( PDF ) Estudos imuno-histoquímico das proteínas p53, p16, Fhit, caspase 3 e antígeno Ki67; e citogenético molecular em lesões benignas e carcinoma de esôfago

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Marilanda Ferreira Bellini

Estudos imuno-histoquímico das proteínas p53, p16,

Fhit, caspase 3 e antígeno Ki67; e citogenético

molecular em lesões benignas e carcinoma de

esôfago

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Campus de São José do Rio Preto

Marilanda Ferreira Bellini

Estudos imuno-histoquímico das proteínas p53, p16,

Fhit, caspase 3 e antígeno Ki67; e citogenético

molecular em lesões benignas e carcinoma de

esôfago

Orientadora: Profa. Dra. Ana Elizabete Silva

Co-Orientadora: Profa. Dra. Marileila Varella-Garcia

São José do Rio Preto – SP

2009

Programa de Pós-Graduação em Genética

Tese apresentada para

obtenção do Título de Doutor

em Genética

ads:

Bellini, Marilanda Ferreira.

Estudos imuno-histoquímico das proteínas p53, p16, Fhit, caspase 3 e

antígeno Ki67; e citogenético molecular em lesões benignas e carcinoma de

esôfago/ Marilanda Ferreira Bellini. - São José do Rio Preto : [s.n.], 2009.

219 f. : il. ; 30 cm.

Orientador: Ana Elizabete Silva

Co-orientador : Marileila Varella-Garcia

Tese (doutorado) – Universidade Estadual Paulista, Instituto de

Biociências, Letras e Ciências Exatas

1. Genética. 2. Chagas, Doença de. 3. Megaesôfago chagásico. 4.

Esofagite crônica. 5. Carcinoma de células escamosas. 6. Esôfago - Doenças.

7. Lesões esofágicas benignas. I. Silva, Ana Elizabete. II. Varella-Garcia,

Marileila. III. Universidade Estadual Paulista, Instituto de Biociências, Letras e

Ciências Exatas. IV. Título.

CDU - 575

Ficha catalográfica elaborada pela Biblioteca do IBILCE

Campus de São José do Rio Preto - UNESP

Marilanda Ferreira Bellini

Estudos imuno-histoquímico das proteínas p53, p16, Fhit, caspase 3 e

antígeno Ki67; e citogenético molecular em lesões benignas e carcinoma

de esôfago

Dissertação apresentada para obtenção do título

de Doutor em Genética, junto ao Programa de

Pós-Graduação em Genética do Instituto de

Biociências, Letras e Ciências Exatas, da

Universidade Estadual Paulista “Júlio de Mesquita

Filho”, Campus de São José do Rio Preto.

BANCA EXAMINADORA

Profa. Dra. Ana Elizabete Silva

Professor Adjunto Livre-Docente

UNESP – São José do Rio Preto

Orientadora

Profa. Dra. Juliana Karina Ruiz Heinrich

Bióloga

Universidade Estadual de Campinas

Profa. Dra. Agnes Cristina Fett Conte

Professor Livre-Docente

Faculdade de Medicina de São José do Rio Preto

Profa. Dra. Silvia Regina Rogatto

Professor Adjunto Livre-Docente

UNESP – Botucatu

Profa. Dra. Paula Rahal

Professor Adjunto Livre-Docente

UNESP – São José do Rio Preto

São José do Rio Preto, 20/02/2009.

O presente trabalho foi realizado no Laboratório de

Citogenética e Biologia Molecular Humana, Departamento de Biologia do

Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto

– SP, da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP;

Laboratório de Imuno-histoquímica e Colorações Especiais, Seção de

Patologia do Hospital de Base de São José do Rio Preto – SP; Laboratório

de Patologia Cirúrgica e Molecular, Hospital Sírio Libanês, São Paulo – SP e

Cytocore, University of Colorado, Health Sciences Center, Aurora – CO –

USA, com bolsa de Doutorado do CNPq, bolsa de estágio de doutoramento

no exterior CAPES e auxílio financeiro da FAPESP.

Dedicatória

Aos meus pais, Pedro Edgard Bellini e Cleonice

Maria Ferreira Bellini, e a minha “irmãzinha” Franciele Ferreira

Bellini, pois não deve ter sido nada fácil me apoiar minhas

peripécias e idéias de cientista lunática.

Agradecimentos

“Senhor,

Dai-me serenidade para aceitar as coisas que não posso

mudar;

Coragem para mudar as que eu posso e

Sabedoria para distinguir umas das outras.”(E. K. Ross,D.Kessler)

A Deus, “por ordenar o mundo de maneira

tão bela e sábia”, permitindo que sempre se abra uma janela, quando todas

as portas parecem estar fechadas;

“Eu tenho tanto pra te falar,

mas com palavras não sei dizer, como é grande o meu amor por você,

Nem mesmo o céu, nem as estrelas, mesmo o sol ou o infinito, não

conseguem ser maior que o meu amor, nem mais bonito” (Vinicius de

Morais) A minha Família pelo AMOR incondicional;

“Imagine how the world could be

So very fine

So happy together” (Garry Bonner e Alan Gordon)

A Juliano Farias da Nóbrega pela parceria,

companheirismo, apoio e incentivo.

“Mestre, posso sair mais tarde hoje?

Ao contrário do que algumas vezes te pedi, hoje quero ficar mais um pouco.

Ficar mais alguns instantes para desfrutar de tua presença e da proteção

que ela me traz.

Ficar, para poder admirar, mais uma vez, a tua destreza, a tua capacidade

de diagnosticar e prognosticar. Ficar pela segurança, pelo poder contar,

contar contigo, amparando minhas dúvidas e, suscitando em mim a

maturidade de um profissional...

Agora, encontro-me, procurando meu estado de aluno, sabendo que em

instantes me tornarei seu colega. Porém, com a certeza de jamais deixar de

ser seu discípulo. Discípulo que guardará na memória seus ensinamentos e

no coração, a gratidão, o respeito e a saudade” (Anônimo)

As Profª Drª Ana Elizabete Silva e Profa.

Dra. Marileila Varella-Garcia pela confiança, ensinamentos e amizade;

A Banca Examinadora, Profa. Dra. Silvia

Regina Rogatto, Profa. Dra. Juliana K.R. Heinririch, Profa. Dra. Agnes C.

Fett-Conte e Profa. Dra. Paula Rahal por terem aceitado a participar da

análise e enriquecimento do trabalho.

Aos meus amigos que “como anjos me levantaram, quando minhas asas

tiveram problemas, me lembrando como voltar a voar” (Mário Quintana);

“O mais altruísta dos amigos que um homem pode ter neste mundo egoísta,

aquele que nunca o abandona e nunca mostra ingratidão ou deslealdade, é

o cão”. (Anônimo)

A todos animais de estimação, que

passaram pelo meu percurso, pelos rabinhos abanando de felicidade, e em

especial a Bella, pela felicidade ao me reencontrar depois de um ano e pelo

companheirismo confidente, meses e meses, sempre ao meu lado, durante

a confecção desta Tese;

“Se tem bigodes de foca, nariz de tamanduá;

Parece meio estranho, heim?

Também um bico de pato e um jeitão de sabiá;

Mas é amigo, não precisa mudar, é tão lindo deixa assim como está”

(Roberto Carlos)

Aline Poersch, Andressa Seri, Arianca

Alvarado, Cíntia Nunes, Claudiane Borba, Edson Lopes, Gislaine

Rodrigues, Mateus Marcos, Milene Lara e Patrícia Batista, por não se

importarem com as diferenças, desavenças e distância;

Isabeth Estevão, Ligia Baracioli, Lucilene

Maschio e Rita Peruquetti, a esquerda festiva, por também “esquecerem o

que falaram sobre ser adulto” (Arnaldo Jabor) e terem dividido suas idiotices

comigo;

Cadu Barbosa, Fernanda Carregaro,

Franciole e Tatiana Caparroz, Gilmar Santos, Janaina Rigonatto,

Leonardo Cabrioti, Marco “Todd” Asanome, Marta Lovato, Mauro

Leonardo, Nayara Delgado, Patrícia Ayub, Rodrigo Juliano, Ronaldo

Costa e Tázio Gaspari, “por me guardarem do lado esquerdo do peito,

mesmo que tempo e a distância digam não” (Milton Nascimento).

Luciana Ondei, Paula Zamaro, Ana

Carolina, Ana Luiza, Profa. Dra. Claudia Regina Bonini Domingos,

Nelson C. Tukamoto Jr, Carlos Zago, por se serem AMIGOS até nos

momentos mais difíceis.

“Don’t stop believin

Hold on to the feelin

Streetlight people” (James Iha)

Diana Villa, Estefânia Muriedas e Elvia

Navarro, por serem as melhores muchachas em terras tão distantes.

Aos meus primos Monique, Wanderson,

Ivan, Bruna, Ligia, Juliano e Michele por me aceitaram como sou e se

divertirem com isso, sempre oferecendo o ombro amigo.

“Pane no sistema, alguém me desconfigurou” (Pitty)

A Flayd Guerreiro e Juliano Farias

Nóbrega por toda assessoria excell, photoshop, corel draw e afins;

“When you’re down and troubled

And you need some loving care

And nothing, nothing is going right

Close your eyes and think of me and soon

I will be there to brighten up even your darkest night

…

Ain’t it good to know you’ve got a friend…” (Carole King)

Marcelo Almeida, Milena Andrade,

Juliano Farias, Manuela Santoro, Rodriguinho, Bruninha, “The Class

Team”, pela diversão durante o prepatório para TOEFL Test and friendship.

"O valor das coisas não está no tempo que elas duram, mas na intensidade

com que acontecem. Por isso, existem momentos inesquecíveis, coisas

inexplicáveis e pessoas incomparáveis”.(Fernando Pessoa)

Aos colegas do Laboratório de

Citogenética e Biologia Molecular Humana, Cidinha Silveira, Daniela

Bizari, Fernanda Manoel-Caetano, Héctor Matioli, Isabella Migliori,

Josué Rodrigues, Juliana Oliveira, Márcia Duarte e Yvana Jorge.

Ao Pessoal do Setor de Patologia do

Hospital de Base, Dinha por repassar meus telefonemas constantes,

Andréia por estar sempre disponível em me auxiliar com os prontuários,

Darlei pela seleção dos blocos.

A Imuno-histoquimica por ter me

presenteado com grandes amigas: Elaine Keid, Elaine Gouveia, Flávia

Gomes e Ana Paula.

Aos meninos do Laboratório de Patologia

do Hospital Sírio Libanês, Felipe, Laisaac, Fernando, Robson,

Alexandre, Agenor; e as meninas também, Rosangela, Mayra, Márcia,

Rosineide, Dra. Fernanda, por dividirem comigo seu espaço e sua simpatia,

em especial ao Isaque Santana e a Josiane Costa pela atenção,

companhia e disposição.

As Profas. Dras. Patrícia Maluf Cury e

Kátia Ramos Moreira Leite pelo diagnóstico patológico das amostras e

auxílio nas análises de imuno-histoquímica.

Ao Prof. Dr. Sebastião Roberto Taboga e

Luis Roberto Faleiros pela colaboração no preparo das lâminas e foto-

documentação.

Ao Setor de Endoscopia do Hospital de

Base, Dr. Kenji Miyazaki e Dr. Henrique Oliveira pela coleta das biópsias

de megaesôfago.

A Profa. Dra. Agnes C. Fett-Conte,

Adriana Barbosa e Cristina do Laboratório de Genética, FAMERP, pela

disponibilização do microscópio de fluorescência;

A Profa. Dra. Eny Maria Goloni Bertollo, a

secretária Eliana, Silvia e João (SAME) pelo auxílio na consulta de

prontuários;

Margaret Skokan, Pornthip Kiatsimkul,

Sujatha Gajapathy e Yun Xiao, “Leila’s Angels”, pela convivência,

ensino-aprendizagem, intercâmbio cultural, horários de almoço, horários

extraordinários, caronas, confidências, confiança, segredos, desabafos,

choros, ombros, caixinhas de lenços, abraços, alegrias, chocolates, sorvetes

e principalmente pela amizade.

A equipe do Cancer Center, University of

Colorado Health Sciences Center, pela convivência harmoniosa,

agradável e muito produtiva, em especial a Maria Pia Morelli, Marialuisa

Valente, Sharvari Gayal, Christopher Koch e Efang Li pela amizade

construída.

Ao Prof. Dr. Antonio José Manzato pela

assessoria estatística.

A Seção de Pós-graduação, em especial a

Rosana Ferro, Rosemar Brena e Silvia Kazama, por estarem sempre

solicitas.

“Cada um que passa em nossa vida, passa sozinho, pois cada pessoa é

única, e nenhuma substitui a outra. Cada um que passa em nossa vida,

passa sozinho, mas não vai só, nem nos deixa só, leva um pouco de nós

mesmos, e deixa um pouco de si mesmo. Há os que levem muito, mas há

os que não deixam nada. Essa é a maior responsabilidade de nossa vida. É

a prova evidente que duas almas não se encontram ao acaso” (Saint –

Exupéry)

Ana Lúcia Dias, Ana Paula Moreno,

Bárbara Bisoli, Brett Radetsky, Christina Mailloux, Cínara Brandão,

Claudio Cordioli, Débora Santana, Eliza Oliveira, Fabián Mendiburu,

Fernando Lago, Flávia Paba, Helena Lelis, Jozélia Ferreira, Junior

Caixeta, Lauriane Giselle, Lidiane Moura, Profa. Dra. Maria Tercília A.

Oliveira, Mário Sérgio Mantovani, Monique Beaudoin, Nancy Joleon,

Hangeseth’s, Montoya’s, Paulsen’s, Família Tinoco, Família Monteiro

Amaral, Família Guerreiro, Família Farias Nóbrega; Sandra, Edimilson e

Maryanne Melo; Nathalie, Nayara, Silvana e José Carlos de Souza;

Nélio e Márcio Farias; Enéas e Mathias Bontempo; Jociléa, Alex e A.J.

Scalisse; Fatinha e Grazielle Prado; Cybelle, Maria Helena e

Hindemburgo Almeida.

“Mas a Escola é a luz que ilumina o caminho da gente” (Carlos Pedro

Ferreira)

A Universidade Estadual Paulista Júlio de

Mesquita Filho – UNESP, ao Instituto de Biociências Letras e Ciências

Exatas – IBILCE, ao Programa de Pós-Graduação em Genética – PPGGen,

pela oportunidade de concluir esse passo tão importante em minha

formação acadêmica.

“Dos filhos deste solo és mãe gentil

Pátria amada

Brasil” (Joaquim Osório Duque Estrada)

Aos órgãos de fomento: CNPq pela bolsa de

estudos, CAPES pela bolsa de estágio de doutoramento no exterior e

FAPESP pelo auxílio financeiro.

A todos aqueles que de maneira direta e

indireta colaboraram para o desenvolvimento deste trabalho.

MUITO OBRIGADA

Epígrafe

“Você não sabe o quanto eu caminhei

Prá chegar até aqui

Percorri milhas e milhas antes de dormir

Eu não cochilei

Os mais belos montes escalei

Nas escuras de frio chorei

A vida ensina e o tempo traz o tom

Pra nascer uma canção

Com a fé do dia-a-dia

Encontrar solução

...

Meu caminho só meu Pai pode mudar”

(Toni Garrido, Lazão, Da Gama e Bino Farias)

Sumário

SUMÁRIO

I. INTRODUÇÃO........................................................................................................... 38

I.1. Lesões Benignas do esôfago ....................................................................... 39

I.2. Carcinogênese do esôfago e alterações genéticas……………………………45

I.3. Alterações Genéticas em esofagite crônica e megaesôfago ................. ……53

I.4. Controle de Proliferação Celular e Apoptose ............................................... 56

II. OBJETIVOS ............................................................................................................. 62

CAPÍTULO I: Genomic imbalances in esophageal squamous cell carcinoma

identified by molecular cytogenetic techniques ............................................................. 64

CAPÍTULO II: p53, p16 and Fhit Proteins Expressions in Chronic Esophagitis and

Chagas Disease ........................................................................................................... 94

CAPÍTULO III: Expression of caspase-3 protein and Ki67 antigen in benign lesions

and esophageal carcinoma and relationship with p53 protein. .................................... 104

CAPÍTULO IV: Chromosomal imbalances are uncommon in Chagasic

megaesophagus ......................................................................................................... 125

III. DISCUSSÃO.......................................................................................................... 159

IV. CONCLUSÕES ..................................................................................................... 167

REFERÊNCIAS BIBLIOGRÁFICAS ............................................................................ 170

APÊNDICE ................................................................................................................. 192

Apêndice 1: Termo de Consentimento Livre e Esclarecido (Sanduíche) .......... 193

Apêndice 2: Questionário ................................................................................. 194

Apêndice 3: Caracterização das amostras de mucosa esofágica normal e

Carcinoma de células escamosa de esôfago ................................................... 195

Apêndice 4: Caracterização das amostras de pacientes com esofagite crônica

........................................................................................................................ 196

Apêndice 5: Caracterização das amostras de pacientes chagásicos sem

megaesôfago. .................................................................................................. 197

Apêndice 6: Caracterização das amostras de pacientes chagásico com

megaesôfago Apêndice……………………………………………………………..198

Apêndice 7: Chromosomal Mapping for the newly developed probes. ............. 201

ANEXOS .................................................................................................................... 202

Anexo 1. Parecer Consubstanciado do Comitê de Ética em Pesquisa

Institucional (Projeto de Doutorado Original). ................................................... 203

Anexo 2. Parecer Consubstanciado do Comitê de Ética em Pesquisa

Institucional (Projeto de Estágio de Doutoramento no Exterior). ...................... 204

Anexo 3. Metodologias para o desenvolvimento de sondas utilizadas na técnica

de Hibridação In Situ Fluorescente (FISH) ....................................................... 205

Lista de Figuras

I. INTRODUÇÃO

Figura 1. Imagens endoscópicas: (A) Esôfago Normal, (B) Esofagite

Crônica......................................................................................................... 40

Figura 2. Esquema de alterações em megaesôfago: dilatação do corpo

esofágico e falta de abertura do esfíncter esofagiano inferior..................... 42

Figura 3. Vias intrínseca e extrínseca de apoptose..................................... 59

Capítulo I

Figure 1 Summary of copy number alterations. in esophageal squamous cell

carcinomas analyzed by comparative genomic hybridization. tumor.…...... 93

Capítulo II

Figure 1. Immunohistochemical stain in esophageal mucosa……………..101

Capítulo III

Figure 1. Immunostaining of Ki67 antigen and CPP32 in esophageal

mucosa.......................................................................................................124

Capítulo IV

Figure 1. Exploratory analysis using three-dimensional plots of the

percentages of loss genes status for individual patients is illustrated…….155

Figure 2. Exploratory analysis using three-dimensional plots of the

percentages of gain and loss genes status for individual patients is

illustrated…………………………………………………………………………156

Figure 3: Exploratory analysis using three-dimensional plots of the

percentages of gain genes status for individual patients is illustrated…….157

Figure 4. FISH images for the unbalanced genes and centromeric targets in

the megaesophagus specimens…………………………………………........158

Lista de Tabelas

Capítulo II

Table I. Demographic and clinicopathological distribution,

immunohistochemical and statistical analysis for the proteins p53, p16 and

Fhit in esophageal mucosa .…………………………………………………….99

Table II. Kappa test to describe the intensity of agreement among the

expression of three proteins simultaneously and association between protein

expression, two by two, by Fisher’s exact test on esophageal mucosa, in

each evaluated group…………………………………………………………..100

Capítulo III

Table 1. Immunohistochemical and statistical analysis for Ki67 antigen and

CPP32 protein in the different groups……………………………………......120

Table 2. Association between expression of Ki67 antigen and CPP32 protein,

by Fisher’s Exact Test (p<0.05)…………………………………...................121

Table 3. Association between expression of p53 protein

[15]

with Ki67 antigen

and CPP32 protein, by Fisher’s Exact Test (p<0.05)……………….…........122

Capítulo IV

Table 1. Mean copy number per cell and standard deviation (SD) for the 12

DNA targets tested in esophageal mucosa, including 9 genes and 3

centromeric sequences.....……………………………………………………..149

Table 2. Comparison among the patient groups based on total counting

results for the targets…………………………………………………………...150

Table 3. Comparison between gene and centromere target……………….150

Table 4. Mean copy number per cell and ranging of each target in distinct

grades of megaesophagus………………………………………..…………...151

Table 5. Frequencies of loss, balanced and gain of the targets in chagasic

megaesophagus (CM) and health patients (NM)……………………………152

Table 6. Frequencies of nuclei with aneusomies of 3, 7 and 9 centromeres in

the different groups …………………………………………………………….153

Lista de abreviaturas e

símbolos

Lista de abreviaturas e símbolos

- Absence of immunostain

+ Weak immunostain

++ Moderate immunostain

+++ Strong immunostain

Mean

* Statistically significant, p<0.05

a, b, c

Statistically significant , p<0.05

AAA

achalasia, adrenocortical insufficiency,

alacrimia

aCGH arranjos de CGH (array CGH)

ADAM8

ADAM metallopeptidase domain 8

AgNOR Argyrophilic nucleolar organizer region

Ala aminoácido Alanina

ANOVA Analysis of Variance

APAF-1

apoptotic peptidase activating factor 1

Arg aminoácido Arginina

B Basal layer of tissue

BAC Bacterial artificial chromosome

Bad proteína Bad

Bak proteína Bak

BCL2

B-cell CLL/lymphoma 2

Bcl-2 proteína Bcl-2 (Bcl-2 protein)

Bcl-X proteína Bcl-X (Bcl-X protein)

BRCA2

breast cancer 2, early onset

Cat. # catalog number

CCND1

cyclin D1

CD Chagas Disease

CDC25B

cell division cycle 25 homolog B (S. pombe)

CDK4

cyclin-dependent kinase 4

CDK6

cyclin-dependent kinase 6

CDKN2A

(P16, INK4A, MTS-1)

cyclin-dependent kinase inhibitor 2A

(melanoma, p16, inhibits CDK4)

CE Chronic Esophagitis

CGH Comparative Genomic Hybridization

cIAP1, 2

baculoviral IAP repeat-containing 2

CK4 Cytokeratin 4

CK14 Cytokeratin 14

CONEP Comitê Nacional de Ética em Pesquisa

CM Chagasic Megaesophagus

CPP32 caspase 3, apoptosis-related cysteine

peptidase

DAB

3,3’-diaminobenzidine

DAPI 4’, 6’-diamino-2-phenylindole

DR Receptores de Morte (Death Receptors)

ECT2

epithelial cell transforming sequence 2

oncogene

EGF epidermal growth factor (beta-urogastrone)

EGFR

epidermal growth factor receptor

[erythroblastic leukemia viral (v-erb-b)

oncogene homolog, avian])

ERBB2

v-erb-b2 erythroblastic leukemia viral

oncogene homolog 2, neuro/glioblastoma

derived oncogene

ESCC Esophageal Squamous Cell Carcinoma

FAD

presenilin 1

FADD

Fas (TNFRSF6)-associated via death

domain

FFPE formalin fixed, paraffin embedded

FGF3

(INT-2)

fibroblast growth factor 3 (murine mammary

tumor virus integration site (v-int-2)

FGF4

fibroblast growth factor 4

FGFR1

fibroblast growth factor receptor 1

FHIT

fragile histidine triad gene

Fhit proteína Fhit

FISH

Fluorescence In Situ Hybridization

FITC Fluorescein microscopy filter

FRA Região de sítio frágil

FRA3B fragile site, aphidicolin type, common,

fra(3)(p14.2)

Peróxido de Hidrogênio

HE Coloração Hematoxilina & Eosina

HGF

hepatocyte growth factor (hepapoietin A;

scatter factor)

HGF-R

hepatocyte growth factor receptor

HLA Human Leukocyte Antigen

IAP

baculoviral IAP repeat-containing

ILP2

baculoviral IAP repeat-containing 8

INCA Instituto Nacional do Câncer

Ki67 Antígeno Ki67

KYSE Serie of esophageal squamous cell

carcinoma cell line

LI Labeling Index

LOH Loss of Heterozygosity

LY6K

lymphocyte antigen 6 complex, locus K

mCGH metaphase CGH

M-FISH Multiplex fluorescence in situ hybridization

MIB

mindbomb homolog

MLPA Multiplex Ligation Dependent Probe

Amplification

MYC

v-myc myelocytomatosis viral oncogene

homolog (avian)

MYEOV

myeloma overexpressed (in a subset of

t(11;14) positive multiple myelomas)

number of specimens positive

immunostained

N Total of specimens

NAIP

NLR family, apoptosis inhibitory protein

NCL-Ki67p monoclonal antibody Ki-67

NCOA3

nuclear receptor coactivator 3

NM Normal mucosa, health patients

NP it was not performed

ns not significant, p>0.05

oligo-array Oligonucleotide array

p15 proteína p15

P15

(CDKN2B, INK4B, MTS-2)

cyclin-dependent kinase inhibitor 2B (p15,

inhibits CDK4)

p16 proteína p16

p53 proteína p53

PCNA proliferating cell nuclear antigen

PCR-SSCP polymerase chain reaction-single strand

conformation polymorphism

PIK3CA

phosphoinositide-3-kinase, catalytic, alpha

polypeptide

PPFIA

protein tyrosine phosphatase, receptor type,

f polypeptide (PTPRF), interacting protein

(liprin)

pRb proteína retinoblastoma

Pro aminoácido Prolina

retinoblastoma

RB1

retinoblastoma 1

RT Room temperature

S Superficial layer of tissue

SD Standard Deviation

SE Standard Error

SHANK2

SH3 and multiple ankyrin repeat domains 2

SKY

Spectral Karyotyping

Smac/Diablo

diablo homolog (Drosophila)

SNP Single Nucleotide Polymorphism

SSC Sodium chloride, sodium citrate solution

SST

somatostatin

TE Serie of esophageal squamous cell

carcinoma cell line

TERC

telomerase RNA component

TFRC

transferrin receptor (p90, CD71)

TGFα

transforming growth factor, alpha

TMEM16A

anoctamin 1, calcium activated chloride

channel

TP53

tumor protein p53

TP63

tumor protein p63

versus

v/v Volume/volume

XIAP

X-linked inhibitor of apoptosis

WT1

Wilms tumor 1

YAMA

Aliase of caspase 3, apoptosis-related

cysteine peptidase

YES-1

v-yes-1 Yamaguchi sarcoma viral oncogene

homolog 1

µ mean

Resumo

RESUMO

O carcinoma de esôfago apresenta um modelo de

progressão tumoral a partir da seqüência esofagite, atrofia, displasia,

carcinoma in situ e carcinoma invasivo, com algumas alterações genéticas

bem estabelecidas nos estágios iniciais e avançados da

carcinogênese.Contudo em lesões benignas precursoras como o

megaesôfago e esofagite crônica os estudos genéticos são escassos.

Portanto, com o objetivo de identificar o envolvimento de algumas proteínas

que participam da regulação do ciclo celular e apoptose, no presente estudo

foi avaliada a expressão das proteínas p53, p16, Fhit, caspase-3 e do

antígeno Ki67, por imuno-histoquímica. Foram utilizados cortes histológicas

de mucosa de pacientes chagásicos crônicos sem (CD) e com megaesôfago

(CM), este último grupo por apresentar maior risco de desenvolvimento de

carcinoma esofágico, e pacientes com esofagite crônica (CE), devido à

relação entre o processo inflamatório e carcinogênese. Estas amostras

foram comparadas com carcinoma de células escamosas de esôfago

(ESCC) e mucosa esofágica histologicamente normal (NM). Também se

avaliou a ocorrência de concordâncias utilizando o Teste Kappa, entre os

casos com a expressão alterada das proteínas nos diferentes grupos, assim

como a ocorrência de associações, entre padrões alterados de expressão

das proteínas com sexo, idade, hábitos tabagistas e etilistas. Outro objetivo

do estudo foi avaliar o padrão de perdas e ganhos cromossômicos de genes

freqüentemente descritos como relacionados com a carcinogênese

esofágica, FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A, YES1,

NCOA3, e centrômeros 3, 7 e 9, como controles, por FISH. A avaliação

imuno-histoquímica revelou que a proporção de casos positivos para a

proteína p53 aumentou progressivamente de acordo com a severidade da

lesão, CD (7,7%), CM (26,1%), CE (52,2%) and ESCC (100%). Entretanto,

as proteínas p16 e Fhit não mostraram diferenças estatisticamente

significantes entre os grupos, mas também, em CE observou-se um número

maior de casos com expressão reduzida dessas proteínas (95,7%; 34,8%,

respectivamente), similar ao ESCC (90,9%; 18,2%, respectivamente). A

análise de concordância entre as expressões das proteínas (Teste Kappa)

não evidenciou concordâncias entre os casos com expressão alterada e

normal, para as três proteínas simultaneamente. Porém, o Teste Exato de

Fisher mostrou associação entre a expressão alterada das proteínas p53 e

p16; exceto em ESCC, único grupo que apresentou associação entre p53 e

Fhit. Enquanto a associação entre a expressão alterada de p16 e Fhit, só

não foi evidenciada em NM. A investigação de alterações nas taxas de

proliferação celular e apoptose evidenciaram que o índice de marcação de

Ki67 (LI) foi similar em todos os grupos, 30,1% em NM, 30,9% em CD,

42,3% em CM; 44,6% em CE e 48,4% em ESCC, no total de 500 células

analisadas por caso, não sendo observadas diferenças significantes. A

frequência de positividade de CPP32 foi similar nos grupos CD (30,8%), CM

(30,4%) e CE (34,8%), mas aumentada em ESCC (55,5%). Também não foi

observada associação entre os níveis de CPP32, Ki67 e expressão da

proteína p53 nos diferentes grupos, nem dessas com os parâmetros de

idade, sexo e hábitos tabagista e etilista. A análise citogenética para

avaliação dos padrões de perdas e ganhos nas camadas superficial e basal

de mucosa esofágica não mostrou diferenças significantes para a maioria

dos alvos investigados, exceto para EGFR no grupo CM, que apresentou

freqüência menor de cópias na camada basal. Considerando-se o número

total de cópias (camadas superficial e basal) apenas o gene CDKN2A e

centrômero 9 apresentaram médias significativamente menores em CM que

em NM. Enquanto, quando foram avaliadas, para cada gene, as freqüências

de núcleos com padrão de perda ou ganho, apenas o gene PIK3CA

apresentou freqüência significativamente maior de núcleos com perdas no

grupo CM em comparação a NM. A partir de análise exploratória usando

“plots” tridimensional das porcentagens das classes de genes com perda ou

ganho, alguns casos de CM mostraram níveis, não-estatisticamente

significantes, mas que se destacaram da frequência média do grupo, de

perdas para genes específicos: TP63, FHIT, PIK3CA, EGFR, CDKN2A,

YES1; CEP9 ou de ganhos para PIK3CA, TP63, FGFR1, MYC, CDKN2A,

NCOA3, e CEP9, sendo a maioria associada aos graus III e IV do

megaesôfago, provavelmente devido ao processo inflamatório acentuado.

Em conclusão, o presente estudo evidência o envolvimento da expressão

aumentada da proteína p53 nos processos iniciais da carcinogênese

esofágica (esofagite crônica e megaesôfago chagásico), sugerindo sua

utilização como marcador em lesões precursoras.De modo contrário, perdas

na expressão de Fhit e p16 não parecem ser significantes, assim como

alterações na expressão do antígeno Ki67 e da proteína caspase-3,

sugerindo que não há evidências de alterações importantes nos níveis de

proliferação celular e apoptose nas lesões benignas e carcinoma, nas

condições estudadas. Além desses dados, observação de que regiões

genômicas com desequilíbrios na carcinogênese esofágica não são

significativamente afetadas em megaesôfago chagásico sugere que não

será possível utilizar tais características como marcadores eficazes de risco

para desenvolvimento de ESCC em megaesôfago.

Palavras-chave: Megaesôfago chagásico, Esofagite crônica, Carcinoma de

células escamosas, apoptose, proliferação celular, ganhos, perdas e

amplificações gênicas, proteínas reguladoras do ciclo celular

Abstract

ABSTRACT

Esophagus carcinoma presents a tumor progression model from the

sequence esophagitis, atrophy, dysplasia, carcinoma in situ and invasive

carcinoma, with some well-established genetic changes in early and

advanced stages of carcinogenesis. In benign precursor lesions such as

megaesophagus and chronic esophagitis genetic studies are scarce.

Therefore, to identify the involvement of certain cell cycle and apoptosis

regulatory proteins, the present study evaluated the expression of p53, p16,

Fhit, caspase-3 and Ki67 antigen by immunohistochemistry. Histological

sections of esophageal mucosa were obtained from chronic chagasic

patients without (CD) and megaesophagus (CM), the latter group presents

higher risk of developing esophageal cancer, and patients with chronic

esophagitis (CE), because the relationship between the inflammatory

process and carcinogenesis. These samples were compared with squamous

cell carcinoma of the esophagus (ESCC) and histologically normal

esophageal mucosa (NM). It also assessed the occurrence of agreement

using the Kappa test, among the cases with altered expression of proteins in

different groups as well as the occurrence of associations, and altered

patterns of protein expression with sex, age, smoking and alcohol habits.

Another aim of the study was to evaluate the pattern of chromosomal gains

and losses of genes frequently described as related to esophageal

carcinogenesis, FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A,

YES1, NCOA3 and centromere 3, 7 and 9, as controls for FISH. The

immunohistochemical evaluation showed that the proportion of cases

positive for p53 protein increased progressively according to the severity of

the injury, CD (7.7%), CM (26.1%), CE (52.2%) and ESCC (100%). However,

the proteins p16 and Fhit showed no statistically significant differences

between groups, but also in CE was observed a greater number of cases

with reduced expression of these proteins (95.7%, 34.8%, respectively),

similar to ESCC (90.9%, 18.2%, respectively). The analysis of correlation

between the expressions of proteins (Kappa test) showed no correlation

between the cases with normal expression changed and, for the three

proteins simultaneously. However, the Fisher's exact test showed an

association between the altered expression of p53 and p16, except in ESCC,

the only group that showed an association between p53 and Fhit. While the

association between altered expression of p16 and Fhit, was not observed

only in NM. Investigation of changes in cell proliferation and apoptosis rates

revealed that the rate of labeling of Ki67 (LI) was similar in all groups, 30.1%

in NM, 30.9% in CD, 42.3% in CM; 44.6% in CE and 48.4% in ESCC, in total

500 cells examined per case, no significant differences were observed. The

frequency of positivity of CPP32 was similar in the groups CD (30.8%), CM

(30.4%) and CE (34.8%) but increased in ESCC (55.5%). Also no

association was found between the levels of CPP32, Ki67 and p53 protein

expression in different groups, or those with the parameters such as age,

sex, smoking and alcohol habits. The cytogenetic analysis to assess

patterns of losses and gains in the superficial and basal layers of

esophageal mucosa showed no significant differences for most of the targets

investigated, except for EGFR in the CM group, which showed lower

frequency of copies in the basal layer. Considering the total number of

copies (superficial and basal layers) only the CDKN2A gene and centromere

9 showed significantly lower averages in CM than in NM. Meanwhile, they

were evaluated for each gene, the frequencies of nuclei with standard loss or

gain, only the PIK3CA gene showed a significantly higher frequency of nuclei

with losses in the CM group compared to NM. From exploratory analysis

using three-dimensional plots of the percentages of genes classes with loss

or gain, some cases of CM showed levels, non-statistically significant, but it

highlighted the group's average frequency of losses for specific genes: TP63,

FHIT, PIK3CA, EGFR, CDKN2A, YES1; CEP9 or gains for PIK3CA, TP63,

FGFR1, MYC, CDKN2A, NCOA3 and CEP9, most of them associated with

grades III and IV of megaesophagus, probably due to the inflammatory

process. In conclusion, this study highlighted the involvement of increased

expression of p53 protein in cases of benign precancerous lesions

(esophagitis and chronic chagasic megaesophagus), suggesting its use as a

marker in precursors lesions. Controversially, losses on the expression of

Fhit and p16 not appear to be significant as well as changes in the

expression of Ki67 antigen and caspase-3 protein, suggesting that there is

no evidence of major changes in the levels of cell proliferation and apoptosis

in carcinoma and benign lesions under the studied conditions. Besides these

data, the findings that genomic imbalances common in esophageal

carcinomas are not affected in megaesophagus suggest that these features

will not be effective biomarkers for risk assessment of esophageal carcinoma

in chagasic megaesophagus.

Key words: chagasic megaesophagus, chronic esophagitis, esophageal

squamous cell carcinoma, apoptosis, cell proliferation, genomics imbalances,

cell cycle regulators proteins

Introdução

INTRODUÇÃO

I.1. Lesões Benignas do esôfago

O esôfago, devido ao seu papel na condução de

substâncias até o estômago, está exposto a condições adversas

responsáveis pela ocorrência de lesões que variam desde uma simples

azia, esofagites e hérnias de hiato até cânceres letais. As lesões benignas

mais frequentemente encontradas neste órgão são as esofagites e a

acalasia ou megaesôfago (BRASILEIRO FILHO, 2006).

A esofagite é uma lesão da mucosa esofágica com

subseqüente inflamação, mundialmente comum. Nos Estados Unidos e

em outros países ocidentais, a esofagite ocorre em aproximadamente 5%

da população adulta e prevalências maiores são encontradas no Irã e

algumas partes da China (CRAWFORD, 2004).

As esofagites podem ocorrer na forma aguda e crônica,

sendo a primeira causada pelo refluxo do conteúdo gástrico, infecção por

bactérias, vírus ou fungos, ingestão de alimentos e líquidos

excessivamente quentes, substâncias cáusticas (BRASILEIRO FILHO,

2006) e ingestão de medicamentos (MINCIS, 1997).

A esofagite crônica (Figura 1) pode ser provocada pelos

mesmos agentes que a aguda e ainda pela estase alimentar (retenção de

alimentos) (CRAWFORD, 2004; BRASILEIRO FILHO, 2006). Essa estase

pode causar a esofagite crônica devido ao contato prolongado do alimento

parcialmente digerido com a mucosa esofágica, aumento do crescimento

bacteriano e irritação química (RIBEIRO et al., 1996). Quanto maior o

grau da dilatação esofágica, maior a proliferação de microorganismos no

líquido de estase, contribuindo para o desenvolvimento da inflamação

(PAJECKI et al., 2002). As alterações fundamentais das esofagites são:

infiltrado inflamatório de mononucleares, hiperplasia do epitélio, erosões e

ulcerações da mucosa, estenose do órgão, hiperemia, friabilidade e graus

variados de fibrose na parede (CRAWFORD, 2004; MADER et al., 2002;

BRASILEIRO FILHO, 2006).

Essas duas principais lesões do esôfago, ou seja, o

refluxo esofágico e a esofagite crônica estão associados a carcinogênese

esofágica. A primeira, possivelmente, aumenta a susceptibilidade ao

desenvolvimento de esôfago de Barret e conseqüentemente ao

adenocarcinoma (JONES et al., 2003), enquanto que a esofagite crônica é

considerada uma alteração precoce no carcinoma de células escamosas

do esôfago (MANDARD et al., 1998).

FIGURA 1. Imagens endoscópicas: (A) Esôfago Normal, (B) Esofagite Crônica (IQB,

Instituto Químico Biológico, 2007)

Exames endoscópicos em grupos de alto risco de

desenvolvimento de câncer, de diferentes países, têm demonstrado a

presença de esofagite crônica em 42% a 84% dos indivíduos, atrofia em

3,8% a 10% e displasia em 2,4% a 8% (CASTELLETTO et al., 1992).

Estes dados sugerem que a história natural do carcinoma esofágico pode

iniciar-se a partir da esofagite (MUÑOZ, 1997). Pacientes com evidência

histológica de esofagite desenvolveram, por um período de até 6,8 anos,

carcinoma de células escamosas do esôfago e câncer esofágico não

específico em 0,15% e 0,3% dos casos respectivamente, indicando um

risco moderadamente aumentado para o desenvolvimento de carcinoma,

mas não para o adenocarcinoma (MURPHY et al., 2005). Por outro lado,

Wang et al. (2005) em acompanhamento clínico por endoscopia durante o

período de 13 anos, não observaram evidências de que a presença de

esofagite aumente o risco de desenvolvimento do carcinoma de esôfago.

Outra lesão do esôfago com potencial pré-canceroso é o

megaesôfago ou dilatação do esôfago (Figura 2), também denominada

acalasia, cardioespasmo e aperistalsia, (VANTRAPPEN; HELLEMANS,

1982 apud KRAICHELY; FARRUGIA, 2006). Ele é caracterizado pela

destruição ou ausência dos plexos nervosos intramurais, determinando a

ausência de peristaltismo ao nível do corpo do órgão e a falta de abertura

do esfíncter esofagiano inferior, em resposta à deglutição. Em

conseqüência, inicia-se a retenção do bolo alimentar (estase esofágica)

na luz do órgão (BRASILEIRO FILHO, 2006), pela dificuldade no

relaxamento do esfíncter esofagiano inferior (KRAICHELY; FARRUGIA,

2006). O sintoma clínico clássico do megaesôfago é a dificuldade para

engolir (disfagia) que ocorre de modo progressivo, evoluindo de alimentos

sólidos para pastosos, até a pessoa engolir apenas líquidos. Podem

ocorrer também a regurgitação e a aspiração noturnas do alimento não

digerido (OLIVEIRA et al., 1998; CRAWFORD, 2004).

Mundialmente, tais características correspondem à lesão

denominada de acalasia, cuja patogênese é pobremente conhecida e

parece envolver alterações degenerativas na inervação neural levando à

dilatação do órgão (CRAWFORD, 2004). A estase alimentar constitui um

estímulo permanente para as contrações do corpo do esôfago na tentativa

de ultrapassar o obstáculo representado pelo esfíncter. No início, essas

contrações são fortes e irregulares levando à hipertrofia das fibras

musculares e espessamento da camada muscular caracterizando,

portanto, a fase inicial da dilatação (megaesôfago). As contrações tornam-

se progressivamente menos intensas e as fibras musculares vão

alongando-se e gradualmente sendo substituídas por tecido conjuntivo,

tornando a dilatação mais acentuada pela atonia completa da musculatura.

Conseqüentemente, provoca a inflamação da mucosa (esofagite), que

pode determinar o aparecimento de acantose, paraceratose e leucoplasia,

lesões possivelmente pré-cancerosas (PINOTTI, 1996; KRAICHELY;

FARRUGIA, 2006).

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Objetivos

II. OBJETIVOS

Considerando que algumas alterações genéticas estão

relacionadas às etapas iniciais da carcinogênese do esôfago, justifica-se

a necessidade de estudos que avaliem alterações na expressão de

proteínas e desequilíbrios gênicos em lesões benignas do esôfago com

potencial pré-canceroso, sendo assim, o presente trabalho teve como

objetivos:

a. Avaliar a expressão das proteínas p53, p16 e Fhit, em mucosa

esofágica de pacientes com lesões benignas como esofagite crônica e

megaesôfago chagásico em comparação com o carcinoma de esôfago;

assim como verificar a ocorrência de concordância e associações no

padrão de expressão das três proteínas em lesões benignas e carcinoma

esofágico;

b. Investigar a ocorrência de alterações em apoptose, por meio da

expressão da caspase-3, e proliferação celular, com o antígeno Ki67, e

associações com a expressão da proteína p53 nos grupos de lesões

benignas e carcinoma do esôfago;

c. Avaliar no grupo de pacientes com megaesôfago chagásico os

padrões de deleção e ganho de genes citados em desequilíbrios gênicos

em carcinoma esofágico identificados a partir de dados da literatura,

assim como associações com a graduação do megaesôfago;

d. Investigar nos grupos de lesões benignas e carcinoma a ocorrência

de associações entre a expressão das proteínas p53, p16, Fhit, caspase-

3 e antígeno Ki67, assim como os desequilíbrios gênicos com parâmetros

como idade, sexo, hábitos tabagista e etilista.

Capítulo I

CAPÍTULO I: Genomic imbalances in esophageal squamous cell

carcinoma identified by molecular cytogenetic techniques

Authors: Marilanda Ferreira Bellini

, Ana Elizabete Silva

Marileila

Varella-Garcia

Affiliations

Laboratório de Citogenética e Biologia Molecular, Department of Biology,

UNESP, São Paulo State University, Campus São José do Rio Preto, SP;

Brazil

Medicine/Medical Oncology, University of Colorado, Health Sciences

Center, Aurora, Colorado, USA

Short running title: Genomic imbalances in esophageal cancer

Key words: Genomic Gains, Genomic Losses, Amplifications, Oncogenes,

CGH, FISH and Esophageal Carcinoma

Corresponding author:

Ana Elizabete Silva,

IBILCE/UNESP – Departamento de Biologia, Rua Cristóvão Colombo,

2265, São José do Rio Preto, SP, Brazil - CEP. 15054-000;

phone 55 - 17 – 32212384.

e-mail:[email protected]sp.br

Artigo de revisão a ser submetido e redigido nas normas da revista

GENETICS AND MOLECULAR BIOLOGY (e-ISSN: 1678-4685)

Resumo

A presente revisão sumariza as alterações

cromossômicas detectadas por técnicas de citogenética molecular em

carcinoma de células escamosas de esôfago (ESCC), a nona neoplasia

maligna mais comum no mundo. A análise genômica total em linhagens

celulares de ESCC e em tumores primários indicou que as regiões de

ganho mais freqüentes são observadas em regiões dos cromossomos 1,

2q, 3q, 5p, 6p, 7, 8q, 9q, 11q, 12p, 14q, 15q, 16, 17, 18p, 19q, 20q, 22q, e

X, com amplificações focais em 1q32, 2p16-22, 3q25-28, 5p13-15.3,

7p12-22, 7q21-22, 8q23-24.2, 9q34, 10q21, 11p11.2, 11q13, 13q32,

14q13-14, 14q21, 14q31-32, 15q22-26, 17p11.2, 18p11.2-11.3, e em

20p11.2. As regiões de perdas recorrentes são 3p, 4, 5q, 6q, 7q, 8p, 9,

10p, 12p, 13, 14p, 15p, 18, 19p, 20, 22, Xp e Y. Ganhos em 5p e 7q, e

deleções em 4p, 9p e 11q são fatores prognósticos significantes para

estadiamento de ESCC. Ganhos em 6p e 20p, e perdas em 10p e 10q são

as alterações mais significantes tanto em carcinomas primários quanto em

metástases, o que sugere que essas regiões contenham oncogenes e

genes supressores tumorais. Ganho em 12p e perda em 3p podem estar

associados comrápida progressão da doença. Essas alterações

genômicas podem representar marcadores de diagnóstico ou prognóstico

aplicáveis na prática clínica, assim como identificarem moleculas alvo

para terapia personalizada.

Palavras-chave: carcinoma de células escamosas de esôfago, deleções,

ganhos, amplificações, citogenética molecular

Abstract

This review summarizes chromosome changes detected

by molecular cytogenetic approaches in esophageal squamous cell

carcinoma (ESCC), the ninth most common malignancy in the world.

Whole genome analyses in ESCC cell lines and tumors indicated that the

most frequent genomic gains were observed at 1, 2q, 3q, 5p, 6p, 7, 8q, 9q,

11q, 12p, 14q, 15q, 16, 17, 18p, 19q, 20q, 22q, and X, with focal

amplifications at 1q32, 2p16-22, 3q25-28, 5p13-15.3, 7p12-22, 7q21-22,

8q23-24.2, 9q34, 10q21, 11p11.2, 11q13, 13q32, 14q13-14, 14q21,

14q31-32, 15q22-26, 17p11.2, 18p11.2-11.3, 20p11.2. The recurrent

losses involved 3p, 4, 5q, 6q, 7q, 8p, 9, 10p, 12p, 13, 14p, 15p, 18, 19p,

20, 22, Xp and Y. Gains at 5p and 7q, and deletion at 4p, 9p, and 11q

were significant prognostic factors for patients with ESCC. Gains at 6p and

20p, and losses at 10p and 10q were the most significant imbalances, both

in primary carcinoma and in metastasis, suggesting these regions could

harbor oncogenes and tumor suppressor genes. Gains at 12p and losses

at 3p could be associated with poor relapse-free survival. These changes

may represent markers with clinical applicability at the diagnosis or

prognosis or may identify molecular targets for personalized therapy.

Key words: esophageal squamous cell carcinoma, deletion, gain,

amplification, molecular cytogenetic

Esophageal Carcinoma

Cancer of the esophagus has been reported as the ninth

most common malignancy in the world, but its incidence varies largely

among regions (Lam et al., 2000), with higher incidence in China, Japan,

Singapore and Puerto Rico (INCA, 2008). The American Cancer Society

estimated around 16,470 new cases of esophageal carcinoma in the USA

population for 2008 (American Cancer Society, 2008). The Brazilian

National Institute of Research in Cancer (INCA) reported that esophageal

cancer was the sixth in the cancer rank of mortality in 2000 with 5,307

deaths (INCA, 2008) and estimated about 10550 new cases in 2008

ranging among geographical areas from 1.04 to 19.07 in males and from

0.39 to 7.58 in 100,000 in females.

The development of human esophageal cancer is a

progressive process, resulting in successive accumulation of genetic

changes that culminate in the malignant transformation (Knudson, 1985;

Somers; Schechter, 1992; Xue et al., 2006). An early indicator of this

process is the increased proliferation of esophageal epithelial cells,

morphologically evolving to stages of basal cell hyperplasia, dysplasia,

carcinoma in situ and invasive carcinoma (Muñoz, 1997; Mandard et al.,

2000; D’Âmico, 2006).

With the exception of the USA the esophageal squamous

cell carcinoma (ESCC) is the prevalent histological type worldwide (except

in USA) and has multifactorial origin. Besides environmental components

(Crawford, 2004), several genetic factors are associated with esophageal

carcinogenesis such as chromosomal aneuploidy, allelic deletions,

activation of oncogenes and inactivation of tumor suppressor genes

(Kuwano et al., 2005). At the cellular level, these factors head to disorders

of the cell proliferation, differentiation and apoptosis (Koch et al., 1994;

McCabe; Dlamini, 2005; D’Amico, 2006; Daigo; Nakamura, 2008;

Khushalani, 2008).

Specific chromosome aberrations have been identified as

markers for diagnosis and prognosis in solid tumors (Tada et al., 2000;

Yen et al., 2000; Shiomi et al., 2003; Qin et al., 2004, 2005ab; Wang et al.,

2006; Qin et al., 2008). Despite reports of numerous chromosome

changes, no particular diagnostic or prognostic chromosomal markers

have been described in esophageal carcinoma. The aim of this review is to

summarize the recurrent chromosomal changes detected by molecular

cytogenetic approaches in ESCC, such as gains and losses in regions that

may harbor oncogenes and tumor suppressor genes. These chromosomal

imbalances may represent markers with clinical applicability in the

diagnostis or prognostic or be supportive of novel targeted therapies.

Molecular Cytogenetic Technologies Identify Genomic Changes in

Cancer

The primary cytogenetic technique used for a better

understanding of the molecular pathogenesis involved in esophageal

carcinogenesis was DNA fluorescence in situ hybridization (FISH). FISH

uses small fragments of DNA as fluorescent probes that bind to

chromosomal sequences of the target DNA to which they show a high

degree of homology (Bauman et al., 1980; Langer et al., 1981) it detects

and localizes specific DNA sequences on chromosomes. FISH probes are

often derived from fragments of DNA on the order of few hundreds to 200

thousand base-pairs that are isolated, purified, amplified and labeled with

fluorochrome-conjugated nucleotides. The DNA probe may be hybridized

to distinct DNA targets such as metaphase chromosomes, interphase

nuclei, and extended chromatin fibers and DNA fragments in a variety of

biological specimens or platforms, including isolated cells, tissue section

and oligonuclotide arrays (Speicher; Carter, 2005) and DNA microarrays

(Solinas-Toldo et al., 1997; Pinkel et al., 1998).

There are numerous variants of the FISH assay; the

variant most effectively used to detect genome wide imbalances is called

Comparative Genomic Hybridization (CGH). CGH was originally

developed as the competitive hybridization of a mix of the test DNA and a

normal reference DNA, labeled with different fluorochromes, to metaphase

spreads of a normal specimen (metaphase CGH - mCGH) (Kallioniemi et

al., 1992; du Manoir et al., 1993). ). Chromosomal regions of the test DNA

with normal copy numbers will show a balanced ratio of hybridization with

the test and the control DNA. Chromosomal regions with excess or lack of

copy numbers in the test DNA will show the predominant color of the

hybridization in the normal template. The DNA used in the assay is

extracted from dividing or non-dividing cells from virtually all types of

tissues even those fixed in formalin, thus the technique allows an

informative overview of archived specimens. The level of resolution of

specific imbalances results in the mCGH assay basically depends on the

condensation of the chromosomes to which the DNA mix is hybridized and

is estimated as ranging from 5 to 10 MB (Kallioniemi et al., 1992; Speicher;

Carter, 2005). It is possible to identify large regions involved in low level of

genomic gain or loss and small regions with focal amplification but overall

the resolution of the mCGH is limited or insufficient for identification of

specific chromosomal bands (Kallioniemi et al., 1992; Speicher; Carter,

2005). Therefore, mCGH studies are used as preliminary tools, to infer

potential genes located in imbalanced regions and to support refined

studies with higher resolution techniques, such as array CGH (aCGH) or

FISH with single gene probes (Nakakuki et al., 2002; Arai et al., 2003)

The aCGH technique detects chromosome copy number

changes at a much higher resolution level than mCGH (Solinas-Toldo et

al., 1997; Pinkel et al., 1998). Instead of using metaphase spreads as

template for hybridization, the aCGH uses a collection of DNA inserts

contained in bacterial artificial chromosomes (BAC arrays) or

oligonucleotides (oligo-arrays) printed on a glass slide. Similarly to the

mCGH, a differentially labeled mix of DNAs from the test sample and a

normal reference control sample is hybridized with the platform and the

ratio of the fluorescence intensity of the test to the reference DNA is

calculated. Using aCGH, copy number changes can be detected at levels

of few hundred kilobases of DNA sequences for BAC arrays or 30 kb for

oligoarrays (Snijders et al., 2001; Fiegler et al., 2003; Medical Genetics

Laboratories, 2008). SNP array is a type of DNA microarray which is used

to detect polymorphisms within a population. A single nucleotide

polymorphism (SNP), a variation at a single site in DNA, is the most

frequent type of variation in the genome. For example, there are around 10

million SNPs that have been identified in the human genome. As SNPs are

highly conserved throughout evolution and within a population, the map of

SNPs serves as an excellent genotypic marker for research (Sherry et al.,

2001).

Other variants of the FISH technology are the Multiplex-

FISH (M-FISH) and the Spectral Karyotyping (SKY). These procedures

allow identification of the chromosomal origin of each chromosomal region

in a metaphase cell, e.g., visualizes all 24 human chromosomes in a single

hybridization (Speicher; Carter, 2005). In both techniques, the probe set is

a pool of differentially labeled DNAs for each of the 24 human

chromosomes. In the M-FISH, images for each fluorochrome are

individually collected and merged, and a combinatorial labeling algorithm

identifies each chromosome, visualized in pre-defined pseudocolour

(Speicher et al., 1996). In the SKY assay, a single image is captured per

cell and an interferometer is used for discrimination of the fluorochrome

spectrum in each image pixel, to which a pseudocolor is assigned

(Schröck et al., 1996). Both techniques have been successful in clarify

complex chromosomal rearrangements in solid tumors (Schröck; Padilla-

Nash, 2000), including ESCC (Yen et al., 2003).

Altogether, the numerous variants of FISH technology

have allowed accurate identification of DNA sequences of interest in the

chromosomes and search of the whole genome total for gains and losses

associated with in the carcinogenesis process.

Genomic Imbalances in ESCC cell lines

ESCC lines are highly abnormal cytogenetically. An

integrated study using mCGH, SKY and FISH with single probes was

performed in 8 ESCC cell lines; the pooled CGH results revealed frequent

gains on almost all chromosome arms (1p, 1q, 3q, 5p, 6p, 7p, 7q, 8q, 9q,

11q, 12p, 14q, 15q, 16p, 16q, 17q, 18p, 19q, 20q, 22q, and Xq), while

frequent losses were found on 3p, 4, 5q, 6q, 7q, 9p, and 18q. SKY

analyses detected 195 translocations, 13 deletions and 2 duplications. The

most frequently amplified genes were PIK3CA and TP63 (3p28). The

protein encoded by PIK3CA represents the catalytic subunit, which uses

ATP to phosphorylate phosphatidylinositoland TP63 encodes, a protein

that works in the development and maintenance of stratified epithelial

tissues, these oncogenes, were amplified in 6 and 5 cell lines, respectively

(Yen et al., 2003; National Center for Biotechnology Information, 2009).

Multiple gains and losses involving different chromosomal

regions were also found by mCGH in 10 ESCC cell lines of the KYSE

series, TE-1–6, -8 – 11, -13, and -15. The most frequent losses were

observed at chromosome arms 3p, 4p and q, 8p, 9p, 18q, and Xp;

whereas the most common gains were noted at 1q, 3q, 5p, 7p, 8q, 9q, 11q,

18p, 20q and Xq. While focal loss was only identified at 11q 23-25, focal

amplifications were detect at 1q32, 2p16-22, 3q25-28, 5p13-15.3, 7p12-22,

7q21-22, 8q23-24.2, 9q34, 10q21, 11p11.2, 11q13, 13q32, 14q13-14,

14q21, 14q31-32, 15q22-26, 17p11.2, 18p11.2-11.3, 20p11.2 (Shinomiya

et al., 1999; Pimkhaokham et al. 2000; Su et al., 2006).

Later, Yang et al. (2008a) characterized cytogenetic

abnormalities in the cell line KYSE 410-4 using M-FISH, detecting

chromosome gains at 2q, 3, 8, 17p, and X. An isochromosome 3q was

visualized in this line, which might be one intermediate mechanism leading

to 3p loss and 3q gain. For the cell line KYSE 180, M-FISH analysis

detected loss of DNA copy number at 4p, 5q, 6q, 9, 10p, 12p, 13, 14p, 15p,

18p, 18q, 20, 22, and Y; chromosomal gains and translocations mainly

involved chromosomes 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q, 11, 12q, 14q, 16,

17q, 19, and Xp. Seven derivative chromosomes involving chromosomes

5, 8, 12, 14, 14, 14, and 17 presented complex translocations, each

involving three or four chromosomes and loss of chromosomes 9, 13, and

Y were also detected (Wu et al., 2006).

Since previous studies of CGH revealed frequent

amplifications in 18p in esophageal cell lines (Shimada et al., 1992;

Pimkhaokham et al., 2000), Nakakuki et al. (2002) screened 29 ESSC cell

lines to discern amplifications by FISH of 14 known genes and 21

uncharacterized transcripts within amplicons in chromosome 18. These

authors also investigated the levels of protein expression of these genes

by southern-, dot- and northern-blotting. Only four known genes, showed

amplification and correlated over-expression, YES1, TYMS, HEC and

TGIF YES1encodes a protein with tyrosine kinase activity, TYMS is critical

for DNA replication and repair, HEC isinvolved in spindle checkpoint

signaling and TGIF is a highly conserved transcription regulator with

potential role in the transmission of nuclear signals during development

and in the adult.. , These results suggest those genes are likely candidate

targets for 18p11.3 amplification and may be associated with esophageal

tumorigenesis.

In summary, these whole genome analyses in ESCC cell

lines indicated that the most frequent genomic gains were observed at 1p,

1q, 2q, 3q, 5p, 6p, 7p, 7q, 8q, 9q, 11q, 12p, 14q, 15q, 16p, 16q, 17p, 17q,

18p, 19q, 20q, 22q, and X, with focal amplifications at 1q32, 2p16-22,

3q25-28, 5p13-15.3, 7p12-22, 7q21-22, 8q23-24.2, 9q34, 10q21, 11p11.2,

11q13, 13q32, 14q13-14, 14q21, 14q31-32, 15q22-26, 17p11.2, 18p11.2-

11.3, 20p11.2. The recurrent losses involved 3p, 4, 5q, 6q, 7q, 8p, 9, 10p,

12p, 13, 14p, 15p, 18, 20, 22, Xp and Y (Figure 1A). The detected

alterations affect most of the genome and involve regions harboring many

known oncogenes and tumor suppressor genes, as well regions not yet

associated with such genes. Although the level of molecular resolution of

the majority of these studies is low and not conclusive, these findings are

promising in assisting the design of refined investigations on the molecular

pathogenesis and development of new therapeutics of ESCC.

Genomic Imbalances in ESCC tumor samples

Despite the better understanding of the risk factors and

cellular derangements associated with esophageal cancer, the clinical

treatment of this disease surprisingly has had little changes and long-term

survival from esophageal cancer remains poor, with 5-years survival rates

about 20% (Hsia et al., 2003).

The combination of different techniques (FISH, mCGH and

aCGH) indicated the chromosomes 1, 3, 7, 9, 11, 18, 19 and 20 shows

high frequency of alterations. Genomic profiles in primary carcinomas

have showed imbalances affecting most of the chromosomes, such as

gains at 1q, 3q, 5p, 7p, 8q, 11q, 13q, 18p, 20q, Xq; and losses at 1p, 3p,

4p, 8p, 9p, 18q, 19, 22q and Y. Focal losses at 9p13, focal gains at the

regions 5p15, 8p12-11.2, 8q24, 11q13, 14q32, and amplifications at 1p34,

2p24, 2q24-34, 3q22-ter, 7p12-22, 8q13-qter, 11p11.2, 11q13, 12p11.2,

13q21-34, 17q12, 20q12-13 and Xq27-28 are also commons findings

(Shinomiya et al., 1999; Pack et al., 1999; Mayama et al., 2000; Yen et al.,

2001; Kamitani et al., 2002; Yen et al., 2003; Kwong et al., 2004; Qin et al.,

2004, 2005ab, 2008; Sugimoto et al., 2007). High-level amplifications were

observed in 30 regions and recurrently involved 7p11.2 and 11q13. EGFR

isa cell surface protein that when binds to its ligand induces receptor

dimerization and tyrosine autophosphorylation, leading cell proliferation

the 2

region harborsCCND1 is a regulator of CDK kinases required for

G1/S cell cycle transition. (Carneiro et al., 2008; National Center for

Biotechnology Information, 2009). Interstitial deletions in 1p, 3p, 5q, 6q,

11q, and 12q were also detected (Pack et al., 1999). These observations

altogether evidenced that accumulation of chromosomal aberrations is

common in ESCC (Figure 1B) and suggest that 1q, 3q, 5p, 6q, 8q,18p,

and 20q, mainly the specific regions 1p34, 2p24, 2q24-34, 3q22-ter, 7p12-

22, 8q13-qter, 11p11.2, 11q13, 12p11.2, 13q21-34, 17q12, 20q12-13 and

Xq27-28, may contain ESCC-related oncogenes; 1p, 3p, 4p, 8p, 9p13, 9q

and 19p may contain ESCC-related tumor suppressor genes involved in

the development and progression of esophageal cancer (Figure 1B).

Early genomic changes and imbalances associated with tumor

staging

It has been known that ESCC arise through multi-step

genetic and cytogenetic alterations. The time sequence of these

alterations, however, remains to be identified. Thus studies that correlate

chromosomal aberrations with stage and clinical outcome of prognostic

significance are necessary to assist in the selection of patients for specific

treatments.

An interesting and recently published study explored the

application of M-FISH for early diagnosis and risk prediction of precursor

lesions of ESCC in tumor and premalignant lesions in 113 patients (Yao et

al. 2008). Elevated aneuploidy rates of chromosomes 3, 8, 10, 12, 17 and

20 were frequently found both in ESCC and in its precursor dysplastic

lesions. These findings support the conclusion that a multi-target FISH

assay investigating chromosomal aneuploidy in esophageal dysplastic

sites may be useful to predict risk to ESCC.

The progression to advanced, metastatic stage is

accompanied by genomic changes, as detected by mCGH analyses in

lymph node metastasis of ESCC. Copy number gains were frequently

detected at 1q, 1p36.32, 3q, 5p, 8q23-qter, 11q13-14, 5p14-pter, 6p, 20q,

7p22.3, 7q, 2p, 12p, 19p13.3 and 20p and DNA amplifications were

detected at 11q13, 2q12, 6p12-6q12, 7q21, 20q11.2 and 20p12. Losses

were detected at 18q, 3p, 9p, 5q14-23, 4q, 13, and 11q22-qter (Qin et al.,

2005a; Wang et al., 2006; Carneiro et al., 2008; Qin et al., 2008).

Gain of 3q, 5p, 1q, 11q13-14 and loss of 4 and 13q were

all significantly correlated with pathologic staging, while gains of 8q, loss of

4p were linked to nodal metastasis and gains of 2p and loss of 4 and

11q14-qter were associated with distant organ metastasis (Qin et al.,

2004). It is likely that gain of 1q, 3q, 5p, , 11q13-14 and loss of 4 and 13q

are the genetic aberrations critical for the development of esophageal

carcinoma, whereas gains of 2p and 8q, and loss of 4 and , 11q14-qter

are considered later events associated with tumor progression and are

thought to confer metastatic potential to esophageal carcinoma.

Furthermore, nodal and distant organ metastases involve different genes

(Qin et al., 2005b). The gains of 3q and 11q13 and losses of 3p, 4q, 5q14-

23, 9p and 18q were detected in both early and advanced stage ESCC.

It was found that deletions of 4p and 13q12–q14 and gain

of 5p were significantly correlated with pathologic staging. Losses of 8p22-

pter and 9p also were found more frequently in patients with advanced

disease. Gain of 8q24-qter was seen more frequently in patients with

grade 3 tumors (Yen et al., 2001). Shiomi et al. (2003) using mCGH,

observed that gains of 3q, 8q, 11q13 and 14q were early events, while

losses of 3p, 5q, 13q and 21q, and gains of 1p and Xq were later events in

progression of individual tumors.

Gains at 6p and 20p, and losses at 10p and 10q are the

most significant imbalances, as in primary carcinoma as in metastasis,

suggesting the regions could have oncongenes and tumor suppressor

genes (Qin et al., 2005a). However, gain in 12p, and loss in 3p could be

associated with poor relapse-free survival (Kwong et al., 2004).

Searching for relevant oncogenes in ESCC

In 41 primary ESCC investigated by technology??(Fujita et

al. 2003); (Xu et al.2007), numerous genes have been seen amplified,

such as the cell cycle-regulator kinase BTAK (20q13.2-q13.3) in 9.8%,

E2F1 (20q11.2), plays a crucial role in the control of cell cycle and action

of tumor suppressor proteins) in 7.3%, NCOA3 (20q12) in 4.9%, and DcR3

(20q13.3), which plays a regulatory role in apoptosis) in 4.9%Xu et al.

(2007) detected NCOA3 overexpression and increased copy number in

46% and 13% of 221 ESCCs, respectively. NCOA3 encodes a nuclear

receptor coactivator that interacts with nuclear hormone receptors to

enhance their transcriptional activator functions. Its overexpression was

observed more frequently in late T stages than in earlier stages, but no

significant association of expression of NCOA3 and lymph node

metastases was observed. These results suggest that overexpression of

NCOA3 caused by genomic gain or other molecular mechanisms might

provide a selective advantage for the development and local invasion of

certain subsets of ESCC.

To investigate allelic imbalance of chromosome 18q in

ESCC, FISH analysis was performed and two of five resected ESCC

samples from patients showed loss of one copy of chromosome 18q and

13 of 46 ESCC samples (28.3%) showed loss of almost the entire 18q

(Ando et al., 2007). The authors suggested that 18q loss may play an

important role in the progression of ESCC.

Some studies have been focused on the identification of

genetic targets for identifying deranged pathways and clinically applicable

markers. Oncogene amplification was examined with DNA microarrays in

20 surgically resected ESCC and 57 oncogenes were detected as

amplified. Alterations in DNA copy numbers detected by microarrays were

compared to those obtained by mCGH. Amplification of 8 oncogenes

(CCND1, FGF3/ FGF4, EMS1, SAS, ERBB2, PDGFRA, MYC, and BCL2)

was detected by DNA microarrays in 9 of 20 tumors. Although ERBB2 was

23-fold higher than the control level in one case, the average magnitude of

gene amplification was approximately 2 - to 4 - fold that of the control level.

EMS1, CCND1, and FGF3/FGF4, which are all located on 11q13, were

amplified in 7, 5, and 4 of 20 ESCC, respectively, and they were co-

amplified in 3 tumors. EMS1 has two roles: it regulates the interactions

between components of adherens-type junctions and organize the

cytoskeleton and cell adhesion structures of epithelia and carcinoma cells

(National Center for Biotechnology Information, 2009). FGF4/FGF3 broad

the mitogenic, cell survival and oncogenic activity. A comparison of

genome DNA microarrays and mCGH data revealed that although most

amplified oncogenes were included in chromosomal regions for which

DNA copy number gains were detected by mCGH, not all amplified genes

on microarrays showed concomitant DNA copy number gains on mCGH,

indicating that the resolution reached by the mCGH technique was limited.

Microarrays of oncogenes are useful for the comprehensive identification

of amplified oncogenes and for analysis of areas of specific amplification

within chromosomal regions with DNA copy number increases detected by

mCGH analysis (Arai et al., 2003).

FISH assays revealed amplification of PLK1 (polo-like

kinase 1 (Drosophila)), an essential gene for the maintenance of genomic

stability during mitosis (Feng et al., 2009), suggesting that PLK1 might be

a useful prognostic marker. Investigation on 108 ESCCs and 9 ESCC cell

lines showed that MDS1 (myelodysplasia syndrome 1) and PRKCI (protein

kinase C, iota), genes implicated in neoplastic transformation, were

frequently gained and a positive correlation was found for PRKCI between

amplification and tumor size, lymph node metastasis and clinical stage.

PRKCI gene amplification was highly correlated with protein

overexpression suggesting that gene amplification is one important

mechanism involved in PRKCI overexpression (Yang et al.

, 2008b).

Overexpression of epidermal growth factor receptor

(EGFR, epidermal growth factor receptor) is observed in many cancers,

sometimes accompanied by gene amplification. By FISH, EGFR gene was

found amplified in ESCC (Sunpaweravong et al., 2005; Hanawa et al.,

2006). Also, EGFR gene overrepresentation (balanced gene and

chromosome 7 polysomy) and HER-2 amplification were common events

(Mimura et al., 2005; Sunpaweravong et al., 2005; Bizari et al., 2006).

Cyclin D1 (CCDN1) is a cell-cycle regulator and an

oncogene implicated in the pathogenesis of numerous tumor types.

Amplification of the CCDN1 gene occurs commonly in ESCC and it was

detected by FISH (Sheyn et al., 1997; Jin et al., 2004; Manoel-Caetano et

al., 2004; Sunpaweravong et al., 2005; Bizari et al., 2006).

Correlation between the overexpression and amplification

of oncogenes with survival has not been extensively investigated, however,

the survival of patients with BTAK or E2F1 amplification was significantly

lower than that of patients without these abnormalities, suggesting that

amplification of those genes is likely to lead to an increase in the number

of malignant ESCC phenotypes and that aberrations can be expected to

be useful as marker of poor prognosis (Fujita et al., 2003).

Concluding Remarks

The molecular cytogenetic approaches have been useful

to demonstrate the extensive genetic complexity in the different stages of

ESCC, supporting the involvement of various key genes in the esophageal

carcinogenesis. Most of the unbalanced chromosomal regions were similar

in primary tumors and cell lines, confirming that cell lines are reliable

models for investigation of molecular mechanisms in effect in patient

specimens.

There were specific imbalanced patterns during tumor

progression. While gains at 1q, 5p, 8q, 14q and losses at 4p, 13q and 18q

are related to early stages of ESCC development; gains at 1p, 2p, 7p22.3,

8q, 8q24-qter, Xq and losses at 8p22-pter, 11q14-qter, 13q and 21q are

associated with advanced stage. Gains at 3q and 11q13, and losses at 3p,

4q, 5q14-23, 9p, 18q were detected in both early and advanced stage of

ESCC. These regions harbor genes relevant for essential cellular

processes (e.g., regulation of transcription, signal transduction, cell

proliferation and cell differentiation) that participate of central signalizing

cascades impacting cancer development. Some imbalances may indicate

a poor prognostic marker such as losses at 4p, 9p, 11q and gains at 1p36-

32, 5p, 7q, 19p13.3. Gene amplifications affected mostly oncogenes like

YES-1, TYMS, HEC, TGIF, NCOA-3, BTAK, DcR-3, E2F1, MYC, EGFR,

EGR2, CCND1/ Cyclin D1, FGF3/FGF4, EMS1, SAS, ERBB2 (Her-2-neu),

PDGFR1, BCL2, MDS1 and PRKCI, while genomic losses lead to

deletions of suppressor tumor genes CDKN2A, MTAP, TP53.

The identification of these critical genes in esophageal

carcinogenesis will assist the development of individualized target therapy,

which is expected to positively impact the clinical outcome of ESCC

patients.

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Legend of figures:

Figure 1. Summary of copy number alterations. in esophageal squamous

cell carcinomas analyzed by comparative genomic hybridization.

Chromosomal regions of gain are represented in red on the right side of

the chromosome ideograms, the amplification regions are represented by

the larger dark red line on the right side of the chromosome ideograms

and regions of loss are represented in green on the left side. A. cell lines;

B. tumor

Loss

Gain

Amplification

Loss

Gain

Amplification

Capítulo II

CAPÍTULO II: p53, p16 and Fhit Proteins Expressions in Chronic

Esophagitis and Chagas Disease

Authors: Marilanda Ferreira Bellini

; Kátia Ramos Moreira Leite

, Patrícia

Maluf Cury

and Ana Elizabete Silva

Authors Addresses:

Department of Biology, UNESP, São Paulo State University, Campus

São José do Rio Preto, SP;

Laboratory of Surgery and Molecular Pathology, Hospital Sírio Libanês,

São Paulo, SP;

Department of Pathology, FAMERP – Medicine School, São José do Rio

Preto, SP, Brazil

Correspondence to: Ana Elizabete Silva, IBILCE/UNESP –

Departamento de Biologia, Rua Cristóvão Colombo, 2265, São José do

Rio Preto, SP, Brazil - CEP. 15054-000.

Tel:+ 551732212384, e-mail: [email protected]

Key Words: p53, p16, Fhit protein, esophagitis, megaesophagus,

esophageal carcinoma.

Running Title: Bellini et al: p53, p16 and Fhit Protein Expressions in

Esophageal Lesions

Supported by Brazilian Agencies FAPESP and CNPq

Artigo original publicado na revista

Anticancer Research (ISSN:1791-7530)

28(6A):3793-3800, 2008.

Resumo

Introdução: Alguns modelos sugerem que o

desenvolvimento do carcinoma esofágico é resultante de uma sequência

de alterações, que envolvem esofagite, atrofia, displasia, carcinoma in situ

e carcinoma invasivo. Enquanto diversas alterações genéticas têm sido

descritas em carcinoma de esôfago, os estudos em lesões benignas com

potencial pré-canceroso ainda são escassos. Material e Métodos: Foi

realizada imuno-histoquíca para as proteínas p53, p16 e Fhit em mucosa

esofágica de pacientes com Doença de Chagas (CD), megaesôfago

chagásico (CM), esofagite crônica (CE), carcinoma de células escamosas

de esôfago (ESCC) e em mucosa normal (NM). Resultados: A proporção

de casos positivos para a proteína p53 aumentou progressivamente de

acordo com a severidade da lesão, CD (7.7%), CM (26.1%), CE (52.2%)

and ESCC (100%). Entretanto, as proteínas p16 e Fhit não mostraram

diferenças estatisticamente significantes entre os grupos. Conclusão: O

presente estudo mostra o envolvimento da proteína p53 super expressa

nos processos iniciais da carcinogênese esofágica, sugerindo sua

utilização como marcador em lesões precursoras, inversamente, perdas

na expressão de Fhit e p16 não foram significantes

Capítulo III

105

CAPÍTULO III: Expression of Ki67 antigen and caspase-3 protein in

benign lesions and esophageal carcinoma and relationship with p53

protein.

Authors: Marilanda Ferreira Bellini

; Patrícia Maluf Cury

, Ana Elizabete

Silva

Authors Addresses:

UNESP, São Paulo State University, Department of Biology, Campus

São José do Rio Preto, SP, Brazil

FAMERP – Medicine School, Department of Pathology, São José do Rio

Preto, SP, Brazil

Correspondences to: Ana Elizabete Silva, IBILCE/UNESP –

Departamento de Biologia, Rua Cristóvão Colombo, 2265, São José do

Rio Preto, SP, Brazil - CEP. 15054-000; phone 55 - 17 - 32212384

e-mail: [email protected]esp.br

Artigo original a ser submetido e redigido nas normas da revista

APOPTOSIS (e-ISSN: 1573-675X)

106

Resumo

O desbalanço entre proliferação e morte celular é um dos

fatores chave da tumorigênese. O presente estudo teve por objetivos

verificar alterações nos níveis de proliferação celular e apoptose em

lesões benignas esofágicas em comparação com carcinoma esofágico e

estabelecer associações com a expressão da proteína p53, estimada em

estudo prévio. Ensaios de immuno-histoquímica foram realizados para o

antígeno Ki67 e proteína caspase-3 (CPP32) em mucosa esofágica de

pacientes com Doença de Chagas sem (CD) e com megaesôfago (CM),

pacientes com esofagite crônica (CE), pacientes com carcinoma esofágico

(ESCC) e em mucosa normal (NM). Associações com a expressão de p53,

estimada em estudo anterior, também foram realizadas. O índice de

marcação de Ki67 (LI) foi similar em todos os grupos (variação de 30,1%

a 48%), não sendo observadas diferenças significantes. A frequência de

positividade de CPP32 foi similar nos grupos CD (30,8%), CM (30,4%) e

CE (34,8%), mas aumentada em ESCC (55,5%), embora sem diferenças

significantes. Nenhuma associação entre os níveis de CPP32, Ki67 e

expressão da proteína p53 foram observadas, nos diferentes grupos.

Desta forma, não há evidências de alterações nos níveis de proliferação

celular e apoptose nas lesões benignas estudadas.

Palavras-chave: CPP32, Ki67, megaesôfago chagásico, esofagite

crônica, carcinoma esofágico.

107

Abstract

One key factor in the tumorigenesis is the imbalance

between cell proliferation and death. The present study aimed to evaluate

alterations of apoptosis and cell proliferation in esophageal benign lesion

comparing to esophageal carcinoma and establish relationship with p53

expression estimated in previous study. Immunohistochemistry was

performed for expression of caspase-3 protein (CPP32) and Ki67 antigen

in the esophageal mucosa from patients with Chagas disease without (CD)

and with megaesophagus (CM), patients with chronic esophagitis (CE),

esophageal carcinoma (ESCC) and in normal mucosa (NM). The Labeling

Index (LI) for Ki67 was similar in every group (range: 30.1% - 48%)and

was not statistically significant. The positive CPP32 immunostaining was

observed in similar frequencies in the groups of CD (30.8%), CM (30.4%)

e CE (34.8%), but increased in ESCC (55.5%), though no significant

differences. No associations among the levels of CPP32, Ki67 and p53

expression were observed in the different groups. Thus are not evident

changes in in benign studied lesions.

Key Words: CPP32, Ki67, chagasic megaesophagus, chronic esophagitis,

esophageal squamous cell carcinoma

108

Introduction

The cancer development involves many genes that control

growth, cell proliferation and homeostasis of the tissue by apoptosis [1].

After an inflammatory process or the toxic effects of carcinogens the cell

proliferation increases the probability of new genetically altered cells [2].

There is a tendency of neoplastic population increase proliferative capacity

and escape the control mechanisms of normal growth. With the increased

cell proliferation, the mutant progeny arises as a result of genomic

instability, where the majority does not survive due to immune selection, or

apoptosis metabolic changes [3].

Many of the biochemical and morphological alterations

that occur during apoptosis is consequence from cysteine proteases family

called caspase, which in turn cleave the various substrate proteins that

account for apoptosis. Caspase-3, also known as CPP32, YAMA, or

apopain, is probably the one that so far best correlates with apoptosis.

Caspase-3–mediated proteolysis, which is initiated by the multiple different

stimuli (endogenous and exogenous) that induce apoptosis, is a critical

element of the apoptotic process [4]. Analysis of the expression of

caspase-3 from primary esophageal squamous cell carcinoma (ESCC)

showed that 60% of the cases were positive [5], and that caspase-3 was

associated with a favorable prognosis. Caspase-3 was suggested as a

new prognostic factor in ESCC indicating that biomarkers related

apoptosis can be used as predictive in advance and prognosis of ESCC [6,

7, 8].

The assessment of cell proliferation by the detection of

Ki67 antigen in neoplastic cell populations has been shown to be of

prognostic value. There is a strong correlation between low or high Ki67

index and low or high-grade histopathology of neoplasms [8]. The

progressive increase of Ki67 antigen from dysplasia to ESCC, suggested

that this antigen is an effective biomarker in the squamous ephitelium of

esophagus [9, 10, 11].

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TP53 tumor suppressor gene, which acts on apoptosis

and cell proliferation, is the major mutated gene in human neoplasm.

Mutations in this gene may fail both to arrest of cell cycle as to cause cell

death in cancer cells, because the mutated form of p53 is ineffective in

triggering apoptosis [12].

Esophageal carcinogenesis follows the multistep model

involving esophagitis, atrophy, dysplasia, carcinoma in situ, and finally

invasive carcinoma [13, 14]. Thus is important to evaluated benign lesions

with precancerous potential as esophagitis and megaesophagus

(esophagus dilation), the later as a consequence of Chagas disease,

which there are few genetic studies that investigated changes of their cell

cycle, as apoptosis and cell proliferation. The aim of this study was to

evaluate the levels of cell proliferation (Ki67) and apoptosis (CPP32) and

their associations with expression of p53 protein, estimated in previous

study [15] in benign and possible precursor lesions, as chagasic

megaesophagus and chronic esophagitis in cancer-free patients and

ESCC patients.

Samples and Methods

Samples

A total of 78 specimens of paraffin embedded esophageal

tissue were obtained from patients who underwent middle and distal

esophageal biopsies, before any chemo- or radiotherapy treatment, at

Pathology Section, Hospital de Base (São José do Rio Preto, SP, Brazil)

at the period between 2003 and 2006. Twenty-three specimens were

proceeding from Chagas Disease patients with chagasic megaesophagus

(CM), 13 with Chagas Disease (CD) but without megaesophagus, 23 from

chronic esophagitis (CE) patients, and 9 from esophageal squamous cell

carcinoma (ESCC). Both patients with Chagas Disease and chronic

110

esophagitis were esophageal cancer-free. Esophageal mucosa from 10

healthy patients were obtained and diagnosed as histologically normal

(NM).

In the NM, with histologically normal esophageal mucosa,

the majority of subjects were female, aged between 26 and 67 years (

SE = 42.4 ± 4.81), of which 40% of them have alcoholic habit and 30%

have smoking habit. Of the 36 chronic chagasic patients, 13 did not have

megaesophagus (CD), the majority being female, aged between 45 and

79 years (

± SE = 61.3 ± 2.85). Among chagasic without

megaesophagus, 30.8% were alcoholic and 38.5% were smokers. In this

group, 53.8% developed other complications related to Chagas disease -

(5 / 13) and megacolon (2 / 13) chagasic cardiomyopathy, and most

(76.9% - 10/13) had mild to moderate esophagitis. The other 23 patients

had chronic chagasic megaesophagus (CM) and most were male, aged 57

to 83 years (

± SE = 64.3 ± 2.08). As the classification of

megaesophagus, 4.3% (1 / 23) of cases were grade I, 13% (3 / 23) grade

II, 43.5% (10/23) grade III and 39.2% (9 / 23) Grade IV. Among patients

with chagasic megaesophagus, 34.8% (8 / 23) had alcoholic habit and

56.5% (13/23) smoking. Among other complications of Chagas' disease,

four patients (17.4%) developed megacolon, and 34.8% have heart

disease. Except for six cases, all other patients had esophagitis: 23.5%

mild, 47.1% moderate and 29.4% severe. In the group CE group, about

half were female or male, aged 40 to 70 years (

± SE = 50.5 ± 1.99).

Only 4.3% of the cases were drinkers and 17.4% were smokers. In

contrast, the group with squamous cell carcinoma of the esophagus was

formed by 8 males and one female, with ages ranging from 42 to 69 years

(

± SE = 56.5 ± 2.68). Of these, most are drinker and smoker (72.7% and

81.8%, respectively). In this group, three patients are chagasic, of which

two had chagasic megaesophagus associated with carcinoma.

111

The study was approved by Brazilian National Research

Ethics Committee (CONEP).

Immunohistochemical Assay

Consecutive 4µm-tick sections were cut from each

trimmed paraffin block, and mounted in glass slides pre-treated with 3-

aminopropyl-triethoxysilane/acetone solution. In brief, following

deparaffinization, sections were re-hydrated, treated with citrate buffer, at

96°C, for 30 min.; and treated with 3% H

in methanol (v/v) for 30 min.

to block the endogenous peroxidases. The sections were then incubated

for 1 hour, at room temperature, with specifics antibodies: CPP32 (clone

JHM-62, Novocastra, Newcastle, UK, 1:100) and Ki67 antigen (clone MM1,

Novocastra, Newcastle, UK, 1:200). The slides were incubated with

secondary antibody; next, slides were incubated polymer to CPP32

(Novolink Polymer, Novocastra, Newcastle, UK,) or streptavidin-biotin

peroxidase to Ki67 (Dako Cytomation Kit, Strept ABComplex/HRP, Dako,

Glostrup, Denmark), following the manufacturer’s instructions. The

immunostaing was visualized with 3,3’ diaminobenzidine

tetrahydrochloride (DAB) containing 0,005% H

, and counterstained

with hematoxylin. Negative controls were established by replacing the

primary antibody with buffer solution. Tonsil tissues were used as positive

controls for both antibodies.

The polyclonal antibody (NCL-Ki67p) labels Ki67 antigen

in the granular components of the nucleolus during late G1, S, G2 and M

phases [9]. The positive immunostain for Ki67 antigen was brown nuclear

staining and the negative was the absence of nuclear staining. Five

hundred cells were counted for each sample. Ki67 was considered positive

when >10% of cells showed nuclear reactivity [16, 17]. The Labeling Index

(LI) for Ki67 antigen (the ratio of cells positively stained for Ki67 on the

total of cells counted for each case [12] was calculated.

112

For CPP32, all tissue extension was examined for each

sample. Immunostaing for CPP32 (brown cytoplasm staining) was graded

by intensity of staining as negative: (-) absent brown staining and (+)

weakly stained; or as positive: (++) moderately stained and (+++) strongly

stained [4].

All analysis was done under a light microscope (x400

magnification). Areas that were poorly preserved, crushed, cauterized,

folded, or retracted were specifically avoided.

Statistical Analysis

Descriptive statistics, Kolmogorov and Smirnov method for

normality distribution, Kruskall-Wallis Test for CPP32 and Ki67; and

Analysis of Variance (ANOVA) with Tukey-Kramer Multiple Comparisons

post-test for Ki67 antigen were used to determined statistical significance.

Fisher’s Exact Test was performed to establish associations between

apoptosis, cell proliferation and p53 expression, two by two. The level of

significance was set as P<0.05. The statistical analysis was carried out

with GraphPad Instat 3 computer software [18].

Results

The LI for Ki67 antigen for each group was as follow:

30.1% of NM, 30.9% of CD, 42.3% of CM; 44.6% of CE and 48.4% of

ESCC in 500 cells analyzed per case (Table 1). The Kolmogorov and

Smirnov method indicated normality distribution and then ANOVA with

Tukey-Kramer Multiple Comparison post-test was performed among the

groups, but none significant difference was observed.

One hundred percent of the cases in the NM, CD, CE and

ESCC groups were classified as Ki67 positive, since they showed more

than 10% of cell positively immunostained for the antigen, while in the CM

113

group 91.3% of cases were Ki67 positive. The statistical analyses for the

positive cases, by Kruskall-Wallis Test, did not indicate any significant

difference among the groups (Table 1).

Positive citoplasmatic immunostaining for CPP32 was

observed in a few cases in the normal mucosa group 20% (2/10), which

was interpreted as normal level of apoptosis. The positive CPP32

immunostaing was observed in similar frequencies in the groups of CD,

CM and CE respectively, 30.8% (4/13), 30.4% (7/23), and 34.8% (8/23),

but increased in ESCC group, 55.5% (5/9) (Table 1). Statistically

significant differences were not observed using the test Kruskall-Wallis test

among the groups.

The establishment of associations between CPP32, Ki67

and p53 expression (based on previous data, [15]) were performed by

Fisher’s Exact Test, indicating that there is no association between the cell

regulation processes studied in the groups analyzed (Table 2), neither

between the altered expression of p53 and CPP32 protein or Ki67 antigen

in each studied group (Table 3). Moreover, significant associations were

not observed between altered expression pattern of Ki67 antigen and

CPP32 protein, with parameters such as age, gender, smoking and

alcoholism (data not shown) at different groups.

Figure 1 illustrates the immunostaining of Ki67 antigen

(nuclear) and CPP32 protein (cytoplasmic) in the esophageal mucosa of

benign lesions and carcinoma.

Discussion

Recent studies have demonstrated that gene

abnormalities of cell-cycle regulators that function in the transition from the

G1 to S phase are associated with life style factors in ESCC [19], however,

associations between these parameters, such as age, gender, smoking

114

and alcoholism, and the status of Ki67 antigen and CPP32 protein were

not observed in the studied groups (data not shown).

In the present study, the Ki67 labeling index (LI) were

higher in CM, CE and ESCC groups, than in the others, although both LI

and positivity for Ki67 was not statistically significant among the different

groups. However, ESCC showed higher expression of CPP32 than the

others groups, indicating high level of apoptosis in tumor tissue. When the

analysis of associations between the molecules (Ki67, CPP32 and p53)

could be performed, no associations were found.

Tumor cell cycle analysis has indicated that tumors with a

higher proliferation rate (>40%) show more aggressive clinical behaviors

compared to tumors with a low proliferation rate (<40%) [20]. The Ki67 LI

has been identified as a parameter of tumor proliferation. ESCC patients

with a high Ki67 LI have lower postoperative survival rates; thus, a high

Ki67 LI is one of the prognostic factors of ESCC [19, 21]. In our study, the

Ki67 LI were greater than 40% in CM (14 cases, 60.8%), CE (11 cases,

47.8%) and ESCC (5 cases, 55.5%) groups, but did not show significant

difference with the NM and CD groups (with Ki67 LI ~30%). Despite some

studies which indicate that the Ki67 LI is a powerful prognostic marker for

patients with esophageal carcinoma [22, 23, 24], the results of the current

study did not displayed increased Ki67 LI in the ESCC group compared to

others groups analyzed.

High expression of tumor proliferation-related factors (Ki67,

PCNA, and AgNOR), abnormalities of adhesion molecule (E-Cadherin,

alpha-Catenin), activation of autocrine mechanism of growth factor (EGFR,

TGFα, EGF), and DNA ploidy pattern, which is thought to be the result of

an accumulation of genomic abnormalities are also prognostic factors for

esophageal cancer [25]. Ki67-% was significantly higher in patients with

erosive esophagitis and in patients with functional heartburn than in

controls, concluding Ki67 evaluation provides quantitative and objective

data on squamous epithelium proliferative activity [9, 11]. Meanwhile, the

115

present study did not find any statistical significant difference among the

benign lesion and ESCC, neither among the normal mucosa. Though, the

expression of Ki67 did not show significant differences in biomarker

expression between carcinoma of the esophagus and its precursor lesions

(mild, moderate and severe dysplasia) [20], what corroborate our results.

We observed an increased expression of CPP32 protein

only in ESCC group (55.5%), but it was not significant. The positive

staining in ESCC group should be arose from the high apoptotic activity in

response to the severe injury, whereas in CD, CM and CE (positive in

~30% cases), it might be occur as a consequence of physiological and

inflammatory process of esophageal epithelium. So the high expression of

CPP32 protein in ESCC, in the current study, may indicate a favorable

prognostic, according some studies that observed a relationship between

moderate to strong CPP32 staining and better prognostic [6]. Moreover,

caspase-3 revealed significant increase in the positivity pattern between

dysplasia and their corresponding invasive cancer portion and may be

involved in the progression from dysplasia to ESCC [26, 27]. In conclusion,

the analysis of the apoptotic protein expression patterns would be valuable

to develop rational strategies for early detection of lesions at risk in

advance as well as for prevention and treatment of ESCC.

Wild type of p53 triggers apoptosis following DNA damage

or induces p21WAF1/Cip1, which causes G1 arrest, allowing time for

damaged DNA to be repaired. Therefore, examining the relationship

among p53, cell proliferation and apoptosis may be critical to determine

the progress of carcinogenesis [12, 28]. Beardsmore et al. (2003) [12]

found relationship between p53 expression and apoptosis, or p53

expression and cell proliferation indexes when ESCC patients were

treated with chemotherapy. But, in our study associations between the

molecules CPP32, Ki67 and p53 could not be performed in all groups,

because, in some cases, the combination of variables was zero. When the

analysis was performed in the groups CD (CPP32 x p53); CM (Ki67 x p53;

116

Ki67 x CPP32 and CPP32 x p53) and CE (CPP32 x p53); we could not

find any relationship among the expression of the molecules evaluated.

These results also can be in consequence of reduced number of cases

evaluated in each group.

The absence of association between Ki67 antigen and

CPP32 protein could be related to an imbalance in cell defense

mechanism, when the organism tries to interrupt the proliferation of

damaged cell, decreasing cell proliferation and increasing the apoptosis

levels, because close coordination of these two phenomena is essential

not only for a regulation and normal physiology, but to disease prevention

[4] and combat.

In conclusion, the role of the Ki67 antigen in ESSC seems

not clear, because some authors believe in the increase of Ki67

expression following the severity of the lesion, while others do not. In the

present study, we did not find any difference among the groups, indicating

the need of further investigations. Our results did not show statistically

difference of the CPP32 expression between the benign lesion groups and

ESCC group. Also were not observed associations among the levels of

CPP32, Ki67 and p53 expression. Thus it is not evident significant

changes in apoptosis and cell proliferation in the studied esophageal

benign lesions, at the tested conditions.

Acknowledgements:

The authors are grateful to Dr. Sebastião Roberto Taboga

and Luiz Roberto Faleiros for help with histological sections and photo

documentation. We also thank Brazilian agencies Fundação de Apoio à

Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de

Pesquisa e Desenvolvimento (CNPq) for financial support.

117

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120

Table 1.

Immunohistochemical and statistical analysis for Ki67 antigen and CPP32 protein in the different groups.

Groups

Ki67 CPP32

Labeling Index (LI) Positive Positive (++/+++)

± SE (%)

Range

n/N %

NM 10 30.1 ± 2.63

18.2 - 44

10/10 100.0

2/10 20.0

CD 13 30.9 ± 2.80

12.0 – 42.0

13/13 100.0

4/13 30.8

CM 23 42.3 ± 3.82

2.4 – 62.8

21/23 91.3

7/23 30.4

CE 23 44.6 ± 3.28

20.6 – 76.4

23/23 100.0

8/23 34.8

ESCC 9 48.4 ± 7.12

21.2 – 72.0

9/9 100.0

5/9 55.5

Statistical Analysis

ANOVA, p = 0.0120

Tukey-Kramer Multiple

Comparison Test, p > 0.05

Kruskall-Wallis Test, p = 0.3035

Kruskall-Wallis Test, p = 0.5731

NM, health patients; CD, Chagas Disease patients without megaesophagus; CM, chagasic megaesophagus; CE, chronic esophagitis; ESSC, esophageal squamous

cell carcinoma; N, total of specimens; n, number of specimens positive immunostained;

, average; SE, standard error. Statistical Analysis: groups following the

same letter are not statistically different, on the same column;

, there is not statistical difference.

120

121

Table

. Association between

expression of Ki67 antigen and CPP32 protein, by Fisher’s Exact Test

(p<0.05).

Groups

Ki67 + Ki67 - Ki67 x CPP32

CPP32 + CPP32 - CPP32 + CPP32 - p value

1 (10%)

9 (90%)

0 (0%)

1 (7.69%)

12 (92.31 %)

0 (0%)

6 (26.08%)

15 (65.22%)

0 (0%)

2 (8.70%)

1.000

12 (52.17%)

11 (47.83%)

0 (0%)

ESCC

5 (55.56%)

4 (44.44%)

0 (0%)

NM, health patients; CD, Chagas Disease patients without megaesophagus; CM, chagasic

megaesophagus; CE, chronic esophagitis; ESSC, esophageal squamous cell carcinoma; NP, it was

not performed because one row or column is filled with zeros and in this situations the analysis is

impossible;

it is not statistically significant

121

122

Table 3

. A

ssociation between expression of Ki67 antigen and CPP32 protein with p53 protein

[

]

, by Fisher’s Exact Test (p<0.05).

Groups

Ki67 + Ki67 -

Ki67xp53

CPP32 + CPP32 -

CPP32xp53

p53 + p53 - p53 + p53 - p value p53 + p53 - p53 + p53 - p value

0 (0%)

10 (0%)

0 (0%)

1 (10%)

0 (0%)

9 (90%)

4 (30.8%)

9 (69.2%)

0 (0%)

4 (30.8%)

1 (7.7%)

8 (61.5%)

1.0000

6 (26.0%)

15 (65.2%)

0 (0%) 0 (0%) NP

2 (8.7%)

5 (21.7%)

4 (17.4%)

12 (52.2%)

1.0000

8 (34.8%)

15 (65.2%)

0 (0%)

3 (13.0%)

5 (21.7%)

9 (39.2%)

6 (26.1%)

0.4003

ESCC

9 (100%)

0 (0%)

5 (55.5%)

0 (0%)

4 (44.4%)

0 (0%)

NM, health patients; CD, Chagas Disease patients without megaesophagus; CM, chagasic megaesophagus; CE, chronic esophagitis; ESSC, esophageal

squamous cell carcinoma; NP, it was not performed because one row or column is filled with zeros and in this situations the analysis is impossible;

it is

not statistically significant.

122

123

Legend of figure:

Figure 1. Immunostaining of Ki67 antigen and CPP32 in esophageal mucosa. A,

Ki67 antigen. B. CPP32 protein. 1., normal mucosa (Ki67+, CPP32 -), 2. chagasic

without megaesophagus (Ki67+, CPP32 ++), 3. chagasic megaesophagus (Ki67+,

CPP32 ++), 4. chronic esophagitis (Ki67+, CPP32 +), 5. esophageal squamous cell

carcinoma (Ki67+, CPP32 +++). Ki67+, more than 10% of cell is positively

immunostained. CPP32 -, absence of immunostaining; CPP32 +, weak

immunostaining; CPP32 ++, moderate immunostaining; CPP32 +++, strong

immunostaining. Magnification 200x

124

Capítulo IV

126

CAPÍTULO IV: Chromosomal imbalances are uncommon in Chagasic

megaesophagus

Authors: Bellini, MF

1,2

; Manzato, AJ

; Silva, AE

; Varella-Garcia, M

Authors Addresses:

UNESP, São Paulo State University, Department of Biology, Campus

São José do Rio Preto, SP, Brazil

University of Colorado Denver, Department of Medicine/Medical

Oncology, Aurora, Colorado, USA

UNESP, São Paulo State University, Department of Computer Sciences

and Statistics, Campus São José do Rio Preto, SP, Brazil

Correspondence to: Ana Elizabete Silva,

IBILCE/UNESP – Departamento de Biologia, Rua Cristóvão Colombo,

2265, São José do Rio Preto, SP, Brazil - CEP. 15054-000;

phone 55 - 17 – 32212384.

e-mail:[email protected]sp.br

Running Title: Chromosomal Imbalances in chagasic megaesophagus

Artigo original a ser submetido e escrito nas normas da revista

CHROMOSOME RESEARCH : CYTOGENETICS, GENOMICS,

CHROMATIN AND THE NUCLEUS (e-ISSN: 1573-6849)

127

Resumo

A doença de Chagas é uma doença parasitária tropical.

Entre os pacientes na fase crônica, 6 a 7% desenvolvem mega síndromes

do esôfago ou cólon. A dilatação do esôfago é conhecida como

megaesôfago chagásico (CM) e uma de suas conseqüências tardias é o

aumento do risco desses pacientes desenvolverem carcinoma esofágico

(ESCC). Baseando-se na relação entre o CM e ESCC, investigamos, em

cortes histológicos da mucosa esofágica de pacientes com CM e mucosa

normal (NM), genes frequentemente não-balanceados descritos em ESCC,

como FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A, YES1,

NCOA3, e centrômeros 3, 7 e 9, como controles, por FISH (Hibridação In

Situ Fluorescente). A análise citogenética nas camadas superficial e basal

da mucosa esofágica não mostrou diferenças significantes para a maioria

dos alvos investigados, exceto para EGFR no grupo CM, que apresentou

freqüência menor de cópias na camada basal. O número total de cópias

de PIK3CA, CDKN2A e CEP9 foi significativamente menor em CM que em

NM. Alguns casos de CM mostraram níveis não-significantes de perdas

para genes específicos: TP63, FHIT, PIK3CA, EGFR, CDKN2A, YES1;

CEP9 ou de ganhos para PIK3CA, TP63, FGFR1, MYC, CDKN2A e

NCOA3. Em conclusão, a observação de que alvos genômicos com

desequilíbrios na carcinogênese esofágica não são significativamente

afetadas em CM sugere que não será possível utilizar tais características

como marcadores eficazes de risco para desenvolvimento de ESCC em

CM.

Palavras-chave: megaesôfago chagásico, carcinoma de células

escamosas de esôfago e FISH

128

Abstract

Chagas' disease is a human tropical parasitic disease. Among the chronic

chagasic individuals, 6 to 7% develop mega syndromes of the esophagus

or colon. The esophagus dilation is known as Chagas megaesophagus

(CM) and one of the serious late consequences of CM is the increased risk

for esophageal carcinoma (ESCC). Based on the association between CM

and ESCC, we proposed to investigate in CM the genomic status of DNA

targets frequently unbalanced in ESCC as FHIT, TP63, PIK3CA, EGFR,

FGFR1, MYC, CDKN2A, YES1 and NCOA3; and the centromere 3,

centromere 7 and centromere 9 were used as control, by FISH. Analyses

in the superficial and basal layers of the epithelial mucosa did not show

differences among them, excepted for the gene EGFR in CM, which was

deleted in the basal layer. The copy number of PIK3CA, CDKN2A and

CEP9 were significantly lower in CM than in NM. Some CM cases had

non-significant levels of deletions in TP63, FHIT, PIK3CA, EGFR,

CDKN2A, YES1; CEP9 or gains at PIK3CA, TP63, FGFR1, MYC,

CDNK2A and NCOA3. In summary, the findings that genomic imbalances

common in esophageal carcinomas are not affected in CM suggest that

these features will not be effective biomarkers for risk assessment of

ESCC in CM

Key Words: chagasic megaesophagus, esophageal squamous cell

carcinoma, FISH

129

Introduction

Chagas' disease is a human tropical parasitic disease

which occurs in the Americas, particularly in South America (Lana; Tafuri,

2000). During the chronic phase, 6 to 7% of chagasic patients develop

mega syndromes (muscular hypertrophy and dilation) of the esophagus or

colon, probably because the protozoan T. cruzi destroys the mioenteric

plexus (Crawford, 2004). In these digestive ways, muscular hypertrophy

and dilation of the esophagus or colon are observed in advanced stages of

the disease (Brasileiro-Filho, 2006).

The megaesophagus is consequence of achalasia

characterized by the destruction or lack of intramural nerve plexus, which

determines the absence of peristalsis and lack of openness of the lower

esophageal sphincter in response to swallowing. In consequence, there is

food retention, called esophageal stasis (Kraichely; Farrugia, 2006), which

can determine the appearance of acanthosis, paraceratose and

leukoplakia, possibly pre-cancerous lesions (Pinotti et al, 1996). One of

the serious late consequences of chagasic megaesophagus is the

increased risk (3% to 8%), of developing esophageal squamous cell

carcinoma (ESCC) compared to when megaesophagus is not present

(Bektas et al, 2001; Brüncher et al., 2001, Gockel et al, 2006). Also, ESCC

develops in chagasic megaesophagus patients at a younger age than in

those without this disease (Crawford, 2004). The detection of cancer in

these patients is complicated because the symptoms are hidden by the

severe dysphagia caused by megaesophagus (Chino et al, 2000), and the

diagnosis is made frequently only in advanced stage (Haubrich; Schaffner;

Berk, 1995) resulting in a poor prognosis (Brücher et al, 2001).

ESCC has been reported as the ninth most common

malignancy and ranks the sixth most frequent cause of death over the

world, but its incidence varies extremely among regions (Health A to Z,

2008; INCA, 2008) The American Cancer Society estimated around

16,470 new cases of esophageal carcinoma in the USA population for

130

2008 (American Cancer Society, 2008). The Brazilian National Institute of

Research in Cancer (INCA) reported that esophageal cancer was the sixth

in the cancer rank of mortality in 2000 with 5,307 deaths (INCA, 2008) and

estimated about 10550 new cases in 2008 of this malignancy ranging

among geographical areas from 1.04 to 19.07 in males and from 0.39 to

7.58 in 100,000 in females (INCA 2008).

Geographical variations and higher incidence in patients

over 40-year old favors a multifactorial origin for ESCC, and the

involvement of environmental factors such as smoking and alcoholic habits

has been long known (Matsuo et al., 2001; Kuwano et al., 2005; Pelucchi

et al., 2008). In addition to the environmental and pathogenic factors,

genetic alterations, such as chromosomal aneuploidies, allelic deletions,

activation of oncogenes and inactivation of tumor suppressor genes are

associated with esophageal carcinogenesis (Kuwano et al., 2005;

Daigo;Nakamura, 2008; D’Amico, 2008). Most of these phenomena are

associated with genomic imbalances, such as gene amplification, over-

representation or loss (Daigo;Nakamura, 2008; D’Amico, 2008).

In ESCC, different techniques of molecular cytogenetic

have shown unbalanced genomic regions implicated with the amplification

of some oncogenes and deletion of tumor suppressor genes (Kuwano et

al., 2005; Daigo;Nakamura, 2008; D’Amico, 2008). In this study were

selected FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A, YES1 and

NCOA3 genes, frequently unbalanced in ESCC (Pack et al., 1999;

Shinomyia et al., 1999; Du Plessis et al., 1999; Pimkhaokham et al., 2000;

Yen et al., 2001; Ishizuka et al., 2002; Nakakuki et al., 2002; Arai et al.,

2003; Shiomi et al., 2003; Yen et al., 2005; Su et al., 2006; Wang et al.,

2006)

The amplification of the tumor suppressor gene TP63

(3p28, whose protein plays in the development and maintenance of

stratified epithelial tissues) has been described by Yen et al. (2003, 2005),

in early stage of ESCC carcinogenesis but down-regulated in advanced

131

stage of disease. (Yen et al., 2005). However, losses at 3p and 9p,

correspondent to the FHIT and CDKN2A respectively, were lost in some

cases of ESCC (Du Plessis et al, 1999, Pack et al, 1999, Shinomiya et al,

1999, Pimkhaokham et al, 2000, Yen et al, 2001, Arai et al, 2003, Shiomi

et al, 2003, Su et al, 2006, Wang et al, 2006).

Amplification of PIK3CA (3q26), whose protein represents

the catalytic subunit, which uses ATP to phosphorylate

phosphatidylinositol, was assessed ESCC (Yen et al. 2003; Yang et al.,

2008, National Center for Biotechnology Information, 2009). FISH using

BAC clones containing oncogene PIK3CA found that gene was amplified

in 6 cell lines. Q-PCR analysis of primary tumors revealed amplification of

PIK3CA in 100% of the cases (Yen et al., 2003) .Positive correlation was

found only between amplification and tumor size, lymph node metastasis

and clinical stage. PIK3CA gene amplification was highly correlated with

protein overexpression, suggesting that gene amplification is one

important mechanism involved in PIK3CA overexpression (Yang et al.,

2008).

Overexpression of membrane receptors has been

reported in various cancers, and it has been suggested that it may be a

poor prognostic factor in cases with solid tumors (Sugiura et al, 2007).

EGFR (7p12) is a cell surface protein that binds to epidermal growth factor.

Binding of the protein to a ligand induces receptor dimerization and

tyrosine autophosphorylation and leads to cell proliferation (National

Center for Biotechnology Information, 2009). Overexpression of EGFR is

observed in many cancers, sometimes accompanied by gene amplification.

By FISH, EGFR gene showed amplification in ESCC (Sunpaweravong et

al., 2005; Hanawa et al., 2006). Also, EGFR gene overrepresentation

(balanced gene and chromosome 7 polysomy) was common event in

ESCC (Mimura et al., 2005; Sunpaweravong et al., 2005). The co-

expression of both aFGF and FGFR1 (8p12) was associated with a larger

tumor area and poorer prognosis what suggests the membrane receptors

132

may promote proliferation of esophageal cancer cells in an angiogenesis-

independent and autocrine manner, and may contribute to rapid growth of

esophageal cancer on recurrence after esophageal resection (Sugiura et

al, 2007).

Ishizuka et al. (2002) and Arai et al. (2003) detected

amplification of MYC (8q24) by aCGH. MYC was found to be amplified in

the 3 ESCC cell lines (Ishizuka et al., 2002) and in 9 of 20 tumors found on

the microarray, but it was located on chromosomal regions that showed no

increase in DNA copy number by CGH (Arai et al., 2003).

YES1 (18p11.31, protein has tyrosine kinase activity)

amplification was detected by FISH, what suggests it is likely to be

candidate target for 18p11.3 amplification and be associated with

esophageal carcinogenesis (Nakakuki et al., 2002, National Center for

Biotechnology Information, 2009).

NCOA3 (20q12), which the protein is a nuclear receptor

coactivator that interacts with nuclear hormone receptors to enhance their

transcriptional activator functions, has been observed overexpressed and

with increased copy number in 46% and 13% of ESCCs, respectively (Xu

et al., 2007). Overexpression of NCOA3 was observed more frequently in

primary ESCCs in late T stages than that in earlier stages, but no

significant association of expression of NCOA3 and status of lymph node

metastases was observed. These results suggest that overexpression of

NCOA3 caused by genomic gain or other molecular mechanisms might

provide a selective advantage for the development and local invasion of

certain subsets of ESCC.

Despite the numerous genetic alterations reported in

esophageal carcinogenesis, studies in benign lesions with precancerous

potential as megaesophagus are scarce. This study investigated the

genomic status in chagasic megaesophagus of genes frequently

unbalanced in ESCC with the goal of identifying a potential panel of

markers of risk to cancer. Among the as FHIT, TP63, PIK3CA, EGFR,

133

FGFR1, MYC, CDKN2A, YES1 and NCOA3 and three DNA targets:

centromere 3, centromere 7 and centromere 9 used as control, by FISH

assay.

Material and Methods

1. Subjects Characterization

Formalin-fixed, paraffin-embedded (FFPE) esophageal

mucosa was obtained from 10 health individuals with histologically normal

esophagus (NM) and 40 patients with diagnosis of chagasic

megaesophagus (CM) who underwent middle and distal esophageal

biopsies from 2000 to 2007 at the Hospital de Base of the São José do

Rio Preto, SP, Brazil. The study was approved by the Institution Research

Ethical Committee (CEP) and by the Brazilian National Research Ethics

Committee (CONEP).

From 40 CM patients, 20 were male and 20 were female

and the mean age was 62.5 years (range: 40 – 83 years). Twenty-nine

were never-alcoholics, 3 were former-alcoholics (at least 5 years) and 8

were current alcoholics (at least 5 years); 18 were never-smokers, 6 were

former-smokers (at least 5 years) and 16 were current smokers (at least 5

years). The histological grade of megaesophagus was classified as I to IV,

based on the retention of contrast, diameter, tonicity of the lower sphincter

and the length of the esophagus body (de Rezende; Lauar; de Oliveira,

1960). This study cohort included 5 patients with megaesophagus grade I,

7 with grade II, 17 with grade III and 11 with grade IV. The group group

was followed-up until the end of this study and none individual developed

esophageal squamous cell carcinoma. The CM group was followed-up

until the end of this study and none individual developed esophageal

squamous cell carcinoma.

The control group (NM) was composed by 10 health

patients who were submitted to endoscopy under suspicion of dyspepsia,

but the histopathological analysis has shown normal esophageal mucosa.

134

In this group, seven individuals were female and three were male, and the

mean age was 42.4 (range: 26 - 67 years), where four were current

alcoholics and 6 were never alcoholics; 3 were current smokers and 7

were never smokers. Despite the groups composed for 10 patients, only

two of them was tested for each probe set.

2. Fluorescence In Situ Hybridization (FISH)

Serial 4µm-thick sections were cut from each trimmed

paraffin block and mounted in glass slides pre-treated with 3-aminopropyl-

triethoxysilane/acetone solution. Tissue sections were incubated at 56°C

overnight, deparaffinized in Citrisolv (Fisher Brand, Cat #22-143975)

washes (three times for 10 min), and dehydrated in 100% ethanol. After

incubation in 2xSSC (Sodium chloride, sodium citrate solution, pH 7.0) at

75°C, sections were digested with proteinase K (0.25 mg/ml in 2xSSC, pH

7.0) at 45°C, rinsed in 2xSSC (pH 7.0) at room temperature for 5 min, and

dehydrated in an ethanol series.

The following DNA probe sets were used: EGFR/CEP7

(Abbott Molecular, Cat. # 32-191053), P16/CEP9 (CDKN2A, Abbott

Molecular, Cat. # 32-190078), c-MYC (Abbott Molecular, Cat. # 32-190006)

and homebrew probes: FGFR1 (RP11-350N15), FHIT (CTD-2196D15) /

centromere 3 (p alpha 3.5), TP63 (RP11 - 373I6) / PIK3CA (RP11 -

245C23) and YES1 (RP11-769O8) / NCOA3 (RP11-456N23). Each probe

set was applied to the selected area on a designated slide. The

hybridization areas were covered with glass coverslip and sealed with

rubber cement. Co-denaturation of chromosomal and probe DNAs was

performed at 85°C for 10 min and hybridization was allowed to occur in a

humidified chamber at 37°C for 24 h for commercial probes and 48h for

homebrew probes. After hybridization, the slides were washed twice in

2xSSC/0.3% NP-40 at 73

C for 2min, rinsed in 2xSSC at RT for 2min,

dehydrated in 70%, 85% and 100% ethanol, allowed to air dry and

135

counterstained with 4’, 6’-diamino-2-phenylindole - DAPI, (0.3 ug/ml in

Vectashield Mounting Medium, Vector, Cat. # H-1200).

Microscopic analyses were performed in epifluorescence

microscope. Fluorescence signals were scored using single band filters for

DAPI, FITC (fluorescein), and Texas red, a double-band pass filter (FITC

and Texas red) and a triple-band pass filter (DAPI, FITC, and Texas Red).

Histological areas previously selected in the HE-stained sections were

identified in the fluorescence in situ hybridization (FISH)-treated slides.

Signals were scored in 100 epithelial nuclei per specimen, in at least four

distinct areas: 50 nuclei in the superficial layer (larger cells, closer to the

lumen) and 50 nuclei in the basal layer (darker HE-stained nuclei, smaller

cells, closer to the muscular layer). The scoring was performed in both

layers to check for differences between proliferate activity, since basal

layer cells are in high proliferative activity, whereas the superficial layer

cells are more mature.

For 3 genes, FHIT, EGFR and CDKN2A the experiments

were performed including centromeric sequences of carrier chromosomes

as an internal control, respectively chromosomes 3, 7 and 9. This design

was necessary for FHIT and CDKN2A since these genes are know for

their loss in cancer specimens.

3. Statistical Analysis

Descriptive statistics were calculated using an Microsoft Excel

macro template previously validated including mean copy number per cell,

standard deviation and frequency of cells with 1, 2 and ≥3 copies of each

DNA target tested, as well as the ratio for gene/control probe when

applicable. Comparisons between the superficial and basal layers were

done by t-student paired test, and between the experimental groups were

performed by t-student unpaired test; in both cases confidence level was

established as 0.05 (Graphpad, 2008). Association between age, gender

and life style factors (smoking and alcoholism), and megaesophagus

136

grade in the groups was compared by ANOVA and the comparisons for

aneusomies were done by χ

test. One exploratory analysis using three-

dimensional plots was performed for the percentages of genes status

classes.

Results

The descriptive indexes (mean and standard deviation) for

the genes FHIT, PIK3CA, TP63, EGFR, FGFR1, MYC, CDKN2A,YES1,

NCOA3 and centromeric sequences of chromosomes 3, 7 and 9, and the

results of the statistical analyses for comparison of mean copy numbers

between the superficial and basal layers in each group are presented in

Table 1. The range of the mean in the CM group was 1.49 - 1.79 in the

superficial layer and 1.45 - 1.79 in the basal layer. In the NM the mean

frequency ranged 1.35 – 1.77 in the superficial layer and 1.61-1.78 in the

basal layer. Superficial and basal layers did not show significant

differences, excepted for EGFR in the CM group (p=0.008), that showed

lower copy number in the basal layer. These results supported the

combination of both layers for comparison of each gene between the CM

and NM groups (Table 2). The total counting of copy number of the

PIK3CA, TP63, FGFR1, MYC, YES1 and NCOA3 genes are not

statistically different, when the groups CM and NM were compared.

However, the copy number of the gene CDKN2A was significantly lower

(p<0,0001) in the CM group compared to NM, the same was observed for

the centromere 9 (CEP9) (p=0.0002).

The gene/centromere ratio, for FHIT/CEN3, EGFR/CEP7

and CDKN2A/CEP9, genes for which a control probe (centromeric

sequence) was included in the assay, in all CM and NM specimens

analyzed was balanced, ranging from 0.96 to 1.00, and statistically

significant difference was not detected between the mean copy number for

each gene and its centromere (Table 3).

137

In the CM group there was no significant differences on

the mean copy number per cell for most genomic target according the

histological grades of megaesophagus (I, II, III and IV) excepted for MYC

and YES1 (Table 4), Similarly, significant associations with parameters

such as age and gender and life style factors (smoking and alcoholism), ,

at different groups, were also not observed (data not shown).

When frequencies of nuclei with balanced copy number or

loss and gain copy number for each target were evaluated (Table 5), only

PIK3CA showed higher frequency of cells with loss in CM compared to NM

(36.4% vs. 28.5%). Statistical analysis was not performed for gain due to

the scarcity of cells with this pattern.

An exploratory analysis using three-dimensional plots of the

percentages of genes status classes (loss, balanced and gain) for

individual patients is illustrated in Figures 1, 2 and 3. Several patients were

identified harboring deletions in specifics targets: CM36 (FHIT), CM13 and

CM19 (PIK3CA), CM 13 (TP63), CM30 (EGFR), CM12 (CDKN2A), CM7

(YES1), and CM16, CM34 and CM36 (CEP9). Other patients have shown

genomic gain for specific targets: CM11 and CM28 (PIK3CA), CM6 and

CM7 (TP63), CM3, CM6 and CM30 (MYC), CM30 (FGFR1), CM11 and

CM16 (CDKN2A), CM36 (NCOA3), and CM11 and CM21 (CEP9). Some

showing changes in more than one target. Interestingly, although no

statistical difference was observed among the megaesophagus grades,

excepted for MYC and YES1, most of the patients detected with loss or

gain in the exploratory analyses were diagnosed with advanced

megaesophagus grade III and IV.

The Figure 4 shows some FISH images for the unbalanced

genes and centromeric targets in the megaesophagus specimens.

Monosomies involving centromeres 3, 7, and 9 were also

investigated in each group (Table 6) and the cells were classified in three

classes: <2 copies, 2 copies and >2 copies. The CM group showed

significantly increased frequencies of cells with only one copy for

138

centromeres 7 and 9 (51% and 44%, respectively) in comparison to the

NM group (28% and 23%, respectively). The CM group also showed

numerically higher frequencies of cells with >2 copies of centromeres 7

and 9 (1.68% and 10.38%, respectively) than the NM group (0.50% and

0.00%, respectively), although it was not possible to perform statistical

analysis.

Discussion

The esophageal squamous cell carcinoma is one of the

most prevalent cancers worldwide and has multifactorial origin, in which

the environment plays very significant role (Health A to Z, 2008),

especially the abuse of alcohol and smoking (Matsuo et al., 2001;

Kuwano et al., 2005; Pelucchi et al., 2008). Besides the participation of

environmental and pathologic factors, several genetic alterations are

associated with ESCC, such as aneuploidies, allelic deletions, activation of

oncogenes and inactivation of tumor suppressor genes (Kuwano et al.,

2005; Daigo; Nakamura, 2008; D’Amico, 2008).

In the present study, the analysis of genomic status in CM

of DNA targets frequently unbalanced in ESCC as FHIT, TP63, PIK3CA,

EGFR, FGFR1, MYC, CDKN2A, YES1 and NCOA3; and the centromeres

3, 7 and 9 used as control, showed that the superficial and basal layers of

esophageal mucosa are similar, except for lower EGFR copy number in

the basal than in the superficial layer. The analysis for gene loss and of

centromeres, indicated only PIK3CA with loss pattern and monosomies of

the chromosomes 7 and 9 in the CM group. Despite the differences were

not statistically significant, we detected CM cases with loss for FHIT,

CDKN2A, EGFR, TP63, PIK3CA, YES1 and centromere 9 and cases with

gain for PIK3CA, TP63, MYC, FGFR1, CDNK2A, NCOA3 and centromere

9 sequences, most of all associated to grades III and IV of

megaesophagus. Although significant differences were detected on the

139

mean copy number only for MYC and YES1 among the histological grades

of megaesophagus.

Human esophageal carcinoma expresses multi-autocrine

growth factors and hormones including EGF. Overexpression of epidermal

growth factor receptor (EGFR) by tumor cells is closely correlated with

tumor invasion and poor prognosis. The accumulation and interaction of

several growth factors produced by tumor cells are necessary for the

progression of human esophageal and gastric carcinomas (Yoshida et al,

1990).

Evidence suggests the existence of a new model of EGFR

signaling pathway in which activated EGFR undergoes nuclear

translocation and subsequently regulates gene expression and potentially

mediates other cellular processes. Likely through its ability to upregulate

gene expression, the nuclear EGFR pathway is associated with the major

characteristics of more aggressive tumors such as increased proliferative

potential, nitric oxide synthesis, and accelerated G1/S cell cycle

progression (Lo; Hung, 2006). Increased expression of nuclear EGFR is

linked to poor clinical outcome in patients with oropharyngeal squamous

cell carcinomas (Psyrri et al, 2005). In this study, decrease in the copy

number of EGFR was found in the basal layer of CM in comparison to the

superficial layer and in one CM case there was EGFR loss (not statistically

significant), what could indicate that EGFR may be deleted in this benign

lesion.

The loss of CDKN2A function, preventing the blocking of

G1 phase appears to be a necessary event for the progression of pre-

cancerous cells to malignancy (Mori et al., 1994; Nuovo et al., 1999;

Li;Yang, 2003). In primary ESCC and cell lines, occurrence of point

mutations or deletions in the CDKN2A gene have been observed in 16%

of cases (Chen et al., 2006), but the most frequent phenomenon

responsible for CDKN2A inactivation is hypermethylation of the promoter

region. In ESCC and pre-neoplastic lesions several studies have shown

140

hypermethylation of CDKN2A with frequencies ranging from 20% to 88%.

This frequency increases gradually with the histological severity of the

disease and is associated with poor prognosis (Fukuoka et al., 2006; Guo

et al., 2006, Roth et al., 2006; Guo et al., 2007; Ito et al., 2007; Fujiwara et

al., 2008; Wang et al., 2008). The loss of p16 protein expression, detected

by immunohistochemical techniques, has been observed in 50% to 97% of

cases of ESCC (Kato et al., 2000; Ralhan et al., 2000; Takeuchi et al.,

2001; Chang et al., 2005; Guo et al., 2007; Fujiwara et al., 2008). The

current study could only detect the fraction of cases in which the loss of

CDKN2A was due to physical deletion. This higher frequency of CDKN2A

and centromere 9 losses in CM compared with NM, observed in the

present study, is also a phenomenon that associates CM with the

esophageal carcinogenesis process, because patients with idiopathic

achalasia and chagasic megaesophagus, with or without esophageal

carcinoma, showed changes in the expression of p16 (Chino et al., 2000)

and Bellini et al. (2008) observed a marginal decrease in the p16

expression in megaesophagus.

TP63 and PIK3CA were reported as oncongenic potential

in a variety of carcinomas (Yen et al, 2005). Gene amplification is one of

the basic mechanisms that lead to overexpression of oncogenes and

PIK3CA and TP63 are described as amplified in ESCC (Pimkhaokoham

et al, 2002; Yen et al, 2005). In our research, we found a statistically

significant difference in deletion of PIK3CA gene in CM, probably

indicating the loss of this gene in the early steps of esophageal

carcinogenesis. Overexpression of TP63 was found in most ESCC (Hibi et

al, 2000, 2001; Glickman et al, 2001; Hu et al, 2002). In a

immunohistochemistry study, TP63 protein was found highly expressed in

ESCC, dysplasia, and even in histologically normal epithelia of esophagus

adjacent to the cancerous tissues (Glickman et al, 2001). We found two

CM cases with numerical gain for PIK3CA, and two case with gains for

TP63, but none had significantly higher copy numbers.

141

Some CM cases were highlighted for deletion of FHIT in

chromosome 3 and EGFR in chromosome 7, however only chromosome

7 loss was statistically significant. Deletion in both FHIT alleles results in

loss of exons and concomitant failure in the transcript (Druck et al., 1997).

These genomic rearrangements alter the mRNA, leading to absence or

reduction of Fhit protein, which is associated with the development of

several kinds of cancer (Mori et al., 2000; Kuroki et al., 2003). Loss of

heterozygosity (LOH) has also been reported in low-grade dysplasia in

regions 3p14-p21 as early events in esophageal carcinogenesis (3p14.2).

The FHIT gene has been described as hypermethylated in 33% to 69.4%

of ESCC cases. Its hypermethylation is also significantly correlated with

deletion and loss of protein expression in esophageal carcinoma (Druck et

al., 1997).

In the present study, 3 cases showed gain for MYC, not

statistically significant. One mCGH study revealed gains of 8q23~ter,

including the MYC locus, but amplification of MYC was infrequent in ESCC

(1/20) by microarray (Arai et al., 2003) corroboting our results. We also

verified 10% (4/40) cases with FGFR1 gain like Ishisuka et al. (2002) who

identified 6% (2/32) of primary ESCC amplified in the locus 8p11. NCOA3

amplification was observed in 4.9% (2/41) of ESCC specimens (Fujita et

al., 2003), amplification/gain of NCOA3 gene was observed in 13%

(15/115) of cases, including high-level amplification in 5 cases (4.3%) and

low-level gain in 10 cases (8.7%) (Xu et al, 2008), similarly, we observed

2,5% of CM patients with gain for this gene.

The CM cases identified with genomics deletion and gain

were, in the most, grade III and IV, although no statistically significant,

excepted for the MYC and YES1 genes. Megaesophagus patients have

high variety in esophageal microbiota, which consists mainly of Gram-

positive anaerobic bacteria, correlated with the dilation grade (Pajecki et

al., 2002; Gockel et al., 2006). These data suggest that the level of dilation

and inflammation of megaesophagus could promote genetic damages and

142

could be related to increased risk of tumor development (Chino et al., 2000;

Bektas et al., 2001; Lehman et al., 2001).

Numerical alterations of chromosomes 7, 11 and 17 and

TP53 deletion, by FISH technique, have been reported in megaesophagus

by our research group in previous study (Manoel-Caetano et al., 2004).

However, recent subset analyses did not find mutations CDKN2A (exons 1

and 2) FHIT (exons 5 and 7), suggesting these events are uncommon in

CM (Manoel-Caetano et al., 2009). Similar data were found for p16 and

Fhit protein expression, with no significant decrease in the level of protein

expression in these patients (Bellini et al., 2008).

In conclusion, despite the relevance of genomic

imbalances involving TP63, EGFR, MYC, FGFR1, YES1 and NCOA3 in

the esophageal carcinogenesis, these genes were not found unbalanced

in chagasic megaesophagus. Only PIK3CA and CDKN2A showed deletion

pattern and losses of chromosomes 7 and 9 could be related to the early

process of carcinogenesis in chagasic megaesophagus. These findings

suggest that genomic imbalances are not promising biomarkers for

assessment of ESCC risk in CM.

Acknowledgements

The authors are grateful to Dr. Kenji Miyazaki and

Henrique Oliveira for collected the biopsies; Dr. Patricia Maluf Cury for

supply the paraffin embedded material; Dr. Sebastião Roberto Taboga

and Luiz Roberto Faleiros for help with histological sections. We also

thank Brazilian agency CAPES and UCCC Cancer Center of University of

Colorado for financial and technical support.

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http://www.graphpad.com/instat. April 3

, 2008.

Health A to Z (2008) Internet Reference. Retrieved from

http://www.healthatoz.com/healthatoz/Atoz/common/standard/transform.js

p?requestURI=/healthatoz/Atoz/ency/esophageal_cancer.jsp. December

8th 2008.

INCA, Instituto Nacional do Câncer Internet Reference. Retrieved from

http://www.inca.org.br. December 8

, 2008.

149

Table 1. Mean copy number per cell and standard deviation (SD) for the 12 DNA targets tested in esophageal mucosa, including 9 genes and 3 centromeric sequences.

Groups

Targets (Average±SD)

FHIT

3p14.2

PIK3CA

3q26.32

TP63

3q28

EGFR

7p12

FGFR1

8p12

MYC

8q24

CDKN2A

9p21

YES1

18p11.31

NCOA3

20q12

CEN3

Centromere 3

CEP7

Centromere

CEP9

Centromere

CM 40

S - Layer

1.77±0.08 1.76±0.09 1.77±0.11

1.52±0.12

1.78±0.01 1.76±0.09 1.49±0.08 1.67±0.13 1.65±0.11 1.77±0.08 1.53±0.12

1.53±0.09

B - Layer

1.78±0.08

1.78±0.08 1.78±0.09

1.45±0.15

1.76±0.10 1.75±0.09 1.50±0.07 1.67±0.12 1.64±0.11 1.79±0.07 1.50±0.16

1.53±0.09

Total

1.76±0.08 1.77±0.07 1.77±0.09

1.49±0.13

a,b

1.77±0.08 1.76±0.08 1.50± 0.06

1.67±0.11 1.64±0.10 1.77±0.07 1.52±0.13

1.53±0.07

Range

1.56-1.90 1.56-1.96 1.56-2.06 1.25-1.80 1.56-2.22 1.58-1.77 1.34-1.59 1.37-1.99 1.37-1.80 1.56-1.90 1.26-1.53 1.33-1.65

S - Layer

1.35±0.49 1.70±0.09 1.70±0.09

1.60±0.00 1.63±0.01 1.66±0.01 1.76±0.14 1.70±0.08 1.66±0.11 1.71±0.07 1.67±0.01

1.77±0.15

B - Layer

1.75±0.04 1.78±0.11 1.78±0.11

1.74±0.01 1.70±0.03 1.74±0.02 1.72±0.01 1.68±0.13 1.61±0.01 1.76±0.08 1.79±0.04

1.77±0.04

Total

1.74±0.04 1.74±0.01 1.74±0.01

1.67±0.01 1.67±0.03 1.70±0.01 1.74±0.07

1.69±0.07 1.64±0.06 1.74±0.08 1.73±0.21

1.77±0.10

Range

1.71-1.77 1.64-1.86 1.64-1.86 1.60-1.75 1.62-1.72 1.69-1.70 1.69-1.79 1.58-1.75 1.59-1.68 1.68-1.79 1.76-1.82 1.70-1.84

CM, Chagasic Megaesophagus; NM, health individuals; SD, standard-deviation; S, superficial; B, basal; Statistical Analysis: t-Student (p<0.05);

a,b,

, p=0.008.

149

150

Table 2. Comparison among the chagasic megaesophagus (CM) and

normal mucosa groups based on total counting results for the targets

Targets

CM NM

Statistical

Analysis

Average ± SD

FHIT

1.77±0.08

1.73±0.22

PIK3CA

1.77±0.07 1.74±0.02 ns

TP63

1.77±0.09 1.74±0.01 ns

EGFR

1.49±0.12 1.67±0.01 ns

FGFR1

1.77±0.08 1.67±0.01 ns

CDKN2A

1.50±0.08

1.74±0.07 * p<0.0001

MYC

1.75±0.07 1.70±0.01 ns

YES-1

1.67±0.11

1.69±0.08 ns

NCOA-3

1.64±0.10 1.64±0.06 ns

CEN3 1.77±0.08

1.74±0.08

CEP7 1.51±0.13 1.73±0.02 ns

CEP9 1.53±0.08 1.77±0.10 * p=0.0002

SD, standard-deviation; Statistical Analysis: t-Student; * p<0.05; ns, p>0.05

Table 3. Comparison between gene and centromere targets

Gene-Centromere

CM NM

Average±SD Ratio Average±SD Ratio

FHIT 1.77±0.08 1.00 1.74±0.04

1.00

CEN3 1.77±0.08

1.74±0.08

t-Student

ns ns

EGFR 1.49±0.12 0.98 1.67±0.01 0.96

CEP7 1.51±0.13 1.73±0.02

t-Student

ns ns

CDKN2A 1.51±0.09 1.00 1.74±0.07 0.98

CEP9 1.54±0.10 1.77±0.10

t-Student

ns ns

chagasic megaesophagus, CM; SD, standard-deviation; normal mucosa, NM;

NP, assay was not performed; Statistical Analysis: t-Student (p<0.05); ns,

p>0.05.

151

Table 4. Mean copy number per cell and ranging of each genomic target in distinct grades of

megaesophagus

Targets

Megaesophagus Grade

ANOVA I II III IV

FHIT

1.66 ± 0.331 1.54±0.319 1.77±0.067 1.80±0.080 p = 0.0810

1.0 - 1.86 1.0 - 1.83 1.66 - 1.88 1.68 - 1.92

PIK3CA

1.75 ± 0.009 1.78 ± 0.057 1.77 ±0.090 1.77 ± 0.067 p = 0.9319

1.74 - 1.76 1.68 - 1.84 1.58 - 1.87 1.65 - 1.84

TP63

1.75 ± 0.033 1.78 ± 0.058 1.77 ± 0.997 1.78 ± 0.096 p = 0.8483

1.71 - 1.79 1.68 - 1.83 1.58 - 1.89 1.65 - 1.99

EGFR

1.50 ± 0.130 1.51 ± 0.100 1.49 ± 0.109 1.47 ± 0.140 p = 0.9467

1.36 - 1.68 1.34 - 1.61 1.3 - 1.73 1.25 - 1.8

FGFR1

1.77 ± 0.060 1.81 ± 0.057 1.76 ± 0.081 1.73 ± 0.120 p = 0.3936

1.7 - 1.84 1.72 - 1.87 1.57 - 1.88 1.58 - 2.01

MYC

1.76 ± 0.062 1.81 ± 0.058 1.75 ± 0.083 1.71 ± 0.070 p = 0.0492 *

1.68 - 1.85

a,b,c

1.72 - 1.87

a,b

1.6 - 1.89

a,b,c

1.58 - 1.81

a,c

CDKN2A

1.50±0.057 1.48 ± 0.035 1.49 ± 0.081 1.51 ± 0.087 p = 0.8833

1.42 - 1.55 1.44 - 1.53 1.34 - 1.59 1.34 - 1.69

YES-1

1.71 ± 0.088 1.64 ± 0.053 1.65 ± 0.088 1.57 ± 0.123 p = 0.0463 *

1.61 - 1.83

1.57 - 1.70

a,b

1.53 - 1.9

a,b

1.37 - 1.78

NCOA-3

1.72 ± 0.085 1.64 ± 0.075 1.68 ± 0.091 1.59 ± 0.136 p = 0.0685

1.61 - 1.83 1.53 - 1.73 1.54 - 1.91 1.37 - 1.8

CEN3

1.80 ± 0.069 1.73 ± 0.106 1.77 ± 0.070 1.79 ± 0.079 p = 0.3806

1.69 - 1.86 1.56 - 1.83 1.66 - 1.88 1.68 - 1.92

CEP7

1.59 ± 0.129 1.52 ± 0.115 1.52 ± 0.097 1.50 ± 0.153 p = 0.5688

1.41 - 1.74 1.34 - 1.64 1.35 - 1.68 1.26 - 1.78

CEP9

1.54 ± 0.068 1.53 ± 0.063 1.53 ± 0.094 1.54 ±0.094 p = 0.945

1.45 - 1.62 1.44 - 1.62 1.33 - 1.63 1.34 - 1.69

, p>0.05; * p < 0.05

152

Table 5. Frequencies of loss, balanced and gain of the genes in chagasic

megaesophagus (CM) and health patients (NM).

Target

Loss Balanced Gain

CM NM χ

test CM NM CM NM

FHIT

0.79

1.34

P= 10.672

2478

98.96

144

96.64

0.24

2.04

PIK3CA

898

36.39

28.50

P=0.000

1530

61.99

139

69.50

1.42

2.00

TP63

885

23.29

29.00

P=2.579

2878

75.74

136

68.00

0.97

3.00

EGFR

298

7.59

6.00

P=1.067

3433

88.03

187

93.50

171

4.38

0.50

FGFR1

928

24.38

34.00

P=4.172

2833

74.41

121

60.50

1.21

0.50

MYC

918

24.00

P=4.812

2849

75.00

135

67.50

0.01

1.00

CDKN2A

183

4.69

3.00

P=17,702

3649

93.56

194

97.00

1.74

0.00

YES1

1381

36.34

31.00

P=2.171

2407

63.34

136

68.00

0.32

1.00

NCOA3

1.86

5.80

P= 3,627

2366

97.81

130

94.20

0.33

0.00

N, number of cell counted; Statistical Analysis, χ

test: *, p<0.05,

p>0.05

153

Table 6. Frequencies of nuclei with aneusomies of 3, 7 and 9 centromeres in the

different groups

Centromere

Statistical

Analysis

N %

CEN 3

< 2 copies

683 21.00

54 27.00

P = 0.470

2 copies

2510 78.44

145 72.50

> 2 copies

7 0.56

1 0.50

CEP 7

< 2 copies

1637 51.16

56 28.00

P = 0.00

2 copies

1509 47.16

143 71.50

> 2 copies

54 1.68

1 0.50

CEP9

< 2 copies

1400 43.75

46 23.00

P = 0.00

2 copies

1468 45.87

154 77.00

> 2 copies

332 10.38

0 0.00

CM, Chagasic Megaesophagus; NM, normal mucosa; N number of cells counted;

Statistical Analysis, χ

test: *, p<0.05,

p>0.05.

154

Legend of figures:

Figure 1. Exploratory analysis using three-dimensional plots of the

percentages of loss genes status for individual patients is illustrated.

EGFR, CM30 ~ 30% of loss; FHIT, CM36 ~10% of loss and YES1, CM7

~60% of loss

Figure 2. Exploratory analysis using three-dimensional plots of the

percentages of gain and loss genes status for individual patients is

illustrated. PIK3CA, CM13 and CM19 ~ 40% of loss, CM28 and CM11,

between 5 and 10% of gain; TP63, CM13 ~ 45% of loss, CM6 and CM7

~10% of gain; CDKN2A, CM12 ~12% of loss, CM 11 and CM 16 ~ 6% of

gain; CEP9, CM16, CM34 and CM 36 ~ 60% of loss, CM1 and CM 21 ~

6% of gain.

Figure 3: Exploratory analysis using three-dimensional plots of the

percentages of gain genes status for individual patients is illustrated.

NCOA3, CM36 ~ 4,5% of gain; MYC, CM3, CM6 and CM30 between 5

and 7.5% of gain; FGFR1, CM30 ~10% of gain.

Figure 4. FISH images for the unbalanced genes and centromeric targets

in the megaesophagus specimens.

CM30

EGFR Mean frequency in the CM

Loss = 6.84%

CM36

Mean frequency in the CM

Loss = 0.63%

FHIT

CM7

155

Mean frequency in the CM

Loss = 36.34%

YES1

Figure 1.

Health patients

Gain

Megaesophagus patients

CM13

CM19

CM11

CM28

CM6

CM7

PIK3CA

Mean frequency in the CM

Loss: 23.63%

Gain: 0.92%

TP63

CM13

CM6

CM7

Mean frequency in CM

Loss: 23.29%

Gain: 1.07 %

CM36, CM16, CM34

156

Health patients

Gain

CM12

CDKN2A

CM11

CM16

Mean frequency in the CM

Loss: 4.76%

Gain: 1.79%

Mean frequency in the CM

Loss: 46.5%

Gain: 1.21%

Centromere 9

CM21

CM1

Figure 2.

Megaesophagus patients

Mean frequency in the CM

Gain = 0.21%

NCOA3

CM36

CM30

CM3

CM6

Mean frequency in the CM

Gain = 0.87%

MYC

CM30

CM19

CM3, CM7

157

CM30

Mean frequency in the CM

Gain = 1.21%

FGFR1

Figure 3.

Health patients

Gain

Megaesophagus patients

CDKN2A

CEP9

A. Loss CDKN2A and centromere 9

EGFR

CEP7

B. Loss of EGFR and centromere 7

158

FHIT

CEN3

C. Loss FHIT and gain of centromere 3

TP63

PIK3CA

D. Gain of TP63 and loss of PIK3CA

Figure 4.

Discussão

160

III. DISCUSSÃO

No presente estudo, a avaliação de alterações na expressão de

proteínas reguladoras do ciclo celular, como p53, p16 e Fhit, e dos níveis de

Ki67 e CPP32, em lesões benignas do esôfago com potencial pré-canceroso

mostrou expressão aumentada da proteína p53 com um aumento crescente

de casos p53 positivos de acordo com a severidade da lesão (megaesôfago,

esofagite e carcinoma). Assim indicando participação desta nas etapas

iniciais da carcinogênese do esôfago. Entretanto, para as proteínas p16 e

Fhit não foram observadas alterações importantes em suas expressões entre

os diferentes grupos. Da mesma forma, também não se observou alterações

significantes nos níveis de proliferação celular e apoptose nos grupos de

lesões benignas, mas apenas uma freqüência maior de casos CPP32

positivos nos carcinomas. Também não foi evidenciada a ocorrência de

concordância na expressão alterada das proteínas p53, p16 e Fhit nos

grupos avaliados, nem associação da expressão alterada da p53 com a

expressão de CPP32 e antígeno Ki67. Nestas avaliações não foi possível

estabelecer associações entre essas proteínas e parâmetros como idade,

sexo e hábitos tabagista e etilista. O estudo citogenético molecular em

pacientes com megaesôfago chagásico, pela técnica FISH, para avaliação

de perdas e ganhos de genes freqüentemente em desequilíbrio em

carcinoma esofágico (FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A,

YES1, NCOA3, e centrômeros 3, 7 e 9), mostrou que o número de cópias

dos genes nas camadas superficial e basal da mucosa esofágica eram

estatisticamente diferentes apenas para EGFR, com menor número de

cópias na camada basal. Neste estudo, observaram-se diferenças

significantes no grupo de megaesôfago como perda de CDKN2A, PIK3CA e

161

dos cromossomos 7 e 9, associados principalmente aos graus III e IV do

megaesôfago, ainda que diferença significativa não tenha sido observada

entre o número de cópias para a maioria dos alvos genômicos estudados e

os graus de megaesôfago, exceto para os genes MYC e YES1.

O gene TP53 desempenha papel importante na regulação do

ciclo celular, mas quando alterado é um dos principais genes envolvidos em

cerca de 50% das neoplasias humanas (ASKER; WIMAN; SELIVANOVA

1999). No presente estudo pode ser constatado um aumento crescente de

casos p53 positivos a partir do grupo de chagásicos sem megaesôfago

(7,7%), com megaesôfago (26,1%), esofagite crônica (52,2%) e carcinoma

esofágico (100%). Fuji et al. (2000), avaliando a natureza pré-maligna de

megaesôfago, analisaram a expressão de p53, por imuno-histoquímica, em

pacientes com carcinoma esofágico que apresentavam megaesôfago e as

regiões de epitélio não maligno com esofagite e/ ou displasia. Os autores

verificaram expressão aumentada de p53 nos tumores, mas não no epitélio

não-neoplásico, como na esofagite e displasia. Contudo, no epitélio de

megaesôfago, observou além de alterações histológicas em vários graus, a

expressão aumentada de p53. Da mesma forma, Fagundes et al. (2005)

também detectaram a imunoexpressão da proteína p53 de forma crescente

em indivíduos com mucosa normal (12%), esofagite moderada (22%),

esofagite severa (33%), displasia (36%) e carcinoma (100%), com

predomínio da expressão em fumantes e etilistas, sugerindo que pacientes

que apresentam esofagite crônica, com hiperexpressão de p53 têm risco

elevado para desenvolver câncer de esôfago. Entretanto, mais recentemente,

Gockel et al. (2006), não encontraram positividade de p53 em nenhum dos

15 pacientes com megaesôfago estudados. ,

Os pacientes com megaesôfago chagásico frequentemente

apresentam esofagite crônica, devido à retenção do bolo alimentar (estase

esofágica) no esôfago dilatado, aumentando o risco de proliferação

162

bacteriana e danos à mucosa (CHINO et al., 2000; FUJI et al., 2000;

PAJECKI et al., 2002). Entre essas bactérias estão caracterizadas aquelas

pertencentes a microbiota normal da boca e orofarínge, que são

responsáveis por criar um ambiente favorável a microrganismos anaeróbios,

capazes de reduzir nitratos em nitritos, gerando carcinógenos, que poderão

desencadear o processo de carcinogênese esofágica (PAJECKI et al., 2002).

No estudo realizado 69% dos pacientes com megaesôfago também

apresentavam esofagite crônica. Portanto, os casos com expressão alterada

de p53, (6/23) em megaesôfago chagásico e (12/23) em esofagite crônica,

podem ter ocorrido em resposta ao processo inflamatório e/ou a injúrias no

DNA, enquanto que em carcinoma de células escamosas de esôfago a

expressão acentuada da proteína nos 11 casos estudados deve estar

relacionada com mutações no gene TP53. A freqüência maior de casos p53

positivos no grupo de esofagite crônica reforça a participação do processo

inflamatório na expressão dessa proteína. Murphy et al. (2005) observaram

um aumento moderado no risco de desenvolvimento de carcinoma de

esôfago, mas não de adenocarcinoma em pacientes com esofagite,

confirmando que a lesão precursora de adenocarcinoma seja o esôfago de

Barret e que esofagite possa ser uma lesão precursora de carcinoma de

células escamosas do esôfago.

A perda ou redução da expressão da proteína p16 pode estar

associada com mutações de ponto, deleções, perda de heterozigose ou

processos que inativam a expressão gênica, como a hipermetilação da

região promotora (HU et al., 2004; KRÜGER et al., 2005; SANTIN et al.,

2005). Em estudo de carcinoma esofágico familial e esporádico, no Irã, foi

observado que 64,3% dos membros da família estudada apresentavam o

gene CDKN2A (MTS-1; P16) com hipermetilação na região promotora. Nos

casos esporádicos, essa freqüência foi maior nas amostras de tecido tumoral,

ou seja, 73,3%, mas nas amostras de soro e sangue, dos mesmos pacientes,

163

a hipermetilação foi de 26,6% e 43,3%, respectivamente (ABBASZADEGAN

et al., 2005). Assim, revelando uma forte associação com o processo de

hipermetilação do gene apenas no tecido tumoral, e indicando que o gene

CDKN2A mutante pode ser utilizado como biomarcador na identificação

precoce de carcinoma esofágico em populações de alto risco e em casos de

história familiar. No presente estudo, entretanto, a análise da expressão da

proteína p16 em pacientes com lesões esofágicas benignas e em carcinoma

esofágico se mostrou significantemente alterada apenas no grupo de

esofagite crônica, visto que foram encontradas perdas na expressão desta

proteína em freqüência elevada também no grupo de mucosa normal.

Entretanto, o estudo citogenético molecular ora realizado mostrou média

menor de número de cópias de CDKN2A e do centrômero 9 no grupo de

megaesôfago, indicando perda desses alvos genômicos nesta lesão.

A expressão anormal da proteína Fhit foi descrita como um

evento freqüente no estágio inicial do desenvolvimento do carcinoma de

esôfago, devido à perda completa ou redução da expressão, aumentando de

acordo com a progressão da severidade das alterações histopatológicas da

mucosa, ou seja, de displasia (média, moderada e severa) para carcinoma in

situ e carcinoma invasivo (MORI et al., 2000; KITAMURA et al., 2001). No

presente estudo, essa expressão anormal foi observada principalmente no

grupo de esofagite, mas a análise estatística empregada não mostrou

diferença entre os grupos estudados. A análise citogenética de perdas e

ganhos destacou um caso de megaesôfago com deleção de FHIT, embora

não tenha sido observado diferenças significantes na média de número de

cópias desse gene e centrômero 3 entre os grupos de megaesôfago e

mucosa normal.

As alterações necessárias para o surgimento de câncer

envolvem vários genes que controlam o crescimento, a proliferação celular e

a homeostase do tecido por meio de apoptose (KOCH et al., 1994). Um dos

164

marcadores de proliferação celular é o antígeno nuclear Ki67 expresso nas

fases G1, S e M do ciclo celular. A expressão aumentada de Ki67 nuclear

tem sido associada à progressão de uma variedade de tumores (LUTTGES

et al., 2000 apud LEBE et al., 2004). Nos grupos de mucosa normal,

chagásicos sem megaesôfago, esofagite crônica e carcinoma 100% dos

casos foram classificados como Ki67 positivos, pois apresentaram mais de

10% de positividade para marcação do antígeno. Enquanto que no grupo de

megaesôfago 91,3% casos foram positivos para Ki67. Porém, a análise

estatística para os casos positivos, não indicou diferenças estatisticamente

significativas entre os grupos.

A avaliação de apoptose utilizando a marcação

citoplasmática da proteína caspase-3 (CPP32) em lesões primárias de ESCC

tem mostrado que 60% dos casos são positivos (HSIA et al., 2003). No

presente estudo, a coloração positiva para CPP32 foi observada em poucos

casos de mucosa normal 20% (2/10), sendo interpretada como nível normal

de apoptose, e em frequências similares entre os grupos CD, CM e CE

respectivamente, 30,8% (4/13), 30,4% (7/23) e 34,8% (8/23), mas

aumentada em ESCC, 55,5% (5/9), corroborando alguns estudos da

literatura sugerindo que este evento esteja relacionado com melhor

prognóstico.

A proteína p53 desempenha uma importante função na

indução de apoptose em resposta a danos no DNA (LEVINE, 1997; STEELE

et al., 1998). As células bloqueadas na fase G

permitem o reparo ou

induzem a apoptose se houver falhas nesse mecanismo, prevenindo assim,

a transmissão de lesões no DNA às células filhas (LANE, 1992, 1994). Nesse

contexto, a inativação da proteína p53 pode promover a desregulação do

ciclo celular e a inibição de apoptose, fornecendo forte vantagem no

crescimento de células tumorais, como evidenciado no presente estudo, em

que observamos alteração na expressão da proteína p53 nas lesões

165

benignas e carcinoma. Entretanto, não foram estabelecidas associações

entre a expressão alterada da proteína p53 com o antígeno Ki67 ou a

proteína CPP32.

Além de fatores ambientais e patogênicos, alterações

genéticas, como aneupolidias, deleções alélicas, ativação de oncogenes e

inativação de genes supressores tumorais estão associadas a carcinogênese

esofágica (KUWANO et al., 2005; DAIGO;NAKAMURA, 2008; D’AMICO,

2008). Estudos de citogenética molecular, como mCGH, aCGH, SKY e FISH

indicam algumas regiões cromossômicas como alvo de alteração durante o

desenvolvimento do câncer. Entre elas podemos citar as regiões 1, 2q, 3q,

5p, 6p, 7, 8q, 9q, 11q, 12p, 14q, 15q, 16, 17, 18p, 19q, 20q, 22q e X, para

ganhos, com amplificações em 1q32, 2p16-22, 3q25-28, 5p13-15.3, 7p12-22,

7q21-22, 8q23-24.2, 9q34, 10q21, 11p11.2, 11q13, 13q32, 14q13-14, 14q21,

14q31-32, 15q22-26, 17p11.2, 18p11.2-11.3, 20p11.2; e as seguintes regiões

envolvidas em perdas 3p, 4, 5q, 6q, 7q, 8p, 9, 10p, 12p, 13, 14p, 15p, 18,

19p, 20, 22, Xp and Y (SHIMADA et al., 1992; PACK et al., 1999;

SHINOMIYA et al., 1999; MAYAMA et al., 2000; PIMKHAOKHAM et al. 2000;

YEN et al., 2001, 2003; KAMITANI et al., 2002; HSIA et al., 2003; KWONG et

al., 2004; QIN et al., 2004, 2005ab, 2008; SU et al., 2006; SUGIMOTO et al.,

2007; YANG et al. 2008). Além dessas regiões, os genes PIK3CA (3q26),

TP63 (3p28), EGFR (7p11), YES1 (18p11.31) e NCOA3 (20q12) foram

encontrados amplificados em freqüências elevadas, enquanto sào

encontradas deleções nos genes supressores de tumor CDKN2A (9p21),

MTAP (9p21) and TP53 (17p13.1) em ESCC (SHIMADA et al., 1992;

PIMKHAOKHAM et al., 2000; NAKAKUKI et al., 2002; YEN et al., 2003; XU et

al., 2007; CARNEIRO et al., 2008).

No presente estudo, a análise de perdas e ganhos, no grupo

de megaesôfago, de genes freqüentemente em desequilíbrio em ESCC,

mostrou que o número de cópias do gene EGFR era menor na camada basal

166

da mucosa esofágica que na superficial, e um caso de megaesôfago foi

destacado para perda desse gene. Outras alterações também foram

evidenciadas como uma média menor de número de cópias de CDKN2A e

centrômero 9, padrão de deleção do gene PIK3CA e perda dos

cromossomos 7 e 9. Apesar dos demais genes investigados não

apresentarem alterações significantes, podemos destacar alguns casos de

megaesôfago com ganho em PIK3CA, TP63, CDKN2A, FGFR1, MYC,

NCOA3 e centrômero 9, ou deleção em FHIT, PIK3CA, EGFR, CDKN2A,

TP63, YES1 e centrômero 9, sempre associados aos graus III e IV de

megaesôfago. Sabe-se que pacientes com megaesôfago apresentam grande

variedade na microbiota esofágica, correlacionadas ao grau de dilatação

esofágica e processo inflamatório (PAJECKI et al., 2002; GOCKEL et al.,

2006).Portanto, a maior incidência de câncer nos pacientes portadores de

megaesôfago pode estar relacionada à ocorrência dessas alterações

inflamatórias da mucosa, desencadeando alterações genéticas (PINOTTI et

al., 1996), como evidenciado neste estudo. Apesar de, até a conclusão deste

estudo, as informações dos prontuários médicos dos pacientes com

megaesôfago não revelaram nenhum caso que tenha evoluído para

carcinoma. Dessa forma, esses achados citogenéticos em megaesôfago,

assim como a expressão alterada da proteína p53 tanto em megaesôfago

como em esofagite crônica são relevantes, pois evidenciam a ocorrência de

alterações genéticas em lesões benignas precursoras como eventos

precoces que devem participar das etapas iniciais da carcinogênese do

esôfago.

Conclusões

168

IV. CONCLUSÕES

No presente estudo, considerando-se as amostras

avaliadas e as técnicas utilizadas, obtemos as seguintes conclusões:

a) A expressão da proteína p53 é aumentada nas lesões benignas

estudadas, obedecendo a uma progressão em relação à severidade

histopatológica da lesão, a partir do megaesôfago, esofagite crônica e

carcinoma indicando sua participação desde os estágios iniciais até

tardios da carcinogênese esofágica;

b) A expressão das proteínas p16 e Fhit não apresentam alterações

significantes nos grupos de lesões benignas e carcinomas avaliados, não

havendo indicação de que desempenhem um papel importante na

progressão das lesões estudadas;

c) Não há concordância no padrão de expressão das proteínas p53,

p16 e Fhit nas lesões benignas e carcinoma, entretanto expressão

alterada das proteínas p53 e p16, p53 e Fhit, p16 e Fhit podem estar

associadas nestas lesões durante o processo de controle do ciclo celular;

d) Não há evidências de alterações nos níveis de expressão do

antígeno Ki67 e da proteína caspase-3, indicando ausência alterações

significantes nas taxas de proliferação celular e apoptose nas lesões

estudadas;

e) Da mesma forma, também não há evidências de associações

entre a expressão alterada das proteínas p53, p16, Fhit, CPP32 e

antígeno Ki67 com parâmetros como sexo, idade, hábitos tabagista e

etilista nas lesões benignas e carcinomas do esôfago avaliados;

169

f) Apesar da relevância de desequilíbrios gênicos em TP63, EGFR,

MYC, FGFR1, YES1 e NCOA3 em carcinoma de esôfago, estes genes

não apresentam padrões de perda ou ganho em megaesôfago chagásico.

Assim não devem encontrar-se desbalanceados neste tipo de lesão do

esôfago;

g) Apenas os genes PIK3CA e CDKN2A apresentam padrão de

deleção em megaesôfago chagásico, juntamente com perda dos

cromossomos 7 e 9, sendo consideradas como alterações iniciais do

processo carcinogênico do esôfago.

h) A observação de que regiões genômicas com desequilíbrios na

carcinogênese esofágica não são significativamente afetadas em

megaesôfago chagásico sugere que não será possível utilizar tais

características como marcadores eficazes de risco para desenvolvimento

de carcinoma em megaesôfago.

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Apêndices

193

Apêndice 1: Termo de Consentimento Livre e Esclarecido (Sanduíche)

TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO

(Conselho Nacional de Saúde, Resolução 196/96)

Eu, Marilanda Ferreira Bellini, RG: 30.212.578-4 SSP/SP, aluna de Doutorado do Programa de Pós-Graduação

em Genética do Instituto de Biociências, Letras e Ciências Exatas – IBILCE / UNESP – Campus São José do Rio

Preto, convido ____________________________________________________________

RG:_________________________, nascido em ____/_____/______ e domiciliado à __________________

_______________________________________________, município de _______________________, usuário (ou

responsável legal pelo usuário_____________________________________________), a participar, como

voluntário, no projeto de pesquisa “Investigação de perdas e ganhos genômicos em pacientes com

Megaesôfago Chagásico, pela técnica FISH (Hibridização in situ fluorescente) e desenvolvimento de

sondas de DNA como marcadores de tumorigênese”, e em extensões deste, que pretende estudar alterações

na expressão do material genético, pelas técnicas CGH e FISH em pessoas que apresentam um tipo de doença

que provoca o aumento do esôfago, causado pela doença de Chagas, por isso sendo conhecida como

“megaesôfago chagásico”. Declaro que o voluntário foi satisfatoriamente esclarecido que: A) o estudo será

realizado utilizando pequenos pedaços (biópsias) de local do esôfago com alteração (megaesôfago chagásico),

que serão colhidos durante o processo de rotina para a realização de exame de endoscopia, solicitado pelo

médico do voluntário, no Serviço de Endoscopia do Hospital de Base de São José do Rio Preto; B) não haverá

nenhum risco adicional para sua saúde, pois a colheita do material será realizada pelos profissionais qualificados

e que precisa fazer a endoscopia independente deste projeto; C) o voluntário poderá consultar as pesquisadoras

responsáveis em qualquer época para esclarecimento de qualquer dúvida; D) está livre para, a qualquer momento,

deixar de participar da pesquisa e que não precisa apresentar justificativas para isso; E) fornecerá informações

através de um questionário realizado durante uma entrevista antes do seu exame e também autoriza a coleta de

dados em seu prontuário médico; F) todas as informações fornecidas pelo voluntário e os resultados obtidos serão

mantidos em sigilo e que estes últimos serão utilizados somente para divulgação em reuniões e revistas científicas,

sem sua identificação, pois o material coletado será identificado por código; G) será informado pelo seu médico

dos resultados obtidos, caso possam auxiliar seu tratamento ou prevenir a progressão da lesão que apresenta no

esôfago, independentemente do fato destes poderem mudar seu consentimento em participar da pesquisa; H) que

a biópsia só será utilizada após o procedimento cotidiano de emblocamento em parafina pelo Setor de Patologia

do Hospital de Base de São José do Rio Preto, responsável pelo seu armazenamento; l) este estudo consiste

numa pesquisa científica importante, pois seus resultados poderão trazer informações básicas, evidenciando a

expressão diferencial de proteínas, envolvidas nos estágios iniciais que ocorrem nestes tipos de lesões do

esôfago (esofagite crônica e megaesôfago chagásico), e assim, se há um risco para o aparecimento de um câncer

do esôfago, portanto podendo auxiliar futuramente no diagnóstico precoce, evolução e tratamento da doença.

Assim, o usuário consente em participar do projeto de pesquisa em questão.

São José do Rio Preto, _____ de _____________ de 200____

____________________________ ___________________________

Usuário/ responsável legal Pesquisador responsável

OBS: Este termo apresenta duas vias, uma destinada ao usuário ou seu representante legal e a outra ao

pesquisador.

Projeto:

Investigação de perdas e ganhos genômicos em pacientes com Megaesôfago Chagásico, pela técnica

FISH (Hibridização in situ fluorescente) e desenvolvimento de sondas de DNA como marcadores de tumorigênese

Pesquisadores: Doutoranda Marilanda Ferreira Bellini

Profa. Dra. Ana Elizabete Silva

Instituição: UNESP – Universidade Estadual Paulista/IBILCE - Instituto de Biociências, Letras e Ciências Exatas

Endereço: R.Cristóvão Colombo, 2265 - Jardim Nazareth – CEP 15054-000 São José do Rio Preto-SP

Fone: 17 3221-2384

E-mail: mbellini@webmail.ibilce.unesp.br

194

Apêndice 2: Questionário

Questionário do Projeto: “Investigação de perdas e ganhos genômicos em pacientes com

megaesôfago chagásico, pela técnica de FISH (Hibridização in situ fluorescente) e

desenvolvimento de sondas de DNA como marcadores de tumorigênese”

Responsáveis: M.sc. Marilanda Ferreira Bellini, Profa. Dra. Ana Elizabete Silva (Departamento

de Biologia – IBILCE/UNESP – São José do Rio Preto – SP) e Dr. Kenji Miyazaki (Setor de

Endoscopia – Hospital de Base, São José do Rio Preto)

IDENTIFICAÇÃO:

Nome:______________________________________________Prontuário:_______________

Data de nascimento: ____/____/_______ Sexo: ( )M ( ) F

Endereço Atual: _____________________________________Fone:____________________

Cidade:____________________________________________Estado:___________________

Procedência: ________________________________________________________________

Profissão Atual:_____________________________________Tempo de atuação:__________

Profissão Anterior:___________________________________Tempo de atuação:__________

DADOS PESSOAIS E FAMILIAIS

Consumo de bebida alcoólica: ( )Sim ( )Não

Há quantos anos? _______________ Tipo de bebida: ___________ dose:________________

Consumo de cigarro: ( )Sim ( )Não

Há quantos anos? _______________ Quantidade (unidade/dia): ________________

Doenças Anteriores no Aparelho Digestório:

( ) esôfago ( ) estômago Outras:___________ Tipo:________________

Tratamentos anteriores para megaesôfago: ( ) Sim ( ) Não Tipo:____________

Cirurgias anteriores: ( ) Sim ( ) Não Tipo:____________

Há história de tumor de esôfago ou outras doenças na família: ( ) Sim ( )Não

Tipo:_______________________________ Grau de Parentesco:_______________________

Data: ____/____/________ Responsável pelo Procedimento:______________________

195

Apêndice 3: Caracterização das amostras de mucosa esofágica normal e

Carcinoma de células escamosa de esôfago

Caracterização de estilo de vida e clinico-patológica nos grupos de pacientes com mucosa

esofágica normal (NM) e com carcinoma de células escamosas de esôfago (ESCC),

acompanhados por 4 anos.

Código

Idade

(anos)

Sexo Etilismo

Tabagismo

Dados Clínicos

Mucosa E

sofá

gica N

ormal

NM1

26 F Não Não Nada Consta

NM2

67 F Não Não

Nada Consta

NM3

33 M Sim Sim Nada Consta

NM4

29 F Não Não Nada Consta

NM5

51 M Sim Sim Nada Consta

NM6

34 M Sim Não Nada Consta

NM7

34 F Sim Não Nada Consta

NM8

66 F Não Não Nada Consta

54 F Não Não Nada Consta

NM10

30 F Não Sim Nada Consta

Carcinoma de células escamosas de esôfago

ESCC1

65 M Sim Sim

Cardiopatia, megacólon,

megaesôfago

ESCC2

56 M Sim Sim Sorologia positiva (Chagas)

ESCC3

69 F Sim Não

Megacólon, megaesôfago,

cardiopatia

ESCC4

42 M Não Sim Tumor intestinal operado

ESCC5

48 M Sim Sim Nada Consta

ESCC6

64 M Sim Sim Nada Consta

ESCC7

47 M Sim Sim Nada Consta

ESCC8

55 M Sim Sim Nada Consta

ESCC9

64 M Não Sim Nada Consta

ESCC10

48 M Sim Sim Nada Consta

ESCC11

63 M Não Não gastrite enantematosa

F, Feminino; M, Masculino

196

Apêndice 4: Caracterização das amostras de pacientes com esofagite

crônica

Caracterização de estilo de vida e clinico-patológica nos grupos de pacientes com

esofagite crônica (CE), acompanhados por 4 anos.

Código

Idade

(anos)

Sexo Etilismo Tabagismo Dados Clínicos

40 F Não Não Nada Consta

CE2

43 F Não Sim Nada Consta

CE3

50 F Não Sim Cardiopatias, obesidade, óbito

CE4

52 M Sim Sim Hérnia Hiato, gastrite leve

CE5

40 M Sim Não Malária

CE6

42 F Não Não Nada Consta

CE7

41 F Não Não Nada Consta

CE8

49 F Não Não Nada Consta

CE9

57 F Não Não Nada Consta

CE10

56 M Não Não Nada Consta

CE11

65 M Não Não Gastrite enantematosa

CE12

51 M Não Não

Gastrite enantematosa, H.

pylori+

CE13

55 M Não Não Nada Consta

CE14

67 M Não Não Cardiopatia

CE15

54 M Não Não Gastrite enantematosa

CE16

46 F Não Não Gastrite discreta

CE17

40 F Não Não Nada Consta

CE18

47 M Não Não

Gastrite leve; úlcera duodenal

recorrente

CE19

42 M Não Não Nada Consta

CE20

70 F Não Não Nada Consta

CE21

45 F Não Sim Carcinoma de cólo uterino

CE22

41 F Não Não Cálculo renal

CE23

68 M Não Sim Câncer de próstata

F, Feminino; M, Masculino

197

Apêndice 5: Caracterização das amostras de pacientes chagásicos sem

megaesôfago.

Caracterização de estilo de vida e clinico-patológica no grupo de paciente chagásico sem

megaesôfago (CD), acompanhados por 4 anos.

Código

Idade

(anos)

Sexo Etilismo Tabagismo Dados Clínicos

CD1

63 M Sim Não

Esôfago de Barret; megacólon

operado

CD2

62 M Sim Não Megacólon; esofagite crônica leve

62 F Não Não Cardiopatia; esofagite crônica leve

72 F Não Sim Esofagite crônica leve

CD5

76 F Não Não Esofagite crônica leve

49 M Não Sim Esofagite crônica moderada

45 F Não Não Megacólon; esofagite crônica leve

CD8

75 F Sim Sim

Esofagite crônica moderada,

gastrite leve e cardiopatia

59 M Sim Sim Esofagite leve

CD10

79 F Não Não Nada Consta

CD11

50 F Não Sim Nada Consta

CD12

59 F Não Não

Megacólon, esofagite crônica

moderada

CD13

57 M Não Não

Megacólon, esofagite de refluxo,

gastrite erosiva

F, Feminino; M, Masculino

198

Apêndice 6: Caracterização das amostras de pacientes chagásico com

megaesôfago

Caracterização de estilo de vida e clinico-patológica no grupo de paciente chagásico com megaesôfago

(CM), acompanhados por 2 a 4 anos.

Código

Original

Código

FISH

Grau

Idade

(anos)

Sexo Etilismo Tabagismo Dados Clínicos

CM1

II 59 M Não Não

Esofagite crônica

moderada

CM2

II 58 F Sim Não

Megacólon operado,

esofagite crônica leve

CM3

CM2

IV 64 F Não Sim Esofagite crônica leve

CM4 CM3

IV 72 M Não Não

Megacólon/ esofagite

crônica severa

CM5 CM4

I 83 M Sim Sim

Megacólon; esofagite

crônica severa; óbito

CM6

CM5

III 60 M Sim Não Esofagite crônica leve

CM7 CM6

III 81 F Sim Sim

Megacólon operado;

cardiopatia; esofagite

crônica leve

CM8

CM7

IV 55 F Não Sim Nada Consta

CM9

CM8

IV 61 M Não Não Cardiopatia

CM10

CM9

IV 78 M Não Sim Esofagite crônica severa

CM11

CM10

III 58 F Não Não Gastrite crônica

CM12 CM11

III 63 M Sim Sim

Gastrite severa e

cardiopatia

CM13

CM12

IV 42 M Não Não Gastrite leve

CM14 CM13

III 69 F Sim Sim

Megaesôfago recidivado;

cardiopatia; esofagite

crônica severa

CM15 CM14

III 70 F Não Sim

Esofagite crônica

moderada

CM16 CM15

IV 68 F Não Não

Esofagite crônica

moderada, gastrite

crônica severa,

cardiopatia

CM17

CM16

IV 83 M Não Sim Esofagite crônica severa

CM18 CM17

III 61 M Não Sim

H pylori +, gastrite

crônica moderada

CM19 CM18

III 64 M Sim Sim

Esofagite crônica

moderada, gastrite

199

crônica discreta

CM20

III 57 M Sim Sim

Gastrite crônica, H

pylori+

CM21 CM20

IV 59 F Não Sim

Cardiopatia, esofagite

crônica moderada

CM22

III 66 F Não Não

Esofagite crônica

moderada, gastrite

enantemática e

cardiopatia

CM23 CM21

II 53 F Não Sim

Esofagite crônica

moderada

CM22

IV 63 F Não

Não

Megaesôfago

Recidivado

CM23

I 57 F Não

Sim

Gastrite, Extração de

vesícula biliar, esofagite

crônica moderada,

cardiopatia

CM24

III 69 M S Sim

Cardiopatia,

Hipertensão, Metaplasia

Intestinal, Gastrite

Crônica Enamtematosa

CM25

III 58 F Não

Não

Constipação Intestinal,

Megacólon, Megabulbo

Duodenal, Gastrite

Enantematosa

CM26

III 40 M Não

Sim

Gastrite Crônica

Discreta, H pylori +,

Esofagite Crônica Leve,

Megacólon e

Megaesôfago

Recidivados

CM27

I 59 F Não

Não

Gastrite enamtematosa,

duodenite erosiva,

metaplasia intestinal

CM28

II 59 F Não

Não

Gastrite, Esofagite

Erosiva

CM29

II 64 M Não

Não

Megaesôfago

Recidivado, Megacólon,

Esofagite Crônica

Moderada, Refluxo

Gastro-esofágico

CM30

III 70 F Não

Sim

Constipação Intestinal,

Megacólon,

Gastrectomia Parcial,

Colectomia subtotal,

gastrite Crônica,

Metaplasia Intestinal,

Esofagite Crônica

Moderada

CM31

III 40 M Não

Não

Constipação instestinal,

esofagite crônica

moderada, gastrite

enantematosa

200

CM32

II 48 F Não

Não

Constipação Instestinal,

Refluxo Gástrico-

esofágico, esofagite

crônica moderada,

pangastrite

enamtematosa

CM33

I 64 F Não

Não

Gastrite Erosiva,

Bulboduodenite

CM34

III 53 M Sim

Não

Esofagite Crônica

Moderada, Refluxo

Gastro-esofágico

CM35

III 57 M Sim

Sim

Nada Consta

CM36

II 66 M Sim

Sim

Constipação Intestinal,

Megacólon, Cardiopatia,

Gastrite, Hérnia de Hiato

CM37

III M Não Não

Nada Consta

CM39

II 76 F Não

Não

Cardiopatia

CM19

III 65 M Sim Sim

Gastrite Crônica, H

pylori+, Metaplasia

Intestinal, Cardiopatia

CM40

II 62 M Não

Sim

Ulcera, Gastrite,

Esofagite Crônica

Erosiva, Constipação

Intestinal

CM1

I 69 F N N

Hérnia de Hiato, Gastrite

Enantemosa, Ulcera,

Esofagite, H pylori +,

Cardiopatia

CM38

IV 56 F Não

Sim Constipação Intestinal

F, Feminino; M, Masculino

201

Apêndice 7: Chromosomal Mapping for the newly developed probes.

A1) FHIT (red) and

FGFR1 (green);

B1) TP63 (red) and

PIK3CA (green);

C1) pα3.5 (green);

D1) YES1 (red) and

NCOA3 (green);

Inverted DAPI images.

A2) FHIT – 3p14.2 and

FGFR1 – 8p12;

B2) TP63 – 3q28 and

PIK3CA – 3q26.32;

C2) pα3.5 – centromere

of chromosome 3;

D2) YES1 – 18p11.31

and NCOA3 – 20q12.

Anexos

203

ANEXOS

Anexo 01. Parecer Consubstanciado do Comitê de Ética em Pesquisa

Institucional (Projeto de Doutorado Original).

204

Anexo 02. Parecer Consubstanciado do Comitê de Ética em Pesquisa

Institucional (Projeto de Estágio de Doutoramento no Exterior).

205

Anexo 03. Metodologias para o desenvolvimento de sondas utilizadas na

técnica de Hibridação In Situ Fluorescente (FISH)

Cytogenetics Core

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206

VI. DNA Cloning - Protocol 1

Establishing cultures, isolating single colonies, and verifying sequences

I. Reagents

1. Agar plates. LB-agar plates must contain the appropriate antibiotic in the correct concentration,

based on the library design.

Antibiotics

a. Ampicillin (Sigma, Cat. # A2804).

Stock solution (1:1000) = 60 mg/ml

Dissolve 1.5 g ampicillin sodium salt in 25 ml of distilled water.

Filter sterilize, dispense in 1ml aliquots in screw cap tubes. Store at –20°C.

Usual final concentration in culture media = 40 ug/ml.

b. Kanamycin sulfate (Gibco, Cat. # 11815-016).

Stock = 25 mg/ml

Dissolve 250 mg in 10 ml of distilled water.

Filter sterilize, dispense in 1 ml aliquots in screw cap tubes. Store at –20°C.

Usual final concentration in culture media = 25 ug/ml.

c. Tetracycline hydrochloride (Gibco, Cat. # 11860-012).

Stock = 12.5 mg/ml

Dissolve 125 mg in 10 ml ethanol/water (50% v/v).

Filter sterilize, dispense in 1ml aliquots in screw cap tubes. Store at -20°C in the dark (wrap tubes

in aluminum foil).

Usual final concentration in culture media = 12.5 ug/ml.

d. Penicillin/Streptomycin (SIGMA, Cat. # P0906). Penicillin G potassium = 5,000,000 Units

(3.34 g/5millions units = 0.668 g/million units). Streptomycin sulfate = 5 g

Stock = 10,000 U of pen & strep

Dissolve the content of the vial in 500 ml distilled water.

Filter sterilize, dispense in 2 ml aliquots in screw cap tubes. Store at –20°C.

Usual final concentration is 1/10 dilution.

e. Chloramphenicol (Sigma, Cat # C-0378)

Stock = 34 mg/ml

Dissolve 340 mg in 10 mL of absolute EtOH. Mix solution completely and aliquot into

microcentrifuge tubes. Store at –20°C.

2. LB-broth – freshly prepared and supplemented with the appropriate antibiotic (see above for

information.).

3. 1% Triton X

4. 20mM Tris HCl 7.5

5. 2mM EDTA

6. BAC clones (recommend

http://www.ensembl.org

http://genome.ucsc.edu

for clone selection)

7. Primers (recommend Integrated DNA Technologies, Coralville, IA 800-328-2661 www.idtdna.com)

specific to BACs

8. GoTaq Green Master Mix (Promega, Cat. # M7122)

9. Sterile 50% Glycerol – Combine equal amounts Sterile water and Glycerol, autoclave

10. 1xPBS

11. Cold 100% ethanol and 70% ethanol.

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207

12. 3 M NaAcetate pH 5.4

13. TE Buffer (10mM Tris-HCl, 0.5mM EDTA, pH 9.0)

14. Ethidium Bromide Stock Solution 10mg/ml

15. Agarose ultraPure (Life Technologies Cat. # 15510-027)

16. 1 x Tris-Acetate Buffer (TAE) – Dilute 1 part 50x stock in 49 parts Milli-Q water

Prepare 50x stock buffer as follows:

242.0 g Tris base

57.1 mL Glacial Acetic Acid

100 mL 0.5M EDTA pH 8.0

Sufficient quantity Milli-Q water for 1L solution]

17. 6x loading dye

18. Markers for size of DNA bands: (a) 100bp High Mass Ladder; (b) 3 ul of Gel Pilot 100 bp ladder

(Qiagen # 239035) or (c) 0.5 ul of 100 bp DNA ladder (Invitrogen # 15628-019)

II. Procedure

A. Selecting clones

1. Select the proper clone(s) for the area of interest using

http://www.ensembl.org

http://genome.ucsc.edu

2. Print a hard copy of the Contig View for the Probe Development binder which includes:

a. Chromosomal map

b. Position of gene of interest

c. Collection of clones available

d. Highlight selected clone

3. Acquire the clone(s) of interest from one of the following:

a. http://

www.CHORI.org

b. http://

www.invitrogen.com

4. Print a hard copy of the documentation for the clone purchase

B. Establishing cultures from stab or stocks (Following steps should be done using sterile technique.)

1. Prepare LB broth minus antibiotic. Autoclave with Liquid240 setting. Aliquot 3 ml of sterile LB

broth into 15ml tube. Incubate the tube overnight at 37°C to test for contamination. If the broth is

clear on day two, proceed with the protocol.

2. Determine the amount of LB broth (3 ml per 15ml tube per clone) you will need and supplement

this amount with the appropriate antibiotic. Label each tube with clone ID, initials and date.

3. Aliquot 3 ml of antibiotic-supplemented LB broth into 15 ml tubes. Stab the original glycerol stock

with a sterile loop, scoop up a few cells and swish loop into the broth. Incubate in shaker waterbath

overnight at 37°C.

4. After culturing overnight, freeze 1 ml of culture in two labeled cryovials: 500ul culture + 500ul

50% sterile glycerol and store at -80°C. Indicate location of frozen vials in Freezer -80°C log.

5. Store the remaining amount at 4°C (good for 2 weeks) wrapped or proceed to the next step.

C. Isolating and setting up mini-cultures from single cell colonies

1. Prepare LB agar minus antibiotic. Autoclave with Liquid240 setting. Pour agar plates (100 x

15mm plates hold 35-40ml), allow to solidify and store all but one plate at 4°C. Incubate the

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agar plate overnight at 37°C to test for contamination. If the plate is clear on day two, proceed

with protocol.

2. Determine the number of agar plates (2 plates per clone) need, apply 75-100ul of antibiotic on

plate surface and spread thoroughly with sterile glass triangle. Allow plates to dry for 2-3

hours. Label each plate with clone, tech initials and date.

3. Stab the original glycerol stock or touch the liquid culture with a sterile loop and streak the

surface of the antibiotic LB-agar plate. Streak plates with a small number of turn-arounds as to

not facilitate overgrowth.

Incubate overnight at 37°C in designated incubator

5. Transfer 5-10 single bacterial colonies using the end of a transfer pipet tip to 15 ml tubes with 3

ml LB-broth with the appropriate antibiotic – one colony per tube. Label each tube with the

clone ID, colony number, initials and date.

6. Store agar plates at 4°C (good for 2 weeks) wrapped in Saran wrap

7. Incubate tubes with loose lids (must allow air flow) in shaker waterbath 8-12h at 37°C.

D. Verifying presence of specific DNA sequences

1. Design primers specific to template

a. Search Ensembl (www.ensembl.org) to insure primers are crossing intron/exon junctions; use

flanking parameters of 500 (flanking sequence) 25 (intron bp); show full intronic sequence

b. Copy and paste selected sequence from Ensembl into Integrated DNA Technologies (IDT)

www.idtdna.com

to select and purchase forward and reverse primer sequences.

1. Optimal primers are 400bp, < 50% C/G content, and have annealing temperature ranges

from 55-60°C

2. Blast primers before purchasing directly from IDT webpage.

a. Select the following Blast parameters:

Database Human genomic + transcript

Optimize for Highly similar sequences (megablast)

b. An optimal Blast exhibits 100% Query Coverage and an E value <1.0 to the

area of interest only. If there is 100% Query Coverage and an E value <1.0 to areas different

from the chosen, new primer sequences must be selected for optimization.

3. Once a sequence Blast is successful, print a hard copy of the Blast table (typically 3-8

pages in length) and label the pages “forward” or “reverse”

4. Print a hard copy of the purchased Primer Set

c. Create and print for the Probe Development binder a Word document that includes the

following: 1. Name of clone; 2. Name of gene/area; 3. Exon/Intron sequence used for

primer design; 4. Forward and Reverse primer - sequences only

2. Prepare PCR templates from single colony cultures lysing the cells as follows:

a. Prepare plasmid lysis solution by combining the following for a total volume of 5ml:

1. 50ul 100% Triton X; 2. 100ul 1M Tris HCl 7.5; 3. 20ul 0.5M EDTA; 4. 4830ul

sterile water

b. In a microcentrifuge tube add 1. 50 ul plasmid lysis solution; 2. 5 ul of cell suspension

c. Heat to 95°C for 5 minutes using the PTC 200 or AB 2720 Thermal Cycler

d. Chill on ice or hold at 4°C until ready for PCR

3. Store remaining single colony cultures at 4

C after parafilming the caps shut (good for two

weeks).

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209

4. Submit the lysates to ‘Touchdown’ PCR (programmed into AB 2720 Thermal Cycler for

primers chosen with annealing temperature range from 55-65°C).

a. Dilute Primers. For IDT primers, add the vial’s nmol value ul x 10 of sterile or nuclease-

free water directly into the primer vial.

b. Dilute Stock. 10ul stock primer + 90ul sterile or nuclease-free water. Be sure to label

diluted primer tubes with primer name, forward or reverse, initials and date.

c. In a 200ul microcentrifuge tube add the following reagents in the order listed below:

Promega GoTaq Green Master Mix = 12.5 ul

Primers = 1 ul each of forward and reverse

Sterile Water = 9 ul

Template = 1.5 ul

Total reaction volume = 25 ul

d. Store vials at -20°C or proceed with next step.

5. Verify size of fragment by submitting specimen to gel electrophoresis using a 1% agarose gel

to confirm that the product band has the expected size (small gel casting tray 50 ml, larger tray

150 ml + add EtBr prior to pouring the gel - ~4-6 ul of 10mg/ml stock EtBr). Allow gel to

cascade for 30-40 mins for a small gel, 45-60 min for a large gel, run at 85-90 volts.

6. In the marker lane, use appropriate size markers (ie 2 ul of 100bp High Mass Ladder, 3 ul of

Gel Pilot 100 bp ladder or 0.5 ul of 100 bp DNA ladder Invitrogen +1 ul 6x dye + sterile water

to complete 6 ul. In the test DNA wells, use 6ul of DNA product (dye is part of GoTaq Green

Master Mix)

7. Compare the results of the gel for each colony per clone and select for further steps one or two

colonies that showed the expected product size and had the sharpest band on the gel.

8. Expand the clone in overnight cultures:

a. For mini-cultures: 3 ml LB broth with proper antibiotic + one loopful of culture at 37°C in

the water bath shaker.

b. For maxi-cultures: Transfer remaining content of 15 ml tube to a 1 liter erlenmeyer and add

an additional 125 ml of TB broth and appropriate quantity of antibiotic.

9. Make aliquots of the selected single colony (in duplicate) to freeze: 500ul single colony culture

+ 500ul 50% sterile glycerol and store at -80°C. Indicate location of frozen vials in Freezer -

80°C log.

10. Submit the remaining culture to the appropriate DNA purification protocol.

Revised April 15, 2008 (LG/MS/SH)

Approved on ____________by______

Read and understood by laboratory personnel

Name Date

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

210

VI. DNA Cloning – Protocol 6

BAC DNA Preparations from Mini-Cultures

I. Reagents

1. QIAamp DNA Mini Kit (Qiagen Cat. # 51304)

2. 1XPBS

3. Cold 100% ethanol and 70% ethanol.

4. 3 M NaAcetate pH 5.4

5. TE Buffer (10mM Tris-HCl, 0.5mM EDTA, pH 9.0)

6. Ethidium Bromide Stock Solution 10mg/ml

7. Agarose ultraPure (Life Technologies Cat. # 15510-027)

8. 1 x Tris-Acetate Buffer (TAE) – Dilute 1 part 50x stock in 49 parts Milli-Q water

Prepare 50x stock buffer as follows: 242.0 g Tris base + 57.1 mL Glacial Acetic Acid +100 mL

0.5M EDTA pH 8.0 + Milli-Q water to complete 1L solution]

9. 6x loading dye

10. Markers for size of DNA bands: (a) 100bp High Mass Ladder; (b) 3 ul of Gel Pilot 100 bp

ladder (Qiagen # 239035) or (c) 0.5 ul of 100 bp DNA ladder (Invitrogen # 15628-019)

II. Procedure Note: Following steps should be done using sterile technique

A. Establishing Cultures and Isolating Single Colonies

1. Complete DNA Cloning Protocol 1: Establishing bacterial cultures, isolating single colonies

and verifying presence of desired sequence

B. DNA Extraction and Purification

Extract and purify DNA from mini-cultures using the QIAamp DNA mini-kit (Appendix pg.49 and

Blood and Body Fluid Spin Protocol pgs.27-29.) The purified DNA is used as template for whole

genome amplification using the Qiagen REPLI-g kit. Even though this mini-kit is not designed for

BAC DNA, experimentation has resulted in sufficient yields for REPLI-g amplification. For greater

BAC DNA yields refer to DNA Protocol 5 using the Nucleobond BAC Maxi-Kit.

1. Centrifuge 2ml of cell suspension for 5 min at 9.0x1000 rpm/g in a microcentrifuge tube. Remove

the supernatant completely and discard, taking care not to disturb the cell pellet.

2. Resuspend cell pellet in 1XPBS to a final volume of 200 µl.

3. Add 20 µl QIAGEN Proteinase K.

4. Add 200 µl Buffer AL to the sample. Mix by pulse-vortexing for 15 s. In order to ensure efficient

lysis, it is essential that the sample and Buffer AL are mixed thoroughly to yield a homogeneous

solution.

5. Incubate in Thermal Block at 56°C for 10 min.

6. Pulse spin the microcentrifuge tube to remove drops from the inside of the lid.

7. Add 200 µl 100% ethanol to the sample, and mix again by pulse-vortexing for 15 s. After mixing,

pulse spin the microcentrifuge tube to collect drops from the inside of the lid.

8. Carefully apply the mixture from step 6 to the QIAamp Spin Column (in a 2 ml collection tube)

without wetting the rim, close the cap, and centrifuge at 9.0x1000 rpm/g in for 1 min. Place the

Cytogenetics Core

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211

QIAamp Spin Column in a clean 2ml collection tube (provided), and discard the tube containing

the filtrate.

9. Carefully open the QIAamp Spin Column and add 500 µl Buffer AW1 without wetting the rim.

Close the cap and centrifuge at 9.0x1000 rpm/g for 1 min. Place the QIAamp Spin Column in a

clean 2 ml collection tube (provided), and discard the collection tube containing the filtrate.

10. Carefully open the QIAamp Spin Column and add 500 µl Buffer AW2 without wetting the rim.

Close the cap and centrifuge at 9.0x1000 rpm/g for 3 min.

11. Place the QIAamp Spin Column in a new 2 ml collection tube (not provided) and discard the

collection tube with the filtrate. Centrifuge at 9.0x1000 rpm/g for 1 min.

12. Place the QIAamp Spin Column in a clean 1.5 ml microcentrifuge tube (not provided), and discard

the collection tube containing the filtrate. Carefully open the QIAamp Spin Column and add 200 µl

Buffer AE. Incubate at room temperature (15–25°C) for 5 min, and then centrifuge at 9.0x1000

rpm/g for 1min. ***IMPORTANT: After centrifugation, keep the supernatant and label

microcentrifuge tube Elute1.

13. Repeat Step 12 in a second clean 1.5 ml microcentrifuge tube. ***IMPORTANT: After

centrifugation, keep the supernatant and label microcentrifuge tube Elute2.

C. DNA Precipitation and Concentration

1.Add content Elute 2 tube to Elute 1 tube for a total volume of 400 µl.

2.Add to the 400 µl reaction:

a. 0.1x the volume of the reaction of 3M Na Acetate = 40 µl

b. 2-2.5x of the volume of reaction of 100% cold EtOH = ~1100 µl

3. Mix gently but thoroughly by inverting the tube and keep at -80°C for 50 min., or at -20°C

overnight.

4. Spin 40 min. in refrigerated microcentrifuge at 11000 rpm. Carefully remove the supernatant.

5. Wash pellet with 70% cold ethanol, add slowly ~200 ul of cold 70% ethanol to the wall of the tube,

invert the tube and spin for 10min in refrigerated microcentrifuge at 11000 rpm.

6. Remove supernatant, wipe the tube walls carefully and dry the pellet at 37°C for ~20 min.

7. Dissolve the pellet in 10-20 ul TE buffer.

8. Incubate the tube in water bath at 37°C for 20 min to completely dissolve the pellet. Mix well.

9. Verify size of fragment by submitting specimen to gel electrophoresis using a 1% agarose gel to

confirm that the product band has the expected size (small gel casting tray 50 ml, larger tray 150

ml + add EtBr prior to pouring the gel - ~4-6 ul of 10mg/ml stock EtBr). Allow gel to cascade for

30-40 mins for a small gel, 45-60 min for a large gel, run at 85-90 volts.

10. In the marker lane, use appropriate size markers (ie 2 ul of 100bp High Mass Ladder, 3 ul of Gel

Pilot 100 bp ladder or 0.5 ul of 100 bp DNA ladder Invitrogen +1 ul 6x dye + sterile water to

complete 6 ul. In the test DNA wells, use 6ul of DNA product (dye is part of GoTaq Green Master

Mix)

11. Annotate the DNA vial with ID, concentration, date, tech initials, and buffer type.

12. Store DNA at -20°C.

Revised April 15, 2008 (LG/MS/SH)

Approved on ____________by______

Read and understood by laboratory personnel

Name Date

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

212

VI. DNA Cloning – Protocol 8

Amplification of DNA Sequences

I. Reagents

1. REPLI-g Midi Kit (Qiagen Cat. No. 150045)

2. Human Control Kit (Qiagen Cat. No. 150090)

3. Ethidium Bromide Stock Solution 10mg/ml

4. Agarose ultraPure (Life Technologies Cat. No. 15510-027)

5. 1 x Tris-Acetate Buffer (TAE) – Dilute 1 part 50x stock in 49 parts Milli-Q water

Prepare 50x stock buffer as follows: 242.0 g Tris base + 57.1 mL Glacial Acetic Acid +100 mL

0.5M EDTA pH 8.0 + Milli-Q water to complete 1L solution]

6. 6x loading dye

7. Markers for size of DNA bands: (a) 100bp High Mass Ladder; (b) 3 ul of Gel Pilot 100 bp ladder

(Qiagen # 239035) or (c) 0.5 ul of 100 bp DNA ladder (Invitrogen # 15628-019)

II. Procedure

This protocol is optimized for BAC DNA templates. The optimal DNA concentration to be entered

into the REPLI-g assay should be 100ng.

1. Prepare Stock Buffer DLB by adding 500 ul nuclease-free water to the tube. Mix thoroughly and

centrifuge briefly. Note: Reconstituted Buffer DLB can be stored for 6 months at –20°C.

2. Prepare sufficient Buffer D1 (denaturation buffer) and Buffer N1 (neutralization buffer) for the total

number of whole genome amplification reactions. Buffer D1 and Buffer N1 should not be stored

longer than 3 months.

Preparation of Buffer D1 Component Volume*

Reconstituted Buffer DLB† 5 ul

Nuclease-free water 35 ul

Preparation of Buffer N1 Component Volume*

Stop Solution 8 ul

Nuclease-free water 72 ul

3. Clearly label a 200ul microcentrifuge tube and pipet 5 or 2.5 ul of template DNA into the tube.

The concentration of template DNA should be 100 ng. Lower starting concentrations of REPLI-g

DNA may lead to inaccurate labeling reactions downstream, although there is evidence of

successful amplification with as little as 11ng.

4. Add 5 or 2.5 ul Buffer D1 to the DNA for denaturation. Mix by vortexing and centrifuge briefly.

5. Incubate the samples at room temperature (15–25°C) for 3 min.

6. Add 10 or 5 ul Buffer N1 to the samples for neutralization. Mix by vortexing and centrifuge briefly.

7. Thaw REPLI-g Midi DNA Polymerase on ice. Thaw all other components at room

temperature, vortex, then centrifuge briefly. The REPLI-g Midi Reaction Buffer may form a

precipitate after thawing. The precipitate will dissolve by vortexing for 10 s.

8. Prepare a master mix on ice. Mix and centrifuge briefly.

Important: Add the master mix components in the order listed. After addition of water and REPLI-g

Midi Reaction Buffer, briefly vortex and centrifuge the mixture before addition of REPLI-g Midi

DNA Polymerase. The master mix should be kept on ice and used immediately upon addition of the

REPLI-g Midi DNA Polymerase.

Preparation of Master Mix per specimen (5ul)

Preparation of Master Mix per specimen (2ul)

REPLI-g Midi Reaction Buffer 29ul REPLI-g Midi Reaction Buffer 29ul

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

213

Nuclease-free water 10ul

REPLI-g Midi DNA Polymerase 1ul REPLI-g Midi DNA Polymerase 1ul

8. Add the 30 or 40 ul of the master mix to 20 or 10 ul of denaturated DNA (step 4).

9. Place the 200 ul microcentrifuge tube into the Applied BioSystems Thermal Cycler and select Run.

Choose ‘repli-g’ and Start. The Thermal Cycler REPLI-g selection is pre-programmed with the

following steps:

a. Incubate at 30°C for:

i. 16 hrs (recommended for non-repetitive sequence DNA)

ii. 6 or 8 hrs (recommended for repetitive sequence DNA)

b. Inactivate REPLI-g Midi DNA Polymerase by heating the sample for 3 min at 65°C.

c. Store amplified DNA at 4°C for short-term storage or –20°C for long-term storage.

10. Dilute the REPLI-g product 1:9 (REPLI-g product to Sterile Water)

11. Verify size of fragment by submitting specimen to gel electrophoresis using a 1% agarose gel to

confirm that the product band has the expected size (small gel casting tray 50 ml, larger tray 150

ml + add EtBr prior to pouring the gel - ~4-6 ul of 10mg/ml stock EtBr). Allow gel to cascade for

30-40 mins for a small gel, 45-60 min for a large gel, run at 85-90 volts.

10. In the marker lane, use appropriate size markers (ie 2 ul of 100bp High Mass Ladder, 3 ul of Gel

Pilot 100 bp ladder or 0.5 ul of 100 bp DNA ladder Invitrogen +1 ul 6x dye + sterile water to

complete 6 ul. In the test DNA wells, use 6ul of DNA product (dye is part of GoTaq Green Master

Mix)

12. Annotate the DNA vial with ID, concentration, date, and initials.

13. Store DNA at -20°C or proceed with labeling.

Revised April 15, 2008 (LG/MS/SH)

Approved on ____________by______

Read and understood by laboratory personnel

Name Date

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

214

IV. DNA Labeling - Protocol 3

DNA Direct Labeling by Nick-Translation

I. Reagents:

1. Nick Translation Kit (Abbott Molecular Cat. # 32-801300)

2. SpectrumGreen (Cat. # 30-803200), SpectrumOrange (Cat. # 30-803000) or SpectrumRed (Cat. #

30-803400) labeled nucleotides

II. Preparing the Reagents (Protect From Light):

1. 0.2 mM SpectrumGreen, SpectrumOrange or SpectrumRed. Add 10 ul of 1mM dUTP stock to 40 ul

nuclease-free water

2. 0.1 mM dTTP. Add 10 ul of 0.3 mM dTTP to 20 ul nuclease-free water

3. 0.1 mM dNTP Mix. Mix together 10 ul each of 0.3 mM dATP, dCTP, and dGTP

III. Procedure (Protect from Light):

1. Prepare pure DNA. Labeling DNA not efficiently purified can result in poor incorporation of

SpectrumGreen, SpectrumOrange or SpectrumRed labeled nucleotides and high background after

hybridization.

2. Label a 0.5 ml microcentrifuge tube with the DNA ID, label to be used, and date. Place the tube on

ice.

3. For each reaction, add the following to the labeled tube in the correct order:

a. 1 ug of DNA (x ul) and dH

0 (17.5 – x ul).

b. 5 ul Nick Translation Buffer

c. 2.5 ul of 0.2 mM SpectrumGreen, SpectrumOrange or SpectrumRed dUTP.

d. 5 ul of 0.1 mM dTTP.

e. 10 ul of 0.1 mM dNTP mix. Vortex briefly and pulse spin.

f. 10 ul of nick translation enzyme.

NOTE: Be sure to take an ice bucket to the freezer and place the nick-translation enzyme in it. Carry

the bucket back to the workstation. Return the enzyme to the freezer immediately after use. Proper

storage and handling procedures will ensure a long life for the enzyme mix.

4. Mix briefly and spin for 2 seconds (pulse spin) in a microcentrifuge.

5. Place the tube in a thermoblock, and incubate at 15°C for 3-20 hours, depending on the size of the

DNA to be labeled and the desired size of the labeled fragments, according to the table below. For

directions to use the thermoblock see Protocol IV. 4.

DNA Initial Size of DNA Final Size of Fragment

Incubation Time

2 ug Plasmid DNA (~0.5-

1kb of

repeated sequences)

~50-100 bp 3 Hours

1 ug ~4.5 kb ~300 bp 4 Hours

1 ug ~7.9 kb ~300 bp 7 Hours

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

215

1 ug ~10 kb ~300 bp 8 Hours

1 ug Cosmids (~40 kb) ~150-200 bp 12 Hours

1 ug P1 DNA (~70-100 kb) ~300 bp 15 Hours

1 ug BAC DNA (~100-200kb) ~300 bp 16 Hours: Cell Lines

18 Hours: Tissue Sect.

1 ug YAC DNA (~200kb-1.5Mb) ~300 bp 20 Hours

1 ug Genomic DNA (for CGH) ~300-1000 bp 3 Hours

6. Spin down labeled DNA for 2 seconds in a microcentrifuge and store at -20

C until needed (up to 2

years).

IV. Comments:

1. Before using the probes, it is convenient to determine the size of the labeled fragments (Protocol

IV.P2). The probe can be left in this state or may be immediately precipitated for FISH (Protocol IV.

P5).

2. For hybridization in formalin-fixed, paraffin-embedded tissue sections, probe fragments should be

smaller and longer incubation times are needed. For BACs, 18 h. instead of 16 h. is optimal.

3. The amount of DNA used in the labeling reaction may be changed depending on the original size

and the optimal final size. For instance, for 5 kb inserts, 2 ug of DNA may be introduced in the

reaction and labeled for 8 hours; this is expected to provide more labeled DNA with the correct final

size. In this case, the precipitation and hybridization is regularly performed taking the final volume of

the labeled DNA as for 1 ug.

Revised April 8, 2008 (LG/SH)

Approved on _____________by ____

Read and understood by laboratory personnel

Name Date

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

216

IV. DNA Labeling - Protocol 5

Probe Preparation for Hybridization

I. Reagents:

1. Cold 100% ethanol and 70% ethanol.

2. 3 M Na Acetate pH 5.4

3. 5 mg/ml salmon/herring sperm DNA.

4. 1 ug/ul Cot 1 DNA (Human, mouse, rat)

5. Hybridization Buffer: 65% formamide hybridization buffer for repetitive sequence; 50% formamide

hybridization buffer for single sequences or commercial mix according to the manufacturer

recommendations.

II. Procedure (Protect from Light):

1. Add to the 50 ul of labeling reaction

a. 50x the amount of labeled DNA (1ug) of salmon sperm (10 ug/ul) = 5 ul

b. 10x the amount of labeled DNA of Cot-1 DNA* (1 ug/ul) = 10 ul

c. 0.1x the volume of the reaction of 3M Na Acetate = 7.5 ul

d. 2-2.5x of the volume of reaction of 100% cold EtOH = ~180 ul

* Cot-1 DNA is only added if the probe is not a repetitive sequence. In addition the reagent is

species-specific (human, mouse, etc.) and its concentration may be adjusted to allow better

signals

2. Mix by inverting the tube and keep at -80°C for 30-60 min., or at -20°C overnight.

* Hold tube at 4

C in cold room for 5 min before spinning to equilibrate.

3. Spin 30 min. in refrigerated microcentrifuge at 11000 rpm. Remove the supernatant.

4. Wash pellet 2 times with 70% cold ethanol (slowly add ~200 ul of cold 70% ethanol to the wall of

the tube, and invert the tube. If the pellet gets loose when you add the ethanol, it must be centrifuged

again.

5. Wipe the tube walls carefully and dry the pellet at 56°C for ~15-30 min.

6. Redissolve the pellet in the desired amount of the appropriate hyb mix. (homebrew 65% formamide

hybridization buffer for repetitive sequence; homebrew 50% formamide hybridization buffer for single

sequences; for commercial hyb mixes follow manufacturer’s instructions).

7. Incubate the tube in a water bath at 37°C for 15-30 min to completely dissolve the pellet. Mix well

and store at -20°C until needed.

Comments: If the probe generates heavy background in the FISH preparations, it may help to

precipitate the labeling reaction twice. In this case, at the first time only the labeled DNA, 3 M Na

Acetate and 100% ethanol is used. Then, the pellet is dissolved in 100 ul of dH

O, and the proper

proportions of carrier and blocking DNA, 3 M Na Acetate and 100% ethanol are added.

III. For Hybridization with Mixture of Probes (for one 22 x 22 mm cover area)

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

217

A. Combination of Unique Sequence (or Alpha Satellite) Probes

1. Mix the following components:

a. Selected amount of each labeled unique sequence DNA [about 100-150 ng for cosmid, PAC

and BAC DNA, 100 to 200 ng for YAC DNA]

b. 10x of Cot-1 Human or the proper species (1 ug/ul) to labeled DNA (do not exceed 30 ug if

mixing several probes)

c. 50x of salmon sperm DNA (5 ug/ul) or an amount that does not exceed a total of 15 ug of

DNA, otherwise the hybridization solution will be too concentrated and the pellet will not dissolve in

hybridization mix.

d. 0.1x the volume of the DNA solution of 3 M Na Acetate

e. 2-2.5x of the volume of reaction of 100% cold EtOH

2. Mix well and incubate at -80°C for 30 min.

3. Microspin at 4°C for 30 min. at 11,000 rpm. Remove the supernatant.

4. Carefully wipe the tube walls and dry the pellet at 56°C for 15-30 min.

5. Carefully rinse the pellet in 70% ethanol (slowly add ~200 ul of cold 70% ethanol to the wall of the

tube, and invert the tube. If the pellet gets loose when you add the ethanol, it must be centrifuged

again.

6. After the pellet is dry, add the desired amount of 50% formamide hybridization mix (minimum 10-

15 ul) to dissolve the pellet. Incubate the tube at 37°C for 10 min. (it may be necessary to incubate in

higher temperature or longer time). Mix by tapping with finger tip; spin 2 sec. in a microcentrifuge to

pellet the droplets.

7. Store at -20°C or proceed for denaturation.

The steps below can be performed during the hybridization (see Protocols in section V. FISH).

8. Calculate the total volume needed for all slides (for example: 6 ul of this mixture is needed for a 15 mm

dia. coverslip). Ratio of probe to dH

O to 50% formamide hybridization buffer or LSI Hybridization

Buffer is 1.0 ul probe to 2.0 ul dH

O to 7.0 ul LSI Hybridization Buffer. Adjust the volumes accordingly

for different coverslips.

9. Denature the probe mixture at 75-80°C for 5-10 min. (place the tube tightly closed, in a bubble rack

and float in a water bath at 80°C for 10 min.).

10. Chill on ice for about 2-5 min. to condense vapor inside the tube. Pulse spin to collect the droplets.

11. Incubate the probe mixture in a water bath at 37°C for 20-30 min. for pre-annealing of the Cot-1

repetitive sequences (if applicable).

12. Chill the mixture on ice for 2-5 min. Pulse spin and keep on ice until use.

B. Combination of Probes with Repetitive and Unique Sequences

1. Proceed with steps 1 to 7 of III.A for repetitive sequence probes (EXCEPT that no Cot-1 DNA is

added and 65% formamide hybridization buffer is used).

Cytogenetics Core

Anschutz Medical Campus, L18-8401A

Mail Stop 8117 P.O. Box 6511 Aurora, CO 80045

Phones: 303-724-3147, 303-724-3148

Fax: 303-724-3889 www.uccc.info/cytogenetics

218

2. Prepare unique sequence probes as defined in III.A.

The steps below can be performed during the hybridization (see Protocols in section V. FISH).

3. After blocking of the repetitive sequences of the unique sequence DNA, mix the appropriate amount

of repetitive and unique sequence probes along with dH

O and LSI Hybridization Buffer if necessary

to obtain the required volume of the final mixture.

4. Denature the probe mixture at 75-80°C for 5-10 min and chill on ice until use.

Revised April 8, 2008 (LG/SH)

Approved on _____________by ____

Read and understood by laboratory personnel

Name Date

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