Dissertations / Theses on the topic 'Precursor T-Cell Lymphoblastic Leukemia-Lymphoma'

Although cure rates have significantly improved for children with T-cell acute lymphoblastic leukemia (T-ALL), 20-30% undergo induction failure or relapse with most succumbing to disease. Leukemia-initiating cells (L-ICs) are hypothesized to be resistant to conventional chemotherapy and radiation and are thereby responsible for disease recurrence. Using an in vivo limiting dilution assay, we previously showed that the murine T-ALL L-IC is quite rare, with only 0.003-0.05% of cells capable of initiating disease, and demonstrated that the L-IC is a subset of the leukemic DN3 thymic progenitor population. Work described in this thesis validates the L-IC assay using two transplantation methods to rule out effects of homing and/or microenvironment on T-ALL L-IC survival and maintenance. Using this assay, we demonstrate that sustained Notch1 signaling is required for T-ALL initiation in vivo and show that treatment with a Notch1 inhibitor reduces or in some cases eliminates the L-IC population. We further analyze the effects of inhibiting c-Myc, a Notch1-regulated gene, on L-IC frequency and uncover an essential role for c-Myc in L-IC survival and expansion. Suppressing c-Myc by using specific shRNAs or a c-Myc inhibitor reduces the L-IC population and interferes with leukemia initiation. Together, these findings reveal a critical role of the Notch1-c-Myc pathway in T-ALL initiation and suggest that therapeutics targeted at this pathway could be used to treat and/or prevent disease relapse in patients.

Thesis (Ph. D.)--West Virginia University, 2010.
Title from document title page. Document formatted into pages; contains x, 102 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008
Title from title page screen (viewed on June 19, 2008). Research advisor: Pamela S. Hinds RN, Ph.D. Document formatted into pages (viii, 185 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 86-98).

Orientador: José Andres Yunes
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-21T05:50:44Z (GMT). No. of bitstreams: 1 Jotta_PatriciaYoshioka_D.pdf: 8922035 bytes, checksum: 3734371a320410ba431d6e6ce6579e55 (MD5) Previous issue date: 2012
Resumo: A leucemia linfóide aguda (LLA) é o câncer mais frequente na infância, e destas, 15% são do tipo T (LLA-T). A hiperativação da via PI3K/Akt tem sido amplamente descrita em tumores e em linhagens celulares de LLA-T. PTEN é o principal regulador negativo dessa via e frequentemente encontra-se inativado em cânceres humanos. Com frequência, pacientes com LLA-T apresentam mutações ativadoras de NOTCH1. NOTCH1 pode regular transcricionalmente PTEN, contudo ainda não está claro como as mutações ativadoras de NOTCH1 influenciariam a expressão de PTEN nas LLA-T. Nós encontramos uma ocorrência de 11 (17,7%) mutações no éxon 7 do PTEN em 62 casos de LLA-T estudados consecutivamente. Contudo, nenhuma mutação foi encontrada na análise de 71 casos de LLA-B derivada. A maioria das mutações de PTEN apresentavam inserções/deleções de mais de 3 nucleotídeos. Não encontramos associação entre mutações em PTEN e o gênero, a idade e a contagem de glóbulos brancos ao diagnóstico. Pacientes com alterações no PTEN apresentaram uma tendência a pior sobrevida global (OS, p=0.07). Dentre os pacientes de LLA-T classificados como alto risco (n=56), aqueles possuindo anormalidades no PTEN mostraram-se associados significativamente a menor OS (p=0.019) e sobrevida livre de leucemia (LFS 47% vs 76%; p=0.045). As curvas de LFS foram significativamente diferentes (p=0.003), mesmo considerando apenas pacientes que atingiram a remissão no dia 28 do tratamento para a análise. Nosso estudo também mostrou que pacientes com mutações em NOTCH1 apresentavam aumento na transcrição de MYC e menor expressão de PTEN mRNA comparados a pacientes com NOTCH1 selvagem. Nós recentemente demonstramos que células de LLA-T apresentavam fosforilação de PTEN mediada por CK2, resultando na estabilização e consequentemente inativação da proteína PTEN. Corroborando ao estudo anterior, os casos de LLA-T analisados, independente do status de mutação em NOTCH1, expressam níveis significativamente mais altos de proteína PTEN do que controles normais. Para avaliar o impacto da regulação transcricional de NOTCH e a inativação postranscricional por CK2 de PTEN, nós tratamos as células de LLA-T com inibidores de gamma-secretase (DAPT e de CK2 (DRB/TBB). Nosso estudo enfatiza a relevância biológica e clínica da regulação do PTEN em LLA-T. E sugerimos o uso combinado de inibidores de gamma-secretase e CK2 devem possuir potencial terapêutico nas LLA-T
Abstract: T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of pediatric ALL. Patients with T-ALL are at increased risk of relapse compared with children treated for B-cell precursor ALL. Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. PTEN is the main negative regulator of the PI3K/Akt survival pathway. T-ALL patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. We report that PTEN exon 7 mutations occurred in 11 (17.7%) out of 62 consecutive pediatric T-cell acute lymphoblastic leukemia (T-ALL) but in none of 71 precursor B-ALL patients. Most PTEN mutations were insertions/deletions of more than 3 nucleotides. No associations were found between PTEN mutation and age, gender, WBC at diagnosis, early response to therapy and remission rate. Patients with PTEN mutation (n=11) had a tendency toward worse overall survival (OS, p=0.07). Remarkably, PTEN mutations were significantly associated with lower OS (p=0.019) and leukemia-free survival (LFS 47% vs 76%, p=0.045) within patients classified in the high risk group (n=56). LFS curves were significantly different (p=0.003) even if only patients who reached remission on day 28 were considered for analysis. We compared patients with or without NOTCH1mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of NOTCH transcriptional and CK2 post-translational inactivation of PTEN, we treated TALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL. And emphasize the biological and clinical relevance of PTEN regulation in pediatric T-ALL
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular

Gain of function NOTCH1 mutations are common in both patients with T-ALL and in mouse models of the disease. Inhibiting the Notch pathway in T-ALL cell lines results in growth arrest and/or apoptosis in vitro, suggesting a requirement for Notch signaling in T-ALL. Therefore, we sought to examine the role of Notch1 signaling in both premalignancy and in the maintenance of leukemic growth. Using a murine model of T-ALL, in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development, our preleukemic studies reveal that Notch1 mutations are early events that contribute to the clonal expansion of DN3 and DN4 progenitors. We also demonstrate that progenitors are maintained within the tumor and are enriched in leukemia-initiating cell (L-IC) activity, suggesting Notch1 may contribute to L-IC self-renewal. By studying the effects of Notch signaling in murine T-ALL cell lines, we also demonstrate that Notch1 promotes the proliferation and survival of leukemic blasts through regulation of Lef1 and the Akt/mTOR pathways. Given that T-ALL cell lines are dependent on Notch signaling in vitro, we investigated the effects of Notch inhibition in vivo. We provide evidence that Notch1 can be successfully targeted in vivo and that Notch inhibition, with γ-secretase inhibitors (GSIs), significantly extends the survival of leukemic mice. We also demonstrate that administration of GSIs in combination with rapamycin inhibits human T-ALL growth and extends survival in a mouse xenograft model. Given that NOTCH1 may be required to maintain both L-IC and bulk leukemic growth, targeting NOTCH1 may prove to be an efficacious targeted therapy for T-ALL patients with aberrant NOTCH1 activation.

A Leucemia Linfocítica Aguda na infância e adolescência é uma neoplasia de precursores linfóides de natureza heterogênea. Foi a primeira neoplasia disseminada a tornar-se curável pela quimioterapia. No Brasil os estudos cooperativos para o seu tratamento foram iniciados em 1980 com a criação do primeiro protocolo para o tratamento desta leucemia, denominado Grupo Brasileiro para o Tratamento da Leucemia na Infância. Na seqüência destes estudos foram observadas Sobrevidas Livre de Eventos de 50%, 58% e 70% nos protocolos 80, 82 e 85 respectivamente. Durante as décadas de 1980 e 1990, com a divulgação dos excelentes resultados alcançados com os regimes dos protocolos do grupo Berlin Frankfurt Münster, uma série de instituições em nosso País passou a adotá-los. Apesar de termos conhecimento dos dados referentes a evolução dos pacientes protocolados no GBTLI e dos demais estudos divulgados pelas instituições de origem, não tínhamos, porém, uma avaliação epidemiológica nem o conhecimento dos índices de sobrevivência alcançados no estado do Rio Grande do Sul. Neste trabalho foram avaliados 1472 pacientes com LLA, 833 (56,59%) do sexo masculino, 639 (43,41%) do sexo feminino, com idades entre zero e 20 anos, média de 7,40 anos (desvio padrão 5,14) e mediana de 5,70 anos (amplitude 0,06 a 20,76), no período de 1980 a 2008 provenientes deste Estado. Os dados foram colhidos individualmente dos prontuários dos pacientes admitidos nas principais instituições hospitalares que mantém assistência a pacientes pediátricos com neoplasias hematológicas. No presente estudo 487 pacientes (39,40%) foram registrados oficialmente nos protocolos do GBTLI; 678 (54,85%) receberam tratamento baseados nos regimes do grupo BFM e 71 (5,75%) por outros regimes incluindo os tratados segundo o protocolo UKALL. Os casos não foram, no entanto, protocolados e, na grande maioria não foram observados a totalidade dos itens exigidos para os pacientes registrados oficialmente. A sobrevida livre de eventos dos pacientes protocolados foi significativamente superior comparada aos não protocolados, 62,41% ± 2,43% e 53,86% ± 2,04% respectivamente, em cinco anos. De acordo com a faixa etária os pacientes que apresentavam idade de 15 a 19 anos tiveram um índice de SLE de 37,98% ± 4,72% em cinco anos, inferior quando comparado aos de zero a 4 anos e 5 a 9 anos respectivamente: 62,78% ± 2,28% e 62,43% ± 2,84%. Foi observada, na população estudada, uma SG de 63,73% ± 1,49% e SLE de 57,27% ± 1,57%, o que sugere uma discussão para implementar projetos com a finalidade de elevar estes índices de sobrevida. Epidemiologicamente a incidência da LLA com progenitores B seguiu o padrão observado em países desenvolvidos com um pico de freqüência absoluta entre as idades de 2 a 4 anos. Houve diferença significativa entre a população proveniente da região urbana ou rural sendo a SLE em cinco anos de 61,76% ± 1,76% e 49,81% ± 4,28% respectivamente. A SLE e a SG em lactentes e portadores de Síndrome de Down foi inferior aos resultados obtidos em instituições dos países desenvolvidos o que torna aconselhável uma revisão das condições para a assistência destes pacientes. Este estudo teve como finalidade principal retratar a situação passada e a atual do tratamento da LLA na infância e adolescência no Rio Grande do Sul e acumular dados aqui não interpretados e que poderão ser analisados posteriormente
Acute lymphocytic leukemia in childhood and adolescence is a neoplastic disease of lymphoid precursor of heterogeneous nature, being the first disseminated neoplasia to become curable through Chemotherapy. In Brazil, cooperative studies for the treatment of ALL started in 1980 with the creation of the first protocol by the Grupo Brasileiro para o Tratamento da Leucemia na Infância. According to these studies, Event Free Survival of 50%, 58% and 70% in the protocols of 1980, 1982 e 1985 respectively was observed. During the 80s and 90s many Brazilian institutions adopted the protocols motivated by the excellent results achieved by the Berlin Frankfurt Münster group. In spite of the knowledge provided by data related to the evolution of patients protocoled by the GBTLI and other studies published by medical institutions, there was not an epidemiologic evaluation or knowledge of survival indexes achieved in the state of Rio Grande do Sul. In this work, 1472 patients with ALL from the state of Rio Grande do Sul were evaluated. Among the subjects, 833 (56,59%) were male, 639 (43,41%) female, with age range between 0- 20 y average age of 7,40 (standard deviation 5,14) and median 5,70 years old (amplitude 0,06 to 20,76). Data was collected from the medical registers of patients with hematologic neoplasia in medical institutions, which offered pediatric assistance for ALL comprising the period between 1980 until 2008. In the present study, 487 patients (39,40%) were officially registered in the protocols of GBTLI; 678 (54,85%) received treatment based on the regime of the BFM group and 71 (5,75%) were treated according to other regimens (including the UKALL protocol). The cases, however, were not protocoled and, in most cases, the totality of items required for the patients registered officially were not observed. The EFS of the patients protocoled were significantly superior to those who were not protocoled (62,41% ± 2,43% and 53,86% ± 2,04% respectively) in five years. In respect to the age range, the patients which were between 15-20 years had an EFS index of 37,98% ± 4,72% in five years, which is inferior to the index of patients 0-4 years and 5-9 years: 62,78% ± 2,28% e 62,543± 2,84% respectively. An overall survival (OS) of 63,73% ± 1,49% and EFS of 57,27% ± 1,57% was observed in the population studied. These results indicate that a discussion for the implementation of projects, which can increase the indexes of cure, should be carried out. Epidemiologically, the incidence of ALL in B progenitors followed the pattern observed in developed countries with an absolute frequency peak in the age range of 2-4 years. The outcome was superior for patients coming from urban area in comparison to those from rural area. EFS and OS in infant and Down syndrome patients were inferior to the results obtained in developed countries, showing how important it is to review the conditions of the assistance provided to these patients. The objective of this work is to present the development of ALL treatment in childhood and adolescence in the state of Rio Grande do Sul, Brazil, from its beginning until the current days providing data, which can be analyzed and interpreted posteriorly

O acometimento do câncer na infância é relativamente raro, com taxas relevantes de incidência de alguns tumores, como a leucemia linfoblástica aguda (LLA) e o tumor de Wilms (TW). Embora o câncer seja uma das dez primeiras causas de óbito de crianças e adolescentes e a primeira por doença a partir dos cinco anos, nas últimas décadas o progresso da terapêutica tem possibilitado um declínio nas taxas de mortalidade e expansão dos prazos de sobrevida. Desta forma, o acompanhamento efetivo no enfrentamento da doença passou a buscar análises mais amplas dos efeitos orgânicos tardios da doença e da terapêutica, incluindo as condições psicossociais do sobrevivente, como nas avaliações da qualidade de vida relacionada à saúde (QVRS). No sentido de ampliar o conhecimento e alternativas para o acompanhamento ambulatorial e periódico da condição de sobrevivência, este estudo buscou comparar o impacto na QVRS do sobrevivente adulto, dada a diferença na terapêutica de escolha para a remissão da LLA (quimioterapia) e do TW (cirurgia e quimioterapia), utilizando uma avaliação a distância da QVRS (SF-36, via telefone).Objetivos: Analisar e comparar a QVRS de sobreviventes adultos de LLA e TW, entre si e em relação a participantes sadios, acompanhados no Ambulatório Fora de Terapia do ITACI-HC-FMUSP, através da aplicação alternativa (via telefone) do SF-36.Casuística e Método: 90 participantes, acima de 18 anos. Grupo controle(CTRL) (30 sujeitos, fisicamente saudáveis, com ausência de diagnóstico prévio de câncer, recém-ingressos em curso superior) e Grupos experimentais (60 sobreviventes - Ambulatório Fora de Terapia - ITACI - HCFMUSP): grupo LLA (GLLA) - 30 sobreviventes LLA e grupo TW (GTW) 30 sobreviventes TW. A avaliação foi realizada através da aplicação, via telefone, do SF- 36. Após compilação dos domínios do SF-36, os resultados foram analisados através do teste de qui-quadrado, teste t-independente e teste de ANOVA. Resultados: Os participantes não apresentaram diferença significativa quanto a idade, a maioria eram solteiros, sem filhos e provenientes de São Paulo. O nível mais elevado de escolaridade do CTRL decorreu do critério de inclusão, mas com relevante proporção de sobreviventes no nível superior. Nos sobreviventes não houve diferença significativa de idade de diagnóstico e tempo de fora de terapia. Quanto a QVRS, houve melhores resultados dos sobreviventes masculinos em relação às sobreviventes e participantes CTRL. Especificamente, GLLA e GTW para Vitalidade e GLLA para Aspectos sociais, Saúde mental e Aspectos emocionais, no último aspecto detectada diferença também para as sobreviventes GTW. Nos sobreviventes com diagnóstico tardio (acima 53 meses) o GLLA apresentou melhores resultados na Capacidade funcional. Na percepção da própria saúde, houve diferença para todos os domínios, exceto nos Aspectos sociais e emocionais, estando as diferenças circunscritas a percepções positivas (boa, muito boa e excelente) da própria saúde pelos sobreviventes e controles. Conclusão: Particularmente no período do estudo, para a amostra selecionada e os aspectos analisados pelo SF-36 pode-se inferir que, apesar de algumas diferenças encontradas, os sobreviventes não apresentaram evidências de comprometimento de QVRS. O SF-36 (via telefone) pode ser um recurso de acesso e avaliação de QVRS de sobreviventes sob acompanhamento ambulatorial
The involvement of childhood cancer is relatively rare, with relevant incidence rates of some cancers such as Acute Lymphoblastic Leukemia (ALL) and Wilms Tumor (WT). Although cancer is one of the top ten causes of death in children and adolescents and the first disease from the age of five, in recent decades the therapeutic progress has made possible a decline in mortality rates and expansion of the survival periods. In this way, the effective monitoring in the confrontation of the disease passed to seek broader analyses of later organic effects from disease and therapy, including the psychosocial conditions of survivor, as in evaluations of healthrelated quality of life (HRQoL). In order to increase knowledge and alternatives for monitoring outpatient and periodic survival condition, this study sought to compare the impact on HRQoL of adult survivor, given the difference in the choice therapy for the remission of ALL (chemotherapy) and WT (surgery) using a remote assessment of HRQoL (SF -36 via telephone call). Objectives: Analyze and compare the HRQoL of adult survivors of ALL and WT between themselves and in relation to healthy participants, followed at the Ambulatory outside ITACI - HC - USP therapy , by alternative application ( by phone calls) of the SF - 36 . Methods: 90 participants , above 18 years. Control group (CTRL): (30 subjects, physically healthy, no history of oncological diagnosis, newly joined in higher education) and experimental groups (60 survivors - Outpatient Therapy - ITACI - HCFMUSP ): ALL group ( GALL ) - 30 ALL survivors and WT group ( GWT ) 30 WT survivors. The evaluation was performed by applying SF-36, via telephone calls. After compilation of the SF -36 domains, the results were analyzed through chi - square test, independent t test and ANOVA test. Results: Participants showed no significant difference regarding age, most were single, childless and from Sao Paulo. CTRL highest level of schooling resulted from inclusion criterion but with relevant proportion of survivors at the top level. In survivors there was no significant difference in age of diagnosis and time outside therapy. As for HRQoL there have been better results of male survivors in relation to female survivors and CTRL participants. Specifically GALL and GWT for vitality domain and GALL for social aspects, mental health and emotional aspects. In the last domain, it was detected also difference female survivors GWT. In survivors with late diagnosis (above 53 months) the GALL presented better results in functional capacity. In the perception of their own health, there were differences for all domains except in social and emotional aspects, with differences confined to positive perceptions (good, very good and excellent ) of own health by survivors and controls. Conclusion: Particularly during the study period, for the selected sample and the analyzed aspects by SF -36 can be inferred that, despite some differences, survivors did not show evidence of impairment of HRQoL . The SF -36 (via telephone calls) can be a resource of access HRQoL evaluation of survivors under ambulatory followup

O câncer pediátrico é raro em números absolutos, porém quando comparado às incidências em adultos vem apresentando aumento nas taxas de incidência, exigindo um preparo do sistema de saúde para acompanhamento. Este acompanhamento não pode ser igual ao do adulto, visto que crianças possuem diferenças fisiológicas nas diferentes faixas etárias pediátricas. Sendo assim, estudos de utilização de medicamentos são importantes nesta população, a fim de promover o uso racional dos mesmos, bem como, garantir seu uso seguro e uma terapêutica eficaz. O presente estudo tem por objetivo avaliar as prescrições de antineoplásicos, na unidade de oncologia pediátrica de um hospital universitário de Porto Alegre, identificando os protocolos mais utilizados, e posteriormente contextualizando-os com o preconizado no referencial teórico. Foi realizado um estudo transversal prospectivo envolvendo internações realizadas na unidade de oncologia pediátrica do Hospital de Clínicas de Porto Alegre (HCPA). Foram analisadas 274 internações, das quais 40 eram primeira internação e 234 reinternações. A maioria dos pacientes tinha idade de 0 a 10 anos, uma discreta prevalência do sexo masculino, de raça/cor branca, residentes na mesorregião metropolitana. A principal forma de financiamento das internações foi o público, e a principal causa de internação foi o tratamento, sendo o mais frequente o quimioterápico. Foram analisados os protocolos utilizados durante as internações de pacientes com diagnóstico de Leucemia Linfocítica Aguda, Retinoblastoma e Sarcoma de Ewing. Os protocolos são conjunto de regras criadas a partir de grupos cooperativos ou da indústria farmacêutica, permitindo um maior conhecimento sobre os medicamentos utilizados na pediatria. A oncologia pediátrica com sua particularidade de ser uma doença culturalmente ligada ao óbito possui maior facilidade de estudos deste porte. Porém, ainda são necessários mais estudos de utilização destes medicamentos, a fim de agregar conhecimento aos protocolos de tratamento.
Pediatric cancer is rare in absolute numbers, but when compared to the incidence in adults has shown an increase in incidence rates, requiring a prepared system health for following. Pediatrics treatment can’t be the same as that of adults, because children have different physiological differences in pediatric age groups. Studies of medication use in this population are very important in order to promote the rational use of drugs, as well as to guarantee their safe use and effective therapy. The present study aims to evaluate the anticancer prescriptions in pediatric oncology unit of a university hospital in Porto Alegre, identifying the most used protocols, and contextualizing them later with the recommendations in the theoretical framework. A prospective cross-sectional study involving hospital admissions in the pediatric oncology unit of the Hospital de Clínicas de Porto Alegre (HCPA) was performed. We analyzed 274 admissions, which 40 were first admission and 234 were readmissions. These admissions most patients were aged 0 to 10 years, white race, a slight male prevalence, residing in Metropolitan Mesoregion. The financing the hospital admissions was public, and the maining cause of hospitalization was treatment, the most frequent being the chemotherapy. We analyzed the protocols used during the admissions of patients diagnosed with Acute Lymphocytic Leukemia, Retinoblastoma and Ewing's Sarcoma. Protocols are studies that allow a greater knowledge about medicines for pediatric use. The pediatric oncology with its characteristic of being a disease has culturally linked with death ease of studies of this size. However, more studies of medication use are still needed to use these drugs in order to knowledge to the treatment protocols.

Introduction: The Central Nervous System (CNS) is an important site of relapse in patients with Acute Lymphoblastic Leukemia (ALL), despite increasing cure rates in the last four decades in young patients. The presence of leukemic blasts in the Cerebrospinal Fluid (CSF) presents prognostic implication and therefore defines changes in the therapeutic protocol, which is why it requires diagnostic accuracy. The emergence of new techniques for identifying CSF leukemia blasts, such as flow cytometry and molecular methods, which are more costly and not uniformly accessible for clinical use, have led to the need to reassess the role of conventional cytology as diagnostic tool. Objectives: To identify the proportion of CSF samples positive for blasts in children and adolescents with ALL, using a standardized cytology technique and with standardization of variables related to the process that could interfere with the result. DESIGN: Prospective, descriptive, uncontrolled study in which samples of CSF obtained by lumbar puncture of patients with ALL that were starting treatment were examined. CSF samples were sent to the laboratory shortly after collection, being processed and cytocentrifugated in cytofunyl in up to four hours after collection. Four slides were prepared, stained and analyzed by a pathologist and a hematologist. RESULTS: Twenty-eight patients with ALL were evaluated, with predominance of male (58.6%), immunophenotype B (82.2%) and 78.5% were stratified as high risk for relapse. Of the 205 CSF samples evaluated, 26 (12.6%) were positive for blasts and among 28 patients, 11 (39.2%) had CSF with neoplastic infiltration. Comparing the groups with and without CNS infiltration, no statistically significant difference was observed for the variables analyzed. CONCLUSIONS: Conventional cytology was effective in the identification of CNS infiltration by blasts, provided there is a vigilance of factors related to collection, processing and analysis of CSF that may interfere in the reliability of the result.
Introdução: O Sistema Nervoso Central (SNC) é importante sítio de recaída em pacientes com Leucemia Linfoblástica Aguda (LLA), apesar das crescentes taxas de cura nas últimas quatro décadas em pacientes jovens. A presença de blastos leucêmicos no Líquido Cefalorraquiano (LCR) apresenta implicação prognóstica e, portanto, define mudanças no protocolo terapêutico, razão pela qual requer acurácia diagnóstica. O surgimento de novas técnicas de identificação de blastos leucêmicos no LCR, como a citometria de fluxo e os métodos moleculares, que têm custo mais elevado e não estão uniformemente acessíveis para uso clínico, trouxe a necessidade de reavaliar-se o papel da citologia convencional como instrumento diagnóstico. Objetivos: Identificar a proporção de exames de LCR positivos para blastos em crianças e adolescentes com LLA, utilizando-se de técnica de citologia padronizada e com padronização de variáveis relacionadas ao processo que pudessem interferir no resultado. Delineamento: Estudo prospectivo, descritivo, não controlado, no qual foram examinadas amostras de LCR obtidas por punção lombar de pacientes com LLA que estavam iniciando o tratamento. As amostras de LCR foram encaminhadas ao laboratório logo após a coleta, sendo processadas e citocentrifugadas em citofunil em até no máximo quatro horas após a coleta. Quatro lâminas foram preparadas, coradas e analisadas por um patologista e um hematologista. Resultados: Foram avaliados 28 pacientes com LLA, havendo predomínio do sexo masculino (58,6%), imunofenótipo B (82,2%) e 78,5% foram estratificados como de alto risco para recaída. Dentre as 205 amostras de LCR avaliadas, 26 (12,6%) foram positivas para blastos e dentre os 28 pacientes, 11 (39,2%) obtiveram algum exame de LCR com infiltração neoplásica. Comparando-se os grupos com e sem infiltração de SNC, não se observou diferença estatisticamente significante para as variáveis analisadas. Conclusão: Citologia convencional foi efetiva na identificação de infiltração de SNC por blastos leucêmicos, desde que haja vigilância dos fatores relacionados a coleta, processamento e análise do LCR que possam interferir na fidedignidade do resultado.
Aracaju, SE

Chung, Po Yin.
Thesis submitted in: December 2006.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 128-155).
Abstracts in English and Chinese.
Thesis Abstract --- p.i
論文摘要 --- p.iv
Acknowledgements --- p.vi
Abbreviations --- p.vii
Thesis Content --- p.xi
List of Figures --- p.xv
List of Tables --- p.xvii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1. --- Normal Hematopoiesis --- p.1
Chapter 1.2. --- Hematological Malignancy and the Aberrant Development of Blood Cells --- p.2
Chapter 1.3. --- Leukemia and Its Classification --- p.3
Chapter 1.4. --- Childhood Acute Lymphoblastic Leukemia (ALL) --- p.5
Chapter 1.4.1. --- Epidem iology --- p.5
Chapter 1.4.2. --- Causes and Risk Factors --- p.6
Chapter 1.4.3. --- Molecular Pathophysiology --- p.7
Chapter 1.4.4. --- Clinical Presentation --- p.9
Chapter 1.4.5. --- Classification --- p.10
Chapter 1.4.5.1. --- Immunophenotyping --- p.10
Chapter 1.4.5.2. --- French-American-British (FAB) Classification --- p.12
Chapter 1.4.6. --- Diagnosis and Prognosis --- p.14
Chapter 1.4.6.1. --- Morphological and Cytochemical Analysis --- p.15
Chapter 1.4.6.2. --- Cytogenetic and Molecular Genetic Characterizations --- p.16
Chapter 1.4.7. --- Treatment --- p.19
Chapter 1.5. --- Overview of Epigenetics --- p.21
Chapter 1.6. --- Concepts ofDNA Methylation --- p.23
Chapter 1.6.1. --- CpG Islands --- p.23
Chapter 1.6.2 --- Mechanisms of DNA Methylation --- p.24
Chapter 1.6.3 --- Physiological Roles of DNA Methylation --- p.28
Chapter 1.6.4 --- Initiation of Aberrant DNA Methylation --- p.30
Chapter 1.7. --- DNA Methylation in Tumorigenesis --- p.31
Chapter 1.7.1. --- Regional Hypermethylation --- p.33
Chapter 1.7.2 --- Global and Regional Hypomethylation --- p.34
Chapter 1.7.3 --- Microatellite Instability and Oncogeneic Mutation --- p.35
Chapter Chapter 2 --- Literature Review --- p.37
Chapter 2.1. --- Aberrant DNA Methylation in Childhood ALL --- p.37
Chapter 2.1.1. --- Cell Cycle --- p.39
Chapter 2.1.2. --- Apoptosis --- p.41
Chapter 2.1.3. --- Tissue Invasion and Metastasis --- p.42
Chapter 2.1.4. --- Transcription Factors and Metabolic Enzymes --- p.44
Chapter 2.1.5. --- Putative Tumor Suppressor Genes --- p.44
Chapter 2.1.6. --- Chromosome Instability --- p.46
Chapter 2.2. --- Methodologies in DNA Methylation Analysis --- p.50
Chapter 2.2.1. --- Principle of Methylation-sensitive Arbitrarily Primed PCR (MS-AP PCR) --- p.50
Chapter 2.2.2. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.53
Chapter 2.2.3. --- Cloned Bisulfite Sequencing --- p.55
Chapter 2.2.4. --- Experimental Use of Demethylating Agents --- p.55
Chapter Chapter 3 --- Background of Research --- p.58
Chapter 3.1. --- Current Methylation Studies in Childhood ALL --- p.58
Chapter 3.2. --- Objectives of Research --- p.60
Chapter 3.3. --- Study Approach and Experimental Design --- p.61
Chapter Chapter 4 --- Materials and Methods --- p.63
Chapter 4.1. --- Clinical Samples and ALL Cell Lines --- p.63
Chapter 4.1.1. --- Clinical Samples from Pediatric Patients with ALL and Normal Healthy Donors --- p.63
Chapter 4.1.2. --- ALL Cell Lines --- p.63
Chapter 4.2. --- Genomic DNA Isolation from Clinical Samples and Cell Lines --- p.64
Chapter 4.2.1. --- Ficoll Gradient Centrifugation --- p.64
Chapter 4.2.2. --- DNA Extraction --- p.64
Chapter 4.3. --- MS-AP PCR --- p.65
Chapter 4.3.1. --- Methylation-sensitive Restriction Enzyme Digestion of Genomic DNA --- p.65
Chapter 4.3.2. --- Arbitrarily Primed Polymerase Chain Reaction --- p.66
Chapter 4.3.3. --- Isolation of Differentially Methylated DNA Fragments --- p.69
Chapter 4.4. --- Cloning of Differentially Methylated DNA Fragments --- p.70
Chapter 4.4.1. --- TA Cloning --- p.70
Chapter 4.4.2. --- Screening of Positive Clones --- p.71
Chapter 4.4.3. --- Preparation of Plasmid DNA by Alkaline Lysis Method --- p.72
Chapter 4.5. --- DNA Sequence Analysis of Differentially Methylated DNA Fragments --- p.72
Chapter 4.5.1. --- Dye-terminator Cycle Sequencing --- p.72
Chapter 4.5.2. --- CpG islands Analysis of Differentially Methylated Sequences --- p.73
Chapter 4.6. --- DNA Methylation Analysis --- p.74
Chapter 4.6.1. --- Sodium Bisulfite Modification --- p.74
Chapter 4.6.2. --- Combined Bisulfite Restriction Analysis --- p.75
Chapter 4.6.3. --- Cloned Bisulfite Genomic Sequencing --- p.76
Chapter 4.7 --- Gene Expression Study --- p.76
Chapter 4.7.1. --- RNA Extraction from Clinical Samples and ALL Cell Lines --- p.76
Chapter 4.1.2. --- Reverse Transcription PCR --- p.77
Chapter 4.7.3. --- Semi-quantitative RT-PCR --- p.78
Chapter 4.7.4. --- 5-aza-2 '-deoxycytidine Demethylation Treatment --- p.79
Chapter Chapter 5 --- Results --- p.80
Chapter 5.1. --- Generation of DNA Methylation Pattern by MS-AP PCR --- p.80
Chapter 5.1.1. --- Differential Methylation Patterns of Childhood ALL --- p.84
Chapter 5.1.2. --- Methylation Patterns of B and T lineages Childhood ALL --- p.86
Chapter 5.2. --- UCSC BLAT Analysis of Differential Methylated DNA Sequences
Chapter 5.3. --- Identification of Candidate Gene --- p.89
Chapter 5.4. --- Fibrillin 2 --- p.90
Chapter 5.4.1. --- FBN2 CpG Islands: UCSC BLAT Search Analysis --- p.90
Chapter 5.4.2. --- Verification ofFBN2 by ALL Cell Lines --- p.91
Chapter 5.4.2.1. --- Semi-quantitative RT-PCR --- p.91
Chapter 5.4.2.2. --- COBRA --- p.92
Chapter 5.4.2.3. --- Cloned Bisulfite Sequencing --- p.94
Chapter 5.4.2.4. --- Demethylation Treatment Resorted FBN2 mRNA Expression in ALL Cell Lines --- p.98
Chapter 5.4.3. --- Studies ofFBN2 in Childhood ALL --- p.99
Chapter 5.4.3.1. --- Methylation Analysis --- p.99
Chapter 5.4.3.2. --- Semi-quantitative RT-PCR --- p.105
Chapter Chapter 6 --- Discussion --- p.107
Chapter 6.1. --- Genome-wide Screening Approach: MS-AP PCR --- p.107
Chapter 6.2. --- Sample Selection in this Study --- p.109
Chapter 6.2.1. --- MS-AP PCR --- p.109
Chapter 6.2.2. --- Methylation Studies --- p.109
Chapter 6.2.3. --- Studies in ALL Cell Lines --- p.110
Chapter 6.3. --- Methylation Patterns in Childhood ALL --- p.111
Chapter 6.4. --- Candidate Genes Selection Strategies in MS-AP PCR --- p.112
Chapter 6.5. --- Fibrillin 2: mRNA Expression and Methylation Studies --- p.113
Chapter 6.5.1 --- ALL Cell Lines --- p.113
Chapter 6.5.2 --- Childhood ALL --- p.113
Chapter 6.5.2.1 --- mRNA Expression and Methylation Studies --- p.113
Chapter 6.5.2.2 --- Statistical Analysis --- p.115
Chapter 6.5.3. --- Possible Roles of FBN2 in Leukemogenesis --- p.116
Chapter 6.6. --- Clinical Application of FBN2 Aberrant Methylation --- p.119
Chapter 6.6.1. --- Tumor Markers --- p.119
Chapter 6.6.2. --- Use of Demethylating Drugs in Chemotherapy --- p.121
Chapter 6.7. --- Limitations of Methylation Studies --- p.122
Chapter 6.7.1. --- MS-AP PCR --- p.122
Chapter 6.7.2. --- Techniques Used in Methylation Study --- p.122
Chapter 6.7.3. --- Problems in Methylation Study --- p.123
Chapter 6.8. --- Future Studies --- p.125
Chapter Chapter 7 --- Conclusion --- p.127
References --- p.128
Appendix --- p.155

by Betty Shuc Han Wills.
Year shown on spine: 1997.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.
Includes bibliographical references (leaves 117-128).
ACKNOWLEDGMENT --- p.i
ABSTRACT --- p.ii
TABLE OF CONTENTS --- p.iv
LIST OF APPENDICES --- p.viii
Chapter CHAPTER 1 --- INTRODUCTION --- p.1
Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6
Parental Responses to the Diagnosis of Acute Lymphocytic Leukaemia (ALL) --- p.7
Disclosure Of the Child's Diagnosis --- p.10
Impact of Cancer Treatment on Parents --- p.14
Sources of Support for Parents --- p.18
Coping Strategies of Parents of Children With ALL --- p.20
Coping With The Uncertainty of the Disease --- p.23
Research Studies Involving Chinese Parents --- p.24
Summary Of Issues From Literature Review --- p.27
Chapter CHAPTER 3 --- METHODOLOGY
Research Design --- p.29
Sampling --- p.31
Data Collection Method --- p.32
Data Collection Procedure --- p.34
Ethical Considerations --- p.38
Pilot Study --- p.40
Data Analysis --- p.42
Issues of Reliability and Validity --- p.45
Validity --- p.45
Reliability --- p.48
Chapter CHAPTER 4 --- RESULTS & DISCUSSION
Introduction --- p.50
Chapter (I). --- Parents' Profile --- p.51
Demographic Characteristics Of The Parents
Chapter (II). --- Major categories Corresponding To Interviewing The Mothers --- p.54
Initial Reactions of the Child's Confirmed Diagnosis --- p.55
Unpreparedness for the child's Diagnosis
Suddenness of the Diagnosis --- p.56
Physical and psychological reactions to the child's Diagnosis --- p.58
Sources of Support for the Mothers --- p.62
The mothers' main source of support
Other sources of support for the mothers --- p.64
Disclosure Of Child's Diagnosis --- p.66
Disclosure of the child's diagnosis to the child
Disclosure of the child's diagnosis to members of the immediate and extended families --- p.68
Disclosure of child's diagnosis to non-family members --- p.70
Uncertainty Brought On By The Illness --- p.71
Waiting for confirmation of diagnosis
Uncertainty about the success of treatment --- p.73
Uncertainty about the child's future --- p.74
Changes In The Family Routine --- p.75
Needed to normalise family life
Chapter (III). --- Major Categories Corresponding to Interviewing The Fathers Initial reactions to the child's confirmed diagnosis of ALL --- p.78
Suddenness of diagnosis --- p.79
Physical and psychological reactions to the diagnosis --- p.80
Disclosure Of The Child's Diagnosis --- p.82
Disclosure of the child's diagnosis to the child
Disclosure of the child's diagnosis to members of the immediate and extended family --- p.85
Disclosure of the child's diagnosis to non-family members --- p.86
Sources Of Support For The Fathers --- p.87
Support from immediate and extended families
Support from medical professionals --- p.89
Support from friends --- p.90
Changes In The Family Routine --- p.91
Coping Strategies Utilised By The Fathers --- p.92
Open communication
Use of religious beliefs and rituals --- p.93
Chapter (IV). --- Comparison Of Categories Found Between The Mothers And The Fathers --- p.95
Initial reactions to the child's confirmed diagnosis of ALL
Disclosure of the child's diagnosis --- p.96
Sources of support for the parents --- p.100
Changes in the family routine --- p.101
Summary of findings --- p.103
Chapter (V). --- Differences between the initial and second interviews --- p.105
Chapter CHAPTER FIVE --- CONCLUSION
Limitations Of The Study --- p.108
Implications For Nursing Practice --- p.112
Recommendations For Future Research --- p.114
Conclusion --- p.115
REFERENCES --- p.117
APPENDIX I - PERSONAL DATA FORM --- p.129
APPENDIX II - INTERVIEW SCHEDULE --- p.130
APPENDIX III - CONSENT FORM --- p.134
APPENDIX IV - SAMPLE SCRIPT OF INTERVIEWS WITH MOTHERS --- p.135
APPENDIX V - MATRICES ON MOTHERS AND FATHERS --- p.140
APPENDIX VI - SAMPLE OF FIELD NOTES FOR MOTHERS AND FATHERS --- p.143

Acute lymphoblastic leukemia (ALL) encompasses a spectrum of lymphoid progenitors that have undergone malignant transformation and clonal proliferation at various stages of differentiation. Some cases of ALL have been documented to have prenatal origins and in particular neonatal exposure to various environmental pollutants is associated with increased disease risk, including childhood lymphoma and leukemia. Dibenzo[def,p]chrysene (DBC) is a polycyclic aromatic hydrocarbon (PAH) and in our laboratory has been established as a transplacental carcinogen in mice, producing aggressive T-cell lymphoblastic lymphomas, lung, liver, uterine, ovarian, and testicular lesions, depending on timing and dose of exposure. Investigation of the transplacental and translactational transfer of DBC was warranted following a cross-foster experiment demonstrating the greatest tumorigenic response occurred in offspring both gestating in and nursed by an exposed female. [¹⁴C]-DBC (GD17) dosing was utilized to examine time-dependent alterations of [¹⁴C] in maternal and fetal tissues, excreta, and residual levels at weaning. Fetal tissue levels of [¹⁴C]-DBC equivalents were 10-fold lower than maternal tissue, and after weaning the residual body burden was roughly equivalent in offspring exposed only in utero or only via lactation. Certain bioactive food components, including indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), and sulforaphane (SFN) from cruciferous vegetables have been shown to target cellular pathways regulating carcinogenesis. In the above mentioned DBC initiated model of carcinogenesis, I3C is an effective transplacental chemopreventive agent. We sought to extend our chemoprevention studies in mice to a human neoplasm in cell culture, analogous to the observed murine T-cell lymphomas. Treatment of the human T-ALL cell line CCRF-CEM (CEM) with I3C reduced cell proliferation and viability only at supraphysiologic concentrations whereas DIM, the primary acid condensation product of I3C, had a marked effect at low micromolar concentrations in vitro and reduced growth of CEM xenografts in vivo. Additional T-ALL lines, selected to represent the heterogeneity of the disease, (CCRF-HSB2, Jurkat, and SUP-T1) responded similarly in vitro, demonstrating a potential therapeutic value of DIM in T-ALL. Given that epigenetic reprograming is especially active during fetal development and that DNA hypermethylation contributes to the etiology of T-ALL we examined genome-wide DNA methylation in CEM. Differential methylation analysis revealed that DIM and I3C alter CpG methylation in unique, yet overlapping, gene targets. DIM treated cells exhibited a dose-dependent decrease in hypermethylation, an observation consistent with an epigenetic mechanism of cancer suppression. Pyroseqencing and RTPCR technologies were utilized to validate changes in DNA methylation and to compare these patterns with a transcriptional response in both novel targets and candidate genes selected from the literature. Collectively, these studies merited returning to the murine transplacental model for further investigation of genetic and epigenetic changes upon maternal dietary intervention with I3C. More importantly we incorporated whole cruciferous vegetable diets (10% broccoli sprouts or 10% Brussels sprouts), SFN diet, or the combination of SFN and I3C, in order to examine matrix and mixture effects. Preliminary analysis suggests a worse prognosis for those animals exposed in utero to SFN or the whole foods, especially males. As this is the first study to administer SFN or whole cruciferous vegetables in a transplacental model of carcinogenesis, our results warrant further study on the concentration dependent influence of these potent phytochemicals during the perinatal window.
Graduation date: 2012

Leung Kam Tong.
Thesis submitted in: September 2006.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 79-95).
Abstracts in English and Chinese.
Abstract --- p.i
Abstract (Chinese) --- p.iii
Acknowledgements --- p.v
Table of contents --- p.vi
List of figures --- p.ix
List of abbreviations --- p.xii
Chapter Chapter 1 --- General Introduction --- p.1
Chapter 1.1 --- Acute lymphoblastic leukemia --- p.1
Chapter 1.2 --- T-cell acute lymphoblastic leukemia --- p.2
Chapter 1.2.1 --- Chemotherapy --- p.2
Chapter 1.2.1.1 --- Induction therapy --- p.2
Chapter 1.2.1.2 --- Intensification therapy --- p.3
Chapter 1.2.1.3 --- Maintenance therapy --- p.3
Chapter 1.2.2 --- Chemoresistance in T-ALL --- p.3
Chapter 1.3 --- Apoptosis and chemoresistance --- p.5
Chapter 1.3.1 --- "Initiation, execution and regulation of apoptosis" --- p.5
Chapter 1.3.1.1 --- Initiation of apoptosis --- p.5
Chapter 1.3.1.2 --- Execution of apoptosis --- p.7
Chapter 1.3.1.3 --- Regulation of apoptosis --- p.7
Chapter 1.3.2 --- Mechanisms of resistance to apoptosis --- p.9
Chapter 1.3.2.1 --- Overexpression of pro-survival proteins --- p.9
Chapter 1.3.2.2 --- Downregulation and mutation of pro-apoptotic proteins --- p.11
Chapter 1.3.2.3 --- Other mechanisms --- p.13
Chapter 1.4 --- Bcl-2 interating mediator of cell death --- p.14
Chapter 1.4.1 --- Role of Bim in apoptosis --- p.16
Chapter 1.4.2 --- Regulation of Bim --- p.17
Chapter 1.4.2.1 --- Transcriptional regulation of Bim --- p.18
Chapter 1.4.2.2 --- Post-transcriptional regulation of Bim --- p.18
Chapter 1.5 --- c-Jun N-terminal kinase --- p.20
Chapter 1.5.1 --- Pro-apoptotic role of JNK --- p.21
Chapter 1.5.2 --- Anti-apoptotic role of JNK --- p.21
Chapter 1.6 --- Hypotheses --- p.22
Chapter Chapter 2 --- Materials and Methods --- p.23
Chapter 2.1 --- Cell culture --- p.23
Chapter 2.2 --- Induction of quantification of apoptosis --- p.24
Chapter 2.3 --- Determination of caspase activities --- p.24
Chapter 2.4 --- Western blotting --- p.25
Chapter 2.4.1 --- Protein extraction and determination of protein concentration --- p.25
Chapter 2.4.2 --- SDS-PAGE and immunodetection --- p.26
Chapter 2.5 --- Cell-free apoptosis reactions --- p.27
Chapter 2.6 --- Analysis of mitochondrial membrane potential --- p.27
Chapter 2.7 --- Transient transfection of Sup-Tl cells --- p.28
Chapter 2.8 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.28
Chapter 2.8.1 --- RNA isolation --- p.28
Chapter 2.8.2 --- Synthesis of first-strand cDNA --- p.29
Chapter 2.8.3 --- Polymerase chain reaction --- p.29
Chapter 2.9 --- Alkaline phosphatase digestion of Bim --- p.30
Chapter Chapter 3 --- Results --- p.31
Chapter 3.1 --- The T-ALL cell line Sup-Tl is resistant to etoposide-induced apoptosis --- p.31
Chapter 3.2 --- Sup-Tl cells are resistant to etoposide-induced caspase activation --- p.40
Chapter 3.3 --- Sup-Tl cells are insusceptible to etoposide-induced mitochondrial alterations --- p.46
Chapter 3.4 --- BimEL is required for etoposide-induced apoptosis in Sup-Tl cells --- p.51
Chapter 3.5 --- The reduced level of BimEL in Sup-Tl cells is owing to the presence of constitutively active JNK --- p.58
Chapter Chapter 4 --- Discussion --- p.67
References --- p.79

Tese de doutoramento, Ciências Biomédicas (Ciências Morfológicas), Universidade de Lisboa, Faculdade de Medicina, 2011
Acute lymphoblastic leukemia (ALL) is the most frequent cancer found in children and results from the clonal expansion of transformed lymphoid precursors. Approximately 15% of pediatric ALL patients present with a T-cell phenotype (T-ALL). Despite the recent improvements in the treatment of T-ALL, there are still a high number of relapses and the intensive chemotherapeutic regiments used are associated with long-term severe complications. In order to develop new therapeutic strategies that can further increase efficacy while reducing side effects, one needs to better understand the pathobiology of T-ALL. In particular, it is necessary to understand how microenvironmental and cell-autonomous mechanisms influence the initiation and the progression of leukemia. The present thesis has the preocupation of exploring the mechanisms by which both an extracellular cue (IL-4) and a cell-intrinsic transcription factor (TAL1) may partake in leukemia development and maintenance. Interleukin-4 (IL-4) is a γ-common chain cytokine produced within the bone marrow microenvironment that is known to promote the in vitro proliferation of T-ALL cells. In Chapter 2, we present evidence that IL-4 induces primary T-ALL cell cycle progression from G0/G1 into S and G2/M, by up-regulating cyclin D2, E and A and down-regulating the cyclin-dependent kinase inhibitor p27kip1. Transfection of T-ALL cells with the VP22-p27kip1 fusion protein, which is able to translocate into the cytoplasm and nucleus of target cells, abrogates IL-4-mediated proliferation. This indicates that p27kip1 downregulation is mandatory for cell cycle progression of T-ALL cells stimulated with IL-4. Furthermore, IL-4 stimulates mTOR activation, as determined by increased phosphorylation of its downstream targets p70S6K, S6 and 4E-BP1. Inhibition of mTOR signaling with rapamycin prevents IL-4-induced T-ALL cell growth, cell cycle progression and proliferation. Our results identify mTOR as a critical regulator of IL-4-mediated effects in T-ALL cells and support the rationale for using mTOR pharmacological inhibitors in T-ALL therapy (Cardoso et al. Leukemia 2009). The basic helix-loop-helix transcription factor TAL1 is aberrantly expressed in up to 65% of T-ALL patients. LMO2, a Lim-only domain protein, is often co-expressed ectopically with TAL1 in this malignancy. These genes appear to have leukemogenic potential, since both TAL1 and LMO2 transgenic mice develop leukemias of T-cell phenotype. However, it is still unclear whether TAL1 is effectively leukemogenic in humans, or whether merely participates as a secondary event in the transformation Abstract xiii process in T-ALL. To address this question, we transduced hematopoietic progenitors with TAL1 and/or LMO2 and co-cultured them with OP9-Dll1 stromal cells, which have the capacity to induce T-cell differentiation in vitro. We found that TAL1 and LMO2 genes deregulate human T-cell differentiation in stromal cell co-cultures. Interestingly, the coordinated expression of both TAL1 and LMO2 led to a relative increase in CD3+CD4+CD8+ T-cell precursors with increased cell size. This observation is particularly interesting given that TAL1-expressing patients normally display a similar phenotype. These preliminary results show that TAL1 and LMO2 can disrupt normal human T-cell development, therefore likely predisposing thymocytes to malignant transformation (Chapter 3). In our effort to characterize the mechanisms by which TAL1 might promote T-cell leukemogenesis, we developed a TAL1 inducible system, by fusing TAL1 with the hormone binding domain (HBD) of the estrogen receptor (ER), which we expressed in a TAL1-negative T-cell line. Upon 4-Hydroxi-Tamoxifen (4OHT) treatment, ER-TAL1 fusion protein is able to translocate into the nucleus and consequently trigger its transcriptional program. Gene expression profiling of 4OHT-treated HPB-ALL cells stably transduced with the ER-TAL1 fusion revealed a total of 26 genes up- or down-regulated by TAL1 activation, in at least two independent experiments. We selected seven of those genes on the basis of their function/potential interest in cancer and confirmed the differential expression of three (CASZ1, DMGDH and OR5M3) by qRT-PCR. Accordingly, transfection of another TAL1-negative T-ALL cell line, P12, with TAL1, also led to increased expression of the validated TAL1 target genes. The possible involvement of CASZ1 in TAL1-mediated anti-apoptotic and proliferative effects in T-ALL cells was subsequently investigated. Knock-down of TAL1 with siRNA in the TAL1-positive T-ALL cell line Jurkat decreased the expression of CASZ1, correlating with loss of cell viability. Moreover, CASZ1 knockdown in Jurkat cells led to functional effects similar to those of TAL1 knockdown, namely a decrease in survival and proliferation. Overall, these studies allowed the identification of three novel TAL1 downstream targets, likely with functional relevance for TAL1-mediated leukemogenic potential (Chapter 4). TAL1 binds to repressive chromatin complexes, namely involving HDAC1, and incubation with HDAC inhibitors (HDACis) promotes apoptosis of leukemia cells derived from TAL1 transgenic mice. In Chapter 5, we evaluated the impact of HDACis on TAL1 from a somewhat different perspective, namely by analyzing their impact on Abstract xiv TAL1 expression rather than transcriptional activity. We found that incubation of T-ALL cells with HDACis strikingly down-regulates TAL1 protein expression. This is due to decreased TAL1 gene transcription in cells with an intact TAL1 locus, and to impaired TAL1 mRNA translation in cells that harbor the TAL1d deletion. Importantly, HDACi-induced apoptosis of T-ALL cells is significantly reversed by TAL1 forced over-expression. Our results indicate that the HDACi-mediated apoptotic program in T-ALL cells is partially dependent on the down-regulation of TAL1 expression, and suggest that integration of HDACis into T-ALL treatment protocol may be of potential therapeutic benefit (Cardoso et al, Leukemia 2011, advance online publication). Taken together, the results described in this thesis highlight the importance that microenviromental factors, such as IL-4, might have in the progression of T-ALL (for instance, by activating mTOR and promoting cell cycle progression), and hint on the importance that cell-autonomous factors, such as TAL1 and LMO2, may have in predisposing T-cells for malignant transformation and promoting survival of T-ALL cells. Importantly, our results further demonstrate that both extracellular cues and intracellular molecular lesions can constitute targets for therapeutic intervention in T-ALL.
A Leucemia Linfoblástica Aguda (LLA) é o cancro mais frequente em crianças, resultando da expansão clonal maligna de precursores linfóides. Aproximadamente 15% dos doentes com LLA apresentam marcadores de células T (LLA-T). Embora os regimes quimioterápicos actualmente em uso sejam bastante eficazes, existe ainda um número significativo de doentes que recidivam. Além disso, os regimes intensivos de quimioterapia estão normalmente associados a efeitos secundários consideráveis a médio e longo prazo. Para melhor perceber a biologia da LLA-T e determinar novos alvos terapêuticos é necessário perceber em que medida factores microambientais e mecanismos intra-celulares influenciam a génese e a progressão da leucemia. A presente tese procura identificar os mecanismos pelos quais tanto um factor extracelular (IL-4) como um factor de transcrição celular (TAL1) podem participar no desenvolvimento e progressão da leucemia. A Interleucina-4 (IL-4) é uma citocina da cadeia comum-γ, produzida na medula óssea, que estimula a proliferação in vitro de células LLA-T. No capítulo 2, demonstramos que a IL-4 induz a progressão do ciclo celular da fase G0/G1 para as fases S e G2/M em células LLA-T primárias, devido ao aumento da expressão das ciclinas D2, E e A e à diminuição de expressão do inibidor de cinases dependentes de ciclinas p27Kip1. A transfecção de células LLA-T com a proteína de fusão VP22-p27Kip1, que é capaz de translocar para o citoplasma e núcleo das células alvo, impede a proliferação mediada por IL-4. Além disso, a IL-4 estimula a activação de mTOR, como demonstra o aumento de fosforilação dos seus alvos p70S6K, S6 e 4E-BP1. A inibição da sinalização mediada por mTOR com rapamicina impede o crescimento celular, a progressão do ciclo celular e a proliferação de células LLA-T estimuladas por IL-4. Estes resultados identificam mTOR como um regulador dos efeitos moleculares e celulares promovidos por IL-4 em células LLA-T e fortalecem a hipótese do uso de inibidores farmacológicos de mTOR no tratamento de doentes com LLA-T (Capítulo 2; Cardoso et al. Leukemia 2009). O factor de transcrição hélice-volta-hélice TAL1 é aberrantemente expresso em quase 65% dos doentes com LLA-T. A proteína “Lim-only domain” LMO2, é geralmente co-expressa com TAL1 neste tipo de leucemia. Estes genes parecem contribuir para a génese da leucemia, visto que ratinhos transgénicos para TAL1 e LMO2 desenvolvem leucemia com fenótipo de células T. No entanto, não se confirmou Resumo x até hoje se TAL1 estará envolvido na génese da leucemia em seres humanos, ou apenas secundariamente activado como resultado do processo de transformação em LLA-T. Para responder a esta questão, transduzimos progenitores hematopoiéticos com TAL1 e/ou LMO2 e co-cultivamos estes progenitores com células estromais OP9-Dll1, que têm a capacidade de induzir a diferenciação de células T in vitro. Descobrimos que os genes TAL1 e LMO2 desregulam a diferenciação de células T em co-cultura com células estromais. A expressão coordenada destes dois genes leva a um pequeno aumento de precursores T CD3+CD4+CD8+ de tamanho celular aumentado. Esta observação é particularmente interessante visto que se sabe que os blastos de pacientes com LLA-T que expressam TAL1 apresentam um imunofenótipo idêntico. Estes resultados preliminares mostram que TAL1 e LMO2 podem perturbar o normal desenvolvimento de células T humanas, possivelmente predispondo os timócitos para transformação maligna (Capítulo 3). Com o intuito de identificar e caracterizar os eventuais alvos transcricionais através dos quais TAL1 poderá gerar leucemia de células T, desenvolvemos um sistema indutível em que a fusão de TAL1 com o domínio de ligação a hormonas (DLH) do receptor de estrogénio (RE) permite a regulação fina da actividade de TAL1 numa linha celular T sem expressão de TAL1 endógeno. Após tratamento com 4-Hidróxi-Tamoxifeno (4HT), a proteína de fusão RE-TAL1 consegue translocar para o núcleo celular e consequentemente activar o seu programa de trancrição. O perfil de expressão da linha celular HPB-ALL estavelmente transduzida com a fusão RE-TAL1 e tratada com 4HT revelou um total de 26 genes cuja expressão aumentou ou diminuiu após activação de TAL1, em pelo menos 2 experiências independentes. Seleccionámos sete genes com base na sua função e potencial interesse em cancro e confirmámos a expressão diferencial de três (CASZ1, DMGDH e OR5M3) por PCR quantitativo em tempo real. A transfecção de TAL1 numa outra linha celular LLA-T sem expressão deste gene (P12), resulta igualmente num aumento da expressão destes genes. O possível envolvimento de CASZ1 nos efeitos anti-apoptóticos e proliferativos mediados por TAL1 em células LLA-T também foi investigado. A diminuição da expressão de TAL1 com siRNA na linha celular Jurkat, que expressa TAL1 abundantemente, diminui significativamente a expressão de CASZ1, e a perda de expressão correlacionacom perda de viabilidade celular. Acresce que a diminuição da expressão de CASZ1 em células Jurkat tem efeitos funcionais semelhantes aos que ocorrem após redução da expressão de TAL1, nomeadamente diminuição da viabilidade celular e proliferação. Resumo xi No geral, estes estudos permitiram a identificação de três novos genes alvo de TAL1, com possível relevância funcional no contexto do potencial poder oncogénico de TAL1 (Capítulo 4) TAL1 parece ter não apenas um papel de regulador positivo mas também de repressor da transcrição. Não é, portanto, de surpreender que tenha sido demonstrado anteriormente que TAL1 se pode associar a complexos de cromatina repressivos, nomeadamente HDAC1, e que a incubação com inibidores de HDAC (iHDAC) induz apoptose em células leucémicas derivadas de ratinhos transgénicos para TAL1. No capítulo 5, avaliamos o impacto dos iHDAC em TAL1 numa perspectiva diferente, nomeadamente analisando o seu impacto na expressão de TAL1 e não no impacto na actividade transcricional. Os nossos estudos revelam que a incubação de células LLA-T com iHDAC diminui drasticamente a expressão da proteína TAL1. Este efeito é devido à diminuição da transcrição do gene TAL1 em células que mantêm o locus TAL1 intacto, mas também devido à diminui da tradução de mRNAs TAL1 em células que contêm a delecção TAL1d. Igualmente importante é o facto da apoptose induzida pelos iHDAC ser inibida pela sobre-expressão de TAL1. Os nossos resultados indicam que o programa apoptótico promovido pelos iHDAC em LLA-T é parcialmente dependente da diminuição da expressão de TAL1 e sugerem que a integração de iHDAC no protocolo de tratamento de doentes LLA-T pode trazer benefícios terapêuticos (Cardoso et al, Leukemia 2011, advance online publication). O conjunto dos estudos descritos nesta dissertação destacam a importância que os factores micro-ambientais, como IL-4, podem ter na progressão de LLA-T (por exemplo, activando mTOR e promovendo a progressão no ciclo celular) e mostram a importância que factores celulares como TAL1, e também LMO2, podem ter na predisposição de células T para a transformação maligna e na sobrevivência das células LLA-T. Finalmente, os resultados desta tese demonstram que tanto factores extracelulares como lesões intracelulares podem constituir alvos promissores para intervenção terapêutica em LLA-T.

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

Leukemia is the most common malignant disease in children patients. In our laboratory (CLIP) a novel subtype of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) with lineage switch during early phase of treatment towards myeloid lineage (swALL) was recently documented. SwALL incidence is almost 4 % of all BCP-ALLs (Slámová et al., 2014). DNA methylation (presence of 5-methylcytosine) is together with post-translational histone modifications and non- coding RNAs an epigenetic mechanism which regulates gene expression without changes of genetic code. DNA methylation is easily detected by bisulphite conversion and subsequent sequencing. The aim of this work was to compare genome-wide DNA methylation patterns between patients with swALL and control BCP-ALLs. The first step in achieving that was revision and improvement of bioinformatic processing protocol for eRRBS data from massive parallel sequencing. To improve the sequence adapter trimming I tested four bioinformatic tools - FAR, cutadapt, Trimmomatic and fastx_clipper. I implemented the fastest and most effective - Trimmomatic into the processing protocol. As a next step I analysed the data with improved protocol and extended the analysis in R programming environment where the comparison of studied groups was performed. The comparison of...