Orientadores: José Andrés Yunes, Ana Carolina de Mattos Zeri / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T13:46:58Z (GMT). No. of bitstreams: 1 Canevarolo_RafaelRenatino_M.pdf: 3376497 bytes, checksum: f3e8edf217e0d57089ffcffbe4a8097a (MD5) Previous issue date: 2012 / Resumo: O uso intensivo e combinado de diferentes quimioterápicos tem permitido a cura de 70-80% das leucemias linfóides agudas (LLA) da infância, sendo que a recaída da doença decorre em grande parte da resistência intrínseca das células leucêmicas à quimioterapia. Alguns dos quimioterápicos utilizados na LLA são inibidores metabólicos, como o metotrexato (MTX), antagonista do ácido fólico, que impede a divisão celular ao inibir a síntese de nucleotídeos. Em uma abordagem metabolômica, foi investigada a associação entre linhagens leucêmicas resistentes ou sensíveis ao MTX e metabólitos biondicadores de cada um destes fenótipos. Seis linhagens celulares B-derivadas e oito T-derivadas foram classificadas como sendo resistentes ou sensíveis ao MTX pelo método do 3-(4,5-dimetiltiazol-2il)-2,5-difenil brometo de tetrazolina (MTT) após 48h de co-cultura com diferentes concentrações da droga. Cinco linhagens foram classificadas como resistentes e nove como sensíveis ao MTX. Após 24h de cultura na presença ou ausência de MTX (ambos em triplicata), os metabólitos intracelulares das linhagens foram acessados por ressonância magnética nuclear (RMN) numa abordagem metabolômica. Ao total, oitenta e quatro metabólitos foram quantificados, dos quais 72 foram também identificados. A análise de componentes principais (PCA) não conseguiu segregar as amostras de acordo com sua resistência, ao passo que a análise discriminante por mínimos quadrados parciais (PLS-DA) foi efetiva nesta separação. Os metabólitos mais relevantes para a construção dos modelos de classificação quanto à resistência ao MTX, tanto para amostras tratadas quanto controles foram: ATP, dimetilglicina, fosfocolina e sarcosina (associados à resistência); carnitina, CB-09 (composto não identificado), colato, fumarato, glicocolato, lactato, malato e succinato (associados à sensibilidade ao MTX). A capacidade de classificar corretamente as amostras em sensíveis ou resistentes foi obtida com a construção de curvas da característica operativa do receptor (ROC) para os metabólitos individualmente. Os metabólitos com desempenho bom ou excelente na análise ROC (AUC>0,8) foram selecionados para comporem "testes diagnósticos" de classificação de amostras. De todas as combinações possíveis dentre os metabólitos selecionados, o teste que considerou a combinação de carnitina, sarcosina e succinato em amostras não tratadas com MTX apresentou sensibilidade de 100% (identificou todas as 15 amostras resistentes) e especificidade de 92,3% ao classificar corretamente 24 de 26 amostras sensíveis. O melhor teste diagnóstico para amostras tratadas com MTX considerou as concentrações de CB-MTX, glicocolato, sarcosina e succinato; apresentou sensibilidade de 100% (identificou as 15 amostras resistentes) e especificidade de 85,2%, equivocando-se na classificação de 4 dentre 27 amostras sensíveis. As concentrações metabólicas diferenciais apontaram para uma superativação dos metabolismos energético e de lipídeos em linhagens sensíveis ao MTX, ao passo que linhagens resistentes teriam superativado o metabolismo da glicina. As análises metabolômicas e de integração bioquímica dos metabólitos revelaram interações gênicas, enzimáticas e metabólicas que podem estar alteradas em linhagens sensíveis ou resistentes ao MTX, bem como permitiram a especulação sobre possíveis alvos moleculares que poderiam tornar sensíveis células resistentes ao quimioterápico / Abstract: The intensive use of different and combined chemotherapics has allowed curing 70-80% of pediatric acute lymphoblastic leukemia (ALL), and the relapse of the disease stems largely from the intrinsic resistance of leukemic cells to chemotherapy. Some of the chemotherapics used in ALL are metabolic inhibitors such as methotrexate (MTX), a folic acid antagonist, which prevents cell division by inhibiting the synthesis of nucleotides. The association between leukemic strains resistant or sensitive to MTX and the metabolites associated with each of these phenotypes were investigated. Six B- and eight T-derived cell lines were classified as resistant or sensitive to MTX by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, after 48h in co-culture with different concentrations of the drug. Five lineages were classified as resistant, and nine as sensitive to MTX. After 24 hours of culture in the presence or absence of MTX (both in triplicates), the intracellular metabolites of the lineages were assessed by nuclear magnetic resonance (NMR), in a metabolomic approach. In total, 84 metabolites were quantified, 72 of which were also identified. The principal component analysis (PCA) did not segregate the samples according to their resistance, whereas the supervised partial least square discriminate analysis (PLS-DA) was effective in this separation. ATP, dimethylglycine, sarcosine and phosphocholine were associated with MTX resistance in both models constructed for treated and untreated samples, whereas carnitine, CB-MTX (unidentified compound), cholate, fumarate, glycocholate, lactate, malate and succinate were associated with sensitivity to MTX. The ability to correctly classify the samples into sensitive or resistant groups was checked with the construction of the receiver operating characteristic (ROC) curves for metabolites individually. Metabolites with good or excellent performance in ROC analysis (AUC> 0.8) were selected to compose "diagnostic tests" for classifying samples. Of all the possible combinations among the selected metabolites, the test composed by the comination of carnitine, sarcosine and succinate in untreated samples exhibited sensitivity of 100% (identified all 15 resistant samples) and specificity of 92.3% in classifying correctly 24 of 26 sensitive samples. The best diagnostic test for samples treated with MTX took into consideration concentrations of CB-MTX, glycocholate, sarcosina, succinato. It had a sensitivity of 100% (identified 15 resistant samples) and specificity of 85.2%, classifying incorrectly 4 out of 27 sensitive samples. Differential metabolic concentrations pointed to an over activation of energy and lipids metabolism in MTX-sensitive strains, whereas resistant strains seemed to have overactive the glycine metabolism. Metabolomic and biochemical integration analysis revealed genetic, enzymatic and metabolic interactions that might be altered in strains sensitive or resistant to MTX, as well as allowed speculations about possible molecular targets on which intervention could make resistant cells susceptible to chemotherapy / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientadores: José Andrés Yunes, Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T20:56:25Z (GMT). No. of bitstreams: 1 Zenatti_PriscilaPini_D.pdf: 14781518 bytes, checksum: 2324b48b89768b42bcd5bf002d30a510 (MD5) Previous issue date: 2012 / Resumo: A IL-7 é uma citocina essencial para o desenvolvimento, sobrevivência, e proliferação dos timócitos prematuros normais no timo e linfócitos T maduros nos órgãos linfoides periféricos. O receptor da IL-7 é um heterodímero constituído pela IL-7Ra (codificado pelo gene IL7R) e IL-2Ry. A IL-2Ry participa também dos receptores da IL-2, IL-4, IL-9, IL-15 e IL-21, sendo por isso conhecida como cadeia comum gama ou yc. A sinalização via IL-7 é imprescindível para o processo de rearranjo V(D)J dos receptores de células T (TCR), pois leva à modificação da cromatina que permite o acesso da recombinase aos loci TCR no DNA Camundongos defeituosos para a cadeia alfa do receptor da IL-7 (IL-7Ra) desenvolvem imunodeficiências devido à falta de células T. A via IL-7/IL-7Ra é também importante para proliferação e sobrevivência da leucemia linfóide aguda de células T (LLA-T). Suspeitando da existência de mutações em IL7R resultando em ganho de função e hiperativação da via IL-7/IL-7Ra, procurou-se por mutações no gene do IL-7Ra em mais de 50 casos de LLA-T pediátrica. Procurou-se também mutações no domínio de autoinibição das JAK1 e JAK3, moléculas associadas ao IL-7Ra e IL-2Ry, respectivamente. Aproximadamente 9% das LLA-T pediátricas apresentaram mutações no IL-7Ra, resritas a uma região estreita do éxon 6, que resultaram, na maioria dos casos, em inserções de uma cisteína no domínio transmembrana/justamembrana da proteína. A presença dessa cisteína leva à homodimerização das cadeias IL-7Ra mediante a formação de pontes de dissulfeto e à ativação constitutiva do IL-7Ra independente de seu ligante, conforme verificado pela fosforilação de JAK1, STAT5, AKT e BAD e análise de mutantes artificiais do IL-7Ra com subtração ou adição de resíduo de cisteína. Verificou-se também que o local de inserção da cisteína é crítico para que a homodimerização das cadeias IL-7Ra mutantes resulte em ativação da via JAK/STAT. Receptores imitados tiveram um efeito transformante nas linhagens celulares Dl e Ba/F3, que sobreviveram na ausência dos fator de crescimento IL-7 e IL-3, respectivamente. Além disso, células Dl transduzidas com IL7R mutante, quando injetadas em camundongos knockout (KO) para IL7, causaram esplenomegalia, metástase e tumor no local da injeção. Resultados semelhantes foram obtidos com a expressão ectópica do IL7R mutante em células progenitoras hematopoiéticas de camundongos knockout para IL7R, JAK3 e/ou IL2RG, demonstrando que o homodímero IL-7Ra mutante atua independentemente dessas moléculas. Em conclusão, mutações no IL7R contribuem para a leucemogenese em 9% das crianças com LLA-T. Espera-se que a melhor caracterização do mecanismo responsável pela ativação constitutiva do IL-7Ra mutante abra caminho para o desenho de novas drogas e anticorpos monoclonais, o que permitirá avaliar o valor terapêutico do bloqueio/inibição do IL-7Ra mutante nas LLA-T / .Abstract: The IL-7, a product of stromal cells, is normally required for T cells development and for survival of mature peripheral T cells. The IL-7R consists of two components, the IL-7Ra (encoded by IL7R) and the common gamma chain (yc), or IL-2Ry, that is shared by receptors for IL-2, IL-4, IL-9, IL-15 and IL-21. The IL-7 signaling has a role in V(D)J recombination in developing T and B cells by controlling access of the V(D)J recombinase to the locus. IL-7Ra deficiency mice showed a diminished T cell number and impaired lymphocyte development. Further, the IL-7/IL-7Ra pathway is important for T-acute lymphoblastic leukemia (T-ALL) proliferation and survival. Hypothesizing that IL7R gain-of-function mutation could be occurring in T-ALL, 50 T-ALL samples were analyzed for mutations. The kinases JAK1 and JAK3, mat are associated with IL-7Ra and yc, respectively, were also studied for mutations. About 9% of childhood T-ALL presented mutations at the transmembrane domain encoded by éxon 6, and all of mem were in-frame insertions and deletions. In all but three cases there was an insertion of cysteine mat is essential for disulfide bond formation and constitutive activation of the receptor independent of IL-7. The constitutive signaling was confirmed by phosphorylation of JAK1, STAT5, AKT and BAD, and analysis of IL-7Ra artificial mutants with or without cysteine. The position of cysteine insertion is very important to disulfide bond formation, to activate the JAK/STAT pathway and to support the proliferation of Ba/F3 and Dl cell lines in the absence of cytokine. Moreover, IL7R mutant transduced Dl cells injected into ILT1' mice caused splenomegaly, metastasis and tumor at the injection site. Similar results were obtained with the ectopic expression of the IL7R mutant in hematopoietic progenitor cells of IL7K1', JAK3'1' and/or IL2RG'1' mice, demonstrating that the mutant homodimer IL-7Ra operates independently of these molecules. In conclusion, mutations in ÚÍQIL7R contribute to leukemo genes is in 9% of children with ALL-T. We hope mat a better comprehension of the mechanism responsible for the constitutive activation of IL-7Ra mutant opens new perspectives into the design of new drugs and monoclonal antibodies, which may turn into valuable therapeutic treatment / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Acute lymphoblastic leukemia (ALL) has poor prognosis in older/elderly adults and in high-risk/relapsed disease. Recommended treatment of ALL was evaluated (study I-IV). Data was obtained from the Swedish Acute Leukemia registries and from patient records. I. We assessed ALL relapse treatment and outcome in 76 patients aged 15-65 years (y). Complete remission (CR) was achieved in 50/71 patients (70%). Of them, 29 underwent allogeneic hematopoietic stem cell transplantation (hSCT). Five year overall survival (OS) was 15%, but close to 50% in 19 patients <35y after hSCT. II. We studied outcome of treatment with the Hyper-CVAD protocol in 19 of 24 patients with T-ALL aged 18-72y. CR was reached in 89%, but 5y leukemia-free survival was only 29%, and 20% in 15 patients not transplanted in CR1. Six patients received hSCT in CR2. Finally, 5y OS in all 19 patients was 47%. The only negative prognostic factor found was age ≥35y. III. We evaluated minimal residual disease (MRD) monitoring in 35 patients with Philadelphia (Ph) negative B-ALL aged 46-79y and treated with the ABCDV protocol. The CR rate was 91%. MRD was measured by flow cytometry in 73% in CR1 (MRD1) and omitted in those >70y or with high-risk ALL. Five patients received hSCT (only one due to MRD). Five year OS in the whole cohort was 47%. Continuous CR but not OS was improved in patients with MRD1 <0.1 %. IV. We studied 155 patients with ALL (Ph+ in 35%) aged 55-85y and treated with remission induction/palliation (124/31). Both, intensive, and palliative treatment resulted in the CR rates of 70/83/16% and 3y OS of 26/32/3%. OS was negatively influenced by age and platelet count ≤35×109/L (but not Ph+). OS was not enhanced by introduction of an age-adapted protocol. We concluded that intensive treatment with subsequent allogeneic hSCT is the most reasonable option in younger patients with ALL recurrence (I). Hyper-CVAD has low relapse-preventing efficacy (II). MRD guided intensification is probably feasible in only a minority of older patients (III). Prognosis in elderly ALL is poor, but no longer impaired by Ph+ (IV).

Orientador: José Andrés Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T07:51:03Z (GMT). No. of bitstreams: 1 Ganazza_MonicaAparecida_D.pdf: 9547403 bytes, checksum: 7d821874cefad54cd4ae846262dbb09a (MD5) Previous issue date: 2014 / Resumo: A leucemia linfóide aguda (LLA) é o câncer mais comum da infância. Os atuais protocolos de tratamento da LLA levam à cura em até 80% dos casos e parte do sucesso se deve à aplicação de diferentes tratamentos para os pacientes estratificados em diferentes grupos de risco. Contudo, pacientes considerados em remissão podem apresentar conteúdo substancial de células neoplásicas, a chamada doença residual mínima (DRM), cuja proliferação está associada com a recaída da doença e que podem estar em níveis indetectáveis pelas técnicas convencionais de análise morfológica. O atual protocolo do Grupo Brasileiro de Tratamento da Leucemia Infantil (GBTLI-LLA-2009) utiliza as análises da DRM para estratificar os pacientes em grupos de risco e assim determinar seu tratamento. Desta maneira o presente estudo objetivou (1) estudar a DRM por método qualitativo e quantitativo (RQ-PCR) em amostras D35 de 120 pacientes prospectivos admitidos para tratamento com o protocolo GBTLI-LLA-2009 no Centro Boldrini entre dezembro de 2009 e julho de 2013 (2) determinar a freqüência e sensibilidade dos rearranjos gênicos de imunoglobulinas e receptores de células T (3) analisar a realocação dos pacientes em grupos de risco em três pontos do tratamento (D8, D15 e D35) (4) buscar associações entre resultados de DRM no D35 e variáveis clínico-biológicas dos pacientes (5) analisar a sobrevida global nos pacientes (6) analisar o grau de concordância entre os resultados obtidos por PCR qualitativo e RQ-PCR no D35. Para a DRM no D15 as análises foram realizadas por citometria de fluxo pela equipe multidisciplinar do Centro Boldrini, já para DRM no D35 as análises foram feitas por PCR qualitativo e RQ-PCR com o uso de primers para a região VDJ das imunoglobulinas e receptores de células T. Observou-se que pelo menos 1 rearranjo gênico foi detectado ao diagnóstico para 98% dos casos estudados através de PCR qualitativo. Os rearranjos mais frequentes ao diagnóstico foram IGH , IGK, TCRG e TCRD para os casos de LLA-B derivada e TCRG, TCRD e Sil-Tal e IGH para os casos de LLA-T. Os primers paciente específicos para RQ-PCR mais sensíveis (sensibilidade 1x10-4) foram aqueles envolvendo rearranjos IGH, TCRD, TCRG e IGK. Associação entre presença de DRM no D35 detectada por RQ-PCR e idade dos pacientes (grupo <1 e ?9 anos) foi observada. Presença de DRM ?10-3 no D35 se mostrou relacionada com sobrevida global em pacientes com LLA-B derivada classificados como BR após análises de DRM no D15. Casos de LLA-B derivada classificados como BR após o D15 em sua maioria não apresentaram DRM nos pontos analisados (D15 e D35) sugerindo que a análise da DRM nesses casos seja dispensável. Já para os casos LLA-B derivada classificados como AR após o D15, 7 pacientes considerados bons respondedores no D15 mostraram-se respondedores lentos no D35 ressaltando a importância da análise da DRM nesse ponto do tratamento. Para casos com LLA-T o número reduzido de pacientes impediu qualquer conclusão a respeito. As duas metodologias aplicadas para as análises da DRM no D35 obtiveram concordância de 80% e 100% para LLA-B derivada e LLA-T, respectivamente / Abstract: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. The recent ALL treatment protocols can achieve the complete remission in 80% of cases and this success is due to different treatments for patients stratified into different risk groups. Therefore, patients considered in remission may have substantial contents of neoplasic cells, the minimal residual disease (MRD). The proliferation of such neoplasic cells is associated with disease relapse and they can be undected by conventional methods. The current protocol of the Brazilian Childhood Leukemia Treatment (GBTLI-LLA-2009) uses the analysis of MRD to stratify patients into risk groups and thus determine their treatment. Thus this study aimed to (1) study the MRD for qualitative and quantitative method (RQ-PCR) at D35 from 120 prospective patients admitted for treatment with protocol GBTLI-LLA-2009 at Centro Boldrini between December 2009 and July 2013 (2) determine the frequency and sensitivity of immunoglobulin and T cell receptor gene rearrangements (3) analysis of the relocation of patients into risk groups in three points of treatment (D8, D15 and D35) (4) find associations between results of MRD at D35 and D15 and clinical-biological characteristics of patients (5) analyze the overall survival in patients (6) to analyze the degree of agreement between the results obtained by qualitative PCR and RQ-PCR in D35. MRD analyzes at D15 were performed by flow cytometry by the multidisciplinary team of the Centro Boldrini, MRD at D35 analyzes were performed by qualitative PCR and RQ-PCR using primers for the VDJ region of the immunoglobulin and T cell receptors. At least one gene rearrangement was detected at diagnosis to 98% of the cases studied by qualitative PCR. The most frequent rearrangements at diagnosis were IGH, IGK, TCRG and TCRD for cases of B-ALL derived and TCRG, TCRD and Sil-Tal and IGH in cases of T-ALL. The most sensitive RQ-PCR patient specific primers (sensitivity 1x10-4) were those involving IGH, TCRD, TCRD and IGK rearrangements. Association between presence of MRD in D35 detected by RQ-PCR and age group (<1 and ? 9 years) was observed. Presence of MRD ? 10-3 in D35 showed to be related with overall survival in patients with B-ALL derived classified as BR after analysis of MRD in D15. Cases of B-ALL derived classified as low risk after the D15 mostly showed no MRD in the analyzed points (D15 and D35) suggesting that the analysis of these cases is dispensable. As for the B-ALL derived cases classified as AR after D15, 7 patients considered good responders in D15 proved to be slow responders in D35 emphasizing the importance of analyzing MRD at this point of treatment. For T-ALL cases the small number of patients did not allow any conclusion. Both methodologies for the analysis of DRM in D35 obtained 80% and 100% of agreement for B-ALL derived and T-ALL respectively / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Introduction:Acute Lymphoid Leukemia (ALL) is the most frequent cancer in young people and, if analyzed together with Non-Hodgkin Lymphoma (NHL), we find that they are responsible for at least one third of all cases of childhood cancer. Present-day therapeutic protocols include high doses of glucocorticoids (GC), drugs associate with high potential for elevating intraocular pressure (IOP) and, consequently provoking damage to the fibers of the optic nerve fibers, a pathology classified as cortisone glaucoma. In genetically susceptible patients, ocular hypertension normally occurs some weeks into the use of a steroid but is generally reversible with the suspension of its use. However, depending on the levels of ocular pressure and the duration of ocular hypertension, it can result in optic neuropathology and, in extreme cases, blindness. Since ALL and NHL are oncological disorders with elevated potential for cure of young people with have high life expectancy, the identification of eventual long-term treatment complications could give support to a delineation still lacking in scientific literature, that is, an ophthalmological protocol for these cases. Objective: The aim of this study was to evaluate the behavior of intraocular pressure in pediatric patients treated with GC for the acute lymphoproliferative neoplasias that are most common during childhood and adolescence.Methods: A systematic review of the theme was carried out, followed by a descriptive, prospective study of children and adolescents of both sexes who were diagnosed with ALL and NHL, and who were registered for beginning chemotherapeutic treatment at the Dr. Oswaldo Leite Oncology Center of Sergipe. The inclusion criteria were: diagnosis of ALL or NHL-T confirmed by immunophenotyping of bone marrow or peripheral blood samples (ALL), or immunohistochemistry of material obtained by open biopsy (NHL); age less than 19; no previous chemotherapy; absence of previous diagnosis compatible with glaucoma or any other disorder envolving change in intraocular pressure; no systemic use of GC in the six months preceding diagnosis of ALL or NHL. Patients whose evaluation of IOP might not have been technically adequate, as well as those who expired during the follow-up period, were excluded. Intraocular pressure was measured before treatment (D0), on the eighth (D8), the fourteenth (D14) and twentieth (D28) treatment day. The IOP results above 21 mm Hg were considered to be ocular hypertension. Results: Results of the systematic review indicate the need for new studies, for the review found a total of only three published articles whose results varied between total control of ocular pressure and visual function, to irreversible blindness. The results of our field research involved 15 patients, two of them with ocular hypertension, and with a statistically significant difference of measurements of IOP between D0 vs D8 and D0 vs D14 (p=0.013). Conclusion: The possibility of silent ocular hypertension, with the consequent risk of irreversible blindness, indicates the need to assess the introduction of a protocol for verification of IOP in patients recently diagnosed with ALL and NHL, including weekly exams, at least until the complete cessation of GC use. / Introdução: Leucemia Linfóide Aguda (ALL) é o câncer mais comumente encontrado entre os jovens e, se analisada em conjunto com o Linfoma não-Hodgkin (NHL), são responsáveis por pelo menos um terço dos casos de câncer infantil. Protocolos terapêuticos atuais incluem altas doses de glicocorticóides (GC), droga associada com alto potencial para elevar a pressão intraocular (IOP) e, consequentemente, provocar danos às fibras do nervo óptico, patologia classificada como glaucoma cortisônico. A hipertensão ocular geralmente ocorre com algumas semanas de uso de GC em pacientes geneticamente susceptíveis, mas é geralmente reversível com a descontinuação do tratamento. Entretanto, dependendo dos níveis pressóricos oculares e do tempo de elevação, pode resultar em neuropatia óptica e, em situações extremas, em cegueira. Por serem a ALL e o NHL doenças oncológicas com potencial elevado de cura, em indivíduos jovens com elevada expectativa de vida, a identificação de eventuais complicações de longo prazo decorrentes do tratamento poderá subsidiar o delineamento de um protocolo oftalmológico para esses casos, ainda inexistente na literatura científica. Objetivo: O objetivo deste estudo foi avaliar o comportamento da pressão intraocular em pacientes pediátricos portadores das mais frequentes neoplasias linfoproliferativas agudas da infância e adolescência, e que são tratados com GC. Métodos: Foi feita uma revisão sistemática sobre o tema estudado, seguida por um estudo descritivo, prospectivo, em crianças e adolescentes de ambos os sexos, com diagnóstico de ALL e NHL, matriculados para início de tratamento quimioterápico no Centro de Oncologia de Sergipe Dr. Oswaldo Leite. Os critérios de inclusão foram: diagnóstico de ALL ou NHL-T, confirmada por imunofenotipagem de amostra de medula óssea ou sangue periférico (ALL) ou imuno-histoquímica de material obtido por biópsia aberta (NHL); idade menor de 19 anos; sem quimioterapia anterior; ausência de diagnóstico prévio compatível com glaucoma ou doença anterior relacionada a qualquer mudança na pressão intra-ocular; não uso sistêmico de GC nos seis meses anteriores ao diagnóstico da ALL ou NHL. Pacientes cuja avaliação da pressão intraocular (PIO) pode não ter sido tecnicamente adequada e os que faleceram durante o período de seguimento foram excluídos. Realizaram-se medidas de pressão intraocular antes do tratamento (D0), no oitavo (D8), décimo quarto (D14) e vigésimo (D28) dias de tratamento. Os resultados da PIO acima de 21 mm de Hg foram considerados como hipertensão ocular Resultados: Os resultados da revisão sistemática apontaram para necessidade de novos estudos, limitando-se a um total de três publicações de relatos de casos envolvendo sete pacientes, com resultados variando de total controle da pressão ocular e conservação da função visual, até cegueira irreversível. Os resultados da pesquisa de campo envolveram 15 pacientes, com dois casos de hipertensão ocular e com diferença estatisticamente significativa entre as médias de PIO entre D0 vs D8 e D0 vs D14 (p = 0,013). Conclusão: A possibilidade de hipertensão ocular silenciosa, com o consequente risco de cegueira irreversível, indica a necessidade de se avaliar a introdução de um protocolo para verificação da IOP em pacientes jovens recentemente diagnosticados com ALL e NHL, incluindo exames semanais, pelo menos até a retirada completa do GC.

Notre groupe a montré que l’hypoxie sévère (0.1% O2) induit un arrêt du cycle cellulaire en G0 des cellules humaines CD34+ et des cellules murines FDCP mix. Peu d’études ont exploré l’existence de Cellules Initiatrices de Leucémie (CIL) dans les LAL et leur rôle dans les rechutes. Notre projet s’est focalisé sur l’effet de l’hypoxie sévère sur la quiescence des CIL dans les LAL, qui pourrait être responsable d’un pourcentage de rechutes. En effet dans la niche hématopoïétique, ou sont localisées les Cellules souches hématopoïétiques et probablement les CIL, la concentration d’oxygène avoisinerait 0,1%.Nous avons utilisé la lignée de LAL NALM6 pour explorer les effets de l’hypoxie sévère sur leur survie, leur cycle cellulaire et leur chimiorésistance. Nos résultats ont mis en évidence qu’une culture à 0.1% O2 durant 7 jours de la lignée NALM6: - inhibe leur prolifération sans surmortalité, - révèle une population restreinte de CIL quiescentes et chimiorésistantes capables d’induire une leucémie dans des souris. Nous avons recherché les relations entre l’hypoxie sévère et quelques caractéristiques des cellules primaires de patients atteints de LAL : existence et rôle de CIL résistantes à l’hypoxie et aux agents thérapeutiques conventionnels des LAL ; localisation de ces cellules résiduelles dans la moelle osseuse des souris xénogreffées. Nos résultats suggèrent que certaines rechutes de LAL pourraient être dues à la persistance à long terme de « quiescent/dormant » CIL dans les niches hypoxiques de la moelle osseuse. Ce modèle est intéressant pour explorer les mécanismes in vitro et in vivo de chimiorésistance dans les LAL et le rôle de l’environnement dans ce phénomène. / Our group showed that severe hypoxia (0.1% O2) induces G0 cell-cycle-arrest of human CD34+ cells and of murine FDCP-mix Cells. Few studies explored the existence of quiescent Leukemia Initiating Cells (LIC) in ALL and their role in primary chemoresistance and relapses. Our project is focused on the effect of very low O2 concentrations in the maintenance of quiescent LIC in ALL, that could be responsible of a percentage of relapses. Indeed in bone marrow niches, where hematopoietic stem cells and probably LIC are located, the O2 concentrations are below 0.1%.In the present study we used the NALM-6 ALL cell line to explore the effects of culture at 0.1% O2 on their survival, cell cycle and chemoresistance. Our results evidence that a 7 days culture of NALM-6 cells at 0.1% O2: - inhibits their proliferation without major cell death; - reveals a restricted LIC population of quiescent and chemoresistant LIC; - maintains quiescent chemoresistant LIC that induce leukemia when injected in immunodeficient mice. We investigated the relationships between severe hypoxia and some characteristics of ALL primary cells obtained from patients: existence and role of quiescent chemoresistant LICs in ALL relapses; location of these residual cells inside the bone marrow of engrafted mice. Our results suggest that some ALL relapses could be due to the long term persistence of “quiescent / dormant” LIC in hypoxic bone marrow niches. This model is of interest for exploring the in vitro and in vivo (xenograft) mechanisms of chemoresistance in ALL and the role of the bone marrow environment in this phenomenon.

Les leucémies aiguës lymphoblastiques de la lignée B (LAL-B) sont les cancers pédiatriques les plus fréquents. Dans ce type de leucémie, l'une des anomalies génétiques les plus fréquentes est la translocation t(12 ;21) aboutissant à la protéine de fusion ETV6-RUNX1. Cette pathologie est décrite comme un modèle à deux « hits ». Le premier, se produit in utero et génère la protéine de fusion. Le second, correspond à l’acquisition d’anomalies génétiques après la naissance. Ces réarrangements génomiques aberrants ont été décrits comme provenant d’une activité anormale de la recombinasse RAG. Notre travail a consisté dans un premier temps à compléter le modèle de leucémogénèse à plusieurs « hits ». En continuant notre étude des LAL B à translocation ETV6-RUNX1, nous nous sommes concentrés sur le rôle de RUNX1, gène dérégulé dans ce type de leucémie.L’ensemble de nos résultats confirme le rôle prépondérant de RUNX1 dans l’hématopoïèse et la leucémogenèse grâce à sa capacité à s’associer à des protéines aux fonctions différentes et grâce à son implication dans la transcription de gènes clé en hématologie. Nos résultats ouvrent donc de nouvelles perspectives dans la compréhension du contrôle de l’activité transcriptionnelle de RUNX1 et dans son rôle dans les hémopathies malignes. / B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer. In this type of leukemia, one of the most common genetic abnormalities is the ETV6-RUNX1 rearrangement. This malignancy is described as a two "hits" model. The first event occurs mainly in utero and generates the fusion gene ETV6-RUNX1. The second event consists in the acquisition of additional genetic abnormalities after birth. These aberrant genomic modifications have been described as resulting from abnormal activity of the RAG recombinase. Our work consisted initially in completing the leukemogenesis model. In continuing our study of ETV6-RUNX1 B-ALL, we focused on the role of RUNX1, an upregulated gene in this type of leukemia. All results confirm the predominant role of RUNX1 in hematopoiesis and leukemogenesis thanks to its ability to associate with proteins with different functions and its involvement in the transcription of key genes in hematology. Our results therefore open new perspectives in understanding the control of transcriptional activity of RUNX1 and its role in malignant hematology.

Therapeutic proteins have become a cornerstone of modern medicine since the FDA approval of recombinant human insulin in 1982. Likewise, biosensors transform chemical detection and disease diagnostics by identifying biomarkers, chemical contaminants, and infective agents. Long-standing methods for creating therapeutics and biosensors employ whole cells such as Escherichia coli (E. coli). Alternatively, cell-free protein synthesis (CFPS) employs the enzymatic reactions necessary for protein production and biosensing within a cell, but in an engineered reactor environment facilitating unprecedented access to and control over biochemical machinery, preservation by cryodesiccation for portable deployment, and functionality in cytotoxic applications. This dissertation reports advances in an E. coli CFPS production platform toward creating therapeutic proteins by this means. First, an endotoxin-free CFPS platform is created by optimizing fermentation and cell-extract harvest of an endotoxin-free E. coli strain. Next, liquid cell growth culture media is specially formulated to change chemical composition during cell culture and provide a streamlined method for producing high-yielding, endotoxin-free E. coli CFPS. Then, novel CFPS bioreactor formats are mathematically validated and developed which employ "hydrofoam" and oxygen to increase therapeutic protein production yield. Additionally, advances are reported in CFPS biosensing technology. First, a chimeric fusion protein incorporating the ligand binding domain of the human estrogen receptor is expressed in CFPS to detect estrogenic chemicals in the presence of human blood and urine. Next, the molecular mechanism of this protein construct is elucidated and the assay readout is optimized with mathematical simulations and CFPS. Then, CFPS is metabolically engineered to create a biosensor of L-glutamine, the most abundant amino acid in the body. Finally, this dissertation reports the development of a synergistic platform for potentially treating Acute Lymphoblastic Leukemia wherein CFPS is engineered to both produce the therapeutic protein crisantaspase and assess its activity in the presence of human serum for improved, potentially even personalized treatment of the disease. It is anticipated that the advances reported herein will contribute to the utility of in vitro or cell-free protein synthesis for therapeutic and diagnostic applications.

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.

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.