RUI MONTEIRO DE BARROS MACIEL

Prevalência de fenótipos e genótipos

no Consórcio Brasileiro de
Neoplasia Endócrina Múltipla Tipo 2
(BRASMEN)

Memória apresentada à Academia Nacional de Medicina

para concorrer à cadeira número 49

(Patrono Enjolras Vampré)

Rio de Janeiro, julho, 2017

1
 RUI MONTEIRO DE BARROS MACIEL

Prevalência de fenótipos e genótipos

no Consórcio Brasileiro de
Neoplasia Endócrina Múltipla Tipo 2
(BRASMEN)

Memória apresentada à Academia Nacional de Medicina

para concorrer à cadeira número 49

(Patrono Enjolras Vampré)

Rio de Janeiro, julho, 2017

2
Índice

Prefácio 4

1. Introdução 8

1.1. Neoplasias Endócrinas Múltiplas do tipo 2 8

1.2. Apresentação clínica do Carcinoma Medular de Tiroide 12

1.3. Diagnóstico do CMT 15

1.4. Genotipagem do gene RET em MEN2 16

1.5. Impacto da avaliação genética no assintomático 20

1.6. Resumo da introdução e objetivos 22

2. Pacientes e Métodos 23

3. Resultados 26

4. Discussão 33

5. Conclusões 41

6. Referências 42

7. Agradecimentos 51

8. Projetos de pesquisa na área de MEN2 e CMT 53

9. Capítulos de livros na área de CMT e MEN2 54

10. Título das Teses Orientadas sobre CMT e MEN2 56

11. Trabalhos publicados na área de CMT e MEN2 58

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PREFÁCIO

A Memória que ora apresentamos à Academia Nacional de Medicina

para concorrer à vaga de Membro Titular, intitulada “Prevalência de fenótipos

e genótipos no Consórcio Brasileiro de Neoplasia Endócrina Múltipla Tipo 2

(BRASMEN)”, apresenta dados inéditos sobre a prevalência de fenótipos e

genótipos da Neoplasia Endócrina Múltipla do tipo 2 (MEN 2, do inglês

Multiple Endocrine Neoplasia type 2) no Brasil, obtidos por meio de estudo

colaborativo realizado em vários centros médicos nacionais. Como

complementação ao trabalho incluímos, ao final, as listas dos projetos de

pesquisa financiados, dos capítulos de livro, das teses orientadas e as 25

publicações de nosso grupo na área de MEN 2 e Carcinoma Medular de

Tiroide.

Nosso interesse referente a esta doença iniciou-se em 2002, quando

fomos procurados pelo emérito cirurgião de Cabeça e Pescoço, Dr. Marcos

Brasilino de Carvalho, a respeito de uma família por ele operada com

diagnóstico de carcinoma medular da tiroide (CMT). O propósito, um homem

de 49 anos, assintomático até a idade de 47 anos, notou o aparecimento de

um nódulo no lobo direito da tiroide e apresentava valores muito elevados de

calcitonina sérica. Com a hipótese de CMT, submeteu-se à tiroidectomia total

com esvaziamento do compartimento central e apresentou, ao exame

anátomo-patológico, o diagnóstico de CMT, hiperplasia de células C e doença

metastática em 3 linfonodos. O paciente relatava que sua mãe tinha sido

submetida à cirurgia da tiroide em 1972, com a idade de 47 anos, com

diagnóstico de carcinoma indiferenciado da tiroide; 30 anos depois, logo

depois da cirurgia do filho, seu exame histológico foi revisto e confirmou-se o

diagnostico de CMT. Além disso, o paciente informava que sua filha também

apresentava nódulo na tiroide.

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 O CMT é um tumor originário das células C ou parafoliculares da tiroide,

que secretam calcitonina; é um tumor raro, sendo responsável por 5-10% dos

casos de câncer de tiroide, na nossa casuística. A maioria dos casos de CMT é

esporádica, mas aproximadamente 40% é associada a MEN2, síndrome

hereditária causada por mutações do gene RET. Como veremos adiante, a

gravidade da doença está ligada ao local da mutação no gene RET.

Pois bem, como o Dr. Marcos Brasilino sabia que estávamos fazendo

estudos moleculares em doenças da tiroide, queria que estudássemos esta

família para determinar se seria um caso de MEN2 e onde estaria esta mutação

do gene RET para que pudesse tomar uma conduta em relação aos demais

familiares. Depois de verificarmos que os pacientes não apresentavam

mutações nos éxons 10 a 15 do gene RET, até então descritos na literatura

como regiões onde se assestavam as mutações (regiões “hot-spot”),

estendemos nossa pesquisa para os demais éxons do RET e reportamos uma

mutação ainda não descrita na literatura no éxon 8 (Gly533Cys), não apenas no

caso índice, mas em 76 indivíduos de 229 membros da família (“A novel germ-

line point mutation in RET Exon 8 (Gly533Cys) in a large kindred with familial

medullary thyroid carcinoma”, Álvares-da-Silva, Maciel, Dias-da-Silva, Toledo,

Carvalho e Cerutti, Journal of Clinical Endocrinology and Metabolism 2003; 88:

5438-5443).

Por essa época grupos europeus e americanos demonstravam que era

muito útil a organização de consórcios nacionais e transnacionais para a

genotipagem do RET, que detecta 92-98% de todos os portadores da

mutação. Como, já mencionado, o CMT ocorre em pelo menos 95% de todos

os pacientes com MEN 2, mas as mutações nos diferentes códons causam

penetrância variável das manifestações da doença nos afetados. Assim,

sabendo-se qual o códon implicado nas famílias portadoras de MEN 2, é

5
possível melhorar o diagnóstico, o estadiamento, o tratamento e o seguimento

dos pacientes, incluir a decisão sobre a cirurgia profilática da tiroide dos

afetados e determinar os indivíduos não afetados da família, além de

selecionar aqueles que mereceriam terapias especiais ou a inclusão em ensaios

clínicos com drogas em experimentação. Desta forma, depois da organização

desses consórcios e do estudo de um grande número de casos, sabe-se, hoje,

quais as mutações que apresentam comportamento clínico mais agressivo e

que merecem uma abordagem terapêutica mais precoce. Além disso, estes

consórcios têm permitido o estabelecimento de métodos padronizados de

diagnóstico molecular e estratégias cirúrgicas e de seguimento mais eficazes.

Assim, inspirado nestas experiências, apresentamos nesta memória os

resultados da prevalência de genótipos e fenótipos de pacientes brasileiros

portadores de MEN2 (BRASMEN), com a participação dos principais grupos

acadêmicos experientes de nosso país. O objetivo é conhecer melhor a

doença em nosso meio para estabelecer práticas mais eficazes, uma vez que

este conhecimento gerou-se a partir de observações padronizadas e

sistemáticas num grande número de casos. Pela diversidade e

heterogeneidade genética de nossa população observamos distribuição dos

códons afetados pelas mutações e comportamentos da neoplasia diferentes

daqueles existentes na Europa.

Para a consecução desta trabalho obtivemos apoio da FAPESP

(Fundação de Amparo à Pesquisa do Estado de São Paulo), por meio de projeto

temático, que abordou diversos aspectos da moléstia e deu-nos o suporte

laboratorial e de pesquisa ("Carcinoma medular da tiroide: revisitação à clínica,

à biologia molecular, à bioquímica e à biologia do desenvolvimento depois dos

achados da genética molecular", processo 06/60402-1, com duração de 2007 a

2011) e do Ministério da Saúde, para o suporte ao atendimento e tratamento

dos pacientes (Centro Integrado das Doenças da Tiroide da Escola Paulista de

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Medicina/Hospital Israelita Albert Einstein, projeto 25000.168513/2008-11 e

2011/14, com duração de 2008 a 2014). A estes projetos sucederam-se três

outros, que têm contribuído para a continuidade dos estudos na área de MEN2:

um Auxílio Regular da FAPESP (“Carcinoma medular de tiroide hereditário:

percepção e atitude de pacientes, familiares e profissionais de saúde sobre

questões bioéticas”, processo 10/51547-1, com duração de 2010 a 2013); um

projeto patrocinado pela FINEP/Grupo Fleury (“Exoma em doenças órfãs”, do

Departamento de Pesquisa e Desenvolvimento do Grupo Fleury, FINEP, projeto

37 P&D, NP-82, FINEP 0914005500); e outro temático da FAPESP, com Janete

Cerutti (“Sequenciamento Completo do Exoma, Paired-end RNA e Genoma:

Novos Insights sobre a Natureza Genética do Câncer de Tiroide na Idade

Adulta e na Faixa Etária Pediátrica e Aplicações na Prática Clínica”, processo

14/06570-6, com duração de 2015 a 2019). Além disso, para a realização do

trabalho houve a irrestrita colaboração de muitos centros brasileiros que

contribuíram com suas casuísticas.

7
1.INTRODUÇÃO

1.1. Neoplasias Endócrinas Múltiplas do tipo 2

As Neoplasias Endócrinas Múltiplas do tipo 2 (MEN 2, do inglês Multiple

Endocrine Neoplasia type 2) são síndromes genéticas autossômicas

dominantes constituídas por associações de tumores de origem endócrina. A

MEN 2 classifica-se em dois subtipos, de acordo com suas manifestações

clínicas: MEN 2A e MEN 2B (1-3). Apesar de serem consideradas doenças

raras, o diagnóstico mais precoce e a descoberta dos portadores

assintomáticos vem aumentando sua prevalência, de cerca de 1 caso para cada

200.000 ou 500.000 habitantes (4) para possivelmente 1: 80.000 habitantes (5).

Recentemente estabeleceu-se, na Noruega, país com dados epidemiológicos

de toda a população, que a incidência de MEN 2A é de 1: 66.438 nascimentos

vivos e a prevalência de 1:79.462 (6).

Sipple descreveu a MEN 2A em 1961, num mesmo paciente que

apresentava carcinoma medular de tiroide (CMT), feocromocitoma e

hiperparatiroidismo primário (7). A MEN 2B, descrita por Williams e Pollock,

em 1966, tem fenótipo distinto, pois inclui ganglioneuromatose, hábito

marfanoide e alterações oculares, além de CMT e de feocromocitoma (8).

A manifestação clínica mais importante e predominante de todos os

subtipos de MEN 2 é o CMT, que ocorre em mais de 95% dos indivíduos; seu

diagnóstico e tratamento precoce são de extrema importância, pois o CMT é a

principal causa de morte desses pacientes (1).

Há pouco mais de 20 anos descobriu-se que a MEN 2 é acarretada por

mutações germinativas do gene RET (9,10), que tem 21 éxons, localiza-se no

cromossomo 10 e codifica uma proteína altamente conservada entre as

espécies, que é um receptor transmembranal que contém tirosinaquinases em

8
sua porção intracelular. Na vigência de uma mutação, o receptor sofre ativação

constitutiva, o que causa a transmissão inapropriada de sinais intracelulares

reguladores da proliferação celular e a consequente formação de tumores (11).

A descoberta desse gene e das mutações que causam MEN 2 revolucionou o

diagnóstico e tratamento desses pacientes, pois proporcionou a identificação

de portadores assintomáticos que poderiam ser tratados profilaticamente.

Além disso, verificou-se que o fenótipo poderia variar na dependência do éxon

e do códon do RET afetado.

Tabela 1. Manifestações clínicas da Neoplasia Endócrina Múltipla (MEN) Tipo 2

Manifestações clínicas Frequência em %

MEN2A Carcinoma medular de tiroide 95-100

Feocromocitoma 50

Hiperpatiroidismo primário 15-20

Líquen Amiloide cutâneo < 10

Doença de Hirschsprung <2

MEN2B Carcinoma medular de tiroide 98-100

Feocromocitoma 50

Caracteres típicos 90

Hábito marfanoide

Neuromas de mucosa

Ganglioneuromatose intestinal

Hipertrofia dos nervos corneanos

A MEN2A é o subtipo mais frequente, representa 95% dos pacientes

com MEN2 e caracteriza-se pela ocorrência de CMT em 95% dos casos. A

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incidência de feocromocitoma e hiperparatiroidismo é variável de acordo com

a mutação encontrada no gene RET. A mutação mais frequentemente

verificada ocorre no códon 634 do éxon 11 e associa-se ao feocromocitoma e

ao hiperparatiroidismo primário em, respectivamente, 50% e 20% dos

pacientes. A MEN2A apresenta quatro variantes: clássica; MEN2A com líquen

cutâneo amiloidótico; MEN2A com doença de Hirschsprung; e carcinoma

medular de tiroide familiar (CMTF) (3) (Tabela 1).

O líquen amiloidótico cutâneo é uma lesão pruriginosa presente na

região interescapular (Figura 1), descrita apenas nos indivíduos portadores de

mutação no códon 634 do RET (12).

Figura 1. Líquen amiloidótico cutâneo em região dorsal de paciente com MEN2A

portadora de mutação no gene RET (códon 634). Fonte: Acervo do autor (1,12)

A doença de Hirschsprung ou aganglioneurose congênita caracteriza-se

pelo desenvolvimento anormal do sistema nervoso entérico, o que acarreta

constipação severa e obstrução intestinal no período neonatal. A causa da

doença são mutações inativadoras do RET; entretanto, por um mecanismo

10
ainda desconhecido, algumas famílias com MEN2A apresentam não apenas as

manifestações de MEN2A, mas também a doença de Hirschsprung (1-3,12).

O CMTF é caracterizado pela presença única do CMT em membros de

uma mesma família. Previamente considerado um subtipo de MEN, atualmente

é considerado uma variação fenotípica do MEN2A, com menor penetrância do

feocromocitoma e hiperparatiroidismo (3). O antigo critério diagnóstico era

desafiador e incluía, em uma de suas definições mais rígidas, a necessidade de

10 familiares afetados, com idade acima de 50 anos, sem história de

feocromocitoma e hiperparatiroidismo (3).

Figura 2. Ganglioneuromas na língua em paciente com MEN 2B. Fonte: Acervo do

autor (3,12)

MEN2B é a forma mais rara, porém, a mais agressiva de MEN2. Ela

caracteriza-se pelo desenvolvimento precoce do CMT em mais de 98% dos

pacientes, de feocromocitoma em 50% dos indivíduos e de caracteres

distintos, como ganglioneuromatose intestinal generalizada, hábito

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marfanoide, neuromas em lábios e terço distal da língua (Figura 2) e alterações

oculares (1-3,11,12). Aproximadamente 75% dos casos de MEN2B são

esporádicos, com mutação “de novo” do gene RET, ou seja, mutação surgida

no caso-índice (13). A ganglioneuromatose intestinal ocorre em 50% dos

pacientes e é causa de constipação, sintoma frequente já no primeiro ano de

vida, que pode causar complicações mais graves (3,13). A presença de

constipação intestinal associada a neuromas em lábios e língua em uma criança

deve levantar a suspeita de MEN2B e deve suscitar a avaliação do CMT por

meio da ultrassonografia (USG) da tiroide e dosagem de calcitonina sérica. As

alterações oculares descritas nos pacientes com MEN2B são choro sem

lágrimas na infância, pálpebras evertidas e espessadas, ptose leve e nervos

corneanos evidentes. As alterações esqueléticas dessa síndrome incluem face

alongada, pé cavo, pectus escavatum, palato arqueado, escoliose e luxação da

epífise femoral (1-3,12-14).

1.2. Apresentação clínica do Carcinoma medular de tiroide

O CMT, descrito em 1959 por Hazard e cols. (15) é, como sabemos, um

tumor neuroendócrino originário das células C ou parafoliculares. Estas células

têm o potencial de secretar diversos peptídeos, incluindo a calcitonina e o

antígeno carcinogênico embrionário (CEA), utilizados como marcadores

tumorais.

O CMT corresponde a cerca de 1 a 2% dos cânceres de tiroide segundo

estatísticas norte-americanas (3). No Brasil, num trabalho comparativo entre a

incidência geral de câncer de tiroide no Estado de São Paulo com os Estados

Unidos da América, a incidência anual por 100.000 indivíduos em São Paulo foi

de 0,56 em mulheres e 0,15 em homens, enquanto que a norte-americana foi

de 0,17 em mulheres e 0,13 em homens (Tabela 2) (16). Assumindo que a

incidência de CMT no Brasil (17) seja semelhante à paulista, em 2016 seriam

12
 diagnosticados cerca de 578 casos novos de CMT em mulheres e 148 em

homens no Brasil.

Tabela 2. Incidência de tumores de tiroide em São Paulo e nos Estados Unidos

São Paulo (n=15.892) SEER (n=42.717) RTI São Paulo /

SEER
Feminino Masculino Feminino Masculino

Casos Incidênciaa Casos Incidênciaa F/M Casos Incidênciaa Casos Incidênciaa F/M Feminino Masculino

Total 13.313 18,43 2.579 4,42 4,17 32.562 11,17 10.155 3,60 3,10 1,65 1,23
Papilífero 9607 13,22 1818 3,05 4,33 28414 9,85 8219 2,92 3,37 1,34 1,04
Folicular 1650 2,32 299 0,54 4,3 2879 0,95 1128 0,40 2,38 2,44 1,35
3,7 1,3
Medular 390 0,56 85 0,15 596 0,17 354 0,13 3,29 1,15
3 1
Anaplásico 93 0,13 33 0,07 1,86 279 0,06 187 0,06 1,0 2,17 1,17
Outros
143 0,21 59 0,11 1,91 242 0,07 151 0,05 1,40 3,0 2,20
específicos
Inespecíficos 1429 2,0 285 0,50 4,0 242 0,07 116 0,04 1,75 28,57 12,50

Abreviaturas: SEER (The Surveillance, Epidemiology, and End Results Program of the

National Cancer Institute) – Programa de Vigilância, Epidemiologia e Resultados Finais

do Instituto Nacional de Câncer dos Estados Unidos da América; RTI – Razão da taxa

de incidência; F/M – Razão Feminino/Masculino a

Valores ajustados por faixa etária

por 100.000/casos-ano (16)

Macroscopicamente, o CMT apresenta-se como uma massa sólida,

branco-acinzentada, frequentemente dura, bem circunscrita, porém não

encapsulada. À microscopia óptica, apresenta células com citoplasma granular

abundante que, à microscopia eletrônica, aparecem como pequenos grânulos

de secreção elétron-densos limitados por membrana. As células podem ser

fusiformes, poligonais ou ovais, com núcleo central. As figuras mitóticas são

pouco frequentes. A presença de amiloide é considerada uma característica do

CMT. O amiloide do CMT difere do encontrado em outras doenças e é

formado, possivelmente, a partir da calcitonina ou da pró-calcitonina (1-3).

Enquanto o CMT esporádico tende a ser unifocal, o CMT hereditário é

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multicêntrico e bilateral. A lesão inicial do CMT hereditário é a hiperplasia das

células C, que progride para CMT microscópico e, posteriormente, para CMT

macroscópico (1-3, 13). O desenvolvimento dessas alterações está diretamente

relacionado à agressividade da mutação do RET. Com mutações mais

agressivas, associadas à MEN2B, estas alterações podem ser observadas já no

1o. mês de vida (1) e em MEN2A por volta dos 3 a 5 anos de idade (1-3).

Com a disponibilidade do teste genético, o modo de apresentação do

paciente com CMT hereditário tem se modificado. A apresentação clínica mais

frequente do paciente-índice, ou seja, daquele indivíduo sem história familiar

de CMT, continua a ser a descoberta de um nódulo tiroidiano com ou sem

linfomegalia cervical palpável ou identificado por meio da USG. A partir do

diagnóstico de CMT na família e da identificação de uma mutação do RET no

paciente-índice, os familiares deverão ser submetidos à avaliação genética e

aqueles portadores de mutação deverão ser avaliados e tratados

precocemente, de preferência antes do desenvolvimento de doença

macroscópica.

Em pacientes que se apresentam com CMT clinicamente aparente, o

envolvimento de linfonodos regionais é muito comum. Aproximadamente 60 a

80% dos pacientes com CMT palpável (> 1 cm) possuem adenomegalia

cervical (1-3,13). O envolvimento de linfonodos do compartimento central

(nível VI) e da cadeia cervical (II-V) ipsilateral ao tumor é o mais frequente;

entretanto, pode ocorrer o envolvimento de linfonodos cervicais contralaterais

em até 40% dos pacientes com doença palpável (1-3,13). Aproximadamente

10% dos pacientes com CMT apresentam, ao diagnóstico, evidência de

doença metastática à distância (1). Portanto, após o diagnóstico de CMT,

recomenda-se que se faça uma avaliação da extensão da doença. Esta

avaliação deve incluir as dosagens de calcitonina e CEA, além de exames de

14
imagem. Uma parte dos pacientes, geralmente com CMT disseminado, pode

apresentar diarreia e “flushing”. Em casos raros de CMT, a produção ectópica

de ACTH pode causar a síndrome de Cushing (1-3).

É importante ressaltar que a forma mais agressiva de CMT hereditário

ocorre em pacientes com MEN2B. Nesses casos, o CMT está frequentemente

associado a metástases nos linfonodos cervicais nos primeiros anos de vida e a

morte por doença metastática pode ocorrer na 2a. ou 3a. décadas de vida (13).

1.3. Diagnóstico do CMT

O diagnóstico do CMT em um paciente com doença clinicamente

aparente é feito pela citologia aspirativa por agulha fina (CAAF) do nódulo

tiroidiano ou dos linfonodos cervicais. Quando a CAAF é suspeita ou

consistente com CMT, recomenda-se dosar a calcitonina e o CEA séricos,

assim como excluir hiperparatiroidismo primário e feocromocitoma. Os

achados citológicos sugestivos de CMT são hipercelularidade, células

parafoliculares fusiformes agrupadas ou isoladas, que podem ser binucleadas

com núcleo excêntrico e cromatina grosseira granular e amiloide. Entretanto, a

CAAF apresenta sensibilidade ao redor de 50% para o diagnóstico de CMT

(18,19). Para melhorar essa taxa, pode-se realizar a dosagem da calcitonina no

aspirado da punção do nódulo suspeito e imunocitoquímica para detectar a

presença de calcitonina, na ausência de tiroglobulina (20).

A avaliação pré-operatória dos indivíduos com CMT inclui exames de

imagem para definição da extensão da doença. O nível da calcitonina basal

auxilia na escolha dos exames de imagem; calcitonina inferior a 500 pg/mL

sugere doença localizada, mas é apropriada realização de USG e/ou TC

cervical para avaliação dos linfonodos. Em pacientes com calcitonina superior a

500 pg/mL, recomenda-se a avaliação de metástases à distância com

15
tomografia de tórax, cintilografia óssea e ressonância magnética ou tomografia

de abdômen, com contraste, para a detecção de metástases hepáticas (1-3).

Sempre que houver o diagnóstico de CMT, recomenda-se a

genotipagem do RET, pois quando da identificação de uma mutação, além de

informações importantes sobre o fenótipo de MEN2, temos a possibilidade da

avaliação dos familiares. Em todos os pacientes suspeitos de CMT em que não

temos o resultado da avaliação do RET no pré-operatório, é conveniente que

se faça o rastreamento de feocromocitoma para diminuir a morbimortalidade

intra e pós-operatória (1-3,13).

1.4. GENOTIPAGEM DO GENE RET EM MEN2

O gene RET foi inicialmente identificado em 1985 (21). Encontra-se no

braço longo do cromossomo 10 (10q11.2) e codifica a proteína RET, um

receptor transmembranal com domínio para tirosinaquinases (TK). A ativação

desta quinase é responsável pela diferenciação e pelo crescimento de vários

tecidos em desenvolvimento, incluindo tecidos derivados dos arcos branquiais

(paratiroide), da crista neural (cérebro, gânglios simpático e parassimpático,

células C da tiroide, medula adrenal e gânglios entéricos), além do sistema

urogenital (22).

Em 1987, dois anos após a identificação inicial do RET no cromossomo

10, o defeito genético causador da NEM 2A foi localizado neste mesmo

cromossomo (23). Menos de uma década depois, em 1993 e 1994, foram

demonstrados, respectivamente, que a NEM 2A (9,10) e a NEM 2B (23-26) são

causadas por mutações germinativas no RET herdadas de forma autossômica

dominante. Atualmente já se identificaram mais de 100 alterações genéticas

envolvendo o RET na gênese das NEM 2, dentre mutações, duplicações,

inserções e deleções (22,27).

16


Figura 3. Formas de ativação do receptor RET. (A) Em condições normais, a ligação do

GNDF (glial cell-derived neurotropic growth fator) ou outro ligante ao receptor ativa a
TK; (B) Na NEM 2A e CMTF, quando ocorre mutação de domínio extracelular no RET
as cisteínas formam pontes dissulfeto ativadoras no receptor mutado, induzindo uma
dimerização independente da presença de ligante; (C) No CMTF, quando ocorre
mutação de domínio intracelular no RET com ativação como monômeros,
independente de ligante; (D) Na NEM 2B, a mutação mais frequentemente observada
(M918T) afeta a qualidade dos sinais intracelulares, alterando a especificidade do
substrato da TK de uma quinase típica de receptor (sempre com metionina na posição
918) para uma quinase típica do citoplasma (com a treonina na posição 918),
causando ativação do receptor sem necessidade de dimerização. Modificado a partir
da referência (27)

Desta forma, mutações ativadoras do RET causam MEN2A, MEN2B e

CMTF, enquanto as inativadoras promovem a doença de Hirschsprung (1-

3,12,22,27).

17
 Início do Início do
Códons com Classificação Tiroidectomia rastreamento rastreamento
Mutações de risco Profilática Feocromocitoma Hiperparatiroidismo
533/611/620/631/ Moderado Ao redor de 16 anos 16 anos
666/768/790/804/ 5 anos
891/912
C 634F/G/R/S/W/Y Alto Até 5 anos 11 anos 11 anos
A883F

M918T Altíssimo Até 1 ano 11 anos _____

Tabela 3. Classificação de risco das mutações do RET associadas a MEN2 e

recomendação de idade de tiroidectomia profilática e início do rastreamento para

feocromocitoma e hiperparatiroidismo

Já se evidenciaram mutações associadas a MEN2 nos éxons 5, 8, 10, 11,

13, 14, 15 e 16 do RET. As mutações mais associadas a MEN2A e CMTF

envolvem os éxons 10 (códons 609, 611, 618 e 620) e 11 (códon 634), que

codificam o domínio extracelular rico em cisteína, com a substituição de uma

cisteína por outro aminoácido (Tabela 3, Figura 4). Em MEN2A, a maioria das

famílias apresentam uma mutação no códon 634; destas, a mais comum resulta

na substituição de cisteína por arginina (C634A) (1-3,13,22,27,28). As mutações

que envolvem o códon 634 são tipicamente associadas não só ao CMT, mas

também ao feocromocitoma, hiperparatiroidismo primário e ao líquen

amiloidótico cutâneo (1-3). Mutações que envolvem o domínio intracelular das

tirosinaquinases também são descritas em famílias com MEN2A e CMTF, mas

menos frequentemente. Mutações no domínio intracelular das tirosinaquinases

são responsáveis por MEN2B (1-3,12,22,27). Mais de 95% dos indivíduos com

MEN2B apresentam uma única mutação que substitui metionina por treonina

no códon 918 (M918T) (1,3,13,22,27).

18
Figura 4. Gene RET, proteína RET e mutações do RET que causam MEN2A, MEN2B e

FMTC. A estrutura do gene RET com éxons 1 a 20 está no centro, em cinza. A

proteína RET está à esquerda, com os resíduos de amino-ácidos 1 a 1114 e as

porções extra-celular (peptide-sinal (SP), domínios similes à caderina (CLDs) e

domínio rico em cisteínas (CRD); transmembranal (TM) e intracellular, com os domínios

tirosinaquinases (TKs). As mutações causadoras de MEN2A e MEN2B são mostradas à

direita em preto e as causadoras de FMTC em vermelho (22).

19
 Mutações germinativas do RET também são descritas em 6 a 7% dos

indivíduos com CMT aparentemente esporádico, ou seja, indivíduos sem

história familiar e com manifestação tardia do CMT (1,3). Por essa razão,

recomenda-se a avaliação genética de todos os pacientes com CMT pois a

identificação de um indivíduo com uma mutação no RET, frequentemente

resulta na identificação de outros portadores da mutação na família (3,13).

A avaliação genética do RET é feita por meio do sequenciamento direto

de produtos de reação em cadeia de polimerase (PCR) provenientes da

amplificação dos éxons 5, 8, 10, 11, 13, 14, 15, 16, região que abrange mais

de 99% das mutações.

1.5. IMPACTO DA AVALIAÇÃO GENÉTICA NA CONDUTA DO

PORTADOR ASSINTOMÁTICO

O que distingue MEN2 de outras síndromes genéticas associadas ao

câncer é o fato de que a avaliação genética altera a conduta do paciente.

Como a tiroidectomia total (TT) é uma intervenção segura, recomenda-se a

realização da TT em todos os portadores de uma mutação do gene RET,

idealmente antes que haja metástases para linfonodos cervicais. A experiência

atual indica que a TT profilática resulta na melhora da morbidade e

mortalidade desses pacientes (3). Os consórcios internacionais já definiram a

idade para se realizar a TT profilática, de acordo com a agressividade da

mutação (Tabela 3, Figura 4 ) (3,13). A mutação M918T associada ao MEN2B é

classificada com risco altíssimo e a TT é indicada durante o primeiro ano de

vida e o esvaziamento do compartimento central (nível VI) deve ser realizado

apenas se houver evidência de metástases linfonodais, evitando-se ao máximo

o risco de comprometer as glândulas paratiroides (3). Crianças com MEN2B

com mais de 1 ano de idade devem ser submetidas à TT com esvaziamento do

20
nível VI.

A mutações no códon 634 associadas a MEN2A e a mutação A883F

associada a MEN 2B são classificadas como risco alto e a TT deve ser realizada

antes dos 5 anos de idade. As demais mutações associadas ao MEN2A são

classificadas como risco moderado, mais indolentes e ainda não há um

consenso quanto à melhor idade a se realizar a TT profilática, pois o

desenvolvimento do CMT é variável e pode ocorrer mais tardiamente.

Atualmente, existem duas recomendações aceitáveis e a decisão de como

proceder deve ser individualizada, de acordo com a evolução clínica do CMT

em cada família. Uma opção é realizar o teste de estímulo da calcitonina com

cálcio endovenoso e indicar a TT quando esse teste se tornar anormal (29),

além de acompanhar anualmente, com calcitonina sérica e USG da tiroide.

Outra opção é adotar a idade de 5 anos para se recomendar a TT profilática

(Tabela 3) (3,13). Nas mutações de risco alto ou moderado, o esvaziamento

linfonodal do compartimento central no momento da tiroidectomia profilática,

deve ser realizado se houver suspeita clínica ou radiológica ou quando os

níveis de calcitonina basal no pré-operatório forem maiores do que 40 pg/mL

(3).

Além da TT profilática, os portadores de MEN2A e MEN2B devem ser

submetidos a um rastreamento anual para excluir feocromocitoma e o

hiperparatiroidismo primário. O rastreamento do feocromocitoma deve ser

iniciado a partir dos 11 anos de idade nos portadores de mutação de altíssimo

risco. Indivíduos com MEN2A associada à mutação de risco alto devem ser

rastreados para feocromocitoma e hiperparatiroidismo a partir dos 11 anos de

idade e em mutações de risco moderado, por sua vez, o início pode ser aos 16

anos (Tabela 3) (3,13).

21
1.6. RESUMO DA INTRODUÇÃO E OBJETIVOS

MEN2 é uma síndrome autossômica dominante caracterizada pelo

desenvolvimento do CMT, feocromocitoma e hiperparatiroidismo primário. A

manifestação principal é o CMT, que é o primeiro tumor a se desenvolver e é a

causa de morte mais frequente. Nos últimos 20 anos, a elucidação da alteração

genética e a experiência obtida com a correlação do genótipo e fenótipo

resultaram em um avanço surpreendente no diagnóstico e na conduta de

pacientes portadores de uma mutação no gene RET.

No indivíduo que se apresenta com CMT, a identificação da mutação

genética indica o risco do paciente desenvolver outras manifestações

associadas à MEN2 e a conhecer melhor o prognóstico da sua doença. Nos

familiares, a análise genética serve para identificar os indivíduos sem mutação,

que não precisarão mais de seguimento, pois não apresentarão risco de

desenvolver as manifestações associadas à MEN2; e detectar precocemente os

indivíduos portadores de mutação, permitindo, dessa maneira, o tratamento

precoce do CMT e do feocromocitoma. Além disso, o conhecimento de que

RET é um receptor tirosinaquinase propiciou o desenvolvimento de novas

abordagens terapêuticas, com potencial de tratar o CMT metastático.

Considerando-se os fatos acima, os objetivos deste estudo são:

1. colecionar os dados de genotipagem do gene RET do maior

número possível de famílias brasileiras com MEN2/FMTC;

2. analisar a distribuição e frequência das mutações do RET;

3. comparar o tipo e a prevalência das mutações do RET com outros

países.

22
2. PACIENTES E MÉTODOS

Os vários centros brasileiros dedicados a MEN2 têm publicado

extensivamente sobre vários aspectos da moléstia; porém, ainda não tinha sido

feita no país uma apresentação relativa à prevalência dos genótipos e

fenótipos das famílias com MEN2 de modo abrangente, objetivo desta

memória.

Convidamos a maioria destes centros, Universidade Federal de São

Paulo (UNIFESP), Universidade Federal do Rio Grande do Sul (UFRGS),

Universidade de São Paulo (São Paulo, FMUSP e Ribeirão Preto, FMRPUSP) ,

Universidade de Fortaleza (UNIFOR), Instituto Nacional do Câncer (INCA),

Universidade Federal do Paraná (UFPR), Universidade Federal de Minas Gerais

(UFMG), Universidade de Campinas (UNICAMP), Universidade Estadual

Paulista (UNESP) e Universidade Federal do Espírito Santo (UFES), a preencher

um questionário padronizado contendo informações a respeito das mutações

do RET e dos fenótipos encontrados nas diversas famílias.

Cada centro realizou a avaliação clínica individual dos pacientes e das

famílias para a classificação fenotípica, de acordo com os critérios propostos

pela American Thyroid Association (3) e pela Sociedade Brasileira de

Endocrinologia e Metabologia (30). Cada centro também estadiou seus

pacientes de acordo com o sistema de classificação TNM (tumor, linfonodos,

metástases da American Joint Committee on Cancer (AJCC)/Tumor Nodes

Metastasis (TNM), conforme apresentado na Tabela 4.

23
Tabela 4. Estadiamento pós-operatório do CMT. Adaptado do AJCC Cancer Staging Manual
(98)

T- Tumor primário N- Metástases linfonodais M- Metástases à distância

Tx- não pode ser avaliado Nx- não pode ser avaliado Mx- não pode ser avaliado
T1- tumor de até 2cm N0- ausente M0- ausente
limitado à tiroide N1a- metástases em linfonodos M1- presente
(T1a: <1cm e T1b: 1- 2cm) no nível VI (pré-traqueal,
T2- tumor >2cm até 4cm paratraqueal, pré-laríngeo)
limitado à tiroide N1b- metástase cervical
T3- tumor >4cm ou com unilateral, bilateral ou
extensão extratiroidiana contralateral ao tumor ou
mínima mediastinal superior
T4a- tumor de qualquer
tamanho que se estende
além da cápsula tiroidiana,
invadindo partes moles,
laringe, traqueia, esôfago
ou nervo laríngeo
recorrente

T4b- tumor invade fáscia

pré-vertebral ou artéria
carótida/vasos mediastinais
Estadiamento Grupos
I T1, N0, M0

II T2, N0, M0
T3, N0, M0

III T1, N1a, M0

T2, N1a, M0
T3, N1a, M0

IV A T4a, N0, M0
T4a, N1a, M0
T1, N1b, M0
T2, N1b, M0
T3, N1b, M0
T4a, N1b, M0

IV B T4b, qualquer N, M0

IV C Qualquer T, qualquer N, M1

24
 Após o recebimento dos questionários dos vários centros, organizamos

as diversas planilhas, estruturadas a partir de um único modelo.

Obtivemos o consentimento informado dos pacientes e das comissões

de ética em pesquisa em todos os centros. Todos os indivíduos receberam

aconselhamento pré e pós-teste genético. Na Universidade Federal de São

Paulo este estudo recebeu a aprovação número CEP 1749/06.

Realizamos a genotipagem do RET por meio do DNA extraído das

amostras de sangue dos pacientes e famílias dos centros da UNIFESP,

UNIFOR, INCA, UFPR, UFMG, UNICAMP e UFES no Laboratório de

Endocrinologia Molecular e Transalacional, de acordo com técnica já descrita,

que analisa todos os éxons (31). Nos demais centros a genotipagem realizou-

se localmente, por meio de técnicas semelhantes, investigando-se as mutações

descritas na literatura. Quando uma mutação era identificada ao caso índice,

os demais membros da família eram sequenciadas apenas no éxon específico

da mutação.

Para a análise estatística dos dados apresentamos primeiramente as

médias, medianas, valores máximos e mínimos; utilizamos os percentuais

considerando o valor total de casos para um fenótipo ou genótipo específico.

Analisamos as variáveis categóricas por meio do teste do qui-quadrado com

correção de Fisher quando apropriado. Utilizamos o software “IBM SPSS

Statistics for Windows, version XX” (IBM Corp., Armonk, NY, EUA).

Consideramos significante p<0,05.

25
3. RESULTADOS

Apresentamos na Tabela 4 os resultados clínicos e moleculares dos 555

pacientes, provenientes de 150 famílias, que indicam 34 mutações

germinativas diferentes em 16 códons dos éxons 8,10,11,13,14,15 e 16 do

gene RET. Conseguimos, em nossa série, determinar a mutação em todos os

pacientes com MEN 2, diferentemente de outros países, que apresentam

pequena proporção de pacientes com genotipagem negativa do RET.

As mutações mais frequentemente encontradas, classificando-as pelo

número de famílias afetadas, ocorrem nos códons 634, éxon 11 (n=56), códon

918, éxon 16 (n=34) e códon 804, éxon 14 (n=20). Quando analisamos as

mutações pelo número absoluto de pacientes com diagnóstico estabelecido

de CMT após a cirurgia, as mutações mais frequentemente encontradas

encontram-se nos códons 634, éxon 11 (n=260, 46,8%), 533, éxon 8 (n=59,

10,6%), 620, éxon 10 (n=56, 10,1%) e 918, éxon 16 (n=52, 9,3%). Dos 260

pacientes afetados por mutação no códon 634, 168 apresentavam a mutação

C634Y (64,6%), 73 a C634R (28,1%), 9 a C634W (3,5%), 7 a C634G (2,7%) e 3 a

C634S (1,2%) (Tabela 4).

Depois do CMT, a associação mais frequentemente verificada entre os

pacientes com MEN2 é o feocromocitoma (21,3% do total dos pacientes com

CMT hereditário). Esta incidência é concentrada no códon 634 (78,8% do total

de pacientes com feocromocitoma e 36% de todos os indivíduos com mutação

no 634), seguido pelo códon 918 (10,2% do total de casos de feocromocitoma

e 36% de todos com mutação no 918) e pelo códon 620 (5,9% do total dos

casos de feocromocitoma e 13% de todos com mutação no 620).

Hiperparatiroidismo e líquen amiloidótico cutâneo são exclusivos dos

pacientes com mutação no códon 634, com 44 e 42 casos, respectivamente.

Por outro lado, casos de doença de Hirschsprung foram vistos apenas nos

portadores de mutação no códon 620 (Tabela 4).

26
 A Tabela 5 mostra a prevalência dos dados do BRASMEN em relação ao

gênero, idade de aparecimento do CMT, classificação do TNM e porcentagem

de lesões T3 e T4, N1 e M1. Na coluna final acrescentamos, para comparação,

os pacientes com CMT esporádico, pois não apresentaram mutações do RET.

Nota-se a preponderância de mulheres, tanto na série de CMT

hereditário (64%), como esporádico (61%). Quanto à idade, a paciente mais

jovem de nossa série tinha 5 anos e apresentava mutação no códon 634.

Infelizmente, porém, para a mutação do códon M918T, a mais agressiva, pois

causa MEN2B, a idade mais jovem ao diagnóstico foi 6 anos, o que demonstra

que nossa comunidade médica não está diagnosticando precocemente esta

moléstia, apesar dos sinais exuberantes do fenótipo de MEN2B. Como visto na

introdução, uma série destas crianças poderia ter sido diagnosticada nos

primeiros anos de vida, sem apresenta lesões tumorais grandes e sem

metástases linfonodais ou à distância. Outras mutações detectadas em crianças

ocorreram nos códons 611, 618, 620, do éxon 10 e 630, do éxon 11,

consideradas de gravidade moderada na nova classificação da ATA, mas que

afetaram as crianças com diagnóstico de CMT na nossa série entre 8 e 10 anos

de idade, com tumores com elevada porcentagem de lesões T3 e T4 ao TNM.

As medianas mais baixas de idade ao diagnóstico aconteceram com as

mutações nos códons M918T, C634 e C630R. Por outro lado, os pacientes

afetados mais tardiamente portavam as mutações nos códons V804L, V804M,

além dos esporádicos (Tabela 5).

Os dados referentes à análise dos CMT de acordo com a classificação

TNM, entre os pacientes que apresentaram número significativo de casos para

análise (n=10), evidenciam que os pacientes portadores das mutações nos

códons C618 e V804L, além daqueles com CMT esporádico, são os que

apresentam proporção mais elevada de lesões T3 e T4 (50%, 42,9% e 30,4%,

respectivamente); além disso, como sabido, já se apresentam, ao diagnóstico,

27
com metástases aos linfonodos (M1). Infelizmente, vários pacientes

apresentaram-se com metástases à distância no momento do diagnóstico,

como 50% daqueles com mutação no códon M918T; 23,8% daqueles com

mutações no códon C620; 15,5% daqueles com mutações no códon C634. Os

pacientes com CMT esporádico apresentaram-se com metástases à distância

em 26,8% dos casos (Tabela 5).

Quando comparamos os resultados do BRASMEN (n=555) com as

maiores casuísticas publicadas na literatura, EUROMEN (n=145), ITAMEN

(n=246) e a Alemã (n=205), vemos várias semelhanças, mas também

divergências (Tabela 6). Assim, enquanto que as mutações nos códons S891,

V804, G768, C609, C611, C630, C634 e M918T apresentam prevalências

similares, algumas tem baixa prevalência (códons L790 e C618) e outras são

registradas principalmente no Brasil, como nos códons G533C, 804L e M918V.

A Tabela 7 mostra os dados da comparação da penetrância do

feocromocitoma na mutação do códon G533C (Tabela 7). Assim, em 193

pacientes portadores da mutação G533C encontrou-se 1 paciente com

feocromocitoma em 119 no Brasil (0,84%), 3 pacientes em 53 na Grécia

(5,66%) (39) e 3 em 21 nos Estados Unidos (14,3%) (40), ou seja, uma diferença

significativa no número de pacientes com feocromocitoma nos Estados Unidos

e Europa quando comparados ao Brasil (Tabela 7). Além disso, diferentemente

do que é encontrado no Brasil, o feocromocitoma foi a manifestação inicial do

MEN2 nos Estados Unidos e na Grécia (Tabela 7).

28
Tabela 4. Distribuição das mutações do RET no Brasil, classificadas pelo codon, exon, estratificação de risco, número de famílias e
número de pacientes afetados por CMT, feocromocitoma (FEO), líquen amiloidótico cutâneo (LAC) e doença de Hirschsprung
(HSCR). * Porcentagem de casos em relação ao número total de pacientes afetados; ** porcentagem de casos com o mesmo
codon afetado
N° famílias N° pacientes com CMT HPTH
Mutação do RET Exon Classificação de risco FEO (%∗/%∗∗) LAC (%∗/%∗∗) HSCR (%∗/%∗∗)
com CMT (%∗) (%∗/%∗∗)
G533C 8 Moderado 1 59 (10.6) 1 (1/2) 0 0 0
C609G/R/S/Y/W Moderado 7 14 (2.5) 1 (1/7) 0 1 (2/7) 0
C611G/R/Y/W Moderado 4 12 (2.2) 1 (1/8) 0 0 0
10
C618F/R/S Moderado 6 11 (2.0) 3 (3/27) 1 (2/9) 0 0
C620G/R/S Moderado 6 56 (10.1) 7 (6/13) 2 (4/4) 0 6 (100/11)
C630R Moderado 1 2 (0.4) 0 0 0 0
D631Y Moderado 0 0 0 0 0 0
11
C634F/G/R/S/W/Y Alto 56 260 (46.8) 93 (79/36) 44 (92/17) 42 (98/16) 0
K666E Moderado 0 0 0 0 0 0
E768D Moderado 6 15 (2.7) 0 0 0 0
13
L790F Moderado 2 3 (0.5) 0 0 0 0
V804L Moderado 7 13 (2.3) 0 0 0 0
14
V804M Moderado 13 34 (6.1) 0 0 0 0
A883F Alto 0 0 0 0 0 0
15
S891A Moderado 7 24 (4.3) 0 0 0 0
R912P Moderado 0 0 0 0 0 0
M918T 16 Moderado 26 33 (5.9) 12 (10/36) 1 (2/3) 0 0
M918V Altíssimo 8 19 (3.4) 0 0 0 0
Total 150 555 118 48 43 6
29
Tabela 5. Prevalência do CMT hereditário de acordo com as várias mutações e do CMT esporádico, considerando gênero, idade,
TNM (7th. AJCC), porcentagem de lesões T3 e T4, N1 e M1
CMT hereditário CMT
Mutação do RET G533C C609 C611 C618 C620 C630R C634 E768D L790F V804L V804M S891A M918T M918V esporádico

Gênero Masculino 24 4 4 1 22 2 112 1 2 3 9 5 13 5 92
Feminino 35 7 8 10 34 0 140 13 1 10 20 18 19 14 197
Idade Mediana 47 39 40 36 21 22 27 41 41 55 44 51 16 51 50
Média 45 39 39 37 24 22 28 49 NA 54 50 43 17 46 49
Mínimo 21 25 10 10 8 10 5 38 NA 35 30 21 6 24 12
Máximo 72 57 52 65 41 34 73 68 NA 77 74 65 33 67 83
7th AJCC TNM T1 31 2 2 2 34 1 113 8 1 2 18 4 9 11 83
T2 15 2 1 3 4 0 56 3 0 2 1 6 10 4 68
T3 4 0 2 4 4 1 15 0 0 2 3 1 3 3 43
T4 0 0 0 1 0 0 7 0 0 1 1 1 4 1 23
N1 16 2 3 4 28 0 109 5 1 6 12 5 22 12 110
M1 0 0 0 1 10 0 24 0 0 0 1 0 11 2 26
% T3 e T4 7,1 0,0 40,0 50,0 9,5 50,0 11,5 0,0 0,0 42,9 17,4 16,7 26,9 21,1 30,4
% N1 35,7 66,7 50,0 44,4 66,7 0,0 56,2 62,5 100,0 66,7 57,1 45,5 78,6 63,2 57,0
% M1 0,0 0,0 0,0 11,1 23,8 0,0 15,5 0,0 0,0 0,0 5,0 0,0 50,0 12,5 26,8
30
Tabela 6. Comparação dos 3 maiores estudos e o BRASMEN
Mutacão
EUROMEN % Itália % Alemanha % BRASMEN %
RET
M918T 4 2.8 20 8.1 32 15.6 33 5.9
M918V 0 0.0 0 0.0 0 0.0 19 3.4
Ala883 0 0.0 1 0.4 0 0.0 0 0.0
Cys634 98 67.6 86 35.0 73 35.6 260 46.8
Asp631 0 0.0 0 0.0 0 0.0 0 0.0
Cys630 1 0.7 4 1.6 1 0.5 2 0.4
Cys620 10 6.9 9 3.7 14 6.8 56 10.1
Cys618 10 6.9 15 6.1 11 5.4 11 2.0
Cys611 4 2.8 1 0.4 6 2.9 12 2.2
Cys609 1 0.7 6 2.4 1 0.5 14 2.5
G533C 0 0.0 0 0.0 0 0.0 59 10.6
Glu768 1 0.7 9 3.7 2 1.0 15 2.7
Leu790 7 4.8 8 3.3 26 12.7 3 0.5
Tyr791 3 2.1 1 0.4 14 6.8 0 0.0
Val804 3 2.1 52 21.1 19 9.3 47 8.5
Ser891 3 2.1 23 9.3 6 2.9 24 4.3
Cys515 0 0.0 1 0.4 0 0.0 0 0.0
Lys666 0 0.0 1 0.4 0 0.0 0 0.0
Met848 0 0.0 1 0.4 0 0.0 0 0.0
Ser904 0 0.0 1 0.4 0 0.0 0 0.0
Thr338 0 0.0 1 0.4 0 0.0 0 0.0
Sem
0 0.0 6 2.4 0 0.0 0
mutação 0.0
TOTAL 145 100 246 100 205 100 555 100
VUS (variações de significado incerto) foram excluídas
 Tabela 7. Prevalência de feocromocitoma em portadores de G533C de 3 continentes
Brasil Grécia EUA p
Total de pacientes estudados 432 192 47 -
n de portadores RET G533C 119 (27,5%) 53 (27,6%) 21 (44,7%) 0,0439
n de famílias 1 15 1 -
N de casos de Feo 1/119 (0,84%) 3/53 (5,66%) 3/21 (14,3%) 0,0064
Idade média do diagnóstico em
anos (intervalo) 63 49,6 (35-66) 41 (29-65) -
Unilateral 1/1 (100%) 2/3 (66,6%) 3/3 (100%) ns
Bilateral 0/1 (0%) 1/3 (33,3) 0/3 (0%) ns
Manifestacão inicial 0/1 (0%) 3/3 (100%) 3/3 (100%) 0,0302

32
4. Discussão

A epidemiologia molecular avalia a contribuição dos fatores de risco

genético na etiologia, distribuição geográfica e prevenção de uma determinada

doença. Poucas áreas da medicina tem sido afetadas de modo tão claro pelo

diagnóstico genético como a MEN2, uma vez que as alterações ocasionadas

pelas mutações do RET apresentam penetrância superior a 95% para o

desenvolvimento do CMT. Assim, quase que invariavelmente, um indivíduo

afetado por mutação do RET desenvolverá CMT. Como o CMT é uma doença

agressiva que pode levar à morte, é essencial que se ache precocemente os

indivíduos afetados assintomáticos para a realização do tratamento profilático

pela tiroidectomia e se evite o estabelecimento da doença.

Outro aspecto que deveria ser considerado nas análises destes dados é

que a maior parte da literatura sempre apresenta os resultados de genotipagem

do RET em relação ao número de famílias. Porém, como demostramos

recentemente, em estudos de ancestralidade e gene fundador (32,33), é

possível que muitas famílias aparentemente não relacionadas apresentem, de

fato, um ancestral comum e, assim, não seriam várias famílias, mas apenas uma.

Um bom exemplo é a família por nós descrita portadora da mutação no códon

G533C (34). A concentração da família numa única cidade, o hábito de se

33
reunirem frequentemente e preocuparam-se com sua genealogia e com ajuda

mútua, fez com que eles se considerassem uma única família, apesar de seu

tamanho imenso, com mais de 700 membros entre afetados e não-afetados na

última avaliação (35). Por outro lado, as 8 famílias independentes descritas por

nós inicialmente como portadoras da mutação M918V são, na verdade,

descendentes de um único ancestral e deveriam ser consideradas como uma

única família; porém, por viverem em cidades diferentes e não se reconhecerem

como parentes, não são considerados como sendo da mesma família (32).

À medida que as várias mutações do gene RET foram sendo descritas e

seus respectivos fenótipos estudados, percebeu-se uma correlação entre

genótipos e fenótipos e que seria possível o estabelecimento de diretrizes

referentes ao momento ideal da tiroidectomia profilática, da percepção de

quais pacientes poderiam ser portadores de hiperparatiroidismo ou

feocromocitoma ou dos que teriam mais chance de apresentar metástases no

início da doença. Em 2001, por ocasião do 7o. International Workshop sobre

MEN2 e MEN1, propôs-se uma diretriz que baseava a indicação da

tiroidectomia profilática pelo genótipo do RET (36), que foi seguida por

refinamentos decorrentes de novos achados que evidenciaram que o

tratamento profilático era, de fato, justificado, pois reduziu a morbidade e a

34
mortalidade destes pacientes. Estas diretrizes evoluíram para os consensos da

Sociedade Americana de Tiroide, ATA, de 2009 e 2015 (3,13).

Porém, com a evolução dos estudos em termos da ampliação do número

de pacientes e de geografias estudadas, vem se percebendo alguma

heterogeneidade entre os fenótipos das diferentes famílias com a mesma

mutação do RET (3). Esta heterogeneidade justifica a necessidade de um

esforço global para a descrição das famílias com MEN2, com o objetivo de

esclarecer as circunstâncias genéticas ou do meio ambiente que estariam

modificando estes fenótipos.

Este estudo apresenta a maior coleção de dados já coletada, com 555

pacientes com MEN2. Diferentemente de outros trabalhos, conseguimos

determinar a etiologia da mutação do RET de todos os pacientes portadores de

MEN2. Uma das explicações pode ser a execução de técnica mais ampliada

para a genotipagem e não restrita apenas aos éxons onde ocorrem as mutações

mais comuns (hot-spots) (37,38). Outro aspecto interessante é que a execução

sistemática da genotipagem de RET em todos os pacientes com CMT

esporádico permitiu o achado de MEN2 em várias famílias de pacientes que

aparentemente pareciam tratar-se, à primeira vista, de casos esporádicos.

Apesar de MEN2 ser uma doença causada por mutação dominante,

nossa casuística mostra mais mulheres do que homens. Este fato pode ser

35
explicado pela procura maior dos serviços de saúde pelas mulheres (39). Além

disso, como consideramos apenas os indivíduos operados e com a confirmação

do CMT após a cirurgia, muitos homens ficam adiando as cirurgias propostas e,

em consequência, também contribuem para a preponderância feminina.

Preocupou-nos a idade tardia em que os pacientes brasileiros foram

diagnosticados, fato que contribui para a maior morbidade da doença. Talvez

pelo fato de ser considerada uma doença rara, os pediatras e endocrinologistas

preocupam-se menos na divulgação da síndrome, o que contrasta com a série

de sinais evidentes que os portadores de MEN2B apresentam desde o

nascimento, como choro sem lágrimas, pálpebras evertidas e espessadas, ptose

leve e nervos corneanos evidentes; além disso, as alterações esqueléticas

incluem face alongada, pé cavo, pectus escavatum, palato arqueado, escoliose

e luxação da epífise femoral (1-3,12-14).

Outro fato negativo em nossa casuística foi o achado de lesões com TNM

apresentando tumores grandes, T3 e T4, além de muitos com metástases

linfonodais e à distância por ocasião do diagnóstico.

Também observamos que algumas mutações nos códons 611 e 618,

considerados, considerados moderados pela classificação de risco da ATA,

demonstram agressividade maior, com tumores grandes e metástases à

apresentação clínica.

A comparação dos resultados do BRASMEN (n=555) com as maiores

casuísticas publicadas na literatura, EUROMEN (n=145), ITAMEN (n=246) e a

Alemã (n=205) (40), mostra semelhanças, mas também divergências (Tabela 6).

36
Assim, enquanto que as mutações nos códons S891, V804, G768, C609, C611,

C630, C634 e M918T apresentam prevalências similares, algumas tem baixa

prevalência (códons L790 e C618) e outras são registradas principalmente no

Brasil, como nos códons G533C, 804L e M918V.

A análise dos nossos dados enseja a apresentação de algumas mutações

especiais no Brasil. A primeira delas, pelo número de afetados, é a mutação

G533C. Desde sua primeira descrição, mencionávamos na discussão (34), que

seria plausível que outros indivíduos vivendo na Europa poderiam carregar esta

mesma mutação, pelo fato da família ser originária de Barcelona e ter emigrado

para a região sudeste do Brasil no final do século XIX, uma vez que o Mar

Mediterrâneo sempre foi um local de muitas correntes migratórias (41).

Interessantemente, pesquisadores gregos descreveram, alguns anos depois,

que esta mutação é a mais prevalente na Grécia (42); a seguir, também uma

família de origem grega radicada nos Estados Unidos, foi descrita como

portadora da mutação G533C (43). Tendo em vista estes achados, especulamos

que estas famílias poderiam ter um ancestral comum, uma vez que desde a

antiguidade houve várias cidades-estado gregas espalhadas pelo Mediterrâneo,

incluindo a Península Ibérica, onde fica Barcelona (41). Confirmamos esta

hipótese ao demonstrar que os pacientes brasileiros (via Barcelona) e os

pacientes gregos têm um ancestral comum (33), o que é fascinante e sugere

que, historicamente, a explicação mais plausível para a origem desta mutação é

que ela ocorreu na Grécia primeiramente e que as correntes migratórias gregas

a levaram para a Península Ibérica; o fato de que a G533C ser a mutação mais

prevalente na Grécia reforça esta hipótese.

Ainda com relação à mutação G533C, outra observação interessante é a

penetrância diferente de feocromocitoma que encontramos nas diferentes

37
populações estudadas. Recentemente, Castinetti e colaboradores (44), num

estudo multicêntrico verificou que a penetrância do feocromocitoma não seria

determinada apenas pelo genótipo do RET. Verificaram num grande número de

pacientes com mutações no RET nos exons 10 e 11 que a penetrância do

feocromocitoma é menor na América do Sul, quando comparada à Europa e

propuseram que outros modificadores genéticos ou ambientais poderiam

explicar esta diferença. Assim, na casuística brasileira a mutação G533C, no

éxon 8, causa principalmente CMT familiar (apenas 1 caso de feocromocitoma

em 119 pacientes), enquanto que na Grécia e Estados Unidos há penetrância

elevada de feocromocitoma (42,43). Como mostrado na Tabela 7, verificamos

diferença significativa de penetrância de feocromocitoma entre as populações

do Brasil e as encontradas na Grécia e nos Estados Unidos em 193 pacientes. O

que é curioso, pois além da mutação ser a mesma, demonstramos

recentemente que os pacientes brasileiros e gregos portadores da mutação no

códon G533C têm um ancestral comum (33). Este fato sugere, então, que o

risco para o desenvolvimento do feocromocitoma na mutação no códon G533C

depende de outros fatores modificadores, como polimorfismos, mutações

adicionais, ação de outros genes ou circunstâncias epigenéticas e/ou

ambientais.

Outro dado importante de nossa casuística foi a descoberta da mutação

M918V no Estado do Ceará (32). O códon 918 é classicamente relacionado à

NEM 2B e tem sido intensamente estudado desde 1994, quando a primeira

mutação representada por uma substituição de metionina por treonina (M918T)

foi descrita (24-26). Em 1997, Cirafici e colaboradores demonstraram que dentre

todas as possíveis substituições no códon 918 apenas a mutação M918T

mostrou uma alta atividade transformadora (45). Em 2011, um grupo italiano

38
descreveu 6 novas mutações associadas a CMT aparentemente esporádico,

sendo uma delas a M918V (46). Essa mutação foi identificada em um paciente

com CMT aparentemente esporádico, de baixo risco e em um familiar deste

paciente que, apesar de ter idade avançada, não apresentava evidências

laboratorial ou estrutural de CMT (46). Usando uma análise computacional, a

qual combina características biofísicas dos aminoácidos e proteínas para

predizer o grau de alteração da atividade biológica da proteína mutada

codificada, a mutação M918V mostrou um escore de 15 se comparada ao

escore de 65 encontrado para a mutação clássica de NEM 2B, a M918T. Pela

análise in vitro, a mutação M918V produziu uma quantidade intermediária de

unidades formadoras de colônias (UFC), entre 15 e 30 unidades por µg de DNA,

semelhante à encontrada em mutações de risco intermediário e baixo, tais

como V804M. Enquanto isso, a mutação M918T mostrou mais de 100 UFC/ µg

de DNA. Neste estudo, diante da apresentação clínica do CMT relacionado à

M918V e dos resultados das análises in silico e in vitro, foi sugerido que essa

rara troca fosse considerada uma mutação moderada (3).

Em nossa opinião, entretanto, o achado mais interessante de nossas

pesquisas referentes à mutação M918V foi o estudo completo de seu efeito

fundador e de sua datação. A princípio pensávamos que se tratasse de famílias

isoladas, mas o estudo dos haplótipos evidenciou que todos estes pacientes

tem um ancestral comum e estudos matemáticos sugeriram a datação desta

mutação para um período de cerca de 15 gerações atrás. Nossos estudos

históricos baseados em narrativas orais e nos arquivos de registros de

nascimento nos cartórios e de batismo nas igrejas confirmaram a existência de

um pioneiro que emigrou de Braga, Portugal, para o Brasil em 1700 e participou

da colonização do nordeste do Ceará ao longo do vale do Rio Jaguaribe, fatos

39
que corroboram nossa hipótese de que esta mutação acompanha a população

dessa região por aproximadamente 350-400 anos e que essas famílias

supostamente diferentes pertencem a uma única família que foi sendo

desmembrada no correr dos séculos.

Os achados que as mutações do gene RET no Brasil guardam certa

semelhança com a população européia, mas também certas divergências, o que

se explica com os dados sobre a ancestralidade genômica dos brasileiros, que

indicam uma variabilidade ancestral elevada e que cada brasileiro tem uma

proporção singular de ancestralidade Ameríndia, Européia e Africana (47,48).

Apesar do número elevado de pacientes incluídos neste trabalho, é

importante que se ressalte que ainda não temos uma visão abrangente das

características das mutações do RET causadoras de MEN 2 no Brasil, uma vez

que os centros que mais contribuíram nesta casuística situam-se nas regiões Sul

e Sudeste; temos um bom volume de pacientes do Nordeste, mas como todos

são do Ceará, estamos diante de um viés em nossa observação. Não

conseguimos informações dos centros das regiões Norte e Centro-Oeste.

40
5.Conclusões

1. Os resultados clínicos e moleculares dos 555 pacientes do BRASMEN

com mutação no gene RET, provenientes de 150 famílias, apresentam 34

mutações germinativas diferentes em 16 códons dos éxons

8,10,11,13,14,15 e 16 do gene RET.

2. As mutações mais encontradas pelo número de famílias ocorrem nos

códons 634, 918 e 804. Pelo número de pacientes nos códons 634, 533,

620 e 918.

3. Depois do CMT, a associação mais frequentemente verificada entre os

pacientes com MEN2 é o feocromocitoma (21,3%).

4. Nota-se a preponderância de mulheres em relação aos homens, tanto na

série de CMT hereditário (64%), como esporádico (61%).

5. Quando comparamos os resultados do BRASMEN com os demais países,

vemos várias semelhanças, mas também divergências. Assim, enquanto

que as mutações nos códons S891, V804, G768, C609, C611, C630, C634

e M918T apresentam prevalências similares, algumas tem baixa

prevalência (códons L790 e C618) e outras são registradas principalmente

no Brasil, como nos códons G533C, 804L e M918V.

6. A penetrância do feocromocitoma nos pacientes com mutação no códon

G533C é maior nos Estados Unidos e Europa do que no Brasil.

41
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Maciel RMB, Cerutti JM. Comprehensive analysis of RET gene should be

performed in patients with MEN 2 syndrome and no apparent genotype-

phenotype correlation: an appraisal of p.Y791F and p.C634Y RET mutations

in five unrelated Brazilian families. J Endocrinol Invest 2013; 36: 975-981

39. Gomes R, Nascimento EF, Araújo FC. Porque os homens buscam menos

os serviços de saúde do que as mulheres? Cad Saude Pub 2007; 23: 565-

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40. Romei C, Mariotti S, Fugazzola L, Taccaliti A, Pacini F, Opocher G,

Mian C, Castellano M, degli Uberti E, Ceccherini I et al. Multiple endocrine

neoplasia type 2 syndromes (MEN 2): results from the ItaMEN network

analysis on the prevalence of different genotypes and phenotypes. Eur J

Endocrinol 2010; 163: 301-308.

41. Braudel F. The Mediterranean and the Mediterranean World in the Age

of Philip II. Berkeley, California, USA: University of California Press, 1995.

42. Sarika HL, Papathoma A, Garofalaki M, Vasileiou V, Vlassopoulou B,

Anastasiou E & Alevizaki M. High prevalence of exon 8 G533C mutation in

apparently sporadic medullary thyroid carcinoma in Greece. Clinical

Endocrinology 2012 77 857–862.

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 43. Castro MR, Thomas BC, Richards ML, Zhang J & Morris JC. Multiple

endocrine neoplasia type 2A due to an exon 8 (G533C) mutation in a large

North American kindred. Thyroid 2013; 23 1547-1552

44. Castinetti F, Maia AL, Peczkowska M, Barontini M, Hasse-Lazar K, Links

TP, Toledo RA, Dvorakova S, Mian C, Bugalho MJ et al. The penetrance of

men2 pheochromocytoma is not only determined by ret mutations.

Endocr Related Cancer 2017 Jun 25. pii: ERC-17-0189.

45. Cirafici AM, Salvatore G, De Vita G, Carlomagno F, Dathan NA,

Visconti R, Melillo RM, Fusco A & Santoro M. Only the substitution of

methionine 918 with a threonine and not with other residues activates RET

transforming potential. Endocrinology 1997; 138: 1450-1455.

46. Cosci B, Vivaldi A, Romei C, Gemignani F, Landi S, Ciampi R, Tacito A,

Molinaro E, Agate L, Bottici V, et al. In silico and in vitro analysis of rare

germline allelic variants of RET oncogene associated with medullary

thyroid cancer. Endocrine Related Cancer 2011; 18 603–612.

47. Pena SDJ, Bastos-Rodrigues L, Pimenta JR, Bydlowski SP. DNA tests

probe the genomic ancestry of Brazilians. Braz j Med Biol Res 2009; 42: 870-

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48. Pena SDJ, Di Pietro G, Fuchshuber-Moraes M, Genro JP, Hutz MH,

Kehdy FSG, Kohlrausch, Magno LAV, Montenegro RC, Moraes MO, Moraes

MO, Moraes MEA, Moraes MR, Ojopi EB, Perini JA, Racciopi C, Ribeiro-dos-

Ribeiro-dos-Santos AKC, Rios-Santos F, Romano-Silva MA, Sortica VA,

VA, Suarez-Kurtz G. The genomic ancestry of individuals from diferente

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 diferente geographical regions of Brazil is more uniform than expected.

expected. PlosOne 2011; 6: e17063, 1-9

50
7. Agradecimentos

Este trabalho teve o financiamento da Fundação de Amparo à Pesquisa do

Estado de São Paulo, FAPESP (processos 2006/60402-1; 2010/51547-1 e

2014/06570-6).

Agradeço o auxílio dos colegas do Núcleo de Neoplasias Endócrinas

Múltiplas da Escola Paulista de Medicina, Universidade Federal de São Paulo

(EPM/UNIFESP), especialmente Cléber Camacho, Susan Lindsey, Flavia Valente,

Ji Yang, João Martins, Magnus Dias da Silva, Janete Cerutti, Alberto Lobo,

Danielle Macelaro, Ilda Kunii, os cirurgiões da disciplina de Cirurgia de Cabeça e

Pescoço da EPM/UNIFESP e os alunos de pós-graduação Adriana Álvares-da-

Silva, Cecilia Martins-Costa, Sharmila Sousa, Priscila Signorini.

Agradeço os colegas dos demais centros que contribuíram com suas

casuísticas e discussões sobre CMT e MEN 2, especialmente Ana Hoff, Ana

Maia, Sergio Toledo, Cecilia Martins-Costa, Fernanda Vaisman, Laura Ward, Léa

Maciel, Hans Graf, Inez França, Delmar Lourenço, Cencita Cordeiro, Celia

Nogueira e Anelise Impellizzeri.

51
8. Coordenação ou participação em projetos de pesquisa na
área de MEN2 e CMT

1. Coordenador do projeto temático "Carcinoma medular da tiroide: revisitação

à clínica, à biologia molecular, à bioquímica e à biologia do desenvolvimento

depois dos achados da genética molecular" (processo 06/60402-1), financiado

pela FAPESP, com duração de 2007 a 2011

2.Coordenador do auxílio à Pesquisa “Carcinoma medular de tiroide

hereditário: percepção e atitude de pacientes, familiares e profissionais de

saúde sobre questões bioéticas” (processo 10/51547-1), financiado pela

FAPESP, com duração de 2010 a 2013

3.Pesquisador principal do Projeto Temático “Sequenciamento Completo do

Exoma, Paired-end RNA e Genoma: Novos Insights sobre a Natureza Genética

do Câncer de Tiroide na Idade Adulta e na Faixa Etária Pediátrica e Aplicações

na Prática Clínica” (processo 14/06570-6, pesquisadora responsável JM Cerutti),

financiado pela FAPESP, com duração de 2015 a 2019

4.Pesquisador do projeto “Exoma em doenças órfãs”, do Departamento de

Pesquisa e Desenvolvimento do Grupo Fleury, financiado pela FINEP, projeto 37

P&D, NP-82, FINEP 0914005500, com duração de 2015 a 2017

52
9. Capítulos de livros publicados na área de MEN2 e CMT
nos últimos 5 anos

1.”Diagnostico y tratamiento del cancer de tiróides”, RMB Maciel, RPM Biscolla, L

Villar, PWS Rosario, in Endocrinología Clínica, 4ª. edición, editado por Lucio Vilar,

Gen Editora, 2012, pg. 267-280

2.”Câncer de Tiroide: classificação e diagnóstico”, PWS Rosário, RMB Maciel, E

Moura, L Vilar, in Endocrinologia Clínica, 5ª. edição, editado por Lucio Vilar, Gen

Editora, 2013, pp 272-280

3.”Tumores de Tiroide”, RMB Maciel, in Tratado de Oncologia, cap. 147, editado

por Paulo M. G. Hoff, Editora Atheneu, 2013, pg 2147-2161

4.“Câncer de tiroide”, RMB Maciel, in Wajchenberg-Tratado de Endocrinologia

Clínica, editado por BL Wajchenberg, AC Lerário, RTB Betti, 2a. Edição, Gen

Editora, 2014, pp 143-153

5.”Alterações moleculares e genéticas do carcinoma medular de tiroide”, RMB

Maciel e CP Camacho, in “Perguntas chave em carcinoma medular de tiroide,

editado por AO Hoff e G de Castro Jr., Permanyer Brasil Publicações, 2014, pp.

23-26

6.”Diagnostico y tratamiento del câncer de tiróides”, RMB Maciel, RPM Biscolla, L

Villar, PWS Rosario, in Endocrinología Clínica, 4ª. edición, editado por Lucio Vilar,

Gen Editora, 2012, pg. 267-280

8.”Neoplasia Endócrina Múltipla Tipo 2”, AL Maia, L Ceolin, RMB Maciel, in Guia

de Oncologia, INCA, 2016

9.”Câncer da tiroide”, RMB Maciel, in Atualização Terapêutica, editado por DR

Borges, AL Colombo, LR Ramos, R Guinsburg, LM Ferreira, 25a. Edição, 2016/17,

capítulo 378, pp

53
10.”Neoplasia endócrina múltipla tipo 2”. AO Hoff, LAC Neves, RMB Maciel, in

Endocrinologia, 2a. ed., editado por MJA Saad, RMB Maciel e BB Mendonça,

Editora Atheneu, São Paulo, 2017, pp 1277-1286

54
10. Orientações de teses na área de na área de MEN2 e CMT

1."Mutações de proto-oncogene RET e incidência de carcinoma medular de

tiróide em pacientes com diagnóstico de doença de Hirschsprung", Tese de

Mestrado apresentada por Fernando Danelon Leonhardt ao Programa de Pós-

Graduação em Otorrinolaringologia e Cirurgia de Cabeça e Pescoço, Universidade

Federal de São Paulo, 2004

2.”Dez anos de seguimento clínico da família com neoplasia endócrina múltipla

2A por mutação no gene RET p.Gly533Cys indicam a necessidade de avaliações

periódicas e individualizadas nos familiares afetados”, Tese de Mestrado

apresentada por Priscila Bernal da Costa Seguro Signorini ao Programa de Pós-

Graduação em Endocrinologia da Escola Paulista de Medicina, Universidade

Federal de São Paulo em 2012.

3. “Quantificação do RNA mensageiro de tiroglobulina, do receptor do hormônio

tiroestimulante e de calcitonina obtidos do sangue periférico de uma população

com função tiroidiana normal“, apresentada por Maria Clara de Carvalho Melo ao

Programa de Pós-Graduação em Medicina Translacional da Escola Paulista de

Medicina, Universidade Federal de São Paulo em 2014.

4."Descrição de uma nova mutação de ponto no oncogene RET, exon 8 (Gly533Cys)

em uma família com carcinoma medular de tiroide", apresentada por Adriana

Madeira Álvares da Silva ao Programa de Pós-Graduação em Endocrinologia da

Escola Paulista de Medicina, Universidade Federal de São Paulo em 2003. Co-

orientação com a Profa. Janete M. Cerutti.

55
5."O carcinoma medular da tiroide e a evolução do diagnóstico laboratorial",

apresentada por Cleber Pinto Camacho ao Programa de Pós-Graduação em

Endocrinologia Clínica da Escola Paulista de Medicina, Universidade Federal de São

Paulo em 2012.

6.”Consórcio Brasileiro de Neoplasia Endócrina Múltipla tipo 2: Medicina

Translacional, Bioética e Sociedade.”, apresentada por Maria Sharmila Alina de

Sousa ao Programa de Pós-Graduação em Endocrinologia Clínica da Escola Paulista

de Medicina, Universidade Federal de São Paulo em 2015.

7.”Diagnóstico clínico-molecular, prognóstico e avaliação da expressão gênica de

vias de tumorigênese para individualização do seguimento clínico de pacientes com

carcinoma medular da tiroide”, apresentada por Susan Chow Lindsey ao Programa

de Pós-Graduação em Endocrinologia Clínica da Escola Paulista de Medicina,

Universidade Federal de São Paulo em 2015. Co-orientação com o Professor

Magnus R. Dias-da-Silva.

8.”Novos aspectos no seguimento de carcinomas diferenciado e medular de

tiroide”, apresentada por Ji Hoon Yang ao Programa de Pós-Graduação em

Endocrinologia Clínica da Escola Paulista de Medicina, Universidade Federal de São

Paulo em 2015.

9.”Carcinoma medular de tiroide no estado do Ceará: epidemiologia, avaliação.5.

clínica e diagnóstico molecular”, apresentada por Maria Cecília Martins-Costa ao

Programa de Pós-Graduação em Endocrinologia Clínica da Escola Paulista de

Medicina, Universidade Federal de São Paulo em 2016.

56
11. Trabalhos Publicados na área de MEN2 e CMT

1."A Novel Germline Point Mutation in RET Exon 8 (Gly533Cys) in a Large

Kindred with Familial Medullary Thyroid Carcinoma". AM Álvares da Silva, RMB

Maciel, MR Dias da Silva, Silvia RC Toledo, MB de Carvalho, JM Cerutti. Journal

of Clinical Endocrinology and Metabolism 2003; 88: 5438-5443.

2."Y791F RET mutation and early onset of medullary thyroid carcinoma in

Brazilian Kindred: evaluation of phenotype-modifying effect of germline

variants". R Tamanaha, CP Camacho, ES Ikejiri, RMB Maciel, JM Cerutti. Clinical

Endocrinology (Oxf) 2007; 67: 806-808.

3."Early diagnosis of Multiple Endocrine Neoplasia Type 2B: a challenge for

physicians". CP Camacho, AO Hoff, SC Lindsey, PS Signorini, FOF Valente, MNL

Oliveira, RPM Biscolla, JM Cerutti, RMB Maciel. Archives of Endocrinology and

Metabolism 2008; 52: 1393-1398.

4."Evaluation of RET polymorphisms in a six-generation family with G533C RET

mutation: specific RET variants may modulate age at onset and clinical

presentation. R Tamanaha, CP Camacho, AC Pereira, AM Álvares da Silva, RMB

Maciel, JM Cerutti. Clinical Endocrinology (Oxf) 2009; 71: 56-64.

5."The RET p.G533C mutation confers predisposition to multiple endocrine

neoplasia type 2A in a Brazilian kindred and is able to induce a malignant

phenotype in vitro and in vivo". on cell proliferation, invasion and metastases".

57
MNL Oliveira, JP Hemerly, AU Bastos, R Tamanaha, FRM Latini, CP Camacho, A

Impellizzeri, RMB Maciel, JM Cerutti. Thyroid 2011; 21: 975-985.

6."Evidence that polymorphisms in detoxification genes modulate the

susceptibility for sporadic medullary thyroid carcinoma”. RB Barbieri, NE Bufalo,

R Secolin, AC Silva, LV Assumpção, Maciel RMB, JM Cerutti, LS Ward. European

Journal of Endocrinology 2012; 166: 241-245.

7."Extended RET gene analysis in patients with apparently sporadic medullary

thyroid cancer: clinical benefits and cost". SC Lindsey, IS Kunii, F Germano-

Neto, MY Sittoni, CP Camacho, FOF Valente, JH Yang, PS Signorini, R Delcelo,

JM Cerutti, RMB Maciel, MR Dias da Silva. Hormones & Cancer 2012; 3: 181-

186.

8."Measurement of calcitonin and CGRP messenger ribonucleic acid refines the

follow-up of patients with medullary thyroid cancer and may replace the

pentagastrin stimulation test". CP Camacho, SC Lindsey, MCC Melo, JH Yang, F

Germano-Neto, FOF Valente, AL Machado, MCO Mamone, TRN Lima, RPM

Biscolla, JGH Vieira, JM Cerutti, MR Dias-da-Silva and RMB Maciel. Thyroid

2013; 23: 308-316.

9.”Genes of detoxification are important modulators of hereditary medullary

thyroid carcinoma risk”. RB Barbieri, NE Bufalo, LL Cunha, VL Assumpção, RMB

Maciel, JM Cerutti, LS Ward. Clinical Endocrinology (Oxf) 2013: 79: 288-293.

58
10."An unusual genotype-phenotype correlation in MEN 2 patients: screening

for RET double germline mutations should be performed to avoid misleading

diagnosis and treatment?" JM Cerutti, RMB Maciel. Clinical Endocrinology (Oxf)

2013; 79: 591-592.

11.”Fine needle aspiration and medullary thyroid carcinoma: the risk of

inadequate preoperative evaluation and initial surgery when relying upon fna

cytology alone”. GF Essig, Jr, K Porter, D Schneider, A Debora, SC Lindsey, G

Busonero, D Fineberg, B Fruci, K Boelaert, JWA Smit, JAA Meijer, L Duntas, N

Sharma, G Costante, S Filetti, RS Sippel, B Biondi, D Topliss, F Pacini, RMB

Maciel, PC Walz, RT Kloos. Endocrine Practice 2013; 19: 920-927.

12."Comprehensive analysis of RET gene should be performed in patients with

MEN 2 syndrome and no apparent genotype-phenotype correlation: an appraisal

of p.Y791F and p.C634Y RET mutations in five unrelated Brazilian families". FOF

Valente, MR Dias da Silva, CP Camacho, IS Kunii, R Tamanaha, RMB Maciel, JM

Cerutti. Journal of Endocrinological Investigation 2013; 36: 975-981.

13."A ten-year clinical update of a large RET p.Gly533Cys kindred with MEN2A

emphasizes the need for an individualized, ongoing risk assessment of relatives

with medullary thyroid carcinoma". PS Signorini, MIC França, CP Camacho, SC

Lindsey, FOF Valente, TS Kasamatsu, AL Machado, CP Salim, R Delcelo, AO

Hoff, JM Cerutti, MR Dias da Silva, RMB Maciel. Clinical Endocrinology (Oxf)

2014; 80: 235-245.

59
14."Genome-Wide Copy Number Analysis in a Family with p.G533C RET

Mutation and Medullary Thyroid Carcinoma Identified Regions Associated with

Higher Predisposition to Lymph Node Metastasis". AN Araujo, LS Moraes, MIC

França, H Hakonarson, J Li, R Pellegrino, RMB Maciel, JM Cerutti. Journal of

Clinical Endocrinology and Metabolism 2014; 99: E 1104-112.

15.”Polymorphisms of cell cycle control genes influence the development of

sporadic medullary thyroid cancer”. RB Barbieri, NE Bufalo, LVM Assumção,

RMB Maciel, JM Cerutti, LS Ward. European Journal of Endocrinology 2014;

171: 761-767.

16. “Development and Application of a Novel Sensitive Immunometric Assay for

Calcitonin in a Large Cohort of Patients with Medullary and Differentiated

Thyroid Cancer, Thyroid Nodules, and Autoimmune Thyroid Diseases”. CP

Camacho, SC Lindsey, TS Kasamatsu , AL Machado, JRM Martins, RPM Biscolla,

MR Dias-da-Silva, JGH Vieira, RMB Maciel. European Thyroid Journal 2014; 3:

117-124.

17.”Integration of a Postoperative Calcitonin Measurement Into An Anatomical

Staging System Improves Initial Risk Stratification in Medullary Thyroid Cancer”.

JH Yang, SC Lindsey, CP Camacho, FOF Valente, F Germano-Neto, AL

Machado, MCO Mamone, F Brodskyn, RPM Biscolla, RM Tuttle, MR Dias-da-

Silva, RMB Maciel. Clin Endocrinol (Oxf) 2015; 83: 938-942.

18.”Comprehensive assessment of the disputed RETY791F variant shows no

association with medullary thyroid carcinoma susceptibility“. RA Toledo, R

Hatanaka, DM Lourenço Jr., SC Lindsey, CP Camacho, M Almeida, JV Lima-Jr, T

60
Sekiya, E Garralda, MS Naslavsky, G Yamamoto, M Lazar, O Meirelles, TJP

Sobreira, ML Lebrao, YAO Duarte, J Blangero, M Zatz, JM Cerutti, RMB Maciel,

SPA Toledo. Endocrine Related Cancer 2015; 22: 65-76.

19.”RET Y791F variant does not increase the risk for MTC”. RA Toledo, RMB

Maciel, Z Erlic, DM Lourenço Jr., JM Cerutti, C Eng, HP Neumann, SP Toledo.

Thyroid 2015; 25: 973-974

20.”RET Y791F: alone or accompanied?”. RA Toledo, DM Loureço Jr., CP

Camacho, SC Lindsey, JM Cerutti, RMB Maciel, SPA Toledo. Archives of

Endocrinology and Metabolism 2015; 59: 476-477.

21.”Macrocalcitonin is a novel pitfall in the routine of serum calcitonin

immunoassay”. TG Alves, TS Kasamatsu, MCZ Meneghetti, A Mendes, IS Kunii,

CP Camacho, MR Dias-da-Silva, RMB Maciel, JGH Vieira, JRM Martins. Journal

of Clinical Endocrinology and Metabolism 2016; 101: 653-658.

22.“Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in

patients with medullary thyroid carcinoma”. FP Nascimento, MG Cardoso, SC

Lindsey, IS Kunii, FOF Valente, MML Kyzis, R Delcelo, CP Camacho, RMB

Maciel, MR Dias-da-Silva. Molecular Medicine Reports 2016; 13: 1653-1660.

23.”Multifocality in sporadic medullary thyroid carcinoma: an international

multicenter study”. Essig Jr GF, Porter K, Schneider D, Arpaia D, Lindsey SC,

Busonero G, Fineberg D, Fruci B, Boelaert K, Smit JW, Meijer JAA, Duntas L,

Sharma N, Costante G, Filetti S, Sippel RS, Biondi B, Topliss DJ, Pacini F, Maciel

RMB, Waltz PC, Kloos RT. Thyroid 2016; 26: 1563-1572.

61
24.”M918V RET mutation causes familial medullary thyroid carcinoma: study of 8

affected kindreds”. MC Martins-Costa, LL Cunha, SC Lindsey, CP Camacho, RP

Dotto, GK Furuzawa, MSA Sousa, TS Kasamatsu, IS Kunii, MM Martins, AL

Machado, JRM Martins, MR Dias-da-Silva, RMB Maciel. Endocrine Related

Cancer 2016; 23: 909-920.

25.”Evidence for the founder effect of RET533 as the common Greek and

Brazilian ancestor spreading multiple endocrine neoplasia 2A. LL Cunha, SC

Lindsey, MI França, L Sarika, A Papathoma, IS Kunii, JM Cerutti, MR Dias-da-

Silva, M Alevizaki, RMB Maciel. European Journal of Endocrinology 2017 176:

515-519.

26.”Assessment of depression, anxiety, quality of life and coping in long-

standing multiple endocrine neoplasia type 2 patients”. KC Rodrigues, RA

Toledo, FL Coutinho, AB Nunes, RMB Maciel, AO Hoff, MC Tavares, SPA

Toledo, DM Lourenço Jr. Thyroid 2017; 27: 693-706.

27.”The combined use of calcitonin doubling time and 18

F-FDG PET/CT

improves prognostic values in medullary thyroid carcinoma: the clinical utility of

18
F-FDG PET/CT”. JH Yang, CP Camacho, SC Lindsey, FOF Valente, DM

Andreoni, LY Yamaga, J Wagner, RPM Biscolla, RMB Maciel. Endocrine Practice

2017 Jun 14. doi: 10.4158/EP171806.OR. [Epub ahead of print]

62
0021-972X/03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(11):5438 –5443
Printed in U.S.A. Copyright © 2003 by The Endocrine Society
doi: 10.1210/jc.2003-030997

A Novel Germ-Line Point Mutation in RET Exon 8

(Gly533Cys) in a Large Kindred with Familial Medullary
Thyroid Carcinoma
ADRIANA M. ÁLVARES DA SILVA, RUI M. B. MACIEL, MAGNUS R. DIAS DA SILVA,
SILVIA R. C. TOLEDO, MARCOS B. DE CARVALHO, AND JANETE M. CERUTTI
Laboratory of Molecular Endocrinology, Division of Endocrinology (A.M.A.D.S., R.M.B.M., M.R.D.D.S., J.M.C.), and J. F.
Perez Genomic Center (R.M.B.M., M.R.D.D.S.), Department of Medicine; Division of Genetics (J.M.C.), Department of
Morphology and Institute of Pediatric Oncology (S.R.C.T.), Department of Pediatrics, Escola Paulista de Medicina, Federal
University of Sao Paulo; Division of Genetics and Biotechnology (A.M.A.D.S.), Santo Andre Foundation; and Service of
Head and Neck Surgery (M.B.D.C.), Heliopolis Hospital, 04039-032 Sao Paulo, Brazil

Familial medullary thyroid carcinoma is related to germ-line hyperplasia (CCH) in patients subjected to surgery, and fam-
mutations in the RET oncogene, mainly in cysteine codon 10 ily members without the mutation are clinically unaffected.
or 11, whereas noncysteine mutations in codons 13–15 are The histological analysis of 35 cases submitted to thyroidec-
rare. We now report a new missense point mutation in exon 8 tomy revealed that 21 patients had MTC after the age of 40 yr
of the RET gene (1597G3 T) corresponding to a Gly533Cys sub- and 8 before the age of 40 yr, 4 presented MTC or CCH before
stitution in the cystein-rich domain of RET protein in 76 pa- the age of 18 yr, 2 died due to MTC at the age of 53 and 60 yr,
tients from a 6-generation Brazilian family with 229 subjects, and CCH was found in a 5-yr-old child, suggesting a clinical
with ascendants from Spain. It is likely that the mutation heterogeneity. To improve the diagnosis of FMTC, analysis of
causes familial medullary thyroid carcinoma (FMTC), be- exon 8 of RET should be considered in families with no iden-
cause no other mutation was found in RET, the mutation co- tified classical RET mutations. (J Clin Endocrinol Metab 88:
segregates with medullary thyroid carcinoma (MTC) or C cell 5438 –5443, 2003)

M EDULLARY THYROID CARCINOMA (MTC) pre-

sents either sporadically or in hereditary forms (1).
In hereditary forms, MTC and/or its precursor lesion, C cell
ing one of its ligands (glial cell line-derived neurotrophic
factor, neurturin, artemin, and persephin) and one of their
cell surface-bound coreceptors (respectively, GFR!-1,
hyperplasia (CCH), is the single component in familial MTC GFR!-2, GFR!-3, and GFR!-4) (1–3, 6).
(FMTC) or is part of the multiple endocrine neoplasia type Almost all mutations in MEN2A involve one of the cys-
2 syndromes (MEN2A and 2B). In MEN2A, in addition to teines in the extracellular domain of RET that are encoded by
MTC, 50% of patients have a pheochromocytoma, whereas exons 11 (codon 634) or 10 (codons 609, 611, 618, and 620).
parathyroid hyperplasia or adenoma is observed in 10 –30% In MEN2B a single mutation has been found in 95% of pa-
of the cases. MEN2B syndrome is recognized by MTC, pheo- tients, Met918Thr, in the tyrosine kinase domain of RET en-
chromocytoma, and developmental abnormalities such as coded by RET exon 16. In FMTC, RET gene mutations involve
marfanoid habitus, skeletal abnormalities, mucosal neuro- either a cysteine codon in exon 10 or exon 11 or, less often,
mas, and diffuse intestinal ganglioneuromatosis; parathy- codons 768, 790, and 791 in exon 13; codon 804 in exon 14;
roid disease is not observed (1– 4). and codon 891 in exon 15 (4, 5, 7, 8). In addition, Pigny et al.
Activating germ line mutations of the RET oncogene pre- (9) described an FMTC with a 9-bp duplication in exon 8,
dispose to hereditary MTC (1–5). The RET gene is located on which creates an additional cysteine residue in the extracel-
chromosome 10q11.2, comprises 21 exons and encodes a lular cysteine-rich domain of RET.
tyrosine kinase receptor. Like other tyrosine kinases recep- In this paper we describe a new missense point mutation
tors, RET protein has extracellular, transmembrane, and cy- in exon 8 of RET gene (1597G3 T) that corresponds to a
toplasmic domains. The extracellular domain includes re- Gly533Cys substitution in the cystein-rich domain of RET
gions with homology to the cadherin family and a large protein in 76 patients from a 6-generation Brazilian family
cysteine-rich region, and the intracellular domain functions with 229 subjects of Caucasian background. In addition,
in the phosphorylation of tyrosine residues involved in the DNA-based analysis of the RET oncogene enables the iden-
interaction with downstream targets and activation of sig- tification of 2 new polymorphisms in intron 8 and 2 already
naling pathways (1–3, 6). Under normal conditions, RET described polymorphisms in exons 7 and 15.
receptor is activated by a multicomponent complex involv-
Subjects and Methods
Family history
Abbreviations: CCH, C Cell hyperplasia; FMTC, familial medullary
thyroid carcinoma; MEN, multiple endocrine neoplasia; MTC, medul- The pedigree of the family is depicted in Fig. 1. This large family is
lary thyroid carcinoma; Pg, pentagastrin; sCT, serum calcitonin; SNP, of Caucasian origin, with ascendants from Barcelona, Spain, who im-
single nucleotide polymorphism. migrated to Brazil in the 19th century, resides mainly in the southeastern

5438

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 29 May 2015. at 11:39 For personal use only. No other uses without permission. . All rights reserved.
Álvares Da Silva et al. • RET Exon 8 Mutation in a Large FMTC Kindred
FIG. 1. Pedigree of the six-generation family with FMTC. Circles and squares denote female and male family members, respectively. Solid circles
indicate subjects with MTC.
region of Brazil, and consists of 6 generations with a total of 229 indi-
viduals. Before undergoing genetic testing, a signed letter of informed
consent was obtained from all patients or their legal guardians. The
study was approved by the ethics and research committee of Univer-
sidade Federal de São Paulo and Hospital Heliópolis and was in agree-
ment with the 1975 Helsinki statement, revised in 1983.
The index case (IV.77; Fig. 1) is a 49-yr-old man, asymptomatic until
the age of 47 yr, when he noticed a nodule in the right lobe of the thyroid
gland. Thyroid ultrasound showed an 18-mm nodule in the left lobe and
a 35-mm nodule in the right lobe. The basal serum calcitonin (sCT) level
was 1080 pg/ml (normal range, !11.5 pg/ml). Total thyroidectomy with
selective neck dissection (central compartment, level VI) was performed,
and histological examination showed the presence of MTC, CCH, and
microscopic metastatic disease in three lymph nodes with extracapsular
extension to adjacent tissues. His last sCT determination, after surgery,
was 181 pg/ml; work-up performed showed no radiological evidence of
metastatic disease.
The proband’s mother, a 78-yr-old woman (III.18) had a thyroid
nodule in 1972 at the age of 47 yr, when she underwent total thyroid-
ectomy and postoperative irradiation for undifferentiated thyroid car-
cinoma. Thirty years later, at the time of her son’s surgery, the paraffin-
embedded tissue from the thyroid tumor was reviewed and revealed
that it was a MTC. Despite being asymptomatic, she has residual MTC,
as her sCT is 2200 pg/ml. She has refused further investigation.
During the genetic screening, the proband’s daughter (V.121), a 22-
yr-old woman, was identified as a gene carrier and had a basal sCT of
385 pg/ml; she underwent total thyroidectomy and neck dissection, and
histological analysis confirmed the presence of MTC with lymph node
metastases. Similarly, the proband’s son (V.122), a 21-yr-old man, was
identified as a gene carrier. He exhibited increased sCT in response to
pentagastrin (Pg; 0.5 "g/kg during 3 min) stimulation (441 pg/ml) and
underwent total thyroidectomy; the pathology was consistent
with MTC.
There is consanguinity in one of the branches of the family; as can be
observed on the right of Fig. 1. Patients II.7 and II.8 were cousins, as were
patients IV.70 and IV.71.
All family members were evaluated by their own physicians, who
referred to us the medical history, physical examination, and results of
sCT, serum calcium, and urinary metanephrines; sCT was determined
by a chemiluminescence immunoassay from Nichols Institute (San Juan
Capistrano, CA; normal values, !11.5 pg/ml). There was no clinical or
biochemical evidence of pheochromocytoma, parathyroid disease, or
other associated clinical features attributed to MEN2.
Control group
One hundred DNA samples were obtained from healthy volunteers,
aged 20 – 65 yr, living in the southeastern region of Brazil.
DNA analysis
Genomic DNA was extracted from peripheral blood lymphocytes
using a GFX Genomic Blood DNA Purification Kit (Amersham Phar-
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J Clin Endocrinol Metab, November 2003, 88(11):5438 –5443 5439
macia Biotech, Piscataway NJ), and DNA from the tumor was extracted
using a phenol/chloroform (Life Technologies, Inc., Grand Island, NY)
standard method. Primers specific for exons 7–19 of the RET gene (Gen-
Bank accession no. AJ243297) were designed and used to generate PCR
products, which were purified and directly sequenced. Briefly, the PCR
reaction was performed employing 200 ng genomic DNA in a 50-"l
volume containing 10 mmol/liter Tris-HCl (pH 8.3), 50 mmol/liter KCl,
200 "mol/liter of each deoxy-NTP, 1.5 mmol/liter MgCl2, 1 U Taq DNA
polymerase (Life Technologies, Inc.), and 25 pmol of each specific primer
(Life Technologies, Inc., Sao Paulo, Brazil). Cycling conditions to gen-
erate PCR products included an initial phase of 5 min at 94 C, followed
by 35 cycles of 45 sec at 94 C, an annealing temperature step of 1 min,
and an extension step of 1 min at 72 C. PCR products were resolved by
electrophoresis in a 1.5% agarose gel stained with ethidium bromide,
purified using the CONCERT Rapid PCR Purification System (Life Tech-
nologies, Inc., Gaithersburg, MD), and quantified using a DNA mass
ladder (Life Technologies, Inc.). Purified PCR products (150 ng) were
submitted to direct sequencing using the ABI PRISM Big Dye Terminator
Cycle Sequencing Ready Reaction Kit and the ABI 3100 sequencer (PE
Applied Biosystems, Foster City, CA). Each sample was sequenced at
least twice and in both directions. The PCR specific primers, product
sizes, and annealing temperatures are summarized in Table 1.
Results
We first screened the proband’s DNA by looking for RET
gene mutations in exons 10, 11, 13, 14, and 15, where most
of the RET mutations were found in MEN2. As we have not
found a mutation that could be associated with the tumor
phenotype, we extended the analysis to exons 7–9, 12, and
16 –19. A novel mutation located at nucleotide 1597 in the
exon 8 of the RET gene was identified in DNA samples
obtained from both peripheral blood and tumor tissue of the
index case (IV.77). This missense point mutation arises in
heterozygosis and causes a substitution of glycine to cysteine
residue at codon 533 (Gly533Cys) in the cysteine-rich extra-
cellular domain of the RET protein; this substitution is a G
to T transversion and is displayed as two peaks in Fig. 2,
differently from the single peak observed in the wild-type
alleles.
Direct sequencing of RET exon 8 PCR products from
genomic DNA of family members showed that 76 of 229
members were carriers for this mutation, and 153 were un-
affected. In addition, 14 members of the family were not
tested. Eleven of those 14 cases died many years before the
genetic testing; 2 died due to MTC with metastatic disease
(II.6, III.20), 1 died due to heart disease, but had MTC (II.7),
and 8 died due to other causes. Three of those 14 cases
5440 J Clin Endocrinol Metab, November 2003, 88(11):5438 –5443 Álvares Da Silva et al. • RET Exon 8 Mutation in a Large FMTC Kindred

refused to perform the genetic test. However, all 14 cases not yet undergone surgery; 3 have scheduled surgery; 10
were definitely carriers, because they or their descendants presented low levels of sCT after stimulation with Pg, and
had MTC (Table 2). surgery was delayed; 24 did not complete the clinical eval-
To date, 35 of the 76 gene carriers have undergone total uation because they had recent molecular diagnosis; 3 re-
thyroidectomy. Histological examination showed the pres- fused further clinical investigation; and 1 refused surgery.
ence of MTC in 29 patients and of CCH in 6 patients. CCH All 153 non-carriers of RET Gly533Cys mutation family mem-
was described in only 5 patients from 29 with MTC, probably bers had normal levels of sCT (normal range, !11.5 pg/ml)
due to methodological differences in processing the tissues and, consequently, were excluded from further clinical
for histology (Table 3). The postoperative calcitonin levels investigation.
were undetectable in 20 patients and elevated in 8. The 7 The clinical course of MTC has been variable in this family
remaining cases were recently submitted to thyroidectomy, (Tables 2 and 3). Before genetic screening, two patients died
and their own physician requested the postoperative sCT due to MTC after protracted illness and metastasis. The first
measurement. Of these 8 patients with elevated sCT, 6 pre- patient was a woman (II.6) who died at 53 yr of age after 14
sented lymph node metastases (III.17, III.18, IV.65, IV.77, yr of disease; the second patient was a man (III.20) who died
IV.87, and V.121), and 2 had no evidence of lymph node at 60 yr of age after 7 yr of disease. Both patients underwent
involvement (IV.68 and IV.91). Forty-one gene carriers have total thyroidectomy, whose histological examination dem-
onstrated the presence of MTC, and before death both had
TABLE 1. Primers used to amplify exons 7–19 of the RET gene evidence of distant metastatic disease. On the other hand,
Annealing several gene carriers died at older ages due to other causes,
Size
Exon Oligonucleotide F/R
(bp)
temperature without clinical manifestations of MTC (II.1, II.10, III.4, III.5,
(C)
III.15, and III.16). In addition, two patients positive for the
7 5"-TACCCTCAGGCCATTACAGG-3" 580 60 Gly533Cys mutation, a 75-yr-old man (III.6) and a 69-yr-old
5"-ACTGCTTTTCTCAAAGGGCA-3"
8 5"-GCTGTTCCCTGTCCTTGG-3" 356 62
man (III.21), are asymptomatic, notwithstanding that they
5"-CTATGCTGGCATCGAGAGC-3" have refused clinical investigation.
9 5"-GATCTGCCTAGGAGGTGGTG-3" 310 60 Among all family members identified by genetic screen-
5"-ACTCTGGCTGAAGTGCCTGT-3" ing, the youngest affected patient is a 5-yr-old girl (V.145)
10 5"-GCAGCATTGTTGGGGGACA-3" 172 59
5"-GTTGACACCTCTGTCGGGCT-3"
who exhibited increased sCT after Pg stimulation (basal sCT,
11 5"-CCTCTGGCGGTGCCAAGCCTC-3" 118 61 8 pg/ml; after Pg, 120 pg/ml; normal, 40 pg/ml). She un-
5"-GACAGCAGCACCGAGACGAT-3" derwent total thyroidectomy, and histological examination
12 5"-CTCTTCTCCCCCTTCCCTC-3" 280 60 showed the presence of CCH. The youngest patient to
5"-CACTGTGCCTGTGCCTGG-3"
13 5"-AACTTGGGCAAGGCGATGCA-3" 275 60
present MTC and lymph node metastases is the 22-yr-old
5"-AGAACAGGGCTGTATGGAGC-3" proband’s daughter (V.121; Table 3).
14 5"-CCTGGCTCCTGGAAGACC-3" 298 60 Additionally, 2 new single nucleotide polymorphisms
5"-CATATGCACGCACCTTCATC-3" (SNPs) were detected in intron 8 of the RET gene, corre-
15 5"-TTCCTACAGCTCGTTCATCG-3" 219 60
5"-TCTTTCCTAGGCTTCCCAAG-3" sponding to an A to G variation at nucleotide 127810
16 5"-CTGAAAGCTCAGGGATAGGG-3" 202 60 (127810A3 G) and an insertion of a C after nucleotide 127813
5"-TAACCTCCACCCCAAGAGAG-3" (127813insC; GenBank accession no. AJ243297) in 11 of 76
17 5"-GAGCCACTCACTGGTCCTT-3" 280 60 affected family members. Also, 2 previously described SNPs
5"-GGGAATGCACACAGATGTCC-3"
18 5"-GGCTGTCCTTCTGAGACCTG-3" 250 60 were detected in the proband DNA samples; these polymor-
5"-GAGTCTCCCCAAGCCACTTT-3" phisms are 1296G3 A in exon 7 and 2712C3 G in exon 15.
19 5"-TAGTTGTGGCACATGGCTTG-3" 310 60 We did not find the 1597G3 T substitution in the genomic
5"-GATAGTGCAGAGGGGACAGC-3" DNA of 100 healthy individuals. However, the 127810A3 G

FIG. 2. Direct sequencing of PCR products from exon

8 of the RET oncogene in a normal control and the
index case. In the normal control a GGC was observed,
whereas in the index case a GGC/TGC was found due
to a G to T transversion. The mutation in heterozygosis
is displayed as two peaks, in contrast to the one peak
observed in the wild-type alleles. The mutation occurs
at nucleotide 1597, which corresponds to codon 533.

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Álvares Da Silva et al. • RET Exon 8 Mutation in a Large FMTC Kindred J Clin Endocrinol Metab, November 2003, 88(11):5438 –5443 5441

TABLE 2. Clinical characteristics of 14 carriers not submitted to genetic screening

Age at diagnosis Lymph node

Patient no. Sex Surgery Histology Follow-up
or death (yr) metastasis
II.1 M 81 No Died, heart
II.6 F 53 Yes Yes MTC Died, MTC with metastasis
II.7 F 72 Yes Yes MTC Died, heart
II.9 M 84 No Well, refused investigation
II.10 M 86 No Died, heart
III.1 F 84 No Well, refused investigation
III.2 F 34 No Died, breast tumor
III.4 M 76 No Died, heart
III.5 M 76 No Died, diabetes mellitus
III.14 M 60 No Died, ear tumor
III.15 M 52 No Died, stroke
III.16 M 75 No Died, dementia
III.20 M 60 Yes Yes MTC Died, MTC with metastasis
IV.1 M 59 No Well, refused investigation
M, Male; F, female.

TABLE 3. Clinical characteristics of 35 gene carriers submitted to total thyroidectomy

Patient Pre-op basal/post Lymph node

Sex Age (yr) Histology Follow-up
no. Pg stimulus metastasis
III.7 F 73 1610 Yes MTC Recent surgery (2 months)
III.12 M 61 92 No MTC Recent surgery (1 month)
III.13 F 68 249 No MTC Undetectable sCT
III.17 M 72 1000 Yes MTC Died, 72 yr, heart, MTC under control
III.18 F 47 ND Yes MTC Proband’s mother, MTC with meta
III.23 F 60 1970 Yes MTC Undetectable sCT
III.32 F 59 188 Yes MTC Undetectable sCT
III.34 M 63 1390 Yes MTC Undetectable sCT
IV.8 F 45 85/857 No MTC Undetectable sCT
IV.11 F 53 50 No MTC Recent surgery (1 wk)
IV.12 F 51 959 Yes MTC Recent surgery (2 months)
IV.13 F 47 1050 No MTC Recent surgery (1 month)
IV.26 F 49 266/1600 No MTC Recent surgery (2 months)
IV.28 M 45 40 No MTC Recent surgery (2 wk)
IV.62 M 43 290 No MTC Undetectable sCT
IV.65 F 50 446 Yes MTC Elevated sCT
IV.67 F 46 343 No MTC Undetectable sCT
IV.68 M 42 88/320 No CCH Elevated sCT
IV.71 M 41 143/1870 No MTC Undetectable sCT
IV.72 M 38 113 No MTC Undetectable sCT
IV.74 M 31 60 No MTC Undetectable sCT
IV.77 M 47 1080 Yes MTC Elevated sCT
IV.80 M 42 90 No MTC Undetectable sCT
IV.87 M 42 511 Yes MTC Elevated sCT
IV.89 M 39 75 No MTC Undetectable sCT
IV.91 F 33 1780 No MTC Elevated sCT
IV.92 F 33 10 No CCH Undetectable sCT
IV.98 F 34 580 No MTC Undetectable sCT
IV.103 F 24 30/331 No MTC Undetectable sCT
V.109 F 18 60/81 No CCH Undetectable sCT
V.112 M 11 14/80 No CCH Undetectable sCT
V.121 F 22 385 Yes MTC Elevated sCT
V.122 M 21 51/441 No MTC Undetectable sCT
V.125 M 15 / No CCH Undetectable sCT
V.145 F 5 8/120 No CCH Undetectable sCT
sCT normal values, less than 11.5 ng/ml. ND, Not done; M, male; F, female.

substitution and 127813insC insertion were found in 23% of 76 carriers. According to the last consensus statement on
them. MEN2 (5), only 1 abnormality on RET exon 8 has been pre-
viously reported by Pigny et al. (9), who identified a FMTC
Discussion family with 4 affected members with a germ-line 9-bp du-
In the present report we describe a novel RET mutation in plication in the same locus, creating an additional cysteine
exon 8, codon 533, which causes substitution of a glycine by residue after codon 531.
a cysteine in the cysteine-rich domain of the RET receptor in Several facts indicate that this mutation is associated with
a 6-generation FMTC family composed of 229 members and the tumor phenotype. First, in the index case, no other germ-

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5442 J Clin Endocrinol Metab, November 2003, 88(11):5438 –5443 Álvares Da Silva et al. • RET Exon 8 Mutation in a Large FMTC Kindred

TABLE 4. Amino acid alignment of the RET thyrosine kinase receptor of six different organisms

Rattus norvegicus RPECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTRTCPDGHCDALESRDINICPQD

Mus musculus RPECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTRTCPDGHCDAVESRDANICPQD
Homo sapiens RLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDINICPQD
Xenopus laevis KSECEECGGLGSLTGRCQWRQGHGEGITTNYSTCTPSLLTCPDGICDAVEMNDSAICPQD
Gallus gallus RGDCESARGLGTTTGRCQWRQGSDKGISKNYSTCSPDLRTCPDSVCDAVESKDASICPQD
Danio rerio RGDCEATRGLGAPTGRCQWRQGRDKGISKRYSTCSPNLGTCPDGYCDAIESKNISICPQD
RET mutant RLECEECGCLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDINICPQD
The mutation Gly533Cys is denoted below the alignment in the RET mutant row. Underlined letters denote conservative amino acid
distribution among these organisms at the codon 533.

line mutation was found in exons 7–19 of the RET gene. (12–21). Actually, part of this Brazilian family has recently
Second, the mutation cosegregates with the MTC or CCH in emigrated to other countries, with two patients living in
all 35 patients already submitted to surgery. Third, those Australia (IV.80 and IV.125) and two living in the United
family members without the mutation are clinically unaf- States (IV.84 and IV.104), justifying the importance of ex-
fected. Fourth, the cysteine-rich domain is extremely con- panding the genetic search over the hot spot region for a
served among different organisms (Table 4), and probably, mutation in the RET gene.
the rupture in the balance of cysteine residues causes con- A clinical heterogeneity is observed in this family with
stitutive phosphorylation of RET protein. However, to dem- FMTC. Interestingly, some observations indicate a more be-
onstrate that Cys533Gly in the RET receptor causes ligand- nign course of this disease. First, 6 gene carriers died from
independent activation, similar to the other cysteine causes not related to MTC. Second, the proband’s mother is
mutations described in MEN2A and FMTC families, in vitro still alive at 78 yr of age after a 30-yr course of indolent
analysis should be performed. It is interesting to note that the metastatic disease (III.18); finally, 20 of 35 surgically treated
described mutation, in which a glycine is changed by a cys- patients are probably cured. However, 2 affected members
teine in the codon 533, is in the same region of the previously died due to MTC at ages 53 and 60 yr (II.6 and III.20), 8 of
described duplication (9), where the 9-bp duplication leads 35 carriers had MTC or CCH before 40 yr of age, and 4
to the introduction of an extra cysteine residue. We therefore carriers had CCH before 18 yr of age. Therefore, follow-up
believe that this mutation causes the disease. of these cases is necessary to better understand the prognosis
According to the consensus statement on MEN2 (5), the of patients with this mutation.
reported kindred can be rigorously characterized as FMTC We expect that the mutation described in this paper will
and not MEN2A, as there are more than 10 carriers in the cover a significant proportion of the families with a hitherto
family, multiple carriers or affected members are over 50 yr undisclosed mutation in the RET gene. To improve genetic
of age, and an adequate medical history has been obtained. testing sensitivity, the analysis of RET exon 8 should be
The results of the molecular studies also revealed the pres- considered in FMTC families with no identified classical
ence of 4 polymorphisms. The follow-up of patients with mutations.
these polymorphisms will allow us to investigate whether
the presence of 1 or any of the SNPs could be associated with Acknowledgments
a different course of the disease, contributing to earlier onset
We thank Nildete Gomes, M.D.; M. Inez C. França, M.D.; José R. V.
of MTC, as recently suggested for other RET mutations (10 – Podestá, M.D.; Antonio Pinto, M.D.; and Fernando Pretti, M.D., for
12). Interestingly, the SNPs in intron 8 of the RET oncogene clinical and histological information about the family. We also thank
was found in 11 of 76 affected family members and in 23 Mauro S. Figueiredo, M.D.; Cassandra Corvelo, Ph.D.; Omar M.
normal controls. As the initial reverse primer was designed Hauache, M.D.; Ana O. Hoff, M.D.; and Gustavo S. Guimarães for
in exactly the same spot of the SNPs, one of alleles was not helpful discussions; Ilda S. Kunii and Rosana Tamanaha for technical
assistance; and Angela Faria for efficient laboratory administration. We
amplified by PCR, suggesting a mutation in homozygosis. A express appreciation to all family members for their prompt
new upstream primer was designed, and the PCR product collaboration.
sequencing showed that the mutation occurred in
heterozygosis. Received June 9, 2003. Accepted August 6, 2003.
The mutation Cys533Gly has never been reported in other Address all correspondence and requests for reprints to: Janete M.
Cerutti, Ph.D., Laboratory of Molecular Endocrinology, Division of En-
FMTC or MEN2A kindreds. This could be secondary to the docrinology, Department of Medicine, Universidade Federal de Sao
fact that exon 8 is not routinely screened or that the mutation Paulo, Rua Pedro de Toledo 781, 12 andar, 04039-032 Sao Paulo SP,
is clustered in this family. This is a large family with a Brazil. E-mail: cerutti-endo@pesquisa.epm.br.
Caucasian background, and it is conceivable that relatives This work was supported by grants from the Sao Paulo State Research
living in Europe might also bear the same mutation; hence, Foundation [FAPESP Grants 99/03688-4 and 01/00246 (to R.M.B.M. and
J.M.C.) and 00/03442-2 (to M.R.D.D.S.)], Brazilian Research Council
it is likely to address a founder effect from Spain, and it is (R.M.B.M. is investigator under Contract 300879/1998 –1999), and
feasible that other relatives also migrated to other countries CAPES-Brazilian Department of Education (J.M.C. is investigator under
by the time the ancestor moved to Brazil at the end of the 19th Contract 1946/01-3).
century. Recent studies of genetic screening from Spain and
countries of South America with a large number of descen- References
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806 Letters to the Editor

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(1999) Chromogranin A as serum marker of neuroendocrine
tumors: comparison with neuron-specific enolase and correlation with
immunohistochemical findings. International Journal of Biological
Markers, 14, 160 – 166.
2 Grossrubatscher, E., Dalino, P., Vignati, F., Gambacorta, M., Pugliese,
R., Boniardi, M., Rossetti, O., Marocchi, A., Bertuzzi, M. & Loli, P.
(2006) The role of chromogranin A in the management of patients
with phaeochromocytoma. Clinical Endocrinology, 65, 287 – 293.
3 Lenders, J.W.M., Eisenhofer, G., Mannelli, M. & Pacak, K. (2005)
Phaeochromocytoma. Lancet, 366, 665 – 675.
4 Giusti, M., Sidoti, M., Augeri, C., Rabitti, C. & Minuto, F. (2004) Effect
of short-term treatment with low dosages of the proton-pump inhib-
itor omeprazole on serum chromogranin A levels in man. European
Journal of Endocrinology, 150, 299 – 303.
5 Sanduleanu, S., Stridsberg, M., Jonkers, D., Hameeteman, W., Bie-
mond, I., Lundqvist, G., Lamers, C. & Stockbrügger, R.W. (1999) Serum
Fig. 1 Pedigree of family with medullary thyroid carcinoma (MTC) and RET
gastrin and chromogranin A during medium- and long term acid
Y791F mutation.
suppressive therapy: a case-control study. Alimentary Pharmacology &
Therapeutics, 13, 145 – 153.
Letter
XXX to the Editor

As routinely performed in all patients with MTC and family

Y791F RET mutation and early onset of medullary
thyroid carcinoma in a Brazilian kindred: evaluation
of phenotype-modifying effect of germline variants
Although there is no doubt that different mutations in the RET
gene play a crucial role in the pathogenesis of all forms of multiple
endocrine neoplasia 2 (MEN2) and that the timing of the thyroid
surgery must be tailored based on the knowledge of the affected
RET codon, a great extent of clinical heterogeneity is seen not only
among families with different mutations but also among family
members with the same mutation.1 These findings suggest that
additional factors may affect the course and severity of the disease.
Several reports have verified the association between the presence
of single-nucleotide polymorphisms (SNPs) of the RET gene and
early onset of disease.2
In this study we report on a four-generation Brazilian family with
medullary thyroid carcinoma (MTC) as the only clinical feature. The
index case (II.3, Fig. 1) was referred to us in 2004 at the age of
37 years for follow-up of a MTC diagnosed in another institution.
She presented a thyroid nodule suspicious for MTC on fine-needle
aspiration in 1994, but had refused surgery. Her family history
revealed that her sister (II.2) and two of her nieces (III.12 and III.13)
underwent total thyroidectomy for thyroid nodules, which were
diagnosed as MTC. Because of her niece’s diagnosis, she underwent
total thyroidectomy with central neck dissection in 1997 and the
histological examination showed bilateral MTC. At first evaluation
she had no clinical evidence of metastastatic disease or the presence
of phaeochromocytoma or hyperparathyroidism. However, 7 years
later, serum calcitonin (sCT) was significantly elevated (627 pg/ml,
normal range 23·0–71·0 pg/ml) and neck dissection (levels II–V)
revealed involvement of 7 of 41 lymph nodes. Postoperatively, sCT
remained elevated (1610 pg/ml, normal 5 pg/ml). Upon family
screening, six other family members (II.1, III.1, III.3, III.6, III.7 and
III.9) underwent thyroidectomy for thyroid nodules suspicious for
MTC (Fig. 1).
history, the index case was submitted to RET mutation screening of
exons 8, 10, 11, and 13 –16, as previously described.3 We found a
TAT>TTT mutation within exon 13 of RET, resulting in an Y791F
substitution in the tyrosine kinase domain of the RET receptor.
Twenty-six family members underwent testing for the RET mutation.
Of the 26 family members 17 were mutation-positive. Among
the affected, 10 carriers had clinical, biochemical and histological
diagnosis of MTC. All affected members were also screened for the
presence of hyperparathyroidism and phaeochromocytoma through
biochemical tests including measurements of serum calcium and
PTH and urinary catecholamines, vanillylmandelic acid and
metanephrines. No biochemical evidence of hyperparathyroidism
and phaeochromocytoma was found.
This is the first description of this rare mutation in a Brazilian family
and represents the largest reported kindred with Y791F mutation. The
affected patients had MTC as the only feature. More importantly, we
here describe two patients younger than 20 years of age and MTC at
diagnosis, the youngest age reported in the literature. Furthermore,
laboratory data showed that initial sCT is elevated in a 5-year-old girl
and in an 8-year-old boy (IV.2 and IV.5). Although sCT was elevated
in patients II.5, II.6, IV.2 and IV.5, they refused surgery (Table 1).
The Y791F mutation is exclusively found in Europe and is more
common in Germany and Austria.4 Interestingly, this family has a
German ancestor. Although it is found in a small number of families
with familial medullary thyroid carcinoma (FMTC) and MEN2A, this
mutation is associated with low penetrance of MTC and very low risk
of associated lesions. The European Multiple Endocrine Neoplasia
(EUROMEN) consensus suggests that in asymptomatic carriers of Y791F
mutation, there is no need of prophylactic thyroidectomy before the
age of 10 years and for central lymph-node dissections before the age
of 20 years.1 Others suggest that surgery should be postponed until
the age of 20 years.5 In fact, the reported age of earliest MTC onset
1
is 20 years and the mean age of index cases reported is 57·4 years.
Because it is not clear why patients with RET mutations have such
a variable course and considering that SNPs may play an important

© 2007 The Authors

Journal compilation © 2007 Blackwell Publishing Ltd, Clinical Endocrinology, 67, 805– 808
© 2007 The Authors
Table 1. Clinical features of patients with RET germline mutation affecting codon 791 and SNPs investigated in this family

Age at Postoperative
Age at first thyroidectomy Preoperative Postoperative diagnosis and Lymph node L769L¶’** S836S¶’** G691S/S904S¶’** IVS1-126¶’**
Patient Sex* visit (years) (years) sCT levels† sCT levels†’‡ tumour size (cm)§ metastases exon 13 exon 14 exon 11/exon 15 intron 1

II.1 F 51 52 NA <2 Bilateral MTC (1·5/0·8) No + – + –

II.2 F 31 35 NA 102 Bilateral MTC (1·9) No + – – –
II.3 F 27 30 NA 1610 Bilateral MTC (1·3/0·8) Yes + – + –
II.4 M 55 No 350 No – – + – + –
II.5 M 58 No 360 No – – + – + –
III.1 M 26 26 NA 72 MTC (1·5) Yes + – + +
III.3 M 32 33 160 NA Bilateral MTC (0·8/0·5) No + – + –
III.6 M 20 21 370 12·2 Bilateral MTC (1·2/1·0) Yes ++ – – –
III.7 F 18 19 340 <2 Bilateral MTC (1·2/0·8) Yes + – – +
III.9 F 15 15 530 2·5 Bilateral MTC (0·8/1·3) Yes ++ – – +
III.10 M 37 No 280 No – – + – – +
III.12 F 27 27 NA <2 Bilateral MTC (2·2/1·5) No + – – +

Journal compilation © 2007 Blackwell Publishing Ltd, Clinical Endocrinology, 67, 805– 808
III.13 F 25 25 NA <2 MTC (1·0) No + – – +
IV.2 F 5 No 8 No – – + – – –
IV.3 F 3 No 2 No – – + – – –
IV.4 M 14 No 12 No – – + – + –
IV.5 M 8 No 54 No – – + – – –

*M, male; F, female.

†Normal calcitonin reference: male 12 pg/ml and female 5 pg/ml. Value was obtained at the patient’s last clinical visit. N/A, not available.
‡Postoperative serum calcitonin (sCT) levels correspond to most recent evaluation.
§MTC, medullary thyroid carcinoma.
¶NlaIII was used to investigate the germline variant IVS1–126G>T within intron, BanI the G691S variant in exon 11, Fnu4HI the L769L variant in exon 13, AluI the S836S variant in exon 14 and RsaI the S904S
variant in exon 15 of RET gene.
**(+) presence of heterozygous SNP; (++) presence of homozygous SNP; (–) absence of SNP.
Letters to the Editor 807
808 Letters to the Editor
role in modifying the age of onset, all affected and nonaffected family
members were screened for five known RET SNPs (Table 1). The
SNPs were selected based on their previous association with clinical
course of sporadic or hereditary MTC, in particular promoting
an early onset of disease. DNA samples from 100 unrelated healthy
individuals were used as our normal, ethnically matched control
group. The presence or absence of each SNP was assessed by poly-
merase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) (Table 1) and analysis in a 2·5% agarose gel or 10%
polyacrilamide gel. The study was approved by the Institutional Ethics
and Research Committee and informed consent was obtained from
participants, their parents or their legal guardians.
We found that germline mutation Y791F and the L769L variant
co-occur, as previously demonstrated.4 The L769L variant was found
in heterozygosis in 15 carriers and in homozygosis in 2 carriers
(Table 1). The median age at diagnosis for heterozygosis was
28·5 years and for homozygosis was 18 years. Of note, the two
patients homozygous for L769L and disease onset before the age
of 20 years had bilateral MTC and lymph node involvement at
diagnosis.
Because most of Y791F/L769L carriers had clinical manifestations
before age of 35 years, we therefore suggest that L769L may have an
effect on the early age onset of the disease. The precise mechanism
by which this variant may affect the age of onset is open to new studies.
Another hypothesis is that L769L may be in linkage disequilibrium
with another variant. Further in vitro analysis and variant association
studies in the future may confirm or reject our hypothesis. However,
we did not find an association between L769L and S836S variants
although it has been suggested in the literature.
In conclusion, we suggest that patients harbouring Y791F mutation
may be considered at risk in an age earlier than 20 years and speculate
that L769L variant of RET may be related to the earlier age of onset
in these patients. These findings have important clinical implications
and may help to optimize a more individualized approach in the
timing and extent of prophylactic thyroidectomy.
Acknowledgements
This project was supported by grants 04/15288-0 and 05/60330-8
given to JMC and from the São Paulo State Research Foundation
Journal compilation © 2007 Blackwell Publishing Ltd, Clinical Endocrinology, 67, 805– 808
(FAPESP). JMC and RMBM are investigators of the Brazilian
Research Council (CNPq) and RT is a recipient of a research fellow-
ship from CAPES. We thank Dr Marco Antonio Braconi Moura who
provided clinical information.
Rosana Tamanaha*, Cléber P. Camacho†, Elza S. Ikejiri†,
Rui M. B. Maciel† and Janete M. Cerutti*,†
*Division of Genetics, Department of Morphology and
†Division of Endocrinology, Department of Medicine,
Federal University of São Paulo, São Paulo, Brazil.
Correspondence: Janete M. Cerutti,
Rua Pedro de Toledo 781, 12° andar,
Federal University of São Paulo, 04039-032 São Paulo, SP, Brazil.
Tel: +55-11-5081-5233; Fax: +55-11-5084-5231;
E-mail: cerutti-endo@pesquisa.epm.br
doi: 10.1111/j.1365-2265.2007.02964.x
References
1 Machens, A., Niccoli-Sire, P., Hoegel, J., Frank-Raue, K., van
Vroonhoven, T.J., Roeher, H.D., Wahl, R.A., Lamesch, P., Raue, F.,
Conte-Devolx, B. & Dralle, H. (2003) Early malignant progression of
hereditary medullary thyroid cancer. New England Journal of Medicine,
349, 1517–1525.
2 Weber, F. & Eng, C. (2005) Editorial: germline variants within RET –
clinical utility or scientific playtoy? Journal of Clinical Endocrinology
and Metabolism, 90, 6334 – 6336.
3 Da Silva, A.M., Maciel, R.M., Da Silva, M.R., Toledo, S.R., De Carvalho,
M.B. & Cerutti, J.M. (2003) A novel germ-line point mutation in RET
exon 8 (Gly (533) Cys) in a large kindred with familial medullary thyroid
carcinoma. Journal of Clinical Endocrinology and Metabolism, 88,
5438 –5443.
4 Baumgartner-Parzer, S.M., Lang, R., Wagner, L., Heinze, G., Niederle,
B., Kaserer, K., Waldhausl, W. & Vierhapper, H. (2005) Polymorphisms
in exon 13 and intron 14 of the RET protooncogene: genetic modifiers
of medullary thyroid carcinoma? Journal of Clinical Endocrinology and
Metabolism, 90, 6232– 6236.
5 Gimm, O., Ukkat, J., Niederle, B.E., Weber, T., Thanh, P.N., Brauckhoff,
M., Niederle, B. & Dralle, H. (2004) Timing and extent of surgery in
patients with familial medullary thyroid carcinoma/multiple endocrine
neoplasia 2A-related RET mutations not affecting codon 634. World
Journal of Surgery, 28, 1312–1316.
© 2007 The Authors
 Early Diagnosis of Multiple Endocrine Neoplasia Type 2B:
a Challenge for Physicians

ABSTRACT perspectives
Background: The hereditary form of medullary thyroid carcinoma may occur
isolated as a familial medullary thyroid carcinoma (FMTC) or as part of Mul-
tiple Endocrine Neoplasia 2A (MEN2A) and 2B (MEN2B). MEN2B is a rare
syndrome, its phenotype may usually, but not always, be noted by the physi-
cian. In the infant none of the MEN2B characteristics are present, except by
early gastrointestinal dysfunction caused by intestinal neuromas. When avail- CLEBER P. CAMACHO
able, genetic analysis confirms the diagnosis and guides pre-operative evalu- ANA O. HOFF
ation and extent of surgery. Here we report four cases of MEN2B in which the
SUSAN C. LINDSEY
late diagnosis had a significant impact in clinical evolution and, potentially, in
overall survival. Case Report: We report four cases, 2 men and 2 women, with PRISCILA S. SIGNORINI
differences in their phenotypes and with a late diagnosis. The first case has a FLÁVIA O. F. VALENTE
history of severe gastrointestinal obstruction requiring a surgery intervention
MARIANA N. L. OLIVEIRA
two days after his birth. The second told had nodules in the oral mucosa and
constipation since childhood. The third case referred a history of constipation ILDA S. KUNII
from birth until 5 months of life. The fourth has had a history of chronic con- ROSA PAULA M. BISCOLLA
stipation since childhood. Discussion: New concepts have emerged since the
JANETE M. CERUTTI
RET oncogene was identified in 1993 as the responsible gene for hereditary
medullary thyroid carcinoma. The majority of MEN2B individuals have M918T RUI M. B. MACIEL
mutation in the exon 16 of RET, with a few cases having a mutation A883F or
the association of V804M with E805K, Y806C or S904C mutations. The con-
sensus classifies the RET mutation in codon 918 as of highest risk and recom- Laboratory of Molecular
mends total thyroidectomy and central lymph node dissection until 6 months Endocrinology, Division of
after birth. A fast and precise diagnosis is essential to reach these goals. The Endocrinology, Department of
identification of early manifestations such as intestinal ganglioneuromatosis Medicine, Federal University of
and oral mucosal neuromas should prompt the physician to initiate an inves- São Paulo (CPC, AOH, SCL, PSS,
tigation for multiple endocrine neoplasia type 2B. Conclusion: The diagnosis FFV, MN, RPMB, JMC, RMBM);
of MEN2B is very important to allow appropriate investigation of associated Fleury-Medicina e Saúde (AOH,
diseases and to allow counseling and appropriate screening of relatives for a RPMB, RMBM), São Paulo, SP, Brazil.
RET mutation. Even patients with MEN2B, which often have typical physical
features, may not be properly recognized and be followed as a sporadic
case. Based on this, all suspicious cases of multiple endocrine neoplasia
should undergo a molecular genetic test. (Arq Bras Endocrinol Metab 2008;
52/8:1393-1398) copyright© ABE&M todos os direitos reservados

Keyword: Medullary thyroid carcinoma; MEN2B; M918T; Phenotype and in-

testinal ganglioneuromatosis

RESUMO

Diagnóstico Precoce de Neoplasia Endócrina Múltipla Tipo 2B: um Desafio

para os Médicos.
A forma hereditária do carcinoma medular da tiróide pode ocorrer de modo
isolado, o carcinoma medular da tiróide familiar (FMTC), ou como parte das
neoplasias endócrinas múltiplas tipo 2A (MEN2A) e 2B (MEN2B). MEN2B é
uma síndrome rara e seu fenótipo é usualmente, mas nem sempre, notado Received in 2/9/2008
pelo médico. Na infância, nenhuma das características de MEN2B estão pre- Accepted in 29/10/2008

Arq Bras Endocrinol Metab 2008;52/8 1393

 Multiple Endocrine Neoplasia Type 2B
Camacho et al.

sentes, exceto pela disfunção gastrintestinal precoce, causada pelos neuromas

intestinais. Quando disponível, a análise genética confirma o diagnóstico e
orienta a avaliação pré-operatória e extensão da cirurgia. Neste artigo, apre-
sentamos quatro casos de MEN2B, nos quais o diagnóstico tardio teve impacto
significativo na evolução clínica e, potencialmente, na mortalidade em geral.
Apresentação dos casos: Apresentamos quatro casos, dois homens e duas
mulheres, com diferenças em seus fenotipos e com diagnóstico tardio. O
primeiro caso tem história de obstrução gastrintestinal importante em que foi
necessária cirurgia dois dias após o nascimento. O segundo paciente apresen-
tava nódulos na mucosa oral e constipação desde a infância. O terceiro referia
história de constipação desde o nascimento até 5 meses de idade. O quarto
tinha história de constipação intestinal desde a infância. Discussão: Novos con-
ceitos emergiram desde que o oncogene RET foi identificado, em 1993, como
o gene responsável pelo carcinoma medular da tiróide hereditário. A maioria
dos indivíduos apresenta a mutação M918T no éxon 16 do RET, enquanto pou-
cos casos apresentam a mutação A883F ou a associação de V804M com E805K,
Y806C ou S904C. O consenso recomenda a tiroidectomia total com dissecção
dos linfonodos no compartimento central até os 6 meses após o nascimento. O
diagnóstico rápido e preciso é essencial para o atingir os objetivos. Conclusão:
O diagnóstico precoce de MEN2B é muito importante para propiciar a investi-
gação apropriada de doenças associadas e para permitir aconselhamento e
rastreamento dos parentes para uma mutação do RET. Pacientes com MEN2B,
que apresentam frequentemente achados típicos ao exame físico, podem não
ser reconhecidos e seguidos como casos esporádicos. Por causa disso, todos
os casos de neoplasia endócrina múltipla devem ser avaliados pelo teste gené-
tico para mutações do RET. (Arq Bras Endocrinol Metab 2008; 52/8:1393-1398)
Descritores: Carcinoma medular de tiróide; MEN2B; M918T; Fenótipo; Gan-
glioneuromatose intestinal

INTRODUCTION Table 1. List of signs and symptoms of MEN2B and their fre-
quencies.

T he hereditary form of medullary thyroid carcinoma

may occur isolated as a familial medullary thyroid
carcinoma (FMTC) or as part of Multiple Endocrine
Clinical characteristics

Mucosal neuromas of the lips, eyelids,

Frequency

100%
Neoplasia 2A (MEN2A) and 2B (MEN2B). The Multi- buccal mucosa, tongue, palate and
ple Endocrine Neoplasia 2B represents an inherited au- intestinal mucous membrane (earliest sign)
tosomal dominant syndrome characterized by medullary Enteric Ganglioneuromatosis 100%
thyroid carcinoma, pheochromocytoma, mucosal neu-
romas, ganglioneuromatosis of the gut and marfanoid Medullary carcinoma of the thyroid (very 90%
habitus (1). Although MEN2B is a rare syndrome, its young child)
copyright© ABE&M todos os direitos reservados

phenotype may usually, but not always, be noted by the Skeletal abnormalities of the spine (lordosis, common
physician. kyphosis, scoliosis), talipes equinovarus,
The clinical diagnosis of MEN2B may be facilitated kyphoscoliosis, joint laxity and) and pes
by the identification of a characteristic phenotype whi- cavus

ch includes distinctive facial features such as thickened Thickened medullated corneal nerves
lips and elongated face, a “marfanoid” body habitus, (slitlamp examination)
mucosal neuromas (of lips, buccal mucosa, tongue,
eyelids, palate and intestinal mucous membrane), thi- Facies with thickened lips
ckened medullated corneal nerves (on slitlamp exami- Marfanoid habitus 65-75%
nation), skeletal abnormalities (kyphoscoliosis, joint
laxity and pes cavus) in addition to medullary carcinoma Pheochromocytoma 45-50%
of the thyroid and pheochromocytoma (2) (Table 1).

1394 Arq Bras Endocrinol Metab 2008;52/8

 Multiple Endocrine Neoplasia Type 2B
Camacho et al.

On the other hand, in the infant, none of these

A B
characteristics are present, except by early gastrointesti-
nal dysfunction caused by intestinal neuromas (3). In-
testinal ganglioneuromatosis can lead to severe
constipation and megacolon simulating Hirschsprung’s
disease (4-7). In fact, Hirschsprung’s disease may also
occur with MEN2B (8).
The early onset and aggressiveness of medullary
thyroid cancer in MEN2B requires an early diagnosis
and treatment. It is critical that these cases are identi-
fied in the first year of life so that early thyroidectomy is
performed. When available, genetic analysis confirms
the diagnosis and guides pre-operative evaluation and
extent of surgery (6). C
Here we report four cases of MEN2B in which the
late diagnosis had a significant impact in clinical evolu-
tion and, potentially, in overall survival.

CASE REPORTS

Figure 1. A) Hip X-Ray of first case showing a long and later-

First case ally-displaced femoral neck; B) fourth patient clinical char-
acteristics; C) mucosal neuromas in the tongue of fourth
A 20 year-old man had a thyroid nodule diagnosed
patient.
when he was 12 years old, following non-specific thro-
at complaints. He subsequently saw an endocrinolo-
gist who made the diagnosis of MTC through a serum
calcitonin of 18750 pg/mL. He underwent total A B
thyroidectomy at the age of 14, but only a partial re-
section was achieved. He then received systemic che-
motherapy with multiple drugs for four years and is
now on imatinib (Gleevec®). During the treatment,
one year after the thyroidectomy, lung nodules were
found in a CT scan. C D
The past medical history was significant for seve-
re gastrointestinal obstruction requiring a surgery
intervention two days after his birth and an ortho-
pedic surgery for a bone fracture after a fall at the copyright© ABE&M todos os direitos reservados

age of 11. There was no family history of endocrine

diseases.
In his last clinical visit, at the age of 20, the physical
examination revealed a normal blood pressure, a wei-
ght of 43.8 kg, height of 181 cm with an arm span of
175 cm and the presence of mucosal neuromas in the
tongue. He brought a hip x-ray which showed femoral
deformities (Figure 1A).
The genetic analysis revealed a mutation of the
RET proto-oncogene, M918T (Figure 2A).
Figure 2. Eletropherograms of the reported cases with exon
16 RET mutation at codon 918 (ATG>ACG).
Second case
A 23 year-old man with complaints of sweating, hyper-
tension (170 x 110 mmHg in more than one ocasion)
and headache for 18 months was diagnosed with a ne-
arly five-centimeter pheochromocytoma in the right
adrenal gland and then underwent adrenalectomy.

Arq Bras Endocrinol Metab 2008;52/8 1395

 Multiple Endocrine Neoplasia Type 2B
Camacho et al.
Prior to the surgery, on physical examination, a
thyroid nodule was noticed. Thyroid ultrasound confir-
med it to be a thyroid nodule in the right lobe, measu-
ring 1.4 cm in diameter and with central calcification.
Fine needle aspiration cytology confirmed a MTC.
Three months after the adrenalectomy, the patient un-
derwent total thyroidectomy with bilateral neck dissec-
tion and pathology revealed a multifocal medullary
carcinoma with lymph-node involvement and extra-
thyroidal extension.
Upon questioning, he told he had nodules in the
oral mucosa and constipation since childhood. On phy-
sical examination mucosal neuromas in the tongue
were observed and there were no other remarkable fe-
atures.
He was then submitted to close clinical follow-up.
An abdominal CT scan revealed bilateral nephrolithia-
sis and an enlarged colon. A MIBG scintigraphy sho-
wed high uptake in left adrenal gland. To better
investigate the MIBG findings, a magnetic resonance
imaging (MRI) was obtained which revealed a 1.1 cm
nodule in the left adrenal gland with characteristics of a
pheochromocytoma. A left adrenalectomy was perfor-
med confirming a pheochromocytoma.
He also had a femoral fracture after an accident.
The DNA analysis revealed a mutation of the co-
don 918 of the RET oncogene (M918T) (Figure
2B).
Third case
A 21 year-old woman presented a thyroid nodule at the
age of 16, which was diagnosed as a medullary thyroid
carcinoma. She underwent a total thyroidectomy and
pathology revealed multifocal medullary thyroid carci-
noma (largest lesion measuring 4.5 x 2.7 cm) associa-
ted with extra-thyroidal invasion, C cell hyperplasia
and lymph-node involvement.
copyright© ABE&M todos os direitos reservados
Several months later she was found to have locally-
recurrent disease and underwent a limited neck dissec-
tion with removal of a small number of lymph-nodes.
Post-operatively, due to the persistence of high serum
calcitonin levels, she was treated with external-beam
radiotherapy.
Three years later, she complained of diarrhea and
weakness. At this time serum calcitonin was significan-
tly abnormal (18004 pg/mL) and a CT scan of the
chest revealed multiple nodules in the lungs confirmed
to be metastatic MTC by a biopsy. Prior to the referral
1396
to our institution, she underwent a left cervical neck
dissection which failed to identify metastatic lymph-
nodes.
She was then referred to our institution for further
evaluation and management. Upon questioning she
admitted to have had episodes of headache, palpitations
and sweating lasting approximately three minutes, two
to three times a week. In addition, she referred a his-
tory of constipation from birth until 5 months of life.
There is no family history of medullary thyroid carcino-
ma, however, her father died at a young age from an
unknown cause.
On physical examination, the blood pressure was
120 x 70 mmHg (lying) and 100 x 70 mmHg (stan-
ding up), height was 171 cm, her arm span was 170
cm. Other remarkable features included a thin and
elongated face, thickened lips and mucosal neuromas
in the tongue.
The laboratory evaluation included high levels of
calcitonin (> 20.000 pg/mL) and CEA (423.7 µg/L)
and normal levels of PTH and calcium. The urinary
metanephrines were measured twice and total metane-
phrines were found to be elevated (1954 µg/24h and
1556 µg/24h, normal values 95 to 425 µg/24h).
A MIBG scan revealed moderate adrenal uptake,
more intense in the left adrenal gland. MRI found sli-
ght thickening of both the adrenal glands with no spe-
cific lesions indicating a pheochromocytoma. Due to
evidence of catecholamine hyper secretion without the
localization of a pheochromocytoma, she was started
on prazosin 1 mg/day and she has remained clinically
stable.
Additionally, the MRI revealed liver lesions suspi-
cious for metastasis and a vertebral lesion also suspi-
cious for metastatic disease. A recent neck ultrasound
revealed a mass in the left thyroid bed measuring 27x16
mm and two suspicious lymph nodes in the right cervi-
cal area. Due to the finding of progressive and widely
metastatic disease she is under evaluation for systemic
therapy.
The DNA analysis revealed a M918T RET muta-
tion (Figure 2C).
Fourth case
A 46 year-old woman was diagnosed with a thyroid no-
dule 30 years ago. She was treated with total thyroidec-
tomy and cervical dissection and received external-beam
radiotherapy one year later.
Arq Bras Endocrinol Metab 2008;52/8

The patient was recently evaluated at our institu-
tion and she then complained of weight loss, diarrhea
(10 bowel movements a day) and dyspnea in the past
year and a half. Before the diarrhea started she had
chronic constipation (since childhood). In the past four
weeks she had been having paroxysmal hypertension
which led her to increase the anti-hypertensive drugs
dosage by her own. Systolic blood pressure levels varied
from 200 mmHg to 50 mmHg and during the hyper-
tensive episodes she describes that she felt “as her chest
was going to explode”. In addition, she refers that she
has been evaluated by an ophthalmologist who obser-
ved “a MEN-related eye manifestation”.
The family history is significant for a grandmother
with history of a goiter, mother who died from diabetes
complications at age 83 and a father who died from
hypertension at the age of 87. She has a 16 year-old
daughter who is healthy and apparently has no thyroid
disease.
Physical examination findings were orthostatic hy-
potension (140 x 90 mmHg lying and 90 x 70 mmHg
standing), height of 1.72m and an arm span of 1.62m,
an elongated face with thickened lips and mucosal neu-
romas in the tongue (Figures 1B and 1C).
Laboratory evaluation revealed a high serum calci-
tonin level (2800 pg/mL), an elevated CEA (132
µg/L) and elevated urinary metanephrines.
MRI found multiples nodules in right lobe of the
liver, cholelithiasis, a mass in the right adrenal gland
measuring 11,7 cm and a mass in the left adrenal mea-
suring 4.7 cm. A CT scan of the chest showed diffuse
interstitial lung abnormalities which are under diag-
nostic evaluation .
DNA analysis revealed a M918T RET mutation
(Figure 2D).
The patient was started on prazosin 1 mg/day and
her blood pressure levels are now normal. She is cur-
rently under evaluation and preparation for bilateral
adrenalectomy.
DISCUSSION
New concepts have emerged since the RET proto-on-
cogene was identified in 1993 as the responsible gene
for hereditary medullary thyroid carcinoma. The iden-
tification of the genotype-phenotype correlations in-
cluding age-related development of disease, association
with other manifestations such as pheochromocytoma
Arq Bras Endocrinol Metab 2008;52/8
Multiple Endocrine Neoplasia Type 2B
Camacho et al.
and hyperparathyroidism and the codon-specific ag-
gressiveness of medullary thyroid carcinoma has enri-
ched the physician’s knowledge of the disease and
facilitated the management of the individual patient
and of kindreds with MEN2. Specifically, it has become
clear that, for MEN2B patients, the chance for a better
outcome is the early diagnosis and treatment and the
proper screening of family members (9, 10).
The majority of MEN2B individuals have M918T
mutation in exon 16, with a few cases having a muta-
tion A883F or the association of V804M with E805K,
Y806C or S904C mutations (11-15). The consensus
established in the last International Workshop on Mul-
tiple Endocrine Neoplasia classifies the RET mutation
in codon 918 as highest risk and recommends total
thyroidectomy and central lymph node dissection until
6 months after birth. A fast and precise diagnosis is es-
sential to reach these goals.
In this report, we describe four patients with
MEN2B and incurable MTC due to late diagnosis. The
diagnosis of MEN2B is frequent late, especially when
there is no family history of MEN2B. In newborns, the
characteristic MEN2B phenotype is often absent and
the only common manifestation is constipation due to
intestinal ganglioneuromatosis. In fact, all our four pa-
tients with MEN2B had severe constipation during in-
fancy. It would be important to include MEN2B as a
differential diagnosis of severe constipation and conge-
nital megacolon as, although rare, an early diagnosis of
MEN2B could lead to appropriate treatment and po-
tential cure (6,16,17).
Other characteristics that could alert physicians of a
MEN2B diagnosis include the finding of thickened
corneal nerve upon an ophthalmologic evaluation as
observed in patient 4 and the presence of skeletal mani-
festations such as scoliosis, kyphosis, lordosis, joint laxi-
ty, slipped capital femoral epiphyses and pes cavus
(18-19). Although only one of the four patients has
copyright© ABE&M todos os direitos reservados
orthopedic complaints, the alterations may be common
and guide the physician to the diagnosis of MEN2B.
The reported patients have characteristics such as
the elongated face with thickened lips and mucosal
neuromas, but not the classical marfanoid habitus, re-
presented by an arm span bigger than the height. The
mucosal neuromas are the earliest signs and are present
in almost all patients with MEN2B. Even in the absen-
ce of other signs or symptoms, the presence of mucosal
neuromas in the early ages should motivate a MEN2B
investigation.
1397
 Multiple Endocrine Neoplasia Type 2B
Camacho et al.
The complete syndrome with mucosal neuromas,
pheochromocytoma and MTC occurs in only 50% of
the cases and when present is already too late for a pro-
phylactic approach. The identification of early manifes-
tations such as intestinal ganglioneuromatosis and oral
mucosal neuromas should prompt the physician to ini-
tiate an investigation for multiple endocrine neoplasia
type 2B.
CONCLUSION
The diagnosis of MEN2B is very important to allow
appropriate investigation of associated diseases and to
allow counseling and appropriate screening of relatives
for a RET mutation. Even patients with MEN2B, whi-
ch often have typical physical features, may not be pro-
perly recognized and be followed as a sporadic case.
Based on this, all suspicious cases of multiple endocrine
neoplasia should undergo a molecular genetic test.
Ackowledgements: This work was supported by grants from the
São Paulo State Research Foundation (Fapesp) to RMBM
(06/60402-1, BRASMEN Study) and to JMC (05/60330-8).
The authors are grateful to J. Gilberto H. Vieira, Teresa S. Ka-
samatsu, and Magnus R. Dias da Silva for helpful discussions and
to Angela Faria for secretarial support. JMC and RMBM are
researchers of the Brazilian Research Council (CNPq). No po-
tential conflict of interest relevant to this article was reported.
We wish to thank all the Endocrinologists and Head and Neck
Surgeons who referred their patients to the Medullary Carcino-
ma Clinic and all the team of the Adrenal Clinic at UNIFESP for
the patients referred and for the assistance in the pheochro-
mocytoma cases. This study is part of activities coordinated by
the BRASMEN Project. The BRASMEN study is a data-basis of
Brazilian patients with MEN2-related diseases generated by a
consortium of several Brazilian universities.
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the month. Multiple endocrine neoplasia 2B syndrome. Arch
Pediatr Adolesc Med. 2001;155:845-6.
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the presenting symptom in de novo multiple endocrine neo-
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4. Erdogan MF, Gulec B, Gursoy A, et al. Multiple endocrine neo-
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Natl Med Assoc. 2006;8:783-6.
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type 2 syndrome presenting with bowel obstruction caused by
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6. Unruh A, Fitze G, Janig U, Bielack S, Lochbuhler H, Coerdt W.
Medullary thyroid carcinoma in a 2-month-old male with multi-
ple endocrine neoplasia 2B and symptoms of pseudo-Hirschs-
prung disease: a case report. J Pediatr Surg. 2007;42:1623-6.
7. Cuthbert JA, Gallagher ND, Turtle JR. Colonic and oesophage-
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endocrine neoplasia type 2 and Hirschsprung disease. J Intern
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9. Machens A, Ukkat J, Brauckhoff M, Gimm O, Dralle H. Advan-
ces in the management of hereditary medullary thyroid can-
cer. J Intern Med. 2005;257:50-9.
10. Machens A, Brauckhoff M, Holzhausen HJ, Thanh PN, Lehnert
H, Dralle H. Codon-specific development of pheochromocyto-
ma in multiple endocrine neoplasia type 2. J Clin Endocrinol
Metab. 2005;90:3999-4003.
11. Cranston AN, Carniti C, Oakhill K, et al. RET is constitutively
activated by novel tandem mutations that alter the active site
resulting in multiple endocrine neoplasia type 2B. Cancer Res.
2006;66:10179-87.
12. Miyauchi A, Futami H, Hai N, et al. Two germline missense
mutations at codons 804 and 806 of the RET proto-oncogene
in the same allele in a patient with multiple endocrine neopla-
sia type 2B without codon 918 mutation. Jpn J Cancer Res.
1999;90:1-5.
13. Gimm O, Marsh DJ, Andrew SD, et al. Germline dinucleotide
mutation in codon 883 of the RET proto-oncogene in multiple
endocrine neoplasia type 2B without codon 918 mutation. J
Clin Endocrinol Metab. 1997;82:3902-4.
14. Smith DP, Houghton C, Ponder BA. Germline mutation of RET
codon 883 in two cases of de novo MEN 2B. Oncoge-
ne.1997;15:1213-7.
15. Menko FH, van der Luijt RB, de Valk IA, et al. Atypical MEN
type 2B associated with two germline RET mutations on the
same allele not involving codon 918. J Clin Endocrinol Metab.
2002;87:393-7.
16. Chang A, Chan WF, Lo CY, Lam KS. Multiple endocrine neoplasia
type 2B in a Chinese patient. Hong Kong Med J. 2004;10:206-9.
17. Byard RW, Thorner PS, Chan HS, Griffiths AM, Cutz E. Patholo-
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18. Maia AL, Gross JL, Punales MK. Multiple endocrine neoplasia
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Correspondence to:
Rui M. B. Maciel
Laboratory of Molecular Endocrinology. Division of
Endocrinology, Department of Medicine, Federal University
of São Paulo
Rua Pedro de Toledo 669, 11° andar
04039-032, São Paulo, SP, Brazil
E-mail: rui.maciel@unifesp.br
Arq Bras Endocrinol Metab 2008;52/8
Clinical Endocrinology (2009) 71, 56–64
ORIGINAL ARTICLE
Evaluation of RET polymorphisms in a six-generation family
Blackwell Publishing Ltd
with G533C RET mutation: specific RET variants may modulate
age at onset and clinical presentation
,
Rosana Tamanaha* †, Cléber P. Camacho*, Alexandre C. Pereira‡, Adriana M. Álvares da Silva§,
Rui M. B. Maciel† and Janete M. Cerutti*,†
Genetic Bases of Thyroid Tumors Laboratory, *Division of Genetics, †Division of Endocrinology, Federal University of São Paulo,
São Paulo, Brazil; ‡Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil and §Head and Neck
Service, Heliópolis Hospital, São Paulo, Brazil
Summary
Context We previously described a six-generation family with
G533C RET mutation and medullary thyroid carcinoma, in the
largest family reported do date. Of particular interest, phenotype
variability regarding the age of onset and clinical presentation of
the disease, was observed.
Objective We evaluate whether single SNPs within RET oncogene
or haplotype comprising the RET variants (defined by Haploview)
could predispose to early development of MTC in this family and
influence the clinical manifestation.
Design Eight SNPs were selected based on their previous associa-
tion with the clinical course of hereditary or sporadic MTC, in
particular promoting an early onset of disease. The variants were
initially tested in 77 G533C-carriers and 100 controls using either
PCR-direct sequencing or PCR–RFLP. Association between a SNP or
haplotype and age at diagnosis or presence of lymph node metastasis
was tested in 34 G533C-carries with MTC. Different bioinformatic
tools were used to evaluate the potential effects on RNA splicing.
Results An association was found between IVS1–126G > T and age
at diagnosis. The variant [IVS8 +82A > G; 85–86 insC] was associated
with the presence of lymph node metastases at diagnosis. In silico
analysis suggested that this variant may induce abnormal splicing. This
in silico analysis predicted that the [IVS8 +82A > G; 85–86 insC] could
alter the splicing by disrupting and/or creating exonic splicing
enhancer motifs.
Conclusions We here identified two RET variants that were
associated with phenotype variability in G533C-carriers, which
highlights the fact that the modifier effect of a variant might depend
on the type of mutation.
Correspondence: Janete Cerutti, Genetic Bases of Thyroid Tumors
Laboratory, Rua Pedro de Toledo 669, 11° andar, Federal University of
São Paulo, São Paulo 04039-032, SP, Brazil. Tel.: +55 11 5081 5233;
Fax: +55 11 5084 5231; E-mail: j.cerutti@unifesp.br
56
doi: 10.1111/j.1365-2265.2008.03491.x
(Received 13 August 2008; returned for revision 8 September 2008;
finally revised 1 November 2008; accepted 18 November 2008)
Introduction
Medullary thyroid carcinoma (MTC) is a tumour arising from
thyroid C cells. MTC is a consistent feature of multiple endocrine
neoplasia type 2 (MEN2), a rare genetic syndrome transmitted in
an autosomal dominant pattern. MEN2 is divided into three
clinical forms: (i) MEN2A is characterized by the presence of MTC
in association with phaeochromocytoma and/or hyperparathy-
roidism, (ii) MEN2B in which MTC is accompanied by phaeochro-
mocytoma and developmental abnormalities and (iii) FMTC when
MTC is the only clinical feature.1,2
MEN2 syndrome is caused by germline activating mutations of
the RET oncogene, a single-pass membrane receptor tyrosine
kinase.3,4 Different mutations in RET oncogene are associated with
different forms of MEN2. Although most of MEN2A and FMTC-
related mutations are clustered in the extra cellular cysteine-rich
domain (exons 8, 10 and 11), noncysteine residues within the
intracellular tyrosine kinase domain have been found in about 1%
of the cases with FMTC (exons 13–16).1,3,5–10 MEN2B is associated
with mutations within tyrosine kinase domain (exon 15 and
exon 16).2,11,12
Although a strong genotype-phenotype correlation has been
described, clinical heterogeneity was observed within families or
among families with a specific RET mutation.13,14
In 2003, our group described a 6-generation family initially
diagnosed as FMTC5 according to guidelines for MEN2 syndrome.15
A G533C substitution in exon 8 of RET was identified as the
mutation-causing FMTC. Of particular interest, phenotype variability
regarding the age of onset and clinical presentation of the disease was
observed. Similarly, clinical heterogeneity was described in two
Greek kindred with G533C RET mutation and familial distribution
of FMTC.9 Recently, a group reported a 66-yeard old male with
bilateral phaeochromocytoma as the first clinical manifestation in a
© 2009 Blackwell Publishing Ltd

patient with G533C RET germline mutation. Thyroidectomy was
performed and histological examination showed the presence of
MTC, indicating that patients with G533C mutation also have a
predisposition for MEN2A.16
The aforementioned findings and other examples of this puzzling
phenomenon, in which identical mutations result in highly variable
combinations of clinical features, suggests a role for genetic modifiers.
Several authors have suggested that SNPs within the RET oncogene
could have interacting, predisposing or modifying roles in the
pathogenesis of MEN2 syndrome and sporadic MTC.14,17–29
The present study evaluated whether SNPs within RET could
predispose to early development of MTC in this family, the largest
family reported do date, and influence the clinical manifestation.
Subjects and methods
Subjects
Our study included 172 patients belonging to a family with MTC
from which 77 individuals were carriers of the G533C mutation in
exon 8 of the RET gene.5 The patient’s clinical history reveals that
41 patients underwent thyroidectomy before referral to our institution
for genetic screening. The patients were treated only on the basis of
clinical and biochemical diagnosis: elevated calcitonin level and a
thyroid nodule palpable on physical examination or visualized
by ultrasound. Final histology confirmed MTC in 34 and C cell
hyperplasia in 7 cases. Age at disease onset was defined as the age at
which clinicopathological diagnosis of MTC was made. Clinical data
for the 34 patients with MTC included in association analysis
are summarized in Table 1. In all remaining carriers (n = 36) pro-
phylactic thyroidectomy was indicated based on genetic screening.
Additionally, 100 unrelated individuals which were gender and
age-matched, were used to investigate the prevalence of these SNPs
in normal individuals from same geographical area. These individuals
were chosen from a sample of individuals ascertained in a transversal
study conducted in the same area to determine the prevalence
of cardiovascular risk factors.30 None had a record history of
malignancy or endocrine disease.
Before study inclusion, all the study participants, their parents or
their legal guardian signed an informed consent. The study was
approved by the ethics and research committee of the Federal Uni-
versity of São Paulo and was conducted according to the principles
expressed in the Declaration of Helsinki.
Selection of SNPs and genotyping
Eight SNPs were selected based on their previous association with
the clinical course of sporadic or hereditary MTC, in particular
promoting an early onset of disease (for references, see Table 1).
Genomic DNA was obtained from peripheral blood by standard
procedures as previously described.5 The fragments covering the
RET variants were amplified using PCR primers and conditions
previously described.5,20 Genotyping was performed using either
PCR-sequencing or polymerase chain reaction-restriction fragment
length polymorphism (PCR-RFLP), as detailed in Table 2. For
PCR-RFLP analysis, an aliquot of PCR product was digested with
© 2009 Blackwell Publishing Ltd, Clinical Endocrinology, 71, 56–64
RET polymorphisms as genetic modifier 57
the appropriate restriction enzyme and analysed as previously
described.26 For sequencing, PCR products were purified using the
Concert Rapid PCR Purification System (Invitrogen, Carlsbad, CA)
and submitted to direct sequencing using the Big Dye™ Terminator
Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster
City, CA) as described.39,40 Each sample was sequenced at least twice
and in both directions.40
Statistical analysis
Hardy–Weinberg equilibrium (HWE) for each SNP was assessed
2
in controls by Chi-Square (χ ) tests. Linkage disequilibrium (LD)
and population haplotype was performed using Genepop 3·4
(http://genepop.curtin.edu.au) and the software package Haploview
(http://broad.mit.edu/mpg/haploview). A χ2 test with Yates’s
correction was used to compare the prevalence of different SNPs in
G533C-carriers and control.
The first objective of our analysis was to find whether SNPs could
affect the age of onset. For this purpose, we used Mann–Whitney
U-test. The logistic regression model was used to study the effect of
SNP on age at onset and receiver operating characteristic (ROC)
curve analysis was used to determine the best cut off for age (the best
cut off value was determined balancing sensitivity and specificity).
For the analysis between SNP and gender we used χ2 tests with
Fischer’s correction. Kaplan-Meyer curves were constructed to
analyse the relationship between the occurrence of MTC and age.
Curves were compared with the log-rank statistic.
The second objective of our analysis was to find whether any of
the SNPs were associated with presence of lymph node metastases.
To investigate an association between SNPs and the presence/absence
of metastasis, logistic regression was used as aforementioned.
P ≤ 0·05 was considered statistically significant.
In silico analysis
The analysis was performed using prediction programs for splice
sites (NNSPLICE: http://www.fruitfly.org/seq_tools/splice.html)41 and
for exonic-splicing enhancer (ESE) (ESEFinder: http://exon.cshl.edu/
ESE/).42 RET genomic sequence including SNPs within intron 8
was extracted from the GenBank (http://www.ncbi.nlm.nih.gov;
accession # AJ243297) and analysed using the default threshold
values. In each case both the wild-type and mutant sequence were
submitted.
Results
Frequency of RET SNPs in G533C-carrier and control
We have investigated eight RET polymorphisms in 77 G533C-
carriers. The genotype distribution at individual SNPs are shown
in Table 2. Additionally, 5 out of 8 polymorphisms were investigated
in 100 unrelated normal individuals from the same geographical
region, which were gender and age-matched (control). The three
remaining SNPs were not investigated as there was not enough mate-
rial available. Genotype data of five SNPs in unrelated controls were
used to build the LD map of the studied region (Fig. 1a). Although
58 R. Tamanaha et al.

Table 1. Clinical features and genotype of patients with G533C mutation included in association analysis

Gender and age at Preoperative Histology/ Multifocality/ [IVS8

Age/ thyroidectomy sCT levels tumour Lymph node IVS1– IVS2 + + 82A > G; [G691S;
surgery (years) (ng/ml) size (cm) metastases 126G > T A45A 9G > A 85–86 insC] S904S] L769L S836S

1 77/Yes F/73 714 MTC/0·7 Yes/No – + + + – + –

2 72/Yes M/68 NA MTC/< 0·1 Yes/No – + + – + – –
3 65/Yes M/61 92 MTC/0·6 Yes/No – + + – – + –
4 78/Yes M/72 1000 MTC/0·7 NA/Yes – – ++ – – – –
5 81/Yes F/47 NA MTC/NA NA/Yes – – + + – + +
6 68/Yes F/61 1970 MTC/2 No/Yes – – ++ – + – –
7 63/Yes F/59 188 MTC/0·8 Yes/Yes + – + – – – –
8 67/Yes M/62 1390 MTC/2·3 Yes/Yes – – ++ + – + +
9 52/Yes F/48 83 MTC/0·3 Yes/No + – + – – – –
10 57/Yes F/54 50 MTC/0·7 Yes/No – – + – – – –
11 55/Yes F/51 963 MTC/1·3 Yes/Yes – – + – – – –
12 51/Yes F/47 1050 MTC/3 No/No – – + – – – –
13 44/Yes M/40 NA MTC/1 Yes/No – – + – + – –
14 55/Yes F/52 NA MTC/0·6 Yes/No – – ++ – + – –
15 53/Yes F/48 266 MTC/0·9 Yes/No – – ++ – + – –
16 50/Yes M/46 40 MTC/0·1 Yes/No – ND ND – + – –
17 43/Yes M/43 53 MTC/0·3 Yes/No – + + – – + –
18 47/Yes M/43 290 MTC/0·9 Yes/No – – ++ – + – –
19 54/Yes F/50 446 MTC/1·7 Yes/Yes – – + – + – –
20 51/Yes F/44 343 MTC/0·7 Yes/No – + + – – – –
21 46/Yes M/41 143 MTC/0·5 Yes/No + – + – – – –
22 43/Yes M/38 113 MTC/0·5 Yes/No + – + – – – –
23 36/Yes M/31 60 MTC/0·5 No/No + – + – – – –
24 54/Yes M/48 1080 MTC/2·5 Yes/Yes + – + + – + –
25 42/Yes M/42 90 MTC/2·7 NA/No + – + – – – –
26 48/Yes M/42 511 MTC/1·4 No/Yes + – + – – – –
27 44/Yes M/39 75 MTC/0·5 No/No + – + – – – –
28 40/Yes F/35 1780 MTC/2·5 Yes/No + – + – – – –
29 37/Yes F/33 53 MTC/0·6 No/No + – + – – – –
30 39/Yes F/34 62 MTC/0·6 Yes/No + – + – – + +
31 29/Yes F/24 30 MTC/0·4 No/No – – ++ – + – –
32 29/Yes M/27 NA MTC/0·6 Yes/No – – + – + – –
33 27/Yes F/21 385 MTC/1 No/Yes + – + + – + +
34 28/Yes M/23 51 MTC/0·2 Yes/No – + + – – – –

sCT, serum calcitonin; MTC, medullary thyroid carcinoma; CCH, C cell hyperplasia. (+) SNP in heterozygosis; (+ +) SNP in homozygosis; (–) Absence of
SNP. (*) surgery was not performed. NA, not available; ND, not determined.

we genotyped the 95 G553C noncarriers, to avoid potential bias, they

were not used for comparison (Table 2).
Of note, only SNPs G691S and S904S were in significant LD in
individuals from the population under study. Therefore, to avoid
redundant information, the results were grouped together and here-
after named [G691S; S904S].
The variants IVS1–126G > T and [G691S; S904S] were under-
represented in cases compared to controls (P < 0·05). The S836S was
over-represented in G533C-carriers when compared to control and
to cases reported in the literature (P = 0·008) (Table 2). This fact
reflects the extent of the genetic heterogeneity that exists in different
populations. It is remarkable that the allelic variant IVS2 +9G > A
co-segregate with G533C RET mutation in 100% cases (P < 0·0001;
information is available upon request from the authors).
Linkage disequilibrium analysis
LD between each pair of loci in each population was assessed by
Genepop 3·4 (http://genepop.curtin.edu.au). Several significant
non-random associations of alleles at two loci were found (informa-
tion is available upon request from the authors). S836S and L769L
appears to be in LD in at least some populations21,31,43 and very often
occurs in combination with IVS1–126G > T.32 In our analysis, a
significant association was seen between S836S and L769L in G533C-
carriers (Fisher’s exact test, P < 0·0001) (information is available
upon request from the authors).
It has been suggested that IVS1–126G > T (intron 1) and A45A
(exon 2) variants are in LD in Hirschsprung disease but are not in
phaeochromocytoma.20 We observed that IVS1–126G > T and A45A

© 2009 Blackwell Publishing Ltd, Clinical Endocrinology, 71, 56–64

 RET polymorphisms as genetic modifier 59

Table 2. Prevalence of RET polymorphisms in our study and previous reported in the literature

Prevalence (%) of RET G533C non-carrier Control G533C carriers

SNPs in the literature* (n = 95) (n = 100) (n = 77) Statistics

Control/MEN2 or WW Wp pp WW Wp pp WW Wp pp G533C vs.

refSNP ID Polymorphism Analysis sporadic MTC (%) (%) (%) (%) (%) (%) (%) (%) (%) Control

rs2565206 g.IVS1–126G > T NlaIII 28–35/29–50 37 52 6 31 69 0 42 34 1 0·002

(39·0) (54·7) (6·3) (31·0) (69·0) 0 (54·5) (44·2) (1·3)
rs1800858 p.A45A† Sequencing 23/21–59 37 56 1 57 14 0 ND
GCG > GCA (39·3) (59·6) (1·1) ND ND ND (80·3) (19·7) 0
rs2435351 g.IVS2 +9G > A† Sequencing 38–44/19–47 72 21 1 0 60 11 ND
(76·6) (22·3) (1·1) ND ND ND 0 (84·5) (15·5)
rs3026750; g.[IVS8 +82A > G; Sequencing ND/19–31 79 16 0 66 11 0 ND
rs34827976 85–86 insC] (83·2) (16·8) 0 ND ND ND (85·7) (14·3) 0
rs1799939 p.[G691S; S904S]‡ BanI/RsaI 14–28/4·5–32 54 41 0 63 33 4 60 17 0 0·048
rs1800863 GGT > AGT (56·8) (43·2) 0 (63·0) (33·0) (4·0) (77·9) (22·1) 0
TCC > TCG
rs1800861 p.L769L Fnu4HI 14–29/19–36 57 29 9 70 29 1 57 19 1 0·672
CTT > CTG (60·0) (30·5) (9·5) (70·0) (29·0) (1·0) (74·0) (24·7) (1·3)
rs1800862 p.S836S AluI 2–8·4/1·2–9·3 71 24 0 96 4 0 64 13 0 0·008
AGC > AGT (74·7) (25·3) 0 (96·0) (4·0) 0 (83·1) (16·9) 0

Control: unrelated individuals from the same geographical region, which were gender and age-matched. RET wild-type allele (W) and single nucleotide
polymorphism alleles (p). The [IVS8 +83 A > G; 86–87 insC] polymorphism was defined according GenBank accession number AJ243297. ND, not determined.
*References: 17, 19, 21, 24, 29, 31, 32, 33–38. †Variants investigated in 71 G533C-carriers. ‡The G691S and S904S co-occur in all cases and controls tested.
As regard, the results were grouped together.

Fig. 1 (a) LD across the 5 RET SNPs in unrelated individuals (control) were examined with Haploview. The colour code on the Haploview plot follows the
standard colour scheme for Haploview: white (|D′| < 1, LOD < 2); shades of pink/red (|D′| < 1, LOD ≥ 2); blue (|D′| = 1, LOD < 2); bright red (|D′| = 1,
LOD ≥ 2). (b) LD structure in individuals carriers of the G 533C mutation.

© 2009 Blackwell Publishing Ltd, Clinical Endocrinology, 71, 56–64

60 R. Tamanaha et al.

co-occur in just two cases of G533C-carriers. A significant association

was observed between the IVS1–126G > T and [G691S; S904S] in
G533C-carriers.
It was previously reported that the variants [IVS8 +82A > G;
85–86 insC] and L769L are in LD.37 We found a highly significant
association between [IVS8 +82A > G; 85–86 insC] and L769L
variants in G533C-carriers.

Association between RET genotypes and clinical features

We investigated whether the polymorphisms could have an effect on

the age at onset or clinical course of disease. The mean age at
diagnosis was 45·5 (range = 21–61; SD = 13·2).
Regarding to age at onset, we found a significant association
between the presence of IVS1–126G > T variant and age at diagnosis.
The mean age of onset of MTC in patients who had the IVS1–
126G > T allele was 39 years while patients without this SNP was
49 years (Mann–Whitney U-test; P = 0·0102). A logistic regression
and ROC curve analysis was performed to define the best cut-off for
age of onset. Thus, the choice of a suitable cut-off can be determined
by quantitative assessment of their degree of accuracy. We found that
42 years old was the best cut-off point (sensitivity of 80%, specificity
of 54% and accuracy of 70%). Therefore we classified the patients
into two groups, i.e. age of onset ≤ 42 (13 patients) and > 42 (21
patients). Most patients classified into the group age at onset ≤ 42
enclose those patients with the IVS1–126G > T allele (n = 10). On
the other hand, 17 patients who were classified into the group of age
at onset > 42 years were negative for this allele (P = 0·0014) (Fig. 2a).
Interestingly, ROC curve and Kaplan-Meyer analysis revealed that
about 92% of G533C-carriers and IVS1–126G > T allele carriers will
develop MTC by the age of 73 years.
In our analysis, we could not confirm the association between age
at onset and the other seven investigated polymorphisms.
Subsequently, we compared the frequencies of each variant in
those patients with MTC and absence of lymph node metastases at
diagnosis to patients who presented lymph node metastasis at
diagnosis. Importantly, all patients had undergone standard surgical
treatment for MTC, including total thyroidectomy and central
lymph node dissection. Among 34 patients who presented MTC, 10
patients presented lymph node metastases at diagnosis. We found
an association between the presence of the [IVS8 +82A > G; 85–86
insC] and lymph node metastasis at diagnosis; 23 of 24 patients who
did not presented lymph node metastasis at diagnosis were negative
for this variant (P = 0·019) (Fig. 2b). No other polymorphism was
associated with presence/absence of metastases.
In silico analysis
Some point mutations can have effects on the structure of the
encoded protein by inactivating or activating an exonic splicing
enhancer (ESE). Since the effects of IVS1–126G > T were previously
analysed,23,24 we here conducted in silico analysis to better under-
stand if [IVS8 +82A > G; 85–86 insC] may have an effect on RNA
splicing and therefore an effect on encoded protein.
According to the splice site prediction (NNSplice), the variant
created neither acceptor nor donor splice sites. The ESEs prediction
Fig. 2 Distribution of G533C-carriers by age of onset (a) and presence or
absence of lymph node metastases (b). The bars represent the frequency of
patient in each group. (a) The G533C-carriers with MTC (n = 34) were
grouped into ≤ 42 and > 42 years groups, as described in results section. Most
of cases classified into the group age at onset ≤ 42 enclose those patients with
IVS1-126G>T (black) while most of cases with > 42 years old are negative
for IVS1-126G>T variant (White) (P = 0·0014). (b) Most of G533C-carriers
with MTC and lymph node metastases were positive for IVS8+82A>G; 85_86
insC variant (black) while most of cases with absence of lymph node
metastases were negative for IVS8+82A>G; 85_86 insC variant (White)
(P = 0·019).
program, an online resource to identify ESEs in query sequences
which are responsive for the binding sites of specific serine/arginine-rich
(SR) proteins involved in the splicing machinery (SF2/ASF, SC35,
SRp40 or SRp55). The [IVS8 +82A > G; 85–86 insC] variant created
one putative ESE for SF2/ASF at position 22 and disrupted one
putative ESE for SC35 at position 30 (given the 5′ end position in
Fig. 3a). Additionally a new putative BranchSite (BP) was identified
(the identified c represents the 85–86 insC) (Fig. 3b).
Discussion
We previously investigated a large kindred with MTC whose ancestors
5
migrated to Brazil from Spain in the 19th century. Even though
these individuals share the same disease-causing mutation in the
RET oncogene, there are significant phenotypic differences regarding
the age of onset of disease and clinical presentation. This large kindred
therefore represents a powerful resource to study factors, such as
genetic modifiers, that modulate the clinical manifestations of the
disease. In the present study, we investigated whether the presence
of polymorphisms within the RET gene was correlated with
the observed phenotype acting thus as genetic modifiers.
Of the eight tested variants, IVS1–126G > T was associated with
earlier age at onset and [IVS8 +82A > G; 85–86 insC] with lymph
node metastasis at diagnosis.

© 2009 Blackwell Publishing Ltd, Clinical Endocrinology, 71, 56–64

 RET polymorphisms as genetic modifier 61

Fig. 3 Graphic output window of ESEfinder analysis of partial sequence of human intron 8, which includes the IVS8 +82A > G; 85–86 insC variant of RET
gene, compared with the corresponding wild-type (Wt). (a) The ESEfinder allow identifying new putative ESEs that are recognized by individual Human SR
proteins (SF2/ASF, SC35, SRp40 and SRp55). It also predict whether the point mutation or polymorphism disrupt such elements. The high of each bar indicates
the score value and its placement on x-axis represent its position along the sequence. According to the distribution of ESEs motifs, a potential ESE SF2/ASF
was created in the variant and a SC35 was disrupted. (b) Several sequence elements within the intron are required for the spliceosome catalyses splicing reaction;
among these the 5′ splice site (5′ SS), the 3′ splice site (3′ SS), and the branch point (BP). We here identified a new putative branch site sequence in the variant.

The IVS1–126G > T variant was previously associated with
sporadic MTC suggesting that either this germline mutation or
haplotype containing this variants predispose to sporadic MTC in a
low penetrance fashion.23,24 Similarly the cited studies, the family
here investigated migrated from Spain.
Although the precise mechanism and pathogenic importance of
this variant is still enigmatic, in silico analysis revealed that a new
binding site for NFAT transcription factor (nuclear factor of
activated T-cells) was created.23,24 Interestingly, NFAT family
proteins have been found to be involved in cell cycle regulation, cell
differentiation, cell survival, angiogenesis, tumour cell invasion and
metastasis.44,45
Further evidence supporting the idea that intron 1 is involved in
the transcriptional regulation came from interspecies comparison
(Pan troglodytes, Rattus norvegicus, Mus musculus and Gallus
gallus).46 The authors demonstrated the occurrence of evolutionary
conserved regions located in the intronic sequence and suggested
that these conserved regions contain binding sites for relevant
transcription factors. Thus far, there is no available data from in vivo
or in vitro analysis regarding this putative binding motif and further
experiments are required to prove the functional significance of this
in silico prediction.
An additional hypothesis is that an unknown functional variant,
which may be in linkage disequilibrium with the haplotype con-
taining the IVS1–126G > T variant, could possibly control the
activity of RET oncogene. In fact, a MTC-specific risk haplotype that
includes IVS1–126G > T and S836S was previously described.23 This
group and others have also suggested that the wild type allele at
IVS1–126G > T is in LD a variant that may confer a protective
effect.22,23,47

© 2009 Blackwell Publishing Ltd, Clinical Endocrinology, 71, 56–64

62 R. Tamanaha et al.
Therefore, scanning of the RET chromosomal region for
polymorphisms in LD with the IVS1–126G > T variant might enable
the identification of the functional polymorphism(s) that may affect
age at diagnosis. In our particular setting, G553C-carries presented
the IVS1–126G > T in significant LD with IVS2 + 9G > A, G691S
and S904S (Fig. 1b), although none of these other variables were
associated with age of diagnosis. Taken together, our data suggest
that, within the RET region studied, the IVS1–126G > T polymorphism
may indeed be causally related to age of onset.
Regarding the [IVS8 + 82A > G; 85–86 insC] variant, we describe
an association between [IVS8 + 82A > G; 85–86 insC] and presence
of lymph node metastases at diagnosis. Although it is still an enigma
how this variant could influence cancer progression and metastasis,
one possible explanation is that the polymorphism could lead to
uncommonly spliced product that would generate a protein with a
different or additional function associated with invasion or migration.
This variant could also affect the use of constitutive splice sites, result
in mRNA degradation and consequently in an allele-specific expression
imbalance. Although is unusual that intronic variant generate
consensus binding motif for a splicing factor, this has been described
in RET and other genes.19,48
The ESEfinder analysis performed here suggests that [IVS8
+82A > G; 85–86 insC] variant not only abrogates a potential SC35
binding site but also creates a putative splicing enhancer motif for
SF2. One or both of these events may be responsible for the aberrant
splicing phenotype. Interestingly, other have previously suggested
that [IVS8 + 82A > G; 85–86 insC] variant could possible affect
splicing.32 Consistent with our in silico findings, other have provided
further in vitro and in vivo evidences that an increased binding of
the SC35 splicing factor, due to an intronic genetic alteration, was
directly and specifically involved in a case of genetic disease.49
Although the RET haplotype G691S/S904S was previously
considered as a genetic modifier of MEN2A,17,25 in the present study
we have not been able to confirm such association. It is worth
mentioning that the authors analysed 35 unrelated Spanish families
with MEN2A and found that individuals with G691S/S904S variant
in homozygosis were, on average, 10 years younger at diagnosis.17
Therefore, the lack of association in our study can be partially
explained by the fact that in our kindred this haplotype was found
in homozygosis in one case. Additionally, Lesuer et al. did not find
an association between G691S/S904S and a modifier effect on age at
onset in MEN2A families.14
Concerning the other variants here investigated, we and other
have reported that the variant L769L may have a modifier effect on
the age at onset in patients with FMTC and germline mutation
F791Y.19,26 No association was found between L769L and early age
of disease onset in patients with germline mutation G533C.
Several hypotheses could explain this apparent discordance in the
literature. First, one potential limitation of many associations studies
is that they are underpower and lack of association could just reflect
small sample size. Nevertheless, underpowered is an issue mainly in
negative-result situations. Our report describes the association
between polymorphism and clinically relevant phenotypes and
therefore lack of statistical power is not an issue in this situation. In
addition, like other association-based derived results, our data
provide only potential insights that should necessarily be followed
in more controlled scenarios, as example a secondary cohort in
which G533C mutation was identified.
Second, if different mutations in the same gene can cause different
phenotypic effects, one could expect that a variant could interact
differently with distinct mutations (within–gene interaction). In
other words, some variants may have subtle effect on a specific RET
mutation while others may have major effect.
Third, it is still not clear if a variant has similar effects in different
environments. Some genetic variants could affect the phenotype but
only under specific environments.
Finally, our data are derived from variants not necessarily causal
in a direct sense. All positively associated variants may be in linkage
disequilibrium with other mutation or a nearby functional variant
that directly modulates the associated phenotype. This scenario
would certainly explain the different results obtained in different
genetic structures (i.e. populations) worldwide.
In summary, we found two RET variants that were associated with
phenotype variability and therefore could be considered as candidate
disease-associated variants. This highlights the fact that the modifier
effect of a variant might depend on the type of mutation.
Regarding the ability to demonstrate how a specific polymorphism
or a haplotype play a role as a genetic modifier of a particular
mutation, this still remains a significant challenge. Functional analysis
will lead toward a better understanding the consequence of such RET
polymorphism and will certainly promote our knowledge of the disease
mechanism.
Acknowledgements
This project was supported by the grants 04/15288–0, 05/60330–8
and 06/60402–1 from the São Paulo State Research Foundation
(FAPESP). JMC and RMBM are investigators of the Brazilian
Research Council (CNPq) and RT and CPC are recipients of a
CAPES fellowship grant.
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© 2009 Blackwell Publishing Ltd, Clinical Endocrinology, 71, 56–64
 THYROID
Volume 21, Number 9, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2010.0190

The RET p.G533C Mutation Confers Predisposition

to Multiple Endocrine Neoplasia Type 2A in a Brazilian
Kindred and Is Able to Induce a Malignant Phenotype
Downloaded by American Thyroid Association (ATA) from online.liebertpub.com at 07/10/17. For personal use only.

In Vitro and In Vivo

Mariana N.L. Oliveira,1 Jefferson P. Hemerly,1 André U. Bastos,1 Rosana Tamanaha,1 Flavia R.M. Latini,1
Cléber P. Camacho,2 Anelise Impellizzeri,3 Rui M.B. Maciel,2 and Janete M. Cerutti1

Background: We have previously described a p.G533C substitution in the rearranged during transfection (RET)
oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming
potential of RET p.G533C mutation.
Methods: Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected
into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both
in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C–carriers in
this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma.
Results: Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels
and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C
increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis,
hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in
PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we
described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they
were absent in the DNA isolated from blood.
Conclusions: Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to
PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian
kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.

Introduction order with high penetrance. MEN 2 syndrome includes three

different clinical variants. MEN 2A is characterized by the

T he oncogene rearranged during transfection (RET )

encodes a single-pass tyrosine kinase transmembrane
receptor. It contains four cadherin-like domains and a juxta-
membrane cysteine rich region in the extracellular domain.
The intracellular domain encompasses two tyrosine kinase
subdomains, which are involved in the activation of several
intracellular signal transduction pathways linked to essential
cell processes, such as cell proliferation, differentiation,
motility, and survival [reviewed by refs. (1,2)].
Germ-line mutations in RET gene have been identified as
the underlying cause of the multiple endocrine neoplasia type
2 (MEN 2) syndrome, an inherited autosomal dominant dis-
presence of medullary thyroid carcinoma (MTC), pheochro-
mocytoma, and hyperparathyroidism. MEN 2B is character-
ized by early onset of MTC, pheochromocytoma and, more
rarely, by ganglioneuromas, thickened cornea nerves, and
marfanoid habitus. Familial MTC (FMTC) is characterized by
MTC as the only clinical feature, although the accurate dis-
tinction between FMTC and MEN 2A is not always clear
[reviewed by refs. (2,3)].
The clinical variability observed in MEN 2 syndrome re-
flects genetic heterogeneity. Most mutations associated with
MEN 2A and FMTC were identified in cysteine residues in the
extracellular domain (exons 10 and 11). Almost all individuals

1
Division of Genetics, Genetic Bases of Thyroid Tumors Laboratory, and 2Division of Endocrinology, Laboratory of Molecular
Endocrinology, Federal University of São Paulo, São Paulo, Brazil.
3
Division of Endocrinology, Federal University of Minas Gerais, Belo Horizonte, Brazil.

975
 976
with MEN 2B have an identical mutation in codon 918 (exon
16) (4), although A883F mutation (exon 16) has been described
in a few cases (5).
Recently, the American Thyroid Association created spe-
cific MTC clinical guidelines that combined the MTC litera-
ture with evidence-based medicine and the knowledge of a
panel of expert clinicians. The guideline recommendation
on the timing of prophylactic thyroidectomy and the extent
of surgery is based in this model that utilizes genotype-
phenotype correlations to stratify mutations into four (A–D)
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risk levels (6).
Although it is established that RET mutation in MEN 2
causes RET constitutive activation and that there is a clear
genotype-phenotype correlation, in vitro studies have dem-
onstrated differences in transforming activity of RET receptor
carrying different mutations. Additionally, for less common
mutations, controversy still exists regarding the timing and
extent of surgical intervention. Hence, functional analysis
could provide further evidence for the transforming potential
of these mutations.
We have previously described a mutation in exon 8 of the
RET gene, which leads to a substitution of p.G533C in a family
with MTC as the only clinical feature (7). This mutation was
later identified in two Greek families with FMTC (8). Ad-
ditionally, the p.G533C mutation was described in unrelated
Greek families with pheochromocytoma as the first clinical
manifestation (9,10). Follow-up of these patients has shown
high levels of basal and stimulated serum calcitonin levels.
Total thyroidectomy was performed, and histological exam-
ination confirmed the presence of MTC. The occurrence of
MTC and pheochromocytoma in these Greek kindred provide
the first evidence that p.G533C mutation leads to the MEN 2A
phenotype (9,10).
We here functionally explored the transforming potential
of RET p.G533C mutation. We also report one patient with
pheochromocytoma in this large Brazilian kindred and as-
sessed the mutational status of RET in pheochromocytoma
tissue.
Materials and Methods
Pathological data
We previously described a six-generation family with RET
p.G533C mutation and MTC as the only clinical manifesta-
tion. Out of 76 p.G533C-carriers, 29 had MTC, and 6 CCH (7).
Further, we reported new cases harboring RET p.G533C
mutation who had MTC before referral to our institution for
genetic screening (11). Herein, pathological TNM (pTNM)
classification at the time of surgery and the presence of local
and distant metastases during follow-up is described in those
for whom follow-up data were available (n ¼ 34).
Cell culture
A differentiated thyroid follicular cell derived from 18-
month-old Fischer rat (PCCL3) was maintained in medium
consisting of Ham F-12 supplemented with 5% FCS (Invitro-
gen Corp., Carlsbad, CA) and a four hormone mixture (4H)
including thyrotrophin (1 mU/mL), hydrocortisone (10 nM),
insulin (10 mg/mL), and apo-transferrin (5 mg/mL). A human
MTC cell line (TT) was maintained in Ham F-12 supple-
mented with 10% FBS.
OLIVEIRA ET AL.
Plasmid constructs and transfection assays
The expressing vector pcDNA3.1 encoding the short RET
wild type (Wt) and RET p.C634Y isoforms were kindly do-
nated by Dr Massimo Santoro. The RET p.G533C (mutant)
was generated from the RET Wt by site-directed mutagenesis
using the QuickChange XL mutagenesis kit according to the
manufacturer’s recommendations (Stratagene, La Jolla, CA).
The primers were 50 AGGAGTGTGGCTGCCTGGGCTCCC
30 (sense) and 50 GGGAGCCCAGGCAGCCACACTCCT 30
(antisense). The mutations were confirmed by direct se-
quencing using the Big DyeTerminator Cycle Sequencing
Ready Reaction Kit and analyzed in the ABI PRISM 3100
genetic analyzer (Applied Biosystems, Foster City, CA) as
described (12).
About 10 mg of each construct were stably transfected into
PCCL3 cells by electroporation (Bio-Rad Laboratories, Inc.,
Hercules, CA). For each transfection experiment, gentamicin-
resistant colonies (300 mg/mL) were pooled and checked for
ectopic expression of RET by quantitative polymerase chain
reaction (qPCR). To this end, total RNA was isolated and re-
verse transcribed to cDNA. An aliquot of cDNA was used in
20 mL PCR reaction containing SYBR Green PCR Master Mix
(Applied Biosystems) and 200 nM of each primer for target
(RET ) or reference gene (Actb) as described (13). qPCR reac-
tions were performed in triplicate, the threshold cycle (Ct)
was obtained using Applied Biosystem software and was
averaged (SD " 1). Gene expression was calculated as de-
scribed (13,14). Primer sequences are described in Supple-
mentary Table S1 (Supplementary Data are available online
at www.liebertonline.com/thy).
Protein analysis
Cells expressing RET mutants and Wt were serum- and H4-
starved overnight. Total cell lysates were prepared as de-
scribed (15). For immunoprecipitation experiments, 2 mg of
protein was incubated with anti-RET (Cell Signaling, Beverly,
MA) at 48C, and immunocomplexes were recovered by ad-
sorption to protein A/G plus-agarose beads (sc-2003, Santa
Cruz Biotechnology) at 48C overnight. Total protein (50 mg) or
imunocomplexes were electrophoresed on 4%–12% SDS-
polyacrylamide gel (Invitrogen Corp.), followed by Western
blot analysis as described (15). The membrane was incubated
overnight at 48C with antiphospho-ERK (pERK) (1:1000),
anti-ERK (1:500), anti-RET (1:500) (Cell Signaling), or anti-
phospho-RET Y1062 (1 mg/mL; R&D Systems, Inc., Minnea-
polis, MN). Goat anti-rabbit secondary antibody (HRP
conjugated) was used at 1:5000 dilution (DAKO, Glostrup,
Denmark). The immune complexes were detected using using
SuperSignal West Pico Chemiluminescent Substrate (Pierce,
Rockford, IL). Band intensities were quantified using Molec-
ular Dynamics Image Quant System (GE Healthcare, Buck-
inghamshire, United Kingdom).
Cell proliferation assay
Cell proliferation rate was measured with MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] as-
say as described (16). In brief, PCCL3 cells (104) expressing
RET mutants or Wt were seeded in quintuplicate. At each time
point, cells were incubated with MTT reagent for 3 hours.
Absorbance was measured at a wavelength of 560 nm.
 FUNCTIONAL CHARACTERIZATION OF RET P.G533C MUTATION
Cell viability and apoptotic assays
4 a 7-week period, after which animals were sacrificed and
For cell viability, PCCL3 cells (10 ) were seeded in quintu-
plicate. At the indicated time, cells were stained with Guava
ViaCount reagent (Guava Technologies, Hayward, CA).
Viable and nonviable cells were differentially stained with
Guava ViaCount reagent and counted by a Guava Mini flow
cytometer according to the manufacturer’s specifications
(Guava Technologies).
To detect apoptosis, the cells were double stained with
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annexin-V and 7-amino-actinomycin D (7-AAD), according to
the manufacturer’s recommendations (Guava Nexin method,
Guava Technologies). Both early apoptotic (annexin-V–
positive) and late apoptotic (annexin-V– and 7-AAD–positive)
cells were included in the analysis. Results were expressed as
the percentage of apoptotic positive cells.
Assay for micronuclei
Cells (7#103) were seeded in slide desk (Nunc, Roskilde,
Denmark). After 4 days, cells were fixed and stained using
rapid panoptic LB (Laborclin, São Paulo, Brazil). Micronuclei
were counted under a light microscope at a total magnifica-
tion of #400 according to standard procedures (17).
Anchorage-independent growth
Anchorage-independent growth was assessed by a
double-layer soft agar assay as described (15). After 4
weeks, cells were stained with MTT (1 mg/mL) and pho-
tographed. Colonies formed in soft agar ($64 cells) were
counted from at least four different areas. Two independent
experiments were performed in triplicate. The TT cell line
that harbors the RET p.C634W substitution was used as
positive control.
Expression of genes potentially modulated
by RET p.G533C
To investigate whether the RET p.G533C alters the ex-
pression of thyroid specific genes in PCCL3 cells, as a conse-
quence of its transforming potential, we next investigated the
expression of genes associated with differentiated thyroid
function by qPCR: sodium iodide symporter (Slc5a5), thyroid
peroxidase (Tpo), thyroglobulin (Tg), and thyroid stimulating
hormone receptor (Tshr). We additionally investigated the
expression of genes previously described as induced (Cathe-
psin B (Ctsb) and Decorin (Dcn)) or repressed (Chemokine (C-
X-C motif ) ligand 12 (CxcL12) and TIMP metallopeptidase
inhibitor 3 (Timp3)) by MEN 2A (p.C634R) and/or MEN 2B
(p.M918T) mutations (18).
Total RNA isolation, cDNA synthesis, and PCR reaction
were performed as previously mentioned. Gene expression
was calculated, as described (19). Primers are described in
Supplementary Table S1.
Tumors xenografts in nude mouse
Seven- to 8-week-old female athymic nude (nu/nu) mice
were maintained according to the guidelines of the Division of
Animal Resources at the Federal University of São Paulo. To
evaluate the tumorigenic potential of RET mutant, cells
(6#106) expressing RET p.G533C and parental cells were in-
jected subcutaneously sc into the flank of each mouse (n ¼ 7)
977
for each experimental group. Mice were then monitored over
necropsy was performed. At necropsy, lung, spleen, and liver
were removed and divided into two identical fractions. One
half was immediately snap frozen in liquid nitrogen and
stored at %808C. The other half was fixed by immersion in a
buffered-formalin solution, cut into serial sections of 5 mm,
and routinely stained with haematoxylin and eosin for his-
tological evaluation. Metastatic involvement was monitored
by analysis of haematoxylin and eosin paraffin sections.
Identification of PCCL3 cells expressing
RET p.G533C in liver metastasis
To provide evidence that cells expressing p.G533C were
able to survive in the circulation, reach the liver, and prolif-
erate into tumor colonies, RET expression was investigated
in mouse liver by reverse transcription-polymerase chain
reaction (RT-PCR). To this end, total RNA was isolated from
liver tissues, reverse transcribed into cDNA, and amplified by
PCR as described (13). The identity of the PCR product was
confirmed by sequence analysis (11,20). Additionally, the
expression of thyroid-specific gene (Tg) was evaluated in liver
specimens from control group (n ¼ 2) or nude mice injected
cells expressing RET p.G533C (n ¼ 2) by qPCR as previously
mentioned. The primers for target gene (Tg) and internal
control (Actb) are summarized in Supplementary Table S1.
DNA isolation and RET mutational screening
To investigate whether an additional mutation within RET
oncogene could be implicated in the tumorigenesis of pheo-
chromocytoma, genomic DNA was isolated from adrenal
tumor using standard methods. Moreover, two MTCs sam-
ples obtained from RET p.G533C–carriers were investigated
for the presence of RET mutations. PCR products of exons 8–
16 of RET gene (GenBank #AJ243297) were submitted to direct
sequencing as described (11). Each sample was sequenced at
least twice and in both directions.
Statistical analysis
In vitro results and qPCR values were log transformed and
submitted to ANOVA test with Student-Newman-Keuls ad-
justment. Differences yielding a p < 0.05 were considered
significant.
Results
Pathological features
Herein we reassess pathological features of 34 RET
p.G533C–carriers with MTC whom follow-up data were
available. Patients were classified according to pTNM system.
Eighteen patients were classified into stage 1, 4 into stage II,
and 12 into stage III (Table 1). Although longer follow-up is
needed, these results pointed out that the phenotype of MTC
in patients with this noncysteine mutation may be more ag-
gressive than originally thought.
Moreover, in our previous study, we reported that
two family members died from advanced MTC with me-
tastasis to lung and liver at the ages of 53 and 60, and MTC
or CCH was diagnosed in four patients before the age of
18 (7,11).
 978 OLIVEIRA ET AL.

Table 1. Pathological Features of 34 p.G533C-Carriers

Year of Size of medullary Extrathyroidal

Cases Pedigreea surgeryb thyroid carcinoma (cm) pTNM Stage extension ML/LD Pheochromocytoma

1 III.7 2002 0.7 T1N0Mx I NO 0/6 no

2 III.9 2003 <0.1 T1N0Mx I NO 0/12 no
3 III.12 2002 0.6 T1N0Mx I NO 0/23 no
4 III.17 NA 0.7 T1N1Mx III NO ND no
5 III.18 NA NA T1N1Mx III NO ND no
6 III.23 NA 2 T1N1Mx III NO ND no
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7 III.32 2001 0.8 T1N1Mx III NO ND yes

8 III.34 2001 2.3 T2N1Mx III NO 3/3 no
9 IV.8 2002 0.3 T1N0Mx II NO 0 no
10 IV.11 2003 0.25 T1N0Mx I NO 0 no
11 IV.12 2002 1.3 T1N1Mx III NO 2/6 no
12 IV.13 NA 3 T2N0Mx II NO 0/ND no
13 IV.19 2003 1,1 T3N1Mx III YES 1/5 no
14 IV.24 2003 0.6 T1N0Mx I NO 0/14 no
15 IV.26 2002 1,3 T3NxMx III YES NA no
16 IV.28 2003 0,5 T1N0Mx I NO 0/4 no
17 IV.52 2004 0.3 T1NxMx I NO NA no
18 IV.62 2002 0.9 T1N0Mx I NO 0/4 no
19 IV.65 2001 1.7 T1N1Mx III NO 3/5 no
20 IV.67 2001 0.7 T1N0Mx I NO 0/17 no
21 IV.71 2001 0.5 T1N0Mx I NO 0/3 no
22 IV.72 2002 0.5 T1N0Mx I NO 0/7 no
23 IV.74 2001 0.5 T1N0Mx I NO 0/7 no
24 IV.77 2001 2.5 T2N1Mx III NO 3/10 no
25 IV.80 NA 2.7 T2N0Mx II NO NA no
26 IV.87 2001 1.4 T3NxMx III YES NA no
27 IV.89 2001 0.5 T1NxMx I NO NA no
28 IV.91 2001 2,5 T2NxMx II NO NA no
29 IV.93 2003 0,6 T1N0Mx I NO 0/8 no
30 IV.98 2001 0.6 T1N0Mx I NO 0/ND no
31 IV.103 2002 0,4 T1N0Mx I NO 0/2 no
32 V.37 NA 0.6 T1NxMx I NO NA no
33 V.121 2001 1 T1N1Mx III NO 1/8 no
34 V.122 2002 0.2 T1N0Mx I NO 0/6 no
a
Numbers are according to the pedigree of the six-generation family previously published (7).
b
All patients underwent surgery between 2001 and 2004, and the most recent follow-up was performed between 2009 and 2010.
NA, not available; ND, not detailed; ML, number of metastatic lymph nodes; LD, number of lymph nodes dissected.

Forced expression of RET p.G533C in PCCL3 cells onstrated that the tyrosine residue 1062 (Y1062) is essential
for the transforming activity of RET mutants, we next in-
To investigate the transforming potential of RET p.G533C
vestigated the phosphorylation level of Y1062 and p42/44
mutation in a thyroid follicular cell, constructs encoding RET
extracellular signal-regulated kinase (pERK 1/2). On star-
mutants (p.G533C or p.C634Y) or RET Wt were permanently
vation, both RET and ERK phosphorylation were higher
transfected into PCCL3 cells. To avoid the interference of
in mutants (i.e., p.G533C and p.C634Y) than observed in
clonal selection, clones from each transfectant were pooled
RET Wt. The results are graphically represented (Fig. 1A).
together. Expression of the exogenous RET mRNA was con-
Forced expression of RET p.G533C resulted in a significant
firmed by qPCR. Our results demonstrated that the expres-
increase in cell proliferation, when compared with RET Wt.
sion of RET was similar in all transfectants. Moreover,
Although at lower levels, ectopic expression of RET p.C634Y
sequencing analysis of PCR products confirmed the presence
increased cell proliferation in comparison with RET Wt
of RET mutants or RET Wt (data not shown). Pooled cells
( p < 0.05; Fig. 1B). These findings are in agreement with those
were used in all in vitro and in vivo assays.
obtained for pRET and pERK.

RET p.G533C increases RET and ERK
phosphorylation and cell proliferation
We next investigated the ability of p.G533C mutation
to convert RET into an oncogene and to stimulate the RET/
MEK/ERK cascade. Seeing that several groups have dem-
RET p.G533C increases cell viability while reduces
apoptosis rate of PCCL3 cells
After 24 hours, the expression of RET p.G533C and Wt
resulted in a significant increase in cell viability and in a de-
crease in the apoptosis rate, when compared with parental
 FUNCTIONAL CHARACTERIZATION OF RET P.G533C MUTATION 979
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FIG. 1. Levels of
rearranged during
transfection (RET) Y1062 and
ERK1/2 phosphorylation in
PCCL3 cells expressing RET
mutants and cell proliferation
of different mutants in com-
parison with wild type (Wt)
(A) Increased RET Y1062 and
ERK phosphorylation were
observed in RET p.C533C and
p.C634Y when compared
with RET wild type. The
results are graphically
represented as density units
for phospho-RET Y1062
(pRET) relative to total RET
and phospho-ERK (pERK)
relative to total ERK. (B)
Proliferation rate of
PCCL3 cells after expression
of RET. The data represent
the mean & SD of absorbance
at 560 nm of at least two
experiments performed in
quintuplicate on each of
4 days (*p < 0.05).

cells. After 48 hours, the expression of RET p.G533C increased cells expressing p.G533C may be associated with tumor pro-
cell viability and reduced the rate of apoptosis when com- gression. The results are graphically represented ( p < 0.01)
pared with RET Wt and parental cells ( p < 0.05; Fig. 2A, B). (Fig. 2C).

RET p.G533C induces micronuclei formation RET p.G533C effects on anchorage-independent

in PCCL3 cells
We observed that RET p.G533C stable expression signifi-
cantly increases the number of cells with micronuclei when
compared with parental cells (2.4-fold) or RET Wt (2-fold),
suggesting that the increased genomic instability observed in
growth
We noted that RET p.G533C significantly increased the
ability of PCCL3 cells to form colonies in semisolid medium,
when compared with PCCL3 cells expressing RET Wt
( p < 0.05; Fig. 3). The human MTC cell line (TT), which not
 980 OLIVEIRA ET AL.

FIG. 2. Effects of RET

p.G533C expression in cell
viability, apoptosis, and
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micronuclei formation. (A)

Mean of percentage & SD of
viable cells shows increased cell
viability of RET-expressing
cells 24 hours after seeding. (B)
Percentage of apoptotic cells
was lower in cells expressing
RET p.G533C than in PCCL3
parental, at indicated times.
Percentage of apoptotic cells is
represented by the mean & SD
of from experiment performed
in quintuplicate. (C)
Representative results of
micronuclei formation pointed
out with arrows (magnification
#1000). Results of two
independent experiments
performed in triplicate are
graphically represented. Bar
graph shows the percentage
(&SD) of micronucleated cells
observed in parental and
RET-expressing PCCL3 cells
(*p < 0.05).

only displays high clonogenicity ability but also forms large
colonies in soft agar, was used as a positive control.
RET p.G533C reduces expression of thyroid specific
markers in PCCL3 cells and modulates expression
of Dcn, Cxcl12, and Timp3
It has been reported that alterations along the RET/RAS/
RAF/MAPK pathway promote loss of the differentiated
phenotype, measured by the expression of thyroid-specific
genes (21). We next investigated whether RET p.G533C affect
the expression of thyroid specific genes. The expression of
Slc5a5 (11.3-fold), Tpo (10.9-fold), and Tg (3.1-fold) was lower
in RET p.G533C expressing cells ( p < 0.05), compared with
parental cells. Although not significant, we also noted that the
expression of Tshr in RET p.G533C expressing cells was lower
than the expression observed in parental cells or cells ex-
pressing RET Wt (Fig. 4A).
Identification of genes modulated by RET p.G533C muta-
tion may help better understand the underlying mechanism
by which p.G533C induces the transformed phenotype. Si-
milar to those previously reported for RET p.C634R and
p.M918T mutations, p.G533C expressing cells induced the
expression of Dcn (4.9-fold) while reduced the expression of
Timp3 (4.9-fold) and Cxcl12 (5-fold), when compared with
parental cells ( p < 0.05) (Fig. 4B). Ctsb was expressed at higher
levels in p.G533C expressing cells; however, the difference
was not statistically significant (data not shown). Although
the differences in signal transduction activated by different
RET mutations still remains unknown, we here described
genes that are modulated by p.G533C in vitro.
Evidence that RET p.G533C–expressing cells were
able to induce metastatic dissemination in nude mice
Since anchorage-independent growth is a strong indicator
of the transformed phenotype, we next studied the tumori-
genic potential of PCCL3 cells expressing RET p.G533C in vivo,
and compared them with PCCL3 parental cells. Although
nude mice injected with PCCL3 expressing RET p.G533C
(n ¼ 7) and parental cells (n ¼ 6) did not form any visible
tumors for up to 7 weeks, histological analysis showed the
 FUNCTIONAL CHARACTERIZATION OF RET P.G533C MUTATION
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presence of large lesions inside liver of animals injected with
RET p.G533C, whereas no lesion was observed in control
group. Intriguingly, in all sections examined, not only the liver
architecture was disturbed but also several foci of arrested
cells could be seen close to the portal vessels (Fig. 5A–C).
These findings suggest that both cell extravasation and cell
infiltration into the liver parenchyma occurred.
To test whether the PCCL3 cells expressing p.G533C were
able to reach the liver and form metastasis, we investigated
the expression of the human RET in liver of both experimental
and control groups. RT-PCR detected the presence of RET
transcripts in the liver of animals injected with cells expres-
sing RET p.G533C. Sequencing analysis of PCR products
confirmed the presence of human RET p.G533C in the liver
(Fig. 5D). No RET transcripts were identified in the liver of
control groups.
We next confirmed there was expression of Tg in the liver of
animals injected with PCCL3 cells expressing p.G533C, whereas
Tg was not expressed in liver of control group (Fig. 5E).
Diagnosis of pheochromocytoma
in a RET p.G533C–carrier
A 58 year-old woman was referred to the Endocrinology
Service of Hospital das Clı́nicas, Federal University of Minas
Gerais, for evaluation of a thyroid nodule (0.8 cm). Serum
calcitonin level was 188 ng/mL (normal values <10 ng/mL).
Laboratory tests excluded hyperparathyroidism and pheo-
chromocytoma, as calcium concentrations and urinary meta-
nephrines levels were normal. Total thyroidectomy with
981
FIG. 3. Effect of RET p.G533C
expression in anchorage-
independent growth. Colonies
larger than 64 cells were counted
after 4 weeks. A representative
picture of RET-expressing cells
and positive control (TT cells) is
shown. Bar graph shows the
mean & SD of two independent
experiments performed in
triplicate (*p < 0.05).
central lymph node dissection was performed. Pathological
findings showed MTC with lymph node metastasis. At the
age of 63, the patient had a hypertensive crisis. Magnetic
resonance imaging (MRI) showed a large mass in the right
adrenal gland measuring 4.3#1.7 cm. An intermediate signal
on T1 and high signal on T2-weighted was identified, sug-
gesting pheochromocytoma. Urinary metanephrines were
normal; normetanephrine: 0.40 (reference level <0.41 mg/24
hours) and metanephrine: 0.11 (reference level <0.31 mg/24
hours). The patient was referred for laparoscopic right adre-
nalectomy, and histopathology examination confirmed the
diagnosis of pheochromocytoma (case 7, Table 1).
Identification of novel RET variants
in pheochromocytoma
In addition to the RET p.G533C mutation, two novel vari-
ants within RET oncogene were found in DNA isolated from
pheochromocytoma: a GGC>GTC at codon 548 (exon 8) that
leads to a G548V substitution and a TCC>ACC at codon 556
(exon 9) that leads to a S556T replacement. These variants
were not detected in the DNA extracted from the blood of the
patient (Fig. 6) and in two MTC from RET p.G533C–carriers
(data not shown).
Discussion
In 2003, our group described a missense mutation in exon 8
of RET gene in a six-generation family with MTC as the only
clinical feature (7). This mutation, which leads to a p.G533C
 982 OLIVEIRA ET AL.
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FIG. 4. RET modulates the

expression of (A) thyroid-specific
genes and (B) expression of genes
previously demonstrated as
regulated by RET mutants in
PCCL3 cells. Bar graph
represents mean & SD of
experiment performed in
quintuplicate (*p < 0.05 vs.
PCCL3; #p < 0.05 vs. Wt).

substitution in the cysteine-rich domain of RET, was later
reported in two Greek families with FMTC (8).
Subsequently, it was reported that two unrelated Greek
families harboring RET p.G533C mutation had pheochro-
mocytoma as the first clinical manifestation (9,10). Ad-
ditionally, the index cases in the reported Greek families had a
mild, late form of MTC. These findings, associated with the
fact that none of family members died from MTC-related
causes, led the authors to suggest that RET p.G533C mutation
is associated with a milder phenotype of the MEN 2A (9,10).
Further, all mutations identified within exon 8 of RET onco-
gene were classified by the American Thyroid Association
management guidelines for patients with MTC as risk level A,
which is the lowest risk level for aggressive MTC (6).
Recently, a new mutation (p.C515S) within exon 8 of RET
oncogene was described (22). Functional analysis showed that
this mutation was unable to induce foci formation in NIH-
3T3 cells, suggesting a reduced oncogenic potential when
compared with MEN 2A mutants. When this manuscript was
in the final stage of preparation, Muzza et al., described three
new variants within exon 8 of RET oncogene (23). The iden-
tified variants (p.A510V and p.E511K and p.C531R) had
higher transforming potential than RET Wt, but were signif-
icantly lower than RET p.C634R. Importantly, the p.E511K
mutation was associated with a slight increase in transform-
ing potential in vitro, but it was associated with distant
metastasis and, therefore, with a more aggressive clinical
behavior. The p.A510V mutation was associated with an
 FUNCTIONAL CHARACTERIZATION OF RET P.G533C MUTATION
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in B and C. Thyroglobulin (Tg) expression was found in the liver isolated from experimental group (n ¼ 2), whereas it was
absent in control group (n ¼ 2). Actin (Actb) was used as internal control.
aggressive phenotype with persistent hypercalcitoninemia.
Altogether, these findings not only highlight the fact that the
prevalence of exon 8 mutations are higher than previously
thought, but also that they can be associated with either
FMTC or MEN 2A and have different biological behavior.
Here, we explored the biological function of p.G533C mu-
tation in PCCL3 cells. RET p.G533C had similar levels of RET
Y1062 and ERK1/2 phosphorylation, when compared with
that of RET with codon 634 mutation (p.C634Y). Importantly,
RET mutants showed higher levels of RET Y1062 and ERK1/2
phosphorylation, compared with RET Wt. Although further
analysis is needed, these findings suggest that p.G533C and
p.C634Y may have similar signaling abilities.
Given that RET/MEK/ERK signaling pathway is an im-
portant intracellular cascade that leads to cell proliferation and
survival, we next investigated whether RET p.G533C alters cell
proliferation. Consistent with RET and ERK1/2 phosphoryla-
tion results, p.G533C and p.C634Y increased cell proliferation
when compared with RET Wt, mainly after 72 hours.
Altogether, these results suggest that the p.G533C muta-
tion has effects similar to that observed for p.C634Y substi-
tution. Investigating the role of other tyrosine residues in the
kinase domain will help better understand the biochemical
and biological proprieties of RET p.G533C mutant.
983
FIG. 5. Representative
image of (A) normal liver
tissue isolated from nude
mouse injected with parental
cells (control group) and
(B) nude mice injected with
PCCL3 cells expressing RET
p.G533C (experimental
group) (original magnifica-
tion #100). (C) Higher
magnification (#200) of the
marked area in B.
(D) Sequencing analysis of
the RET transcript expressed
in liver of nude mice injected
with PCCL3 cells expressing
human RET p.G533C.
(E) Expression of thyroid-
specific gene in liver shown
We next showed that p.G533C significantly increased cell
viability, while reducing apoptosis.
It has been demonstrated that transformation of thyroid
PCCL3 cells with genes coding for effectors along the MAPK
pathway (i.e., RAS and BRAF) induces genomic instability
(24,25). These findings, along with the observation that MEN
2-associated tumors from the same patient can have different
genetic alterations, raised the possibility that RET p.G533C
could induce micronuclei formation, as a consequence of ge-
nomic instability. We demonstrated that RET p.G533C sig-
nificantly increases the number of cells with micronuclei,
suggesting that the expression of RET mutant in PCCL3 cells
may promote tumor progression by increasing genetic insta-
bility.
Although it formed less and smaller colonies in semisolid
medium than the positive control, we noted that p.G533C
formed more colonies in soft agar than Wt. These findings
suggest that p.G533C triggers a more invasive phenotype
than Wt.
In the current study, in vivo characterization showed that
RET p.G533C cells were not able to form visible tumors in
nude mice. Similar results were also found by Santoro et al.
(26). However, we observed a widespread infiltration of
PCCL3 cells expressing RET p.G533C within the liver. This
FIG. 6. Two novel variants (A) G548V
and (B) S556T within RET oncogene
were found in DNA isolated from
pheochromocytoma (bottom panel). These
variants were not detected in the DNA
extracted from the blood of the patient (upper
panel).
 984
result, in association to the colony formation assay, suggests
that the cells may be capable of surviving without attaching to
a substratum, a prerequisite for metastasis. RT-PCR analysis
with subsequent DNA sequencing of the PCR product and
expression of thyroid-specific gene in the liver confirmed that
the liver-infiltrating cells expressed human RET p.G533C. Of
note, in patients with MTC, the liver is a major site of meta-
static disease.
Interestingly, metastatic colonies in the liver were mainly
located near the main blood vessels of the liver portal vein.
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Importantly, the more metastatic cells, the greater the likeli-
hood that these cells will be resistant to various therapeutic
modalities. Therefore, screening of potential RET inhibitors
for RET p.G533C is required.
Although clinical and laboratory follow-up reveal that the
several RET p.G533C–carriers had a benign course and be-
came disease-free, 12 of 34 had lymph node metastases or
invasion at the time of diagnosis, two family members died
from metastatic disease to lung and liver, and MTC or CCH
was diagnosed in four patients before age of 18 years.
We also sought to determine whether RET p.G533C could
affect the expression of thyroid specific genes in vitro, as a
consequence of transformed phenotype. RET p.G533C, simi-
larly to RET Wt, reduces the expression of thyroid specific
genes and, therefore, may play a role in reversing the differ-
entiated state of PCCL3 cells. It is worth mentioning that the
expressions of Tpo and Tshr were lower in cells expressing
p.G533C when compared with those expressing RET Wt.
To further elucidate how MEN 2 mutations promote tu-
morigenesis, we next investigated the expression of genes
previously identified as differentially expressed in NIH3T3 -
cells expressing MEN 2A and MEN 2B mutant proteins in
comparison with parental cells (18). Similar to p.C634R and
p.M918T mutants, RET p.G533C induced the expression of
Dcn while reduced the expression of Timp3. However, a
similar effect was observed in cells expressing RET Wt. These
findings suggest that exogenous expression of RET may
modulate the expression of a number of genes. Regarding
Cxcl12, it has been demonstrated that ectopic expression of
RET/PTC1 rearrangement triggers the expression of inflam-
mation-invasion related genes such as CXCL12 (27). Intrigu-
ingly, we described a reduced expression of Cxcl12 with
expression of the RET mutant. Our findings are comparable
with those of Watanabe et al., who verified that RET p.C634R
and p.M918T mutants repressed the expression of Cxcl12 (18).
In the current study, we also report a 63-year-old woman in
whom pheochromocytoma was diagnosed during follow-up.
Although RET p.G533C have been previously associated with
MEN 2A phenotype in Greek kindred (9,10), our finding re-
inforces the need for screening for pheochromocytoma and
primary hyperparathyroidism in p.G533C-carriers.
Interestingly, in unrelated Greek families, pheochromocy-
toma was the presenting feature, whereas in our family it was
reported in only 1 out of 76 p.G533C-carriers. These findings
suggest that the pheochromocytoma penetrance may differ
between Brazilian and Greek families. However, the molec-
ular mechanisms associated with this variability in clinical
expression are poorly understood. It has been suggested that a
second hit which causes a dominant effect of the mutant RET
allele may be a potential mechanism for pheochromocytoma
tumorigenesis in patients with MEN 2A (28). We here de-
scribed two novel variants (p.G548V and p.S556T) within RET
OLIVEIRA ET AL.
oncogene in pheochromocytoma that were absent in the blood
cells from the same patient and in MTC from two patients
with RET p.G533C mutation. Consistent with previous stud-
ies (28), we provide additional evidence that additional ge-
netic events may be associated with tumorigenesis of MEN
2A-associated pheochromocytoma. Although further analysis
is needed to demonstrate the transforming potential of these
variants, it highlights the need for screening for mutations in
non-‘‘hot spot’’ regions.
In summary, we functionally explore the biological and
biochemical effect of the RET p.G533C mutation in PCCL3 cells.
Overall, our in vitro and in vivo analysis indicates that this
mutation appears to confer more malignant properties than
was initially thought. We also describe one patient with
pheochromocytoma in a large Brazilian kindred and identified
two novel RET variants in DNA isolated from the pheochro-
mocytoma. Longer follow-up of the patients and further
studies on signaling pathways activated by RET p.G533C,
p.G548V, and p.S556T mutations, in comparison with other
noncysteine and cysteine RET mutants, will provide new in-
sights into the mechanism associated with the pathogenesis of
p.G533C-MEN 2A associated tumors.
Acknowledgments
This work was supported by The São Paulo State Research
Foundation (FAPESP) (grants 05/60330-8, 06/60402-1 and
09/11257-7). J.M.C. and R.M.B.M. are investigators of the
Brazilian Research Council (CNPq). M.N.L.O., F.R.M.L.,
A.U.B. and J.P.H. are scholars from FAPESP. R.T. is scholar
from CAPES.
Disclosure Statement
The authors declare that there is no conflict of interest
which could be perceived as prejudicing the impartiality of
the research reported.
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Address correspondence to:
Janete M. Cerutti, Ph.D.
Division of Genetics
Genetic Bases of Thyroid Tumors Laboratory
Universidade Federal de São Paulo
Rua Pedro de Toledo 669, 118 andar.
São Paulo 04039-032, SP
Brazil
E-mail: j.cerutti@unifesp.br
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 This article has been cited by:

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Magnus R Dias-da-Silva, Maria Alevizaki, Rui M B Maciel. 2017. Evidence for the founder effect of RET533 as the common Greek
and Brazilian ancestor spreading multiple endocrine neoplasia 2A. European Journal of Endocrinology 176:5, 515-519. [CrossRef]
2. Rodrigues Karine C. , 1, 2 Toledo Rodrigo A. , 1, * Coutinho Flavia L. , 1 Nunes Adriana B. , 3 Maciel Rui M.B. , 4 Hoff Ana
O. , 2 Tavares Marcos C. , 5 Toledo Sergio P. A. , 1, 4 and Lourenço Delmar M. Jr. 1, 2 1Endocrine Genetics Unit (LIM-25),
Endocrinology Division, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil. 2Endocrine
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3Department of Endocrinology, Federal University of Rio Grande do Norte (UFRN), Natal, Brazil. 4Translational and Molecular
Endocrinology Laboratory, Endocrinology Division, Federal University of Sao Paulo (UNIFESP), São Paulo, Brazil. 5Head and
Neck Surgery Division, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil. . 2017. Assessment
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27:5, 693-706. [Abstract] [Full Text HTML] [Full Text PDF] [Full Text PDF with Links] [Supplemental Material]
3. Mehdi Hedayati, Marjan Zarif Yeganeh, Sara Sheikholeslami, Farinaz Afsari. 2016. Diversity of mutations in the RET proto-
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5. Wells Samuel A. Jr. , 1, * Asa Sylvia L. , 2 Dralle Henning , 3 Elisei Rossella , 4 Evans Douglas B. , 5 Gagel Robert F. , 6
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Bruce , 11 Rosenthal M. Sara , 12 Santoro Massimo , 13 Schlumberger Martin , 14 Shah Manisha , 15 and Waguespack Steven
G. 6 1Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 2Department of Pathology,
University Health Network, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario,
Canada. 3Department of General, Visceral, and Vascular Surgery, University Hospital, University of Halle-Wittenberg, Halle/
Saale, Germany. 4Department of Endocrinology, University of Pisa, Pisa, Italy. 5Department of Surgery, Medical College of
Wisconsin, Milwaukee, Wisconsin. 6Department of Endocrine Neoplasia and Hormonal Disorders, Division of Internal Medicine,
The University of Texas MD Anderson Cancer Center, Houston, Texas. 7Department of Radiation Oncology, Memorial Sloan-
Kettering Cancer Center, New York, New York. 8Department of Surgery, Washington University School of Medicine, St.
Louis, Missouri. 9Section of Endocrinology and Metabolism, Department of Internal Medicine, Endocrinology and Metabolism
and Biochemistry, University of Siena, Policlinico Santa Maria alle Scotte, Siena, Italy. 10Endocrine Practice, Moleculargenetic
Laboratory, Medical Faculty, University of Heidelberg, Heidelberg, Germany. 11University of Sydney School of Medicine, Sydney,
New South Wales, Australia. 12Departments of Internal Medicine, Pediatrics and Behavioral Science, University of Kentucky,
Lexington, Kentucky. 13Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita' di Napoli “Federico II,”
Napoli, Italy. 14Institut Gustave Roussy, Service de Medecine Nucleaire, Université of Paris-Sud, Villejuif, France. 15Division of
Medical Oncology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio. . 2015. Revised American
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6. Priscila S. Signorini, Maria Inez C. França, Cleber P. Camacho, Susan C. Lindsey, Flávia O. F. Valente, Teresa S. Kasamatsu,
Alberto L. Machado, Camila P. Salim, Rosana Delcelo, Ana O. Hoff, Janete M. Cerutti, Magnus R. Dias-da-Silva, Rui M. B.
Maciel. 2014. A ten-year clinical update of a large RET p.Gly533Cys kindred with medullary thyroid carcinoma emphasizes the
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7. Theodora Pappa, Maria Alevizaki. 2014. The value of genetic screening in medullary thyroid cancer. Expert Review of Endocrinology
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3Department of Surgery, Mayo Clinic College of Medicine, Rochester, Minnesota. 4Department of Anatomic and Clinical
Pathology, Mayo Clinic College of Medicine, Rochester, Minnesota. . 2013. Multiple Endocrine Neoplasia Type 2A Due to an
Exon 8 (G533C) Mutation in a Large North American Kindred. Thyroid 23:12, 1547-1552. [Abstract] [Full Text HTML] [Full
Text PDF] [Full Text PDF with Links]
9. R Casey, S Prendeville, C Joyce, D O'Halloran. 2013. First reported case in Ireland of MEN2A due to a rare mutation in exon 8
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[CrossRef]
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 AUTHOR COPY ONLY
European Journal of Endocrinology (2012) 166 241–245 ISSN 0804-4643

CLINICAL STUDY

Evidence that polymorphisms in detoxification genes modulate

the susceptibility for sporadic medullary thyroid carcinoma
R B Barbieri1, N E Bufalo1, R Secolin5, A C N Silva1, L V M Assumpção2, R M B Maciel3, J M Cerutti4 and L S Ward1
1
Laboratory of Molecular Genetics Cancer, Faculty of Medical Sciences and 2Thyroid Cancer Unit, Department of Endocrinology, University of Campinas
(UNICAMP), PO Box 6111, Campinas, São Paulo, Brazil, 3Laboratory of Molecular Endocrinology and 4Genetic Bases of Thyroid Tumor Laboratory,
Federal University of São Paulo (UNIFESF), São Paulo, Brazil and 5Laboratory of Molecular Genetics, Department of Medical Genetics, University of
Campinas (UNICAMP), Campinas, São Paulo, Brazil
(Correspondence should be addressed to L S Ward; Email: ward@fcm.unicamp.br)

Abstract
Aim: Polymorphic low-penetrance genes have been consistently associated with the susceptibility to a
series of human tumors, including differentiated thyroid cancer.
Methods: To determine their role in medullary thyroid cancer (MTC), we used TaqMan SNP method
to genotype 47 sporadic MTC (s-MTC) and a control group of 578 healthy individuals for
CYP1A2*F, CYP1A1m1, GSTP1, NAT2 and 72TP53. A logistic regression analysis showed that
NAT2C/C (ORZ3.87; 95% CIZ2.11–7.10; PZ2.2!10K5) and TP53C/C genotypes (ORZ3.87; 95%
CIZ1.78–6.10; PZ2.8!10K4) inheritance increased the risk of s-MTC. A stepwise regression
analysis indicated that TP53C/C genotype contributes with 8.07% of the s-MTC risk.
Results: We were unable to identify any relationship between NAT2 and TP53 polymorphisms
suggesting they are independent factors of risk to s-MTC. In addition, there was no association between
the investigated genes and clinical or pathological features of aggressiveness of the tumors or the
outcome of MTC patients.
Conclusion: In conclusion, we demonstrated that detoxification genes and apoptotic and cell cycle control
genes are involved in the susceptibility of s-MTC and may modulate the susceptibility to the disease.

European Journal of Endocrinology 166 241–245

Introduction same RET mutation, there is considerable phenotypic

The prevalence of medullary thyroid carcinoma (MTC)
among thyroid cancers varies from 3% to no more than
8% in different series of patients, but MTC are
responsible for 13.4% of the deaths caused by thyroid
tumors (1–2). The identification of germline RET
mutations differentiates the sporadic from the heredi-
tary or familial form of the disease, a form that accounts
for only 20–25% of the cases, occurring in a ratio of one
case for every 30 000 individuals (1–2).
Currently available genetic tests for identification of
RET mutations offer a prospective successful therapy,
even before C-cell hyperplasia evolves to medullary
thyroid carcinoma, and disease prognostic accuracy. In
addition, the genetic screening of patients with apparent
sporadic MTC (s-MTC) allows the identification of a
relevant percentage of hidden familial MTC, preclinical
diagnosis and prompt treatment of unsuspected affected
family members. In effect, somatic RET mutations have
been documented in 40–50% of MTC cases (3). Modern
guidelines determine the timing and extent of surgery
according to aggressiveness and age of onset of
inherited MTC, which differ depending on the specific
genetic mutation. However, even in patients with the
variability (4–6). Some studies have suggested that
polymorphisms in the RET gene, perhaps combined
with other low-penetrance genes, could explain this
variability. Recently, the CDKN1B V109G poly-
morphism was associated with a more favorable
s-MTC progression than the wild-type allele, suggesting
an additional role for low-penetrance genes in the
prognosis of MTC patients (7).
In fact, polymorphic low-penetrance genes have been
consistently associated with the phenotype of a series of
human tumors, including differentiated thyroid cancer
(8–11). A growing number of genes encoding enzymes
involved in the biotransformation of toxicants has been
identified and cloned, leading to increased knowledge of
allelic variants in genes and genetic defects that may
result in a differential susceptibility towards poisonous
environmental elements. Moreover, low-penetrating
polymorphisms in metabolism genes tend to be much
more common in the population than allelic variants of
high-penetrating cancer genes. For that reason, these
genes are of considerable importance from a public
health point of view.
Most carcinogens we are exposed to during our
lifetime are metabolized by the cytochrome P450 family

q 2012 European Society of Endocrinology DOI: 10.1530/EJE-11-0843

Online version via www.eje-online.org
 AUTHOR COPY ONLY
242 R B Barbieri and others
of enzymes, including CYP1A2*F, CYP1A1m1 and
others. The products of these enzymes are further
yielded to the action of enzymes such as GSTP1 and
arylamine N-acetyltransferase 2 (NAT2), which can
promote detoxification and excretion of these
compounds in bile or urine. If this detoxification does
not happen or is incomplete, these toxicants may cause
genetic damage by different processes of DNA
interaction, triggering a carcinogenesis process.
However, a series of genes involved in cell cycle control
and apoptosis, such as TP53, can repair the damaged
cell or lead it to death, preventing uncontrolled cell
growth and a consequent cancer. Polymorphisms in
these genes can lead to an aberrant protein activity,
affecting cell function.
Different inherited genetic profiles can provide specific
protection or determine a risk for the development of a
particular cancer type. Along with environmental
factors, which probably serve as triggers of diseases,
this inherited profile of detoxifying and repairing
systems may modulate the specific phenotype of each
patient (12). In addition, polymorphisms of relevant
xenobiotic metabolizing enzymes may be used as
toxicological susceptibility markers, as our group and
others have demonstrated in differentiated thyroid
carcinomas and many other human tumors (12).
This study aimed to investigate the role and outcome
of germline inheritance of polymorphisms in some of the
most important genes related to differentiated thyroid
carcinomas susceptibility such as CYP1A2*F,
CYP1A1m1, GSTP1 codon 105, NAT2 C282T and
TP53 codon 72 genes in patients with s-MTC.
Materials and methods
Patients
This study was approved by the Ethics and Research
Committees of both Federal University of São Paulo
(UNIFESP) and University of Campinas (UNICAMP) and
was in agreement with the 1975 Declaration of Helsinki
revised in 1983. A signed letter of informed consent was
obtained from each individual included in this analysis.
We studied a total of 625 individuals, including 47
patients with s-MTC, and 578 individuals without MTC
selected from an iodine-sufficient area. All patients were
sequenced for the complete RET gene and familial MTC
was excluded. None of the s-MTC patients had any
other type of malignant tumor. Individuals with a
history of past thyroid disease, radiation exposure,
specific environment risk or occupational exposure
risks and antecedents of malignancy were excluded.
Data regarding: lifetime occupational history; dietary
habits; alcohol, coffee and drug consumption; medical
history with an emphasis on previous and/or current
thyroid diseases; use of exogenous hormones and
concomitant medications; reproductive history and
www.eje-online.org
EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166
family history of cancer and other anamnestic data
were obtained using a structured questionnaire. Owing
to the limited reliable data obtained as to the duration in
years of smoking, at what age smoking started, quantity
smoked and number of years since smoking stopped,
patients and controls were grouped into the categories
never-smoked and ever-smoked. This last group com-
prised individuals who consumed at least 20 packages,
20 cigarettes-per-pack during 1 year for the prior
5 years. All data, including nodule size, tumor
histological features and laboratory examinations,
were confirmed in patient records.
RET gene sequencing
Genomic DNA was isolated from peripheral blood
leucocytes by standard phenol techniques. PCR
products of exons 8, 10, 11, 13, 14, 15 and 16 of the
RET gene were purified using the Concert Rapid PCR
Purification System (Invitrogen) and submitted to direct
sequencing using the Big Dye Terminator Cycle
Sequencing Ready Reaction Kit (Applied Biosystems,
Foster City, CA, USA) as described previously (4). Each
sample was sequenced at least twice and in both
directions.
Polymorphisms genotyping
Five polymorphisms were genotyped: CYP1A2*F
(rs762551), CYP1A1m1 (rs 4646903), NAT2 C282T
(rs1041983), GSTP1 codon 105 (rs 1695) and TP53
codon 72 (rs 1042522) using TaqMan system (Applied
Biosystems). Fluorescence signals were detected by
the 7500 system sequence detection software
(Applied Biosystems). The sequence-specific primers
were: CYP1A2*F (C_8881221_40), NAT2 C282T
(C_8684085_20), GSTP1 codon 105 (C_3237198_20)
and TP53 codon 72 (C_2403545_10). The gene
CYP1A1m1 primer was designed according to the
following information: Forward 5 0 -GCACTGGTAC-
CATTTTGTTTCACT-3 0 ; Reverse 5 0 -GCTGAGGTGGGA-
GAATCGT-3 0 . The corresponding sequences of
fluorescence signals were VIC – CACCTCCTGGGCTCA
and FAM – ACCTCCCGGGCTCA.
TaqMan PCR and genotyping analyses were performed
on Applied Biosystems 9600 Emulation System (Applied
Biosystems). The reaction mixtures were amplified in 2 ml
of genomic DNA (10 ng/ml), 2.5 ml of 2! TaqMan
Universal Master Mix, 0.25 ml of 40! primer/probe mix
and 0.25 ml of ddH2O in a volume of 5 ml. PCR cycling
conditions were as follows: one cycle at 60 8C for 1 min as
initial step; one cycle at 95 8C for 20 min; 40 cycles at
92 8C for 3 min and at 60 8C for 30 s; and one cycle at
60 8C for 1 min as the annealing step. The results were
analyzed on Applied Biosystems 9600 Emulation System
using the allelic discrimination assay program.
 AUTHOR COPY ONLY
EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166 Detoxification genes and susceptibility for s-MTC 243

Statistical analysis Table 2 Genotyping characteristics of the 49 patients with sporadic

medullary thyroid cancer (s-MTC) compared with the group of
We used t-test and Fisher’s exact test to evaluate controls. Data are presented as n (%).
differences between the groups concerning age, gender
and smoking, using R environment (13). Hardy– Genes s-MTC Controls HWE P value
Weinberg equilibrium (HWE) was analyzed from CYP1A2 47 (100) 339 (100) 0.869
genotypes by ARLEQUIN Software (14). These analyses A/A 22 (46.1) 162 (47.8)
were performed by SAS Statistical Software (Statistical C/A 19 (40.4) 147 (43.3)
Analysis System, version 9.1.3, 2002–2003). Logistic C/C 6 (12.6) 30 (8.9)
CYP1A1m1 46 (100) 552 (100) 0.583
and stepwise regressions were used to analyze associ- C/C 2 (4.3) 15 (2.7)
ation between polymorphisms and MTC by R Statistical T/C 19 (41.3) 160 (28.9)
Software (13). Post-hoc statistical power of the sample T/T 25 (54.4) 377 (68.4)
was evaluated using GPower Software (15), with a NAT2 C282T 46 (100) 181 (100) 0.400
statistical significant level of 0.05. C/C 21 (45.6) 87 (48.1)
C/T 20 (43.4) 81 (44.7)
T/T 5 (11.0) 13 (7.1)
GSTP1 46 (100) 347 (100) 0.002
A/A 20 (43.4) 200 (57.6)
A/G 19 (41.3) 113 (32.5)
Results G/G 7 (15.3) 34 (9.9)
TP53 codon 72 45 (100) 278 (100) 0.131
As shown in Table 1, patients and controls were similar C/C 18 (40.0) 31 (11.2)
regarding age (PZ0.8576), gender (PZ0.7869) and G/C 21 (46.6) 149 (53.6)
smoking habits (PZ0.3546). Our sample presented G/G 6 (13.4) 98 (35.2)
97.05% of statistical power to detect genetic association
between polymorphisms and MTC by logistic regression
analysis.
contributes with 8.07% of the s-MTC phenotype, whereas
Among the polymorphisms studied, GSTP1 was not
1.53% is due to the age of tumor onset as represented
in HWE. This fact may be due to the relatively small size
in Fig. 1.
of the groups studied and the high genetic heterogeneity
We were unable to establish any relationship between
of the Brazilian population, composed of relatively
the profile of the studied genes and patients’ clinical
recent groups of immigrants from Europe, Africa,
characteristics, lifetime occupational history; dietary
Asia, all mixed to the indigenous populations (16).
habits; alcohol, coffee and drug consumption or out-
Therefore, although GSTP1 was associated with the risk
come. A sample size calculation indicated that a much
of s-MTC development, as shown in Table 2, this
larger cohort of patients would be needed to reach the
polymorphism was not included in further statistical
statistical power to detect main effects or interactions or
analysis.
the precision to quantify them.
Table 3 depicts the results of the logistic regression
analysis that demonstrates an association between s-MTC
and NAT2C/C genotypes (P valueZ2.2!10K5; ORZ
3.87; 95% CIZ2.11–7.10); NAT2T/T (P valueZ Discussion
3!10K7; ORZ0.15; 95% CIZ0.06–0.36); TP53C/C
(P valueZ2.8!10K4; ORZ3.87; 95% CIZ1.78–6.10) Germline RET mutations confer a high risk of
and TP53G/G genotypes (P valueZ2.8!10K4; ORZ developing MTC, but they do not account for all cases;
0.28; 95% CIZ0.13–0.62). A stepwise regression neither do they explain the variability in presentation
analysis demonstrated that TP53C/C genotype and outcome in clinical patients. A sizeable fraction of
the remaining heritability that still needs to be identified
Table 1 Clinical characteristics of the sporadic medullary thyroid is thought to be due to cumulative effects of suscep-
carcinoma (s-MTC) patients compared with the group of controls. tibility alleles associated with low- to moderate-
penetrance genes, in accordance with a model of
Clinical features s-MTC Controls P polygenic inheritance. A series of detoxifying and
Age (yearsGS.D.) 41.01G15.94 39.87G16.31 0.8576 repairing genes that may contribute to this model
Gender (n (%)) 0.7869 have been identified in differentiated thyroid cancer and
Male 32 (68.1) 414 (72.0) other tumors (8–11). In fact, differences in the cellular
Female 15 (31.9) 161 (28.0) mechanisms of activation and detoxification of carcino-
Smoking (n (%)) 0.3546
Yes 7 (14.9) 96 (24.2) genic chemicals may confer different degrees of
No 40 (85.1) 301 (75.8) susceptibility to cancer to each individual, accounting
Alcohol consumption 0.453 for the observed phenotypic variations (9–11).
(n (%)) This study demonstrates, for the first time to our
Yes 7 (24.1) 96 (31.7)
No 40 (75.9) 301 (68.3)
knowledge, an association between NAT2 and TP53
detoxifying genes and s-MTC phenotype. In fact, the

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 AUTHOR COPY ONLY
244 R B Barbieri and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166

8.07%
1.53%
TP53C/C
Age of onset
Others factors
90.40%
Figure 1 Graphic representation of the relative contribution of the
investigated factors to sporadic medullary thyroid carcinoma
susceptibility according to a stepwise regression analysis.
inheritance of a C homozygous allele for NAT2 C282T
gene increases the risk for the development of s-MTC by
more than three times. Epidemiological studies have
shown that a significant part of all cancers are related to
environmental factors, considering tobacco smoke and
diet as the main attributable exposures in a long and
increasing list of carcinogenic elements (17). NAT gene
acts as an inactivating enzyme, catalyzing the conju-
gation of carcinogenic substances such as ionising
radiation and tobacco (18). The enzyme is responsible
for the N-acetylation of arylamine and hydrazine
xenobiotics, and has been implicated in the suscep-
tibility to various cancers including differentiated
thyroid carcinomas (11).
Furthermore, the presence of C homozygous allele for
TP53 gene also increases by more than three times the
susceptibility to s-MTC. Cell cycle and apoptosis
regulators directly involved in the initiation of malig-
nant cell proliferation have long been preferred targets
as cancer risk markers (19). The Pro allele was also
associated with an increased risk of differentiated
thyroid cancer in previous reports (10).
Although the genetic risks of these polymorphisms
are relatively modest, their high frequency in the
population suggests that they may have a considerable
impact on the incidence of s-MTC. In fact, polymorph-
isms of genes that codify enzymes involved in the
detoxification of toxicants may contribute to the
variable susceptibility to a series of carcinogenic
compounds. The identification of a profile of suscep-
tibility to s-MTC might allow the recognition of a group
of individuals at risk, hence deserving a more careful
observation.
A sizeable fraction of the remaining heritability that
still needs to be identified is thought to be due to
cumulative effects of susceptibility alleles associated
with low- to moderate-penetrance genes, in accordance
with a polygenic model of inheritance. A series of
detoxifying and repairing genes that may contribute to
this model have been identified in differentiated thyroid
cancer and other tumors (8–11).
Unfortunately, pathways of carcinogen metabolism
are complex and mediated by the activity of multiple
genes. In addition, these polymorphisms may have
additive or opposite effects; likewise, different toxicants
may produce composite and complex effects. Therefore,
definite conclusions depend on studies with larger
sample sizes that determine the risk estimates associated
with other variants, gene–gene and gene–environment
interactions. Biological microchips designed to identify
polymorphisms in a large series of xenobiotic-metaboliz-
ing, apoptotic and cell cycle control genes may allow the
investigation of multiple contributing factors to the
susceptibility to s-MTC and help understand their
relationship (20).

Table 3 Logistic regression analysis of the association among different genotypes and the OR
for a sporadic medullary thyroid carcinoma development risk.

95% CI
SNP/genotype b P value OR Low High

CYP1A2*F
AA K0.03687 0.9049780 0.96 0.53 1.77
CA K0.11322 0.7173899 0.89 0.48 1.65
CC 0.46334 0.3352942 1.59 0.63 3.98
CYP1A1m1
CC 0.52213 0.4042537 1.69 0.52 5.47
CT 0.51064 0.1029917 1.67 0.91 3.05
TT K0.56144 0.0660261 0.57 0.31 1.03
NAT2 C282T
CC 1.35409 0.0000220 3.87 2.11 7.10
CT 0.35142 0.2530759 1.42 0.78 2.59
TT K1.92965 0.0000003 0.15 0.06 0.36
TP53 codon 72
CC 1.19101 0.0002856 3.29 1.78 6.10
GC 0.07304 0.8092102 1.08 0.59 1.95
GG K1.27589 0.0004093 0.28 0.13 0.62

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 AUTHOR COPY ONLY
EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by the State of São Paulo Research
Foundation (FAPESP) under the grant number 07067-5. L S Ward,
R M B Maiel, and J M Cerutti are researchers of the National Council
for Scientific and Technological Development (CNPq).
Acknowledgements
We thank the team of statisticians of the Faculty of Medical Sciences
and Etna Macário, this paper reviser, for all valuable suggestions and
insights.
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Received 29 September 2011
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Accepted 2 November 2011
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HORM CANC (2012) 3:181–186
DOI 10.1007/s12672-012-0109-7

ORIGINAL PAPER

Extended RET Gene Analysis in Patients with Apparently

Sporadic Medullary Thyroid Cancer: Clinical
Benefits and Cost
Susan C. Lindsey & Ilda S. Kunii &
Fausto Germano-Neto & Misaki Y. Sittoni &
Cléber P. Camacho & Flávia O. F. Valente & Ji H. Yang &
Priscila S. Signorini & Rosana Delcelo &
Janete M. Cerutti & Rui M. B. Maciel &
Magnus R. Dias-da-Silva

Received: 23 January 2012 / Accepted: 12 March 2012 / Published online: 31 May 2012
# Springer Science+Business Media, LLC 2012

Abstract RET sequencing has become an important tool in
medullary thyroid cancer (MTC) evaluation and should be
performed even in the absence of family history of MTC.
The most commonly studied exons in index cases are 8, 10,
11, and 13–16. To address the ATA guidelines regarding the
sequencing of the entire coding region of RET, we selected
50 patients with sporadic MTC (sMTC) without mutations
Electronic supplementary material The online version of this article
(doi:10.1007/s12672-012-0109-7) contains supplementary material,
which is available to authorized users.
S. C. Lindsey : I. S. Kunii : F. Germano-Neto : M. Y. Sittoni :
C. P. Camacho : F. O. F. Valente : J. H. Yang : P. S. Signorini :
R. M. B. Maciel (*) : M. R. Dias-da-Silva
Laboratory of Molecular and Translational Endocrinology,
Department of Medicine, Escola Paulista de Medicina,
Universidade Federal de São Paulo,
São Paulo, São Paulo 04039-032, Brazil
e-mail: rui.maciel@unifesp.br
M. R. Dias-da-Silva
e-mail: diasdasilvamr@gmail.com
in the hot spot regions of RET for extended investigation of
exons 1–7, 9, 12, 17, 18, and 19. Twenty-seven of 50
patients presented with one or more features suggesting famil-
ial disease. We found only a new RET variant (p.Gly550Glu)
in one patient with MTC. Several polymorphisms were ob-
served, and their frequency was histogram scaled by exons
and introns. Eight patients were also included for somatic
mutation search. We estimated the sequencing cost by strati-
fying into four investigation approaches: (1) hot spot exons in
a new patient, (2) the remaining exons if the hot spots are
negative in a patient with suspected familial disease, (3) a
relative of a carrier for a known RET mutation, and (4) tumor
sequencing. In spite of the increasing number of variants
being described in MTC, it appears that there is no direct
clinical benefit in extending RET germ line analysis beyond
the hot spot regions in sMTC. The cost evaluation in apparent
sMTC using a tiered approach may help clinicians make more
suitable decisions regarding the benefits of investigating only
the hot spots against the entire coding region of RET.

R. Delcelo
Department of Pathology, Escola Paulista de Medicina,
Universidade Federal de São Paulo,
Introduction
São Paulo, Brazil
Medullary thyroid cancer (MTC) originates from parafollic-
J. M. Cerutti ular calcitonin-producing cells (C cells) and accounts for 3–
Genetic Bases of Thyroid Tumors Laboratory, Department of
Morphology and Genetics, Escola Paulista de Medicina,
7 % of all thyroid cancers. It manifests as a sporadic tumor
Universidade Federal de São Paulo, in 75–80 % of patients and in the remainder as an autosomal
São Paulo, São Paulo 04039-032, Brazil dominant inherited disease [1]. The hereditary forms of the
182 HORM CANC (2012) 3:181–186

disease are caused by germ line mutations in the RET which indicated that familial disease is more likely to be
oncogene (REarranged during Transfection), resulting in present when the tumor is diagnosed at the age of 34 years
familial MTC (FMTC), multiple endocrine neoplasia or under (Supplementary Fig. A). Based on this finding and
(MEN) 2A, and MEN 2B. In addition to a family history including a safety margin, we considered individuals
of MTC or MEN 2, other features suggesting familial dis- 35 years old or younger as suspected of having familial
ease include multifocal medullary tumors, C cell hyperpla- disease and selected them to undergo comprehensive RET
sia (CCH), and young age at diagnosis [2]. As for sporadic molecular evaluation. The other 23 individuals were con-
tumors, it is known that somatic RET mutations are present secutive patients seen for sporadic MTC. All subjects signed
in as many as 64 % of cases [4, 9, 10]. a consent form in accordance to the policies of the univer-
Germ line RET testing should be offered to all patients sity’s ethical committee.
presenting with MTC, primary CCH, and MEN 2 and to
people with a family history of FMTC or MEN 2. Other
situations that should prompt RET testing are the presence of
cutaneous lichen amyloidosis in the central upper back and
individuals with a diagnosis of Hirschsprung’s disease [8]. A
RET analysis allows the early detection of asymptomatic
carriers and has become very useful for screening at risk
relatives of affected patients [13], as mutation carriers have a
high risk of developing MTC and, thus, can benefit from
prophylactic thyroidectomy.
When investigating index cases, some centers initially
test only exons 10 and 11; others additionally sequence
exons 13, 14, 15, and 16, and a few also investigate
exons 5 and 8 for mutations in the RET gene [8, 12]. In B
our laboratory, we routinely sequence exons 8, 10, 11, Positive for p.Gly550Glu
Glu Ile Thr
and 13–16. Nevertheless, when the initial genetic anal-
MTC
ysis is negative in the presence of MEN 2 or when
there is a discrepancy between the genotype and the Negative for p.Gly550Glu

phenotype, the American Thyroid Association guidelines

recommend sequencing the entire coding region of RET
C D
[8]. Given this recommendation and the clinical rele- C T GT GC A GA GA T C A C C A C T GT GCA GA GA T C A C C A
r Cys Ala Glu Ile Thr r Cys Ala Glu Ile Thr
vance of this issue, in this study, we aim to evaluate the
benefits of sequencing the entire coding region of RET
in patients with apparently sporadic MTC. In addition,
we address the sequencing of selected exons using DNA
from tumor tissue from sporadic MTC patients. E Glycine

Homo sapiens EWRQGDGKGITRNFSTC

Patients and Methods Mus musculus EWRQGDGKGITRNFSTC
Rattus norvegicus EWRQGDGKGITRNFSTC
Bos taurus EWRQGDGKGITRNFSTC
Patients Canis lupus familiaris EWRQGDGKGITRNFSTC
Gallus gallus QWRQGSGKGITTNYSTC
From our cohort followed at the Thyroid Clinic, Hospital Danio rerio QWRQGRDKGISKRYSTC
:**** ***: .:***
São Paulo, Escola Paulista de Medicina, Federal University
of São Paulo, we selected 50 patients with MTC and no Fig. 1 Evaluation of the family presenting the p.Gly550Glu variant. a
mutations in exons 8, 10, 11, 13, 14, 15, or 16 to undergo Pedigree chart; b electropherogram of germ line sequencing of the
index case; c, d electropherogram of germ line sequencing of her
further investigation for other mutations in RET. Of the 50 mother and sister; e sequence alignment of human RET protein resi-
patients, 27 also presented with one or more features sug- dues in which the position of the conserved glycine at residue 550 is
gesting familial disease, such as a young age of onset of indicated (arrow). Multiple sequence alignment was generated with
MTC (16 patients), tumor multifocality (13 patients), or the Clustal Omega software available at the European Bioinformatics
Institute website (http://www.ebi.ac.uk/Tools/msa/clustalo/). Asterisk
presence of CCH (10 patients). We defined young age of indicate residues in the column are identical in all sequences in the
onset based on a cutoff that was established by a statistical alignment, colon indicates conserved substitutions, and period indi-
analysis using an ROC curve performed by our group, cates semiconserved substitutions observed in the alignment
HORM CANC (2012) 3:181–186
Molecular Analysis
Genomic DNA was extracted from peripheral blood
using an in-house method. Tumor DNA from eight
patients who underwent thyroidectomy was extracted
from paraffin-embedded tumor tissues through deparaf-
finization with xylol or n-octane heated baths followed
by standard protocols. DNA was amplified through
polymerase chain reaction with Platinum Taq DNA
Polymerase (Invitrogen, Carlsbad, CA, USA), and spe-
cific primers for the entire coding region of RET and
for tumor hot spots were designed using the public
primer design method available at http://ihg.gsf.de/ihg/
ExonPrimer.html in a way to avoid SNP regions in
accordance to the reference sequences from the Univer-
sity of California Santa Cruz database (Supplementary
Table A).
PCR products were purified using Illustra GFX PCR
DNA and Gel Purification Kit (GE Healthcare, Bucking-
hamshire, UK) and submitted to direct sequencing by the
Sanger method, using Big Dye™ Terminator Cycle Se-
quencing Ready Reaction Kit, ABI PRISM 3100 Genetic
Analyzer (Applied Biosystems, Foster City CA, USA).
Each exon was sequenced at least twice and in both
directions.
The sequences were analyzed by two separate evaluators
using BioEdit Sequence Alignment Editor and CLC Main
Workbench 6 (http://www.clcbio.com) and compared to ref-
erence data available at the NCBI GenBank (RefSeq
NG_007489) and the Ensembl Genome Browser. Somatic
mutations were referred using the Catalog of Somatic
Fig. 2 Histogram of the frequency of polymorphisms. y-axis: total
number of times the polymorphisms were detected in each exon and
intron, which are represented in the x-axis. Exon 1—rs10900296,
rs10900297; intron 1—rs12267460; exon 2—rs1800858; intron 2—
rs2435351; exon 3—rs1800859; intron 3—rs35906041, rs2472739,
rs115766280; intron 4—rs2435352, rs2742243; intron 5—rs1864404,
rs3026742; exon 6—new; intron 6—rs9282835; exon 7—rs1800860;
183
Mutations in Cancer database (http://www.sanger.ac.uk/ge-
netics/CGP/cosmic/).
Results
Fifty patients with MTC negative for mutations in the hot
spot exons of RET were analyzed for mutations in exons 1,
2, 3, 4, 5, 6, 7, 9, 12, 17, 18, and 19. In one of these cases, a
patient who had been diagnosed with a 1.3-cm MTC staged
T1bN0M0 at the age of 32, a p.Gly550Glu variant was
identified in the acceptor splicing region of exon 9
(Fig. 1). The patient’s disease is now in clinical and bio-
chemical remission. Her mother and one of her sisters tested
positive for this variant (Fig. 1), but their serum calcitonin
was undetectable as well as the ultrasound examination
revealed no thyroid nodules. All the other patients investi-
gated were negative for RET mutations in all exons. Several
polymorphisms were also found, both intronic and exonic,
as depicted in Fig. 2 (details in Supplementary Table B).
We were able to investigate tumor tissue from eight of the
patients who underwent total thyroidectomy. Sequencing
revealed mutations in five of these samples (Fig. 3); one
of the identified mutations is the most frequent one, p.
Met918Thr, whereas the other mutations have not been
described previously. The presence of these mutations was
confirmed by cloning the PCR products into pCR4 vector
(Invitrogen) followed by sequencing. A p.Cys620Arg mu-
tation was detected in one of the clones from the tumor
sample from one of the patients (Fig. 3). The clinical char-
acteristics of these patients are outlined in Table 1.
intron 8—rs3026750, rs111463326; intron 9—new; exon 11—
rs1799939; intron 11—rs2256550; intron 12—rs760466; exon 13—
rs1800861; intron 13—rs112200125, rs111264957, rs3026767; exon
14—rs1800862; intron 14—rs111306965, rs11238441; exon 15—
rs1800863; intron 15—rs3026771; intron 16—rs3026772; intron 17—
new; exon 18—rs17158558; intron 18—rs2742236; intron 19—
rs2075912
184 HORM CANC (2012) 3:181–186

Fig. 3 RET somatic sequencing

analysis. a–f Electropherograms A p.Met918Thr B p.Val706Ala
of MTC tissue sequencing
AAA TGGACGGCAA T TG GT C T C CG CGGAT G CCT
of five patients

C p.Gly894Ser D p.Glu615Lys

GGA T T TCAGC T TG TCC CCC TGAGAAGGAGAAG

E p.Gln681Stop F p.Cys620Arg

GCCCGCC TAGGCC T TC GTGCT TCCGCGAGCCC

Discussion biochemical investigation of the family so far suggests this is

a variant unlikely to be related to MTC. This led us to presume
In this study, 1 out of 50 patients with sporadic MTC presented that sequencing the entire coding region of RET in this study
with a variant in RET that has not been described before. It is did not bring immediate clinical benefits, at the cost of greatly
not clear whether this newly identified variant has a pathogenic increasing the price of testing. Nevertheless, it is very impor-
role. The nucleotide change occurs in a position that has the tant that the patient carrying the p.Gly550Glu variant and her
potential to alter RNA splicing, and its analysis through the relatives be carefully monitored for signs and symptoms of
Variant Effect Predictor (www.ensembl.org) suggests that it is MTC or MEN.
probably damaging. In addition, this sequence is highly con- In addition, this study demonstrated the presence of a
served in several species, at both the nucleotide and amino acid large number of polymorphisms throughout the RET gene;
levels (Fig. 1). Despite these assumptions, the clinical and to our knowledge, such a broad investigation has never been
published. Whether there is a causative association between
Table 1 Clinical features of the patients who underwent tumor se- these polymorphic RET nucleotides and disease remains to
quencing and the detected mutations be clarified [3, 5, 6, 14]. Interestingly, one of these poly-
Patient’s clinical features Exon Mutation morphic regions has been identified as a fragile site
concerning DNA breaks [7].
25 years old; pT2N1bMx 16 p.Met918Thr Regarding the novel somatic mutations detected in our
27 years old; pT1N1aMx – – study, the patients bearing them will be closely observed
56 years old; pT4N1bMx – –
58 years old; pT3N0Mx; 11 p.Val706Ala Table 2 Estimated costs for a stratified RET mutational analysis
7-cm tumor
32 years old; pT2N0Mx; CCH 15 p.Gly894Ser RET sequencing coverage Technical–operational
cost
59 years old; pT1mN1Mx 10 p.Glu615Lys,
p.Cys620Arg
Hot spots in a new patient USD 720.00
48 years old; pT2mN1aMx 11 p.Gln681Stop
Additional exons if selected exons negative USD 831.00
69 years old; pT3N0Mx – –
Testing of a relative for a specific USD 305.00
known mutation
Age at diagnosis, pathological TNM staging—AJCC 2010
Tumor testing (from 1 to 4 exons) USD 305.00–513.00
CCH C cell hyperplasia
HORM CANC (2012) 3:181–186
during clinical follow-up and the mutations will be subject
of further molecular and functional investigations. It is
known that somatic RET mutations in MTC are present in
as many as 64 % of cases and occur mainly in exons 16
(Met918Thr in up to 79 % of the cases) and 11 (up to
21.2 %), followed by exons 10 and 15 in similar proportions
[4, 9, 10]. Based on these frequencies, tissue analysis can be
performed through a tiered approach—first sequence exon
16; only if negative, proceed to exon 11; if no mutations are
found, then analyze exons 10 and 15. This tiered approach
would add less cost to the investigation than extended germ
line sequencing would. Along with the fact that somatic
mutations have been related to disease prognosis in some
studies [4, 10], this information is currently used in clinical
trials to evaluate any possible influence on the patients’
response to new target drug therapies, and somatic mutation
analysis may be useful in the near future. Thus, although
currently somatic mutation analysis has little clinical benefit
to the MTC patients and their families, it offers a more
complete molecular diagnosis that can be used in clinical
decision making.
Considering the importance of the genetic screening for
RET mutations and, on the other hand, the scarcity of trained
labs in many areas and the cost for an accurate molecular
diagnosis, we estimated the cost of this type of screening in
an academic institution, taking into account our experience
over the past 10 years in testing RET in more than 1,000
individuals. We stratified this analysis into four situations:
sequencing select exons in a new patient, sequencing the
remaining exons if the hot spots are negative in a patient
with suspected familial disease, sequencing a relative of a
carrier for a known RET mutation, and tumor sequencing
(Table 2). The cost analysis for such cases may help clini-
cians make more suitable decisions regarding the benefits of
investigating only one specific RET mutation, investigating
hot spot exons, examining the entire coding region or
performing tissue analysis for prognosis assessment, and
predicting drug response. It is important to note that, espe-
cially in diseases caused by multiple genes or by genes with
a longer coding sequence, whole exome sequencing might
become a useful tool and, as its application increases, its
cost will likely reduce in the forthcoming years [11].
In spite of the increasing number of variants being de-
scribed in MTC and MTC tumor tissue, it appears there is no
direct clinical benefit in extending RET analysis beyond the
hot spot regions, reinforcing the recommendation of the
American Thyroid Association guidelines. However, when
assisting a young adult whose tumor histopathology reveals
multifocal disease or CCH, which are more related to inher-
itance, complete RET testing might be able to unmask a
hidden familial MTC. Therefore, the challenge for endocri-
nologists and biochemical and molecular diagnostic labora-
tories will be to determine which test(s) to offer and how to
185
best test for these mutations in a way that is relevant to
patient care and has a reasonable cost–benefit trade off.
Acknowledgments The authors thank the team of the Laboratory
of Molecular and Translational Endocrinology, especially Teresa
Kasamatsu, José Gilberto Vieira, João Roberto Martins, Maria
Izabel Chiamolera, and Maria Sharmila de Sousa, and the team
of the Thyroid Clinic, particularly Maria da Conceição Mamone,
Alberto Machado, Reinaldo Furlanetto, Luiza Matsumura, Jairo
Hidal, Rosa Paula Biscolla, Maria Cecília Costa and Danielle
Andreoni, as well as the Head and Neck Surgery group. We also
thank Fernando Soares and Gilberto Furuzawa for the daily tech-
nical assistance. The authors’ research is supported by São Paulo
State Research Foundation (FAPESP) 2009/50575-4 (to S.C.L.),
2010/08981-2 (to M.S.Y.), 2006/54922-2 (to J.M.C.), 2006/60402-
1 (to R.M.B.M. and M.R.D.S.), and Fleury Group (12518). R.M.
B.M. and J.M.C. are investigators of the Brazilian Research
Council. R.M.B.M. is an investigator of the Fleury Group.
Disclosure The authors have no conflict of interest to disclose.
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 THYROID
Volume 23, Number 3, 2013
ª Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2012.0361

Measurement of Calcitonin
and Calcitonin Gene–Related Peptide mRNA Refines
the Management of Patients with Medullary Thyroid Cancer
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and May Replace Calcitonin-Stimulation Tests

Cléber P. Camacho,1 Susan C. Lindsey,1 Maria Clara C. Melo,1 Ji H. Yang,1 Fausto Germano-Neto,1
Flávia de O.F. Valente,1 Thiago R.N. Lima,2 Rosa Paula M. Biscolla,1 José G.H. Vieira,1
Janete M. Cerutti,2 Magnus R. Dias-da-Silva,1 and Rui M.B. Maciel1

Background: Serum calcitonin (sCT) is the main tumor marker for medullary thyroid cancer (MTC), but it has
certain limitations. Various sCT assays may have important intra-assay or interassay variation and may yield
different and sometimes conflicting results. A pentagastrin- or calcium-stimulation calcitonin (CT) test may be
desirable in some situations. Alternatively, or in the absence of the stimulation test, mRNA detection offers the
advantages of being more comfortable and less invasive; it only requires blood collection and has no side effects.
The objective of this study was to investigate the applicability of measuring calcitonin-related polypeptide alpha
(CALCA) gene transcripts (CT-CALCA and calcitonin gene–related peptide [CGRP]-CALCA) in patients with
MTC and in relatives diagnosed with a RET mutation and to test mRNA as an alternative diagnostic tool for the
calcitonin-stimulation test.
Methods: Twenty-three healthy controls and 26 individuals evaluated for MTC were selected, including patients
with sporadic or hereditary MTC and RET mutation–carrying relatives. For molecular analysis, RNA was
extracted from peripheral blood, followed by cDNA synthesis using 3.5 lg of total RNA. Quantitative real-time
polymerase chain reaction (RT-qPCR) was performed with SYBR Green and 200 nM of each primer for the two
specific mRNA targets (CT-CALCA or CGRP-CALCA) and normalized with the ribosomal protein S8 as the
reference gene.
Results: We detected CALCA transcripts in the blood samples and observed a positive correlation between them
(r = 0.946, p < 0.0001). Both mRNAs also correlated with sCT (CT-CALCA, r = 0.713, p < 0.0001; CGRP-CALCA,
r = 0.714, p < 0.0001). The relative expression of CT-CALCA and CGRP-CALCA presented higher clinical sensi-
tivity (86.67 and 100, respectively), specificity (97.06 and 97.06), positive predictive value (92.86 and 93.75), and
negative predictive value (94.29 and 100), than did sCT (73.33, 82.35, 64.71, and 87.50, respectively). In addition,
the CALCA transcript measurement mirrored the response to the pentagastrin test.
Conclusion: We demonstrate that the measurement of CALCA gene transcripts in the bloodstream is feasible and
may refine the management of patients with MTC and RET mutation–carrying relatives. We propose consid-
ering the application of this diagnostic tool as an alternative to the calcitonin-stimulation test.

Introduction syndrome (named multiple endocrine neoplasia [MEN]), a

dominantly inherited disorder that is caused by an activating

C alcitonin is the major tumor marker for medullary

thyroid carcinoma (MTC), and it is highly produced,
dynamically stored, and rapidly secreted into the blood by the
normal calcium-sensing parafollicular C cells and by the C
cell–derived MTC (1,2). MTC may occur either sporadically
(accounting for almost 75% of the cases) or as part of a familial
mutation in the RET oncogene (3,4).
The technology to measure serum calcitonin (sCT) has
evolved during the past four decades (2,5–9). sCT is com-
monly used for the diagnosis and follow-up of MTC patients
(10), and its doubling time is currently considered to be a
reliable prognostic indicator and one of the major criteria in

1
Laboratory of Molecular and Translational Endocrinology, Department of Medicine, and 2Department of Genetics, Escola Paulista de
Medicina, Federal University of São Paulo, São Paulo, Brazil.

308
 CALCITONIN AND CGRP mRNA IN THE MANAGEMENT OF MTC
the management of persistent tumors (11). Moreover, sCT is
used as a biochemical screening tool for MTC in relatives with
RET mutations, frequently assisting with planning the mo-
dality of the surgical approach towards prophylactic or thera-
peutic thyroidectomy (10). Calcitonin measurements can also
be of great value in the washout of fine-needle aspiration (FNA)
biopsies during the investigation of thyroid nodules because it
increases the accuracy of the diagnostic procedure (12).
By contrast, the various sCT assays may have important
intra-assay or interassay variation and may yield different
Downloaded by American Thyroid Association (ATA) from online.liebertpub.com at 07/10/17. For personal use only.
and sometimes conflicting results (13,14). Beyond these ca-
veats related to the assay reproducibility, a hook effect in
samples with high levels of sCT and a cross-reaction with
procalcitonin may happen, although these phenomena
are uncommon with two-site monoclonal antibody assays
(15–19). On the other hand, heterophilic antibodies (with a
prevalence of 1.3% to 3.7%) or anti-calcitonin interference may
occur and can sometimes be misleading (20–23).
A pentagastrin- or calcium-stimulation calcitonin test may
be desirable in screening for MTC in RET mutation-carrying
relatives or in the postoperative follow-up of MTC patients.
Alternatively, or in the absence of the stimulation test, mRNA
detection offers the advantages of being more comfortable
and less invasive because it only requires blood collection and
lacks other side effects.
The aims of this work were to investigate the applicabil-
ity of the measurement of calcitonin-related polypeptide
alpha (CALCA) gene transcripts, particularly calcitonin (CT-
CALCA) with the co-transcribed calcitonin gene–related
peptide (CGRP-CALCA) mRNA, and to refine the manage-
ment of patients with MTC and RET mutation–carrying rela-
tives by suggesting an alternative molecular diagnostic tool to
the calcitonin-stimulation test.
Patients and Methods
Patients
We selected 26 patients who were consecutively evaluated
in our MEN outpatient clinic (Thyroid Unit), at the Division of
Endocrinology, Department of Medicine, Escola Paulista de
Medicina, Federal University of São Paulo, in São Paulo,
Brazil. Fourteen had already been diagnosed with MTC and
had undergone total thyroidectomy; six of these patients had
an identified RET germ-line mutation and eight were typical
sporadic cases. The remaining 12 patients were relatives of
patients with a RET germline mutation and were found to be
positive after genetic screening of the proband. In addition,
we also studied 23 healthy individuals without any thyroid
disorders as controls.
After careful clinical analyses, biochemical (sCT and carcino-
embryonic antigen) and imaging studies, these 26 patients
were divided into three groups: RET mutation–carrying rel-
atives (n = 12), MTC patients with no evidence of disease after
surgery (n = 5), and MTC patients with biochemical and/or
structural disease (n = 9).
The RET mutation–carrying relatives had a blood sample
collected before surgery, and disease status (with or without)
was defined only after thyroidectomy and confirmatory his-
topathological analysis. The disease status of MTC patients
with no evidence of disease after surgery and MTC patients
with biochemical and/or structural disease was determined
only after a minimal period of 5 years of follow-up and after
309
clinical examination and imaging studies (cervical ultrasound
and, when indicated according to the American Thyroid As-
sociation guidelines, computed tomography and magnetic
resonance imaging) (10,24). The 23 healthy controls without a
thyroid disorder were considered to be without disease based
on clinical and ultrasound examination.
Blood samples were collected for simultaneous sCT mea-
surement and mRNA extraction. Signed letters of informed
consent were obtained from all of the patients, and the clinical
and molecular diagnostic studies were approved by the uni-
versity’s ethics and research committee (protocol number
1749/06) and conformed to the 1975 Declaration of Helsinki
as revised in 1983.
Positive and negative controls for the mRNA
measurement assay
TT, an MTC cell line obtained from ATCC (number CRL-
2005) through the Rio de Janeiro Cell Bank, was used as a
positive control for the CT-CALCA and CGRP-CALCA
mRNAs. The latter also served as the internal control for
validation of the CALCA major-expressed transcripts. The
cells were grown in Dulbecco’s modified Eagle’s medium
(Invitrogen Corp., Carlsbad, CA) that was supplemented with
10% fetal bovine serum (Invitrogen), 100 U/mL of penicillin,
and 100 lg/mL streptomycin, and then maintained in a hu-
midified incubator containing 5% carbon dioxide at 37!C, as
previously described (21,22). Dissected tissue from a follicular
carcinoma was used as a negative control.
sCT assay
We used an immunofluorometric assay developed in our
laboratory for the measurement of sCT. This assay is based on
two anti-calcitonin monoclonal antibodies, one of which is
linked to the solid phase and is specific for the 11–23 amino
acid sequence (E1P10), and the other of which is biotinylated
and specific for the 17–32 amino acid region ( J1P9). The as-
say’s analytic sensitivity is 1 pg/mL, with an upper-normal
range limit of 18.4 pg/mL for men and 7.8 pg/mL for women.
Pentagastrin-stimulation calcitonin test
Twelve RET mutation–carrying relatives prior to thyroid-
ectomy and four patients who had been already thyroidecto-
mized because of MTC underwent a pentagastrin-stimulation
test (Table 1). Samples for sCT measurement were drawn
through an indwelling catheter before and 2, 5, 10, and 15
minutes after an intravenous bolus of 0.5 lg/kg of pentagastrin
(PentagastrinTM, Cambridge Laboratories Ltd, Newcastle,
United Kingdom). All of the samples were centrifuged, and the
serum aliquots were immediately stored at -20!C. The test was
considered to be positive when there was at least a threefold
increase in the sCT level and/or a value above 100 pg/mL after
the stimulus (25,26).
The blood samples for CALCA transcripts measurement
were collected before the pentagastrin infusion through the
same catheter.
RNA blood extraction, cDNA synthesis
and quantitative real-time polymerase chain reaction
Blood samples (4 mL) from each patient were collected in
tubes containing EDTA, and total-RNA isolation and cDNA
 310 CAMACHO ET AL.

Table 1. Clinical, Pathological, Biochemical, and Molecular Features of the 49 Subjects Enrolled in the Study

Clinical
Pentagastrin- staging and
RET germline Basal sCT stimulated CT mRNA CGRP mRNA pathological
Patient no. Sex mutation (pg/mL) sCT (pg/mL) (RE) (RE) analysis

Thyroid-healthy controls
1–23 10 male, n/a 1–21 n/a Undetectable 0.01–4.46 n/a
13 female to 3.50
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RET mutation-carrying relatives

24 Male p.Val804Met 1 11.1 0.53 1.03 n/a
25 Female p.Cys609Ser 6.4 6.3 0.48 1.39 n/a
26 Male p.Val804Met 3.9 10.6 5.14 3.52 No MTC
27 Female p.Cys634Arg 1090 n/a 104.97 63.38 n/a
28 Female p.Glu768Asp 26 432 18.23 15.62 T1aNxMx, CCH
29 Male p.Tyr791Asn 2.7 8.4 1.48 2.55 n/a
30 Male p.Cys634Arg 50 434 56.6 19.67 T1aNxMx, CCH
31 Female p.Cys634Tyr 10.4 n/a 0.80 1.12 n/a
p.Tyr791Phe
32 Female p.Glu768Asp 4.6 57 5.11 5.66 CCH
33 Female p.Cys634Tyr 26.3 52 0.42 2.34 No MTC
p.Tyr791Phe
34 Female p.Cys634Tyr 7.3 68 2.74 28.78 MTC, T1NxMx
p.Tyr791Phe
35 Male p.Cys634Gly 29.2 65 3.23 3.27 No MTC or CCH
MTC after surgery without evidence of disease
36 Male None 3.9 2.4 1.05 3.44 T1bN0Mx
37 Female p.Cys634Gly 1.9 n/a 0.69 0.4 T1amN0Mx
38 Male p.Cys634Arg 3.5 n/a 33.77 16.25 T2mN1bMx
39 Female None 1.4 4.1 1.10 2.04 T1bN0Mx
40 Female None 1 1 2.50 1.17 T1bNxMx
MTC with biochemical and/or structural disease
41 Male None 5777 n/a 429,451.53 162,134.05 T3N1bM1
42 Female None 18 n/a 1912.6 1463.82 T2N1Mx
43 Male p.Cys609Ser 156 n/a 1.51 3.68 n/a
44 Male None 67,200 n/a 94,514.64 320,551.38 T3N1Mx
45 Female p.Met918Thr 20,000 n/a 17.49 11.68 T3mN1M1
46 Female None 1078 n/a 8.91 5.2 T1bN1bM1
47 Male None 6410 n/a 533.61 297.21 T4amN1bM1
48 Female p.Cys634Arg 7.3 77 8.35 6.18 T2mN1M0
49 Female p.Met918Thr 2834 n/a 83.64 55.78 n/a
Cell line and tissue controls
PC n/a TT cell line n/a n/a 271,130.58 163,785.92
NC n/a None n/a n/a 0 0 Follicular
carcinoma

Clinical status, germ-line RET analysis, basal and pentagastrin-stimulated sCT (pg/mL), relative expression (RE) of the CT-CALCA and CGRP-
CALCA mRNAs, and pathological findings with the TNM staging (24) of each individual. Positive (PC) and negative (NC) controls and the RE of
CT-CALCA and CGRP-CALCA are shown. Basal sCT cutoffs are 18.4 pg/mL for men and 7.8 pg/mL for women. CT-CALCA RE cutoff is 4.3 and
CGRP-CALCA is 3.6. A positive cell line (PC) and a negative tissue control (NC) are included at the bottom of the table.
n/a, not available or not applicable; CT, calcitonin; sCT, serum CT; CGRP, calcitonin gene–related peptide; CALCA, calcitonin-related
polypeptide alpha; MTC, medullary thyroid cancer; CCH, C-cell hyperplasia.

synthesis were performed as previously reported (27–29),
with the exception of using 3.5 lg of total RNA for the cDNA
synthesis. The cDNA was quantified using the NanoVue Plus
spectrophotometer (GE Healthcare, Buckinghamshire, United
Kingdom) and was considered adequate when the A260/
A280 ratio was between 1.8 and 2.0. The total RNA was
treated with DNAse from the TURBO DNA-free kit (Ambion,
Austin, TX) and was reverse-transcribed to cDNA using the
Super-Script III Reverse Transcriptase kit (Invitrogen) with
oligo (dT) 12–18 primers (Invitrogen) and 1 U of RNaseOUT"
Recombinant Ribonuclease Inhibitor (Invitrogen) in a reaction
volume of 30 lL and diluted with UltraPure DNAse/RNAse-
Free Distilled Water (Invitrogen) to final volume of 60 lL. An
aliquot of 2 lL of the newly synthesized cDNA was used in 20 lL
polymerase chain reaction (PCR) amplifications containing
10 lL of SYBR Green PCR Master Mix and 200 nM of each pri-
mer for the two specific mRNA targets (CT-CALCA or CGRP-
CALCA) or for the reference gene (ribosomal protein S8, RPS8).
All of the primers were designed using the Primer3 software
(http://frodo.wi.mit.edu), avoiding single nucleotide polymor-
phisms within the primer binding sites and similar transcripts,
as follows: CTF 5¢-ATC TAA GCG GTG CGG TAA TC-3¢; CTR
 CALCITONIN AND CGRP mRNA IN THE MANAGEMENT OF MTC
5¢-CTT GTT GAA GTC CTG CGT GT-3¢; CGRPF 5¢-CCC
AGA AGA GAG CCT GTG ACA-3¢; CGRPR 5¢-CTT CAC
CAC ACC CCC TGA TC-3¢; RPS8 5¢-AAC AAG AAA TAC
CGT GCC C-3¢; and RPS8 3¢-GTA CGA ACC AGC TCG TTA
TTA G-5.
The quantitative real-time PCR (qRT-PCR) amplifications
for each subject were performed in triplicate within 40 cycles
using the 7500 Real Time PCR System (Applied Biosystems,
Foster City, CA), according to the manufacturer’s protocol.
All 49 samples (in triplicate) were run under the same PCR
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cycling conditions. The threshold cycle (Cq, formerly Ct) was
obtained using the Applied Biosystems software and was
averaged with SD £ 1. The CT-CALCA and CGRP-CALCA
mRNA expression was standardized using blood samples
collected from individuals prior to thyroidectomy in whom
the histopathological analysis ruled out both MTC and C-cell
hyperplasia (CCH), as well as thyroiditis. The relative ex-
pression (RE) for CT-CALCA and CGRP-CALCA mRNA was
calculated by the formula 2(Cs - Rs)/2(Cn - Rn), where Cs is
the Ct cycle number for CT-CALCA or CGRP-CALCA in the
samples, Rs is the Ct value found for RPS8 of each sample, Cn
is the mean value of CT-CALCA or CGRP-CALCA Ct value
of individuals without evidence of disease, and Rn is the
RPS8 Ct value in the same individuals. This protocol is cur-
rently used by our group for studying other thyroid tran-
scripts (27–29).
Statistical analysis
All of the biochemical (sCT) and molecular data (RT-qPCR)
values were log-transformed before statistical analysis. The
RT-qPCR cutoff values for the CT-CALCA and CGRP-CALCA
mRNA were calculated using a receiving operating charac-
teristic (ROC) curve. Sensitivity, specificity, positive predictive
value (PPV), and negative predictive value (NPV) were cal-
culated using the formulas of Galen and Gambino (30,31). The
statistical correlation strength between the CT-CALCA and
CGRP-CALCA transcripts and between each transcript and
the sCT was calculated using the Pearson test. The two-group
analysis was performed using the unpaired Student’s t test; for
more than two groups, we used analysis of variance, including
a post hoc analysis with a Bonferroni correction. All of the data
were analyzed using the StatView software, and we consid-
ered a p value of 0.05 to be statistically significant.
Results
CT-CALCA mRNA can be detected in the blood stream
As a proof of principle, we were able to detect and utilize the
measurement of the CT-CALCA mRNA and its partner tran-
script CGRP-CALCA mRNA in the blood of patients with MTC
and in those relatives who tested positive for RET mutations
and were thus at risk for early C-cell disease or MTC. In addi-
tion, we detected CT-CALCA in 8 out of 23 thyroid-healthy
controls and CGRP-CALCA in all 23. CALCA transcripts were
detected in all 26 patients and in the TT cultured cells (the
positive control). We were able to measure a very low RE for the
CT-CALCA and CGRP-CALCA mRNAs and the values were
0.002 (median 2.62 and maximum 429,451) and 0.001 (median
2.3800 and maximum 32,0551), respectively (Table 1).
In the follicular thyroid carcinoma tissue used as negative
control, we detected traces of CALCA transcripts. Thus, after
311
RE calculation using the RPS8 internal control, the RE was
considered to be zero (CT-CALCA, RE = 0.000005; CGRP-
CALCA, RE = 0.000011; Table 1).
CT-CALCA and CGRP-CALCA mRNAs are correlated
We observed a highly positive correlation between the
CT-CALCA mRNA and CGRP-CALCA mRNA (r = 0.946,
p < 0.0001; Fig. 1A). The RE distribution analysis through an
ROC curve could predict the RE cutoffs for the CT-CALCA
and CGRP-CALCA mRNAs, which were 4.3 and 3.6, re-
spectively. The area under the curve was 0.923 for CT-CALCA
and 0.964 for CALC-CGRP. Moreover, the majority of the
subjects yielded CT-CALCA mRNAs with higher RE values
than those of the CGRP-CALCA mRNAs.
Based on the defined transcriptional RE cutoffs that were
obtained from the ROC curve and the sCT concentration, we
noted that the CT-CALCA mRNA diverged from the sCT in
10 out of 49 cases (20.4%) and that the CGRP-CALCA mRNA
diverged in 10 out of 49 cases (20.4%). Furthermore, the CT-
CALCA and CGRP-CALCA mRNAs diverged in three indi-
viduals (11%). The RE of CT-CALCA and CGRP-CALCA in
each group of stratified patients is presented in Figure 2, along
with the disease status reclassification after thyroidectomy of
the RET mutation–carrying relatives and the further follow-
up of all of the MTC patients.
Upon predictive analysis testing, the CT-CALCA mRNA
RE yielded a higher PPV and higher NPV than did the sCT.
Moreover, CGRP-CALCA yielded a higher PPV and NPV
than CT-CALCA (Table 2). After further analyses of the in-
dividuals who presented no evidence of MTC, we could not
find any significant differences based on the presence or ab-
sence of the thyroid gland when measuring the CT mRNA
( p = 0.3144) or the CGRP mRNA ( p = 0.2367).
CALCA mRNA correlates with sCT and may replace
the calcitonin-stimulation test
The CT-CALCA and CGRP-CALCA mRNAs also corre-
lated with the biochemical sCT (Figs. 1B, 1C). There was a
positive correlation between the CT-CALCA mRNA and the
sCT (r = 0.713, p < 0.0001) and between the CGRP-CALCA
mRNA and the sCT (r = 0.714, p < 0.0001). With regard to only
the disease-free individuals, the RE of the CGRP-CALCA
mRNA was 1.3-fold higher than that of the CT-CALCA
mRNA, but when we considered patients with biochemical
and/or structural disease, the RE of the CT-CALCA mRNA
was 1.5-fold higher than that of the CGRP-CALCA mRNA.
Four individuals exhibited falsely elevated sCT levels; two
were healthy controls without evidence of disease and two
presented negative CALCA mRNA with no response in the
pentagastrin-stimulation test and no MTC or CCH in the
surgically removed thyroid tissue. sCT from three healthy
controls presented a nonlinear response to the dilution with
mouse serum, which may suggest circulating antibody assay
interference.
The CALCA transcript measurement, particularly CGRP-
CALCA, mirrors the response to the pentagastrin-stimulation
test in all 11 patients tested and agrees in quantification (Table
2). We also compared the patients who responded to the
stimulation test with those who did not. After analyzing each
transcript, a significant difference between the two groups
was observed (Fig. 3). We identified a small overlap between
 312 CAMACHO ET AL.
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FIG. 2. Relative expression of CT-CALCA (A) and CGRP-

CALCA (B) in healthy controls, RET mutation-carrying rel-
atives, medullary thyroid cancer (MTC) patients with no
evidence of disease after surgery, and MTC patients with
biochemical and/or structural disease. The healthy controls
are represented by white dots, and the gray dots represent
individuals with no evidence of the disease. The black dots
represent patients with confirmed MTC or C-cell hyperplasia
(CCH) after thyroidectomy in the second group; a patient
later diagnosed with tumor recurrence in the third group;
and patients with evidence of disease in the fourth group.
The dashed line represents the receiving operating charac-
teristic (ROC) curve cutoff of the RE of CT-CALCA (4.3) and
CGRP-CALCA (3.6). The subjects are in the same order as in
Table 1. < LOD, below level of detection.

FIG. 1. CALCA transcript intercorrelation and their corre- Table 2. Summary of the Comparative Statistical
lation with sCT. CT-CALCA and CGRP-CALCA correlate Analysis of CT-CALCA and/or CGRP-CALCA
with each other (A), CT-CALCA mRNA reflects its peptide mRNA and Their Prospective Use
counterpart measured in the serum (sCT) (B), and CGRP-
CALCA mRNA also correlates with sCT (C). CT, calcitonin; Sensitivity Specificity PPV NPV
CGRP, calcitonin gene–related peptide; CALCA, calcitonin-
related polypeptide alpha. Serum calcitonin (sCT) 73.33 82.35 64.71 87.50
CT-CALCA mRNA 86.67 97.06 92.86 94.29
the 90th percentile of the nonresponsive group and the 10th CGRP-CALCA mRNA 100 97.06 93.75 100
percentile of the group that was responsive to the pentagastrin- Post-pentagastrin test 100 100 100 100
stimulation test for the CT-CALCA RE, but there was no Transcripts measured while detectable in the blood stream, basal
overlap between these groups for the CGRP-CALCA RE and pentagastrin-stimulated sCT.
(Fig. 3). PPV, positive predictive value; NPV, negative predictive value.
 CALCITONIN AND CGRP mRNA IN THE MANAGEMENT OF MTC
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FIG. 3. Comparative analysis of the CALCA transcripts and
their group response to the pentagastrin-stimulation test. A
comparative plot of the relative expression of CT-CALCA (A)
and CGRP-CALCA (B) mRNAs between the groups of in-
dividuals with and without response to the pentagastrin
test—demonstrating the transcripts’ ability to distinguish the
two groups.
Discussion
The revised American Thyroid Association (ATA) guide-
lines for patients with thyroid nodules and differentiated
thyroid cancer do not recommend sCT measurement in
screening patients with thyroid nodules (32). The major
problem is the lack of specificity, with a high incidence of
false-positive results (23,33). The ATA guidelines for MTC
management recommend sCT measurement (without the
pentagastrin-stimulation test) during the follow-up of RET-
mutation carriers and during the follow-up of MTC patients
after surgery (10). In patients with thyroid nodular disease
and elevated basal sCT, in RET-mutation carriers prior to
prophylactic surgery, and during the follow-up of MTC pa-
tients, the calcitonin-stimulation test is performed in some
centers as a strategy to increase sensitivity and specificity
(10,34). Although sCT is a good biomarker to diagnose and
follow the majority of MTC patients, it may not be sensitive
enough to anticipate the diagnosis of small MTCs or CCH
while performing family screening (35).
It has been demonstrated that transcriptional splicing of the
CALCA gene forms three isoforms (36). The first isoform is
translated and cleaved to form calcitonin and CCP-I (formerly
known as PDN-21 or katacalcin), the second isoform forms
calcitonin and CCP-II, and the third gives rise to CGRP (36).
The CALCA gene has six exons, each with well-established
313
contributions to splicing isoforms: exon 1 is not translated;
exon 2 encodes the signal peptide; exon 3 forms the amino-
terminal peptide; exon 4 encodes calcitonin; and exon 5, CGRP,
and exon 6 comprise part of the CGRP but are not translated.
Based on the CALCA splicing characteristics, we were able to
design accurate and specific primers for these transcripts,
which were subsequently confirmed by sequencing.
CGRP is a peptide synthesized in the peripheral and central
nervous system of mammals and in others species (37). CGRP
is also produced in perivascular nerve terminals and acts as a
potent vasodilator (38–40). In sepsis, an increase in the pro-
duction of CGRP and CT mRNA occurs in adipose tissue and
transiently by adherent monocytes (41). In rats, CT and CGRP
mRNA are found in normal thyroid tissue (42). The CGRP
transcript and its peptide have been detected in MTC tumor
tissue (43–45). Moreover, the CGRP peptide has been detected
in the plasma of MTC patients, also with response after pen-
tagastrin stimulation (46).
Herein, we have proposed an alternative tool that is similar
to the one already in use in the management of differentiated
thyroid cancer, using thyroglobulin and thyrotropin receptor
mRNAs in the follow-up of patients who have tested positive
for anti-thyroglobulin antibodies (47–49). In two previous
studies, blood CT mRNA was detected by RT-PCR (50,51). In
addition, CT mRNA has also proven useful in FNA biopsies of
thyroid nodules for the diagnosis of MTC (52). Following
these findings, our work is the first attempt to use RT-qPCR to
measure CALCA gene transcripts in the blood, and also the
first attempt at using CGRP mRNA quantification. Moreover,
the present study refined the laboratory tools for use in the
management of patients with MTC and of their relatives who
are diagnosed with RET mutations.
Because the levels of CGRP-CALCA and CT-CALCA
mRNA are closely related, and both are positively correlated
with the sCT measurements, one can argue that very few
spurious non-CALCA transcripts might have occurred. We
believe that this positive correlation does not necessarily re-
flect interdependence because other posttranscriptional
events might play a role in this mechanism.
Interestingly, patient 34 (Table 1), who was a RET mutation–
carrying relative with a basal sCT within the normal range
(7.8 pg/mL) and a calcitonin of 68 pg/mL in the stimula-
tion test, presented a high RE of CGRP-CALCA, but a normal
RE of CT-CALCA. He underwent thyroidectomy, and MTC
was histopathologically confirmed, which may suggest a
higher sensitivity of CGRP-CALCA mRNA. After thyroid-
ectomy, patient 38 (Table 1) had low levels of sCT and a high
RE of CT-CALCA and CGRP-CALCA mRNAs. However, his
most recent tests revealed higher levels of sCT (15 and 8.9 pg/
mL); therefore, an FNA biopsy of a 0.7 cm cervical lymph
node that we had been closely monitoring was performed,
and the cytopathological analysis of this node revealed
metastatic MTC. This result suggests that the CT-CALCA
and CGRP-CALCA mRNAs could have enabled early detec-
tion of tumor recurrence in this case. Conversely, patient 43
(Table 1) exhibited an elevated sCT with a normal RE of the
CT-CALCA mRNA and a slightly elevated RE of the CGRP-
CALCA mRNA. Thus far, this patient has not exhibited
any sign of macroscopic disease after thyroidectomy for
MTC, which might suggest other veiled sCT assay interfer-
ences, such as heterophilic antibody or a rare CT peptide
conformation (52).
 314
We believe that sCT lacks sensitivity and specificity in some
clinical situations. In cases with discrepancies between the clinical
presentation and the sCT during follow-up, the CALCA mRNAs
may help to refine the diagnosis of patients who are appar-
ently disease-free. CALCA transcript quantification showed the
capability to distinguish between the group that responded to the
pentagastrin-stimulation test and the nonresponsive group.
The stability and the reproducibility of the CT-CALCA
mRNA appear to be greater compared with those of its pep-
tide, as suggested in our study. Therefore, we believe that
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the detection of both transcripts are feasible options for the
follow-up of atypical MTC patients and for the staging of
asymptomatic RET mutation carriers. Ultimately, CALCA
transcript measurements could replace the laborious stimula-
tion test as a more comfortable method; however, studies with
larger series of patients are clearly necessary to better define the
use of the measurements in clinical practice. In light of our
findings, we suggest this molecular diagnostic tool as an al-
ternative to the stimulation test for those individuals who have
sCT levels that are borderline or mildly above the normal range
as well as for asymptomatic RET mutation–carrying relatives.
Although previous studies suggest that other tissues could
produce CT mRNA, based on our results and the correlation
with sCT, we believe that mRNA production by the C cell and
by the MTC is much higher, and that this type of interference is
insufficient to affect the use of CT or CGRP mRNA as potential
diagnostic tools (53). We demonstrate that, in the MTC-free
patients, the presence of the thyroid gland does not affect the
diagnostic power of measuring the CT or CGRP transcripts.
Acknowledgments
The authors thank the team of the Laboratory of Molecular
and Translational Endocrinology, especially Teresa Kasamatsu,
Ilda Kunii, Priscila Signorini, Mariana Oliveira, João Roberto
Martins, Maria da Conceição Mamone, Alberto Machado,
Maria Izabel Chiamolera, Reinaldo Furlanetto, Luiza Matsu-
mura, and Jairo Hidal, as well as the Head and Neck Surgery
team. We also thank Gilberto Furuzawa for daily technical
assistance. The authors’ research is supported by the São Paulo
State Research Foundation-FAPESP grants 2006/60402-1 and
2010/51547-1 (to R.M.B.M. and M.R.D.S), 2009/11257-7 and
2011/0787-2 (to J.M.C.), and 2011/20747-8 (to M.R.D.S.), by a
grant from the Fleury Group (12518) and by a grant from the
Brazilian Ministry of Health (25000.168513/ 2008-11). S.C.L. is a
MD PhD scholar from FAPESP. M.C.C.M and F.G.N. are PhD
students from CAPES (Coordenação de Aperfeiçoamento de
Pessoal de Nı́vel Superior). R.M.B.M. and J.M.C. are investi-
gators of the Brazilian Research Council. R.P.M.B., J.G.H.V.,
and R.M.B.M. are also investigators of the Fleury Group.
Author Disclosure Statement
The authors have no conflict of interest to declare.
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Address correspondence to:
Magnus R. Dias da Silva, MD, PhD
Laboratório de Endocrinologia Molecular e Translacional
Rua Pedro de Toledo, 669, 11o andar
Universidade Federal de São Paulo
04039-032, São Paulo, SP
Brazil
E-mail: mrdsilva@unifesp.br
 This article has been cited by:

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Humberto Dellê, Cleber P. Camacho. 2016. Correlation between indoleamine 2,3 dioxygenase mRNA and CDKN2A/p16 mRNA:
a combined strategy to cervical cancer diagnosis. Medical Oncology 33:11. . [CrossRef]
2. Kloos Richard T., Monroe Robert J., Traweek S. Thomas, Lanman Richard B., Kennedy Giulia C.. 2016. A Genomic Alternative
to Identify Medullary Thyroid Cancer Preoperatively in Thyroid Nodules with Indeterminate Cytology. Thyroid 26:6, 785-793.
[Abstract] [Full Text HTML] [Full Text PDF] [Full Text PDF with Links] [Supplemental Material]
Downloaded by American Thyroid Association (ATA) from online.liebertpub.com at 07/10/17. For personal use only.

3. Petros Perros, Kristien Boelaert, Steve Colley, Carol Evans, Rhodri M Evans, Georgina Gerrard BA, Jackie Gilbert, Barney
Harrison, Sarah J Johnson, Thomas E Giles, Laura Moss, Val Lewington, Kate Newbold, Judith Taylor, Rajesh V Thakker,
John Watkinson, Graham R. Williams. 2014. Guidelines for the management of thyroid cancer. Clinical Endocrinology 81, 1-122.
[CrossRef]
4. Priscila S. Signorini, Maria Inez C. França, Cleber P. Camacho, Susan C. Lindsey, Flávia O. F. Valente, Teresa S. Kasamatsu,
Alberto L. Machado, Camila P. Salim, Rosana Delcelo, Ana O. Hoff, Janete M. Cerutti, Magnus R. Dias-da-Silva, Rui M. B.
Maciel. 2014. A ten-year clinical update of a large RET p.Gly533Cys kindred with medullary thyroid carcinoma emphasizes the
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Clinical Endocrinology (2013) 79, 288–293
ORIGINAL ARTICLE
Genes of detoxification are important modulators of hereditary
medullary thyroid carcinoma risk
R. B. Barbieri*, N. E. Bufalo*, L. L. Cunha*, L. V. M. Assumpc!a
~o†, R. M. B. Maciel‡, J. M. Cerutti§ and
L. S. Ward*
*Faculty of Medical Sciences, Laboratory of Cancer Molecular Genetics, University of Campinas (FCM-Unicamp), Campinas,
†Laboratory of Molecular Endocrinology, Federal University of S~ao Paulo (Unifesf), S~ao Paulo, ‡Genetic Bases of Thyroid Tumor
Laboratory, Federal University of S~ao Paulo (Unifesf), S~ao Paulo and §Faculty of Medical Sciences, Laboratory of Cancer Molecular
Genetics, University of Campinas (FCM-Unicamp), Campinas, SP, Brazil
Summary
Context Different inherited profiles of genes involved in cellular
mechanisms of activation and detoxification of carcinogenic
products can provide specific protection or determine the risk
for cancer. Low-penetrance polymorphic genes related to the
biotransformation of environmental toxins have been associated
with susceptibility to and the phenotype of, human tumours.
Objective To investigate the role of germline inheritance of poly-
morphisms in CYP1A2*F, CYP1A1 m1, GSTP1, NAT2 and TP53
genes in hereditary medullary thyroid carcinoma (HMTC) patients.
Design This study was developed in University of Campinas
(Unicamp).
Patients We studied 132 patients with HMTC, 88 first-degree
relatives of HMTC patients and 575 control individuals.
Measurements All patients with MTC and their relatives were
sequenced for the RET gene and five genes were genotyped using
TaqMan! system.
Results We observed that the inheritance of CYP1A2*F (OR = 2!10;
95% CI = 1!11–3!97; P = 0!022), GSTP1 (OR = 4!41; 95% CI = 2!47–
7!88; P < 0!001) and NAT2 (OR = 2!54; 95% CI = 1!16–5!58;
P = 0!020) variants increased the risk for HMTC. In addition, multiple
regression analysis showed that the inheritance of GSTP1 polymor-
phisms was associated with the diagnosis in older patients (B = 8!0229;
95% IC = " 5!5735; P = 0!0054). Concerning the group of HTMC
relatives, CYP1A2*F (OR = 2:40; 95% CI = 1!19–4!86; P = 0!015),
CYP1A1 m1 (OR = 2!79; 95% CI = 1:04–7!51; P = 0!042), GSTP1
(OR = 2!86; 95% IC = 1!53–5!32; P < 0!001) and NAT2 (OR = 2!25;
95% IC = 1!20–4!22; P = 0!012) were associated with HMTC risk.
Conclusions We have demonstrated that the inheritance of
specific genes determining the individual response to environ-
mental toxins may contribute to the risk and phenotypic vari-
ability that exists in patients with HMTC. Moreover, we
identified a group at risk in relatives of HMTC patients.
Correspondence: Laura Sterian, Ward, Laboratory of Cancer Molecular
Genetics, Faculty of Medical Sciences, PO Box 6111, Campinas, SP-Brazil. Tel.:
+55 19 3521 9081; Fax: +55 19 3521 9081; E-mail: ward@fcm.unicamp.br
288
doi: 10.1111/cen.12136
(Received 2 October 2012; returned for revision 29 October 2012;
finally revised 22 November 2012; accepted 17 December 2012)
Introduction
Medullary thyroid carcinoma (MTC) accounts for 3–4% of all
new thyroid malignancies diagnosed each year in the USA, but
contributes to up to 14% of all thyroid-cancer-related deaths.1,2
The average survival for MTC is far lower than that for more
common thyroid cancers, in large part because of a high propor-
tion of late-stage diagnoses.3,4
RET (REarranged during Transfection) proto-oncogene muta-
tions are responsible for the hereditary type of MTC (HMTC)5
and are associated with nearly complete penetrance of the MTC
phenotype.6 Genetic testing of the RET gene identifies disease-
causing mutations in more than 95% of individuals with multi-
ple endocrine neoplasia (MEN) type 2A and MEN 2B and in
about 88% of individuals with familial MTC (FMTC) – these
autosomal dominant disorders are associated with a high life-
time risk of MTC.7 In addition, codon-specific phenotypes for
RET mutation detection provide the basis for individualized risk
evaluation and prophylactic thyroidectomy in carriers.8,9 How-
ever, the clinical characteristics and the progression of MTC
patients present considerable variability. MTC typically occurs in
early childhood in MEN 2B, predominantly in early adulthood
in MEN 2A and in middle age for FMTC. Moreover, even indi-
viduals harbouring the same RET mutation may present consid-
erable phenotypic variability, suggesting that other factors are
important determinants of risk and/or may modulate tumour
development. A series of studies has focused on the multiple
polymorphic variants of RET, which have consistently been asso-
ciated with the risk for development and progression of MTC,
and there is evidence of a synergistic effect of these polymor-
phisms on hereditary MTC.10,11 However, the influence of other
genes on hereditary MTC risk and clinical characteristics has
been poorly explored so far.
© 2012 John Wiley & Sons Ltd

Low-penetrance polymorphic genes related to the biotransfor-
mation of environmental toxins have been associated with the
susceptibility and the phenotype of most human tumours,
including differentiated thyroid cancers.12–15 We recently pre-
sented evidence that arylamine N-acetyltransferase 2 (NAT2),
but neither CYP nor GSTP polymorphisms, were associated with
the risk of sporadic MTC.16 In addition, we found that the
inheritance of a TP53 gene C homozygous allele was able to
incresase the susceptibility to sporadic MTC more than three-
fold, suggesting that biotransformation and cell cycle controller
genes could modulate the susceptibility to MTC.16
The present study aimed to investigate the role of germline
inheritance of polymorphisms in CYP1A2*F, CYP1A1 m1,
GSTP1 codon 105, NAT2 and TP53 codon 72 genes in the devel-
opment of HMTC patients. In addition, we intended to deter-
mine if these genes could be associated with a specific profile of
risk in the relatives of HMTC subjects.
Materials and methods
Patients
This study was approved by the Research Ethics Committees of
both Federal University of S~ao Paulo (Unifesp) and University
of Campinas (Unicamp) and was in agreement with the 1975
Declaration of Helsinki revised in 1983. A signed letter of
informed consent was obtained from each individual included in
this analysis.
We studied a total of 795 persons, comprising 132 patients
with HMTC, 88 first-degree relatives of HMTC patients and 575
control individuals without any type of neoplasia. The patients
presented with a thyroid nodule, palpable on physical examina-
tion or visualized by ultrasound, and/or had relatives with pro-
ven or suspected diagnosis of MTC. MTC was confirmed by
cytology and/or by elevated serum calcitonin levels. Patients who
had prophylactic thyroidectomy were not included. Age of dis-
ease onset was defined by the age at which the clinicopathologi-
cal diagnosis of MTC was ascertained. All patients with MTC
and their relatives were sequenced for the RET gene. The 88
first-degree relatives of HMTC patients did not have mutations
in the RET gene or any other type of malignant tumour. Indi-
viduals with a history of past thyroid disease, taking specific
drugs or medications, with evidence or history of other tumours
not related to multiple endocrine neoplasia syndromes, exposed
to specific environment or occupational risk factors were
excluded. Ethnicity was determined by interviews, in accordance
with the Brazilian Institute of Geography and Statistics, but we
further grouped individuals into Caucasians and Non-Cauca-
sians for statistical comparison.
An in-person questionnaire, previously described,16 was used
to collect demographic information, data regarding lifetime
occupational history, dietary habits, alcohol, coffee and other
medication or drug consumption, medical history with an
emphasis on previous and/or current thyroid diseases, use of
exogenous hormones and concomitant medications, reproduc-
tive history and family history of cancer and other anamnestic
© 2012 John Wiley & Sons Ltd
Clinical Endocrinology (2013), 79, 288–293
Low-penetrance genes and thyroid carcinoma 289
data.15–17 All data, including nodule size, tumour histological
features and laboratory results, were confirmed inpatient
records.
RET gene sequencing and polymorphism genotyping
Genomic DNA was isolated from peripheral blood leukocytes using
standard phenol techniques. PCR products of exons 8, 10, 11, 13,
14, 15 and 16 of the RET gene were purified using the Concert
Rapid PCR Purification System (Invitrogen, Carlsbad, CA) and
submitted to direct sequencing, using the Big DyeTM Terminator
Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster
City, CA), as previously described.18 Five genes were genotyped:
CYP1A2*F (rs 762551), CYP1A1 m1 (rs 4646903), NAT2 C282T (rs
1041983), GSTP1 codon 105 (rs 1695), TP53 codon 72 (rs 1042522)
using TaqMan! system (Applied Biosystems, Foster City, Califor-
nia, USA), as previously described.16 These SNPs were chosen
because they have previously been associated with sporadic
medullary and/or differentiated thyroid carcinoma risk by our
group and others. 12–16
Statistical analysis
We applied t-test and Fisher’s exact test to evaluate differences
between groups concerning age, gender and smoking, using the R
environment. 19 The ANOVA test was used to compare variables
that presented Gaussian distribution among three or more
groups. Hardy–Weinberg equilibrium (HWE) was analysed from
genotypes by ARLEQUIN software.20 These analyses were performed
using SAS statistical software (Statistical Analysis System, version
9!1!3, 2002–2003).19 Multiple logistic regression analysis was per-
formed to analyse associations between polymorphisms and
MTC, using Winstat statistical software.21 Variables were consid-
ered eligible for multiple logistic regression adjustment when uni-
variate analysis evidenced at least a trend of association (P < 0!1).
Post-hoc statistical power of the sample was evaluated using
GPower software, with a statistical significance level of 0!05
(GPower 3.1, Heinrich Heine University, Düsseldorf, Germany).
Results
Hereditary MTC patients were predominantly male and presented
a large age range, as summarized in Table 1. The control popula-
tion comprised mostly men; however, as the patient groups showed
differences concerning age and gender (P = 0!0001), all logistic
regression analyses were performed with corrections for these fac-
tors as well as ethnicity. The group of HMTC relatives included 40
male patients and 48 female patients aged 17–84 years
(38!78 " 15!41 years).
Influence of low-penetrance genes on the susceptibility
to HMTC
The RET gene mutations identified in the HMTC patients group
are detailed in Table 2. Univariate logistic regression analysis
indicated that the presence of CYP1A2*F, GSTP1 and NAT2
290 R. B. Barbieri et al.

Table 1. Characteristics of clinical patients with hereditary medullary We also observed a predominance of a T homozygous geno-
thyroid carcinoma (HMTC) compared with the control group type for NAT2 C282T in HMTC patients when compared with
controls. The presence of this polymorphism is associated with
Clinical faster acetylation22 and increased significantly the risk of devel-
factors HMTC Controls P-value
oping MTC (OR = 2!54; 95% CI = 1!16–5!58; P = 0!020). Mul-
tivariate logistic regression analysis adjusted for gender, age and
Age (years + SD) 34!61 " 18!37 39!87 " 16!31 0!005
Gender Male 71 (53!8%) 414 (72!0%) 0!0001 ethnicity confirmed the importance of the inheritance of this
Female 61 (46!2%) 161 (28!0%) gene variant (OR = 3!42; 95% CI = 1!28–9!13; P = 0!014), as
Ethnicity Caucasian 120 (96!0%) 469 (82!3%) 0!0023 detailed in Table 4.
Non-Caucasian 5 (4!0%) 101 (17!7%) The GG GSTP1 genotype was also overrepresented in HMTC
(24!4%) patients when compared with controls (9!8%;
P < 0!001), increasing substantially the risk of developing
Table 2. RET gene mutations identified in 138 patients with hereditary
HMTC (OR = 4!41; 95% CI = 2!47–7!88; P < 0!001). Multivari-
medullary thyroid carcinoma
ate logistic regression analysis adjusted for gender, age and eth-
No. of
nicity confirmed the importance of inheritance of a GG
cases No. of families genotype in the GSTP1 gene for the risk of developing HMTC
(OR = 5!57; 95% CI = 2!13–14!57; P < 0!001). We were not
Exons Codons Mutations N % N able to demonstrate any relationship between CYP1A1 m1 and
TP53 genetic profiles and susceptibility to HMTC (P = 0!757
Exon 8 533 G533C 81 58!7 1 and P = 0!102 respectively).
Exon 10 618 C618R 3 2!2 2
609 C609R 1 0!7 1
Exon 11 634 C634G 21 15!2 5 Genotype association with features of aggressiveness in
C634Y 3 2!2 2 patients with HMTC
C634R 3 2!2 1
Exon 13 791 L791F 24 17!4 1 An ANOVA test demonstrated that patients with different
Exon 14 804 V804M 2 1!4 1 CYP1A2*F genotypes presented different tumour sizes
(P = 0!0180). A t-test showed that the inheritance of a polymor-
phic homozygous or heterozygous allele was associated with
variants was associated with susceptibility to HMTC. CYP1A2*F smaller tumour size (P = 0!0413). Multiple regression analysis
gene polymorphisms were overrepresented in the group of adjusted for age at diagnosis, ethnicity, gender and CYP1A2*F
HMTC when compared with controls, and the inheritance of a status showed that only non-Caucasian ethnicity was associated
CYP1A2*F C allele more than doubled the risk of developing a with larger tumours (P = 0!0041).
HMTC (OR = 2!10; 95% CI = 1!11–97; P = 0!022), as detailed Distant metastases were diagnosed in 22!8% of patients. Inves-
in Table 3. tigating the genetic profile of this subset of patients, we could

Table 3. Comparison between genotyping profiles of CYP1A2*F, NAT2, GSTP1, CYP1A1 m1 and TP53 of patients with hereditary medullary thyroid
carcinoma (HMTC) and controls, using univariate logistic regression analysis

Genes Genotypes HMTC n/(%) Controls n/(%) P-value OR 95% CI – OR

CYP1A2*F A/A 54 (43!9) 162 (47!8) 0!022 2!1 1!11–3!97

C/A 48 (39!0) 147 (43!4)
C/C 21 (17!1) 30 (8!8)
NAT2 C282T C/C 50 (38!2) 87 (48!0) 0!02 2!54 1!16–5!58
C/T 62 (47!3) 81 (44!8)
T/T 19 (14!5) 13 (7!2)
GSTP1 A/A 44 (32!4) 200 (57!6) <0!001 4!41 2!47–7!88
A/G 59 (43!4) 113 (32!6)
G/G 33 (24!2) 34 (9!8)
CYP1A1 m1 T/T 92 (77!3) 377 (68!3) 0!757 1!22 0!35–4!30
T/C 24 (20!2) 160 (29)
C/C 3 (2!5) 15 (2!7)
TP53 G/G 56 (42!1) 98 (35!2) 0!102 1!97 0!87–4!43
G/C 68 (51!1) 149 (53!6)
C/C 9 (6!8) 31 (11!2)

Bold values mean significant values.

© 2012 John Wiley & Sons Ltd

Clinical Endocrinology (2013), 79, 288–293
 Low-penetrance genes and thyroid carcinoma 291

Table 4. Comparison between genotyping profiles of CYP1A2*F, NAT2, GSTP1, CYP1A1 m1 and TP53 of patients with hereditary medullary thyroid
carcinoma (HMTC) and controls, using multivariate logistic regression analysis adjusted for gender, age and ethnicity

Genes Genotypes HMTC n/(%) Controls n/(%) OR 95% CI – OR P-value

NAT2 C282T C/C 50 (38!2) 87 (48!0) 3!42 1!28–9!13 0!014

C/T 62 (47!3) 81 (44!8)
T/T 19 (14!5) 13 (7!2)
GSTP1 A/A 44 (32!4) 200 (57!6) 5!57 2!13–14!57 <0!001
A/G 59 (43!4) 113 (32!6)
G/G 33 (24!2) 34 (9!8)
CYP1A2*F A/A 54 (43!9) 162 (47!8) 0!616 0!27–1!40 0!246
C/A 48 (39!0) 147 (43!4)
C/C 21 (17!1) 30 (8!8)
CYP1A1 m1 T/T 92 (77!3) 377 (68!3) 0!877 0!44–1!73 0!704
T/C 24 (20!2) 160 (29)
C/C 3 (2!5) 15 (2!7)
TP53 G/G 56 (42!1) 98 (35!2) 2!55 0!99–6!61 0!053
G/C 68 (51!1) 149 (53!6)
C/C 9 (6!8) 31 (11!2)

Bold values mean significant values.

observe that patients with distant metastases were older
(48!154 " 18!339) than those who did not have distant metasta-
ses (35!932 " 15!899; P = 0!0235) at diagnosis. Multiple logistic
regression analysis showed that age at diagnosis was associated
with the presence of distant metastases (P = 0!0232) for the
presence of NAT2 polymorphisms.
An ANOVA test showed that age at diagnosis was not equally
distributed according to GSTP1 genotypes (P = 0!0086). In fact,
patients who had polymorphic homozygous and heterozygous
alleles were diagnosed later (37!648 " 18!854 years old) than
wild-type homozygous patients (29!057 " 16!150 years old;
P = 0!0229). Multiple logistic regression analysis adjusted for
gender confirmed that GSTP1 inheritance was associated with
later age at diagnosis (B = 8!0229; 95% IC = " 5!5735;
P = 0!0054).
Genetic profile of relatives of HMTC patients
Table 5 compares genotyping profiles of first-degree relatives of
HMTC patients and the control group. Univariate regression
analysis showed that the inheritance of CYP1A2*F and CYP1A1
m1 gene C alleles in homozygosis was more frequent among
HMTC relatives than in controls (8!8% vs 19%; P = 0!015 and
2!7% vs 7%; P = 0!042 respectively), increasing the risk of
HMTC (OR = 2!40; 95% CI = 1!19–4!86; P = 0!015 and
OR = 2!79; 95% CI = 1!04–7!51; P = 0!042 respectively).

Table 5. Comparison between genotyping profiles of relatives of HMTC patients and controls, using univariate logistic regression analysis

Genes Category Relatives% Controls% OR 95% CI – OR P-value

CYP1A2*F A/A 43 47!8 2!40 1!19–4!86 0!015

C/A 38 43!4
C/C 19 8!8
CYP1A1 m1 T/T 63!6 68!3 2!79 1!04–7!51 0!042
T/C 29!4 29
C/C 7 2!7
NAT2 C282T C/C 31!8 48 4!06 1!76–9!40 0!001
C/T 48!9 44!8
T/T 19!3 7!2
GSTP1 A/A 32!6 57!6 2!84 1!36–5!92 0!005
A/G 51!7 32!6
G/G 15!7 9!8
TP53 C/C 34!9 35!3 0!42 0!14–1!29 0!13
C/G 60!5 53!6
G/G 4!7 11!2

Bold values mean significant values.

© 2012 John Wiley & Sons Ltd

Clinical Endocrinology (2013), 79, 288–293
292 R. B. Barbieri et al.

Table 6. Comparison between genotyping profiles of relatives of HMTC patients and controls, using multivariate logistic regression analysis adjusted
for gender, age and ethnicity

Genes Category Relatives% Controls% OR 95% CI – OR P-value

NAT2 C282T Absent 31!8 48!0 2!25 1!20–4!22 0!012

Allele T Present 38!2 52!0
GSTP1 Absent 31!8 57!6 2!86 1!53–5!32 <0!001
Allele G Present 68!2 42!4
TP53 Absent 4!7 11!2 4!05 1!25–13!16 0!020
Allele G Present 95!3 88!8
CYP1A1 m1 Absent 7!0 2!7 0!644 0!17–2!58 0!554
Allele T Present 93!0 97!3
CYP1A2*F Absent 19!0 8!8 0!648 0!27–1!55 0!331
Allele A Present 81!0 91!2

Bold values mean significant values.

The presence of the NAT2 C282T allele in homozygosis was
also more frequent in relatives than in controls (19!3% vs 7!2%;
P = 0!001), increasing the chance of developing HMTC
(OR = 4!06; 95% IC = 1!76–9!40; P = 0!001). Multivariate
analysis adjusted for sex, age and ethnicity confirmed the impor-
tance of the inheritance of a T-allele in the susceptibility to
HMTC (OR = 2!25; 95% IC = 1!20–4!22; P = 0!012), as detailed
in Table 6.
Likewise, the inheritance of a GG GSTP1 genotype was more
frequent in HMTC relatives (15!7%) than in controls (9!8%;
P < 0!001), increasing the susceptibility to HMTC (OR = 2!84;
95% IC = 1!36–5!92; P = 0!005). Multivariate analysis adjusted
for sex, age and ethnicity confirmed the importance of the
inheritance of a G-allele in GSTP1 gene in the susceptibility to
MTC (OR = 2!86; 95% IC = 1!53–5!32; P < 0!001).
Univariate regression analysis did not show any association
with TP53 gene (P = 0!13), but multivariate regression analysis
showed that the inheritance of the G TP53 codon 72 allele was
also related to HMTC susceptibility (OR = 4!05; 95% IC = 1!25–
13!16; P = 0!020).
Discussion
Taking advantage of a relatively large, well-characterized control
population and a group of 132 HTMC patients that included 81
patients all from a single large family living in the same region,
hence sharing both a similar genetic background and environ-
mental exposure pattern, we demonstrated, for the first time, to
our knowledge, that polymorphisms in genes involved in the
response to environmental carcinogens are associated with the
risk of an hereditary medullary tumour and to its phenotypic
characteristics.
Different inherited profiles of genes involved in the cellular
mechanisms of activation and detoxification of carcinogenic
chemicals may confer specific protection or determine an
increased risk of developing a particular cancer type. Along with
the exposure to environmental factors, which probably serve as
disease triggers, this inherited profile of detoxifying and repairing
systems may modulate the specific phenotype of each patient.23 In
addition, polymorphisms of relevant xenobiotic metabolizing
enzymes may be used as toxicological susceptibility markers, as
our group and others have demonstrated in differentiated thyroid
carcinomas and many other human tumours.23 There is a growing
amount of evidence indicating that genetic variants not related to
RET may influence the clinical course of MTC patients.11
Recent studies of low penetrance genes in MTC have demon-
strated the influence of these genes in disease development,11,16
the CDKN1B V109G polymorphism in particular might influ-
ence the clinical course of patients with sporadic MTC. The
assessment of the CDKN1B genotype might be a new tool for
MTC prognosis along with other known markers, such as the
last postoperative serum calcitonin and the detection of RET
mutations.11
We previously demonstrated that NAT2 and TP53 detoxify-
ing genes were associated with susceptibility to sporadic MTC16
and, in the present study, we showed that polymorphic alleles
of CYP1A2*F, GSTP1 and NAT2 gene may not only contribute
to susceptibility to HMTC, but also modulate patients’ clinical
presentation. In fact, we demonstrated that CYP1A2*F inheri-
tance was associated with tumour size, whereas GSTP1 poly-
morphisms increased the risk for developing HMTC in older
individuals. The genetic risks of these polymorphisms are rela-
tively modest; however, they may have a considerable impact
on MTC development, as they occur with high frequency in
the population.16
In addition, we showed that CYP1A2*F, CYP1A1 m1, NAT2
C282T and GSTP1 genetic profiles could identify a group of
higher risk in proband relatives. Further observations are needed
to prove the clinical utility of these data, allowing more careful
observation and, perhaps, early identification and a better prog-
nosis of these individuals.
In conclusion, to our knowledge, we have demonstrated for
the first time that the inheritance of specific low-penetrance
genes that determine the individual response to environmental
toxicants may contribute to phenotypic variability in hereditary
medullary thyroid carcinoma.

© 2012 John Wiley & Sons Ltd

Clinical Endocrinology (2013), 79, 288–293

Acknowledgements
We thank the team of statisticians and our group from the Lab-
oratory of Molecular Genetics of Cancer (GEMOCA) of the Fac-
ulty of Medical Sciences, and Etna Mac!ario, who revised this
manuscript, for all valuable suggestions and insights.
Funding
This study was supported by the State of S~ao Paulo Research
Foundation (Fapesp) under the grant number 07 067-5. LSW,
RMBM and JMC are researchers of the National Council for Sci-
entific and Technological Development (CNPq).
Declaration of interest
The authors declared no conflicts of interest.
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1 Ball, D.W. (2007) Medullary thyroid cancer: monitoring and
therapy. Endocrinology and Metabolism Clinics of North America,
36, 823–837.
2 Hundahl, S.A., Fleming, I.D., Fremgen, A.M. et al. (1998) A
National Cancer Data Base report on 53,856 cases of thyroid car-
cinoma treated in the U.S., 1985–1995. Cancer, 83, 2638–2648.
3 Roman, S., Lin, R. & Sosa, J.A. (2006) Prognosis of medullary
thyroid carcinoma: demographic, clinical, and pathologic predic-
tors of survival in 1252 cases. Cancer, 107, 2134–2142.
4 Romei, C., Cosci, B., Renzini, G. et al. (2011) RET genetic
screening of sporadic medullary thyroid cancer (MTC) allows
the preclinical diagnosis of unsuspected gene carriers and the
identification of a relevant percentage of hidden familial MTC
(FMTC). Clinical Endocrinology(Oxford), 74, 241–247.
5 Elisei, R., Romei, C., Cosci, B. et al. (2007) RET genetic screen-
ing in patients with medullary thyroid cancer and their relatives:
experience with 807 individuals at one center. The Journal of
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6 Frank-Raue, K., Buhr, H., Dralle, H. et al. (2006) Long-term
outcome in 46 gene carriers of hereditary medullary thyroid
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ual RET genotype. European Journal of Endocrinology, 155,
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7 Brandi, M.L., Gagel, R.F., Angeli, A. et al. (2001) Guidelines for
diagnosis and therapy of MEN type 1 and type 2. The Journal of
Clinical Endocrinology and Metabolism, 86, 5658–5671.
8 Tuttle, R.M., Ball, D.W., Byrd, D. et al. (2010) Medullary carci-
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9 Zhou, P., Liu, J., Cheng, S.W. et al. (2012) Hereditary medullary
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effect of RET polymorphisms on sporadic medullary thyroid car-
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11 Pasquali, D., Circelli, L., Faggiano, A. et al. (2011) CDKN1B
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12 Bufalo, N.E., Leite, J.L., Guilhen, A.C. et al. (2006) Smoking and
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CYP1A1 allelic variants. Endocrine-Related Cancer, 13, 1185–1193.
13 Granja, F., Morari, J., Morari, E.C. et al. (2004) Proline homozy-
gosity in codon 72 of p53 is a factor of susceptibility for thyroid
cancer. Cancer Letters, 210, 151–157.
14 Granja, F., Morari, J., Morari, E.C. et al. (2004) GST profiling
may be useful in the screening for thyroid nodule malignancy.
Cancer Letters, 209, 129–137.
15 Guilhen, A.C., Bufalo, N.E., Morari, E.C. et al. (2009) Role of
the N-acetyltransferase 2 detoxification system in thyroid cancer
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16 Barbieri, R.B., Bufalo, N.E., Secolin, R. et al. (2012) Evidence
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17 de Lima Junior, M.M., Reis, L.O., Guilhen, A.C. et al. (2012)
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18 Tamanaha, R., Camacho, C.P., Pereira, A.C. et al. (2009) Evalua-
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19 Hothorn, T. & Leisch, F. (2011) Case studies in reproducibility.
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Clinical Endocrinology (2013)
LETTER TO THE EDITOR
An unusual genotype-phenotype correlation in MEN
2 patients: should screening for RET double germline
mutations be performed to avoid misleading
diagnosis and treatment?
We read with particular interest the letter by Conzo et al. pub-
lished in a recent issue of Clinical Endocrinology.1 In an attempt
to identify new and possibly undetected RET mutations at the
time of first diagnosis, the authors performed further genetic
testing on DNA extracted from peripheral blood in a patient
bearing a RET double mutation (p.C634R and p.A640G) who
had an unusual clinical course of disease. In addition to the two
previously described germline RET mutations in exon 11, the
analysis revealed a third novel p.M700L RET mutation within
exon 11 of RET gene. They share the lessons they learned and
raise several interesting points.
In this context, we believe that our own findings add to this
scenario. In 2007, we reported a four-generation Brazilian family
with a p.Y791F RET mutation and an unusual, more aggressive,
disease course2 than expected for p.Y791F carriers. At that time,
we attributed the unpredicted clinical history to the presence of
a single nucleotide polymorphism (SNP) in the RET gene.2
Later, the pathogenic role played by the Y791F mutation in
MEN 2 was questioned.3 Subsequently, it was shown that
p.Y791F can co-exist with a p.C634Y RET mutation4 and that
double germline RET mutations may occur in patients with
uncommon clinical manifestations.1
We therefore examined whether an additional mutation
within the RET gene could be in linkage disequilibrium with the
p.Y791F substitution. Expanded analysis to exons not previously
investigated showed no additional mutation in the RET gene.
However, following primer redesign for RET hotspot regions
(exons 8, 10, 11, 13–16) and sample retesting, an additional
mutation (p.C634Y) in the RET gene was found. The p.C634Y/
p.Y791F RET double mutation segregates with the disease. It is
difficult to explain why the RET p. C634Y mutation was not
identified in our previous analysis, given that the set of primers
and PCR and sequencing protocols have previously been used to
find mutations at codon 634 in other MEN 2 patients.
As several SNPs have been described within the RET gene in
recent years, we believe that it is prudent to double check the
primers used for possible intronic or exonic SNP–primer
mismatches. The quality of reagents used, quality of DNA and
equipment precision are all known to produce genotyping
errors. One should also consider that the distribution of alleles
varies between different populations. Although there is no
simple solution, their occurrence and effect can be reduced by
considering the most likely potential causes of error.
© 2013 John Wiley & Sons Ltd
Importantly, over the past decade, thousands of index patients
and family members diagnosed with MEN 2 have undergone
testing to elucidate the genetic cause of the disorder. We still do
not know whether RET double mutations are more common
than initially thought. However, one should keep in mind the
take-home message pointed out by Conzo et al. and consider
retesting those MEN 2A patients in whom the original genetic
studies were carried out some time ago and the clinical course is
unusual. We would like to share the lessons we have learned
and hope that they may help those working in this field, to carry
out the most appropriate test and achieve the optimal clinical
management for patients with RET mutations and MEN 2
syndrome.
Disclosure
All authors declare that there is no conflict of interest.
Janete M. Cerutti*,† and Rui M. B. Maciel†
*Genetic Bases of Thyroid Tumors Laboratory, Division of
Genetics, Department of Morphology and Genetics, Escola
Paulista de Medicina, Universidade Federal de S~ao Paulo, S~ao
Paulo, SP, Brazil, †Laboratory of Molecular and Translational
Endocrinology, Division of Endocrinology, Department of Medicine,
Escola Paulista de Medicina, Universidade Federal de S~ao Paulo,
S~ao Paulo, SP, Brazil
E-mail: j.cerutti@unifesp.br
doi: 10.1111/cen.12155
References
1 Conzo, G., Circelli, L., Pasquali, D. et al. (2012) Lessons to be
learned from the clinical management of a MEN 2A patient bear-
ing a novel 634/640/700 mutation of the RET proto-oncogene.
Clinical Endocrinology, 77, 934–936.
2 Tamanaha, R., Camacho, C.P., Ikejiri, E.S. et al. (2007) Y791F
RET mutation and early onset of medullary thyroid carcinoma in
a Brazilian kindred: evaluation of phenotype-modifying effect of
germline variants. Clinical Endocrinology, 67, 806–808.
3 Erlic, Z., Hoffmann, M.M., Sullivan, M. et al. (2010) Pathogenic-
ity of DNA variants and double mutations in multiple endocrine
neoplasia type 2 and von Hippel-Lindau syndrome. Journal of
Clinical Endocrinology and Metabolism, 95, 308–313.
4 Toledo, R.A., Wagner, S.M., Coutinho, F.L. et al. (2010) High
penetrance of pheochromocytoma associated with the novel
C634Y/Y791F double germline mutation in the RET protoonco-
gene. Journal of Clinical Endocrinology and Metabolism, 95, 1318–
1327.
1
ENDOCRINE PRACTICE Rapid Electronic Article in Press
Rapid Electronic Articles in Press are preprinted manuscripts that have been reviewed and accepted for publication, but have
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DOI:10.4158/EP13143.OR
© 2013 AACE.
Original Article EP13143.OR

FINE NEEDLE ASPIRATION AND MEDULLARY THYROID CARCINOMA: THE

RISK OF INADEQUATE PREOPERATIVE EVALUATION AND INITIAL SURGERY
WHEN RELYING UPON FNAB CYTOLOGY ALONE

Running title: FNAB AND MEDULLARY CARCINOMA

Garth F. Essig, Jr, MD1; Kyle Porter, MAS2; David Schneider, MS, MD3; Arpaia Debora, MD4;
Susan C Lindsey, MD5; Giulia Busonero, MD6; Daniel Fineberg, BMedSci, MBBS7; Barbara
Fruci, MD8; Kristien Boelaert, MD, PhD, MRCP9 Johannes W. Smit, MD, PhD10; Johannes
Arnoldus Anthonius Meijer, MD11; Leonidas Duntas, MD12; Neil Sharma, MBChB, MRCS,
DOHNS9; Giuseppe Costante, MD13; Sebastiano Filetti, MD14; Rebecca S. Sippel, MD3;
Bernadette Biondi, MD4; Duncan J. Topliss, MD7; Furio Pacini, MD15; Rui M. B. Maciel, MD,
PhD5; Patrick C Walz, MD1; Richard T. Kloos, MD16*

From the 1Department of Otolaryngology-Head and Neck Surgery, Ohio State University,
Columbus, Ohio, 2Center for Biostatistics, Ohio State University; 3Section of Endocrine Surgery,
University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin,4
Department of Clinical Medicine and Surgery University of Naples Frederico II, Naples, Italy,
5
Division of Endocrinology, Laboratory of Molecular Endocrinology, Department of Medicine,
Federal University of Sao Paulo, São Paulo, Brazil, 6Section of Endocrinology and Metabolism,
Department of Medical. Surgical and Neorological Sciences, University of Siena, Siena,
Italy,7Department of Endocrinology & Diabetes, Alfred Health, Monash University, Melbourne,
Australia; 8Dipartimento di Scienze della Salute, University of Catanzaro Magna Græcia,
Catanzaro, Italy; 9School of Clinical and Experimental Medicine, Centre for Endocrinology,
Diabetes & Metabolism, Institute of Biomedical Research University of Birmingham,
Birmingham, United Kingdom;10Department of Internal Medicine, Radboud University
Nijmegen Medical Centre, Department of Internal Medicine, Nijmegen, The Netherlands,
11
Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, the Netherlands;
12
Department of Endocrinology and Internal Medicine, University of Athens, Department of
Endocrinology and Internal Medicine, Athens, Greece, 13Department of Medicine, Institut Jules
Bordet, Bruxelles, Belgium, 14Dipartimento Di Medicina Interna, University of Roma La
Sapienza, Roma, Italy, 15Department of Medical, Surgical and Neurological Sciences, University
of Siena, Siena, Italy, 16Divisions of Endocrinology, Diabetes and Metabolism, and Nuclear
Medicine The Ohio State University College of Medicine; Divisions of Endocrinology, Diabetes
and Metabolism, and Nuclear Medicine, Columbus, Ohio.
*Current affiliation: Veracyte Inc., 7000 Shoreline Court, Suite 250, South San Francisco,
California USA. 94080 email: Richard.Kloos@veracyte.com
1
DOI:10.4158/EP13143.OR
© 2013 AACE.
Address correspondence to Richard T. Kloos MD, 7000 Shoreline Court, Suite 250, South San
Francisco, CA 94080.
E-mail:Richard.Kloos@Veracyte.com

2
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© 2013 AACE.
ABSTRACT

Objectives: To evaluate the diagnostic accuracy of fine needle aspiration biopsy (FNAB)

to preoperatively diagnose medullary thyroid cancer (MTC) among multiple international centers

and evaluate how the cytological diagnosis alone could impact patient management.

Methods: A retrospective chart review of sporadic medullary thyroid carcinoma (sMTC)

patients from 12 institutions over the last 29 years was performed. FNAB cytology results were

compared to final pathologic diagnoses to calculate FNAB sensitivity. To evaluate the impact of

cytology sensitivity for MTC according to current practice, and to avoid confounding results by

local treatment protocols, changes in treatment patterns over time, and the influence of ancillary

findings (e.g. serum calcitonin), therapeutic interventions based on the FNAB cytology alone

were projected into one of 4 treatment categories: total thyroidectomy (TT) and central neck

dissection (CND), TT without CND, diagnostic hemithyroidectomy, or observation.

Results: Three hundred thirteen patients from 4 continents and 7 countries were

included, 245 of whom underwent FNAB. FNAB cytology revealed MTC in 43.7% and possible

MTC in an additional 2.4%. One hundred thirteen (46.1%) patients with surgical pathology

revealing sMTC had FNAB findings that supported TT with CND while 37 (15.1%) supported

TT alone. In the remaining cases, diagnostic hemithyroidectomy and observation were projected

in 32.7% and 6.1%, respectively.

Conclusions: FNAB is an important diagnostic tool in the evaluation of thyroid nodules,

but the low sensitivity of cytological evaluation alone in sMTC limits its ability to command an

optimal pre-operative evaluation and initial surgery in over half of the affected patients.

Key words: Fine-needle biopsy, medullary thyroid carcinoma, humans, thyroid nodule,

thyroid neoplasm
3
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© 2013 AACE.
Abbreviations:

AJCC = American Joint Committee on Cancer; CND = central neck dissection; FNAB =

fine needle aspiration biopsy; IRB = Institutional Review Board; MTC = medullary thyroid

cancer; MEN 2 = multiple endocrine neoplasia type 2; RET = REarranged during Transfection;

sMTC = sporadic medullary thyroid carcinoma; TT = total thyroidectomy; TNM = tumor node

metastases

INTRODUCTION

For the past several decades, management of thyroid nodules has relied upon fine needle

aspiration biopsy (FNAB) to differentiate benign lesions that may be observed from malignant

lesions that require surgical treatment (1-6). The routine use of FNAB has reduced the number of

unnecessary surgeries for thyroid nodules, and has doubled the percentage of thyroid cancers

found in operated pathologic specimens (7, 8). In patients with medullary thyroid carcinoma

(MTC), definitive cure rests heavily upon early diagnosis and appropriate initial treatment (9,

10). It is generally believed that FNAB has low sensitivity to diagnose MTC, yet relatively few

studies of this topic are published. Of 9 identified studies, two suggest good FNA sensitivity for

MTC (11, 12), while the others indicate diminished FNAB sensitivity and the potential for

misdiagnosis or inadequate initial treatment (10, 13-18). The largest series included 91 patients

(12), and 4 of these 9 studies included less than 20 patients. With 245 patients, our study is much

larger and is almost equal in size to the sum of the 9 referenced publications.

MTC accounts for approximately 2.2% of all thyroid cancer cases in a database of 43,644 thyroid

cancer cases diagnosed from 1992 through 2006 in the United States (19) and historically is observed

sporadically in approximately 75%-80% of patients while the remainder have a hereditary form
4
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© 2013 AACE.
(MEN 2) (2) transmitted with autosomal dominant patterns of inheritance by mutation of the

REarranged during Transfection (RET) proto-oncogene (20-24).

The pre-operative identification of MTC allows for pre-operative evaluation for MEN 2

and for pre-operative oncologic staging as both of these evaluations may directly alter patient

management (2). Identification of concomitant pheochromocytoma and/or hyperparathyroidism

is expected to alter patient care, while the pre-operative discovery of distant or locoregional

metastases may alter the goals and extent of initial thyroid surgery (2). Devascularized

parathyroid glands are autographed into the neck in patients with sporadic MTC (sMTC), while

they are transplanted into the forearm in patients harboring RET mutations with a significant risk

of future risk of hyperparathyroidism (2). Additionally, because MTC metastasizes to local

lymph nodes early in the disease course, and because surgical resection is the only known

treatment option that offers the possibility of cure, current guidelines suggest a prophylactic

CND for MTC (2), while the role of this procedure for epithelial derived differentiated thyroid

cancer remains controversial (25).

In light of this, the optimal pre-operative evaluation of thyroid nodules should allow for

early detection of MTC, preoperative evaluation for MEN syndromes and the appropriate initial

surgical treatment. The objective of this investigation was to evaluate the diagnostic accuracy

FNAB alone as a tool to preoperatively diagnose MTC in a large multicenter and international

experience. To achieve this objective we specifically did not include adjunctive information such

as calcitonin measurements.

METHODS

Institutional Review Board (IRB) approval and center recruitment

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© 2013 AACE.
 IRB approval was obtained from the primary authorks institution prior to proceeding.

Next, a data collection sheet was generated with explicit instructions for de-indentified data

collection to standardize data input. Fifty-three institutions in 11 countries were invited to

participate in data collection. IRB approval was obtained from each participating institution per

their institutional protocols. Subsequently, the data were reviewed for internal agreement and

standardized for data analysis. The AJCC 7th edition staging manual was utilized for tumor

staging (26).

Data collection

Retrospective chart review was performed on all patients with a diagnosis of MTC at

each institution and data were collected for all patients with sMTC. Data collected are outlined

in Table 1. Only sMTC cases were included to avoid potential bias by the cytopathologist who

may interpret the FNA specimen differently if they knew it was collected from a patient with a

personal or family history of MEN2. FNAB cytology results were reviewed and grouped

according to those that diagnosed or raised the possibility of MTC versus those that did not. This

grouping is not influenced by the changes in FNA classification that occurred during the years of

this study, including the recent introduction of the Bethesda classification (27).

Statistical analysis

FNAB sensitivities for MTC diagnosis were calculated as the number of MTC or possible

MTC diagnoses divided by the total number of MTC cases, with 95% confidence intervals

calculated using the exact binomial distribution. Patient ages were categorized into quartile

groups for summarization and analysis. Chi-square tests were used to evaluate associations of

FNAB sensitivity with TNM staging, patient age, and regional site. For overall chi-square tests

significant at the =0.05 level, post hoc chi-square comparisons between each pair of categories
6
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© 2013 AACE.
were performed, using Holmks step down procedure to adjust for multiple comparisons. For

nodal status, in addition to comparing FNAB sensitivity for MTC diagnosis among nodal stages,

performance of lymph node dissection [determined (N0-N1b) versus undetermined nodal status

(Nx)] was compared between FNAB MTC diagnosis and no MTC diagnosis. In addition to

univariate chi-square analyses, a multivariate logistic regression model including the

independent variables city, age quartile, T-stage, N-stage, and M-stage was fit to FNAB MTC

diagnosis outcome to evaluate associations while controlling for covariates. All analyses

included only sMTC patients having a pre-operative FNAB. Data were analyzed using

SAS/STAT® software Version 9.2 (Cary, NC).

Assignment to surgical categories

To independently interpret the results of FNAB sensitivity according to currently

accepted treatment strategies, and to avoid confounding results by local treatment protocols,

changes in treatment patterns over time, or the influence of ancillary findings (e.g. serum

calcitonin, lymphadenopathy), assumptions regarding surgical intervention based on the FNAB

cytology alone were made as follows:

Total thyroidectomy with central neck dissection: Criteria for inclusion in this category were

FNAB diagnoses of MTC or possible MTC. Additionally, the FNAB result of pspindle cell

neoplasmk was also included in this category as this diagnosis was assumed to trigger a more

aggressive surgical approach.

Total thyroidectomy without central neck dissection: Criteria for inclusion in this category were

FNAB results of papillary or anaplastic thyroid carcinoma, FNAB suspicious for malignancy,

malignancy, and malignant epithelial neoplasm.

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© 2013 AACE.
Diagnostic lobectomy without central neck dissection: Criteria for inclusion in this category were

FNAB results of follicular or Hurthle cell pathology, thyroid neoplasia, lymphoma, atypia, and

results listed as inconclusive, indeterminate, and insufficient.

Observation: Criteria for inclusion in this category were FNAB results designated as benign,

colloid nodule, or goiter.

RESULTS

Subject recruitment

Of the 53 institutions invited, 13 agreed to participate and 12 ultimately submitted data

sheets. Responding institutions represented 7 nations and 4 continents. Three hundred thirteen

patients with sMTC were identified from these centers presenting for management from 1983-

2011. Average age at first surgical intervention was 52±15 years (range 19 to 101 years). While

81.2% of patients had negative RET proto-oncogene testing supporting sMTC, the remaining

18.8% were untested and classified as sMTC based upon negative family history and absence of

other findings of MEN syndromes.

FNAB results

Of the 313 sMTC patients, 245 (78.3%) underwent preoperative FNAB. FNAB revealed

a sensitivity of 45.7% for the diagnosis of MTC (43.7%) or possible MTC (2%). No change in

FNAB sensitivity over time was detected (data not shown). Other biopsy findings are listed in

Table 2. Classifying these results based upon the projected surgical intervention as outlined in

the Methods section, 46.1% of patients ultimately proven to have sMTC would have undergone

TT with CND, 15.1% TT without CND, 32.7% diagnostic hemithyroidectomy without CND, and

6.1% observation (Figure 1).

Staging analysis
8
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© 2013 AACE.
 We performed an analysis to investigate if FNA sensitivity was associated with the

currently utilized TNM staging system that categorizes tumors according to tumor size and

extent (T), nodal metastases (N), and distant metastases (M). When evaluating FNAB sensitivity

in terms of AJCC 7th edition TNM staging, no differences were identified in terms of FNAB

sensitivity with regard to Tumor stage (Table 3), or Metastatic status (Table 4). Nodal stage was

significantly associated with MTC diagnosis [X2(4) = 13.2, p = 0.01]. This difference was

centered on undetermined versus determined nodal stage, with cytology of MTC or possible

MTC in 25% of undetermined (Nx) versus 51% of determined (nodal stages N0-N1b combined),

p=0.007 (Table 5). Comparing the proportion of cases with undetermined nodal stage suggested

that when FNAB did not suggest MTC, a lymph node dissection was less likely to occur (71%

N0-N1b when MTC not suggested versus 88% with MTC or possible MTC cytology, p<0.001).

Age analysis

Age quartiles were significantly associated with FNAB diagnosis of MTC [X2(3) = 9.2,

p=0.03]. However, after adjusting for multiple comparisons, there were no significant

differences in FNAB sensitivity for MTC among pairs of age quartiles.

Regional analysis

The sensitivity of FNAB among the sites surveyed ranged broadly (0-100%) (Table 6).

Though comparisons between individual locations revealed significant differences in sensitivity

[X2(10) = 43.3, p<0.001], there were no significant pairwise differences when adjusting for

multiple comparisons.

Multivariate model

In the multivariate model for MTC diagnosis, city (p=0.02), age quartile (p=0.02), and N-

stage (p=0.001) remained significantly associated with MTC diagnosis. After adjusting for
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© 2013 AACE.
multiple comparisons, there remained no significant pairwise differences for city of FNAB or

patient age quartile. Determined N-stage (N0, N1a, or N1b) had significantly greater odds of

FNAB MTC or possible MTC cytology than undetermined N-stage (Nx) [OR = 5.8 (95% CI =

2.3, 14.2), p < 0.001].

DISCUSSION

FNAB cytology has improved the ability to identify benign thyroid nodules to avoid

diagnostic surgery, and facilitated the preoperative diagnosis of thyroid carcinoma. Diagnosing

cancer pre-operatively allows for an appropriate pre-operative evaluation, counseling, and the

performance of the best treatment at the first operation. For these reasons, FNAB is widely used

when evaluating thyroid nodules.

Total thyroidectomy and central neck dissection with or without selective neck dissection

of the lateral neck compartments is the recommended treatment for patients with MTC (2).

Although FNAB plays an integral role in diagnosing differentiated thyroid cancers, it has been

shown to have lower diagnostic accuracy for the specific diagnosis of MTC in this multicenter

and international series, in smaller single center series (10, 13-18), and in individual case reports

(28). Lower diagnostic accuracy may lead to misdiagnosis and suboptimal initial treatment, or to

a significantly delayed diagnosis if surgery is deferred. Inadequate initial surgery would require

additional surgery, which may be more difficult due to scarring and disruption of tissue planes. A

significant delay in the diagnosis may allow the disease to advance, which could require a more

extensive or difficult surgery, and possibly a longer hospitalization. More advanced disease

would be expected to affect the patientks prognosis. Advanced age at diagnosis, extent of primary

tumor, nodal disease, and distant metastases all predict an adverse outcome and support the

desire to avoid a significant delay in diagnosis (2). In our series, 245 sMTC patients underwent
10
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© 2013 AACE.
FNAB, and the cytological findings suggested MTC or possible MTC in only 45.7%. Thus, the

majority of sMTC patients in our series would not be expected to receive an optimal preoperative

evaluation or initial operation if decision making were based on their FNAB cytology alone.

In the current study, only 46.1% of patients were projected to receive the recommended

therapy for MTC based upon the initial FNAB alone. Of the remaining patients, 15.1% were

projected to undergo a thyroidectomy without a central neck dissection, while 32.7% were

projected to undergo a diagnostic lobectomy, and 6.1% were projected to undergo non-operative

intervention. These findings highlight the limitations of traditional FNAB cytology for the

diagnosis of MTC. The finding that Nx sMTC patients were less likely to have an FNAB

suggestive or diagnostic of MTC is consistent with the idea that without a pre-operative

diagnosis of MTC, these patients are unlikely to received optimal pre-operative evaluations or

receive the optimal surgical treatment. For those patients not treated optimally at the initial

surgery, additional surgery may be important based on the findings of Panigrahi et al who found

that MTC patients receiving surgery discordant with American Thyroid Association

recommendations had shorter survival than those receiving surgery according to the

recommendations which included a total thyroidectomy and lymph node resection (29).

Similar to our results, Bugalho described 67 MTC patients in one of the largest series

published previously and found that cytology suggested MTC in 63%, was suspicious for

malignancy in 21%, benign in 9%, and indeterminate in 7% (13). In our sMTC series we found

benign FNAB cytology in 6.1% of cases. While a few studies report benign FNAB cytology in

1% or less of MTC cases (11, 12) , others report rates as high as 17%-25% (10, 15, 17).

The limitation of thyroid FNAB for the pre-operative diagnosis of MTC has driven the

search for alternative methods to diagnose MTC with greater efficacy. Methods currently
11
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© 2013 AACE.
available include various cytological specimen staining techniques, immunostaining for

calcitonin (30), serum calcitonin screening (10, 18, 31-34), measurement of calcitonin in the

FNAB needle washout (35) and quantification of mRNA gene expression from FNAB samples

with support vector machine identification of a signature suggestive of MTC (36). Among these,

serum calcitonin screening has been the most clinically validated and utilized based on its higher

sensitivity for MTC than FNAB. However, calcitonin screening is criticized for its low positive

predictive value and to date, no method has gained worldwide acceptance as cost-effective to

improve the outcome of patients with nodular thyroid disease.

The strengths of the current study of the sensitivity of FNAB alone for sMTC include the

large number of patients accrued from 12 centers across 7 countries. This diversity provides

balance to regional discrepancies in cytological experience and thresholds for the cytological

diagnosis of MTC. The weaknesses of the current study include its retrospective nature,

unblinded status of the cytopathologist to ancillary information, and lack of detail regarding the

FNAB methodology for each patient (e.g. ultrasound guidance, needle gauge, number of needle

passes, aspiration technique, and use of cytologic stains or immunostains). Still, the finding that

most patients with sMTC are not recognized by FNAB cytology alone is sobering.

CONCLUSION

FNAB is an important diagnostic tool in the evaluation of thyroid nodules, but it

demonstrates poor sensitivity in the preoperative diagnosis of sMTC. This shortcoming limits the

ability of FNAB to command an optimal preoperative evaluation and initial surgery in over half

of the patients with sMTC, and illustrates the need for additional tools to accurately identify

these patients preoperatively.

DISCLOSURES
12
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© 2013 AACE.
 Dr. Kloos is an employee and stockholder of Veracyte, Inc. This study was conceived

and initiated prior to the initiation of this relationship. Veracyte provided no funding, input, or

support for this study.

13
DOI:10.4158/EP13143.OR
© 2013 AACE.
Table 1. Data collected on each patient with sporadic medullary thyroid cancer.
Data Points Collected
Age (at initial surgery)
RET oncogene testing status
Preoperative Clinical Diagnosis
-Basis for preoperative diagnosis
Preoperative FNAB diagnosis
Preoperative N stage
-Basis for N staging
Extent of thyroidectomy
Extent of central neck dissection
Extent of lateral neck dissection
TNM stage based upon AJCC 7th ed
Extracapsular invasion
Lymphovascular invasion
Primary tumor maximum dimension
Number and location of tumor foci
Co-existence of additional thyroid
pathology

14
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© 2013 AACE.
Table 2. Projected clinical action (A-D) based upon only the FNAB cytology result.
FNAB result Number Percentage
Not performed 68 21.7*
Performed 245 78.3*
A. Total Thyroidectomy with CND 113 46.1
MTC 107 43.7
Possible MTC 5 2.0
Spindle cell neoplasm 1 0.4
B. Total Thyroidectomy without CND 37 15.1
PTC 6 2.4
ATC 1 0.4
Malignant 15 6.1
Suspicious 13 5.3
Malignant epithelial neoplasm 2 0.8
C. Diagnostic Hemithyroidectomy without CND 80 32.7
Follicular 19 7.8
Atypical 2 0.8
Hurthle Cell 4 1.6
Inconclusive 5 2.0
Indeterminate 37 15.1
Insufficient 10 4.1
Thyroid neoplasia 2 0.8
Lymphoma 1 0.4
D. Observation/Benign cytology 15 6.1
*- indicates percentage of total patients (313). Remaining percentages reflect the proportion of
each category from those with FNAB data available (n=245)

15
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© 2013 AACE.
Table 3. FNAB cytology sensitivity for MTC according to Tumor Node Metastases (TNM) T

stage.

MTC positive/ 95% CI for

T stage
pre-op FNABs % positive
0-1a* 28/64 (44%) 31-57
1b 23/44 (52%) 37-68
2 21/54 (39%) 26-53
3 23/47 (49%) 34-64
4a** 12/25 (48%) 28-69
4b 3/6 (50%) 12-88
n/a 1/4 (25%) 1-81
x 1/1 (100%) 3-100
* Includes one T0 patient and one T1 unspecified patient (not a or b)
** Includes three T4 unspecified patients (not a or b)
n/a- data not available
No significant differences

16
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© 2013 AACE.
Table 4. FNAB cytology sensitivity for MTC according to Tumor Node Metastases (TNM) M
stage
MTC positive/ 95% CI for
M stage
pre-op FNABs % positive
0 40/100 (40%) 30-50
1 21/36 (58%) 41-74
x 51/109 (47%) 37-57
M stage 0 vs. 1, p=0.08

17
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© 2013 AACE.
Table 5. FNAB cytology sensitivity for MTC according to Tumor Node Metastases (TNM) N
stage
MTC positive/ 95% CI for
N stage
pre-op FNABs % positive
0 34/70 (49%) 36-61
1a* 11/24 (46%) 26-67
1b 53/98 (54%) 44-64
n/a 1/1 (100%) 3-100
x 13/52 (25%) 14-39
* Includes 5 N1 patients with a or b status not specified
No significant differences among N0, N1a, N1b.
Sensitivity for x significantly lower than N0, N1a, and N1b (p=0.007).

18
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© 2013 AACE.
Table 6. FNAB cytology sensitivity for MTC according to geographic region.
MTC positive/ 95% CI for
Country Site
pre-op FNABs % positive

Australia Melbourne 10/16 (63%) 35-85

Brazil San Paulo 31/62 (50%) 37-63

Greece Athens 7/7 (100%) 59-100

Holland Leiden 8/20 (40%) 19-64
Total 38/82 (46%) 35-58
Catanzaro 1/10 (10%) 0-45
Italy Naples 8/10 (80%) 44-97
Rome 12/20 (60%) 36-81
Siena 17/42 (40%) 26-57
United
Kingdom Birmingham 0/16 (0%) 0-21
Total 18/42 (43%) 28-59
United States Columbus 9/30 (30%) 15-49
Madison 9/12 (75%) 43-95

19
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© 2013 AACE.
FIGURE TITLES AND LEGENDS
Figure 1.
Title: Projected clinical action based only on FNAB cytology.

Figure 2.
Title: FNAB cytology sensitivity for MTC according to patient age.
Legend:
MTC positive/ 95% CI for
Age Quartile Age Range
pre-op FNABs % positive
1 18.7 to 41.3 32/61 (52%) 39-65

2 41.3 to 52.7 21/61 (34%) 23-48

3 52.7 to 64.1 23/61 (38%) 26-51

4 64.1 to 100.7 35/61 (57%) 44-70

20
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© 2013 AACE.
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 J. Endocrinol. Invest. 36: 975-981, 2013
DOI: 10.3275/8997

Comprehensive analysis of RET gene should be performed

in patients with multiple endocrine neoplasia type 2 (MEN 2)
syndrome and no apparent genotype-phenotype correlation:
An appraisal of p.Y791F and p.C634Y RET mutations in five
unrelated Brazilian families
F.O.F. Valente1, M.R. Dias da Silva1, C.P. Camacho1, I.S. Kunii1, A.U. Bastos2, C.C.N. da Fonseca2,
H.P.C. Simião1, R. Tamanaha2, R.M.B. Maciel1, and J.M. Cerutti1,2
1Laboratoryof Molecular and Translational Endocrinology, Division of Endocrinology, Department of Medicine, Escola Paulista de
Medicina, Universidade Federal de São Paulo, São Paulo, SP; 2Genetic Basis of Thyroid Tumor Laboratory, Division of Genetics,
Department of Morphology and Genetics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil

ABSTRACT. Background: We previously identified a four- gene was identified in the reported family. The double mu-
generation family with medullary thyroid cancer (MTC) and tation occurred in cis and segregated with the phenotype.
a germline p.Y791F RET mutation whose cancer lacked a Through the Brazilian Genetic Screening Program devel-
strong genotype-phenotype correlation. The entire gene oped at our institution, we additionally report the combi-

s
coding region of the RET gene should be sequenced when nation of these two mutations (p.C634Y/p.Y791F) in the

t i
genotype-phenotype discrepancies are observed in patients RET gene in four other unrelated families. The overall pen-

ur
with multiple endocrine neoplasia type 2 (MEN 2), even if a etrance of MTC and pheochromocytoma in patients with
RET hotspot mutation has been identified. Methods: A new the p.C634Y/p.Y791F mutations was 79% and 13%, re-

K
genetic test was performed in the index case of this family spectively. Conclusion: Our data emphasises that a com-

e
with the p.Y791F RET germline mutation. The entire cod- prehensive analysis of the RET gene may reveal multiple

c
ing region of the RET gene was investigated by direct se- germline mutations in MEN 2 patients who exhibit an atyp-

i
quencing of PCR products. Once a mutation was identified, ical clinical course of the disease.

t r Y
the target exon was sequenced in all at-risk relatives. Re-

i L
sults: An additional p.C634Y germline mutation in the RET
(J. Endocrinol. Invest. 36: 975-981, 2013)
©2013, Editrice Kurtis

E d E ON
3 ,
INTRODUCTION

AL
U S national Workshop on MEN published the first consensus

1
Multiple endocrine neoplasia type 2 (MEN 2) is an auto- that classified RET mutations into three risk levels (7). Sub-

© 20
N
somal dominant-inherited tumour syndrome that can be sequently, the American Thyroid Association (ATA) in-

S O
classified into three clinical phenotypes (reviewed by San- corporated the current medullary thyroid carcinoma

PER
toro et al.) (1). Germline mutations in the RET gene have (MTC) literature with evidence-based medicine to sum-
been identified as the underlying basis of MEN 2 syn- marise the state-of-the-art knowledge into the new MTC

FOR
drome. In MEN 2A and familial medullary thyroid carcino-
ma (FMTC) families, most of the mutations occur in one of
the six cysteine codons within exon 10 and exon 11. Non-
cysteine mutations located within exons 5, 8, 10, 11, 13-15
have also been reported (2-5). Almost all individuals with
MEN 2B exhibit p.M918T mutation in exon 16 (6). In ad-
dition, mutations within exon 14 (p.V804M), in conjunction
with a second RET mutation located within exons 13, 14 or
15, and mutations within exon 15 (p.A883F) have also been
described in a few cases of MEN 2B (4).
For the most prevalent mutations, a striking genotype-
phenotype correlation has been observed. Therefore,
based on genotype-phenotype correlation, the 7th Inter-
Key-words: MEN 2A, RET, p.C634Y, p.Y791F, medullary thyroid carcinoma.
Correspondence: J. Cerutti, PhD, Genetic Basis of Thyroid Tumour Laboratory, Rua
Pedro de Toledo 669, 11° andar, Universidade Federal de São Paulo, 04039-032,
São Paulo, SP, Brazil.
E-mail: j.cerutti@unifesp.br
Accepted May 9, 2013.
First published online May 30, 2013.
management guidelines (4). Since patients with MEN 2A-
associated mutations at codon 634 develop MTC at
younger ages and exhibit higher rates of pheochromo-
cytoma (PHEO) and primary hyperparathyroidism (PHPT),
codon 634 mutations were categorised within a separate
risk level, and all RET mutations were stratified into four
risk levels (A to D).
Although the ATA updated the classification system to
incorporate all known mutations, the pathogenic roles of
recently described rare RET mutations and variants of un-
certain significance (VUS) remain less clear. Further, the
presence of double mutations in the RET gene necessi-
tates better classification and management.
Within this field of double RET mutations, we recently
faced a new dilemma related to the RET p.Y791F muta-
tion. This mutation was initially identified in a patient with
apparently sporadic MTC. Because no additional muta-
tions were described in exons 10, 11 and 13-16 of RET
gene, this RET p.Y791F mutation was considered a new
hotspot for MEN 2 syndrome (8). Although this mutation
was also associated with susceptibility to apparently spo-
radic PHEO and Hirschsprung’s disease (9), the RET

975
F.O.F. Valente, M.R. Dias da Silva, C.P. Camacho, et al.

1 2

1 2 3 4 5 6 7 1 2 3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6

1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5

1 2 3 4 5

s
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

t i
ur
1 2 3

K
1 2

i c e
r
1 2 3 4

d i t ON LY
E
1 2 3 4 5 6 7 8 9 10 11 12

E
3 , AL
U S
1
1

© 20 O N
S
Fig. 1 - Pedigree of investigated families (1-5) with RET germline double mutation at codons p.C634Y/p.Y791F. The black arrows indi-

PER
cate index cases. Among the tested patients, 39 (39/64; 61%) were positive for p.C634Y/p.Y791F double mutation. There is a high pen-
etrance of MTC (79%; 31/39), compared to PHEO (13%; 5/39). Interestingly, CLA was described in six cases (15%; 6/39) and PHPT in three
(8%; 3/39).

FOR
p.Y791F mutation was considered ATA-A, the lowest risk
mutation for MTC and PHEO (10-12). Recently, Erlic et al.
These findings challenge those obtained in our previous
study, in which we described a large family with the
raised important issues regarding the pathogenicity of this p.Y791F RET mutation and MTC as the only clinical fea-
rare RET mutation. The authors identified the p.Y791F ture (16). Remarkably, MTC exhibited a more aggressive
mutation in 8/1,000 normal controls (0.8%). Moreover, clinical course of disease than expected for a RET
clinical re-evaluation of cases with the RET p.Y791F mu- p.Y791F carrier (16).
tation demonstrated that none of those patients who un- To correlate the unusual course of disease in this fami-
derwent prophylactic thyroidectomy exhibited MTC. ly with an additional still undetected mutation within
Therefore, the authors suggested that p.Y791F is not a the RET gene, a new molecular genetic test was per-
pathogenic mutation for MEN 2 (13). formed in the index case. Targeted analysis of each ex-
Recently, a double mutation of the RET gene at codons on of the RET gene revealed an additional germline mu-
634 and 791 was reported in families with MEN 2A. The tation within exon 11 of the RET gene (p.C634Y). We
authors suggested that patients with the double germline additionally describe four unrelated Brazilian families
mutation (p.C634Y/p.Y791F) carry a codon 634-like pat- with MEN 2 syndrome and a RET p.C634Y/p.Y791F
tern of MTC and increased susceptibility of PHEO (14). double mutation. Differing from previous reports (14),
Another group previously described two families with our p.C634Y/p.Y791F-mutated patients exhibited a low
MEN 2A and MEN 2B and the p.Y791F mutation in com- penetrance of PHEO.
bination with mutations in exons 10 (p.C620F) and 16 This study further supports the observation that RET
(p.M918T), respectively (15). p.Y791F mutation might not be pathogenic. Furthermore,

976
 RET p.C643Y/p.Y791F double mutation

it illustrates that a double mutation in the RET gene may Family 2

not be as rare as expected. Retesting MEN 2 patients
whose clinical disease course is unusual may help clini-
cal management.
PATIENTS AND METHODS
Patients
From 2002 to 2012, 95 index cases of families with MTC and
292 cases of apparently sporadic MTC were screened for
germline mutations in exons 8, 10, 11 and 13-16 of the RET
gene (RefSeq: NM_020975.4) as part of the Brazilian Genetic
Screening Program at the Laboratory of Molecular and Trans-
lational Endocrinology, Division of Endocrinology, Federal Uni-
versity of São Paulo, Brazil. The index cases, from the five un-
related families with MEN 2 who were included in this study
and at-risk relatives, were identified through this on-going pro-
gram.
Patients had undergone standard surgical treatment for MTC,
including total thyroidectomy and central lymph node dissec-
tion, according to the ATA guidelines (4). The families’ histories
The index case (II.2) was a 43-year-old male with a thyroid nodule
diagnosed as suspicious for MTC on FNA (Table 1, Fig. 1). Labo-
ratory tests revealed high serum calcitonin (4,985 pg/ml) and el-
evated urinary levels of catecholamines and metanephrines. Skin
biopsy was performed to confirm the diagnosis of CLA. The pa-
tient underwent bilateral adrenalectomy for PHEO. Subsequent-
ly, he underwent total thyroidectomy with central neck dissection.
Final histology revealed multifocal MTC, with the largest nodule
measuring 2.9 cm (T2N0M0). After thyroidectomy, serum calci-
tonin levels remained undetectable. During follow-up, the patient
reported a history of headache, and a pituitary macroadenoma
(1.2 cm) was identified and surgically resected. Histological anal-
yses revealed a prolactinoma, although serum prolactin levels
were only mildly elevated. After 5 years of follow-up, he remains
free of disease with undetectable calcitonin levels. Family history
revealed that his mother died during labour.
Family 3
Retrospective analysis revealed that the index case (II.2) was a
30-yr-old male who underwent total thyroidectomy and central

s
are detailed below, and the pedigrees illustrated in Figure 1. All neck dissection for the diagnosis of metastatic MTC on FNA cy-

t i
diagnostic procedures were performed in accordance with the tology (Table 1, Fig. 1). Final histology confirmed MTC. Cervical

ur
regulations of the local ethics committee. Informed consent was lymph node dissection was followed by systemic chemotherapy,
obtained from all investigated subjects. and external neck beam radiation was performed. Over two

K
years of follow-up, calcitonin levels remained persistently ele-
Family 1

e
vated (>36,000 pg/ml), but no distant metastases have been

c
The pedigree of this family was partially described by Tamana- identified as of the last follow-up. Family history revealed that his

r i
ha et al. (16). The index patient (II.4) was diagnosed with mul- mother had died from acute myocardial infarction at the age of

t LY
48 and that his uncle died from advanced thyroid cancer. The

i
tifocal MTC (T1N1M0) (Table 1, Fig. 1). Although she har-

d
patient was then referred to our institution for the RET mutation

N
boured a mutation classified as ATA-A risk, her plasma calci-

E O
tonin levels continued to rise. Despite biochemical evidence screening program. During follow-up, patient exhibited both

E
,
of disease, imaging studies did not identify the site of persis- clinical and biochemical evidence of PHEO. Although his sister,

S
3 U
tent or recurrent disease. The patient’s history revealed that who lives overseas, was diagnosed with a large PHEO during

1 AL
her mother had developed PHEO, and one sister and two pregnancy (II.2), she did not undergo RET screening. The index

© 20
nieces had undergone operations for thyroid cancer. RET case was lost to follow-up.

O N
screening of at-risk relatives revealed that the p.Y791F muta-

S
tion was present in 17 family members, 12 of whom had bio- Family 4

PER
chemical, clinical and histological evidence of MTC. Annual The index case (III.14) was a 17-yr-old girl who noticed a left
biochemical screening has been conducted for other tumours neck enlargement and underwent total thyroidectomy (Table 1,

FOR
associated with MEN 2. Thus far, none of the RET p.Y791F car-
riers followed have developed clinical or biochemical evidence
of PHEO or PHPT. The index case exhibits persistent bio-
chemical disease, with calcitonin levels of approximately 1,700
pg/ml, even 15 yr after the first surgery.
Fig. 1). The pathology report revealed multifocal MTC, with the
largest nodule measuring 3 cm (T2NxMx). Two years later, a
right cervical dissection was performed due to lymph node
metastases, but the patient continued to exhibit elevated serum
calcitonin (134 pg/ml). She was referred to our institution for

Table 1 - Clinical data of index cases harbouring p.Cys634Tyr/p.Tyr791Phe double mutation.

Family/Index Case Age*/ Gender Presentation Histology TNM PHEO PHPT CLA Comments
1/ II.4 31/F FNA of Multifocal T1N1M0 No No No Biochemical
thyroid nodule MTC disease
2/ II.2 43/M FNA of Multifocal T2N0M0 Yes No Yes Macroprolactinoma
thyroid nodule MTC
3/ II.2 30/M Thyroid nodule and Multifocal TxN1M1 Yes No No Liver
cervical LN metastasis MTC metastasis
4/ III.14 17/F Neck enlargement Multifocal T2N1Mx No No No Recurrent
MTC LN metastasis
5/ II.3 32/M Paroxysms and Multifocal T1N0M0 Yes No No
weight loss MTC
*Age of surgery. LN: lymph nodes; PHEO: pheochromocytoma; PHPT: Primary hyperparathyroidism; CLA: cutaneous lichen amyloidosis; MTC: medullary
thyroid carcinoma; FNA: fine-needle aspiration.

977
F.O.F. Valente, M.R. Dias da Silva, C.P. Camacho, et al.
RET gene mutation analysis. Her aunt (II.5, Fig. 1) had under-
gone total thyroidectomy 10 yr before for a thyroid nodule that
had been misdiagnosed as follicular thyroid carcinoma (FTC).
At-risk relatives were tested for RET mutation and calcitonin lev-
els and screened for PHEO and PHPT. In two cases, skin biop-
sy was performed to confirm the diagnosis of CLA. Three pa-
tients were diagnosed with PHEO, nine were diagnosed with
MTC, three were diagnosed with PHPT and two were diagnosed
with CLA (Fig. 1).
Family 5
The index patient (II.3) was a 32-year-old man who was referred
for palpitation, sweating, fatigue and an unexplained loss of 10
kg over four months. FNA cytology of a thyroid nodule sug-
gested MTC (Table 1, Fig. 1). The patient underwent total thy-
roidectomy with cervical neck dissection. Final histology con-
firmed multifocal MTC (T1N0M0). He remains free of disease
with undetectable calcitonin levels. Family history revealed that
his older brother (II.2) had been misdiagnosed with FTC with ar-
eas of Hürthle cells and that his sister (II.1) had MTC. The index
case and at-risk relatives were tested for RET mutations and
screened for other MEN 2-associated tumours. Three family
members (II.1, II.2 and III.6) exhibited skin lesions that were con-
sistent with CLA.
K
DNA isolation, PCR amplification
and direct sequencing
c e
To retest the index case of family 1, genomic DNA was isolated
i
r
from fresh whole blood as described (17). DNA quality and
t
LY
quantity were confirmed. DNA from the index cases of families
i
2-5 (Fig. 1) and relatives were isolated and amplified by PCR, as
d ON
described (17). PCR products were purifed and submitted to di-
E E
rect sequencing using the BigDye Terminator Cycle Sequenc-
, S
ing Kit (Applied Biosystems, Foster City, CA, USA) as described
3 AL
U tation was identified in all members who harboured the
(18). Each exon was sequenced at least twice and in both di-
© 20
1
rections.
S O N
RET mutational analysis
PER
All coding regions of the RET gene that had not been previously
investigated at the time of the first diagnosis were sequenced in
FOR
the index case of family 1 (II.4). PCR products were purified and
submitted to direct sequencing as previously described (19).
Moreover, a new set of PCR primers for the “hotspot” regions of
the RET gene (exons 8, 10, 11 and 13-16) were designed and used
to re-sequence the index case of family 1 (Fig. 1, Table 1). PCR
primers were designed using http://ihg.gsf.de/ihg/ExonPrimers.html
to avoid SNPs in the primer-binding sites in accordance with the ref-
erence sequences from the University of California Santa Cruz
Database. For more details, please see reference (19). The index
cases of families 2-5 (Fig. 1, Table 1) were submitted to direct se-
quencing as described above. When a RET germline mutation
was identified in the index cases, all the at-risk family members
were sequenced for the appropriate exon.
RNA isolation, cDNA synthesis, cloning
and sequencing
To confirm that the p.C634Y (exon 11) and p.Y791F (exon 13)
RET mutations were in the same allele, PCR fragments encom-
passing exons 10-14 of the RET gene were cloned and se-
quenced. Since the introns were too large to be amplified, cD-
NA was used as the template. To this end, RNA was isolated
from fresh frozen MTC tissue obtained from individual II.2 of
978
family 1 (Fig. 1). Total RNA was isolated using TRIZOL reagent,
as previously described (20, 21). An aliquot of the cDNA reaction
was used as the template for PCR amplification, as described
above. Two primers (10F: 5’-CTTCCCTGAGGAGGAGAAGTG-
3’ and 14R: 5’-ATTTGGCGTACTCCACGATG-3’) were designed
to amplify a 617-bp segment. PCR products were purified and
directly introduced into the pCR4-TOPO TA vector, according to
the manufacturer's recommendations (Invitrogen Corp.). At least
10 ampicillin-resistant colonies were selected from each trans-
formant. Plasmid DNA was purified using the Qiagen Miniprep
Kit (Qiagen Inc., Valencia, CA) and sequenced using the BigDye
Terminator Cycle Sequencing Kit (22).
Haplotype analysis
Two polymorphic microsatellite markers (D10S196 and
D10S1652) flanking the RET locus at chromosome 10q11 were
used to exclude a common chromosomal ancestry in the five
apparently unrelated families with the p.C634Y/p.Y791F muta-
tions and MEN 2 syndrome. PCR reactions were performed us-
ing a fluorescent dye-labelled primer. The ABI 3100 sequencer
detected the fluorescence. ABI PRISM software GeneScan (Ap-
s
plied Biosystems) was used for gel analysis.
t i
ur
RESULTS
Identification of the RET germline double mutation
p.C634Y/p.Y791F in families with MEN 2 syndrome
When we retested the index patient of family 1, we iden-
tified an additional mutation in the RET gene. The
TGC>TAC mutation, which leads to the substitution of a
cysteine at codon 634 for a tyrosine (p.C634Y), was iden-
tified within exon 11 of the RET gene (Fig. 2A). There-
fore, exon 11 was re-sequenced in all family members
with the newly designed set of primers. The p.C634Y mu-
RET p.Y791F substitution. Importantly, all former cases
who were negative for p.Y791F were also negative for
p.C634Y. Therefore, the double mutation within the RET
gene (p.C634Y/p.Y791F) co-segregated with the disease
in this family (Fig. 1). We additionally found four appar-
ently unrelated families with the RET p.C634Y/p.Y791F
double mutation through the Brazilian Genetic Screen-
ing Program (Fig. 1). These findings further support the
observation that RET p.Y791F mutation might not be
pathogenic. Importantly, no overlap was found among
the families reported in this study with those families with
the RET p.C634Y/p.Y791F double mutation who were
previously reported in Brazil (14).
In our study, a high penetrance of MTC (79%) was found
in the p.C634Y/p.Y791F carriers compared with PHEO
(13%) (Fig. 1). A high penetrance of PHEO (100%) was
previously described in the double-mutated index cas-
es, with an overall penetrance of 84% when all family
members who were positive for the p.C634Y/p.Y791F
double mutation were included (14).
The RET germline double mutation occurs in one
of the paired homologous chromosomes
The genotype-phenotype correlation observed in this
study indicates that the two mutations in the RET gene
(p.C634Y and p.Y791F) are on the same allele. To deter-
mine whether the two point mutations located at exons 11
 RET p.C643Y/p.Y791F double mutation

Fig. 2 - Detection of germline double mu-

tations in the RET gene. PCR-direct se-
quencing shows that the TGC>TAC mu-
tation leads to p.C634Y substitution (A)
and TAT>TTT mutation leads to p.Y791F
substitution (B) in germline DNA isolated
from peripheral blood. To show that both
mutations are placed in the same allele,
we used cDNA as the template. PCR re-
action was performed using specific set
of primers located in exons 10 and 14.
PCR product was cloned into pCR4 Topo
vector. Direct sequencing of cloned PCR
products indicates that both mutations
occurred in cis orientation. Right panel
shows the RET RefSeq RNA sequence
(NM_020975.4) (C1), sequencing electro-
pherogram of wild-type allele (C2), and the
double-mutant (p.C634Y/p.Y791F) allele
(C3). Due to size limitation of stretched
electropherogram between mutations,
part of the sequence was omitted and rep-
resented by the sign (…).

t i s
ur
and 13 occurred in cis orientation (on the same DNA MTC tissues (no.=49). The p.Y791F mutation was not
strand) or trans orientation (on different DNA strands), identified in any of the MTC samples tested (data not

K
PCR fragments encompassing exons 10-14 were cloned shown). Although not tested in control samples, these

e
and sequenced (Fig. 2). Approximately half of the colonies data suggest that the p.Y791F single mutation is a very

c
exclusively exhibited the wild-type allele (Fig. 2A). The rare variant.

i
other half of the colonies had both mutations, thus con-

t r
firming that both mutations occur on the same DNA Haplotype analysis of two microsatellite markers

i LY
strand (i.e., on the same allele; Fig. 2B). Therefore, both The haplotype analysis of two microsatellite markers

d ON
the genotype-phenotype correlation and the cloned PCR

E E
fragment encompassing both mutations demonstrate
(D10S196 and D10S1652) of the five index cases and
family members suggests that the families do not have

, S
that the germline double mutation segregates with the a common ancestor (data not shown).

3
disease.

1 AL
U
© 20
N
Prevalence of the p.Y791F RET single mutation DISCUSSION

S O
The p.Y791F single mutation was not identified in 90 in- We previously identified a four-generation kindred with a

PER
dex cases of families with MTC (excluding the index cas- p.Y791F RET mutation and an unexpected clinical dis-
es presented in this study) or in 292 patients with appar- ease manifestation (i.e., an early age of onset and a more

FOR
ently sporadic MTC screened through the Brazilian Ge- aggressive form of MTC) (16). Because the p.Y791F mu-
netic Screening Program at our institution. We also in- tation has been described as non-pathogenic (13), it was
cluded data from studies performed in Brazil that inves- essential to better determine the role of the p.Y791F sub-
tigated germline mutations and/or SNPs within exon 13 stitution in this family.
of the RET gene (Table 2). Among the 920 chromosomes As double germline RET mutations may occur in pa-
tested (no.=460 cases), none exhibited the p.Y791F sin- tients with uncommon clinical manifestations (23) and
gle mutation. We additionally tested DNA isolated from RET p.C634Y/p.Y791F was identified in MEN 2A families
(14), we examined whether an additional mutation with-
in the RET gene could be in linkage disequilibrium with
Table 2 - Frequency of p.Y791F single mutation in studies per- the p.Y791F variant. However, the expanded analysis,
formed in Brazil. which included exons that were not previously investi-
Family members who gated (16), did not detect other mutations within the
Study Index cases (%) were positive for RET gene.
p.C634Y/p.Y791F double mutation In an effort to identify a mutation that might not have
(26)* 0/11 ND been detected in our first analysis and, therefore, could
(14)* 0/4 0/15 explain the unusual phenotype observed in this family,
(27)# 0/23 ND newly designed sets of primers were used to re-sequence
This study* 0/382 (0%)§ 0/25 the “hotspots” of mutation in the RET gene. Interesting-
Total 0/420 0/40 ly, in addition to the p.Y791F germline mutation, a nov-
el p.C634Y RET mutation was identified in the index case.
ND: not determined. *exon 13 was analyzed by direct PCR sequencing;
#exon 13 was analyzed by denaturing gradient gel electrophoresis (DGGE) While this manuscript was under review, a group de-
and sequencing; §Index sporadic (no.=292) and familial cases (no.=90). scribed a case with MEN 2A, an atypical course of dis-

979
F.O.F. Valente, M.R. Dias da Silva, C.P. Camacho, et al.
ease and two heterozygous germline mutations within
exon 11 of the RET gene (p.C634R/p.A640G). Since the
patient exhibited an unusual clinical pattern, the authors
performed a new genetic test on DNA extracted from
peripheral blood. Significantly, in addition to the two
germline mutations found at the time of the first diag-
nosis, the authors described a novel mutation (p.M700L)
in exon 11 of the RET gene (24).
It is difficult to clearly identify why the mutations were
not identified during the first diagnosis in both our study
and the study by Conzo et al. One possibility is that the
rate of genotyping errors might be higher than predict-
ed (25). The main cause of genotyping errors is allele
dropout due to the low quality and quantity of DNA and
the occurrence of SNPs on the target site sequence (25).
In this study, to eliminate as many errors as possible, both
the quality and quantity of DNA were double-checked,
and a new set of primers for the hotspots regions was
used.
Additionally, although all hotspots for mutations in the
RET gene are sequenced in some centers, RET se-
quencing may have been finished when one mutation is
found. This procedure may miss the detection of an ad-
ditional (double) RET mutation. Therefore, re-sequenc-
ing MEN 2 patients who have mutations at codon 634
K
and who lack genotype-phenotype correlation may clar-
e
ify this issue and will help to determine the prevalence
c
of double mutations that affect these codons and to im-
i
prove the clinical management of these families.
t r
Remarkably, to date, five unrelated families with MEN 2
i LY
and the p.C634Y/p.Y791F double mutation have been
d N
O
identified through the Brazilian Genetic Screening Pro-
E
E
gram performed in our institution. Unlike previous reports
, S
(14), we did not observe an increased penetrance of
3 U
PHEO in the p.C634Y/p/Y791F carriers in our study in
1 AL
comparison with codon 634 single mutation carriers. In
© 20
N
accordance with what has been described for other mu-
O
S
tations at codon 634, six p.C634Y/p.Y791F carriers ex-
PER
hibited CLA.
Although the pathogenic role of the p.C634Y mutation in
FOR
MEN 2 patients is established, it remains unclear whether
the p.Y791F mutation could lead to a higher pathogenic-
ity due to an additive effect or instead attenuate the effect
of the p.C634Y mutation. However, p.C634Y/p.Y791F car-
riers may exhibit a 634 codon-like pattern of MTC and,
therefore, should be treated according to the guidelines
for the p.C634Y mutation.
This study highlights the importance of meticulous
screening for RET double germline mutations, particu-
larly in families in which there is no apparent genotype-
phenotype correlation.
ACKNOWLEDGMENTS
This work was supported by the São Paulo State Research Foundation
(FAPESP) (grant numbers 06/54922-2 and 09/11257-7). JMC and RMBM
are investigators of the Brazilian Research Council (CNPq). AUB is a schol-
ar from FAPESP.
Disclosure statement
The authors declare that there is no potential conflict of interest that could
be perceived as prejudicing the impartiality of the research reported.
980
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1. Santoro M, Melillo RM, Carlomagno F, Vecchio G, Fusco A.
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2. Castellone MD, Verrienti A, Magendra Rao D, et al. A novel de no-
vo germ-line V292M mutation in the extracellular region of RET in
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529-34.
3. Muzza M, Cordella D, Bombled J, et al. Four novel RET germline
variants in exons 8 and 11 display an oncogenic potential in vitro.
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4. Kloos RT, Eng C, Evans DB, et al. Medullary thyroid cancer: man-
agement guidelines of the American Thyroid Association. Thyroid
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4725-9.
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7. Brandi ML, Gagel RF, Angeli A, et al. Guidelines for diagnosis and
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8. Berndt I, Reuter M, Saller B, et al. A new hot spot for mutations in
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ma and multiple endocrine neoplasia type 2A. J Clin Endocrinol
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9. Vaclavikova E, Dvorakova S, Sykorova V, et al. RET mutation
Tyr791Phe: the genetic cause of different diseases derived from
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10. Arum SM, Dahia PL, Schneider K, Braverman LE. A RET mutation
with decreased penetrance in the family of a patient with a "spo-
radic" pheochromocytoma. Endocrine 2005, 28: 193-8.
11. Brauer VF, Scholz GH, Neumann S, et al. RET germline mutation in
codon 791 in a family representing 3 generations from age 5 to
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12. Fitze G, Schierz M, Bredow J, et al. Various penetrance of familial
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codon 790/791 germline mutations. Ann Surg 2002, 236: 570-5.
13. Erlic Z, Hoffmann MM, Sullivan M, et al. Pathogenicity of DNA vari-
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and von Hippel-Lindau syndrome. J Clin Endocrinol Metab 2010,
95: 308-13.
14. Toledo RA, Wagner SM, Coutinho FL, et al. High penetrance of
pheochromocytoma associated with the novel C634Y/Y791F dou-
ble germline mutation in the RET protooncogene. J Clin Endocrinol
Metab 2010, 95: 1318-27.
15. Dvorakova S, Vaclavikova E, Ryska A, et al. Double germline mu-
tations in the RET Proto-oncogene in MEN 2A and MEN 2B kin-
dreds. Exp Clin Endocrinol Diabetes 2006, 114: 192-6.
16. Tamanaha R, Camacho CP, Ikejiri ES, Maciel RM, Cerutti JM. Y791F
RET mutation and early onset of medullary thyroid carcinoma in a
Brazilian kindred: evaluation of phenotype-modifying effect of
germline variants. Clin Endocrinol (Oxf) 2007, 67: 806-8.
17. Tamanaha R, Camacho CP, Pereira AC, et al. Evaluation of RET
polymorphisms in a six-generation family with G533C RET muta-
tion: specific RET variants may modulate age at onset and clinical
presentation. Clin Endocrinol (Oxf) 2009, 71: 56-64.
18. Hemerly JP, Bastos AU, Cerutti JM. Identification of several novel
non-p.R132 IDH1 variants in thyroid carcinomas. Eur J Endocrinol
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19. Lindsey SC, Kunii IS, Germano-Neto F, et al. Extended RET gene
analysis in patients with apparently sporadic medullary thyroid can-
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20. Oler G, Nakabashi CD, Biscolla RP, Cerutti JM. Seven-year follow-
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21. Cerutti JM, Oler G, Michaluart P jr, et al. Molecular profiling of
matched samples identifies biomarkers of papillary thyroid carci-
noma lymph node metastasis. Cancer Res 2007, 67: 7885-92.
22. Oliveira MN, Hemerly JP, Bastos AU, et al. The RET p.G533C mu-
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23. Qi XP, Ma JM, Du ZF, et al. RET germline mutations identified by
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e20353.
24. Conzo G, Circelli L, Pasquali D, et al. Lessons to be learned from
the clinical management of a MEN 2A patient bearing a novel
634/640/700 mutation of the RET proto-oncogene. Clin Endocrinol
2012, 77: 934-6.
25. Pompanon F, Bonin A, Bellemain E, Taberlet P. Genotyping errors:
causes, consequences and solutions. Nat Rev Genet 2005, 6: 847-59.
26. Magalhaes PK, de Castro M, Elias LL, Soares EG, Maciel LM.
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t i s
K ur
i c e
i t r LY
E d E ON
3 , L U S
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1 S O N A
PER R
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Clinical Endocrinology (2014) 80, 235–245 doi: 10.1111/cen.12264

ORIGINAL ARTICLE

A ten-year clinical update of a large RET p.Gly533Cys kindred

with medullary thyroid carcinoma emphasizes the need for an
individualized assessment of affected relatives
Priscila S. Signorini*, Maria Inez C. Franc!a*, Cleber P. Camacho*, Susan C. Lindsey*, Fla
!via O. F. Valente*,
Teresa S. Kasamatsu*, Alberto L. Machado*, Camila P. Salim*, Rosana Delcelo†, Ana O. Hoff*,§, Janete M.
Cerutti‡, Magnus R. Dias-da-Silva* and Rui M. B. Maciel*

*Department of Medicine, Laboratory of Molecular and Translational Endocrinology, Escola Paulista de Medicina, Universidade
Federal de S~ao Paulo, †Department of Pathology, Escola Paulista de Medicina, Universidade Federal de S~ao Paulo, ‡Departments of
Morphology and Genetics, Escola Paulista de Medicina, Universidade Federal de S~ao Paulo and §Instituto do C^ancer do Estado de
S~ao Paulo (ICESP), Faculdade de Medicina da Universidade de S~ao Paulo, S~ao Paulo, Brazil

the fact that it allows us to delineate the natural history of RET

Summary 533 MEN2A 10 years after its first description.

Objective Reviewing the clinical outcomes of a large kindred
with a RET p.Gly533Cys mutation, 10 years after the first
description of this kindred, has provided an important set of
clinical data for healthcare decision-making.
Design and Patients We identified 728 RET533 Brazilian rela-
tives, spread out over 7 generations. We performed clinical
examination, biochemical and imaging analyses in the proband
and in 103 carriers.
Measurement and Results The proband has been followed
without evidence of structural disease in the last 10 years but
with elevated calcitonin. The clinical and surgical features of 60
thyroidectomized RET533 relatives were also described. Forty-six
patients had MTC (21–72 years), and 11 patients had C-cell
hyperplasia (CCH) (5–42 years). Twelve MTC patients with
lymph node metastases had a tumour size of 0!7–2!8 cm. Calci-
tonin level and CEA were correlated with disease stage, and
none of the patients presented with an altered PTH or meta-
nephrine. A 63-year-old woman developed pheochromocytoma
and breast cancer. Two other RET533 relatives developed lung
squamous cell carcinoma and melanoma.
Conclusions A vast clinical variability in RET533 presentation
was observed, ranging from only an elevated calcitonin level
(3%) to local metastatic disease (25%). Many individuals were
cured (42%) and the majority had controlled chronic disease
(56%), reinforcing the need for individualized ongoing risk
stratification assessment. The importance of this update relies on
Correspondence: Magnus R. Dias da Silva, Laboratory of Molecular and
Translational Endocrinology, Escola Paulista de Medicina, Universidade
Federal de S~ao Paulo, Rua Pedro de Toledo 669, 11° andar, 04039-032
S~ao Paulo, SP, Brazil. Tel.: +55 11 5084 5231;
E-mail: mrdsilva@unifesp.br
(Received 27 December 2012; returned for revision 21 January
2013; finally revised 17 May 2013; accepted 4 June 2013)
Introduction
Multiple endocrine neoplasia type 2 (MEN-2) is an autosomal
dominant inherited syndrome with tumours of endocrine
glands, including thyroid carcinoma and pheochromocytoma.
MEN-2 was first described by Sipple in 1961,1 followed by the
histopathological identification of the stromal amyloid content
in thyroid parafollicular C-cells. In 1965, the term ‘medullary
thyroid cancer’ (MTC) was established,2 and an association with
parathyroid gland hyperplasia was subsequently identified.3
These findings are the major components of the constellation of
this familial, multitumour endocrine pathology.2,4–6 In almost
all families with MEN-2, the oncogenic process is caused by an
activating, germline RET mutation.7
The phenotypic variation in MEN-2 is diverse and partly
related to a mutated codon in the RET oncogene.8 Ten years
ago, our group identified a large kindred with familial medullary
thyroid carcinoma (FMTC) carrying a missense mutation in
exon 8 of the RET oncogene, which corresponds to a glycine-
to-cysteine substitution at codon 533.9 We then reported a
pheochromocytoma in a RET533 patient, indicating that this
mutation leads to an MEN2A phenotype.10 We have also
reported the possible role of interfering SNPs in modifying the
clinical outcome in this large family cohort.11 Here, we present
an update on a clinically detailed follow-up, 10 years after its
first description, of the largest reported family with a RET muta-
tion and discuss the epidemiological, clinical and surgical find-
ings based on the RET mutation classification system developed

© 2013 John Wiley & Sons Ltd 235

236 P. S. Signorini et al.
by the American Thyroid Association (ATA).12,13 In view of the
variability of the clinical presentation, penetrance and tumour
aggressiveness, we also comment on the need for an individual-
ized risk assessment for patients with MTC carrying p.Gly533Cys
mutation.
Patients and methods
Brazilian RET533 kindred
We have been following a Brazilian family with individuals
afflicted by MEN-2 syndrome due to a p.Gly533Cys RET onco-
gene mutation. This 728-member family has a Caucasian origin
with ancestors from Barcelona, Spain, who immigrated to Brazil
at the beginning of the 20th century. Most of the family mem-
bers reside in south-eastern Brazil and are systematically
followed at an outpatient thyroid clinic either in Vitoria or in
S~ao Paulo by a single team of physicians. A signed letter of
informed consent was obtained from all patients or their legal
guardians. Our study was approved by the research ethics com-
mittee of the Universidade Federal de S~ao Paulo.
Biochemical laboratory tests
Serum calcitonin (sCT) was measured using a chemilumines-
cence assay developed by our laboratory, which has an analyt-
ical sensitivity of 1 pg/ml and a cut-off of 7!8 pg/ml for
females and 18!4 pg/ml for males.14 Serum CEA was measured
using an electrochemiluminescence assay (normal value:
<5!0 lg/l for smokers and <3!0 for nonsmokers). The total
urine metanephrine level was measured by HPLC (normal
range: 150–510 lg/g of creatinine for females and 110–480 lg/g
of creatinine for males, Chromsystems, Munich, Germany).
Serum PTH was measured using an electrochemiluminescence
assay (normal range: 15–65 pg/ml, Roche Diagnostics, Mann-
heim, Germany).
Histopathological findings
The histological studies were first performed using haematoxy-
lin–eosin (HE) staining. To improve the accuracy of the calcito-
nin immunostaining, we treated tissues with the same antibody
used to measure sCT (E1P10, UNIFESP). We also studied other
MTC biomarkers using commercial antibodies that recognize
chromogranin A (Dako; 1:8000), synaptophysin (Dako; 1:1000),
monoclonal CEA (Dako; 1:3000), thyroglobulin (Dako; 1:1200)
and Ki 67 (Spring; 1:250). The slide staining was performed on
4-lm paraffin sections of formalin-fixed tissue. Sections of a
normal thyroid gland were used as a negative control and for
background reduction.
RET molecular analysis
After peripheral blood germline DNA extraction, we performed
a specific RET gene PCR, and the products were directly
sequenced using the Big Dye Terminator/ABI 3100 sequencer
(PE Applied Biosystems, Foster City, CA, USA), as previously
described.15,16 A total of 432 patients underwent germline testing
for RET 533 mutation (exon 8). The index case instead under-
went comprehensive RET coding gene sequencing to eliminate
the possibility of a double mutation outside the RET mutation
hotspot.
Statistical analysis
The results are predominantly given as frequencies, and Spear-
man’s correlation coefficients were used for statistical analysis.
The data were analysed using GraphPad Prism 5 software (La
Jolla, CA, USA). We considered a p value of less than 0!05 to be
statistically significant.
Results
Over 728 subjects and patients were assessed from the family
carrying the RET533 mutation for initial clinical evaluation.
Members of this family who are at risk to have inherited this
mutation were invited to enrol in the molecular diagnostic
study, including 432 agreeing to have genetic testing by the
end of this study. Of these family members, one-third were
mutation carriers (119/432). Patients and relatives were
assessed as summarized in the flow chart depicted in Fig. 1.
Based on the initial identification of the family in 2002 and
the scientific publication in 2003,9 we have actively expanded
the genetic testing by 35%. A full circular RET533 pedigree is
depicted in Fig. 2a with a zoom of the proband’s immediate
family in the centre (Fig. 2b). Within this period, the
participants underwent neck and adrenal ultrasound for
structural disease screening and blood and urine collection for
biochemical evaluations, including the measurement of calcito-
nin, CEA, calcium, PTH and urinary metanephrine. The
major epidemiological, clinical and surgical features of the
RET533 kindred patients and relatives are summarized in
Table 1.
RET533 proband follow-up
The index case is 59 years old and was asymptomatic until
47 years of age, when he noticed a nodule in the right lobe of
his thyroid gland. Thyroid ultrasound showed a 35-mm nodule
in the right lobe and an 18-mm nodule in the left lobe. His
basal sCT level was 1080 pg/ml. A total thyroidectomy with
selective neck node dissection in the central compartment (level
VI) was performed. The histological examination showed the
presence of MTC, CCH and microscopic metastatic disease in
three lymph nodes (LN) with extracapsular extension into adja-
cent tissues.9 He underwent cervical external beam radiation.
To date, although his median sCT level has remained
unchanged at approximately 150 pg/ml, his routine metastatic
restaging has shown no radiological evidence of persistent
structural disease. He was evaluated on average every
4–6 months during the first 2 years after surgery and then
every year for the past 8 years.
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Clinical Endocrinology (2014), 80, 235–245
 Long-term follow-up of a large RET533 MEN-2 family 237

728 subjects and patients addressed for

clinical evaluation

313 non-carriers 119 carriers of RET533 296 non-obligatory carriers (ie,

mutation subjects not genetically tested)

59 patients awaiting 60 underwent

surgery thyroidectomy

1–36 years (19), 3–72 years (60),

median 14 median 42

MTC CCH Normal

(46) (11) (3)
21–72 years, 5–42 years, 3–25 years,
median 47 median 17 median 8

TNM staging system (available data for histopathology revision)

I II III IVa/IVb/IVc
(16) (8) (15) (0)
23–72 years, 35–69 years, 21–72 years,
median 40·5 median 47 median 48

Calcitonin Calcitonin Calcitonin Calcitonin

2·2–33·0 pg/ml <1–6·7 pg/ml <1–9·0 pg/ml 2–1000 pg/ml
median 5·9 median 1 median 1 median 29

Mean follow-up period of 9·9 years

(range 0·6–35)

Fig. 1 Flow diagram reporting the summary of follow-up and major clinical outcomes of the Brazilian RET533 cohort.

neck node dissection at 21 years of age. Histopathological

Thyroidectomized RET533 affected relatives:
histopathological findings and occurrence of other
tumours
Based upon the current clinical review, 60 of the 103 carriers
have undergone total thyroidectomy (see Fig. 1). Forty-six
patients had medullary thyroid carcinoma (MTC) that was con-
firmed histopathologically, with the age at the time of diagnosis
between 21 and 72 years. The clinical and biochemical follow-up
results of these 60 thyroidectomized patients are summarized in
Table 2. An elevated sCT had a good predictive value for identi-
fying MTC (Fig. 3a). The distribution of basal sCT positively
correlated with the age of onset for the MTC (r = 0!48,
P < 0!01) (Fig. 3b). To identify better prognostic variables for
the age extremes of the disease, a more detailed clinical report
of the youngest and the oldest RET533 patient presenting with
MTC is described as follows.
The youngest RET533 patient diagnosed with MTC was the
proband’s daughter. She underwent total thyroidectomy with
tumour analysis confirmed the presence of MTC with LN metas-
tases. Five years later, she developed cervical LN metastases,
confirmed by cytology fine-needle aspiration biopsy (FNAB).
Hence, she underwent a second neck node surgical revision. Her
second to last sCT level was still 358 pg/ml, and she again had an
abnormal neck US and FNAB that confirmed recurrent meta-
static MTC. Because of her age and disease apparently limited to
the cervical LNs, she underwent a third surgical neck dissection
6 months later when sCT level was 21!5 pg/ml.
The proband’s aunt, who was later identified as a gene carrier
and had a basal calcitonin level of 499 pg/ml, is the oldest
patient in this RET533 family. She underwent total thyroidec-
tomy with a neck node dissection at 72 years of age. Histopath-
ological analysis of the tumour confirmed MTC, but, in contrast
to her first-degree relatives, without LN metastases. She has been
asymptomatic, and her last neck US, which was performed when
she was 81 years old, was unremarkable. Her last sCT level was
less than 1 pg/ml. During the routine examinations after sur-

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Clinical Endocrinology (2014), 80, 235–245
238 P. S. Signorini et al.

(a)

(b)

Fig. 2 Schematic pedigree representing the Brazilian RET533 kindred (a) displaying the proband’s closest relatives (b). Affected individuals are
designated by the symbols shown in the key legend at the bottom centre of the figure. The proband is identified with a solid black arrow.

gery, she has not shown any evidence of biochemical or struc-
tural disease.
To date, among all 60 patients who underwent surgery after
being found to have the RET533 mutation and an abnormal sCT,
eleven patients presented with C-cell hyperplasia (CCH), with the
onset varying between 5 and 42 years of age. These clinical, bio-
chemical and histopathological findings are also summarized in
Table 2. The relevant clinical information regarding both patients
with CCH at the opposite age extremes is further described.
A 5-year-old girl, who tested positive for the mutation and
presented with a slightly normal basal sCT level of 8 pg/ml, also
exhibited an increased sCT of 120 pg/ml after pentagastrin (PG)
stimulation. She underwent a total thyroidectomy, and histologi-
cal examination showed the presence of CCH, as previously
reported.9 Today, she is 14 years old, and her last sCT level was
<1 pg/ml. She is the youngest affected patient of this family
presenting only with CCH and without disease 9 years after
prophylactic surgery.

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 Long-term follow-up of a large RET533 MEN-2 family 239

Table 1. Summary of the major epidemiological, clinical and surgical underwent total thyroidectomy, and his tumour histology only
features of RET533 carriers. Age, gender, type of surgery, major showed CCH. His sCT level has been less than 1 pg/ml. During
histopathology findings, AJCC TNM staging system, calcitonin-stratified the last 2 years, he presented with a chronic cough that was
histopathological group, follow-up period and current clinical status are
initially attributed to tobacco smoke exposure. Upon further
depicted as the total number, median and range when the clinical data
were available investigation, he was diagnosed with lung squamous cell carci-
noma (SCC), confirmed by histological tumour analysis after
Values (range) thoracic surgery. He has been free from MTC but is still receiv-
Number of ing chemotherapy for SCC and being followed closely at the
Features subjects (%) Median Range outpatient pulmonary clinic.
Among the 11 patients who tested positive for the mutation,
Thyroidectomized relatives (Total: 60) underwent thyroidectomy and were found to only have CCH,
Age (years) one patient also developed a second neural crest–related tumour,
Mean 40!5 melanoma, which was diagnosed 10 years after the thyroidec-
Median (range) 42 (3–72)
tomy. Another symptom was observed in a 32-year-old man
Gender
Male 25 (41!6) with RET533 who presented with chronic constipation and a
Female 35 (58!4) presumptive diagnosis of late-onset megacolon; however, his
Surgery colon biopsy was normal.
Total Tx 19 After being diagnosed with MTC with LN metastases, a
Total Tx + central lymph 32 63-year-old woman was also diagnosed with a pheochromocy-
node dissection toma during her fifth year of follow-up, after presenting with a
Total Tx + lateral lymph 9
sudden hypertensive crisis. Abdominal MRI showed a large mass
node dissection
Histopathology in the right adrenal gland, measuring 4!3 9 1!7 cm, with an
Normal 3 intermediate T1 and a high T2 signal suggesting pheochromocy-
CCH 11 toma. The patient was referred for laparoscopic right adrenalec-
MTC 46 tomy. The tumour removed confirmed the histopathological
TNM staging system (AJCC) diagnosis of pheochromocytoma.10 Two years after the last sur-
I 16 gery, we found that she had been diagnosed with breast cancer
II 8
and is receiving chemotherapy.
III 15
IVa/IVb/IVc 0 Three RET533 affected relatives had no histological abnormali-
Calcitonin (pg/ml) stratified by histopathological group ties related to MTC. Two children of these relatives had mildly ele-
Pre-Tx vated calcitonin, and their parents voluntarily opted for surgery,
Normal/CCH 7 60 1!9–90 at 8 and 3 years of age. The third affected relative had normal
MTCp N0 23 113 30–1780 basal and pentagastrin-stimulated sCT nevertheless, he decided to
MTCp N1 10 737 188–1970 undergo prophylactic thyroidectomy at 25 years of age.
Post-Tx
Twelve patients with MTC had LN metastases, and all of their
Normal/CCH 10 2 0–7!8
MTC pN0 26 1 0–29 tumours were ≥0!7 centimetres. The deaths of two of the pro-
MTC pN1 9 21!5 2–208 band’s relatives attributed to MTC metastasis that were reported
Clinical status up to the end of study to us at the beginning of our systematic follow-up included a
Follow-up (years) 10 0!3–35 53-year-old woman and a 60-year-old man who most likely died
Remission* 21 (42) due to distant metastatic disease in the liver and lungs 14 and
Persistent biochemical 28 (56) 7 years after the MTC diagnosis, respectively.
disease (alone)
In our last task force evaluation held in Vitoria, in the State
Persistent biochemical and 1 (2)
structural disease of Espirito Santo, 163 more individuals were tested for sCT. The
Death from disease 2† median sCT was 3!7 pg/ml (<1–32 pg/ml) in 107 noncarriers,
Relatives awaiting thyroidectomy (Total: 59) 44 pg/mL (2!20–688 pg/ml) in 21 carriers who had not yet
Age (years) 19 14 1–36 undergone thyroidectomy and 26!0 pg/mL (<1–358 pg/ml) in 35
Gender: number (%) carriers post-thyroidectomy. In this last group, 18 of the 35
Male 12 (63) individuals had undetectable calcitonin levels (≤1 pg/ml), 11 had
Female 7 (27)
sCT level between 2!5 and 50 pg/ml and 4 individuals had sCT
Calcitonin 5!9 2!2–33
greater than 50 pg/ml (91–358 pg/ml). In addition, CEA was
*Remission when calcitonin ≤1 pg/ml. measured in the carriers as a potential alternative MTC marker.
†Before the beginning of this study. The mean CEA was 2!48 l/l (0–10!7 l/l) in 33 post-thyroidec-
tomy carriers and 2!6 lg (0!7–18!3) in 19 carriers who had not
In contrast, the oldest patient presenting with CCH was a yet undergone thyroidectomy.
42-year-old man who had a high basal calcitonin level (73 pg/ Of the 17 RET533 who were approved for surgery after
ml) and a positive pentagastrin stimulation test (321 pg/ml). He undergoing a neck US, only 3 (18%, 31 to 43 years of age) had

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240 P. S. Signorini et al.

Table 2. Current clinical follow-up of all 60 thyroidectomized RET533 relatives. Detailed clinical and surgical follow-up of the first 60 RET533 carriers
who underwent thyroidectomy and had a confirmed clinical status at the conclusion of the study. We present the age at disease onset, postsurgical
calcitonin, the presence of CCH and Pheo, MTC tumour size, pTNM staging and the extension of local tumour invasion

Age of Onset Tumour Local Since initial Calcitonin

Patients Gender (years) CCH Size (cm) pTNM ML_LD invasion Stage surgery (years) after TT Pheo

1 F 72 0!7 T1N0Mx 0_6 No I 10 <1 No

2 M 69 <0!1 T1N0Mx 0_12 No II 9 <1 No
3 M 61 0!6 T1N0Mx 0_23 No I 10 – No
4 M 72 0!7 T1N1Mx No III NA – No
5 F 47 NA T1N1Mx No III 35 88 No
6 F 60 2 T1N1Mx NI No III 11 13!5 No
7 F 57 0!8 T1N1Mx No III 11 2 Yes
8 M 62 2!3 T2N1Mx 3_3 No III 11 7 No
9 F 48 0!3 T1N0Mx 0_NI No II 10 <1 No
10 F 54 0!25 T2N0Mx 0_NI No II 9 6!4 No
11 F 51 1!3 T1N1Mx 2_6 No III 10 9!2 No
12 F 47 3 T2N0Mx No II NA 1 No
13 M 40 1!1 T3N1Mx 1_5 Yes III 9 – No
14 F 51 0!6 T1N0Mx 0_14 No I 9 2!5 No
15 F 48 1!3 T3NxMx NA Yes III 10 <1 No
16 M 40 0!5 T1N0Mx 0_4 No I 9 – No
17 M 45 0!3 T1NxMx NA No – 8 <1 No
18 M 43 0!9 T1N0Mx 0_4 No II 10 9 No
19 F 48 1!7 T1N1Mx 3_5 No III 11 208 No
20 F 45 0!7 T1N0Mx 0_17 No I 11 5!7 No
21 M 41 0!5 T1N0Mx 0_3 No I 11 1 No
22 M 38 0!5 T1N0Mx 0_7 No I 10 6!7 No
23 M 31 0!5 T1N0Mx 0_7 No I 11 <1 No
24 M 48 2!5 T2N1Mx 3_10 No III 11 156 No
25 M 42 2!7 T2N0Mx No II NA – No
26 M 42 1!4 T3NxMx NA Yes III 11 22!5 No
27 M 29 0!5 T1NxMx NA No – 11 0 No
28 F 35 2!5 T2NxMx NA No II 11 1 No
29 F 32 0!6 T1N0Mx 0_8 No – 9 0 No
30 F 34 0!6 T1N0Mx 0_NI No I 11 2 No
31 F 24 0!4 T1N0Mx 0_2 No I 10 0 No
32 M 27 0!6 T1NxMx NA No III NA 29 No
33 F 21 1 T1N1Mx 1_8 No III 11 21!5 No
34 M 23 0!2 T1N0Mx 0_6 No I 10 0 No
35 M 42 0!3 T1N0Mx 0_7 No I 5 <1 No
36 F 25 7 7!8 No
37 F 59 NA 5 1!7 No
38 F 36 0!3 T1N0Mx No I 6 6 No
39 F 17 Yes 8 3!7 No
40 F 19 Yes 10 <1 No
41 M 12 Yes 10 <1 No
42 M 52 NA 8 91 No
43 F 17 Yes 8 <1 No
44 M 42 Yes 11 0 No
45 F 5 Yes 10 2 No
46 M 32 Yes 1 <1 No
47 F 3 1 2 No
48 F 33 Yes 11 <1 No
49 F 16 Yes 8 – No
50 F 10 Yes 5 <1 No
51 F 68 0!7 T1N0Mx No I 10 – No
52 F 38 2!8 T2N1Mx No III 1 2 No
53 M Yes NA – No
54 F 68 NA T1N1Mx Yes III 18 1000 No

(continued)

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 Long-term follow-up of a large RET533 MEN-2 family 241

Table 2. (continued)

Age of Onset Tumour Local Since initial Calcitonin

Patients Gender (years) CCH Size (cm) pTNM ML_LD invasion Stage surgery (years) after TT Pheo

55 F 41 0!8 T1N0Mx 1 2 No
56 F 8 1 2 No
57 F 58 1 T1NxMx No I 3 2 No
58 M 47 3 T2N0Mx 0_5 No II 0!6 2 No
59 F 56 NA NA No
60 M 57 1!5 T1N1Mx 1_12 No III 0!3 No

ML, metastatic lymph node out of LD, lymph nodes dissected; NI, not informed; NA, not available.

additional post-thyroidectomy carriers have also undergone neck

US and LN FNAB. One of these patients, the proband’s daugh-
ter who was described above, had a confirmed LN metastasis.
The other patient was a 68-year-old woman, who, despite having
an elevated calcitonin and a positive FNAB, still refuses surgery.
Neither the RET533 kindred’s proband nor other carriers have
had primary hyperparathyroidism in the past 10 years of follow-
up.

Discussion

Delineating the natural history of the Brazilian RET533

Fig. 3 Distribution of serum calcitonin among thyroidectomized
RET533 carriers. Basal sCT at the time of either molecular or clinical
diagnosis (a) and its scatter distribution based on the age at diagnosis
(b). Filled circles represent patients with MTC, and open circles those
with only C-cell hyperplasia (CCH).
thyroid nodules and underwent a fine-needle aspiration biopsy
(FNAB). One biopsy showed an indeterminate result from a
2!6-cm nodule, but the patient had a normal sCT, so she
deferred a thyroidectomy for now. The other two FNABs were
positive for MTC. These patients have already undergone thy-
roidectomy, which confirmed MTC histologically. Two
MTC family
Our report represents a clinical evaluation of one of the largest
families carrying a RET oncogene mutation. This kindred com-
prises 432 relatives among a total of 728 subjects who were ini-
tially assessed by the same team of physicians over the last ten
years. This set of clinical information broadens our understand-
ing of the clinical outcomes of FMTC and MEN-2 syndrome
and reinforces the need for an individualized, ongoing stratifica-
tion of patients with MTC.
Although MTC is still the most common cause of death in
patients with MEN2A, a decrease in the incidence of persistent or
recurrent disease has been observed, even after considering the
enormous outcome variability. This variability is mainly due to
the surgeon’s experience, which affects the completeness of the
initial surgery, as previously described in retrospective
studies.17,18 This work and others have suggested that a longer fol-
low-up period is necessary to confirm that the subjects are cured.5
Another factor that worsens outcomes in oncological patients
is the presence of a second tumour, an event that is not rare in
MEN-2 compared to primary MTC.19 In 1996, another large
MTC reported family brought to attention the need of screening
for other endocrinopathies in FMTC families with cysteine
codon mutations of the RET oncogene, because these may not
be MTC-only families.20 In 2007, a Greek group first reported
another RET533 family outside of Brazil, in which they reclassi-
fied the kindred as having MEN-2.21 In 2008, this mutation was
also associated with MEN2A by another Greek group.6 More
recently, Sarika et al.22 also identified RET533 mutation in 10 of
129 patients with sporadic MTC (7!75%) who were negative for
RET hotspot mutations, emphasizing the need to include exon-8

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242 P. S. Signorini et al.

mutation screening systematically in patients diagnosed with
apparently sporadic MTC.
Despite identifying just one patient with a pheochromocytoma
in our large clinical study, we have actively followed 119
RET533 carriers who have had no evidence of adrenal involve-
ment based on periodic biochemical and image screening at least
annually. While this article was in revision, Castro et al.,23 pub-
lished the first RET533 family from United States presenting
with MEN2A in several affected members. Surprisingly, they
found that pheochromocytoma was more common in their
patients than previously reported in Brazil and Greece (3 of 21
genetic testing carriers). This lower incidence of pheochromocy-
toma in our studied family compared to other families has
raised the hypothesis of a RET double mutation. Thus, we have
searched for an interfering second mutation other than RET533
in our patient with a pheochromocytoma; however, the sequence
analysis of 19 coding exons of RET was negative for additional
mutations.10
Most of the reviewed histopathology when available showed the
classic appearance of MTC comprising with solid sheets and pack-
ets of tumour cells traversed by fibrovascular septa and abun-
dantly stained amorphous deposits, similar to what we observe in
other patients. For instance, representative histopathological find-
ings observed in RET533 tissues are depicted in Fig. 4, with a
spindle cell MTC variant (4a) surrounded by C-cell hyperplasia
(4b), cervical lymph node metastases from MTC (4d) and pheo-
chromocytoma of the right adrenal gland (4e).
During the surveillance of this cohort, we found one case of
melanoma in association with RET533-MEN2A syndrome.
Because melanoma is also a neural crest–related tumour, this
association seems to be likely although rare.24 Additionally, a
patient with MTC had a second tumour diagnosed as lung
squamous cell carcinoma. Rotondi et al.25 describe a familial
association between MTC and bronchial carcinoid tumours and
papillary thyroid carcinoma. An association between MTC and
breast cancer was described by Andry et al.26 in 1993.
Individualizing ATA risk-A RET533 carriers
The ATA created a new classification system (groups A, B, C
and D) for RET mutations based on the biological aggressiveness
of the MTC.27 RET533 has been classified as the ‘least risk’

(a) (b)

(c) (d)

(e)

Fig. 4 Representative histopathological findings of tissues from the RET533 FMTC patients. After surgery, a RET533 relative was diagnosed with classic
medullary thyroid carcinoma (MTC) with a spindle cell variant (a) (H&E, 9 400) and surrounding C-cell hyperplasia (CCH) (b). MTC and CCH
presenting with strong positive immunostaining for calcitonin (b) (9 100) and a corresponding image with CCH and immunostaining for CEA
(9 100) (c). (d) Cervical lymph node metastases from MTC (H&E, 9 100) and (e) a pheochromocytoma of the right adrenal (H&E, 9 100).

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Clinical Endocrinology (2014), 80, 235–245

group (i.e. ATA-A), for which they suggest that prophylactic
total thyroidectomy can wait beyond 5 years of age in the setting
of a normal annual calcitonin level, benign annual neck US and
a less aggressive MTC family history. Our 10-year clinical update
supports a more sensible timing for prophylactic surgery.
Because we did not find any carrier less than 21 years of age
with MTC, we recommend that thyroidectomy can be per-
formed after the age of 10 if the other tests are normal. Besides,
the natural history of Brazilian RET533 affected relatives rein-
forces that the tumour progression is greatly variable, but
towards a good prognosis. We consider that prophylactic thy-
roidectomy in identified gene carriers under 20 years of age is
reasonable; however, RET533 carriers and patient’s management
should rely more on an individual’s clinical variables.
We agree with the recommendation by the last consensus that
the basal sCT level and age seem to be good parameters for
determining the timing of a prophylactic surgical procedure in
familial MTC patients. However, in this case, we disagree with
the sCT cut-off value of 40 pg/ml for the decision to undergo a
prophylactic or therapeutic approach. Our RET533 affected
relatives, who underwent surgery without using this cut-off, all
fortunately have had a good outcome. Likely, having an experi-
enced surgical team overcame most of the biochemical and pre-
surgical risk factors, such as the type of mutation, the calcitonin
level, the presence of LN metastases or local extension and
tumour size, leading to a higher rate of cure or controlled
disease.
The youngest RET533 patient with confirmed MTC was
21 years old and the youngest patient with CCH was 5 years old
at the time of diagnosis. Their favourable postsurgical outcomes
thus far suggest that these patients have a great chance of being
cured and reinforce the safety of the ATA recommendation
discussed above, which states that thyroidectomy can be performed
after 5 years of age if the sCT level and neck US are normal.
Our series of patients with an ATA-A risk mutation also
confirms the benign course of the disease, even when they
undergo thyroidectomy at an older age. A French group
described the follow-up of 15 patients with a codon 790 muta-
tion,28 in which the 5 patients (45–76 years) who underwent a
thyroidectomy and LN (LN) dissection were cured. The same
group also published a large study in which 170 patients with a
RET germline mutation underwent a thyroidectomy before age
21.29 One-third of these patients had an ATA-A (19!5%) or
ATA-B (13!5%) RET mutation. None of the patients with a
preoperative CT < 31 ng/ml had persistent or recurrent disease.
In addition, a tumour <10 mm and the absence of LN metasta-
sis (N0) were good predictors of disease-free survival, which
was 100% for patients with an ATA-A mutation and 95!5% for
patients with an ATA-B mutation. Therefore, the authors admit
that the decision to perform thyroidectomy should consider
other parameters, such as the parents’ preference for the timing
of surgery and different practices among institutions. In
another American study, the youngest age of MTC in the ATA-
A and ATA-B carriers was 15 years;30 thus, this classification
was useful in determining the appropriate time for a prophylac-
tic thyroidectomy.
© 2013 John Wiley & Sons Ltd
Clinical Endocrinology (2014), 80, 235–245
Long-term follow-up of a large RET533 MEN-2 family 243
Primary hyperparathyroidism (PHPT) has been reported in
15–30% of patients with MEN2A,27 and in two large studies, the
median age at the time of diagnosis was 38 years.31,32 In our
series, none of the RET533 patients have been diagnosed with
PHPT. Based on our experience that no RET533 carrier has
developed PHPT, we suggest that for these patients, PHPT
screening should begin at 20 years of age and be repeated every
5 years. In summary, RET533 patients when followed asymp-
tomatic should be still regularly screened for PHEO and PHPT
every 5 years, as it is suggested to other low-penetrance muta-
tions causing MEN2A.
The most useful markers when following MTC patients are
still the sCT and CEA levels.33 Notably, in this report, the levels
of these two biochemical markers strongly correlated with the
MTC stage. Therefore, CT and CEA measurements have been
incorporated routinely as part of the initial evaluation and the
follow-up of RET533 carriers. Post-thyroidectomy carriers with
elevated sCT systematically undergo imaging studies to stage
and/or exclude distant metastatic disease. So far, none of these
patients have developed distant metastases.
We believe that our RET533 clinical update reinforces the need
for individualized, risk-adjusted patient evaluation and, as a result,
provides a strong argument for designing better risk assessment
criteria and management strategies for patients with MEN2 syn-
drome. Not only for thyroid cancers but also oncology as a whole,
following the ongoing clinical outcome has become an important
tool for healthcare decision-making, especially for identifying
treatments that target specific phases of the disease. Therefore,
using an individualized assessment, we can clearly benefit both the
patients and the health system by encouraging a deeper physician–
surgeon participation in this process and most likely generating
better cost-effectiveness. Additional studies on other large MEN2A
families are needed to help determine the criteria for the ongoing
stratification of patients with MTC.
Acknowledgements
The authors thank the team from the Laboratory of Molecular
and Translational Endocrinology, especially Adriana Madeira-
da-Silva, Ilda Kunii, Jo~ao R. M. Martins, Conceic!~ao Mamone,
Reinaldo Furlanetto, Luiza Matsumura, Jairo Hidal and Anelise
Nogueira, for helpful discussions and the Head and Neck Sur-
gery team, particularly Drs. Flavio C. Hojaij, Marcel Palumbo
and Fabio Brodskyn. We are grateful to Mariana Oliveira,
Fernando Soares and Gilberto Furuzawa for technical assistance
and Angela M. Faria and Yeda Queiroga for efficient laboratory
administration. In addition, we would like to acknowledge Drs.
Nildete Gomes, Jos!e R. V. Podest!a, Antonio Pinto and Fernando
Pretti, for clinical and histological information about the family.
We also thank Gilmar Miranda from Siratec Ltda for imaging
design. We express appreciation to all family members for their
prompt collaboration. The authors’ research grant was sup-
ported by the S~ao Paulo State Research Foundation/FAPESP
(2006/60402-1 and 2010/51547-1 to R.M.B.M. and M.R.D.S. and
2006/54922-2 to J.M.C.) and the Fleury Group (12518). S.C.L is
supported by a fellowship grant from FAPESP (2009/50575-5).
244 P. S. Signorini et al.
R.M.B.M. is an investigator of the Fleury Group. R.M.B.M. and
J.M.C. are investigators of the Brazilian National Research
Council (CNPq).
Conflict of interests
All authors have nothing to disclose.
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 Genome-Wide Copy Number Analysis in a Family with
p.G533C RET Mutation and Medullary Thyroid
Carcinoma Identified Regions Potentially Associated
with a Higher Predisposition to Lymph Node
Metastasis

Aline N. Araujo, Lais Moraes, Maria Inez C. França, Hakon Hakonarson, Jin Li,
Renata Pellegrino, Rui M. B. Maciel, and Janete M. Cerutti
Genetic Bases of Thyroid Tumors Laboratory, Division of Genetics, Department of Morphology and
Genetics, Universidade Federal de São Paulo, SP, Brazil (A.N.A., L. M., J.M.C.); Laboratory of Molecular
and Translational Endocrinology, Division of Endocrinology, Department of Medicine, Universidade
Federal de São Paulo, SP, Brazil (M.I.C.F., R.M.B.M.); Center for Applied Genomics, The Children’s
Hospital of Philadelphia, Research Institute, Philadelphia, PA, USA (H.H., J.L., R.P.); Department of
Pediatrics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA (H.H.).

Context: Our group described a p.G533C RET gene mutation in a large family with multiple
endocrine neoplasia type 2 (MEN 2) syndrome. Clinical heterogeneity, primarily associated with
the presence of lymph node metastases, was observed among the p.G533C-carriers.

Objective: The aim of this study was to use single nucleotide polymorphism (SNP)-array tech-
nology to identify copy number variations (CNVs), which are present in the constitutional DNA
and associated with the established clinical and pathological features of aggressive medullary
thyroid carcinoma (MTC), primarily presence of lymph node metastasis.

Design: Fifteen p.G533C carriers with MTC were chosen for the initial screening. The subjects
were divided into 2 groups according the presence (n!8) or absence (n!7) of lymph node
metastasis. Peripheral blood DNA was independently hybridized using a Genome-Wide SNP
Array 6.0 platform. The results were analyzed using both Genotyping Console and PennCNV
software. To identify the possible candidate regions associated with the presence of lymph node
metastasis, cases (metastatic MTC) were compared with controls (non-metastatic MTC). The
identified CNVs were validated by quantitative polymerase chain reaction (qPCR) in an extended
cohort (n!32).

Results: Using 2 different algorithms, we identified 9 CNVs regions that may contribute to
susceptibility to lymph node metastasis. The validation step confirmed that a CNV loss impacting
the FMN2 gene was potentially associated with a greater predisposition to lymph node metas-
tasis in this family (P!0.0179). Finally, we sought to investigate whether the development of
lymph node metastasis might not depend upon a single CNV but rather a combination of various
CNVs. These analyses defined a CNV pattern related to a more aggressive phenotype in this
family, with CNV deletions being enriched in the metastatic group (P!0.0057).

Conclusion: Although hereditable specific RET mutations are important to determine cancer risk,
germline CNVs in disease-affected individuals may predispose them to MTC aggressiveness.

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations:

Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received July 29, 2013. Accepted February 27, 2014.

doi: 10.1210/jc.2013-2993 J Clin Endocrinol Metab jcem.endojournals.org 1

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2 Genome-Wide Copy Number Analysis of MTC J Clin Endocrinol Metab

M edullary thyroid carcinoma (MTC) stems from the

neural crest-derived calcitonin (CT)-secreting
parafollicular C cells of the thyroid gland. MTC represents
at onset and the aggressiveness of MTC, as measured as
the presence of lymph node metastasis.
It has been speculated that other genetic modifier loci
3%–10% of all thyroid cancers. Although most (75%– can be potentially associated with milder phenotypes and
80%) patients exhibit sporadic disease, MTC can occur as even intrafamilial phenotypic variability. Current studies
a part of an autosomal dominant inherited multiple en- have demonstrated that copy number variations (CNVs)
docrine neoplasia (MEN) type 2 (MEN 2) syndrome are the most prevalent type of structural variation in the
(20%–25%) (Reviewed by (1, 2)). Three distinct clinical human genome and represent a significant source of ge-
MEN 2 subtypes have been identified. MEN 2A is the most netic diversity (12). CNVs are classified as DNA segments
common subtype and is characterized by the presence of that are between 1 kb and several megabases in size and are
MTC, pheochromocytoma (PHEO), and primary hyper- present at a variable copy number in comparison with a
parathyroidism (PHPT). MEN 2B is characterized by the reference genome (13–15). Although they were initially
presence of aggressive MTC, PHEO, marfanoid habitus, described as benign genetic changes that did not cause a
distinct mucosal neuromas on the tongue, lips, and sub- clinically recognized phenotype, heritable genomic CNVs
conjunctival areas and diffuse ganglioneuroma of the gas- can modulate gene expression and predispose an individ-
trointestinal (GI) tract. Familial MTC (FMTC) is charac- ual to disease (14, 16). Although CNVs are found to be
terized by the presence of multigenerational transmission common in the genome, their associations with prognosis
of MTC in families in which no members exhibit adrenal factors, such as the presence of lymph node metastasis,
or parathyroid gland involvement (1, 3). tumor grade, tumor size, and early age of onset, are cur-
The REarrangement during Transfection (RET) gene, rently understudied.
In the present study, using the Affymetrix Genome-
which maps to chromosome 10q11.2 and encodes a trans-
Wide Human SNP array 6.0, which features more than 1.8
membrane tyrosine kinase receptor, is the only gene
million probes, we investigated whether CNVs contribute
known to be associated with MEN 2 syndromes. To date,
to a greater predisposition to the presence of lymph node
over 200 germline mutations in the RET gene have been
metastases in a family with the MEN 2A phenotype and
identified in MEN 2 families (Reviewed by (1)). It is well
p.G533C RET mutation.
known that there is a strong genotype-phenotype corre-
lation, as specific RET codon mutations correlate with the
phenotypic expression of hereditary MTC. Indeed, using
Subjects and Methods
available evidence from the literature and expert opinions,
the American Thyroid Association developed a specific Subjects
MTC Clinical Guideline (3). However, clinical heteroge- Approximately 432 family members were previously tested
neity has been observed among families or even within for the RET p.G533C mutation; 119 members were RET carri-
families with the same RET mutation. ers, and 60 patients underwent thyroidectomy. Histological ex-
aminations confirmed MTC in 46 patients. Over a mean 10-year
Previously, we described a large Brazilian family with
follow-up, lymph node metastases were confirmed in 13 patients
FMTC and the RET p.G533C mutation in exon 8 of the and were absent in 33 (11). For the current study, patients were
RET gene (4). Later, this RET mutation was observed in selected based on the clinical aggressiveness of the MTC, mea-
2 Greek kindred with FMTC (5) and in a North American sured as the presence/absence of lymph node metastasis. The
family (6). A more complex scenario has emerged because inclusion criteria included patients who had undergone total thy-
roidectomy and central neck dissection, were treated only on the
PHEO was identified as the first clinical manifestation in basis of clinical and/or biochemical diagnosis (elevated calci-
unrelated Greek families with RET p.G533C mutations. A tonin levels and/or a palpable thyroid nodule on physical exam-
follow-up of the Greek patients confirmed the presence of ination or visualized by ultrasound, computed tomography (CT)
MTC, providing the first evidence that the RET p.G533C [CT] scans or magnetic resonance imaging (MRI) [MRI)]) and
mutation leads to MEN 2A phenotype (7, 8). Recently, were followed-up annually with clinical examination, basal/
stimulated calcitonin, urinary catecholamine, serum ionized cal-
one of the Brazilian family members who was a RET cium, PTH, and CT or MRI.
p.G533C carrier developed PHEO. Thus, our results fur- According to the inclusion criteria, the p.G533C carriers
ther support the evidence that this mutation is associated (n!15) were sorted into 2 groups. The first group comprised
with MEN 2A syndrome (9). patients with metastatic MTC (n!8), with a mean age of onset
In addition to the variability in phenotype observed of 48.63 years (standard deviation [SD]"13.05). The second
group included patients with nonmetastatic MTC (n!7), with a
among families with the RET p.G533C mutation, intra- mean age of onset of 34.14 years (SD " 6.12). The PCR exper-
familial variability has also been observed (4, 10, 11). This imental validation set included the initial individuals and 17 ad-
phenotypic variability is largely related to the patient’s age ditional p.G533C carriers, including both patients with meta-

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doi: 10.1210/jc.2013-2993 jcem.endojournals.org 3

static MTC (n!9) and nonmetastatic MTC (n!23). The mean
age at onset for the metastatic and nonmetastatic groups was
47.44 years (SD " 12.71; range, 21– 62 years) and 41.91 years
(SD " 12.15; range, 23–72), respectively (Table 1). Ethical ap-
proval for this study was obtained from the Federal University of
São Paulo Research Ethics Committee.
Previously reported clinical and pathological features, such as
the age at onset (defined as the age at which the clinic-patho-
logical diagnosis of MTC was made), gender, tumor size, local
invasion (defined as invasion of adjacent structures), presence of
lymph node metastasis, pTNM, and serum Calcitonin (sCT),
were assessed as previously described (4, 9, 11).
DNA Isolation
Each patient had previously contributed blood at the time of
the RET screening diagnosis. The DNA was extracted using a
standard phenol/chloroform method, as described previously
(4). All stored DNA samples were requantified using a Nano-
Drop spectrophotometer (Thermo Fisher Scientific Inc.,
Waltham, MA). DNA quality control (QC) gel analysis was per-
formed. Only samples with high molecular weight genomic DNA
were used.
Copy Number Variation Analysis
The Affymetrix Genome-Wide Human SNP Array 6.0 (Affy
6.0) is an array platform that aims to perform both high-density
SNP genotyping and high-resolution CNV discovery simultane-
ously (Affymetrix, Santa Clara, CA, USA). The array is designed
to interrogate 1.8 million genetic markers, containing 906,600
single nucleotide polymorphisms (SNPs) and 946,000 copy
number probes. Fifteen patients were genotyped using Affy 6.0.
DNA (250 ng) from each patient was digested using restriction
enzymes. Linker ligation, amplification, fragmentation, label-
ing, and array hybridization were performed following the man-
ufacturer’s standard protocol (Affymetrix). To exclude experi-
mental errors, prior to hybridization, only samples that passed
through the 3 Affy 6.0 Quality Control (QC) checkpoints were
hybridized on GeneChip arrays. Arrays were scanned on the
Affymetrix GeneChip Scanner 3000 7G. The sample images
(CEL. Files) were acquired using Affymetrix GeneChip Com-
mand Console software (AGCC).
CNV detection methods
To identify germline CNVs that may be potentially associated
with a higher risk for the development of a more aggressive MTC
in patients with MEN 2A and RET p.G533C carriers, 8 meta-
static MTC (cases) were compared with 7 nonmetastatic MTC
(controls). The Affy 6.0 data from the 270 HapMap samples
were used as a reference genome.
To maximize the CNV discovery, CNV calling was per-
formed using 2 algorithms, which are freely available and widely
used for CNV analysis of data generated using the Affy 6.0 plat-
form. The complete dataset was initially analyzed by visual in-
spection using the Genotyping Console 4.1 (GTC) and Chro-
mosome Analysis Suite (ChAS) software (Affymetrix).
Genotyping calls (SNPs and CNVs probes) were generated using
the Birdseed3 v1 or v2 algorithm. All samples passed the soft-
ware’s QC with an average call rate of 98.6%, which is higher
than the default threshold of 86%. The calculation of the copy
number (CN) state was established on the Genotyping Console
using the Canary algorithm. To identify gains (amplification) or
losses (deletion), each sample’s copy number was estimated in
comparison with the HapMap diploid genotypes. To avoid in-
dividual heterogeneities, we considered only those events (dele-
tion/amplification) occurring in at least 3 of 8 patients from the

Table 1. Clinical and Pathological Features of the Patient Set.

Metastatic Non-Metastatic
MTC (n ! 9) MTC (n ! 23) Total (n ! 32) P value
Age of Onset* 47.44 " 41.91 " 43.47 " 0.1361
(mean"SD) 12.71 12.15 12.36
Gender
Male 3/9 (33%) 11/23 (47.83%) 14/32 (43.75%) 0.6942
Female 6/9 (66%) 12/23 (52.17%) 18/32 (56.25%)
Tumor Size 1.756 " 0.8432 " 1.108 " 0.0023*
0.6912 0.8139 0.8766
Local Invasion
Yes 1/9 (11%) 1/21 (4.76%) 2/30 (6.67%) 0.5172
No 8/9 (89%) 20/21 (95.24%) 28/30 (93.33%)
Stage
I and II 0/9 (0%) 13/15 (86.67%) 13/24 (54.17%) #0.0001*
III 9/9 (100%) 2/15 (13.33%) 11/24 (45.83%)
Pheochromocytoma
Yes 1/9 (11.11%) 0/17 (0%) 1/26 (3.85%) 0.3462
No 8/9 (88.89%) 17/17 (100%) 25/26 (96.15%)
Preoperative sCT 866.6 " 310.2 " 481.4 " 0.0042*
levels 602.6 453.0 556.6
(ng/mL; mean "
SD)
sCT levels after TT 49.08 " 3.565 " 17.69 " 0.0010*
(ng/mL; mean " 76.82 6.523 46.63
SD)

*Statistically significant.

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4 Genome-Wide Copy Number Analysis of MTC
metastatic MTC group as allelic alterations (!30% of cases)
(17).
Additionally, the dataset was also assessed using the
PennCNV software (University of Pennsylvania, Philadelphia,
and The Children´s Hospital of Philadelphia, USA) (18). The
Affymetrix CEL files were preprocessed according to the proto-
cols on the PennCNV website (http://www.openbioinformatic-
s.org/penncnv/penncnv tutorial affy gw6.html) to calcu-
late the log R ratio (LRR) and the B allele frequency (BAF). The
Hapmap CEU samples genotyped on Affy 6.0 were used with our
samples to generate canonical genotype clusters. CNV calling
was conducted with PennCNV, a method based on a Hidden
Markov model (HMM), which correlates the found intensities
with the copy number (CN) state of each locus. PennCNV called
CNVs containing at least 3 SNPs and/or CNV probes that were
deleted (losses) or duplicated (gains) in our dataset. The
affygw6.hmm and affygw6.hg18.pfb files implemented in
PennCNV were used for CNV calling. Moreover, using Par-
seCNV (19), we converted the CN stats to PLINK ped files. Using
these ped files, we generated the statistical p-values (derived from
Fisher’s exact test) implemented in PLINK (20) to compare the
copy number-based allele frequency differences in the metastatic
vs the nonmetastatic cases.
A copy number state of 2 per individual was considered nor-
mal. To determine the copy number changes, we used the HMM
model, which performs segmentation of the log2 ratio intensity
data and, for each segment, predicts copy number up to 5 states
representing the following: homozygous deletion (CN state ! 0),
hemizygous deletion or single copy loss (CN state ! 1), neutral
copy number or normal diploid (CN state ! 2), single copy gain
(CN state ! 3), and amplified (CN state ! 4).
Copy Number Validation
Quantitative real-time PCR (qPCR) was used to validate the
copy number state for CNVs exhibiting differences between the
cases and controls. In this validation step, a larger series of
p.G533C-carriers with MTC (n!32) were tested (Table 1).
Primers, designed using the Primer3 (21) software, were located
within an overlapping CNV region (ie, a common region for all
patients who tested positive for the specific CNV, supplemental
Figure 1, supplemental Table 1). If the overlapping CNV region
encompassed a gene, the primer was designed within the gene
region.
The PCR reaction was performed in triplicate using 30 ng of
each gDNA sample in a 12-"l PCR volume containing 1X SYBR
Green PCR Master Mix (PE Applied Biosystems, Foster City,
CA, USA) and 3 pmol of each specific primer for the target locus/
gene. All PCR reactions were performed using a standard PCR
program in the ABI Prism 7500 Sequence Detection System (PE
Applied Biosystems). ACTB (#-actin) was used as a control for
normalization (reference gene). The copy number was calculated
using the comparative Ct method (2(-$Ct), where $Ct!Cttest sam-
ple–Ctreference control) (22). Standard curves were initially per-
formed to determine the PCR efficiency (E) for each primer pair
using the established equation: E!(10-1/slope –1)x100 (23). A
standard curve slope of –3.2 to –3.6 indicated 90 to 110% effi-
ciency, which allowed comparisons between different reactions.
Combination of Multiples CNVs
It has been suggested that a disease phenotype may depend
upon a combination of several CNVs rather than a single CNV.
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J Clin Endocrinol Metab
We thus evaluated whether multiple CNVs may exhibit a specific
pattern associated with a more aggressive MTC. To this end, we
used the CN states obtained from qPCR of each patient (n!32)
and analyzed the differences between deletion and amplification
frequencies in the metastatic and nonmetastatic groups.
Statistical Analysis
We compared the qualitative clinical features (gender, local
invasion, MTC stage and pheochromocytoma occurrence) be-
tween the metastatic and nonmetastatic group using the 2-sided
Fisher’s exact test. We also compared the association between
the presence of an observed CNV and metastasis, gender, local
invasion and stage by the 2-sided Fisher’s exact test. For the
quantitative clinical features, the Shapiro-Wilk test was used to
verify the normality of distribution. As the samples did not ex-
hibit a normal distribution, a 2-tailed Mann-Whitney test was
performed to compare the clinical, biochemical, and patholog-
ical features between the metastatic and nonmetastatic groups
(age of onset, tumor size, preoperative and postoperative calci-
tonin level). The Mann-Whitney test was also used to correlate
the presence of a specific CNV with the tumor size and preop-
erative and postoperative calcitonin level. In the combination
analysis, Fisher’s exact test (2-sided) was used to analyze the
difference between the deletion and amplification frequencies in
the metastatic and nonmetastatic groups.
StatView 4.5 (Abacus Concepts, Berkeley, CA, USA) and
GraphPad Prism 5.01 (GraphPad Software, La Jolla, CA, USA)
software were used for the data analyses, and P # .05 was con-
sidered to be statistically significant.
Complementary Analysis
To better understand the biological functions of the genes
related to the CNVs identified, we uploaded the gene names/
symbols to the NIH Database for Annotation, Visualization and
Integrated Discovery (DAVID) data analysis suite (http://david-
.abcc.ncifcrf.gov). In particular, we searched for overrepresen-
tation in Gene Ontology (GO) classification, such as Cellular
Component and Biological Process. The DAVID functional clus-
tering tool was not used because the number of selected genes
was small to generate enrichment values and pathways.
Results
Identifying Lymph Node Metastasis-associated
CNVs
To identify CNVs possibly associated with a higher
predisposition to lymph node metastasis, we compared the
metastatic with the nonmetastatic group. Using GTC and
ChAS software (Affymetrix), based on GRCh37/hg19, we
identified 8 regions with CNV present in at least 3 of 8
metastatic MTC cases (%30% of cases) and rare or absent
in most nonmetastatic MTC cases (Table 2). Using
PennCNV, which use genomic positions of the markers
correspond to the human reference NCBI36/hg18, we
identified 3 CNV regions that were significantly different
in metastatic (cases) vs nonmetastatic (control). Statistical
significance for each CNV was assessed by Fisher’s exact
doi: 10.1210/jc.2013-2993 jcem.endojournals.org 5

Table 2. Relevant CNVs detected: A comparison of the Metastatic and Non-metastatic Groups.
Alteration Detected

Cytoband CNVR Coordinates Size Marker Reference Genes Distance Metastatic Non-Metastatic P value
(kb) Count from Gene (n ! 8) (n ! 7)
(kb)
Genotyping Losses 1q21.3 chr1:152555175–152586528 31.4 23 LCE3C (NM 178434.2) 0 3/8 (37.5%) 0/7 (0%)
Console LCE3B (NM 178433.1)
(GRCh37/hg19)
1q31.3 chr1:196738610 –196802060 63.4 78 CFHR3 (NG 015993.1) 0 4/8 (50%) 2/7 (28.57%)
CFHR1 (NG 013060.1)
1q43 chr1:240391234 –240394127 2.9 8 FMN2 (NM 020066.4) 0 4/8 (50%) 0/7 (0%)
2q22.3 chr2:146865961–146866922 1.0 11 PABPC1P2 (NR 026904.1) 477.7 4/8 (50%) 1/7 (14.29%)
3q22.1 chr3:129775845–129795080 19.2 45 ALG1L2 5.6 7/8 (87.5%) 1/7 (14.29%)
(NM 001136152.1)
Gains
2p11.2 chr2:86948834 – 86970406 21.6 4 RMND5A (NM 022780.3) 0 4/8 (50%) 1/7 (14.29%)
7q34 chr7:142476706 –142490975 14.3 35 PRSS3P2 (NR 001296.3) 0 3/8 (37.5%) 0/7 (0%)
PRSS2 (NG 008322.1)
11q11 chr11:55353110 –55447453 94.3 54 OR4C11 0 5/8 (62.5%) 2/7 (28.57%)
(NM 001004700.2)
OR4P4 (NM 001004124.1)
OR4S2 (NM 001004059.2)
OR4C6 (NM 001004704.1)
PennCNV Losses
(NCBI36/hg18)
1q31.3 chr1:194994473–195068695 74.2 94 CFHR3 (NG 015993.1) 0 3/8 (37.5%) 0/7 (0%) 0.0447*
CFHR1 (NG 013060.1)
2q22.3 chr2:146580874 –146583404 2.5 24 PABPC1P2 (NR 026904.1) 477.7 4/8 (50%) 0/7 (0%) 0.0027*
4q26 chr4:115394772–115403839 9.1 30 ARSJ (NM 024590.3) 274.4 3/8 (37.5%) 0/7 (0%) 0.0185*

*Statistically significant. The highlighted CNVs were identified using both CNV calling algorithms.

test (Table 2). Combining the results from the 2 algorithms tastasis (Figure 2). The CN states were classified, as pre-
yielded 9 metastatic-specific CNVs (6 losses and 3 gains) viously mentioned. Each patient was classified according
(Table 2, supplemental Figure 2). Important, CNV regions to the occurrences of amplification and deletion (Patient
on chromosome 1 (1q31.3) and chromosome 2 (2q22.3) CNV consensus; Figure 2). A different pattern was ob-
were identified by both algorithms. Most CNVs observed served between patients from the metastatic and nonmeta-
were copy number losses (66%), and all identified CNVs static groups (P ! .0085). CNV deletions were signifi-
are listed in the Database of Genomic Variants. Overall, cantly enriched in the metastatic group (P ! .0057),
most CNV regions (45%) were associated with multiple whereas amplification was enriched in the nonmetastatic
genes located within a single CNV region, few CNVs
group (P ! .0179).
(22%) contain only 1 RefSeq, and some (33%) CNV re-
Because the RMND5A gene exhibited an opposite pat-
gions mapped closed to a gene (Distance from Gene % 0)
tern compared with other CNVs, being amplified in the
(Table 2).
metastatic group and deleted in the nonmetastatic group,
Validation of CNV Losses and Gains by qPCR the gene was excluded from this analysis.
We performed qPCR validation to confirm the individ-
ual copy number variants in a large series of RET Gene Ontology and Functional Annotation of CNV
p.G533C-carriers with MTC (Table 1). Although the al- DAVID was used to determine the gene ontology
gorithms of the SNP array analysis allowed the prediction (http://david.abcc.ncifcrf.gov/) for those CNVs that con-
of CN States: 0, 1, 2, 3, and 4 (Figure 1, A and C), to tained genes. The most representative GO for each gene
perform the statistical analysis, the samples were classified symbol is presented in supplemental Table 2.
as loss (CN state ! 0 and 1), normal (CN ! 2), and gain
(CN state ! 3 and 4) (Table 3). An analysis of each region
revealed that the loss of the 1q43 region, which contains Discussion
the FMN2 gene, was significantly associated with the pres-
ence of lymph node metastasis (P ! .0179) (Table 3; Fig- Evidence reported in the literature clearly demonstrates
ure 1, A and B). We additionally verified the association of that CNVs play an important role in phenotypic expres-
CNVs with clinical-pathological features (gender, tumor sion. Consequently, although hereditable specific RET
size, local invasion, MTC stage, serum calcitonin level). mutations are important in determining the MTC risk in
We observed an association between the loss of the 1q21.3 families with MEN 2 syndrome, germline CNVs in dis-
region, which comprise the Late Cornified Envelope Pro- ease-affected individuals may predispose to MTC
tein 3C (LCE3C) gene, and tumor size (P ! .0222) (Table aggressiveness.
4; Figures 1, C and D).
The aim of the current study was to search for genomic
Combination of multiple CNVs may be correlated regions that might contribute to the development of lymph
with the development of lymph node metastasis node metastasis in a MEN 2A family in the context of the
Furthermore, we investigated whether multiples CNVs p.G533C RET mutation. To identify the possible candi-
could be associated with the presence of lymph node me- date CNV regions associated with a higher predisposition

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6 Genome-Wide Copy Number Analysis of MTC J Clin Endocrinol Metab

Table 3. The frequencies of 9 Selected CNVs in the 32 MTC Patients, Obtained Using qPCR.
Alteration Detected
Locus/Gene CN Type Metastatic (n Non-metastatic P value
! 9) (n ! 23)
LCE3C Loss 7/9 (77.78%) 10/23 (43.48%) 0.1220
CFHR1 Loss 2/9 (22.22%) 3/23 (13.04%) 0.6042
FMN2 Loss 8/9 (88.89%) 9/23 (39.13%) 0.0179*
2q22.3 Loss 7/9 (77.78%) 13/23 (56.52%) 0.4224
3q22.1 Loss 4/9 (44.44%) 4/23 (17.39%) 0.1760
4q26 Loss 7/9 (77.78%) 13/23 (56.52%) 0.4224
RMND5A Gain 6/9 (66.67%) 16/23 (69.57%) 1
PRSS3P2 Gain 5/9 (55.56%) 15/23 (65.22%) 0.6960
OR4S2 Gain 2/9 (22.22%) 13/23 (56.52%) 0.1220
Fisher’s exact test. *statistically significant.

to lymph node metastasis in this family, a high-resolution tive analysis to identify overlapping regions as candidates
SNP array was used to screen metastatic and nonmeta- for further evaluation, given that multiple estimates from
static MTC patients. CNV calling was performed using different software/algorithms for the same CNV event in-
Genotyping Console 4.1 and Chromosome Analysis Suite creases the confidence of the calls made.
software and PennCNV software, which use distinct al- In this study, we identified 9 CNV regions that were
gorithms. The comparison of CNV events in a dataset recurrent in patients with metastasis and, therefore, po-
using different methods of detection calls can be useful in tentially associated with higher predisposition to lymph
assessing the accuracy and consistency of the algorithms node metastases. Two CNV regions, located at chromo-
used. However, in our data, we did not intend to focus on somes 1q31.3 and 2q22.3, were identified by both Geno-
software comparisons; instead, we performed a conserva- typing Console and PennCNV.

Figure 1. An illustration of the Chromosome Analysis Suite software (Affymetrix) used to visualize the Genotyping Console (Affymetrix) results.
A. The figure presents a representation of the patient’s data for a candidate CNV deletion on 1q43, including the Log2 ratio, which represents the
normalized intensity related to a reference (diploid) and the copy number state (HMM-derived) based on Log2. B. The copy number state of the
FMN2 gene, which is localized on 1q43, obtained by qPCR for all patients. C. An illustration of the result obtained for a candidate CNV deletion,
which is localized on 1q21.3. D, The mean size of tumor was greater in patients who had LCE3C loss (1.371cm) than those without LCE3C loss
(0.6192 cm).

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doi: 10.1210/jc.2013-2993 jcem.endojournals.org 7

Table 4. The Correlation between LCE3C Loss and the Clinical and Pathological features.
Patients without LCE3C Patients with LCE3C P
Variable deletion (n ! 15) deletion (n ! 17) value
Gender
Male 6/15 (40%) 8/17 (47.06%) 0.7345
Female 9/15 (60%) 9/17 (52.94%)
Tumor Size (cm; 0.6192 " 0.3956 1.371 " 0.9123 0.0222*
mean"SD)
Local Invasion
Yes 0/14 (0%) 2/16 (12.5%) 0.4851
No 14/14 (100%) 14/16 (87.5%)
Stage
I 5/12 (41.67%) 5/16 (31.25%) 0.6979
II 4/12 (33.33%) 3/16 (18.75%) 0.4184
III 3/12 (25%) 8/16 (50%) 0.2530
Preoperative sCT Levels 311.1 " 309.8 627.4 " 681.8 0.3961
(ng/mL;
mean"SD)
sCT Levels after TT 15.59 " 39.33 19.94 " 54.83 0.4134
(ng/mL;
mean"SD)
sCT, serum calcitonin; TT, total thyroidectomy.
*Statistically significant.

It has been suggested that CNVs identified from SNP cellular disorganization and, therefore, can contribute to
genotyping data must be validated as completely as pos- the metastatic process, requires further investigation. An-
sible in order to avoid false negative and false positive other question that remains to be answered is whether
identification of CNV (24). Therefore, the identified similar effects occur in other families with MEN 2 syn-
CNVs were further validated by genomic qPCR in an ex- drome and other known pathogenic mutations in the RET
tended sample set. gene.
A CNV region located within the FMN2 gene was We also observed that the LCE3C loss is associated
found to be consistently deleted in the patients from the with a larger tumor size. The deletion of this gene has
metastatic group and in few patients from nonmetastatic previously been associated with psoriasis (27–29), with a
group. There is relatively little information on the function possible role in the process of keratinization (30).
of the FMN2 protein. The protein was initially described Previous studies using CGH or array-CGH investigated
as a protein that plays a role in actin cytoskeletal organi- tumor-associated copy number changes in sporadic and
zation and/or the establishment of cell polarity during mei- hereditary MTC. The authors observed that most somatic
otic processes. FMN2 was recently determined to be CNVs exhibited allelic losses. The most frequent allelic
highly induced by ARF in a p53-independent manner (25). losses in occurred at 7q36, 12p13.31, 13q12, and
The presence of FMN2 protein was found to inhibit p21 19p13.3–11(17). The authors’ findings underscore the
degradation, resulting in increased p21 levels and, conse- significance of the allelic losses and the possible roles of
quently, cell-cycle arrest. After the increased expression of tumor suppressor genes together with RET mutations in
p21 causes the cell-cycle arrest, FMN2 deletion could re- MTCs.
sult in p21 loss of expression and, consequently, cell-cycle Somatic chromosomal imbalances have also been ob-
progression. Additionally, it has been suggested that served in sporadic or hereditary MTC. Ciampi et al re-
FMN2 may affect other stages of the cell cycle. The FMN2 ported that approximately 30% of MTC cases harbored
gene was also associated with tumor susceptibility in the RET copy number alterations in both hereditary and spo-
colon (26). This region is highly conserved between mice radic MTC. In particular, chromosome 10 copy number
and humans, which is consistent with the hypothesis that gains were more frequently observed in sporadic MTC,
this gene may play an important role in tumorigenesis. whereas RET gene amplification was observed in hered-
The role of FMN2 in thyroid cancer is unknown, and itary cases (31). Another study demonstrated that the
this report is the first to detail an association with the RET-mutated sporadic tumors exhibited the greatest
metastatic process. Whether this deletion could lead to the number of imbalances. The most common imbalances in
expression of a nonfunctional or deleterious truncated both sporadic and hereditary MTC were losses at 1p,
protein or spliced variant isoform, which ultimately causes 3q26, 9q13, and 22q (32). Although the identified regions

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8 Genome-Wide Copy Number Analysis of MTC J Clin Endocrinol Metab

differed from those identified in our study, these findings the metastatic process. One of the key points of our study
are consistent with ours, as they also observed more de- is that we used a high-resolution array platform that de-
letions than amplifications. tects CNV at a higher resolution than conventional chro-
With respect to metastatic process, other groups have mosome-based CGH.
used array-CGH to map chromosome imbalances in pri- It has been suggested that a phenotype may not depend
mary MTC and their respective metastases and MTC cell upon a single CNV but rather on a combination of several
lines (33). Although the number of chromosomal altera- CNVs. Thus, a specific CNV is likely to contribute to small
tions increased in metastasis, the authors did not observe or moderate phenotypic effects, whereas combined CNVs
any specific chromosomal gains or losses associated with could result in more considerable effects (24, 34). In our
study, the analysis of multiple CNVs was able to define the
pattern of CNVs related to the presence of lymph node
metastasis in this family. We identified the regions in
which losses were enriched in the metastatic group, sug-
gesting that they may increase the predisposition to lymph
node metastasis.
This study is the first to explore the contribution of
germline CNVs using the high-resolution Affymetrix Ge-
nome-wide Human SNP Array to predict a more aggres-
sive phenotype in RET-positive MEN 2A family. Our
findings support the hypothesis that CNVs may help to
define tumor aggressiveness in this family. The future chal-
lenge will be to investigate whether the CNVs identified
here are also associated with the presence of lymph node
metastasis in Greek and North American families with
RET p.G533C germline mutations and to determine
whether theses CNVs are associated lymph node metas-
tasis in families with other RET mutations.

Acknowledgments
Address all correspondence and requests for reprints to: Janete
M. Cerutti, Ph. D., Genetic Bases of Thyroid Tumor Laboratory,
Rua Pedro de Toledo 669 - 11° andar, Universidade Federal de
São Paulo, 04039 – 032, São Paulo/SP, Brazil, Phone: &55–11-
5576 – 4979., E-mail: j.cerutti@unifesp.br.
This work was supported by Funding Support: research
grants from The São Paulo State Research Foundation (FAPESP),
grant numbers 10/51547–1, 11/10787 and 12/02902–9. A.N.A.
and L. M. are FAPESP scholars. J.M.C. and R.M.B.M. are in-
vestigators with the Brazilian Research Council (CNPq).
Disclosure Summary: The authors have reported no conflicts
of interest.

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 R B Barbieri and others Cell cycle control genes and 171:6 761–767
Clinical Study sporadic MTC

Polymorphisms of cell cycle control genes

influence the development of sporadic
medullary thyroid carcinoma
R B Barbieri, N E Bufalo, R Secolin, L V M Assumpção, R M B Maciel1,
J M Cerutti1 and L S Ward Correspondence
should be addressed
University of Campinas (FCM - Unicamp), 126, Tessalia Vieira de Camargo, Street. Cidade Universitaria Zeferino Vaz, to L S Ward
Campinas – São Paulo, 13083-887 Brazil and 1Federal University of Sao Paulo (Unifesp), 669, Pedro Toledo Street, Email
São Paulo-SP 04039-032, Brazil ward@fcm.unicamp.br

Abstract
Background: The role of key cell cycle regulation genes such as, CDKN1B, CDKN2A, CDKN2B, and CDKN2C in sporadic
medullary thyroid carcinoma (s-MTC) is still largely unknown.
Methods: In order to evaluate the influence of inherited polymorphisms of these genes on the pathogenesis of s-MTC, we
European Journal of Endocrinology

used TaqMan SNP genotyping to examine 45 s-MTC patients carefully matched with 98 controls.
Results: A multivariate logistic regression analysis demonstrated that CDKN1B and CDKN2A genes were related to s-MTC
susceptibility. The rs2066827*GTCGG CDKN1B genotype was more frequent in s-MTC patients (62.22%) than in controls
(40.21%), increasing the susceptibility to s-MTC (ORZ2.47; 95% CIZ1.048–5.833; PZ0.038). By contrast, the rs11515*CGCGG of
CDKN2A gene was more frequent in the controls (32.65%) than in patients (15.56%), reducing the risk for s-MTC (ORZ0.174; 95%
CIZ0.048–0.627; PZ0.0075). A stepwise regression analysis indicated that two genotypes together could explain 11% of the total
s-MTC risk. In addition, a relationship was found between disease progression and the presence of alterations in the CDKN1A
(rs1801270), CDKN2C (rs12885), and CDKN2B (rs1063192) genes. WTrs1801270 CDKN1A patients presented extrathyroidal tumor
extension more frequently (92%) than polymorphic CDKN1A rs1801270 patients (50%; PZ0.0376). Patients with the WT CDKN2C
gene (rs12885) presented larger tumors (2.9G1.8 cm) than polymorphic patients (1.5G0.7 cm; PZ0.0324). On the other hand,
patients with the polymorphic CDKN2B gene (rs1063192) presented distant metastases (36.3%; PZ0.0261).
Conclusion: In summary, we demonstrated that CDKN1B and CDKN2A genes are associated with susceptibility, whereas the
inherited genetic profile of CDKN1A, CDKN2B, and CDKN2C is associated with aggressive features of tumors. This study
suggests that profiling cell cycle genes may help define the risk and characterize s-MTC aggressiveness.

European Journal of
Endocrinology
(2014) 171, 761–767

Introduction
Although responsible for no more than 3–5% of all thyroid
cancers, medullary thyroid carcinoma (MTC) is responsible
for more than 14% of thyroid cancer-related deaths (1, 2).
The majority of MTC cases (O75%) are sporadic MTC
(s-MTC), even though up to 40% of these so-called sporadic
cases may be caused by somatic RET mutations (3). Despite
the importance of the RET receptor, it is clear that other
signal transduction pathways, tyrosine kinase receptors, and
tumors suppressor genes are involved in MTC tumorigenesis
and progression (4). In fact, the molecular basis of s-MTC is
still poorly understood (5). It is plausible that, similar to
other malignancies, s-MTC is caused by multiple common
genetic variants in different susceptibility genes commonly
called ‘low-penetrance genes’ (6).
Dysregulation of the cell cycle is a hallmark of many
cancers (7, 8, 9). Control and timing of the cell cycle
involve checkpoints and regulatory pathways that
ensure the fidelity of DNA replication and chromosome

www.eje-online.org ! 2014 European Society of Endocrinology Published by Bioscientifica Ltd.

DOI: 10.1530/EJE-14-0461 Printed in Great Britain
 Clinical Study R B Barbieri and others Cell cycle control genes and 171:6 762
sporadic MTC

segregation (10). Alterations of genes involved in the
G1 phase of the cell cycle, including the cyclins, cyclin-
dependent kinases (CDKs), and CDK inhibitors (CDKIs),
are common events in neoplastic development of a series
of different types of tumors (11). Their role in s-MTC is still
largely unknown.
Thyroid tumors show low expression of the CDKI P27
(Kip1), and recent evidence has demonstrated that P27 is
downregulated by the active RET mutant (12). These data
suggest that a decreased P27 (CDKN1B) activity is an
important event during thyroid tumorigenesis. However,
p27K/K mice develop MEN-like tumors only in combination
with the loss of another CDKI, p18 (Ink4c). Hence, it is
possible that p18 (Cdkn2c) and p27 are functional collabor-
ators in the suppression of tumorigenesis, and that the loss
of both may also be important to MTC development (12).
There have been some reports associating polymor-
phisms of cell cycle genes with s-MTC (13, 14, 15, 16). The
CDKN1B V109G polymorphism was reported to influence
the clinical course of patients presenting sporadic MTC
European Journal of Endocrinology
malignancy were excluded. An in-person questionnaire,
described previously, was used to collect demographic and
health condition information (17). All data, including
nodule size, tumor histological features, and laboratory
test results, were confirmed using the hospital records of
the patient. All patients were managed using the same
standard protocol according to the recommendations of
current guidelines (18, 19).
Genotyping
DNA was extracted from peripheral blood leukocytes
obtained from both patients and controls using a standard
phenol–chloroform protocol. Nine polymorphisms were
genotyped: CDKN1A (rs1801270 and rs1059234), CDKN1B
(rs2066827 and rs34330), CDKN2A (rs11515), CDKN2B
(rs2069426, rs3731239, and rs1063192), and CDKN2C
(rs12885) using the TaqMan system (Applied Biosystems),
as described previously (17). These SNPs were chosen

because they have previously been associated with thyroid

(15). In addition, cell cycle and apoptosis regulators have
long been recognized as critical in the initiation of
malignant cell proliferation and this study has recently
demonstrated the influence of the presence of a
C homozygous allele for the TP53 gene on the suscep-
tibility to s-MTC (17).
This study aimed to evaluate the role of genetic
variations in key cell cycle regulation genes CDKN1A,
CDKN1B, CDKN2A, CDKN2B, and CDKN2C in sporadic
medullary thyroid cancer pathogenesis.
Patients and methods
This study was approved by the Ethics and Research
Committees of the Federal University of São Paulo
(Unifesp) and the State University of Campinas (Unicamp).
carcinoma or other tumor risks as described in Table 1
(13, 15, 16, 20, 21, 22).
Statistical analysis
The t-test and the Fisher exact test were employed in order
to evaluate differences in age and sex using the SAS
statistical software (Statistical Analysis System, version
9.1.3, 2002–2003). For comparison of continuous or
orderable variables between two groups, we applied the
Mann–Whitney U test. The Hardy–Weinberg equilibrium
(HWE) and linkage disequilibrium between SNPs were
performed using the HAPLOVIEW software (23). Logistic
and stepwise regressions were used to analyze the
association between polymorphisms and genes using the

Table 1 Sequence-specific primers used for genotyping

Patients CDKN1B, CDKN1A, CDKN2C, CDKN2A, and CDKN2B genes and
the corresponding reference of their description in the risk of
A total of 45 patients diagnosed with s-MTC based on
other tumors.
cytology and elevated serum calcitonin levels, and who
have signed an informed consent form, were enrolled in Genes SNP Registration applied
this study. As described previously, none of the s-MTC
p27 – CDKN1B rs2066827 C_11916245_10
patients had any other types of malignant tumor or any rs34330 C_2402292_10
history of thyroid tumor in first-degree relatives (14). All p21 – CDKN1A rs1801270 C_14977_20
patients were sequenced for the complete RET gene and no p18 – CDKN2C rs12885 C_1452499_10
p16 – CDKN2A rs11515 C_12096259_10
mutation was identified. Ninety-eight healthy individuals rs3088440 C_16008027_10
from the same region and matched for sex and age with p15 – CDKN2B rs2069426 C_15858974_10
rs3731239 C_27974751_10
the enrolled patients served as controls. Individuals with
rs1063192 C_2618046_10
a history of past thyroid disease and antecedents of

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 Clinical Study R B Barbieri and others Cell cycle control genes and 171:6 763
sporadic MTC

R software (24). All P values were adjusted for multiple
comparisons by the Bonferroni correction. Post-hoc
statistical power of the sample was evaluated using the
GPower software (25), with a statistically significant level
of %0.05 for all tests.
Results
Sporadic MTC patients (15 males and 30 females; 42.20G
12.30 years old) and controls (30 males and 68 females;
40.30G11.20 years old) were similar concerning age
(PZ0.356) and sex (PZ0.745). The rs1059234 poly-
morphism of CDKN1A was not in the HWE, possibly
because of the relatively small size of the studied groups
and the high genetic heterogeneity of the Brazilian
population. Thereby, this polymorphism was excluded
from further statistical analyses.
Influence of cell cycle control gene polymorphisms
European Journal of Endocrinology
rs2066827*GT (PZ0.0014; ORZ3.48; 95% CIZ1.67–7.29)
and rs2066827*TT (PZ0.0299; ORZ0.41; 95% CIZ0.20–
0.86), in the CDKN1B gene; and rs11515*C/C (PZ0.0324;
ORZ2.51; 95% CIZ1.02–6.16) and rs11515*CG
(PZ0.0432; ORZ0.42; 95% CIZ0.17–1.03), in the
CDKN2A gene.
A multivariate logistic regression analysis confirmed
the importance of the inheritance of CDKN1B and
CDKN2A gene variants (ORZ2.472 and ORZ0.174
respectively), as detailed in Table 3.
The rs2066827*GTCGG CDKN1B genotype was over-
represented in s-MTC patients (62.22%) when compared
with controls (40.21%; PZ0.038), increasing substantially
the susceptibility to s-MTC (ORZ2.47; 95% CIZ1.048–
5.833; PZ0.038). By contrast, the polymorphic
rs11515*CGCGG of the CDKN2A gene was more expressed
in the control population (32.65%) than in s-MTC patients
(15.56%; PZ0.0075), reducing the risk of developing
s-MTC (ORZ0.174; 95% CIZ0.048–0.627; PZ0.0075).

A stepwise regression analysis indicated that, in

on the susceptibility to s-MTC
our sample, CDKN1B rs2066827*GT genotype contributed
A univariate logistic regression, given in Table 2, showed to 8% for the total risk of developing an s-MTC, whereas
associations between s-MTC and the following genotypes: CDKN2A rs11515*CC contributed to 3%. These two

Table 2 Genotyping characteristics of patients with sporadic medullary thyroid cancer (s-MTC) compared with the group of
controls.

95% CI

Genes SNP Genotype s-MTC (%) Controls (%) P valuea OR High Low

CDKN1B rs2066827 GG 37.78 59.79 0.3155 0.39 0.09 1.68

GT 57.78 27.84 0.0014 3.48 1.67 7.29
TT 4.44 12.37 0.0299 0.41 0.20 0.86
rs34330 CC 60.00 51.04 0.5940 1.46 0.71 2.98
CT 37.78 39.58 1.0000 0.95 0.46 1.96
TT 2.22 9.38 0.2070 0.28 0.05 1.74
CDKN1A rs1801270 AA 82.93 67.01 1.0000 1.17 0.28 4.84
AC 17.07 27.84 0.4381 0.59 0.25 1.41
CC 0.00 5.15 0.5577 1.53 0.69 3.39
CDKN2C rs12885 GG 70.73 77.32 0.3949 0.71 0.32 1.57
GT 26.83 22.68 0.5631 1.27 0.56 2.86
TT 2.44 0.00 0.2058 6.64 0.07 622.36
CDKN2A rs11515 CC 84.44 67.35 0.0324 2.51 1.02 6.16
CG 15.56 31.63 0.0432 0.42 0.17 1.03
GG 0.00 1.02 0.8335 0.71 0.01 66.93
CDKN2B rs2069426 AA 86.67 75.53 1.0000 0.75 0.23 2.42
CA 4.44 14.89 0.1915 0.31 0.07 1.28
CC 8.89 9.57 0.1858 2.34 0.90 6.05
rs3731239 AA 55.00 57.89 1.0000 0.72 0.36 1.46
AG 45.00 30.53 0.8044 1.51 0.73 3.15
GG 0.00 11.58 1.0000 0.94 0.31 2.82
rs1063192 CC 55.56 53.13 0.4257 2.14 0.77 5.96
TC 26.67 37.50 0.6055 0.61 0.28 1.33
TT 17.78 9.38 1.0000 1.10 0.54 2.24

a
Corrected by the Bonferroni adjustment.

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 Clinical Study R B Barbieri and others Cell cycle control genes and 171:6 764
sporadic MTC

Table 3 Comparison between genotyping profiles CDKN1B and CDKN2A of patients with sporadic medullary thyroid carcinoma
(s-MTC) and control population, using multivariate logistic regression analysis.

95% CI
a
Genes SNP Genotype s-MTC (%) Controls (%) P value OR High Low

CDKN1B rs2066827 GG 37.78 59.79 0.0388 2.472 1.048 5.833

GTCTT 62.22 40.21
CDKN2A rs11515 CC 84.44 67.35 0.0075 0.174 0.048 0.627
CGCGG 15.56 32.65

a
corrected by Bonferroni adjustment.

genotypes together could explain 11% of the total
s-MTC risk (Fig. 1).
Genotype relationship with features of
aggressiveness in patients with s-MTC
For these analyses, patients were divided into two groups:
WT (homozygous normal alleles) and polymorphic (a
European Journal of Endocrinology
Recent studies have indicated that SNPs of genes in
cell cycle control play an important role in carcinogenesis
and may lead to altered susceptibility to different cancers
(26, 27, 28). An association of CDKN1B (rs2066827) and
CDKN2A (rs11515) gene variants was found with the
presence of s-MTC. It was demonstrated that the
rs2066827*GTCGG CDKN1B genotype was over-
represented in s-MTC patients when compared with

combination of homozygous polymorphicCheterozygous controls, increasing the susceptibility to this tumor.

alleles), for each gene. Clinical and pathological charac-
teristics, including age, sex, tumor stage (T, N, and M), and
size, extra thyroidal extension, serum calcitonin levels,
and recurrence in patients were analyzed and compared
with their genetic profile.
The Mann–Whitney U test demonstrated that patients
with WT gene presented extrathyroidal tumor extension
more frequently (92%) than patients with polymorphic
gene in rs1801270 CDKN1A gene (50%; PZ0.0376).
Patients with WT CDKN2C gene (rs12885) presented
larger tumors (2.9G1.8 cm) than patients with poly-
morphic gene (1.5G0.7 cm; PZ0.0324). By contrast,
only patients with polymorphic CDKN2B gene
(rs1063192) presented distant metastases (36.3%;
PZ0.0261).
We were unable to find any other associations
between the profile of the studied genes and the clinical
or pathological characteristics of patients.
The codon 109 in the CDKN1B gene SNP results in a
valine to glycine (TOG) substitution (V109G – rs2066827)
(29). The G allele of the CDKN1B gene (rs2066827) has
been associated with an increased risk for thyroid (13),
squamous cell (28), prostate, and breast cancers (27, 30).
However, whether this polymorphism is associated with
a better or worse prognosis remains uncertain (13, 21,
28, 31). Pasqualini et al. also found differences in the
frequency of the WT (TT – 53.6%) and polymorphic alleles
8%
3%
rs2066827*G/T
rs11515*C/C
Other factors
89%

Discussion
This case–control study is, to the best of our knowledge,
the first to investigate the association of CDKN1B,
CDKN1A, CDKN2C, CDKN2A, and CDKN2B polymorph-
isms and the susceptibility to s-MTC. Besides the demon-
stration that CDKN1B and CDKN2A genes were related to Figure 1
s-MTC susceptibility, this study also found evidences that Graphical representation of the relative contribution of the
CDKN1A, CDKN2B, and CDKN2C gene variants are CDKN1B and CDKN2A genes to sporadic medullary thyroid
associated with tumor features of aggressiveness. carcinoma risk according to a stepwise regression analysis.

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 Clinical Study R B Barbieri and others
(TGCGG – 46.4%) between patients and controls
(PZ0.048), suggesting a risk of the disease in individuals
bearing the CDKN1B rs2066827 polymorphic allele (13).
The V109Gl polymorphism falls within a region (amino
acids 97–151) that physically interacts with the activation
of the domain-binding protein, which triggers the
proteolytic degradation of P27 (32). In fact, a reduced
P27 expression has been associated with a poor clinical
prognosis in head and neck tumors (33). In addition, it has
been demonstrated that amino acid changes around
position 108, such as the ones induced by V109G
polymorphism, can reduce P27kip1 cytosolic transloca-
tion, increasing its nuclear stability (31, 34). However, no
evidence has been provided to support the association of
CDKN1B polymorphisms with tumor progression or out-
come in MTC (15).
In the study population, polymorphic rs11515*CGC
GG of CDKN2A gene was expressed more in the control
population than in s-MTC patients, hence presenting a
protective effect to s-MTC development. CDKN2A gene
European Journal of Endocrinology
polymorphisms have largely been investigated in
different types of cancers (20, 22, 35). The SNP (rs11515
– C98A) CDKN2A gene determines a non-synonymous
serine-to-arginine substitution located in the 3 0 -UTR of
exon 3 (36). In fact, similarly, we observed for s-MTC,
CDKN2A gene rs11515 polymorphism was also considered
a protective factor against cervical cancer (35). CDKN2A
gene (rs11515) polymorphism may affect the DNA-
binding zinc finger motif, thus modifying p21 expression
and function (37). Additionally, the overexpression of p21
has been demonstrated to prevent mammalian cell
proliferation and inhibit all cyclin–CDK complexes,
suggesting that p21 is a universal inhibitor of cyclin–
CDK complexes (38).
We also found a relationship between tumor aggres-
siveness and the presence of variants of the CDKN1A
(rs1801270), CDKN2C (rs12885), and CDKN2B
(rs1063192) genes.
Patients presenting polymorphic CDKN1A (rs1801270)
less frequently have extrathyroidal tumor extension,
suggesting that the inheritance of this CDKN1A poly-
morphism may be associated with a better outcome. A
study of cervical cancer in a Chinese population also
showed a significant association between the polymorphic
allele of the CDKN1A (rs1801270) gene and a decreased risk
of disease (39). CDKN1A (rs1801270) polymorphisms were
demonstrated to increase the risk of second primary
malignancy in patients with index squamous cell
carcinoma of head and neck (31). The SNP (rs1801270)
within the CDKN1A codon 31 results in a substitution of
Cell cycle control genes and 171:6 765
sporadic MTC
arginine for serine in a conserved region of the protein
and may affect the DNA-binding affinity and thus the
physiological function of p21 (40). The p21 protein plays
a role in cell cycle regulation. It binds to cyclin–CDK
complexes to arrest cell cycle progression at the G1 phase.
The transcription of p21 is partially regulated by p53,
which binds to the p21 promoter and induces expression of
p21. Therefore, alterations in the functional domains as
well as in the promoter region of p21 could be affected by
SNPs and affect p21 functionality (41). CDKN1A is often
misregulated in human cancers, but depending on the
cellular context and other circumstances, it can act both as
a tumor suppressor and as an oncogene (42).
The molecular mechanism underlying a possible
protective effect of the CDKN1A (rs1801270) and
CDKN2C (rs12885) polymorphic alleles in cancer is
unclear. The CDKN2C gene, involved in cell cycle
regulation, is located at 1p32. A frequent finding in MTC
tumors is the loss of heterozygosity at chromosome 1p,
indicating a potential tumor suppressor gene in this locus
(4). Homozygous deletion of the CDKN2C gene was also
observed in the human MTC cell line (TT) bearing a RET
mutation (16). These data indicate that the CDKN2C gene
may act as a tumor suppressor and may facilitate MTC
development (4).
Regarding CDKN2B gene polymorphism (rs1063192),
we found distant metastases only in polymorphic patients,
suggesting an association of this polymorphism with
tumor aggressiveness. The polymorphism rs1063192 is
located within a gene encoding CDKN2B, which is also
known as p15 (CDKN2B), a well-known tumor suppressor
gene involved in the retinoblastoma (Rb) pathway (43).
This region on chromosome 9p21 was implicated as a
hotspot locus showing the association with various
diseases, including myocardial infarction (44, 45), coron-
ary artery disease (46, 47), type 2 diabetes (48, 49), glioma
(50), and endometriosis (51); however, there is no report
of a possible effect in thyroid cancer.
In conclusion, we found evidence that variations in
some cell cycle genes are associated with both suscep-
tibility and progression of s-MTC. As usually observed in
studies concerning the effects of polymorphisms, no one
demonstrated a highly significant association (52).
Although functional evaluation and larger studies are
needed to confirm the observed associations, our findings
contribute to the identification of the various factors
involved in the pathogenesis of s-MTC. It is suggested that
profiling cell cycle genes may help define the risk for
s-MTC and characterize tumor aggressiveness.
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 Clinical Study R B Barbieri and others
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by the State of São Paulo Research Foundation
(FAPESP) under the grant number 07067-5. L S Ward, R M B Maciel, and
J M Cerutti are researchers of the National Council for Scientific
and Technological Development (CNPq).
Acknowledgements
The authors thank the team of statisticians of the Faculty of Medical
Sciences and Espaço da Escrita – Coordenadoria Geral da Universidade –
UNICAMP – for the language services provided and for all their valuable
suggestions and insights.
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Received 6 June 2014
Revised version received 5 September 2014
Accepted 2 October 2014
www.eje-online.org
 Clinical Thyroidology / Original Paper

Eur Thyroid J 2014;3:117–124 Received: November 5, 2013

Accepted after revision: April 18, 2014
DOI: 10.1159/000363055
Published online: June 18, 2014

Development and Application of a Novel

Sensitive Immunometric Assay for Calcitonin in
a Large Cohort of Patients with Medullary and
Differentiated Thyroid Cancer, Thyroid Nodules,
and Autoimmune Thyroid Diseases
Cléber P. Camacho a Susan C. Lindsey a Teresa S. Kasamatsu a
Alberto L. Machado a, b João Roberto M. Martins a Rosa Paula M. Biscolla a, b
Magnus R. Dias da Silva a José Gilberto H. Vieira a, b Rui M.B. Maciel a, b
a
Laboratory of Molecular and Translational Endocrinology, Division of Endocrinology, Department of Medicine,
Escola Paulista de Medicina, Universidade Federal de São Paulo, and b Fleury Medicine and Health, São Paulo, Brazil

Key Words
Calcitonin · Medullary thyroid carcinoma · Thyroid nodule ·
Autoimmune thyroid disease · Immunometric assay
Abstract
Background: Serum calcitonin (sCT) is a useful biomarker for
medullary thyroid cancer (MTC). Consensus has not been
reached concerning sCT measurements in the evaluation of
nodular thyroid disease (NTD). Objective and Methods: We
developed a new immunofluorometric assay for sCT and
have validated it in samples from 794 patients [203 with
MTC, 205 with autoimmune thyroid disease (ATD), 248 with
NTD, 80 with differentiated thyroid cancer (DTC) ‘free of dis-
ease’, 58 with chronic renal failure (CRF)] and 178 normal in-
dividuals, including samples after pentagastrin tests and
samples from the washout of 92 FNA procedures in patients
with NTD or MTC. We also compared some samples from pa-
tients with low or high calcitonin levels using both this assay
and the Nichols Institute Diagnostics (NID) assay. Results:
The assay’s analytical sensitivity was 1.0 pg/ml. Considering
MTC patients prior to surgery, the cut-off values for the 95%
reference range were 11.1 pg/ml for males and 5.5 pg/ml for
females and employing the ROC curve were 18.4 pg/ml for
males and 7.8 pg/ml for females. sCT in patients with MTC
was strongly correlated with disease status. Patients with
NTD and ATD did not present false-positive results. sCT mea-
surements were significantly correlated with age (excluding
MTC and CRF). The NID test had a strong correlation with our
assay. A hook effect was observed only with concentrations
>200,000 pg/ml. Conclusions: We developed a novel sCT as-
say and validated it in healthy subjects, as well as in a large
cohort of patients with MTC, NTD, ATD, DTC, and CRF.
© 2014 European Thyroid Association
Published by S. Karger AG, Basel
Introduction
Serum calcitonin (sCT) is a useful biomarker for med-
ullary thyroid cancer (MTC) and is employed for its di-
agnosis and follow-up monitoring [1]. In addition, some
guidelines advocate sCT measurements for the differen-
tial diagnosis of nodular thyroid disease (NTD) while
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Universidade Federal de Sao Paulo

© 2014 European Thyroid Association Rui M.B. Maciel, MD, PhD

Published by S. Karger AG, Basel Laboratory of Molecular and Translational Endocrinology, Division of Endocrinology
2235–0640/14/0032–0117$39.50/0 Department of Medicine, Escola Paulista de Medicina, Universidade Federal de
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E-Mail karger@karger.com
São Paulo, Rua Pedro de Toledo 669, 11th Floor, São Paulo, SP 04039-033 (Brazil)
www.karger.com/etj
E-Mail rui.maciel @ unifesp.br
other guidelines assume a neutral or a contrary position
regarding the use of sCT for NTD due to false-positive
results, cost-effectiveness, and large reference range of as-
says [2–8].
The available sCT immunometric assays present large
reference ranges [9–12]. Procalcitonin cross-reactivity in
infections, inflammatory reactions and hyperparathy-
roidism, appears to be minimal [13]. Nevertheless, in oth-
er situations, such as autoimmune thyroid diseases (ATD)
or chronic renal failure (CRF), the corresponding mild
elevations in basal sCT result in indeterminate results re-
garding MTC diagnosis [14, 15].
We developed a novel assay because the gold standard
assay for measuring sCT is no longer available [10].
Therefore, in this study we have validated a novel immu-
nometric two-site sCT assay in a large cohort of patients,
including different clinical scenarios, as MTC and differ-
entiated thyroid cancer (DTC), NTD, ATD, and CRF, be-
sides samples stimulated after the pentagastrin test and
from the washout of fine-needle aspiration cytology
(FNAC).
Subjects and Methods
Subjects and Samples
We collected blood samples from 794 patients (2–84 years old,
617 women and 177 men) and from 178 normal individuals (3–80
years old, 131 women and 47 men) who were followed at the Divi-
sion of Endocrinology, Department of Medicine, Federal Univer-
sity of São Paulo, in São Paulo, Brazil. A signed letter of informed
consent was obtained from all patients or their legal guardians. The
study was approved by the Research Ethics Committee of the uni-
versity.
The patients were: (a) 203 who had MTC before and after sur-
gical treatment, 125 of whom had no evidence of disease after
treatment and 68 of whom had elevated sCT prior to or after sur-
gery (with or without evidence of metastasis); (b) 533 who had
thyroid disease other than MTC, 205 with ATD, 248 with NTD and
80 with DTC ‘free of disease’, and (c) 58 who had stable CRF and
no evidence of thyroid disease. Additionally, we studied 178
healthy individuals without thyroid disease (no history, normal
physical examination, normal thyroid tests and normal neck US).
Samples from Pentagastrin Test
We performed the pentagastrin test on 45 MTC patients with:
(a) positive RET mutations before prophylactic surgery; (b) with
persistent detectable sCT levels after surgery, and (c) with NTD
with previously high levels of sCT. To perform the pentagastrin
test, patients fasted for 8 h, and blood samples were drawn before
and 2, 5, 10 and 15 min after an intravenous bolus of 0.5 g/kg pen-
tagastrin (PentagastrinTM; Cambridge Laboratories Ltd, Newcastle,
UK) [16]. All 225 samples derived from these tests were centri-
fuged, and serum aliquots were stored at –20 ° C until sCT mea-
surements.
118 Eur Thyroid J 2014;3:117–124
DOI: 10.1159/000363055
Samples from FNAC Washout
FNAC procedures were performed in patients with NTD or in
lymph nodes of MTC patients, yielding a total of 92 samples for
CT analysis from the FNAC washouts. After the smear was pre-
pared for cytology, the needle was washed with 1 ml of 0.9% NaCl,
and the sample was stored at –20 ° C until the CT measurements.
Calcitonin Assay
The immunofluorometric two-site assay is based on the bind-
ing of human calcitonin by monoclonal antibodies (mAbs) ob-
tained from hybridomas produced from spleen cells of mice that
had been immunized with calcitonin peptides [17, 18]. Screening
(using human calcitonin at >97% purity; Scripps Laboratories, San
Diego, Calif., USA), selection, ascites production, purification and
IgG subtyping were previously described [19]. After titer and spec-
ificity analysis, we selected two cell lines, one specific for amino
acids 11–23 and the other specific for amino acids 17–32.
The assay is based on the first mAb bound to the wells of the
microtiter plates to serve as the solid phase, and the second mAb
biotinylated (Sulfo-NHS-LC-Biotin Kit; Pierce, Rockford, Ill.,
USA). Microtiter plates were coated with the first mAb in PBS at a
final concentration of 10 μg/ml, by adding 200 μl to each well and
letting them incubate overnight at 4 ° C. Next, the plate was washed
and blocked with buffer (50 mM Tris pH 7.75, 0.5% BSA and 0.05%
bovine γ-globulin) at 37 ° C for 1 h. The standard curve was based
on the WHO International Standard Calcitonin, Human NIBSC
89/620 prepared by serial dilution in human serum undetectable
for sCT. After washing the plate, 150 μl of the standard curve, or
controls, or samples were added. Next, 50 μl of the biotinylated
second mAb (1/3,000) was added, supplemented with 5% mouse
serum. The plate was shaken for 1 h at room temperature in the
dark and then incubated for 18 h at 4 ° C. After washing, we added
200 μl of europium-labeled streptavidin (1/2,000) (PerkinElmer,
USA) to each well and incubated the plate for 30 min at room tem-
perature with agitation before washing and adding enhancing so-
lution for time-resolved fluorescence reading.
Comparative Analysis
We compared the samples from 23 patients with low or high
calcitonin levels using both our novel assay and the Nichols Insti-
tute Diagnostics (NID) sCT assay.
Statistical Analysis
The results are presented as frequencies with the median, min-
imum and maximum values. The 95% confidence interval was cal-
culated after log transformation of the data, and the final results
were back-transformed to the original scale [19]. The cut-off value
was calculated using the ROC curve statistical approach. Spear-
man’s correlations were calculated, and the two-group analysis for
gender was performed using the Mann-Whitney U test. The data
were analyzed using SPSS 13 for Windows (SPSS, Inc., Chicago,
Ill., USA). A p value of 0.05 was considered significant.
Results
We present a novel method to quantify CT levels that
was utilized to assay and validate 1,289 samples (972 bas-
al serum samples from patients and healthy individuals,
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Universidade Federal de Sao Paulo
Camacho et al.
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 Female Male Female Male
600 60

500 50

400 40
Frequency

Frequency
300 30

200 20

100 10

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 0 20 40 60 80 100 0 20 40 60 80 100
a sCT (pg/ml) sCT (pg/ml) b Age (years) Age (years)

3
Median sCT (pg/ml)

1
Fig. 1. a Histogram of sCT (pg/ml) measurements in female and
male individuals without MTC or CRF. b Age histogram of female
and male individuals without MTC or CRF. c Bar graph with me- 0
dian sCT (pg/ml) on the vertical axis and age on the horizontal axis •20 12–40 41–60 >60
in female (white bars) and male (black bars) individuals without c Age (years)
MTC or CRF.

225 stimulated serum samples from pentagastrin tests,
and 92 samples from FNAC washouts).
The analytical sensitivity of the assay is 1.0 pg/ml, with
an intra-assay coefficient of variation of 2.1–6.4% and an
inter-assay coefficient of variation of 7.1–9.4%. In normal
male subjects, 97.9% exhibited sCT levels <18.4 pg/ml,
and 91.6% of the normal female subjects presented with
sCT levels <7.8 pg/ml (fig. 1a; tables 1–4). The 95% refer-
ence range value for the entire cohort was 6.9 pg/ml, with
a cut-off value of 9.9 pg/ml within the ROC curve. When
we analyzed the male and female groups separately, the
95% reference interval values were 11.1 and 5.5 pg/ml,
respectively. Considering MTC patients prior to surgery
and the use of the ROC curve, the cut-off value for the
male group was 18.4 and 7.8 pg/ml for the female group
(fig. 2; tables 1–4).
Patients with NTD exhibited a maximum sCT concen-
tration of 15.6 pg/ml in males with a median of 1.0 pg/ml.
All the male patients had levels <18.4 pg/ml, and 99.1% of
the female patients had sCT concentrations ≤7.8 pg/ml
(tables 1–4).
Patients with ATD exhibited a minimum sCT concen-
tration of 1.0 pg/ml, a maximum of 15.1 pg/ml, and a me-
dian of 1.0 pg/ml. Among ATD patients, 100% of the
males presented with levels <18.4 pg/ml, and 98.8% of the
females demonstrated levels <7.8 pg/ml (tables 1–4).
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Serum Calcitonin Measurement Using a Eur Thyroid J 2014;3:117–124 119

Novel Immunometric Assay DOI: 10.1159/000363055
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Table 1. Minimal, maximal and median CT values (in pg/ml) and Table 2. Number and percentage (in parentheses) of normal male
total number of subjects analyzed for normal subjects and patients subjects and male patients with NTD, ATD, CRF, and DTC with
with NTD, ATD, CRF, and DTC undetectable values, under the 95% reference range (11.1 pg/ml)
and the ROC curve cut-off (18.4 pg/ml)
Min Max Med Total
≤1 pg/ml ≤11.1 pg/ml ≤18.4 pg/ml
Normal subjects
Male 1.0 19.8 2.4 47 Male subjects only
Female 1.0 33.0 1.0 131 Normal subjects 11 (23.4) 43 (91.5) 47 (100)
Total 1.0 33.0 1.4 178 NTD patients 12 (52.2) 21 (91.3) 22 (100)
ATD patients 34 (75.6) 44 (100) 44 (100)
NTD patients CRF patients 7 (28.0) 22 (88) 23 (92)
Male 1.0 15.6 1.0 22 DTC patients 7 (63.6) 11 (100) 11 (100)
Female 1.0 12.7 1.0 226
Total 1.0 64.0 1.0 248
ATD patients
Male 1.0 5.0 1.0 44
Female 1.0 15.1 1.0 161 Table 3. Number and percentage (in parentheses) of normal fe-
Total 1.0 15.1 1.0 205 male subjects and female patients with NTD, ATD, CRF, and DTC
with undetectable values under the 95% reference range (5.5 pg/
CRF patients ml) and the ROC curve cut-off (7.8 pg/ml)
Male 1.0 21.7 1.9 25
Female 1.0 18.4 1.0 33 ≤1 pg/ml ≤5.5 pg/ml ≤7.8 pg/ml
Total 1.0 21.7 1.0 58
DTC patients Female subjects only
Male 1.0 6.3 1.0 11 Normal subjects 69 (52.3) 112 (85.5) 120 (91.6)
Female 1.0 9.1 1.0 69 NTD patients 200 (88.5) 222 (98.7) 223 (99.1)
Total 1.0 9.1 1.0 80 ATD patients 140 (87.5) 159 (98.8) 159 (98.8)
CRF patients 26 (76.5) 31 (93.9) 31 (93.9)
DTC patients 64 (92.8) 66 (95.7) 68 (98.6)

Patients with DTC were considered as a negative con-

trol group, since they were submitted to total thyroidec- Table 4. Number and percentage (in parentheses) of normal sub-
tomy, were ‘free of disease’ and, therefore, were expected jects and patients with NTD, ATD, CRF, and DTC without gender
to have undetectable sCT values. However, 9 out of 80 discrimination with undetectable values, values under the 95% ref-
erence range (6.9 pg/ml) and the ROC curve cut-off (9.9 pg/ml)
patients displayed sCT levels between 1 and 10 pg/ml (ta-
ble 2). The maximum value observed in this group was ≤1 pg/ml ≤6.9 pg/ml ≤9.9 pg/ml
9.1 pg/ml (tables 1–4).
The patients with stable CRF had a minimum sCT All subjects
concentration of 1.0 pg/ml, a maximum of 21.7 pg/ml, Normal subjects 69 (52.3) 155 (87.0) 165 (92.7)
NTD patients 200 (88.5) 242 (97.6) 246 (99.2)
and a median of 3.2 pg/ml (tables 1–4). In this group, 92%
ATD patients 140 (87.5) 203 (99.0) 203 (99.0)
of the male patients presented with levels ≤18.4 pg/ml, CRF patients 26 (76.5) 51 (87.9) 53 (91.4)
and 93.9% of the female patients demonstrated levels <7.8 DTC patients 64 (92.8) 78 (97.5) 80 (100)
pg/ml.
All of the sCT measurements, excluding those from
the MTC and CRF patients, were significantly correlated
with age (rs –0.225, p = 0.01). The age histogram displays
the higher concentration of individuals between 40 and The NID test utilized capture antibodies against pro-
60 years (fig. 1b). The highest median value in women was tein regions similar to those employed in our assay.
observed in those patients <20 years of age, whereas the Therefore, when the same samples were analyzed using
highest median value in men was observed in the 20- to our assay and the NID test system, we identified a strong
40-year-old age group (fig. 1c). The highest absolute val- correlation between results (fig. 3).
ues occurred in female patients <20 years of age and male We also analyzed samples from 45 patients who un-
patients between 40 and 60 years of age. derwent a pentagastrin stimulation test, 29 of whom car-
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120 Eur Thyroid J 2014;3:117–124 Camacho et al.

DOI: 10.1159/000363055
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 1.0 1.0

0.8 0.8

0.6 0.6

Sensitivity
Sensitivity

0.4 0.4

0.2 0.2

0 0
0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
a 1 – specificity (AUC: 0.995) b 1 – specificity (AUC: 0.993)

Fig. 2. ROC curve of the male (a) and female (b) group, to identify the cut-off level for sCT with the highest ac-
curacy to differentiate between individuals without and with MTC. AUC = Area under the curve.

ried a RET mutation prior to thyroidectomy, 12 of whom

had undergone thyroidectomy for MTC treatment, and 4
of whom were suspected to harbor MTC. The maximum 200
sCT concentration that was observed after pentagastrin
stimulation was 784 pg/ml. After stimulation, a threefold
increase in the basal calcitonin value or an sCT concen- 150
tration >100 pg/ml was considered a positive response;
sCT (pg/ml)

based on these criteria, 22 of the 45 patients were positive

for the pentagastrin stimulation test (12 mutation carri- 100
ers, 7 MTC patients post-surgery, and 3 patients with sus-
pected MTC). The 7 RET mutation carriers underwent
thyroidectomy, and the histology was confirmed to be ei- 50
ther MTC or C-cell hyperplasia. There were 23 subjects
who did not respond to the stimulation test, but this result rs = 0.93, p < 0.01
was compatible with the results of the clinical follow-up. y = 0.8x
0
All of the FNAC washout samples were diluted prior
0 50 100 150 200 250
to analysis to avoid the hook effect. Calcitonin levels were
NID sCT (pg/ml)
measured in 50 lymph nodes; the minimum observed lev-
el of 1.0 pg/ml was identified in a lymph node with benign
cytological findings, whereas the maximum concentra-
tion of 3,875,000 pg/ml occurred in a metastatic lymph Fig. 3. Correlation between the measurement of sCT (pg/ml) in 23
samples with our assay (vertical axis) and with the NID test system
node from a patient with MTC. None of the benign lymph (horizontal axis).
nodes exhibited calcitonin levels >1.0 pg/ml. Addition-
ally, 5 of the 9 cases with values >1,638 pg/ml were from
samples that were non-diagnostic or unsatisfactory by cy-
tological determination. To calculate the cut-off value for
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Serum Calcitonin Measurement Using a Eur Thyroid J 2014;3:117–124 121

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FNAC washouts, we use 37 washouts with positive or
negative cytological results through a ROC curve. The
cut-off for a positive metastatic lymph node was 270 pg/
ml. All of the 11 positive metastatic samples had CT levels
>558 pg/ml, and, by investigating these positive lymph
node FNAC washouts, we were able to estimate that
200,000 pg/ml is the concentration of calcitonin at which
a hook effect may be observed.
In the 42 washouts performed on thyroid nodules, 6
subjects with benign cytology had calcitonin concentra-
tions <2.2 pg/ml, and 4 of the 6 subjects with calcitonin
levels >1,600 pg/ml were positive by cytology for malig-
nant disease. There were also 2 patients with positive cy-
tology who presented with washout calcitonin concentra-
tions of 2.1 and 2.7 pg/ml, but the surgical pathology re-
ports did not confirm the diagnosis of MTC suggested by
the FNA. Furthermore, 1 of the 8 samples that were insuf-
ficient for or indeterminate by cytological analysis dis-
played a washout calcitonin concentration of 2,076 pg/
ml, with a surgical pathology report confirming MTC.
Discussion
Since immunoassays were developed, the detection of
biological substances has become less time-consuming,
more sensitive and more specific [20]. Our single-step,
two-site assay utilizing mAbs was designed to obtain
maximum sensitivity and specificity without interference
or cross-reactivity.
When validating and determining the reference range
for a new assay, it is necessary to evaluate both healthy
and unhealthy populations. Although we selected a
healthy group to determine normal sCT levels and to
compare these levels with those of MTC patients, we be-
lieve that the most important problem facing our daily
practice is the application of sCT measurements to ex-
clude the presence of MTC in patients with NTD and
ATD. Furthermore, the sCT concentration can be quan-
titated in patients for whom MTC is not suspected, i.e. in
patients with diabetes mellitus type 2 or in non-obese
subjects treated with the human glucagon-like peptide-1
(GLP-1) analog liraglutide. Because the GLP-1 receptor
agonist has been associated with increased sCT levels and
tumor formation in rodents, the sCT concentration has
been analyzed in over 5,000 patients who were receiving
either liraglutide or control therapy, but an effect of the
GLP-1 analog on sCT levels was not demonstrated [21].
Previous reports have shown significant differences in
sCT levels between male and female subjects [2, 22–24],
122 Eur Thyroid J 2014;3:117–124
DOI: 10.1159/000363055
which is in accordance with our results. A post-mortem
study has shown that physiologically, men have twice as
many C cells as women [25]. Additionally, the reference
ranges for the NID assay account for gender differences,
with values that are comparable to ours (<11.5 ng/l for
men and <4.6 ng/l for women) [22]. The age of the patient
is another complicating factor, and the negative correla-
tion between sCT levels and age that was observed in our
study is supported by studies demonstrating higher levels
of sCT in children than in adults [2, 22, 24].
In spite of several reports suggesting the routine mea-
surement of sCT in the evaluation of thyroid nodules [2,
4, 5, 26–30], there is some resistance in adopting this ap-
proach because of concerns regarding false-positive re-
sults [6, 29, 30]. However, in this study, we demonstrated
that 100% of the male NTD patients without MTC and
more than 99% of the female NTD patients without MTC
presented with sCT levels below the calculated cut-off for
their respective groups (≤18.4 and ≤7.8 pg/ml, respec-
tively). In the ATD group, 100% of the male patients and
98.8% of the female patients exhibited sCT concentra-
tions below the respective cut-off values.
Other area of resistance for the routine use of sCT
measurements in the evaluation of NTD is related to cost-
effectiveness. However, after the convincing study by
Cheung et al. [31], who performed sensitivity analyses
and found the cost-effectiveness of calcitonin screening
to be effective and very similar to other screening strate-
gies, as TSH, colonoscopy, and mammography screening,
it is becoming clear that this issue was also answered. In
this regard, our assay was developed in house and some
of the costs, as personnel and the development of mono-
clonals, were included in the research proposal. The cal-
culated cost for its determination is USD 5.00 per sample.
Only a few studies of sCT screening for MTC in NTD
have included CRF patients [3, 14, 31, 32]. Among the pa-
tients at our center with stable kidney disease, less than 4%
of the males and 6.1% of the females demonstrated sCT
levels above the cut-off for each group, which is similar to
a previous study [14]. However, another study has shown
that the prevalence of hypercalcitoninemia in dialysis pa-
tients amounts to 46% and that it is more common in male
than in female patients [31]; this group has also demon-
strated a reversibility of hypercalcitoninemia after kidney
transplantation [32]. In a recent paper, Kratzsch et al. [10]
have shown that higher CRF stages were associated with
increased sCT concentrations, but many of these patients
were also treated with proton pump inhibitor therapy, a
clinical condition known to be associated with hypercal-
citoninemia. Thus, we believe that different results would
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be observed in our study if unstable CRF patients under-
going hemodialysis were included.
In the DTC-treated group, where we expected to have
undetectable sCT levels, we detected a low sCT concentra-
tion in 9/80 patients. Other studies have also detected sCT
in DTC patients, but the results were interpreted as re-
lated to cross-reactivity with non-thyroid substances [10].
Nevertheless, patients who have undergone a total thy-
roidectomy and radioiodine therapy are not necessarily
completely free of thyroid tissue because ablation failure
may occur in nearly 25% of these cases [33]. Therefore,
these findings suggest that low sCT levels may also be de-
tected in MTC patients after surgery, due to remnant nor-
mal thyroid tissue, containing normal C cells, even in the
absence of a tumor, as previously suggested [3].
To evaluate the occurrence of the hook effect, we mea-
sured the high sCT values from the pentagastrin stimula-
tion tests, as well as those in the FNA washouts. We ob-
served the hook effect in samples with high calcitonin val-
ues from the FNA washouts but not in those from the
pentagastrin test. Thus, we now routinely dilute samples
from FNA washouts before analysis [34].
Another concern is the prevalence of interference in
calcitonin assays stemming from heterophilic antibodies
in the blood samples, which was reported to occur 3.7%
of the time [35]. This type of interference is relevant to
laboratory practice, and the addition of mouse serum
could potentially prevent misleading results. As such,
when we suspected a false-positive result (due to a dis-
crepancy between the clinical and the laboratory find-
ings), we first repeated the assay on a new sample to rule
out pre-analytical errors. If the high level was reproduced,
we diluted the sample with mouse serum; if the value then
decreased in a non-linear manner, interference by hetero-
philic antibodies was suspected [36].
We also utilized our assay to measure calcitonin levels
in FNAC washout samples from thyroid nodules and
lymph nodes. The concentration of CT in the FNAC
washout samples was more accurate than the cytology re-
sults, and our assay was particularly useful for samples
with non-diagnostic or unsatisfactory material for or
yielding indeterminate results by cytology.
In conclusion, we developed and validated a novel CT
assay, using it in healthy subjects, as well as in a large co-
hort of patients with MTC, DTC, NTD, ATD and CRF.
We established cut-off values, one based on the 95% ref-
erence range (11.1 pg/ml for males vs. 5.5 pg/ml for fe-
males) and the other on the ROC curve (18.4 pg/ml for
males vs. 7.8 pg/ml for females). Our results have con-
firmed previous data showing higher values in children,
Serum Calcitonin Measurement Using a
Novel Immunometric Assay
as well as in adult males, compared with adult females.
Furthermore, this study has suggested that sCT can be
used as a screening for MTC in patients with NTD, as well
as in patients with ATD, since the large majority of pa-
tients with these diseases have shown values below the
reference ranges. This new assay is also very useful for
elevated values of sCT, as in stimulation tests and in
washout samples from FNAC. In addition, we demon-
strated a strong correlation between our assay and the
NID assay, which was previously utilized worldwide.
Therefore, this new assay has been validated.
Acknowledgements
This work was supported by research grants from the São Pau-
lo State Research Foundation-FAPESP to R.M.B.M. and to
M.R.D.S. (2006/60402-1 and 2010/51547-1) and by a FAPESP Fel-
lowship Grant to S.C.L. (2009/50575-4). R.M.B.M. is investigator
of the Brazilian Research Council; R.P.M.B., J.G.H.V. and R.M.B.M.
are also investigators of the Fleury Group. The authors are very
grateful to Dr. M. Conceição Mamone for her expertise in thyroid
cytopathology, to the Medullary Thyroid Carcinoma Study Group,
Drs. Flavia Valente, Ji H. Hiang, Fausto Germano-Neto, Priscila
Signorini, Rosana Delcelo, Janete Cerutti, Mariana Frossard, Mar-
iana N. L. Oliveira, Ilda S. Kunii, Sharmila Sousa and Ana O. Hoff
for their helpful suggestions and information about patients and
to Angela M. Faria and Yeda Queiroga for their efficient labora-
tory administration. We are very grateful to the Head and Neck
Surgical Team, particularly Drs. Flavio C. Hojaij, Marcel Palumbo
and Fabio Brodskyn.
Disclosure Statement
The authors have no conflicts of interest to disclose.
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Clinical Endocrinology (2014)
ORIGINAL ARTICLE
Integration of a postoperative calcitonin measurement into an
anatomical staging system improves initial risk stratification in
medullary thyroid cancer
Ji H. Yang*,1, Susan C. Lindsey*,1, Cle
!ber P. Camacho*, Fla
!via O. F. Valente*, Fausto Germano-Neto*,
Alberto L. Machado*, Maria Conceic!a ~o O. C. Mamone*, Fa !bio Brodskyn†, Rosa Paula M. Biscolla*,
Robert Michael Tuttle‡, Magnus R. Dias-da-Silva* and Rui M. B. Maciel*,2
,2
*Laboratory of Molecular and Translational Endocrinology, Division of Endocrinology, Department of Medicine, Escola Paulista de
Medicina, Universidade Federal de S~ao Paulo, †Department of Otorhinolaryngology and Head and Neck Surgery, Escola Paulista de
Medicina, Universidade Federal de S~ao Paulo, S~ao Paulo, SP, Brazil and ‡Endocrinology Service, Memorial Sloan-Kettering Cancer
Center, New York, NY, USA
Summary
Objective Staging systems applied to medullary thyroid cancer
(MTC) rely on initial clinical and pathological features and do
not consider the response to treatment. To determine whether
MTC staging can be improved by incorporating the first postop-
erative calcitonin measurement.
Patients and measurements Eighty-five patients being moni-
tored for MTC (median follow-up 5 years) were retrospectively
classified according to both the American Joint Committee on
Cancer (AJCC) and the proposed combined risk stratification
system (low, intermediate and high risk), which incorporates the
first postoperative calcitonin measurement, using the outcomes
no evidence of disease (NED), biochemical evidence of disease,
structurally identifiable disease and death.
Results Ninety per cent of AJCC I patients were classified as
NED at final follow-up. When we added a postoperative calcito-
nin measurement, 95% low-risk patients were classified as NED
at final follow-up. AJCC stages I and IV were associated, respec-
tively, with no occurrence and a high rate (63%) of structurally
identifiable disease. Stages II and III yielded similar predictions
of structurally identifiable disease, 13% and 14%, respectively.
When we included the postoperative calcitonin level, the patients
with structural evidence of disease included none from the low-
risk group, 10% from the intermediate group and 63% from the
high-risk group. The proportion of variance explained analysis
Correspondence: Rui M. B. Maciel, Laboratory of Molecular and Trans-
lational Endocrinology, Division of Endocrinology, Department of Medi-
cine, Escola Paulista de Medicina, Universidade Federal de S~ao Paulo,
Rua Pedro de Toledo 669, 11! andar, 04039-033 Sao Paulo, SP, Brazil.
Tel.: +55 11 5084 5231; E-mail: rui.maciel@unifesp.br
1
These authors contributed equally to this work.
2
These authors contributed equally to this work.
© 2014 John Wiley & Sons Ltd
doi: 10.1111/cen.12657
(PVE) was better for the combined risk stratification system
(54%) than for the AJCC system alone (32%).
Conclusion Including the first postoperative calcitonin mea-
surement with the anatomical staging system can better predict
the clinical outcome of patients with MTC and refine the fol-
low-up of these patients.
(Received 4 July 2014; returned for revision 19 August 2014; finally
revised 7 October 2014; accepted 30 October 2014)
Introduction
Endocrinologists and surgeons have expressed a growing interest
in both reviewing thyroid cancer management paradigms and
basing treatment choices and follow-up recommendations on
individualized risk assessments.1,2 It is important to develop
similar strategies that improve the initial risk estimates in med-
ullary thyroid carcinoma (MTC).3
The American Joint Committee on Cancer (AJCC) staging sys-
tem has been widely applied to MTC, relying on the TNM system
(tumour size, extrathyroidal extension, nodal and distant metasta-
ses).4 Other staging systems rely primarily on the pathological fea-
tures of the disease.5 These variables are a static assessment of the
patient at the time of the initial surgery. In fact, most staging sys-
tems were designed to predict mortality and not recurrent disease
or the response to additional therapy. The American Thyroid
Association (ATA) recommends evaluating other factors, such as
postoperative serum calcitonin, carcinoembryonic antigen (CEA)
and calcitonin and CEA doubling times, to improve the postoper-
ative follow-up of individuals with MTC.6
Few studies have examined dynamic risk stratification in
MTC. Tuttle and Ganly recently proposed an evaluation of the
response to therapy in MTC and emphasized the importance of
assessing the response to therapy to modify initial risk esti-
mates.2 Therefore, we designed this dynamic stratification study
1
2 J. H. Yang et al.
to determine whether postoperative staging based on anatomical
variables in MTC could be improved by incorporating the first
postoperative calcitonin measurement.
Patients and methods
Subjects
We retrospectively reviewed the medical records of 128 patients
with MTC who were followed at the Multiple Endocrine Neo-
plasia outpatient clinic in the Division of Endocrinology,
Department of Medicine, Escola Paulista de Medicina, Univer-
sidade Federal de S~ao Paulo, in S~ao Paulo, Brazil, between Feb-
ruary 2007 and June 2013. Forty-three patients were excluded
from this study because their records did not include sufficient
information for risk stratification; we therefore present the
results of 85 patients with complete follow-up. Seventy-one
(84%) patients had been referred to our centre after thyroidec-
tomy.
During the initial evaluation, blood samples were collected for
sCt measurements and RET gene analysis; signed letters of
informed consent were also obtained. All of the clinical and
molecular diagnostic studies were approved by the university’s
ethics research committee.
Laboratory tests
Serum calcitonin was measured using an ‘in-house’ immunoflu-
orometric assay; the analytical sensitivity of the assay is 1 pg/
ml.7 For stratification, we considered the first postoperative cal-
citonin measurement, provided it had been collected at least
1 month after surgery and within the first year.
An analysis for germline mutations in the RET gene hot spots
(exons 8, 10, 11, 13, 14, 15 and 16) was performed after extract-
ing genomic DNA from peripheral blood, performing polymer-
ase chain reaction amplification and sequencing of the amplified
product.8
Initial risk stratification
All patients were staged according to the 7th edition AJCC stag-
ing system.4 Stages I and II comprise intrathyroidal tumours or
those with minimal extrathyroidal extension (pT1 and pT2/pT3,
respectively) with no lymph node involvement or distant metas-
tases. Stage III comprises tumours with central neck lymph node
metastases (pN1a). Stage IV comprises tumours with major ex-
trathyroidal extension (pT4a, stage IVa and pT4b, stage IVb),
those with lateral neck lymph node metastases (N1b, stage IVa)
and those with distant metastases (stage IVc). Patients were sub-
sequently stratified as being at risk for recurrent/persistent dis-
ease in three groups based on our proposed combined risk
stratification staging system, which combines the first postopera-
tive calcitonin level as an indicator of the initial treatment effi-
cacy with the AJCC staging. Thus, the patients were classified as
low risk when presenting I/II AJCC staging and postoperative
calcitonin <10 pg/ml; intermediate risk when presenting III
AJCC staging or postoperative calcitonin between 10 and
150 pg/ml; and high risk when presenting IV AJCC staging or
calcitonin >150 pg/ml.
Serial assessments and evaluation of clinical outcomes
The follow-up evaluations were conducted mainly in accordance
with the 2009 ATA MTC management guidelines.6 At each visit,
the patients were evaluated through clinical assessment, calcito-
nin and CEA measurements and neck ultrasound, as well as
MRI, CT and 18-FDG-PET scans when indicated. All patients
were classified into one of the following clinical outcomes: (i)
no evidence of disease (NED) if serum calcitonin was <10 pg/
ml, and there was no detectable structural disease; (ii) biochemi-
cal evidence of disease if calcitonin was >10 pg/ml, and there
was no detectable structural disease; (iii) structurally identifiable
disease if disease was identified in cross-sectional imaging stud-
ies; and (iv) death.
Statistical analysis
The variables were presented as median, mean and standard
deviation values. A value of P < 0!05 was considered to be sta-
tistically significant. To compare the two stratification risk sys-
tems, an ROC curve was initially established to categorize the
groups. Using SPSS software (version 13.0, SPSS Inc., Chicago,
IL, USA), a univariate regression was performed to calculate the
parameters for the proportion of variance explained (PVE)
analysis, using the Omega Squared formula.
Results
A total of 53 (62%) patients presented with sporadic tumours
and 32 (38%) patients presented with familial tumours (12
index cases and 20 family members). Among the familial cases,
20 patients had a mutation in codon 634 of the RET gene (ATA
risk level C), one patient presented MEN2B (mutation in codon
918, ATA risk level D) and the remaining patients had less
aggressive mutations (ATA risk level A). Patients with familial
tumours were younger at the time of diagnosis (age,
35 " 18 years) than were patients with sporadic tumours (age,
49 " 14 years) (P < 0!01). Three of the family members diag-
nosed through genetic screening underwent prophylactic thy-
roidectomy but pathological analysis revealed a microscopic
MTC (0!2–0!4 cm). The clinical and pathological features, risk
stratification and outcomes for the 85 patients enrolled in this
study are summarized in Table 1.
Based on the proposed risk stratification adding the first post-
operative calcitonin to tumour staging, 47% of the patients were
classified as low risk; 11!8% of the patients were classified as inter-
mediate risk; and 41!2% of the patients were classified as high risk.
There was no significant difference in age at the time of diagnosis
among the three initial risk groups, as well as in the type of disease
(familial vs sporadic). The postoperative calcitonin measurement
was collected within the first 3 months after the initial treatment
in 65% of the patients and by 6 months in 73% of the patients.
© 2014 John Wiley & Sons Ltd
Clinical Endocrinology (2014), 0, 1–5
 Combined risk stratification in MTC 3

Table 1. Epidemiological and clinical description of patients with MTC When we added a postoperative calcitonin measurement to
the AJCC system, 38 (95%) of the 40 patients classified as low
n risk were NED at the last visit; 4 (40%) of the 10 patients classi-
fied as intermediate risk were NED; and only 1 (3%) of the 35
Gender patients classified as high risk was NED (Table 2B). Patients
Female 64% 56
who presented structural evidence of disease at the last clinical
Male 36% 29
Age at time of diagnosis (years) assessment represented 10% of the intermediate-risk group and
Mean " SD 43 " 17 85 63% of the high-risk group and were absent from the low-risk
Median 44 group. This system also predicted death from the disease and
Range 7–74 biochemical evidence of disease.
Tumour type To evaluate the outcome prediction, we determined the PVE
Sporadic 62% 53 for each system. The observed variance in predicting evidence of
Hereditary 38% 32
disease (NED vs recurrent/persistent disease) was 32% for the
Pre-operative calcitonin (pg/ml) 32
Median 529 AJCC system and 54% for the combined risk stratification system.
Range 8!3–43840
Tumour size
Discussion
pT1 50!6% 42
pT2 25!3% 21 The MTC risk assessment using the AJCC system and the first
pT3 10!8% 9 postoperative serum calcitonin value for clinical outcome predic-
pT4 13!3% 11
tion demonstrated better PVE than AJCC alone because of its
Lymph nodes
pN0 41!2% 35
incorporation of a variable related to the therapeutic response.
pN1a 10!6% 9 Even though studies with a longer period of follow-up are
pN1b 32!9% 28 necessary to confirm these findings, this suggests that rather
pNx 15!3% 13 than relying only on static parameters that do not change over
AJCC staging the clinical course, including a marker that reflects the response
Stage I 36!5% 31 to the initial surgery could provide a more reliable outcome
Stage II 17!6% 15
prediction. Performing restratification that includes the response
Stage III 8!2% 7
Stage IV 37!7% 32
to therapy has already proven to be useful in differentiated
Risk for recurrent/persistent disease thyroid cancer1,9 because it enables more individualized patient
Low 47!0% 40 management.
Intermediate 11!8% 10 In the present study, the cut-off for classifying patients as low
High 41!2% 35 risk or as NED was defined as calcitonin <10 pg/ml.7 However,
Follow-up (years) because the functional sensitivity of calcitonin assays may vary,
Mean " SD 6!4 " 3!8 85
other institutions might consider establishing their own cut-offs
Median 5!2
Range 1–26
for postoperative stratification. Patients were classified as high risk
Status at the last visit when the serum calcitonin level is >150 pg/ml, because a higher
No evidence of disease 50!6% 43 calcitonin indicates a higher likelihood of distant metastases.6,10
Biochemical evidence of disease only 20% 17 CEA is also an established tumour marker for MTC and could be
Structurally identifiable disease 27% 23 included in the initial risk stratification along with calcitonin.2
Death from disease 2!4% 2 Although rare, in cases of nonsecretory MTC or low-level calcito-
nin in the presence of tumour lesion, we suggest classifying the
SD, standard deviation; AJCC, American Joint Committee on Cancer.
patient as high risk, based on the presence of structural disease.
Other factors known to be related to prognosis should also be
There was no significant difference between sporadic and familial considered. The stage of disease, extrathyroidal extension and
MTC (P = 0!0601) in terms of the outcome. age are cited in the literature as independent factors influencing
Among the patients who developed structural evidence of dis- a biochemical cure.11–13 Additionally, low pre-operative serum
ease, 12 patients underwent additional surgery, and three of calcitonin values14–16 and lymph node involvement2,4,5,17 showed
these patients were then reclassified as presenting biochemical correlations as predictors of clinical remission in a univariate
evidence of disease. A schematic flowchart describing the follow- analysis. Somatic RET mutations in sporadic MTCs have been
up of the cohort is presented in Fig. 1. associated with a worse prognosis in some studies,18 although
Among 31 patients classified as AJCC I, 28 (90%) were NED this finding was not confirmed in other studies.19 In addition,
at the last visit, as was only 1 (3%) of the 32 patients classified the calculated serum calcitonin doubling time is widely reported
as stage IV disease (Table 2A). For the outcomes regarding bio- in the literature to be a reliable prognostic tool in the
chemical evidence of disease and structurally identifiable disease, management of MTC.6,20
the AJCC system was unable to predict a clear trend. The two The management of MTC can be refined by adopting a
patients who died were in stage IV (6% of this group). combined risk stratification system, rather than relying only on

© 2014 John Wiley & Sons Ltd

Clinical Endocrinology (2014), 0, 1–5
4 J. H. Yang et al.

Fig. 1 Schematic representation of follow-up

assessment of patients with MTC. Clinical course
of 85 patients for whom the combined initial risk
stratification was retrospectively performed: initial
risk stratification (in the first year), serial
assessments and final assessment at the end of the
study. Three of twelve patients who underwent
additional surgery during follow-up presented with
biochemical evidence of disease at the last visit.

Table 2. AJCC staging system and outcome (A. Upper panel) and combined risk stratification system and outcome (B. Bottom panel), both at the last
follow-up visit

(A)

AJCC staging (n = 85) Stage I (n = 31) Stage II (n = 15) Stage III (n = 7) Stage IV (n = 32)

No evidence of disease, % (n = 43) 90 (n = 28) 67 (n = 10) 57 (n = 4) 3 (n = 1)

Biochemical evidence of disease only, % (n = 17) 10 (n = 3) 20 (n = 3) 29 (n = 2) 28 (n = 9)
Structurally identifiable disease, % (n = 23) 0 (n = 0) 13 (n = 2) 14 (n = 1) 63 (n = 20)
Death from disease, % (n = 2) 0 (n = 0) 0 (n = 0) 0 (n = 0) 6 (n = 2)

(B)

Combined risk stratification (n = 85) Low risk (n = 40) Intermediate risk (n = 10) High risk (n = 35)

No evidence of disease, % (n = 43) 95 (n = 38) 40 (n = 4) 3 (n = 1)

Biochemical evidence of disease only, % (n = 17) 5 (n = 2) 50 (n = 5) 28 (n = 10)
Structurally identifiable disease, % (n = 23) 0 (n = 0) 10 (n = 1) 63 (n = 22)
Death from disease, % (n = 2) 0 (n = 0) 0 (n = 0) 6 (n = 2)

pathological staging. Using an early measurement of calcitonin
from the first postoperative months (ideally around the first
3 months) provides important prognostic information that can
be used to guide early follow-up recommendations that can be
subsequently modified based on serial calcitonin values and dou-
bling times obtained over time. Carrying out dynamic risk
assessment is important because it provides better prognostic
information that can be used to guide the frequency of clinical
evaluations and the need for other tests and imaging studies,
reducing unnecessary work-up in low-risk patients and closely
monitoring higher-risk patients.
Acknowledgements
The authors thank the team of the Laboratory of Molecular and
Translational Endocrinology, especially Ilda Kunii, Teresa
Kasamatsu, Gilberto Furuzawa, Priscila Signorini, Maria Inez
Franc!a, Rosana Delcelo, Danielle Andreoni and Jo~ao Roberto
Martins. The research of the authors is supported by the S~ao
Paulo State Research Foundation (FAPESP) grants 2006/60402-1
and 2010/51547-1 (to R.M.B.M. and M.R.D.S.), 2009/50575-4
(research-fellowship grant to S.C.L.); a fellowship grant from
CAPES – Coordenac!~ao de Aperfeic!oamento de Pessoal de N!ıvel
Superior (to F.G.N.); grant 12518 from the Fleury Group; and
grant 25000.168513/2008-11 from the Brazilian Ministry of
Health. R.P.M.B. and R.M.B.M. are investigators of the Fleury
Group.
Disclosure statement
The authors have no conflict of interest to declare.
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 R A Toledo et al. MTC not related to RET Y791F 22:1 65–76
Research
Open Access

Comprehensive assessment of the

disputed RET Y791F variant shows no
association with medullary thyroid
carcinoma susceptibility

Rodrigo A Toledo1, Roxanne Hatakana1, Delmar M Lourenço Jr1, Susan C Lindsey6,

Cleber P Camacho6, Marcio Almeida9, José V Lima Jr10, Tomoko Sekiya1,
Elena Garralda8, Michel S Naslavsky4, Guilherme L Yamamoto4, Monize Lazar4,
Osorio Meirelles11, Tiago J P Sobreira7, Maria Lucia Lebrao3, Yeda A O Duarte2,
John Blangero9, Mayana Zatz4, Janete M Cerutti5, Rui M B Maciel6 and
Sergio P A Toledo1,6
1
Endocrine Genetics Unit (Laboratório de Investigação Médica/LIM-25) of Hospital das Clı́nicas,
University of São Paulo School of Medicine, São Paulo, São Paulo 05403-010, Brazil
2
Nursing School, 3School of Public Health, and 4Human Genome Research Center, University of São Paulo,
São Paulo, São Paulo, Brazil
Endocrine-Related Cancer

5
Division of Genetics, Genetic Bases of Thyroid Tumors Laboratory, Department of Morphology and Genetics, and
6
Division of Endocrinology, Laboratory of Molecular and Translational Endocrinology, Department of Medicine,
Federal University of São Paulo, São Paulo, São Paulo, Brazil
7
Brazilian National Laboratory of Biosciences, Campinas, São Paulo, Brazil Correspondence
8
Centro Integral Oncológico Clara Campal, Hospital Universitário Sanchinarro, Madrid, Spain should be addressed
9
Department of Genetics, Texas Biomedical Research Institute, AT&T Genomic Computing Center, to R A Toledo
San Antonio, Texas, USA Emails
10
Endocrinology Division, Santa Casa Hospital, São Paulo, São Paulo, Brazil toledorodrigo@usp.br or
11
Laboratory of Epidemiology and Population Sciences, National Institute on Aging, Bethesda, Maryland, USA toledorodrigo79@gmail.com

Abstract
Accurate interpretation of germline mutations of the rearranged during transfection (RET) Key Words
proto-oncogene is vital for the proper recommendation of preventive thyroidectomy in " multiple endocrine
medullary thyroid carcinoma (MTC)-prone carriers. To gain information regarding the most neoplasias

disputed variant of RET, ATA-A Y791F, we sequenced blood DNA samples from a cohort " RET oncogene

of 2904 cancer-free elderly individuals (1261 via Sanger sequencing and 1643 via whole- " MEN2

exome/genome sequencing). We also accessed the exome sequences of an additional 8069 " medullary thyroid carcinoma

individuals from non-cancer-related laboratories and public databanks as well as genetic " genetics

results from the Catalogue of Somatic Mutations in Cancer (COSMIC) project. The mean
allelic frequency observed in the controls was 0.0031, with higher occurrences in Central
European populations (0.006/0.008). The prevalence of RET Y791F in the control databases
was extremely high compared with the 40 known RET pathogenic mutations (PZ0.00003),
while no somatic occurrence has been reported in tumours. In this study, we report new,
unrelated Brazilian individuals with germline RET Y791F-only: two tumour-free elderly
controls; two individuals with sporadic MTC whose Y791F-carrying relatives did not show
any evidence of tumours; and a 74-year-old phaeochromocytoma patient without MTC.
Furthermore, we showed that the co-occurrence of Y791F with the strong RET C634Y
mutation explains the aggressive MTC phenotypes observed in a large affected family that
was initially reported as Y791F-only. Our literature review revealed that limited analyses

http://erc.endocrinology-journals.org q 2015 The authors This work is licensed under a Creative Commons
DOI: 10.1530/ERC-14-0491 Published by Bioscientifica Ltd Attribution 3.0 Unported License.
Printed in Great Britain
 Research R A Toledo et al.
have led to the misclassification of RET Y791F as a probable pathogenic variant and,
consequently, to the occurrence of unnecessary thyroidectomies. The current study will have
a substantial clinical influence, as it reveals, in a comprehensive manner, that RET Y791F only
shows no association with MTC susceptibility.
Introduction
MEN2 (MIM #164761) is a dominantly inherited multi-
glandular tumour syndrome that presents with a high
penetrance of medullary thyroid carcinoma (MTC;
observed in virtually 100% of cases), phaeochromocytoma
(50%) and parathyroid adenoma and/or hyperplasia (20%)
(Eng et al. 1996). Germline gain-of-function mutations of
the rearranged during transfection (RET) proto-oncogene,
mostly occurring in exons 8, 10, 11, 13, 14, 15 and 16,
activate RET via autophosphorylation of the trans-
membrane receptor tyrosine kinase; these mutant
receptors are highly expressed in MEN2-related tissues,
Endocrine-Related Cancer
leading to tumour growth. The International Consortium
on MTC of 1996 (Eng et al. 1996), the 2001 Consensus on
MENs (Brandi et al. 2001) and the updated American
Thyroid Association (ATA) Consensus on MTC (Kloos et al.
2009) recommend that individuals carrying a germline
disease-causing RET mutation should be subjected to early
prophylactic total thyroidectomy, which is currently the
only effective approach for preventing the progression of
MTC. For the purpose of molecular diagnosis and for
adequate surgical management of patients and their
relatives, it is crucial to know whether a RET gene variant
identified via genetic testing is a benign polymorphism, a
variant of unknown clinical significance or a pathological
disease-causing mutation (Toledo et al. 2006).
Biased genetic analyses of highly selected patients, in
addition to relatively small numbers of controls, may
result in a misunderstanding and misclassification of the
pathogenicity of rare variants of medically actionable
genes (Weber & Eng 2005). In this context, there is
currently a great debate in the literature regarding the
potential pathogenicity of the c.2372AOT Y791F variant
in exon 13 of RET (Berndt et al. 1998, Fitze et al. 2002,
2004, Gimm et al. 2002, Brauer et al. 2004, Plaza-Menacho
et al. 2007, Tamanaha et al. 2007, Vestergaard et al. 2007,
Eng 2010, Erlic et al. 2010, Cerutti & Maciel 2013,
Pe˛czkowska et al. 2013, Rich et al. 2014).
Recently, we described nine MEN2A families carrying
both the classical RET C634Y mutation and the RET Y791F
variant, which revealed a previously unrecognised role for
http://erc.endocrinology-journals.org q 2015 The authors
DOI: 10.1530/ERC-14-0491 Printed in Great Britain
MTC not related to RET Y791F 22:1 66
Endocrine-Related Cancer
(2015) 22, 65–76
Y791F in the MEN2 syndrome. Although further analysis
is still needed, our results indicate that Y791F may modify
the effects of other concurrent RET mutations and may
therefore act as a modifier variant (Toledo et al. 2010).
However, the role of RET Y791F-only in MTC/MEN2-
related pathogenesis and susceptibility is currently a
matter of dispute (Vestergaard et al. 2007, Kloos et al.
2009, Eng 2010, Erlic et al. 2010, Rich et al. 2014 and
Supplementary References, see section on supplementary
data given at the end of this article).
The current study represents the largest analysis
focusing on the RET (ATA-A) Y791F variant performed to
date, including new data from Sanger and next-generation
sequencing analyses of 2904 healthy adult/elderly individ-
uals. We also accessed genetic and genomic findings from
multiple public databases, resulting in the analysis of a total
of 11 544 healthy individuals and 18 163 cancer samples.
Subjects
Written informed consent was obtained from the
subjects, in accordance with the protocols approved by
the Institutional Review Board of each participating centre.
Cohorts of new investigated controls
In this study, we report data from 2904 previously
uninvestigated individuals from three tumour-free adult/
elderly cohorts. The first cohort comprised 1261 healthy
adult/elderly Brazilians (54% females and 46% males),
with a mean age of 65.2 years. Blood DNA samples from
these individuals were subjected to Sanger sequencing
of RET exon 13 in our laboratory at the University of Sao
Paulo School of Medicine. This cohort included samples
from the School of Public Health of the University of Sao
Paulo and control samples from the Experimental Oncol-
ogy Laboratory (LIM-24) of the University of Sao Paulo
School of Medicine. The ethnicity of the tumour-free
adult/elderly subjects was 79% White/White Latino and
10% African and mixed Black/Caucasian.
Published by Bioscientifica Ltd.
 Research R A Toledo et al.
The second cohort has been designated the SABE60C
cohort and is a control group that was assembled and
subjected to whole-exome sequencing by the Human
Genome and Stem Cell Research Center (HUG-CELL) of
the University of Sao Paulo. SABE60C consists of
604 Brazilian individuals from Sao Paulo City (214 males
and 390 females), with minimum and maximum ages of
63.3 and 92.2 respectively, and an average age of 74.2G6.9
years. The majority of the individuals in this cohort
were White (57.62%), followed by mixed Black/Caucasian
(18.38%), Black (7.45%), Asian (1.99%) and Amerindian
(0.33%). Approximately 7% (6.62%) of the individuals
answered positively to belonging to another ethnicity,
and 7.62% did not answer this question.
The third cohort investigated in this study is the
T2D-GENES cohort, which comes from a large collabo-
rative study composed of 1039 completely sequenced
individuals. The primary focus of this study group is the
discovery of new genes associated with the development
of type 2 diabetes using the information provided by large
human pedigrees. Each sequenced pedigree contains
Endocrine-Related Cancer
between 22 and 86 individuals distributed across three to
five generations. The sample population is composed of
Mexican–American (48.9% males) individuals living
in San Antonio, Texas (SATX), who are part of the large
San Antonio Family Study (SAFS) project, which has been
ongoing for more than 25 years, with a primary focus on
cardiovascular diseases.
Public databases
Genetic and genomic data from the following non-tumour-
enriched public databases were accessed: i) the NHLBI GO
Exome Sequencing Project; ii) the database of single
nucleotide polymorphisms (dbSNP) of the National Center
for Biotechnology Information (NCBI), National Library of
Medicine and iii) Ensembl (European Bioinformatics
Institute, EBI, and European Molecular Biology Laboratory,
EMBL), from the Wellcome Trust Sanger Institute (WTSI).
The genotyping results for the 1000 European–American
controls reported by Erlic et al. (2010) were also included.
In addition, genetic and genomic findings from the
cancer-specific Catalogue of Somatic Mutations in Cancer
(COSMIC) database were also accessed, while variant
classifications were obtained from the databases of the
ARUP MEN2/RET program of the Department of Path-
ology, University of Utah (Margraf et al. 2009).
All of the data from the databases were obtained
between 20 July and 23 July 2014, and the websites for
each database are listed at the end of the article.
http://erc.endocrinology-journals.org q 2015 The authors
DOI: 10.1530/ERC-14-0491 Printed in Great Britain
MTC not related to RET Y791F 22:1 67
Literature review
The PubMed and Google Scholar databases were used for
the literature review. Space limitations prevented us from
providing more thorough coverage of the topic, and we
apologise to those whose work could not be cited.
An extended list of the studies on RET Y791F is included
as online supplementary data.
Methods
DNA extraction, PCR and Sanger sequencing
Blood DNA was extracted using the salting out method
and a Qiagen kit (Qiagen). PCR amplification of RET exon
13 was carried out using 10 ng of gDNA and the following
primers: F: 5 0 -AACTTGGGCAAGGCGATGCA-3 0 and R:
5 0 -AGAACAGGGCTGTATGGAGC-3 0 . The PCR products
were run on a 1% agarose gel, and specific 277 bp
amplicons (Supplementary Fig. 3, see section on supple-
mentary data given at the end of this article) were then
purified using the ExoSap-IT enzyme (USB Co., Cleveland,
OH, USA). BigDye Terminator v.3.1 cycle sequencing
(Applied Biosystems) was employed to sequence the
reaction products, and the samples were run on an ABI
Prism 3130xl – Genetic Analyzer (Applied Biosystems).
Whole-exome and whole-genome sequencing
Whole-exome sequence data were generated using Agilent
SureSelect v2 technology for targeted exon capture, and
the obtained reads were aligned using the Burrows-
Wheeler Aligner (BWH) tool. Piccard was employed to
convert, sort and index the aligned data files; the sequence
quality scores were recalibrated using Genome Analysis
Toolkit (GATK); and annotation of the variants was
performed using Annovar.
Whole-genome sequencing (WGS) of 586 individuals
was performed externally by Complete Genomics, Inc.
(CGI, Mountain View, CA, USA) via a sequence-by-ligation
method. The paired-end sequencing reads (70 bp) were
mapped using the human reference genome (V 37.2), with
a mean coverage of 60!. Variant calling was performed
by CGI using version 2.0.3.1 of their proprietary pipeline.
The false discovery rate estimates for SNP calls on the CGI
platform ranged from 0.2 to 0.6%. The variant calls within
the WGS were processed using CGA Tools software
(version 1.5.0, build 31), made available by CGI. The
WGS information was employed together with the
pedigree information and a previously generated gen-
ome-wide association chip for effective imputation of
Published by Bioscientifica Ltd.
 Research R A Toledo et al. MTC not related to RET Y791F 22:1 68

offspring using the MACH Bayesian imputation algorithm
(Li 2010). The final sample comprised 1039 individuals
genotyped using nearly 23 million SNPs.
Protein structure analysis
The structure of the RET receptor was generated based on
the crystal structure of the RET tyrosine kinase domain
bound to adenosine that has been recently deposited in the
RCSB-PDB protein databank (ID: 4CKJ, Plaza-Menacho
et al. 2014) and using the software program YASARA (Krieger
et al. 2009). The RET domains were determined using the
Conserved Domain Database (CDD), and a figure was
generated with DOG 2.0 (Ren et al. 2009).
In silico pathogenic prediction
Four packages with different algorithms were used to
investigate the pathogenicity of RET Y791F in silico: SIFT;
PMUT; Align-GVGD; and POLYPHEN2 (the websites are
listed at the end of the article).
Endocrine-Related Cancer
report from Europe (Berndt et al. 1998), the three European–
American cohorts of control individuals presented the
highest frequencies, which ranged from 0.0017 to 0.008.
The three Latin cohorts together with the African–American
population presented the lowest frequencies of RET Y791F,
ranging from 0 to 0.0007 (Supplementary Table 1). Notably,
both the original Y791F MTC cases reported by Berndt et al.
(1998) and the screen of 1000 controls conducted by Erlic
et al. (2010), which identified the highest allelic frequency
among the controls, are from Germany. In addition, as
discussed later in this study, the largest cohort of patients
carrying the Y791F variant also comes from Germany,
indicating a probably higher prevalence of the Y791F
variant in this country. Interestingly, one of the healthy
Y791F-only Brazilian individuals reported in this study and
the two unrelated Brazilian cases with sporadic MTC who
also carried the variant (descriptions of these individuals are
provided later in this report) were direct descendants of
Germans. In addition, at least two out of the nine Brazilian
MEN2A families with C634Y/Y791F that we had previously
reported were of German descent (Toledo et al. 2010,

Valente et al. 2013). However, no cases harbouring

C634Y/Y791F have been reported outside of Brazil thus far.
Statistical analysis
Owing to possible incomplete assessment of the
Statistical analysis was performed by O Meirelles using phenotypic features of affected individuals or late disease
IBM SPSS (www-01.ibm.com/software/analytics/spss/). onset, the control databases may conceivably include
patients with rare diseases such as (familial) MTC and
MEN2 syndrome who carry pathogenic RET variants. To
Results explore the chance of this situation occurring, we assessed
whether the Exome Sequencing Project (ESP) exome
RET Y791F in the tumour-free cohorts variation database (which is not a tumour-enriched data-
The analyses of 22 138 alleles from the control subjects bank) included any classical disease-associated RET
revealed that Y791F is a very rare SNP, with a frequency that mutations. No alleles corresponding to strong RET
varies greatly among the analysed populations (Fig. 1 and mutations, classified as an MTC level-3 risk by the consensus
Supplementary Table 1, see section on supplementary data on MENs or as levels C-D by the ATA MTC guidelines, were
given at the end of this article). In accordance with an initial found in the ESP control databases (Fig. 2 and Supplementary
Fig. 1, see section on supplementary data given at the end of
0.009
RET Y791F frequencies in different this article). Notably, one allele of the low-risk V804M
cohorts of healthy individuals
0.008 mutation, which is classified as MTC risk 1/ATA-A, was found
0.007
0.006 among the 13 000 RET alleles from the ESP database. Valine
0.005
0.004 804 is a gatekeeper residue that is critical for ATP binding and
0.003
0.002
activation of the RET kinase receptor. Protein conformation
0.001 analyses have revealed that the V804M and V804L mutations
0
Latin Latin African– Latin adult European– European– European– modify the ATP-binding pockets of RET, facilitating ATP
Mexicans erlderly American Brazilians American* American& American#
Brazilians binding and conferring resistance to the multikinase
inhibitor vandetanib, which is approved for the treatment
Figure 1 of advanced MTC (Carlomagno et al. 2004, Wells et al. 2010).
RET Y791F frequencies in different control populations/cohorts. The The identification of heterozygous RET V804M in the
highest frequencies are observed in the populations from central Europe,
especially Germany. Details of the populations are provided in controls may be due to the relatively mild phenotype or
Supplementary Table 1. late onset of MTC that is frequently associated with this

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 Research
Comparison of the total alleles of MTC-associated RET
mutations and Y791F in ESP exome variant server (controls)
18
16
14
12
10
8
6 HC-FMUSP for routine check-ups, and her medical report
4
2
0 serious clinical features. She died in 2008 at the age of 94.
A883F
M918T
C634R/G/F/S/W/Y
C630R/F/S/Y
C611R/G/F/S/W/Y
C618R/G/F/S/Y
C620R/G/F/S/W/Y
D D C B B B
Figure 2
No RET ATA-B, C or D alleles were found in the ESP control databases. Only
one instance of the V804M allele (ATA-A) was found among the 13 000
sequenced alleles, indicating a very low frequency of bona fide MTC-
related RET mutations in healthy individuals. In contrast, 15 alleles of Y791F
were found within the ESP control databases, indicating that the
frequencies of this specific variant behave differently, possibly as an
accompanying genetic modifier or as a benign polymorphism.
Endocrine-Related Cancer
variant (Feldman et al. 2000). The ESP dataset also included
one allele of each of the three RET variants G321K, K666E,
and R866W. These three last variants are very rare and are
noted on the ATA guidelines as ‘mutations based on limited
families/case reports and may represent variants of unknown
significance’ (Kloos et al. 2009).
In contrast to the absence/very low frequency of bona
fide RET-activating mutations in the ESP dataset, the
Y791F variant was present in 14 individuals, one of
whom was homozygous (N allelesZ15). This allelic
frequency is significantly higher than that observed
among the remaining RET mutations (PZ0.00003).
Previously unreported tumour-free elderly individuals
harbouring RET Y791F
In this study, we report two Brazilian RET Y791F carriers who
were identified during the genetic screening of the healthy
elderly controls by Sanger sequencing performed in our
laboratory. The first subject is a 76-year-old woman who has
never developed tumours and presents no health compli-
cations. She has been followed by the Hospital das Clı́nicas
in Sao Paulo (HC-FMUSP) for routine check-ups. Her
calcitonin level was undetectable (!2 pg/ml) in September
2014, and she reports no history of tumours in her family.
Her father was German and died at the age of 75 due to a
heart attack. Her 45-year-old daughter (an only child) was
invited to come to the hospital to provide blood for
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C609F/R/G/S/Y
G533C
B B
R A Toledo et al. MTC not related to RET Y791F 22:1 69
biochemical examinations and genetic testing; her calcito-
nin level was !2 pg/ml, and sequencing analysis revealed
that she harboured the WT allele.
Less information is available regarding the second
subject with RET Y791F. She was also followed by
shows no tumour development or presence of other
R912P
E768D
L790F
V804L
V804M
Y791F
In accordance with our data on elderly individuals
carrying RET Y791F without any history of tumours, a
recent study that analysed the exome data of 44
centenarian Ashkenazi Jews has found two individuals
A A A A A A A (?) (2/44Z0.045) harbouring the RET Y791F variant
(Freudenberg-Hua et al. 2014).
Evaluation of RET Y791F in MTC, MEN2, hyperpara-
thyroidism (HPT) and phaeochromocytoma shows no
association with susceptibility to endocrine neoplasia
Berndt et al. (1998) first reported the presence of the Y791F
variant in MTC patients in 1998. In addition to the
mutational hotspots in exons 10 and 11, the authors
reported exon 13 as a new potential hotspot following the
identification of the L790F and Y791F variants in patients
with familial MTC. In the original paper, the two RET
Y791F families were small and included a combined total
of only three clinically affected individuals (Berndt et al.
1998). The association linking Y791F to familial
MTC/MEN2 is based on limited and selected cohorts.
More importantly, there is no convincing data in the
literature showing co-segregation of Y791F with MTC
(Supplementary References).
Additionally, Gimm et al. (2002) analysed German
patients with mutations in codons 790 and 791 and
suggested a mild form of the disease.
Through a review of the literature, we found two large
families reported to carry RET Y791F; one family is from
Denmark with Central Europe descendants, and the other
is from Brazil. Vestergaard et al. (2007) initially identified
a Danish young case with goitre carrying RET Y791F and
subsequently performed genetic screening of a total of 27
family members. It is noteworthy that 15 of the family
members were Y791F carriers, and none exhibited
abnormal clinical or biochemical results. The calcitonin
levels of the carriers and non-carriers in this family did not
differ significantly, and total thyroidectomy was post-
poned (Vestergaard et al. 2007). The second kindred were
a large Brazilian RET Y791F family with 17 carriers, ten of
whom were diagnosed with MTC, including cases with the
early and aggressive phenotype (Tamanaha et al. 2007).
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 Research R A Toledo et al.
Although this family was initially described as an Y791F-
only family, a subsequent analysis indicated that these
patients also harboured the bona fide activating germline
RET C634Y mutation (Cerutti & Maciel 2013, Valente et al.
2013). These results are in accordance with the previous
report of C634Y-Y791F cis being found in four Brazilian
MEN2A families (Toledo et al. 2010).
The largest reported cohort of patients carrying RET
Y791F was described by Frank-Raue and colleagues and
comprised 22 German MTC/MEN2 cases and 34 screened
relatives. The authors concluded that Y791F carriers
develop milder clinical features and achieve higher cure
rates compared with carriers of the codon 790 and 804
mutations (Frank-Raue et al. 2008). No control samples
from the German population were analysed in this study.
Patients with Y791F who display hyperparathyroidism
and no MTC have been reported as well (Vierhapper et al.
2005). Another study identified RET Y791F in German
patients with glioblastoma multiforme and gastric and
pancreatic cancers who showed no clinical features of
MTC or MEN2 (Rückert et al. 2011).
Endocrine-Related Cancer
In accordance with the results of studies by Erlic et al.
(2010) and Pe˛czkowska et al. (2013) showing occasional
phaeochromocytoma cases with co-occurrence of the RET
Y791F variant and pathogenic variants in other phaeo-
chromocytoma-related genes, we identified a 68-year-old
male with phaeochromocytoma carrying the RET Y791F
variant and the new germline variant D236N in the SDHB
gene. Nevertheless, the pathogenicity of the latter variant
is still uncertain.
Two new MTC patients harbouring RET Y791F without
a family history of endocrine neoplasias
In this study, we report a 37-year-old female followed by the
MEN clinic of the Federal University of Sao Paulo (UNIFESP)
who presented a thyroid nodule with cytological results
indicative of Hürthle cell neoplasm and an extremely high
calcitonin level (2079 pg/ml, normalZ8.5/5.0 pg/ml).
During physical examination, the patient showed no
cutaneous lichen amyloidosis, marfanoid habitus or
mucosal neuromas. She underwent total thyroidectomy
with prophylactic central neck dissection. A pathological
analysis revealed a 2.5 cm MTC on the left thyroid lobe and
lymphocytic thyroiditis with clusters of hyperplastic
Hürthle cells. The tumour was confined to the thyroid,
and the 20 lymph nodes that were analysed showed no
evidence of metastasis. A genetic analysis of all of the RET
hotspot exons revealed the Y791F variant. Three years after
surgery, the calcitonin levels of this patient remain
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MTC not related to RET Y791F 22:1 70
undetectable (!2 pg/ml); her carcinoembryonic antigen
(CEA) level was 1.6 ng/ml, and cervical ultrasonography
showed no sign of relapse. Additionally, her clinical and
biochemical screening results were negative for phaeo-
chromocytoma and hyperparathyroidism.
Screening of this patient’s family revealed no history of
endocrine tumours. Her 32-year-old sister also carries the
RET Y791F variant and presented a thyroid ultrasound
exhibiting thyroiditis and no nodules and biochemical
examinations showing no evidence of MTC (calcitonin
levels !1 pg/ml) or primary hyperparathyroidism. She was
followed by another service and inadvertently underwent
thyroidectomy. Pathological analysis found no evidence of
MTC or C-cell hyperplasia. Her mother (59 years old) and
her two sons (18 and 11 years old) did not harbour the RET
Y791F variant and presented low calcitonin levels. Her
father, who was probably the obligatory carrier, died in a car
accident at the age of 46 years with no signs of the disease.
He was from Germany. The lack of a family history of MTC
in this family with three carriers argues that the RET Y791F
variant is not an endocrine-neoplasia-susceptibility
variant. Unfortunately, similar to this family, other
asymptomatic relatives with the Y791F variant have also
undergone unnecessary thyroidectomies (Supplementary
References).
Recently, we have identified another patient with
MTC harbouring the RET Y791F variant from the city
of Curitiba, Brazil. The patient, a 35-year-old woman, was
diagnosed with a thyroid nodule. The patient underwent
a thyroidectomy, and the final histological analysis
revealed MTC. An extended RET analysis (exons 1–12
and 14–21) was performed, and no other mutations were
identified. The patient has a 9-year-old daughter exhibit-
ing normal thyroid ultrasound results. The patient also has
a German background.
A new elderly phaeochromocytoma patient harbouring
RET Y791F without MTC
A RET Y791F germline variant was observed in a patient
being followed at Santa Casa Hospital in Sao Paulo, who
was referred to our laboratory for genetic testing at the
University of Sao Paulo. The remaining RET hotspot exons
were sequenced, and no variant was identified. The patient
was a 74-year-old man diagnosed with a left adrenal
incidentaloma (9!8.3!6.5 cm). He had shown hyperten-
sion for the past 14 years, in addition to exhibiting chronic
atrial fibrillation and subclinical autoimmune hyper-
thyroidism. His surgical pathology was diagnostic of
phaeochromocytoma (8.5 cm and 220 g), and the results
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 Research R A Toledo et al. MTC not related to RET Y791F 22:1 71

of his thyroid ultrasound examination were compatible
with thyroiditis without nodules. His calcitonin and
calcium/parathyroid hormone levels were normal. No
family history of tumours was reported, and the sequencing
results for the TMEM127 gene showed no mutations. This
case is another example of an elderly individual harbouring
the RET Y791F variant without any increased risk for MTC.
RET Y791F in human tumours
The number of RET ATA mutations observed in the
COSMIC genetic and genomic dataset increased substan-
tially according to the mutation’s aggressiveness: 94.1% of
the ATA RET mutations occurring in cancers belong to the
ATA C–D classification (strong mutations), while only 1.4%
are weak ATA-A RET mutations (Fig. 3 and Supplementary
Tables 2 and 3, see section on supplementary data given at
the end of this article). Among the ATA-A risk mutations,
the most common variant is V804M, while the remaining
variants are very rare and with an uncertain functional
effect according to the ATA classification.
Endocrine-Related Cancer
regarding somatic or germline status). Of the 1549 MTCs in
the COSMIC dataset, 625 (40.3%) exhibited a RET
mutation, none of which were Y791F, indicating that
there is not a frequent association between this variant and
MTC tumourigenesis (Supplementary Table 4, see section
on supplementary data given at the end of this article).
In addition to the lack of evidence regarding a ‘driver’
property of RET Y791F in human tumours, two reports
describing results indicative of a ‘passenger’ role for Y791F.
The first case is an Y791F MTC patient in whom the most
aggressive RET mutation, M918T, was identified somatically,
which is most probably the cause of MTC (Fitze et al. 2002).
The second case has been recently reported by Carlino et al.
(2014) who described a patient with pancreatic carcinoma
presenting the typical KRAS G12D mutation and the RET
Y791F variant. There is no information as to whether the
Y791F mutation was germline or somatic in this patient, but
as KRAS G12D is a very well-described pancreatic cancer-
driver mutation, the results strongly indicate a minor/
non-pathological role for RET Y791F. Interestingly, the
patient is Caucasian, was born in Australia and has

Among the entire COSMIC dataset of 18 163 tumour a German surname (Carlino, electronic communication).
samples with available RET genotypes, only one
(a lymphoid neoplasm, ID: COSM1159820) presented the RET Y791F in HSCR
RET Y791F variant (0.002% – no information available
RET Y791F was first described in a case of Hirschsprung’s
disease (HSCR) (Seri et al. 1997). HSCR is a genetically
ATA classification RET mutations on COSMIC
heterogeneous disorder, and inactivation (nonsense and
ATA-A 8 (1.4%) frameshift) mutations in RET are its primary cause (Edery
ATA-B 26 (4.5%) et al. 1994). In families harbouring activating RET
ATA-C 64 (11.2%) mutations occurring primarily in extracellular domains
ATA-D 474 (82.9%) (i.e. exon 10), such as C620R, it has been classically
Total 572 reported that HSCR develops in up to 7% of carriers. Since
the initial report, other Y791F-carrier HSCR cases with
no evidence of MTC have been reported, raising the
500
question as to whether this variant is actually involved
400 in tumourigenesis (Virtanen et al. 2013).
300

200
In silico RET Y791F analyses

100 All four of the software programs used in this study

predicted low or very low pathogenicity for the RET Y791F
0
variant (data not shown). Although the software programs
ATA-A ATA-B ATA-C ATA-D
employ different algorithms to calculate potential patho-
genicity, they all use evolutionary conservation, which
Figure 3
The number of RET mutations observed in the COSMIC cancer genetic and indicated that an amino acid change at Tyrosine 791 is
genomic dataset increases substantially according to the ATA mutation more likely to be tolerated, rather than being lethal or
aggressiveness classification: 94.1% of the RET mutations found in cancers pathogenic. Our assessment of the RET protein structure
belong to the ATA C–D classification (strong mutations), while only 1.4%
consist of weak ATA-A RET mutations. One of the 8 (12.5%) ATA-A variants indicate that Y791F localises to the N-lobe, in b 4, and is
is Y791F, corresponding to 0.17% of the mutations. close to the ATP-binding site (Supplementary Fig. 3),

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 Research R A Toledo et al.
which is a region that does not commonly contain strong
RET mutations.
In vitro RET Y791F assays
Plaza-Menacho et al. (2007) investigated the RET Y791F
variant in vitro and reported ligand-independent acti-
vation. According to the results of this previous study,
Y791F is an autophosphorylated monomeric activated
receptor that leads to the constitutive activation of STAT3.
However, the mechanism of STAT3 activation occurs via
Src, JAK1 and JAK2, which differs from the classical RET
C634R activation mechanism (Plaza-Menacho et al. 2007).
In a later study, Hyndman et al. (2013) analysed in vitro
RET variants that had been previously reported in HSCR
cases and showed that Y791F behaved similarly to the WT
protein in many respects, with similar RET/pERK/pSTAT3
phosphorylation, surface localisation, and RNA and protein
stability being observed. Nevertheless, unlike to the RET WT
protein and similarly to other HSCR variants, Y791F was
associated with reduced levels of cell migration and no
Endocrine-Related Cancer
ability to promote colony formation (Hyndman et al. 2013).
Although this last study did not include bona fide RET-
activating mutations for comparison, the results indicate
WT-like functional and molecular effects of Y791F, rather
than Y791F acting as a gain-of-function mutation.
A third study by Cosci et al. (2011) combined in silico
and in vitro assays to analyse known pathogenic mutations
of RET, such as C634R and M918T and ‘rare medium/low-
risk’ variants, including Y791F. Correlations between the
results obtained using different approaches were observed
primarily for the stronger variants, while the overall
results and classification of the weaker variants remained
unclear. The in silico classification of RET Y791F was
deleterious, although focus and soft agar assays indicated
Y791F to be a weak and probably non-pathogenic
variant (Cosci et al. 2011). In contrast, the high prolife-
ration rates associated with Y791F, C634R and M918T
achieved statistical significance, but these results were
inconclusive, as several probably non-pathogenic variants,
such as V648I, also achieved statistical significance.
The three studies of RET cited above, together with
many others in the literature, show how difficult and
sometimes capricious it can be to characterise a weak
variant using in vitro or in silico approaches, especially in
genes related to genotypes with major medical impli-
cations, such as the recommendation for total thyroid-
ectomy in the case of RET. To the best of our knowledge,
there is no animal model of the RET Y791F variant.
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MTC not related to RET Y791F 22:1 72
RET Y791F as a potential phenotypic modifying variant
in cases with pathogenic RET mutations
The data that we were able to generate and review in the
current study indicate a clear lack of pathogenicity of RET
Y791F-only. However, the possible role of Y791F as a
phenotype-modifying variant cannot be discarded. In fact,
we have recently reported four MEN2A families carrying
the classical RET C634Y mutation and Y791F in cis (Toledo
2010). The patients presented aggressive MTC (codon
C634-like pattern) associated with an increased suscep-
tibility to unusually large bilateral phaeochromocytomas.
This finding is indicative of a modifier effect of Y791F
when associated with classical disease-causing RET
mutations (Toledo 2010). In the same study, we described
in vitro analyses performed in Dr Lois Mulligan’s laboratory
(ON, Canada), demonstrating that the double C634Y/
Y791F RET receptor is significantly more highly phos-
phorylated than either the WT receptor or the single
C634Y and Y791F RET mutants (Toledo et al. 2010).
Interestingly, an association between RET C634Y and
Y791F has been recently observed in five additional Brazilian
families (Cerutti & Maciel 2013, Valente et al. 2013). In
addition to C634Y/Y791F, the presence of Y791F has been
reported in patients harbouring other disease-associated
RET mutations, such as C620F, V804L and M918T
(Jindrichova et al. 2004). Although current knowledge
shows no relevant clinical effect, the possible mechanism
of such co-occurrence seems worth exploring in the future.
Known RET missense variants identified through
whole-exome/genome sequencing
Four variants classified as ATA-A mutations (S649L,
V804M, R844Q and S891A) (Kloos et al. 2009) were rarely
found in the whole-exome/genome sequences of the
tumour-free individuals assessed in the current study.
The V804M and S891A variants are also included in the
RET list as MTC level 1 mutations (Brandi et al. 2001). As
mentioned earlier, V804M is a well-described gatekeeper
mutation associated with a mild, late-onset phenotype,
which could explain its rare occurrence in the presumably
healthy cohort (1/13 000 alleles in the ESP dataset,
0.00007). The frequencies of V804M and Y791F in the
controls were very different and were compatible with a
true, low-risk mutation and a rare non-pathogenic variant
respectively (Fig. 2 and Supplementary Fig. 2, see section
on supplementary data given at the end of this article). In
addition, a novel change involving Valine 804 (c.2411COT;
p.V804A) was, to the best of our knowledge, identified
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 Research R A Toledo et al. MTC not related to RET Y791F 22:1 73

Table 1 The nine new variants of the RET proto-oncogene identified via whole-exome and whole-genome sequencing in the
current study

Genotype
Casuistic Nucleotide Codon RET protein dbSNP or ESP SIFT PolyPhen2 n frequency

SABE60C C1352T 451 T451M* Not reported D B 1 0.0008

SATX T1418A 473 L473Q Not reported D D 3 0.0014
SATX A1462T 488 T488S Not reported T B 1 0.0004
SABE60C C1573T 525 R525W Not reported D D 1 0.0008
SABE60C C1678T 560 P560S Not reported D D 1 0.0008
SABE60C G1799T 600 R600L Not reported D B 1 0.0008
SATX C2077T 693 R693C Not reported D D 2 0.0001
SABE60C C2225T 742 T742M Not reported D D 1 0.0008
SABE60C A2255G 752 Y752C Not reported T D 1 0.0008
SATX T2411C 804 V804A Not reported D D 1 0.0005

Please see the discussion and interpretation of these findings in the main text. The T451M variant is not present in the control databases (dbSNP and ESP),
but it was identified in two endometrium carcinomas (COSMIC project IDs: 1584958 and 918112). The other variants have not been reported in COSMIC.
D, damaging; B, benign; T, tolerated; SABE60C, University of Sao Paulo; SATX, San Antonio, Texas.

for the first time in this study (Table 1, allelic frequency
of 0.00001 in the SATX control cohorts). An in silico
analysis using both SIFT and Polyphen2 indicated the
V804M and V804A changes to be pathogenic.
Endocrine-Related Cancer
classified as damaging by both software programs; and
one variant was classified as benign/tolerated by both
software programs (Table 1). Despite the rarity of these
variants and the in silico predictions, the fact that our

In accordance with our results for S649L, seven
individuals from the ESP dataset were also identified as
harbouring this variant, which has been presumably
associated with late-onset, non-aggressive MTC (Colombo-
Benkmann et al. 2008).
RET V292M was also found in the SABE60C cohort
(1 out of 1218 of the sequenced alleles, 0.0008). This
variant is classified as pathogenic in the ARUP MEN2 and
dbSNP/NCBI databases, but it has not been included in the
MTC guidelines (Eng et al. 1996, Kloos et al. 2009).
Currently, limited evidence is available regarding the
pathogenicity of this variant.
The previously reported RET Y791N variant was
identified in two control individuals analysed via Sanger
sequencing (2/2522, 0.008): a 76-year-old male and a
55-year-old female. There is no evidence of pathogenicity
of this variant in the literature; it was initially reported
in a case of HSCR and later in the sister of an MTC case
harbouring RET R770Q (Ruiz-Ferrer et al. 2006, Frank-Raue
et al. 2010).
Novel RET missense variants identified via
whole-exome/genome sequencing
Nine of the RET missense variants identified herein via
massive parallel DNA sequencing have not been reported
in the dbSNP/NCBI or ESP databanks and, to the best of
our knowledge, are novel (Table 1). Eight of the variants
were classified as damaging by Polyphen2 or SIFT; six were
population was composed of healthy adult/elderly indi-
viduals with no familial history of tumours indicates that
these variants are probably benign polymorphisms or low-
penetrance genetic changes. If new evidence appears in
the future indicating pathogenicity in these variants,
especially V804A, which occurs at a known RET hotspot,
we will contact the carriers and offer genetic counselling,
molecular testing and specific clinical screening.
Conclusions of the current comprehensive
assessment of RET Y791F
1. There is no robust evidence from either our data or
the literature indicating that RET Y791F-only may
cause familial MTC or MEN2 syndrome (reports
addressing very small families should not be
considered to be informative). RET Y791F, like other
non-pathogenic variants of RET, can be found in
sporadic MTC patients, but its presence alone does
not indicate an increased MTC risk.
2. The frequencies of the RET Y791F variant detected in
large cohorts of healthy individuals are characteristic
of the behaviour of a rare benign polymorphism,
rather than a medically actionable pathogenic
mutation of the RET proto-oncogene.
Lessons from findings 1 and 2:
† Asymptomatic germline RET Y791F-only carriers
(with no other disease-causing mutations of RET)
should not undergo preventive total thyroidectomy

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 Research R A Toledo et al.
based on the genetics. Familial risk, clinical out-
comes, and biochemical and imaging data are the
most valuable resources for managing these cases.
† There is no indication that genetic and clinical
screening of family members of typical sporadic
MTC cases harbouring RET Y791F-only is beneficial.
3. The patients with familial MTC/MEN2 who were
initially reported to be RET Y791F-only carriers were
also found to harbour other associated disease-causing
RET variants (C634Y in cis), leading to the disease.
Lessons from finding 3:
† Genetic testing for RET is only complete when
sequencing of all of the hotspot mutations in
exons 8 and 10–16 is successfully performed. That
is, sequencing of the remaining exons should not
cease when the first suspect variant is identified.
† Sequencing or re-sequencing of the hotspot exons of
the RET gene (and possibly the full gene depending
on the phenotype) of familial MTC/MEN2 patients
initially described as RET Y791F-only carriers can
reveal medically actionable pathogenic mutations
Endocrine-Related Cancer
in the RET proto-oncogene.
† Limitations of PCR, such as allelic drop-out (PCR
failure of one allele) and/or preferential amplifi-
cation (hypo-amplification of one allele), may be
responsible for false-negative RET genetic results.
Therefore, the use of a second set of primers (with
different sequences than those used for the initial
PCR and sequencing run) is recommended for the
re-sequencing of questionable cases.
4. Phaeochromocytoma patients initially reported to be
RET Y791F-only carriers can harbour variants of other
genes, e.g. VHL or SDHx, that are most probably the
cause of the disease.
Lesson from finding 4:
† Established clinical predictors and algorithms
should be used to sequence phaeochromocytoma-
susceptibility genes in phaeochromocytoma cases
that are initially reported as RET Y791F-only.
5. Biased genetic analysis of highly selected patients and
a relatively small number of controls can result in
misunderstanding and misclassification of the patho-
genicity of rare variants in medically actionable genes.
Lesson from finding 5:
† The analysis of large and informative datasets from
tumour-free individuals constitutes an accessible
and useful tool for improving the interpretation
of a molecular diagnosis and aiding in genetic
counselling and the clinical management and
treatment of carriers.
http://erc.endocrinology-journals.org q 2015 The authors
DOI: 10.1530/ERC-14-0491 Printed in Great Britain
MTC not related to RET Y791F 22:1 74
Final comments about the era of NGS
The scientific and medical community working on RET-
related diseases needs to be aware of the implications
related to the dramatic increase in the number of new
variants of this gene being identified in the current next-
generation sequencing era. In fact, nine new RET variants
were identified in the present study. Moreover, it is
imperative to use caution when interpreting genetic
variants with limited information. At the same time,
access to genetic data from large cohorts presents an
opportunity to re-evaluate the clinical behaviour of
disputed RET variants, such as Y791F.
Websites
1. NHLBI ESP Exome Sequencing Project: http://evs.gs.
washington.edu.
2. dbSNP/NCBI: http://www.ncbi.nlm.nih.gov/SNP.
3. Ensembl EBI/EMBL/WTSI: http://www.ensembl.org.
4. COSMIC: http://cancer.sanger.ac.uk/cancergenome/
projects/cosmic.
5. ARUP RET database, Univ. Utah: http://arup.utah.
edu/database/MEN2/MEN2_display.php.
6. SIFT: sift.jcvi.org.
7. PMUT: http://mmb2.pcb.ub.es:8080/PMut/.
8. POLYPHEN2: http://genetics.bwh.harvard.edu/pph2/.
9. Align-GVGD: http://agvgd.iarc.fr/.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/
ERC-14-0491.
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by funds from the Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (CNPq), grant number
401.990/2010-9 and the Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) grants number 2013/01476-9 (to S P A Toledo) and
2006/60402-1 and 2010/51547-1 (to R M B Maciel). S P A Toledo, J M Cerutti
and R M B Maciel are investigators with the CNPq. R A Toledo has received
a Ciências Sem Fronteiras CNPq postdoctoral fellowship and is currently
working at the Centro Nacional de Investigaciones Oncológicas (CNIO) in
Madrid. R Hatakana, T Sekiya, S C Lindsey, D M Lourenço Jr, and R A Toledo
received research fellowships from FAPESP. S P A Toledo is a Senior Professor
in the Division of Endocrinology, Department of Medicine, Federal
University of São Paulo, SP, Brazil, with a grant from the Programa
Published by Bioscientifica Ltd.
 Research R A Toledo et al.
de Visitante Nacional Senior – Edital 028/2013 – from the Coordenadoria
de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES), Brazil.
Acknowledgements
The authors would like to acknowledge all of the subjects and volunteers
who participated in this study. They would also like to thank Dr Roger
Chammas (Experimental Oncology Laboratory/LIM-24, University of Sao
Paulo School of Medicine) and Dr Patricia L Dahia (University of Texas
Health Science Center at San Antonio) for their support.
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Received in final form 24 November 2014
Accepted 25 November 2014
Made available online as an Accepted Preprint
25 November 2014
Published by Bioscientifica Ltd.
 THYROID
Volume 25, Number 8, 2015
LETTER TO THE EDITOR
ª Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2015.0168

RET Y791F Variant Does Not Increase the Risk

for Medullary Thyroid Carcinoma
Downloaded by American Thyroid Association (ATA) from online.liebertpub.com at 07/10/17. For personal use only.

Rodrigo A. Toledo,1 Rui M.B. Maciel,2 Zoran Erlic,3 Delmar M. Lourenço Jr.,1 Janete M. Cerutti,4
Charis Eng,5 Hartmut P. Neumann,6 and Sergio P. Toledo1,2

Dear Editor: tance, at that time, of Y791F as a new MTC susceptibility

We read with interest the revised 2015 American Thyroid
Association (ATA) Guidelines for the Management of
Medullary Thyroid Carcinoma (MTC) recently published in
Thyroid by Wells et al. (1), in which the authors updated the
2009 ATA guidelines for MTC.
As with the previous guidelines, the revised 2015 ATA
guidelines gave great attention to the heritability of MTC,
which was intensely discussed in recommendations 1–12, as
well as throughout the text. Of note, the ATA mutation risk
categories were changed from D, C, B–A to ‘‘highest risk’’
(HST, M918T mutation), ‘‘high risk’’ (H, C634 mutations),
and ‘‘moderate risk’’ (MOD, non-C634 or M918 mutations),
respectively. As established previously, the suggested timing
for thyroidectomy depends on the strength of the RET mu-
tation. The revised 2015 ATA consensus recommends that
thyroidectomy should be performed in the first year/months
of age in patients harboring a M918T RET mutation and
before five years of age in patients with a C634 RET muta-
tion. Thyroidectomy in patients carrying RET mutations
classified as MOD was recommended to be performed during
childhood or young adulthood, depending primarily on serum
calcitonin levels (1).
In this letter, we would like to point out relatively new data
about the RET Y791F variant, which has not been commented
on in the 2015 ATA guidelines, and our understanding of its
implications in the management of MTC patients.
The Y791F variant was first identified in MTC patients
in an extended genetic analysis that included exon 13 of the
RET gene and was reported as a new potential pathogenic
mutation. A number of subsequent studies confirmed the
occurrence of this variant in cases with MTC, especially in
sporadic patients from central Europe presenting with a
less aggressive phenotype. These findings led to accep-
allele associated with variable and a mostly mild form of
the disease.
However, several large, well-controlled recent studies
have clearly demonstrated that RET Y791F (alone) does not
increase the risk for MTC. Erlic et al. first reported a family in
which the index patient who presented with a head and neck
paraganglioma harbored the SDHC R72C missense mutation,
while the Y791F variant was observed in three tumor-free
adult relatives (2). In addition, very similar frequencies of
Y791F were found comparing 1475 endocrine tumor patients
(0.9%) and 1000 population-based controls (0.8%) (2). The
same group later reported a coincidental RET Y791F variant
in a patient with bilateral metachronous pheochromocytomas
(and no MTC) who also carried a truncating germline MAX
mutation associated with a biallelic knockdown in the tumor
samples (3).
Recently, a comprehensive study analyzed the prevalence
of RET Y791F in 11,544 healthy individuals from multiple
genetic backgrounds and 18,163 cancer samples, and the
results support the conclusion that Y791F is indeed a rare
genetic polymorphism instead of a disease-causing mutation
(4). In addition, this study also performed an extended review
of the literature and was able to identify adult cases harboring
Y791F that inadvertently underwent thyroidectomy and had
no histopathological signs of MTC (4).
In conclusion, there is robust evidence showing that the
RET Y791F alone must be considered a nonpathogenic
polymorphism of RET not associated with increased herita-
bility of MTC (2–4).
On the other hand, cases with co-occurrence of RET
Y791F and a bona fide mutation in any other endocrine tumor
susceptibility gene should be clinically managed according
to the recommendations of the bona fide disease-causing
mutation.

1
Endocrine Genetics Unit (Laboratório de Investigação Médica/LIM-25) of Hospital das Clı́nicas, University of São Paulo School of
Medicine, São Paulo, Brazil.
3
Department of Hepatology and Gastroenterology, Interdisciplinary Centre of Metabolism: Endocrinology, Diabetes, and Metabolism,
Charité-Universitätsmedizin Berlin, Berlin, Germany.
4
Division of Genetics, Genetic Bases of Thyroid Tumors Laboratory, Department of Morphology and Genetics, and 2Division of
Endocrinology, Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Federal University of São Paulo,
Brazil.
5
Genomic Medicine Institute, Lerner Research Institute and Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio.
6
Section for Preventive Medicine, Department of Nephrology and General Medicine, University Medical Centre, Albert Ludwigs
University of Freiburg, Freiburg, Germany.

973
 974
Acknowledgments
Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq), grant number 401.990/2010-9 and the
Fundação de Amparo a Pesquisa do Estado de São Paulo
(FAPESP) grants number 2013/01476-9 (to S P A Toledo)
and 2006/60402-1 and 2010/51547-1 (to R.M.B.M.).
R.A.T. has received a CNPq postdoctoral fellowship and is
currently working at the Centro Nacional de Investigaciones
Oncológicas (CNIO) in Madrid.
Downloaded by American Thyroid Association (ATA) from online.liebertpub.com at 07/10/17. For personal use only.
S.P.A.T. is a Senior Professor in the Division of En-
docrinology, Department of Medicine, Federal University of
São Paulo, SP, Brazil, with a grant from the Programa de
Visitante Nacional Sênior—Edital 028/2013—from the
Coordenadoria de Aperfeiçoamentos de Pessoal de Nı́vel
Superior (CAPES), Brazil.
Author Disclosure Statement
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the re-
search reported.
References
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Raue K, Robinson B, Rosenthal MS, Santoro M, Schlum-
berger M, Shah M, Waguespack SG 2015 Revised American
Thyroid Association Guidelines for the Management of
Medullary Thyroid Carcinoma The American Thyroid As-
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Carcinoma. Thyroid 25:567–610.
LETTER TO THE EDITOR
2. Erlic Z, Hoffmann MM, Sullivan M, Franke G, Peczkowska
M, Harsch I, Schott M, Gabbert HE, Valimäki M, Preuss SF,
Hasse-Lazar K, Waligorski D, Robledo M, Januszewicz A,
Eng C, Neumann HP 2010 Pathogenicity of DNA variants
and double mutations in multiple endocrine neoplasia type 2
and von Hippel-Lindau syndrome. J Clin Endocrinol Metab
95:308–313.
3. Pe˛czkowska M, Kowalska A, Sygut J, Waligórski D, Mal-
inoc A, Janaszek-Sitkowska H, Prejbisz A, Januszewicz A,
Neumann HP 2013 Testing new susceptibility genes in the
cohort of apparently sporadic phaeochromocytoma/para-
ganglioma patients with clinical characteristics of hereditary
syndromes. Clin Endocrinol 79:817–823.
4. Toledo RA, Hatakana R, Lourenço DM Jr, Lindsey SC,
Camacho CP, Almeida M, Lima JV Jr, Sekiya T, Garralda E,
Naslavsky MS, Yamamoto GL, Lazar M, Meirelles O, So-
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Cerutti JM, Maciel RM, Toledo SP 2015 Comprehensive
assessment of the disputed RET Y791F variant shows no
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Address correspondence to:
Rodrigo Almeida Toledo, MSc, PhD
Endocrine Genetics Unit (LIM-25),
Department of Endocrinology
University of São Paulo, School of Medicine
Av. Dr. Arnaldo 455—Cerqueira César
São Paulo, SP
01246-903
Brazil
E-mail: toledorodrigo@usp.br;
toledorodrigo79@gmail.com
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Endocrinology Division, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil. 2Endocrine
Oncology Division, Institute of Cancer of the State of São Paulo, University of São Paulo School of Medicine, São Paulo, Brazil.
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3Department of Endocrinology, Federal University of Rio Grande do Norte (UFRN), Natal, Brazil. 4Translational and Molecular
Endocrinology Laboratory, Endocrinology Division, Federal University of Sao Paulo (UNIFESP), São Paulo, Brazil. 5Head and
Neck Surgery Division, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil. . 2017. Assessment
of Depression, Anxiety, Quality of Life, and Coping in Long-Standing Multiple Endocrine Neoplasia Type 2 Patients. Thyroid
27:5, 693-706. [Abstract] [Full Text HTML] [Full Text PDF] [Full Text PDF with Links] [Supplemental Material]
 letter to the editor

RET Y791F: alone or accompanied?

Rodrigo A. Toledo1, Delmar M. Loureço Jr.1, Cleber Camacho2,

Susan Lindsey3, Janete Cerutti1, Rui M. B. Maciel3, Sergio P. A. Toledo1

Dear Editor,

W e read with interest the article “Diagnosis, treatment, and follow-up of med-
ullary thyroid carcinoma: recommendations by the Thyroid Department of
the Brazilian Society of Endocrinology and Metabolism” recently published in this
1
Faculdade de Medicina da
Universidade de São Paulo
(FMUSP), São Paulo, S, Brasil
2
Programa de Pós-Graduação em
Medicina, Universidade Nove de
Journal (1). Julho (Uninove), São Paulo, S, Brasil
The article was written by Brazilian endocrinologists and surgeons and consists of a 3
Escola Paulista de Medicina,
Universidade Federal de São Paulo
consensus of the Thyroid Department of the Brazilian Society of Endocrinology and Me- (Unifesp), São Paulo, SP, Brasil
tabolism (SBEM). As a full version of it is also available in Portuguese language and free
Correspondence to:
for download, it is definitely a valuable reference to Brazilian doctors in residency train- Rodrigo A. Toledo
ings and medical specialists in the fields of endocrinology, surgery, oncology and others. Dr. Arnaldo, 455
05409011 – São Paulo, SP, Brasil
Virtually all individuals carrying a germline mutation in the RET proto-oncogene toledorodrigo@usp.br
toledorodrigo79@gmail.com
will develop medullary thyroid carcinoma (MTC) and therefore total prophylactic
Received on Apr/8/2015
thyroidectomy is indicated at early ages, based on the specific RET mutations (1-3). Accepted on Apr/21/2015
In this context, we would like to make a comment regarding to the Y791F variant DOI: 10.1590/2359-3997000000060

located at the exon 13 of RET. Although initial data regarding this variant indicated
that it could be a weak mutation associated with mild forms of the disease (4,5), re-
cent studies have shown no association of this variant with an increased susceptibility
to MTC. Erlic and cols. (6) and Toledo and cols. (7) have analyzed the frequencies of
RET Y791F in large cohorts of tumor free individuals and showed that the variant be-
haves as a rare non-pathogenic polymorphism rather than a disease-causing mutation.
In addition, a thorough review of the literature does not document a link of Y791F
(alone) with familial MTC. On the contrary, the reported Y791F carriers who inadver-
tently underwent thyroidectomy presented no histopathological signs of disease (5,6).
These data indicate that thyroidectomy should not be recommended to cases with
genetic testing results showing RET Y791F (alone). However, especial attention
should be taken by Brazilian doctors, geneticists and surgeons, as the RET Y791F
variant has been identified in combination with the strong RET C634Y (exon 11) mu-
tation in more than 15 Brazilian families with MTC/MEN2 (7,8). Noteworthy, this
combination changes completely the clinical scenario, and patients with RET C634Y/
Y791F should be treated accordingly to the recommendations to cases carrying RET
C634 mutations, in which early total thyroidectomy is needed to be performed (1-3).
Therefore, when receiving a genetic testing showing the presence of the RET Y791F
variant, it is crucial to make sure that the remaining exons of the gene were completely
sequenced as well, to fully certify whether is a Y791F-alone or a RET C634Y/Y791F
patient (6). 
In conclusion, we would like to congratulate the authors for the SBEM consensus
Copyright© AE&M all rights reserved.

and invite all the Brazilian medical doctors and geneticists to read our extended study
and review of the literature about RET Y791F, a variant that need to be well unders-
tood by the health professionals of our country to the better management of Brazilian
patients with MTC and their relatives (6).

476 Arch Endocrinol Metab. 2015;59/5


Funding: Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), grant number 401.990/2010-9 and the
Fundação de Amparo à Pesquisa do Estado de São Paulo (Fa-
pesp) grants number 2013/01476-9 (to Sergio P. A. Toledo)
and 2006/60402-1 and 2010/51547-1 (to Rui M. B. Maciel).
Rodrigo A. Toledo has received a Ciências Sem Fronteiras CNPq
postdoctoral fellowship and is currently working at the Centro
Nacional de Investigaciones Oncológicas (CNIO) in Madrid.
Sergio P. A. Toledo is a Senior Professor in the Division of En-
docrinology, Department of Medicine, Federal University of São
Paulo, SP, Brazil, with a grant from the Programa de Visitante
Nacional Sênior – Edital 028/2013 – from the Coordenado-
ria de Aperfeiçoamentos de Pessoal de Nível Superior (Capes),
Brazil.
Disclosure: no potential conflict of interest relevant to this article
was reported.
REFERENCES
1. Maia AL, Siqueira DR, Kulcsar MA, Tincani AJ, Mazeto GM, Ma-
ciel LM. Diagnosis, treatment, and follow-up of medullary thyroid
carcinoma: recommendations by the Thyroid Department of the
Brazilian Society of Endocrinology and Metabolism. Arq Bras En-
docrinol Metabol. 2014;58(7):667-700.
2. 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.
Arch Endocrinol Metab. 2015;59/5
RET Y791F: alone or accompanied?
3. American Thyroid Association Guidelines Task Force; Kloos RT,
Eng C, Evans DB, Francis GL, Gagel RF, Gharib H, et al. Medullary
thyroid cancer: management guidelines of the American Thyroid
Association. Thyroid. 2009;19(6):565-612.
4. Berndt I, Reuter M, Saller B, Frank-Raue K, Groth P, Grussendorf M,
et al. A new hot spot for mutations in the ret protooncogene caus-
ing familial medullary thyroid carcinoma and multiple endocrine
neoplasia type 2A. J Clin Endocrinol Metab. 1998;83(3):770-4.
5. Gimm O, Niederle BE, Weber T, Bockhorn M, Ukkat J, Brauckhoff
M, et al. RET proto-oncogene mutations affecting codon 790/791:
A mild form of multiple endocrine neoplasia type 2A syndrome?
Surgery. 2002;132(6):952-9.
6. Erlic Z, Hoffmann MM, Sullivan M, Franke G, Peczkowska M,
Harsch I, et al. Pathogenicity of DNA variants and double mu-
tations in multiple endocrine neoplasia type 2 and von Hippel-
Lindau syndrome. J Clin Endocrinol Metab. 2010;95(1):308-13.
7. Toledo RA, Hatakana R, Lourenço DM Jr, Lindsey SC, Camacho
CP, Almeida M, et al. Comprehensive assessment of the disputed
RET Y791F variant shows no association with medullary thyroid
carcinoma susceptibility. Endocr Relat Cancer. 2015;22(1):65-76.
8. Toledo RA, Wagner SM, Coutinho FL, Lourenço DM Jr, Azevedo
JA, Longuini VC, et al. High penetrance of pheochromocytoma
associated with the novel C634Y/Y791F double germline mu-
tation in the RET protooncogene. J Clin Endocrinol Metab.
2010;95(3):1318-27.
9. Valente FO, Dias da Silva MR, Camacho CP, Kunii IS, Bastos AU,
da Fonseca CC, et al. Comprehensive analysis of RET gene should
be performed in patients with multiple endocrine neoplasia type 2
(MEN 2) syndrome and no apparent genotype-phenotype correla-
tion: an appraisal of p.Y791F and p.C634Y RET mutations in five
unrelated Brazilian families. J Endocrinol Invest. 2013;36(11):975-81.
Copyright© AE&M all rights reserved.
477
 ORIGINAL ARTICLE

Macrocalcitonin is a novel pitfall in the routine of

serum calcitonin immunoassay

Thalita G. Alves1,2, Teresa S. Kasamatsu1,

Ji H. Yang1, Maria Cecília Z. Meneghetti2, Aline Mendes2, Ilda S. Kunii1,
Susan C. Lindsey1, Cléber P. Camacho1, Magnus R. Dias da Silva1,
Rui M. B. Maciel1, José Gilberto H. Vieira1, João Roberto M. Martins1,2
1
Thyroid Disease Center and Laboratory of Molecular and Translational Endocrinology, Division of
Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo,
Brazil. Rua Pedro de Toledo, 669 11th floor, 04039 – 032, São Paulo, SP, Brazil, Tel: !55 11 5084 5231.;
2
Molecular Biology Division, Department of Biochemistry, Escola Paulista de Medicina, Universidade
Federal de São Paulo, Brazil. Rua Três de Maio, 100, fourth floor, 04044 – 020, São Paulo, SP, Brazil, Tel:
!55 11 5579 –3175.

Context: Calcitonin (CT) is a sensitive marker of medullary thyroid carcinoma (MTC) and is used for
primary diagnosis and follow-up after thyroidectomy. However, persistently elevated CT is ob-
served even after complete surgical removal without evidence of a recurrent or persistent tumor.

Objective: To investigate the presence of assay interference in the serum CT of MTC patients who
are apparent without a structural disease.

Patients and Method: We studied three index MTC cases for CT assay interference and 14 patients
with metastatic MTC. The CT level was measured using an immunofluorometric assay. Screening
for assay interference was performed by determination of CT levels before and after serum treat-
ment with polyethylene glycol. Additionally, samples were analyzed by chromatography on ultra-
performance liquid chromatography and protein A Sepharose.

Results: Patients with biochemical and structural disease showed CT mean recovery of 84.1% after
PEG treatment, whereas patients suspected of interference showed recovery from 2–7%. The
elution profile on HPLC showed that the immunometric CT from these three patients behaved like
a high molecular weight aggregate (MW"300 kDa). Additionally, when these samples were ap-
plied to the protein A-Sepharose, CT immunoreactivity was retained on the column and was only
released after lowering the pH.

Conclusions: For the first time, our results show the presence of a novel pitfall in the CT immu-
noassay: “macrocalcitonin”. Its etiology, frequency and meaning remain to be defined, but its
recognition is of interest and can help clinicians avoid unnecessary diagnostic investigations and
treatment during the follow-up of MTC.

M easurement of serum calcitonin (CT) is a major

marker of medullary thyroid carcinoma (MTC),
used for both the primary diagnosis, to predict the out-
elevated risk for multiple endocrine neoplasia (MEN)
(MEN 2), both basal and stimulated serum CT could also
help to guide the timing of thyroidectomy and indicate the
come (1), and it can additionally be used as an aid in the need for a more extensive initial surgical approach (3).
planning of long-term follow-up after thyroidectomy (2, However, such strategies remain controversial (4), espe-
3). Additionally, in RET mutation carriers, who are at

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations:

Printed in USA
Copyright © 2015 by the Endocrine Society
Received August 10, 2015. Accepted December 2, 2015.

doi: 10.1210/jc.2015-3137 J Clin Endocrinol Metab press.endocrine.org/journal/jcem 1

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2 Macrocalcitonin- a new pitfall in the immunoassay J Clin Endocrinol Metab

cially regarding the limitations of assay methods and the suspect lymph nodes on cervical ultrasound, and her first bio-
interpretations of results. chemical analysis showed a slightly increased serum CT (118
pg/mL). Further biochemical analyzes showed a serum CT rang-
Indeed, persistently elevated CT can be observed even
ing from 138 –262 pg/mL. Carcinoembryonic antigen measure-
after complete surgery with no evidence of a persistent or ments were all normal. Neck and thorax tomography, abdomen
recurrent tumor in the follow-up, leading to potential mis- and pelvis magnetic resonance imaging (MRI) and a bone scan
diagnosis. Despite recent improvements in the immuno- showed no abnormalities. During three years of follow-up, the
chemiluminometric (ICLA) CT assays that minimize patient remained very well with no clinical or imaging evidence
of a residual or recurrent tumor but maintains persistently in-
cross-reactivity with procalcitonin and CT-related pep-
creased serum CT levels.
tides, some challenges remain, especially concerning the
presence of heterophilic antibodies in patient serum, Patient B
which can produce elevated CT (5– 8). In 2011, a 16-year-old boy carrying the V804M RET muta-
Thus, some challenges arise in clinical practice. When tion was referred for evaluation. Physical examination and cer-
the postoperative serum CT is undetectable, the risk of vical ultrasound were normal. Basal CT was mildly increased (35
persistent or recurrent residual disease is low, and further pg/mL). A pentagastrin stimulation test was performed and
showed a blunted response (28.4, 35.0, 37.0, 30.0 and 25.7
CT-stimulating tests or imaging techniques are not imme-
pg/mL at 0, 2, 5, 10 and 15 minutes, respectively). Screening for
diately required. In contrast, if CT levels exceed 500 pg/ hyperparathyroidism and pheochromocytoma were negative.
mL, radiographically identifiable distant metastases are Despite the low risk of ATA stratification at the time (ATA risk
almost always present (9). However, upon modestly in- level A), the patient and his family were selected for prophylactic
creased CT levels (#150 pg/mL), the presence of local or thyroidectomy. He underwent surgery in May 2011, and histo-
pathology was normal, including the absence of C-cell hyper-
distant metastasis by radiographic imaging is difficult to
plasia (CCH). During three years of follow-up, the patient main-
detect and requires additional and often expensive tests tained normal cervical ultrasound but had persistently increased
during follow-up (10). An additional challenge occurs serum CT (30 – 40 pg/mL).
when CT levels are persistently increased but unchanged
over the time. Patient C
Here, we describe three patients with MTC whose se- A 40-year-old woman carrier of the E768D RET mutation
rum CT in the late postoperative period was modestly was referred to us in 2010 for evaluation. She had already been
submitted to prophylactic thyroidectomy in January 2010, and
increased but showed no evidence of residual tumor. They histopathology revealed a 0.6 cm diameter MTC and bilateral
were diagnosed as having a new interference in the CT CCH. Postoperative cervical ultrasound was completely normal
immunoassay, which was characterized by falsely in- with no evidence of mass or suspect lymph nodes. Screening for
creased values and herein termed “macrocalcitonin hyperparathyroidism and pheochromocytoma was negative.
(macro-CT)”. The first serum CT showed a value of 40.3 pg/mL. Serum car-
cinoembryonic antigen (CEA) was within the normal range. Af-
ter three years of follow-up, there has been no evidence of clinical
or imaging recurrence of MTC, but the patient’s CT serum re-
Patients and Methods mains moderately increased (35–50 pg/mL).

We studied three index MTC cases suspected of having CT assay
interference (A, B, and C) and, for comparison, 14 patients with
MTC, who were followed at the Division of Endocrinology, De-
partment of Medicine, Escola Paulista de Medicina, Federal Uni-
versity of São Paulo, São Paulo, Brazil. The study was approved
by the Ethical Committee of UNIFESP/EPM (CAAE:
12 820 313.4.0000.5505), and written informed consent was
obtained from each subject.
Patient A
In 2003, a 45-year-old white woman attended with a history
of multinodular goiter. Cytology was benign, but the patient was
submitted to total thyroidectomy due to recent increased volume
of the thyroid gland. Histopathology showed an isolated MTC
(2.0 cm) with no evidence of capsular, vascular or extrathyroidal
invasion. During the postoperative period, serum CT and cer-
vical ultrasound were unremarkable. In April 2010, the patient
was referred to us for further evaluation. Revision of histopa-
thology confirmed the diagnosis of MTC. The presence of a
genomic mutation in the gene RET was negative. There were no
Calcitonin measurement and polyethylene glycol
(PEG) treatment
Serum CT levels were measured by an in-house two-site im-
munofluorometric assay using two mouse monoclonal antibod-
ies against two different epitopes of CT. This assay has an ana-
lytical sensitivity of 1.0 pg/mL, and the 95% reference values are
5.5 pg/mL and 11.1 pg/mL for females and males, respectively
(11). In thyroidectomized patients who have been treated for
differentiated thyroid carcinoma, more than 97.5% had a CT #
6.9 pg/mL (11). Screening for assay interference was carried out
by the determination of CT levels before and after treating the
serum with PEG (PEG-6000, Labsynth Products Labs Ltd., Sao
Paulo, Brazil). The PEG protocol was performed as previously
described (12, 13). Briefly, 250 !L of 250 g/L PEG-6000 solution
was added to 250 !L of each serum sample, vortex mixed for 1
minute, centrifuged at 9500 g for 5 minutes at 25°C, and the CT
level measured in the supernatant in triplicate. Recovery (%) was
calculated by the original CT value. We also analyzed the lin-
earity of serum calcitonin measurements after serial dilution with
an equal amount of mouse serum to check the possible interfer-

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doi: 10.1210/jc.2015-3137 press.endocrine.org/journal/jcem 3

ence of heterophilic antibodies. Suspect samples CT was also Thyroglobulin autoantibodies, thyroperoxidase
analyzed by a commercially available electrochemiluminescence autoantibodies and procalcitonin measurement
immunoassay (Elecsys Calcitonin, Roche Diagnostics, Mann- In the serum samples suspect for interference we performed
heim, Germany). thyroglobulin autoantibodies (TG-Ab) and thyroperoxidase au-
toantibodies (TPO-Ab) assays using electrochemiluminescence
Gel filtration on ultraperformance liquid immunoassay (Roche Diagnostics, Mannheim, Germany) and
chromatography (UPLC) procalcitonin measurement by an enzyme-linked immunosor-
The three serum samples suspected of interference, a sample bent assay (ELISA) (USCN Life Science Inc, Houston, TX, USA).
obtained from the washout from a fine needle aspiration of a
metastatic MTC lymph node, and two serum samples from pa-
tients with metastatic disease were analyzed by gel filtration Results
chromatography. For this analysis, samples were chromato-
graphed on a Biosep S-3000 column 30 $ 6.7 mm (Phenomenex, Patients with biochemical and structural disease presented
Torrance, CA, USA) coupled to a UPLC system (AKTA, GE a CT mean recovery of 84.1% (ranging from 51%–135%)
Healthcare, Uppsala, Sweden). After calibrating the column with
after PEG treatment (Table 1). Interestingly, the three sus-
standard molecular weights (GE Healthcare, Uppsala, Sweden),
serum samples (200 !L) were applied and eluted with a 0.05 M pect patients for interference showed very low CT recov-
phosphate-buffered saline (PBS) at pH 7.4, and a flow rate of 0.5 ery, ranging from 2%–7%. In comparison with the in-
mL/min. Aliquots of 0.5 mL were collected separately for CT house assay, serum levels of CT determined by commercial
measurement. method showed lower values of 67, 2 and 4 pg/mL for
cases A, B and C, respectively.
Affinity chromatography on protein A Sepharose Analyzes by gel filtration on a Biosep S-3000 column
Possible interferences were also characterized on protein A-
showed that CT obtained from washout of fine needle
Sepharose CL-4B (GE Healthcare, Uppsala Sweden), which spe-
cifically binds immunoglobulins. Initially, 2 mL of protein A-
aspiration of a metastatic MTC lymph node (Figure 1A)
Sepharose was packed into a plastic column and equilibrated in had an immunometric CT elution profile between 20 –23
PBS. Serum sample (0.5 mL) and the same volume of standard minutes, which corresponds to peptides of low molecular
CT (WHO International Reference Standard for Human Calci- weight (LMW). As expected, the two serum samples from
tonin 89/620, National Institute for Biological Standards and patients with metastatic MTC (patients 10 and 11)
Control were applied separately to the column and eluted with showed a similar retention time for immunometric CT
PBS; then, 0.5 mL aliquots (n % 10) were collected for subsequent
when compared with the CT washout sample (Figure 1B
CT measurement. After this procedure, the column was washed
10 times with PBS followed by the addition of 5 mL of 0.1 M and 1C). Surprisingly, the patient with low recovery (Fig-
glycine at pH 2.8. Finally, 0.5 mL aliquots (n % 10) were col- ure 1D) had almost all immunometric CT eluted as a high
lected, and each of them was assayed for CT. molecular weight aggregate (MW " 300 kDa).

Table 1. Clinical and laboratory data of patients with MTC enrolled in the CT immunoassay interference study.
Age RET Surgery Disease CT CT – PEG Recovery
Patient Gender (years) mutation (years) status (pg/mL) (pg/mL) (%)
A F 66 Negative 12 B 261 9 3.4
B F 40 E761D 4 B 36 0.6 1.7
C M 16 V804M 4 B 38 2.8 7.4
1 F 22 C634Y 2 B/S 21 754 18 567 85.3
2 F 28 Negative 5 B/S 19 800 18 729 94.6
3 M 48 Negative 9 B/S 14 070 12 985 92.3
4 M 35 Negative 9 B/S 11 795 7928 67.2
5 M 62 Negative 9 B/S 11 271 7642 67.8
6 F 44 S891A 4 B/S 10 087 7644 75.8
7 F 46 C634Y 1 B/S 9193 5414 58.9
8 F 44 Negative 7 B/S 941 848 90.1
9 F 67 Negative 14 B/S 744 593 79.7
10 F 57 S891A 4 B/S 429 438 102.1
11 F 22 C634Y 6 B/S 249 338 135.7
12 M 57 S891A NP B/S 114 59 51.7
13 M 41 C634Y 3 B/S 87 78 89.7
14 F 39 Negative 6 B/S 57 49 86.0
Blood serum from patients A, B and C suspected for calcitonin immunofluorometric assay interference. RET mutation: identified germ line RET
mutation. Clinical status B: only biochemical disease, and B/S presenting biochemical and structural disease. Time since thyroidectomy in years. CT:
calcitonin (pg/mL) as measured by the in-house assay. CT-PEG: calcitonin (pg/mL) after polyethylene glycol treatment.

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4 Macrocalcitonin- a new pitfall in the immunoassay J Clin Endocrinol Metab

Figure 2 shows the elution profiles of CT that bind and umn and was only released after the addition of 0.1 M
did not bind protein A Sepharose. As expected, standard glycine-HCl (Figure 2D-F), clearly indicating an immuno-
CT (2A) and serum from patients with metastatic MTC globulin-CT complex.
(2B and 2C) showed immunoreactivity for CT in early Sera from the three reported cases were negative for
elution steps, confirming that CT was not retained in the TG-Ab, TPO-Ab and procalcitonin.
column. However, when samples suspect of assay inter-
ference were applied to the protein A-Sepharose, CT im-
munoreactivity was almost completely retained on the col-

Figure 1. Representative elution profiles of immunometric serum calcitonin observed through ultraperformance liquid chromatography. Elution
distribution from a sample obtained from the washout of fine needle aspiration of a metastatic medullary thyroid carcinoma lymph node (A) and
serum from patients with high (B and C) and low (D) CT recovery after polyethylene glycol treatment. Sera samples were chromatographed on a
Biosep S-3000 column 30 $ 6.7 mm (Phenomenex, Torrance, CA, USA) coupled to an ultraperformance liquid chromatographer.

Figure 2. Calcitonin immunoreactivity after elution via protein A-Sepharose affinity chromatography. WHO International Reference Standard for
Human Calcitonin 89/620, National Institute for Biological Standards and Control (A), serum from patients with metastatic MTC (B and C) and
patients with MTC carrying interference in CT assay (D-F) obtained through the column and the elution on protein A Sepharose.

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doi: 10.1210/jc.2015-3137 press.endocrine.org/journal/jcem 5

Discussion vented by adding an excess of mouse serum during the

routine CT immunometric assay.
To our knowledge, the present study demonstrates for the Another possibility for the unexpectedly high level of
first time the presence of a new interference in the serum calcitonin in these three reported cases could be the pres-
CT immunoassay, herein termed macro-CT. This new in- ence of antibodies against CT in the sera. In fact, the in-
terference results in a falsely increased CT measurement terference of endogenous antibodies during routine im-
and represents a new challenge for MTC management. munoassays has already been described for some proteins
CT is recognized as the cornerstone for the manage- and peptides, especially for prolactin, insulin, GH, LH and
ment of MTC. Screening for MTC in nodular thyroid dis- TSH (21–23), but thus far not for CT. The nature of this
ease using serum CT measurements has identified this tu- phenomenon involves the formation of macroaggregates
mor in 0.3–1.5% of cases (4, 10). In this setting, some between the peptide and immunoglobulins, mainly IgG,
authors argue for CT serum testing in the context of thy- but other molecular modifications, including glycosyla-
roid nodule evaluation to allow for the early diagnosis of tion and covalent/noncovalent binding, are also possible
a potentially more aggressive tumor (1, 14, 15) and to (24). Our findings showed a similar mechanism as dem-
improve the sensitivity of fine-needle aspiration cytology, onstrated by three strategies: demonstration of low recov-
which will actually confirm the diagnosis of MTC in less ery after PEG treatment, the presence of immunoreactivity
than 50% of patients (16). However, this issue is contro- for CT as a macroaggregate in the gel filtration chroma-
versial, and many questions remain unanswered, espe- tography and the presence of CT-immunoglobulin com-
cially regarding cost-effectiveness, the availability of plexes by protein A-Sepharose analysis. However, some
pentagastrin for stimulation testing and the difficult in- care may be needed when finding unexpected high levels
terpretation of false-positive serum CT levels (3, 4). In this of CT. The macro-CT phenomenon may be patient’s sam-
regard, when evaluating preoperative thyroid nodules ple and assay system dependent. An example of this was
with serum CT determination, clinicians should be aware the discrepancy observed between serum calcitonin values
that variable elevations in CT levels may also underlie obtained by the in-house method compared with the com-
several clinical conditions, such as autoimmune thyroid- mercial test. These disagreements have been also observed
itis, chronic renal failure, hypergastrinemia, hyperpara- in the description of other autoimmune macroaggregates
thyroidism, and inflammatory diseases, and numerous tu- phenomena, such as for macroprolactin (25–27), macro-
mors, especially those derived from neuroendocrine tissue TSH (21, 28) and macro-FSH (29). Therefore, it is possible
from the intestine and lungs (3, 17). Depending on the that the same phenomenon is occurring with macro-CT.
immunoassay performed, approximately 10% of normal This could occur due to differences in the affinity of CT for
subjects might have CT levels greater than 10 pg/mL (3). endogenous anti-CT antibody or the antibodies used by
Additionally, the serum CT level varies according to age; each different CT immunoassay. Another possibility is
sex; physical activity; and the use of common drugs such that, for different serum samples, there were anti-CT an-
as proton pump blockers, beta blockers and calcium (17). tibodies directed against different epitopes, which could
During the follow-up of thyroidectomized patients for also influence CT recognition by a specific method (27).
MTC, certain concerns remain because interference in the Therefore, if the endogenous anti-CT antibody binds to a
CT immunoassay cannot be entirely avoided (3). specific CT peptide site required for recognition of an im-
Thus, despite the high reliability and sensitivity of the munoassay, this method will not recognize CT adequately.
new ICLA for CT measurement (18, 19), some concerns Although the exact etiology and frequency of mac-
persist, especially regarding the presence of falsely ele- ro-CT phenomenon remain to be explored, its discovery
vated serum CT levels. With these new immunoassays, opens new perspectives for refining the management of
cross-reactivity with procalcitonin and other related pep- patients with MTC. Additional importance for this work
tides has been almost completely abolished, but the pres- arises if a CT measurement was used in the initial man-
ence of heterophilic antibodies resulting in falsely in- agement of thyroid nodules, as proposed by some authors
creased levels is not negligible and occurs in up to 3.7% of (1, 14, 15). In these cases, additional caution must be
cases (20). taken, especially if the CT measurement was only mod-
In the present study, sera from the three reported cases erately increased (#150 pg/mL). Finally, rather than a
were negative for antithyroid antibodies and procalci- simple immunological artifact, macro-CT recognition is
tonin. Furthermore, the presence of heterophilic antibod- noteworthy because it can help clinicians avoid unneces-
ies were also excluded once the CT results maintained sary diagnostic investigations and treatments during MTC
linearity after dilution; this interference was also pre- follow-up.

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6 Macrocalcitonin- a new pitfall in the immunoassay
Acknowledgments
This work was supported by research grants from the São Paulo
State Research Foundation-FAPESP to RMBM, JGHV, JRMM
and MRDS (2006/60402–1 and 2010/51547–1), by a FAPESP
Fellowship Grant to TGA (2010/19478), The Federal Agency of
Support and Evaluation of Postgraduate Education (Coordena-
ção de Aperfeiçoamento de Pessoal de Nível Superior – CAPES)
and National Council for Scientific and Technological Develop-
ment (CNPq). JGHV and RMBM are investigators of the Fleury
Group. The authors are very grateful to Dr. M. Conceição Ma-
mone for her expertise in thyroid cytopathology, to Alberto L.
Machado for his expertise in neck US, to Gilberto K. Furuzawa
for laboratory help, to Drs. Flávia F. Valente for information
about patients, and to Angela M. Faria and Yeda Queiroga for
efficient laboratory administration. We are very grateful to the
Head and Neck Surgical team, particularly Drs. Flavio C. Hojaij,
Marcel Palumbo and Fabio Brodskyn.
Address all correspondence and requests for reprints to: João
R. M. Martins, MD, Department of Medicine and Department
of Biochemistry, Escola Paulista de Medicina, Universidade Fed-
eral de São Paulo, Brazil. Rua Pedro de Toledo, 669 11th floor,
04 039 – 032, São Paulo, SP, Brazil, Phone/Fax: !55–11-5084 –
5231, Email: jrmmartins.bioq@epm.br.
Disclosure Statements: The authors have nothing to
disclosure
This work was supported by FAPESP, CAPES, CNPq.
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 MOLECULAR MEDICINE REPORTS

Analysis of somatic mutations in BRAF, CDKN2A/p16 and

PI3KCA in patients with medullary thyroid carcinoma
FABRÍCIO P. NASCIMENTO1, MIRIAN G. CARDOSO1,2, SUSAN C. LINDSEY1,
ILDA S. KUNII1, FLÁVIA O.F. VALENTE1, MARINA M.L. KIZYS1, ROSANA DELCELO3,
CLÉBER P. CAMACHO1, RUI M.B. MACIEL1 and MAGNUS R. DIAS‑DA‑SILVA1,2

1
Laboratory of Molecular and Translational Endocrinology, Department of Medicine;
Departments of 2Biochemistry and 3Pathology, Escola Paulista de Medicina,
Universidade Federal de São Paulo, Sao Paulo 04039-032, Brazil

Received December 31, 2014; Accepted October 14, 2015

DOI: 10.3892/mmr.2015.4731

Abstract. Medullary thyroid carcinoma (MTC), a neuroen-
docrine tumor originating from thyroid parafollicular cells,
has been demonstrated to be associated with mutations in
RET, HRAS, KRAS and NRAS. However, the role of other
genes involved in the oncogenesis of neural crest tumors
remains to be fully investigated in MTC. The current study
aimed to investigate the presence of somatic mutations in
BRAF, CDKN2A and PI3KCA in MTC, and to investigate
the correlation with disease progression. DNA was isolated
from paraffin-embedded tumors and blood samples from
patients with MTC, and the hotspot somatic mutations were
sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn
and p.His94Tyr, and polymorphisms within the 3' untranslated
region (UTR) of CDKN2A (rs11515 and rs3088440) were
identified, however, no mutations were observed in other
genes. It was suggested that somatic point mutations in BRAF,
CDKN2A and PI3KCA do not participate in the oncogenesis
of MTC. Further studies are required in order to clarify the
contribution of the polymorphisms identified in the 3'UTR of
CDKN2A in MTC.
Introduction
Medullary thyroid carcinoma (MTC), a neuroendocrine tumor
originating from thyroid parafollicular cells, accounts for
~4% of thyroid cancer cases (1). The majority are sporadic
cases, however, 20-25% occur as a hereditary syndrome termed
multiple endocrine neoplasia type 2 (MEN 2A and MEN 2B)
Correspondence to: Professor Magnus R. Dias-da-Silva, Laboratory
of Molecular and Translational Endocrinology, Department of
Medicine, Escola Paulista de Medicina, Universidade Federal de São
Paulo, 669 Rua Pedro de Toledo, São Paulo 04039‑032, Brazil
E-mail: mrdsilva@unifesp.br
Key words: medullary thyroid cancer, somatic mutation, RET,
BRAF, RAS, CDKN2A, PI3KCA
and as familial MTC, both of which are associated with germ-
line mutations in the RET oncogene (2).
Mutations in the RET oncogene have previously been identi-
fied in the tumor tissue of up to 64% of sporadic MTC cases (3).
In addition, RAS gene mutations are observed in 10% of
RET-negative cases and are associated with a subset of tumors
with less aggressive behavior (4). While certain studies identified
that ~90% of sporadic MTCs exhibited mutually exclusive muta-
tions in RET, HRAS and KRAS (4-8), Moura et al (3) reported
the presence of the RAS mutation in one case with RET-positive
sporadic MTC and Rapa et al (9) identified no RAS mutations
in 49 examined cases. Nevertheless, the clinical phenotype of
sporadic and inherited MTCs is heterogeneous even in the pres-
ence of the same mutation; however the molecular mechanisms
underlying the pathology remain to be fully elucidated.
In addition, it remains unclear whether there is a modula-
tory role in MTC tumor progression for additional genes such
as BRAF, CDKN2A and PI3KCA. These genes participate in
the tumorigenesis of several types of human malignancies such
as tumors derived from neural crest cells, including melanoma,
pheochromocytoma and paraganglioma (10-12).
BR A F, li ke R ET and R A S, is involved in the
mitogen-activated protein kinase pathway and has a
well-established role in the pathogenesis of malignancies such
as melanoma and papillary thyroid cancer (13). Nevertheless,
the contribution in the tumorigenesis of MTC remains contro-
versial. A previous study reported a high prevalence of the
p.Val600Glu BRAF mutation in sporadic MTC cases (14);
however, subsequent studies did not confirm this observa-
tion (3,9,15,16).
An additional tumor suppressor gene, CDKN2A/p16INK4A, is
involved in the G1/S transition in the cell cycle. Mutations and
deletions have been identified in melanoma, and polymorphisms
in its 3' untranslated region (UTR) have been associated with
earlier progression from primary to metastatic disease (17). By
contrast, polymorphisms in another tumor suppressor gene,
CDKN1B, which is in the same CDKN family, are associated
with improved outcomes (18).
Additionally, PI3KCA is a gene that serves an important
role in signaling pathways and cell growth, and contributes to
tumorigenesis in several types of human malignancy (19,20).
2 NASCIMENTO et al: ANALYSIS OF SOMATIC MUTATIONS IN BRAF, CDKN2A AND PI3KCA IN MTC

Table I. Primers used in the present study.

Gene Forward primer Reverse primer

BRAF exon 15 5'‑AACTCAGCAGCATCTCAGGG‑3' 5'‑CTTCATAATGCTTGCTCTGATAG‑3'

CDKN2A exon 1 5'‑ACCCTGGCTCTGACCATTC‑3' 5'‑CAGGTCACGGGCAGAC‑3'
CDKN2A exon 2 5'‑GACCTCAGGTTTCTAACGCC‑3' 5'‑CATATATCTACGTTAAAAGGCAGGAC‑3'
PI3KCA exon 9 5'‑TGGCAGTCAAACCTTCTCTC‑3' 5'‑GAGAAAGTATCTACCTAAATCCACAGA‑3'
PI3KCA exon 20 5'‑AAATGTTTTGGTGTTCTTAATTTATTC‑3' 5'‑GCAGCCAGAACTCTTTATTTTG‑3'
C‑kit exon 9 5'‑GCCAGGGCTTTTGTTTTCTT‑3' 5'‑AGCCTAAACATCCCCTTAAATTG‑3'
C‑kit exon 11 5'‑AACCATTTATTTGTTCTCTCTCCA‑3' 5'‑CCACTGGAGTTCCTTAAAGTCA‑3'
C‑kit exon 17 5'‑TGGTTTTCTTTTCTCCTCCAAC‑3' 5'‑GGACTGTCAAGCAGAGAATGG‑3'
HRAS exon 2 5'‑GGCAGGAGACCCTGTAGGAG‑3' 5'‑AGCTGCTGGCACCTGGAC‑3'
HRAS exon 3 5'‑GTCCCTGAGCCCTGTCCTC‑3' 5'‑CAGCCTCACGGGGTTCAC‑3'
HRAS exon 4 5'‑CTCTCGCTTTCCACCTCTCA‑3' 5'‑GGGTGGAGAGCTGCCTCA‑3'
KRAS exon 2 5'‑TTAACCTTATGTGTGACATGTTCTAA‑3' 5'‑GGTCCTGCACCAGTAATATGC‑3'
KRAS exon 3 5'‑AGACTGTGTTTCTCCCTTCTCA‑3' 5'‑TGGCATTAGCAAAGACTCAAA‑3'
KRAS exon 4 5'‑GATATTTGTGTTACTAATGACTGTGCT‑3' 5'‑TTATGATTTTGCAGAAAACAGATC‑3'
NRAS exon 2 5'‑TCGCCAATTAACCCTGATTAC‑3' 5'‑TCCGACAAGTGAGAGACAGG‑3'
NRAS exon 3 5'‑TGGGCTTGAATAGTTAGATGC‑3' 5'‑AGTGTGGTAACCTCATTTCCC‑3'

However, the role of this gene in the tumorigenesis of MTC
remains to be fully understood.
Therefore, the current study aimed to verify the prevalence
of somatic mutations in BRAF, CDKN2A and PI3KCA, which
have already been described in other neural crest-derived
tumors, and to determine the possible supporting role of these
genes in the tumorigenesis of MTC.
Patients and methods
Patients and tissue samples. From 128 patients with MTC
assessed at the Multiple Endocrine Neoplasia outpatient
clinic at the Universidade Federal de Sao Paulo (Sao Paulo,
Brazil) between February 2007 and June 2013, formalin‑fixed
paraffin‑embedded (FFPE) tumor tissues were selected from
31 patients on the basis of the availability of tumor tissues,
with no other selection criteria. DNA extraction was subse-
quently performed, using an in-house method as previously
described (21). Subsequent to DNA extraction, 20 samples
(from 13 males and 7 females; mean age, 40.55±16.74 years)
provided the appropriate quantity and quality of DNA. The study
was approved by the Ethics and Research Committee of the
Un ive r si d a d e F e d e r a l d e Sa o P a u lo ( p r o t o c ol
number 1945/10), and all patients provided informed consent.
Additionally, 1,092 genotypes of variant frequencies (single
nucleotide polymorphisms; SNPs) were obtained from the
1000 Genomes database (http://www.1000genomes.org/) as a
population genetics control.
DNA extraction and genotyping. DNA from peripheral
blood and somatic DNA from 10-µm sections of FFPE
MTC tissues was extracted using an in-house method as
previously described (21). Polymerase chain reaction (PCR)
was performed to amplify DNA corresponding to hotspot
exons 2, 3 and 4 of HRAS; 2, 3 and 4 of KRAS; 2 and 3 of
NRAS; 15 of BRAF; 9 and 20 of PI3KCA; and exons 2, 3 and
the 3'UTR of the CDKN2A gene. The sequences of the
primers are listed in Table I. The reactions were performed
using 10 pM of each specific primer, 2.5 µl PCR buffer,
200 µM dNTP, 1.5 µM MgCl2 and 0.2 units Taq DNA poly-
merase (Invitrogen; Thermo Fisher Scientific, Waltham, MA,
USA) in a 25-µl total reaction volume. The cycling condi-
tions were as follows: 5 min at 95˚C, 38 cycles of 45 sec at
95˚C, 45 sec for annealing and 1 min at 72˚C, and a final
elongation for 10 min at 72˚C. The PCR products were puri-
fied using the Illustra GFX PCR DNA and Gel Purification kit
(GE Healthcare Life Sciences, Chalfont, UK) and were subject
to sequencing using the Sanger method, with the Big Dye™
Terminator Cycle Sequencing Ready Reaction kit and the
ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems;
Thermo Fisher Scientific). Gel electrophoresis of the PCR
products was performed to analyze product quality and yield
using a 1.8% agarose gel and a DNA ladder.
In silico analysis of HRAS mutations and CDKN2A polymor-
phisms. Mutational analysis of HRAS was performed by the
use of Project HOPE to obtain structural information from the
analysis of PDB‑file 1CTQ (22). The in silico analysis for the
CDKN2A polymorphisms was performed using the Functional
Single Nucleotide Polymorphism database (http://compbio.
cs.queensu.ca/F-SNP/) as previously described (23). This
database provides information regarding potential deleterious
effects of SNPs with respect to splicing, transcription, transla-
tion and post-translation based on SNP functional significance
(FS). The FS score for neutral SNPs is 0.1764, whereas the FS
score for disease-associated SNPs is in the range of 0.5-1.
Statistical analysis. The allele and genotype frequencies were
compared between patients with MTC and the 1000 Genomes
database controls using a χ2 test. The clinicopathological features
of patients carrying each of the polymorphisms rs11515 and
rs3088440 were compared with those of patients without such
 MOLECULAR MEDICINE REPORTS 3

Table II. Summary of patient clinicopathological features and molecular analysis.

Age at Germline RET Somatic Somatic H-, K-, Somatic

Patient Gender diagnosis (y) pTNMa allele RET allele NRAS allele CDKN2A

1 M 28 T2N1bMx WT WT HRAS_p.Asp33Asn rs11515

2 F 25 T3N1bMx WT p.Met918Thr - WT
3 M 38 T1N1aMx WT WT NA rs11515/rs3088440
4 M 56 T3N1bMx WT WT WT WT
5 F 49 T2NxMx WT p.Gln681Stop ‑ WT
6 M 69 T2N0Mx WT WT HRAS_p.Gln61Arg rs3088440
7 M 27 T4N1Mx WT WT WT WT
8 M 51 T3N1bMx WT p.Met918Thr - rs11515
9 F 56 T1N1bMx WT WT HRAS_p.Asp33Asn WT
10 M 41 T4N1bMx WT WT HRAS_p.His94Tyr WT
11 M 27 T1N1aMx p.Cys634Arg ‑ ‑ rs11515/rs3088440
12 F 21 T1N1aMx p.Gly533Cys ‑ ‑ rs11515
13 M 61 T1N1aMx p.Gly533Cys ‑ ‑ WT
14 F 22 T2N0Mx p.Cys634Arg ‑ ‑ rs11515/rs3088440
15 M 43 T2N0Mx p.Cys634Arg ‑ ‑ rs11515
16 M 72 T1N0Mx p.Cys634Arg ‑ ‑ WT
17 M 45 T1N1aMx p.Cys634Arg ‑ ‑ rs3088440
18 F 31 T1NxMx p.Cys634Arg ‑ ‑ rs3088440
19 F 15 T1N1aMx p.Cys634Arg ‑ ‑ rs3088440
20 M 40 T1N0Mx p.Gly533Cys ‑ ‑ WT
a
TNM (Tumor, Node, Metastasis)/American Joint Committee on Cancer staging system. M, male; F, female; y, years; NA, not available; WT,
wild-type.

polymorphisms using the χ2 test or the Student's unpaired t-test
as appropriate. P<0.05 was considered to indicate a statistically
significant difference, and the Hardy-Weinberg equilibrium
was evaluated. Statistical analyses were performed using SPSS,
version 22.0 (IBM SPSS, Armonk, NY, USA) and GraphPad
Prism, version 3.0 (GraphPad Software, Inc., La Jolla, CA, USA).
Results
Screening of the RET, HRAS, KRAS and NRAS genes.
Mutational screening of the RET gene was performed
on all 20 patients. A total of 10 cases were identified to be
familial tumors as confirmed by the presence of a germline
mutation. In total, 30% of the sporadic cases (3/10) presented
with a RET somatic mutation. The clinicopathological features
and molecular analysis, including tumor staging based on the
American Joint Committee in Cancer staging system (24), are
summarized in Table II.
To investigate exclusive causative mutations in cases of
sporadic MTC other than RET mutations, HRAS, KRAS
and NRAS were screened for somatic mutations in the hotspots.
The majority of these patients had been previously analyzed
for RET germline mutations as part of our routine evalua-
tion, and for RET somatic mutations in a previous study (25)
Two novel HRAS mutations, p.Asp33Asn and p.His94Tyr, were
detected in RET-negative MTC tumors. Mutational analysis
using Project HOPE suggests that the p.His94Tyr mutation is
deleterious, and that the p.Asp33Asn mutation is likely to be
damaging (Fig. 1). No differences in the clinical presentation
or histological observations were noted between patients with
MTC that had a mutation in the RAS gene (Table II).
No somatic mutations were identified in exon 15 of BRAF
or in exons 9 and 20 of PI3KCA. Patient 9 was not analyzed for
somatic mutations in PI3KCA due to an insufficient number of
tumor samples.
Despite not having identified somatic mutations in
CDKN2A hotspots, two polymorphisms in the 3'UTR regula-
tory region, 500 C→G (rs11515) and 540 C→T (rs3088440),
were identified in the patients observed. The heterozygotic
pattern of the two SNPs was observed in the same propor-
tion, 7/20 MTC (35%). The genotype distribution was identified
to be in the Hardy-Weinberg equilibrium and was not identi-
fied to exhibit linkage disequilibrium. To investigate whether
the observed polymorphisms were limited to a somatic event,
they were further analyzed in the peripheral blood, which
confirmed germline inheritance. The in silico analysis
demonstrated that the CDKN2A polymorphisms rs11515 and
rs3088440 are located in the transcriptional regulatory region
and that the nucleotide alterations may affect the binding of
transcription factors.
In seven cases, it was possible to detect the presence of
these polymorphisms in the secondary tumors in the lymph
nodes (tumor metastases), however no differences between
the genotypes of the primary and secondary tumors were
observed, indicating that there was no additional somatic event
in CDKN2A involved in the metastatic process. This analysis
4 NASCIMENTO et al: ANALYSIS OF SOMATIC MUTATIONS IN BRAF, CDKN2A AND PI3KCA IN MTC

A B

C D

E F

G H

Figure 1. Mutational analysis of the HRAS somatic mutations p.Asp33Asn and p.His94Tyr. Electropherogram of tumor tissues (A) 1 and (B) 10; (C and D) sequence
alignment of human HRAS protein residues in which the position of the conserved amino acids are indicated (arrows); multiple sequence alignment was
generated with Clustal Omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/), *indicates that the residues in the column were identical in all sequences
in the alignment; schematic structures of the (E) original and (F) mutant amino acids in the two HRAS mutations; (G, H) structure of the HRAS proteins in
ribbon-presentation; gray, protein; magenta, side chain of the mutation (p.Asp33Asn and p.His94Tyr).

was additionally performed for BRAF and PI3KCA in meta-
static tissues.
No associations between the polymorphisms and the clini-
copathological features observed were identified (Table III).
In addition, the frequency of the SNPs was compared with
a population genetics control, and there was no significant
difference between the two populations (Table IV).
Discussion
The adjuvant role of additional genes in the tumorigenesis of
MTC was investigated in the current study through analysis of
tumor tissues from 20 patients. Screening in hotspot regions
of BRAF, CDKN2A and PI3KCA did not identify any somatic
mutations in the coding region. In addition, the results of the
current study were not in agreement with the BRAF muta-
tion frequency of 68.2% observed by Goutas et al (14). This
suggests that BRAF does not serve an important role in the
tumorigenesis of MTC. The observations of the current study
concerning MTC are consistent with a previous study that
demonstrated that somatic mutations in genes other than RET
and RAS are very rare or even absent (5). Notably, the present
study identified two novel HRAS mutations.
Additionally, two common polymorphisms in the 3'-UTR
non-coding region of the gene CDKN2A were identi-
fied, rs11515 and rs3088440 (26). It is known that protein
synthesis can be modulated by regulatory elements located
in the 5'-UTR and 3'-UTR regions. The 3'-UTR, the site of
the polymorphisms identified in the current study, serves an
important role in translation and mRNA stability. Alterations
in this region may be associated with the onset or progression
of disease (27).
These polymorphisms have been investigated in various
tumor types including urinary bladder neoplasm (28),
esophageal adenocarcinoma (29) and cervical cancer (30) as
presented in Table V. The two identified polymorphisms have
 MOLECULAR MEDICINE REPORTS 5

Table III. Correlation between CDKN2A SNPs and clinicopathological features in the patient cohort.

rs11515 (n=20) rs3088440 (n=20)

Clinicopathological --------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------
feature CC (n=13) CG (n=7) P‑value CC (n=13) CT (n=7) P‑value

Gender 0.526 0.474

Male (n=13) 8/13 (61.5%) 5/7 (71.4%) 9/13 (69.2%) 4/7 (57.1%)
Female (n=7) 5/13 (38.5%) 2/7 (28.6%) 4/13 (30.7%) 3/7 (42.9%)
Age at diagnosis 0.088a 0.272a
Mean ± SD (y) 45.41±17.49 31.53±11.36 44.062±17.49 31.53±11.36
Tumor type 0.500 0.175
Sporadic (n=10) 7/13 (53.8%) 3/7 (42.9%) 8/13 (61.5%) 2/7 (28.5%)
Familial (n=10) 6/13 (46.1%) 4/7 (57.1%) 5/13 (38.5%) 5/7 (62.5%)
T category 0.464 0.291
T1 7/13 (53.8%) 2/7(28.5%) 5/13 (38.5%) 4/7 (57.1%)
T2 2/13 (15.3%) 3/7 (42.9%) 3/13 (23.1%) 2/7 (28.5%)
T3 2/13 (15.3%) 2/7 (28.5%) 3/13 (23.1%) 1/7 (14.4%)
T4 2/13 (15.3%) 0/7 (0%) 2/13 (15.3%) 0/7 (0%)
Tumor size 0.421a 0.689a
Mean ± SD (cm) 1.954±1.11 2.34±1.03 2.315±1.22 1.671±0.59
<2 8/13 (61.5%) 2/7(28.6%) 0.378 7/13 (53.8%) 4/7 (57.1%) 0.339
≥2 5/13 (38.5%) 5/7 (71.4%) 6/13 (46.1%) 3/7 (42.9%)
Lymph node metastases 0.742 0.742
N0 4/13 (30.8%) 5/7 (71.4%) 3/13 (23.07%) 3/7 (42.9%)
N1 9/13 (69.2%) 2/7 (28.5%) 10/13 (76.9%) 4/7 (57.1%)
AJCC stage 0.742 0.742
I and II 4/13 (30.7%) 2/7 (28.5%) 4/13 (30.7%) 2/7 (28.5%)
III and IV 9/13 (69.2%) 5/7 (71.4%) 9/13 (69.2%) 5/7 (71.4%)

P-values were obtained using the χ2 test; acontinuous variables analyzed with Student's t-test. SNPs, single nucleotide polymorphisms; SD,
standard deviation; y, years; AJCC, American Joint Committee on Cancer.

Table IV. Comparative analysis of the frequency of the non-coding CDKN2A germ line single nucleotide polymorphisms in
patients with MTC and the control.

A, rs11515

Genotype frequency Allele frequency

------------------------------------------------------------------------ -----------------------------------------------------
Population CC CG GG C (32) G (8) P‑value

MTC 0.60 0.40 ‑ 0.80 0.20 0.25

1,000 genomesa 0.79 0.19 0.02 0.88 0.12

B, rs3088440

Genotype frequency Allele frequency

------------------------------------------------------------------------ -----------------------------------------------------
Population CC CT TT C (31) T (9) P-value

MTC 0.55 0.45 ‑ 0.78 0.22 0.65

1,000 genomesa 0.73 0.24 0.03 0.85 0.15
a
Sequences obtained from the 1000 Genomes database used as a population control. MTC, medullary thyroid carcinoma. The numbers in
parentheses represent the frequency of each allele type in this locus in the studied cohort.
6 NASCIMENTO et al: ANALYSIS OF SOMATIC MUTATIONS IN BRAF, CDKN2A AND PI3KCA IN MTC

Table V. Summary of the studies on CDKN2A polymorphisms in different tumor types.

Source, year (ref) rs11515 (%) rs3088440 (%) Tumor type n Sample Method used

Sauroja et al, 2000 (17) 16.67 16.67 Melanoma 48 Frozen/FFPE tissue PCR-SSCP/
sequencing
Kumar et al, 2001 (26) 25 27.27 Melanoma 229 FFPE tissue PCR-SSCP
Sakano et al, 2003 (28) 18.1 12.9 Bladder 309 Blood PCR-SSCP
Geddert et al, 2005 (29) 13.3 - ADC 315 FFPE tissue PCR-RFLP
Chansaenroj, et al 2013 (30) 7.1 17.9 Cervical 56 Cervical swab Sequencing
Straume et al, 2002 (31) 25 23 Melanoma 185 FFPE tissue PCR-SSCP/
sequencing
Boonstra et al, 2011 (32) 22.07 - EAC 214 FFPE tissue Sequencing
21.05 - ESCC 97 FFPE tissue Sequencing
Pinheiro et al, 2014 (33) 15.63 ‑ HNSCC 96 FFPE tissue PCR‑RFLP
Jin et al, 2012 (34) ‑ 16.7 SGC 156 Blood PCR‑RFLP
Polakova et al, 2008 (35) 25.98 13.07 Colorectal 612 Blood PCR‑RFLP
Royds et al, 2011 (36) 31.78 ‑ GBM 107 Blood Sequencing
Thakur et al, 2012 (37) 13.64 ‑ Cervical 150 Fresh tissue PCR‑RFLP
Zhang et al, 2011 (38) - 17.0 SCCHN 1,287 Blood PCR-RFLP
Zhang et al, 2013 (39) - 20.5 DTC 303 Blood PCR-RFLP
- 20.9 PTC 273 Blood PCR-RFLP
De Giorgi et al, 2014 (40) 16.67 ‑ Melanoma 12 Blood Sequencing
Song et al, 2014 (41) - 33.88 SCCOP 552 Blood PCR-RFLP
Nascimento et al, 2015a 35 35 MTC 20 FFPE tissue + blood Sequencing
a
Indicates the current study. ADC, gastric and esophageal adenocarcinomas; EAC, esophageal adenocarcinoma; ESCC, esophageal squamous
cell carcinoma; GBM, glioblastoma multiforme; SCCHN, squamous cell carcinoma of the head and neck; SGC, salivary gland carcinoma;
DTC, differentiated thyroid carcinoma; PTC, papillary thyroid cancer; HNSCC, head and neck squamous cell carcinoma; SCCOP, squamous
cell carcinoma of the oropharynx; FFPE, formalin‑fixed paraffin‑embedded; PCR; polymerase chain reaction; SSCP, single‑strand conforma-
tion polymorphism; RFLP, restriction fragment length polymorphism.

been previously associated with an earlier progression from
primary to metastatic disease in the case of melanoma (17),
and rs3088440 was associated with the mechanism of tumor
invasion in bladder cancer (28). Controversially, this poly-
morphism has been previously associated with a sub-group
with reduced vertical growth of melanoma and a favorable
outcome (31). However, additional studies have not identified
a clinical correlation with tumor behavior (30,32,33).
Using in silico analysis, the current study identified
that the polymorphisms rs11515 and rs3088440 are located
within a transcriptional regulatory region, and the alteration
of nucleotides can affect the binding of potential transcrip-
tional factors. For example, the presence of the C allele in
rs3088440 favors the binding of the transcription factor
c-Myb, which potentially results in the transcriptional
repression of the CDKN2A gene, compromising its normal
function in cell cycle control (42). However, no association
was identified between this polymorphism and the clinico-
pathological parameters investigated in the cohort studied
(Table III).
In conclusion, it is suggested that BRAF, CDKN2A and
PI3KCA, listed as potential adjuvants in the tumorigenesis
of MTC, do not participate through somatic mutations as
modulators of oncogenesis. To the best of our knowledge, the
current study is the first to investigate these two CDKN2A
polymorphisms in the pathophysiology of MTC. Therefore,
CDKN2A and its regulatory regions and the additional genes
involved in tumorigenesis warrant further investigation in
MTC.
Acknowledgements
The authors would like to thank the team of the Laboratory
of Molecular and Translational Endocrinology, particu-
larly Ms. Teresa Kasamatsu, Mr. Gilberto Furuzawa,
D r Jo ã o Ro b e r t o M a r t i n s, D r Ji Ho o n Ya ng,
Dr Fausto Germano Neto and Mr. Fernando Soares. The authors
would additionally like to acknowledge Mr. Gilmar Miranda
from Siratec Ltd. for graphic art design. The current study was
supported grants from the Sao Paulo State Research Foundation
(grant nos. 2012/11036‑3, 2012/02465‑8, 2012/01628‑0,
2009/50575-4, 2012/00079-3 and 2011/20747-8).
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THYROID
Volume 26, Number 11, 2016
ª Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2016.0255

Multifocality in Sporadic Medullary Thyroid Carcinoma:

An International Multicenter Study

Garth F. Essig Jr.,1 Kyle Porter,2 David Schneider,3 Debora Arpaia,4 Susan C. Lindsey,5 Giulia Busonero,6
Daniel Fineberg,7 Barbara Fruci,8 Kristien Boelaert,9 Johannes W. Smit,10 Johannes Arnoldus Anthonius Meijer,11
Leonidas H. Duntas,12 Neil Sharma,13 Giuseppe Costante,14 Sebastiano Filetti,15 Rebecca S. Sippel,3
Bernadette Biondi,4 Duncan J. Topliss,7 Furio Pacini,6 Rui M.B. Maciel,5 Patrick C. Walz,1 and Richard T. Kloos16,*

Background: Current surgical standard of care in sporadic medullary thyroid carcinoma (sMTC) consists of a
minimum of total thyroidectomy with central neck dissection. Some have suggested thyroid lobectomy with
isthmusectomy and central neck dissection for patients with sMTC, given their lower frequency of bilateral
disease, although this topic has not been thoroughly studied. This study assessed the prevalence of multifocality
in sMTC via a large international multi-institutional retrospective review to quantify this prevalence, including
the impact of geography, to assess more accurately the risks associated with alternative surgical approaches.
Methods: A retrospective chart review of sMTC patients from 11 institutions over 29 years (1983–2011) was
undertaken. Data regarding focality, extent of disease, RET germline analysis plus family and clinical history
for multiple endocrine neoplasia type 2 (MEN2), and demographic data were collected and analyzed.
Results: Patients from four continents and seven countries were included in the sample. Data for 313 patients with
documented sMTC were collected. Of these, 81.2% were confirmed with negative RET germline testing, while the
remaining 18.8% demonstrated a negative family history and no manifestations of MEN2 syndromes other than MTC.
Bilateral disease was identified in 17/306 (5.6%) patients, while multifocal disease was noted in 50/312 (16.0%)
sMTC patients. When only accounting for germline negative patients, these rates were not significantly different
(5.6% and 17%, respectively). Among them, when disease was unifocal in the ipsilateral lobe and isthmus, bilateral
disease was present in 6/212 (2.8%) cases. When disease was multifocal in the ipsilateral lobe or isthmus, then
bilateral disease was present in 8/37 (21.6%) cases ( p < 0.001). No geographic differences in focality were identified.
Conclusions: The 5.6% prevalence of bilateral foci in sMTC suggests that total thyroidectomy should remain the
standard of care for initial surgery, as less complete thyroid surgery may fail to address fully the primary site of
disease. Whether ipsilateral tumor focality should be an independent factor determining the need for completion
thyroidectomy when sMTC is diagnosed after hemithyroidectomy remains to be determined.

Keywords: multifocality, medullary thyroid carcinoma, humans, thyroid neoplasm, surgery

1
Department of Otolaryngology—Head and Neck Surgery; 2Center for Biostatistics; The Ohio State University Wexner Medical Center,
Columbus, Ohio.
3
Section of Endocrine Surgery, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
4
Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy.
5
Division of Endocrinology, Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Federal University of
Sao Paulo, São Paulo, Brazil.
6
Section of Endocrinology and Metabolism, Department of Medical, Surgical, and Neurological Sciences, University of Siena, Siena, Italy.
7
Department of Endocrinology and Diabetes, Alfred Health, Monash University, Melbourne, Australia.
8
Département of Endocrinology and Nephrology, Pierre Oudot Hospital, Bourgoin-Jallieu, France.
9
School of Clinical and Experimental Medicine, Centre for Endocrinology, Diabetes, and Metabolism, Institute of Biomedical Research;
13
Institute of Head and Neck Studies and Education; University of Birmingham, Birmingham, United Kingdom.
10
Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands.
11
Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, The Netherlands.
12
Evgenidion Hospital, Unit of Endocrinology, Diabetes and Metabolism, Thyroid Section, University of Athens, Athens, Greece.
14
Department of Medicine, Institut Jules Bordet, Brussels, Belgium.
15
Dipartimento Di Medicina Interna, University of Roma La Sapienza, Rome, Italy.
16
Veracyte, Inc., South San Francisco, California.
*Previous affiliation: Divisions of Endocrinology, Diabetes and Metabolism, and Nuclear Medicine, The Ohio State University Wexner
Medical Center, Columbus, Ohio.

1563
1564
Introduction
M edullary thyroid carcinoma (MTC) accounted for
2.2% of all thyroid cancer in a database of 43,644 thy-
roid cancer cases from 1992 to 2006 in the United States (1),
and historically is observed sporadically in approximately 75–
80% of patients while the remainder have a hereditary form
(multiple endocrine neoplasia type 2 [MEN2], including the
familial MTC variant) (2) transmitted with autosomal dom-
inant patterns of inheritance by mutation of the REarranged
during Transfection (RET) proto-oncogene (3–7).
Surgery is the only curative treatment for both hereditary
and sporadic forms of MTC, and its chance of success is
greatest when the disease is confined to the thyroid, or when
minimal lymph node metastatic disease is present (8). Total
thyroidectomy, central neck dissection, and therapeutic dis-
section of involved lateral neck compartments, with or without
prophylactic dissection of clinically uninvolved compartments
(levels II–V), is the current recommended treatment for pa-
tients with MTC (2,9). Because the hereditary form of MTC is
due to an oncogenic germline mutation in the RET proto-
oncogene, virtually all patients are at risk for bilateral and
multifocal disease (10–12), which justifies the need for total
thyroidectomy, whether prophylactic (ideal) or therapeutic.
Therapeutic total thyroidectomy in addition to lymph node
dissection has also been generally recommended for patients
with sporadic MTC (sMTC). However, it has been noted that
tumor multifocality and bilaterality occurs less frequently
compared with the hereditary form (12). Based upon these
findings, the argument has been made for unilateral thyroid-
ectomy (lobectomy and isthmusectomy) with a lymph node
dissection for sMTC that is confined to one lobe (13), short-
ening operating time and potentially lowering the risks of
hypoparathyroidism, hypothyroidism, recurrent laryngeal
nerve injury, voice changes, and other surgical complications
compared with total thyroidectomy (14–21).
The prevalence of bilateral foci in sMTC has been reported
to range widely from 0% to 66.7% (13,22–37), though these
reports were not all based upon germline DNA testing to
exclude MEN2. Several studies from single institutions have
more systematically applied RET mutation testing and stan-
dardized pathological analyses to investigate the occurrence
of multifocal or bilateral disease more accurately among
sMTC patients. Still, most of these patient cohorts were
small, and some inconsistent results were seen (Table 1). The
objective of this investigation was to evaluate the prevalence,
and possible correlation, of multifocality and bilaterality in
sMTC. A large, international, multi-institutional retrospective
investigation was conducted to quantify this occurrence in a
large and diverse population to limit random or biased findings
from small cohorts and to test the impact of geography. These
analyses allow the role of alternative surgical approaches in the
current treatment of sMTC to be considered.
Methods
Institutional Review Board approval
and center recruitment
Institutional Review Board (IRB) approval was obtained
from the primary author’s institution prior to proceeding.
Next, a data collection sheet was generated with explicit in-
structions for de-identified data collection to standardize data
ESSIG ET AL.
input. Fifty-three institutions in 11 countries were invited to
participate in data collection. IRB approval was obtained from
each participating institution per their institutional protocols.
Subsequently, the data were reviewed for internal agreement
and standardized for data analysis. The seventh edition of the
American Joint Committee on Cancer’s (AJCC) staging man-
ual was utilized for tumor staging (38).
Data collection, definitions, and inclusion criteria
Retrospective chart review was performed on all patients
with a diagnosis of MTC at each institution, and data were
collected for all patients with sMTC. sMTC was defined as
MTC in either (i) the presence of a negative RET germline
analysis, or (ii) the absence of a family history of MTC and no
evidence of other manifestations of MEN syndromes. De-
mographic data are reviewed in Table 2. Bilateral disease in
sMTC was defined as the presence of the sMTC diagnosis in
foci in both lobes of the thyroid gland. Cases of multifocal
disease within one lobe and the isthmus were not included in
this definition. Only cases in which a pathologist assessed
both thyroid lobes were included in the assessment of bilat-
eral disease, excluding patients who did not undergo surgery
after diagnosis of sMTC due to medical comorbidities and
patients who underwent unilateral thyroid lobectomy without
completion thyroidectomy. Multifocal disease in sMTC was
defined as the presence of the sMTC diagnosis in more than
one pathologic focus within the thyroid gland, either in the
same lobe or the contralateral lobe. By definition, all bilateral
cases were considered multifocal. Only patients who did not
undergo surgery after diagnosis of sMTC were excluded.
Statistical analysis
Bilateral and multifocal sMTC cases were identified. The
number and percentage of each (denominator = all included
sMTC cases based on above described criteria) are reported
with exact binomial confidence intervals for the percentage
calculated for each. Counts and percentages with exact bi-
nomial confidence intervals are also reported by study site,
FNA result, T stage, N stage, M stage, and age quartile. Within
each of these categories, chi-square tests were performed to
assess differences in bilateral and multifocal disease. Sig-
nificant omnibus chi-square tests were followed up with all
pairwise comparisons by chi-square or Fisher’s exact tests, as
appropriate. All tests were evaluated at the a = 0.05 signifi-
cance level. Data were analyzed using SAS/STAT! v9.2 (SAS
Institute, Inc., Cary, NC).
Results
Subject recruitment
Of the 53 institutions invited, 13 agreed to participate,
and 11 ultimately submitted data. Responding institutions
represented seven nations and four continents. From these
centers presenting for management from 1983 to 2011, 313
patients with biopsy-proven sMTC were identified. One pa-
tient elected for nonoperative management of biopsy-proven
sMTC and was excluded from this analysis. The remaining
312 operated patients were included in analyses of multi-
focality. Average age at first surgical intervention was 52 – 15
years (range 19–101 years). While 81.2% of patients had
negative RET proto-oncogene testing supporting sMTC, the
MULTIFOCALITY IN SPORADIC MEDULLARY CARCINOMA 1565

Table 1. Summary of Literature Evaluating Multifocal and/or Bilateral sMTC

Publication
Lead Total Total % Bilateral % Multifocal country of
author Year patients sMTC disease disease Definition of sMTC origin
Zedenius (27) 1995 46 46 n/a 4.3 Clinically sporadic, no familial occurrence; Sweden
two with multifocality and C-cell
hyperplasia; germline tested exons 10,
11, 16, and negative
Scopsi (22) 1996 109 93 21.5 43 Negative familial screening for MTC or Italy
pheochromocytoma, and basal or
pentagastrin-stimulated calcitonin
Beressi (23) 1998 899 83 3.9a 20 Isolated MTC, no familial history of MTC France
or MEN2, no bilateral C-cell hyperplasia
Kebebew (24) 2000 104 58 66.7 n/a No relative with MEN2; 17% without United States
germline mutation in exons 10, 11,
13, and 14
Dolan (28) 2000 38 23b 0 0 Not explicitly stated Ireland
Weber (29) 2001 36 16 n/a 19 Not explicitly stated Germany
Kaserer (30) 2001 50 34 8.8 8.8 Not carriers of mutations in exons 10, 11, Austria
and 13–16
Miyauchi (13) 2002 94 40 0 18.3c No familial occurrence, MEN2 or germline Japan
RET mutation
Miyauchi (13) 2002 94 20 5 18.3c Apparently sporadic, no familial occurrence Japan
or MEN2, but no RET testing
Scollo (31) 2003 101 54 13 n/a Absent germline mutation when screened, France
negative familial MTC and pentagastrin
screening
Guyétant (32) 2003 66 27 3.7 14.8 Not carriers of mutations in exons 8, 10, France
11, 13–16
Hamy (33) 2005 43 43 n/a 25.6 Not carriers of mutations in exons 10, France
11, 13–16
Chow (34) 2005 22 19 10.5 21.1 All 12 patients with multifocal or bilateral Hong Kong
disease germline tested (three mutated)
Gulben (25) 2006 32 32 28 n/a Physical examination, absence of Turkey
pheochromocytoma, no familial
occurrence, normal serum calcium
Machens (37) 2007 232 126 4.8 8 Not carriers of mutations in exons 10, Germany
11, and 13–16
Scheuba (35) 2007 97 88 21.6 22.7 Not carriers of mutations in exons 5, 8, Austria
10, 11, and 13–16
Moura (36) 2009 52 51 n/a 25 Not carriers of mutations in exons 5, 8, Portugal
and 10–16
Current Study 2016 312 312 5.6 16 No familial occurrence, MEN2 or International
germline RET mutation (81.2% tested)
a
Only micro sMTC included in analysis, bilaterality in 3/76 who underwent bilateral surgery.
b
Twenty-three patients were noted to have sMTC; only nine underwent total thyroidectomy, allowing evaluation of both focality and
laterality.
c
Reported only among the combined cohorts (n = 60) as intraglandular metastases.
sMTC, sporadic medullary thyroid carcinoma; MEN2, multiple endocrine neoplasia type 2.

remaining 18.8% were untested and classified as sMTC based
upon negative family history and absence of other findings of
MEN syndromes. Six patients underwent thyroid lobectomy
alone and were excluded from analysis of bilateral disease
(n = 306; Fig. 1).
Bilateral disease
Of the 306 sMTC patients who underwent bilateral thyroid
surgery, 17 (5.6%) [confidence interval (CI 3.3–8.8%] dem-
onstrated pathologic evidence of bilateral disease. Of these,
documentation of the dimensions of the secondary tumor
were only included for 10/17 specimens. For the subset of
bilateral tumors, the median size of the primary site was
1.6 cm (range 0.3–7.2 cm), while the largest contralateral
MTC focus was 0.8 cm (range 0.06–4.8 cm). When only ac-
counting for germline RET negative patients, bilateral disease
was present in 14/249 patients (5.6% [CI 3.1–9.3%]). In pa-
tients without germline RET mutations, when disease was
unifocal in the ipsilateral lobe and isthmus, bilateral disease
1566 ESSIG ET AL.

Table 2. Demographic, Pathologic, and Treatment Information Collected for Each Subject
Median age at initial surgery 51 years (range 19–101 years)
RET oncogene testing status
Negative 253 (81.1)
Not tested 59 (18.9)
Preoperative clinical diagnosis
MTC 106 (34)
Possible MTC 6 (1.9)
Othera 200 (64.1)
Extent of thyroidectomy
Total thyroidectomy 256 (82.1)
Lobectomy with completion 50 (16)
Lobectomy only 6 (1.9)
Extent of central neck dissection
Prophylactic Ipsilateral 93 (29.8); contralateral 73 (23.4)
Therapeutic Ipsilateral 87 (27.9); contralateral 76 (24.4)
Not done Ipsilateral 93 (29.8); contralateral 124 (39.7)
Unable to assess Ipsilateral 39 (12.5); contralateral 39 (12.5)
Extent of lateral neck dissection
Prophylactic Ipsilateral 40 (12.8); contralateral 15 (4.8)
Therapeutic Ipsilateral 90 (28.8); contralateral 32 (10.3)
Not done Ipsilateral 162 (51.9); contralateral 252 (80.8)
Unable to assess Ipsilateral 20 (6.4); contralateral 13 (4.2)
TNM stage based upon AJCC 7th edition All tumors: n = 312; multifocal: n = 50; bilateral: n = 17
Stage I All tumors: 93 (29.8); multifocal: 7 (14); bilateral: 3 (17.6)
Stage II All tumors: 58 (18.6); multifocal: 0 (0); bilateral: 0 (0)
Stage III All tumors: 22 (7.1); multifocal: 5 (10); bilateral: 1 (5.9)
Stage IVa All tumors: 87 (27.9); multifocal: 21 (42); bilateral: 8 (47.1)
Stage IVb All tumors: 4 (1.3); multifocal: 2 (4); bilateral: 1 (5.9)
Stage IVc All tumors: 44 (14.1); multifocal: 14 (28); bilateral: 3 (17.6)
Unable to stage All tumors: 4 (1.3); multifocal: 1 (2); bilateral: 1 (5.9)
Extrathyroidal extension (at any focus)
Present 83 (26.6)
Not present 215 (68.9)
Unknown 14 (4.4)
Lymphovascular invasion
Present 109 (34.9)
Not present 158 (50.6)
Unknown 45 (14.4)
Tumor maximum dimension
Primary tumors 20 mm (range 1–120 mm) in 286 (91.7%); not recorded in 26 (8.3%)
Contralateral tumors 8 mm (range 0.6–48 mm) in 9 (52.9%); not recorded in 8 (47.1%)
Number and location of tumor focib
Ipsilateral N = 286/312c; median = 1; range 0–11d
Isthmus N = 9/17c; median = 0; range 0–2
Contralateral N = 10/17c; median = 0; range 0–3
Dimensions of sMTC focib
Ipsilateral N = 301/286e; median = 18; range 0–120d
Isthmus N = 10/9e; median = 6; range 2–40
Contralateral N = 11/9e; median = 6; range 0.6–48
Values are presented as n (%) or median (range) as appropriate.
a
Details are provided on the subset of 245 patients who underwent preoperative fine-needle aspiration in Essig et al. (39).
b
Calculated from patients with specific information regarding size and number of foci available.
c
Number of patients with specific data reported/number of patients with disease reported in the location (e.g., 17 patients had disease
reported in the contralateral lobe, but only 10 reported the exact number and size of foci present).
d
Several patients with primary tumor in isthmus with no reported lobe focus were assigned ‘‘0’’ for the number and size of ipsilateral foci.
e
Number of foci with specific data reported/number of patients with discrete data reported. (e.g., the exact number and size of foci in the
contralateral lobe were reported in 9 patients, which totaled to 11 foci). The numerator exceeds the denominator due to multifocality in the
location.
TNM, tumor, node, metastasis staging system; AJCC, American Joint Committee on Cancer.
MULTIFOCALITY IN SPORADIC MEDULLARY CARCINOMA 1567

FIG 1. Schematic account-

ing for all patients identified
in this retrospective multi-
center study.

was present in 6/212 (2.8%) cases. When disease was mul- Age analysis. Age quartiles were not significantly asso-
tifocal in the ipsilateral lobe or isthmus, then bilateral disease ciated with prevalence of bilateral sMTC. However, the
was present in 8/37 (21.6%) cases ( p < 0.001). majority of bilateral sMTC (11/17; 64.7%) was noted in the
younger half of the sample.

Staging analysis. When prevalence of bilateral sMTC Regional analysis. The prevalence of bilateral sMTC
was analyzed in terms of the seventh edition of AJCC TNM among the sites surveyed ranged broadly (0–17.7%; Table 4),
staging, there was no linear association with increasing T but there was no statistically significant difference in preva-
stage and prevalence of bilateral disease. However, a statis- lence among the sites ( p = 0.72).
tically significant increase in the prevalence of bilateral
sMTC was noted when comparing T4a, T4b, and T3 sMTC Multifocal disease
with T2 disease ( p = 0.02 for each comparison; Table 3).
There was no significant difference between T2 and lower For analysis of multifocal disease, the original 313 patients
stage disease in the prevalence of bilateral disease. There was were reviewed, and only the single patient who elected
no association between nodal status or metastatic status and nonoperative management was excluded. Of the remaining
prevalence of bilateral sMTC. 312 patients, 50 were found to have multifocal disease (16%
[CI 12–21%]; Fig. 1). Of these 50 multifocal patients, 17 had
bilateral disease, 42 were multifocal only on the ipsilateral
side, and one was multifocal ipsilaterally but did not undergo
Table 3. Bilateral sMTC Analysis by T Stage resection of the contralateral lobe to allow bilaterality to be
determined. When only accounting for known germline RET
T stage Bilateral/all sMTC CI negative patients, multifocal disease (not bilateral) was present
0–1a a
5/77 (6.5%) 2–15% in 11.5% (29/253) of patients, and unifocal tumors were found
1b 1/51 (2.0%) 0–10% in 83% (210/253) of patients.
2 0/71 (0%) 0–5%
3 5/61 (8.2%)* 3–18% Staging analysis. When prevalence of multifocal sMTC
4aa 3/26 (11.5%)* 2–30% was analyzed in terms of the seventh edition of the AJCC TNM
4b 2/11 (18.2%)* 2–52% staging (38), there was no linear association with increasing T
X 1/9 (11.1%) 0–48% stage and prevalence of bilateral disease. However, a statisti-
Staging based upon AJCC 7th edition. Comparisons not statis- cally significant increase in the prevalence of multifocal sMTC
tically significant unless stated. was noted when comparing T4a, T4b, and T3 sMTC with T2
a
T0–1a includes one patient with a nodal focus of sMTC without disease ( p = 0.001, 0.002, and 0.004, respectively). Multi-
disease identified in the thyroid, and one patient quantified as T1 but focality was also significantly more common in T4b disease
not further characterized. T4a includes four patients quantified as
T4 but not further characterized. compared with T3, T1b, and T0–1a disease ( p = 0.03, 0.04,
*p = 0.02. and 0.001, respectively). Remaining comparisons between T
CI, confidence interval; X, stage unknown. stages were not significantly different (Table 5). Multifocal
1568 ESSIG ET AL.

Table 4. Bilateral sMTC Analysis by Region Table 6. Multifocal sMTC Analysis by Nodal
and Metastatic Staging
Bilateral/
Country Site all sMTC CI Multifocal/all sMTC CI
Australia Melbourne 3/17 (17.7%) 4–43% N stage
Brazil Sao Pauloa 3/70 (4.3%) 1–12% 0 4/85 (5%) 0–8%
Greece Athens 0/7 (0%) 0–41% 1aa 5/32 (16%) 0–16%
Holland Multiple sites 0/21 (0%) 0–16% 1b 37/121 (31%)* 5–17%
Italy Catanzaro 0/10 (0%) 0–31% X 4/74 (5%) 0–9%
Naples 0/11 (0%) 0–28% M stage
Rome 4/34 (11.8%) 3–27% 0 15/121 (12%) 7–20%
Sienna 3/56 (5.4%) 1–15% 1 16/44 (36%)** 22–52%
United Kingdom Birmingham 1/21 (4.8%) 0–24% X 19/147 (13%) 8–20%
United States Columbus, OH 2/48 (4.2%) 1–14%
Madison, WI 1/11 (9.1%) 0–41% Staging based upon AJCC 7th edition. Comparisons not statis-
tically significant unless stated.
a
There was no statistically significant difference in prevalence N1a includes five patients quantified as N1 but not further
among the sites ( p = 0.72). characterized.
a
Exons 8, 10, 11, and 13–16 of the RET gene were sequenced in *p < 0.001; **p = 0.001.
their entirety. Forty-five of these patients underwent testing exons
1–7, 9, 12, 17, 18, and 19, and part of a 50 sMTC patient study that
found no additional causative mutations (40).
ment for patients with MTC (2). This is well supported in
patients with hereditary MTC who present with clinical dis-
disease was also significantly more common in N1b compared ease or significantly elevated serum calcitonin levels, as there
with N0 patients ( p < 0.001). There were no other significant is a high prevalence of tumor bilaterality and multifocality,
associations with nodal status (Table 6). With respect to met- necessitating removal of the thyroid gland in its entirety (2).
astatic disease, there was a significantly greater prevalence of Total thyroidectomy is also recommended in sMTC, despite
multifocality in those with distant metastatic disease compared the fact that the disease tends to be a unicentric process
with those without ( p = 0.001; Table 6). confined to one lobe. This is historically justified by the
possibilities of bilateral primary disease or intraglandular
Age analysis. The prevalence of multifocal sMTC in the metastases, the possibility of inherited disease when RET
youngest quartile of the sample (26%) was significantly greater proto-oncogene testing has not been performed or was neg-
than the third and fourth quartiles (8% and 9%, respectively; ative, and to optimize surgical access for bilateral central
p = 0.003 and p = 0.01 for each comparison). neck lymph node dissection (10–12). Still, some authors have
recommended hemithyroidectomy (lobectomy with isthmu-
sectomy) with central and ipsilateral lateral neck dissection
Regional analysis. The prevalence of multifocal sMTC based on the absence of RET proto-oncogene mutation and on
among the sites surveyed ranged broadly (0–24%), but there the macroscopic extent of the primary tumor (13,41). Ito et al.
was no statistically significant difference in prevalence among managed a series of patients with this approach in Japan, and
the sites ( p = 0.23; Table 7). reported clinical outcomes better than those from Western
countries, and none of their patients showed disease
Discussion
Total thyroidectomy with central neck dissection, with or
without lateral neck dissection, is a well-established treat- Table 7. Multifocal sMTC Analysis by Region
Multifocal/
Table 5. Multifocal sMTC Analysis by T Stage Country Site all sMTC CI

T stage Multifocal/all sMTC CI Australia Melbourne 4/17 (24%) 7–50%

Brazil Sao Pauloa 10/71 (14%) 7–24%
0–1aa 11/79 (14%) 7–24% Greece Athens 1/7 (14%) 0–58%
1b 6/52 (12%) 4–23% Holland Multiple sites 1/21 (5%) 0–24%
2 4/72 (6%) 1–14% Italy Catanzaro 0/10 (0%) 0–31%
3 14/62 (23%)* 13–35% Naples 0/12 (0%) 0–26%
4aa 8/26 (31%)** 14–52% Rome 8/35 (23%) 10–40%
4b 6/11 (55%)*** 23–83% Sienna 11/56 (20%) 10–32%
X 1/10 (10%) 0–45% United Kingdom Birmingham 1/21 (5%) 0–24%
United States Columbus, OH 11/49 (22%) 12–37%
Staging based upon AJCC 7th edition. Comparisons not statis- Madison, WI 3/13 (23%) 5–54%
tically significant unless stated.
a
T0–1a includes one patient with a nodal focus of sMTC without No statistically significant difference in prevalence among the
disease identified in the thyroid, and one patient quantified as T1 but sites ( p = 0.23).
a
not further characterized. T4a includes four patients quantified as Exons 8, 10, 11, and 13–16 of the RET gene were sequenced in
T4 but not further characterized. their entirety. Forty-five of these patients underwent testing exons
*p = 0.004; **p = 0.002; ***p = 0.03, <0.001, 0.04, and 0.001 1–7, 9, 12, 17, 18, and 19 and part of a 50 sMTC patient study that
compared with T3, T2, T1b, and T1a, respectively. found no additional causative mutations (40).
MULTIFOCALITY IN SPORADIC MEDULLARY CARCINOMA
recurrence in the thyroid remnant (41). These authors noted
that previous treatment recommendations focused more on
the debate of using central and lateral nodal dissection, while
their approach instead focused on the extent of thyroidec-
tomy (while all patients underwent ipsilateral central and
lateral neck dissections). Total thyroidectomy (as well as
bilateral central neck dissection) places the bilateral recurrent
and superior laryngeal nerves and all parathyroid glands
at risk. In comparison, hemithyroidectomy (lobectomy with
isthmusectomy) and ipsilateral central neck dissection places
only the ipsilateral nerves at risk and lowers the risk of
permanent hypoparathyroidism by leaving undisturbed
parathyroid tissue on the contralateral side. Further, hemi-
thyroidectomy significantly reduces the risk of long-term
hypothyroidism requiring thyroid hormone replacement
compared with bilateral thyroidectomy (21). In the surgical
treatment for sMTC, these surgical risks must be weighed
against the risk of persistent disease. Regarding the currently
recommended extent of thyroid surgery according to guide-
lines, the American Thyroid Association (ATA) and the
National Comprehensive Cancer Network (NCCN) recom-
mend total thyroidectomy as opposed to hemithyroidectomy
(2,9). In patients diagnosed with apparent sMTC following
a hemithyroidectomy, these guidelines indicate that RET
germline mutation status, serum calcitonin, and the results of
imaging studies should determine the role of completion
thyroidectomy (2,9). It is important to note that this approach
does not include tumor focality in this consideration. This
may be particularly relevant for patients with multifocal
disease in the ipsilateral lobe or isthmus, as 21.6% of them
were found to be harboring disease in the contralateral lobe.
While serum calcitonin and ultrasonography may safely in-
dicate the need for completion thyroidectomy (immediately
or subsequently) in similar patients treated with a hemi-
thyroidectomy, especially those with macroscopic MTC in
the remaining lobe, it is unknown if patient outcomes are
impaired by not including tumor multifocality as an inde-
pendent clinical factor to guide the completion thyroidec-
tomy decision. In the present cohort, the median size of the
largest contralateral MTC focus was 8 mm (range 0.6–
48 mm), suggesting that a significant fraction of patients with
bilateral disease would have had negative or inconclusive
preoperative ultrasound findings of the contralateral lobe. It is
also important to recognize that bilateral disease did occur,
even in sMTC patients with unifocal disease in the ipsilateral
lobe and isthmus, suggesting that all patients who chose to be
treated with an initial lobectomy require at least ongoing
careful surveillance.
The consequences of persistent disease are believed to be
significant. Complete surgical resection when the disease is
confined to the neck is currently the only curative treatment
of MTC (sporadic or hereditary) (11,42). Residual primary
tumor in a remaining thyroid lobe could potentially give rise
to metastatic foci, a disease state associated with a signifi-
cantly reduced rate of biochemical cure. Duh et al. reported
that patients who underwent less surgery than routine total
thyroidectomy and central neck dissection have been shown
to require more reoperations (43). In a study evaluating
prognostic factors in sMTC, Gulben et al. reported in a
multivariate analysis that the extent of the primary surgical
resection was an independent risk for survival (25). Patients
with incomplete resection had a 4.8 times higher risk of death
1569
than those who had a complete resection. In their sample,
28% of tumors were noted to be bilateral. Panigrahi et al.
reported that patients receiving surgery discordant with ATA
guideline recommendations had shorter survival than those
receiving surgery according to recommendations (44). Still,
not all studies support these findings. Esfandiari et al. re-
ported that for tumors £2 cm without distant metastases,
overall survival was not altered by surgical intervention,
while all surgical interventions similarly improved survival
compared to no surgery for tumors >2 cm without distant
metastases (45).
The frequency of bilateral disease in sMTC varies widely
from 0% to 66.7%, as illustrated in Table 1, although the
frequency was lower in series with more thorough germline
RET proto-oncogene testing (13,22–36). The present findings
show that 17/306 (5.6%) patients had bilateral disease, which
is comparable to the median value of 7.2% among the pre-
vious reports shown in Table 1. To account for any geo-
graphical variances in focality and bilaterality, 313 patients
with biopsy proven sMTC were identified from 11 centers
within seven nations and four continents. The prevalence of
bilateral sMTC among the sites surveyed ranged broadly (0–
18%; Table 4). There were no statistically significant dif-
ferences in prevalence among the geographic locations. Al-
though there was no linear association with increasing T
stage and prevalence of bilateral disease, there was a statis-
tically significant increase in the prevalence of bilateral
sMTC when comparing advanced T stage lesions with T2
tumors. Still, among T2 and lower tumors, the prevalence of
bilaterality was 4.5%.
Multifocal tumors have also been shown to be indepen-
dent risk factors for nodal metastasis, and may explain why
tumor size alone does not always predict lymph node me-
tastasis (23,37). Multifocal disease was found to be signifi-
cantly more common in N1b compared with N0 patients
( p < 0.001). With respect to distant metastatic disease, there
was a significantly greater prevalence of multifocality in
those with metastatic disease compared with those without.
The prevalence of multifocal sMTC in the youngest quartile
of the sample (26%) was significantly greater than the third
and fourth quartiles. One may speculate that this is due to
unrecognized hereditary MTC, despite their negative family
histories, or negative germline RET proto-oncogene analysis.
Lindsey et al. studied 50 patients for this question (40), 45 of
whom are included in this study. Their 50 patient cohort had
been screened by sequencing exons 8, 10, 11, and 13–16 of
the RET gene in their entirety. Still, 27 patients had one or
more features suggesting familial disease, such as a young
age of onset of MTC (<35 years of age; 16 patients), tumor
multifocality (13 patients), or the presence of C cell hyper-
plasia (10 patients). Four patients had both young age at di-
agnosis (in these cases <30 years old) and multifocal disease.
Those 50 patients underwent extended RET testing of exons
1–7, 9, 12, and 17–19 with the identification of no new
causative mutations. The present study identified multifocal
tumors in 50/312 (16%) patients, which is consistent with the
median value of 18.3% among the prior studies shown in
Table 1 (range 0–43%). There were no statistically significant
differences in prevalence among the geographic locations. As
with bilateral disease, there was no linear association with
increasing T stage and prevalence of multifocal disease. As
with bilateral disease, there was a statistically significant
1570
increase in the prevalence of multifocal sMTC when com-
paring advanced versus early T-staged tumors.
The strengths of the current study of the rate of multi-
focality and bilaterality for clinically sMTC include the large
number of patients accrued from 11 centers across seven
countries, most of whom had negative RET germline testing.
To the authors’ knowledge, this is the largest study of its kind.
The geographic diversity of the patients provides balance to
institutional discrepancies and establishes a broad benchmark
for the prevalence of multifocal and bilateral disease in
sMTC. Preoperative analysis of RET gene mutation is an
important point of differentiation, as up to 17% of patients
with apparent sMTC with no family history or diagnostic
criteria for MEN2A have been found to harbor germline RET
gene mutations (26). While separate results for the entire
cohort and those with known negative analyses for germline
RET mutations are provided, these results were not statisti-
cally different. Only 3/17 patients with bilateral disease did
not undergo germline RET mutational analysis.
The limitations of the current study include its retrospec-
tive nature and the lack of standardized protocol in assessing
surgical specimens (such as number of tissue sections ex-
amined or use of calcitonin immunohistochemistry) or extent
of RET gene analysis (e.g., rare or newly identified variants
associated with inherited MTC may not have been investi-
gated). Most familial MTC cases are identified when testing
the most commonly affected exons, so additional testing for
rare mutations is unlikely to change the findings meaning-
fully. In Tables 4 and 7, it should be noted that the Sao Paulo
site contributed the largest fraction of patients to this study
and routinely sequenced exons 8, 10, 11, and 13–16 of the
RET gene in their entirety, and 45 of these patients also un-
derwent extended testing of exons 1–7, 9, 12, 17, 18, and 19
(40). The Sao Paulo rates of bilaterality and multifocality are
consistent with those of the entire cohort, and add credibility
and generalizability to the findings. Additional limitations
include the lack of standardized use and extent of lymph node
surgery in sMTC among the participating centers, and the N
stage may have been underestimated in patients who did not
undergo maximal lymph node dissection. Finally, 312 pa-
tients were included in the analyses of multifocality, and 306
patients in the analyses of bilaterality. The difference was
that six patients underwent only hemithyroidectomy (Fig. 1).
Unifocal disease was found in five of these patients, and
multifocal disease in the remaining patient. These five (or six)
patients could have been excluded from the multifocality
analyses, since the status of the contralateral lobe was un-
known, although it is unlikely that the findings would have
changed significantly, as the unifocal patients would have
required finding disease in the contralateral lobe to change
their focality status, and the data demonstrate that the prev-
alence of this occurring is low.
Conclusion
On average, 1/18 patients with sMTC have bilateral tu-
mor foci in the thyroid gland. This finding supports the ATA
and NCCN guideline recommendations that total thyroid-
ectomy should remain the initial standard of care as opposed
to hemithyroidectomy. In patients diagnosed with appar-
ent sMTC following a hemithyroidectomy, these guidelines
indicate that RET germline mutation status, serum calcito-
ESSIG ET AL.
nin, and the results of imaging studies should determine the
role of completion thyroidectomy (2, 9). sMTC patients
with multifocal MTC in the ipsilateral thyroid lobe or
isthmus have a significant prevalence of MTC in the con-
tralateral lobe. It remains unknown if ipsilateral tumor fo-
cality should be included as an independent factor when
considering completion thyroidectomy.
Author Disclosure Statement
Dr. Kloos is an employee and equity owner in Veracyte,
Inc. This study was conceived and initiated prior to the ini-
tiation of this relationship. Veracyte provided no funding,
input, or support for this study.
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Address correspondence to:
Richard T. Kloos, MD
Veracyte, Inc.
6000 Shoreline Court, Suite 300
South San Francisco, CA 94080
E-mail: Richard.Kloos@Veracyte.com
 AUTHOR COPY ONLY M C Martins-Costa et al. RET M918V causes familial MTC 23:12 909–920
Research

M918V RET mutation causes familial

medullary thyroid carcinoma: study of
8 affected kindreds

M Cecília Martins-Costa1,2,3, Lucas L Cunha1, Susan C Lindsey1, Cleber P Camacho1,

Renata P Dotto1, Gilberto K Furuzawa1, M Sharmila A Sousa1, Teresa S Kasamatsu1,
Ilda S Kunii1, Márcio M Martins1, Alberto L Machado1,4, João R M Martins1,
Magnus R Dias-da-Silva1 and Rui M B Maciel1,4 Correspondence
should be addressed
1Department of Medicine, Thyroid Diseases Center and Laboratory of Molecular and Translational Endocrinology, to R M B Maciel or
Division of Endocrinology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil M R Dias-da-Silva
2Center for Endocrinology and Metabology, Hospital Geral de Fortaleza, Fortaleza, CE, Brazil
Email
3Department of Medicine, Universidade de Fortaleza, Fortaleza, CE, Brazil
rui.maciel@unifesp.br or
4Fleury Medicine and Health, São Paulo, SP, Brazil
mrdsilva@unifesp.br

Abstract
Endocrine-Related Cancer

Germline mutations in codon 918 of exon 16 of the RET gene (M918T) are classically Key Words
associated with multiple endocrine neoplasia type 2B (MEN 2B) with highly aggressive f medullary thyroid
medullary thyroid cancer (MTC), pheochromocytoma and a unique phenotype. The carcinoma

objectives of this study are to describe the rare M918V RET mutation discovered in f RET mutation

8 MTC kindreds from Brazil lacking the MEN 2B phenotype classically observed in f RET M918V

M918T patients and to investigate the presence of a founder effect for this germline f founder effect

mutation. Eight apparently sporadic MTC cases were diagnosed with the germline
M918V RET mutation. Subsequently, their relatives underwent clinical and genetic
assessment (n = 113), and M918V was found in 42 of them. Until today, 20/50 M918V
carriers underwent thyroidectomy and all presented MTC/C-cell hyperplasia; the
remainder carriers are on clinical follow-up. None of the M918V carriers presented
clinical features of MEN 2B. Their clinical presentation was heterogeneous, and the age
at tumor diagnosis ranged from 24 to 59 years. Lymph node metastases were present in
12/20 patients, and presumable distant metastases in 2/20; in contrast, we observed a
carrier of up to 87 years of age without evidence of MTC. Ethnographic fieldwork and
haplotype analyses suggested that the founder mutation first settled in that area fifteen
generations ago and originated from Portugal. Our study is the first to demonstrate the
RET M918V mutation co-segregating in 8 familial MTC kindreds with validated evidence
of a founder effect. We suggest that M918V MTC should be clinically considered an
Endocrine-Related Cancer
American Thyroid Association (ATA) moderate-risk category. (2016) 23, 909–920

Introduction
Activating RET oncogene germline mutations causes
multiple endocrine neoplasia type 2A (MEN 2A) and
type 2B (MEN 2B). Virtually, all patients with a RET
mutation will develop medullary thyroid cancer (MTC).
In the absence of associated pheochromocytoma (PHEO)
and primary hyperparathyroidism (HPTH), MEN 2A is
subclassified as familial medullary thyroid carcinoma
(FMTC) (Wells et al. 2015).

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 AUTHOR COPY ONLY
Research M C Martins-Costa et al. RET M918V causes familial MTC 23:12 910

The understanding of the genotype/phenotype Clinical evaluation and follow-up of M918V carriers
correlation of RET mutations has increased over recent
Clinical evaluation consisted of medical history
decades due to the widespread practice of RET sequencing
and physical examination. We particularly looked
and careful long-term clinical follow-up of affected
for syndromic features and the MEN 2B phenotype,
kindreds (Signorini et  al. 2014, Wells et  al. 2015). This
including PHEO, marfanoid habitus, mucosal neuromas
knowledge has allowed recommendations on the timing
and ganglioneuromatosis (Camacho et al. 2008).
of prophylactic thyroidectomy and surgery extent based
We performed peripheral blood collection to
on stratified mutation risk levels (Brandi et  al. 2001,
assess calcitonin, carcinoembryonic antigen (CEA),
Kloos et  al. 2009, Wells et  al. 2015). In fact, increased
thyroid-stimulating hormone (TSH), free thyroxine
genetic screening for RET mutations, particularly for
(FT4), parathyroid hormone (PTH) and total calcium
apparently sporadic MTC, has also led to the possibility
measurements. For those who presented PTH levels above
of discovering new mutations of which the transforming
the upper limit of the range, we measured 25-hydroxy-
activity remains uncertain (Chen et al. 2010).
vitamin D. We also measured plasma metanephrines, as
Germline mutations in codon 918 in exon 16 of the
well as 24-h urinary catecholamines and metanephrines
RET gene (M918T) are characteristically associated with
for biochemical investigation of PHEO.
multiple endocrine neoplasia type 2B (MEN 2B) with
Cervical and abdominal ultrasound (US) was
highly aggressive MTC, PHEO and a unique phenotype
performed with a LOGIQ scanner (GE Healthcare Bio-
(Carlson et al. 1994, Hofstra et al. 1994, Eng et al. 1994,
Science) using a 12-MHz transducer integrated with color-
Wells et al. 2013, 2015, Frank-Raue & Raue 2015). To date,
Doppler examination. Thyroid nodules with suspicious
the M918V variation was described in only one patient
features for malignancy were submitted to US-guided fine
with MTC in a large Italian cohort and in one family
needle aspiration biopsy (FNAB) using a 23-gauge needle
Endocrine-Related Cancer

member who had not developed MTC at the time of

(25 × 0.6 mm) attached to a 10-mL syringe.
publication (Cosci et al. 2011).
For patients with an MTC diagnosis, the clinical
Here, we describe this rare methionine-to-valine
management, including imaging investigation, was
substitution in the 918 codon of the RET gene in eight
performed in accordance with American Thyroid
MTC kindreds without the MEN2B phenotype classically
Association (ATA) guidelines (Kloos et  al. 2009, Wells
observed in M918T patients. Moreover, we report the
et  al. 2015). However, because the M918V mutation
clinical, molecular and histological features of such
has not yet been classified in any MTC guideline, we
families and the presence of a founder effect for this
clinically approached the carriers based on serum
germline mutation.
calcitonin and CEA measurements, as well as the FNAB
results of detected thyroid nodules; only those patients
with positive results for these tests were referred for
Patients and methods
surgery. After histological confirmation of MTC, and
Index patients, their relatives and controls classification of the neoplasia according to the TNM
(tumor, node, metastasis) staging system (American
As part of the Brazilian Research Consortium for Multiple
Journal Committee on Cancer 2010), those patients
Endocrine Neoplasia (BRASMEN), we evaluated 60 index
were followed in accordance with the ATA guidelines
cases of MTC (31 sporadic, 28 MEN 2A and 1 MEN 2B)
(Wells et al. 2015). The remaining carriers who present
in Ceará, a Northeastern State of Brazil. Eight apparently
normal calcitonin and cervical ultrasound examination
sporadic MTC patients were identified with the M918V
were maintained on a careful follow-up.
RET mutation. Then, we have invited the relatives at-risk
to enter in this study and, at the time of this report, 113
(female n = 84; male n = 29) members were assessed.
Laboratory studies
Additionally, 40 thyroid-healthy individuals
originating from the same state but unrelated to the Basal plasma calcitonin was measured using an
patients were investigated as a control group. in-house immunofluorometric assay, with cut-off values
The study protocol was approved by the internal of 18.4 pg/mL for males and 7.8 pg/mL for females
review board of the Universidade Federal de São Paulo (Camacho et al. 2014). CEA, TSH and FT4 were measured
(CAAE: 16441414.9.1001.5505111), and all patients by electrochemiluminescent immunoassays (Elecsys
received genetic counseling before and after RET testing. analyzer, Roche Diagnostics GmbH). The normal ranges

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DOI: 10.1530/ERC-16-0141 Printed in Great Britain
 AUTHOR COPY ONLY
Research M C Martins-Costa et al.
were as follows: CEA, smokers: <5 ng/mL, non-smokers:
<3 ng/mL; TSH, 0.27–4.2 mIU/L; FT4, 0.93–1.7 ng/dL; and
PTH, 15–65 ng/mL. Total calcium was measured using a
colorimetric assay (Olympus AU400 analyzer, Beckman
Coulter Biomedical K.K., Shizuoka, Japan). The normal
ranges were 8.8–10.6 mg/dL for adults and 8.5–11 mg/dL
for children. The 25-hydroxyvitamin D levels were
measured using a immunochemiluminescent assay.
The normal range was 30–60 ng/mL (Maeda et  al.
2014). Plasma metanephrines were measured using
liquid chromatography–tandem mass spectrometry
(XEVO TQ-S, Milford, MA, USA), and the normal range
was <0.5 nmol/L for metanephrine and <0.9 nmol/L
for normetanephrine. Urinary catecholamines and
metanephrines were measured using high-performance
liquid chromatography (Chromsystems Instruments
& Chemicals GmgH, Munich, Germany). The normal
ranges for catecholamines were as follows: up to
97 µg/24 h for norepinephrine, up to 500 µg/24 h for
dopamine and up to 27 µg/24 h for epinephrine. The
normal ranges for metanephrines were as follows: up
Endocrine-Related Cancer
to 320 µg/24 h for metanephrine and up to 390 µg/24 h
normetanephrine.
Genomic DNA was extracted from peripheral
blood leukocytes using an in-house protocol
(Kizys et  al. 2012). Sequence analysis of hot-spot
bearing exons 8, 10, 11 and 13–16 was performed; in
addition, extended RET gene analysis was performed
in all M918V carriers who had histopathological
confirmation of MTC/CCH to exclude mutations in
other exons (Lindsey et  al. 2012). Briefly, each exon
was sequenced at least twice and in both directions.
The cycling conditions were as follows: 5 min at 94°C,
followed by 38 cycles of 20 s each at 94°C and 30 s
at 60°C and an extension step of 2 min at 72°C. PCR
products were direct-sequenced using an ABI Prism Big
Dye Terminator Cycle Sequencing Ready Reaction Kit
(Applied Biosystems). The sequences were analyzed
using the BioEdit Sequence Alignment Editor and
CLC Main Workbench 6 (http://www.clcbio.com)
and compared with reference data available in the
NCBI GenBank (RefSeq NG_007489) and the Ensembl
Genome Browser (Lindsey et al. 2012).
Genealogical fieldwork
The observation that the highest concentration of
patients with the M918V RET mutation occurred in the
same region in the northeast area of the State of Ceará
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RET M918V causes familial MTC 23:12 911
in association with the anecdotal perception of high
consanguinity among inhabitants led us to perform a
genealogical investigation of the families, including
data collection from these families in the local church
and municipality archives, such as birth, marriage and
death registries.
Founder effect and mutation age estimation analysis
We hypothesized that a founder effect would explain
the high frequency of the M918V RET mutation in those
patients from northeastern Ceará. Haplotype analysis
of four RET-flanking microsatellite markers, D10S197,
D10S196, D10S1652 and D10S537, was performed to
track the founder effect (Qi et al. 2011). The first marker
was placed at position 50.04 cM, and the other three
markers were at positions 70.07, 80.61 and 89.16 cM.
The chromosome map distances were derived from the
deCODE map (https://www.ncbi.nlm.nih.gov/probe/).
GenePop was used to perform the Hardy-Weinberg
exact test (Rousset 2008). Haplotypes of the patients
and controls were reconstructed using the statistical
software package PHASE, version 2.1 (Stephens et al. 2001,
Stephens & Donnelly 2003, Stephens & Scheet 2005). We
constructed the phylogenetic tree of the studied families
with POPTREE2 software by estimating the genetic
distance measures (Takezaki et al. 2010).
The proportion of haplotypes commonly observed
in all carriers was reduced with time. This allowed us
to estimate the age of the founder mutation. Disease
mapping using Linkage disEquilibrium (DMLE) version
2.3 (Reeve & Rannala 2002) estimated the age of M918V
RET mutation based on the disease chromosomes
sampled and the population growth rate. Because the
M918V RET mutation was recently described (Cosci
et  al. 2011), the proportion of chromosomes sampled
was difficult to calculate. Therefore, three different
analyses were performed using a different calculation
for the proportion of mutation-carrying chromosomes
sampled: 0.015, 0.01 and 0.005 (Caleca et  al. 2014).
The population growth rate was estimated as described
previously (Papi et al. 2009).
Statistical analysis
The primary data are presented as the mean ± standard
deviation (S.D.), median and minimal and maximal values.
SPSS software (version 23; SPSS) was used to perform the
analysis. A P value <0.05 was considered significant.
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 AUTHOR COPY ONLY
Research M C Martins-Costa et al.
Results
Clinical and genetic diagnosis of FMTC in eight kindreds
carrying the RET M918V mutation
After the detection of the germline M918V RET mutation
in 8 apparently unrelated MTC indexes cases, we extended
the analysis to their relatives (Fig.  1). Consequently, we
have studied 121 individuals (83 women and 38 men),
being 8 index cases and 113 at-risk relatives. From these
113 at-risk relatives screened, 42 were diagnosed with
this mutation and 71 were non-carriers. In 2 families (F3
and F6), the mutation was identified in 2 generations. In
3 families (F1, F4 and F5), the mutation was identified in
Endocrine-Related Cancer
Figure 1
Pedigree of eight apparently unrelated MTC families identified through
the study. Indexes cases are identified by black arrows. Full black icons
represent either affected MTC proband or relatives. The sons of F7.3
are adopted.
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DOI: 10.1530/ERC-16-0141 Printed in Great Britain
RET M918V causes familial MTC 23:12 912
3 generations. For 3 index cases (F2, F7 and F8), only one
individual is noticeably affected because the other family
members have not answered our call for the clinical and
genetic evaluation to date (Fig. 1).
At the moment of RET screening, 4 of these 42
M918V-positive relatives had already been submitted to
total thyroidectomy, although they were not aware that
their thyroid disease had a hereditary component and
that it was related to a common bond with index patients
(patients F1.6, F1.7, F1.8 and F1.9). The remainder
38 relatives positive for M918V had not performed any
surgical procedure at the time of RET screening.
The clinical and biochemical features of all M918V
carriers are shown in Table  1. At the initial assessment
after RET screening, 42% (14/33) of the patients presented
serum calcitonin levels above the limit of reference; 20%
(6/30) had elevated CEA. Two of 25 patients presented
elevations of PTH, both related to decreased levels of
vitamin D (18 and 21 ng/mL). None of the M918V carriers
presented hypercalcemia or elevation of plasma and 24-h
urinary metanephrines.
Therefore, from the 50 M918V carriers, 8 were index
cases, 4 were relatives who had already performed total
thyroidectomy at the time of RET screening and 38 were
relatives who came to RET screening without a history
of previous thyroidectomy. From the 38 relatives of this
latter group, we have proposed surgical treatment in
8 (F1.1, F1.2, F1.3, F1.4, F1.5, F1.10, F4.1 and F6.1)
because we have noticed elevated serum calcitonin and
suspicious thyroid nodules or cervical lymph nodes by
cervical ultrasound, some of them associated to suspicious
or positive results on cytology after FNAB (Table 2). After
surgery, we have observed MTC or C-cell hyperplasia
(CCH) in all 20 M918V operated carriers (Table 2).
The clinical presentation of MTC in the index cases
varied considerably, particularly for the age at diagnosis
and the TNM (tumor, node and metastasis) staging
(Table  2). For the index cases, the youngest patient was
diagnosed with MTC at 24 years of age (F6); however, at
age 16, he noticed a nodule on the left side of his neck; the
lesion was investigated 8  years later because the nodule
persistently increased in volume. He presented the most
advanced postoperative TNM stage of this cohort. The
oldest M918V index case was diagnosed at 59 years of age
(T2mN1bM0), and the follow-up did not reveal evidence
of biochemical or structural disease (F5). Additionally,
one of the M918V index cases (F8) was diagnosed at
age 41  years with a 1-cm MTC confined to the thyroid
gland. To date, she has no sign of metastatic lymph nodes,
unlike the other index cases (7/8), which had lymph node
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 Endocrine-Related Cancer
Research

DOI: 10.1530/ERC-16-0141
Table 1 Clinical and biochemical features of M918V mutation carriers.

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Relatives (n = 42)
Features at the first clinical All M918V carriers Index cases Already TT at RET screening TT after RET screening No TT after RET screening
visit after RET screening (n = 50) (n = 8) (n = 4) (n = 8) (n = 30) P

Age (years)
Median (range) 41 (7–87) 54.5 (24–59) 51.5 (50–54) 36.5 (28–68) 36.5 (7–87) 0.24a
Mean ± S.D. 42.6 (±17.3) 48.2 (±12.2) 51.7 (±1.7) 41.5 (±14.5) 40.1 (±19.8)
Gender 0.75b
Male 17 2 1 2 12
Female 33 6 3 6 18
Calcitonin level (pg/mL) n = 33 n=2 NA n=8 n = 23 0.007c
Median (range) 11.7 (1–4640) 2866 (1092–4640) 121.5 (1.3–2695) 7.1 (1.0–35.8)
M C Martins-Costa et al.

Mean ± S.D. 281 (±913.2) 447.5 (±916.3) 9.7 (±8.8)

CEA (ng/mL) n = 30 NA NA n=7 n = 23 0.08c

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Median (range) 1.4 (0.2–105) 3.9 (0.3–105) 1.2 (0.2–4.5)
Mean ± S.D. 6.2 (±19.2) 21.4 (±37.8) 1.6 (±1.1)
PTH level (ng/mL) n = 25 NA NA n=6 n = 19 0.07c
Median (range) 44.6 (24.3–76.3) 59.6 (29.6–76.3) 39.7 (24.3–70.5)
Mean ± S.D. 46.5 (±14.4) 56.2 (±15.5) 43.9 (±13.2)
Total calcium (mg/dL) n = 26 NA NA n=6 n = 20 0.58c
Median (range) 9.5 (8.2–10) 9.5 (9.2–9.9) 9.4 (8.2–10)
Mean ± S.D. 9.4 (±0.4) 9.5 (±0.2) 9.4 (±0.4)
Thyroid nodule on the US
Yes 22 8 4 8 2 <0.0001b

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No 23 0 0 0 23
AUTHOR COPY ONLY

CT, calcitonin; CEA, carcinoembryonic antigen; n, number of patients; PTH, parathyroid hormone; S.D., standard deviation; TT, total thyroidectomy; US, ultrasound.
RET M918V causes familial MTC

Reference ranges: Basal plasma calcitonin, males: <18.4 pg/mL, females: <7.8 pg/mL; CEA, smokers: <5.0 ng/mL, non-smokers <3.0 ng/mL; PTH: 15–65 ng/mL; Total calcium, adults: 8.8–10.6 mg/dL,
children: 8.5–11.0 mg/dL.
aANOVA test, Kruskal–Wallis, Dunn test among index cases and subgroups of relatives; bChi-square test between thyroidectomized and non-thyroidectomized relatives after RET screening;

cMann–Whitney test between thyroidectomized and non-thyroidectomized relatives after RET screening.
23:12
913
 Endocrine-Related Cancer

Table 2 Clinical and laboratory findings of M918V RET index cases and relatives with confirmation of MTC/CCH by histopathology.
Research

Age at Total Time since

diagnosis thyroidectomy initial Time of Current

DOI: 10.1530/ERC-16-0141
Index Affected (years/ Initial at RET symptoms FNAB cytology Preoperative Clinical TNM follow-up sCt
patients relatives gender) symptoms screening (months) (Bethesda) sCt (pg/mL) Surgery extension staging (months) (pg/mL)

F1 54/F Thyroid nodule Yes 2 NA NA TT + central and T3m N0 M0 73 4.3

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right lateral
F1.1 34/F – No II 20 TT + central ND T1am N0 M0 50 <2.0
F1.2 31/M – No V (PTC) 2695 TT + central/right T1bm N1b M0 50 132
lateral ND
F1.3 30/M – No VI (MTC) 116 TT + central ND T1am N0 M0 50 2
F1.4 28/F – No III 148 TT + central ND T1am N1a M0 50 2
F1.5 42/F – No IV 11.7 TT T1am N0 M0 50 <2.0
F1.6 51/F Thyroid nodule Yes 24 II NA TT + central/left T3m N1b M0 67 8.2
lateral ND
F1.7 50/F Thyroid nodule Yes 17 II NA TT T3m N0 M0 108 50.3
F1.8 52/M – Yes IV NA TT and central T3m N1a M0a 32 219
ND
M C Martins-Costa et al.

F1.9 54/F – Yes VI (PTC) NA TT CCH onlyb 36 <2.0

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F1.10 45/F – No III 81.5 TT + central ND CCH onlyc 50 <2.0
F2 – 58/F – Yes – V NA TT + central/right T2m N1b M0 88 140
lateral ND
F3 – 55/F Disturbs of Yes 2 IV NA TT + central/right T1b N1a M0d 73 6.2
swallowing lateral ND
F4 – 40/F Hoarseness and Yes 3 VI (MTC) 1092 TT + central/ T3m N1b M1e 97 506
cervical nodules bilateral ND
F4.1 68/F – No – VI (MTC) 286 TT + central/level T1bm N1b M0 68 2
VII ND
F5 – 59/F Thyroid nodule Yes 2 NA NA TT + central/ T2m N1b M0 157 2
bilateral ND

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F6 – 24/M Lateral cervical mass Yes 96 NA NA TT + central/ T4am N1b M1e 124 1698
growth bilateral ND
AUTHOR COPY ONLY

F6.1 59/F – No – VI (MTC) 323 TT + central ND T1bm N0 M0 45 6.2

RET M918V causes familial MTC

F7 – 55/M Hoarseness Yes 60 V 4640 TT + central/ T3m N1b M0 54 371

bilateral ND
F8 – 41/F – Yes – IV NA TT T1 N0 M0 240 7.5

CCH, C-cell hyperplasia; FNAB, fine needle aspiration biopsy; MTC, medullary thyroid carcinoma; NA, not available; ND, node dissection; PTC, papillary thyroid carcinoma; sCt, serum calcitonin; TNM,
23:12

tumor, nodal, metastasis (staging according to the American Joint Committee on Cancer-AJCC).
aF1.8 had also a 0.6 cm PTC; bF1.9 had been previously submitted to total thyroidectomy because of a FNAB cytology positive for PTC. Histopathology review and immunohistochemistry confirmed the

PTC (pT3NxMx) and besides no foci of MTC were found, CCH was identified; cF1.10’s thyroid nodule was in fact a pT1N0Mx PTC. Although no foci of MTC were found, CCH was identified; dthis patient
presented areas of CCH associated to a focus of MTC; epresumable metastases in patients presenting sCt >500 mg/dL. The imaging abnormalities found in these patients were not biopsied.
914
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Research M C Martins-Costa et al. RET M918V causes familial MTC 23:12 915

involvement at the time of the initial diagnosis. Two

patients (F4 and F6), besides lymph node involvement,
also presented persistently very elevated calcitonin levels
and distant lesions on imaging methods. The F4 index
patient presented a 1-cm highly vascularized hepatic
nodule seen by abdominal magnetic resonance imaging
(MRI), and the last serum calcitonin on the follow-up is
506 pg/mL. Patient F6 has been diagnosed with cervical
and thoracic vertebrae lesions suggestive of metastases
3 years after the thyroidectomy by MRI, and the last serum
calcitonin on the follow-up is 1698 pg/mL. No increase in
the size of those presumable metastatic lesions of both
patients was noted during the follow-up. The remaining
index patients presented at their last clinical assessment
the following status: one had elevated calcitonin levels
(140 pg/mL) and cervical lateral lymph nodes (F2);
one had elevated calcitonin levels (371 pg/mL) with no
structural evidence of disease (F7); 3 had persisted low
calcitonin levels (<10 pg/mL) (F1, F3 and F8) and one had
undetectable calcitonin (F5) (Table  2). MTC was present Figure 2
in all surgical specimens and in 7/8 patients presenting Tracking evidence for RET M918V founder effect. Physical map of South
Endocrine-Related Cancer

multifocal tumor. It is noteworthy that some cases were
presumably cured, despite relatively late thyroid surgery
(F1, F3, F5 and F8).
To date, 12 relatives carrying the M918V mutation
have undergone total thyroidectomy, with confirmation
of MTC and/or CCH in the histopathology. As mentioned,
4 of these patients had been already submitted to
thyroidectomy when they came up to perform RET
screening; 3 of them (patients F1.6, F1.7 and F1.8, Table 2)
were already aware of MTC diagnosis after RET screening,
and one of them (patient F1.9, Table 2) had been submitted
to thyroidectomy because of a thyroid nodule positive
America showing the geographic position of Brazil with the localization
of the surrounding physical area of Jaguaribe’s valley with its river and
Apodi plateau (hatched area) at the Northeastern State of Ceará.
for papillary thyroid carcinoma (PTC). Histopathological
review of F1.9 patient confirmed the PTC besides C-cell
hyperplasia. In addition, patients F1.8 and F1.10 also
demonstrated small PTCs on surgical specimens (Table 2).
In 7/12 relatives carrying the M918V mutation with
MTC/CCH, the disease was restricted to the thyroid gland,
and the remainder presented metastatic lymph nodes at
diagnosis. Seven of 12 relatives achieved undetectable
serum calcitonin in the postoperative evaluation.

Figure 3
Genotype–phenotype co-segregation in RET
M918V largest family. We represent family 1 with
the identification of the affected MTC index case
by black arrow and relatives. Full black icons
represent either the affected MTC proband or
relatives, and half-filled icons represent relatives
affected by CCH only. Empty icons with positive
(+) signaling represent patients who were tested
positive for M918V without clinical evidence of
MTC/CCH. Empty icons with negative (−) signaling
represent patients who were tested negative for
M918V. Empty icons without upper signaling
represent individuals who were not submitted to
RET screening. *represents the presumed pioneer
immigrated to northeastern Brazil from Portugal.

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Research M C Martins-Costa et al. RET M918V causes familial MTC 23:12 916

The clinical and laboratory findings, as well as the surgical
extent of all patients with confirmed MTC/CCH are
summarized in Table 2.
For the other 30 M918V carriers, we have decided,
after discussions with the medical team and the
patients, that a careful follow-up would be a better
recommendation than prophylactic surgery because
they present normal calcitonin and CEA levels, and no
suspicious nodules/lymph nodes on cervical ultrasound.
We did not observe statistically significant difference
of gender and age between both groups of patients
(indicated for surgical treatment, n = 8 vs clinical
follow-up, n = 30). However, the group who performed
surgical treatment after RET screening had higher levels
of serum calcitonin and a higher frequency of thyroid
nodules on the ultrasound (Table 1).
Until the last follow-up visit (up to 5  years of
observation), no mutation carrier had presented clinical
manifestations or laboratory tests indicating HPTP
or PHEO, neither cutaneous lichen amyloidosis nor
Hirschsprung’s disease. Importantly, clinical assessment
Endocrine-Related Cancer
along the Jaguaribe River valley, where he established
several settlements.
The haplotype analysis of our founder effect
investigation confirmed the qualitative ethnographic
data collected. All studied loci were in Hardy–Weinberg
equilibrium. Seven patients (33.3%) from 4 apparently
unrelated families shared a common haplotype (D10S197-
RET-D10S196-D10S1652: 168-M918V-98-170; Fig.  4A).
However, only 2 wild-type chromosomes in the control
group presented this haplotype (P < 0.001). In fact,
statistically significant differences in allele frequencies
between the wild-type and the M918V RET chromosomes
were observed for markers located closer to RET, in
particular D10S197, D10S196 and D10S537. This finding

of all MTC patients and mutation carriers did not reveal

any typical features of MEN 2B, such as musculoskeletal
abnormalities; neuromas in the lips, the tongue or the
conjunctiva; or ganglioneuromatosis of the intestine.
None of the 40 control individuals presented with
the M918V RET mutation.

Tracking the RET M918V founder effect and age

of mutation

Due to the observation that the highest concentration

of patients with the M918V RET mutation was in the
region that comprises the Jaguaribe River valley and the
Apodi plateau (Fig.  2) in the northeast area of the State
of Ceará and because the F1 and F6 live in nearby cities
(<25 miles) of this region, we investigated the presence of a
founder effect.
The ethnographic investigation delved into the
historical archives of the region with the highest
concentration of M918V-affected patients, studying the
family ancestor data in the churches and oral life history Figure 4
Tracking founder effect. (A) Haplotype branching trees derived from
narratives. After performing a wide pedigree, we observed haplotype analysis of the seven mentioned patients. Family haplotypes
that some of the M918V MTC patients from this region are indicated with the corresponding family ID numbers. The markers
who were unaware of MTC in other family members used are shown together with their position in the deCODE genetic
map. Boxed numbers correspond to common allele in respective loci.
belonged to the same family (F1.6, F1.7 and F1.8) (B) Histograms of estimated age of M918V RET mutation obtained
(Fig.  3). In 1700s, a pioneer immigrated to northeastern from DMLE software. The age of mutation is given in number of
Brazil from Braga, Portugal, colonizing this region in the generations. This histogram was obtained using growth rate of 0.4411
(25 years/generation) and the proportion of sampled chromosome of
northeast of Ceará. Both historical registries and oral life 0.015. (C) Phylogenetic tree of all families (numbered from #1 to #8)
history narratives suggested that this colonizer migrated and the control group (Ct).

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Research M C Martins-Costa et al.
supports our hypothesis that the M918V RET mutation
was settled in Ceará state as a founder mutation.
The State of Ceará’s population currently includes
8,904,459 people (Instituto Brasileiro de Geografia
e Estatística 2015). Historical and demographic data
indicate that the population was 721,686 in 1872
(Instituto Brasileiro de Geografia e Estatística 1872).
Assuming 25  years/generation, the estimated growth
rate was 0.4411, and assuming 30  years/generation, the
estimated growth rate was 0.5294. Figure 4B presents a set
of histograms for age estimation. Briefly, we estimated that
the founder mutation was settled in Ceará approximately
15 generations ago, which corresponds to 376  years
(considering the year/generation 25  years) or 451  years
(considering the year/generation 30 years).
The phylogenetic tree (Fig. 4C) revealed that control
group shared poor genetic similarity with the patients.
Our tree findings revealed that all families coalesce in a
common knot, corresponding to a putative most recent
common ancestor. Most importantly, our data indicate
that different families may be related to each other.
Endocrine-Related Cancer
Discussion
By studying 8 kindreds affected by the RET M918V
mutation, we report for the first time that this mutation
co-segregates with familial MTC and is inherited with
complete penetrance pattern. The RET 918 codon is
classically related to the MEN 2B syndrome and has been
extensively studied since the first mutation in this codon
was reported (Carlson et  al. 1994, Hofstra et  al. 1994,
Eng et al. 1994, Cirafici et al. 1997, Wells et al. 2015). In
contrast to the M918T mutation, which causes MEN 2B
syndrome with a more aggressive MTC, we did not detect
any MEN 2B features in the M918V carriers.
M918V was described for the first time in 2011
in one MTC patient and in one of 3 screened relatives
of this patient, who have not developed MTC despite
advanced age. In addition, it was characterized as a
low transforming mutation by in silico and in vitro
analysis (Cosci et  al. 2011). Cirafici and coworkers had
previously demonstrated that only the mutation M918T
exhibited a highly transforming activity from all possible
substitutions for methionine 918, including RET M918V,
which presented a low transforming potential (Cirafici
et al. 1997). More recently, Plaza-Menacho’s study revealed
that C-terminus oncogenic RET kinase mutants become
phosphorylated earlier than the wild-type RET, due to a
combination of enhanced kinase activity with ATP affinity,
which mechanism is related to a better intermolecular
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RET M918V causes familial MTC 23:12 917
substrate phosphorylation. Moreover, RET M918T
leads to cis-inhibitory machinery involving tethering
contacts between the glycine-rich loop, activation loop,
alpha C-helix and may elicit transactivation in the way
of promoting intermolecular autophosphorylation
by improving substrate presentation, and therefore,
uncontrolled oncogenic activity (Plaza-Menacho et  al.
2014). Would RET M918V shed a light on understanding
the phenotypic oncogenic spectrum of MEN2B and
FMTC? Further scrutinizing studies on M918V mutant
mechanism of  intermolecular autophosphorylation are
still needed.
We evaluated 8 MTC patients referred to us for RET
sequencing in whom we detected the M918V mutation
and 12 at-risk relatives in whom MTC/CCH was diagnosed
after the identification of this mutation. In contrast to
the M918T mutation, which causes MEN 2B syndrome
with a more aggressive MTC, we did not detect any MEN
2B features in the M918V carriers and a more favorable
outcome compared with classical highest-risk M918T
mutation carriers. In fact, the clinical presentation was
quite heterogeneous, particularly for the age at diagnosis,
the TNM staging and the follow-up after thyroidectomy
(Table  2). The advanced TNM stage in 2 index cases (F6
and F7, Table 2) was possibly related to the long interval
from the manifestation of the initial symptoms to surgical
treatment; their follow-up was also remarkable for the
highest levels of postoperative serum calcitonin and CEA,
suggesting the persistence of disease.
For the relatives whose diagnosis of MTC was perfor-
med after the genetic screening, we observed that the
mothers (F4.1 and F6.1) of 2 probands had less advanced
disease than their children. This finding reinforces the
importance of RET mutation screening to diagnose the
tumor before the onset of symptoms. Interestingly, we
observed that patient F1.8 was diagnosed at 52 years of age
with a tumor (T3mN1aM0) and a micro PTC, whereas his
sister (F1.9), who was also submitted to total thyroidectomy
due to PTC, presented only CCH. Additionally, an 87-year-
old male with a normal calcitonin level and thyroid
ultrasound without any nodules recently died from an
unrelated condition, whereas his grandson was diagnosed
with a multifocal MTC and lateral lymph node metastases
at 31 years of age (F1.2) (Table 2). We believe that genetics
and environmental factors may contribute to the wide
clinical heterogeneity of our patients (Rigotto et al. 2012).
It is well known that, even in large kindreds, the
pattern of disease presentation may not become evident
until the evaluation of several family members, and
even then, it may vary compared with reports of other
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Research M C Martins-Costa et al.
families with the same RET mutation (Wells et al. 2013).
Taking into consideration that we have observed only
20 patients with M918V MTC/CCH to date, cervical
lymph node metastases were found in 12/20 patients,
including 3 patients younger than 35  years (F1.2, F1.4,
and F6), and probable distant metastases were noted in
2/20, suggesting a more aggressive behavior of M918V
MTC. Patients F4 and F6 presented structural lesions
in liver and spinal vertebrae, respectively, which could
correspond to distant metastases as they are associated
to persistent high levels of calcitonin (above 500 pg/mL).
However, false-positive results cannot be totally excluded,
as these abnormalities were not biopsied.
On the other hand, we observed a relatively favorable
presentation among the majority of carriers. Therefore,
we believe that assessment and follow-up of a greater
number of patients carrying M918V is necessary to get a
better understanding about the behavior of MTC related
to this novel mutation. In general, a substantial number
of patients with distant metastases of MTC may have
indolent disease and quiescent or slow-growing lesions
Endocrine-Related Cancer
over years of routine observation (Cabanillas et al. 2014).
So far, 20 out of 50 M918V carriers underwent total
thyroidectomy and all of them presented MTC/CCH in
surgical specimens (Table  2). It is possible that 30 M918V
remaining carriers, who were not submitted to surgical
treatment, may present microscopic MTC/CCH whose size
did not influence basal calcitonin levels or the formation
of a thyroid nodule on US imaging yet. Therefore, we
faced an issue that was discussed among the medical
team as to how we would deal with M918V carriers
who did not present any test suggesting the presence of
MTC/CCH. Presently, there is no recommendation in
guidelines about prophylactic thyroidectomy for M918V,
so we have decided by a cautious follow-up using basal
calcitonin and neck US performed at every 6-month interval.
In fact, when we initially found the first patients
with M918V, we were not sure that M918V was a
mutation causing disease as up to the present time, the
only mutation described in codon 918 was the M918T,
classically associated to the MEN 2B phenotype. For those
reasons, we opted to perform RET extended analysis in all
M918V who had histopathological confirmation of MTC
or CCH. Also, M918V had not been found in 40 thyroid-
healthy controls from the same country state.
Moreover, we observed that 2 patients carrying M918V
mutation had CCH and PTC (F1.9 and F1.10, Table 2) and
1 patient (F1.8, Table 2) had MTC and PTC. The association
of PTC and MTC occasionally occurs, which comes in a
variable rate (Biscolla et al. 2004, Machens & Dralle 2012),
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RET M918V causes familial MTC 23:12 918
depending on the environmental conditions or differences
in the study populations (Wells et al. 2015).
We further investigated the ethnographic and
quantitative aspects of the dispersion of the M918V RET
mutation. All of our data support the hypothesis that
a founder effect is responsible for the dispersion of the
M918V RET mutation along the Jaguaribe River throughout
the State of Ceará, consistent with the higher prevalence
of MTC around the northeast of the state. Therefore, our
findings substantiate both anecdotal and community
health-based evidence collected from the participants
during the ethnographic fieldwork. We propose that the
Brazilian growth rate followed distinct phases in history
and that the constant growth rate portrayed here is a mere
mathematical extrapolation. It is also noteworthy that the
historical registries of the population in the studied region
were not precisely accounted for as we journeyed into the
past. However, we encountered consistent information
with the aid of the oral life history narratives and the
document analysis, which corroborated to our hypothesis
that this mutation has accompanied the population of
Ceará for approximately 350–400 years and that these 8
supposedly distinct kindreds are one single family that
has been dismembered through the years.
Considering the results of biochemical and/or
imaging tests used for the investigation of MTC, and
the histopathological results for those patients who
were submitted to surgical treatment, we observed a low
penetrance of this neoplasia among M918V carriers (~40%
(20/50)). Also, evaluating the median age of the relatives
who were M918V carriers according to their status at the
RET screening (already thyroidectomized at RET screening
with MTC/CCH confirmation by histopathology:
51.5  years; thyroidectomized after RET screening with
MTC/CCH confirmation by histopathology: 36.5  years
vs non-thyroidectomized after RET screening: 36.5 years,
Table  1), we observed a relatively lower progression of
MTC in all subgroups of carriers.
The majority of the MTC patients presented a favorable
clinical course, even though there are high frequency of
lymph node metastases at initial diagnosis and presumable
distant metastasis in 2 patients. In conclusion, based
on the heterogeneity of clinical presentation of M918V
mutation in our case series, we propose that this mutation
should be classified as an ATA moderate-risk mutation and
that M918V carriers must undergo total thyroidectomy
and neck dissection as appropriate, whenever elevated
serum calcitonin or suspicious thyroid nodules or cervical
lymph nodes are found. For the M918V carriers without
biochemical or imaging tests suggestive of MTC, we believe
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Research M C Martins-Costa et al.
that prophylactic thyroidectomy could be postponed
beyond 10 years of age (Wells et al. 2013, 2015).
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by research grants from the Sao Paulo
State Research Foundation (FAPESP) to R M B M, J R M M, M R D-S
(2006/60402-1) and to R M B M (2010/51547-1 and 2014/06570-6)
by FAPESP fellowship grants to S C L (2009/50575-4) and to M S A S
(2010/51546-5 and 2012/21942-1) and by a Fleury Group Research Grant
(12518). R M B M and A L M are investigators of the Fleury Group.
Author contribution statement
Drs Dias-da-Silva and Maciel had full access to all the data in the study and
take responsibility for the integrity of the data and the accuracy of the data
analysis. Drs Martins, Dias-da-Silva and Maciel contributed to study concept
and design. Martins-Costa, Cunha, Lindsey, Camacho, Dotto, Furuzawa,
Sousa, Kasamatsu, Kunii, Martins, Machado, Martins, Dias-da-Silva
and Maciel contributed to the acquisition, analysis or interpretation
of data. Martins-Costa, Martins, Dias-da Silva and Maciel drafted the
Endocrine-Related Cancer
manuscript. Cunha, Sousa, Martins, Dias-da-Silva, and Maciel critically
revised the manuscript for important intellectual content. Cunha and
Camacho contributed to statistical analysis. Martins, Dias-da-Silva and
Maciel obtained funding. Administrative, technical, or material support
was given by Furuzawa, Kasamatsu and Kunii. Study was supervised by
Martins, Dias-da-Silva and Maciel.
Acknowledgments
The authors are thankful to the Coordination for the Improvement
of Higher Education Personnel, Brazil-Ministry of Education (CAPES)
for the support to our Post-Graduation Program. The authors are thankful
to the team of the Laboratory of Molecular and Translational Endocrinology.
The authors are also thankful to the surgeons in Ceará who performed the
surgeries in our patients: André P Cortez, Marcelo E Holanda, Fernando
Porto Carreiro-Filho, J Wilson M de Farias, Sérgio B Lima and Luís A Albano
Ferreira. Also, the authors acknowledge all patients and relatives who
kindly hosted our regional BRASMEN task force in northeast Ceará.
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Received in final form 5 October 2016
Accepted 7 October 2016
Published by Bioscientifica Ltd.
 AUTHOR COPY ONLY L L Cunha and others Founder effect of RET533 in 176:5 515–519
Clinical Study MEN2A patients

Evidence for the founder effect of RET533 as

the common Greek and Brazilian ancestor
spreading multiple endocrine neoplasia 2A
Lucas L Cunha1, Susan C Lindsey1, Maria Inez C França1, Leda Sarika3,
Alexandra Papathoma3, Ilda S Kunii1, Janete M Cerutti2, Magnus R Dias-da-Silva1,
Maria Alevizaki3 and Rui M B Maciel1
Correspondence
1
Departments of Medicine and 2Morphology and Genetics, Escola Paulista de Medicina, Universidade should be addressed
Federal de São Paulo, São Paulo, Brazil, and 3Endocrine Unit, Department of Medical Therapeutics, to R M B Maciel
School of Medicine, National and Kapodistrian University of Athens, Athens, Greece Email
rui.maciel@unifesp.br

Abstract
Objectives: About one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to
mutations in the RET gene. A rare mutation in exon 8 (G533C) of RET, previously described in a large Brazilian family
European Journal of Endocrinology

with MEN2A, also appeared to be clustering in Greece, whereas it was rarely reported in other ethnic groups. The aim
of this study was to identify a possible common ancestry between these carriers.
Patients and methods: Twelve RET G533C mutation carriers, four randomly selected from the Brazilian cohort and
eight from apparently unrelated Greek families, were studied for a possible common ancestral origin. RET flanking
microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used.
Results: Genomic DNA analysis using these markers showed that many of these apparently unrelated individuals
shared a common haplotype indicating a common ancestral origin.
Conclusion: Our data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8
of RET gene originate from a common ancestor. Due to historical reasons, we speculate that the more plausible
explanation for the origin of this mutation is in Greece.

European Journal of
Endocrinology
(2017) 176, 515–519

Introduction
More than 25% of medullary carcinoma patients (MTC)
have familial disease, which is due to germ line mutations
of the RET gene. The clinical presentation varies according
to the type of RET mutation. After the routine application
of genetic analysis in all MTC cases (1), it became
apparent that some apparently sporadic cases were in fact
familial as they carried a RET mutation. Furthermore, a
wider distribution of mutations across the RET gene was
identified, which were different from those originally
described. In this context, we recently described a large
family with medullary thyroid carcinoma (MTC) carrying
a missense mutation in exon 8 of RET, which corresponds
to a glycine-to-cysteine substitution at codon 533 (2); this
was later confirmed to be MEN2A. This family comprises
728 members of Caucasian origin with ancestors from
Catalunia, Spain, who immigrated to Brazil at the end
of the 19th century. Most of the family members live
in Southeastern Brazil and have been followed at an
outpatient Endocrine Unit either in Vitoria or São Paulo
by the same team of physicians for over fifteen years.
During this long-term follow-up, molecular and clinical
characteristics of these patients have been described in
detail (3, 4). However, the ancestry related to the origin of
G533C RET mutation remains to be elucidated.

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DOI: 10.1530/EJE-16-1021 Printed in Great Britain
 AUTHOR COPY ONLY
Clinical Study L L Cunha and others Founder effect of RET533 in 176:5 516
MEN2A patients

Figure 1
(A) Schematic distribution of RET
microsatellite markers in representative
family members of apparently unrelated
kindreds from Brazil and Greece.
Microsatellite markers D10S197, D10S196,
D10S1652 and D10S537, and their distance
in cm, distributed throughout RET
genomic region as haplotype analysis.
(B) Phylogenetic tree of Brazilian and
Greek families and controls.

In 2007, Bethanis et al. reported a Greek patient with using four RET flanking microsatellite markers (D10S197,
European Journal of Endocrinology

MEN 2A harboring the same RET G533C mutation (5). A
few years later, Sarika et al. identified this mutation in 10
out of 129 patients with apparently sporadic medullary
thyroid carcinoma in Greece (6). Gathering these
results, we hypothesized whether Brazilian and Greek
patients carrying the same RET mutation could share a
common ancestor.
We proceeded with the investigation of putative
founder effect as a point of coalescence between Brazilian
and Greek affected families.
Patients and methods
Twelve patients with the G533C RET mutation were
enrolled in the study: 4 randomly selected from the
Brazilian cohort and 8 randomly selected from apparently
unrelated Greek families and followed at the Endocrine
Unit of Athens University Medical School (Athens,
Greece). A control group comprising 29 unrelated
D10S196, D10S1652 and D10S537) was performed, as
described by Qi  et  al. (8), to track a founder effect. The
first marker was placed at position 50.04 cm and the other
three at positions 70.07, 80.61 and 89.16 cm (Fig.  1A).
The chromosome map distances were derived from the
deCODE map (URL:http://www.ncbi.nlm.nih.gov/probe).
Haplotypes of patients and controls were reconstructed
using the statistical software package PHASE, version
2.1 (http://stephenslab.uchicago.edu/software.html)
(9). We constructed the phylogenetic tree of the studied
families with POPTREE2 software by estimating the
genetic distance between them (10). Chi-square test was
used to yield differences in allele distribution among
Greek patients, Brazilian patients and Brazilian controls.
Table 1 Clinical characteristics of the cohort of
G533C patients.
Age at Status at the
Patient diagnosis pTNM last follow-up
number Origin (years) Gender staging visit

thyroid-healthy Brazilian individuals was also studied. 1 Brazil 57 F T1N0Mx NED

Blood samples were obtained from all participants, and all 2 Brazil 44 F T2N1aMx NED
3 Brazil 33 M No MTC NED
laboratory procedures were performed at the Laboratory 4 Brazil 53 M T3N1aM0 NED
of Molecular and Translational Endocrinology at the 5 Greece 36 F T1N0M0 NED
Federal University of Sao Paulo – UNIFESP, Brazil. The 6 Greece 34 F T1bN0M0 NED
7 Greece 55 M T1N0M0 NED
study protocol was approved by the internal review board
8 Greece 37 M T1N0M0 NED
of UNIFESP (São Paulo, Brazil) and University of Athens 9 Greece 35 F T1N0M0 NED
(Athens, Greece). All patients received genetic counseling 10 Greece 35 F T1N0M0 NED
before and after RET testing. 11 Greece 24 F T3N1aM0 NED
12 Greece 50 F T1N0M0 NED
Genomic DNA was extracted from peripheral-blood
leucocytes as previously reported (7). Haplotype analysis NED, no evidence of disease.

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Clinical Study L L Cunha and others Founder effect of RET533 in 176:5 517
MEN2A patients

Table 2 Polymorphic microsatellite markers and allele presented similar distribution of RET polymorphic
frequency in Brazilian and Greek patients with medullary microsatellite markers compared to Greek patients,
thyroid carcinoma bearing the RET G533C mutation. suggesting that patients from both nationalities may share
a unique genetic signature (Table  2). When comparing
RET G533C RET G533C Greek
Brazilian patients patients Brazilian and Greek affected patients to the control
Marker Allele n % n % P value group, we observed a different distribution of D10S197
D10S197 162 0 0 3 37.5 0.223 (P = 0.016), D10S196 (P = 0.042), D10S1652 (P < 0.001) and
166 0 0 1 12.5 D10S537 alleles (P = 0.003; Table 3). These results reveal a
168 4 100 4 50 genetic progenitor similarity between Brazilian and Greek
D10S196 96 3 75 4 50 0.634
RET G533C patients regarding RET flanking regions that
98 0 0 1 12.5
100 1 25 3 37.5 was not observed in unrelated thyroid-healthy controls.
D10S1652 157 1 25 0 0 0.312 According to the analysis of the phylogenetic tree
166 0 0 1 12.5 of the MTC patients and controls, Brazilian and Greek
170 0 0 2 25
173 3 75 5 62.5 patients pertained to a common root, corresponding to
D10S537 139 2 50 1 12.5 0.262 a putative most recent common ancestor (Fig.  1B). As a
144 1 25 0 0 matter of fact, the control group shared reduced genetic
148 1 25 5 62.5
similarity with the MTC patients.
152 0 0 1 12.5
154 0 0 1 12.5

Discussion
European Journal of Endocrinology

GenePop was used to perform Hardy–Weinberg exact test

(11), and all loci were in Hardy–Weinberg equilibrium.
Results
This study included 8 Greek patients (2 men and 6 women)
and 4 Brazilian patients (2 men and 2 women). Clinical
and biochemical data of these patients are summarized in
Table 1. As can be seen from the table, all the examined
cases had excellent long-term follow-up compatible with
the relatively mild phenotype that has already been
reported for carriers of this mutation (12). Furthermore,
the age at presentation was in all cases relatively late.
Four MTC patients (3 from Greece and one from
Brazil) shared a common haplotype (D10S197-RET-
D10S196-D10S1652: 168-RETG533C-100-173-148, Fig. 1A
and Table 2), which was not seen in chromosomes bearing
wild-type alleles (P < 0.005). In fact, Brazilian patients
After the original identification that the exon 8 G533C
RET mutation is associated with inherited MTC in our
large Brazilian pedigree, there followed very few relative
reports from the Mediterranean region and especially
from Greece. Interestingly, the one family harboring this
mutation in the USA proved to be of Greek ethnic origin
(13). Our data suggest that Brazilian patients with MTC
caused by the G533C RET mutation have an ancestor
common to the Greek patients that share the same
RET genotype. Indeed, by tracking the founder effect,
we brought scientific shreds of evidence to this sense.
Probably, the G533C mutation observed in Brazilian
patients, originally from Catalunia, Spain, was settled by
a migratory movement in Europe, either from Greece or
from those same who established the Greek population.
Since ancient times, more than thirty Greek city-
states had several colonies over the Mediterranean Sea,

Table 3 Comparison of RET haplotype frequency among Brazilian and Greek RET533 patients with thyroid-healthy controls.

RET G533C Brazilian

Marker Allele patients RET G533C Greek patients Thyroid-healthy controls P valuea P valueb P valuec

D10S197 168 4 4 18 0.013 0.424 0.016

Other alleles 0 8 40
D10S196 96 3 4 14 0.059 0.199 0.042
Other alleles 1 4 44
D10S1652 173 3 5 1 <0.001 <0.001 <0.001
Other alleles 1 3 57
D10S537 148 1 5 8 0.472 0.004 0.003
Other alleles 3 3 50

a
Brazilian patients vs controls; bGreek patients vs controls; cBrazilian patients vs Greek patients vs controls.

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 AUTHOR COPY ONLY
Clinical Study L L Cunha and others
including in the Iberian Peninsula, where Catalunia
is located (14). In our first description of the G533C
RET mutation, we speculated that ‘this large family
has a Caucasian background from Catalunia, and it is
conceivable that relatives living in Europe might also
bear the same mutation; hence, it is likely to address a
founder effect from Spain, and it is feasible that other
relatives also migrated to other countries by the time
the ancestor moved to Brazil at the end of the 19th
century’ (2).
The fact that Brazilian (from Catalunia, Spain) and
Greek patients have a common ancestor is fascinating and
suggests that, historically, the more plausible explanation
for the origin of this mutation is in Greece as Greek
migratory currents have occurred in the Mediterranean
Sea for many centuries; furthermore, the substantial
proportion of the G533C RET mutation among the
hereditary MTC patients in Greece reinforces this
hypothesis (6).
It is worth noting that the world is a melting pot, in
European Journal of Endocrinology
which our genetically heterogeneous population can be
described as a complex fusion of ethnicities, nationalities
and cultures. In this scenario, cultural assimilation in
the setting of immigration is important not only for
the maintenance of national unity but also for the
understanding of the genetic distribution of deleterious
alleles. This ethnographic consideration highlights the
impact of the present study on public health.
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
The authors’ research was supported by grants from the São Paulo State
Research Foundation/FAPESP (2006/60402-1 and 2010/51547-1 to R M B M
and M R D S and 2014/06570-6 to J M C) and from the Fleury Group (12518).
S C L received a research fellowship grant from FAPESP (2009/50575-4). R M
B M is an investigator of the Fleury Group. J M C is an investigator of the
Brazilian National Research Council (CNPq).
Author contribution statement
Lucas L Cunha: Collection and assembly of data, data analysis and
interpretation and manuscript writing; Susan C Lindsey: Clinical data
analysis, interpretation and manuscript writing; Maria Inez C França:
Provision of study patients; Leda Sarika: Provision of study patients,
clinical data analysis and interpretation; Alexandra Papathoma: Technical
support; Ilda S Kunii: Technical support; Janete M Cerutti: Data analysis
and interpretation and manuscript writing; Magnus R Dias-da-Silva:
Design, data analysis and interpretation and manuscript writing; Maria
www.eje-online.org
Founder effect of RET533 in 176:5 518
MEN2A patients
Alevizaki: Conception and design and manuscript writing; Rui M B Maciel:
Conception and design and manuscript writing.
Acknowledgements
The authors thank the team from the Laboratory of Molecular and
Translational Endocrinology, especially Dr Cléber Camacho.
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Chen XL, Chen CY et al. RET germline mutations identified by
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10 Latter BD. Selection in finite populations with multiple alleles. 3.
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 AUTHOR COPY ONLY
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Received 13 December 2016

Accepted 30 January 2017
European Journal of Endocrinology

www.eje-online.org
 THYROID
Volume 27, Number 5, 2017
ª Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2016.0148

Assessment of Depression, Anxiety, Quality of Life,

and Coping in Long-Standing Multiple Endocrine
Neoplasia Type 2 Patients
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Karine C. Rodrigues,1,2 Rodrigo A. Toledo,1,* Flavia L. Coutinho,1

Adriana B. Nunes,3 Rui M.B. Maciel,4 Ana O. Hoff,2 Marcos C. Tavares,5
Sergio P. A. Toledo,1,4 and Delmar M. Lourenço, Jr.1,2

Background: Data on psychological harm in multiple endocrine neoplasia type 2 (MEN2) are scarce.
Objectives: The aim of this study was to assess anxiety, depression, quality of life, and coping in long-standing
MEN2 patients.
Patients and Methods: Patients were 43 adults (age ‡18 years) with clinical and genetic diagnosis of MEN2
and long-term follow-up (10.6 – 8.2 years; range 1–33 years). This was a cross-sectional study with quali-
tative and quantitative psychological assessment using semi-directed interviews and HADS, EORTC QLQ
C30, and MINI-MAC scales. Adopting clinical criteria from 2015 ATA Guidelines on MEN2, biochemical
cure (39%; 16/41), persistence/recurrence (61%; 25/41), and stable chronic disease (22/41) of medullary
thyroid carcinoma (MTC) were scored. Pheochromocytoma affected 19 (44%) patients, with previous ad-
renalectomy in 17 of them.
Results: Overall, anxiety (42%; mean score 11 – 2.9; range 8–18; anxiety is defined as a score ‡8) and de-
pression (26%; mean score 11 – 3.8; range 8–20; depression is defined as a score ‡8) symptoms were frequent.
Patients who transmitted RET mutations to a child had higher scores for weakness-discouragement/anxious
preoccupation and lower scores for cognitive, emotional, and physical functioning ( p < 0.05). Feelings of guilt
were present in 35% of patients with mutation-positive children. Lower mean score values for depression and
anxiety and higher scores for role, cognitive, and emotional functioning were noticed in 33 patients who were
well-informed about their disease ( p < 0.05). Fighting spirit was more frequently found in patients with multiple
surgical procedures ( p = 0.019) and controlled chronic adrenal insufficiency ( p = 0.024). Patients with MEN2-
related stress-inducing factors had lower scores for fighting spirit and cognitive functioning and higher scores for
insomnia and dyspnea ( p < 0.05). Eleven patients required sustained psychotherapeutic treatment. Mean global
health status was relatively good in MEN2 cases (68.1 – 22.3), and the cured group had higher physical func-
tioning ( p = 0.021).
Conclusions: Psychological distress is likely chronic in MEN2 patients. This study identified diverse MEN2-
related factors (degree of information on disease, mutation-positive children, number of surgeries, comorbidities,
stress-inducing factors, and cure) interfering positively or negatively with the results of the psychometrics scales.
The active investigation of these factors and the applied psychological assessment protocol are useful to identify
MEN2 patients requiring psychological assistance.

Keywords: MEN2, anxiety, depression, quality of life, coping, medullary thyroid carcinoma

1
Endocrine Genetics Unit (LIM-25), Endocrinology Division; 5Head and Neck Surgery Division; Hospital das Clı́nicas, University of
São Paulo School of Medicine, São Paulo, Brazil.
2
Endocrine Oncology Division, Institute of Cancer of the State of São Paulo, University of São Paulo School of Medicine, São Paulo,
Brazil.
3
Department of Endocrinology, Federal University of Rio Grande do Norte (UFRN), Natal, Brazil.
4
Translational and Molecular Endocrinology Laboratory, Endocrinology Division, Federal University of Sao Paulo (UNIFESP),
São Paulo, Brazil.
*Currently at the Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid, Spain.

693
 694
Introduction
C ancer patients may frequently develop psycho-
logical effects such as anxiety and depression as a con-
sequence of fear of recurrence, stress over surgery, and low
self-esteem (1). Thus, standard-of-care guidelines for distress
management in oncologic patients have been established (2).
Presently, advances in therapy and early diagnosis have
significantly increased the cure rate of several types of can-
cer, or have allowed patients to live with stable disease for
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prolonged periods.
In familial cancer syndromes, psychosocial challenges in-
clude the stigmatization and burden conferred by positive
mutation carrier status, fear of social discrimination, decision
making on reproduction, issues related to genetic testing in
relatives and offspring, feelings of guilt for transmission of a
germline mutation to descendants, and direct influences on a
couple relationship or between parents and children, among
others. Moreover, these factors may potentially amplify psy-
chological distress in patients with hereditary cancer (3–5).
Recently, sporadic and multiple endocrine neoplasia type 2
(MEN2)-related medullary thyroid carcinoma (MTC) have
been extensively reviewed (6–11). Contrasting with most
cancers, MTC is often a slow-growing carcinoma. Patients
usually live prolonged periods with stable advanced disease
and frequently have a higher global health status and poten-
tial long-term survival. In MTC associated with MEN2, in
which the diagnosis is often performed at earlier stages than
observed with the sporadic form of MTC (6,7,12–17), pa-
tients and relatives can be facing the psychosocial challenges
associated with familial cancer syndromes outlined above. In
this scenario, MEN2-related MTC is a good model to study
the psychological impact on the quality of life (QoL) and
psychological adjustments in patients living with cancer as a
chronic disease.
In addition, treatment-related complications may occur in
a substantial number of MEN2-related MTC patients, such
as hypoparathyroidism after total thyroidectomy, adrenal
insufficiency due to bilateral adrenalectomy, and postsurgical
hypothyroidism. These clinical conditions, even if appro-
priately treated with supplementation therapy, are usually
associated with lower health-related quality of life (HRQoL)
(18–22).
Early psychological studies in MEN2 have focused on the
distress related to genetic counseling and the disclosure of
RET genetic testing results to patients and their at-risk de-
scendants (3,4,23–25). However, scarce data are available on
the psychological consequences of MEN2 independent of
genetic testing disclosure (26,27).
The current study assessed anxiety and depressive symp-
toms, HRQoL, and psychological adjustment in a subset of 43
MEN2 patients living for years with MTC or at potential risk
of MTC recurrence.
Material and Methods
Patients
The study was conducted between November 2011 and
March 2015 and was approved by the local ethics committee
(CAPPesq) of the University of São Paulo School of Medi-
cine and the national ethics committee (CONEP). A signed
informed consent was obtained from all studied individuals.
RODRIGUES ET AL.
Only patients with at least one year of clinical and genetic
diagnosis and follow-up of MEN2 were invited to participate.
Thus, a chronicity status was secured in all patients, tenta-
tively excluding immediate and direct influences of the dis-
closure of genetic test results at the time of the psychological
interview. Other eligibility criteria included the following:
patients with stable apparent or occult metastatic disease,
cervical recurrent MTC or slowly progressive distant me-
tastasis, and biochemically cured patients after total thy-
roidectomy (basal serum calcitonin levels <2 pg/mL).
The exclusion criteria were as follows: negative RET sta-
tus, young RET-mutation carrier individuals (<18 years),
mental disease, brain metastasis, recent chemotherapy and/or
radiotherapy (<6 months), end-stage advanced disease at the
time of interview, quickly progressive advanced MTC, un-
controlled hypothyroidism, hypoparathyroidism, or adrenal
insufficiency.
All patients were participants of the MEN2 clinical
screening program performed by the authors’ group since
1985 at the Endocrine Genetics Unit. RET genetic testing and
genetic counseling have been offered to all MEN2 patients
and their at-risk relatives since 1999. Overall, 43 adult pa-
tients from unrelated 12 families, following the inclusion and
exclusion criteria, agreed to participate: 11 index cases and
32 family members with clinical and genetic diagnosis of
MEN2 (Table 1). A large family was defined as a family with
five or more affected members.
The clinical criteria recently established by the 2015
American Thyroid Association (ATA) Guidelines for MTC
were applied to define MEN2A, MEN2B, and the four phe-
notypic variants of MEN2A: classical MEN2A, MEN2A
associated with Hirschsprung Disease, MEN2A with cuta-
neous lichen amyloidosis, and isolated familial MTC (7).
Methods
In the present cross-sectional study, semi-directed inter-
views, psychological assessment instruments, and review and
analysis of medical records were applied.
Semi-directed interview. Patients were classified ac-
cording to sex, age, marital status, education level, occupa-
tion, and number of children. Feelings of guilt associated
with the possibility of transmitting the genetic mutation,
current disease status (active disease or cured), and satis-
faction degree with information previously offered about the
disease were addressed. Three psychological scales usually
applied to cancer patients were selected. The tests were ini-
tially developed in English (28–30) and then translated to and
statistically validated in Portuguese (31–33).
Hospital Anxiety and Depression scale. The Hospital
Anxiety and Depression scale (HADS) defines the presence
or absence of distress through application of a question-
naire including 14 items: seven questions directed to in-
vestigate depression (HADS-D) and seven to assess anxiety
(HADS-A). The total scores for each subscale—depression
and anxiety—ranges from 0 to 21. Scores of ‡8 in both
subscales are suggestive of an anxiety disorder or depres-
sion. The scale has the advantage of being appropriate for
cancer patients, allowing the exclusion of physical signs
 Table 1. Clinical and Genetic Characteristics of 43 MEN2 Patients
Characteristics n (%)
Sex
Male 19 (44)
Female 24 (56)
Syndrome and phenotypic variants/genotype
1. MEN2A (12 families) 42 (98)
Typical (classical) MEN2A (3 families) 11 (26)
c.1858T>C, p.C620R (1 family) 1 (2)
c.1900T>C, p.C634R (1 family) 8 (19)
c.1901G>A, p.C634Ya (1 family) 2 (5)
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MEN2A and CLA (5 families) 7/17 patients with CLA 17 (39)

c.1900T>C, p.C634R (2 families) 3 (7)
c.1901G>A, p.C634Y (1 family) 2 (5)
c.1901G>A, p.C634Ya (1 family) 7 (16)
c.1901G>A, p.C634Ya (1 family) 5 (11)
MEN2A and HD (2 families) 2/11 patients with HD 11 (26)
c.1858T>C, p.C620R:
MEN2A/HD (1 family) 4 (10)
MEN2/HD (1 family) 7 (16)
MEN2A (1 family) 3 (7)
c.2410G>A, p.V804M (MTC only)
2. MEN2B (1 family) 1 (2)
c.2753T>C, p.M918T
MTC 43 (100)
Previous surgery 41/43 (95)
Cured (cancer survivors) 16/41 (39)
Not cured (chronic oncologic patients): 25/41 (61)
Stable metastatic disease (occult or distant) 22/41 (54)
Cervical recurrent/ distant slowly progressive disease 3/41 (7)
Type of surgeries
Prophylactic thyroidectomy 1/41 (2)
Total thyroidectomy 21/41 (51)
Total thyroidectomy + cervical lymph node dissection 19/41 (47)
Additional surgical procedures of cervical lymph node dissection 16/41 (41)
Total cervical surgeries: 57
Persistent hypoparathyroidism 12/41 (29)
No previous surgery (1 case awaiting for surgery; 1 case refused it) 2/43 (5)
Pheo 18 (42)
Bilateral Pheo 12/18 (67)
Unilateral Pheo 6/18 (33)
Previous surgery 16/18 (89)
Types of surgery
Unilateral adrenalectomy 4/16 (25)
Bilateral adrenalectomy 12/16 (75)
Synchronous 5/12 (42)
Asynchronous 7/12 (58)
Total number of adrenal surgeries: 23
Adrenal insufficiency in glucocorticoid/mineralocorticoid therapy 12/18 (67)
Waiting for surgery 2/18 (11)
Hirschsprung disease 2/43 (5%)
Distal colectomy + corrective surgery of intestinal transit
Number of surgeries
0 1 (2)
1 18 (42)
2 12 (28)
3 10 (24)
4 1 (2)
6 1 (2)
Total number of surgeries: 82
a
The p.C634Y mutation and the allelic variant p.Y791F segregated in MEN2 patients of four families (59).
MEN2, multiple endocrine neoplasia type 2; MEN2A, MEN type 2A; MEN2B, MEN type 2B; MTC, medullary thyroid cancer; Pheo,
pheochromocytoma; CLA, cutaneous lichen amyloidosis; HD, Hirschsprung Disease; n, number; Reference sequence, NC_000010.10 for
genomic DNA and NM_020630.4 for cDNA.

695
 696
and symptoms that could interfere in assessing depression
and anxiety (28,31,34,35).
European Organization for Research and Treatment of
Cancer Quality of Life Questionnaire C30. The European
Organization for Research and Treatment of Cancer Quality
of Life Questionnaire C30 (EORTC QLQ C30) is a psycho-
logical assessment tool widely used to evaluate QoL in cancer
patients (29,36). It consists of 30 questions for assessing
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general aspects of HRQoL in cancer patients. The results are
converted to a linear score ranging from 0 to 100. For func-
tional and global health domains, 0 means the worst HRQoL
status and 100 represents the best status. For the symptoms
scale, higher scores correspond to higher intensity of symp-
toms and lower HRQoL status (29,33,36,37). Additionally,
patients were assessed with the Eastern Cooperative Oncol-
ogy Group (ECOG) performance status scale (29).
Scale of Mental Adjustment to Cancer: Factorial Struc-
ture. The MINI-MAC was developed to assess different
types and degrees of psychological adjustment during life-
time of oncologic patients. It is based on 29 questions related
with reactions of patients to cancer, which are answered
following a Likert scale of four points reflecting levels of
agreement: level 1, this is not remotely applicable to me;
level 2, it is not applicable to me; level 3, it applies to me;
and level 4, it applies fully to me. The questionnaire defines
five subscales: discouragement-weakness, anxious preoccu-
pation, fighting spirit, cognitive avoidance, and fatalism. The
highest score corresponds to the greatest endorsement of the
adjustment response (30,32). Demographic/clinical charac-
teristics and genetic profiles of the 43 MEN2 patients were
obtained through a review of medical records and semi-
directed interviews.
Data analysis
Data analysis was performed qualitatively and quantita-
tively. The issues of semi-directed interviews were analyzed
using the technique of content analysis. Quantitative data
were analyzed using SPSS for Windows v16.0 (SPSS, Inc.,
Chicago, IL). Spearman correlation was applied to analyze
relations between the scales of anxiety, depression, fighting
spirit, and the other scales. Tests were performed with a
significance level of 5%.
Results
Clinical and genetic profile
The clinical and genetic characteristics of the 43 MEN2
patients are shown in Table 1. A MEN2A phenotype was
present in 98% (42/43) of cases, whereas a MEN2B syn-
drome was identified in one (2%) case. The latter patient with
MEN2B had a milder clinical presentation of MTC, charac-
terized by a slower progression and lower aggressiveness
than that expected for patients harboring a M918T mutation.
Despite the late MTC diagnosis (at 17 years of age) and
therapy (total thyroidectomy + cervical lymph node dissec-
tion), this patient had stable and persistent biochemical dis-
ease characterized by mildly elevated calcitonin levels
(33 pg/mL) for a long period of follow-up (7 years). A typical
MEN2A phenotype was present in 11 patients from three
RODRIGUES ET AL.
families harboring RET mutations: p.C634R (n = 8), p.C634Y
(n = 2), and p.C620R (n = 1). MEN2A associated with cuta-
neous lichen amyloidosis was seen in five families involv-
ing 17 affected cases: 14 patients carrying a p.C634Y and
three carrying a p.C634R mutation, seven of whom exhibited
cutaneous lichen amyloidosis. The phenotypic variant of
MEN2A associated with Hirschsprung Disease was iden-
tified in one patient in each of two unrelated families har-
boring a p.C620R mutation (Table 1). In the first family
with associated Hirschsprung Disease, pheochromocytoma
(Pheo) was present in one of the four patients with MTC,
while it was absent in all seven affected members with MTC
in the second MEN2 family (38). The affected members of
one family with a p.V804M mutation developed only MTC,
and the three subjects were >50 years of age from two gen-
erations (Table 1).
MTC was detected in all 43 patients, of whom 41 (95%)
had been previously submitted to total thyroidectomy: 39%
(16/41) were biochemically cured of MTC after surgery, and
61% (25/41) had persistent/recurrent disease. Thus, previous
surgical treatment of MTC was not performed in only two
patients: one (p.C634Y) previously underwent bilateral ad-
renalectomy for Pheo and was waiting for thyroidectomy,
whereas the other (p.V804M), with mildly high basal calci-
tonin levels and positive calcium test, refused surgery.
Twelve patients required long-term therapy with oral calcium
and calcitriol due to persistent postsurgical hypoparathy-
roidism (29%; 12/41; Table 1).
Pheo was diagnosed in 18 (42%) patients, and 16 of
whom were previously submitted to adrenalectomy: 12 bi-
lateral (5 synchronous; 7 asynchronous) and four unilateral.
The two remaining patients have recently been diagnosed
with unilateral disease, and psychological interviews pre-
ceded adrenalectomy. All 12 patients with adrenal insuffi-
ciency secondary to bilateral adrenalectomy were treated
with glucocorticoid/mineralocorticoid substitution and were
clinically well controlled at the time of the psychological
evaluation (Table 1).
Two patients underwent intestinal surgery for Hirsch-
sprung Disease during the first year of life. Overall, 41 pa-
tients were submitted to 82 surgical procedures for MEN2
(M = 2.0 surgery/patient; range 1–6 surgeries; Table 1).
MEN2-related MTC: long-term follow-up
Long-term follow-up of MEN2-related MTC cases was
documented (Table 2). The duration of the follow-up, con-
sidered as the time interval between the first surgery and
psychological interviews, was 10.6 – 8.2 years (range 1–33
years). Most patients (34; 81%) had the first surgery 3–33
years before the psychological interview. Furthermore, il-
lustrating the relatively long follow-up, the mean time in-
terval between the last surgery and the psychological
interview was 5.5 – 4.6 years (range 1–15 years). Most pa-
tients (28; 67%) had the last surgery 3–15 years before the
psychological interview (Table 2).
Most patients with persistent MTC (85%; 22/25) had stable
disease characterized by occult biochemical disease or
structural/biochemical disease with clear evidence of stable
distant metastasis. Three other patients presented slowly
progressive disease defined by cervical recurrence and/or
slow progression of distant metastasis. Therapies such as
 PSYCHOLOGICAL DISTRESS, QOL, AND COPING IN MEN2 697

Table 2. Potentially Stressful Factors, Psychological Complaints, Disorders, and Adjustment

Types Observed in 43 Chronic MEN2 Patients, Following the Administration of Psychological
Assessment Instruments (Semi-Directed Interview, HADS, and MINI-MAC)
Long-standing follow-up, Mage; range (years)
a. Psychological interview 39.3 – 14.7; 19–70
b. First surgery 28.1 – 15.1; 5–67
c. Last surgery 33.3 – 15.7; 8–67
Correlations: a versus b ( p < 0.001); a versus c ( p < 0.001)
Time interval between first surgery and psychological interview: 10.6 – 8.2; 1–33
Time interval between last surgery and psychological interview: 5.5 – 4.6; 1–15
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Semi-directed interview
1. Stressful potentially factors, n (%)
Patients with RET mutation-positive children 17 (40)
Expectancy for results of genetic testing 7 (16)
Waiting for the own surgery 4 (9)
Waiting for surgery of their children 1 (2)
Slowly progressive distant metastasis 2 (5)
Total 30 (70)
2. Feelings of guilt from transmission of the mutation to their children, n (%)
Presence of guilt 6 (35)
Absence of guilt 11 (65)
HADS, n (%)
Anxiety symptoms (score ‡8) 18 (42%)
Score: 11 – 2.9, range 8–18
Depression symptoms (score ‡8) 11 (26%)
Score: 11 – 3.8, range 8–20
Clinically relevant anxiety symptoms (score ‡11) 12 (28%)
Score: 13 – 2.6, range 11–18
Clinically relevant depression symptoms (score ‡11) 5 (12%)
Score: 14 – 3.6, range 11–20
MINI-MAC (scale from 1 to 4),a M – SD
Weakness-discouragement 1.5 – 0.84
Anxious preoccupation 1.9 – 0.95
Fighting spirit 3.3 – 0.77
Cognitive avoidance 2.56 – 1.17
Fatalism 3.1 – 0.90
a
An average value closer to the maximum value (of 4) represents a higher level of coping used by the cancer patient, as follows: level 1,
this is not remotely applicable to me; level 2, this is not applicable to me; level 3, this applies to me; level 4, this applies fully to me.
HADS, Hospital Anxiety and Depression scale; MINI-MAC, scale of mental adjustment to cancer: factorial structure; SD, standard
deviation.

chemotherapy, radiotherapy, or inclusion in clinical trials
with targeted therapies were not recommended for the latter
cases. At the time of interview, only four patients with pre-
vious MEN2-related surgeries were still waiting for surgical
procedures (MTC 2; Pheo 2). One patient with local recur-
rence waited for cervical surgery, another for thyroidectomy
and parathyroidectomy after subtotal adrenalectomy, and two
patients for unilateral adrenalectomy. One patient refused
surgery for MTC (Table 1).
Overall, 25 MEN2 patients had uncured MTC (stable 22,
recurrent/slowly progressive 3), whereas 16 were biochemi-
cally cured patients. Another two patients had no previous
treatment for MTC (Table 1).
Psychological profile
Semi-directed interview. In the semi-directed interview,
31 (72%) patients reported fear of dying when they received
the genetic diagnosis. At the time of the psychological in-
terview, 30 (70%) MEN2 patients had children, and 24 of
them reported that RET genetic testing had been offered to
them. All except one agreed with RET genetic testing for their
children. Each of the other six (18%) affected parents with
children younger than three years of age received genetic
counseling based on the recommendations issued in the
2009 ATA Guidelines (14). Seventeen (70%) parents had at
least one RET mutation-positive child. One was waiting for
the genetic testing result. Six other parents had mutation-
negative children. Overall, 46 at-risk children were tested,
and half of them were positive RET carriers. Most parents
(13/17; 76%) expressed strong feelings of fear of losing their
children at the time of genetic testing disclosure. Feelings of
guilt of having transmitted the genetic mutation to their
children were present in one third of transmitting parents
(6/17; 35%; Table 2). At the time of the psychological in-
terview of the 17 MEN2 parents with a RET mutation-
positive child, all their children except one had a previous
history of at least one MEN2-related surgery.
HADS anxiety scale. High scores for anxiety symptoms,
defined as a HADS score ‡8, were present in 18 cases (42%;
score 11 – 2.9; range 8–18): 12 (28%) patients had scores
 698 RODRIGUES ET AL.

‡11, indicating the presence of clinically relevant anxiety Table 3. Quality of Life Results Obtained
symptoms, whereas another six (14%) patients exhibited for 43 MEN2 Patients Using EORTC QLQ C30
symptoms of anxiety disorders (8 £ score <11). In the re-
maining 25 MEN2 patients, the mean HADS score was EORTC QLQ C30 M – SD
4 – 2.2 (range 0–7; Table 2). Global health status/QoLa 68.1 – 22.3
Functional scales
HADS depression scale. High scores compatible with Role functioninga 72.4 – 32.2
depression symptoms, defined as a HADS score ‡8, were Cognitive functioninga 57.7 – 36.1
Physical functioninga 79.3 – 21.2
detected in 11 cases (26%; score 11 – 3.8; range 8–20).
Emotional functioninga 59.7 – 32.8
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Scores ‡11 were found in five (12%) patients, documenting
clinically relevant depression symptoms, whereas symptoms
indicative of depression disorders were present in six patients
(14%; 8 £ score <11). Low score values were documented in
the remaining 32 patients (74%; score 3 – 2.0; range 0–7;
Table 2).
Overall, distress (anxiety and/or depression) was present in
20 (46%) cases, taking a HADS score ‡8 as the cutoff, and of
these, 13 (30%) had HADS scores ‡11.
MINI-MAC
Mean values of the different types of coping in the 43
MEN2 patients evaluated by MINI-MAC are shown in Ta-
ble 2. Coping behaviors such as fighting spirit (3.3 – 0.77),
cognitive avoidance (2.56 – 1.17), and fatalism (3.1 – 0.90)
were higher than weakness-discouragement (1.5 – 0.84) and
anxious preoccupation (1.9 – 0.95; Table 2). Very high score
values (‡3) were more frequent for fighting spirit (86%; 37/
43) and fatalism (65%; 28/43) in comparison with cognitive
avoidance (42%; 18/43), anxious preoccupation (16%; 7/43),
and weakness-discouragement (9%; 4/43). All 13 patients
with the maximum score of four for fatalism had high scores
(range 3.25–4.0) for fighting spirit.
EORTC QLQ C30
Most of the 43 MEN2 patients were in a satisfactory global
state of health (68.1 – 22.3) when assessed by EORTC QLQ
C30, considering both physical and emotional aspects (Ta-
ble 3). Patients were classified as ECOG 0 (29/43; 67%) or 1
(14/43; 33%).
Correlation between psychological
assessment instruments
A strong direct correlation between anxiety and depressive
symptoms was detected (r = 0.702; p < 0.001). Both symp-
toms had direct correlations with weakness-discouragement,
anxious preoccupation, cognitive avoidance, fatigue, pain,
insomnia, and constipation (Supplementary Table S1; Sup-
plementary Data are available online at www.liebertpub
.com/thy). Inverse correlations were detected between anxi-
ety/depression symptoms and scales of global health status
and functional subscales (role, cognitive, social, and emo-
tional functioning; p < 0.05; Supplementary Table S2). A
correlation between fighting spirit and fatalism was also
observed (r = 0.369; p = 0.015). However, no correlation be-
tween anxiety/depression symptoms and fighting spirit was
observed ( p > 0.05).
Eleven (25%) patients required individual psychothera-
peutic follow-up after the initial evaluation.
Social functioninga 81.4 – 28.9
Symptom scales
Painb 35.3 – 36.6
Fatigueb 31.3 – 29.5
Insomniab 27.8 – 35.9
Constipationb 23.6 – 36.7
Diarrheab 12.7 – 20.7
Nausea and vomitingb 12.2 – 21.2
Dyspneab 27.3 – 35.7
Appetite lossb 19.7 – 35
Financial difficultiesb 17.4 – 30.5
a
Scores range from 0 to 100, with a higher score representing a
higher level of function.
bScores range from 0 to 100, with a higher score representing a
higher level of symptoms.
EORTC QLQ C30, European Organization for Research and
Treatment of Cancer Quality of Life Questionnaire C30; QoL,
quality of life.
Cured versus uncured MTC patients
Higher scores for physical functioning (87 – 21 versus
77 – 18; p = 0.021) and cognitive avoidance (odds ratio
[OR] = 2.68; p = 0.024) were observed in the cured group
compared with uncured cases. Furthermore, a non-significant
tendency to a higher prevalence of working activities was
observed in cured (81%) compared with uncured (52%) pa-
tients ( p = 0.054). There was no correlation between ECOG
performance and state of cure in the patients ( p = 0.084).
Combined or independent analysis of potential
factors of psychological effect
Presence of children. Twenty-five patients had children
either harboring mutations or waiting for genetic testing
disclosure or genetic screening. These affected parents
were compared with 18 patients with no children or with
RET-negative children. The first group had higher scores for
insomnia ( p = 0.004), dyspnea ( p = 0.005), anxious preoccu-
pation ( p = 0.008), and weakness-discouragement ( p = 0.049).
Furthermore, scores were lower for cognitive ( p = 0.001),
emotional ( p = 0.028), and physical functioning ( p = 0.038) in
the first subset of cases (Table 4).
Information on disease. Thirty-three MEN2 patients
(77%) reported they were well informed on MEN2 by the
multidisciplinary team. These patients had lower mean scores
for fatigue ( p = 0.002), weakness-discouragement ( p = 0.012),
anxiety ( p = 0.022), depression ( p = 0.031), anxious preoccu-
pation ( p = 0.031), and constipation ( p = 0.044), as well as
higher scores for role ( p = 0.010), cognitive ( p = 0.031), and
emotional functioning ( p = 0.009; Table 5).
 Table 4. Psychological Harm in MEN2 Patients as a Result of the Presence/Absence of Children,
Knowledge of RET Genetic Status of the Offspring (RET-Positive Children or RET-Negative
Children), and Expectancy for RET Genetic Testing Disclosure
Variable Childrena M SD Minimum Maximum n p
Age (years) Yes 42.56 13.99 22.0 70.0 25 0.083
No 34.72 14.70 19.0 66.0 18
HADS: anxiety Yes 8.12 5.00 1.0 18.0 25 0.081
No 5.39 3.48 0.0 11.0 18
HADS: depression Yes 5.64 4.95 0.0 20.0 25 0.400
No 4.11 3.39 0.0 12.0 18
Weakness-discouragement Yes 1.70 1.01 1.0 4.0 25 0.049
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No 1.19 0.44 1.0 2.8 18

Anxious preoccupation Yes 2.23 1.03 1.0 4.0 25 0.008
No 1.49 0.66 1.0 3.0 18
Role functioning Yes 64.00 36.86 0.0 100.0 25 0.056
No 86.11 19.17 33.3 100.0 18
Cognitive functioning Yes 42.00 38.22 0.0 100.0 25 0.001
No 80.56 19.17 50.0 100.0 18
Fatigue Yes 38.67 31.61 0.0 100.0 25 0.056
No 20.99 25.54 0.0 88.9 18
Insomnia Yes 40.80 39.74 0.0 100.0 25 0.004
No 9.26 22.30 0.0 66.7 18
Physical functioning Yes 74.93 21.54 33.3 100.0 25 0.038
No 88.52 13.30 60.0 100.0 18
Emotional functioning Yes 50.91 33.63 0.0 100.0 25 0.028
No 73.38 28.91 4.2 100.0 18
Dyspnea Yes 40.00 39.67 0.0 100.0 25 0.005
No 9.26 22.30 0.0 66.7 18
Statistically significant ( p < 0.05) values are shown in bold; those in italic bordered statistical significance.
a
MEN2 patients with children either harboring RET mutations or waiting for genetic testing disclosure or for genetic screening (yes);
MEN2 patients with no children or with RET-negative children (no).

Table 5. Correlations Between Information Degree on MEN2 Disease and Psychometric

Indexes of the HADS, MINI-MAC, and EORTC QLQ-C30
Information
Variable degree on MEN2a M SD Minimum Maximum n p
Age (years) Yes 40.18 14.57 22.0 70.0 33 0.470
No 36.30 15.32 19.0 68.0 10
HADS: anxiety Yes 6.03 4.16 0.0 15.0 33 0.022
No 10.10 4.77 4.0 18.0 10
HADS: depression Yes 4.15 3.65 0.0 15.0 33 0.031
No 7.80 5.57 3.0 20.0 10
Weakness-discouragement Yes 1.33 0.76 1.0 4.0 33 0.012
No 2.00 0.98 1.0 3.8 10
Anxious preoccupation Yes 1.75 0.91 1.0 4.0 33 0.031
No 2.49 0.95 1.0 4.0 10
Role functioning Yes 80.81 26.39 0.0 100.0 33 0.010
No 48.33 38.85 0.0 100.0 10
Cognitive functioning Yes 65.15 33.69 0.0 100.0 33 0.031
No 35.00 38.85 0.0 83.3 10
Fatigue Yes 22.90 24.68 0.0 77.8 33 0.002
No 58.89 31.45 11.1 100.0 10
Constipation Yes 14.14 27.68 0.0 100.0 33 0.044
No 53.33 50.18 0.0 100.0 10
Emotional functioning Yes 67.73 30.51 4.2 100.0 33 0.009
No 35.83 31.68 0.0 91.7 10
Statistically significant ( p < 0.05) values are shown in bold. Preferentially, the correlations with statistical significance were represented
in table.
a
Patients declared, during the semi-directed interview, if they felt satisfied and well-informed (yes) or not (no) about MEN2 disease.

699
 700 RODRIGUES ET AL.

Factors inducing stress. Overall, 30 MEN2 patients
presented one or more potential stress-inducing factors: pa-
tients (40%; 17/43) with RET mutation-positive children
(50%; 23/46), patients waiting for surgery (9%; 4/43) or
surgery for their children (2%; 1/43), parents awaiting genetic
testing of their children (16%; 7/43), and presence of slowly
progressive distant metastasis (5%; 2/43). These stress-
inducing factors were absent in 13 subjects (30%; Table 2). Of
note, there were no differences in global health status or
anxiety/depression scores between these two groups. In pa-
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of mutation-positive children, ECOG status and presence/
absence of MEN2-related stress-inducing factors, number of
surgeries, information on disease, and MEN2-related co-
morbidities (adrenal insufficiency and hypoparathyroidism).
Twenty-three (56%) patients were married, 17 (40%) single,
one (2%) divorced, and one (2%) widowed. The most fre-
quent non-MEN2-related comorbidities were systemic arte-
rial hypertension (21%; 9/43), dyslipidemia (21%; 9/43), and
diabetes (4.7%; 2/43).
Most (24/38; 56%) of the patients were actively working

tients with stress-inducing factors, scores were higher for
fighting spirit (3.6 – 0.36 vs. 2.9 – 0.94; p = 0.028), insomnia
(36.2 – 39.2 vs. 7.7 – 20; p = 0.034), and dyspnea (35.6 – 39
vs. 7.7 – 20; p = 0.034), and lower for cognitive functioning
(48.9 – 38.7 vs. 79.5 – 20.6; p = 0.019). Stress-inducing fac-
tors were significantly more frequent in older MEN2 patients
and women (67% vs. 33%; p = 0.029; Table 6).
Treatment-related comorbidities. HADS, EORTC QLQ
C30, and MINI-MAC score values did not differ in patients
with or without hypoparathyroidism. Fighting spirit was
higher in patients who had undergone three or more surgical
procedures (3.8 – 0.4 vs. 3.3 – 0.71; p = 0.019) or who had
clinically controlled chronic adrenal insufficiency secondary
to bilateral adrenalectomy (3.6 – 0.9 vs. 3.3 – 0.6; p = 0.024;
Supplementary Table S3). The presence of treatment-related
comorbidities (hypoparathyroidism and adrenal insufficien-
cy) or number of MEN2-related surgical procedures did not
interfere with working activities.
Other correlations. Data for demographic profile (age,
sex, job, marital status, and education level), patient status
(index case or family member), and family size were corre-
lated with results obtained from psychometric scales (HADS,
MINI-MAC, and EORTC QLQC 30), disease state (cured
or uncured), presence/absence of children, presence/absence
and had higher physical functioning (86 – 16 vs. 70 – 23;
p = 0.04) than inactive subjects di (unemployed, off work,
and not retired). In turn, the latter group (33%; 14/38) pre-
sented with a higher scale of pain (57 – 37 vs. 26 – 34;
p = 0.02). The small group consisting of students (n = 3) or
retired individuals (n = 2) were excluded from this analysis.
The global health status (73 – 22 vs. 59 – 16; p = 0.04) was
lower in the group of index cases (11/43) than it was in family
members (32/43).
The mean age of affected members of large MEN2 fami-
lies was lower than that observed in small families (36 – 13
years vs. 46 – 15 years; p = 0.03). Statistically significant
correlations were observed in two items of the symptoms
scale (EORTC QLQ C30), and they were more prevalent in
small families: dyspnea ( p = 0.02) and insomnia ( p = 0.02).
No other correlations were statistically significant.
Qualitative analysis
During the semi-directed interview, qualitative psycho-
logic investigation evaluated the individual experiences of
the patients that were consistent with data obtained from
psychometric scales. Thus, patients reported daily events and
facts associated with anxiety and depression symptoms and
insecurity regarding their own future and their children. In
addition, diverse attitudes and daily decisions were clearly
associated with the different types of coping observed with

Table 6. Correlations Between Stressful Factorsa in MEN2 Patients and Psychometric

Indexes of the HADS, MINI-MAC, and EORTC QLQ-C30
Variable Stressful factors M SD Minimum Maximum n p
Age (years) Yes 42.40 14.27 22.0 70.0 30 0.032
No 32.08 13.36 19.0 65.0 13
HADS: anxiety Yes 7.60 4.87 1.0 18.0 30 0.265
No 5.54 3.64 0.0 11.0 13
HADS: depression Yes 5.20 4.77 0.0 20.0 30 0.948
No 4.54 3.48 0.0 12.0 13
Fighting spirit Yes 3.60 0.36 3.0 4.0 30 0.028
No 2.92 0.94 1.0 4.0 13
Cognitive functioning Yes 48.89 38.64 0.0 100.0 30 0.019
No 79.49 20.59 50.0 100.0 13
Insomnia Yes 36.22 39.21 0.0 100.0 30 0.034
No 7.69 19.97 0.0 66.7 13
Dyspnea Yes 35.56 39.08 0.0 100.0 30 0.034
No 7.69 19.97 0.0 66.7 13
Statistically significant ( p < 0.05) values are shown in bold. Preferentially, the correlations with statistical significance were represented
in table.
a
Patients with or without potential stressful factors. The following stressful factors were considered: parents with RET mutation-positive
children, cases waiting for his own surgery or of their children, short-term expectancy for genetic testing of their children, and presence of
slowly progressive distant metastasis.
 PSYCHOLOGICAL DISTRESS, QOL, AND COPING IN MEN2
the MINI-MAC scale. However, most parents (16/17; 94%),
during the qualitative interview, revealed documenting
feelings of guilt for transmitting the mutation to their chil-
dren, which contrasted with 35% of them affirming this guilt
during the semi-directed interview.
Discussion
Previous psychological studies in MEN2
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Available psychological studies in MEN2 patients are
scarce and have mostly focused on distress preceding the
disclosure of RET genetic testing and reactions of patients
and their family members when the mutation-positive carrier
status was established (3,4,23–25). Grosfeld et al. quantita-
tively and qualitatively documented distress in affected
MEN2 family members and partners as result of anticipation
of genetic testing disclosure, ambivalent feelings of couples
about the decision making for performing genetic testing on
their offspring, tackling marital problems and changes in the
relationship between parents and children caused for feelings
of guilt for genetic transmission, and excessive preoccupa-
tion with disease-related signs in mutation-carrying offspring
(24,25). In addition, to the authors’ knowledge, only two
other studies have been published applying psychometric
scales in MEN2 patients. The first analyzed distress in 35
MEN2 patients and 40 cases with sporadic MTC applying
HADS and HRQoL using the subjective quality of life profile
(SQLP) (26). Subsequently, the authors compared HRQoL
findings from the same MEN2 cohort with a control popu-
lation (27).
In contrast, the present cross-sectional study focused on
psychological distress (anxiety and depression), HRQoL, and
coping in long-standing MEN2 patients using semi-directed
interviews and psychometric scales (HADS, EORTC QLQ
C30, and MINI-MAC). The MINI-MAC scale has not pre-
viously been used to investigate mechanisms of psycholog-
ical adjustment in MEN2. Furthermore, instead of SQLP, the
EORTC QLQ C30 scale was chosen to evaluate HRQoL, as
this test is widely applied in cancer patients (29,33,36,37,39–
41). In addition, the EORTC QLQ C30 scale has been con-
sidered satisfactory when directed specifically to long-term
cancer survivors, such as the present MTC cases (36).
Distress in MEN2 and other inherited cancer patients
Qualitative psychological assessment in MEN2 cases re-
vealed high basal distress indexes (50%) preceding genetic
counseling and genetic testing disclosure (3). A lower and
variable prevalence of baseline psychological distress (6–
39%) was documented in other hereditary cancer syndromes
(Von Hippel–Lindau, Li–Fraumeni, hereditary breast and
ovarian cancer, hereditary nonpolyposis colon cancer, fa-
milial adenomatous polyposis) (5,42,43). A wide variation in
the prevalence of psychological distress (anxiety 0.9–49%;
depression 0–46%) has been documented in cancer patients.
These data may be due to the use of different psychometric
scales and study designs (cross-sectional vs. prospective),
specific characteristics of each cancer type, sex, age, staging,
cultural differences, and time interval since diagnosis (1).
Degrees of basal distress could not be approached in the
present cases, since in all patients genetic counseling and
testing preceded the application of psychometric scales.
701
Short-term distress outcome in hereditary cancer
patients and MEN2
Despite high baseline distress indexes, a significant short-
term reduction of symptoms reaching normal levels is typi-
cally observed in hereditary cancer syndromes one year after
genetic testing disclosure (5,26). Concordantly, a significant
distress reduction was reported in MEN2 one year after ge-
netic testing disclosure (3). The short-term distress outcome
could not be explored in the present study, as MEN2 patients
had long-term follow-up (10.6 – 8.2 years; range 1–33 years)
with genetic testing disclosure several years before the ap-
plication of the psychometric scales.
Long-term distress outcome in MEN2
Overall, long-term results regarding distress in hereditary
cancer syndromes are contradictory (5). Long-term distress
rate observed in the MEN2-related MTC patients in this study
(anxiety and/or depression: 46%; HAD score ‡8) was defi-
nitely more frequent than the short-term distress rate previ-
ously reported in MEN2 cases (20%) one year after disclosure
of genetic testing results (3). These findings indicate a chronic
distress state in the present cases. Using a HADS score >11 as a
cutoff, Freyer et al. (26) identified that 48% of their patients
with MTC suffered from distress. Changing the HADS score to
‡8 resulted in the distress rate affecting 73% of patients.
Formal comparisons with the present study could not be per-
formed, as there were no available data on long-term follow-up
in the MEN2 patients reported by Freyer et al. (26). Thus,
different from the present study, interferences on short-term
distress rate after genetic testing disclosure previously reported
by Grosfeld et al. (3) could not be excluded in the study by
Freyer et al. (26).
Possible reasons for a high prevalence of long-term distress
in the present MEN2 cases may not be fully explained by
feeling isolated and a loss of perspective for the future com-
monly seen in cancer-patient series. This conclusion is sup-
ported by the fact that most of patients were actively working,
had satisfactory HRQoL patterns, and were biochemically
cured or had stable disease. Thus, it is likely that other factors
are potentially contributing to the chronic distress state in
MEN2, for example the impact of having mutation-positive
offspring, the degree of information on the syndromic disease,
and influence of MEN2-related comorbidities.
Impact of having children or mutation-positive children
It was found that having a child carrying a RET mutation or
expecting results of genetic testing in the offspring are asso-
ciated with higher scores for weakness-discouragement and
anxiety in affected parents. In contrast, patients without chil-
dren or with non-carrier children were found to have higher
HRQoL scores (cognitive, emotional, and physical function-
ing), an observation that further illustrates the state of high
chronic distress in parents with genetically affected children.
Consistent with these observations, patients with hereditary
MEN2-related MTC present lower levels of satisfaction and
HRQoL than sporadic MTC cases and control individuals,
documenting a negative psychological impact of harboring a
germline RET mutation (27). Additionally, increasing levels of
anxiety were reported in parents of at-risk children after the
disclosure of positive genetic testing results (24).
 702
Taken together, these data support the findings of a chronic
anxiety state in MEN2 patients and lower HRQoL scores in
parents who transmitted the RET mutation to their children,
contrasting with previous studies showing a gradual and
progressive decrease in psychological concerns and better
adaptation one year after MEN2 diagnosis and in other fa-
milial cancer syndromes such as hereditary breast and ovar-
ian cancer (HBOC) and Lynch syndrome (3,5,26).
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Information about MEN2
In the present series, most patients felt they were well
informed about MEN2 by medical staff, which probably led
to strong compliance with medical management and accep-
tance of genetic testing (95%) for their children. Of note, a
shared decision-making approach has been tentatively used
in all cases by the study group in patients affected by genetic
syndromes of predisposition to endocrine neoplasia (44–46).
These results reinforce those of Cleiren et al. on the high
degree of patient satisfaction obtained with specialized
multidisciplinary support (47). The present data indicate less
psychological harm, improved psychological adjustments,
and better HRQoL scores in well-informed patients. Hence,
the importance of offering high-quality information to pa-
tients as a potential tool to secure better psychometric indexes
must be emphasized.
MEN2-related comorbidities
The impact of MEN2 treatment-related comorbidities such
as hypothyroidism, hypoparathyroidism, and adrenal insuf-
ficiency have not been analyzed in early psychological
studies in MEN2. These conditions can be associated with
lower HRQoL patterns, even in patients with adequate hor-
monal substitution (18–22). In the present study, patients
were only interviewed if their potential comorbidities were
well controlled. Thus, all 41 MEN2-related MTC patients
operated prior to the psychological interview had compen-
sated post-thyroidectomy hypothyroidism. Moreover, com-
paring MEN2 subsets with or without hypoparathyroidism
and/or adrenal insufficiency, no significant differences were
observed, except for a higher fighting spirit in patients with
adrenal insufficiency.
Overall, most MEN2-related clinical comorbidities are di-
rect consequences of multiple surgeries and they would have a
potential to influence or amplify psychological harm. However,
we observed no correlation between the number of MEN2-
related surgeries and psychological indexes, except for higher
fighting spirit in patients with three or more surgeries.
A previous study documented no influence of thyroidec-
tomy on distress in MEN2-related MTC cases (26). In addi-
tion, high levels of frustration and latent dissatisfaction were
present in MEN2-related MTC patients with or without thy-
roidectomy (26). It was not possible to analyze the influence
of surgery on psychological concerns because almost all cases
underwent thyroidectomy prior to evaluation (41/43; 95%).
QoL in MTC/MEN2
The application of different scales of HRQoL prevented
comparisons between the study by Freyer et al. and the
present data (26,27).
RODRIGUES ET AL.
Cured and uncured patients
MTC/MEN2 is still frequently diagnosed at advanced
stages in which cure rates are lower, as seen in many of the
present cases (61%). Conversely, cure rates are usually much
higher when genetic screening is performed and if at-risk
relatives are diagnosed at an early stage, as seen in 16 (39%)
of the present patients.
Of note, applying EORTC QLQ C30, the global HRQoL
scores for several types of cancer have been reported to be
nearly equal to those of the general population. In contrast,
markedly worse HRQoL mean scores have been reported in
cancer patients or survivors compared with scores for the
general population if specific subscales of the EORTC QLQ
C30 are considered as functioning scales, symptom scales,
and symptom items (39,41).
Despite the lack of healthy unaffected controls, higher
physical functioning was documented in the cured patients,
while the global HRQoL scores were similar in both groups.
These findings are in line with the data of Hinz et al. and
support the key role of analyzing specific components of
HRQoL as functioning scales (39). Additionally, the higher
physical functioning in the cured patients is also illustrated by
the tendency to mildly higher frequency of working activities
( p = 0.054). Furthermore, cognitive avoidance was most evi-
dent in the cured group ( p = 0.024). This psychological defense
mechanism was appropriate for this subset, as it did not inter-
fere with patient compliance with the offered medical care.
High frequencies of psychological concerns found in pa-
tients with persistent disease and cancer survivors (cured pa-
tients) may be due to multiple reasons. Among them, patients
knew about the low tumor cure rates in older patients, restricted
non-surgical therapeutic tools for MTC, frequent requirement
for multiple surgeries for disease control, and the fact that MTC
can be resistant to chemo- and radiotherapy. Several patients
also experienced the death of affected relatives. All these factors
could result in disbelief and eventually may explain the high
frequency of fatalism in the present patient series. However, the
potential negative impact of fatalism probably was counter-
balanced by satisfactory QoL, a high physical functioning, and
appropriate information on actual disease state favoring strong
fighting spirit and, consequently, working activities and excel-
lent compliance with medical care.
The similar frequencies of psychological distress (anxiety
and depression) in the cured and uncured MTC patients are
likely due to a high prevalence of stable disease in the latter
group. Of note, all patients had a satisfactory performance
status based on ECOG and HRQoL scales, although HRQoL
subscales revealed a higher performance status in the cured
group.
Stress-inducing factors
Several potentially stress-inducing factors were identified,
alone or in combination, in 30 MEN2 patients, despite similar
scores for HRQoL and distress compared with patients without
such factors. The finding of higher indexes of insomnia and
dyspnea and lower cognitive functioning suggests that these
patients are more susceptible to psychological harm. In con-
trast, higher scores for fighting spirit underscore an appropriate
adaptive response mechanism in these patients. The more ad-
vanced age associated with increasing stress-inducing factors
may be explained at least in part by a high prevalence of parents
 PSYCHOLOGICAL DISTRESS, QOL, AND COPING IN MEN2
with RET mutation-positive children or parents expecting ge-
netic screening results for their children.
Some potential stress-inducing factors verified in the
MEN2 patients were of limited duration, for example waiting
for genetic testing or surgery, while others were persistent
such as the presence of RET mutation-positive children and
the constant fear of disease recurrence or progression.
The excess of catecholamine secreted by Pheos may be
considered an important stress-inducing factor in MEN2, and
it could potentially contribute to psychological distress in
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these patients. Korpershoek et al. (48) reported that adrenal
medulla hyperplasia represents micro-Pheos in MEN2, and
early surgery should be considered. The impact of Pheos as a
stress-inducing factor seems to be minimized in the present
cases, as there were only two patients with recently diagnosed
Pheos waiting for adrenalectomy and no case with a sus-
pected micro-Pheos.
Prospective studies applying psychometric scales to
MEN2 patients are needed to study the impact of stress-
inducing factors on psychological distress and HRQoL, and
to identify scenarios requiring closer psychological surveil-
lance and attendance. Furthermore, cultural differences and
religious and ethical views may also impact psychological
scores and should be considered in future investigations.
Guilt
During semi-directed interviews, feelings of guilt were
present in 35% of patients with mutation-positive children.
However, during qualitative psychological interviews, such
feelings were strongly present in most parents with mutation-
positive children. Thus, a process of cognitive avoidance, a
common coping mechanism in these patients, influenced the
answer during the semi-directed questionnaire, obscuring the
feelings of guilt of most parents.
RET mutations and psychological profile
The strong genotype–phenotype correlation in MEN2 and its
impact on clinical management, early diagnosis, and decisions
on surgical procedures, including prophylactic thyroidectomy,
is widely known (7,12,14,17,38,49–56). One may hypothesize
that RET mutations in different risk categories may be associ-
ated with different psychological outcomes. Most patients har-
bored exon 10 and 11 RET mutations associated with an earlier
onset of MTC. In contrast, nearly half of the patients reported by
Freyer et al. harbored RET 804 codon mutations, which are
usually associated with a later MTC onset, lower tumor ag-
gressiveness, and a better prognosis (26). The two studies cannot
be compared for this reason and because the study by Freyer
et al. also included patients with sporadic MTC.
Overall, 175 genetic RET variants have been described
so far (57,58). While the clinical impact of many mutations
is well established, other sequence variants may be poly-
morphisms or variants of unknown significance (VUS)
(7,17,59,60). A clear distinction of pathogenic and benign
variants or VUS is warranted to avoid misclassification,
erroneous genetic diagnosis, inappropriate genetic coun-
seling, unnecessary prophylactic thyroidectomies, and un-
desirable physical and psychological harm.
Brauer et al. stressed the difficulties in dealing with ge-
netic counseling and clinical management of patients har-
boring the RETY791F variant, which was initially classified
703
as having a moderate aggressiveness risk (61). The data
(62,63) and other observations (64) support that limited
DNA sequencing analysis has led to the misclassification
of RETY791F as a pathogenic variant and unnecessary thy-
roidectomies (59,62–64).
Moreover, recent studies identified peculiarities and diffi-
culties in genetic counseling and the potential for psycho-
logical harm in patients harboring VUS in the BRCA genes
(65). A growing number of VUS in the RET gene have been
documented (7,57,58). However, there is no study on the
psychological impact in individuals with identified VUS or
misclassified variants in the RET gene.
Considering the present psychological scenario, interven-
tions in patient interest groups and specialized psychologic
support would have the potential to reduce the high indexes
of chronic distress, feelings of guilt for transmission to off-
spring, negative coping as fatalism, and amplification of
positive coping behaviors as fighting spirit. The value of
systematic psychological assessment in MEN2 patients is
supported by the need for individual psychotherapy in 25% of
the studied patients. The patients were followed for at least
six months on a weekly basis for 50-minute sessions, and this
support and counseling had a positive impact.
Finally, opportunities for the future should be highlighted.
They include the support by patient interest groups that allow
individual and social concerns to be addressed. Aside from
obtaining general information about the disease, participation
in interest groups can decrease patients’ feeling of isolation.
Furthermore, such groups provide an opportunity to ex-
change and compare experiences, and they allow complex
questions about the disease to be addressed.
In summary, high levels of chronic distress in MEN2 pa-
tients are documented, despite a relatively good QoL. This
finding is most probably associated with several stress-
inducing factors, some of them of a persisting nature, in these
patients. In addition, cured cases may have higher physical
functioning and higher frequency of working activities.
Conclusion
The psychological assessment protocol developed for and
directed to the care of MEN2 patients has proven effective to
identify individuals requiring psychological assistance and in-
dividual psychotherapeutic treatment. Symptoms of anxiety
and depression should be actively investigated and monitored
in MEN2 patients, given their high prevalence and long dura-
tion. Coping behaviors were frequent in the MEN2 patients
studied here, especially fatalism and fighting spirit. The pres-
ence of MEN2-related stress-inducing factors may result in the
occurrence of adverse psychological effects and may require
targeted psychological support. Specialized multidisciplinary
teams involving genetic counselors, physicians, surgeons, and
psychologists with expertise in MEN2 may improve the quality
of medical care of the patients and their family members.
Acknowledgments
We would like to thank all patients and their family’s
members for agreeing to participate of this study. We also
thank Maria G. Cavalcanti (Endocrine Genetics Unit), for
providing social support. We are very grateful to Prof. Ste-
phen J. Marx and to the editor of Thyroid for valuable sug-
gestions, commentaries, criticism, and corrections.
 704
K.C.R. is the recipient of a FAPESP fellowship (2012-
04698-0). R.A.T. has received a Ciencias Sem Fronteiras
CNPq postdoctoral fellowship and is currently at the Centro
Nacional de Investigaciones Oncologicas (CNIO), Madrid.
S.P.A.T. is presently a CAPES (Coordenadoria de Aperfei-
çoamento de Pessoal de Nı́vel Superior) National Senior
Visiting Professor (PVNS program) at the Federal University
of Sao Paulo, Brazil. This investigation was supported by
FAPESP (2013/01476-9) and CNPq (401990/2010-9) grants
to S.P.A.T. and a FAPESP grant (2013/19810-2) to D.M.L.J.
Downloaded by American Thyroid Association (ATA) from online.liebertpub.com at 07/10/17. For personal use only.
Author Disclosure Statement
The authors have nothing to declare.
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Address correspondence to:
Delmar M. Lourenço, Jr., MD, PhD
Endocrine Genetics Unit (LIM25)
Endocrinology
University of São Paulo School of Medicine
Av. Dr. Arnaldo, 455, 5" andar
Cerqueira Cesar
São Paulo, SP, 01246-903
Brazil
E-mail: delmarmuniz@usp.br
 Original Article
THE COMBINED USE OF CALCITONIN DOUBLING TIME AND 18F-FDG
PET/CT IMPROVES PROGNOSTIC VALUES IN MEDULLARY THYROID
CARCINOMA: THE CLINICAL UTILITY OF 18F-FDG PET/CT
Ji H. Yang, MD, PhD1; Cléber P. Camacho, MD, PhD1; Susan C. Lindsey, MD, PhD1;
Flavia O.F. Valente, MD1; Danielle M. Andreoni, MD1; Lilian Y. Yamaga, MD, PhD2;
Jairo Wagner, MD2; Rosa Paula M. Biscolla, MD, PhD1; Rui M.B. Maciel, MD, PhD1
ABSTRACT
Objective: Calcitonin and carcinoembryonic antigen
(CEA) doubling times are established prognostic markers
in medullary thyroid cancer (MTC). On the other hand,
18F-fluorodeoxyglucose (FDG) positron emission tomogra-
phy/computed tomography (PET/CT) shows an increased
rate of detection with high blood tumor marker levels in
several cancers. This study aimed to analyze the ability of
18F-FDG PET/CT to determine prognosis in the follow-up
of patients with MTC.
Methods: Medical records of 17 patients with MTC
who underwent 18F-FDG PET/CT were analyzed retro-
spectively. All patients were classified into two groups:
stable disease or progressive disease.
Results: Eight patients presented with progressive
disease, and all of them showed 18F-FDG uptake (100%),
compared to only 3 of 9 patients who presented in stable
condition (33%). 18F-FDG PET/CT results were able to
distinguish progressive from stable disease (P = .009).
Calcitonin levels >4,020 pg/mL (P = .0004), CEA levels
>26.8 ng/mL (P = .04), and a calcitonin doubling time
<24.1 months (P = .015) were associated with progres-
Submitted for publication February 7, 2017
Accepted for publication May 14, 2017
From the 1Multiple Endocrine Neoplasia Outpatient Clinic and Laboratory
of Molecular and Translational Endocrinology, Division of Endocrinology,
Department of Medicine, Escola Paulista de Medicina, Universidade Federal
de São Paulo, São Paulo, Brazil, and 2Imaging Department, Hospital Israelita
Albert Einstein, São Paulo, Brazil.
Address correspondence to Dr. Rui M.B. Maciel, Laboratório de
Endocrinologia Molecular e Translacional, Rua Pedro de Toledo, 669, 11°
andar, 04039-032, São Paulo, SP, Brazil.
E-mail: rui.maciel@unifesp.br
Published as a Rapid Electronic Article in Press at http://www.endocrine
practice.org on June 14, 2017. DOI: 10.4158/EP171806.OR
To purchase reprints of this article, please visit: www.aace.com/reprints.
Copyright © 2017 AACE.
sive disease in our cohort. The proportion of variance
explained that predicted progressive disease was 32% for
18F-FDG uptake, 27.1% for a calcitonin doubling time of
24.1 months, and 41.2% for doubling time plus 18F-FDG
PET/CT.
Conclusion: 18F-FDG uptake was able to distinguish
progressive from stable disease. However, this tool should
not replace the validated calcitonin doubling time, but
rather the combination of information could improve the
clinical re-assessment and better identify high-risk patients
who require more careful surveillance. (Endocr Pract.
2017;23:xxx-xxx)
Abbreviations:
CEA = carcinoembryonic antigen; CT = computed
tomography; 18F-FDG = 18F-fluorodeoxyglucose; MTC
= medullary thyroid cancer; PET = positron emission
tomography; PVE = proportion of variance explained;
sCT = serum calcitonin; SUV = standard uptake value;
US = ultrasound
INTRODUCTION
Medullary thyroid cancer (MTC) originates from C
cells and produces calcitonin and carcinoembryonic anti-
gen (CEA), which are markers for the follow-up of patients
with MTC (1). Serum calcitonin (sCT) and CEA reflect
tumor burden, and their decline after primary surgery indi-
cates a better prognosis (2,3). Undetectable levels of serum
calcitonin (sCT) after surgery predict a survival rate of
98% within 10 years (4), but unfortunately, less than 50%
of patients attain this condition (4-6), with most retaining
biochemical or structural disease. In fact, in the presence
of distant metastasis, the 5-year survival rate drops to 35 to
60% (7,8).
Although alternative approaches such as chemoembo-
lization, chemotherapy, external-beam radiation therapy,
and treatment with multikinase inhibitors may improve
symptoms and reduce tumor burden, they are not curative
ENDOCRINE PRACTICE Vol 23 No. 8 August 2017 1
2 18F-FDG PET/CT in MTC, Endocr Pract. 2017;23(No. 8)
treatments, and their use has some limitations due to side
effects (9,10). Thus, an early diagnosis that enables surgi-
cal intervention before local or metastatic dissemination is
the most important prognostic factor in these patients (11).
Several other predictors regarding MTC prognosis have
been reported in the literature. Low pre-operative sCT
levels (4,12,13) and the absence of lymph node involve-
ment (4,14,15) have been assessed as determinants of good
prognosis in univariate analysis. On multivariate analysis,
age (4,5,16), tumor staging (4,5,14), postoperative sCT
(6), and sCT/CEA doubling time (17) were characterized
as independent markers to estimate prognosis.
18F-Fluorodeoxyglucose (18F-FDG) positron emission
tomography/computed tomography (PET/CT) is an imag-
ing technique routinely used in the detection of most cancer
lesions because of their high glycolytic rate. It has been
established that the detection rate of metastases increas-
es with blood tumor marker levels (18-20). On the other
hand, there is a clear association between shorter sCT and
CEA doubling times with tumor growth in MTC (17,21).
Concerning this point, some studies (22-24) have proposed
that a higher rate of PET-positive MTC lesions might be
observed in patients with shorter doubling times. However,
the association between 18F-FDG PET/CT and progressive
disease has not yet been fully established. Therefore, the
aim of this study was to analyze the ability of 18F-FDG
PET/CT alone and in combination with established predic-
tive factors to determine progressive disease in a single
reference center in Brazil.
METHODS
Patients and Clinical Assessments
From a total of 142 patients with MTC followed at
the Multiple Endocrine Neoplasia Outpatient Clinic of
the Universidade Federal de São Paulo, 24 underwent
18F-FDG PET/CT. Patients presenting elevated or rising
sCT and CEA levels or structural disease underwent
18F-FDG PET/CT to uncover structural evidence of metas-
tases or for restaging, respectively. Seven were excluded
due to incomplete data, and therefore, the medical records
of 17 patients (13 structural and 4 biochemical disease)
were analyzed retrospectively.
The patients were followed through serial assess-
ments. Routine evaluation included a clinical examination
and sCT and CEA measurements with doubling time calcu-
lations. All patients underwent cervical ultrasound (US) as
a routine serial assessment, and suspicious cervical lesions
were submitted to US-guided fine-needle aspiration cytol-
ogy (FNAC) (25). Conventional imaging, such as magnetic
resonance imaging and CT, were performed primarily to
assess tumor staging when necessary. sCT was measured
using an immunofluorometric assay with an analytical
sensitivity of 1.0 pg/mL (26). Serum CEA was measured
using an electrochemiluminescence assay (normal values,
<3.0 ng/mL for nonsmokers and <5.0 ng/mL for smokers).
Two patients were excluded from the analysis of CEA and
its doubling time due to secondary morbidity that could
have influenced those data in one and due to imprecise
values arising from the sample dilutions in another. The
median interval between sCT and CEA measurements and
18F-FDG PET/CT images was 41 days (range, 0 to 220
days) and 46 days (range, 0 to 220 days), respectively.
Both doubling times were calculated using the ATA on-line
calculator (http://www.thyroid.org/professionals/calcula-
tors/thyroid-cancer-carcinoma, accessed March 30, 2016)
using four measures of each marker collected between
2.5 years before performing 18F-FDG PET/CT and up to
2 months after the scan. None of the patients underwent
systemic (tyrosine kinase inhibitors, chemotherapy) or
local (radiotherapy or surgical procedure) therapy during
this study. The median time of follow-up was 80.6 months
(range, 46 to 180 months) from the MTC diagnosis and 36
months (range, 26 to 39 months) from the 18F-FDG PET/
CT scan.
The study was approved by the Institutional Review
Board (approval number 565.528).
18F-FDG PET/CT
The scan was performed by the protocol described by
Yamaga et al (27). To analyze 18F-FDG PET/CT results,
we used the clinical follow-up data and combined them
with other imaging modality results and available cyto-
logic/histologic reports. For predictive value analysis,
the following criteria were considered to represent posi-
tive 18F-FDG uptake for local recurrence or metastasis:
(1) positive histopathology results; (2) the presence of a
detectable lesion at the corresponding site on conventional
imaging studies or an increase in lesion size on follow-up
imaging when the histopathology results were not avail-
able; (3) patients with biochemical disease; or (4) any
extra-articular high bone uptake after excluding recent
surgery or trauma (22,28). By contrast, the results were
considered negative when there was no 18F-FDG uptake.
Progressive Versus Stable Disease
According to combined and serial data from conven-
tional imaging, the patient’s clinical status was classified
as a stable or progressive disease. Progressive disease
was defined as a persistent increase in tumor size over the
course of follow-up based on RECIST criteria (version
1.1), and stable disease was defined as lesions showing
stable characteristics. All of the patients with biochemi-
cal disease were classified as stable disease because they
presented constant levels of sCT and CEA.
Predictors of Progressive Disease
To study the determinants of progressive disease, the
following parameters were analyzed: (1) sCT and CEA
doubling times; (2) absolute sCT and CEA levels; and (3)
18F-FDG PET/CT.
 18F-FDG PET/CT in MTC, Endocr Pract. 2017;23(No. 8) 3

Statistical Analysis and 83,760 pg/mL) and 28 ng/mL (minimum/maximum,

For continuous variables, the differences between
the stable and progressive groups were assessed using the
Mann-Whitney U test. The receiver operating character-
istic curve was used for continuous variables to calculate
the best cutoff point that corresponded to disease progres-
sion. The chi-square test or Fisher’s exact test was used to
determine the differences in the frequencies of categorical
variables. Odds ratios with 95% confidence intervals were
established to measure the association between predictors
and outcome. The predictive value of the variables alone
and in combination was calculated by the proportion of
variance explained (PVE). A P value <.05 was considered
statistically significant.
RESULTS
Table 1 shows the patients’ demographic and clinical
data. There were 10 sporadic and 7 familial MTC patients.
All 17 patients (12 women) underwent total thyroidecto-
my, and 16 patients were also submitted to lateral lymph
node resection. The postoperative median levels of sCT
and CEA were 3,040 pg/mL (minimum/maximum, 283
2 and 1,000 ng/mL), respectively. Four patients had
biochemical disease, 13 had structural metastatic diseases,
and 8 presented progressive disease (patients 1 through 8),
two of whom died during the follow-up period (Table 1,
patients 1 and 2).
sCT and CEA
Measurements and Doubling Times
Both the absolute level of sCT and the sCT doubling
time of the 8 patients who developed progressive illness
were significantly different from those of the 9 patients with
stable disease: 14,405 pg/mL (minimum/maximum, 4,200
and 83,760 pg/mL) versus 1,347 pg/mL (minimum/maxi-
mum, 283 and 47,800 pg/mL) for the median sCT level (P
= .0015) and 13.1 months (minimum/maximum, 5.7 and
27.8 months) vs. 27.8 months (minimum/maximum, 12.5
and 106 months) for the median sCT doubling time (P =
.005) (Fig. 1 A and B). Regarding CEA doubling times, the
difference between 23.8 months (minimum/maximum, 8.9
and 90.7 months) for patients with progressive disease and
54 months (minimum/maximum, 17.9 and 754 months) for
patients with stable disease was not significant (P = .45).

Table 1
Demographic and Clinical Data
DT DT Clinical MTC Patient Current
Pt G A1 A2 Type CT CT CEA CEA TNM status status PV status
1 M 55 62 Spor 6,020 8 28 9 T3N1bM1 Struct Prog + Death
2 M 56 59 Spor 17,890 5.7 21 11 T4N1bM1 Struct Prog + Death
3 M 50 56 Spor 67,200 22 668 44 T3N1bM1 Struct Prog + Struct
4 F 44 56 Spor 757 15 5 97 T3N1bM0 Struct Prog + Struct
5 F 31 35 Spor 11,210 11.7 33 23.8 T1N1bM0 Struct Prog + Struct
6 M 56 59 Spor 4,200 28 88 63 T4N1bM1 Struct Prog + Struct
7 F 8 21 MEN2A 83,760 11.7 1000 a T2N0M1 Struct Prog + Struct
8 M 27 29 Spor 17,600 19 121 91 T4N1bM1 Struct Prog + Struct
9 F 35 38 Spor 439 89 6 52 T4N1aM0 Bioch Stable − Bioch
10 F 73 76 MEN2A 642 48 a a T4N0M0 Bioch Stable − Stable
11 F 54 56 MEN2A 283 28 2 18 T2N1bM0 Bioch Stable − Stable
12 M 40 48 Spor 1,347 106 13 754 T4N1bM0 Bioch Stable − Stable
13 F 54 58 MEN2A 1,361 40 39 53 T2N1bM0 Struct Stable − Struct
14 F 64 67 MEN2A 611 16 15 55 T1N1aM0 Struct Stable − Struct
15 M 20 26 MEN2B 1,436 58 12 63 T4N1bM0 Struct Stable + Struct
16 F 23 27 Spor 3,040 12.5 3 16 T3N1bM1 Struct Stable + Struct
17 F 55 63 Spor 3,840 26 21 316 T3N1bM0 Struct Stable + Struct
A1 = age at diagnosis (years); A2 = age at 18F-fluorodeoxyglucose–positron emission tomography/computed tomography
(18F-FDG PET/CT) (years); Bioch = biochemical disease; CEA = carcinoembryonic antigen (ng/mL); CT = calcitonin (pg/
mL); DT CT = doubling time of CT (months); DT CEA = doubling time of CEA (months); F = female; FN = false negative; G =
gender; M = male; MEN2A = multiple endocrine neoplasia 2A; MEN2B = multiple endocrine neoplasia 2B; MTC = medullary
thyroid cancer; No = patient number; Prog = progressive disease; Pt = patient; PV = predictive value of 18F-FDG PET/CT; Spor =
sporadic; Struct = structural disease; TN = true negative; TP = true positive.
aNot reliable data.
4 18F-FDG PET/CT in MTC, Endocr Pract. 2017;23(No. 8)

sCT levels >4,020 pg/mL (P = .0004), CEA levels

>26.8 ng/mL (P = .04), and sCT doubling time shorter than
24.1 months (P = .015) were associated with progressive
disease in our cohort. By contrast, there was no associa-
tion between the progression of disease and CEA doubling
time. When we used the doubling time cutoff of 24 months,
as established in the literature, only sCT doubling time (but
not CEA doubling time) distinguished progressive from
stable disease (P = .015) (Table 2).

Fig. 1. Box-plot diagrams for serum calcitonin level (A) and
calcitonin doubling time (B) in the progressive- and stable-
disease groups.
18F-FDG PET/CT
From the 17 18F-FDG PET/CT scans performed,
we found 11 true positive and 6 negative results (Table
1). The mean standard uptake value (SUV) level of the
positive lesions was 4.6 (range, 2.5 to 10.3). Four nega-
tive cases occurred in patients with biochemical disease
(patients 9 through 12). Despite high sCT levels (283
to 1,347 pg/mL), no structural focus of metastasis was
displayed. After a median of 29.4 months (range, 29 to
34 months) of follow-up, these patients showed stable
sCT and CEA levels without structural evidence of MTC
metastases. Remarkably, 18F-FDG PET/CT uncovered a
synchronous primary neoplasm in 2 patients: 18F-FDG
uptake in enlarged mesenteric lymph nodes due to a
non-Hodgkin lymphoma (patient 9) and in the sigmoid
due to a colorectal carcinoma (patient 10). Both patients
are currently receiving treatment for their second tumor.
However, 18F-FDG PET/CT was not able to identify 1.0 to
1.1 cm metastatic cervical lesions confirmed by FNAC in
2 patients who presented sCT levels of 611 and 1,361 pg/
mL (patients 13 and 14).

Table 2
Association Between Predictors and Progressive Disease
B. Stable C. Progressive D. Chi-square E. Odds ratio F. PVE
A. Cutoff disease (n) disease (n) (P) (95% CI) (%)
Calcitonin DT >24 7 1 24.5
.015 27.1
(months) <24 2 7 (1.8-336)

CEA DT >24 6 4 2.3

.6 1.8
(months) <24 2 3 (0.3-20)

Serum calcitonin >4,020 0 7 95

.0004 64.9
(pg/mL) <4,020 9 1 (3.4-2683)

Serum CEA >26.8 1 6 21

.04 24.8
(ng/mL) <26.8 7 2 (1.5-293)

Calcitonin DT >24.1 7 1 24.5

.015 27.1
(months) <24.1 2 7 (1.8-336)

CEA DT >48 6 3 4
.3 5.7
(months) <48 2 4 (0.4-36)

18F-FDG
Yes 3 8 32
uptake .009 32
No 6 0 (1.4-725)
Abbreviations: CEA = carcinoembryonic antigen; CI = confidence interval; DT = doubling time; 18F-FDG =
18F-fluorodeoxyglucose; PVE = proportion of variance explained.

The sCT cutoff of 1,399 pg/mL was associated with
positive 18F-FDG uptake (P = .0005). The median sCT level
in the 11 positive 18F-FDG PET/CT patients was 6,020
pg/mL (minimum/maximum, 757 and 83,760 pg/mL),
compared to 627 pg/mL (minimum/maximum, 283 and
1,347 pg/mL) found in the remaining 6 negative patients
(P = .0006). Additionally, the median sCT doubling time
of 14.8 months (minimum/maximum, 5.7 and 58 months)
in positive 18F-FDG PET/CT patients was shorter than
the 44 months (minimum/maximum, 15.6 and 106.4
months) observed in patients without 18F-FDG uptake
(P = .01) (Table 3).
None of the analyses regarding CEA distinguished
positive from negative 18F-FDG PET/CT patients, includ-
ing the CEA cutoff for positive scans (17.7 ng/mL), serum
CEA level (median, 12.6 ng/mL [minimum/maximum, 2
and 39] vs. 33 ng/mL [minimum/maximum, 5.4 and 1,000])
or CEA doubling time (median, 53.5 months [minimum/
maximum, 8.9 and 316] vs. 53.2 [minimum/maximum, 18
and 754]) (Table 3).
All 8 patients who presented with progressive disease
showed 18F-FDG uptake (100%), compared to only 3 of
9 patients who presented with a stable condition (33%).
Thus, 18F-FDG PET/CT results were able to distinguish
progressive from stable disease (P = .009) (Table 2,
column D).
Eventually, after analyzing all predictors, the PVE that
corresponded to progressive disease was 64.9% for a sCT
cutoff of 4,020 pg/mL, 32% for 18F-FDG uptake, 27.1%
for a sCT doubling time of 24.1 months, and 24.8% for
a CEA cutoff of 26.8 ng/mL (Table 2, column F). When
we considered the combination of those predictors, the
PVE was 64.9% for sCT plus 18F-FDG PET/CT, 44.3% for
sCT plus doubling time, and 41.2% for doubling time plus
18F-FDG PET/CT.
DISCUSSION
The transition from stable to progressive disease is
frequently unpredictable in MTC (29), and patients may
live for years with a stable condition and then suddenly
exhibit progressive disease (8). Currently, short sCT and
CEA doubling times are considered the best available
indicators to assess tumor behavior, MTC recurrence, and
cancer mortality (30,31). Barbet et al (17) demonstrated a
5-year 100% survival rate when sCT and CEA doubling
times were longer than 24 months, whereas sCT and CEA
doubling times shorter than 24 months represented a worse
clinical outcome. In our cohort, 24.1 months represented the
cutoff for sCT doubling time that corresponded to progres-
sive disease, which was close to the value of 24 months
established in the literature to predict worse prognosis
(30). However, we did not find corresponding results for
the CEA doubling time. This difference might be partially
explained by the heterogeneity in sCT and CEA production
18F-FDG PET/CT in MTC, Endocr Pract. 2017;23(No. 8) 5
Table 3
Association Between Predictors and 18F-FDG Uptake
18F-FDG 18F-FDG P
uptake + uptake − value
Serum calcitonin, 6,020 627
.0006
pg/mL (min-max) (757-83,760) (283-1,347)
Serum CEA, 12.6 33
>.05
ng/mL (min-max) (2-39) (5.4-1,000)
Calcitonin DT, 14.8 44
months (min-max) (5.7-58) (15.6-106.4)
.01
CEA DT, 53.5 53.2
>.05
months (min-max) (8.9-316) (18-754)
Abbreviations: CEA = carcinoembryonic antigen; DT =
doubling time; 18F-FDG = 18F-fluorodeoxyglucose.
by tumor cells, as the synthesis of these markers is neither
parallel nor correlated, generating a divergence of serum
levels (32).
Regarding the cutoff levels of CT and CEA, CT
levels >4,020 pg/mL and 26.8 ng/mL for CEA were asso-
ciated with clinical progression. It is worth emphasizing
that caution is required in interpreting this information.
Moreover, there are some questions regarding the use
of a single static marker to predict disease progression.
First, absolute values reflect tumor burden, rather than the
precise growth rate, and there is considerable variability
and fluctuation in daily serum concentrations (17). Second,
extremely high values of sCT and CEA, as shown in our
cohort, could increase the cutoff for predicting outcome.
Third, less-differentiated tumors may lose the ability to
synthesize sCT (33). Therefore, care is needed regard-
ing the use of these cutoffs in daily clinical practice to
avoid misjudgments.
At the same time, a higher absolute sCT level and
a shorter sCT doubling time were observed in positive
18F-FDG PET/CT patients. Several publications support a
similar association between 18F-FDG uptake and unfavor-
able prognosis in MTC, while a negative 18F-FDG PET/
CT result has been related to longer sCT doubling time
and less-aggressive tumor behavior (23,29,34,35). Another
point concerns the cutoff for sCT levels for true-positive
18F-FDG uptake. The literature depicts better sensitivity
when sCT levels are >815 to 1,000 pg/mL (22,28,35,36),
whereas this value is of limited use when it is <500 pg/
mL. Our cutoff was 1,399 pg/mL, which is higher than
the values observed in the literature. The small number of
patients and extremely high values of sCT and CEA, as
discussed above, might explain this finding.
Considering the ability of 18F-FDG uptake to distin-
guish between progressive and stable disease, this study
found an association between 18F-FDG uptake and progres-
sion in 100% of patients with progressive disease. Several
studies have suggested that 18F-FDG PET/CT might be
more sensitive in a rapidly growing tumor, playing an
6 18F-FDG PET/CT in MTC, Endocr Pract. 2017;23(No. 8)
important role in increasing sCT and shortening doubling
time in patients. Moreover, 18F-FDG PET/CT might be
especially representative of aggressive tumor behavior,
which presents more active glucose metabolism and higher
18F-FDG uptake. Furthermore, although there is no stab-
lished SUV cutoff as indicative of MTC lesions, Singh
et al (37) studied 47 patients with thyroid cancer (3 with
MTC) and found that a lesional SUV increase in subse-
quent 18F-FDG PET/CT scans was positively correlated
with increase in lesion area and tumor growth, working as
a prognostic marker.
The American Thyroid Association MTC guidelines
recommend that patients with significant tumor burden and
symptomatic or progressive metastatic disease according
to RECIST could timely be considered for systemic thera-
py with agents targeting both re-arranged in transcription
and vascular endothelial growth factor receptor tyrosine
kinases such as vandetanib and cabozantinib (38). In this
context, we suggest that the combination of sCT doubling
time and 18F-FDG PET/CT might be used as a marker of
disease progression in MTC. As we demonstrated here, the
combined data presented a PVE of 41%, which was better
than the sCT doubling time (PVE of 27.1%) or 18F-FDG
PET/CT (PVE of 32%) alone.
The present study has some limitations. First, it was
a retrospective study with a relatively small number of
patients included. A slow growth rate of neuroendocrine
tumors might attenuate 18F-FDG uptake, requiring a longer
follow-up period. A slow growth rate might be one of the
causes for negative 18F-FDG uptake in the 4 patients with
biochemical disease and the small size of the MTC lesions,
which are both known as limitations of 18F-FDG PET/
CT scans (39). Additionally, we obtained no pathologic
confirmation of any lesions with 18F-FDG uptake because
some patients did not undergo surgery. In some patients,
the clinical management verified the conclusions made by
Koopmans et al (40) that patients with MTC have a good life
expectancy and undergo routine imaging to assess possible
tumor growth; unless for a specific clinical purpose, it is not
ethical to use the invasive procedure to obtain a cytologic or
histologic diagnosis of all suspicious lesions.
CONCLUSION
In conclusion, our results suggest that 18F-FDG PET/
CT can identify aggressive and progressive MTC disease
and that the combination of 18F-FDG PET/CT uptake and
sCT doubling time could improve the clinical re-assess-
ment and provide better identification of high-risk patients
who require more careful surveillance.
ACKNOWLEDGMENT
We thank the team of the Laboratory of Molecular and
Translational Endocrinology, especially Ilda Kunii, Teresa
Kasamatsu, João Roberto Martins, Magnus Dias-da-Silva,
and Jairo Hidal. The research of the authors is supported
by São Paulo State Research Foundation (FAPESP) grants
2006/60402-1 and 2010/51547-1 to R.M.B.M.; grant
12518 from the Fleury Group to R.M.B.M.; and grant
25000.168513/2008-11 from the Brazilian Ministry of
Health to R.M.B.M. S.C.L. received a fellowship grant
from FAPESP (2009/50575-4); F.O.F.V. received a fellow-
ship grant from CAPES-Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior.
DISCLOSURE
The authors have no multiplicity of interest to disclose.
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