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Agradecimentos: The present work was supported by the Brazilian funding agencies: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenacao de Aperfeicoamento de Pessoal de N?vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cient?fico e Tecnologico (CNPq). We thank Dr Jose Vassallo (A.C. Camargo) for performing IHC analysis, and the A. C. Camargo Hospital for providing tissue microarrays Abstract: This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE MINAS GERAIS - FAPEMIG FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado

A punção aspirativa com agulha fina guiada por ultrassom (PAAF-US) é o principal método propedêutico através do exame citológico de nódulos tireoidianos. Contudo, 10 a 30% dos casos apresentam como resultados amostras inadequadas ou indeterminadas, requerendo nova PAAF-US ou biópsia com agulha grossa dirigida por US (BAG-US). No presente trabalho avaliamos a performance do marcador CaT12, previamente identificado como marcador de tumores tireoidianos malignos, para auxiliar na detecção de carcinomas em fragmentos de nódulos tireoidianos através da Citometria de Fluxo. O resultado obtido foi comparado com o anátomo-patológico, e testes diagnósticos foram feitos para avaliar a capacidade do marcador em identificar os tecidos contendo malignidade. Análises prévias de citometria de fluxo do marcador com linhagens tumorais foram otimizadas e indicaram que a marcação externa sem permeabilização celular era adequada para a detecção do tumor. Posteriormente, avaliou-se o processamento das biópsias, que foram submetidas à digestão com colagenase para posterior marcação e detecção. Foram coletadas 29 amostras de portadores de nódulos tireoidianos submetidos a PAAF-US com estratificação Bethesda. Após detecção de células tumorais, estabeleceu-se os cut-offs utilizados para os valores de positividade do CaT12, e os parâmetros diagnósticos foram sensibilidade de 86,67% e especificidade de 78,57%, com acurácia 82,76%, acima da apresentada nos exames anátomo-patológicos pós-cirúrgicos 75,8% e das PAAFs com 53,5%. Nossos resultados sugerem que a nova tecnologia poderá ser usada na análise de material aspirado tanto por PAAF-US quanto por BAG-US, sem depender dos parâmetros citológicos ou histológicos e podem significar uma mudança de paradigmas para o diagnóstico do câncer de tireoide. Ultrasound-guided Fine Needle Aspiration (USG-FNA) followed by cytological examination is a common procedure performed for diagnosis of thyroid nodules. However, 10 to 30% of the cases present indeterminate results, requiring the use of US-guided core needle biopsies. In the present work we evaluated the performance of the CaT12 biomarker, previously identified as a marker of malignant thyroid tumors, to aid in the detection of carcinomas in fragments of thyroid nodules through Flow Cytometry. Results were compared with histopathological analyses, and diagnostic tests were performed to evaluate the marker's ability to distinguish malignant tissues. Previous flow cytometric analyzes of the marker with tumor cell lines were optimized and indicated that cell labeling without permeabilization was adequate for tumor detection. Subsequently, the biopsies were processed and submitted to digestion with collagenase prior to labeling and detection. Twenty-nine samples from patients with thyroid nodules submitted to USG-FNB were collected. After detection of tumor cells, cut-off value was calculated and positivity was established. Diagnostic parameters present sensitivity of 86.67%, specificity of 78.57%, and accuracy of 82.76%, much higher than the accuracy presented by the histopathology (75.8%), and USG-FNB (53.5%) analysis. Our results suggest that the novel technology can be used in the analyses of biopsies without depending on cytological or histological parameters, and may lead us to a paradigm shift for the diagnosis of thyroid cancer. Tese (Doutorado)