Novos biomarcadores para o diagnóstico não invasivo do carcinoma oral de células escamosas

CHAPTER I: Early detection of oral squamous cell carcinoma (OSCC) is an important factor to reduce mortality rates and treatment success is directly related to its diagnosis at early stages. However, unfortunately, most oral carcinomas are diagnosed in late stages of the disease. This is the first study that describes an scFv fragment, selected by Phage Display against total salivary proteins, that effectively recognize antigens with diagnostic potential for early detection of oral cancer. ELISA immunoassays in 30 samples of saliva from OSCC patients and 30 healthy subjects showed that the scFv D09 recognize antigens present in tumoral samples (p <0.0001). The diagnostic test showed high discriminative power with a significant ROC curve (AUC=0.97) and 100% specificity. Imunohistochemistry tests revealed that the selected antibody also showed higher affinity to tissues related to well differentiated oral carcinomas, such as keratin pearls. The two-dimensional proteomic analysis revealed that the scFv D09 recognizes two spots in saliva samples (~50 kDa; pI 5.75 e 5.86) and only one in tissue samples (~28 kDa and pI 4.68). Final characterization of the peptides sequences is under investigation through mass spectrometry. These data, although preliminary, may significantly alter the diagnosis of oral cancer, once they provide strong evidence that saliva is a powerful clinical tool for early detection through a non-invasive and highly sensitive diagnostic method. CHAPTER II: Several studies have been suggesting annexin A1 protein as an active player in tumorigenesis of many organs. Nevertheless, its tumor biomarker role has been mainly studied in tissues by immunohistochemistry or cell culture. Hence, in this investigation, the peripheral blood from 27 oral squamous cell carcinoma (OSCC) patients and 25 negative control individuals were examined by quantitative real-time PCR. Down-regulated ANXA1 expression at mRNA level was observed in OSCC samples (p = 0.026). Significantly diminished mRNA levels correlated to age, sex and the anatomical site of the tumor lesion were observed. Moreover, the ROC curve analysis revealed the performance of ANXA1 expression as a suitable biomarker for patients with oral cavity cancer, especially those with 60 years of age or older and/or women. For the first time, ANXA1 mRNA is revealed as blood-based biomarker, and its adoption for complementary non-invasive diagnosis of OSCC is suggested. These results suggest that, beyond the anti-inflammatory function, annexin A1 may also play a tumor suppressor role in peripheral blood cells, such as leukocytes.Doutor em Genética e BioquímicaCAPÍTULO I: A detecção precoce do carcinoma oral de céulas escamosas (OSCC) é fator importante na diminuição das taxas de mortalidade e o sucesso do tratamento relaciona-se diretamente com seu diagnóstico em fases iniciais. Contudo, infelizmente, a maioria dos carcinomas orais é diagnosticada em fases tardias da doença. No presente trabalho, pela primeira vez, é descrito um fragmento scFv, selecionado por Phage Display contra proteínas totais salivares, que reconhece efetivamente antígenos com potencial diagnóstico para a detecção precoce do câncer oral. A análise de reatividade por meio do teste imunoenzimático ELISA em 30 amostras de saliva de pacientes com OSCC e 30 indivíduos saudáveis, mostrou que o scFv D09 reconhece antígenos presentes na saliva tumoral (p