Atualmente, a Leucemia Linfocítica Crônica (LLC) pode ser dividida em dois grupos: um com mutações somáticas no gene da região variável da cadeia pesada da imunoglobulina (MIgVH) e outro sem mutações (NMIgVH). Alguns estudos mostraram que a expressão de CD38 na superfície das células B de LLC pode estar correlacionada com o estado mutacional do gene VHIg, entretanto, esses controversos. Estudos recentes mostraram que a expressão da proteína tirosina quinase Zap-70 está melhor associada com o estado mutacional do gene IgVH. O objetivo deste estudo foi avaliar a expressão de Zap-70 e CD38, por citometria de fluxo, nas células CD19+ de pacientes com LLC e correlacioná-los com o estádio clínico (EC), sobrevida livre de tratamento (SLT) e sobrevida global (SG). A expressão de Zap-70 e CD38 foi avaliada, em 144 de pacientes com LLC classificados nos estádios clínicos A, B e C de acordo com os critérios de Binet: 59 (41%) do EC-A, 38 (26%) do EC-B e 47 (33%) do EC-C. Foi observada menor positividade para Zap-70 e CD38 nos pacientes do EC-A do que nos EC-B e C. Quando avaliada a SLT nos pacientes do EC-A, os casos Zap-70+ assim como os CD38+ apresentaram menor SLT. A média de SG dos pacientes Zap-70+ e CD38+ foi menor quando comparado com os Zap-70- e CD38- entretanto quando correlacionada com o EC não foi observada diferença estatisticamente significante entre a expressão desses marcadores e o EC-A, B ou C. Pela analise combinada de CD38 e Zap-70, dividimos os pacientes em dois grupos (Zap-70-/CD38- e Zap-70+ ou CD38+). Observamos que a expressão positiva desses dois marcadores estava associada ao EC, uma vez que a grande maioria dos pacientes dos estádios B (74%) e C (66%) expressam Zap-70 ou CD38. Entretanto, os pacientes do EC-A, Zap-70+ ou CD38+, apresentaram SG menor quando comparado com os Zap-70-/CD38-. Essa diferença não foi observada nos pacientes do EC-B e do EC-C. Também foi observada menor SLT nos pacientes no EC-A, Zap-70+ ou CD38+. Esses resultados sugerem que análise combinada de Zap-70 e CD38 podem ser empregadas na avaliação dos pacientes do EC-A para se acompanhar a evolução clinica desse grupo de pacientes. Porém, estudos adicionais devem ser realizados para se validar a utilização clínica desses marcadores. / Actually, chronic lymphocytic leukemia (CLL) can be divided in two subsets: one with somatically mutated immunoglobulin heavy-chain variable-region genes (MIgVH) and other with unmutated sequences. (UMIgVH). Some studies have shown that CD38 expression in CLL cells are correlated with IgVH mutational status. However, the value of CD38 as surrogate IgVH mutational status is controversial. Recent studies, have found that Zap-70 protein tyrosine kinase expression is strongly associated with the mutational status IgVH. The aim of this study was to evaluate the Zap-70 and CD38 expression, for flow cytometry, in CD19+ LLC cells and correlate with the Binet’s staging system, treatment-free survival (TFS) and a overall survival (OS). Zap-70 and CD38 was evaluated, in 144 CLL patients that was classified in A, B and C Binet’s staging system: 59 (41%) in stage A, 38 (26%) in B and 47 (33%) in C. We observed low Zap-70 and CD38 expression in stage A patients than in stage B and C cases. When we analyzed the TFS in stage A patients Zap-70+ and CD38+ patients showed shorter TFS than Zap-70- and CD38-. Then we observed that the OS of Zap-70+ and CD38+ patients was, also, shorter than Zap-70- and CD38- cases. However, statistical differences was not found when Zap-70 and CD38 expression was correlated with stage A, B or C Binet’s staging system. To understand the associated Zap-70 and CD38 expression, we divided the CLL patients in two subgroups (Zap-70-/CD38 - and Zap-70+ or CD38+). We observed that CD38+ or Zap-70+ was associated Binet’s staging system, once most of stage B (74%) and C (66%) patients are Zap-70+ or CD38+. However, stage A patients, Zap-70+ or CD38+, showed shorter OS than Zap-70-/CD38-. These differences were not observed in stage B and C patients. Shorter TFS was also observed in the Zap-70+ or CD38+ stage A patients. These results suggest that combined analysis of Zap-70 and CD38 can be used to evaluate stage A patients to observe the clinical evolution of the disease. Nevertheless, other studies must be carried to confirm the clinical use of these markers.

Chronic Lymphocytic Leukemia is the primary lymphoid neoplasm in adults and and it is especially manifested in the elderly. Because it is a heterogeneous disease it awakens great interest regarding its prognosis. Rai and Binet developed staging systems to predict the evolution of the disease and currently, the analysis of expression of CD38 and Zap-70 has been investigated as a prognostic factor for indicating presence or absence of the mutation in the gene IgVH, so, the objective of this study was to analyze the clinical and laboratory profiles of patients with Chronic Lymphocytic Leukemia taking as reference the clinical staging of Rai and Binet and quantification of CD38 and Zap-70 expression as prognosis factors. We searched the medical records of 64 patients treated at University Hospital of Santa Maria and the variables considered were swollen lymph nodes, presence or absence of hepatomegaly and / or splenomegaly, hematological evaluation of peripheral blood and immunophenotype. The data obtained were correlated with the staging of Rai (1975) and Binet (1981), the expression of CD38 and Zap-70 and clinical stage. The results showed no association between ataging Rai and Binet and the expression of CD38, Zap-70 and Binet clinical staging. / A Leucemia Linfocítica Crônica é a principal neoplasia linfóide em adultos e se manifeta principalmente em indivíduos idosos. Por ser uma doença heterogênea, desperta grande interesse quanto ao seu prognóstico. Rai e Binet desenvolveram sistemas de estadiamento capazes de prever a evolução da doença e atualmente, a análise da expressão de CD38 e Zap- 70 tem sido investigada como fator prognóstico por indicar presença ou ausência da mutação no gene IgVH, assim, o objetivo deste estudo foi analisar o perfil clínico-laboratorial dos pacientes com Leucema Linfocítica Crônica, tomando como referência os estadiamentos clínicos de Rai e Binet e a quantificação da expressão de CD38 e Zap-70 como fatores prognóstico. Foram pesquisados 64 prontuários médicos de pacientes atendidos no Hospital Universitário de Santa Maria e as variáveis consideradas foram aumento de linfonodos, presença ou ausência de hepatomegalia e/ou esplenomegalia, avaliação hematológica de sangue periférico e imunofenótipo. Os dados obtidos foram correlacionados com o estadiamento de Rai (1975) e Binet (1981), a expressão de CD38 e Zap-70 com o estádio clínico de Binet. Os resultados demonstraram que não há associação entre o estadiamento de Raí e Binet e a expressão de CD38, Zap-70 com o estadiamento clínico de Binet.

Atualmente, a Leucemia Linfocítica Crônica (LLC) pode ser dividida em dois grupos: um com mutações somáticas no gene da região variável da cadeia pesada da imunoglobulina (MIgVH) e outro sem mutações (NMIgVH). Alguns estudos mostraram que a expressão de CD38 na superfície das células B de LLC pode estar correlacionada com o estado mutacional do gene VHIg, entretanto, esses controversos. Estudos recentes mostraram que a expressão da proteína tirosina quinase Zap-70 está melhor associada com o estado mutacional do gene IgVH. O objetivo deste estudo foi avaliar a expressão de Zap-70 e CD38, por citometria de fluxo, nas células CD19+ de pacientes com LLC e correlacioná-los com o estádio clínico (EC), sobrevida livre de tratamento (SLT) e sobrevida global (SG). A expressão de Zap-70 e CD38 foi avaliada, em 144 de pacientes com LLC classificados nos estádios clínicos A, B e C de acordo com os critérios de Binet: 59 (41%) do EC-A, 38 (26%) do EC-B e 47 (33%) do EC-C. Foi observada menor positividade para Zap-70 e CD38 nos pacientes do EC-A do que nos EC-B e C. Quando avaliada a SLT nos pacientes do EC-A, os casos Zap-70+ assim como os CD38+ apresentaram menor SLT. A média de SG dos pacientes Zap-70+ e CD38+ foi menor quando comparado com os Zap-70- e CD38- entretanto quando correlacionada com o EC não foi observada diferença estatisticamente significante entre a expressão desses marcadores e o EC-A, B ou C. Pela analise combinada de CD38 e Zap-70, dividimos os pacientes em dois grupos (Zap-70-/CD38- e Zap-70+ ou CD38+). Observamos que a expressão positiva desses dois marcadores estava associada ao EC, uma vez que a grande maioria dos pacientes dos estádios B (74%) e C (66%) expressam Zap-70 ou CD38. Entretanto, os pacientes do EC-A, Zap-70+ ou CD38+, apresentaram SG menor quando comparado com os Zap-70-/CD38-. Essa diferença não foi observada nos pacientes do EC-B e do EC-C. Também foi observada menor SLT nos pacientes no EC-A, Zap-70+ ou CD38+. Esses resultados sugerem que análise combinada de Zap-70 e CD38 podem ser empregadas na avaliação dos pacientes do EC-A para se acompanhar a evolução clinica desse grupo de pacientes. Porém, estudos adicionais devem ser realizados para se validar a utilização clínica desses marcadores. / Actually, chronic lymphocytic leukemia (CLL) can be divided in two subsets: one with somatically mutated immunoglobulin heavy-chain variable-region genes (MIgVH) and other with unmutated sequences. (UMIgVH). Some studies have shown that CD38 expression in CLL cells are correlated with IgVH mutational status. However, the value of CD38 as surrogate IgVH mutational status is controversial. Recent studies, have found that Zap-70 protein tyrosine kinase expression is strongly associated with the mutational status IgVH. The aim of this study was to evaluate the Zap-70 and CD38 expression, for flow cytometry, in CD19+ LLC cells and correlate with the Binet’s staging system, treatment-free survival (TFS) and a overall survival (OS). Zap-70 and CD38 was evaluated, in 144 CLL patients that was classified in A, B and C Binet’s staging system: 59 (41%) in stage A, 38 (26%) in B and 47 (33%) in C. We observed low Zap-70 and CD38 expression in stage A patients than in stage B and C cases. When we analyzed the TFS in stage A patients Zap-70+ and CD38+ patients showed shorter TFS than Zap-70- and CD38-. Then we observed that the OS of Zap-70+ and CD38+ patients was, also, shorter than Zap-70- and CD38- cases. However, statistical differences was not found when Zap-70 and CD38 expression was correlated with stage A, B or C Binet’s staging system. To understand the associated Zap-70 and CD38 expression, we divided the CLL patients in two subgroups (Zap-70-/CD38 - and Zap-70+ or CD38+). We observed that CD38+ or Zap-70+ was associated Binet’s staging system, once most of stage B (74%) and C (66%) patients are Zap-70+ or CD38+. However, stage A patients, Zap-70+ or CD38+, showed shorter OS than Zap-70-/CD38-. These differences were not observed in stage B and C patients. Shorter TFS was also observed in the Zap-70+ or CD38+ stage A patients. These results suggest that combined analysis of Zap-70 and CD38 can be used to evaluate stage A patients to observe the clinical evolution of the disease. Nevertheless, other studies must be carried to confirm the clinical use of these markers.

T lymphocytes express either a preTCR, or a clonotyoic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs result in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility.Througout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulates the subtleties found in men.In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.

T lymphocytes express either a preTCR, or a clonotypic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs results in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, and adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility. Throughout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulate the subtleties found in men. In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.