Immunoexpression of cleaved caspase-3 shows lower apoptotic area indices in lip carcinomas than in intraoral cancer

Objective This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites. Material and Methods A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type. Results From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015). Conclusions Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors

Análise comparativa das proteínas expressas na saliva de pacientes com leucoplasia oral e carcinoma de boca: uma abordagem proteômica

Oral cancer is a worldwide problem. More than 90% of oral cancer cases are oral squamous cell carcinomas (OSCC), which is aggressive and particularly affects chronic smokers and drinkers. Tumor pathogenesis in non-smokers/drinkers is not well understood. Currently, OSCC is diagnosed through physical examination and biopsies. Visual inspection or dye staining of the mouth is useful for early detection of oral cancer and precancerous lesions, such as leukoplakia, but still the majority of OSCC are diagnosed at late stages. 2D-gel protein profiling has been used to identify proteins in OSCC tissue and saliva specimens. Analysis of saliva has the advantages of being safe, easy to access and non-invasive. The objective of this study was to evaluate proteins present in saliva of patients with oral leukoplakia (OL)and patients with tongue and floor of mouth (TF)SCC compared to a control group using proteomic techniques to better understand the molecular pathogenesis of OSCC, enabling the tumor to be identified at an early stage in individuals with or without risk factors. A total of 39 primary TFSCC in clinical stage I and II were identified during 2007 and 2008 at Instituto Nacional de Câncer (INCA), and 15 patients with OL were gathered from Odontoclínica Central do Exército and INCA. Patients were submitted to anamnesis and oral inspection. Paraffin embedded tissues were analyzed for diagnosis confirmation, WHO dysplasia evaluation, tumor grading, and histologic risk assessment. Whole saliva samples from ten healthy individuals were employed as a control group and compared to patients with OL as well as TFSCC patients with (n=10) and without (n=10) cancer associated oral habits(smoking and drinking). Cancer groups were compared with each other. Saliva was submitted to centrifugation and the supernatant stored at 80 °C. Saliva from each group was pooled (n=10) and precipitated with cold acetone. After 2D electrophoresis the differential protein components were analyzed with Image Master Software and tandem matrix assisted laser desorption/ionization time-of-flight mass spectrometry for protein identification. Descriptive statistics were calculated and a p-value of less than.05 was considered significant. TFSCC patients who smoked and drinked were mainly man, over 45 years old, while non-smokers/drinkers were female with high education level and tongue tumors. Among clinicopathologic features analyzed, only tumoral lymphocytic infiltrate showed association with neck metastasis absence. A total of 48 protein spots representing 20 proteins were identified in LO and TFSCC. Cytokeratin 10 and lysozyme were leukoplakia specific while apolipoprotein-A1 was significantly raised in LO. Beta actin was increased in smokers/drinkers TFSCC. Proapolipoprotein was specific for non-drinkers/smokers TFSCC compared to control, as well as tropomyosin in the same group when compared to smokers/drinkers TFSCC. In conclusion, both usually high- and low-abundant proteins were identified in saliva of TFSCC and OL patients and can be used in the future as a panel for early detection of OSCC. Differences in TFSCC in patients with and without oral habits must be further investigatedFundação de Amparo a Pesquisa do Estado do Rio de JaneiroO câncer de boca é um problema mundial e mais de 90% dessas lesões são carcinomas de células escamosas (CCE), uma doença agressiva que afeta principalmente tabagistas e etilistas. Em não usuários dessas substâncias sua patogênese não é bem compreendida. O CCE de boca se diagnostica por exames físicos e biópsia, mas sua prevenção pode ocorrer com a identificação de lesões precursoras, como a leucoplasia, através da inspeção visual e uso de corantes, mas a maioria ainda é diagnosticada em estádios avançados. A eletroforese 2D tem sido usada para identificar proteínas no tecido e na saliva de pacientes com CCE de boca. A análise da saliva é segura, de fácil acesso e não-invasiva. Esse estudo se propôs a avaliar as proteínas presentes na saliva de pacientes com leucoplasia oral (LO) e com CCE de língua e assoalho (LA), quando comparados a um grupo controle, através de técnicas proteômicas, aumentando a compreensão sobre a patogênese molecular do CCE de boca, para permitir o diagnóstico precoce do tumor em indivíduos com ou sem fatores de risco. Ao todo, 39 pacientes com CCELA em estádio clínico I e II foram identificados em 2007 e 2008 no Instituto Nacional de Câncer (INCA) e 15 pacientes com LO foram selecionados da Odontoclínica Central do Exército e do INCA. Foram feitos anamnese e exame oral dos pacientes. As lâminas foram revisadas para confirmação do diagnóstico, avaliação da displasia e da gradação segundo a OMS, e aplicação da avaliação histopatológica de risco. As amostras de saliva total de dez indivíduos saudáveis foram usadas como controle e comparadas a dez pacientes com LO, dez pacientes com CCELA tabagistas e etilistas e dez não tabagistas nem etilistas. Também foram comparados entre si os dois grupos com CCE. A saliva foi centrifugada e o sobrenadante misturado a inibidores de proteólise para ser armazenado a -80 °C. A saliva de cada grupo (n=10) foi reunida e precipitada com acetona. Após eletroforese 2D, as manchas com diferença de expressão foram analisadas com o programa Image Master, sendo as proteínas identificadas por espectrometria de massas por dessorção-ionização de laser em matriz tempo de vôo. Análise estatística descritiva foi realizada e valores <0,05 considerados significativos. Os pacientes com CCELA tabagistas/etilistas eram homens acima dos 45 anos, enquanto não tabagistas/etilistas eram mulheres, com maior nível de escolaridade e com tumores de língua. Dentre os aspectos clínico-patológicos analisados, somente o infiltrado linfocitário tumoral teve relação com ausência de metástases cervicais. 48 manchas representando 20 proteínas foram identificadas nas análises de LO e CCELA. A ceratina 10 e a lisozima foram exclusivas da leucoplasia, enquanto a apolipoproteína-A1 apresentou aumento significativo nesse grupo. Beta-actina estava aumentada nos tabagistas/etilistas. Proapolipoproteína foi exclusiva de não tabagistas/etilistas comparados ao controle, assim como tropomiosina no mesmo grupo quando comparado aos tabagistas/etilistas. Concluindo, proteínas classicamente muito e pouco abundantes foram identificadas com sucesso na saliva de CCELA e LO e podem ser usadas futuramente como um painel para detecção precoce do CCE de boca. As diferenças entre CCELA com e sem influência de tabaco e álcool precisam ser melhor investigada

Immunoexpression of cleaved caspase-3 shows lower apoptotic area indices in lip carcinomas than in intraoral cancer

ABSTRACT Objective This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites. Material and Methods A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type. Results From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015). Conclusions Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors