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

No description available.

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

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

<p>The array-based form of comparative genomic hybridization (array-CGH) is a new methodology that has shown to be of significant importance. This thesis focuses on the development of array-CGH with the aim to define candidate regions/genes on chromosome 22 in a wide spectrum of cancer-related conditions. In <b>paper I</b>, we developed and applied the first comprehensive genomic microarray, representing human chromosome 22, for analysis of DNA copy number. Using this array-based approach, we identified gene copy number alterations, including heterozygous/homozygous deletions, amplifications, IGLV/IGLC locus instability and the breakpoints of imbalanced translocation, in several 22q-associated disorders. In <b>paper II</b>, we applied the same array to perform DNA copy number profiling of a series of ovarian carcinoma. cDNA arrays were also used in this study to correlate gene expression levels with DNA-copy number. In the course of this analysis, we determined a small 3.5 Mb candidate 22q telomeric region and suggested a number of specific candidate genes. <b>Paper III</b> described the comprehensive and high-resolution analysis of chromosome 22 in a large set of various stage breast cancers. Multiple distinct patterns of genetic aberrations were observed. The smallest identified candidate locus was 220 kb in size and mapped to a gene-rich region in the vicinity of telomere of 22q. Intriguing result of this study was the detection of high frequency (26.6%) of intra-tumoral clonal variation in gene copy number profiles, which should be viewed as a high number, considering that we study in detail only a single human chromosome. In <b>paper IV</b>, we profiled a series of 28 Wilms tumor samples using 22q-array in order to assess specific regions affected with DNA dosage-alterations. The distribution of aberrations defined a complex amplifier genotype and delimited two tumor suppressor/oncogene candidate loci. These results open up for several avenues for continued research of these tumor forms. These findings also demonstrate the power of array-CGH in the precise determination of minute DNA copy number alterations and strengthen the notion that further studies, preferentially in the context of the entire human genome, are needed.</p>

The array-based form of comparative genomic hybridization (array-CGH) is a new methodology that has shown to be of significant importance. This thesis focuses on the development of array-CGH with the aim to define candidate regions/genes on chromosome 22 in a wide spectrum of cancer-related conditions. In <b>paper I</b>, we developed and applied the first comprehensive genomic microarray, representing human chromosome 22, for analysis of DNA copy number. Using this array-based approach, we identified gene copy number alterations, including heterozygous/homozygous deletions, amplifications, IGLV/IGLC locus instability and the breakpoints of imbalanced translocation, in several 22q-associated disorders. In <b>paper II</b>, we applied the same array to perform DNA copy number profiling of a series of ovarian carcinoma. cDNA arrays were also used in this study to correlate gene expression levels with DNA-copy number. In the course of this analysis, we determined a small 3.5 Mb candidate 22q telomeric region and suggested a number of specific candidate genes. <b>Paper III</b> described the comprehensive and high-resolution analysis of chromosome 22 in a large set of various stage breast cancers. Multiple distinct patterns of genetic aberrations were observed. The smallest identified candidate locus was 220 kb in size and mapped to a gene-rich region in the vicinity of telomere of 22q. Intriguing result of this study was the detection of high frequency (26.6%) of intra-tumoral clonal variation in gene copy number profiles, which should be viewed as a high number, considering that we study in detail only a single human chromosome. In <b>paper IV</b>, we profiled a series of 28 Wilms tumor samples using 22q-array in order to assess specific regions affected with DNA dosage-alterations. The distribution of aberrations defined a complex amplifier genotype and delimited two tumor suppressor/oncogene candidate loci. These results open up for several avenues for continued research of these tumor forms. These findings also demonstrate the power of array-CGH in the precise determination of minute DNA copy number alterations and strengthen the notion that further studies, preferentially in the context of the entire human genome, are needed.

The PAX gene family of transcription factors plays a prominent role during embryogenesis however can be aberrantly re-activated during tumorigenesis and contributes to the malignant phenotype. / During embryonic kidney development, PAX2 exerts an anti-apoptotic function however its expression typically attenuates during the post-natal period. On the other hand, PAX2 aberrant expression is observed in the majority of Renal Cell Carcinomas (RCC). RCC is resistant to chemotherapy; up-regulation of anti-apoptotic genes is recognized to contribute to tumor resistance to chemotherapy. We hypothesized that the anti-apoptotic effect of the PAX2 gene that is expressed in RCC cells contributes to RCC and their resistance to chemotherapy-induced cell death. / Human embryonic kidney (HEK293) cells transfected with a PAX2 expression vector and exposed to cisplatin, were protected from apoptosis compared to control cells. Conversely, murine collecting duct cells stably transfected with PAX2 antisense cDNA had twofold increases in cisplatin-induced apoptosis. Similarly, PAX2 knockdown using PAX2 siRNA in RCC cells CAKI-1 and ACHN enhances cisplatin-induced apoptosis in vitro. / To test the combination of PAX2 expression silencing and cisplatin treatment in vivo we developed a model of renal tumors by injecting ACHN cells as a xenograft under the skin of nude mice. I showed that a PAX2 shRNA successfully knocks down PAX2 mRNA and protein levels in a RCC cell line (ACHN). ACHN cells stably transfected with shRNAs targeted against the PAX2 homeodomain, are more susceptible to cisplatin-induced caspase-3 activation than the control ACHN cell line. Furthermore, growth of subcutaneous ACHN/shPAX2 xenografts in nude mice is significantly more responsive to cisplatin therapy than control of ACHN cell tumors. This work proposes PAX2 as a potential therapeutic gene target in metastatic renal cell carcinoma and suggests that adjunctive PAX2 knockdown may enhance the efficacy of chemotherapeutic agents such as cisplatin. / Wilms tumor, the most common pediatric renal cancer, is thought to arise from a progenitor cell of the metanephric mesenchyme that fails to complete nephrogenesis. In addition to its characteristic triphasic histology, WT can exhibit myogenic differentiation. Myogenic programming during muscle development is controlled by a PAX3 transcription factor determinant for muscle development; unexpectedly PAX3 transcriptional activity has been recently identified in the embryonic mouse kidney. These observations led us to hypothesize that PAX3 plays a role during kidney development. Furthermore, we predict that if PAX3 expression is verified during renal development, PAX3 may also be expressed in Wilms tumor with a myogenic component. / I showed that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of the developing mouse kidney. In a panel of 20 Wilms tumors, PAX3 was identified in tumor samples with myogenic histopathology. Furthermore, mutations of WT1 were consistently associated with PAX3 expression in Wilms tumors and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. / This work supports a novel model of normal renal development in which progenitor cells of the metanephric blastema express PAX3 when targeted toward the stromal cell fate. Suppression of PAX3 is integral to the mesenchyme-to-epithelium transition, which defines the nephrogenic cell fate and may be accomplished, in part, by WT1. Conversely, failure to suppress PAX3 may account for the myogenic phenotype in a subset of WT1-negative Wilms tumors.

WT1 has two main isoforms: WT1(-KTS) and WT1(+KTS). Both are known to bind to a DNA consensus sequence with different affinities, and are thus postulated to play overlapping but distinct functional roles in the cell. WT1 is also known to bind to certain RNA moieties as well as to various protein partners (e.g. Ciao-1). This study focuses on the development of large scale protein purification protocols for WT1 zinc finger (ZF) proteins as well as Ciao-1. By using a combination of his-tag affinity and size exclusion chromatography we were able to purify milligram quantities of these proteins. It was also the intention to obtain crystals of the WT1 ZF protein in complex with any one of its known binding partners, in particular the protein Ciao-1 (a WD40 protein) and the 14 mer consensus sequence of DNA (known as WTE). In conjunction with structural studies it was determined that a previously made SELEX RNA library was not selective for the (+KTS) isoform of WT1 ZF, and therefore no RNA candidate could be identified for future structural studies.

WT1 has two main isoforms: WT1(-KTS) and WT1(+KTS). Both are known to bind to a DNA consensus sequence with different affinities, and are thus postulated to play overlapping but distinct functional roles in the cell. WT1 is also known to bind to certain RNA moieties as well as to various protein partners (e.g. Ciao-1). This study focuses on the development of large scale protein purification protocols for WT1 zinc finger (ZF) proteins as well as Ciao-1. By using a combination of his-tag affinity and size exclusion chromatography we were able to purify milligram quantities of these proteins. It was also the intention to obtain crystals of the WT1 ZF protein in complex with any one of its known binding partners, in particular the protein Ciao-1 (a WD40 protein) and the 14 mer consensus sequence of DNA (known as WTE). In conjunction with structural studies it was determined that a previously made SELEX RNA library was not selective for the (+KTS) isoform of WT1 ZF, and therefore no RNA candidate could be identified for future structural studies.