Indução da morte celular imunogênica em linhagens celulares de leucoplasia e carcinoma de células escamosas oral sob tratamento de artemisinina e cisplatina / Induction of immunogenic cell death in leukoplakia and oral squamous cell carcinoma cell lines under artemisinin and cisplatin treatment

Novos fármacos, como a artemisinina (ART), podem ser promissores no tratamento de lesões potencialmente malignas (LPM) e podem ser úteis quando usados em associação com outros quimioterápicos, especialmente na redução dos seus efeitos colaterais. A leucoplasia oral (LO) é a LPM mais comum da cavidade bucal e, pode evoluir para um carcinoma de células escamosas oral (CCEO). Não há terapia para evitar a sua transformação maligna, a quimioprevenção pode iniciar a morte celular imunogênica (MCI) que ativa o sistema imunológico para que reconheça e elimine as células malignas ou pré-malignas, sendo um potencial tratamento para as LPM. O presente estudo objetivou avaliar se a ART e a cisplatina (CSP) associadas ou não seriam capazes de induzir a MCI em linhagens celulares de LO (DOK) e CCEO SCC180). Material e métodos: As linhagens celulares HaCat (controle), DOK e SCC-180 foram tratadas por ART e CSP de forma combinada ou isolada, a fim de analisar a citotoxicidade e a genotoxicidade destes fármacos, além da capacidade destes em reduzir a migração celular e, se os compostos seriam capazes de induzir a expressão da proteína box de alta mobilidade (HGMB-1), caspase 3, 8, 9, e Calreticulina (CALR). Resultados: Em todas as linhagens celulares a CSP e CSP+ART causaram uma resposta dose dependente, apresentando maior citotoxicidade com doses mais altas, o que não foi observado com a ART. A formação de micronúcleos não foi observada no teste de genotoxicidade. A taxa de migração foi reduzida com as concentrações de IC50 de CSP e ART+CSP para as células de CCEO. Não foram encontradas expressões significativas de proteínas relacionadas à MCI ou apoptose nas linhagens de LO e CCEO, tratadas com ART, CSP ou ART+CSP, indicando que outro tipo de morte celular possa ter ocorrido. Conclusão: A MCI e a apoptose não foram evidenciadas como forma de morte celular após as linhagens de LO e CCEO receberem tratamentos com ART, CSP e a associação de ambas. Efeitos genotóxicos não foram observados nas doses testadas. O tratamento de CSP e ART+CSP foi capaz de reduzir a migração de células de CCEO. Também concluímos que novos estudos são necessários para elucidar se a ART e CSP podem ocasionar a MCI ou apoptose em linhagens celulares de LO e CCEO (AU)

New drugs, such as artemisinin (ART), may be promising in the treatment of potentially malignant lesions (PML) and may be useful when used in combination with other chemotherapy drugs, especially in reducing their side effects. Oral leukoplakia (OL) is the most common LPM of the oral cavity and can progress to oral squamous cell carcinoma (OSCC). There is no therapy to prevent its malignant transformation, chemoprevention can initiate immunogenic cell death (ICM) that activates the immune system to recognize and eliminate malignant or pre-malignant cells, being a potential treatment for LPM. The present study aimed to evaluate whether or not ART and cisplatin (CSP) combined would be capable of inducing MCI in LO (DOK) and CCEO SCC-180) cell lines. Material and methods: HaCat (control), DOK and SCC-180 cell lines were treated by ART and CSP in combination or alone, in order to analyze the cytotoxicity and genotoxicity of these drugs, in addition to their ability to reduce cell and, whether the compounds would be able to induce the expression of high mobility box protein (HGMB-1), caspase 3, 8, 9, and Calreticulin (CALR). Results: In cytotoxicity at higher doses, which was not observed with ART. The formation of micronuclei was not observed in the genotoxicity test. The migration rate was reduced with the IC50 concentrations of CSP and ART+CSP for OSCC cells. No significant expressions of proteins related to MCI or apoptosis were found in the LO and CCEO lines, treated with ART, CSP or ART+CSP, indicating that another type of cell death may have occurred. Conclusion: MCI and apoptosis were not evidenced as a form of cell death after the LO and CCEO lines received treatments with ART, CSP and the combination of both. Genotoxic effects were not observed at the doses tested. CSP and ART+CSP treatment was able to reduce OSCC cell migration. We also conclude that new studies are necessary to elucidate whether ART and CSP can cause MCI or apoptosis in LO and CCEO cell lines.(AU)

Estudo da imunoexpressão das proteínas ING3 e ciclina D1 nas displasias epiteliais e carcinomas epidermoides de língua / Study of the immunoexpression of ING3 and cyclin D1 proteins in epithelial dysplasias and squamous cell carcinomas of the tongue

O carcinoma epidermoide oral (CEO) é uma neoplasia maligna de patogenia complexa, já a displasia epitelial oral (DEO) é uma entidade que, na dependência de sua evolução, exibe riscos de transformação maligna para o CEO. A expressão alterada de genes supressores tumorais e proteínas reguladoras do ciclo celular apresentam relação com a etiopatogênese de diferentes cânceres. Entre os genes supressores tumorais destaca-se a família de inibidores de crescimento (inhibitor of growth - ING), que impedem o crescimento celular descontrolado. Dentre as proteínas reguladoras do ciclo celular podemos citar a Ciclina D1, que atua na progressão da fase S para a fase G1, permitindo a proliferação celular. O objetivo do presente estudo foi analisar a imunoexpressão das proteínas ING3 e Ciclina D1 em 28 espécimes de DEO e 25 de CEO localizados em língua, bem como avaliar morfologicamente e reclassificar as lesões segundo os critérios estabelecidos pela Organização Mundial da Saúde (OMS), além de modelo binário (Kujan et al. 2006), para DEO, e modelo buds-depth of invansion (BD) (Almagunsh et al. 2015) para CEO. A imunoexpressão das proteínas foi analisada de forma quantitativa, considerando a localização celular (citoplasmática e/ou nuclear para ING3 e nuclear para Ciclina D1) presentes no componente epitelial. A expressão das proteínas foi comparada entre os dois grupos de lesões, bem como com os parâmetros clínico-patológicos das lesões estudadas, através dos testes estatísticos Mann-Whitney (U) e teste de correlação de Spearman adotando um nível de significância de 5% para todos os testes considerando o valor de p ≤ 0,05. Dos 28 casos de DEO analisados 53,6% apresentaram-se como lesões de alto risco segundo o modelo binário, assim como dos 25 casos de CEO analisados foram considerados escores de alto risco (48%) de acordo com o modelo BD. Não foram encontradas associações estatisticamente significativas entre as variáveis morfológicas das lesões estudadas e as características clínicas (p>0,05). Com relação a expressão de Ciclina D1, esta foi significativamente maior em DEO quando comparada aos casos de CEO (p<0,05). A expressão de ING3 núcleo-citoplasmática foi significativamente menor nos casos de CEO, quando comparada aos casos de DEO (p<0,05), já a expressão de ING3, restrita ao citoplasma, foi maior nos casos de DEO, quando comparadas aos casos de CEO (p<0,05). Os resultados do presente estudo sugerem que a expressão das proteínas não tem relação com as gradações das lesões, porém, há notável diminuição da expressão nuclear de ING3 conforme a progressão maligna, indicando função supressora tumoral alterada desta proteína em DEO e CEOs. Destacamos, ainda, o aumento da expressão de Ciclina D1 em DEOs, mostrando que as lesões detêm uma taxa de proliferação celular significativa (AU).

Oral squamous cell carcinoma (OSCC) is a malignant neoplasm of complex pathogenesis. In turn, oral epithelial dysplasia (OED) is an entity that, depending on its evolution, exhibits varied risks of malignant transformation to OSCC. The altered expression of tumor suppressor genes and cell cycle regulatory proteins are related to the etiopathogenesis of different malignant tumors. Among the tumor suppressor genes, the family of inhibitors of growth (ING) stands out, which prevents uncontrolled cell formation. Amid the cell cycle regulatory proteins, Cyclin D1 acts in the progression from S to G1 phase, allowing cell proliferation. The aim of the present study was to analyze the immunoexpression of ING3 and Cyclin D1 proteins in 28 OED and 25 OSCC specimens, located on the tongue, as well as to morphologically evaluate and to reclassify the lesions following the criteria established by World Health Organization (WHO) and the model proposed by Kujan et al. (2006), for OED; for OSCC, the system proposed by Almagunsh et al. (2015) was used. Protein immunoexpression was quantitatively evaluated, considering the cellular location (cytoplasmic and/or nuclear for ING3 and nuclear for Cyclin D1), present in the epithelial component. Protein expression was compared between the two groups of samples, as well as with the clinical-pathological parameters of the lesions studied, through the Mann-Whitney (U) statistical tests and the Spearman correlation test, adopting a significance level of 5% for all tests considering the value of p≤ 0.05. Of the 28 cases of OED analyzed, 53.6% were high-risk lesions according to the binary model and of the 25 cases of OSCC analyzed, 48% were considered high-risk scores according to the BD model. No statistically significant associations were found between the morphological variables of the lesions studied and the clinical characteristics (p>0.05). A statistically significant difference was observed between the absence of ING3 expression and the grading of OEDs, according to the WHO (p<0.05). Regarding the expression of Cyclin D1, it was significantly higher in OED, when compared to OSCC cases (p<0.05). Nucleocytoplasmic ING3 expression was significantly lower in OSCC cases when compared to OED cases (p<0.05). Cytoplasm-restricted ING3 expression was higher in OED cases when compared to OSCC cases (p<0.05). The results of the present study suggest that, although there is no evidence of a significant relationship between the expression of proteins and the histopathological gradations of the lesions, there is a notable decrease in the nuclear expression of ING3, with an increase in the severity of the lesions, indicating an altered tumor suppressor function of this protein in OED and OSCCs. We also highlight the increased expression of Cyclin D1 in OEDs, showing that these lesions have a significant rate of cell proliferation (AU).

Imunoexpressão da Yes-Associated Protein (YAP) em hiperceratoses e displasias epiteliais orais / Immunoexpression of Yes-Associated Protein (YAP) in hyperkeratosis and oral epithelial dysplasia

A busca por um biomarcador que auxilie na predição de risco de transformação maligna das desordens orais potencialmente malignas (DOPMs) representa um grande desafio, já que pode auxiliar no manejo precoce e adequado dos pacientes. Este estudo avaliou a imunoexpressão da Yes-Associated Protein (YAP) em lesões intraorais leucoplásicas, eritroplásicas ou leucoeritroplásicas, com diagnóstico histopatológico de hiperceratose ou displasia epitelial oral (DEO), correlacionando essa imunoexpressão com o grau de severidade morfológica dessas lesões. A amostra foi composta por 20 casos de hiperceratoses e 53 casos de DEOs, além de 10 casos de mucosa oral normal (grupo controle). Para a avaliação do grau de displasia, foram utilizadas as gradações da OMS (EL-NAGGAR et al., 2017) e o Sistema Binário (KUJAN et al., 2006), sendo o perfil imunoistoquímico da proteína YAP avaliado por meio de escores, que variaram entre 0 e 3, com base em sua localização intracelular (citoplasmática ou nuclear) e por sua distribuição no tecido epitelial. Para a análise entre os parâmetros estudados foram realizados os testes estatísticos Qui-quadrado de Pearson, Exato de Fisher, além de testes não paramétricos, (nível de significância de 95%). Displasias leves foram enquadradas (100%) no baixo risco de transformação maligna, enquanto as moderadas dividiram-se entre baixo (47%) e alto risco (53%), sendo as displasias severas, em sua maior parte (71%), classificadas como de alto risco (p < 0,001). O grupo controle exibiu imunomarcação de escore 0 (80%), as hiperceratoses e displasias leves (em 80% das vezes) exibiram escore 1, já nas displasias moderadas (63%) e severas (79%), foram predominantes os escores 2 e 3; com padrão de imunomarcação nuclear associado ao alto risco de transformação maligna sugerido pelo Sistema binário (p = 0,002). A imunoexpressão da YAP foi semelhante entre hiperceratoses e displasias leves, o que deve suscitar maior atenção dos profissionais frente aos casos de hiperceratose, além disto a expressão da YAP aparenta dicotomizar as DOPMs entre as lesões com baixo risco de transformação maligna e as lesões com alto risco, o que pode sugerir no futuro, sua utilização como potencial marcador preditivo de progressão destas lesões (AU).

The search for a biomarker that helps to predict the risk of malignant transformation of oral potentially malignant disorders (OPMDs) represents a great challenge, as it can help in the early and adequate management of patients. This study evaluated the immunoexpression of Yes-Associated Protein (YAP) in intraoral leukoplastic, erythroplastic or leukoerythroplastic lesions, with histopathological diagnosis of hyperkeratosis or oral epithelial dysplasia (OED), correlating this immunoexpression with the degree of morphological severity of these lesions. The sample consisted of 20 cases of hyperkeratosis and 53 cases of OED, in addition to 10 cases of normal oral mucosa (control group). To assess the degree of dysplasia, the WHO grading (EL-NAGGAR et al., 2017) and the Binary System (KUJAN et al., 2006) were used, and the immunohistochemical profile of the YAP protein was evaluated through scores, which ranged from 0 to 3, based on their intracellular location (cytoplasmic or nuclear) and their distribution in the epithelial tissue. For the analysis of the studied parameters, Pearson's Chi-square and Fisher's Exact statistical tests were performed, in addition to non-parametric tests (significance level of 95%). Mild dysplasias were classified (100%) in the low risk of malignant transformation, while the moderate ones were divided between low (47%) and high risk (53%), with severe dysplasias, for the most part (71%), classified as high risk (p < 0.001). The control group exhibited score 0 of immunostaining (80%), hyperkeratosis and mild dysplasias (80% of booth) exhibit score 1, whereas in moderate (63%) and severe dysplasia (79%), the predominant scores was 2 and 3; with a pattern of nuclear immunostaining associated with the high risk of malignant transformation suggested by the Binary System (p = 0.002). The immunoexpression of YAP was similar between hyperkeratosis and mild dysplasias, which should attract greater attention from professionals in cases of hyperkeratosis. Furthermore, the expression of YAP appears to dichotomize OPMDs between lesions with low risk of malignant transformation and lesions with high risk, which may suggest, in the future, its use as a potential predictive marker of the progression for these lesions (AU).

Randomized comparative study in the treatment of oral leucoplakia with laser and conventional surgery / Estudo comparativo randomizado no tratamento da leucoplasia oral com laser e cirurgia convencional

Objective: High-energy lasers are used as an alternative to surgical treatment of potentially malignant disorders in the oral cavity. The present article aims to make a prospective randomised comparative clinical assessment of the effect of laser surgery and conventional surgery in the treatment of oral leukoplakia (OL). Material and methods: In the study were included 89 patients with histologically confirmed oral leukoplakia lesions. Laser excision of the lesions using Er YAG laser was performed in 36 of the patients, while standard surgical excision was used in 53 of the cases. Following clinical assessment comparing the two treatment methods was conducted based on: pain, wound healing, infection and recurrence of the lesions. Results: A statistically significant difference between two groups according pain in the postoperative period was found. Patients treated with laser ablation experienced far less pain than those treated with surgical excision. The healing time was significantly faster in the group treated with Er YAG laser, and regarding the occurrence of postoperative infections, the results of the two methods did not differ significantly. Recurrence was observed earlier in the group treated with laser ablation, but the levels align over a longer period of time. Conclusion: Er YAG laser ablation is a contemporary method for the treatment of oral leukoplakia without dysplasia, providing similar success, compared to conventional surgical excision, with less postoperative discomfort for the patients. (AU)

Objetivo: Os lasers de alta potência são utilizados como alternativa ao tratamento cirúrgico de doenças potencialmente malignas da cavidade oral. O presente artigo tem como objetivo fazer uma avaliação clínica prospectiva e randomizada comparativa do efeito da cirurgia a laser e da cirurgia convencional no tratamento da leucoplasia oral (LO). Material e Métodos: No estudo foram incluídos 89 pacientes com lesões de leucoplasia oral confirmadas histologicamente. A excisão das lesões com laser Er YAG foi realizada em 36 dos pacientes, enquanto a excisão cirúrgica padrão foi utilizada em 53 dos casos. A avaliação clínica seguinte comparando os dois métodos de tratamento foi realizada com base em: dor, cicatrização da ferida, infecção e recorrência das lesões. Resultados: Foi encontrada diferença estatisticamente significante entre os dois grupos de acordo com a dor no pós-operatório. Os pacientes tratados com ablação a laser experimentaram muito menos dor do que aqueles tratados com excisão cirúrgica. O tempo de cicatrização foi significativamente mais rápido no grupo tratado com laser Er YAG e, em relação à ocorrência de infecções pós-operatórias, os resultados dos dois métodos não diferiram significativamente. A recorrência foi observada mais cedo no grupo tratado com ablação a laser, mas os níveis se alinham por um longo período de tempo. Conclusão: A ablação a laser Er YAG é um método contemporâneo para o tratamento da leucoplasia oral sem displasia, proporcionando sucesso semelhante ao da excisão cirúrgica convencional, com menor desconforto pós-operatório para os pacientes. (AU)

Alteration in Serum Lipid Profile Pattern in Oral Squamous Cell Carcinoma and Potentially Malignant Disorders

ABSTRACT Objective: To evaluate and compare lipid profile level in oral submucous fibrosis (OSMF), oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) patients. Material and Methods: Thirty histopathologically diagnosed subjects each of OL, OSMF, OSCC were recruited along with 30 healthy controls. 5ml of venous blood is collected and estimated using standard diagnostic kits. Results: The mean of Total cholesterol level in controls was 219.03 mg%, in OSCC, OL and OSMF was 142.89 ± 10.21mg%, 155.44 ± 17.63 mg% and 180.60 ± 13.25 mg%, respectively. The mean low-density lipid level in controls was 137.24 mg and in OSCC, OL and OSMF groups were 109.28 ± 2.16 mg%, 126.63 ± 0.85 mg% and 119.15 ± 0.93 mg%, respectively. The mean of high-density lipid level in controls, OSCC, OL and OSMF was 42.87 ± 0.42 mg%, 36.50 ± 2.31 mg%, 21.13 ± 0.77 mg% and 28.37 ± 1.11mg%, respectively. The mean of very low density lipids level in controls, OSCC, OL and OSMF was 30.12 ± 1.51 mg%, 17.24 ± 0.80 mg%, 22.25 ± 0.93 mg% and 25.89 ± 0.43 mg%, respectively. The mean triglyceride level in controls, OSCC, OL and OSMF was 118.80 ± 9.47 mg%, 91.2 ± 3.03 mg%, 105.05 ± 2.96 mg% and 106.19 ± 3.09 mg%, respectively. Conclusion: Lipid profile levels could be early indicators of precancer and cancer.

Correlation Between Clinical and Histopathologic Diagnosis of Oral Potentially Malignant Disorder and Oral Squamous Cell Carcinoma

ABSTRACT Objective: To determine the frequency of oral potentially malignant disorders and Oral Squamous Cell Carcinoma (OSCC) and evaluate the consistency between their clinical and pathological features. Material and Methods: This retrospective study was conducted on records with a diagnosis of oral leukoplakia, oral erythroplakia, erythroleukoplakia, actinic cheilitis, lichen planus, and OSCC in the Pathology Department of Kerman dental school from September 1997 to September 2017. Data were analyzed in SPSS 21 at the significance level of ≤5%. Results: There were 378 cases of oral potentially malignant disorders and 70 cases of OSCC with a mean age of 46.82 ± 15.24 years. Buccal mucosa was the most frequent site, and lichen planus the most common lesion. Females were significantly older than males in leukoplakia and carcinoma in situ lesions. Clinical diagnosis and histopathology were consistent in 69.03% of cases. Conclusion: Clinical and histopathological diagnoses were consistent in 69.03% of records. The highest degree of clinical compliance with histopathology was observed in OSCC. Dentists should pay attention to oral potentially malignant disorders for early diagnosis to prevent their transformation to malignancy.

CD4+, CD8+ and FOXP3+ lymphocytes infiltrating oral leukoplakia: an immunohistochemical analysis / Linfócitos CD4+, CD8+ e FOXP3+ que se infiltram na leucoplasia oral: uma análise imunohistoquimica

Objetivo: Avaliar a infiltraçãdo dos linfócitos CD4+ , CD8 + e FOXP3+ e sua correlação com caracteristicas sociodemográfica, clinicopatologicas e estilo de vida de pacientes com leucoplasias bucais. Pacientes e métodos: Oitenta pacientes com diagnóstico de leucoplasia bucal foram incluidos no estudo. Análises retrospectivas foram realizadas para verificar as características sociodemográficos, clinicopatológicos e estilo de vida dos pacientes. O infiltrado linfocitário foi caracterizado por imunoistoquímica com antígenos contra de CD4+ , CD8 + e FOXP3+ . Resultados: Dos 80 pacientes incluidos neste estudo, (60%) eram homens e a idade variou de 25 a 82 anos com idade média de 58,6 anos.Trinta e oito (47.5%) eram idosos, Trinta e dois (40%) eram adultos de meia idade e apenas dez (10%) adultos jovens. Sessenta e um dos pacientes eram fumantes (76.2%) e quarenta e seis eram etilistas (57.5%). Vinte e sete (35.5%) das lesões apresentaram algum grau de displasia epitelial. O grau de displasia epitelial apresentou correlação positiva com a intensidade do consumo do alcool (p=0.008). Houve correlação positiva entre os linfócitos CD4+ e CD8+ (p=0.005). Conclusão: O infiltrado linfocitário não foi relacionado com nenhuma característica clinicopatológica das lecoplasias bucais. Entretanto, o grau de displasia está relacionado ao estilo de vida dos pacientes(AU)

Objective: To evaluate the infiltration of CD4+ , CD8+ and FOXP3+ lymphocytes and their correlation with sociodemographic, clinicopathological and lifestyle characteristics of patients with oral leukoplakia. Patients and methods: Eighty patients diagnosed with oral leukoplakia were included in the study. Retrospective analyses were performed in order to verify the sociodemographic, clinicopathologic and lifestyle characteristics. The lymphocytic infiltrate characterization was performed by immunohistochemistry with antibodies against CD4+, CD8+, and FOXP3+ markers. Results: Of 80 patients included in the study, 60% were men, and their age ranged from 25 to 82 years, with a mean of 58.6. Thirty-eight patients (47.5%) were elderly, Thirty-two (40%) middle-aged, and only ten (10%) young adults. Sixty-one of the patients were smokers (76.2%) and forty-six were alcoholics (57.5%). Twenty-seven (35.5%) of the lesions presented some degree of dysplasia. The degree of epithelial dysplasia was correlated with the intensity of alcohol consumption (p=0.008). A positive correlation was found between CD4+ and CD8+ lymphocytes (p=0.005). Conclusion: The lymphocytic infiltrate of oral leukoplakia was not correlated with any clinicopathologic characteristic. However, the degree of epithelial dysplasia was correlated with the lifestyle of the patients(AU)

DEC1: a potential biomarker of malignant transformation in oral leukoplakia

Abstract The purpose of this study was to analyze the differential expression of DEC1 in oral normal mucosa (NM), oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC). Surgically excised specimens from patients with OLK (n = 47), OSCC (n = 30) and oral normal mucosa (n=11) were immunostained for DEC1. The expression of DEC1 protein was evaluated, and its association with the clinicopathological features was analyzed. The expression of DEC1 in NM, OLK and OSCC tissues increased in turn, and significant differences were observed among the groups (P < 0.0001). In terms of the association between DEC1 expression and epithelial dysplasia, DEC1 expression was lower in hyperkeratosis without dysplasia (H-OLK) than in OLK with moderate to severe dysplasia (S-OLK), and these differences were significant (p < 0.05). The expression of DEC1 in OSCC with OLK was significantly higher than that in OSCC without OLK (p < 0.01). Therefore, DEC1 could be a potential biomarker of malignant transformation in the carcinogenesis of OSCC, which may provide a new research direction for the transformation of oral potentially malignant disorders (OPMDs) into OSCC.

BMI-1 expression increases in oral leukoplakias and correlates with cell proliferation

Abstract Oral leukoplakia (OL) is a white lesion of an indeterminate risk not related to any excluded (other) known diseases or disorders that carry no increased risk for cancer. Many biological markers have been used in an attempt to predict malignant transformation; however, no reliable markers have been established so far. Objective To evaluate cell proliferation and immortalization in OL, comparing non-dysplastic (Non-dys OL) and dysplastic OL (Dys OL). Methodology This is a cross-sectional observational study. Paraffin-embedded tissue blocks of 28 specimens of Non-dys OL, 33 of Dys OL, 9 of normal oral mucosa (NOM), 17 of inflammatory hyperplasia (IH), and 19 of oral squamous cell carcinomas (OSCC) were stained for Ki-67 and BMI-1 using immunohistochemistry. Results A gradual increase in BMI-1 and K-i67 expression was found in oral carcinogenesis. The immunolabeling for those markers was higher in OSCC when compared with the other groups (Kruskal-Wallis, p<0.05). Ki-67 expression percentage was higher in OL and in IH when compared with NOM (Kruskal-Wallis/Dunn, p<0.05). Increased expression of BMI-1 was also observed in OL when compared with NOM (Kruskal-Wallis/Dunn, p<0.05). No differences were observed in expression of both markers when non-dysplastic and dysplastic leukoplakias were compared. A significant positive correlation between Ki-67 and BMI-1 was found (Spearman correlation coefficient, R=0.26, p=0.01). High-grade epithelial dysplasia was associated with malignant transformation (Chi-squared, p=0.03). Conclusions These findings indicate that BMI-1 expression increases in early oral carcinogenesis and is possibly associated with the occurrence of dysplastic changes. Furthermore, our findings indicate that both Ki-67 and BMI-1 are directly correlated and play a role in initiation and progression of OSCC.

Fresh Frozen Amniotic Membrane and Buccal Pad of Fat for Reconstruction of Oral Mucosal Defect After Surgical Excision of Leukoplakia

Objective: To compare the use of Fresh Frozen Amniotic Membrane (FFAM) and Buccal Pad of Fat (BPF) for reconstruction of oral mucosal defect after surgical excision of leukoplakia. Material and Methods: Twenty patients were randomly selected and divided into two groups. Group 1 use amniotic membrane graft and Group 2 BPF. Both groups were evaluated preoperatively and postoperatively. Incisal opening, epithelialization and fibrosis were evaluated after one month of surgery. Chi square and Student t tests were used. Results: According to the presence of smoking habits, the highest frequencies were for smoking (30%) and betel leaf areca nut with tobacco (30%). Regarding the diameter of oral leukoplakia, in 40% of the participants it was 2x3 cm2. In Group1, after one month of surgery preoperative and postoperative inter-incisal opening values were 44.20 ± 3.37 and 42.05 ± 3.47 (p<0.001). In Group 2, preoperative and postoperative inter-incisal opening values were 44.09 ± 3.32 and 43.01±3.38 (p>0.05). When FFAM was used complete epithelialization in 70% and incomplete epithelialization in 30% patients. When BPF was used the results were almost similar. Fibrosis occurred in 30% in Group 1. There were no complications like flap necrosis, infectiona and hematoma formation. Conclusion: Incisal opening was significantly better in Fresh Frozen Amniotic Membrane Group, epithelialization and fibrosis were almost same in both groups after surgical excision of oral leukoplakia.

Oral Candidal Carriage Among Patients with Oral Potential Malignant Disorders: A Case-Control Study

Objective: To determine the prevalence of Candida species in the saliva of patients with clinically suspected oral potentially malignant disorders (OPMD) and healthy cohorts. Material and Methods: Unstimulated saliva was collected from patients with OPMD (n=100) and age and sex matched healthy subjects (n=170). The samples were inoculated onto Sabouraud Dextrose Agar and incubated for a week. The colonies of the isolates were enumerated using a colony counter. The isolates were identified using standard phenotypic methods. The significance of oral candidal carriage was calculated using Independent T test. Odds and Risk ratio was calculated using Pearson's Chi-square test. Results: Oral candida carriage was present in 51% of patients with OPMD while healthy cohorts had a prevalence of 20.6%. A good statistical significance was observed for the prevalence of oral candidal carriage for patients with OPMD in comparison to healthy cohorts (p=0.013). Significant Odds and risk ratio was observed for the prevalence of Candida species among OPMD. Majority of the isolates in both groups were C. albicans. Colony forming units were high among patients with OPMD. Conclusion: A significant association of oral candidal carriage to oral potentially malignant disorders in comparison to healthy cohorts was observed. Candidal species may be potent risk factor for transition of OPMD to oral Squamous Cell Carcinoma.

Evaluation of p16ink4a expression and presence of HPV-16 DNA by real time PCR in patients with oral leukoplakia / Avaliação da expressão da p16Ink4a e a presença do DNA do HPV-16 pela Real time PCR em pacientes com leucoplasia bucal

Objetivo: Avaliar a expressão do p16Ink4a por imunohistoquímica e a presença do HPV16 pela Real time PCR em tecido fresco, plasma e saliva de pacientes com leucoplasia bucal (LB) e hiperplasia fibrosa inflamatória (HFI). Material e métodos: Foram incluídos 67 pacientes com o diagnóstico de LB e 44 pacientes com diagnóstico de HFI no estudo. Foram coletados dados sociodemográficos, clinicopatológicos, amostras de tecido fresco, sangue e saliva, que foram armazenados em um freezer a -80o C para realização de análise molecular. Também foi utilizado o tecido parafinizado para realização da imunohistoquímica para a p16Ink4a. As amostras de materiais biológicos obtidos dos pacientes foram submetidas à detecção do DNA do HPV-16 pela Real time PCR. O tecido parafinizado dos mesmos pacientes foram utilizados para avaliar a expressão da p16Ink4a pela imunohistoquímica. Resultados: Dos 67 pacientes incluídos de LB no estudo, 55,2% eram do sexo masculino com uma média de idade de 57,1 anos. Os pacientes idosos (>45 anos) compuseram 86,6% da amostra. As localizações anatômicas mais acometidas por LB foram a mucosa jugal (35,8%) e língua (20,9%). Sobre o tabagismo, 71,8% dos pacientes eram fumantes, onde a maioria (47,8%) foi classificada como tabagista leve. Em relação ao consumo de álcool, 47,4% eram alcoolistas, sendo a sua maioria classificada como alcoolista leve (59,3%). O grupo HFI foi composto por 54,5% pacientes do sexo masculino com uma média de idade de 57,3 anos. 90,9% dos pacientes eram idosos (>45 anos). A ocorrência das lesões foram em mucosa jugal (31,8%) e rebordo alveolar (25%). A expressão para p16Ink4a na LB foi fraca (41,8%) e para o grupo HFI, majoritariamente a expressão foi forte (68,2%). Real time PCR não detectou HPV-16 em nenhumas das amostras analisadas. Conclusão: Os achados em nosso estudo mostraram que a detecção de HPV de alto risco em tecido fresco, plasma e saliva para LB e HFI não se correlacionaram com a superexpressão da p16Ink4a(AU)

Objective: To evaluate the expression of p16Ink4a by immunohistochemistry and the presence of HPV-16 by Real time PCR in fresh tissue, blood plasma, and saliva of patients with oral leukoplakia (OL) and inflammatory fibrous hyperplasia (IFH). Material and methods: Sixty-seven patients with the diagnosis of OL and 44 patients with the diagnosis of IFH were included in the study. Sociodemographic, clinicopathological, fresh tissue, blood, and saliva samples were collected and stored at - 80o C for molecular analysis. Paraffinized-embedded tissue was also used to perform the immunohistochemistry for p16Ink4a. Samples of biological material obtained from the patients were submitted to HPV-16 DNA detection by real time PCR, both with specific probes. Paraffinized-embedded tissue from the same patients were used to evaluate the expression of p16ink4a by immunohistochemistry. Results: Out of the 67 included OL patients, 55.2% were male with a mean age of 57.1 years. Elderly patients (> 45 years) comprised 86.6% of the sample. The prevalence of lesions was highest in the buccal mucosa (35.8%) followed by the mobile tongue (20.9%). 71.8% were smokers and the majority (47.8%) was classified as light smoker. Regarding alcohol consumption, 47.4% were alcoholics, most of whom were classified as light alcoholics (59.3%). The IFH group was composed of 54.5% of male patients with a mean age of 57.3 years. 90.9% of the sample were elderly patients (> 45 years). The highest occurrence of the lesions was in buccal mucosa (31.8%) and alveolar ridge (25%). The expression for p16Ink4a in LB was mostly weak (41.8%) and for the IFH group, the expression was mostly strong (68.2%). There was no positivity in any of the samples for HPV-16 by real time PCR. Conclusion: Our findings showed the HR-HPV detection in fresh tissue, plasma blood and saliva for OL and IFH does not correlate with p16Ink4a immunoexpression in paraffinized-embedded tissue(AU)

Comparação da prevalência do HPV-16 em pacientes com carcinoma espinocelular de boca e lesões potencialmente malignas bucais / Comparison of the HPV-16 prevalence in patients with oral squamous cell carcinoma and oral potentially malignant disorders

O papilomavírus humano (HPV) subtipo 16 é um fator de risco para o desenvolvimento do carcinoma espinocelular (CEC) de orofaringe. No entanto, o papel do mesmo na carcinogênese oral, bem como a associação com as lesões potencialmente malignas, permanece controverso. O objetivo deste estudo foi comparar a prevalência do HPV-16, em amostras de tecido fresco, obtidas de 27 pacientes com CEC oral, 37 pacientes com leucoplasia bucal, 24 pacientes com líquen plano bucal (LPB) e 32 pacientes controle, correlacionando a presença do HPV com as variáveis clínico-patológicas em uma população da região noroeste do estado de São Paulo - Brasil. Realizouse a extração do DNA das amostras e a verificação da presença do HPV-16 por meio da Real-Time Polymerase Chain Reaction. Todas as amostras foram negativas para o HPV-16 nos quatro grupos estudados. Conclui-se que a ausência do HPV-16 nas amostras de CEC bucal, leucoplasia e LPB indica que a infecção pelo mesmo não é comum e não representa um fator de risco importante na carcinogênese oral na população da região noroeste do estado de São Paulo ­ Brasil(AU)

Human Papillomavirus (HPV), specially subtype 16, is a known risk factor for the oropharyngeal squamous cell carcinoma (SCC) development. However, HPV role in oral carcinogenesis, as well as in potentially malignant oral lesions remains controversial. The goal of the present study was to compare the HPV16 prevalence, in fresh tissue samples obtained from 27 oral SCC patients, 34 oral leukoplakia (OL) patients, 24 oral lichen planus (OLP) patients and 32 control patients, correlating HPV presence with the clinicopathological variables in a population from northwest region of the Sao Paulo state - Brazil. DNA extraction was carried out and all samples were submitted to Real-Time PCR for HPV-16 DNA detection. We found that all fresh tissue samples of oral SCC, OL, OLP and oral normal mucosa were negative for HPV-16. We conclude that HPV-16 absence in oral SCC, OL and OLP samples indicates that its infection is uncommon and does not represent an important risk factor in oral carcinogenesis in the population from northwest region of the Sao Paulo state ­ Brazil(AU)

HPV-16 DNA detection in fresh tissue, saliva and plasma of patients with oral leukoplakia by real time PCR / Detecção do DNA do HPV-16 em tecido fresco, saliva e plasma sanguíneo de pacientes com leucoplasia bucal pela Real Time PCR

Objetivo: Avaliar a presença do HPV-16 em tecido fresco, saliva e plasma sanguíneo de pacientes com leucoplasia bucal pela real time PCR na região noroeste do estado de São Paulo, Brasil. Pacientes e métodos: Trinta e sete pacientes com diagnóstico de leucoplasia bucal foram incluídos no estudo. Destes, foram obtidos dados sociodemográficos, clinicopatológicos, estilo de vida e amostras de tecido fresco, sangue e saliva que foram armazenados a - 80ºC para posterior análise molecular. Os materiais obtidos destes pacientes foram submetidos à detecção do DNA viral pela técnica da real time PCR com sonda específica para o HPV-16. Resultados: Dos 37 pacientes incluídos no estudo, 64,8% eram homens e a idade variou de 25 a 82 anos, com uma média de 58,72 anos. Dezesseis pacientes (43,2%) eram idosos e 43,2%, adultos de meia idade, e apenas 13,6%, adultos jovens. A maioria dos pacientes era fumante (72,9%), sendo que 16,3% eram ex-fumantes e 10,8%, não fumantes. Da mesma forma, a maioria (62,2%) era etilista, 21,6%, ex-etilistas e 16,2%, não-etilistas. Vinte e sete por cento das lesões apresentaram algum grau de displasia epitelial. A detecção do HPV-16 pela PCR em tempo real não foi positiva para nenhuma amostra, resultando em um índice de 0% de detecção. Conclusão: O HPV-16 não foi identificado na população estudada. No entanto, outros subtipos do HPV de baixo e alto risco podem estar associados à ocorrência de leucoplasia bucal nesta população, o que requer novas investigações. Estudos epidemiológicos mais amplos são necessários para esclarecer a variabilidade geográfica na prevalência do HPV no carcinoma espinocelular de cabeça e pescoço e lesões bucais potencialmente malignizáveis(AU)

Objective: To evaluate the prevalence of HPV-16 DNA detection in fresh tissue, saliva and blood plasma from patients with oral leukoplakia by the real time PCR in the northwest region of the São Paulo state, Brazil. Patients and methods: Thirty-seven patients diagnosed with oral leukoplakia were included in the study. Sociodemographic, clinicopathologic and lifestyle data, fresh tissue, saliva and blood plasma samples were collected. Biologic material was stored at -80ºC and then submitted to viral DNA detection by the real time PCR technique with a probe specific for HPV-16. Results: Of the 37 patients included in the study, 64.8% were men, and the age ranged from 25 to 82 years, with a mean of 58.72. Sixteen patients (43.2%) were elderly, 43.2% were middle-aged adults, and only 13.6% were young adults. Most patients were smokers (72.9%), 16.3% were former smokers, and 10.8% were non-smokers. Most patients (62.2%) were current drinkers, 21.6% were ex-drinkers and 16.2% were non-drinkers. Twenty seven percent of the lesions presented some degree of dysplasia. HPV-16 detection by real-time PCR was not positive for any sample, resulting in a 0% detection rate. Conclusion: The HPV-16 was not identified in the population studied. However, other low and high-risk HPV subtypes might be associated to the occurrence of oral leukoplakia in this population, which requires further investigations. Broader epidemiological studies are required to clarify the geographic variability in the prevalence of HPV in head and neck squamous cell carcinoma and oral potentially malignant lesions(AU)

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.

The presence of Helicobacter pylori in oral cavities of patients with leukoplakia and oral lichen planus

ABSTRACT Objective Helicobacter pylori infection is one of the most common bacterial infections in men. This gastrointestinal pathogen is closely related to gastritis, peptic ulcers, and the increased risk of gastric cancer. Numerous studies have indicated oral cavities as possible Helicobacter pylori reservoirs. Helicobacter pylori has been detected both in supragingival and subgingival plaques, and also in saliva. In addition, the relationship between lesions of oral mucosa and the presence of H. pylori has been evaluated and described in some studies. The aim of this study was to assess the presence of Helicobacter pylori DNA in the oral cavity of patients with oral leukoplakia and oral lichen planus. Material and Methods The study included 54 patients with oral leukoplakia, 72 with oral lichen planus lesions, and 40 healthy controls. The presence of Helicobacter pylori in oral cavity samples was analyzed using a single-step Polymerase Chain Reaction (PCR) method. All patients underwent a periodontal examination and the following clinical parameters were collected: pocket depth, bleeding, and plaque indexes. The periodontal status was assessed using the Offenbacher classification. Results In most patients, pathological lesions were in typical sites on the buccal mucosa (leukoplakia in 88%, and oral lichen planus in 93% of patients). The DNA of the Helicobacter pylori was present in 20% of patients with leukoplakia and 23% of patients with lichen planus. We did not find the DNA of H. pylori in healthy controls. The periodontal status described by periodontal indices was worse in the investigated group than in the control group. Conclusion These findings suggest that the H. pylori presence in oral cavities may be related with leukoplakia and lichen planus oral lesions.

Altered ß-catenin expression in oral mucosal dysplasia: a comparative study

Objective The current study aimed to investigate the β-catenin expression in oral leukoplakia (OL) with different degrees of epithelial dysplasia and normal oral mucosa.Material and Methods Formalin-fixed, paraffin-embedded tissue samples of 39 OL (mild dysplasia n=19, moderate dysplasia n=13, and severe dysplasia n=7), and 10 normal oral mucosa (control group) were submitted to immunohistochemical reactions to anti-β-catenin primary antibody. A qualitative β-catenin analysis was performed based on the percentage of positive cells. The cellular location and the epithelial layer were also considered. The Chi-square test and the Fisher’s exact test were used to verify possible differences in the β-catenin expression among the OL groups. A p-value of <0.05 was considered statistically significant.Results Membranous expression of β-catenin in parabasal and basal layers was gradually lost in the higher degrees of epithelial dysplasia. In normal oral mucosa, β-catenin was detected only in the cytoplasmic membrane. However, a significant increase in cytoplasmic β-catenin could be observed between mild and moderate dysplasia (Fisher Exact test - p<0.001) and between mild and severe dysplasia (p<0.001).Conclusions The β-catenin cytoplasmic expression observed in this study may represent the initial stage of modifications in the E-cadherin-catenin complex, along with morphological cellular changes.

Avaliação da expressão do KI-67 e do BMI-1 em leucoplasias bucais displásicas e não displásicas / Evaluation of Ki-67 and BMI-1 expression in non-dysplastic and dysplastic oral leukoplakias

Leucoplasia bucal (LB) é uma desordem potencialmente maligna, com risco de transformação maligna que varia de 0,13% a 17,5%. Muitos estudos vêm buscando estabelecer biomarcadores capazes de predizer o potencial de transformação maligna dessa lesão. O Ki-67 é uma proteína não-histônica nuclear que tem sido amplamente utilizada para avaliar proliferação celular. O BMI-1 é uma proteína considerada um marcador essencial para a manutenção das propriedades de autorrenovação e da tumorigenicidade do carcinoma espinocelular. O objetivo principal desse estudo observacional transversal foi avaliar proliferação e imortalização celulares em LB a partir da marcação imunoistoquímica do Ki-67 e do BMI-1, comparando lesões displásicas com não displásicas. Casos de LB não displásica - LBND (n=28), LB displásica - LBD (n=33) foram selecionados a partir de prontuários de pacientes e comparados com mucosa clinicamente normal - MN (n=9), hiperplasia inflamatória - HI (n=17) e carcinoma espinocelular - CEC (n=19). Os diagnósticos histopatológicos foram confirmados a partir da revisão de cortes histológicos corados por hematoxilina e eosina. Adicionalmente, cortes histológicos foram submetidos à técnica imunoistoquímica para avaliação de Ki-67 e BMI-1. Para a quantificação foi considerado o percentual de células positivas por 1000 células para o CEC e 1500 células para os demais grupos. O percentual de imunomarcação de Ki-67 e de BMI-1, quando avaliadas todas as camadas epiteliais em conjunto, foi mais alto no CEC quando comparado aos demais grupos (Kruskal-Wallis, p<0.05). A expressão de Ki-67 foi maior em LBND, LBD e HI quando comparada com MN (Kruskal-Wallis, p<0.05)...

Oral leukoplakia (OL) is potentially malignant disorder, with a risk of malignant transformation that ranges from 0.13% to 17.5%. Many biological markers have been used as an attempt to predict malignant transformation, but no reliable markers have been established so far. The Ki-67 is a nuclear non-histone regarded as reliable marker of proliferating cells .BMI-1 is a protein considered as an essential marker for maintenance of properties self-renewability and tumorigenicity of squamous cell carcinoma. The main aim this cross-sectional observational study was to evaluate cell proliferation and immortalization in oral leukoplakia, comparing non-dysplastic and dysplastic lesions. Cases of non-dysplastic – Non-dys OL (n=28), dysplastic – Dys OL (n=33) records were selected and compared with normal oral mucosa – NOM (n=9), inflammatory hyperplasia –IH (n=17), and oral squamous cell carcinoma - OSCC (n=19). The histopathological diagnosis was confirmed by the revision of Hematoxilin and Eosin stained slides. Additionally, histological sections were submitted to immunohistochemical technique for evaluation of Ki-67 and BMI-1. The labeling index was determined by counting the labeled nuclei of 1000 cells for OSCC cases and 1500 for the others comparison groups. Ki-67 and BMI-1 immunolabeling percentage were higher in OSCC in comparison of others groups when all epithelial layers were evaluated together (Kruskal-Wallis, p<0.05). Ki-67 immunolabeling increased in Non-dys OL, Dys OL and IH when compared to NOM (Kruskal-Wallis, p<0.05). Furthermore, BMI-1 immunolabeling was higher in Dys OL relation to NOM in the analysis of all epithelial layers together (Kruskal-Wallis, p<0.05)...

Análise histopatológica de lesões leucoqueratósicas da mucosa oral avaliadas pelos exames de luminescência, cromoscopia e microscopia confocal reflectante / Histological analysis identified and assessed by tests of luminescence, chromoendoscopy and confocal reflectance leukokeratosis lesions

O câncer de mucosa oral é um problema de saúde pública, com maior incidência em homens acima de 50 anos. Uma das manifestações clínicas mais precoces do câncer da mucosa oral são as lesões leucoqueratósica. O aspecto clínico não homogêneo e o tamanho maior de 200mm2, em mucosa não queratinizada como a do assoalho da boca e ventre da língua, são aspectos que implicam na possibilidade de evolução da lesão. A dificuldade do diagnóstico das lesões precoces está na seleção do local a ser biopsiado principalmente frente a lesões extensas e heterogêneas. O objetivo desta pesquisa é avaliar se a utilização do exame clínico juntamente com os métodos auxiliares de diagnóstico das lesões leucoqueratósica da mucosa oral (VELscope®, Azul de toluidina e Solução de lugol) contribui para uma maior precisão do diagnóstico de displasias nestas lesões quando comparado ao histopatológico. E, identificar os padrões morfológicos destas lesões quando avaliadas por meio da microscopia confocal reflectante. Foram selecionados 30 pacientes, maiores de 18 anos, portadores de lesão clínica compatível com leucoplasia oral triados no ambulatório da disciplina de Estomatologia Clínica da Faculdade de Odontologia da Universidade de São Paulo e que precisavam ser submetidos à biópsia para o estabelecimento do diagnóstico final. Foram realizados os testes de Azul de Toluidina, Solução de Lugol, VELscope®, microscopia confocal reflectante e posteriormente, a biópsia incisional para obtenção do diagnóstico final. Os pacientes incluídos possuíam média de idade 60,66 anos, sendo 70% (21/30) do gênero feminino e 30% (9/30) do gênero masculino. O tabagismo foi relatado por 16,7% (5/30) dos pacientes, sendo 60% (3/5) homens. A associação do tabagismo e etilismo foi relatada em 10% (3/30) dos pacientes, o tabagismo isoladamente por 6,6% (2/30) e o de etilismo por 3,3%(1/30)...

Cancer of the oral mucosa is a public health problem, with higher incidence in men above 50 years. One of the earliest manifestations of cancer of the oral mucosa lesions are leukokeratosis. The inhomogeneous clinical aspect and the larger size of 200mm2 in non-keratinized mucosa as the floor of the mouth and constipation of the tongue, are aspects that imply the possibility of evolution of the lesion. The difficulty of diagnosis of early lesions is in the selection of the site to be biopsied primarily against large and heterogeneous lesions. The objective of this research is to evaluate the use of clinical examination along with diagnostic aids leucoqueratósica of oral lesions (VELscope ®, Toluidine blue and Lugol solution) methods contributes to greater accuracy of diagnosis of dysplasia in these lesions when compared the histopathological. And identify the morphological patterns of these lesions when evaluated by reflectance confocal microscopy. 30 patients older than 18 years, with clinical lesion compatible with oral leukoplakia screened in the outpatient discipline of Clinical Dentistry, Faculty of Dentistry, University of São Paulo and that needed to be biopsied to establish the final diagnosis were selected. Tests toluidine blue, Lugol's solution, VELscope ®, reflectance confocal microscopy (RCM) and subsequently, incisional biopsy to obtain the final diagnosis were performed. Patients enrolled had a mean age 60.66 years, 70% (21/30) were female and 30% (9/30) were male. Smoking was reported by 16.7% (5/30) of patients, 60% (3/5) homens.A association of smoking and alcohol use was reported in 10% (3/30) of patients, smoking alone by 6.6% (2/30) and of alcoholism by 3.3% (1/30)...