Orientadores: Carmino Antonio de Souza, Katia Borgia Barbosa Pagnano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T20:51:16Z (GMT). No. of bitstreams: 1 Mascarenhas_CintiadoCouto_M.pdf: 7297123 bytes, checksum: a5c6ff86609313924a25638b32816a31 (MD5) Previous issue date: 2009 / Resumo: O desenvolvimento da Leucemia Mielóide Crônica (LMC) tem como característica a formação do cromossomo Philadelphia que envolve a quebra do gene BCR gerando um rearranjo molecular denominado BCR-ABL, cujo produto final é uma proteína de fusão citoplasmática que determina a patogenia da doença. Esta proteína é uma tirosina quinase (TK) que possui capacidade de auto-ativação e para a inativação desta proteína, foram desenvolvidos os inibidores da tirosina quinase (ITK), que tem a capacidade de se ligar no mesmo sítio de ligação da molécula de ATP. Esta ligação impede a transferência dos grupos fosfatos aos substratos subseqüentes, bloqueando a cascata de transdução de sinais e prevenindo a ativação das vias mitogênica dependente da quinase Bcr-Abl e anti-apoptóticas levando à morte do fenótipo BCR-ABL.Um dos principais mecanismos de resistência ao tratamento com ITK são as mutações pontuais, sendo a T315I foco de estudos mais detalhados por tornar a proteína mutante altamente insensível a todas as drogas inibidoras da proteína TK disponíveis atualmente Foi utilizado neste trabalho a técnica de D-HPLC para fazer screening de mutações nos pacientes com LMC com resposta sub ótima ou falha de tratamento de acordo com os critérios da Leukemia Net. Para o screening do éxon 6 foram selecionados 93 pacientes com LMC: 5 eram intolerantes, 67 resistentes e 21 com resposta subótima. Como controle negativo foi usado o sangue periférico doadores de sangue do Hemocentro da UNICAMP. Para o screening de mutações de todo o gene BCR-ABL foram estudados 37 pacientes com LMC e como controle negativo, usamos a linhagem celular HL60 que não possui a translocação BCR-ABL. No screening do éxon 6, 23 amostras (25%) mostraram um perfil de eluição no D-HPLC anormal em relação ao controle, o que sugeriu a presença de mutação. A sobrevida global (OS) para todo grupo foi de 80% em uma mediana de tempo de observação de 30 meses. OS para pacientes sem mutações foi de 87% e para os pacientes com mutações foi de 56% em uma mediana de tempo de observação 37 e 10 meses, respectivamente (p <0,0001, RR = 68). No screening de todo o gene BCR-ABL 17 (46%) tiveram perfil cromatográfico diferente do controle Como estávamos estabelecendo a padronização do método, procedemos com o seqüenciamento de todas as amostras e os resultados obtidos foram comparados com a seqüência depositada no banco de dados GenBank (U07563). Das 17 amostras com alteração do perfil cromatográfico, observamos a presença de mutação em 13 amostras. Acreditamos que isso se deva a sensibilidade do método de D-HPLC que é capaz de identificar tanto polimorfismos quanto mutações com maior eficiência que o seqüenciamento. Em resumo, o D-HPLC demonstrou ser um método sensível e prático para o acompanhamento do aparecimento de mutações no domínio da quinase na rotina clínica. Mutações nessa região estudada são clinicamente relevantes e podem conferir um pior prognóstico. A detecção precoce pode ser uma ferramenta importante para otimizar a terapêutica na LMC. / Abstract: The development of chronic myeloid leukemia (CML) is the formation of the characteristic Philadelphia chromosome involving breach of the BCR gene generating a molecular rearrangement called BCR-ABL, whose final product is a cytoplasmic fusion protein that determines the pathogenesis of the disease. This is a protein tyrosine kinase (TK) that has self-ativaçãoe to inactivate this protein have developed the inhibitors of tyrosine kinase (ITK), which has ability to connect on the same site of binding of molecule of ATP. This connection prevents the transfer of phosphate groups to substrates subsequent, blocking the cascade of signal transduction and preventing the activation of mitogenic pathways dependent kinase BCR-ABL and anti leading to apoptotic death phenotype of BCR-ABL.One major mechanisms of resistance to treatment with ITK are mutations off, and the T315I focus of more detailed studies by making mutant protein highly insensitive to all drugs Inhibit TK protein currently available was used in this work to D-HPLC technique to screening for mutations in patients with CML with sub-optimal response or failure of treatment according to the criteria Leukemia Net For the screening of exon 6 were selected 93 CML patients: 5 were intolerant, 67 resistant and 21 with answer sub-optimal. The negative control we used the peripheral blood donors Blood from the blood of UNICAMP. For the screening of mutations throughout the BCR-ABL gene were studied 37 patients with CML and control negative, we used the HL60 cell line that does not have the translocation BCR-ABL. In the screening of exon 6, 23 samples (25%) showed a profile of the D-HPLC elution abnormal in the control, which suggested the presence of mutation. The overall survival (OS) for whole group was 80% in a median time of observation of 30 months. OS for patients with mutations was 87% and for patients with mutations was 56% in the median observation time of 37 and 10 months respectively (p <0.0001, RR = 68). In screening the entire gene BCR-ABL 17 (46%) had chromatographic profile different from the control we were setting the standardization of methods, procedures with the sequencing of all samples and the results were compared with the sequence deposited in the GenBank database (U07563). Of the 17 samples with change the chromatographic profile, we observed the presence of mutation in 13 samples. We believe that this is due to sensitivity of the method of D-HPLC is able to identify the mutations both polymorphisms with greater efficiency to the sequencing. In summary, the D-HPLC has proved a sensitive and practical method for monitoring the appearance of mutations in the kinase domain in the clinical routine. Mutations studied in this region are clinically relevant and may confer worse prognosis. Early detection can be a tool important to optimize therapy in CML. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Orientadores: Carmino Antonio de Souza, Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T11:16:23Z (GMT). No. of bitstreams: 1 Mascarenhas_CintiadoCouto_D.pdf: 4558328 bytes, checksum: f219d85bc4e15d72a39a7d3242be9ffb (MD5) Previous issue date: 2013 / Resumo: A elucidação dos mecanismos moleculares envolvidos na fisiopatologia e tratamento das doenças hematológicas, bem como no entendimento do perfil de expressão gênica das linhagens celulares leucêmicas, tem sido objeto de numerosas investigações. Com o uso da técnica SSH (Subtractive Supression Hybridization ou Biblioteca Subtrativa Supressiva) foi possível identificar importantes genes que se encontram diferencialmente expressos em granulócitos de pacientes com Leucemia Mielóide Crônica e indivíduos controle. Foram encontrados 39 genes superexpressos e 173 com expressão diminuída em células de LMC. Ao relacionar esses genes com vias metabólicas que estão reguladas positiva (expressão aumentada) ou negativamente (expressão diminuída) nessa doença, verificou-se que a maioria dos genes estavam relacionados com a regulação de NF-kB, AKT, o Interferon e a IL-4 em células de controle. Entre os genes superexpressos encontrados na LMC, foi observado o SEPT5, RUNX1, MIER1, KPNA6 e FLT3, enquanto PAN3, TOB1 e ITCH estavam com expressão diminuída nessa doença em comparação com indivíduos controle. O TOB1 se mostrou promissor, uma vez que é um gene supressor tumoral, pode estar envolvido na proliferação de células leucêmicas e interage com vários outros genes encontrados neste estudo. Assim, devido à grande heterogeneidade de funções relacionadas com a expressão desse gene, foi investigada a relação entre a expressão de mRNA e as respostas aos ITK's na LMC. A avaliação foi realizada por PCR em tempo real em doentes com CCgR, PCgR, MINCgR e NOCgR após tratamento com TKI's e os resultados foram comparados com a expressão em granulócitos de indivíduos controle, observando que os pacientes NOCgR têm uma expressão de TOB1 significativamente inferior em comparação com doadores saudáveis e pacientes que alcançaram RCgC. Ao comparar pacientes não resistentes e resistentes a diferença foi significativa. Esses resultados sugerem que a expressão diminuída de TOB1 em pacientes NOCgR pode ser indicativo de desregulação da apoptose e de vias de sinalização importantes nessa doença incluindo a via da AKT, conduzindo assim a resistência a ITK's nesses pacientes. Outro objetivo deste trabalho foi caracterizar a função dos genes TOB1 e SEPT5 nos processos celulares e vias de sinalização de apoptose, proliferação, migração e ciclo celular em linhagens celulares leucêmicas. Ao realizar o silenciamento desses genes (utilizando partículas lentivirais) notou-se que o silenciamento de TOB1, como já descrito na literatura em outras doenças, interfere na proliferação celular, clonogenicidade, apoptose, ciclo celular e expressão de proteínas importantes da cascata de sinalização, o que salienta sua importância em células BCR-ABL positivas. Já o gene SEPT5 ao ser silenciado leva a algumas alterações como a apoptose e ciclo celular. Nesse contexto, o silenciamento destes genes chama atenção para as possibilidades de controle da proliferação celular, apoptose, ciclo celular e clonogenicidade em células BCR-ABL positivas. Foi realizada a avaliação da expressão desses genes em células de sangue periférico e medula óssea de pacientes com LMC e indivíduos controles, linhagens celulares de câncer humano e linhagens de murino. Os resultados mostraram um aumento significativo na expressão do gene SEPT5 em todos os tipos celulares analisados em pacientes com LMC. O mesmo padrão foi observado em células de murino que possuem a mutação T315I e em células humanas que possuem a translocação t(9;22) e estão relacionadas com a fase blástica da doença [K562, KU812, NALM]. Quando avaliada a expressão do gene TOB1 nota-se diminuição em todos os tipos celulares analisados em pacientes com LMC e em células BaF3T315I. Também foi observada uma baixa expressão em células com a t(9;22) e estão relacionadas com a fase blástica da doença[K562, KU812, NALM] quando comparadas a expressão em medula óssea controle. Outro resultado interessante foi obtido a partir da análise de adesão celular em granulócitos de pacientes com LMC e controles, evidenciando a diminuição da adesão em granulócitos de pacientes com LMC em relação aos de controles, levando a hipótese de que essa alteração nas propriedades adesivas dos granulócitos em pacientes com LMC pode estar diretamente ligada à liberação de células jovens pela matriz da medula óssea. A criação de estratégias que levam ao melhor entendimento da fisiopatologia da doença e avanço no tratamento da LMC deve ser focada em vários genes alvos e não apenas no BCR-ABL, pois no desenvolvimento da LMC há a ativação e desativação de várias vias de sinalização celular. Os resultados deste estudo podem ajudar na melhor compreensão dessas vias e também para identificar outros genes e vias úteis para a melhora no manejo terapêutico e criação de novas drogas para o tratamento dessa doença / Abstract: The elucidation of the molecular mechanisms involved in the pathophysiology and treatment of blood disorders, as well as the understanding of genes expression profiling of leukemia cell lines has been the focus of numerous investigations. The use of the SSH (Suppression Subtractive Hybridization Library or Suppression Subtractive) technique made available the identification of important genes which are differentially expressed in granulocytes from patients with chronic myeloid leukemia (CML) and healthy controls. 39 genes overexpressed were found, and 173 with decreased expression in CML cells. When correlating these genes with metabolic pathways that are regulated positively (increased expression) or negatively (decreased expression) in this disease, it was found that most of the genes were related to the regulation of NF-kB, AKT, Interferon and IL-4 in control cells. The following genes were found overexpressed in CML: SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were found with decreased expression in this disease compared with controls. The TOB1 gene showed promising since it is a tumor suppressor, may be involved in the proliferation of leukaemic cells and interacts with several others genes found in this study. Thus, due to the great heterogeneity of functions related to this gene, was investigated the relationship between mRNA expression and TKI's responses. The evaluation was performed by real time PCR in patients with CCgR, PCgR, MINCgR and NOCgR after treatment with TKI and healthy controls. Was observed that patients that have NOCgR, the TOB1 expression is significantly lower compared with healthy donors and patients who achieved CCgR. When comparing non-resistant and resistant patients the difference also was significant. These results suggest that reduced expression of TOB1 in NOCgR patients may indicate apoptosis deregulation and changes in important signaling pathways of CML including the Akt pathway, thereby leading to TKI's resistance of these patients. Another aim of this work was to characterize the function of TOB1 and SEPT5 in cellular processes and signaling pathways of apoptosis, proliferation, migration and cell cycle in leukemic cell lines. After the silencing of these genes (using lentiviral particles), was noted that the silencing TOB1 - as described in the literature for other diseases - interferes in cell proliferation, clonogenicity, apoptosis, cell cycle and in expression of important proteins at signaling cascade, which emphasizes its importance in BCR-ABL positive cells. The SEPT5 silencing leads to some changes such as apoptosis and cell cycle. In this context, the silencing of these genes leads to attention of possibilities of control of cell proliferation, apoptosis, cell cycle and cell clonogenicity in BCR-ABL positive cells. Was assessed the expression of these genes in cells from peripheral blood and bone marrow of CML patients and controls, as well in human and murine cell lines. Results showed a significant increase in SEPT5 gene expression in patients with CML in all cell types evaluated. The same profile was observed in murine cells BAF3T315I and in human cells having the translocation t (9; 22) been related to blast crisis [K562, KU812, NALM]. When measuring expression of TOB1, was noted decrease in all cell types studied in CML patients and cells BaF3T315I. Another interesting result was obtained from the analysis of cell adhesion at granulocytes in CML patients and controls which showed decreased adhesion of granulocytes in CML patients compared to controls, leading to the hypothesis that the change in adhesive properties at CML can be directly linked to the release of young cells by bone marrow. The creation of strategies that lead to better understanding of the pathophysiology of the disease and advance in the treatment of CML should be focused on several target genes and not only in BCR-ABL, since in the development of CML there is an activation and deactivation of multiple signaling pathways . The results of this study may help to better understand these pathways and also to identify other genes and pathways useful for improving the management and development of new therapeutic drugs to treat this disease / Doutorado / Clinica Medica / Doutora em Clínica Médica

Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.

CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.

La leucémie myéloïde chronique (LMC) est une maladie myéloproliférative, résultant d'une translocation réciproque entre les chromosomes 9 et 22, donnant naissance à une kinase de fusion à activité continue, la BCR-ABL. Les inhibiteurs de la tyrosine kinase (ITK) sont le traitement de choix de la LMC, mais ces inhibiteurs n'offrent pas de cure radicale aux patients, qui, une fois le traitement interrompu, présentent une rechute et une exacerbation de leur condition. En plus, les patients peuvent développer une résistance ou une intolérance aux ITK, d'où la nécessité de nouvelles modalités thérapeutiques. Les imidazoquinoxalines sont de nouveaux composés à visée anticancéreuse, dont la structure est basée sur celle de l'imiquimod, un immunomodulateur connu. EAPB0203, un composé du groupe des imidazo[1,2-α]quinoxalines, a fait preuve d'un pouvoir anticancéreux sur des cellules de mélanome, in vitro et in vivo, ainsi que sur des cellules de leucémie liée au virus HTLV-1, in vitro. EAPB0503 est un composé de la même famille, à pouvoir environ 10 fois supérieur à celui de l'EAPB0203 sur les cellules de mélanome. Dans le cadre de ce projet de thèse, l'activité de l'EAPB0203 a été comparée à celle de l'EAPB0503, et leur effet sur la prolifération de trois lignées cellulaires de LMC a confirmé que l'EAPB0503 est plus efficace que l'EAPB0203. Pour ce, l'EAPB0503 a été adopté pour la suite des expérimentations que nous avons entreprises dans le but de caractériser le mécanisme d'action par lequel ce composé inhibe la croissance cellulaire. Les CI50 de l'EAPB0503 ont été déterminées pour chacune des trois lignées, et adoptées tout au long du travail. On a démontré que l'EAPB0503 provoque un arrêt du cycle cellulaire en PréG0 (accumulation de cellules apoptotiques) et en G2/M. Une nette augmentation du niveau de phosphorylation de l'histone-3 suggère un arrêt des cellules en début de mitose. Cet arrêt du cycle cellulaire s'accompagne d'une diminution du potentiel membranaire mitochondrial, d'une activation modérée du cytochrome c et du clivage de PARP (suggérant l'implication de caspases). On a également démontré que l'EAPB0503 induit l'apoptose comme le montre la fragmentation de l'ADN. Des résultats préliminaires proposent aussi une stabilisation de la protéine pro-apoptotique, bax, et une diminution du niveau de la protéine anti-apoptotique Bcl-2, augmentant ainsi le rapport Bax/Bcl-2 et renversant ainsi l'équilibre du côté apoptotique. En plus, EAPB0503 a potentialisé l'effet de l'imatinib (le premier ITK), et, à deux, ces deux agents ont eu un effet synergique sur la croissance des cellules de LMC. Il a été également très intéressant de montrer que l'EAPB0503 dégrade l'oncoprotéine, BCR-ABL, dans les cellules sensibles et résistantes à l'imatinib. Ensemble, ces résultats suggèrent un effet prometteur de l'EAPB0503 sur la LMC, en monothérapie ou en association avec les ITK. / Chronic myeloid leukemia (CML) is a myeloproliferative disorder that originates in a reciprocal translocation, resulting in the Philadelphia chromosome that harbors the bcr-abl oncogene. This fusion gene codes for a constitutively active tyrosine kinase, BCR-ABL that confers leukemogenesis. Tyrosine kinase inhibitor (TKI), have revolutionized the therapeutic management of CML. Imatinib, the first generation TKI, is highly effective in inducing remissions and prolonging the survival of CML patients. However, failure of this therapy occurs in almost one-third of the patients due to intolerance to treatment, lack of therapeutic response or resistance. Moreover, CML stem cells (LIC) remain insensitive to this therapy, which almost inevitably leads to relapse upon treatment discontinuation. Imidazoquinoxalines are imiquimod derivatives with direct immunomodulatory effect and antitumor activity on melanoma and T-cell lymphoma, attributed to growth inhibition and induction of caspase-dependent apoptosis. We investigated the effects of EAPB0203 and EAPB0503, novel imidazoquinoxaline derivatives, on human CML cell lines. We show that EAPB0203 and EAPB0503 inhibit cell growth of three CML cell lines in a dose- and time-dependent manner. Compared to the previously described EAPB0203, EAPB0503 has a better inhibitory activity on CML cells. We demonstrated that EAPB0503 induced a specific cell cycle arrest in mitosis in CML cells, evidenced by decreased levels of cyclin D1 and increased phosphorylation of histone-3. This was accompanied by the direct activation of apoptosis as demonstrated by increased PreG0 population, DNA breakage, poly(ADP-ribose) polymerase cleavage and breakdown of mitochondrial membrane potential. Preliminary results have shown a stabilization of the pro-apoptotic molecule, bax, versus a decrease in anti-apoptotic bcl-2, shifting the ratio towards the induction of apoptosis. Interestingly, EAPB0503-treated cells had decreased levels of BCR-ABL oncoprotein. The combination of EAPB0503 with imatinib synergizes to inhibit CML cells proliferation and most importantly, EABP0503 inhibited the proliferation of imatinib-resistant CML cells offering a promising therapeutic modality that alone or in combination with TKI would circumvent mutations and resistance to TKI and improve the prognosis of CML.

A introdução do Mesilato de Imatinibe (MI) como primeiro inibidor específico de BCR-ABL1 na prática clínica revolucionou o tratamento da Leucemia Mieloide Crônica (LMC), tornando-se a terapia padrão para o tratamento desta doença. Porém, cerca de 30% dos pacientes com LMC não respondem à terapia com MI e um número substancial destes casos de resistência não tem causa conhecida. O MI interage com transportadores de membrana ABCG2 e SLCO1A2. Este estudo teve como objetivo investigar a relação da expressão gênica de ABCG2 e de SLCO1A2 com marcadores de resposta ao tratamento com MI, em indivíduos com LMC e avaliar a influência dos polimorfismos ABCG2 c.421C>A e ABCG2 c.-19-99G>A na resposta ao MI. Foram incluídos 118 pacientes com LMC os quais foram classificados em dois grupos: Grupo Respondedor, constituído por 70 pacientes com resposta citogenética completa com a dose padrão de MI (400 mg/dia) por até 18 meses e, Grupo não Respondedor constituído por 48 pacientes sem resposta citogenética completa à dose inicial de 400 mg/dia de MI ou que perderam esta resposta ao longo do tratamento e foram reescalonados para doses de 600 ou 800 mg/dia. A resposta ao tratamento foi avaliada segundo os critérios da European LeukemiaNet. Foram excluídos pacientes com alterações citogenéticas diferentes do cromossomo Ph e mutações no gene BCR-ABL1. Amostras de sangue periférico foram utilizadas para: extração do RNA total para quantificação dos transcritos BCR-ABL1 e expressão gênica de ABCG2 e SLCO1A2; extração de DNA e análise citogenética de banda G. A expressão do gene ABCG2 e SLCO1A2 e as análises dos polimorfismos foram feitas por PCR em tempo real. A expressão de ABCG2 foi maior no grupo de não respondedores ao MI (P=0,028). Este resultado foi influenciado pelos pacientes com resistência primária (N= 34 P=0,029), mas não pelos que apresentaram resistência secundária (N=14 P=0,249) quando comparado com respondedores (N=70). A elevada expressão do gene ABCG2 foi também associada àqueles pacientes que não tiveram resposta molecular maior (número de transcritos BCR-ABL1 &#8804; 0,1%) (P=0,027) quando todos os pacientes foram analisados. O gene estudado não foi associado com a resposta molecular completa (número de transcritos BCR-ABL1 &#8804; 0,032%). Com relação ao gene SLCO1A2 não foi possível determinar sua expressão devido à baixa concentração do RNA obtido. Os polimorfismos c.421C>A e c.-19-99G>A não foram associados com a expressão do gene ABCG2 e a resposta ao MI. A RMC (no grupo de respondedores) foi associada com o genótipo 421CC e houve tendência a maior frequencia de portadores do genótipo -19-99GG neste mesmo grupo. Portadores do genótipo -19-99AA apresentaram tendência ao risco de ter LMC. Os resultados deste estudo nos permitem concluir que a maior expressão de ABCG2 está associada com a resistência primária ao MI podendo então ser um mediador da resistência ao MI. Os polimorfismos do gene ABCG2 não influenciaram na expressão gênica de ABCG2, mas impactaram na RMC no grupo respondedor ao MI. / The introduction of imatinib mesylate (IM) as the first specific inhibitor of BCR-ABL1 in clinical practice has revolutionized the treatment of chronic myeloid leukemia (CML), becoming the standard therapy for this disease. However, about 20% of CML patients do not respond to therapy with IM and a substantial number of these cases of resistance have no known cause. The MI interacts with membrane transporters ABCG2 and SLCO1A2. The aim of this study was to investigate the relationship of ABCG2 and SLCO1A2 gene expression with markers of response to MI in individuals with CML and evaluate the influence of polymorphisms ABCG2 c.421C> A and c. ABCG2-19-99G> A in response to the MI. One hundred and eighteen patients in chronic phase of CML were studied and classified in two groups: Responder Group comprised 70 patients who had a complete cytogenetic response within 18 months of treatment. The non-responder group comprised 48 patients who did not have a complete cytogenetic response with the initial dose (400 mg/day) of IM or who relapsed during treatment and were submitted to higher doses of 600 or 800 mg/day. Criteria of failed response to treatment were established by European LeukemiaNet. Patients with cytogenetic patterns other than the Philadelphia chromosome and patients with mutations in the BCR-ABL1 gene were excluded from this study. Blood samples were obtained for: total RNA extraction for quantification of BCR-ABL1 and gene expression of ABCG2 and SLCO1A2; genomic DNA extraction and band G cytogenetic analysis. The gene expression and the analysis of the polymorphisms were performed by real time PCR. Expression of ABCG2 in non-responder group was higher than in responder group (P=0.028). This result was influenced by patients with primary resistance (n= 34 P=0.029) but not secondary resistance (n=14 P=0.249) when compared with responders (n=70). The higher expression of ABCG2 gene was also associated with those patients who had major molecular response (number of BCR-ABL1 . 0.1%) (P=0.027) when all patients were analyzed. The studied gene was not associated with the complete molecular response (number of BCR-ABL1 .0.0032). Regarding to the gene SLCO1A2 was not possible to determine its expression due to low concentration of RNA obtained. The c.421C>A e c.-19-99G>A were not associated neither with the ABCG2 gene expression and MI response. CMR in responders group was associated with the 421CC genotype ant there was a trend for higher frequency of carriers of genotype -19-99GG in the same group. Carriers of 19-99AA genotype tended to the risk of having CML. The results of this study allow us to conclude that the higher expression of ABCG2 is associated with primary resistance to IM and may be a mediator of resistance to IM. The ABCG2 polymorphisms did not influence the gene expression of ABCG2 but impacted in CMR of the responders to IM.

A Leucemia Mielóide Crônica (LMC) é uma doença rara. No entanto, os avanços nas pesquisas básica e clínica nos últimos anos, colocaram a LMC em evidência sendo hoje uma neoplasia maligna potencialmente curável. O diagnóstico e tratamento desta doença são, no entanto, extremamente caros. Não havendo dados sistemáticos nem registros de incidência da LMC no Rio Grande do Sul ou no Brasil, o levantamento de dados baseado em registros dos centros de referência se justifica também para planejar ações em saúde. Entre 1996 e 2000, 276 casos foram diagnosticados. A estimativa de casos novos anuais foi de aproximadamente 0,6:100.000 habitantes, e a idade média no momento do diagnóstico foi 42 anos e 4 meses (±16 anos e 2 meses). Quanto ao tratamento e evolução destes pacientes, dos 257 avaliados, 56 (21,8%) foram submetidos ao transplante alogênico de medula óssea, com taxa de sobrevida em 5 anos de 75% e 27% para as fases crônica e acelerada/blástica, respectivamente. O tempo médio de sobrevida para os 257 pacientes foi de 47,7 meses (IC 43,3 - 52,1). Comparando ao relatado na literatura, encontramos um menor número anual de novos casos e também uma média de idade no diagnóstico mais baixa. Isto poderia ser explicado pela menor referência de idosos a serviços terciários de saúde. Para os pacientes transplantados, os resultados foram semelhantes aos relatados na literatura. / Although rare, the advances made in basic and clinical research throughout the last years have thrown a spotlight on CML. Diagnosis and treatment of CML is of high cost. Since there is no systematic data or information about the incidence of CML in Rio Grande do Sul or Brazil, the data obtained from reference centers serve to estimate the number of CML cases in our state to better plan health actions. Between 1996 and 2000, 276 cases were diagnosed. The annual estimate of new cases was approximately of 0,6:100,000 inhabitants, and the median age at diagnosis was 42 years and 4 months (±16 years and 2 months). The mean overall survival time for the 257 patients was 47,7 months (CI 43,3-52,1). That could be explained by the lack of referral for older patients. Regarding treatment and evolution, of the 257 valuable patients, 56 (21,8%) were submitted to allogeneic BMT with a five-year survival of 75% and 27% for chronic and accelerated/blastic phases, respectively. In conclusion, we found a lower estimated incidence and a lower median age at diagnosis. For the transplanted patients the results were similar to those reported in the literature.

Chronic Myeloid Leukemia is a kind of blood cancer with around 1 incidence per 100 000 persons/year. After the development of an effective treatment, imatinib, in the late 1990:s, the survival percentage has increased drastically. The high survival has turned the attention to different kinds of treatment responses, which in turn are good prognostic factors to future health status. In this thesis, the focus is on whether or not the patient has achieved a so called major molecular response after 12 month, or not. More precisely, the aim is to find prognostic factors to the 12 month response. In order to find prognostic factors for this binary response variable, a multivariate logistic regression analysis is conducted, with the goal of finding a parsimonious logistic model that describes the data. The analysis is done from a merged dataset from three earlier studies. The prognostic factors in the final model are treatment, 3 month response, and enlarged spleen. However, the residual analysis indicates that the model is incomplete, implying that further research needs to be done.

Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, a minute chromosome that leads to the creation of the fusion gene BCR/ABL and the transcription of the fusion protein BCR/ABL in transformed cells. The constitutively active tyrosine kinase BCR/ABL confers enhanced proliferation and survival on leukemic cells. CML has in only a few decades gone from being a disease with very bad prognosis to being a disease that can be effectively treated with oral tyrosine kinase inhibitors (TKIs). TKIs are drugs inhibiting BCR/ABL as well as other tyrosine kinases. In this thesis, the focus has been on the immune system of CML patients, on immune escape mechanisms present in untreated patients and on how these are affected by TKI therapy. We have found that newly diagnosed, untreated CML patients exert different kinds of immune escape mechanisms. Patients belonging to the Sokal high-risk group had higher levels of myeloid-derived suppressor cells (MDSCs) as well as high levels of the programmed death receptor 1 (PD-1)-expressing cytotoxic T cells compared to control subjects. Moreover, CML patients had higher levels of myeloid cells expressing the ligand for PD-1, PD-L1. CML patients as well as patients with B cell malignacies had high levels of soluble CD25 in blood plasma. In B cell malignacies, sCD25 was found to be released from T regulatory cells (Tregs). Treatment with the TKIs imatinib or dasatinib decreased the levels of MDSCs in peripheral blood. Tregs on the other hand increased during TKI therapy. The immunostimulatory molecule CD40 as well as NK cells increased during therapy, indicating an immunostimulatory effect of TKIs. When evaluating immune responses, multiplex techniques for quantification of proteins such as cytokines and chemokines are becoming increasingly popular. With these techniques a lot of information can be gained from a small sample volume and complex networks can be more easily studied than when using for example the singleplex ELISA. When comparing different multiplex platforms we found that the absolute protein concentration measured by one platform rarely correlated with the absolute concentration measured by another platform. However, relative quantification was better correlated.

Imatinib mesylate is indicated for the treatment of chronic myeloid leukemia (CML) and unresectable and/or metastatic malignant gastrointestinal stromal tumors (GIST). By the application of this anticancer drug, problems occurs in terms of stability and activity. Computer assisted design (CAD) works showed that the modification of the B and C part molecule can increase the effectivity of the drug. The new derivatives of the drug will be obtained to change some part of the B and C segments. The total synthesis of a new imatinib mesylate will be done and the activity tests are going to be determined in collaboration with other groups.