Introdução: A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. No estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que é utilizada como marcador diagnóstico para a SB. Essas alterações são causadas por um defeito no mecanismo de reparo do DNA, decorrente de uma mutação no gene BLM. Objetivos: Realizar o estudo citogenético de trocas entre cromátides irmãs para o diagnóstico de pacientes com suspeita clínica de SB; caracterizar os aspectos clínicos e avaliar a evolução de pacientes com SB. Métodos: Foram estudados nove pacientes (4 M e 5 F) pertencentes a oito famílias com suspeita clínica de SB utilizando preparações cromossômicas tratadas com 5- bromo-2-desoxiuridina (BrdU) e coloração Hoechst - Giemsa para visualização diferencial das cromátides irmãs e análise de freqüência de TCI. Resultados e Discussão: Todos os pacientes foram positivos para a pesquisa de TCI cuja freqüência variou de 45,2 a 61,3 TCI/metáfase. A idade dos pacientes ao diagnóstico variou de 1a1m até 11a (média de 4a6m). O principal motivo do encaminhamento foi o déficit de crescimento e apenas um paciente foi encaminhado por apresentar lesões cutâneas. Todos apresentaram deficiência de crescimento pré e pós-natal, microcefalia e hipoplasia malar. O eritema esteve presente em 8/9 pacientes. Manchas café-au-lait e/ou manchas hipocrômicas foram observadas em sete pacientes. Um paciente apresentou agenesia unilateral da fíbula, encurtamento da tíbia e agenesia do 5° artelho, associado à hipoplasia renal. As infecções de repetição foram relatadas em 8/9 pacientes, sendo principalmente pneumonia e diarreia. Deficiência de imunoglobulinas foi observada em 6/9 pacientes, principalmente: deficiência de IgG (3/6), de IgA (2/6) e de IgM (1/6). A consanguinidade entre os pais foi encontrada em 4/8 famílias, apenas uma família apresentou dois filhos afetados. Duas pacientes (2/9) evoluíram com tumor de Wilms (TW), uma aos 3a6m e a outra aos 3a11m. Houve recidiva em uma paciente que faleceu aos cinco anos. A outra paciente evoluiu bem e atualmente está com 20 anos. Conclusão: O diagnóstico da SB deve ser feito precocemente baseado na avaliação clínica. A pesquisa citogenética de TCI, que é de baixo custo e fácil aplicação, é fundamental para a confirmação diagnóstica. A freqüência aumentada para o desenvolvimento de neoplasias em idade precoce, alerta para um rastreamento das neoplasias mais comuns como linfoma, leucemia e tumor de Wilms. / Introduction: Bloom syndrome (BS) is a rare chromosomal instability syndrome, transmitted by autosomal recessive inheritance. It´s characterized by pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectatic erythema on the face and impaired immune system. BS patients present an increased predisposition to develop cancer at early age, which is the main cause of death. In the cytogenetic exam is observed an increase of spontaneous breaks and sister chromatid exchange (SCE) that is used as a diagnostic biomarker for the BS. These changes are caused by a defect in DNA repair mechanism, due to mutations in the BLM gene. Objectives: Perform the cytogenetic study of sister chromatid exchange for the clinical diagnosis of BS patients; to characterize the clinical aspects and assess the follow-up of patients. Methods: Nine patients (4 M, 5 F) from eight families with clinical diagnoses of BS were studied using standard chromosome preparations treated with 5-bromo-2-deoxyuridine (BrdU) and Hoechst-Giemsa differential staining for visualization and analysis of frequency of SCE. Results and Discussion: All patients were positive for the presence of SCE with the frequency ranged from 45.2 to 61.3 SCE/metaphase. The age at diagnosis ranged from 1y1mo to 11y (mean 4y6mo). The main reason for referral was growth deficit except one due to skin lesions. All patients presented pre and post-natal growth deficiency, microcephaly and malar hypoplasia. The erythema was present in 8/9 patients. Cafe-au-lait spots and/or hypochromic spots were observed in seven patients. One patient had unilateral agenesis of the fibula, shortening tibia and agenesis of the 5th toe associated with renal hypoplasia. The recurrent infections were reported in 8/9 patients, mainly pneumonia and diarrhea. Immunoglobulin deficiency was observed in 6/9 patients such as IgG (3/6), IgA (2/6) and IgM (1/6). The parental consanguinity was found in 4/8 families, one family had two affected. Two patients (2/9) developed Wilms tumor (WT), one at 3y6mo and another at 3y11mo. There was recurrence in one patient who died at five years. The other patient is well at 20 years old. Conclusion: The diagnosis of BS should be done early based in clinical findings. The cytogenetic for SCE exam is essential for diagnostic confirmation, which is low cost and easy application. The screening for the most common malignancies such as lymphoma, leukemia and WT must be done due to increased predisposition for cancer development at an early age

Introdução: A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. No estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que é utilizada como marcador diagnóstico para a SB. Essas alterações são causadas por um defeito no mecanismo de reparo do DNA, decorrente de uma mutação no gene BLM. Objetivos: Realizar o estudo citogenético de trocas entre cromátides irmãs para o diagnóstico de pacientes com suspeita clínica de SB; caracterizar os aspectos clínicos e avaliar a evolução de pacientes com SB. Métodos: Foram estudados nove pacientes (4 M e 5 F) pertencentes a oito famílias com suspeita clínica de SB utilizando preparações cromossômicas tratadas com 5- bromo-2-desoxiuridina (BrdU) e coloração Hoechst - Giemsa para visualização diferencial das cromátides irmãs e análise de freqüência de TCI. Resultados e Discussão: Todos os pacientes foram positivos para a pesquisa de TCI cuja freqüência variou de 45,2 a 61,3 TCI/metáfase. A idade dos pacientes ao diagnóstico variou de 1a1m até 11a (média de 4a6m). O principal motivo do encaminhamento foi o déficit de crescimento e apenas um paciente foi encaminhado por apresentar lesões cutâneas. Todos apresentaram deficiência de crescimento pré e pós-natal, microcefalia e hipoplasia malar. O eritema esteve presente em 8/9 pacientes. Manchas café-au-lait e/ou manchas hipocrômicas foram observadas em sete pacientes. Um paciente apresentou agenesia unilateral da fíbula, encurtamento da tíbia e agenesia do 5° artelho, associado à hipoplasia renal. As infecções de repetição foram relatadas em 8/9 pacientes, sendo principalmente pneumonia e diarreia. Deficiência de imunoglobulinas foi observada em 6/9 pacientes, principalmente: deficiência de IgG (3/6), de IgA (2/6) e de IgM (1/6). A consanguinidade entre os pais foi encontrada em 4/8 famílias, apenas uma família apresentou dois filhos afetados. Duas pacientes (2/9) evoluíram com tumor de Wilms (TW), uma aos 3a6m e a outra aos 3a11m. Houve recidiva em uma paciente que faleceu aos cinco anos. A outra paciente evoluiu bem e atualmente está com 20 anos. Conclusão: O diagnóstico da SB deve ser feito precocemente baseado na avaliação clínica. A pesquisa citogenética de TCI, que é de baixo custo e fácil aplicação, é fundamental para a confirmação diagnóstica. A freqüência aumentada para o desenvolvimento de neoplasias em idade precoce, alerta para um rastreamento das neoplasias mais comuns como linfoma, leucemia e tumor de Wilms. / Introduction: Bloom syndrome (BS) is a rare chromosomal instability syndrome, transmitted by autosomal recessive inheritance. It´s characterized by pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectatic erythema on the face and impaired immune system. BS patients present an increased predisposition to develop cancer at early age, which is the main cause of death. In the cytogenetic exam is observed an increase of spontaneous breaks and sister chromatid exchange (SCE) that is used as a diagnostic biomarker for the BS. These changes are caused by a defect in DNA repair mechanism, due to mutations in the BLM gene. Objectives: Perform the cytogenetic study of sister chromatid exchange for the clinical diagnosis of BS patients; to characterize the clinical aspects and assess the follow-up of patients. Methods: Nine patients (4 M, 5 F) from eight families with clinical diagnoses of BS were studied using standard chromosome preparations treated with 5-bromo-2-deoxyuridine (BrdU) and Hoechst-Giemsa differential staining for visualization and analysis of frequency of SCE. Results and Discussion: All patients were positive for the presence of SCE with the frequency ranged from 45.2 to 61.3 SCE/metaphase. The age at diagnosis ranged from 1y1mo to 11y (mean 4y6mo). The main reason for referral was growth deficit except one due to skin lesions. All patients presented pre and post-natal growth deficiency, microcephaly and malar hypoplasia. The erythema was present in 8/9 patients. Cafe-au-lait spots and/or hypochromic spots were observed in seven patients. One patient had unilateral agenesis of the fibula, shortening tibia and agenesis of the 5th toe associated with renal hypoplasia. The recurrent infections were reported in 8/9 patients, mainly pneumonia and diarrhea. Immunoglobulin deficiency was observed in 6/9 patients such as IgG (3/6), IgA (2/6) and IgM (1/6). The parental consanguinity was found in 4/8 families, one family had two affected. Two patients (2/9) developed Wilms tumor (WT), one at 3y6mo and another at 3y11mo. There was recurrence in one patient who died at five years. The other patient is well at 20 years old. Conclusion: The diagnosis of BS should be done early based in clinical findings. The cytogenetic for SCE exam is essential for diagnostic confirmation, which is low cost and easy application. The screening for the most common malignancies such as lymphoma, leukemia and WT must be done due to increased predisposition for cancer development at an early age

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

Das ADAMTS16-Gen kodiert für eine Metalloproteinase, deren Funktion und Regulation bislang nicht beschrieben sind. Die ADAMTSs werden von Zellen verschiedener Organsysteme sezerniert und sind für den Abbau extrazellulärer Matrixbestandteile und die Prozessierung von Oberflächenrezeptoren, Signalmolekülen oder Wachstumsfaktoren verantwortlich. In der vorliegenden Arbeit wurden die gewebespezifische Lokalisation von Adamts16 und die möglichen Funktionen der Metalloproteinase im Urogenitalsystem untersucht. Weiterhin konnte die Regulation der Adamts16-Expression durch das Wilms-Tumor Protein beschrieben werden. In verschiedenen Zelllinien des Urogenitalsystems konnte eine Wt1-abhängige Adamts16-Expression festgestellt werden. Zudem erfolgte im Urogenitalsystem eine Koexpression von Adamts16 und Wt1 in embryonalen und adulten Podozyten, somatische Zellen der XX-Gonadenanlage und Granulosazellen und Epithelzellen des adulten Ovars. Im Testis war Adamts16 ohne signifikante Wt1-Koexpression in Spermatozyten und elongierten Spermatiden lokalisiert. Außerhalb des Urogenitalsystems waren Adamts16 und Wt1 im Epikard koexprimiert. Ein Wt1-Knockdown in Epikardzellen und embryonalen Nieren zeigte jeweils einen Rückgang des Adamts16-Expressionsniveaus. Ein Adamts16-Knockdown in embryonalen Nieren resultierte in verminderten Ureterverästelungen, was eine funktionelle Rolle von Adamts16 in der murinen Nierenentwicklung ex vivo andeutet. Der Wt1-Knockdown in Gonadenkulturen zeigte, dass Wt1 die Adamts16-Expression in XY-Gonaden hemmt, in XX-Gonaden hingegen aktiviert. Innerhalb des Adamts16-Gens konnten drei Wt1-Konsensusmotive identifiziert werden. Mit Hilfe von EMSAs und ChIPs konnte die Bindung der Wt1(-KTS)-Isoform an diese Konsensusmotive belegt werden. Ein Reportergenassay zeigte die Aktivierung des Adamts16-Promotors durch Wt1(-KTS) in Granulosazellen, wobei eine Verkürzung der Adamts16-Promotorsequenz zu einer Reduktion der Promotoraktivität führte. / The Adamts16 gene encodes for a metalloproteinase, whose function and regulation is hardly explored. ADAMTSs are secreted by different cells of various organs and are responsible for breaking down extracellular matrix compounds and processing signaling molecules, growth factors and surface receptors. In this work the tissue specific localization of Adamts16 and its possible function and regulation within the genito-urinary system were analyzed. Furthermore the regulation of Adamts16 through the wilms tumor transcription factor Wt1 is described. Different cell lines derived from the genito-urinary system showed a Wt1-dependent mRNA expression of Adamts16. In addition both proteins were co-expressed in embryonic and adult podocytes, somatic cells of the embryonic XX-gonad and granulosa and epithelial cells of the adult ovary. The testes showed a Wt1-independent Adamts16 expression in spermatocytes and elongated spermatids. Outside the genito-urinary system Adamts16 and Wt1 were co-expressed in the epicardium. Knockdown of Wt1 in both epicardial cells and embryonic kidney explants showed a decrease in the Adamts16 mRNA expression level. In turn the Knockdown of Adamts16 led to an inhibited branching morphogenesis in embryonic kidney explants. This indicates a functional role of Adamts16 in the ex vivo kidney development. Knockdown of Wt1 in cultured embryonic gonads revealed that Wt1 inhibits the expression of Adamts16 in XY-gonads but activates it in XX-gonads. Three Wt1 consensus motives were identified within the Adamts16 gene. Using EMSA and ChIP the binding of the Wt1(-KTS)-isoform to all three consensus motives was verified. The ability of Wt1 to activate the Adamts16 promoter was confirmed through reporter gene assays in granulosa cells.

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

Les leucémies aiguës myéloïdes (LAM) sont des hémopathies malignes hétérogènes déclenchées, dans la plupart des cas, par des anomalies génétiques (mutations, translocations ou inversions). Elles se caractérisent par un blocage de la différenciation de certains progéniteurs ou précurseurs hématopoïétiques (blastes) et leur prolifération clonale incontrôlée provoquant leur accumulation dans la moelle osseuse. Le traitement actuel de ces patients repose essentiellement sur l’utilisation d’agents de chimiothérapie (cytarabine associée à une anthracycline) permettant d’éliminer les cellules leucémiques et d’obtenir une rémission complète (RC) (définie morphologiquement comme une moelle osseuse normale avec moins de 5% de blastes). Cette RC est obtenue chez une majorité des patients mais plus d’un patient sur deux va rechuter quelques mois après l’arrêt du traitement. Ces rechutes attestent de la persistance de cellules leucémiques résiduelles après le traitement, que l’on appelle maladie résiduelle mesurable (MRD). Celle-ci a été mise en évidence grâce au développement de technologies performantes et sensibles tels que la cytométrie en flux multi-paramétrique et la PCR en temps réel (qPCR) permettant ainsi la détection de profils d’expression ou d’anomalies génétiques associés aux LAM. A ce jour, plusieurs mécanismes ont été décrits pour expliquer la présence de cette MRD. Celle-ci peut être causée par une résistance au traitement de certains sous-clones leucémiques (anomalies génétiques intrinsèques leur conférant une résistance ou un phénotype quiescent) ou par la présence de cellules souches leucémiques (naturellement quiescentes). Le système immunitaire pourrait également jouer un rôle en induisant la quiescence de certaines cellules les rendant résistantes aux chimiothérapies conventionnelles, ou en contrôlant leur croissance tumorale par l’établissement d’un état d’équilibre entre leur prolifération et leur lyse. Les modèles murins de LAM actuellement utilisés permettent d’étudier la leucémogenèse et l’efficacité thérapeutique de certains composés mais font abstraction du rôle de la réponse immunitaire dans ces processus du fait de leur immunodéficience. De plus, aucun modèle murin de MRD leucémique n’existe pour étudier les causes de la persistance cancéreuse après traitement par chimiothérapie. Ainsi, le but de cette thèse a été de développer un modèle murin syngénique et immunocompétent de MRD leucémique sur-exprimant ou non le gène Wilms’ Tumor 1 (WT1). WT1 est un des rares antigènes décrits dans les LAM et une réponse lymphocytaire cellulaire et humorale dirigée contre cette protéine a été décrite chez ces patients. La création de ce modèle sur-exprimant ou non WT1 permettra ainsi d’étudier le rôle de la réponse immunitaire spécifique de celui-ci dans la persistance leucémique. Pour développer ce modèle nous avons, dans un premier temps, caractérisé phénotypiquement et génotypiquement des sous-clones isolés de la lignée leucémique C1498 capable d’induire une LAM de type myélo-monocytaire chez des souris immunocompétentes C57BL/6J. Dans un deuxième temps, certains sous-clones ont été sélectionnés pour leur sensibilité à la cytarabine et transfectés de manière à exprimer stablement une protéine fluorescente (ZsGreen) en association ou non avec la protéine WT1. Enfin, ce modèle de MRD leucémique a été obtenu en modulant la quantité de cellules leucémiques injectée ainsi que la cinétique et la dose d’injection de la cytarabine. La MRD a été suivie par cytométrie en flux (expression ZsGreen) et par qPCR (expression ZsGreen et/ou de Wt1) dans le sang et la moelle osseuse des souris survivantes grâce au traitement [...]. / Acute myeloid leukemia (AML) is a genetic disorder leading to a blockade of differentiation and a clonal expansion of hematopoietic progenitors or precursors (called blasts) which accumulate in the bone marrow and then invade the blood stream. Conventional treatment relies on the use of chemotherapy agents (cytarabine in combination with an anthracycline) to eliminate leukemia cells and achieve complete remission (defined as normal bone marrow morphology with less than 5% blasts). This complete remission is achieved in a majority of patients but more than 50% of them will relapse several months after the treatment. These relapses indicate the presence of residual leukemic cells after treatment, known as measurable residual disease (MRD). It has been highlighted by the development of efficient and sensitive molecular biology technologies such as multi-parameter flow cytometry and real-time PCR allowing the detection of AML-associated expression patterns and genetic abnormalities. Several mechanisms have been described that can explain the presence of this MRD. It may be caused by the resistance to treatment of certain leukemic sub-clones (resistance-conferring mutations or quiescent phenotype) or the presence of leukemic stem cells. Finally, the immune system could also induce the quiescence of certain leukemic cells rendering them resistant to conventional chemotherapies, or control their growth leading to a state of equilibrium between their proliferation and lysis. Several AML mouse models allow the study of leukemogenesis and the testing of new therapeutic agents for leukemic cells eradication. However, they are mostly based on the transfer of human leukemic cells in immune-deficient mice and do not provide information about the role of the immune system in the leukemic cell survival, sub-clonal expansion or persistence. Moreover, there is still no available leukemia MRD mouse model allowing the study of leukemic cell persistence after chemotherapy treatment. According to these findings, the aim of this thesis was to develop a syngeneic and immune-competent mouse model of leukemia MRD overexpressing or not the Wilms' Tumor 1 (WT1) gene. The WT1 protein is described as an antigen associated with AML and is targeted by specific lymphocyte cellular and humoral responses in AML-affected patients. Creating a syngeneic and immune-competent leukemia MRD mouse model overexpressing or not this antigen will allow determining the role of this specific immune response in the cancer cell persistence. To set up this model, we first phenotyped and genotyped sub-clones isolated from the murine C1498 leukemic cell line able to induce a myelo-monocytic AML in immune-competent C57BL/6J mice. In a second step, certain sub-clones were selected for their sensitivity to cytarabine treatment and transfected to stably express the fluorescent ZsGreen protein with or without the WT1 antigen. Lastly, the MRD mouse model was obtained after modulation of various parameters such as the amount of leukemic cells administered, the kinetics and injection doses of chemotherapy. The leukemia MRD was monitored by flow cytometry (expression of the ZsGreen protein) and by real-time PCR (expression of the ZsGreen and/or Wt1 genes) in the peripheral blood and the bone marrow of treated and surviving mice. Thus, we generated a syngeneic and immune-competent leukemia MRD mouse model useful to study the immune mechanisms involved in the persistence of leukemic cell after treatment and to test new (immune)-therapeutic strategies targeting these residual cells.

Wilms’ tumor gene 1 (WT1) is a zinc finger transcriptional regulator with crucial functions in embryonic development. Originally WT1 was described as a tumor suppressor gene, but later studies have shown oncogenic properties of WT1 in a variety of tumors. Because of its dual functions in tumorigenesis, WT1 has been described as a chameleon gene. In this thesis, the significance of WT1 as a biomarker was investigated in acute myeloid leukemia (AML), clear cell renal cell carcinoma (ccRCC), ovarian carcinoma (OC) and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Previous studies have suggested that expression of WT1 is a potential marker for detection of minimal residual disease (MRD) in AML. We aimed to define expression of WT1 as an MRD marker in AML. In adult AML patients, we found that a reduction of WT1 expression in bone marrow (≥ 1-log) detected less than 1 month after diagnosis was associated with an improved overall survival (OS) and freedom from relapse (FFR). In peripheral blood, a reduction of WT1 expression (≥ 2-log) detected between 1 and 6 months after treatment initiation was associated with an improved OS and FFR. WT1 harbor pathogenic genetic variants in a considerable proportion of AML and T-lymphoblastic leukemia (T-ALL), but mutations have not been reported in BCP-ALL. We aimed to evaluate the clinical impact of WT1 mutations and single nucleotide polymorphisms (SNPs) in BCP-ALL. Pathogenic mutations in the WT1 gene were rarely seen in childhood BCP-ALL. However, five WT1 SNPs were identified. In survival analyses, WT1 SNP rs1799925 was found to be associated with worse OS, indicating that WT1 SNP rs1799925 may be a useful marker for clinical outcome in childhood BCP-ALL. We also explored whether WT1 mutations and SNPs in ccRCC could be used as biomarkers for risk and treatment stratification. We therefore examined whether SNPs or mutations in WT1 were associated with WT1 expression and clinical outcome. Sequencing analysis revealed that none of the previously reported WT1 mutations were found in ccRCC; however, we identified six different WT1 SNPs. Our data suggest that pathogenic WT1 mutations are not involved in ccRCC, and the prognostic significance of WT1 SNPs in ccRCC is considerably weak. However, a favorable OS and disease-specific survival were found in the few cases harboring the homozygous minor allele. OC has a poor prognosis, and early effective screening markers are lacking. Serous OCs are known to express the WT1 protein. Overexpressed oncogenic proteins can be considered potential candidate antigens for cancer vaccines and T-cell therapy. It was therefore of great interest to investigate whether anti-WT1 IgG antibody (Ab) measurements in plasma could serve as biomarkers of anti-OC response. We found limited prognostic impact, but the results indicated that anti-WT1 IgG Ab measurements in plasma and WT1 staining in tissue specimens could be potential biomarkers for patient outcome in the high-risk subtypes of OCs. In conclusion, the results of this thesis indicate that WT1 gene expression can provide information about MRD of patients with AML, and WT1 SNP rs1799925 may be used as a biomarker for predicting clinical outcome in childhood BCP-ALL. In ccRCC, the prognostic significance of WT1 SNPs is weak and limited to the subgroup of patients that are homozygous for the minor allele. In OCs anti-WT1 IgG Ab measurement in plasma and WT1 staining in tissue specimens could possibly be used as biomarkers for predicting patient outcome in the high-risk subtypes of OCs.

Στόχος της διδακτορικής διατριβής είναι η ανάπτυξη ενός τετραδιάστατου προσομοιωτικού μοντέλου της ελεύθερης ανάπτυξης και απόκρισης όγκων νεφροβλαστώματος σε σχήματα χημειοθεραπείας. Το μοντέλο αναπτύχθηκε στα πλαίσια των εξής ευρωπαϊκών προγραμμάτων: ACGT (Advancing Clinicogenomic Trials on Cancer, FP6-2005-IST-026996), p-Medicine (From data sharing and integration via VPH models to Personalized medicine, FP7-ICT-2009-6-270089) και TUMOR (Transatlantic Tumour Model Repositories, FP7-ICT-2009.5.4). Το μοντέλο είναι κατά βάση διακριτό και κλινικά προσανατολισμένο . Κάνει σε μεγάλη έκταση, χρήση διακριτών οντοτήτων και διακριτών γεγονότων ενώ αναφέρεται σε πολλές χωροχρονικές κλίμακες της βιολογίας του καρκίνου. . Αποτελεί μια «από επάνω προς τα κάτω» (top-down) προσομοιωτική προσέγγιση. Ξεκινώντας από τα μακροσκοπικά απεικονιστικά δεδομένα ασθενών (υψηλό επίπεδο βιοπλοκότητας) και συνεχίζοντας προς τα επίπεδα χαμηλότερης πολυπλοκότητας. Ο κλινικός προσανατολισμός του μοντέλου αποτέλεσε τη βασική αρχή κατά την ανάπτυξή του. Τα διαθέσιμα ιατρικά δεδομένα αποσκοπούν στην εξατομίκευση της προσομοίωσης της συμπεριφοράς του όγκου για κάθε ασθενή. Το μοντέλο που αναπτύχθηκε στα πλαίσια του διδακτορικού, αποτελεί μέρος μιας σύνθετης αλγοριθμικής κατασκευής και ενός συστήματος βιοϊατρικής τεχνολογίας, του “Ογκοπροσομοιωτή”. Έχουν πραγματοποιηθεί εκτεταμένοι έλεγχοι αξιοπιστίας του μοντέλου και παραμετρικές μελέτες. Η ανάλυση ευαισθησίας του μοντέλου αποκάλυψε ποιοί βιολογικοί μηχανισμοί παίζουν σημαντικό ρόλο στην εξέλιξη του όγκου. Αυτά τα συμπεράσματα μπορούν να παρέχουν περαιτέρω κατανόηση στη δυναμική της βιολογίας του καρκίνου. Τα αρχικά αποτελέσματα των προσομοιώσεων της ελεύθερης ανάπτυξης και απόκρισης σε χημειοθεραπεία τυχαίου όγκου (ελλειψοειδούς) ενισχύουν σημαντικά την αξιοπιστία του μοντέλου. H ήδη δημοσιευμένη προσαρμογή του μοντέλου σε πραγματικά περιστατικά κλινικών δοκιμών (SIOP 2001/GPOH) αποτελεί μια βάση δημιουργίας εμπιστοσύνης της επιστημονικής κοινότητας στο προσομοιωτικό δυναμικό της προσέγγισης. Για την κλινική προσαρμογή του συγκεκριμένου Ογκοπροσομοιωτή χρησιμοποιούνται τα διαθέσιμα κλινικά δεδομένα (απεικονιστικά, ιστοπαθολογικά), προ- και μετα-εγχειρητικά, σε συνδυασμό με τη διαθέσιμη πληροφορία από τη βιβλιογραφία. Η δυνατότητα να αξιοποιούνται άμεσα πρόσθετα δεδομένα στα πλαίσια κλινικών δοκιμών, περιορίζοντας έτσι το “παράθυρο” των πιθανών λύσεων αποτελεί ένα ιδιαίτερα ξεχωριστό χαρακτηριστικό του παρουσιαζόμενου μοντέλου. Σημειώνεται ότι το μοντέλο βρίσκεται κάτω υπό συνεχή βελτιστοποίηση και επέκταση στα πλαίσια ευρωπαϊκών προγραμμάτων υπό υλοποίηση και κλινικών δοκιμών. / The aim of this thesis is to develop a 4D spatiotemporal simulation model of the free growth of Wilms’ tumours and its response to chemotherapeutic regimens. The model has been developed within the framework of three EC-funded projects: ACGT (Advancing Clinicogenomic Trials on Cancer, FP6-2005-IST-026996), p-Medicine (From data sharing and integration via VPH models to Personalized medicine, FP7-ICT-2009-6-270089) and TUMOR (Transatlantic Tumour Model Repositories, FP7-ICT-2009.5.4). It is is a predominantly discrete entity -discrete event, clinically-oriented multiscale cancer model. A ‘‘top-down’’ simulation approach has been formulated. The approach method starts from the macroscopic imaging data representing a high scale/level of tumour biology and proceeds towards lower scales/levels. The clinical orientation of the model has been a fundamental guiding principle throughout its development. Available medical data is exploited, in order to support patient-individualized modelling. The model developed has been embedded in a complex algorithmic system and a bioengineering tool denoted by the term “Oncosimulator”. A thorough cross-method sensitivity analysis of the model has been performed, revealing the most determinant biological mechanisms in terms of tumour aggressiveness and therapy outcome. The outcome of this work has provided important insight into the biology of cancer dynamics. Initial results of free growth and response to chemotherapy have been produced and judged as reasonable, thus supporting the validity of the model Successful model adaptation to real clinical cases in the context of the SIOP/GPOH clinical trial guided by a sensitivity analysis has been achieved and published. In order to validate the model, the available pre- and post-surgery clinical data (imaging and histopathological) are used in combination with available literature data. The potential to readily exploit additional data available in the context of clinical trials, thereby narrowing the window of possible solutions, is a particularly distinctive feature of the model. The model is under continuous refinement, optimization and extension in the framework of pertinent clinical trials.

Myeloid leukemias include malignant diseases characterized by clonal expansion of the myeloid cell lineage. While in case of chronic myeloid leukemia (CML), the main cause of the disease has already been identified - t(9;22) and the aktivity of the fusion product of the translocation BCR-ABL, acute myeloid leukemia (AML) has been associated with plenty of different translocations and mutations. The aim of this work was to contribute to the improvement of monitoring of patients with myeloid leukemias via detailed study of the panleukemic marker Wilms tumor gene 1 (wt1) expression. Prognostic value of wt1 expression has been proved for AML patients, however, it has not yet been confirmed for CML patients. Expression of different wt1 variants (more then 36 protein products) is known very poorly in both, AML and CML as well as in normal hematopoiesis. Most of the study is focused on CML, only limited parts are dedicated to AML. In the first part of the work, we clearly proved prognostic value of total wt1 mRNA expression for CML patients. Statistical evaluations revealed critical wt1 values which enable to specify prognosis of patients responding non-optimally to imatinib. Bcr-abl looses much of its prognostic value in these patients. Further, we have designed and optimized PCRs for selected wt1...

La tumeur de Wilms (WT) est la tumeur du rein la plus fréquente chez l'enfant âgé de moins de 5 ans. Bien que la majorité des enfants avec WT soit aujourd'hui soignée, 10 à 15% rechutent. L'identification de nouveaux marqueurs pronostic au diagnostic est donc nécessaire. Nous avons centralisé le matériel biologique de toutes les WTs françaises inclus dans le protocole SIOP-2001. Le blastème, la composante histologique la plus agressive de ces tumeurs, a été caractérisé par une analyse qualitative (architecture et aspect cytologique, index mitotique et prolifératif) et quantitative (volume et pourcentage). En collaboration avec les équipes européennes du Renal Tumor Study Group nous avons caractérisé les anomalies structurales de la WT par Multiple Ligation Probe Amplification (MLPA). De plus, nous nous sommes focalisés sur la région 11p15, où le gène IGF2 est localisé, en analysant les anomalies structurales et de méthylation au niveau de différents loci (IGF2-DMR0, ICR1, ICR2, H19), par MLPA et par ASMM RTQ- PCR (TaqMan allele-specific methylated multiplex real-time quantitative PCR). Nous avons de plus étudié l'expression d'IGF2 par RT-QPCR. Une dernière partie de notre étude a porté sur la caractérisation de la réponse immune intratumorale par immunohistochimie (CD3, CD4, CD8, PD1, PDL1). En conclusion, nous avons identifié des potentiels marqueurs pronostiques concernant la prise en charge des WTs: volume et pourcentage de blastème, index mitotique et de prolifération, perte de méthylation d'IGF2-DMR0 et rapport CD4/CD8. Ce travail pourrait contribuer à la détermination d'une nouvelle classification bio-pathologique de la WT. / Wilms tumor (WT) is the most common tumor of the kidney in children aged under 5 years. Although the majority of children with WT survives, 10-15% relapse. The identification of new prognosis markers at diagnosis is necessary. We centralized the biological material of all French WTs included in the SIOP-2001 protocol. The blastema, the most aggressive histological component of these tumors, was characterized by qualitative (architectural and cytological aspect, proliferative and mitotic index) and quantitative analysis (amount and percentage). In collaboration with European teams Renal Tumor Study Group we characterized the structural abnormalities of the WT by Multiple Ligation Probe Amplification (MLPA). In addition, we focused on the 11p15 region, where the IGF2 gene is located, analyzing the structural abnormalities and methylation at different loci (IGF2-DMR0, ICR1, ICR2, H19) by MLPA and ASMM RTQ - PCR (TaqMan allele-specific methylated multiplex quantitative real-time PCR). We also studied the expression of IGF2 by RT-QPCR. A final part of our study focused on the characterization of intratumoral immune response by immunohistochemistry (CD3, CD4, CD8, PD1, PDL1). In conclusion, we identified potential prognostic markers for the management of WTs: volume and percentage of blastema, mitotic and proliferation index, loss of IGF2 methylation-DMR0 and CD4 / CD8 ratio. This could contribute to the determination of a new bio-pathological classification of WT.