1 António Manuel Ferreira de Gouveia Tumores Mesenquimatosos do Tubo Digestivo Estudo de parâmetros clínico-patológicos e de prognóstico, com especial ênfase nos Tumores Estromais Gastrointestinais Dissertação de candidatura ao grau de Doutor apresentada à Faculdade de Medicina da Universidade do Porto Fevereiro de 2011
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1
António Manuel Ferreira de Gouveia
Tumores Mesenquimatosos do Tubo Digestivo
Estudo de parâmetros clínico-patológicos e de prognóstico, com especial ênfase nos Tumores Estromais Gastrointestinais
Dissertação de candidatura ao grau de Doutor
apresentada à Faculdade de Medicina da Universidade do Porto
Fevereiro de 2011
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Orientador: Prof. Doutor José Manuel Pedrosa Baptista Lopes Co-orientador: Prof. Doutor Silvestre Porfírio Ramos Carneiro
Artº 48, § 3º - A Faculdade não responde pelas doutrinas expandidas na dissertação (Regulamento
da Faculdade de Medicina do Porto, 29 de Janeiro de 1931, Decreto nº 19337)
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JÚRI
Presidente: Reitor da Universidade do Porto
Vogais: Doutor Henrique Manuel Bicha Castelo, Professor Catedrático da Faculdade
de Medicina da Universidade de Lisboa;
Doutor José Manuel Pedrosa Baptista Lopes, Professor Associado da
Faculdade de Medicina da Universidade do Porto, e orientador da tese;
Doutor António Taveira Gomes, Professor Associado Convidado da
Faculdade de Medicina da Universidade do Porto;
Doutor João António Pinto de Sousa, Professor Associado Convidado
da Faculdade de Medicina da Universidade do Porto;
Doutor Rui Manuel Vieira Reis, Professor Auxiliar da Escola de Ciências da
Saúde da Universidade do Minho;
Doutora Ana Paula Soares Dias Ferreira, Professora Auxiliar da Faculdade
de Medicina da Universidade do Porto.
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CORPO CATEDRÁTICO DA FACULDADE DE MEDICINA DO PORTO
Professores Catedráticos Jubilados ou Aposentados
Doutor Abel José Sampaio da Costa Tavares
Doutor Abel Vitorino Trigo Cabral
Doutor Alexandre Alberto Guerra de Sousa Pinto
Doutor Amândio Gomes Sampaio Tavares
Doutor António Augusto Lopes Vaz
Doutor António Carvalho Almeida Coimbra
Doutor António Fernandes da Fonseca
Doutor António Fernandes de Oliveira Barbosa Ribeiro Braga
Doutor António Germano Pina da Silva Leal
Doutor António José Pacheco Palha
Doutor António Luís Tomé da Rocha Ribeiro
Doutor António Manuel Sampaio de Araújo Teixeira
Doutor Belmiro dos Santos Patrício
Doutor Cândido Alves Hipólito Reis
Doutor Carlos Rodrigo de Magalhães Ramalhão
Doutor Cassiano Pena de Abreu e Lima
Doutor Daniel dos Santos Pinto Serrão
Doutor Eduardo Jorge da Cunha Rodrigues Pereira
Doutor Fernando de Carvalho Cerqueira Magro Gomes Ferreira
Doutor Fernando Tavarela Veloso
Doutor Francisco Sousa Lé
Doutor Henrique José Ferreira Gonçalves Lecour de Menezes
Doutor José Augusto Fleming Torrinha
Doutor José Carvalho de Oliveira
Doutor José Fernando de Barros Castro Correia
Doutor José Luís Medina Vieira
Doutor José Manuel da Costa Mesquita Guimarães
Doutor Levi Eugénio Ribeiro Guerra
Doutor Luís Alberto Martins Gomes de Almeida
Doutor Manuel Augusto Cardoso de Oliveira
Doutor Manuel Machado Rodrigues Gomes
Doutor Manuel Maria Paula Barbosa
Doutora Maria da Conceição Fernandes Marques e Magalhães
Doutora Maria Isabel Amorim de Azevedo
Doutor Mário José Cerqueira Gomes Braga
Doutor Serafim Correia Pinto Guimarães
Doutor Valdemar Miguel Botelho Santos Cardoso
Doutor Walter Friedrich Alfred Osswald
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Professores Catedráticos Efectivos
Doutor Alberto Coimbra Sobrinho Simões
Doutor Manuel Mergulhão Castro Tavares
Doutora Maria Amélia Duarte Ferreira
Doutor José Agostinho Marques Lopes
Doutor Patrício Manuel Vieira Araújo Soares Silva
Doutor Daniel Filipe Lima Moura
Doutor Alberto Manuel Barros da Silva
Doutor José Manuel Lopes Teixeira Amarante
Doutor José Henrique Dias Pinto Barros
Doutora Maria Fátima Machado Henriques Carneiro
Doutora Isabel Maria Amorim Pereira Ramos
Doutora Deolinda Maria Valente Alves Lima Teixeira
Doutora Maria Dulce Cordeiro Madeira
Doutor Altamiro Manuel Rodrigues Costa Pereira
Doutor Rui Manuel Almeida Mota Cardoso
Doutor António Carlos Freitas Ribeiro Saraiva
Doutor Álvaro Jerónimo Leal Machado de Aguiar
Doutor José Carlos Neves da Cunha Areias
Doutor Manuel Jesus Falcão Pestana Vasconcelos
Doutor João Francisco Montenegro Andrade Lima Bernardes
Doutora Maria Leonor Martins Soares David
Doutor Rui Manuel Lopes Nunes
Doutor José Eduardo Torres Eckenroth Guimarães
Doutor Francisco Fernando Rocha Gonçalves
Doutor José Manuel Pereira Dias de Castro Lopes
Doutor Manuel António Caldeira Pais Clemente
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À Graça, ao João, à Luísa e à Carolina
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AGRADECIMENTOS
Ao Prof. Doutor José Manuel Lopes, meu orientador e principal impulsionador deste projecto, pela
amizade e pelo incentivo constantemente demonstrados. Pela transmissão do conhecimento
científico, compreensão e, acima de tudo, pelo elevado grau de exigência, o meu sincero
reconhecimento.
Ao Prof. Doutor Amadeu Pimenta, meu primeiro orientador e anterior Director de Serviço na
Cirurgia Geral do Hospital de S. João, pela capacidade de transmitir, com generosidade e tolerância,
as aptidões técnicas e humanas ímpares que sempre soube ostentar. Pelo crescimento pessoal e
profissional, o meu profundo e eterno agradecimento.
À Dra. Olga Martinho, à Dra. Ana Gomes e ao Prof. Doutor Rui M. Reis, pela colaboração
empenhada e pela valiosa participação na elaboração dos trabalhos.
Ao Prof. Doutor Valdemar Cardoso e ao Prof. Doutor M. Cardoso de Oliveira, meus anteriores
Directores de Serviço na Cirurgia 4, Cirurgia B e Cirurgia Geral do Hospital de S. João, pelo apoio
convicto do projecto e por sempre me terem incentivado na procura do conhecimento.
Ao Prof. Doutor Silvestre Carneiro, por ter aceitado incondicionalmente a co-orientação na fase
final dos trabalhos, agradeço a disponibilidade e o encorajamento.
Ao Dr. Costa Maia, meu actual Director de Serviço na Cirurgia Geral do Hospital de S. João, pelo
estímulo e por me ter facultado as condições necessárias à conclusão deste projecto.
À Prof. Doutora Fátima Carneiro, Directora do Serviço de Anatomia Patológica do Hospital de S.
João, e ao Prof. Doutor Sobrinho Simões, Director do Serviço de Anatomia Patológica da
Faculdade de Medicina do Porto e do IPATIMUP, pelo acolhimento e pela forma como sempre
facilitaram o acesso aos meios necessários ao desenrolar dos trabalhos.
À Dra. Paula Silva, pela amizade e pela inestimável e sempre abnegada colaboração.
À Dra. Ana F. Capelinha, ao Dr. Dionísio de la Cruz e a todos os co-autores, pela valiosa
contribuição e partilha de ideias.
À Prof. Doutora Cristina Santos e ao Prof. Doutor Armando Teixeira-Pinto, pela ajuda preciosa na
análise dos dados.
Ao Dr. Sousa Rodrigues, ao Dr. Aníbal Liberal, ao Dr. Aníbal Justiniano, ao Dr. Bernardo
Bonifácio e ao Prof. Doutor Moutinho Ribeiro, pelo exemplo de competência e pela minha
iniciação em Cirurgia.
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Aos elementos da Unidade Esófago-Gastro-Duodenal do Serviço de Cirurgia Geral, presentes e
passados, pelo apoio, colaboração e amizade dispensados.
A todos os meus colegas, especialistas e internos, do Serviço de Cirurgia Geral no Hospital de S.
João, pelo interesse e cooperação nas diferentes actividades desenvolvidas.
Às secretárias dos Serviços de Cirurgia B, de Cirurgia Geral e de Anatomia Patológica do Hospital
de S. João, pela dedicação e pelo auxílio sempre solícito.
À Novartis Oncology (Portugal), pelo financiamento de parte dos custos envolvidos na execução do
projecto, e ao Sr. José Seara, por toda a atenção dispensada.
O trabalho só foi possível devido à necessária disponibilização de meios por parte de várias
Instituições, das quais destaco: a Faculdade de Medicina da Universidade do Porto, o Hospital de S.
João, E.P.E., o IPATIMUP e a Escola de Ciências da Saúde da Universidade do Minho. O meu
voto de gratidão pela hospitalidade e pelas condições de trabalho que sempre souberam
proporcionar.
Aos meus Pais, pelo modelo de trabalho e integridade que me souberam transmitir, e pelo apoio
emocional e dedicação de toda uma vida.
Aos meus irmãos e aos meus familiares pelo incentivo e pelo apoio incondicional recebido ao longo
dos anos.
Aos meus filhos, pela alegria que despertam e pela paciência revelada nos muitos momentos de
ausência do pai.
À minha mulher, que em todos os momentos sinto ao meu lado, pelo estímulo, a compreensão e o
carinho.
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Ao abrigo do Art. 8º do Decreto-Lei nº 388/70 fazem parte integrante desta dissertação os
seguintes trabalhos publicados:
Gouveia AM, Pimenta AP, Capelinha AF, de la Cruz D, Silva P, Lopes JM. Surgical margin status
and prognosis of gastrointestinal stromal tumor. World J. Surg. 2008 Nov, Vol 32 (11): 2375-82.
a Definido como metastização ou morte relacionada com o GIST b Número de casos limitado c Dados insuficientes Tabela adaptada de Miettinen et al.184 Dados baseados no follow-up prolongado de 1,055 GISTs gástricos, 629 do intestino delgado, 144 duodenais e 111 rectais38, 96.
Um nomograma prognóstico para estimativa da sobrevida livre de doença após ressecção cirúrgica
macroscópica completa de GISTs primários localizados foi proposto por Gold et al.188. É um
método que utiliza a dimensão, o índice mitótico e a localização dos tumores, para prever a
probabilidade de sobrevida livre de recidiva (SLR). São atribuídos pontos em cada caso, numa
escala baseada na localização (gástrico vs. intestino delgado vs. cólon/recto vs. extragastrointestinal),
na dimensão (variável contínua não-linear) e no índice mitótico (< 5 vs. ≥5/50 CGA) dos tumores.
O total de pontos indica a SLR aos 2 e 5 anos. O nomograma permitiu estimar uma probabilidade
de concordância de 0,78, equivalente à obtida com o sistema AFIP, e superior à do sistema NIH no
mesmo estudo138. Desconhece-se a precisão do nomograma para estimar a SLR a longo prazo em
GISTs indolentes e progressão tardia188. As estimativas da SLR obtidas com o nomograma podem
ser mais úteis para a selecção dos doentes candidatos a tratamento adjuvante com inibidores
tirosina-cinásicos138.
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Foram propostos outros sistemas de estratificação de risco dos GISTs. Vários autores apresentaram
versões do sistema de estratificação de risco NIH, incluindo outros factores de prognóstico. O
risco de recidiva pode ser estimado com base não só no índice mitótico, dimensão e localização do
tumor, mas também na qualidade das margens cirúrgicas e na ocorrência de ruptura do tumor24.
Apesar da controvérsia existente, a ressecção cirúrgica com margens microscópicas positivas (R1)
pode expor os doentes a risco de recidiva tumoral loco-regional. Rutkowski et al.189 descreveram
que a ressecção não radical (R1) e a ruptura do tumor associam-se a resultados desfavoráveis31.
Joensuu et al. utilizaram o sistema NIH como base e incluiram a presença de ruptura tumoral como
factor de risco para estimar comportamento agressivo, independente do tamanho e do índice
mitótico dos tumores186. Outra modificação da proposta de Joensuu foi a inclusão dos tumores
não-gástricos, do grupo de risco intermédio (classificação NIH), no grupo de risco elevado,
corroborando a importância da classificação de Miettinen/AFIP.
A ocorrência de ruptura tumoral intra-abdominal, antes ou durante a ressecção cirúrgica, tem sido
associada a evolução clínica desfavorável, com metastização peritoneal190, 191. A ruptura do tumor
deve ser sempre valorizada porque traduz risco de evolução desfavorável, independente de qualquer
outro factor prognóstico24.
Para além da dimensão do tumor, do índice mitótico e da localização do tumor, do estado das
margens cirúrgicas e da ruptura tumoral, têm sido investigados outros parâmetros preditivos de
recidiva181. A proliferação tumoral estimada com Ki-67 foi descrita como factor prognóstico
independente192-194. A invasão difusa da mucosa é uma ocorrência rara, associada a evolução mais
agressiva da doença181, 195, 196. A presença de necrose tem sido descrita com valor prognóstico em
algumas séries38, 197, 198. A aneuploidia é um factor de agressividade193, 199, 200, e alguns estudos têm
sugerido que a expressão da telomerase é um factor de mau prognóstico190, 201. A expressão ou o
padrão de expressão KIT e a expressão de CD34 não parecem ser factores independentes de
prognóstico dos doentes com GIST202.
Estadiamento clínico dos GISTs
Woodall et al. propuseram um método de estadiamento dos GISTs baseado num sistema tumor-
grade-metastasis (TGM)203. Os autores, usando resultados do SEER (2537 casos), determinaram a
dimensão 7cm como a mais útil para a previsão do comportamento clínico dos GISTs do que os
valores previamente usados (2 cm, 5cm e 10cm). No entanto, uma percentagem destes casos foi
diagnosticada antes de 2000, sem avaliação da expressão KIT, motivo pelo qual a série estudada
poderá não corresponder totalmente a casos de GIST. Além disso, o método utilizado para a
classificação do grau de diferenciação dos GISTs, em quatro categorias e de forma semelhante à
dos sarcomas dos tecidos moles, não parece ser a mais adequada.
A sétima edição da classificação TNM da International Union Against Cancer (UICC) publicada em
2010 incluiu pela primeira vez a classificação e o estadiamento dos GISTs204. O objectivo principal
da classificação TNM é facilitar uma abordagem homogénea e padronizada dos tumores e o
estabelecimento de follow-up uniforme dos doentes com base no estádio do tumor185.
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A classificação TNM utiliza critérios da avaliação de risco para caracterizar os tumores em quatro
categorias T, combinando o índice mitótico e a localização do tumor para definir os estádios
clínicos. Em geral, dada a raridade das metástases ganglionares linfáticas nos GISTs, é recomendada
a classificação de todos os casos em que os gânglios linfáticos regionais não foram estudados, como
pN0. A presença de metástases ganglionares linfáticas, tal como a metastização à distância,
corresponde a um estadio IV.
A classificação TNM reproduz, genericamente, os 8 subgrupos de prognóstico definidos pela
classificação de Miettinen/AFIP4. Deste modo, pode-se argumentar que a utilização dos mesmos
critérios de risco para determinar os estádios TNM representa uma agregação diferente dos grupos
de risco estabelecidos por vários grupos de trabalho nos últimos anos185. Um dos aspectos mais
sensíveis é a subdivisão da categoria “alto risco” em dois ou três estádios (II, III ou III-B),
sabendo-se que o valor desta subclassificação dos tumores de alto risco continua a aguardar
validação.
Na proposta de classificação de Miettinen/AFIP os tumores pequenos (< 2 cm) e mitoticamente
inactivos não apresentam risco de progressão e poderão ser considerados como de comportamento
clínico benigno, independente da localização anatómica4. Sendo assim, a inclusão deste grupo de
tumores numa classificação TNM de tumores “malignos” pode ser controversa.
Outra controvérsia relaciona-se com o tipo de crescimento dos GISTs. A classificação TNM não
estabelece a relevância da infiltração da subserosa, do epíploon/mesentério, da invasão do
peritoneu visceral pelas células tumorais, da ruptura tumoral, nem da multiplicidade de tumores
primários no epíploon/mesentério.
É, por isso, evidente a necessidade de um sistema consensual e validado de
classificação/estadiamento que inclua a heterogeneidade do comportamento biológico dos GISTs.
9 - O papel da análise molecular no prognóstico e no tratamento não-cirúrgico dos GISTs
Apesar das semelhanças é importante considerar as diferenças entre GISTs com mutação do KIT e
mutação do PDGFRA e os casos com sequências normais de KIT e de PDGFRA (wild-type). A
resposta clínica objectiva ao imatinib depende da presença e do tipo de mutação do RTK
implicado. Foi proposta uma classificação molecular dos GISTs10, 90, que destaca as diferenças
moleculares destes tumores e fornece uma referência rápida para outras síndromes com que podem
associar-se (Tabela 2).
Com base em quatro ensaios clínicos (fase I-III) que investigaram mais de 700 GISTs com
genótipos diferentes, as taxas de resposta objectiva ao imatinib nos GISTs com mutações no exão
11 e no exão 9 do gene KIT, e nos GISTs wild-type foram, respectivamente, 72-86%, 38-48% e 28%9,
21, 87, 152, 205, 206. Os GISTs com mutação no gene PDGFRA respondem ao imatinib, com a excepção
da mutação D842V no exão 189, 83, 87, 113. As taxas de resposta ao imatinib mais favoráveis [mediana
do tempo livre de progressão da doença mais elevada (~ 24 meses) e mediana da sobrevida mais
longa (~ 63 meses)]9, 87, 206 são descritas nos GISTs com mutações no exão 11 do KIT.
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Tabela 2: Classificação molecular dos GISTs
Frequência Comentários
KIT
Exão 11 67% A maioria é sensível ao imatinib
Exão 9 10% Requer dose mais elevada de imatinib
Exão 13 & 17 3%
PDGFRA
Exão 12 & 14 2% Sensível ao imatinib
Exão 18 5% D842V é resistente ao imatinib
Wild type 15% Mutações BRAF ou KRAS raras
GIST Familiar Raros Mutações KIT ou PDGFRA germinativas
Carney-Stratakis Raros Mutações SDHB, SDHC, SDHD
GIST Pediátrico Raros Mutações KIT e PDGFRA são raras
Tríade de Carney Raros Sem mutações KIT ou PDGFRA
GIST associado a NF-1 Raros Mutações KIT ou PDGFRA pouco comuns
Adaptado de Corless, C.10
Os doentes com mutações no exão 11 do gene KIT são habitualmente tratados com imatinib, numa
dose diária de 400 mg, e a escalada da dose para 800 mg/dia tem sido recomendada nos casos de
progressão da doença durante o tratamento. Os doentes que têm mutação no exão 9 do KIT
apresentam sobrevida livre de progressão da doença mais longa quando tratados com a dose inicial
de 800 mg/dia87, 89, 206. De acordo com o consenso actual, o estado mutacional KIT deve ser
avaliado por rotina em GISTs inoperáveis, com administração de 800 mg/dia de imatinib nos casos
em se identifica mutação no exão 9 do KIT24, 138.
A realização da análise molecular de rotina em todos os GISTs continua a ser um tema muito
controverso. Os resultados dos principais centros de referência Europeus e dos Estados Unidos
fornecem evidência a favor da realização destes testes nos GISTs irressecáveis e/ou metastáticos,
beneficiando os doentes que têm mutação no exão 9 do KIT. A análise mutacional pode também
ser considerada na decisão de realizar tratamento adjuvante com imatinib em GISTs primários de
risco “intermédio e alto"24, 138. Deste modo, podem ser excluídos GISTs com mutações resistentes
ao imatinib (ex: mutações D842V do PDGFRA). Estes testes, na opção neoadjuvante, permitem
ajustar a decisão terapêutica de forma mais adequada.
Embora o tratamento de primeira linha dos doentes com GIST metastático/irressecável seja, de
acordo com o estado actual da arte, a utilização do imatinib, é possível que no futuro este regime
terapêutico possa ser alterado para outros inibidores tirosina-cinásicos ou terapêuticas alternativas,
com base no estudo molecular dos tumores89, 207.
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10 - Tratamento
10. a) Doença primária localizada
É consensual o tratamento de GISTs primários de dimensão ≥ 2 cm e sem evidência de
disseminação peritoneal ou metastização à distância com ressecção cirúrgica macroscópica
completa24, 138. No entanto, quando são detectados “nódulos subepiteliais” esófago-gástricos ou
duodenais com diâmetro <2 cm, a abordagem padrão dos doentes consiste na avaliação com
ecografia endoscópica e na vigilância activa, porque muitos destes nódulos, quando correspondem a
GISTs, são tumores de baixo risco biológico1, 184, ou com significado clínico desconhecido. A
exérese cirúrgica é reservada para os doentes cujo tumor aumenta de dimensão ou é sintomático.
Os resultados de uma análise retrospectiva recente208 indicam que apenas alguns (3/23; 13,0%) dos
tumores pequenos sem características eco-endoscópicas de risco (maior dimensão, limites
extraluminais irregulares, padrão ecográfico heterogéneo, presença de áreas císticas e focos
hiperecogénicos) progridem durante o seguimento de longo prazo com eco-endoscopia. Como
alternativa, a decisão pode ser partilhada com o doente, no sentido de se poder optar por uma
avaliação histológica inicial (biópsia com agulha) ou pela excisão, quando a morbilidade não é
substancial. Nos “nódulos” intra-abdominais não passíveis de avaliação endoscópica, a
exérese/ressecção laparoscópica/laparotómica é a abordagem padrão. Nos “nódulos” rectais (ou
do espaço recto-vaginal), a abordagem deve ser a realização de biópsia/exérese, após avaliação por
ecografia endoscópica, independente da dimensão do tumor, porque os GISTs nesta localização
têm risco biológico mais elevado e as implicações de uma intervenção cirúrgica nesta região são
mais críticas, sobretudo nos tumores de grandes dimensões24.
As orientações da ESMO e da NCCN indicam que os tumores com dimensões > 2 cm devem ser
ressecados24, 138, porque, sendo GISTs, implicam maior risco de comportamento agressivo.
Para doentes com GIST primário localizado, a ressecção cirúrgica continua a ser a única
possibilidade de cura209. Normalmente causa pouca morbilidade em tumores <10 cm limitados ao
estômago ou intestino delgado. Ao contrário dos carcinomas GIs , os GIST não se originam nas
camadas epiteliais e apresentam biologia e comportamento diferente, com implicações distintas
relativamente às margens cirúrgicas e à linfadenectomia dos gânglios linfáticos loco-regionais.
Dependendo do órgão onde se origina, da localização exacta, e das dimensões do tumor, podem ser
necessárias abordagens cirúrgicas diferentes. O objectivo do tratamento é a ressecção completa do
GIST, com margens microscópicas negativas (R0) e preservando a pseudo-cápsula intacta (i.e.
evitando a ruptura tumoral)24, 138.
Como o GIST não tem geralmente padrão infiltrativo intraparietal, raramente é necessário efectuar
ressecções alargadas27. As recomendações actuais para as margens cirúrgicas têm por base a
experiência, o consenso, e a aplicação de conceitos patobiológicos sobre GIST24, 138. Não há dados
prospectivos conclusivos sobre a extensão das margens de ressecção e o risco de recidiva local ou à
distância do GIST.
A ressecção “em cunha” é a opção mais utilizada nos GISTs do estômago e a ressecção segmentar
nos GISTs do intestino delgado. Nos GISTs de grandes dimensões, na pequena curvatura e/ou
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com envolvimento pilórico, pode não ser possível a ressecção “em cunha”, sendo mais adequada a
gastrectomia distal. A gastrectomia total não é habitualmente necessária, devendo ser considerada
em função da localização (junção esófago-gástrica) e/ou a extensão do tumor.
Os GISTs do recto são incomuns e o diagnóstico definitivo é, frequentemente, obtido aquando do
estudo anátomo-patológico da peça operatória. O GIST rectal de pequenas dimensões, localizado
no terço inferior, pode ser removido com ressecção parietal completa, com abordagem trans-anal
ou trans-esfinctérica210. Esse tipo de abordagem deve ser efectuada com os cuidados necessários,
porque foram descritas taxas menores de ressecção R0 (32% vs. 82%) e taxas maiores de recidiva
local (77% vs. 31%), quando comparadas com a ressecção anterior baixa do recto, que é o
procedimento recomendado para GISTs dos terços superior e médio211, 212.
A técnica cirúrgica utilizada para ressecar os GISTs tem implicações determinantes na ocorrência de
recidiva tumoral. A ruptura do tumor deve ser estritamente evitada, especialmente quando tem
Fig. 4: Aspecto macroscópico de ressecção de GIST gástrico com invasão do cólon transverso.
grandes áreas císticas ou necróticas. A enucleação simples dos GISTs é considerada uma opção
inadequada, porque pode não remover parte da pseudo-cápsula que contém células tumorais viáveis
e associa-se a maior frequência de ruptura tumoral. Por estes motivos, a enucleação não é
aconselhável, mesmo quando o objectivo é preservar uma estrutura vital213.
Quando o GIST apresenta grandes dimensões pode ser submetido a tratamento pré-operatório
com imatinib (neoadjuvante), com o objectivo de se obter condições de ressecabilidade do tumor
que é, muitas vezes, necrótico e friável214. Esta opção pode facilitar a ressecção cirúrgica com
52
preservação da função ou do órgão, particularmente em GISTs da junção esófago-gástrica, da
segunda porção do duodeno e do 1/3 inferior do recto24, 138. Quando há invasão de órgãos
adjacentes, a ressecção “em bloco” pode ser uma alternativa. A ressecção incompleta do tumor
deve ser efectuada apenas como opção terapêutica paliativa, em casos de hemorragia, dor ou
sintomas relevantes, secundários ao efeito de massa.
Muitos autores descreveram que os GISTs metastizam raramente para os gânglios linfáticos,
mesmo nos casos de alto risco. Bucher et al.215 descreveram 5/80 doentes (6%) com GIST
localizado com metástases hematogéneas, com ou sem envolvimento ganglionar linfático. Na série
de Rutkowski et al.189 (n=335) descreve-se quatro casos (1,2%) com metastização ganglionar. Os
estudos publicados indicam que não se justifica a realização de linfadenectomia de rotina no
tratamento cirúrgico dos GISTs, excepto quando é detectado envolvimento ganglionar
macroscópico24, 27, 138, 215-217.
Tumor residual microscópico (R1)
De acordo com as orientações da ESMO e da NCCN, nos casos em que foi efectuada ressecção
microscopicamente incompleta (R1) pode ser considerado o alargamento de margens quando a
localização exacta da lesão é identificavel e o risco de morbilidade cirúrgica é baixo.
Quando a ressecção R0 resulta previsivelmente em complicações funcionais ou co-morbilidades
importantes, e o tratamento médico neoadjuvante não é eficaz ou não pode ser administrado, a
decisão de realizar uma ressecção R1 deve ser discutida com o doente. A ressecção R1 pode ser
aceitável em GISTs de baixo risco. Não há estudos que demonstrem a associação de ressecção R1
com sobrevida mais curta dos doentes218.
Cirurgia Laparoscópica
A cirurgia laparoscópica no GIST está a ser progressivamente mais utilizada nos últimos anos. O
diagnóstico endoscópico melhorou a capacidade de identificar GISTs gástricos de pequenas
dimensões com baixo risco de agressividade1, 184. A abordagem minimamente invasiva tem vindo a
ser adoptada na generalidade destes tumores, devido aos benefícios potenciais decorrentes de se
evitar a laparotomia dos doentes. Recomenda-se que a técnica deve seguir rigorosamente os
princípios oncológicos da cirurgia aberta: ressecção completa do tumor com margens livres (R0),
evitando a disseminação de células tumorais na cavidade peritoneal24, 138, 219. Nos GISTs de grandes
dimensões, a ressecção R1 pode complicar-se com ruptura intra-operatória do tumor e
disseminação peritoneal. Por este motivo, a ressecção laparoscópica tem sido desaconselhada em
doentes com GISTs de grandes dimensões173, 218. Novitsky et al.220 sugeriram que esta
recomendação devia ser revista, porque não foi baseada em evidências, mas traduz apenas uma
medida preventiva para cirurgiões inexperientes neste procedimento. Diversos autores propuseram
a adopção de orientações mais alargadas para a cirurgia laparoscópica nos GISTs221, 222. Várias séries
descrevem a realização de ressecção por via laparoscópica de tumores com dimensões entre 0,3-
12,5 cm218, 223, sugerindo a laparoscopia na ressecção de GISTs, sobretudo em localização gástrica.
53
Não há estudos de ensaios clínicos controlados randomizados e prospectivos que permitam validar
estas opções.
A NCCN (2007) considera aceitável a ressecção laparoscópica de tumores até 5 cm de dimensão, e
que os tumores > 5 cm, dependendo da localização e da morfologia, podem ser ressecados por via
laparoscópica ou por técnicas de laparoscopia hand-assisted21, 138, 220, 223.
Antes de iniciar a ressecção do tumor, deve ser realizada uma exploração formal da cavidade
abdominal para excluir a presença de metastização no peritoneu ou no fígado. A ecografia intra-
operatória pode ser útil na avaliação de metástases hepáticas e, nas lesões suspeitas, para orientar a
realização de biópsias. A endoscopia intra-operatória tem sido frequentemente utilizada para a
localização dos GISTs de pequenas dimensões e na selecção da técnica de ressecção mais adequada.
Para evitar o risco de ruptura, os GISTs não devem ser manipulados directamente com os
instrumentos laparoscópicos220. Embora não existam dados disponíveis, a utilização de saco de
extracção da peça operatória parece essencial para evitar a disseminação de células tumorais na
cavidade abdominal ou no orifício da porta respectiva, e eventualmente metastização24, 220, 224.
Nas diversas séries publicadas, têm sido utilizadas diferentes abordagens, dependendo de vários
factores (ex: dimensão, localização e forma macroscópica do tumor), para o tratamento dos GISTs
gástricos: ressecções “em cunha” ou segmentares por via laparoscópica, laparoscópica-endoscópica
(intragástrica) ou laparoscópica hand-assisted220. Os GISTs da parede anterior e da pequena e grande
curvatura do estômago são geralmente submetidos a ressecção “em cunha”, com máquina de
anastomose linear endoscópica. As lesões de maiores dimensões podem ser ressecadas com
margens livres, utilizando o bisturi ultrassónico220. Os tumores da parede posterior são muitas vezes
abordados pela retrocavidade dos epíploons, mas a abordagem trans-gástrica, com gastrotomia
anterior, constitui alternativa válida, especialmente nos GISTs localizados próximo da junção
esófago-gástrica225-228. Esta opção é, no entanto, tecnicamente mais exigente226, e há relatos de
ressecções incompletas213 e de complicações pós-operatórias, como estenoses e “fugas” na linha de
sutura224. A abordagem combinada endoscópica-laparoscópica intragástrica tem sido descrita como
método alternativo no tratamento dos GISTs da junção esófago-gástrica220, 224.
A localização do tumor não deve ser considerada contra-indicação absoluta para cirurgia
minimamente invasiva, desde que assegurada a experiência técnica necessária e todas as precauções
indispensáveis224. No entanto, nos GISTs com maiores dimensões e/ou com localizações
desfavoráveis, como a pequena curvatura ou a junção esófago-gástrica, pode não ser possível
efectuar ressecção “em cunha” com margens livres de tumor, podendo ser necessário optar por
gastrectomia subtotal ou total. Nestes casos, o tratamento neoadjuvante com imatinib, como
sugerido nas recomendações ESMO e NCCN, pode ser uma opção válida, para redução da
dimensão do tumor que permita a cirurgia conservadora do órgão224. Contudo, a exequibilidade e
os resultados deste tipo de abordagens são ainda objecto de avaliação em curso229.
A cirurgia laparoscópica pode aplicar-se a GISTs com localizações diversas, como é o exemplo dos
GISTs rectais de pequenas dimensões. No entanto, os dados disponíveis relativamente a ressecções
laparoscópicas de GISTs noutras localizações (extragástricas) são escassos138.
54
Os resultados globais publicados da cirurgia laparoscópica descrevem que as complicações intra-
operatórias e pós-operatórias são relativamente raras, ocorrendo, respectivamente, em 6,8% e 7,7%
dos doentes218. As ressecções decorrem com perdas mínimas de sangue, satisfazendo os tempos de
cirurgia e períodos curtos de estadia hospitalar223, 224. Evita-se também a morbilidade relacionada
com a ferida operatória da laparotomia220. A "curva de aprendizagem" nos procedimentos
laparoscópicos faz presumir que, com a maior experiência técnica, os tempos operatórios serão
progressivamente melhorados230, 231.
Apesar dos dados de follow-up dos doentes serem escassos, não excedendo ~5 anos218, algumas
séries descreveram segurança oncológica na abordagem laparoscópica220, 232, 233, com eficácia e taxas
de recidiva semelhantes às obtidas com cirurgia convencional.
A aplicabilidade da abordagem laparoscópica deve assentar numa variedade de factores, incluindo
as características do doente, a dimensão e a forma macroscópica do tumor, o padrão de invasão e a
localização do tumor, bem como a experiência e qualificação em cirurgia laparoscópica do
cirurgião220.
Os dados da literatura indicam que as ressecções laparoscópicas ou assistidas por laparoscopia são
exequíveis e associam-se a taxas de recidiva reduzidas, períodos curtos de internamento e
morbilidade baixa213, 217, 220, 232, 234, 235. Esta abordagem deve ser recomendada como opção de
escolha para a maioria dos doentes com GISTs gástricos de pequenas e médias dimensões220, 224.
GIST primário localmente avançado
Nos GISTs localmente avançados, não metastáticos, pode ser impossível a realização de ressecção
R0. Nestes casos, deve considerar-se a citorredução tumoral com terapêutica neoadjuvante com
imatinib. Esta abordagem pode facilitar a obtenção de margens cirúrgicas R0 e permitir uma
cirurgia menos mutilante, com melhores resultados funcionais, de acordo com orientações da
ESMO e da NCCN . Esta recomendação baseia-se em publicações de dados retrospectivos, não
randomizados236, 237. O tratamento primário com imatinib, para citorredução do tumor, pode ser
considerado nos GISTs em que se prevê um risco elevado de hemorragia ou ruptura tumoral
durante a cirurgia. A resposta terapêutica máxima é atingida geralmente após 6-12 meses de
tratamento. A intervenção cirúrgica subsequente pode, na maioria dos casos, ser realizada com
segurança24, 214, 238. No entanto, nem sempre é necessário esperar pela resposta máxima antes de
realizar a cirurgia. A análise mutacional pode auxiliar a ponderar a terapêutica neoadjuvante dos
GISTs com menos sensibilidade ao imatinib (por exemplo, com mutações D842V do PDGFRA), e
permitir adoptar o esquema terapêutico mais adequado. A PET ou PET-TC, ou a avaliação da
densidade do tumor com TC pode ser particularmente útil na avaliação rápida da resposta à
terapêutica, não condicionando atraso na intervenção cirúrgica dos GISTs que não respondem ao
tratamento239-242.
É imprescindível estabelecer um plano terapêutico multidisciplinar, envolvendo patologistas,
radiologistas, cirurgiões e médicos oncologistas. A partilha de experiências, disponível em centros
55
de referência para sarcomas e GISTs e/ou em redes de referenciação de doentes oncológicos, deve
ser entendida como fundamental no tratamento dos GISTs24.
Tratamento adjuvante
Após ressecção, o risco de recidiva de GISTs pode ser substancial180, 243, conforme definido pela
classificação de risco (classificação de Miettinen/AFIP). Tendo em conta a eficácia do imatinib na
doença metastática, o seu uso em tratamento adjuvante tem sido avaliado em vários estudos244-246.
Um ensaio clínico intergrupo de fase II (Z9000) do American College of Surgeons Oncology Group
(ACOSOG) avaliou o tratamento adjuvante com imatinib, administrado numa dose de 400 mg/dia
durante 12 meses, após ressecção completa do tumor primário em GISTs de risco elevado (n=107).
Os GISTs de alto risco foram definidos como tumores com dimensões> 10 cm, com ruptura ou
hemorragia intraperitoneal, ou tumores multifocais (> 5). Os resultados descrevem que o imatinib é
bem tolerado no contexto adjuvante, prolonga a sobrevida livre de recidiva e está associado a
sobrevida global melhor, em comparação com controlos históricos244.
Foi concluído um ensaio clínico intergrupo do ACOSOG (Z9001), de fase III, randomizado,
duplamente cego, em que os doentes receberam imatinib (400 mg/dia) ou placebo durante 1 ano,
após efectuarem uma ressecção macroscópica completa de GISTs primários KIT-positivos e com
diâmetro ≥3 cm. Os doentes foram apenas estratificados segundo o tamanho do tumor (3 – 6 cm, 6
– 10 cm, and ≥ 10 cm). Com um tempo mediano de follow-up (713 doentes) de 19,7 meses, o estudo
descreve que o tratamento adjuvante com imatinib aumentou significativamente a sobrevida livre de
recidiva dos doentes, relativamente ao grupo placebo (98% vs. 83%). Resultados idênticos foram
descritos nos diferentes subgrupos de estratificação, segundo a dimensão do tumor, mas a maior
diferença entre o braço terapêutico e o braço placebo foi observada nos GISTs de risco mais
elevado, com diâmetro ≥10 cm. Não foram observadas diferenças na sobrevida global entre os dois
braços do estudo246. Estes resultados apoiaram decisivamente o papel do imatinib no tratamento
adjuvante do GIST. No entanto, é necessário um período de follow-up mais longo para conclusões
definitivas sobre algumas questões fundamentais: taxa de recidiva absoluta após um intervalo de
tempo de follow-up maior; tempo de atraso no aparecimento da recidiva; e, no caso de recidiva,
tempo decorrido até ocorrer resistência secundária ao imatinib. Com base neste estudo, o imatinib
foi aprovado, como terapêutica adjuvante no GIST, pela FDA (Federal Drug Administration) e pela
EMA (European Medicine Agency). Embora ainda não exista consenso geral relativamente a que
subgrupo de doentes deve ser administrado o tratamento adjuvante, é amplamente aceite que deve
ser proposto aos doentes com GISTs de risco elevado. Segundo as recomendações actuais, o
imatinib adjuvante pode ser proposto como opção para doentes com um risco substancial de
recidiva (ESMO)24, mais concretamente, para doentes com GISTs de risco intermédio ou elevado
(NCCN)138.
A análise mutacional dos GISTs pode ser relevante para complementar a avaliação de risco na
selecção dos doentes que são previsivelmente mais susceptíveis de beneficiar com o tratamento
adjuvante. Os resultados dos estudos disponíveis sustentam o tratamento adjuvante durante um
período de 12 meses. Estão em curso dois ensaios clínicos fase III (EORTC 62024; SSG
56
XVIII/AIO), para avaliar períodos de tratamento mais longos (0 vs. 2 e 1 vs. 3 anos,
respectivamente)247, 248. Foi recentemente iniciado um estudo fase II de tratamento adjuvante,
denominado Post-resection Evaluation of Recurrence-free Survival for GastroIntestinal Stromal Tumors with
Adjuvant Imatinib (PERSIST)-5 (ClinicalTrials.gov identifier: NCT00867113) para avaliar a sobrevida
livre de recidiva em doentes com GIST de alto risco tratados com imatinib (400 mg/dia) durante 5
anos após ressecção cirúrgica.
No caso de ruptura pré ou per-operatória do tumor, com disseminação de células tumorais na
cavidade peritoneal, pode assumir-se um estado de doença peritoneal oculta. Este estado coloca os
doentes perante um risco elevado de recidiva peritoneal e, por esse motivo, podem considerar-se
candidatos a tratamento com imatinib. A duração ideal do tratamento nestes casos é ainda
desconhecida24.
Follow-up dos doentes
Não existem dados publicados conclusivos sobre qual o seguimento de rotina ideal para os doentes
com GIST localizado submetidos a tratamento cirúrgico. Os esquemas de follow-up variam de
instituição para instituição. A avaliação de risco baseada no índice mitótico, na dimensão e na
localização do tumor, pode ajudar na selecção da melhor estratégia de seguimento. Nos doentes
com GISTs de risco alto, a recidiva ocorre geralmente nos primeiros 2-3 anos após ressecção. Nos
GISTs de risco baixo, a recidiva é menos provável e surge, na maior parte dos casos, após 2-3 anos.
Como as metástases podem ocorrer 10-15 anos após a cirurgia, o seguimento dos doentes deve ser
prolongado 12.
Existindo actualmente um tratamento eficaz para os GISTs recidivados e/ou metastáticos, algumas
instituições de referência propõem a realização de TC todos os 3-4 meses durante 3 anos, nos
doentes com GISTs de risco intermédio ou alto; depois, de 6/6 meses até aos 5 anos; e
posteriormente com periodicidade anual. Nos doentes com tumores de risco baixo, é proposta a
realização de TC de 6/6 meses durante os primeiros 5 anos. Os doentes com GISTs de risco muito
baixo não necessitam provavelmente de seguimento de rotina, embora o risco de comportamento
agressivo não deva ser considerado nulo24, 138.
10. b) Doença recidivada e metastática
Reconhece-se que o tratamento cirúrgico isolado não é curativo nos casos de GIST avançado, com
doença recidivada e/ou metastática, mesmo quando se removem todas as lesões metastáticas
abdominais.
Na recidiva, cerca de 2/3 dos doentes têm envolvimento hepático e 1/2 doença peritoneal27. As
metástases extra-abdominais no pulmão ou no esqueleto são mais raras, podendo desenvolver-se
em estadios mais avançados.
57
As metástases hepáticas são geralmente multifocais, difusas e difíceis de ressecar. Cerca de 26% dos
doentes, submetidos a metastasectomia249, desenvolvem recidiva após ressecção hepática174. A
exérese das lesões peritoneais associa-se geralmente a recidiva subsequente.
A quimioterapia convencional para o tratamento do GIST tem uma taxa diminuta (cerca de 5%) de
resposta249-252. A radioterapia tem também valor reduzido, devido à localização dos tumores e à
limitação nas doses que podem ser utilizadas202. A embolização da artéria hepática e a cirurgia de
citorredução, seguida de quimioterapia intraperitoneal, foram também objecto de investigação, mas
os resultados obtidos são desencorajadores253, 254.
O tratamento de escolha do GIST recidivado/metastático consiste, com poucas excepções, na
administração do mesilato de imatinib (ESMO, NCNN). Os doentes com GIST primário e doença
metastática síncrona de baixo volume podem ser seleccionados num primeiro tempo para ressecção
cirúrgica, especialmente se forem sintomáticos. No entanto, as orientações de consenso
recomendam a associação ao tratamento com imatinib, mesmo nos casos em que toda a doença
macroscópica é ressecada24.
O uso do mesilato de imatinib revolucionou o tratamento dos GISTs. O imatinib é um inibidor
selectivo de tirosina-cínases específicas, incluindo o KIT, PDGFRα, ARG, c-FMS, ABL e BCR-
ABL253. O imatinib causa uma inibição competitiva no local de ligação do ATP do receptor KIT,
levando à inibição da auto-fosforilação e subsequente interrupção das vias de sinalização envolvidas
na proliferação e na sobrevida celular255. O imatinib foi inicialmente desenvolvido como um
inibidor do PDGFRα. A sua primeira utilização terapêutica foi no tratamento da leucemia mielóide
crónica (LMC), em que uma proteína de fusão BCR-ABL (Breakpoint cluster region-Abelson gene protein)
causa desregulação da actividade tirosina-cinásica256. Descreveu-se que o imatinib induz uma
resposta completa em quase todos os doentes com LMC em fase crónica, tendo sido aprovado pela
FDA para o tratamento dos doentes com LMC em 2001257.
Em 2001, Joensuu et al. publicaram a experiência com imatinib num único doente com GIST
metastático23. Os resultados confirmados nas imagens seriadas da RMN e da PET foram muito
promissores. Esta publicação de um caso clínico desencadeou a realização de vários ensaios
clínicos, sendo actualmente descrito que até 80% dos doentes com GIST metastático têm resposta
parcial ou estabilidade da doença após terapêutica com imatinib56. O imatinib é geralmente bem
tolerado e os efeitos laterais incluem edemas, erupção cutânea, diarreia, náuseas, dores abdominais e
fadiga.
Eficácia e tolerabilidade do imatinib como terapêutica de 1ª linha
A eficácia e a tolerabilidade com 400 e 800 mg/dia de imatinib na terapêutica de 1ª linha dos GISTs
têm sido descritas em ensaios clínicos fase II/III.
Num estudo fase II, multicêntrico e randomizado (B2222), com imatinib na dose de 400 ou 600
mg/dia em doentes com GIST avançado258, a taxa de resposta, baseada nos critérios Southwest
Oncology Group (SWOG)259, foi de 53,7% (n=79) com resposta parcial e 27,9% (n=41) com doença
estável, não se obtendo resposta completa em nenhum doente. Nos resultados a longo prazo deste
58
estudo206 (tabela 3) descreve-se que, com uma mediana de seguimento de 71 meses, as taxas de
resposta, a mediana da sobrevida livre de progressão (SLP) e a mediana da sobrevida global (SG)
foram idênticas em ambos os braços terapêuticos. Neste estudo, a mediana da resposta terapêutica
foi de 29 meses e a mediana da SG 57 meses.
Tabela 3: Resultados de ensaios clínicos fase II e III com os inibidores tirosina-cinásicos (ITC) imatinib e sunitinib, em doentes com GIST localmente avançado ou metastático
SLP (mediana): 96 semanas. SG aos 3 anos estimada: 73,6%
European-Australasian (Verweij, Casali et al. 2004)
Fase III-R (946)
Imatinib, 400 vs. 800
RC: 5%, RP: 45%, DE: 32% vs. RC: 6%, RP: 48%, DE: 32%
SG a 1 e 2 anos: 85% vs. 86% e 69% vs. 74%. SLP aos 2 anos: 50% vs. 56% (p=0,026).
Intergroup S0033 (Blanke, Rankin et al. 2008)
Fase III-R (746)
Imatinib, 400 vs. 800
RC: 5%, RP: 40%, DE: 25% vs. RC: 3%, RP: 42%, DE: 22%
SLP e SG (medianas): 18 vs. 20 meses e 55 vs. 51 meses (p=NS)
Sunitinib phase III (Demetri, van Oosterom et al. 2006)
Fase III-R (312)a
Sunitinib, 50mg/kg (esq. 4/2) vs. placebo
RP: 7%, DE: 58% vs. RP: 0%, DE: 48%
SLP (mediana): 24,1 vs. 6 semanas (p<0,0001)
EORTC- European Organisation for Research and Treatment of Cancer, STBSG- Soft Tissue and Bone Sarcoma Group, RC- resposta completa, RP- resposta parcial, DE- doença estável, SG- sobrevida global, SLP- sobrevida livre de progressão, TPT- tempo para progressão do tumor, R- randomizado, N- número de doentes, NS- não significativo. a - terapêutica de 2ª linha.
Outro estudo fase II, iniciado pelo EORTC Soft Tissue and Bone Sarcoma Group260, corroborou os
resultados obtidos com o imatinib (tabela 3). Este ensaio avaliou a eficácia de 400 mg de imatinib
administrado duas vezes/dia. Aos 12 meses, 73% dos casos estavam livres de progressão da
doença. Neste estudo, os efeitos laterais associados à dose de 800 mg/dia mais comuns foram:
Esophageal GIST: Therapeutic implications of an uncommon presentation of a rare tumor
A. M. Gouveia,1,3 A. P. Pimenta,1,3 J. M. Lopes,2,3 A. F. Capelinha,2 S. S. Ferreira,1 C. Valbuena,2 M. C. Oliveira1,3
1Serviço de Cirurgia B and 2Pathology Department, Oporto Medical School and Hospital de São João and 3Institute of Molecular Pathology and Immunology of the University of Oporto, (IPATIMUP), Oporto, Portugal
SUMMARY. Gastrointestinal stromal tumors (GISTs) are rarely reported in the esophagus. The authorsreport a patient with an esophageal GIST, incidentally found after an echocardiogram. CT scan and endo-scopic ultrasonography showed the tumor in the dependence of the muscularis propria of the esophageal wall.An Ivor–Lewis esophagectomy was performed. The tumor was well-circumscribed involving the submucosal andthe muscular layers of the esophagus, measuring 13.5 ×××× 8.5 ×××× 7.6 cm, without involving the surgical margins.Histologically, the tumor consisted of spindle cells, with low mitotic index (2/50 HPF), that were immuno-reactive for KIT (CD117) and CD34, consistent with GIST of high risk of aggressive behavior. No adjuvanttherapy was given to the patient, who is alive and without evidence of disease 1 year after surgery. Sinceesophageal GISTs are rarely reported in the literature and usually have a poor prognosis, the diagnostic differ-entiation of these tumors from other more common mesenchymal neoplasms is essential, both for therapeuticand prognostic reasons.
Gastrointestinal stromal tumors (GIST) aredefined as c-KIT (CD117, stem cell factor receptor)– positive mesenchymal spindle or epithelioid celltumors in the gastrointestinal (GI) tract. Depend-ing upon the histologic findings, GISTs wereclassified for many years as leiomyomas, leiomyo-blastomas and leiomyosarcomas, as a result of theirapparent origin in the muscularis propria of theGI tract wall. Although rare, they are the mostcommon mesenchymal tumors of the GI tract andaccount for up to 3% of GI tract malignant tumors.GISTs predominate in the stomach (50–70%) andsmall bowel (20–33%).
In the esophagus, squamous carcinoma and adeno-carcinoma are the common malignant tumorsand leiomyoma the most frequent mesenchymalneoplasm. GISTs have been documented very rarely(< 5%) in the esophagus. Therefore, the diagnosticdifferentiation of GISTs from other mesenchymalneoplasms, particularly in the esophagus, is essen-
tial not only because the former group has a highrisk of malignant behavior, but is usually respon-sive to a new recently targeted therapy, STI-571(imatinib mesylate) – a receptor tyrosine kinaseinhibitor of the activated KIT protein, as fre-quently observed in GISTs.
We report herein an esophageal GIST incident-ally found in a patient who underwent an echocar-diogram. Clinical presentation, diagnosis, and riskclassification of aggressive behavior will be dis-cussed, as well as the available therapeutic optionsfor these tumors.
Patient report
In the course of a hypertension investigation a59-year-old man underwent an echocardiogramthat raised the suspicion of an aortic dilatation. Hehad no digestive symptoms and his past medical his-tory was unremarkable. Clinical examination wasirrelevant and his hematological and biochemicalprofile was normal. A CT scan revealed a well-outlined tumor in the posterior mediastinum,apparently originating in the left lateral esophagealwall without involving the lumen of the esophagus(Fig. 1a). The tumor was independent from the
Address correspondence to: Dr António M. Gouveia, Serviço de Cirurgia B, Hospital de São João, 4202–451 Porto, Portugal. Email: [email protected]
Esophageal GIST 71
aorta, and extended from the tracheal bifurcationto the esophagogastric junction: it was a solidlesion, capturing contrast heterogeneously with apoorly outlined central hipocapturing region. Anesophagogastroscopy showed deformity of the eso-phageal wall extending from 35 cm to 40 cm fromthe incisors, apparently caused by compression of asubepithelial mass (Fig. 1c); no lesions were foundin the stomach. Endoscopic ultrasonography con-firmed the tumor features revealed by the CT scanand showed that the tumor was located in themuscularis propria (Fig. 1b).
An Ivor–Lewis esophagectomy was performed tocompletely resect the tumor and the patient had anuneventful postoperative recovery, without adjuvantchemotherapy or radiotherapy.
The patient is asymptomatic and with no clinicalevidence of tumor recurrence or metastasis after12 months of follow-up.
Pathologic features
The tumor was a well-circumscribed gray-whitefibrous mass measuring 13.5 × 8.5 × 7.6 cm, involv-ing the submucosa and the muscular layers, andsparing the mucosa of the esophagus (Fig. 1d). Thecut surface showed foci of hemorrhage, necrosisand cystic areas in the tumor.
Histologically, the lesion was composed of amoderately cellular proliferation of spindle cellsembedded in a collagenous matrix (Fig. 2b) withfoci of hyalinization. The cells had cytoplasmic vacuo-lization, low pleomorphism and there were somebizarre cells (Fig. 2b-inset). Mitotic index was 2/50high power fields (HPF: 0.152 mm2). There were
areas of tumor hemorrhage and necrosis. There wasno invasion of the mucosa (Fig. 2a) and the surgi-cal margins were free of tumor. Immunohisto-chemical studies were performed and tumor cellsstained diffusely for KIT (CD117; Fig. 2d), and CD34(Fig. 2c), and focally for HHF35. None of the cellsstained for pS100 or desmin. Proliferation index,using Ki-67, was less than 1%.
The diagnosis of esophageal GIST, with high riskof aggressive behavior, was performed using the cri-teria of the International Consensus on GIST.1
DISCUSSION
Gastrointestinal stromal tumors (GISTs) are themost common mesenchymal tumors of the humangastrointestinal (GI) tract, but they are rare (1–3%)in the esophagus.2,3
‘Gastrointestinal stromal tumor’ used to be acollective term referring to primary mesenchymaltumors of the GI tract, but now it is consideredto be a particular tumor that originates from theinterstitial cell of Cajal (ICC), or its precursor, inthe GI tract wall and has expression of the tyrosinekinase receptor KIT in almost all cases.4–7
In 1998 the presence of gain-of-function muta-tions in the c-KIT proto-oncogene were found inGISTs.4 Those mutations are thought to be respon-sible for constitutive ligand-independent activationof KIT and subsequent tumor pathogenesis.1,4,8,9
More recently, PDGFRA activating mutations weredescribed in the small group of GISTs (about 10%)lacking KIT mutations.10
The specific diagnosis of GIST before biopsy orsurgery is quite difficult in any location. Althoughthe diagnosis can be often suspected histologically,
Fig. 1 Esophageal GIST: (a) CT scan disclosing well-circumscribed tumor in the posterior mediastinum; (b) endoscopic ultrasonography showing the intraparietal esophageal location of the tumor; (c) endoscopy showing the endoluminal esophageal aspect of the tumor, without ulcerating the mucosa; and (d) macroscopy of the tumor, after longitudinal section of the esophagus. Note the smooth appearance of the mucosa over the tumor.
Fig. 2 Esophageal GIST: (a) histologic low power view showing submucosal involvement by the tumor; (b) tumor spindle cells and collagenous stroma, inset: bizarre tumor cells; (c) CD34 immunoreactivity of tumor cells; and (d) CD117 (KIT) immunoreactivity of tumor cells.
72 Diseases of the Esophagus
the term GIST is usually applied, as in our case, forneoplasms displaying KIT (CD117) immunoposi-tivity, with very rare exceptions.1
In a published series of 68 patients with esopha-geal mesenchymal tumors,11 Miettinen et al. found48 (71%) leiomyomas and 17 (25%) GISTs. TheGISTs were located in the distal esophagus anddysphagia was the typical symptom. In two patientsthe tumor was an incidental radiologic finding, asin our case, in spite of the large dimension of thetumor. It is noteworthy that this was the only casewith esophageal localization found by us in a seriesof 78 GISTs.12
According to the consensus approach for defin-ing risk of aggressive behavior, GISTs are classifiedinto low-, intermediate- and high-risk groups,depending on the size and mitotic index of thetumor.1 There is a tendency to consider smallGISTs (< 2 cm in diameter) with low mitotic activ-ity as having a good prognosis. However, a fewGISTs apparently lacking mitotic activity maymetastasize, which raises difficulties in predictingthe behavior of GISTs on an individual basis.Therefore, we concur that it is important to stressthat all patients with GIST must be carefully andregularly followed up for an indefinite period, inlight of the uncertainties expressed above and thedocumented tendency of these tumors to pursue anindolent clinical course with significant risk of laterelapse.1 Some authors reported that esophagealGISTs are often diagnosed late and tend to beassociated with a poor prognosis.1,13 In the paperpublished by Miettinen et al.,11 12 out of 17esophageal GISTs were classified as high risk, as inour case.
Complete surgical resection is the standard oftreatment for primary GIST, with no need for widemargins or regional lymphadenectomy. In our case,due to the large dimension of the tumor, an Ivor–Lewis esophagectomy was performed and the surgi-cal margins were free of tumor. Survival of patientswith GIST, after resection of all gross disease,varies considerably in published series. Five-yearsurvival rates for these patients range from 35% to65%, depending on the inclusion of patients withdisseminated disease.3,14,15
Gastrointestinal stromal tumors appear to serveas a model for molecular-based diagnosis and treat-ment of solid tumors and for this reason patholo-gists will play a vital role in the diagnosis andtreatment of these cases.20,22–24 Treatment of GISTshas changed dramatically since the introduction ofimatinib mesylate (Glivec/Gleevec; Novartis Onco-logy). This is a small molecule that selectivelyinhibits the enzymatic activity of the ABL andBCR-ABL fusion protein, platelet derived growthfactor receptor, and KIT tyrosine kinases. Imatinibmesylate inhibits the mutated KIT receptor
observed in most GISTs, leading to the onset ofapoptosis and decreased proliferation of tumorcells. Preliminary results of several trials indicatethat imatinib is an effective and safe treatment inpatients with unresectable, recurrent, and metastatictumors.16–21 The role of imatinib in the adjuvant set-ting has also been the subject of recent interest.Bumming et al. reported four patients treated formicroscopic positive margins with a recurrence-freeinterval of 7–13 months.25 The American College ofSurgeons Oncology Group (ACOSOG) is sponsor-ing two ongoing adjuvant trials, in patients withprimary tumors presenting selected risk criteria(larger tumors; intraperitoneal tumor rupture orhemorrhage; multifocal tumors).
Although a radical excision of the tumor wasperformed in all but one patient of the Miettinenet al. series,11 nine patients of this series died of dis-ease, with a median survival of 29 months, stressingthe aggression of GISTs in esophageal localization.Thus, it would be interesting to consider the useof imatinib in the adjuvant setting, particularly inlarge and high-risk GISTs of the esophagus.
References
1 Fletcher C D M, Berman J J, Corless C et al. Diagnosis ofgastrointestinal stromal tumors: a consensus approach. HumPathol 2002; 33: 459–65.
2 Miettinen M, Lasota J. Gastrointestinal stromal tumors(GISTs). Definition, occurrence, pathology, differential diag-nosis and molecular genetics. Pol J Pathol 2003; 54: 3–24.
3 DeMatteo R P, Lewis J J, Leung D, Mudan S S, Woodruff JM, Brennan M F. Two hundred gastrointestinal stromaltumors: recurrence patterns and prognostic factors for sur-vival. Ann Surg 2000; 231: 51–8.
4 Hirota S, Isozaki K, Moriyama Y et al. Gain-of-functionmutations of c-kit in human gastrointestinal stromal tumors.Science 1998; 279: 577–80.
5 Kindblom L G, Remotti H E, Aldenborg F, Meis-KindblomJ M. Gastrointestinal pacemaker cell tumor (GIPACT): gas-trointestinal stromal tumors show phenotypic characteristicsof the interstitial cells of Cajal. Am J Pathol 1998; 152:1259–69.
6 Sircar K, Hewlett B R, Huizinga J D, Chorneyko K, BerezinI, Riddell R H. Interstitial cells of Cajal as precursors ofgastrointestinal stromal tumors. Am J Surg Pathol 1999; 23:377–89.
7 Sarlomo-Rikala Kovatich A J, Barusevicius A, Miettinen M.CD117: a sensitive marker for gastrointestinal stromal tumor thatis more specific than CD34. Mod Pathol 1998; 11: 728–34.
8 Lux M L, Rubin B P, Biase T L et al. KIT extracellular andkinase domain mutations in gastrointestinal stromal tumors.Am J Pathol 2000; 156: 791–5.
9 Rubin B P, Fletcher J A, Fletcher C D M. Molecularinsights into the histogenesis and pathogenesis of gastrointes-tinal stromal tumors. Int J Sur Pathol 2000; 8: 5–10.
10 Heinrich M C, Corless C L, Duensing A et al. PDGFRAactivating mutations in gastrointestinal stromal tumors. Sci-ence 2003; 299: 708–10.
11 Miettinen M, Sarlomo-Rikala M, Sobin L H, Lasota J.Esophageal stromal tumors: a clinicopathologic, immuno-histochemical, and molecular genetic study of 17 cases andcomparison with esophageal leiomyomas and leiomyosarco-mas. Am J Surg Pathol 2000; 24: 211–22.
12 Gouveia A, Silva P, Costa S, Pimenta A, Oliveira M C, Lopes JM. GIST: The Hospital S. João (Porto) series during the last13 years (abstract). Hepatogastroenterology 2003; 50: LXIII.
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13 Miettinen M, El-Rifai W H L, Sobin L, Lasota J. Evalua-tion of malignancy and prognosis of gastrointestinal stromaltumors: a review. Hum Pathol 2002; 33: 478–83.
14 Ng E H, Pollock R E, Munsell M F, Atkinson E N,Romsdahl M M. Prognostic factors influencing survival ingastrointestinal leiomyosarcomas. Implications for surgicalmanagement and staging. Ann Surg 1992; 215: 68–77.
15 Roberts P J, Eisenberg B. Clinical presentation of gastro-intestinal stromal tumors and treatment of operable disease.Eur J Cancer 2002; 38: S37–S38.
16 Joensuu H, Roberts P J, Sarlomo-Rikala M et al. Effect ofthe tyrosine kinase inhibitor STI571 in a patient with ametastatic gastrointestinal stromal tumor. N Engl J Med2001; 344: 1052–6.
17 van Oosterom A T, Judson I, Verweij J et al. Safety andefficacy of imatinib (STI571) in metastatic gastrointestinalstromal tumours: a phase I study. Lancet 2001; 358: 1421–3.
18 Demetri G D, von Mehren M, Blanke C D et al. Efficacyand safety of imatinib mesylate in advanced gastrointestinalstromal tumors. N Engl J Med 2002; 347: 472–80.
19 Heinrich M C, Blanke C D, Druker B J, Corless C L. Inhibi-tion of KIT tyrosine kinase activity: a novel molecular
approach to the treatment of KIT-positive malignancies. JClin Oncol 2002; 20: 1692–703.
20 DeMatteo R P, Heinrich M C, El-Rifai W M, Demetri G.Clinical management of gastrointestinal stromal tumors:before and after STI-571. Hum Pathol 2002; 33: 466–77.
21 Blanke C D, von Mehren M, Joensuu H et al. Evaluation ofthe safety and efficacy of an oral molecularly-targetedtherapy, STI-571, in patients with unresectable or metastaticgastrointestinal stromal tumors (GISTS) expressing c-KIT(CD117) (Abstract). Presented at the 37th Annual Meetingof the American Society of Clinical Oncology, San Francisco,CA, May 12–15, 2001.
22 Blanke C D, Eisenberg B L, Heinrich M C. Gastrointestinalstromal tumors. Curr Treat Options Oncol 2001; 2: 485–91.
23 Kitamura Y, Hirota S, Nishida T. Gastrointestinal stromaltumors (GIST): a model for molecule-based diagnosis andtreatment of solid tumors. Cancer Sci 2003; 94: 315–20.
24 O’Leary T, Berman J J. Gastrointestinal stromal tumors:answers and questions. Hum Pathol 2002; 33: 456–8.
25 Bümming P, Andersson J, Meis-Kindblom J M et al. Neo-adjuvant, adjuvant and palliative treatment of gastrointestinalstromal tumours (GIST) with imatinib: a centre-based studyof 17 patients. Br J Cancer 2003; 89: 460–4.
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Molecular alterations of KIT and PDGFRA in GISTs:evaluation of a Portuguese series
A L Gomes,1 A Gouveia,2,3,5 A F Capelinha,2,4,5 D de la Cruz,4 P Silva,2,4,5 R M Reis,1
A Pimenta,3,4,5 J M Lopes2,4,5
1 Life and Health SciencesResearch Institute (ICVS),School of Health Sciences,University of Minho, Braga,Portugal; 2 IPATIMUP, Instituteof Molecular Pathology andImmunology of the University ofPorto, Porto, Portugal;3 Department of Surgery, PortoUniversity, Porto, Portugal;4 Department of Pathology, H.S.Joao, Porto University, Porto,Portugal; 5 Medical Faculty ofPorto University, Porto, Portugal
Correspondence to:Professor Dr Jose Manuel Lopes,Rua Roberto Frias, s/n, 4200-465 Porto, Portugal; [email protected]
ALG and AG contributed equallyto this study.
Accepted 10 April 2007Published Online First9 November 2007
This paper is freely availableonline under the BMJ Journalsunlocked scheme, see http://jcp.bmj.com/info/unlocked.dtl
ABSTRACTAim: To assess KIT and PDGFRA mutations frequencies ina Portuguese series of gastrointestinal stromal tumours(GISTs).Methods: 78 GISTs were evaluated for CD117 expressionand screened for mutations in KIT (exons 9, 11, 13, 14and 17) and PDGFRA (exons 12, 14 and 18) genes.Results: KIT activating mutations were identified in 44(56%) of the 78 GISTs. Forty cases (91%) presented amutation in KIT exon 11, and 4 (9%) in exon 9. One caseshowed a 4 bp deletion in intron 14. PDGFRA mutationswere observed in 5 cases (6%): 2 (3%) in exon 12 and 3(4%) in exon 18. Survival analysis was performed in 63 ofthe 78 GISTs. The presence of mutated KIT wassignificantly correlated with shorter survival of patients(p = 0.0460), and inversely associated with epithelioidhistological type of GISTs (p = 0.0064).Conclusions: Overall, the incidence of both KIT andPDGFRA mutations in these Portuguese series was 63%,being in agreement with other studies, mainly of Iberianpopulations. The great majority of mutations were locatedin KIT exon 11, statistically associated with worseprognosis and indicative of favourable response toimatinib-based therapy in this Portuguese series of GISTs.
Gastrointestinal stromal tumours (GISTs)although rare, are considered to be the mostfrequent gastrointestinal mesenchymal tumoursin humans.1 A Scandinavian study estimated theincidence of GISTs to be between 20 and 40 permillion.2 In Portugal, as far as we know, anepidemiological study is yet to be done.
The cellular origin of GISTs is not fully under-stood, but they are thought to arise from inter-stitial cells of Cajal or their precursors, due to theirsimilar positive KIT (CD117) and CD34 stainingand negative staining for both desmin and S-100protein immunostaining.1 3 GISTs are rarely foundoutside the gastrointestinal tract, being mostcommonly found in the stomach (40–70%), smallintestine (20–50%) and colon or rectum (5–15%).1 2 4 Nowadays, the diagnosis of GISTs ispartially dependent on tumour cells overexpressionof CD117 together with CD34.5 The expression ofsuch immunohistochemistry features is useful todifferentiate GISTs from other mesenchymaltumours of the gastrointestinal tract, namelyleiomyomas and leiomyosarcomas, nerve sheathtumours, and other primary and metastatictumours possibly occurring in this location.1 2 4
KIT belongs to the class III receptor tyrosinekinases (RTKs), which also include platelet-derivedgrowth factor A and B (PDGFRA, PDGFRB),colony stimulating factor-1 receptor (CSF1R) and
FMS-related tyrosine kinase 3 (FLT3).6 These RTKsare characterised by the presence of an extracellulardomain, a transmembrane domain, a juxtamem-brane domain, and an intracellular domain wherethe two kinase domains are lodged.7 RTK activa-tion occurs when by ligand binding, the receptordimerises and suffers conformational transforma-tions, which induce activation of the kinasedomains. These, in turn, lead to activation ofimportant intracellular signalling pathways, suchas RAS/mitogen activated protein kinase (RAS/MAPK), phosphoinositide-3 kinase (PI3K), andsignal transducers and activators of transcription(STAT), which regulate many physiological func-tions such as cell survival, proliferation, differentia-tion, adhesion and apoptosis.7 8
GISTs are molecularly characterised by muta-tions in KIT oncogene, located in the long arm ofchromosome 4 (4q11–12).9 There is a broadspectrum of KIT mutations in GISTs, ranging from20% to 80%, most of them being located in thejuxtamembrane domain (exon 11), followed bymutations in the extracellular domain (exon 9),and seldom in the kinase (exon 13 and 17) and ATPpocket (exon 14) domains.10–12 Later studiesreported the presence of activating mutations inthe PDGFRA oncogene in wild-type KIT bearingGISTs.13 14 PDGFRA is also located at 4q11–12 andexhibits similar RTK cellular functions.13 14 Thehotspot regions in this gene lie in the juxtamem-brane (exon 12) and kinase (exons 14 and 18)domains, and have been reported in 5–12% ofcases.12 14 The frequency of KIT/PDGFRA muta-tions in GISTs varies from series to series, probablyreflecting epidemiological and methodological dif-ferences in the various studies on record.10 12
Until recently, the treatment of GISTs waslimited to surgical removal of the tumour.Unfortunately, even in patients where the tumourwas completely and successfully removed, therewas a high probability of recurrence.1 The devel-opment of imatinib mesylate (Glivec/Gleevec,Novartis, Basel, Switzerland), a selective inhibitorof RTKs, has brought new hope for GIST patients.Imatinib targets KIT by competing with its ATPbinding site, preventing further phosphorylationsof downstream intracellular signalling moleculesresponsible for its oncogenic properties.15 16 Severalstudies have showed the importance of KIT andPDGFRA molecular status in the imatinibresponse.10 12 It has been reported that patientswith tumours harbouring exon 11 KIT mutationsare more likely to respond to imatinib therapy thanthose with either exon 9 KIT mutations orundetectable mutations.10 12
In Portugal, the incidence of both KIT and PDGFRAmutations in GISTs is, to the best of our knowledge, unknown.Since different genotypic features give rise to different drugresponses and thus different prognosis, it becomes important todefine which patients will positively respond to imatinibtreatment. Therefore, we characterised the occurrence of KITand PDGFRA mutations in a series of Portuguese GIST patients.
MATERIALS AND METHODS
Tissue samplesSeventy-eight formalin-fixed and paraffin-embedded consecu-tively diagnosed primary, previously untreated, sporadic GISTs,classified according to World Health Organization criteria5 andrisk group,17 were retrieved from files (1989–2005) from thePathology Department of S. Joao Hospital, Porto, Portugal. Allpatients were Caucasian and of Portuguese origin, with a meanage of 61.7 years (range 20–88). Thirty-eight (48.7%) patientswere female and 40 (51.3%) were male. Follow-up data,managed according to the guidelines of the European Societyof Medical Oncology,18 were available in 63 patients (range 0.2–206.0 months, mean 122.8 (12.1) months, median 132.6 (26.8)months) in September 2006.
ImmunohistochemistryThe immunohistochemistry procedure was performed accordingto the streptavidin–biotin–peroxidase complex principle, usingrabbit polyclonal anti-human antibodies raised against CD117(dilution 1:500; clone A 4502, DAKO, Carpinteria, Denmark),actin (dilution 1:100; clone HHF35, DAKO), desmin (dilution1:50; Zymed Laboratories, San Francisco, California, USA), S100protein (dilution 1:1000; DAKO) and endothelial cell markerCD34 (dilution 1:40; clone QBEnd/10, NovoCastra Laboratories,Newcastle-upon-Tyne, UK). Briefly, deparaffinised and rehy-drated slides were subjected to 10 min incubation in 3% hydrogenperoxide in methanol, in order to inhibit endogenous peroxidase.No antigen retrieval was used. After incubation with primary
antibody at room temperature for 30 min, the secondarybiotinylated goat anti-polyvalent antibody was applied for10 min, followed by incubation with streptavidin–peroxidasecomplex. The immune reaction was visualised by DAB as achromogen (Ultravision Detection System Anti-polyvalent, HRP/DAB; Lab Vision, Fremont, California, USA). Any (strong/weak,focal, moderate or diffuse) membrane (CD117) and/or cytoplasm(CD117, actin, desmin, and CD34), and nuclear (S100 protein)immunoreactivity of the cells was considered as positive staining.Appropriated positive and negative controls were included in eachrun: interstitial cells of Cajal in a section of normal intestine wereused as positive control for CD117, smooth layers for actin anddesmin, small nerves for S100 protein, and vessels for CD34. Fornegative controls, primary antibodies were omitted. Mast cells,smooth layers, small nerves, and vessels were used as internalpositive controls in the cases tested. All sections were counter-stained with haematoxylin.
DNA isolationSelected areas containing at least 85% of tumour tissue weremacrodissected into a microfuge tube using a sterile needle(Neolus, 25 G, 0.5 mm). DNA isolation was performed asdescribed previously.19 Briefly, the dissected tissue was depar-affinised by a serial extraction with xylol and ethanol (100%–70%–50%) and allowed to air-dry. DNA was extracted usingQiagen’s QIAamp DNA Micro Kit (Qiagen, Hilden, Germany),following the manufacturer’s instructions. DNA samples werestored at 220uC for further analysis.
KIT mutation analysisKIT mutation analysis was performed as described pre-viously.19 20 DNA was subjected to PCR amplifications followedby direct sequencing for exon 11, and pre-screening by singlestrand conformational polymorphism (SSCP) analysis for exons9, 13, 14 and 17, followed by direct sequencing of SSCP positivecases. Briefly, the PCR reaction was carried in a final volume of
Figure 1 Morphological andimmunohistochemical features ofgastrointestinal stromal tumours (GISTs).(A) spindle cell and (B) epithelioid tumourcells with (C) membranar/cytoplasmicand (D) cytoplasmic/paranuclear dotimmunoreactivity for CD117. Noteimmunoreactivity of interstitial cells ofCajal (C, inset). H&E and ABCimmunohistochemistry (2006).
25 ml, under the following conditions: 16 buffer (Bioron,Ludwigshafen, Germany); 1.5 mM MgCl2 (Bioron); 200 mMdNTPs (Fermentas, Hanover, Maryland, USA); 0.5 mM primers(previously described by Corless et al,21 except for exon 14: 59-TCTCAC CTT CTT TCT AAC CTT TTC TT-39 (forward); 59-CCCATG AAC TGC CTG TCA AC-39 (reverse); MWG-Biotech,Ebersberg, Germany); and 1 unit of Super Hot Taq Polymerase(Bioron, Germany). SSCP analysis of exons 9, 13, 14 and 17 wasperformed in a 16 MDE gel (MDE: mutation detectionenhancement, Cambrex, Charles City, Iowa, USA), with 6%glycerol addition in the exon 13 analysis, and 3% glycerol additionin exon 14 analysis. PCR product (20 ml) was incubated at 95uCfor 10 min with an equal volume of formamide loading buffer(98% formamide, 10 mM EDTA, and 1 mg/ml bromophenol blueand xilene cyanol). SSCP gels were run at 20uC. Samples with aSSCP pattern different from the normal pattern were directlysequenced. All cases were confirmed twice with a new PCRamplification, SSCP and direct sequencing analysis.
PDGFRA mutation analysisTumours bearing a wild-type KIT gene were further screened forhotspot PDGFRA mutations (exons 12, 14 and 18) as previouslydescribed.19 20 Briefly, the PCR reaction was carried out in a finalvolume of 25 ml, under the following conditions: 16 buffer(Bioron); 1.5 mM MgCl2 (Bioron); 200 mM dNTPs (Fermentas);0.5 mM primers (previously described by Heinrich et al13; MWG-Biotech) and 1 unit of Super Hot Taq Polymerase (Bioron). PCRwas followed by direct sequencing. All cases were confirmed twicewith a new PCR amplification and direct sequencing analysis.
Statistical analysisThe available clinical and molecular data were analysed withStatView for Windows, V.5.0. Overall survival time analysis using
Kaplan–Meyer and log rank tests was performed with SPSS forWindows, V.14.0. Probability values ,0.05 were consideredsignificant.
RESULTS
ImmunohistochemistryStrong membrane and/or cytoplasm tumour cells immunoreactiv-ity for CD117 was found in variably focal, moderate or diffuseareas in 72 (92%) GIST cases (fig 1). In three cases, CD117immunoreactivity was weak. Six GIST cases (8%) did not showCD117 immunoreactive tumour cells. Interstitial cells of Cajal andmast cells, used as internal positive controls, were always variablyobserved in each case. Table 1 summarises statistical analysis ofCD117 immunostaining and clinical–pathological features.CD117 immunoreactivity was significantly associated(p = 0.015) with spindle cell and epithelioid GIST subtypes. Thefrequency and expression features of the other antibodies wasvariable from case to case, and within the same tumour, as follows:actin (51%), desmin (6%), S100 protein (18%), and CD34 (73%);immunoexpression was observed in focal areas/rare tumour cellsfor actin, desmin, and S100 protein, whereas CD34 immunoex-pression was found in moderate or diffuse areas (data not shown).Four of the six CD117 negative GISTs expressed CD34 withoutany tumour cell expression for the other markers tested.
KIT mutation analysisMutation screening analysis revealed that 44 of 78 GISTs (56%)presented KIT activating mutations (table 2). Forty casesshowed mutation in exon 11 (91%, 40/44) and four cases inexon 9 (9%, 4/44). Among the exon 11 mutations, we observed3–54 bp in-frame deletions in 24 tumours (60%, 24/40), eitheralone (62%, 15/24) or associated with missense mutations orinsertions (38%, 9/24), single base substitutions in 15 tumours(38%, 15/40) and an in-frame insertion associated with a pointmutation in 1 tumour (2%, 1/40). Additionally, a silentmutation (Y570Y) was detected in two GISTs. The exon 9sequence alterations consisted of Ala–Tyr duplication betweencodons 502 and 503 in three GIST cases, and a point mutation(G470R) in one case. One silent mutation was detected in bothexons 13 and 17 (P627P and S865S, respectively). Also, a 4 bpdeletion was detected affecting the intronic sequence followingexon 14 (IVS14+24:del4). In addition, to exclude the possibilityof false-negatives in the SSCP screening at exons 9, 13, 14 and17, 10 KIT wild-type GISTs were direct sequenced for all exons.No additional mutations were identified.
Table 3 shows statistical analysis of KIT mutations andclinical–pathological features. No correlation was detectedbetween KIT mutation status and CD117 expression(p = 0.39). However, all but two GISTs harbouring KITmutation were positive for CD117 expression. Additionally,the three GISTs with weak CD117 immunoreactivity depictedwild-type KIT. A statistically significant correlation wasobtained between the epithelioid morphology and lack of KITmutation (p = 0.0064). The presence of mutated KIT wassignificantly associated with shorter survival of patients(p = 0.0460) (fig 2). No correlation was obtained between anyspecific type of KIT mutation (point mutation, deletion, ormixed mutation), or its location (exon 9 or exon 11), andpatient survival (data not shown).
PDGFRA mutation analysisIn KIT wild-type GISTs, PDGFRA activating mutations wereidentified in five cases; two in exon 12, and three in exon 18
Table 1 Correlation analysis of CD117 with clinical and pathologicalfeatures of gastrointestinal stromal tumours (n = 63)
(table 4). The mutations in exon 12 consisted of a pointmutation (D583G) and an in-frame deletion (583del586). Twocases disclosed a point mutation (D842V) in exon 18; there wasanother case with a point mutation (I843T) together with an in-frame deletion (844del847). In addition, we identified two silentmutations, one in exon 12 (D577D) and another in exon 18(I834I). No mutation was observed in exon 14 of PDGFRA.Furthermore, mutational analysis of exons 12 and 18 showedthe presence of a known homozygous substitution A.G(polymorphism R) in the third position of the codon for proline567 in exon 12, and an insertion in intron 18 (IVS18-50insA).
All GISTs with PDGFRA mutations showed CD117 immu-noreactive tumour cells.
DISCUSSIONThe intensive cancer research in the last decade has highlightedthe fundamental role of RTKs, in particular of KIT andPDGFRA in GIST pathogenesis.10 These two RTKs are of greatvalue for therapeutic management as a result of the develop-ment of RTK inhibitors, such as imatinib and sunitinib.22–24
There is, however, insufficient epidemiological data on thefrequency and type of mutations in the KIT and PDGFRA genesin GISTs from countries in southern Europe countries, such asPortugal.
In this study, we have shown that 92% of GISTs expressCD117, irrespective of the topography, age or gender, inaccordance with previous studies in other populations.25 26 Nostatistically significant correlation was depicted between CD117expression and presence of KIT mutations (p = 0.3933). In fact,two of the six CD117-negative GISTs contained a KIT mutation(a missense mutation in exon 9, and a three base-pair deletion inexon 11). Other authors have also encountered KIT mutationsin CD117-negative GIST cases.27 Our molecular study wasuseful for the definitive diagnosis of GIST in 2/6 CD117negative cases. The frequency of the CD117-negative wild-typecases for KIT and PDGFRA mutations found in our series (5%),fits with results described in the literature.27
We showed the presence of KIT mutations in 56% of GISTcases, 91% (40/44) being located in exon 11. These frequenciesare in accordance with previously published ranges for otherpopulations (30–80%), particularly those of the IberianPeninsula.1 10 28 In 75% (30/40) of these cases, mutations were
clustered in the region between codons 550 and 561, known tobe the most frequently altered section of exon 11, with 57% (17/30) affecting codon 557 or 558. These two codons are reportedto be associated with the metastatic behaviour of GISTs.10 29
However, of these 17 GIST cases, only four recurred (4/17,24%). Even though it has been previously reported that all pointmutations occur exclusively in codons 557, 559, 560 and 576, wehave additionally encountered a novel point mutation in codon570 (Y570F).1 Mutations in KIT exon 9 have been correlatedwith a small intestinal topography, but only one of our fourGIST cases harbouring a mutation in this exon was located inthe small intestine.1 10 In the present study, and in agreementwith previous reports, KIT mutation positive status was shownto be associated with worse GIST prognosis, translated intoshorter patient survival.10
Figure 2 Kaplan–Meier curve for the 63 patients with gastrointestinalstromal tumours, regarding KIT alterations. Patients having a wild-typeKIT (n = 27) have a better prognosis than patients having tumoursharbouring mutated KIT (n = 36) (p = 0.046).
Table 3 Correlation of KIT mutations with clinical-pathological featuresof gastrointestinal stromal tumours (n = 63)
Concerning PDGFRA, mutations were detected in 6% (5/78)of our cases, corresponding to 15% of KIT wild-type GISTs.Two of these mutations are known to be imatinib-resistant(D842V).22 An association between gastric location and presenceof PDGFRA mutation has been reported.30 In our series,although the number of cases with mutations in PDGFRA islow for statistical evaluation (n = 5), 80% (4/5) of mutationsoccurred in the stomach.
It is now well established that the response of GIST patientsto imatinib-based therapy is dependent not only on thepresence, but also on the type of KIT and PDGFRA mutationexhibited.10 16 22 Specifically, mutations affecting the juxtamem-brane domain (exon 11, partial response in up to 84% of cases)or the extracellular domain (exon 9, partial response in up to48%) predict objective response to imatinib.10 16 22 On the otherhand, it is also known that some mutations are responsible forimatinib resistance, namely in KIT V654A and W670I (exon 13),D816V and T823D (exon 17), and PDGFRA D842V (exon18).16 31 32 Of these resistant mutations, only D842V mutationwas detected in two GISTs in our series. Recently, the US Foodand Drug Administration approved a new RTK inhibitor,sunitinib (Sutent, Pfizer, New York, USA) as a second-linetherapy for GIST patients who experience disease progression inspite of increased doses of imatinib, mainly due to primary oracquired secondary imatinib-resistant mutations, or who areunable to tolerate treatment with imatinib.24 33 Therefore, withthese two RTK inhibitors available, there is an imperative needto redefine GIST pathological (diagnosis/prognostic) evaluation,as well as to consider molecular characterisation of both KITand PDGFRA, in order to achieve an efficient and predictivetailored therapeutic management for each individual patient.
In conclusion, we have reported for the first time thefrequency of KIT and PDGFRA mutations in a large series ofPortuguese GIST patients. We have shown the presence of KITmutations in 56% of cases and PDGFRA mutations in 6% ofcases. In addition, the presence of mutated KIT was associatedwith a shorter patient survival. The great majority of KITactivating mutations (91%) were located in exon 11, indicativeof a favourable response to imatinib-based therapy in themanagement of these patients. Finally, our results might beuseful to integrate a multi-institutional consortium database forthe clarification of the epidemiology, biology and managementof GIST patients.
Funding: Supported by NOVARTIS Oncology, Portugal. ALG is the recipient offellowship grant (SFRH/BI/15257/2004) from FCT, Lisbon, Portugal.
Competing interests: None declared.
REFERENCES1. Rubin BP. Gastrointestinal stromal tumours: an update. Histopathology 2006;48:83–
3. Miettinen M, Virolainen M, Maarit-Sarlomo-Rikala. Gastrointestinal stromaltumors—value of CD34 antigen in their identification and separation from trueleiomyomas and schwannomas. Am J Surg Pathol 1995;19:207–16.
4. Hirota S. Gastrointestinal stromal tumors: their origin and cause. Int J Clin Oncol2001;6:1–5.
5. Miettinen M. Blay JY, Sobin LH. Mesenchymal tumours of the stomach. In:Hamilton SR and Aaltonen LA, eds. Pathology and genetics of tumours of the digestivesystem. Lyon: IARC Press, 2000:62–5.
6. Kitamura Y, Hirotab S. Kit as a human oncogenic tyrosine kinase. Cell Mol Life Sci2004;61:2924–31.
7. Roskoski R. Structure and regulation of Kit protein-tyrosine kinase—the stem cellfactor receptor. Biochem Biophys Res Commun 2005;338:1307–15.
8. Ronnstrand L. Signal transduction via the stem cell factor receptor/c-Kit. Cell MolLife Sci 2004;61:2535–48.
9. Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-function mutations of c-kit in humangastrointestinal stromal tumors. Science 1998;279:577–80.
10. Miettinen M, Lasota J. Gastrointestinal stromal tumors: review on morphology,molecular pathology, prognosis, and differential diagnosis. Arch Pathol Lab Med2006;130:1466–78.
11. Tamborini E, Bonadiman L, Greco A, et al. A new mutation in the KIT ATP pocketcauses acquired resistance to imatinib in a gastrointestinal stromal tumor patient.Gastroenterology 2004;127:294–9.
12. Tornillo L, Terracciano LM. An update on molecular genetics of gastrointestinalstromal tumours. J Clin Pathol 2006;59:557–63.
13. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations ingastrointestinal stromal tumors. Science 2003;299:708–10.
14. Corless CL, Schroeder A, Griffith D, et al. PDGFRA mutations in gastrointestinalstromal tumors: frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol2005;23:5357–64.
15. Baker DE. Imatinib mesylate. Rev Gastroenterol Disord 2002;2:75–86.16. Debiec-Rychter M, Dumez H, Judson I, et al. Use of c-KIT/PDGFRA mutational
analysis to predict the clinical response to imatinib in patients with advancedgastrointestinal stromal tumours entered on phase I and II studies of the EORTC SoftTissue and Bone Sarcoma Group. Eur J Cancer 2004;40:689–95.
17. Fletcher CD, Berman JJ, Corless C, et al. Diagnosis of gastrointestinal stromaltumors: a consensus approach. Int J Surg Pathol 2002;10:81–9.
18. Blay JY, Bonvalot S, Casali P, et al. Consensus meeting for the management ofgastrointestinal stromal tumors. Report of the GIST Consensus Conference of 20–21March 2004, under the auspices of ESMO. Ann Oncol 2005;16:566–78.
19. Gomes AL, Bardales RH, Milanezi F, et al. Molecular analysis of c-Kit and PDGFRA inGISTs diagnosed by EUS. Am J Clin Pathol 2007;127:1–8.
20. Reis RM, Martins A, Ribeiro SA, et al. Molecular characterization of PDGFR-alpha/PDGF-A and c-KIT/SCF in gliosarcomas. Cell Oncol 2005;27:319–26.
21. Corless CL, McGreevey L, Haley A, et al. KIT mutations are common in incidentalgastrointestinal stromal tumors one centimeter or less in size. Am J Pathol2002;160:1567–72.
22. Heinrich MC, Corless CL, Demetri GD, et al. Kinase mutations and imatinib responsein patients with metastatic gastrointestinal stromal tumor. J Clin Oncol2003;21:4342–9.
23. Zalcberg JR, Verweij J, Casali PG, et al. Outcome of patients with advanced gastro-intestinal stromal tumours crossing over to a daily imatinib dose of 800 mg afterprogression on 400 mg. Eur J Cancer 2005;41:1751–7.
24. Joensuu H. Sunitinib for imatinib-resistant GIST. Lancet 2006;368:1303–4.25. Miettinen M, Sobin LH, Lasota J. Gastrointestinal stromal tumors of the stomach: a
clinicopathologic, immunohistochemical, and molecular genetic study of 1765 caseswith long-term follow-up. Am J Surg Pathol 2005;29:52–68.
26. Feng F, Liu XH, Xie Q, et al. Expression and mutation of c-kit gene in gastrointestinalstromal tumors. World J Gastroenterol 2003;9:2548–51.
27. Medeiros F, Corless CL, Duensing A, et al. KIT-negative gastrointestinal stromaltumors: proof of concept and therapeutic implications. Am J Surg Pathol2004;28:889–94.
28. Martin J, Poveda A, Llombart-Bosch A, et al. Deletions affecting codons 557–558 ofthe c-KIT gene indicate a poor prognosis in patients with completely resectedgastrointestinal stromal tumors: a study by the Spanish Group for Sarcoma Research(GEIS). J Clin Oncol 2005;23:6190–8.
29. Wardelmann E, Losen I, Hans V, et al. Deletion of Trp-557 and Lys-558 in thejuxtamembrane domain of the c-kit protooncogene is associated with metastaticbehavior of gastrointestinal stromal tumors. Int J Cancer 2003;106:887–95.
30. Penzel R, Aulmann S, Moock M, et al. The location of KIT and PDGFRA genemutations in gastrointestinal stromal tumours is site and phenotype associated. J ClinPathol 2005;58:634–9.
31. Frost MJ, Ferrao PT, Hughes TP, et al. Juxtamembrane mutant V560GKit is moresensitive to imatinib (STI571) compared with wild-type c-kit whereas the kinasedomain mutant D816VKit is resistant. Mol Cancer Ther 2002;1:1115–24.
32. Wardelmann E, Merkelbach-Bruse S, Pauls K, et al. Polyclonal evolution of multiplesecondary KIT mutations in gastrointestinal stromal tumors under treatment withimatinib mesylate. Clin Cancer Res 2006;12:1743–9.
33. Prenen H, Cools J, Mentens N, et al. Efficacy of the kinase inhibitor SU11248against gastrointestinal stromal tumor mutants refractory to imatinib mesylate. ClinCancer Res 2006;12:2622–7.
Take-home messages
c The frequency of KIT and PDGFRA activating mutations hasbeen described in a series of Portuguese gastrointestinalstromal tumours (GISTs).
c Of the Portuguese patients with GISTs, 56% harboured KITmutations and 6% exhibited PDGFRA mutations.
c The presence of KIT mutations in GISTs was associated with aworse patient prognosis; however, these mutations areindicative of favourable response to imatinib-based therapy.
2008 61: 203-208 originally published online September 7,J Clin Pathol A L Gomes, A Gouveia, A F Capelinha, et al. GISTs: evaluation of a Portuguese series
inPDGFRA and KITMolecular alterations of
http://jcp.bmj.com/content/61/2/203.full.htmlUpdated information and services can be found at:
Low frequency of MAP kinase pathway alterationsin KIT and PDGFRA wild-type GISTs
Olga Martinho,1 Antonio Gouveia,2,3,4 Marta Viana-Pereira,1 Paula Silva,2,4
Amadeu Pimenta,2,3,4 Rui Manuel Reis1 & Jose Manuel Lopes2,4,5
1Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga,2IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, 3Department of Surgery,
H.S. Joao, 4Medical Faculty of Porto University, and 5Department of Pathology, H.S. Joao, Porto, Portugal
Date of submission 10 July 2008Accepted for publication 16 December 2008
Martinho O, Gouveia A, Viana-Pereira M, Silva P, Pimenta A, Reis R M & Lopes J M
(2009) Histopathology 55, 53–62
Low frequency of MAP kinase pathway alterations in KIT and PDGFRA wild-type GISTs
Aims: Gastrointestinal stromal tumours (GISTs) arecommonly driven by oncogenic mutations in KIT andPDGFRA. However, 10–40% of these patients are wild-type for these genes. The prognostic significance ofwild-type GISTs is controversial, and they rarelyrespond to imatinib. The aim of this study was toelucidate the molecular lesions underlying wild-typeGISTs tumorigenesis.Methods and results: Twenty-nine KIT and PDGFRAwild-type GISTs were re-assessed for the presence of‘cryptic’ KIT exon 11 duplications. Using a specificpolymerase chain reaction assay, three previouslyundetected mutations were identified. In the remaining26 wild-type GISTs, KIT, stem cell factor (SCF),phospho-KIT and phospho-ERK expression was evalu-
ated by immunohistochemistry. Samples were screenedfor gain-of-function mutations in the mitogen-activatedprotein kinase (MAPK) cascade. KIT and SCFco-expression associated with KIT activation wasobserved in approximately 30% of cases. Furthermore,phospho-ERK expression showed that MAPK is acti-vated in approximately 30% of cases. None of RASfamily (H-, K- and N-RAS) oncogenes exhibited acti-vating mutations, whereas BRAF mutations werefound in approximately 4% of cases.Conclusions: In the absence of RAS mutations, MAPKcould be activated through SCF ⁄ KIT autocrine ⁄ para-crine mechanisms and ⁄ or mutated BRAF in a subset ofKIT ⁄ PDGFRA wild-type GISTs.
Gastrointestinal stromal tumours (GISTs) are the mostcommon primary mesenchymal tumours of the diges-tive tract.1,2 According to the World Health Organiza-tion (WHO) classification, GIST diagnosis should be
reserved for KIT (CD117)-positive mesenchymaltumours of the gastrointestinal tract. However, itis currently accepted that a small fraction of GISTs(5–10%) are CD117 negative.3–7 KIT is an oncoproteinthat belongs to the class III receptor tyrosine kinase(RTK) subfamily, being the stem cell factor (SCF) theligand, also known as KIT ligand, steel factor or mastcell growth factor.8–10 Upon binding of SCF to KIThomodimers, it induces dimerization and autophos-phorylation, which leads to activation of several
Address for correspondence: J M Lopes, Rua Dr Roberto Frias,
intracellular signalling cascades such as mitogen-acti-vated protein kinase (MAPK) (RAS ⁄ RAF ⁄ MEK ⁄ ERK),PI3K ⁄ AKT and STAT, regulating cellular proliferationand survival of cells.9,10
In GISTs, KIT overexpression is mainly driven bygain-of-function mutations of KIT gene, with a fre-quency that varies between studies from 30% to80%.11,12 The great majority of KIT mutations occurat exon 11, followed by exon 9 and rarely at exons 13,14 and 17.11,12 In addition, KIT wild-type GISTs canexhibit activating mutations of other class III RTKgenes, the platelet-derived growth factor receptor a(PDGFRA), which affects exon 18 more often, andrarely exons 12 and 14.12–14 It has been shown thatGIST patients harbouring KIT and PDGFRA activatingmutations are effectively treated with specific tyrosinekinase inhibitors such as Imatinib mesilate (Glivec�;Gleevec�).15,16 KIT and PDGFRA mutation frequencyvariation in GISTs may depend to some extent ontumour location, methodology and type of tissue used(frozen or formalin-fixed, paraffin-embedded).11,12,14,17
Recently, Lasota et al. have suggested that KIT exon11 duplications may be undetected in formalin-fixedparaffin-embedded samples, due to the preferentialpolymerase chain reaction (PCR) amplification of thewild-type KIT allele over the large fragment mutantallele.18 In fact, using a PCR assay specific for the 30
region affected by duplications at exon 11, the authorsshowed the presence of these types of mutation in 4 ⁄ 16previously considered wild-type KIT formalin-fixedparaffin-embedded GISTs.18
Contrasting with the extensive knowledge of KIT andPDGFRA activation by gene mutations, few studieshave investigated the disruption of KIT signallingindependently of these genetic alterations.19–22 TheMAPK (RAS ⁄ RAF ⁄ MEK ⁄ ERK) pathway, one of theKIT downstream cascades, is highly preserved andimplicated in cell growth, differentiation, migration andsurvival.23 Activated RTK phosphorylates RAS, whichbinds RAF-1 kinase that phosphorylates MEK1 ⁄ 2,leading to ERK1 ⁄ 2 activation. Phosphorylated ERKtranslocates into nucleus and regulates gene expressionthrough several transcription factors such as c-Myc,CREB and AP-1, ultimately leading to changes in geneexpression.23 In several types of solid tumours, such aspancreatic, colonic, papillary thyroid and melanomas,this signalling pathway can be constitutively up-regulated by activating mutations of RAS family genes(N-RAS, K-RAS and H-RAS), or by downstream RAFfamily member gene BRAF.9,23–26 Importantly, con-stitutive activation of this RAS ⁄ RAF ⁄ ERK cascadecould modulate patient response to anti-RTK therapies,independently of the upstream RTK status.
Recently, we assessed KIT and PDGFRA mutationstatus in a formalin-fixed paraffin-embedded series ofGISTs.27 In the present study we aimed to clarify thepoorly characterized molecular picture of KIT andPDGFRA wild-type GISTs. Initially, the presence of‘cryptic’ KIT exon 11 duplication mutations wasre-evaluated. Then, the activation status of KIT andthe pattern of KIT ⁄ SCF autocrine ⁄ paracrine stimula-tion loops were assessed. We also evaluated alterationsof the MAPK signalling pathway by the analysis of RASfamily and BRAF mutations and the expression levelsof phosphorylated ERK. Doing so, we aimed to identifynew potential prognostic and therapeutic markers forKIT and PDGFRA wild-type GISTs.
Materials and methods
tissue samples
Twenty-nine formalin-fixed paraffin-embedded primarysporadic GISTs, previously characterized immuno-histochemically for CD117, actin, S100, desminand CD34, and molecularly for KIT and PDGFRAmutations, were retrieved from the Pathology Depart-ment of S. Joao Hospital files (1989–2005), Porto,Portugal.27 All patients were Caucasian of Portugueseorigin. Tumours were classified according to WHO,and the parameters analysed in each case included:age, gender, primary tumour site, tumour size,histological type, mitotic index and risk group.3,28
Follow-up data were available in all patients, as ofDecember 2007, and collected through direct inter-view with patients or their relatives, and by review ofin-hospital patient files.
dna isolation
Selected areas containing ‡85% of tumour tissue weremacrodissected into a microfuge tube using a sterileneedle (Neolus, 25 G, 0.5 mm; Leuven, Belgium) andDNA isolation was performed using Qiagen’s QIAamp�
DNA Micro kit (Qiagen, Hilden, Germany), as previ-ously described.27
mutation analysis of spec if ic 30
region of kit
exon 1 1 , h-ras, k-ras, n-ras and braf
Polymerase chain reaction-single-strandconformation polymorphismPre-screening of the hotspot regions affecting KIT (30
region of exon 11), H-RAS, K-RAS, N-RAS [exons 1(codons 12–13) and 2 (codon 61)] and BRAF (exons11 and 15) genes was performed by PCR–single-strand
conformation polymorphism (SSCP), followed by directDNA sequencing of samples that showed a mobilityshift in the PCR-SSCP analysis.
Briefly, the PCR was carried out in a total volume of25 ll, consisting of 1 ll of DNA solution, 0.3 lm ofboth sense and antisense primers (MWG-Biotech,Ebersberg, Germany), 200 lm of dNTPs (Fermentas,Glen Burnie, MD, USA), 1.5 mm of MgCl2 (Bioron,Ludwigshafen, Germany), 1· Taq Buffer incomplete(Bioron) and 1 U of Taq Superhot DNA Polymerase(Bioron). The reaction consisted of an initial denatur-ation at 96�C for 10 min, followed by 40 cycles ofdenaturation at 96�C for 45 s, annealing at 55–60�Cfor 45 s and extension at 72�C for 45 s, followed by afinal extension for 10 min at 72�C, in a Thermocycler(Bio-Rad, Hercules, CA, USA). Primer sequences for allgenes were as previously described.18,29,30
PCR products were mixed with an equivalent volumeof denaturing loading buffer [98% formamide, 10 mm
ethylenediamine tetraaceticacid (EDTA), 1 mg ⁄ mlbromophenol blue and xylene cyanol]. After denatur-ing at 98�C for 10 min and quenching on ice, 20 ll ofthe mixture was loaded onto a 1· Mutation DetectionEnhancement (MDE) gel (Cambrex, East Rutherford,NJ, USA) and 6% of glycerol for KIT, without glycerolfor K-RAS and with 3% glycerol for H-RAS and N-RASgenes and on a 0.8· MDE gel without glycerol forBRAF. The run was performed at 20�C for 16 h.Samples with a SSCP pattern different from the normalwere further directly sequenced (Stabvida, Oeiras,Portugal), as described.27 All cases were confirmedtwice with a new and independent PCR amplificationfollowed by direct sequencing.
immunohistochemistry analysis
of phospho-kit, scf and phospho-erk
Representative 3-lm-thick sections were subjected toimmunohistochemical analysis according to the strep-tavidin–biotin peroxidase complex system (UltraVisionLarge Volume Detection System Anti-Polyvalent,Horseradish Peroxidase; Lab Vision Corporation, Fre-mont, CA, USA). Immunohistochemistry procedureswere carried out as already published by us and otherauthors,31–35 with some modifications. Briefly, depa-raffinized and rehydrated slides were submitted to heat-induced antigen retrieval for 20 min at 98�C with10 mm citrate buffer (pH 6.0) for phospho-KIT andphospho-ERK, and with 1 mm EDTA buffer (pH 7.8) forSCF. After incubation with the primary antibodiesraised against phospho-kit Tyr703 (dilution 1:30;incubation ON at 4�C; clone ZMD.243; Zymed Labora-tories, San Francisco, CA, USA), SCF (dilution 1:200;
incubation ON at 4�C; clone G-3; Santa Cruz Biotech-nology, Santa Cruz, CA, USA) and phospho-p44 ⁄ 42MAPK Thr202 ⁄ Tyr204 (dilution 1:100; incubationON at 4�C; clone 20G11; Cell Signalling Technology,Beverly, MA, USA), the secondary biotinylated goatanti-polyvalent antibody was applied for 10 min fol-lowed by incubation with the streptavidin–peroxidasecomplex. The immune reaction was visualized by 3,30-diamonobenzidine as a chromogen. All sections werecounterstained with Gill-2 haematoxylin. Appropriatepositive controls were included in each run: humancolonic tissue showing extracellular staining of glan-dular cells was used for SCF, GIST with KIT genemutation for phospho-KIT, and a cell block of a breastcarcinoma cell line (MDA-MB-435) with BRAF muta-tion (V600E) was used for phospho-ERK. For negativecontrols, primary antibodies were omitted and alsoreplaced with a universal negative control antibody(carcinoembryonic antigen, rabbit antihuman; DakoCorp., Carpinteria, CA, USA). Furhtermore, CD117)leiomyoma cases36 were used as negative controls forphospho-KIT expression, as described.31
Tumour samples were evaluated for both extent andintensity of immunoreactivity. The extent of immuno-reactivity was evaluated on a scale of 0–3 (0, absence ofpositive cells; 1, <25% positive cells; 2, 26–50% positivecells; and 3, >50% positive cells) and the intensity alsoon a scale of 0–3 (0, negative; 1, weak; 2, moderate; 3,strong). The final score used was the sum of both extentand intensity scores, with values between 0 and 2 beingclassified as negative, 3 and 4 as moderately positive,and 5 and 6 as strongly positive.
statistical analysis
The available clinicopathological and immunohisto-chemical data were analysed with SPSS software forWindows, version 14.0 (SPSS Inc., Chicago, IL, USA).Correlations between categorical variables were per-formed using Fisher0s exact test. Disease-specific survival(DSS) was calculated from the time of diagnosis untildeath related to the disease, or censored at the time oflatest follow-up. Cumulative survival probabilities werecalculated using the Kaplan–Meier method. Differencesbetween survival rates were tested with the log rank test.A P-value < 0.05 was considered to be significant.
Results
re-evaluation of kit mutations
Twenty-nine of 78 previously described27 wild-type KITand PDGFRA GISTs were screened for KIT mutations
using a PCR assay designed to amplify a region in the30 part of KIT exon 11 commonly affected by duplica-tions.18 The PCR-SSCP followed by direct sequencinganalysis allowed identification of three novel casesharbouring KIT exon 11 duplications: case G6(579_583dup), case G36 (578_591dup) and caseG58 (573_580dup), which ranged from 14 to 42nucleotides (Figure 1A). Consequently, the frequencyof wild-type KIT and PDGFRA GISTs in our series was33.3% (26 ⁄ 78).
h-ras, k-ras, n-ras and braf mutations
In the remaining 26 KIT and PDGFRA wild-typeGISTs, we performed analysis of the hotspot regions ofRAS family and BRAF genes. The study of codon12 ⁄ 13 of H-RAS gene did not reveal any mutation(Table 1). We identified only a T fi C substitution atcodon 27 in two cases, which originates a silentmutation (H27H). The analysis of the other hotspotregion, codon 61, did not show any mutation(Table 1). No genetic alterations were identified inthe hotspot regions of the other RAS family genes,K-RAS and N-RAS (Table 1).
The mutation screening of BRAF exons 11 and 15showed the presence of the hotspot V600E mutation inone of 26 cases (3.8%) (Table 1 and Figure 1B). Inorder to determine whether BRAF V600E mutationwas also present in GISTs exhibiting KIT or PDGFRAmutations, we extended the analysis of BRAF exon 15
to the remaining cases of the series. No additionalmutation was identified in the 52 KIT or PDGFRAmutated GISTs. Overall, the frequency of BRAF muta-tion in our GIST series was 1.3% (1 ⁄ 78). The V600EBRAF mutated case (G9) was a bona fide intermediate-risk spindle cell GIST from the small bowel of a 71-yearold man (Figure 2).
express ion of kit ( cd 1 1 7 ) , phosphorylated-kit
and scf
Table 1 summarizes CD117, phospho-KIT and SCFexpression. CD117 immunoreactivity was observed in23 (88.5%) of 26 wild-type KIT and PDGFRA GISTs.27
In order to evaluate whether KIT was activated inthese tumours, we performed immunohistochemicalanalysis with an antibody to KIT phosphorylatedresidues Tyr703. Strong cytoplasmic immunoreactivity(Figure 3A) was observed in two, and moderatepositivity in three GISTs. To determine whether thisKIT activation was associated with an autocrine ⁄ para-crine stimulation loop, we evaluated the expression ofSCF. Positive cytoplasmic SCF expression was observedin 20 (76.9%) of the 26 cases, 10 depicting moderateand 10 strong positivity (Figure 3B). Co-expression ofSCF ⁄ CD117 was present in 65.4% of the cases. Allphospho-KIT positive GISTs showed co-expression ofSCF ⁄ CD117.
express ion of phosphorylated erk
In order to study MAPK pathway activation, weevaluated the expression of ERK1 ⁄ 2 using an antibodyfor the p44 ⁄ 42 MAPK phosphorylated at residuesThr202 ⁄ Tyr204 (ERK1 and ERK2). We found moder-ate phospho-ERK expression in eight cases (30.8%)(Figure 3C). Phospho-ERK stained both nuclear andcytoplasmic compartments. As expected, the caseharbouring BRAF activating mutation (case G9) alsoshowed phospho-ERK positivity. Furthermore, weobserved a significant correlation between phospho-KIT and phospho-ERK expression (P = 0.02). All casesexpressing both phospho-KIT and phospho-ERKshowed co-expression of SCF.
clinicopathological features
and statist ical analysis
Table 2 summarizes the clinicopathological parametersof the 26 re-evaluated KIT and PDGFRA wild-typeGISTs. The mean and median age of patients atdiagnosis was 59.9 and 62.5 years, respectively(range 20–82 years), and the male ⁄ female ratio was
A
A
B
A A A A A A A A A A ANT
T T T T T T T T T T TA A A A A A A AC C C CCG G G G G G G G G
T T T T TGC G G G GC C C C C C C
Figure 1. A, DNA sequencing of KIT exon 11 573_580dup
mutation in case G58; arrow indicates beginning of duplicated
DNA sequence. B, DNA sequencing of BRAF exon 15 V600E
mutation from case G9; arrow indicates the mutated base.
1.6. The 5-year cumulative DSS was 83.6%, with amedian follow-up time of 62.07 months (mean83.95 ± 61.10; range 1–204 months). All but twopatients were submitted to surgical resection of primaryGIST. None of the nine (34.6%) deceased patients, one
within the postoperative (1 month) period, had anautopsy. In four patients the cause of death was dueto the tumour disease. In the remaining patients, oneis with and 16 are without evidence of disease.Three patients are currently under imatinib treatment
Table 1. Immunohistochemistry and molecular analysis of wild-type gastrointestinal stromal tumours
without evidence of disease (n = 2) or with stabledisease (n = 1).
On statistical univariate analysis, we found that ashorter DSS was significantly associated with tumour
size >100 mm (P = 0.005) and high-risk GISTs(P = 0.027). Age, gender, primary site of tumour,histological type and mitotic index were not significantprognostic markers (Table 2). Regarding immuno-
A B
C
Figure 2. Pathological features of V600E BRAF mutated gastrointestinal stromal tumour (case G9), showing low power of intestinal submucosal
histochemical parameters, no significant correlationwas found between the analysed molecules and clinico-pathological features (Table 2).
Discussion
The majority of GISTs are driven by KIT and PDGFRAoncogenic mutations.7,15 Nevertheless, a subset of GISTpatients, varying from 10% to 40%, are wild-type forboth KIT and PDGFRA genes.4,7,15,27,37 The prognosticfactors of wild-type GISTs are still a matter of debate.Some studies have shown a better prognosis of wild-type GISTs when compared with mutated cases,12,27
although a recent population-based study has failed toconfirm these findings.38 In the present series, wefound that clinicopathological features commonlyassociated with overall GIST patients’ outcome, i.e.largest tumour size and high-risk grade, also constitutepoor prognostic factors in wild-type GISTs. It has beenshown that patients with wild-type GIST rarely respondto imatinib,39,40 and their response to sunitinib-basedtherapy is still unclear.7,15 Thus, these patients raiseimportant therapeutic challenges. Another unexploredand puzzling issue is the molecular lesions underlyingKIT and PDGFRA wild-type GIST tumours.
In the present study, we have investigated the role ofSCF ⁄ KIT autocrine or paracrine stimulation loops, andthe contribution of MAPK (RAS ⁄ RAF ⁄ MEK ⁄ ERK)cascade alterations in a subset of previously describedKIT ⁄ PDGFRA wild-type GISTs.27 In the light of newfindings of undetectable KIT exon 11 duplication typeof mutations in formalin-fixed paraffin-embedded tis-sues,18 we initially re-assessed all wild-type cases. Withthis approach, we were able to detect three new caseswith duplication-type of mutations in exon 11, leadingto 33% of wild-type GISTs in this Portuguese series,which fits with other series, namely from the IberianPeninsula.37
The co-expression of both ligand and receptor inapproximately 65% of cases and the expressionof phospho-KIT in approximately 30% of SCF ⁄ KITco-expressing cases support the possibility of auto-crine ⁄ paracrine mechanisms in wild-type GISTs. Ourfindings are in agreement with a recent report thatfound SCF ⁄ KIT co-expression in the majority of GISTs,independently of KIT and PDGFRA mutation status.35
KIT activation, assessed by Western blot in cell linesand frozen tissues, has previously been reported in 70–80% of wild-type GISTs with the same proportion andlevel found in KIT ⁄ PDGFRA mutated GISTs.20,35 Sim-ilar to our study, others have also observed thatKIT ⁄ SCF co-expression does not always lead to KITactivation.35 The observed discrepancy in the number
Table 2. Clinicopathological parameters and correlation withdisease-specific survival of wild-type gastrointestinal stromaltumours
Parameters (n = 26) N % P-value*
Age (years)£60 12 46.2 NS
>60 14 53.8
GenderMale 16 61.5 NS
Female 10 38.5
Tumour localStomach 16 61.5 NS
Small bowel 9 34.6
Other 1 3.8
Tumour size (mm)£50 7 26.9 0.005
>50 £ 100 13 50.0
>100 6 23.1
Histological typeSpindle 18 69.2 NS
Epithelioid 5 19.2
Mixed 3 11.5
Mitotic index (50 ⁄ HPF)£5 19 73.1 NS
>5 7 26.9
Risk gradeVL ⁄ L ⁄ I 16 61.5 0.027
H 10 38.5
p-KITPositive 5 19.2 NS
Negative 21 80.8
SCFPositive 20 76.9 NS
Negative 6 23.1
p-ERKPositive 8 30.8 NS
Negative 18 69.2
N, number of cases; NS, not significant values (P > 0,05);HPF, high-power field; VL, very low; L, low; I, intermediate;H, high risk; p-KIT, phosphorylated KIT; SCF, stem cell factor;p-ERK, phosphorylated ERK.
of cases with KIT activation may be explained by thedifferences in methodologies and tissue fixation of thetumours. In our study, the antibody used recognizesKIT phosphorylation at tyrosine 703, whereas in theaforementioned studies the antibody recognizes tyro-sine 721.20,35 Despite the well-known advantages ofimmunohistochemical evaluation on formalin-fixedparaffin-embedded tissues, this method is less consis-tent than Western blot analysis for the evaluation ofprotein phosphorylation status. Therefore, furtherstudies are warranted to determine the frequency ofKIT activation and, most importantly, the efficacy ofavailable treatments in wild-type GISTs.
Despite the high frequency of RAS family genemutation in several types of neoplasms such aspancreatic, papillary thyroid, colonic and non-smallcell lung cancer (NSCLC),23,41 none of the 26 analysedGISTs exhibited non-synonymous mutation of theH-RAS, K-RAS and N-RAS hotspot regions. The anal-ysis of BRAF, another important oncogene of theMAPK cascade pathway, showed the presence ofhotspot-activating V600E mutation in one wild-typecase (approximately 4%). The V600E mutationaccounts for 90% of BRAF mutations in humancancers and is associated with higher kinase activity,which stimulates ERK activity independently of RASstatus.42 In our series, no BRAF mutation was iden-tified in KIT or PDGFRA mutated GISTs, suggestingthat BRAF, KIT and PDGFRA mutations are mutuallyexclusive. So far, there are no reports on the analysis ofRAS and BRAF gene mutations in GISTs.
To elucidate further the role of MAPK pathway inwild-type GISTs, we evaluated phospho-ERK expres-sion, which was observed in 30% of our wild-typecases. Previous studies with GIST cell lines havesuggested that ERK activation is dependent on KITsignalling19 and that some KIT wild-type cell lines alsoexhibit ERK activation.21 Our results are in agreementwith these findings, since phospho-ERK expression wassignificantly (P = 0.02) correlated with phospho-KITexpression. However, phospho-ERK expression in 50%of our cases without phospho-KIT expression suggeststhat KIT-independent ERK activation is probably drivenby other pathways (e.g. AKT ⁄ PI3K and JAK ⁄ STAT) inwild-type GISTs.16,20
Currently, some drugs that target the RAS ⁄ RAF andMEK molecules are under clinical trials and others inpreclinical assays. Hitherto, no ERK1 or ERK2 inhib-itors have been reported.41 The most successful anti-RAF inhibitor is Sorafenib (Nexavar�). Sorafenib is apotent inhibitor of both wild-type and mutant (V600E)BRAF and has proven to target other RTKs also,including KIT, vascular endothelial growth factor
receptor (VEGFR)-2, VEGFR-3, PDGFRB, i.e. it is amulti-kinase inhibitor.43 The Food and Drug Adminis-tration has approved Sorafenib for advanced renal cellcarcinoma. Sorafenib is also being evaluated forNSCLC, prostatic, breast, pancreatic and melanomatumours.43,44 Several studies have shown that Sorafe-nib anti-tumour activity is associated with an anti-angiogenic effect.43,44 In contrast to Sorafenib, smallmolecule inhibitors of MEK1 ⁄ 2 are highly specificinhibitors. AZD6244 (ARRY-142886) preclinical stu-dies has been very promising, thus leading to furtherclinical trials.45,46 Interestingly, Solit and colleagueshave reported that BRAF mutant tumours are verysensitive to MEK inhibition.47
In conclusion, our study constitutes the most com-prehensive analysis of MAPK deregulation in KIT andPDGFRA wild-type GISTs. We have described theabsence of RAS mutations, and suggest that MAPKpathway can be activated through SCF ⁄ KIT auto-crine ⁄ paracrine mechanisms and ⁄ or mutated BRAF ina subset of wild-type GISTs. Despite the low frequencyof MAPK molecular alterations, additional studies arewarranted to evaluate the potential role of MAPKpathway targeting in wild-type (KIT and PDGFRA)GISTs.
Note: While the present paper was under revision, astudy from Agaram NP and colleagues, also reportedthe presence of the V600E hotspot BRAF mutation ina small fraction of KIT and PDGFRA wild-type GISTs.48
Acknowledgements
The authors thank Drª Joana Paredes (IPATIMUP,Porto, Portugal) for kindly providing the MDA-MB-435breast tumour cell line. O.M. is the recipient of a PhDfellowship (SFRH ⁄ BD ⁄ 36463 ⁄ 2007) from FCT, Por-tugal. This study was partially supported by NovartisOncology, Portugal.
Loss of RKIP expression is associated with poor survivalin GISTs
Olga Martinho & António Gouveia & Paula Silva &
Amadeu Pimenta & Rui Manuel Reis &
José Manuel Lopes
Received: 8 June 2009 /Revised: 17 July 2009 /Accepted: 30 July 2009 /Published online: 25 August 2009# Springer-Verlag 2009
Abstract Gastrointestinal stromal tumours (GISTs) are raremesenchymal tumours of the digestive tract and arecommonly driven by oncogenic mutations in KIT andPDGFRA genes. Tumour size, location, mitotic index andKIT/PDGFRA mutations are the most important prognosticparameters in GISTs. However, additional studies screeningfor new molecular prognostic markers in GISTs aremissing. Raf kinase inhibitor protein (RKIP) has beenconsidered as a suppressor of metastasis and a prognosticmarker in several neoplasms. In the present study we aimedto examine whether RKIP expression is associated withGIST clinical–pathological features. Using immunohisto-
chemistry, we determined RKIP expression levels in a well-characterised series of 70 GISTs. We found that RKIP isexpressed in the great majority of cases, and absent inapproximately 9% of GISTs. Additionally, we found thatloss of RKIP expression was not due to the promotermethylation as assessed by methylation-specific PCR. Lossof RKIP expression was associated with poor disease-specific survival and with tumour necrosis in GISTs.Furthermore, a statistical tendency was observed betweenthe positive RKIP expression and absence of metastasis. Sofar, this is the first study assessing RKIP expression levelsin GISTs. We conclude that loss of RKIP expression couldhave an important role as prognostic marker in GISTs.
Keywords RKIP. Expression . Survival . GISTs
Introduction
Gastrointestinal stromal tumours (GISTs) are rare mesen-chymal tumours of the digestive tract [1, 2]. Gain-of-function mutations in KIT and PDGFRA oncogenes havebeen identified in a great majority of GISTs and areconsidered to be one of the first molecular events in theirpathogenesis [3, 4]. The tyrosine kinase inhibitor imatinibmesylate is the gold standard in the treatment of metastaticGISTs, leading up to 75% response rates [5]. Severalstudies have shown the importance of KIT and PDGFRAmolecular status in imatinib response [6, 7].
Tumour size, location and mitotic index are the mostimportant prognostic parameters of GISTs [8, 9]. Severalauthors have proven the usefulness of these and otherclassification parameters in GISTs clinical follow-up [10,11]. Moreover, other studies have shown that molecular
Olga Martinho and António Gouveia contributed equally to the study.
O. Martinho : R. M. ReisLife and Health Sciences Research Institute (ICVS),School of Health Sciences, University of Minho,Braga, Portugal
A. Gouveia : P. Silva :A. Pimenta : J. M. Lopes (*)IPATIMUP, Institute of Molecular Pathologyand Immunology of the University of Porto,Rua Dr. Roberto Frias, s/n,4200-465 Porto, Portugale-mail: [email protected]
A. Gouveia :A. PimentaDepartment of Surgery,Porto, Portugal
J. M. LopesDepartment of Pathology, H.S. João,Porto, Portugal
A. Gouveia : P. Silva :A. Pimenta : J. M. LopesMedical Faculty of Porto University,Porto, Portugal
genetic markers, such as the type of KIT and PDGFRAmutations, might also have prognostic value [12, 13].However, a part of GISTs behave unpredictably leading tothe need of screening new molecular prognostic markers.
The MAP kinase (RAS/RAF/MEK/ERK) pathway, oneof the KIT downstream cascades, is highly preserved andimplicated in cell growth, differentiation, migration andsurvival [14]. Activated RTK phosphorylates RAS, whichbinds RAF-1 kinase that phosphorylates MEK1/2, leadingto ERK1/2 activation. Phosphorylated ERKs translocatesinto nucleus and regulates gene expression through severaltranscription factors like c-Myc, CREB and AP-1, ulti-mately leading to changes in gene expression [14].Recently, we and others observed activation of this pathwayin GISTs by presence of BRAF activating mutations [15,16]. In addition, we described the absence of RASmutations, and suggest that MAP kinase could also beactivated through SCF/KIT autocrine/paracrine mechanismsin a subset of KIT/PDGFRA wild-type GISTs [16].
Raf kinase inhibitory protein (RKIP; also known asPEBP, for phosphatidylethanolamine-binding protein) wasoriginally identified as an endogenous inhibitor of the RAS/RAF/MEK/ERK pathway by interfering with the phosphor-ylation and activation of MEK by Raf-1 [17, 18].Subsequently, RKIP was shown also to suppress theactivation of the nuclear factor Kappa B (NFkB) cellsurvival pathway by blocking the inactivation of theinhibitor of NFkB, namely IkB [19]. In mammals, RKIPis also a negative regulator of G-protein coupled receptors[GPCRs] by inhibiting GRK-2 [20], and may be involvedin regulating the partitioning of chromosomes and mitosisprogression through RKIP binding to centrosomal andkinetochore regions of metaphase chromosomes [21]. Thecollective evidence indicates that RKIP regulates theactivity and mediates the crosstalk between several impor-tant cellular signalling pathways including cell differentia-tion, cell cycle, apoptosis and cell migration. Attenuation ofRKIP function is implicated in several human diseases,such as neurologic diseases and metastases in cancer [22,23].
RKIP is a widely expressed and highly conservedprotein [24–26], which is downregulated in severaltumours, including highly metastatic prostate carcinoma,breast, colon and gastric carcinoma, hepatocellular carci-noma, melanoma, insulinoma and ovarian carcinoma [27–36]. Furthermore, RKIP is also a prognostic marker inprostate, colorectal and gastric carcinomas [34, 36–38].The molecular mechanisms underlying RKIP down-regulation in cancer is not yet fully understood. Someauthors suggested RKIP promoter methylation as apotential RKIP silencing event [21, 39].
In the present study we aimed to clarify the role of RKIPin the prognosis of GISTs.
Materials and methods
Tissue samples
Seventy formalin-fixed paraffin-embedded primary spo-radic GISTs, classified according to WHO criteria and riskgroup [40], previously characterised immunohistochemi-cally for CD117, actin, S100, desmin and CD34, andmolecularly for KIT and PDGFRA mutations, wereretrieved from the Pathology Department of S. JoãoHospital files (1989–2005), Porto, Portugal [10, 41]. Allpatients were Caucasian and of Portuguese origin, with amean age of 62.1 years (range 20–88). Thirty-six (51.4%)patients were female and 34 (48.6%) were male. Mostfrequent tumour location was gastric (n=40) and smallintestine (n=23); other location were: colon (n=2), rectum(n=1), oesophagus (n=1) and omentum/mesentery (n=3).Three GIST patients with surgically resected tumours weretreated with imatinib (400 mg daily, with escalation to600 mg daily whenever indicated), because of tumourrecurrence. Of these patients, two are alive with stabledisease and one died due to the disease. Follow-up datawas available in all patients, as at December 2007, andcollected through direct interview with patients or theirrelatives, and by review of in-hospital patient files. Themedian follow-up time of patients was 54.1 months(range, 1–206). The diagnosis of metastases, includingthe two cases with very limited biopsy material, was basedon definitive imagiological evidence obtained during thefollow-up of the patients.
Immunohistochemistry analysis for RKIP
Representative 3-µm thick sections were subjected toimmunohistochemical analysis according to the streptavidin–biotin peroxidase complex system (UltraVision Large VolumeDetection System Anti-Polyvalent, HRP; Lab Vision Corpo-ration). Briefly, deparaffinised and rehydrated slides weresubmitted to heat-induced antigen retrieval for 20 min at 98°Cwith 10 mM citrate buffer (pH6.0). After incubation with theprimary antibodies raised against RKIP (dilution 1:600;incubation 2 h at RT; Upstate Biotechnology, Lake Placid,NY), the secondary biotinylated goat anti-polyvalent antibodywas applied for 10 min followed by incubation with thestreptavidin–peroxidase complex. The immune reaction wasvisualised by 3,3′-diamonobenzidine as a chromogen. Allsections were counterstained with Gill-2 haematoxylin. Fornegative controls, primary antibody was replaced by auniversal negative control antibody (CEA, rabbit anti-human,DAKO Corporation, Carpinteria, CA). A prostate carcinomawas used as positive control.
Tumour samples were evaluated for both extension andintensity of the immunoreactions. The score used was the
278 Virchows Arch (2009) 455:277–284
sum of the percentage of positive cells (0, negative; 1, lessthan 25% positive cells; 2, 26% to 50% positive cells and 3,more than 50% positive cells) and the staining intensity (0,negative; 1, weak; 2, moderate and 3, strong). Scoresbetween 0 and 2 were classified as negative, 3 and 4 asmoderate positive and 5 and 6 as strongly positive.
DNA isolation
Selected areas contained at least 85% of tumour tissue weremacrodissected into a microfuge tube using a sterile needle(Neolus, 25G-0.5 mm) and DNA isolation was performedusing Qiagen’s QIAamp® DNA Micro Kit, as previouslydescribed [42].
Methylation analysis of RKIP promoter
DNA methylation pattern in the promoter region of theRKIP gene was determined by methylation-specific PCR(MSP), as previously described [39] with some modifica-tions. Briefly, bisulphite treatment of 200 ng DNA wasdone using EZ DNA Methylation Golf Kit (Zymo ResearchCorporation, USA) according to the manufactures instruc-
tions. The PCR was carried out in a total volume of 15µl,consisting of 1µl of bisulphite modified DNA, 0.2µM ofboth sense and anti-sense primers (MWG-Biotech, Ebers-berg, Germany), 200µM of dNTPs (Fermentas, USA),1,5 mM of MgCl2 (Bioron, Germany), 1× Taq Bufferincomplete (Bioron, Germany) and 1 U of Taq SuperhotDNA Polymerase (Bioron, Germany). The reaction con-sisted of an initial denaturation at 95°C for 10 min,followed by 40 cycles with denaturation at 95°C for 30 s,annealing at 52–58°C for 30 s and extension at 72°C for30 s, followed by a final extension for 10 min at 72°C, in aThermocycler (BioRad). To MSP reaction, were usedspecific primers to distinguish methylated DNA (204 bpPCR product) from unmethylated DNA (205 bp PCRproduct), as described [39]. CpGenome Universal Methyl-ated DNA (Chemicon International, USA) was used asmethylated control. Blood DNA of a young healthyindividual was used as unmethylated control.
Statistical analysis
The available clinical–pathological and immunohistochem-istry data were analysed with SPSS software for Windows,
Fig. 1 Immunohistochemistry analysis of RKIP in GISTs. a Weak expression (×200), b strong expression (×200), c negative expression (×200), dpositive expression of RKIP in normal adjacent tissue (×200)
Virchows Arch (2009) 455:277–284 279
version 15.0. Correlations between categorical variableswere performed using Fischer’s exact test. Disease-specificsurvival (DSS) was calculated from the time of diagnosisuntil death related with the disease, or censored at the timeof latest follow-up, as described [10]. Cumulative survivalprobabilities were calculated using the Kaplan–Meiermethod. Differences between survival rates were testedwith the log-rank test. p value inferior to 0.05 wasconsidered significant.
Fig. 2 Agarose gel (2%) showing MSP result for methylation analysisof RKIP gene in the two cases with RKIP loss of expression (G30 andG60). Un unmethylated, M methylated, L 100 bp ladder
Parameter RKIP expression
N Negative (%) Positive (%) pa
CD117 expression
Negative 6 2 (33.3) 4 (66.7) 0.079
Positive 64 4 (6.3) 60 (93.7)
KIT/PDGFRA mutations
Mutant 44 4 (9.1) 40 (90.9) 1.000
Wild-type 26 2 (7.7) 24 (92.3)
Age (years)
≤60 30 2 (6.7) 28 (93.3) 0.694
>60 40 4 (10) 36 (90)
Gender
Male 34 3 (8.8) 31 (91.2) 1.000
Female 36 3 (8.3) 33 (91.7)
Tumour local
Small intestine 23 0 (0) 23 (100) 0.051
Gastric 40 4 (10) 36 (90)
Other 7 2 (28.6) 5 (71.4)
Risk grade
Very low/low/intermediate 41 2b (4.9) 39 (95.1) 0.224
High 29 4 (13.8) 25 (86.2)
Cellular morphology
Spindle 45 4 (8.9) 41 (91.1) 0.842
Epithelioid 8 0 (0) 8 (100)
Mixed 17 2 (11.8) 15 (88.2)
Tumour size (cm)
≤5 29 1 (3.4) 28 (96.6) 0.268
>5 to ≤10 23 2 (8.7) 21 (91.3)
>10 18 3 (16.7) 15 (83.3)
Mitotic index (50/HPF)
≤5 50 3 (6) 47 (94) 0.343
>5 20 3 (15) 17 (85)
Necrosis
Absent 29 0 (0) 29 (100) 0.038*
Present 41 6 (14.6) 35 (85.4)
Metastasis
Absent 58 3 (5.2) 55 (94.8) 0.058
Present 12 3 (25) 9 (75)
Mean survival time (months ± SD) 70 60.60±20.07 167.87±10.35 0.023*c
Table 1 Association betweenRKIP expression and clinical–pathological parameters inGISTs (n=70)
N number of cases, HPF highpower field (×400)
*p<0.05, statistically significantvaluesa Fischer´s exact testb One case low and one caseintermediate riskc Log-rank test
280 Virchows Arch (2009) 455:277–284
Results
RKIP expression
In the present study, we used a series of 70 GISTs whichcomprises 91.4% of CD117 positive cases and 63% of KIT/PDGFRA mutated GISTs, as previously characterised [41].Immunohistochemical approach was done to detect RKIPprotein expression and distribution in GIST cases. Weobserved cytoplasmatic expression of RKIP in 64 (91.5%)of the 70 GISTs studied: 30 cases showed moderate and 34strong positivity (Fig. 1a and b). The remaining 8.5% (6/70)of cases were considered as negative for RKIP expression(Fig. 1c). In a subset of cases, it was possible to analyse thenormal adjacent tissue that showed to be strongly positivefor RKIP expression (Fig. 1d).
RKIP methylation
To determine whether absence of RKIP expression was due togene promoter hypermethylation, we performed methylation-specifc PCR in all six cases without RKIP expression. None ofthe cases exhibited RKIP promoter hypermethylation (Fig. 2).
Clinical–pathological features and statistical analysis
After univariate statistical analysis we observed that there isno correlation between RKIP expression and CD117staining and KIT/PDGFRA mutations status (Table 1).Concerning clinical–pathological data, no significant corre-lations (p>0.05) were found between RKIP expression andage, gender, risk grade, cellular morphology, tumour sizeand mitotic index (Table 1). RKIP negativity was correlatedwith presence of necrosis (p=0.038). There was a tendency(p=0.051) for absence of RKIP expression in gastric GISTsand a tendency (p=0.058) for the development of metas-tasis in RKIP negative cases (Table 1).
Regarding clinical–pathological parameters and disease-specific survival (univariate analysis), we found that a shorterDSS was significantly associated with male gender (p=0.014),high-risk GISTs (p<0.001), tumour size >10 cm (p=0.001),mitotic index >5 (p=0.009) and development of metastasis(p<0.001). Loss of RKIP expression was significantly (p=0.023) associated with poor DSS (Table 1 and Fig. 3). Onlythree cases were treated with imatinib-based therapy, ham-pering any statistical analysis. The low number of casesprecluded multivariate analysis of the studied parameters.
Discussion
Unresectable or metastatic GIST is a fatal disease resistantto conventional cytotoxic chemo and radiotherapy. The
median survival time for patients with metastatic GIST isapproximately 20 months, and 9–12 months for patientswith local recurrences [43]. Treatment with imatinibmesylate is the first option for metastatic disease [44].However, further molecular characterization is warrantedfor the treatment of primary and secondary imatinib-resistant GISTs. Furthermore, excepting KIT and PDGFRAmutations, no other molecular alterations have beenconsistently associated with prognosis of GISTs [12, 13].
RKIP is considered to be a signal transduction modulatorand a metastasis suppressor [45]. The first evidence camefrom cell lines derived from metastatic prostate carcinomas,which display decreased levels of RKIP as compared withprimary tumour cell lines [27]. In human breast carcinoma[32] and metastatic colorectal carcinoma [34, 46], RKIPexpression is consistently lost in lymph node metastases butnot in primary tumours, supporting the suggestion thatRKIP is downregulated in metastatic development. More-over, overexpression of RKIP in vitro and in vivo prostateand melanoma tumour models suggest that RKIP representan important suppressor of metastasis by decreasingvascular invasion [27, 29]. Consistent with the role ofRKIP as a potent suppressor of metastatic development,several studies described that highly metastatic prostatecarcinoma [27], malignant melanoma [29], breast cancerlymph node metastases [32], insulinoma [31], colorectalcarcinoma [46], hepatocarcinoma [33], ovarian carcinoma[35], Merckel cell carcinoma [47] and thyroid carcinoma[48] display frequently a marked decrease in RKIPexpression.
Work by Eves et al. [49] also indicates that the absenceof RKIP may increase the genetic instability of the cell
Fig. 3 Disease-specific survival (DSS) according to RKIP expressionin GISTs (n=70). Cumulative survival is significantly lower in caseswith RKIP loss of expression (p=0.023)
Virchows Arch (2009) 455:277–284 281
while work with hepatoma cells suggests that the absenceof RKIP may increase the rate of cell division [33].
Furthermore, loss of cytoplasmatic RKIP has also beenassociated with colorectal carcinoma recurrence [34] andwith poor prognosis in prostate, colorectal and gastricadenocarcinomas [34, 36–38]. Recently, it was proposedthat the level of RKIP in the blood can be used as aprognostic marker for prostate cancer patients [38]. Thus,loss of RKIP expression may be considered as a marker oftumour progression.
In our series, well-established prognostic markers (malegender, high risk, tumour size >10 cm, mitotic index >5 anddevelopment of metastasis) were significantly associatedwith DSS [10]. Regarding RKIP expression, our results inGISTs has shown that a negative cytoplasmatic expression,found in approximately 9% of the cases, is significantlycorrelated with the presence of necrosis and with DSS ofGIST patients. Furthermore, a tendency was observedbetween the presence of RKIP expression and absence ofmetastasis. So far, there are no reported studies of RKIPexpression in GISTs. We recently demonstrated that MAPKpathway alterations occur at low frequency in KIT andPDGFRA wild-type GISTs, corroborating the low percent-age of RKIP absence of expression in the present study[16]. Further studies with a largest series are needed tovalidate RKIP expression as a prognostic marker in GISTs.Furthermore, it will be also interesting in the future toassess the expression levels of RKIP in the metastaticbiopsies of GISTs.
Despite the increasing evidence that RKIP is lost duringtumour progression, especially in metastasis, the mecha-nisms of RKIP down-regulation remains to be unraveled[21]. To further elucidate the genetic event that underliesRKIP down-regulation in GISTs, we analysed the methyl-ation status of the CpG islands of RKIP promoter in RKIPnegative GISTs. All six GISTs without RKIP expressionwere found unmethylated for RKIP promoter. Formerstudies concerning RKIP methylation are discrepant. RKIPmethylation was described in a cohort of 12 patients withhyperplastic polyposis coli [39]. Noteworthy, the sameauthors, using 28 colorectal carcinomas, reported thatmethylation of the RKIP promoter was not responsible forloss of RKIP expression [46]. Recently, Al-Mulla et al. [21]reported that 72.5% of 40 RKIP negative colorectalcarcinomas were methylated at the RKIP promoter, andsuggested it as the major mechanism by which RKIP issilenced [21].
The role of RKIP in tumour progression and inmetastases development raises hope for tailored therapeuticapproaches, using drug-induced modulation of RKIPexpression, to control tumour aggressiveness. Locostatinhas already been shown to abrogate the ability of RKIP toinhibit Raf-1 [50]. In some prostate and breast carcinoma
cell lines, increasing levels of RKIP expression re-sensitisecells to drug-induced apoptosis. In drug-sensitive cell lines,down-regulation of RKIP led to the resistance to DNA-damaging drugs [9-nitrocamptothecin, taxol and cisplatin][28]. Rituximab up-regulates RKIP, which has been shownto sensitise non-Hodgkin’s lymphoma cell lines tochemotherapeutic-induced apoptosis [51]. Both inhibitionof the MEK–ERK pathway and inhibition of the NFkBpathway were suggested as possible mechanisms for thesedrug-sensitising effects [51]. Recently, RKIP expressionlevels in pituitary adenomas were found to correlate withboth acute and long-term clinical response to octreotide[52]. Furthermore, Bonavida et al. demonstrated that nitricoxide mediated chemo/immunosensitization via inhibitionof NFkB may also involve the induction of RKIP. Inductionof RKIP inhibits anti-apoptotic pathways that regulatetumour cell sensitivity to apoptotic stimuli [53].
In the present study we describe for the first time thatGIST RKIP expression levels correlate with clinical–pathological parameters in univariate analysis. We foundthat the loss of RKIP expression was independent ofpromoter methylation in a subset of GISTs. Most important,loss of RKIP was associated with tumour necrosis and witha poor survival, i.e., a potential prognostic marker for GISTpatients. Further studies with larger series and with in vivo/in vitro tumour models are warranted to evaluate the role ofRKIP in metastatic development and survival of GISTpatients.
Acknowledgements OM is recipient of a PhD fellowship (SFRH/BD/36463/2007) from FCT, Portugal. This study was partiallysupported by NOVARTIS Oncology, Portugal.
Conflicts of interest statement We declare that we have no conflictof interest.
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284 Virchows Arch (2009) 455:277–284
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DISCUSSÃO E CONCLUSÕES
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DISCUSSÃO / CONCLUSÕES
Uma vez que nos resultados da tese se incluem os trabalhos publicados com discussão, decidiu-se
simplificar o mais possível o conteúdo deste capítulo nesta dissertação. Para maior facilidade de
exposição, decidiu-se também seguir a mesma sequência do capítulo de objectivos.
I. Objectivo: Analisar o valor prognóstico da qualidade das margens cirúrgicas no tratamento dos
doentes com GIST primário.
As opções cirúrgicas mais utilizadas no tratamento dos doentes com GISTs primários localizados
são a ressecção “em cunha”, nos GISTs do estômago, e a ressecção segmentar, nos GISTs do
intestino delgado, sem necessidade de linfadenectomia de rotina. Como estes tumores não têm
geralmente padrão infiltrativo intraparietal, estas opções são, de facto, consideradas adequadas ao
tratamento da maioria dos doentes com GIST. No entanto, dependendo do órgão onde se origina o
GIST, da localização exacta e da dimensão do tumor, podem ser necessárias abordagens cirúrgicas
distintas: ressecção parcial ou total de órgão, ou eventual remoção de tecidos adjacentes.
A ressecção cirúrgica no GIST deve garantir uma margem completa de tecido normal em redor do
tumor primário. A revisão das principais séries publicadas permite constatar que vários autores se
referem a ressecção macroscópica completa27, 190, 243, 319-322 dos tumores, enquanto outros
especificam a ressecção R0191, 215, 323-327 como procedimento padrão no tratamento cirúrgico dos
GISTs. Alguns autores defendem que o estado microscópico das margens cirúrgicas (positivo ou
negativo), em contraste com os resultados obtidos noutros tumores sólidos malignos, não
influencia a sobrevida dos doentes, nem mesmo a recidiva do GIST27, 319, 328. Num estudo de 200
doentes com GIST, DeMatteo et al.27 sugerem que as margens microscópicas não influenciam
significativamente a evolução dos tumores e que a recidiva ocorre mais provavelmente como
resultado das características biológicas intrínsecas do tumor. Contudo, nas séries publicadas, o
número relativamente pequeno de casos com margens microscópicas positivas, após ressecção
macroscópica completa, não permite um esclarecimento cabal sobre a adequação desta sugestão.
De facto, as séries incluem um número substancial de GISTs de grandes dimensões e de alto risco
biológico, nos quais a ressecção completa pode não evitar a recidiva (particularmente à distância) do
tumor nem a sobrevida mais curta dos doentes. A análise dos resultados do estudo de DeMatteo et
al. confirma que a maioria das ressecções cirúrgicas foi realizada em GISTs volumosos e de alto
risco biológico215, 329. O valor da margem microscópica negativa (por exemplo, GISTs> 10 cm) é
muito discutível, pois é possível admitir que os tumores podem libertar células, não detectáveis,
para a cavidade peritoneal27, 320. Adicionalmente, alguns destes resultados podem estar enviesados
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pelo efeito do tratamento adjuvante realizado nos GISTs avançados ou submetidos a ressecção
incompleta27, 330.
Outros autores sugerem que o resultado negativo (R0) na avaliação das margens cirúrgicas
microscópicas pode modificar o prognóstico dos GISTs215, 324, 325, 329, 331-336. No entanto, estes
resultados podem também ter sido influenciados pelo número de ressecções incompletas em GISTs
de alto risco biológico329.
Na nossa série de 104 casos submetidos a cirurgia de ressecção, foi realizada ressecção completa
macroscópica (R0 ou R1) em 92,3% (n=96) dos GISTs e verificou-se a existência de margens
microscópicas negativas (R0) em 75% (n=78) dos casos. A ressecção cirúrgica dos GISTs primários
associou-se a sobrevida específica aos 5 anos de 87,7% e a sobrevida livre de recidiva aos 5 anos de
89,8%, valores que se comparam favoravelmente com os resultados de outros estudos publicados.
A taxa de recidiva de GIST foi significativamente menor nos casos com ressecção R0, quando
comparada com a dos casos com ressecção R1 (p = 0,045), mas apenas a presença de tumor
residual macroscópico (R2) se associou de forma significativa (análise multivariada) a progressão da
doença e a sobrevida mais curta dos doentes (p = 0,013). Tal como nos estudos de DeMatteo et al.27
e Pierie et al.319, o nosso estudo sugere que a ressecção macroscópica do tumor tem impacto
positivo no prognóstico, sendo mais curta a sobrevida específica nos doentes com GISTs em que
foram obtidas margens cirúrgicas R2.
Apesar da controvérsia existente, como abordado neste trabalho, a ressecção cirúrgica com margens
microscópicas positivas (R1) pode expor os doentes a um risco elevado de recidiva tumoral loco-
regional. O objectivo da cirurgia em todos os casos de GIST primário deve ser, segundo as
recomendações de consenso actuais: ressecção completa do tumor, com margens microscópicas
negativas (R0), e preservando a pseudo-cápsula intacta (i.e. evitando a ruptura tumoral)24, 138.
Os dados da literatura, baseados em resultados de análise retrospectiva, mostram que as ressecções
por via laparoscópica, ou assistidas por laparoscopia, são exequíveis e associam-se a taxas de
recidiva reduzidas, períodos curtos de internamento e morbilidade baixa213, 217, 220, 232, 234, 235. Esta
abordagem deve ser considerada a opção de escolha, por exemplo, para a maioria dos doentes com
GISTs gástricos de pequenas e médias dimensões220, 224. Recomenda-se, no entanto, que a técnica
deve obedecer estritamente aos princípios oncológicos acima definidos24, 138, 219.
A abordagem cirúrgica recomendada para os GISTs do intestino delgado é a ressecção segmentar
com margens macroscópicas de 2 a 3 cm, e para GISTs gástricos considera-se suficiente uma
margem de ressecção de 1 a 2 cm218, 225, 326, 337. O exame intra-operatório de amostras de tecido peri-
tumoral por um anátomo-patologista deve ser obrigatório quando existe a possibilidade de as
margens cirúrgicas serem positivas.
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De acordo com as orientações actuais da ESMO e da NCCN, nos casos em que se realiza uma
ressecção microscópica incompleta (R1) é de considerar o alargamento de margens quando a
localização exacta da lesão pode ser identificada e o risco de morbilidade cirúrgica é baixo.
As recomendações actuais para as margens cirúrgicas têm por base a experiência, o consenso e a
aplicação dos conhecimentos biológicos sobre GIST24, 138. De facto, não há dados prospectivos que
permitam concluir sobre o impacto da qualidade das margens de ressecção (R0 e R1),
designadamente as microscópicas, no risco de recidiva (local ou à distância) do GIST.
Conclusão:
As principais séries publicadas permitem confirmar que não há consenso geral sobre o impacto do
estado microscópico das margens cirúrgicas (positivo/R1 ou negativo/R0) no prognóstico dos
doentes com GIST. Na nossa série, foi realizada ressecção completa macroscópica (R0 ou R1) em
92,3% dos GISTs. A taxa de recidiva foi significativamente menor nos casos com ressecção R0,
mas na análise multivariada apenas a presença de tumor residual macroscópico (R2) se associou
significativamente a sobrevida mais curta dos doentes. De acordo com as recomendações de
consenso actuais, os nossos resultados sublinham o valor prognóstico da ressecção macroscópica
completa dos tumores, com o objectivo de se obterem margens microscópicas negativas e evitar a
ruptura do tumor. Neste contexto, pode ser considerado o exame intra-operatório de amostras peri-
tumorais e o alargamento de margens, após ressecção microscópica incompleta (R1). Os resultados
publicados indicam que a ressecção laparoscópica ou assistida por laparoscopia, é exequível e
apresenta, na maioria dos casos de GISTs gástricos, segurança e eficácia comparável à da cirurgia
convencional.
No entanto, não existem dados prospectivos que permitam concluir sobre o impacto da extensão
das margens de ressecção, designadamente microscópica, no risco de recidiva (local ou à distância)
de GIST.
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II. Objectivo: Avaliar os procedimentos de diagnóstico de GIST no esófago, que é uma localização
rara deste tipo de tumores, e rever as opções terapêuticas mais adequadas nesta localização.
Os GISTs são os tumores mesenquimatosos mais comuns do tracto gastrointestinal, mas são
raramente diagnosticados (1-3%) no esófago27, 338. O diagnóstico específico de GIST antes da
biópsia ou cirurgia é bastante difícil em qualquer localização, mas constitui um desafio no esófago,
devido às semelhanças na apresentação clínica, endoscópica e radiográfica com o leiomioma
esofágico, que é um tumor mais comum nesta localização. A ultrassonografia endoscópica pode ser
útil no diagnóstico destes casos e confirmar que o tumor se origina da parede do esófago.
Adicionalmente, a realização de biópsia com agulha fina através da ultrassonografia endoscópica
permite frequentemente a realização de um diagnóstico, designadamente pela identificação de
células fusiformes/epitelióides que expressam os marcadores imuno-histoquímicos característicos
de GIST ou, em alternativa, de outros tipos celulares que excluem o diagnóstico de GIST339-341.
Quando são detectados nódulos subepiteliais esofágicos, gástricos ou duodenais com diâmetro < 2
cm, a abordagem recomendada consiste na avaliação com ecografia endoscópica e vigilância activa
do doente. Estes nódulos, quando correspondem a GISTs, são tumores habitualmente de baixo
risco biológico1, 184. Nestas circunstâncias, a exérese cirúrgica é reservada para os doentes cujo
tumor aumenta de dimensões ou é sintomático. Adicionalmente, quando estes nódulos apresentem
Neste trabalho, a avaliação da contribuição de alterações na via de sinalização MAPK nos GISTs
wild-type consistiu na análise mutacional dos genes da família RAS e BRAF e na avaliação dos níveis
de expressão imuno-histoquímica de ERK fosforilada. Apesar da elevada frequência de mutações
nos genes da família RAS em vários tipos de tumores, incluindo os carcinomas do pâncreas, o
carcinoma papilar da tireóide, o carcinoma do cólon e o carcinoma do pulmão de células não-
pequenas356, 360, não se identificou em nenhum dos 26 GISTs desta série mutações do H-RAS, do
K-RAS, nem das regiões hotspot do N-RAS. A análise do BRAF, outro oncogene importante da via
MAPK, permitiu identificar a presença da mutação activante V600E em cerca de 4% (um caso) dos
GISTs wild-type desta série. A mutação V600E é responsável por 90% das mutações do BRAF nos
tumores malignos humanos e associa-se a maior actividade cinásica, que induz a activação de ERK,
independente do estado de activação de RAS361. Por outro lado, não se identificou nesta série
qualquer mutação do BRAF nos GISTs com mutações do KIT ou do PDGFRA, sugerindo que as
mutações do BRAF, KIT e PDGFRA são mutuamente exclusivas.
Quando o presente trabalho foi iniciado, não existiam publicações sobre a análise de mutações do
RAS ou BRAF em GISTs. Posteriormente, outros estudos descreveram mutações V600E no exão
15 do BRAF em 5-13% dos GISTs wild-type de adultos (5%= 3/61 casos115, 7%= 2/28 casos114,
13%= 9/70 casos116). Os GISTs com mutação do BRAF parecem localizar-se preferencialmente no
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intestino delgado, sendo ainda controversa a sua associação com o comportamento biológico em
GISTs de alto risco.
Para melhor esclarecimento do papel da via da MAPK em GISTs wild-type, avaliou-se a expressão de
fosfo-ERK que foi identificada em 30% dos casos wild-type da nossa série. Estudos previamente
publicados sugeriram que a activação de ERK (extracellular signal-regulated kinase) em GISTs depende
da sinalização KIT63 e que algumas linhas celulares KIT wild-type também têm activação de ERK76.
Os nossos resultados estão de acordo com estes resultados publicados, salientando-se que
identificamos uma correlação significativa (p=0,02) entre a expressão de fosfo-ERK e a expressão
de fosfo-KIT. No entanto, a expressão de fosfo-ERK em 50% dos casos da nossa série sem
expressão de fosfo-KIT, sugere que a activação de ERK independente da activação de KIT nos
GISTs wild-type é causada provavelmente por outras vias (ex: AKT/PI3K e JAK/STAT) de
sinalização intracelular16, 353.
Os factores de prognóstico dos GISTs wild-type são ainda controversos. Alguns estudos sugerem um
prognóstico melhor nos GISTs wild-type, quando comparados com os tumores com mutações55,
sendo que estes resultados não foram confirmados num estudo de base populacional362. Nesta
nossa série, verificámos que parâmetros clínico-patológicos habitualmente associados a
comportamento agressivo nos doentes com GIST, incluindo a maior dimensão do tumor e o grupo
de alto risco de agressividade, são também factores de mau prognóstico em doentes com GISTs
wild-type.
Existem trabalhos publicados de ensaios clínicos fase II e III descrevendo que os doentes com
GISTs wild-type raramente respondem ao tratamento com imatinib9, 87, 205, 258. Estes doentes
constituem um desafio terapêutico muito importante. As evidências mais recentes sugerem que o
sunitinib pode ser particularmente eficaz no tratamento dos GISTs com mutações do KIT no exão
9 e nos GISTs com genótipo wild-type (sem mutações do KIT / PDGFRA)89, 207, 272, mas as
indicações terapêuticas precisas para este último subgrupo de doentes não estão ainda claramente
estabelecidas.
A activação constitutiva da via de sinalização intracelular RAS/RAF/MEK/ERK pode também
regular a resposta dos doentes a terapêuticas dirigidas aos receptores tirosina-cinásicos,
independente do estado a montante dos receptores tirosina-cinásicos.
Alguns fármacos dirigidos contra moléculas RAS/RAF/MEK são actualmente objecto de estudo
em ensaios clínicos ou pré-clínicos em curso. Não foram ainda descritos inibidores de ERK1 ou
ERK2 com aplicabilidade clinicamente relevante360. O inibidor anti-RAF sorafenib (Nexavar ®) é
um inibidor tirosina-cinásico potente do BRAF wild-type e do BRAF com mutação V600E, e
descreveu-se que é um inibidor multi-cinásico que inibe outros receptores tirosina-cinásicos,
incluindo o KIT, o receptor do factor de crescimento endotelial vascular (VEGFR) -2, o VEGFR-3
e o PDGFRB363. A FDA aprovou o sorafenib para o tratamento do carcinoma de células renais e
do hepatocarcinoma avançados, estando também a ser avaliado no carcinoma do pulmão de células
não-pequenas, nos carcinomas da próstata, da mama e do pâncreas e no melanoma363, 364. Vários
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estudos descreveram a associação da actividade anti-tumoral do sorafenib a um efeito anti-
angiogénico363, 364.
Em contraste com o sorafenib, as pequenas moléculas inibidoras da MEK1/2 (MAPK/ERK kinase
1 / 2) são consideradas inibidores mais específicos. Os estudos pré-clínicos com AZD6244
(ARRY-142886) revelaram resultados promissores e levaram ao desenvolvimento de ensaios
clínicos adicionais365, 366. Neste contexto, é interessante sublinhar que Solit et al. descreveram que os
tumores com mutação do BRAF são muito sensíveis à inibição cinásica de MEK367.
Os resultados promissores recentemente descritos com o inibidor da cínase RAF PLX4032
(Plexxikon), também conhecido como RG7204, em doentes com melanomas que têm mutação
V600E, sugerem a possibilidade de se obter respostas semelhantes noutros tipos de tumores que
partilhem dependência oncogénica do gene RAF na sua patogénese368, 369.
Conclusão:
A via da MAPK (RAS/RAF/MEK/ERK) a jusante do receptor KIT está implicada na patogénese
de vários tipos de tumores sólidos. O nosso estudo constitui uma análise compreensiva da
desregulação da via MAPK nos GISTs wild-type para os genes KIT e PDGFRA. Na ausência de
mutações RAS sugere-se que a via da MAPK pode ser activada através de mecanismos
autócrinos/parácrinos de SCF/KIT e/ou por mutação do gene BRAF num subgrupo de GISTs
wild-type. A mutação activante V600E do BRAF foi observada num (4%) dos GISTs wild-type da
nossa série. Posteriormente, outros estudos descreveram mutações V600E no exão 15 do BRAF
em 5-13% dos GISTs wild-type de adultos.
Alguns fármacos dirigidos contra moléculas RAS/RAF/MEK são actualmente objecto de estudo
em ensaios clínicos ou pré-clínicos. As mutações activantes V600E nos GISTs wild-type levantam a
possibilidade de terapêutica com inibidores da cínase RAF [ex: PLX4032 (Plexxikon)] neste
subgrupo de tumores. Apesar da reduzida frequência das alterações moleculares da via da MAPK,
são necessários estudos adicionais que avaliem o potencial do tratamento dirigido a moléculas desta
via em GISTs (KIT e PDGFRA) wild-type.
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V. Objectivo: Avaliar a expressão de RKIP, a sua associação com diferentes parâmetros clínico-
patológicos, e esclarecer o papel deste marcador em doentes com GISTs primários.
O GIST irressecável e/ou metastático é uma doença potencialmente fatal e reconhecidamente
resistente à quimioterapia convencional e à radioterapia. A mediana de sobrevida dos doentes com
GIST metastático é de aproximadamente 20 meses e nos doentes com recidiva local é de 9-12
meses27. O mesilato de imatinib é uma opção de primeira escolha para o tratamento da doença
metastática258. No entanto, continua a ser necessária uma caracterização molecular mais completa e
adequada dos tumores para o tratamento mais eficaz dos GISTs com resistência primária e/ou
secundária ao imatinib. Nestas circunstâncias, com a excepção das mutações do KIT e do
PDGFRA, actualmente não existe outra alteração molecular preditiva, de forma consistente, do
prognóstico dos doentes com GIST101, 326.
A proteína RKIP é considerada um modulador de transdução de sinal (Fig. 5) e um supressor de
metastização tumoral370. A primeira evidência surgiu no estudo de Fu et al., em que se descreveu
que as linhas celulares derivadas de metástases de carcinomas da próstata tinham diminuição dos
níveis da expressão de RKIP, quando comparadas com linhas celulares do tumor primário371. No
Fig. 5: RKIP e via de sinalização da MAPK.
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carcinoma da mama372 e no carcinoma colo-rectal metastático372, 373 foi também descrita a perda da
expressão de RKIP nas metástases ganglionares linfáticas, mas não nos tumores primários. Estes
resultados suportam a possibilidade da expressão RKIP poder ser sujeita a regulação negativa nos
tumores em que se desenvolve doença metastática. Adicionalmente, a sobre-expressão de RKIP in
vitro e in vivo em modelos de tumores da próstata e de melanomas sugere que esta proteína
representa um importante supressor da metastização, diminuindo a invasão vascular371, 374. Vários
estudos corroboraram o papel de RKIP como supressor potente da patogénese das metástases de
tumores, descrevendo frequentemente diminuição acentuada da expressão de RKIP: carcinoma da
próstata metastático371, melanoma maligno374, metástases ganglionares linfáticas do carcinoma da
mama372, insulinoma375, carcinoma colo-rectal373, hepatocarcinoma376, carcinoma do ovário377,
carcinoma de células de Merkel378 e carcinoma da tireóide379.
O estudo de Eves et al.380 sugere também que a ausência de RKIP pode aumentar a instabilidade
genética das células e, no trabalho de Lee et al.376, em células de hepatoma, sugere-se que a ausência
de RKIP pode aumentar a taxa de divisão celular. A perda de expressão citoplasmática de RKIP
tem sido também associada a recidiva tumoral, no carcinoma colo-rectal381, e a mau prognóstico,
nos adenocarcinomas da próstata, colo-rectal e do estômago381-384. Foi recentemente proposto que
o nível sérico de RKIP pode ser usado como marcador de prognóstico em doentes com cancro da
próstata384. A perda de expressão RKIP pode ser, desta forma, considerado um marcador de
progressão tumoral.
Na nossa série de doentes submetidos a cirurgia de ressecção (n=104; trabalho I), marcadores de
prognóstico bem estabelecidos como o diâmetro do tumor > 10 cm, o índice mitótico > 5
mitoses/CGA, a classificação de alto risco, e o estado das margens cirúrgicas (R2), além do
desenvolvimento de metástases, associaram-se significativamente com a SE dos doentes (análise
univariada). No presente estudo (n=70) identificou-se ausência de expressão citoplasmática de
RKIP em 8,5% dos casos (6/70), significativamente associada com a presença de necrose (p=0,038)
e com a SE (p=0,023) dos doentes com GIST. Adicionalmente, observou-se uma tendência para a
associação entre a presença de expressão RKIP e a ausência de metastização. Não existem outros
estudos publicados sobre a expressão de RKIP em GISTs, com base na pesquisa bibliográfica mais
recente. Descreveu-se no estudo prévio da nossa série (trabalho IV) que a frequência de alterações
da via MAPK em GISTs KIT e PDGFRA wild-type é baixa, o que está de acordo com a percentagem
baixa de casos sem expressão RKIP observada no presente estudo385. No entanto, serão necessários
estudos adicionais, com séries maiores, para validação da expressão RKIP como eventual marcador
de prognóstico em doentes com GIST. Além disso, será também importante avaliar os níveis de
expressão RKIP em biópsias de lesões metastáticas de GISTs.
Apesar da evidência crescente de que a expressão de RKIP se perde durante a progressão do tumor,
especialmente nas lesões metastáticas, continuam por esclarecer os mecanismos da regulação
negativa de RKIP386. Para esclarecer melhor o evento genético subjacente à regulação negativa de
RKIP nos GISTs, analisou-se o estado de metilação das regiões CpG (cytosine-phosphate-guanine) do
promotor do RKIP nos GISTs sem expressão deste marcador da nossa série. Não foi observada
metilação do promotor do RKIP em nenhum dos seis GISTs sem expressão de RKIP. Nos estudos
previamente publicados, os resultados sobre a metilação do RKIP são discrepantes. A metilação do
RKIP foi descrita num grupo de 12 doentes com polipose hiperplásica do cólon318. Curiosamente,
149
os mesmos autores constataram que a metilação do promotor do RKIP não era responsável pela
perda de expressão de RKIP em 28 casos de carcinoma colo-rectal373. Recentemente, Al-Mulla et
al.386 descreveram que 72,5% dos 40 carcinomas colo-rectais sem expressão de RKIP apresentavam
metilação do promotor do RKIP e sugeriram que este seria o mecanismo responsável pela
expressão de RKIP negativa386.
O papel de RKIP na progressão tumoral e no desenvolvimento de metástases pode permitir novas
abordagens terapêuticas específicas, baseadas na modulação da expressão de RKIP induzida por
fármacos, para controlo da agressividade tumoral. O locostatin parece poder anular a capacidade de
RKIP inibir a cínase Raf-1387. Os níveis aumentados de expressão RKIP, em linhas celulares de
carcinoma da próstata e da mama, re-sensibilizam as células para a apoptose induzida por fármacos.
Nas linhas celulares sensíveis à acção dos fármacos, a regulação negativa de RKIP induziu o
desenvolvimento de resistência a agentes quimioterápicos que lesam o ADN (9-nitrocamptothecin,
taxol e cisplatina)388. O anticorpo monoclonal rituximab regula positivamente a proteína RKIP, e
pode sensibilizar linhas celulares de linfoma não-Hodgkin para a apoptose induzida por agentes
quimioterápicos389. A inibição da via MEK–ERK e a inibição da via NFkB (nuclear factor kappa-light-
chain-enhancer of activated B cells) foram propostas como mecanismos implicados neste efeito de
quimio-sensibilização389. Recentemente, foi descrita a relação dos níveis de expressão RKIP em
adenomas hipofisários com a resposta clínica a curto e longo prazo ao tratamento com
octreotido390. Bonavida et al. descreveram que a quimio/imuno-sensibilização mediada pelo óxido
nítrico, através da inibição da NFkB, pode também envolver a indução de RKIP. A indução de
RKIP inibe as vias anti-apoptóticas que regulam a sensibilidade das células tumorais aos estímulos
apoptóticos391.
Conclusão:
No presente estudo, descreveu-se pela primeira vez uma associação significativa entre os níveis de
expressão tumoral de RKIP e parâmetros clínico-patológicos em doentes com GIST. Verificou-se
que a perda de expressão de RKIP num subgrupo de GISTs (6/70) parece ser independente da
metilação do promotor. Salienta-se que a perda de RKIP na nossa série se associou
significativamente a necrose do tumor e a sobrevida mais curta dos doentes. A participação de
RKIP na progressão tumoral e no desenvolvimento de metástases sugere que este marcador pode
ter potencial prognóstico, e pode permitir novas abordagens terapêuticas específicas, baseadas na
modulação da expressão de RKIP em doentes com GIST. No entanto, são ainda necessários
estudos adicionais, com séries maiores, e modelos tumorais in vivo e in vitro, que permitam clarificar
o papel do RKIP na progressão da doença e na sobrevida dos doentes.
150
151
PERSPECTIVAS FUTURAS
Nos estudos subsequentes dar-se-á continuidade à análise da sobrevida dos doentes referenciados e
seguidos em regime de consulta externa, baseada numa actualização constante dos dados da
avaliação clínica e imagiológica de cada caso.
Procurar-se-á a detecção precoce de recidivas e o início do tratamento atempado mais adequado. A
caracterização molecular de cada GIST pode revelar-se fundamental para adaptar a estratégia
terapêutica concreta, baseada nas mutações específicas identificadas.
Neste sentido, adquire particular interesse ponderar a avaliação da utilização de inibidores tirosina-
cinásicos (ex: imatinib, sunitinib, e novas terapêuticas emergentes) em contexto neoadjuvante e
adjuvante, pelo potencial de redução das recidivas locais e/ou da doença metastática.
Nos casos de GIST avançado já em tratamento com ITKs, será importante avaliar a resposta
terapêutica e, em casos seleccionados, ponderar o benefício da opção cirúrgica complementar. A
cirurgia de citorredução pode ser útil em casos de progressão localizada do tumor e, minorando o
potencial desenvolvimento de mutações secundárias, em doentes com GISTs que respondem ao
tratamento com ITKs. A ressecção cirúrgica no GIST metastizado pode ser, assim, considerada
como opção adicional no arsenal terapêutico de segunda /terceira linhas, em doentes seleccionados.
Sempre que possível, será sistematicamente obtido material criopreservado para banco de tumor e
tecido não tumoral, com consentimento informado dos doentes, para estudos moleculares que
permitam contribuir para o esclarecimento dos mecanismos da resistência aos inibidores tirosina-
cinásicos e da progressão da doença.
Com estes estudos, e com a análise da expressão imuno-histoquímica dos tumores, pretende-se dar
continuidade, designadamente, ao estudo das diferentes proteínas envolvidas nas vias de sinalização
intracelular (ex: PI3K e MAPK) implicadas na patogénese dos GISTs, que possam permitir
identificar biomarcadores de resposta e potenciais alvos para novas terapêuticas dirigidas em casos
de resistência aos tratamentos actualmente aprovados.
152
153
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