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UNIVERSIDADE ESTADUAL DE MONTES CLAROS
Ludmilla Regina de Souza
Análise da expressão imunoistoquímica das proteínas p53, APE1, hMSH2 e
ERCC1 em Queilite Actínica e Carcinoma de Células Escamosas de Lábio
Montes Claros
2010
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Ludmilla Regina de Souza
Análise da expressão imunoistoquímica das proteínas p53, APE1, hMSH2 e
ERCC1 em Queilite Actínica e Carcinoma de Células Escamosas de Lábio
Orientador: Prof. Dr. Alfredo Maurício Batista de Paula
Corientador: Prof. Dr. André Luiz Sena Guimarães
Montes Claros
2010
Dissertação apresentada ao Programa de Pós-
graduação em Ciências em Saúde da
Universidade Estadual de Montes Claros,
como parte das exigências para a obtenção
do título de Mestre em Ciências da Saúde.
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Souza, Ludmilla Regina de.
S719a Análise da expressão imunoistoquímica das proteínas p53, APE1,
hMSH2 e ERCC1 em Queilite Actínica e Carcinoma de Células
Escamosas de Lábio [manuscrito] / Ludmilla Regina de Souza. – 2010.
160 f.
Bibliografia : f. 153-159.
Dissertação (mestrado) - Universidade Estadual de Montes Claros –
Unimontes, Programa de Pós-Graduação em Ciências da Saúde/
PPGCS, 2010.
Orientador: Prof. Dr. Alfredo Maurício Batista de Paula.
Coorientador: Prof. Dr. André Luiz Sena Guimarães.
1. Queilite actínica. 2. Carcinoma - Lábios. 3. Reparo de DNA. I.
Paula, Alfredo Maurício Batista de. II. Guimarães, André Luiz Sena.
III. Universidade Estadual de Montes Claros. IV. Título.
Catalogação Biblioteca Central Prof. Antonio Jorge
UNIVERSIDADE ESTADUAL DE MONTES CLAROS
Reitor Paulo César Gonçalves de Almeida
Vice-reitor João dos Reis Canela
Pró-reitor de Pesquisa e Pós-Graduação Sílvia Nietsche
Coordenadoria de Pós-Graduação Hercílio Martelli Júnior
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenação Paulo Rogério Ferreti Bonan
Subcoordenador João Felício Rodrigues Neto
Dedico essa vitória à minha maravilhosa família, sempre presente e incentivadora de todas as
minhas conquistas... aos meus pais Leandro e Eliane, às minhas irmãs Lorena e Larissa, à
querida Tia Neusa e ao meu amor Célio.
AGRADECIMENTOS
É com enorme satisfação que agradeço a Deus por mais essa etapa concluída com êxito, por
todas as providências em minha vida e pelas pessoas maravilhosas que sempre colocou em
meu caminho.
Mesmo diante de todas as adversidades da vida, não poderia dizer que me faltou amor, porque
fui agraciada por Deus com uma família linda. Pai, Mãe, Lore, Tia Neusa e Vó, sempre se
alegrando a cada passo dado e intercedendo a Deus por mim. Sou muito grata ao apoio
irrestrito e pelas demonstrações de orgulho nos pequenos e grandes acontecimentos. A
satisfação de vocês alimenta minha vontade de correr atrás dos meus sonhos e fazer sempre o
melhor. Sintam-se sempre amados, mesmo que distantes. A Célio, por seu amor,
companheirismo e ensinamentos; por fazer as minhas dificuldades se tornarem mais suaves e
por todas as abdicações em prol da nossa felicidade. À Larissinha, Tinha e Ed, por
tranquilizarem meu coração ao saber que as pessoas que mais amo ganharam mais um brilho
em suas vidas. A todos os tios e primos, pelos pequenos gestos que tomam grandes dimensões.
À minha para sempre amiga Amandinha. Sinal de Deus na vida de todos aqueles que com ela
convivem. Para sempre lembrada como minha melhor parceira de laboratório. Aos amigos “Os
Grandes”, exemplos de que grandes amizades continuam para sempre. À família David por ser
tão acolhedora e pela contagiante felicidade que sempre renovou minhas forças. Às colegas de
república, pelo aprendizado.
Ao meu orientador Alfredo e ao sempre presente André, que em algum momento dessa
caminhada se tornaram, além de orientadores, também grandes amigos. Sou muito grata a
vocês por todos os ensinamentos, pela formação profissional e pessoal, pelo exemplo de
trabalho em equipe e por acreditarem em mim.
Aos colegas de laboratório que contribuíram cada um da sua maneira, pelo aprendizado diante
das diferenças, e por suavizarem as dificuldades enfrentadas. Ao Eri e ao Tinho pela amizade
sincera e gratuita. À Lu, pela amizade e pelas experiências trocadas; aprendi muito durante
nossa convivência e me lembrarei sempre da nossa parceria com muito carinho. Aos meus
companheiros da carcinogênese labial, Thiago, Camilinha, Carol, Géfter e Eri, por estarem
sempre prontos a somarem para a melhoria do trabalho e, sobretudo, pela consideração de
todos vocês. Ao Carlos e ao Marcos, pelo aprendizado profissional e pessoal, por termos
conseguido transformar os momentos de amor e ódio em amizade. Luquinhas, Érica, Pat,
Érica, Rosa, Leandro e Fabiano, pelo carinho que sempre me trataram. E como diz Leandro, já
está na hora de ir embora... e a “arcaica” do laboratório levará cada um de vocês para sempre
nas suas recordações. Muito sucesso às Florzinhas!
Aos colegas da pós-graduação, em especial aos colegas do mestrado acadêmico Lucyana,
Naiara, Pedro, Luiza, Leonardo, Daniela, Sheila e Maria Fernanda, por terem tornado o
período do mestrado mais agradável e pelo aprendizado com as diferentes formações.
Aos coordenadores do Programa de Pós-graduação em Ciências da Saúde, pela dedicação e
oportunidade de crescimento.
Aos professores da pós-graduação, pelos inestimáveis conhecimentos transmitidos, pelo
profissionalismo e, principalmente, pela dedicação ao programa.
Às secretárias Maria do Carmo e Kátia pela presteza, orientações e conselhos.
A toda equipe do HU por sempre atender aos meus pedidos “pra ontem”, pelas conversas, pela
simplicidade, por todos os serviços prestados.
Ao reitor da Universidade Estadual de Montes Claros, Paulo César Gonçalves de Almeida,
pela oportunidade de cursar graduação e pós-graduação em uma Universidade pública.
À CAPES pela bolsa de pesquisa, por me permitir dedicar com exclusividade a uma atividade
que exerço com muito amor.
Sonho que se sonha só
É só um sonho que se sonha só;
Mas sonho que se sonha junto é
realidade.
Raul Seixas
RESUMO
Introdução: As abordagens dos mecanismos de controle da expressão gênica na progressão do
tumor em lábio são bastante escassas. Objetivos: Avaliar a expressão imunoistoquímica das
proteínas p53, APE1, hMSH2 e ERCC1 em Mucosa Labial (LM), Queilite Actínica (QA) e
Carcinoma de Células Escamosas de Lábio (CCEL), e investigar o valor prognóstico dessas
proteínas nas gradações morfológicas da QA e do CCEL. Métodos: Secções histológicas de
LM, QA e CCEL, obtidas a partir de amostras fixadas em formaldeído e embebidas em
parafina, foram preparadas para avaliações morfológicas em coloração de hematoxilina-eosina
e avaliações imunoistoquímicas da proteína supressora de tumor p53 e das proteínas de reparo
de DNA APE1, hMSH2 e ERCC1. Os dados encontrados foram analisados estatisticamente.
Resultados: Expressões progressivas de p53 e APE1 foram encontradas durante a progressão
do tumor em lábio. hMSH2 e ERCC1 apresentaram expressões aumentadas em QA e
decréscimos das expressões em CCEL. Em CCEL, alta expressão de APE1 foi associada à
maior agressividade do fronte invasivo. Os demais marcadores não apresentaram relação com
as gradações morfológicas da QA e do CCEL. Conclusões: As expressões imunoistoquímicas
das proteínas p53, APE1, hMSH2 e ERCC1 parecem estar associadas à progressão do tumor e
podem ajudar a discriminar a QA do CCEL. Regulações positivas das expressões de p53 e
APE1 são requeridas para os eventos da carcinogênese de lábio. Contrariamente, regulação
negativa das expressões de hMSH2 e ERCC1 parece ser indicativa da transição das lesões de
QA para CCEL. Expressão imunoistoquímica diferencial de APE1 é encontrada entre os graus
de agressividade morfológica do fronte invasivo.
Palavras-chave: Queilite actínica. Carcinoma de células escamosas de lábio. Carcinogênese de
lábio. p53. APE1. hMSH2. ERCC1. Reparo de DNA. Gene supressor de tumor.
Imunoistoquímica.
ABSTRACT
Introduction: The approaches control pathways of gene expression in lip tumor progression are
very scarce. Aims: To evaluate the immunohistochemical expression of p53, APE1, hMSH2
and ERCC1 proteins in Lip Mucosa (LM), Actinic Cheilitis (AC) and Lip Squamous Cell
Carcinoma (LSCC), and to investigate the value prognostic these proteins in morphological
grades of AC and LSCC. Methods: Histological sections of LM, AC and LSCC, obtained from
fixed samples in formaldehyde and embedded in paraffin, were prepared to morphological
analysis in haematoxylin-eosin and immunohistochemical evaluation of tumor suppressor
protein p53 and DNA repair proteins APE1, hMSH2 and ERCC1 proteins. Data found were
statistically analyzed. Results: Progressive expressions of p53 and APE1 were found during lip
tumor progression. hMSH2 and ERCC1 showed increased expression in AC and decrease of
expression in LSCC. In LSCC, APE1 high expression was associated to higher invasive front
aggressiveness. The others markers had not relation with AC and LSCC morphological grades.
Conclusions: Immunohistochemical expression of p53, APE1, hMSH2 and ERCC1 proteins
seems to be associated at tumor progression and can be held to discriminate AC of LSCC.
Upregulated of p53 and APE1 expressions are required for the events of lip carcinogenesis.
Contrarily, downregulated expressions of hMSH2 and ERCC1 seem to be indicative of
transition from AC to LSCC lesions. Differential immunohistochemical expression of APE1 is
founded among the grades of invasive front morphological aggressiveness.
Key words: Actinic cheilitis. Lip squamous cell carcinoma. Lip carcinogenesis. p53. APE1.
hMSH2. ERCC1. DNA repair. Tumor suppression gene. Immunohistochemical.
LISTA DE ABREVIATURAS E SIGLAS
6-4PP 6-4 fotoprodutos
AP Apurínico/apirimidínico
Asp Ácido aspártico
CCE Carcinoma de Células Escamosas
CCEL Carcinoma de Células Escamosas de Lábio
CDP Dímero de pirimidina
CpG Segmentos ricos em citosina e guanina
GGR Reparo genômico global
Glu Ácido glutâmico
ICL Ligações cruzadas
kb Quilo bases
kDA Quilo daltons
LM Mucosa Labial
MMR Mismatch repair (Reparo de bases mal pareadas)
nm Nanômetros
QA Queilite Actínica
REB Reparo por Excisão de Bases
REN Reparo por Excisão de Nucleotídeos
TCR Reparo acoplado à transcrição
UV Ultravioleta
UVA Ultravioleta A
UVB Ultravioleta B
UVC Ultravioleta C
SUMÁRIO
1 INTRODUÇÃO
13
2 REFERENCIAL TEÓRICO
15
2.1 Aspectos epidemiológicos, etiopatogenéticos e clínicos da Queilite Actínica
15
2.2 Aspectos epidemiológicos, etiopatogenéticos e clínicos do Carcinoma de Células
Escamosas de Lábio
18
2.3 Proteína supressora de tumor p53
21
2.4 Proteína de reparo APE1
24
2.5 Proteína de reparo ERCC1
26
2.6 Proteína de reparo hMSH2
29
3 OBJETIVOS
33
3.1 Objetivo geral
33
3.2 Objetivos específicos
33
4 PRODUTOS
34
4.1 Artigo 1
34
4.2 Artigo 2
34
4.3 Artigo 3
34
4.4 Artigo 4
34
5 CONSIDERAÇÕES FINAIS
59
REFERÊNCIAS
60
APÊNDICES
91
Apêndice A
92
Apêndice B
113
Apêndice C
133
ANEXOS
161
Anexo A
161
13
1 INTRODUÇÃO
A carcinogênese labial é um modelo de fotocarcinogênese decorrente da exposição contínua à
radiação ultravioleta, em especial no comprimento de onda UVB, caracterizada por uma
progressão de alterações moleculares, histológicas e clínicas (1-3). O carcinoma de células
escamosas de lábio (CCEL) é a condição patológica maligna mais prevalente entre as
neoplasias labiais, que pode originar-se de mucosa labial aparentemente saudável ou a partir de
lesões cancerizáveis, principalmente a partir de queilite actínica (QA) (2;4). Estima-se que
aproximadamente 95% dos casos de carcinoma de células escamosas de lábio tenham evoluído
de queilite actínica (5). A aparência clínica da QA revela a ocorrência de danos superficiais a
lesões avançadas, ocorrendo em muitos casos dificuldade na diferenciação da QA para o
CCEL inicial (2).
Exposição à radiação ultravioleta (UV), na forma de radiação solar ou artificial, representa um
risco potencial à saúde humana (6). A mutagênese por UV é caracterizada por uma alta
frequência de mutações de transição em sequências de bases pirimidínicas próximas,
implicando no aumento da formação de fotoprodutos diméricos, como dímeros de pirimidina
(CDPs) e 6-4 fotoprodutos (6-4PP) (7;8). Danos oxidativos, ligações cruzadas no DNA e
deleções cromossômicas são alterações adicionais frequentemente encontradas (9;10). O
espectro UVB da radiação solar é responsável pela maioria das alterações de conformação da
cromatina nos tumores em pele (11;12). A genotoxicidade UVA tem sido atribuída
principalmente à excitação de moléculas reativas de oxigênio que podem gerar alterações na
sequência codante e quebras em fitas simples de DNA (13;14). Mutações no gene supressor de
tumor TP53 induzidas por UV são bastante relatadas (15-17).
Como preconizado pelo dogma central da carcinogênese, a expansão clonal das células ocorre
como consequência da ativação ou inibição de genes cruciais para o controle do ciclo,
manutenção da integridade, proliferação e diferenciação. Esses genes foram classificados como
gatekeepers, genes que controlam a proliferação celular e regulam a apoptose, como TP53, e
caretakers, os genes responsáveis pela manutenção da integriadade do genoma (18). As
alterações genéticas ocasionadas pela radiação UV são preferencialmente removidas pelos
14
genes caretakers pertencentes às vias de reparo por excisão de bases (REB), reparo por excisão
de nucleotídeos (REN) e reparo de bases mal pareadas (MMR). A proteína APE1 é uma
proteína de reparo REB indispensável no reparo aos sítios AP induzidos pela radiação UV,
agindo também na ativação de fatores de transcrição necessários ao controle do ciclo celular
(19). ERCC1 é uma proteína de reparo REN que realiza a incisão de sequências de
nucleotídeos que alteram a estrutura da hélice do DNA e bloqueiam a transcrição e replicação
normais (20). A proteína hMSH2 age primariamente na remoção de pareamentos errôneos
entre bases nitrogenadas e, em resposta ao dano UV, é estimulada a ativar o bloqueio do ciclo e
a apoptose dependente ou independentemente da p53 (21). A inativação de uma dessas
proteínas, por exemplo, pode não iniciar o tumor em si, mas cria um ambiente de instabilidade
genética que alimenta a transformação das células e permite a ocorrência de novas mutações.
A idéia de que o câncer oral é muitas vezes precedido por uma lesão precursora não invasiva é
geralmente aceita (22). A compreensão da natureza e do comportamento das lesões em mucosa
oral têm se limitado a avaliações histopatológicas, com resultados nem sempre satisfatórios
(23). Dificuldades nos diagnósticos das displasias, em especial nos riscos de malignidade das
mesmas, têm suscitado a necessidade da opinião de mais de um profissional especializado para
a obtenção de resultados mais confiáveis (23;24). Diante disso, a presença de alterações
moleculares, como perda alélica, tem auxiliado no diagnóstico de lesões cancerizáveis orais
com alto risco de transformação maligna (25;26). Da mesma forma, inúmeros biomarcadores
têm sido utilizados na avaliação da progressão neoplásica (27-30). Portanto, análises das
principais vias moleculares no controle da expressão gênica aos danos induzidos pela radiação
solar são essenciais para a compreensão do mecanismo da carcinogênese. Na pesquisa por
marcadores que possam auxiliar na detecção da queilite acttínica com alto risco de malignidade
e a evolução morfológica do carcinoma de lábio, o presente estudo teve por objetivo
compreender o papel das proteínas p53, APE1, ERCC1 e hMSH2 nos diferentes estágios
morfológicos da progressão do tumor em lábio.
15
2 REFERENCIAL TEÓRICO
2.1 Aspectos epidemiológicos, etiopatogenéticos e clínicos da Queilite Actínica
Queilite actínica é um termo clínico usado para descrever alterações inflamatórias dos lábios
causadas pela exposição ao componente ultravioleta da radiação solar. A QA recebeu atenção
de pesquisadores pela primeira vez em 1923 (31). Sessenta anos mais tarde, a exposição
cumulativa à radiação ultravioleta foi associada à ocorrência da doença (32). Posteriormente,
foi considerada a principal lesão potencialmente maligna do lábio, podendo afetar a superfície
do vermelhão labial de maneira focal ou multicêntrica (33). O índice de transformação
maligna dessa lesão não é conclusivo, extendendo-se entre 10 a 20% (34-37).
A QA acomete principalmente homens acima de cinquenta anos de idade. O contexto
ocupacional exposto ao sol e o tipo de pele são os mais importantes fatores de risco. Pessoas
caucasianas são mais facilmente acometidas devido à menor proteção conferida pela
pigmentação da pele. Hábito de fumo, susceptibilidade genética, imunossupressão e irritação
crônica do lábio são fatores adicionais que podem contribuir para o desenvolvimento da lesão
(6;33;38-40). O lábio inferior é mais acometido que o lábio superior, provavelmente em
decorrência da maior incidência direta da radiação solar (41). Como o epitélio labial é mais
fino que o epitélio da pele, ele apresenta menos pigmentação melânica e poucas secreções de
glândulas sebáceas e sudoríparas, o que confere menor proteção à radiação actínica nesse sítio
(36). O perfil epidemiológico dos portadores de QA é bastante similar ao perfil dos portadores
de CCEL (42).
Clinicamente a QA apresenta-se como uma lesão esbranquiçada hiperceratótica, com
formação de crostas, descamação com eritema e perda da plasticidade e da definição das
margens do vermelhão labial. Pode apresentar-se leucoplásica, eritroplásica ou
leucoeritroplásica (40). Manisfestações de atrofia, ressecamento, sangramento intermitente e
fissuras labiais também podem ser evidenciadas (39;43). Na maioria das vezes a lesão
apresenta-se assintomática, difusa e pobremente demarcada, podendo apresentar, em alguns
16
casos, sintomas como ardor e prurido (38). Lesões únicas com sinais clínicos bem demarcados
são sugestivas de maior agressividade morfológica. A apresentação clínica da QA nem
sempre se diferencia do CCEL inicial (2).
As alterações histopatológicas da QA variam de hiperceratose, com ou sem displasia, a
carcinoma in situ (44). As principais alterações histológicas são espessamento do epitélio e da
camada de queratina, ulceração, acantose, degeneração basofílica do colágeno (elastose solar),
dilatação dos vasos sanguíneos, intenso infiltrado inflamatório e leve a severa displasia
(39;45;46). A ocorrência de displasia, considerada a alteração morfológica mais prevalente
entre as lesões de QA, e o grau em que ela se encontra no epitélio são geralmente aceitos
como os mais importantes preditores da transformação maligna (22;38-40;47). Visto que
alterações epiteliais aumentadas associam-se com figuras de acantose e espessamento da
camada de queratina, sugere-se que as alterações epiteliais se iniciam na camada basal e
expandem para as camadas superiores à medida que a exposição solar ou a outro agente
agressor permanece (39). O processo inflamatório intenso no cório da QA crônica e a
permanência de ulceração podem ser considerados sinais de alerta para a possibilidade de um
carcinoma espinocelular invasivo no tecido adjacente (37).
As classificações das displasias não têm sido fielmente reprodutíveis, apresentando níveis de
concordância bastante variáveis, principalmente diante das classificações das displasias
moderadas (23;48-50). Além disso, estudos têm agrupado diferentes subdivisões das
displasias como forma de avaliação da agressividade morfológica e do risco de transformação
maligna. No entanto esses agrupamentos ocorrem de diferentes formas entre os trabalhos
descritos na literatura. Em alguns estudos, ausência de alteração displásica é agrupada com
displasia leve (51). Em outros, displasia leve é estudada em conjunto com moderada,
enquanto displasia grave e carcinoma in situ constituem outro grupo (50). Diante da
dificuldade em categorizar as displasias leve, moderda e grave e em estabelecer o risco de
transformação maligna entre elas, Kujan e colaboradores (52), com base nos critérios da
Organização Mundial de Saude (2005), propuseram um novo sistema de gradação binária para
a classificação da displasia, subdividindo em baixo risco e alto risco. A sensibilidade e
especificidade do novo sistema foram de 85% e 80%, respectivamente, e, além disso, ele foi
considerado um indicador de prognóstico reprodutível. A subjetividade na avaliação, o
17
desconhecimento e a falta de calibração para o uso dos critérios de classificação também são
atribuições para a falta de reprodutibilidade das gradações displásicas (22;53).
A agressividade das alterações histológicas e o risco de malignidade da lesão não podem ser
avaliados clinicamente, e qualquer lesão suspeita deve ser biopsiada e examinada por
especialistas (39). Com o intuito de auxiliar na predição da transformação maligna da QA,
estudos citológicos, moleculares e imunoistoquímicos vêm sendo realizados, no entanto, ainda
dissociados da prática clínica. A superexpressão de COX-2, PAR-2 e o aumento da densidade
de mastócitos positivos para triptase podem contribuir para a formação da elastose solar em
QA e associam-se ao início do processo neoplásico (54;55). Alterações no mecanismo p53
também foram evidenciadas na QA e essa proteína foi apontada como um marcador de
fotodano e um preditor das fases iniciais da carcinogênese (56-58). Alto índice de mutações
no gene do receptor-3 de fator de crescimento de fibroblastos (FGFR3) em lesões de QA e
CCEL sugere um papel desse receptor nas desordens epiteliais e na patogênese dessas lesões
(59).
As condutas terapêuticas para a QA visam impedir que ela sofra transformação maligna. Nos
quadros em que a histologia não demonstra atipia epitelial, pode-se optar pelo uso de
protetores labiais e outras medidas que minimizem o índice de exposição solar. Alguns casos
podem apresentar regressão espontaneamente e a grande maioria requer remoção da lesão
(60;61). Os tratamentos mais empregados são vermelhionectomia, crioterapia, tratamento a
laser com dióxido de carbono, eletrodissecção e aplicação de 5-fluoracil tópico e descamação
química com ácido trifluoracético (62-64). Tratamento com terapia fotodinâmica usando
ácido 5-aminolevulínico também tem apresentado resultado satisfatório (65). A orientação
quanto aos fatores de risco e o acompanhamento clínico periódico dos indivíduos são
fundamentais.
18
2.2 Aspectos epidemiológicos, etiopatogenéticos e clínicos do Carcinoma de Células
Escamosas de Lábio
O carcinoma de células escamosas ou espinocelular (CCE) é um tumor maligno constituído
pela proliferação atípica de células da camada espinhosa (66). Cerca de 90% dos carcinomas
que acomentem o lábio correspondem a CCE (4;67). O CCEL pode desenvolver-se a partir da
mucosa labial aparentemente saudável ou a partir de lesões potencialmente malignas, como a
queilite actínica, a queilite glandular e as leucoplasias labiais (4;43;68). A QA é considerada a
principal lesão cancerizável do lábio (33). A relevância da ocorrência de QA prévia ao
surgimento do tumor foi retratada por pesquisadores que evidenciaram que os tumores do
lábio que se originavam de QA apresentavam melhores prognósticos, com menores índices
metastáticos; entretanto a ocorrência de recidiva foi independente da presença de QA, o que
provavelmente relaciona-se a tratamentos mal sucedidos (69).
As incidências de CCEL são bastante discrepantes entre as diferentes áreas geográficas,
parcialmente explicadas pelas variações étnicas e culturais e o grau de exposição aos fatores
de risco. As maiores incidências mundiais de câncer de lábio são relatadas na população
branca do Canadá e da Austrália. Mais de 50% dos cânceres orais na Austrália são localizados
no lábio, enquanto em partes da Ásia e da África o CCEL é uma lesão praticamente
inexistente (70;71). O acometimento em lábio inferior corresponde a mais de 80% dos casos,
aproximadamente 5% ocorrem em lábio superior e entre 6-10% em comissuras (72). CCEL
superior costuma ser mais agressivo que em lábio inferior (73).
CCEL surge como consequência de múltiplos eventos moleculares induzidos por
carcinógenos, cujo principal agente causador é a radiação solar (74). A radiação UV, em
especial a radiação UVB (comprimento de onda de 290-320 nm), destaca-se como o principal
carcinógeno ambiental (3). Um importante caráter mutagênico também é atribuído à radiação
UVA (320-400 nm), no entanto, em função do seu maior comprimento de onda, esta apresenta
menor poder de penetração nas células que UVB (75). Os mais relevantes fatores associados
ao desenvolvimento da doença são a pigmentação da pele e a intensidade da exposição solar
no ambiente de trabalho (71;74). As ocupações mais atingidas são aquelas referentes a altos
19
picos de incidência solar, tais como fazendeiros, marinheiros, empreiteiros, garis e
trabalhadores rurais (76;77). Consumo de tabaco, exposição a microorganismos oncogênicos,
imunossupressão e predisposição genética também têm sido implicados na etiopatogênese da
lesão (72;74). Sugere-se que o caráter hereditário para o câncer de lábio seja associado, em
especial, a fatores de risco familiares comuns (78). A importância relativa de cada fator e a
relação quantitativa complexa entre a quantia e duração devem ser consideradas (79;80).
Os homens são os mais acometidos pela neoplasia, 80-90% dos casos, mais precisamente
aqueles com histórico de exposição solar e que se encontram nas sétima e oitava décadas de
vida (3;81-83). Notadamente, a menor incidência de câncer de lábio entre as mulheres é
atribuída ao uso frequente de agentes protetores como batom e à menor exposição ao ar livre
(82;84). O CCEL em negros é uma doença rara, e predominantemente de idosos do sexo
masculino. Albinismo e xeroderma pigmentoso predispõe à ocorrência de câncer de lábio em
indivíduos jovens. A deficiência na proteção conferida pela melanina pode explicar as altas
incidências nesses grupos (74;85;86).
O CCEL é uma lesão assintomática e de crescimento lento, cuja evolução pode variar de 20 a
30 anos (87). A aparência típica do CCEL é de uma lesão ulcerada com margens
proeminentes, embora lesões exofíticas com margens aparentemente limitadas possam surgir
(73). O diagnóstico precoce torna-a a mais curável malignidade em região de cabeça e
pescoço, apesar do atraso no diagnóstico se extender de 5 meses a 2 anos (72;74;88;89). Uma
possível causa para o relativo atraso no diagnóstico do CCEL é o acometimento frequente do
lábio por enfermidades como herpes, mucocele, leucoplasias, queilites, granulomas, entre
outras, sugerindo que a alta frequência de lesões benignas possa levar o paciente a postergar a
procura profissional (90).
O comportamento do CCEL não segue os mesmos padrões descritos para a pele e para a
cavidade oral (91). Em relação à pele, a neoplasia no lábio tem maior probabilidade de
desenvolver metástase, todavia é de melhor prognóstico quando comparada ao câncer intra-
oral (63). A maioria dos casos são diagnosticados em estágio clínico inicial, com prevalência
de tamanhos tumorais T1/T2, metástases cervicais infrequentes e raras metástases à distância
20
(72;92;93). Na população brasileira, 81% dos indivíduos apresentam lesões T1/T2 e 79% são
clinicamente negativos para metástase cervical (94). Em caso de metástases à distância, cuja
prevalênca é de 0,5-2%, os sítios mais frequentemente acometidos são pulmão, fígado e pele.
Estágios avançados e recorrência locorregional são fortes indicadores do risco de metástase à
distância (3;95). Tamanho tumoral, superexpressão imunoistoquímica das proteínas p27
kip1
e
ciclina D1 e interação entre p27
kip1
e a espessura do tumor são considerados preditores de
metástases regionais (96;97). Histologicamente, as lesões apresentam-se bem a
moderadamente diferenciadas (74;92). Elevadas expressões das proteínas p53 e p21
WAF1CIP1
são detectadas no fronte invasivo tumoral, e um aumento de p53, ciclina D1 e PCNA
associam-se à agressividade morfológica da lesão (56;98). A avaliação de proteínas de reparo
em CCEL não é abordada na literatura.
Os tratamentos cirúrgico ou radioterápico em lesões T1/T2 de CCEL têm sido igualmente
efetivos (99;100). As desvantagens da radioterapia são a necessidade de tratamento
prolongado, a inespecificidade pelo órgão-alvo e a necessidade de um longo período de tempo
para proteger o lábio dos fatores ambientais (81). A maior desvantagem da cirurgia é que esta
é um procedimento invasivo e resulta em perda de tecidos não envolvidos (101). A
inadequação da remoção cirúrgica resulta em recorrência local (92). Praticamente todos os
casos de carcinomas avançados requerem remoção cirúrgica em combinação com radioterapia
adjuvante. Caso uma nova incisão seja inviável, a radioterapia é adotada para controle local
(92;102). A disseminação metastática reduz acentuadamente a possibilidade de cura, com um
impacto negativo sobre a sobrevida do paciente (81). Outros parâmetros como tamanho do
tumor, profundidade da invasão, diferenciação histológica e envolvimento perineural se
correlacionam a falhas no tratamento local (103).
O lábio é o único sítio da cavidade oral que apresenta sobrevida maior que cinco anos. Estudo
de câncer intra-oral nos Estados Unidos revelou que a frequência de sobrevida de cinco anos é
mais elevada em homens brancos, sendo quase duas vezes maior em brancos que em negros.
No entanto, ressalta-se que a incidência de câncer de lábio entre os negros é baixa (104). A
taxa de sobrevida de cinco anos é significativamente influenciada pela diferenciação
histológica do tumor primário. Tempo de sobrevida maior que cinco anos é encontrado em
21
91,0% dos indivíduos que apresentam tumores bem diferenciados. Para tumores moderado e
pobremente diferenciados, a frequência muda para 75,4% e 39,3%, respectivamente (79).
Apesar da alta taxa de cura comparada às demais neoplasias de cabeça e pescoço, alguns
estudos relatam índices de recidiva de 5 a 20% e mortalidade de 5 a 10% (102). O
acompanhamento do paciente e o tratamento cervical são passos fundamentais na conduta dos
indivíduos com CCEL (105).
2.3 Proteína supressora de tumor p53
O gene TP53 compreende 11 éxons e 10 íntons, em um domínio cromossomal de 20 kb
localizado no cromossomo 17p13.105-p12 (106). O produto desse gene é uma proteína com
53 kDa, p53, que age principalmente sobre o controle do ciclo celular nos pontos de
checagem (checkpoint), evitando assim a proliferação de células que apresentem danos ao
material genético (107;108).
O domínio de ligação da porção central da p53 ao DNA requer um grande entrelaçamento β-
pregueado para adequado posicionamento e orientação dos elementos estruturais que
interagem com o DNA. Mutações de ponto podem retirar da proteína a especificidade ao
DNA e, além disso, proteínas mutantes podem agir como domínios negativos ao formar
heterodímeros que inibem a ligação da p53 selvagem (109).
Em situações normais, a proteína p53 selvagem é sintetizada no citoplasma e em seguida
retorna para o núcleo, onde, após realizar sua atividade, é inibida pela ligação com a proteína
MDM2. MDM2 ativa o retorno da p53 para o citoplasma, além da sua ubiquitinação e
posterior degradação nos proteossomos (110). A fim de manter os checkpoints G1 e G2, a
proteína p14
ARF
sequestra MDM2 e mantém ativa a p53 (111). A superexpressão MDM2 e o
controle da localização intracelular podem inativar a p53 selvagem (112;113). Fosforilação,
glicosilação e ligações de moléculas diversas ao RNAm da p53 podem também atuar no
controle dessa proteína (110).
22
Vários sinais ativam a proteína p53, como danos ao material genético por agentes
oncogênicos, genotóxicos e estresse oxidativo, e imediatamente eleva-se o nível da proteína
selvagem (114;115). Diante de um dano que estimule o bloqueio do ciclo na fase G1/S, p53
estimula a ativação transcricional do gene p21, que bloqueará a ligação ciclina-CDK (116).
Na ausência da ligação ciclina-CDK, a proteína pRb se manterá associada ao fator de
transcrição E2F, e a subsequente transcrição dos genes cujas proteínas são necessárias à fase
de síntese torna-se silenciada. Dessa forma, o bloqueio do ciclo celular permitirá tempo para
reparo do dano (108;117). O bloqueio do ciclo na fase G2 depende da inibição das ligações
ciclina B/cdc2 e topoisomerase II/cdc2, que estimulam a entrada das células na fase S. O
bloqueio ciclina B/cdc2 ocorre por intermédio da ativação da transcrição dos genes 14-3-3σ e
Gadd45 por p53, cuja função 14-3-3σ é ligar-se ao complexo ciclina B/cdc2 e exportá-lo para
o núcleo, ao passo que Gadd45 age dissociando esse complexo (118;119). A inibição do
complexo topoisomerase II/cdc2 é proveniente da supressão da topoisomerase II por p53
(118). Caso o reparo fracasse, p53 estimula genes pró-apoptóticos como BAX, PIG3, Puma,
Noxa, Faz e PARP e reprime a transcrição de genes anti-apoptóticos como BCL-2, ativando o
“suicídio celular”. Falhas ao mecanismo de ação da p53 favorecem a transmissão de mutações
às células filhas, permitindo que mutações adicionais se acumulem no genoma e, em
determinado momento, tornam-se suficientes para desencadear a transformação celular (120).
Mutações TP53 ocorrem ao longo de todo o gene, com uma forte predominância nos
domínios de ligação ao DNA. A grande maioria delas acomete os éxons 5 a 10 nos tumores
humanos (121). As mutações podem decorrer de uma mudança de sentido (missence), 80%
dos casos, em que há troca de um aminoácido por outro, resultando em proteínas aberrantes e
mais estáveis, e por deleções do gene, inserções ou síntese truncada, 20%, em que não há
aumento da vida média e nem acúmulo da proteína (122). O produto gênico normal apresenta
um curto período de meia-vida, na ordem de 20 a 30 minutos, ao passo que o produto da
proteína mutada é mais estável e se associa com o acúmulo dessa proteína no núcleo (123). O
espectro de danos decorrentes de alteração na p53 é bastante amplo, podendo causar
inativação de genes pro ou anti-apoptóticos, efeito mitogênico descontrolado em
consequência de mutações de ganho de função sobre o estímulo a fatores de crescimento,
aumento da atividade da topoisomerase I (causa quebra na fita de DNA), inativação da
regulação transcricional de genes de reparo, dentre outros (119). A expressão da p53 mutada
parece ser a mudança genética mais comum nos cânceres humanos, respondendo por cerca de
23
50% dos tumores (119;124-128). A maioria das mutações que ocorrem no TP53 em tumores
de pele são indicativas de mutações induzidas pela radiação UV, destacando as desaminações
de bases 5-metil-citosina e as mutações de transição C para T ou CC para TT em sequências
CpG (8;129;130). Recentemente foi proposto que hot spots mutacionais no códon 278
parecem ser específicas para o CCE cutâneo (131).
Embora a literatura retrate que em diferentes sítios anatômicos as alterações do gene TP53 e
do seu produto gênico ocorram prevalentemente em fases tardias da carcinogênese, vários
casos de marcação positiva para a forma mutada da p53 são encontrados na fase de displasia e
morfologia normal da epiderme (51;132-135). Em mucosa normal e lesões displásicas, as
marcações positivas para p53 têm sido restritas às camadas basal e parabasal do epitélio,
sugerindo que os danos químicos, fisiológicos ou microbiológicos induzidos ao DNA podem
ser reparados previamente à replicação. Expressão p53 em todas as camadas epiteliais é
encontrada predominantemente em lesões cancerizáveis e carcinoma (1;136;137). Além disso,
o acúmulo dessa proteína tem sido associado ao crescimento tumoral (135;138).
Outro mecanismo de supressão tumoral é realizado pela p53 tipo selvagem ao inibir a
angiogênese, de forma que ao induzir a síntese da molécula antiangiogênica, a
trombospondina-1, há uma indução à involução da vascularização tumoral e consequente
redução da nutrição e disseminação neoplásica. Diversos estudos têm examinado o efeito da
p53 na expressão desses fatores, observando-se alta correlação entre o aumento da densidade
microvascular e as alterações da p53 (1;139;140). Numerosas pesquisas têm sido realizadas
com o objetivo de analisar seu potencial para utilização clínica, em especial como elemento
de valor prognóstico. Embora o grande número de evidências sugira que exames com a
detecção da p53 mutada venham a ser, em breve, utilizados na prática clínica, não existe ainda
um consenso sobre uma metodologia padrão a ser aplicada como procedimento de rotina. Há
necessidade de uma melhor definição quanto aos métodos de imunoistoquímica ou na
pesquisa de anticorpos séricos. Outro importante aspecto a ser analisado é a variabilidade dos
efeitos da mutação da proteína p53 de acordo com o segmento comprometido no gene (141).
24
2.4 Proteína de reparo APE1
O mecanismo de Reparo por Excisão de Bases (REB) foi descoberto por Lindahl e publicado
pela primeira vez em 1974. O mecanismo básico de REB foi primeiramente elucidado em
Escherichia coli, ao descrever uma atividade enzimática que catalisava a liberação de bases
uracila (142). Estudos subsequentes demonstraram que o mecanismo REB, inicialmente
proposto por Lindahl, foi altamente conservado ao longo da evolução, com grande homologia
entre Escherichia coli e mamíferos, o que contribui para os grandes avanços na compreensão
do mecanismo de excisão de bases (143-145).
O sistema de reparo de bases em células humanas pode ocorrer por substituição de uma única
base, REB curto, ou por substituição de mais de uma base, também chamado REB longo
(146;147). Várias hipóteses tentam explicar o motivo para se proceder por uma dessas vias,
cujas hipóteses da concentração de ATP próxima ao sítio AP (nomenclatura referente a sítio
apurínico/apirimidínico) e o reconhecimento do sítio pela DNA polimerase β são as mais
reconhecidas. Sugere-se que o mecanismo REB curto dependa da alta concentração de ATP
próxima ao sítio AP e do reconhecimento do sítio AP pela DNA polimerase β, ao passo que
REB longo parece ser efetuado mediante baixas concentrações de ATP e ausência de
reconhecimento da fenda AP pela DNA polimerase β (148;149).
O mecanismo REB especificamente remove alterações de bases únicas que tenham sido
erroneamente metiladas, oxidadas ou reduzidas, e que apresentem danos provenientes de
reações de alquilação, desaminação, depurinação ou depirimidinação (145;150). O primeiro
passo REB é mediado por glicosilases, responsáveis pela hidrólise da ligação N-glicosídica
entre a desoxirribose e a base danificada ou incorreta. Esse processo resulta na geração de
sítios apurínicos e apirimidínicos. As AP endonucleases humanas reconhecem os sítios AP
gerados pelas DNA glicosilases ou por perda espontânea de bases e realizam hidrólise da
ligação fosfodiéster adjacente ao sítio AP na porção 5’, gerando um terminal 3’OH e um
5’fosfato. A proteína APE1 é geralmente abundantemente expressa (aproximadamente 10
5
-
10
6
moléculas por célula) e destaca-se pela maior prevalência entre as AP endonucleases,
onde representa a quase totalidade das atividades de incisão dos sítios AP (151). Subsequente
25
à atividade endonucleásica, a DNA polimerase desprende o terminal 5’fosfato e insere uma
nova base à fenda, e a DNA ligase catalisa a formação das ligações fosfodiéster, completando
assim o mecanismo de reparo (152). Algumas particularidades na base desse mecanismo
distinguem REB curto de REB longo. A persistência dos sítios AP no DNA resulta em
bloqueio da replicação, mutações citotóxicas e instabilidade genética (153).
O gene HAP1 localiza-se no cromossomo 14q11.2-q12, responsável por codificar a proteína
AP endonuclease I, também conhecida como APE1, APEX1, Ref-1 e HAP-1 (154).
Adicionalmente à função de reparo de bases alteradas, a proteína APE1 desempenha
importante papel no reparo de quebras em fitas simples de DNA. Essas quebras são
provenientes de ataques às desoxirriboses, na maioria das vezes provocadas por espécies
reativas de oxigênio. Nesses casos, os grupamentos 3’OH e 5’fosfato necessários ao
reconhecimento pelas DNA polimerases são modificados em 3’fosfato, 3’fosfoglicoaldeído,
3’fosfoglicolato, 5’OH e 5’fosfodesoxirribose derivativa (155;156). Nesse sub-mecanismo
REB, as proteínas APE1 e PNK (polinucleotídeo quinase) restauram os grupamentos
terminais danificados e permitem que a fenda seja reconstituída (157). APE1 também tem se
mostrado importante na regulação gênica de diferentes fatores de transcrição, tais como p53,
AP-1, NF-кB e HIF-1α, alguns deles envolvidos na sobrevivência celular e apoptose
(154;158). Superexpressão APE1 aumenta a habilidade da p53 em transativar uma série de
promotores alvos e em estimular p21 (159). A ativação AP-1 é criticamente envolvida na
regulação do RNA mensageiro e da proteína ERCC1, visto que essa última contém em sua
região promotora um sítio de ligação AP-1. A modulação AP-1 pode intermediar a resposta ao
dano e consequente citotoxicidade à cisplatina (160). A ativação da ligação de fatores de
transcrição ao DNA por intermédio da proteína APE1 pode ocorrer de forma direta ou
indireta. De forma direta, a proteína promove a redução de resíduos de cisteína nos sítios de
ligação das proteínas alvo, ao passo que indiretamente as proteínas APE1 agem reduzindo
outras proteínas de regulação redox, como glutationa e tioredoxina, que consequentemente
responderão à ativação dos fatores de transcrição. A atividade redox indireta de APE1 ficou
conhecida como chaperona redox (158).
Diante da habilidade multifuncional de APE1, percebe-se que ela exerce atividade de reparo
preferencialmente em núcleo, mas atividades em citoplasma e mitocôndria também têm sido
26
caracterizadas recentemente (161). Sua síntese e translocação nuclear são induzidas por
estresse oxidativo, hipóxia e agentes que danificam o DNA (28;154;162). Foi descoberto
recentemente que a proteína p53 selvagem regula negativamente APE1 por intermédio de
ligações a sítios da região promotora dessa proteína, incluindo sítios de ligação Sp1. Sabe-se
que p53 age como um fator pró-apoptótico em resposta ao estresse, ao passo que APE1 é uma
proteína a favor do reparo dos danos e sobrevivência das células (125;156). Essa repressão da
expressão de APE1 via p53 pode ocorrer como um mecanismo de repressão a genes
antiapoptóticos em situações de estresse (163). A expressão reduzida de APE1 durante a
apoptose tem sido verificada em vários estudos, como nos eventos de maior sensibilidade às
terapias (164-166).
Algumas variantes polimórficas apresentam atividade APE1 reduzida (167). Indivíduos
portando o alelo Glu no polimorfismo de transversão TG (Asp148Glu) apresentam maior
sensibilidade à radiação ionizante que os indivíduos que apresentam o alelo Asp (168).
Elevada expressão APE1 é um evento comum a várias neoplasias (28;165;169;170).
2.5 Proteína de reparo ERCC1
O mecanismo de reparo por excisão de nucleotídeos (REN) é um importante caminho
bioquímico pelo qual são removidas diversas variedades de danos ao DNA, direcionando-se
àquelas classes de lesões que distorcem a hélice e interferem no pareamento de bases,
geralmente obstruindo a transcrição e a replicação normais (171). Esse mecanismo de reparo
subdivide-se em dois distintos sub-mecanismos, reparo genômico global (GGR), responsável
por remover lesões em ambas as fitas do genoma, e reparo acoplado à transcrição (TCR),
cuja função é remover danos na fita transcrita do DNA (172).
O gene ERCC1 (Excision Repair Cross Complementation Group 1) constitui um importante
componente dos genes de reparo de DNA. Foi o primeiro gene clonado que age
especificamente nas vias de reparo por excisão de nucleotídeos. Localiza-se no cromossomo
19q13.2-q13.3 e sua região codificadora apresenta 1,1 kb e 10 éxons. A região correspondente
27
ao éxon 8 parece expressar a função de incisão da região alterada do DNA (173-175). REN é
um processo complexo que requer a participação de cerca de 30 proteínas diferentes. As
proteínas XPC-HR23B e XPE reconhecem as torções no DNA, e o fator de transcrição TFIIH
realiza a abertura da fita para a verificação do dano e reunião do complexo de reparo. As
proteínas XPA e RPA são direcionadas para a estrutura desnaturada e reconhecem o fragmento
danificado. Encontrado o dano, as endonucleases XPF/ERCC1 e XPG são recrutadas e fazem a
dupla incisão na cadeia (20;176;177). ERCC1 posiciona a endonuclease XPF, que catalisa a
incisão do DNA na porção 5´ da fita lesionada, ao passo que XPG catalisa a incisão da
extremidade 3’. O domínio central da proteína ERCC1 apresenta sítios de ligação para DNA de
fita simples e de fita dupla, bem como um sítio de ligação para a proteína XPA. A ligação à
XPA é importante para promover um elo entre a maquinaria de reparo e XPF. A ligação
ERCC1/XPF é efetuada pelos domínios C-terminal de ambas as proteínas. As duas
subunidades ERCC1 e XPF são instáveis em estado dissociado, sendo a organização dimérica
crítica para a estabilidade e a atividade catalítica XPF (178;179). Após a incisão da fita, as
DNAs polimerases δ e ε removem os nucleotídeos e ressintetizam o DNA usando a fita não
danificada como molde, e a DNA ligase liga as extremidades à sequência original (180).
Alterações do ERCC1 resultam em graves falhas ao mecanismo de reparo (29;181).
As lesões mais comumente processadas por REN incluem as alterações induzidas por luz
ultravioleta e os adutos formados no DNA por agentes químicos, como hidrocarbonetos
aromáticos e cisplatina (20;182;183). A radiação ultravioleta, em especial nos comprimentos
de onda UVB e UVC, induz a formação de dímeros de pirimidina ciclobutano e 6-4
fotoprodutos, lesões que provocam torções na hélice de DNA (11;12). UVA também pode
induzir a formação de CPDs, mas através de um mecanismo indireto apresenta importância
primária na formação de bases oxidadas (13;184). CDPs e 6-4PP são formados por ligações
estáveis entre as posições 5 e 6 de duas bases pirimidinas adjacentes e 6 e 4 de duas
pirimidinas próximas, respectivamente (12;185). A deficiência do processo de reparo a essas
lesões traduz em quadros diversos de instabilidades genéticas, que em sua maioria são
extremamente sensíveis à radiação UV e mais vulneráveis às alterações neoplásicas (186).
Enquanto a reduzida expressão de ERCC1 tem sido associada ao risco aumentado de câncer
(187), a elevada expressão dessa proteína associa-se à pior resposta ao tratamento
quimioterápico e pior sobrevida nesse grupo de indivíduos (188;189). O processo de reparo do
DNA danificado parece ser crítico para a resistência celular às drogas e, consequentemente,
28
para a sobrevida dos indivíduos afetados (190;191). Vários estudos epidemiológicos
demonstram associações entre genes de reparo e o risco de desenvolvimento de câncer, mas
uma relação de causalidade não pode ser estabelecida. Portanto, a baixa expressão de ERCC1
observada em pacientes com câncer pode ser causa ou consequência do mesmo (192).
A ocorrência de polimorfismos no gene ERCC1 tem sido estudada frente ao risco de
desenvolvimento de vários tipos de câncer (193;194). Muitos estudos têm focado em dois
polimorfismos. O polimorfismo C8092A pode afetar a estabilidade do DNA e o polimorfismo
T19007C pode está associado com níveis reduzidos do RNA mensageiro e da proteína
(195;196).
Estudos sugerem que o reparo às ligações cruzadas, lesões que bloqueiam a replicação ou
transcrição por impedirem que a fita de DNA se desenrole e separe, seja realizado pelos
mecanismos de reparo REN e reparo por recombinação (197;198). No entanto, estudos prévios
mostraram não haver hipersensibilidade a agentes causadores de ligações cruzadas em células
animais deficientes em diversas proteínas REN, mas diante da ausência de ERCC1 ou XPF foi
percebida hipersensibilidade a esses agentes. Esse resultado indica que o complexo
ERCC1/XPF está envolvido no reparo às ligações cruzadas, mas o envolvimento desse
complexo independe do mecanismo REN (199-201). Acredita-se que o complexo ERCC1/XPF
apresenta atividade endonucleásica no reparo às ligações cruzadas (202).
Numerosos estudos têm demonstrado o papel da p53 em REN (203-206). Essa possibilidade
surgiu pela observação de que as ativações das proteínas XPE (DDB2/p48) e XPC-HR23B por
p48 são dependentes da ativação p53. Ao término do evento de reparo, p53 interage com o
complexo TFIIH e inibe a atividade das helicases (203;205). A diminuição do reparo GGR é
notificada em caso de inibição p53, mas a relação dessa proteína com o reparo TCR ainda é
bastante controversa (206;207).
29
2.6 Proteína de reparo hMSH2
A proteína hMSH2 integra o sistema de reparo de bases mal pareadas (MMR – do inglês
mismatch repair), cuja função primária é direcionar a maquinaria de reparo para a fita recém-
sintetizada, a fim de remover pareamentos errôneos entre bases nitrogenadas e pequenas alças
de inserção/deleção decorrentes de erros de alinhamento de nucleotídeos. Esse reparo é
efetuado quando algumas bases conseguem escapar da revisão realizada pela DNA polimerase
no momento da replicação ou por danos no mecanismo de recombinação (208;209). Um alto
grau de conservação evolucionária do sistema MMR permitiu uma melhor compreensão desse
mecanismo (210).
O sistema de reparo MMR humano é composto pelos heterodímeros codificados pelos genes
MUT, MutSα, MutSβ, MutLα, MutLβ e MutLγ. MutSα (hMSH2/hMSH6) reconhece bases
mal pareadas, principalmente G-T, e um ou dois nucleotídeos extrahelicoidais em locais de
alças de inserção ou deleção (211). MutSβ (hMSH2/hMSH3) reconhece preferencialmente
alças de inserção/deleção longas (dois a cinco nucleotídeos), mas também reconhece
pequenas alças (212). Na ausência de hMSH2, toda função de reconhecimento mismatch
encontra-se prejudicada. Após o reconhecimento, os heterodímeros MutLα (hMLH1/hPMS2)
e MutLβ (hMLH1/hPMS1) conectam os passos do reconhecimento aos passos do reparo.
MutLγ (hMLH1/hMLH3) apresenta importante papel na meiose (210). As enzimas PCNA,
EXO1, MED1 e FEN-1 ligam-se ao complexo de reconhecimento e removem os erros de
pareamento, havendo uma distinção da base danificada pela ausência do sinal de metilação na
fita recém sintetizada (211). Os novos nucleotídeos são inseridos pelas proteínas polimerases
δ e ε (213).
Além disso, o sistema MMR é indispensável na remoção de pequenas alças ao longo do DNA,
formadas principalmente em regiões microssatélites (sequências repetitivas de nucleotídeos
no DNA). Durante a síntese de DNA nessas sequências, o primer ou a fita molde podem
ocasionalmente dissociar-se e reanelarem incorretamente, dando origem a uma molécula de
DNA cuja sequência na fita molde difere da fita transcrita (214). Devido a essa falha de
reanelamento, forma-se uma estrutura parcialmente helicoidal, conhecida como alça de
30
inserção/deleção. Na ausência do mecanismo de reparo MMR, as alças de inserção/deleção e
os erros de pareamento permanecem e favorecem o fenótipo neoplásico (215).
Proteínas MMR também afetam a eficiência e a fidelidade das recombinações mitóticas e
meióticas (216;217). Elas bloqueiam o processo de troca entre fitas homólogas (fitas
similares, mas não idênticas) diante da detecção de um dano na fita no momento da
recombinação mitótica. MutS e MutL também inibem a recombinação entre sequências
diferentes, por se ligarem às extremidades das fitas (218). A consequência do defeito MMR
na meiose não está bem esclarecida, mas sabe-se que a recombinação meiótica requer uma
classe diferente de heterodímeros MutS, hMSH4/hMSH5, importante na fidelidade da
segregação meiótica e formação dos gametas (161;219;220). hMSH4 e hMSH5 não interagem
com hMSH2, hMSH3 ou hMSH6, sugerindo que essas proteínas são funcionalmente
diferentes das proteínas que participam do reparo MMR (219).
Pesquisas recentes relatam que hMSH2 é requerido para o processo de reparo de ligações
cruzadas ICL (do inglês Interstrand Cross-Links), as mais deletérias lesões ao DNA que
efetivamente impedem que o DNA se desenrole e separe, bloqueando a transcrição e a
replicação (201;221). O complexo de reparo de excisão de nucleotídeos XPC-RAD23B
reconhece as ligações cruzadas no DNA, e as endonucleases ERCC1-XPF e XPG realizam a
incisão da fita nas extremidades 5’ e 3’ próximas ao dano (222). Proteínas do complexo
MutSβ (hMSH2-hMSH3) também se ligam aos sítios de ligação cruzada no DNA,
demonstrando que ICLs podem ser processadas pelo mecanismo de reparo por recombinação
homóloga após a introdução de uma quebra na fita dupla do DNA nas proximidades ICL. Este
reparo é dependente de proteínas de reparo MMR, REN, proteínas de recombinação
homóloga e reparo de quebra de fita dupla (201;221;223).
Agentes que danificam bases nitrogenadas na fita molde, como por exemplo, agentes que
causam metilação em guanina, causam erro de pareamento que são detectadas pelo complexo
MutSα. Na correção do pareamento errôneo, uma nova base é adicionada à fita recém
sintetizada pela DNA polimerase. No entanto, como o dano foi inserido na fita molde, novas
bases continuamente substituirão a base pareada à base danificada e o erro de pareamento será
31
perpetuado. Dessa forma, o sistema MMR é repetidamente desencadeado, até o momento que
proteínas MMR sinalizam o bloqueio da forquilha de replicação (224). ATM (ataxia
telangiectasia) e ATR (ataxia telangiectasia mutado) fosforilam p53 e p73 a fim de ativar a
cascata cinase e sinalização do checkpoint, devido ao reparo sem sucesso, determinando o
bloqueio do ciclo ou morte celular programada. ATM e ATR também ativam as proteínas
hMSH2 e hMLH1 para fosforilarem p53 (225). Nesse contexto, o bloqueio do ciclo pode
também ser determinado por quebras na fita de DNA decorrentes dos repetidos ciclos de
reparo (215). Como as células deficientes em MMR não tentam processar o erro de
pareamento, elas sobrevivem à custa do dano (226). Quando o gene hMSH2 apresenta-se
mutado, o ciclo celular exibe uma menor fase G1, acompanhada de uma maior exposição ao
dano e um menor nível de eliminação das células (218;227). Evitar a apoptose é uma das
marcas de desenvolvimento do câncer e progressão, e estudos têm mostrado que a apoptose
pode ser estimulada pela expressão de hMSH2, mesmo na ausência de p53 (228;229). Esse
mecanismo explica muitos casos de resistência celular aos tratamentos antineoplásicos
empregados. Células deficientes em MMR são mais resistentes à morte e, portanto, mais
tolerantes aos tratamentos (230). Similarmente, deficiência MMR tem sido associada à
resistência celular à radiação ultravioleta (218;225). Em contraste aos mecanismos anteriores,
células deficientes em MMR são mais sensíveis à morte desencadeadas por ligações cruzadas
na fita (201).
O reparo MMR também está envolvido nos danos causados por espécies reativas de oxigênio
(144;231). Quando um dano ao DNA é induzido por radiação UV, as etapas iniciais da
remoção do dano são realizadas pelos sistemas de reparo de excisão bases (REB) e pelo
reparo de excisão de nucleotídeos (REN). Reconstituída a fita, as proteínas hMSH2 e hMSH6
agem ligando-se aos pares de bases não pareadas para clivá-las, no sentido de conter o tipo de
lesão formada pela radiação UV (232;233). Quando o dano causado pela radiação provoca
quebra na fita dupla, o mecanismo de recombinação homóloga é acionado, e o envolvimento
de proteínas MMR (MutSα) torna-se fundamental na prevenção de hiper-recombinações
(234). A diminuição e a expressão heterogênea da proteína hMSH2 são comuns na maioria
dos casos de carcinoma de células escamosas, em especial na transição da lesão cancerizável
para carcinoma, o que se associa a uma redução na indução da apoptose (218;235).
Hipermetilação na região promotora de hMSH2 e hMLH1 pode ter um papel na redução na
32
expressão dessas proteínas e na carcinogênese oral, e pode correlacionar-se ao
desenvolvimento de múltiplas malignidades orais (236).
33
3 OBJETIVOS
3.1 Objetivo geral
Avaliar a expressão imunoistoquímica das proteínas p53, APE1, hMSH2 e ERCC1 nos
diferentes estágios morfológicos da progressão do tumor em lábio.
3.2 Objetivos específicos:
Realizar a gradação morfológica e o risco de transformação maligna das lesões de queilite
actínica.
Realizar as gradações morfológicas de todo parênquima tumoral o do fronte invasivo nas
amostras de carcinoma de células escamosas de lábio.
Analisar a expressão imunoistoquímica das proteínas p53, APE1, hMSH2 e ERCC1 de
acordo com o risco de transformação maligna das lesões de queilite actínica.
Analisar a expressão imunoistoquímica das proteínas p53, APE1, hMSH2 e ERCC1 de
acordo com as gradações morfológicas das lesões de carcinoma de células escamosas de
lábio.
34
4 PRODUTOS
4.1 Artigo 1: Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in
Actinic Cheilitis and Lip Squamous Cell Carcinoma, formatado segundo as normas para
publicação do periódico Histopathology.
4.2 Artigo 2: Demographic Aspects and Clinicopathological Analysis of Actinic cheilitis in a
Brazilian Population, formatado segundo as normas para publicação do periódico Oral
Diseases. (Apêndice A)
4.3 Artigo 3: Demographic and Clinicopathological Analysis of Lip Squamous Cell
Carcinoma in a Brazilian Population, formatado segundo as normas para publicação do
periódico Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology.
(Apêndice B)
4.4 Artigo 4: Association of mast cell, eosinophil leukocyte and microvessel densities in
actinic cheilitis and lip squamous cell carcinoma, formatado segundo as normas para
publicação do periódico Histopathology. (Apêndice C)
O produto primário da dissertação foi o artigo 1. Os demais produtos alcançados foram
obtidos visando uma maior compreensão da progressão do tumor em lábio.
35
Immunohistochemical analysis of p53, APE1, hMSH2, and ERCC1 proteins in Actinic
Cheilitis and Lip Squamous Cell Carcinoma.
Ludmilla R. Souza
1
; Thiago Fonseca-Silva
1
; Camila S. Pereira
1
; Erivelton P. Santos
1
;
Lucianne C. Lima
2
; Heloísa A. Carvalho
3
; Ricardo S Gomez
3
; André L. S. Guimarães
4
;
& Alfredo M. B. De Paula
4
1
Postgraduate.Health Sciences Program. State University of Montes Claros, Montes Claros, Brazil.
2
Postgraduate. Department of Medicine. State University of Montes Claros, Montes Claros, Brazil.
3
MD, PhD. Lecturer. School of Medicine. Department of Radiotherapy. University of São Paulo. São
Paulo, Brazil.
4
DDS, PhD, Lecturer. Department of Clinic, Pathology and Surgery. School of Dentistry. Federal
University of Minas Gerais, Belo Horizonte, Brazil.
5
DDS, PhD, Lecturer. Health Sciences Program. State University of Montes Claros, Montes Claros,
Brazil.
RUNNING TITLE: p53,MSH2,ERCC1,APE in lip carcinogenesis
Key words: Actinic cheilitis, Lip squamous cell carcinoma, lip carcinogenesis, p53,
DNA repair proteins.
Address for correspondence: A.M.B. De Paula, Laboratório de Pesquisa em Saúde,
Hospital Universitário Clemente de Faria, Universidade Estadual de Montes Claros, Av.
Cula Mangabeira, 562 Santo Expedito, Montes Claros, 39401-001, Minas Gerais,
Brazil. e-mail: [email protected]
Tel: 51-21-38 32248327. Fax: 55-21-38 32298500
36
Abstract
Introduction A network of interacting cellular components is known to mediate the
regulatory role of tumor suppressor proteins in genomic stability. One of these
componets is considered to be the DNA repair machinery.
Aims: To compare the immunohistochemical expressions of tumor suppressor protein
p53, and DNA repair proteins APE1, hMSH2 and ERCC1 in normal lip mucosa with
actinic cheilitis and lip squamous cell carcinoma.
Methods and Results: Archived tissue specimens of Lip mucosa (LM), Actinic cheilitis
(AC), and Lip squamous cell carcinoma (LSCC) were submitted to morphological
analysis and immunohistochemical reactions. Data found were statistically analyzed.
Progressive increase of p53 and APE1 tissue expressions were observed with increasing
malignancy of the epithelial tissue during lip tumour progression. Controversially, we
found a significant loss of hMSH2 and ERCC1 expressions from AC to LSCC groups.
In LSCC, a higher APE1 expression was associated to high aggressiveness of invasive
front tumor grade.
Conclusions: Immunohistochemical expressions of p53, APE1, hMSH2 and ERCC1
proteins seem to be associated to lip tumor progression and can be held to discriminate
the premalignant and cancer lip diseases. Still, APE1 expression was associated with
LSCC lesions with higher morphological aggressiveness.
37
Introduction
The lip squamous cell carcinoma (LSCC) may develop from its premalignant
condition, the Actinic cheilitis (AC), or, infrequently, from the healthy lip mucosa
1, 2
.
Although it has been estimated that almost 95% of LSCC originate from AC
3, 4
, data
about the malignance transformation rate from AC to LSCC are not conclusive
4-6
. The
lip carcinogenesis results mainly from the chronic exposition of lip to ultraviolet
radiation (UVR) of solar radiation, especially UVB and UVA
7-9
. However, actinic
radiation may indeed not be the only risk factor for lip carcinogenesis. Socioeconomic
factors, lifestyle factors such as smoking, alcohol drinking, and dietary habits,
immunosuppression, and a background of inherited resistance or genetic susceptibility
might produce a synergistic effect
3, 7-12
. Exposure to both endogenous and exogenous
genotoxic agents cause cell cycle disturbances allowing repair of DNA damage, whose
activities are central to maintaining normal cellular functions
13, 14
.
TP53 is a well-known tumor suppressor gene. Its protein, the p53, is a
transcription factor that binds to DNA specific sequences to activate intermediate genes
of antiproliferative response, including the cell cycle blocking, apoptose, damage repair
and differentiation
15-17
. The overexpression of abnormal p53 protein has been reported
and represented the mutations of TP53 gene in upper aerodigestive tract squamous cell
carcinoma
18-20
. Deregulated p53 has also been investigated as a potential predictor of
malignant transformation in premalignant oral lesions
21-24
including premalignant lip
disease
25-30
.
Following exposure to genotoxic agents, ATM/ATR kinases, established as main
molecular sensors for UV-induced DNA damage, activate proteins responsible for cell
cycle control and removal of DNA damage
31
. DNA repair pathways are usually specific
38
for a class of given damage: mismatch repair pathway (MMR); homologous
recombination (HR), and nonhomologous end joining (NHEJ); base excision repair
(BER), and nucleotide excision repair (NER)
32
.
Mismatch repair (MMR) are highly conserved DNA caretaker proteins
represented by MutS and MutL. Their primary role of MMR is to avoid genomic
mutations occurring in microsatellite regions. In these regions, the template and
daughter strands are particularly prone to misalignment during DNA polymerase
sliding. Defective human MMR mechanism manifests itself as the phenomenon termed
microsatellite instability. When genomic mutations target microsatellite sequences
located at the coding region of genes controlling cell growth and differentiation, the cell
undergoes neoplastic transformation
33-35
. MutS heterodimers detect mismatches in
DNA duplexes and initiates the MMR machinery and MutL heterodimers make a
connection between the recognition and excision of the mismatch from the strand within
which it is contained
36, 37
. hMSH2 acts in the recognition of mismatched base pairs of
DNA damaged, mainly G-T, and in the recognition mismatched base pairs opposite to
cyclobutane pyrimidine dimers or pyrimidone photoproducts. Besides, hMSH2 interacts
with NER proteins to contain DNA interstrand cross-links UVR induced
38
.
NER is a highly conserved DNA repair pathway that deals with a wide range of
DNA lesions promoted by UVR-induced photoproducts, bulky mono-adducts, cross-
links, and oxidative damage. The basic steps of NER proteins are DNA damage
recognition; lesion demarcation and verification; assembly of a preincision complex;
DNA opening by helicases; dual incision by endonucleases typically a few bases away
from the lesion; release of the excised oligomer, repair synthesis to fill in the resulting
gap, and connection of strand
32, 39-43
. The ERCC1-XPF complex represents an
endonuclease that catalyzes 5’ incision of damaged site, and concomitant 3’ incision to
39
damage is catalyzed by XPG
31, 39
. XPF–ERCC1 is also known to be involved in
recombinational DNA repair and in the repair of DNA interstrand cross-links
44-47
. Pre
clinical studies discussed previously suggest that ERCC1 may be an important
predictive and/ or prognostic marker in human cancer
34, 46, 48-50
.
The BER pathway is responsible for primary repair base that arise due to
oxidative stress, alkylation, deamination and depurination/depyrimidination damages or
from spontaneous initiated hydrolysis of oxidative DNA damage. BER involves two
major classes of repair enzymes, the DNA glycosylases and APEs. Glycosylases
remove damaged bases, creating the apurinic/apyrimidinic sites that may block DNA
replication, leading to cytotoxic mutations or genetic instability. APE1
(apurinic/apyrymidinic endonuclease/redox effector-1), a multifunctional enzyme, is
responsible for removal of basic residues to generate 3’OH and 5’deoxyribose
phosphate termini, whose 3’OH termini is utilized by DNA β-polymerase for complete
repair synthesis
51, 51-55
. APE1 has also been involved in redox regulation, performed of
manner direct on transcription factors (such as, fos, jun, nuclear factor-kB, and p53)
enhances their DNA-binding activity or by indirectly facilitate the activation of these
factors by others reducing molecules
55,56
. Notoriously, little is known about BER
functions in malignant tumors, especially in head and neck sites
54, 55
.
Disruption of mechanisms that regulate cell-cycle checkpoints and DNA repair
result in genomic instability and contributes to the progression from dysplasia to cancer
in multicellular organisms. The identification of reliable biomarkers will lead to a better
understanding of the pathophysiology of lip disease progression. Moreover, these
biomarkers could be used as therapeutic targets in future therapeutic strategies. The aim
of this study was to analyze the immunohistochemical expression of p53, APE1,
hMSH2, and ERCC1 proteins in samples of lip mucosa, AC, and LSCC in order to
40
investigate the role of these proteins at different morphological stages of lip tumor
progression.
Samples and Methods
Ethical approval for this study was obtained from the relevant local ethics
committees (Unimontes/Coep-1113/2008).
Tissue Specimens
This retrospective study was performed on archived tissue blocks from
surgically resected samples of LSCC (n= 27; group male-to-female ratio= 8.0:1; group
mean age= 63.41 ± 15.90 years), of AC (n= 30; group male-to-female ratio= 3.2:1;
group mean age= 54.07 ± 16.40 years), and biopsies of lip mucosa (LM) used as
controls (n= 15; group male-to-female ratio 1.1:1; group mean age 20.40 ± 10.33 years).
Data relating to patients and samples were obtained from the Department of Dentistry at
the State University of Montes Claros, Minas Gerais, Brazil. The diagnoses of patients
presenting AC or LSCC lesions were confirmed by clinical examination and
histopathological analysis.
Anatomical site and clinical staging
Anatomical sites included in this study were 1- Mouth and perioral region (C00 -
lower and upper lip), according to International Classification of Diseases for Oncology
57
. All LSCC patients were classified according to the International Union against
41
Cancer - TNM Classification of Malignant Tumours
58
on the basis of the primary site
(C00). In the LSCC group, 29.6% of cases were in stage I, 37.0% were in stage II,
14.8% were in stage III and 18.5% were in stage IV.
Morphological staging
Five-µm-thick sections were cut, deparaffinized, and stained with hematoxylin
and eosin for histological examination. Morphological analysis was carried out by an
oral pathologist (AMB De Paula), without prior knowledge of the demographic or
clinical characteristics relating to the samples. Epithelial dysplasia degree in AC lesions
and cell differentiation degree of LSCC samples were graded using World Health
Organization (WHO) morphological methods
57
. Additionally, the analysis of risk of
malignant transformation of AC samples was based on criteria of Kujan et al.
59
. For
LSCC was also employed the analysis of invasive front grading system modified by
Bryne (1992)
60
, in which tumor cell characteristics were solely graded within the least
differentiated parts of the most invasive three to four cell layers at the advancing front
of tumors. All samples of LM exhibited morphologically normal epithelium tissue,
although in some samples a discrete mononuclear inflammatory infiltrate was detected.
Immunohistochemical reactions
Paraffin sections of 3µm-thick were mounted in slides, treated and submitted to
immunohistochemical method for p53, APE1, hMSH2 and ERCC1 proteins. The
primary antibodies against mouse monoclonal antibody anti-p53 (clone DO7,
Novocastra Lab., New Castle, UK), hMSH2 (clone 25D12, Novocastra Lab., New
Castle, UK), ERCC1 (clone 8F1, Abcam, Cambridge, UK) and APE1 proteins (clone
42
2104, Abcam, Cambridge, UK) was detected with the aid of an LSAB™ visualisation
kit (K0690; Dako, Glostrup, Denmark) employing the chromogen diaminobenzidine for
colour development. Slides were finally counterstained with Mayer’s hematoxylin and
mounted. Positive controls were sections of oral carcinoma that has previously shown to
be positive for antibodies investigated. Negative controls were obtained by substituting
normal whole rabbit serum (X0902; Dako) for the primary antibodies. Only cells that
presented brown nuclear staining were considered positive. The immunohistochemical
expression of biomarkers was evaluated in Olympus
®
BH2 microscope (x10 ocular and
x40 objective lens). The ocular lattice (grid) with 100 points composed of 10 horizontal
and 10 vertical test lines measuring 0.092 mm
2
was superimposed on the fields to be
measured. Immunohistochemical analyses of hMSH2, ERCC1, and APE1 proteins were
performed by obtaining the percentage of positive staining cells in all fields counted (12
fields for each specimen). The medians of the staining percentages of hMSH2, ERCC1
and APE1 proteins were used as cutoff to define the cases as low (≤ median) and high
(> median) values for all samples, considering as high expression when the biomarkers
expression were higher than median value
61
. Positive p53 expression was defined when
more than 10% of cells stained positively
62
.
Statistical analysis
All data were transferred to software SPSS
®
17.0 (SPSS Inc., Chicago, IL, USA)
and submitted to specific tests with statistic confidence 95% (p<0.05). Kappa statistic
was used to assess intra-examiner reproducibility (AMB De Paula) relating to
morphological grades of AC (n= 15) and LSCC (n= 15). Kappa test revealed good
concordance for AC (WHO, k= 0.571, p= 0.058; Risk of Malignant Transformation, k=
43
0.857, p= 0.001) and LSCC groups (WHO, 1995, k= 0.877, p= 0.000; Invasive Front
Grading, k= 0.492, p= 0.024).
The p53 expression in LM, AC and LSCC was examined by
2
-test with the
Fisher and Pearson exact tests. Kolmogorov-Smirnov test was used in order to see
whether the distributions of continuous variables were normal or not. Analyses of
hMSH2, ERCC1 and APE1 assumed non-parametrical distribution and were compared
between groups using Mann-Whitney statistical test. Comparative analysis of
biomarkers expressions according to morphological degree were examined by
2
-test
with the Fisher and Pearson exact tests.
Results
Figure 1 presents immunohistochemical expressions of biomarkers evaluated in
LM, AC and LSCC samples. Lip mucosa did not show any p53 tissue expression. With
respect to AC samples, p53 staining was predominantly found in basal, parabasal, and
suprabasal epithelial tissue. In LSCC group, it was shown a diffuse and accumulated
distribution of p53 staining in malignant parenchyma, but especially in areas of invasive
front. Tissue expression of APE1 protein was frequently noted in basal and parabasal
layer of LM samples. However, this expression consistently demonstrated strong
nuclear staining from basal layer to surface epithelium in about a half of AC cases,
independent of dysplastic changes of epithelial tissue. An increase of APE1 expression
was found in all parenchyma of LSCC lesions. Expression of hMSH2 was observed
most prominently just above the basal cell layer of the epithelium. Localization of
hMSH2 was exclusively nuclear and very prominent in the most proliferating parts of
44
the LM, AC, and LSCC epithelium. Similarly, ERCC1 expression was detected in all
epithelial layers in the majority of LM, AC and LSCC samples.
The p53 protein expression was detected in 60.0% and 92.6% of cases of AC
and LSCC, respectively. For APE1 protein, the mean percentages (mean ± SD) of tissue
expression in LM, AC and LSCC samples were 17.35 ± 30.04, 31.96 ± 39.56, and 58.19
± 38.27. hMSH2 expression in LM, AC and LSCC groups were 74.90 ± 35.44, 86.34 ±
22.23, and 81.34 ± 22.37, respectively. ERCC1 tissue expression in LM, AC, and LSCC
samples were 50.63 ± 39.79, 72.35 ± 28.44 and 53.43 ± 27.25.
Analysis of p53, APE1, hMSH2, and ERCC1 tissue expressions was
significantly different between AC and LSCC groups (p<0.05). The expression of p53
and APE1 in LM was significantly lower than LSCC (p=0.000 and p=0.001,
respectively). Similarly, the expression of p53 and APE1 in AC was significantly lower
than LSCC samples (p=0.004 and p=0.017, respectively). In addition, AC showed also
higher expression of p53 compared to LM (p=0.000). Controversially, decreased
expression of ERCC1 was found in AC compared to LSCC group (p=0.003). In similar
way, decreased expression of hMSH2 expression was observed in LSCC compared to
AC (p=0.016) (Figure 2).
The analyses of biomarkers expression according to the morphological grading
systems for both AC and LSCC samples did not show statistical significance between
groups, except for APE1 protein in LSCC group. In this group, it was found a high
expression of APE1 protein in samples with higher morphological aggressiveness,
according to invasive front tumor grade (Table 1).
45
Discussion
It has been postulated that genetic damage affecting activation of oncogenes
and/or inactivation of tumour suppressor genes, in conjunction with an impaired
capacity of DNA repair mechanisms, may be a factor in the development of squamous
cell carcinoma of the upper aerodigestive tract
13, 14, 63
.
Mutant p53 can be detected immunohistochemically based on the long half-life
of mutant versus wild-type p53
15
. It has been shown that p53-positive cells in normal
lip mucosa of individuals with history of chronic solar exposure occur only in 20% of
cases
64
. In our study, control lip samples did not exhibit p53-positive cells. It is
acceptable to infer that control cases have less cumulative exposure to the carcinogenic
factors since, in this group, the patients had the average age lower than AC and LSCC
individuals. Our data also showed that immunohistochemical detection of mutant p53
protein was observed in both premalignant and neoplastic disease of lip. In this way, as
shown here and by other works
25, 26, 28, 30
, the important role of mutational inactivation
of p53 during the initial lip tumourigenic steps, provided by both environmental factors
and genetic predisposition. The inactivation of p53 in lip carcinogenesis has been
associated to deregulations of p21, bcl-2, and Bax expressions with consequent
reduction of apoptosis
25, 28
. It is well known that p53 is essential in protecting cells
from UVR-induced DNA damage
65, 66
. Both UVA and UVB radiations are proved to
produce DNA damage directly and indirectly through oxidative stress, promoting
mutation in key genes associated to cell proliferation and/or apoptotic cell death
mechanisms
65, 67, 68
. It has been noted that TP53 gene seems to be a good target for UVR
and keratinocytes of lip seem to be particularly prone to this mutation
69
. Recently, it
was shown that p53 has also had an antioxidant function, up-regulating several genes,
46
such as DNA repair genes, that protect cells against oxidative stress
41, 70
. Therefore,
providing the basis for other aberrations, tissue overexpression of mutated p53 is likely
to be one of the first fundamental aberrations for multi-step process of lip
carcinogenesis.
The deleterious effects of UVR on cellular targets involve photosensitization and
the generation of reactive oxygen species, whose major effects is to promote DNA
damage
55
. APE1 represents a primary defense pathway against oxidative DNA damage
41
, whose repair efficiency is affected by cellular levels of APE1, in a manner rate-
limiting
71
. Similarly to findings of p53 in this current study, our results showed that
APE1 tissue expression increased progressively in epithelial cells from lip mucosa to lip
cancer groups. So, this biomarker also seems to represent an early event of lip tumor
progression. Other works have also implicated a high expression of APE1 in the
cervical
72
, and prostate
73
carcinogenesis. Additionally, our results showed a higher
expression of APE1 in LSCC samples of high morphological aggressiveness. The
increased APE1 expression in more invasive LSCC lesions, and maybe with high
metastatic potential, and so, with high biological aggressiveness seems to be associated
with both DNA repair and redox function alterations. In head and neck cancer, the high
expression of APE1 was associated with advanced nodal disease and resistance to
chemoradiotherapy
74
. A high tissue expression of APE1 has also been associated to
higher morphological aggressiveness in ovarian epithelial tumors and hepatocarcinoma
75
. Further studies are necessary to clarify if deregulated tissue expression of APE1 in
LSCC has prognostic significance.
Reduced DNA repair capacity in normal tissue may result in genomic instability
and constitute a risk factor for cancer development
76
. The critical role of NER in the
maintenance of genome integrity is underpinned by the fact that germline mutations in
47
several of the NER genes cause Xeroderma pigmentosum, a disorder characterized by
extreme sensitivity to sunlight and a predisposition to develop skin cancer and other
malignancies
77
. Physiologically, high level of ERCC1 tissue expression associated with
enhanced DNA repair activity and DNA damages accumulate faster in individuals with
suboptimal repair capacity
78
. A reduced expression of ERCC1 mRNA levels was
associated with a more than two-fold increased risk of head and neck squamous cell
carcinoma
79
. Our results showed that a high ERCC1 expression was noted during
transition from control group to AC lesions. However, decreased expression of ERCC1
occurred in the lip cancer group. It has been demonstrated that decreased ERCC1
function may be caused by continuous pathological action of endogenous or exogenous
genotoxic agents, that may affect gene transcription and stability of transcripts
34, 48, 80
.
Once established an ERCC1-deficient scenario, the lip transformed cells will have a
worse mutation repair and this could contribute importantly to the initiation
phenomenon of tumor lip.
MMR proteins are important mediators for the maintenance of genomic stability,
restoring pairing postreplicative errors, blocking the cell cycle and promoting apoptosis
in damage response
33, 81, 82
. It has also been reported that defective MMR proteins is a
contributing factor in the development of squamous cell carcinoma of head and neck
because of occurrence of microsatellite instability
13,14,83,84
. This microsatellite instability
is determined, in the large majority of cases, by inactivation of hMSH2 and hMLH1
genes, which prevent protein expression
81,46
. Therefore, the immunohistochemical
analysis of MSH2 protein in tumor tissues may be a useful screen for the detection of
defective hMSH2 mediated mismatch repair
85
. Overall, our findings showed a high
tissue expression of hMSH2 in all groups studied. It seems that hMSH2 expression is
probably not affected during the initial steps of lip carcinogenesis. In fact, hMSH2 gene
48
did not undergo molecular suppression, appearing active and functionally
overexpressed, as demonstrated by the high percentage of cell positivity in all groups.
However, during transition from premalignant to lip cancer, it was noted a significant
decrease in hMSH2 tissue expression. This downregulation expression of hMSH2
protein has been noted in other studies of human carcinogenesis, including head and
neck squamous cell
83, 86
. This finding has been associated with occurrence of more
aggressive phenotype, constituting a hallmark of potential mutator phenotype for this
type of neoplasm. In UVB-induced skin tumor, it has been suggested that the down-
expression of hMSH2 during tumor progression occurs as consequence of elevated
proliferation index associated with altered programmed cell death
87
. The rising
mutational risk of microsatellite sequences, observed when the replication rate
increases, might act as an activation input for MMR genes that are under the control of
other genes regulating cellular proliferation
86
. It will be important to verify in future
studies if a higher expression of hMSH2 in premalignant lip lesions occurs due to an
abnormal increase in the cellular proliferation and apoptosis reduction of preinvasive lip
cells compared to the control samples.
In conclusion, our findings confirm that tumor lip progression represents a
pathological process promoted by accumulated damages, which favor genetic e/or
epigenetic alterations in tumor suppressor and DNA repair proteins. The tissue
expression of p53, APE1, hMSH2 and ERCC1 proteins could help to discriminate lip
premalignant disease to lip cancer. APE1 expression was associated with LSCC lesions
with higher morphological aggressiveness. Still, we also speculate that p53, APE1,
hMSH2, and ERCC1 proteins would be potential targets for gene therapies of lip cancer
in the future.
49
Competing interests
None to declare.
Acknowledgements
This work was supported by the Fundação de Amparo a Pesquisa no Estado de
Minas Gerais (Fapemig) and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (Capes).
50
Figure 1 – p53, ERCC1, hMSH2 and APE1 immunohistochemical expression in lip
mucosa (LM), actinic cheilitis (AC) and lip squamous cell carcinoma (LSCC) samples.
Slides stained diaminobenzidine and counterstained with Mayer’s haematoxylin,
magnification 200 X and 400 X.
51
Figure 2 – Immunohistochemical expressions of p53, APE1, ERCC1, and hMSH2 in lip
mucosa (LM), actinic cheilitis (AC) and lip squamous cell carcinoma (LSCC) samples.
Bars indicate standard error and the differences were significant statistically at p<0.05.
52
Table
Table 1 – Analysis of p53, hMSH2, ERCC1 and APE1 proteins expression and morphological grades of AC and LSCC.
p53
hMSH2
ERCC1
APE1
Morphologic Parameters
Negative (%)
Positive (%)
p value
Low (%)
High (%)
p value
Low (%)
High (%)
p value
Low (%)
High (%)
p value
Actinic cheilitis
WHO Grade
Absent/Mild (n=12)
04 (33.3)
08 (44.4)
0.543
06 (40.0)
06 (40.0)
1.000
06 (40.0)
06 (40.0)
1.000
06 (40.0)
06 (40.0)
1.000
Moderate/Severe (n=18)
08 (66.7)
10 (55.6)
09 (60.0)
09 (60.0)
09 (60.0)
09 (60.0)
09 (60.0)
09 (60.0)
AC Morphological Risk Grade
Low risk (n=15)
06 (50.0)
09 (50.0)
1.000
08 (53.3)
07 (46.7)
0.715
09 (60.0)
06 (40.0)
0.237
07 (46.7)
08 (53.3)
0.715
High risk (n=15)
06 (50.0)
09 (50.0)
07(46.7)
08 (53.3)
06 (40.0)
09 (60.0)
08 (53.3)
07 (46.7)
Lip squamous cell carcinoma
WHO Grade
Well differentiated (n=16)
01 (50.0)
15 (60.0)
0.659
07 (50.0)
09 (69.2)
0.507
06 (42.9)
10 (76.9)
0.103
09 (64.3)
07 (53.8)
0.837
Moderate (n=04)
0 (0.0)
04 (16.0)
03 (21.4)
01 (7.7)
02 (14.3)
02 (15.4)
02 (14.3)
02 (15.4)
Poorly differentiated (n=07)
01 (50.0)
06 (24.0)
04 (28.6)
03 (23.1)
06 (42.9)
01 (7.7)
03 (21.4)
04 (30.8)
LSCC Invasive Front Grade
4-8 (n=11)
01 (50.0)
10 (40.0)
0.782
05 (35.7)
06 (46.2)
0.841
04 (28.6)
07 (53.8)
0.406
05 (35.7)
06 (46.2)
0.009*
9-12 (n=11)
01 (50.0)
10 (40.0)
06 (42.9)
05 (38.5)
07 (50.0)
04 (30.8)
09 (64.3)
02 (15.4)
13-16 (n=05)
0 (0.0)
05 (20.0)
03 (21.4)
02 (15.4)
03 (21.4)
02 (15.4)
0 (0.00)
05 (38.5)
53
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59
5 CONSIDERAÇÕES FINAIS
Os índices de baixo risco e alto risco de transformação maligna em lesões de QA foram
igualmente evidenciados. A maioria das amostras de CCEL exibiu morfologia bem
diferenciada e agressividade do fronte invasivo entre leve e moderada. As expressões
imunoistoquímicas das proteínas p53, APE1 e ERCC1 foram associadas ao processo da
carcinogênese labial, e a expressão de hMSH2 esteve associada à transição da QA para o
CCEL. As avaliações das proteínas p53, APE1, hMSH2 e ERCC1 podem auxiliar nos
diagnósticos de QA e CCEL, cujas expressões de p53 e APE1 apresentaram índices
crescentes na transição da QA para o CCEL, e decréscimos nas expresões de hMSH2 e
ERCC1 foram encontrados em CCEL. A expressão da forma mutada da proteína p53 foi
diferentemente expressa na mucosa labial saudável comparada à QA. Expressão aumentada
da proteína APE1 foi associada à evolução do grau de invasividade do fronte tumoral.
60
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92
APÊNDICES
Apêndice A – Artigo 2
Demographic Aspects and Clinicopathological Analysis of Actinic cheilitis in a Brazilian
Population.
Fonseca-Silva, T
1
; Santos, CCO
1
; Souza, LR
1
; Haikal, DS
2
; Bonan, PRF
3
; Guimarães, ALS
3
;
De Paula, AMB
3
1
Postgraduate. Health Sciences Program. State University of Montes Claros, Montes Claros,
Brazil.
2
Lecturer. DDS. Department of Dentistry. State University of Montes Claros, Montes Claros,
Brazil.
3
DDS, PhD, Lecturer. Health Sciences Program. State University of Montes Claros, Montes
Claros, Brazil.
Running Title: AC: clinicopathological analysis.
Key-words: Actinic cheilitis; epidemiology; clinical aspects; morphological staging;
prognostic.
Address for correspondence:
Dr. Alfredo Maurício Batista De Paula
Laboratório de Pesquisa em Saúde. Hospital Universitário Clemente de Faria. Universidade
Estadual de Montes Claros - Unimontes. Av. Cula Mangabeira, 562- Bairro Santo Expedito.
Montes Claros - Minas Gerais - Brazil.
CEP: 39401-001.
Phone: +51-21-38 32248327. Fax: +55-21-38 32298500
e-mail: ambpatologi@gmail.com
93
Abstract This study analyzed health records data of Actinic cheilitis (AC) patients (n=61). It was
investigated whether any association exists between severity of clinical appearance and
morphological changes of AC lesions with demographic and risk factors of disease. Formalin
fixed and paraffin-embedded AC archived specimens were stained with H&E for morphological
grading analysis. Differences between groups were examined by χ
2
-test and exact Fisher test.
Results showed that AC was more prevalent in older people, with outdoor activities. The male-to-
female ratio was 3.7:1. Lower lip was affected predominantly. A history of chronic sunlight
chronic exposure, tobacco and alcohol drinking habits were noted. Patients exhibited
predominately nonhomogeneous lesion. Morphologically, it was identified a high frequency of
mild/moderate dysplastic changes and low risk of malignant transformation. It was noted an
association between chronic solar exposure and well-differentiated lesions (p=0.015). Severe
dysplastic epithelial changes and high risk of malignant transformation were associated with
tobacco-users (p=0.037 and p=0.020, respectively). Analysis of demographic, and
clinicopathological parameters of AC can provide a background to better understanding of lip
premalignant disease in order to obtain an effective diagnosis, prevention, treatment, and follow-
up.
94
Introduction
The Actinic cheilitis (AC) is a potentially malignance disorder of the vermillion of the lip
that results mainly from the chronic exposition to ultraviolet (UV) component of solar radiation,
especially UVB (Cavalcante et al. 2008) and other climatic factors such as climate, temperature
and humidity (Xavier et al. 2009). However, other exogenous causes, as tobacco use, seem to
determine the occurrence of illness (Cavalcante, Anbinder, & Carvalho 2008;Jaber et al.
1999;Markopoulos et al. 2004). Besides, imbalances in genetic mechanisms that affect immune
system and cell cycle have been recently documented (Martinez et al. 2008;Xavier et al. 2009).
Frequently, AC occurs in white men who work in areas exposed to solar UV radiation.
The highest incidence of disease has been observed towards the fourth and fifth decades of life.
Lower lip is usually more affected than upper lip (Kaugars et al. 1999). AC lesion manifests as an
asymptomatic white hyperkeratotic disorder that can present crusting, erosions, and, sometimes, a
chronic bleeding. The affected lip turns to a dry, wrinkled and descamative semimucosa, and
exhibits a white to gray color in its surface, with interspersed red areas (Rogers, III and Bekic
1997) and, frequently, the lesion may be either well or ill defined. The histopathological features
included acanthosis, basophilic change of the connective tissue (solar elastosis), chronic
inflammation within the connective tissue, perivascular inflammation, and thickness of the
keratin layer (Kaugars et al. 1999). However, the most important morphological findings refer to
epithelial tissue alterations. The histological features of the epithelium in AC may vary from
hyperkeratosis to varying grades of dysplasia ranging from mild to severe until carcinoma in situ
or even LSCC. Neoplastic parenchyma of LSCC is characterized by small islands, cords or nests
95
of malignant epithelium invading into the underlying connective tissues with different grades of
inflammation, which it is chronic predominantly (Chou et al. 2009).
AC is an important potentially premalignant condition involving the lip. Therefore,
special attention must be directed to AC patients with intent to prevent the occurrence of lip
squamous cell carcinoma (LSCC) (Epstein et al. 2002). Some studies have described, in the
literature, the percentages of malignant transformation of this lesion, whose values range from
approximately 10-20% of the cases (Xavier et al. 2009). It is estimated that 95% of lower lip
cancer cases originate from AC (Santos et al. 2003). Lip carcinogenesis may result from various
exogenous and endogenous risk factors acting in an independent, antagonistic, or synergistically
way to produce early dysplastic epithelial alterations. The presence of epithelial dysplasia
evaluated by morphological examination is one of the prognostic predictors of malignant
transformation of the potentially malignant lesions (Brennan et al. 2007;Lee et al. 2006).
The AC has great relevance due to its considerable incidences in tropical countries and its
potential malignant transformation. The knowledge of local patients’ profile is important to
ascertain the magnitude of the problem and to identify predisposing factors and ways of
controlling the disease. Still, although AC is a recognized precancerous lesion of the lip, not
many studies in the literature have examined the association of epidemiological, clinical and
histopathological spectrum. Therefore, the aim of this descriptive, cross-sectional study was to
evaluate the epidemiological and clinicopathological data of AC patients in a Brazilian sample.
Besides, we investigated associations between these parameters according to severity of clinical
appearance and epithelial dysplastic change in AC samples.
96
Subjects and Methods
Patients
This study evaluated a series of AC patients (n= 61) who were treated at the Clinic of
Stomatology of the State University of Montes Claros, between 2002 and 2009, in Montes Claros
city, Minas Gerais state, Brazil (latitude: 16° 44' 06' - longitude: 43° 51' 42''). The health records
of these patients were retrieved and demographic, risk factors, and clinicopathological data were
analyzed. Socio-demographic, risk factors, and clinical factors were recorded. Soon after clinical
diagnosis, all AC patients were treated with lip sunscreen during the twenty-one days period.
After this time, if lesions remained, a complete excision of disease was carried out to treatment
and evaluation of its morphological aspects. All patients were submitted to regular follow-up.
Ethical approval for this study was obtained from the local ethics committees (Unimontes/Coep,
protocol 629/2007).
Clinical classification and anatomical site
Anatomical sites included in this study were lower and upper lip. Clinically, AC lesions
were classified in homogeneous and non-homogeneous (Figure 1A, B). Homogeneous AC
lesions were considered as a predominantly white lesion of uniform flat, thin appearance that may
exhibit shallow cracks and has a smooth, wrinkled, or corrugated surface with a constant texture
throughout this area. Non-homogeneous AC lesions were defined as a predominantly white or
white and red lesion that may be irregularly flat, nodular, or exophytic, and further subdivided
into nodular, ulcerated, verrucous, and speckled types (Lee et al. 2006). If the clinical appearance
97
of a lesion did not allow an unequivocal categorization, agreement was further achieved after
discussion between specialists.
Definitions of terms used for ethnicity (skin color), tobacco and alcohol drinking habits
Although ethnicity could not be truly established, respecting the hazards of judging
Brazilians by color, race and geographical origin as demonstrated in the past findings (Parra et al.
2003), we analyzed the parameter skin color classifying patients as white and nonwhite. Cases
considered as exposed to chronic solar exposure had cumulative and intense solar exposure at the
job environment, such as farmers, employees of building construction, or garbage collector. Non-
exposure has not occupational antecedents of intense sunlight chronic exposure, such as teacher
or dentist. It was also considered the time and the duration of solar exposition. The tobacco habit,
including type of tobacco use (industrial cigarettes, cigars, and associations), number of years of
smoking, and the average daily consumption of cigarettes were investigated. The drink habit
(intake of beer and cachaça, a very popular and inexpensive sugar cane spirit with 35-45% ethyl
alcohol content), was investigated. A can of light beer (i.e. 330 ml) or a drink of cachaça (i.e.
40ml) contains 12 g of ethanol. Alcohol drinking habit was just classified as present or absent.
Ex-drinkers and ex-smokers were subjects who had abstained from any type of drinking and
smoking for at least one year. Smokers and ex-smokers were grouped combined. Similarly, with
regard to alcohol intake, user patients of alcohol beverage were grouped with ex-drinkers.
Tissue Specimens and Morphological Grading
Archived tissue blocks were obtained from Oral Pathology Laboratory of the State
University of Montes Claros from surgically resected samples of AC (n=28). Hematoxylin and
98
eosin-stained sections of AC lesions were examined under a light microscope. All specimens
were analyzed by the same examiner (AMB, De Paula), without prior information about the
epidemiological and clinical findings about each sample. It was noted, in all samples, basophilic
connective tissue changes consistent with solar elastosis. Other morphological changes were also
noted, such as hyperplasia or epithelial atrophy, hyper-para(ortho)keratosis, (hiper)granulosis,
and an inflammatory infiltrate, usually mononuclear chronic in the connective tissue. All cases
showed epithelial change equal to or greater than mild epithelial (Figure 1C, D). The epithelial
dysplasia was graded using two morphological methods: World Health Organization (2005)
criteria, and morphological analysis of risk of malignant transformation (low and high risk),
according criteria of Kujan et al. (2007).
Statistical analysis
Kappa statistic was used to assess intraexaminer reproducibility (AMB De Paula) for
morphological grading of AC samples (n=15) from our files. Two weeks later, in order to
calibrate the same observer, the same morphological criteria were considered and the samples
were regraded. The intraexaminer concordance and p value for measurements of morphological
grading of AC (k=0.726, p<0.05) revealed a good agreement result. Kolmogorov-Smirnov test
was used in order to evaluate whether the distribution of continuous variable (age) was normal or
not. For this variable, the distribution of data exhibited a normal distribution and was compared
using the Student´s t-tests and ANOVA statistical tests. The other associations (non-parametrics)
were examined using the
2
-statistical tests with Pearson and Fisher’s exact tests. Statistical
analysis showing confidence interval above 95% (p<0.05) was considered significant. All tests
99
for significance were two-sided. All statistical analyses were performed with the statistical pack
SPSS
®
(SPSS Inc., Chicago, IL, USA), version 13.0 for Windows
®
.
Results
This study included 61 cases clinically diagnosed as AC, which 28 cases were
morphologically confirmed. The male-to-female ratio was 3.7:1 with a mean age (± standard
deviation) of 56.38 (± 17.13) years, ranging from 16 to 98 years, respectively). Mean age for men
and women were 57.35 (± 17.33) and 52.77 (± 16.52) years, respectively. The AC involved
mainly the lower lip (96.7%). Majority of patients were tobacco and alcohol drinking users
(62.3% and 55.7%, respectively), with a chronic history of tobacco consumption (57.9%). The
average daily consumption of cigarettes/day was noted in 60.5% of individuals. Forty-nine
patients (80.3%) reported a chronic solar exposure history for less than eight hours daily, during a
period of more than twenty years. At the moment of clinical diagnosis, AC presented as non-
homogeneous lesions (78.7%). All AC patients were prescribed labial sunscreen as first choice of
treatment. However, twenty-eight patients (45.9%) showed non-regression of lesion after
treatment. In this group, the AC lesions were submitted to morphological examination. It was
noted a predominance of mild and moderate dysplasia cases (42.9% and 39.3%, respectively) and
lesions with lower risk of malignant transformation (53.6%) (Table 1).
Table 2 shows the comparative analysis of the demographic data, and risk factors between
homogenous and non-homogeneous AC clinical appearance. It was not found a statistically
100
significant difference between epidemiological factors and the clinical appearance of AC lesions
(homogeneous and non-homogeneous lesions).
The association between the morphological analysis of AC samples and epidemiological
and clinical factors of lip disease is presented in table 3. It was noted an association between
occurrence of chronic solar exposure and lesions with low epithelial dysplastic changes
(p=0.015). Severe dysplastic epithelial changes and lesions with high risk of malignant
transformation were associated with tobacco-users patients (p=0.037 and p=0.020, respectively).
Discussion
AC is widely recognized as a precancerous lesion of the lip and represents an important
public health problem in many countries around the world (Kaugars et al. 1999;Lindqvist et al.
1981;Main and Pavone 1994;Markopoulos, Albanidou-Farmaki, & Kayavis 2004). However, it is
important to consider specific regional contexts, in order to verify the influence of typical risk
factors, the habits of population, and the environmental conditions in which individuals have
been exposed to.
The classical profile for a patient at high risk for AC is white older men who exhibit
chronic history of solar exposure, the most important etiologic factor for disease (Cataldo &
Doku 1981;Kaugars et al. 1999;Lundeen et al. 1985;Main & Pavone 1994;Markopoulos,
Albanidou-Farmaki, & Kayavis 2004). Concerning gender, females seem to be less likely to
suffer from AC because of frequent lipstick use which can protect the lip from sunlight,
especially the products that contain sunscreen. Additionally, females still engage themselves in
less farm or outdoor activities (Markopoulos, Albanidou-Farmaki, & Kayavis 2004). However,
101
this job setting for this group has changed noticeably in the last decades. At present, females are
exposed to sunlight as often as males due to their similar job activities. Although our study is
retrospective and has been too short in duration to ascertain whether there is a trend toward more
women being diagnosed, this group must also be considered in public health strategies. As
verified in our study, AC disease was not exclusive to fair-skinned individuals. Other studies
have also been identifying a high incidence of AC in non-white individuals (Cavalcante,
Anbinder, & Carvalho 2008;Goracci et al. 1981).
Optical radiation within the electromagnetic spectrum includes ultraviolet radiation (UV),
visible light, and infrared radiation. UVR is characterized as the radiation between 100 and 400
nm in length (World Health Organization 1994). It is characterized according to wave length into
ultraviolet A (315-400nm), B (280-315nm), and C (100-280nm). UVA is responsible for skin
aging, and has more recently been implicated, along with UVB, in the development of skin
cancers. The major source of UV is solar radiation, although exposure to artificial sources in the
workplace and tanning salons are becoming relevant in terms of effects on human health
(Gallagher and Lee 2006;Marrot and Meunier 2008). The epidermis of the skin has the ability to
absorb UV radiation without damage to epithelial cells because of its thicker and higher melanin
content. When UV and visible radiations reach the skin, one part is reflected, and the other part is
absorbed in its various layers. UVB are mainly absorbed by epidermal cells components (eg,
proteins or DNA), whereas UVA radiation penetrates deeply into the skin, reaching the basal
layer of the epidermis and even dermal fibroblasts (Marrot & Meunier 2008). However, the lip
has less protection than the skin because the epithelium is thinner, usually lacks the thicker
keratin covering of skin, has less melanin, and has fewer secretions from sebaceous and sweat
glands (Kaugars et al. 1999;Nicolau & Baelus 1964). Curiously, a series of studies has revealed
102
that sunlight is probably of minor importance as a risk factor in AC and lip cancer (Lindqvist,
Teppo, & Pukkala 1981;Lindqvist and Teppo 1978). Chronic exposure to sunlight is commonly
noted in patients with AC (Cataldo & Doku 1981;De Visscher and van, I 1998;Kaugars et al.
1999;Main & Pavone 1994;Nicolau & BAELUS 1964;Streilein 1992). In current study, we found
an important participation of the traditional risk factor for AC occurrence. The northern region of
Minas Gerais state in Brazil assumes importance for disease because of high solar radiation level,
typical of tropical regions. Besides, this region maintains its economy based on agriculture
activities, and therefore, many people work subjected to long periods under sun radiation
exposure (Rola et al. 2009).
In this study, it was showed a higher occurrence of lip disease on lower lip. A few
considerations have been reported in literature about possible reasons for the higher occurrence of
AC in this anatomical site than upper lip. Anatomically, lower lip shows a lesser solar protection
promoted by nose and frequently, it is naturally everted. In both situations, lower lip turns more
susceptible to the sunlight exposure. Still, it is probable that tobacco and alcoholic beverage
products might also act more frequently for occurrence of disease in lower lip. It has been
suggested that the etiology of upper lip cancer could be regarded as a separate entity and different
from the lower lip (Lindqvist & Teppo 1978). However, further studies need to be conducted to
clarify this question.
AC represents a clinical lesion with a spectrum of morphological findings (Kaugars et al.
1999). The presence and degree of epithelial dysplasia represents one of the various prognostic
predictors of malignant transformation of lesions (Brennan et al. 2007;Cavalcante, Anbinder, &
Carvalho 2008;Kaugars et al. 1999;Main & Pavone 1994;Markopoulos, Albanidou-Farmaki, &
Kayavis 2004; Robinson 1989). Nevertheless, it is recognized that occasionally non-dysplastic
103
lesions may turn into cancer, while not all dysplastic lesions become malignant (Brennan et al.
2007). Besides, spontaneous regressions of oral dysplasias have also been reported, especially
after discontinuation of habits associated with cancer risk (e.g., smoking tobacco), and changes in
lifestyle (i.e., habit or dietary intervention), respectively (Brennan et al. 2007). Similar to other
works (Cavalcante, Anbinder, & Carvalho 2008;Kaugars et al. 1999;Markopoulos, Albanidou-
Farmaki, & Kayavis 2004), all samples of this study presented with some degree of epithelial
dysplasia.
In this current study, we did not find associations between epidemiological and
morphological variables with the clinical appearance of AC lesion. The early clinical signs of AC
disease are subtle. Due to the slow evolution of disease, patients commonly attribute the
alterations to the process of aging and neglect the lesion until the malignant transformation
occurs and lip cancer reaches advanced tumor stage (Cavalcante, Anbinder, & Carvalho
2008;Rogers, III & Bekic 1997). It is interesting to note that, even when late clinical signs of
disease appear, the severity of clinical change is not necessarily related to the occurrence and
degree of dysplastic alteration of epithelial tissue (Kaugars et al. 1999).
Tobacco smoking is the most important etiologic factor in oral potentially premalignant
lesions and squamous cell carcinoma of the upper aerodigestive (Banoczy et al. 2001;De Paula et
al. 2009;Reichart 2001). Similarly, cigarette smoking has been mentioned as a fundamental
etiologic factor for AC (Cataldo & Doku 1981;Cavalcante, Anbinder, & Carvalho 2008;Freitas et
al. 2008). In our study, we identified the tobacco use among the majority of AC patients. Still,
our results showed a higher occurrence of more serious degree of epithelial dysplasia between
AC lesions in smoker than nonsmoker users. Lee et al. (2006) showed that smoking habit had
significant impact on the presence of epithelial dysplasia in oral leukoplakia, the most important
104
potentially premalignant lesion of the mouth. Tobacco may induce an alteration in oncogenes and
tumor suppressor genes, which subsequently leads to dysplastic changes verified during oral
carcinogenesis (Chen et al. 2008). Our results also showed that a higher morphological grade of
malignant transformation was found in the AC lesion of smokers. It has been hypothesized that
strong carcinogenic factors such as smoking can shorten the transformation process from AC to
frank cancer or even skip the stage of dysplastic lesion formation (Lee et al. 2006). On the other
hand, some studies have demonstrated that smoking-related oral potentially premalignant
conditions seem to have less malignant potential than those not associated with smoking (Einhorn
and Wersall 1967;Schepman et al. 1998;Silverman S Jr et al. 1984). Regarding alcohol drinking
habit, although it has been noted the occurrence of this risk habit between AC patients in this
current study, it was not found any association between this habit and severity of clinical
appearance and dysplastic changes of lesion.
Unexpectedly, our results showed a high severity of dysplastic alterations in AC lesions of
individuals without history of chronic solar exposure. The reasons for this controversial finding
are not clear. UV radiation can be absorbed by a wide range of organic molecules and can thus
initiate photochemical reactions that alter these absorbing molecules (chromophores) or others in
subsequent chemical reactions. UVA and UVB radiations are proved to produce DNA damage
directly and indirectly through oxidative stress, which may contribute to genetic imbalances
occurrence and promotion of architectural tissue disturbance accompanied by cytological atypia
(Kambayashi et al. 2003). In this way, it may be that, in the absence of UVR damage, there could
be other exogenous and endogenous etiologic factors acting in the affected lip. Through results
found in this study, it is reasonable to accept that chronic exposure to sunlight associated with
105
other exogenous factors, such as smoking and alcohol drinking habits, in a background of genetic
susceptibility, must produce a synergistic, multiplicative effect on etiopathogenesis of AC.
However, a more likely explanation is that from the viewpoint of multistage lip carcinogenesis,
factors affecting the occurrence of lip disease may be different from those responsible for the
severity of dysplastic changes exhibited by epithelial tissue during the lip carcinogenesis.
In conclusion, our data showed that traditional risk factors for AC disease influenced the
severity of morphological grading of lesions. Analysis of clinicopathological parameters of AC
can provide a background to better understand the potentially premalignant condition of lip.
106
Acknowledgments
This study was supported by Foundation for the Support of Research of Minas Gerais
State (Fapemig).
Conflict of interest
None was declared.
107
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110
Tables
Table 1 - Descriptive analysis of epidemiological and clinicopathological characteristics of
AC sample.
Variables
n
%
Variables
n
%
Gender
Skin Color
Male
48
78.7
White
25
41.0
Female
13
21.3
Non-white
36
59.0
Residence
Sunlight Chronic Exposure
Urban
27
44.3
Yes
49
80.3
Rural
34
55.7
No
12
19.7
Time of Sunlight Exposure
Duration of Solar Exposure
≤8 hours/day
27
55.1
≤20 years
15
20.6
>8 hours/day
22
44.9
>20 years
34
69.4
Tobacco Habit
Type of Tobacco
Ever
38
62.3
Industrial cigarettes
17
44.7
Never
23
37.7
Straw cigarettes
08
21.1
Association
13
34.2
Quantity of Tobacco
≤10 cigarettes/day
15
39.5
Duration of Tobacco Habit
>10 cigarettes/day
23
60.5
≤20 years
16
42.1
>20 years
22
57.9
Alcohol Drinking Habit
Ever
34
55.7
Anatomical Site
Never
27
44.3
Lower lip
59
96.7
Upper lip
02
3.2
Clinical
Appearance
Lesion Regression after
Treatment
Non-homogeneous
48
78.7
Yes
33
54.1
Homogeneous
13
21.3
No
28
45.9
Epithelial
Dysplasia
Risk of Malignant
Transformation
Mild
12
42.9
Low risk
15
53.6
Moderate
11
39.3
High risk
13
46.4
Severe
05
17.8
111
Table 2- Association between epidemiological and clinical appearance of AC lesions (n=61).
Values were calculated by
2
-statistical test.
+
Association compared with Student´s t-statistical test.
* p value significant.
Clinical Appearance
Parameters
Homogeneous
Nonhomogeneous
p value
Age
+
53.54
53.96
0.937
Gender
Male
11 (84.6%)
37 (77.1%)
0.556
Female
02 (15.4%)
11 (22.9%)
Skin Color
White
05 (38.5)
20 (41.7)
0.835
Non-white
08 (61.5)
28 (58.3)
Sunlight Chronic Exposure
Yes
12 (92.3%)
37 (77.1%)
0.221
No
01 (7.7%)
11 (22.9%)
Time of Solar Exposure
≤8 hours/day
08 (66.7%)
19 (51.4%)
0.345
>8 hours/day
04 (33.3%)
18 (48.6%)
Duration Solar Exposition
≤20 years
04 (33.3%)
11 (29.7%)
0.814
>20 years
08 (66.7%)
26 (70.3%)
Tobacco Habit
Ever
10 (76.9%)
28 (58.3%)
0.220
Never
03 (23.1%)
20 (41.7%)
Quantity of Tobacco
≤10 cigarette/day
04 (40.0%)
11 (39.3%)
0.968
>10 cigarette/day
06 (60.0%)
17 (60.7%)
Duration of Tobacco Habit
≤20 years
05 (50.0%)
11 (39.3%)
0.556
>20 years
05 (50.0%)
17 (60.7%)
Alcohol Drink Habit
Ever
10 (76.9%)
24 (50.0%)
0.083
Never
03 (23.1%)
24 (50.0%)
112
Table 3- Association between epidemiological factors and morphologic grading for AC lesions resistant to
sunscreen treatment.
Values were calculated by
2
-statistical tests with Pearson and Fisher’s exact tests.
+
Association compared with Anova for epithelial dysplasia grading and Student´s t-test for analysis of risk of malignant transformation grading.
* p value significant.
Epithelial Dysplasia
Grading
Risk of Malignant
Transformation Grading
Parameters
Mild
Moderate
Severe
p value
Low
High
p value
Age
+
52.64
57.55
56.80
0.980
55.73
58.00
0.743
Gender
Male
10 (83.3%)
10 (90.9%)
04 (80.0%)
0.806
12 (80.0%)
12 (92.3%)
0.600
Female
02 (16.7%)
01 (9.1%)
01 (20.0%)
03 (20.0%)
01 (7.7%)
Skin Color
White
07 (58.3)
04 (36.4)
03 (60.0)
0.509
08 (57.1)
06 (46.2)
0.705
Non-white
05 (41.7)
07 (63.6)
02 (40.0)
07 (46.7)
07 (53.8)
Sunlight Chronic Exposure
Yes
10 (83.3%)
11 (100.0%)
02 (40.0%)
0.015*
13 (86.7%)
10 (76.9%)
0.639
No
02 (16.7%)
00 (0.0%)
03 (60.0%)
02 (13.3%)
03 (23.1%)
Time of Solar Exposure
≤8 hours/day
04 (40.0%)
07 (63.6%)
00 (0.0%)
0.204
06 (46.2%)
05 (50.0%)
0.855
>8 hours/day
06 (60.0%)
04 (36.4%)
2 (100.0%)
07 (53.8%)
05 (50.0%)
Duration of Solar Exposure
≤20 years
05 (50.0%)
02 (18.2%)
00 (0.0%)
0.177
05 (38.5%)
02 (20.0%)
0.405
>20 years
05 (50.0%)
09 (81.8%)
2 (100.0%)
08 (61.5%)
08 (80.0%)
Tobacco Habit
Ever
04 (33.3%)
09 (81.8%)
04 (80.0%)
0.037*
06 (40.0%)
11 (84.6%)
0.016*
Never
08 (66.7%)
02 (18.2%)
01 (20.0%)
09 (60.0%)
02 (15.4%)
Quantity Tobacco
≤10 Cigarette
02 (50.0%)
03 (33.3%)
03 (75.0%)
0.378
02 (33.3%)
06 (54.4%)
0.620
>10 Cigarette
02 (50.0%)
06 (66.7%)
01 (25.0%)
04 (66.7%)
05 (45.5%)
Duration of Tobacco Habit
≤20 years
01 (25.0%)
02 (22.2%)
03 (75.0%)
0.164
02 (33.3%)
04 (36.4%)
1.000
>20 years
03 (75.0%)
07 (77.8%)
01 (25.0%)
04 (66.7%)
07 (63.6%)
Alcohol Drinking Habit
Ever
06 (50.0%)
06 (54.5%)
04 (80.0%)
0.510
09 (60.0%)
07 (53.8%)
0.743
Never
06 (50.0%)
05 (45.5%)
01 (20.0%)
06 (40.0%)
06 (46.2%)
Clinical Appearance
Homogeneous
01 (25.0%)
02 (50.0%)
01 (25.0%)
0.735
02 (50.0%)
02 (50.0%)
1.000
Non-homogenous
11 (45.8%)
09 (37.5%)
04 (16.7%)
13 (54.2%)
11 (45.8%)
113
Apêndice B – Artigo 3
Epidemiological Study and Clinicopathological Analysis of Lip Squamous Cell Carcinoma in
a Brazilian Population.
Souza, LR
1
; Silva, TF
1
; Santos, CCO
1
, Corrêa, GTB
1
; Haikal, DS
2
; Santos, FBG
3
; Cardoso,
CM
3
; Guimarães, ALS
4
; De Paula, AMB
4
1
Postgraduate. Health Science Program. State University of Montes Claros, Montes Claros,
Brazil.
2
Lecturer. DDS. Department of Dentistry. State University of Montes Claros, Montes Claros,
Brazil.
3
Lecturer. DDS. Department of Medicine. State University of Montes Claros, Montes Claros,
Brazil.
4
Lecturer. DDS. Health Science Program. Department of Dentistry. State University of
Montes Claros, Montes Claros, Brazil.
Brief running title: Demographic and Clinical Analysis of LSCC.
Keywords: Squamous cell carcinoma; lip; epidemiology, risk factors; clinipathologic
parameters.
Address for correspondence:
Dr. Alfredo Maurício Batista De Paula
Laboratório de Pesquisa em Saúde. Hospital Universitário Clemente de Faria. Universidade
Estadual de Montes Claros - Unimontes. Av. Cula Mangabeira, 562- Bairro Santo Expedito.
Montes Claros - Minas Gerais - Brazil.
CEP: 39401-001.
Phone: +51-21-38 32248327. Fax: +55-21-38 32298500
e-mail: ambpatologi@gmail.com
114
Abstract This retrospective, cross-sectional study evaluated the association between
epidemiological and clinicopathological profile of 30 primaries lip squamous cell carcinoma
(LSCC) in a Brazilian population. LSCC represented 10.8% and 3.5% of all oral cavity and
head and neck squamous cell carcinoma, respectively. LSCC was more prevalent in elderly
men, with male-to-female ratio of 5:1. Lower lip was more affected (83.3%) and it was
statistically associated with chronic solar exposure (p=0.019). It was observed high rates of
tobacco (80%) and alcohol drinking habits (60%). Early TNM staging, T1/T2 tumor size, and
non-metastatic disease were more predominant. Morphologically, it was identified a high
frequency of well differentiated tumor samples. Worse Karnofsky performance status was
associated with cervical metastasis (p=0.035). T3/T4 lesions and female patients group were
associated with LSCC lesions with high severity of the morphological aggressiveness
(p=0.004 and p=0.023, respectively). Analyses of epidemiological, clinical and morphological
data of LSCC patients add a better understanding about the profile of LSCC patients of a
specific population. Our findings can be useful for public health planning, stimulating the
creation of health promotion strategies in order to orientate the population and health
professionals.
115
Introduction
Lip squamous cell carcinoma (LSCC) is a common malignancy of the mouth, and it is
responsible for over 30% of all oral squamous cell carcinomas
1
. In some countries, the
incidence of disease can reach rates of 50%, considering all oral cancers
2
. LSCC occurs in
the lower lip about 80% of all cases and results mainly of chronic exposition to ultraviolet
(UV) component of the solar radiation, specially UVB
3
. Main factors associated with disease
development are outdoor activity and skin color. However, actinic radiation may indeed not
be the only risk factor for lip carcinogenesis. Factors socioeconomic and lifestyle factors such
as smoking and dietary habits, immunosuppression, and genetic predisposition might produce
a synergistic effect
, 4-7
.
LSCC may evolve from lip premalignant conditions, as Actinic cheilitis (AC), or even
from the healty lip
8
. As most malignancies, the occurrence of LSCC increases with age
progression
9
. The time needed for sun-induced changes of the lip to evolve into cancer varies
from 20 to 30 years, but such evolution can occur in less time in some individuals
10
.
Although LSCC occurs frequently in a site of easy visualization by own patients and health
professionals, the delay in diagnosis ranges from 5 months to 2 years, especially justified by
the fact that the disease is generally asymptomatic and the lip is often affected by other non-
neoplastic diseases frequently. These facts seem to explain why the average time of patient
delay is somewhat late for this type of cancer
11-13
. LSCC shows survival rate higher than 5-
years, exhibiting a better prognosis compared to same disease in other sites of upper
aerodigestive tract
14
. The early detection of disease has been considered the better prognostic
factor for survival increase of individuals
15
.
116
The LSCC has great relevance by considerable incidences on tropical countries. The
difference in epidemiology of disease seems to be influenced by local socio-demographic and
cultural characteristics. This background is important to ascertain the magnitude of the public
health problem. Therefore, the purpose of this descriptive, cross-sectional study was to
delineate the epidemiological and clinicopathological profile of a LSCC Brazilian sample.
Besides, we investigated possible associations between epidemiological variables and
clinicopathological staging of LSCC group.
Patients and Methods
Study Design, Patients and Ethical Aspects
This descriptive, cross-sectional study was performed analyzing a series of 30 fully
reviewed cases of primary LSCC selected from 724 fully reviewed cases of primary upper
aerodigestive tract squamous cell carcinoma, confirmed histologically
16
. This sample was
obtained from public and private health centers in Montes Claros city - Minas Gerais state,
Brazil (latitude: 16° 44' 06' - longitude: 43° 51' 42'') (Figure 1), between 1996 and 2008. Data
were gathered from the medical and dentistry health records for this investigation as follows:
age, gender, anatomical site, ethnicity, cancer family history, Karnofsky performance status
scale (KPS), tobacco and alcohol drinking habits, and tumor clinical staging (TNM). The
protocol for the majority of patients included surgical resection of the primary tumour
associated with a conservative or radical neck dissection in cases of suspect lymph nodes
metastases as evidenced by clinical or imaginological exams. None of the patients examined
presented distant metastases. The group of young patients was composed by the subjects who
117
were aged 45 years or less. Ethical approval was obtained from the relevant local ethics
committees (Unimontes/COEP, protocol 1113/2008).
Anatomical Site and Clinicopathological Staging
The anatomical sites reviewed in this study included 1- Mouth and perioral region - lip
(C00). All patients were staged according to the UICC TNM Classification of Malignant
Tumors (1997)
17
. LSCC was classified according to the primary site as described in the
International Classification of Diseases (ICD-10) for Oncology (WHO, 1990). Two
histological grading were used: (1) WHO’s grading system (divided squamous cell carcinoma
into well, moderately and poorly differentiated). (2) The invasive front grade (IFG) modified
by Bryne (1991)
18
considers the most invasive three to four cell layers at the advancing front
of tumors. It consists of morphological parameters: degree of keratinization, nuclear
pleomorphism, pattern of invasion and inflammatory cell infiltration, which were scored from
1-4 individually. The scores for each morphological feature were summed into a total
malignancy score. At end, the samples were categorized as follows: 4-8, 9-12, and 13-16
groups. High values for IFG has been associated with independent prognosis significance for
oral squamous cell carcinoma (Bettendorf et al., 2004).
Definitions of Terms Used for Ethnicity, Family Cancer History, KPS, and Tobacco and
Alcohol Drinking Habits
Although ethnicity could not be truly established, respecting the findings about the
hazards of judging Brazilians by color, race and geographical origin
19
. This variable was
analyzed classifying patients according to skin color as white and non-white. Cases
considered as exposed to chronic solar exposure had cumulative and intense solar exposure in
118
the job’s environment, such as farmers, employers of building construction, or garbage
collectors. Non-exposure has not occupational antecedents of intense and chronic solar
exposition, such as teacher or dentist. All patients were asked about the occurrence of cancer
in a first relative. Cancer term was defined using WHO definition as ‘‘an uncontrolled growth
and spread of cells that may affect almost any tissue of the body’’. Performance status is a
global assessment of the patients’ actual level of function and ability of self-care. All the
patients were categorized according to the Karnofsky performance status scale index (KPS)
20
.
It describes the ability of patients in to perform normal activity and do active work, and
whether there is any need for assistance of oncologic patients. The scale is rated on 0-100, in
steps of 10 grade. At 100 all is well; at 0 the patient is dead
20
. The drink habit (intake from
overall alcohol, beer and cachaça, a very popular and inexpensive sugar cane spirit with 35-
45% ethyl alcohol content), was also investigated. A can of light beer (i.e. 330 ml) or a drink
of cachaça (i.e. 40ml) contains 12 g of ethanol. Alcohol drinking habit was classified as
present or absent. In similar way, the tobacco habit was too investigated as present or absent.
Ex-drinkers and ex-smokers were subjects who had abstained from any type of drinking and
smoking for at least one year. Non-smokers and ex-smokers were grouped combined.
Similarly, with regard to alcohol intake, those patients that never drank alcohol were
considered non-drinkers and were grouped with ex-drinkers.
Statistical analyses
Intraexaminer agreement was evaluated using the kappa statistics. Kappa test was used
to assess intraexaminer reproducibility (AMB De Paula) for morphological grading in LSCC
samples (n=15, in each group). Two weeks later, in order to calibrate the same examiner, the
same morphological criteria were considered and the samples were graded again and
119
submitted to statistical analysis. Descriptive statistics were calculated. The associations
between epidemiological and clinicopathological parameters were examined by chi-square
test and Fisher´s exact tests. For statistical reasons, the clinical dependent variables
considered in this study were cervical metastasis (absent and present), tumor size (T1/T2 and
T3/T4), and TNM staging (early - I/II and late - III/IV). Morphological dependent variables
were represented by morphological grading criteria of WHO and Bryne et al.
17, 18
Statistical
analysis showing a confidence above 95% (p<0.05) were considered significant. All tests for
significance were two-sided. All statistical analyses were performed with the statistical pack
SPSS
®
(SPSS Inc., Chicago, IL, USA), version 13.0 for Windows
®
.
Results
Intraexaminer concordances were classified as good for WHO (k=0.877, p=0.000),
and Bryne (k=0.492, p=0.024) morphological grades.
The LSCC lesions represented 10.8% of all oral cavity squamous cell carcinomas and
perioral region and 3.5% of the totality cases with localization in the upper aerodigestive tract.
The LSCC involved mainly the lower lip (83.3%). The male-to-female ratio was 5:1, with a
mean age at the diagnosis of 58.8 (± 15.57) years, ranging from 25 to 92 years). The majority
of individuals were represented by older patients (76.7%). Age parameter among female
gender varied from 56 to 86 years (mean 67.6 years), and in the male group it varied from 25
to 92 years (mean 57.1 years). The descriptive statistics of LSCC group is presented in Table
1. There were 21 (70%) patients whose occupations were classified as exposed to chronic sun
radiation. It was noted significant association between chronic solar exposure in the job’s
environment and incidence of LSCC in lower lip (p=0.019) (data not shown). A half of LSCC
120
patients (n=15) were evaluated as capable in performing normal activities according to KPS
index. All these subjects received the maximum score of 100 by the evaluation of KPS. The
comparative evaluations between the epidemiological variables with size tumor, cervical
metastasis, and TNM clinic staging of LSCC patients are presented in Table 2. The
occurrence of cervical metastasis was associated statistically with worse KPS scores
(p=0.035).
The evaluation of epidemiological and clinical variables is presented in Table 3. It was
noticed that the LSCC samples of female group showed a higher morphological severity for
WHO grading (p=0.004).
Morphological and clinical variables were also analyzed each other. It was observed a
significant association between T3/T4 lesions and LSCC samples with loss of morphological
differentiation (p=0.023).
Discussion
Lip squamous cell carcinoma is an important public health problem in many countries
of the world
3, 15, 21-23
. However, according to regional context, it is important to realize the
influence of specific risk factors, considering the habits and the environmental conditions in
which individuals have been exposed. The LSCC has its origin related mainly to chronic solar
exposure
23-25, 37, 46
. Additionally, it has been showed that occupational antecedents provided
us a good idea of sun exposition grade
26
. In the current study, we found an association
between chronic solar exposure in the job’s environment and incidence of LSCC (data not
shown). The northern region of Minas Gerais state, Brazil, assumes importance for disease
because of high solar radiation level, typical of tropical regions. This region maintain its
121
economy based on agriculture activities, and therefore, many people work subjected to long
periods under sun radiation exposure
27
. Regarding the typical socio-demographic profile of
LSCC patients, the frequent lack of information about the etiology of disease has contributed
for most intensive exposure to its risk factors
11
.
In our study, nearly all cases of LSCC were located on lower lip. The literature
highlights that the majority of LSCC cases occurs in the lower lip, probably due to its
position, which it receives a higher exposure of solar radiation
4, 28
. Still, it is probable that
tobacco and alcohol beverage products might also acts more frequently for occurrence of
disease in lower lip. Interesting, it has been suggested that the etiology of upper lip cancer
could be regarded as a separate entity and different of the lower lip
29
. However, further
studies need to be made to clarify this question. Women, as noted in other studies, were less
affected by disease than men
13
. This finding has been associated with the frequent use of
lipstick, a known lip protector against solar exposure
3, 4, 23
. The high ratio male:female could
be attributed to the fact that most outdoor workers still are men. However, this work scenario
has been changed noticeably in the last decades and women have been exposed to sunlight as
often as males due to their similar working habits.
Currently, it has been noted evidences that display the association between tobacco
and alcohol drinking habits and the occurrence of LSCC
4, 11, 13, 16, 30, 31
. Although the
occurrence of tobacco and alcohol drinking habits were expressive in our sample, these
findings seem to be more important for early events of lip carcinogenesis than influencing the
clinicopathological parameters of lip disease. Future studies are needed to clarify the potential
contribution of alcohol comsumption for the LSCC ethiopathogenesis.
The hypothesis that cancer may be a time-dependent process
32
is consistent with the
findings of our study, mainly considering the similar time of exposure to risk factors in both
122
young and older patients investigated in this study. The LSCC incidence increases
progressively with age, being the disease very uncommon in young people. Exception for
individuals with some types of genetic alteration, as syndromes, such as Xeroderma
pigmentosum, that often exhibits squamous cell carcinoma in skin and mucosa during the first
and second decades of life
12, 33, 34
. As the cancer family history was not an important factor
influencing the clinicopathological parameters of LSCC in this study (p=0.542; data not
shown), it seems that environmental factors associated to lifestyle with cumulative levels of
the most important risk factors found were critical for the onset of lip disease. In some cases,
the hereditary character of LSCC has been suggested associated in particular with common
family risk factors
35
.
Our results showed that LSCC patients frequently exhibited, at the diagnosis moment,
early TNM clinical staging and absence of cervical metastasis. This finding is probably due to
anatomical position and slow growth of the lip lesion and its metastatic nodes. Lip is also of
easier access compared to other sites of oral cavity. The occurrence of disease in this site
usually stimulates an early search for health care centers. The literature indicates that LSCC is
associated with the lowest incidence of cervical metastasis among all the cancers of the oral
cavity, especially when referring to small lesions
36,37
. It was also noted that young patients
predominately exhibited T1/T2 lesions and absence of cervical metastasis during diagnosis of
disease. It seems that young individuals looking for health centers more quickly when they
perceive some early lip alteration, especially for aesthetical reason.
Reduced global health has been found in cancer patients
38-40
. KPS is a physician-
assigned score focused on the patient’s ability to perform basic tasks
41
. The KPS has been
used use performance status as a guide to treatment plans for individual oncologic patients by
clinicians because of its relationship and prognosis
40, 42
. In current study, it was shown that
123
the occurrence of cervical metastasis was associated with KPS scores ≤80. This finding
emphasizes the importance of health state and the need of adequate care for the patients. As
frequently noted, TNM staging exhibits powerful impact on prognosis of head and neck
squamous cell carcinoma patients
43
. The association between lower KPS scores and cervical
metastasis could potentially and negatively contribute for LSCC patients prognosis. This
relationship between these variables seems to be associated with duration of disease and
problems that are common with advanced staging of disease. Further studies are important to
evaluate if KPS scale can capture related and clinically relevant information for prognosis in
LSCC.
The importance of the differentiation grade in determining the prognosis for
squamous cell carcinoma was introduced by Broders (1920)
44
in LSCC samples. Well-
differentiated morphologic grades were prevalent in our samples of LSCC. In general, well-
differentiate LSCC generally do promote a better prognosis
4, 15, 45
. Our data showed that
T3/T4 lesions were associated with morphological findings of tumors poorly differentiated. It
is probable that undifferentiated lesions posses malignant cells with higher proliferative rates
and, so, this fact could contribute to the occurrence of great tumor. Other interesting finding
in this study was that women had a higher number of undifferentiated LSCC lesions. In many
studies, as well as in this, average age of cancer diagnosis in females was 10 years larger than
in males
6, 15
. Therefore, considering the phenomenon of cancer progression, it seems
reasonable to suggest that older LSCC lesions present more undifferentiated malignant cells.
This current study has presented limitations, such as sample size, especially for
statistical reasons. Besides, the typical difficulties of cross-sectional study design in to make
causal inference. On the other hand, it adds a better understanding about the profile of LSCC
patients of a specific population. Our findings can be useful for public health planning,
124
stimulating the creation of health promotion strategies in order to orientate the population and
health professionals. Particularly in Brazil, where this type of health program represents the
main government strategy to improve primary health care in the communities by providing a
comprehensive range of preventive and curative health care services.
125
Conflict of interest
None was declared.
Acknowledgments
This study was supported by Foundation for the Support of Research of Minas Gerais
State (Fapemig).
126
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Tables
Table 1- Descriptive analysis of epidemiological, risk factors, clinical, and
morphological variables of the LSCC patients in study.
Parameters
n
%
Parameters
n
%
Gender
Age
Male
25
83.3
Young
07
23.3
Female
05
16.7
Older
23
76.7
Skin color
Chronic Solar Exposure
White
13
43.3
Exposed
21
70.0
Non-white
17
56.7
Non-exposed
09
30.0
Family History of Cancer
KPS
Absent
20
66.7
90-100
15
50.0
Present
10
33.3
70-80
15
50.0
Tobacco Use
Alcohol Drinking Use
Never
06
20.0
Never
12
40.0
Ever
24
80.0
Ever
18
60.0
Tumor Site
T Parameter
Upper lip
05
16.7
T1/T2
23
76.7
Lower lip
25
83.3
T3/T4
07
23.3
N Parameter
TNM Staging
Absent (N0)
22
73.3
Early (I/II)
23
76.7
Present (N1/N2/N3)
08
26.7
Late (III/IV)
07
23.3
WHO Morphological
Grading
Invasive Front
Grading (IFG)
Well Differentiated
17
56.7
4-8
13
43.4
Moderately Differentiated
06
20.0
9-12
10
33.3
Poorly Differentiated
07
23.3
13-16
07
23.3
131
Table 2 - Association between independent variables and clinical aspects of LSCC sample.
Size Tumor (T)
Cervical Metastasis (N)
TNM Staging
Parameters
T1/T2
T3/T4
p value
Absent
Present
p value
Early
Late
p value
Age
Young
07 (30.4)
0 (0.0)
0.096
05 (22.7)
02 (25.0)
0.896
05 (23.8)
02 (22.2)
1.000
Older
16 (69.6)
07 (100)
17 (77.3)
06 (75.0)
16 (76.2)
07 (77.8)
Gender
Female
04 (17.4)
01 (14.3)
1.000
04 (18.2)
01 (12.5)
1.000
04 (19.0)
01 (11.1)
1.000
Male
19 (82.6)
06 (85.7)
18 (81.9)
07 (87.5)
17 (81.0)
08 (88.9)
Skin color
White
10 (43.5)
03 (42.9)
1.000
09 (40.9)
04 (50.0)
0.698
09 (42.9)
04 (44.4)
0.936
Non-White
13 (56.5)
04 (57.1)
13 (59.1)
04 (50.0)
12 (57.1)
05 (55.6)
Tumor Site
Upper Lip
04 (17.4)
01 (14.3)
1.000
04 (18.2)
01 (12.5)
1.000
04 (19.0)
01 (11.1)
1.000
Lower Lip
19 (82.6)
06 (85.7)
18 (81.1)
07 (87.5)
17 (81.0)
08 (88.9)
Family History of
Cancer
Absent
15 (65.2)
05 (71.4)
1.000
15 (68.2)
05 (62.5)
0.770
14 (66.7)
06 (66.7)
1.000
Present
08 (34.8)
02 (28.6)
07 (31.8)
03 (37.5)
07 (33.3)
03 (33.3)
KPS
90-100
14 (60.9)
01 (14.3)
0.080
14 (63.6)
01 (12.5)
0.035*
13 (61.9)
02 (22.2)
0.109
70-80
09 (39.1)
06 (85.7)
08 (36.4)
07 (87.5)
08 (38.1)
07 (77.8)
Chronic Solar
Exposure
Yes
16 (69.6)
05 (71.4)
1.000
16 (72.7)
05 (62.5)
0.589
15 (71.4)
06 (66.7)
0.794
No
07 (30.4)
02 (28.6)
06 (27.3)
03 (37.5)
06 (28.6)
03 (33.3)
Tobacco Habit
Ever
18 (78.3)
06 (85.7)
1.000
18 (78.3)
06 (85.7)
1.000
17 (81.0)
07 (77.8)
1.000
Never
05 (21.7)
01 (14.3)
05 (21.7)
01 (14.3)
04 (19.0)
02 (22.2)
Alcohol Drinking
Habit
Ever
14 (60.9)
04 (57.1)
1.000
14 (60.9)
04 (57.1)
1.000
12 (57.1)
06 (66.7)
0.626
Never
09 (39.1)
03 (42.9)
09 (39.1)
03 (42.9)
09 (42.9)
03 (33.3)
Values calculated by
2
and exact Fisher statistical tests.
* p value significant (p<0.05).
132
Table 3 - Association between independent variables and morphological grading of LSCC sample.
WHO
IFG
Parameters
I
II
III
p value
4-8
9-12
13-16
p value
Age
Young
04 (23.5)
03 (50.0)
0 (0.0)
0.105
04 (30.8)
02 (20.0)
01 (14.3)
0.676
Older
13 (76.5)
03 (50.0)
07 (100)
09 (69.2)
08 (80.0)
06 (85.7)
Gender
Female
01 (5.9)
0 (0.0)
04 (57.1)
0.004*
01 (7.7)
02 (20.0)
02 (28.6)
0.461
Male
16 (94.1)
06 (100)
03 (42.9)
12 (92.3)
08 (80.0)
05 (71.4)
Skin color
White
08 (47.1)
02 (33.3)
03 (42.9)
0.843
06 (46.2)
03 (30.0)
04 (57.1)
0.520
Non-White
09 (52.9)
04 (66.7)
04 (57.1)
07 (53.8)
07 (70.0)
03 (42.9)
Tumor Site
Upper Lip
02 (11.8)
01 (16.7)
02 (28.6)
0.604
02 (15.4)
01 (10.0)
02 (28.6)
0.592
Lower Lip
15 (88.2)
05 (83.3)
05 (71.4)
11 (84.6)
09 (90.0)
05 (71.4)
Family History of
Cancer
Absent
13 (76.5)
02 (33.3)
05 (71.4)
0.149
10 (76.9)
06 (60.0)
04 (57.1)
0.577
Present
04 (23.5)
04 (66.7)
02 (28.6)
03 (23.1)
04 (40.0)
03 (42.9)
KPS
90-100
11 (64.7)
02 (33.3)
02 (28.6)
0.181
09 (69.2)
04 (40.0)
02 (28.6)
0.165
70-80
06 (35.3)
04 (66.7)
05 (71.4)
04 (30.8)
06 (60.0)
05 (71.4)
Chronic Solar
Exposure
Yes
11 (64.7)
04 (66.7)
06 (85.7)
0.582
09 (69.2)
08 (80.0)
04 (57.1)
0.597
No
06 (35.3)
02 (33.3)
01 (14.3)
04 (30.8)
02 (20.0)
03 (42.9)
Tobacco Habit
Ever
15 (88.2)
05 (83.3)
04 (57.1)
0.218
10 (76.9)
08 (80.0)
06 (85.7)
0.896
Never
02 (11.8)
01 (16.7)
03 (42.9)
03 (23.1)
02 (20.0)
01 (14.3)
Alcohol Drinking
Habit
Ever
10 (58.8)
04 (66.7)
04 (57.1)
0.930
08 (61.5)
04 (40.0)
06 (85.7)
0.165
Never
07 (41.2)
02 (33.3)
03 (42.9)
05 (38.5)
06 (60.0)
01 (14.3)
Values calculated by
2
and exact Fisher statistical tests.
* p value significant (p<0.05).
133
Apêndice C – Artigo 4
Association of mast cell, eosinophil leukocyte and microvessel densities in actinic cheilitis
and lip squamous cell carcinoma
Ludmilla R. Souza,
1
Thiago F. Silva,
1
Carolina C. O. Santos,
1
Marcos V. M. Oliveira,
1
Rodrigo Corrêa-Oliveira,
2
André L. S. Guimarães,
1
& Alfredo M.B. De Paula
1
1
Health Science Programme, State University of Montes Claros, 39401-001, Montes Claros,
MG, Brazil
2
Laboratory of Molecular and Cellular Immunology, Research Center Renée Rachou –
Fiocruz, 30190-002 , Belo Horizonte, MG, Brazil
Running Title: MC, EL and MVD in lip carcinogenesis
Keywords: actinic cheilitis, lip squamous cell carcinoma, lip carcinogenesis, eosinophil
leukocyte, histochemistry, immunohistochemistry, mast cell, microvessel density.
Address for correspondence:
Dr. Alfredo Maurício Batista De Paula
Laboratório de Pesquisa em Saúde. Hospital Universitário Clemente de Faria. Universidade
Estadual de Montes Claros - Unimontes. Av. Cula Mangabeira, 562- Bairro Santo Expedito.
Montes Claros - Minas Gerais - Brazil.
CEP: 39401-001.
Phone: +51-21-38 32248327. Fax: +55-21-38 32298500
e-mail: ambpatologi@gmail.com
134
Abstract Aims: To determine the contributions of mast cells (MC), eosinophil leucocytes (EL)
and microvessel density (MVD) in lip carcinogenesis, and to establish the relationships between
these biomarkers and their possible prognostic value in lip squamous cell carcinoma (LSCC).
Methods and results: Archived tissue specimens of lip mucosa, actinic cheilitis lesions and LSCC
were formalin fixed, paraffin-embedded, sectioned and stained with toluidine blue and
haematoxylin-eosin to identify MC and EL, and with anti-CD31 antibody for the MVD study.
The average densities of biomarkers were determined in all samples. Progressive increases in
MC, EL and microvessel densities were observed during lip tumour development. Correlation
analysis revealed positive associations between the biomarkers during tumour progression. In
LSCC samples, significant associations were found between MVD values and the occurrence of
cervical metastasis and late TNM staging. In contrast, no relationships could be established
between MC or EL densities and socio-demographic and clinicopathologic parameters.
Conclusions: MC, EL and microvessel densities increased during lip carcinogenesis, and for MC
and EL this may be related to the stimulation of tumoural angiogenesis. MVD could be a useful
prognostic factor in LSCC, whilst tumoural microvessel-targeted approaches may represent new
therapeutic modalities for pre-malignant and malignant disease of the lip.
135
Introduction
Lip squamous cell carcinoma (LSCC) and its premalignant lesion, actinic cheilitis (AC),
are principally caused by chronic exposure to ultraviolet (UV) B radiation (280-315 nm). The
major source of UV B is solar radiation, but the effect on human health of exposure to radiation
from artificial sources in the workplace and in tanning salons is becoming increasingly
important.
1,2
Additionally, a range of factors have been identified as having an association with
the development of both diseases, and these include environmental (i.e. prolonged exposure to
sunlight and rural domicile), behavioural (i.e. use of tobacco), occupational and genetic
influences, as well as socioeconomic status and viral infections.
3,4
The rate of development of a tumour is regulated by a delicate balance between the pro-
and anti-tumourigenic activities promoted by the neoplastic cells themselves, as well as by cells
in the surrounding microenvironment. Local inflammation at the site of tumour growth results in
the accumulation of a variety of cell types that are usually linked to the kinetics of oncogenesis.
5
Moreover, it has been demonstrated that UV radiation not only contributes to the initiation and
promotion of oncogenesis, via effects on cellular DNA and intracellular signal transduction, but
also interferes with the immunity of the host against cancer cells.
1, 2, 6, 7
Several studies have shown that tumour angiogenesis, i.e. the formation of new blood
vessels from pre-existing vascular structures in the tumour microenvironment,
8,9
plays a crucial
role in tumour progression and as a prognostic indicator of human solid cancers.
10-13
The process
of vessel formation is a complex event comprising degradation of the extracellular matrix,
migration and proliferation of endothelial cells, formation of new vessels, and synthesis of
extracellular matrix. Tumour angiogenesis is mediated in both tumoural and tissue-resident
136
inflammatory cells through the release of numerous signalling and growth factors, but the exact
mechanism is still not fully understood despite intense investigation.
8
Moreover, with respect to
carcinomas in the head and neck, the role of tumour angiogenesis has not been established
unambiguously.
14-20
Much research attention has been focussed on the effects of mast cells (MC) and
eosinophil leukocytes (EL) on tumours in consideration of their possible roles in the development
of tumour angiogenesis. MC are long-lived cells that are widely distributed within an organism,
particularly in connective tissues, and are generally located beneath epithelial surfaces.
21-22
Such
cells are now recognised as an early and persistent inflammatory cell type in many human
tumours, and the accumulation of MC is reportedly associated with tumour progression and
prognosis.
23-26
MC may act directly by stimulating the migration and proliferation of endothelial
cells or indirectly by degrading the connective tissue matrix and activating collagenases in order
to provide space for the formation of neovascular sprouts.
27,28
Eosinophil leukocytes (EL) are a
distinct lineage of granulocytes that arise in the bone marrow, circulate in the blood and emigrate
into peripheral tissues.
29,30
EL can stimulate tumoural angiogenesis through synthesis and release
of potent angiogenic factors.
31-34
Additionally, tumour-associated tissue eosinophilia (TATE)
35
has been claimed to be involved in the biological behaviour and prognosis of various human
malignancies, most especially of squamous cell carcinomas located at sites on the head and
neck.
36-40
Inflammatory cells act cooperatively and synergistically with both stromal cells and
malignant cells by stimulating endothelial cell proliferation and blood vessel formation.
13,31,41
It
is presumed that the histological identification and quantification of specific inflammatory cells
and microvessel densities (MVD) in tumours could provide an estimate of the interaction
137
between these biomarkers and tumoural angiogenesis, and of their consequences in oncogenesis
and tumour prognosis. Moreover, studies of molecular disturbances that occur in potentially
malignant lesions related to the acquisition of one malignant phenotype can assist in the
development of diagnostic tools, prognostic assays and more efficient treatment.
42
On this basis,
the aim of the present study was evaluate MC, EL and microvessel densities, and the
relationships between these biomarkers, at different stages of lip carcinogenesis. Additionally,
associations between these biomarkers and the socio-demographic and clinicopathologic
parameters of LSCC patients were investigated.
Samples and Methods
Ethical approval for this study was obtained from the relevant local ethics committees
(Unimontes/COEP-1187/2008).
TISSUE SPECIMENS
Data relating to patients and samples derived therefrom were obtained from the
Department of Dentistry at the State University of Montes Claros, Minas Gerais, Brazil. The
health records of patients were retrieved and socio-demographic and clinical data were analysed.
Archived tissue blocks from surgically resected samples of LSCC (n = 29; group male-to-female
ratio = 6.2:1;group mean age = 63.34 ± 15.48 years), of AC (n = 29; group male-to-female ratio =
4.8:1; group mean age = 54.39 ± 18.17 years), and biopsies of lip mucosa (LM) used as controls
(n = 13; group male-to-female ratio 1.2:1; group mean age 20.62 ± 10.73 years), were analysed.
The diagnoses of patients presenting AC or LSCC lesions were confirmed by clinical
examination and histopathological analysis. In the LSCC group, the prognostic analysis was
138
carried out on the basis of clinicopathologic parameters. All LSCC patients were submitted to
surgical treatment, and none had received radiotherapy or chemotherapy prior to surgery.
CLINICAL AND MORPHOLOGICAL CLASSIFICATION
All LSCC patients were classified according to the International Union against Cancer
(UICC) - TNM Classification of Malignant Tumours
43
on the basis of the primary site, as
described in the International Classification of Diseases for Oncology (C00).
44
In the LSCC
group, 20.7% of cases were in stage I, 48.3% were in stage II, 13.8% were in stage III and 17.2%
were in stage IV. Morphological grading of the LSCC group was based on World Health
Organization (WHO) criteria, and the morphological analysis was carried out by an oral
pathologist (AMB De Paula) without prior knowledge of the demographic or clinical
characteristics relating to the samples. The final grades established were: well differentiated - n =
19, moderately differentiated - n = 2, and poorly differentiated - n = 8. The microscopic features
of the AC samples were analysed and graded as low risk (n = 12) and high risk (n = 17).
45
All
samples of LM exhibited morphologically normal epithelium tissue, although in some samples a
discrete mononuclear inflammatory infiltrate was detected.
HISTOLOGICAL AND HISTOCHEMICAL STAINING
For the purposes of morphological and histochemical analysis, samples were fixed in
formalin, embedded in paraffin, serially sectioned at 5 µm, and evaluated under a conventional
light microscope. Sections were stained with hematoxylin and eosin for EL density analysis. In
order to visualise MC, two sections of each sample were stained with 1% toluidine blue and
139
counterstained with 5% methanol yellow for 5 min, following which the sections were
dehydrated, cleared and mounted with synthetic balsam.
IMMUNOHISTOCHEMICAL STAINING FOR CD31 ANTIGEN
For the immunohistochemical evaluation of MVD, sections were mounted on
organosilane- coated slides. The primary mouse monoclonal antibody against CD31 antigen
(Novocastra Laboratories, Newcastle-upon-Tyne, UK) was detected with the aid of an LSAB™
visualisation kit (product # K0690; Dako, Glostrup, Denmark) employing the chromogen
diaminobenzidine for colour development. Slides were finally counterstained with Mayer’s
hematoxylin and mounted. Negative controls were obtained by substituting normal whole rabbit
serum (product # X0902; Dako) for the primary antibodies. A sample of oral hemangioma
(previously shown to be strongly CD31-positive) was used as a positive control, whilst small-
and intermediate-sized vessels located away from normal stroma and lesions served as an internal
positive control. Only stroma cells in capillary-sized vessels, the cytoplasm of which was stained
brown, were considered positive.
DETERMINATION OF MC, EL AND MICROVESSEL DENSITIES
MC were identified on the basis of cytoplasm with intense purple granules and nuclei
presenting a bluish morphologic aspect. EL were characterised as cells exhibiting lobulated
nuclei and intensely eosinophilic cytoplasmic granules. For quantitative analysis, an Olympus
®
BH2 microscope (×10 ocular and ×40 objective lenses) was employed, and an ocular lattice (area
0.092 mm
2
) with 100 points composed of 10 horizontal and 10 vertical test lines was
superimposed on the test field to be measured. A total area of 1.84 mm
2
was evaluated for each of
140
the samples, and this corresponded to 20 randomly selected high-power microscopic fields
located in regions of high MC and EL densities. In the LM and AC samples, MC and EL
densities were determined in the subepithelial (including epithelium/connective tissue junctions),
connective and submucosal zones. In the case of LSCC samples, densities were assessed in the
connective tissue underlying the invasive front areas of tumoural parenchyma.
Estimation of MVD was performed using a method in which “hot spots” of high vessel
density were selected by immunohistochemical staining for CD-31 antigen.
27
Individual
microvessels were identified on the basis of stained endothelial cells or transversally sectioned
tubes with a single layer of endothelial cells either with or without a thin base membrane. A
single continuous vessel that appeared to involve two or more CD31 positive foci was counted as
one microvessel. Initially, the sample was inspected at low magnification in order to become
familiar with its size and shape and to identify the most appropriate fields for counting. MVD
was then performed on three fields in the tumour sample presenting the highest vascular
densities. Within each “hot spot”, five high-power microscopic fields were analysed providing a
total of 15 analysed fields per sample. MVD counting was performed by an investigator who had
no knowledge of the clinical data associated with the sample.
STATISTICAL ANALYSIS
Statistical analyses were performed with the aid of SPSS
®
statistical pack (version 17.0
for Windows
®
; SPSS, Chicago, IL, USA). Differences between populations were considered to
be significant when the confidence level was > 95% (p < 0.05).
The Kappa statistic (κ) was applied in order to assess intra-examiner reproducibility
relating to the morphological grading of AC and LSCC samples. Archived samples (n = 15 for
141
each group) were graded by the same examiner on two separate occasions, with an interval of 2
weeks, according to the same morphological criteria, and the results were submitted to statistical
analysis. The concordance values obtained (AC samples, κ = 0.857, p < 0.05; LSCC samples, κ =
0.877, p < 0.05) revealed good agreement between the two assessments.
All of the continuous variables studied were nonparametrically distributed according to
the Kolmogorov-Smirnov test. Between group differences in mean values of MC, EL and
microvessel densities were evaluated using Kruskal-Wallis statistical tests, combined with the
Bonferroni correction (α = 0.006). The Spearman correlation test was employed to assess
relations between biomarkers. Associations between MC, EL and microvessel densities, and the
socio-demographic and clinicopathologic findings for the AC and LSCC groups were
investigated using Mann-Whitney and Kruskal-Wallis statistical tests.
Results
Figure 1 presents examples of MC, EL and microvessels identified in LM, AC and LSCC
samples. MC were often observed in small groups around normal mucosal tissue and lesions. In
the LM samples, MC were not randomly distributed in submucosal zones but were localised
close to nerves and blood vessels (Figure 1A). In AC and LSCC samples, MC were generally
scattered throughout the stroma adjacent to dysplastic/neoplastic tissues, and were also located
near to or around the blood capillaries and nerves (Figures 1B and C). EL were identified in LM
samples at low, but detectable, levels primarily located in subepithelial tissue (Figure 1D). In
dysplastic and neoplastic samples, EL were observed in the form of a sequential and progressive
infiltration into the stroma of lesions, attaining the highest density in the submucosa. In AC
lesions, EL were frequently noted adjacent to basophilic amorphous areas that were characteristic
142
of solar elastosis (Figure 1E). Microvessels positive for CD31 antigen were scarce and distributed
uniformly in the stroma of LM samples (Figure 1G). In AC lesions, these structures were
preferentially observed in the lamina propria (Figure 1H), whilst in LSCC a higher expression of
CD31 antigen was detected in the tumoural stroma adjacent to the invasive front area (Figure 1).
Densities (cells or microvessels/mm
2
) evaluated in LM samples were in the ranges 0 to
51.63 (mean 24.80 ± 19.60) for MC, 5.43 to 17.93 (mean 12.66 ± 4.25) for EL, and 3.87 to 7.47
(mean 5.49 ± 1.15) for MVD. In AC samples, the densities ranged from 0 to 216.30 (mean 53.33
± 53.81) for MC, from 4.89 to 71.19 (mean 28.31 ± 17.07) for EL, and from 0.33 to 14.40 (mean
7.43 ± 3.55) for MVD, whilst the respective ranges for LSCC samples were 5.43 to 203.80 (mean
118.44 ± 42.40), 47.83 to 190.76 (mean 117.58 ± 37.36) and 2.07 to 21.13 (mean 11.54 ± 4.90).
The density scores for MC, EL and microvessels exhibited statistically significant increases with
the pathologic progression of lip carcinogenesis (i.e. according to the transition from LM to AC
and ultimately to LSCC). With respect to MC density, significant differences were observed
between the LM and LSCC groups and between the AC and LSCC groups, whilst for EL density
and MVD there were significant differences between all groups of samples (p<0.05) (Figure 2).
Additionally, correlation analysis revealed positive associations between all biomarkers, namely,
MC/EL (r = 0.584, p < 0.05), MC/MVD (r = 0.445, p < 0.05) and EL/MVD (r = 0.528, p < 0.05)
(Figure 2).
In contrast, MC, EL and microvessel densities were not associated with morphological
gradings for AC epithelial dysplasia. Moreover, no relationships could be established in the
LSCC group between MC or EL densities and socio-demographic and clinicopathologic
parameters. However, significant associations were found between MVD values and the
occurrence of cervical metastasis and late TNM staging (p < 0.05) (Table 1).
143
Discussion
Tumour-infiltrating cells at the periphery of the tumour, and especially at the sites of
obvious invasion, have been considered to play an important role in tumour progression and the
biological behaviour of various human malignancies. Several studies have revealed that the
infiltration of immune cells into the tumour microenvironment can induce contradictory actions
that may contribute to, inhibit or have no influence on, tumour progression and prognosis.
5,46-48
However, information relating to the influence of MC and EL on cancer progression and,
consequently, their potential prognostic value, is somewhat ambiguous. Thus MC, which are
naturally cytotoxic, are known to suppress tumour growth and to release anti-tumour
compounds,
24
whilst EL play a role in antibody-dependent cell-mediated cytotoxicity, and in the
synthesis and release of cytokines that could contribute, either direct or indirectly, to tumour
cytotoxicity.
37,49,50
In contrast some studies have demonstrated that MC and EL can also exert
important pro-tumourigenic effects.
51,52
It appears, therefore, that MC and EL develop diverse
activities according to the different conditions found in the tumour microenvironment, but the
nature of the factors that determine whether such cells will support anti- or pro-tumourigenic
processes are currently unclear.
In the present study, increases in the densities of MC, EL and microvessels were related
with lip tumour progression, suggesting that these occurrences may be important in the
determination of lip carcinogenesis. The types and quantities of cells that constitute the immune
infiltrates in pre-cancer and solid tumours are related to the local production of chemotactic
cytokines synthesised and released by tumour, immune and stromal cells.
5, 53-55
The sequential
migration of MC and the presence of high densities of these cells in progressive oral mucosal
144
dysplasia and subsequent development of squamous cell carcinoma, point to the influence of
tumour-favouring effects.
23,26,56
The cytokine stem cell factor (SCF) and its c-kit receptor
stimulate the directional motility of both mucosal and connective tissue-type MC.
57
In the case of
EL, chemotactic factors such as eotaxin, which exert a selective action on EL, have also been
claimed to play a role in the mechanism of in situ EL infiltration and maintenance. A number of
other factors, including lymphocyte chemoattractant factor (LCF), interleukin-2 (IL-2), IL-3, IL-
5, chemokine (C-C motif) ligand 5 (CCL5 or RANTES) and even IL-4, appear to regulate the
expression of vascular cell adhesion molecule-1 (VCAM-1), which is an important receptor in EL
transmigration through the endothelium.
37,40,50,58
In this manner, the results here reported could be
associated with the migration processes and the accumulation sites of MC and EL during lip
carcinogenesis since, once located in the tumour microenvironment, these cells could exert pro-
tumourigenic activities that would serve to stimulate the progression of lip tumour. Moreover,
UV radiation is known to suppress host immunity against the development of tumour cells.
1,2,7
It
is possible, therefore, that photodamage of lip tissue could promote changes in the
microenvironment causing the region to become more appropriate for the migration and
maintenance of MC and EL during solar carcinogenesis. These hypotheses, however, require
confirmation by further study.
MVD is reportedly increased significantly in a relatively large spectrum of pre-malignant
squamous cell lesions, including oral mucosa, and apparently performs an important role in the
transition of normal tissue to the pre-cancerous state and, eventually, to full-blown cancer.
11,13
The results of the present study are in agreement with earlier reports of increased MVD during
oral carcinogenesis leading to intense vascularisation in malignant tumours.
14,19,59,60
Pre-invasive
malignant cells associated with oral squamous cell carcinoma are known to remain dormant until
145
they become angiogenic, and this is followed by a phase of rapid tumour growth. It is possible
that increased MVD in connective tissue in pre-malignant and malignant tumours of the upper
aerodigestive tract reflects the increasing nutrient requirements of actively growing transforming
cells.
A further interesting finding of the present study was the significant and positive
relationship between MC and EL densities and angiogenesis during lip tumour progression.
Inflammatory cells communicate via a complex network of intercellular signalling pathways
typically mediated by surface adhesion molecules, cytokines and cytokine receptors. Recent
studies have shown that MC and EL participate in a complex self-perpetuating cycle, such that
EL produces mediators responsible for MC differentiation, activation, proliferation and
survival,
30
whilst activated MC release mediators, such as IL-5 and granulocyte-monocyte
colony-stimulating factor,
61
that favour EL recruitment and activation. The present results support
the hypothesis that both MC and EL may upregulate angiogenesis from the initial to the end
stages of lip carcinogenesis by acting as modulators of the microenvironment and thus making
these regions more permissive to cancer progression.
Whilst the exact mechanisms by which MC and EL promote angiogenesis remain unclear,
it is known that these cells express a range of potent pro-angiogenic factors associated with blood
vessels in developing tumours. Thus, MC accumulate at the boundary between healthy tissues
and malignancies and are often found in close association with blood vessels prior to the onset of
angiogenesis in tumours.
15,27,28,62
MC are rich in metalloproteases, such as tryptase and
plasminogen activator, that contribute to the degradation of the extracellular matrix, which is the
first step in neoangiogenesis and tumour invasiveness.
62
Moreover, MC synthesise and release
potent angiogenic cytokines, such as vascular endothelial growth factor (VEGF), fibroblast
146
growth factor-β (FGF-β), transforming growth factor-β (TGF-β), nerve growth factor (NGF),
tumour necrosis factor-α (TNF-α) and IL-8, and the serine proteases tryptase and chymase. On
this basis, it has been suggested that MC act as the “angiogenic switch” during the early stages of
tumour development.
63
Similarly, it has been proposed that EL play a regulatory role in pro-
angiogenesis by stimulating matrix remodelling and endothelial cell migration, proliferation and
subsequent sprout formation. The most important selective mediator of angiogenesis released or
induced by EL is VEGF, but the cells also release granulocyte-macrophage-colony stimulating
factor, FGF-β, NGF, TNF-α, IL-8, and eotaxin.
32-34,64
A number of reports have suggested that high MC and EL densities may be associated
with tumour behaviour in head and neck squamous cell carcinoma.
26,37,51,65-67
In the present study,
however, neither MC nor EL appeared to exert any influence on the biological behaviour of
LSCC. It would seem, therefore, that the determination of increased MC and EL densities in
tumour tissue from pathological specimens associated with good or bad prognosis is insufficient
to explain the actual role of these cells in tumoural tissues. An accurate insight into the role of
inflammatory cells in tumours may be obtained from the use of activation or inhibition markers,
together with a consideration of the local microenvironmental stimuli in the regulation of the
functions of these cells.
Blood vessels play a crucial role in malignant tumour prognosis.
10,13
Having developed an
intrinsic vascular network, the neoplastic mass is apparently able to grow indefinitely both in situ
and at distant sites to the extent that the intrinsic vascular network enables its cells to enter the
vascular bed and colonise other organs.
9
It has been verified in the present study that a higher
MVD was associated with the poorest prognosis in the LSCC group and was linked with cervical
metastasis and late TNM clinical stage. A number of studies on head and neck squamous cell
147
carcinomas have also revealed higher densities of tumour microvessels in association with
metastatic disease,
15,16
recurrence,
17
and lowest survival.
20
Such findings may signify important
mechanisms through which angiogenesis can stimulate tumour development and metastasis by
providing an efficient vascular supply and also an escape route for loco-regional and distant
metastasis. Additionally, it is possible that well-vascularised tumours appear to metastasise more
easily by virtue of facilitated access to intravascular transportation.
15
A number of explanations are available to account for the conflicting results obtained in
this and in earlier studies. Thus, it may be that MC and EL initially infiltrate cancerous tissue in
order to suppress tumour activities, but that the cancer cells subsequently stimulate the
angiogenic properties of MC and EL while suppressing their cytotoxic functions. Furthermore,
cells are generally not functional unless activated, but the quantitative analysis of inflammatory
cells per se is not capable of discriminating between activated and resting cells. In this context,
the methodologies employed to identify and categorises blood vessels and inflammatory cells in
published studies have varied widely.
In conclusion, the present findings confirm increased MC, EL and microvessel densities
during lip carcinogenesis and, for MC and EL, this may be related to the stimulation of tumoural
angiogenesis. Since tumour angiogenesis is generally accepted as being beneficial for tumour
growth, and since both MC and EL seem to have an indirect effect on accelerating lip tumour
progression, it is suggested that the reversal of this process, i.e. enhancing the cytotoxic functions
of these cells and suppressing their angiogenic activities, could afford a novel strategy in
developing treatments for LSCC. MVD alone seems to influence the clinical behaviour of LSCC
and is clearly associated with the occurrence of cervical metastasis and late TNM staging. On this
basis, it is likely that MVD could be a useful prognostic factor in LSCC, and that tumoural
148
microvessel-targeted approaches may represent new therapeutic modalities for pre-malignant and
malignant disease of lip.
149
Competing interests
None to declare.
Acknowledgments
The authors are grateful for support from Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (FAPEMIG).
150
Figures
Figure 1. Mast cells, eosinophil leukocytes and immunohistochemical expression of CD31
antigen (microvessels) in lip mucosa (LM; panels A, D and G), actinic cheilitis (AC; panels B, E
and H) and lip squamous cell carcinoma (LSCC; panels C, F and I) samples. Panels A, B and C:
slides stained with toluidine blue, magnification 400 X, arrowheads indicate mast cells; panels D,
E and F: slides stained with haematoxylin-eosin, magnification 400 X, arrowheads indicate
eosinophil leukocytes; panels G, H and I: colour developed with diaminobenzidine and
counterstained with Mayer’s haematoxylin, magnification 400 X.
151
Figure 2. Mean densities (± standard error) of mast cells (MC) and eosinophil leukocytes (EL),
and microvessel densities (MVD) in lip mucosa (LM), actinic cheilitis (AC) and lip squamous
cell carcinoma (LSCC) samples. Bars indicate standard error and the differences were significant
statistically at p<0.05.
152
Table 1. Association between densities of mast cells (MC) and eosinophil leukocytes (EL) and microvessel
densities (MVD), and the epidemiological, clinical and morphological parameters of the lip squamous cell
carcinoma group.
MC
EL
MVD
Parameters
Mean values
p values
Mean values
p values
Mean values
p values
Age
Young (n = 5)
108.37
0.707
36.80
0.862
4.99
0.175
Old (n = 24)
120.54
116.94
12.09
Family history of cancer
No (n = 20)
118.26
0.724
116.74
0.706
11.53
0.832
Yes (n = 9)
118.84
119.44
11.56
Smoking habit
Yes (n = 26)
117.03
0.616
121.55
0.115
11.73
0.474
No (n = 03)
130.61
83.15
9.87
Alcohol drinking habit
Yes (n = 18)
114.52
0.574
121.80
0.500
10.85
0.406
No (n = 11)
124.85
110.67
12.67
Recurrence
No (n = 22)
124.92
0.146
121.07
0.541
12.10
0.262
Yes (n = 7)
98.06
106.60
9.80
T parameter
T1/T2 (n = 21)
108.43
0.067
113.22
0.283
10.59
0.071
T3/T4 (n = 8)
144.70
129.00
14.04
N parameter
N0 (n = 20)
108.18
0.070
110.92
0.144
10.03
0.005*
N1,N2,N3 (n = 9)
141.24
132.37
14.89
TNM
I/II (n = 20)
109.13
0.150
110.38
0.099
10.22
0.018*
III/IV (n = 9)
139.13
133.57
14.47
WHO Grading
Well differentiated (n = 16)
119.85
0.765
120.71
0.875
12.52
0.225
Intermediate (n = 6)
129.62
118.48
8.55
Undifferentiated (n = 7)
112.29
109.92
9.97
* Values bearing asterisks are statistically significant (p < 0.05) according to Mann-Whitney and Kruskal-
Wallis tests.
153
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