Polymorphisms in methylenetetrahydrofolate reductase and cystathionine beta-synthase in oral cancer - a case-control study in southeastern Brazilians / Polimorfismos em metilenotetrahidrofolato redutase, cistationina beta-sintase no câncer de boca - um estudo de caso-controle no Sudeste brasileiro

ABSTRACT INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a serious public health problem, due to its high mortality rate and worldwide rising incidence. OSCC susceptibility is mediated by interactions between genetic and environmental factors. Studies suggest that genetic variants encoding enzymes involved in folate metabolism may modulate OSCC risk by altering DNA synthesis/repair and methylation process. OBJECTIVE: The goals of this study were to evaluate the association of three genotypic polymorphism (MTHFR C677T, MTHFR A1298C and CBS 844ins68) and oral cancer risk in southeastern Brazilians and evaluate the interactions between polymorphisms and clinical histopathological parameters. METHODS: This case-control study included 101 cases and 102 controls in the state of Espírito Santo, Brazil. MTHFR genotyping was done by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) and CBS genotyping by PCR (polymerase chain reaction) analysis. RESULTS: MTHFR C677T polymorphism was associated with lymph node involvement. Genotype CT + TT acted as a protective factor. MTHFR A1298C AC + CC genotype was associated with tumor differentiation, and possibly with a better prognosis. In risk analysis, no correlation was observed between genotypes and OSCC. CONCLUSION: We concluded that MTHFR C677T, MTHFR A1298C and CBS 844ins68 polymorphisms were not associated with OSCC risk in southeastern Brazilians; however, we suggest a prognosis effect associated with MTHFR C677T and A1298C polymorphisms in OSCC.

Resumo Introdução: O carcinoma espinocelular oral (CECO) trata-se de um importante problema de saúde pública, devido à elevada taxa de mortalidade e incidência crescente em todo o mundo. A susceptibilidade ao CECO é mediada por interações entre fatores genéticos e ambientais. Estudos sugerem que as variantes genéticas que codificam as enzimas envolvidas no metabolismo do folato podem modular o risco de CECO, alterando a síntese/reparação do DNA e o processo de metilação. Objetivo: Os objetivos deste estudo foram avaliar a associação de três polimorfismos genotípicos (MTHFR C677T, MTHFR A1298C e CBS 844ins68) e o risco de câncer oral em brasileiros da região Sudeste, e avaliar as interações entre polimorfismos e parâmetros clínico-histopatológicos. Método: Este estudo de caso-controle incluiu 101 casos e 102 controles no estado do Espírito Santo, Brasil. A genotipagem do polimorfismo MTHFR foi realizada por PCR-RFLP (Reação de Polimerase em Cadeia - Polimorfismo no Comprimento de Fragmento de Restrição) e a do CBS por análise da PCR (Reação de Polimerase em Cadeia). Resultados: O polimorfismo MTHFR C677T foi associado ao envolvimento de gânglios linfáticos. O genótipo CT + TT atuou como um fator protetor. O genótipo MTHFR A1298C AC + CC foi associado à diferenciação do tumor e, possivelmente, a um prognóstico melhor. Na análise de risco, a correlação entre os genótipos e o CECO não foi observada. Conclusão: Concluímos que os polimorfismos MTHFR C677T, MTHFR A1298C e CBS 844ins68 não estão associados ao risco de CECO nos brasileiros da região Sudeste; no entanto, sugerimos um efeito prognóstico associado aos polimorfismos MTHFR C677T e A1298C em CECO.

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.

Immunohistochemical evaluation of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth

The aim of this study was to evaluate the immunoexpression of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth and to correlate the results with demographic, survival, clinical (TNM staging) and histopathological variables (tumor grade, perineural invasion, embolization and bone invasion). Data from medical records and diagnoses of 41 patients were reviewed. Histological sections were subjected to immunostaining using primary antibodies for human MMP-2, MMP-9 and CD31 and streptavidin-biotin-immunoperoxidase system. Histomorphometric analyses quantified positivity for MMPs (20 fields per slide, 100 points grade, ×200) and for CD31 (microvessels <50 µm in the area of the highest vascularization, 5 fields per slide, 100 points grade, ×400). Statistical design was composed by non-parametric Mann-Whitney U test (investigating the association between numerical variables and immunostainings), chi-square frequency test (in contingency tables), Fisher's exact test (when at least one expected frequency was less than 5 in 2×2 tables), Kaplan-Meier method (estimated probabilities of overall survival) and Iogrank test (comparison of survival curves), all with a significance level of 5%. There was a statistically significant correlation between immunostaining for MMP-2 and lymph node metastasis. Factors associated negatively with survival were N stage, histopathological grade, perineural invasion and immunostaining for MMP-9. There was no significant association between immunoexpression of CD31 and the other variables. The intensity of immunostaining for MMP-2 can be indicative of metastasis in lymph nodes and for MMP-9 of a lower probability of survival.

O objetivo deste estudo foi avaliar a imunoexpressão de MMP-2, MMP-9 e CD31/densidade microvascular em carcinomas espinocelulares de soalho bucal e correlacionar os resultados com variáveis demográficas, de sobrevida, clínicas (estadiamento TNM) e histopatológicas (grau de diferenciação tumoral, invasão perineural, embolização e invasão óssea). Dados de prontuários e de diagnósticos de 41 pacientes foram revisados. Cortes histológicos foram submetidos à imunomarcação usando anticorpos primários para MMP-2, MMP-9 e CD31 humanos e sistema streptoavidina-biotina-imunoperoxidase. Análise histomorfométrica quantificou a positividade para MMPs (20 campos, grade de 100 pontos por lâmina, ×200) e para CD31 (microvasos <50 µm na área de maior vascularização, 5 campos, grade de 100 pontos por lâmina, ×400). O planejamento estatístico foi composto pelo teste não paramétrico U de Mann-Whitney (verificação da associação entre variáveis numéricas e imunomarcações), teste de frequências do qui-quadrado (em tabelas de contingência), teste exato de Fisher (quando pelo menos uma frequência esperada foi menor do que 5 em tabelas 2×2), método de Kaplan-Meier (estimativa de probabilidades de sobrevida global) e teste de Iogrank (comparação das curvas de sobrevida), todos com nível de significância de 5%. Houve correlação estatisticamente significante entre imunomarcação para MMP-2 e metástase em linfonodo. Os fatores relacionados negativamente com a sobrevida foram estadiamento N, gradação histopatológica, invasão perineural e imunomarcação de MMP-9. Não houve associação significativa entre imunoexpressão de CD31 e as demais variáveis. A intensidade de imunomarcação para MMP-2 pode ser indicativa de metástase em linfonodo e para MMP-9 de uma menor probabilidade de sobrevida.

Immunohistochemical detection of human telomerase reverse transcriptase in oral cancer and pre-cancer.

Purpose: Telomerase is a specialized ribonucleoprotein complex that stabilizes telomeres by adding "TAG" repeats to the end of chromosomes. The catalytic subunit of telomerase is human telomerase reverse transcriptase (hTERT), whose expression is the critical determinant of telomerase activity. Telomeres and telomerases play an important role in the longevity of cell and are known to conform "immortalization" on neoplastic cells. Although there exists a lot of information on telomerase in oral cancer, very little is known about their expression in leukoplakia and oral submucous fibrosis (OSF). This study addresses this lacuna. Materials and Methods: In this preliminary study, immunohistochemistry (IHC) was used to detect the expression of hTERT protein in oral squamous cell carcinoma (OSCC) (n=30), leukoplakia (n=15), OSF (n=15) and normal oral mucosa (n=10). The cellular localization of immunostain, intensity of stain, mean nuclear labeling index (LI) and mean nuclear labeling score (LS) of hTERT protein were studied. A total number of 1000 cells were counted in each slide. All the data were analyzed using SPSS software version 10.0.2. The cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity and LI were compared across the groups using Pearson's χ2 test. The mean LI and LS for OSF, leukoplakia, OSCC and normal were compared using analysis of variance (ANOVA). A P-value <0.05 was considered to be statistically significant. Results: The mean nuclear LI increased from OSF (22.46±4.53), through normal (28.3±12.3) to OSCC (47.56±21.30) (P=0.002) and from normal (28.3±12.3), through leukoplakia (44.06±14.6), to OSCC (47.56±21.30) (P=0.00). The mean nuclear labeling score was observed to increase from OSF (37.8±15), through normal (64.9±30.7), to OSCC samples (106.9±29.77) (P=0.00) and from normal (64.9±30.7), through leukoplakia (85.6±25.1) to OSCC samples (106.9±29.77) (P=0.00). Conclusion: There was increased expression of hTERT protein in OSCC and leukoplakia samples when compared to normal oral mucosa. The cellular localization, LI and LS in OSF were significantly different from OSCC and leukoplakia.

Análise comparativa da imunoexpressão da proteína p53 (clones DO-7 e PAb-240) em carcinomas de células escamosas intrabucais e labiais / Comparative analysis of p53 protein immunostaining (antibodies DO-7 and PAb-240) in oral cavity and lip squamous cell carcinomas

INTRODUÇÃO: A carcinogênese caracteriza-se como um processo multifatorial, e a inativação da proteína p53 é uma alteração genética comumente observada nos carcinomas de células escamosas de boca (CCEB). OBJETIVO: Analisar e comparar a imunoexpressão da proteína p53, por meio dos clones DO-7 e PAb-240, em CCEB com localização intrabucal e em lábio inferior. MATERIAL E MÉTODOS: Foram selecionados 40 casos de CCEB, sendo 20 de localização intrabucal e 20 em lábio inferior. Foi realizado um estudo imuno-histoquímico utilizando os anticorpos anti-p53 clone DO-7 e PAb-240. A imunoquantificação foi realizada por meio de análise digital de imagem, e os resultados, submetidos a tratamentos estatísticos. RESULTADOS: A imunoexpressão da proteína p53 foi verificada com o anticorpo DO-7 em 13 casos (65 por cento) de carcinoma intrabucal e em 19 (95 por cento) de carcinoma de lábio inferior. Imunorreatividade para o anticorpo PAb-240 foi observada em 9 casos (45 por cento) de lesões intrabucais e em 15 (75 por cento) localizados em lábio inferior. Não foram observadas, segundo o teste de Mann-Whitney, diferenças estatisticamente significativas (p > 0,05) na expressão da proteína p53 entre as duas localizações estudadas, independentemente do anticorpo avaliado. Foram identificadas, pelo teste de Wilcoxon, diferenças estatisticamente significativas entre a expressão dos clones DO-7 e PAb-240 em cada um dos grupos analisados (valor p = 0,013 - lábio inferior; valor p = 0,016 - intrabucal). CONCLUSÕES: A expressão da proteína p53 observada nos CCEB, com localizações intrabucais e labiais, sugere a ocorrência de mutações no gene TP53. As diferenças quantitativas obtidas entre os anticorpos estudados, independentemente da localização das lesões, refletem uma especificidade distinta entre os clones DO-7 e PAb-240. O desenvolvimento de mais estudos será fundamental para estabelecer o anticorpo mais adequado para proteína p53 em CCEB.

BACKGROUND: Carcinogenesis is a multifactorial process and inactivation of p53 protein is a genetic change commonly observed in oral squamous cell carcinomas (OSCC). OBJECTIVES: To analyze and compare the expression of p53 protein through antibodies DO-7 and PAb-240 in OSCC samples located in the oral cavity and lower lip. MATERIAL AND METHODS: Forty cases of OSCC were selected and divided into oral cavity and lower lip groups (20 cases each). Immunohistochemical technique was performed using antibodies DO-7 and PAb-240. Quantification of the cases was performed through digital image analysis and underwent specific statistical treatments. RESULTS: Expression of p53 protein was verified with DO-7 antibody in 13 cases (65 percent) of oral cavity carcinomas and in 19 cases (95 percent) of lower lip carcinoma. PAb-240 positivity was detected in 9 cases (45 percent) of oral cavity lesions and in 15 cases (75 percent) located in the lower lip. According to Mann-Whitney test, there were no statistically significant differences between the expressions of p53 protein in both groups, regardless of the antibody used. According to Wilcoxon test, there were statistically significant differences between the expression of DO-7 antibody and PAb-240 in each of the analyzed groups (p-value = 0.013; lower lip p-value = 0.016 - oral cavity). CONCLUSIONS: The expression of p53 protein was observed both in the oral cavity and lip OSCC, which suggests the occurrence of mutations in TP53 gene. The quantitative differences between the antibodies studied, regardless of the site of the lesions, reflect different specificity between clones DO-7 and PAb-240. Further studies are required to establish the best antibody for p53 protein in oral squamous cell carcinomas.

CYP1A1 and GSTP1 polymorphisms in an oral cancer case-control study

CYP1A1 and GSTP1 polymorphisms have been associated with a higher risk to develop several cancers, including oral squamous cell carcinoma (OSCC), which is closely related to tobacco and alcohol consumption. Both genes code for enzymes that have an important role in activating or detoxifying carcinogenic elements found in tobacco and other compounds, and polymorphic variants of these genes may result in alterations of the enzymatic activity. The CYP1A1 gene codes for the enzyme aryl hydrocarbon hydroxylase, which is responsible for the metabolism of polycyclic aromatic hydrocarbons. The investigated polymorphism, Ile/Val, seems to increase the activity of the enzyme in homozygous individuals, leading to an accumulation of carcinogens. The Ile/Val polymorphism occurs because of an A->G transition at exon 7, resulting in the CYP1A1*2B allele. The GSTP1*B variant shows an A->G transition at exon 5, changing the amino acid Ile to Val, with a reduced catalytic activity of the enzyme. Due to this reduction, the carriers of mutant alleles lost the capability to metabolize carcinogens, which could be responsible for a higher susceptibility to cancer. We conducted a case-control study in a group of 72 cases with newly diagnosed OSCC and 60 healthy controls matched for age, gender, smoking habits, and ethnicity. We used PCR methods to identify the allelic variants CYP1A1*2B and GSTP1*B. The data obtained showed no statistically significant association of allelic or genotypic variants of CYP1A1*2B (OR = 1.06; 95 percent CI = 0.49-2.29) and GSTP1*B (OR = 1.40; 95 percent CI = 0.70-2.79) with OSCC.

Lipid peroxidation and antioxidant enzyme status in oral carcinoma patients.

OBJECTIVE: To measure the lipid peroxidation and endogenous antioxidant enzyme status in oral carcinoma and the protective role of exogenous antioxidants. MATERIAL AND METHODS: 20 new cases of histologically proven oral squamous cell carcinoma, 20 of leukoplakia and 20 age and sex matched healthy conrols were included. Intra oral pH of patients and controlled were measured by quantitative litmus paper test and serum was analysed for malonialdehyde (MDA), super oxide bismutase (SOD), catalase and glutathione peroxidase (GP). Patients with leukoplakia were treated with exogenous antioxidants for 3 months and the same were reassessed. RESULTS: Oral pH of oral cancer patients was neutral (PH-7) but that of leukoplakia and controls were mildly acidic (6.64 and 6.58 respectively). Serum malonialdehyde levels were highest in oral cancer group. With antioxidant enzymes super oxide bismutase, catalase and glutathione peroxidase different pattern was noticed. Antioxidant enzymes remained almost the same (P > 0.005 each) in patients with leukoplakia after 3 months of vitamin A,C and E. but there was marginal increase in catalase level (P<0.05). CONCLUSION: This study shows the positive benefit of vitamin (A,C,E) and nutrition supplementation on the antioxidant enzyme defense system hence prevention of oral carcinogenesis in patients with leukoplakia.

Histochemical studies on the distribution of certain dehydrogenases in squamous cell carcinoma of cheek.

Histochemical distribution of lactate, isocitrate and succinate dehydrogenases in normal oral epithelium and in well differentiated squamous cell carcinoma of cheek region was studied. Lactate dehydrogenase, isocitrate dehydrogenase, and succinate dehydrogenase activity was found to be more in the malignant cells. Succinate and lactate dehydrogenase presented a conspicuous pattern in which the cells located at the periphery of the malignant sheets in well differentiated squamous cell carcinoma showed moderate to intense activity as compared to cells in the central portion. The results are discussed and it is suggested that the enhanced glycolysis and subdued function of the tricarboxylic acid cycle is not a universal criterion during malignancy as reported in the previous investigations.