Made available in DSpace on 2016-08-19T18:16:04Z (GMT). No. of bitstreams: 1 dissertacao Thaiana.pdf: 2704967 bytes, checksum: e27a6cd767b0d6ac2256bec85730e19a (MD5) Previous issue date: 2012-08-13 / Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children and adolescents younger than fifteen years, with incidence peak between two and five years old. Several parameters have been evaluated for their importance in the prognostic evaluation of pediatric patients with ALL, of these, the evaluation of the anomalous myeloid expression prognosis of these pathologies. The aim of this work was to analyze the influence of myeloid anomalous expression in prognosis of children and adolescents with ALL of the state of Maranhão. It was evaluated 65 patients under 18 years diagnosed with ALL at the Instituto de Oncologia Maranhense Aldenora Belo (IMOAB) in São Luís, Maranhão. Laboratory data were obtained at diagnosis, made on the basis of morphological/ cytochemical and immunophenotype criteria. Other data were obtained from medical records. The sample was divided into groups with and without aberrant expression of myeloid antigens CD13 and CD33 for analysis with prognostic variables. The results indicated that the average age in the sample was 6,5 years, with the majority (69,2%) it was male children. The B-ALL was the most frequent type (83,1%). The mean percentage of blasts in our series was 75,8% in bone marrow and 46,6% in peripheral blood. Over ninety percent (90,8%) patients had L1 type morphology. The profile of blood test indicated the mean values of 32.373 leukocytes/mm3; 8,26 g / dl hemoglobin and 52.498 platelets/mm3. The anomalous myeloid expression occurred in 49,2% of the sample. The GBTLI-99 classification showed 56,9% of subjects with low risk of recurrence. At the end of the induction treatment period, 72,3% of children have responded positively in remission. The platelet count was significantly lower in without myeloid anomalous expression group (33.627 platelets/mm3, p = 0.01) compared to group that expressed this marker. About eighty-nine percent (88.9%) children with ALL-B and without myeloid aberrant expression had less than 50.000 platelets/mm3 (p = 0.01). Children with expression of CD33 were significantly older than those who did not express this marker (9,4 years, p = 0.01). It is concluded that the platelet count may be an important parameter to be analyzed in the prognosis of children with myeloid aberrant expression. The CD33 may be important in population with ALL and aberrant expression, since the CD13 was present in almost all cases. / A leucemia linfóide aguda (LLA) representa a neoplasia hematológica mais frequente em crianças e adolescentes menores de quinze anos, com pico de incidência entre dois e cinco anos de idade. Diversos parâmetros têm sido avaliados quanto à sua importância na avaliação prognóstica da população pediátrica com LLA, dentre estes, a avaliação da expressão anômala mielóide no prognóstico destas patologias. O objetivo deste trabalho foi analisar a influência da expressão anômala mielóide no prognóstico de crianças e adolescentes com LLA do estado do Maranhão. Foram avaliados 65 pacientes menores de 18 anos com diagnóstico de LLA assistidos no Instituto Maranhense de Oncologia Aldenora Belo (IMOAB), em São Luís, Maranhão. Os dados laboratoriais foram obtidos ao diagnóstico, realizado com base nos critérios morfológicos/citoquímicos e imunofenotípicos. Os demais dados foram obtidos a partir de prontuários. A amostra foi dividida em grupos com e sem expressão anômala mieloide dos antígenos CD13 e CD33 para análise com as variáveis prognósticas. Os resultados obtidos indicaram que a média de idade na amostra foi de 6,5 anos, sendo que a maioria (69,2%) se tratava de crianças do sexo masculino. A LLA-B foi o tipo mais frequente (83,1%). A porcentagem média de blastos em nossa casuística foi de 75,8% na medula óssea e de 46,6% em sangue periférico. Mais de noventa por cento (90,8%) dos pacientes apresentava morfologia do tipo L1. O perfil do hemograma indicou os valores médios de 32.373 leucócitos/mm3; 8,26 g/dl de hemoglobina e 52.498 plaquetas/mm3. A expressão anômala mielóide ocorreu em 49,2% da casuística. A classificação GBTLI-99 indicou 56,9% de sujeitos com baixo risco de recidiva. No final do período de indução do tratamento, 72,3% das crianças responderam positivamente com remissão da doença. A contagem de plaquetas foi estatisticamente inferior no grupo sem expressão anômala mielóide (33.627 plaquetas/mm3, p = 0,01) em relação ao grupo que expressou este marcador. Cerca de oitenta e nove por cento (88,9%) das crianças com LLA-B e sem expressão anômala mielóide apresentavam menos de 50.000 plaquetas/mm3 (p = 0,01). Crianças com expressão do CD33 foram significativamente mais velhas do que aquelas que não expressavam este marcador (9,4 anos, p = 0,01). Conclui-se que a contagem de plaquetas pode ser um importante parâmetro a ser analisado no prognóstico de crianças sem expressão anômala mielóide. O CD33 pode ser importante na caracterização da população com LLA e expressão anômala, já que o CD13 esteve presente em quase todos os casos.

Les leucémies aigües lymphobastiques (LAL) représentent un tiers des leucémies et constituent le cancer pédiatrique le plus fréquent chez l’enfant. Les LAL de type T (LAL-T)sont caractérisées par l’expansion anormale de progéniteurs de lymphocytes T. Aujourd’hui,la réponse curative aux traitements est proche de 80% chez l’enfant et 50% chez l’adulte. La rechute reste donc fréquente et souvent de mauvais pronostic. Pour ces raisons,l’identification de nouvelles voies de signalisation en vue de développer de nouvelles stratégies thérapeutiques est cruciale afin d’améliorer le traitement des LAL-T.Les résultats précédents du laboratoire ont révélé l’activation soutenue de la voie calcineurine (Cn)/NFAT dans des échantillons humains de lymphomes et de LAL, ainsi que dans des modèles murins de ces pathologies. Le laboratoire a ensuite montré que Cn est intrinsèquement requise pour la capacité des cellules leucémiques de LAL-T à propager la maladie (activité LIC « Leukemia Initiating Cells ») dans un modèle murin de LAL-T induit parun allèle activé de NOTCH1 (ICN1). Puisque l’inhibition pharmacologique de Cn induit de nombreux effets secondaires, la recherche de cibles thérapeutiques en aval de Cn constitue un axe de recherche important. J’ai participé à une étude du laboratoire montrant que l’expression à la surface cellulaire de CXCR4 est régulée par Cn et requise pour la migration des cellules de LAL-T, mais non suffisante pour rétablir le potentiel de ré-initiation suggérant que d’autres effecteurs doivent être impliqués dans cette activité.Les facteurs de transcription NFAT (NFAT1, NFAT2 et NFAT4) sont des effecteurs importants de Cn en réponse à la signalisation calcique lors du développement des thymocytes, mais également dans les lymphocytes T. L’essentiel de ce travail de thèse a utilisé des LAL-T induites par ICN1 dans lesquelles l’inactivation génique des trois facteurs NFAT par recombinaison homologue. Nous avons ainsi montré que (i) les facteurs NFAT sont requis en aval de Cn pour le potentiel LIC des LAL-T-ICN1 in vivo, (ii) leur inactivation altère la survie, la prolifération et la migration des cellules de LAL-T in vitro, (iii) NFAT1,NFAT2 et NFAT4 ont une fonction largement redondante dans les LAL-T. Nous avons également par une approche transcriptomique identifié deux gènes dont l’expression estsous contrôle des facteurs NFAT et impliqués dans la régulation de la survie et de la prolifération des LAL-T in vitro : CDKN1A et MAFB.Tout comme la voie Cn/NFAT, les CaMKs sont des protéines kinases activées en aval de la signalisation calcique dans les lymphocytes T. Nous avons montré par une approche pharmacologique que l’inhibition des CaMKs dans les LAL-T-ICN1 in vitro altère la survie etla prolifération des cellules leucémiques. L’inhibition spécifique par une approche d’ARN interférence de deux isoenzymes CaMKIIγ et CaMKIIδ suggèrent que ces protéines jouent dans le maintien des cellules leucémiques in vitro. / T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T cell progenitors. Despite initial response to chemotherapy, relapses remain frequent in children and adults. Previous results identify sustained activation of Calcineurin (Cn)/NFAT signaling pathway in human T-ALL and murine T-ALL models. Importantly, they also demonstrated Cn is essential for T-ALL Leukemia Initiating Cells (LIC) activity in a murine model of T-ALL induced by an activated allele of NOTCH1 (ICN1). Since pharmacologic inhibition of Cn induces side effects, we aim to identify downstream effectors involved in T-ALL. NFAT (Nuclear Factor of Activated T cells) factors play crucial roles downstream Cn during development and activation of T cells. To address their role in T-ALL, we generated mouse ICN1-induced T-ALL in which NFAT genes can be inactivated either single or in combination following Cre-mediated gene deletion. We demonstrated that (i) NFAT factors are required downstream Cn for LIC activity in T-ALL in vivo (ii) ex vivo NFAT factors deletion alters survival, proliferation and migration of T-ALL (iii) NFAT1, 2 and 4 have a largely redundant function in T-ALL. Moreover, the NFAT-dependant transcriptome allowed to identify important targets (CDKN1A, MAFB) involved in T-ALL survival and proliferation in vitro. Calmodulin-dependant kinases (CaMK) are kinases activated by calcium signaling in T cells. We showed that pharmacologic inhibition of CaMKs in ICN1-induced T-ALL alters survival and proliferation of T-ALL in vitro. Beside, specific inhibition by RNA interference of CaMKIIg and CaMKIId suggests a putative role of these kinases in T-ALL maintenance.

Mon travail de thèse concerne l’étude des leucémies aiguës lymphoblastiques T (LAL-T). Il se décline en deux projets. Le premier, Le Saracatinib affecte les LAL-T humaines en ciblant la kinase LCK dans les cellules riches en radeaux lipidiques, nous a permis d’identifier une nouvelle voie métabolique importante pour la prolifération des cellules leucémiques. Nous avons montré que la kinase LCK est intégrée dans les radeaux lipidiques et impliquée dans la croissance des cellules leucémiques. L’inhibiteur de LCK Saracatinib affecte les cellules de LAL-T in vitro et in vivo en ciblant particulièrement les cellules les plus agressives ayant beaucoup de radeaux lipidiques à leur surface. Ces résultats permettent d’envisager une nouvelle stratégie thérapeutique pour traiter les LAL-T et ont fait l’objet d’une publication parue dans Leukemia en janvier 2018. Le second projet, Les leucémies aiguës lymphoblastiques T produisent l’interleukine 7 de manière autocrine, démontre pour la première fois que pour la plupart des LAL-T, les cellules sont capables de sécréter elles-mêmes la cytokine IL-7. Nous avons complété cette étude par une analyse des mécanismes épigénétiques impliqués dans la régulation de cette sécrétion autocrine. Nos résultats montrent qu’elle est activée par la fixation des facteurs de transcription IRF-1 (Interferon Regulatory Factor 1) et IRF-2 au niveau du promoteur du gène IL 7, lorsque celui-ci est peu méthylé. Grâce à l’inactivation du gène IL-7 dans un de nos modèles de LAL-T, nous avons pu démontrer que la sécrétion autocrine favorise le développement de la leucémie chez la souris xénogreffée en impactant la prise de greffe et le nombre de cellules initiatrices de leucémie. Ainsi, la régulation épigénétique de la sécrétion autocrine d’IL-7 pourrait être impliquée dans les premières étapes de la leucémogenèse des LAL-T. / My PhD work concerns T-cells acute lymphoblastic leukemia (T-ALL) and includes two projects. The first one, Saracatinib impairs maintenance of human T-ALL by targeting the LCK tyrosine kinase in cells displaying high level of lipid rafts, allow us to identify a new signaling pathway important for the proliferation of T-ALL cells. We showed that LCK is localized into lipid rafts and is involved in the growth of T-ALL cells. The LCK inhibitor Saracatinib affects T-ALL cells in vitro and in vivo by targeting the most aggressive cells displaying high level of lipid rafts. These results highlight a new therapeutic strategy to treat T-ALL and were published in Leukemia in January 2018. The second project, T cell acute lymphoblastic leukemia produces autocrine interleukin 7, demonstrated for the first time that T-ALL cells are able to produce IL-7 cytokine. We performed an analysis of epigenetic mechanisms involved in the regulation of this autocrine secretion. Our results showed that when the IL-7 gene promoter is low methylated, Interferon Regulatory Factor 1 (IRF-1) and (IRF-2) transcription factors bind IRF-E sequence and upregulate IL-7 gene transcription. Thanks to IL 7 gene inactivation in one of our T ALL models, we demonstrated that autocrine secretion promotes leukemia development on xenografted mice through increasing engraftment cells capacity and leukemia initiating cells number. Thus, epigenetic regulation of IL-7 autocrine secretion may be involved in the leukemogenesis of T-ALL.

<p>The aims of the studies described in this thesis were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL).</p><p>Leukemic cells from 370 children with newly diagnosed ALL were tested by the Fluorometric Microculture Cytotoxicity Assay to measure their resistance to each of ten standard cytotoxic drugs. In the high-risk group, increased in vitro resistance to each of the drugs dexamethasone, etoposide and doxorubicin was associated with a worse clinical outcome. Combining the results for these drugs yielded a drug resistance score, showing a relative risk of relapse in the most resistant group that was 9.8 times higher than in the most sensitive group. In the standard-risk and intermediate-risk groups, final evaluation must await longer follow-up.</p><p>The new cytotoxic agent CHS 828 was equally active in vitro in samples from children with acute myeloblastic leukemia (AML) and ALL, with 50% cell kill at concentrations achievable in vivo. In AML samples CHS 828 also displayed high frequencies of synergistic interactions with four standard drugs. The well-known differences in clinical outcome between Down´s syndrome (DS) and non-DS children with acute leukemia may partly be explained by our finding of differences in drug resistance at the cellular level.</p><p>Pharmacokinetic studies were performed at the start of induction treatment of ALL. Doxorubicin was assayed by reversed-phase liquid chromatography with fluorometric detection, and vincristine by high performance liquid chromatography with electrochemical detection. Plasma doxorubicin concentrations were measured in 107 children after 23 h of a 24-h infusion. The median steady-state concentration in children 4-6 years old, a group known to have a favorable outcome of treatment, was about 50% higher than in those 1-2 and >6 years old Vincristine pharmacokinetics was evaluated in 98 children. There was no correlation between age and total body clearance or any other pharmacokinetic parameters.</p><p>In vitro testing of cellular drug resistance might be useful in predicting the outcome in high-risk ALL. The further exploration of CHS 828 in childhood leukemia seems warranted. There is no pharmacokinetic rationale for the common practice of administering relatively lower doses of vincristine to adolescents than to younger children.</p>

<p>Treatment results and clinical characteristics in adult acute lymphoblastic leukaemia (ALL) were evaluated regarding three issues: a new treatment with cytarabine up-front, stem cell transplantation and a comparison between adult and paediatric treatment protocols. All studies were conducted on a national basis. Furthermore, activity of imatinib was investigated by in vitro cytotoxicity assay. </p><p>The national protocol was evaluated in 153 adult ALL patients. A high complete remission rate, 86%, was achieved with 29% overall survival at 3-years. Favourable outcome was identified in patients < 40 years with precursor B phenotype and continuous complete remission was higher for precursor B compared to T-ALL. </p><p>Stem cell transplantation was evaluated in 187 patients. No differences in outcome between allogeneic and autologous transplantation were found, with the exception of Philadelphia-positive ALL, in which allogeneic transplantation was preferable. Limited chronic graft-versus-host disease (compared to none) resulted in superior disease free survival. </p><p>The paediatric NOPHO-92 and the Adult protocols were evaluated for 243 ALL-patients. Superior remission rate and survival were achieved for 10-18 year-olds treated according to the Paediatric protocol compared to both 15-25 and 25-40 year-olds treated according to the Adult protocol. Treatment protocol was a significant prognostic factor for patients aged 15-20 years. </p><p>Fluorometric Microculture Cytotoxicity Assey was used to analyze 15 tumour cell samples from ALL patients. High concordance was determined between in vitro sensitivity to imatinib and presence of BCR-ABL. Daunorubicin, prednisolone and cytarabine had the greatest benefit from a combination with imatinib. </p><p>The national adult treatment protocol’s results were consistent with international trials regarding precursor B ALL but may be under performing for T-ALL. Adolescents may benefit from treatment according to the Paediatric protocol. No difference in outcome between allogeneic and autologous stem cell transplantation was determined except for Philadelphia-positive patients, despite the indication of a graft-versus-leukaemia effect.</p>

Nearly 80% of children treated for cancer are expected to survive, but not without cost. Survivors face unprecedented challenges associated with long-term consequences of treatment, also called late effects. Approximately half of children treated for cancer are at risk for experiencing cognitive late effects, which typically emerge several years post diagnosis. The nature and extent of cognitive late effects appear to be developmental and related to patient, disease, and treatment variables. However, the relationships between these variables is not well understood because there have been few prospective and longitudinal studies that report on the contributions of these variables over time. This dissertation examined the effects of patient, disease, and treatment variables, as well as their interactions over time on neurocognitive functioning in childhood cancer survivors. It comprises part of a large prospective, randomized clinical trial designed to examine changes in cognitive function over three years as a function of different levels of monitoring of school-based intervention based on individual educational plans (IEPs). This dissertation uniquely contributed a new measure (the Treatment Intensity Rating Scale) that was used to systematically classify treatment severity across different types of cancer and cancer treatments. Participants included 61 children ages 7 to 12 years at enrollment who were two to five years from completion of treatment for a brain tumor, leukemia, or lymphoma. Participants received yearly neuropsychological evaluations for a follow-up period of 3 years. Results of these evaluations were used to develop IEPs. Participants were randomized to have their IEPs monitored on a quarterly or annual basis for the duration of the study. Contrary to the progressive decline in neurocognitive functioning that is typically anticipated in pediatric cancer survivors, analyses revealed relative stability of performance on neurocognitive measures over time. Higher neurocognitive performance was noted in children whose IEPs were monitored more frequently versus less frequently. Results also supported gender-specific risk for late effects, with lower performance on select neurocognitive measures in females compared to males. Results of this study provide encouraging evidence of the positive effects of school-based interventions and their close monitoring. This has important implications for quality of life as these children survive well beyond childhood into adulthood.

The aims of the studies described in this thesis were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL). Leukemic cells from 370 children with newly diagnosed ALL were tested by the Fluorometric Microculture Cytotoxicity Assay to measure their resistance to each of ten standard cytotoxic drugs. In the high-risk group, increased in vitro resistance to each of the drugs dexamethasone, etoposide and doxorubicin was associated with a worse clinical outcome. Combining the results for these drugs yielded a drug resistance score, showing a relative risk of relapse in the most resistant group that was 9.8 times higher than in the most sensitive group. In the standard-risk and intermediate-risk groups, final evaluation must await longer follow-up. The new cytotoxic agent CHS 828 was equally active in vitro in samples from children with acute myeloblastic leukemia (AML) and ALL, with 50% cell kill at concentrations achievable in vivo. In AML samples CHS 828 also displayed high frequencies of synergistic interactions with four standard drugs. The well-known differences in clinical outcome between Down´s syndrome (DS) and non-DS children with acute leukemia may partly be explained by our finding of differences in drug resistance at the cellular level. Pharmacokinetic studies were performed at the start of induction treatment of ALL. Doxorubicin was assayed by reversed-phase liquid chromatography with fluorometric detection, and vincristine by high performance liquid chromatography with electrochemical detection. Plasma doxorubicin concentrations were measured in 107 children after 23 h of a 24-h infusion. The median steady-state concentration in children 4-6 years old, a group known to have a favorable outcome of treatment, was about 50% higher than in those 1-2 and &gt;6 years old Vincristine pharmacokinetics was evaluated in 98 children. There was no correlation between age and total body clearance or any other pharmacokinetic parameters. In vitro testing of cellular drug resistance might be useful in predicting the outcome in high-risk ALL. The further exploration of CHS 828 in childhood leukemia seems warranted. There is no pharmacokinetic rationale for the common practice of administering relatively lower doses of vincristine to adolescents than to younger children.

Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR). In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment. The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection. In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary. In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer affecting children in the world and in our country. The exact molecular etiology of the disease still remains to be elucidated. This study hypothesized that four genes, namely CYP2E1*5B, *6, and *7B, NQO1*2 SNPs, GSTM1 null and GSTT1 null, alone or in combination, could contribute to the risk of development of childhood ALL. Also interactions of these polymorphisms with non-genetic risk factors were investigated. The genotyping of these polymorphisms were done on 209 healthy subjects, and 185 patients with childhood ALL, in Turkish population. Venous blood samples were collected and genomic DNA was isolated from these samples. Genotyping was done by PCR-RFLP techniques. In the case-control analyses for the risk of development of childhood ALL, only GSTT1 null was found to be associated with the development of disease (OR= 1.8, p=0.01). CYP2E1*5B and *6 combination showed an increased risk of 2.7 fold (p= 0.04). Also co-presence of CYP2E1*6-GSTT1 and CYP2E1*7B-GSTT1 polymorphisms increased the risk significantly above 4.0 fold. The risk increased more to 7.6 fold, when CYP2E1*5B,*6 and GSTT1 null were considered together, with borderline significance (p=0.04). When interaction of exposure to cigarette smoke and genetic polymorphisms were investigated, NQO1*2 and GSTM1 null were turned out to be significant risk factors for the development of disease when the parental or child&rsquo / s postnatal exposure to cigarette smoke was considered. This study presented several new findings to the literature in terms of genetic epidemiology of childhood ALL. The present work would also contribute to public health in determining the susceptibility of the Turkish population to childhood ALL.

No description available.