Clinical trials are currently exploring the combinations of P ARP inhibitors and immunotherapies in the treatment of ovarian cancer, but their effects on the ovarian tumour microenvironment (TME) remain unclear. Here, we investigate how olaparib, PD-L1 monoclonal antibodies and their combination can influence TME composition and survival of tumour-bearing mice. We further explored how BRCA mutations can influence the response to therapy. Olaparib and combination therapies similarly improved the median survival of Brca1- and Brca2-deficient tumour-bearing mice. Anti-PD-L1 monotherapy improved the survival of mice with Brca1-null tumours, but not Brca2-null tumours. A detailed analysis of the TME revealed that the olaparib monotherapy resulted in a large number of immunosuppressive and immunomodulatory effects in the more inflamed Brca1-deficient TME but not Brca2-deficient tumours. Anti-PD-L1 treatment was mostly immunosuppressive, resulting in a systemic reduction of cytokines and a compensatory increase of PD-L1. The results of the combination therapy generally resembled the effects of one or both of the monotherapies, along with unique changes observed in certain immune populations. In-silico analysis of RNA-seq also revealed numerous differences between Brca-mutated tumour models. In summary, these findings shed light on the influence of novel therapeutics and BRCA mutations on the ovarian TME.

No description available.

O câncer de próstata (CaP) é o tumor maligno mais frequente e uma das principais causas de morte por câncer na população masculina no mundo e no Brasil. Três fatores são de fundamental importância no prognóstico da doença: o estadiamento, o grau histológico (avaliado pelo escore de Gleason) e o antígeno prostático específico (Prostate Specific Antigen - PSA). Há fatores adicionais descritos que podem influenciar na evolução do câncer, como invasão perineural, invasão angiolinfática, acometimento da cápsula, lateralidade do tumor, estágio tumoral e invasão de linfonodos. Embora a associação entre mutações do BRCA2 e o risco de câncer de próstata esteja bem documentado, pouco se sabe sobre o papel do BRCA2 na progressão do câncer de próstata após o diagnóstico inicial. O presente trabalho se propõe a melhor elucidar o papel da proteína BRCA2 nos carcinomas prostáticos comparando os resultados da expressão proteica de BRCA2 com dados clinicopatológicos de pacientes acometidos por Câncer de Próstata, além de avaliar a expressão de BRCA2 com escore de Gleason >=7 após a prostatectomia, tanto na classificação de Gleason segundo Epstein 2005, quanto na classificação do grau de Gleason definida em 2014 com suas respectivas biópsias por agulha. Dos arquivos do Serviço de Patologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, foram selecionados 125 blocos de parafina com amostras de câncer de próstata diagnosticados entre 2005 e 2010. As lâminas coradas com hematoxilina e eosina (H&E) foram utilizadas para a construção de microarranjos teciduais (TMAs). Nesses TMAs foi realizado estudo imunohistoquímico para BRCA2. A proteína BRCA2 com ponto de corte de 15% (classificação antiga) foi superexpressa em 118 casos (94,4%), enquanto a expressão de BRCA2 com cut-off de 56% (classificação nova) foi expressa em 63 (51,2%) de 125 casos. A superexpressão de BRCA2 associada a classificação antiga de Gleason correlacionou-se com bilateralidade do tumor e com estágio tumoral, em contrapartida a expressão de BRCA2 associada a atual classificação se correlacionou apenas com lesão intraepitelial prostática (PIN). Nossos resultados indicam que a expressão de BRCA2 pode ser um biomarcador importante de progressão tumoral nos carcinomas de próstata. / Prostate cancer (PCa) is the most common malignancy and a major cause of cancer death in male population in the world and in Brazil. Three factors are of fundamental importance in the prognosis of the disease: the staging, histological grade (measured by Gleason score) and the Prostate Specific Antigen (PSA). There are additional factors described that may influence the development of cancer, such as perineural invasion, angiolymphatic invasion, capsule involvement, tumor laterality, tumor stage and lymph node invasion. Although the association between mutations in Breast Cancer Gene 2 (BRCA2) and the risk of prostate cancer is well documented, little is known about the role of BRCA2 in the progression of prostate cancer after initial diagnosis. This study aims to elucidate the role of BRCA2 protein in prostate carcinomas comparing the results of the BRCA2 protein expression with clinicopathological data of patients with prostate cancer. For that reason, we evaluated the expression of BRCA2 with Gleason score >=7 after prostatectomy, both in the second Epstein 2005 Gleason score, and in the classification of Gleason grade set in 2014 with their respective needle biopsies. 125 paraffin blocks with prostate cancer samples diagnosed between 2005 and 2010 were selected from the archives of Hospital Pathology Service of the Ribeirao Preto Medical School, University of Sao Paulo. The slides stained with hematoxylin and eosin (H&E) were used for the construction of the tissue microarrays (TMAs). These TMAs was performed immunohistochemical study for BRCA2. The BRCA2 protein with a cut-off point of 15% (old classification) was overexpressed in 118 cases (94.4%), while the expression of BRCA2 to cut off 56% (new classification) was expressed in 63 (51.2%) of 125 cases. The BRCA2 associated overexpression of the old classification of Gleason presented correlation with tumor bilateralism and tumor stage, however BRCA2 expression associated with the current classification was correlated only with Prostatic Intraepithelial Lesion (PIN). Our results indicate that BRCA2 expression can be an important marker of tumor progression in prostate carcinomas.

Les femmes porteuses d’une mutation du gène BRCA1 ou BRCA2 ont un risque de cancer du sein (CS) très élevé dont les estimations varient beaucoup d’une étude à l’autre. L’objectif principal de cette étude est de mieux estimer le risque de CS associé aux gènes BRCA1/2 en tenant compte de la variabilité des mutations et des facteurs « environnementaux/style de vie » et de leur éventuelle interaction. Nous avons analysé les données de la cohorte GENEPSO composée de femmes porteuses d’une mutation du gène BRCA1 ou BRCA2 à l’aide d’un modèle de Cox pondéré. L’analyse des facteurs de risque gynéco-obstétrique et de « style de vie » a permis de mettre en évidence une association entre le risque de CS et les radiations ionisantes, la consommation de tabac, l’indice de masse corporelle, l’âge aux premières règles, la parité, les interruptions de grossesse, la contraception orale, la ménopause et les traitements hormonaux substitutifs. Cette étude confirme l’existence d’une zone centrale à moindre risque de CS dans les gènes BRCA1/2 et de décrire une nouvelle région à haut risque située dans la région 3’ du gène BRCA2. Cette étude montre également une interaction entre la localisation des mutations et la parité ainsi que la ménopause. Cette étude montre l’importance de la prise en compte simultanée des facteurs de risque « non génétiques » et de la localisation des mutations dans les gènes BRCA1/2 dans l’estimation des risques de CS. Si nos résultats sont confirmés sur de plus larges données, cette étude pourrait aider ces femmes dans le choix du type de stratégie de surveillance ou de prévention le mieux adapté à leur situation. / Breast cancer is the most frequent and mortal cancer among French women. Mutations in BRCA1 and BRCA2 confer a high risk of breast cancer. The diversity of BRCA1 and BRCA2 mutations and of gyneco-obstetrical and life style risk factors may modify the magnitude of this risk. The objective of this study is to assess breast cancer risk taking into account gyneco-obstetrical and life style factors, location of mutation in BRCA1 and BRCA2 genes and there potential interaction.The data from 1337 women BRCA1 and BRCA2 mutation carriers of the French study GENEPSO have been analysed by using a weighted Cox regression model to take into account the bias of recruitment. Our results show an association between breast cancer risk and X-ray exposures, tobacco, body mass index, age at menarche, parity, interrupted pregnancies, oral contraceptives, menopausal status and hormone replacement therapy. We confirm the existence of a central low breast cancer risk region in BRCA1 (LR1 region between codons 374 and 1161) and in BRCA2 (LR2 region between codons 957 and 1827). In addition, a new high breast cancer risk region is found in the 3’ region of BRCA2 (HR2 region between codons 2546 and 2968). Moreover, our results suggest a variation in breast cancer risk associated with parity and menopausal status according to the location of the mutation in BRCA1 and BRCA2. Our findings show that, taking into account environmental and lifestyle modifiers, mutation position might be important for the clinical management of BRCA1 and BRCA2 mutation carriers and could also be helpful in understanding how BRCA1 and BRCA2 genes are involved in breast cancer.

Introdução: Mutações nos genes BRCA1 e BRCA2 são responsáveis por cerca de 50% dos casos de câncer de mama e/ou ovário hereditários. Atualmente não conhecemos o perfil de mutações destes genes na população brasileira, com exceção de mutações fundadoras que ocorrem em grupos étnicos específicos. Objetivos: Detectar mutações e polimorfismos nos genes BRCA1 e BRCA2 em 73 pacientes com câncer de mama selecionadas para o teste genético. Casuística e métodos: Realizamos o sequenciamento direto e o teste de MLPA para os genes BRCA1 e BRCA2 em 73 indivíduos, sendo 63 pacientes com câncer de mama com risco maior ou igual a 10% de acordo com os critérios de Frank, Evans e BRCAPRO, dois pacientes com câncer de ovário e oito indivíduos saudáveis com forte histórico familiar de câncer ligado a mutações em BRCA1 e/ou BRCA2. Resultados: Encontramos 60 mutações no gene BRCA1: 13 alterações missense (incluindo a deletéria R71G), sete mutações sinônimas, uma mutação frameshift (a deletéria 5382insC), uma mutação nonsense (a deletéria R1751X), uma deleção in frame, uma alteração 3UTR e 36 variantes intrônicas. Em BRCA2 encontramos 57 mutações, entre as quais 26 mutações missense, uma alteração 5UTR, 11 mutações sinônimas, 14 variantes intrônicas, duas mutações nonsense (as deletérias R2318X e R3128X) e três mutações frameshift deletérias (5844del5, 6633del5 e 6610insTT). Nenhuma mutação foi detectada pelo teste de MLPA. Discussão e considerações finais: Nove de 73 indivíduos estudados são portadores de mutações deletérias, sendo que a mutação fundadora Ashkenazi 5382insC foi encontrada em duas pacientes não aparentadas e que outro grupo de pesquisa já reportou sua alta frequência numa população paulistana. As alterações de significado clínico desconhecido foram encontradas em toda a extensão dos genes BRCA1 e BRCA2 e são inúmeras. Algumas apareceram em somente uma paciente, o que nos leva a pensar que talvez uma ou algumas destas mutações tenham algum efeito patogênico, como a mutação 6610insTT, que gera uma proteína incompleta e foi encontrada em três gerações de uma família. A técnica de MLPA não detectou grandes rearranjos em ambos os genes, mostrando que este tipo de alteração genética não é freqüente em nossa coorte e que talvez esta seja uma característica mais prevalente em populações menos miscigenadas. Salientamos, portanto, a importância de ampliar este estudo e de estimular pesquisas futuras, visando um aconselhamento genético eficiente, com a diminuição do número de casos inconclusivos gerados pelas variantes de significado indeterminado e o acompanhamento clínico das famílias / Introduction: Mutation in BRCA1 and BRCA2 genes are responsible for more than 50% of hereditarian breast and ovarian cancer cases. Nowadays, we still dont know the Brazilian mutation profile for these genes, except when founder mutations occur in specific ethnic groups. Objetives: Detection of mutation and polymorphisms in BRCA1 and BRCA2 genes in 73 breast cancer patients selected for genetic testing. Casuistic and methods: we have realized direct sequencing of BRCA1 and BRCA2 in 73 patients, whose 63 have had breast cancer and showed at least 10% of risk according to Frank, Evans and BRCAPRO, two patients with ovarian cancer and eight healthy individuals of strong family history of cancer linked to mutations in BRCA1 and BRCA2. Results: We have found 60 mutations in BRCA1: 13 missense mutations (including the deleterious R71G), seven synonymous mutations, one frameshift mutation (the deleterious 5382insC), one nonsense mutation (the deleterious R1751X), one in-frame deletion, one 3UTR mutation and 36 intronic variants. In BRCA2 we have found 57 mutations: 26 missense mutations, one 5UTR mutation, 11 synonymous mutations, 14 intronic variants, two nonsense mutations (the deleterious R2318X and R3128X) and three frameshift mutations (5844del5, 6610insTT and 6633del5). No mutation was detected by MLPA technique. Discussion and final considerations: Nine of 73 studied individuals carry deleterious mutations. Among them, the Ashkenazi founder mutation 5382insC has been found in two unrelated patients and it was previously reported by another research group for its high prevalence on a population from São Paulo. Alterations of unknown clinical significance have been found all over BRCA1 and BRCA2 gene extension and are countless. Some of them are shown only in one patient, leading us to think that maybe one or a few might have a pathogenic effect, like 6610insTT, which leads to a BRCA2 incomplete protein and was shown in 3 generations of a family. MLPA technique have not detected large genomic rearrangements in both genes, showing that this kind of mutation is not frequent on our cohort and maybe this genetic alteration characterizes less miscigenated populations. So, we emphasize the importance of enlarge this study and stimulate future researches, aiming an efficient genetic counseling, decreasing inconclusive cases generated by unknown clinical significance variants, and follow up of affected families

O câncer de próstata (CaP) é o tumor maligno mais frequente e uma das principais causas de morte por câncer na população masculina no mundo e no Brasil. Três fatores são de fundamental importância no prognóstico da doença: o estadiamento, o grau histológico (avaliado pelo escore de Gleason) e o antígeno prostático específico (Prostate Specific Antigen - PSA). Há fatores adicionais descritos que podem influenciar na evolução do câncer, como invasão perineural, invasão angiolinfática, acometimento da cápsula, lateralidade do tumor, estágio tumoral e invasão de linfonodos. Embora a associação entre mutações do BRCA2 e o risco de câncer de próstata esteja bem documentado, pouco se sabe sobre o papel do BRCA2 na progressão do câncer de próstata após o diagnóstico inicial. O presente trabalho se propõe a melhor elucidar o papel da proteína BRCA2 nos carcinomas prostáticos comparando os resultados da expressão proteica de BRCA2 com dados clinicopatológicos de pacientes acometidos por Câncer de Próstata, além de avaliar a expressão de BRCA2 com escore de Gleason >=7 após a prostatectomia, tanto na classificação de Gleason segundo Epstein 2005, quanto na classificação do grau de Gleason definida em 2014 com suas respectivas biópsias por agulha. Dos arquivos do Serviço de Patologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, foram selecionados 125 blocos de parafina com amostras de câncer de próstata diagnosticados entre 2005 e 2010. As lâminas coradas com hematoxilina e eosina (H&E) foram utilizadas para a construção de microarranjos teciduais (TMAs). Nesses TMAs foi realizado estudo imunohistoquímico para BRCA2. A proteína BRCA2 com ponto de corte de 15% (classificação antiga) foi superexpressa em 118 casos (94,4%), enquanto a expressão de BRCA2 com cut-off de 56% (classificação nova) foi expressa em 63 (51,2%) de 125 casos. A superexpressão de BRCA2 associada a classificação antiga de Gleason correlacionou-se com bilateralidade do tumor e com estágio tumoral, em contrapartida a expressão de BRCA2 associada a atual classificação se correlacionou apenas com lesão intraepitelial prostática (PIN). Nossos resultados indicam que a expressão de BRCA2 pode ser um biomarcador importante de progressão tumoral nos carcinomas de próstata. / Prostate cancer (PCa) is the most common malignancy and a major cause of cancer death in male population in the world and in Brazil. Three factors are of fundamental importance in the prognosis of the disease: the staging, histological grade (measured by Gleason score) and the Prostate Specific Antigen (PSA). There are additional factors described that may influence the development of cancer, such as perineural invasion, angiolymphatic invasion, capsule involvement, tumor laterality, tumor stage and lymph node invasion. Although the association between mutations in Breast Cancer Gene 2 (BRCA2) and the risk of prostate cancer is well documented, little is known about the role of BRCA2 in the progression of prostate cancer after initial diagnosis. This study aims to elucidate the role of BRCA2 protein in prostate carcinomas comparing the results of the BRCA2 protein expression with clinicopathological data of patients with prostate cancer. For that reason, we evaluated the expression of BRCA2 with Gleason score >=7 after prostatectomy, both in the second Epstein 2005 Gleason score, and in the classification of Gleason grade set in 2014 with their respective needle biopsies. 125 paraffin blocks with prostate cancer samples diagnosed between 2005 and 2010 were selected from the archives of Hospital Pathology Service of the Ribeirao Preto Medical School, University of Sao Paulo. The slides stained with hematoxylin and eosin (H&E) were used for the construction of the tissue microarrays (TMAs). These TMAs was performed immunohistochemical study for BRCA2. The BRCA2 protein with a cut-off point of 15% (old classification) was overexpressed in 118 cases (94.4%), while the expression of BRCA2 to cut off 56% (new classification) was expressed in 63 (51.2%) of 125 cases. The BRCA2 associated overexpression of the old classification of Gleason presented correlation with tumor bilateralism and tumor stage, however BRCA2 expression associated with the current classification was correlated only with Prostatic Intraepithelial Lesion (PIN). Our results indicate that BRCA2 expression can be an important marker of tumor progression in prostate carcinomas.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-19T10:42:04Z No. of bitstreams: 2 Dissertação - Rebeca Mota Goveia - 2018.pdf: 2457051 bytes, checksum: 721dc25f2c29d54fc3bd4f0f13c5fc83 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-19T11:18:57Z (GMT) No. of bitstreams: 2 Dissertação - Rebeca Mota Goveia - 2018.pdf: 2457051 bytes, checksum: 721dc25f2c29d54fc3bd4f0f13c5fc83 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-07-19T11:18:57Z (GMT). No. of bitstreams: 2 Dissertação - Rebeca Mota Goveia - 2018.pdf: 2457051 bytes, checksum: 721dc25f2c29d54fc3bd4f0f13c5fc83 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: Breast cancer is the second most common cancer in the world, and the most common among women, only 5% to 10% are hereditary and half of them are caused by hereditary breast and ovarian cancer (HBOC) , caused by variations in the BRCA1 and BRCA2 genes. Objectives: The present study aimed to identify the prevalence of deletions and duplications in BRCA1 and BRCA2 genes in breast cancer patients in Goiás, Brazil. Materials and methods: We evaluated 46 breast cancer patients who met National Comprehensive Cancer Network (NCCN) criteria for HBOC syndrome screening. A 4 ml blood sample was collected for DNA extraction using commercial kit and the MLPA (Multiplex Ligation Dependent Probe Amplification) technique was performed using the SALSA MLPA P002 BRCA1 and SALSA MLPA P045 BRCA2 / CHECK2 kits. Results and discussion: The majority of the patients were female (97.83%) and the mean age of the patients was 37.52 years. In this group, 43.47% of the patients were younger than 35 years at the time of diagnosis and 35% of them were diagnosed with triple negative tumors. The most common molecular subtype was luminal A (46.2%) followed by triple negative tumors (28.20%). No patient was found with rearrangements in BRCA1. In the BRCA2 gene, one patient (2.12%) presented a false positive result for the heterozygous deletion of exon 27, which may have been caused by the presence of a small change in the probe binding region. Conclusion: This was the first study performed to analyze large deletions and duplications in patients from the central-western region of Brazil. We can conclude that the frequency of large deletions and duplications in the BRCA1 and BRCA2 genes in the Goian population is low. / Introdução: O câncer de mama é o segundo tipo de câncer mais freqüente no mundo, e o mais comum entre as mulheres, apenas 5% a 10% são hereditários e metade deles são causados ​​pela síndrome do câncer de mama e ovário hereditário (HBOC), causada por variações nos genes BRCA1 e BRCA2. Objetivos: O presente estudo teve como objetivo identificar a prevalência de deleções e duplicações nos genes BRCA1 e BRCA2 em pacientes com câncer de mama no estado de Goiás, Brasil. Materiais e métodos: Avaliamos 46 pacientes com câncer de mama que atenderam aos critérios do National Comprehensive Cancer Network (NCCN) para pesquisa da síndrome HBOC. Foi coletada uma amostra de sangue de 4 ml para extração de DNA usando kit comercial e a técnica MLPA (Multiplex Ligation Dependent Probe Amplification) foi realizada usando os kits SALSA MLPA P002 BRCA1 e SALSA MLPA P045 BRCA2 / CHECK2. Resultados e discussão: A maioria dos pacientes era do sexo feminino (97.83%) e a idade média dos pacientes era de 37,52 anos. Neste grupo 43.47% dos pacientes possuíam idade inferior a 35 anos no momento do diagnóstico sendo que 35% destes foram diagnosticados com tumores triplo negativo. O subtipo molecular mais comum foi o luminal A (46.2%) seguido de tumores triplo negativos (28.20%). Nenhum paciente foi encontrado com rearranjos no gene BRCA1. No gene BRCA2, um paciente (2,12%) apresentou um resultado falso positivo para a deleção em heterozigoze do éxon 27, fato que pode ter sido ocasionado pela presença de uma pequena alteração na região de ligação da sonda. Conclusão: Este foi o primeiro estudo realizado para análise de grandes deleções e duplicações em pacientes da região centro-oeste do Brasil. Podemos concluir que a frequência de grandes deleções e duplicações nos genes BRCA1 e BRCA2 na população goiana é baixa.

Introdução: Mutações nos genes BRCA1 e BRCA2 são responsáveis por cerca de 50% dos casos de câncer de mama e/ou ovário hereditários. Atualmente não conhecemos o perfil de mutações destes genes na população brasileira, com exceção de mutações fundadoras que ocorrem em grupos étnicos específicos. Objetivos: Detectar mutações e polimorfismos nos genes BRCA1 e BRCA2 em 73 pacientes com câncer de mama selecionadas para o teste genético. Casuística e métodos: Realizamos o sequenciamento direto e o teste de MLPA para os genes BRCA1 e BRCA2 em 73 indivíduos, sendo 63 pacientes com câncer de mama com risco maior ou igual a 10% de acordo com os critérios de Frank, Evans e BRCAPRO, dois pacientes com câncer de ovário e oito indivíduos saudáveis com forte histórico familiar de câncer ligado a mutações em BRCA1 e/ou BRCA2. Resultados: Encontramos 60 mutações no gene BRCA1: 13 alterações missense (incluindo a deletéria R71G), sete mutações sinônimas, uma mutação frameshift (a deletéria 5382insC), uma mutação nonsense (a deletéria R1751X), uma deleção in frame, uma alteração 3UTR e 36 variantes intrônicas. Em BRCA2 encontramos 57 mutações, entre as quais 26 mutações missense, uma alteração 5UTR, 11 mutações sinônimas, 14 variantes intrônicas, duas mutações nonsense (as deletérias R2318X e R3128X) e três mutações frameshift deletérias (5844del5, 6633del5 e 6610insTT). Nenhuma mutação foi detectada pelo teste de MLPA. Discussão e considerações finais: Nove de 73 indivíduos estudados são portadores de mutações deletérias, sendo que a mutação fundadora Ashkenazi 5382insC foi encontrada em duas pacientes não aparentadas e que outro grupo de pesquisa já reportou sua alta frequência numa população paulistana. As alterações de significado clínico desconhecido foram encontradas em toda a extensão dos genes BRCA1 e BRCA2 e são inúmeras. Algumas apareceram em somente uma paciente, o que nos leva a pensar que talvez uma ou algumas destas mutações tenham algum efeito patogênico, como a mutação 6610insTT, que gera uma proteína incompleta e foi encontrada em três gerações de uma família. A técnica de MLPA não detectou grandes rearranjos em ambos os genes, mostrando que este tipo de alteração genética não é freqüente em nossa coorte e que talvez esta seja uma característica mais prevalente em populações menos miscigenadas. Salientamos, portanto, a importância de ampliar este estudo e de estimular pesquisas futuras, visando um aconselhamento genético eficiente, com a diminuição do número de casos inconclusivos gerados pelas variantes de significado indeterminado e o acompanhamento clínico das famílias / Introduction: Mutation in BRCA1 and BRCA2 genes are responsible for more than 50% of hereditarian breast and ovarian cancer cases. Nowadays, we still dont know the Brazilian mutation profile for these genes, except when founder mutations occur in specific ethnic groups. Objetives: Detection of mutation and polymorphisms in BRCA1 and BRCA2 genes in 73 breast cancer patients selected for genetic testing. Casuistic and methods: we have realized direct sequencing of BRCA1 and BRCA2 in 73 patients, whose 63 have had breast cancer and showed at least 10% of risk according to Frank, Evans and BRCAPRO, two patients with ovarian cancer and eight healthy individuals of strong family history of cancer linked to mutations in BRCA1 and BRCA2. Results: We have found 60 mutations in BRCA1: 13 missense mutations (including the deleterious R71G), seven synonymous mutations, one frameshift mutation (the deleterious 5382insC), one nonsense mutation (the deleterious R1751X), one in-frame deletion, one 3UTR mutation and 36 intronic variants. In BRCA2 we have found 57 mutations: 26 missense mutations, one 5UTR mutation, 11 synonymous mutations, 14 intronic variants, two nonsense mutations (the deleterious R2318X and R3128X) and three frameshift mutations (5844del5, 6610insTT and 6633del5). No mutation was detected by MLPA technique. Discussion and final considerations: Nine of 73 studied individuals carry deleterious mutations. Among them, the Ashkenazi founder mutation 5382insC has been found in two unrelated patients and it was previously reported by another research group for its high prevalence on a population from São Paulo. Alterations of unknown clinical significance have been found all over BRCA1 and BRCA2 gene extension and are countless. Some of them are shown only in one patient, leading us to think that maybe one or a few might have a pathogenic effect, like 6610insTT, which leads to a BRCA2 incomplete protein and was shown in 3 generations of a family. MLPA technique have not detected large genomic rearrangements in both genes, showing that this kind of mutation is not frequent on our cohort and maybe this genetic alteration characterizes less miscigenated populations. So, we emphasize the importance of enlarge this study and stimulate future researches, aiming an efficient genetic counseling, decreasing inconclusive cases generated by unknown clinical significance variants, and follow up of affected families

En phase somatique, plusieurs mécanismes de réparations de l’ADNinterviennent pour réparer les cassures double brin (CDB) de l’ADN. Enphase méiotique, les CDB de l’ADN engendrées de façon programmées parSpo11 sont réparées par la recombinaison homologue (RH) dont les acteursprincipaux sont Rad51 et Dmc1 aidés de Brca2. Chez Arabidopsis, en absencede Brca2, le déroulement méiotique est perturbé, les chromosomes nes’associent pas en bivalents, ils apparaissent emmêlés. Ainsi, en absencede Brca2, la recombinaison homologue pourrait ne plus être fonctionnelleet les anomalies chromosomiques observées pourraient être le résultat deréparations aberrantes des CDB de l’ADN effectuée par d’autres mécanismesde réparation de l’ADN. Nous avons montrés, chez Arabidopsis, que le Nonhomologous End Joining (NHEJ) et/ou le Single Strand Annealing (SSA),mécanismes de réparation des CDB de l’ADN en phase somatique,n’intervenaient pas en phase méiotique dans la formation des anomaliesobservées en absence de Brca2. Toujours dans l’hypothèse où ces figuresméiotiques soient le résultat de liaisons covalentes, nous avons regardési les ADN-ligases ne pourraient pas être impliquées. Ainsi, nous avons pumontrer que la Ligase 6, ADN-ligase spécifique des plantes, n’avait pas derôle dans les anomalies chromosomiques observées en méiose en absence deBrca2. D’ailleurs la Ligase 6 ne semble pas non plus intervenir dans lesfigures chromosomiques observées chez les mutants rad51 et mnd1. Le rôlede la Ligase 6 n’ayant pas été déterminé lorsque nous avons démarré cetravail, nous avons voulu identifier son le rôle en étudiant le mutantcorrespondant. Le mutant ligase 6 ne présente pas de sensibilité auxstress génotoxiques utilisés ce qui indique que la Ligase 6 ne semble pasintervenir dans la réparation de l’ADN. La mutation dans le gène LIGASE Iest létal à l’état homozygote, de plus nous avons pu observer uneségrégation anormale chez l’hétérozygote mutant pour le gène LIGASE I. Lalétalité du mutant ligase I a été contournée par l’utilisation d’unsystème ARNi pour éteindre l’expression du gène LIGASE I uniquement enméiose. Cependant, l’implication de la Ligase I, dans les anomaliesméiotiques observées en absence de Brca2 n’a pas pu être déterminée.Enfin, nous avons confirmé que, chez Arabidopsis, Xrcc4 avait un rôle dansle NHEJ via son interaction avec la Ligase IV et via la sensibilité dumutant xrcc4 à différents stress génotoxiques. En revanche, Xrcc4-like nesemble pas interagir avec les acteurs du complexe de ligation du NHEJ etle mutant ne présente pas de sensibilité aux stress génotoxique, indiquantque cette protéine n’est pas impliquée dans le NHEJ et plus généralementdans les mécanismes de réparation de l’ADN. / In somatic cells, several mechanisms are involved in the repair of DNAdouble strand breaks (DSB). In meiotic cells, programmed DSBs are causedby Spo11 and repaired by homologous recombination (HR), whose main playersare Rad51 and Dmc1 aided by Brca2. In Arabidopsis, in the absence ofBrca2, meiosis is disturbed, chromosomes do not organize into bivalents,they appear stuck and entangled together. Thus, in the absence of Brca2,HR may be no functional and the chromosomal anomalies we observecouldresult from the aberrant repair of the DNA DSBs due to other mechanisms ofDNA repair. We have shown in Arabidopsis that the homologous end joining(NHEJ) and/or Single Strand Annealing (SSA), mechanisms of DNA DSB repairthat are active in the somatic phase, were not involved in the formationof the meiotic anomalies observed in the absence of Brca2 in meioticcells. Still assuming that these figures are the result of meioticcovalent bond, we checked whether DNA ligases could be involved. Thus, wehave shown that 6 Ligase, DNA ligase specific plants, had no role in thechromosomal abnormalities observed in meiosis in the absence of Brca2.Besides the Ligase 6 does not seem to interfere with the meiotic figuresobserved in rad51 and mnd1 mutants. We wanted to identify the Ligase 6role in studying its mutant. Ligase 6 mutant did not show sensitivity togenotoxic stress. The Ligase 6 does not seem to be involved in DNA repair.The lethality of the ligase I mutant was bypassed with a RNAi constructaimed at extinguishing the gene expression of LIGASE I atmeiosis only.However, the involvement of Ligase I in the meiotic anomalies observed inthe absence of Brca2 could not be determined. Finally, we confirmed that,in Arabidopsis, Xrcc4 has a role in NHEJ through its interaction withligase IV and the sensitivity of the xrcc4 mutant to different genotoxicstress. In contrast, Xrcc4-like does not appear to interact with playersin the NHEJ ligation complex and the mutant shows no sensitivity togenotoxic stress. These result indicated that this protein is not involvedin NHEJ and, more generally in the mechanisms of DNA repair.

Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.