analysis of the expression of proteins related … · fregnani, dr. andré caroli, dr. fernando...
TRANSCRIPT
UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE ODONTOLOGIA DE PIRACICABA
GLEYSON KLEBER DO AMARAL SILVA
ANÁLISE DA EXPRESSÃO DE PROTEÍNAS RELACIONADAS AO REPARO DO DNA
E PROLIFERAÇÃO CELULAR EM AMELOBLASTOMA E CARCINOMA
AMELOBLÁSTICO
ANALYSIS OF THE EXPRESSION OF PROTEINS RELATED TO DNA
REPAIR AND CELLULAR PROLIFERATION IN AMELOBLASTOMA
AND AMELOBLASTIC CARCINOMA
PIRACICABA
2017
GLEYSON KLEBER DO AMARAL SILVA
ANÁLISE DA EXPRESSÃO DE PROTEÍNAS RELACIONADAS AO REPARO DO DNA
E PROLIFERAÇÃO CELULAR EM AMELOBLASTOMA E CARCINOMA
AMELOBLÁSTICO
ANALYSIS OF THE EXPRESSION OF PROTEINS RELATED TO DNA REPAIR AND
CELLULAR PROLIFERATION IN AMELOBLASTOMA AND AMELOBLASTIC
CARCINOMA
Dissertação apresentada à Faculdade de
Odontologia de Piracicaba da Universidade
Estadual de Campinas como parte dos
requisitos exigidos para a obtenção do título
de Mestre em Estomatopatologia, na Área de
Patologia.
Orientador: Prof. Dr. Pablo Agustin Vargas
Coorientador: Prof. Dr. Felipe Paiva Fonseca
ESTE EXEMPLAR CORRESPONDE À
VERSÃO FINAL DA DISSERTAÇÃO
DEFENDIDA PELO ALUNO GLEYSON
KLEBER DO AMARAL SILVA, E
ORIENTADA PELO PROF. DR. PABLO
AGUSTIN VARGAS.
PIRACICABA
2017
DEDICATÓRIA
Dedico este trabalho aos meus pais, Ednamay Amaral Silva e Walter Benedicto da
Silva, que sempre me estimularam e nunca mediram esforços para minha formação
educacional e moral.
À minha irmã Greyce Kellen do Amaral Silva, por todo apoio e companheirismo
durante nossas caminhadas de idas e vindas da escola e pelo apoio e conselhos ao logo da
minha vida. Estas e outras lembranças estarão presentes eternamente em minha mente.
À minha tia Maura Cassia Ferreira de Macedo, por estar presente em grande parte
da minha vida me permitindo experimentar de atividades e momentos felizes.
À minha amada Danielle Ferreira Sobral de Souza, pelo companheirismo, apoio e
estímulo na realização dos meus sonhos.
AGRADECIMENTOS
À Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas
(Unicamp), na pessoa de seu diretor, Prof. Dr. Guilherme Elias Pessanha.
À Profa. Dra. Cínthia Pereira Machado Tabchoury, coordenadora do Programa de
Pós-Graduação da Faculdade de Odontologia de Piracicaba da Universidade Estadual de
Campinas (Unicamp).
Ao Prof. Dr. Márcio Ajudarte Lopes, coordenador do Programa de Pós-Graduação
em Estomatopatologia, da Faculdade de Odontologia de Piracicaba da Universidade Estadual
de Campinas (Unicamp).
Ao meu orientador, Prof. Dr. Pablo Agustin Vargas, agradeço por todas as
oportunidades de aprendizagem, crescimento e por ser um exemplo de profissional sempre
tratando com os assuntos com seriedade, mas também permitindo momentos de descontração
em ocasiões oportunas. Obrigado por suas orientações e confiança no meu trabalho.
Ao meu coorientador, Prof. Dr. Felipe Paiva Fonseca, por todas as trocas de
experiências, ensinamentos e acompanhamento durante esta fase tão importante em minha
vida. Sou grato por sua grande contribuição no meu amadurecimento profissional.
Agradeço ao Prof. Dr. Oslei Almeida pela oportunidade e seus ensinamentos
durante nossas práticas na Rotina e por sempre estimular os seus alunos a estudar e buscar o
conhecimento, uma demonstração clássica de andragogia. Ao Professor Dr. Alan Roger, pelos
ensinamentos sempre que possíveis, especialmente quando nos encontramos no Orocentro.
Aos Professores Drs. Jacks Jorge, Ricardo Colleta e Edgar Graner, agradeço igualmente pelos
ensinamentos transmitidos durante a pós-graduação. À todos os demais colaboradores na
realização deste projeto e publicação dos artigos: Profa. Manoela Martins, Prof. Eduardo
Fregnani, Dr. André Caroli, Dr. Fernando Soares, Dr. Hélder Pontes, Dra. Vivian Wagner,
À Fabiana e ao Adriano por toda a ajuda e momentos de aprendizado e
descontração nos laboratórios.
Os meus mais sinceros agradecimentos a todos os colegas e amigos do curso pela
companhia, momentos de trocas de experiências pessoais e profissionais, especialmente à
Debora Lima, Leonardo Reis, Debora Campanela, Celeste Sánchez e Ciro Dantas pela
companhia e trocas de experiências e conhecimentos teóricos e práticos, muitas vezes
verdadeiras aulas, e por tornarem os dias em Piracicaba mais agradáveis.
À Fundação de Amparo à Pesquisa do Estado de São Paulo pela concessão de
bolsa de estudo (processo FAPESP: 2015/21520-8).
AGRADECIMENTOS ESPECIAIS
Agradeço a Deus, nosso Pai Celestial, por todas as provas, expiações e desafios
como instrumentos para minha evolução moral e espiritual. Lhe agradeço pelo simples dom
da existência.
Aos meus pais, professores da vida e de profissão, agradeço por servirem de fonte
de inspiração e por todo o amor e carinho me ofertado. Sei que não foi fácil para mim chegar
até aqui, mas reconheço que vocês tiveram de abrir mão de muitas coisas e sonhos para que
eu pudesse conquistar os meus.
Agradeço a minha irmã, Greyce Amaral, e a minha tia, Maura Macêdo, por todo o
carinho o carinho, amor e contribuição para a minha formação moral. Muitas lembranças me
vêm à mente neste momento, como quando íamos e voltávamos da escola cantando músicas e
fazendo brincadeiras para nos distrair. São tantas coisas para agradecer que certamente as
palavras não conseguem descrever.
À Danielle Ferreira, pelo companheirismo, seu amor, compreensão e paciência
principalmente nestes últimos dias. Sabemos que nós temos uma grande e linda história e que
existem muitos amigos que fazem parte dela. Hoje quero agradecer especialmente por
acreditar em mim, no meu potencial, principalmente quando muitas vezes eu mesmo duvido.
Obrigado por me fazer acreditar nos meus sonhos e buscar lutar por ele.
Aos entes queridos que jamais saíram da lembrança, sinto na obrigação de
agradecer todo o amor e carinho por mim. A distância física ainda dói, mas nos momentos
mais difíceis sei que vocês intercederam por mim de alguma maneira. De todos, faço o
agradecimento especial para minha querida avó Socorro, a qual tenho a certeza que continua a
me abençoar. Obrigado minha amada avó.
Agradeço aos professores e amigos Danyel Elias da Cruz Perez e Flavia Maria da
Cruz Perez pelos ensinamentos ao longo da graduação e pela inspiração como exemplos de
profissionais, professores e orientadores. Ao professor Danyel faço um agradecimento
especial por toda a consideração e orientação enquanto fui seu aluno durante a graduação e no
programa de Iniciação Científica. Por esses e outros motivos sou eternamente grato.
RESUMO
O ameloblastoma é um tumor odontogênico benigno, porém com comportamento local
agressivo devido seu potencial de infiltração e destruição óssea, afetando preferencialmente a
região posterior de mandíbula. A sua contraparte maligna, o carcinoma ameloblástico, pode
surgir de novo ou desenvolver-se de um ameloblastoma prévio, estando associado a um alto
índice de recidivas e metástases linfonodal e a distância. Embora sejam raros, ambos
representam os tumores odontogênico benigno e maligno mais frequentemente diagnosticados
nos principais serviços de Patologia Oral. Os mecanismos moleculares envolvidos com a
etiopatogenia destes tumores são pouco conhecidos, e apesar de alterações no sistema de
reparo de erros de pareamento de bases do DNA, conhecido como sistema mismatch (MMR)
favorecerem o desenvolvimento de diferentes neoplasias humanas, a importância destes no
desenvolvimento do ameloblastoma e do carcinoma ameloblástico ainda carece de estudos. O
MMR humano compreende o conjunto de proteínas divididas em dois grupos, hMutS e
hMutL. Sua principal função é de corrigir erros de pareamentos e de loops de inserção e
deleção das bases nitrogenadas no DNA. Uma variedade de neoplasias e condições
potencialmente malignas possuem alteração desse sistema. Os objetivos deste trabalho estão
divididos em capítulos e compreendem: (i) conceituar a função das proteínas do sistema
mismatch nas lesões neoplásicas humanas, com ênfase nas que acometem a região oral; (ii)
analisar quantitativamente a imunoexpressão das proteínas hMutS em germes dentais
humanos, ameloblastomas e carcinomas ameloblásticos, correlacionando seus resultados com
a expressão de BRAF-V600E e com a recorrência tumoral. Foi observado que expressão
imunohistoquímica das proteínas hMutS em 10 amostras de germes dentais humanos, 39
ameloblastomas e 2 carcinomas ameloblásticos apresentam um decréscimo sequencial nos
níveis de expressão nos grupos analisados, especialmente das proteínas hMSH2 e hMSH6 em
ameloblastomas (p=0.0059) em relação ao germe dental humano (p<0.0001). A expressão
imunohistoquímica das proteínas hMutS em 73 amostras de ameloblastomas contidas em um
bloco de 1.0 mm de tissue microarray permitiu associar seus resultados com a taxa de
recorrências e presença da mutação BRAF-V600E. Os casos de ameloblastomas com
sobrexpressão das proteínas hMSH2, hMSH3 apresentaram a presença de BRAF-V600E
(p<0.05) e foram associados com a recorrência da lesão (p=0.035). Estes dados sugerem que a
sobrexpressão de hMutS pode estar indiretamente relacionada mecanismos moleculares e
genéticos envolvidos na progressão e recorrência tumoral.
Palavras-chave: ameloblastoma; tumores odontogênicos; hMSH2; hMSH3; hMSH6; proteínas
mismatch.
ABSTRACT
Ameloblastoma is a benign odontogenic tumor, but with aggressive local behavior due to its
potential for infiltration and bone destruction, preferentially affecting the posterior region of
the mandible. Its malignant counterpart, ameloblastic carcinoma, may arise de novo or
develop from a previous ameloblastoma, being associated with a high rate of relapses and
lymph node metastases and distance. Although rare, they both represent the benign and
malignant odontogenic tumors most frequently diagnosed in the main Oral Pathology
services. The molecular mechanisms involved in the etiopathogenesis of these tumors are
poorly understood, and although alterations in the DNA bases pairings repair system, known
as the mismatch system (MMR), favor the development of different human neoplasms, the
importance of these in the development of ameloblastoma and ameloblastic carcinoma still
lacks studies. Human MMR comprises the set of proteins divided into two groups, hMutS and
hMutL. Its main function is to correct errors of pairings and loops of insertion and deletion of
the bases in the DNA. Several neoplasms and potentially malignant conditions have alteration
of this system. The objectives of this work are divided into chapters and include: (i)
conceptualizing the function of the proteins of the mismatch system in human neoplastic
lesions, with emphasis on those that affect the oral region; (ii) quantitatively analyze the
immunoexpression of hMutS proteins in human dental germs, ameloblastomas and
ameloblastic carcinomas, correlating their results with the expression of BRAF-V600E and
with tumor recurrence. Immunohistochemical expression of hMutS proteins in 10 human
dental germ samples, 39 ameloblastomas and 2 ameloblastic carcinomas showed a sequential
decrease in expression levels in the analyzed groups, especially the hMSH2 and hMSH6
proteins in ameloblastomas (p = 0.0059) in relation to human dental germ (p <0.0001). The
immunohistochemical expression of hMutS proteins in 73 samples of ameloblastomas
contained in a 1.0 mm block of tissue microarray allowed to associate their results with the
rate of recurrences and the presence of the BRAF-V600E mutation. The cases of
ameloblastomas with overexpression of the hMSH2, hMSH3 proteins showed the presence of
BRAF-V600E (p <0.05) and were associated with lesion recurrence (p = 0.035). These data
suggest that the overexpression of hMutS may be indirectly related molecular and genetic
mechanisms involved in tumor progression and recurrence.
Keywords: ameloblastoma; odontogenic tumors; hMSH2; hMSH3; hMSH6; mismatch
proteins.
SUMÁRIO
1 INTRODUÇÃO 11
1.1 Ameloblastoma 11
1.2 Carcinoma ameloblástico 11
1.3 Sistema de reparo mismatch 12
2 ARTIGOS 14
2.1 Artigo: Mismatch repair system proteins in oral benign and malignant lesions 14
2.2 Artigo: Prognostic significance of hMSH2, hMSH3 and hMSH6 expression in
ameloblastoma 27
3 DISCUSSÃO 50
4 CONCLUSÃO 52
REFERÊNCIAS 53
ANEXOS 56
Anexo 1: Certificado de Direitos Autorais da Journal of Oral Pathology and
Medicine 56
Anexo 2: Carta de aceite para publicação do Journal of Oral Surgery, Oral
Medicine, Oral Pathology, Oral Radiology 57
Anexo 3: Certificado do Comitê de Ética em Pesquisa FOP/Unicamp 58
11
1 INTRODUÇÃO
1.1 Ameloblastoma
O ameloblastoma é um tumor odontogênico epitelial sem a formação de tecido
mineralizado que se origina de remanescentes de tecido odontogênico como a lâmina dentária,
órgão do esmalte, restos de Malassez e do revestimento epitelial de cistos odontogênicos,
especialmente os cistos dentígeros. (Dhanuthai et al., 2012; Masthan et al., 2015; McClary et
al., 2016). Primeiramente descrito por Cusack (1827), esta rara neoplasia acomete os ossos
gnáticos e representa cerca de 1% de todos os tumores orais, variando entre 9% – 11% dentre
os tumores odontogênicos (Masthan et al., 2015). Apresenta pico de ocorrência entre os 30-60
anos de idade, sem predileção pelo gênero e com incidência global de 0,5 casos/milhão/ano
(Fregnani et al., 2002; McClary et al., 2016). As manifestações clínicas apresentam
sintomatologia discreta, normalmente associada a um crescimento de volume indolor e lento,
porém localmente agressivo e infiltrativo, resultando em alta taxa de recorrência. Possui forte
predileção pela mandíbula em relação à maxila (13:1), preferencialmente a região posterior e
em terceiros molares não erupcionado (Dhanuthai et al., 2012). É radiograficamente
observado como imagens radiolúcidas uniloculares ou multiloculares, podendo apresentar
deslocamentos e reabsorções dentárias (Dhanuthai et al., 2012; McClary et al., 2016). Quando
associado às características microscópicas, pode ser classificado como ameloblastoma
unicístico, sólido ou multicistico (a maioria dos casos) ou periférico, apresentando seus
padrões histológicos variáveis (Dhanuthai et al., 2012; Masthan et al., 2015; McClary et al.,
2016).
A abordagem cirúrgica representa o tratamento padrão para o ameloblastoma,
porém os índices de recidiva da lesão estão fortemente relacionados com a modalidade
terapêutica empregada. No ameloblastoma unicístico, a taxa de recorrência corresponde a 4%
para o tratamento radical, como a ressecção em bloco, contra 17% do tratamento conservador,
como a curetagem da lesão, enquanto que no multicístico temos um índice de recorrências de
8% quando utilizado tratamento radical contra 38% quando tratamentos conservadores são
realizados (Antonoglou and Sándor, 2015).
1.2 Carcinoma ameloblástico
Os tumores odontogênicos malignos são neoplasias raras que correspondem a
menos de 6% de todos os tumores odontogênicos nas principais séries epidemiológicas
publicadas. Dentre estas lesões raras, o carcinoma ameloblástico corresponde ao tumor
odontogênico maligno mais comumente diagnosticado, com uma incidência estimada de
apenas 1,79 casos para cada 10 milhões de pessoas/ano. Esta neoplasia combina as
12
características histológicas do ameloblastoma com diferentes graus de atipia celular. Pode
surgir de novo, sem uma neoplasia benigna prévia, ou desenvolver-se de um cisto ou tumor
odontogênico benigno pré-existente, como um ameloblastoma, recebendo a denominação de
carcinoma ameloblástico secundário (Hall et al., 2007; Pirklbauer et al., 2012).
Embora sua apresentação clínica seja variável, uma tumefação local de
crescimento rápido, associada a dor e parestesia, são as manifestações mais relatadas. Possui
ligeira predileção pelo sexo masculino, com idade média de 40 anos, tendo a região posterior
da mandíbula como o sítio mais afetado. Radiograficamente apresenta imagens radiolúcidas
mal definidas, com eventuais focos radiopacos sugestivos de calcificações distróficas,
podendo causar reabsorções e deslocamentos dentários (Benlyazid et al., 2007). A análise
histológica do carcinoma ameloblástico demonstra regiões de diferenciação ameloblástica
com graus variáveis de pleomorfismo e atipia celular, além de figuras de mitose atípicas,
necrose tecidual e eventuais focos de invasão perineural (Fitzpatrick et al., 2015; Lucca et al.,
2010).
Pouco se conhece sobre suas alterações genéticas ou moleculares, o que limita o
aprimoramento da abordagem terapêutica desta entidade. Portanto, os pacientes são tratados
por meio de abordagem cirúrgicas radicais, como a remoção em bloco do tumor ou a ampla
ressecção com margens de segurança, causando um elevado grau de morbidade terapêutica.
Além disso, altos índices de recorrência que variam de 28,3% a 90% são relatados na
literatura dependendo em especial da abordagem cirúrgica empregada. A frequência de
metástases a distância chega a 22% e os locais mais acometidos são o pulmão, cérebro e
múltiplos ossos. O prognóstico é considerado sombrio com taxas de sobrevida após cinco
anos próximas de 70%, diminuindo para cerca de 20% em pacientes com metástases
(Benlyazid et al., 2007; Hall et al., 2007).
1.3 Sistema de reparo mismatch
Os mecanismos genéticos envolvidos nessas manifestações neoplásicas ainda são
pouco compreendidos. Uma das linhas de estudo pouco explorada está relacionada com o
sistema de reparo de bases nitrogenadas mal pareadas do DNA, conhecido como sistema
mismatch (MMR). O mecanismo deste sistema é mediado pela associação de proteínas que
mantêm a fidelidade do material genético replicado, corrigindo erros no pareamento de bases
e impedindo a instabilidade de microssatélites. O sistema humano é homólogo ao encontrado
em na bactéria Escherichia coli (E. coli), sendo denominados homólogos MutS (hMutS) e
MutL (hMutL) (Hsieh and Yamane 2008). Em tumores odontogênicos, o estudo deste
mecanismo foi apenas abordado por Castrilli et al. (2001) de forma descritiva através de
13
estudo imunohistoquímico em 25 casos de ameloblastomas. Neste estudo, os autores
identificaram a expressão positiva das proteínas hMSH2 e hMLH1 (componentes dos grupos
hMutS e hMutL, respectivamente), mas não realizaram estudos quantitativos ou comparativos
com germes dentais humanos, por exemplo.
Neste contexto, propusemos um estudo mais aprofundado da expressão
imunohistoquímica das proteínas relacionadas ao sistema mismatch em ameloblastomas e
carcinomas ameloblásticos, tendo como controle o epitélio ameloblástico normal de germes
dentais humanos, comparando seus resultados e correlacionando-os com as características
clínicas e potencial prognóstico.
14
2 ARTIGOS
2.1 Artigo: Mismatch repair system proteins in oral benign and malignant lesions
Artigo publicado no Journal of Oral Pathology and Medicine, 2017 Apr;46(4):241-245. doi:
10.1111/jop.12484. Epub 2016 Aug 10. (Anexo 1)
Gleyson Kleber do Amaral Silva1
Manoela Domingues Martins2
Hélder Antônio Rebelo Pontes3
Eduardo Rodrigues Fregnani4
Márcio Ajudarte Lopes1
Felipe Paiva Fonseca1
Pablo Agustin Vargas1
1. Department of Oral Diagnosis - Piracicaba Dental School – University of Campinas
(Piracicaba/Brazil)
2. Department of Pathology - School of Dentistry - Federal University of Rio Grande do
Sul (Porto Alegre/Brazil).
3. Service of Buccal Pathology – João de Barros Barreto University Hospital – Federal
University of Pará (Belém/Brazil).
4. Department of Oral Medicine – Sírio-Libanês Hospital (São Paulo/Brazil).
Corresponding Author:
Prof. Dr. Pablo Agustin Vargas
Piracicaba Dental School – University of Campinas (UNICAMP)
Department of Oral Diagnosis – Pathology
Av. Limeira, 901 ZipCode: 13414-903 Piracicaba – São Paulo – Brazil
Tel.: +55 19 21065319 E-mail: [email protected]
Keywords: Mismatch repair system, MutS, MutL, MSH, MLH, oral cancer, leukoplakia.
15
Abstract
Different environmental agents may cause DNA mutations by disrupting its double-
strand structure; however, even normal DNA polymerase function may synthesize
mismatch nucleotide bases, occasionally demonstrating failure in its proofreading
activity. To overcome this issue, mismatch repair (MMR) system, a group of proteins
specialized in finding mispairing bases and small loops of insertion or deletion,
works to avoid the occurrence of mutations that could ultimately lead to innumerous
human diseases. In the last decades, the role of MMR proteins in oral carcinogenesis
and in the development of other oral cavity neoplasms has grown, but their importance
in the pathogenesis and their prognostic potential for patients affected by oral
malignancies, especially oral squamous cell carcinoma (OSCC), remain unclear.
Therefore, in this manuscript we aimed to review and critically discuss the currently
available data on MMR proteins expression in oral potentially malignant lesions, in
OSCC, and in other oral neoplasms to better understand their relevance in these lesions.
Keywords: leukoplakia; mismatch repair system; MLH; MSH; MutL; MutS; oral cancer
Introduction
It is known that different endogenous and exogenous agents may cause DNA
mutations by disrupting its double-strand structure (1); however, DNA damage can also be
originated because of natural replication errors, including failures of DNA polymerase
activity (2) that may incorporate mismatched nucleotide bases, predisposing individuals to
innumerous human diseases (3).
Twelve different single-base incorporated errors can be found (4). Because these
genetic errors may pass unnoticed by the proofreading activity of DNA polymerase, the post-
replicated DNA strand is additionally checked by a mismatch repair (MMR) system that
consists of a group of proteins specialized in finding not only mispairing bases, but also small
loops of insertion or deletion, and ultimately providing their corrections (5).
Important new data were provided on the structure and function of MMR in the
human genome stability, but its importance on the development of different malignant and
potentially malignant human diseases has only recently been investigated. Therefore, in this
manuscript we aimed to review the current data available on MMR expression pattern and its
importance for the progression of oral squamous cell carcinoma (OSCC) and oral potentially
malignant lesions, as well as to provide an overview on their importance for the pathogenesis
of other oral cavity neoplasms.
16
MMR system and your proteins
Mismatch repair system was initially investigated in Escherichia coli, being
classified in four different groups as MutS, MutL, MutH, and MutU (6), whereas following
studies in eukaryotic cells defined only two homologous groups, hMutS and hMutL (7),
which include the somatic human proteins hMSH2 (gen locus on 2p21), hMSH3 (5q14.1),
hMSH4 (1p31), hMSH5 (6p21.3), hMSH6 (2p16), hMLH1 (3p21), hPMS1 (2q31.1), hPMS2
(7q22.2), and hMLH3 (14q24.3) (8, 9).
Although bacterial MutS and MutL groups are formed by homodimers, in
eukaryotic cells these groups comprise heterodimers composed by two related, but distinct
protein subunits (10). hMutS contains the heterodimers hMSH2- hMSH6 (called hMutSα) and
hMSH2-hMSH3 (called hMutSβ), whereas hMutL contains the heterodimers hMLH1-hPMS2
(hMutLa), hMLH1-hPMS1 (hMutLb), and hMLH1-hMLH3 (hMutLγ) (5).
As a post-replication event, hMutS scans the new DNA strand looking for
mismatches and loops of insertion or deletion. However, hMutSα more efficiently recognizes
base–base mismatches and insertion/deletion mispairs in which one strand contains one or
two unpaired nucleotides, whereas hMutSβ preferentially identifies loops of insertion/deletion
of two to over 10 nucleotides (10). Following the recognition of a mispaired, hMutS
undergoes an ATP-induced conformational change that activates it and promotes its
interaction with hMutL proteins. Subsequently, Proliferating Cell Nuclear Antigen (PCNA)
activates hMutL to incise the daughter strand hundreds of base distant from the mismatch.
Once hMutL nicks the DNA, hMutS activates EXO1 (an exonuclease) to progressively excise
the DNA containing the mismatch or the insertion/deletion loop, while DNA polymerases d/e
initiate the new strand synthesis from the nicked point (11) (Fig. 1).
However, MMR genes can also be disrupted by mutations or hypermethylations,
predisposing cells to acquire an increased amount of DNA mismatches and potentially
developing a neoplastic phenotype. This is better exemplified by Lynch syndrome and its
increased incidence of hereditary non-polyposis colorectal cancer (HNPCC) (12).
In addition, studies demonstrating abnormal expression of MMR components in
breast (13), prostate (14), urothelial (15), and lung cancers (16) have also been described,
usually showing a lower expression of MMR proteins.
Similarly, alterations in the MMR complex have also been correlated with the
onset and progression of oral potentially malignant lesions and OSCC, with some new
evidences that these proteins could play a role as prognostic determinants (Table 1). All these
studies support additional investigations to better understand the biological implication of
MMR mutation and epigenetic modifications in the neoplastic context.
17
MMR expression in oral and lip potentially malignant lesions
Leukoplakia is the most common potentially malignant lesion of the oral cavity,
and several studies looked forward to assessing the MMR protein expression pattern in this
lesion. Caldeira et al. (17) evaluated hMLH1 immunoexpression in different grades of
dysplasia, demonstrating a lower expression in more dysplastic lesions. These results were
confirmed by the same group that showed a lower expression of hMLH1 in more dysplastic
OL, also revealing an inverse correlation with p53 and AgNOR expression (18). These
authors suggested that hMLH1 alterations would represent an early molecular event in oral
carcinogenesis, what was supported by Jessri et al. (19) who confirmed a reduced expression
of hMLH1, hPMS2, and hMSH2 in OL and in OSCC when compared to normal oral mucosa,
also attributing a diagnostic role for these proteins. Sengupta et al. (20) showed that the
promoter regions of hMLH1 and hMSH2 were hypermethylated in 63% of their OL samples,
and this epigenetic feature correlated with the presence of microsatellite instability in
dysplastic cells and with tobacco use by the patients. Fernandes et al. (21) also described
altered hMLH1 expression in oral epithelium associated with tobacco use, with a higher
expression in smokers than non-smokers.
Similar results were described for actinic cheilitis (AC), the most common
potentially malignant lesion of the lip. Sarmento et al. (22) immunohistochemically analyzed
forty cases of lower lip AC demonstrating that hMLH1 and hMSH2 were less expressed in
more dysplastic lesions. These results were also obtained by Oliveira et al. (23) who showed
higher expression of hMLH1 in AC with mild dysplasia, whereas Souza et al. (24) found a
lower expression of hMSH2 in lip cancer than in AC.
Taken together, currently available studies that investigated MMR proteins in oral
and lip potentially malignant lesions have predominantly evaluated hMLH1 and hMSH2
proteins, always using immunohistochemical technique.
Although this methodology precludes the functional state of the proteins, most of
the results suggest that they are downregulated in more dysplastic lesions, making patients
more susceptible to additional DNA mutations and consequent malignant transformation.
However, further analyses with different laboratorial techniques are necessary to confirm this
assumption and to better understand the role played by MMR system in the pathogenesis of
OL and AC.
MMR expression in oral squamous cell carcinoma
Oral cancer is the sixth most common malignancy in the world (25), and
squamous cell carcinoma accounts for more than 90% of all cases (26). Different authors have
18
attempted to determine the importance of MMR proteins for OSCC pathogenesis, and Lo
Muzio et al. (27) results represent one of the earliest evidences that a reduction in the
expression of hMSH2 and hMLH1 could be associated with OSCC development.
This initial observation was subsequently confirmed by Jessri et al. (19) that
revealed a progressive reduction of hMLH1, hMSH2, and hPMS2 from mild, moderate, and
severe dysplasia to OSCC. In a following study, Jessri et al. (25) further confirmed the lower
expression of hMLH1, hMSH2, hPMS2, and hMSH6 in more dysplastic OL and even more in
OSCC, also highlighting the diagnostic utility of hMSH6 in the identification of carcinoma in
situ. Sarmento et al. (22) confirmed the lower expression of hMLH1 and hMSH2 in OSCC
and Helal et al. (28) additionally identified a significant correlation between the low hMSH2
and p53 expressions in OSCC, suggesting that hMSH2 would play a role in the late stages of
oral carcinogenesis.
In addition to mutational status of MMR proteins, their frequently described lower
expression in OSCC can also be explained by epigenetic events. Czerninski et al. (29)
demonstrated that 50% of their OSCC cases had hypermethylation of the promoter region of
hMSH2 and hMLH1, similar to the results of González-Ramírez et al. (30) who observed that
76% of their OSCC cases presented hyper- methylation of the promoter regions of hMLH1,
whereas Sengupta et al. (20) showed that tobacco use increased susceptibility to hMLH1 and
hMSH2 hypermethylation. Tawfik et al. (31) assessed the expression and the methylation
pattern of hMLH1 promoter, observing that 30.6% of their Head and Neck Squamous Cell
Carcinoma (HNSCC) cases (including oral cavity) presented a low hMLH1 expression, with
86.7% of the cases having hypermethylation of the promoter region. On the other hand, Wang
et al. (32) failed to correlate hMLH1 and hMSH2 methylation, exon mutation, and gene
expression with the presence of microsatellite instability in patients with HNSCC.
Genetic polymorphisms of MMR genes in patients affected by OSCC were also
investigated, and some studies showed that single nucleotide polymorphism (SNP) of hMLH1
and hMSH3 can be associated with OSCC predisposition (33, 34). Nogueira et al. (35)
showed that inherited hMLH1 c.-93G>A, hMSH2 c.21119C>G, hMSH3 c.3133G>A, and
EXO1 c.1765G>A polymorphisms are predictors of clinical outcomes of patients affected by
HNSCC.
Few authors attempted to demonstrate the clinical significance of MMR proteins
in patients affected by OSCC. Cell lineages with low expression of hMSH2 and hMLH1 were
shown to be more resistant to the chemotherapeutic effect of cisplatin (36, 37), whereas our
recent study evidenced that lower hMSH2 expression was associated with higher T stage and
that both hMSH2 and hMSH6 expression predicted a poor prognosis (38).
19
In contrast to the observed for potentially malignant lesions, although
immunohistochemical studies still predominate in the evaluation of MMR system in OSCC,
there are several genetic analyses currently available. However, functional assays and
different in vitro and in vivo analyses are still necessary to fully understand the role of MMR
proteins in the development of OSCC. Moreover, only one study investigated the prognostic
potential of MMR proteins, demanding additional analyses. Finally, some reports used
squamous cell carcinomas from different head and neck regions in their studies, what could
preclude stronger conclusions given the biological heterogeneity of these lesions. On the other
hand, available evidences point toward an important role played by the loss of MMR system
to OSCC pathogenesis.
MMR expression in other oral neoplasms
Mismatch repair proteins have also been investigated in other oral neoplasms like
in oral melanoma where hMLH1, hMSH2, hPMS1, and hPMS2 expressions were shown to be
frequently decreased (39, 40). Regarding salivary gland tumors, Tobón-Arroyave et al. (41)
suggested that the lower expression of hMLH1 and hMSH2 in cases of pleomorphic adenoma
would indicate a higher risk for malignant transformation of this tumor. However, no
significant differences were observed between benign and malignant salivary gland tumors in
two other studies (42, 43), whereas salivary gland adenocarcinomas demonstrated higher
expression of hMSH2 and hMLH1 when compared with pleomorphic adenomas in Castrilli et
al. (43) investigation. These results demonstrate divergent conclusions regarding MMR
proteins in salivary gland tumorigenesis.
Regarding odontogenic tumors, MMR proteins were investigated only in
ameloblastomas. Castrilli et al. (44) evaluated the expression of hMSH2 and hMLH1 proteins
by immunohistochemistry in 25 cases, observing a nuclear positivity in all of them,
speculating that the development and progression of ameloblastomas would not depend on a
defect in the MMR system.
Conclusion
Mismatch repair system is an important group of proteins for DNA repair and
maintenance of the genomic stability. Disruption of this system has been suggested to be
important in the pathogenesis of different oral lesions, including potentially malignant
disorders and malignant neoplasms. However, most of the studies currently available are
limited to demonstrate the immunoexpression of these proteins (more frequently of hMSH2
and hMLH1), and the size of the samples investigated is usually small, rarely correlating their
20
expression patterns with clinicopathological parameters. Therefore, although we have
evidences that the loss of MMR proteins is important for the development of several oral
lesions, further studies are warranted to better understand their importance as possible
therapeutic targets, especially in the OSCC context, where these proteins also remain to be
further validated as prognostic markers.
References
1. Ribezzo F, Shiloh Y, Schumacher B. Systemic DNA damage responses in aging and
diseases. Semin Cancer Biol 2016; 37– 38: 26–35.
2. Loeb LA, Cheng KC. Errors in DNA synthesis: a source of spontaneous mutations.
Mutat Res 1990; 238: 297–304.
3. Preston BD, Albertson TM, Herr AJ. DNA replication fidelity and cancer. Semin
Cancer Biol 2010; 20: 281–93.
4. Johnson SJ, Beese LS. Structures of mismatch replication errors observed in a DNA
polymerase. Cell 2004; 116: 803–16.
5. Hsieh P, Yamane K. DNA mismatch repair: molecular mechanism, cancer, and ageing.
Mech Ageing Dev 2008; 129: 391–407.
6. Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and
cancer biology. Annu Rev Biochem 1996; 65: 101–33.
7. Kolodner RD, Marsischky GT. Eukaryotic DNA mismatch repair. Curr Opin Genet Dev
1999; 9: 89–96.
8. Lipkin SM, Wang V, Jacoby R, et al. MLH3: a DNA mismatch repair gene associated
with mammalian microsatel- lite instability. Nat Genet 2000; 24: 27–35.
9. Clark N, Wu X, Her C. MutS homologues hMSH4 and hMSH5: genetic variations,
functions, and implications in human diseases. Curr Genomics 2013; 14: 81–90.
10. Modrich P. Mechanisms in eukaryotic mismatch repair. J Biol Chem 2006; 281: 30305–
9.
11. Erie DA, Weninger KR. Single molecule studies of DNA mismatch repair. DNA Repair
(Amst) 2014; 20: 71–81.
12. Peltom€aki P. Lynch syndrome genes. Fam Cancer 2005; 4: 227–32.
13. Naqvi RA, Hussain A, Deo SS, et al. Hypermethylation analysis of mismatch repair
genes (hmlh1 and hmsh2) in locally advanced breast cancers in Indian women. Hum
Pathol 2008; 39: 672–80.
14. Dominguez-Valentin M, Joost P, Therkildsen C, Jonsson M, Rambech E, Nilbert M.
Frequent mismatch-repair defects link prostate cancer to Lynch syndrome. BMC Urol
21
2016; 16: 15.
15. Ehsani L, Osunkoya AO. Expression of MLH1 and MSH2 in urothelial carcinoma of
the renal pelvis. Tumour Biol 2014; 35: 8743–7.
16. Slováková P, Majerová L, Matáková T, Skerenová M, Kavcová E, Halasová E.
Mismatch repair gene polymorphisms and association with lung cancer development.
Adv Exp Med Biol 2015; 833: 15–22.
17. Caldeira PC, Abreu MHNG, Batista AC. DO CARMO MAV. hMLH1
immunoexpression is related to the degree of epithelial dysplasia in oral leukoplakia. J
Oral Pathol Med 2011; 40: 153–9.
18. Caldeira PC, Aguiar MCF, Mesquita RA. DO CARMO MAV. Oral leukoplakias with
different degrees of dysplasia: com- parative study of hMLH1, p53, and AgNOR. J Oral
Pathol Med 2011; 40: 305–11.
19. Jessri M, Dalley AJ, Farah CS. MutSa and MutLa immuno- expression analysis in
diagnostic grading of oral epithelial dysplasia and squamous cell carcinoma. Oral Surg
Oral Med Oral Pathol Oral Radiol 2015; 119: 74–82.
20. Sengupta S, Chakrabarti S, Roy A, Panda CK, Roychoudhury S. Inactivation of human
mutL homolog 1 and mutS homolog 2 genes in head and neck squamous cell carcinoma
tumors and leukoplakia samples by promoter hypermethylation and its relation with
microsatellite instability phenotype. Cancer 2007; 109: 703–12.
21. Fernandes AM, de Souza VRDC, Springer CRA, et al. Tobacco and inflammation
effects in immunoexpression of hMSH2 and hMLH1 in epithelium of oral mucosa.
Anticancer Res 2007; 27: 2433–7.
22. Sarmento DJS, de Almeida WL, Miguel MCC, et al. Immuno- histochemical analysis of
mismatch proteins in carcinogenesis of the lower lip. Histopathology 2013; 63: 371–7.
23. de Oliveira DHIP, de Sousa Lopes MLD, de Santana Sarmento DJ, Queiroz LMG, da
Costa Miguel MC, da Silveira EJD. Relationship between the epithelial expression of
hMLH1, MDM2, and p63 and lower lip carcinogenesis. J Oral Pathol Med 2014; 43:
357–63.
24. Souza LR, Fonseca-Silva T, Pereira CS, et al. Immunohisto- chemical analysis of p53,
APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell
carcinoma. Histopathology 2011; 58: 352–60.
25. Jessri M, Dalley AJ, Farah CS. hMSH6: a potential diagnostic marker for oral
carcinoma in situ. J Clin Pathol 2015; 68: 86– 90.
26. Pontes FSC, Carneiro JT, Fonseca FP, da Silva TSP, Pontes HAR, Pinto DS. Squamous
cell carcinoma of the tongue and floor of the mouth. J Craniofac Surg 2011; 22: 925–30.
22
27. Lo Muzio L, Nocini P, Mignogna MD, et al. Immunocyto-chemical detection of
hMSH2 and hMLH1 expression in oral SCC. Anticancer Res 1999; 19: 933–40.
28. Helal TEA, Fadel MT, El-Thobbani AK, El-Sarhi AM. Immunoexpression of p53 and
hMSH2 in oral squamous cell carcinoma and oral dysplastic lesions in Yemen:
relationship to oral risk habits and prognostic factors. Oral Oncol 2012; 48: 120–4.
29. Czerninski R, Krichevsky S, Ashhab Y, Gazit D, Patel V, Ben- Yehuda D. Promoter
hypermethylation of mismatch repair genes, hMLH1 and hMSH2 in oral squamous cell
carcinoma. Oral Dis 2009; 15: 206–13.
30. González-Ramírez I, Ramírez-Amador V, Irigoyen-Camacho ME, et al. hMLH1
promoter methylation is an early event in oral cancer. Oral Oncol 2011; 47: 22–6.
31. Tawfik HM, El-Maqsoud NMRA, Hak BHAA. EL-SHERBINY YM. Head and neck
squamous cell carcinoma: mismatch repair immunohistochemistry and promoter
hypermethylation of hMLH1 gene. Am J Otolaryngol 2011; 32: 528–36.
32. Wang Y, Irish J, Macmillan C, et al. High frequency of microsatellite instability in
young patients with head-and-neck squamous-cell carcinoma: lack of involvement of
the mismatch repair genes hMLH1 AND hMSH2. Int J Cancer 2001; 93: 353–60.
33. Jha R, Gaur P, Sharma SC, Das SN. Single nucleotide polymorphism in hMLH1
promoter and risk of tobacco-related oral carcinoma in high-risk Asian Indians. Gene
2013; 526: 223–7.
34. Mondal P, Datta S, Maiti GP, et al. Comprehensive SNP scan of DNA repair and DNA
damage response genes reveal multiple susceptibility loci conferring risk to tobacco
associated leukoplakia and oral cancer. PLoS One 2013; 8: e56952.
35. Nogueira GAS, Louren,co GJ, Oliveira CBM, et al. Association between genetic
polymorphisms in DNA mismatch repair-related genes with risk and prognosis of head
and neck squamous cell carcinoma. Int J Cancer 2015; 137: 810–8.
36. Fujieda S, Tanaka N, Sunaga H, et al. Expression of hMSH2 correlates with in vitro
chemosensitivity to CDDP cytotoxicity in oral and oropharyngeal carcinoma. Cancer
Lett 1998; 132: 37–44.
37. Adachi M, Ijichi K, Hasegawa Y, Nakamura H, Ogawa T, Kanematsu N. Human MLH1
status can potentially predict cisplatin sensitivity but not microsatellite instability in
head and neck squamous cell carcinoma cells. Exp Ther Med 2010; 1: 93–6.
38. Wagner VP, Webber LP, Salvadori G, et al. Overexpression of MutSa complex proteins
predicts poor prognosis in oral squamous cell carcinoma. Medicine (Baltimore) 2016;
95: e3725.
39. Korabiowska M, Brinck U, Ruschenburg I, Honig JF, Stachura J, Droese M. Loss of
23
DNA-mismatch repair gene expression in oral melanomas. Oncol Rep 1999; 6: 921–3.
40. Lo Muzio L, Nocini P, Mignogna MD, et al. Immunocyto- chemical detection of
hMSH2 and hMLH1 expression in oral melanoma. Anticancer Res 2000; 20: 741–8.
41. Tobón-Arroyave SI, Flórez-Moreno GA, Jaramillo-Cárdenas JF, et al. Expression of
hMLH1 and hMSH2 proteins in pleomorphic adenoma of minor salivary glands:
relationship with clinical and histologic findings. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 2009; 108: 227–36.
42. Ohki K, Kumamoto H, Ichinohasama R, et al. Genetic analysis of DNA microsatellite
loci in salivary gland tumours: comparison with immunohistochemical detection of
hMSH2 and p53 proteins. Int J Oral Maxillofac Surg 2001; 30: 538– 44.
43. Castrilli G, Fabiano A, la Torre G, et al. Expression of hMSH2 and hMLH1 proteins of
the human DNA mismatch repair system in salivary gland tumors. J Oral Pathol Med
2002; 31: 234–8.
44. Castrilli G, Piantelli M, Artese L, et al. Expression of hMSH2 and hMLH1 proteins of
the human DNA mismatch repair system in ameloblastoma. J Oral Pathol Med 2001;
30: 305–8.
Acknowledgement
This research has been supported by grants from the São Paulo State Research
Foundation (FAPESP 2015/21520-8 and FAPESP 2015/16056-0).
Conflict of interest statement
The authors state that they have no potential conflict of interest that could bias the
results obtained in this study.
Ethics committee approval
Not applied.
24
Figure 1: Schematic illustration of MMR system, exemplifying hMutSα (hMSH2 and
hMSH6) and hMutLa (hMLH1 and hPMS2) function. (A) Mismatch base (red triangle) was
incorporated in the DNA strand during replication and not recognized by DNA polymerase.
(B) hMutSα recog- nized the error and recruited hMutLa to form a complex. (C) PCNA
activates hMutLa endonuclease function to incise the daughter strand both distally and
proximally to the mispairing position. (D) hMutSα activates EXO1 to remove the excised
DNA containing the mismatch. (E) DNA polymerase with PCNA initiates the new DNA
synthesis from the nicked region.
25
Table 1. Results obtained by the currently available studies that investigated MMR proteins in oral lesions.
Authors Lesion investigated MMR proteins
investigated
Main results obtained
Fujieda et al.,
1998
OSCC hMSH2 Lower expression of hMSH2 showed more resistance to
cisplatin.
Lo Muzio et al.,
1999
OSCC hMSH2/hMLH1 Reduction of these proteins may be related with OSCC.
Korabiowska et
al., 1999
Oral melanoma hMSH2/hMLH1/hPMS1/hPMS2 Lower expression correlated with high aneuploidy ratio.
Lo Muzio et al.,
2000
Oral melanoma hMSH2/hMLH1 Low expression of the proteins.
Wang et al., 2001 OSCC hMLH1/hMSH2 There is no association between MMR proteins and MSI.
Ohki et al., 2001 SGT hMSH2 hMSH2 expression is not significantly different between benign
and malignant tumors.
Castrilli et al.,
2001
Ameloblastoma hMSH2/hMLH1 All cases were positive for the proteins.
Castrilli et al.,
2002
SGT hMSH2/hMLH1 hMSH2 and hMLH1 expressions are not significantly different
between benign and malignant tumors.
Sengupta et al.,
2007
Leukoplakia/HNSCC hMSH2/hMLH1 Hypermethylation of the promoter regions and positive
correlation with tobacco use.
Czerninski et al.,
2009
OSCC hMSH2/hMLH1 OSCC presented hypermethylation of hMSH2 and hMLH1
promoters.
Tobón-Arroyave
et al., 2009
PA hMSH2/hMLH1 Lower expression correlated with a higher risk for malignant
transformation.
Adachi et al.,
2010
HNSCC hMLH1 Low expression of hMLH1 showed more resistance to cisplatin.
Souza et al., 2011 AC hMSH2 Lower expression in severe dysplasia.
González-
Ramírez et al.,
2011
OSCC hMLH1 Presence of hypermethylation of hMLH1.
Tawfik et al., HNSCC hMLH1 86.7% of the sample showed hypermethylation.
26
2011
Caldeira et al.,
2011a
Leukoplakia hMLH1 Lower expression in severe dysplasia.
Caldeira et al.,
2011b
Leukoplakia hMLH1 Lower expression in severe dysplasia.
Helal et al., 2012 OSCC hMSH2 Lower expression in OSCC.
Sarmento et al.,
2013
AC/OSCC hMLH1/hMSH2 Lower expression in OSCC.
Jha et al., 2013 OSCC hMLH1 Polymorphism in this gene may be related with OSCC
predisposition.
Mondal et al.,
2013
Leukoplakia/OSCC hMSH3 Polymorphism in hMSH3 may be related with OSCC
predisposition.
de Oliveira et al.,
2014
AC hMLH1 Lower expression in severe dysplasia.
Nogueira et al.,
2015
OSCC hMLH1/hMSH2/hMSH3/EXO1 Polymorphisms in the MMR system may be related with OSCC
predisposition.
Jessri et al.,
2015a
OSCC hMSH2/hMSH6/hMLH1/hPMS2 Focal lack of hMSH6 indicates carcinoma in situ.
Jessri et al.,
2015b
Leukoplakia/OSCC hMSH2/hMSH6/hMLH1/hPMS2 Lower expression in severe dysplasia (except hMSH6).
Wagner et al.,
2016
OSCC hMSH2/hMSH6 Independent prognostic markers
AC: Actinic cheilitis; PA: Pleomorphic adenoma; Oral squamous cell carcinoma; HNSCC: Head and neck squamous cell carcinoma; SGT: Salivary gland tumors.
27
2.2 Artigo: Prognostic significance of hMSH2, hMSH3 and hMSH6 expression in
ameloblastoma
Artigo aceito para publicação no periódico Oral Surgery, Oral Medicine, Oral Pathology, Oral
Radiology. (Anexo 2)
Gleyson Kleber do Amaral-Silva1, Celeste Sánchez-Romero1, Vivian Petersen Wagner2,
Manoela Domingues Martins2, Hélder Antônio Rebelo Pontes3, Eduardo Rodrigues Fregnani4,
Fernando Augusto Soares5, Oslei Paes de Almeida1, André Caroli Rocha6, Alan Roger
Santos-Silva1, Felipe Paiva Fonseca7 and Pablo Agustin Vargas1
1. Department of Oral Diagnosis - Piracicaba Dental School – University of Campinas
(Piracicaba/Brazil)
2. Department of Pathology - School of Dentistry - Federal University of Rio Grande do Sul
(Porto Alegre/Brazil).
3. Service of Buccal Pathology – João de Barros Barreto University Hospital – Federal
University of Pará (Belém/Brazil).
4. Oral Medicine Department – Sírio-Libanês Hospital (São Paulo/Brazil).
5. Department of Pathology, A. C. Camargo Cancer Center (São Paulo/Brazil).
6. Medical School, Clinics Hospital, University of São Paulo (São Paulo/Brazil).
7. Department of Oral Surgery and Pathology – School of Dentistry – Universidade Federal
de Minas Gerais (Belo Horizonte/Brazil).
Corresponding Author:
Prof. Dr. Pablo Agustin Vargas, FRCPath
Piracicaba Dental School – University of Campinas (UNICAMP)
Department of Oral Diagnosis – Oral Pathology
Av. Limeira, 901 ZipCode: 13414-903 Piracicaba – São Paulo – Brazil
Tel.: +55 19 21065319 E-mail: [email protected]
Conflict of interest statement
The authors state that they have no potential conflict of interest that could bias the results
obtained in this study. This research has been supported by grants from the São Paulo State
Research Foundation (FAPESP 2015/21520-8).
Word count for the abstract: 177 Manuscript word count: 3162
Number of references: 44 Number of figures/tables: 6 figures and 1 table
28
Abstract
Aims: To investigate hMutS proteins in human dental germ, ameloblastomas and
ameloblastic carcinoma, and to determine whether the expression of these proteins has any
prognostic potential.
Methods and results: Ten cases of human dental germs, 39 ameloblastomas and 2
ameloblastic carcinomas were used to determine the distribution of the proteins during the
carcinogenesis process. Simultaneously, another sample of 73 ameloblastomas was arranged
in tissue microarray and their clinical, microscopic and radiographic features, treatment
outcome, presence of BRAF-V600E mutation and follow-up data, were assessed to determine
the prognostic relevance of the proteins. Immunohistochemistry was performed using
antibodies against hMutS (hMSH2, hMSH3, hMSH6) and Ki67. hMSH2 and hMSH6 were
significantly down-expressed in ameloblastomas (p=0.0059) than in human dental germs
(p<0.0001). hMSH2, hMSH3 and the protein combinations were significantly associated with
BRAF-V600E mutation (p<0.05). Simultaneous over-expression of hMutS was associated
with recurrences (p=0.035); however, the proteins did not predict the disease-free survival of
patients (p>0.05).
Conclusions: hMutS proteins are down-regulated in ameloblastoma; moreover, simultaneous
over-expression of these proteins in ameloblastoma was associated with recurrence, but did
not predict disease-free survival.
Keywords: ameloblastoma; odontogenic tumors; hMSH2; hMSH3; hMSH6; mismatch
protein.
29
Introduction
Ameloblastoma is one of the most important odontogenic tumors given its high
frequency, local aggressiveness and infiltrative potential1-3. Surgery remains the gold standard
of treatment, ranging from conservative to aggressive approaches that lead to a recurrence rate
varying from 6% to over 51%, and different degrees of morbidity.4 Although malignant
odontogenic tumors are rarely identified, representing less than 6% of all odontogenic tumors,
ameloblastic carcinoma is the most common subtype. It may arise de novo or secondarily to a
previous benign ameloblastoma.5,6 Adult males are more affected and mandible is the most
involved location.7 Wide surgical resection is the recommended management, but there is a
recurrences rate of up to 90% and distant metastases are present in approximately 22% of the
cases.5,7
Ameloblastoma and ameloblastic carcinoma pathogenesis is poorly understood,
but a growing number of studies have attempted to define the molecular events that may drive
their development and clinical behavior. Recently, much attention has been paid to the
Mitogen activated protein kinases (MAPK) pathway mutations, especially in the B-Raf
(BRAF) oncogene,8–10 which was shown to be altered in approximately 60% of
ameloblastomas, potentially determining an increased ameloblastoma aggressiveness.11
In an attempt to avoid genetic mutations or decrease their frequency, humans
contain a complex group of proteins organized in the so-called mismatch repair system
(MMR), which is responsible for identifying and correcting mismatch and small loops of
deletion and insertion in DNA bases.12,13 The MMR system consists of hMutS with its
heterodimers hHMSH2-hMSH6 (called hMutSα) and hHMSH2-hMSH3 (called hMutSβ), and
of hMutL with its heterodimers hMLH1-hPMS2 (hMutLα), hMLH1-hPMS1 (hMutLβ) and
hMLH1-hMLH3 (hMutLγ).12 The failure in this system, either by mutations or by epigenetic
events, may lead to the development of pathological conditions, including different human
cancers,14,15 however, its importance for the pathogenesis of odontogenic tumors
remainsunclear and demands to be better understood.16 Therefore, the aim of this study is to
investigate the expression pattern of hMSH2, hMSH3 and hMSH6 in human dental germ,
ameloblastomas and ameloblastic carcinomas, and to determine whether the expression of
these proteins has any prognostic potential for patients affected by ameloblastomas.
30
Material and Methods
Samples
For investigating the different expression patterns of hMutS proteins in benign
and malignant tumors and in their normal counterpart, 10 formalin-fixed paraffin-embedded
human dental germs present in surgical specimens sent for microscopic analysis of other
lesions, 39 cases of ameloblastomas and 2 cases of ameloblastic carcinomas were retrieved
from the files of three Brazilian institutions (Federal University of Pará – Belém; Federal
University of Rio Grande do Sul – Porto Alegre; and University of Campinas – Piracicaba) in
a 12-year period from January 2003 to January 2015. New 5 μm histological sections stained
with haematoxylin and eosin (H&E) were used to confirm the original diagnoses by two oral
pathologists. Clinical data regarding sex, age, tumor location, predominant microscopic
pattern and radiographic features were also retrieved from the patients’ dental charts.
Simultaneously, another set of 93 cases of ameloblastomas with full
clinicopathological data available (sex, age, tumor location, tumor size, tumor duration,
vestibular/palatine cortical disruption, basal cortical disruption, predominant microscopic
pattern, radiographic pattern, recurrences, follow-up data and BRAF-V600E
immunohistochemical status) were retrieved from the files of the Department of Pathology of
the A. C. Camargo Cancer Center (São Paulo – Brazil) and organized in a tissue microarray
(TMA) block to investigate the prognostic potential of hMutS proteins. The disease-free
survival (DFS) was obtained by calculating the time between the treatment date and the date
of recurrence or the last follow-up data obtained.The study was approved by the Ethics
Committee of the Piracicaba Dental School, University of Campinas (protocol 54172016.8).
Tissue microarray
The construction of the TMA block was previously described.11 Briefly, after the
microscopic review of the 93 ameloblastoma cases, areas of interest in each case were
selected from their respective H&E stained sections, and a TMA block was constructed
(Beecher Instruments, Silver Springs, USA). The areas identified in the donor paraffin block
were punctured twice with a 1.0 mm needle, and the two cylinders obtained were transferred
to the receptor paraffin block. A case was considered appropriate when at least 25% of the
total area of the cylinder with neoplastic tissue was available for analysis. Sequential 4 µm
thick sections were cut and collected on adhesive slides. A map specifying the exact position
of each core was made and a cylinder of normal liver tissue was included in the lower left
31
corner of the TMA block for orientation. TMA protocol used for this study was previously
validated by our group.1
Immunohistochemistry
Immunohistochemical reactions were carried out in 3µm histological sections of
all cases. Samples were dewaxed with xylene and then hydrated in an ethanol series. Antigen
retrieval was performed using citrate solution (pH 6.0) in a pressure cooker for 3 minutes, and
the endogenous peroxidase activity was blocked using 10% hydrogen peroxide. After washing
in PBS buffer (pH7.4), slides were incubated overnight with primary antibodies against
hMSH2 (Polyclonal, Santa Cruz Technology, California, USA, diluted 1:100), hMSH6
(Polyclonal, Cell Marque, California, USA, diluted 1:50), hMSH3 (Polyclonal rabbit
antihuman, Novus Biologicals, Newcastle, UK) and Ki-67 (Monoclonal, clone MIB1,
DakoCytomation, Glostrup, Denmark, diluted 1:100). All slides were subsequently exposed
toavidin-biotin complex, horseradish peroxidase reagents (LSAB Kit-DakoCytomation), and
diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO, USA), and subsequently
counter-stained with Carazzi haematoxylin. Positive control sections were used for each
antibody, including colonic samples for hMSH2, hMSH3 and hMSH6, and oral squamous cell
carcinoma (OSCC) samples for Ki-67. Negative controls were obtained by omitting the
primary antibodies.
Conventional quantitative analysis
In the human dental germ, the 39 cases of ameloblastomas and the 2 ameloblastic
carcinomas, the expression of hMSH2, hMSH3, hMSH6 and Ki-67 was obtained by one
observer by counting the percentage of positive cells in a total of 1,000 cells per case in
hotspot areas, using photomicrographs in 400x taken with a DFC345 Photomicroscope (Leica
DMR, Leica Microsystems, Wetzlar, Germany) and assessed with ImageJ software (version
1.51c, National Institutes of Health, Bethesda, MD, USA).
Digital analysis
The immunohistochemical reactions against hMSH2, hMSH3 and hMSH6 done in
the ameloblastoma cases of the TMA sections were then scanned into high-resolution images
using the Aperio Scanscope CS Slide Scanner (Aperio Technologies Inc, Vista, CA). The
digital images obtained in .svs format were visualized using the ImageScope software (Aperio
Technologies Inc., Vista, CA). hMSH2, hMSH3 and hMSH6 nuclear stainings were analyzed
32
using the Nuclear V9 algorithm (Aperio Technologies Inc, Vista, CA) with the following
input parameters: averaging radius: 0.9; curvature threshold: 2.5; lower threshold: 0; upper
threshold: 230; minimum nuclear size: 22; maximum nuclear size: 165; minimum roundness:
0.3; minimum compactness: 0.1; minimum elongation: 0.2; clear area objective: 240; and an
intensity threshold ranging from 0 to 230, in which strong staining was considered from 0
to185 and weak staining was from 185 to 230. At least 1000 cells were quantified in each case
and the percentage of positive cells was determined.
Statistical analysis
Initially, D’Agostino–Pearson test was used to determine the normality
distribution of the obtained data. Subsequently, to compare the immunoexpression of hMSH2,
hMSH3, hMSH6 and Ki67 between human dental germ and ameloblastoma, the unpaired
Student t-test and Mann–Whitney test were used. To investigate the association of hMSH2,
hMSH3 and hMSH6 with clinicopathological parameters, results were categorized as above
and below the mean positivity and the two-tailed Fisher’s exact test and the chi-square test
were used. Pearson and Spearman tests were used to investigate the correlation of hMSH2,
hMSH3 and hMSH6 with the expression of Ki67. Survival analysis was calculated using the
Kaplan–Meier method and the Log-rank univariate test was used to compare the groups.
Because only two cases of ameloblastic carcinoma could be retrieved, the results obtained in
this lesion were only descriptively described. GraphPad Prism version 5.01 (La Jolla, CA,
USA) and IBM SPSS version 20 (Chicago, IL, USA) software were used for statistical
analyses and a pvalue ≤ 0.05 using a 95% confidence interval was considered statistically
significant.
Results
Expression of hMSH2, hMSH3, hMSH6 and Ki67 in human dental germ, ameloblastoma
and ameloblastic carcinoma
In the ameloblastoma cases, males and females were affected in 17 cases each,
and in 5 cases the gender was not available. The mean age of this sample was 26 years (range
20–30 years) and the posterior region of the mandible was the most affected (77% or 30
cases) followed by the posterior region of maxilla (23% or 9 cases). Tumors predominantly
presented as multilocular images (38% or 15 cases), whereas in 36% (14 cases) of the cases
the radiographic exam revealed a unilocular image and in 25.6% of the cases data were not
available (10 cases). Microscopically, 41% of the cases (16/39 cases) predominantly
33
presented the follicular pattern, whereas 59% of the cases (23/39 cases) predominantly
presented the plexiform pattern. Due to its rarity, only two cases of ameloblastic carcinoma
could be retrieved, one of them affecting a 28-year-old female patient whose details have been
described previously17 and the other affecting the posterior mandible of a 23-year-old male
patient.
Evaluating hotspot areas of the epithelial component of the human dental germs a
Ki67 proliferative index of 6.67% was obtained and positive cells were present both in the
ameloblastic layer and in the central stellate reticulum area. In ameloblastomas the
proliferative index was 3.76% and positive cells were predominantly located in the basal layer
of the neoplastic epithelial nests. This variation was significantly different (p < 0.0005). The
mean proliferative index of the ameloblastic carcinoma cases achieved 63% (Figure 1).
The expression of hMSH2, hMSH3 and hMSH6 proteins was found in all
investigated cases with similar distribution patterns, but different rates. All proteins were
observed in both epithelial and mesenchymal compartments of the human dental germs. In the
epithelial component the proteins were predominantly presented in the ameloblastic layer,
although some cells in the stellate reticulum were also positively stained, achieving in the
hotspot areas of the epithelium an immunoreactivity of 60.2%, 60.2% and 60.6% to hMSH2,
hMSH3 and hMSH6, respectively (Figures 2A, 3A and 4A). In ameloblastoma cases, the
proteins were also present in both peripheral and central areas of the neoplastic epithelial
nests and strands. Positive nuclei of granular cells were rarely observed and the stromal cells
were negative. Positivity in ameloblastomas achieved 43.2%, 59.5% and 40.9% to hMSH2,
hMSH3 and hMSH6, respectively (Figures 2B, 3B and 4B). Although only two cases of
ameloblastic carcinoma were analyzed, we observed a mean positivity of 38.5%, 39.5% and
41.8% to hMSH2, hMSH3 and hMSH6, respectively, also lower than those observed in the
normaltissue, but not too different from the benign tumor (Figures 2C, 3C and 4C).
Statistically, we observed that the expression of all three proteins was decreased in
ameloblastomas if compared to their normal counterpart, and significance was obtained for
hMSH2 (p = 0.0059) and hMSH6 (p < 0.0001), whereas hMSH3 did not achieve statistical
significance (p = 0.8836) (Figures 2D, 3D and 4D). Statistical analyses were not performed
for ameloblastic carcinoma.
The expression of hMSH2, hMSH3 and hMSH6 proteins did not correlate with
Ki67 proliferative index in human dental germs (p = 0.56, p = 0.59 and p = 0.43, respectively)
and in ameloblastomas (p = 0.50, p = 0.51 and p = 0.42, respectively).
34
Correlation of hHMSH2, hMSH3 and hMSH6 with clinicopathological and follow-up
features of ameloblastoma
To investigate whether the expression of hMSH2, hMSH3 and hMSH6 proteins
would have any prognostic significance for patients affected by ameloblastoma, we used
TMA sections initially containing 93 cases. However, because the TMA block had been used
in previous studies and because of the aggressiveness of the immunohistochemical reactions
to the neoplastic tissue, 73 cases remained in the TMA sections to be quantified. The
clinicopathological and follow-up data regarding this final sample have been described
previously.11
All cases arranged in the TMA sections showed nuclear positivity for the three
markers in both peripheral and central areas of the nest and islands, with no staining in the
stromal cells. The sections were then digitized and the reactions quantified using the Nuclear
V9 algorithm (Aperio Technologies Inc, Vista, CA), the results of which were categorized as
above and below the mean positivity to be correlated with the clinicopathological parameters
and follow-up status of the patients. Table 1 summarizes the results obtained. Briefly, we
observed that hMSH2 and hMSH3 were significantly associated with cases presenting BRAF-
V600E mutation (p = 0.007 and p = 0.021, respectively) [data on BRAF V600E expression in
this same sample have been previously described – Fregnani et al.11], whereas hMSH6 did not
show any association. Moreover, when we organized the cases expressing high levels of
hMSH2/hMSH3 (hMutSβ), hMSH2/hMSH6 (hMutSα) and hMSH2/hMSH3/hMSH6
simultaneously, we also observed a significant association with BRAF-V600E status (p =
0.009; p = 0.007 and p = 0.002, respectively).
Although the expression of each protein individually did not demonstrate a
significant association with the presence of recurrences, when all three proteins were
simultaneously overexpressed a statistical significance was obtained (p = 0.035). No other
significant association with clinicopathological parameters was found. Finally, using
univariate analysis we were unable to find a significant difference in the DFS rates of patients
with higher and lower expression of hMSH2, hMSH3, hMSH6 and their combinations (p >
0.05), although it could be seen that in all these situations those patients with a lower
expression presented a higher DFS rate (Figures 5 and 6).
Discussion
To avoid the accumulation of genetic mutations, humans count with a complex
group of proteins that form the mismatch repair system (MMR), which is responsible for
35
identifying and correcting errors in the pairing process of nucleotide bases during the DNA
replication.12,16 Here, we investigated the importance of hMutS proteins for ameloblastoma
pathogenesis and behavior because the components of this system were shown to be
deregulated in different human diseases14,18-24. Our results demonstrated that hMSH2 and
hMSH3 expression is associated with BRAF-V600E mutation and that hMSH2, hMSH3 and
hMSH6 simultaneous over-expression is associated with more recurrences, although the
proteins did not predict the DFS rate of these patients.
Very little is known about MMR proteins in odontogenic tumors. The only
research in this context is from Castrilli et al.,25 which observed the expression of hMSH2 and
hMLH1 in 25 ameloblastoma cases. However, the authors did not use a control group to
determine whether the proteins were over- or down-expressed, and did not attempt to
determine their clinical significance. In the present study, using a sample of human dental
germs in different stages of development we observed that the proteins are down-regulated in
ameloblastomas; moreover, the two cases of ameloblastic carcinoma also showed a lower
expression of hMSH2, hMSH3 and hMSH6, suggesting that this system would be down-
regulated in the tumorigenesis process of ameloblastoma/ameloblastic carcinoma, possibly
allowing the tumors to undergo more genetic mutations.
Similar to our results, investigating oral carcinogenesis Jessri et al.19 observed a
lower expression of hMLH1, hMSH2 and hPMS2 in OSCC than in oral leukoplakia, which
has also been documented by Sarmento et al.26 during lip carcinogenesis. On the other hand,
Xuan et al.27 observed an opposite distribution of hMSH2 during gall bladder carcinogenesis,
describing a higher expression in the malignancy and a lower expression in its preceding
nonneoplastic conditions. Taken together, these results suggest that the expression pattern of
MMR molecules during human tumorigenesis may differ depending on the tissue analyzed.
In accordance with some previous reports where hMutS proteins have not
predicted the survival rates of patients affected by different neoplasms,28,29 we did not observe
a correlation of hMutS expression patterns with prognostic factors in ameloblastoma either;
nevertheless, Wagner et al.18 demonstrated a significant prognostic role for hMSH2 and
hMSH6 in OSCC, Stark et al.30 found a prognostic potential for hMSH6 in glioblastoma and
Perrin et al.31 also showed that hMSH2 is a prognostic indicator for colon cancer. Thus, our
results and these studies illustrate the variable prognostic implications of MMR proteins in
different human neoplasms.
In some malignancies altered expression of MMR proteins was shown to be
associated with clinicopathological parameters such as tumor size, lymph node metastasis,
36
clinical stage and chemotherapy resistance.18,32,33 In line with these reports, in our research a
significant association was demonstrated between the simultaneous over-expression of
hMSH2/hMSH3/hMSH6 and the presence of recurrences. As previously hypothesized,34 we
may also speculate that cases presenting hMutS over-expression could carry other molecular
events that would lead to a tumor with a higher potential of recurrences, stimulating the
expression of hMutS proteins as an attempt to repair other genetic errors. Therefore, hMutS
over-expression may represent an indicator of other molecular events.
In the last few years new insights into the molecular characteristics of
ameloblastoma have been obtained and different groups, including ours, have demonstrated
the importance of BRAF-V600E mutation for ameloblastoma development and behavior.9,11,35
Recent reports showed that 46 to 82% of ameloblastomas carry the BRAF-V600E
mutation9,36,37 and using the same sample used in this study, we demonstrated that BRAF-
V600E immunoexpression seems to be an important determinant of aggressiveness.11 Here,
we observed that except for hMSH6, the other proteins and their respective combinations
were all significantly associated with the presence of BRAF-V600E mutation, but we cannot
determine whether the presence of the BRAF mutation led to a higher expression of the
proteins or whether the lower expression of hMutS proteins in ameloblastomas, if compared
to human dental germs, would favor the occurrence of BRAF-V600E mutation. Interestingly,
in colorectal cancer Miyakura et al.38 and Koinuma et al.39 had already demonstrated that
BRAF somatic mutations are strongly associated with hMLH1 promoter methylation, whereas
Waldmann et al.40 showed that BRAF-V600E mutation was present in 67% of their colorectal
sample deficient for hMLH1 and Aparicio et al.41 showed that BRAF-V600E mutation was
very frequent in elderly patients affected by colorectal cancer deficient for MMR proteins.
These reports confirmed the importance of MMR system disturbance for the occurrence of
BRAF mutationsin colorectal cancer, but more studies are necessary to determine whether this
direct association is also true in ameloblastomas.
We also investigated the possible correlation of hMutS proteins expression with a
higher proliferative index measured by Ki67. Similar to previous analyzes that could not find
a significant correlation between MMR proteins and the proliferative potential of other
tumors,42 we did not obtain such correlation in ameloblastomas either. On the other hand,
Helal et al.43 demonstrated a significant correlation of hMSH2 with p53 expression in OSCC
and Köster et al.44 in breast cancer, suggesting that hMutS proteins would be more important
for the control of the apoptosis of tumor cells than for their proliferative potential.
37
Despite the new data described in this study, it has the limitation of not having
investigated the functional status of the proteins, not allowing us to determine whether this
protein system is properly acting for DNA maintenance. Moreover, due to the rarity of
ameloblastic carcinoma, only a very small sample could be used, precluding statistical
analyzes and the acquisition of stronger results.
In conclusion, hMutS proteins were down-regulated in ameloblastoma; moreover,
simultaneous over-expression of these proteins in ameloblastoma was associated with
recurrence, but did not predict disease-free survival.
38
References
1. Neves-Silva R, Fonseca FP, de Jesus AS, et al. Tissue microarray use for
immunohistochemical study of ameloblastoma. J Oral Pathol Med. 2016;45(9):704-711.
doi:10.1111/jop.12428.
2. Fregnani ER, Fillipi RZ, Oliveira CRGCM, Vargas PA, Almeida OP. Odontomas and
ameloblastomas: variable prevalences around the world? Oral Oncol. 2002;38(8):807-
808. doi:10.1016/S1368-8375(02)00050-7.
3. Oomens MAEM, van der Waal I. Epidemiology of ameloblastomas of the jaws; a report
from the Netherlands. Med Oral Patol Oral Cir Bucal. 2014;19(6):e581-3.
http://www.ncbi.nlm.nih.gov/pubmed/25350592.
4. Milman T, Ying G-S, Pan W, LiVolsi V. Ameloblastoma: 25 Year Experience at a Single
Institution. Head Neck Pathol. June 2016. doi:10.1007/s12105-016-0734-5.
5. Hall JM, Weathers DR, Unni KK. Ameloblastic carcinoma: an analysis of 14 cases. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;103(6):799-807.
doi:10.1016/j.tripleo.2006.11.048.
6. Pirklbauer K, Kozakowski N, Russmueller G, Ewers R, Klug C. Manifestation of an
ameloblastic carcinoma ten years after follicular cyst enucleation in the mandibular
ramus. J Craniomaxillofac Surg. 2012;40(4):362-365. doi:10.1016/j.jcms.2011.05.016.
7. Benlyazid A, Lacroix-Triki M, Aziza R, Gomez-Brouchet A, Guichard M, Sarini J.
Ameloblastic carcinoma of the maxilla: case report and review of the literature. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod. 2007;104(6):e17-24.
doi:10.1016/j.tripleo.2007.05.026.
8. Brown NA, Betz BL. Ameloblastoma: A Review of Recent Molecular Pathogenetic
Discoveries. Biomark Cancer. 2015;7(Suppl 2):19-24. doi:10.4137/BIC.S29329.
9. Diniz MG, Gomes CC, Guimarães BVA, et al. Assessment of BRAFV600E and
SMOF412E mutations in epithelial odontogenic tumours. Tumour Biol.2015;36(7):5649-
5653. doi:10.1007/s13277-015-3238-0.
10. Tan S, Pollack JR, Kaplan MJ, Colevas AD, West RB. BRAF inhibitor treatment of
primary BRAF-mutant ameloblastoma with pathologic assessment of response. Oral Surg
Oral Med Oral Pathol Oral Radiol. 2016;122(1):e5-7. doi:10.1016/j.oooo.2015.12.016.
11. Fregnani ER, Perez DE da C, Paes de Almeida O, et al. BRAF-V600E expression.
correlates with ameloblastoma aggressiveness. Histopathology. September 2016:1-12.
doi:10.1111/his.13095.
39
12. Hsieh P, Yamane K. DNA mismatch repair: molecular mechanism, cancer, and ageing.
Mech Ageing Dev. 2008;129(7-8):391-407. doi:10.1016/j.mad.2008.02.012.
13. Erie DA, Weninger KR. Single molecule studies of DNA mismatch repair. DNA Repair
(Amst). 2014;20:71-81. doi:10.1016/j.dnarep.2014.03.007.
14. Slováková P, Majerová L, Matáková T, Skereňová M, Kavcová E, Halašová E. Mismatch
repair gene polymorphisms and association with lung cancer development. Adv Exp Med
Biol. 2015;833:15-22. doi:10.1007/5584_2014_83.
15. Dominguez-Valentin M, Joost P, Therkildsen C, Jonsson M, Rambech E, Nilbert M.
Frequent mismatch-repair defects link prostate cancer to Lynch syndrome. BMC Urol.
2016;16:15. doi:10.1186/s12894-016-0130-1.
16. Amaral-Silva GK do, Martins MD, Pontes HAR, et al. Mismatch repair system proteins
in oral benign and malignant lesions. J Oral Pathol Med. 2016;(3).
doi:10.1111/jop.12484.
17. Fonseca FP, de Almeida OP, Vargas PA, Gonçalves F, Corrêa Pontes FS, Rebelo Pontes
HA. Ameloblastic carcinoma (secondary type) with extensive squamous differentiation
areas and dedifferentiated regions. Oral Surg Oral Med Oral Pathol Oral Radiol.
2016;121(6):e154-61. doi:10.1016/j.oooo.2015.09.021.
18. Wagner VP, Webber LP, Salvadori G, et al. Overexpression of MutSα ComplexProteins
Predicts Poor Prognosis in Oral Squamous Cell Carcinoma. Medicine (Baltimore).
2016;95(22):e3725. doi:10.1097/MD.0000000000003725.
19. Jessri M, Dalley AJ, Farah CS. MutSα and MutLα immunoexpression analysis in
diagnostic grading of oral epithelial dysplasia and squamous cell carcinoma. Oral Surg
Oral Med Oral Pathol Oral Radiol. 2015;119(1):74-82. doi:10.1016/j.oooo.2014.06.017.
20. Peltomäki P. Lynch syndrome genes. Fam Cancer. 2005;4(3):227-232.
doi:10.1007/s10689-004-7993-0.
21. Fishel R. Mismatch repair. J Biol Chem. 2015;290(44):26395-26403.
doi:10.1074/jbc.R115.660142.
22. Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Milestones of Lynch
syndrome: 1895-2015. Nat Rev Cancer. 2015;15(3):181-194. doi:10.1038/nrc3878.
23. John AM, Schwartz RA. Muir-Torre syndrome (MTS): An update and approach to
diagnosis and management. J Am Acad Dermatol. 2016;74(3):558-566.
doi:10.1016/j.jaad.2015.09.074.
40
24. Westenend PJ, Schütte R, Hoogmans MMCP, Wagner A, Dinjens WNM. Breast cancer
in an MSH2 gene mutation carrier. Hum Pathol. 2005;36(12):1322-1326.
doi:10.1016/j.humpath.2005.08.025.
25. Castrilli G, Piantelli M, Artese L, et al. Expression of hMSH2 and hMLH1 proteins of the
human DNA mismatch repair system in ameloblastoma. J Oral Pathol Med.
2001;30(5):305-308. http://www.ncbi.nlm.nih.gov/pubmed/12076327.
26. Sarmento DJ de S, de Almeida WL, Miguel MC da C, et al. Immunohistochemical
analysis of mismatch proteins in carcinogenesis of the lower lip. Histopathology.
2013;63(3):371-377. doi:10.1111/his.12197.
27. Xuan YH, Choi YL, Shin YK, et al. An immunohistochemical study of the expression of
cell-cycle-regulated proteins p53, cyclin D1, RB, p27, Ki67 and MSH2 ingallbladder
carcinoma and its precursor lesions. Histol Histopathol. 2005;20(1):59-66.
http://www.ncbi.nlm.nih.gov/pubmed/15578423.
28. Fromont G, Rouprêt M, Amira N, et al. Tissue microarray analysis of the prognostic
value of E-cadherin, Ki67, p53, p27, survivin and MSH2 expression in upper urinary tract
transitional cell carcinoma. Eur Urol. 2005;48(5):764-770.
doi:10.1016/j.eururo.2005.07.005.
29. Cooper WA, Kohonen-Corish MRJ, Chan C, et al. Prognostic significance of DNA repair
proteins MLH1, MSH2 and MGMT expression in non-small-cell lung cancer and
precursor lesions. Histopathology. 2008;52(5):613-622. doi:10.1111/j.1365-
2559.2008.02999.x.
30. Stark AM, Doukas A, Hugo H-H, Mehdorn HM. The expression of mismatch repair
proteins MLH1, MSH2 and MSH6 correlates with the Ki67 proliferation index and
survival in patients with recurrent glioblastoma. Neurol Res. 2010;32(8):816-820.
doi:10.1179/016164110X12645013515052.
31. Perrin J, Gouvernet J, Parriaux D, et al. MSH2 and MLH1 immunodetection and the
prognosis of colon cancer. Int J Oncol. 2001;19(5):891-895.
http://www.ncbi.nlm.nih.gov/pubmed/11604984.
32. Fujieda S, Tanaka N, Sunaga H, et al. Expression of hMSH2 correlates with in vitro
chemosensitivity to CDDP cytotoxicity in oral and oropharyngeal carcinoma. Cancer
Lett. 1998;132(1-2):37-44. doi:10.1016/S0304-3835(98)00157-8.
33. Kato M, Takano M, Miyamoto M, et al. DNA mismatch repair-related protein loss as a
prognostic factor in endometrial cancers. J Gynecol Oncol. 2015;26(1):40-45.
doi:10.3802/jgo.2015.26.1.40.
41
34. Theocharis S, Klijanienko J, Giaginis C, et al. Expression of DNA repair proteins,
MSH2, MLH1 and MGMT in mobile tongue squamous cell carcinoma: associations with
clinicopathological parameters and patients’ survival. J oral Pathol Med.2011;40(3):218-
226. doi:10.1111/j.1600-0714.2010.00945.x.
35. Brown NA, Rolland D, McHugh JB, et al. Activating FGFR2-RAS-BRAF mutations in
ameloblastoma. Clin Cancer Res. 2014;20(21):5517-5526. doi:10.1158/1078-0432.CCR-
14-1069.
36. Sweeney RT, McClary AC, Myers BR, et al. Identification of recurrent SMO and BRAF
mutations in ameloblastomas. Nat Genet. 2014;46(7):722-725. doi:10.1038/ng.2986.
37. Kurppa KJ, Catón J, Morgan PR, et al. High frequency of BRAF V600E mutations in
ameloblastoma. J Pathol. 2014;232(5):492-498. doi:10.1002/path.4317.
38. Miyakura Y, Sugano K, Konishi F, et al. Extensive methylation of hMLH1 promoter
region predominates in proximal colon cancer with microsatellite instability.
Gastroenterology. 2001;121(6):1300-1309.
http://www.ncbi.nlm.nih.gov/pubmed/11729109.
39. Koinuma K, Shitoh K, Miyakura Y, et al. Mutations of BRAF are associated with
extensive hMLH1 promoter methylation in sporadic colorectal carcinomas. Int J cancer.
2004;108(2):237-242. doi:10.1002/ijc.11523.
40. Waldmann E, Ferlitsch M, Binder N, et al. Tumor and Patient Characteristics of
Individuals with Mismatch Repair Deficient Colorectal Cancer. Digestion.
2015;91(4):286-293. doi:10.1159/000381284.
41. Aparicio T, Schischmanoff O, Poupardin C, et al. High prevalence of deficient mismatch
repair phenotype and the V600E BRAF mutation in elderly patients with colorectal
cancer. J Geriatr Oncol. 2014;5(4):384-388. doi:10.1016/j.jgo.2014.08.002.
42. Li M, Liu L, Wang Z, et al. Overexpression of hMSH2 and hMLH1 protein in certain
gastric cancers and their surrounding mucosae. Oncol Rep. 2008;19(2):401-406.
http://www.ncbi.nlm.nih.gov/pubmed/18202787.
43. Helal TEA, Fadel MT, El-Thobbani AK, El-Sarhi AM. Immunoexpression of p53
andhMSH2 in oral squamous cell carcinoma and oral dysplastic lesions in Yemen:
Relationship to oral risk habits and prognostic factors. Oral Oncol. 2012;48(2):120-124.
doi:10.1016/j.oraloncology.2011.08.024.
44. Köster F, Schröer A, Fischer D, Horn A-K, Diedrich K, Friedrich M.
Immunohistochemistry of proteins for DNA mismatch repair in correlation to prognostic
42
factors of mammarian cancer. Oncol Rep. 2007;17(5):1223-1227.
http://www.ncbi.nlm.nih.gov/pubmed/17390069.
43
Figures legends
Figure 1. Ki67 expression. A) In the epithelial component of the human dental germs Ki67
was predominantly observed in the peripheral palisade cells (DAB; 400x). B) Only some cells
were positive in ameloblastoma, more commonly in the basal cells (DAB, 400x). C) In
ameloblastic carcinoma Ki67 immunostaining was high and observed in peripheral and
central areas of epithelial nests and strands (DAB, 400x). D) Ki67 immunoexpression was
significantly higher in human dental germs than in ameloblastomas (Mann-Whitney test. p =
0.0005). Because of the small sample size of ameloblastic carcinoma, statistical analysis could
not be done for this group.
44
Figure 2. hMSH2 expression. A) Nuclear expression of hMSH2 protein in human dental
germs (DAB, 400x). B) In ameloblastoma the nuclear expression was seen both in the
peripheral and central areas of the neoplastic islands and strands (DAB, 400x). C) hMSH2
was also observed in ameloblastic carcinomas (DAB, 400x). D) hMHS2 was significantly
reduced in ameloblastomas than in human dental germs (Mann-Whitney test. p = 0.0059).
Because of the small sample size of ameloblastic carcinoma, statistical analysis could not be
done for this group.
45
Figure 3. hMSH3 expression. A) Positive expression in human dental germs (DAB, 400x). B)
Nuclear positivity in both peripheral and central cells of neoplastic islands and strands of
ameloblastoma (DAB, 400x). C) Both cases of ameloblastic carcinomas were also positive for
the protein (DAB, 400x). D) There was no significant difference in the expression of hMSH3
between human dental germs and ameloblastoma (Unpaired Student t-Test. p = 0.8836).
Because of the small sample size of ameloblastic carcinoma, statistical analysis could not be
done for this group.
46
Figure 4. hMSH6 expression. A) Peripheral and central cells of the epithelial component of
human dental cells were positive for hMSH6 (DAB, 400x). B) Ameloblastoma was diffusely
positive (DAB, 400x). C) In ameloblastic carcinoma positivity was seen in both cases
analyzed (DAB, 400x). D) hMSH6 expression was significantly reduced in ameloblastomas
than in human dental germs (Unpaired Student t-Test. p < 0.0001). Because of the small
sample size of ameloblastic carcinoma, statistical analysis could not be done for this group.
47
Figure 5. Analysis of hMSH2, hMSH3 and hMSH6 expressions as prognostic determinants
for patients affected by ameloblastoma. Results showed that patients expressing lower levels
of the proteins presented higher survival rates, but not significantly different from those
expressing higher levels of the proteins. A) High and B) low expression of hMSH2 in
ameloblastomas. C) Kaplan-Meyer survival curves were not statistically different according
to Log-rank univariate analysis (p = 0.137). D) High and E) low expression of hMSH3 in
ameloblastomas. F) Survival curves were not significantly different (p = 0.627). G) High and
H) low expression of hMSH6 in ameloblastomas. I) No statistical difference was seen
between survival rates of patients expressing different levels of this protein (p = 0.543). Using
digital analysis, blue nuclei represent negative cells and orange nuclei represent positive cells.
48
Figure 6. Analysis of the importance of hMSH2, hMSH3 and hMSH6 combination for
determining the survival rates of patients affected by ameloblastomas. Although patients
expressing higher levels of the proteins exhibited a shorter survival than those expressing
lower levels of the proteins combined, no statistical significance could be obtained using the
univariate analysis. A) Survival curves obtained when hMSH2 and hMSH3 were
simultaneously over-expressed (p = 0.495). B) Survival curves obtained when hMSH2 and
hMSH6 were simultaneously over-expressed (p = 0.180). C) Survival curves obtained when
hMSH2, hMSH3 and hMSH6 were simultaneously over-expressed (p = 0.148).
49
Table 1. Analysis of the association between clinicopathological parameters obtained from the 73 cases of ameloblastoma with the expression of
hMSH2, hMSH3, hMSH6 and their combination (Chi-square and Fisher’s exact tests).
hMSH2 hMSH3 hMSH6 hMSH2/hMSH3 hMSH2/hMSH6 hMSH2/hMSH3/hMSH6
Sex p = 0.890
p = 0.402
p = 0.629
p = 0.569
p = 1.000
p = 0.673
Age p = 0.834
p = 0.687
p = 0.207
p = 0.456
p = 0.336
p = 0.533
Tumor site p = 0.784
p = 0.720
p = 0.664
p = 0.724
p = 0.203
p = 0.661
Tumor size p = 0.344
p = 0.161
p = 0.091
p = 0.863
p = 0.794
p = 0.726
Tumor duration p = 0.192
p = 0.538
p = 0.925
p = 0.105
p = 0.833
p = 0.582
Microscopic features p = 0.340
p = 0.517
p = 0.411
p = 0.706
p = 0.461
p = 0.249
Radiographic features p = 0.535
p = 0.873
p = 0.492
p = 0.628
p = 0.592
p = 0.503
Vestibular/palatine cortical
disruption
p = 0.819
p = 0.576
p = 0.325
p = 0.857
p = 0.358
p = 0.456
Basal cortical disruption
p = 0.594
p = 0.346
p = 0.191
p = 0.365
p = 0.218
p = 0.218
Recurrences p = 0.106
p = 0.417
p = 0.227
p = 0.368 p = 0.056
p = 0.035*
BRAF V600E status p = 0.007* p = 0.021* p = 0.077 p = 0.009*
p = 0.007*
p = 0.002*
* Statistically significant p value.
50
3 DISCUSSÃO
O ameloblastomas é o principal tumor odontogênico benigno comumente
diagnosticado nos serviços de diagnóstico oral (Nalabolu et al., 2017). Apresenta
características de agressividade local, além de índice importante de recorrência baseado na
modalidade terapêutica empregada. A progressão do tumor para o carcinoma ameloblástico
secundário é possível, apesar de ser uma ocorrência rara (Dhanuthai et al., 2012; Milman et
al., 2016). Assim como outros estudos, nos identificamos uma predominância do padrão
histológico sólido nas amostras de ameloblastomas (Dhanuthai et al., 2012; Fregnani et al.,
2010).
Os mecanismos de desenvolvimento destes tumores ainda não estão
suficientemente definidos, embora recentes estudos tenham identificado a expressão da
mutação BRAF-V600E em ameloblastomas, associando sua presença com o maior potencial
de agressividade do tumor (Fregnani et al., 2016). Esta mutação corresponde à substituição do
aminoácido valina (V) por ácido glutâmico (E) em decorrência da inserção de um nucleotídeo
timina no lugar de adenina no códon 600, gerando um mismatch do tipo transversão de
purinas (Davies et al., 2002). De acordo com Rossetti et al. (2015), este erro de pareamento é
o mais crítico por criar pontes de hidrogênio promiscuas.
Como avaliado em nossa primeira publicação, os seres humanos possuem um
sistema de proteínas homólogo ao identificado nas bactérias E. coli, onde apresentam a função
de identificação de erros de pareamento correto das bases do DNA após a replicação, além de
inserção ou deleção de bases. Nosso estudo também demostrou uma variedade de lesões orais
que apresentam alterações importantes nos níveis de expressão destas proteínas (Amaral-Silva
et al., 2017).
No nosso segundo estudo nós identificamos a expressão imunohistoquímica das
proteínas hMutS em germes dentais humanos normais, bem como a redução progressiva desta
expressão em ameloblastomas, com exceção da proteína hMSH3 (p = 0.8836). Não foi
possível obter uma análise estatística desta relação com o carcinoma ameloblástico devido ao
número reduzido de amostras (n = 2). Os casos de ameloblastomas que apresentaram
sobrexpressões das proteínas hMSH2 e hMSH3 isoladamente mostraram significativa
associação com a presença da mutação BRAF-V600E (p = 0.007 e p = 0.021,
respectivamente). Quando analisada a expressão simultânea de hMSH2/hMSH3 (hMutSβ),
hMSH2/hMSH6 (hMutSα) e hMSH2/hMSH3/hMSH6, também encontramos a associação
com BRAF-V600E (p = 0.009; p = 0.007 e p = 0.002, respectivamente).
51
Alguns autores relacionam a deficiência de proteínas MMR com BRAF-V600E
em câncer colorretal esporádico, correlacionando os achados com o prognóstico e tempo de
sobrevida (Aparicio et al., 2014; Luey et al., 2014; Sinicrope et al., 2016; Toon et al., 2014;
Waldmann et al., 2015; Wang et al., 2016). Nós encontramos que, apenas de reduzidas, as
sobrexpressões simultâneas em relação as médias das proteínas hMSH2/hMSH3/hMSH6
apresentaram, além da relação estatística com BRAF-V600E, uma importante correlação
clínica com a presença de tumores recorrentes.
Assim como Theocharis et al. (2011), nós acreditamos que a sobrexpressão de
hMutS poderiam estar estimulada diante de outros eventos moleculares associados ao
potencial de recorrência, na tentativa de reparar possíveis erros de pareamento de bases desses
gens também sobrexpressos.
Não houve correlação estatística relevante entre a expressão das proteínas hMutS
e Ki67. Apenas observamos que a nossa expressão média de Ki67 nos ameloblastomas
(3,76%) estava um pouco abaixo da faixa relatada por Meer et al. (2003) (entre 4,6% a 8,8%).
52
4 CONCLUSÃO
Com base nos resultados obtidos em nosso estudo, podemos concluir que:
1) Alterações no sistema mismatch estão presentes em diversas formas de
condições potencialmente malignas e em neoplasias humanas. A ausência
da expressão das suas proteínas são fatores diagnósticos para a Síndrome
de Lynch. Muitas condições patológicas apresentam redução na expressão
das proteínas, sendo isso potencialmente relacionado com eventos
epigenéticos;
2) A expressão imunohistoquímica média das proteínas hMutS estão
sequencialmente reduzidas no ameloblastoma e no carcinoma
ameloblástico em relação ao epitélio odontogênico do germe dental
humano normal;
3) A sobrexpressão das proteínas hMutS no ameloblastoma, sejam em
conjunto ou isoladas (exceto hMSH6), apresentam relação estatisticamente
significante com a presença da mutação BRAF-V600E;
4) A sobrexpressão simultânea das proteínas hMSH2/hMSH3/hMSH6 em
ameloblastomas apresentam potencial indicador para a recorrência
tumoral.
53
REFERÊNCIAS*
Amaral-Silva GK do, Martins MD, Pontes HAR, Fregnani ER, Lopes MA, Fonseca FP, et al.
Mismatch repair system proteins in oral benign and malignant lesions. J Oral Pathol Med
2017;46:241–5. doi:10.1111/jop.12484.
Antonoglou GN, Sándor GK. Recurrence rates of intraosseous ameloblastomas of the jaws: a
systematic review of conservative versus aggressive treatment approaches and meta-analysis
of non-randomized studies. J Craniomaxillofac Surg 2015;43:149–57.
doi:10.1016/j.jcms.2014.10.027.
Aparicio T, Schischmanoff O, Poupardin C, Mary F, Soufir N, Barrat C, et al. High
prevalence of deficient mismatch repair phenotype and the V600E BRAF mutation in elderly
patients with colorectal cancer. J Geriatr Oncol 2014;5:384–8. doi:10.1016/j.jgo.2014.08.002.
Benlyazid A, Lacroix-Triki M, Aziza R, Gomez-Brouchet A, Guichard M, Sarini J.
Ameloblastic carcinoma of the maxilla: case report and review of the literature. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 2007;104:e17-24.
doi:10.1016/j.tripleo.2007.05.026.
Castrilli G, Piantelli M, Artese L, Perfetti G, Rubini C, Fioroni M, et al. Expression of
hMSH2 and hMLH1 proteins of the human DNA mismatch repair system in ameloblastoma. J
Oral Pathol Med 2001;30:305–8.
Cusack JW. Report of the amputations of the lower jaw. Dubl Hosp Rec 1827;4:1–38.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF
gene in human cancer. Nature 2002;417:949–54. doi:10.1038/nature00766.
Dhanuthai K, Chantarangsu S, Rojanawatsirivej S, Phattarataratip E, Darling M, Jackson-
Boeters L, et al. Ameloblastoma: a multicentric study. Oral Surg Oral Med Oral Pathol Oral
Radiol 2012;113:782–8. doi:10.1016/j.oooo.2012.01.011.
Fitzpatrick SG, Hirsch SA, Listinsky CM, Lyu DJ-H, Baur DA. Ameloblastic carcinoma with
features of ghost cell odontogenic carcinoma in a patient with suspected Gardner syndrome.
Oral Surg Oral Med Oral Pathol Oral Radiol 2015;119:e241-5.
doi:10.1016/j.oooo.2014.09.028.
*De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of
Medical Journal Editors - Vancouver Group. Abreviatura dos periódicos em conformidade com o PubMed.
54
Fregnani ER, da Cruz Perez DE, de Almeida OP, Kowalski LP, Soares FA, de Abreu Alves F.
Clinicopathological study and treatment outcomes of 121 cases of ameloblastomas. Int J Oral
Maxillofac Surg 2010;39:145–9. doi:10.1016/j.ijom.2009.11.022.
Fregnani ER, Fillipi RZ, Oliveira CRGCM, Vargas PA, Almeida OP. Odontomas and
ameloblastomas: variable prevalences around the world? Oral Oncol 2002;38:807–8.
doi:10.1016/S1368-8375(02)00050-7.
Fregnani ER, Perez DE da C, Paes de Almeida O, Fonseca FP, Soares FA, Castro-Junior G, et
al. BRAF-V600E expression correlates with ameloblastoma aggressiveness. Histopathology
2016:1–12. doi:10.1111/his.13095.
Hall JM, Weathers DR, Unni KK. Ameloblastic carcinoma: an analysis of 14 cases. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 2007;103:799–807.
doi:10.1016/j.tripleo.2006.11.048.
Lucca M, D’Innocenzo R, Kraus JA, Gagari E, Hall J, Shastri K. Ameloblastic carcinoma of
the maxilla: a report of 2 cases. J Oral Maxillofac Surg 2010;68:2564–9.
doi:10.1016/j.joms.2009.09.088.
Luey N, Toon CW, Sioson L, Clarkson A, Watson N, Cussigh C, et al. A further investigation
of combined mismatch repair and BRAFV600E mutation specific immunohistochemistry as a
predictor of overall survival in colorectal carcinoma. PLoS One 2014;9:e106105.
doi:10.1371/journal.pone.0106105.
Masthan KMK, Anitha N, Krupaa J, Manikkam S. Ameloblastoma. J Pharm Bioallied Sci
2015;7:S167–S170. doi:10.4103/0975-7406.155891.
McClary AC, West RB, McClary AC, Pollack JR, Fischbein NJ, Holsinger CF, et al.
Ameloblastoma: a clinical review and trends in management. Eur Arch Oto-Rhino-
Laryngology 2016;273:1649–61. doi:10.1007/s00405-015-3631-8.
Meer S, Galpin JS, Altini M, Coleman H, Ali H. Proliferating cell nuclear antigen and Ki67
immunoreactivity in ameloblastomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2003;95:213–21. doi:10.1067/moe.2003.62.
Milman T, Ying G-S, Pan W, LiVolsi V. Ameloblastoma: 25 Year Experience at a Single
Institution. Head Neck Pathol 2016. doi:10.1007/s12105-016-0734-5.
Nalabolu GRK, Mohiddin A, Hiremath SKS, Manyam R, Bharath TS, Raju PR.
55
Epidemiological study of odontogenic tumours: An institutional experience. J Infect Public
Health 2017;10:324–30. doi:10.1016/j.jiph.2016.05.014.
Pirklbauer K, Kozakowski N, Russmueller G, Ewers R, Klug C. Manifestation of an
ameloblastic carcinoma ten years after follicular cyst enucleation in the mandibular ramus. J
Craniomaxillofac Surg 2012;40:362–5. doi:10.1016/j.jcms.2011.05.016.
Rossetti G, Dans PD, Gomez-Pinto I, Ivani I, Gonzalez C, Orozco M. The structural impact of
DNA mismatches. Nucleic Acids Res 2015;43:4309–21. doi:10.1093/nar/gkv254.
Sinicrope FA, Shi Q, Allegra CJ, Smyrk TC, Thibodeau SN, Goldberg RM, et al. Association
of DNA Mismatch Repair and Mutations in BRAF and KRAS With Survival After
Recurrence in Stage III Colon Cancers : A Secondary Analysis of 2 Randomized Clinical
Trials. JAMA Oncol 2016. doi:10.1001/jamaoncol.2016.5469.
Theocharis S, Klijanienko J, Giaginis C, Rodriguez J, Jouffroy T, Girod A, et al. Expression
of DNA repair proteins, MSH2, MLH1 and MGMT in mobile tongue squamous cell
carcinoma: associations with clinicopathological parameters and patients’ survival. J Oral
Pathol Med 2011;40:218–26. doi:10.1111/j.1600-0714.2010.00945.x.
Toon CW, Chou A, DeSilva K, Chan J, Patterson J, Clarkson A, et al. BRAFV600E
immunohistochemistry in conjunction with mismatch repair status predicts survival in patients
with colorectal cancer. Mod Pathol 2014;27:644–50. doi:10.1038/modpathol.2013.200.
Waldmann E, Ferlitsch M, Binder N, Sellner F, Karner J, Heinisch B, et al. Tumor and Patient
Characteristics of Individuals with Mismatch Repair Deficient Colorectal Cancer. Digestion
2015;91:286–93. doi:10.1159/000381284.
Wang J, Andrici J, Sioson L, Clarkson A, Sheen A, Farzin M, et al. Loss of INI1 expression
in colorectal carcinoma is associated with high tumor grade, poor survival, BRAFV600E
mutation, and mismatch repair deficiency. Hum Pathol 2016;55:83–90.
doi:10.1016/j.humpath.2016.04.018.
56
ANEXOS
Anexo 1: Certificado de Direitos Autorais da Journal of Oral Pathology and Medicine.
57
Anexo 2: Carta de aceite para publicação do Journal of Oral Surgery, Oral Medicine, Oral
Pathology, Oral Radiology.
58
Anexo 3: Certificado do Comitê de Ética em Pesquisa FOP/Unicamp.