocrine neoplasia type I: surgical trends and results of a 256-patient series from Groupe d’Etude des Ne´oplasies Endocriniennes Multiples Study Group. World J Surg.

2001;25(7):886-90, http://dx.doi.org/10.1007/s00268-001-0046-z.

Falchetti A, Cilotti A, Vaggelli L, Masi L, Amedei A, Cioppi F, et al. A patient with MEN1-associated hyperparathyroidism, responsive to cinacalcet. Nat Clin Pract Endocrinol Metab. 2008;4(6):351-7, http:// dx.doi.org/10.1038/ncpendmet0888.

Moyes VJ, Monson JP, Chew SL, Akker SA. Clinical use of cinacalcet in MEN1 hyperparathyroidism. Int J Endocrinol. 2010;2010:906163. Epub 2010 May 26.

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REVIEW

Diagnostic work-up of gastroenteropancreatic neuroendocrine tumors ¨ berg Kjell O Uppsala University, Department of Medical Sciences, University Hospital, Department of Endocrine Oncology, Uppsala, Sweden.

Neuroendocrine tumors are a heterogeneous group of malignancies that present a diagnostic challenge. The majority of patients (more than 60%) present with metastatic disease at diagnosis. The diagnosis is based on histopathology, imaging, and circulating biomarkers. The histopathology should contain specific neuroendocrine markers such as chromogranin A, synaptophysin, and neuron-specific enolase and also an estimate of the proliferation by Ki-67 (MIB1). Standard imaging procedures consist of computed tomography or magnetic resonance imaging together with somatostatin receptor scintigraphy. 68Ga-DOTA-octreotate scans will in the future replace somatostatin receptor scintigraphy because they have higher specificity and sensitivity. Other positron imaging tomographic scanning tracers that will come into clinical use are 18F-DOPA and 11C-5HTP. Neuroendocrine tumors secrete many different peptides and amines that can be used as circulating biomarkers. The most useful general marker is chromogranin A, which is both a diagnostic and prognostic marker in most neuroendocrine tumors. However, there is still a need for improved biomarkers for early detection and follow-up of patients during treatment. In addition, molecular imaging can be further developed for both detection and evaluation of treatment.

KEYWORDS: Chromogranin A; 68Ga-DOTATOC; Somatostatin Receptor Scintigraphy; Tumor Node Metastasis Staging; Grading. ¨ berg K. Diagnostic work-up of gastroenteropancreatic neuroendocrine tumors. Clinics. 2012;67(S1):109-112.

O

E-mail: adm-oberg@medsci.uu.se Tel.: +46 18 611 3875

in the SEER database (3). In such centres, 5-year survival rates in patients with metastatic midgut NETs exceed 50% (4,5). In a multivariate analysis of patients with well-differentiated tumors and moderately differentiated NETs from the SEER database, disease stage, primary tumor site, histologic grade, sex, race, age and year at diagnosis were predictors of outcome (p,0.001). In my centre, in a multivariate analysis of 354 patients with pancreatic NETs, the prognostic factors were TNM stage, World Health Organization (WHO) classification, Ki67, and radical surgery (6). Liver tumor burden, or number of metastases, tumor slope, extrahepatic disease, co-morbidities and performance status represent additional prognostic parameters (3,7). Retrospective data indicate that circulating chromogranin A (CgA) is of prognostic value; highly elevated levels were associated with limited survival (8,9). Other prognostic markers that are available (e.g., CK19, PTEN, TSC-2 expression in tumor tissue) require further validation (10,11).

INTRODUCTION Neuroendocrine tumors (NETs) comprise a heterogeneous group of neoplasms that are frequently metastatic at the time of diagnosis, and distance of metastatic disease is, next to grading, one of the most important prognostic factors (1–3). The availability of modern imaging methods for diagnosis and staging of NETs has improved at the same time as the spectrum of therapeutic options in the management of metastatic disease has increased during recent years. The frequency of metastatic disease varies depending on the type of tumor, but in specialized centres, 80–90% of patients who present with small intestinal NETs (carcinoids) and 60–70% of patients with pancreatic NETs have liver metastases. Histology is the strongest predictor of survival.

In the most recent SEER (Surveillance Epidemiology and End Results) database analyses, median survival in distant metastatic disease was 33 months in patients with G1/G2 NETs and only 5 months in patients with poorly differentiated tumors (neuroendocrine carcinoma (NEC) G3) (1).

In specialized centres for the treatment of NETs, overall 5year survival rates for patients with stage IV, pancreatic and small intestinal NETs are much higher than those reported

DIAGNOSTIC WORK-UP The initial diagnostic approach in patients with NETs includes histological examination, which is always required before therapeutic decisions are made. Clinicians should also consider performing repetitive biopsies to reassess the prognosis if the disease course changes significantly. The following investigations are also required: (a) immunohistochemical markers and detailed histological analysis; (b) assessment of the primary tumor and the extent of extrahepatic spread by imaging, including patterns of

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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elements. The classification itself uses a common definition frame that is based on grading and specific staging tools. The definitions are NET for the previous ‘‘carcinoid’’/welldifferentiated endocrine tumor/carcinoma, and neuroendocrine carcinoma (NEC) for the previous small cell/poorly differentiated carcinoma.

hepatic metastases; and (c) biochemical assessment of functionality and general tumor markers.

HISTOPATHOLOGY The neuroendocrine signature of a cell is defined by the expression of general and specific neuroendocrine markers.

General neuroendocrine markers are observed in all cell types, and include the cytosol antigens neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5), as well as the secretory vesicle antigens of chromogranin family A for large dense core vesicle (LDCV) and synaptophysin for small synaptic vesicles (SSV). Other neuroendocrine tissue markers are the ATP-dependent vesicular monoamine transporter isoforms (VMAT1 and VMAT2), neuroendocrine secretory protein 55 (NESP55), synaptic vesicle protein 2 (SV2) in both LDCVs and SSV, and neural cell adhesion molecule (N-CAM) (12,13).

Immunohistochemical determination of CgA and synaptophysin as well as proliferation marker Ki-67 (MIB-1) is mandatory. In patients with multiple endocrine neoplasia type 1, specific markers such as gastrin, insulin, and pancreatic polypeptide (PP) should be determined. For patients with unknown primary tumors, TTF1 (bronchial/lung), CDX2 (intestinal serotonin-midgut), and PP/Islet-1/Glucagon (pancreatic) can be used to guide the search for the primary tumor (14,15).

TUMOR STAGING AND GRADING Staging is performed with the familiar tumor node metastasis (TNM) approach according to the anatomical location of the tumors, and this approach is recommended by the WHO, the American Joint Committee on Cancer and the International Union Against Cancer. However, at variance with the European Neuroendocrine Tumor Society proposal, the WHO-approved TNM staging system is conceived for ‘‘carcinoid’’ only, and some parameters for the appendix and the pancreas are different (see Table 1) (17–20).

Grading is performed by definition of proliferation using both the mitotic count and the Ki-67 index, as proposed by the European Neuroendocrine Tumor Society. Notably, both the WHO and the American Joint Committee on Cancer endorse such a grading system.

IMAGING A standard computed tomographic (CT) scan of the chest, abdomen, and pelvis or magnetic resonance image is mandatory, and should be complemented by somatostatin receptor scintigraphy including single photon emission computer tomography (SPECT)-SRS and triphasic CT (21,22). Positron emission tomographic (PET) scanning using 68Ga-somatostatin analogue (68Ga-DOTATOC-PET/ CT) is an alternative if available, as it has higher resolution than additional somatostatin receptor scintigraphy.

CLASSIFICATION The current WHO 2010 classification introduces the definition ‘‘neoplasm’’ to encompass low- to high-grade neuroendocrine tumors (16). At variance with the WHO 2000 classification, the neuroendocrine connotation is enforced, in recognition of the expression of antigens shared with nerve

Table 1 - The American Joint Committee on Cancer (AJCC) and European Neuroendocrine Tumors Society (ENETS) staging classifications for pancreatic neuroendocrine tumors with cross-tabulation of stage distributions.

AJCC Staging Classification T1

Tumor limited to the pancreas, ,2 cm Tumor limited to the pancreas, .2 cm Tumor extends beyond the pancreas, but not involving the celiac axis or SMA Tumor involves the celiac axis or SMA No regional LN metastasis Regional LN metastasis No distant metastasis Distant metastasis

T2 T3 T4 N0 N1 M0 M1 Stage IA IB IIA IIB III IV

AJCC AJCC AJCC AJCC

ENETS Staging Classification

I II III IV

T1 T2 T3 T4 N0 N1 M0 M1

Tumor limited to the pancreas, ,2 cm Tumor limited to the pancreas, 2-4 cm Tumor limited to the pancreas, .4 cm, or invading duodenum or CBD Tumor invades adjacent structures No regional LN metastasis Regional LN metastasis No distant metastasis Distant metastasis

T

N

M

Stage

T

N

M

T1 T2 T3 T1-3 T4 Any T

N0 N0 N0 N1 N0-1 Any N

M0 M0 M0 M0 M0 M1

I IIA IIB IIIA IIIB IV

T1 T2 T3 T4 Any T Any T

N0 N0 N0 N0 N1 Any N

M0 M0 M0 M0 M0 M1

ENETS I

ENETS II

ENETS III

ENETS IV

25 0 0 0

59 4 0 0

0 37 18 0

0 0 0 282

CBD = common bile duct; LN = lymph node; SMA = superior mesenteric artery.

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Gastroenteropancreatic neuroendocrine tumors ¨ berg K O

68Ga-DOTATOC-PET/CT may help to identify the primary tumor, and is a reliable method for early detection of bone metastases in patients with NETs (23–25). For the detection of small pancreatic NETs, endoscopical ultrasonography seems superior to PET/CT (26). In general, PET should be replaced by PET/CT; 18F-DOPA PET/CT and 5-HTP-PET/ CT are promising diagnostic tools, and may be considered if they are available and if somatostatin receptor imaging is negative (27,28). However, their use in the standard workup may not be recommended at this time. Although recent studies indicate a prognostic value of FDG-PET in welldifferentiated NETs, it is not recommended as a routine imaging method (29). Further studies are needed to support its role as a prognostic tool. In special situations, for example if liver transplantation is being considered, FDGPET/CT can be considered for G2 tumors as well as 18FDOPA-PET/CT or 5HTP-PET/CT if available. Investigation of the large bowel may be useful, either by means of a colonoscopy plus ileoscopy, or by means of a colon CT with a neutral enema. In a retrospective analysis of 123 patients with NET liver metastases of unknown primary, in 47% the primary tumor was localized in the small or large intestine by lower endoscopy (30). If the primary tumor is suspected to be in the small intestine, double balloon enteroscopy or video capsule endoscopy may be performed if available and considered necessary for the therapeutic management. If the CT study of liver metastases is inconclusive, T2-weighted thin-slice dynamic gadolinium-enhanced magnetic resonance imaging, or, if available, contrast-enhanced ultrasonography, should be performed. Magnetic resonance imaging is considered superior to CT in the detection and follow-up of liver metastases, and is a preferable choice in clinical trials (21).

The imaging report should include segmental information on the distribution of liver metastases. Although there are no standardized imaging techniques to reliably measure liver tumor burden, an experienced radiologist can use such reports to estimate the percentage of liver tumor involved.

(i.e., right-sided heart failure due to tricuspid regurgitation and/or pulmonic stenosis because of valve fibrosis (probably) caused by elevated circulating serotonin) (34).

There are, however, several limitations to the use of CgA as a biomarker in GEP-NETs. Treatment with proton-pump inhibitors can cause a secondary increase in CgA as a result of the increased gastrin production. Chronic atrophic gastritis may also cause elevated CgA. Impaired renal function may cause accumulation of the peptide, which also results in falsely elevated levels. Many patients with midgut carcinoids are initially misdiagnosed with irritable bowel syndrome, sometimes several years before the correct diagnosis is made (35). There have been reports of elevated CgA in irritable bowel syndrome and in inflammatory bowel disease. CgA is thus not of value as a screening test in the evaluation of unclear diarrhea.

There has been impressive progress in the field of biomarkers as well as molecular imaging. However, we still need more sensitive markers for early detection and follow-up. Furthermore, new markers delineating sensitivity to various therapies are warranted. Molecular imaging is in its early stages and has the potential to be a significant tool in the management of patients with NETs.

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CIRCULATING MARKERS IN CLINICAL PRACTICE The minimal biochemical work-up for NETs includes circulating chromogranin A and assessment of a specific marker to assess functionality, such as urinary 5-HIAA evaluation in carcinoid syndrome. Additional assessment of insulin, C-peptide, (proinsulin), gastrin, pancreatic polypeptide, vasoactive intestinal peptide, glucagon, and calcitonin should be useful, depending on the functional status of the tumor, clinical symptoms, and histological features.

Chromogranins are a family of glycoproteins found in many hormone-producing organs, and early on they were discovered to be elevated in the plasma of patients with endocrine tumors. Plasma chromogranin A (CgA) has been reported to be a prognostic biomarker in GEP-NETs, correlating with hepatic tumor burden and with shorter survival. In the setting of radically operated midgut carcinoids, elevation of CgA has been reported to be both a diagnostic marker and an early marker of recurrent disease. A decrease in CgA levels has been used as a marker of response to treatment in clinical trials, for which biochemical response usually is defined as a $50% reduction of CgA (31–33). The combination of CgA levels and levels of N-terminal pro-brain natriuretic peptide (NtproBNP) correlate significantly with carcinoid heart disease

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15. Srivastava A, Hornick JL. Immunohistochemical staining for CDX-2, PDX-1, NESP-55, and TTF-1 can help distinguish gastrointestinal carcinoid tumors from pancreatic endocrine and pulmonary carcinoid tumors. Am J Surg Pathol. 2009;33(4):626-32.

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Multiple endocrine neoplasia type 2: achievements and current challenges Andreas Machens, Henning Dralle Martin-Luther-University Halle-Wittenberg, Department of General, Visceral and Vascular Surgery, Halle (Saale)/Germany.

Incremental advances in medical technology, such as the development of sensitive hormonal assays for routine clinical care, are the drivers of medical progress. This principle is exemplified by the creation of the concept of multiple endocrine neoplasia type 2, encompassing medullary thyroid cancer, pheochromocytoma, and primary hyperparathyroidism, which did not emerge before the early 1960s. This review sets out to highlight key achievements, such as joint biochemical and DNA-based screening of individuals at risk of developing multiple endocrine neoplasia type 2, before casting a spotlight on current challenges which include: (i) ill-defined upper limits of calcitonin assays for infants and young children, rendering it difficult to implement the biochemical part of the integrated DNA-based/biochemical concept; (ii) our increasingly mobile society in which different service providers are caring for one individual at various stages in the disease process. With familial relationships disintegrating as a result of geographic dispersion, information about the history of the origin family may become sketchy or just unavailable. This is when DNA-based gene tests come into play, confirming or excluding an individual’s genetic predisposition to multiple endocrine neoplasia type 2 even before there is any biochemical or clinical evidence of the disease. However, the unrivaled molecular genetic progress in multiple endocrine neoplasia type 2 does not come without a price. Screening may uncover unknown gene sequence variants representing either harmless polymorphisms or pathogenic mutations. In this setting, functional characterization of mutant cells in vitro may generate helpful ancillary evidence with regard to the pathogenicity of gene variants in comparison with established mutations.

KEYWORDS: Medullary thyroid carcinoma; Pheochromocytoma; Primary hyperparathyroidism; RET protooncogene; Age-related progression; Calcitonin screening; DNA-based screening.

Machens A, Dralle H. Multiple endocrine neoplasia type 2: achievements and current challenges. Clinics. 2012;67(S1):113-118.

E-mail: AndreasMachens@aol.com Tel.: 49 345 5572314

Before this landmark event, individuals operated on for MTC used to be diagnosed with papillary, follicular, or undifferentiated thyroid cancer. After completing their fourth decade of life, some of these patients succumbed to metastatic MTC or to acute hypertensive crisis owing to adrenergic hormonal excess from a previously undetected pheochromocytoma. In the absence of a postmortem examination, these deaths were frequently ascribed to acute cardiovascular or cerebrovascular events. Without clinical– pathologic characterization of the disease, targeted interventions are difficult, if not impossible, to carry out at an early stage in the disease process.

INTRODUCTION Incremental advances in medical technology, such as the development of sensitive hormonal assays for routine clinical care, are the drivers of medical progress. This principle is exemplified by the creation of the concept of multiple endocrine neoplasia type 2 (MEN2), which did not emerge before the early 1960s (1). This hereditable syndrome combines three distinct disease entities into one clinical syndrome: medullary thyroid cancer (MTC); pheochromocytoma; and primary hyperparathyroidism (2,3).

MEN2B patients who do not have primary hyperparathyroidism also develop conjunctival, corneal, buccal, lip and tongue neuromas, intestinal ganglioneuromas (dubbed ‘‘pseudo-Hirschsprung’s disease’’), and skeletal anomalies subsumed under the designation of ‘‘Marfanoid habitus’’ as they grow older (2–4).

BIOCHEMICAL SCREENING The widespread availability of commercial calcitonin and catecholamine assays in the 1960s completely changed the scene, enabling biochemical screening of asymptomatic individuals from MEN2 families who were suspected to harbor C-cell disease and adrenal medullary disease (5). At the very beginning, calcitonin screening, augmented through stimulation with intravenous bolus injections of pentagastrin or calcium gluconate short infusions, was largely cross-sectional, including kindreds of various age groups at different stages of tumor development (5). With the increasing inclusion of asymptomatic infants and young

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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children at risk of developing MEN2 tumors, the focus of the screening programs shifted toward longitudinal observation of family members (6). This prompted biochemical screening of individuals at risk of developing MEN2 over many years who were slated for preemptive thyroidectomy as soon as basal serum calcitonin levels exceeded the upper normal limit of the assay. With the exception of a few falsepositives from physiologic C-cell disease of the thyroid or calcitonin-secreting small cell lung cancer or pancreatic neuroendocrine cancer (7), this approach worked fairly well, reducing the number of patients newly diagnosed with advanced MTC (5,6). As a result, family members from the younger generations were found to have more localized disease (‘‘anticipation phenomenon’’), as opposed to the metastatic disease that used to haunt earlier generations.

This breakthrough in the management of MEN2 patients is beautifully illustrated in Figure 1 with the pedigree of an MEN2A family of Transylvanian Saxons (ethnic Germans whose ancestors colonized Sibiu (Hermannstadt) county in present-day Romania during the 13th century). The first two affected individuals, I-2 and II-3, developed huge neck lumps (presumably MTC) and severe hypertension (presumably due to pheochromocytoma), causing aphonia (presumably as a result of recurrent laryngeal nerve invasion) and hemiplegia at the age of 42 years (presumably following cathecholamine excess) in individual II-3. Both

MTC and pheochromocytoma went unrecognized as such by the rural Communist healthcare system, so that the diagnosis of MEN2A was not made at the time (Figure 1, shaded area). After the fall of the Eastern bloc in 1989, the whole family migrated to Germany where individuals III-7 and IV-9 were found to harbor node-positive MTC and pheochromocytoma at the age of 36 and 14 years in 1994 and 1995, respectively (Figure 1). Unfortunately, several family members declined screening.

DNA-BASED SCREENING The 1993 and 1994 discoveries of RET (REarranged during Transfection) as the susceptibility gene for MEN2 ushered in the molecular era (8–10). Owing to the need for genetic association studies for unequivocal cases of MEN type 2, higher risk mutations in codon 918 (the classic MEN2B mutation; American Thyroid Association/ATA D), codon 634 (the classic MEN2A mutation; ATA C) and codons 609, 611, 618, 620, and 630 (ATA B) were overrepresented in the International MEN2 Consortium (2). The subsequent scanning of the RET proto-oncogene for additional mutations identified more RET mutations in codons 768, 790, 791, 804, and 891 between 1995 and 1998 (11–14). These milder mutations were often associated with familial MTC only, an abortive form of MEN2.

Figure 1 - Multigenerational Ethnic German C634R Family from Romania.

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790, 791, 804, and 891); primary hyperparathyroidism as early as 5 years (carriers of mutations in codon 634), 34–41 years (carriers of mutations in codons 609, 611, 618, and 620), and 38–54 years (carriers of mutations in codons 533, 768, 790, 791, 804, and 891), but for unknown reasons, never in MEN2B patients (carriers of mutations in codon 918).

With more data set to be forthcoming, the current ATA assignments of the rare RET mutations in codon 630 (currently ATA-B) and 883 (currently ATA D) may need to be revised.

GENOTYPE–PHENOTYPE CORRELATIONS Transformation from hyperplasia to neoplasia The RET proto-oncogene encodes for a transmembrane tyrosine kinase receptor. The constitutive (i.e., genetically encoded) activation of the mutated RET receptor protein is believed to cause, in decreasing frequency, hyperplasia of the parafollicular C-cells, adrenal medullary cells, and parathyroid chief cells (3). A second mutation in one of these neuroendocrine cells (‘‘second hit’’) is thought to cause MTC (C-cell cancer), pheochromocytoma, and primary hyperparathyroidism. Because the acquisition of somatic mutations by these cells reflects the play of chance, the development of the various MEN2 components can vary tremendously even among members of the same family, compromising predictions regarding the age by which the various MEN2 components will have developed. As a rule, the weaker the transforming activity of the inherited RET mutation, the more variable will be the clinical presentation (phenotype) of the various MEN2 components (3,15,16), with steep gradients from highest risk ATA-D to very highrisk ATA-C, high-risk ATA-B, and lowest risk ATA-A mutations (Table 1). Carriers of the last two groups of mutations may develop familial MTC only.

Youngest age of node-positive MTC Another literature review of the youngest age of carriers harboring lymph node metastases from MTC revealed a comparable pattern (16). Barring rare exceptions, lymph node metastases have not yet developed before age 2 years (carriers of mutations in codon 918), not before age 5 years (carriers of mutations in codon 634) and 15 years (carriers of mutations in codons 630) and not before age 20 years in the remaining RET carriers.

Window of opportunity for cure The lead time provided by early screening of infants and young children from RET families defines a ‘‘window of opportunity’’, setting the time frame within which total thyroidectomy alone will eliminate all C-cell disease (3). If this time frame has been exceeded, systematic dissection of the central and lateral lymph nodes on either side of the neck is unavoidable. In this situation, the patient’s prospects of cure fall from 95% with no lymph node metastases to 57–31% with 1–10 lymph node metastases and 0–4% with .10 lymph node metastases (29,30).

Youngest age of tumor development A systematic review of the literature (2,15–28), focused on the youngest carrier age at which a given MEN2-related tumor was ever reported, yielded the following chart of earliest tumor development (Table 1): medullary thyroid cancer as early as age 2 months (carriers of mutations in codon 918), 10 months (carriers of mutations in codon 634), 12 months (carriers of mutations in codon 630), 4–7 years (carriers of mutations in codons 609, 611, 618 and 620) and 9–21 years (carriers of mutations in codons 533, 768, 790, 791, 804 and 891); pheochromocytoma as early as age 12 years (carriers of mutations in codon 918 and 634), 19–30 years (carriers of mutations in codons 609, 611, 618, and 620), and 28–59 years (carriers of mutations in codons 768,

INTEGRATED DNA-BASED/BIOCHEMICAL CONCEPT OF EARLY THYROIDECTOMY The impossibility of anticipating the development of the various MEN2 components has prompted both overtreatment

Table 1 - RET genotype–phenotype relationships and ATA recommendations for thyroidectomy.

RET genotype

ATA group

D C B

A

Exon

Codon

16 15* 11 11{ 10 10 10 10 8 13 13 13 14 15

918 883* 634 630{ 620 618 611 609 533 768 790 791 804 891

Relative frequency of genotype among RET families (%)

RET phenotype: youngest age at tissue diagnosis (years) (2,15–28)

Germany (n = 140) (19)

MTC

PCC

pHPT

0.2 39* 0.8 1{ 5 5 7 4 21 9 10 15 6, 12 39

12 39* 12 – 19 19 30 19 35 59 28 38 28–33 39

– N/A 5 32 N/A 41 40 34 N/A N/A N/A 38 54 N/A

15 0 41 0.7 7 5 1 0.7 0{ 1 12 7 6 2

Italy (n = 235) (22) 8 0.4 37 2 4 6 0.4 3 0{ 4 3 ,1 22 10

ATA recommendations for thyroidectomy (31) DNA based

b+stCT based

ASAP (#1 year)

if q

,5 years ,5 years (may delay if b+stCT WNL)

if q if q

May delay .5 years if b+stCT WNL

if q

ASAP = as soon as possible; ATA = American Thyroid Association; b+stCT = basal and stimulated serum calcitonin levels; MTC = medullary thyroid cancer; PCC = pheochromocytoma; pHPT = primary hyperparathyroidism; RET = REarranged during Transfection. * May need regrouping if confirmed in subsequent studies (ATA group A?). { May need regrouping if confirmed in subsequent studies (ATA group C?). { May be more common in Brazilians of Catalan/Spanish ancestry (17) and the Greek population (20).

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Figure 2 - German RET Families with 2 Index Patients.

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communication had occurred. This disruption delayed the diagnosis of hereditary MTC in the second index patient by 7 years in the C611F family (Figure 2a) and 19 years in the C620S family (Figure 2b). This significant delay may have contributed to our inability to biochemically cure the second index patient, giving the C-cell disease 7 and 19 additional years to spread beyond curability. There is a paucity of research into the reasons behind some gene carriers’ unwillingness to share genetic risk with individuals at risk within their families. Hypothetical reasons include repression, feelings of guilt and resentment, emotional distress, and poor familial interaction.

This is when DNA-based screening, requiring only a venous blood draw, comes into play. Barring sample mixup, the RET gene test confirms or rules out an individual’s genetic predisposition to MEN2 even before there is any biochemical or clinical evidence of disease. This is most relevant in the event of adopted children or broken families in whom information about the history of the origin family is sketchy or just unavailable.

However, this unrivaled molecular genetic progress does not come without a price. DNA-based screening may occasionally uncover previously unknown RET sequence variants dubbed ‘‘variants of unknown significance’’ (VUS), which may represent harmless polymorphisms or pathogenic mutations. This distinction is difficult, if not impossible, to make when one sole family member is clinically affected so that there is no demonstrable segregation of the trait with the disease. This scenario is common in small and younger families whose members conceivably may not have been given sufficient time to develop the disease. In this setting, functional characterization of RET mutant cells in vitro has been harnessed to generate ancillary evidence regarding the pathogenicity of unknown sequence variants in comparison with established RET mutations (35–38). This experimental approach, albeit promising, will need further validation before being considered for routine clinical use.

(based on the above worst-case scenarios) and undertreatment (practicing the law of averages) of gene carriers. Although the respective RET mutation sets the stage for tumor development, defining a time corridor, this corridor often is too wide to be useful for individual decisions with regard to the timing of prophylactic thyroidectomy.

This imperfection of the DNA-based approach prompted researchers to build on the strengths of the DNA-based and biochemical screening programs, giving rise to the integrated DNA-based/biochemical concept of early thyroidectomy (16). Herein, biochemical information (basal serum calcitonin levels) is utilized to spot imminent or early malignant transformation from C-cell hyperplasia to MTC while it is still confined to the thyroid gland. There is good evidence to suggest that MTC has not developed as long as basal calcitonin levels remain within normal limits.

No more than one-third of pediatric RET gene carriers with increased basal calcitonin levels are believed to harbor occult MTC (16). It may be interesting to note that lymph node metastases have not yet been reported with basal serum calcitonin levels #40 pg/ml (upper normal limit ,10 pg/ml) (16,26,31).

What precisely constitutes a normal or elevated serum calcitonin level for infants and young children remains to be determined. As a rule, calcitonin levels tend to be higher in infants younger than 6 months but are thought to approximate to adult levels by the age of 3 years (32). In male and female children older than 6 months (i.e., aged 1–9 years), normal calcitonin values as high as 17 pg/ml and 8.5 pg/ml, respectively, have been noted, exceeding the manufacturer’s upper normal limits of ,11.5 pg/ml for male adults and ,4.6 pg/ml for female adults (33). The application of adult reference data to young children for whom upper normal limits are rarely, if ever, available from the manufacturer of the calcitonin assay may yield incorrect information for children whose seemingly abnormal test results may actually be within the normal limits for that age group. This renders it challenging to implement the calcitonin-based part of the ATA guidelines (Table 1; right panel) for young children unless calcitonin serum levels are quite high.

AUTHOR CONTRIBUTIONS Machens A contributed to the conception and design of the manuscript; collection, analysis and interpretation of data; drafting of the article; and final approval of the version to be published. Dralle H was responsible for the collection, analysis and interpretation of data; critical revision of the article for important intellectual content; and final approval of the version to be published.

CURRENT CHALLENGES In our increasingly mobile society, clinical care for MEN2 has become fragmented, with different service providers caring for one individual at various stages in the disease process. Geographical dispersion of family members, some of whom may be living abroad, can add to a screening program’s difficulty in reaching out to all relatives who may have inherited the trait running in the family. These issues frustrated screening efforts directed at all 86 members of a large French L790F RET family. Of these potential gene carriers, no more than 22 family members (26%) ended up being tested despite the authors’ vigorous attempts at complete ascertainment (34). Moreover, not every newly identified gene carrier is prepared to share personal genetic information with his/her next of kin.

This attitude explains why there can be more than one index patient within an extended RET family whose members, living in silos as it were, underwent RET screening after developing MTC independently of one another. Figure 2 depicts the pedigrees of two unrelated RET families in whom a longstanding breakdown of

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DOI:10.6061/clinics/2012(Sup01)20

REVIEW

Temozolomide in aggressive pituitary adenomas and carcinomas Leon D. Ortiz,I Luis V. Syro,II Bernd W. Scheithauer (in memoriam),III Fabio Rotondo,IV Humberto Uribe,V Camilo E. Fadul,VI Eva Horvath,IV Kalman KovacsIV I

Instituto de Cancerologia, Clinica Las Americas, Division of Neuro-oncology, Medellin, Colombia. II Hospital Pablo Tobon Uribe and Clinica Medellin, Department of Neurosurgery, Medellin, Colombia. III Mayo Clinic, Department of Laboratory Medicine and Pathology, Rochester, Minnesota, USA. IV University of Toronto, St Michael’s Hospital, Department of Laboratory Medicine, Toronto, Ontario, Canada. V Clinica SOMA, Department of Neurosurgery, Medellin, Colombia. VI Dartmouth Hitchcock Medical Center, Department of Medicine (Section of Hematology/Oncology) and Neurology, Lebanon, New Hampshire, USA.

Temozolomide is an alkylating agent used in the treatment of gliomas and, more recently, aggressive pituitary adenomas and carcinomas. Temozolomide methylates DNA and, thereby, has antitumor effects. O6-methylguanineDNA methyltransferase, a DNA repair enzyme, removes the alkylating adducts that are induced by temozolomide, thereby counteracting its effects. A Medline search for all of the available publications regarding the use of temozolomide for the treatment of pituitary tumors was performed. To date, 46 cases of adenohypophysial tumors that were treated with temozolomide, including 30 adenomas and 16 carcinomas, have been reported. Eighteen of the 30 (60%) adenomas and 11 of the 16 (69%) carcinomas responded favorably to treatment. One patient with multiple endocrine neoplasia type 1 and an aggressive prolactin-producing adenoma was also treated and demonstrated a good response. No significant complications have been attributed to temozolomide therapy. Thus, temozolomide is an effective treatment for the majority of aggressive adenomas and carcinomas. Evidence indicates that there is an inverse correlation between levels of O6-methylguanine-DNA methyltransferase immunoexpression and therapeutic response. Alternatively, high-level O6-methylguanine-DNA methyltransferase immunoexpression correlates with an unfavorable response. Here, we review the use of temozolomide for treating pituitary neoplasms.

KEYWORDS: Pituitary adenoma; Pituitary carcinoma; MGMT; Temozolomide; Review.

Ortiz LD, Syro LV, Scheithauer BW, Rotondo F, Uribe H, Fadul CE, et al. Temozolomide in aggressive pituitary adenomas and carcinomas. Clinics.

2012;67(S1):119-123.

E-mail: lvsyro@une.net.co Tel.: 57 4 231 9099

carcinomas are of large size, invasive and associated with craniospinal and/or systemic metastases. Multiple treatment approaches, including surgery, external beam radiotherapy, radiosurgery, adjuvant pharmacological therapy and various chemotherapeutic approaches, are palliative at best, achieving only temporary effects (2,3). Progression of the disease after the diagnosis of pituitary carcinoma is variable but often inexorable. Approximately 75% of patients with systemic metastasis die within 1 year of documented spread (4).

Recent publications have reported the efficacy of temozolomide for treating aggressive pituitary adenomas and carcinomas (5–29). In this review, we discuss the indications, results and side effects of temozolomide therapy.

INTRODUCTION Patients with aggressive pituitary adenomas are difficult to clinically manage. Frequently, these adenomas are large, rapidly growing and invasive to their surroundings. These tumors also present problems due to incomplete resection and recurrence. Reoperation, pharmacological treatment and radiotherapy are routinely employed, often in combination. Nevertheless, many patients experience tumor regrowth (1). Endocrinologically active adenomas, particularly prolactin (PRL)- and growth hormone (GH)-producing tumors, may become resistant to medical therapy, thus requiring multiple resections. Radiation and conventional chemotherapy are often employed in an attempt to achieve local tumor control, but the results are often disappointing.

Pituitary carcinomas are rare and present particular diagnostic and therapeutic challenges. All of these

Temozolomide Temozolomide is a second-generation alkylating chemotherapeutic agent related to a series of imidazotetrazines that were originally synthesized in 1987 (30). Orally administered, it readily crosses the blood-brain barrier. At physiological pH, it undergoes rapid conversion to methyltriazeno-imidazole-carboxamide (MTIC), the active agent. It exerts its cytotoxic effects through methylation of DNA at the O6 position of guanine (31), which then mispairs with

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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fibrosis, reduced mitotic activity, Ki-67 labeling and, surprisingly, neuronal transformation. MGMT immunoexpression was completely absent. Subsequently, the case of a 41-year-old patient with an aggressive silent subtype 2 corticotroph adenoma was reported (10). It showed no morphological changes after temozolomide treatment. The tumor cell nuclei were immunopositive for MGMT. Based upon these results, it was suggested that MGMT immunoexpression may predict the responsiveness of temozolomide therapy (10).

To the best of our knowledge, to date 30 cases of pituitary adenoma have been treated with temozolomide (7-16,1820,22-26,28,29). Patient ages varied from 20 to 71 years (mean: 51 years). Aside from one tumor that was incidentally discovered in a patient with concomitant glioblastoma multiforme, all tumors were morphologically studied. The group included ten adrenocorticotropic hormone (ACTH)producing tumors, nine PRL-producing tumors, seven clinically non-functioning adenomas, two silent ACTH adenomas, and two GH-producing adenomas. The time between clinical presentation and onset of temozolomide treatment was between 2 and 23 years (mean: 10.7 years).

Twenty-six of the 30 tumors had been irradiated previously.

Aside from the incidentally discovered tumor in one patient, all other patients had undergone at least one operative procedure, the number ranging from 0 to 6 procedures (mean: 2.7). The response rate was 60% (18/30 patients). Debono et al. (11) reported the first case of a pituitary adenoma associated with multiple endocrine neoplasia type 1 (MEN1) that was treated with temozolomide. The 47-year-old man had primary hyperparathyroidism and a lesion at the head of the pancreas. Genetic analysis confirmed MEN1. The dopamine-resistant, prolactin-producing pituitary adenoma responded dramatically to temozolomide, demonstrating clinical, biochemical, and radiological improvements.

MGMT immunoexpression was documented in 23 of the 30 patients. A high level of labeling was seen in six instances, an intermediate level in six instances, and a low level in 11 instances. Only one of six patients with high MGMT immunoexpression responded to temozolomide.

Two tumors with intermediate MGMT immunoexpression did not respond, whereas nine of the 11 tumors with lowlevel MGMT immunoexpression responded favorably.

thymine during the next cycle of DNA replication. The sequence of mismatch-repair events lead to apoptosis.

Temozolomide is accepted as an essential component of adjuvant therapy for the treatment of glioblastoma multiforme and other tumors of the central nervous system (32–34). Recent reports indicate its efficacy for treating advanced-stage malignant neuroendocrine neoplasia (35), melanoma (36,37), and colorectal carcinoma (38).

The standard therapeutic dose of temozolomide is 150– 200 mg/m2 for five of every 28 days (5/28). Depletion of O6methylguanine-DNA methyltransferase (MGMT) has been proposed as a means of tumor response to temozolomide (39). Experimental and clinical data have shown that the temozolomide response is schedule-dependent and that alternative dosing regimens may enhance its efficacy (40,41). The anti-angiogenic effect of temozolomide can be optimized by administering low doses of the drug on a frequent or continuous schedule without extended interruptions (i.e., ‘‘metronomic’’ chemotherapy). Temozolomide can also be administered on a continuous, daily metronomic schedule, thus achieving MGMT depletion and improving the clinical response. This dose is generally 50 mg/m2/day without interruption (28/28). Other temozolomide schedules recommend administration every other week (7/14) or a 21consecutive-day regimen that is administered every 28 days (21/28). Both are considered dose-dense regimens based on the Norton-Simon model of cell proliferation, which states that a dose of chemotherapy will have a fixed cell-kill rate regardless of the size of the tumor (39). Thus, decreasing the time interval between doses (i.e., increasing the dose density) improves efficacy by minimizing the opportunity for cellular regrowth between cycles (41). In these dose-dense regimens, temozolomide is administered at a dose of 150 mg/m2/day for 7 days every other week (7/14) or at a dose of 85–100 mg/ m2/day for 21 consecutive days (21/28) (40). In reported pituitary cases, temozolomide was administered as monotherapy in all but three cases. The standard 5/28 regimen was used in almost all instances except in the series of Bush et al.

(23), in which a dose-dense, 21/28 schedule was used, and one case progressed to carcinoma, which was then treated using the metronomic 28/28 schedule (29). Temozolomide absorption is only minimally affected by food. Furthermore, no serious side effects have been reported. Common, nonhematological adverse effects are present, including nausea, vomiting, fatigue, headache and constipation, most of which are mild to moderate in severity.

As previously stated, MGMT is a DNA repair protein that reverses the effects of temozolomide therapy (42) by removing alkylating adducts, thereby counteracting its effects (43) and conferring resistance to the agent (44).

Low-level expression in a wide variety of human tumors is thought to result from epigenetic silencing by hypermethylation of the MGMT gene promoter (44,45). Low-level MGMT expression is a predictive marker of a favorable clinical outcome in patients with temozolomide-treated glioblastomas (46–48). Herein, we extend this observation to aggressive adenohypophysial tumors.

Pituitary carcinomas Pituitary carcinomas are difficult to manage given their relentless growth and metastatic spread, despite the effects of multimodality therapy. The latter includes repeated surgeries, pharmacological manipulation, radiotherapy, and conventional chemotherapy (3,49), including various combinations of cis-platinum, etoposide and/or paclitaxel, but these have failed to achieve favorable clinical responses (2).

Initial reports of the successful use of temozolomide to treat pituitary carcinomas appeared in 2006 (5,6). To date, 16 cases have been treated (5,6,13,16,17,21-23,25). The time between disease presentation to temozolomide administration was 5–23 years (mean: 10.7 years). The group included nine PRL-producing tumors, five ACTH-producing tumors, three clinically non-functional tumors, and three silent corticotroph carcinomas. Eleven of the 16 patients (69%) experienced a clinical and radiological response to temozolomide. Assessment of MGMT immunoexpression was only

Pituitary adenomas The first temozolomide-treated pituitary adenoma patient was reported in 2006 (7,8). The tumor, which was investigated using histological, immunohistochemical and electron microscopic techniques, showed significant posttherapeutic effects, including hemorrhage, necrosis, focal

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available in 11 patients; only one patient with high-level immunoexpression responded to treatment.

clinical improvement and tumor shrinkage following treatment (23). It is of note that some tumors with no promoter methylation also respond to therapy (22,23). These observations challenge the notion that MGMT promoter methylation is a reliable predictor of treatment response. Indeed, two recent studies on MGMT promoter methylation and the temozolomide response in eight aggressive adenomas and seven pituitary carcinomas found no association between promoter methylation status and response (22,23). Salehi et al. (55) did not find a close correlation between MGMT immunoexpression and MGMT promoter methylation in a study on aggressive pituitary adenomas and carcinomas.

Thus, the utility of MGMT promoter methylation for the selection of patients who might respond well to temozolomide treatment remains controversial and an active focus of further research. The problem is that many tumors possess an admixture of MGMT immunopositive and immunenegative cells. Obviously, in such instances, it is difficult to predict overall the responsive to temozolomide.

To date, MGMT immunoexpression has been documented in 23 adenomas and 11 carcinomas. In 18 tumors, MGMT labeling was low and correlated with a good therapeutic response to temozolomide (14/18 cases). In eight instances, MGMT staining was intermediate: five tumors responded well and three did not. In eight patients with high MGMT immunoexpression, only one responded positively. Thus, tumors featuring low- and intermediatelevel MGMT immunoexpression (n = 26), when compared with those with high MGMT immunoexpression (n = 8), showed a good response to temozolomide in 19/26 (73%) versus 1/8 (13%) cases (p = 0.0039; two-tailed Fisher’s exact test).

Morphological changes Morphological comparisons of pathology before and after temozolomide treatment were performed in three cases: all were patients with adenomas. One was a non-responder who exhibited no changes (10) and two were responders (8,23) whose tumors after treatment demonstrated hemorrhage, necrosis, focal fibrosis, inflammatory infiltrates, fewer mitoses, and a low Ki-67 labeling index, as well as neuronal transformation. The latter is considered to be a metaplastic phenomenon and has been observed in a variety of untreated endocrine neoplasms, including GH cell adenomas (50).

DISCUSSION Response to treatment In patients who respond to temozolomide, three basic patterns of radiographic changes are seen on MRI: tumor necrosis and hemorrhage (7,8), cystic change (19), and shrinkage (9,11-14). Such changes may be seen as early as two months after the onset of treatment.

In the tumors that respond to temozolomide, the clinical response is rapid and associated with prompt decreases in chiasmatic compression and mass effects. In patients with endocrine-active PRL- and ACTH-producing tumors, an almost immediate reduction in plasma hormone levels becomes apparent after commencement of therapy. Thus, it is possible to quickly evaluate the treatment response.

MGMT immunoexpression and MGMT promoter methylation

Suggested indications for using temozolomide treatment

The inverse relationship between MGMT immunoexpression and temozolomide response was first reported in two patients with aggressive pituitary adenomas (10). The observation was subsequently confirmed by McCormack et al. who assessed MGMT immunoreactivity in a PRL cell carcinoma and an aggressive GH-producing adenoma (13).

In both studies, tumors with low-level MGMT immunoexpression demonstrated clinical and radiological responses to temozolomide therapy. Other authors have reported similar findings (51). Thus, the demonstration of MGMT immunoreactivity appears to be useful for predicting the response of aggressive pituitary adenomas and carcinomas to temozolomide treatment.

The reports of Kovacs et al. (10) and McCormack et al.

(13,51) underscore the inverse relationship between MGMT immunoexpression and the efficacy of temozolomide therapy, and this relationship has also been noted in highgrade gliomas (46-48,52). Whereas the standard method for evaluating MGMT status in gliomas is the identification of promoter methylation (47), immunohistochemical detection of MGMT expression is an attractive alternative. It represents an inexpensive, readily accessible technology that is available to most laboratories (53,54). Whereas most reports pertaining to gliomas and temozolomide therapy are based on promoter methylation status, data relating to pituitary neoplasms are based on MGMT immunoexpression (24,51).

Although several recent reports of aggressive pituitary tumors that were successfully treated with temozolomide have reported absent or low immunohistochemical expression of MGMT as a factor, one patient whose tumors exhibited higher levels of MGMT staining also experienced

Based on the published cases and the reported response rates, temozolomide therapy can be recommended when other options fail, particularly in the following instances: (1) aggressive PRL-producing tumors that are resistant to bromocriptine or cabergoline and continue to grow after surgery and radiotherapy; (2) aggressive ACTH-producing tumors, especially Crooke cell and Nelson syndrome variants that cannot be cured by surgery and radiotherapy; (3) recurrent, clinically non-functional tumors exhibiting continued growth after repeated surgeries and radiotherapy; (4) pituitary carcinomas.

Temozolomide has been documented as valuable for the treatment of aggressive pituitary adenomas and carcinomas.

The clinical and radiological responses are encouraging: 60% of aggressive adenomas and 69% of pituitary carcinomas respond well to treatment. An inverse correlation exists between MGMT immunoexpression and the therapeutic response to temozolomide. Based upon our literature review, a significant proportion of the adenohypophysial tumors that are responsive to temozolomide show low-level MGMT immunoexpression, whereas only one tumor showing high-level immunoexpression was found to respond.

The use of immunohistochemistry for determining MGMT immunoexpression appears to be a promising guide for therapeutic decision-making (13,53). Obviously, all available methods for assessing the likelihood of a positive temozolomide response should be utilized. We feel strongly that the determination of MGMT immunoreactivity is of clinical value and that MGMT promoter methylation status should

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be determined as well. The molecular mechanisms that affect MGMT expression remain to be fully elucidated.

Targeted modulation of MGMT may be used in patients who may otherwise not respond to temozolomide therapy.

Future therapies that incorporate different dosing regimens or other drugs may improve the tumor responsiveness.

DISCLOSURE The authors have no personal financial or institutional interest in any pharmaceutical agents or testing devices described herein.

ACKNOWLEDGMENTS The authors are grateful to the Jarislowsky and Lloyd Carr Harris Foundation for their continued support. The authors thank Mrs. Denise Chase from the Mayo Clinic for her excellent assistance and secretarial support.

AUTHOR CONTRIBUTIONS Ortiz LD, Syro LV, Scheithauer BW, and Kovacs K conceived the study and wrote the manuscript. Ortiz LD, Syro LV, and Rotondo F searched the literature and extracted the data. Rotondo F, Uribe H, Fadul CE, and Horvath E contributed to the initial version of the manuscript. All authors contributed to the critical revision of the manuscript and approved the final version.

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DOI:10.6061/clinics/2012(Sup01)21

REVIEW

Kinase inhibitors for advanced medullary thyroid carcinoma Martin Schlumberger, Marie-He´le`ne Massicotte, Camila L. Nascimento, Ce´cile Chougnet, Eric Baudin, Sophie Leboulleux University Paris-Sud XI, Institut Gustave Roussy, Department of Nuclear Medicine and Endocrine Oncology, Centre de Re´fe´rence ‘‘Tumeurs Re´fractaires de la Thyroı¨de’’, Villejuif, France.

The recent availability of molecular targeted therapies leads to a reconsideration of the treatment strategy for patients with distant metastases from medullary thyroid carcinoma. In patients with progressive disease, treatment with kinase inhibitors should be offered.

KEYWORDS: Thyroid Neoplasms; Molecular Targeted Therapy.

Schlumberger M, Massicotte MH, Nascimento CL, Chougnet C, Baudin E, Leboulleux S. Kinase inhibitors for advanced medullary thyroid carcinoma. Clinics. 2012;67(S1):125-129.

E-mail: schlumbg@igr.fr Tel.: 33142116095

benefits of novel therapies may be largely outweighed by drug toxicities and the rigors of clinical trial participation.

Also, there is no evidence that the efficacy of these novel therapies is higher at an early stage than later when metastases are larger in size, and treatment can be postponed in most patients until progression has been documented by imaging.

Imaging should emphasize identification of all clinically relevant sites of disease, including tumors large enough to be serially assessed to determine response to therapy as well as those that might require additional localized intervention, such as surgery, external radiation therapy, radiofrequency ablation, cryotherapy, cement injection, or embolization. These treatments should be performed whenever needed before the initiation of any systemic treatment, and they may enable systemic treatment to be postponed in some patients.

Diagnostic procedures in metastatic MTC patients should include spiral computed tomographic scanning or magnetic resonance imaging of the brain, ultrasonography of the neck, contrast-enhanced spiral computed tomographic scanning of the neck and chest, triple-phase computed tomographic scanning or preferably magnetic resonance imaging of the liver (because liver metastases may be difficult to visualize with computed tomography during anti-angiogenic treatment), bone scintigraphy and magnetic resonance imaging of the spine and pelvis. Fluorodeoxyglucose (FDG) uptake on positron emission tomography scanning is usually low, and for this reason FDG positron emission tomography scanning is usually poorly sensitive and cannot be used to assess progression rate or response to treatment (1,3).

In the absence of treatment, imaging is repeated every six months, and progression rate is assessed using Response Evaluation Criteria in Solid Tumor (RECIST) (4,5). Patients with measurable lesions and documented progression in a

INTRODUCTION Medullary thyroid carcinoma (MTC) accounts for 5–8% of all thyroid cancers (1,2). Distant metastases are observed at presentation in 7–23% of MTC patients. In patients with elevated calcitonin levels after initial treatment, clinical recurrence will occur at different time intervals after surgery, depending on the amount of persistent disease and the progression rate. Recurrent disease in the neck and mediastinum is frequently amenable to surgery, and some patients may also benefit from external radiation therapy.

Distant metastases are the main cause of MTC-related death.

They often affect multiple organs, including lungs, bones, and liver, and more rarely brain, skin, and breast, and they are frequently associated with a neck recurrence (3). In patients with recurrent disease, a good quality of life is usually maintained, but diarrhea may be debilitating. Slow tumor growth is common and, in retrospective series, survival after the discovery of distant metastases was around 25% at 5 years and 10% at 10 years, but recent series suggest that survival rates might be higher with earlier discovery of metastatic disease.

Selection of patients for clinical trials Patients with metastatic MTC must be accurately characterized concerning all clinical prognostic indicators, including age, performance status, histology, disease extent and location, and progression rate. The importance of this latter parameter cannot be overemphasized, as many patients with metastatic MTC can be asymptomatically stable for long periods of time, and in such patients, the Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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given time interval (between 6 and 15 months) should be considered candidates for systemic treatment. Progression rate can also be evaluated by the doubling times of serum markers, calcitonin and carcinoembryonic antigen levels (6,7), but progression should always be confirmed by imaging (4,5).

Molecular Targeted Therapies Several molecular abnormalities thought to be important in thyroid oncogenesis and/or progression have been defined in MTC and represent potential targets for therapy (1,2,14). The aims of treatments are first to improve the quality of life and second to prolong overall survival.

Because it is difficult to demonstrate an improvement in overall survival in patients with slowly progressing disease and a long life expectancy, surrogate parameters have been used: the response rate studied in phase II trials is poorly related to overall survival; progression-free survival studied in randomized phase III trials is better related to overall survival. At the present time, results of only one phase III trial are available.

Standard Systemic Treatments Symptomatic treatments of diarrhea include loperamide, diphenoxylate/atropine, and codeine (1). Earlier clinical trials of cytotoxic chemotherapies suffered many shortcomings. Due to both the rarity of apparent benefit and the significant toxicity of the treatments, physicians enrolled only patients with a large tumor burden and rapidly progressive metastatic disease. The few prospective trials that were reported did not include sufficient numbers of patients to demonstrate benefits or reject false-negative conclusions. None used the nowstandard RECIST (4,5), and many trials reported results mixing together patients with differentiated thyroid cancer, MTC and anaplastic thyroid cancer. The most frequently tested agent in thyroid cancer patients is doxorubicin, used either alone or in combination with cisplatin. Tumor response rates range from 0% to 22% in MTC patients, with all responses being partial and only lasting a few months (8,9).

As MTC is a well-differentiated endocrine tumor, various combinations of 59-fluorouracil, dacarbazine, streptozocin, cyclophosphamide, and vincristine have been used and produced some responses (in about 20% of patients) with symptomatic improvement in some (10). Newer cytotoxic drugs, such as taxanes, gemcitabine, or irinotecan, have not been reported in significant series of MTC patients.

Treatment with bi-specific antibodies directed against carcinoembryonic antigen and diethylene triamine pentaacetic acid (DTPA) and with DTPA labeled with 13I or with octreotide labeled with yttrium-90 prolonged overall survival but did not induce objective responses in patients with large metastases (11,12), and hematological and renal toxicities were significant. Interferon a and somatostatin analogs used either alone or in combination in MTC patients only produced an inconstant and transient effect on diarrhea. Dendritic cell immunotherapy may be effective but is still under evaluation (13).

In conclusion, these therapeutic methods provide low response rates in patients with advanced and progressive refractory MTC.

Molecular targets In most familial forms of MTC, one of a variety of germline RET (rearranged during transfection) mutations is found. The proto-oncogene receptor RET is a membrane receptor with tyrosine kinase activity, and these mutations activate the kinase function that triggers downstream mitogenic and survival signaling. Somatic mutations in RET are also found in 30–50% of sporadic MTC tumors, and of these, 80% are mutations at codon 918. Once activated, RET stimulates several intracellular signaling cascades that include the GTPase-Ras/serine/threonine-protein kinase Braf (BRAF)/mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/ alpha serine/threonine-protein kinase (AKT) pathways.

Besides RET, BRAF mutations have only been rarely reported in MTC, but RAS mutations have been found in 68% of MTC tumors without RET mutation and rarely in MTC harboring a RET mutation (15). Finally, little is known about loss of function of tumor suppressors in MTC (1,2).

Angiogenesis serves a critical role in the development of these hypervascularized tumors and provides another set of potential molecular targets for therapy. Vascular endothelial growth factors (VEGF) and VEGF receptors (VEGFR-1 (Flt1) and VEGFR-2 (Flk-1, KDR)) are often overexpressed in thyroid cancer tissues, both in tumor cells and supporting vascular endothelium, and they also trigger the MAPK signaling pathway (17). Receptors for the fibroblast growth factor and for the platelet-derived growth factor are also often overexpressed and may play a role in tumor angiogenesis.

Table 1 - Kinase inhibitor activities relevant to thyroid carcinomas.

Inhibitory Concentration 50% (nm)

Drug

Axitinib Sunitinib Motesanib Sorafenib Vandetanib Pazopanib Cabozantinib (XL184) Lenvatinib (E7080)

VEGFR1

VEGFR2

VEGFR3

RET

1.2 2 2

0.25 9 3 90 40 30 0.035 4

0.29 17 6 20 110 47

41 59 49 100

10 22

5

4 35

BRAF

Other targets

PDGFR, CKIT 6 EGFR PDGFR, C-KIT C-MET, C-KIT PDGFR, FGFR

VEGFR1 = vascular endothelial growth factor 1, VEGFR2 = vascular endothelial growth factor 2, VEGFR3 = vascular endothelial growth factor 3, BRAF = GTPase-Ras/serine/threonine-protein kinase B-raf.

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Kinase inhibitors and thyroid cancer Schlumberger M et al.

A large randomized phase III trial comparing progressionfree survival (PFS) in patients treated with vandetanib (300 mg/day) or placebo has been completed on 331 patients with locally advanced or metastatic MTC (22). The median PFS was significantly prolonged from 19.3 months in the placebo arm to a predicted median of .30.5 months (median not yet reached) in the vandetanib arm (Hazard Ratio 0.46; p,10–4); partial responses were observed in 45% of patients treated with vandetanib, with a predicted median duration of response of 22 months. Benefits of vandetanib treatment were observed in all subgroups of patients, regardless of RET mutation status, progression rate and tumor burden. Sufficient events to analyze overall survival have not occurred yet. The serum calcitonin and carcinoembryonic antigen levels decreased by $50% and over a minimum of four weeks in 69% and 52% of patients, respectively. However, calcitonin production by MTC is controlled by the RET signalling pathway, and RET kinase inhibitors may decrease calcitonin production independent of tumor mass changes (23). Time to worsening of pain was significantly improved by vandetanib.

In some patients, diarrhea was improved rapidly after initiation of therapy, and this often allowed patients to resume a normal social life. Adverse events, including diarrhea, fatigue, rash, hypertension, and prolongation of the QTc interval on electrocardiogram, were mainly classified as grade 1 or 2. However, 12% of patients receiving vandetanib discontinued treatment and 35% required that their dose of vandetanib be reduced because of an adverse event.

Cabozantinib (XL-184). In a phase I trial, cabozantinib induced a partial response in 17 of 34 evaluable MTC patients, of whom 10 had a confirmed partial response, and another 15 patients had stable disease (18). Partial responses were observed regardless of RET mutation status, and in both treatment-naŨve patients and patients who had previously been treated with kinase inhibitors, suggesting that there is no cross resistance with other compounds. On the basis of these favorable results, a randomized phase III trial of cabozantinib (175 mg/day) versus placebo is ongoing in patients with progressive MTC (NCT00704730).

Motesanib. In a phase II trial, motesanib (starting at 125 mg/day) induced a partial response in only two of 91 patients, but another 43% had stable disease longer than 24 weeks. The low efficacy of the drug may be attributed to its malabsorption related to diarrhea (24).

Molecular targeted therapies used in thyroid cancer The RET kinase appears to be a promising target for molecular therapy of MTC (2,14). ATP-competitive inhibitors that exert an inhibitory effect on the RET kinase prevent the activation of the downstream proteins (Table 1). Owing to the close structural relationship between the kinases in RET and VEGFR, most VEGFR kinases inhibitors also inhibit RET kinase. For example, vandetanib effectively inhibits VEGFR2, VEGFR3, epidermal growth factor receptor (EGFR) and RET kinases. X-ray crystallographic analysis of the vandetanibRET kinase complex demonstrates that the drug docks into the ATP-binding pocket of the kinase, inhibiting the wild-type enzyme and most of the activated mutant forms. Sorafenib, sunitinib, motesanib, lenvatinib (E7080), and cabozantinib (XL-184) are also multi-kinase inhibitors that share the ability to inhibit RET and VEGFR, along with other kinases; in contrast, axitinib and pazopanib seem to act only as antiVEGFR agents. C-met is overexpressed in medullary thyroid cancer as a result of RET activation, and cabozantinib that inhibits both RET and c-met is being tested in MTC (16,18).

Results of clinical trials Available results in MTC (Table 2) patients from phase I, II, and III trials have clearly confirmed the efficacy of these compounds. To date only vandetanib has achieved US Food and Drug Administration regulatory approval and more recently European Medicines Agency approval for therapy of advanced and progressive MTC, but some treatment guidelines recommend use of available agents for selected patients with progressive metastatic disease on the basis of the phase II results (1,19).

Vandetanib at the maximal tolerated dose (300 mg/day) included 30 patients with hereditary MTC who had germline RET mutations. Partial response was observed in 10 patients, among whom six had a confirmed partial response, and stable disease longer than 24 weeks was observed in another 16 patients (20). Another phase II trial with vandetanib (100 mg/day) included 19 hereditary MTC patients, and a partial response was observed in three patients and stable disease lasting 24 weeks or longer in a further 10 patients, demonstrating anti-tumor activity in this setting. However, it is not clear whether the lower dose induced less toxicity, and two patients discontinued the study because of a vandetanib-related adverse event (21).

Table 2 - Results obtained in patients with medullary thyroid carcinoma with kinase inhibitors.

Author Vandetanib

Wells (2010) Robinson (2010) Wells (2010)

Cabozantinib Motesanib Axitinib Sunitinib

Kurzrock (2010) Schlumberger (2009) Cohen (2008) De Souza (2010) Carr (2010) Kober (2007) Lam (2010) Bible (2010) DeGroot (2007) Frank-Raue (2007)

Sorafenib Pazopanib Imatinib

Patients (n)

PR (%)

SD .6 months (%)

Median PFS (months)

Median OS (months)

Dose reduction for toxicity (%)

Withdrawal for toxicity (%)

30 19 331

30/20 c 16 44

53 NE 53

27.9 NE 30.5 (v) vs. 19.3 (placebo)

NE 10 .36

73 15 35

23 11 12

34 83 11 25 6 5 19 14 15 9

50/29 c 2 18 32 50 40 10 7 0 0

44 43 27 46 33 aaa 43 57 27 56

11 17 NE NE aaa 17.9

NE .36 NE NE aaa NE

NE 38 NE NE aaa 76

NE 13

aaa 16

27

20

PFS = progression-free survival, OS = overall survival, c = confirmed; NE = not evaluated.

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even phase I trials that are testing the newest therapies should be considered for patients with progressive thyroid cancer, as these protocols may allow early identification of possibly effective drugs (37).

The drugs used up to now have similar mechanisms of action: all are anti-angiogenic and most also target the RET kinase. The relative role of the inhibition of each target or of their combined inhibition is currently unknown, but because axitinib and pazopanib are thought not to inhibit RET kinase, responses suggest an important role for the anti-angiogenic effects of these compounds. Also, responses to vandetanib or cabozantinib have been observed in patients without RET mutation, probably indicating that targeting mutant RET may not be necessary in all MTC tumors.

Future progress will be made in three directions. Firstly, by improving the knowledge of targets present in each individual tumor and by increasing the number of drugs directed against each of these targets, it may be possible to offer thyroid cancer patients a personalized selection of therapies. Secondly, there is an urgent need to test other drugs in thyroid cancer patients and to understand the basis of tumor response. Further trials should also search for other treatment modalities, including combination with cytotoxic chemotherapy or sequential treatment modalities or immunotherapy (21). Finally, to increase the accrual of patients, to optimize the experimental design of the protocol, to improve the characterization of tumor tissues, and to improve the tolerance of treatment, the collaborative participation of a multidisciplinary team of endocrinologists, oncologists, nuclear physicians, surgeons, pathologists, laboratory researchers, and statisticians should be strongly encouraged through national and international networks.

Search strategy: All papers written in English found on PubMed between 2000 and 2010 using the key words ‘‘metastatic thyroid cancer, anaplastic thyroid cancer, targeted therapies’’ were analyzed.

Sorafenib. In a phase II trial, sorafenib (400 mg/twice daily) induced a partial response in two of 21 patients, and another nine patients had stable disease for more than 15 months; calcitonin levels decreased over 50% in nine patients (25). In another trial with five MTC patients, two had a partial response (26). A phase I trial of sorafenib combined with the farnesyl transferase inhibitor tipifarnib yielded partial response in five patients and stable disease in another five patients, with a median PFS of 15 months (27).

Axitinib. In a phase II trial, axitinib (5 mg twice daily) induced a partial response in two of 11 patients and another three patients had stable disease lasting at least 16 weeks (28).

Sunitinib. In a phase II trial, sunitinib (50 mg/day, 4 weeks on and 2 weeks off) induced a partial response in eight of 25 patients and another 46% had disease stabilization for more than 24 weeks (29). In a smaller trial, sunitinib (37.5 mg/day continuously) induced a partial response in three of six patients (30).

Pazopanib. In a phase II trial, pazopanib (800 mg/day) induced a partial response in one of 14 patients and another eight patients had stable disease (31).

Lenvatinib (E7080). E7080 showed promising results in several MTC patients in a phase I trial (32), and a phase II trial in progressive MTC is ongoing (NCT00784303).

Imatinib. Imatinib was used in two trials (starting dose 600 mg/day) on nine and 15 MTC patients, respectively, and no tumor response was observed (33,34). In a phase I trial combining imatinib with dacarbazine and capecitabine, no response was observed in seven MTC patients (35).

Toxicities of molecularly targeted therapies Adverse effects from these targeted therapies are significant, including fatigue, hypertension, QTc prolongation, anorexia, diarrhea, cytopenias, and skin toxicities. These short- or median-term side effects may lead to dose reduction in 11–73% of patients and to withdrawal of drug in 7–25%. Serum thyroid-stimulating hormone levels should be regularly monitored as they may increase during treatment with any of these kinase inhibitors; such an effect should lead to an increase in the daily levothyroxine treatment dose (36). Given that these treatment modalities may be given for months or even years, further work to minimize toxicities is needed.

Conflicts of interest: MS: Amgen, Astra-Zeneca, Bayer, Exelixis, Eisai, Genzyme, Ipsen Pharma, Roche.

AUTHOR CONTRIBUTIONS All authors were responsible for the writing and approval of the manuscript.

CONCLUSION AND PERSPECTIVES

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Although response criteria in contemporary trials differ markedly from those in trials evaluating cytotoxic chemotherapy, the anti-tumor efficacy of these agents in MTC patients is likely to be much greater than that of earlier chemotherapies. Benefits demonstrated with vandetanib in improving PFS and delaying worsening of pain counterbalance its adverse effects. Results of the ongoing phase III trial with cabozantinib are also expected to confirm the promising results obtained in the phase I trial. To date only vandetanib has been labeled by the US Food and Drug Administration for use in the rare MTC patients with a large tumor burden in whom progression has been documented, and it should be used as first-line treatment for this indication. Patients in whom the use of vandetanib is contraindicated or who experience toxicity or resistance should preferably be included in prospective trials, and

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DOI:10.6061/clinics/2012(Sup01)22

REVIEW

Total parathyroidectomy in a large cohort of cases with hyperparathyroidism associated with multiple endocrine neoplasia type 1: experience from a single academic center Fabio Luiz de Menezes Montenegro,I Delmar Muniz Lourenc¸o Junior,II Marcos Roberto Tavares,I Sergio Samir Arap,I Climerio Pereira Nascimento Junior,I Ledo Mazzei Massoni Neto,I Andre´ D’Alessandro,I Rodrigo Almeida Toledo,II Fla´via Lima Coutinho,II Lenine Garcia Branda˜o,I Gilberto de Britto e Silva Filho,I Ano´i Castro Cordeiro,I Sergio Pereira Almeida ToledoII I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Head and Neck Surgery Section, Department of Surgery, Sa˜o Paulo/SP, Brazil.

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Endocrine Genetics Unit, Endocrinology Division, Sa˜o Paulo/SP, Brazil.

II

Most cases of sporadic primary hyperparathyroidism present disturbances in a single parathyroid gland and the surgery of choice is adenomectomy. Conversely, hyperparathyroidism associated with multiple endocrine neoplasia type 1 (hyperparathyroidism/multiple endocrine neoplasia type 1) is an asynchronic, asymmetrical multiglandular disease and it is surgically approached by either subtotal parathyroidectomy or total parathyroidectomy followed by parathyroid auto-implant to the forearm. In skilful hands, the efficacy of both approaches is similar and both should be complemented by prophylactic thymectomy. In a single academic center, 83 cases of hyperparathyroidism/ multiple endocrine neoplasia type 1 were operated on from 1987 to 2010 and our first surgical choice was total parathyroidectomy followed by parathyroid auto-implant to the non-dominant forearm and, since 1997, associated transcervical thymectomy to prevent thymic carcinoid. Overall, 40% of patients were given calcium replacement (mean intake 1.6 g/day) during the first months after surgery, and this fell to 28% in patients with longer follow-up.

These findings indicate that several months may be needed in order to achieve a proper secretion by the parathyroid auto-implant. Hyperparathyroidism recurrence was observed in up to 15% of cases several years after the initial surgery. Thus, long-term follow-up is recommended for such cases. We conclude that, despite a tendency to subtotal parathyroidectomy worldwide, total parathyroidectomy followed by parathyroid auto-implant is a valid surgical option to treat hyperparathyroidism/multiple endocrine neoplasia type 1. Larger comparative systematic studies are needed to define the best surgical approach to hyperparathyroidism/multiple endocrine neoplasia type 1.

KEYWORDS: Hyperparathyroidism; Parathyroidectomy; MEN1; Parathyroid glands; Parathyroid neoplasms; Hypercalcemia.

Montenegro FLM, Lourenc¸o DM Jr, Tavares MR, Arap SS, Nascimento Jr CP, Massoni Neto LM, et al. Total parathyroidectomy in a large cohort of cases with hyperparathyroidism associated with multiple endocrine neoplasia type 1: experience from a single academic center. Clinics. 2012;67(S1):131-139.

E-mail: fabiomonte@uol.com.br Tel.: 55 11 2661 6425

and represented by parathyroid hyperplasia or adenoma (8); it occurs equally in males and females, and it frequently occurs at ages under 30 years (1,2). Despite relatively mild elevations in serum parathormone (PTH) and total/ionized calcium usually seen in HPT/MEN1, there have been recent reports of early onset, and progressive, frequent, extensive and severe bone and renal complications (7,9) In recent years, routine genetic screening performed in atrisk family members of index MEN1 cases has significantly increased the recognition rate of young asymptomatic patients presenting at early metabolic stages of HPT in our and other institutions (10,11). Furthermore, in recent decades, the clinical picture of sporadic or familial HPT has changed dramatically and most cases are more frequently recognized and diagnosed early, at asymptomatic stages (12,13).

INTRODUCTION Primary hyperparathyroidism (HPT) is highly prevalent in patients with multiple endocrine neoplasia type 1 (MEN1), and its penetrance is almost complete by the age of 40 years (1–7). HPT associated with MEN1 (HPT/MEN1) is an asynchronic, asymmetric multiglandular and multifocal disorder; the vast majority of cases are benign tumors

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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making or ruling out a diagnosis of MEN1 before neck exploration in each patient with apparently sporadic HPT. An adequate preoperative diagnosis may help the surgeon to choose the best surgical option (24,29,30). It is well documented that measurement of intraoperative PTH may fail in patients undergoing parathyroidectomy for whom MEN1 is not suspected (31). Moreover, early diagnosis of MEN1 is critical to the adequate management of other MEN1-related tumors that have a clinical impact (3). In this context, mutation analysis of the MEN1 gene in apparently sporadic cases of HPT in which the patient is younger than 30 years of age may be helpful in the surgical management of HPT (1).

Genetic testing is crucial to confirm the familial character of HPT disease within clinically affected family members identified in MEN1 kindreds genetically characterized by a MEN1 germline mutation. Although rare, relatives presenting primary HPT but not harboring a germline MEN1 mutation have been reported. According to MEN1 clinical criteria, these patients would have been diagnosed as a MEN1 affected case (1). However, they are classified as sporadic HPT cases and should be surgically treated accordingly. Such cases represent MEN1 phenocopies, and do not need further surveillance for MEN1-related neoplasias.

Parathyroid hyperplasia or adenoma accounts for the vast majority of HPT cases in MEN1, as carcinoma seems to be an extremely rare event in this condition (32). The prevalence of parathyroid carcinomas in MEN1 tends to be much lower than in sporadic cases of HPT (33).

Accordingly, we have seen no cases of parathyroid carcinoma in our HPT/MEN1 series, compared with higher frequencies observed in our sporadic HPT cases, in which we observed a 1.8–6.1% frequency of parathyroid carcinoma (34,35).

Medical non-surgical strategies The medical treatment of HPT associated with MEN1 is still under investigation. The experience with cinacalcet in HPT is limited to few cases, and no long-term follow-ups of disease end-points are available, despite short-term (up to 24 weeks) reductions of calcium and PTH (14–16).

Also, the use of percutaneous ethanol injection in cases of HPT recurrence after subtotal parathyroidectomy in HPT/ MEN1 patients resulted in short-term eucalcemia. However, hypercalcemia recurred in 90% of cases after 32 months of therapy (17).

Furthermore, high-intensity focused ultrasound applied to sporadic, uniglandular HPT is under investigation, although temporary palsy of the recurrent laryngeal nerve in one out of four cases has been reported. Recovery was noted only 40 days after therapy (18), and the role of highintensity focused ultrasound in multiglandular parathyroid disease is yet to be defined.

Long-term annual clinical follow-up has been recommended for selected sporadic cases of HPT, because bone mineral density may remain stable up to 10 years after diagnosis (19,20). To date, it is not clear whether this recommendation can be extended to HPT/MEN1.

Surgical treatment Surgery is the classic therapy for primary HPT in MEN1.

However, the criteria to indicate parathyroidectomy in these cases remain under debate. The International Consortium/ National Institutes of Health Consensus on MENs (1) recommended that indications for parathyroidectomy in MEN1 should follow the same criteria used for sporadic asymptomatic primary HPT (21,22).

Most HPT/MEN1 patients are younger than 50 years at diagnosis, and thus fulfill the indication for parathyroid surgery based solely on this criterion. However, it may not be easy to convince an asymptomatic patient that he/she will benefit from an operation that can be considered palliative, as recurrence rates are usually high (23). In some young asymptomatic cases, the treatment may be perceived as worse than the disease itself (24).

What is the best surgical approach to HPT in MEN1?

The best surgical approach to HPT in MEN1 is under debate, and includes resection of tissues other than parathyroid glands themselves.

Preventive thymectomy. Presently, preventive thymectomy is highly recommended to be performed during either subtotal or total parathyroidectomy in HPT/MEN1 patients.

Since its proposal in 1997 (36), there is a general agreement that transcervical thymectomy should be performed during parathyroidectomy in all MEN1 cases, in order to: (a) detect supernumerary parathyroid glands, (b) prevent thymic carcinoids (occurrence in MEN1 ranges from 2.6% (37) to 8% (38)), and (c) to disclose intrathymic parathyroid tissue when less than four parathyroid glands are identified during neck exploration (39). It has been estimated that transcervical thymectomy usually excises only 30–40% of the thymus (39). Consequently, thymic carcinoids have developed in a few cases, even after preventive transcervical resection, and this has caused its value to be questioned (25).

Accordingly, we have adopted routine transcervical thymectomy during parathyroid intervention in HPT/ MEN1 cases since 1997. To date, no thymic carcinoid has been detected in the thymic specimens, and none of our patients has developed thymic carcinoid after this procedure.

However, before 1997, in a specific HPT/MEN1 patient submitted to parathyroidectomy not associated with transcervical thymectomy, a thymic carcinoid with metastases was

Surgery in HPT/MEN1 The surgical treatment of HPT/MEN1 is a challenge to the surgeon for several reasons. First, in the vast majority of cases, HPT/MEN1 is a multiglandular disease that usually occurs asymmetrically and asynchronically. Second, the frequency of supernumerary parathyroid glands is considerable. Furthermore, the surgeon is frequently confronted with parathyroid glands with macroscopic features resembling normal characteristics and he/she must decide how much tissue should be taken, evaluating the risk for permanent hypoparathyroidism versus disease persistence or early recurrence. One parathyroid gland may appear normal macroscopically, but may cause persistent hypercalcemia (24). Finally, although questionable as a 100% effective preventive strategy for thymic carcinoid tumors (25,26), transcervical thymectomy has been recommended to be carried out during parathyroidectomy in HPT/MEN1 cases (27).

Presurgical diagnosis of MEN1 As the first manifestation of MEN1 is usually HPT, and was found to increase .70% in one of our series (28), clinicians and surgeons must be strictly committed to

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intraoperative PTH sampling, the skillfulness of the surgeon is crucial. Therefore, parathyroidectomy in MEN1 patients is usually performed to a better standard in specialized tertiary academic centers where medical personnel and residents are highly trained (55,56). Moreover, it is also important that MEN1 patients can be followed-up in specialized outpatient services in order to receive support for both oncological and end-organ diseases (43,45,47,48,51, 57–59).

Case series. From 1987 to August, 2011, 83 HPT/MEN1 patients were submitted to 99 parathyroid interventions at the Hospital das Clı´nicas, University of Sa˜o Paulo School of Medicine. There were 43 females and 40 males and the age at the parathyroid surgery varied from 17 to 74 years old (mean, 40.8 years). No difference in age distribution according to gender was noticed, as shown in Figure 1.

Almost 75% of the patients were younger than 50 years of age and this finding may be related to the criteria used to indicate parathyroidectomy for these patients. In this HPT/ MEN1 series, no child carrying the MEN1 gene had an indication for parathyroidectomy, although young HPT cases have been operated on in our group for other reasons, such as neonatal HPT (60).

Figure 2 shows that there has been an increasing number of HPT/MEN1 patients in recent years, most probably due to the active clinical and biochemical search for MEN1 diagnosis, as well as to the routine familial genetic MEN1 screening procedure performed by our group (3,6,7,10,11, 28,61). In a recent study, HPT/MEN1 accounted for 17% of all cases undergoing parathyroidectomy at our institution (62).

Seventy-five out of the 83 cases of HPT/MEN1 (90.4%) had their initial operation at our institution between 1987 and 2011. Of the other eight cases, two underwent

documented (27). Absence of thymic carcinoid with metastases in prophylactic thymectomy specimens has been reported in large series by others (30,39).

Subtotal versus total parathyroidectomy. Regarding the metabolic control of the HPT in MEN1, in the past many groups have favored more aggressive strategies, such as total parathyroidectomy followed by immediate parathyroid autotransplantation (40–43). However, in the last 20 years, several authors have advocated subtotal parathyroidectomy as the most suitable strategy in HPT/MEN1 cases (39,44–47).

Currently, only a few authors recommend total parathyroidectomy with autotransplantation (30,48–50). In fact, for many years, it was claimed that only a prospective randomized trial would help to answer this issue; however, as far as we know, no such study has been published to date.

Data on this topic vary according to the group, but subtotal resection seems to be associated with a lesser risk of hypoparathyroidism. In 57 HPT/MEN1 cases, 34 were subjected to subtotal parathyroidectomy and 23 to total parathyroidectomy and autotransplantation. Rates of HPT persistency or recurrence were higher in the first group, while post-surgical hypoparathyroidism was more prevalent in the second group (51). In a large case series comprising 92 HPT/MEN1 patients treated over a period of 42 years, the 10-year actual recurrence-free interval data were 49% for subtotal parathyroidectomy and 84% for total parathyroidectomy and autotransplantation (47). In that series, post-surgical hypoparathyroidism rates were 26% for subtotal parathyroidectomy and 46% for total parathyroidectomy, but no significant difference was documented (p = 0.18). Notwithstanding, it was concluded that the high incidence of severe hypoparathyroidism after total parathyroidectomy with autotransplantation favored subtotal resection as the procedure of choice at the initial operation.

It is worthwhile noting that a limited number of cases (n = 16) underwent total parathyroidectomy and parathyroid autotransplantation in that study (47). However, in another recent HPT/MEN1 series (n = 47), patients submitted to total parathyroidectomy and immediate autotransplantation presented with 22% postsurgical permanent hypoparathyroidism, whereas this rate was 45% in cases submitted to subtotal parathyroidectomy (30).

The rate of permanent hypoparathyroidism may be different in patients undergoing a first cervical exploration or a secondary cervical surgery. Thus, Tonelli et al. observed hypoparathyroidism in 10 out of 45 patients (22%) after the first surgery and in three out of six patients (50%) after a second cervical operation (48). Also, the rate of postsurgical hypoparathyroidism may be affected by the time of follow–up of the patient. Accordingly, there are reports of cryopreserved autografted parathyroid tissues achieving adequate PTH secretion only 2–4 years after implantation (45).

A recent meta-analysis suggested that subtotal parathyroidectomy is superior to total parathyroidectomy with immediate autotransplantation (52). Conversely, other authors favor a more aggressive initial operation (50,53). In this context, it has been claimed that further high-quality evidence for the best surgical approach for HPT in MEN1 is lacking.

Experience from a single academic center In our experience, surgical treatment of HPT in MEN1 is a highly complex procedure, and the medical residents in our institution have been trained intensively in this technique (54,55). Despite improvements in imaging techniques and

Figure 1 - Age distribution according to gender in 83 cases of hyperparathyroidism/multiple endocrine neoplasia type 1.

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(5/47). This latter rate may in fact be slightly higher because a longer period of follow-up could not be performed in a few cases.

Total parathyroidectomy and autograft. The standard initial operation for HPT/MEN1 cases at our institution has been total parathyroidectomy followed by immediate parathyroid autograft to the non-dominant forearm, associated with transcervical thymectomy. Of note, although other groups have abandoned cryopreservation of parathyroid fragments because of the good results obtained with subtotal parathyroidectomy (46), we continue to advise cryopreservation whenever this procedure is possible. Parathyroid cryopreservation may have some limiting factors (63), but it is recommended when available, irrespective of the type of parathyroid surgery, since subtotal resection involves also a significant risk of permanent hypoparathyroidism (24,30).

Our experience with long-term cryopreserved tissue in secondary HPT (64) is similar to that reported by others in MEN1/HPT (30).

Initially, parathyroid autografts consisted of 20 fragments (each fragment was approximately 261 mm) that were transplanted in individual pockets. Presently, based in our experience in secondary HPT, 30 parathyroid gland fragments are usually implanted in a single pocket (65).

Intra-operative PTH. Intra-operative parathyroid hormone (ioPTH) measurements have been obtained in more recent cases, although in our protocol all glands should be searched and excised irrespective of their macroscopic appearance. In our experience, ioPTH in HPT/MEN1 cases has presented some limitations. Indeed, preoperative diagnosis of MEN1 is much more informative to the surgeon than imaging studies

Figure 2 - Emerging recognition and surgical treatment of patients with hyperparathyroidism/multiple endocrine neoplasia type 1 at our institution.

parathyroidectomy in our institution before 1987 and data from their first operation are unavailable. Surgeons initially involved in treating these two HPT/MEN1 cases might have been unaware of MEN1 at the time, excising only enlarged parathyroid glands. The remaining six cases underwent parathyroidectomy in other institutions, and their previous medical records were not available (Figures 3 and 4).

We had a reoperative rate due to HPT persistence of 8.0% (6/75) and an HPT recurrence rate of 6.75% (5/75), including all cases. However, taking into account only those patients with more than 5 years of follow-up (i.e., patients operated on up to 2006), the gross recurrence rate was 10.6%

Figure 3 - Distribution of patients with hyperparathyroidism/multiple endocrine neoplasia type 1 who had not had a previous neck operation (1987 to 2011).

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Figure 4 - Distribution of patients with hyperparathyroidism/multiple endocrine neoplasia type 1 who had had a previous neck operation.

observed: two retropharyngeal right superior parathyroid glands (one patient with right thyroid lobe agenesia), one intrathyroidal gland, one superior gland, and one located at the aortic arch in medium mediastinum and excised by thoracoscopy. Two further cases presented with late HPT recurrence due supernumerary parathyroid glands. One was located at the cervical region and the other at the mediastinum and, in this latter case, total thymectomy was performed by sternotomy. A rate as high as 33% for supernumerary glands has been reported in MEN1/HPT cases, including intrathyroidal supernumerary glands in 2.5% of patients (8). In our current experience, no patient with MEN1/HPT has presented a supernumerary intrathyroidal gland; however, it has been observed in some patients with renal HPT at our center (67). Our rate of intrathymic supernumerary glands was 4.9% (unpublished data). This rate is rather inferior to the rate of almost 30% reported by others (8). However, in a large series of routine bilateral cervical thymectomy in renal HPT, the frequency of supernumerary thymic parathyroids was 6.5% (68). A similar rate of 6% has been described in MEN1/HPT (39).

Of note, two specific cases were called our attention, and they are commented on below.

Non-functional tumor progression. The first case of nonfunctional tumor progression was a female patient who was initially submitted to cervical surgery in 1993 and presented MEN1-related tumors: a non-functioning pituitary adenoma, multiple pancreatic endocrine tumors, as well as a goiter. No

or ioPTH. Thus, it can inform: (a) whether additional high hyperfunctional parathyroid tissue was left behind (which has not happened yet in our group); or (b) whether the exploration can be finished, despite the fact that fewer than four glands have been identified after an extensive search.

For academic purposes, we usually measure PTH at the following times: at the induction of anesthesia (basal), after identification of all parathyroid tissue (pre-excision), 10 min after the resection of the largest parathyroid gland, and again 10 min and 15 min after the excision of the remaining parathyroid tissue and thymus. In some circumstances, a decrease .50% of ioPTH has been observed after the excision of the largest parathyroid gland (Figure 5). This finding has been described previously (66), and may explain failures of ioPTH in the surgical management of HPT/ MEN1 patients (31).

Reoperations by the same team. Fourteen out of the 75 HPT patients (18.7%) were reoperated in a total number of 16 reoperative procedures (Figure 3). Not all neck reoperations were related to HPT. Two consisted of total thyroidectomy and neck dissection for associated metastatic papillary thyroid carcinoma. In five cases, the parathyroid autograft at the forearm was excised due to recurrent HPT. One patient with non-functioning tumors was operated on due to goiter, and recurrence occurred later (see details of this case below). In five cases (6.7%), fewer than four glands could be initially identified at the first operation, and early HPT recurrence or persistence was

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exons and their boundaries, no germline mutations were found characterizing a phenocopy, as previously reported in this condition (69). Thus, the surgical choice for this specific patient was a single parathyroid resection, with resolution of HPT. The patient is still under observation, without any sign of recurrence five years after the operation.

Reoperations from other centers. As mentioned, six of our HPT/MEN1 cases were initially operated on in other centers. Two were previously subjected to total thyroidectomy due to papillary thyroid carcinoma; a simultaneous intraoperative single parathyroid adenoma was found by the first surgeon. The two reoperations involved resection of the three additional parathyroid glands, central neck dissection and thymectomy. The third patient had been subjected to an intentional subtotal parathyroidectomy due to HPT/MEN1 at his primary center seven years before. His remnant was excised and partially autografted.

Three other cases had been subjected previously to surgery due to ‘‘a parathyroid adenoma’’; in these cases, excision of the remaining parathyroid glands was carried out, followed by parathyroid autografts and thymectomy in one of the patients. One of these patients had been previously subjected to two cervical surgical explorations in another center; however, HPT persisted due an undescended left inferior parathyroid gland located close to the submandibular gland. This residual gland was discovered after a careful review of sestamibi scintigraphy. A first quick view may be misinterpreted as a negative result in planar projection (Figure 6). We excised this remaining parathyroid gland and part of it was immediately auto-implanted to the non-dominant forearm.

Thyroid cancer in MEN1/HPT. Papillary thyroid carcinoma was found in five out of 83 cases (6%). This frequency was similar to that observed in sporadic HPT cases (9%) and carcinomas incidentally found in compressive multinodular goiter (11%) treated at the same institution (70). In four out of five cases, pathological data were available: all cases had microcarcinomas, which were multicentric in two instances. Microscopic metastases were present in two cases, and one had unilateral neck lymph node metastasis. One of these patients had apparently been subjected to high-dose radiotherapy due to a pituitary tumor many years before the neck operation.

Recurrence and hypoparathyroidism. Recently, we attempted to contact 71 out of the 85 patients operated on from 1987 to 2010 in our institution, and information of 45

Figure 5 - A marked decrease of intra-operative parathyroid hormone (ioPTH) after excision of the largest parathyroid gland only, in a case of hyperparathyroidism/multiple endocrine neoplasia type 1. Persistence or early hyperparathyroidism recurrence may occur if surgery is guided solely by ioPTH.

calcium abnormality was present at that time. During the partial thyroidectomy (lobectomy), an enlarged parathyroid was excised, although total parathyroidectomy was not performed at that time. She had no evidence of HPT until 13 years after parathyroid adenomectomy, when HPT ensued and she was subjected to reoperation. This case illustrates that hyperparathyroidism may ensue many years after an apparent non-functional enlargement of the parathyroid gland.

Relative of a MEN1 patient, with HPT due to a phenocopy. Another case, not included in the present

series, is a person with HPT who was a relative of a MEN1 patient. This case came to the hospital with a clinical diagnosis of HPT/MEN1. However, after sequencing all MEN1 and p27

Figure 6 - Sestamibi scintigraphy after two previous neck interventions in a patient with hyperparathyroidism/multiple endocrine neoplasia type 1 who had been treated initially at another hospital. There was a focal radiopharmacological concentration close to the left submandibular gland due to an undescended left inferior parathyroid.

Figure 7 - Numbers (%) of patients with hyperparathyroidism/ multiple endocrine neoplasia type 1 requiring calcium supplements according to time elapsed since the initial treatment (1998 to 2010).

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Total parathyroidectomy in cases with hyperparathyroidism associated with MEN1 Montenegro FLM et al. collection and analysis of data, the draft and critical review of the manuscript, and approval of the final version of the manuscript. Tavares MR, Branda˜o LG and Silva Filho GB conceived, drafted and critically reviewed the manuscript, and approved the final version of the manuscript.

was available. As a first approach, a compact questionnaire was given to these patients in order to update the information on post-surgical therapy with the calcium supplement. Ten out of the 45 patients had died because of neoplasias other than MEN1-related tumors (22.2%). This observation might serve to re-emphasize the importance of early MEN1 detection and treatment, with the aim of decreasing morbidity and mortality, with reference to MEN1-related tumors in MEN1 (3). Five out of the 35 remaining cases (14.3%) had recurrent HPT disease; 14 others (40.0%) required calcium and/or calcitriol replacement therapy; and the remaining 16 cases did not take either calcium or calcitriol (45.7%). It is worthwhile noting that in HPT/MEN1 cases calcium requirement rates are impacted by the time elapsed since the parathyroidectomy. In Figure 7, the percentages of patients requiring calcium supplements were obtained at different periods after parathyroid intervention. We found that the longer the follow-up, the lower the frequency of patients requiring calcium supplement. Accordingly, in one of our HPT/MEN1 series, parathyroid auto-implants took from 6 to 15 months to secrete sufficient amounts of PTH in order to lead to normal calcemia (58).

For each individual HPT/MEN1 patient, it is necessary to decide which is the best operation to achieve the ultimate goal of maintaining normocalcemia and avoiding both iatrogenic hypocalcemia and operative complications; and facilitating future surgery for recurrent disease (71,72).

In many cases, calcium/calcitriol supplemental therapy could be stopped 15 months after total parathyroidectomy followed by parathyroid auto-implant, but not in all patients (58). A question to be addressed is the amount of calcium supplement required for each case. In our patients taking calcium supplement for more than one year, the mean intake was 1.6 g/day (range, 0.5–3.0 g/day), and 50% were taking 1.5 g/day or less.

Our present observations are in accordance with others: parathyroid autografts may take long periods, sometimes up to seven years, to achieve adequate function, and PTH levels increase with time (23,58).

Subtotal and total parathyroidectomy followed by immediate parathyroid autotransplantation are surgical approaches that are currently accepted to treat cases with HPT associated with MEN1. In our single academic center, total parathyroidectomy followed by parathyroid autotransplantation to the non-dominant forearm associated with preventive transcervical thymectomy has been the surgical approach of choice for cases of HPT/MEN1. This strategy has been successful in achieving low rates of HPT recurrence. The considerable number of calcium-dependent patients, who were mainly observed in the first nine months after operation, tended to decrease during longterm follow-up.

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ACKNOWLEDGMENTS RAT is a recipient of FAPESP post-doctoral fellowships (2009/15386-6 and 11942/2009). SPAT is partially supported by a CNPq fellowship and a CNPq grant (401990/2010-9).

AUTHOR CONTRIBUTIONS Montenegro FLM, Lourenc¸o DM Jr, Nascimento Junior CP, Arap SS, Massoni Neto LM, D’Alessandro A, Toledo RA, Coutinho FL, Cordeiro AC, and Toledo SPA conceived the study and were responsible for the

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DOI:10.6061/clinics/2012(Sup01)23

REVIEW

Primary hyperparathyroidism in multiple endocrine neoplasia type 1: when to perform surgery?

Francesca Giusti,I Francesco Tonelli,II Maria Luisa BrandiI I Bone and Mineral Metabolism Unit, Department of Internal Medicine, University Hospital of Careggi, Florence, Italy.

Physiopathology, University Hospital of Careggi, Florence, Italy.

II

Department of Clinical

Primary hyperparathyroidism is a common endocrinological disorder. In rare circumstances, it is associated with familial syndromes, such as multiple endocrine neoplasia type 1. This syndrome is caused by a germline mutation in the multiple endocrine neoplasia type 1 gene encoding the tumor-suppressor protein menin. Usually, primary hyperparathyroidism is the initial clinical expression in carriers of multiple endocrine neoplasia type 1 mutations, occurring in more than 90% of patients and appearing at a young age (20–25 years). Multiple endocrine neoplasia type 1/primary hyperparathyroidism is generally accompanied by multiglandular disease, clinically manifesting with hypercalcemia, although it can remain asymptomatic for a long time and consequently not always be recognized early. Surgery is the recommended treatment. The goal of this short review is to discuss the timing of surgery in patients when primary hyperparathyroidism is associated with multiple endocrine neoplasia type 1.

KEYWORDS: Primary hyperparathyroidism; Multiple endocrine neoplasia 1; PHPT-MEN1; Subtotal parathyroidectomy; Total parathyroidectomy.

Giusti F, Tonelli F, Brandi ML. Primary hyperparathyroidism in multiple endocrine neoplasia type 1: when to perform surgery? Clinics. 2012;67(S1):141-144.

E-mail: m.brandi@dmi.unifi.it Tel.: 39 055 7946304

that is widely used is ‘‘a case with tumor in two of the three principal organs: anterior pituitary, parathyroid, and enteropancreatic endocrine tissue’’ (4,5).

Familial MEN1 is defined as one MEN1 case plus one first-degree relative with one of the three principal tumors or involvement of only one organ and a MEN1 diseasecausing germline mutation (6).

The MEN1 gene was identified in 1997 and is the only gene known to be associated with this syndrome (7,8).

Genetic testing with direct sequencing of the MEN1 gene is widely available and provides the best method of diagnosis; it can detect MEN1 gene mutations in about 90% of patients with MEN1 within 4–6 weeks. Germline MEN1 mutation is identifiable in 90% of typical MEN1 families and in only 6–10% of isolated cases (9). Some patients without an identified mutation may have large deletions that are not recognized by polymerase chain reaction or have mutations in the tested open reading frame and intron–exon junctions (10,11). Recently it became possible to search for large deletions or duplications in the MEN1 gene using the multiplex ligation-dependent probe amplification assay (12).

Genetic testing makes possible: (a) the identification of asymptomatic adults with a family history of MEN1; (b) the prenatal diagnosis of MEN1; and (c) the elimination of noncarriers by clinical screening (13).

INTRODUCTION Primary hyperparathyroidism (PHPT) is a condition caused by hyperfunction of the parathyroid tissue, usually involving only one gland. However, it is often multiglandular when expressed within complex hereditary syndromes, such as multiple endocrine neoplasia type 1 (MEN1), MEN2A, familial isolated hyperparathyroidism, autosomal dominant mild hyperparathyroidism, neonatal severe hyperparathyroidism, familial hypocalciuric hypercalcemia, and the hyperparathyroidism–jaw tumor syndrome (1,2).

Surgery is the principal treatment for these forms of familial PHPT, apart from in cases associated with familial hypocalciuric hypercalcemia. In this short review, we discuss the timing of surgery in patients with PHPT associated with MEN1.

MEN1 SYNDROME MEN1 syndrome (MIM#131100) is a rare hereditary cancer syndrome encompassing a variety of more than 20 endocrine and nonendocrine tumors (3). MEN1 is relatively rare (approximately 1 in 30,000) and the definition of MEN1

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

MEN1-PHPT PHPT in MEN1 is the most common form of endocrinopathy and it represents 2–4% of all forms of PHPT. It is the

No potential conflict of interest was reported.

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earliest endocrine expression in most patients, clinically relevant neoplasia is uncommon before puberty, occurring in 90% of MEN1 individuals between 20 and 25 years of age, although mild-to-moderate hyperparathyroidism often emerges during adolescence with mild hypercalcemia.

All individuals are affected by 50 years of age (14). Its progression is usually gradual but significant hypercalcemia is occasionally evident in early adolescence.

The signs and symptoms of hypercalcemia include kidney stones, low bone mass, constipation, nausea, vomiting, polyuria, dehydration, hypercalciuria, hypertension, shortened QT interval, lethargy, depression, confusion, and anorexia (14).

The medical therapies of MEN1-related PHPT include drugs such as bisphosphonates and calcimimetics. Currently, these drugs have a limited role, being used primarily in the presence of recurrent PHPT or when there are contraindications to surgery (15–17).

The treatment of choice in MEN1-related PHPT is surgery but in this regard some considerations deserve attention.

capable of identifying supernumerary glands that would otherwise go unnoticed (24).

Biochemistry and bone mineral density In young MEN1 patients, PHPT is often asymptomatic; mild ionized hypercalcemia with parathyroid hormone (PTH) within the normal range is typical (25).

However, it should be remembered that PHPT is defined as an excess of PTH with consequent increase in bone turnover, leading to a reversible loss of cortical and trabecular bone caused by an expansion of the remodeling space and a loss of cortical for increased endocortical resorption (26). MEN1 hyperparathyroid patients show more precocious and severe bone loss compared with patients with sporadic PHPT (27–31).

Surgical approaches The optimal surgical approach in PHPT in MEN1 is still under discussion. The choice being between: subtotal parathyroidectomy with removal of at least three to three and a half glands; and total parathyroidectomy with removal of all parathyroid glands and autologous parathyroid tissue graft.

Subtotal parathyroidectomy requires the identification of all parathyroid glands and the leaving of a remnant the size of a normal parathyroid, approximately 20–30 mg. The dissection must be meticulous to preserve the delicate single end-artery vascular supply to the parathyroid remnant. The parathyroid tissue left in situ should be prepared before resection of the remaining glands, to avoid bleeding and ischemia. Moreover, this residue must be marked with a non-absorbable suture or a surgical clip for future identification. It may be useful to suture the residual remnant gland away from the recurrent laryngeal nerve to facilitate reoperative surgery in case of persistent or recurrent disease (18,32,33).

Total parathyroidectomy provides identification and resection of all four glands followed by a parathyroid heterotopic auto-implant in the non-dominant forearm (20– 25 pieces, 1 mm each, of fresh parathyroid tissue implanted in one to five pockets created in the brachioradialis muscle).

This surgical approach must be accompanied by intraoperative histologic examination, to qualify and confirm the presence of parathyroid tissue in the removed tissues, and by rapid intraoperative PTH evaluation (18,32,33).

Reoperative debulking surgery of the forearm graft for recurrent disease can be performed under local anesthesia.

Prophylactic transcervical thymectomy should be performed in all patients to remove possible intrathymic supernumerary parathyroid glands and prevent the development of thymic carcinoid.

Both procedures should include a meticulous search for ectopic parathyroid tissue, which is commonly encountered within the thymus, mediastinum, carotid sheath, and in the tracheo-esophageal groove (21–23).

Extensive surgery is burdened by an increased risk of permanent hypoparathyroidism (hypocalcemia) and recurrent laryngeal nerve injury. Acute hypoparathyroidism may cause mild to severe neuromuscular symptoms ranging from neuromuscular irritability to seizures. Less extensive surgery yields a higher risk of recurrent disease requiring re-intervention, which increases the risk of more complications. In this latter intervention, multiple operations are

Age The age of onset of familial hyperparathyroidism is clearly anticipated at two–three decades compared with the non-syndromic form. The occurrence of hyperparathyroidism at a young age carries a high suspicion of MEN1 syndrome, supported by family history and an association with other endocrinopathies typical of MEN1 syndrome (13). Preclinical diagnosis of MEN1 gene mutations should be offered to all patients with suspected genetic disease (18).

With presymptomatic detection of genetic predisposition to this syndrome, patients can be followed with periodic clinical tumor surveillance for expression of the disease as well as for recurrence after treatment or progression of the disease; knowledge of carrier status enables early intervention (3).

Genotype–phenotype correlation regarding the severity of PHPT in MEN1 cannot be identified (18,19) but knowledge decreases the uncertainty and anxiety in the patient and allows focused surveillance by the physician.

Parathyroid tissue Generally the PHPT is caused by a multiglandular disease and the parathyroid glands can become hyperplastic or develop adenomas. The growth of the glands is asynchronous and asymmetric (18), as each gland is considered to be a monoclonal lesion in which the germline mutation in the MEN1 gene confers on the parathyroid tissue a high susceptibility for the development of a tumor after the second somatic mutation (2,20).

Morphologically, parathyroid glands in MEN1 may appear macroscopically normal, also because they can differ in terms of volume, weight, and size (18).

Parathyroid adenomas in MEN1 can be ectopic, often located in the thymus, rarely within the thyroid gland, in the anterior mediastinum, in the pericardium, or surrounding the trachea, the esophagus, and the carotid artery (21–23).

Supernumerary glands are frequently found in up to 20% of MEN1 patients, although some authors do not report supernumerary glands (18). A possible explanation for this discrepancy may be the misinterpretation of the size, shape or position of the parathyroid glands in MEN1 (18) because of different surgical strategies used; some of which are more

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of progressively challenging reoperations. Late surgical interventions are easier because of the glands’ more visible enlargement, but hypercalcemic complications increase.

The diagnosis of MEN1-associated PHPT should be confirmed by genetic testing. Carriers of MEN1 mutations require regular screening, regardless of the presence of hyperparathyroidism from the second decade of life, it being difficult to establish a genotype–phenotype relation.

When gene carriers are studied prospectively, biochemical evidence of neoplasia can be detected as early as 5–10 years before clinically evident disease, allowing for early surgical intervention and therefore dramatically reducing both the morbidity and the mortality related to the syndrome.

Surgery remains the main method of PHPT management in MEN1, despite the high recurrence rate with the need for repeat procedures. Some controversies surround the optimal surgical technique for MEN1 PHPT. The optimal timing for surgery remains controversial and must be assessed case by case without forgetting that the complications of PHPT in MEN1 are early in onset and more severe than in sporadic PHPT. Medical therapy with the goal of bone preservation is an option for patients without symptoms, and calcimimetics effectively normalize calcium levels only with specific indications.

often required with a consequent increase in surgical morbidity (18,32,33).

It should be noted that in the hands of an expert surgeon the rate of persistent disease in MEN1-associated PHPT is less than 20%, but this may rise to 40–60% for surgeons with less experience. For example, some MEN1 patients may have only a single macroscopically abnormal gland and might undergo inadequate surgery (34).

DISCUSSION The optimal treatment of PHPT in MEN1 is parathyroidectomy, as it reduces the risk of kidney stones, fractures (improved bone mineral density), and potential cardiovascular morbidity, improving quality of life and reducing gastrin production in MEN1 patients who also have a gastrinoma (35).

Some authors think that parathyroid surgery in patients with MEN1 should be thought of as a debulking or palliative procedure because long-term recurrence is inevitable. Surgery being indicated to treat and prevent the complications of hyperparathyroidism (24).

There are also disputes about surgical timing in patient with MEN1. The surgical indication for PHPT is obvious in patients with symptoms or with severe hypercalcemia; and in these circumstances there appears to be a good risk/ benefit balance. However, if MEN1 syndrome has not been diagnosed preoperatively and if an experienced surgeon is not available, then surgical treatment might not be appropriate (18).

In young patients with asymptomatic, mild hypercalcemia the timing of surgery is controversial, given that recurrence rates increase in relation to the follow-up time.

According to our experience, biochemical screening for hyperparathyroidism is recommended, as well as for the other MEN1-related tumors, and it should be performed regularly in carriers of an MEN1 mutation. This is because clinical manifestation can be mild for long periods of time, but a lack of regular screening may result in the delay of diagnosis and in the development of complications (36).

Total serum calcium concentration corrected for albumin level or ionized calcium fraction was proposed as a single sufficient screening test for hyperparathyroidism in MEN1 (37). However, increased PTH has been observed in MEN1 patients with mild PHPT even in the absence of hypercalcemia, making it necessary to include serum PTH concentration measurement in the screening program (38).

Imaging is not necessary for the diagnosis of parathyroid disease in MEN1 for known multiglandular involvement, but the presence of an experienced MEN1 surgeon is certainly recommended (14).

Assessment of bone mass loss by densitometry is essential in PHPT, mostly in MEN1 patients who have PHPT during the bone mass accretion period (26–31). Lourenc¸o et al. (39) clearly indicated that bone demineralization in MEN1 PHPT patients was early in onset, progressive, frequent, extensive, and severe. Surgery is the most effective way to preserve bone mass in patients with MEN1 PHPT (20).

Therefore, surgery is recommended in young asymptomatic patients in whom the serum calcium values are more than 1 mg/dl over the upper-normal limit and whose bone mineral density values are lower than –2.5 T-score.

Early parathyroidectomy predisposes the patient to an earlier recurrence of hyperparathyroidism and the possibility

ACKNOWLEDGMENTS This work was supported by F.I.R.M.O. Fondazione Raffaella Becagli (to MLB).

AUTHOR CONTRIBUTIONS All the authors were responsible for the manuscript writing.

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CLINICS 2012;67(S1):145-148

DOI:10.6061/clinics/2012(Sup01)24

REVIEW

Surgical treatment of pancreatic endocrine tumors in multiple endocrine neoplasia type 1 Marcel Cerqueira Cesar Machado Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Surgery, Sa˜o Paulo/SP, Brazil.

Surgical approaches to pancreatic endocrine tumors associated with multiple endocrine neoplasia type 1 may differ greatly from those applied to sporadic pancreatic endocrine tumors. Presurgical diagnosis of multiple endocrine neoplasia type 1 is therefore crucial to plan a proper intervention. Of note, hyperparathyroidism/multiple endocrine neoplasia type 1 should be surgically treated before pancreatic endocrine tumors/multiple endocrine neoplasia type 1 resection, apart from insulinoma. Non-functioning pancreatic endocrine tumors/multiple endocrine neoplasia type 1 .1 cm have a high risk of malignancy and should be treated by a pancreatic resection associated with lymphadenectomy. The vast majority of patients with gastrinoma/multiple endocrine neoplasia type 1 present with tumor lesions at the duodenum, so the surgery of choice is subtotal or total pancreatoduodenectomy followed by regional lymphadenectomy. The usual surgical treatment for insulinoma/multiple endocrine neoplasia type 1 is distal pancreatectomy up to the mesenteric vein with or without spleen preservation, associated with enucleation of tumor lesions in the pancreatic head. Surgical procedures for glucagonomas, somatostatinomas, and vipomas/ multiple endocrine neoplasia type 1 are similar to those applied to sporadic pancreatic endocrine tumors. Some of these surgical strategies for pancreatic endocrine tumors/multiple endocrine neoplasia type 1 still remain controversial as to their proper extension and timing. Furthermore, surgical resection of single hepatic metastasis secondary to pancreatic endocrine tumors/multiple endocrine neoplasia type 1 may be curative and even in multiple liver metastases surgical resection is possible. Hepatic trans-arterial chemo-embolization is usually associated with surgical resection. Liver transplantation may be needed for select cases. Finally, pre-surgical clinical and genetic diagnosis of multiple endocrine neoplasia type 1 syndrome and localization of multiple endocrine neoplasia type 1related tumors are crucial for determining the best surgical strategies in each individual case with pancreatic endocrine tumors.

KEYWORDS: MEN1; Pancreatic Tumors; Endocrine Tumors; Pancreatic Endocrine Tumors; Neuroendocrine Tumors.

Machado MCC. Surgical treatment of pancreatic endocrine tumors in multiple endocrine neoplasia type 1. Clinics. 2012;67(S1):145-148

E-mail: mccm37@uol.com.br Tel.: 55 11 3289-1188

INTRODUCTION

PETs/MEN1 VERSUS S-PETs The PETs associated with MEN1 (PETs/MEN1) may differ markedly from sporadic PETs (S-PETs) and are characterized by: (i) an earlier disease onset; (ii) a propensity to present higher risk for malignancy; (iii) multiple tumor lesions scattered throughout the pancreas and duodenum, instead of a single pancreatic nodule as in S-PETs; (iv) variable hormone production; (v) most patients having a familial history of PETs; and (vi) most cases (,90%) harboring an inactivating germline mutation in the MEN1 tumor suppressor gene (1). Based on the clinical differences between PETs/ MEN1 and S-PETs, distinct surgical approaches are used for each of these conditions.

Surgical treatment of tumor lesions in PETs/MEN1 may be controversial but its rationale is based on cure of the clinical syndrome and avoidance of malignant tumor progression (2).

In MEN1 patients, primary hyperparathyroidism is frequently the first clinical manifestation and is usually the first condition to be surgically treated. This was recently verified in a large MEN1 family caused by a founding MEN1 gene mutation (3,4). The reduction in calcium/parathyroid hormone serum levels after total parathyroidectomy followed by

Pancreatic neuroendocrine tumors (PETs) originate from pancreatic islet tissue. Pancreatic endocrine tumors may produce clinical symptoms as a result of excessive production of one or more hormones such as gastrin, insulin, somatostatin, glucagon, and vasoactive intestinal polypeptide (functioning PETs) or may be silent, producing only pancreatic polypeptides (non-functioning PETs).

Multiple endocrine neoplasia type 1 (MEN1) is a complex inherited condition, which may comprise up to 20 different types of endocrine and non-endocrine tumors, although the three most prevalent conditions are hyperparathyroidism, PETs, and pituitary tumors (1). MEN1 leads to increased morbidity and mortality rates, mainly as a result of PETs and thymic carcinoids (1,2).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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parathyroid auto-implant in patients with MEN1 is beneficial to the metabolic status, but it also leads to decreasing secretion of several hormones, such as gastrin, which in turn may cause a significant improvement of the clinical symptoms secondary to the hormonal excess (5–7). Parathyroidectomy in MEN1 should therefore be performed before operation for PETs, apart from in insulinoma.

As mentioned, surgical strategies applied to PETs/MEN1 differ greatly from the usual approaches used in patients with S-PETs. Clinical and genetic diagnosis of MEN1 should be made or ruled out before surgery in each patient with MEN1, as reported previously (3,8). This procedure will allow the identification of the best surgical approach to each individual case of PET, which may potentially lead to lower morbidity and mortality rates in patients with PETassociated MEN1.

We also performed methodical screening for the MEN1 tumor suppressor gene in our MEN1 families, which should allow the early diagnosis and treatment of MEN1 in relatives carrying MEN1 germline mutations and a trend towards lower morbidity rates (9).

NON-FUNCTIONING PETS In a recent study, Goudet et al. (12) verified that PETs, including the non-functioning PETs, and thymic carcinoids are frequent causes of death in patients with MEN1.

At diagnosis, non-functioning PETs are frequently malignant, usually larger and with a worse prognosis than functioning PETs. Tumor size was positively correlated with malignancy and lesions .2.0 cm have been reported to have a higher risk for malignancy (13). In addition, other authors showed that non-functioning PETs with diameters .1.0 cm are already prone to metastasize (14). Based on these findings, pancreatic resection associated with lymphadenectomy has been recommended in these cases (14). Moreover, as liver metastases are frequently found in PETs.1.0 cm, these tumors should be carefully investigated and operated on as soon as possible (12,14). Concordantly, extended distal pancreatectomy associated with enucleation of PETs.1 cm located at the pancreatic head has also been recommended, in an attempt to prevent liver metastases (15).

Conversely, total pancreatectomy, associated or not with duodenectomy, has not been indicated because of the possible decrease in the patient’s quality of life, although this surgical intervention may be used in selected PET/ MEN1 patients (16).

PREOPERATIVE EVALUATION Computed tomography, magnetic resonance imaging, and ultrasonography can miss PETs,1 cm. Octreotide scintigraphy is an efficient diagnostic tool but this depends on the size of the lesion and the presence of somatostatin receptors in the tumor cells. Recently, gallium-labeled somatostatin analog peptide has been used to detect MEN1-related tumors (10).

Endoscopic ultrasonography is the most useful tool for tumor diagnosis and localization in PETs/MEN1, as well as for depiction of the anatomic relationship with the main pancreatic duct, which is important for the proper surgical treatment. Endoscopic ultrasonography is able to detect PETs,1 cm, allowing early diagnosis (Figure 1). Invasive techniques for the diagnosis and localization of gastrinomas or insulinomas have been reported, although their accuracy has not been reproduced in all centers (11).

Furthermore, in our experience, intraoperative palpation, inspection, and ultrasonography are efficient tools to localize most lesions (11).

GASTRINOMAS The vast majority of sporadic gastrinomas are represented by single tumors located at the pancreas and the current surgical approach to these lesions is tumor enucleation.

Conversely, the majority of gastrinomas/MEN1 are multiple, asynchronic tumors mostly spread throughout the duodenum and less frequently found in the pancreas.

These frequently malignant tumors (,60%) are mostly associated with multiple and small gastric carcinoids (1,3,14).

Controversies related to the surgical approach to gastrinoma/MEN1 may exist and are mostly related to the timing and extension of the surgical procedure. These tumors usually have an unpredictable course, difficult preoperative localization and present as multiple duodenal tumors.

Although PETs usually lead to limited survival, some patients with metastatic PETs may survive for long periods of time with clinical treatment.

Some authors recommend surgical treatment only for cases with gastrinomas/MEN1 .3.0 cm, whereas others indicate an early surgical intervention for all gastrinoma/ MEN1 patients, as soon as the diagnosis is made (14,17–19).

This controversy may be related to the fact that there appear to be two different patterns of gastrinomas in MEN1 patients: the first tend to have an indolent course with or without metastasis, whereas the second pattern is characterized by rapid tumor progression. Although there are no definite markers for the adequate identification of these two potential gastrinoma subsets, the aggressiveness of these tumors could be evaluated by the following parameters: serum levels of gastrin, tumor histological differentiation, Ki-67 positivity, a high mitotic number, and the presence of progesterone receptors, as reported recently (19).

Pancreatoduodenectomy with regional lymphadenectomy Limited surgical resection with excision of duodenal tumors, excision of pancreatic cephalic lesions, and distal

Figure 1 - Endoscopic ultrasonography. Arrow shows a small pancreatic neuroendocrine tumor.

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pancreatectomy have been proposed as surgical alternatives for the treatment of gastrinomas/MEN1 (18). However, as reported in the literature and also supported by our own observations, such surgical approaches may be frequently followed by a low cure rate and a high recurrence rate.

Considering that most gastrinomas/MEN1 are located in the duodenum, a more radical surgical intervention is proposed for these patients including pancreatoduodenectomy followed by regional lymphadenectomy (17,20).

Pancreatoduodenectomy may be performed with pylorus preservation or including gastric resection (Whipple’s technique). It is important to actively search for the presence of duodenal tumors even in the first 1–2 cm of the duodenum. Despite the functional advantages of the pylorus-preserving technique, it may be safer to perform Whipple’s procedure in gastrinoma/MEN1 patients.

In some cases, total pancreatectomy is the treatment of choice, in an attempt at the complete removal of all tumor lesions (Figure 2). Radical resection may also be considered because the sensitivity of the methods used in detecting tumor lesions is low. Also, intraoperative gastrin measurements may be helpful to improve our capacity for determining the extent of resection (14).

Figure 3 - Distal pancreatectomy in a patient with insulinomas associated with multiple endocrine neoplasia type 1. Eleven small tumors were found. Serum glucose returned to normal after surgery.

pancreatic head, a pylorus-preserving pancreatoduodenectomy should be performed. Higher recurrence rates have been observed after surgical resection of insulinoma/ MEN1, compared with sporadic insulinomas (21–23).

The confirmation of complete tumor removal is of paramount importance. Intraoperative determinations of serum glucose and insulin levels for evaluating the presence of tumor tissue left behind have been reported but their accuracy is low.

In our experience, intraoperative ultrasonography associated with monitoring of serum levels of glucose/insulin is a useful tool to confirm the completeness of tumor resection (11).

INSULINOMAS For sporadic insulinomas the diagnosis, preoperative localization, and surgical approach are well established and surgery consists of tumor enucleation (11).

In insulinoma/MEN1, the surgical management is completely different because in this condition, there are multiple tumors spread throughout the pancreas, including tumors with potential for malignancy. Enucleation is not the best treatment for these lesions because high incidences of recurrence or persistence of hypoglycemia have been reported after simple tumor enucleation.

The current therapeutic surgery for insulinoma/MEN1 is distal pancreatectomy up to the mesenteric vein with or without spleen preservation, associated with enucleation of lesions located in the head of the pancreas (Figure 3). This procedure frequently has few postoperative complications (11,14). However, in cases with major involvement of the

GLUCAGONOMAS, SOMATOSTATINOMAS, AND VIPOMAS These three types of tumors occur rarely in MEN1 patients. Specific guidelines for the surgical treatment of these lesions are not currently available so the surgical procedures usually follow basically the same criteria used for non-functioning or functioning PETs described above.

Most of these tumors are malignant and metastatic lesions are usually present in PETs.3 cm. Radical surgical resection is the proposed treatment for these latter lesions.

HEPATIC METASTASES As stated by the National Comprehensive Cancer Network guideline and the consensus guidelines by the European NET Study Group: surgical treatment is the most effective therapeutic method with which to treat hepatic metastases from PETs/MEN1 (24–26).

Surgical resection of a single hepatic metastasis may be curative. Even in cases with multiple liver metastases, surgical resection is possible. In this latter situation, portal vein embolization is used as a strategy to increase the left lobe size and metastatic lesions can then be removed in a first-stage operation (Figure 4). This procedure is useful as cytoreductive surgery in patients with a clinical syndrome that is refractory to clinical treatment. Hepatic trans-arterial chemo-embolization can also be used as an ablation tool,

Figure 2 - Octroscan showing gastrinomas disseminated throughout the pancreas. Total pancreatectomy was performed in this case.

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9.

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11.

12.

13.

Figure 4 - Patient with bilateral liver metastases. After portal vein embolization of the right liver, an extended right hepatectomy was performed with enucleation of the left lobe metastases.

14.

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15.

CONCLUSION

16.

Surgical strategies applied to PETs/MEN1 may greatly differ from those used in patients with S-PETs. Clinical and genetic diagnosis of MEN1 should be performed or ruled out before surgery in all patients presenting with apparently sporadic PETs. The presurgical differential diagnosis between S-PETs and PETs/MEN1 will allow surgeons to choose the best surgical approach to each individual case, aiming to lower morbidity and mortality rates, or even obtain cure, in patients with PETs associated with MEN1.

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DOI:10.6061/clinics/2012(Sup01)25

REVIEW

Surgical approach to medullary thyroid carcinoma associated with multiple endocrine neoplasia type 2 Marcos R. Tavares,I Se´rgio P. A. Toledo,II Fa´bio L. M. Montenegro,I Raquel A. Moyses,I Rodrigo A. Toledo,II Tomoko Sekyia,II Claudio R. Cernea,I Lenine G. Branda˜oI I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Head and Neck Surgery Division, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Endocrine Division, Sa˜o Paulo/SP, Brazil.

We briefly review the surgical approaches to medullary thyroid carcinoma associated with multiple endocrine neoplasia type 2 (medullary thyroid carcinoma/multiple endocrine neoplasia type 2). The recommended surgical approaches are usually based on the age of the affected carrier/patient, tumor staging and the specific rearranged during transfection codon mutation. We have focused mainly on young children with no apparent disease who are carrying a germline rearranged during transfection mutation. Successful management of medullary thyroid carcinoma in these cases depends on early diagnosis and treatment. Total thyroidectomy should be performed before 6 months of age in infants carrying the rearranged during transfection 918 codon mutation, by the age of 3 years in rearranged during transfection 634 mutation carriers, at 5 years of age in carriers with level 3 risk rearranged during transfection mutations, and by the age of 10 years in level 4 risk rearranged during transfection mutations. Patients with thyroid tumor .5 mm detected by ultrasound, and basal calcitonin levels .40 pg/ml, frequently have cervical and upper mediastinal lymph node metastasis. In the latter patients, total thyroidectomy should be complemented by extensive lymph node dissection. Also, we briefly review our data from a large familial medullary thyroid carcinoma genealogy harboring a germline rearranged during transfection Cys620Arg mutation.

All 14 screened carriers of the rearranged during transfection Cys620Arg mutation who underwent total thyroidectomy before the age of 12 years presented persistently undetectable serum levels of calcitonin (,2 pg/ml) during the follow-up period of 2–6 years. Although it is recommended that preventive total thyroidectomy in rearranged during transfection codon 620 mutation carriers is performed before the age of 5 years, in this particular family the surgical intervention performed before the age of 12 years led to an apparent biochemical cure.

KEYWORDS: Carcinoma; Medullary; Multiple Endocrine Neoplasia; Surgery.

Tavares MR, Toledo SPA, Montenegro FLM, Moyses RA, Toledo RA, Sekyia T, et al. Surgical approach to medullary thyroid carcinoma associated with multiple endocrine neoplasia type 2. Clinics. 2012;67(S1):149-154.

E-mail: mtavares@apm.org.br Tel.: 55 11 3061-7000

The well-defined genetic origin of the condition and the possibility of prophylactic surgical treatment of MTC make it a model for early diagnosis, genetic counseling, surgical prevention and therapy, as well as cancer research (9,10).

This uncommon tumor has its origin in the thyroid C-cells and accounts for 4–5% of all thyroid cancer tumors in the USA, and it is the main cause of death in MEN2 patients (11,12). MTC may occur as a dominant inherited condition caused by a germline mutation in the RET gene in 20–30% of cases of MEN2, although S-MTC is the more prevalent form of the disease.

MTC presents different levels of aggressiveness: (a) it is extremely aggressive and usually occurs during the first year of life in MEN2B (MTC, hyperparathyroidism, pheochromocytoma, and mucosal neuromas); (b) it is highly aggressive in MEN2A (MTC, hyperparathyroidism, and pheochromocytoma) mostly in cases harboring a RET 634 codon mutation; and (c) it is often moderately aggressive in familial MTC (FMTC), as well as in S-MTC (13). Therefore, the behavior of MTC in MEN2 may present extreme variations, ranging from an indolent MTC, as frequently seen in cases harboring intracellular ‘‘weak’’ mutations, to

INTRODUCTION In the era of genomic medicine, multiple endocrine neoplasia (MEN) represents a model paradigm of the strong impact of genetic testing on clinical practice (1–4). An example of this is the impressive impact of mutation analysis of the RET proto-oncogene, which leads to MEN type 2 (MEN2A), on the day-to-day activity of clinicians, endocrinologists, neck surgeons, and urologists (5,6). As a result of this, RET germline mutation carriers detected at an early age are able to undergo preventive total thyroidectomy (TT), which has a high ‘‘cure’’ rate. This surgical approach may ultimately lead to prevention of highly malignant medullary thyroid carcinoma (MTC), either in its inherited or sporadic (S-MTC) form (5–8).

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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Recommended age for prophylactic thyroid surgery. The age at which TT in RET-positive mutation carriers is performed plays a crucial role in the success of MTC treatment. The recommended ages for TT in MEN2 are presently well defined and they are based on the strong genotype–phenotype correlation in MEN2 (1,5,15). Thus, surgery depends basically on the RET codon mutation involved, although the behavior of the disease in a specific family should also be considered in some cases.

At the Seventh International Workshop on MEN at the National Institutes of Health (NIH) (5), a classification of the risk of MTC was established based on the different degrees of MTC aggressiveness, which in turn depend directly on the codon in which the germline RET mutation has occurred. Briefly, this consensus recommended that carriers of mutations in extracellular RET codons, such as codons 634, 620, 618, and 609, should be operated on before the age of 5 years. Individuals carrying mutations in intracellular RET codons, such as codon 804, should undergo TT before the age of 10 years. Patients carrying some specific RET mutations, such as those in codons 790 and 791, may have the surgery postponed, and be observed under a strict follow-up. Carriers with a RET 918 mutation, which leads to MEN2B, should be operated on by 6 months of age. The purpose of this classification system was to offer a guideline to clinical endocrinologists and neck surgeons.

Recently, the American Thyroid Association (ATA) consensus on MTC added new recommendations (15).

Thus, individuals carrying the highly aggressive germline RET mutations in codon 634 should undergo preventive thyroid surgery before 5 years of age, possibly around 3 years of age, as MTC has been reported at ages as low as 3 years in RET 634 codon mutation carriers (10).

Several guidelines for the diagnosis and management of SMTC and MTC/MEN2 have been published by different medical authorities such as the International MEN2 Consortium (1), the NIH Workshop on MEN (5), the American Society of Clinical Oncology (ASCO) (22), the ATA (MTC Clinical Guidelines) (15), and the Brazilian Guidelines-MTC (23), among others. The recommendations are broadly similar, although small variations may be present.

General comments. Ideally, all surgical thyroid intervention in MTC/MEN2 cases should be preventive.

However, in clinical practice, surgical interventions in MTC may vary from prophylactic to palliative approaches depending on the age at the MTC diagnosis and treatment, the disease stage and the RET codon mutation.

Also, as it is mandatory in all surgical thyroid intervention, a careful neck dissection is needed to avoid parathyroid function damage, which may ultimately lead to permanent hypoparathyroidism. Accordingly, special attention is necessary to protect the vascular supply of the parathyroid glands. Also, this would decrease the chance of cervical nerve lesion, which may lead to transient or permanent nerve palsy. It is worth noting that possible thyroid remnants left behind with thyroid C-cells during a TT procedure, such as remnants in the posterior thyroid capsule, may lead to persistent MTC, and may increase the chance for recurrent disease (Tavares et al., unpublished observations).

TT associated with removal of cervical and mediastinal lymph node metastases should be performed by skilled surgeons in tertiary centers with expertise in the management of MTC cases.

highly aggressive MTC in cases with RET mutations in codon 634 and mainly in codon 918 (5,14,15).

Lymph node metastasis in the neck and upper mediastinum is a frequent finding in MTC cases that are diagnosed late, and metastases are present in about 70% of MTC patients who have a palpable thyroid nodule (.1.0 cm diameter). At this stage of the disease, biochemical ‘‘cure’’ rates drop to 30% or less (16,17). Thus, in RET mutation carriers, all efforts should be focused on early diagnosis and surgical treatment of MTC (5,14,15). The only effective treatment for this condition is TT, although several drugs acting on the RET receptor protein kinase have been tested in clinical trials (18).

Calcitonin measurements Calcitonin (Ct) is produced by thyroid C-cells and is the tumor marker for MTC. Currently, basal serum Ct measurements and the calcium-stimulated Ct test have been mainly used during the post-surgical follow-up of cases of inherited MTC (5,17). The calcium-stimulated Ct test may be used to predict MTC in patients who present without a thyroid nodule at ultrasound and who have repeatedly normal basal Ct values (males, ,8.5 pg/ml; females, ,5.0 pg/ml). The higher risk of MTC in cases with abnormally high Ct levels after a calcium-stimulation test could further support an indication for prophylactic TT, possibly with dissection of the central compartment of the neck. However, some specialists argue that calcium-stimulated Ct testing is rarely needed in the diagnosis or follow-up of MTC, because new Ct assays have high sensitivity and specificity, and detection values are as low as 1–2 pg/ml (15).

Conversely, Ct measurements may be highly helpful in the diagnosis of S-MTC cases, which are often diagnosed late. In this context, routine Ct measurements in patients with thyroid nodules may contribute to an earlier diagnosis of MTC, although the prevalence of MTC in thyroid nodules is about 1.0% (19,20).

Genetic testing Genetic RET testing should be offered to all patients with MTC (6), since between 1% and 7% of apparently sporadic MTC cases harbor a germline RET mutation, and thus need to be reclassified as inherited cases. Also, genetic RET testing should be offered to all at-risk (50% risk) first-degree relatives of patients with familial MTC, in order to disclose potential carriers with positive mutations (1,5). Further to this, genetic testing should be offered to all cases with phaeochromocytoma, and possibly to patients with hyperparathyroidism who are younger than 30 years of age (5,21).

In at-risk relatives, genetic testing for MTC predisposition should be performed at a very early age, as prophylactic surgical intervention is able to prevent MTC in the vast majority, but not all, of the RET-positive mutation carriers who are below the age of 3–5 years (10).

Genetic RET testing is usually based on mutation analysis of seven hot-spot exons: 8, 10, 11, 13, 14, 15, and 16. It allows a genetic diagnosis at the pre-neoplastic early stage of MTC, which corresponds to thyroid C-cell hyperplasia (5,15).

Surgical approach Our main aim is to focus on recommended surgical approaches in young children carrying a germline RET mutation.

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pole of the thyroid gland and above the brachiocephalic vein and aortic arch (Figure 1). Surgical access to level VII without splitting the manubrium sternum is laborious, as it is a narrow cavity containing important vessels and other anatomical structures and also some parathyroid glands (26).

We start the central neck dissection by identifying the left common carotid artery at the level of the hyoid bone. It proceeds caudally on the anteromedial rim of the carotid artery until it reaches the superior border of the left innominate vein. From this point on, dissection goes horizontally toward the right side following the superior border of the vein, until it comes to the innominate artery.

Dissection follows cranially in the anterior wall of the brachiocephalic and right carotid arteries and finishes at the level of the hyoid bone at the right side of the neck. Lymph nodes are removed en bloc in the level of the prevertebral fascia, with partial resection of the upper part of the thymus.

Recurrent laryngeal nerves and parathyroid glands have been identified previously during TT, and these should be preserved.

TT associated with elective level VI central neck compartment dissection is recommended in MEN2B cases harboring the RET 918 mutation presenting with Ct .40 pg/ml, or evidence of lymph node metastasis by ultrasound, even when TT is to be performed during the first year of life. TT associated with level VI central neck compartment dissection is also recommended in MEN2A/FMTC cases before the age of 5 years with Ct .40 pg/ml or there is evidence of lymph node metastasis.

TT complemented by prophylactic dissection of the central neck may potentially avoid the risk of reoperation of this neck compartment (5,15). This procedure is recommended for most RET-positive carriers older than 5 years who have a thyroid nodule ,5 mm observed by ultrasound, but with Ct levels .40 pg/ml. The selection of patients to avoid dissection of the central neck is based on the increased risk for postsurgical hypoparathyroidism, a complication with harmful long-term consequences in children. Thus, the surgeon must pay special attention to parathyroid glands and parathyroid vasculature during central neck intervention.

Lateral neck dissection. Complimentary dissection of the lateral neck is performed when jugulo-carotideal lymph node metastases are identified by neck ultrasound. Lymph nodes with a diameter .1.0 cm and round in shape are suspicious and must be investigated for MTC metastasis by

Previous investigation for pheochromocytoma is mandatory to avoid the potential risk of adrenal crisis during surgery of MTC, since pheochromocytoma usually occurs in 50% of MEN2A cases (5). Also, hyperparathyroidism needs to be investigated, since it affects 20–30% of MEN2A patients.

Prophylactic treatment. Familial genetic screening of the RET proto-oncogene frequently allows planned prophylactic surgery in MEN2 (15).

Total thyroidectomy only. TT has a minimal morbidity.

It is recommended for most children aged 3–5 years with RET-positive mutated MEN2A, and even beyond this age in those presenting with normal basal serum Ct (,10 pg/ml), no thyroid nodule (or nodules ,5 mm), or lymph node metastasis in either side of the neck detected by ultrasound.

TT involves the excision of all thyroid tissue, including the superior pole, isthmus, pyramidal remnants, posterior capsule, tubercle of Zuckerkandl, and thyrothymic thyroid rests (15).

In MEN2B, TT is recommended for children younger than 6 months of age with no evidence of disease or thyroid nodule .5 mm and no lymph node metastasis. However, MEN2B cases may present lymph node metastasis at a very young age. Thus, when TT is performed after the first year of life, it is usually associated with cervical lymph node dissection.

In patients with hereditary MTC, disease-free survival is best predicted by tumor, node, metastases (TNM) staging and a preoperative basal Ct level below 30 pg/ml. Basal Ct, class D genotype, and age constitute the key determinants in deciding the timing of preoperative surgery (24).

TT and dissection of the central compartment of the neck (levels VI and VII). The central neck compartment lies in

and near the median sagittal plane of the neck and comprises lymph nodes in the pre- and paratracheal area, and precricoid (Delphian) and perithyroidal areas. The central neck is divided into sublevels (25,26) that comprise the following: the prelaryngeal lymph nodes, between the hyoid bone and the superior margin of isthmus of the thyroid gland; the region lateral to the right and left recurrent laryngeal nerve; the pretracheal sublevel, anterior to the trachea from the inferior pole of the thyroid gland, and above the brachiocephalic vein and aortic arch, which includes level VII of the neck. There are few anatomical landmarks and limited appropriate functional studies to individualize the group of lymph nodes located between the recurrent laryngeal nerves below the inferior

Figure 1 - Sublevels of the central compartment of the neck. Spaces of the central compartment of the neck: prelaryngeal (1); lateral to the recurrent laryngeal nerves on the right (2) and left (3) sides; pretracheal, between the recurrent laryngeal nerves (4).

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members, it was shown that the chance of achieving a biochemical cure in MTC is clearly dependent on the size of the primary tumor.

Conversely, in one series of children with MEN2A and FMTC who underwent prophylactic TT at age 4 years or over, 11% experienced biochemical persistence or recurrence, and all of them were operated on at age 13 years or above (12).

MTC diagnosed during thyroid surgery. TT is adequate when MTC with a diameter ,5 mm is identified by frozen section during surgery for a thyroid nodule. Central neck dissection and examination of both lateral compartments of the neck are necessary only if a positive lymph node is detected. Lymph nodes .1 cm diameter, round in shape and/or with a hard consistency are considered suspicious, and must be excised and subjected to frozen section. Neck dissection of levels II–V is mandatory in the side of the neck with the positive lymph node. Lateral neck dissection is indicated only for disease that has been detected. Although comprehensive modified neck dissection of levels II–V is the best treatment, selective dissection of the compromised level may be considered for these patients. Elective dissection is performed for MTC with a diameter .5 mm. In an attempt to detect S-MTC or the index cases of MTC/MEN2 families before surgery, the European Thyroid Association (ETA) recommends that serum Ct should be routinely measured in patients with thyroid nodules and clinical risk factors for MTC (20). This procedure can benefit some people, as they are able to better plan their surgery.

MTC diagnosed after thyroid surgery. In approximately 10–15% of cases of MTC, diagnosis is obtained by pathological analysis after thyroidectomy for a thyroid nodule (29). Since neck dissection is performed for positive or suspicious lymph nodes, and also in well-differentiated thyroid carcinomas, reoperation should not be the first step considered in this difficult situation. Investigation should begin with serum basal Ct level, neck ultrasound, and analysis of RET proto-oncogene mutation. A normal Ct level indicates that there is a very low chance of residual MTC, and no other treatment is necessary. Standard follow-up is sufficient in these cases.

Elevated postsurgical basal Ct levels indicate persistent disease (15), and there is a trend for Ct levels to correlate with disease stages (17). Tiny thyroid tissue amounts left behind, most frequently undetectable, can result in Ct levels ,40 pg/ml. Ct levels .400 pg/ml probably indicate distant metastases (15).

A diligent search for metastases in the neck, mediastinum, bones, lung, and liver is accomplished by ultrasound and computed tomography (CT) of these segments. Suspicious nodules should be further examined by ultrasound or CTguided fine needle aspiration biopsy with cytological study.

Immunocytochemistry analysis for Ct, and Ct measurements in the washout, are the next steps to be considered in cases with a negative cytology sample. Patients whose examinations are normal are carefully followed up with basal Ct and neck ultrasound. If a rise in Ct is noticed, a further CT is helpful to establish the disease staging (15).

Parathyroid glands. High priority must be given to preserving parathyroid function (15). Normal parathyroid glands should be left in situ with an adequate vascular pedicle whenever possible. Accidentally resected or devascularized normal parathyroid glands must be immediately autografted in the neck of MTC patients who

a performing cytology. It is important to emphasize that lymph nodes with diameters ,1.0 cm may already have minimal MTC metastases and cytological accuracy may be not sufficiently high to detect these (8). Thus, a negative cytology result should be followed by immunocytochemistry for Ct and measurement of Ct levels in the washout obtained using a fine needle aspiration biopsy.

Standard modified neck dissection preserving the sternocleidomastoid muscle, jugular vein, and accessory nerve is the best approach if there has been no previous neck dissection. Elective dissection of the lateral compartment of the neck is not indicated for MTC, unless follow-up is not affordable. The low cure rate obtained with the extended neck procedure is attributed to distant metastases.

Moreover, dissection of the lateral neck can be safely performed a second time, if necessary.

Surgery for advanced disease. In MTC with a palpable thyroid nodule .1.0 cm, central compartment, bilateral and/or contralateral lymph node metastases are present in up to 50–70% of cases (5). In most cases, TT followed by dissection of central and both lateral neck compartments are indicated. This procedure frequently takes a long time and should be carried out carefully.

Thus, the decision to perform routine, extensive neck lymph node dissection should be taken ideally after confirmation of lymph node disease.

It is worthwhile mentioning that thyroid surgery is the only current potentially curative therapeutic method for MTC, although new drugs acting upon the RET protein kinase receptor have been actively investigated (18).

Persistent and recurrent disease. Persistent or recurrent MTC disease has been reported in up to 2–10% of young patients after prophylactic TT, mostly in cases that had operations after the ages of 8–13 years (12,27,28). In agreement with this, there was no persistent or recurrent MTC in a series of 50 children who underwent prophylactic TT and central neck dissection before the age 8 years (27).

In an MTC series, tumors up to 5 mm were found in seven of eight children, aged from 3 to 13 years, with a RET 634 codon mutation. After TT, a 3-year-old boy developed hypoparathyroidism, a condition that that requires ongoing calcium intake and also treatment with calcitriol; this condition has also occurred in 30% of our adult MTC/ MEN2 patients after TT (Tavares et al., unpublished data).

Documented loco-regional recurrences can be treated by reoperation of the central neck compartment and/or selective neck dissection of the compromised lateral neck lymph node echelons. Selective dissection is preferred to extensive neck lymph node dissection, based on the high probability that there are distant metastases in these cases, and these render elective lymph node resection ineffective.

Isolated distant metastasis can be treated by surgery, and radiation therapy and chemotherapy are used as palliative measures. New drugs targeting the RET tyrosine kinase receptor are in phase III clinical trials, and they have shown good initial results (18).

Remission. There was no persistent or recurrent MTC in a series of 50 children when they underwent prophylactic thyroidectomy and central neck dissection before age 8 years (27). In a large series, Pelizzo et al. reported that all patients undergoing TT at stages I and II were totally cured (13). Also, in a family with FMTC resulting from the Cys620Arg mutation, all patients undergoing TT below or at 12 years of age were cured biochemically (28). In family

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are RET negative, and in MEN2B and FMTC patients. In contrast, in patients with MEN2A, the parathyroid glands should be routinely autografted to a heterotopic site in the neck or forearm. Furthermore, the location for implantation of the excised parathyroid tissue is determined by the type of RET mutation present. Patients with a strong family history of HPT or those harboring a RET mutation associated with an increased risk of HPT (i.e., at codon 634) should have the parathyroid implant placed in the forearm. If the MTC patient has MEN2B, FMTC, or a mutation with a low risk of HPT, the graft can be placed in the sternocleidomastoid muscle (23).

Surgical neck exploration to remove a parathyroid adenoma is indicated only after secure localization of the enlarged gland. Normal glands are left in situ. Excision of the four glands is indicated only if all glands are clearly abnormal.

Prophylactic treatment of HPT is not usually performed in all academic centers, as HPT is absent in cases of FMTC and MEN2B, although it occurs in 20–30% of MEN2A patients. Moreover, HPT associated with MEN2A frequently presents as a single parathyroid adenoma that, in many instances, can be localized by image studies such as ultrasound and/or sestamibi scintigraphy (5).

Follow-up. Long-term postoperative follow-up is recommended in MTC, either to confirm biochemical cure or monitor the patient for persistent or recurrent MTC disease.

Due to the high sensitivity and specificity of the methods for Ct measurements, persistently undetectable basal levels of Ct (,2 pg/ml) indicate absence or remission of MTC, whereas detectable Ct levels usually indicate the presence of MTC disease.

However, up to 5% of MTC cases with undetectable basal Ct levels who undergo TT may show abnormally high calcium-stimulated Ct values (.80 pg/ml). These latter cases cannot be considered to be cured, and they should be followed up carefully. Accordingly, recurrence may occasionally occur 5–7 years after the initial surgical treatment, even though there was a prolonged period in which Ct remained undetectable (,2 pg/ml) (5).

During follow-up, a neck ultrasound can identify suspicious lymph node metastases. A negative ultrasound eliminates the risk of neck metastasis, and indicates that a search for distant metastases may be needed. It is worthwhile pointing out that several conditions other than MTC may be associated with increased Ct levels, and these conditions should be ruled out (19).

As there is a correlation between basal Ct levels and MTC disease staging, chest and abdominal CT are indicated to search for distant metastases in cases with basal Ct levels .400 pg/ml. MTC disease may be frequently difficult to locate, and all suspicious lymph nodes should be routinely sampled by fine-needle aspiration biopsy followed by immunocytochemistry analysis for Ct. The complete set of recommended investigations is given in the ATA guidelines (15).

Genetics Unit in the Department of Endocrinology began in the early 1990s (30,31) and has continued up to the present day (28,36). Here, we would like to briefly mention our observation in a large family with FMTC from the north-east area Brazil, with more than 35 affected individuals harboring the germline heterozygous RET Cys620Arg mutation. So far, in this family, we have obtained persistent biochemical remission (Ct ,2 pg/ml) in 14 RET-positive carriers who underwent TT before or at 12 years of age, although RET 620 codon mutation carriers should be ideally undergo TT by the age of 5 years (5,15). Partial data obtained from this family have been published previously, as part of an international consortium on RET exon 10 mutation (28).

In conclusion, we have briefly reviewed some specific aspects of the surgical approaches to MTC, mostly those recommended for young asymptomatic children carrying a germline RET mutation.

ACKNOWLEDGMENTS FUNDING: SPAT is a recipient of a CNPq fellowship (401990/2010-9), TS is a recipient of a FAPESP doctoral fellowship (11942/2009), and RAT is a recipient of FAPESP post-doctoral fellowship (2009/15386-6).

AUTHOR CONTRIBUTIONS Tavares MR is the main author and was responsible for the treatment of patients, manuscript revision and manuscript writing. Toledo SPA was responsible for the diagnoses of patients, manuscript writing and revision, bibliography and translation. Montenegro FLM, Moyses RA, Toledo RA were responsible for the manuscript revision and bibliography. Cernea CR was responsible for manuscript revision, bibliography and translation revision. Branda˜o LG and Sekyia T were responsible for the manuscript revision.

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CLINICS 2012;67(S1):155-160

DOI:10.6061/clinics/2012(Sup01)26

REVIEW

Surgical approach in patients with hyperparathyroidism in multiple endocrine neoplasia type 1: total versus partial parathyroidectomy Francesco Tonelli,I Francesco Giudici,I Tiziana Cavalli,I Maria Luisa BrandiII I

University of Florence, Department of Clinical Physiopathology, Surgical Unit, Florence, Italy. II University of Florence, Department of Internal Medicine, Florence, Italy.

Usually, primary hyperparathyroidism is the first endocrinopathy to be diagnosed in patients with multiple endocrine neoplasia type 1, and is also the most common one. The timing of the surgery and strategy in multiple endocrine neoplasia type 1/hyperparathyroidism are still under debate. The aims of surgery are to: 1) correct hypercalcemia, thus preventing persistent or recurrent hyperparathyroidism; 2) avoid persistent hypoparathyroidism; and 3) facilitate the surgical treatment of possible recurrences. Currently, two types of surgical approach are indicated: 1) subtotal parathyroidectomy with removal of at least 3–3K glands; and 2) total parathyroidectomy with grafting of autologous parathyroid tissue. Transcervical thymectomy must be performed with both of these procedures. Unsuccessful surgical treatment of hyperparathyroidism is more frequently observed in multiple endocrine neoplasia type 1 than in sporadic hyperparathyroidism. The recurrence rate is strongly influenced by: 1) the lack of a pre-operative multiple endocrine neoplasia type 1 diagnosis; 2) the surgeon’s experience; 3) the timing of surgery; 4) the possibility of performing intra-operative confirmation (histologic examination, rapid parathyroid hormone assay) of the curative potential of the surgical procedure; and, 5) the surgical strategy. Persistent hyperparathyroidism seems to be more frequent after subtotal parathyroidectomy than after total parathyroidectomy with autologous graft of parathyroid tissue. Conversely, recurrent hyperparathyroidism has a similar frequency in the two surgical strategies. To plan further operations, it is very helpful to know all the available data about previous surgery and to undertake accurate identification of the site of recurrence.

KEYWORDS: Primary hyperparathyroidism; MEN1; Surgery; Total parathyroidectomy; Subtotal parathyroidectomy.

Tonelli F, Giudici F, Cavalli T, Brandi ML. Surgical approach in patients with hyperparathyroidism in multiple endocrine neoplasia type 1: total versus partial parathyroidectomy. Clinics. 2012;67(S1):155-160.

E-mail: francesco.tonelli@unifi.it Tel.: +39-055-7947559

However, controlled clinical trials comparing SPTX and TPTX are lacking. Review of the published surgical data shows that persistent HPT is more frequent after SPTX than after TPTX, and recurrent hyperparathyroidism has a similar percentage in both surgical options. The recurrence rate is strongly influenced by the lack of a pre-operative diagnosis of MEN1, the surgeon’s experience, the timing of the surgery, and the possibility of performing intraoperative confirmation [histologic examination and rapid parathyroid hormone (PTH) assay] of the potential curative capacity of the surgical procedure. To plan further operations, it is very helpful to have all the available data about previous surgery and to undertake accurate identification of the possible site of the recurrence.

Penetrance in HPT/MEN1 is nearly 100% at the age of 50 years; the age of onset of biochemical alteration is usually 20–25 years (2).

As in sporadic HPT, diagnosis is usually prompted by the presence of hypercalcemia, with elevated serum levels of ionized calcium and PTH. Parathyroid gland involvement in MEN1 is asynchronous and asymmetrical; the change in volume varies, but the maximum/minimum mean volume ratio is 9.6 (3). Macroscopically normal parathyroid glands

INTRODUCTION Usually, primary hyperparathyroidism (HPT) is the first endocrinopathy to be diagnosed in patients with multiple endocrine neoplasia type 1 (MEN1), and is the most common one (1). The optimal surgical timing and strategy in HPT/MEN1 are still under debate. The aims of surgery are to: correct hypercalcemia, and thus prevent persistent or recurrent hyperparathyroidism; avoid persistent hypoparathyroidism; and facilitate the surgical treatment of possible recurrences. Currently, two types of surgical approaches are indicated: subtotal parathyroidectomy (SPTX) with removal of at least 3–3.5 glands; and total parathyroidectomy with autologous graft of parathyroid tissue (TPTX). Transcervical thymectomy must be performed with both procedures.

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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Less than subtotal parathyroidectomy, with removal of enlarged glands only, is associated with a high persistence/ recurrence rate, so it is no longer used for diagnosed MEN1HPT. Therefore, two surgical approaches are in routine use: SPTX, with removal of at least 3–3.5 glands, or TPTX. Transcervical thymectomy must be performed with both procedures as the thymus can harbor an ectopic parathyroid gland; thymectomy is also helpful in preventing thymic carcinoids possibly arising during the evolution of the syndrome.

Subtotal parathyroidectomy (SPTX). SPTX is defined as the removal of three glands and part of the fourth (usually the least involved one), preserving not more than 50 mg of parathyroid tissue. The fourth gland can also be completely preserved if normal in volume. SPTX is used with the main aim of avoiding permanent hypoparathyroidism and also of reducing the risk of early postoperative hypocalcemia.

SPTX becomes the surgical choice when a fourth parathyroid cannot be found out during surgery. This occurs in up to 20% of cases, even when treatment occurs in specialized centers (14,15). Nevertheless, there are many criticisms leveled at SPTX as, in most cases, all the parathyroid glands are found to be pathologic and it is difficult to establish which of them is less involved and can be preserved.

Furthermore, it can be difficult to preserve enough parathyroid tissue, particularly in the case of small glands.

During this maneuver, the fine parathyroid vessels can easily be damaged, with consequent permanent hypoparathyroidism. In addition, surgery on these potentially pathologic glands could expose the patient to the risk of favoring the seeding of parathyroid cells into the nearest structures, thus favoring cervical recurrence of HPT (16).

All these considerations may explain the poor outcomes shown in several published studies, with high rates either of persistent and early recurrent HPT or of hypoparathyroidism (Table 1) (4,5,13–15,17–29).

make up 12–55% of all glands; sometimes the intra-operative findings may consist of just one or two enlarged glands (4–6).

Moreover, the frequency of supernumerary glands (up to 20% in HPT/MEN1) (6,7) and of ectopic glands (often intrathymic, within the thyroid gland, in the anterior mediastinum) is higher than expected (5,8). Some reports describe the presence of parathyroid nests (embryonic parathyroid rests, a direct result of ontogenesis) embedded within the fatty tissue surrounding the trachea, esophagus, and carotid artery (8–11). Genetic testing is the gold standard to confirm diagnosis of MEN1, and should be performed in any young patient with hyperparathyroidism, even in the absence of a clear familial history of MEN1 syndrome.

Timing of surgery The optimal timing for surgery in HPT/MEN1 is still under debate. It is possible to follow the same indications used for sporadic HPT, taking into account that cases of HPT/MEN1 often have only mildly elevated calcium and PTH levels. However, even when MEN1-related HPT has a long asymptomatic phase, it is important to note that it is the cause of a detectable bone mass decrease at a young age (12). Correction of hypercalcemia by prompt intervention is helpful in patients who show a decrease in bone mineral density, as it can prevent renal complications and may be of benefit for other concomitant endocrinopathies, such as hypergastrinemia (13).

In the case of concomitant multiple endocrinopathies in patients with HPT/MEN1, there is agreement among authors on the need to treat the HPT first, with the exception of cases of insulinoma.

Surgical strategy The aims of surgery are to correct hypercalcemia, preventing persistent or recurrent HPT; avoid persistent hypoparathyroidism; and facilitate the surgical treatment of possible recurrences.

Table 1 - Results after subtotal parathyroidectomy (SPTX) in multiple endocrine neoplasia type 1 (MEN1)hyperparathyroidism (HPT).

Author

Year

Period

Pts

Mean follow-up (yrs)

Persistent HPT (%)

Recurrent HPT (%)

Hypoparathyroidism (%)

Edis et al. (23) Prinz et al. (24) Van Heerden et al. (25) Goretzki et al. (26) Hellman et al. (17) Kraimps et al. (5) O’ Riordain et al. (15) Janson et al. (18) Thompson et al. (27) Grant at al. (28) Nilsson et al. (19) Hellman et al. (20) Goudet et al. (14) Dotzenrath et al. (21) Arnalsteen et al. (4) Elaraj et al. (22) Hubbard et al. (29) Norton et al. (13)

1979 1981 1983 1991 1992 1992 1993 1994 1994 1994 1994 1998 2001 2001 2002 2003 2006 2008

1959–76 1955–76 1960–83 1986–90 1982–91 1966–88 1970–91 1971–92 1972–92 1980–93 1971–92 1969–96 1986–97 1986–98 1992–01 1960–02 1974–02 1970–05

55 12 45 18 11 14 54 4 14 15 2 9 73 25 66 63 21 41

3.9 9.5 N.A. N.A. 11.9 8 10 9.9 20 4.7 9 7.3 N.A. 10 10 10 5 7.9

13 33 6.6 11 0 14 0 0 7 0 0 22 16.8 N.A. N.A. N.A. 0 12

0 0 6.6 0 27.3 36 16.4* 25 7 13.3 0 44 N.A. 8* 33* 51* 5 44

35 25 13 0 27.3 10 8 0 0 0 0 0 N.A. 12 12.7 26 10 10

*

Actuarial estimation.

N.A. = not applicable.

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Table 2 - Results after total parathyroidectomy (TPTX) in multiple endocrine neoplasia type 1 (MEN1)hyperparathyroidism (HPT).

Author

Year

Period

Pts

Mean follow-up (yrs)

Wells et al. (31) Malmaeus et al. (36) Hellman et al. (17) Janson et al. (18) Dralle et al. (35) Nilsson et al. (19) Hellmann et al. (20) Elaraj et al. (22) Hubbard et al. (29) Norton et al. (13) Tonelli et al. (6)

1980 1986 1992 1994 1994 1994 1998 2003 2006 2008 2007

1973–80 1961–85 1982–91 1971–92 1976–92 1971–92 1969–96 1960–02 1974–02 1970–05 1990–06

36 18 23 6 4 6 15 16 4 9 45

7 6.5 6.1 9.9 6.3 9 10.2 10 14 9.9 6.5

Persistent HPT (%)

Recurrent HPT (%)

Hypoparathyroidism (%)

3 0 0 0 0 0 0 N.A. 0 0 0

30 0 22 0 0 0 20 16* 50 55 11

5.6 26 30 0 50 0 47 46 25 22 22

* Actuarial estimation.

N.A. = not applicable.

Total parathyroidectomy (TPTX). TPTX consists of total parathyroidectomy with transcervical thymectomy and autologous transplantation of parathyroid tissue. The aim of this technique is to radically remove all the parathyroid tissue, including the occult gland potentially present within the thymus and the surrounding fatty tissues, to avoid cervical recurrences that are difficult to treat, and to reduce the risk of permanent hypoparathyroidism by means of a graft, usually in the nondominant forearm.

The graft can be obtained at the end of surgery from fresh autologous tissue preserved at 4 ˚C in Ringer lactate solution or days later (once it has been ascertained that the HPT has been cured) using cryopreserved tissue, using the technique described by Sonoda et al. (30) and Wells et al.

(31). The tissue to graft should be chosen from the gland that appears most normal in volume, colour, and texture after analysis of frozen sections. The graft fragments should be the smallest possible (around 1 mm3) to facilitate the graft implant (32). The optimal grafting site is generally the brachio-radialis muscle of the nondominant forearm. This procedure will allow monitoring of the success of the autotransplantation through bilateral blood sampling from each basilic vein for measurement of PTH levels. After incision of the fascia of the brachio-radialis muscle, its fibers are bluntly divided in order to create small spaces in which to embed 2–3 fragments of parathyroid tissue. The fascia is then sutured with nonabsorbable wires to mark the site. The number of fragments that should be grafted is

still under debate, with various recommendations in the medical literature. Commonly 20–25 fragments (or more in the case of cryopreserved tissue) are considered the optimal number (33). Other series report 5–10 transplanted fragments (23,24), but with higher rates (50–60%) of nonworking grafts, either with fresh or cryopreserved parathyroid tissue (Table 2) (13,17–20,22,28–30,33,35,36). It is still unclear whether the success rate is associated with the technical procedure or with the parathyroid histopathology.

Interestingly, in the study by Janson and Tisell, no permanent hypoparathyroidism was seen and rapid parathyroid function restoration was observed after grafting parathyroid tissue within the abdominal subcutaneous fat in seven patients with MEN1 (18). In the past few years our center has moved towards implantation of eight fresh fragments instead of more than 12. This choice did not increase the number of cases of permanent hypoparathyroidism. We also implanted the autograft in the subcutaneous tissue of the nondominant forearm with the same optimal results. These data support the importance of local factors (i.e. angiogenesis) for the successful implantation of the graft (Table 2) (13,17–20,22,28,30,35,36).

Cause of failure of treatment of MEN1-HPT Unsuccessful surgical treatment of HPT is more frequently observed in MEN1 than in sporadic HPT. Persistent hypoparathyroidism seems to be more frequent after SPTX than after TPTX (9% vs. 0.03%), and the cause may be related to hyperfunctioning parathyroid tissue in the portion of the gland preserved during SPTX. Conversely, recurrent hyperparathyroidism has a similar percentage in the two surgical strategies (18.6% vs. 18.5%); it is strongly dependent on the length of follow-up, and the cause may be related to the hyperfunctioning of the tissue if an excessive amount has been left in place after SPTX, or to the presence of supernumerary glands in cases treated with TPTX. There is no evidence in the literature of persistent or recurrent HPT/MEN1 cases caused by parathyreomatosis, although such cases have been reported in sporadic primary HPT (1– 2%) and secondary HPT (12%) (37–39).

The recurrence rate is strongly influenced by: 1. Pre-operative diagnosis of MEN 1 significantly reduces the rate of surgical failures (5,40) and pre-operative

Table 3 - Intraoperative parathyroid hormone (PTH) monitoring for prediction of multi-glandular parathyroid disease after removal of the first pathological parathyroid gland.

Author Clerici et al. (44) Jaskowiak et al. (45) Kivlen et al. (43){ Arnalsteen et al. (4){ Thompson et al. (46)

Decrease of intra-operative PTH from baseline

False positive percentage*

.50% at 109 .50% at 109 .50% at 109 .50% at 59 .70% at 209

75% 50% 14% 5% 0%

*

Decrease below the cut-off point in the presence of other enlarged gland/s. { Patients with MEN1 only.

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Figure 1 - Blood sampling after the removal of each parathyroid gland until the parathyroid hormone (PTH) values are practically undetectable in patients with HPT/MEN1 (6).

detection of a MEN1 gene mutation is essential when the clinical history is suggestive of MEN1.

2. HPT/MEN1 persistence rates correlate with the experience of the surgeon, being significantly lower for surgeons working centers with high referral rates of HPT (17).

3. The optimal timing of surgery in asymptomatic cases with mild hypercalcemia is still debated. In fact, postponing surgery for some patients with MEN1 may increase the chances of identifying all the involved parathyroid glands, as over time, they become enlarged, making surgery easier and more effective.

4. Intra-operative histologic examination is useful to confirm the presence of parathyroid tissue in the specimen, especially in doubtful cases when ectopic glands are found. Histologic confirmation may be of value when grafting fresh parathyroid tissue is programmed, making it possible to avoid grafting of fragments of tissue with pathologic histologic features.

5. Rapid intra-operative PTH measurement has become important in clinical practice to guide the surgery (41,42).

Its sensitivity is reported as nearly 95% in surgical treatment of sporadic adenoma (4,42,43). When used for the discrimination of HPT caused by hyperplasia or multiglandular disease, the rapid intra-operative PTH assay has shown substantially lower reliability, with a high rate of false positive values (43–45), probably due to the presence of glands suppressed (even if potentially neoplastic) by the most pathologic one. In fact, 10 minutes after removal of the first pathologic gland, PTH values can decrease by more than 50% from baseline. Using other criteria, such as evaluation after 20 minutes and a cut-off of 70%, Thompson et al. did not observe any false positive cases (Table 3) (46). In our opinion, the main role of intra-operative PTH should be to confirm removal of all the parathyroid tissue, so that the search for other parathyroid glands can be stopped and the graft of fresh parathyroid tissue performed. In our experience, stepwise venous sampling after the removal of each parathyroid gland until the PTH values are almost undetectable (i.e. near to the sensitivity limit of the PTH assay used) is a reliable method to confirm effective parathyroidectomy and to indicate that parathyroid autotransplantation can proceed (Figure 1) (6).

If a forearm graft is present, circulating PTH levels should be monitored after arm ischemia for 30 minutes produced by a tourniquet, in order to establish if such values decrease until they become undetectable (47). Re-operation is characterized by a substantial risk of inferior laryngeal nerve injury; therefore, it is indicated only when the recurrence is symptomatic, with high urinary calcium loss and significant bone mass reduction.

In the case of cervical or mediastinal recurrence, the surgical option is to remove the entire residual parathyroid tissue and then to perform heterotopic autologous transplantation. This technique carries a low risk of persistent HPT. After TPTX with autograft, recurrence rates of 38% and 36%, respectively, were reported by Helmann et al. (20) and Kivlen et al. (43). However, TPTX without autograft is a possible choice for these patients after adequate informed consent, as they will need lifelong substitutive therapy with calcium and vitamin D. If recurrence is diagnosed at the graft site, the removal of transplanted tissue is the treatment of choice, even if the best results cannot be always achieved; a 42% cure rate was reported by Kivlen et al. (43).

Recently, a pharmacologic approach was proposed for the cure of sporadic primary or secondary HPT, with the introduction of calcimimetics, a new class of drugs with the property of reducing PTH release (48,49). The efficacy of these molecules in HPT/MEN1, and particularly in its recurrence, needs to be proven through further studies and longer follow-up.

AUTHOR CONTRIBUTIONS Tonelli F is the principal author of the discussion about the best surgical approach for MEN1 patients. He operated and followed the clinical course of MEN1 patients affected by hyperparathyroidism treated in our Centre.

Giudici F has reviewed literature, analyzing the differences between different types of surgery in patients with hyperparathyroidism in MEN1in terms of results and complications. Cavalli T has reviewed the literature on this subject and has contributed to the drafting of the article. Brandi ML has reviewed literature, contributing to the Article and the accuracy of the English language; she carried out the clinical and metabolic follow up of all the patients operated in our Centre.

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DOI:10.6061/clinics/2012(Sup01)27

REVIEW

Retroperitoneoscopic adrenalectomy in pheochromocytoma Marcelo Hisano, Fabio Carvalho Vicentini, Miguel Srougi Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo.

Since the first laparoscopic adrenalectomy, the technique has evolved and it has become the standard of care for many adrenal diseases, including pheochromocytoma. Two laparoscopic accesses to the adrenal have been developed: transperitoneal and retroperitoneal. Retroperitoneoscopic adrenalectomy may be recommended for the treatment of pheochromocytoma with the same peri-operative outcomes of the transperitoneal approach because it allows direct access to the adrenal glands without increasing the operative risks. Although technically more demanding than the transperitoneal approach, retroperitoneoscopy can shorten the mean operative time, which is critical for cases with pheochromocytoma where minimizing the potential for intra-operative hemodynamic changes is essential. Blood loss and the convalescence time can be also shortened by this approach. There is no absolute indication for either the transperitoneal or retroperitoneal approach; however, the latter procedure may be the best option for patients who have undergone previous abdominal surgery and obese patients. Also, retroperitoneoscopic adrenalectomy is a good alternative for treating cases with inherited pheochromocytomas, such as multiple endocrine neoplasia type 2A, in which the pheochromocytoma is highly prevalent and frequently occurs bilaterally.

KEYWORDS: Adrenalectomy; Laparoscopy; Retroperitoneoscopy; Pheochromocytoma; MEN2A. Hisano M, Vicentini FC, Srougi M. Retroperitoneoscopic adrenalectomy in pheochromocytoma. Clinics. 2012;67(S1):161-167.

E-mail: mhisano@usp.br Tel.: 55 11 2661-0000

gained popularity because it allows direct access to the gland and avoids unexpected lesions to intra-abdominal organs. In some academic centers (18), it has become the surgery of choice for adrenalectomy, although it requires a strict working space and an unusual anatomical recognition compared with transperitoneal access.

Of note, retroperitoneal adrenalectomy may be especially useful for treating cases with inherited PHEO, especially those with multiple endocrine neoplasia type 2 (MEN2A) in which PHEO frequently occurs concomitantly with medullary thyroid carcinoma and primary hyperparathyroidism.

The prevalence of PHEO with MEN2A is 50% and frequently occurs bilaterally (50–60%), either synchronic or asynchronically (19). Recently, PHEO was reported to be highly prevalent in MEN2A patients harboring the double 634/791 mutation in the RET proto-oncogene (20).

In the present paper, we briefly described the revised retroperitoneoscopic adrenalectomy technique and the outcomes of adrenal lesions that have been operated on using this approach.

INTRODUCTION Pheochromocytoma (PHEO) is a neuroendocrine tumor that derives from chromaffin cells of the adrenal medulla (1). It is a challenging disease that can be cured by removing the affected adrenal gland (2). Open adrenalectomy has been the standard of care in this situation (1). Laparoscopic adrenalectomy was first described in 1992 by Gagner et al.

(3) using the transperitoneal approach. Since then, the procedure has been continuously developed and has become the gold standard of care (4–6). Laparoscopic adrenalectomy has evolved from the transperitoneal approach to the retroperitoneal approach, and even singlesite access and natural orifice transluminal endoscopic surgery (NOTES) have been developed (7–10).

Retroperitoneoscopy was first described in 1969 by Bartel (11) as a diagnostic tool. In the following years, it has evolved to a minimally invasive diagnostic procedure and finally became a useful route for operative procedures by the early 90s (12–15).

Retroperitoneoscopic adrenalectomy was developed in 1993 (16), initially only for small benign lesions and recently for lesions .5 cm and even malignancies (17). This technique

Surgical technique After confirming the diagnosis of PHEO, the patient needs to be clinically prepared for adrenalectomy in order to avoid the possibility of a dangerous intraoperative adrenal crisis. This preparation usually requires at least 4– 6 weeks of using either amlodipine, or preferentially prazosin, a selective a1-antagonist. Initially, 1 mg/day is given at bedtime for 10–15 days. Then, 1 mg of prazosin should be added for 7–10 days until a dose of approximately

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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half-jackknife position, with the hip, joints and knees bent at 75–90 ˚ (Figure 2). The aim of this position is to increase the space between the last rib and the iliac crest.

The first incision is usually made just below or at the tip of the last rib, and the retroperitoneal space is reached by sharp or blunt dissection of the abdominal wall. A retroperitoneal space can be created using the index finger or balloon dissection, and the latter option is more suitable for obese patients. Other ports can be placed under finger guidance or by retroperitoneoscopic direct vision. A Hasson-type trocar or a blunt-tip trocar (BLUNT TIPH, Covidien) with an inflatable balloon can be used during the first incision in order to avoid or minimize gas leakage. The distribution of the laparoscopic ports varies among different institutions, and the endoscopic angle depends on the preference of the surgeon. Care should be taken to avoid the subcostal nerves.

CO2 pressure can usually be maintained between 10 and 15 mmHg, although a CO2 pressure of 20–25 mmHg can be safely applied for patients in the prone position (30).

After reaching the retroperitoneum, the surgical tactics involve the identification of psoas muscle for special orientation and either vascular access to the adrenal gland by following the inferior vena cava (on the right side) or the renal vein (on the left side), or posterior access to the adrenal by opening Gerota’s fascia, mainly for the prone position. For the right side, the adrenal vein is usually situated posterolaterally to the inferior vena cava (Figure 3); for the left side, the adrenal vein is usually located on the upper surface of the renal vein (Figure 4). In case of PHEO, it is recommended to perform an early ligation of the main adrenal vein in order to minimize potential intraoperative hypertension due to catecholamine release during adrenal manipulation (31–33). Alternatively, some prefer to dissect the gland before adrenal vein ligation (34–36). The inferior and medial aspects of the gland are then dissected, and the small inferior and medial adrenal arteries and veins are separated by clips or sectioned using ultrasonic scissors (Figures 5 and 6). It is important to always avoid direct gland and tumor manipulation during the dissection. The superior aspect of the gland is dissected, followed by the lateral aspects, which are usually poorly vascularized. The specimen is extracted through the first incision using an appropriate retrieval bag system. Placing an aspirative drain for the retroperitoneal space is optional, and the trocars are removed under retroperitoneoscopic observation. The fascia of the port sites that are more than 10 mm are closed, followed by skin closure using an absorbable material.

10 mg is reached. Prazosin has a short effective lifespan and, thus, should be given two or three times a day. Also, the ingestion of large amounts of liquids (.2.5 L/day) and a salty diet in order to expand the extracellular volume and avoid possible postsurgical hypotension or even hypovolemic shock is recommended (21,22). b-adrenoceptor antagonists (e.g., propanolol) should be added only 2–3 days before surgery, if necessary (e.g., the occurrence of tachycardia) (1,2,23). Usually, computerized tomography or magnetic resonance imaging has been already performed during the diagnostic phase of PHEO and will be useful for choosing specific surgical techniques. For the success of the surgical procedure, it is crucial to obtain a careful surgical history of all PHEO patients who are candidates for retroperitoneoscopic adrenalectomy. The reason for this is because a previous retroperitoneal intervention may represent a relative contraindication for this procedure.

Conversely, previous upper abdominal surgery can make the procedure more attractive.

Surgery is performed with the patient under general anesthesia, with laryngeal intubation and assisted ventilation. A peripheral arterial line and a central venous catheter are inserted for cardiovascular control in all cases. An orogastric tube is routinely placed for the procedure only.

The bladder remains empty by using a Foley catheter ¨ SCHH, Teleflex).

(RU There are two basic positions that the patient should be placed into for a retroperitoneocopic adrenalectomy: the lateral posterior and the prone position (24–28), and the surgeon should choose which position he/she prefers. In the lateral position, the patient is placed in total lateral decubitus with hyperextension of the affected side by elevating the lumbar bridge of the table and straightening the ipsilateral lower extremity in order to increase the space between the 12th costal arch and the iliac crest (18,28,29) (Figure 1). In the prone position, the patient is placed in a

RESULTS There are few studies concerning the retroperitoneal approach to treat adrenal PHEO, although most of them are not exclusively related to PHEO cases. Earlier studies were based on case series to show the feasibility and safety of this type of treatment. Also, comparative studies of the transperitoneal and retroperitoneal routes, and between the open and retroperitoneal routes, are available, as commented below.

Clinical series Laparoscopy itself has been shown to be a safe procedure and does not increase the cardiovascular risk of treating patients with PHEO (37–39). Some studies do not report any difference between open and laparoscopic approaches in regards to intraoperative hemodynamic changes and

Figure 1 - Lateral position. The patient is placed in a hyperextension position. 1 – first incision is made below the last rib to create a retroperitoneal space; 2 – second port, 1 cm above the iliac crest, where the 0 ˚ endoscope enters; 3 – working port located 4 cm anterior to trocar number 2.

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Figure 2 - Prone position. The patient is placed in a half-jackknife position with the hip, joints and the knees bent 75–90 ˚. Trocar disposition and camera entrance are illustrated.

complications (37). Nau et al. (38) compared these parameters for the transperitoneal laparoscopic approach to treat PHEO and other adrenal diseases and detected only an increased use of antihypertensive drugs during the procedure for patients with PHEO (p,0.001). They did not find any other increased cardiovascular risk or complication in this group, even when considering the fact that PHEO patients present a significantly higher classification according to the American Society of Anesthesiologists (ASA) (p,0.001). It is important to stress that hypertension must be controlled before the procedure in order to achieve a positive clinical outcome.

Walz et al. described a series of 560 retroperitoneal adrenalectomies that were performed via the posterior approach; 119 of the procedures were for PHEO (30). The mean operative time of all of these cases was 67 min; the operative time for PHEO was significantly longer than for other adrenal diseases (80 vs. 62 min, respectively; p,0.001).

There was one conversion to the laparoscopic lateral approach due to severe obesity among the PHEO cases.

Overall, intraoperative complications were noticed in 4.2% of cases; severe bleeding that required a transfusion occurred in one patient who was undergoing a partial adrenalectomy. The main complication encountered during follow-up was hypoesthesia or relaxation of the abdominal wall in 8.5% of the 560 cases.

In 2007, Zhang et al. (18) published a series of 824 retroperitoneoscopic adrenalectomies that were performed via the lateral retroperitoneal approach, of which 62 of these procedures were for PHEO. In these cases, there was one conversion to open surgery (1.6%) due to large lesions and adhesions. The mean operative time for all of these cases was 45 min. No major bleeding or transfusion was reported.

A prospective study conducted by Walz et al. (39) treated both PHEO and paraganglioma patients. Of 161 treated tumors, 113 (70.2%) were unilateral PHEOs that were treated by retroperitoneoscopy. The median operative time in these cases was 82 min (range: 20–300 min); for the first half of these patients, the median time for the procedure was 110 min, but the second half required only 55 min (p,0.001).

Li et al. (36) described the largest retrospective series specifically for describing lateral retroperitoneoscopic adrenalectomy that included 131 procedures, including 11 for extra-adrenal PHEO. The authors divided their experiences into three phases and compared their laparoscopic data to

open-access cases. In the first phase, laparoscopy accounted for 37% of the cases, and in the third phase laparoscopy was responsible for 83% of the cases. The median operative times in the first, second, and third phases were 105, 85, and 75 min, respectively. Median blood loss in the same phases was 450, 140, and 70 mL, respectively. Fourteen patients (10.7%) had no complications and most of them (71.4%) were considered Clavien grade I complications.

Comparative Studies A non-randomized retrospective study comparing retroperitoneal access to the open approach for adrenal PHEO (40) reported statistical differences that favored the retroperitoneal access when comparing mean blood loss (140 vs.

592 ml, p,0.05), mean hospital stay (4.4 vs. 9.8 days, p,0.05), and analgesic requirements. The authors concluded that, although technically demanding, retroperitoneoscopic adrenalectomy is a good option for the treatment of PHEO. Other studies have compared the transperitoneal and retroperitoneal routes for adrenalectomy, but not specifically for PHEO cases. Baba et al. (41) compared three approaches for reaching the adrenal gland: transperitoneal, lateral retroperitoneal, and lumbar retroperitoneal access.

The lumbar approach demonstrated the shortest average operative time: 142 versus 194 min for the lateral retroperitoneal route and 252 min for the transperitoneal route (p,0.02). Of note, the first alternative allows direct access to the gland, with no need to retract other abdominal or retroperitoneal organs. Furthermore, blood loss was significantly lower for both lateral retroperitoneal (22 ml) and lumbar access (32 ml) than transperitoneal access (101 ml) (p,0.01). In 1998, Miyake et al. (42) retrospectively compared these surgical routes of access and verified that operative times were longer and postoperative pain was higher for transperitoneal laparoscopy, although there was no statistical difference. Suzuki et al. (24) compared three different approaches to the adrenal gland: anterior transperitoneal, lateral transperitoneal, and lateral retroperitoneal.

They found a mean operative time of 175, 118, and 176 min, respectively. The lateral transperitoneal approach was significantly favored (p,0.0001). No significant difference was found regarding blood loss. Shoulder pain and paralytic ileus occurred in patients of all groups, but in the retroperitoneal approach it occurred only when the peritoneum was opened. Suzuki et al. (24) recommended that experienced surgeons should treat small lesions using

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Figure 3 - Right-side retroperitoneoscopic view. AV: adrenal vein. IVC: inferior vena cava. P: psoas muscle.

The mean operative time (117 vs. 84 min, respectively; p,0.05), blood loss (340 vs. 200 ml, respectively; p,0.05), hospital stay (7.8 vs. 4.8 days, respectively; p,0.05), and complication rates were significantly higher in the transperitoneal group. Blood pressure fluctuations, long-term blood pressure control, metabolic and imaging follow-up were similar in both groups; however, the recurrence rates were not verified.

the minimally invasive retroperitoneal approach to avoid complications related to peritoneum insufflation.

Other authors did not find any significant differences regarding transperitoneal versus retroperitoneal access.

Fernandez-Cruz et al. (43) documented no difference regarding blood loss or hemodynamic parameters between both approaches, in addition to operative time, blood loss and complications. Gockel et al. (33) reported that the median operative time is lower in the transperitoneal approach (85 min) compared with the retroperitoneal approach (120 min) (p,0.001), particularly when the right side was compared. Also, a shorter learning curve was observed for the transperitoneal approach compared with the retroperitoneal approach. These results were explained by earlier recognition of anatomic landmarks during transperitoneal access.

Rubinstein et al. (44) conducted a randomized prospective comparison between transperitoneal and retroperitoneal routes for adrenalectomy. Patients from both groups were comparable with respect to age, body mass index, tumor size, and ASA classification, and no significant differences were noticed. Thus, median operative time, blood loss, and specimen weight were similar in both groups, as well as analgesic requirements, hospital stay, and complication rates. The mean convalescence time was 4.7 weeks for the transperitoneal approach and 2.3 weeks for the retroperitoneal approach (p = 0.02). Two patients in the transperitoneal group developed with port site-related hernias.

A retrospective study comparing the transperitoneal and retroperitoneal approaches for treating PHEO analyzed 99 patients with unilateral adrenal lesions and comparable demographic data (45). There were 40 patients in the transperitoneal group and 59 in the retroperitoneal group.

Special situations Previous abdominal surgery or radiation therapy does not seem to affect the clinical outcome of the retroperitoneal approach. Viterbo et al. (46) compared retroperitoneal access to the kidneys and adrenals in patients with and without previous abdominal surgery but was unable to verify significant differences regarding operative time, blood loss, or convalescence. Retroperitoneal access is the preferred route for patients with a history of prior abdominal surgery as a way for surgeons to avoid visceral and vascular complications (10,28,44,47,48).

Obese patients with PHEO can be preferentially managed with retroperitoneoscopic adrenalectomy. There are two reported cases that clearly document the feasibility and relative safety of this approach (49,50). Because of the direct access to the gland, some authors consider obesity as an indication to perform the retroperitoneal approach (28,44,51).

A high body mass index is correlated with a longer operative time, mainly for those .25 kg/m2 (33,51).

Tumor size must also be taken into consideration when planning the surgical approach. Due to more restricted space, retroperitoneoscopy is usually indicated when adrenal tumors are ,7 cm (43,52), although some experienced centers have operated on larger tumors (18,30,53,54).

Figure 4 - Left-side retroperitoneoscopic view. A: adrenal. AV: adrenal vein. LV: lumbar vein. RA: renal artery. RV: renal vein. Note the laparoscopic clips for the inferior and medial adrenal arteries.

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Figure 6 - Left-side retroperitoneoscopic adrenalectomy after adrenal vein section. Adrenal arteries indicated by the arrows. A: adrenal. AO: aorta. AV: adrenal vein. RA: renal artery. RV: renal vein.

Figure 5 - Right-side retroperitoneoscopic adrenalectomy after adrenal vein section. Adrenal arteries indicated by the arrows. A: adrenal. AV: adrenal vein. IVC: inferior vena cava. P: psoas muscle.

supervision before being able to perform the procedure in under a mean operative time of 90 min.

Retroperitoneoscopic adrenalectomy can be indicated for the treatment of PHEO with the same peri-operative outcomes as the transperitoneal approach because it allows direct access to the adrenal glands with no increase in the operative risks.

Although technically more demanding than the transperitoneal approach, retroperitoneoscopy can shorten the mean operative time, which is useful for cases of PHEO in order to minimize intraoperative hemodynamic changes. Blood loss and convalescence time can be also reduced by this approach.

There is no absolute indication for using either the transperitoneal or retroperitoneal approaches; however, the latter procedure may be the better option for cases with a previous abdominal surgeries and obese patients.

For both approaches, tumor size .5 cm can increase the operative times (33,55). When there is suspicion of malignant PHEO, the surgical procedure must be considered. In such cases, the open procedure should be favored over the laparoscopic approach (56), mainly if radiological findings indicate local invasion or, as is the case of large tumors (57), some studies document good outcomes in cases of malignant, organ-confined tumors (52,57). Also, there is a relative contraindication for retroperitoneoscopy if there has been previous retroperitoneal manipulation or scarring (44).

In these cases, the transperitoneal approach would be more suitable.

Laparoscopic adrenalectomy can also be performed on children. Most cases are conducted by the transperitoneal approach. A non-randomized retrospective multicenter study showed that this method is a feasible and secure procedure for young patients, with a 10% conversion rate (58). Furthermore, when referring to the retroperitoneal approach, there are few studies indicating that the surgical positions for adults can also be applied to children.

However, there are no available data related to retroperitoneal treatment specifically for PHEO. Most cases are included in non-specific retroperitoneal adrenalectomy series (59,60), or even included in adult series, indicating the feasibility of the method with no major complications, although right-sided procedures can be technically more demanding (61).

An important issue concerning the retroperitoneoscopic technique is how to teach the procedure to those who do not routinely perform it. A study conducted by Barczynski and Walz (62) reported the experience of a pioneering group in this technique who introduced it to an inexperienced group.

The experienced group continuously supervised the student group and offered direct surgical assistance during the initial cases. They compared the first cases of each group and found a significant impact on the operative time and conversions in the learning group. The recommendation was that previous visits and contacts to experienced centers, as well as initial assistance from an experienced surgeon, can flatten the learning curve of the apprentice surgeons, as reflected by safer outcomes for patients. It was estimated that the apprentice had to operate on 20–25 cases under

AUTHOR CONTRIBUTIONS Hisano M, Vicentini FC, and Srougi M have equal participation in this review article. All three authors worked on technical aspect of the surgery, as well as prepared the illustrations, and reviewed the published literature of the subject.

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DOI:10.6061/clinics/2012(Sup01)28

REVIEW

Post-surgical follow-up of primary hyperparathyroidism associated with multiple endocrine neoplasia type 1 Flavia L. Coutinho,I Delmar M. Lourenco Jr.,I Rodrigo A. Toledo,I Fabio L. M. Montenegro,II Sergio P. A. ToledoI I Endocrine Genetics Unit (LIM-25), Endocrinology Division, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. II Head and Neck Surgery Department, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo.

The bone mineral density increments in patients with sporadic primary hyperparathyroidism after parathyroidectomy have been studied by several investigators, but few have investigated this topic in primary hyperparathyroidism associated with multiple endocrine neoplasia type 1. Further, as far as we know, only two studies have consistently evaluated bone mineral density values after parathyroidectomy in cases of primary hyperparathyroidism associated with multiple endocrine neoplasia type 1. Here we revised the impact of parathyroidectomy (particularly total parathyroidectomy followed by autologous parathyroid implant into the forearm) on bone mineral density values in patients with primary hyperparathyroidism associated with multiple endocrine neoplasia type 1. Significant increases in bone mineral density in the lumbar spine and femoral neck values were found, although no short-term (15 months) improvement in bone mineral density at the proximal third of the distal radius was observed.

Additionally, short-term and medium-term calcium and parathyroid hormone values after parathyroidectomy in patients with primary hyperparathyroidism associated with multiple endocrine neoplasia type 1 are discussed. In most cases, this surgical approach was able to restore normal calcium/parathyroid hormone levels and ultimately lead to discontinuation of calcium and calcitriol supplementation.

KEYWORDS: Primary Hyperparathyroidism; MEN1; Bone Mineral Density; Parathyroid Hormone; Total Parathyroidectomy.

Coutinho FL, Lourenco DM Jr, Toledo RA, Montenegro FL, Toledo SPA. Post-surgical follow-up of primary hyperparathyroidism associated with multiple endocrine neoplasia type 1. Clinics. 2012;67(S1):169-172.

E-mail: flacoutinho07@yahoo.com.br Tel.: 55 11 3061 7252

multiglandular parathyroid neoplasia (hyperplasia versus adenoma), has an earlier disease onset (20 versus 40 years of age), has a higher recurrence rate after parathyroidectomy (PTX), and is often less aggressive (2,5,7). A potentially more virulent form of HPT has also been reported in association with Zollinger–Ellison syndrome (8). We have found an increase in the frequency of HPT/ MEN1in recent years, since familial genetic screening for the MEN1 gene was introduced into routine use in our hospital (9–11).

INTRODUCTION Primary hyperparathyroidism (HPT) is a common endocrine disorder, affecting 0.3% of the general population and 1–3% of postmenopausal women (1). It is characterized by hypercalcemia and elevated or inappropriately normal levels of parathyroid hormone (PTH). Most patients (80– 95%) present with the sporadic form of the disease (s-HPT), whereas familial cases may be associated with multiple endocrine neoplasia type 1 (MEN1) and MEN type 2, jaw tumor syndrome, neonatal severe primary HPT, and familial isolated HPT (2–4). HPT disease associated with MEN1 (HPT/MEN1) differs in many aspects from s-HPT, including its clinical, pathological, and genetic characteristics, and it is therefore treated with different surgical protocols, and exhibits different cure rates and outcomes (2,5,6). In contrast to s-HPT, HPT/MEN1 presents as a

PTX IN HPT/MEN1 Instead of adenomectomy, the procedure recommended for s-HPT, two basic surgical procedures have been performed for the treatment of patients with HPT/MEN1: subtotal partial parathyroidectomy (SPTX) and total parathyroidectomy (TPTX). SPTX consists of the removal of at least 3–3.5 glands, leaving a normal parathyroid remnant of 20–30 mg, whereas TPTX consists of the removal of all four glands followed by autologous heterotopic implant of parathyroid tissue in the muscles of the non-dominant forearm (8,12-16). Ultimately, in the hands of skilled surgeons, both methods have been reported to achieve

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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X-ray absorptiometry (DXA, Hologic QDR-4500 S/N 45130, Waltham, MA) (34). Significant improvements in BMD were verified in all cases 15 months after PTX at both the LS and FN, but not at 1/3 DR. Hence, 15 months after PTX, the LS values increased from 0.843 to 0.909 g/cm2, with a BMD increase of +8.4¡9.84% (p = 0.001). The FN values rose from 0.745 to 0.798 g/cm2, and BMD increased by +6.9¡3.17% (p = 0.0001). Conversely, no significant changes were seen in the 1/3 DR BMD values 15 months after PTX (34).

It was noted that pre-surgical bone mineral mass was already compromised in the 16 patients with HPT/MEN1.

Hence, there was a high prevalence of reduced BMD (Zscore , – 2.0) for the 1/3 DR (50%), LS (43.7%), ultradistal radius (UDR; 43.7%), FN (25%), and total femur (TF) (18.7%) in these cases.

euparathyroidism, maintaining correct PTH secretion and calcium metabolism, and preventing clinical complications.

BONE MINERAL DENSITY IN HPT/MEN1 Very few data on bone status in patients with HPT/ MEN1 are available in the literature, which limits comparisons. Burgess et al. documented high rates of early and severe bone mineral loss in the femoral neck (FN) and lumbar spine (LS) of 29 women in a large MEN1 family from Tasmania (17). Of note, the authors used analysis of the bone mineral density (BMD) to evaluate bone status in the studied cases.

Similarly, in 2008, we confirmed the same demineralization pattern in affected cases from an extended MEN1 family harboring a founding MEN1 germline heterozygous mutation. We also identified the proximal one-third of the distal radius (1/3 DR) as an early and severely compromised bone site in this MEN1 family (18). Patients with MEN1-related HPT usually have severe bone and kidney involvement despite a milder biochemical presentation compared with their s-HPT counterparts (19).

In 2010, our preliminary findings were confirmed in a larger series of HPT/MEN1 cases that we were able to study. In this latter investigation, we found that urolithiasis occurred early (,30 years) and frequently (75%) and that it was usually associated with kidney-related co-morbidities (50%), as well as with renal insufficiency in the older group (.50 years) (33%). We also verified that bone mineralrelated and urolithiasis-related renal complications in HPT/ MEN1 were of early onset, frequent, extensive, severe, and progressive (20). Notably, the trabecular bone mineral content in the studied cases was not protected, in contrast to the previously reported trend in s-HPT (21-25).

Furthermore, it was recently suggested that the Menin protein, coded by the MEN1 gene, may interfere in osteoblast differentiation (26–28), and may also have a direct demineralizing effect on trabecular bone in animal models (29).

The impact of PTX in s-HPT has been widely studied, and significant short-term bone mineral recovery of the LS and FN has been documented (22,30,31). Conversely, 1/3 DR BMD was previously thought to have no marked recovery one year after PTX (31), whereas long-term mineral recovery of the LS, FN and also 1/3 DR has been documented (22,25,30-33).

BMD DATA IN s-HPT and HPT/MEN1 The BMD values after PTX in our study were between those reported by Burgess et al. (17) in HPT/MEN1 cases, and those seen in s-HPT (32). Furthermore, in contrast to the short-term and long-term 1/3 DR BMD recovery reported

by Rubin et al. (32) in cases with s-HPT, we could not document significant change in 1/3 DR BMD (-0.6¡15.73%) during follow-up of our cases (Table 1).

These data indicate a trend for potential differences in BMD recovery after surgery in patients with HPT/MEN1 compared with patients with s-HPT.

PTH AND CALCIUM AFTER PTX IN HPT/MEN1 In this study, immediately after PTX, all cases presented transient signals and symptoms of hypocalcemia, associated with low serum calcium and PTH values. Therapy with oral calcium (,2–3 g/day) and calcitriol (,1 mg/day) supplements was instituted in all cases to achieve normal calcemia.

This treatment was maintained for 4–5 months (with an average time of 8.3 months), although progressively lower therapeutic doses were needed over this time. The postsurgical pattern of increasing serum levels of PTH secreted by the parathyroid implant is shown in Figure 1. A slow biochemical (calcium, PTH) recovery after successful total PTX followed by autologous parathyroid implantation was verified in this series of patients. Fully viable parathyroid Table 1 - Mean percentage change in bone mineral density at three sites in patients with hyperparathyroidism associated with multiple endocrine neoplasia type 1 (HPT/MEN1) and those with sporadic HPT, after parathyroidectomy.

BMD AFTER PTX IN HPT/MEN1 Data on bone mineral recovery after PTX in the specific subset of patients with HPT/MEN1 have been published in only two papers so far (17,34).

Data from Burgess et al.

This first study addressed the LS and FN BMD before and after PTX in five women with HPT/MEN1. In this limited number of patients, significant recovery of BMD was seen in the femoral neck (3.2¡2.9%) and lumbar spine (5.2¡2.5%) after PTX (17).

LS (%) FN (%) 1/3 DR (%) Post-surgical mean time (months)

Burgess (17) (HPT/MEN1)

Coutinho (34) (HPT/MEN1)

Rubin (32) (s-HPT)

5.2 3.2 NA 18

8.4 6.9 –0.6 15

9 5 4 12

LS, lumbar spine; FN, femoral neck; 1/3 DR, distal third of radius; NA, data not evaluated.

In all cases studied by Coutinho et al. (34) total parathyroidectomy was followed by autologous parathyroid implant, and bone mineral density values were assessed using dual-energy X-ray absorptiometry (Hologic apparatus).

Our data We assessed the BMD changes in 16 patients with HPT/ MEN1 11 years after total PTX followed by autologous parathyroid implant in the forearm, using dual-energy

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Post-surgical follow-up of HPT/MEN1 Coutinho FL et al.

Figure 1 - Mean serum levels of parathyroid hormone (PTH; mean ¡ SEM) before and after total parathyroidectomy (PTX) followed by autologous parathyroid implant in the non-dominant forearm in 16 cases of hyperparathyroidism associated with multiple endocrine neoplasia type 1. Serum samples were collected from both non-dominant (A) and dominant (B) forearms. These data are based on those of Coutinho et al. (34).

implants with consistent mean PTH secretion values (32.6¡9.1 ng/ml) were obtained 9 months after PTX (34).

Based on these data, we conclude that in patients with HPT/MEN1, bone demineralization is an important complication and should be routinely evaluated before PTX, and patients should be followed up for several years. Calcium and calcitriol supplementation is frequently needed for several months after PTX until efficient function of the autologous parathyroid implant is achieved.

In conclusion, in our experience there is a marked shortterm improvement in BMD at the LS and FN in most patients with HPT/MEN1 after total PTX followed by autologous parathyroid implant into the forearm, although BMD at the 1/3 DR may take longer to recover (.18 months) after surgery. Hence, LS and FN BMDs are the better sites to investigate BMD after PTX in patients with HPT/MEN1.

AUTHOR CONTRIBUTIONS Coutinho FL was responsible for the clinical assessment, data collection and analysis, and manuscript writing. Lourenco DM Jr is the doctor who contributed to the clinical assessment of patients with MEN1. Toledo RA is the biologist who performed the molecular diagnosis of patients in the study. Montenegro FL is the surgeon responsible for the surgical treatment of all patients in the study. Toledo SPA guided the study.

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CLINICS 2012;67(S1):173-178

DOI:10.6061/clinics/2012(Sup01)29

REVIEW

Surgical management of pancreatico-duodenal tumors in multiple endocrine neoplasia syndrome type 1 ˚ kerstro¨m, Peter Sta˚lberg, Per Hellman Go¨ran A Department of Surgery, University Hospital Uppsala, Uppsala, Sweden.

Pancreatico-duodenal tumors are the second most common endocrinopathy in multiple endocrine neoplasia syndrome type 1, and have a pronounced effect on life expectancy as the principal cause of disease-related death.

Previous discussions about surgical management have focused mainly on syndromes of hormone excess and, in particular, the management of multiple endocrine neoplasia syndrome type 1-related Zollinger–Ellison syndrome.

Since hormonal syndromes tend to occur late and indicate the presence of metastases, screening with biochemical markers and endoscopic ultrasound is recommended for early detection of pancreatico-duodenal tumors, and with early surgery before metastases have developed. Surgery is recommended in patients with or without hormonal syndromes in the absence of disseminated liver metastases. The suggested operation includes distal 80% subtotal pancreatic resection together with enucleation of tumors in the head of the pancreas, and in cases with Zollinger– Ellison syndrome, excision of duodenal gastrinomas together with clearance of regional lymph node metastases.

This strategy, with early and aggressive surgery before metastases have developed, is believed to reduce the risks for tumor recurrence and malignant progression.

KEYWORDS: Multiple Endocrine Neoplasia Type 1; Neuroendocrine Pancreatico-Duodenal Tumors; Surgical Treatment.

A˚kerstro¨m G, Sta˚lberg P, Hellman P. Surgical management of pancreatico-duodenal tumors in multiple endocrine neoplasia syndrome type 1. Clinics.

2012;67(S1):173-178.

E-mail: goran.akerstrom@surgsci.uu.se Tel.: 46 18 6114600

the second allele is silenced by somatic mutation. The gene encodes for Menin, with an important role for DNA replication and transcriptional regulation (6). The gene is complex, with more than 1,000 identified mutations, without strong genotype–phenotype correlations and also with variable disease expression within families (7). Genetic diagnosis is obtained by complete MEN1 gene sequencing, which can reveal mutations in 70–90% of typical MEN1 cases, and a recently introduced multiplex ligation-dependent assay (MLPA), which can detect large deletions in another 4% (8). If mutation is unknown in the family, the genetic diagnosis can be difficult and negative genetic testing cannot exclude the syndrome. Without positive genetic diagnosis, MEN1 is clinically diagnosed if a patient has tumors in two of the three classical endocrine organs (parathyroid, pancreas/duodenum, or pituitary), or has family history of MEN1 and one such tumor (2).

Genetic family screening is important since unaffected family members may be spared unnecessary investigations and anxiety (3). Screening for MEN1 endocrine tumors is recommended in gene carriers, since biochemical abnormalities can often be revealed decades before clinical symptoms appear (2,9). If screening is delayed until clinical symptoms develop, morbidity and mortality may be encountered, especially from neuroendocrine pancreaticoduodenal (NEN-PET) and thymic tumors (2,3,9–11).

Screening to reveal any of the three classical endocrinopathies should begin in children during the first decade of life (Table 1) (11,12).

INTRODUCTION Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant hereditary syndrome with high penetrance, where 50% of children of an affected parent inherit the trait, and virtually all gene carriers develop features of the syndrome (1–3). The syndrome is rare, with a prevalence of two or three cases per 100,000 population, being equally common in males and females (2,3). MEN1 patients classically present with tumors of the parathyroid, the endocrine pancreas/duodenum and the anterior pituitary. The trait also implies increased risk of adrenocortical lesions (with hyperplasia, adenoma, rarely carcinoma in up to 30% of patients), increased incidence of foregut carcinoids (occurring in 7%, in the thymus, bronchial tree, and the stomach), and lower incidence of multiple lipoma, ependymoma, leiomyoma, meningeoma, facial angiofibroma, and collagenoma (2–5).

Genetic diagnosis MEN1 is caused by inactivating mutations of the MEN1 tumor suppressor gene on chromosome 11q13, germline mutation has inactivated one allele, and tumors occur when

Copyright ß 2012 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

No potential conflict of interest was reported.

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Table 1 - MEN1 screening. Revised from Brandi et al (12).

Tumor

Age that screening began (years)

Biochemical tests (annually)

Parathyroid Gastrinoma Insulinoma Non-functioning PETs

10 20 5 20

Serum calcium (PTH) Serum gastrin Fasting serum glucose/insulin/proinsulin PP, proinsulin, insulin, glucagon, VIP, chromogranin A

Anterior pituitary Foregut carcinoid

5 20

Prolactin, IGF-1 None

Imaging (every 3 years) None None None Endoscopic US (OctreoScan, CT) Brain MRI CT

of pancreatic hormones even in the absence of a syndrome of hormone excess, most commonly showing raised serum values of PP and chromogranin A, sometimes gastrin, insulin and proinsulin, occasionally glucagon, vasoactive intestinal peptide (VIP) or calcitonin (2,4,11). Only a minority of the microtumors acquire the potential to grow to clinically relevant lesions, since each patient will during their lifetime experience only few large tumors (2,4,11,18).

Cure requires total pancreatico-duodenectomy, which is rarely considered as initial procedure because of significant morbidity and mortality associated with resulting severe diabetes and exocrine pancreatic insufficiency (2,3,11).

Extended survival has been reported in MEN1 patients with PETs, even in the presence of metastases, suggesting that the disease may often have an overall indolent course.

However, disease progression varies widely between and within families, and individual patients have PETs with exceedingly more malignant behavior (2,4,19). Although suggested by some authors, no convincing and confirmed genotype–phenotype relationships have yet been demonstrated (2,4,9,19,24). The MEN1 PETs have been claimed to have favorable prognosis compared with sporadic tumors, though earlier diagnosis of MEN1 tumors, or in case of ZES, disparate survival for patients with duodenal and pancreatic gastrinomas, appear to explain this difference (11,21,25).

With adjustment for younger age due to screening detection in MEN1, similar survival expectancy has been revealed for MEN1-associated and sporadic PETs (11,21,25).

Malignant progression of PETs and thymic carcinoids has been the principal cause of premature disease-related death in MEN1, and the pancreatic malignancy has been identified as the major cause of death, with nearly half of affected patients dying before 50 years of age, mainly with liver metastases (2,9,11,26–29). When first recognized in 1989 that duodenal gastrinomas was the most common cause of MEN1 ZES, Norman Thompson introduced the procedure for distal, subtotal (80–85%) pancreatic resection, together with enucleation of pancreatic head tumors, duodenotomy for excision of duodenal gastrinomas and careful dissection of lymph gland metastases around the pancreatic head (11,30,31). In our experience, PETs could be detected by biochemical screening decades before development of a clinical syndrome of hormone excess (such as ZES), and we have suggested earlier surgery for malignancy prevention, not only in the MEN1 ZES patients, but also in patients with non-functioning MEN1 lesions (2,3,9,11,18,32). Screening studies revealed that 30–50% of patients already had metastases when a clinical syndrome of hormone excess, most often ZES, had developed, and for two decades we have therefore recommended timely repeated biochemical screening for PETs in MEN1 carriers to achieve early diagnosis, and early surgery for malignancy prevention

It is most important to emphasize that primary hyperparathyroidism (pHPT) has been the most common and generally the first detected endocrinopathy in MEN1, often diagnosed at ,20 years of age, and affecting more than 95% of patients at 40 years of age (2,3,9–12). MEN1-pHPT is typically associated with hyperplasia/multiglandular parathyroid disease, and should be suspected in all cases with multiglandular involvement or recurrent HPT (13). Younger HPT patients (,40 years) may often (,10%) be index cases for MEN1 kindreds, and are liberally subjected to parathyroid surgery due to long-term risk for osteoporosis and renal complications (2,9–16). The presence of the MEN1 syndrome may be revealed by screening with serum calcium or careful penetration of the family history in patients with pancreaticoduodenal tumors, pituitary tumors, or foregut carcinoids (2,3,9,11). Hypercalcemia caused by HPT stimulates gastrin, and early parathyroid surgery is recommended in patients with MEN1-associated Zollinger–Ellison syndrome (ZES), together with liberal use of proton pump inhibitors (PPIs) (16,17).

Pancreatico-duodenal neuroendocrine tumors (PETs) PETs are the second most common endocrinopathy in MEN1, with a prevalence of 35–40% at the age of 50 years, with higher figures if patients are subjected to more careful screening programs (2,3,11,18,19). Rarely, patients will have PETs as the first endocrinopathy recognized in childhood.

The MEN1 endocrinopathy consists of numerous microadenomas typically spread throughout the entire pancreas, which vary in size from slightly larger than a normal islet to a few millimeters in diameter, and with generally only few concomitant larger tumors (2,3,8,9,11,20,21). Pancreatic islets of normal size, cell morphology, and arrangement invariably surround the microtumors and often occur together with areas of nesidioblastosis with exocrine duct proliferations, and clusters of endocrine cells, which has been claimed to support the origin of microadenomas from pancreatic duct precursor cells rather than the pancreatic islets (2,11,21). Microtumors lack the normal islet cell organization, and frequently show immunoreactivity for multiple hormones, most commonly pancreatic polypeptide (PP), but there may also be reactivity for glucagon, insulin, proinsulin, somatostatin, or sometimes only chromogranin A (21,22). Duodenal microtumors, which have been identified in 50% of patients with the endocrinopathy, stain for serotonin, gastrin, and somatostatin (23). Gastrin immunoreactivity has generally been absent in pancreatic microadenomas, and only occasionally demonstrated in larger pancreatic tumors (21), in which PP or only chromogranin A reactivity has been most common (2,21). The presence of endocrinopathy can be diagnosed by serum measurements

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Surgery and pancreatico-duodenal tumors in MEN1 ˚ kerstro¨m G et al.

A

In patients with hypoglycaemia, a single tumor $5 mm is expected to cause hyperinsulinism, and the tumor can often be revealed by EUS. Non-functioning tumors may, however, occur concomitantly and the source of insulin (or proinsulin) excess may occasionally have to be determined by selective intra-arterial calcium-injection test (SAS test), which can regionalize the hypersecretion and identify rare multifocal insulinomas (11,41). Favorable cure rate after surgery has been reported in patients with MEN1 insulinomas, but concomitant distal (80%) pancreatic resection is recommended to remove concomitant non-functioning tumors and minimize risk of recurrence (11,19,40–42).

Tumor enucleation has only been reported with increased risk for recurrence of non-functioning tumors or new insulinoma (2,11,19,40–42). The most important is to emphasize that the malignancy rate is higher with MEN1 associated than with sporadic insulinoma, and may be recognized by metastases with also moderately large MEN1 insulinomas (11).

Rare MEN1 patients with vipoma or glucagonoma syndromes usually present with large PETs with high risk of malignancy, and should be treated with radical surgery (11,19,44,45). Due to severe hormone symptoms, patients with these tumors may require liver resection, treatment with repeated radiofrequency (RF) ablation or even resection of lung metastases (46).

(2,3,9,11,18,20,21,29,32). Consequently, in our center, the non-functioning PETs have become the most common tumor entity requiring surgery in ,50% of MEN1 patents (19,29,32).

Surgery for non-functioning PETs Raised biochemical markers, especially serum PP values, have together with raised values of chromogranin A become increasingly important for early detection of non-functioning PETs (2,3,9,11,29,32). Patients may also have raised insulin/proinsulin, glucagon, VIP or calcitonin values, without a hormone excess syndrome. Most authors agree that patients with non-functioning tumors larger than 2– 3 cm should undergo surgery, but the size limit has been the subject of controversy (19). Studies of non-functioning MEN1 PETs in the French GTE register revealed a low (4%) metastases rate for tumors #10 mm, and notably higher metastases rate (15–52%) for larger tumors (33–35).

However, reporting a 15% mortality risk from pancreatic surgery, the authors recommended surgery for non-functioning PETs greater than or equal to 2 cm (33–35). We and other authors recommend surgery for non-functioning MEN1 PETs around or greater than 10 mm, since the metastases rate is unacceptably high for larger tumors, and surgery has been carried in rather large series without mortality (11,19,36,37).

Before surgery, contrast-enhanced computed tomography (CT) is routinely performed to clarify the anatomy and possible presence of liver metastases (11). C-5-hydroxytryptophane (5HTP)-positron emission tomography (PET) has been efficiently used in our department to reveal small PETs and lymph node metastases (38). Endoscopic ultrasound (EUS) has become the most important method for early detection of PETs, with the ability to show the relation to the pancreatic and the bile ducts, and is now used for routine screening follow-up of MEN1 patients (11,19,36,37,39).

The surgical procedure generally consists of distal 80% subtotal pancreatic resection with the pancreas divided to the left of the portal vein, and enucleation of possible pancreatic head tumors. Occasionally patients present with larger or multiple tumors, and more extensive procedures may be required. Smaller tumors (,5 mm) deep in the pancreatic head, or close to the bile or pancreatic ducts, are left if enucleation is considered hazardous. Duodenotomy is not done in patients without a rise in serum gastrin.

Surgery for ZES For MEN1 ZES patients surgery remains controversial because persistent normalization of raised gastrin levels is rarely achieved, and long survival can also be expected without operation (2,3,11,19,47,48). Some surgeons have advocated surgery when gastrin excess could be regionalized by the SAS (Imamura) test, others only when tumors .2–3 cm have been visualized (30,31,48–50).

We have also proposed surgery in the absence of liver metastases without pre-operative tumor localization or regionalization, since the vast majority (,90%) of MEN1 ZES patients have single, or multiple, small duodenal tumors as the cause of gastrin excess, and smaller tumors generally require duodenotomy for visualization (2,11, 21,23,30,31). Also, the smaller duodenal tumors are often associated with conspicuously larger regional lymph node metastases which may easily be mistaken for the primary tumor when occurring within the peripancreatic fasciae capsule (11,51). Ultimately, there is often delay before liver metastases develop in ,10–20% of patients, which may provide a favorable interval for intervention, where lymph node metastases may be excised together with the primary lesion (11,52). Gastrin-secreting pancreatic tumors are uncommon in MEN1, but have been reported to be large and they may be associated with earlier liver metastases, as reported for patients with sporadic pancreatic gastrinoma (19,50). Survival is favorable in patients with small duodenal gastrinomas, even in the presence of lymph node metastases, but is worse for patients with larger duodenal or pancreatic gastrinomas (19). Surgical excision of gastrinoma and lymph gland metastases may normalize gastrin excess, but the effect is time-limited, since virtually all patients will recur with hypergastrinemia, sometimes after several years’ delay (3,11,19,47,48,53,54). Since ZES patients with liver metastases have significantly shorter survival, and liver metastases possibly develop less frequently in operated patients, we perform surgery to reduce the risk of further

Surgery for PETs with syndromes of hormone excess ZES has been the most common hormone syndrome in MEN1, which ultimately may be present in 30–50% of patients, and implies that 30% or more of ZES patients have the MEN1 syndrome (2,3,11,19,36). Insulinoma causing the hypoglycemia syndrome has been revealed in 4–10%, vipoma in 3–5% and symptomatic glucagonoma has been exceptionally rare (,1%) (2,3,11,19,36).

Surgery for insulinomas, vipomas and glucagonomas There is general consensus to submit MEN1 patients with insulinoma and a hypoglycaemia syndrome to surgery after biochemical diagnosis and fasting test verification, more or less irrespective of tumor size, since no efficient medical treatment option is available (2,11,18,19,39–43).

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progression (3,11,19,50,53). Milder recurrent hypergastrinemia in ZES can be efficiently controlled by PPI, and surgery has in our opinion the additional important goal to delay malignant development, also by removal of concomitant non-functioning tumors (2,3,11,18). Non-functioning tumors of conspicuous size have in our experience been almost invariably present when MEN1 patients have been subjected to surgery with ZES, and may be a more likely cause of spread with liver metastases.

Our general policy is to subject MEN1 patients with raised serum gastrin and ZES verified by gastric acid hypersecretion (pH,2 in gastric aspirate) to exploration with duodenotomy for visualization (palpation is essential) of single or multiple duodenal gastrinomas. Distal (80%) pancreatic resection is carried out for removal of concomitant non-functioning tumors together with careful dissection of regional metastases, and enucleation of possible tumors in the head of the pancreas (3,11,18).

Pancreatico-duodenectomy (PD) has been performed in limited series of MEN1 patients and shown to offer better possibilities for cure of ZES in selected patients than excision of duodenal gastrinomas combined with subtotal pancreatic resection (3,11,19,37,43). However, in our experience concomitant non-functioning tumors have virtually always necessitated distal pancreatic resection in the ZES patients (3,11,18) Moreover, if reoperation is required for recurrent pancreatic tumor this may be exceedingly difficult after previous pancreatico-jejunostomy as part of PD. After PD, treatment of liver metastases with embolization or RF ablation may be hazardous and should be avoided due to risk for serious ascending infection via the hepaticojejunostomy (46). Still PD is occasionally required as the primary procedure in MEN1 patients with a large pancreatic head or duodenal tumors, and sometimes also required at reoperation for recurrent duodenal or pancreatic head tumors (55). In our experience such recurrences have been treated with re-resection of the pancreatic neck or new enucleations rather than total pancreatectomy, but this may ultimately be required (55). Pancreas-preserving duodenectomy has been reported as an elegant technique to remove multiple duodenal gastrinomas entirely, but it is a difficult procedure suggested to benefit selected patients with multifocal duodenal gastrinomas, with the problem of leaving behind the possible common concomitant nonfunctioning pancreatic tumors (56–58).

High gastrin and long-standing gastrin excess may, in 5– 30% of MEN1-ZES patients, result in development of type 2 gastric carcinoids in the gastric body and fundus and occasionally in the antrum (51). These tumors are generally multiple and often larger than 1.5 cm, and may occupy virtually the entire fundic mucosa. Occasional tumors are larger (.4 cm). Lymph node metastases occur in ,30%, and liver metastases in 10–20%. Smaller lesions may sometimes regress if eugastrinemia is achieved, and the remaining tumors should be locally excised (59). Some MEN1 patients have died from such malignant gastric carcinoids with metastases, and therefore gastrectomy may sometimes be required for the large gastric carcinoids (59,60).

dissected to the aorta. The pancreatic tail and body are explored via the lesser sack, with retroperitoneum incised below the pancreas, to allow blunt dissection of the distal parts. The entire pancreas is bidigitally palpated and scanned with intraoperative ultrasonography (IOUS), revealing lesions larger than 3-4 mm, and facilitating safe enucleation by showing relations between tumors and ductal structures (2,3,11). Metastatic lymph glands are searched for around the splenic and celiac vessels, in the hepatoduodenal ligament, and especially within the dorsal fasciae capsule of the pancreatic head.

A pancreatic head tumor is generally enucleated by cautious dissection with careful ligation/clipping of vessels and any pancreatic duct tributaries. A distal 80% body and tail resection is undertaken by transecting the pancreatic neck to the left of the porto-mesenteric vein. Any enucleated area in the pancreatic head is left open, with drainage carefully applied.

Since the majority of MEN1 patients require prophylactic cancer operation, and efficient removal of lymph node metastases, with predilection in the hilus of the spleen, splenectomy rather than spleen preservation is generally recommended.

Duodenotomy is more efficient than duodenoscopy or EUS to visualize gastrinomas in MEN1-ZES, which can be multiple in ,50% of patients (58). Routine duodenal exploration is performed in patients with the ZES syndrome via longitudinal duodenotomy in the descending part of the duodenum. Even small tumors can be identified by palpation after digital inversion of the proximal and distal parts of the duodenum. Duodenal tumors smaller than 5 mm can be enucleated with the mucosa; larger tumors require limited excision of the duodenal wall.

Postoperative follow-up/reoperation. Surgery for the MEN1 pancreatico-duodenal endocrinopathy rarely results in lifelong cure, and recurrence should be expected (2,3,11,19,53,54). The patients should be subjected to, in general, yearly follow-up with biochemical markers and radiological investigations, including EUS. Reoperation is considered when a lesion of arbitrarily ,10 mm is visualized concomitant with a rise in biochemical markers, or a patient develops a clinical syndrome of hormone excess. Reoperations have in our experience been mainly performed as resections or enucleations of new tumors, and have been uncomplicated and compatible with long survival and generally preserved pancreatic function. We have experienced no mortality, or pancreatic fistulas requiring reoperation, after primary or reoperative surgery in our MEN1 PETs patients. Total pancreatectomy may be required for recurrent, rapidly growing, or unusually large tumors, and is even considered when there is a strong history of markedly malignant pancreatic tumors in the family (2,3,11,55). We have with few exceptions avoided this, due to the resulting generally severe diabetes that ensues, and complete removal of the pancreatic head and duodenum to achieve total pancreatectomy, after previous subtotal pancreatic resection, has, according to reports from other groups, sometimes been difficult or complicated (19,54). Liver metastases are treated with liver resection when possible, and RF ablation. In patients with unresectable liver spread or other distant metastases, oncologic treatment is given, with a common response to combinations of streptozotocin and 5-fluorouracil or doxorubicin;

Technical aspects of MEN1 pancreatico-duodenal exploration In MEN1 patients the entire pancreas is explored by bilateral subcostal incision. The duodenum and the pancreatic head are mobilized with ventral and dorsal surfaces

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34. Triponez F, Dosseh D, Goudet P, Cougard P, Bauters C, Murat A, et al.

Epidemiology data on 108 MEN 1 patients from the GTE with isolated nonfunctioning tumors of the pancreas. Ann Surg. 2006;243(2):265–72.

35. Goudet P, Murat A, Binquet C, Cardot-Bauters C, Costa A, Ruszniewski P, et al. Risk factors and causes of death in MEN 1 disease. A GTE (Groupe d’Etude des Tumeurs Endocrines) cohort study among 758 patients. World J Surg. 2010;34(2):249–55.

36. Doherty GM, Thompson NW. Multiple endocrine neoplasia type 1: duodenopancreatic tumours. J Intern Med. 2003;253(6):590–8.

37. Bartsch DK, Fendrich V, Langer P, Celik I, Kann PH, Rothmund M. Outcome of duodenopancreatic resections in patients with multiple endocrine neoplasia type 1. Ann Surg. 2005;242(5):757–66. ¨ hrlefors H, Sundin A, Garske U, Juhlin C, Oberg K, Skogseid B, et al.

38. O Whole-body 11C-5-hydroxytryptophan positron emission tomography as a universal imaging technique for neuroendocrine tumors: comparison with somatostatin receptor scintigraphy and computed tomography.

J Clin Endocrinol Metab. 2005;90(6):3392–3400. ˚ kerstro¨ m G, Skogseid B. Endoscopic 39. Hellman P, Hennings J, A ultrasound for evaluation of pancreatic tumors in multiple endocrine neoplasia type 1. Br J Surg. 2005;92(12):1508–12.

40. O’Riordian DS, O’Brian T, van Heerden JA, , Service FJ, Grant CS. Surgical management of insulinoma associated with multiple endocrine neoplasia type 1. World J Surg. 1994;18(4):488–94.

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recently radioreceptor therapy with Luthetium has also been considered.

Several patient series support the active management strategy for MEN1 PETs by reporting reduced death risk in operated patients (19,24,37,43,54,56–58,61–63). However, strong evidence for an improvement in survival is lacking.

It is crucial to emphasize that liberal indications for pancreatico-duodenal surgery, and aggressive resection surgery, have to be undertaken cautiously with low morbidity and virtually absent mortality, since extended survival can often be expected without surgery (11,55,61,64).

AUTHOR CONTRIBUTIONS skerstro¨m G was responsible for the manuscript writing, layout and references. Sta˚lberg P provided assistance to the manuscript writing and permission requests. Hellman P provided assistance to the manuscript writing.

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