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UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE CIÊNCIAS MÉDICAS.
SUSANA OLIVEIRA BOTELHO RAMALHO
AVALIAÇÃO PROGNÓSTICA DE COMPONENTES ASSOCIADOS À TRANSIÇÃO
EPITÉLIO-MESENQUIMAL EM MULHERES COM CARCINOMA SEROSO DE ALTO
GRAU DE OVÁRIO
PROGNOSTIC EVALUATION OF COMPONENTS ASSOCIATED WITH EPITHELIAL-
MESENCHYMAL TRANSITION IN WOMEN WITH HIGH-GRADE SEROUS OVARIAN
CARCINOMA
CAMPINAS
2017
SUSANA OLIVEIRA BOTELHO RAMALHO
AVALIAÇÃO PROGNÓSTICA DE COMPONENTES ASSOCIADOS À TRANSIÇÃO
EPITÉLIO-MESENQUIMAL EM MULHERES COM CARCINOMA SEROSO DE ALTO
GRAU DE OVÁRIO
PROGNOSTIC EVALUATION OF COMPONENTS ASSOCIATED WITH EPITHELIAL-
MESENCHYMAL TRANSITION IN WOMEN WITH HIGH-GRADE SEROUS OVARIAN
CARCINOMA
Tese apresentada ao Programa de Pós-Graduação em Tocoginecologia da
Faculdade de Ciências Médicas da Universidade Estadual de Campinas para
a obtenção do título de Doutora em Ciências da Saúde, na Área de
Oncologia Ginecológica e Mamária.
Thesis presented to the Obstetrics and Gynecology Post Graduation
Program of School of Medical Sciences from State University of Campinas
for obtaining the Ph.D. degree in Health Sciences in Gynecologic Oncology
and Breast Cancer Area.
ORIENTADORA: PROFA. DRA. SOPHIE FRANÇOISE MAURICETTE DERCHAIN
COORIENTADOR: PROF. DR. LUIS OTÁVIO ZANATTA SARIAN
ESTE EXEMPLAR CORRESPONDE À VERSÃO
FINAL DA DISSERTAÇÃO DEFENDIDA PELA
ALUNA SUSANA OLIVEIRA BOTELHO RAMALHO ORIENTADA PELA
PROFA. DRA. SOPHIE FRANÇOISE MAURICETTE DERCHAIN
CAMPINAS
2017
BANCA EXAMINADORA DA DEFESA DE DOUTORADO
SUSANA OLIVEIRA BOTELHO RAMALHO
ORIENTADORA: PROFA. DRA. SOPHIE FRANÇOISE MAURICETTE DERCHAIN
COORIENTADOR: PROF. DR. LUIS OTAVIO ZANATTA SARIAN
MEMBROS:
1. PROFA. DRA. SOPHIE FRANÇOISE MAURICETTE DERCHAIN
2. PROF. DR. LUIZ CARLOS ZEFERINO
3. PROF. DR. JOSÉ BARRETO CAMPELLO CARVALHEIRA
4. PROF. DR. MAX SENNA MANO
5. PROFA. DRA. MARIA DEL PILAR ESTEVEZ DIZ
Programa de Pós-Graduação em Tocoginecologia da Faculdade de Ciências Médicas
da Universidade Estadual de Campinas.
A ata de defesa com as respectivas assinaturas dos membros da banca examinadora
encontra-se no processo de vida acadêmica do aluno.
DATA DA DEFESA: 07/04/2017
DEDICATÓRIA
Aos meus pais Cristina e Eduardo Ramalho que me transmitiram valores, sendo grandes
exemplos de esforço, dedicação e realizações;
Ao meu esposo Juliano Gibertoni, pelo amor e cumplicidade;
À nossa filhota, nosso amorzinho, a doce Catarina Ramalho Gibertoni, sempre nosso encanto
e inspiração.
AGRADECIMENTOS
A minha orientadora, Profa. Dra. Sophie Françoise Mauricette Derchain, pela amizade e revisão
cuidadosa desta tese.
Ao meu coorientador, Prof. Dr. Luis Otávio Zanatta Sarian, pelo incentivo e revisão dos artigos.
Aos membros da banca de qualificação, Prof Dr.Luiz Carlos Zeferino e Prof. Dr. Jose Barreto
Campello Carvalheira que contribuíram para a elaboração final desta tese.
À Profa. Dra. Liliana Aparecida Lucci De Angelo Andrade, pelo suporte e análise anátomo-
patológica e imunoistoquímica.
À Profa. Dra. Iscia Teresinha Lopes Cendes, pela gentileza e generosidade em disponibilizar o
Laboratório de Genética Molecular para realização dos experimentos.
Ao Prof. Dr. Gustavo Antônio de Souza, pela orientação do mestrado e contínuo estímulo para
finalização deste doutorado.
Ao Prof. Dr. Luiz Carlos Teixeira, pelo apoio, estímulo e confiança em meu trabalho.
Ao Prof. Dr. Cassio Cardoso Filho, pelo incentivo na elaboração desta Tese.
À Dra. Geisilene Russano de Paiva Silva, pela amizade e pela generosidade em disponibilizar
o Laboratório de Patologia Experimental do CAISM para realização dos
experimentos.
Ao Rodrigo de Andrade Natal, pelo seu papel fundamental no planejamento, na realização da
parte laboratorial e na análise estatística desta tese.
À Marina Pavanello, pela paciência e contribuição essencial na avaliação dos casos desta
Tese.
À Amanda Canato Ferracini, pela ajuda na elaboração do banco de dados e na realização do q-RT-
PCR.
Ao colegas Luis Felipe Assad Sallum, pela contribuição na coleta de dados.
À colega Adriana Yoshida, pelo incentivo e otimismo nesta jornado.
Aos colegas Uanderson Resende e Leonardo Roberto da Silva, pela amizade e
companheirismo nos momentos difíceis.
Aos funcionários do SAME, da ASTEC, da secretaria da divisão de oncologia e do ambulatório
de quimioterapia do CAISM, pela disponibilidade e solicitude.
Estudo parcialmente financiado pela Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP) processo número 2012/15059-8, Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) processo número 306583/2014-3, Fundo
de Apoio ao Ensino, à Pesquisa e Extensão (FAEPEX) processos número 519.292 e 519.294
RESUMO
Introdução: o carcinoma seroso de alto grau de ovário (CSAGO) é uma doença heterogênea
que apresenta alta mortalidade. Inicialmente, a maioria das mulheres responde à quimioterapia
com platina, porém rapidamente, muitas se tornam resistentes à droga e evoluem com recidiva
e óbito. O maior conhecimento das vias responsáveis pelos mecanismos de invasão e
metástase, em mulheres com CSAGO, poderá ajudar na identificação de biomarcadores
prognósticos e desenvolvimento de novas terapias alvo. A transição epitélio-mesenquimal
(TEM) é um importante processo celular relacionado com invasão e metástases. Alguns
componentes proteicos, tais como o discoidin domain receptor 2 (DDR2), atuando por meio
da extracelular signal-regulated kinase 1/2 (erk1/2), e o co-ativador transcricional yes-
associated protein (YAP), atuando na via Hippo, estão associados a TEM. Nessas vias, os
microRNAs miR-182, miR-96 e miR-9 estão descritos como reguladores pós-transcricionais.
Objetivo: avaliar a expressão do DDR2, do YAP e dos miR-182, miR-96 e miR-9 em blocos
de parafina de mulheres com CSAGO, e sua associação com características clínicas, do tumor,
resposta à platina e sobrevida. Metodologia: foram incluídas 63 mulheres com CSAGO
estádios III/IV, submetidas à quimioterapia com platina de 1996 a 2013 e acompanhadas até
2016 no Hospital da Mulher Prof. Dr. José Aristodemo Pinotti, Brasil. Todas tinham blocos de
parafina e dados completos no prontuário. A expressão do DDR2 e do YAP foi avaliada por
imunoistoquímica em lâminas de microarranjo de tecido (TMA) e os níveis de microRNAs
foram avaliados por reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR).
Para comparação entre a expressão do DDR2 e do YAP com idade, nível de CA125, estádio,
doença residual pós-cirurgia e resposta à platina foram utilizados os testes de Mann-Whitney,
qui-quadrado e Fisher. A sobrevida livre de progressão (SLP) e a sobrevida global (SG) foram
calculadas por regressão de Cox. As curvas de SLP e SG foram estimadas pelo teste de
Kaplan-Meier e comparadas pelo teste de Log-Rank. A expressão dos níveis dos microRNAs
e do DDR2 e YAP foi comparada pelo teste t. Resultados: a expressão do DDR2 foi alta em
8(13,7%) mulheres. Não houve associação entre a expressão do DDR2 e a idade, estádio,
CA125, doença residual pós-cirurgia e resposta à quimioterapia. A SLP foi significativamente
menor nas mulheres cujos tumores apresentaram alta expressão de DDR2 (p=0,03), mas não a
SG (p=0,49). O nível de expressão do miR-182 foi significativamente menor nas mulheres
com alta expressão de DDR2 (p<0,001), mas não o nível de expressão do miR-96 (p=0,067).
Alta expressão nuclear do YAP com baixa expressão citoplasmática foi encontrada em
15(24,5%) mulheres. Não houve associação entre a expressão do YAP e as características da
doença ou sobrevida. O nível de expressão do miR-9 não se associou com a expressão do
YAP. Conclusão: baixos níveis de expressão do miR-182 foram associados com alta expressão
do DDR2 a qual se relacionou com menor SLP. Esses achados sugerem que a ativação da
erk1/2 pelo DDR2 foi relevante para a TEM nesses casos de CSAGO. O papel da via Hippo
permaneceu indeterminado.
Palavras-chave: neoplasias ovarianas, transdução de sinal, platina.
ABSTRACT
Introduction: high-grade serous ovarian carcinoma (HGSOC) is a heterogeneous disease with
high mortality. Initially, most women respond to platinum-based chemotherapy, but rapidly,
many become resistant to the drug and progress with relapse and death. Better knowledge of
the pathways responsible for the mechanisms of invasion and metastasis in women with
HGSOC may help the identification of prognostic biomarkers and the development of new
target therapies. The epithelial-mesenchymal transition (EMT) is an important cellular process
related to invasion and metastasis. Some protein components such as the discoidin domain
receptor 2 (DDR2), acting through the extracellular signal-regulated kinase 1/2 (erk 1/2), and
the transcriptional co-activator yes-associated protein (YAP), acting in Hippo pathway, are
associated with EMT. In these pathways, microRNAs such as miR-182, miR-96 and miR-9
are described as post-transcriptional regulators. Objective: to evaluate the expression of
DDR2, YAP and miR-182, miR-96 and miR-9 in formalin fixed paraffin embedded blocks of
women with HGSOC, and its association with clinical and pathological characteristics,
platinum response and survival. Methodology: 63 women with HGSOC stages III/IV, who
underwent platinum-based chemotherapy from 1996 to 2013, followed up until 2016 at
Women's Hospital Prof. Dr. José Aristodemo Pinotti, Brazil, were included. All women had
paraffin blocks and complete clinical data on their charts. DDR2 and YAP expression were
assessed by immunohistochemistry on tissue microarray (TMA) slides and the microRNAs
were evaluated by quantitative real time Polymerase Chain Reaction (qRT-PCR). For the
comparison of DDR2 and YAP expression with age, CA125 level, stage, post-surgery residual
disease and platinum response, Mann-Whitney, qui-square and Fisher tests were used.
Progression-free survival (PFS) and overall survival (OS) were calculated by Cox regression.
PFS and OS curves were estimated by Kaplan-Meier and compared by Log-Rank test.
Expression of microRNA levels and DDR2 and YAP were compared by t-test. Results: DDR2
expression was high in 8(13.7%) women. There was no association between DDR2
expression and age, stage, CA125, post-surgery residual disease and response to
chemotherapy. PFS was significantly worse in women whose tumors had high DDR2
expression (p=0.03), but not OS (p=0.49). MiR-182 expression level was lower in women
with high DDR2 expression (p<0.001), but not the expression level of miR-96 (p=0.067).
High nuclear expression of YAP with low cytoplasmic expression was found in 15(24.5%)
women. There was no association between YAP expression and clinical characteristics or
survival. MiR-9 expression level was not associated with YAP expression. Conclusion: low
expression levels of miR-182 were associated with high expression of DDR2, which was
associated with poorer DFS. These findings suggest that the activation of erk1/2 by DDR2
was relevant to the EMT of these women with HGSOC. The role of Hippo pathway remained
indeterminate.
Keywords: ovarian neoplasms, signal transduction, platinum.
LISTA DE ABREVIATURAS E SIGLAS
ABCB1 - Adenosine triphosphate binding cassette subfamily B member 1 gene
ASCO - American Society of Clinical Oncology
AUC - Area under curve
BCL2L11 - Bcl2-like 11 gene
BRCA 1 - Breast cancer 1
BRCA 2 - Breast cancer 2
CA - California
CA 125 - Cancer antigen 125
CAISM - Centro de Atenção Integral à Saúde da Mulher
CCNE1 - Cyclin E1 Gene
CEP - Comissão de Ética em Pesquisa
CI - Confidence interval
cpYAP - Cytoplasmic phosphorilated yes associated protein
CSAGO - Carcinoma Seroso de Alto Grau de Ovário
DAB - 3,3´-Diaminobenzidine
DDR1 - Discoidin domain receptor 1
DDR2 - Discoidin domain receptor 2
DMSO - Dimetilsulfóxido
DNA - Ácido desoxirribonucleico
e cols - E colaboradores
EMT - Epithelial-Mesenchymal Transition
erk1/2 - Extracellular signal-regulated kinase 1/2
et al - E outro(s); e outra(s)
FDA - Food and Drug Administration
FFPE - Formalin-Fixed Paraffin-Embedded
FIGO - Federação Internacional de Ginecologia e Obstetrícia
FOXO1 - Forkhead box protein O1
g - Grama
GOG - Gynecologic Oncology Group
H&E - Hematoxilina & eosina
HGSOC - High grade serous ovarian cancer
IC - Intervalo de confiança,
Inc - Incorporation
INCA - Instituto Nacional de Câncer
LATS - Large tumor supressor
miR-182 - Micro Ribonucleic Acid-182
miR-9 - Micro Ribonucleic Acid-9
miR-96 - Micro Ribonucleic Acid-96
miRNA - Micro Ribonucleic Acid
ml - Mililitro
mm - Milímetros
mM - MiliMol
mRNA - Messenger Ribonucleic Acid
MST - Mamalian ste20-like protein kinase
MYC - Myelocytomatosis viral Gene
NF1 - Neurofibromatosis Gene
NJ - New Jersey
nYAP - Nuclear yes associated protein
ºC - Grau Celsius
OS - Overall Survival
PALB2 - Partner and localizer of BRCA2
PARP1 - Poly adenosine diphosphate-ribose polymerase-1
PBS - Phosphate-Buffered Saline
PDZ - Psd-95(Post Synaptic Density Protein), DigA(Drosopli Disc Large Tumor
Suppressor) and ZO1(Zonula Occlu Protein)
PFS - Progression Free Survival
Ph - Potencial hidrogeniônico
p-value - Significância estatística
QNA - Quimioterapia neoadjuvante
qRT-PCR - Quantitative Real Time Polymerase Chain Reaction; Transcrição
Reversa e Reação em Cadeia da Polimerase em Tempo Real
ROC - Receiver Operating Characteristic
SD - Standard Deviation
SG - Sobrevida Global
SLP - Sobrevida Livre de Progressão
Snail1 - Snail 1 (drosophila homolog), zinc finger protein
snRNA - Small nuclear ribonucleic acid
STIC - Serous tubal intraepithelial carcinoma
TAZ - Transcriptional co-activator with PDZ-binding motif
TCGA - The Cancer Genome Atlas
TCLE - Termo de consentimento livre e esclarecido
TEAD - Tea domain-containing transcription factor
TEM - Transição Epitélio Mesenquimal
TERT - Telomerase reverse transcriptase Gene
TMA - Microarranjo de tecidos;Tissue MicroArray
TME - Transição mesenquimal-epitelial
TP53 - Tumor protein 53 Gene
TWIST - Twist-related protein
Unicamp - Universidade Estadual de Campinas
USA - United States of America
UV - Radiação Ultravioleta
VT - Vermont
YAP - Yes associated protein
ZEB 1/2 - Zinc finger E-box binding homeobox 1/2
LISTA DE SÍMBOLOS
% - Por cento
µl - Microlitro
µl/ml - Microlitro por mililitro
µm - Micromilímetro
SUMÁRIO
1. INTRODUÇÃO .................................................................................................................. 15
2. OBJETIVOS ....................................................................................................................... 25
2.1. Objetivo Geral .............................................................................................................. 25
2.1. Objetivos Específicos ................................................................................................... 25
3. METODOLOGIA ............................................................................................................... 26
3.1. Seleção dos Sujeitos ..................................................................................................... 26
3.2. Avaliação Clínica e Seguimento .................................................................................. 28
3.3. Marcadores ................................................................................................................... 28
3.3.1. Expressão Proteica ........................................................................................................ 28
3.3.1.1. Construção do Microarranjo de Tecidos (tissue microarray, TMA) .......................... 28
3.3.1.2. Preparação do Material para Estudo Imunoistoquímico ............................................ 29
3.3.1.3. Interpretação e Escore Imunoistoquímico ................................................................. 30
3.3.2. Expressão de MicroRNA .......................................................................................... 30
3.3.2.1. Extração do RNA ....................................................................................................... 30
3.3.2.2. Transcrição Reversa e Reação em Cadeia da Polimerase em Tempo Real (qRT-PCR)
.................................................................................................................................. 31
3.4. Análise Estatística ........................................................................................................ 31
4. RESULTADOS ................................................................................................................... 32
Artigo 1: Discoidin domain receptor 2 (DDR2) is a predictor of worse outcome in women
with high-grade serous ovarian carcinoma ......................................................................... 33
Artigo 2: Yes associated protein (YAP) in high grade serous ovarian carcinoma .............. 51
5. DISCUSSÃO GERAL ........................................................................................................ 73
6. CONCLUSÃO .................................................................................................................... 77
7. REFERÊNCIAS ................................................................................................................. 78
8. ANEXOS ............................................................................................................................ 88
Anexo 1 - Ficha para coleta de dados clínicos .................................................................... 88
Anexo 2 - Carta de aprovação do projeto no CEP-Unicamp .............................................. 89
Anexo 3 - Adendo a carta de aprovação do CEP-Unicamp ................................................ 92
15
1. INTRODUÇÃO
O câncer de ovário é a terceira neoplasia ginecológica mais incidente no mundo,
com 238.719 casos ao ano e é também a maior causa de óbito com 151.917 mortes [1]. Na
Europa, o número de casos novos de câncer esperado para 2012 era de 65.538 com 42.704
mortes [2]. Nos Estados Unidos, para 2016 são estimados 22.280 casos novos e 14.240 mortes
[3]. No Brasil foram estimados 6.150 casos novos de câncer de ovário para o ano de 2016,
com risco de 5,95 casos a cada 100 mil mulheres. Especificamente para o estado de São
Paulo, foram estimados 1.590 casos novos de câncer de ovário no ano de 2016 [4].
Os cânceres primários de ovário são classificados em três grupos: epiteliais
(90%), tumores de cordões sexuais e estroma (8%) e de células germinativas (3%) [5]. Já os
tumores metastáticos no ovário correspondem a 5% a 15% dos casos e, geralmente, o tumor
primário é do trato gastrointestinal, mama ou endométrio. Dentre os carcinomas existem
diversos subtipos que apresentam diferentes características clinico-patológicas, moleculares e
comportamentais. São os subtipos: serosos, endometrióides, células claras, mucinosos, mistos
e indiferenciados [5].
A maioria dos carcinomas de ovário é esporádica, somente 10% a 20% dos casos
tem caráter hereditário. A síndrome do câncer de mama e ovário corresponde a 90% dos
cânceres hereditários e é de herança autossômica dominante, relacionada a mutações dos genes
Breast Cancer 1 Gene (BRCA1) e Breast Cancer 2 Gene (BRCA2) [6, 7]. Mulheres com
mutações nestes genes possuem um risco de até 46% de vir a desenvolverem câncer de ovário
durante a vida [8].
Ao observar que os carcinomas de ovário constituem um grupo heterogêneo e
distinto em suas características clinico patológicas, uma proposição dualista de patogênese
identificou as categorias tipo I e tipo II [9-11]. Este modelo relaciona os tipos histológicos
com suas prováveis lesões precursoras. O tipo I engloba os carcinomas serosos de baixo grau,
carcinomas endometrióides, mucinosos, células claras e tumores malignos de Brenner. Estes
carcinomas apresentam progressão lenta a partir de tumores benignos, para tumores
borderline até tumores malignos. Geralmente se apresentam como grandes lesões císticas
unilaterais de crescimento lento. Quando restritos ao ovário os tumores tipo I apresentam
excelente prognóstico porém, se diagnosticados em estádios avançados o prognóstico é
reservado. Os tumores tipo I são responsáveis por 10% dos óbitos por câncer de ovário. Os
tumores tipo II compreendem os carcinomas serosos de alto grau, indiferenciados e
carcinossarcoma. Estes tumores são agressivos, de crescimento rápido e disseminação
16
peritoneal precoce. Assim, a maioria dos casos apresenta ao diagnóstico doença avançada com
ascite [11].
Algumas teorias sobre a origem e prováveis lesões precursoras do câncer do
ovário foram propostas. A mais recente delas, é que a doença teria início em órgãos extra-
ovarianos, acometendo o ovário secundariamente. O carcinoma seroso surgiria através do
deslocamento de células epiteliais tubárias para o epitélio de superfície ovariano, que exposto
durante a ovulação resulta, na formação de cisto de inclusão. A implantação direta das
chamadas serous tubal intraepithelial carcinoma (STIC) nos cistos de inclusão do ovário
durante a ovulação levaria a lesões precursoras do carcinoma seroso de alto grau de ovário
(CSAGO) [12-14].
O CSAGO é o subtipo de carcinoma de ovário mais comum e, é o responsável
pela grande maioria dos óbitos. Os CSAGO são divididos morfologicamente em tipo usual e
variante sólida, pseudoendometrióide e transicional (SET). O tipo usual é composto por
massas sólidas de células de padrão papilar, glandular e cribiforme com necrose associada. Os
CSAGO tipo SET se apresentam como massas sólidas de células que simulam carcinomas
endometrióides e de células transicionais, alto índice mitótico e mais frequentemente
infiltrado linfocitário peri tumoral. Os CSAGO tipo SET ocorrem em pacientes jovens, são
mais frequentemente associados a mutações do BRCA1 e tem melhor prognóstico [11].
O tratamento atual padrão para mulheres com CSAGO consiste em cirurgia
citoredutora seguida de quimioterapia baseada em platina e paclitaxel [15, 16]. A cirurgia
citoredutora ótima associada à quimioterapia baseada em platina é o principal fator
prognóstico [17-19]. Em pacientes com ampla disseminação tumoral, uma estratégia cirúrgica
agressiva e abrangente, incluindo cirurgia radical pélvica, ressecção intestinal e remoção de
lesões em abdome superior (diafragma, baço e fígado) é freqüentemente necessária para se
obter uma citoredução ótima [20,21]. No entanto, o esforço de alcançar cirurgia citoredutora
ótima aumenta a morbidade e mortalidade peri-operatória.
Assim, buscou-se estratégias alternativas que equilibrassem eficácia e segurança
[22]. Neste contexto, a quimioterapia neoadjuvante (QNA) vem sendo empregada como
alternativa de tratamento. Estudos randomizados que avaliaram a QNA mostraram resultados de
não inferioridade [23-25]. Em recente revisão das diretrizes da American Society of Clinical
Oncology (ASCO) a QNA foi incluída como uma opção de tratamento inicial [26]. Entretanto,
apesar dos resultados de não inferioridade, há uma tendência em considerar ideal iniciar o
tratamento com cirurgia citoredutora [27]. Vergote e cols. [23] não observaram diferença na
sobrevida comparando mulheres com carcinoma de ovário avançado submetidas à cirurgia
17
citoredutora inicial, com aquelas submetidas à QNA. Entretanto, van Meurs e cols. [28], numa
análise secundária, observaram que as mulheres com doença em estádio IIIC ou doença
metastática de menor volume apresentaram melhor sobrevida com cirurgia primária. Mulheres
com doença em estádio IV e com grandes volume de doença metastática apresentaram melhor
sobrevida com QNA. Assim, a cirurgia citoredutora inicial pareceu mais eficaz em mulheres
com CSAGO avançado, com menor volume de doença, na qual a citoredução ótima foi mais
factível. Em mulheres com grande volume de doença metastática, a QNA seria a melhor opção
por não atrasar o início do tratamento. Deste modo, a avaliação preoperatória é essencial para
definir os casos em que não é possivel atingir citoredução ótima [29].
A quimioterapia baseada em platina e paclitaxel é considerada padrão ouro para a
primeira linha de tratamento [30-33]. O protocolo de administração vem sendo reavaliado com
estratégias de infusão em dose densa. Katsumata e cols. [34] demonstraram uma melhora da
sobrevida livre de progressão (SLP) de cerca de 10 meses e da sobrevida global (SG) de cerca
de 40 meses utilizando dose densa. Entretanto, um estudo de meta-análises, comparando dose
densa com dose convencional, demonstrou benefício somente em SLP e não em SG. [35].
A resposta à quimioterapia com platina e paclitaxel é o fator determinante da
sobrevida. Apesar das altas taxas de resposta inicial à quimioterapia, muitas mulheres
apresentam resistência primária ou adquirida as drogas [36]. Estima-se que pacientes resistentes
à platina tenham uma SLP média de 3-4 meses com SG média de 12 meses. Essas mulheres
também apresentam baixas taxas de resposta a outras linhas de tratamento com drogas
citotóxicas alternativas como topotecano, gencitabina e paclitaxel [37].
A principal teoria que explica a resistência à platina é a teoria clonal. Infere-se que
no momento do diagnóstico, pequenas populações de células intrinsecamente resistentes à
platina já estejam presentes no tumor. Assim, embora o tratamento citotóxico inicial possa ser
capaz de destruir os clones sensíveis à quimioterapia, ele também seleciona os clones
resistentes [38]. De acordo com Patch e cols. [39] a resposta à platina se classifica em quatro
categorias: 1) refratária: a doença progride na vigência da quimioterapia inicial; 2) resistente:
tem progressão da doença nos primeiros seis meses após o termino da quimioterapia inicial;
3) sensível primário: não há evidência de progressão da doença nos seis meses após o término
da quimioterapia inicial, ou há normalização do cancer antigen 125 (CA125), ou há 50% de
diminuição do CA125 (desde que tenha CA125 aumentado no inicio) após múltiplas linhas de
quimioterapia baseada em platina; 4) resistência adquirida: não há resposta à platina na
doença recidivada, após ter tido resposta em tratamentos anteriores.
18
A partir de novas técnicas de biologia molecular, iniciou-se um grande movimento
mundial com intuito de sequenciar vários tumores, afim de encontrar medicamentos mais
eficazes [40]. A genética dos CSAGO começou a ser desvendada no projeto The Cancer
Genome Atlas (TCGA) [41]. O genoma de 489 CSAGOs foi sequenciado com uma análise
completa do exoma de 316 casos. Foi avaliada a expressão de ácido ribonucleico (RNA)
mensagerio (mRNA), microRNA, metilação da região promotora e número de cópias de ácido
desoxirribonucleico (DNA). Nesta análise, foram identificados quatro subtipos transcricionais
baseados na expressão de mRNA, três subtipos baseados na expressão de microRNA, quatro
subtipos baseados na região promotora metilada e uma assinatura de transcrição associada à
sobrevida. Mutações germinativas dos genes BRCA1 e BRCA2 foram encontradas em 9% e
8% dos casos e mutações somáticas foram encontradas em mais 3%. Foram notadas inúmeras
aberrações em número de cópias de DNA principalmente os seguintes genes-alvo: Cyclin E1
Gene (CCNE1), Myelocytomatosis viral Gene (MYC), Telomerase Reverse Transcriptase
Gene (TERT) e Neurofibromatosis Gene (NF1). Esses achados sugerem que os CSAGO
evoluem a partir de erros na via de reparo do DNA, seguido de instabilidade cromossômica e
alterações no número de cópias. Baseado nos resultados do TCGA, Patch e cols. [39]
compararam o sequenciamento de mulheres com CSAGO sensíveis, resistentes e refratárias. A
mutação no gene TP53 foi encontrada em todos os CSAGO e 50% apresentaram mutações em
genes associados à via de reparo do DNA, a recombinação homóloga. Nas mulheres
resistentes à platina foram encontradas poucas mutações com potencial para desenvolvimento
de drogas-alvo. Em 20% dos casos de mulheres com CSAGO resistentes à platina Patch e
cols. [39] observaram uma associação com a amplificação do gene CCNE1. A amplificação
do gene CCNE1 é uma alteração de pior prognóstico e mutuamente exclusiva de defeitos na
via da recombinação homóloga. Mulheres com CSAGO com mutações BRCA1 e BRCA2
apresentam melhor sobrevida. Outros mecanismos de resistência à platina observados em
mulheres com CSAGO recidivados são: reversão da mutação BRCA1 e BRCA2, perda da
metilação da região do promotor BRCA1, mutação nos genes Forkhead Box Protein O1
(FOXO1) e BCL2-Like 11 (BCL2L11) e aumento da expressão do gene Adenosine
Triphosphate Binding Cassette Subfamily B member 1 Gene (ABCB1).
Algumas terapias alvo desenvolvidas para mulheres com CSAGO buscam
bloquear vias de sinalização que induzem crescimento, disseminação e resistência à
quimioterapia. Dentre estas novas terapias, destacam-se as drogas antiangiogênicas como o
bevacizumab [42] e os inibidores de Poly Adenosine Diphosphate-Ribose Polymerase-1
(PARP1) como o olaparib [43].
19
A adição do bevacizumab à quimioterapia baseada em platina no tratamento de
primeira linha do câncer de ovário, foi investigada na Europa, Austrália e Canadá pelo
Gynecologic Cancer Intergroup (GCIG) [44] e nos Estados Unidos da América pelo
Gynecologic Oncology Group (GOG) [45]. O bevacizumab, como manutenção após o término
da quimioterapia, se associou com aumento significativo da SLP levando a sua aprovação.
Paralelamente, observou-se que o bevacizumab também aumentou significativamente a SLP
em mulheres com carcinoma de ovário recorrente, sensíveis a platina [46]. E finalmente, nos
carcinomas de ovário resistentes a platina, demonstrou-se que bevacizumab associado a
quimioterapia também aumentou significativamente a SLP [47].
Já, os inibidores de PARP como o olaparib, atuam especialmente em CSAGO com
mutações germinativas ou somáticas nos genes BRCA1 ou BRCA2. A PARP é uma proteína
nuclear que ativa mecanismos de reparo do DNA via excisão de base. Inicialmente, os
inibidores de PARP foram desenvolvidos como agentes quimio-sensibilizadores, e sua
atividade como agente único não era reconhecida [48]. Em 2005, Bryant e cols. [49] e Farmer
e cols. [50] observaram que as linhagens de células BRCA1 ou BRCA2 heterozigóticas eram
100 a 1000 vezes menos sensíveis aos inibidores de PARP do que as células deficientes em
BRCA1. Concluiram que as células deficientes em BRCA eram selectivamente sensíveis à
inibição da PARP por um mecanismo de letalidade sintética. Neste mecanismo as células
cancerosas são seletivamente sensíveis à inativação de dois genes ou de duas vias, já que a
inativação de qualquer dos genes ou das vias sozinha não é letal. Os inibidores da PARP
bloqueiam o reparo de quebra de fita única de DNA. Eles inibem a via de reparo de excisão de
base o que conduz à morte celular. Se a célula não pode iniciar a recombinação homóloga,
como é o caso dos tumores com mutação BRCA1 ou BRCA2, recorre a vias mais propensas a
erros. A união por extremidade não homóloga ou alinhamento de fita única podem causar
mutações cromossômicas grosseiras, inibição de crescimento e eventual morte celular [48].
Mulheres com CSAGO e mutação no BRCA1 ou BRAC2 apresentam uma
elevada sensibilidade aos inibidores da via de reparo de excisão de bases, como os inibidores
de PARP [48]. O olaparib foi o primeiro inibidor de PARP a ser introduzido na prática clínica
para o tratamento de CSAGO recidivado. Ele foi estudado como terapia de manutenção em
mulheres com CSAGO recidivados, sensíveis à platina, que receberam dois ou mais regimes
baseados em platina e que tiveram uma resposta parcial ou completa ao seu mais recente
regime baseado em platina [51, 52]. Observou-se um aumento significativo da SLP em
mulheres tratadas com olaparib como terapia de manutenção, quando comparada com o grupo
placebo. Esse aumento da SLP foi significativamente maior nas mulheres com mutação do
20
BRCA. Entretanto, não houve diferença da SG independentemente da mutação do BRCA.
Oza e cols. [53] observaram um aumento significativo da SLP, sem impacto na SG, em
mulheres com CSAGO utilizando olaparib como droga concomitante à quimioterapia,
seguido de manutenção até progressão. Assim o olaparib também foi aprovado como
manutenção até progressão para mulheres com CSAGO, sensíveis a platina, com BRCA
mutado que já receberam três ou mais linhas de quimioterapia com platina [54].
Apesar do aumento da SLP observado com bevacizumab e olaparib, ainda não se
obteve aumento da SG. Reconhece-se hoje a natureza complexa e heterogênea dos CSAGO,
que apresentam um comportamento agressivo e disseminação metastática precoce. A
identificação de biomarcadores e seu papel em vias de sinalização associadas à invasão e
metástase, é uma estratégia promissora para o desenvolvimento de novas drogas alvo, com
potencial aumento da sobrevida das mulheres com CSAGO.
Os mecanismos de invasão e metástase são duas características intrínsecas às
células cancerosas [55]. Estas possibilitam crescimento tumoral e disseminação metastática.
Os mecanismos de invasão e metástases ocorrem em etapas sendo a transição epitélio-
mesenquimal (TEM) o processo pelo qual as células epiteliais adquirem habilidades de
invadir, resistir a apoptose e se disseminar. Em uma primeira fase as células epiteliais perdem
tanto a polaridade quanto o contato células a célula. A e-caderina é a molécula responsável
pela adesão célula a célula. Assim uma das características da primeira fase de ativação da
TEM é a perda da expressão de e-caderina. Ainda na primeira fase de invasão as células
cancerosas escapam do tumor primário e evadem para o parênquima normal vizinho. Já, na
segunda fase de metastatização, as células entram na circulação linfática e sanguínea. Na
circulação invadem o parênquima dos tecidos distantes formando pequenos nódulos
chamados micrometástases [55-57].
Nas lesões micrometastáticas ocorre a colonização. Para o sucesso da colonização
é necessária a adaptação das células cancerosas a um novo microambiente. A TEM é um
processo reversível, que pode ser transitoriamente ativado ou inativado durante os
mecanismos de invasão e metástase. Assim, durante a colonização ocorre o processo reverso
chamado transição mesenquimal-epitelial (TME). É através da TME que as células tumorais
adquirem habilidades para se desenvolver e se tornar macrometástases. Observa-se que as
células tumorais que compõe as macrometástases apresentam um padrão histopatológico
similar às células do tumor primário que nunca passaram pelo processo de TEM [55-57].
A expressão de fatores de transcrição associados à TEM ocorre em resposta à
ativação de diferentes vias de sinalização. Estas vias de sinalização se compõem de cascatas
21
intracelulares de quinases que induzem fatores que ativam a transcrição de genes-alvo
associados à TEM. Diversos fatores de transição como snail 1 drosophila homolog, zinc
finger protein (snail1), twist-related protein (twist) e zinc finger e-box binding homeobox 1/2
(Zeb1/2) foram descritos como atuantes em vias de sinalização induzindo a TEM. Estes
fatores de transcrição são importantes especialmente como estimuladores do mecanismo de
invasão, já que sua expressão se associa à supressão da e-caderina e à disseminação de
metástases [57]. O papel de várias vias de sinalização e fatores de transcrição a elas
associadas, vem sendo estudado na TEM. Entre elas se destacam a via Hippo [58] e a proteína
extracellular signal-regulated kinase 1/2 (erk1/2) [59].
A via Hippo promove proliferação celular, apoptose e auto-renovação das células-
tronco. Quando ativada a via Hippo age como supressora tumoral [60]. É uma cascata de
quinases cujo objetivo final é a fosforilação, retenção citoplasmática e degradação de seu co-
ativador transcricional, a yes-associated protein (YAP) [61]. Quando desregulada a via Hippo
contribui para o desenvolvimento do câncer [62]. Estudos in vitro demonstraram que a
hiperexpressão do YAP induz a TEM, inibe a apoptose e aumenta o número de células-tronco
[63, 64].
A erk1/2 é uma quinase com um domínio de ligação para o fator de transcrição
snail1 [65]. Estudos in vitro demonstraram que quando ativada a erk1/2 fosforila o fator de
transcrição snail1 induzindo sua acumulação nuclear. A acumulação nuclear do snail1 está
associada à perda da expressão da e-caderina e à indução da TEM [58, 66]. O discoidin
domain receptor 2 (DDR2) é um receptor tirosino quinase ativado pelo colágeno tipo I da
matriz extracelular. Após a ativação pelo colágeno, o DDR2 aciona importantes componentes
de sinalização como a sarcoma quinase (src) e a erk1/2 quinase [67,68]. Em câncer da mama
o DDR2 estabiliza a snail1 por meio da ativação das quinases src e erk, promovendo in vitro
invasão e migração, e, in vivo, metástases. Assim, em carcinomas da mama a expressão de
DDR2 mantém o nível e a atividade da snail1 sustentando a TEM [69, 70].
Diversos reguladores da TEM têm sido estudados, entre eles a recém-descoberta
classe dos microRNAs [56]. Os microRNAs são RNAs não codificadores, compostos por
aproximadamente 22 nucleotídeos. Seu mecanismo de ação inclui a ligação à região 3' não-
traduzida de seus mRNAs alvos, induzindo a degradação do mRNA alvo ou inibindo a
tradução. Eles assim atuam em nível pós transcricional, regulando a expressão de genes [71].
Os microRNAs regulam a expressão de centenas de genes de modo a atuar como figuras
centrais de múltiplas vias de sinalização. Análises de bioinformática estimam que existam
mais de 1.800 microRNAs que regulam até 60% da expressão gênica (transcriptoma) [72].
22
Os microRNAs estão frequentemente desregulados no câncer, e esta desregulação
parece ter um papel fundamental para sua ocorrência, progressão e disseminação. Estima-se
que a expressão aberrante ocorra porque os genes que codificam os microRNAs estão
presentes em locais do genoma humano susceptíveis a rearranjos cromossômicos,
amplificações e deleções gênicas, também chamadas regiões cancerígenas do genoma [73].
Outros fatores como regulações epigenéticas e anormalidades em genes que regulam os
microRNAs contribuem para a expressão alterada dos microRNAs em tecidos tumorais. A
expressão aberrante pode estar associada a atividade pro ou anti-tumoral. Vários microRNAs
agem diretamente sobre fatores de transcrição e componentes das vias de sinalização
associadas à TEM [56]. Estes microRNAs podem atuar como oncogenes ou como supressores
tumorais [74].
Portanto, a TEM é um processo complexo que pode ser iniciado por múltiplas
sinalizações que ativam fatores de transcrição, receptores de tirosino quinase e
microRNAs [55,56,58,61]. Deste modo focamos nosso estudo em duas proteínas (DDR2 e
YAP), e em 3 microRNAs (miR-182, miR-96 e miR-9) componentes de vias de
sinalização associadas à TEM.
O DDR2 interage com componentes estruturais da matrix extracelular que, em
nivel celular, controlam processos fundamentais de proliferação, diferenciação, migração e
sobrevivência celular [58]. A expressão aberrante do DDR2 é encontrada em vários tumores
sólidos como pulmão, tumores de cabeça e pescoço e mama além de ovário [75-78].
Especialmente em carcinomas mamários [75], em carcinomas de cabeça e pescoço [76] e em
carcinoma de pulmão [77], a expressão desregulada de DDR2 se correlaciona com pior
prognóstico. Em carcinomas da mama triplo negativo, a alta expressão do DDR2 com a baixa
expressão do DDR1 se associou a uma pior sobrevida [75].
O papel da expressão do DDR2 em carcinomas de ovário é duvidoso.
Recentemente Fan e col. [78] avaliaram a expressão de DDR2 em mulheres com carcinomas
de ovário. Observaram uma associação significativa do DDR2 com estádio e metástases
peritoneais. A expressão do DDR2 se associou como fator independente de pior SG. Por estar
associada a um pior prognóstico, o DDR2 é um potencial alvo terapêutico a ser estudado no
tratamento de carcinomas. Em câncer de mama o mecanismo pelo qual o DDR2 atua parece
ser sua ativação via colágeno tipo1 e a fosforilação do fator de transcrição snail1 [58]. O
snail1 suprime o miR-182 para iniciar o processo de invasão e metastases induzindo a TEM.
Por outro lado o miR-182 inibe snail1 para restabelecer a identidade epitelial para colonização
e formação de macrometástase, induzindo a TME [79-83].
23
O YAP é um co-ativador transcricional importante da via de sinalização Hippo
[61,84]. A Hippo é uma via que quando ativa induz uma cascata de fosforilação que retém o
YAP no citoplasma. O YAP retido no citoplasma não consegue se mover para o núcleo. A
via Hippo inativada ocorre quando o YAP não está fosforilado e portanto transloca-se para o
núcleo. O YAP nuclear interage com o fator de transcrição tea domain-containing
transcription factor (TEAD) e ativa genes proliferativos e anti-apoptóticos induzindo a
TEM [85-89].
A expressão do YAP e YAP fosforilado vem sendo descrita como um fator de pior
prognóstico [88]. Em carcinomas metaplásicos da mama a expressão nuclear do YAP se
associa à perda da expressão de e-caderina e a um fenótipo relacionado à TEM [90]. Em
carcinomas gástricos Kang e cols. [91] demonstraram por análises in vitro e in vivo que a alta
expressão de YAP e seu acúmulo no núcleo se associa com pior sobrevida livre de doença. Em
carcinoma de colón, a alta expressão de YAP apresentou uma tendência a menor sobrevida e,
a co-expressão de YAP e transcriptional co-activator with PDZ-binding motif (TAZ) se
correlacionaram com pior prognóstico [92]. Em câncer de ovário Xia e cols. [88] e Hall e
cols. [87] avaliaram a ativação da YAP em culturas de células e em amostras de tecido
parafinado. Observaram que a ativação do YAP promoveu o crescimento tumoral, resistência
à platina, perda da inibição por contato e aumento da migração celular. Especialmente a alta
expressão do YAP nuclear (nYAP) combinada à baixa expressão do YAP fosforilado
citoplasmático (cpYAP) se associaram com uma sobrevida em 5 anos 50% menor. A alta
expressão do cpYAP foi um fator independente de melhor prognóstico e a alta expressão de
nYAP se associou à recidiva, resistência à platina e pior sobrevida [87, 88, 93]. Os níveis de
expressão do miR-9 tem sido associados com a via Hippo em câncer da mama e em câncer
gástrico [94, 95].
O tecido parafinado permite a avaliação de proteínas nucleares e citoplasmáticas
assim como detecção dos níveis de microRNA [78, 96]. A imunoistoquímica é um método de
baixo custo e validado em microarranjo de tecido parafinado [97]. Tem protocolos
sedimentados em vários laboratórios de anatomia patológica e apresenta alta
reprodutibilidade. Entretanto há uma grande variedade na sensibilidade dos reagentes,
levando a resultados falso positivos e falso negativos, assim como variações inter e intra
observador [75-78, 87, 88, 90-92].
A transcrição reversa e reação em cadeia da polimerase (PCR) em tempo real
(qRT-PCR) é um dos métodos mais utilizados para detectar a expressão relativa de um
microRNA específico [96]. A qRT-PCR envolve a transcrição reversa, reação em cadeia de
24
polimerase e análise de seus produtos no momento que se formam. A variabilidade
experimental está associada às habilidades do operador, especialmente na fase RT. Ainda
assim por meio de um experimento bem planejado, aplicado com controles adequados, a qRT-
PCR é um dos métodos mais sensíveis e eficientes para medida da expressão gênica [98].
O Hospital da Mulher Prof. Dr. José Aristodemo Pinotti - CAISM - Unicamp é um
hospital de referência no atendimento terciário. Possui um serviço de ginecologia oncológica
onde realiza desde o diagnóstico até o tratamento e seguimento de um número expressivo de
mulheres com carcinoma de ovário. A avaliação de componentes das vias responsáveis pelos
mecanismos de invasão e metástase em mulheres com CSAGO poderá ajudar na identificação
de biomarcadores prognósticos e no potencial desenvolvimento de novas terapias alvo
moleculares. A TEM é um importante mecanismo celular relacionado com invasão e
metástases. Optamos por estudar a expressão proteica do DDR2 e do YAP, assim como os
níveis de expressão dos miR-182, miR-96 e miR-9 em mulheres com CSAGO e sua
associação com características clínicas, do tumor, resposta á platina e sobrevida dessas
mulheres brasileiras, atendidas neste serviço de referência.
25
2. OBJETIVOS
2.1. Objetivo Geral
Avaliar a expressão do receptor tirosino quinase DDR2 e do co-ativador
transcricional YAP assim como dos reguladores pós transcricionais miR-182, miR-96 e miR-9
relacionados com a TEM em blocos de parafina de mulheres com CSAGO, e sua associação
com características clínicas, do tumor, resposta à platina e sobrevida dessas mulheres.
2.1. Objetivos Específicos
2.2.1. Avaliar a relação da expressão do DDR2 com as características clínicas, do tumor,
resposta à platina e sobrevida em mulheres com CSAGO e a relação entre a expressão de
DDR2 e os níveis de miR-182 e miR-96.
2.2.2. Avaliar a relação entre a alta expressão do nYAP com baixa expressão de cpYAP com as
características clínicas, do tumor, resposta à platina e sobrevida em mulheres com CSAGO e a
relação entre a expressão da YAP e os níveis de miR-9.
26
3. METODOLOGIA
Esta tese consiste em dois estudos inter-relacionados, realizados no Hospital da
Mulher Prof. Dr. José Aristodemo Pinotti, Centro de Atenção Integral à Saúde da Mulher
(CAISM), Universidade Estadual de Campinas, Unicamp. A metodologia geral para os dois
estudos é apresentada a seguir, enquanto os detalhes de cada um estão especificados nas
respectivas publicações.
3.1. Seleção dos Sujeitos
Para este estudo de coorte reconstituído, foram selecionados os blocos de parafina
e os prontuários de 114 mulheres tratadas por CSAGO no Hospital Dr José Aristodemo
Pinotti, Centro de Atenção Integral à Saúde da Mulher (CAISM) de 1996 até 2013 e
acompanhados até 2016. Foram incluídas 63 mulheres com blocos de parafina e com dados
completos no prontuário. Este estudo foi aprovado pelo Comitê de Ética e Pesquisa (CEP
1086/2009 e CEP 710.452/2014). Todos os espécimes patológicos foram coletados durante a
cirurgia primária ou por biópsia de fragmentos aberta ou transcutânea realizada antes da
quimioterapia. Foram analisados por um patologista especialista em patologia ginecológica de
acordo com as diretrizes da Classificação Internacional de Tumores da Organização Mundial
de Saúde [5]. Todas as mulheres foram estadiadas de acordo com a classificação da Federação
Internacional de Ginecologia e Obstetrícia FIGO [99]. Foram excluídos casos com doença em
estádio I e II (12 mulheres), segundo tumor primário (2 mulheres), que fizeram menos de 3
ciclos de quimioterapia baseada em platina (14 mulheres), sem bloco de parafina disponível
(17 mulheres), e prontuários não disponíveis (6 mulheres). Assim, foram incluídos os blocos
de parafina 63 mulheres com CSAGO e dados completos no prontuário. Devido à dificuldade
técnica na leitura da imunoistoquímica do DDR2, cinco mulheres foram excluídas desse
primeiro estudo. A leitura do YAP foi inadequadas em dois blocos que foram excluídos do
segundo estudo. A seguir a figura ilustra as pacientes selecionadas, incluídas e excluídas em
cada estudo.
27
Fluxograma de seleção das mulheres do artigo 1 e artigo 2
28
3.2. Avaliação Clínica e Seguimento
A idade, estádio, nível sérico de CA125, doença residual pós cirurgia, ciclos de
quimioterapia baseada em platina, resposta quimioterapia baseada em platina, sobrevida livre
de progressão e sobrevida global foram coletados dos prontuários das mulheres. A resposta a
platina foi classificada de acordo com Patch e cols [39] 1) refratárias: a doença progride na
vigência da quimioterapia inicial; 2) resistente: tem progressão da doença nos primeiros seis
meses após o termino do tratamento inicial; 3) sensível primário: mulheres sem evidência de
progressão da doença nos seis meses após o término da quimioterapia ou mulheres com
normalização do cancer antigen 125 (CA125), ou 50% de diminuição do CA125 (desde que
tenha CA125 aumentado no inicio) em uma ou múltiplas linhas de quimioterapia baseada em
platina; 4) resistência adquirida: mulheres que não respondem mais a platina na doença
recidivada, após terem respondido a platina em tratamento anterior. A sensibilidade inicial é
inferida se a paciente com doença residual macroscópica após cirurgia não teve progressão
por seis meses após o termino da quimioterapia e/ou teve uma resposta completa ou maior que
50% para terapia de segunda ou terceira linha [39]. Para resposta a platina o tempo de
resposta foi estimado em meses a partir do final do tratamento primário de quimioterapia
baseada em platina até a data da progressão. A sobrevida foi avaliada em relação a sobrevida
livre de progressão (SLP) e sobrevida global (SG). Para a SLP e SG, o tempo de sobrevida foi
estimado em meses, da data do diagnóstico até a última consulta, progressão ou óbito. A
progressão/recidiva foi avaliada através de descrição do exame clínico no prontuário, exames
de imagem ou nível do CA125.
3.3. Marcadores
3.3.1. Expressão Proteica
3.3.1.1. Construção do Microarranjo de Tecidos (tissue microarray, TMA)
Após selecionar as lâminas histológicas originais do tumor coradas rotineiramente
com hematoxilina & eosina (H&E) duas áreas representativas do tumor de cada caso, foram
marcadas com tinta permanente em vidro. Foi confeccionada uma planilha para registro dos
números dos blocos de parafina. Foi realizada a perfuração das duas áreas marcadas nos
blocos de parafina com uma agulha do aparelho de TMA (Beecher Instruments Microarray
Technology, Silver Spring, CA, USA), na espessura de 2,0mm. Estes cilindros de tecidos
retirados foram ordenados sobre o bloco receptor do aparelho de TMA com distância de
29
0,2mm entre si. Após a confecção dos blocos de TMA foram realizados cortes histológicos em
lâminas silanizadas (Starfrost), recobertas com adesivo e submetidas à fixação por UV
(Instrumedics Inc, Hackensack, NJ, USA). Posteriormente, as lâminas foram submetidas a
um banho de parafina e armazenadas em freezer a -20°C, até o momento do uso para reação
imunoistoquímica. O processamento e a análise do TMA foi realizado de acordo com o
protocolo validado em patologia de ovário [97].
3.3.1.2. Preparação do Material para Estudo Imunoistoquímico
As lâminas sinalizadas foram hidratadas em álcool etílico em concentrações
decrescentes (100, 80, 50%) e lavadas com água destilada corrente. A atividade da peroxidase
endógena foi bloqueada com três banhos, cada um com duração de 3 minutos, em água
oxigenada a 10 volumes, seguidos de lavagem em água corrente e destilada. Para recuperação
antigênica utilizou-se a panela à vapor Pascal Dako, com o objetivo de “desmascarar” os
antígenos. As lâminas foram imersas em tampão citrato de sódio pH 6,0 (10mM) à 95°C
durante 30 minutos. A seguir, resfriadas em temperatura ambiente durante 20 minutos e
lavadas em água corrente e destilada. Após esta etapa, os cortes histológicos foram incubados
em câmara úmida à 4°C, por uma noite, com seguintes anticorpos primários específicos: para
detectar o DDR2 foi utilizado o anticorpo monoclonal rábico anti DDR2: (Abcam,
Cambridge, USA 1:1500); para detectar o YAP nuclear foi utilizado o anticorpo monoclonal
de coelho anti YAP (Abcam, Cambridge, USA1:100) e; para detectar o YAP citoplasmática
fosforilada foi utilizado o anticorpo monoclonal de coelho anti YAP phospho S127 (Abcam,
Cambridge, USA1:350). Após a incubação, as lâminas foram lavadas três vezes em
Phosphate-Buffered Saline (PBS), sob agitação (solução salina tamponada com fosfato pH 7,4
a 7,6) e secadas. Como sistema de detecção, as lâminas são incubadas com ADVANCETM
HRP
Detection System (Dako) a 37°C durante 1 hora e a seguir, submetidas a três lavagens em PBS
sob agitação. Após a incubação, a revelação foi feita com substrato cromogênico 3,3´-
Diaminobenzidine(DAB), SIGMA, código D5637) na proporção de 0,06g para 100ml em
PBS, 500μl de água oxigenada 3% e 1ml de dimetilsulfóxido (DMSO) à 37°C durante 5
minutos. Finalmente, as lâminas foram lavadas em água corrente e contra-coradas com
hematoxilina de Harris durante 30 a 60 segundos. Os cortes foram desidratados em banhos de
álcool etílico em concentrações crescentes e diafanizadas em três banhos de xilol para, a
seguir, serem montadas em lamínula e resina (Entellan). Controles internos e externos
positivos e negativos foram utilizados para validar as reações imunistoquímicas.
30
3.3.1.3. Interpretação e Escore Imunoistoquímico
A leitura do DDR2 foi realizada de acordo com o percentual e a intensidade das
células. O controle positive foi realizado no tecido miocárdico. Foi analisada a expressão
proteica citoplasmática do DDR2 de acordo com a seguinte escala: (1) percentual de células
coradas ≤5%, escore 0; 6-25%, escore 1; 25-50%, escore 2; 51-75%, escore 3; e >75%, escore
4; e (2) intensidade da células: sem coloração, escore 0; pálida, escore 1; amarela escore 2; e
marrom, escore 3. Os valores dos escores (1) e (2) foram multiplicados e a coloração final foi
graduada como ausente (escore 0), fraca (escore 1-4), moderada (escore 5-8) ou forte (escore
9-12) [78]. Foi definido através da análise da curva Receiver operating characteristic (ROC)
que escores baixo/médio compreende 0-8 e escore 9-12 compreende o escore alto.
A coloração do YAP considerou uma associação de YAP nuclear (nYAP) e YAP
citoplasmático fosforilado (cpYAP). O controle positivo foi realizado no tecido de
adenocarcinoma de próstata. Para o nYAP foi considerada apenas o percentual de células com
núcleos corados: casos com mais de 10% das células com núcleo corado foram classificados
como alta expressão. Casos negativos ou menos de 10% das células com núcleo corado foram
classificados como baixa expressão [90]. Para o cpYAP foram considerados o percentual de
células coradas e a intensidade: (1) percentual de células coradas 0%, escore 0; >0-1%, escore
1; >1-10%, escore 2; >10-33%, escore 3; >33-66%, escore 4; e >66-100%, escore 5 (2)
intensidade da células: sem coloração, escore 0; pálida, escore 1; amarela escore 2; e marrom,
escore 3. Os valores dos escores (1) e (2) foram somados [87] e a coloração final foi graduada
como cpYAP baixa expressão (escore 0 a 6) e cpYAP alta expressão (escore 7-8), definido
através da análise da curva ROC. De acordo com a função e atividade presumida na via Hippo
foram definidos dois grupos: o primeiro com alta expressão do nYAP e baixa expressão do
cpYAP. E o grupo 2 que compreende todas as outras combinações (nYAP alta expressão com
o cpYAP alta expressão; nYAP baixa expressão com o cpYAP baixa expressão e o nYAP baixa
expressão com o cpYAP alta expressão).
3.3.2. Expressão de MicroRNA
3.3.2.1. Extração do RNA
Foram realizados quatro cortes de 20µm em cada blocos de parafina selecionado.
Estes cortes foram submetidos a desparafinização com Xylene a 55°C, e depois lavados em
etanol. Para a extração do RNA total foi utilizado o RecoverAllTM
Total Nucleic Acid Isolation
Kit for Formalin-fixed, paraffin-embedded (FFPE) seguindo as orientações do fabricante com
31
as seguintes modificações: a digestão foi realizada com um acréscimo de 8µL de proteinase K
em 100µg/mL (Invitrogen, Carlsbad, CA, USA). A quantidade e qualidade do RNA foi
avaliada por espectrofotômetro Epoch (BioTek Instruments, Winooski, VT, USA) usando as
razões 260/280 e 260/230 como controle de qualidade. A quantidade e qualidade do RNA
avaliada em todas as amostras foi adequada.
3.3.2.2. Transcrição Reversa e Reação em Cadeia da Polimerase em Tempo Real (qRT-
PCR)
A retro transcrição de 10μg de RNA total(5μL de RNA total em 2 μg/μL) para
DNA complementar foi realizada utilizando o Kit de retro transcrição MicroRNA
TaqMan®(Applied Bio-systems, Foster City, CA) de acordo com as instruções do fabricante.
O conjunto de primers de miR-182, miR-96, miR-9 e o controlo endógeno(U6 snRNA) foram
adquiridos da Applied Biosystems(Applied Bio-systems, Foster City, CA, EUA) para retro
transcrição. A retro transcrição foi realizada utilizando Mastercycler Epgradiente
S(Eppendorf, Hamburgo, Alemanha). Uma solução contendo 10μL de TaqMan® Fast
Advanced Master Mix (Bio-sistemas aplicados, Foster City, CA, EUA), 1μL x20x de Gene
Expression Assay, 1μL de cDNA e 8μL de água isenta de RNA, somando um total de 20μL,
foi processada numa placa óptica de 96 poços a 50°C durante 2 min, seguidos de 95°C
durante 30s e depois 40 ciclos de 95°C durante 10s e 60°C durante 30s realizados em sistema
7500(Roche, Alemanha). O sinal foi coletado no ponto final de cada ciclo. Todas as reações
foram realizadas em triplicado para cada caso e as expressões relativas de miR-182, miR-96,
miR-9 foram normalizadas utilizando o método 2 –∆∆
CT.
3.4. Análise Estatística
Todas as análises foram realizadas usando o programa R (Environment for
Statistical Computing Software) [100]. A comparação entre a expressão do DDR2 e da YAP
com a idade e o nível sérico de CA125 foi realizada usando o teste de Mann-Whitney. A
comparação entre a expressão do DDR2 e da YAP e o estádio, a doença residual pós cirurgia,
os ciclos de platina e a resposta platina foi realizada usando o teste de Fisher ou qui-quadrado.
SLP e a SG foram calculadas por regressão proporcional de Cox, ajustadas pelo estádio, nível
sérico de CA125, doença residual pós cirurgia e resposta a platina. Para avaliação da relação
entre a expressão dos níveis de miR e a expressão do DDR2 e YAP foi utilizado o teste t. As
curvas SLP e SG foram estimadas pelo método Kaplan-Meier e comparadas pelo teste Log-
Rank. Para significância estatística foi considerado um p<0,05.
32
4. RESULTADOS
Artigo 1: Discoidin domain receptor 2 (DDR2) is a predictor of worse outcome in women
with high-grade serous ovarian carcinoma
Artigo 2: Yes associated protein (YAP) in high grade serous ovarian carcinoma
33
Artigo 1: Discoidin domain receptor 2 (DDR2) is a predictor of worse outcome in women
with high-grade serous ovarian carcinoma
Comprovante de submissão do artigo
From: [email protected] <[email protected]> on behalf of Gynecologic Oncology <[email protected]> Sent: Wednesday, March 1, 2017 10:50 PM To: [email protected] Cc:[email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; Subject: Gynecologic Oncology: Submission Confirmation Title: Discoidin domain receptor 2 (DDR2) is a predictor of worse outcome in women with high-grade serous ovarian carcinoma. Corresponding Author: Prof Sophie Derchain Authors: Liliana Andrade, MD,PhD; Rodrigo A Natal, Post Grad; Marina Pavanello, Msc; Amanda C Ferracini, Pharmaceutic; Luis Felipe Sallum, MD; Cassio Cardoso Filho, MD,PhD; Luis Otávio Sarian, MD, PhD; Sophie Derchain, MD,PhD Research Paper Dear Prof. Derchain, This is to confirm that the above-mentioned manuscript has been received for consideration in Gynecologic Oncology. Your manuscript will undergo a quick screening process to ensure that it meets all submission requirements. Please note that if your manuscript does not meet all submission requirements, it will be returned to you without being seen by an editor or reviewers. You will be able to check on the progress of your manuscript by logging on to the Elsevier Editorial System for Gynecologic Oncology as an author: https://ees.elsevier.com/ygyno/
34
Discoidin domain receptor 2 (DDR2) is a predictor of worse outcome in women with high-
grade serous ovarian carcinoma.
Authors
Susana Ramalho, MD, MSca; Liliana A L De Angelo Andrade, MD, PhD
b;Rodrigo de
Andrade Natal, Post Grad c; Marina Pavanello, Biologist
a; Amanda Canato Ferracini
Pharmaceuticd, Luis Felipe Sallum, MD
a, Cassio Cardoso Filho MD, PhD
a , Luis Otávio
Sarian, MD, PhDa; Sophie Derchain, MD, PhD
a †
a Department of Obstetrics and Gynecology, State University of Campinas, Campinas, Faculty
of Medical Sciences, Campinas, São Paulo, Brazil.
b Department of Pathology, University of Campinas, Campinas, Faculty of Medical Sciences,
Campinas, São Paulo, Brazil
c Laboratory of Investigative and Molecular Pathology, LAPE – Faculty of Medical Sciences
– State University of Campinas. Rua Tessália Vieira de Camargo, 126, Zip code: 13083-970 –
Campinas, São Paulo, Brazil.
d Postgraduated Program in Medical Sciences, State University of Campinas, Campinas,
Faculty of Medical Sciences, Campinas, São Paulo, Brazil.
† Correspondence to: Sophie Derchain Department of Obstetrics and Gynecology – Faculty of
Medical Sciences – State University of Campinas. Rua Tessália Vieira de Camargo, 126, Zip
code: 13083-970 – Campinas, São Paulo, Brazil. E-mail: [email protected]. Phone
+55 19 35219305
This study was partially funded by National Council for Scientific and Technological
Development (CNPq) number 306583//2014-3, the São Paulo Research Foundation
(FAPESP) number 2012/15059-8 funded this study and Support Fund for Teaching, Research
and Extension (FAEPEX) processes number 519.292 and 519.294
35
Abstract
Objective: High-grade serous ovarian carcinoma (HGSOC) is a aggressive disease with early
metastatic dissemination. The epithelial-mesenchymal transition (EMT) is an important
cellular process related to invasion and metastasis. The receptor tyrosine kinase, discoidin
domain receptor 2 (DDR2), acting through the extracellular signal-regulated kinase1/2
(erk1/2) is associated with EMT. To evaluate the relation of DDR2 expression and miR-182
and miR-96 expression levels with progression free and overall survival in women with high-
grade serous ovarian carcinoma (HGSOC). Methods: for this reconstituted cohort study, 58
women with stage III/IV HGSOC diagnosed and treated from 1996 to 2013 and followed up
until 2016 were included. Formalin fixed paraffin embedded tissue was used to evaluate the
protein expression of DDR2 and to evaluate microRNA expression levels The expression of
miR-182 and miR-96 were verified using quantitative real-time PCR (qRT-PCR). Results:
DDR2 expression was high in 8(13.7%) women and low/medium in 50(86.3%). There was no
association between DDR2 expression and age, CA125 level, stage, post-surgery residual
disease and clinical response to platinum-based chemotherapy. However, progression free
survival was significantly worse in women whose tumors had high DDR2 expression
(p=0.03), but not overall survival (p=0.49). MiR-182 expression levels were lower in DDR2
high expression group (p<0.001), but not miR-96 (p=0.067). Conclusion: Taking together low
miR-182 expression levels were associated with high DDR2 expression which confers worse
progression free survival in women with HGSCOC. Our findings should indicate that the
activation of erk1/2 by DDR2 is relevant to the EMT of these HGSOC.
Key words: serous subtype, epithelial ovarian cancer; prognosis, drug resistance, epithelial-
mesenchymal transition.
36
Introduction
Ovarian carcinoma is a common cause of death among women with gynecological
cancer. High grade serous ovarian carcinoma (HGSOC) is responsible for most deaths [1],
since the 5-year survival rates is around 30-40% [2, 3]. Most cases of HGSOC are diagnosed
at advanced stages and the standard approach is cytoreductive surgery and platinum-based
chemotherapy or neoadjuvant chemotherapy followed by surgery [4]. Despite high response
rates to first line treatment with platinum based chemotherapy, most patients with HGSOC
will relapse or progress [5]. Therefore, the identification of prognostic and predictive
biomarkers in HGSOC may help define drug targets in selected populations.
Discoidin domain receptor 2 (DDR2) belongs to the tyrosine kinase receptor
family that mediate cell interactions with structural components of the extracellular matrix
(ECM) [6, 7]. DDR2 is activated essentially through fibrillar collagen type I, which is one of
the most abundant components of the ECM [8, 9]. The ECM contributes to cancer cell
migration, acts as an immunologic barrier, forms a niche for new metastatic cells and provides
survival and proliferation signals [10, 11]. Activated DDR2 regulates transcription pathways
that increase the quantity of metalloproteinases in the ECM and foster epithelial-mesenchymal
transition (EMT)[11-13]. DDR2 promotes EMT through extracelular signal-regulated
kinase1/2 (erk1/2) that in turn causes the stabilization of snail1. These processes sustain an
EMT phenotype, leading to proliferation and metastasis. Several studies have identified a
large amount of miRNA involved in EMT [14-16]. There is a dynamic reciprocal suppression
between miR-182 and snail1, a key transcriptional factor of EMT. In breast cancer murine
model, a mutual interaction between miR-182 and snail1 may be related to colonization and
lung metastasis [17].
Cytoplasmic DDR2 expression has been studied in different sets of cancer
patients. In head and neck squamous carcinoma, DDR2 expression was associated with high
grade tumors but no prognostic value was demonstrated [18]. In hepatocellular carcinoma
(HCC) DDR2 expression was found to be an independent prognostic factor. Patients whose
tumors present higher DDR2 expression had a lower 5-year disease free and overall survival
(OS) [19]. In breast cancer high expression of DDR2 was associated with high tumor grade
and triple negative subtype [20]. DDR2 was also associated with breast cancer proliferation,
migration and metastasis [11].
To our knowledge, there is only one study addressing DDR2 expression in
ovarian cancer [21]. Using real-time quantitative PCR (qRT-PCR), those authors found an
association between higher DDR2 expression levels in ovarian carcinoma compared to normal
37
ovarian tissue. They also found a significant association between higher cytoplasmic DDR2
immunohistochemical expression with advanced stage and poor OS. This study was designed
to evaluate the relation of DDR2 expression and miR-182 and miR-96 levels with progression
free survival (PFS) and OS in a large set of patients with advanced stage, HGSOC.
Subjects and methods
Patients and tissue specimens
For this reconstituted cohort study, the consecutive formalin fixed paraffin
embedded (FFPE) tissue sample and the respective files of 114 patients diagnosed and treated
at the Women's Hospital Prof. Dr. José Aristodemo Pinotti, State University, Campinas, Brazil
from 1996 to 2013 and followed up to 2016 were retrieved. This study was approved by the
local institutional ethics committee (CEP 1086/2009 and CEP 710.452/2014). All pathological
specimens, collected during primary surgery or percutaneous biopsy before chemotherapy
were analysed by an expert gynaecological pathologist according to the guidelines of the
World Health [22] and staged according to FIGO classification [23]. Exclusion criteria was
stage I/II (12 women), second primary cancer (2 women), less than 3 cycles of platinum based
chemotherapy (14 women), no available FFPE tissue sample (17 women) and missed files (6
women). FFPE tissue samples from the remaining 63 HGSOC patients with complete data
were selected. Due to technical difficulties, adequate immunohistochemistry reading was not
possible for five patients.
Age, stage, CA125, post-surgery residual disease, cycles of platinum-based
chemotherapy, platinum response, PFS and OS data were obtained from patient files. For PFS
and OS time was estimated in months, from the date of diagnosis to the last follow-up visit,
recurrence or death. For platinum response, time was estimated in months, from the end of
primary platinum-based chemotherapy treatment to the date of progression. Platinum response
was classified as recommended by Patch et al. [24]: i) primary refractory, patients progressed
during primary treatment; ii) primary resistant, patients progressed in less than six months
after the end of primary treatment; iii) primary chemosensitive patients progressed after six
months of the end of primary treatment and iv) acquired resistant, patients failed to respond to
chemotherapy for progressed/relapsed disease having previously demonstrated sensitivity to
treatment. The progression/relapse was assessed through clinical examination, imaging and
CA125 level.
38
Immunohistochemistry
Slides stained with hematoxylin and eosin from the original FFPE blocks were
analyzed for the selection of representative tumor regions. Tissue microarray (TMA, Beecher
Instruments Microarray Technology, Silver Spring, CA, USA) was built and sections from
TMA were placed on electrically charged slides for immunohistochemical procedures.
Sections were deparaffinized with xylol and dehydrated in alcohol series. Washes in hydrogen
peroxide were performed, followed by distilled water washes. For antigen retrieval, we used a
commercially available pressure cooker (T-fal®), in which slides were immersed in citrate
buffer pH 6.0 for 30 minutes. The slides were dried at room temperature and washed in distilled
water. After that, the sections were incubated in a moist chamber, with monoclonal mouse
antibody against DDR2 (Abcam, Cambridge, USA 1:1500) at 4ºC, overnight. The slides were
then washed in PBS, pH 7.4. As detection system, the slides were incubated in ADVANCE™
HRP Detection System (Dako) at 37ºC for 1 hour, and washed in PBS. After, DAB
chromogenic substrate (3´-diaminobenzidine, SIGMA, St Louis, MA, USA) was applied at a
proportion 0.06g to 100ml of PBS, 500μl hydrogen 3% peroxide and 1ml dimethylsulfoxide
(DMSO) at 37ºC for 5 minutes. Finally, the slide was washed in tap water and counterstained
with Harris’ hematoxylin for 30 to 60seconds. After being dehydrated, the slide was mounted
in resin (Entellan®, Merck, Darmstadt, Germany). TMA analysis was performed according to
protocols fully validated in ovarian carcinoma [25]. Internal/external, positive/negative
controls were used for validation of the reactions. Positive control was done in myocardial
(heart) tissue. Immunostaining was separately reviewed and scored by an expert gynaecologic
pathologist.
Image analysis
DDR2 protein expression was analyzed in the cytoplasm. The proportion and
intensity of positively stained cancer cells were assessed. The extensional standards taken
were as follows: (1) number of positive stained cells ≤5%, scored 0; 6-25%, scored 1; 25-
50%, scored 2; 51-75%, scored 3; and >75%, scored 4; and (2) intensity of stain: colorless,
scored 0; pallideflavens, scored 1; yellow scored 2; and brown, scored 3. The extensional
standards (1) and (2) were multiplied, and the staining grade was stratified as absent (0 score),
weak (1-4 score), moderate (5-8 score) or strong (9-12 score) [18, 21] as shown in figure 1.
Intensity and proportion of positive cells were multiplied and thus the scoring system was
defined as low/medium expression for scores of 0-8, and as high expression for scores of 9-
12. This cut off point was determined using ROC curve.
39
RNA extraction
Four 20µm thickness FFPE sections from each sample were submitted to
deparaffinization with xylene at 55°C, and then washed in ethanol. Total RNA was extracted
using the RecoverAll™Total Nucleic Acid Isolation Kit (Ambion, Kruss, Denmark) was used
according to manufacturer´s instructions with the following modifications: digestion with
lyses solution was performed with the addition of 8µL proteinase K at 100µg/mL (Invitrogen,
Carlsbad, CA, USA) at 55°C overnight. Quantity and quality of total RNA was assessed with
Epoch spectrophotometer (BioTek Instruments, Winooski, VT, USA) using 260/280 and
260/230 ratios as quality controls. All samples had adequate RNA quantity and quality.
Reverse transcription and quantitative real-time polymerase chain reaction analysis
Reverse transcription of 10µg of total RNA (5µL of total RNA at 2µg/µL) to
cDNA was done using TaqMan® MicroRNA Reverse Transcription Kit (Applied Bio-
systems, Foster City, CA) per the manufacturer’s instructions. Primer set of miR-182, miR-96
and endogenous control (U6 snRNA) for specific reverse transcription were purchased from
Applied Biosystems (Applied Bio-systems, Foster City, CA, USA). Retro transcription was
carried out using Mastercycler Epgradiente S (Eppendorf, Hamburg, Germany). TaqMan®
Fast Advanced Master Mix (Applied Bio-systems, Foster City, CA, USA) containing 10µL of
TaqMan® Fast Advanced Master Mix, 1µL of 20x Gene Expression Assay, 1µL of cDNA and
8µL of RNase-free water, total of 20μL volume were processed in a 96-well optical plate at
50°C for 2 min, followed by 95°C for 30s and then 40 cycles of 95°C for 10s and 60°C for
30s performed in 7500 system (Roche, Germany). The signal was collected at the endpoint of
every cycle. All reactions were run in triplicate for each case and the relative expression miR-
182 and miR-96 was normalized using the 2 –∆∆CT
method.
Statistical analysis
The comparison of DDR2 expression was performed using the Mann-Whitney
test. Fisher exact tests were used to compare categorical data. Median with 95% confidence
interval (95%CI) PFS and OS in months and years were calculated and compared using the
Log-rank test. Cox proportional hazard ratios with 95%CI were used to analyze PFS and OS.
MiR-182 and miR-96 relative expression were normalized to U6 snRNA. Shapiro-Wilk test
was used to assess data normality. The comparison of miR-182 and miR-96 in the DDR2
expression status was performed using t-tests. A p-value lower than 0.05 was considered
significant. All statistical analyses were performed using the R Project for Statistical
Computing [26].
40
Results
Among 58 patients with HGSOC, 50(86.3%) presented with low/medium
expression of DDR2 and 8(13.7%) presented with high expression. Median age, FIGO stage,
CA125 level, post-surgery residual disease and response to platinum based chemotherapy did
not differ according to DDR2 expression (table 1). All patients with high expression of DDR2
progressed within 15 months and none was alive within 5 years. Analyzing patients with
low/medium DDR2 expression, 29% were still free of progression after 5 years and 35% were
alive (table 2). After adjusted for FIGO Stage, CA125 level, post-surgery residual disease and
platinum response, PFS was significantly lower in patients with high expression of DDR2
[HR=2.69 (95%CI) 1.1 to 6.6], but OS was not associated with DDR2 expression [HR=1.6
(95%CI) 0.4 to 5.7] (table 3).
As depicted in figure 2 miR-182 expression levels were significantly lower in
patients harboring tumors with higher expression of DDR2 (p<0.001). MiR-96 expression
levels were not associated with DDR2 expression (p=0.067).
Discussion
In this study, we investigated DDR2 expression in patients with stage III/IV
HGSOC who received platinum based chemotherapy. High DDR2 expression was neither
associated with FIGO stage, CA125 levels, post-surgery residual disease nor platinum
response. High DDR2 expression was associated with worse PFS but not with OS. There was
a significant association between lower levels of miR-182 in DDR2 high expression group.
Tyrosine kinase receptor aberrant expression and function cooperatively
contribute to aggressive behavior and progression in several cancers [12]. Therefore, high
DDR2 expression has been investigated as a prognostic factor in different tumors. Xu et al.
[18] analyzed DDR2 expression in 79 head and neck squamous cell carcinomas (HNSCC).
qRT-PCR results showed that DDR2 expression levels were significantly higher in HNSCC
tumor samples compared with samples of adjacent normal tissue. In addition, DDR2
expression levels were increased in high-grade tumors. In the same study, Xu et al.[18]
demonstrated that DDR2 protein expression was identified in 34(43%) patients of HNSCC
and was significantly associated with higher pathological stage and lymph node metastases.
However, Xu et al.[18] consider tumors with moderate or strong immunostaining as high
DDR2 expression (scored 5-12). They concluded that DDR2 expression is a hallmark of high-
grade and metastatic HNSCC. In our analyses, only high-grade, advanced stage ovarian
carcinomas were included. In addition, using ROC curve analyses, we defined high DDR2
41
expression as a staining score between 9 and 12. Using this cutoff point, only 8(13.7%)
patients were positive for DDR2. The methodological dissimilarities notwithstanding, our
study and Xu´s concur in that high DDR2 expression is associated with worse PFS.
In hepatocellular carcinoma samples, higher DDR2 protein expression was
associated with vascular invasion, multiple tumors, high-grade tumors and advanced disease
stage [19]. After multiple analyses, DDR2 expression proved to be an independent prognostic
factor for PFS and OS. Toy et al. [20] examined DDR2 expression in 198 invasive breast
carcinomas. High DDR2 expression was identified in 110(55%) samples and was associated
with high-grade tumors, triple-negative subtype and worse survival.
In our study, high expression of DDR2 was significantly associated with lower
PFS but not with OS. In a recent published cohort of ovarian cancer patients, Fan et al. [21]
concluded that DDR2 expression was an independent prognostic marker of OS. In their study,
tumors with moderate or strong immunostaining (scored 4-12) were labeled as high DDR2
expression; therefore, among 103 ovarian cancer with mixed histological types, around 60%
(63) had DDR2 high expression. One of the major boundary of the clinical use of DDR2 is the
lack of standardization regarding staining and image analysis. Authors disagreed in terms of
cutoff points for DDR2 positivity, and many studies do not underscore DDR2 positivity
thresholds on ROC curve analysis, or any other technical argument [18-21].
In our study, miR-182 levels were significantly lower in tumors with high DDR2
expression. MiR-182 levels were not independently associated with PFS, but tumors with
lower miR-182 and higher DDR2 expression levels had a worse PFS. MiR-182 and miR-96
belong to the miR183 family [15]. Yang et al. [14], examining 459 cases of HGSOC in TCGA
data, identified miR-182 along with other eight microRNAs as part of a regulatory network
for the mesenchymal subtype. The mesenchymal subtype was determined through microRNA
expression analysis as a predictor of poor patient overall survival. Since miR-182 expression
is associated with increased survival in the TCGA cohort, in our analysis miR182 lower
expression levels were associated with high DDR2 expression, which in turn is a group of
women displaying worse PFS.
As described by Zhang et al.[13] and Gonzalez et al. [11], DDR2 activation in
breast cancer cells promotes extracellular signal-regulated kinase 1/2(erk1/2) cascade activity.
Activated erk1/2 directly phosphorylates snail1 leading to snail1 nuclear accumulation and
reduced ubiquitination. DDR2 expression is associated with nuclear snail1 and absence of E-
cadherin expression, which represent the occurrence of EMT. Snail1 suppresses miR-182 at
42
the stage of EMT to start metastasis whereas miR-182 inhibits snail1 to reestablish epithelial
identity for colonization and macro metastasis formation [17].
MiR-182 is described as consistently up-regulated in several cancer types
including ovarian cancer [27]. Liu et al. [28] described that overexpression of miR-182 in
ovarian cancer cell lines would enhance progression through deregulation of miR-182
downstream targets HMGA2 and MTSS1. Wang et al. [29] concluded that, in ovarian cancer
cells, miR-182 acts as an oncogenic miRNA by negatively targeting programmed cell death
4(PDCD4). Xu et al.[18] showed that in orthotropic xenografts of nude mice anti miR-182
reduces ovarian cancer burden, invasion and metastasis. While some authors described
miR182 role as promoting cell growth, invasion and chemoresistance, it must be emphasized
that, the above results were mainly derived from in vitro studies, performed on ovarian cancer
cells, in tumor types other than only HGSOC.
In conclusion, although we did not assess gene expression of DDR2, we depicted a
significant number of consecutive HGSOC women with available FFPE sample and annotated
clinical data. To the best of our knowledge, this is the largest study cohort of Brazilian women
with HGSOC, in which all cases were reviewed by an expert pathologist, DDR2 expression
was performed and platinum resistance was assessed. The current study suggests that in stage
III/IV patients with HGSOC submitted to platinum-based chemotherapy, DDR2 expression
was an independent prognostic factor for PFS, and that lower miR-182 expression levels were
associated with high DDR2 expression.
Conflict of interest: The authors declare that there is no conflict of interest regarding the
publication of this article.
43
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46
Table 1: High grade serous ovarian carcinoma (HGSOC) patient distribution according to
clinical features and discoidin domain receptor 2 (DDR2) expression.
DDR2 expression
Clinical features Low/medium High p
Age (years) mean and +/- SD 58.1 (+/-11.1) 64.9 (+/-8.4) 0.11#
Stage [n(%)]
III 50(100%) 7(87%) 0.14*
IV 0(0%) 1(13%)
CA125 mean and Standard Deviation 2260.3 (+/-5473.7) 794 (+/- 888.7) 0.28#
Post-surgery residual disease [n(%)]
No 23(46) 3(38) 0.72*
Yes 27(54) 5(62)
Cycle of platinum [n(%)]
3 to 5 3(6) 1(13) 0.33*
>= 6 47(94) 7(87)
Platinum response[n(%)]
Refractory/resistant 15(30) 5(62) 0.11*
Sensitive/acquired resistance 35(70 3(38)
#Mann Whitney; *Fisher SD= Standard Deviation; Discoidin domain receptor 2 (DDR2) expression
low/medium= score 0-8; High=score 9-12.
47
Table 2: Progression free survival, overall survival and discoidin domain receptor (DDR2)
expression. Median (with 95%CI) PFS and OS in months and years were calculated.
Number
of
patients
(%)
Median PFS
months
(95%CI)
PFS rates
(95%CI)
2 years
PFS rates
(95%CI)
5 years
PFS rates
(95%CI)
10 years
p
Low/medium
DDR2
50
40.6
(0.7 to 172)
0.47
(0.36 to 0.62)
0.35
(0.24 to 0.49)
0.29
(0.19 to 0.45)
<0.001
High DDR2 8 6.7
(1.6 to 14.9)
- - -
Number
of
patients
(%)
Median OS
months
(95%IC)
OS rates
(95%IC)
2 years
OS rates
(95%IC)
5 years
OS rates
(95%IC)
10 years
p
Low/medium
DDR2
50
63.0
(12.3 to 170.5)
0.84
(0.75 to 0.93)
0.57
(0.45 to 0.71)
0.35
(0.22 to 0.55)
0.154
High DDR2 8 26.4
(17.9 to 34.6)
0.75
(0.50 to 1.00)
- -
PFS= progression free survival. OS=overall survival. p = Log- rank test.
48
Table 3: Progression free survival and overall survival in women with high grade serous
ovarian carcinoma according to discoidin domain receptor 2 (DDR2) expression.
DDR2
expression
n Median PFS
(95%CI)
HR
(95%CI) p*
Median OS
(95%CI)
HR
(95%CI) p*
Low/medium 50 29.5
(0.5 to 133)
reference 54.9
(11.3 to 138.5)
reference
High 8 6.74
(5.6 to 15.0)
2.69
(1.1 to 6.6)
0.03 26.4
(17.4 to 34.5)
1.6
(0.4 to 5.7)
0.49
*Adjusted by stage, CA 125 level, post-surgery residual disease and platinum response. PFS=progression free
survival, OS=overall survival. HR=Cox proportional hazard ratio. 95%CI =95% confidence interval.
49
Figure 1 - Representative images discoidin domain receptor 2 (DDR2) expression through
immunohistochemistry assays in high grade serous ovarian carcinoma (A) absent score 0; (B)
weak score 1-4 (C) moderate score 5-8 (D) strong score 9-12 DDR2 expression (X100).
50
Figure 2 – Representative image of miR182 levels(A) and miR-96 levels(B) according to
discoidin domain receptor 2 (DDR2) immunohistochemistry expression groups: low/medium
and high DDR2.
A B
51
Artigo 2: Yes associated protein (YAP) in high grade serous ovarian carcinoma
Comprovante de submissão do artigo
From: [email protected] <[email protected]> on behalf of Medical Oncology (MEDO) <[email protected]> Sent: Wednesday, March 1, 2017 3:34 AM Subject: MEDO-D-17-00360 - Submission Confirmation Dear Prof Derchain, Thank you for submitting your manuscript, Yes associated protein (YAP) expression in high grade serous ovarian carcinoma, to Medical Oncology. The submission id is: MEDO-D-17-00360 Please refer to this number in any future correspondence. During the review process, you can keep track of the status of your manuscript by accessing the following web site: http://medo.edmgr.com/
52
Yes associated protein (YAP) expression in high grade serous ovarian carcinoma.
Authors
Susana Ramalho, MD, MSca; Luis Otávio Sarian, MD, PhDa; Rodrigo de Andrade Natal,
Post Gradc; Liliana A L De Angelo Andrade, MD, PhDb; Marina Pavanello, Biologista;
Amanda Canato Ferracini Pharmaceuticd; Luis Felipe Sallum, MD, MSca; Cassio Cardoso
Filho MD, PhDa; Sophie Derchain, MD, PhD a †
a Department of Obstetrics and Gynecology, State University of Campinas, Campinas,
Faculty of Medical Sciences, Campinas, São Paulo, Brazil.
b Department of Pathology, University of Campinas, Campinas, Faculty of Medical Sciences,
Campinas, São Paulo, Brazil
c Laboratory of Investigative and Molecular Pathology, LAPE – Faculty of Medical Sciences
– State University of Campinas. Rua Tessália Vieira de Camargo, 126, Zip code: 13083-970 –
Campinas, São Paulo, Brazil.
d Postgraduated Program in Medical Sciences, State University of Campinas, Campinas,
Faculty of Medical Sciences, Campinas, São Paulo, Brazil.
† Correspondence to: Sophie Derchain. Department of Obstetrics and Gynecology – Faculty
of Medical Sciences – State University of Campinas. Rua Tessália Vieira de Camargo, 126,
Zip code: 13083-970 – Campinas, São Paulo, Brazil. E-mail: [email protected]
Phone +55 19 35219305
This study was partially funded by National Council for Scientific and Technological
Development (CNPq) number 306583//2014-3, the São Paulo Research Foundation (FAPESP)
number 2012/15059-8 funded this study and Support Fund for Teaching, Research and
Extension (FAEPEX) processes number 519.292 and 519.294
53
Abstract
Yes associated protein Yap (YAP) is a key transcriptional co-activator of the Hippo pathway.
Nuclear YAP expression is associated with epithelial-mesenchymal transition (EMT). MiR-9
is a microRNA that targets e-cadherin whose loss expression is a hallmark of EMT. This study
was designed to evaluate (YAP) expression, miR-9 expression levels, patient outcomes and
survival in high grade serous ovarian carcinoma (HGSOC). Data of 61 women, stage III/IV
HGSOC diagnosed and treated from 1996 to 2013 and followed up to 2016 were included.
Formalin fixed paraffin embedded tissue were used to evaluate the immunohistochemical
expression of nuclear yes associated protein (nYAP) and cytoplasmic phosphorylated yes
associated protein (cpYAP). MiR-9 expression levels was assessed by quantitative real-time
PCR (qRT-PCR). Among the 61 women included 15 (24.5%) had high nYAP and low cpYAP
expression. There were no association between YAP expression and age, stage, CA125 level,
post-surgery residual disease and clinical response to platinum-based chemotherapy. In the
univariate analysis progression free survival [(26.6 vs 25.4 Hazard Ratio (HR) 1.04 (0.51 -
1.82);p = 0.91)] and overall survival [(55.6 vs 48.2 HR 0.70 (0.65 - 3.11); p = 0.37)] did not
differ between high nYAP and low cpYAP and others combinations. MiR-9 expression levels
were not associated with YAP expression (0.157). Our findings suggest that there is no
association between high nYAP and low cpYAP expression and survival. MiR-9 expression
levels did not correlate with YAP expression. In women with HGSCOC possibly miR-9 and
Hippo signaling pathway have an unclear role.
Key words: epithelial ovarian cancer; yes-associated protein, platinum resistance, survival.
54
Introdução
Epithelial ovarian cancer is the first cause of death among gynecological cancers
[1]. The most common and aggressive histological type of epithelial ovarian cancer is high
grade serous ovarian carcinoma (HGSOC). Typically, HGSOC is diagnosed at advanced
stages due to the anatomic pelvic location, lack of alarm symptoms and no effective screening
methods [2]. Stage III and IV cases comprise more than 70% of all diagnosed HGSOC, and 5-
year survival is only 30% [3, 4]. Since most cases have disseminated disease in the peritoneal
cavity at diagnosis, current standard treatment protocols generally mandate either
cytoreductive surgery (if feasible), followed by adjuvant platinum based chemotherapy, or
neoadjuvant chemotherapy followed by surgery [5]. However, at least one third of HGSOC
patients show upfront resistance to platinum-based treatments, and another third acquire
resistance at some point during treatment [6, 7].
Agents targeting molecules that drive tumor development and/or progression have
been shown to improve the prognosis of many cancers such as breast, lung, hematologic and
melanoma. In ovarian cancer, however, only a few targeted agents, such as Bevacizumab and
Olaparib, have been proposed and tested as treatments associated to or after platinum-based
chemotherapy. In a few subsets of patients, these treatments yielded better progression-free
survival (PFS), although no benefit in overall survival (OS) has been shown so far [8, 9].
Therefore, there is a critical need to identify oncogenic signaling pathways in HGSOC and
expand the arsenal of drugs capable of improving the clinical outcomes of patients with the
disease [10, 11].
The yes associated protein (YAP) is a key transcriptional co-activator of the Hippo
pathway [12]. Yap is positioned in the 11q22 genomic region which is amplified in several
cancers including ovarian cancer. Therefore, YAP has been studied as a candidate oncogene
[13]. The Hippo pathway is a signaling transduction pathway that consists in a core kinase
cascade including various components (MST1/2, LATS1/2, SAVl and YAP). Hippo pathway
is considered to be triggered when MST and LATS kinase are activated. MST1/2 activation
induces phosphorylation and activation of LATS1/2. Activated LATS1/2 phosphorylates YAP.
YAP Phosphorylation results in YAP cytoplasmic (cpYAP) retention, YAP nuclear (nYAP)
exportation and cpYAP degradation by proteasome. Thus triggered Hippo pathway inhibits
YAP activity. However, YAP accumulated in the nucleus (nYAP) interacts with TEA domain
transcription factor (TEAD). High levels of nYAP combined with low levels of cpYAP are
associated with an inactivated Hippo pathway [14-16]. nYAP in a inactivated Hippo pathway
mediates the expression of genes associated with proliferation, apoptosis inhibition and
55
epithelial to mesenchymal transition (EMT) [15, 16]. EMT is the process where epithelial
cells lose both polarity and cell-to-cell contact, acquiring motility and invasion which allows
discharge from the primary tumor into neighboring normal parenchyma and enter into
circulation to initiate metastasis. The activation of EMT increases tumor ability to migrate and
spread [17].
Similarly, the reverse process, the mesenchymal to epithelial transition, is
necessary for metastasis outgrowth [17, 18]. The EMT triggering leads to the activation of
transcriptional factors like E74-like factor 3 (ELF3), snail1/snail2, zeb1/zeb2, that suppress
the expression of mesenchyme markers like E-cadherin [18, 19]. Recent evidence postulates
that numerous microRNA coordinate EMT either by targeting transcriptional factors or
activating EMT signaling pathways as Hippo signaling pathway [20, 21]. Among those
microRNA´s, miR-9 expression level has been associated with Hippo pathway in breast [22]
and gastric cancer cells [23].
Lately, in vitro and in vivo studies described YAP expression to be associated with
adverse PFS and OS in numerous cancers such as gastric cancer [24], colorectal [25], breast
[26, 27] and liver [28]. In ovarian cancer cell lines, YAP activity was positively correlated
with ovarian cancer colony formation capability, cell proliferation, cisplatin and taxol
resistance, migration and anchorage, independent growth and distant metastasis [29]. In vivo
studies showed that low cpYAP expression with consequent high nYAP expression was an
independent factor associated with poor overall survival in patients with ovarian carcinoma
[13]. In another cohort including patients with ovarian carcinoma, Xia et al. [29] also found
an association between high nYAP with poor overall survival. It is worth noting, however,
that these studies were based on a large amalgam of subtypes of ovarian carcinomas, which
may be intrinsically resistant to platinum-based chemotherapy. We thus decided to evaluate
YAP expression, miR-9 expression levels, patient outcomes, such as response to platinum-
based treatment and survival in a set of Brazilian women with HGSOC.
Materials and methods
Patients and tissue specimens
For this reconstituted cohort study, the consecutive formalin fixed paraffin
embedded (FFPE) tissue sample and the respective files of 114 women diagnosed and treated
at the women´s Hospital of Campinas State University, Campinas, Brazil from 1996 to 2013
and followed up to 2016 were selected. This study was approved by the local institutional
ethics committee (CEP 1086/2009 and CEP 710.452/2014). All pathological specimens,
56
collected during primary surgery or before neoadjuvant chemotherapy were analysed by an
expert gynaecological pathologist according to the guidelines of the World Health
Organization International Classification of Ovarian Tumours [30] and staged according to
FIGO classification [31]. Exclusion criteria was stage I and II (12 women), second primary
cancer (2 women), no chemotherapy or less than 3 cycles of platinum based chemotherapy
(14 women), no available FFPE tissue sample before chemotherapy (17 women) and missed
files (6 women). FFPE tissue samples from the remaining 63 HGSOC women with complete
data were selected. Due to technical difficulty 2 patients did not had adequate
immunohistochemistry reading. Age, stage, CA125, post-surgery residual disease, cycles of
platinum based chemotherapy, platinum response, PFS and OS data were obtained from
patients files. For platinum response time was estimated in months, from the end of primary
platinum based chemotherapy treatment to the date of progression. The platinum response
was classified as recommended by Patch et al. [32] as followed: primary refractory were
women who progressed during primary treatment; primary resistant were women who
progressed in less than six months after the end of primary treatment; primary chemosensitive
were women who progressed after six months of the end of primary treatment and acquired
resistant were women that failed to respond to chemotherapy for progressed/relapsed disease
having previously demonstrated sensitivity to earlier lines. For PFS and OS time was estimated
in months, from the date of diagnosis to the last follow-up visit, recurrence or death. The
progression/relapse was assessed through clinical examination, imaging and CA125 level.
Immunohistochemistry
The selected antibodies used were monoclonal rabbit antibody against YAP
(Abcam, Cambridge, USA 1:100) and monoclonal rabbit antibody against YAP phospho-
S127 (Abcam, Cambridge, USA 1:350). Slides stained with hematoxylin and eosin from the
original FFPE blocks were analyzed for the selection of representative tumor regions. Tissue
microarray (TMA, Beecher Instruments Microarray Technology, Silver Spring, CA, USA)
was built and sections from TMA were placed on electrically charged slides for
immunohistochemical procedures. Sections were deparaffinized with xylol and dehydrated in
alcohol series. Washes in hydrogen peroxide were performed, followed by distilled water
washes. For antigen retrieval, we used a commercially available pressure cooker (T-fal®), in
which slides were immersed in Edta buffer for YAP antibody and citrate buffer for
phosphorylated YAP antibody pH 6.0 for 30 minutes. The slides were dried at room temperature and
washed in distilled water. After that, the sections were incubated in a moist chamber, with
57
monoclonal rabbit antibody against YAP and with monoclonal rabbit antibody against YAP
phospho-S127, at 4ºC, overnight. Positive control was done in prostate adenocarcinoma
tissue. The slides were then washed in PBS, pH7.4. As detection system, the slides were
incubated in ADVANCE™ HRP Detection System (Dako) at 37ºC for 1 hour, and washed in
PBS. After, DAB chromogenic substrate (3´-diaminobenzidine, SIGMA, St Louis, MA, USA)
was applied at a proportion 0.06g to 100ml of PBS, 500μl hydrogen 3% peroxide and 1ml
dimethylsulfoxide (DMSO) at 37ºC for 5 minutes. Finally, the slide was washed in tap water
and counterstained with Harris’ hematoxylin for 30 to 60 seconds. After being dehydrated, the
slide was mounted in resin (Entellan®, Merck, Darmstadt, Germany). TMA analysis was
performed according to protocols fully validated in ovarian carcinoma [33]. Internal/external,
positive/negative controls were used for validation of the reactions. Immunostaining for YAP
and YAP phospho-S127 were separately reviewed and scored by an expert gynaecologic
pathologist.
Image analysis
The analysis was assessed according to the proportion and intensity of positively
stained cancer cells. TMA immunostaining for YAP was read considering YAP nuclear
expression. Nuclear YAP (nYAP) protein expression was analyzed and percentages of cells
with nuclear staining were estimated. Cases with more than 10% positive tumor nuclei were
considered high and zero or less than 10% were considered low [26]. TMA immunostaining
for YAP phospho-S was read considering YAP cytoplasmic expression. Cytoplasmic
phosphorylated YAP (cpYAP) was analyzed and the extensional standards taken were as
follows: (1) number of positive stained cells 0%, scored 0; >0-1%, scored 1; >1-10%, scored
2; >10-33%, scored 3; >33-66%, scored 4; and >66-100%, scored 5 (2) intensity of stain:
colorless, scored 0; pallideflavens, scored 1; yellow scored 2; and brown, scored 3. The
extensional standards (1) and (2) were combined, and the staining grade was stratified as low
(0-6 score) and high (7-8 score) [13] as shown in figure 1. This cut off point was determined
by ROC curve.
RNA extraction
Four 20µm thickness FFPE sections from each sample were submitted to
deparaffinization with xylene at 55°C, and then washed in ethanol. Total RNA was extracted
using the RecoverAll™. (Ambion, Kruss, Denmark) Total Nucleic Acid Isolation Kit was used
according to manufacturer´s instructions with the following modifications: digestion with lyses
58
solution was performed with the addition of 8 µL proteinase K at 100µg/mL (Invitrogen,
Carlsbad, CA, USA) at 55°C overnight. Quantity and quality of total RNA was assessed with
Epoch spectrophotometer (BioTek Instruments, Winooski, VT, USA) using 260/280 and
260/230 ratios as quality controls. All samples had adequate RNA quantity and quality.
Reverse transcription and quantitative real-time polymerase chain reaction analysis
Reverse transcription of 10µg of total RNA (5µL of total RNA at 2µg/µL) to
cDNA was done using TaqMan® MicroRNA Reverse Transcription Kit (Applied Bio-
systems, Foster City, CA) per the manufacturer’s instructions. Primer set of miR-9 and
endogenous control (U6 snRNA) for specific reverse transcription were purchased from
Applied Biosystems (Applied Bio-systems, Foster City, CA, USA). Retro transcription was
carried out using Mastercycler Epgradiente S (Eppendorf, Hamburg, Germany). TaqMan®
Fast Advanced Master Mix (Applied Bio-systems, Foster City, CA, USA) containing 10µL of
TaqMan® Fast Advanced Master Mix, 1µL of 20x Gene Expression Assay, 1µL of cDNA and
8µL of RNase-free water, total of 20μL volume were processed in a 96-well optical plate at
50°C for 2 min, followed by 95°C for 30s and then 40 cycles of 95°C for 10s and 60°C for
30s performed in 7500 system (Roche, Germany). The signal was collected at the endpoint of
every cycle. All reactions were run in triplicate for each case and the relative expression miR-
9 was normalized using the 2 –∆∆CT
method.
Statistical analysis
Initially, a descriptive analysis of all collected variables was carried out. YAP
expression was classified into 2 groups based on the combination of nYAP and cpYAP
intensity score: the first group had high nYAP and low cpYAP expression and the second
group were other combinations of nYAP and cpYAP expression patterns. Cases with high
nYAP and low cpYAP were considered Hippo inactivated and other combinations were
considered unknown. Means and standard deviation were calculated for age and CA125 level.
Comparison of groups was performed using Mann-Whitney test. Fisher exact test or qui-
square was used to compare categorical data. The survival curves were estimated by Kaplan-
Meier method and compared by using Log-Rank test. Hazard ratio (HR) with 95% confidence
intervals were calculated. A p-value lower than 0.05 was considered significant. All statistical
analyses were performed using the R Project for Statistical Computing [34]. MiR-9 relative
expression was normalized to U6 snRNA. Shapiro-Wilk test was used to assess the data
normality. The comparison of miR-9 in YAP expression status was performed using t-tests.
59
Results
As described in table 1, among the 61 patients with HGSOC, 15 (24.5%) patients
had high nYAP and low cpYAP (24.5%). The other 46 (75.5%) patients had different
combinations of nYAP and cpYAP expression. Median age, FIGO stage, CA125 level, post-
surgery residual disease and cycles of platinum based chemotherapy did not differ according
to YAP expression. There was also no difference in platinum response comparing the YAP
expression categories (p=1) (table 2).
In the univariate analysis, progression free survival (26.6 vs 25.4 HR 1.04 (95%
IC 0.51 to 1.82; p = 0.91) and overall survival (55.6 vs 48.2 HR 0.70 (95% IC 0.65 to 3.11; p
= 0.37) did not differ across YAP expression categories (table 3 and figure 2).
As Shown in figure 3, miR-9 expression levels were not associated with YAP
expression categories (p=0.157).
Discussion
We investigated nYAP and cpYAP expression in a large set of patients with
advanced HGSOC submitted to platinum based chemotherapy. High nYAP and low cpYAP
expression were not associated with platinum response, either PFS or OS. In addition, miR-9
copy number was not associated with YAP expression.
YAP has often been described as an oncogene in several different tumors with an
increasing correlation between YAP expression and survival [35]. Wang et al [25] investigated
YAP expression 139 cases of colorectal cancer. Their results showed that YAP was
overexpressed in roughly 50% of cases; when overexpression occurred in the nucleus, those
authors detected an association with shorter OS (p<0.001). In that study,
immunohistochemistry analysis considered both cytoplasmic and nuclear YAP expression.
Since only nuclear YAP is able to mediate expression of genes associated with cancer
proliferation and survival, Wang et al. [25] results miss the Hippo pathway core signaling
dynamics. Kang et al [24] evaluated YAP expression in a cohort of 101 gastric cancers. High
nuclear YAP correlated with poor disease specific survival, mainly in patients with early stage
disease. Kang et al [24] findings reiterated the functional significance of YAP nuclear
localization. When YAP is in the nucleus it is presumably transcriptionally active and
therefore high nYAP staining advocate for high YAP activity. However, YAP cytoplasmic
expression can only be evaluated once phosphorylated YAP antibody is used. In the Hippo
pathway, YAP Phosphorylation results in YAP cytoplasmic (cpYAP) retention. Thus, only
cpYAP expression can adequately quantify YAP activity in the cytoplasm. Kim et al. [27]
60
performed YAP and pYAP expression analyses in 678 FFPE breast cancer tissue specimens. In
univariate analysis, nYAP expression was associated with shorter OS. Although Kim et al.[27]
examined nuclear and cytoplasmic YAP expression with YAP and pYAP antibodies, combined
analysis of high nYAP and low cpYAP was not performed.
In human ovarian cancer, Hall et al. [13] demonstrated that in immortalized
ovarian surface epithelial cells YAP was located predominantly in the nuclei and pYAP was
predominantly in the cytoplasm. Hall et al. [13] used YAP and pYAP antibodies to evaluate
protein expression of YAP in the nuclei (nYAP) and expression of pYAP in the cytoplasm
(cpYAP), exactly as we did. They studied a cohort of 70 women ovarian carcinomas and
found that high nYAP expression was a significant prognostic marker of poor patient survival.
The combined analysis of nYAP and cpYAP high/low categories showed that YAP highest
activity was expressed in tumors with high nYAP and low cpYAP. Patients whose tumors
expressed high nYAP and low cpYap fared significantly worse than patients with other
combined category of nYAP and cp YAP. Hall et cols. also postulated that YAP subcellular
distribution (High nYAP and low cpYAP) was a strong independent predictor of disease
specific survival for ovarian cancer. Hall et cols also found that overexpression of YAP in
immortalized ovarian surface epithelial cells resulted in increased proliferation, resistance to
cisplatin induced apoptosis, cell migration and anchorage independent growth.
Xia et al. [29] performed protein expression analyses of YAP and pYAP
expression; slides were evaluated based on cytoplasmic and nuclear staining. In that study,
YAP and pYAP expression were not associated with poor prognosis. However, when
evaluating YAP antibody alone, patients with high nYAP versus low nYAP and patients with
high nYAP and low cYAP had worse survival when compared with patients in other
categories. When evaluating high YAP with high pYAP (considering cytoplasmic and nuclear
expression) with other categories, patients with high YAP and high nYAP expression had
significantly inferior outcome. Xia et al. [29] proposed that high YAP expression rather than
its subcellular distribution was associated with patient survival. Xia et al. [29] also did in vitro
and in vivo studies and, in concordance with Hall et al. [13] findings, showed that YAP
promotes ovarian cancer cell proliferation, enhances resistance to chemotherapy drugs, cell
migration and anchorage independent growth.
In contrast to Hall et al. [13] and Xia et al. [29], we did not find a relationship
between YAP expression and survival in stage III/IV HGSOC. Both Xia and Hall cohorts
incorporated tissue samples of epithelial ovarian cancer with different histology types,
including high-grade and low-grade ovarian carcinoma, clear cell carcinoma, endometrioid
61
carcinoma and mucinous carcinoma. A recently reviewed dualistic model [4] divides
epithelial ovarian cancer according to carcinogenesis and genetic data in type I and Type II
tumors. The new model divides type I tumors into three groups: endometriosis related tumors
that included endometriod, clear cell and seromucinous carcinoma, low grade serous
carcinoma and mucinous carcinoma and Brenner tumors. Type II tumors are most part high
grade serous carcinoma. Type I Tumors develop from benign lesions that undergo malignant
transformation. Type II tumors arise from intraepithelial carcinomas in the fallopian tube and
disseminate with aggressive behavior as carcinomas involving ovaries and peritoneum.
Dualistic model has highlighted the heterogeneity of ovarian cancer with type I and type II
comprehending entirely different diseases. Consequently studies that aim to identifie predict
and prognostic factors should focus either on type I or type II disease due to their distinct
clinical behavior and platinum response.
In our study. miR-9 expression levels were not associated with YAP expression on
Hippo pathway. In breast cancer miR-9 act as an oncomiR by negatively regulating tumor
suppressor LIFR and Ecadherin, inactivating Hippo pathway [22]. In gastric cancer cells,
miR-9 also acted as an oncomiR by targeting CUL4A and indirectly regulating LATS1-Hippo
signaling pathway promoting cell proliferation and invasion [23]. Chen et al. [22] showed that
in breast cancer cells MiR- 9 can target two metastasis suppressors: the leukemia inhibitory
factor receptor (LIFRI) and E-cadherin. E-cadherin maintains adherence junctions and
sequesters β-catenin at cytoplasmic membrane. LFRI promotes membrane localization of
scribble which promotes YAP phosphorylation and functional inactivation. As a result LFRI
promotes Hippo signaling activation. In gastric cancer Deng et al. [23] demonstrated that
miR-9 overexpression down regulated a transcriptional factor (CUL4A) and activate LATS1
retaining YAP in cytoplasm, which means that Hippo signaling pathway is activated and
therefore there is a suppression of cell proliferation and invasion.
In ovarian cancer, miR-9 has been controversially described either as a oncomiR
or as tumor suppressor miR. Zhao et al. [36] in their study revealed this double-face role of
miR-9 in ovarian tumor cells. They analyzed the expression of nine microRNAs by
performing real-time polymerase chain reaction in 46 primary ovarian tumor cells from
surgically resected patients with distinct sensitivity to platinum based chemotherapy. MiR-9
expression was significantly higher in platinum sensitive patients and tumor cells from
platinum sensitive patients were more tumorigenic than those from platinum resistant patients.
Laios et al. [37] analyzed miR-9 expression levels in 22 FFPE and 18 fresh frozen samples of
primary and recurrent ovarian cancer. Using real-time polymerase chain reaction technic,
62
miR-9 was significantly down regulated in recurrent cancers when compared to primary.
Recently, Yanaihara et al [38] in a cohort of HGSC and ovarian cancer clear cell (OCCC)
showed that miR-9 presented with higher expression in OCCC. MiR-9 overexpression
directly target E-cadherin thereby inducing an EMT. Therefore, although we did not find an
association between miR-9 expression levels and YAP expression on Hippo pathway,
microRNAs can play different roles in the development and progression of cancers
conditional on target genes.
One of the major boundary of YAP immunohistochemical analysis is that YAP is a
transcriptional co-activator in the Hippo pathway. As a transcriptional co-activator, YAP can
be either phosphorylated and retained in the cytoplasm or active in the nucleus. Thus, YAP
expression can be read in the cytoplasm and nucleus, and there is no defined standard pattern
regarding staining and image analysis. In conclusion, although YAP expression was not
confirmed with in vitro and in vivo tests, we have a significant cohort of HGSOC patients with
available FFPE samples and adequate follow-up. This is the largest study of Brazilian patients
with HGSOC, in which all cases were reviewed by an expert pathologist, nYAP and cpYAP,
expression, miR-9 expression levels were performed and platinum resistance was assessed.
We concluded that in in this large set of patients with advanced HGSOC
submitted to platinum based chemotherapy, high nYAP and low cpYAP expression were not
associated with platinum response, miR-9 expression levels, progression free or overall
survival.
Conflict of interest: The authors declare that there is no conflict of interest regarding the
publication of this article.
63
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Table 1: High grade serous ovarian carcinoma (HGSOC) patient distribution according to
Nuclear Yes associated protein (nYAP) and Cytoplasmic phosphorylated Yes-associated
protein (cpYAP) categories.
nYAP expression
cpYAP expression [%] Low (%) High (%)
Low [%] 13 (21.3) 15 (24.6)
High [%] 15 (24.6) 18 (29.5)
YAP=Yes-associated protein; nYAP=nuclear YAP; cpYAP=cytoplasmic phosphorylated YAP; Upregulated YAP
considers high nYAP plus low cpYAP. All others combination of nYAP and cpYAP expression were considered
YAP unknown.
68
Table 2: High grade serous ovarian carcinoma (HGSOC) patient distribution according to
clinical features and Yes-associated protein (YAP) expression categories.
High nYAP and low cpYAP
15 cases
Others combinations of
nYAP and cpYAP
46 cases
p
Age (years) mean +/- SD 59.26(+/-11.9)
59.45(+/-10.75) 0.841#
Stage [n(%)]
III 14 (93) 45(98)
0.434*
IV 1(7) 1(2)
CA125 mean
+/- SD
1090.9 (+/-1258.6) 2300.6 (+/-5694.1)
0.506#
Post-surgery residual
disease [n(%)]
No 9(60) 19(30) 0.335†
Yes 6(40) 27(70)
Cycle of platinum [n(%)]
3 to 5 1(7) 4(9) 1.000*
>= 6 14(93) 42(91)
Platinum response[n(%)]
Refractory/resistant 5(33) 17(27) 1.000†
Sensitive/acquired
resistance
10(67) 29(63)
#Mann Whitney; *Fisher; † qui-square; SD=Standard Deviation; YAP=yes-associated protein; nYAP=nuclear
YAP; cpYAP=cytoplasmatic phosphorylated YAP; Cases with high nYAP and low cpYAP were considered
Hippo inactivated and others combinations were considered unknown.
69
Table 3: Progression free survival and overall survival in women with high grade serous
ovarian carcinoma according to yes-associated protein (YAP) expression categories
YAP
n
Median PFS
(95%IC)
HR
(95%IC) p
Median OS
(95%IC)
HR
(95%IC) p
High nYAP and low
cpYAP
15 26.6
(2.13 to 3.36)
1.04
(0.51 to 1.82)
0.91 55.6
(14.2 to 21.5)
0.70
(0.65 to 3.11)
0.37
Others combinations
of nYAP and cpYAP
46 25.4
(0.38 to 1.46)
Reference 48.2
(14.1 to 17.9)
Reference
PFS=progression free survival, OS=overall survival. HR=hazard ratio. Cases with high nYAP and low cpYAP
were considered hippo inactivated and others combinations were considered unknown. During follow-up 13
patients with high nYAP and low cpYAP expression progressed and 8 died. In others combinations of nYAP and
cpYAP, 37 patients progressed and 30 died.
70
Figure 1 - Representative images of nYAP and cpYAP expression through
immunohistochemistry assays in high grade serous ovarian carcinoma (A) High nYAP; (B)
Low nYAP; (C) High cpYAP and (D) Low cpYAP (X100). YAP=Yes-associated protein;
nYAP=nuclear YAP; cpYAP=cytoplasmatic phosphorylated YAP.
71
Figure 2 - Progression free survival probability (p=0.91 – figure 2A) and overall survival
probability (p=0.37 – figure 2B) according to Yes-associated protein (YAP) expression in
patients with high grade serous ovarian carcinoma. Cases with high nYAP and low cpYAP
were considered hippo inactivated and others combinations were considered unknown.
B A
72
Figure 3 – Representative image of miR-9 levels according to according to Yes-associated
protein (YAP) expression in patients with high grade serous ovarian carcinoma. Cases with
high nYAP and low cpYAP were considered hippo inactivated and others combinations were
considered unknown.
73
5. DISCUSSÃO GERAL
Neste trabalho, para o qual foram selecionados CSAGO diagnosticados em
estádio III/IV, observou-se que a expressão de DDR2 não se associou com características
clinicas ou com resposta à quimioterapia baseada em platina. A maior expressão do DDR2 se
associou independentemente e significamente com pior SLP, porém não se associou com a
SG. Baixos níveis de miR-182 foram associados com alta expressão de DDR2. O miR-96 não
se associou com a expressão do DDR2. A expressão do YAP não se associou com a sobrevida
em pacientes com CSAGO. O miR-9 não se associou com a a expressão do YAP.
O DDR2 e o YAP são marcadores que vem sendo estudados como relevantes para
tumorigênese e progressão dos CSAGO [78, 87, 88]. Sabe-se que o tumor tem a capacidade
aderir, invadir a membrana basal, e migrar via canais linfáticos ou vasculares. A regulação
deste processo ocorre pela interação das células tumorais e dos componentes do estroma,
sendo muito influenciado pelo microambiente tumoral. O DDR2 é um receptor tirosino
quinase que quando ativado pelo colágeno induz proliferação e metástases [101]. No presente
estudo o DDR2 teve um significativo papel prognóstico em mulheres com CSAGO, com
impacto na SLP. A alta expressão do DDR2 parece ser um evento inicial da tumorigênese que
predispõe a recidiva [102]. O impacto da expressão do DDR2 se dilui na SG pois esse é um
evento influenciado por diversos fatores. A análise da expressão do YAP na presente coorte de
mulheres com CSAGO não se associou com a sobrevida. Esses resultados não são
concordantes com Hall e cols. [87] e Xia e cols. [88]. Nesses estudos anteriores a análise
imunoistoquímica incluiu variados tipos de carcinoma de ovário que apresentam
características moleculares distintas. Além disso, não houve seleção dos casos diagnosticados
em estádio mais avançado ou determinação da sensibilidade à platina. Xia e cols. [88]
realizaram imunoistoquímica em blocos de parafina de pacientes com carcinoma de ovário e
identificaram uma associação com sobrevida. Porém, Xia e cols. [88] consideraram a
expressão do YAP total e não a expressão do nYAP ou do cpYAP. Assim, Xia e cols. [88]
concluiram que a alta expressão de YAP, e não distribuições subcelulares está associada à
sobrevida. Diferentemente, Hall e cols. [87] em sua análise de 70 blocos de parafina de
pacientes com carcinoma de ovário postularam que a combinação da expressão alta do nYAP
e expressão baixa do cpYAP teve em uma associação significativa com a sobrevida. Esta
associação indica que quando o YAP está localizado no núcleo, ele está realizando ativamente
a transcrição. De maneira contrária, quando o YAP está fosforilado e retido no citoplasma a
transcrição está inativada e, portanto a coloração intensa, citoplasmática de pYAP sugere
74
baixa atividade de YAP. Assim, infere-se que quando o YAP está ativado a via da Hippo, que é
uma via supressora tumoral, está desativada; já quando o YAP esta desativado a via da Hippo
está ativada. Os mecanismos de regulação do YAP ainda não são bem conhecidos. Uma
hipótese é que exista um pool constante do cpYAP que regule o nível do nYAP. Assim, mesmo
quando o nYAP está alto, a fosforilação citoplasmática não está totalmente inibida [103]. A
elucidação deste mecanismo será importante se considerarmos YAP como um alvo adequado
para intervenções terapêuticas.
DDR2 e YAP são marcadores descritos em estudos pré-clínicos como importantes
para crescimento e progressão tumoral além de possíveis alvo de terapias em
desenvolvimento [77, 87, 88, 104]. Um estudo recente reportou a eficácia do veterporfin
como supressor do complexo YAP/TEAD. Em experimentos in vitro e in vivo em modelo
animal observaram a supressão do complexo YAP/TEAD quando expostos a veterporfin
[105]. A veterporfin parece uma droga promissora em câncer de ovário. O DDR2 também é
considerado um alvo terapêutico por ser um receptor com atividade tirosino quinase.
Paralelamente, mutações oncogênicas já foram relatadas em carcinoma de pulmão não
pequenas células com particular eficácia do dasatinib [77].
Estes estudos anteriores são modelos de pesquisa básica em câncer que utilizam
culturas de células e modelos animais. As culturas de células são responsáveis por grandes
avanços no conhecimento da biologia tumoral. É o método que primeiro valida biomarcadores
e testa drogas alvo. Estudos in vitro conseguem determinar a funcionalidade e selecionar as
drogas que serão posteriormente testadas em estudos clínicos com humanos. Porém como o
carcinoma de ovário é muito heterogêneo, com características moleculares distintas e nem
todas as culturas de células são representativas de todas as variedades de carcinomas de
ovário. Várias culturas de células de ovário comumente utilizadas são classificadas de
maneira incorreta [106]. Recentemente foi publicada uma validação de 25 linhagens de
culturas de células com a correta identificação do subtipo de carcinoma de ovário [107].
Estudos in vivo, com inserção de células tumorais de carcinomas de ovário obtidos de
mulheres, em ratos, tem propiciado melhor entendimento da interação do tumor com o
microambiente e a resistência a drogas [108].
No presente estudo a imunoistoquímica foi escolhida por detectar expressão
proteica em tecido parafinado. A expressão proteica é expressão final e geralmente funcional
de uma via de sinalização celular. A imunoistoquimica é uma técnica consagrada e inserida na
prática diária. É uma reação antígeno-anticorpo que utiliza anticorpos marcados com
substância cromógena, que identificam proteínas específicas, visualizadas e quantificadas por
75
microscopia óptica. Na rotina de um serviço de patologia já existe uma padronização para
vários anticorpos; em carcinomas serosos de alto grau marcadores como p53 e BRCA são
usados para definição diagnóstica e terapêutica [109, 110]. Apesar da imunoistoquimica ser
uma técnica consagrada, a análise final e quantificação da coloração pode ser afetada por
fenômenos de autólise, variações na fixação e variações inter-observadores. No presente
estudo, uma das dificuldades foi a falta de padronização na análise da expressão de DDR2 e
do YAP. Especialmente para a expressão do YAP, fosforilado ou não, a falta de um padrão
definido de positividade na coloração destes anticorpos, que coram núcleo e citoplasma,
elevou a dificuldade do patologista na interpretação dos resultados. A falta de padronização da
leitura do DDR2 e do YAP é portanto um empecilho à sua inserção na prática diária. A qRT-
PCR é uma metodologia atualmente amplamente difundida na pesquisa e no diagnóstico. É
um método que apresenta alta sensibilidade e especificidade por identificar a molécula-alvo
em soluções com vários componentes. A qRT-PCR é o método de escolha para estudos
quantitativos de microRNA pela alta capacidade de automação e precisão de quantificação
[96, 98].
As mulheres com CSAGO apresentam boa resposta inicial a quimioterapia
baseada em platina, porém com recidivas precoces decorrentes da resistência à esse
quimioterápico. A resistência à platina determina um pior prognostico e baixa sobrevida
dessas mulheres[16, 17, 39]. Segundo Dong e cols [111], antes de utilizar na prática clínica,
informações derivadas de estudos moleculares, vários pontos-críticos devem ser considerados:
1) são necessários estudos de coortes prospectivos incluindo grande numero de mulheres para
validar os biomarcadores potenciais. Os resultados destes biomarcadores devem ser
reprodutíveis e consistentes entre vários laboratórios. 2) as amostras biológicas para pesquisa
devem ser adequadamente obtidas, armazenadas e processadas, seguindo os protocolos e
padrões exigidos. Os registros clinico-patológicos relacionados devem ser claros e precisos,
incluindo um seguimento adequado. 3) novos métodos não invasivos de detecção precoce,
diagnóstico e avaliação de resposta à terapêutica devem ser desenvolvidos. 4) para o
desenvolvimento de terapia alvo, devem ser realizados experimentos que elucidem as funções
dos potenciais alvos. Modelos mais consistentes devem ser desenvolvidos para identificar
assinaturas moleculares associadas aos diferentes fenótipos clínicos. Deste modo, será
possível coordenar combinações de terapia alvo, terapia epigenética e terapia convencional,
que possam interferir positivamente na sobrevida das mulheres com câncer de ovário.
O presente estudo foi realizado no Hospital da Mulher Prof. Dr. José Aristodemo
Pinotti - CAISM - Unicamp que é um hospital de referência no atendimento à saúde terciária.
76
No setor de ginecologia oncológica as mulheres com CSAGO são tratados conforme
protocolo padrão de cirurgia e quimioterapia baseada em platina. Minha perspectiva a partir
da realização deste trabalho como médica do CAISM, é planejar um modelo de atenção que
contemple a coleta de material biológico (sangue, ascite e tecido fresco e parafinado) em
todas as mulheres com carcinoma de ovário de modo a termos um biobanco alinhado a um
banco de dados clínicos que possa servir de base para estudos de mecanismos de resistência a
drogas.
77
6. CONCLUSÃO
6.1. A expressão do DDR2 não esteve associada nenhuma variável clinico-
patológica ou resposta a platina. A maior expressão do DDR2 esteve associada com pior SLP
na analise uni e multivariada mas não se associou com a SG em mulheres com CSAGO.
Baixos níveis de miR-182 foram associados com alta expressão do DDR2. O miR-96 não se
associou com a expressão do DDR2.
6.2. Em mulheres com CSAGO a alta expressão do nYAP com baixa expressão de
cpYAP não esteve associada com nenhuma variável clinico-patológica ou resposta a platina. A
expressão do YAP não se associou com sobrevida em mulheres com CSAGO. O miR-9 não se
associou com a expressão do YAP.
78
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88
8. ANEXOS
Anexo 1 - Ficha para coleta de dados clínicos
89
Anexo 2 - Carta de aprovação do projeto no CEP-Unicamp
90
91
92
Anexo 3 - Adendo a carta de aprovação do CEP-Unicamp
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