Dissertations / Theses on the topic 'Neoplasm DNA'

Orientadores: Jose Antonio Simões, Sophie Françoise Mauricette Derchain
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Objetivo: Avaliar a recuperação e estabilidade do DNA para detecção do papillomavírus humano (HPV) através da Reação em Cadeia da Polimerase (PCR) utilizando amostras estocadas por até 24 meses em Universal Collection Medium (UCM) com reagente desnaturante. Métodos: Sessenta amostras de citologia da cérvix uterina positivas para HPV, - que foram coletadas em UCM com resultado de Papanicolaou NIC 2 e NIC 3 entre os anos de 2003 e 2005 e que foram estocadas - foram utilizadas para este estudo. Todas as amostras haviam sido congeladas a ¿20°C após a adição do reagente desnaturante (0,05% de azida sódica + solução de NaOH) e da retirada da alíquota necessária para a realização do teste de Captura¿Híbrida 2 (CH 2) para identificação do DNA-HPV. O tempo de estocagem das amostras utilizadas foi de 6, 12 e 24 meses (20 amostras para cada tempo de estocagem). A extração do DNA foi realizada de acordo com protocolo específico para este tipo de material. A técnica de PCR foi realizada para confirmação da presença e da integridade do DNA através da detecção da ß-globina humana utilizando-se primers de consenso G73 e G74, e a detecção do DNA-HPV foi realizada utilizando-se os primers de consenso PGMY09 e PGMY11. Resultados: O DNA extraído do material desnaturado foi recuperado em 57 das 60 (95%) amostras estudadas. O DNA-HPV só não pôde ser detectado por PCR em uma destas amostras recuperadas. Conclusão: A recuperação e a estabilidade do DNA-HPV foi excelente após dois anos de estocagem do material cervical colhido em UCM com reagente desnaturan
Abstract: Objective: To evaluate the recovery and stability of DNA for the detection of HPV by Polymerase Chain Reaction (PCR) from clinical specimens stored for up to 24 months in Universal Collection Medium (UCM) with denatured reagent. Materials and methods: Sixty stores HPV-positive cervical smears collected from women with CIN 2 or CIN 3 diagnosis at Pap smear cytology between 2003 and 2005 were utilized to study. All samples were stored at ¿20°C after add of the denaturing reagent (sódica azida 0,05% and solution NaOH) and removing the aliquot required for carrying out the hybrid capture 2 assay for the identification of HPV-DNA, the samples were stored for 6, 12 or 24 months (20 samples for each storage time). DNA-HPV extraction was performed according to a protocol specifically designed for this type of material. The presence and quality of DNA was confirmed by human ß-globin detection using the consensus primers G73 and G74 and HPV was detected using the consensus primers PGMY09 and PGMY11 through the technique of PCR. Results: The DNA extracted from the denatured material was recovered in 57 out of 60 (95%) of the samples studied. DNA-HPV failed to be detected in one of the recovered samples. Conclusions: The recovery and stability of DNA-HPV from cervical samples stored for up to two years in UCM were excellent
Mestrado
Ciencias Biomedicas
Mestre em Tocoginecologia

Orientador: Laura Sterian Ward
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Para investigar a utilidade clínica de dois genes supressores tumorais envolvidos no controle da proliferação e sobrevivência celular, nós estudamos por imunoistoquímica as proteínas ATM e PTEN em 272 pacientes diagnosticados carcinoma diferenciado da tireóide (142 carcinomas papilíferos do tipo clássico, 72 carcinomas papilíferos variante folicular, 17 carcinomas papilíferos de células altas e 41 carcinomas foliculares); 106 amostras de tecido de pacientes diagnosticados com doenças tiroidianas benignas (55 adenomas foliculares e 51 com bócio), 18 tecidos de tiróide normal extraídos de lobo contra-lateral de indivíduos operados por adenoma folicular. A expressão quantitativa de ATM foi avaliada por Real-Time PCR em 87 CP. Ainda, analisamos o perfil genotípico para três polimorfismos de ATM (D1853N, S707P e S49C) em 199 pacientes (164 carcinomas papilíferos e 35 carcinomas foliculares) e 219 indivíduos controles. Os pacientes foram seguidos por 53,8±41 meses utilizando-se um protocolo padrão. 179 pacientes foram classificados como livre de doença e 48 pacientes tiveram má evolução (recidivas e 12 mortes). A análise da regressão logística múltipla ajustada para sexo e faixa etária mostrou que a expressão da proteína ATM foi mais freqüente entre os pacientes que apresentavam tumores menos agressivos (81%) e que evoluíam livres de doença (63%) (p=0.016; p=0.0276 respectivamente). Maior incidência de casos que expressavam a proteína PTEN também foi observada em pacientes que não tiveram metástase na evolução (75%) (p= 0.0437). O alelo heterozigoto para o polimorfismo D1853N de ATM foi mais prevalente entre os controles (64%) do que nos indivíduos com câncer (36%) (p=0.0364) Estes dados indicam que ATM e PTEN podem ser úteis na identificação de agressividade e na classificação de prognóstico do CDT.
Abstract: In order to investigate the clinical utility of two tumor-suppressing genes involved in the control of cell proliferation and survival, we've studied proteins ATM and PTEN through immunohistochemistry in 272 differentiated thyroid carcinoma diagnosed patients (142 classical papillary thyroid carcinomas type, 72 papillary thyroid carcinomas follicular variant, 17 papillary thyroid carcinomas tall cells variant and 41 follicular carcinomas), 106 tissue samples from patients diagnosed with benign thyroid diseases (55 follicular thyroid adenomas and 51 with goiter), 18 normal thyroid tissue samples extracted from the counter lateral lobe of individuals operated for follicular thyroid adenomas. The quantitative expression of ATM gene was available for Real-Time PCR in 87 papillary carcinomas. In order to analyze the genotypic profile, we've also studied three ATM polymorphisms (D1853N, S707P e S49C) in 199 patients (164 papillary carcinomas and 35 follicular carcinomas) and 219 control individuals. Patients were accompanied for 53,8±41 months, using a standard protocol. 179 patients were tagged as disease-free and 48 patients had bad outcome (12 deaths). The analysis of multiple logistic regression adjusted for gender and age has showed that the ATM protein expression was more frequent among patients that didn't have metastasis when diagnosed (81%) and that were free of disease (63%) (p=0.016; p=0.0276 respectively). The major incidence of cases who expression PTEN protein, also was observed in patients that didn't have metastasis during follow- up (75%) (p= 0.0437). The heterozygous alleles for D1853N polymorphism was more prevailing among the controls (64%) than in individuals with cancer (36%) (p=0.0364). Nevertheless these results demonstrated that ATM and PTEN protein expression can be useful in identifying patients with aggressiveness and the classification of prognosis in differentiated thyroid carcinoma.
Doutorado
Clinica Medica
Doutor em Clínica Médica

Dissertation (Ph.D.)--University of Toledo, 2008.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: pages 73-77, 93-95, 109-111, 145-172.

Interactions between the retinoic acid receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In acute promyelocytic leukemia (APL), RARalpha is fused with the promyelocytic leukemia (PML) gene, resulting in the expression of the fusion protein PML/RARalpha. Here, I report that topoisomerase II beta (topoIIbeta) associates with and negatively modulates PML/RARalpha and RARalpha transcriptional activity, and increased levels and association of topoIIbeta cause resistance to retinoic acid (RA) in APL cell lines. Knock down of topoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA-gene expression. Overexpression of topoIIbeta, in clones from an RA-sensitive cell line, conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicate that topoIIbeta is bound to an RA-response element, and inhibition of topoIIbeta causes hyper-acetylation of histone 3 at lysine 9 and activation of transcription. These results identify a novel mechanism of resistance in APL and provide further insights to the role of topoIIbeta in gene regulation and differentiation.
Studies to determine the mechanism by which topoIIbeta protein is regulated found that levels of protein kinase C delta (PKCdelta) correlated with topoIIbeta protein expression. Moreover, activation of PKCdelta, by RA or PMA, led to an increase of topoIIbeta protein levels. Most notably, in NB4-MR2 cells, we observed increased phosphorylation levels of threonine 505 on PKCdelta, a marker of activation. Inhibition of PKCdelta was able to overcome the topoIIbeta repressive effects on RA-target genes. In addition, the combination of RA and PKCdelta inhibition led to increased expression of the granulocytic marker, CD11c, in NB4 and NB4-MR2 cells. These results suggest that PKCdelta regulates topoIIbeta expression, and a constitutively active PKCdelta in the NB4-MR2 cell line leads to overexpression of topoIIbeta.
In conclusion, these studies demonstrate that topoIIbeta associates with RARalpha, binds to RAREs and plays a critical role in RA dependent transcriptional regulation and granulocytic differentiation. In addition, I show that topoIIbeta overexpression leads to RA resistance and provide evidence that topoIIbeta protein levels are regulated via a mechanism involving the PKCdelta pathway. This work has contributed to an enhanced understanding of the role of topoIIbeta in gene regulation and brings novel perspectives in the treatment of RA-resistance in APL.

INTRODUÇÃO: A resposta ao tratamento no carcinoma epidermóide de cabeça e pescoço (CECCP) varia significantemente em diferentes casos e muitos pacientes não respondem ao tratamento e são expostos aos seus efeitos. A cisplatina é o quimioterápico mais utilizado no tratamento de CECCP e a quimioradioterapia é o método terapêutico usado nos carcinomas localmente avançado. Neste trabalho foram estudados possíveis marcadores de resposta a quimioradioterapia e sobrevida em pacientes portadores de CECCP, dentre eles a osteopontina (OPN) que tem sido associada à agressividade tumoral em vários cânceres e também tem sido relacionada a sobrevida, formam estudados também alguns polimorfismos em genes que estão relacionados com a cisplatina, seja na detoxificação deste fármaco, Glutationas S transferase (GSTP1, GSTT1 e GSTM1), seja no reparo dos danos causados no DNA pela via de excisão de nucleotídeos (XPD -751 e ERCC 118). CASUÍSTICA E MÉTODOS: Amostras de 69 pacientes localmente avançados submetidos à quimioterapia adjuvante ou exclusiva com cisplatina tiveram a sua OPN dosada pelo imunoensaio elisa em coletas realizadas antes e depois do término do tratamento. Para a análise dos polimorfismos, amostras de 95 pacientes localmente avançados tratados com quimioradioterapia com cisplatina exclusiva foram analisadas por PCR RFLP. RESULTADOS: Com relação à OPN, dos 69 pacientes estudados, a concentração da OPN antes do início da quimioradioterapia no grupo como um todo, foi de 102,5 ng/mL com uma mediana de 82,1 ng/mL. O correspondente valor da OPN após o tratamento n=46 foi de 104,0 ng/mL e mediana de 92,9 ng/mL. A OPN se mostrou mais elevada nos pacientes com maior tamanho tumoral, p=0,009 (ANOVA). Em análises correlacionado reposta ao tratamento e concentração de OPN, observamos que os pacientes que obtiveram resposta completa apresentaram menores níveis de OPN do que aqueles que não responderam ao tratamento. Quando realizamos uma análise multivariada notamos correlação entre baixa OPN antes do tratamento e uma melhor sobrevida global. Na análise dos polimorfismos (n=95), observamos que para os genes de reparo de DNA, XPD e ERCC, o genótipo mais freqüente foi C/T (n=43) e A/A (n=44), respectivamente. Para a GSTP1 a maior freqüência foi de A/G (47,4%) e para a GSTT1 e M1, vimos que a maioria dos pacientes, 83,2% mostrou ter GSTT1 funcional, enquanto 58,9% tiveram GSTM1 não funcional. Neste grupo de pacientes, não notamos nenhuma associação significante entre os genótipos dos pacientes e a resposta a quimioradioterapia, assim como não foi possível uma correlação entre a sobrevida global e os genótipos. CONCLUSÃO: Em síntese, a OPN após o término do tratamento pareceu estar associada com a resposta ao tratamento e com uma melhor sobrevida no grupo estudado e em relação aos polimorfismos, um aumento do número de amostras possa talvez mostrar alguma associação com resposta ao tratamento e a sobrevida global em pacientes com CECCP localmente avançados.
INTRODUCTION: The response to treatment in head and neck squamous cell carcinoma (HNSCC) varies significantly in different cases and many patients do not respond to treatment and are exposed collateral effects. Cisplatin is a chemotherapeutic used to treat HNSCC and chemoradioterapy is the major strategy used in locally advanced carcinomas. In this work, we studied potential markers for chemoradioterapy response and survival in HNSCC patients, such as osteopontin (OPN), which has been associated with tumor aggressiveness and survival. Furthermore, it was studied some genetic polymorphisms related to cisplatin detoxification (glutathione - S transferase, subtypes GSTP1, GSTT1 and GSTM1), as well genes involved in the repair of DNA damage by nucleotide excision (ERCC and XPD -751 - 118). METHODS: Plasmatic OPN levels, before and end of treatment, were measured in 69 patients with locally advanced tumors submitted to adjuvant chemotherapy with cisplatin only by ELISA. For polymorphism analysis, samples from 95 patients with locally advanced tumors treated with cisplatin alone were analyzed by PCR - RFLP. RESULTS: The OPN levels before the chemoradioterapy in the group (n=69) was 102.5 ng/mL with a median of 82.1 ng/mL. The corresponding value of OPN after treatment (n= 46) was 104.0 ng/mL and a median of 92.9 ng/mL. The OPN was higher in patients with larger tumor size, p = 0.009 (ANOVA). In tests correlated to treatment response and concentration of OPN, we observed that patients who achieved complete response had lower levels of OPN than those who did not respond to treatment. The multivariate analysis revealed that lower OPN levels before treatment significant a better overall survival. In the analysis of the polymorphisms (n = 95) the frequency of genotype for the DNA repair genes (XPD and ERCC), was C / T (n = 43) and A / A (n = 44), respectively. The most frequent genotype for GSTP1 was A/ G (47.4%), in 83.2% of the patients, the GSTT1 was functional, while in 58.9% of patients presented GSTM1 non-functional. In this group of patients, there was no any significant association between the genotypes and chemoradiotherapy response nor overall survival. CONCLUSION: In summary, plasmatic OPN levels after treatment cisplatin seemed to be associated with treatment response and better survival. However, larger sample size would be demonstrating some association with treatment response and overall survival in patients with locally advanced HNSCC.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Objetivos: avaliar o desempenho da citologia oncÃtica convencional e da captura de hÃbridos II na detecÃÃo de lesÃes cervicais neoplÃsicas e prÃ-neoplÃsicas. Sujeitos e MÃtodos: foram recrutadas aleatoriamente 1685 mulheres, da demanda espontÃnea de postos de saÃde da rede pÃblica, em cinco municÃpios do estado do CearÃ. As pacientes, apÃs assinarem termo de consentimento, responderam a um questionÃrio prÃ-elaborado e, a seguir, foram submetidas à coleta de material para CO, CH II e à realizaÃÃo de colposcopia, que, sendo positiva, levou à imediata biÃpsia dirigida das Ãreas anormais. Os dados foram digitados no programa Microsoft Excel 2000 e analisados no SPSS-for Windows, versÃo 10.0. O desempenho da CO e CH II foram calculados atravÃs da sensibilidade, especificidade, dos valores preditivos positivo e negativo e dos respectivos intervalos de confianÃa de 95%. Considerou-se, para anÃlise, como padrÃo ouro negativo, o resultado da colposcopia negativo ou resultado negativo no exame histopatolÃgico e, como padrÃo ouro positivo, o resultado positivo do histopatolÃgico. Avaliaram-se dois pontos de corte distintos: qualquer achado prÃ-neoplÃsico e neoplÃsico do colo uterino e achados de lesÃes intra-epiteliais de alto grau ou cÃncer. Resultados: 56 mulheres (3,4%) apresentaram atipias celulares na CO, sendo a CH II positiva em 315 (19%). Embora 337(20,32%) mulheres tenham sido positivas em um dos testes, somente 19(1,1%) foram positivas nos dois. Entre as 150 que tiveram colposcopia positiva somente em 53 foram encontradas lesÃes no exame histopatolÃgico, sendo a prevalÃncia estimada de 3,2% para qualquer lesÃo e de 0,4% para lesÃes de alto grau/cÃncer. Considerando o ponto de corte o achado de qualquer lesÃo prÃ-neoplÃsica ou neoplÃsicas, a sensibilidade encontrada para a CO e a CH II foi de 30,2% e de 71,7%, respectivamente. A especificidade dos testes mencionados foi de 97,5% e de 82,7%. O VPP e VPN da CO foram de 28,6% e de 97,7%, respectivamente. Jà o VPP e VPN da CH foram 12,1% e 98,9%. Considerando o ponto de corte lesÃes de alto grau ou cÃncer, temos: sensibilidade e especificidade da CO de 28,6% e de 99,9%, enquanto os VPP e VPN foram de 54,8% e de 99,7%, respectivamente. A CH II alcanÃou 100% de sensibilidade e 81,3% de especificidade. Os VPP e VPN ficaram em 2,2% e 100%. ConclusÃo: o teste de detecÃÃo do DNA-HPV pela CH II foi mais sensÃvel, porÃm menos especÃfico que a CO. Quando associado à CO, melhora significativamente a detecÃÃo das lesÃes cervicais, principalmente as de alto grau e cÃncer. Para este grupo de lesÃes, a CH II isolada apresentou melhor especificidade sem perda da sensibilidade, mostrando-se um bom teste para o rastreamento primÃrio.
Objective: to compare the usual Pap smear (Papanicolaou) and the Hybrid Capture II tests in detecting cervical intraepithelial neoplasia in women of Ceara State. Subjects and Methods: 1685 women were enrolled from routine practice in five municipalities of the main Cearà State Health Regions. The whole study was explained to the volunteers, who accepted to participate by signing an informed consent form. The study procedures included filling a questionaire and a cervical sample collection, done by a physician, for cytology and HPV-DNA Hybrid Capture, followed by a complete colposcopic evaluation with directed biopsy if necessary. Data were analyzed in Statistical Package for Social Sciences - SPSS - for Windows 10.0. The accuracy of both tests â Pap smear and Hybrid Capture II - was evaluated by using the sensitivity, specificity, positive predictive value, negative predictive value and the respective 95% confidence intervals. The negative colposcopic examination or negative histological result were considered gold standard for negative results. Positive histological results were considered gold standard for positive results. Results: 56 women (3,4%) had abnormal pap smear. Hybrid Capture tests were positive in 315 women (19%). Despite 337 (20,32%) tests had positive results for one of the two tests, only 19 (1,1%) were positive in both tests. Lesions were detected in 53 women among those 150 considered positive in colposcopic examination. The prevalence for any lesion was estimated in 3,2% and for high grade lesions and cancer in 0,4%. Using the cut-off point as the finding of any cervical lesion, the sensitivity of pap smear and HC II was 30,2% and 71,7%, respectively. The specificity for pap smear and HC II was 97,5% and 82,7%, respectively. The positive and negative predictive value for pap smear was 28,6% and 97,7%, respectively. The positive and negative predictive value for HC II was 12,1% and 98,9%, respectively. By using the cut-off value as high grade cervical lesions and cancer, the sensitivity and specificity for pap smear were 28,6% and 99,9%, respectively, and the positive predictive value and negative predictive value for the same test were 54,8% and 99,7%. The sensitivity and specificity for HC II were 100% and 81,3%, respectively, as well as 2,2% and 100% for positive and negative predictive value. Conclusions: hybrid Capture II test was more sensitive than pap smear, however Hybrid Capture II test was less specific than pap smear. When both tests were used together for detecting cervical lesions the results improved significantly, mainly high grade lesion and cancer. For this group of lesions, HC II alone, presented better specificity, without loss of the sensitivity, apparently itâs a good test for primary sceening.

INTRODUÇÃO: O estudo das vias moleculares e das alterações específicas responsáveis pela progressão desfavorável de pacientes com CCR parece essencial para o desenvolvimento de terapias mais efetivas. OBJETIVO: Comparar a expressão quantitativa dos genes TACSTD2, Ku70, KRAS mutante e SERIN1 em amostras de tecidos normal e tumoral de pacientes com CCR e relacionar sua expressão com variáveis clínico-patológicas. MÉTODOS: Foram estudados 37 pacientes com CCR submetidos à ressecção cirúrgica entre julho de 2005 e julho de 2009 e cujas amostras congeladas de tecidos tumoral e normal foram armazenadas em um banco de tecidos. Através da RT-PCR foi sintetizado o cDNA a partir do RNA extraído das amostras teciduais. A expressão dos genes TACSTD2, KRAS mutante, Ku70 e SERIN1 foi quantificada pela técnica de PCR em tempo real. RESULTADOS: A expressão do KRAS mutante foi maior no tecido tumoral do que no normal (p = 0,024). A expressão tumoral dos genes Ku70, TACSTD2 e SERIN1 foi respectivamente menor, igual e maior que o tecido normal, porém sem significância estatística. Associação estatisticamente significativa também foi observada entre idade e expressão de KRAS mutante no tecido normal e tumores pouco diferenciados e expressão de Ku70 no tecido normal. Não foram observadas outras associações estatisticamente significativas. CONCLUSÕES: A expressão do KRAS mutante no tecido tumoral é maior do que no tecido normal (p = 0,024) na casuística de 37 pacientes com CCR estudados através da técnica de PCR em tempo real.
INTRODUCTION: Knowledge of the molecular pathways and of the specific alterations responsible for the unfavorable progression of patients with CCR appears essential for the development of more effective therapies. PURPOSE: To compare the quantitative expression of the genes TACSTD2, mutant KRAS, Ku70 and SERIN1 in samples of normal and tumoral tissues of patients with CCR and to relate their expression to clinicopathologic characteristics. METHODS: 37 patients with CCR were studied. The patients had been operated on between July 2005 and July 2009, and their frozen samples of tumoral and normal tissues had been stored in a tissue bank. The expression of the genes TACSTD2, mutant KRAS, Ku70 and SERIN1 was quantified through the technique of real time polymerase chain reaction. RESULTS: The mutant KRAS expression was higher in the tumoral tissue than in the normal tissue (p = 0,024). Although not significant, the tumoral expression of the genes Ku70, TACSTD2 and SERIN1 was respectively lower, equal to, and higher than in the normal tissue. Statistically significant association was also observed between age and mutant KRAS expression in normal tissue and between poorly-differentiated tumors and Ku70 expression in normal tissue. No other statistically significant associations were observed. CONCLUSIONS: Tumoral tissues express mutant KRAS at higher levels than normal tissues in the casuistic of 37 patients with CCR studied through the technique of PCR real time.

Introdução: Apesar da identificação de fatores prognósticos clínicos e patológicos, muitos pacientes portadores de carcinoma de células renais (CCR) apresentam metástases ao diagnóstico e outros irão desenvolver recorrência local ou à distância durante o seguimento. Novos fatores prognósticos e de origem molecular têm sido avaliados no CCR, destacando-se o PTEN como um dos principais genes envolvidos na carcinogênese renal. Objetivos: Avaliar os fatores clínicos e anatomopatológicos mais significativos nas taxas de sobrevida, identificar a frequência de hipermetilação do gene PTEN através da técnica do pirosequenciamento, o impacto da hipermetilação do gene nas taxas de sobrevida global (SG) e livre de doença (SLD), como também, a associação da presença de hipermetilação com os principais fatores prognóticos. Material e métodos: Foram avaliados 137 pacientes portadores de CCR submetidos a tratamento cirúrgico do tumor primário entre 1997 e 2009. Foram considerados os dados epidemiológicos, clínicos, anatomopatológicos, de estadiamento (TNM 2004) e os obtidos da reação de pirosequenciamento. Resultados: O tempo de seguimento médio foi de 32,3 meses e mediana de 28,8 meses. Considerando o estadimento clínico, foram fatores independentes para a SG: idade (p<0,01), ASA (p=0,02), margens cirúrgicas (p=0,04), grau de Fuhrman (p=0,01), estádio clínico (p<0,001) e subtipo histológico (p<0,01). No modelo múltiplo a SLD foi influenciada únicamente pelo estádio clínico (p<0,001). Dos 137 casos analisados, hipermetilação do gene foi detectada em cinco casos (3,6%). Devido a baixa freqüência detectada optou-se por não realizar a associação da metilação do PTEN com os fatores prognósticos. Em relação às taxas de SG e SLD, de acordo com o perfil de hipermetilação do PTEN, não houve a ocorrência de nenhum evento, ou seja, morte, morte por CCR ou recorrência da doença para os cinco casos que apresentavam hipermetilação. Conclusões: A hipermetilação do xv PTEN foi detectada com baixa frequência, sugerindo a participação de outros genes ou mecanismos moleculares diferentes da metilação na inativação deste gene frequentemente envolvido na carcinogênese renal. As taxas de sobrevida não foram influenciadas pelo perfil de hipermetilação do PTEN, permanecendo o estadiamento clínico do TNM como a principal variável determinante da evolução e do risco de recidiva pelo CCR
Introduction: Despite the identification of clinical and pathological prognostic factors, many patients with renal cell carcinoma (RCC) have metastases at diagnosis and others will develop local or distant recurrence during follow-up. New prognostic factors and of molecular origin have been evaluated in RCC, highlighting PTEN, one of the main genes involved in renal carcinogenesis. Objetives: To assess the most significant clinical and pathological factors in survival rates, and identify the frequency of hypermethylation of the PTEN gene by the pyrosequencing technique, the impact of gene hypermethylation on overall survival (OS) rates and disease free interval (DFS), as well as associating presence of hypermethylation with main prognostic factors. Methods: We evaluated 137 patients with RCC that underwent surgical treatment of primary tumor between 1997 and 2009. We considered the epidemiological, clinical, pathological, staging (TNM 2004) data and those obtained from pyrosequencing. Results: Mean follow-up was of 32.3 months and the median of 28.8 months. Considering the clinical TNM stage, the OS was influenced in the multiple model by age (p < 0.01), ASA (p = 0.02), surgical margins (p = 0.04), Fuhrman´s grade (p = 0,01), clinical stage (p <0.001) and cell subtype (p < 0.01). DFS were influenced in multivariate analysis only by presence of clinical stage (p <0.001). Of the 137 cases examined, gene hypermethylation was detected in five cases (3,6%). Because of this low frequency perceived, we elected not to carry out the association of PTEN methylation with prognostic factors. Regarding OS and DFS rates, according to the hypermethylation of PTEN profile, no event occurred, that is to say death, death from RCC or disease recurrence in the five cases with hypermethylation. Conclusions: Hypermethylation of PTEN was detected with low frequency suggesting involvement of other genes or different molecular mechanisms of methylation upon inactivation of this gene, frequently involved in renal xvii carcinogenesis. Survival rates were not influenced by the hypermethylation of PTEN profile, with clinical TNM staging remaining as the main determinant for development and risk of RCC recurrence

Introdução: A incidência de tumores adrenocorticais em crianças é particularmente elevada nas regiões sudeste e sul do Brasil, correlacionandose com a ocorrência da mutação germinativa p.R337H do supressor tumoral p53, entretanto, o carcinoma adrenocortical é uma neoplasia endócrina maligna rara em todo o mundo com uma incidência aproximada de 0,5 - 2 casos por milhão por ano. Esta condição é uma doença heterogênea, apresentando frequentemente comportamento clínico agressivo e letal. A cascata de sinalização Wnt é uma via importante de transdução de sinal em cânceres humanos e tem sido implicada na tumorigênese adrenocortical. A atividade desta via de sinalização é dependente da quantidade de beta-catenina citoplasmática e nuclear. Mutações ativadoras no gene da beta-catenina (CTNNB1) foram relatadas em diversas neoplasias humanas. Estudos demonstraram que mutações no gene CTNNB1 são os defeitos genéticos mais frequentemente encontrados em adenomas e em carcinomas adrenocorticais. O estudo destas mutações demonstrou que as alterações no gene CTNNB1 localizam-se principalmente exon 3, que codifica a porção amino terminal da beta- catenina. Objetivos: determinar a ocorrência e a frequência das mutações somáticas no exon 3 do gene CTNNB1. Adicionalmente, determinar a imunorreatividade de beta-catenina e de p53 em tumores adrenocorticais benignos e malignos de crianças e adultos. Correlacionar os resultados da análise de mutações gênicas e os dados de imunorreatividade com as características hormonais, a mutação p.R337H do p53, o diagnóstico histológico e a evolução dos tumores adrenocorticais de crianças e adultos. Métodos: Neste estudo, a análise de imunohistoquímica para beta-catenina e p53 foi realizada em 103 tumores adrenocorticais benignos e malignos (40 crianças e 63 adultos), estando as amostras histológicas alocadas em micromatriz tecidual (TMA). A pesquisa de mutações no exon 3 do gene CTNNB1 foi determinada por seqüenciamento automático em 64 tumores adrenocorticais. Resultados: a imunorreatividade para beta-catenina em citoplasma e/ou núcleo foi evidenciada de maneira similar nos tumores adrenocorticais benignos e malignos de crianças e de adultos (15% e 23,8%, respectivamente). O percentual das células neoplásicas imunorreativas para beta-catenina em citoplasma e/ou núcleo não foi significativamente diferente entre os tumores clinicamente benignos e malignos pediátricos (15,6% vs. 12,5%, respectivamente; p=0,93) e entre adenomas e carcinomas adrenocorticais de adultos (28,5% vs. 17,8%, respectivamente; p=0,38). A síndrome endócrina causada pelo perfil de secreção hormonal foi similar entre os tumores adrenocorticais com presença ou ausência de acúmulo citoplasmático e/ou nuclear de beta-catenina em crianças e adultos. A associação entre acúmulo anormal de beta-catenina e diminuição de sobrevida foi avaliada nos pacientes adultos portadores de carcinomas adrenocorticais isoladamente (n=25), sendo observada na curva de Kaplan- Meier uma tendência de significância (log-rank p=0,07). A análise do gene CTNNB1 revelou mutações somáticas em heterozigose em 10 tumores adrenocorticais (4 crianças e 6 adultos). As mutações encontradas no gene CTNNB1 foram, sobretudo do tipo missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Outras mutações encontradas compreenderam: a inserção de um único nucleotídeo (p.E9GfsX14), dando origem a uma desvio de leitura do exon 3; além da deleção dos três nucleotídeos do códon 45 (p.Ser45del). Todos os tumores com mutações somáticas no gene CTNNB1 mostraram acúmulo anormal para ?-catenina, com exceção de um caso. A presença de alterações no gene CTNNB1 não se associou ao tamanho do tumor (Teste de Mann-Whitney: p=0,75), desfecho desfavorável tanto no grupo pediátrico (log-rank p=0,29) como no grupo de pacientes adultos (log-rank p=0,77). Todos os pacientes portadores da mutação germinativa do gene TP53 apresentaram imunorreatividade nuclear de p53 nas células tumorais. Não foi encontrada correlação entre a presença de acúmulo anormal de beta-catenina e imunorreatividade nuclear de p53, considerando os grupos de crianças e de adultos portadores de tumores adrenocorticais. Adicionalmente, não foi observada correlação entre mutações no gene CTNNB1, bem como acúmulo anormal de beta-catenina, com a imunorreatividade nuclear de p53 no grupo de tumores adrenocorticais de pacientes adultos, porém, interessantemente, avaliando isoladamente o grupo de tumores adrenocorticais pediátricos, foi observada relação entre a presença de mutações no gene CTNNB1 e a presença de acúmulo nuclear de p53 (X2: p=0,009). Conclusões: Estes dados confirmam a participação da via Wnt na tumorigênese adrenocortical de crianças e de adultos, que apresenta uma prevalência de ativação semelhante entre crianças e adultos. O acúmulo citoplasmático e/ou nuclear de beta-catenina provavelmente é um marcador biológico de mau prognóstico do carcinoma de adrenocortical de adultos. Adicionalmente, observamos evidências de uma correlação positiva entre mutações no gene CTNNB1 e acúmulo nuclear de p53 em tumores adrenocorticais pediátricos, confirmando uma possível relação destas duas vias na tumorigênese do córtex da glândula suprarrenal
Introduction: The incidence of adrenocortical tumors in children is particularly high in the southeastern and southern regions of Brazil, correlating with the occurrence of p.R337H p53 tumor suppressor germline mutation. However, adrenocortical carcinoma is a worldwide rare endocrine malignancy with an approximate incidence of 0.5 to 2 cases per million per year. This condition is a heterogeneous disease and is often lethal. The Wnt signaling pathway is an important signal transduction pathway in human cancers and has been implicated in adrenocortical tumorigenesis. The activity of this signaling pathway is dependent on the amount of nuclear and cytoplasmic beta-catenin. Activating mutations of ?-catenin (CTNNB1) gene have been reported in several human malignancies. Studies have shown that CTNNB1 mutations are the most common genetic defect found in adrenocortical adenomas and carcinomas. The study of these mutations demonstrated that the changes in CTNNB1 gene are mainly located in exon 3, which encodes the amino terminal portion of the beta- catenin. Objectives: to determine the occurrence and frequency of CTNNB1 somatic mutations and the abnormal beta-catenin and p53 accumulation in benign and malignant adrenocortical tumors in both children and adults. We also evaluated the correlation of the gene mutations analysis and immunohistochemistry data with the hormonal characteristics, the p.R337H germline mutation, the histological diagnosis and the prognosis of adrenocortical tumors in children and adults. Methods: In this study, immunohistochemistry for beta-catenin and p53 was performed in 103 benign and malignant (40 children and 63 adults) adrenocortical tumors. The histological samples were allocated in a tissue microarray (TMA). The study of the CTNNB1 gene was performed by direct sequencing of 64 adrenocortical tumors. Results: The beta-catenin abnormal accumulation was similar in benign and malignant adrenocortical tumors of children and adults (15 % and 23.8 %, respectively). The percentage of cells with beta-catenin abnormal accumulation was not significantly different between benign and malignant pediatric adrenocortical tumors (15.6% vs. 12.5 %, respectively; P=0.93) and between adrenocortical adenomas and carcinomas in adults (28.5% vs 17.8 %, respectively; p=0.38). The endocrine syndrome caused by hormonal tumor secretion was similar in patients with and without beta-catenin abnormal accumulation both in pediatric and adult patients. The association between beta-catenin abnormal accumulation and decreased survival was evaluated in adult patients with adrenocortical carcinomas (n=25) and a trend toward significance was observed (log-rank p=0,07). The analysis of the CTNNB1 gene revealed heterozygous somatic mutations in 10 adrenocortical tumors (6 adults and 4 children). The mutations found in CTNNB1 gene were mainly missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Other mutations found included: a single nucleotide insertion (p.E9GfsX14) and a deletion within codon 45 of exon 3 of CTNNB1 gene, (p.Ser45del). All tumors with somatic mutations in the CTNNB1 gene showed abnormal beta -catenin accumulation, except for one case. The mutations in CTNNB1 gene was not associated with tumor size (Mann - Whitney: p=0.75), unfavorable outcome in both pediatric (log -rank p=0.29) and adult group of patients (log-rank p=0.77). All patients with TP53 germline mutation showed p53 nuclear accumulation in the tumor cells. No correlation was found between the presence of beta-catenin abnormal accumulation and p53 nuclear accumulation in adrenocortical tumor cells of children and adults. In addition, no correlation was observed between CTNNB1 mutations, as well as beta-catenin abnormal accumulation, with p53 nuclear accumulation in adults adrenocortical tumors. Interestingly, the evaluation of pediatric adrenocortical tumors revealed a relationship between the occurrence of CTNNB1 mutations and the presence of p53 nuclear accumulation (X2: p=0.009). Conclusions: These data confirm the involvement of the Wnt pathway in adrenocortical tumorigenesis of children and adults, which has a prevalence similar activation between children and adults. We observed that abnormal beta-catenin accumulation in adults adrenocortical carcinoma is probably associated with a dismal prognosis. Additionally, we found evidence of a positive relationship between CTNNB1 mutations and p53 nuclear accumulation in pediatric adrenocortical tumors, confirming a possible connection of these two pathways in the pediatric adrenocortical tumorigenesis

DNA methylation is increasingly recognised as a significant epigenetic event that may initiate and drive the process of neoplasia in humans. In the colon, DNA methylation of key genes is common in a subset of colorectal cancers. The extent to which DNA methylation at various genes contributes to initiation of colorectal neoplasms is less clear. This study sought to clarify the biological and clinicopathological significance of methylation of various genes in the development of sporadic and familial colorectal neoplasia. Quantitative methylation-specific PCR (qMSP) assays (capable of detecting down to a measureable proportion of 0.1% of the total input DNA) were developed to determine the presence of CpG methylation at a given gene. Methylation of MLH1-C was found in the apparently normal mucosa samples from seven of 104 (7%) of individuals with sporadic colorectal cancer (CRC) showing microsatellite instability (MSI). No methylation of MLH1-C was found in the biological samples of individuals with microsatellite stable (MSS) counterparts (n=131). MLH1-C methylation may be a field defect that predisposes to the development of sporadic colorectal neoplasia, particularly those demonstrating MSI. Methylation of three of five genes within the 3p22 region including AB002340, MLH1, ITGA9, PLCD1 and DLEC1 (regional 3p22 methylation) was found in 83% of sporadic MSI (n=86) and 12% of MSS cancers demonstrating BRAF V600E mutation (n=42). Regional 3p22 correlated strongly with CpG island methylator phenotype (CIMP), and other clinicopathological characteristics typical of CIMP. Thus, regional 3p22 methylation and CIMP may be overlapping phenomena. Regional 3p22 methylation and the BRAF V600E mutation were found in normal colonic mucosa of four individuals with sporadic MSI CRC, and these cases also had multiple synchronous serrated polyps. These molecular aberrancies may predispose some individuals to the development of metachronous serrated neoplasia. Germline epimutations of APC do not contribute towards the development of FAP, AFAP, or hyperplastic polyposis syndromes. However, APC methylation in normal colonic mucosa of these individuals may represent a field defect in the development of futher neoplasms. In conclusion, different patterns of DNA methylation in normal colonic mucosa may represent a field defect important in the development of different subtypes of colorectal neoplasia.

I optimized a microarray method for identifying genome-wide CpG island methylation changes called Microarray-based Methylation Assessment of Single Samples (MMASS). A prospective sample set of matched normal colorectal mucosa, adenoma and adenocarcinoma tissues were collected as well as a retrospective set of primary colorectal carcinomas and where present their metastatic tissues. All cases were assayed for microsatellite instability. Genome-wide methylation patterns were profiled using MMASS and were correlated with their corresponding genome-wide expression changes for a subset of the cases. Differentially methylated and expressed targets were identified during neoplastic progression of CRC. DNA methylation was measured in a set of 10 CpG islands known to be important in CRC, using quantitative pyrosequencing assays, in 261 tissue samples from 138 colectomy specimens removed for CRC and 9 for diverticular disease. Recursive partitioning (RP) was used to identify threshold methylation levels that discriminated normal tissue from neoplastic tissues with 100% sensitivity (95% CI: 93.2-100.0) and 90.5% specificity (95% CI: 69.6-98.8). DNA methyltransferase DNMT3B has been implicated in the progression of colorectal neoplasia. Immunohistochemical analysis of the expression of DNMT3B was positively correlated with methylation of SFRP2 (r = 0.415, p ≤ 0.001) and negatively correlated with methylation of IGF2 DMR0 (r = -0.262, p = 0.014). SFRP2 and IGF2 CpG islands methylation changes are proposed to be potential biomarkers for CRC screening. Faecal DNA can be tested for methylation levels of SFRP2adn IGF2 to identify high risk patients. The UK NHS bowel cancer screening programme is the ideal environment in which to take these findings forward.

Introduction: Colorectal cancer is a major clinical and public health problem. Early stage colorectal cancer is amenable to surgical intervention with a high cure rate. Screening for the disease has now been rolled out throughout the UK, although current screening modality of faecal occult blood testing (FOBT) is suboptimal. Specific genetic alterations found in primary tumours, including mutations in proto-oncogenes, microsatellite instability and loss of heterozygosity (LOH), have been shown to be detectable in the host plasma. Colorectal cancer is an excellent paradigm in which to investigate the application of assays to detect tumour-related plasma DNA. There is substantial data regarding specific mutations and their frequency in colorectal cancer tissue and adenomas. The ability to detect early stage colorectal neoplasia using DNA plasma assays holds considerable promise as a non-invasive screening modality. Materials and Methods: Assay performance was assessed using colorectal cancer cell lines and archived material from genetic studies. Assays were then applied to a prospective cohort of 124 colorectal neoplasia cases and controls. Plasma DNA was quantified using a sensitive DNA binding fluorescent dye and detection platform. A well-characterised tumour-specific mutation was used as the target for the first assay. This mutation was in a poly A tract of the transforming growth factor beta receptor II (TGFpRII) gene and was assessed using a restriction fragment length polymorphism (RFLP) assay. Microsatellite analysis was performed using 3 polymorphic markers relevant to colorectal neoplasia in matched tumour, normal and plasma DNA samples. A novel assay was developed exploiting real-time fluorescent PCR amplification of single nucleotide polymorphisms in the adenomatous polyposis coli (APC) gene as a means to detect tumour-specific allelic imbalance. Assay performance was determined in spiking experiments, and performance assessed in clinical samples. A further iteration of the assay was developed to quantify tumour specific alleles in plasma by counting individual alleles PCR amplified from plasma DNA of cases and controls. Results: Quantification of total plasma DNA revealed a significant difference between cases and controls (area under the receiver operator curve: 0.7). Despite intensive efforts to overcome the problem, the TGFpRII RFLP assay was affected by technical difficulties when applied to plasma DNA. The required high number of PCR cycles introduced artefact and limited its applicability. Microsatellite analysis demonstrated LOH in matched tumour and plasma samples with maximal sensitivity of 67% but specificity was only 32%. The quantitative PCR assay to detect APC LOH was able to detect 3-5ng of homozygous DNA introduced into 1 ml of heterozygous plasma, and accurately quantified LOH in tumour tissue. However, it was not able to discriminate cases from controls by assessment of plasma DNA. Counting of alleles in plasma DNA from a test case demonstrated matching LOH to that seen in the primary tumour. Allele counting of a further cohort suggested that this approach has a low false positive rate. Discussion: Analysis of plasma DNA is technically challenging due to a low abundance of partially fragmented mutant DNA sequences admixed with normal DNA in plasma with DNAases and PCR inhibitors. However total quantity of plasma DNA was higher in cancer cases compared to controls with good specificity for cancer at high DNA concentrations. Fluorescent microsatellite analysis of plasma DNA demonstrated encouraging overall sensitivity but poor specificity that waslikely at least partly a reflection of low DNA abundance and hence sampling error within aliquots of plasma DNA. A quantitative PCR approach was developed and validated with relevant levels of in vitro sensitivity, and improved discrimination of LOH in some clinical samples. Adaptation of this approach to count alleles individually is labour intensive and expensive but appears to address issues of sampling error due to abundance and hence improves specificity. These data, whilst highlighting some of the technical challenges in the field, demonstrate associations of plasma DNA in colorectal neoplasia that warrant further investigation.

ChlR1 is a DNA helicase implicated in diverse cellular processes including sister chromatid cohesion and DNA replication and repair. However, the mechanism by which ChlR1 participates in these processes is unknown. Data presented in this thesis show that siRNA-mediated depletion of ChlR1 causes increased sensitivity to chemically-induced replication stress. Treatment of ChlR1-depleted cells with hydroxyurea results in increased mono-ubiquitination of PCNA and increased chromatin-associated RPA, indicating stalled DNA replication. Furthermore, ChlR1 is recruited to chromatin following hydroxyurea treatment, supporting a role in the stabilisation of forks during replication stress. Fibroblasts derived from a Warsaw Breakage Syndrome (WABS) patient caused by mutation of ChlR1 (G57R) have both defective sister chromatid cohesion and G2 checkpoint following radiation-induced damage. Complementation with wild-type ChlR1 rescued this mutant phenotype while a known helicase dead mutant of ChlR1 (K50R) or the WABS-associated mutants G57R or ΔK897 did not. However, increased and prolonged Chk1 activation was observed in both K50R and ΔK897 complemented cells after treatment with hydroxyurea while the G57R was comparable to wild-type. These data suggest that the novel WABS mutation (G57R) may retain some wild-type ChlR1 activity and offer important insight into the molecular basis of the WABS phenotype.

New massively parallel sequencing technologies offer opportunities to profile genomes and transcriptomes for copy number variations, polymorphisms, somatic point mutations, chromosomal rearrangements and can capture gene expression and splicing information. A suite of methods was developed to analyze both RNA-seq and whole genome/exome sequence data from malignant cells for the purpose of identifying somatic point mutations and fusion transcripts. This work reports the application of these and other tools to gain insights into the somatic mutations involved in two common classes of lymphoid malignancies, namely non Hodgkin lymphoma and acute lymphoblastic leukemia. Analysis of multiple cases by a combination of RNA-seq, genome and exome sequencing revealed genes significantly mutated in non Hodgkin lymphoma including many not previously known to be mutated in these or any other cancers. These included multiple genes involved in altering the methylation or acetylation state of histones such as EZH2, MLL2, CREBBP and MEF2B, suggesting a previously unappreciated role of deregulated or altered epigenetic gene regulation in lymphomagenesis. Some of the mutated genes, such as MLL2, had clear patterns of inactivating mutations, indicating they act as tumour suppressors in NHL. Others had mutation hot spots that can be indicative of an oncogenic gain of function and this was proven to be the case for the mutation hot spot identified in EZH2. Analysis of acute lymphoblastic leukemia revealed both novel point mutations and fusion transcripts. The latter included fusions that potentially deregulate known oncogenes such as JAK2 and ABL1. These data may indicate new treatment options for patients with ALL and NHL and lend new insights into the molecular nature of these diseases.

The heterogeneity of salivary gland neoplasms have made classification and prognostication of these tumours sometimes difficult, and the in­troduction of techniques, such as enzyme and carbohydrate histochemis­try and electron microscopy have only to a certain extent increased our knowledge in these respects. In the present study immunohistochemical methods have been used to identify intermediate filament proteins (IFP) in normal fetal and adult parotid glands, as well as in salivary neo­plasms. The intermediate filaments (IF) make up the cytoskeleton in eucaryotic cells. Epithelial tissue contains IF composed of different cytokeratins (CK 1-19) whilst mesenchymal tissue generally contains IF composed of vimentin, and the IFP pattern is very stable even during cell transformation. It would thus be possible to further clarify the histogenesis of salivary neoplasms by identifying IFP, in addition the IFP pattern would probably be useful in tumour typing. Furthermore, ultrastructural cytochemical studies, microspectorphotometry on nuclear DNA as well as enzyme secretory studies of certain tumour types were carried out, in order to further characterize the biology of salivary neoplasms. The immunohistochemical investigations showed that in normal parotid tissue, the different cell types differed in IFP expression: acinar cells express mainly CK 18 and myoepithelial cells mainly CK 17 and 19, whilst duct cells contained a broad range of CK. Vimentin could in ad­dition to CK be detected in myoepithelial cells and basal cells of ex­cretory ducts. Fetal parotid cells showed a similar CK pattern as mature duct cells. In addition, vimentin could be found in some basal cells of the terminal tubules of the fetal glands. Salivary neoplasms could be divided into three types with regard to their IFP pattern:  Acinic cell carcinomas showed a CK-pattern similar to normal acinar cells but a co-expression of CK and vimentin was present in some cells.  Adenoid cystic carcinomas, mixed tumours and basal cell adenomas showed a CK-pattern of normal duct or myoepithelial cells. The peri­pheral cells were also vimentin positive. 3. Mucoepidermoid carcinomas and adenocarcinomas had a similar CK-pattern as duct cells, and no tu­mour cells contained vimentin. This indicates that typing of IFP may be useful for subgrouping of salivary neoplasms. By stereological measurements, the cells of acinic cell carcinomas were found to be very similar to normal parotid acinar cells. Furthermore, they contained amylase and after stimulation by norepiphrine a secre­tory response was induced, with a rise in intracellular cAMP as well as a release of amylase. By single cell measurements of nuclear DNA con­tent, no difference was found between acinic cell carcinomas with de­finite metastasis and those without recurrence, both in paraffin sec­tions and cytological smears.
digitalisering@umu.se

Mitochondria are cytoplasmic organelles that are found in almost all mammalian cells. Mitochondria contain their own genome, mitochondrial DNA (mtDNA) that encodes 13 subunits of the electron transfer chain, which is the primary generator of cellular energy. Precise regulation of mtDNA copy number is essential for normal cell function and also the differentiation of stem cells into specialized cell types. Abnormal regulation of mtDNA copy number is associated with cellular dysfunction, mitochondrial disease and more recently cancer. Glioblastoma multiforme (GBM) is a highly malignant subgroup of brain tumors that exhibit similar characteristics to human neural stem cells (hNSCs) including multipotency and the expression of the stem cell factors. It is unknown how GBM cells regulate their mtDNA copy number during differentiation and whether this differs to hNSCs. Furthermore, it is unknown what role mtDNA plays in the gene expression profiles and the tumorigenicity of GBM. To address these issues, GBM cells and hNSCs were differentiated for 28 days and their mtDNA copy number and gene expression were analyzed. In addition, GBM cells were progressively depleted of their mtDNA using the depletion agent, 2'-3'-dideoxycytidine, and their in vivo tumorigenicity assessed. hNSCs and GBM cell lines regulated their copy number in a differential manner during differentiation. hNSCs progressively expanded their mtDNA copy number and adopted a differentiated phenotype whilst GBM cells failed to mimic these processes and their differentiation was incomplete. In addition, progressive depletion of mtDNA copy number in GBM cells resulted in reduced proliferation rates and the down regulation of stem cell factors. In vivo, mtDNA depleted GBM cells formed tumors at a reduced rate and frequency relative to nondepleted cells. These outcomes demonstrate that mtDNA copy number is abnormally regulated in GBM cells and hinders their ability to complete differentiation. The failure of mtDNA-depleted GBM cells to consistently generate tumors strongly suggests that maintenance of mtDNA copy number is essential for GBM cells to be tumorigenic.

DNA methylation helps regulate transcriptional activity and is widely studied in cancer biology. This investigation aimed to establish the significance of Human Papillomavirus (HPV) DNA methylation in HPV-associated disease both in terms of basic biology and as a potential biomarker. Assays to assess DNA methylation and gene expression were developed and evaluated. Pyrosequencing was used to assess DNA methylation of four regions of the HPV16 genome (E2, L1/L2, enhancer, promoter). Gene expression was assessed using quantitative PCR with assays for E2, E6 and E7. HPV integration was assessed using Detection of Integrated Papillomavirus Sequences (DIPS). The relationship between HPV methylation, gene expression and integration was explored in vitro and in vivo using cell cultures and clinical cohorts. A variety of sample materials were used including short term and immortal cell lines, cervical cancer biopsies, cytology samples and Vulval Intraepithelial Neoplasia (VIN) biopsies. In general, hypermethylation of the HPV genome was associated with low HPV gene expression and the presence of integrated HPV genomes. To better understand the potential clinical utility of HPV DNA methylation, the relationship between HPV DNA methylation and various stages of cervical disease was determined. The HPV genome was progressively hypermethylated with increasing severity of cervical disease and certain regions of the HPV genome were more affected than others. A longitudinal study was also performed in order to determine a relationship between HPV methylation and clinical outcome. Differences in HPV methylation among patients who had persistent HPV infection and low grade disease, persistent infection and high grade disease and patients that cleared HPV infections were observed. Throughout the study the potential application of a HPV biomarker was considered and the correct biomarker design procedures were referred to. Several of the early biomarker development steps were successfully achieved.

In the course of this study it was established that CtlP associates in vivo with, and directly binds to, the DNA damage mediator proteins containing BRCT domains: 53BP1, MDC1, TopBP1 and NBS1. The binding sites on all of these proteins have been mapped establishing which regions of CtlP interact directly with 53BP1, MDC1, TopBP1 and NBS1 and vice versa. This implies that CtlP is involved in the DNA damage response on many levels interacting with proteins that mediate different aspects of DNA damage sensing and signalling. CtIP's nuclear localization before and after DNA damage was determined and its colocalization with the other DNA damaged responsive proteins examined. It was found that CtIP associates in vivo with DNA damage sensor proteins such as the MRN complex (Mre11, Rad50 and NBS1) and RPA70, signal transducer proteins such as the PIKK kinases ATM, ATR and SMG1 and the mediator proteins 53BP1 and MDC1. All of those proteins are involved in detecting and repairing double stranded or single stranded lesions. Moreover CtIP has a major influence on phosphorylation events induced during repair processes and thus could be an inducer of kinase activity. The deletion of CtlP causes impairment of phosphorylation of important proteins that take part in the DNA damage response pathways such as NBS1, ATM, 53BP1, Chk1 and RPA70. However, impaired phosphorylation only occurs in response to single stranded DNA lesions or the collapse of replication forks. This implies that it is the ATR pathway that is malfunctioning in CtIP depleted cells. CtIP was previously reported to be phosphorylated on Ser664 and Ser745 by the ATM kinase in response to the DNA double stranded breaks. I have established two possible new phosphorylation sites: Ser506 and Ser555. They could be potentially involved in CtlP's redistribution to the DNA damage sites but this remains to be elucidated.

Germ cell tumours (GeTs)affect both paediatric and adult populations, and can occur either in gonadal or extragonadal regions along the body's ventral midline. These tumours can be broadly categorized into two subgroups, seminomatous (SEM) or nonseminomatous (N-SEM). The latter can be further subcategorized into embryonal carcinoma (EC), teratoma, yolk sac tumour (YST) and choriocarcinoma (eC) according to their differentiation. As in many other tumours, DNA methylation has been proposed to be involved in GCTdevelopment. However to date, most studies were performed using adult testicular GCTs. Furthermore, these studies only include a handful of genes in their analysis. Thus, the roles of DNA methylation in paediatric and extragonadal GeTs have not been explored. Therefore, this project attempted to fill this gap in knowledge by performing methylation analysis in a cohort of paediatric GCT samples and GCTcell lines. Although paediatric GCTsmostly consist of teratomas, seminomas or YSTs, only the latter two were included in the methylation analysis as they were the only samples in the available tumour bank. Using the methylation level of L1NE-l repeat elements as a measurement of global genome methylation, we found that both paediatric seminoma and YST samples displayed global hypomethylation as compared to somatic controls. However, when methylation at gene promoter regions was investigated using Illumina Golden Gate methylation arrays, seminoma and YST exhibited very different methylation features. YSTs were found to be highly methylated at many of the sites investigated. Surprlslnglv, we found that the methylation features in seminoma were similar to the somatic controls. From this analysis, we identified 85 genes that were differentially methylated in the VSTs. However, by correlating our methylation data with the expression array data performed by our collaborators on the same samples, only eight of these genes (PYCARO, CASPB, C02, HOAC9, TFAP2C, ETV1, EV/2A, HLA-F) were differentially expressed. As in previous GCTstudies, our analysis was focused on the methylation at epG islands. During the course of this project technological advancement led to the creation of new methylation arrays that offer wider genome coverage. One example is the Infinium Methylation 450K array that covers more than 450,000 CpG sites and includes regions flanking the CpG islands such as the CpG shores and CpG shelves. Since no previous GCT studies have attempted to investigate methylation in those regions, we utilized this methylation array on four GCTcell lines; TCAM2 (seminoma), NT2Dl (teratocarcinoma), GCT27 (embryonal carcinoma) and GCT44 (yolk sactumour). Similar to previous GCT studies, we found that nonseminomatous GCT cell lines displayed higher methylation at the CpG islands as compared to the seminoma cell lines. Strikingly, expanding our analysis to other regions (CpG shores and shelves etc.) revealed that each GCT subtype exhibited distinct methylation features. Both ECand teratoma cell lines displayed higher methylation than the seminoma and YST cell lines at all regions. Interestingly, the YST cell line only showed higher methylation than the seminoma cell line at the CpG islands and to a lesser extent at the CpG shores while the seminoma cell line exhibited higher methylation at the CpG shelves as compared to the YST cell line. This is the first time such features have been reported for GCTs. From this Infinium methylation data, we have also identified a high number of hypermethylated genes including those that are uniquely methylated for each cell line. By correlating this methylation data with Affymetrix gene expression data, 98 genes that were differentially methylated and differentially expressed in the YST cell line have been identified. However, further analysis needs to be performed to understand the role of these genes in YST development. As in other types of tumour, the hypermethylation observed in the YST cell line might be caused by many epigenetic modifiers. Using real-time RT-PCR on three epigenetic modifiers (DNMT38, EZH2, SUZ12), we found that DNMT38 was highly expressed in the YST samples and cell line as compared to the seminoma samples and cell line. This suggests that DNMT38 might contribute to YST hypermethylation and resulting differences in their biology. However, knockdown of DNA methyltransferases (DNMTs) and DNMT38 using 5-azadeoxycytidine and microRNA-29b respectively, did not seem to have any effect on the response of all four GCT cell lines towards cisplatin. On the other hand, both knockdowns only caused little effect on cell migration; affecting only the seminoma and YST cell lines. Nonetheless, further analysis is still needed to fully assess the role of DNA methylation in regulating cell behaviour. In summary, paediatric YSTs displayed hypermethylation at many promoter regions as compared to seminomas. Meanwhile, methylation analysis at regions outside of CpG islands in GCTcell lines revealed unique methylation features for each GCT subtype which might indicate different underlying mechanisms in their development. Further analysis on genes found to be differentially methylated and differentially expressed in both paediatric and GCTcell lines are now needed to fully establish their role in GCT development.

Thesis (Ph.D.) - University of Glasgow, 2007.
Ph.D. thesis submitted to the Faculty of Medicine, Division of Cancer Sciences and Molecular Pathology, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Casein Kinase 2 (CK2) is a ubiquitous serine/threonine kinase. Due to its pleiotropic nature CK2 is involved in a multitude of cellular pathways including cell survival, proliferation and apoptosis. Therefore it has come as no surprise that a requirement for CK2 activity has been identified during the repair of DNA damage. Here we have described a novel role for CK2 in the response to DNA double-strand breaks. We have shown the mediator of DNA damage checkpoint 1 (MDC1) is constitutively phosphorylated by CK2, which is required for an interaction with the MRE11/RAD50/NBS1 (MRN) complex, via the FHA domain of NBS1. Moreover, disruption of this interaction resulted in loss of MRN foci following ionizing radiation and a partial G2/M checkpoint defect. Furthermore, the identification of three siblings presenting NBS/Seckel-like phenotypes with a unique mutation in the BRCT domain of NBS1, that phenocopied some of our observations, provided additional evidence for the importance of phospho-dependent interactions within the cell. Lastly, the identification of putative CK2 target residues in MRE11 and our preliminary data suggest that the kinase may play further roles in regulating the activity of the MRN complex that lie outside its activity in DNA double-strand break repair.

Autophagy is an evolutionarily conserved process that is important for the maintenance of cellular homeostasis and also genomic integrity. Autophagy is a self-digestive process that takes place in the cytoplasm, however recent studies by Eileen White and colleagues demonstrated that defective autophagy leads to accumulation of DNA damage in vitro and in vivo [1] [2]. This discovery leads to a question: whether there is increased DNA damage incidences or defective DNA damage repair in autophagy deficient cells. Autophagy and DNA damage are two important areas for cancer research. The aim of this project is to provide a better understanding of the role of autophagy plays in DNA damage and DNA damage response. The activation of Chk1 facilitates its degradation. The results presented in this project illustrate that autophagy deficient cells exhibit elevated proteasomal activities and autophagy inhibition leads to activation of Chk1. These combined factors contribute to increased degradation of Chk1 in autophagy deficient cells. This was manifested first as decreased phospho-Chk1 in response to DNA damage, later on when the loss of autophagy effect is more pronounced; decrease in total Chk1 protein level was observed. Chk1 is a crucial DNA damage response mediator that plays roles in cell cycle checkpoints and DNA damage repair. Cells without autophagy appear to have intact cell cycle checkpoints in response to starvation or DNA damaging agents; however they show deficiency in homologous recombination (HR) repair pathways. Autophagy deficient cells display increased spontaneous cell death and formation of micronuclei. Defective HR pathways in autophagy deficient cells lead to hyper-dependency on non-homologous end-joining (NHEJ) process. Since HR and NHEJ are the two main ways of repairing double strand breaks (DSB), it is not surprising that inhibition of NHEJ following DSB inducing agents in autophagy deficient cells results in persistence of damage lesions and increased cell death. 3 This project demonstrated that loss or inhibition of autophagy leads to defective DNA damage response pathways. We established that Chk1 is de-regulated in autophagy deficient cells and this has differential downstream effects on DNA damage response. These findings potentially provide a novel synthetic lethal strategy to selectively kill autophagy-deficient cells, which are implicated in a number of diseases including certain cancers.

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Os tumores cerebrais primários mais comuns são denominados gliomas. Eles são definidos patologicamente pela presença de características histológicas e imuno-histoquímicas que evidenciam diferenciação glial. De acordo com a suposta linhagem de origem, eles são classificados como astrocitomas, oligodendrogliomas ou ependimomas. Dentre eles, os astrocitomas são os mais comuns e agressivos. O tratamento atualmente utilizado inclui remoção cirúrgica seguida de quimioterapia com temozolamida (TMZ) e radioterapia, porém sua eficácia é muito baixa devido à alta resistência das células tumorais. Buscando encontrar genes associados com a elevada resistência dos astrocitomas, realizamos um estudo anterior de expressão gênica diferencial utilizando uma coleção de genes de reparo de DNA. Nesta análise foram identificados sete genes significantemente superexpressos em glioblastoma multiforme (GBM), o tipo mais agressivo de astrocitoma. Estes genes são: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L e XRCC2. Através de RT-PCR quantitativo, avaliamos os níveis de expressão destes genes em um painel expandido de 54 casos clínicos de astrocitomas de diferentes graus de malignidade e em 5 linhagens celulares de GBM. Todos os genes analisados mostraram-se mais expressos nos astrocitomas, com exceção de RAD54L em amostras de astrocitoma de grau II. Além disso, a superexpressão dos 7 genes avaliada isoladamente não exerce influência direta na sobrevida dos pacientes. Evidenciou-se ainda a superexpressão mais acentuada de EXO1 e NEIL3, que foram selecionados para realização de ensaios funcionais de silenciamento, e avaliação do ciclo celular e taxas de apoptose/morte efetiva das células. Estes ensaios foram realizados com as linhagens celulares T98G e U138MG, que apresentaram maiores níveis de expressão destes genes. Nos ensaios funcionais, observamos que o silenciamento...
Gliomas are the most common type of primary brain cancers. They are pathologically defined by the presence of histological and immunehistochemical characteristics that evidence glial differentiation. According to the hypothetical cell of origin they are classified in: astrocytomas, oligodendrogliomas and ependimomas. Among them, astrocytomas are the more common and aggressive type. The treatment currently used for GBM includes surgical resection of tumor followed by chemotherapy with temozolamide (TMZ) and radiotherapy, but this protocol is still insufficient due to the high resistance of cancer cells. Searching for repair genes associated with the high resistance of astrocytomas, we developed a previous study of differential gene expression using a collection of DNA repair genes. In this analysis, we identified seven genes significantly overexpressed in glioblastoma multiforme (GBM), namely: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L and XRCC2. Using quantitative RT-PCR, we evaluated the expression of these genes in an expanded panel of samples with 54 clinical cases of different grade astrocytomas and five GBM cell lines. All genes showed expression significantly higher in astrocytomas, except RAD54L in grade II astrocytomas. Moreover, the overexpression of this 7 genes evaluated individually doesn't exert direct influence upon patient's survival rate. Remarkably, EXO1 and NEIL3 showed the higher fold changes and were chosen for functional silencing assays. This experiments were performed with T98G and U138MG cell lines that showed the higher expression levels among the GBM cell lines analyzed. In the functional assays, we observed that the silencing of EXO1 or NEIL3 doesn't induce changes in the apoptosis and cell death rates and doesn't change the distribution of cells in cycle. Beyond this, the silencing of this two genes doesn't sentisizes cells to ionizing radiation.

Orientador : Valeria Valente
Banca: Cleslei Fernando Zanelli
Banca: Ana Lúcia Fachin Saltoratto
Resumo: Os tumores cerebrais primários mais comuns são denominados gliomas. Eles são definidos patologicamente pela presença de características histológicas e imuno-histoquímicas que evidenciam diferenciação glial. De acordo com a suposta linhagem de origem, eles são classificados como astrocitomas, oligodendrogliomas ou ependimomas. Dentre eles, os astrocitomas são os mais comuns e agressivos. O tratamento atualmente utilizado inclui remoção cirúrgica seguida de quimioterapia com temozolamida (TMZ) e radioterapia, porém sua eficácia é muito baixa devido à alta resistência das células tumorais. Buscando encontrar genes associados com a elevada resistência dos astrocitomas, realizamos um estudo anterior de expressão gênica diferencial utilizando uma coleção de genes de reparo de DNA. Nesta análise foram identificados sete genes significantemente superexpressos em glioblastoma multiforme (GBM), o tipo mais agressivo de astrocitoma. Estes genes são: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L e XRCC2. Através de RT-PCR quantitativo, avaliamos os níveis de expressão destes genes em um painel expandido de 54 casos clínicos de astrocitomas de diferentes graus de malignidade e em 5 linhagens celulares de GBM. Todos os genes analisados mostraram-se mais expressos nos astrocitomas, com exceção de RAD54L em amostras de astrocitoma de grau II. Além disso, a superexpressão dos 7 genes avaliada isoladamente não exerce influência direta na sobrevida dos pacientes. Evidenciou-se ainda a superexpressão mais acentuada de EXO1 e NEIL3, que foram selecionados para realização de ensaios funcionais de silenciamento, e avaliação do ciclo celular e taxas de apoptose/morte efetiva das células. Estes ensaios foram realizados com as linhagens celulares T98G e U138MG, que apresentaram maiores níveis de expressão destes genes. Nos ensaios funcionais, observamos que o silenciamento...
Abstract: Gliomas are the most common type of primary brain cancers. They are pathologically defined by the presence of histological and immunehistochemical characteristics that evidence glial differentiation. According to the hypothetical cell of origin they are classified in: astrocytomas, oligodendrogliomas and ependimomas. Among them, astrocytomas are the more common and aggressive type. The treatment currently used for GBM includes surgical resection of tumor followed by chemotherapy with temozolamide (TMZ) and radiotherapy, but this protocol is still insufficient due to the high resistance of cancer cells. Searching for repair genes associated with the high resistance of astrocytomas, we developed a previous study of differential gene expression using a collection of DNA repair genes. In this analysis, we identified seven genes significantly overexpressed in glioblastoma multiforme (GBM), namely: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L and XRCC2. Using quantitative RT-PCR, we evaluated the expression of these genes in an expanded panel of samples with 54 clinical cases of different grade astrocytomas and five GBM cell lines. All genes showed expression significantly higher in astrocytomas, except RAD54L in grade II astrocytomas. Moreover, the overexpression of this 7 genes evaluated individually doesn't exert direct influence upon patient's survival rate. Remarkably, EXO1 and NEIL3 showed the higher fold changes and were chosen for functional silencing assays. This experiments were performed with T98G and U138MG cell lines that showed the higher expression levels among the GBM cell lines analyzed. In the functional assays, we observed that the silencing of EXO1 or NEIL3 doesn't induce changes in the apoptosis and cell death rates and doesn't change the distribution of cells in cycle. Beyond this, the silencing of this two genes doesn't sentisizes cells to ionizing radiation.
Mestre

To date, the success of cancer vaccines in human clinical trials has been limited. One of the reasons for this is the immunological tolerance to tumour antigens found in cancer patients. A novel DNA fusion vaccine design which links a pathogen-derived domain (DOM) of fragment C from tetanus toxin to a peptide epitope from a tumour antigen has been developed in our laboratory. The microbial sequence is able to activate a non-tolerised pool of helper T cells, providing T-cell help for immune induction against the linked tumour-specific sequence. The main aim of this project was to produce a therapeutic DNA vaccine against human papillomavirus (HPV)-associated cancers. A number of DNA fusion vaccines against the E7 antigen from HPV16 were constructed, including pDOM.E749-57, which encodes a well described H-2Db-binding epitope from E7 fused to the DOM sequence. CD8+ T-cell responses to the vaccines were demonstrated using flow cytometry and functional assays. Importantly, these responses were stronger than those induced by a published synthetic long peptide strategy. In vivo tumour challenge experiments showed that DNA vaccines had a protective and therapeutic effect. The vaccines were then tested in transgenic mice which develop spontaneous E7-expressing tumours in a setting of tolerance. DNA vaccine-mediated E7-specific CD8 + T-cell responses were successfully induced in these mice, together with a reduction in the mass of spontaneous tumours. This is the first demonstration of pDOM-epitope DNA vaccine-mediated therapy for spontaneous tumours and bodes well for translation into the clinic. One limiting factor for DNA vaccination in humans may be the delivery system. Electroporation (EP) is one approach which may overcome this. Therefore, a secondary aim of this project was to investigate the impact of EP on immune responses to DNA vaccination in more detail. EP proved essential for generating T-cell and antibody responses to the pDOM.E7 49-57 vaccine in sub-optimal conditions. This information will be crucial for the planning of therapeutic vaccination protocols in patients.

This research has been carried out to study the UAE population structure, particularly as the UAE has an unusual population composition. Transient workers from Indian sub-continental outnumber the native Arabic population by approximately three (60%) to one (20%). Other Arab populations make up approximately 15% of the population, the majority are from Egypt, Yemen, Palestine and Sudan. The relatively high levels of consanguineous marriages (between close relatives such as first cousin) between UAE native population added more complexity to the population. Therefore, it was important to analysis the population before applying any new tools to forensic analysis and paternity testing in the UAE. Two polymorphic systems were chosen (VNTRs and STRs) to investigate the population substructure. Five single loci VNTR/HinfI probes MS1, MS31, MS43A, YNH24 and G3 were used to profile 173 individuals from the UAE native Arabic population, 154 individuals from Indian and 112 individuals from Pakistani populations. The FST was calculated by comparing the UAE Arabic population to both Indian and Pakistani populations. The highest value of (0.0062) was observed between the UAE Arabic and Indian populations. No evidence of substructure was observed when Indian population compared to Pakistani population (FST = -0.003). In addition, eight sample STR loci D5S818, D7S820, D13S317, D16S539, vWA, THO1, TPOX and CSF1PO (GenePrintTM PowerPlexTM 1.2 System) were used to profile 229 UAE native Arab (100 from Sharjah and 129 from Abu Dhabi), 194 Indian, 197 Pakistani and 121 Egyptian individuals. The data were analysed to estimate a number of forensic and paternity parameters, including matching probability, discrimination power, probability of paternal exclusion and typical paternity index in order to assess the application of these two systems for forensic and paternity tests in the UAE. The five VNTR loci and eight STR loci together were proved to be very powerful tool for forensic analysis and determining paternity within the UAE.

Background: Lung cancer is the most common cause of cancer death worldwide and is estimated to account for more than 1,380,000 deaths per year. Lung cancer can be separated into two major histological types: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC), accounting for approximately 15% and 85% of cases respectively. Epidermal Growth Factor Receptor (EGFR) is a tyrosine-kinase receptor. In NSCLC EGFR overexpression is found in over 80% of cases, and EGFR copy number gain (CNG) or amplification is found in nearly 60% of them. Tumours with EGFR mutations can be treated using anti-EGFR drugs; however currently genetic analysis has to be performed on tissue which is obtained by biopsy. Aims: This project aims to investigate alternative methods of obtaining tumour DNA for genetic analysis, to potentially improve or support the current diagnostic process. This project will investigate both new testing methods (molecular assays) and new sources of tumour DNA (cell free DNA from plasma). Methods: A number of methods were employed during this project. Initially EGFR and KRAS mutation detection was attempted using a novel Peptide Nucleic Acid Polymerase Chain Reaction (PNA-PCR) assay devised by GeneFirst Ltd (Oxford, UK). The second approach utilised custom designed TaqMan Array 384 well plate assays for the detection of EGFR, KRAS, NRAS and BRAF mutations. 40 clinical EDTA blood samples were obtained for the investigation of the use cfDNA for oncogenic mutation detection. Plasma DNA extracted using two automated platforms (Qiagen EZ1 and Promega Maxwell). The extracted DNA was analysed using the Ion Torrent Next Generation Sequencing (NGS) platform. Results: The GeneFirst novel PNA PCR assays appeared to tolerate low concentration FFPE DNA samples but had a very high false positive rate and the endogenous control assay failed regularly (0- 33.3% failure rate over different assay versions). The TaqMan Array assay was very successful at detecting EGFR, KRAS, NRAS and BRAF mutations from FFPE tissue, displaying 97.62% and 94.74% concordance with previously used diagnostic assays (Qiagen Therascreen EGFR RGQ PCR and Thermo Fisher KRAS castPCR). For the automated isolation of cfDNA, the Promega Maxwell instrument gave consistently superior results to the Qiagen EZ1. CfDNA was successfully used to detect oncogenic mutations using both PCR and NGS assays. Conclusion: This project has utilised a number of approaches in order to investigate new approaches for the detection of clinically actionable oncogenic mutations, both in FFPE tissue (obtained through surgery or biopsy) and the relatively new cfDNA analyte. Two PCR techniques were compared using DNA from FFPE tissue, and the TaqMan Array assay was shown to be vastly superior. The TaqMan Array was subsequently adopted as the primary diagnostic assay in UHCW Pathology. CfDNA (despite the limited number of samples) showed great potential as an alternative for tissue for detection actionable cancer mutations. The Ion Torrent Next Generation Sequencing system proved to be the most sensitive and powerful technique of the ones utilised here, and will prove an invaluable asset for future development of this work.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Neoplasia é um processo anormal que compromete a proliferação e diferenciação celular de início localizado, podendo disseminar pelo organismo do hospedeiro levando a sua morte. As características desse processo devem ser investigadas para se estabelecer um protocolo terapêutico, para isso, uma importante ferramenta é o Teste do Cometa que detecta a lesão do DNA. Quanto ao protocolo terapêutico a Terapia Fotodinâmica (TFD) é uma nova modalidade utilizada para o tratamento de neoplasia. Consiste na indução da citotoxicidade, através da interação da luz de comprimento de onda adequado, de um fotossensibilizador e do oxigênio molecular presente nas células. Com o objetivo de quantificar a lesão no DNA das neoplasias mamárias pelo Teste do Cometa; avaliar a resposta desse tumor à Terapia Fotodinâmica como tratamento único e adjuvante a cirurgia utilizamos 20 ratas Sprague-Dawley divididas em quatro grupos durante 12 semanas: os grupos G1 a G4 receberam dose única de DMBA 50 mg/kg por gavagem para indução do tumor de mama. Quando da manifestação da neoplasia aplicou-se nos animais de todos os grupos citados o TNM clínico, exame citológico e Teste do Cometa. O G1 foi acompanhado clinicamente, o G2 submetido à TFD, o G3 à cirurgia associada a TFD, G4 à cirurgia. Os animais do grupo 2 foram submetidos a uma única sessão da TFD, após 8 semanas, tiveram o estadiamento clínico e lesão no DNA menores quando comparado ao grupo controle. Os animais do grupo 3 não apresentaram recidivas até o final de 12 semanas quando foram comparados com os animais do grupo 4 o qual apresentou 60% de taxa de recidiva após 11 semanas, embora os animais do grupo 3 tenham apresentado menor lesão no DNA. A TFD pode ser usada no modelo experimental de tumor de mama como terapia única e adjuvante a cirurgia para redução da evolução do tumor e evitar recidivas respectivamente.
Neoplasm is a process that results from an the abnormal proliferation and cell differentiation initially localized, but can metastize leading to death. The characteristics of this process should be investigated to establish a therapeutic protocol Comet assay is an important tool that detects the DNA damage. Photodynamic Therapy (PDT) is a therapeutic modality that can be used to treat cancer, where the cytotoxic effect is induced by the interaction of light at adequate wavelength, a photosensitizer and molecular oxygen present in cells. Aiming to quantify the DNA damage by Comet assay in mammary tumor assess the tumor response after Photodynamic Therapy as a single treatment or adjunvant modality to surgery, 20 Sprague-Dawley rats were used. The animals were divided into four groups all animals received a single dose of DMBA, 50 mg / kg by gavage to induce the mammary tumors the TNM clinical, cytological and Comet assay classifications were performed. The animals of were clinically monitored, the animals were treated by PDT, the animals were treated with surgery and PDT, and the animals of only surgery. The animals in group 2 were subjected to a single session of PDT, and after 8 weeks, clinical staging and DNA damage were less when compared to the control group. The animals in group 3 showed no tumor recidive until the end of the 12 th week. The animals of group 4 showed 60% rate of tumor recurrence after 11 weeks. The animals in group 3 showed less DNA damage when compared to group 4. PDT can be used in an experimental model of mammary tumor as single and as adjuvant therapy to reduce the development of tumors and to prevent recurrences, respectively.

Orientador: Noeme Sousa Rocha
Banca: Claudia Valeria Seullner Brandão
Banca: Cristina Kurachi
Resumo: Neoplasia é um processo anormal que compromete a proliferação e diferenciação celular de início localizado, podendo disseminar pelo organismo do hospedeiro levando a sua morte. As características desse processo devem ser investigadas para se estabelecer um protocolo terapêutico, para isso, uma importante ferramenta é o Teste do Cometa que detecta a lesão do DNA. Quanto ao protocolo terapêutico a Terapia Fotodinâmica (TFD) é uma nova modalidade utilizada para o tratamento de neoplasia. Consiste na indução da citotoxicidade, através da interação da luz de comprimento de onda adequado, de um fotossensibilizador e do oxigênio molecular presente nas células. Com o objetivo de quantificar a lesão no DNA das neoplasias mamárias pelo Teste do Cometa; avaliar a resposta desse tumor à Terapia Fotodinâmica como tratamento único e adjuvante a cirurgia utilizamos 20 ratas Sprague-Dawley divididas em quatro grupos durante 12 semanas: os grupos G1 a G4 receberam dose única de DMBA 50 mg/kg por gavagem para indução do tumor de mama. Quando da manifestação da neoplasia aplicou-se nos animais de todos os grupos citados o TNM clínico, exame citológico e Teste do Cometa. O G1 foi acompanhado clinicamente, o G2 submetido à TFD, o G3 à cirurgia associada a TFD, G4 à cirurgia. Os animais do grupo 2 foram submetidos a uma única sessão da TFD, após 8 semanas, tiveram o estadiamento clínico e lesão no DNA menores quando comparado ao grupo controle. Os animais do grupo 3 não apresentaram recidivas até o final de 12 semanas quando foram comparados com os animais do grupo 4 o qual apresentou 60% de taxa de recidiva após 11 semanas, embora os animais do grupo 3 tenham apresentado menor lesão no DNA. A TFD pode ser usada no modelo experimental de tumor de mama como terapia única e adjuvante a cirurgia para redução da evolução do tumor e evitar recidivas respectivamente.
Abstract: Neoplasm is a process that results from an the abnormal proliferation and cell differentiation initially localized, but can metastize leading to death. The characteristics of this process should be investigated to establish a therapeutic protocol Comet assay is an important tool that detects the DNA damage. Photodynamic Therapy (PDT) is a therapeutic modality that can be used to treat cancer, where the cytotoxic effect is induced by the interaction of light at adequate wavelength, a photosensitizer and molecular oxygen present in cells. Aiming to quantify the DNA damage by Comet assay in mammary tumor assess the tumor response after Photodynamic Therapy as a single treatment or adjunvant modality to surgery, 20 Sprague-Dawley rats were used. The animals were divided into four groups all animals received a single dose of DMBA, 50 mg / kg by gavage to induce the mammary tumors the TNM clinical, cytological and Comet assay classifications were performed. The animals of were clinically monitored, the animals were treated by PDT, the animals were treated with surgery and PDT, and the animals of only surgery. The animals in group 2 were subjected to a single session of PDT, and after 8 weeks, clinical staging and DNA damage were less when compared to the control group. The animals in group 3 showed no tumor recidive until the end of the 12 th week. The animals of group 4 showed 60% rate of tumor recurrence after 11 weeks. The animals in group 3 showed less DNA damage when compared to group 4. PDT can be used in an experimental model of mammary tumor as single and as adjuvant therapy to reduce the development of tumors and to prevent recurrences, respectively.
Mestre